**Part 2**

**Medical Biotechnology** 

72 Innovations in Biotechnology

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### *In Vivo* **Circular RNA Expression by the Permuted Intron-Exon Method**

So Umekage, Tomoe Uehara, Yoshinobu Fujita, Hiromichi Suzuki and Yo Kikuchi *Dept. of Environmental and Life Sciences, Toyohashi University of Technology Japan* 

#### **1. Introduction**

Functional RNAs, *e.g.*, aptamers (Lee *et al*., 2005; Que-Gewirth & Sullenger, 2007), ribozymes (Malhbacher *et al*., 2010), antisense oligonucleotides (Hnik *et al*., 2009), and double-stranded RNA (dsRNA) (Watts & Corey, 2010), hold promise for use as RNA drugs in the near future. However, the linear form of RNA without chemical modifications is rapidly degraded in both human serum and cell extracts due to endogenous nucleases. Therefore, it will likely be necessary to chemically modify these RNA drugs (Pestourie *et al*., 2005; Watts *et al*., 2008) to protect them from nuclease-dependent degradation. In fact, the recently developed aptamer drug pegaptanib sodium (Macugen®; Pfizer) for use against macular degradation consists of 2'-F- or 2'-OCH3-substituted nucleotides, thus preventing its rapid degradation in the ocular environment. Although at present it is the only commercially available RNA drug, we infer from the selling price of Macugen® that similar novel chemically modified RNA drugs are likely to be expensive because production of a chemically modified RNA molecule and scaling up the production yield of the RNA are expensive in principle. Therefore, the development of not only inexpensive but also durable RNA drugs will facilitate the widespread use of easily administered RNA drugs. To address the problems outlined above, our research group has considered *in vivo* circular RNA expression as a model for inexpensive RNA drug production because circular RNA molecules are resistant to exoribonucleases without any chemical modifications under cellular conditions. Therefore, the circular form of RNA would be a promising RNA drug candidate without requiring chemical modification.

Circular RNA can be produced both *in vitro* and *in vivo* using two methodologies. The first makes use of ligase to ligate both ends of the linear form of RNA transcripts (Chen & Sarnow, 1998; Beaudry & Perreault, 1995), while the second uses a spontaneous group I intron selfsplicing system, designated as the permuted intron-exon (PIE) method (Puttaraju & Been, 1992). The latter technique is the only methodology available for *in vivo* circular RNA production because it has no requirement for proteinaceous components, such as ligases. Therefore, the PIE method is a promising economical methodology for producing circular RNA drugs. In this chapter, we describe our circular streptavidin RNA aptamer expression by the PIE method as a model for RNA drug production (Umekage & Kikuchi, 2006, 2007, 2009a, 2009b). Then, we discuss our recent improvements in the circular RNA expression technique, *i.e.*, the **t**andem **o**ne-way transcription of **P**IE (TOP) method, to achieve higher yields of *in vivo*

circular RNA expression. In this system, we achieved production of approximately 0.19 mg of circular RNA from a 1-L culture of the *Escherichia coli* strain JM101Tr. To our knowledge, this is the highest circular RNA expression yield reported to date. Finally, we will discuss *in vivo* circular RNA expression by the marine phototrophic bacterium *Rhodovulum sulfidophilum*. This bacterium produces RNA both within the cell and in the culture medium in nature and produces no RNases in the culture medium (Suzuki *et al*., 2010), whereas strong RNase activity is observed in the culture medium of a conventional *E*. *coli* strain that can be used for RNA production. Therefore, we speculated that *Rdv*. *sulfidophilum* would be a suitable strain for RNA production in the culture medium bypassing the total RNA extraction procedure to break the cell membrane, such as the acid guanidinium thiocyanate phenol chloroform (AGPC) method (Chomczynski & Sacchi, 1987).

#### **2. Group I intron self-splicing and the permuted intron-exon (PIE) method**

Group I intron self-splicing RNA from the ciliate *Tetrahymena* was the first discovered ribozyme (Cech *et al*., 1981). The group I intron sequence has been widely detected in eukaryotes (Cech *et al*., 1981), prokaryotes (Xu *et al*., 1990) and some bacteriophages (Ehrenman *et al*., 1986). This self-splicing does not require any proteinaceous components but does require the presence of Mg2+ and guanosine nucleotides (Cech *et al*., 1981). After self-splicing, the concomitant ligation of the two exons takes place (Fig. 1). This self-splicing mechanism consists of a well-defined two-step transesterification mechanism, and the sequential self-splicing steps take place after formation of the higher-order intron architecture. In the first step, a guanosine nucleotide attacks the phosphate at the 5' splicing site and scission occurs between the upstream exon and the intron, and the guanosine nucleotide is then ligated to the 5' side of the intron. Next, the hydroxyl group of the 3' end of the upstream exon shows nucleophilic attack of the downstream splicing site of the phosphorus, and intron circularisation and exon ligation occur. Therefore, it is assumed that both the 5' end of the 5' half exon and 3' end of the 3' half exon are somehow ligated before self-splicing occurs, and the resulting spliced exon product has a circular conformation. Several biochemical (Galloway-Salbo *et al*., 1990) and structural investigations of group I intron self-splicing (Stahley & Atrobel, 2006) indicated that the peripheral region of the intron architecture and internal open reading frame (ORF) sequence does not participate in formation of the intron architecture and the self-splicing event mentioned above. Theses investigations allowed us to permute the order of the intron and exon sequence without distorting the tertiary structure of the permuted intron architecture. Puttaraju & Been (1992) first reported that circular permutation of the group I intron from both the *Anabena* pretRNA intron and the *Tetrahymena* intron generated a circular RNA exon *in vitro*. Another PIE from the T4 phage group I intron was later shown to be applicable for generating the circular exon (Ford & Ares, 1994). As the exon sequence does not participate in the selfsplicing reaction, the exon sequence in the PIE sequence is replaced with another foreign sequence. Based on this concept, several circular RNAs have been developed by the PIE method, *i.e.*, the tat-activated response (TAR) RNA (Puttaraju & Been, 1995; Bohjanen *et al*., 1996; Bohjanen *et al*., 1997), rev responsive element (Puttaraju & Been, 1995), HDV ribozyme (Puttaraju *et al*., 1993; Puttaraju & Been, 1996), tRNA (Puttaraju & Been, 1992), *Bacillus subtilis* PRNA (Puttaraju & Been; 1996), mRNA encoding GFP (Perriman & Ares, 1998), yeast actin exon (Ford & Ares, 1994), hammerhead ribozyme (Ochi *et al*., 2009), and streptavidin RNA aptamer (Umekage & Kikuchi, 2006, 2007, 2009a, 2009b) (Table 1).

circular RNA expression. In this system, we achieved production of approximately 0.19 mg of circular RNA from a 1-L culture of the *Escherichia coli* strain JM101Tr. To our knowledge, this is the highest circular RNA expression yield reported to date. Finally, we will discuss *in vivo* circular RNA expression by the marine phototrophic bacterium *Rhodovulum sulfidophilum*. This bacterium produces RNA both within the cell and in the culture medium in nature and produces no RNases in the culture medium (Suzuki *et al*., 2010), whereas strong RNase activity is observed in the culture medium of a conventional *E*. *coli* strain that can be used for RNA production. Therefore, we speculated that *Rdv*. *sulfidophilum* would be a suitable strain for RNA production in the culture medium bypassing the total RNA extraction procedure to break the cell membrane, such as the acid guanidinium thiocyanate phenol chloroform

**2. Group I intron self-splicing and the permuted intron-exon (PIE) method** 

RNA aptamer (Umekage & Kikuchi, 2006, 2007, 2009a, 2009b) (Table 1).

Group I intron self-splicing RNA from the ciliate *Tetrahymena* was the first discovered ribozyme (Cech *et al*., 1981). The group I intron sequence has been widely detected in eukaryotes (Cech *et al*., 1981), prokaryotes (Xu *et al*., 1990) and some bacteriophages (Ehrenman *et al*., 1986). This self-splicing does not require any proteinaceous components but does require the presence of Mg2+ and guanosine nucleotides (Cech *et al*., 1981). After self-splicing, the concomitant ligation of the two exons takes place (Fig. 1). This self-splicing mechanism consists of a well-defined two-step transesterification mechanism, and the sequential self-splicing steps take place after formation of the higher-order intron architecture. In the first step, a guanosine nucleotide attacks the phosphate at the 5' splicing site and scission occurs between the upstream exon and the intron, and the guanosine nucleotide is then ligated to the 5' side of the intron. Next, the hydroxyl group of the 3' end of the upstream exon shows nucleophilic attack of the downstream splicing site of the phosphorus, and intron circularisation and exon ligation occur. Therefore, it is assumed that both the 5' end of the 5' half exon and 3' end of the 3' half exon are somehow ligated before self-splicing occurs, and the resulting spliced exon product has a circular conformation. Several biochemical (Galloway-Salbo *et al*., 1990) and structural investigations of group I intron self-splicing (Stahley & Atrobel, 2006) indicated that the peripheral region of the intron architecture and internal open reading frame (ORF) sequence does not participate in formation of the intron architecture and the self-splicing event mentioned above. Theses investigations allowed us to permute the order of the intron and exon sequence without distorting the tertiary structure of the permuted intron architecture. Puttaraju & Been (1992) first reported that circular permutation of the group I intron from both the *Anabena* pretRNA intron and the *Tetrahymena* intron generated a circular RNA exon *in vitro*. Another PIE from the T4 phage group I intron was later shown to be applicable for generating the circular exon (Ford & Ares, 1994). As the exon sequence does not participate in the selfsplicing reaction, the exon sequence in the PIE sequence is replaced with another foreign sequence. Based on this concept, several circular RNAs have been developed by the PIE method, *i.e.*, the tat-activated response (TAR) RNA (Puttaraju & Been, 1995; Bohjanen *et al*., 1996; Bohjanen *et al*., 1997), rev responsive element (Puttaraju & Been, 1995), HDV ribozyme (Puttaraju *et al*., 1993; Puttaraju & Been, 1996), tRNA (Puttaraju & Been, 1992), *Bacillus subtilis* PRNA (Puttaraju & Been; 1996), mRNA encoding GFP (Perriman & Ares, 1998), yeast actin exon (Ford & Ares, 1994), hammerhead ribozyme (Ochi *et al*., 2009), and streptavidin

(AGPC) method (Chomczynski & Sacchi, 1987).

Fig. 1. Group I intron self-splicing RNA and the permuted intron-exon (PIE). Predicted secondary structure of the *td* group I intron sequence (upper left side) and the PIE sequence (upper right side). The pink and black lines show the intron sequence and the green line indicates the internal ORF sequence. Coloured horizontal lines shown in the middle of this figure illustrate the circular permutation of the *td* intron. In the normal *td* intron (left side), after transesterification, the exon sequence is ligated. In the permuted *td* intron (right side), the exon sequence is circularised and a split intron sequence appears. Circularisation of the exon sequence by the PIE method also requires magnesium ions and guanosine nucleotides.

These results confirmed the availability of the PIE method to yield a wide variety of circular RNAs. As circularisation is driven only by magnesium ions and guanosine nucleotides, the circularisation of RNA in *E. coli* (Puttaraju & Been, 1996; Perriman & Ares, 1998; Umekage & Kikuchi, 2007, 2009a, 2009b) and *Saccharomyces cerevisiae* (Ford & Ares, 1994; Puttaraju & Been, 1996) have been demonstrated, and the *in vivo* expressed circular RNAs reported above are functionary active. Our group also showed that the circular streptavidin RNA aptamer produced both *in vitro* and in *E*. *coli* (Umekage & Kikuchi, 2006, 20007, 2009a, 2009b), and the expressed circular streptavidin RNA aptamer was purified from the total RNA fraction by the solid-phase DNA probe method (Suzuki *et al*., 2002). This is the first evidence that both *in vitro* and *in vivo* circularisation of an RNA aptamer and the *in vivo* circularised RNA generated by the PIE method can be purified (Umekage & Kikuchi, 2009a).


Table 1. Summary of circular RNA production. "Category of circularised RNA": Source of the circularised exon sequence. "Intron": Source of the intron sequence used for constructing the PIE sequence. "Expression": Circular RNA production *in vitro* or *in vivo* (the expression host strain is listed).

On the other hand, we found that this circularisation affected the original activity of the linear form of functional RNAs. The dissociation constant (*K*d) of the circular streptavidin RNA aptamer increased (Umekage & Kikuchi, 2009a) and ribozyme activity of the hammerhead ribozyme decreased (Ochi *et al*., 2009). These observations suggest that structural constraints were induced by circularisation. Although it is also important to take into consideration the circular RNA structure before constructing the PIE sequence, it is difficult to predict the tertiary structure of the circularised RNA molecule.

Therefore, optimisation of the circularised sequence would be required involving randomising the spacer sequence, inserting the poly(A) sequence, *etc*. We succeeded in recovering the functional activity of the circular hammerhead ribozyme by adding a poly(A) spacer between the ribozyme sequence and the indispensable linkage sequence derived from the exon sequence for circularisation by the PIE method (Ochi *et al*., 2009), and the recovered ribozyme activity of the circular hammerhead ribozyme was dependent on the length of the poly(A) spacer (Ochi *et al*., 2009).

HDV ribozyme Anabaena *in vitro* Puttaraju *et al*., 1993;

element RNA Anabaena *in vitro* Puttaraju & Been, 1995

hammerhead ribozyme T4 phage *in vitro* Ochi *et al*., 2009

Table 1. Summary of circular RNA production. "Category of circularised RNA": Source of the circularised exon sequence. "Intron": Source of the intron sequence used for constructing the PIE sequence. "Expression": Circular RNA production *in vitro* or *in vivo* (the expression

On the other hand, we found that this circularisation affected the original activity of the linear form of functional RNAs. The dissociation constant (*K*d) of the circular streptavidin RNA aptamer increased (Umekage & Kikuchi, 2009a) and ribozyme activity of the hammerhead ribozyme decreased (Ochi *et al*., 2009). These observations suggest that structural constraints were induced by circularisation. Although it is also important to take into consideration the circular RNA structure before constructing the PIE sequence, it is

Therefore, optimisation of the circularised sequence would be required involving randomising the spacer sequence, inserting the poly(A) sequence, *etc*. We succeeded in recovering the functional activity of the circular hammerhead ribozyme by adding a poly(A) spacer between the ribozyme sequence and the indispensable linkage sequence derived from the exon sequence for circularisation by the PIE method (Ochi *et al*., 2009), and the recovered ribozyme activity of the circular hammerhead ribozyme was dependent on the

*in vitro* & *E*. *coli* DH5α & *S. cerevisiae* IH1097

*in vitro* & *E*. *coli* DH5α& *S. cerevisiae* IH1097

*in vitro* & *E*. *coli* JM109(DE3) & *E*. *coli* JM101Tr

*in vitro* Puttaraju & Been, 1992

*<sup>E</sup>*. *coli* BL21(DE3) Puttaraju & Been, 1996

*<sup>E</sup>*. *coli* BL21(DE3) Perriman & Ares, 1998

DSM 1374T this study (see 2.4)

Puttaraju & Been, 1996

Ford & Ares, 1994

Ford & Ares, 1994

Puttaraju & Been, 1995 Bohjanen *et al*., 1996; Bohjanen *et al*., 1997

Umekage & Kikuchi, 2006, 2007, 2009a, 2009b

circularised RNA Intron Expression Reference

Anabaena & Tetrahymena

RNA Anabaena *in vitro* 

GFP ORF T4 phage *in vitro* &

streptavidin aptamer T4 phage *Rdv. sulfidophilum*

difficult to predict the tertiary structure of the circularised RNA molecule.

*B. subtilis* PRNA Anabaena *in vitro* &

Category of

tRNA exon from *Anabaena* PCC7120

Rev-responsive

tat-activated response

host strain is listed).

*td* exon from T4 phage T4 phage

actin ORF from yeast T4 phage

streptavidin aptamer T4 phage

length of the poly(A) spacer (Ochi *et al*., 2009).

Fig. 2. Schematic representation of the plasmid pGEM-3E5T7t, and the predicted secondary structure of the circular streptavidin RNA aptamer produced from the plasmid. (A) The figure shows pGEM-3E5T7t. The PIE sequence is located between the T7 promoter sequence and the T7 terminator sequence. The PIE sequence consists of the 3' half intron, streptavidin aptamer sequence and 5' half intron sequence. The intron sequence is derived from the *td* intron of bacteriophage T4. (B) Predicted secondary structure of the circular streptavidin RNA aptamer. Upper and lower case letters indicate the aptamer sequence and exon sequence derived from the original exon sequence of the *td* gene, respectively. Streptavidin RNA is derived from the S1 aptamer reported by Srisawat & Engelke (2001). The thick arrow represents the self-ligated junction. The thin arrow (anticlockwise) indicates the orientation of the circular RNA from 5' to 3'. The thin line and black dot represent Watson– Crick base pairing and G-U Wobble base pairing, respectively.

#### **2.1** *In vivo* **circular streptavidin RNA aptamer expression by the PIE method**

To demonstrate *in vivo* circular RNA expression, our group designed the circular streptavidin RNA aptamer as a model RNA dug and the PIE sequence for production of the circular streptavidin RNA aptamer. The PIE sequence consists of the 3' half intron, aptamer sequence and 5' half intron sequence in this order (Fig. 2A), with omission of the internal ORF sequence (Fig. 1, shown as the green-coloured line) in the *td* intron for this PIE construction that does not participate in the self-splicing reaction of the intron. We constructed a circular streptavidin RNA aptamer expression vector, pGEM-3E5T7t, which consists of three parts: the T7 RNA promoter, the PIE sequence and the T7 terminator sequence in the multicloning site in the standard cloning vector, pGEM-3Z (Promega) (Fig. 2A). The streptavidin RNA aptamer sequence in the PIE sequence was derived from the S1 aptamer sequence reported by Srisawat & Engelke (2001), and the intron sequence derived from the *td* intron of bacteriophage T4. Both ends of the resulting circular streptavidin RNA aptamer sequence were ligated by an indispensable linker sequence derived from a partial exon sequence of the *td* gene of bacteriophage T4 (Fig. 2b, lower case).

To express the circular streptavidin RNA aptamer *in vivo*, pGEM-3E5T7t was transformed into JM109(DE3) (*endA*1 *recA*1, *gyr*A96, *thi*-1, *hsdR*17 (rK- , mK+), *relA*1, *supE*44, (*lac*-*proAB*), [F', *traD*36, *proAB*, *lacI*qZ, M15], (DE3)) (Promega), which encodes an isopropyl-β-Dthiogalactopyranoside (IPTG)-inducible T7 RNA polymerase on its genomic sequence. The full-growth culture of JM109(DE3) harbouring pGEM-3E5T7t was transferred into 1 L of fresh LB broth and cultured until the optical density at 600 nm (OD600) reached 0.7 at 30C. Then, IPTG was added to a final concentration of 0.4 mM and circular RNA expression was induced by cultivation for 2 h at 30C with vigorous shaking. After extraction of the total RNA, which included the circular RNA, using the AGPC method to break the cell membrane (Chomczynski & Sacchi, 1987), the circular RNA expression in the total RNA fraction was monitored by ethidium bromide staining and Northern blotting analysis.

We performed two-dimensional (2D) denaturing polyacrylamide gel electrophoresis (2D PAGE) (Schumacher, 1983; Feldstein, 2007) to monitor whether the circular RNA was present in the recovered total RNA fraction. This 2D-electrophoresis is based on the differences in migration behaviour between linear and circular RNA under denaturing gel conditions. Unlike linear RNA, migration of a circular RNA molecule of the same length varies with the acrylamide and/or bis-acrylamide concentration on denaturing polyacrylamide electrophoresis (PAGE). Therefore, after 2D denaturing gelelectrophoresis, the linear RNA fraction has migrated in the diagonal direction, whereas the circular RNA appears beside the diagonal line of the linear molecule. Both ethidium bromide staining and Northern blotting analysis showed a single spot beside the diagonal migration line, indicating that the circular RNA was present in the total RNA fraction. This spot was eluted and subjected to partial alkaline digestion. The partially digested nicked circular RNA migrated faster than the intact circular molecule on denaturing 10% PAGE, confirming that the eluted RNA was a circular form. The degradation products of circular RNA were not detected by Northern blotting analysis. This clearly showed that the circular RNA expressed in *E*. *coli* cells was protected against exonuclease-induced degradation, such as that induced by ribonuclease II (RNase II) (Frazão *et al*., 2006). The expression level of the circular RNA aptamer was determined to be 2.5 ± 0.46 ng per 1 μg of total RNA by Northern blotting analysis. The yield of the circular RNA aptamer in that total RNA was estimated to be approximately 24 μg.

Next, we developed a circular RNA purification method for future inexpensive and economical RNA production and purification. We showed that the circular streptavidin RNA aptamer was successfully purified with the solid-phase DNA probe technique (Suzuki *et al*., 2002). To purify the circular RNA produced by the PIE method, we designed a 5' biotinylated DNA probe that can hybridise with the circular RNA and the circular RNA-DNA hybrid can be easily trapped using a streptavidin-coated column. The trapped circular RNA-DNA hybrid is also denatured with a high concentration of urea (7 M) solution and the circular RNA is eluted. We can also choose the elution buffer such that the DNA probe

sequence in the multicloning site in the standard cloning vector, pGEM-3Z (Promega) (Fig. 2A). The streptavidin RNA aptamer sequence in the PIE sequence was derived from the S1 aptamer sequence reported by Srisawat & Engelke (2001), and the intron sequence derived from the *td* intron of bacteriophage T4. Both ends of the resulting circular streptavidin RNA aptamer sequence were ligated by an indispensable linker sequence derived from a partial

To express the circular streptavidin RNA aptamer *in vivo*, pGEM-3E5T7t was transformed into JM109(DE3) (*endA*1 *recA*1, *gyr*A96, *thi*-1, *hsdR*17 (rK-, mK+), *relA*1, *supE*44, (*lac*-*proAB*), [F', *traD*36, *proAB*, *lacI*qZ, M15], (DE3)) (Promega), which encodes an isopropyl-β-Dthiogalactopyranoside (IPTG)-inducible T7 RNA polymerase on its genomic sequence. The full-growth culture of JM109(DE3) harbouring pGEM-3E5T7t was transferred into 1 L of fresh LB broth and cultured until the optical density at 600 nm (OD600) reached 0.7 at 30C. Then, IPTG was added to a final concentration of 0.4 mM and circular RNA expression was induced by cultivation for 2 h at 30C with vigorous shaking. After extraction of the total RNA, which included the circular RNA, using the AGPC method to break the cell membrane (Chomczynski & Sacchi, 1987), the circular RNA expression in the total RNA fraction was monitored by ethidium bromide staining and Northern blotting analysis.

We performed two-dimensional (2D) denaturing polyacrylamide gel electrophoresis (2D PAGE) (Schumacher, 1983; Feldstein, 2007) to monitor whether the circular RNA was present in the recovered total RNA fraction. This 2D-electrophoresis is based on the differences in migration behaviour between linear and circular RNA under denaturing gel conditions. Unlike linear RNA, migration of a circular RNA molecule of the same length varies with the acrylamide and/or bis-acrylamide concentration on denaturing polyacrylamide electrophoresis (PAGE). Therefore, after 2D denaturing gelelectrophoresis, the linear RNA fraction has migrated in the diagonal direction, whereas the circular RNA appears beside the diagonal line of the linear molecule. Both ethidium bromide staining and Northern blotting analysis showed a single spot beside the diagonal migration line, indicating that the circular RNA was present in the total RNA fraction. This spot was eluted and subjected to partial alkaline digestion. The partially digested nicked circular RNA migrated faster than the intact circular molecule on denaturing 10% PAGE, confirming that the eluted RNA was a circular form. The degradation products of circular RNA were not detected by Northern blotting analysis. This clearly showed that the circular RNA expressed in *E*. *coli* cells was protected against exonuclease-induced degradation, such as that induced by ribonuclease II (RNase II) (Frazão *et al*., 2006). The expression level of the circular RNA aptamer was determined to be 2.5 ± 0.46 ng per 1 μg of total RNA by Northern blotting analysis. The yield of the circular RNA aptamer in that

Next, we developed a circular RNA purification method for future inexpensive and economical RNA production and purification. We showed that the circular streptavidin RNA aptamer was successfully purified with the solid-phase DNA probe technique (Suzuki *et al*., 2002). To purify the circular RNA produced by the PIE method, we designed a 5' biotinylated DNA probe that can hybridise with the circular RNA and the circular RNA-DNA hybrid can be easily trapped using a streptavidin-coated column. The trapped circular RNA-DNA hybrid is also denatured with a high concentration of urea (7 M) solution and the circular RNA is eluted. We can also choose the elution buffer such that the DNA probe

exon sequence of the *td* gene of bacteriophage T4 (Fig. 2b, lower case).

total RNA was estimated to be approximately 24 μg.

still binds to the solid-phase and it can be reused for another round of RNA purification (data not shown). Using a streptavidin-coated column (GE Healthcare), the circular streptavidin RNA aptamer was eluted under denaturing conditions and yielded 21 μg of the circular RNA (about 88% recovery) from 1 L of *E*. *coli* cell culture. Electrophoretic mobility shift assay (EMSA) also showed that the purified circular streptavidin RNA aptamer from JM109(DE3) retained its binding properties toward streptavidin.

To verify the suitability of the circular RNA for future RNA therapeutic uses, we measured the half-life of the purified circular RNA aptamer in HeLa cell extracts as a model of intracellular conditions. The estimated half-life of the purified circular streptavidin RNA aptamer was at least 1,386 min, while that of the S1 aptamer, which is the linear form of the streptavidin RNA aptamer, was 43 min. These observations suggested that the circular RNA escapes exoribonuclease-dependent RNA degradation under intracellular conditions. However, the circular RNA degraded completely within 15 s in 25% human serum. This is reasonable because human serum contains the RNaseA family ribonucleases (Haupenthal *et al*., 2006; Haupenthal *et al*., 2007; Turner *et al*., 2007). These findings indicated that the circular RNA would be useful under cellular conditions only when delivered into the cell in a precise manner, *e.g.*, by using cationic liposomes (Sioud & Sorensen, 2003; Sorensen *et al*., 2003) or virus vector systems (Mi *et al*., 2006), to prevent RNaseA family ribonuclease-dependent degradation.

#### **2.2 Constitutive** *in vivo* **circular streptavidin RNA aptamer expression by the PIE method**

We then considered the constitutive circular RNA expression, as the previous expression procedure requires monitoring of the optical density for optimal IPTG induction (see 2.1). For constitutive expression of the RNA sequence in *E*. *coli*, we followed the procedure of Ponchon & Dardel (2004). They reported that the M3 vector containing the strong constitutively active lipoprotein (*lpp*) promoter, which is one of the strongest promoters in *E. coli* (Movva *et al*., 1978; Inoue *et al*., 1985), is applicable for *in vivo* RNA expression in the *E*. *coli* strain JM101Tr (Δ(*lac pro*), *supE*, *thi*, *recA56*, *srl-300*.:Tn*10*, (*F'*, *traD36*, *proAB*, *lacIq*, *lacZ*, Δ*M15*)). In addition, total RNA expression in JM101Tr is higher than that of JM109(DE3) (our unpublished observation).

Before constructing the constitutive PIE expression plasmid, we replaced the original tRNAMet sequence between the *lpp* promoter and *rrn*C terminator sequence in the M3 vector with the PIE sequence from pGEM-3E5T7t. The resulting expression vector is designated as pM3-3E5. The PIE sequence was amplified from the PIE sequence in pGEM-3E5. After transformation of pM3-3E5 into the JM101Tr strain, cell density (OD600) was measured at several time points during cultivation and 1-mL aliquots were collected from 200 mL of 2×YT medium. Total RNA was recovered by ISOGEN (Nippon Gene) and Northern blotting analysis was performed. At various time points in culture from early logarithmic phase to stationary phase, circular RNA was visible in each lane on electrophoretic analysis even with ethidium bromide staining. The presence of circular RNA, but not the nicked form, was clearly detected on Northern blotting analysis and the amount of circular RNA increased with cell growth. These results suggested that the *lpp* promoter was active and drove expression of the PIE sequence without any induction. The stain JM101Tr is positive for ribonucleases, such as ribonuclease II (Frazão *et al*., 2006). Therefore, these observations indicated that the circular RNA also accumulated in the *E*. *coli* JM101Tr strain, escaping degradation by exonucleases as seen in the previous expression system described in Section 2.1. The resulting yield of circular RNA after 18 h of cultivation at 30°C was estimated to be 3.6 ± 0.15 ng per 1 μg of total RNA, which was approximately 1.5-fold higher than that of the previous method (Umekage & Kikuchi, 2009a) (see 2.1). These observations indicated effective constitutive circular RNA expression in this system.

#### **2.3 Improving circular RNA expression with the tandem one-way transcription of PIE (TOP) technique**

To augment the circular RNA expression in *E*. *coli*, we developed the TOP (**t**andem **o**ne-way transcription of **P**IE) technique, which is a simple methodology for increasing the copy number of the PIE sequence in a single plasmid. The TOP technique is shown schematically in Fig. 3A. With this technique, it is easy to amplify the copy number by sequential insertion of the transcriptional unit in a single plasmid (Fig. 3B). First, we amplified the transcriptional unit, which consists of the *lpp* promoter, PIE sequence and *rrn*C terminator in pM3-3E5 (see Section 2.1) with both the 5' flanking sequence containing *Kpn*I–*Xho*I sites and the 3' flanking sequence containing a *Sal*I site. Next, we digested the amplified sequence with *Kpn*I and *Sal*I, and the resulting fragment was inserted into the M3 plasmid doubledigested with *Kpn*I and *Xho*I. The digested *Xho*I site on the M3 plasmid and the *Sal*I site on the amplified fragment can hybridise with mutual 3' protruding ends of the palindromic TCGA sequence, and the resulting ligated fragment forms the sequence GTCGAG, which can be digested with neither *Xho*I nor *Sal*I (Fig. 3B). Therefore, the inserted sequence is as follows: 5'-*Kpn*I-*Xho*I-*lpp* promoter-PIE sequence-*rrn*C terminator sequence-GTCGAG site-3' (Fig. 3C). Thus, the subsequent transcriptional unit can be inserted at the *Kpn*I–*Xho*I site. We constructed four series of pTOP vectors using M3 designated as pTOP(I), pTOP(II), pTOP(III) and pTOP(IV) in parallel with the number of inserted transcriptional units.

This pTOP plasmid has a constitutive *lpp* promoter and therefore the constitutive expression of the PIE sequence in JM101Tr is expected, similar to that using the constitutive expression plasmid pM3-3E5 described in Section 2.2. To demonstrate the availability of the TOP technique, we then analysed the circular streptavidin RNA aptamer expression in *E*. *coli* by Northern blotting analysis and we detected that the circular RNA expression was expressed in all pTOP vectors (pTOP(I), (II), (III) and (IV) ) (Fig. 3D).

As shown in the Fig. 3D., the circular RNA expression increased until two tandem insertions of the PIE, and the expression yields were almost the same using pTOP(II) and pTOP(III) (Table 2). These results indicated that the TOP system is a potentially useful and simple methodology for increasing circular RNA expression in *E*. *coli*. The circular RNA expression using pTOP(II) was estimated to be about 9.7 ± 1.0 ng per 1 μg of total RNA after 18 h of cultivation and this yield was approximately 2.7-fold higher than that of the expression procedure using the pM3-3E5 system as described in Section 2.2. In addition, the circular RNA expression in 1 L of culture medium was estimated to be approximately 0.19 mg, which is the highest yield of circular RNA expression in *E*. *coli* reported to date. In contrast, expression of the circular RNA dropped dramatically when using pTOP(IV); the reason for this drop in expression level is not yet clear. To address this problem, we collected pTOP(IV) after 18 h of cultivation in JM101Tr and the plasmid was single-digested with *Hin*dIII and then subjected to 1% agarose gel electrophoresis. A few single-digested pTOP(IV) fragments

indicated that the circular RNA also accumulated in the *E*. *coli* JM101Tr strain, escaping degradation by exonucleases as seen in the previous expression system described in Section 2.1. The resulting yield of circular RNA after 18 h of cultivation at 30°C was estimated to be 3.6 ± 0.15 ng per 1 μg of total RNA, which was approximately 1.5-fold higher than that of the previous method (Umekage & Kikuchi, 2009a) (see 2.1). These observations indicated

**2.3 Improving circular RNA expression with the tandem one-way transcription of PIE** 

To augment the circular RNA expression in *E*. *coli*, we developed the TOP (**t**andem **o**ne-way transcription of **P**IE) technique, which is a simple methodology for increasing the copy number of the PIE sequence in a single plasmid. The TOP technique is shown schematically in Fig. 3A. With this technique, it is easy to amplify the copy number by sequential insertion of the transcriptional unit in a single plasmid (Fig. 3B). First, we amplified the transcriptional unit, which consists of the *lpp* promoter, PIE sequence and *rrn*C terminator in pM3-3E5 (see Section 2.1) with both the 5' flanking sequence containing *Kpn*I–*Xho*I sites and the 3' flanking sequence containing a *Sal*I site. Next, we digested the amplified sequence with *Kpn*I and *Sal*I, and the resulting fragment was inserted into the M3 plasmid doubledigested with *Kpn*I and *Xho*I. The digested *Xho*I site on the M3 plasmid and the *Sal*I site on the amplified fragment can hybridise with mutual 3' protruding ends of the palindromic TCGA sequence, and the resulting ligated fragment forms the sequence GTCGAG, which can be digested with neither *Xho*I nor *Sal*I (Fig. 3B). Therefore, the inserted sequence is as follows: 5'-*Kpn*I-*Xho*I-*lpp* promoter-PIE sequence-*rrn*C terminator sequence-GTCGAG site-3' (Fig. 3C). Thus, the subsequent transcriptional unit can be inserted at the *Kpn*I–*Xho*I site. We constructed four series of pTOP vectors using M3 designated as pTOP(I), pTOP(II), pTOP(III) and pTOP(IV) in parallel with the number of inserted transcriptional units.

This pTOP plasmid has a constitutive *lpp* promoter and therefore the constitutive expression of the PIE sequence in JM101Tr is expected, similar to that using the constitutive expression plasmid pM3-3E5 described in Section 2.2. To demonstrate the availability of the TOP technique, we then analysed the circular streptavidin RNA aptamer expression in *E*. *coli* by Northern blotting analysis and we detected that the circular RNA expression was expressed

As shown in the Fig. 3D., the circular RNA expression increased until two tandem insertions of the PIE, and the expression yields were almost the same using pTOP(II) and pTOP(III) (Table 2). These results indicated that the TOP system is a potentially useful and simple methodology for increasing circular RNA expression in *E*. *coli*. The circular RNA expression using pTOP(II) was estimated to be about 9.7 ± 1.0 ng per 1 μg of total RNA after 18 h of cultivation and this yield was approximately 2.7-fold higher than that of the expression procedure using the pM3-3E5 system as described in Section 2.2. In addition, the circular RNA expression in 1 L of culture medium was estimated to be approximately 0.19 mg, which is the highest yield of circular RNA expression in *E*. *coli* reported to date. In contrast, expression of the circular RNA dropped dramatically when using pTOP(IV); the reason for this drop in expression level is not yet clear. To address this problem, we collected pTOP(IV) after 18 h of cultivation in JM101Tr and the plasmid was single-digested with *Hin*dIII and then subjected to 1% agarose gel electrophoresis. A few single-digested pTOP(IV) fragments

effective constitutive circular RNA expression in this system.

in all pTOP vectors (pTOP(I), (II), (III) and (IV) ) (Fig. 3D).

**(TOP) technique** 

Fig. 3. Construction of the pTOP vectors, and the availability of the TOP method for generating circular RNA in JM101Tr. (A) Outline of the TOP method. (B) Illustration of sequential insertion of the PIE sequence into the same plasmid. First, *Kpn*I and *Xho*I double digested plasmid and *Kpn*I and *Sal*I double digested insertion sequence were prepared. Both the *Kpn*I site from the plasmid and the insertion sequence are ligated and the *Xho*I-digested site in the plasmid and the *Sal*I-digested site in the insertion sequence are ligated, resulting in the sequence GTCGAG at the 3' side of the inserted site. (C) Nucleotide sequence of one unit of the TOP system. Arrows represent splicing positions of this PIE sequence: yellow, the PIE sequence; blue box, *lpp* promoter sequence; italicised sequence in the blue box, –35 and –

10 regions of the *lpp* promoter; red upper case letters, aptamer sequence and *rrn*C terminator sequence; lower case letters in the yellow region, intron sequence of the *td* gene; bold lower case letters, exon sequence of the *td* gene; bold, circularised sequence; boxed sequence, ligated sites. (D) Northern blotting analysis of the circular RNA expression by each pTOP series. Total RNA derived from JM101Tr containing the *in vivo* expressed circular streptavidin RNA aptamer was fractionated by 10% denaturing PAGE. In addition, the circular RNA expression monitored using the 32P-labelled complementary oligo-DNA probe of the aptamer sequence (5'-CCAATATTAAACGGTAGACCCAAGAAAACATC-3'). 5S rRNA was monitored as an internal control using the 32P-labelled complementary oligo-DNA probe sequence (5'- GCGCTACGGCGTTCACTTC-3'). Arrows indicate the migration positions of the circular RNA (circular), nicked RNA (nicked) and 5S rRNA. Circular RNA control marker (M) was prepared by *in vitro* transcription (Umekage & Kikuchi, 2009a). "-", Total RNA from JM101Tr; "M3", negative control of the TOP system lacking the PIE sequence. Roman numerals I, II, III and IV represent the total RNA from JM101Tr harbouring pTOP(I), pTOP(II), pTOP(III) and pTOP(IV), respectively.

showed unexpected migration behaviour (data not shown), suggesting that it was difficult for pTOP(IV) to undergo replication in JM101Tr during 18 h of cultivation. Although the expressional host strain JM101tr has the *recA56* mutant, which results in defects in recombination, this genetic mutation is not sufficient to confer stability on pTOP(IV). This instability of pTOP(IV) in JM101Tr indicates the necessity for optimisation of the TOP technique for further augmentation of circular RNA expression; *e.g.*, optimisation of the intervening sequence between the two transcriptional units, considering the direction of transcription, changing the expressional host to a strain lacking another gene that results in defective recombination, such as *sbcB*, *C* or another *rec* gene (Palmer *et al*., 1995), and optimising the copy number of PIE sequences in the single transcriptional unit to avoid accumulation of *lpp* promoter in the single plasmid.

#### **2.4 Circular RNA expression by the marine phototrophic bacterium** *Rhodovulum sulfidophilum*

Finally, we would like to discuss our new project to develop an economical and efficient method for RNA production using the marine phototrophic bacterium *Rdv*. *sulfidophilum*  (Fig. 4), taking advantage of its unique characteristics in that nucleic acids are produced extracellularly (Suzuki *et al*., 2010). In addition this bacterium produces no RNases in the culture medium (Suzuki *et al*., 2010). Although the mechanism of extracellular RNA production by this bacterium has not been fully characterised, this extracellular RNA expression system represents an economical and efficient methodology for RNA production as it is only necessary to collect the culture medium containing extracellularly produced RNA and purify the RNA of interest with a column bypassing the need for a cell extraction procedure using phenol or various other extraction reagents to rupture the cell membrane.

We began by constructing the engineered circular RNA expression plasmid, pRCSA, based on the broad-host range plasmid pCF1010 (Lee & Kaplan, 1995). The PIE sequence was amplified from pGEM-3E5T7t, and the *rrn*A promoter and *puf* terminator sequence were amplified from the genomic DNA of *Rdv*. *sulfidophilum* DSM 1374T (Hansen & Veldkamp, 1973; Hiraishi & Ueda, 1994). The resulting amplified DNA fragments were inserted into pCF1010 to give pRCSA, which was then transformed into *Rdv*. *sulfidophilum* DSM 1374T by conjugation using the mobilising *E*. *coli* strain S-17 as a plasmid donor (Simon *et al*., 1983). The heat shock transformation method can also be used (unpublished observation) (Fornari & Kaplan, 1982). The transformed *Rdv*. *sulfidophilum* DSM 1374T was cultured under anaerobic conditions under incandescent illumination (about 5,000 lx) for 12 – 16 h at 25°C in PYS-M medium (Nagashima *et al*., 1997, Suzuki *et al*., 2010). Cultured cells were harvested and the total intracellular RNA was extracted with the AGPC method. The estimated yield of the intracellular circular RNA was approximately 1.3 ng per 1 L of culture medium by Northern blotting analysis. On the other hand, the circular RNA expression in the culture medium was barely detected by Northern blotting analysis; however, RT-PCR analysis demonstrated the existence of circular RNA in the cultured medium (data not shown). At present, neither intracellular nor extracellular expression of the circular RNA aptamer can be achieved at practical levels for economic and efficient circular RNA expression, and the overall improvement of RNA expression using this bacterium is strongly promoted.

Fig. 4. Overview for circular RNA expression using *Rdv*. *sulfidophilum* DSM 1374T. Circular RNA expression plasmid, pRCSA, was transformed into *Rdv*. *sulfidophilum* DSM 1374T by conjugation using the mobilising *E*. *coli* strain S-17 (Simon *et al*., 1983) or by direct transformation using the heat shock method (Fornari & Kaplan, 1982). The transformed *Rdv*. *sulfidophilum* was grown under anaerobic-light conditions. The PIE sequence in pRCSA was transcribed with the endogenous RNA polymerase and circular RNA was generated from the PIE sequence. The circular RNA produced inside the cell was released extracellularly into the culture medium.

#### **3. Conclusions**

84 Innovations in Biotechnology

10 regions of the *lpp* promoter; red upper case letters, aptamer sequence and *rrn*C terminator sequence; lower case letters in the yellow region, intron sequence of the *td* gene; bold lower case letters, exon sequence of the *td* gene; bold, circularised sequence; boxed sequence, ligated sites. (D) Northern blotting analysis of the circular RNA expression by each pTOP

showed unexpected migration behaviour (data not shown), suggesting that it was difficult for pTOP(IV) to undergo replication in JM101Tr during 18 h of cultivation. Although the expressional host strain JM101tr has the *recA56* mutant, which results in defects in recombination, this genetic mutation is not sufficient to confer stability on pTOP(IV). This instability of pTOP(IV) in JM101Tr indicates the necessity for optimisation of the TOP technique for further augmentation of circular RNA expression; *e.g.*, optimisation of the intervening sequence between the two transcriptional units, considering the direction of transcription, changing the expressional host to a strain lacking another gene that results in defective recombination, such as *sbcB*, *C* or another *rec* gene (Palmer *et al*., 1995), and optimising the copy number of PIE sequences in the single transcriptional unit to avoid

**2.4 Circular RNA expression by the marine phototrophic bacterium** *Rhodovulum* 

Finally, we would like to discuss our new project to develop an economical and efficient method for RNA production using the marine phototrophic bacterium *Rdv*. *sulfidophilum*  (Fig. 4), taking advantage of its unique characteristics in that nucleic acids are produced extracellularly (Suzuki *et al*., 2010). In addition this bacterium produces no RNases in the culture medium (Suzuki *et al*., 2010). Although the mechanism of extracellular RNA production by this bacterium has not been fully characterised, this extracellular RNA expression system represents an economical and efficient methodology for RNA production as it is only necessary to collect the culture medium containing extracellularly produced RNA and purify the RNA of interest with a column bypassing the need for a cell extraction procedure using phenol or various other extraction reagents to rupture the cell membrane. We began by constructing the engineered circular RNA expression plasmid, pRCSA, based on the broad-host range plasmid pCF1010 (Lee & Kaplan, 1995). The PIE sequence was amplified from pGEM-3E5T7t, and the *rrn*A promoter and *puf* terminator sequence were amplified from the genomic DNA of *Rdv*. *sulfidophilum* DSM 1374T (Hansen & Veldkamp, 1973; Hiraishi & Ueda, 1994). The resulting amplified DNA fragments were inserted into pCF1010 to give pRCSA, which was then transformed into *Rdv*. *sulfidophilum* DSM 1374T by

series. Total RNA derived from JM101Tr containing the *in vivo* expressed circular streptavidin RNA aptamer was fractionated by 10% denaturing PAGE. In addition, the circular RNA expression monitored using the 32P-labelled complementary oligo-DNA probe of the aptamer sequence (5'-CCAATATTAAACGGTAGACCCAAGAAAACATC-3'). 5S rRNA was monitored as an internal control using the 32P-labelled complementary oligo-DNA probe sequence (5'- GCGCTACGGCGTTCACTTC-3'). Arrows indicate the migration positions of the circular RNA (circular), nicked RNA (nicked) and 5S rRNA. Circular RNA control marker (M) was prepared by *in vitro* transcription (Umekage & Kikuchi, 2009a). "-", Total RNA from JM101Tr; "M3", negative control of the TOP system lacking the PIE sequence. Roman numerals I, II, III and IV represent the total RNA from JM101Tr

harbouring pTOP(I), pTOP(II), pTOP(III) and pTOP(IV), respectively.

accumulation of *lpp* promoter in the single plasmid.

*sulfidophilum*

Our circular streptavidin RNA aptamer expression system described in Sections 2.1, 2.2 and 2.3 is summarised in Table 2. To our knowledge, the TOP method is the most effective means of circular RNA expression, and the *in vivo* constitutive RNA expression is suitable for circular RNA expression, as the spontaneously expressed circular RNA can exist stably within the cell avoiding endogenous exoribonuclease-dependent degradation. By using the circular streptavidin RNA aptamer expression plasmid pTOP(II) and *E*. *coli* JM101Tr as a host stain, the expression yield of the circular RNA was estimated to be approximately 0.19 mg per 1 L of culture. Although the TOP method requires further improvement to augment circular RNA expression, it is notable that this method easily increased the level of circular RNA expression by simple multiplying the copy number of transcription units in the single plasmid. Therefore, we assumed that the TOP strategy will be more effective especially using a low copy number plasmid, because increasing the plasmid copy number by genetic engineering is not easy. We also presented the solid-phase DNA probe method as a simple purification procedure for *in vivo* expressed circular RNA, because this technique does not require electrophoresis for purifying the circular RNA (Umekage & Kikuchi, 2009a).

