Regenerative Therapies for Inherited Blood Disorders

遗传性血液疾病的再生疗法

• 批准号: 9157455
• 负责人: Andre LaRochelle
• 金额: $ 166.4万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9157455
• 关键词: 3-Dimensional; Affect; Animals; Autologous; Autologous Transplantation; Biological Assay; Bioreactors; Blood; Bone Marrow; CD34 gene; CSF3 gene; CXCR4 gene; Cell Aging; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell division; Cells; ChIP-seq; Child; Clinical; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Coculture Techniques; Collaborations; Collection; Cyclic GMP; Cytoskeleton; DNA Repair Pathway; DNMT3a; Data; Derivation procedure; Detection; Development; Diamond-Blackfan anemia; Disease; Dose; Down-Regulation; Employee Strikes; Endothelial Cells; Engraftment; Fanconi' s Anemia; Fibronectins; Floods; Gene Expression; Gene Transfer; Generations; Genes; Genetic; Gold; Growth; Guide RNA; Hematological Disease; Hematology; Hematopoietic; Hematopoietic Stem Cell Transplantation; Hematopoietic System; Hematopoietic stem cells; Home environment; Hospitals; Human; Human body; Hypoxia; ITGB2 gene; Immunoglobulin G; Inborn Genetic Diseases; Individual; Inherited; Institutes; Investigation; Knowledge; Laboratories; Legal patent; Leukocyte adhesion deficiency - Type 1; Life; Ligands; Lipids; Lipofectamine; Maintenance; Medical; Megakaryocytes; Modeling; Modification; Mus; Mutation; Oxygen measurement, partial pressure, arterial; PTPRC gene; Pathway interactions; Patients; Phenotype; Plasmids; Preparation; Process; Production; Protocols documentation; Publications; Publishing; Research; Research Personnel; Retroviral Vector; Retroviridae; Safety; Saint Jude Children' s Research Hospital; Sendai virus; Serum; Silk; Spumavirus; Staging; Stem Cell Development; Stem cells; Structure; Suspension substance; Suspensions; System; Techniques; Technology; Testing; Therapy Clinical Trials; Time; Tissues; Toxic effect; Transfection; Translating; Transplantation; Tube; United States Food and Drug Administration; Universities; Viral Vector; Virus; Washington; Western Blotting; base; biophysical properties; bone marrow failure syndrome; c-myc Genes; cell age; clinical application; falls; flexibility; gene correction; gene therapy; gene therapy clinical trial; gene transfer vector; genetic approach; genome editing; homologous recombination; in vivo; induced pluripotent stem cell; interest; large scale production; macromolecule; meetings; methylation pattern; notch protein; novel; novel strategies; peripheral blood; prevent; progenitor; programs; regenerative; regenerative therapy; research study; scaffold; scale up; self-renewal; small molecule; stem cell biology; stem cell technology; therapeutic transgene; transcription factor; transcriptome sequencing; vector

1. Objective 1: Develop approaches for the genetic correction of human HSCs Development of a clinical trial for LAD-1. A clinical trial for gene therapy of LAD-1 using first-in-human foamy viral vectors is in preparation. Documents are expected to be submitted to the Food and Drug Administration (FDA) in the fall 2015. In preparation for this trial, we have performed process-development and scale-up of FVV production (using the ΔΦMSCV-GFP and ΔΦMSCV-hCD18 FVV) in collaboration with investigators at CCHMC Vector Production Facility (VPF) (Dr. Johannes C.M. van der