Commandeering an RNA-Editing Enzyme to Correct DNA Nonsense Mutations Causing Duchenne Muscular Dystrophy
Abstract
Duchenne muscular dystrophy (DMD) — an X-linked recessive disease affecting 1 in about 4,000 newborn males – is a severely debilitating neuromuscular disease resulting in a life expectancy of about 30 years. DMD causes progressive muscle degeneration due to an absent or defective dystrophin protein, coded for by the DMD gene, the largest gene of the human genome. Mutations in this DMD gene causes muscular dystrophy, for which there is no cure. Approximately 15% of all DMD cases are the result of a single point mutation where one single base of DNA (of the 2.2 million bases in the DMD gene) is mutated, causing the muscle cell to produce an incomplete or truncated dystrophin protein in the muscle, which leads to the progressive skeletal muscle degeneration in patients. This type of mutation is called a nonsense mutation, which introduces a premature STOP codon in the gene, instructing the cell to terminate the dystrophin production before the entire protein is synthesized. If the single base of DNA, which was altered, causing this nonsense mutation, can be repaired back to its original DNA base, the DMD patient would effectively be cured and would not require any additional drugs for the remainder of their life. If the DNA is restored in skeletal muscle satellite cells, then these cells can fuse with existing muscle fibers to regenerate and repair the damaged fibers. Then the muscle cell would produce the complete full-length dystrophin protein, resulting in normal muscle function. The last few years has seen an explosion of DNA gene editing tools and techniques. The majority of these utilize a large bacterial protein called CRISPR-Cas9, which can edit DNA by cutting out the defective gene and inserting a normal one in its place. However, because this type of editing cuts DNA, the method can have unintended results from uncontrolled DNA repair, which can have harmful consequences. Another recently developed method can edit a single DNA base without cutting the DNA. While this can prevent unintended DNA damage due to cutting, this method still utilizes a large CRISPR-Cas9 bacterial protein fused to other bacterial proteins, which can cause immunogenic reactions and reduce efficiency. Additionally, this technique can result in editing bases flanking the intended DNA base producing collateral damage that can also have unintended repercussions. This proposal’s main objective is to develop an innovative DNA base editor that mitigates the problems associated with CRISPR-Cas9 by commandeering a human RNA editing enzyme and redeploying it to serve as a DNA adenosine base editor, with single-base precision, to correct hundreds of DMD-causing nonsense mutations. Since this method relies on a protein already found in muscle cells, a foreign bacterial protein does not have be delivered into the cell together with additional RNA and possibly a piece of normal DNA fragment of the DMD gene to replace the damaged DNA. The method proposed here would simply require the delivery of smaller fragments of RNA or modified RNA, called oligonucleotides, to bind to the mutated DNA. Then the human RNA editing protein called ADAR can repair the defective DNA adenosine to guanosine. Because all stop codons contain an adenosine, this method in theory should be able to repair all DMD-causing nonsense mutations, curing 15% of muscular dystrophy patients. Preliminary data have been collected that indeed shows ADAR can change the adenosine of DNA to a guanosine analog, which is converted to guanosine at the next round of DNA replication. In this method, we would design the sequence and assembly of RNA oligonucleotides to selectively target of the region of the DMD gene to be repaired, which would not interact with other regions of DNA resulting in off-target DNA changes. The RNA oligonucleotides would still be required to be delivered or administered to the muscle cells, but the Food and Drug Administration has already approv
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Dec 28, 2022
- Source ID
- W81XWH2210893
Entities
People
- Andrew J Fisher
Organizations
- United States Army
- University of California, Davis