*3.2.4. Herpes Simplex Virus Vectors (HSV)*

and minus strands packaged with equal efficiency (Daly, 2003). No human disease has been associated with AAV infection which is an important feature when thinking in AAV as gene vectors. Six serotypes of AAV have been described with AAV2 being the most widely used for gene-transfer studies (Hermonat et al., 1984). AAV2 cell entry is mediated by binding to heparin sulfate proteoglycans and αvβ5 integrin; fibroblast growth factor receptor-1 (FGFR-1) may also be involved. The distribution of these molecules on many different cell types can explain the prolonged *in vivo* expression following AAV treatment seen in the liver, brain, skeletal muscle, lung, and hematopoietic stem cells of animal models (Daly, 2003). AAV vectors contain no viral genes that could elicit undesirable immune or inflammatory responses. The primary host reaction that might have an unwanted impact is the production of neutralizing

174 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

One major concern when developing AAV vectors for gene delivery is that DNA constructs larger than the wild-type 4.7 kb sequence do not package well and vector titers decrease sharply thus constituting an insertional limitation for the cDNAs. Another important issue is the frequently seen integration of AAV genome in the host cell chromosomes. However, the available evidence indicates that integration of wild type AAV *in vivo* does not reflect the experimental *in vitro* observations, but appears to be a rather rare event and AAV genomes mostly persist as episomes, as has also been demonstrated for AAV vectors (Carter et al., 2009).

Retroviruses are lipid-enveloped viruses; with nucleocapsids containing two copies of a linear, positive-stranded 7–11 kb RNA genome. The family *Retroviridae* contains various viruses that have shown potential utility for gene therapy, such as the **gammaretrovirus** *(or simple retrovi‐ rus)*, *spumaviruses* and **lentiviruses** *(or complex retrovirus)*. Following attachment and receptormediated entry into host cells, viral reverse transcriptase and integrase enzymes mediate reverse transcription and integration of the virus genome into the host-cell chromatin. Retroviral vectors have the ability for stable integration and allow long-term expression so that theoretically a single administration could have a sustained, potentially even, lifelong curative effect (Schambach et al., 2009). As for any viral vector, replication-deficiency is a condition. To achieve this goal, the retroviral coding sequences have to be removed, which creates at least 6 kb space for the transgene of interest. Since neither structural proteins nor replication enzymes are encoded by the target cell, the generation of replication-competent virus is prevented. The gammaretroviruses cannot infect quiescent, non-dividing cells, which is a handicap of the vectors derived from these retroviruses. However, this can be overcome by the use of lentiviral

For most RV, taking advantage of the insertional mechanism, the simplest application is in the production of cell lines that express a transgene introduced on a retroviral vector. For modified, transgenic animals, the lentivirus group must be used because gammaretrovectors are silenced during embryonic development. RV vectors can also be used in the delivery of toxic genes to cancer cells, which are actively dividing. Another area of application is gene discovery. The integration of the viral genome can reveal function by insertional inactivation of a gene in the

antibodies against the viral particles.

*3.2.3. Retroviral vectors (RV)*

vectors.

host cell chromosome (Somia, 2003).

HSV-1 is a double-stranded DNA virus,with a capsid surrounded by a dense layer of proteins -the tegument- enveloped in a lipid bilayer with surface proteins. It has evolved to persist in a lifelong nonintegrated latent state without causing disease in the immune-competent host. Among the herpes family Herpes Simplex Virus type 1 (HSV-1) is an attractive vehicle because in natural infection, the virus establishes latency in neurons, a state in which viral genomes may persist for the life of the host as intranuclear episomal elements. Although the wild-type virus may be reactivated from latency under the influence of a variety of stresses, completely replication-defective and non-lytic viruses can be design (Goins et al., 2003). HSV-1 has a broad host range and does not require cell division for infection and gene expression. Accordingly, HSV may be generally useful for gene transfer to a variety of normal and disease tissues. The overall size of the HSV-1 genome (152 kb) represents an attractive feature for employing the vector for the transfer of large amounts of exogenous genetic sequences. Approximately onehalf of the HSV-1 coding sequences are nonessential for virus replication in cell culture. At least 44 kb of HSV sequence can potentially be removed in order to accommodate a transgene (Wolfe et al., 2009).

The obstacles that need to be addressed in order to take advantage of the full potential of these vectors include elimination of residual vector toxicity, design of promoter cassettes that provide sufficient level and duration of transgene expression, and targeting of transgene expression to specific cell populations through the use of tissue-specific promoters, or by altering the virus host range through modifying receptor utilization for attachment and entry (Wolfe et al., 2009).

#### *3.2.5. Sendai Virus Vectors (SeV)*

Since its isolation in 1953 in Japan, Sendai virus (SeV) has been widely used as a research tool in cell biology and in the industry, but the application of SeV as a recombinant viral vector has been investigated only recently. Sendai virus (SeV) is a nonsegmented negative-strand RNA virus belonging to the *Paramyxoviridae* family. As SeV can infect various animal cells with an exceptionally broad host range and is not pathogenic to humans, various applications have been explored for SeV as a recombinant viral vector capable of transient but strong gene expression (Nakanishi and Otsu, 2012). Its RNA nature is advantageous for applications in which chromosomal integration of exogenous genes can be undesirable. These viral vectors are currently being tested in regenerative medicine to reprogram cell genomes to a pluripotent state with a surprisingly high efficiency (Nishimura et al., 2011; MacArthur et al., 2012) and as recombinant viral vaccines for influenza prevention (Le et al., 2011).

