Hybrid Dual Vector Approach

The trans-splicing vectors can effectively deliver a large expression cassette if an optimal gene-splitting site can be identified and used. Similarly, the overlapping vectors also have the potential for efficient expression of a large gene should there exist a highly recombinogenic region in the target gene. A generic approach that can meet the need of any transgene would certainly be more appealing. With this in mind, we developed a novel hybrid vector system (Fig. 12.3) (Ghosh et al. 2008). In the hybrid system, transgene reconstitution can be accomplished by both ITR-dependent and ITR-independent pathways. A highly recombinogenic foreign DNA sequence is engineered into the intron regions in the trans-splicing vectors. The foreign DNA sequence can then mediate efficient homologous recombination (Fig. 12.3a, the overlapping pathway). As a result, transgene reconstitution is guaranteed irrespective of the gene-splitting site or the recombinogenic potential of the transgene. Alternatively, transgene reconstitution can also occur via ITR-mediated recombination (Fig. 12.3a, the trans-splicing pathway). Using the LacZ reporter gene as a template, we found that the performance of the hybrid vectors significantly surpassed that of other dual vector systems (Fig. 12.3b) (Ghosh et al. 2008). Preliminary studies with the 6-kb AH2-R19 mini-dystrophin gene also yielded promising results (Ghosh et al. 2008).

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