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In Cellular Studies, Gene Editing Rescues Parkinson's Disease Phenotypes

Talan, Jamie

doi: 10.1097/01.NT.0000552946.92586.cb
At the Bench
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In a gene editing experiment involving human induced pluripotent stem cells from a patient with a familial form of Parkinson's disease, turning up DNA methylation led to a downregulation of SNCA messenger-RNA and reduced alpha-synuclein protein levels by about a third — sufficient to reverse the mitochondrial dysfunction, cellular vulnerability, and neuronal death that are part of the disease's pathological phenotype.

Scientists at Duke University used gene editing techniques to target DNA methylation on a region of a gene implicated in Parkinson's disease (PD) — SNCA — and reduced levels of alpha-synuclein transcript and protein.

Elevated levels of wild-type alpha-synuclein are thought to drive dopaminergic neuronal loss and in turn susceptibility to PD. The researchers were intrigued by earlier studies that had shown that the DNA-methylation state within a region located in intron 1 of SNCA gene affects alpha-synuclein expression. Others in the field have reported disease-related changes in DNA methylation in this region of SNCA intron 1, they said.

In the current experiment, turning up DNA methylation led to a downregulation of SNCA messenger-RNA and reduced alpha-synuclein protein levels by about a third — sufficient to reverse the mitochondrial dysfunction, cellular vulnerability, and neuronal death that are part of PD's pathological phenotype.

DNA methylation is an epigenetic phenomenon, said Ornit Chiba-Falek, PhD, associate professor of neurology at Duke and senior author of the study in the November 7, 2018, issue of Molecular Therapy. “It does not alter the DNA sequence but is added to the DNA to regulate the genome activity, and it controls transcription and gene expression.

“It is important to maintain physiologically normal levels of alpha-synuclein,” said Dr. Chiba-Falek. “This epigenome editing technique rescued the Parkinson's phenotypes at a cellular level,” she said.

The researchers are planning to test this approach in a rat model of PD. “This approach would be highly attractive for developing ‘smart drugs’ as disease-modifying therapies and preventative interventions for PD, Alzheimer's disease, and other neurological diseases and pathologies associated with dysregulation of gene expression,” Dr. Chiba-Falek added.

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Study Design

For the study, the research team used reprogrammed stem cells derived from a patient with an early-onset familial form of PD that involves triplication of the SNCA locus. Studies have shown that SNCA human induced pluripotent stem cell-derived (iPSC) dopamine neurons have a two-fold increase in SNCA mRNA and protein expression compared with healthy neurons.

Dr. Chiba-Falek thought the SNCA locus would be a good target. The lower the DNA methylation, the higher the messenger-RNA and protein expression, she reasoned.

Dr. Chiba-Falek partnered with Boris Kantor, PhD, a viral vector specialist and assistant research professor in neurobiology, to do the research. His expertise in designing lentiviral delivery vehicles for gene therapy moved the project forward. He used CRISPR-deactivated Cas9 fused with the catalytic domain of DNA-methyltransferase 3A and added it to the human iPSC-derived dopamine neurons.

Increasing DNA methylation downregulated SNCA messenger-RNA by 30 percent (p=0.006) and that led to a 25 percent reduction in protein levels compared to those in cells carrying the control transgene (without a synthetic gRNA molecule that binds to the target, intron 1).

Another analysis of the dopamine neurons only (detected by immunofluorescent double staining) showed a 36 percent reduction in SNCA protein levels.

The investigators also tested the dopamine neurons and found that there was a significant rescue of cellular perturbations characteristic of the PD patient-derived neurons with the SNCA triplication. There was a reduction in reactive oxygen species and an increase in the viability of the cells.

Delivering the lentivirus DNA methylation package to target specifically the regulatory element in SNCA intron 1 was a clever way of directly targeting SNCA transcripts with another technique called RNA interference, [in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules], she added. “Those studies led to too much depletion of alpha-synuclein and that had its own neurotoxic effects.”

The new approach would allow them to have tight regulation of the transcription program of SNCA gene (by targeting DNA methylation), Dr. Chiba-Falek said.

“DNA methylation represents a stable epigenetic mark with a potential for long-term effects on genome expression,” she and her fellow scientists wrote in the paper.

Worried that there could be off-target effects of the technique, they looked at the global DNA-methylation levels and found no changes in the neurons that contained the DNA-methylation editing tool compared to the parental dopamine neuron-progenitor cells. They also examined the expression levels of 20 randomly selected genes and found similar transcript profiles in the DNA-methylated edited neurons and those without the DNA-methylated editing system.

Now, the Duke scientists are using the dopamine neuron-progenitor cells to test whether they can fine-tune the expression levels of alpha-synuclein by editing the DNA methylation state at this region of SNCA intron.

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The Advantage of the Delivery Platform

Dr. Kantor also is studying other ways to introduce gene modifications in precise targeted regions across the genome. He said that lentivirus has a checkered history and has been shown to integrate its genome with the host. He is now modifying lentiviral platforms so that they do not integrate into the host.

Dr. Kantor noted that novel non-integrating vectors would significantly enhance the safety of lentiviral delivery platforms. “One of the advantages of nonintegrating lentiviruses is that they have extremely low integration capacity and, therefore, the risk associated with insertions of the viral genomes into ‘unsafe harbors,’ that is, within the human genome, is very low,” said Dr. Kantor. “Another advantage of this delivery platform is its capability to carry bulky inserts like those needed for expression of CRISRP/Cas9 components,” he added.

So, when would a patient benefit from this personalized therapy? “Once the alpha-synuclein Lewy bodies have been formed it is too late,” explained Dr. Chiba-Falek. “I think we would have to start in the preclinical stages, which means we need better biomarkers than we have now.”

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Expert Commentary

“Lowering pathologic alpha-synuclein levels in PD gets at the core issue in most forms of PD,” said Ted M. Dawson, MD, PhD, director of the Institute for Cell Engineering and director of the Morris K. Udall Parkinson's Disease Research Center, interim director of the movement disorder division and the Parkinson's Disease and Movement Disorder Center at Johns Hopkins University School of Medicine.

“CRISPR approaches are exciting, but I am just not sure how one can modify these approaches to impact a disease like PD that involves the entire nervous system,” Dr. Dawson added. “We need approaches that target the entire nervous system.”

“The investigators showed that altering DNA methylation can change the expression of alpha-synuclein,” said Robert Nussbaum, MD, chief medical officer at Invitae, a genetic testing company in California. “The basic idea is to epigenetically alter alpha-synuclein just enough but not too much. Combining CRISPR technology and targeting DNA methylation works to do that.”

“The [current study] is interesting. It is still basic research, though,” Dr. Nussbaum said. “We need to understand whether there are any off-site targets. Plus, how are scientists going to deliver epigenetic editing to all the cells in which you want to modify alpha-synuclein expression?”

“Nonetheless,” Dr. Nussbaum continued, “this work takes an innovative approach that builds on the large body of basic science findings on the role of alpha synuclein in PD to ask if we can start to translate these results into useful interventions. I look forward to more progress in exploring how manipulation of alpha- synuclein levels can affect the onset or progression of this disease.”

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The study authors and independent sources reported no disclosures relevant to the study.

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Link Up for More Information

•. Kantor B, Tagliaferro L, Gu J, et al Downregulation of SNCA expression by targeted editing of DNA methylation: A potential strategy for precision therapy in PD Mol Ther 2018; 26(11):2638–2649.
© 2019 American Academy of Neurology