#### **3.3. Recombinant adenoviral vectors**

Although the pathologies associated with wild-type Adenovirus (AdV) infections are gener‐ ally mild, there is a potential risk of using fully replication-competent AdV for gene transfer because the inflammatory host responses may alter organ function. There is also the possibility of overwhelming infection if AdV replication is allowed to progress when there are deficiencies in the host defense system. These situations became then a major reason to develop recombi‐ nant, non-pathogenic viral particles.

Regardless experimentation with different viral gene delivery systems, adenoviral vectors continue to be widely used for gene transfer strategies (Kovesdi et al., 1997; De Gruijl et al, 2012; Youngjoo et al, 2013; Fishbein I et al, 2013). They were also the first viral vector system to be developed. However this was more by chance than intentionally while working in the production of live adenoviral vaccines propagated in monkey cell lines. Infection of tissue culture cells with AdV vaccine accidentally contaminated with simian virus 40 (SV40) resulted in the production of the SV40 T antigen, even after removal of the SV40 virions from the AdV stocks by immunodepletion. Analysis of this adenovirus revealed that the T-antigen gene from SV40 had recombined into the E3 region of the Ad genome. This demonstrated the possibility that AdV could carry foreign genes and express them as well as demonstrating the dispensa‐ bility of the E3 genes for *in vitro* replication (Roy-Chowdhury and Horwitz, 2002; Campos et al., 2007).

The first wild type adenoviruses subjected to "vectoring" process were AdVs derived from the human serotypes 5 (Ad5) and 2 (Ad2). First-generation replication-deficient Ad5 vectors were developed by deleting the E1 genes, necessary for expression of E2 and late genes required for AdV DNA synthesis, capsid protein expression, and viral replication. Further deletions included the E3 genes which are involved in the evasion of host immune defenses but dispensable for replication of the virus *in vitro*. Therefore, because of this experimental manipulation leading to viral replication impairment, there was the need to develop a biological system capable of providing the genes required for the virus propagation. This led to the creation of the HEK 293 cell line, which was transfected with sheared adenovirus-type 5 (Ad5) genomic DNA and stably expresses the E1 genes (Graham et al., 1977).

One of the most spectacular results in gene therapy using HD-AdV was published by Kim et al., 2001. They used a gutless AdV harboring the apoE gene to treat apoE-deficient C57BL/6 mice which display spontaneous hypercholesterolemia. These mice received a single intrave‐ nous (i.v) injection of HD-AdV-ApoE at the 12th week of age. This isolated intervention managed to normalized plasma apoE concentrations and therefore diminish the plasma cholesterol level to values found in wild-type mice. However, the most outstanding finding involves the duration of the protective effect of this gene therapy, being of at least 2.5 years,

Many of these AdV vectors produced in accordance with regulated quality standards, are now being used for human clinical trials. For detailed information the reader is referred to the

The skeletal muscle gene transfer approach using Adenoviral Vectors has created controversy. There are several studies with heterogeneous efficiency rates and, in some cases, divergent outcomes. On this section we will intend to present and discuss these results as well as to introduce a novel technology that might overcome the difficulties experienced in the trans‐

following reviews: Russell, 2000; Józkowicz and Dulak, 2005; Campos, 2007.

**3.4. Adenoviral vectors in tissue-specific gene transfer: The skeletal muscle**

the lifespan of these mice.

**Ad vector generation**

second E2 (complete

third most of the

or partial)

genome. ITR and packaging seq. retained ("gutless", HC or HD-Ad)

**AdV genome deletions**

**foreign DNA insertion capacity**

**Table 1.** Comparison of adenoviral vectors generations (Russell, 2000).

**propagation in cell lines**

8kb 293-C2 (express E2) AE1-2a

35-37 kb 293 cells expressing Cre;

require helper virus

first E1 and/or E3 6.5 kb HEK293 (express E1) transient significant Possibility of the

**type of transgene expression**

transient important;

especially after repeated administration

long-lasting low or none No production of

**host immune response**

**risks**

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production of replicationcompetent adenovirus (RCA)

No production of

RCA

RCA

duction of this tissue.

Although the first-generation AdV vectors are generally considered replication defective, there is some low level expression of viral antigens that limits the duration of transgene expression *in vivo*, due to elimination of transduced cells by the cellular immune system (McConnell and Imperiale, 2004). To avoid this response and allow long-term episomal expression a new recombinant vector was developed. They are referred as "gutless" or high-capacity Adenovi‐ rus (HC-AdV) lack all viral coding sequences except the *cis*-elements required for the genome replication and encapsidation. Therefore, they need to be assisted by Helper Adenoviruses that provide all necessary replication genes in *trans*; therefore, they are also called Helper-Dependent adeno vectors (HD-AdV) (Amalfitano, 1999). In addition, these vectors have a much higher packaging capacity of ~35 kb of foreign DNA, enabling the expression of large transgenes or the inclusion of human genomic regulatory elements (Palmer and Ng, 2005). See Table 1 for comparison of the profile of different vector generations.

A variety of applications can be found for these vectors. The most widely explored are Gene Therapy for prostate, colon, cervix, ovary and CNS tumors, for genetic diseases such as Hemophilia, Duchene Muscular Dystrophy, Familial Hypercholesterolemia, Autoimmune Diabetes; viral vaccines; supplementary therapy for degenerative conditions as Parkinson, Alzheimer and Rheumatic Arthritis and production of recombinant proteins among others.


