Self-Complementary AAV5 Vector Facilitates Rapid Transgene Expression in Photoreceptor and Retinal Pigmental Epithelium Cells of the Mouse

Abstract

Purpose: : To clarify whether transduction efficiency and specificity of AAV5 vector containing self-complementary duplex strands (sc) in the retina are similar to that of traditional single strand (ss) AAV5 vectors.

Methods: : One microliter of scAAV5-smCBA-GFP vector (1X10⁹ genome containing particles/ml) and ssAAV5-smCBA-GFP vector (1X10⁹ genome containing particles/ml) were subretinally or intravitreally (through cornea) injected into right and left eyes of adult C57BL/6 mice, respectively. At post-injection day (PID) 1, 2, 5, 7, 10, 14, 21, and 35, eyes were enucleated and neuroretinal wholemount, retinal pigmental epithelium (RPE) wholemount and eyecup sections were prepared to evaluate the GFP expression by fluorescent microscopy.

Results: : GFP expression using subretinal scAAV5 was first detected in both RPE and photoreceptor (PR) cells at PID 2, and strong GFP fluorescence was detected in most RPE and PR cells at PID 35 from either RPE, neuroretinal wholemounts or transverse retinal sections. In contrast, GFP expression using subretinal ssAAV5 was first detected in RPE cells at PID 7 and in PR cells at PID 10; at PID 35, GFP expression from ssAAV5 was similar to that of scAAV5 at PID 14 in RPE cells and weaker than that of scAAV5 at PID 10 in PR cells by evaluating the fluorescence intensity and distribution pattern of GFP-containing cells from either RPE, neuroretinal wholemounts or transverse retinal sections. No GFP fluorescence was detected in any retinal cells after trans-corneal intravitreal delivery of either scAAV5-GFP vector or ssAAV5-GFP vector from either RPE, neuroretinal wholemounts or transverse retinal sections.

Conclusions: : scAAV5 vector has a more rapid transgene expression onset and stronger overall transgene expression than those of ssAAV5 vector although they have similar transduction specificities to RPE and PR cells. scAAV5 vector may therefore be considered as primary vector candidates for RPE and/or PR cells related gene therapy, particularly for those diseases requiring rapid and robust transgene expression to prevent RPE and/or PR cells from early degeneration soon after birth.

Keywords: gene transfer/gene therapy • gene/expression • retina