Single nucleotide seed modification restores in vivo tolerability of a toxic artificial miRNA sequence in the mouse brain.

Alex Mas Monteys,Matt S Wilson,Matt J Sowada,Jodi L Mcbride,Brett D Dufour,Beverly L Davidson,Ryan M Spengler,Clayton K Oakley

doi:10.1093/nar/gku979

Abstract

Huntington's disease is a fatal neurodegenerative disease caused by polyglutamine-expansion in huntingtin (HTT). Recent work showed that gene silencing approaches, including RNA interference (RNAi), improve disease readouts in mice. To advance RNAi to the clinic, we designed miHDS1, with robust knockdown of human HTT and minimized silencing of unintended transcripts. In Rhesus macaque, AAV delivery of miHDS1 to the putamen reduced HTT expression with no adverse effects on neurological status including fine and gross motor skills, no immune activation and no induction of neuropathology out to 6 weeks post injection. Others showed safety of a different HTT-targeting RNAi in monkeys for 6 months. Application of miHDS1 to Huntington's patients requires further safety testing in normal rodents, despite the fact that it was optimized for humans. To satisfy this regulatory requirement, we evaluated normal mice after AAV.miHDS1 injection. In contrast to monkeys, neurological deficits occurred acutely in mice brain and was attributed to off-target silencing through interactions of miHDS1 with the 3′UTR of other transcripts. While we resolved miHDS1 toxicity in mouse brain and maintained miHDS1-silencing efficacy, these studies highlight that optimizing nucleic acid-based medicines for safety in humans presents challenges for safety testing in rodents or other distantly related species.

Highlights

Huntington’s disease (HD) is a neurodegenerative disorder caused by CAG repeat expansion (>36 repeats) within the first exon of huntingtin
When AAV vectors expressing miHDS1 were injected into the putamen of non-human primates (NHPs), HTT levels were significantly reduced and there were no signs of neuronal degeneration, immune responses or motor deficits [17]
We set out to test the safety of an RNA interference (RNAi) trigger in mice, which was designed for safety in humans and was shown to be safe in non-human primates [17]

Summary

Introduction

Huntington’s disease (HD) is a neurodegenerative disorder caused by CAG repeat expansion (>36 repeats) within the first exon of huntingtin. Earlier work with transgenic mice using a tetracyclineresponsive system to express a pathogenic human huntingtin (HTT) fragment demonstrated that deleterious effects from the mutant protein, including the development of HTT-containing inclusions, reactive astrocytosis, decreases in dopamine D1 receptor levels, striatal atrophy and progressive motor symptoms, resolved with cessation of expression [4,5]. These data imply that there is a window of opportunity to treat HD and support the notion that some symptoms may be reversible upon mutant HTT reduction. The development of approaches to reduce gene expression using gene-silencing technologies, such us RNA interference (RNAi), hold great promise as a therapy for HD [6,7,8,9]

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