Gene Delivery Using Viral Vectors - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 247

host, degrades host mRNA, helps in viral replication, and regulates gene expression
of viral proteins. The viral genome immediately travels to the nucleus, but the VHS
protein remains in the cytoplasm.
The capsid and the receptors on the surface of the virus are formed by the help of
late proteins. Packaging of the viral particles, including the genome, core, and capsid,
occurs in the nucleus of the cell. Concatemers of the viral genome are generally sep-
arated by cleavage and are placed into preformed capsids. HSV-1 normally under-
goes a process of primary and secondary envelopment, and the primary envelope is
acquired by budding into the inner nuclear membrane of the cell. This then fuses
with the outer nuclear membrane, releasing a naked capsid into the cytoplasm. The
virus acquires its final envelope by budding into cytoplasmic vesicles [191-193] .
5.5.2 Design and Construction of Herpes Simplex Viral Vectors
Generally, two main strategies for HSV-based vectors are in use today: geneti-
cally engineered viruses and plasmid-derived “amplicon” vectors. The first approach
involves the construction of recombinant viruses containing deletions in one or more
viral genes whose expression is crucial for viral replication. These viruses are incapa-
ble of producing a productive viral infection (replication incompetent) in normal cells.
For that, they require a complementing cell line that can supply the deleted protein(s)
to the virus in trans for replication. Sometimes foreign genes can be inserted into these
mutated viral genomes, with the goal of producing a virus vector that will infect the tar-
get cell and express the foreign gene without killing the cell (due to viral replication).
The second approach involves the use of plasmid-derived vectors containing HSV-1
having the origins of DNA replication and DNA packaging signals that enable multiple
copies of the vector genomes to be packaged into helper virus virions.
Helper viruses can be either recombinant viruses containing a deletion within a nec-
essary viral gene or viruses containing temperature-sensitive mutations that prevent rep-
lication at 37°C. In the former case, the replication of the helper virus and packaging
of the amplicon vector DNA must arise in a cell line that is capable of complementing
the mutations in the helper virus. Plasmid-derived vectors (amplicons) are more advanta-
geous because the DNA constructs can be easily manipulated to test endogenous, foreign,
antisense, or promoter gene expression in the target cell. Although the delivery efficiency
of these multiple copy vectors is high, the primary disadvantage of this system is the
fluctuating helper virus-amplicon ratio with passage, which may result in some infected
cells not receiving the amplicon genome. Viral titers must be monitored to ensure high
amplicon delivery and experimental reproducibility in the absence of wild-type recom-
binants [194] . In spite of the vector system used, two primary aims must be achieved to
facilitate long-term gene expression in neuronal cells. The first aim involves the construct
of mutant vectors that themselves are noncytotoxic to cells. Several studies have noted
active expression of a foreign gene by HSV vector constructs that subsequently became
inactivated due to cytotoxic effects induced by vector systems [186,194] .
The second aim involves designing stable, active promoters that are capable of
expressing appropriate levels of the foreign protein. The specific promoter involved
in individual therapies may change according to the type, status, and activity of the