Triple Vectors Expand AAV Transfer Capacity in the Retina

Abstract

Retinal gene transfer with adeno-associated viral (AAV) vectors holds great promise for the treatment of inherited retinal degenerations (IRDs). One limit of AAV is its transfer capacity of about 5 kb, which can be expanded to about 9 kb, using dual AAV vectors. This strategy would still not suffice for treatment of IRDs such as Usher syndrome type 1D or Alström syndrome type I (ALMS) due to mutations in CDH23 or ALMS1, respectively. To overcome this limitation, we generated triple AAV vectors, with a maximal transfer capacity of about 14 kb. Transcriptomic analysis following triple AAV transduction showed the expected full-length products along a number of aberrant transcripts. However, only the full-length transcripts are efficiently translated in vivo. We additionally showed that approximately 4% of mouse photoreceptors are transduced by triple AAV vectors and showed correct localization of recombinant ALMS1. The low-photoreceptor transduction levels might justify the modest and transient improvement we observe in the retina of a mouse model of ALMS. However, the levels of transduction mediated by triple AAV vectors in pig retina reached 40% of those observed with single vectors, and this bodes well for further improving the efficiency of triple AAV vectors in the retina.

A dual‐AAV approach restores fast exocytosis and partially rescues auditory function in deaf otoferlin knock‐out mice

“…The correct reassembly of the full-length expression cassette in the nuclei of target cells results in the production of full-length mRNA and protein (Yan et al, 2000;Duan et al, 2001;Chamberlain et al, 2016). To date, dual-and triple-AAV vectors have demonstrated efficacy in photoreceptors and muscle cells (Duan et al, 2001;Ghosh et al, 2008;Trapani et al, 2014;Maddalena et al, 2018), but have not been established for IHCs.…”

Gene therapy and the adeno‐associated virus in the treatment of genetic and acquired ophthalmic diseases in humans: Trials, future directions and safety considerations

“…This group also tested an AAV8 serotype and found that there was more expression of the transgene in photoreceptors when compared to AAV2 serotypes. In the case of USH 1D (CDH23 mutation) and Alström syndrome type 1 (ALMS1 mutation), larger payloads are necessary, and recently triple AAV vector strategies have allowed transfer capacities up to about 14 kb, albeit with substantially reduced photoreceptor transduction efficiencies . Further investigation will be needed to determine the utility of dual and triple vectors for delivering larger genes into the eyes.…”

“…In the case of USH 1D (CDH23 mutation) and Alström syndrome type 1 (ALMS1 mutation), larger payloads are necessary, and recently triple AAV vector strategies have allowed transfer capacities up to about 14 kb, albeit with substantially reduced photoreceptor transduction efficiencies. 120 Further investigation will be needed to determine the utility of dual and triple vectors for delivering larger genes into the eyes. If their reduced transduction efficiencies cannot be overcome, alternative methods such as viral vectors with larger capacities like lentiviruses, or gene editing techniques such as CRISPR/Cas strategies may be employed; however, both involve risks of direct alteration of the host genome.…”

Highly efficient and super-bright neurocircuit tracing using vector mixing-based virus cocktail

“…Furthermore, MAP-ENVIVIDERS also provides great potential to facilitate the coexpression of multiple genes and reduce the harmful effects for clinical gene therapy [51][52][53][54][55][56] . labeling.…”