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Enhanced systemic delivery of antisense oligonucleotides using cell-penetrating peptide to reverse RNA toxicity in DM1
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International audience. "IntroductionAntisense oligonucleotides (AON) targeting expanded-CUG transcripts to either degrade the pathogenic RNAs or interfere with abnormal binding/sequestration of RNA-binding proteins like MBNL1 have shown very promising results in DM1 models. Thus, injection of steric blocking PMO-CAG AON in DM1 mice reverses molecular changes and myotonia induced by CUGexp-RNA. However efficient skeletal muscles delivery of AON following systemic administration is still an ongoing challenge for muscular diseases. MethodHere we described the use of a PMO-CAG AON conjugated to an arginine-rich cell-penetrating peptide (CPP) designated as Pip6a, to enhance its systemic delivery. ResultsAs preliminary experiments, we confirmed in vitro using human DM1 muscle cells that Pip6a-PMO-CAG-treatment induces the release of MBNL1 from nuclear foci and its relocalization into the nucleus, which leads to the normalization of DM1 splicing defects including SOS1, DMD, MBNL1 and LDB3 genes. Next we assess Pip6a-PMO-CAG in vivo using the DM1 HSA-LR transgenic model expressing CUGexp-RNA in skeletal muscles. Intravenous (IV) injection of a single dose of Pip6a-PMO-CAG (12.5 mg/kg) results in a significant correction of alternative splicing defects in treated HSA-LR mice. Subsequent experiments revealed that a complete correction of DM1 splicing defects as well as myotonia in skeletal muscles of HSA-LR mice is reached after three IV injections. This correction is also accompanied with a significant decrease in the level of foci. The beneficial effect is maintained for several weeks after the last injection without adverse effects. Finally, analysis of the transcriptome by RNA-sequencing shows that the pip6a-CAG7 treatment induces a global correction of the diseased transcriptome of HSA-LR mice, at both gene expression and alternative splicing levels. DiscussionThis study demonstrates that Pip6a-CPP-conjugated PMO-CAG reverses CUGexp-RNA toxicity in DM1 mice and represents an efficient tool for AON delivery in skeletal muscles for DM1."
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