Amplicons as an Alternative Vector System

Often referred to as ''defective'' HSV-1 vectors, amplicons are plasmids engineered to contain both an HSV origin of replication and packaging signals and a bacterial origin of replication (54). Amplicons are propagated in bacteria and then cotransfected with a defective HSV ''helper'' virus to create a mixed population of HSV particles containing either the defective HSV helper genome or concatemers of the plasmid packaged within an HSV capsid. In concept, the production of virion-packaged amplicons uses transient complementation of the entire HSV genome to provide replication machinery and viral structural components. A comparison of replication-defective genomic HSV vectors and helper virusfree amplicons are shown in Figs. 5A and B). Amplicons have been used to express reporter genes (55-61) or biologically active peptides (55,62-70) transiently in tissue culture systems.

In vivo, prolonged expression of both a lacZ reporter gene (59,71-75) and of the TH gene (71) following amplicon injection into brain have been reported. However, the production of amplicons requires repeated passaging of the amplicon/ helper virus preparation, which results in the emergence of recombinant wild-type virus that, although estimated to occur at the low frequency of 10~5 (71,72), results in the death of 10% of infected animals in experiments in vivo (71). The production of true helper virus-free amplicon preparations using multiple restriction fragments of the helper virus genome that lack packaging signals has recently been reported (76). However, the maximal yield obtained with that method has remained low (< 107 pfu/mL), and expression in vivo has not been fully tested (76). The presence of cytotoxic helper virus and the generation of replication-competent contaminants represent technical hurdles to the effective production and use of amplicons in human patients. Helper-free ampli-cons will likely require the development of new helper systems to make their use practical enough for human applications.

Figure 4 Comparison of viral vector payload capacities. Schematic diagram of various viral vector genomes currently in use for gene transfer and therapy studies, including the overall size of the entire vector genome. The HSV-1 vector, which contains a 38-kb deletion of sequences comprising the joint region and the entire unique short (US) segment of the viral genome (ICP4", ICP22~, ICP27~), can accommodate foreign trangene sequenes that are larger than lentivirus or AAV vectors and equivalent in size to the complete adenoviral (AdV) genome.

Figure 4 Comparison of viral vector payload capacities. Schematic diagram of various viral vector genomes currently in use for gene transfer and therapy studies, including the overall size of the entire vector genome. The HSV-1 vector, which contains a 38-kb deletion of sequences comprising the joint region and the entire unique short (US) segment of the viral genome (ICP4", ICP22~, ICP27~), can accommodate foreign trangene sequenes that are larger than lentivirus or AAV vectors and equivalent in size to the complete adenoviral (AdV) genome.

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