Abca4 Knockdown in the Cone-Rich Rodent Psammomys Obesus Leads to Stargardt's Disease-Like Progressive Retinal Degeneration.

Purpose: Mutations in the gene ABCA4 coding for photoreceptor-specific ATP-binding cassette subfamily A member 4, are responsible for Stargardts disease type 1 (STGD1), the most common form of inherited macular degeneration. We recently showed that injection of viral vectors expressing CRISPR/Cas9 tools directed against Abca4 into young Sand Rat (Psammomys obesus) eyes led to extensive structural and functional retinal degeneration resembling STGD1. Here we provide further evidence that this is highly likely due to specific knockdown of Abca4 and not off-target errors. Methods: We performed subretinal injections of Adeno-Associated Virus-CRISPR/Cas9-Abca4 constructs into postnatal (∼P15) Psammomys obesus. Eyes were examined by noninvasive exploration (ocular coherence tomography, fundus and electroretinography) at 15-60 days after injection. Additionally, subgroups were euthanized over the same time period, and ocular tissue was used for immunochemical analyses. Results: RNAscope analysis of injected eyes showed knockdown of Abca4, rhodopsin and cone transducin mRNA in transduced regions; neighbouring tissue that was not transduced showed robust expression of all three. Injection of control AAV, expressing CAS9 alone, induced only mild glial activation. Statistically significant decreases in visual responses to light flashes were only seen in eyes injected with the fully active CRISPR/Cas9-Abca4 probes. Conclusions: Taken together, these data rule out off-target effects as responsible for the observed degeneration, and indicate that Psammomys obesus faithfully recapitulates many of the features seen in human STGD1, thus positioning it as an important research opportunity to further explore genotype-phenotype relationships and test putative therapeutic approaches.