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 α-synuclein (SNCA) gene has been implicated in the etiology of Parkinson's disease (PD) and dementia with Lewy bodies (DLB).

Genetic analysis of α-synuclein 3' untranslated region and its corresponding microRNAs in relation to Parkinson's disease compared to dementia with Lewy bodies.

The  intronic single nucleotide polymorphism (SNP), rs356168, has been associated with Parkinson's disease (PD) in large genome wide association studies (GWAS). Recently, the PD-risk allele, rs356168-G was shown to increase -mRNA expression using genome edited human induced pluripotent stem cells (iPSC)-derived neurons. In this study, as means of validation, we tested the effect of rs356168 on total -mRNA levels using brain tissues, temporal and frontal cortex, from healthy control donors. Carriers of the rs356168-G allele demonstrated a borderline significant decrease of -mRNA levels in temporal brain tissues ( = 0.02) compared to individuals homozygous for the 'A' allele. Similar trend, but weak, was observed in the analysis of frontal cortex samples, however, this analysis did not reach statistical significance. These results conflict with the recently reported effect of  SNP rs356168 described above. Our study conveys the need to carefully interpret the precise molecular mechanism by which rs356168, or another tightly linked variant, affects the regulation of  expression. The regulatory mechanisms that contribute to the observed associations between PD and the -3' linkage disequilibrium region warrant further investigations.

Interpreting Gene Expression Effects of Disease-Associated Variants: A Lesson from  rs356168.

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.

Downregulation of SNCA Expression by Targeted Editing of DNA Methylation: A Potential Strategy for Precision Therapy in PD.

Human-induced Pluripotent Stem Cell (hiPSC)-derived models have advanced the study of neurodegenerative diseases, including Parkinson's disease (PD). While age is the strongest risk factor for these disorders, hiPSC-derived models represent rejuvenated neurons. We developed hiPSC-derived Aged dopaminergic and cholinergic neurons to model PD and related synucleinopathies. Our new method induces aging through a `semi-natural' process, by passaging multiple times at the Neural Precursor Cell stage, prior to final differentiation. Characterization of isogenic hiPSC-derived neurons using heterochromatin and nuclear envelope markers, as well as DNA damage and global DNA methylation, validated our age-inducing method. Next, we compared neurons derived from a patient with SNCA-triplication (SNCA-Tri) and a Control. The SNCA-Tri neurons displayed exacerbated nuclear aging, showing advanced aging signatures already at the Juvenile stage. Noteworthy, the Aged SNCA-Tri neurons showed more α-synuclein aggregates per cell versus the Juvenile. We suggest a link between the effects of aging and SNCA overexpression on neuronal nuclear architecture.

Article	Year
Genetic analysis of α-synuclein 3' untranslated region and its corresponding microRNAs in relation to Parkinson's disease compared to dementia with Lewy bodies. Alzheimer's & dementia : the journal of the Alzheimer's Association\| PubMed ID: 28431219	2017
Interpreting Gene Expression Effects of Disease-Associated Variants: A Lesson from rs356168. Frontiers in genetics\| PubMed ID: 28979294	2017
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
Multiplication of the SNCA locus exacerbates neuronal nuclear aging. Human molecular genetics\| PubMed ID: 30304516	2019

Lidia Tagliafierro

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