The most remarkable advantage of circularising functional RNAs is protection from exoribonuclease-induced degradation without the need for chemical modifications, such as use of 2'-protected nucleotides (*e.g.*, 2'-fluoro, 2'-*O*-methyl, LNA) (Schmidt *et al*., 2004; Burmeister *et al*., 2005; Di Primo *et al*., 2007; Pieken *et al*., 1991) or phosphorothioate linkages (Kang *et al*., 2007). Although chemical synthesis of RNA molecules is currently the main methodology used for synthetic RNA production, the *in vivo* circular RNA production technique described in this chapter is a promising method for future RNA drug production because it is both economical and the product can be purified simply. In addition, circular RNA without any chemical modification would be safer than chemically modified RNA for therapeutic human use.

This PIE method can be applied in any species because it requires only magnesium ions and guanosine nucleotides. However, the expression of circular RNA inside human cells or other mammalian cells in culture has not been examined. Therefore, we are currently examining circular RNA expression in human cells based on this method for future development of gene therapy methodologies. We assume that PIE transcription and concomitant RNA circularisation take place in the nucleus, and therefore the circular functional RNA (including aptamers, ribozymes, dsRNA *etc.*) expression within the nucleus will represent a novel gene regulation method targeting nuclear events, such as transcription (Battaglia *et al*., 2010), RNA splicing (van Alphen *et al*., 2009), telomere repairing (Folini *et al*., 2009) and chromatin modification (Tsai *et al*., 2011).


Table 2. Summary of circular RNA expression. "IPTG" and "constitutive" indicate that the circular RNA expression was induced by the addition of IPTG and constitutive expression of the circular RNA by the constitutive *lpp* promoter, respectively. "Yield" represents the circular RNA expression yield (ng) per 1 μg of total RNA recovered from the harvested cells. The data include standard deviations (±), which were derived from three independent experiments (*n* = 3).

#### **4. Acknowledgements**

The authors thank Dr. L. Ponchon (French National Center for Scientific Research, CNRS, Paris, France) for *E*. *coli* strain JM101Tr and the expression plasmid M3, and Dr. K. Matsuura (Tokyo Metropolitan University, Tokyo, Japan) for *Rdv*. *sulfidophilum*. This work was supported by an NISR Research Grant (to S.U.) and a Grant for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to Y.K.).

#### **5. References**

86 Innovations in Biotechnology

plasmid. Therefore, we assumed that the TOP strategy will be more effective especially using a low copy number plasmid, because increasing the plasmid copy number by genetic engineering is not easy. We also presented the solid-phase DNA probe method as a simple purification procedure for *in vivo* expressed circular RNA, because this technique does not

The most remarkable advantage of circularising functional RNAs is protection from exoribonuclease-induced degradation without the need for chemical modifications, such as use of 2'-protected nucleotides (*e.g.*, 2'-fluoro, 2'-*O*-methyl, LNA) (Schmidt *et al*., 2004; Burmeister *et al*., 2005; Di Primo *et al*., 2007; Pieken *et al*., 1991) or phosphorothioate linkages (Kang *et al*., 2007). Although chemical synthesis of RNA molecules is currently the main methodology used for synthetic RNA production, the *in vivo* circular RNA production technique described in this chapter is a promising method for future RNA drug production because it is both economical and the product can be purified simply. In addition, circular RNA without any chemical modification would be safer than chemically modified RNA for

This PIE method can be applied in any species because it requires only magnesium ions and guanosine nucleotides. However, the expression of circular RNA inside human cells or other mammalian cells in culture has not been examined. Therefore, we are currently examining circular RNA expression in human cells based on this method for future development of gene therapy methodologies. We assume that PIE transcription and concomitant RNA circularisation take place in the nucleus, and therefore the circular functional RNA (including aptamers, ribozymes, dsRNA *etc.*) expression within the nucleus will represent a novel gene regulation method targeting nuclear events, such as transcription (Battaglia *et al*., 2010), RNA splicing (van Alphen *et al*., 2009), telomere repairing (Folini *et al*., 2009) and

pGEM-3E5T7t JM109(DE3) IPTG 2.5 ± 0.46 Umekage & Kikuchi, 2009a pM3-3E5 JM101Tr constitutive 3.6 ± 0.15 Umekage & Kikuchi, 2009b

Table 2. Summary of circular RNA expression. "IPTG" and "constitutive" indicate that the circular RNA expression was induced by the addition of IPTG and constitutive expression of the circular RNA by the constitutive *lpp* promoter, respectively. "Yield" represents the circular RNA expression yield (ng) per 1 μg of total RNA recovered from the harvested cells. The data include standard deviations (±), which were derived from three independent

The authors thank Dr. L. Ponchon (French National Center for Scientific Research, CNRS, Paris, France) for *E*. *coli* strain JM101Tr and the expression plasmid M3, and Dr. K. Matsuura (Tokyo Metropolitan University, Tokyo, Japan) for *Rdv*. *sulfidophilum*. This work was

pTOP(I) JM101Tr constitutive 5.0 ± 1.5 this study pTOP(II) JM101Tr constitutive 9.7 ± 1.0 this study pTOP(III) JM101Tr constitutive 9.0 ± 1.8 this study pTOP(IV) JM101Tr constitutive 1.8 ± 0.70 this study

(ng/μg) Reference

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experiments (*n* = 3).

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### **DNA Mimicry by Antirestriction and Pentapeptide Repeat (PPR) Proteins**

Gennadii Zavilgelsky and Vera Kotova

*State Research Institute of Genetics and Selection of Industrial Microorganisms ("GosNIIgenetika"), Moscow Russia* 

#### **1. Introduction**

90 Innovations in Biotechnology

Turner, J.J.; Jones, S.W.; Moschos, S.A.; Lindsay, M.A. & Gait, M.J. (2007). MALDI-TOF mass

Umekage, S. & Kikuchi, Y., (2006). Production of circular form of streptavidin RNA aptamer

Umekage, S. & Kikuchi, Y., (2007). Production of circular streptavidin RNA aptamer in vivo.

Umekage, S. & Kikuchi, Y., (2009). In vitro and in vivo production and purification of

Umekage, S. & Kikuchi, Y., (2009). In vivo circular RNA production using a constitutive promoter for high-level expression. J. Biosci. Bioeng., Vol. 108, No. 4, pp. 354-356 van Alphen R.J., Wiemer, E.A., Burger, H. & Eskens, F.A. (2009). The spliceosome as target

Watts, J.K. & Corey, D.R., *Bioorg*. *Med*. *Chem*. *Lett*., (2010). Clinical status of duplex RNA.

Watts, J.K.; Deleavey, G.F. & Damha, M.J., *Drug Discov*. *Today*, (2008), Chemically modified

Xu, M.Q.; Kathe, S.D.; Goodrich-Blair, H.; Nierzwicki-Bauer, S.A. & Shub, D.A. (1990).

Bacterial origin of a chloroplast intron: conserved self-splicing group I introns in

for anticancer treatment. *Br*. *J*. *Cancer*, Vol. 100, No. 2, pp. 228-232

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cyanobacteria. *Science*, Vol. 250, No. 4987, pp. 1566-1570

*in vitro*. Nucleic Acids Symp. Ser. (Oxf), Vol. 50, pp. 323-324

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*Mol*. *BioSystems*, Vol. 3, pp. 43-50

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spectral analysis of siRNA degradation in serum confirms an RNase A-like activity.

circular RNA aptamer. J. Biotechnol., Vol. 139, No. 4, pp. 265-272, (2008 Epub ahead

Protein mimicry of DNA is a recently discovered direct mechanism of regulation of DNAdependent enzyme activity by means of proteins that mimic DNA structure and interact with a target enzyme and completely inhibit (or modulate) its activity. DNA-mimicking inhibitor proteins bind directly to the enzyme and thus blocks or alters the activity of the latter. Protein mimicry of DNA was first described in Ugi derived from PBS2 bacteriophage of *Bacillus subtilis* (Mol et al, 1995). This protein of 84 amino acid residues with a total charge of (–12) inhibits uracil-DNA glycosylase (UDG), an enzyme involved in DNA repair (Mol et al, 1995; Putnam & Tainer, 2005). Subsequently, this type of protein mimicry was found in the ribosomal elongation factor EF-G (tRNA-like motif), and in the dTAFII 230 component of eukaryotic transcription factor TFIID (DNA-like domain) (Liu et al., 1998). The family of DNA mimetics further includes DinI, a negative SOS response regulator in *E. coli* (Ramirez et al., 2000), and a nucleosome forming protein HI1450 of *Haemophilus influenzae* (Parsons et al., 2004). However, in most of these cases, only a part of the protein molecule is DNA-like, in contrast to antirestriction and pentapeptide repeat (PPR) proteins, whose entire structure mimics the B-form of DNA. For instance, the X-ray structure of Ugi reveals a domain similar to the B-form of DNA, but the molecule as a whole is globular. Note that, in Ugi, the crucial negative charges are those of E20, E28, and E31 in the N domain (Mol et al.,1995).

Horizontal gene transfer is a fundamental mechanism for driving diversity and evolution. Transmission of DNA to bacterial cells that are not direct descendants of the donor is often achieved via mobile genetic elements such as plasmids, conjugative transposons and bacteriophages. Mobilization of these elements can lead in the spread of antimicrobial resistance in clinical environments and in the wider community.

Over 50% of eubacteria and archaea contain the genes for one or more of the four classes of known DNA restriction and restriction-modification (RM) systems (Roberts et al., 2005). RM systems work by recognizing specific DNA sequences and triggering an endonuclease activity which rapidly cleaves the foreign DNA allowing facile destruction by exonucleases (Bickle & Kruger,1993; Murray, 2000; Loenen, 2003).

Mobile genetic elements such as plasmids, transposons and bacteriophage contain the specific genes encoding anti-RM systems. Activation of anti-RM system weakens or negates the RM defence system allowing further horizontal gene transfer (Wilkins, 1995; Zavilgelsky, 2000; Murray, 2002; Tock & Dryden, 2005).

The genes encoding antirestriction proteins are situated on conjugational plasmids (*ard*A gene) and some bacteriophages (*ocr* and *dar*A genes). Antirestriction proteins inhibit the type I restriction-modification enzymes and thus protect unmodified DNA of plasmids and bacteriophages from degradation. Genes *ard* (*a*lleviation of *r*estriction of DNA) facilitate the natural DNA transfer between various types of bacteria ensuring overcoming intercellular restriction barriers (horizontal genes transfer). Genes *ocr* (bacteriophage T7) and *dar*A (bacteriophage P1) significantly increase the infection efficiency by phages of the bacterial cells.

Antirestriction proteins ArdA and Ocr belong to the group of very acidic proteins and contain a characteristic sequence of negative charges (Asp and Glu). X-ray diffraction study of proteins ArdA and Ocr carried out demonstrated that these proteins were like the B-form of DNA (Walkinshaw et al., 2002; McMahon et al., 2009). Therefore the antirestriction proteins operate on the principle of concurrent inhibition replacing DNA in the complex with the enzyme (DNA mimicry).

DNA-mimetic antirestriction proteins ArdA and Ocr can be electroporated into cells along with transforming DNA and protect unmodified DNA from degradation. As a result the antirestriction proteins improve transformation efficiency. The highly charged, very acidic proteins Ocr and ArdA can be used as a purification handle similar to other fusion tags. A monomeric mutant of the Ocr protein was used as a novel fusion tag which displayed solubilizing activity with a variety of different passenger proteins (DelProposto et al., 2009).

The pentapeptide repeat is a recently discovered protein fold. MfpA and Qnr (A,B,C,D,S) are two newly characterized pentapeptide repeat proteins (PPRs) that interact with type II topoisomerase (DNA gyrase) and confer bacterial resistance to the drugs quinolone and fluoroquinolone [Hegde et al., 2005; Hedge et al., 2011). The *mfp*A gene is chromosome borne in *Mycobacterium tuberculosis (*Hegde et al., 2005; Montero et al., 2001), while *qnr* genes are plasmid borne in Gram-negative enterobacteria (Martinez-Martinez, L. et al.,1998; Tran et al., 2005; Cattoin & Nordmann, 2009; Rodriguez-Martinez et al. 2011). The size, shape, and surface potential of MfpA and Qnr proteins mimics duplex DNA (Hegde et al., 2005; Vetting et al., 2009; Hegde et al., 2011).

#### **2. Type I restriction-modification systems**

Restriction–modification (RM) systems form a barrier protecting a cell from the penetration by foreign DNA (Murray, 2000; Loenen, 2003). In the modern understanding, RM enzymes are a part of the "immigration control system", which discriminates between its own and foreign DNA entering the cell (Murray, 2002). The system is based on two conjugated enzymatic activities: those of restriction endonucleases and DNA methyltransferases. RM enzymes recognize a specific nucleotide sequence in the DNA, and the restriction endonuclease cleaves the double strand of unmodified DNA. The host DNA is protected from enzymatic cleavage by specific methylation of the recognition sites produced by DNA methyltransferases. RM enzymes are classified in four types. We shall now discuss the features of type I RM systems, since it is these systems that are efficiently inhibited by antirestriction proteins. Figure 1 schematically represents the activity of a type I enzyme, e.g., EcoKI. EcoKI comprises five subunits (R2M2S): two R subunits are restriction

the RM defence system allowing further horizontal gene transfer (Wilkins, 1995;

The genes encoding antirestriction proteins are situated on conjugational plasmids (*ard*A gene) and some bacteriophages (*ocr* and *dar*A genes). Antirestriction proteins inhibit the type I restriction-modification enzymes and thus protect unmodified DNA of plasmids and bacteriophages from degradation. Genes *ard* (*a*lleviation of *r*estriction of DNA) facilitate the natural DNA transfer between various types of bacteria ensuring overcoming intercellular restriction barriers (horizontal genes transfer). Genes *ocr* (bacteriophage T7) and *dar*A (bacteriophage P1) significantly increase the infection efficiency by phages of the bacterial cells. Antirestriction proteins ArdA and Ocr belong to the group of very acidic proteins and contain a characteristic sequence of negative charges (Asp and Glu). X-ray diffraction study of proteins ArdA and Ocr carried out demonstrated that these proteins were like the B-form of DNA (Walkinshaw et al., 2002; McMahon et al., 2009). Therefore the antirestriction proteins operate on the principle of concurrent inhibition replacing DNA in the complex with the

DNA-mimetic antirestriction proteins ArdA and Ocr can be electroporated into cells along with transforming DNA and protect unmodified DNA from degradation. As a result the antirestriction proteins improve transformation efficiency. The highly charged, very acidic proteins Ocr and ArdA can be used as a purification handle similar to other fusion tags. A monomeric mutant of the Ocr protein was used as a novel fusion tag which displayed solubilizing activity with a variety of different passenger proteins (DelProposto et al., 2009). The pentapeptide repeat is a recently discovered protein fold. MfpA and Qnr (A,B,C,D,S) are two newly characterized pentapeptide repeat proteins (PPRs) that interact with type II topoisomerase (DNA gyrase) and confer bacterial resistance to the drugs quinolone and fluoroquinolone [Hegde et al., 2005; Hedge et al., 2011). The *mfp*A gene is chromosome borne in *Mycobacterium tuberculosis (*Hegde et al., 2005; Montero et al., 2001), while *qnr* genes are plasmid borne in Gram-negative enterobacteria (Martinez-Martinez, L. et al.,1998; Tran et al., 2005; Cattoin & Nordmann, 2009; Rodriguez-Martinez et al. 2011). The size, shape, and surface potential of MfpA and Qnr proteins mimics duplex DNA (Hegde et al., 2005; Vetting

Restriction–modification (RM) systems form a barrier protecting a cell from the penetration by foreign DNA (Murray, 2000; Loenen, 2003). In the modern understanding, RM enzymes are a part of the "immigration control system", which discriminates between its own and foreign DNA entering the cell (Murray, 2002). The system is based on two conjugated enzymatic activities: those of restriction endonucleases and DNA methyltransferases. RM enzymes recognize a specific nucleotide sequence in the DNA, and the restriction endonuclease cleaves the double strand of unmodified DNA. The host DNA is protected from enzymatic cleavage by specific methylation of the recognition sites produced by DNA methyltransferases. RM enzymes are classified in four types. We shall now discuss the features of type I RM systems, since it is these systems that are efficiently inhibited by antirestriction proteins. Figure 1 schematically represents the activity of a type I enzyme, e.g., EcoKI. EcoKI comprises five subunits (R2M2S): two R subunits are restriction

Zavilgelsky, 2000; Murray, 2002; Tock & Dryden, 2005).

enzyme (DNA mimicry).

et al., 2009; Hegde et al., 2011).

**2. Type I restriction-modification systems** 

endonucleases that cleave the double helix of unmodified DNA, two M subunits are methyltransferases that methylate adenine residues at the recognition site, and an S subunit recognizes a specific DNA site (sK) and forms a stable complex with it.

Fig. 1. Activity of a type I restriction–modification enzyme. 1, Both DNA strands at the sK site are methylated. The enzyme–DNA complex dissociates. 2, One of DNA strands at the sK site is methylated. The methylase (M) methylates the adenyl residue of the other strand, and the complex dissociates. 3, Both DNA strands at the sK site are unmethylated. The enzyme initiates DNA translocation through the R subunits accompanied by the formation of a supercoiled loop and subsequent double-stranded DNA break.

The sK site is "hyphenated", i.e., only seven outmost nucleotides of the 13 bp long recognition sequence are conserved (e.g. EcoKI recognizes 5'-AACNNNNNNGTGC-3'). According to the footprinting data, EcoKI covers 66 bp of the DNA sequence. Further events depend on the sK status. If both DNA strands at the site are methylated, the complex dissociates.

If only one strand is methylated, the methylase M methylates the respective adenyl residue, and the complex dissociates. If both DNA strands are unmethylated, the DNA helix is translocated through the R subunits, while the S subunit remains bound to the sK site. The endonuclease R randomly cleaves the DNA strands at a considerable distance from the sK site. This is the principal difference between the type I RM enzymes and type II restriction endonucleases, which introduce a double-strand DNA break directly at the recognition site or at a specific distance from it. The translocation process itself is associated with considerable energy expenditure in the form of ATP. As a result, type I RM enzymes are ATP-dependent, whereas type II enzymes are not. Another characterizing feature of the EcoKI–sK complex is that the S subunit binds only to the outmost conserved nucleotides of the site. As a result, the double stranded DNA undergoes significant deformation, acquiring a kink of approximately 34°, which sets additional energy demands. Nucleotide sequences of the recognition sites vary and are specific for each type I enzyme (EcoK, EcoB, EcoA, EcoD, Eco124, StyLT, StySP, CfrAI, and many others). Based on their homology and the possibility of subunit exchange, type I RM systems are classified into four families: IA, IB, IC, ID. Restriction is efficient against foreign DNA irrespective of the way it is introduced into the cell: by injection from a phage, transformation, or conjugative transmission. Thus, type I RM systems constitute a socalled restriction barrier that prevents interspecies horizontal gene transfer.

#### **3. Conjugative plasmids and transposons, bacteriophages, and antirestriction**

Natural horizontal gene transfer between bacteria is mediated primarily by transmissible plasmids, conjugative transposones, and bacteriophages (Wilkins, 1995). Evolution of all transmissible plasmids, conjugative transposones and some bacteriophages gave rise to systems enabling them to overcome restriction barriers. This phenomenon has been termed antirestriction (Zavilgelsky, 2000; Tock & Dryden, 2005). An investigation of antirestriction mechanisms employed by transmissible plasmids showed that the process involves a specialized antirestriction protein encoded by the *ard*A gene (*a*lleviation of *r*estriction of *D*NA). *ard*A genes were first discovered in plasmids of the incompatibility group N in 1984– 1985 (Belogurov et al.,1985), and later in other types of plasmids (Kotova et al., 1988; Delver et al., 1991). In 1991–1995, *ard*A genes were sequenced and the primary structure of ArdA proteins was determined (Delver et al., 1991; Chilley & Wilkins, 1995). Genes *ard*A are located in the leader region of the plasmid sequence, which lies next to *ori*T and is the first to enter the host cell in the course of conjugative transfer. The *ori*T site, the origin of plasmid conjugative replication, is located at the boundary of the *tra* operon with the rest of plasmid.

The conjugative transposon Tn916 of the bacterial pathogen *Enterococcus faecalis* contains *orf*18 gene, which is located within position region and encodes an ArdA antirestriction protein (Serfiotis-Mitsa et al., 2008). Genes of the *ard*A family encode small, very acidic proteins comprised of 160–170 amino acid residues and bearing a characteristic total negative charge of (–20 to –30) which act as specific highly efficient inhibitors of cellular type I RM enzymes. ArdA proteins inhibit restriction endonucleases of different families (IA, IB, IC, and ID) and with different recognition site sequences with nearly the same efficiency. Thanks to this property of ArdA, transmissible plasmids can overcome the restriction barriers through horizontal transmission from the donor cell into bacteria of various species and genera.

Some bacteriophages also possess genes encoding antirestriction proteins, such as *0.3(ocr )*  (phage T7) and *dar*A (phage P1) (Dunn et al., 1981; Kruger et al., 1983; Iida et al., 1988). These genes increase the efficiency of phage infection.

Antirestriction proteins, both of plasmid (ArdA) and phage origin (Ocr), inhibit only type I RM enzymes, whose genes (*hsd*RMS) are usually located on the bacterial chromosome, but not type II restriction endonucleases, the genesof which are normally located on plasmids.

#### **4. DNA mimicry by antirestriction proteins**

94 Innovations in Biotechnology

endonucleases, which introduce a double-strand DNA break directly at the recognition site or at a specific distance from it. The translocation process itself is associated with considerable energy expenditure in the form of ATP. As a result, type I RM enzymes are ATP-dependent, whereas type II enzymes are not. Another characterizing feature of the EcoKI–sK complex is that the S subunit binds only to the outmost conserved nucleotides of the site. As a result, the double stranded DNA undergoes significant deformation, acquiring a kink of approximately 34°, which sets additional energy demands. Nucleotide sequences of the recognition sites vary and are specific for each type I enzyme (EcoK, EcoB, EcoA, EcoD, Eco124, StyLT, StySP, CfrAI, and many others). Based on their homology and the possibility of subunit exchange, type I RM systems are classified into four families: IA, IB, IC, ID. Restriction is efficient against foreign DNA irrespective of the way it is introduced into the cell: by injection from a phage, transformation, or conjugative transmission. Thus, type I RM systems constitute a socalled

Natural horizontal gene transfer between bacteria is mediated primarily by transmissible plasmids, conjugative transposones, and bacteriophages (Wilkins, 1995). Evolution of all transmissible plasmids, conjugative transposones and some bacteriophages gave rise to systems enabling them to overcome restriction barriers. This phenomenon has been termed antirestriction (Zavilgelsky, 2000; Tock & Dryden, 2005). An investigation of antirestriction mechanisms employed by transmissible plasmids showed that the process involves a specialized antirestriction protein encoded by the *ard*A gene (*a*lleviation of *r*estriction of *D*NA). *ard*A genes were first discovered in plasmids of the incompatibility group N in 1984– 1985 (Belogurov et al.,1985), and later in other types of plasmids (Kotova et al., 1988; Delver et al., 1991). In 1991–1995, *ard*A genes were sequenced and the primary structure of ArdA proteins was determined (Delver et al., 1991; Chilley & Wilkins, 1995). Genes *ard*A are located in the leader region of the plasmid sequence, which lies next to *ori*T and is the first to enter the host cell in the course of conjugative transfer. The *ori*T site, the origin of plasmid conjugative replication, is located at the boundary of the *tra* operon with the rest of plasmid. The conjugative transposon Tn916 of the bacterial pathogen *Enterococcus faecalis* contains *orf*18 gene, which is located within position region and encodes an ArdA antirestriction protein (Serfiotis-Mitsa et al., 2008). Genes of the *ard*A family encode small, very acidic proteins comprised of 160–170 amino acid residues and bearing a characteristic total negative charge of (–20 to –30) which act as specific highly efficient inhibitors of cellular type I RM enzymes. ArdA proteins inhibit restriction endonucleases of different families (IA, IB, IC, and ID) and with different recognition site sequences with nearly the same efficiency. Thanks to this property of ArdA, transmissible plasmids can overcome the restriction barriers through

horizontal transmission from the donor cell into bacteria of various species and genera.

These genes increase the efficiency of phage infection.

Some bacteriophages also possess genes encoding antirestriction proteins, such as *0.3(ocr )*  (phage T7) and *dar*A (phage P1) (Dunn et al., 1981; Kruger et al., 1983; Iida et al., 1988).

Antirestriction proteins, both of plasmid (ArdA) and phage origin (Ocr), inhibit only type I RM enzymes, whose genes (*hsd*RMS) are usually located on the bacterial chromosome, but not type II restriction endonucleases, the genesof which are normally located on plasmids.

restriction barrier that prevents interspecies horizontal gene transfer.

**antirestriction** 

**3. Conjugative plasmids and transposons, bacteriophages, and** 

It has been supposed that antirestriction proteins of the ArdA family, as well as Ocr are modulator proteins with a structure similar to that of the B-form DNA, and the characteristic surface distribution of negatively charged D and E residues (aspartic and glutamic acids) imitates the distribution of negatively charged phosphate groups along the DNA double helix (Zavilgelsky, 2000). That is, antirestriction proteins imitate the DNA structure, which is currently termed "protein mimicry of DNA". The spatial structure of the smallest antirestriction protein, Ocr of phage T7 (116 amino acids), was published in 2002 (Walkinshaw et al., 2002). As shown by X-ray crystallography, the spatial structure of Ocr was similar to the B-form of DNA (Fig. 2). The major stem of the Ocr monomer is constituted by three α-helices: A (residues 7–24), B (residues 34– 44), and a long, somewhat bent one, D (residues 73–106); the helices form a tightly packed bunch with strictly regularly positioned negatively charged D and E carboxyls along the stem axis, nearly reproducing the distribution of negatively charged phosphate groups along DNA double helix. The short α-helix C (residues 49–57) is a part of the interface determining the contact of monomers and stable dimer formation.

The structure of the Ocr dimer, both in solution and in crystal form, is similar in length and charge distribution to 24 bp of DNA double helix. The contact of monomers is established by a Van der Waals interaction between hydrophobic clusters within the C α-helices in the middle of the polypeptide: A50, F53, S54, M56, A57, and V77.

Fig. 2. Spatial structure of the (Ocr)2 protein dimer. Shown is the positioning of α-helices A, B, C, D, and amino acid residues 53F and 57A in the hydrophobic cluster 52IFSVMAS, which determines the Van der Waals attraction of the monomers.

The spatial structure of the ArdA protein from the conjugative transposon Tn916 (166 amino acids), was published in 2009 (McMahon et al., 2009). As was shown by X-ray crystallography, ArdA protein has a extremely elongated curved cylindrical structure witn defined helical groowes. The high density of Asp and Glu residues on the surface follow a helical pattern and the whole protein mimics a 42-base pair stretch of B-form DNA making ArdA dimer by far the largest DNA mimic known (Fig. 3). Each monomer of this dimeric structure can be decomposed into three domains: the N-terminal domain 1 (residues 3-61), the central domain 2 (residues 62-103) and the C-terminal domain 3 (residues 104-165). The N-terminal domain 1 consists of a three-stranded anti-parallel β-sheet and one short α -helix interspersed with three large loops of 10 or more residues. The central domain 2 of ArdA is a four α–helix bundle. The C-terminal domain 3 has a three-stranded β -sheet and three αhelices packed together in a manner that creates a groove in the structure 11 angstrem wide. Analysis of the electrostatic surface of ArdA shows that 2 and 3 domains have a profoundly negative potential (the pI of ArdA is 4). The ArdA dimer, like the monomer, is highly elongated and curved (Fig. 3). The chord that connects the extreme ends has the length of 140 angstrem. The pattern of negative charge even extends across the dimer interface through the conserved residues D109, D111, D112, D115, E122, E123 and E129.

This distribution and conservation of charged residues is evidence for the necessity of dimer formation for protein function and suggests that ArdA across all species will have similar structural requirements. The dimer interface contains the anti-restriction motif (amino acids 126-140 in the Tn916 ArdA protein) identified previously (Belogurov & Delver, 1995) conserved as well.

Fig. 3. Spatial structure of the (ArdA)2 protein dimer.

The ArdA dimer appears to mimic about 42 bp of bent B-form DNA. This is comparable in length to the footprint of the EcoKI Type IA RM enzyme, without its cofactors, on DNA. In comparison, the Ocr dimer from phage T7 mimics only about 24 bp., similar in length to the 30 bp footprint of the Type I RM enzyme in the presence of its cofactors and to the footprint of the MTase core, M.EcoKI, of the Type I RM enzyme. The typical DNA target for a Type I RM enzyme is 14 bp long and bipartite, e.g. EcoKI recognizes 5'-AACNNNNNNGTGC-3', and lies centrally in the experimental DNA footprint. It was built the M.EcoKI-ArdA model: domain 3 overlaps the EcoKI target sequence, domain 2 contacts the extremites of the DNA-binding groove in M.EcoKI and domain 3 projects beyond the M.EcoKI structure. Domain 1 is not essential for antirestriction as it can be deleted (Delver et al., 1991) indicating that the key aspect of antirestriction by ArdA is the binding to the MTase core using domains 2 and 3.

The mimicry of DNA enables antirestriction proteins to compete with DNA for binding with the RM enzyme and thus to inhibit DNA degradation (restriction) and methylation (modification). From the point of view of classical enzymatic catalysis, antirestriction is a case of competitive inhibition based on structural similarity between the enzyme substrate and the inhibitor molecule. The relative positioning of monomers in the (Ocr)2 dimer is typical: the angle between their longitudinal axes is approximately 34° (Fig. 2). This dimer structure is nearly equivalent to the kinked DNA double helix structure that is formed at the recognition site of the type I RM enzyme–DNA complex (Murray, 2000). Consequently, Ocr does not require additional energy to bind to EcoKI, and efficiently displaces doublestranded DNA from the complex (the complex formation constant for Ocr–EcoKI is approximately 100 times higher than for DNA–EcoKI) (Atanasiu et al., 2002).

#### **5. Antirestriction and antimodification activities of ArdA and Ocr proteins**

Both ArdA and Ocr inhibit ATP-dependent type I RM enzymes. However, the great difference between the life cycles of transmissible plasmids (symbiosis with a bacterial cell) and

elongated and curved (Fig. 3). The chord that connects the extreme ends has the length of 140 angstrem. The pattern of negative charge even extends across the dimer interface

This distribution and conservation of charged residues is evidence for the necessity of dimer formation for protein function and suggests that ArdA across all species will have similar structural requirements. The dimer interface contains the anti-restriction motif (amino acids 126-140 in the Tn916 ArdA protein) identified previously (Belogurov & Delver, 1995)

The ArdA dimer appears to mimic about 42 bp of bent B-form DNA. This is comparable in length to the footprint of the EcoKI Type IA RM enzyme, without its cofactors, on DNA. In comparison, the Ocr dimer from phage T7 mimics only about 24 bp., similar in length to the 30 bp footprint of the Type I RM enzyme in the presence of its cofactors and to the footprint of the MTase core, M.EcoKI, of the Type I RM enzyme. The typical DNA target for a Type I RM enzyme is 14 bp long and bipartite, e.g. EcoKI recognizes 5'-AACNNNNNNGTGC-3', and lies centrally in the experimental DNA footprint. It was built the M.EcoKI-ArdA model: domain 3 overlaps the EcoKI target sequence, domain 2 contacts the extremites of the DNA-binding groove in M.EcoKI and domain 3 projects beyond the M.EcoKI structure. Domain 1 is not essential for antirestriction as it can be deleted (Delver et al., 1991) indicating that the key aspect of antirestriction by ArdA is the binding to the MTase core using domains 2 and 3.

The mimicry of DNA enables antirestriction proteins to compete with DNA for binding with the RM enzyme and thus to inhibit DNA degradation (restriction) and methylation (modification). From the point of view of classical enzymatic catalysis, antirestriction is a case of competitive inhibition based on structural similarity between the enzyme substrate and the inhibitor molecule. The relative positioning of monomers in the (Ocr)2 dimer is typical: the angle between their longitudinal axes is approximately 34° (Fig. 2). This dimer structure is nearly equivalent to the kinked DNA double helix structure that is formed at the recognition site of the type I RM enzyme–DNA complex (Murray, 2000). Consequently, Ocr does not require additional energy to bind to EcoKI, and efficiently displaces doublestranded DNA from the complex (the complex formation constant for Ocr–EcoKI is

approximately 100 times higher than for DNA–EcoKI) (Atanasiu et al., 2002).

**5. Antirestriction and antimodification activities of ArdA and Ocr proteins** 

Both ArdA and Ocr inhibit ATP-dependent type I RM enzymes. However, the great difference between the life cycles of transmissible plasmids (symbiosis with a bacterial cell) and

through the conserved residues D109, D111, D112, D115, E122, E123 and E129.

Fig. 3. Spatial structure of the (ArdA)2 protein dimer.

conserved as well.

bacteriophages (infection and lysis of bacteria) makes it interesting to compare the inhibition efficiencies of these proteins. For this purpose, we cloned *ard*A and *ocr* under a strictly regulated promoter. To quantify the intracellular concentration of the antirestriction proteins, we developed a bioluminescence method that utilizes the *Photorhabdus luminescens luxCDABE*  genes as reporters. The *luxCDABE* genes were cloned in the pZE21 and pZS33 vectors under the control of the PltetO\_1 promoter. The hybrid plasmids were introduced in MG1655Z1 cells. Expression of the *lux* genes was induced by adding anhydrotetracycline in the medium, and the bioluminescence intensity was measured. Since the bioluminescence intensity is directly proportional to the luciferase concentration and the sensitivity of the bioluminescence method is high, it is possible to estimate the enzyme concentration in the cell within a broad range, starting with extremely low concentrations. A calibration plot was constructed to characterize the intracellular content of the enzyme (in relative units (RU)) as a function of the inductor (anhydrotetracycline) concentration (Fig. 4). The luciferase content in MG1655Z1 cells varied from 1 (in the absence of anhydrotetracycline) to 5000 (20 ng/ml anhydrotetracycline or more) RU. It is natural to assume that the relative contents of the proteins synthesized from the *ardA*  and *0.3(ocr)* genes cloned in the pZE21 and pZS33 vectors vary within the same range as the luciferase content under the same expression conditions.

To measure the antirestriction activities of the ArdA and Ocr proteins, titration with phage λ.0 was performed for MG1655Z1 cells carrying a hybrid plasmid with the *ardA* or *0.3(ocr)* gene; cells without the hybrid plasmid were used as a control. Since the genome of strain MG1655Z1 contains the *hsdRMS* genes, which code for the *Eco*KI restriction–modification enzyme, the phage λ.0 seeding efficiency was approximately four orders of magnitude lower than in the case of control strain TG\_1. However, when MG1655Z1 cells contained a plasmid with the cloned *ardA* or *0.3(ocr)* gene, the phage seeding efficiency changed depending on the production of the antirestriction protein. As the protein production increased, the phage seeding efficiency grew from 10–4 (no inhibition) to 1 (complete inhibition of restriction–modification enzymes).

Fig. 4. Luciferase content (relative units, RU) in *E. coli* MG1655Z1 cells containing the pZS33\_*lux* or pZE21\_*lux* plasmid as a function of anhydrotetracycline content. The *P. luminescens luxCDABE* genes were cloned in the pZS33 and pZE21 vectors under the control of the P1tetO\_1 promoter. The luciferase content in the presence of the pZS33\_*lux* plasmid and the absence of the inductor anhydrotetracycline was taken as unity.

ArdA ColIb-P9, Ocr T7 antirestriction and antimodification activities were avaluated as a function of the inhibitor concentration, that enabled us to estimate the relative difference in dissociation constants ( *K* d ) that describe the interaction efficiency for ArdA or Ocr and EcoKI (Fig. 5) (Zavilgelsky et al., 2008).

Fig. 5. Antirestriction activity of ArdA ColIb-P9, Ocr T7, and Ocr mutant F53D A57E as a function of their intracellular levels. X-axis: intracellular antirestriction protein concentration (relative units). Y-axis: Antirestriction activity (unmodified λ DNA was used as an EcoKI target). Dotted lines indicate the *K*d points. Circles, native Ocr; squares, Ocr F53D A57E; triangles, ArdA.

The antimodification activity of the ArdA and Ocr proteins was inferred from the seeding efficiency of phage λMG1655Z1 (phage λ.0 propagated for one cycle in MG1655Z1 cells carrying a plasmid with the *ardA* or *0.3(ocr)* gene) on strains AB1167 r+m+ and TG1 r–m–. The ratio between the phage titers on these strains reflected the extent of phage DNA modification (methylation). The *ardA* and *0.3(ocr)* genes were cloned in the pZE21 and pZS33 vectors with the strongly regulated PltetO-1 promoter; the results are summarized in Tables 1 and 2. The intracellular concentrations of the ArdA and Ocr proteins were estimated from the calibration plot constructed by the bioluminescence method (Fig. 1). The ArdA and Ocr proteins substantially differed in the capability of inhibiting the *Eco*KI enzyme. The Ocr protein almost completely inhibited the *Eco*KI restriction–modification system, affecting both restriction and modification activities of the enzyme in a broad Ocr concentration range. The effect was already detectable when Ocr was present at several tens of molecules per cell (1 RU corresponds approximately to ten molecules of the inhibitor protein per cell) (Table 1).

In the case of the ColIb\_P9 ArdA protein, the efficiency of inhibition of the restriction activity of the *Eco*KI enzymes started to decrease when the protein concentration was approximately half its threshold value (which corresponded to complete inhibition of *Eco*KI activity), that is, when ArdA occurred at 10000– 15000 molecules per cell. Inhibition of modification activity of the *Eco*KI enzyme started at higher intracellular ArdA concentrations, at approximately 45000–50000 ArdA molecules per cell (Table 2).

The antirestriction and antimodification activities of the ArdA and Ocr proteins as functions of their intracellular concentrations (in RU) are shown in Fig. 6. While the Ocr protein

ArdA ColIb-P9, Ocr T7 antirestriction and antimodification activities were avaluated as a function of the inhibitor concentration, that enabled us to estimate the relative difference in dissociation constants ( *K* d ) that describe the interaction efficiency for ArdA or Ocr and

Fig. 5. Antirestriction activity of ArdA ColIb-P9, Ocr T7, and Ocr mutant F53D A57E as a function of their intracellular levels. X-axis: intracellular antirestriction protein concentration (relative units). Y-axis: Antirestriction activity (unmodified λ DNA was used as an EcoKI target). Dotted lines indicate the *K*d points. Circles, native Ocr; squares, Ocr F53D A57E;

The antimodification activity of the ArdA and Ocr proteins was inferred from the seeding efficiency of phage λMG1655Z1 (phage λ.0 propagated for one cycle in MG1655Z1 cells carrying a plasmid with the *ardA* or *0.3(ocr)* gene) on strains AB1167 r+m+ and TG1 r–m–. The ratio between the phage titers on these strains reflected the extent of phage DNA modification (methylation). The *ardA* and *0.3(ocr)* genes were cloned in the pZE21 and pZS33 vectors with the strongly regulated PltetO-1 promoter; the results are summarized in Tables 1 and 2. The intracellular concentrations of the ArdA and Ocr proteins were estimated from the calibration plot constructed by the bioluminescence method (Fig. 1). The ArdA and Ocr proteins substantially differed in the capability of inhibiting the *Eco*KI enzyme. The Ocr protein almost completely inhibited the *Eco*KI restriction–modification system, affecting both restriction and modification activities of the enzyme in a broad Ocr concentration range. The effect was already detectable when Ocr was present at several tens of molecules per cell (1 RU

corresponds approximately to ten molecules of the inhibitor protein per cell) (Table 1).

concentrations, at approximately 45000–50000 ArdA molecules per cell (Table 2).

In the case of the ColIb\_P9 ArdA protein, the efficiency of inhibition of the restriction activity of the *Eco*KI enzymes started to decrease when the protein concentration was approximately half its threshold value (which corresponded to complete inhibition of *Eco*KI activity), that is, when ArdA occurred at 10000– 15000 molecules per cell. Inhibition of modification activity of the *Eco*KI enzyme started at higher intracellular ArdA

The antirestriction and antimodification activities of the ArdA and Ocr proteins as functions of their intracellular concentrations (in RU) are shown in Fig. 6. While the Ocr protein

EcoKI (Fig. 5) (Zavilgelsky et al., 2008).

triangles, ArdA.

inhibited both activities of the *Eco*KI enzyme with similar efficiencies and acted already at extremely low concentrations in the cell, the antirestriction and antimodification activity curves substantially differed in the case of the ArdA protein. As estimations showed, the dissociation constant *K*d(met) characteristic of ArdA\_dependent inhibition of methylase activity of the *Eco*KI enzyme was tenfold higher than Kd(rest).

The difference in inhibitory properties of the Ocr and ArdA proteins toward type I restriction–modification enzymes is probably determined by the difference in life cycle between phages and transmissible plasmids; i.e., a phage kills the cell, while a plasmid becomes part of cell genetic material.

The ArdA proteins lose their capability of inhibiting modification activity of *Eco*KI\_like proteins relatively easy. For instance, the ArdA antirestriction proteins encoded by the R16 (incB) and R64 (incI1) transmissible plasmids inhibit restriction activity of the *Eco*KI enzyme, but do not affect its modification activity [25, 26]. Yet the proteins are highly homologous to the ColIb\_P9 ArdA protein. In the 166 amino acid residues, differences are observed only in four positions with R64 ArdA and in nine positions with R16 ArdA. We have earlier found that certain single or double substitutions of hydrophobic amino acid resdues for negatively charged residues (D and E) in the region of the antirestriction motif abolish antimodification activity of ArdA encoded by the pKM101(incN) transmissible plasmid, while its its antirestriction activity is still preserved [17].

In this work, we used site\_directed mutagenesis and constructed the ColIb\_P9 ArdA mutant that contained three amino acid substitutions in the C\_terminal domain; hydrophobic residues were replaced with a more hydrophobic one: F156I, F158I, and V163I.Activities of the mutant protein are characterized in Table 3. As is seen, the mutant protein inhibited antirestriction activity of the *Eco*KI enzyme, but lost the inhibitory effect on its modification activity.

Likewise, certain amino acid substitutions transform the Ocr protein into an antirestriction protein that inhibits only antirestriction activity of the *Eco*KI enzyme. X\_ray analysis of the Ocr protein in crystal demonstrates that a contact of the monomers in the (Ocr)2 homodimer is due to hydrophobic interactions between F53 and A57, which are in the hydrophobic fragment 52\_IFSVMAS\_ in a short α\_helix [11]. We constructed an Ocr mutant with two substitutions, F53D and A57E, assuming that repulsion of negative charges (D…E) would lead to dissociation of the dimer. The *0.3(ocr)* gene with a single or double mutation was cloned in the pUC18 vector. The Ocr F53D A 57E double mutant was tested for functional activity and proved to efficiently inhibit only *Eco*KI restriction activity without affecting methylase activity of the enzyme (Table 4, data on the antirestriction activity of the proteins are omitted). Note that the single amino acid substitutions of the interface region did not affect the antimodification activity of the Ocr protein (Table 4). Like the Ocr protein, the ArdA proteins are active in a homodimeric form. This is true for both the native ColIb\_P9 ArdA protein and the R64 ArdA mutant, which is incapable of inhibiting methylase activity of the enzymes.

Based on the data obtained for the Ocr and ArdA mutant proteins, we assume that the antirestriction proteins form complexes of two types with a type I restriction–modification type, which consists of five subunits (R2M2S) [27]. When an antirestriction protein interacts with the S subunit, which recognizes a specific site in DNA, the DNA strand is displaced, and both restriction and modification activities of the enzyme are inhibited. When an antirestriction protein interacts with the R subunit, which is responsible for ATP\_dependent translocation and endonucleolytic cleavage of nonmethylated DNA, only restriction activity of the enzyme is inhibited. To check this hypothesis, it was important to construct the Ocr mutants that were incapable of inhibiting methylase activity of the enzymes and preserved the effect on their restriction activity. Such properties were observed for the Ocr F53D A57E mutant, which was constructed in this work and had two substitutions of negatively charged amino acid residues for hydrophobic residues in the interface region of the (Ocr)2 homodimer. Thus, the model of type I restriction– modification enzymes with two different binding sites for antirestriction proteins is applicable not only to the ArdA proteins, whose genes are in transmissive plasmids, but also to the Ocr proteins, whose genes are in bacteriophage genomes.


Notes: \* The *0.3* (*ocr*) gene was cloned either in the pZE33 vector (row 1) or in the pZE21 vector (other rows) under the control of the P1tetO\_1 promoter.

\*\* Here and in Table 3: Restriction or modification alleviation factor *R* = *K*+ / *K*–, where *K*– is the coefficient of restriction for MG1655Z1 cells without the plasmid containing the *0.3* (*ocr*) gene and *K*+ is the coefficient of restriction for MG1655Z1 cells carrying the plasmid.

Table 1. Antimodification and antirestriction activities of the Ocr protein as dependent on its intracellular concentration\*


Table 2. Antimodification and antirestriction activities of the ColIb\_P9 ArdA protein as dependent on its intracellular concentration\*

the enzyme is inhibited. To check this hypothesis, it was important to construct the Ocr mutants that were incapable of inhibiting methylase activity of the enzymes and preserved the effect on their restriction activity. Such properties were observed for the Ocr F53D A57E mutant, which was constructed in this work and had two substitutions of negatively charged amino acid residues for hydrophobic residues in the interface region of the (Ocr)2 homodimer. Thus, the model of type I restriction– modification enzymes with two different binding sites for antirestriction proteins is applicable not only to the ArdA proteins, whose genes are in transmissive plasmids, but also to the Ocr proteins, whose genes are in bacteriophage genomes.