Loo, Dr. Carolyn Lutzko and Dr. Punam Malik) and Dr. David Russell (University of Washington). A considerable effort was needed to develop processes for large-scale concentration and purification of this extremely serum dependent virus. Since FV is a non-pathogenic virus, its isolation and purification techniques had to be developed. Assays required for assuring safety and potency of ΔΦMSCV-hCD18 FVV were developed, and some of the final safety assays still need further development to a cGMP-grade assay. The protocol for production of clinical grade FVV stocks for gene therapy of LAD-1 has been optimized. Optimization of the CRISPR/Cas9 genome editing technology in human HSCs. We have investigated multiple approaches for transfection of human CD34+ cells that have resulted in low efficiency of delivery or unacceptable cell toxicity, including cationic lipids, nucleofection, DMRIE-C, PEI, CellfectinII, 293fectin, Superfect, Effectine, Endofectin-Plus, Glycofect, Escort V, and Lipofectamine 3000. We are currently focusing on alternative approaches, including large-scale production of non-integrating retroviral vectors carrying Cas9 and gRNA cassettes, and induced transduction by osmocytosis and propanebetaine (iTOP, Cell 2015). A recent publication has indicated that HSCs with CXCR4 haploinsufficiency have a competitive repopulation advantage, perhaps due to enhanced proliferation. We have developed and amplified the Cas9/gRNA plasmids necessary for targeted introduction of a gene of interest (GFP and CD18) in the CXCR4 locus via homologous recombination (HR) DNA repair pathways. Activation of HR pathways requires active cell division; we have therefore tested the ability of a recently described small molecule (UM729) to increase HSC proliferation/self-renewal. Preliminary data indicate successful amplification of CD34+ cells, setting the stage for testing Cas9/gRNA for targeting the CSCR4 locus once approaches for delivering macromolecules in human CD34+ cells (step 1 above) have been optimized. In FY2016, we plan to assess the impact of CXCR4 haploinsufficiency on human HSC engraftment and proliferation using the gold-standard immuno-deficient (NSG) murine model. 2. Objective 2: Develop approaches for expansion of genetically corrected human HSCs Investigation of pathways regulating HSC self-renewal and differentiation We have determined that G-CSF mobilized human CD34+ cells cultured ex vivo for 21 days in the presence of immobilized Notch (Delta-1ext-IgG) ligand results in 5-fold expansion of long-term repopulating HSCs when cultured under hypoxic conditions (1.5% O2) compared to cultures performed under normoxia (21% O2) where maintenance but no expansion of HSCs was observed. These data suggest synergy between hypoxia and Notch pathways. We have also initiated investigations testing whether transient downregulation of DNMT3a expression using SiRNA in human CD34+ cells may prevent differentiation of HSCs and favor their self-renewal during short-term culture. Conditions for optimal detection of DNMT3A expression by Q-PCR and Western blots have been developed. A first attempt at transiently downregulating DNMT3A expression has failed using commercially available SiRNA. We are testing an alternative SiRNA (Silencer Select, Life Technologies). By exploiting the extensive HSC amplification after transplantation, we