A study led by Glenn Yiu at the University of California, Davis, has demonstrated that CRISPR-Cas9 gene editing can effectively reduce VEGF levels in monkeys, a key factor in abnormal blood vessel growth associated with choroidal neovascularisation (CNV). The research, published in Molecular Therapy, highlights the potential of CRISPR-Cas9 for treating wet age-related macular degeneration (AMD) but also underscores the risk of retinal toxicity at high adeno-associated viral (AAV) vector doses.
The researchers used AAV vectors to deliver CRISPR-Cas9 and guide RNA (gRNA) targeting a conserved region of the VEGFA gene. The aim was to achieve long-term suppression of VEGF, a critical step in preventing the progression of CNV. Four animals received sub-retinal injections of Cas9 and gRNA vectors at a 1:1 ratio in both eyes, with active and empty gRNA vectors administered to contralateral eyes.
Promising VEGF Reduction at Low Doses
CRISPR-mediated VEGFA ablation showed promise in reducing VEGF levels, particularly in eyes treated with low AAV doses (6 x 1011 vector genomes/eye). VEGF protein concentrations dropped by 33% in these eyes, leading to a corresponding decrease in CNV severity, as demonstrated by fluorescein angiography (FA) imaging of laser-induced lesions.
Retinal Toxicity at High Doses
However, the study also revealed significant retinal toxicity at high AAV doses (6 x 1012 vector genomes/eye). Retinal imaging showed subfoveal deposits and concentric macular rings, corresponding to outer retinal thinning, especially in the photoreceptor layer. Histological analysis confirmed sub-retinal fibrosis marked by retinal pigment epithelial (RPE) cells and fibrotic materials.
Interestingly, eyes receiving the higher AAV dose showed consistently low VEGF levels, regardless of whether they were treated with active or empty gRNA. This suggests that the retinal disruption may have damaged a larger number of VEGF-producing cells. According to Glenn Yiu, "While we don't know the exact mechanism for the retinal damage, we believe it is related to the high Cas9 expression as we have not observed toxicity using AAVs expressing other transgenes."
Inflammation and Immune Response
The retinal damage appeared unrelated to VEGFA gene editing itself, as similar effects were observed in control eyes receiving AAV vectors without the active gRNA. Immunostaining showed activation of glial and microglial cells, suggesting local inflammation, while systemic immune responses remained minimal. Elevated levels of local inflammation markers, particularly CCL2 and CXCL10, were found in the treated eyes.
Implications for Future Therapies
The study highlights the potential of CRISPR-Cas9 for long-term VEGF suppression and CNV treatment. However, it also emphasizes the need to optimize the AAV delivery system to minimize retinal damage. Lower vector doses or alternative delivery methods could offer safer options for future therapeutic strategies. As Yiu notes, high expression of SpCas9, a bacterial protein, even within the eye, could potentially trigger local inflammation.
The research underscores the importance of careful dose optimization and delivery strategies when using CRISPR-Cas9 for ocular gene therapy. Further studies are needed to refine the approach and mitigate the risk of retinal toxicity.
