Regenerative Therapies for Inherited Blood Disorders-Gene therapy

遗传性血液疾病的再生疗法-基因疗法

• 批准号: 9357240
• 负责人: Andre LaRochelle
• 金额: $ 39.15万
• 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/9357240
• 关键词: Affect; American; Animals; Autologous Transplantation; Biological Assay; Blood; Bone Marrow; CD34 gene; CRISPR/Cas technology; CXCR4 gene; Cell Aging; Cell Culture Techniques; Cell Survival; Cell Therapy; Cell surface; Cells; Child; Clinical; Clinical Trials; Codon Nucleotides; Collaborations; Cryopreservation; Cyclic GMP; DNA Repair Pathway; Data; Dependovirus; Derivation procedure; Development; Dimethyl Sulfoxide; Disease; Employee Strikes; Engraftment; Enrollment; Event; Floods; Flow Cytometry; Gene Transfer; Genes; Genetic; Gold; HIV Receptors; HIV-1; Hematological Disease; Hematopoietic stem cells; Homing; Hospitals; Hour; Human; Human body; ITGB2 gene; Inborn Genetic Diseases; Infection; Inherited; Insertional Mutagenesis; Knock-out; Knowledge; Lead; Leukocyte adhesion deficiency - Type 1; Lymphoid; MS4A1 gene; Manuscripts; Measures; Mediating; Medical; Mus; Myelogenous; Nonhomologous DNA End Joining; PTPRC gene; Pathway interactions; Pattern; Preparation; Process; Production; Publications; Publishing; Reporting; Research Personnel; Retroviridae; Safety; Serotyping; Serum; Site; Societies; Spumavirus; Stem cells; T-Lymphocyte; Techniques; Technology; Testing; Therapy Clinical Trials; Tissues; Toxic effect; Transgenes; Translating; Transplantation; Viral Vector; Virus; Work; alanine aminopeptidase; arm; base; bone marrow failure syndrome; cell age; cellular transduction; clinical application; clinically relevant; clinically significant; curative treatments; flexibility; gene therapy; gene therapy clinical trial; gene transfer vector; genome editing; homologous recombination; induced pluripotent stem cell; integration site; interest; knockout gene; mouse model; novel; novel strategies; programs; regenerative; regenerative therapy; research clinical testing; research study; scale up; stem cell biology; transduction efficiency; treatment group; vector

Summary 1. Objective 1.1: Development of a clinical trial for LAD-1. A clinical trial for gene therapy of LAD-1 using first-in-human foamy viral vectors (FVV) is in preparation and enrollment is expected to start in FY17. In preparation for this trial, we have performed: a) Process-development and scale-up of FVV production Process-development and scale-up was performed using the MSCV-GFP and MSCV-hCD18 FVV in collaboration with investigators at Cincinnati Childrens Hospital-Vector Production Facility. 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 MSCV-hCD18 FVV were developed, and some of the final safety assays still need further development to a cGMP-grade assay. Production of clinical grade FVV stocks for gene therapy of LAD-1 is ongoing. The process-development and scale-up were published in FY16 (Nasimuzzaman et al. Mol Ther Meth & Clin Devel 2016). b) Pre-clinical testing of FVV transduction in CD34+ cells derived from a subject with severe LAD CD34+ cells were transduced for 16 hours at MOI of 0, 1, 2, and 5 with a FVV expressing a human codon optimized CD18 transgene. Flow cytometry of CD34+ cells cultured for 3 days after transduction demonstrated CD18+ cell surface expression in 33-45% of cells. Higher MOIs resulted in decreased cell survival with no significant benefit in transduction efficiency, suggesting an optimal MOI of 1-2. NSG mice were transplanted with MSCV-hCD18-transduced human LAD-1 HSPCs (1 x 105 cells/mouse), and human cell engraftment was measured in murine BM 5 months after transplantation using flow cytometry. Human CD45+ cells were detected in all mice (average 1%). Mice