0.0 (vector pZS33) 1 2000 2000 0.0 (vector pZE21) 4 5000 5000

\*\* Here and in Table 3: Restriction or modification alleviation factor *R* = *K*+ / *K*–, where *K*– is the coefficient of restriction for MG1655Z1 cells without the plasmid containing the *0.3* (*ocr*) gene and *K*+ is

Table 1. Antimodification and antirestriction activities of the Ocr protein as dependent on its

0.0 (vector pZS33) 1 Not determined Not determined

0.2 6 1 6 0.5 8 1 10 1.0 12 1 20 2.0 30 1 120 5.0 150 4 400 7.5 500 10 1000 10.0 1000 100 2500 15.0 4000 400 5000 20.0 5000 1000 5000

Table 2. Antimodification and antirestriction activities of the ColIb\_P9 ArdA protein as

0.0 (vector pZE21) 4 1 5

EcoKI modification alleviation factor (R) for ArdA

0.2 6 5000 5000 0.5 8 5000 5000 1.0 12 5000 5000 2.0 30 5000 5000 5.0 150 5000 5000 10.0 1000 5000 5000 20.0 5000 5000 5000 40.0 5000 5000 5000 Notes: \* The *0.3* (*ocr*) gene was cloned either in the pZE33 vector (row 1) or in the pZE21 vector (other

EcoKI modification alleviation factor (R) for Ocr\*\*

EcoKI restriction alleviation factor (R) for Ocr

EcoKI restriction alleviation factor (R) for ArdA\*\*

Ocr concentration in the cell, RU

Anhydrotetracyclin, ng/ml

rows) under the control of the P1tetO\_1 promoter.

dependent on its intracellular concentration\*

intracellular concentration\*

Anhydrotetracyclin, ng/ml

the coefficient of restriction for MG1655Z1 cells carrying the plasmid.

Ard concentration in the cell, RU

We conclude that the dimeric form of an antirestriction protein is essential for inhibiting both activities of a type I restriction–modification system, while the monomeric form is sufficient for inhibition of its restriction activity.

• The *ardA* gene was cloned in the pZE21 vector under the control of the P1 tetO\_1 promoter.

Fig. 6. Antirestriction and antimodification activities of the ColIb\_P9 ArdA and T7 Ocr proteins as functions of their intracellular concentrations. Curves: *1*, antimodifi cation activity of Ocr; *2*, antirestriction activity of Ocr;*3*, antimodification activity of ArdA; *4*, antirestriction activity of ArdA.

ArdA and Ocr differ considerably in their ability to inhibit the methylase (modification) activity of EcoKI-like enzymes. As a rule, if *ard*A and *0.3(ocr)* genes are governed by a strong promoter, antirestriction and antimodification activities of ArdA and Ocr are established simultaneously ( Delver et al., 1991; Chilley & Wilkins, 1995;Atanasiu et al., 2002). Some data suggest, however, that the inhibition of endonuclease and methylase activities depends on different interactions of ArdA proteins with type I RM enzymes. For instance, some natural ArdA proteins inhibit only the endonuclease activity of EcoKI. The respective genes are located in transmissible plasmids R16 (incB) (Thomas et al., 2003) and R64 (incI1) (Zavilgelsky et al., 2004). Furthermore, *in vitro* quantification of the ArdA– EcoKI complex showed that ArdA interacts more efficiently with the complete enzyme R2M2S than with its methylase form M2S, which can only modify DNA (Nekrasov et al., 2007). In contrast to ArdA proteins, Ocr from phage T7 binds to the entire EcoKI enzyme and to its methylase form with nearly equal affinities (Atanasiu et al., 2002), and, therefore, even in very low concentrations it inhibits both the endonuclease and the methylase activities of the enzyme (Fig. 5). This property of Ocr is probably related to the difference between the life cycles of a phage and of a transmissible plasmid: a phage kills the host cell, whereas a plasmid becomes part of its genetic material. However, a double amino acid substitution in the 52IFSVMAS hydrophobic cluster of the Ocr interface (an Ocr homodimer is formed by a Van der Waals interaction between these clusters), that is, a substitution of acidic 53D and 57E for hydrophobic 53F and 57A (Fig. 6), causes the mutant protein Ocr F53D A57E to lose the antimethylation while retaining the antirestriction activity against EcoKI. In addition, the mutant protein Ocr F53D A57E has a *K*d of 10 –7M, which is 1000 times higher than the *K*d of the native Ocr form (Fig. 6) (Zavilgelsky et al., 2009).


Notes: \* Phage λ.0 was used to infect *E. coli* JM109 r–m+ cells. A phage lysate obtained after one reproduction cycle (λjm109) was titrated on strains TG\_1 and AB1157.

\*\* The coefficient of restriction *K* (column 3), which was used to estimate the antirestriction activity of the ArdA proteins, was determined as the ratio of the titer of phage λ.0 on strain AB1157 to the titer of the same phage on strain TG\_1 r–m–.

Table 3. Effects of the ArdA (ColIb\_P9) protein and its F156I F158I V163I mutant on *Eco*KI restriction and *Eco*KI modification in *E. coli* K\_12 AB1157 r+ m+ and MJ109 r–m+\* cells upon the cloning of the corresponding genes in the pUC18 vector


\* Phage λ.0 was used to infect *E. coli* JM109 r– m+ cells. A phage lysate obtained after one reproduction cycle (λjm109) was titrated on strains TG\_1 and AB1157. The results were averaged over five replicate experiments.

Table 4. Effects of the native and mutant T7 Ocr proteins on *Eco*KI\_dependent modification in *E. coli* K\_12 JM109 r– m+ cells\*

*K*d is determined by intracellular protein concentration characterized with a 50% decrease in the inhibition of EcoKI endonuclease activity. For Ocr, this level was approximately 1700 times lower than for ArdA. According to in vitro data, the Ocr–EcoKI complex formation had a *K*d of 10 –10 M (Atanasiu et al., 2002). Therefore, the *K*d for ArdA–EcoKI complex formation is 1.7×10–7M.

The fact that endonuclease and methylase EcoKI activities are inhibited by ArdA or Ocr separately suggests that antirestriction proteins can bind type I enzymes in two ways: the complex formation of the first type inhibits both endonuclease and methylase activity of the enzyme, whereas in the complex of the second type, endonuclease activity is blocked while methylase activity is retained. As a working hypothesis, we propose the following model of interaction between antirestriction proteins (ArdA and Ocr) and type I RM enzymes (Fig. 7). ArdA and Ocr can form a complex both with the S-subunit that contacts with the *s*K site on

pUC18 Absent 2.0 x 10-4 1 pVB2(pUC18) ArdA F1561 F1581 V1631 1 1 pSR3(pUC18) ArdA native 1 2.0 x 10-4 Notes: \* Phage λ.0 was used to infect *E. coli* JM109 r–m+ cells. A phage lysate obtained after one

\*\* The coefficient of restriction *K* (column 3), which was used to estimate the antirestriction activity of the ArdA proteins, was determined as the ratio of the titer of phage λ.0 on strain AB1157 to the titer of

Table 3. Effects of the ArdA (ColIb\_P9) protein and its F156I F158I V163I mutant on *Eco*KI restriction and *Eco*KI modification in *E. coli* K\_12 AB1157 r+ m+ and MJ109 r–m+\* cells upon

pUC18 Absent 1 1 pSR8 Ocr native 1 2.0 x 10-4 pSR9 Ocr F53D 1 2.0 x 10-4 pSR10 Ocr A57E 1 2.0 x 10-4 pSR11 Ocr F53D A57E 1 1 \* Phage λ.0 was used to infect *E. coli* JM109 r– m+ cells. A phage lysate obtained after one reproduction cycle (λjm109) was titrated on strains TG\_1 and AB1157. The results were averaged over five replicate

Table 4. Effects of the native and mutant T7 Ocr proteins on *Eco*KI\_dependent modification

*K*d is determined by intracellular protein concentration characterized with a 50% decrease in the inhibition of EcoKI endonuclease activity. For Ocr, this level was approximately 1700 times lower than for ArdA. According to in vitro data, the Ocr–EcoKI complex formation had a *K*d of 10 –10 M (Atanasiu et al., 2002). Therefore, the *K*d for ArdA–EcoKI complex

The fact that endonuclease and methylase EcoKI activities are inhibited by ArdA or Ocr separately suggests that antirestriction proteins can bind type I enzymes in two ways: the complex formation of the first type inhibits both endonuclease and methylase activity of the enzyme, whereas in the complex of the second type, endonuclease activity is blocked while methylase activity is retained. As a working hypothesis, we propose the following model of interaction between antirestriction proteins (ArdA and Ocr) and type I RM enzymes (Fig. 7). ArdA and Ocr can form a complex both with the S-subunit that contacts with the *s*K site on

Coefficient of restriction (K) of phage λ.0 on AB1157 r+m+(\*\*)

Coefficient of restriction (K) of phage λjm109 on TG1 r-m-

Coefficient of restriction (K) of phage λjm109 on AB1157 r+m+

Coefficient of restriction (K) of phage λjm109 on AB1157 r+m+

Plasmid Protein

the same phage on strain TG\_1 r–m–.

Plasmid Protein

in *E. coli* K\_12 JM109 r– m+ cells\*

formation is 1.7×10–7M.

experiments.

reproduction cycle (λjm109) was titrated on strains TG\_1 and AB1157.

the cloning of the corresponding genes in the pUC18 vector

DNA, and with the R-subunit responsible for the translocation and cleavage of unmodified DNA. The binding of ArdA or Ocr to the S-subunit simultaneously inhibits both endonuclease and methylase activity by displacing DNA from its complex with the R2M2S enzyme (Fig. 7, *1*). However, the binding can be easily disrupted if, as a result of amino acid substitutions, the protein is not in the dimeric form, or if the angle between the longitudinal axes of the monomers differs from the critical 34°. As a consequence, it becomes energetically unfavorable for a DNA-mimic protein to displace kinked DNA from its complex with the S-subunit. On the other hand, the interaction of ArdA or Ocr with the Rsubunit probably does not depend on the particular dimer structure, since the R-subunit is responsible for DNA strand translocation and the respective complex is not site-specific. Thus ArdA and Ocr inhibit only the endonuclease activity of the enzyme, while its methylase activity is preserved: DNA can still bind to the S-subunit, and the M-subunit specifically methylates adenyl residues at the sK site (Fig. 7, *2*).

Fig. 7. Putative scheme of ArdA or Ocr interaction with a type I RM enzyme (R2M2S). *1*, An ArdA/Ocr complex with the S subunit: unmodified DNA is entirely displaced. Both endonuclease and methylase activities are inhibited (r– m– - phenotype). *2*, An ArdA/Ocr complex with the R subunit: a DNA strand is displaced from the translocation center. Only endonuclease activity is inhibited (r– m+ phenotype). Endonuclease and methylase activities of a type I RM enzyme are designated as "r" and "m" respectively.

*In vitro* experiments showed that the R2M2S form of EcoKI binds two (Ocr)2 dimers, while the methylase form M2 S binds only one (Atanasiu et al., 2002). This result fits well into the above model of inhibition by antirestriction proteins. As the ArdA binding constant is higher for M2S than for R2M2S, moderate levels of ArdA synthesized under natural conditions inhibit only the endonuclease activity of type I RM enzymes so as to protect the plasmid DNA in transmission, but do not affect the methylase activity which is crucial for maintaining the integrity of the plasmid and the host chromosome. The native Ocr form from phage T7 binds to RM enzymes, simultaneously inhibiting both the endonuclease and the methylase activity, and, therefore, interacts with the S-subunit. There is an obvious reason for the Ocr activity being so high (*K*d = 10–10 M): in the course of infection, the phage DNA is immediately attacked by cellular endonucleases.

#### **6. Pentapeptide repeat proteins (ppr proteins)**

#### **6.1 Inhibitors of DNA gyrase**

Quinolones and also fluoroquinolones are synthetic derivatives of nalidixic acid; they belong to a group of antibiotics with wide spectrum of action and high activity and inhibit DNA gyrase. Quinolones bind to the gyrase–DNA complex. This results in stabilization of the covalent enzyme tyrosyl-DNA phosphate ester (a transient reaction intermediate) and causes death of bacteria. Quinolones have been successfully used for inactivation of *Mycobacterium tuberculosis* cells. During the first years of clinical use of quinolones, findings of *M. tuberculosis* strains resistant to quinolones were rather rare events. Studies of the nature of resistance to quinolones in the laboratory strains of *M. tuberculosis* and the related strain *M. smegmotis* have shown that this effect is determined by missense mutations (amino acid substitutions) in A-chain of DNA gyrase, or it represents the result of regulatory mutation potentiating expression of a protein pump responsible for the extracellular efflux of toxic compounds. However, the wide use of quinolones in medical practice resulted in the discovery of a new type of quinolone resistance. It was shown that the gene determining such type of resistance in *M. smegmotis* and *M. tuberculosis* encodes the MfpA protein, a specific inhibitor of DNA gyrase (Hegde et al., 2005; Montero et al., 2001). The MfpA proteins of *M. tuberculosis* and *M. smegmotis* consist of 183 and 192 residues correspondently; they share 67% identity. In 1998, the resistance to quinolones found in *Klebsiella pneumoniae* was shown to be encoded by the *qnr*A gene and transferred by the conjugated plasmid (Martinez-Martinez et al., 1998). Subsequent investigations have established that *qnr* genes have a worldwide distribution in a range of bacterial pathogens, mainly Gram-negative opportunist (particularly *Enterobacteriaceae* )(Robicsek et al., 2006). Sequence comparison of plasmids isolated from clinical Gram-negative strains differentiates five distinct *qnr* subfamilies *qnr*A, *qnr*B, *qnr*S (Jacoby et al., 2008), and most recently *qnr*C and *qnr*D (Wang et al., 2009; Cavaco et al., 2009). The proteins encoded by these genes exhibit the same function of DNA gyrase inhibition.

MfpA and QnrABCDS proteins belong to the pentapeptide repeat protein (PRP) family. Amino acid sequences of these proteins contain a repeated pentapeptide with the consensus [S, T, A, V][D, N][L, F][S, T, R][G]. MfpA consists of 183 amino acid residues and in these pentapeptides each second amino acid is D or N and each third amino acid is L or F. Table1 shows that MfpA protein consists of 30 pentapeptides, which determine characteristic features of its spatial structure. Figure 6a (taken from (Vetting et al., 2006) shows the spatial structure of the MfpA protein; it consists of a righ-handed β-helix, which corresponds to Bform DNA in size, shape, and electrostatics. In solutions, MfpA forms a dimer due to hydrophobic contact of several amino acids located at the C-end of an α-helical site. The monomeric MfpA consists of eight coils, and four repeated pentapeptides form four sides of a quadrant (1-4) (Table 5). Such spatial structure was named RHQBH (right-handed quadrilateral beta-helix) or "Rfr" (Repeated five-residues). The dimer (MfpA)2 has a rod-like shape 100 angstrem in length and 27 angstrem in diameter. The total charge of the dimer is (–10), but the negative charges are distributed non-randomly. This results in (MfpA)2 dimer, which mimicks a 30 bp segment of B-form duplex DNA. Docking analysis revealed the existence of tight contact between (MfpA)2 dimer and A2 dimer of the DNA gyrase A subunit (Fig. 8) due to electrostatic complementation between strongly cationic "seat" of the A2 dimer interface and a strongly anionic surface of the (MfpA)2 dimer.

Structural analysis of the *Aeromonas hydrophila,* AhQnr protein is shown that it contain two prominent loops (1 and 2) that project from the PRP structure (Xiong et al., 2011). Deletion mutagenesis demonstrates that both contribute to the protection of *Escherichia coli* DNA gyrase from quinolones. A model for the Qnr:DNA gyrase interaction was suggested, where loop1 interacts with the gyrase A "tower" and loop2 with the gyrase B TOPRIM domains.

Structural similarity between MfpA and Qnr proteins and DNA duplex of the gyrase substrate determines the effectiveness of competitive inhibition of the gyrase; this represents the molecular basis of bacterial resistance to quinolone antibiotics. It should be noted that in contrast to gyrase inhibition by quinolones, the inhibition of gyrase by MfpA and Qnr proteins is not accompanied by cell chromosome degradation. Consequently, the presence of the genes *mfp*A or *qnr* in the bacterial genome is very important because the "fee" for the rescue from the inactivating effect of antibiotics is delayed development of the cell. It is possible that the main function of DNA mimic inhibitors of gyrase consists in modulation of DNA supercoiling, which may potentiate supercoiling at the stage of DNA replication and decrease the rate of supercoiling when the level of chromosome compactness becomes optimal in a particular cell.

#### **6.2 Another PRP family proteins**

104 Innovations in Biotechnology

reason for the Ocr activity being so high (*K*d = 10–10 M): in the course of infection, the phage

Quinolones and also fluoroquinolones are synthetic derivatives of nalidixic acid; they belong to a group of antibiotics with wide spectrum of action and high activity and inhibit DNA gyrase. Quinolones bind to the gyrase–DNA complex. This results in stabilization of the covalent enzyme tyrosyl-DNA phosphate ester (a transient reaction intermediate) and causes death of bacteria. Quinolones have been successfully used for inactivation of *Mycobacterium tuberculosis* cells. During the first years of clinical use of quinolones, findings of *M. tuberculosis* strains resistant to quinolones were rather rare events. Studies of the nature of resistance to quinolones in the laboratory strains of *M. tuberculosis* and the related strain *M. smegmotis* have shown that this effect is determined by missense mutations (amino acid substitutions) in A-chain of DNA gyrase, or it represents the result of regulatory mutation potentiating expression of a protein pump responsible for the extracellular efflux of toxic compounds. However, the wide use of quinolones in medical practice resulted in the discovery of a new type of quinolone resistance. It was shown that the gene determining such type of resistance in *M. smegmotis* and *M. tuberculosis* encodes the MfpA protein, a specific inhibitor of DNA gyrase (Hegde et al., 2005; Montero et al., 2001). The MfpA proteins of *M. tuberculosis* and *M. smegmotis* consist of 183 and 192 residues correspondently; they share 67% identity. In 1998, the resistance to quinolones found in *Klebsiella pneumoniae* was shown to be encoded by the *qnr*A gene and transferred by the conjugated plasmid (Martinez-Martinez et al., 1998). Subsequent investigations have established that *qnr* genes have a worldwide distribution in a range of bacterial pathogens, mainly Gram-negative opportunist (particularly *Enterobacteriaceae* )(Robicsek et al., 2006). Sequence comparison of plasmids isolated from clinical Gram-negative strains differentiates five distinct *qnr* subfamilies *qnr*A, *qnr*B, *qnr*S (Jacoby et al., 2008), and most recently *qnr*C and *qnr*D (Wang et al., 2009; Cavaco et al., 2009). The proteins encoded by these genes exhibit the same function

MfpA and QnrABCDS proteins belong to the pentapeptide repeat protein (PRP) family. Amino acid sequences of these proteins contain a repeated pentapeptide with the consensus [S, T, A, V][D, N][L, F][S, T, R][G]. MfpA consists of 183 amino acid residues and in these pentapeptides each second amino acid is D or N and each third amino acid is L or F. Table1 shows that MfpA protein consists of 30 pentapeptides, which determine characteristic features of its spatial structure. Figure 6a (taken from (Vetting et al., 2006) shows the spatial structure of the MfpA protein; it consists of a righ-handed β-helix, which corresponds to Bform DNA in size, shape, and electrostatics. In solutions, MfpA forms a dimer due to hydrophobic contact of several amino acids located at the C-end of an α-helical site. The monomeric MfpA consists of eight coils, and four repeated pentapeptides form four sides of a quadrant (1-4) (Table 5). Such spatial structure was named RHQBH (right-handed quadrilateral beta-helix) or "Rfr" (Repeated five-residues). The dimer (MfpA)2 has a rod-like shape 100 angstrem in length and 27 angstrem in diameter. The total charge of the dimer is (–10), but the negative charges are distributed non-randomly. This results in (MfpA)2 dimer, which mimicks a 30 bp segment of B-form duplex DNA. Docking analysis revealed the

DNA is immediately attacked by cellular endonucleases.

**6. Pentapeptide repeat proteins (ppr proteins)** 

**6.1 Inhibitors of DNA gyrase** 

of DNA gyrase inhibition.

The first protein of the PRP family was originally found in *Anabaena* cyanobacteria (Black et al., 1995). The HglK protein (encoded by the *hgl*K gene and consisting of 727 residues) contains a series of 36 tandem pentapeptides with the consensus sequence ADLSG. Using methods of bioinfor matics, a group of proteins belonging to PRP family has been identified in *Synechocystis cyanobacteria*; there are 15 proteins with series of tandem pentapeptide repeats varying from 13 to 44 (Bateman et al., 1998). By now the proteins of the PRP family have been found in almost all living organisms excluding yeasts. According to data analysis (Vetting et al., 2006), 525 proteins (484 prokaryotic and 41 eukaryotic) with the pentapeptide motif have been identified. Sequencing of the genome of the cyanobacterium *Cyanothece sp.* PCC 51142 revealed 35 pentapeptide- containing proteins. It was determined (Buchko et al., 2006a) the spatial structure of the Rfr32 protein, which consists of 167 residues. The authors demonstrated that the 21 tandem pentapeptide repeats (with the consensus motif A(N/D)LXX) fold into a right-handed quadrilateral β- helix, or Rfr-fold (as in the case of the MfpA protein); this structure imitates the rod-like structure of B-form DNA. The Rfr structure is also typical for another protein, Rfr23, encoded by a gene that has also been found in the genome of *Cyanothece sp.* PCC 51142 (Buchko et al., 2006b). The real functions of the pentapeptide-containing proteins found in cyanobacteria remain unknown. Some proteins determining immunity of bacteria to their own synthesized antibiotics also belong to the PRP family. These include the McbG protein (encoded by a *mcb*G gene located in the operon responsible for biosynthesis of microcin B17 (Pierrat & Maxwell, 2005) and the OxrA protein, which determines the resistance of *Bacillus megatherium* to oxetanocin A (Morita et al., 1999). In contrast to quinolones, microcin B17 interacts with B-subunit of DNA gyrase. A significant group of pentapeptide repeat family proteins has complex structure and contains

Fig. 8. Ribbon diagram of the *Mycobacterium tuberculosis* MfpA dimer. The four faced of the quadrilateral β-helix are colored green (face 1), blue (face 2), yellow (face 3) and red (face 4).

Fig. 8. Ribbon diagram of the *Mycobacterium tuberculosis* MfpA dimer. The four faced of the quadrilateral β-helix are colored green (face 1), blue (face 2), yellow (face 3) and red (face 4). several domains, including those with catalytic functions. However, the functional role of the pentapeptide repeats in this group remains unknown. But if the putative catalytic function of such protein consists of posttranslational modification of some DNA-binding protein (e.g. histone acetylation), one can suggest that binding of the target protein to the pentapeptide domain would significantly increase selectivity of such a modification reaction.


Table 5. Position of pentapeptides along the axis of the MfpA protein molecule.

#### **7. Application of new DNA mimetics**

Since genes encoding DNA mimics (e.g. *ard*A and *qnr*ABCDS) are located on transmission elements, transposons, and plasmids, this promotes their wide distribution among bacteria of various species and genera.

Thus it is important to investigate in detail the structure of such proteins and the mechanisms of their action. The most illustrative example is the distribution of *qnr*ABCDS genes responsible for the resistance to quinolone antibiotics among clinical bacterial strains. The search for and analysis of genes encoding DNA mimics and representing constituents of transmission elements are important tasks. Below we consider some putative variants of use of DNA-mimicking proteins. The DNA mimics may be successfully used for substitution of DNA during elucidation of spatial structure of the DNA-dependent enzymes by means of Xray analysis (Dryden, 2006). In some cases, it is difficult to obtain crystals of the complexes of the DNA dependent enzymes and DNA and it is possible that substitution of DNA by the DNA mimics may solve this problem. There are examples illustrating successful use of such substitutions: Ugi–UDG (Putnam &Tainer, 2005). It is suggested that substitution of DNA by the Ocr protein might be used for crystallization of Ocr in its complex with an S-subunit of EcoKI. Therefore, it should be noted that spatial structure of S-subunit of two type I restriction–modification enzymes has been determined (Kim et al., 2005; Calisto et al., 2005). The DNA mimics can be used in affinity chromatography. Affinity columns with a DNA mimics can be used with high effectiveness for detection and purification of various types of DNA-dependent enzymes. Use of radioactive or fluorescent labels will increase the sensitivity of such method. The perspectives of *in vitro* construction of new types of DNA mimics (i.e. generation of proteins with different "design" and new functions) may be quite wide. These include potential tasks of constructing of DNA mimics, inhibiting or modulating activity of specific groups of DNA-dependent enzymes and tasks related to site directed changes in the structure of already known DNA mimics. Such works are rather successful. For example, using site-directed mutagenesis we have modified the structure of ArdA and Ocr; the modified proteins selectively inhibit the endonuclease (restriction) activity of type I restriction–modification enzymes without any influence on their methylase (modification) activity (Zavilgelsky et al., 2011). The use of such type of antirestriction proteins in gene engineering gives an opportunity to develop stable strains with hybrid plasmids because the process of specific modification of chromosome DNA remains unimpaired. The protein Ocr has already been used as an effective factor promoting significant increase of bacterial transformation by plasmids. Adding a small amount of the Ocr protein to solution with plasmid DNA causes significant (by several orders of magnitude) increase in effectiveness of cell transformation during electroporation. In this case unmethylated DNA and host bacteria with active type I restriction–modification system are used. The Ocr protein (as well as plasmid DNA) easily penetrates inside cells and immediately protects unmethylated DNA against degradation (EPICENTRE Forum 9, 8, htpp//www.epibio.com/forum.asp).

The highly charged, very acidic proteins Ocr and ArdA may be used as a purification handle similar to other fusion tags. A monomeric mutant of the Ocr protein (13.8 kDa, very acidic, pI = 3.8) was used as a novel fusion tag whith displays solubilizing activity with a variety of different passenger proteins (DelProposto et al., 2009).

In general, perspectives of the use of the DNA mimics might be related diagnostics and therapy of various diseases (e.g. for inhibition of specific enzymes and corresponding biochemical processes in cells).

#### **8. Conclusion**

Modern data on the mechanisms of the modulation of the DNA - binding enzymes by protein mimicry of DNA are reviewed. It has recently been demonstrated that DNA-binding enzymes can be controlled by the direct binding of a control protein to the DNA-binding site on the enzyme. The structures of these control proteins have been discovered to mimic the structure and electrostatics of DNA. Such DNA-mimics might be able to target bacterial restriction systems (Ocr, ArdA), drug resistance systems (MfpA, QnrABCS), as well as replication, recombination, and repair. It puts forward a range of potential uses of new DNA mimics in applied biotechnology.

Figure 9 shows structures of Ocr and MfpA monomers and B-form DNA. Their comparison emphasizes the extraordinary capacities of living nature to develop unique forms crucial for adaptation. The most surprising thing is that nature has chosen different ways for design of proteins mimicking the DNA duplex. In one case (e.g. Ocr) these are tightly packed αhelices, in the other it is a right-handed β-helix (MfpA). Existence of significant negative charge (of the whole macromolecule or particular domain) required for similarity with the DNA polyanion is a common feature of DNA mimics. However, this is a necessary but not sufficient precondition. At the moment the only reliable method for detection of DNA mimics is X-ray analysis.

Fig. 9. The structures of Ocr(a) and MfpA(c) monomers and B-form of DNA(b)

#### **9. References**

108 Innovations in Biotechnology

sensitivity of such method. The perspectives of *in vitro* construction of new types of DNA mimics (i.e. generation of proteins with different "design" and new functions) may be quite wide. These include potential tasks of constructing of DNA mimics, inhibiting or modulating activity of specific groups of DNA-dependent enzymes and tasks related to site directed changes in the structure of already known DNA mimics. Such works are rather successful. For example, using site-directed mutagenesis we have modified the structure of ArdA and Ocr; the modified proteins selectively inhibit the endonuclease (restriction) activity of type I restriction–modification enzymes without any influence on their methylase (modification) activity (Zavilgelsky et al., 2011). The use of such type of antirestriction proteins in gene engineering gives an opportunity to develop stable strains with hybrid plasmids because the process of specific modification of chromosome DNA remains unimpaired. The protein Ocr has already been used as an effective factor promoting significant increase of bacterial transformation by plasmids. Adding a small amount of the Ocr protein to solution with plasmid DNA causes significant (by several orders of magnitude) increase in effectiveness of cell transformation during electroporation. In this case unmethylated DNA and host bacteria with active type I restriction–modification system are used. The Ocr protein (as well as plasmid DNA) easily penetrates inside cells and immediately protects unmethylated DNA against degradation (EPICENTRE Forum 9, 8,

The highly charged, very acidic proteins Ocr and ArdA may be used as a purification handle similar to other fusion tags. A monomeric mutant of the Ocr protein (13.8 kDa, very acidic, pI = 3.8) was used as a novel fusion tag whith displays solubilizing activity with a

In general, perspectives of the use of the DNA mimics might be related diagnostics and therapy of various diseases (e.g. for inhibition of specific enzymes and corresponding

Modern data on the mechanisms of the modulation of the DNA - binding enzymes by protein mimicry of DNA are reviewed. It has recently been demonstrated that DNA-binding enzymes can be controlled by the direct binding of a control protein to the DNA-binding site on the enzyme. The structures of these control proteins have been discovered to mimic the structure and electrostatics of DNA. Such DNA-mimics might be able to target bacterial restriction systems (Ocr, ArdA), drug resistance systems (MfpA, QnrABCS), as well as replication, recombination, and repair. It puts forward a range of potential uses of new DNA

Figure 9 shows structures of Ocr and MfpA monomers and B-form DNA. Their comparison emphasizes the extraordinary capacities of living nature to develop unique forms crucial for adaptation. The most surprising thing is that nature has chosen different ways for design of proteins mimicking the DNA duplex. In one case (e.g. Ocr) these are tightly packed αhelices, in the other it is a right-handed β-helix (MfpA). Existence of significant negative charge (of the whole macromolecule or particular domain) required for similarity with the DNA polyanion is a common feature of DNA mimics. However, this is a necessary but not sufficient precondition. At the moment the only reliable method for detection of DNA

variety of different passenger proteins (DelProposto et al., 2009).

htpp//www.epibio.com/forum.asp).

biochemical processes in cells).

mimics in applied biotechnology.

mimics is X-ray analysis.

**8. Conclusion** 


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### **Platelet Rich Plasma (PRP) Biotechnology: Concepts and Therapeutic Applications in Orthopedics and Sports Medicine**

Mikel Sánchez1, Isabel Andia1,2, Eduardo Anitua3 and Pello Sánchez1 *1Mikel Sánchez Arthroscopic Surgery Unit, Vitoria-Gasteiz 2Biocruces Research Institute, Vizcaya 3Eduardo Anitua Foundation, Vitoria-Gasteiz Spain* 

#### **1. Introduction**

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149-182

Regenerative medicine is the augmentation or substitution of diseased or injured cells or tissues by one of two means: (1) an improvement in the ability of endogenous cells to reform damaged tissue or (2) the use of exogenous cells or tissues to replace damaged cells or tissues. Advances in regenerative medicine essentially depend on improving our understanding of cell biology and molecular signaling. Cell signaling is complex and incompletely understood due to the multiple interactions and cross-talk among system components. The human body has some 100 trillion cells, which in the healthy state coordinate their actions through an exchange of chemical signals to maintain body homeostasis. Every cell phenotype secretes signaling proteins that influence their own behavior (autocrine) or the behavior of other neighboring cells (paracrine) through interactions with specific transmembrane receptors located in the cellular membrane. Currently, a great deal of research is directed towards improving our understanding of intercellular communication and the intracellular transduction of these signals; in the field of regenerative medicine, this knowledge will help to disentangle the mysteries of tissue repair and to achieve proper tissue repair and regeneration. Moreover, to reach this goal we must integrate all the information and understanding derived from basic research into novel therapies that yield quicker and more efficient tissue regeneration.

Within the last decade, the development of platelet-rich plasma (PRP) technology has emerged. The impact of the discoveries regarding the potential of PRP healing has fueled the optimism about autologous regenerative medicine. Indeed, the emergence and application of PRP technology, i.e., autologous molecular pool, has revolutionized the field of regenerative medicine in part due to the repair capacities of growth factors (GFs) and cytokines secreted by platelets. The easy preparation protocols, biosafety and versatility of PRP preparations have stimulated translational research and interest by both the scientific and medical communities. PRP therapies represent a major breakthrough in the treatment of many medical conditions and are currently one of the hottest topics in regenerative medicine because of their important implications for our future health. Discovery and contributions in the field have not only improved the clinical treatment of many patients with different clinical conditions but, from a multimolecular perspective, have opened the field of PRP science to cellular and molecular exploration of healing mechanisms. This technology provides the opportunity of moving molecular knowledge off the shelves and into practice, making it relevant in a clinical context, and achieves a true marriage between what we have learned through research and clinical applications.

This chapter will provide an overview of the potential therapeutic use of platelets and plasma for the release of signaling proteins in regenerative medicine. For the purposes of this chapter, the basic principles of healing and the role of platelets as molecular reservoirs will be discussed. A detailed description of the potential technological relevance of PRP biotechnology is followed by a section on applications of PRP therapies in numerous clinical conditions and medical fields with a special emphasis on orthopedics and sports medicine. There is no question that the key to both future advances in PRP science and its application in the treatment of disease and trauma lies in a better understanding of repair processes.

#### **2. A picture of healing mechanisms**

The most effective way to improve tissue repair is to understand normal healing mechanisms after a perturbation due to disease, which then becomes the basis for improving patient care and health. Healing mechanisms are, to a great extent, shared by the different tissues of the body and can be depicted by overlapping and successive phases characterized by a preponderance of cell signaling from various systems. The spatially and temporally dynamic nature of healing mechanisms presents a challenge to the identification of critical mechanisms. Firstly, hemostasis is accomplished through a network of processes that include the platelet system and the coagulation cascade; such processes arrest bleeding and set in motion the inflammatory response.

#### **2.1 Early inflammatory response**

Inflammation and blood coagulation are intimately linked. Acute inflammation, the complex systemic early defense system, is the first reaction of the innate immune system (platelet, leukocytes and macrophages) to injury. Direct exposure of cells to physical, mechanical or chemical trauma has immunological consequences relative to the degree of injury, i.e., the apoptotic or necrotic condition of resident fibroblasts. Accordingly, local regulatory mechanisms adjust the magnitude of the response so that inflammatory processes are localized to areas of damage, and the amount and duration of immune cell infiltration are adequate to phagocyte apoptotic/necrotic cells. In addition, endothelial cells, which are actively involved in healing, limit clot formation to the sites of injury. Activated platelets and leukocytes within this clot then release growth factors and numerous cytokines, establishing the onset of inflammation.

Eventually, spatially and temporally changing patterns of various leukocyte subsets transmigrate across the endothelium. Circulating neutrophils are rapidly captured by selectins that are presented by endothelial cells; they then invade the wounded tissue in response to chemical signals. The lifespan of neutrophils in the injured tissue is about two days, during which they perceive signals from the environment and respond by secreting cytokines (Borregaard et al., 2007). Furthermore, neutrophils release stored substances carried in different granule subsets, including reactive oxygen species, cationic peptides or

with different clinical conditions but, from a multimolecular perspective, have opened the field of PRP science to cellular and molecular exploration of healing mechanisms. This technology provides the opportunity of moving molecular knowledge off the shelves and into practice, making it relevant in a clinical context, and achieves a true marriage between

This chapter will provide an overview of the potential therapeutic use of platelets and plasma for the release of signaling proteins in regenerative medicine. For the purposes of this chapter, the basic principles of healing and the role of platelets as molecular reservoirs will be discussed. A detailed description of the potential technological relevance of PRP biotechnology is followed by a section on applications of PRP therapies in numerous clinical conditions and medical fields with a special emphasis on orthopedics and sports medicine. There is no question that the key to both future advances in PRP science and its application in the treatment of disease and trauma lies in a better understanding of repair processes.

The most effective way to improve tissue repair is to understand normal healing mechanisms after a perturbation due to disease, which then becomes the basis for improving patient care and health. Healing mechanisms are, to a great extent, shared by the different tissues of the body and can be depicted by overlapping and successive phases characterized by a preponderance of cell signaling from various systems. The spatially and temporally dynamic nature of healing mechanisms presents a challenge to the identification of critical mechanisms. Firstly, hemostasis is accomplished through a network of processes that include the platelet system and the coagulation cascade; such processes arrest bleeding

Inflammation and blood coagulation are intimately linked. Acute inflammation, the complex systemic early defense system, is the first reaction of the innate immune system (platelet, leukocytes and macrophages) to injury. Direct exposure of cells to physical, mechanical or chemical trauma has immunological consequences relative to the degree of injury, i.e., the apoptotic or necrotic condition of resident fibroblasts. Accordingly, local regulatory mechanisms adjust the magnitude of the response so that inflammatory processes are localized to areas of damage, and the amount and duration of immune cell infiltration are adequate to phagocyte apoptotic/necrotic cells. In addition, endothelial cells, which are actively involved in healing, limit clot formation to the sites of injury. Activated platelets and leukocytes within this clot then release growth factors and numerous

Eventually, spatially and temporally changing patterns of various leukocyte subsets transmigrate across the endothelium. Circulating neutrophils are rapidly captured by selectins that are presented by endothelial cells; they then invade the wounded tissue in response to chemical signals. The lifespan of neutrophils in the injured tissue is about two days, during which they perceive signals from the environment and respond by secreting cytokines (Borregaard et al., 2007). Furthermore, neutrophils release stored substances carried in different granule subsets, including reactive oxygen species, cationic peptides or

what we have learned through research and clinical applications.

**2. A picture of healing mechanisms** 

and set in motion the inflammatory response.

cytokines, establishing the onset of inflammation.

**2.1 Early inflammatory response** 

proteases. The key role of neutrophils is to clear the early rush of contaminating bacteria; in a sterile wound, such as surgical incisions that are experimentally induced, neutrophil absence does not perturb the healing process.

Monocyte recruitment and infiltration at the injury site happens days later and is highly regulated by adhesion molecules expressed by endothelial cells and by chemokines and other substances released by platelets, neutrophils (Soehnlein et al., 2009) and apoptotic/necrotic cells (Nathan, 2006). Commanded by signals present in the environment, monocytes turn into macrophages (the dedicated phagocytes) and induce major changes in gene expression and cell function. Indeed, the severity of tissue injury may determine the different states of macrophage activation. "Innate" activation occurs through lipolysaccharide or interferon-γ (IFN-γ) and is associated with a pro-inflammatory state [the production of interleukin-6 (IL-6), interleukin-1β (IL-1β) and tumor necrosis factor-α (TNFα). Alternatively, "classical" activation occurs through IL-4/IL-23 and is associated with the synthesis of healing factors including transforming growth factors (TGF-β and TGF–α), basic fibroblastic growth factor (bFGF), platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF) (Krysko et al., 2006).

Recent research suggests that these features of inflammation may determine the difference between efficient repair and the failure to repair. For example, in animal experiments, neutropenia accelerated the closure of incision wounds (Dovi et al., 2003) but did not affect the healing of surgically repaired tendons. Macrophage depletion impaired skin wounding by reducing collagen deposition and angiogenesis and also impaired the response to wounding in diabetic mice. Other studies suggest that targeting macrophage activation may provide a new therapeutic approach to protect tissues from ischemia and promote repair. Notwithstanding, macrophage depletion significantly improved the morphology and biomechanical properties of the tendon-bone interface after experimental anterior cruciate ligament (ACL) surgery. Thus, there are large gaps in the understanding of how neutrophils and macrophages influence repair. The difficulty in understanding the inflammatory response stems, in part, from biological redundancy: one molecule may have several functional roles, and different molecules may perform overlapping functions.

#### **2.2 Trophic phase**

New tissue formation occurs 2-10 days after injury and is characterized by cellular proliferation and the migration of different cell types. New blood vessels are formed by a process known as angiogenesis, and later, the sprouts of capillaries along with fibroblasts and macrophages replace the fibrin matrix with granulation tissue that forms the new substrate for cell migration

#### **2.2.1 Cell proliferation and migration**

The proliferative phase begins with the formation of a fibrin, fibronectin glycosiaminoglycan, and hyaluronic acid matrix that is initially populated with macrophages and platelets. The various cytokines secreted by these cells enhance cell migration into the site using the fibrin and fibronectin matrix as a scaffold. Progenitors of differentiated cell types, such as bone, cartilage, muscle, nerve sheath and connective tissue cells, are thought to contribute to a collection of proliferating progenitor cells. Alternatively, progenitor stem-cell-like for tissue niches migrate, divide and differentiate into tissue fibroblasts. Fibroblasts move through the extracellular matrix by binding fibronectin, vitronectin and fibrin via their arginine-glycineaspartic acid amino acid sequence recognized by their integrin receptors. The fibroblasts proliferate in response to GFs and cytokines and become the predominant cell type by the third to fifth day following injury. Fibroblasts also secrete extracellular zinc dependent endopeptidases called metalloproteinases (MMPs), which facilitate their movement through the matrix and help with the removal of damaged matrix components. Once the fibroblasts have entered the wound, they produce collagen, proteoglycans and other components. Fibroblast activities are predominantly regulated by GFs such as PDGF and TGF-β. PDGF secreted by platelets and macrophages stimulates fibroblast proliferation, chemotaxis and collagenase expression. TGF-β has pleiotropic actions that are context-dependent.

#### **2.2.2 Angiogenesis**

Angiogenesis occurs with the formation of new capillary networks through endothelial cell migration and division. Endothelial cells are activated to initiate angiogenesis such that new blood vessels are initiated to promote blood flow to support the high metabolic activity in the newly deposited tissue. Angiogenesis is regulated by a combination of local stimulatory factors such as VEGF and anti-angiogenic factors such as angiostatin, endostatin, and thrombospondin. Local factors that stimulate angiogenesis include low oxygen tension, low pH and high lactate levels. Soluble mediators such as bFGF, HGF, TGF- β and VEGF also stimulate endothelial cells to produce vessels. Tissue oxygen levels directly regulate angiogenesis through hypoxia inducible factor (HIF), which binds oxygen. When there is a decrease in oxygen levels surrounding capillary endothelial cells, HIF-1 levels increase and stimulate VEGF transcription to promote angiogenesis. Animal studies have shed some light on the natural pattern of GF expression during this stage. For example, signaling of VEGF-A via the endothelial receptors VEGFR1 and VEGFR2 is present at the healing site early after tissue injury. Other growth factors including TGF-β, PDGF-BB, and angiopoietin-1, which are important for vessel stabilization, are expressed later at the healing site.

The new vasculature allows the delivery of nutrients and the removal of by-products. As noted above, granulation tissue consists of a dense network of blood vessels and capillaries, elevated cellular density of fibroblasts and macrophages and randomly organized collagen fibers. The metabolic rate of this tissue is high and reflects the activity required for cell migration, division and protein synthesis, which emphasizes the importance of adequate nutrition and oxygen to properly heal the wound. Granulation tissue is particularly abundant and accompanies the process of wound healing by secondary intention.

#### **2.2.3 Synthesis of the extracellular matrix**

The high concentration of growth factors and cytokines initially secreted by platelets and leukocytes and later amplified by macrophages induces a rapid increase in specific cell populations, including migrating fibroblasts and resident cells. The number of stromal cells increases in parallel with angiogenesis, which is readily evident in the hypoxic environment produced by the injury. So, the production of extracellular matrix molecules grows in proportion with increasing cell number.

Growth factors, including TGF-β1, PDGF, BDNF, bFGF and type-I insulin-like growth factor (IGF-I), function at various stages during the healing process and produce different

niches migrate, divide and differentiate into tissue fibroblasts. Fibroblasts move through the extracellular matrix by binding fibronectin, vitronectin and fibrin via their arginine-glycineaspartic acid amino acid sequence recognized by their integrin receptors. The fibroblasts proliferate in response to GFs and cytokines and become the predominant cell type by the third to fifth day following injury. Fibroblasts also secrete extracellular zinc dependent endopeptidases called metalloproteinases (MMPs), which facilitate their movement through the matrix and help with the removal of damaged matrix components. Once the fibroblasts have entered the wound, they produce collagen, proteoglycans and other components. Fibroblast activities are predominantly regulated by GFs such as PDGF and TGF-β. PDGF secreted by platelets and macrophages stimulates fibroblast proliferation, chemotaxis and

Angiogenesis occurs with the formation of new capillary networks through endothelial cell migration and division. Endothelial cells are activated to initiate angiogenesis such that new blood vessels are initiated to promote blood flow to support the high metabolic activity in the newly deposited tissue. Angiogenesis is regulated by a combination of local stimulatory factors such as VEGF and anti-angiogenic factors such as angiostatin, endostatin, and thrombospondin. Local factors that stimulate angiogenesis include low oxygen tension, low pH and high lactate levels. Soluble mediators such as bFGF, HGF, TGF- β and VEGF also stimulate endothelial cells to produce vessels. Tissue oxygen levels directly regulate angiogenesis through hypoxia inducible factor (HIF), which binds oxygen. When there is a decrease in oxygen levels surrounding capillary endothelial cells, HIF-1 levels increase and stimulate VEGF transcription to promote angiogenesis. Animal studies have shed some light on the natural pattern of GF expression during this stage. For example, signaling of VEGF-A via the endothelial receptors VEGFR1 and VEGFR2 is present at the healing site early after tissue injury. Other growth factors including TGF-β, PDGF-BB, and angiopoietin-1, which

The new vasculature allows the delivery of nutrients and the removal of by-products. As noted above, granulation tissue consists of a dense network of blood vessels and capillaries, elevated cellular density of fibroblasts and macrophages and randomly organized collagen fibers. The metabolic rate of this tissue is high and reflects the activity required for cell migration, division and protein synthesis, which emphasizes the importance of adequate nutrition and oxygen to properly heal the wound. Granulation tissue is particularly

The high concentration of growth factors and cytokines initially secreted by platelets and leukocytes and later amplified by macrophages induces a rapid increase in specific cell populations, including migrating fibroblasts and resident cells. The number of stromal cells increases in parallel with angiogenesis, which is readily evident in the hypoxic environment produced by the injury. So, the production of extracellular matrix molecules grows in

Growth factors, including TGF-β1, PDGF, BDNF, bFGF and type-I insulin-like growth factor (IGF-I), function at various stages during the healing process and produce different

collagenase expression. TGF-β has pleiotropic actions that are context-dependent.

are important for vessel stabilization, are expressed later at the healing site.

abundant and accompanies the process of wound healing by secondary intention.

**2.2.3 Synthesis of the extracellular matrix** 

proportion with increasing cell number.

