High-throughput optimization of gene editing systems for treating Amyotrophic lateral sclerosis

Project Summary Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurological disease in which motor neurons degenerate, thereby leading to paralysis and death due to respiratory failure. The incidence of ALS differs based on ancestral origin, with ~5.5 cases per 100,000 persons in the United States. In addition to motor function

impairment, a subset of patients (~15%) suffers from frontotemporal dementia (FTD), leading to low quality of life and extreme emotional strain on the patient’s caregivers. From a genetic perspective, ALS pathogenesis is commonly driven by GGGGCC (G4C2) repeat expansions in the C9ORF72 gene, which cause a disease subtype

of ALS referred to as c9ALS/FTD. The repeat expansions lead to C9ORF72 haploinsufficiency, accumulation of repeat RNA, and the production of aggregation-prone proteins composed of repeating dipeptides. While antisense oligonucleotides and RNA interference (RNAi) approaches have been used to target the causal

mutations of ALS, these approaches are limited due to their transient effect, lack of specificity, and inability to multiplex. Gene editing technologies have emerged as powerful approaches to target causal drivers of rare diseases and engineer cell-based therapeutics. Therefore, there is a critical need for a gene editing-based

therapy that can safely and efficiently deplete the disease-causing mutant C9ORF72. The original CRISPR/Cas9 system that targets DNA is limited in this context since repeat expansions are at the kilobase scale and cannot be efficiently deleted with current Cas9-based technologies. The overall goal of this proposal is to develop a

gene therapy for ALS using the Acrobat Genomics proprietary enARGN gene editing system, which is more compact than the CRISPR/Cas9 system and more deliverable. Preliminary studies have demonstrated the activity of Acrobat Genomics ARGN gene editing system. In this Phase I SBIR project, Acrobat Genomics plans

to develop a high-throughput protein engineering platform (AcrobaTx) to enhance ARGN gene editing activity. The protein engineering approach is enabled by advancements in high-throughput oligonucleotide synthesis, sequencing technologies, and protein language models. Notably, the approach utilizes pooled screening and is

highly parallel to enable the study of thousands of proteins in a single experiment (Specific Aim 1). With an optimized ARGN protein, a novel editing strategy will be developed to deplete expression of mutant C9ORF72 and restore expression of the wild-type allele (Specific Aim 2). In summary, by protein engineering the ARGN

system, Acrobat Genomics will develop a new therapy for ALS, which is currently a devastating and incurable disease with few treatment options. In addition to the proposed research investigations, this application will lay the foundation for the entrepreneurial career development of the PI. The PI is the co-inventor of foundational

technologies used in this proposal and has co-founded Acrobat Genomics. The PI aims to transition from academia to commercialize the enARGN system as a therapeutic for genetic diseases and has identified mentors with significant business and scientific expertise to aid in this endeavor.