Developing Remyelination Strategies for Demyelinating Optic Neuropathies Using Human Pluripotent Stem Cells

PROJECT SUMMARY/ABSTRACT Optic nerve demyelinating diseases such as Optic Neuritis (ON) cause acute reduction of visual acuity and often chronic visual loss. The estimated incidence of ON in the USA is 6.4/100,000, and 50% of the 2.5 million multiple sclerosis (MS) patients worldwide are estimated to develop one or more episodes of ON during the course of

their disease. Myelination of the optic nerve is carried out by a specialized cell, the oligodendrocyte (OL), that coats and protects axons and promotes neural conduction. Demyelination can cause apoptosis of the OLs, axonal damage, and neuronal cell death. Remyelination-based treatments have the potential to help protect

retinal ganglion cell (RGC) neurons and reduce chronic vision loss, and perhaps help restore lost vision. The long-term goal of this project is to identify molecular pathways involved in differentiation and maturation of OLs that can be exploited for promoting remyelination of the demyelinated optic nerve. To this end, the PI has

developed human pluripotent stem cell (hPSC) reporters for RGCs and OLs, and performed single cell RNA- sequencing and a preliminary high-throughput screen (HTS) to identify regulators of OL differentiation and maturation. Based upon this preliminary data, the PI proposes to: 1) expand the screening effort to identify

additional compounds and pathways involved in OL maturation, 2) examine the transcription factors and pathways identified from the preliminary work for their role in OL differentiation, and 3) further identify and systemically characterize microRNAs (miRNAs) involved in OL specification and maturation. To perform the

proposed studies, the PI will develop innovative tools such as hPSC-reporters for OL differentiation with inducible CRISPRi (interference) and CRISPRa (activation) system, and a functional in-vitro hOL/hRGC co-culture system for assessing hRGC myelination by hOLs. In the mentored phase, in Dr. Don Zack’s lab, the PI will carry out

genome-editing to generate the CRISPRi and CRISPRa lines and acquire training at Wilmer’s HCS facility where small molecule screening to probe for signaling pathways involved in OL maturation will be performed. In the second year, under the guidance of Dr. Jay Baraban (an expert in microRNA biology), the miRNA studies on

OLs will be initiated. The mentored phase will also be supplemented by training with Ingo Ruczinski (biostatistics collaborator), who will provide assistance with the pathway analysis and hit validation of HTS. Furthermore, the PI’s co-mentor, Dr. Peter Calabresi, who is a leading scientist in demyelinating diseases, will regularly meet,

advise and help him prioritize the genes and pathways for further examination. Additionally, during the mentored phase, the PI will regularly meet with his advisory committee, attend scientific conferences, and continue his career development. The PI is in an ideal environment for the proposed research and for his career development

as Dr. Zack has an established hPSC lab, state-of-the-art HCS facilities, and collaborations with renowned neuroscientists and vision-scientists. This will help PI to set-up good collaborations, learn new techniques, and build an independent research laboratory at a well-established academic institution.