**2.2.2 Angiogenesis** 

outcomes depending on the conditions. For instance, PDGF, a chemotactic and mitotic factor for fibroblasts, also induces the synthesis of collagen type I. TGF-β1, which peaks early in injuries, is essential for the recruitment and maintenance of progenitor cells during neo-tissue formation, and its function might be necessary during healing. Additionally, the interactions of TGF-β1 with other TGF-β isoforms, namely TGF-β2 and TGF-β3, mediate which type of collagen is synthesized in the healing tissue. IGF-I anabolic and anti-apoptotic activities are regulated by IGF-I binding proteins (BP), IGFBP-2, IGFBP-3, and IGFBP-4, which are also present in the early healing response. In both humans and animals, the expression of IGF-I and TGF-β1 preceded the stimulation of collagen synthesis, a relevant issue in tissue healing. The bioactivity of these growth factors is regulated not only at the receptor level but through activation of TGF-β complex and IGF-binding proteins.

#### **2.3 Tissue remodeling and scarring**

Finally, the tissue enters into the last phase of healing, a long remodeling phase in which granulation tissue matures into a scar. Collagen accumulation reaches a maximum at 2-3 weeks after injury, and the transition to remodeling begins. There is a balance between synthesis, deposition and degradation during this phase. Small capillaries aggregate into larger blood vessels, and there is an overall decrease in the water content of the wound. Similarly, cell density and the overall metabolic activity of the wound decrease. The most dramatic change occurs in the overall type, amount and organization of the collagen fibers, resulting in an increased tensile strength of the tissue. Initially, there is increased deposition of collagen type III, also referred to as reticular collagen, that is gradually replaced by collagen type I. Collagen fibers are cross-linked by the enzyme lysyl oxidase, which is secreted by fibroblasts in the extracellular matrix. The normal adult 4:1 ratio of type I to type III collagen is restored during remodeling. Equilibrium is established as new collagen is formed and collagen type III is degraded. The MMPs, collagenases, gelatinases and stromelysins, control the degradation of extracellular matrix components to facilitate cell migration into the wound, angiogenesis and overall tissue remodeling.

In each of the described healing phases, the specific signaling activity is silenced or counterbalanced by other endogenous signals that serve to limit the duration and to promote progression to a new stage. During all these stages, local and migratory cells synthesize different patterns of GFs and cytokines in an attempt to cope with the temporal demands of the healing tissue. Consequently, therapeutic approaches to manipulate healing may need to integrate multiple cell types and large signaling networks that are necessary for the dynamic communication between cells. The need to target various signaling pathways simultaneously demands the administration of a balanced combination of mediators instead of administering a purified isolated protein, which could not cope with the multiple requirements of the injured tissue. Therefore, the ability to release signaling molecules in a spatiotemporal manner that mimics the needs of the injured tissue has become a challenge in the scientific and medical fields.

#### **2.4 Pathologic tissue healing**

There are many categories of impediments for wound healing. These include local or systemic impediments. The former include tissue viability, seroma and/or hematoma, infection, insufficient blood supply and/or mechanical factors. For example, adequate blood supply must exist to provide nourishment and oxygenation to healing tissues. A lack of blood supply may lead to tissue ischemia and an increased risk of infection. Tissues do not heal if there are more than 105 bacteria per gram of tissue. Hence, necrotic tissue must be debrided to avoid the risk of infection and because it interferes with normal healing.

Mostly clinical differences between chronic and acute healing tissues are thought to be explained in part by alterations in the local biochemical environment. The observation that increased amounts of TGF-β1 were present in hypertrophic scars led to clinical efforts to block scar formation through administration of antibodies against TGF-β1 and other pro-inflammatory mediators. Recent evidence also suggests that changes in the molecular environment of the cells in the wound may change the fate of healing tissues or organs. It is becoming increasingly apparent that growth factors and cytokines play a variety of key roles during normal tissue repair, and many have also been shown to act therapeutically in situations where normal healing is impaired. Although tremendous strides have been made in delineating the myriad of factors involved in normal and pathological healing, it has become clear that single-agent therapies, such as administration of growth factors, have only a moderate impact on tissue repair in the clinical setting, probably due to the redundancy and plasticity of the components of tissue repair or their rapid degradation at the injured site.

In this context, the emergence of PRP biotechnology as a way to harness tissue regeneration for medical needs has fueled the optimism surrounding cell-signaling based regenerative medicine. A deeper understanding will accelerate the development of PRP therapies.

#### **3. Platelets: Molecular contribution to healing**

The ultimate solution to tissue healing is likely to be the administration of multimolecular preparations with the ability to elaborate the full complexity of biological signaling, including all the environmental cues that are needed to regulate the biological mechanisms described above. Platelets are a natural source of growth factors and cytokines involved in tissue healing. Until now, it has not been possible to provide a pool of molecular signals and the temporary cell scaffold necessary to initiate healing in the same therapeutic agent. Substantial progress in the understanding of platelet biology has revealed much about the complexity of PRP therapies. Additional insights come from combining the information from the plasma proteome.

#### **3.1 Platelet biology**

Our understanding of fundamental aspects of platelet biology and function has been enriched in the last decades. Platelets are discoid cellular elements that are heterogeneous in size and have the smallest density of all blood cells, at 2 μm in diameter (a leukocyte is about 20 μm in diameter). They are anucleate and originate in the bone marrow as bulges along the length of pseudopodial extensions of megakaryocytes. The so termed proplatelets are then fragmented into individual platelets and released into the blood stream where they travel for about 7-10 days before removal from circulation after senescence and are replaced with younger platelets possessing greater functional capabilities. Platelets are replete with secretory granules, which are critical to platelet function. Among the three types of granules, dense granules, alpha-granules and lysosomes, the alpha-granule is the most abundant. There are approximately 50-80 alpha-granules per platelet, although they are heterogeneous with regard to cargo (Villeneuve et al., 2009). For example, anti-angiogenic proteins are packaged in different alpha-granules subpopulations than pro-angiogenic proteins. Moreover, there is some evidence that secretion of pro- versus anti-angiogenic stores may be agonist-specific (Italiano et al., 2008). The total protein content of platelets includes not only soluble proteins that are released into the extracellular space but also membrane bound proteins that become expressed on the platelet surface. Many of the proteins found in αgranules are also present in plasma. In a recent survey of the platelet membrane proteome, 629 membrane proteins were detected (Maynard et al., 2007). Overall proteomic studies suggest that more than 1048 soluble proteins are present in the supernatant of platelets.

#### **3.2 Platelet function**

118 Innovations in Biotechnology

infection, insufficient blood supply and/or mechanical factors. For example, adequate blood supply must exist to provide nourishment and oxygenation to healing tissues. A lack of blood supply may lead to tissue ischemia and an increased risk of infection. Tissues do not heal if there are more than 105 bacteria per gram of tissue. Hence, necrotic tissue must be

Mostly clinical differences between chronic and acute healing tissues are thought to be explained in part by alterations in the local biochemical environment. The observation that increased amounts of TGF-β1 were present in hypertrophic scars led to clinical efforts to block scar formation through administration of antibodies against TGF-β1 and other pro-inflammatory mediators. Recent evidence also suggests that changes in the molecular environment of the cells in the wound may change the fate of healing tissues or organs. It is becoming increasingly apparent that growth factors and cytokines play a variety of key roles during normal tissue repair, and many have also been shown to act therapeutically in situations where normal healing is impaired. Although tremendous strides have been made in delineating the myriad of factors involved in normal and pathological healing, it has become clear that single-agent therapies, such as administration of growth factors, have only a moderate impact on tissue repair in the clinical setting, probably due to the redundancy and plasticity of the components of tissue repair or their rapid degradation at

In this context, the emergence of PRP biotechnology as a way to harness tissue regeneration for medical needs has fueled the optimism surrounding cell-signaling based regenerative

The ultimate solution to tissue healing is likely to be the administration of multimolecular preparations with the ability to elaborate the full complexity of biological signaling, including all the environmental cues that are needed to regulate the biological mechanisms described above. Platelets are a natural source of growth factors and cytokines involved in tissue healing. Until now, it has not been possible to provide a pool of molecular signals and the temporary cell scaffold necessary to initiate healing in the same therapeutic agent. Substantial progress in the understanding of platelet biology has revealed much about the complexity of PRP therapies. Additional insights come from combining the information

Our understanding of fundamental aspects of platelet biology and function has been enriched in the last decades. Platelets are discoid cellular elements that are heterogeneous in size and have the smallest density of all blood cells, at 2 μm in diameter (a leukocyte is about 20 μm in diameter). They are anucleate and originate in the bone marrow as bulges along the length of pseudopodial extensions of megakaryocytes. The so termed proplatelets are then fragmented into individual platelets and released into the blood stream where they travel for about 7-10 days before removal from circulation after senescence and are replaced with younger platelets possessing greater functional capabilities. Platelets are replete with secretory granules, which are critical to platelet function. Among the three types of granules,

medicine. A deeper understanding will accelerate the development of PRP therapies.

**3. Platelets: Molecular contribution to healing** 

debrided to avoid the risk of infection and because it interferes with normal healing.

the injured site.

from the plasma proteome.

**3.1 Platelet biology** 

Not long ago, platelets were merely considered to function as haemostatic agents. However, as researchers broadened their understanding of platelets, many more facets were identified. Around 1980, platelets were recognized for their healing function. More than a decade later, the involvement of platelets in angiogenesis was discovered. Subsequently, Folkman showed that angiogenesis regulating proteins were selectively pumped into the budding pro-platelets from the mother megakaryocyte and that PF-4 is captured by platelets in tumor-bearing animals. Further developments using PRPs as a therapeutic biotechnology in the past few years have allowed the direct observation of platelet secretomes, not within, but outside the blood stream that interact with various injured tissues and organs.

In the physiological process of wound healing, platelets embedded within blood clots serve as a primary source of biologically active factors. Therefore, the PRP concept is straightforward. As platelets are a major source of healing factors within blood clots, the idea that concentrating platelets at the injured site could accelerate and optimize healing mechanisms opens the door for the development of PRP therapies. For example, typically after muscle strains or contusion, the hematoma that originates as a consequence of vessel disruption contains about 94% red blood cells, a small amount of platelets (4%) and less than 1% leukocytes. The rational for the use of PRPs involves replacing the blood clot with adhesive PRP, thus minimizing the presence of red blood cells (about 95% in volume) while increasing platelet concentration at the injury site. In doing so, we would achieve a supra-physiological concentration of platelet and plasma proteins that accelerates the repair process by direct or indirect mechanisms, i.e., by attracting immune cells via chemotaxis or enhancing further synthesis of healing proteins by local cells. Moreover, the ability to release these signaling factors in a spatiotemporal manner using the fibrin scaffold perfectly meets the needs of the injured tissue over time.

However, the present knowledge of both PRP therapies and healing mechanisms needs to be better explored to translate such knowledge into improved biological therapies.

#### **4. Platelet-rich plasma biotechnology: New tools for tissue repair**

#### **4.1 The history: A three decades perspective**

From a historical point of view, the first blood bank PRP preparations began during the 1960s and become routine preparations through the 1970s. In the 1980s, the advent of regenerative medicine aiming to rapidly translate the science into patient care using the patient's own resources opened the door to the use of platelets as vehicles for the delivery of a balanced pool of healing factors. At that time, platelets were found to release wound healing substances that initiated the repair of injured tissues and vessels in cutaneous ulcers (Margolis et al., 2001). Later in the 1990s, platelets were introduced into maxillofacial surgery as autologous modifications of potent adhesives known as fibrin glues. The use of platelets was particularly fortuitous given that the main initial interest was to take advantage of the adhesive and haemostatic properties of the homologous fibrin during bone surgery. A realization of the clinical potential of PRP-therapies followed the positive clinical observations, such as enhanced bone formation and anti-inflammatory functions, during oral and maxillofacial applications (Whitman et al., 1997; Marx et al., 1998; Anitua E, 1999). At the beginning of the millennium, PRP was used for the first time to treat knee injuries in arthroscopic surgery (Sánchez et al., 2003 a and b), and later it was extended to the treatment of tendons (Sánchez et al., 2007), muscles injuries (Sánchez et al., 2005), osteoarthritic knees (Sánchez et al., 2008) and hips (Sánchez et al., 2011) and for use in chondropathies (Kon et al., 2010). Below we show the temporal sequence of the development of PRP therapies.

Fig. 1. Temporal sequence of the development of PRP technologies

#### **4.2 Terminology**

Long before any therapeutic application was imagined, the term PRP, which described plasma with a platelet count above the peripheral blood, was coined by hematologists. In 2007, the novel connotation of PRP was introduced to the Medical Subject Heading database (MeSH): PRP refers to a product consisting of PLATELETS concentrated in a limited volume of PLASMA used in various surgical tissue regeneration procedures where the GROWTH FACTORS in the platelets enhance wound healing and regeneration. At present (2011), the field is growing more complex, and the primacy of growth factors is now shared by new classes of platelet released biomolecules, which are also critical in healing.

#### **4.3 PRP preparation**

Peripheral blood is the supply source for the preparation of PRPs; the mean number of circulating platelets is 200,000 plt/µl. For PRP preparation, peripheral blood is drawn from the

resources opened the door to the use of platelets as vehicles for the delivery of a balanced pool of healing factors. At that time, platelets were found to release wound healing substances that initiated the repair of injured tissues and vessels in cutaneous ulcers (Margolis et al., 2001). Later in the 1990s, platelets were introduced into maxillofacial surgery as autologous modifications of potent adhesives known as fibrin glues. The use of platelets was particularly fortuitous given that the main initial interest was to take advantage of the adhesive and haemostatic properties of the homologous fibrin during bone surgery. A realization of the clinical potential of PRP-therapies followed the positive clinical observations, such as enhanced bone formation and anti-inflammatory functions, during oral and maxillofacial applications (Whitman et al., 1997; Marx et al., 1998; Anitua E, 1999). At the beginning of the millennium, PRP was used for the first time to treat knee injuries in arthroscopic surgery (Sánchez et al., 2003 a and b), and later it was extended to the treatment of tendons (Sánchez et al., 2007), muscles injuries (Sánchez et al., 2005), osteoarthritic knees (Sánchez et al., 2008) and hips (Sánchez et al., 2011) and for use in chondropathies (Kon et al., 2010). Below we show the

temporal sequence of the development of PRP therapies.

Fig. 1. Temporal sequence of the development of PRP technologies

classes of platelet released biomolecules, which are also critical in healing.

Long before any therapeutic application was imagined, the term PRP, which described plasma with a platelet count above the peripheral blood, was coined by hematologists. In 2007, the novel connotation of PRP was introduced to the Medical Subject Heading database (MeSH): PRP refers to a product consisting of PLATELETS concentrated in a limited volume of PLASMA used in various surgical tissue regeneration procedures where the GROWTH FACTORS in the platelets enhance wound healing and regeneration. At present (2011), the field is growing more complex, and the primacy of growth factors is now shared by new

Peripheral blood is the supply source for the preparation of PRPs; the mean number of circulating platelets is 200,000 plt/µl. For PRP preparation, peripheral blood is drawn from the

**4.2 Terminology** 

**4.3 PRP preparation** 

patient under sterile conditions, with or without anticoagulants, and the plasma is prepared by centrifugation or filtration. The volume can be adapted to the clinical needs, ranging from 10 to 100 mL. Essentially, the methods of producing PRPs determine the composition and concentration of leukocytes, erythrocytes and platelets in a given plasma volume. There are three methods: 1) the double spinning method using automated machines and commercial kits, 2) the single spinning method using conventional laboratory centrifuges followed by manual PRP separation, and 3) selective blood filtration using commercially available technology. Single spinning yields a 1-3 fold change in platelet concentration over baseline levels, and double spinning yields a 4-8 fold change in platelet concentration over baseline levels. Double spinning also concentrates leukocytes. Accordingly, platelet concentrates have been categorized as pure platelet-rich plasma (P-PRP), in which leukocytes are purposely eliminated from the PRP, and leukocyte and platelet-rich plasma (L-PRP), which contains a high concentration of leukocytes (Dohan et al., 2009).

Fig. 2. Methods of producing PRP determine the composition and concentration of leukocytes, and platelets in a given plasma volume

At present, there is much debate surrounding four central questions of clinicians: (1) is the number of platelets important, (2) is the presence of leukocytes important, (3) when should PRP be activated, and (4) how should PRP be activated. The clinical variability observed throughout the studies points out that some techniques might not produce a sufficient number of functional platelets to produce the expected outcome. Similarly, there is no consistency in the methods of application of this therapy, the timing of treatment, the number of injections per series or the volume of injections. This has precluded the establishment of the standards necessary to integrate the extensive relevant literature in basic and clinical science. For example, double spinning techniques yield a PRP concentrate with a volume of about 10% of the volume of blood withdrawn (i.e., 20 mL of whole blood would result in 2 mL of PRP). In contrast, 40-50% of the blood volume is obtained after single spinning. Also, each method leads to a different product with differing biological properties and potential uses. Currently, it is unclear whether these differences have any clinical relevance. Some authors have suggested that PRP preparations containing only moderately elevated platelet concentrations induce optimal biological benefit, whereas lower platelet concentrations produce suboptimal effects and higher concentrations produce inhibitory effects. According to others, the 'therapeutic dose' of PRP is at least 4-6 times higher than the normal platelet count. To add to the discussion, the actual growth factor content does not correlate with the platelet count in whole blood or in PRP when leukocytes are present in the preparation, and there is no evidence that gender or age affects platelet count or growth factor concentrations. However, age may influence the number of receptors on local cells interacting with the plasma signals.

#### **4.4 PRP activation and fibrin delivery**

Because these procedures are considered to be an autograft by the regulatory authorities of most countries, the plasma should be prepared and immediately used at the point of care, and the plasma should not be stored. Prior to application, platelets can be slowly activated by setting in motion the coagulation cascade with the addition of calcium chloride, a necessary cofactor for prothrombin conversion to thrombin. Alternatively, coagulation and platelets can be instantly activated by adding a standard solution of bovine or human thrombin with 10% calcium chloride to the PRP. After plasma activation, the fibrin scaffold can be formed *in vivo* or *ex vivo*: the latter is suitable for implantation in surgery or in ulcer care and provides a gradual release of growth factors in the area where it has been applied. Depending upon the activation mechanism, induced by CaCl2, collagen or thrombin can achieve a sudden burst of GFs or a gradual release. Indeed, a central question in biology and cell signaling is how extracellular factors elicit a complex set of signaling events to achieve specific cellular functions.

Figure below shows fibrin which is a natural biopolymer involved in the coagulation cascade formed upon fibrinogen cleavage by thrombin. It acts as a reservoir for growth factors, cells and enzymes during wound healing and provides a scaffold for the synthesis of the extracellular matrix. Fibrin scaffolds provide nature's cues for tissue regeneration. Fibrin is a key scaffold material for the delivery of biomolecules, and it mimics natural processes and provides adequate exposure time to maximize biological interactions.

The kinetics of signaling may be influenced not only by distinct cell surface receptors but also by the method that their cognate ligands are secreted or delivered. A receptor may be acutely activated by an immediate increase in ligand concentration, a process mimicked in most pharmacological studies. In many cellular processes in vivo, however, cells encounter a gradual increase in the concentration of extracellular factors, i.e., constitutively secreted factors need to accumulate over time to reach a threshold set by the affinity of the receptor.

consistency in the methods of application of this therapy, the timing of treatment, the number of injections per series or the volume of injections. This has precluded the establishment of the standards necessary to integrate the extensive relevant literature in basic and clinical science. For example, double spinning techniques yield a PRP concentrate with a volume of about 10% of the volume of blood withdrawn (i.e., 20 mL of whole blood would result in 2 mL of PRP). In contrast, 40-50% of the blood volume is obtained after single spinning. Also, each method leads to a different product with differing biological properties and potential uses. Currently, it is unclear whether these differences have any clinical relevance. Some authors have suggested that PRP preparations containing only moderately elevated platelet concentrations induce optimal biological benefit, whereas lower platelet concentrations produce suboptimal effects and higher concentrations produce inhibitory effects. According to others, the 'therapeutic dose' of PRP is at least 4-6 times higher than the normal platelet count. To add to the discussion, the actual growth factor content does not correlate with the platelet count in whole blood or in PRP when leukocytes are present in the preparation, and there is no evidence that gender or age affects platelet count or growth factor concentrations. However, age may influence the number of receptors

Because these procedures are considered to be an autograft by the regulatory authorities of most countries, the plasma should be prepared and immediately used at the point of care, and the plasma should not be stored. Prior to application, platelets can be slowly activated by setting in motion the coagulation cascade with the addition of calcium chloride, a necessary cofactor for prothrombin conversion to thrombin. Alternatively, coagulation and platelets can be instantly activated by adding a standard solution of bovine or human thrombin with 10% calcium chloride to the PRP. After plasma activation, the fibrin scaffold can be formed *in vivo* or *ex vivo*: the latter is suitable for implantation in surgery or in ulcer care and provides a gradual release of growth factors in the area where it has been applied. Depending upon the activation mechanism, induced by CaCl2, collagen or thrombin can achieve a sudden burst of GFs or a gradual release. Indeed, a central question in biology and cell signaling is how extracellular factors elicit a

Figure below shows fibrin which is a natural biopolymer involved in the coagulation cascade formed upon fibrinogen cleavage by thrombin. It acts as a reservoir for growth factors, cells and enzymes during wound healing and provides a scaffold for the synthesis of the extracellular matrix. Fibrin scaffolds provide nature's cues for tissue regeneration. Fibrin is a key scaffold material for the delivery of biomolecules, and it mimics natural

The kinetics of signaling may be influenced not only by distinct cell surface receptors but also by the method that their cognate ligands are secreted or delivered. A receptor may be acutely activated by an immediate increase in ligand concentration, a process mimicked in most pharmacological studies. In many cellular processes in vivo, however, cells encounter a gradual increase in the concentration of extracellular factors, i.e., constitutively secreted factors need to accumulate over time to reach a threshold set by the affinity of the receptor.

processes and provides adequate exposure time to maximize biological interactions.

complex set of signaling events to achieve specific cellular functions.

on local cells interacting with the plasma signals.

**4.4 PRP activation and fibrin delivery** 

Fig. 3. Fibrin is a temporary scaffold for cell adhesion at the injured site and also functions as a vehicle for the delivery of growth factors and cytokines

Questions about safety still linger regarding the routine use of PRP. Any concerns regarding the transmission of diseases such as HIV, hepatitis, or Creutzfeldt-Jakob disease, or of the development of immunogenic reactions, a concern with the use of allografts or xenografts, are by definition not applicable due to the autologous nature of PRP. However, some systems use purified bovine thrombin to activate the platelets. This may produce coagulopathies, and most commercial systems now use recombinant human thrombin.

Some authors have raised the issue of genetic instability and have hypothesized that the use of PRP may lead to the development of neoplasms. Growth factors act on receptors located on the cell membranes rather than on the cell nucleus and activate normal gene expression via intracellular signaling proteins, which promote normal, not abnormal, gene expression. Growth factors are not directly mutagenic, and their activities in normal wound healing are highly regulated by various feedback control mechanisms. Furthermore, up to now, no systemic effect on circulating growth factors has been shown after PRP application.

Some antimicrobial activity of PRP (platelet-leukocyte gel) against *Staphylococcus aureus* has been shown in vitro and in vivo, although it is not comparable to systemic antibiotic treatment.

#### **4.5 Technological relevance of PRP biotechnology**

The medical industry is benefiting from a robust demand for technologically advanced products that accommodate the increasingly active baby boomer (people born between the 1940s and 1960s) life style as well as the sedentary lifestyle that accompanies the escalating levels of obesity. Expansion is rapidly occurring in the bone growth factor and protein segments, termed orthobiologics. In fact, PRP technologies are now very important in at least two main market segments: (1) bone repair, which includes the use of regular PRP or PRP composites made of PRP mixed with structural biomaterials or bone grafts and (2) soft tissue repair, which includes the development of techniques for applying PRPs and the surgical tools needed to correctly apply the different physicalchemical configurations of the biomaterial. An estimated 30% of new products under development are "combo products" that involve medical devices embedded with pharmaceutical or biologics components. Ambulatory PRP treatments benefit from pointof-care ultrasonography; ultrasound guidance improves success in PRP per-cutaneous and intra-articular procedures.

Recently, the medical industry has realized the potential of autologous products. Thus, although not fully developed yet, autologous technologies are readily available, and the present leading firms that control the orthopedic industry and market, Zimmer Holdings, Stryker, Biomet, Arthrex, DePuy, Smith & Nephew and Synthes, have introduced PRP devices. In the last few years, several semi-automatic machines have been developed for the centrifugal separation of PRP for therapeutic use. The process of PRP preparation is relatively straight forward and can be performed in the clinic or in the operating room. In most cases, it can be completed within minutes. The cost to both medical practitioners and patients varies widely depending on the method used to produce the PRP.

#### **5. Therapeutic applications**

The versatility and biocompatibility of PRP biotechnology has stimulated its therapeutic use in many different fields (see Figure below), including orthopedics, sports medicine, ophthalmology, dentistry, and cosmetic, plastic and maxillofacial surgery. Here we present some of the most interesting therapeutic applications with a special emphasis on musculoskeletal applications.

Fig. 4. Application of PRP therapies in the different medical fields: management of muscle injuries in sports medicine, open orthopedic surgery, arthroscopic surgery, ulcer care, peripheral nerve repair or the treatment of corneal ulcers in ophthalmology

#### **Demand for musculoskeletal care**

124 Innovations in Biotechnology

important in at least two main market segments: (1) bone repair, which includes the use of regular PRP or PRP composites made of PRP mixed with structural biomaterials or bone grafts and (2) soft tissue repair, which includes the development of techniques for applying PRPs and the surgical tools needed to correctly apply the different physicalchemical configurations of the biomaterial. An estimated 30% of new products under development are "combo products" that involve medical devices embedded with pharmaceutical or biologics components. Ambulatory PRP treatments benefit from pointof-care ultrasonography; ultrasound guidance improves success in PRP per-cutaneous

Recently, the medical industry has realized the potential of autologous products. Thus, although not fully developed yet, autologous technologies are readily available, and the present leading firms that control the orthopedic industry and market, Zimmer Holdings, Stryker, Biomet, Arthrex, DePuy, Smith & Nephew and Synthes, have introduced PRP devices. In the last few years, several semi-automatic machines have been developed for the centrifugal separation of PRP for therapeutic use. The process of PRP preparation is relatively straight forward and can be performed in the clinic or in the operating room. In most cases, it can be completed within minutes. The cost to both medical practitioners and

The versatility and biocompatibility of PRP biotechnology has stimulated its therapeutic use in many different fields (see Figure below), including orthopedics, sports medicine, ophthalmology, dentistry, and cosmetic, plastic and maxillofacial surgery. Here we present some of the most interesting therapeutic applications with a special emphasis on

Fig. 4. Application of PRP therapies in the different medical fields: management of muscle injuries in sports medicine, open orthopedic surgery, arthroscopic surgery, ulcer care,

peripheral nerve repair or the treatment of corneal ulcers in ophthalmology

patients varies widely depending on the method used to produce the PRP.

and intra-articular procedures.

**5. Therapeutic applications** 

musculoskeletal applications.

Musculoskeletal disorders, which affect millions of people worldwide, can lead to chronic pain and physical disability. According to leading pain experts, more people around the world experience musculoskeletal pain than any other type of pain (Global year against musculoskeletal pain, Oct 2009-Oct 2010, www.iasp-pain.org ). In the United States alone, 2.5 million orthopedic reconstructions, including bone, cartilage, ligament and tendon reconstructions, are performed annually. With an aging population and a prolonged life expectancy, an increase in the number of patients suffering from musculoskeletal disorders such as osteoporosis and arthritis are expected in the future. The former is currently suffered by 10 million Americans over the age of 50, and the latter is a chronic musculoskeletal disease that affects 1 in 3 adult Americans. In addition, every year sporting activities result in a variety of injuries to cartilage, ligaments and especially muscles. Taken together, these musculoskeletal diseases increase patient morbidity and disability and the social and economical consequences are staggering. As a result, the period of 2000-2010 has been named "the bone and joint decade" in an effort to increase the attention of scientists regarding the problems related to these disorders and to promote advancement in these fields [Anitua et al., 2006].

#### **5.1 Orthopedic applications**

#### **5.1.1 Bone repair**

More than 6 million bone fractures are reported annually in the USA, of which 5-10% have impaired healing that causes pain and disability. To improve patient-care results, scientists are making great efforts to create bone substitutes and to develop ways of improving bone healing. The use of platelet rich preparations may help to fulfill some of these requirements, particularly as an aid to bone regeneration. In fact, in vitro studies have clearly demonstrated that platelet derived growth factors stimulate the proliferation of human trabecular bone cells and the differentiation of human osteoblast-like cells. Studies have confirmed that the local application of PRPs is especially important in pathological conditions in which bone healing is weakened due to an inadequate blood supply, such as that observed in atrophic nonunion fractures. Both percutaneous injection and surgical augmentation with freshly prepared PRP have been shown to normalize fracture callus (Sánchez et al., 2008). The hundreds of soluble proteins released from both plasma and platelets include VEGF-A, PDGF, FGF, EGF, HGF, and IGF. These angiogenic activators collectively promote vessel wall permeability and promote the growth and proliferation of endothelial cells (Nurden et al., 2008).These findings are consistent with those seen in diabetic patients with a Charcot foot who showed improved healing and fewer complications after ankle fusion treated with fresh PRP. In contrast, previously frozen and thawed PRP supplementation in long bone nonunions treated with external fixation failed to provide clinical usefulness. In orthopedic trauma to date, there are not enough clinical studies to make definite conclusions. However, in some clinical conditions, the development of newly grown bone may be a realistic target if PRP is applied with cells or scaffolds. In fact, the effectiveness of bone grafting can be enhanced by creating custom-made biomaterials that will meet specific structural and biological tissue requirements in different anatomical locations. In this context, a wide array of composite biomaterials can be created by mixing PRP with either artificial or natural biomaterials. Moreover, the use of PRP improves the handling, adhesion and adaptation of the composite graft. This is in part because these biological products may act as a biologic glue to hold together the matrix particles. Apart from facilitating the handling and manipulation, the combination of both materials may have synergistic effects on bone regeneration. For example, when patients with solitary bone cysts were treated with allogenic grafts and PRP, the cysts were filled with newly formed bone after 12 months (Pedzizs et al., 2010). In a randomized control trial among people undergoing a medial, opening-wedge osteotomy of the proximal tibia, the use of an allograft with PRP showed better radiographic osseointegration at all stages of follow-up (Dallari D et al., 2007)

Developing engineered tissue is another interesting approach for bone regeneration. This may be feasible after combining mesenchymal stem cells (MSCs) and scaffold-like platelet rich plasma preparations. In fact, isolated cells, growth factors and biocompatible supporting scaffolds have generally been considered essential prerequisites to tissue engineering approaches. In the last few years, several attempts have been reported especially for bone regeneration but also for cartilage and periodontal tissue engineering. For example, the potential bone regeneration capacity of an MSC and platelet rich plasma mixture (MSC/PRP) was analyzed and compared with other approaches, including a natural deproteinized bovine bone, an autologous bone and the platelet rich product alone. Compared with the other treatments, the results of histology and mechanical properties showed that the MSC/PRP combination provided greater bone maturation and early stage bone regeneration. This mixed preparation has also been successfully used for bone regeneration in several patients. Encouraging results were observed in clinical studies exclusively concerning children. For instance, in the distraction of long bones, Kitoh et al. (2007) reported less complications in children treated with PRP plus MSCs than in children that did not receive PRP and MSC augmentation. The same authors reported an enhanced healing index in a controlled series of children with achondroplasia or hypochondroplasia undergoing limb-lengthening procedures. Even so, achieving control of bone healing is difficult, and the challenges associated with PRP therapies are enormous, extending beyond the present knowledge.

#### **5.1.2 Joint repair**

Arthroscopy is a minimally invasive procedure that allows doctors to treat joint injuries and disease through small incisions in the skin. The concept of not having to perform extensive soft tissue dissection is appealing because the recovery is quicker and less painful than open techniques. The use of PRP in arthroscopic surgery was initially introduced by Sanchez et al. (2003) in the treatment of a cartilage avulsion in the knee of a young athlete and in the reconstruction of the anterior cruciate ligament, as explained below (Sánchez et al., 2003). In recent work, Guadilla et al. (2011) showed how the arthroscopic management of the femoral head may be enhanced by the application of PRP in several ways. First, by grafting the necrotic area with trabecular bone mixed with PRP to induce angiogenesis and to enhance cell survival and function. Second, platelet-rich plasma can be applied within the intra-articular space to improve the conditions of synovial cells, chondrocytes, and subchondral osteoblasts [Andia et al, 2011].

Other authors have shown that the perioperative application of platelet rich plasma and fibrin sealant in arthroplasties reduces blood transfusion requirements, the length of the hospital stay, and the incidence of blood leakage and arthrofibrosis while it improves the range of motion. Another step forward would be to explore the analgesic and antiinflammatory effects of PRP. Additional potential benefits, including blood loss, shorter hospital stay, and faster recovery time, should also be investigated.

*PRP and cartilage engineering ex-vivo:* Evidence of the effects of PRP on cellular proliferation and differentiation comes mainly from studies of tissue engineering. For example, chondral lesions represent a clinical challenge due to the limited capacity of chondrocytes to proliferate in vivo. Thus, autologous cells can be harvested from a small tissue biopsy and sufficiently expanded ex-vivo for re-implantation. When articular chondrocytes are the cellular source, PRP improves ex-vivo proliferation but also causes de-differentiation. Importantly, PRP-expanded cells retain their capacity to re-differentiate and synthesize cartilage-specific proteins when transferred to a 3D environment.

The cultivation of stem cells is another alternative that is under clinical investigation for the treatment of osteoarthritis; given their capacity to differentiate into chondrocytes and secrete a wide array of biologically active factors that support cell proliferation and tissue formation. The sources of these stem cells include the bone marrow and the synovial fluid. In addition, the Hoffa fat pad contains stem cells with chondrogenic potential. Stem cells derived from the meniscus, synovium, Hoffa fat, synovial fat and ACL share similar gene expression profiles. Culturing these cells under hypoxic conditions has been shown to enhance their differentiation into cartilage-like tissue.

To avoid contact of the cells with bovine products and to implement GMP-compatible protocols, PRP releasates or lysates provide a feasible alternative to fetal calf or bovine serum in the expansion of these cells for cartilage engineering purposes. The addition of PRP (compared to fetal calf serum) improves cellular expansion and imparts a differentiation capacity towards the osteogenic, chondrogenic and adipogenic lineage. In addition, PRPs can be used as carriers for chondrocyte delivery during re-implantation.

#### **Osteoarthritis**

126 Innovations in Biotechnology

improves the handling, adhesion and adaptation of the composite graft. This is in part because these biological products may act as a biologic glue to hold together the matrix particles. Apart from facilitating the handling and manipulation, the combination of both materials may have synergistic effects on bone regeneration. For example, when patients with solitary bone cysts were treated with allogenic grafts and PRP, the cysts were filled with newly formed bone after 12 months (Pedzizs et al., 2010). In a randomized control trial among people undergoing a medial, opening-wedge osteotomy of the proximal tibia, the use of an allograft with PRP showed better radiographic osseointegration at all stages of

Developing engineered tissue is another interesting approach for bone regeneration. This may be feasible after combining mesenchymal stem cells (MSCs) and scaffold-like platelet rich plasma preparations. In fact, isolated cells, growth factors and biocompatible supporting scaffolds have generally been considered essential prerequisites to tissue engineering approaches. In the last few years, several attempts have been reported especially for bone regeneration but also for cartilage and periodontal tissue engineering. For example, the potential bone regeneration capacity of an MSC and platelet rich plasma mixture (MSC/PRP) was analyzed and compared with other approaches, including a natural deproteinized bovine bone, an autologous bone and the platelet rich product alone. Compared with the other treatments, the results of histology and mechanical properties showed that the MSC/PRP combination provided greater bone maturation and early stage bone regeneration. This mixed preparation has also been successfully used for bone regeneration in several patients. Encouraging results were observed in clinical studies exclusively concerning children. For instance, in the distraction of long bones, Kitoh et al. (2007) reported less complications in children treated with PRP plus MSCs than in children that did not receive PRP and MSC augmentation. The same authors reported an enhanced healing index in a controlled series of children with achondroplasia or hypochondroplasia undergoing limb-lengthening procedures. Even so, achieving control of bone healing is difficult, and the challenges associated with PRP therapies are

Arthroscopy is a minimally invasive procedure that allows doctors to treat joint injuries and disease through small incisions in the skin. The concept of not having to perform extensive soft tissue dissection is appealing because the recovery is quicker and less painful than open techniques. The use of PRP in arthroscopic surgery was initially introduced by Sanchez et al. (2003) in the treatment of a cartilage avulsion in the knee of a young athlete and in the reconstruction of the anterior cruciate ligament, as explained below (Sánchez et al., 2003). In recent work, Guadilla et al. (2011) showed how the arthroscopic management of the femoral head may be enhanced by the application of PRP in several ways. First, by grafting the necrotic area with trabecular bone mixed with PRP to induce angiogenesis and to enhance cell survival and function. Second, platelet-rich plasma can be applied within the intra-articular space to improve the conditions of

Other authors have shown that the perioperative application of platelet rich plasma and fibrin sealant in arthroplasties reduces blood transfusion requirements, the length of the

synovial cells, chondrocytes, and subchondral osteoblasts [Andia et al, 2011].

follow-up (Dallari D et al., 2007)

enormous, extending beyond the present knowledge.

**5.1.2 Joint repair** 

The dramatic increase in the incidence and prevalence of joint pathology over the past two decades has focused attention on therapeutic interventions that can reverse or ameliorate progressive joint damage and pathology. Degenerative osteoarthritis (OA) is the most common form of arthritis and affects nearly 27 million adults in the US (Lawrence et al., 2008). Despite the vast amount of molecular knowledge accrued during the last few years, a major breakthrough in OA therapy has not emerged. A large part of the problem is that researchers do not know enough about the biology of OA to identify the right targets. The disease is the result of a long chain of events, but some of the links in that chain are still a mystery; nobody is certain which link to cut in order to stop disease progression. Limiting factors in the current efforts are to some extent attributed to a poor understanding of the molecular basis of the disease progression and the lack of dynamic biomarkers that reflect specific biological or pathological processes. Hence, with the exception of surgery, all approaches are merely palliative. The conservative management of OA and chondropathies with PRP biotechnologies is becoming increasingly popular, but clinical evidence is preliminary and modest and is limited mostly to observational case studies that have used patient-reported outcomes as end points. Our preliminary clinical results in a retrospective cohort study of knee OA showed that intra-articular injection of PRP decreased pain and enhanced function compared to hyaluronic acid (HA) injections (Sánchez et al., 2008). In a case series study that involved 115 young patients with low degrees of articular degeneration in the knee, Kon et al. (2010) reported reduced pain and improved function that was maintained at 12 months but not 24 months after treatment (Filardo et al., 2011). Sampson et al. (2010), also in a small case series (n=13), reported significant pain and symptom relief but did not find any significant change in the daily activities or quality of life of the patients treated. PRP injections for hip OA produced clinically significant reductions in pain and function, although this was only seen in 40% of the patients studied (Sánchez et al., 2011). When discussing PRP therapies, differences between the preparations and the readministration procedures used should be acknowledged. Although pure PRP and leukocyte PRP formulations are not comparable in terms of leukocyte content, platelet count or plasma volume, the resulting improvements in pain and function were not exclusive to any one formulation. The pursuit to identify a unifying therapy for OA would be enhanced by refining the end points in future clinical studies.

#### **5.2 Sport medicine**

Sports related soft tissue injuries cause athletes to lose a significant amount of time from their sport and represent a significant burden to society in terms of health care resources, personal disability and activity restriction. In 2002, an estimated 15.8 billion dollars in total health care expenditures was used for the medical management of these injuries (Yu WW 2005). Soft tissue disorders, including muscle, tendon, ligament and joint capsular injuries, represent more than 50% of all the musculoskeletal injuries reported each year in the USA. Primary care studies have shown that 16% of the general population suffers from shoulder pain, whereas elbow tendinopathy affects 1-2% of the population. The importance of this problem is substantial because the field of sports medicine influences millions of people from athletes to those who participate in recreational sports or simply exercise to stay healthy and active.

#### **5.2.1 Muscle injuries**

Muscle injuries resulting from extrinsic or intrinsic mechanisms are extremely common in sports, accounting for about 35-45% of all injuries. Contact sports and sports that require the generation of large eccentric forces present the highest risk. The vulnerability of soccer players to strains and contusions is a substantial problem for professional players and their clubs; such injuries involve significant time lost from training and competition. Due to the increasing demands of the competitive soccer season, muscle treatments able to accelerate the recovery time without adversely affecting the recurrence rate (i.e., those that can minimize the scarring response) are of paramount importance [Andia et al, 2011].

At present, no drugs have been developed that hasten the restoration of muscle function after injury. Therefore, in the absence of any available evidence-based treatments, injection therapies may be an important option to help professional athletes. At the 2nd World Congress of Regenerative Medicine, Sanchez (2005) reported for the first time the application of leukocyte-free PRP to 21 muscle injuries of different severities and different anatomical locations. Small tears progressed well with a single application, whereas more severe tears required 2-3 ultrasound-guided injections. The injected volume depended on tear severity. These athletes, who played in first division teams of the Spanish Soccer League, resumed normal training activities in half the time needed by matched historical controls. Using the same leukocyte-free PRP preparation, Wee (2009) reported good outcomes (1 week to return to pre-injury activities) after three weekly US-guided injections to treat an adductor longus strain in a professional bodybuilder.

Another autologous blood derived biotechnology is named ACS (Autologous Conditioned Serum). This technology consists of an autologous liquid serum conditioned by the incubation of whole blood with glass beads. It contains signaling proteins that include interleukin-1b (IL-1b), tumor necrosis factor-alpha (TNF-a), IL-7, FGF-2, interleukin 1 receptor antagonist (IL-1Ra), HGF, platelet derived growth factor (PDGF-AB), transforming growth factor (TGF-β1) and IGF-1. Wright-Carpenter (2004) assessed the effects of ACS injections in a non-blinded, non-randomized case control study. The experimental group was treated with ACS, and the control group, which was analyzed retrospectively, included patients who had received Traumeel®/Actovegin®. Traumeel is a homeopathic formulation that contains both botanical and mineral ingredients in homeopathic concentrations. It is purported to suppress the release of inflammatory mediators and to stimulate the release of anti-inflammatory cytokines. Actovegin is a deproteinized calve blood hemodialysate that consists of a physiological mix of amino acids. The RICE principle was employed for initial care in both groups. The primary measured outcome was the time needed to resume full sporting activities. The experimental group returned to competition after 16.6 days, whereas the control group took 22.3 days. In addition, MRI scans taken at 16 days in both groups confirmed that regression of the edema/bleeding was faster in the ACS group. Both treatments were safe.

#### **5.2.2 Tendon pathology**

128 Innovations in Biotechnology

enhanced function compared to hyaluronic acid (HA) injections (Sánchez et al., 2008). In a case series study that involved 115 young patients with low degrees of articular degeneration in the knee, Kon et al. (2010) reported reduced pain and improved function that was maintained at 12 months but not 24 months after treatment (Filardo et al., 2011). Sampson et al. (2010), also in a small case series (n=13), reported significant pain and symptom relief but did not find any significant change in the daily activities or quality of life of the patients treated. PRP injections for hip OA produced clinically significant reductions in pain and function, although this was only seen in 40% of the patients studied (Sánchez et al., 2011). When discussing PRP therapies, differences between the preparations and the readministration procedures used should be acknowledged. Although pure PRP and leukocyte PRP formulations are not comparable in terms of leukocyte content, platelet count or plasma volume, the resulting improvements in pain and function were not exclusive to any one formulation. The pursuit to identify a unifying therapy for OA would be enhanced

Sports related soft tissue injuries cause athletes to lose a significant amount of time from their sport and represent a significant burden to society in terms of health care resources, personal disability and activity restriction. In 2002, an estimated 15.8 billion dollars in total health care expenditures was used for the medical management of these injuries (Yu WW 2005). Soft tissue disorders, including muscle, tendon, ligament and joint capsular injuries, represent more than 50% of all the musculoskeletal injuries reported each year in the USA. Primary care studies have shown that 16% of the general population suffers from shoulder pain, whereas elbow tendinopathy affects 1-2% of the population. The importance of this problem is substantial because the field of sports medicine influences millions of people from athletes to those who participate in recreational sports or simply exercise to stay healthy and active.

Muscle injuries resulting from extrinsic or intrinsic mechanisms are extremely common in sports, accounting for about 35-45% of all injuries. Contact sports and sports that require the generation of large eccentric forces present the highest risk. The vulnerability of soccer players to strains and contusions is a substantial problem for professional players and their clubs; such injuries involve significant time lost from training and competition. Due to the increasing demands of the competitive soccer season, muscle treatments able to accelerate the recovery time without adversely affecting the recurrence rate (i.e., those that can

At present, no drugs have been developed that hasten the restoration of muscle function after injury. Therefore, in the absence of any available evidence-based treatments, injection therapies may be an important option to help professional athletes. At the 2nd World Congress of Regenerative Medicine, Sanchez (2005) reported for the first time the application of leukocyte-free PRP to 21 muscle injuries of different severities and different anatomical locations. Small tears progressed well with a single application, whereas more severe tears required 2-3 ultrasound-guided injections. The injected volume depended on tear severity. These athletes, who played in first division teams of the Spanish Soccer

minimize the scarring response) are of paramount importance [Andia et al, 2011].

by refining the end points in future clinical studies.