aim to identify novel factors/pathways involved in HSC self-renewal. We have performed preliminary experiments to identify conditions for optimal HSC amplification after transplantation, including cell dose, timing of cell collection after transplantation, and the impact of serial transplantations. We found that lower cell doses (e.g. 1x106 cells) result in superior HSC expansion in vivo compared to larger cell doses (e.g. 1x107 cells). The optimal timing for cell collection was 1 week; longer times in vivo (e.g. 2, 3, or 4 weeks) resulted in progressive differentiation to more mature progeny. Serial transplantations have led to engraftment levels too low to allow reliable detection and selection of HSCs. In FY16, we will further optimize conditions to detect sufficient number of HSCs after transplantation, namely transplanting larger cell doses and shortening time of cell collection to 3 days in serial transplant experiments. Pending optimization of these conditions, we will compare gene expression and methylation patterns between HSC at steady state (before transplant) and after transplant, using RNA-Seq, and CHIP-Seq approaches. Development of a programmable 3-dimensional bone marrow niche for expansion of human HSCs We have recently started to adapt a programmable 3D silk scaffold system developed by Dr. Kaplan at Tufts University for expansion of HSCs ex vivo. IN FY15, we have optimized most components of this system, including: 1- The basic silk scaffold to mimic the 3D bone marrow microenvironment; 2- The tube structures to mimic bone marrow vasculature; 3- The various cellular and extra-cellular matrix components (e.g. megakaryocytes, endothelial cells, fibronectin); 4- The biophysical properties of the system (e.g. bioreactor flow rate, oxygen tension, etc). 3. Objective 3: Develop approaches for efficient differentiation of human iPSCs into functional HSCs Development of a culture system for hematopoietic differentiation of normal human iPSCs Generation of normal human iPSC lines. We have established several iPSC lines derived from G-CSF mobilized peripheral blood (MPB) CD34+CD38- and CD34+CD38+ of normal control individuals using a non-integrating Sendai virus delivery system expressing the transcription factors Sox2, Klf4, c-Myc and Oct2. Differentiation of normal human iPSCs into hematopoietic cells. We have established a novel system for de novo generation of easily accessible suspension human hematopoietic cells (CD45+CD34+) from iPSCs. A patent application (# PCT/US2014/058583) describing this protocol has been approved and published on April 9, 2015. Characterization of normal human iPSC-derived hematopoietic cells. Up to 60% of iPSC-differentiated cells have a CD45+CD34+ phenotype compared to 10-15% CD45+CD34+ using current co-culture or EB-based protocols. These cells could form colonies in clonogenic progenitor assays, albeit at reduced capacity compared to primary CD34+ cells. These cells, however, failed to home to the bone marrow of immuno-deficient (NSG) animals and did not result in long-term engraftment after transplantation. Differentiation of genetically corrected iPSCs derived from patients with inherited bone marrow failure syndromes into transplantable HSCs We have obtained original and genetically corrected iPSC lines derived from individuals with inherited bone marrow failure syndromes (Fanconi Anemia and Diamond-Blackfan Anemia) from the laboratories of Dr. Juan Carlos Izpisua-Belmonte (Salk Institute) and Dr. MJ Weiss (St. Jude Childrens Research Hospital), respectively. Culture conditions for optimal growth of these lines are being optimized. In FY16, the differentiation protocol developed for normal iPSCs will be evaluated for hematopoietic differentiation of patient-derived iPSCs.

