**3. Viral gene delivery systems**

## **3.1. General overview of viruses as vectors for gene delivery**

For centuries the health sciences have invested sweat and tears in order to fight against viral infections affecting humans, animals and plants. It is reasonable to imagine that the idea of viruses as therapeutic agents was quite a shock when first presented. The enormous advances in Molecular Biology, Biochemistry, Genomics and Human Medicine, among others, have provided to the Virology field the necessary tools for manipulating them on their behalf.

These recombinant vectors are viruses where the genome has been altered in a controlled way by experimental manipulation. For any procedure to generate a recombinant virus the starting point is to clone and manipulate its genome. Thus DNA virus genomes may be cloned directly while RNA virus genomes may be cloned as cDNA. These molecules can then be modified by site-specific alteration, or more drastically, segments may be removed and replaced with foreign DNA sequences. Then the process must be completed by recreating infectious virus particles. This requires specific techniques and is not yet possible for all virus types (Dimmock et al., 2007).

Regarding the biological value of these viral vectors, there are constantly novel potential applications such as vaccines, carriers of nucleic acid sequences for regulating gene expression and agents for gene therapy. In order to become a therapeutic agent the DNA has to be carried into the cell and ultimately reach the nucleus; therefore it is mandatory to be provided of an strategy for membrane cross and lysosomal scape. This is something that naked DNA is very poorly equipped to achieve. By contrast, the nucleic acid that is inside an infectious virus particle can avoid these issues. First, viruses have evolved specific interactions with cell surface molecules that lead to their efficient entry and, second, if that entry involves arrival in the cytoplasm within an endocytic vesicle, then viruses have mechanisms to allow efficient escape. This process of virus-mediated gene delivery into a cell is known as transduction (Dimmock et al., 2007).

It is clear then that, to be potentially useful as a gene delivery vector, a virus should have a number of specific features (Figure 2).

adenoviruses have been isolated and characterized giving birth to the *Adenoviridae* family of

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**Figure 2.** What does it take to be a useful viral vector? (Adapted from Dimmock N, Easton A and Leppard K; 2007)

The focus on AdV for gene transfer was based on basic research. The establishing of the biology of AdV and their capacity to efficiently deliver the viral genome to the target cells became relevant then. More importantly, since AdV was not oncogenic in humans and the genomes of common AdV were completely defined and easy to modify, the production of recombinant AdV (RAdV) was achieved. In the context of gene delivery, serotypes 5 and 2 of the subgroup C have been used the most because their structure and biology is well described and there are convenient biologic reagents available to produce recombinant subgroup C gene transfer vectors in large quantities. Regarding safety, AdVs of subgroup C can cause minor to mild respiratory infections sometimes associated with conjunctival compromise (Ginsberg et al.,

AdV virions consist of a ~36 kb linear double-stranded DNA genome encased within a non-

over 50 members.

1994).

enveloped icosahedral particle (Figure 3).

However, there is no virus that can meet all the criteria for an ideal gene delivery vector and there are some significant drawbacks that will be addressed in the following sections. Thus, each application is likely to need its own vector, chosen and then tailored to fulfill the precise requirements.

#### **3.2. Current viral vectors systems**

#### *3.2.1. Adenoviral vectors (Ad)*

The discovery and initial description of Adenoviruses (AdV) took place in the early 1950s. They were first isolated from human adenoid tissue cultures (Rowe et al., 1953). Since then several different serotypes of human, avian, reptilian, amphibian and other mammalian


**3. Viral gene delivery systems**

et al., 2007).

et al., 2007).

requirements.

number of specific features (Figure 2).

**3.2. Current viral vectors systems**

*3.2.1. Adenoviral vectors (Ad)*

**3.1. General overview of viruses as vectors for gene delivery**

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

For centuries the health sciences have invested sweat and tears in order to fight against viral infections affecting humans, animals and plants. It is reasonable to imagine that the idea of viruses as therapeutic agents was quite a shock when first presented. The enormous advances in Molecular Biology, Biochemistry, Genomics and Human Medicine, among others, have provided to the Virology field the necessary tools for manipulating them on their behalf.

