PMO-based let-7c site blocking oligonucleotide (SBO) mediated utrophin upregulation in mdx mice, a therapeutic approach for Duchenne muscular dystrophy (DMD)

Abstract

Upregulation of utrophin, a dystrophin related protein, is considered a promising therapeutic approach for Duchenne muscular dystrophy (DMD). Utrophin expression is repressed at the post-transcriptional level by a set of miRNAs, among which let-7c is evolutionarily highly conserved. We designed PMO-based SBOs complementary to the let-7c binding site in UTRN 3′UTR, with the goal of inhibiting let-7c interaction with UTRN mRNA and thus upregulating utrophin. We used the C2C12UTRN5′luc3′ reporter cell line in which the 5′- and 3′-UTRs of human UTRN sequences flank luciferase, for reporter assays and the C2C12 cell line for utrophin western blots, to independently evaluate the site blocking efficiency of a series of let-7c PMOs in vitro. Treatment of one-month old mdx mice with the most effective let-7c PMO (i.e. S56) resulted in ca. two-fold higher utrophin protein expression in skeletal muscles and the improvement in dystrophic pathophysiology in mdx mice, in vivo. In summary, we show that PMO-based let-7c SBO has potential applicability for upregulating utrophin expression as a therapeutic approach for DMD.

Duchenne muscular dystrophy cell culture models created by CRISPR/Cas9 gene editing and their application in drug screening

“…Ezutromid/SMT-C1100 was the first utrophin modulator evaluated in clinical assays but was recently abandoned due to lack of evidence of utrophin restoration, nor clinical improvement demonstrated in patients 21 , 22 . Alternatively, other studies proposed new strategies to upregulate UTRN by blocking the inhibitory target region of microRNAs repressing UTRN expression 23 – 25 . Recently, utrophin upregulation has been efficiently achieved using gene therapy in multiple animal models with non-immunogenic side effects 26 .…”

Matricellular Protein CCN5 Gene Transfer Ameliorates Cardiac and Skeletal Dysfunction in mdx/utrn (±) Haploinsufficient Mice by Reducing Fibrosis and Upregulating Utrophin Expression

“…Utrophin is similar in structure and function to dystrophin, and multiple reports have shown that utrophin compensates for the loss of dystrophin ( 45 – 48 ). In previous studies, utrophin upregulation by small molecules in DMD mouse models successfully ameliorated the symptoms of muscle weakness ( 49 , 50 ). Since then, many studies have focused on developing novel small molecules that enhance utrophin expression ( 51 ).…”

Promising therapeutic approaches of utrophin replacing dystrophin in the treatment of Duchenne muscular dystrophy

“…The miRNA, especially the complementary binding site in the 3’UTR of mRNA, can interfere with mRNA stability and protein translation. To reduce the transcriptional and translational regulation of utrophin by miRNA, 2′-O-methyl-phosphorothioate (2OMePS) site-blocking oligonucleotides (SBOs) are used to block the let-7c in 3’UTR of UTRN in some studies [68] . At the let-7c binding site, utrophin protein expression is up-regulated to improve muscle contraction in mdx mice.…”