Multiexon skipping to restore dystrophin protein expression using tethered RNPs

University Of California Berkeley, Berkeley CA

Investigators

Abstract

Multiexon skipping to restore dystrophin protein expression using tethered RNPs Project Summary Mutations in the dystrophin (DMD) gene often lead to frameshift mutations in the mature messenger RNA, causing nonsense mediated decay of the mRNA and loss of dystrophin protein in muscle cells. The most severe of these mutations cause Duchenne muscular dystrophy (DMD), an X-linked disease in males that leads to progressive loss of muscle function and early death. Given the large variety of mutations within the dystrophin gene and its very large size, it has not been possible to derive a general treatment for DMD. One promising strategy to treat a subset of patients is to trigger exon skipping during pre-mRNA splicing, thereby restoring the reading frame in the mature mRNA. Although the resulting dystrophin protein may be shorter than the wild-type version, some of these shorter versions are reasonably functional and can lessen the disease phenotype. Many labs and companies are exploring exon skipping induced by antisense oligonucleotides, exemplified by phosphorodiamidate morpholino oligomers (PMOs), some of which are now in clinical trials. Although the FDA has approved one PMO that leads to skipping of exon 51, this approval is provisional on demonstrating a clear clinical benefit. Further, each single antisense oligonucleotide can only treat a small percentage of patients. To overcome these limitations, one promising strategy would be to induce skipping of exons 45-55 in the DMD mRNA. If successful, such a treatment would generate a truncated dystrophin protein seen in Becker muscular dystrophy (BMD) patients that leads to a much milder phenotype, and could be used to treat ~45% of DMD patients. Although it is possible to induce skipping of exons 45-55 using a cocktail of 10 modified PMOs, these treatments could cause toxicity and induce variably spliced mRNAs due to inefficient exon skipping. We propose to take a fundamentally new approach to induce multiexon skipping. We will program bacterial Argonautes with guide RNAs targeting exons 45 and 55 to form RNA-protein complexes (RNPs) to induce exon skipping. To achieve multiexon skipping, we will tether the RNPs to each other. Tethering the RNPs should bring exons 44 and 56 into closer proximity, which we propose should increase skipping of exons 45-55. In the longer term, the use of tethered guide-RNA/Argonaute RNPs could be a general means to induce multiexon skipping to treat DMD.