Effective delivery of large genes to the retina by dual AAV vectors

Abstract

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.

Gene Therapy of ABCA4-Associated Diseases

“…The results from these initial trials suggest that retinal gene therapy with AAV is safe in humans, that vision can be improved in patients that have suffered from severe impairment of visual function, in some cases for decades, and that readministration of AAV to the subretinal space is feasible, effective, and safe. This, as well as the recent evidence of the efficacy of dual AAV-mediated ABCA4 delivery in mice (Trapani et al 2014), strongly supports further investigation using AAV for treatment of diseases caused by ABCA4 mutation.…”

“…In the overlapping approach, the dual AAV genomes share overlapping sequences, thus the reconstitution of the large gene expression cassette relies on homologous recombination (Duan et al 2001). The third dual AAV approach (hybrid dual) is a combination of the two previous approaches and it is based on the addition of a highly recombinogenic exogenous sequence to the trans-splicing vectors, to increase their recombination efficiency (Ghosh et al 2008(Ghosh et al , 2011Trapani et al 2014). This recombinogenic sequence is placed downstream from the SD signal in the 5 0 -half vector and upstream of the SA signal in the 3 0 -half vector, so that, after recombination, it is spliced out from the mature RNA (Table 1).…”

“…This recombinogenic sequence is placed downstream from the SD signal in the 5 0 -half vector and upstream of the SA signal in the 3 0 -half vector, so that, after recombination, it is spliced out from the mature RNA (Table 1). The recombinogenic sequences used so far to induce the recombination between dual AAV hybrid vectors derive from regions of either the alkaline phosphatase gene (AP) (Ghosh et al 2008(Ghosh et al , 2011 or the F1 phage genome (AK) (Trapani et al 2014). We recently compared side-by-side the efficiency of the various oversized, dual AAV overlapping, trans-splicing, and hybrid strategies for AAVmediated large gene transduction in the mouse and pig retina (Trapani et al 2014), and found that dual AAV trans-splicing and hybrid AKvectors efficiently reconstitute the large ABCA4 gene in mouse PRs.…”

“…The recombinogenic sequences used so far to induce the recombination between dual AAV hybrid vectors derive from regions of either the alkaline phosphatase gene (AP) (Ghosh et al 2008(Ghosh et al , 2011 or the F1 phage genome (AK) (Trapani et al 2014). We recently compared side-by-side the efficiency of the various oversized, dual AAV overlapping, trans-splicing, and hybrid strategies for AAVmediated large gene transduction in the mouse and pig retina (Trapani et al 2014), and found that dual AAV trans-splicing and hybrid AKvectors efficiently reconstitute the large ABCA4 gene in mouse PRs. Notably, subretinal administration of dual AAV trans-splicing and hybrid AK vectors improved the phenotype of a mouse model of STGD1, providing evidence for the efficacy of these strategies for ABCA4 gene therapy.…”

A Fragmented Adeno-Associated Viral Dual Vector Strategy for Treatment of Diseases Caused by Mutations in Large Genes Leads to Expression of Hybrid Transcripts

“…The success of the approach relies on the majority of AAV particles containing large enough fragments of the oversized transgene such that complementary overlaps between truncated transgenes exist. Within the host nucleus these have been predicted to regenerate the original oversized transgene through homologous recombination [7] or annealing of plus and minus strand complementary regions prior to second-strand synthesis [8], Trapani et al have shown success with the fAAV approach in HEK293 cells [9] and Hirsch et al suggested fAAV vectors are a better gene therapy approach for delivering large genes to the retina and skeletal muscle than an alternative trans-splicing dual vector approach [10]. The mechanisms by which transgenes recombine are still being elucidated but both non-homologous end joining (NHEJ) [11] and homologous recombination [10] has been implicated.…”