NGS-guided screening identifies AAV2-I.1 and AAV2-I.12 as potent, broad-tropism retinal gene therapy vectors in primates.
Background
Gene therapy using adeno-associated virus (AAV) vectors holds significant promise for treating inherited retinal diseases (IRDs) and protecting retinal ganglion cells (RGCs) from degeneration. However, current AAV vectors face limitations, including potential safety risks following intravitreal (IVT) administration, restricted packaging capacity, and insufficient cell-type specificity. Refining AAV vector technology to achieve potent, safe, and targeted retinal bioactivity remains a critical challenge, necessitating improved strategies for identifying novel, retina-tropic capsids.
Study Design
Researchers performed iterative in vivo NGS-guided screening of AAV2 peptide-display libraries in both mice and non-human primates (NHPs). The study integrated barcoded functional analytics and single-nuclei RNA sequencing to identify retina-tropic AAV variants. They tested the top-performing mouse capsid, AAV2-GAYPKSP, in a human retina-on-chip model. In NHPs, a barcoded evaluation of 70 retina-tropic variants was conducted following IVT delivery, comparing their performance against benchmark vectors AAV2 and AAV2-7m8.
Results
The top-performing capsid in mice, AAV2-GAYPKSP, robustly drove eGFP expression in a human retina-on-chip model. However, this variant failed to drive gene expression in the NHP retina following IVT delivery, highlighting species-specific differences. In NHPs, the barcoded evaluation of 70 retina-tropic variants revealed a complex correlation between viral genome enrichment and transgene transcriptional output. This provided insights that enhance the mechanistic interpretation and methodological design of AAV library selection strategies. Nonetheless, several NHP variants, including AAV2-I.1 and AAV2-I.12, demonstrated broad retinal transduction and potent expression.
These novel variants significantly outperformed established benchmarks,
AAV2andAAV2-7m8, in the NHP retina.Single-nucleus RNA sequencingfurther confirmed the broad cell-type tropism of these top-performing variants, indicating their potential for widespread gene delivery within the retina.
Key Findings
- NGS-guided screening identified novel AAV variants with enhanced retinal tropism and expression.
- AAV2-GAYPKSP showed robust
eGFPexpression in human retina-on-chip but failed in NHP retina. - AAV2-I.1 and AAV2-I.12 demonstrated broad retinal transduction and potent expression in NHPs.
- Novel NHP variants outperformed benchmark
AAV2andAAV2-7m8in retinal gene expression. Single-nucleus RNA sequencingconfirmed broad cell-type tropism for top-performing variants.
Why It Matters
These findings expand the toolkit for retinal gene therapy, offering novel AAV variants like AAV2-I.1 and AAV2-I.12 that demonstrate superior transduction and expression in primates compared to current standards. This could lead to more effective and potentially safer treatments for inherited retinal diseases (IRDs) and other conditions requiring retinal ganglion cell (RGC) gene modulation. The study also underscores the importance of optimized, NGS-guided screening methodologies in vector discovery, suggesting that future research should prioritize primate models for translation. These new AAV capsids represent a step closer to clinically viable gene therapies with improved retinal targeting and efficacy.
aav
gene-therapy
retinal-diseases
non-human-primate
in-vivo
ngs