**Table 1.** Comparison of adenoviral vectors generations (Russell, 2000).

in the host defense system. These situations became then a major reason to develop recombi‐

Regardless experimentation with different viral gene delivery systems, adenoviral vectors continue to be widely used for gene transfer strategies (Kovesdi et al., 1997; De Gruijl et al, 2012; Youngjoo et al, 2013; Fishbein I et al, 2013). They were also the first viral vector system to be developed. However this was more by chance than intentionally while working in the production of live adenoviral vaccines propagated in monkey cell lines. Infection of tissue culture cells with AdV vaccine accidentally contaminated with simian virus 40 (SV40) resulted in the production of the SV40 T antigen, even after removal of the SV40 virions from the AdV stocks by immunodepletion. Analysis of this adenovirus revealed that the T-antigen gene from SV40 had recombined into the E3 region of the Ad genome. This demonstrated the possibility that AdV could carry foreign genes and express them as well as demonstrating the dispensa‐ bility of the E3 genes for *in vitro* replication (Roy-Chowdhury and Horwitz, 2002; Campos et

The first wild type adenoviruses subjected to "vectoring" process were AdVs derived from the human serotypes 5 (Ad5) and 2 (Ad2). First-generation replication-deficient Ad5 vectors were developed by deleting the E1 genes, necessary for expression of E2 and late genes required for AdV DNA synthesis, capsid protein expression, and viral replication. Further deletions included the E3 genes which are involved in the evasion of host immune defenses but dispensable for replication of the virus *in vitro*. Therefore, because of this experimental manipulation leading to viral replication impairment, there was the need to develop a biological system capable of providing the genes required for the virus propagation. This led to the creation of the HEK 293 cell line, which was transfected with sheared adenovirus-type

Although the first-generation AdV vectors are generally considered replication defective, there is some low level expression of viral antigens that limits the duration of transgene expression *in vivo*, due to elimination of transduced cells by the cellular immune system (McConnell and Imperiale, 2004). To avoid this response and allow long-term episomal expression a new recombinant vector was developed. They are referred as "gutless" or high-capacity Adenovi‐ rus (HC-AdV) lack all viral coding sequences except the *cis*-elements required for the genome replication and encapsidation. Therefore, they need to be assisted by Helper Adenoviruses that provide all necessary replication genes in *trans*; therefore, they are also called Helper-Dependent adeno vectors (HD-AdV) (Amalfitano, 1999). In addition, these vectors have a much higher packaging capacity of ~35 kb of foreign DNA, enabling the expression of large transgenes or the inclusion of human genomic regulatory elements (Palmer and Ng, 2005). See

A variety of applications can be found for these vectors. The most widely explored are Gene Therapy for prostate, colon, cervix, ovary and CNS tumors, for genetic diseases such as Hemophilia, Duchene Muscular Dystrophy, Familial Hypercholesterolemia, Autoimmune Diabetes; viral vaccines; supplementary therapy for degenerative conditions as Parkinson, Alzheimer and Rheumatic Arthritis and production of recombinant proteins among others.

5 (Ad5) genomic DNA and stably expresses the E1 genes (Graham et al., 1977).

Table 1 for comparison of the profile of different vector generations.

nant, non-pathogenic viral particles.

176 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

al., 2007).

One of the most spectacular results in gene therapy using HD-AdV was published by Kim et al., 2001. They used a gutless AdV harboring the apoE gene to treat apoE-deficient C57BL/6 mice which display spontaneous hypercholesterolemia. These mice received a single intrave‐ nous (i.v) injection of HD-AdV-ApoE at the 12th week of age. This isolated intervention managed to normalized plasma apoE concentrations and therefore diminish the plasma cholesterol level to values found in wild-type mice. However, the most outstanding finding involves the duration of the protective effect of this gene therapy, being of at least 2.5 years, the lifespan of these mice.

Many of these AdV vectors produced in accordance with regulated quality standards, are now being used for human clinical trials. For detailed information the reader is referred to the following reviews: Russell, 2000; Józkowicz and Dulak, 2005; Campos, 2007.

#### **3.4. Adenoviral vectors in tissue-specific gene transfer: The skeletal muscle**

The skeletal muscle gene transfer approach using Adenoviral Vectors has created controversy. There are several studies with heterogeneous efficiency rates and, in some cases, divergent outcomes. On this section we will intend to present and discuss these results as well as to introduce a novel technology that might overcome the difficulties experienced in the trans‐ duction of this tissue.

Direct gene transfer into skeletal muscle cells *in vitro* and *in vivo* using either plasmid DNA or recombinant viruses has medical applications in vaccination and gene therapy and also has been widely used in studies of developmental and physiological regulation of muscle gene expression (Hallauer et al., 2000). Many factors regarding muscle tissue barriers, immune response, systemic dissemination, potential toxicity and specific properties of each viral system need to be taken into account when selecting the proper approach for skeletal muscle. It is well known that skeletal muscle is a highly developed and organized tissue in which the constituent myofibers become post-mitotic in fetal life. The mononucleated myogenic precursor cells (satellite cells) that are located between the extracellular matrix and the plasma membrane of myofibers are known to be capable of fusing together or with preexisting myofibers in response to various types of stimuli, mainly to injury (Chargé et al., 2004). These satellite cells are relatively easy to isolate and cultivate *in vitro* and can also be efficiently transduced using virtually any viral vector in contrast with the mature multinucleated myofibers. Thus, is believed that viral transduction during skeletal muscle maturation might require mitotically active myoblasts (van Deutekom et al., 1998). However, because some of the viral vectors have been shown to transduce post-mitotic, immature myofibers *in vitro* and *in vivo* other factors are also likely to be involved in the poor level of viral transduction of mature myofibers (Wolff et al., 1990; Acsadi et al., 1994 and Huard et al., 1996;).

