ARMMs as non-viral vehicles for the delivery of genome editors to treat Stargardt disease

Abstract

Purpose : Stargardt disease (SD) is an inherited retinal disease (IRD) characterized by reduction in central vision and visual acuity. Currently SD patients have no treatment options. Loss-of-function mutations in the ABCA4 gene are the most common causes of the disease. Genome editing has recently emerged as an attractive therapeutic strategy, though effective packaging and delivery of gene editing components still present significant hurdles. We have developed a non-viral platform of engineered extracellular vesicles called Arrestin domain containing protein 1 [ARRDC1]-mediated microvesicles (ARMMs) that enables efficient packaging of genome editors. We have also shown ARMMs' uptake in the retinal pigment epithelium (RPE) and photoreceptors via subretinal administration in rodents, minipigs, and nonhuman primates. In this study, we evaluated a therapeutic approach using ARMMs to correct a specific ABCA4 mutation that causes SD.

Methods : ARMMs were generated by packaging a base editor with either guide RNA (gRNA) targeting the wild type sequence or a common ABCA4 mutation in Stargardt disease. Protein and RNA loading in ARMMs were analyzed using Western blotting and quantitative PCR (qPCR). We further generated an engineered cell line harboring the targeted pathological ABCA4 mutation. Genome editing efficiency was evaluated by Sanger and Next-generation sequencing (NGS) at the mutation site. In addition, we also used mouse, monkey and human cell lines and explant systems to evaluate the effect of ARMMs-delivered base editor at a surrogate site in wild-type ABCA4. Editing at this site results in a synonymous change in the mRNA and serves as proxy for editing efficiency at the targeted locus.

Results : Western blotting and qPCR results showed that both the base editor and gRNAs were effectively packaged in ARMMs. Treatment with ARMMs resulted in functional delivery of therapeutic genome editors and achieved up to 80% base editing in the targeted region of ABCA4 gene in in vitro and ex vivo models.

Conclusions : Our data collectively demonstrate functional delivery of therapeutic genome editors in in vitro and ex vivo models via ARMMs and lay the groundwork for in vivo evaluation of ARMMs-mediated base editing of the ABCA4 locus in photoreceptors and RPE as a strategy to treat SD patients.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.