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

描述（由申请人提供）：Rag1基因的亚型突变是人类Omenn综合征（OS）的主要原因，这是一种独特的联合免疫缺陷，与严重的自身免疫表现相关。OS患者遭受压倒性的感染，并伴有严重的t细胞浸润和器官损伤。除非采用同种异体造血干细胞移植（HSCT）治疗，否则这种疾病会迅速致命，然而，当没有匹配的供体可用时，这种手术的结果不太令人满意。根据来自hla相同供者的HSCT的经验，可以预期，基于基因校正的高纯度hsc的骨髓移植治疗Rag1亚型突变的新方法将非常有益。为了设计这样的治疗方法，首先至关重要的是：a)定义在病毒载体背景下适当表达Rag1基因的离散基因组元件，b)在体外和体内表征Rag1次形蛋白及其与野生型Rag1分子竞争的能力，c)建立用于拯救新生成的Rag1次形小鼠的同基因骨髓移植模型，这些小鼠的特征是T和b淋巴细胞发育严重受损，与活化T细胞的外周扩增有关。在具有半形Rag缺陷的人身上观察到。该提案提供了初步数据，显示了用于表达Rag1的新慢病毒载体的设计及其用于高纯度hsc的转导，以及新开发的半胚Rag1小鼠的初步表征。拟建的研究项目将重点关注Rag1亚型的三个方面，这将显著加快基于慢病毒治疗Rag相关联合免疫缺陷的临床开发进程。将在体内研究携带多种基因组元件驱动Rag1表达的慢病毒载体诱导特异性和适当基因表达的能力。野生型Rag1蛋白在与拟态对应物竞争时诱导功能重排的能力将通过体外重排实验和从Rag1拟态小鼠分离的前b细胞进行研究。最后，本提案的目的1和目的2的结果将作为临床前研究的基础，旨在通过慢病毒转导造血干细胞来拯救Rag1亚型小鼠。总的来说，这项研究将是重要的，以促进发展新的形式的治疗联合免疫缺陷，由于拟态的RAG基因缺陷，基于基因转移。公共卫生相关性：Rag1突变引起的免疫缺陷是造成大量死亡率的原因，可用的治疗方法仍然有限。本研究为基于基因移植校正造血干细胞治疗Rag相关免疫缺陷的新方法的发展奠定了基础。

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.