Despite this wide range of viral delivery systems, *Adenoviral (AdV) vectors* are probably the most prominent ones in this matter and have been extensible use to deliver genes into skeletal muscle. However, several obstacles have been identified in the application of adenovirus as gene delivery vehicles to skeletal muscle (Acsadi et al., 1994 and van Deutekom et al., 1998). The major limitations facing first generation adenoviral gene transfer to skeletal muscle are (1) the lack of transgene persistence due to the immune rejection of transduced myofibers; (2) the relatively low insert carrying capacity; (3) the reduced viral transducibility during muscle maturation; and (4) repeated administration associated with the production of neutralizing antibodies is limited to the viral capsid (Cao et al., 2001). During experiments of AdV gene transfer in animals of different ages it became clear that the transduction efficiency was related with the maturation state of the muscle. While the skeletal muscle of newborn mice achieved high levels of AdV infection, the mature muscle from adult animals was significantly less susceptible to infection under the same conditions (Huard et al., 1995). Here the high adeno‐ viral transduction of newborn myofibers could be explained due partly to transduction of myoblasts and partly to the higher levels of CAR in these myofibers (Nalbantoglu et al., 1999). Several studies have shown that in developing human, mouse and rat muscle, expres‐ sion of the primary AdV membrane receptor CAR is severely downregulated even at early ages with CAR mRNA being barely detectable in adult myofibers (Nalbantoglu et al., 1999). Furthermore, it has been demonstrated that forced expression of CAR in myotubes by different approaches, such as RAdV encoding hCAR or transgenic mice overexpressing the receptor, overcomes the poor AdV mediated transducibility of these cells (Nalbantoglu et al., 2001 and Kimura et al., 2001). On the other hand, basal lamina and glycocalyx surrounding mature skeletal muscle cells appear to be an anatomical barrier that may limit the access and distri‐ bution of exogenously introduced virus. (van Deutekom et al., 1998 and Cao et al., 2001). It has been reported that the extracellular matrix of mature myofibers may form a physical barrier and prevent the passage of some viral particles that are too big to pass through its pores, which are estimated at 40 nm in size. Adenoviral particles are about 70 nm and 100 nm in diameter and appear to be too large to penetrate the pores of the basal lamina (Cao et al., 2001). Re‐ gardless these difficulties some authors have published high rates of skeletal muscle trans‐ duction using AdV. In 2002, Sapru et al., was able to achieve nearly 100% of transduced fibers in the adult rat Soleus and more than 80% in the Tibialis Anterior muscle when infected with an adenoviral vector harbouring the cDNA of the GFP under the control of the CMV promoter (AdVCMV-GFP). These authors claim that the viral titer used was an important factor since they could increase the number of transduced fiber when the viral dose was doubled. Other major factor regarding skeletal muscle infection with AdV and AAV seems to be the fiber composition of the muscle, with suggested preferential transduction of slow fibers (Pruchnic et al., 2000 and Sapru et al., 2002). New AdV vectors lacking all viral genes, the *Helper-Dependent AdV vectors*, show a markedly decreased immunogenicity and hence, an improved persistence of transgene expression in muscle *in vivo* (Bilbao et al., 2005). These observations suggest that the limitations regarding the immunogenicity with the use of adenoviral vectors are being overcome. However, the inability of adenoviral vectors to efficiently transduce mature myofibers remains a major hurdle facing the widespread application of adenoviral gene

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transfer to skeletal muscle (van Deutekom et al., 1998).

*Retroviral vectors (RV)* can infect dividing myoblasts with a high efficiency although they remain incapable of infecting post mitotic myotubes or myofibers (Miller et al., 1990 and Salvatori et al., 1993). In addition, the ability to become stably integrated into the host cell genome, which can provide long-term, stable expression of the delivered gene, may also represent a risk for insertional mutagenesis. As mentioned in Section 3.2 of the present chapter, other limitations to the use of retroviruses are the gene insert capacity (less than 7 kb) and the relatively low production titers (105 –106 plaque-forming units per milliliter (pfu/mL)). The recombinant vectors obtained from the *Herpes Simplex Virus type 1 (HSV-1)* can persist in the host cell in a nonintegrated state and be prepared at adequately high titers (107 –109 pfu / ml). They are capable of transducing muscle cells in most maturation stages while carrying large DNA fragments. However, are still unable to highly penetrate and transduce mature myofibers (Huard et al., 1997). *Adeno-associated derived viral vectors (AAV)* have also been used to approach muscle cells. Although a long-term gene expression (up to 18 months) and a high efficiency of mature myofibers transduction have been observed in mouse skeletal muscle, the application of adeno-associated viral vectors for gene therapy may be limited by their restrictive gene insert capacity (Pruchnic et al., 2000). In the last few years a novel gene transfer viral vector has been used into skeletal muscle, the *recombinant Sendai virus (SeV) vectors*. As explained before, the wild SeV is a non-segmented negative-strand RNA virus belonging to the *Para‐ myxoviridae* family that can infect various animal cells with an exceptionally broad host range. Shiotani et al., 2001 accomplished a significant overexpression of hIGF-1 in the adult rat Tibialis Anterior muscle when injected the tissue with a recombinant SeV vector (hIGF-1/SeV). They indicate a favorable gene delivery to mitotic myoblasts, post-mitotic immature and mature myofibers.

