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：
8939814
负责人：
John Tisdale
金额：
$ 51.78万
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/8939814
关键词：
Address Anemia Animal Experimentation Animal Model Animals Autologous B-Lymphocytes Biological Models Bone Marrow Transplantation Busulfan Capsid Cells Clinical Clinical Trials Clonal Hematopoietic Stem Cell Collaborations Development Disease Disease model Dose Drug Formulations Drug Kinetics Engraftment Enhancers Erythroid Gene Expression Gene Transfer Gene Transfer Techniques Genes Globin HIV Hematopoiesis Hematopoietic stem cells Hemoglobinopathies Human Individual Infusion procedures Insertional Mutagenesis Intravenous Laboratories Lentivirus Vector Macaca mulatta Marrow Measures Methods Modeling Monitor Mus Myelogenous Myelosuppression Oral Patients Peripheral Blood Stem Cell Phylogenetic Analysis Plague Plasmids Population Pre-Clinical Model Preclinical Testing Production Reagent Regulatory Element Reporter Retroviral Vector Risk Rodent Model SIV Series Sickle Cell Anemia Single-Gene Defect Site Source Staging Stem cells T-Lymphocyte Technology Testing Thalassemia Transplantation Viral Vector Whole-Body Irradiation absorption base beta Globin beta Thalassemia chemotherapeutic agent clinical application clinically relevant conditioning cross reactivity dosage gene transfer vector genetic manipulation genetically modified cells in vivo in vivo Model irradiation lentiviral-mediated neutrophil nonhuman primate peripheral blood pre-clinical promoter 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, the Sadelain group succeeded 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, setting the stage for preclinical testing in the large animal model. In collaboration with the Sadelain laboratory, 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 set 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.

地中海贫血和血红蛋白病代表了一种异质性贫血组，其特征是一个或多种环球蛋白 - 分子亚基的缺乏/减少或异常产生，以及旨在取代缺失或有缺陷的全球基因的策略，长期以来一直被认为是潜在的治愈性的。实际上，携带球蛋白基因的逆转录病毒载体是在鼠模型中进行测试的第一个基因转移载体之一，但是低基因转移速率和较差的球蛋白基因表达却困扰着该领域。此外，事实证明，啮齿动物模型不足以建模人类造血。在大型动物中，通过系统地测试转导方法在竞争性重生模型中的转导方法取得了重大进展，在消融状态后，具有高剂量辐照后可以实现的长期体内基因转移水平为5-10％或更高。发现髓样和红细胞菌落之间的共同整合位点以及周围血液T和B细胞群体以及某些克隆对髓样后代的长期贡献满足了对真正造血干细胞转导的严格标准，以及克隆动力学以及支持的静脉内静脉模型。克隆跟踪技术的发展也已被证明在评估插入诱变与整合逆转录病毒载体的风险中很重要。同时，萨德兰（Sadelain）小组成功地获得了高滴度，稳定的病毒载体，这些媒介忠实地传递了人类β-珠蛋白基因以及足以改善疾病的关键调节元素，这在大型动物模型中为临床前测试的阶段树立了繁琐的疾病。与Sadelain实验室合作，我们现在通过对携带人β-蛋白质的慢病毒基因转移载体以及恒河猴猕猴模型中的关键调节序列进行临床前测试。但是，在临床应用之前，仍有许多其他问题要解决。我们最近建立了稳态骨髓，这是镰状细胞疾病中唯一实用的干细胞源，它是非人类灵长类动物中遗传操作的可行靶标，但是尚待建立足够的适应性植入所需的条件类型和程度。我们先前已经表明，即使表达异构基因，低剂量照射足以使鼠模型中临床相关的转基因细胞植入水平。这种辐照剂量允许在非人类灵长类动物中通过转基因细胞长期植入，但水平太低而无法期望临床益处。将辐照剂量增加到与骨髓性接壤的水平只会导致适度的改善。 Busulfan是一种烷基化的化学治疗剂，长期以来一直被用作全身照射的替代剂，用于调节骨髓移植。但是，口服配方的吸收不稳定，需要对单个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。我们最近评估了鼠模型中布鲁芬的新近可用的静脉注射配方，结果表明，在非乳化剂量下，可以在高达80％的水平下达到依赖剂量的植入。通过延迟输注到中性粒细胞纳迪尔当天，可以进一步改善。现在正在非人类灵长类动物模型中对该药物进行测试，以确定足够的剂量以允许在足以临床应用的水平下植入转基因细胞。最近已经用含有β球蛋白基因和关键调节元件的慢病毒载体转导的自体外周血干细胞移植了三个恒河猴。尽管最初的植入良好，但长期遗传改性细胞的长期水平低于表型校正所必需的，并且现在正在进行此应用的其他措施。利用了源自HIV和SIV的质粒的组合，产生了带有SIV衣壳序列的嵌合载体，从而有效地在竞争重生模型中可以有效地转导恒河猴的重生细胞。该嵌合载体的出现使我们能够再次使用非人类灵长类动物模型作为环球蛋白基因转移的临床前模型，利用基于HIV的媒介来传递球蛋白基因及其调节元素。现在，我们已经在模型中针对基因转移进行了优化的条件，并正在探索对向量的许多变化，包括方向，绝缘，其他增强剂，隔热器和其他策略，以增加每个载体拷贝数量的β-珠蛋白量，这是矢量修饰的颗粒的胎儿中的reythroid后代中的数量。现在，已经用慢病毒载体移植了第一系列动物，这些向态载体编码由红细胞特异性启动子/增强子驱动的GFP，其水平与该模型中标准报告媒介的水平相匹配。现在已经用编码人β-珠蛋白的载体移植了第二组动物，其结果相似。这些努力将支持该疾病的临床试验，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一种手段。