In our recent manuscript entitled Downregulation of SNCA expression by targeted editing of DNA-methylation: A potential strategy for precision therapy in PD, recently published in the journal Molecular Therapy journal (via Cell Press), we developed new therapeutic strategies targeting the regulation of SNCA expression. Elevated levels of SNCA have been implicated in the pathogenesis of Parkinson’s disease (PD). The gene encodes a protein called alpha-synuclein, which, when expressed in high levels, can be toxic as it tends to generate and be assembled in pathological aggregates in the brain of a Parkinson’s patient via formation of so-called Lewy bodies — the hallmark of this devastating neurological disease.
In our publication, we have developed a new approach to reduce the pathological levels of alpha-synuclein. The approach is based on epigenetic targeting that results in the modifications in gene functions without affecting gene composition per se. In an attempt to alleviate the pathological overexpression of alpha-synuclein, we employed highly innovative technology, called CRISPR/Cas9 which most recently has been repurposed to modulate gene expression in a precise fashion.
We used an inactive form of Cas9 namely dCas9 which we fused with the catalytic (active) domain of an enzyme called DNA-methyltransferase 3A (DNMT3A). This enzyme is responsible for DNA methylation by means of adding methyl group that consists of one carbon and three hydrogen atoms — to a specific part of the SNCA gene (called intron 1). This modification instructs the DNA to create a closed “heterochromatin structure” that is generally inaccessible to transcription factors and RNA polymerase – the enzyme responsible for rewriting genetic information from DNA to RNA molecules. Lowering RNA synthesis results in the reduction of overall alpha-synuclein levels.
We constructed and applied the system to stem cell-derived dopamine-producing neurons — those primarily degenerated in Parkinson’s — from a patient with the SNCA gene triplication, which has been associated with increased production and aggregation of alpha-synuclein in Parkinson’s disease. To deliver dCas9-DNMT3A system to the cells, we used an optimized vehicle called a lentiviral vector. The vector has been evolved in Dr. Kantor’s laboratory to efficiently transport the therapeutic cargo inside the cells. By targeting DNA methylation at SNCA-intron 1, we were able to lower alpha-synuclein mRNA and protein levels. This reduction protected dopamine-producing neurons from characteristic disease-related changes. Specifically, Parkinson’s disease associates with dysfunction in mitochondria (the cells’ powerhouses that produce energy) which leads to pathological over-production of reactive oxygen species that damage all components of the cell, including proteins, lipids, and DNA- eventually leading to the cell death.
Our novel system protected neurons from this damage and other disease-related cellular-phenotypes characteristics of PD. The potential of CRISPR/dCas9 technology as a novel epigenetic-based therapeutic approach is huge and prospectively, our innovative platform that allows the regulation of gene expression programs to be fine-tuned would be highly attractive for developing ‘next generation drugs’ as prevention and/or disease-modifying interventions for PD, Alzheimer’s disease and other neurodegenerative diseases and pathologies associated with dysregulation of gene expression. In aiming to advance this system to the clinic, we now attempting to validate this therapeutic approach in vivo utilizing animal models of PD.
These findings are described in the article entitled Downregulation of SNCA expression by targeted editing of DNA-methylation: A potential strategy for precision therapy in PD, recently published in the journal Molecular Therapy. This work was conducted by Boris Kantor, Lidia Tagliafierro, Jeffrey Gu, Madison Elena Zamora, Ekaterina Ilich, Carole Grenier, Zhiqing Y. Huang, Susan Murphy, and Ornit Chiba-Falek from Duke University.