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Durable HTT silencing using non-evolved dCas9 epigenome editors in patient-derived cells

ABSTRACT: Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a trinucleotide repeat expansion in exon 1 of the huntingtin (HTT) gene. Nuclease-deficient Cas9 protein (dCas9) epigenetic editing for targeted gene regulation is a promising therapeutic approach for HD through downregulation of the causative gene, HTT. A screen of several dCas9 variants with expanded PAM recognition was fused to KRAB and DNMT3A/L to assess the ability to downregulate total HTT. Surprisingly, only SpdCas9 could significantly downregulate HTT, while expanded PAM recognition variants dxCas9 and dCas9-VQR were less efficient or unable to reduce HTT expression. Using our lead construct with SpdCas9, DNA methylation changes were assessed through reduced representation bisulfite sequencing, showing high on-target increases in DNA methylation and few off-targets. In addition, HTT silencing was mitotically stable for up to 6 weeks in a rapidly dividing cell line. Finally, significant downregulation of HTT was achieved in patient-derived neuronal stem cells, showing the efficacy of this system in a disease-relevant cell type. This approach represents a novel therapeutic pathway for the treatment of HD.

ORGANISM(S): Homo sapiens

PROVIDER: GSE283225 | GEO | 2025/04/01

REPOSITORIES: GEO

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Project description:In Huntington’s disease (HD), expanded HTT CAG repeat length correlates strongly with age at motor onset, indicating that it determines the rate of the disease process leading to diagnostic clinical manifestations. Similarly, in normal individuals, HTT CAG repeat length is correlated with biochemical differences that reveal it as a functional polymorphism. Here, we tested the hypothesis that gene expression signatures can capture continuous, length-dependent effects of the HTT CAG repeat. Using gene expression datasets for 107 HD and control lymphoblastoid cell lines, we constructed mathematical models in an iterative manner, based upon CAG correlated gene expression patterns in randomly chosen training samples, and tested their predictive power in test samples. Predicted CAG repeat lengths were significantly correlated with experimentally determined CAG repeat lengths, whereas models based upon randomly permuted CAGs were not at all predictive. Predictions from different batches of mRNA for the same cell lines were significantly correlated, implying that CAG length-correlated gene expression is reproducible. Notably, HTT expression was not itself correlated with HTT CAG repeat length. Taken together, these findings confirm the concept of a gene expression signature representing the continuous effect of HTT CAG length and not primarily dependent on the level of huntingtin expression. Such global and unbiased approaches, applied to additional cell types and tissues, may facilitate the discovery of therapies for HD by providing a comprehensive view of molecular changes triggered by HTT CAG repeat length for use in screening for and testing compounds that reverse effects of the HTT CAG expansion. To evaluate the continuous analytical approach as a strategy to discover the molecular consequences of the HTT CAG repeat, genome-wide gene expression datasets were generated from a panel of 107 human lymphoblastoid cell lines with HTT CAGs spanning the entire spectrum of allele sizes.

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Durable HTT silencing using non-evolved dCas9 epigenome editors in patient-derived cells

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