Emma Sullivan HMB496Y Final Report, April 3, 2020 CRISPR-Cas9 Directed Removal of Duplications in Pelizaeus Merzbacher Disease Emma Sullivan, Human Biology Program, University of Toronto and Eleonora Maino, Genetics and Genome Biology, SickKids Pelizaeus-Merzbacher Disease (PMD) is a rare X-linked pediatric disorder affecting 1in 100,000 boys. The disease is caused by mutations of the PLP1 gene, encoding Proteolipid Protein 1, a ​ ​ key structural protein of the myelin that encapsulates axons for efficient cell-cell communication in the nervous system. Mutations in PLP1 result in abnormal myelination, resulting in severe ​ ​ motor and cognitive impairment, and limited life expectancy. Genomic duplications of various sizes, that contain the entire PLP1 gene, account for 70% of PMD cases. Currently, there is no ​ ​ cure for PMD, with treatment options limited to symptom management. In a previous study, we successfully tested a novel single guide CRISPR-Cas9 genome editing strategy that removed the PLP1 duplication in a PMD mouse model and led to an improvement in disease manifestations. ​ This project aims to utilize this approach to correct PLP1 duplications in three PMD patient cell ​ ​ lines. We characterized the nature of the duplications through Whole Genome Sequencing and identified a common duplicated region among 2 patient cell lines, then designed intergenic and intronic guides and assessed their efficacy in HEK293 cells. The most active guides were selected and will be transfected in wildtype fibroblasts before testing them in patient fibroblasts. Ultimately, a universal guide will be selected and injected in a PMD mouse model to check its efficacy in an in-vivo system. If the cells prove to be functional, they could be utilized for cerebral transplant in individuals affected by PMD. Successful completion of this project will lay the foundation for the development of novel therapies for the majority of PMD patients and potential avenues for the treatment of genetic disorders caused by genomic duplications. Introduction Leukodystrophies are a collection of genetic disorders resulting in the abnormal development or destruction of the white matter, or myelin, of the central nervous system (CNS) (1,2). Myelin, a lipid-rich substance, normally encapsulates the axons of neurons, acting as an insulator to promote rapid cell-cell communication. When myelin production or activity is impaired, the nervous system cannot function properly (1), as in Pelizaeus-Merzbacher disease (PMD), an X-linked leukodystrophy caused by a mutation in the proteolipid protein 1 (PLP1) gene (3, 4, 5). ​ ​ PMD is a rare condition, with an incidence rate of approximately 1:200,000 - 1:500,000 (2), primarily affecting males. PLP1, expressed exclusively in oligodendrocytes of the CNS (2), is a ​ ​ dosage sensitive gene that is responsible for the major protein component of myelin (1, 6). Genomic duplications containing the entire PLP1 gene are the most common cause of PMD, ​ ​ accounting for nearly 70% of all cases (1, 5, 7). These duplications most commonly occur as tandem head-to-tail repeats, and the duplicated region varies in size, ranging from approximately 56 kb to 11 Mb (5, 8). Although the mechanism is unknown, PLP1 overexpression is likely the ​ ​ cause of the hypomyelination seen in PMD patients (5). PMD patients exhibit phenotypic variability, although almost all cases are characterized by early neurological deficits and progressive degeneration (3, 9, 10). PMD was first decribed by Friedrich Pelizaeus in 1885, and later by Ludwig Merzbacher, in children displaying cognitive ​ impairments, spastic quadriplegia, nystagmus or rapid uncontrollable lateral eye movements, Emma Sullivan, HMB496Y Final Report, April 3, 2020 ataxia, hypotonia, and motor delays (3, 11). Based on how the disease manifests, PMD is classified into two primary types. In the most common form, patients with classic PMD exhibit nystagmus, hypotonia, and sometimes motor delays in childhood, and other symptoms including spasticity and ataxia appear in late adolescence. Most patients suffer from life-threatening and eventually fatal complications before they reach age 30 (10). A more severe and less common form, connatal PMD, presents earlier in infancy, and patients have a poor prognosis with a life expectancy that does not exceed early childhood (1, 8, 11). In suspected PMD patients with the aforementioned clinical manifestations, a diagnosis is typically made based on the results of ​ magnetic resonance imaging (MRI) showing abnormal white matter in the CNS (9, 11, 12), and a confirmed change in the PLP1 gene or copy number (4, 13), which distinguishes PMD from ​ ​ other leukodystrophies. Due to the rarity of the disease, patients with no familial history of