**5.2 Sport medicine** 

**5.2.1 Muscle injuries** 

Chronic pain in tendons is very common and studies show that overuse, underloading and overloading, all contribute to tendon injuries and pain. More than 30-50% of the injuries among professional and recreational athletes are overuse tendon injuries resulting in the onset of pain and discomfort. Data collected from sedentary people showed that tendinosis is not necessarily a consequence of overuse. Nevertheless, the odds of having tendinopathy among elite endurance athletes are one in two (Kujala et al., 2005). Thus, the development of innovative strategies to treat tendon injuries is an essential task, but it requires a more thorough understanding of the underlying cellular and molecular mechanisms. The use of platelet rich preparations in this context may be focused on restoring the normal tissue composition while avoiding further degeneration. When we evaluated the effects of the pool of growth factors released from PRP on tendon cells, the results showed that human tendon cells increased their proliferation rate and were stimulated to release VEGF and HGF. The former promotes angiogenesis, which is directly related with tendon healing capability; the latter is a potent antifibrotic agent that can reduce scar formation around tendon tissues. Other studies have reported that injections of platelet rich plasma one week postoperatively increased tendon regenerate strength. The clinical translation of this approach was assayed in a pioneer study involving professional and recreational athletes. PRP was injected into the tendon fibers after the tendon was sutured. After closing the paratenon and before closing the overlying skin, the affected area was covered with the fibrin scaffold. The results showed that those receiving the PRP-therapy experienced a significant acceleration in functional recovery compared with a matched group that underwent conventional surgery. Moreover, the effects induced by PRP therapies had long-term consequences such as decreased cross-sectional area of the Achilles tendon after 18 months [Sánchez et al., 2007]. The feasibility and biosafety of PRP therapies made their application possible not only in surgeries but in the conservative management of tendon problems.

Currently, conservative management with PRP injections and its research attention are increasing [Andia et al, 2011]. Recently, three studies on PRP injection, of which two were on patients with chronic patellar tendinopathy [62,65] receiving three injections of leukocyte-platelet concentrate (double centrifugation), were reported. Significant improvements in the Tegner scores were described in one of the two studies. In addition, improvement in pain and function was reported after a single PRP injection in patients with epycondylitis [Mishra et al., 2006]. More recently, two double-blind, randomized clinical trials were performed on patients with lateral epycondylitis [Peerbooms et al., 2010] and chronic Achilles tendinopathy [De Vos et al., 2010], respectively. In both studies, the experimental treatment consisted of a single injection of an identical buffered PRP. The clinical results were significant for patients with lateral epycondylitis, for which PRP reduced pain and improved function. In contrast, in patients with Achilles tendinopathy, PRP injection did not reduce pain or improve activity [De Vos et al., 2010]. It seems improbable that a single injection could stop or reverse an ongoing degenerative process. Instead repeated injections appear to be more efficient in degenerative pathologies. No complications were reported after PRP treatments.

#### **5.2.3 ACL reconstruction**

Finally, a great deal of effort has been paid to the development of novel medical tools for the repair of injured anterior cruciate ligaments (ACL). The ACL is one of the four major ligaments connecting the bones of the human knee. A torn ACL is a common injury and is typical among the active younger population. The injury requires surgical intervention to stabilize the knee and to prevent cartilage and meniscal injuries, which lead to degenerative joint disease. ACL reconstruction, namely ACL tissue engineering, involves the manipulation of cells and tissues to replace the injured ligament; this process is a complex undertaking and involves many mechanical and biological challenges. It requires both the application of mechanical knowledge and an understanding of how cells are maintained and grow into functioning tissues to replace defective or injured ligaments. At present, the most common options in ACL replacement are allografts or autografts. A novel approach using PRP technologies seeks to facilitate ACL healing by mimicking the native tissue and improving tissue function with the appropriate cues (see Figure below), ultimately leading to better patient care.

Cell cultures and animal research, in addition to human clinical studies, drive the main hypotheses for the application of PRP biotechnology in ACL reconstruction. These applications involve first promoting bone-bone and bone-tendon healing, and second, influencing the pattern of change within the autograft body (ligamentization). Finally, the application of PRP-therapies will help in donor site healing. Graft fixation is the weakest link in ACL reconstruction because knee laxity develops during the immediate postoperative period until biologic fixation occurs within the bone tunnel. Classically, graft stabilization is achieved more rapidly with a bone plug-patella tendon-bone (BPTB) graft than with the hamstring. The BPTB graft becomes anchored to the bone wall via appositional bone formation, and in these circumstances, the use of PRP may aid in the formation of the callus and may accelerate bone fusion (Sánchez et al., 2010). In a

decreased cross-sectional area of the Achilles tendon after 18 months [Sánchez et al., 2007]. The feasibility and biosafety of PRP therapies made their application possible not only in

Currently, conservative management with PRP injections and its research attention are increasing [Andia et al, 2011]. Recently, three studies on PRP injection, of which two were on patients with chronic patellar tendinopathy [62,65] receiving three injections of leukocyte-platelet concentrate (double centrifugation), were reported. Significant improvements in the Tegner scores were described in one of the two studies. In addition, improvement in pain and function was reported after a single PRP injection in patients with epycondylitis [Mishra et al., 2006]. More recently, two double-blind, randomized clinical trials were performed on patients with lateral epycondylitis [Peerbooms et al., 2010] and chronic Achilles tendinopathy [De Vos et al., 2010], respectively. In both studies, the experimental treatment consisted of a single injection of an identical buffered PRP. The clinical results were significant for patients with lateral epycondylitis, for which PRP reduced pain and improved function. In contrast, in patients with Achilles tendinopathy, PRP injection did not reduce pain or improve activity [De Vos et al., 2010]. It seems improbable that a single injection could stop or reverse an ongoing degenerative process. Instead repeated injections appear to be more efficient in degenerative pathologies. No

Finally, a great deal of effort has been paid to the development of novel medical tools for the repair of injured anterior cruciate ligaments (ACL). The ACL is one of the four major ligaments connecting the bones of the human knee. A torn ACL is a common injury and is typical among the active younger population. The injury requires surgical intervention to stabilize the knee and to prevent cartilage and meniscal injuries, which lead to degenerative joint disease. ACL reconstruction, namely ACL tissue engineering, involves the manipulation of cells and tissues to replace the injured ligament; this process is a complex undertaking and involves many mechanical and biological challenges. It requires both the application of mechanical knowledge and an understanding of how cells are maintained and grow into functioning tissues to replace defective or injured ligaments. At present, the most common options in ACL replacement are allografts or autografts. A novel approach using PRP technologies seeks to facilitate ACL healing by mimicking the native tissue and improving tissue function with the

Cell cultures and animal research, in addition to human clinical studies, drive the main hypotheses for the application of PRP biotechnology in ACL reconstruction. These applications involve first promoting bone-bone and bone-tendon healing, and second, influencing the pattern of change within the autograft body (ligamentization). Finally, the application of PRP-therapies will help in donor site healing. Graft fixation is the weakest link in ACL reconstruction because knee laxity develops during the immediate postoperative period until biologic fixation occurs within the bone tunnel. Classically, graft stabilization is achieved more rapidly with a bone plug-patella tendon-bone (BPTB) graft than with the hamstring. The BPTB graft becomes anchored to the bone wall via appositional bone formation, and in these circumstances, the use of PRP may aid in the formation of the callus and may accelerate bone fusion (Sánchez et al., 2010). In a

appropriate cues (see Figure below), ultimately leading to better patient care.

surgeries but in the conservative management of tendon problems.

complications were reported after PRP treatments.

**5.2.3 ACL reconstruction** 

preliminary study, Sánchez et al. (2003) described a procedure for treating bone tunnels and for conditioning the graft prior to implantation with PRP. They compared a group of 50 patients treated with surgery and pure PRP with another group of 50 patients who underwent surgery alone. The two groups were matched for age and graft type. The authors reported better integration of PRP-treated grafts within the tunnels, as assessed by X-rays, and a larger number of completely stable knees in the PRP group. Other authors have explored the influence of autologous bone plugs, either alone or combined with PRP therapies, on the promotion of femoral bone-tendon healing. They reported that bone plugs, but not PRP-therapies, significantly prevented femoral tunnel widening.

Fig. 5. Transfer of autologous GFs and cytokines to the tendon graft, applying the principles of tissue engineering and using PRP biotechnology to estimulate biological mechanisms such as angiogenesis.

The appropriate function of ACL grafts, essential for normal knee biomechanical functioning, entails a successful intra-articular graft ligamentization. One exciting option to enhance ligamentization is to simultaneously transfer multiple cytokines and growth factors (including PDGF, TGF-β1 and VEGF, among others) to the graft by applying an endogenous PRP. Autografts could be loaded in situ with a balanced pool of signaling molecules. These molecules would have the potential to not only activate the graft tenocytes but also to attract cells, such as endothelial or stem cells, from adjacent niches (such as the synovium and/or the intrapatellar pad) to the graft structures using the synovial fluid for passage. The corroboration and clinical translation of this notion may be enhanced healing and intrasynovial adaptation of the tendon graft to the synovial milieu. Recently, we have compared the gross appearance and microscopic qualities of the PRP-treated and untreated grafts during the remodeling period (6-24 months). Gross morphology was evaluated using second-look arthroscopy focusing on graft thickness, apparent tension and synovium coverage. The overall arthroscopic evaluation provided evidence that a higher percentage of the grafts rated as excellent in the PRP group (57% versus 33%). No grafts were scored as poor in the PRP group, but 20% of the controls showed poor morphology. At the same time, PRP treatment influenced the histological characteristics of the tendon graft, which resulted in tissue that was more mature than in the controls. Histology displayed newly formed connective tissue enveloping the graft in 77.3% of the PRP-treated grafts and in 40% of the controls (Sanchez et al., 2010). Other authors have used a compressed gelatin sponge soaked with leukocyte and platelet-rich concentrate (GPS system by Biomet Biologic, Warsaw, USA) sutured to the intra-articular part of the graft, which confirmed the acceleration of the maturation of the grafts treated with PRP as assessed by magnetic resonance imaging.

#### **5.3 Cutaneous ulcers**

Clinical differences between acute and chronic wounds are in part explained by alterations in the local biochemical environment. For example, acute wounds are associated with a greater mitogenic activity than chronic wounds.

Fig. 6. Chronic ulcers are treated with several applications of PRP in order to enhance cell proliferation, and the formation of granulation tissue

Chronic wounds are associated with a higher level of pro-inflammatory cytokines than acute wounds. As chronic wounds begin to heal, they progress to a less inflammatory state. Elevated protease activities in chronic wounds may directly contribute to poor healing by degrading the proteins necessary for normal wound healing. Chronic wounds can be defined as those failing to proceed through an orderly and timely process to produce anatomic and functional integrity. Practically, a chronic wound is one that has failed to heal within 3 months. The cellular, biochemical and molecular events that characterize chronic wounds have been well defined, including a prolonged inflammatory phase, cellular senescence, deficiency of growth factors and/or their receptors, deficient fibrin production and

the grafts rated as excellent in the PRP group (57% versus 33%). No grafts were scored as poor in the PRP group, but 20% of the controls showed poor morphology. At the same time, PRP treatment influenced the histological characteristics of the tendon graft, which resulted in tissue that was more mature than in the controls. Histology displayed newly formed connective tissue enveloping the graft in 77.3% of the PRP-treated grafts and in 40% of the controls (Sanchez et al., 2010). Other authors have used a compressed gelatin sponge soaked with leukocyte and platelet-rich concentrate (GPS system by Biomet Biologic, Warsaw, USA) sutured to the intra-articular part of the graft, which confirmed the acceleration of the maturation of the grafts treated with PRP as assessed by magnetic resonance imaging.

Clinical differences between acute and chronic wounds are in part explained by alterations in the local biochemical environment. For example, acute wounds are associated with a

Fig. 6. Chronic ulcers are treated with several applications of PRP in order to enhance cell

Chronic wounds are associated with a higher level of pro-inflammatory cytokines than acute wounds. As chronic wounds begin to heal, they progress to a less inflammatory state. Elevated protease activities in chronic wounds may directly contribute to poor healing by degrading the proteins necessary for normal wound healing. Chronic wounds can be defined as those failing to proceed through an orderly and timely process to produce anatomic and functional integrity. Practically, a chronic wound is one that has failed to heal within 3 months. The cellular, biochemical and molecular events that characterize chronic wounds have been well defined, including a prolonged inflammatory phase, cellular senescence, deficiency of growth factors and/or their receptors, deficient fibrin production and

**5.3 Cutaneous ulcers** 

greater mitogenic activity than chronic wounds.

proliferation, and the formation of granulation tissue

high levels of proteases. In normally healing wounds, acute inflammation with neutrophil infiltration brings neutrophil-derived matrix protease enzymes that debride the wound and pave the way for new tissue deposition and remodeling. In chronic wounds, the orderliness of the healing process is disrupted by some underlying abnormality that prolongs the inflammatory phase and produces a cascade of tissue responses that perpetuates the nonhealing state. Repeated trauma, foreign bodies, pressure necrosis, infection, ischemia and tissue hypoxia also amplify the chronic inflammatory state, which is characterized by excess neutrophils, macrophages and lymphocytes. Fragments of dead tissue, bacterial products and foreign bodies are powerful chemoattractants that sustain a continuous influx of inflammatory cells, which in turn produce a variety of growth factors, cytokines, and matrix-degrading enzymes. Among the most potent of these enzymes are elastase and MMPs, which are present in large quantities in chronic wounds. Given the low levels of TIMPs, the MMP/TIMP balance is distorted; thus, the excess of proteolytic enzymes shifts the balance towards ECM destruction and the degradation of signaling proteins. Therefore, any effective intervention must include a strategy for disrupting this cycle and setting the wound on a permanent path towards healing. Historically, the first clinical application of platelet derived preparations was conducted in chronic leg ulcers in which wounds were filled with collagen embedded in platelet secreted proteins. This initial product, known as PDWHF (platelet-derived wound healing factors) stimulated the formation of the vascularized connective tissue found in healing wounds. Thereafter, various other types of platelet products have been assayed in several pilot studies, case series and clinical trials.

Growth factors are crucial for timely wound healing; inadequate levels of growth factors may be an important factor contributing to the chronicity of the wound, which may be degraded in excess by cellular or bacterial proteases. Initially, Margolis et al. (2001) showed that platelet releasates were more effective than standard therapy. Subsequently, PRP formulations were refined and primarily applied as fibrin membranes for the treatment of non-healing ulcers. More recently, the use of PRP in the management of chronic diabetic foot ulcers has been successful (Setta HS 2011). Moreover, PRP provides advantages in skin grafting for recalcitrant ulcers (Chem Tim et al., 2010). Allogenic platelet preparations have been used recently to treat recalcitrant ulcers in very elderly hypomobile patients for whom autologous blood processing may be difficult (Greppi et al., 2011). Finally, the use of PRP gel resulted in an improved quality of life and a lower cost of care over a 5-year period than other treatment modalities for patients with non-healing diabetic foot ulcers. Although actual treatment outcomes may differ from those modeled, PRP gel represents a potentially attractive treatment alternative for insurers and health care providers to address the cost burden and health effects of non-healing diabetic foot ulcers (Dougherty EJ 2008)

#### **5.4 Other therapeutic approaches**

The potential therapeutic value and versatility of platelet rich products has stimulated research in additional medical fields. PRP biotechnology holds promise as a healing preparation in surgical procedures and in the treatment of many different diseases. The use of PRPs for cell delivery and tissue engineering permit insights into the development of novel therapies. For example, autologous fat grafting, also known as fat transfer or fat injection, has long been a staple of cosmetic and reconstructive surgery. Fat grafts have proven very effective in the reconstruction of soft tissue defects, particularly for facial plastic and reconstructive procedures. However, there has always been one significant disadvantage associated with autologous fat grafting: the unpredictable and often inconsistent graft survival rate. Promising new evidence has shown that PRP can enhance the fat graft survival rate. Moreover, nasolabial folds, superficial rhytids and acne scars have been successfully treated with injections of autologous PR fibrin matrices (Sclafani AP 2010). Additionally, PRP can be associated with novel dermatologic procedures as an aid in healing. For example, PRP is an effective method for enhancing wound healing and reducing transient adverse effects after fractional carbon dioxide laser resurfacing (Na et al., 2011).

Another remarkable application of PRP is in ophthalmology. Several successful examples include the use of PRP releasates as eye drops for the treatment of a broad spectrum of corneal persistent epithelial defects (Lopez-plandolit et al., 2010). Furthermore, the use of autologous platelet rich plasma was shown to be very effective in the treatment of patients suffering from dry eye symptoms; it improved both patient symptoms and major clinical signs [Alio et al., 2007]. Platelet rich plasma also promotes healing of dormant corneal ulcers even in eyes that are threatened by corneal perforation, and it is a reliable and effective therapeutic tool for the enhancement of epithelial wound healing on the ocular surface.

Fig. 7. The four domains of PRP science. Improved understanding of the biology of PRPs and repair mechanisms have emerged as a potential way of improving PRP formulations and applications. The identification of critical molecules that interact with healing will be critical in developing new approaches to treatments.

Other interesting recent approaches using PRP biotechnology include the successful application of platelet rich plasma in peripheral nerve regeneration (Sariguney et al., 2008) and the use of PRP biotechnology to treat damaged myocardial tissue. Utilizing a murine myocardial permanent ligation and ischemia/reperfusion model, a proprietary PRP formulation demonstrated a positive effect in left ventricular cardiac function. The use of PRP for skin rejuvenation is another application of PRP biotechnology.

### **6. Conclusion**

134 Innovations in Biotechnology

procedures. However, there has always been one significant disadvantage associated with autologous fat grafting: the unpredictable and often inconsistent graft survival rate. Promising new evidence has shown that PRP can enhance the fat graft survival rate. Moreover, nasolabial folds, superficial rhytids and acne scars have been successfully treated with injections of autologous PR fibrin matrices (Sclafani AP 2010). Additionally, PRP can be associated with novel dermatologic procedures as an aid in healing. For example, PRP is an effective method for enhancing wound healing and reducing transient adverse effects after fractional carbon

Another remarkable application of PRP is in ophthalmology. Several successful examples include the use of PRP releasates as eye drops for the treatment of a broad spectrum of corneal persistent epithelial defects (Lopez-plandolit et al., 2010). Furthermore, the use of autologous platelet rich plasma was shown to be very effective in the treatment of patients suffering from dry eye symptoms; it improved both patient symptoms and major clinical signs [Alio et al., 2007]. Platelet rich plasma also promotes healing of dormant corneal ulcers even in eyes that are threatened by corneal perforation, and it is a reliable and effective therapeutic tool for the enhancement of epithelial wound healing on the ocular surface.

Fig. 7. The four domains of PRP science. Improved understanding of the biology of PRPs and repair mechanisms have emerged as a potential way of improving PRP formulations and applications. The identification of critical molecules that interact with healing will be

Other interesting recent approaches using PRP biotechnology include the successful application of platelet rich plasma in peripheral nerve regeneration (Sariguney et al., 2008) and the use of PRP biotechnology to treat damaged myocardial tissue. Utilizing a murine myocardial permanent ligation and ischemia/reperfusion model, a proprietary PRP formulation demonstrated a positive effect in left ventricular cardiac function. The use of

critical in developing new approaches to treatments.

PRP for skin rejuvenation is another application of PRP biotechnology.

dioxide laser resurfacing (Na et al., 2011).

Realistically, a substantial amount of research is needed to bring PRP technologies to the bedside, as clinical and laboratory findings that indicate its potential benefits must be followed by comprehensive clinical studies to demonstrate efficacy. Demonstrating effectiveness in different pathologies will be critical for the widespread adoption of PRP technology, including re-imbursement. Below is a schematic representation that illustrates the four domains of PRP science.

Because of the safety of these products, basic science, clinical discovery and patient-oriented research should be interdependent rather than successive steps. The substantial challenges of incorporating such research into clinical care must be pursued if the potential of PRPs is to be realized. Although PRP therapies have many compositions and procedures for application, they all try to maximize the cell signals that may enhance tissue healing. Our increased understanding of the healing mechanisms that result in tissue repair is paving the way towards the optimization of healing therapies

#### **7. Acknowledgements**

The authors wish to thank the "Unidad de Cirugia Artroscópica", UCA and BTI research teams for their work in the development of PRP biotechnology in orthopedics and sport medicine. We apologize to the authors whose work we could not cite because of the limit ing the number of references.

#### **8. References**


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### **Polymers in the Pharmaceutical Applications - Natural and Bioactive Initiators and Catalysts in the Synthesis of Biodegradable and Bioresorbable Polyesters and Polycarbonates**

#### Ewa Oledzka and Marcin Sobczak

*Department of Inorganic and Analytical Chemistry Medical University of Warsaw, Faculty of Pharmacy, Warsaw Poland* 

#### **1. Introduction**

138 Innovations in Biotechnology

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doi:10.1093/rheumatology/ker303

preparation rich in growth factors for the treatment of knee OA: a retrospective

Treated With Autologous Preparation Rich in Growth Factors. *Journal of Orthopedic* 

complications with a Preparation Rich in Growth Factors: A study of two-cases.

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Healing Environment in Anterior Cruciate Ligament Reconstruction. *Sports* 

injections for the treatment of osteoarthritis of the hip. *Rheumatology*

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metalloproteinases in platelets and megakaryocytes: A novel organization for these

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United States high school soccer injuries, 2005-2007. *American Journal of Sports* 

Biopolymers, synthetic polymers and their derivatives are commonly used in medicine and pharmacy. Significant progress attained in the polymer chemistry and technology has boosted the dynamic development of the medicinal engineering.

Recently, particular interest of scientists has been focused on biomedical polymers, especially those used for drug delivery systems, therapeutic systems and macromolecular prodrugs. The aforementioned applications have opened new exciting prospects for medicine, because specially designed polymers are capable of delivering medicinal substances to the target diseased tissues and cells together with dosing those drugs according to controlled specified pharmacodynamics. Particular attention has recently been paid to chemistry of biocompatiable and biodegradable polymers, because they have an advantage of being readily hydrolyzed into removable and non-toxic products, which can be subsequently eliminated by metabolic pathways. Furthermore, the biomedical polymers have to be synthesized now using friendly for the environment and safe for human health, effective natural initiators, co-initiators and/or catalysts.

Therefore, the main objective of this work is to discuss various polymers recommended for the pharmaceutical applications and then to describe natural compounds used as initiators, catalysts and co-initiators in the synthesis of biodegradable and bioresorbable polyesters and polycarbonates.

#### **2. Polymers in the pharmaceutical applications**

Macromolecules are applied in pharmacy as the pharmacological substances, blood substitutes, drug delivery and therapeutic systems, in the synthesis of macromolecular prodrugs and in the technology of prolonged release drug formulations.

#### **2.1 Polymers with the pharmacological effects and polymeric blood substitutes**

One of the most interesting polymers used in pharmacy, are those exerting a pharmacological effect. DIVEMA, copolymer of divinyl ether-maleic anhydride (Florjanczyk & Penczek, 1998; Papamatheakis et al., 1978) is an example of such compound with antitumoral and antiviral properties. Its action probably includes the stimulation of the glycoprotein production, which suppresses viral RNA translocation in cells and division of cancer cells.

Furthermore, the polymers are often applied as swelling, relaxation and sliding agents. Methylcellulose taken orally is not absorbed from the alimentary tract. However, it detains water on swelling and in consequence causes relaxation of the stercorous mass (Tonnesen & Karlsen, 2002; Zejc & Gorczyca, 2002).

A copolymer of ethylene and propylene glycols has found an application in the therapy of constipations (Tonnesen & Karlsen, 2002; Zejc & Gorczyca, 2002). This non-ionic, surfaceactive polymer is unable to penetrate through the gut walls because of large average molecular weight. However, it causes relaxation and hydration of the stercorous mass by the reduction of the surface tension.

A linear polymer of uronic acids - alginic acid (mannuronic acid conjugated β-1,4 and Lguluronoic acid glycosidically conjugated α-1,4) is mainly obtained from the *Laminaria algae*. This polymer neutralizes hydrochloric acid (Janicki et al., 2002; Zejc & Gorczyca, 2002). Its action relies on detaining of water in stomach followed by reduction of irritations and pain.

A polyvinylpyrrolidone has found an application as anti-diarrhoeal drug (Tonnesen & Karlsen, 2002; Zejc & Gorczyca, 2002). Its amphoteric properties normalize pH in stomach and intestines through acids or bases adsorption, which are usually raised as result of fermentation or putrefaction.

The synthetic hormones with the protein structure play an important role in the modern pharmacology (Zejc & Gorczyca, 2002). The Buserelin, Goserelin, Leuprorelin and Triptorelin are known as synthetic analogues of Gonadoliberin (the hormone of hypothalamus). These oligopeptides are obtained by exchanging of some amino acids in Gonadoliberin molecule and then are used to treat prostate and breast cancers or endometriosis. Another example is synthetic analogue of Somatoliberin used for treating children with some forms of GH deficiency. The synthetic analogue of Somatostatin - Octreotide is applied to treat the alimentary tract (Zejc & Gorczyca, 2002).

Corticotrophins are examples of synthetic hormones of the anterior pituitary, often applied in the therapy of rheumatoid diseases and severe asthma. Thus, Oxitocin, Vasopressin and Ornipressin are belong to the group of hormones of posterior lobe of the hypophysis (Zejc & Gorczyca, 2002). First of them causes uterine contractions, second can contract the smooth muscles of the blood vessels while Ornipressin is often added to the anaesthetics. Moreover causes the vessels contraction.

The peptide antibiotics are the relatively numerous group of the natural oligomers. They are composed of peptide-bounded amino acids to form cyclic, linear or cyclic-linear structures (Markiewicz & Kwiatkowski, 2001; Patrick, 2003; Zejc & Gorczyca, 2002). They may act the Gram-negative (Polymyxin) and Gram-positive (Gramicidin, Prostinamycin) bacteria as well as fungi and protozoa.

One of the most interesting polymers used in pharmacy, are those exerting a pharmacological effect. DIVEMA, copolymer of divinyl ether-maleic anhydride (Florjanczyk & Penczek, 1998; Papamatheakis et al., 1978) is an example of such compound with antitumoral and antiviral properties. Its action probably includes the stimulation of the glycoprotein production, which suppresses viral RNA translocation in cells and division of

Furthermore, the polymers are often applied as swelling, relaxation and sliding agents. Methylcellulose taken orally is not absorbed from the alimentary tract. However, it detains water on swelling and in consequence causes relaxation of the stercorous mass (Tonnesen &

A copolymer of ethylene and propylene glycols has found an application in the therapy of constipations (Tonnesen & Karlsen, 2002; Zejc & Gorczyca, 2002). This non-ionic, surfaceactive polymer is unable to penetrate through the gut walls because of large average molecular weight. However, it causes relaxation and hydration of the stercorous mass by

A linear polymer of uronic acids - alginic acid (mannuronic acid conjugated β-1,4 and Lguluronoic acid glycosidically conjugated α-1,4) is mainly obtained from the *Laminaria algae*. This polymer neutralizes hydrochloric acid (Janicki et al., 2002; Zejc & Gorczyca, 2002). Its action relies on detaining of water in stomach followed by reduction of irritations and pain. A polyvinylpyrrolidone has found an application as anti-diarrhoeal drug (Tonnesen & Karlsen, 2002; Zejc & Gorczyca, 2002). Its amphoteric properties normalize pH in stomach and intestines through acids or bases adsorption, which are usually raised as result of

The synthetic hormones with the protein structure play an important role in the modern pharmacology (Zejc & Gorczyca, 2002). The Buserelin, Goserelin, Leuprorelin and Triptorelin are known as synthetic analogues of Gonadoliberin (the hormone of hypothalamus). These oligopeptides are obtained by exchanging of some amino acids in Gonadoliberin molecule and then are used to treat prostate and breast cancers or endometriosis. Another example is synthetic analogue of Somatoliberin used for treating children with some forms of GH deficiency. The synthetic analogue of Somatostatin -

Corticotrophins are examples of synthetic hormones of the anterior pituitary, often applied in the therapy of rheumatoid diseases and severe asthma. Thus, Oxitocin, Vasopressin and Ornipressin are belong to the group of hormones of posterior lobe of the hypophysis (Zejc & Gorczyca, 2002). First of them causes uterine contractions, second can contract the smooth muscles of the blood vessels while Ornipressin is often added to the anaesthetics. Moreover

The peptide antibiotics are the relatively numerous group of the natural oligomers. They are composed of peptide-bounded amino acids to form cyclic, linear or cyclic-linear structures (Markiewicz & Kwiatkowski, 2001; Patrick, 2003; Zejc & Gorczyca, 2002). They may act the Gram-negative (Polymyxin) and Gram-positive (Gramicidin, Prostinamycin) bacteria as well

Octreotide is applied to treat the alimentary tract (Zejc & Gorczyca, 2002).

**2.1 Polymers with the pharmacological effects and polymeric blood substitutes** 

cancer cells.

Karlsen, 2002; Zejc & Gorczyca, 2002).

the reduction of the surface tension.

fermentation or putrefaction.

causes the vessels contraction.

as fungi and protozoa.

A cyclosporine A – branched and cyclic oligopeptide composed of 11 amino acids is an important macromolecular immunosuppressive drug (Markiewicz & Kwiatkowski, 2001; Zejca & Gorczyca, 2002). Cyclosporine A selectively inhibits lymphocytes T function, thus is widely used as an immune barrier tolerance agent in the transplantology.

Macromolecular inhibitors that absorb the cholesterol from the intestines are also known; form them insoluble in water polymers, which produce complexes with the bile acids. To this polymer group belongs: copolymer of divinylbenzene and styrene substituted with quaternary trimethylammonium group and copolymer of diethyltriamine and epichlorohydrin (Zejc & Gorczyca, 2002).

A heparin, obtained from the animal tissues (mainly livers and lungs) (Zejc & Gorczyca, 2002) is next example of the natural polysaccharide used as the therapeutic agent. The heparin effects on the all blood clotting phases. Usually is used to treat arterial embolism and thrombosis, heart failure and before surgical operations.

A very important group of the biomedical polymers is macromolecular blood substitutes. They are accountable for the regular osmotic pressure and viscosity, closed to the osmotic pressure and blood viscosity; usually used in the anaphylactic shock, heart failure, intoxication, burns, toxic diarrhoea, embolic-thrombotic complications as well as microcirculation impairment.

A polyvinylpyrrolidone was the first synthetic polymer used as the blood substitutes. Its solutions were mainly used to treat the shock after the burns and, in the case when the blood transfusion was not indicated (Janicki et al., 2002; Florjanczyk & Penczek, 1998; Zejc & Gorczyca, 2002). Likewise, the solutions of polyvinyl alcohol have found the applications as the blood substitutes. However, they were withdrawn from the list of the blood substitutes as result of their undesirable side effects.

The blood susbstitutes with the therapeutic action has also been elaborated as result of incorporation of some therapeutic agents (e.g. penicillin, pelentanic acid, p-aminosalicylic chloride) into polyvinyl alcohol (Janicki et al., 2002; Zejc & Gorczyca, 2002).

Currently, the solutions of polysaccharides (e.g. dextran), modified starch derivatives and modified gelatin products (polygeline, oksopolygelin, liquid gelatin) are commonly used as the blood substititues (Janicki et al., 2002; Zejc & Gorczyca, 2002). The dextran with the average molecular weight ranged from 40 000 to 70 000 Da is used as 6 or 10% solution. This polysaccharide is produced by fermentation of the sucrose solutions in the presence of the *Leuconostoc mesenteroides* bacteria. Obtained glucose is polymerized to dextran in the presence of enzymes.

A hydroksyethyl starch is obtained by hydrolysis of high-amylopectine starch in acidic environment (Zejc & Gorczyca, 2002). The reaction products are neutralized followed by the reaction with ethylene oxide. The starch substituted with hydroxyethyl group is then produced in this reaction.

A polygeline is obtained from the reaction of diisocyanate with the gelatin. As result, linked urea groups are produced, whereas liquid gelatin is produced in the reaction with succinic anhydride (Janicki et al., 2002; Zejc & Gorczyca, 2002).

#### **2.2 Macromolecular prodrugs**

A prodrug is a modified therapeutic agent, which is metabolized into active precursor in human body (Janicki et al., 2002). Over the recent years, the conception of macromolecular prodrug has appeared as macromolecule that has therapeutic agents in the structure; the released drug becomes pharmacologically active during hydrolytic biodegradation of the polymer (Ouchi & Ohya, 1995). In general, the therapeutic agent could be incorporate into polymer chain, might be end-capped or may form a pendant group of the macromolecular chain (Figure 1).

Fig. 1. Structure of the macromolecular prodrugs

A prodrug is a modified therapeutic agent, which is metabolized into active precursor in human body (Janicki et al., 2002). Over the recent years, the conception of macromolecular prodrug has appeared as macromolecule that has therapeutic agents in the structure; the released drug becomes pharmacologically active during hydrolytic biodegradation of the polymer (Ouchi & Ohya, 1995). In general, the therapeutic agent could be incorporate into polymer chain, might be end-capped or may form a pendant group of the macromolecular

**2.2 Macromolecular prodrugs** 

Fig. 1. Structure of the macromolecular prodrugs

chain (Figure 1).

Macromolecular prodrugs are mainly used in the cancer therapy. For example, 5 fluorouracil can be applied locally or orally in the therapy of the alimentary tract, urinary bladder and prostate gland cancers. The conjugations of this therapeutic agent as a pendant group to polyethylene glycol (Ouchi et al., 1986, 1992) or to vinyl polymer chain as substituent form examples of its macromolecular prodrugs (Ouchi et al., 1988).

The pharmacokinetics of the macromolecular prodrugs is mainly determined by the structure of the polymer (the rate of hydrolysis under the given conditions and the susceptibility to degradation in the presence of enzymes), its average molecular weight (the ability to the accumulation in blood, lymph, spleen, liver and other organs) or crystallinity (the rate of biodegradation).

The polymers must meet specific criteria to be applied in the synthesis of the macromolecular prodrugs. Namely, macromolecules and their metabolic decomposition products cannot be cumulate in the human body, to be toxic and the most important; the drug should be released from the macromolecule as result of the metabolic processes. The list of the macromolecular prodrugs developed so far is broadly presented in monograph (Ouchi & Ohya, 1995).

#### **2.3 Polymers in the technology of prolonged release drug formulations**

Macromolecules have also found the application in the technology of prolonged release drug formulations. They are mainly intended to ensure the constant concentration of the therapeutic agent in the certain time (e.g. 8-24 hours), in the patient body. The group of these drugs, therefore, can eliminate the drug multiple dosing during a day and reduce total daily dose of it. The prolonged drug forms are usually applied in the therapy of cardiac and alimentary tract diseases, coronary vessels, diabetics, and psychiatric disorders.

The absorption of the therapeutic agent using prolonged release drug forms can be reduced by coating, incorporation, complexation or bonding on the ionites (Janicki et al., 2002). Polymers applied in this technology, could be generally divided into biodegradable and non-biodegradable. Biodegradable macromolecules are definitely more preferred from the toxicological point of view. In the technology of prolonged release drug formulation, natural polymers and their modified derivative (e.g.: starch, cellulose) as well as synthetic polymers are used e.g.: polyethylene, polypropylene, polyvinyl chloride, polyvinyl alcohol, polyvinyl acetate, polyacrylic acid, polycarbophile, polyacrylamides, polyacrylates, polyethylene glycol, poly(amino amide)s, polyurethanes, siloxanes, homoor copolymers of lactide and glycolide, poly(ε-caprolactone), polyorthoesters (Cardamone et al., 1997; Ertan et al., 1997; Huang et al., 1994; Lan et al., 1996; Matthews et al., 1996; Merkli et al., 1998; Ouchi & Ohya, 1995; Schierholz 1997; Sintzel et al., 1996; Uhrich et al., 1999; Ulbrich et al., 1996).

The crystals, pellets and granules of the drug might be coated with several polymer layers, according to the expected release rate. The therapeutic agent is gradually released as result of the polymer erosion or diffusion or is rinsing out from the polymer coating (Figure 2) (Uhrich et al., 1999).

Methylcellulose, polyvinylpyrrolidone and polyvinyl alcohol are predominantly applied as the coating substances. The analogous effect can be obtained by coating of the therapeutic agent with polymeric layers, soluble in different parts of the alimentary tract or under enzymes.

The drug release based on the diffusion takes place when polymers insoluble in the alimentary tract (e.g.: ethyl cellulose, nitrocellulose, cellulose acetate, acrylic and methacrylic ester copolymers) are applied as the coating agents. The coating tablets containing porophors (acrylic and methacrylic ester copolymers, starch, cellulose acetate phthalate or microcrystalline cellulose) are also used. The solubility of these tablets is increased as the effect of porophors dissolution and swelling.

Fig. 2. The mechanism of the controlled release of the therapeutic agent

The incorporation method is relying on the suspension of the therapeutic agent on the prolonged released carrier. Most often as the carriers are used: hydrophobic polymers (e.g.: methylcellulose, acrylic acid polymers) as well as lipopholic polymers and some carriers insoluble in the alimentary tract (e.g.: polyvinyl chloride, polyethylene, cellulose acetate, ethyl cellulose, polystyrene, polyamide, silicone resin and acrylic and metacrylic acids ester copolymers). For instance, when the hydrophilic carrier is used, the tablet is consecutively swelled after passing the alimentary tract followed by creation of high viscous hydogels, which prolonged the drug release. The drug release suspended on the lipophilic carrier is dependant on pH and the presence of enzymes. Matrix tablets contained water-insoluble carriers, however, are stable in the alimentary tract environment. Therefore, the drug is gradually release via the capillaries.

The complexation method involves the creation of poor soluble, therapeutic agent-polymer complexes. The drug is released due to the gradual decomposition of this complex. This technique is also used to produce skin and mucosa antiseptics (iodophors). The iodophors are the complexes of iodine with water-soluble polymers, which perform a role of carrier. They are high active against bacteria, viruses, fungi and protozoa.

The bonding of the drug on the ionites method is usually applied for acidic or basic drugs. It relies on release of the drug based on ion exchange in the alimentary tract.

#### **2.4 Polymers in the therapeutic systems technology**

144 Innovations in Biotechnology

therapeutic agent with polymeric layers, soluble in different parts of the alimentary tract

The drug release based on the diffusion takes place when polymers insoluble in the alimentary tract (e.g.: ethyl cellulose, nitrocellulose, cellulose acetate, acrylic and methacrylic ester copolymers) are applied as the coating agents. The coating tablets containing porophors (acrylic and methacrylic ester copolymers, starch, cellulose acetate phthalate or microcrystalline cellulose) are also used. The solubility of these tablets is increased as the

or under enzymes.

effect of porophors dissolution and swelling.

gradually release via the capillaries.

Fig. 2. The mechanism of the controlled release of the therapeutic agent

The incorporation method is relying on the suspension of the therapeutic agent on the prolonged released carrier. Most often as the carriers are used: hydrophobic polymers (e.g.: methylcellulose, acrylic acid polymers) as well as lipopholic polymers and some carriers insoluble in the alimentary tract (e.g.: polyvinyl chloride, polyethylene, cellulose acetate, ethyl cellulose, polystyrene, polyamide, silicone resin and acrylic and metacrylic acids ester copolymers). For instance, when the hydrophilic carrier is used, the tablet is consecutively swelled after passing the alimentary tract followed by creation of high viscous hydogels, which prolonged the drug release. The drug release suspended on the lipophilic carrier is dependant on pH and the presence of enzymes. Matrix tablets contained water-insoluble carriers, however, are stable in the alimentary tract environment. Therefore, the drug is

The complexation method involves the creation of poor soluble, therapeutic agent-polymer complexes. The drug is released due to the gradual decomposition of this complex. This technique is also used to produce skin and mucosa antiseptics (iodophors). The iodophors The polymers used in the therapeutic systems are the drug forms that are dosing or releasing drug in the exact time with the controlled rate (Janicki et al., 2002; Müller & Hildebrand, 1998). They are designed to ensure constant concentration of the therapeutic agent in the body (Figure 3).

Fig. 3. Drug release profile from the conventional tablets and polymeric therapeutic systems

Therapeutic systems are commonly used in medicine due to their high efficiency in comparison to the conventional drug forms and prolonged release tablets. Considering the way of administration and the location of the drug absorption there are: oral, transdermal, ocular, intra-uterine, implantation and infusion therapeutic systems (Table 1). According to the construction of the element dosing the drug, there are: membrane, matrix and microvessels transdermal therapeutic systems (Knoch & Merkle, 1987; Müller & Hildebrand, 1998), usually used to treat stenocardia, inflammations, motion sickness, chronic hypertensive disease, in the hormonal and anti-nicotinic therapies (De Mey et al., 1989; Fagerström et al., 1993; Hadgraft, 1996; Ho & Chien, 1993; Liedtke et al., 1989; Lin et al., 1993; Man et al., 1993; Monkhouse & Hug, 1988; Sanders, 1996). The novelty comprises the ultrasonic transdermal therapeutic systems and the microelectronics transdermal therapeutic systems, where the drug is released from the polymer carrier under the frequency electric field influence (Prausnitz et al., 1994; Santus & Baker, 1993; Simonin, 1995).

In the ocular therapeutic systems, the drug is released to the lachrymal fluid through the membrane. The intra-uterine therapeutic systems are mostly used in the contraception, whereas implantation therapeutic systems are usually applied under the skin. In their case, the drug release is carried out through the slow diffusion from the polymeric systems to the tissue.


Table 1. The therapeutic systems examples

#### **3. Natural and bioactive initiators and catalysts in the synthesis of biodegradable and bioresorbable polyesters and polycarbonates**

Biodegradable and bioresorbable polymers such as polyglycolide (PG), polylactide (PLA), poly(ε-caprolactone) (PCL), poly(trimethylene carbonate) (PTMC) and copolymers of glycolide (GL), L-lactides (LA), *rac*-lactide (*rac*-LA), ε-caprolactone (CL), trimethylene carbonate (TMC) or others cyclic esters and carbonates are very often used as polymeric prodrugs, drug delivery or therapeutic systems. Aliphatic polyesters and polycarbonates are degraded *in vivo* by hydrolytic deesterification into glycolic, lactic or other acid monomers. The latter species become involved in the carboxylic acid cycle and are subsequently excreted as carbon dioxide and water. Furthermore, biodegradable and bioresorbable drug forms exhibit unique pharmacokinetics, body distribution and pharmacological efficacy.

In the ocular therapeutic systems, the drug is released to the lachrymal fluid through the membrane. The intra-uterine therapeutic systems are mostly used in the contraception, whereas implantation therapeutic systems are usually applied under the skin. In their case, the drug release is carried out through the slow diffusion from the polymeric

**Therapeutic system Polymer Drug** 

copolymers of acetate vinyl and ethyl, poliacrylate, silicone, polyurethanes, polyolefines, polyethylene glycol

polyvinyl alkohol, polyacetale vinyl, polyamides, polyethylene glycol, poliacrylate, silicone, homo- or copolymers of lactide, glicolide and ε-

system copolymers of acetate vinyl and ethyl Pilocarpina

system silicone Progesterone

**3. Natural and bioactive initiators and catalysts in the synthesis of biodegradable and bioresorbable polyesters and polycarbonates** 

copolymers of lactide and glicolide,

Biodegradable and bioresorbable polymers such as polyglycolide (PG), polylactide (PLA), poly(ε-caprolactone) (PCL), poly(trimethylene carbonate) (PTMC) and copolymers of glycolide (GL), L-lactides (LA), *rac*-lactide (*rac*-LA), ε-caprolactone (CL), trimethylene carbonate (TMC) or others cyclic esters and carbonates are very often used as polymeric prodrugs, drug delivery or therapeutic systems. Aliphatic polyesters and polycarbonates are degraded *in vivo* by hydrolytic deesterification into glycolic, lactic or other acid monomers. The latter species become involved in the carboxylic acid cycle and are subsequently excreted as carbon dioxide and water. Furthermore, biodegradable and bioresorbable drug forms exhibit unique pharmacokinetics, body distribution and

silicone Estradiol, Goserelin

caprolactone

Acetate Noretisterone, Buprenorphine, Clonidyne,

Estradiol, Fentanyl, Flurbiprofen, Hyoscine, Isosorbide dinitrate,

Nicotyne, Nitroglycerin, Testosterone

Acetazolamide, Glipizide, Metoprolol,

Nifedipine, Okseprenolol KCl, Li2SO4, FeSO4

systems to the tissue.

Transdermal therapeutic system

Oral therapeutic

Ocular therapeutic

Uterus therapeutic

pharmacological efficacy.

Table 1. The therapeutic systems examples

Implantation therapeutic system

system

There are two methods of the synthesis of aliphatic polyesters or polycarbonates, namely polycondensation of diols, dicarboxylic acids or hydroxycarboxylic acids and ring-opening polymerization (ROP) of cyclic monomers (Platel, 2009; Labet & Thielemans, 2009). The polycondensation is hampered by typical limitations of step polymerization. The polymers obtained in this process are characterized by a high polydispersity. ROP gives polymeric products with the higher molecular weight and lower polydispersity. Therefore, is more preferred route to obtain aliphatic polyesters or polycarbonates (Platel, 2009).

The ROP of cyclic esters, carbonates or ether-esters initiated or catalyzed by the metal complexes or organic compounds yields high molecular weight polymers with the excellent conversion. The metal compounds are used commercially due to their selectivity, rate and lack of side reactions. On the other hand, for some biomedical or pharmaceutical applications, metal residues (Zn, Al, Sn) are undesirable (Albertsson & Varma, 2003; Albertsson & Srivastava, 2008; Varma et al., 2005).

#### **3.1 Natural catalysts of ring-opening polymerization of cyclic esters and carbonates**

The application of enzymes as catalysts of ROP seems to be a perspective direction in the polymer research. Macromolecules with well-defined structures can be formed by enzymecatalyzed processes. On the other hand, the use of enzymes has some disadvantages, such as high cost, large quantity of enzymes required for ROP and relatively low molecular weight of the obtained polymers. However, the metal-free method of polymerization and suitable molecular weights of the resulted polymers are desirable conditions for the pharmaceutical applications, especially for the design of new drug delivery systems.

Fig. 4. e-ROP of cyclic esters, ether-esters or carbonates

The primary research on the enzyme ring-opening polymerization (e-ROP) has been carried out for CL. Currently major works concern ROP of six- and seven-membered cyclic esters, cyclic ether-estres or carbonates.

