Therapeutic potential of Cpf1-based gene editing for myeloproliferative neoplasms

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Project Summary Myeloproliferative neoplasms (MPNs) are groups of hematological diseases overproducing blood cells. A single gain-of-function point mutation in JAK2 gene (1849GèT in exon 14), named JAK2V617F, has been identified in the majority of MPN patients. MPNs are transplantable with hematopoietic stem cells (HSCs) carrying JAK2V617F gene. While JAK2 inhibitors are promising drugs for inhibiting MPN-induced complications, they cannot cure MPNs. Until now, allogeneic HSC transplantation (allo-HSCT) has been the only curative treatment for MPNs. However, a majority of patients suffer from a shortage of HLA-matched donors and some allo-HSCT recipients develop graft-versus-host disease (GvHD) that is a prominent cause of death and has important implications on immunosuppressive medications. Hence, it would be of immense clinical benefit to develop safe gene editing strategies to inactivate JAK2V617F mutant HSCs from MPN patients containing both healthy and mutant HSCs. If these strategies are successful, it will be possible to curatively treat MPN patients using autologous HSC transplantation (auto-HSCT), without a need to find a matching donor in the patient's family and to avoid the risk of GvHD. In this proposal, we hypothesize that JAK2V617F-specific guide RNA (gRNA) will direct Cpf1 nuclease-mediated DNA cleavage of JAK2V617F gene, leading to the inactivation of MPN-initiating HSCs. To test this hypothesis, we will pursue two specific aims: i) Inactivation of MPN cells by removing JAK2V617F exon 14 using CRISPR/Cpf1 gene editing, and ii) Assess the therapeutic potential of CRISPR/Cpf1-based auto-HSCT in a xenograft model of MPN. In aim 1, we will assess the effects of JAK2V617F-specific CRISPR/Cpf1 gene editing on the colony-forming capacity of MPN cell lines and on ex vivo inactivation of HSCs-harboring JAK2V617F in MPN CD34+ HSC pool. In aim 2, we will first generate xenograft models of MPN by engrafting MPN patient-derived CD34+ cells. Next, we will test the therapeutic potential of high-dose chemotherapy followed by auto-HSCT with MPN HSCs edited by JAK2V617F-CRISPR/Cpf1 gene editing. These studies will demonstrate the feasibility of CRISPR/Cpf1 gene editing-based auto-HSCT joint with high-dose chemotherapy for the treatment of patients with MPNs. Regarding expected outcomes, we anticipate that we will demonstrate effective gene editing strategies for inactivating MPN-initiating HSCs. Such results will have an important positive impact because these approaches are translatable for developing gene editing- based auto-HSCT therapeutic strategy for MPN patients and possible for other hematopoietic malignancies.