GENE THERAPY IN INDUCTION OF TRANSPLANT TOLERANCE

尽管最近取得了成功，但临床进展的主要障碍是 移植仍然是同种异体移植排斥和发病率， 非特异性免疫抑制导致的死亡率。 因此，我们认为， 实现捐助方对外国直接投资长期不作出反应 抗原将构成重要的临床进展。 该第一 该提案将研究基因转移技术的潜在用途， 诱导抗原特异性移植耐受。 更确切地说，这些 研究将确定是否可以诱导耐受性， 用自体骨髓细胞重建辐射小鼠， 哪些异种MHC基因已被引入并表达。 含有人HLA-A2基因的逆转录病毒表达载体将 构建并引入包装细胞系。 的菌落 产生高滴度的复制缺陷型重组逆转录病毒， 然后与小鼠骨髓细胞共培养， 转移 然后将经辐照的C57 B1/6（B6）小鼠移植 转化以表达人HLA-A2基因的自体骨细胞。 然后检查长期骨髓重建B6小鼠的 HLA-A2抗原的免疫反应性，通过1）体外免疫 使用来自细胞系或转基因细胞的淋巴细胞刺激的测定 在B6背景下表达HLA-A2基因的小鼠和2）通过 HLA-A2转基因或第三方皮肤移植 对照供体小鼠移植到重建的B6受体上。 成功 这些实验的完成应该提供深入了解异种 并将推动进一步的工作， 研究更复杂的组织相容性屏障的耐受性 用于临床应用。

Despite recent successes, a major impediment to progress in clinical transplantation continues to be allograft rejection and morbidity and mortality resulting from non-specific immunosuppression. Therefore, achievement of long lasting donor-specific unresponsiveness to foreign antigens would constitute an important clinical advance. This FIRST proposal will examine the potential use of gene transfer technology to induce antigen-specific transplantation tolerance. More precisely, these studies will determine whether tolerance can be induced in lethally irradiated mice reconstituted with autologous bone marrow cells into which xenogeneic MHC genes have been introduced and expressed. Retroviral expression vectors, which contain the human HLA-A2 gene will be constructed and introduced into packaging cell lines. Colonies which produce high titer, replication-defective recombinant retroviruses will then be co-cultured with mouse bone marrow cells to attain optimal gene transfer. Irradiated C57B1/6 (B6) mice will then be transplanted with autologous bone cells transformed to express the human HLA-A2 gene. Long-term bone marrow reconstituted B6 mice will then be examined for immunologic reactivity to the HLA-A2 antigen by 1) in vitro immunologic assays using lymphocytes for stimulation from cell lines or transgenic mice expressing the HLA-A2 gene on the B6 background and 2) by transplantation of skin grafts from HLA-A2 transgenic or third-party control donor mice onto reconstituted B6 recipients. The successful completion of these experiments should provide insight into xenogeneic tolerance induction and will provide an impetus to further work designed to study tolerance across more complicated histocompatibility barriers for clinical application.