Improving Engraftment of Hematopoietic Stem Cell Gene Therapy

改善造血干细胞基因治疗的植入

基本信息

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
项目类别：
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

项目摘要

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足或毒性很强的疾病。开发有效的 HSC-GT 的需求仍然未得到满足，该 HSC-GT 可以实现更高水平的植入，同时降低预处理方案毒性且没有免疫原性风险。我们建议使用 HSC-GT 狗模型来研究这些关键问题，这些问题可以转化为血液学和其他疾病治疗的重大进展。该提案的目的旨在提高自体 HSC-GT 的功效和安全性，(1) 通过输注更多数量的离体扩增基因修饰 (GM)-HSC 来增加植入，(2) 通过消除或减少毒性来降低毒性GM-HSC 输注前的化疗/放疗调理，以及 (3) 诱导对 GM-HSC 及其后代产生的治疗蛋白/新抗原的免疫耐受。这些研究旨在取得可转化为改善人类 HSC-GT 的结果。目标 1. 通过离体 CD34+ 细胞 2 周扩增来增加自体 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。使用高通量测序来跟踪长期移植后 GM-HSC 后代的植入。在狗竞争性增殖测定中比较 14 天扩增 GM-HSC 与 3 天培养 GM-HSC。目标 2. 减少 GM-HSC 长期植入所需的细胞毒性预处理方案。为了增加离体扩增 GM-HSC 的竞争性再增殖优势，在低剂量全身照射 (TBI) 之前用 CXCR4 拮抗剂 plerixafor (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。我们将检验以下假设：GM-HSC 比普乐沙福动员的或 TKI 处理的内源性 HSC 具有竞争优势。如果成功，我们将结合普乐沙福 + TKI 来评估这种非细胞毒性方案是否可以在没有 TBI 的情况下实现 GM-HSC 植入。 Plerixafor + TKI 随后输注 GM-HSC 可重复多次，以进一步增加 GM-HSC 的植入。目标 3. 通过 CTLA4-Ig 共刺激阻断和药物免疫抑制（环孢素和吗替麦考酚酯）建立对 GM-HSC 表达的新抗原的免疫耐受。最后，我们将测试最佳的 GM-HSC 移植方案，以纠正具有 R 型 PK 基因的丙酮酸激酶 (PK) 缺陷犬的红细胞疾病，从而实现贫血的功能性治愈。完成这三个目标后，我们将定义高度可转化的方法来增加 GM-HSC 的植入、减少预处理方案毒性并诱导对 GM-HSC 的免疫耐受