transplanted with mock-transduced (MOI=0) LAD-1 CD34+ cells showed a 3.4-fold, 2-fold and 1.4-fold lower engraftment compared to mice injected with CD34+ cells transduced with MSCV-hCD18 at MOI=1 (p<0.01), 2 (p<0.05) and 5 (p>0.05), respectively, suggesting a selective homing/engraftment or survival/proliferative advantage of CD18+ cells. The inverse relationship between engraftment levels and MOI correlated with a gradual decrease in cell survival with increasing MOI, most likely due to toxicity from DMSO (required for FVV cryopreservation) during transduction; cell viabilities of 91%, 84%, 82% and 69% were obtained at MOI 0, 1, 2 and 5, respectively, indicating that further increase in MOI would lead to increasing toxicity. High-level, clinically relevant gene marking levels were obtained; the percentages of human cells expressing CD18 in the murine BM 5 months post-transplantation were 36.0 3.9%, 33.9 5.1%, and 44.5 1.6% at MOI 1, 2 and 5, respectively. Quantitative PCR analysis of vector integrants within engrafted human cells indicated a single integration event occurred in the majority of long-term repopulating HSPCs at all MOI tested. Flow cytometry-based lineage analysis of bone marrow from mice transplanted with MSCV-hCD18-transduced LAD-1 CD34+ cells revealed human CD18+ cells in both CD13+ myeloid (35.5 6.9%) and CD20+ lymphoid (39.8 35.7%) compartments. Interestingly, human myeloid engraftment was superior in recipient mice engrafted with human CD18+ cells (81.5 4.3%) compared to animals transplanted with non-transduced (CD18-) LAD-1 cells (65.3 11.3%). Integration site analysis of engrafted human cells revealed a polyclonal pattern of integration with no evidence of insertional mutagenesis 5 months post-transplantation. Thus, MSCV-hCD18-mediated transduction of human LAD-1 CD34+ cells leads to clinically significant levels of CD18 expression, supporting the use of this CD18-expressing FVV in a human clinical trial. This work was presented at the American Society of Cell and Gene Therapy in FY16. A manuscript is in preparation. 2. Objective 1.2: Optimization of the CRISPR/Cas9 genome editing technology in human HSCs. We have optimized genome editing via NHEJ pathways in primary CD34+ cells in FY16. Efficiencies of INDEL formation and gene knockout of 50-70% are routinely obtained. Based on a recent publication indicating that HSCs with CXCR4 haploinsufficiency have a competitive repopulating advantage, we have performed monoallelic knockout of CXCR4 in normal human CD34+ cells. The impact of CXCR4 haploinsufficiency on human HSC engraftment and proliferation was assessed using the gold-standard immuno-deficient (NSG) murine model. In contrast to the observation made with murine HSCs, no increase in engraftment was observed several months after transplantation of CXCR4-haploinsufficient human CD34+ cells compared to control cells. Disruption of CXCR4 did not negatively impact overall engraftment or skew lineage differentiation. These data suggest that CXCR4 may be of interest as a safe-harbor site for genome editing. Genetic perturbation of CXCR4, a co-receptor for HIV entry, may also be of interest to protect CD34-derived T cells from infection with CXCR4-trophic HIV-1 strains. In FY17, experiments will be conducted to optimize targeted introduction of a gene of interest (e.g. GFP) in the CXCR4 and other safe harbor loci via homologous recombination (HR) DNA repair pathways. Adeno-associated virus (AAV) serotype 6 will be used to deliver homology arms to CXCR4 and other genes to favor HR pathways. Search for novel serotypes of AAV capable of efficient transduction of human HSPCs is underway to further enhance HR-triggered genome editing in human CD34+ cells.

