Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 248

neuronal cell of interest. Initially, strong promoter systems such as the human CMV
IE promoter, the SV40 enhancer, and the RSV LTR were used to drive gene expres-
sion. Such promoter systems were capable of expression, but they were only active
transiently (1 week) and did not result in long-term gene expression. Neuronal-
specific promoters such as the neurofilament and neuronal-specific enolase promot-
ers, which are supposed to be constitutively active in neurons, also produced only
transient expression in several HSV vector constructs [195,196] .
At the stage of HSV latency, the only viral transcripts constantly detected are the
latency associated transcripts (LATs), and the possibility that the LATs are consti-
tutively expressed in latently infected neurons has made them strong candidates for
long-term gene expression in neuronal systems. The identification of transcriptional
activators and suppression mechanisms, which may determine functionality in any
promoter system, is a difficult task, considering the modulation that takes place in
specific cell types and culture systems. Moreover, it has been shown that plasmid-
derived vectors utilizing HSV-1 promoters are more resistant to short-term inacti-
vation and capable of long-term gene expression [197] . One probable justification
could be the high copy number of amplicon molecules delivered to individual cells
[198] and the low-level IE gene activity during latency [199] . In spite of the process
of sustained activity, IE promoters may serve as useful promoter systems in experi-
mental gene transfer vectors. Multiple deleted genomic HSV vectors, by deletion of
ICP4, are adequate to create a vector that is incapable of replicating, and the remain-
ing IE gene products (ICP0, ICP22, ICP27, and ICP47) are abundantly expressed.
The majority of these gene products can drastically alter cell metabolism and gene
expression, and may be toxic to cells. Vector toxicity has been determined by engi-
neering virus mutants deleted for all the IE genes [200,201] .
5.5.3 Application of Herpes Viral Vectors
5.5.3.1 Targeting HSV-Attenuated Vectors
Targeted gene therapy is generally required to prevent damage of healthy tissues and
decrease the risk of germ line transduction and to design vectors that can be admini-
stered intravenously. Genetic alternations to the genome of HSV-1 vectors have been
generated to preferentially target viral infection and/or replication to tumor cells as
opposed to normal cells [202] . Targeting viral infection to particular cells can be
achieved by modifying the initial steps of the virus life cycle, that is, adsorption
and penetration. Modification of the HSV-1 host range has proved a formidable task
because HSV-1 infection is a complex process involving the action of several glyco-
proteins in cell attachment, entry, and cell-to-cell spread.
The first approach to target replication of the attenuated virus is attained by elimi-
nating viral functions essential for replication in normal cells. These mutations give
the virus the attenuated phenotype, which leads to replication only in permitted cells
such as dividing tumor cells or cells with defects in specific cancer pathways [203] .
However, a limitation of this strategy is that many normal tissues also have high
mitotic indices, and such viruses may not discriminate between rapidly proliferating