A preclinical large animal model for globin gene transfer

珠蛋白基因转移的临床前大型动物模型

基本信息

批准号：
10467904
负责人：
John Tisdale
金额：
$ 116.21万
依托单位：
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
依托单位国家：
美国
项目类别：
财政年份：
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/10467904
关键词：
Address Anemia Animal Experimentation Animal Model Animals Autologous B-Lymphocytes Biological Models Bone Marrow Transplantation Busulfan Capsid Cells Clinical Clinical Trials Communication Disease Disease model Dose Drug Kinetics Engraftment Enhancers Erythroid Fetal Hemoglobin Formulation Gene Expression Gene Transfer Gene Transfer Techniques Genes Globin HIV Hematopoiesis Hematopoietic stem cells Hemoglobinopathies Human Infusion procedures Insertional Mutagenesis Intravenous Journals Lentivirus Vector Macaca mulatta Manuscripts Marrow Measures Methods Modeling Monitor Myelogenous Myelosuppression Nature Oral Peripheral Blood Stem Cell Phenotype Phylogenetic Analysis Plasmids Population Pre-Clinical Model Preclinical Testing Production Publishing Reagent Regulatory Element Reporter Reporting Retroviral Vector Rhesus Risk Rodent Model SIV Series Sickle Cell Sickle Cell Anemia Single-Gene Defect Source T-Lymphocyte Technology Transfer Testing Thalassemia Transplantation Viral Vector Whole-Body Irradiation Work absorption base beta Globin beta Thalassemia cellular transduction chemotherapeutic agent clinical application clinical development clinical investigation clinically relevant conditioning cross reactivity dosage endonuclease gene transfer vector genetic manipulation genetically modified cells human model in vivo in vivo Model individual patient integration site irradiation lentiviral-mediated mouse model neutrophil nonhuman primate peripheral blood pre-clinical preclinical development promoter stem cell genes stem cells success technique development transgene expression vector

项目摘要

The thalassemias and hemoglobinopathies represent a heterogeneous group of anemias characterized by absent/reduced or abnormal production of one or more of the globin-molecule subunits, respectively, and strategies which aim to replace the absent or defective globin gene have long been envisioned as potentially curative. Indeed, retroviral vectors carrying globin genes were among the first gene transfer vectors to be tested in murine models, but low gene transfer rates and poor globin gene expression plagued the field. Furthermore, rodent models proved insufficient to model human hematopoieisis. In large animals, significant advances in gene transfer technology have been made by systematically testing transduction methods in a competitive repopulation model, with long-term in-vivo gene transfer levels of 5-10% or higher now achievable after ablative condition with high dose irradiation. The finding of common integration sites among myeloid and erythroid colonies as well as peripheral blood T and B cell populations along with the prolonged contribution of some clones to myeloid progeny satisfied strict criteria for transduction of true hematopoietic stem cells, and the clonal dynamics supported a stochastic model of in vivo hematopoiesis. The development of techniques for clonal tracking have also proven important in assessing the risk of insertional mutagenesis with integrating retroviral vectors. Concurrent with this progress, success in attaining high titer, stable viral vectors which faithfully deliver the human beta-globin gene along with key regulatory elements sufficient to ameliorate disease in a murine model of beta-thalassemia and thalassemia were reported, setting the stage for preclinical testing in the large animal model. We have now moved forward with preclinical testing of lentiviral gene transfer vectors carrying human beta-globin along with key regulatory sequences in the rhesus macaque model. A number of other issues, however, remain to be addressed prior to clinical application. We have recently established steady state marrow, the only practical stem cell source in sickle cell disease, as a viable target for genetic manipulation in the nonhuman primate, yet the type and degree of conditioning required to achieve adequate engraftment remains to be established. We have previously shown that low dose irradiation is sufficient to allow clinically relevant levels of engraftment of genetically modified cells in the murine model, even when xenogeneic genes are expressed. Such irradiation doses allowed for long term engraftment by genetically modified cells in the nonhuman primate, but at levels too low to expect clinical benefit. Increasing the irradiation dose to levels bordering myeloablative resulted in only modest improvement. Busulfan is an alkylating chemotherapeutic agent that has long been used as an alternative agent to total body irradiation for conditioning for bone marrow transplantation. However, erratic absorption of the oral formulation necessitated close pharmacokinetic monitoring of individual patients to achieve predictable myelosuppression. We have recently evaluated a newly available intravenous formulation of busulfan in the murine model, and the results demonstrate that dose dependent engraftment can be achieved at levels of up to 80% at nonmyeloative doses. Further improvement can be achieved by delaying infusion to the day of the neutrophil nadir. This agent is now being tested in the nonhuman primate model in an attempt determine the dosage adequate to allow engraftment of genetically modified cells at levels sufficient for clinical application. Three rhesus macaques have recently been transplanted with autologous peripheral blood stem cells transduced with a lentiviral vector carrying the beta globin gene and key regulatory elements. Though initial engraftment was robust, long term levels of genetically modified cells are below that necessary for phenotypic correction and additional measures to produce vectors for this application are now underway. Utilization a combination of plasmids deriving from both HIV and SIV, a chimeric vector carrying the SIV capsid sequence was produced, enabling efficient transduction of rhesus repopulating cells in the competitive repopulation model. The advent of this chimeric vector has allowed us to again use the nonhuman primate model as a preclinical model for globin gene transfer, utilizing HIV based vectors to deliver the globin gene and its regulatory elements. We have now optimized conditions for gene transfer in the model, and are exploring a number of changes to the vectors including orientation, insulation, additional enhancers, insulators and other strategies to increase the amount of beta-globin per vector copy number among erythroid progeny of vector modified rhesus repopulating cells. The first series of animals have now been transplanted with lentiviral vectors encoding GFP driven by erythroid specific promoters/enhancers, with levels matching that of standard reporter vectors in this model. A second series of animals have now been transplanted with vectors encoding human beta-globin with similar results. These efforts will support clinical trials in the disorder and the model will continue to serve as a means to address any issues that arise in the clinical trials in humans. A manuscript describing this work was published in Nature Communications. We have now also begun to test gene editing strategies in the rhesus with encouraging results. Our initial studies testing editing at the BCL11A erythroid enhancer as a means of reactivating fetal hemoglobin was just published in the Journal of Clinical Investigation. An extension of this work including editing at other loci to promote a more robust activation of fetal hemoglobin has proven successful. Additionally, other loci have been identified as promising repressors of fetal hemoglobin which can be targeted for clinical application.