1。目标1：开发人类HSC遗传校正的方法 开发LAD-1的临床试验。 使用首先人类泡沫病毒载体的LAD-1基因治疗的临床试验正在制备中。预计文件应在2015年秋季提交给食品药品监督管理局（FDA）。为了准备该试验，我们已经进行了FVV生产的过程开发和规模扩大（使用ΔφMSCV-GFP和ΔφMSCV-GFP和ΔφMSCV-HCD18 FVV）与CCHMC Vector Productor Productor Productor Production Facility（C. C. vpff）（C. vpff）（C. vpff）（JONNE）（JONNE） Lutzko和Punam Malik博士）和David Russell博士（华盛顿大学）。为了开发大规模浓度并纯化这种极度血清依赖性病毒的过程需要大量努力。由于FV是一种非致病病毒，因此必须开发其隔离和纯化技术。开发了确保安全性和效力ΔφMSCV-HCD18 FVV所需的测定，并且一些最终安全测定仍需要进一步开发CGMP级测定。生产LAD-1基因治疗的临床级FVV库存的方案已得到优化。 在人HSC中的CRISPR/CAS9基因组编辑技术的优化。 We have investigated multiple approaches for transfection of human CD34+ cells that have resulted in low efficiency of delivery or unacceptable cell toxicity, including cationic lipids, nucleofection, DMRIE-C, PEI, CellfectinII, 293fectin, Superfect, Effectine, Endofectin-Plus, Glycofect, Escort V, and Lipofectamine 3000. We are currently focusing on alternative方法，包括携带Cas9和GRNA盒的非整合逆转录病毒载体的大规模产生，以及渗透刺细胞增多症和丙烷链蛋白的转导（ITOP，Cell，2015）。 最近的出版物表明，具有CXCR4单倍度不足的HSC具有竞争性的重新填充优势，这可能是由于增殖的增强所致。我们通过同源重组（HR）DNA修复途径在CXCR4基因座中靶向引入感兴趣基因（GFP和CD18）所需的CAS9/GRNA质粒。 HR途径的激活需要主动细胞分裂；因此，我们已经测试了最近描述的小分子（UM729）增加HSC增殖/自我更新的能力。初步数据表明，CD34+细胞的成功扩增，为靶向CSCR4基因座的Cas9/GRNA奠定了阶段，一旦接近在人CD34+细胞中输送大分子（上面的步骤1）的方法已被优化。在2016财年，我们计划使用金色标准的免疫缺陷型（NSG）鼠模型评估CXCR4单倍不足对人HSC植入和增殖的影响。 2。目标2：开发扩展遗传校正的人类HSC的方法 调查调节HSC自我更新和分化的途径 我们已经确定G-CSF在存在固定的Notch（Delta-1ext-igg）的存在下动员了人类CD34+细胞21天，导致在低氧条件下进行培养时长期培养的HSC的长期培养的HSC膨胀5倍（1.5％O2（1.5％O2）与Normoxia在Normoxia下进行的培养（21％O2）的培养（1.5％O2），但在HASC中进行了HSC（21％O2）的培养。这些数据表明缺氧和缺口途径之间的协同作用。 我们还开始了研究测试在人CD34+细胞中使用siRNA对DNMT3A表达的短暂下调是否可能阻止HSC的分化，并在短期培养过程中有利于其自我更新。已经开发了Q-PCR和Western印迹最佳检测DNMT3A表达的条件。首次尝试使用市售siRNA瞬时下调DNMT3A表达的尝试失败了。我们正在测试另一种siRNA（Silencer Select，Life Technologies）。 通过利用移植后广泛的HSC扩增，我们旨在确定与HSC自我更新有关的新因素/途径。我们已经进行了初步实验，以识别移植后最佳HSC扩增的条件，包括细胞剂量，移植后细胞收集时间以及串行移植的影响。我们发现，与较大的细胞剂量（例如1x107细胞）相比，较低的细胞剂量（例如1x106细胞）会导致体内HSC上升的膨胀。细胞收集的最佳时机为1周；体内更长的时间（例如2、3或4周）导致逐渐分化为更成熟的后代。连续移植导致植入水平太低，无法可靠的检测和选择HSC。在2016财年，我们将进一步优化条件，以检测移植后足够数量的HSC，即在串行移植实验中将较大的细胞剂量和将细胞收集时间缩短到3天。在对这些条件的优化之前，我们将使用RNA-Seq和CHIP-Seq方法比较HSC（移植前）和移植后HSC之间的基因表达和甲基化模式。 开发可编程的三维骨髓利基市场，用于扩展人类HSC 我们最近开始适应由塔夫茨大学（Tufts University）Kaplan博士开发的可编程3D丝绸支架系统，用于扩展HSC的Ex Vivo。在2015财年，我们优化了该系统的大多数组件，包括：1-基本的丝绸支架，以模仿3D骨髓微环境； 2-模仿骨髓脉管系统的管结构； 3-各种细胞和细胞外基质成分（例如巨核细胞，内皮细胞，纤连蛋白）； 4-系统的生物物理特性（例如生物反应器流速，氧张力等）。 3。目标3：开发有效分化人IPSC为功能HSC的方法 开发用于正常人IPSC造血分化的培养系统 正常人IPSC线的产生。我们已经建立了几条IPSC线，这些IPSC系使用非整合的sendai病毒输送系统，表达转录因子SOX2，KLF4，klf4，c-Myc和oct2。 正常人IPSC分化为造血细胞。我们已经建立了一个新的系统，用于从IPSCS开始从头产生易于获得的易于获得的悬浮悬浮液（CD45+CD34+）。描述该协议的专利申请（＃PCT/US2014/058583）已于2015年4月9日批准并发布。 正常人IPSC衍生的造血细胞的表征。使用当前共培养或基于EB的协议，高达60％的IPSC分化细胞具有CD45+ CD34+表型，与10-15％CD45+ CD34+相比。这些细胞可以在克隆祖细胞测定法中形成菌落，尽管与原代CD34+细胞相比，能力降低。但是，这些细胞未能进入免疫缺陷（NSG）动物的骨髓，并且在移植后没有导致长期植入。 从遗传性骨髓衰竭综合症中衍生成的遗传校正的IPSC的分化为可移植的HSC 我们已经从遗传性骨髓衰竭综合征（Fanconi贫血和钻石 - 黑色贫血）中得出的原始和遗传校正的IPSC系，从Juan Carlos Izpisua Izpisua Belmonte博士（Salk Institute）和MJ Weiss（St. Jude Childrens Hospital）的实验室的实验室中。这些线路最佳生长的培养条件正在优化。在2016财年，将评估针对正常IPSC的分化方案，以评估患者衍生的IPSC的造血分化。