A preclinical large animal model for globin gene transfe

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球蛋白分子亚基的缺失/减少或异常产生，并且长期以来，旨在替代缺失或缺陷的球蛋白基因的策略被认为是潜在的治愈性的。事实上，携带珠蛋白基因的逆转录病毒载体是首批在小鼠模型中测试的基因转移载体之一，但低基因转移率和低珠蛋白基因表达困扰着该领域。此外，啮齿动物模型证明不足以模拟人类造血。在大型动物中，通过在竞争性再增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，在高剂量辐照的消融条件下，目前可实现5-10%或更高的长期体内基因转移水平1，2。在髓系和红系集落以及外周血T和B细胞群体中发现共同整合位点，沿着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，并且克隆动力学支持体内造血的随机模型3 -5。克隆追踪技术的发展也证明了在评估整合逆转录病毒载体的插入诱变风险方面的重要性6，7。与此同时，Sadelain小组成功地获得了高滴度，稳定的病毒载体，忠实地提供人类？O-珠蛋白基因沿着关键调控元件足以改善疾病的小鼠模型？O-地中海贫血8，为大型动物模型的临床前试验奠定基础。与Sadelain实验室合作，我们现在已经推进了携带人类？恒河猴模型中的O-珠蛋白沿着关键调控序列。然而，在临床应用之前，还有一些其他问题有待解决。我们最近已经建立了稳态骨髓，镰状细胞病中唯一实用的干细胞来源，作为非人灵长类动物中遗传操作的可行靶点9，但实现充分植入所需的调节类型和程度仍有待确定。我们以前已经证明，低剂量辐射足以使临床相关水平的转基因细胞在小鼠模型中植入，即使当异种基因表达时10。这样的辐射剂量允许遗传修饰的细胞在非人灵长类动物中长期植入，但水平太低而不能预期临床益处。将辐射剂量增加到接近清髓性的水平仅导致适度的改善11。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，用于骨髓移植的预处理。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白消安静脉注射制剂，结果表明，在非骨髓性剂量下，剂量依赖性植入率可达80%。通过将输注延迟至中性粒细胞最低值当天，可实现进一步改善。目前正在非人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用于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，这些干细胞用携带β珠蛋白基因和关键调控元件的慢病毒载体转导。这些当前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/受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 ?O-globin gene along with key regulatory elements sufficient to ameliorate disease in a murine model of ?O-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 ?O-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.