地中海贫血和血红蛋白病代表了一组异质性贫血，其特征分别是一个或多个珠蛋白分子亚基的缺失/减少或异常产生，而旨在替换缺失或有缺陷的珠蛋白基因的策略长期以来被认为具有潜在的治愈作用。事实上，携带珠蛋白基因的逆转录病毒载体是第一个在小鼠模型中进行测试的基因转移载体，但基因转移率低和珠蛋白基因表达差困扰着该领域。此外，啮齿动物模型被证明不足以模拟人类造血作用。在大型动物中，通过在竞争性繁殖模型中系统地测试转导方法，基因转移技术取得了重大进展，在高剂量照射的消融条件下，目前可以实现 5-10% 或更高的长期体内基因转移水平。骨髓和红系集落以及外周血 T 和 B 细胞群之间共同整合位点的发现，以及一些克隆对骨髓后代的长期贡献，满足了真正造血干细胞转导的严格标准，并且克隆动力学支持随机性体内造血模型。克隆追踪技术的发展也被证明对于评估整合逆转录病毒载体插入突变的风险很重要。与此同时，据报道，成功获得了高滴度、稳定的病毒载体，该载体忠实地传递了人β-珠蛋白基因以及足以改善β-地中海贫血和地中海贫血小鼠模型疾病的关键调控元件，为临床前研究奠定了基础。在大型动物模型中进行测试。我们现在已经在恒河猴模型中推进了携带人β珠蛋白以及关键调控序列的慢病毒基因转移载体的临床前测试。然而，在临床应用之前还有许多其他问题有待解决。我们最近建立了稳态骨髓，这是镰状细胞病中唯一实用的干细胞来源，作为非人类灵长类动物基因操作的可行目标，但实现充分植入所需的调节类型和程度仍有待确定。我们之前已经证明，即使在表达异种基因时，低剂量照射也足以使转基因细胞在小鼠模型中的植入达到临床相关水平。这样的辐射剂量允许转基因细胞在非人类灵长类动物中长期植入，但水平太低，无法预期临床益处。将照射剂量增加到接近清髓性的水平仅导致适度的改善。白消安是一种烷化化疗剂，长期以来一直被用作全身照射的替代药物，用于骨髓移植的调理。然而，口服制剂的吸收不稳定，需要对个体患者进行密切的药代动力学监测，以实现可预测的骨髓抑制。我们最近在小鼠模型中评估了一种新推出的白消安静脉制剂，结果表明，在非骨髓剂量下，剂量依赖性植入可达到高达 80% 的水平。通过将输注延迟至中性粒细胞最低点当天可以实现进一步的改善。目前正在非人类灵长类动物模型中测试该试剂，试图确定足以使转基因细胞以足以临床应用的水平植入的剂量。最近，三只恒河猴被移植了自体外周血干细胞，这些干细胞是用携带β珠蛋白基因和关键调控元件的慢病毒载体转导的。尽管最初的植入是稳健的，但转基因细胞的长期水平低于表型校正所需的水平，并且目前正在采取额外措施来生产用于该应用的载体。利用源自 HIV 和 SIV 的质粒组合，产生了携带 SIV 衣壳序列的嵌合载体，从而能够在竞争性再增殖模型中有效转导恒河猴再增殖细胞。这种嵌合载体的出现使我们能够再次使用非人灵长类动物模型作为珠蛋白基因转移的临床前模型，利用基于HIV的载体来传递珠蛋白基因及其调控元件。我们现在已经优化了模型中基因转移的条件，并正在探索对载体的许多改变，包括方向、绝缘、额外的增强子、绝缘体和其他策略，以增加红系后代中每个载体拷贝数的β-珠蛋白量。载体修饰的恒河猴再生细胞。第一批动物现已移植了由红细胞特异性启动子/增强子驱动的编码 GFP 的慢病毒载体，其水平与该模型中的标准报告载体相匹配。现在，第二批动物被移植了编码人β-珠蛋白的载体，结果相似。这些努力将支持该疾病的临床试验，并且该模型将继续作为解决人类临床试验中出现的任何问题的手段。描述这项工作的手稿发表在《自然通讯》上。我们现在也开始在恒河猴中测试基因编辑策略，并取得了令人鼓舞的结果。我们测试 BCL11A 红细胞增强剂编辑作为重新激活胎儿血红蛋白的手段的初步研究刚刚发表在《临床研究杂志》上。这项工作的延伸，包括在其他位点进行编辑，以促进胎儿血红蛋白更强有力的激活，已被证明是成功的。此外，其他位点已被确定为有前景的胎儿血红蛋白抑制因子，可用于临床应用。