Abstract
Purpose :
Recombinant adeno-associated virus (rAAV) vectors have become promising tools for mediating gene transfer to retinal neurons for the treatment of monogenic inherited retinal diseases. Unfortunately, due to the limited 4.8 kb coding capacity of rAAV and the large size of many therapeutic transgene cassettes, numerous retinal diseases cannot currently be treated using rAAV-mediated gene therapy. To circumvent this limitation, transduction with two or more vectors, each containing separate gene fragments, has been utilized. Although these studies have shown success, in vivo efficiencies remain around 10%. Due to this fact, our study aims to investigate the cellular transduction efficacy when multiple reporter transgenes are delivered to the murine retina.
Methods :
Female C57BL/6J mice (N=9) received bilateral intravitreal injections of one, two, or three recombinant AAV (rAAV) vectors packaging a ubiquitously expressing mCherry, green fluorescent protein (GFP), and/or blue fluorescent protein (BFP) transgene cassettes respectively (N=3/group). Dual fluorescence confocal scanning laser ophthalmoscopy (cSLO) was performed 4 weeks post-injection to assess transgene expression in vivo. Retinae were harvested and dissociated allowing quantification of transduction efficiency via flow cytometry.
Results :
cSLO imaging revealed dual injection of rAAV2/2[MAX].CBA.GFP and rAAV2/2[MAX].CBA.mCherry (N=6 eyes) resulted in widespread retinal transduction centered on the optic near head. The pattern of GFP and mChery expression was observed to substantial overlap, indicating efficient co-transduction following delivery of multiple vectors. Preliminary flow cytometry analysis of dissociated retinae revealed that up to 62.0% (±9.2%) of GFP+ve cells also co-expressed mCherry, indicating that co-transduction with multiple vectors is only partially efficient. Analysis of triple injected eyes in ongoing.
Conclusions :
This study indicates that the ability of multiple rAAV vectors to transduce individual retinal neurons following administration via a single injection is substantially higher than the recombination efficiencies previously reported, suggesting that co-transduction with rAAV vectors containing multiple fragments is not a limiting factor.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.