**5. Pharmacokinetics and biodistribution**

Various approaches have been undertaken to overcome the interaction of vectors with blood components to avoid aggregation as well as embolisms. Moreover for most strategies, the phagocytic clearing system of the organism must be eluded. Pharmacokinetic analysis has

Modular Multifunctional Protein Vectors for Gene Therapy 607

modular protein vectors were made by Wu and colleagues. By injecting intravenously the asialoorosomucoid glycoprotein-polylysine vector (ASOR-PL, see Table 1), they targeted hepatocytes and were able to partially and temporary correct analbuminemia and hypercholesterolemia by overexpressing human serum albumin or functional LDL receptor respectively (Wilson *et al.* 1992; Wu *et al.* 1991; Wu and Wu 1987). In another study, neuroprotection from an acute brain injury was achieved after direct intracerebral injection of the NLSCt vector overexpressing the antioxidant enzyme Cu/ZnSOD (see Table 1). In this experimental setting the overexpression of the therapeutic protein could not only induce reduced infarct volume but also functional improvement of the animals (Peluffo *et al.* 2006). Here, the vector was injected directly into the lesioned brain by a tightly controlled microinjector, using a similar protocol reported for the injection of cells into the human Parkinsonian brain (Brundin *et al.*), or for the intracerebral injection of adeno-associated viral vectors for the treatment of infantile lysosomal storage disease (Worgall *et al.* 2008). Thus, this direct intracerebral injection approach could show some benefits in clinical cases of focal traumatic or ischemic injuries, were a delimited area is lesioned and were in some cases even a decompressing craniectomy is needed leaving a direct entrance to the brain parenchyma. Modular protein vectors have also been used for neuroprotection after acute peripheral nerve transection. For example, Barati and colleagues (Barati *et al.* 2006) delivered a polylysine-based polyplex targeting p75NTR positive cells accomplishing the plasmid encoding for GDNF after a peripheral nerve transection (see Table 1). They showed an almost complete reversal in neuronal death caused by GDNF transgene expression. Though this is a very interesting study, the authors performed a subtle pre-lesion to the nerve one week before the nerve transection injury to upregulate p75NTR receptor, and thus the same experiment should be repeated but under more clinically relevant conditions. In another preclinical setting, the intravenous injection (once a day during 3 consecutive days) of the RVG-9R vector accomplished to the antiviral siRNA siFvEJ (see Table 1) was able to induce 80% survival of animals 30 days after their inoculation with a fatal flavivirus (Kumar *et al.* 2007). Thought many modular vectors have been shown to mediate over-expression or down-regulation of reporter genes, they need to be tested *in vivo* in clinically relevant

More complex modular protein vectors including all the important domains for efficient nucleic acid delivery need to be engineered. They should include domains for DNA attachment and condensation, cell attachment and endocytosis, endosomal escape, cytosol trafficking towards the nucleus, nuclear import, and DNA release. HNRK (Domingo-Espín *et al.* 2011), fkAbp75-ipr (Berhanu and Rush 2008), and the fusogenic-karyophilic-NT-polyplex (Navarro-Quiroga *et al.* 2002) (see Table 1) are three prototypes of this increasingly complex vectors, but additional domains have to be inserted. In addition, an interesting strategy could be the exploitation of several domains that have dual functions, like poly-his with DNA attachment and endosomal escape properties, like melittin with endosomal escape properties and nuclear import potential, or histones with DNA attachment and nuclear import potential. Considering the fact that for example several cellular nuclear proteins have several nuclear localization domains, the introduction of several domains with the same function in the same vector may further increase their efficiency. In fact, dual targeting of cancer cell lines using both transferrin and RGD domains showed synergistic effects (Nie *et al.*). Furthermore, the

models for the establishment of their real potential.

**7. Conclusion** 

shown that physicochemical properties of the vectors such as molecular weight, electrical charge and immunogenicity (or pre-existing antibodies in the organism) are important determinants for the *in vivo* success of the treatment. In addition, the volume and shape of the final vector is also important as it determines if the complex will be internalized into the cell and the cell nucleus. P32plasmid DNA is rapidly eliminated from the circulation after intra-venous injection in mice (Kawabata *et al.* 1995), mainly by a scavenger receptor mechanism-mediated uptake by hepatic phagocytes (Kawabata *et al.* 1995; Takakura *et al.* 1999). Thus, the *in vivo* plasmid delivery needs to modify its physicochemical properties by condensing carriers. One interesting example is the Mannose-Poly-Lysine vector (Man-PL), which was designed to accommodate plasmid DNA for the mannose receptor-mediated transfection of liver endothelial cells and Kuppfer phagocytes. After intravenous injection, the Man-PL-P32DNA vector disappeared from the plasma with a half-life of 1 minute, being 80% of the radioactivity recovered from the liver at 10 minutes (mainly in the mannose receptor+ target cells) and less than 1% at lungs or kidneys at 1 hour after (Nishikawa *et al.* 2000a). This vector had a size of 220nm and a zeta potential of 12,1mV, while the DNA alone had a size of 200nm and a zeta potential of -36,4. This type of study clearly shows the importance of the condensation of plasmid DNA into small less charged particles. In accordance, it has been described that positively charged DNA complexes can activate the alternative complement pathway (Plank *et al.* 1996). The conjugation of vectors with hydrophilic polymers has been shown to decrease their interaction with plasmatic proteins and blood cells, increasing their half-life in circulation. One of the most used polymers is poly (ethylene glycol) (PEG). In an interesting study, Ogris and colleagues compared the blood stability of DNA/transferrin/PEI vectors with or without covalently linked PEG. The non-PEGylated vectors aggregated in plasma, bound several plasmatic proteins like IgM, fibrinogen, fibronectin, and complement C3, and also induced erythrocyte aggregation (Ogris *et al.* 1999). Interestingly, the PEGylated vector showed stable complex size, reduced surface charge, reduced binding of plasmatic proteins and erythrocyte aggregation, and most important, increased *in vivo* circulation half-life combined with enhanced transfection selectivity towards tumours.

