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: 8888188
• 负责人: GEORGE Earl GEORGES
• 金额: $ 79.85万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8888188
• 关键词: AMD3100; Anemia; Animal Model; Autologous; Biological Assay; Bone Marrow; Busulfan; C-KIT Gene; CD34 gene; CXCR4 gene; Canis familiaris; Cell physiology; Cells; Clinic; Clinical; Cytotoxic Chemotherapy; Disease; Dose; Drug Combinations; Engraftment; Erythrocytes; Erythroid; Gene therapy trial; Gene-Modified; Genes; Gray unit of radiation dose; Green Fluorescent Proteins; Health; Hematopoietic; Hematopoietic stem cells; Hereditary Disease; High-Throughput Nucleotide Sequencing; Home environment; Hour; Human; Immune; Immune Tolerance; Immune response; Inborn Errors of Metabolism; Individual; Infusion procedures; Lead; Lentivirus Vector; Malignant Neoplasms; Measures; Methods; Modeling; Molecular; Outcome; Pancytopenia; Patients; Pharmaceutical Preparations; 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; Transplant Recipients; Transplantation; Tyrosine Kinase Inhibitor; Whole-Body Irradiation; cellular transduction; chemotherapy; conditioning; cytotoxic; design; fludarabine; gene therapy; human disease; improved; in vivo; inhibitor/antagonist; prevent; public health relevance; pyruvate kinase deficiency; reconstitution; small molecule; stem cell differentiation; stem cell niche

 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. We propose to use the dog model of HSC-GT to study 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 greatly reducing chemo/radiotherapy conditioning prior to GM-HSC infusion. 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. Identify the optimal combination of small molecules/drugs during the ex vivo expansion of GM-HSC. For each recipient dog, compare two expansion methods in an in vivo competitive repopulation assay. One half of the CD34+ cells will be marked with green fluorescent protein (GFP) expressing lentiviral vector (LV) and the other half with mCherry (mC) expressing LV. After 9.2 Gray total body irradiation (TBI) and GM-HSC infusion, measure the hematopoietic reconstitution of green vs. red cells. This will identify the drug combination and expansion method that achieves optimal sustained engraftment of GM-HSC. To assess the clonal diversity of the progeny of individual HSCs after transplantation, each transduced cell will have a unique molecular "barcode" LV. Use high throughput sequencing to track GM-HSC progeny after transplantation long term. 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; administer the CXCR4 antagonist plerixafor (AMD3100) to increase the unoccupied HSC niches in the bone marrow just prior to low-dose TBI followed by infusion of GM-HSC. Next, test 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 or TKI-treated endogenous irradiated HSC. If successful, we would combine plerixafor + TKI to assess if this non-cytotoxic regimen could achieve GM-HSC engraftment with less TBI. Plerixafor + TKI followed by GM-HSC infusion may be repeated to further increase engraftment of GM-HSC. 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 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，同时降低预适应方案的毒性。我们建议使用HSC-GT的犬模型来研究关键问题，这些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。这项建议的目的是提高自体HSC-GT的有效性和安全性：(1)通过输注更多的体外扩增基因修饰(GM)-HSC来增加植入率；(2)通过在输注GM-HSC之前极大地减少化疗/放射治疗来降低毒性。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。确定GM-HSC体外扩增过程中小分子/药物的最佳组合。对于每一只受体犬，在体内竞争性再种群试验中比较两种扩增方法。一半的CD34+细胞将标记表达慢病毒载体(LV)的绿色荧光蛋白(GFP)，另一半标记表达LV的mCherry(MC)。在9.2度全身照射(TBI)和GM-HSC输注后，测量绿色和红细胞的造血重建情况。这将确定实现GM-HSC最佳持续植入的药物组合和扩张方法。为了评估移植后单个HSCs后代的克隆多样性，每个被转导的细胞都将有一个独特的分子条形码LV。使用高通量测序对移植后的GM-HSC后代进行长期跟踪。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的竞争再植入优势，在小剂量脑损伤后输注GM-HSC之前，应用CXCR4拮抗剂plerixafor(AMD3100)增加骨髓中未被占据的HSC生态位。接下来，测试试剂盒(CD117)特异性酪氨酸激酶抑制剂(TKI)是否能增加GM-HSC的植入。我们将检验这样的假设，即GM-HSC比Plerixafor或TKI治疗的内源性照射HSC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能以更少的TBI实现GM-HSC植入。为进一步增加GM-HSC的植入率，可重复应用Plerixafor+TKI，然后输注GM-HSC。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。在完成这些目标后，我们将定义高度可翻译的方法来增加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。