These recombinant vectors are viruses where the genome has been altered in a controlled way by experimental manipulation. For any procedure to generate a recombinant virus the starting point is to clone and manipulate its genome. Thus DNA virus genomes may be cloned directly while RNA virus genomes may be cloned as cDNA. These molecules can then be modified by site-specific alteration, or more drastically, segments may be removed and replaced with foreign DNA sequences. Then the process must be completed by recreating infectious virus particles. This requires specific techniques and is not yet possible for all virus types (Dimmock

Regarding the biological value of these viral vectors, there are constantly novel potential applications such as vaccines, carriers of nucleic acid sequences for regulating gene expression and agents for gene therapy. In order to become a therapeutic agent the DNA has to be carried into the cell and ultimately reach the nucleus; therefore it is mandatory to be provided of an strategy for membrane cross and lysosomal scape. This is something that naked DNA is very poorly equipped to achieve. By contrast, the nucleic acid that is inside an infectious virus particle can avoid these issues. First, viruses have evolved specific interactions with cell surface molecules that lead to their efficient entry and, second, if that entry involves arrival in the cytoplasm within an endocytic vesicle, then viruses have mechanisms to allow efficient escape. This process of virus-mediated gene delivery into a cell is known as transduction (Dimmock

It is clear then that, to be potentially useful as a gene delivery vector, a virus should have a

However, there is no virus that can meet all the criteria for an ideal gene delivery vector and there are some significant drawbacks that will be addressed in the following sections. Thus, each application is likely to need its own vector, chosen and then tailored to fulfill the precise

The discovery and initial description of Adenoviruses (AdV) took place in the early 1950s. They were first isolated from human adenoid tissue cultures (Rowe et al., 1953). Since then several different serotypes of human, avian, reptilian, amphibian and other mammalian

**Figure 2.** What does it take to be a useful viral vector? (Adapted from Dimmock N, Easton A and Leppard K; 2007)

adenoviruses have been isolated and characterized giving birth to the *Adenoviridae* family of over 50 members.

The focus on AdV for gene transfer was based on basic research. The establishing of the biology of AdV and their capacity to efficiently deliver the viral genome to the target cells became relevant then. More importantly, since AdV was not oncogenic in humans and the genomes of common AdV were completely defined and easy to modify, the production of recombinant AdV (RAdV) was achieved. In the context of gene delivery, serotypes 5 and 2 of the subgroup C have been used the most because their structure and biology is well described and there are convenient biologic reagents available to produce recombinant subgroup C gene transfer vectors in large quantities. Regarding safety, AdVs of subgroup C can cause minor to mild respiratory infections sometimes associated with conjunctival compromise (Ginsberg et al., 1994).

AdV virions consist of a ~36 kb linear double-stranded DNA genome encased within a nonenveloped icosahedral particle (Figure 3).

**Figure 3.** Adenovirus particle structure

The experimental manipulation of its genome has allowed the production of a diversity of recombinant viral particles where most of the replication genes are removed. The deletion of the E1 and E3 regions in first-generation AdV allows ~7 kb of foreign DNA to be inserted into the vector genome (Volpers and Kochanek, 2004). Another feature of these vectors is that they can be grown to extremely high titers in the HEK 293 cell line, with burst sizes typically between 103 -104 viral particles (VP) per cell and final concentrations reaching 1013 VP/ml, after CsCl density gradient centrifugation. Whereas AdV vectors can achieve high expression in many target organs when used *in vivo*, expression of the transgene is limited to days or weeks, mainly because innate and adaptive immune host defenses against the virus. For applications where persistent expression is required to achieve a therapeutic goal, the modern, third-generation, high capacity AdV vectors have become the most efficient alternative (Hackett NR and Crystal RG, 2009). Other important obstacle in the use of RAd for gene transfer is the process of cell attachment and internalization used by the viral particles. The target cell must express the cell membrane receptor CAR (Coxsackie-Adenovirus Receptor) in order to be susceptible to the adenoviral infection (Figure 4).

For those transduction-refractory tissues, modern virology has developed modified-tropism RAds with modifications in fiber/high affinity receptor or the penton–integrin of the capsid. In an extensive survey of the tropism of AdV5-derived vectors but with fibers derived from different serotypes, the fiber genes of AdV16 were found to be better at targeting fibroblasts and chondrocytes, AdV35 at targeting dendritic cells and melanocytes and AdV50 better at targeting myoblasts and hematopoietic stem cells (Havenga et al., 2002). The addition of an oligolysine motif to the C-terminus of the fiber protein, giving the virus an affinity for polyanions such as heparin sulphate, profoundly affects the range of cell types that can be

infected *in vitro* allowing cells lacking CAR to be transduced by RAdVs (Bouri et al., 1999). For a more detailed description of Adenoviral vectors see section 3.3 of the present chapter.