X-linked inheritance are often mistakenly diagnosed with cerebral palsy or other neuropathies (14), further contributing to the already small population of identified individuals with PMD. This limited sample size has made general studies of PMD difficult, and few analyses have been done on PMD patients, so PLP1 mutations at the microscopic level have not been well characterized. ​ ​ Currently, therapeutic approaches are aimed at alleviating symptoms of PMD, as there is no cure for this disease (3, 7, 9), although there are several new approaches being pursued. As described ​ in a 2019 study by Gupta et al, neural stem cell transplantation has been explored as a treatment course for PMD, where hypomyelinating neural cells are replenished with CNS stem cells expanded in culture. Patients in this study all showed positive neural changes monitored via MRI, and all were healthy four years post transplant. However, some patients developed antibodies to the transplanted cells, demonstrating that this procedure can elicit an immune response with the potential for complications (2). In recent years, genome engineering methods ​ have been suggested as treatments for inherited disorders like PMD (15). Clustered regularly ​ interspaced short palindromic repeats (CRISPR) has emerged as an ideal candidate for this ​ approach. CRISPR-Cas systems are found in bacteria, and are used as immune systems to protect the organism from foreign DNA. CRISPR, remnants of harmful viruses, can guide a Cas9 enzyme to identical regions of the DNA where the Cas9 endonuclease acts as molecular scissors, creating double stranded breaks in DNA and effectively removing the intervening target sequence (16, 17). Previous research has shown that by pairing this system with a single guide RNA in vitro and more recently in vivo, it is possible to excise disease duplications in Duchenne muscular dystrophy patient cells, another X-linked neuromuscular disease (15, 18, 19). The customizable nature of this method allows for the targeted removal of any area of interest in the genome; in this case, the duplication region seen in most incidences of PMD. Our project focuses on the CRISPR-Cas9-mediated removal of genomic duplications in PMD patient cells. Due to ​ the heterogeneous nature of PLP1 duplications, the minimal common duplicated region will be ​ ​ identified through sequencing of the duplications in each patient cell line. An RNA guide will be designed to be used with CRISPR-Cas9 to excise the critical portion of the PLP1 duplication ​ ​ responsible for the disease phenotype. In this way, we hope to develop an approach that can be applied to all PMD patients with PLP1 duplications regardless of duplication size, and ​ ​ potentially also to other genetic disorders with similar duplication genotypes. To date, we have performed whole genome sequencing (WGS) on three fibroblast cell lines, successfully amplified the duplication junction in two cell lines, identified a common duplicated region Emma Sullivan, HMB496Y Final Report, April 3, 2020 among them, and designed and tested 20 guides in HEK293 cells. Our next steps are to test the most active guides in WT fibroblasts, and then in patient cells. We also hope to amplify the duplication junction in the third cell line, to see how that duplication lines up against the common duplicated region we have identified, and potentially further narrow down a conserved region responsible for the phenotypic manifestation of PMD. Methods Whole Genome Sequencing (WGS) and Analysis WGS was performed using the Illumina HiSeq X platform by The Centre for Applied Genomics in Toronto, producing 150 bp paired-end reads. Output FASTQ files were aligned to the hg19 genome and analyzed using the Integrative Genomics Viewer (IGV) Software. Duplicated regions were identified by an increase in reads mapping to the area. Exact genomic locations of the PLP1 duplications were determined by identifying the location of soft clipped reads, as ​ ​ previously described (20, 21) (Fig. 1a). 3’ forward and 5’ reverse primers were designed to around the duplication junction (patient 1: 3’F-TCTCACATGCCAAAAACAGG, ​ 5’R-CTCTCCTGCCCCAATCTCTA; patient 3: 3’F-CCCTGTTTGCCTCGGTATTA, 5’R-GGCAATGGCTTGAGAGTAGG) using the Primer 3 Plus Website, and PCR amplification ​ at the following cycling conditions (annealing temperature of 59°C, extension time 15s, and 35 ​ ​ cycles), followed by Sanger sequencing was used to confirm the duplication arrangement. Cell Culture Fibroblast cells from three PMD patients with duplication mutations were received from the ​ biobank of Robert Debré Hospital, Paris, courtesy of Dr. Odile Boespflug-Tanguy and Dr. Anne Baron. Cells were maintained in DMEM with 10% fetal bovine serum (FBS), 1% penicillin- streptomycin, and