HUMAN CTL RESPONSES TO ADENOVIRUS GENE THERAPY VECTORS

本研究的目的是明确产生人腺病毒特异性细胞毒性T淋巴细胞(CTL)的机制，以帮助设计新的策略来避免对腺病毒基因治疗载体的免疫反应。腺病毒作为基因治疗载体正在接受广泛的研究，用于治疗广泛的心、肺和血液疾病，包括囊性纤维化、血友病和动脉粥样硬化。然而，动物模型数据表明，腺病毒载体的免疫原性干扰了腺病毒介导的基因治疗的效果。将早期区域1(E1)缺失腺病毒载体注射到小鼠(腺病毒感染受到自然限制的宿主)，会在几周内产生腺病毒特异性CTL，破坏腺病毒转导的细胞。对这一问题的进一步分析需要研究对腺病毒的人CTL反应。我们成功地从健康成人外周血单核细胞中扩增了记忆腺病毒特异性CTL，并证明这些反应是主要组织相容性复合体(MHC)限制的，并由CD8 T细胞介导。根据我们的研究，记忆性细胞免疫反应的存在很可能会对腺病毒介导的人类基因治疗构成一个主要的额外障碍。然而，我们推测，人腺病毒特异性CTL可能针对有限数量的免疫优势表位。因此，有可能通过消除这些表位来降低腺病毒载体的免疫原性。作为第二种方法，有可能利用腺病毒开发的机制来逃避宿主免疫反应。Ad早期区3(E3)编码帮助细胞对CTL和肿瘤坏死因子产生抵抗力的蛋白质，但该区域在大多数腺病毒载体中缺失或低表达。我们推测，E3区蛋白的结构性表达可能有助于降低腺病毒载体的免疫原性。我们建议通过分析体外抗腺病毒的人CTL反应来解决这些假说。这些研究将为设计更有效的腺病毒基因治疗载体提供实验基础，用于未来的人体试验。

The goal of this proposal is to define the mechanisms involved in the generation of human adenovirus-specific cytotoxic T lymphocytes (CTLs) in order to help design new strategies for evading the immune response to adenovirus gene therapy vectors. Adenoviruses are under extensive investigation as gene therapy vectors for a broad spectrum of heart, lung, and blood diseases including cystic fibrosis, hemophilia, and atherosclerosis. However, data from animal models indicate that the immunogenicity of adenovirus vectors interferes with the efficacy of adenovirus-mediated gene therapy. Administration of early region 1(E1)-deleted adenovirus vectors to mice, a host in which adenovirus infection is naturally restricted, results in the generation of adenovirus-specific CTLs which destroy adenovirus-transduced cells within a few weeks. Further analysis of this problem requires study of human CTL responses against adenovirus. We have successfully amplified memory adenovirus-specific CTLs in peripheral blood mononuclear cells from healthy adults and documented that these responses are major histocompatiblity complex (MHC)-restricted and mediated by CD8+ T cells. Based on our studies, it is likely that the presence of memory cellular immune responses will pose a major additional obstacle for adenovirus-mediated gene therapy in man. We postulate, however, that human adenovirus-specific CTLs may be targeted against a limited number of immunodominant epitopes. Therefore, it may be possible to reduce the immunogenicity of adenovirus vectors by elimination of such epitopes. As a second approach, it may be possible to take advantage of mechanisms which adenovirus has developed to evade host immune responses. The Ad early region 3 (E3) codes for proteins which help make cells resistant to CTLs and tumor necrosis factor, but this region is deleted or poorly expressed from most adenovirus vectors. We postulate that constitutive expression of E3 region proteins may help reduce the immunogenicity of adenovirus vectors. We propose to address these hypotheses by analysis of human CTL responses against adenovirus in vitro. These studies will provide an experimental basis for the design of more effective adenovirus gene therapy vectors for future human trials.