**Figure 4.** Recombinant adenovirus enters cells via CAR-mediated binding allowing internalization via receptor-medi‐ ated endocytosis through clathrin-coated vesicles. Inside the cytoplasm, the endocytosed adenoviral vector escapes from the endosomes, disassembles the capsid and the viral DNA enter into the nucleus through the nuclear envelope pore complex. The viral DNA is not incorporated into the host cell genome, but rather assumes an epichromosomal location, where it can still use the transcriptional and translational machinery of the host cell to synthesize recombi‐

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AAV is a human parvovirus within the genus *Dependovirus.* It was originally observed as a contaminant of laboratory preparations of adenovirus (Carter et al., 2009). Viral particles are small (20–30 nm) and non-enveloped, containing single-stranded DNA molecules with plus

*3.2.2. Adeno-associated virus Vectors (AAV)*

nant protein (From: Pankajakshan Divya and Devendra K. Agrawal, 2013).

**Figure 4.** Recombinant adenovirus enters cells via CAR-mediated binding allowing internalization via receptor-medi‐ ated endocytosis through clathrin-coated vesicles. Inside the cytoplasm, the endocytosed adenoviral vector escapes from the endosomes, disassembles the capsid and the viral DNA enter into the nucleus through the nuclear envelope pore complex. The viral DNA is not incorporated into the host cell genome, but rather assumes an epichromosomal location, where it can still use the transcriptional and translational machinery of the host cell to synthesize recombi‐ nant protein (From: Pankajakshan Divya and Devendra K. Agrawal, 2013).

infected *in vitro* allowing cells lacking CAR to be transduced by RAdVs (Bouri et al., 1999). For a more detailed description of Adenoviral vectors see section 3.3 of the present chapter.

#### *3.2.2. Adeno-associated virus Vectors (AAV)*

**Figure 3.** Adenovirus particle structure

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

adenoviral infection (Figure 4).

103 -104

The experimental manipulation of its genome has allowed the production of a diversity of recombinant viral particles where most of the replication genes are removed. The deletion of the E1 and E3 regions in first-generation AdV allows ~7 kb of foreign DNA to be inserted into the vector genome (Volpers and Kochanek, 2004). Another feature of these vectors is that they can be grown to extremely high titers in the HEK 293 cell line, with burst sizes typically between

For those transduction-refractory tissues, modern virology has developed modified-tropism RAds with modifications in fiber/high affinity receptor or the penton–integrin of the capsid. In an extensive survey of the tropism of AdV5-derived vectors but with fibers derived from different serotypes, the fiber genes of AdV16 were found to be better at targeting fibroblasts and chondrocytes, AdV35 at targeting dendritic cells and melanocytes and AdV50 better at targeting myoblasts and hematopoietic stem cells (Havenga et al., 2002). The addition of an oligolysine motif to the C-terminus of the fiber protein, giving the virus an affinity for polyanions such as heparin sulphate, profoundly affects the range of cell types that can be

 viral particles (VP) per cell and final concentrations reaching 1013 VP/ml, after CsCl density gradient centrifugation. Whereas AdV vectors can achieve high expression in many target organs when used *in vivo*, expression of the transgene is limited to days or weeks, mainly because innate and adaptive immune host defenses against the virus. For applications where persistent expression is required to achieve a therapeutic goal, the modern, third-generation, high capacity AdV vectors have become the most efficient alternative (Hackett NR and Crystal RG, 2009). Other important obstacle in the use of RAd for gene transfer is the process of cell attachment and internalization used by the viral particles. The target cell must express the cell membrane receptor CAR (Coxsackie-Adenovirus Receptor) in order to be susceptible to the

> AAV is a human parvovirus within the genus *Dependovirus.* It was originally observed as a contaminant of laboratory preparations of adenovirus (Carter et al., 2009). Viral particles are small (20–30 nm) and non-enveloped, containing single-stranded DNA molecules with plus

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 antibodies against the viral particles.

*3.2.4. Herpes Simplex Virus Vectors (HSV)*

(Wolfe et al., 2009).

(Wolfe et al., 2009).

*3.2.5. Sendai Virus Vectors (SeV)*

**3.3. Recombinant adenoviral vectors**

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

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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

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

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

recombinant viral vaccines for influenza prevention (Le et al., 2011).

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).

#### *3.2.3. Retroviral vectors (RV)*

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 vectors.

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 host cell chromosome (Somia, 2003).
