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

描述（由申请人提供）：β-血红蛋白病是全球最常见的遗传性疾病，是开发安全有效的造血基因治疗方法的重要范例。在过去的几年里，已经朝着这个目标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校正，最终以第一个I/II期临床试验的慢病毒载体的目的是基因治疗的遗传疾病。虽然第一位接受治疗的患者在移植后4个月（评估的最新时间）显示出转移的珠蛋白基因的持续表达，但我们现在要问的是，即使通过目前的措施只能获得与转导的造血干细胞（HSC）的部分嵌合体，在亚骨髓清除的受者中，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，我们将设计一种通过新型神经生长因子受体（NGF-R）变体进行遗传校正的HSC的离体选择程序，该变体没有残留活性，并且与磁场中临床适用的大量细胞纯化相容。重要的是，我们将评估是否可以通过新的HOX融合蛋白，直接穿透细胞膜，诱导HSC的维持和扩增，而没有潜在的致癌风险所带来的遗传物质的转移，减轻以前不可避免的损失，在这样的程序中发生在体外的HSC内容。在特定目标2中，我们将研究是否可以通过共表达天然促红细胞生成素（Epo）受体变体（对内源性Epo具有增强的敏感性）来提供遗传校正的红细胞的自控体内扩增。基本原理是基于这种遗传变异引起的家族性红细胞增多症的良性自然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动物，食蟹猴，其细胞被人慢病毒载体转导与人细胞相似。该模型将避免大多数非人灵长类动物对HIV载体的低依赖性，从而使得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上述移植策略进行关键评价成为可能。

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.