A New, Non–Imunogenic Gene Therapy Vector for the Human Trabecular Meshwork: scAAV (self–complementary Adeno–Associated Virus)

Abstract

Abstract: : Purpose: Because glaucoma is a chronic disease, gene therapy regimens to control elevated IOP would need vectors that provide long–term expression in the trabecular meshwork (TM) without eliciting an inflammatory response. Such vectors, like AAV2, which corrected blindness in a retinal degeneration animal model, are to date unable to transduce the TM. The transduction limiting step in the TM is unknown. Our goal is to investigate whether newly developed AAV vectors overcome the non–permissive status of the TM cells and could be potentially used for gene therapy of glaucoma. Methods:A specialized AAV2 plasmid, carrying an eGFP reporter gene driven by the CMV promoter, was constructed by deleting 28 bases of one of the ITRs. This construct generates a single–stranded viral genome with a wild type ITR at each end and a mutated ITR in the middle of the sense and non–sense strands of GFP cDNA. The self–complementary scAAV.GFP virus was generated by triple transfection of the new construct plus packaging and Ad helper plasmids in 293 cells. The scAAV.GFP viral genome folds into a double–stranded (ds) molecule after infection. Primary HTM cells were infected with equal number of viral particles of either AAV.GFP or scAAV.GPF viruses. Post–mortem human anterior segments were perfused at 3 µl/min constant flow for 24 h. One eye of each pair was then injected with AAV.GFP while the contralateral was injected with equal number of particles of scAAV.GFP (n=3, two doses). Transduction was monitored by daily fluorescence microscopy of the infected cells and by fluorescence analysis of frozen tissue sections at 8 days post–infection. Results: HTM cells infected with AAV.GFP showed little to none transduction up to 5 days post–infection. About 90% of the HTM cells infected with scAAV.GFP showed positive transduction starting 18 h post–infection and maintained to the end of the 5 days experiment. Histological sections from opposite quadrants from intact human perfused TM tissue showed very efficient transduction by scAAV.GFP, while barely transduction was observed on the eyes infected with AAV.GFP. Transduction was dose dependent. Conclusions:By bypassing host–mediated viral ds DNA synthesis, the new scAAV successfully overcomes the barrier of AAV transgene expression in the human TM. Since AAV–based vectors are currently the least immunogenic and longer expression vectors for in vivo eye gene transfer, scAAVs hold great promise for prolonged gene delivery to the TM and treatment of a chronic disease such as glaucoma.

Keywords: gene transfer/gene therapy • adenovirus • trabecular meshwork