Correction of a splicing defect in a mouse model of congenital muscular dystrophy type 1A using a homology-directed-repair-independent mechanism

Abstract

Splice-site defects account for about 10% of pathogenic mutations that cause Mendelian diseases. Prevalence is higher in neuromuscular disorders (NMDs), owing to the unusually large size and multi-exonic nature of genes encoding muscle structural proteins. Therapeutic genome editing to correct disease-causing splice-site mutations has been accomplished only through the homology-directed repair pathway, which is extremely inefficient in postmitotic tissues such as skeletal muscle. Here we describe a strategy using nonhomologous end-joining (NHEJ) to correct a pathogenic splice-site mutation. As a proof of principle, we focus on congenital muscular dystrophy type 1A (MDC1A), which is characterized by severe muscle wasting and paralysis. Specifically, we correct a splice-site mutation that causes the exclusion of exon 2 from Lama2 mRNA and the truncation of Lama2 protein in the dy/dy mouse model of MDC1A. Through systemic delivery of adeno-associated virus (AAV) carrying clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing components, we simultaneously excise an intronic region containing the mutation and create a functional donor splice site through NHEJ. This strategy leads to the inclusion of exon 2 in the Lama2 transcript and restoration of full-length Lama2 protein. Treated dy/dy mice display substantial improvement in muscle histopathology and function without signs of paralysis.

Impeding Transcription of Expanded Microsatellite Repeats by Deactivated Cas9

“…Here, we observed that rescue of a subset of nuclei was sufficient to yield partial rescue of myotonia. However, myotonia can be modeled as a loss-of-function event in which Clcn1 protein is lost, and there is evidence that in other recessive muscle diseases, gene editing of a small subset of nuclei can restore sufficient protein expression across muscle fibers to yield therapeutic benefit (Kemaladewi et al, 2017, Tabebordbar et al, 2016). Conversely, mis-splicing events yielding pathogenic isoforms with dominant-negative behavior may require full elimination to mitigate deleterious consequences.…”

Reversal of molecular pathology by RNA-targeting Cas9 in a myotonic dystrophy mouse model

“…Immune response to Cas9 involving AAV-mediated, long-term expression in tissue is a major consideration for translation of any CRISPR-based therapeutic to the clinic. Recent efforts involving delivery of Cas9 to adult muscle have assessed immune response to varying degrees (27,28) and detailed assessments have revealed adaptive immune response to Cas9 (29). We assessed gene expression changes between RCas9-and Mbnltreated mice to assess the immune response to Cas9 protein and observed enrichment in adaptive immune response-linked genes ( Figure 3G) and no striking alterations in gene expression linked to innate immune response ( Figure S2).…”

A mutation-independent approach via transcriptional upregulation of a disease modifier gene rescues muscular dystrophy in vivo

“…We have previously demonstrated that an early intervention using CRISPR/Cas9-mediated correction of a splicing defect resulted in robust Lama2 restoration and prevention of disease manifestation 4 . Here we showed that upregulation of Lama1, when initiated at pre-disease-onset, leads to similar prevention.…”

“…23, 2018; Animal Use Protocol number 16-0234H. Intramuscular and temporal vein injections were preformed as previously described 4 . Systemic administration experiments were performed on 3-week-old dy 2j /dy 2j mice through tail vein injection.…”

“…Systemic administration experiments were performed on 3-week-old dy 2j /dy 2j mice through tail vein injection. All functional tests were performed as previously described 4 .…”