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Microbes & Immunity Genetic therapy with HSV-1 vectors

virus-independent packaging system, its helper function is non-replicating and unintegrated vector DNA in host cells,
provided by a series of cosmids with overlapping regions and whether amplicon vectors can be widely used must
carrying the HSV-1 genome (with the pac packaging signal overcome these difficulties. The amplicon production and
removed). Recently, bacterial artificial chromosome purification procedures also need further improvement.
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(BAC) technology has been used to clone the entire HSV-1 At present, we still cannot fully understand which viral
genome lacking the pac signal into the BAC, which can proteins are involved in regulating gene expression, thus
efficiently replicate DNA in bacteria and effectively exert the directly affecting amplicon carrier gene expression.
function of HSV-1 after entering cells, greatly simplifying
the system and significantly improving its efficiency. 4.2. Replication-deficient vector
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There is also a Cre/loxP site-specific recombination system Replication-deficient vectors selectively mutate or delete
that is missing the packaging signal for helper viruses some genes essential for growth in cell culture so that the
and replicates amplicons equally efficiently. Therefore, virus cannot replicate in normal cells unless complemented
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amplicon vectors have emerged as very useful tools in in trans in transformed cell lines. HSV-1 encodes at least
neurological studies. 32 84 genes, half of which are non-essential. Close to 30 kb in
In addition, there are many derived HSV-1 amplicon the genome can be replaced by foreign genes, which makes
vectors, such as HSV-1/adeno-associated virus (AAV) it possible for HSV-1 to carry one to several foreign genes
hybrid amplicons (Figure 4), which combine HSV-1’s for gene therapy.
ability to carry large volumes of DNA as well as site- Several replication-deficient vectors have been
directed integration of AAV and sustained expression of successfully constructed. Deletions were performed in
transgenes. HSV/AAV amplicons contain most of the different combinations using different IE genes. The first-
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properties of the virus, and adeno-associated virus can generation replication-deficient HSV-1 vector lacking ICP4
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integrate its genomic DNA into human chromosome 19, was named d120. Although this defective virus reduces
thereby increasing its stability. These methods have shown pathogenicity and efficiently transmits and transiently
that amplicons can safely deliver large fragments of DNA expresses reporters in the brain, it is toxic to cultured
into the mammalian nuclear environment, turning many neuronal cells, limiting its application. The codeletion
years of dreams into reality, and investigators have applied of five IE genes eliminates cytotoxicity and thus allows
this feature to multiple experimental systems. 32 the vector gene to be continuously expressed in cells, 43-45
further indicating that the cytotoxicity caused by the first-
Non-helper-dependent amplicon systems have been generation deletion is caused by the other four IEs. In
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widely used in many experimental models of gene therapy neuronal cells, the deletion allows sustained expression of
to study neural dysregulation and vaccine development. exogenous genes that are expressed starting with the ICP0
As gene transfer vectors, amplicons, such as neuro- IE promoter or the HCMV IE promoter. In the second
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factors such as nerve growth factor and brain-delivered generation, ICP4 and ICP27 are commonly deleted. Its
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neurotrophic factor, anti-apoptotic proteins, heat shock advantage lies in the absence of expression of viral early
proteins, or antioxidant enzymes, have been used to and late genes, providing sufficient space for independent
deliver and express exogenous genes for brain injury expression of different exogenous genes. As a third-
therapy. 40,41 Amplicons expressing neurotransmitters or generation HSV-1 viral vector, three non-essential genes,
neuroreceptors have also been used in studies of behavioral ICP6, ICP47, and γ34.5, are simultaneously knocked out
traits such as learning and memory. However, transgenes in G47∆, which has efficient and high biosafety oncolytic
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are usually expressed transiently, largely due to the loss of ability. 49
4.3. Replication attenuated vector
Deletion of some non-essential genes allows the virus to
replicate efficiently in vitro, but replication is blocked in vivo.
Several viral genes that affect viral replication, cytotoxicity,
and immune invasion have been identified, and these genes
usually interact with multiple cytokines, thereby optimizing
viral replication conditions and inhibiting cell growth. In
view of these characteristics, investigators have successfully
Figure 4. Schematic representation of the HSV-1 amplicon (left) and the constructed an HSV-1 deletion that restricts replication
HSV/AAV hybrid plasmid (right) in normal quiescent cells and efficiently replicates in both
Abbreviations: AAV: Adeno-associated virus; HSV: Herpes simplex virus;
ITR: Inverted terminal repeats. tumor cells and dividing cells.

Volume 2 Issue 2 (2025) 23 doi: 10.36922/mi.7947