AAV-CRISPR Persistence in the Eye of the Beholder

Despite advances in genome editing technologies based on the adeno-associated virus (AAV)-CRISPR system, there are still concerns about the long-term persistence of recombinant AAV vectors in several organs (liver, muscle, eye) possibly leading to cytotoxicity or genotoxicity related to off-target effects. Indeed, there are still unanswered questions about long-lasting in vivo AAV persistence as a linear or circular DNA that is not targeted by epigenetic silencing in many tissues. In 2017, Kim et al.1 reported an editing approach based on AAV-CjCas9 to downregulate Vegfa or the hypoxia-inducible transcription factor Hif1a in mice displaying age-related macular degeneration (AMD)-related pathological choroidal neovascularization (CNV) induced by laser treatment. Although partial knockdown of either Vegfa or Hif1a provided benefits and reduced the area of CNV, local opsin dysfunction near the Vegfa-edited cells of murine retinal pigment epithelium (RPE) was observed. Conversely, no cone dysfunction was reported upon Hif1a partial knockdown. Lastly, no genome-wide off-target indels, evaluated 6 weeks after intravitreal injection of AAV-CjCas9 vector, were scored, indicating that prolonged expression of AAV-CjCas9 in vivo did not aggravate the genotoxic risk associated with the CjCas9 nuclease. In this issue of Molecular Therapy, the authors now report a long-term (14 months) safety study on C57BL/6J mice intravitreally injected with AAV-CjCas9 vectors targeting Vegfa or Hif1a genes.2 The findings continue to show that the AAV-CRISPR system in the eyes is long lasting, effective, and safe.