Therapeutic potential of Cpf1-based gene editing for myeloproliferative neoplasms

项目摘要 骨髓增生性肿瘤（MPN）是一组血液疾病过度生产血细胞。一 JAK 2基因的一个功能获得性点突变（外显子14的1849 GèT），命名为JAK 2 V617 F， 在大多数MPN患者中发现。MPN可与造血干细胞（HSC）一起移植 携带JAK 2 V617 F基因。虽然JAK 2抑制剂是抑制MPN诱导的并发症的有希望的药物， 他们不能治愈MPN到目前为止，异基因造血干细胞移植（allo-HSCT）一直是唯一的治疗方法。 治疗MPN。然而，大多数患者缺乏HLA匹配的供体， allo-HSCT接受者发生移植物抗宿主病（GvHD），这是死亡的主要原因， 对免疫抑制药物的重要影响。因此，这将是巨大的临床效益， 开发安全的基因编辑策略，以从含有JAK 2 V617 F突变的MPN患者中扩增出JAK 2 V617 F突变的HSC， 健康和突变的HSC。如果这些策略是成功的，它将有可能治愈治疗MPN患者 使用自体HSC移植（auto-HSCT），而不需要在患者的骨髓中找到匹配的供体。 家庭和避免GVHD的风险。在这个提议中，我们假设JAK 2 V617 F特异性引导RNA （gRNA）将指导Cpf 1核酸酶介导的JAK 2 V617 F基因的DNA切割，导致JAK 2 V617 F基因的失活。 MPN启动HSC。为了检验这一假设，我们将追求两个具体目标： 使用CRISPR/Cpf 1基因编辑去除JAK 2 V617 F外显子14，和ii）评估 MPN异种移植模型中基于CRISPR/Cpf 1的自体HSCT。在目标1中，我们将评估 JAK 2 V617 F特异性CRISPR/Cpf 1基因编辑对MPN细胞系集落形成能力和离体 MPN CD 34 + HSC库中携带JAK 2 V617 F的HSC的失活。在目标2中，我们将首先生成异种移植物， 通过移植MPN患者来源的CD 34+细胞建立MPN模型。接下来，我们将测试 JAK 2 V617 F-CRISPR/Cpf 1基因编辑的MPN HSC的高剂量化疗后自体HSCT 编辑.这些研究将证明基于CRISPR/Cpf 1基因编辑的自体造血干细胞联合移植的可行性， 大剂量化疗用于治疗MPN患者。关于预期结果，我们预计 我们将展示有效的基因编辑策略来灭活MPN启动的HSC。这样的结果将 具有重要的积极影响，因为这些方法可用于开发基因编辑- MPN患者的自体造血干细胞移植治疗策略，并可能用于其他造血系统恶性肿瘤。

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.