Study of Rag1 hypomorphic mice and their rescue by lentiviral gene transfer

描述(由申请人提供)：Rag1基因的低形态突变是人类Omenn综合征(OS)的主要原因，Omenn综合征是一种与严重的自身免疫表现相关的独特的联合免疫缺陷。OS患者患有压倒性感染，并伴有严重的T细胞浸润和器官损伤。除非通过异基因造血干细胞移植(HSCT)治疗，否则这种疾病很快就会致命，然而当没有匹配的供者时，这种方法的结果就不那么令人满意了。根据人类白细胞抗原相合供者的HSCT经验，可以预见，基于基因矫正的高纯度HSCs的骨髓移植治疗Rag1亚型突变的新方法将是非常有益的。为了设计这样的治疗方法，首先至关重要的是：a)定义离散的基因组元件以在病毒载体的背景下适当地表达Rag1基因，b)鉴定Rag1亚型蛋白在体外和体内的特征及其与野生型Rag1分子竞争的能力，以及c)建立同基因骨髓移植模型来拯救新产生的Rag1低型型小鼠，其特征是T和B淋巴细胞发育严重障碍，伴随着激活的T细胞的外周扩张，正如在患有低形型RAG缺陷的人类中观察到的那样。除了一个新开发的亚型Rag1小鼠的初步特征外，该方案还提供了用于表达Rag1的新的慢病毒载体的设计以及它们用于高纯度HSCs转导的初步数据。拟议的研究计划将集中在Rag1亚型的三个方面，这将显著加快以慢病毒为基础的治疗RAG相关联合免疫缺陷的临床开发。携带驱动Rag1表达的各种基因组元件的慢病毒载体将在体内研究其诱导特定和适当基因表达的能力。野生型Rag1蛋白在与其亚型蛋白竞争时诱导功能重排的能力将通过体外重排试验和从Rag1亚型小鼠分离的前B细胞中进行研究。最后，这项提案的目标1和目标2的结果将作为旨在通过慢病毒转导HSCs拯救Rag1低构型小鼠的临床前研究的基础。总体而言，这项研究将有助于基于基因转移开发治疗由于RAG基因亚型缺陷引起的联合免疫缺陷的新形式。公共卫生相关性：Rag1突变导致的免疫缺陷是相当大的死亡率的原因，可用治疗方法仍然有限。这一建议为开发一种新的治疗RAG相关性免疫缺陷的方法奠定了基础，该方法基于基因转移纠正造血干细胞。

DESCRIPTION (provided by applicant): Hypomorphic mutations in the Rag1 gene are the main cause of Omenn syndrome (OS) in humans, a unique combined immunodeficiency associated with severe autoimmune manifestations. OS patients suffer from overwhelming infections accompanied with severe T-cell infiltrate and organ damage. The disease is rapidly fatal unless treated by allogeneic hematopoietic stem cell transplantation (HSCT), however the results of this procedure are less satisfactory when no matched donors are available. Based on the experience of HSCT from HLA-identical donors, it can be anticipated that a new treatment for Rag1 hypomorphic mutants based on bone marrow transplantation of genetically corrected highly purified HSCs would be highly beneficial. In order to design such therapeutic approach it is first crucial to: a) define discrete genomic elements for appropriate Rag1 gene expression in a context of a viral vector, b) characterize the Rag1 hypomorphic protein in vitro and in vivo and its ability to compete with the wild-type Rag1 molecule, and c) establish a syngeneic bone marrow transplantation model for the rescue of newly generated Rag1 hypomorphic mice, that are characterized by a severe impairment in T- and B-lymphocyte development, associated with peripheral expansion of activated T cells, as observed in humans with hypomorphic Rag defects. This proposal presents preliminary data showing the design of new lentiviral vectors for the expression of Rag1 and their use for transduction of highly purified HSCs, in addition to the initial characterization of a newly developed hypomorphic Rag1 mouse. The proposed research program will focus on three aspects of Rag1 hypomorphism, which will significantly accelerate the advancement of the clinical development of a lentiviral based therapy for Rag related combined immunodeficiency. Lentiviral vectors carrying various genomic elements driving Rag1 expression will be studied in vivo for their ability to induce specific and appropriate gene expression. The ability of wild-type Rag1 protein to induce functional rearrangement when competing with its hypomorphic counterpart will be studied using an in vitro rearrangement assay and in pre-B cells isolated from Rag1 hypomorphic mice. Finally, results from aims 1 and 2 of this proposal will serve as the basis for preclinical studies aimed at rescuing Rag1 hypomorphic mice using lentiviral transduction of HSCs. Overall, this study will be important to facilitate development of novel forms of treatment for combined immunodeficiency due to hypomorphic RAG gene defects, based on gene transfer. PUBLIC HEALTH RELEVANCE: Immunodeficiencies due to Rag1 mutations are responsible for considerable mortality and available treatments are still limited. This proposal establishes the basis for the development of a new therapeutic approach for Rag related immunodeficiencies based on correction of hematopoietic stem cells by gene transfer.