Oligonucleotides Targeting DNA Repeats Downregulate Huntingtin Gene Expression in Huntington's Patient-Derived Neural Model System.

Tea Umek,Henrik Zetterberg,C I Edvard Smith,Jesper Wengel,Rula Zain,Thomas Olsson,Osama Saher,Olof Gissberg,Karin E Lundin,Eman M Zaghloul,Dzeneta Vizlin-Hodzic,Eric Hanse

doi:10.1089/nat.2021.0021

Abstract

Huntington's disease (HD) is one of the most common, dominantly inherited neurodegenerative disorders. It affects the striatum, cerebral cortex, and other subcortical structures leading to involuntary movement abnormalities, emotional disturbances, and cognitive impairments. HD is caused by a CAG•CTG trinucleotide-repeat expansion in exon 1 of the huntingtin (HTT) gene leading to the formation of mutant HTT (mtHTT) protein aggregates. Besides the toxicity of the mutated protein, there is also evidence that mtHTT transcripts contribute to the disease. Thus, the reduction of both mutated mRNA and protein would be most beneficial as a treatment. Previously, we designed a novel anti-gene oligonucleotide (AGO)-based strategy directly targeting the HTT trinucleotide-repeats in DNA and reported downregulation of mRNA and protein in HD patient fibroblasts. In this study, we differentiate HD patient-derived induced pluripotent stem cells to investigate the efficacy of the AGO, a DNA/Locked Nucleic Acid mixmer with phosphorothioate backbone, to modulate HTT transcription during neural in vitro development. For the first time, we demonstrate downregulation of HTT mRNA following both naked and magnetofected delivery into neural stem cells (NSCs) and show that neither emergence of neural rosette structures nor self-renewal of NSCs is compromised. Furthermore, the inhibition potency of both HTT mRNA and protein without off-target effects is confirmed in neurons. These results further validate an anti-gene approach for the treatment of HD.

Highlights

Huntington’s disease (HD) is one of the most common dominantly inherited neurodegenerative disorders
Integration-free WT and HD induced pluripotent stem cells (iPSCs) lines were exposed to a stepwise differentiation protocol mimicking human neural development (Fig. 1A) to be used as a model for investigating the efficiency of the CAG19 ON to downregulate HTT gene expression in the early stages of neural development
The pluripotency of iPSC lines was confirmed using immunofluorescence and confocal microscopy. Both WT and HD iPSC lines were positive for SSEA4, a glycolipid carbohydrate antigen expressed on the surface of human pluripotent cells, and the cell lines exhibited intense nuclear immunostaining for pluripotency marker OCT4 (Fig. 1B: panel i, and Supplementary Fig. S1A)

Summary

Introduction

Huntington’s disease (HD) is one of the most common dominantly inherited neurodegenerative disorders. It affects the striatum, cerebral cortex, and other subcortical structures, leading to clinical symptoms such as involuntary movement abnormalities, emotional disturbance, and cognitive impairment. The therapies currently available to HD patients offer only moderate symptom relief, and the affected individuals typically die 15–20 years postdiagnosis due to complications such as pneumonia, dysphagia, heart disease, or suicide

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Conclusion