Cell selection strategies for the gene therapy of the beta-hemoglobinopathies

描述(申请人提供)：β-血红蛋白病是世界上最普遍的遗传性疾病，是发展安全和有效的造血基因治疗方法的重要范例。在过去的几年里，为实现这一目标已经取得了实质性的进展，从最初的载体设计到相关小鼠模型的持续纠正，最终达到了慢病毒载体的第一阶段I/II临床试验，目的是基因治疗一种遗传病。虽然第一个接受治疗的患者在移植后4个月显示转移的珠蛋白基因持续表达，这是最近评估的时间，但我们现在正在询问，即使按目前的方法只能获得与转导的造血干细胞(HSCs)的部分嵌合，亚髓消融受者是否可以安全地在几乎所有红细胞中实现治疗性珠蛋白表达。在具体目标1中，我们将通过一种新型的神经生长因子受体(NGF-R)突变体设计一种体外筛选转基因HSCs的方法，该突变体不具有残留活性，与临床上适用的大鼠骨髓细胞在磁场中的纯化兼容。重要的是，我们将评估在这样的过程中，在体外发生的先前不可避免的HSC含量的损失是否可以通过新的HOX融合蛋白来缓解，这种融合蛋白直接穿透细胞膜诱导HSC的维持和扩张，而不会因为转移遗传物质而带来潜在的致癌风险。在特定的目标2中，我们将研究是否可以通过共表达对内源性EPO具有更高敏感性的天然促红细胞生成素(EPO)受体变体来提供体内自控的转基因红细胞扩增。其理论基础是基于这种基因变异引起的家族性红细胞增多症的良性自然病史和广泛的初步数据。在具体目标3中，我们将转向非人灵长类猕猴，它的细胞可被人类慢病毒载体转导的能力与人类细胞相似。该模型将绕过大多数非人类灵长类动物对HIV载体的低渗透率，从而使在与人类密切相关的大型动物模型中对新型珠蛋白慢病毒载体和前述移植策略进行关键评估成为可能。

DESCRIPTION (provided by applicant): The beta-hemoglobinopathies are the most prevalent genetic disorders worldwide and serve as an important paradigm for the development of safe and effective approaches to hematopoietic gene therapy. In the past several years, substantial advances have been made towards this goal, from original vector design to the sustained correction of relevant mouse models, culminating with the first Phase I/II clinical trial of a lentiviral vector aimed at the gene therapy of a genetic disease. While the first patient treated show sustained expression of the transferred globin gene 4 months post-transplantation, the latest time assessed, we are now asking whether therapeutic globin expression within virtually all red blood cells can be safely achieved in sub-myeloablated recipients even when only partial chimerism with transduced hematopoietic stem cells (HSCs) would be obtained by current measures. In Specific Aim 1, we will devise an ex-vivo selection procedure for genetically corrected HSCs by means of a novel nerve growth factor receptor (NGF-R) variant devoid of residual activity and compatible with clinically-applicable bulk cell purification in a magnetic field. Importantly, we will assess whether the previously unavoidable loss of HSC content that occurs in vitro during such a procedure can be alleviated by means of novel HOX fusion proteins that penetrate cell membranes directly to induce HSC maintenance and expansion without the potentially oncogenic risk posed by the transfer of genetic material. In Specific Aim 2, we will investigate whether self-controlled in vivo amplification of genetically corrected red blood cells can be provided by co-expression of a natural erythropoietin (Epo) receptor variant with enhanced sensitivity to endogenous Epo. The rationale is based on the benign natural history of the familial erythrocytosis caused by this genetic variant and extensive preliminary data. In Specific Aim 3, we will turn to a non-human primate, Macaca fascicularis, whose cell transducibility by human lentiviral vectors is similar to that of human cells. This model will circumvent the low permissivity of most non-human primates for HIV vectors and make thus possible the critical evaluation of novel globin lentiviral vectors and aforementioned transplantation strategies in a large animal model closely related to humans.