Despite this wide range of viral delivery systems, *Adenoviral (AdV) vectors* are probably the most prominent ones in this matter and have been extensible use to deliver genes into skeletal muscle. However, several obstacles have been identified in the application of adenovirus as gene delivery vehicles to skeletal muscle (Acsadi et al., 1994 and van Deutekom et al., 1998). The major limitations facing first generation adenoviral gene transfer to skeletal muscle are (1) the lack of transgene persistence due to the immune rejection of transduced myofibers; (2) the relatively low insert carrying capacity; (3) the reduced viral transducibility during muscle maturation; and (4) repeated administration associated with the production of neutralizing antibodies is limited to the viral capsid (Cao et al., 2001). During experiments of AdV gene transfer in animals of different ages it became clear that the transduction efficiency was related with the maturation state of the muscle. While the skeletal muscle of newborn mice achieved high levels of AdV infection, the mature muscle from adult animals was significantly less susceptible to infection under the same conditions (Huard et al., 1995). Here the high adeno‐ viral transduction of newborn myofibers could be explained due partly to transduction of myoblasts and partly to the higher levels of CAR in these myofibers (Nalbantoglu et al., 1999). Several studies have shown that in developing human, mouse and rat muscle, expres‐ sion of the primary AdV membrane receptor CAR is severely downregulated even at early ages with CAR mRNA being barely detectable in adult myofibers (Nalbantoglu et al., 1999). Furthermore, it has been demonstrated that forced expression of CAR in myotubes by different approaches, such as RAdV encoding hCAR or transgenic mice overexpressing the receptor, overcomes the poor AdV mediated transducibility of these cells (Nalbantoglu et al., 2001 and Kimura et al., 2001). On the other hand, basal lamina and glycocalyx surrounding mature skeletal muscle cells appear to be an anatomical barrier that may limit the access and distri‐ bution of exogenously introduced virus. (van Deutekom et al., 1998 and Cao et al., 2001). It has been reported that the extracellular matrix of mature myofibers may form a physical barrier and prevent the passage of some viral particles that are too big to pass through its pores, which are estimated at 40 nm in size. Adenoviral particles are about 70 nm and 100 nm in diameter and appear to be too large to penetrate the pores of the basal lamina (Cao et al., 2001). Re‐ gardless these difficulties some authors have published high rates of skeletal muscle trans‐ duction using AdV. In 2002, Sapru et al., was able to achieve nearly 100% of transduced fibers in the adult rat Soleus and more than 80% in the Tibialis Anterior muscle when infected with an adenoviral vector harbouring the cDNA of the GFP under the control of the CMV promoter (AdVCMV-GFP). These authors claim that the viral titer used was an important factor since they could increase the number of transduced fiber when the viral dose was doubled. Other major factor regarding skeletal muscle infection with AdV and AAV seems to be the fiber composition of the muscle, with suggested preferential transduction of slow fibers (Pruchnic et al., 2000 and Sapru et al., 2002). New AdV vectors lacking all viral genes, the *Helper-Dependent AdV vectors*, show a markedly decreased immunogenicity and hence, an improved persistence of transgene expression in muscle *in vivo* (Bilbao et al., 2005). These observations suggest that the limitations regarding the immunogenicity with the use of adenoviral vectors are being overcome. However, the inability of adenoviral vectors to efficiently transduce mature myofibers remains a major hurdle facing the widespread application of adenoviral gene transfer to skeletal muscle (van Deutekom et al., 1998).

Direct gene transfer into skeletal muscle cells *in vitro* and *in vivo* using either plasmid DNA or recombinant viruses has medical applications in vaccination and gene therapy and also has been widely used in studies of developmental and physiological regulation of muscle gene expression (Hallauer et al., 2000). Many factors regarding muscle tissue barriers, immune response, systemic dissemination, potential toxicity and specific properties of each viral system need to be taken into account when selecting the proper approach for skeletal muscle. It is well known that skeletal muscle is a highly developed and organized tissue in which the constituent myofibers become post-mitotic in fetal life. The mononucleated myogenic precursor cells (satellite cells) that are located between the extracellular matrix and the plasma membrane of myofibers are known to be capable of fusing together or with preexisting myofibers in response to various types of stimuli, mainly to injury (Chargé et al., 2004). These satellite cells are relatively easy to isolate and cultivate *in vitro* and can also be efficiently transduced using virtually any viral vector in contrast with the mature multinucleated myofibers. Thus, is believed that viral transduction during skeletal muscle maturation might require mitotically active myoblasts (van Deutekom et al., 1998). However, because some of the viral vectors have been shown to transduce post-mitotic, immature myofibers *in vitro* and *in vivo* other factors are also likely to be involved in the poor level of viral transduction of mature myofibers (Wolff

178 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

*Retroviral vectors (RV)* can infect dividing myoblasts with a high efficiency although they remain incapable of infecting post mitotic myotubes or myofibers (Miller et al., 1990 and Salvatori et al., 1993). In addition, the ability to become stably integrated into the host cell genome, which can provide long-term, stable expression of the delivered gene, may also represent a risk for insertional mutagenesis. As mentioned in Section 3.2 of the present chapter, other limitations to the use of retroviruses are the gene insert capacity (less than 7 kb) and the

recombinant vectors obtained from the *Herpes Simplex Virus type 1 (HSV-1)* can persist in the

They are capable of transducing muscle cells in most maturation stages while carrying large DNA fragments. However, are still unable to highly penetrate and transduce mature myofibers (Huard et al., 1997). *Adeno-associated derived viral vectors (AAV)* have also been used to approach muscle cells. Although a long-term gene expression (up to 18 months) and a high efficiency of mature myofibers transduction have been observed in mouse skeletal muscle, the application of adeno-associated viral vectors for gene therapy may be limited by their restrictive gene insert capacity (Pruchnic et al., 2000). In the last few years a novel gene transfer viral vector has been used into skeletal muscle, the *recombinant Sendai virus (SeV) vectors*. As explained before, the wild SeV is a non-segmented negative-strand RNA virus belonging to the *Para‐ myxoviridae* family that can infect various animal cells with an exceptionally broad host range. Shiotani et al., 2001 accomplished a significant overexpression of hIGF-1 in the adult rat Tibialis Anterior muscle when injected the tissue with a recombinant SeV vector (hIGF-1/SeV). They indicate a favorable gene delivery to mitotic myoblasts, post-mitotic immature and mature

plaque-forming units per milliliter (pfu/mL)). The

–109

pfu / ml).

