Optimizing lipid nanoparticles for retinal gene editing in the NHP

PROJECT SUMMARY Inherited retinal dystrophies (IRDs) are a group of genetically and clinically heterogenous diseases, inherited in an autosomal dominant, recessive, or X-linked pattern. With an estimated incidence of 1:2000-1:3000, IRDs are the leading cause of vision loss in persons aged 15 to 45. To date, mutations in 280 distinct genes have

been associated with retinal pathology. Gene augmentation, editing, and silencing are the most attractive therapeutic strategies for these patients as they correct the causative genomic malfunction. Currently, there is only one FDA approved gene augmentation therapy for one IRD in this large family of retinal degenerations.

Our long-term goal is to generate novel gene editing platforms for IRDs. The most clinically advanced gene editing therapeutic for retinal degeneration is EDIT-101, which uses a viral vector (AAV) to deliver the Cas9 endonuclease and two guide RNAs that target the CEP290 gene in Leber Congenital Amaurosis type 10

patients. While this is the first in-vivo CRISPR/Cas9 clinical trial underway to treat retinal degeneration, the trial is currently paused, suggesting there is room for improvement in the efficacy of this product either through modulating the gene editing tools, or the delivery platform. Delivering the Cas9 endonuclease in the form of

mRNA, which leads to transient, robust protein expression, would mitigate safety concerns associated with AAV-mediated Cas9. These safety concerns include persistent expression of Cas9 endonuclease and AAV integration into the Cas9-induced double strand breaks. Lipid-based nanoparticles (LNPs) are the most

clinically advanced non-viral platform that can encapsulate mRNA and deliver genome editors. Systemic administration of LNPs, that encapsulate Cas9 mRNA and a guide RNA targeting transthyretin (TTR), has led to a 90% reduction in misfolded TTR protein in amyloidosis patients. To translate these therapeutic gains

observed in the liver to the retina, we first measured gene editing events following subretinal administration of an LNP encapsulating Cas9 mRNA and guide RNA in Ai9 mice. In this proposal, we show significant LNP- mediated gene editing in the murine retina. Additionally, we were one of the first groups to deliver LNPs to the

subretinal space of rhesus macaques and demonstrate their ability to transfect photoreceptors. To advance the development of LNP-mediated gene editing therapies for IRDs, there are three critical gaps of knowledge we propose to address in the most clinically relevant model, the nonhuman primate (NHP): 1) determine which

physiochemical features of LNPs facilitate photoreceptor expression of gene editors, 2) evaluate the immunogenicity of LNPs in the subretinal space, and 3) quantify in-vivo gene editing efficiency in the photoreceptors. Successful completion of these aims will generate novel LNP platforms that mediate the

expression of gene editors in NHP photoreceptors. This will lead to an understanding of in-vivo gene editing in large animals that can be translated to specific IRD mutations. Overall, these studies will advance the development of LNP gene editing therapeutics for IRDs.