Abstract
The long-term goal of our laboratory is to create replication defective HSV-1 based viral vectors that are suitable to support the clinical development of therapeutic gene delivery applications. In order to reach this goal three considerations that must be satisfied include; (1) proven efficacy of vector upon delivery to target cells, (2) safety of vector stock preparations, and (3) feasibility of clinical vector stock production. Efficacy is primarily dependent upon effective transgene expression for an appropriate duration without interference with host cell processes that might affect target cell viability. Thus among the most important considerations for vector design are strategies to eliminate vector toxicity and to provide for appropriate transgene expression. Various replication defective HSV-1 viral mutants based on the manipulation of immediate early genes have been described previously. However, the simultaneous evaluation of various combinations of these mutations and the impact on transgene expression has not been reported. We have created an expansive panel of replication defective IE gene mutant HSV-1 viruses and complementing cell lines to produce them. The IE gene ICP0 appears to mediate vector toxicity and also play an important role in the efficient expression of exogenous transgenes from either the ICP0 IE promoter or the widely used HCMV promoter. Large viral genomic deletions have no effect on virus mediated toxicity unless they result in the reduction of ICP0 expression while these large deletions appear to have a deleterious effect on transgene expression in the absence of ICP0 expression. Current productive capabilities of ICP0 mutants are now very similar to the growth characteristics of our previously described IC4/ICP22/ICP27 mutants due to improvements in ICP0/ICP4/ICP27 or ICP0/ICP4/ICP22/ICP27 complementing cells lines and growth conditions. Growth capabilities, vector mediated toxicity, transgene expression, and complementing cell line construction will be described in further detail.
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