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异质性贫血，其特征分别是一种或多种珠蛋白分子亚基的缺失/减少或异常产生，长期以来，旨在取代缺失或有缺陷的珠蛋白基因的策略一直被认为是潜在的治疗方法。的确，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测试的基因转移载体之一，但基因转移率低和珠蛋白基因表达差困扰着该领域。此外，啮齿类动物模型被证明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，基因转移技术取得了重大进展，在高剂量照射的消融条件下，现在可以实现5-10%或更高的长期体内基因转移水平1,2。髓系和红系以及外周血T细胞和B细胞群之间共同整合位点的发现，以及一些克隆对髓系后代的长期贡献，满足了真正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3-5。克隆跟踪技术的发展也被证明对整合逆转录病毒载体的插入突变风险评估很重要6,7。与此同时，Sadelain团队成功地获得了高滴度、稳定的病毒载体，这些病毒载体忠实地传递了人类&#61538；-珠蛋白基因以及关键的调控元件，足以改善&#61538；-地中海贫血小鼠模型中的疾病8，为在大型动物模型中进行临床前测试奠定了基础。通过与Sadelain实验室的合作，我们现在已经在恒河猴模型中进行了携带人类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，仍有许多其他问题有待解决。我们最近已经建立了稳定状态的骨髓，这是镰状细胞病中唯一可行的干细胞来源，作为非人灵长类动物基因操作的可行靶点，但实现充分植入所需的调节类型和程度仍有待确定。我们之前的研究表明，低剂量照射足以使转基因细胞在小鼠模型中植入临床相关水平，即使在异种基因表达的情况下也是如此。这种辐照剂量允许转基因细胞在非人灵长类动物体内长期植入，但剂量太低，无法预期临床效益。将照射剂量增加到接近清骨髓的水平，结果只有适度的改善。布磺胺是一种烷基化化疗药物，长期以来一直被用作骨髓移植治疗中全身照射的替代药物。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制剂，结果表明，在非骨髓剂量下，剂量依赖性植入可以达到80%的水平。进一步的改善可以通过延迟输注达到中性粒细胞的最低点。这种药物目前正在非人灵长类动物模型中进行测试，试图确定足够的剂量，以使转基因细胞的植入达到足以临床应用的水平。最近，3只恒河猴被移植了自体外周血干细胞，这些干细胞被携带-珠蛋白基因和关键调控元件的慢病毒载体转导。目前的这些研究将用于支持珠蛋白疾病的最终临床研究，其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 irradiation1,2. 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 hematopoiesis3-5. The development of techniques for clonal tracking have also proven important in assessing the risk of insertional mutagenesis with integrating retroviral vectors6,7. Concurrent with this progress, the Sadelain group succeeded in attaining high titer, stable viral vectors which faithfully deliver the human -globin gene along with key regulatory elements sufficient to ameliorate disease in a murine model of -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 -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 expressed10. 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 improvement11. 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. 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.