Novel Cas9/gRNA Ribonucleoprotein Bionanoparticles for Safe and Efficient Inactivation of ALS Disease-Causing Mutations
Abstract
Amyotrophic lateral sclerosis (ALS) is a motor neuron degenerative disease with no cure and short life expectancy. Among ALS cases with known genetic causes, some ALS-causing mutations gain neurotoxic activity independent of their physiological function. Twenty percent of familial ALS cases are caused by mutations in human superoxide dismutase 1 (SOD1) gene, whose physiological function is to detoxify superoxide, a type of reactive oxygen species. About 10% of sporadic ALS cases and 40% of familial ALS cases are caused by hexanucleotide GGGGCC repeat expansion within the non-coding sequence of a gene named C9ORF72. Normal subject also have repeats in this gene, but the copy numbers of the repeats are below 23. The expanded repeats will produce RNA and peptide aggregates that are toxic to motor neurons. For ALS cases caused by mutations in these two genes, inhibiting the production of the toxic products by antisense oligonucleotides will prevent or at least slow down neuron death. This strategy is in clinical trials to treat ALS cases caused by SOD1 mutations. One disadvantage with this strategy is that the patients have to receive the treatment repeatedly since it does not target the molecular pathogenic factor. Recently a novel type of endonuclease, CRISPR/Cas9, was discovered. These RNA-guided endonucleases make it possible to specifically make cuts in human genome. After these types of DNA damages, neurons will repair the damages by directly ligating the broken ends. This repair process is inaccurate and will often generate deletions or insertions, which provides opportunities for us to disrupt the disease-causing DNA. In the case of expanded repeats in C9ORF72 gene, which is the most common ALS-causing mutation, making one cut on each side of the repeats will remove the expanded repeats and permanently correct the mutation. The advantage of using this strategy to treat ALS is that repeated treatment is no longer needed. Although promising, we have to solve the issue related to delivery of the novel endonuclease. Due to the possibility of making cuts at unintended sites and inducing immune responses, the endonuclease should not stay in the body long term. Currently only viral vectors can efficiently deliver genes to the central nervous system (CNS). However they mediate long-term expression of the delivered genes. This proposal will develop a novel adeno associated virus (AAV) capsid-based delivery system for delivering endonucleases to the CNS to remove repeat expansions in C9ORF72 gene. AAV vector has been approved for gene therapy of neural degenerative diseases. Our system will have the delivery efficiency of conventional AAV vectors but only mediate short expression of the delivered endonucleases. Although we will test the system for treating ALS caused by repeat expansion in C9ORF72 gene in this proposal, the strategy, if it works, will be easily adapted for treating ALS caused by SOD1 mutations. This strategy eliminates the molecular pathogenic factors causing ALS, and the effects will be long term. It may develop a cure for ALS patients caused by these mutations. Many other neural degenerative diseases, such as Huntington disease and spinal cerebral ataxia, are also caused by repeat expansion; our strategy can also be used to develop treatments for those diseases. Even though our delivery system enables transient endonuclease action, using these novel endonucleases to treat these ALS cases still has the risk of making DNA damages at unintended genome locations. But this risk can be minimized by developing high fidelity endonucleases, and any new developments in this field will be incorporated into our strategy. In addition, the neurons are terminally differentiated and the risk of off-target caused tumorigenesis is low. This project aims to develop a novel delivery system and to use the novel delivery system to develop novel treatment for ALS cases caused by repeat expansion
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Mar 10, 2021
- Source ID
- W81XWH2010265
Entities
People
- Baisong Lu
Organizations
- United States Army
- Wake Forest University