Gene Therapy for Athabascan SCID

描述（由申请人提供）：SCIDA（T-B-NK+说阿萨巴斯卡语的美洲土著人的严重联合免疫缺陷）是一种由缺乏Artemis蛋白引起的人类原发性免疫缺陷，该蛋白是V（D）J基因重排中非同源末端连接所需的。目前，SCIDA的唯一有效疗法是异基因骨髓移植，其发病率和死亡率高。最近，合作研究者Mort科万博士的实验室通过同源破坏胚胎干细胞中的小鼠Scida基因，产生了SCIDA小鼠模型。在这里，我们建议使用这个Scida+小鼠模型作为一个测试系统，以评估两种不同的基因治疗方法SCIDA。在目标1中，基于HIV-1的慢病毒载体将被设计用于在淋巴细胞群体中优化Scida cDNA序列的表达。这些载体将首先在Scida -/-小鼠胚胎成纤维细胞（MEF）中测试非同源末端连接活性的转导和恢复。然后，这些载体将用于将Scida基因导入Scida -/-骨髓细胞，评估受体动物的阳性转导细胞群的存在和选择性生长以及免疫缺陷的校正。在目标2中，基于睡美人转座子系统的非病毒载体将被设计用于引入和优化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、富集干细胞的骨髓和富集普通淋巴祖细胞的骨髓）中，随后评估移植到Scida -/-受体后的植入、选择性生长和免疫缺陷校正。这些研究的结果预计将提供深入了解的条件下，引入SCIDA基因到适当的淋巴造血祖细胞可以导致纠正免疫缺陷的scida -/-动物，与应用这种基因转移方法治疗SCIDA在人类的最终目标。

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.