An interesting vector for improving pharmacokinetics could be the use of natural circulating molecules, like the low-density lipoprotein (LDL). In fact it has been shown that LDL can act as a vector when mixed with plasmid DNA and injected intravenously, reaching several organs including the brain, heart, kidneys and spleen (Guevara *et al.*). The LDL molecule is composed of a highly hydrophobic core, surrounded by a shell of phospholipids and unesterified cholesterol, as well as a single copy of Apo B100 protein (Segrest *et al.* 2001). The B100 protein contains several motifs that explain the vector profile of the LDL: i) a motif that enable nucleic acid binding, ii) a motif that mediate cellular uptake, and iii) a motif that is apparently involved in transferring DNA into the cell nucleus (Guevara *et al.*). Interestingly, low-density particles composed of lipid, Apo B100, RNA, and core protein of hepatitis C virus were reported in the plasmas of individuals infected with this virus (Andre *et al.* 2002). This and other studies suggested that virus might utilize the potential of the LDL particle to act as a vector as a mechanism for persistent chronic infection.

#### **6. Preclinical studies**

Many interesting preclinical studies have been performed with modular multifunctional protein vectors (see Table 1). The first studies showing *in vivo* functional effects using

shown that physicochemical properties of the vectors such as molecular weight, electrical charge and immunogenicity (or pre-existing antibodies in the organism) are important determinants for the *in vivo* success of the treatment. In addition, the volume and shape of the final vector is also important as it determines if the complex will be internalized into the cell and the cell nucleus. P32plasmid DNA is rapidly eliminated from the circulation after intra-venous injection in mice (Kawabata *et al.* 1995), mainly by a scavenger receptor mechanism-mediated uptake by hepatic phagocytes (Kawabata *et al.* 1995; Takakura *et al.* 1999). Thus, the *in vivo* plasmid delivery needs to modify its physicochemical properties by condensing carriers. One interesting example is the Mannose-Poly-Lysine vector (Man-PL), which was designed to accommodate plasmid DNA for the mannose receptor-mediated transfection of liver endothelial cells and Kuppfer phagocytes. After intravenous injection, the Man-PL-P32DNA vector disappeared from the plasma with a half-life of 1 minute, being 80% of the radioactivity recovered from the liver at 10 minutes (mainly in the mannose receptor+ target cells) and less than 1% at lungs or kidneys at 1 hour after (Nishikawa *et al.* 2000a). This vector had a size of 220nm and a zeta potential of 12,1mV, while the DNA alone had a size of 200nm and a zeta potential of -36,4. This type of study clearly shows the importance of the condensation of plasmid DNA into small less charged particles. In accordance, it has been described that positively charged DNA complexes can activate the alternative complement pathway (Plank *et al.* 1996). The conjugation of vectors with hydrophilic polymers has been shown to decrease their interaction with plasmatic proteins and blood cells, increasing their half-life in circulation. One of the most used polymers is poly (ethylene glycol) (PEG). In an interesting study, Ogris and colleagues compared the blood stability of DNA/transferrin/PEI vectors with or without covalently linked PEG. The non-PEGylated vectors aggregated in plasma, bound several plasmatic proteins like IgM, fibrinogen, fibronectin, and complement C3, and also induced erythrocyte aggregation (Ogris *et al.* 1999). Interestingly, the PEGylated vector showed stable complex size, reduced surface charge, reduced binding of plasmatic proteins and erythrocyte aggregation, and most important, increased *in vivo* circulation half-life combined with enhanced transfection

