Aav

Reovirus

General biology Genetic material Lytic?

Integration?

Features

Transgene capacity Titer

Virion stability

Specific antiviral agent available Ease of genetic manipulation Immunogenicity

Wild-type virus infects nonreplicating cells

Virulence of wild-type virus ds DNA Yes

30 kb

1010 PFU/mL High

Difficult

Moderate Yes

Yes ds RNA

No—requires transgene for tumor killing

Yes—nonspecific integration leading to risk of malignant transformation

106 PFU/mL

Low—need to graft packaging cell line

Easy

Low No

No ds DNA

No 10 kb

1012 PFU/mL

High

Easy

High

Slight ss DNA

Yes—site-specific integration

106 PFU/mL

High No

Easy

Low Yes

No ds RNA Yes

Not investigated 109 PFU/mL

High

Not investigated

Moderate No

Abbreviations: HSV-1, herpes simplex virus-1; AAV, adeno-associated virus; ds, double-stranded; ss, single-stranded.

tributed to the death of an 18-yr-old patient after an arterial infusion of a replication-defective adenovirus vector during a gene therapy trial for ornithine transcarbamylase deficiency. This death was attributed to a massive cytokine response to the adenovirus vector, resulting in disseminated intravascular coagulation.

Despite these concerns, adenoviruses are used in a large number of gene therapy trials (3). They are the most commonly used vector in cancer gene therapy trials (32% of these trials), and the second most commonly used vector in brain tumor gene therapy trials (16% of these trials).

Herpesviruses

HSV-1 is an enveloped, double-stranded linear DNA virus whose genome spans 152 kb, encoding more than 80 genes.

HSV-1 has been developed as a vector either by removing some of the essential genes needed for viral replication (recombinant HSV) or by removing the entire viral genome except for a small 300-bp sequence that provides packaging function (amplicon vector). This packaging signal when incorporated into any bacterial plasmid will allow this plasmid to be packaged into an infectious virion.

HSV-1 offers a number of advantages as a vector (Table 1). These advantages include: (1) the ease with which high titers (typically 1010 infectious particles/mL) can be generated; (2) potential for incorporating a large payload of foreign DNA—approx 30 kb of the HSV-1 genome have been estimated to be replaceable by foreign genes with minimal effects on titers or replication; (3) neurotropism, rendering gene delivery to the CNS more effective; (4) sensitivity to antiherpetic agents like ganciclovir, providing a safety mechanism by which viral replication could be abrogated; and (5) the fact that HSV-1 never integrates and persists as an episome even during latency, ensuring that the risk of insertional mutagenesis posed by retroviral vectors is not an issue with HSV-1 vectors. Furthermore, the lack of integration with HSV-1 vectors is not a concern with cancer gene therapy, as immediate tumor killing probably does not require long-term gene expression.

However, there are four challenges that arise when one is working with HSV-1 vectors. First, genetic manipulation of HSV-1 is difficult owing to the large size of the viral DNA. A second potential obstacle when using HSV-1 recombinant vectors is the fact that most humans have preexisting herpes immunity, which could potentially impair gene delivery. Approximately 60-90% of the adult population has been exposed to HSV-1, as determined by the detection of viral DNA and antibodies to HSV-1 in serum (5). Potential neurotoxicity is a third problem inherent to HSV-1 vectors. HSV-1 is a neurotropic human pathogen that can cause a life-threatening encephalitis from primary infection or from reactivation of latent virus. The introduction of HSV-1 vectors could lead to one of two scenarios that could cause a potentially fatal encephalitis: (1) recombination with latent wild-type HSV-1 could restore full replicative capacity to the introduced vector; or (2) the latent wild-type HSV-1 present in most humans could be reactivated by application of HSV-1 vectors. Finally, the ability of HSV-

1 vectors to infect both dividing and nondividing cells is undesirable for cancer therapy, which requires selective targeting of replicating cells.

Adeno-Associated Virus

Wild-type AAV and AAV vectors depend on the presence of adenovirus for their replication and lytic infection. The vectors can infect quiescent and proliferating cells. Long-term expression can be achieved owing to site-specific integration on human chromosome 19q and transgene amplification. Integration is more efficient in proliferating cells. Aside from the dependence on helper virus, limitations include a small transgene capacity (4.7 kb) and low titers (106 infectious particles/mL).

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