Lipases could also catalyze ROP of cyclic monomers, with different ring sizes as well as monomers containing substituents in the ring: α-methyl-β-propiolactone (1), β–butyrolactone (2), γ-caprolactone (3), α-methyl-valerolactone (4), 1,4-dioxan-2-one (5), δ-caprolactone (6), γethyl-ε-caprolactone (7), ε-heptanolactone (8), δ-decalactone (9), δ-dodecalactone (10), αmethyl-12-dodecanolide (11), α-methyl-15- pentadecanolide (12), L-lactide (13), D-lactide (14), D,L-lactide (15), 1,4-dioxepan-2-one (16), 1,5-dioxepan-2-one (17), 2-methylene-4-oxa-12 dodecanolide (18), 1,3-dioxan-2-one (19), 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one (20), 5-benzyloxy-1,3-dioxan-2-one (21), 1-methyl-1,3-dioxan-2-one (22), cyclobis (hexamethylene carbonate) (23), 2,2'-dimethyl-1,3-dioxan-2-one (24) (Figure 5) (Albertsson & Varma, 2003; Albertsson & Srivastava, 2008; Labet & Thielemans, 2009; Platel, 2009; Varma et al., 2005).

Many families of enzymes were used in ROP of cyclic esters or carbonate: *Aspergillus niger*, *Pseudomonas species*, immobilized *Pseudomonas species*, *Candida rugosa*, *Candida antarctica* (Novozyme-435), *Candida cylindracea*, thermophilic *Esterase lipase* CloneZyme ESL-001, cutinase from *Humicola insolens*, immobilized *Pseudomonas species* on celite, *Porcine pancreatic lipase*, immobilized *Porcine pancreatic*, Lipozyme IM or immobilized lipase from *Thermomyces lanuginose*, *Mucor javanicus*, *Mucor meihei*, *Pseudomonas aeruginosa*, *Pseudomonas cepacia*, *Pseudomonas fluorescens*, *Porcine pancreatic* lipase, *Penicillium rorueforti*, *Tritirachium alkaline proteinase*, *Rhizopus delemer*, *Rhizopus japonicus*, surfactant coated Lipase from *Aspergillus niger,* surfactant coated Lipase from *Pseudomonas species*, surfactant coated Lipase from *Candida rugoza*, surfactant coated Lipase from *Mucor javanicus*, surfactant coated *Pseudomonas species* (Barrera-Rivera et al., 2009; Córdova et al., 1999; Divakar, 2004; Dong, 1998, 1999; Gorke et al., 2007; Henderson et al., 1996; Kobayashi, 2001a, 2001b, 2009; MacDonald et al., 1995; Marcilla et al., 2006; Matsumoto et al., 1999; Mei et al., 2003; Namekawa et al., 1999; Rokicki, 2000; Sivalingam & Madras, 2004; Van Der Mee et al., 2006).

Lipases can accommodate a wide variety of synthetic substrates and still be able to show stereo- and regio-selectivity. They have evolved unusually stable structures that may survive effect of the organic solvents. The lipase-catalyzed hydrolysis in water can be easily reversed in non-aqueous media or bulk into ester synthesis or transesterification (Albertsson & Varma, 2003; Albertsson & Srivastava, 2008; Labet & Thielemans, 2009; Platel, 2009; Varma et al., 2005).

The e-ROP can be carried out in bulk, in organic media and at various interfaces. Enzymecatalyzed reactions proceed under different reaction conditions (i.e. temperature, pressure, time). As an example, e-ROP of cyclic monomers was performed using lipase as catalyst for 2-720 h. *M*n of the resulting polymers was ranged from 1000 to 90 000 Da, when *M*w was in the range from 6 000 to 170 000 Da. The yield of the obtained polymers varied from 10 to 100%. The preferred lipase system generally used is a physically immobilized form of *Candida Antarctica*, commercially available as Novozyme-435 (Barrera-Rivera et al., 2009; Córdova et al., 1999; Divakar, 2004; Dong, 1998, 1999; Gorke et al., 2007; Henderson et al., 1996; Kobayashi, 2001a, 2001b, 2009; MacDonald et al., 1995; Marcilla et al., 2006; Matsumoto et al., 1999; Mei et al., 2003; Namekawa et al., 1999; Sivalingam & Madras, 2004; Van Der Mee et al., 2006).

The primary research on the enzyme ring-opening polymerization (e-ROP) has been carried out for CL. Currently major works concern ROP of six- and seven-membered cyclic esters,

Lipases could also catalyze ROP of cyclic monomers, with different ring sizes as well as monomers containing substituents in the ring: α-methyl-β-propiolactone (1), β–butyrolactone (2), γ-caprolactone (3), α-methyl-valerolactone (4), 1,4-dioxan-2-one (5), δ-caprolactone (6), γethyl-ε-caprolactone (7), ε-heptanolactone (8), δ-decalactone (9), δ-dodecalactone (10), αmethyl-12-dodecanolide (11), α-methyl-15- pentadecanolide (12), L-lactide (13), D-lactide (14), D,L-lactide (15), 1,4-dioxepan-2-one (16), 1,5-dioxepan-2-one (17), 2-methylene-4-oxa-12 dodecanolide (18), 1,3-dioxan-2-one (19), 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one (20), 5-benzyloxy-1,3-dioxan-2-one (21), 1-methyl-1,3-dioxan-2-one (22), cyclobis (hexamethylene carbonate) (23), 2,2'-dimethyl-1,3-dioxan-2-one (24) (Figure 5) (Albertsson & Varma, 2003; Albertsson & Srivastava, 2008; Labet & Thielemans, 2009; Platel, 2009; Varma et al., 2005).

Many families of enzymes were used in ROP of cyclic esters or carbonate: *Aspergillus niger*, *Pseudomonas species*, immobilized *Pseudomonas species*, *Candida rugosa*, *Candida antarctica* (Novozyme-435), *Candida cylindracea*, thermophilic *Esterase lipase* CloneZyme ESL-001, cutinase from *Humicola insolens*, immobilized *Pseudomonas species* on celite, *Porcine pancreatic lipase*, immobilized *Porcine pancreatic*, Lipozyme IM or immobilized lipase from *Thermomyces lanuginose*, *Mucor javanicus*, *Mucor meihei*, *Pseudomonas aeruginosa*, *Pseudomonas cepacia*, *Pseudomonas fluorescens*, *Porcine pancreatic* lipase, *Penicillium rorueforti*, *Tritirachium alkaline proteinase*, *Rhizopus delemer*, *Rhizopus japonicus*, surfactant coated Lipase from *Aspergillus niger,* surfactant coated Lipase from *Pseudomonas species*, surfactant coated Lipase from *Candida rugoza*, surfactant coated Lipase from *Mucor javanicus*, surfactant coated *Pseudomonas species* (Barrera-Rivera et al., 2009; Córdova et al., 1999; Divakar, 2004; Dong, 1998, 1999; Gorke et al., 2007; Henderson et al., 1996; Kobayashi, 2001a, 2001b, 2009; MacDonald et al., 1995; Marcilla et al., 2006; Matsumoto et al., 1999; Mei et al., 2003; Namekawa et al., 1999; Rokicki, 2000; Sivalingam & Madras, 2004; Van Der Mee et al., 2006). Lipases can accommodate a wide variety of synthetic substrates and still be able to show stereo- and regio-selectivity. They have evolved unusually stable structures that may survive effect of the organic solvents. The lipase-catalyzed hydrolysis in water can be easily reversed in non-aqueous media or bulk into ester synthesis or transesterification (Albertsson & Varma, 2003; Albertsson & Srivastava, 2008; Labet & Thielemans, 2009; Platel, 2009;

The e-ROP can be carried out in bulk, in organic media and at various interfaces. Enzymecatalyzed reactions proceed under different reaction conditions (i.e. temperature, pressure, time). As an example, e-ROP of cyclic monomers was performed using lipase as catalyst for 2-720 h. *M*n of the resulting polymers was ranged from 1000 to 90 000 Da, when *M*w was in the range from 6 000 to 170 000 Da. The yield of the obtained polymers varied from 10 to 100%. The preferred lipase system generally used is a physically immobilized form of *Candida Antarctica*, commercially available as Novozyme-435 (Barrera-Rivera et al., 2009; Córdova et al., 1999; Divakar, 2004; Dong, 1998, 1999; Gorke et al., 2007; Henderson et al., 1996; Kobayashi, 2001a, 2001b, 2009; MacDonald et al., 1995; Marcilla et al., 2006; Matsumoto et al., 1999; Mei et al., 2003; Namekawa et al., 1999;

Sivalingam & Madras, 2004; Van Der Mee et al., 2006).

cyclic ether-estres or carbonates.

Varma et al., 2005).

Fig. 5. Representative monomers for e-ROP

The mechanism of e-ROP of cyclic esters using lipases as catalyst has been proposed by several authors. Monomer activated e-ROP (Scheme 1) involves the activation of the monomer molecules by an enzyme followed by the attack of the activated monomer onto the polymer chain end (Albertsson & Srivastava, 2008; MacDonald et al., 1995; Namekawa et al., 1999).

The ROP of cyclic carbonates catalyzed by enzyme or enzyme derivatives, in which polyesters, poly(ether-ester)s and polycarbonates terminated by hydroxyl groups are obtained, seems very attractive from the pharmaceutical or medical point of view.

Bisht and coworkers proposed a mechanism for chain initiation and propagation for lipasecatalyzed trimethylene carbonate polymerization, based on the symmetrical structure of these products and the end-group structure of high molecular weight chains (Scheme 2) (Bisht et al., 1996).

Scheme 2. The mechanism of e-ROP of cyclic carbonates

Bisht and coworkers proposed a mechanism for chain initiation and propagation for lipasecatalyzed trimethylene carbonate polymerization, based on the symmetrical structure of these products and the end-group structure of high molecular weight chains (Scheme 2)

Scheme 1. The mechanism of e-ROP of cyclic esters

Scheme 2. The mechanism of e-ROP of cyclic carbonates

(Bisht et al., 1996).

#### **3.2 Natural initiators and organocatalysts of ring-opening polymerization of cyclic esters and carbonates**

Recently, many modification approaches on biodegradable and bioresorbable polymers were carried out to meet the requirements of specific medical and pharmaceutical applications. Between them, incorporation of bioactive or biocompatible compounds such as lipids, amino acids into polymer chain or using of natural products as organocatalysts has received considerable attention.

The guanidine is a natural base, existing in human body and some vegetables. Some guanidine derivatives are the components of the therapeutic agents (Kinnel et al., 1998; Ramarao et al., 1993). Application of guanidine derivatives as organocatalysts for the synthesis of biodegradable polymers is an attractive way in the materials science technology. Li and coworkers reported the use of hexabutyl guanidinum acetatae in the living ROP of lactides (LAs) (Li et al., 2004). The polymerization was performed in bulk, producing polylactides (PLAs) with moderate molecular weight and narrow polydispersity. Strong guanidine bases: TBD (1,5,7-Triazabicyclo[4.4.0]dec-5-ene), MTBD (7-Methyl-1,5,7 triazabicyclo[4.4.0]dec-5-ene) and DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene) were applied as effective organocatalysts for ROP of LA, δ-valerolactone (VL) and CL by Lohmeijer`s and coworkers (Lohmeijer et al., 2006). They found that TBD was polymerized LA, VL and CL in the controlled manner while MTBD and DBU polymerized only LA. For VL and CL the addition of thiourea co-catalyst was required. Wang and coworkers were used creatinine as catalysts of ROP of LA, examining the influence of temperature, time and creatinine dosage on the polymerization and properties of the produced biodegradable polymer (Wang et al., 2003). Based on the obtained results they proposed that creatinine is initiated ROP of LA according to the coordination-insertion mechanism. The biogenetic guanidine carboxylates: creatinine acetate (CRA) and creatinine glycolate (CRG) were synthesized and then effectively utilized as single-component initiators of ROP of LAs (Li et al., 2009). The mechanism of ROP was proposed based of the experimental investigation. In our laboratory, other guanidine derivatives: arginine and citrulline were successfully applied as initiators of ROP of LA and CL (Oledzka et al., 2011). The incorporation of α-amino acid molecules into the polymer chain was confirmed using 1H, 13C NMR and FT-IR spectroscopy and MALDI TOF MS spectrometry.

Various carboxylic acids (lactic, tartaric, hexanoic, propionic and citric acids) and natural amino acids (glycine, proline and serine) were engaged as catalysts in living ROP of CL and VL (Casas et al., 2004). The reactions were performed without solvent with the efficient way of recovering of the catalysts. Moreover, the authors found that the order of catalytic efficiency of the organic acid catalysts in ROP was as follows: tartaric acid (pKa=2.98)>citric acid (pKa=3.08)>lactic acid (pKa=3.14)>proline (pKa=1.95).

The fatty acids are found naturally in the human body. They are considered biologically safe and are generally considered suitable candidates for the preparation of biodegradable polymers (Teomim & Domb, 2001).

ROP of CL by organic acids catalyst and oleic acid derivatives initiator systems was investigated by Oledzka and coworker (Oledzka & Narine, 2001). They have found that the polymerizations were efficiently catalyzed by succinic and fumaric acid. The incorporation of fatty acid molecules resulted in less crystallinity and lower melting points of the obtained polymer samples. Furthermore, the degradation rate of the received polymers was slower when compared to CL homopolymer, but increase in porosity of the polymers was observed over time.

Natural amino acids are essential components in human nutrition. The studies performed by Liu and coworkers showed that they are also effective as initiators of ROP of CL (Liu & Liu, 2004). Authors found hat the number-average molecular weights of the obtained polymers did not exceed 5700 Da and that used amino acids were incorporated into macromolecule chain (Figure 6). In our laboratory, more detailed studies, involving an extended range of amino-acid initiators applied to ROP of CL and *rac*-LA were previously performed (Sobczak et al., 2008). The polymerization of CL and *rac*-LA was carried out in bulk, at 120–160 ºC. L-alanine, Lcysteine, L-leucine, L-methionine, L-phenylalanine, L-proline, L-threonine, L-aspartic acid, Lglutamic acid, L-histidine, L-lysine were used as initiators of ROP. Aliphatic polyesters have synthesized with high yield (even ca. 100 % in some cases).

Fig. 6. ROP of cyclic esters using natural amino acids as initiators

ROP of aliphatic cyclic carbonates using natural amino acids was also investigated by Liu and coworkers (Liu et al., 2008). The biodegradable polymers with incorporated amino acids molecules were effectively obtained. The presence of natural amino acids in the polymer chain was proved by nuclear magnetic resonance spectroscopy.

Polyamidoamine (PAMAM) dendrimers are highly hyperbranched synthetic polymers with well-defined structure that allows accurate control of shape, size and functionality of terminal groups (Tomalia et al., 1985). The PAMAM dendrimers have useful applications in pharmaceutical technology e.g. as carriers in drug delivery systems, which can cross cell walls by both paracellular and transcellular pathways (Jevprasesphant et al., 2003). In our laboratory, novel star-shaped biodegradable polyesters were synthesized by ROP of CL using PAMAM dendrimer initiator without any metal catalysts (Oledzka et al., 2011). The nonlinear structure as well as physicochemical properties of the obtained biodegradable polymers were confirmed by nuclear magnetic resonance, gel permeation chromatography, thermal gravimetric analysis and differential scanning calorymetry.

#### **3.3 Natural co-initiators of ring-opening polymerization of cyclic esters, ether-esters and carbonates**

Metal catalyzed ROP of cyclic esters, ether-esters or carbonates has become the object of intensive studies with respect to their practical applications in the synthesis of biomaterials.

polymer samples. Furthermore, the degradation rate of the received polymers was slower when compared to CL homopolymer, but increase in porosity of the polymers was

Natural amino acids are essential components in human nutrition. The studies performed by Liu and coworkers showed that they are also effective as initiators of ROP of CL (Liu & Liu, 2004). Authors found hat the number-average molecular weights of the obtained polymers did not exceed 5700 Da and that used amino acids were incorporated into macromolecule chain (Figure 6). In our laboratory, more detailed studies, involving an extended range of amino-acid initiators applied to ROP of CL and *rac*-LA were previously performed (Sobczak et al., 2008). The polymerization of CL and *rac*-LA was carried out in bulk, at 120–160 ºC. L-alanine, Lcysteine, L-leucine, L-methionine, L-phenylalanine, L-proline, L-threonine, L-aspartic acid, Lglutamic acid, L-histidine, L-lysine were used as initiators of ROP. Aliphatic polyesters have

ROP of aliphatic cyclic carbonates using natural amino acids was also investigated by Liu and coworkers (Liu et al., 2008). The biodegradable polymers with incorporated amino acids molecules were effectively obtained. The presence of natural amino acids in the polymer

Polyamidoamine (PAMAM) dendrimers are highly hyperbranched synthetic polymers with well-defined structure that allows accurate control of shape, size and functionality of terminal groups (Tomalia et al., 1985). The PAMAM dendrimers have useful applications in pharmaceutical technology e.g. as carriers in drug delivery systems, which can cross cell walls by both paracellular and transcellular pathways (Jevprasesphant et al., 2003). In our laboratory, novel star-shaped biodegradable polyesters were synthesized by ROP of CL using PAMAM dendrimer initiator without any metal catalysts (Oledzka et al., 2011). The nonlinear structure as well as physicochemical properties of the obtained biodegradable polymers were confirmed by nuclear magnetic resonance, gel permeation chromatography,

**3.3 Natural co-initiators of ring-opening polymerization of cyclic esters, ether-esters** 

Metal catalyzed ROP of cyclic esters, ether-esters or carbonates has become the object of intensive studies with respect to their practical applications in the synthesis of biomaterials.

synthesized with high yield (even ca. 100 % in some cases).

Fig. 6. ROP of cyclic esters using natural amino acids as initiators

chain was proved by nuclear magnetic resonance spectroscopy.

thermal gravimetric analysis and differential scanning calorymetry.

observed over time.

**and carbonates** 

Tin(II) 2-ethylhexanoate (SnOct2) is commonly used as a commercial catalyst for the ROP of cyclic monomers. It is effective, relatively cheap, non-toxic, soluble in the most commonly used organic solvents (Labet & Thielemans; 2009). SnOct2 is considered to have a toxicity much lower than other metal compounds, and it is allowed to be used as a food additive in a number of countries.

SnOct2 must be used together with a nucleophilic compound (generally an alcohol) to initiate the reaction if a controlled synthesis of the polymer is to be obtained. The main drawback of SnOct2 is that it requires high temperature, which leads inter- and intramolecular esterification (Labet & Thielemans; 2009).

According to Kowalski's hypothesis, the first step of the polymerization consists of the production of the active species by reacting the alcohol with the catalyst. The more alcohol is added, the more the equilibrium is displaced towards the right and the more active species are created. With increasing carboxylic acid concentration, the equilibrium shifts to the left and less active species are present in the medium (Scheme 3) (Kowalski et al., 1998). Mechanism of CL and LA polymerization initiated with SnOct2/C4H9NH2 system has also been described (Duda et al., 2005).

Scheme 3. The formation of active centres in the reaction of Sn(Oct)2 with alcohol (co-initiator)

SnOct2 has also been combined with ureidopyrimidinone-alcohol (UPy) compounds. Using the good soluble alcohols, bearing a 1-ethylpentyl moiety, the ROP was significantly more controlled (Celiz & Scherman, 2008).

Sobczak and Kolodziejski have studied SnOct2/L-carnitine (CA) catalytic system. CA is a hydrophilic amino acid derivative, naturally occurring in human cell. Low-molecular weight PCL, PLA and copolymers of CL and *rac*-LA were obtained by the ROP of cyclic esters in the presence of SnOct2/CA. The molecular mass values averaged over the obtained polyesters were roughly in agreement with the theoretical molecular weights calculated from the feed ratio of the cyclic esters to CA (Sobczak & Kolodziejski, 2009).

Zhang and coworkers used cholesterol (CHL) as an initiator and SnOct2 as a catalyst of ROP of CL (Zhang et al., 2005). The polymerization was carried out under rigorously anhydrous conditions. The optimized ring-opening polymerization conditions have been identified to be 8 h at 140 ºC. The molecular weight of CHL-PCL has increased with decreasing cholesterol/CL feed ratio. Incorporation of the cholesteryl moiety into polymer chain has leaded to a slower enzymatic degradation rate. Whereas, Cai and coworkers utilized PAMAM dendrimer as initiator of ROP of LLA (Cai et al., 2003). The star-shaped biodegradable polymers with the average molecular weight about 70000 Da were successfully obtained in that work. The authors also found that the synthesized polymers showed a faster degradation rate than linear homopolymer because of its shortened polymer chains.

### **4. Conclusions**

The pharmaceutical technology is one of the most important fields of using of polymers. From this review, it is clear that macromolecules have been extremely active research area over the last years. In addition is worth to note, that the progress of modern pharmaceutical technology is not feasible without utilization of natural and synthetic polymers. The discovering of new drug forms, e.g.: new therapeutic systems and macromolecular prodgrugs is simply demanded by the market and industry presently. The elaboration of new medical and pharmaceutical specimens will also require intensive investigations in chemistry and biomedical polymer areas.

As is also evident from this discussion, the spectacular improvement has been achieved with natural compounds applied as initiators, catalysts, organocatalysts or co-initiators of polymerization of cyclic esters, ether-esters and carbonates. The utilized compounds are primarily friendly for environment, safe, non-toxic and irreplaceable for the synthesis of polymers for the pharmaceutical applications. Promising avenues of research have also emerged for the enzymatic approach. Increasing interest has also been dedicated to the polymers containing natural compounds in macromolecules that have been incorporated into though the polymerization process. Clearly, the future development of biodegradable and bioresorbable polymers will be based on discovering macromolecules with not only appropriate chemical, physical and mechanical properties but also suitable biological properties.

#### **5. Acknowledgment**

We would like to thank Professor M. Naruszewicz for financial support.

#### **6. References**


The pharmaceutical technology is one of the most important fields of using of polymers. From this review, it is clear that macromolecules have been extremely active research area over the last years. In addition is worth to note, that the progress of modern pharmaceutical technology is not feasible without utilization of natural and synthetic polymers. The discovering of new drug forms, e.g.: new therapeutic systems and macromolecular prodgrugs is simply demanded by the market and industry presently. The elaboration of new medical and pharmaceutical specimens will also require intensive investigations in

As is also evident from this discussion, the spectacular improvement has been achieved with natural compounds applied as initiators, catalysts, organocatalysts or co-initiators of polymerization of cyclic esters, ether-esters and carbonates. The utilized compounds are primarily friendly for environment, safe, non-toxic and irreplaceable for the synthesis of polymers for the pharmaceutical applications. Promising avenues of research have also emerged for the enzymatic approach. Increasing interest has also been dedicated to the polymers containing natural compounds in macromolecules that have been incorporated into though the polymerization process. Clearly, the future development of biodegradable and bioresorbable polymers will be based on discovering macromolecules with not only appropriate chemical, physical and mechanical properties but also suitable biological

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## **Translating 2A Research into Practice**

### Garry A. Luke

*University of St Andrews, Scotland UK* 

#### **1. Introduction**

160 Innovations in Biotechnology

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> Viruses have evolved a number of unconventional translation strategies to amplify the coding potential of their condensed genetic information. Leaky stop codons may be readthrough to produce either the predicted translation product, or at a very low level an extended "read-through" protein. Overlapping (*e.g.* –UAAUG-; -UGAUG-; AUGA-), or highly proximal stop/start codons may give rise to termination accompanied by a low level of re-initiation. There are a number of cases where a single mRNA is translated into more than one protein by recoding, where the rules for decoding are altered through specific sites and signals in the mRNA such as frameshifting and readthrough. Ribosomal "skipping", first identified in the foot-and-mouth disease virus (FMDV), represents yet another translational trick to deliver multiple gene products from limited primary sequence. Briefly, when a ribosome encounters 2A within an open reading frame (ORF), the synthesis of a specific peptide bond is "skipped". The process gives rise to two alternative outcomes: either (i) translation terminates at the end of 2A, or (ii) translation of the downstream sequence occurs. In this manner discrete translation products can be synthesized from a single ORF (for in-depth reviews of recoding see Atkins & Gesteland, 2010).

> 2A and "2A-like" sequences have been thoroughly studied in the last 25 years. These results, as well as our current understanding of the underlying mechanism, are summarized in the first section of this review. In the next section, important considerations in the design of 2A peptide-linked vectors are discussed. The 2A peptide system has worked in all eukaryotic systems tested and has been used with some spectacular successes in a variety of biotechnology applications. In the final section we provide an overview of the literature highlighting some of these successes.

#### **2. Basic research**

#### **2.1 The fmdv genome**

The FMDV genome organization is similar to that of other picornaviruses, comprising a large single ORF flanked by highly structured 5' and 3' untranslated regions (UTRs) (Fig. 1). The 5' UTR, of approximately 1,300 nucleotides (nt) contains sequence elements controlling the replication of viral RNA, packaging of RNA into capsids, and translation of the viral polyprotein. Preceding the ORF is a type II internal ribosome entry site (IRES), crucial for the cap-independent initiation of translation (for reviews see Jackson et al., 1990; Martĩnez-Salas & Ryan, 2010). The 3' UTR is about 90 nt long and is thought to contain cis-acting elements

Fig. 1. The FMDV Genome. The FMDV genome is organized like a cellular mRNA: a 5' untranslated region (5'UTR), a single open reading frame (ORF), a 3' untranslated region (3'UTR) and a poly(A) tail. The polyprotein (~2,300aa) undergoes three "primary", cotranslational cleavages; Lpro cleaves at its own C-terminus, 2A mediates "cleavage" at its own C-terminus and 3Cpro cleaves between [2BC] and 3A. The 2A oligopeptide is only 18aa long, mediating a "cleavage" by a translational effect "ribosome skipping".

required for efficient genome replication (Agol et al., 1999). Moreover, the 3' end of mRNA has also turned out to be surprisingly important in regulating translation (Wells et al., 1998). The ORF encodes a large protein precursor (polyprotein) which can be divided into three regions, designated P1, P2, and P3. These correspond to the N-terminal capsid protein precursor (P1, containing four capsid proteins 1A-1D), the middle of the polyprotein containing three of the nonstructural proteins (P2, the three proteins 2A-2C), and the most Cterminal segment of the polyprotein containing four non-structural proteins (P3, proteins 3A-3D) (Palmenberg, 1987). The full-length translation product is never observed within infected cells due to co-translational, intramolecular, cleavages mediated by Lpro, 2A and 3Cpro domains within the polyprotein (for reviews see, Belsham, 2005; Ryan et al., 2004). Besides releasing itself from the polyprotein, Lpro, in common with 2Apro of the entero- and rhinoviruses, also cleaves the translation initiation factor eIF4G (Glaser & Skern, 2000). This results in the inactivation of cap-dependent translation leading to the shutoff of cellular protein synthesis. The 2A oligopeptide is responsible for the primary cleavage which separates the region comprising the capsid proteins from domains downstream of 2A concerned with the replication of the virus (Ryan et al., 1991; Ryan & Drew, 1994). All picornaviruses encode 3Cpro, which carries out a primary cleavage between 2C and 3A and secondary processing of the [P1- 2A], [2BC] and P3 precursors. In FMDV, 3Cpro also cleaves between 2B and 2C (for review see

Fig. 1. The FMDV Genome. The FMDV genome is organized like a cellular mRNA: a 5' untranslated region (5'UTR), a single open reading frame (ORF), a 3' untranslated region (3'UTR) and a poly(A) tail. The polyprotein (~2,300aa) undergoes three "primary", cotranslational cleavages; Lpro cleaves at its own C-terminus, 2A mediates "cleavage" at its own C-terminus and 3Cpro cleaves between [2BC] and 3A. The 2A oligopeptide is only 18aa

required for efficient genome replication (Agol et al., 1999). Moreover, the 3' end of mRNA has also turned out to be surprisingly important in regulating translation (Wells et al., 1998). The ORF encodes a large protein precursor (polyprotein) which can be divided into three regions, designated P1, P2, and P3. These correspond to the N-terminal capsid protein precursor (P1, containing four capsid proteins 1A-1D), the middle of the polyprotein containing three of the nonstructural proteins (P2, the three proteins 2A-2C), and the most Cterminal segment of the polyprotein containing four non-structural proteins (P3, proteins 3A-3D) (Palmenberg, 1987). The full-length translation product is never observed within infected cells due to co-translational, intramolecular, cleavages mediated by Lpro, 2A and 3Cpro domains within the polyprotein (for reviews see, Belsham, 2005; Ryan et al., 2004). Besides releasing itself from the polyprotein, Lpro, in common with 2Apro of the entero- and rhinoviruses, also cleaves the translation initiation factor eIF4G (Glaser & Skern, 2000). This results in the inactivation of cap-dependent translation leading to the shutoff of cellular protein synthesis. The 2A oligopeptide is responsible for the primary cleavage which separates the region comprising the capsid proteins from domains downstream of 2A concerned with the replication of the virus (Ryan et al., 1991; Ryan & Drew, 1994). All picornaviruses encode 3Cpro, which carries out a primary cleavage between 2C and 3A and secondary processing of the [P1- 2A], [2BC] and P3 precursors. In FMDV, 3Cpro also cleaves between 2B and 2C (for review see

long, mediating a "cleavage" by a translational effect "ribosome skipping".

Martĩnez-Salas & Ryan, 2010). Aside from the processing sites within the viral polyprotein itself, the enzyme also modifies host cell proteins (Belsham et al., 2000; Li et al., 2001).

Fig. 2. Analysis of 2A-mediated "cleavage". Artificial polyprotein cDNA constructs comprising the reporter proteins green fluorescent protein (GFP) and β-glucuronidase (GUS) (left panel). SDS-PAGE of radiolabeled *in vitro* translation products (right panel). The control pGFPGUS construct produces only a single translation product – the [GFP-GUS] fusion protein. The translation profile from the pGFP2AGUS construct shows 3 major products: uncleaved [GFP2AGUS] and the cleavage products [GFP2A] and [GUS]. The profile from pGUS2AGFP also shows 3 major products: uncleaved [GUS2AGFP] and the cleavage products [GUS-2A] and [GFP]. The cleavage products upstream of 2A are highlighted in red, showing the molar excess over the downstream products shown in blue.

Secondary 3Cpro cleavage of the [1D2A] precursor protein between 1D and 2A shows the FMDV 2A segment is only 18aa long (-LLNFDLLKLAGDVESNPG-) (Belsham, 1993). Analysis of recombinant polyproteins and artificial polyprotein systems in which 2A was inserted between two reporter proteins showed that 2A alone, plus the N-terminal proline of protein 2B, was sufficient to mediate a highly efficient co-translational "cleavage" at the C-terminus of 2A (Ryan & Drew, 1994; Ryan et al., 1991; de Felipe et al., 2003). Translation *in vitro*, together with careful quantification of the products (Fig.2), provided the major finding that a molar excess of protein encoded upstream of 2A accumulated over that downstream – an observation at variance with proteolytic processing (Ryan et al., 1989; Donnelly et al., 2001a).

Extensive protein degradation studies, examining the effects of non-specific premature termination of transcription/translation, have shown that none of these effects account for this imbalance (Ryan et al., 1999). Addition of puromycin at low concentration to translation reactions programmed with mRNA encoding a 2A containing reporter yields significant product with a size corresponding to the protein up to the 2A site, indicating a pause in translation at this position (Donnelly et al., 2001a). Employing a "toe-printing" approach, Doronina and colleagues confirmed that ribosomes pause at the end of the 2A coding sequence (-NPGP-), with glycine and proline in the P- and A- sites, respectively (Doronina et al., 2008b). This front end loading was due to different rates of biosynthesis of each portion of the ORF and constitutes a novel type of recoding (Baranov et al., 2002; Brown & Ryan, 2010).

#### **2.2 The cleavage mechanism**

The 2A region of the FMDV encodes a sequence that mediates self-processing by a novel translational effect variously referred to as "ribosome skipping" (Ryan et al., 1999), "stop-go" (Atkins et al., 2007) and "stop carry-on" translation (Doronina et al., 2008a). 2A-mediated cleavage occurs between the C-terminal glycine and the proline of the downstream protein 2B (-LLNFDLLKLAGDVESNPGP-). The upstream protein contains a short 2A peptide Cterminal fusion, whereas the downstream protein includes a single proline residue on its Nterminus (Ryan and Drew, 1994; Ryan et al., 1991). The translational model of 2A cleavage activity posited is shown in Figure 3. Briefly, the nascent 2A peptide interacts with the exit pore of the ribosome such that the C-terminal portion (-ESNPGP-) is sterically constrained within the peptidyl transferase centre of the ribosome. This inhibits nucleophilic attack of the ester linkage between 2A and tRNAgly by prolyl-tRNA in the A site - effectively stalling, or pausing, translation (Ryan et al., 1999; Donnelly et al., 2001a). It has been shown that this block is relieved by the action of translation release factors eRF1 and eRF3, hydrolysing the ester linkage and releasing the nascent protein (Doronina et al., 2008a & b). Thus two major outcomes are possible; either translation terminates at this point, or, translation effectively 'reinitiates' to synthesize the downstream sequences. The latter case would entail; (a) egress of eRF1/3 from the A site, (b) ingress of prolyl-tRNA into the A site, (c) translocation of prolyltRNA to the P site and (d) entry of the next aminoacyl-tRNA (for in-depth reviews of the model see Ryan et al., 2002; Martĩnez-Salas & Ryan, 2010; Brown & Ryan, 2010).

#### **2.3 The occurrence of 2A and 2A-like sequences**

Examining other picornavirus genome sequences showed the DxExNPGP motif to be present in several genera of the *Picornaviridae*: aphtho- cardio-, tescho-, erbo- and certain parechoviruses. Although cardioviruses have much longer natural 2A segments (133 to 143 amino acids) than aphthoviruses, work with *Encephalomyocarditis virus* (EMCV) and *Theiler's murine encephalitis virus* (TMEV) has shown that most of the additional 2A protein is dispensable for primary cleavage activity (Hahn & Palmenberg, 1996; Donnelly et al., 1997). Probing databases for the presence of the motif showed that "2A-like" sequences were also present in a range of non-picornavirus systems. These include a wide range of insect positive-strand RNA viruses belonging to the *Dicistroviridae* and *Tetraviridae* families and the unassigned Iflavirus genus and double-stranded RNA viruses of the *Reoviridae* (insect Cypoviruses and mammalian type C rotaviruses) (Hahn & Palmenberg, 1996; Donnelly et al., 2001b). They are also found in four nonsegmented dsRNA viruses of the *Totiviridae* (Isawa et al., 2011). Analysis of the translation products showed that in all cases these 2As had "cleavage" activity (Luke et al., 2008).

imbalance (Ryan et al., 1999). Addition of puromycin at low concentration to translation reactions programmed with mRNA encoding a 2A containing reporter yields significant product with a size corresponding to the protein up to the 2A site, indicating a pause in translation at this position (Donnelly et al., 2001a). Employing a "toe-printing" approach, Doronina and colleagues confirmed that ribosomes pause at the end of the 2A coding sequence (-NPGP-), with glycine and proline in the P- and A- sites, respectively (Doronina et al., 2008b). This front end loading was due to different rates of biosynthesis of each portion of the ORF and constitutes a novel type of recoding (Baranov et al., 2002; Brown & Ryan, 2010).

The 2A region of the FMDV encodes a sequence that mediates self-processing by a novel translational effect variously referred to as "ribosome skipping" (Ryan et al., 1999), "stop-go" (Atkins et al., 2007) and "stop carry-on" translation (Doronina et al., 2008a). 2A-mediated cleavage occurs between the C-terminal glycine and the proline of the downstream protein 2B (-LLNFDLLKLAGDVESNPGP-). The upstream protein contains a short 2A peptide Cterminal fusion, whereas the downstream protein includes a single proline residue on its Nterminus (Ryan and Drew, 1994; Ryan et al., 1991). The translational model of 2A cleavage activity posited is shown in Figure 3. Briefly, the nascent 2A peptide interacts with the exit pore of the ribosome such that the C-terminal portion (-ESNPGP-) is sterically constrained within the peptidyl transferase centre of the ribosome. This inhibits nucleophilic attack of the ester linkage between 2A and tRNAgly by prolyl-tRNA in the A site - effectively stalling, or pausing, translation (Ryan et al., 1999; Donnelly et al., 2001a). It has been shown that this block is relieved by the action of translation release factors eRF1 and eRF3, hydrolysing the ester linkage and releasing the nascent protein (Doronina et al., 2008a & b). Thus two major outcomes are possible; either translation terminates at this point, or, translation effectively 'reinitiates' to synthesize the downstream sequences. The latter case would entail; (a) egress of eRF1/3 from the A site, (b) ingress of prolyl-tRNA into the A site, (c) translocation of prolyltRNA to the P site and (d) entry of the next aminoacyl-tRNA (for in-depth reviews of the

model see Ryan et al., 2002; Martĩnez-Salas & Ryan, 2010; Brown & Ryan, 2010).

Examining other picornavirus genome sequences showed the DxExNPGP motif to be present in several genera of the *Picornaviridae*: aphtho- cardio-, tescho-, erbo- and certain parechoviruses. Although cardioviruses have much longer natural 2A segments (133 to 143 amino acids) than aphthoviruses, work with *Encephalomyocarditis virus* (EMCV) and *Theiler's murine encephalitis virus* (TMEV) has shown that most of the additional 2A protein is dispensable for primary cleavage activity (Hahn & Palmenberg, 1996; Donnelly et al., 1997). Probing databases for the presence of the motif showed that "2A-like" sequences were also present in a range of non-picornavirus systems. These include a wide range of insect positive-strand RNA viruses belonging to the *Dicistroviridae* and *Tetraviridae* families and the unassigned Iflavirus genus and double-stranded RNA viruses of the *Reoviridae* (insect Cypoviruses and mammalian type C rotaviruses) (Hahn & Palmenberg, 1996; Donnelly et al., 2001b). They are also found in four nonsegmented dsRNA viruses of the *Totiviridae* (Isawa et al., 2011). Analysis of the translation products showed that in all cases these 2As

**2.3 The occurrence of 2A and 2A-like sequences** 

had "cleavage" activity (Luke et al., 2008).

**2.2 The cleavage mechanism** 

Fig. 3. Schematic representation of the translational model of 2A-mediated "cleavage".

Along with the RNA virus 2As, active 2A-like sequences were also detected in the N-terminal region of the ORFs of non-LTR retrotransposons of *Trypanosoma cruzi* and *T.brucei* – L1Tc and igni, respectively (Donnelly et al., 2001b; Heras et al., 2006). Recently we identified a range of 2As in the purple sea urchin *Stronglocentrotus purpuratus*, then demonstrated their cleavage activities (unpublished data). In this case, 2A-like sequences appear in (i) several copies of non-LTR-retroelements (like trypanosomes) and (ii) the N-terminus of nucleotide binding oligomerization domain (NOD)-like, or CATERPILLER proteins (cited in Brown & Ryan, 2010). It appears, therefore, that this method of controlling protein biogenesis is not confined to viruses or genomic sequences comprising insertion(s) of virus-related sequences (retroelements). 2A and "2A-like" sequences have been shown to function in cells from a wide variety of eukaryotes, ranging from yeast (de Felipe et al., 2003) to plants (Halpin et al., 1999) to insects (Roosien et al., 1990) to mammals (Ryan & Drew, 1994). The only requirement for 2A peptide-based cleavage appears to be translation by 80S ribosomes. The reported proteolysis activity of 1D-2A in *E.coli* cells (Dechamma et al., 2008) was not detected in equivalent constructions in our laboratory showing "cleavage" specificity for eukaryotic systems alone (Donnelly et al., 1997). Although the FMDV 2A sequence (hereafter referred to as "F2A") has been the most widely used, biotechnologists should be aware that many 2A-like sequences have been utilized successfully, including equine rhinitis A virus (ERAV, "E2A"), porcine teschovirus-1 (PTV-1, "P2A") and *Thosea asigna* virus (TaV, "T2A") (Szymczak et al., 2004; Arnold et al., 2004; Osborn et al., 2005; Szymczak & Vignali, 2005; Huang et al., 2006; Scholten et al., 2006; Hart et al., 2008; Sommer et al., 2008; Yang et al., 2008).

#### **3. General considerations when using 2A peptide sequences**

#### **3.1 Expression of multiple genes**

Conventional approaches for the production of multicistronic vectors include the use of IRES elements, multiple promoters, fusion proteins, *etc* (for a review see de Felipe, 2002). Adverse side-effects with multiple promoters on viral vectors include interference between promoters, promoter suppression and rearrangement (Cullen et al., 1984; Emerman & Temin, 1986). IRESes provided the first method of creating eukaryotic polycistronic mRNAs. The internal ribosome entry site serves as a launching pad for internal initiation of translation, allowing expression of two or more genes from a single transcript (for review see Komar & Hatzoglou, 2005). Since genes are under the control of the same promoter and integrated into the same place within the genome, transgenes expressed in this way are coordinately regulated. In bicistronic systems, detection of the product encoded by the second cistron is evidence that the first cistron is also being expressed. This approach has been used successfully in gene therapy research in animal systems, and IRESes from different viruses have been tested and shown to function in plant systems (Urwin et al., 2000 & 2002; Dorokhov et al., 2002; Jaag et al., 2003; Bouabe et al., 2008).

On the other hand, there are a couple of limitations using IRES elements. Firstly, the IRES is a relatively large sequence (~500bp) that can cause problems in packaging, especially for size-restricted viral and nonviral vectors. For instance, retro- and lentiviral vectors possess packaging capacities of 8kb and adeno-associated viruses can accommodate <5kb (Thomas et al., 2003). Secondly, expression of the downstream gene can be as much as 10 fold lower than the upstream gene (Mizuguchi et al. 2000; Flasshove et al., 2000; Hasegawa et al., 2007; Ha et al., 2010). In some instances, this can be useful for expressing fluorescent markers or conferring drug resistance during selection (Ngoi et al., 2004). Nevertheless, the obvious advantages of using the 2A sequence vis-á-vis the IRES are its smaller size (~60-70bp) and the stoichiometric production of both upstream and downstream protein products as measured by: i) chloramphenicol acetyltransferase (CAT) and -glucuronidase (GUS) enzyme activity (Halpin et al., 1999); ii) cell free translation *in vitro* and Western blot (Ryan & Drew, 1994; Donnelly et al., 2001a & b; de Felipe et al., 2003; Torres et al., 2010)*;* iii) GFP/FACS with antibiotic resistance (Lorens et al., 2004); iv) co-fluorescence reporting (de Felipe & Ryan, 2004; Samalova et al., 2006); v) fluorescence resonance energy transfer (FRET) analysis (Szymczak et al., 2004) and vi) protein segregation in genetically engineered animals (Provost et al., 2007; Trichas et al., 2008). Further, if multiple gene expression is required, different members of the 2A peptide family can be selected to disrupt sequence homology to help maintain foreign gene insert stability.

#### **3.2 Subcellular targeting of proteins from a 2A polyprotein**

A merit of this expression strategy is that individual components of the 2A-polyprotein can be targeted to a range of different sub-cellular sites using both co- and post-translational signal sequences (El Amrani et al., 2004; Lorens et al., 2004; Szymczak et al., 2004). We discovered, however, a major problem with co-expression of some proteins targeted to, or passing through, the mammalian endoplasmic reticulum (ER). When a 2A-based polyprotein comprising an upstream protein bearing an N-terminal signal sequence was followed by a protein lacking any signal sequence, both proteins were translocated into the

Conventional approaches for the production of multicistronic vectors include the use of IRES elements, multiple promoters, fusion proteins, *etc* (for a review see de Felipe, 2002). Adverse side-effects with multiple promoters on viral vectors include interference between promoters, promoter suppression and rearrangement (Cullen et al., 1984; Emerman & Temin, 1986). IRESes provided the first method of creating eukaryotic polycistronic mRNAs. The internal ribosome entry site serves as a launching pad for internal initiation of translation, allowing expression of two or more genes from a single transcript (for review see Komar & Hatzoglou, 2005). Since genes are under the control of the same promoter and integrated into the same place within the genome, transgenes expressed in this way are coordinately regulated. In bicistronic systems, detection of the product encoded by the second cistron is evidence that the first cistron is also being expressed. This approach has been used successfully in gene therapy research in animal systems, and IRESes from different viruses have been tested and shown to function in plant systems (Urwin et al., 2000 & 2002;

On the other hand, there are a couple of limitations using IRES elements. Firstly, the IRES is a relatively large sequence (~500bp) that can cause problems in packaging, especially for size-restricted viral and nonviral vectors. For instance, retro- and lentiviral vectors possess packaging capacities of 8kb and adeno-associated viruses can accommodate <5kb (Thomas et al., 2003). Secondly, expression of the downstream gene can be as much as 10 fold lower than the upstream gene (Mizuguchi et al. 2000; Flasshove et al., 2000; Hasegawa et al., 2007; Ha et al., 2010). In some instances, this can be useful for expressing fluorescent markers or conferring drug resistance during selection (Ngoi et al., 2004). Nevertheless, the obvious advantages of using the 2A sequence vis-á-vis the IRES are its smaller size (~60-70bp) and the stoichiometric production of both upstream and downstream protein products as measured by: i) chloramphenicol acetyltransferase (CAT) and -glucuronidase (GUS) enzyme activity (Halpin et al., 1999); ii) cell free translation *in vitro* and Western blot (Ryan & Drew, 1994; Donnelly et al., 2001a & b; de Felipe et al., 2003; Torres et al., 2010)*;* iii) GFP/FACS with antibiotic resistance (Lorens et al., 2004); iv) co-fluorescence reporting (de Felipe & Ryan, 2004; Samalova et al., 2006); v) fluorescence resonance energy transfer (FRET) analysis (Szymczak et al., 2004) and vi) protein segregation in genetically engineered animals (Provost et al., 2007; Trichas et al., 2008). Further, if multiple gene expression is required, different members of the 2A peptide family can be selected to disrupt sequence

A merit of this expression strategy is that individual components of the 2A-polyprotein can be targeted to a range of different sub-cellular sites using both co- and post-translational signal sequences (El Amrani et al., 2004; Lorens et al., 2004; Szymczak et al., 2004). We discovered, however, a major problem with co-expression of some proteins targeted to, or passing through, the mammalian endoplasmic reticulum (ER). When a 2A-based polyprotein comprising an upstream protein bearing an N-terminal signal sequence was followed by a protein lacking any signal sequence, both proteins were translocated into the

**3. General considerations when using 2A peptide sequences** 

Dorokhov et al., 2002; Jaag et al., 2003; Bouabe et al., 2008).

homology to help maintain foreign gene insert stability.

