Gene Therapy for Athabascan SCID

描述（由申请人提供）：SCIDA （T-B-NK+阿萨巴斯坎印第安人严重联合免疫缺陷）是一种人类原发性免疫缺陷，由缺乏Artemis蛋白引起，该蛋白是V(D)J基因重排中非同源末端连接所必需的。目前，唯一有效的治疗方法是同种异体骨髓移植，但其发病率和死亡率都很高。最近，合作研究者Mort Cowan博士的实验室通过同源破坏小鼠胚胎干细胞中的SCIDA基因，产生了小鼠SCIDA模型。在这里，我们建议使用Scida+小鼠模型作为测试系统来评估两种不同的Scida基因治疗方法。在Aim 1中，将设计基于HIV-1的慢病毒载体，以优化Scida cDNA序列在淋巴样细胞群体中的表达。这些载体将首先在Scida -/-小鼠胚胎成纤维细胞（MEF's）中进行转导和恢复非同源末端连接活性的测试。然后使用这些载体将Scida基因导入Scida -/-骨髓细胞，评估受体动物阳性转导细胞群的存在和选择性生长，以及纠正免疫缺陷。在Aim 2中，将设计基于睡美人转座子系统的非病毒载体，用于导入和优化Scida编码序列的表达，首先测试在Scida -/-小鼠胚胎成纤维细胞中转座的可行性。然后将这些载体电穿孔到Scida -/-骨髓细胞（未富集的骨髓、干细胞富集的骨髓和普通淋巴样祖细胞富集的骨髓）中，随后评估移植、选择性生长和移植到Scida -/-受体后的免疫缺陷纠正。这些研究的结果有望为将SCIDA基因引入适当的淋巴造血祖细胞可导致纠正SCIDA -/-动物免疫缺陷的条件提供见解，最终目标是将这种基因转移方法应用于治疗人类SCIDA。

DESCRIPTION (provided by applicant): SCIDA (T-B-NK+ severe combined immunodeficiency of Athabascan-speaking Native Americans) is a primary immune deficiency of humans resulting from absence of Artemis protein, required for nonhomologous end-joining in V(D)J gene rearrangement. Currently, the only effective therapy for SCIDA is allogeneic bone marrow transplant, for which there is a high risk of morbidity and mortality. Recently, the laboratory of collaborating investigator Dr. Mort Cowan, generated a mouse model of SCIDA by homologous disruption of the murine Scida gene in embryonic stem cells. Here we propose to use this Scida+ mouse model as a test system to evaluate two different gene therapy approaches for SCIDA. In Aim 1, lentiviral vectors based on HIV-1 will be designed for optimized expression of the Scida cDNA sequence in lymphoid cell populations. These vectors will first be tested in Scida -/-mouse embryo fibroblasts (MEF's) for transduction and restoration of non-homologous end-joining activity. These vectors will then be used to introduce the Scida gene into Scida -/- marrow cells, evaluating recipient animals for the presence and selective outgrowth of positively transduced cell populations as well as for correction of immunodefiency. In Aim 2, nonviral vectors based on the Sleeping Beauty transposon system will be designed for introduction and optimized expression of the Scida coding sequence, first testing for the feasibility of transposition in Scida -/- mouse embryo fibroblasts. These vectors will then be electroporated into Scida -/- marrow cells (unenriched marrow, marrow enriched for stem cells, and marrow enriched for common lymphoid progenitor cells) with subsequent evaluation for engraftment, selective outgrowth and correction of immunodeficiency after transplantation into Scida -/- recipients. Results from these studies are anticipated to provide insight into the conditions under which introduction of the SCIDA gene into appropriate lympho-hematopoietic progenitors can lead to correction of immunodeficiency in scida -/- animals, with the ultimate goal of applying such gene transfer approaches to the treatment of SCIDA in humans.