Expanding AAV packaging capacity with trans-splicing or overlapping vectors: A quantitative comparison

Recombinant adeno-associated (rAAV) viral vectors hold great therapeutic po tential for human diseases. However, a relatively small packaging capacity (less than 5 kb) has limited the application of rAAV for certain diseases s uch as cystic fibrosis and Duchenne muscular dystrophy. Here we compared tw o mechanistically distinct approaches to overcome packaging restraints with rAAV vectors. The trans-splicing approach reconstitutes gene expression fr om two independent rAAV vectors, each encoding unique, nonoverlapping halve s of a transgene. This process requires intermolecular concatamerization an d subsequent splicing between independent vectors. A distinct overlapping v ector approach uses homologous recombination between overlapping regions in two independent vectors. Using the p-galactosidase gene as template, trans -splicing approaches were threefold (in primary fibroblasts) and 12-fold (i n muscle tissue) more effective in generating full-length transgene product s than the overlapping vector approach. Nevertheless, the efficiency of tra ns-splicing remained moderate at approximately 4.3% (for muscle) and 7% (fo r fibroblasts) of that seen with a single vector encoding the full-length t ransgene. The efficiency of trans-splicing was augmented 185-fold by adenov iral E4, but not E2a, gene products. This augmentation was much less pronou nced with the overlapping vectoring approach (12-fold). Transsplicing and o verlapping vector approaches are two viable alternatives to expand rAAV pac kaging capacity.