概括 1。目标1.1：开发LAD-1的临床试验。 使用第一含人类泡沫病毒载体（FVV）的LAD-1基因治疗的临床试验预计将于2017财年开始。为了准备这项试验，我们已经进行了： a）FVV生产的过程发展和扩大规模 使用MSCV-GFP和MSCV-HCD18 FVV与辛辛那提儿童医院 - 载体生产设施的研究人员合作，使用MSCV-GFP和MSCV-HCD18 FVV进行了过程开发和规模。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MSCV-HCD18 FVV的安全性和效力所需的测定，一些最终的安全测定仍需要进一步开发CGMP级测定法。正在进行的LAD-1基因治疗临床级FVV库存的产生。该过程发展和扩大规模发表在16财年（Nasimuzzaman等人，Mol Ther Meth＆Clin Devel 2016）。 b）在患有严重LAD的受试者的CD34+细胞中FVV转导的临床前测试 CD34+细胞在0、1、2和5的MOI中转导16小时，而FVV表达人体密码子优化的CD18转基因。转导后3天培养的CD34+细胞的流式细胞仪显示CD18+细胞表面表达在33-45％的细胞中。较高的MOI导致细胞存活降低，而在转导效率方面没有显着益处，这表明最佳MOI为1-2。将NSG小鼠用MSCV-HCD18转导的人LAD-1 HSPC（1 x 105个细胞/小鼠）移植，并在移植后5个月在鼠BM中测量了人类细胞的植入。在所有小鼠中都检测到人CD45+细胞（平均1％）。与在MOI = 1（p <0.05），2（p <0.05）和5（p <0.05）和5（p <0.05）和5（p <0.05）和5（p <0.05）和5（p <0.05）和5（p <0.05），相比，用模拟转导（MOI = 0）LAD-1 CD34+细胞移植的小鼠表现出3.4倍，2倍和1.4倍的植入术。 CD18+细胞的存活/增殖优势。植入水平与MOI之间的反相关关系与MOI的增加与细胞存活的逐渐降低相关，这很可能是由于DMSO的毒性（FVV冷冻保存所必需的）在转导过程中；在MOI 0、1、2和5分别获得了91％，84％，82％和69％的细胞活力，这表明MOI进一步​​增加将导致毒性增加。获得了高级，临床相关的基因标记水平；移植后5个月在鼠BM中表达CD18的人类细胞的百分比分别为36.0 3.9％，33.9 5.1％和44.5 1.6％，分别为MOI 1、2和5分别为44.5 1.6％。对植入的人类细胞中载体整合物的定量PCR分析表明，在所有MOI测试的大多数长期重现HSPC中，都发生了单个整合事件。基于流式细胞仪的基于流式细胞仪的谱系分析，对用MSCV-HCD18转导的LAD-1 CD34+细胞移植的小鼠的骨髓分析显示，CD13+髓样（35.5 6.9％）和CD20+淋巴样（39.8 35.7％）的CD13+髓样（35.5 6.9％）中的人CD18+细胞显示出人CD18+细胞。有趣的是，与未转二糖（CD18-）LAD-1细胞（65.3 11.3％）的动物相比，与人CD18+细胞的受体小鼠（81.5 4.3％）相比，人髓样植入率高（81.5 4.3％）。植入的人类细胞的整合位点分析揭示了整合的多克隆模式，没有插入后5个月的插入诱变的证据。 因此，MSCV-HCD18介导的人类LAD-1 CD34+细胞的转导导致CD18表达水平显着，在人类临床试验中支持使用该CD18表达FVV的使用。这项工作是在2016财年的美国细胞和基因治疗学会上提出的。手稿正在准备。 2。目标1.2：人类HSC中CRISPR/CAS9基因组编辑技术的优化。 我们已经通过NHEJ途径在16财年的原代CD34+细胞中优化了基因组编辑。常规获得了50-70％的indel形成和基因敲除效率。基于最近的出版物表明，具有CXCR4单倍频率的HSC具有竞争性的重现优势，我们在正常的人CD34+细胞中进行了CXCR4的单相关基因敲除。使用金标准免疫缺陷型（NSG）鼠模型评估了CXCR4单倍不足对人HSC植入和增殖的影响。与用鼠HSC进行的观察相反，与对照细胞相比，CXCR4-甲氟蛋白富含人CD34+细胞的几个月后，植入未观察到植入的增加。 CXCR4的破坏不会对整体植入或偏差分化产生负面影响。这些数据表明，CXCR4可能是用于基因组编辑的避风港站点。 CXCR4的遗传扰动是一种用于HIV进入的共受体的遗传扰动，也可能是保护CD34衍生的T细胞免于感染CXCR4-营养性HIV-1菌株的感兴趣。 在2017财年，将通过同源重组（HR）DNA修复途径进行实验，以优化CXCR4和其他安全港基因座中目标基因（例如GFP）的靶向引入。腺相关病毒（AAV）血清型6将用于为CXCR4和其他基因提供同源性臂，以偏爱HR途径。正在搜索能够有效转导人HSPC的AAV的新型血清型，以进一步增强人CD34+细胞中HR触发的基因组编辑。