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，基因转移技术已经取得了重大进展，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。发现髓系和红系集落以及外周血T和B细胞群体之间的共同整合位点，沿着一些克隆对髓系后代的长期贡献，满足真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。克隆追踪技术的发展也被证明在评估整合逆转录病毒载体的插入突变风险方面很重要。与此同时，Sadelain小组成功获得了高滴度、稳定的病毒载体，该载体忠实地递送人β-珠蛋白基因沿着足以改善β-地中海贫血小鼠模型中的疾病的关键调控元件，为在大型动物模型中进行临床前试验奠定了基础。 与Sadelain实验室合作，我们现在已经在恒河猴模型中推进了携带人β-珠蛋白沿着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，还有一些其他问题有待解决。我们最近建立了稳态骨髓，唯一实用的干细胞来源，在镰状细胞病，作为一个可行的目标，在非人灵长类动物的遗传操作，但类型和程度的条件所需的实现充分的植入仍然有待建立。 我们以前已经表明，低剂量照射足以使临床相关水平的移植的转基因细胞在小鼠模型中，即使当异种基因表达。 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。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。 虽然初始植入是稳健的，但遗传修饰细胞的长期水平低于表型校正所需的水平，并且现在正在采取额外措施来生产用于该应用的载体。 利用来自HIV和SIV的质粒的组合，产生携带SIV衣壳序列的嵌合载体，使得能够在竞争性再增殖模型中有效转导恒河猴再增殖细胞。 这种嵌合载体的出现使我们能够再次使用非人灵长类动物模型作为珠蛋白基因转移的临床前模型，利用基于HIV的载体来递送珠蛋白基因及其调控元件。 我们现在已经优化了模型中基因转移的条件，并且正在探索对载体的许多改变，包括定向、绝缘、额外的增强子、绝缘子和其他策略，以增加载体修饰的恒河猴再增殖细胞的红系后代中每个载体拷贝数的β-珠蛋白的量。 第一系列的动物现在已经移植了由红细胞特异性启动子/增强子驱动的编码GFP的慢病毒载体，其水平与该模型中的标准报告载体的水平相匹配。 第二组动物现在已经被移植了编码人类β-珠蛋白的载体，得到了类似的结果。 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手段。

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.