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

描述（由申请人提供）：β -血红蛋白病是世界范围内最普遍的遗传性疾病，是开发安全有效的造血基因治疗方法的重要范例。在过去的几年中，从最初的载体设计到相关小鼠模型的持续修正，在针对遗传疾病的基因治疗的慢病毒载体的第一个I/II期临床试验中取得了重大进展。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，但我们现在要问的是，在亚髓清除受体中，即使仅与转导的造血干细胞（hsc）部分嵌合，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，我们将设计一种体外选择程序，通过一种新的神经生长因子受体（NGF-R）变体，该变体缺乏残留活性，并与临床应用的磁场散装细胞纯化兼容。重要的是，我们将评估在此过程中体外发生的先前不可避免的HSC含量损失是否可以通过新型HOX融合蛋白直接穿透细胞膜来缓解，从而诱导HSC维持和扩增，而不会产生遗传物质转移带来的潜在致癌风险。在Specific Aim 2中，我们将研究一种对内源性促红细胞生成素（Epo）敏感性增强的天然促红细胞生成素（Epo）受体变体的共表达是否可以提供基因校正红细胞的体内自我控制扩增。其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。在特异性目标3中，我们将转向非人类灵长类动物，Macaca fascularis，其细胞可通过人类慢病毒载体进行转导，类似于人类细胞。该模型将绕过大多数非人类灵长类动物对HIV载体的低许可性，从而可以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估。

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.