Durable HTT silencing using non-evolved dCas9 epigenome editors in patient-derived cells
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ABSTRACT:
PROVIDER: PRJNA1192536 | ENA |
REPOSITORIES: ENA
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Project description:Huntington’s Disease (HD) is an autosomal dominant neurodegenerative disorder caused by a trinucleotide repeat in exon 1 of the Huntingtin (HTT) gene. We used dCas9 epigenetic editing to downregulate HTT as a therapeutic approach for HD. A screen of multiple dCas9 variants fused to KRAB and DNMT3A/L with increasingly expanded PAM targeting was conducted to assess the ability to downregulate total HTT. Surprisingly, only spdCas9 could significantly downregulate HTT, while expanded PAM site variants dxCas9 and dCas9-VQR were less efficient in reducing HTT expression due to decreased binding efficiency of dCas9 variants. We further investigated DNA methylation changes through reduced representation bisulfite sequencing, showing high on-target increases in DNA methylation and few off-targets. In addition, we demonstrate mitotically stable HTT silencing of up to 6 weeks in vitro in a rapidly dividing cell line. Finally, we showed significant downregulation of HTT in patient-derived neuronal stem cells, showing the applicability in a disease-relevant system. This approach holds great promise for those suffering from disorders caused by gain of function mutations like HD.
2025-04-01 | GSE283225 | GEO
Project description:We report the generation of CRISPR-dCas9 DNA methyltransferases to mediate targeted DNA methylation. Using the dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B methyltransferases, we have demonstrated that these two methyltransferase can mediate targeted methylation in three human genes tested: uPA, TGFBR3, and CDKN2A in human HEK293T cells. We also showed that these methyltransferases could mediate gene inhibition. five samples co-transfected with five uPA sgRNAs and each of the four dCas9 fusions, or control transfection with pUC19 plasmid
2016-06-01 | E-GEOD-74935 | biostudies-arrayexpress
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.
2023-12-20 | GSE240937 | 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 | GSE226200 | 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 | GSE226188 | 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 | GSE240935 | 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 | 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 | GSE240933 | 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 | GSE240932 | GEO