Duchenne muscular dystrophy (DMD)—a fatal inherited muscular dystrophy—is caused by loss of Dystrophin,
a protein that maintains muscle integrity. Corticosteroids slow DMD progression but cause side effects.
Addressing the root cause of DMD may improve patient health without needing corticosteroids. Many DMD-
causing mutations disrupt the dystrophin mRNA reading frame, resulting in non-functional protein. Strategies
that skip the out-of-frame exon to restore the reading frame and produce semi-functional protein for improved
muscle function could correct 64% of DMD mutations. FDA-approved antisense oligonucleotide drugs can skip
select exons in dystrophin mRNA, but require lifelong infusions and only work in a small group of patients. Using
CRISPR to edit dystrophin would require just one treatment. CRISPR-mediated ablation of splice sites to cause
exon skipping can increase Dystrophin in DMD models. Yet, editing in unintended tissues is a safety concern for
Cas9 therapies. An ideal platform for DMD would restrict editing to muscle tissue to maximize therapeutic benefit.
Efforts to achieve tissue-specific editing often rely on delivery via adeno-associated viruses (AAVs) with tissue
tropism; yet, it is rarely absolute. Tissue-specific editing was recently achieved using tissue-specific miRNAs to
regulate expression of Cas9 inhibitors [anti-CRISPR (Acr) proteins] via miRNA target sites (TS) in the 3’ UTR of
Acr mRNA. When the platform is systemically delivered to mice via AAV, Acr-TS targeted by liver-specific miRNA
allows editing only in the liver. Unlike tissue-specific promoters, this Acr-TS strategy could be adapted to one or
multiple muscle tissues affected in DMD, as long as muscle-specific (myo)-miRNA can repress an Acr.
With support from Erik Sontheimer (CRISPR, Acr), Eric Olson (DMD), Wen Xue (in vivo CRISPR delivery), Phillip
Zamore (miRNA), Guangping Gao (AAV), and Zhiping Weng (bioinformatics), this proposal seeks to develop a
muscle-specific editing platform to treat DMD. The myo-miRNA, miR-1, can repress an Acr in muscle cell lines
to achieve muscle-specific editing. To fine-tune specificity of editing in muscle tissues for DMD, Aim 1 will test
the ability of myo-miRs varying in abundance and muscle-type specificity to repress Acr and drive muscle-specific
editing in mouse cell lines. The myo-miR construct supporting highest muscle-specific editing will be delivered
to a DMD mouse model, and in vivo muscle function as well as dystrophin exon skipping, Dystrophin protein,
and miRNA level in muscle tissues and liver will be measured. Aim 2 will test the compatibility of additional Cas9
orthologs in the Acr-TS system to enable targeting of more sequences, and develop a single AAV delivery system
for improved safety. An Acr inhibiting the Cas9s to be tested has been identified. The ability of miR-1 to repress
this Acr and drive muscle specific editing by each Cas9 will be tested in cells. A single vector encoding the Acr-
TS system will be designed and packaged into AAV, and muscle-specific editing will be compared to a dual AAV
system in mice. This work will develop a flexible, safe, muscle-specific CRISPR platform with the potential to be
used for any combination of muscle tissues to treat patients with DMD, or other genetic muscle disorders.