lentiviral-vector-platform-for-efficient-delivery-epigenome-editing

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

Duke University Medical Center

The TOMM40 poly-T is a polymorphism in intron 6 of the TOMM40 gene, which is adjacent to and in linkage disequilibrium with APOE. Roses et al. identified the association between the length of TOMM40 poly-T with the risk and age of onset of late-onset Alzheimer's disease (LOAD). Following the original discovery, additional studies found associations between the TOMM40 poly-T and LOAD-related phenotypes independent of APOE genotypes, while others did not replicate these associations. Furthermore, the identity of the TOMM40 poly-T risk allele has been controversial between different LOAD-related phenotypes. Here, we propose a framework to address the conflicting findings with respect to the TOMM40 poly-T allele associations with LOAD phenotypes and their functional effects. The framework is used to interpret previous studies as means to gain insights regarding the nature of the risk allele, very long versus short. We suggest that the identity of the TOMM40 poly-T risk allele depends on the phenotype being evaluated, the ages of the study subjects at the time of assessment, and the context of the APOE genotypes. In concluding remarks, we outline future studies that will inform the mechanistic interpretation of the genetic data.

The effects of the TOMM40 poly-T alleles on Alzheimer's disease phenotypes.

Short structural variants-variants other than single nucleotide polymorphisms-are hypothesized to contribute to many complex diseases, possibly by modulating gene expression. However, the molecular mechanisms by which noncoding short structural variants exert their effects on gene regulation have not been discovered. Here, we study simple sequence repeats (SSRs), a common class of short structural variants. Previously, we showed that repetitive sequences can directly influence the binding of transcription factors to their proximate recognition sites, a mechanism we termed non-consensus binding. In this study, we focus on the SSR termed Rep1, which was associated with Parkinson's disease (PD) and has been implicated in the cis-regulation of the PD-risk SNCA gene. We show that Rep1 acts via the non-consensus binding mechanism to affect the binding of transcription factors from the GATA and ELK families to their specific sites located right next to the Rep1 repeat. Next, we performed an expression analysis to further our understanding regarding the GATA and ELK family members that are potentially relevant for SNCA transcriptional regulation in health and disease. Our analysis indicates a potential role for GATA2, consistent with previous reports. Our study proposes non-consensus transcription factor binding as a potential mechanism through which noncoding repeat variants could exert their pathogenic effects by regulating gene expression.

Toward deciphering the mechanistic role of variations in the Rep1 repeat site in the transcription regulation of SNCA gene.

Elevated levels of SNCA have been implicated in the pathogenesis of Parkinson's disease (PD), while normal physiological levels of SNCA are needed to maintain neuronal function. We ought to develop new therapeutic strategies targeting the regulation of SNCA expression. DNA methylation at SNCA intron 1 regulates SNCA transcription, and PD brains showed differential methylation levels compared to controls. Thus, DNA methylation at SNCA intron 1 is an attractive target for fine-tuned downregulation of SNCA levels. Here we developed a system, comprising an all-in-one lentiviral vector, for targeted DNA methylation editing within intron 1. The system is based on CRISPR-deactivated Cas9 (dCas9) fused with the catalytic domain of DNA-methyltransferase 3A (DNMT3A). Applying the system to human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons from a PD patient with the SNCA triplication resulted in fine downregulation of SNCA mRNA and protein mediated by targeted DNA methylation at intron 1. Furthermore, the reduction in SNCA levels by the guide RNA (gRNA)-dCas9-DMNT3A system rescued disease-related cellular phenotype characteristics of the SNCA triplication hiPSC-derived dopaminergic neurons, e.g., mitochondrial ROS production and cellular viability. We established that DNA hypermethylation at SNCA intron 1 allows an effective and sufficient tight downregulation of SNCA expression levels, suggesting the potential of this target sequence combined with the CRISPR-dCas9 technology as a novel epigenetic-based therapeutic approach for PD.

Article	Year
The effects of the TOMM40 poly-T alleles on Alzheimer's disease phenotypes. Alzheimer's & dementia : the journal of the Alzheimer's Association\| PubMed ID: 29524426	2018
Toward deciphering the mechanistic role of variations in the Rep1 repeat site in the transcription regulation of SNCA gene. Neurogenetics\| PubMed ID: 29730780	2018
Downregulation of SNCA Expression by Targeted Editing of DNA Methylation: A Potential Strategy for Precision Therapy in PD. Molecular therapy : the journal of the American Society of Gene Therapy\| PubMed ID: 30266652	2018

Ornit Chiba-Falek

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