A preclinical large animal model for globin gene transfer

地中海贫血和血红蛋白病代表了一组不同类型的贫血，其特征分别是一个或多个珠蛋白分子亚单位的缺失/减少或异常产生，长期以来，旨在取代缺失或缺陷的珠蛋白基因的策略一直被认为是潜在的治愈方法。事实上，携带珠蛋白基因的逆转录病毒载体是第一批在小鼠模型中进行测试的基因转移载体，但低基因转移率和糟糕的珠蛋白基因表达一直困扰着该领域。此外，事实证明，啮齿动物模型不足以模拟人类造血。在大型动物中，通过在竞争性再种群模型中系统地测试转导方法，基因转移技术已经取得了重大进展，在大剂量照射的消融条件下，体内长期基因转移水平现在可以达到5-10%或更高。髓系和红系集落以及外周血T和B细胞群之间共同整合部位的发现，以及一些克隆对髓系后代的长期贡献，满足了真正的造血干细胞转导的严格标准，克隆动力学支持体内造血的随机模型。克隆跟踪技术的发展在评估整合逆转录病毒载体的插入突变风险方面也被证明是重要的。在取得这一进展的同时，Sadelain团队成功地获得了高滴度、稳定的病毒载体，该载体能够忠实地传递人β-珠蛋白基因以及足以改善β-地中海贫血8小鼠模型疾病的关键调控元件，为在大型动物模型中进行临床前测试奠定了基础。在与Sadelain实验室的合作下，我们现在已经在恒河猴模型中推进了携带人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，仍有许多其他问题需要解决。我们最近已经建立了稳定状态的骨髓，这是镰状细胞疾病中唯一实用的干细胞来源，作为在非人类灵长类动物中进行遗传操作的可行靶点，但实现充分植入所需的条件类型和程度仍有待确定。我们之前已经证明，即使表达了异种基因，低剂量辐射也足以在小鼠模型中实现临床相关水平的转基因细胞植入。这样的辐射剂量允许转基因细胞在非人类灵长类动物体内长期植入，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剂的水平，只会产生轻微的改善。白消安是一种烷基化化疗药物，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，口服制剂的不稳定吸收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的静脉注射白消安配方，结果表明，在非髓鞘剂量下，可实现高达80%的剂量依赖植入。将输液推迟到中性粒细胞最低点的那一天，可以进一步改善病情。该试剂目前正在非人类灵长类动物模型中进行测试，试图确定足以使转基因细胞在足够的水平上植入临床应用的剂量。最近，三只恒河猴被移植了自体外周血干细胞，这种干细胞是由携带贝塔珠蛋白基因和关键调控元件的慢病毒载体转导而来的。虽然最初的植入是稳健的，但长期的转基因细胞水平低于表型纠正所需的水平，目前正在采取额外的措施来为这一应用生产载体。利用来自HIV和SIV的质粒的组合，产生了携带SIV衣壳序列的嵌合载体，使得能够在竞争再种群模型中有效地转导恒河猴再繁殖细胞。这些目前的研究将用于支持珠蛋白紊乱的最终临床研究，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/收益比的情况下最大限度地提高成功的可能性。

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-thalassemia8, 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 primate9, 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.