RNA-Directed Therapy for C9ORF72-Linked ALS
Abstract
Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig s disease, is an invariably fatal disease characterized by the progressive death of motor neurons in the spinal cord. Approximately 6,000 Americans are diagnosed each year and are presented with the dire prognosis of 2-5 years of remaining life expectancy. For unknown reasons, the risk of Veterans of the military to develop ALS is about twice as high as that of the general population. Currently, there are no treatments that can halt, let alone reverse, the course of the disease. A key insight of research into the causes of ALS is that around 10% of patients carry genetic mutations, most often inherited from either parent, that increase their risk of developing ALS or are considered to be disease-causing. One of these mutations is located in a gene called C9ORF72, which encodes a protein whose function largely remains a mystery. Recent studies have revealed that the mutation consists of a massive expansion of a six-nucleotide DNA sequence and that it is located in an intron, a portion of the gene that is transcribed into RNA but not translated into protein. This repetitive sequence causes the C9ORF72 RNA molecules to aggregate, forming dense so-called foci that are microscopically visible when stained for the RNA in patient neuronal cells. The repeats are also non-conventionally translated into repeat-containing peptides, which themselves are prone to aggregation. It is believed that these RNA and/or protein aggregates trigger molecular cascades that perturb normal motor neuron function and ultimately lead to their demise. Regardless of the exact mechanism by which these RNAs cause cell death, which is an active area of investigation, we believe that a drug that degrades these toxic RNA species has the potential to stall disease progression in patients affected by the C9ORF72 mutation. To this end, we recently designed a protein-RNA complex, modified from a bacterial DNA-cleaving system known as CRISPR/Cas9, that is able to specifically degrade repeat-containing RNAs. We have delivered this complex, which we call RCas9, to human cells that artificially produce the repeat RNAs and showed that RCas9 efficiently eliminated the RNA foci. In this study, we aim to develop RCas9 into a therapeutic candidate. One of the safest and most efficient ways to produce RNA or proteins in cells of the human body is to use adeno-associated virus (AAV)-based vectors, which can be engineered to deliver DNA cargoes that are then transcribed into RNA and/or translated into protein by the cells. However, AAV has a limited capacity for such extraneous cargoes, so our first aim is to design and generate RCas9 variants that fit into the AAV vector while maintaining RCas9 s capacity to efficiently and specifically degrade the target RNAs. We will test these variants in neurons that we can produce in the petri dish from skin cells of patients with C9ORF72 repeat expansions. We will next produce RCas9 AAV vectors and inject them into the spinal cord of mice. This part of the study is aimed at investigating if RCas9 delivery results in distribution of RCas9 across the entire spinal cord, and to see if there might be concern for immune responses triggered by RCas9 or the AAV vectors. We will also assess if RCas9 targets other RNAs. Lastly, we will determine if RCas9 can efficiently degrade C9ORF72 repeats and eliminate RNA and protein foci in mice that have been engineered to produce these RNAs. If that is the case, we will determine whether or not this treatment also results in reversal of several behavioral phenotypes, including cognitive impairment, that are relevant to ALS and that we have observed in this ALS mouse model. If successful, our 2-year study will collect the bulk of the efficacy and safety data necessary for a pre-investigational new drug (IND) filing with the Food and Drug Administration. We anticipate that if our study supports a favorable safety and efficacy profile fo
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jul 16, 2019
- Source ID
- W81XWH1910181
Entities
People
- Gene W. Yeo
Organizations
- United States Army
- University of California, San Diego