OR11

Stargardt disease (STGD1) is an incurable juvenile macular dystrophy caused by mutations in ABCA4. As ABCA4 coding sequence is about 7kb and over 1000 mutations have been reported as pathogenic, a mutation-independent gene editing approach could overcome this issue. Therefore, we are developing an approach to correct an entire domain, representing a hotspot of mutations, in the ABCA4 gene. This is the nucleotide binding domain (NBD2) necessary to catalyze ATP hydrolysis and thus power the activity of the ABCA4 transporter.
We have optimized the sequence of the NBD2 domain for expression from an AAV vector. Combining CRISPR-Cas9 and a Homology-Independent Targeted Integration (HITI) strategy, we aim to insert the engineered copy of the NBD2 domain upstream of the ABCA4 transmembrane domains in both human cells and a rat model of STGD1. Electroporation of HEK cells using RNP-Cas9 and a specific guide combined with the engineered NBD2 sequence was initially carried out. Afterwards, the strategy could be tested in vivo using a Stargardt rat model. AAV vectors coding for SaCas9-GFP and guides were compared following subretinal injection. One month post-injection retinas were collected, FACS sorted and a TIDE analysis was conducted to evaluate Cas9 efficiency. Sanger-sequencing verified the insertion of the mini-domain fragment at the junction of interest, in vitro validating our approach in human cells. Five distinct AAV vectors carrying SaCas9 and a guide RNA were compared in the rat model until a 50% cutting efficiency was measured by the TIDE software, with one specific AAV vector and guideRNA combination.  Following the first promising results in HEK cells, we are now testing this strategy in STGD1 patient-derived iPS cells. In parallel, we are co-injecting the selected nuclease-expressing vector and the engineered NBD2 mini-domain to evaluate its level of insertion and its potential effect on bisretinoid levels, a key marker of STGD1. This proof-of-concept will open up the possibility of treating multiple Stargardt patients with a single gene therapy product.