Recombinant adeno-associated viral vectors (rAAV)-vector elements in ocular gene therapy clinical trials and transgene expression and bioactivity assays

Buck et al. (2020). Int J Mol Sci DOI: 10.3390/ijms21124197 Citations: 101

Ocular AAV vectors were engineered without a systematic account of which cassette parts clinical trials actually used. This review catalogued 33 rAAV cassettes from 57 trials, covering six capsids, sixteen promoters, and six polyadenylation signals.

Parts used

AAV2-7M8 SerotypeCAG PromoterCBA PromoterCBh PromoterCMV PromoterhRho Promoter

Key findings

  • The meta-analysis identified 33 unique rAAV gene cassettes deployed across 57 ocular gene therapy clinical trials between 2007-2020, revealing systematic use of 6 capsid variants (AAV2, AAV2-tYF, AAV2-7m8, AAV4, AAV5, AAV8), 16 distinct promoters, and 6 polyadenylation sequences. AAV2 variants dominated early trials (>70% before 2015) while AAV5 and AAV8 captured 45% of clinical products after 2015, reflecting improved RPE transduction efficiency that reduced required viral doses below 10^12 vector genomes to minimize inflammation.

  • Systematic promoter engineering reduced rAAV cassette sizes while maintaining expression levels, with shortened ubiquitous promoters (smCBA 953bp, CBh 800bp, mini-CAG 250bp) replacing full-length CAG (1600bp) and tissue-specific promoters achieving 150× potency gains through enhancer fusion and codon optimization. The NA65p promoter (750bp) demonstrated 150-fold higher RPE expression versus CBA in mouse models while maintaining cell specificity, and photoreceptor-targeting hGRK1 (294bp) efficiently transduced rods and cones in non-human primates with comparable efficacy to 2100bp native promoters.

  • The review catalogued validated transgene expression assays across human iPSC-derived retinal organoids, primary tissue explants, and cell lines, establishing that rAAV-CMV and rAAV-CAG vectors maintained expression for 2-12 weeks in diseased retinal models without silencing. Human donor retinal explants exhibited serotype-specific tropism with AAV2 transducing ganglion cells, AAV5 targeting RPE cells at 3-fold higher efficiency than AAV2, and AAV8 preferentially infecting photoreceptors, validating cross-species translational predictability for clinical trial design.

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Abstract

Inherited retinal dystrophies and optic neuropathies cause chronic disabling loss of visual function. The development of recombinant adeno-associated viral vectors (rAAV) gene therapies in all disease fields have been promising, but the translation to the clinic has been slow. The safety and efficacy profiles of rAAV are linked to the dose of applied vectors. DNA changes in the rAAV gene cassette affect potency, the expression pattern (cell-specificity), and the production yield. Here, we present a library of rAAV vectors and elements that provide a workflow to design novel vectors. We first performed a meta-analysis on recombinant rAAV elements in clinical trials (2007-2020) for ocular gene therapies. We analyzed 33 unique rAAV gene cassettes used in 57 ocular clinical trials. The rAAV gene therapy vectors used six unique capsid variants, 16 different promoters, and six unique polyadenylation sequences. Further, we compiled a list of promoters, enhancers, and other sequences used in current rAAV gene cassettes in preclinical studies. Then, we give an update on pro-viral plasmid backbones used to produce the gene therapy vectors, inverted terminal repeats, production yield, and rAAV safety considerations. Finally, we assess rAAV transgene and bioactivity assays applied to cells or organoids in vitro, explants ex vivo, and clinical studies.