GENE THERAPY FOR CANINI X-SCID

X连锁严重联合免疫缺陷（X-SCID）是最常见的 一种由常见的γ基因突变引起的联合免疫缺陷病 细胞因子受体链 人类和犬的X-SCID由一个块标记 在胸腺生成、外周T和NK淋巴细胞减少症以及 具有缺陷抗体应答的表型B细胞。 小鼠敲除 与人类和狗的不同之处在于，受影响的小鼠 T和B淋巴细胞减少。 骨髓移植（BMT），通过引入 正常造血干细胞（HSC），可用于治疗患者 X-SCID由于接受者的免疫功能低下， 由于对正常HSC的选择性优势，组织相容性BMT通常可以 在没有预先进行细胞消融化疗或放疗的情况下进行。 最 患者没有组织相容性供体， 用组织不相容的BMT治疗，这是不太成功的。 移植经遗传修饰的自体HSC以表达 缺陷基因（干细胞基因疗法）是一种替代方法， 可能最终被证明上级组织不相容的BMT。 的 目前用逆转录病毒载体转导HSC的技术是 并导致低数量转导HSC， 移植 基因治疗ADA缺乏症的临床试验 SCID已经证明逆转录病毒转导的HSC可以植入 不进行细胞消融，产生转导的T、B和骨髓细胞 表达正常水平的ADA 载体阳性细胞的频率 T细胞的增加是因为赋予T细胞的选择性优势。 通过表达ADA转导细胞。 犬X染色体的基因治疗 在犬X-SCID中， 在表型上与人类相同， 可以测试用于临床试验的相同载体和试剂 在狗的模型中。 与ADA缺乏症一样， 有利于表达正常基因产物的T细胞祖细胞， 这可能允许恢复免疫功能，尽管 相对低水平的HSC转导。 研究将分析 转导、植入和功能校正的免疫 系统中接受来自X-细胞的自体CD 34+骨髓细胞的X-SCID犬 在用含有以下的逆转录病毒载体转导后的SCID狗： 人gammac cDNA。 这些研究将使用生物化学和 和免疫学分析，转导的造血细胞的体外培养， 祖细胞，以及将转导的HSC体内移植到X-SCID中 狗来测试基因疗法 载体LTR的修饰将是 测试给予非激活依赖性表达的能力， 防止载体沉默。 我们将测试， 基因治疗后的胸腺生成细胞因子IL-7将加速 通常减缓HSC的功能性免疫的发展。

X-linked severe combined immune deficiency (X-SCID) is the most common form of SCID caused by mutations in the gene for the common gammac cytokine receptor chain. Human and canine X-SCID are marked by a block in thymopoiesis, peripheral T and NK lymphopenia, and presence of phenotypic B cells with defective antibody responses. Murine knockouts for gammac differ from humans and dogs in that the affected mice have both T and B lymphopenia. Bone marrow transplant (BMT), by introducing normal hematopoietic stem cells (HSC), can be used to cure patients with X-SCID. Because of the immunoincompetence of the recipients and the selective advantage to the normal HSC, histocompatible BMT can generally be performed without prior cytoablative chemo- or radiotherapy. Most patients do not have histocompatible donors and receive alternate therapy with histoincompatible BMT, which is less successful. Transplantation of autologous HSC genetically modified to express the defective gene (stem cell gene therapy) is an alternative approach which may ultimately prove to be superior to histoincompatible BMT. The present techniques for transducing HSC with retroviral vectors are inefficient and result in low numbers of transduced HSC for transplantation. Our clinical trial of gene therapy for ADA deficient SCID has demonstrated that retrovirally transduced HSC can engraft without cytoablation, generating transduced T, B, and myeloid cells expressing normal levels of ADA. The frequency of vector-positive cells T cells has risen because of the selective advantage conferred to the transduced cells by the expression of ADA. Gene therapy of canine X- SCID is an ideal large animal pre-clinical model, in that canine X-SCID is phenotypically identical to that of humans, and it is likely that the same vectors and reagents to be used in a clinical trial could be tested in the dog model. As in ADA deficiency, there is an expected selective advantage to T cell progenitors expressing the normal gene product, which may allow restoration of immune function in spite of the relatively low levels of HSC transduction. The studies will analyze transduction, engraftment, and functional correction of the immune system in X-SCID dogs receiving autologous CD34+ marrow cells from X- SCID dogs after transduction with retroviral vectors containing the human gammac cDNA. The studies will use a combination of biochemical and immunologic analyses, in vitro culture of transduced hematopoietic progenitors, and in vivo transplantation of transduced HSC into X-SCID dogs to test gene therapy. Modifications of the vector LTR will be tested for the ability to give activation-independent expression and to prevent vector silencing. We will test whether the administration of the thymopoietic cytokine IL-7 after gene therapy will accelerate the usually slow development of functional immunity from HSC.