Improving Engraftment of Hematopoietic Stem Cell Gene Therapy

改善造血干细胞基因治疗的植入

• 批准号: 8903565
• 负责人: GEORGE Earl GEORGES
• 金额: $ 83.75万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8903565
• 关键词: AMD3100; Allogenic; Anemia; Animal Model; Autologous; Biological Assay; Bone Marrow; Busulfan; C-KIT Gene; CD34 gene; CTLA4-Ig; CXCR4 gene; Canis familiaris; Cell physiology; Cells; Clinic; Clinical; Cyclosporine; Cytotoxic Chemotherapy; Cytotoxic T-Lymphocyte-Associated Protein 4; Disease; Dose; Engraftment; Erythroid; Fluorescence; Gene-Modified; Genes; Gray unit of radiation dose; Health; Hematopoietic stem cells; Hereditary Disease; High-Throughput Nucleotide Sequencing; Home environment; Hour; Human; Immune; Immune Tolerance; Immune response; Immunoglobulins; Immunosuppression; Inborn Errors of Metabolism; Infusion procedures; Lead; Lentivirus Vector; Malignant Neoplasms; Methods; Modeling; Molecular; Outcome; Pancytopenia; Patients; Pharmaceutical Preparations; Process; Proteins; Proto-Oncogene Protein c-kit; Pyruvate Kinase; Radiation; Radiation therapy; Regimen; Research Design; Risk; Safety; Second Primary Cancers; Stem cell transplant; Testing; Time; Toxic effect; Translating; Translations; Transplantation; Tyrosine Kinase Inhibitor; Whole-Body Irradiation; base; cellular transduction; chemotherapy; conditioning; cytotoxic; design; experience; fludarabine; gene therapy; hematopoietic cell transplantation; human disease; immunogenic; immunogenicity; improved; in vivo; mycophenolate mofetil; prevent; public health relevance; pyruvate kinase deficiency; stem cell differentiation; therapeutic gene; therapeutic protein

DESCRIPTION (provided by applicant): Hematopoietic stem cell (HSC) gene therapy (GT) holds promise for curing diseases for which there is currently inadequate or highly toxic treatment. There remains an unmet need to develop effective HSC-GT that achieves a greater level of engraftment with reduced conditioning regimen toxicity and without risk of immunogenicity. We propose to use the dog model of HSC-GT to study these key problems that can translate into significant advances for treatment of hematological and other diseases. The aims of this proposal are designed to improve the efficacy and safety of autologous HSC-GT with (1) increased engraftment by infusion of greater numbers of ex vivo expanded gene modified (GM)-HSC, (2) reduced toxicity by eliminating or reducing chemo/radiotherapy conditioning prior to GM-HSC infusion, and (3) inducing immune tolerance to the therapeutic protein / neoantigen produced by GM-HSC and their progeny. The studies are designed to achieve results that will be translated into improving human HSC-GT. Aim 1. Increase the cell dose of autologous GM-HSC with a 2-week ex vivo expansion of CD34+ cells. During expansion, transduce the CD34+ cells with a non-immunogenic molecular "barcode" lentiviral vector. Use high throughput sequencing to track engraftment of GM-HSC progeny after transplantation long term. Compare 14-day expanded GM-HSC to 3-day cultured GM-HSC in dog competitive repopulation assays. Aim 2. Reduce the cytotoxic conditioning regimen needed for long term engraftment of GM-HSC. To increase the competitive repopulating advantage of ex vivo expanded GM-HSC, mobilize endogenous HSC with the CXCR4 antagonist plerixafor (AMD3100) just prior to low-dose total body irradiation (TBI) followed by infusion of GM-HSC. Next, assess if KIT (CD117)-specific tyrosine kinase inhibitor (TKI) can increase engraftment of GM-HSC. We will test the hypothesis that the GM-HSCs have a competitive advantage over the plerixafor mobilized, or TKI-treated endogenous HSC. If successful, we would combine plerixafor + TKI to assess if this non-cytotoxic regimen could achieve GM-HSC engraftment without TBI. Plerixafor + TKI followed by GM-HSC infusion may be repeated multiple times to further increase engraftment of GM-HSC. Aim 3. Establish immune tolerance to GM-HSC expressed neoantigens with CTLA4-Ig costimulatory blockade and pharmacologic immunosuppression (cyclosporine and mycophenolate mofetil). Finally, we will test the optimal GM-HSC transplant regimen to correct the erythroid disease in pyruvate kinase (PK) deficiency dogs with the R-type PK gene to achieve a functional cure of anemia. Upon completion of these three aims, we will have defined highly translatable approaches to increase engraftment of GM-HSC, reduce conditioning regimen toxicity and induce immune tolerance to GM-HSC

描述(由申请人提供)：造血干细胞(HSC)基因疗法(GT)有望治愈目前治疗不充分或毒性很高的疾病。仍然需要开发有效的HSC-GT，以实现更高水平的植入，降低预适应方案的毒性，并且没有免疫原性风险。我们建议使用HSC-GT的犬模型来研究这些关键问题，这些关键问题可以转化为血液学和其他疾病的治疗方面的重大进展。这项建议的目的是通过(1)通过输注更多的体外扩增基因修饰(GM)-HSC来增加植入，(2)通过在输注GM-HSC之前取消或减少化疗/放疗来降低毒性，以及(3)诱导对GM-HSC及其后代产生的治疗性蛋白/新抗原的免疫耐受，来提高自体HSC-GT的疗效和安全性。这些研究旨在实现将转化为改善人类HSC-GT的结果。目的1.通过体外扩增CD34+细胞2周，增加自体GM-HSC的细胞剂量。在扩增过程中，用非免疫原性分子“条形码”慢病毒载体转导CD34+细胞。用高通量测序技术长期追踪GM-HSC子代移植后的植入情况。比较14天扩增的GM-HSC和3天培养的GM-HSC在犬竞争再繁殖实验中的作用。目的2.减少GM-HSC长期植入所需的细胞毒性调节方案。为了增加体外扩增的GM-HSC的竞争再填充优势，在小剂量全身照射(TBI)之前，用CXCR4拮抗剂plerxafor(AMD3100)动员内源性HSC，然后输注GM-HSC。接下来，评估KIT(CD117)特异性酪氨酸激酶抑制剂(TKI)是否可以增加GM-HSC的植入。我们将检验这样的假设，即GM-HSC在动员或TKI治疗的内源性HSC方面比Plerixa具有竞争优势。如果成功，我们将结合plerixafor+TKI来评估这种非细胞毒性方案是否可以在没有脑损伤的情况下实现GM-HSC植入。为进一步增加GM-HSC的植入率，可重复多次输注Plerixafor+TKI，然后输注GM-HSC。目的3.用CTLA4-Ig共刺激阻断和药物免疫抑制(环孢素和霉酚酸酯)建立对GM-HSC表达的新抗原的免疫耐受。最后，我们将测试最优的GM-HSC移植方案，以纠正R型PK基因缺乏犬的红系疾病，实现贫血的功能性治愈。在完成这三个目标后，我们将确定高度可翻译的方法，以增加GM-HSC的植入，降低预适应方案的毒性，并诱导对GM-HSC的免疫耐受