**3.2 Subcellular targeting of proteins from a 2A polyprotein** 

**3.1 Expression of multiple genes** 

ER (de Felipe & Ryan, 2004). We have identified the source of this problem - the "slipstreaming" effect was due to inhibition of the 2A reaction (formation of fusion protein) by the C-terminal region (immediately upstream of 2A) of some proteins when translocated into the ER – and suggest possible solutions (de Felipe et al., 2010).

The residues that influence cleavage are predicted to reside within the translocon; this length may allow interactions between the nascent peptide and the ribosome that lead to inhibition of the 2A reaction (Ménétret et al., 2000; Beckmann et al., 2001; de Felipe et al., 2010). Solutions to the problem include the use of longer versions of 2A with extra sequences derived from the capsid protein ("1D") (Ryan et al., 1991; Groot Bramel-Verheije et al., 2000; Donnelly et al., 2001b; Klump et al., 2001). Specifically, N-terminal extension of 2A by 5aa of 1D improved "cleavage", but extension by 14aa of 1D or longer (21 and 39aa) produced complete "cleavage" and an equal stoichiometry of the up- and downstream translation products (Donnelly et al., 2001b, see fig. 4). These observations are consistent with our model in which 2A activity is a product of it's interaction with the exit tunnel of the ribosome which is thought to accommodate 30-40aa (Hardesty & Kramer, 2001). Further,

Fig. 4. Translation *in vitro*. Translation products derived from constructs encoding the wildtype 2A sequence are shown together with products derived from constructs encoding Nterminally extended forms of 2A.

the order in which the genes are expressed within the vector needs to be considered. By swapping the order of proteins in several artificial polyproteins the stoichiometry was affected by the gene upstream of 2A (Ma & Mitra, 2002; Lengler et al., 2005; Chinnasamy et al., 2006; Rothwell et al., 2010). Cleavage activity was independent of the immediate downstream sequence (Ryan et al., 1991; Ma & Mitra., 2002). A number of studies show that cleavage efficiency is improved by incorporation of a flexible Gly-Ser-Gly or Ser-Gly-Ser-Gly linker sequence between the upstream protein and the 2A peptide (Lorens et al., 2004; Szymczak et al., 2004; Holst et al., 2006a & b; Provost et al., 2007; Wargo et al., 2009). A noteworthy caveat to attach to this review is that Yan and colleagues argue slipstreaming translocation does not occur in mammalian cells; that is, the second protein downstream of 2A still requires a signal sequence for secretory or membrane-anchored expression (Yan et al., 2010).

#### **3.3 The unwanted tags**

Cleavage occurs at the end of the 2A peptide sequence, therefore most of the 2A remains attached to the C-terminus of the upstream protein. This may affect the activity of some proteins (*e.g.* if their function is affected by the addition of other tags such as Myc, His, *etc*). In the case of proteins translocated into the ER, a strategy was adopted to include a furin proteinase cleavage site (-RAKR-) between the upstream protein and 2A (Fang et al., 2005). Furin is a cellular endoprotease localized on the trans-Golgi networks of virtually all cell types (Steiner, 1998). Upon entering the lumen of the ER, 2A was trimmed away from the upstream protein (in this case antibody heavy chain), leaving only a 2aa C-terminal extension (-RA). In a follow-on study, the use of alternative furin cleavage sequences consisting of only basic amino acids, which can be efficiently cut by carboxypeptidases (- RRRR-, -RKRR-, -RRKR-), resulted in the expression of antibodies with no residual amino acids (Fang et al., 2007). Proteins expressed in plants could have their 2A extensions removed by endogenous proteinases acting on similar hybrid linker peptides. A polyprotein precursor consisting of two different marker proteins connected by a linker peptide of Impatiens balsamina (-SNAADEVAT-) followed by F2A was successfully processed in *Arabidopsis thaliana* (François et al., 2002; François et al., 2004).

For biomedical applications using 2A, a concern stems from the addition of 2A-derived sequences to the upstream protein - this protein may act as a carrier to stimulate an anti-2A immune response. Any potential carrier-effects could be abolished by removal of 2A. It should be noted that this "unwanted" tag does confer two advantages. First, antibodies to the 2A-peptide have been generated, allowing detection and/or immunoprecipitation of "upstream" protein products derived from 2A-containing transgenes (de Felipe et al, 2006 & 2010). Second, a shift in protein size is observed in 2A-tagged proteins which can be useful if mutant and endogenous proteins are co-expressed and need to be identified (Szmczak et al., 2004; de Felipe et al., 2010). To our knowledge, the presence of a proline attached to the amino-terminus of the second protein, as a relic of the 2A self-cleaving process, does not interfere significantly with activity and trafficking; it does, however, confer high protein stability (Varshavsky, 1992).

#### **4. Translational studies**

#### **4.1 Gene expression** *in planta*

Currently, there are several options available for the introduction of multiple transgenes *in planta*. The different methods include sexual crossing, re-transformation, single-plasmid or

et al., 2006; Rothwell et al., 2010). Cleavage activity was independent of the immediate downstream sequence (Ryan et al., 1991; Ma & Mitra., 2002). A number of studies show that cleavage efficiency is improved by incorporation of a flexible Gly-Ser-Gly or Ser-Gly-Ser-Gly linker sequence between the upstream protein and the 2A peptide (Lorens et al., 2004; Szymczak et al., 2004; Holst et al., 2006a & b; Provost et al., 2007; Wargo et al., 2009). A noteworthy caveat to attach to this review is that Yan and colleagues argue slipstreaming translocation does not occur in mammalian cells; that is, the second protein downstream of 2A still requires a signal sequence for secretory or membrane-anchored

Cleavage occurs at the end of the 2A peptide sequence, therefore most of the 2A remains attached to the C-terminus of the upstream protein. This may affect the activity of some proteins (*e.g.* if their function is affected by the addition of other tags such as Myc, His, *etc*). In the case of proteins translocated into the ER, a strategy was adopted to include a furin proteinase cleavage site (-RAKR-) between the upstream protein and 2A (Fang et al., 2005). Furin is a cellular endoprotease localized on the trans-Golgi networks of virtually all cell types (Steiner, 1998). Upon entering the lumen of the ER, 2A was trimmed away from the upstream protein (in this case antibody heavy chain), leaving only a 2aa C-terminal extension (-RA). In a follow-on study, the use of alternative furin cleavage sequences consisting of only basic amino acids, which can be efficiently cut by carboxypeptidases (- RRRR-, -RKRR-, -RRKR-), resulted in the expression of antibodies with no residual amino acids (Fang et al., 2007). Proteins expressed in plants could have their 2A extensions removed by endogenous proteinases acting on similar hybrid linker peptides. A polyprotein precursor consisting of two different marker proteins connected by a linker peptide of Impatiens balsamina (-SNAADEVAT-) followed by F2A was successfully processed in

For biomedical applications using 2A, a concern stems from the addition of 2A-derived sequences to the upstream protein - this protein may act as a carrier to stimulate an anti-2A immune response. Any potential carrier-effects could be abolished by removal of 2A. It should be noted that this "unwanted" tag does confer two advantages. First, antibodies to the 2A-peptide have been generated, allowing detection and/or immunoprecipitation of "upstream" protein products derived from 2A-containing transgenes (de Felipe et al, 2006 & 2010). Second, a shift in protein size is observed in 2A-tagged proteins which can be useful if mutant and endogenous proteins are co-expressed and need to be identified (Szmczak et al., 2004; de Felipe et al., 2010). To our knowledge, the presence of a proline attached to the amino-terminus of the second protein, as a relic of the 2A self-cleaving process, does not interfere significantly with activity and trafficking; it does, however, confer high protein

Currently, there are several options available for the introduction of multiple transgenes *in planta*. The different methods include sexual crossing, re-transformation, single-plasmid or

*Arabidopsis thaliana* (François et al., 2002; François et al., 2004).

expression (Yan et al., 2010).

stability (Varshavsky, 1992).

**4. Translational studies 4.1 Gene expression** *in planta*

**3.3 The unwanted tags** 

multiple-plasmid co-transformation, and IRES-based transformation. The pros and cons of each have been reviewed previously (François et al., 2002; Halpin, 2005; Luke et al., 2006 & 2010). However, these procedures all suffer from a lack of coordinated expression of the different transgenes. As an alternative, the coding sequences of the genes of interest can be linked *via* 2A in a single transcription unit (Halpin et al., 1999; Ma & Mitra, 2002). The first types of genetically modified organisms created using 2A to co-express multiple proteins were plants, initially as a research tool, but also to improve drought-resistance (Kwon et al., 2004); disease-resistance (François et al., 2004; Geu-Flores et al., 2009) and nutritional qualities (Randall et al., 2004). Plant virus vectors based on potato virus X (PVX), cowpea mosaic virus (CPMV), pepino mosaic virus (PepMV), and bean pod mottle virus (BPMV) have been engineered with F2A and used to produce functional recombinant proteins including vaccines and antibodies (Smolenska et al., 1998; Gopinath et al., 2000; Marconi et al., 2006; Zhang et al., 2010; Sempere et al., 2011).

Metabolic and combinatorial engineering of carotenoid biosynthetic pathways in plants, including those synthesizing important industrial and pharmaceutical products, provide excellent examples of the utility of this approach (Ralley et al., 2004; Ha et al., 2010). Carotenoids have attracted interest not only as a source of pigmentation but also for their beneficial effects on human health. One of the most widely known carotenoids is -carotene, which serves as a dietary precursor of vitamin A. In developing countries, where vitamin A deficiency prevails, a promising intervention to existing strategies is to fortify the major staple food, rice, with provitamin A. Golden rice (*Oryza sativa*, GR) is the generic name given to genetically modified rice that produces -carotene in the endosperm (Ye et al., 2000, GR1; Paine et al., 2005, GR2). Engineering the provitamin A (-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm requires two carotenoid biosynthetic genes, phytoene synthase (*psy*) and carotene desaturase (*crtl*) (Lu & Li, 2008). As a step towards the coordinate expression of the two genes, *psy* from *Capsicum* and *crtl* from *Pantoea*, were linked *via* synthetic 2A (*psy-F2A-crtl*) or IRES (*psy-IRES-crtl*) sequences and placed under the control of the rice endosperm-specific globulin promoter (Ha et al., 2010). Collectively, the results demonstrated that the 2A construct performed better than the IRES construct in terms of carotenoid production. In addition, the use of a single promoter (GR1 and 2 require two promoters) reduces the chance of gene silencing and provides more space for transgene stacking.

#### **4.2 Optical imaging of gene expression**

In order to monitor transgene delivery and expression by optical imaging, the coding region is fused to a fluorescent/luminescent reporter. Another approach is to detect the expressed protein through its activity (conversion of a substrate in a fluorescent product as with galactosidase). We (Halpin et al., 1999; Funston et al., 2008; de Felipe et al., 2010) and others (Samalova et al., 2006 & 2008; Hasegawa et al., 2007; Torres et al., 2010) have successfully used the 2A sequence in a number of *in vitro* and *in vivo* heterologous systems to achieve production of various combinations of fluorescent proteins and proteins requiring discrete co- and post-translational subcellular localization. The zebrafish (*Danio rerio*) has proved to be an excellent vertebrate model system for basic and biomedical science and comparative genomics. The lauded advantage of zebrafish embryos being transparent lends itself remarkably well to the use of fluorescence. To demonstrate the utility of the 2A system in zebrafish, reporter constructs employing eGFP and mCherry separated by the P2A sequence were designed to segregate fluorescent proteins to distinct cellular locations (Provost et al., 2007). Tissue-specific expression of both fluorophores in stably transformed embryos shows this approach could facilitate continuous expression of multiple proteins products at various stages of development in zebrafish. Likewise, Trichas et al (2008) used a bi-cistronic reporter construct containing a single coding sequence for a membrane localized red fluorescent protein (Myr-TdTomato) and a nuclear localized green fluorescent protein (H2B-GFP) separated by the T2A sequence to test 2A function in transgenic mice. Mutually exclusive localization of TdTomato and EGFP to the membrane and nucleus was observed in cultured cells and endogenous vertebrate cells, consistent with complete 2A-mediated processing. For the transgenic mice produced in this study, targeted expression was apparent in all tissues examined throughout development and into adulthood and remained constant across several generations.

*In vivo* bioluminescent imaging (BLI) allows a low-cost, noninvasive, and real-time analysis of biological processes at the molecular level in living systems. Cao and colleagues used BLI to visualize engraftment, survival, and rejection of transplanted tissues from a transgenic donor mouse that constitutively expresses luciferase (Cao et al., 2005). The donor mouse has a transgene comprised of a hybrid CMV--actin promoter, a firefly luciferase gene, a F2A and GFP gene. Isolated haematopoietic stem cells (HSC) from these mice express luciferase at the highest level among different haematopoietic cell types, and all haematopoietic lineages tested (with the exception of erythroblasts and red blood cells) express the reporter gene. As a virtually unlimited source of labelled cells this mouse line represents a valuable resource for stem cell and transplantation studies.

#### **4.3 Immunotherapies**

#### **4.3.1 Cancer immunotherapy using heat shock protein**

In an effort to extend the scope of immunotherapy for the control of advanced ovarian cancer, BLI was used to measure tumour load and distribution in mice vaccinated with irradiated tumour cells secreting heat shock protein 70 (Hsp70) (Chang et al., 2007). Hsps, including Hsp70, are highly effective in potentiating immune responses *via* interaction with several surface receptors on antigen-presenting cells (APCs). Hsp-specific receptors efficiently transport the chaperoned peptide into the major histocompatibility complex (MHC) class 1 cross-presentation pathway leading to recognition and activation of cytotoxic T cells (Udono & Srivastava, 1993; Massa et al., 2004). A retrovirus encoding sHsp70-T2A-GFP was used to introduce the gene for secreted hsp70 directly into mouse ovarian surface epithelial cells (MOSEC) that express luciferase. In summary, the tumour-secreted Hsp70 was capable of generating a potent antigen-specific "cytotoxic" CD8+ T-cell response and CD40 was identified as a likely receptor for Hsp70-mediated cross-presentation.

#### **4.3.2 Immunotherapy using monoclonal antibodies**

Advances in recent years delineating the specific components of the immune system that contribute to immune responsiveness point to an important regulatory role for immunomodulators. Monoclonal antibodies (mAbs) are an important class of therapeutic agents for the treatment of cancer, autoimmune disorders, and infectious diseases. Although

zebrafish, reporter constructs employing eGFP and mCherry separated by the P2A sequence were designed to segregate fluorescent proteins to distinct cellular locations (Provost et al., 2007). Tissue-specific expression of both fluorophores in stably transformed embryos shows this approach could facilitate continuous expression of multiple proteins products at various stages of development in zebrafish. Likewise, Trichas et al (2008) used a bi-cistronic reporter construct containing a single coding sequence for a membrane localized red fluorescent protein (Myr-TdTomato) and a nuclear localized green fluorescent protein (H2B-GFP) separated by the T2A sequence to test 2A function in transgenic mice. Mutually exclusive localization of TdTomato and EGFP to the membrane and nucleus was observed in cultured cells and endogenous vertebrate cells, consistent with complete 2A-mediated processing. For the transgenic mice produced in this study, targeted expression was apparent in all tissues examined throughout development and into adulthood and remained constant

*In vivo* bioluminescent imaging (BLI) allows a low-cost, noninvasive, and real-time analysis of biological processes at the molecular level in living systems. Cao and colleagues used BLI to visualize engraftment, survival, and rejection of transplanted tissues from a transgenic donor mouse that constitutively expresses luciferase (Cao et al., 2005). The donor mouse has a transgene comprised of a hybrid CMV--actin promoter, a firefly luciferase gene, a F2A and GFP gene. Isolated haematopoietic stem cells (HSC) from these mice express luciferase at the highest level among different haematopoietic cell types, and all haematopoietic lineages tested (with the exception of erythroblasts and red blood cells) express the reporter gene. As a virtually unlimited source of labelled cells this mouse line represents a valuable

In an effort to extend the scope of immunotherapy for the control of advanced ovarian cancer, BLI was used to measure tumour load and distribution in mice vaccinated with irradiated tumour cells secreting heat shock protein 70 (Hsp70) (Chang et al., 2007). Hsps, including Hsp70, are highly effective in potentiating immune responses *via* interaction with several surface receptors on antigen-presenting cells (APCs). Hsp-specific receptors efficiently transport the chaperoned peptide into the major histocompatibility complex (MHC) class 1 cross-presentation pathway leading to recognition and activation of cytotoxic T cells (Udono & Srivastava, 1993; Massa et al., 2004). A retrovirus encoding sHsp70-T2A-GFP was used to introduce the gene for secreted hsp70 directly into mouse ovarian surface epithelial cells (MOSEC) that express luciferase. In summary, the tumour-secreted Hsp70 was capable of generating a potent antigen-specific "cytotoxic" CD8+ T-cell response and

CD40 was identified as a likely receptor for Hsp70-mediated cross-presentation.

Advances in recent years delineating the specific components of the immune system that contribute to immune responsiveness point to an important regulatory role for immunomodulators. Monoclonal antibodies (mAbs) are an important class of therapeutic agents for the treatment of cancer, autoimmune disorders, and infectious diseases. Although

across several generations.

**4.3 Immunotherapies** 

resource for stem cell and transplantation studies.

**4.3.1 Cancer immunotherapy using heat shock protein** 

**4.3.2 Immunotherapy using monoclonal antibodies** 

satisfactory for short-term applications, antibody intravenous infusion is not appropriate in many long-term treatments. Fang et al. (2007) describe a recombinant adeno-associated virus (rAAV) gene delivery system that allows regulated long-term expression of native fulllength mAbs *in vivo*. In this study a F2A sequence adjacent to a furin cleavage site (ΔK)RKRR was used to link the antibody heavy and light chain sequences. Notably, the gene expression system included a rapamycin-regulated promoter that can be used to stop mAb production if treatment needs to be terminated. This system potentially offers patients a lifelong mAb therapy that requires only a single administration of an rAAV vector.

Cytotoxic T-lymphocyte-associated antigen (CTLA-4), also known as CD152, is a coinhibitory molecule that functions to regulate T-cell activation. Antibodies that block the interaction of CTLA-4 with its ligands CD80 (B7-1) and CD86 (B7-2) can enhance immune responses, including anti-tumour immunity (for a brief review, see Chambers et al., 2001). Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a bone marrow growth factor for APCs, which has also been shown to enhance anti-tumour immune responses. Both preclinical animial models and early clinical development indicate synergy between GM-CSF tumour cell vaccination and CTLA-4 blockade (Hurwitz et al., 2000; Hodi et al., 2003; Quezada et al., 2006). To avoid anti-CTLA-4 side effects, tumour cell lines expressing the full-length F2A anti-CTLA-4 mAb in addition to GM-CSF, were administered locally at the immunization site (Simmons et al., 2008). Preliminary results suggest that the delivery of mAbs or proteins locally from immunotherapy cells should prove useful based on promising anti-tumour responses and the reduction of toxicity or adverse immune events associated with systemic exposure.

#### **4.3.3 Cytokines and immunotherapy**

The cytokine Interleukin-12 (IL-12) is a growth and maturation factor acting on both the innate and adaptive arms of the immune system. It is produced primarily by APCs and exerts immunoregulatory effects on natural killer (NK) and T cells (Kobayashi et al., 1989; Wolf et al., 1991). The APC-derived IL-12 consists of two subunits, p40 and p35, which are covalently linked (Kobayashi et al., 1989). The expression of this cytokine has been complicated by the observation that p40 homodimers (in excess of the heterodimer) exhibit antagonistic activity (Trinchieri et al., 2003). To ensure the equal expression of both subunits, biologically active IL-12 protein was produced using F2A as a linker between the p40 and p35 subunits (Collins et al., 1998; Kokuho et al., 1999; Chaplin et al., 1999; de Rose et al., 2000; Premraj et al., 2006). Numerous studies have been done which clearly indicate that plasmid expressed F2A IL-12 can modulate and augment the immune response elicited by DNA vaccination against mycobacterial infections (Triccas et al., 2002; Palendira et al., 2002; Martin et al., 2003). Additionally, it has been reported that IL-23 (but not IL-27) increased protection after *M. tuberculosis* challenge (Wozniak et al., 2006). In this study, the genes encoding p19 and p40 chains of IL-23 and EB13 and p28 chains of IL-27 were cloned on either side of the F2A protein.

Enhanced persistence of adoptively transferred tumour-infiltrating lymphocytes has been demonstrated by the administration of growth cytokines such as IL-2 and IL-15 (for reviews see Westwood & Kershaw, 2010; Ngo et al., 2011). However, systemic toxicity and expansion of unwanted cell subsets, such as regulatory T cells limit the use of these cytokines when administered systemically. Transgenic expression of IL-2 and IL-15 has been shown to increase antigen-specific T cell expansion *in vivo* and enhance antitumour activity without systemic toxicity in preclinical models (Quintarelli et al., 2007). The 3 genes coexpressed in the cytokine encoding vectors (*iCasp-9,CD34,* and *IL-2* or *IL-15*) were linked using F2A. The truncated form of human CD34 was used as a selectable marker of transduced cells and the inclusion of an iCasp-9 "safety-switch" ensured long-term safety of adoptively transferred lymphocytes.

#### **4.4. Gene therapy**

#### **4.4.1** *In vivo* **gene therapy**

Gene therapy can be defined as the introduction of nucleic acids to somatic cells for a therapeutic purpose (Ylä-Herttuala & Alitalo, 2003). Compared to traditional medicine, gene therapy offers unique possibilities to treat the genetic causes of diseases, such as fatal enzyme deficiencies. Mucopolysaccharidosis type 1 (MPS-1; Hurler syndrome) is a congenital deficiency of -L-iduronidase (IDUA), leading to lysosomal storage of glycosaminoglycans. As accumulation and storage continue, tissue and organ damage becomes manifest as loss of function. Patients with MPS-1 present early in life with rapidly progressing disease that usually results in death due to neurological/CNS deterioration and/or cardiovascular/respiratory problems (Neufeld, 1991).

Fig. 5. Schematic of the tricistronic vector construct containing the therapeutic human iduronidase (IDUA) gene along with the firefly luciferase and DsRed2 reporter genes is shown at the top. (A) Whole-organ DsRed2 expression. (B) Cellular DsRed2 expression. (C) Whole-body *in vivo* luciferase imaging. Representative animals of tricistronic plasmidinjected (left), control (*IDUA* injected, middle), and monocistronic luciferase-injected (right) recipients are shown (adapted from Osborn et al., 2005). (For interpretation of the references to colour in this figure, the reader is referred to the web version of this article.)

antigen-specific T cell expansion *in vivo* and enhance antitumour activity without systemic toxicity in preclinical models (Quintarelli et al., 2007). The 3 genes coexpressed in the cytokine

form of human CD34 was used as a selectable marker of transduced cells and the inclusion of an iCasp-9 "safety-switch" ensured long-term safety of adoptively transferred lymphocytes.

Gene therapy can be defined as the introduction of nucleic acids to somatic cells for a therapeutic purpose (Ylä-Herttuala & Alitalo, 2003). Compared to traditional medicine, gene therapy offers unique possibilities to treat the genetic causes of diseases, such as fatal enzyme deficiencies. Mucopolysaccharidosis type 1 (MPS-1; Hurler syndrome) is a congenital deficiency of -L-iduronidase (IDUA), leading to lysosomal storage of glycosaminoglycans. As accumulation and storage continue, tissue and organ damage becomes manifest as loss of function. Patients with MPS-1 present early in life with rapidly progressing disease that usually results in death due to neurological/CNS deterioration

Fig. 5. Schematic of the tricistronic vector construct containing the therapeutic human iduronidase (IDUA) gene along with the firefly luciferase and DsRed2 reporter genes is shown at the top. (A) Whole-organ DsRed2 expression. (B) Cellular DsRed2 expression. (C) Whole-body *in vivo* luciferase imaging. Representative animals of tricistronic plasmidinjected (left), control (*IDUA* injected, middle), and monocistronic luciferase-injected (right) recipients are shown (adapted from Osborn et al., 2005). (For interpretation of the references

to colour in this figure, the reader is referred to the web version of this article.)

*CD34,* and *IL-2* or *IL-15*) were linked using F2A. The truncated

encoding vectors (*iCasp-9,*

**4.4.1** *In vivo* **gene therapy** 

**4.4. Gene therapy** 

and/or cardiovascular/respiratory problems (Neufeld, 1991).

Enzyme may be delivered by enzyme replacement therapy (ERT), haematopoietic cell transplantation (HCT) or by gene therapy vectors (Tolar & Orchard, 2008). P2A and T2A were utilized to construct a tricistronic vector bearing the human iduronidase (IDUA) gene along with the firefly luciferase and DsRed2 reporter genes (*IDUA*-P2A-luciferase-T2A-DsRed). Efficient cleavage was observed and all three proteins were functional *in vitro* and *in vivo*, leading to high-level therapeutic gene expression in NOD/*scid* mice that could be tracked by non-invasive whole-body luciferase imaging and at the cellular level using DsRed2 (Fig.5. Osborn et al., 2005).

#### **4.4.2** *Ex vivo* **gene therapy**

To improve patient safety and increase the gene transfer efficiency, target cells are taken from the patient, gene-engineered and then adoptively transferred into the patient. Redirecting T cell specificity by T cell receptor (TCR) gene transfer is emerging as an attractive strategy to treat patients suffering from malignant and viral diseases. TCR, together with the CD3and signaling subunits, determines the specific CD4+ and CD8+ T cell responses to antigens bound to MHC molecules (Call & Wucherpfennig, 2005; Rudolph et al., 2006). Using the TCR:CD3 complex as a test system, Szymczak and coworkers reported expression of all four proteins that make up CD3 and the two proteins required to make up TCR using just two retroviral vectors (CD3-2A and TCR-F2A) (Szymczak et al., 2004; reviewed in Radcliffe & Mitrophanous, 2004). Following the seminal paper of Szymczak et al in 2004, several groups have reported efficient TCR expression using 2A peptide linkers to combine TCR and - chain genes (Holst et al., 2006a & b; Scholten et al., 2006; Yang et al., 2008; Leisegang et al., 2008; Wargo et al., 2009). An important consideration in redirecting T cells using TCR genes is the tendency of introduced TCR genes to mispair with endogenous TCR - and - chains. In this regard, "murinized" receptors improved HLA-A2/LMP2-TCR expression on the surface of human T cells and downregulated expression of endogenous TCRs (Hart et al., 2008).

The feasibility of TCR gene therapy was recently demonstrated in the first bench to bedside experiments with TCR gene-modified T cells in melanoma patients. Johnson et al., (2009) treated metastatic melanoma patients with autologous T cells genetically modified with retroviral vectors to express high-avidity TCRs recognizing tumour-associated antigens MART-1 (MART-1*TCR-*furinT2A-MART-1*TCR* and gp100 (gp100*TCR*IRESgp100*TCR*). Objective cancer regression was observed in 30% - 19% respectively, of patients who received these high affinity TCRs. However, in the study with TCR targeting MART, some patients also experienced toxicity to normal melanocytes in the skin, eye and ear. Another interesting recent study details the first clinical trial involving the adoptive transfer of engineered lymphocytes with optimal TCR complementary determining regions (CDRs) directed against NY-ESO-1, a cancer-testis antigen frequently expressed in melanoma as well as a wide range of non-melanoma epithelial malignancies (Robbins et al., 2011). In contrast to MART-1 and gp100, which are expressed in normal tissues as well as tumours, NY-ESO-1 expression is limited to neoplastic cells and germ line tissue (Chen et al., 1997). The - and -chains were expressed in retroviral constructs that contained a furin cleavage site followed by a SGSG spacer and the P2A sequence between the two gene products (Robbins et al., 2008). Response rates of 45% and 67% were observed in patients with melanoma and synovial cell sarcoma, respectively, all of whom had progressive disease after extensive prior treatment.

#### **4.5 Induced pluripotent stem cell generation**

Embryonic stem (ES) cells have the ability to differentiate into any cell type of the body and to grow indefinitely while maintaining pluripotency. Remarkably, adult somatic cells can be reprogrammed and returned to the naive state of pluripotency seen in embryonic stem cells by ectopic expression of a defined set of transcription factors: Oct 3/4, Sox2, KLF4 and c-Myc (Takahashi & Yamanaka, 2006; Takahashi et al., 2007; for review see Das & Pal, 2010). The delivery of these "Yamanaka factors" to create induced pluripotent stem (iPS) cells has typically required multiple individual viral vectors, carrying the risk of both insertional mutagenesis and viral reactivation (Takahashi and Yamanaka, 2006; Aoi et al., 2008).

Fig. 6. Schematic diagram of reprogramming cassette. The four reprogramming factors *c-Myc*, *Klf4*, *Oct4* and *Sox2* were fused in-frame *via* 2A sequences and coexpressed as a single ORF and inset: the CAG enhancer/promoter was used to drive the 2A-linked reprogramming cassette and *mOrange* marker, flanked by *loxP* sites (Kaji et al., 2009).

Preliminary work by Okita et al. (2008 & 2010) achieved reprogramming of murine embryonic fibroblasts using repeated transient expressions of two plasmids - one encoding Oct 3/4, Sox2, KLF4 separated by F2A and the other encoding c-Myc. Although the efficiency of iPS cell generation was low, no vector DNA was stably integrated into the iPS cell genome. Sommer et al. (2008) and Carey et al. (2009) reported the derivation of iPS cells from adult skin fibroblasts using polycistronic lentiviral vectors. Sommer's team used a

Embryonic stem (ES) cells have the ability to differentiate into any cell type of the body and to grow indefinitely while maintaining pluripotency. Remarkably, adult somatic cells can be reprogrammed and returned to the naive state of pluripotency seen in embryonic stem cells by ectopic expression of a defined set of transcription factors: Oct 3/4, Sox2, KLF4 and c-Myc (Takahashi & Yamanaka, 2006; Takahashi et al., 2007; for review see Das & Pal, 2010). The delivery of these "Yamanaka factors" to create induced pluripotent stem (iPS) cells has typically required multiple individual viral vectors, carrying the risk of both insertional

mutagenesis and viral reactivation (Takahashi and Yamanaka, 2006; Aoi et al., 2008).

Fig. 6. Schematic diagram of reprogramming cassette. The four reprogramming factors *c-Myc*, *Klf4*, *Oct4* and *Sox2* were fused in-frame *via* 2A sequences and coexpressed as a single

reprogramming cassette and *mOrange* marker, flanked by *loxP* sites (Kaji et al., 2009).

Preliminary work by Okita et al. (2008 & 2010) achieved reprogramming of murine embryonic fibroblasts using repeated transient expressions of two plasmids - one encoding Oct 3/4, Sox2, KLF4 separated by F2A and the other encoding c-Myc. Although the efficiency of iPS cell generation was low, no vector DNA was stably integrated into the iPS cell genome. Sommer et al. (2008) and Carey et al. (2009) reported the derivation of iPS cells from adult skin fibroblasts using polycistronic lentiviral vectors. Sommer's team used a

ORF and inset: the CAG enhancer/promoter was used to drive the 2A-linked

**4.5 Induced pluripotent stem cell generation** 

single multicistronic mRNA containing an IRES element separating two fusion cistrons - Oct4 and Sox2 linked *via* F2A and KLF4 and c-Myc linked *via* E2A. In a different way, the Carey group delivered the four factors in a single vector: Oct4, Sox2, KLF4, and c-Myc separated by three different 2A peptides (P2A, T2A and E2A, respectively). Both groups demonstrated reprogramming of fibroblasts to an ES cell-like state, however, in neither case was the polycistronic vector deleted from the genome. Therefore, attempts were made to minimize genome integration by removal of the inserted genes after the reprogramming process was switched-on (Chang et al., 2009; Kaji et al., 2009; Woltjen et al., 2009; Yusa et al., 2009) and more recently by using mRNA/miRNA of the four factors rather than DNA vectors (Warren et al., 2010; Yakubov et al., 2010; Miyoshi et al., 2011). The efficient reprogramming of murine and human embryonic fibroblasts and the traceless removal of factors joined with viral 2A sequences by using the Cre/LoxP or *piggyBac* transposon/transposase systems mark important advances towards achieving clinically acceptable methods of deriving iPS cells (see Fig.6).

#### **5. Looking ahead**

Exciting work of many laboratories in the last few years has clearly established the importance of 2A for co-expression technology. Our increasing knowledge about the cleavage mechanism indicates 2A is not just a novel method of controlling protein biogenesis, but that a crucial aspect of its function is to act as a translational "sensor". Protein synthesis in eukaryotes consumes a high proportion of cellular energy, most of which is used in elongation. During times of energy and/or nutrient deprivation, 2A could act to terminate translation in a stop codon-independent manner – devoting the remainder of the cell's resources into translating only that portion of the ORF upstream of 2A. We envisage that 2A-mediated cleavage could find extra utility in the biomedical and biotechnology fields as a reporter for translational stress.

#### **6. Acknowledgements**

The long term support of the Wellcome Trust and the Biotechnology and Biological Sciences Research Council is gratefully acknowledged. The University of St Andrews is a charity registered in Scotland no. SCO13532

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## **Controlling Cell Migration with Micropatterns**

Taro Toyota1,2,3, Yuichi Wakamoto2,3, Kumiko Hayashi4 and Kiyoshi Ohnuma5 *1Department of Basic Science, Graduate School of Arts and Sciences The University of Tokyo 2Research Center for Complex Systems Biology, The University of Tokyo 3Precursory Research of Embryonic Science and Technology (PRESTO) Japan Science and Technology Agency (JST) 4Department of Applied Physics, Graduate School of Engineering Tohoku University 5Top Runner Incubation Center for Academia-Industry Fusion Nagaoka University of Technology Japan* 

#### **1. Introduction**

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Recent advances in soft lithography techniques now allow various designs of micrometresized chambers to be easily fabricated on cell-culture vessels. By culturing cells in different micropatterns, the relationship between geometry and cell response has been studied (Fig. 1c,d). For example, spindle orientation, growth, differentiation, and migration have been shown to be related to micropattern shape. Recently, several groups, including ourselves, have reported that mammalian cells exhibit biased cell movement on asymmetrical periodic micropatterned surfaces. Although it is little wonder that cells migrate asymmetrically in asymmetrical micropatterns, the direction in which they move is not immediately obvious.

In this chapter, we describe biased cell movement in asymmetrical micropatterns. These studies offer new insights into the migration of cells in response to geometry of their surrounding environment, and we suggest strategies for designing artificial scaffolds that direct cell migration.

#### **1.1 Biological significance of cell migration**

#### **1.1.1 Cell movement: A basic characteristic of life**

Movement is a basic characteristic of cells (both unicellular organisms and the various cells of multicellular organisms) that is almost as important as self-renewal (Bray 2001). Some types of cells are extremely motile, while others lack strong motility and are capable of no more than passive movements caused by surrounding forces. Motile cells may sometimes change direction of their own accord in response to changes in their internal state (Oosawa 2001; Nakaoka et al. 2009), but normally they change direction in response to external stimuli, such as chemicals (chemotaxis) and light (phototaxis). For unicellular organisms, the ability to migrate to an environment suited to survival and proliferation is a matter of life and death. Bacteria search for an environment suited to survival by swimming. Social amoebae usually migrate independently of each other, but if the environment deteriorates, they gather to form fruiting bodies (Goldbeter 1996; Gregor et al. 2010). The fact that a great many of the cells of multicellular organisms (particularly animal cells) are capable of migration is also important. In the body plan of multicellular organisms, cells need to be able to do more than just proliferate and differentiate. In the process of development, cells need to migrate to the correct position so that they can adopt their proper shape and properly function (Keller et al. 2008). Cells also need to migrate *en masse* to specific locations to assist in the healing of wounds, to perform immune system functions, and to conduct other aspects of body maintenance (Friedl et al. 2004; Schneider et al. 2010). This suggests that motility is one of the universal characteristics of cells that enable the survival of life forms.

Fig. 1. Schematic illustration of cell migration in a multicellular body (a) and in wound healing (b). *In vitro* cell migration observed in asymmetric micropatterns connected in a linear (c) and circular manner (d).

#### **1.1.2 Migration mechanisms**

188 Innovations in Biotechnology

Movement is a basic characteristic of cells (both unicellular organisms and the various cells of multicellular organisms) that is almost as important as self-renewal (Bray 2001). Some types of cells are extremely motile, while others lack strong motility and are capable of no more than passive movements caused by surrounding forces. Motile cells may sometimes change direction of their own accord in response to changes in their internal state (Oosawa 2001; Nakaoka et al. 2009), but normally they change direction in response to external stimuli, such as chemicals (chemotaxis) and light (phototaxis). For unicellular organisms, the ability to migrate to an environment suited to survival and proliferation is a matter of life and death. Bacteria search for an environment suited to survival by swimming. Social amoebae usually migrate independently of each other, but if the environment deteriorates, they gather to form fruiting bodies (Goldbeter 1996; Gregor et al. 2010). The fact that a great many of the cells of multicellular organisms (particularly animal cells) are capable of migration is also important. In the body plan of multicellular organisms, cells need to be able to do more than just proliferate and differentiate. In the process of development, cells need to migrate to the correct position so that they can adopt their proper shape and properly function (Keller et al. 2008). Cells also need to migrate *en masse* to specific locations to assist in the healing of wounds, to perform immune system functions, and to conduct other aspects of body maintenance (Friedl et al. 2004; Schneider et al. 2010). This suggests that motility is one of the universal

**1.1 Biological significance of cell migration** 

**1.1.1 Cell movement: A basic characteristic of life** 

characteristics of cells that enable the survival of life forms.

linear (c) and circular manner (d).

Fig. 1. Schematic illustration of cell migration in a multicellular body (a) and in wound healing (b). *In vitro* cell migration observed in asymmetric micropatterns connected in a The way cells move differs markedly according to whether they are non-adherent or adherent cells (Eisenbach et al. 2004). Many non-adherent cells propel themselves using cilia or flagella. The structure of motor-protein complexes, energy balances, and protein response networks related to ciliary and flagellar locomotion have been analysed, and these locomotion mechanisms are becoming increasingly well understood. However, there is much that is still unknown about the movement of adherent cells because they lack specific means of movement such as cilia and flagella. This makes it difficult to clearly separate the different aspects of cell movement, such as deformation, migration, and division.

Fig. 2. Schematic illustration of adherent-cell migration integrating cytoskeleton and focal adhesion molecules complex, called as focal adhesions, in a fibroblast (A) and a neuronal cell (B) in overhead (a) and cross-sectional view (b–e). STEP 1 (a, b): Polarization of cell. The cell changes from a spherical or hemispherical shape to become anisotropic. STEP 2 (c): Generation of force driving the locomotion. Fibroblasts form filopodia and lamellipodia, and neuronal cells extend neurites. STEP 3 (d): Fixing the leading edge to the substrate. STEP 4 (e): Diving cell body.

However, there are common features in the movements of adherent cells and the basic mechanisms behind such movements are thought to be the same. Adherent cell migration is a result of the integrated dynamics of the cytoskeleton and adhesions molecules. The cytoskeleton runs throughout the cell body and acts as a "skeleton" and "motor" for the cell. The cytoskeleton is a protein complex composed of actin filaments, intermediate filaments, and microtubules. The cells adhere to the extracellular matrix, substrates, and other cells' surfaces with adhesion molecules including integrin to migrate, to survive, and to acquire extracellular information. The cells adhere to the extracellular matrix to transmit signals from extra-cell to intra-cell and vice versa. Therefore, adhesion to substrates through adhesions molecules acts as an "input–output adaptor". During migration, adherent cells kinetically anchor themselves to rearrange their cytoskeleton. Leading fronts, such as lamellipodia in fibroblasts and leading processes in neurons, are repeatedly formed through the extension of the cytoskeleton, which then adhere to the local environment. The posterior side of the cell is then released and retracted (Fig. 2). This process is being investigated by a number of research groups using a diverse range of observation methods because there are still many aspects that are not well understood(Smilenov et al. 1999; Flaherty et al. 2007; Hu et al. 2007).

#### **1.1.3 Asymmetries in external stimuli determine migration direction**

The migration direction of cells is determined by a number of different stimuli. The tendency of cells to change direction in response to the direction or gradient (spatial asymmetry) of external stimuli is known as *taxis*. The suffix "-taxis" is attached to prefixes representing specific stimuli to create words such as chemotaxis (movement in response to a chemical stimulus), magnetotaxis (magnetic stimulus), hydrotaxis (water), phototaxis (light), rheotaxis (water current), thermotaxis (temperature), and thigmotaxis (touch). Among the many types of taxes, chemotaxis, which is the tendency to move towards a higher or lower concentration of a specific chemical substance, is well known (Eisenbach et al. 2004). The tendency to move in the direction of a stimulus according to the gradient of the stimulus is known as positive taxis, and movement away from a stimulus is known as negative taxis. It is very important for bacteria, for example, to search for food (sugar) by swimming towards higher concentrations of food, and to avoid poison by swimming towards lower concentrations of poison. When social amoebae form fruiting bodies in response to a deteriorating environment, they are known to move towards higher concentrations of 3',5'-cyclic adenosine monophosphate (cyclic AMP) (Goldbeter 1996; Gregor et al. 2010). Even the cells of multicellular organisms display various taxes. For example, neurons extend neurites towards higher concentrations of netrin, with the cell body following suit and migrating in the same direction (Round and Stein 2007). Neutrophils, which are a type of white blood cell that eliminate invading bacteria by englobing them (phagocytosis), are capable of detecting very slight differences in concentration (1% difference between opposite sides of the neutrophil) of *N*-formylmethionyl-leucylphenylalanine (FMLP), a protein derived from bacteria, and move towards higher FMLP concentrations (Weiner et al. 1999). Cells can thus change the direction of their movements according to asymmetries in external stimuli, and this ability is extremely important for survival.

However, there are common features in the movements of adherent cells and the basic mechanisms behind such movements are thought to be the same. Adherent cell migration is a result of the integrated dynamics of the cytoskeleton and adhesions molecules. The cytoskeleton runs throughout the cell body and acts as a "skeleton" and "motor" for the cell. The cytoskeleton is a protein complex composed of actin filaments, intermediate filaments, and microtubules. The cells adhere to the extracellular matrix, substrates, and other cells' surfaces with adhesion molecules including integrin to migrate, to survive, and to acquire extracellular information. The cells adhere to the extracellular matrix to transmit signals from extra-cell to intra-cell and vice versa. Therefore, adhesion to substrates through adhesions molecules acts as an "input–output adaptor". During migration, adherent cells kinetically anchor themselves to rearrange their cytoskeleton. Leading fronts, such as lamellipodia in fibroblasts and leading processes in neurons, are repeatedly formed through the extension of the cytoskeleton, which then adhere to the local environment. The posterior side of the cell is then released and retracted (Fig. 2). This process is being investigated by a number of research groups using a diverse range of observation methods because there are still many aspects that are not well

understood(Smilenov et al. 1999; Flaherty et al. 2007; Hu et al. 2007).

**1.1.3 Asymmetries in external stimuli determine migration direction** 

is extremely important for survival.

The migration direction of cells is determined by a number of different stimuli. The tendency of cells to change direction in response to the direction or gradient (spatial asymmetry) of external stimuli is known as *taxis*. The suffix "-taxis" is attached to prefixes representing specific stimuli to create words such as chemotaxis (movement in response to a chemical stimulus), magnetotaxis (magnetic stimulus), hydrotaxis (water), phototaxis (light), rheotaxis (water current), thermotaxis (temperature), and thigmotaxis (touch). Among the many types of taxes, chemotaxis, which is the tendency to move towards a higher or lower concentration of a specific chemical substance, is well known (Eisenbach et al. 2004). The tendency to move in the direction of a stimulus according to the gradient of the stimulus is known as positive taxis, and movement away from a stimulus is known as negative taxis. It is very important for bacteria, for example, to search for food (sugar) by swimming towards higher concentrations of food, and to avoid poison by swimming towards lower concentrations of poison. When social amoebae form fruiting bodies in response to a deteriorating environment, they are known to move towards higher concentrations of 3',5'-cyclic adenosine monophosphate (cyclic AMP) (Goldbeter 1996; Gregor et al. 2010). Even the cells of multicellular organisms display various taxes. For example, neurons extend neurites towards higher concentrations of netrin, with the cell body following suit and migrating in the same direction (Round and Stein 2007). Neutrophils, which are a type of white blood cell that eliminate invading bacteria by englobing them (phagocytosis), are capable of detecting very slight differences in concentration (1% difference between opposite sides of the neutrophil) of *N*-formylmethionyl-leucylphenylalanine (FMLP), a protein derived from bacteria, and move towards higher FMLP concentrations (Weiner et al. 1999). Cells can thus change the direction of their movements according to asymmetries in external stimuli, and this ability

#### **1.2 Control of cell migration by using micropatterns**

#### **1.2.1 Cell migration around spatial obstacles**

Much of the research on taxes of adherent cells described in the previous section is based on the results of observation under a microscope of cells adhering to a flat substrate applied to glass. However, in reality cells face a plethora of spatial obstacles (such as surrounding cells, soil and plants in nature, and bone and other connective tissues within the body) that make it difficult for them to migrate freely to their destinations (Fig. 1a). As such, cells need to not only recognize the surrounding geometry mechanically, but also process this geometrical information to determine the direction of their next migration (Ingber 2003). However, research on the relationship between geometry and cell migration has so far been limited. With respect to tactile sensibility (thigmesthesia), some animals are known to display the phenomenon of thigmotaxis, but this refers to the tendency of rats and other animals to hug the edges of walls and so forth when moving, and not to the kind of cell movements with respect to physical obstacles that we are discussing here. Investigation of how cells sense spatial restrictions and respond to them requires the creation of a geometry featuring various shapes on a cellular scale (several micrometres), but while structures on a millimetre scale can be crafted using apparatus such as ordinary lathes and milling machines, creation of structures on a smaller scale is difficult, and this is why research has been limited.

#### **1.2.2 Micro-contact printing**

Recent advances in photolithography and other microfabrication techniques have made it possible to create structures that are not toxic to cells and feature all kinds of cell-sized spatial patterns. Of these micropattern techniques, micro-contact printing, a soft lithography technique developed by Whitesides et al. at Harvard University, is particularly well-suited to small-scale research at universities, and is accordingly popular in the field of cellular engineering (Kumar and Whitesides 1993; Kane et al. 1999). Micro-contact printing involves first fabricating a finely patterned master that is then used to produce finely patterned stamps made of the thermosetting silicone elastomer polydimethylsiloxane (PDMS). The stamps are then used to print the patterns associated with cell adherence. Although making masters involves microfabrication techniques such photolithography that requires access to clean rooms and photolithographic equipment, masters do not have to be made in the place where subsequent processes are performed, and so can be made by other research laboratories or companies. Moreover, the subsequent processes can all be performed in a cellular biology laboratory, and enough micro-patterned culture vessels for several experiments can be produced with ease.

