Allele-Specific Silencing of Mutant Huntingtin in Rodent Brain and Human Stem Cells

Drouet, Valérie; Ruiz, Marta; Zala, Diana; Feyeux, Maxime; Aurégan, Gwenaëlle; Cambon, Karine; Troquier, Laetitia; Carpentier, Johann; Aubert, S.; Mérienne, Nicolas; Bourgois-Rocha, Fany; Hässig, Raymonde; Rey, M.; Dufour, Noëlle; Saudou, Frédéric; Perrier, Anselme L.; Hantraye, Philippe; Déglon, Nicole

doi:10.1371/journal.pone.0099341

Cited by 50 publications

(56 citation statements)

References 49 publications

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“…Although post-hoc testing demonstrated statistically significant allelic discrimination following treatment with the rs362273 anti-G siRNA, mean off-target knockdown of this degree is clearly too great to consider the particular siRNAs to be truly selective. These data are consistent with the poor discrimination previously seen when testing siRNA against rs362307 in cell lines and animal models1113.…”

Section: Resultssupporting

confidence: 89%

Allele-Selective Suppression of Mutant Huntingtin in Primary Human Blood Cells

Miller

Pfister

Liu

et al. 2017

Sci Rep

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Post-transcriptional gene silencing is a promising therapy for the monogenic, autosomal dominant, Huntington’s disease (HD). However, wild-type huntingtin (HTT) has important cellular functions, so the ideal strategy would selectively lower mutant HTT while sparing wild-type. HD patients were genotyped for heterozygosity at three SNP sites, before phasing each SNP allele to wild-type or mutant HTT. Primary ex vivo myeloid cells were isolated from heterozygous patients and transfected with SNP-targeted siRNA, using glucan particles taken up by phagocytosis. Highly selective mRNA knockdown was achieved when targeting each allele of rs362331 in exon 50 of the HTT transcript; this selectivity was also present on protein studies. However, similar selectivity was not observed when targeting rs362273 or rs362307. Furthermore, HD myeloid cells are hyper-reactive compared to control. Allele-selective suppression of either wild-type or mutant HTT produced a significant, equivalent reduction in the cytokine response of HD myeloid cells to LPS, suggesting that wild-type HTT has a novel immune function. We demonstrate a sequential therapeutic process comprising genotyping and mutant HTT-linkage of SNPs, followed by personalised allele-selective suppression in a small patient cohort. We further show that allele-selectivity in ex vivo patient cells is highly SNP-dependent, with implications for clinical trial target selection.

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Section: Resultssupporting

confidence: 89%

Allele-Selective Suppression of Mutant Huntingtin in Primary Human Blood Cells

Miller

Pfister

Liu

et al. 2017

Sci Rep

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“…In HD, the expanded CAG repeat encodes a polyglutamine (polyQ) tract in the N-terminal region of huntingtin (HTT) and leads to a wide range of cellular dysfunctions (1). The gain of toxic function of mutant huntingtin (mHTT) has led to considerable efforts to use siRNA, antisense oligonucleotides, or CRISPR/Cas9 to selectively suppress the expression of mHTT (2)(3)(4). Indeed, siRNA and antisense oligonucleotides have shown promising therapeutic effects in HD mice that express transgenic mHTT (2,5).…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease

Chang

Yang

et al. 2017

Journal of Clinical Investigation

328

242

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“…Mechanistic studies of vectorized shRNA have paved the way for powerful preclinical proof-of-concept studies that support the therapeutic relevance of HTT-lowering strategies 14, 15, 16, 17, 22, 23, 24, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49. However, assessment of the biosafety of the gene therapy product is still a major challenge for clinical application in HD.…”

Section: Introductionmentioning

confidence: 99%

Preclinical Evaluation of a Lentiviral Vector for Huntingtin Silencing

Cambon

Zimmer²,

Martineau

et al. 2017

Molecular Therapy - Methods & Clinical Development

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Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder resulting from a polyglutamine expansion in the huntingtin (HTT) protein. There is currently no cure for this disease, but recent studies suggest that RNAi to downregulate the expression of both normal and mutant HTT is a promising therapeutic approach. We previously developed a small hairpin RNA (shRNA), vectorized in an HIV-1-derived lentiviral vector (LV), that reduced pathology in an HD rodent model. Here, we modified this vector for preclinical development by using a tat-independent third-generation LV (pCCL) backbone and removing the original reporter genes. We demonstrate that this novel vector efficiently downregulated HTT expression in vitro in striatal neurons derived from induced pluripotent stem cells (iPSCs) of HD patients. It reduced two major pathological HD hallmarks while triggering a minimal inflammatory response, up to 6 weeks after injection, when administered by stereotaxic surgery in the striatum of an in vivo rodent HD model. Further assessment of this shRNA vector in vitro showed proper processing by the endogenous silencing machinery, and we analyzed gene expression changes to identify potential off-targets. These preclinical data suggest that this new shRNA vector fulfills primary biosafety and efficiency requirements for further development in the clinic as a cure for HD.

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Allele-Specific Silencing of Mutant Huntingtin in Rodent Brain and Human Stem Cells

Cited by 50 publications

References 49 publications

Allele-Selective Suppression of Mutant Huntingtin in Primary Human Blood Cells

Allele-Selective Suppression of Mutant Huntingtin in Primary Human Blood Cells

CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease

Preclinical Evaluation of a Lentiviral Vector for Huntingtin Silencing

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