Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison.
Recombinant adeno-associated (rAAV) viral vectors hold great therapeutic potential for human diseases. However, a relatively small packaging capacity (less than 5 kb) has limited the application of rAAV for certain diseases such as cystic fibrosis and Duchenne muscular dystrophy. Here we compared two mechanistically distinct approaches to overcome packaging restraints with rAAV vectors. The trans-splicing approach reconstitutes gene expression from two independent rAAV vectors, each encoding unique, nonoverlapping halves of a transgene. This process requires intermolecular concatamerization and subsequent splicing between independent vectors. A distinct overlapping vector approach uses homologous recombination between overlapping regions in two independent vectors. Using the beta-galactosidase gene as template, trans-splicing approaches were threefold (in primary fibroblasts) and 12-fold (in muscle tissue) more effective in generating full-length transgene products than the overlapping vector approach. Nevertheless, the efficiency of trans-splicing remained moderate at approximately 4.3% (for muscle) and 7% (for fibroblasts) of that seen with a single vector encoding the full-length transgene. The efficiency of trans-splicing was augmented 1185-fold by adenoviral E4, but not E2a, gene products. This augmentation was much less pronounced with the overlapping vectoring approach (12-fold). Trans-splicing and overlapping vector approaches are two viable alternatives to expand rAAV packaging capacity.
Duan
D
D
Department of Anatomy & Cell Biology, Center for Gene Therapy of Cystic Fibrosis and Other Genetic Diseases, The University of Iowa, Iowa City, 52242, USA. dongshen-duan@uiowa.edu
Yue
Y
Y
Engelhardt
J F
JF
D017502:Adenovirus E2 Proteins / Q000235:genetics / Q000378:metabolism; D017504:Adenovirus E4 Proteins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D000229:Dependovirus / Q000235:genetics* / Q000254:growth & development* / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism / Q000821:virology; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D009132:Muscles / Q000378:metabolism / Q000821:virology; D011533:Proviruses / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D020040:Trans-Splicing / Q000235:genetics*; D019076:Transgenes / Q000235:genetics; D019065:Virus Assembly; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D017502:Adenovirus E2 Proteins; D017504:Adenovirus E4 Proteins; D001616:beta-Galactosidase