–106

host cell in a nonintegrated state and be prepared at adequately high titers (107

et al., 1990; Acsadi et al., 1994 and Huard et al., 1996;).

relatively low production titers (105

myofibers.

On the next section we will introduce the novel combination of Magnetic Nanoparticles and Recombinant Adenoviral Vectors as an efficient alternative for gene delivery in transductionresistant differentiated skeletal muscle cells.

(or zeta-) potential of all types of viral particles in aqueous media allow their assembling with cationic species and particles due to electrostatically induced aggregation (Scherer et al., 2002). The resulting magnetic-viral vectors complexes are then forced into sedimentation over

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As detailed in Section 2.3, one of the most remarkable aspects of magnetofection enhancing viral gene transfer is the lack of need for virus interaction with cellular receptors. This has a particular significance for adenoviral vectors. It is known that for all the Wild-Type (except Subgroup B) and Recombinant Adenovirus cell entry takes place through receptor-mediated endocytosis via the Coxsackie–Adenovirus Receptor (CAR) (Meier and Greber, 2003). Various cell types differ widely in their level of CAR expression, and this may be a limiting factor for the transduction efficiency achievable with Adenovirus (Chorny et al., 2006). When AdVs are complex with MNPs all the viral surface proteins become hidden and unreachable for the CAR receptor. The cell entry in then achieved by unspecific clathrin-mediated endocytosis (Plank

In order to find the optimal magnetic vector formulations for plasmid, siRNA and viral vector delivery, Plank and co-workers have aimed at maximal association with the magnetic com‐ ponent but avoiding an excess of magnetic particles (Plank et al., 2011). They have published that an excess of magnetic nanoparticles can inhibit transfection/transduction efficiency and cause toxicity (Tresilwised et al., 2010 and Sanchez-Antequera et al., 2011). Therefore, finding the optimal MNPs-to-nucleic acid ratios (about 0.5–1 units of iron weight per unit of the nucleic acid weight for triplexes with an enhancer) as well as MNPs-to-virus ratios (2.5–10 fg iron per virus particle further referred to as fg Fe/VP) have turned out useful for a variety of magnetic nanoparticle types (Sanchez-Antequera et al., 2011). The complexes formulated in this way were efficient and hardly toxic in delivery of DNA and siRNA as well in delivery of adenoviral and lentiviral vectors *in vitro* and *ex vivo*. Particularly to this regard there are several studies that have demonstrated the higher efficiency of this method when compare with traditional viral transduction using Adenovirus, Adeno-associated Virus, Baculovirus, Lentivirus and Retrovirus (Chan et al., 2005; Chorny et al., 2009; Hughes et al., 2001; Kaikkonen et al., 2008; Mah et al., 2000 and 2002; Morizono et al., 2009; Raty et al., 2004; Tresilwised et al., 2010 and

Regarding our experimental field, development of reliable techniques for manipulation of gene expression in mature skeletal muscle fibers is critical for understanding molecular mechanisms involved in both physiology and physiopathology. As explained before, differentiated skeletal muscle myotubes and myofibers are refractory to most standard protocols for gene transfer in vitro and in vivo and the use of adenoviral vectors offers relatively low efficiency. It is believed that a maturation-dependent loss of the CAR receptor together with structural and biochemical changes are responsible for these decreased transduction efficiencies (Nalbantoglu et al., 1999). It has been proposed that these limitations can be overcome by achieving adenoviral cellular uptake via a CAR independent pathway using genetic modifications of the capsid proteins or chemical modifications of the virus. However, these strategies are not sufficient for rapid infection of the cells at the target site, as the delivery process itself is diffusion-limited (Haim et al., 2005 and Schillinger et al., 2005). Here, magnetofection provided us a powerful,

the cell monolayer when a magnetic field is applied.

et al., 2011).

2012).

#### **3.5. Use of magnetic nanoparticles and magnetic fields to enhance viral vector-based gene delivery**

Nowadays, the novel association of non-viral or viral vector-based gene delivery with nanotechnology offers the possibility to develop more efficient gene transfer strategies for a number of applications. In 2002 the concept of Magnetofection was first published by Scherer et al. Here, the Magnetic Drug Targeting (MDT) approach (Widder et al., 1978) classically used to concentrate magnetically responsive therapeutic complexes in target areas of the body by means of external gradient magnetic fields was applied for gene delivery. Therefore, Magne‐ tofection is based on the association of Magnetic Nanoparticles (MNPs) with non-viral or viral vectors in order to optimize gene delivery when exposed to a magnetic field (Scherer et al., 2002).

