Epigenome editing durability varies widely across cardiovascular disease target genes
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA999342 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR25442083_1.fastq.gz | Fastqsanger.gz | |||
| SRR25442083_2.fastq.gz | Fastqsanger.gz | |||
| SRR25442084_1.fastq.gz | Fastqsanger.gz | |||
| SRR25442084_2.fastq.gz | Fastqsanger.gz | |||
| SRR25442085_1.fastq.gz | Fastqsanger.gz |
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Project description:Background: Hepatic knockdown of the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene or the angiopoietin-like 3 (ANGPTL3) gene has been demonstrated to reduce blood low-density lipoprotein cholesterol (LDL-C) levels, and hepatic knockdown of the angiotensinogen (AGT) gene has been demonstrated to reduce blood pressure. Genome editing can productively target each of these three genes in hepatocytes in the liver, offering the possibility of durable “one-and-done” therapies for hypercholesterolemia and hypertension. However, concerns around making permanent gene sequence changes via DNA strand breaks might hinder acceptance of these therapies. Epigenome editing offers an alternative approach to gene inactivation, via silencing of gene expression by methylation of the promoter region, but the long-term durability of epigenome editing remains to be established. Objectives and Results: We assessed the ability of epigenome editing to durably reduce the expression of the human PCSK9, ANGPTL3, and AGT genes in HuH-7 hepatoma cells. Cells treated with the CRISPRoff epigenome editor and PCSK9 guide RNAs were maintained for up to 124 cell doublings and demonstrated durable knockdown of gene expression and increased CpG dinucleotide methylation in the promoter, exon 1, and intron 1 regions. In contrast, cells treated with CRISPRoff and ANGPTL3 guide RNAs experienced only transient knockdown of gene expression. Cells treated with CRISPRoff and AGT guide RNAs also experienced transient knockdown of gene expression; although initially there was increased CpG methylation throughout the early part of the gene, this methylation was geographically heterogeneous—transient in the promoter, and stable in intron 1.Conclusions: This work demonstrates precise and durable gene regulation via methylation, supporting a new therapeutic approach for protection against cardiovascular disease via knockdown of genes such as PCSK9. However, the durability of knockdown with methylation changes is not generalizable across target genes, likely limiting the therapeutic potential of epigenome editing compared to other modalities.
2024-07-25 | GSE239465 | GEO
Project description:BackgroundHepatic knockdown of the proprotein convertase subtilisin/kexin type 9 ( PCSK9 ) gene or the angiopoietin-like 3 ( ANGPTL3 ) gene has been demonstrated to reduce blood low-density lipoprotein cholesterol (LDL-C) levels, and hepatic knockdown of the angiotensinogen ( AGT ) gene has been demonstrated to reduce blood pressure. Genome editing can productively target each of these three genes in hepatocytes in the liver, offering the possibility of durable "one-and-done" therapies for hypercholesterolemia and hypertension. However, concerns around making permanent gene sequence changes via DNA strand breaks might hinder acceptance of these therapies. Epigenome editing offers an alternative approach to gene inactivation, via silencing of gene expression by methylation of the promoter region, but the long-term durability of epigenome editing remains to be established.MethodsWe assessed the ability of epigenome editing to durably reduce the expression of the human PCSK9, ANGPTL3 , and AGT genes in HuH-7 hepatoma cells. Using the CRISPRoff epigenome editor, we identified guide RNAs that produced efficient gene knockdown immediately after transfection. We assessed the durability of gene expression and methylation changes through serial cell passages.ResultsCells treated with CRISPRoff and PCSK9 guide RNAs were maintained for up to 124 cell doublings and demonstrated durable knockdown of gene expression and increased CpG dinucleotide methylation in the promoter, exon 1, and intron 1 regions. In contrast, cells treated with CRISPRoff and ANGPTL3 guide RNAs experienced only transient knockdown of gene expression. Cells treated with CRISPRoff and AGT guide RNAs also experienced transient knockdown of gene expression; although initially there was increased CpG methylation throughout the early part of the gene, this methylation was geographically heterogeneous-transient in the promoter, and stable in intron 1.ConclusionsThis work demonstrates precise and durable gene regulation via methylation, supporting a new therapeutic approach for protection against cardiovascular disease via knockdown of genes such as PCSK9 . However, the durability of knockdown with methylation changes is not generalizable across target genes, likely limiting the therapeutic potential of epigenome editing compared to other modalities.
