Evaluating Efficiencies of Dual AAV Approaches for Retinal Targeting

Abstract

Retinal gene therapy has come a long way in the last few decades and the development and improvement of new gene delivery technologies has been exponential. The recent promising results from the first clinical trials for inherited retinal degeneration due to mutations in RPE65 have provided a major breakthrough in the field and have helped cement the use of recombinant adeno-associated viruses (AAV) as the major tool for retinal gene supplementation. One of the key problems of AAV however, is its limited capacity for packaging genomic information to a maximum of around 4.8 kb. Previous studies have demonstrated that homologous recombination and/or inverted terminal repeat (ITR) mediated concatemerization of two overlapping AAV vectors can partially overcome the size limitation and help deliver larger transgenes. The aim of this study was to investigate and compare the use of different AAV dual-vector approaches in the mouse retina using a systematic approach comparing efficiencies in vitro and in vivo using a unique oversized reporter construct. We show that the hybrid approach relying on vector genome concatemerization by highly recombinogenic sequences and ITRs sequence overlap offers the best levels of reconstitution both in vitro and in vivo compared to trans-splicing and overlap strategies. Our data also demonstrate that dose and vector serotype do not affect reconstitution efficiency but a discrepancy between mRNA and protein expression data suggests a bottleneck affecting translation.

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

“…In order to achieve this goal, the process of joining the two vector genomes in the correct orientation, the transcription efficiency, and the translation of the full‐length transcript will need to be enhanced, e.g., by using codon‐optimized cDNA. Nevertheless, improving the large transgene reconstitution efficacy by simply increasing the amount of available vector genomes does not improve protein expression levels as shown for dual‐AAV vectors in the retina (Carvalho et al , ). Since the IHC transduction rates of our single AAV2/6 eGFP‐expressing virus reached 99%, it is unlikely that other AAV serotypes such as AAV2/Anc80 (Landegger et al , ; Suzuki et al , ) will increase the amount of fully functional otoferlin protein levels in IHCs.…”

Can Adeno-Associated Viral Vectors Deliver Effectively Large Genes?

“…70 Similarly, a study comparing different strategies for dysferlin delivery to the muscle identified the overlapping as the most efficient approach, although the hybrid vectors relied on a region of homology derived from an intron of the dysferlin gene, which might have been not sufficiently recombinogenic. 41 Differently, in the retina, low levels of overlapping vectors-mediated transduction have been generally found in the terminally differentiated photoreceptors across different studies, 52,71,72 whereas more efficient reconstitution was found in the retinal pigmented epithelium. 52 Optimization of the overlapping region and use of serotypes with high retinal transduction ability were found to be prerequisites to achieve sustained levels of transgene expression.…”

“…53,73 In the absence of identification of optimal overlapping regions in the transgene or in the presence of poor splitting sites, the hybrid approach has been found to significantly increase the efficiency of transgene reconstitution compared with overlapping or trans-splicing vectors. 51,52 In the retina, various studies have shown the ability of hybrid vectors to reconstitute large transgenes, 52,[72][73][74] at levels that were higher compared with the other dual AAV strategies side-by-side tested 52,72,73 and that resulted in improvement of the retinal phenotype of animal models of inherited retinal diseases. 52,75 As a confirmation, prompted by the promising results obtained in the delivery of the large MYO7A gene to the retina of the Usher 1B mouse model, 52 a phase I/II clinical trial has been planned to investigate the efficacy of hybrid vectors in Usher 1B patients (https:// cordis.europa.eu/project/rcn/212674_it.html).…”

Suprachoroidal Delivery of Viral and Nonviral Gene Therapy for Retinal Diseases

“…Recent AAV technological advances are under preclinical investigation and are being considered for clinical development. This technology includes dual AAV [53][54][55] and triple AAV, 56 in which case, a large transgene is split into 2 or 3 separate vectors to ultimately reconstitute to a full-length gene, as well as intein-mediated protein trans-splicing, 57 which reconstitute large proteins from shorter precursor polypeptides (separately encoded by 2 or more independent AAV vectors). Although convincible preclinical data have been generated through subretinal administration, 58 these platforms have not yet been assessed by the SC route.…”