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

• 批准号: 8149537
• 负责人: John Tisdale
• 金额: $ 91.16万
• 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/8149537
• 关键词: Animal Model; Gene Transfer; Globin; pre-clinical

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. These current studies will be used in support of eventual clinical studies in globin disorders with the ultimate goal of providing preclinical safety and efficacy data in order to maximize the likelihood of success in the context of an acceptable risk/benefit ratio.

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，萨德兰团队成功获得了高滴度、稳定的病毒载体，该载体忠实地传递了人类β-珠蛋白基因以及足以改善β-地中海贫血鼠模型疾病的关键调控元件，为临床前测试奠定了基础在大型动物模型中。 我们与萨德兰实验室合作，目前已在恒河猴模型中推进携带人β珠蛋白和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。 目前的这些研究将用于支持珠蛋白疾病的最终临床研究，最终目标是提供临床前安全性和有效性数据，以便在可接受的风险/效益比的情况下最大限度地提高成功的可能性。