Gene Therapy for Athabascan SCID

描述(申请人提供)：SIDIA(T-B-NK+阿萨巴斯卡语印第安人严重联合免疫缺陷)是一种由于缺乏Artemis蛋白而导致的人类原发免疫缺陷，需要在V(D)J基因重排中进行非同源末端连接。目前，SIDIA的唯一有效治疗方法是异基因骨髓移植，其发病率和死亡率都很高。最近，合作研究人员Mort Cowan博士的实验室通过同源破坏胚胎干细胞中的小鼠SsidA基因，产生了Ssida的小鼠模型。在这里，我们建议使用这个SIDIA+小鼠模型作为测试系统来评估两种不同的SIDIA基因治疗方法。在目标1中，以HIV-1为基础的慢病毒载体将被设计用于在淋巴细胞群体中优化表达SIDIA cDNA序列。这些载体将首先在SIDIA-/-小鼠胚胎成纤维细胞(MEF)中进行测试，以转导和恢复非同源末端连接活性。然后，这些载体将被用于将SSIDA基因导入SSIDA/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，以及用于纠正免疫功能障碍。在目标2中，将设计基于睡美人转座子系统的非病毒载体，以导入并优化表达SIDIA编码序列，首先测试转座在SIDIA-/-小鼠胚胎成纤维细胞中的可行性。然后，这些载体将被电穿孔进入SIDIA-/-骨髓细胞(未扩增的骨髓、富集干细胞的骨髓和富含普通淋巴祖细胞的骨髓)，随后用于植入、选择性生长和移植到SIDIA-/-受者体内后纠正免疫缺陷的评估。这些研究的结果有望深入了解在何种条件下，将SIDIA基因导入适当的淋巴造血祖细胞可以纠正SIDIA-/-动物的免疫缺陷，最终目标是将这种基因转移方法应用于人类SIDIA的治疗。

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.