Practical considerations of recombinant adeno-associated virus-mediated gene transfer for treatment of retinal degenerations.

Background: Photoreceptor (PR) and retinal pigment epithelium (RPE) are the principal cell targets in retinal gene therapy. Recombinant adeno-associated virus (rAAV) has emerged as a very promising vector for gene therapy in hereditary retinal diseases. Gene transfer at different stages of the disease is a practical consideration for future clinical application.

Methods: A rAAV carrying the enhanced green fluorescent protein gene driven by a cytomegalovirus promoter was produced by either co-infecting the 293 cell line with E1-defective adenovirus and purified by CsCl(2) density gradient (CsCl(2)-rAAV), or by transfecting with an adenoviral helper plasmid and purified by iodixanol density gradient followed by heparin column chromatography (heparin-rAAV). The impact of different virus preparations on the patterns of transgene expression was investigated after subretinal injection. Furthermore, rAAV-mediated gene transfer was evaluated at both early and advanced stages of retinal degeneration in four disease models including the RCS rat, rd, RPE(65) (-)/(-) and cathepsin D mutant mice that are associated with PR- or RPE-related gene defects.

Results: CsCl(2)-rAAV predominantly transduced RPE and with less efficiency in PR. In contrast, heparin-rAAV predominantly transduced PR but with much less efficiency in RPE. Subretinal injection of either rAAV preparation induced no changes to retinal morphology and retinal-choroidal vasculature. The product of transgene, however, could be observed in multiple tracts in the brain. In the four disease models, target cells were efficiently transduced not only at the early stage, but also at the late stage of disease as long as the target cells were present.

Conclusions: Different preparations of rAAV have an impact on the patterns of transgene expression after subretinal injection. Patients at advanced stages of retinal degeneration may still benefit from rAAV-mediated gene therapy. The possible side effects of transgenic products on the central nervous system should be carefully monitored once therapeutic genes are employed.