Extremely high precision masters can be created by using the silicon wafers that have become synonymous with semiconductor technologies. A technique using SU-8, a UV-curable resin, can be employed to make masters more easily (Ehrfeld et al. 1999). A spin coater is used to coat a silicon wafer or glass slide with a film of SU-8, and films with a thickness of several micrometres to over 100 μm can be created with excellent reproducibility. These thin SU-8 films are cured by irradiating with UV through a patterned mask, after which the uncured parts are washed away, leaving a three-dimensional pattern (Fig. 3a). SU-8 is not so strong, and thus the master can get chipped after repeated casting with PDMS, but because this method enables the production of micrometre-scale masters with high aspect ratios and low cell toxicity using relatively simple apparatus, it is very widely used.

After pouring the PDMS onto masters created in this way and curing at 60°C overnight, the microfabricated stamp can be removed from the master (Fig. 3b,c). Because PDMS keeps its shape very well on thermosetting, it is a superb material for reproducing sub-micrometre structures, and is also known to be non-toxic to cells. Also, PDMS is a pliant material, making it easy to remove stamps from the master and enabling good contact with the surface to be stamped to ensure even printing.

A substance for controlling cell adhesion is applied as "ink" to the PDMS stamp and then stamped onto the culture substrate (Fig. 3d,e). Whitesides et al. utilized self-assembled monolayers (SAMs) by printing with alkanethiol which have a variety of reactive functional groups to anchor cell adhesion related molecules, and created cell adhesion islands. There are two main micropatterning methods—printing with extracellular matrix substances that promote cell adhesion (e.g. collagen, fibronectin, laminin) (Scholl et al. 2000; Kaji et al. 2003; Hou et al. 2009), or printing with substances that impede cell adhesion (Yang et al. 2005; Saravia et al. 2007; Ohnuma et al. 2009). As we explain later, we used the latter method (Fig. 3f). Microfabrication techniques like these have enabled us to create cell adhesion patterns with a variety of geometric shapes and investigate the way that geometric patterns affect cell movement.

Fig. 3. Schematic illustration of micro-contact printing using a PDMS stamp: (a) fabricating a patterned master from a UV-curable resin by using UV-irradiation through a patterned mask, (b) applying silicone resin, (c) curing the resin to produce a patterned stamp, (d) dipping the patterned surface of the stamp in an ink, (e) printing the ink onto a culture dish, and (f) culturing the cells on the dish.

#### **1.3 Biased movement in asymmetrical micropatterns**

#### **1.3.1 Asymmetry of cell shape and movement**

How does the shape of the space in which a cell can move affect its movement? In the case of chemotaxis, the stimulating substance creates a concentration gradient (spatial asymmetry), and the cell moves in line with that concentration gradient (asymmetrical movement). From this, it is easy to suppose that the asymmetry of the space in which a

After pouring the PDMS onto masters created in this way and curing at 60°C overnight, the microfabricated stamp can be removed from the master (Fig. 3b,c). Because PDMS keeps its shape very well on thermosetting, it is a superb material for reproducing sub-micrometre structures, and is also known to be non-toxic to cells. Also, PDMS is a pliant material, making it easy to remove stamps from the master and enabling good contact with the

A substance for controlling cell adhesion is applied as "ink" to the PDMS stamp and then stamped onto the culture substrate (Fig. 3d,e). Whitesides et al. utilized self-assembled monolayers (SAMs) by printing with alkanethiol which have a variety of reactive functional groups to anchor cell adhesion related molecules, and created cell adhesion islands. There are two main micropatterning methods—printing with extracellular matrix substances that promote cell adhesion (e.g. collagen, fibronectin, laminin) (Scholl et al. 2000; Kaji et al. 2003; Hou et al. 2009), or printing with substances that impede cell adhesion (Yang et al. 2005; Saravia et al. 2007; Ohnuma et al. 2009). As we explain later, we used the latter method (Fig. 3f). Microfabrication techniques like these have enabled us to create cell adhesion patterns with a variety of geometric shapes and investigate the way

Fig. 3. Schematic illustration of micro-contact printing using a PDMS stamp: (a) fabricating a patterned master from a UV-curable resin by using UV-irradiation through a patterned mask, (b) applying silicone resin, (c) curing the resin to produce a patterned stamp, (d) dipping the patterned surface of the stamp in an ink, (e) printing the ink onto a culture dish,

How does the shape of the space in which a cell can move affect its movement? In the case of chemotaxis, the stimulating substance creates a concentration gradient (spatial asymmetry), and the cell moves in line with that concentration gradient (asymmetrical movement). From this, it is easy to suppose that the asymmetry of the space in which a

surface to be stamped to ensure even printing.

that geometric patterns affect cell movement.

and (f) culturing the cells on the dish.

**1.3 Biased movement in asymmetrical micropatterns** 

**1.3.1 Asymmetry of cell shape and movement** 

cell can move imparts a bias on the cell's movement. It is also known that the shape of cells that can move freely on a standard cell culture plate (one that enables cells to adhere and move uniformly) becomes asymmetric. For example, migrating fibroblasts are teardrop-shaped, with their front ends spreading out while the rear ends taper (Fig. 2a); migrating keratocytes, the epithelial cells of the epidermis of fish and frogs are half-moonshaped (Svitkina et al. 1997; Keren et al. 2009) and neurons put out neurites in the direction in which they are moving (Fig. 2b) (Yamasaki et al. 2010). Adherent cells thus show a strong correlation between the direction of migration and the shape of the cell. Creating a cell adhesion island with an asymmetric geometry is accordingly likely to cause both cell shape and cell migration direction to become asymmetric in line with the asymmetry of the island (Fig. 1c,d). However, just as there are positive and negative taxes, spatial asymmetry needs to be actually measured to determine the direction in which it will bias cell movement.

#### **1.3.2 Cells in a teardrop-shaped micropattern**

With respect to the direction of cell movement and geometric pattern of cell adhesion sites, some very interesting research has been carried out on the teardrop shape that fibroblasts assume when moving through an unconfined space. In 2003, Brock et al. reported that cells confined within a polygonal shape tend to form lamellipodia at the corners of the polygon (Brock et al. 2003). Lamellipodia are formed when actin filaments create a mesh and the cell membrane advances, and tend to be formed at the fronts of cells when they are migrating (Fig. 2a). Fibroblasts in an unconfined space tend to advance with the blunt end of the teardrop to the front, but Brock et al.'s observations suggested that cells might also tend to advance towards sharp corners when geometrically confined.

In 2005, Jiang et al. published a very interesting paper on research to elucidate the relationship between cell shape and direction of movement (Jiang et al. 2005). They confined fibroblasts to a teardrop shape, and then used an electric pulse to release them from constraint and observed the direction in which they moved. They found that the fibroblasts moved in the direction of their blunt ends. This result suggested that the shape of the motile cell as a whole was a more important determinant of direction than the ease with which lamellipodia are formed at corners within the geometry. Following this, in 2007, Kumar et al. conducted observations on the direction taken by NIH3T3 fibroblasts in a chain of teardrop-shaped cell adhesion islands (Fig. 1d)(Kumar et al. 2007). They observed the direction of cell movements under various conditions, adjusting the arrangement of the teardrop islands, and the distance separating them, joining them in some cases, and leaving a gap of several micrometres in others, and so forth. They found that the direction of cell movements showed no bias towards either blunt end or sharp end of the teardrop-shaped islands, and that cells chose to move in whichever direction another cell adhesion island lay at the end of the longitudinal axis of each teardropshaped island. This suggested that the cell adhesion sites have a major effect on the direction of cell movement. In 2010, in experiments using epithelial cells, Kushiro et al. used the same teardrop-shaped cell adhesion island geometry as Kumar et al. to investigate how modifying the expression of the gene that controls the formation of lamellipodia affects cell movement (Kushiro et al. 2010). Unlike fibroblasts, epithelial cells moving in an unconfined space actively form broad lamellipodia at the front end of the direction in which they are moving. Kushiro et al.'s findings regarding the direction of cell movement differed markedly from those of Kumar et al. for fibroblasts, but nevertheless showed that the direction of cell movement changes according to the degree of expression of the gene related to lamellipodia formation, and to the arrangement of teardrop-shaped islands and the distance between them.

#### **1.3.3 Cells in ratchet-shaped micropattern**

In 2009, Mahmud et al. investigated the same kind of movements using a slightly different geometry, one that used a ratchet-shaped micropattern (chained triangles connected in a linear manner) (Fig. 1c) rather than teardrop shapes as adhesion islands, and also included physical obstacles (Mahmud et al. 2009). They, too, observed bias in the direction of cell movement, and showed that this bias depends upon the type of cell involved. We explain in more detail later, but our group also created a geometrical pattern in which we combined triangles to form a ratchet shape. When we used this geometry to investigate the direction of movement of neuron-like cells, we observed a bias in movement and also found that the location at which the tips of neurites are formed is critical.

Some of these studies were conducted independently during much the same period. Conclusions that can be drawn from the above research using asymmetric geometrical patterns of about the same size as cells are: (1) many different cell types show bias in the direction of their movements; (2) the direction of cell movement changes according to the shape of the geometrical pattern in which cells can move, type of cell, and gene expression; and (3) bias in the direction of cell movement is related to the formation of lamellipodia and neurites, which are thought to be closely involved in cell movement. As such, while we can use geometrical patterns to bias the direction of cell movement, there is still much that we do not know about bias direction and the mechanisms involved in determining it.

#### **1.3.4 Brownian ratchet theory**

When discussing the bias direction of cell movement, we have not considered stochastic motion resulting from the spontaneous fluctuation of internal state of cell, which is known to be important in cell migrations (Oosawa 2001; Nakaoka et al. 2009). Here, we consider cell migration in an asymmetric geometrical pattern, taking stochastic motion into account. A Brownian particle, which exhibits stochastic motion due to thermal fluctuations, can be caused move directionally in a spatially asymmetric energy barrier under a non-equilibrium condition, as represented by a flashing ratchet (Fig. 4a)(Reimann 2002). In the case of so called rocking ratchet in which an oscillating force is applied to a Brownian particle (Fig. 4b), directional motion of the particle is also observed. This direction is known to be reversed by changes in the amplitude or period of the oscillating force (Bartussek et al. 1994; Reimann 2002). According to an experiment by Mahmud et al. (Mahmud et al. 2009), the transition probability for a cell in spatially asymmetric micropattern is described by a onedimensional Brownian model. So, is there a possibility that the cause of the directional motion of a cell is analogous to that of Brownian ratchets? Furthermore, is a reversal of a cell's direction able to be observed when a signal is oscillating in the presence of an asymmetric geometry as is seen in the rocking ratchet (Fig. 4b)? It is up to future research to

direction in which they are moving. Kushiro et al.'s findings regarding the direction of cell movement differed markedly from those of Kumar et al. for fibroblasts, but nevertheless showed that the direction of cell movement changes according to the degree of expression of the gene related to lamellipodia formation, and to the arrangement of

In 2009, Mahmud et al. investigated the same kind of movements using a slightly different geometry, one that used a ratchet-shaped micropattern (chained triangles connected in a linear manner) (Fig. 1c) rather than teardrop shapes as adhesion islands, and also included physical obstacles (Mahmud et al. 2009). They, too, observed bias in the direction of cell movement, and showed that this bias depends upon the type of cell involved. We explain in more detail later, but our group also created a geometrical pattern in which we combined triangles to form a ratchet shape. When we used this geometry to investigate the direction of movement of neuron-like cells, we observed a bias in movement and also found that the

Some of these studies were conducted independently during much the same period. Conclusions that can be drawn from the above research using asymmetric geometrical patterns of about the same size as cells are: (1) many different cell types show bias in the direction of their movements; (2) the direction of cell movement changes according to the shape of the geometrical pattern in which cells can move, type of cell, and gene expression; and (3) bias in the direction of cell movement is related to the formation of lamellipodia and neurites, which are thought to be closely involved in cell movement. As such, while we can use geometrical patterns to bias the direction of cell movement, there is still much that we do not know about bias direction and the mechanisms involved in

When discussing the bias direction of cell movement, we have not considered stochastic motion resulting from the spontaneous fluctuation of internal state of cell, which is known to be important in cell migrations (Oosawa 2001; Nakaoka et al. 2009). Here, we consider cell migration in an asymmetric geometrical pattern, taking stochastic motion into account. A Brownian particle, which exhibits stochastic motion due to thermal fluctuations, can be caused move directionally in a spatially asymmetric energy barrier under a non-equilibrium condition, as represented by a flashing ratchet (Fig. 4a)(Reimann 2002). In the case of so called rocking ratchet in which an oscillating force is applied to a Brownian particle (Fig. 4b), directional motion of the particle is also observed. This direction is known to be reversed by changes in the amplitude or period of the oscillating force (Bartussek et al. 1994; Reimann 2002). According to an experiment by Mahmud et al. (Mahmud et al. 2009), the transition probability for a cell in spatially asymmetric micropattern is described by a onedimensional Brownian model. So, is there a possibility that the cause of the directional motion of a cell is analogous to that of Brownian ratchets? Furthermore, is a reversal of a cell's direction able to be observed when a signal is oscillating in the presence of an asymmetric geometry as is seen in the rocking ratchet (Fig. 4b)? It is up to future research to

teardrop-shaped islands and the distance between them.

location at which the tips of neurites are formed is critical.

**1.3.3 Cells in ratchet-shaped micropattern** 

determining it.

**1.3.4 Brownian ratchet theory** 

determine whether cell migration is dictated by cell shape and/or by asymmetries in the surrounding space.

Fig. 4. Illustrated scheme of directional motion caused in a flashing ratchet (a) and in a rocking ratchet (b). In a flashing ratchet, a spatially asymmetric energy barrier, which a Brownian particle is subject to, switches in time stochastically. In a rocking ratchet, an oscillating force is applied to a Brownian particle besides the force exerted by a spatially asymmetric energy barrier.

#### **1.4 Extrapolation to cell populations from single-cell migration analysis**

Cells in multicellular organisms or wild environments are not independent entities and inevitably interact with the surrounding cells. Therefore, the effects of cell-to-cell interactions through physical contact, chemical signalling, nutrition competition, etc. must be considered to understand the roles of cellular movement and migration in natural contexts. Even if the movements of individual cells are characterized by simple rules, unexpected collective behaviours may emerge at the cell population level due to cell-tocell interactions.

One of the most understood systems of collective migration is the fruiting-body formation of social amoebae. Upon starvation, thousands of individual cells co-ordinately migrate and aggregate to form fruiting bodies via signalling with cyclic AMP (Goldbeter 1996; Gregor et al. 2010). Fruiting bodies encapsulate spores that can survive severe stress environments for an extended period of time making such collective migration crucial for the survival of the species in harsh environments. Understanding the mechanism of this collective migration requires consideration of the effects of cell-to-cell interactions.

Collective migration also exists in bacteria. Lambert et al. developed a microfluidic device that allows the measurement of the efficiency of chemotactic migration by employing funnel-shaped barriers, and demonstrated that bacteria migrated by chemotaxis beyond the barriers to an area of higher nutrition only when cell density was high (Lambert et al. 2010). Another intriguing study on bacterial migration was reported by Park et al. in which they studied the time-evolution of spatial distributions of bacterial densities in a microfabricated maze (Park et al. 2003a; Park et al. 2003b). Despite the complex topology of the environment, the bacteria migrated and aggregated in a few confined position through chemotaxis towards self-produced signals to create a high cell density. A high cell density is required for the formation of a biofilm, which is resistant to many kinds of stress. A biofilm is a bacterial community-based life-cycle mode that is known to contribute to the virulence of pathogens in bacterial infection (O'Toole et al. 2000; Lewis 2005).

When cells proliferate and divide during migration, another complexity arises. Differences between local environments generally induce different division rates in subpopulations. If subpopulations A and B in different locations have the division rates *k*<sup>A</sup> and *k*B (>*k*A), the difference between the cell densities of the subpopulations grows with exp[(*k*B − *k*A)*t*], where *t* is time. Therefore, different division rates in a cell population can significantly affect the spatial distribution of cell density when the time-scale of observation is comparable to or longer than the mean doubling time of the cell population. This means that changes in spatial distribution cannot be attributed solely to the effect of cell migration. In phenomena such as embryogenesis or cancer metastasis, or during an immune response, cellular proliferation and migration proceed simultaneously. Cellular proliferation and migration are usually studied separately, but their coupling is an important subject for future research.

#### **1.5 Biotechnological advantages of cell migration control by micropatterns**

This kind of cell migration research is of course important from the life science perspective of elucidating the mechanisms behind fundamental cell functions, but it is also important from the perspective of applying the ability to control the direction of cell migration to cellular engineering and medical technology. As mentioned earlier, the migration of individual cells that make up multicellular organisms is a matter of great significance to the development and maintenance of functions of those organisms. The ability to control cell migration could lead to new or improved treatments for developmental disorders, tissue dysfunctions, healing of wounds, cancer metastasis, and so forth. Recently, the relationship between scaffold elasticity, which is closely related to cell migration, and the direction of differentiation has come to be discussed in relation to the induction of stem cell differentiation (Engler et al. 2006). Stem cells are known to be influenced by a huge number of endogenous factors (genes, RNAi, etc.) and exogenous factors such as chemicals and culture environment. The role that cell migration performs in differentiation is likely to attract growing interest.

The control of cell migration also has an important role to play in wound treatment and tissue regeneration (Friedl et al. 2004; Schneider et al. 2010). The migration not only of fibroblasts mentioned above, but also osteoblasts and osteoclasts in bone, and the cells involved in angiogenesis of blood vessels determine the form of those respective tissues, and are an important factor in the expression of the functions of those tissues.

Contributing to this kind of cellular engineering and medical treatment by equipping tissues with asymmetric spaces capable of controlling cell migration is indispensable to the further development of tailor-made treatment and advanced medical technology. Up to now, the focus of attention has been on cell adhesion substances that ensure that cells remain with and maintain the structure of the tissue to which they belong, but moving forward, development in the area of shaping spatial arrangements of those cells is likely to become an increasingly important endeavour.

#### **2. Current studies on cell migration control by our group**

#### **2.1 Introduction**

196 Innovations in Biotechnology

towards self-produced signals to create a high cell density. A high cell density is required for the formation of a biofilm, which is resistant to many kinds of stress. A biofilm is a bacterial community-based life-cycle mode that is known to contribute to the virulence of

When cells proliferate and divide during migration, another complexity arises. Differences between local environments generally induce different division rates in subpopulations. If subpopulations A and B in different locations have the division rates *k*<sup>A</sup> and *k*B (>*k*A), the difference between the cell densities of the subpopulations grows with exp[(*k*B − *k*A)*t*], where *t* is time. Therefore, different division rates in a cell population can significantly affect the spatial distribution of cell density when the time-scale of observation is comparable to or longer than the mean doubling time of the cell population. This means that changes in spatial distribution cannot be attributed solely to the effect of cell migration. In phenomena such as embryogenesis or cancer metastasis, or during an immune response, cellular proliferation and migration proceed simultaneously. Cellular proliferation and migration are usually studied separately, but their coupling is

**1.5 Biotechnological advantages of cell migration control by micropatterns** 

This kind of cell migration research is of course important from the life science perspective of elucidating the mechanisms behind fundamental cell functions, but it is also important from the perspective of applying the ability to control the direction of cell migration to cellular engineering and medical technology. As mentioned earlier, the migration of individual cells that make up multicellular organisms is a matter of great significance to the development and maintenance of functions of those organisms. The ability to control cell migration could lead to new or improved treatments for developmental disorders, tissue dysfunctions, healing of wounds, cancer metastasis, and so forth. Recently, the relationship between scaffold elasticity, which is closely related to cell migration, and the direction of differentiation has come to be discussed in relation to the induction of stem cell differentiation (Engler et al. 2006). Stem cells are known to be influenced by a huge number of endogenous factors (genes, RNAi, etc.) and exogenous factors such as chemicals and culture environment. The role that cell migration performs

The control of cell migration also has an important role to play in wound treatment and tissue regeneration (Friedl et al. 2004; Schneider et al. 2010). The migration not only of fibroblasts mentioned above, but also osteoblasts and osteoclasts in bone, and the cells involved in angiogenesis of blood vessels determine the form of those respective tissues,

Contributing to this kind of cellular engineering and medical treatment by equipping tissues with asymmetric spaces capable of controlling cell migration is indispensable to the further development of tailor-made treatment and advanced medical technology. Up to now, the focus of attention has been on cell adhesion substances that ensure that cells remain with and maintain the structure of the tissue to which they belong, but moving forward, development in the area of shaping spatial arrangements of those cells is likely to become an

and are an important factor in the expression of the functions of those tissues.

pathogens in bacterial infection (O'Toole et al. 2000; Lewis 2005).

an important subject for future research.

in differentiation is likely to attract growing interest.

increasingly important endeavour.

To determine the mechanisms of cell migration within cell-sized geometry, we focused on optically tracing two different types of cells that move in completely different manners. We used the rat adrenal pheochromocytoma cell line, PC12, and the fibroblast cell line, NIH3T3. Upon the addition of nerve growth factor, PC12 cells differentiate into sympathetic neuronlike cells with long, extended neurites (Greene et al. 1982; Ohnuma et al. 2006). Although PC12 cells migrate slowly, they are useful for the study of migration via long neurites. NIH3T3 cells are able to move very fast on glass slides and are frequently used as model cells in cell migration studies (Kumar et al. 2007).

To optically trace the migration of these cells in cell-sized geometry over a long period of time and to analyse the dynamics of cell–substrate contact sites (neurite tips for PC12 cells and focal adhesions for fibroblasts), which work both as input sensors for information regarding the local environment and as action sites for locomotion, we employed two different technologies. The first was a micro-contact printing technique using PDMS as the material for both the stamps and also the cell-repellent ink. We enable to keep the cells inside the PDMS micro-chamber for over 18 days (Ohnuma et al. 2009). The second technology was reflection interference contrast microscopy (RICM) (Curtis 1964). Using RICM, the distance between the glass and the cell surface membrane can be visualized as high-contrast images, which are images of the interference between the reflection of light off the glass–medium interface and off the medium–cell interface.

Analysis of the data showed that both PC12 and NIH3T3 exhibited biased migration in asymmetric micropatterns and that migration is likely to proceed from the sharp end of one micropattern unit to the blunt end of the adjacent micropattern unit. The contact sites of each migrating cell on the glass surface, however, expanded in both directions. The mechanism behind biased cell migration has still not been uncovered, but the current experimental setup will give us useful data for the control of cell migration.

#### **2.2 PC12 migration control in a ratchet-shaped micropattern**

#### **2.2.1 Neuronal cell migration**

Long-distance and directional migration of neuronal cells is a critical step in the developing and regenerating nervous system. Some neuronal cells migrate several millimetres to their final destinations. For example, inhibitory neurons originate in the ganglionic eminences, migrate radially to the cortex, and migrate parallel in the cortex surface to their destinations (Marín and Rubenstein 2001). The cellular mechanisms underlying these directional migratory activities have been extensively studied from the basis of chemotaxis (Ayala et al. 2007; Zheng and Poo 2007); however, chemokine gradients decrease with distance and determination of destination by each neuronal cell likely involves chemokine crosstalk. It was found that scaffolds, such as radial glia and blood vessels, provide routes that guide migrating neurons to their destinations (Rakic 1972; Gasser and Hatten 1990; Bovetti et al. 2007). These scaffolds act as "rails" to produce error-free, long-distance migration. However, physical and chemical circumstances, such as connective tissue, prevent cells from freely migrating towards their destination. Cells not only mechanically sense the local geometry, but they also integrate this mechanical information into their migration (Ingber 2003). The relationship between geometry and cell migration has not been well studied.

#### **2.2.2 Experimental set-up**

To investigate the relationship between neuronal cells' local geometry and their migration, and, thus, uncover a potential control methodology, we performed a simple *in vitro* experiment (Ohnuma et al. 2009). We focused on periodic structures, which are abundant *in vivo*, by fabricating a ratchet-wheel shaped (gear-type) micropattern, which consists of a series of connected triangles. The micropattern was made by printing a PMDS film onto a collagen-coated culture dish (Fig. 3). Our working hypothesis was that neuronal cells would be able to migrate directionally on a periodic scaffold structure if the periodic unit was asymmetric. We tested the hypothesis using cultured PC12 cells that were attached only to the collagen-coated area between the core and the ratchet-shaped outer frame of the microchamber. Because the chamber was designed so that cell migration in the radial direction was restricted and the gap between the teeth and the core was equivalent to the size of a cell body, the cells migrated almost one-dimensionally in the tangential direction. We made both a left (L) and right (R) micropattern (Fig. 5a). The L and R chambers were linesymmetrical to one another and arranged alternately to serve as control chambers for one another. The core diameter, tooth depth, and gap between the teeth and core were approximately 100 µm, 40 µm, and 30 µm, respectively. PC12 cells were plated in neurite outgrowth medium including nerve growth factor in which the cells gradually extend long neurites over approximately 10 days (Greene et al. 1982; Ohnuma et al. 2006). It was previously reported that neuronal cells migrate following neurite extension (Hatten 2002), so it was expected that cell migration in the chambers would differ between cells with short neurites and cells with long neurites (Fig. 5b). Therefore, time-lapse micrographs of the cells in the micropattern were acquired twice: the first acquisition was for 70 hours starting from 1 day after plating, when the cells usually have short neurites; and the second acquisition was for 70 hours starting from 12 days after plating, when the cells usually have long neurites. We defined cell migration in the direction in which the ratchet teeth were tapered as positive migration (Fig. 5c).

#### **2.2.3 Results and discussion**

Using the PDMS printed L and R micropatterns, we found the same biased migration in both types of chambers. The PC12 cells in the L and R micropatterns migrated the same distance in a positive direction in both the first and second micrograph acquisition periods. These results suggested that the direction of migration is biased by chamber geometry, and supports our working hypothesis that the periodic nature of the asymmetrical scaffold determines migration direction. Next, we analysed the position of the cell body and the neurite tips because cell migration was strongly related to neurite formation. Although the mean length of the longest neurite was independent of microchamber geometry, the timecourse trace of the neurite tips showed that they remained around the tips of the ratchet teeth. We also found that as cells migrated in a positive direction they tended to extend their neurites about one tooth ahead of the cell body and place the neurite tip at the tip of the tooth. The cell body then passed by the neurite tip as it migrated (Fig. 5c). It appears that PC12 cells use the neurite tips as a hook to "climb" the ratchet-shaped geometry. One

but they also integrate this mechanical information into their migration (Ingber 2003). The

To investigate the relationship between neuronal cells' local geometry and their migration, and, thus, uncover a potential control methodology, we performed a simple *in vitro* experiment (Ohnuma et al. 2009). We focused on periodic structures, which are abundant *in vivo*, by fabricating a ratchet-wheel shaped (gear-type) micropattern, which consists of a series of connected triangles. The micropattern was made by printing a PMDS film onto a collagen-coated culture dish (Fig. 3). Our working hypothesis was that neuronal cells would be able to migrate directionally on a periodic scaffold structure if the periodic unit was asymmetric. We tested the hypothesis using cultured PC12 cells that were attached only to the collagen-coated area between the core and the ratchet-shaped outer frame of the microchamber. Because the chamber was designed so that cell migration in the radial direction was restricted and the gap between the teeth and the core was equivalent to the size of a cell body, the cells migrated almost one-dimensionally in the tangential direction. We made both a left (L) and right (R) micropattern (Fig. 5a). The L and R chambers were linesymmetrical to one another and arranged alternately to serve as control chambers for one another. The core diameter, tooth depth, and gap between the teeth and core were approximately 100 µm, 40 µm, and 30 µm, respectively. PC12 cells were plated in neurite outgrowth medium including nerve growth factor in which the cells gradually extend long neurites over approximately 10 days (Greene et al. 1982; Ohnuma et al. 2006). It was previously reported that neuronal cells migrate following neurite extension (Hatten 2002), so it was expected that cell migration in the chambers would differ between cells with short neurites and cells with long neurites (Fig. 5b). Therefore, time-lapse micrographs of the cells in the micropattern were acquired twice: the first acquisition was for 70 hours starting from 1 day after plating, when the cells usually have short neurites; and the second acquisition was for 70 hours starting from 12 days after plating, when the cells usually have long neurites. We defined cell migration in the direction in which the ratchet teeth were tapered

Using the PDMS printed L and R micropatterns, we found the same biased migration in both types of chambers. The PC12 cells in the L and R micropatterns migrated the same distance in a positive direction in both the first and second micrograph acquisition periods. These results suggested that the direction of migration is biased by chamber geometry, and supports our working hypothesis that the periodic nature of the asymmetrical scaffold determines migration direction. Next, we analysed the position of the cell body and the neurite tips because cell migration was strongly related to neurite formation. Although the mean length of the longest neurite was independent of microchamber geometry, the timecourse trace of the neurite tips showed that they remained around the tips of the ratchet teeth. We also found that as cells migrated in a positive direction they tended to extend their neurites about one tooth ahead of the cell body and place the neurite tip at the tip of the tooth. The cell body then passed by the neurite tip as it migrated (Fig. 5c). It appears that PC12 cells use the neurite tips as a hook to "climb" the ratchet-shaped geometry. One

relationship between geometry and cell migration has not been well studied.

**2.2.2 Experimental set-up** 

as positive migration (Fig. 5c).

**2.2.3 Results and discussion** 

possible explanation for these results is that since the positively directed neurite bends along the tooth edge while the negatively directed neurite extends in a straight line, the tangential component of the maximal tension of the positive-direction neurite is higher than that of the negative-direction neurite. This results in the probability of continuous forward migration being higher than that of backward migration. Alternatively, the results can be described as thermally fluctuating spring-beads in a rocking ratchet (see 1.3.4).

Fig. 5. Schematic illustration (a) and phase contrast microscopy image (b) of a PC12 cell extending its neurites in a gear-type micropattern. (c) The PC12 cell migrates in a positive direction (arrow).

#### **2.3 Imaging of focal adhesions of NIH3T3 fibroblasts in a teardrop-type microchamber**

#### **2.3.1 Focal adhesions in cell migration**

As previously mentioned, focal adhesions, which are membrane protein complexes, act as "input–output adaptors". Focal adhesions interact with substrates such as the extracellular matrix to allow the cell to adhere, migrate, and acquire extracellular information. Integrin is a major component of focal adhesions. Integrin is a family of trans-membrane proteins that connect physicochemically between the extracellular matrix and the cytoskeleton proteins inside of the cell to anchor cells and to transmit signals from extra-cell to intra-cell and vice versa. Outside-in signals, which are transmitted from outside to inside the cell, activate the adherent affinity of the complex, especially integrin. Inside-out signals via internal signal transduction proteins also activate integrin. Adherent affinity is regulated by both quantitative (density of the complex and ligand on the substrate) and qualitative properties (attractive interaction of the complex and the substrate). Mature focal complexes that have strongly adhered to a substrate are called focal adhesions.

Cell migration is the integrated dynamics of the cytoskeleton and focal adhesions, but how do cells actually migrate using this dynamics? The following is an accepted mechanism for the movement of adherent cells such as fibroblasts on substrates *in vitro*:

STEP 1: Polarization of cell. The shape of the cell changes from spherical or hemispherical to become anisotropic. Cdc42, PIP3, integrin, and microtubules are important in changing the shape of the cell. The cell membrane at the anterior part of the cell, the leading edge, starts to extend and produce actin filaments called filopodia.

STEP 2: Generation of force driving locomotion. Actin filaments are richly synthesized at the leading edge, and filopodia and lamellipodia are formed in association with the activation of Rac1, generating the force to extend the leading edge. The direction of the filopodia and lamellipodia is determined by the Rho protein family and/or the actinbinding protein family.

STEP 3: Fixing the leading edge to the substrate. At the leading edge, focal complexes are formed by the activation of integrin, which is stimulated by the inside-out and outside-in signals from both the cytoskeleton and the substrate. Focal complexes are also led by Rac1 and Cdc42 activity. The focal complexes mature to become focal adhesions at the leading edge. RhoA, which is activated at the posterior side of cell, is also involved in the formation of focal adhesions, so focal adhesions are also formed at the posterior side of the cell.

STEP 4: Diving cell body by generation of tension. Focal adhesions are the contact sites of the cytoskeleton and substrate outside of the cell. This becomes a point of tension through the cytoskeleton. The focal adhesions formed at the anterior and posterior sides are linked by the cytoskeleton as stress fibres force the cell body to shrink. The tension is regulated by actin motor proteins, myosin, and Rho-kinases (Smith et al. 2008).

STEP 5: Decomposition of the focal adhesions at the posterior side. Although the cell can decompose the focal adhesions at either the anterior or posterior side via RhoA, focal adhesion kinase, Src, or microtubules, the cell selects the focal adhesions at the posterior side for decomposition. This results in the posterior side of the cell shrinking due to the tension created by stress fibres in the cytoskeleton.

On the basis of these mechanisms, the physicochemistry of the migrating cell's surroundings potentially affects the migration direction. This aspect of cell migration should be investigated through the dynamics of the focal adhesions formed at both the anterior and posterior sides of the cell.

#### **2.3.2 Imaging of focal adhesions of NIH3T3 fibroblasts using RICM**

RICM can be used to observe focal adhesions. In the 1970s, electron microscopy revealed that the gap between cells and substrates was less than 30 nm (Abercrombie et al. 1971; Revel and Wolken 1973), however, the cells that were observed were fixed (not living) and the observation chamber was under a vacuum. Therefore, the development of optical microscopy for the observation of the focal adhesions of living cells was considered to be the next step. The principles of RICM were established by Curtis in 1964, (Curtis 1964) who regarded the medium between the glass and cell as a thin film, which allowed the observation of the distance between the glass and the cell surface as a high-contrast

Cell migration is the integrated dynamics of the cytoskeleton and focal adhesions, but how do cells actually migrate using this dynamics? The following is an accepted mechanism for

STEP 1: Polarization of cell. The shape of the cell changes from spherical or hemispherical to become anisotropic. Cdc42, PIP3, integrin, and microtubules are important in changing the shape of the cell. The cell membrane at the anterior part of the cell, the leading edge, starts to

STEP 2: Generation of force driving locomotion. Actin filaments are richly synthesized at the leading edge, and filopodia and lamellipodia are formed in association with the activation of Rac1, generating the force to extend the leading edge. The direction of the filopodia and lamellipodia is determined by the Rho protein family and/or the actin-

STEP 3: Fixing the leading edge to the substrate. At the leading edge, focal complexes are formed by the activation of integrin, which is stimulated by the inside-out and outside-in signals from both the cytoskeleton and the substrate. Focal complexes are also led by Rac1 and Cdc42 activity. The focal complexes mature to become focal adhesions at the leading edge. RhoA, which is activated at the posterior side of cell, is also involved in the formation of focal adhesions, so focal adhesions are also formed at the posterior side of

STEP 4: Diving cell body by generation of tension. Focal adhesions are the contact sites of the cytoskeleton and substrate outside of the cell. This becomes a point of tension through the cytoskeleton. The focal adhesions formed at the anterior and posterior sides are linked by the cytoskeleton as stress fibres force the cell body to shrink. The tension is regulated by

STEP 5: Decomposition of the focal adhesions at the posterior side. Although the cell can decompose the focal adhesions at either the anterior or posterior side via RhoA, focal adhesion kinase, Src, or microtubules, the cell selects the focal adhesions at the posterior side for decomposition. This results in the posterior side of the cell shrinking due to the

On the basis of these mechanisms, the physicochemistry of the migrating cell's surroundings potentially affects the migration direction. This aspect of cell migration should be investigated through the dynamics of the focal adhesions formed at both the anterior and

RICM can be used to observe focal adhesions. In the 1970s, electron microscopy revealed that the gap between cells and substrates was less than 30 nm (Abercrombie et al. 1971; Revel and Wolken 1973), however, the cells that were observed were fixed (not living) and the observation chamber was under a vacuum. Therefore, the development of optical microscopy for the observation of the focal adhesions of living cells was considered to be the next step. The principles of RICM were established by Curtis in 1964, (Curtis 1964) who regarded the medium between the glass and cell as a thin film, which allowed the observation of the distance between the glass and the cell surface as a high-contrast

actin motor proteins, myosin, and Rho-kinases (Smith et al. 2008).

**2.3.2 Imaging of focal adhesions of NIH3T3 fibroblasts using RICM** 

tension created by stress fibres in the cytoskeleton.

the movement of adherent cells such as fibroblasts on substrates *in vitro*:

extend and produce actin filaments called filopodia.

binding protein family.

posterior sides of the cell.

the cell.

images through the interference of light reflected from glass–medium and medium–cell membrane interfaces. The thickness of the thin medium "layer" is evaluated using the reflective index of the medium, cell membrane, and glass, and the wavenumber and angle of incident light (Bereiter-Hahn et al. 1979; Simson et al. 1998). Izzard and Lochner reported that the nearest distance between cell membranes and substrates is approximately 10 nm, which is shown as dark areas in RICM images. These are focal adhesions (or focal contacts) (Izzard and Lochner 1976). Sackmann et al. and others significantly developed RICM using a model cell membrane that is composed of giant vesicles bearing membrane protein or that has been modified with polymers (Bruinsma et al. 2000; Smith et al. 2008; Limozin and Sengupta 2009; Streicher et al. 2009). RICM has drawn much attention as a non-probing microscopy for observing focal adhesions (Yin et al. 2003; Sengupta et al. 2006; Théry et al. 2006).

Let us briefly summarize the principles of RICM (Fig. 6a). The intensities of the incident light, the light reflected from the interface of the medium and the surface of the glass substrate, and the light reflected from the interface of the medium and the cell membrane are depicted by *I*0, *I*01, and *I*12, respectively. The intensity profile *I(x)* of the interference between *I*01 and *I*12 is obtained by

$$I(\mathbf{x}) = I\_{01} + I\_{02} + 2\left(I\_{01}I\_{02}\right)^{1/2}\left[2kh(\mathbf{x})\cos\theta + \delta\right] \tag{1}$$

where *k* is the wavenumber of light the phase of which is shifted with *δ,* and *h(x)* is the distance of the cell membrane from the glass surface. Using the Fresnel equation with the reflection amplitude coefficients of each interface (*r*01, *r*12), *I*01 and *I*12 are substituted as follows: *I*01 = *r*012 *I*0 and *I*12 = (1-*r*012)*r*122*I*0. Therefore, the maximum (*I*max) and minimum intensity (*I*min) of the interference are obtained from *I*max = *I*01+*I*12+2(*I*01*I*12)1/2 and *I*min = *I*01+*I*12- 2(*I*01*I*12)1/2, which allows the deduction of the following equation (*n*: refractive index):

$$h(\mathbf{x}) = \frac{\mathcal{A}}{4\pi n} \left[ \arccos\left\{ \frac{2I(\mathbf{x}) - (I\_{\text{max}} + I\_{\text{min}})}{I\_{\text{max}} - I\_{\text{min}}} \right\} + \mathcal{S} \right] \tag{2}$$

When evaluating *h(x)* of a living cell, the light distribution function should be included. The RICM pattern of the cell gives us the height of the cell membrane and the focal adhesions are the darkest areas with *m* = 0 in the following equation (λ: wavelength of light):

$$2\operatorname{i}2kh\left(\mathbf{x}\right)\cos\theta+\mathcal{S}=\mathcal{A}m\,.\tag{3}$$

#### **2.3.3 Experimental set-up**

Since RICM requires a glass substrate surface, which is weakly cell-adherent, the high cell repellency of the micropattern becomes necessary. In order to obtain images of focal adhesions of cells migrating within micropatterns, we again adopted the micro-contact printing technique to construct a PDMS micropattern consisting of a series of connected teardrop shapes (Fig. 1d) (Kumar et al. 2007). The width of the neck between two teardrop shapes was about 6 µm, which was not notably larger than that of the actual design (5 µm). This convinces us that the current technique for constructing the micropattern worked well.

RICM was conducted with a halogen lamp with a 530 to 550 nm optical band-pass filter as the light source, two polarizing filters, and an objective lens with a quarter-wave plate. The microscope set-up was combined with a culture chamber managed by a temperature and humidity control box, and the cells were kept alive for several days in the chambers (Fig. 6b). This RICM set-up enables observation of the cells and the edges of micropatterns (Fig. 6d).

Fig. 6. (a) Schematic illustration of reflection interference contrast microscopy. (b) RICM setup with a cell culturing system. (c,d) RICM images of NIH3T3 cultured on glass (c) or within the micropatterns (white dashed line) (d). Darkest areas in the images of NIH3T3 cell correspond to areas in most contact with the glass surface, i.e. focal adhesion.

#### **2.3.4 Results and discussion**

To validate the RICM set-up, we used a latex bead suspension as described (Rädler and Sackmann 1992; Rädler and Sackmann 1993; Kühner and Sackmann 1996; Heinrich et al. 2008). In brief, latex beads were suspended in 200 mM NaCl solution and then observed with the RICM set-up. Using equation (2), the heights of the beads from the glass surface were estimated to be about 10 nm. According to the Derjaguin–Landau–Verwey–Overbeek theory, the height of latex beads in a high ionic-strength suspension is several nanometers. Therefore, we deemed the performance of the RICM set-up to be sufficiently accurate. Immunofluorescence staining revealed that the dark spots in the RICM images were also areas of the cell that contained focal adhesion protein complex (Geiger 1979; Smilenov et al. 1999), suggesting that our RICM set-up was able to accurately visualize focal adhesions.

In RICM images, focal adhesions are dark and lamellipodia at the edge of cells are bright (Fig. 6c). Although the lamellipodia were extended onto the PDMS micropattern, they did not form focal adhesions, suggesting that the cells were restricted to the teardrop-shaped island in the PDMS micropattern. Formation and degradation of focal adhesions at the front and rear of migrating NIH3T3 fibroblasts in the teardrop-type micropattern (Fig. 1d) were clearly seen with our RICM set-up. Anticlockwise biased-migration of NIH3T3 cells in the teardrop-type micropattern was also observed (Figs. 1d, 6d), which was consistent with results previously reported (Kumar et al. 2007). The focal adhesions of each migrating cell in the micropattern, however, expanded both in clockwise and anticlockwise directions. The mechanism behind biased cell migration has still not been uncovered, but the PMDS micropattern and RICM set-up will give us useful data for the control of cell migration.

### **3. Conclusion**

202 Innovations in Biotechnology

RICM was conducted with a halogen lamp with a 530 to 550 nm optical band-pass filter as the light source, two polarizing filters, and an objective lens with a quarter-wave plate. The microscope set-up was combined with a culture chamber managed by a temperature and humidity control box, and the cells were kept alive for several days in the chambers (Fig. 6b). This RICM set-up enables observation of the cells and the edges of micropatterns (Fig. 6d).

Fig. 6. (a) Schematic illustration of reflection interference contrast microscopy. (b) RICM setup with a cell culturing system. (c,d) RICM images of NIH3T3 cultured on glass (c) or within the micropatterns (white dashed line) (d). Darkest areas in the images of NIH3T3 cell

To validate the RICM set-up, we used a latex bead suspension as described (Rädler and Sackmann 1992; Rädler and Sackmann 1993; Kühner and Sackmann 1996; Heinrich et al. 2008). In brief, latex beads were suspended in 200 mM NaCl solution and then observed with the RICM set-up. Using equation (2), the heights of the beads from the glass surface were estimated to be about 10 nm. According to the Derjaguin–Landau–Verwey–Overbeek theory, the height of latex beads in a high ionic-strength suspension is several nanometers. Therefore, we deemed the performance of the RICM set-up to be sufficiently accurate. Immunofluorescence staining revealed that the dark spots in the RICM images were also areas of the cell that contained focal adhesion protein complex (Geiger 1979; Smilenov et al. 1999), suggesting that our RICM set-up was able to accurately visualize focal adhesions.

correspond to areas in most contact with the glass surface, i.e. focal adhesion.

**2.3.4 Results and discussion** 

We have described an *in vitro* experimental model of cell migration guided by mechanical information of the local geometry. The fact that not only fibroblasts NIH3T3 but also neuronal PC12 cells, robustly exhibited biased movement within the micropatterns is indeed a surprise. Biased movement from the blunt end to the sharp end of the micropatterns resembles colloidal motion in a ratchet pattern. Further progress in the RICM imaging of focal adhesions will no doubt reveal the precise mechanism of cell migration and control within micropatterns.

Results from our group's study are expected to contribute the science of cell migration and the understanding of multi-cellular organisms. For example, the question of whether cell migration results from probabilistic (Brownian) or deterministic components of factors internal or external to cells will be solved when the current set-up is combined with fluorescent microscopy and protein-specific probes. The resultant knowledge on cell migration may also stimulate the research field of soft micromachines which can involve sensory motor coupling (Borckmans et al. 2009; Toyota et al. 2009; Masubuchi et al. 2011). At present, there is no evidence that scaffolds with asymmetrical surface structures exist *in vivo*. However, both repetitive structures, including the somite and the cortical layer, and asymmetric protein distributions are abundant *in vivo*. These studies offer new insights into the migration of cells controlled by mechanical stimulation and suggest strategies for designing artificial scaffolds that direct cell migration.

#### **4. Acknowledgments**

Mr Tomohiro Nakanishi (Chiba University), Prof. Masanori Fujinami (Chiba University) and Prof. Makoto Asashima (The University of Tokyo, National Institute of Advanced Industrial Science and Technology) are acknowledged for the discussion on the mechanism of cell migration, the micropattern manufacturing, and the optical setups. TT was financially supported by the Izumi Science and Technology Foundation and by a Grant-in-Aid for Scientific Research (Young Scientist B) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan. KO was financially supported by the Program to Disseminate Tenure Tracking System from the Japan Science and Technology Agency. TT and KO were financially supported by a Grant-in-Aid for Scientific Research on Priority Areas "System cell engineering by multi-scale manipulation" (20034015) from MEXT, Japan.

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