{"id":131,"date":"2020-11-19T19:05:29","date_gmt":"2020-11-19T19:05:29","guid":{"rendered":"https:\/\/epimvp.med.umich.edu\/?post_type=creative_projects&p=131"},"modified":"2022-03-14T19:25:52","modified_gmt":"2022-03-14T19:25:52","slug":"project-2-proteins-to-cell-systems","status":"publish","type":"creative_projects","link":"https:\/\/epimvp.med.umich.edu\/research\/project-2-proteins-to-cell-systems\/","title":{"rendered":"Project 2: Proteins to Cell Systems"},"content":{"rendered":"\n
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Along with the expanding list of potential epilepsy genes comes the challenge of genetic variants of uncertain significance (VUS). <\/strong><\/h3>\n

VUS numbers are rising as more patients undergo NGS, but we lack the tools to determine VUS pathogenicity and disease mechanisms. To address these shortfalls, the EpiMVP will optimize cutting-edge multiplatform assays for epilepsy gene function.
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The <\/span>long-term goal<\/b> of this project is to deliver an <\/span>in vitro<\/span><\/i> testing pipeline with defined phenotypes in human neuronal models to assay clinically relevant VUS for non-ion-channel epilepsy genes. Project 2 will test variants for the chosen genes in complex structural and functional assays, using human pluripotent stem cell (hPSC) knockout lines generated by the Human Epilepsy Tools Core (HETC)<\/strong>. Human <\/span>in vitro<\/span><\/i> models will include 1) <\/span>2-D hPSC cultures<\/b>: small molecule differentiation into excitatory or inhibitory cortical neurons, excitatory and inhibitory induced neurons (iNeurons) generated by forced transcription factor expression; and 2) Excitatory, inhibitory and fusion (combined excitatory and inhibitory) <\/span>brain organoid cultures<\/b>.<\/span><\/p>\n<\/div>\n<\/div>\n\n\n

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Assays of VUS rescue (or deleterious gain-of-function) effects will include morphology, gene expression and neuronal\/network activity. The latter includes multielectrode array [MEA] and patch clamp recordings, calcium imaging and depth electrode local field potential [LFP] recordings (in brain organoids).<\/span><\/p>\n<\/div>\n<\/div>\n\n\n

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Our immediate goals\u00a0are to optimize assays for 1-2 genes per year, determine VUS pathogenicity in vitro<\/i> for these genes and, in concert with the Variant and Gene Curation Core (VGCC), refine the VUS list for further in vivo<\/i> testing in Project 3. Two sets of milestones\u00a0are proposed:<\/h3>\n

To determine VUS pathogenicity using 2-D hPSC assays (Milestone 1) and brain organoid models (Milestone 2). <\/b>The HETC, Parent and Ross labs have experience generating cortical neurons via small molecule and iNeuron differentiation, and with multiple brain organoid culture protocols. We will express non-ion channel epilepsy gene variants chosen by the <\/span>GVCC<\/b> and <\/span>Project 1<\/b>, and via constructs generated by the <\/span>HETC,<\/b> in a knockout hPSC background<\/span>. Assays will\u00a0 include <\/span>neuronal morphology, gene expression, calcium imaging and electrophysiology in 2-D and brain organoid cultures. <\/span><\/p>\n

These studies will provide the following <\/span>deliverables<\/i><\/b>: 1) multiple optimized, cross-validated (between Parent and Ross labs) hPSC platforms to interrogate epilepsy genes; 2) determination of <\/span>in vitro<\/span><\/i> human neuronal VUS pathogenicity for at least 5 non-ion channel epilepsy genes; 3) human neuronal models for each epilepsy gene; and 4) optimized platforms for future mechanistic and precision therapeutic studies.<\/span><\/p>\n<\/div>\n<\/div>","protected":false},"excerpt":{"rendered":"

The ability to rapidly sequence exomes\/genomes with next- generation DNA sequencing (NGS) has accelerated epilepsy gene discovery, and the list of epilepsy genes has grown well beyond ion channels to those that affect a wide array of cellular functions. <\/p>\n","protected":false},"featured_media":132,"template":"","acf":[],"_links":{"self":[{"href":"https:\/\/epimvp.med.umich.edu\/wp-json\/wp\/v2\/creative_projects\/131"}],"collection":[{"href":"https:\/\/epimvp.med.umich.edu\/wp-json\/wp\/v2\/creative_projects"}],"about":[{"href":"https:\/\/epimvp.med.umich.edu\/wp-json\/wp\/v2\/types\/creative_projects"}],"wp:featuredmedia":[{"embeddable":true,"href":"https:\/\/epimvp.med.umich.edu\/wp-json\/wp\/v2\/media\/132"}],"wp:attachment":[{"href":"https:\/\/epimvp.med.umich.edu\/wp-json\/wp\/v2\/media?parent=131"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}