There are currently several synthetic formulations of MNPs commercially available for biomedical applications such as cell separation, drug/gene delivery, magnetic resonance imaging (MRI) and hyperthermia (Gupta et al., 2005). Despite the differences, they all need to comprise some basic functionality to allow them to be associated with a gene delivery vector. Furthermore, the magnetic properties of these particles have to be sufficient to concentrate the vector at the target cells under a magnetic force and the formulation has to be biocompatible enough for application in living cells or organisms (Plank et al., 2011). Their general structure is based on a magnetic core of magnetite (Fe3O4) or maghemite (g-Fe2O3) coated with synthetic polymers that provides both protection and biological functionality. Occasionally specific organic linkers are added to this structure to generate new attachment sites for drugs or gene vectors (Yallapu et al., 2010). (Figure 5).

**Figure 5.** General Structure of a Magnetic Nanoparticle

For association with nucleic acids and/or viral particles, coatings comprised of cationic polymers such as polyethylenimine (PEI) are particularly useful (Mykhaylyk et al., 2007). The negatively charged phosphate backbone of nucleic acids as well as the negative electrokinetic (or zeta-) potential of all types of viral particles in aqueous media allow their assembling with cationic species and particles due to electrostatically induced aggregation (Scherer et al., 2002). The resulting magnetic-viral vectors complexes are then forced into sedimentation over the cell monolayer when a magnetic field is applied.

On the next section we will introduce the novel combination of Magnetic Nanoparticles and Recombinant Adenoviral Vectors as an efficient alternative for gene delivery in transduction-

**3.5. Use of magnetic nanoparticles and magnetic fields to enhance viral vector-based gene**

Nowadays, the novel association of non-viral or viral vector-based gene delivery with nanotechnology offers the possibility to develop more efficient gene transfer strategies for a number of applications. In 2002 the concept of Magnetofection was first published by Scherer et al. Here, the Magnetic Drug Targeting (MDT) approach (Widder et al., 1978) classically used to concentrate magnetically responsive therapeutic complexes in target areas of the body by means of external gradient magnetic fields was applied for gene delivery. Therefore, Magne‐ tofection is based on the association of Magnetic Nanoparticles (MNPs) with non-viral or viral vectors in order to optimize gene delivery when exposed to a magnetic field (Scherer et al.,

There are currently several synthetic formulations of MNPs commercially available for biomedical applications such as cell separation, drug/gene delivery, magnetic resonance imaging (MRI) and hyperthermia (Gupta et al., 2005). Despite the differences, they all need to comprise some basic functionality to allow them to be associated with a gene delivery vector. Furthermore, the magnetic properties of these particles have to be sufficient to concentrate the vector at the target cells under a magnetic force and the formulation has to be biocompatible enough for application in living cells or organisms (Plank et al., 2011). Their general structure is based on a magnetic core of magnetite (Fe3O4) or maghemite (g-Fe2O3) coated with synthetic polymers that provides both protection and biological functionality. Occasionally specific organic linkers are added to this structure to generate new attachment sites for drugs

resistant differentiated skeletal muscle cells.

180 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

or gene vectors (Yallapu et al., 2010). (Figure 5).

Magnetic Core

Polymer Coating

**Figure 5.** General Structure of a Magnetic Nanoparticle

Organic Linker

For association with nucleic acids and/or viral particles, coatings comprised of cationic polymers such as polyethylenimine (PEI) are particularly useful (Mykhaylyk et al., 2007). The negatively charged phosphate backbone of nucleic acids as well as the negative electrokinetic

Drug/Gene Vector

**delivery**

2002).

As detailed in Section 2.3, one of the most remarkable aspects of magnetofection enhancing viral gene transfer is the lack of need for virus interaction with cellular receptors. This has a particular significance for adenoviral vectors. It is known that for all the Wild-Type (except Subgroup B) and Recombinant Adenovirus cell entry takes place through receptor-mediated endocytosis via the Coxsackie–Adenovirus Receptor (CAR) (Meier and Greber, 2003). Various cell types differ widely in their level of CAR expression, and this may be a limiting factor for the transduction efficiency achievable with Adenovirus (Chorny et al., 2006). When AdVs are complex with MNPs all the viral surface proteins become hidden and unreachable for the CAR receptor. The cell entry in then achieved by unspecific clathrin-mediated endocytosis (Plank et al., 2011).

In order to find the optimal magnetic vector formulations for plasmid, siRNA and viral vector delivery, Plank and co-workers have aimed at maximal association with the magnetic com‐ ponent but avoiding an excess of magnetic particles (Plank et al., 2011). They have published that an excess of magnetic nanoparticles can inhibit transfection/transduction efficiency and cause toxicity (Tresilwised et al., 2010 and Sanchez-Antequera et al., 2011). Therefore, finding the optimal MNPs-to-nucleic acid ratios (about 0.5–1 units of iron weight per unit of the nucleic acid weight for triplexes with an enhancer) as well as MNPs-to-virus ratios (2.5–10 fg iron per virus particle further referred to as fg Fe/VP) have turned out useful for a variety of magnetic nanoparticle types (Sanchez-Antequera et al., 2011). The complexes formulated in this way were efficient and hardly toxic in delivery of DNA and siRNA as well in delivery of adenoviral and lentiviral vectors *in vitro* and *ex vivo*. Particularly to this regard there are several studies that have demonstrated the higher efficiency of this method when compare with traditional viral transduction using Adenovirus, Adeno-associated Virus, Baculovirus, Lentivirus and Retrovirus (Chan et al., 2005; Chorny et al., 2009; Hughes et al., 2001; Kaikkonen et al., 2008; Mah et al., 2000 and 2002; Morizono et al., 2009; Raty et al., 2004; Tresilwised et al., 2010 and 2012).

