Clinical OMICS

NOV-DEC 2018

Healthcare magazine for research scientists, labs, pathologists, hospitals, cancer centers, physicians and biopharma companies providing news articles, expert interviews and videos about molecular diagnostics in precision medicine

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www.clinicalomics.com November/December 2018 Clinical OMICs 35 system is based on CRISPR-dCas9 fused with the catalytic domain of DNA methyltransferase 3A (DNMT3A) an enzyme that can methylate from scratch. The system was delivered to dopaminergic neurons derived from human induced plurip- otent stem cells (hiPSCs) from a PD patient. As a result, the expression of SNCA was modified and disease-related cellu- lar-phenotypes characteristics of the neurons (i.e. mitochon- drial ROS-production and cellular viability) were reversed. There are multiple advantages to using this system. As Kantor explained, one advantage is that the system is "all in one"—meaning the deactivated Cas9 enzyme, the guide RNA, and the methylating enzyme are all delivered together. Perhaps the most exciting result from using this system was the ability to fine tune the expression of α-synuclein (decreasing it by roughly 30%), something that has not been achieved in the past. In order to test the effects of the decreased α-synuclein levels—and the impact that they have on the cell— the cells were exposed to rotenone to induce oxidative stress. The research- ers found that their system amelio- rated the sensitivity of the cells to the stress conditions. "The ability to translate the knowl- edge that has accumulated through basic science research about a disease mechanism, specifically a gene regu- lation mechanism, toward next-gen- eration drug discovery" is what Chiba-Falek finds most exciting about this work. Although the paper focuses on PD, Chiba-Falek is quick to point out that they are working to "develop tools that could be used as a therapy or intervention" for multi- ple different diseases caused by gene dysregulation. When asked what is next, Chiba-Falek explains that they are mov- ing their system "beyond α-synuclein and PD," noting that they are "continuing both vertically, by moving this devel- opment into in vivo animal models, but also expanding hor- izontally, by branching out into other neurodegenerative diseases such as Alzheimer 's disease (AD)." She adds that their ability to achieve fine regulation expands the potential to precisely treat individual patients based on the extent of SNCA dysregulation. "Delivery platforms based on lentiviral vectors are highly efficient for CRISPR-dCas9 delivery," Kantor explained. However, one caveat of employing retroviruses, is that they can integrate their genomes into the host chromosome. This could be dangerous if these integrations occur near certain genes, and could potentially cause cancer if it lands near an oncogene. "Even though this negative outcome has been demonstrated for simple retroviral vectors, excluding len- tiviruses, the concern is real. Therefore, developing safer delivery methods is incredibly important," Kantor added. The team is currently testing a modified, integrase-deficient, version of the lentivirus vector. This delivery vehicle has a mutation in the enzyme needed for integration, and for that reason cannot integrate into the genome. This work lays the foundation for clinical applications which are sorely needed for neurodegenerative diseases. When asked to give an estimate of when this may become useful to patients, Chiba-Falek answered, "there is still a long way to go, but, it is very feasible within our lifetime." The Duke team developed an all-in-one lentiviral vector for targeted DNA-methylation editing within intron 1.

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