RISPR-Mediated Knock out of GPR۳۷L۱ in Oligodendrocyte Progenitor Cells: A Promising Therapeutic Target for Inducing Remyelination

Background: In demyelinating diseases such as multiple sclerosisOligodendrocytes are lost. An up-and-coming restorativestrategy is inducing remyelination. Oligodendrocyte ProgenitorCells differentiation to oligodendrocytes (myelin-producingcells) is impeded by various factors. Doing gene set enrichmentanalysis using DAVID and Panther websites it was shown thatGpr۳۷l۱ is highly expressed in oligodendrocyte progenitor cellsin comparison to oligodendrocytes (more than ۳۰۰ fold changein expression). The selective orexin ۲ receptor (HCRTR۲) antagonistjnj-۱۰۳۹۷۰۴۹ has been shown to inhibit this orphanGPCR. In a previous unpublished study it was shown that inhibitionof HCRTR۲ would increase Proliferation and Differentiationof cortical neural stem cells toward oligodendrocytes.To further analyzed the effect of this inhibitor and also to investigatethe potential of GPR۳L۱ is a constitutively expressedreceptor) in induction of oligodendrogenesis we intended onknocking out this gene using CRISPR/Cas۹ in oligodendrocyteprogenitor cells.Materials and Methods: Gene set enrichment and pathwayanalysis was done using Panther and David websites. Morethan ۴۰% of marketed drugs target G-protein coupled receptors.Top ۵۰۰ G-protein Coupled receptor Genes expressed in Oligodendrocyteprogenitor cells in comparison to oligodendrocyteswas selected. Oligodendrocyte progenitor cells were isolatedusing FACS with pdgralpha monoclonal antibody and culturedwith ۲۰ ng/μl bFGF, ۱۰ ng/μl PDGFAA, ۱% B۲۷ and DMEM/F۱۲ medium. Guide rna was designed using CRISPRko. Vectorscontaining spcas۹ and GPR۳۷L۱ guide RNA is going to betransducted to Oligodendrocyte Progenitor Cells using lentiviralvectors.Results: ۱۵ most enriched GPCRs in Oligodendroycte progenitorcells was chosen out of the ۵۰۰ expressed genes (Chrm۱,Chrm۲, Hrh۱, Chrna۴, Gpr۳۷L۱, Ednrb, P۲ry۱, Gpr۱۷, Gpr۳۷,Gpr۱۶۲, Gpr ۱۹, Gpr۳۴, Gpr۵۶, Gpr۶۲). Gpr۱۷ is highly expressedin newly formed oligodendrocytes with more than ۹۰۰fold change in expression in comparison to mature myelinatingOligodendrocytes. Endothelin b receptor and Gpr۳۷ has alreadybeen shown to negatively regulated Oligodendrocyte differentiation.GPR۳۷L۱ has more than ۳۰۰ fold change in expressionin oligodendrocyte progenitor cells in comparison to oligodendrocytes.Further analysis hasn’t been done yet.Conclusion: GPR۳۷L۱ is a promising target in inducing oligodendrocytedifferentiation. Further analysis to confirm thishypothesis using CRISPR-mediated knockout and the suggestedinhibitor of this receptor (JNJ۱۰۳۹۷۰۴۹) is underway.Also to validate the suggested orexin ۲ receptor inhibitor as aligand for GPR۳۷L۱ ( a constitutive orphan GPCR) in an articlepublished in ۲۰۱۷, and to inspect our previous result showingincreased proliferation and oligodendrocyte differentiation ofcortical neural stem cells the current ongoing experiment is beingcarried out.

کلیدواژه ها:

CRISPR/Cas۹ ، Oligodendrocytes ، GPR۳۷L۱ ، Remyelination