Regarding our experimental field, development of reliable techniques for manipulation of gene expression in mature skeletal muscle fibers is critical for understanding molecular mechanisms involved in both physiology and physiopathology. As explained before, differentiated skeletal muscle myotubes and myofibers are refractory to most standard protocols for gene transfer in vitro and in vivo and the use of adenoviral vectors offers relatively low efficiency. It is believed that a maturation-dependent loss of the CAR receptor together with structural and biochemical changes are responsible for these decreased transduction efficiencies (Nalbantoglu et al., 1999). It has been proposed that these limitations can be overcome by achieving adenoviral cellular uptake via a CAR independent pathway using genetic modifications of the capsid proteins or chemical modifications of the virus. However, these strategies are not sufficient for rapid infection of the cells at the target site, as the delivery process itself is diffusion-limited (Haim et al., 2005 and Schillinger et al., 2005). Here, magnetofection provided us a powerful, accessible and efficient tool for transducing differentiated myotubes of the C2C12 cell line (Pereyra A et al., 2011-Posters Sessions). A first generation (E1/E3-deleted), serotype 5, Recombinant Adenoviral vector harboring de cDNA of the Green Fluorescent Protein (RAdV-GFP) under the control of the CMV promoter was constructed in our laboratory. This vector was incubated with Atto550PEI-Mag2, a magnetic nanoparticle conjugated with a red fluo‐ rescent dye that allows particle tracking during the cellular uptake and internalization. Then the [RAdV-GFP-Atto550PEI-Mag2] complexes were incorporated to the supernatant of the mature myotubes cultures. The magnetic field required for sedimentation was provided by a commercial plate (Oz Biosciences®, Marseille, France) composed of cylindrical-permanent-Nd-Fe-B magnets. The same protocol was tested in undifferentiated C2C12 myoblast cultures and conventional RAdV transduction experiments using the same viral multiplicity of infection (MOI) were also performed for efficiency comparisons. (Figure 6)

**Figure 6.** Magnetofection in cell culture. This basic scheme shows the process of magnetofection in cultured cells. The pre-incubated RAdV-MNPs complexes are introduced to the cell culture. Then the culture plate is exposed to a mag‐ netic field created by the magnetic plate placed under the cells.

As showed in Figure 7, the poor RadV-GFP transduction of mature myotubes was overcome by magnetofection. In myoblasts, were the conventional transduction protocols show an acceptable efficiency, the magnetofection method displayed an enhancer effect. The intracel‐ lular localization of the magnetic nanoparticles can be seen in Figure 8.

1

1

**Myoblasts+RAd-GFP+Atto550- PEI-Mag2 with exposure to magnetic field**

Gene Delivery Systems

183

http://dx.doi.org/10.5772/56869

**Myoblasts+RAd-GFP MOI 60**

2011-Posters Sessions).

**Myoblasts+RAd-GFP+Atto550- PEI-Mag2 without exposure to magnetic field**

**Figure 7.** Magnetofection in mature C2C12 myotubes and myoblasts. The conventional RAdV transduction system was compared against magnetofection. The images were obtained 48 hs after incubation. The green fluorescence corresponds to the expression of the GFP protein encoded by the viral genome. Magnification 40X (Pereyra A et al.,

**Figure 8.** This picture was taken with a Double Band filter in order to appreciate the cellular co-localization of the green and red (Atto550-PEI-Mag2) fluorescence. Magnification 40X (Pereyra A et al., 2011-Posters Sessions).

1

accessible and efficient tool for transducing differentiated myotubes of the C2C12 cell line (Pereyra A et al., 2011-Posters Sessions). A first generation (E1/E3-deleted), serotype 5, Recombinant Adenoviral vector harboring de cDNA of the Green Fluorescent Protein (RAdV-GFP) under the control of the CMV promoter was constructed in our laboratory. This vector was incubated with Atto550PEI-Mag2, a magnetic nanoparticle conjugated with a red fluo‐ rescent dye that allows particle tracking during the cellular uptake and internalization. Then the [RAdV-GFP-Atto550PEI-Mag2] complexes were incorporated to the supernatant of the mature myotubes cultures. The magnetic field required for sedimentation was provided by a commercial plate (Oz Biosciences®, Marseille, France) composed of cylindrical-permanent-Nd-Fe-B magnets. The same protocol was tested in undifferentiated C2C12 myoblast cultures and conventional RAdV transduction experiments using the same viral multiplicity of infection

**Figure 6.** Magnetofection in cell culture. This basic scheme shows the process of magnetofection in cultured cells. The pre-incubated RAdV-MNPs complexes are introduced to the cell culture. Then the culture plate is exposed to a mag‐

As showed in Figure 7, the poor RadV-GFP transduction of mature myotubes was overcome by magnetofection. In myoblasts, were the conventional transduction protocols show an acceptable efficiency, the magnetofection method displayed an enhancer effect. The intracel‐

lular localization of the magnetic nanoparticles can be seen in Figure 8.

netic field created by the magnetic plate placed under the cells.

(MOI) were also performed for efficiency comparisons. (Figure 6)

182 Current Issues in Molecular Virology - Viral Genetics and Biotechnological Applications

**Figure 7.** Magnetofection in mature C2C12 myotubes and myoblasts. The conventional RAdV transduction system was compared against magnetofection. The images were obtained 48 hs after incubation. The green fluorescence corresponds to the expression of the GFP protein encoded by the viral genome. Magnification 40X (Pereyra A et al., 2011-Posters Sessions).

1 **Figure 8.** This picture was taken with a Double Band filter in order to appreciate the cellular co-localization of the green and red (Atto550-PEI-Mag2) fluorescence. Magnification 40X (Pereyra A et al., 2011-Posters Sessions).
