Streamlined development of an IND with the silica nanocapsule loaded with Cas9 genome editors to disrupt the dominant BEST1 mutant allele

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PROJECT SUMMARY– FOLLOWER PROJECT 3. Best Vitelliform Macular Dystrophy, or Best disease (BD), is a relatively common inherited macular degenerative disorder that results in retinal pigment epithelium (RPE) dysfunction and progressive loss of central vision. BD is caused by over 200 different missense mutations in the BEST1 gene. Currently, there are no treatments for autosomal dominant BD due in part to the absence of relevant animal models. To overcome the difficulties in modeling the effects of many pathogenic BEST1 mutations, we utilize patient-specific iPSC-RPE models to develop a biological system that can rapidly screen genome editing leads. As in Follower Project 2, we hypothesize that Cas9-mediated indel formation by the leads in the mutant BEST1 allele will cause frameshifts leading to premature stop codons and resulting in nonsense- mediated decay of the edited transcript, removing the dominant-negative effect and restoring function to the wild- type allele. Our objective in Project 3 is to develop an SNC product, SNC-201, containing a Cas9 payload to specifically target BEST1 mutant alleles and compare various approaches using ribonucleoproteins (RNPs) and Cas9 mRNA/sgRNAs. To achieve this, we will pursue four aims. First, we will generate dual BEST1 allele BD iPSC-RPE reporter systems to facilitate on/off-target analysis of mutant allele targeting. We have developed a BD dual reporter iPSC line that has the mutant BEST1 allele linked to a 3’ tdTomato reporter and the wild-type BEST1 allele linked to a 3’ GFP reporter. Via fluorescent imaging, we can monitor the expression of both the targeted mutant BEST1 allele and the wild-type allele, which is the top ‘off-target’ site for our strategy. Plus, we can monitor channel function on sorted cells. We will adapt this reporter iPSC line to create a wild-type and eight additional mutant lines. Second, we compare the modular editor components of the lead SNC-201s. Using the dual reporter systems transfected with SNC formulations containing RNPs or Cas9 mRNA/sgRNA, we will rapidly screen SNC formulations to disrupt each mutant allele in dual reporter iPSC-RPEs. A comparison of the editing efficiency of Cas9 mRNA/sgRNA and RNP will be achieved through this work. Third, we scale up the synthesis of the SNC RNP leads and rely on Lead Project 1 for protocols to scale up mRNA/sgRNA leads. Finally, we generate a preclinical package for SNC-201 targeting selected mutant BEST1 alleles. After evaluating the safety of an SNC-201 formulation in nonhuman primates, we will complete one INTERACT meeting with the FDA for a product that intends to treat BD patients with multiple different mutations. After obtaining feedback, we will draft a clinical development plan for a pre-IND meeting. Successful completion of our aims will provide a rigorous, stepwise approach to developing an IND for targeting specific mutations – a strategy that could be expanded for all individuals with BD – and address central questions pertaining to the application of gene editing for genotypically heterogeneous dominant diseases. It will also advance the utility of iPSC models as custom preclinical tools to rapidly develop somatic cell genome editing strategies.