The vectors went in two-by-two: Transduction efficiency and tolerability of dual and triple rAAV vector delivery following intravitreal injection for genome-editing applications.

Abstract

Genome or prime editing has become a promising tool for the treatment of hereditary disorders affecting the inner retina, such as dominant optic neuropathies. In vivo delivery of gene editors, such as Cas9, is typically achieved using recombinant adeno-associated virus (rAAV) vectors, which have a broad range of cellular tropisms and are well tolerated following intravitreal administration. Owing to the large size of gene editing constructs and the limited carrying capacity of rAAV (<5.1kb) it is unfortunately usually necessary to split therapeutic transgene cassettes across multiple co-administered vector genomes. While the efficiency with which multiple vector genomes recombine following cellular entry has been studied extensively, another potentially limiting factor is the likelihood of target cells (e.g. retinal ganglion cells) receiving two or more vectors containing genomes that correspond to the full-length expression cassette when recombined. In this study we examine the efficiency with which two or more vector genomes transduce various retinal cell types following intravitreal administration. rAAV2/2[MAX] vectors expressing individual fluorescent reporters (GFP, BFP or mCherry) were co-injected intravitreally singly or in combination (dual or triple), allowing the extent of co-transduction to be assessed through multimodal in vivo imaging, electroretinography, flow cytometry and post-mortem histology. We find that intravitreal co-administration of vectors containing multiple genomes is well tolerated - with no observed alterations in retinal thickness or ERG amplitudes - but that co-transduction efficiency decreases significantly with increasing genome number. As such co-transduction of multiple vectors may be a major bottleneck limiting gene editing of inherited disorders affecting the inner retina.