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