Therapeutic potential of Cpf1-based gene editing for myeloproliferative neoplasms

基于 Cpf1 的基因编辑对骨髓增殖性肿瘤的治疗潜力

• 批准号: 9806809
• 负责人: WEN-SHU WU
• 金额: $ 17.39万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9806809
• 关键词: Adenovirus Vector; Adenoviruses; Allogenic; American; Autologous; Behavioral Research; Blood Cells; CD34 gene; Cause of Death; Cell Line; Cells; Clinical; Clustered Regularly Interspaced Short Palindromic Repeats; DNA; Development; Disease; Engraftment; Exons; Family; Future; Gene Delivery; Genes; Goals; Guide RNA; Health; Hematological Disease; Hematopoietic Neoplasms; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; High Dose Chemotherapy; Human; Immunosuppressive Agents; Introns; JAK2 gene; Joints; Lentivirus Vector; Mediating; Mission; Mus; Mutation; Myeloproliferative disease; NR5A2 gene; Names; Neoplasm Transplantation; Outcome; Patients; Personal Satisfaction; Pharmaceutical Preparations; Phenylalanine; Point Mutation; Positioning Attribute; Public Health; Publications; Research; Risk; Testing; Therapeutic; Transplantation; United States National Institutes of Health; Valine; Xenograft Model; base; curative treatments; endonuclease; experience; gain of function; gene transplantation for gene therapy; graft vs host disease; improved; in vivo; inhibitor/antagonist; mouse model; mutant; nuclease; public health relevance; social; stem cell therapy; therapeutic evaluation; therapeutic gene; therapeutic target

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.

项目概要 骨髓增生性肿瘤 (MPN) 是一组血细胞过度产生的血液疾病。一个 JAK2 基因中的单个功能获得性点突变（外显子 14 中的 1849GèT），命名为 JAK2V617F，已被 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JAK2V617F基因。虽然 JAK2 抑制剂是抑制 MPN 引起的并发症的有前途的药物， 他们无法治愈 MPN。迄今为止，同种异体造血干细胞移植（allo-HSCT）是唯一的治疗方法 MPN 的治疗。然而，大多数患者都面临着 HLA 匹配供体短缺的问题，一些患者 同种异体造血干细胞移植（allo-HSCT）受者会出现移植物抗宿主病（GvHD），这是导致死亡的一个主要原因，并且 对免疫抑制药物具有重要意义。因此，这将给临床带来巨大的益处。 开发安全的基因编辑策略，灭活来自含有这两种物质的 MPN 患者的 JAK2V617F 突变型 HSC 健康和突变的 HSC。如果这些策略成功，就有可能治愈 MPN 患者 使用自体造血干细胞移植（auto-HSCT），无需在患者体内寻找匹配的供体 家庭并避免 GvHD 的风险。在此提议中，我们假设 JAK2V617F 特异性指导 RNA (gRNA) 将指导 Cpf1 核酸酶介导的 JAK2V617F 基因 DNA 切割，导致 JAK2V617F 基因失活 MPN 启动 HSC。为了检验这一假设，我们将追求两个具体目标：i) 通过以下方法灭活 MPN 细胞： 使用 CRISPR/Cpf1 基因编辑去除 JAK2V617F 外显子 14，以及 ii) 评估以下药物的治疗潜力 MPN 异种移植模型中基于 CRISPR/Cpf1 的自动 HSCT。在目标 1 中，我们将评估以下措施的影响： JAK2V617F 特异性 CRISPR/Cpf1 基因编辑对 MPN 细胞系和离体集落形成能力的影响 MPN CD34+ HSC 池中含有 JAK2V617F 的 HSC 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 CD34+ 细胞来建立 MPN 模型。接下来，我们将测试其治疗潜力 高剂量化疗后使用 JAK2V617F-CRISPR/Cpf1 基因编辑的 MPN HSC 进行自动 HSCT 编辑。这些研究将证明基于 CRISPR/Cpf1 基因编辑的自动 HSCT 联合 用于治疗 MPN 患者的高剂量化疗。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，因为这些方法可以转化为开发基因编辑 - 基于自动 HSCT 的 MPN 患者治疗策略以及其他造血系统恶性肿瘤的可能治疗策略。