An interesting vector for improving pharmacokinetics could be the use of natural circulating molecules, like the low-density lipoprotein (LDL). In fact it has been shown that LDL can act as a vector when mixed with plasmid DNA and injected intravenously, reaching several organs including the brain, heart, kidneys and spleen (Guevara *et al.*). The LDL molecule is composed of a highly hydrophobic core, surrounded by a shell of phospholipids and unesterified cholesterol, as well as a single copy of Apo B100 protein (Segrest *et al.* 2001). The B100 protein contains several motifs that explain the vector profile of the LDL: i) a motif that enable nucleic acid binding, ii) a motif that mediate cellular uptake, and iii) a motif that is apparently involved in transferring DNA into the cell nucleus (Guevara *et al.*). Interestingly, low-density particles composed of lipid, Apo B100, RNA, and core protein of hepatitis C virus were reported in the plasmas of individuals infected with this virus (Andre *et al.* 2002). This and other studies suggested that virus might utilize the potential of the LDL

Many interesting preclinical studies have been performed with modular multifunctional protein vectors (see Table 1). The first studies showing *in vivo* functional effects using

particle to act as a vector as a mechanism for persistent chronic infection.

selectivity towards tumours.

**6. Preclinical studies** 

modular protein vectors were made by Wu and colleagues. By injecting intravenously the asialoorosomucoid glycoprotein-polylysine vector (ASOR-PL, see Table 1), they targeted hepatocytes and were able to partially and temporary correct analbuminemia and hypercholesterolemia by overexpressing human serum albumin or functional LDL receptor respectively (Wilson *et al.* 1992; Wu *et al.* 1991; Wu and Wu 1987). In another study, neuroprotection from an acute brain injury was achieved after direct intracerebral injection of the NLSCt vector overexpressing the antioxidant enzyme Cu/ZnSOD (see Table 1). In this experimental setting the overexpression of the therapeutic protein could not only induce reduced infarct volume but also functional improvement of the animals (Peluffo *et al.* 2006). Here, the vector was injected directly into the lesioned brain by a tightly controlled microinjector, using a similar protocol reported for the injection of cells into the human Parkinsonian brain (Brundin *et al.*), or for the intracerebral injection of adeno-associated viral vectors for the treatment of infantile lysosomal storage disease (Worgall *et al.* 2008). Thus, this direct intracerebral injection approach could show some benefits in clinical cases of focal traumatic or ischemic injuries, were a delimited area is lesioned and were in some cases even a decompressing craniectomy is needed leaving a direct entrance to the brain parenchyma. Modular protein vectors have also been used for neuroprotection after acute peripheral nerve transection. For example, Barati and colleagues (Barati *et al.* 2006) delivered a polylysine-based polyplex targeting p75NTR positive cells accomplishing the plasmid encoding for GDNF after a peripheral nerve transection (see Table 1). They showed an almost complete reversal in neuronal death caused by GDNF transgene expression. Though this is a very interesting study, the authors performed a subtle pre-lesion to the nerve one week before the nerve transection injury to upregulate p75NTR receptor, and thus the same experiment should be repeated but under more clinically relevant conditions. In another preclinical setting, the intravenous injection (once a day during 3 consecutive days) of the RVG-9R vector accomplished to the antiviral siRNA siFvEJ (see Table 1) was able to induce 80% survival of animals 30 days after their inoculation with a fatal flavivirus (Kumar *et al.* 2007). Thought many modular vectors have been shown to mediate over-expression or down-regulation of reporter genes, they need to be tested *in vivo* in clinically relevant models for the establishment of their real potential.

### **7. Conclusion**

More complex modular protein vectors including all the important domains for efficient nucleic acid delivery need to be engineered. They should include domains for DNA attachment and condensation, cell attachment and endocytosis, endosomal escape, cytosol trafficking towards the nucleus, nuclear import, and DNA release. HNRK (Domingo-Espín *et al.* 2011), fkAbp75-ipr (Berhanu and Rush 2008), and the fusogenic-karyophilic-NT-polyplex (Navarro-Quiroga *et al.* 2002) (see Table 1) are three prototypes of this increasingly complex vectors, but additional domains have to be inserted. In addition, an interesting strategy could be the exploitation of several domains that have dual functions, like poly-his with DNA attachment and endosomal escape properties, like melittin with endosomal escape properties and nuclear import potential, or histones with DNA attachment and nuclear import potential. Considering the fact that for example several cellular nuclear proteins have several nuclear localization domains, the introduction of several domains with the same function in the same vector may further increase their efficiency. In fact, dual targeting of cancer cell lines using both transferrin and RGD domains showed synergistic effects (Nie *et al.*). Furthermore, the

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combination of engineered modular protein vectors with engineered plasmids for long termregulated expression *in vivo* will be essential. For instance, the pEPI DNA vector was the rst prototype of episomal vector whose function relies exclusively on chromosomal elements, replicating autonomously in low copy numbers in all cells tested (Piechaczek *et al.* 1999). This vector was further engineered to show regulated expression and to be removed from transduced cells when transgene expression is no longer needed (Rupprecht *et al.*). Finally, vectors have to be tested *in vivo*, but evaluating biological effects and not only reporter gene expression, and the comparison of different vectors in a same *in vivo* experimental setting will also contribute to the selection of the best prototypes.
