Durable HTT silencing using non-evolved dCas9 epigenome editors in patient-derived cells
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ABSTRACT:
PROVIDER: PRJNA1192536 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR31559258_1.fastq.gz | Fastqsanger.gz | |||
| SRR31559258_2.fastq.gz | Fastqsanger.gz | |||
| SRR31559259_1.fastq.gz | Fastqsanger.gz | |||
| SRR31559259_2.fastq.gz | Fastqsanger.gz | |||
| SRR31559260_1.fastq.gz | Fastqsanger.gz |
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Project description: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.
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Project description:Programmable epigenetic editors that achieve long-term gene expression modulation hold immense therapeutic potential. However, the large size of current CRISPR-based epigenome editors has limited their widespread application. This study describes the engineering of compact, mRNA-delivered epigenetic repressors, termed CRISPR OFF-EE, using SpyCas9, intein-based split-SpyCas9 or the smaller SF01 Cas variant, to achieve highly efficient and durable silencing of endogenous genes. We developed "superfolder" V2 mRNA constructs with optimized codon usage, UTRs, and nuclear localization signals, which enhanced mRNA stability, translational output, and LNP packaging. These CRISPR OFF-EE V2 editors demonstrated significantly improved and durable gene silencing across multiple targets in variable cell lines. Remarkably, a single intravenous administration of lipid nanoparticles (LNPs) encapsulating the optimized OFF-EE-V2 mRNA and selected sgRNAs targeting PCSK9 in mice led to an approximately 90% reduction in circulating PCSK9 protein and a 55% reduction in LDL-C levels, with effects persisting for at least 180 days. Whole-genome bisulfite sequencing confirmed the high specificity of these editors, with SF01-based editors exhibiting fewer off-target methylation events than SpCas9-based editors. The platform also demonstrated multiplexing capability and a favorable safety profile with liver-restricted activity. These findings establish a robust foundation for advancing in vivo therapeutics based on precise and durable epigenetic silencing using transiently delivered mRNA editors.
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Project description:Permanent epigenetic silencing with programmable editors equipped with transcriptional repressors holds great promise for the treatment of human diseases. However, to unlock its full therapeutic potential, a formal proof of durable epigenetic silencing upon transient editors’ delivery in vivo is needed. To this end, we targeted here Pcsk9, a hepatocyte-expressed gene involved in cholesterol homeostasis. In vitro screening of different editors’ designs indicated zinc finger proteins as the best-performing DNA binding platform to efficiently silence murine Pcsk9, with minimal genome-wide transcriptional and epigenetic perturbations. A single administration of lipid nanoparticles loaded with the editors’ mRNAs halved circulating levels of Pcsk9 for nearly 1 year in mice. Notably, Pcsk9 silencing and accompanying epigenetic repressive marks persisted following forced also upon liver regeneration, further corroborating heritability of the newly installed epigenetic state. This study lays the foundation for the development of in vivo therapeutics modalities based on epigenetic silencing.
2023-12-20 | GSE240934 | GEO
Project description:Permanent epigenetic silencing with programmable editors equipped with transcriptional repressors holds great promise for the treatment of human diseases. However, to unlock its full therapeutic potential, a formal proof of durable epigenetic silencing upon transient editors’ delivery in vivo is needed. To this end, we targeted here Pcsk9, a hepatocyte-expressed gene involved in cholesterol homeostasis. In vitro screening of different editors’ designs indicated zinc finger proteins as the best-performing DNA binding platform to efficiently silence murine Pcsk9, with minimal genome-wide transcriptional and epigenetic perturbations. A single administration of lipid nanoparticles loaded with the editors’ mRNAs halved circulating levels of Pcsk9 for nearly 1 year in mice. Notably, Pcsk9 silencing and accompanying epigenetic repressive marks persisted following forced also upon liver regeneration, further corroborating heritability of the newly installed epigenetic state. This study lays the foundation for the development of in vivo therapeutics modalities based on epigenetic silencing.
2023-12-20 | GSE240939 | GEO
Project description:Permanent epigenetic silencing with programmable editors equipped with transcriptional repressors holds great promise for the treatment of human diseases. However, to unlock its full therapeutic potential, a formal proof of durable epigenetic silencing upon transient editors’ delivery in vivo is needed. To this end, we targeted here Pcsk9, a hepatocyte-expressed gene involved in cholesterol homeostasis. In vitro screening of different editors’ designs indicated zinc finger proteins as the best-performing DNA binding platform to efficiently silence murine Pcsk9, with minimal genome-wide transcriptional and epigenetic perturbations. A single administration of lipid nanoparticles loaded with the editors’ mRNAs halved circulating levels of Pcsk9 for nearly 1 year in mice. Notably, Pcsk9 silencing and accompanying epigenetic repressive marks persisted following forced also upon liver regeneration, further corroborating heritability of the newly installed epigenetic state. This study lays the foundation for the development of in vivo therapeutics modalities based on epigenetic silencing.
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