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Project description:Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated CRISPR-associated nucleases (Cas) are among the most promising technologies for the treatment of hemoglobinopathies including Sickle Cell Disease (SCD). We are only beginning to identify the molecular variables that influence the specificity and the efficiency of CRISPR- directed gene editing, including the position of the cleavage site and the inherent variability among patient samples selected for CRISPR-directed gene editing. Here, we target the beta globin gene in human CD34+ cells to assess the impact of these two variables and find that both contribute to the global diversity of genetic outcomes. Our study demonstrates a unique genetic profile of indels that is generated based on where along the beta globin gene attempts are made to correct the SCD single base mutation. Interestingly, even within the same patient sample, the location of where along the beta globin gene the DNA is cut, HDR activity varies widely. Our data establish a framework upon which realistic protocols inform strategies for gene editing for SCD overcoming the practical hurdles that often impede clinical success.
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Project description:Mounting evidence has called into question our understanding of the role that the central dogma of molecular biology plays in human pathology. The conventional view that elucidating the mechanisms for translating genes into proteins can account for a panoply of diseases has proven incomplete. Landmark studies point to epigenetics as a missing piece of the puzzle. However, technological limitations have hindered the study of specific roles for histone post-translational modifications, DNA modifications, and non-coding RNAs in regulation of the epigenome and chromatin structure. This feature highlights CRISPR systems, including CRISPR-Cas9, as novel tools for targeted epigenome editing. It summarizes recent developments in the field, including integration of optogenetic and functional genomic approaches to explore new therapeutic opportunities, and underscores the importance of mitigating current limitations in the field. This comprehensive, analytical assessment identifies current research gaps, forecasts future research opportunities, and argues that as epigenome editing technologies mature, overcoming critical challenges in delivery, specificity, and fidelity should clear the path to bring these technologies into the clinic.
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Project description:BACKGROUND:Variation in healthcare delivery and outcomes in neonatal intensive care units (NICUs) may be partly explained by differences in safety culture. OBJECTIVE:To describe NICU care giver assessments of safety culture, explore variability within and between NICUs on safety culture domains, and test for association with care giver characteristics. METHODS:NICU care givers in 12 hospitals were surveyed using the Safety Attitudes Questionnaire (SAQ), which has six scales: teamwork climate, safety climate, job satisfaction, stress recognition, perception of management and working conditions. Scale means, SDs and percent positives (percent agreement) were calculated for each NICU. RESULTS:There was substantial variation in safety culture domains among NICUs. Composite mean score across the six domains ranged from 56.3 to 77.8 on a 100-point scale and NICUs in the top four NICUs were significantly different from the bottom four (p<0.001). Across the six domains, respondent assessments varied widely, but were least positive on perceptions of management (3%-80% positive; mean 33.3%) and stress recognition (18%-61% positive; mean 41.3%). Comparisons of SAQ scale scores between NICUs and a previously published adult ICU cohort generally revealed higher scores for NICUs. Composite scores for physicians were 8.2 (p=0.04) and 9.5 (p=0.02) points higher than for nurses and ancillary personnel. CONCLUSION:There is significant variation and scope for improvement in safety culture among these NICUs. The NICU variation was similar to variation in adult ICUs, but NICU scores were generally higher. Future studies should validate whether safety culture measured with the SAQ correlates with clinical and operational outcomes in NICUs.
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Project description:The epigenome defines the unique gene expression patterns and resulting cellular behaviors in different cell types. Epigenome dysregulation has been directly linked to various human diseases. Epigenome editing enabling genome locus-specific targeting of epigenome modifiers to directly alter specific local epigenome modifications offers a revolutionary tool for mechanistic studies in epigenome regulation as well as the development of novel epigenome therapies. Inducible and reversible epigenome editing provides unique temporal control critical for understanding the dynamics and kinetics of epigenome regulation. This review summarizes the progress in the development of spatiotemporal-specific tools using small molecules or light as inducers to achieve the conditional control of epigenome editing and their applications in epigenetic research.
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Project description:Initial sensitivity to psychostimulants can predict subsequent use and abuse in humans. Acute locomotor activation in response to psychostimulants is commonly used as an animal model of initial drug sensitivity and has been shown to have a substantial genetic component. Identifying the specific genetic differences that lead to phenotypic differences in initial drug sensitivity can advance our understanding of the processes that lead to addiction. Phenotyping inbred mouse strain panels are frequently used as a first step for studying the genetic architecture of complex traits. We assessed locomotor activation following a single, acute 20 mg/kg dose of cocaine (COC) in males from 45 inbred mouse strains and observed significant phenotypic variation across strains indicating a substantial genetic component. We also measured levels of COC, the active metabolite, norcocaine and the major inactive metabolite, benzoylecgonine, in plasma and brain in the same set of inbred strains. Pharmacokinetic (PK) and behavioral data were significantly correlated, but at a level that indicates that PK alone does not account for the behavioral differences observed across strains. Phenotypic data from this reference population of inbred strains can be utilized in studies aimed at examining the role of psychostimulant-induced locomotor activation on drug reward and reinforcement and to test theories about addiction processes. Moreover, these data serve as a starting point for identifying genes that alter sensitivity to the locomotor stimulatory effects of COC.
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