GENE THERAPY FOR CANINI X-SCID

X连锁严重联合免疫缺陷(X-SCID)是最常见的 常见伽马基因突变引起的SCID形式 细胞因子受体链。人类和狗的X-SCID由一个块标记 在胸腺生成，外周T和NK淋巴细胞减少，以及存在 抗体反应有缺陷的表型B细胞。小鼠基因敲除 因为Gammac与人类和狗的不同之处在于受影响的小鼠有 T和B淋巴细胞减少。骨髓移植，通过引入 正常造血干细胞(HSC)，可用于治疗慢性粒细胞白血病患者 X-SCID。由于受者的免疫功能不全和 对正常HSC的选择性优势，组织相容的BMT一般可以 在没有事先细胞切除的化疗或放疗的情况下进行。多数 患者没有组织相容的捐赠者，并接受替代 组织不合的骨髓移植治疗不太成功。 自体HSC基因修饰后的移植表达 缺陷基因(干细胞基因治疗)是一种替代方法，它 可能最终被证明优于组织不相容的骨髓移植。这个 目前用逆转录病毒载体转导HSC的技术有 效率低下，并导致转导的HSC数量较少 移植。ADA缺乏症基因治疗的临床研究 SCID已经证明，逆转录病毒转导的HSC可以植入 在不切除细胞的情况下，产生转导的T、B和髓系细胞 表达正常的ADA水平。载体阳性细胞的频率 T细胞的上升是因为被赋予了选择性优势 通过ADA的表达转导细胞。犬X-DNA的基因治疗 在犬X-SCID中，SCID是一种理想的大动物临床前模型 在表型上与人类相同，很可能是 临床试验中使用的相同载体和试剂可以进行测试 在狗的模型中。就像在ADA缺乏症中一样，预计会有选择性 对T细胞祖细胞表达正常基因产物的优势， 这可能允许恢复免疫功能，而不管 HSC转导水平相对较低。这些研究将分析 免疫的转导、植入和功能矫正 X-SCID犬接受X-CD34+自体骨髓细胞的系统 转导SCID犬的逆转录病毒载体 人GAMAc基因。这些研究将结合使用生化 和免疫学分析，转导造血细胞的体外培养 造血祖细胞和转导HSC体内移植的研究 狗来测试基因疗法。向量Ltr的修改将是 测试给出不依赖于激活的表达和 防止载体静音。我们将测试政府是否会 基因治疗后胸腺生成细胞因子IL-7将加速 通常来自HSC的功能免疫发展缓慢。

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.