Activity of Fully Deleted Helper-Virus Independent Adenoviral Gene Therapy Vector

描述(申请人提供)：许多疾病都是由基因缺陷引起的。基因治疗的目的是通过引入正常基因或修复基因缺陷来有效地治疗或治愈这些疾病。为了优化主要基于腺病毒和逆转录病毒的基因转移载体，人们进行了大量的研究工作。然而，基因治疗最初的希望已经被常用病毒载体的生物学所破坏：逆转录病毒具有内在的突变性和致癌性，因为它们整合到人类基因组中；来自腺病毒的载体诱导强烈的体液和细胞免疫反应，从而否定了治疗的成功。Isgenis正在采取双管齐下的方法来解决腺病毒载体的免疫原性问题。首先，其他人也表明，与含有更多病毒基因的载体相比，缺失病毒基因的腺病毒载体(即完全缺失的(FD，无勇气))的免疫原性较低。尽管进行了大量的研究，但有效生产FD腺病毒载体仍然需要使用辅助病毒来传递关键的腺病毒基因。Isgenis从与FDA(细胞和基因治疗部副主任Stephanie Simek博士)的讨论中了解到，使用‘辅助’病毒来生产FD腺病毒载体被认为是有问题的，因为目前的纯化系统不能提供‘辅助’病毒污染水平低于1/3 x 1010颗粒的制剂。Isgenis已经生产了一种新的FD腺病毒载体生产系统，该系统避免了使用‘辅助’病毒和复制高效重组子。Isgenis将使用这种新的载体架构来生产用于治疗血友病A和鸟氨酸转氨酶缺乏症(OTCD)的临床基因治疗载体。这些载体的活性将在临床前动物模型中进行测试。其次，为了克服任何剩余的免疫原性，Isgenis开发了创新的免疫抑制基因转移载体。这些利用了高度特异性，但有效的免疫抑制自然的“否决权”免疫抑制现象。简而言之，在T细胞表面表达CD8的细胞对其表面其他抗原做出反应的T细胞施加“否决权”。这些载体的活性将在临床前动物模型中进行测试。这些新腺病毒载体的开发是使基因治疗重回正轨的重要一步。 公共卫生相关性：基因治疗的目标是通过引入或修复缺陷基因来治愈疾病。在血友病A和OTCD的情况下，丢失的酶将直接输送到常驻的肝细胞。基因治疗载体必须导致目标组织中持续的基因表达，才能提供根治性治疗。针对载体蛋白的免疫反应通常会否定任何治疗效果。因此，人们正在寻求能够生产不再包含载体衍生基因(即病毒基因)的有效基因治疗载体的策略。在腺病毒载体的情况下，设计了这种完全删除的载体。在那里，高效的生产依赖于辅助病毒的使用。然而，FDA认为使用辅助病毒是有问题的。目前的纯化系统不提供辅助病毒污染水平低于1/3x1010的制剂。在这一应用中，我们建议研究一组新的辅助性病毒完全缺失腺病毒基因转移载体在体内的作用。

DESCRIPTION (provided by applicant): Numerous diseases are caused by gene defects. The goal of gene therapy is the effective treatment or cure of these diseases by the introduction of the normal gene or repair of the gene defect. A large research effort has been mounted to optimize gene transfer vectors mostly based on adenoviruses and retroviruses. Yet, the initial promise of gene therapy has been undermined by the biology of the commonly used viral vectors: retroviruses are intrinsically mutagenic and oncogenic as they integrate into the human genome; vectors derived from adenoviruses induce vigorous humoral and cellular immune responses that negate the therapeutic success. Isogenis is taking a two pronged approach to the problem of immunogenicity in Adenoviral vectors. First, others have also shown that Adenoviral vectors deleted of viral genes, i.e. fully deleted (fd, gutless) are less immunogenic than vectors containing more viral genes. Despite intensive research, the efficient production of fd Adenoviral vectors still requires the use of 'helper' viruses to deliver crucial Adenoviral genes. Isogenis had learned from its discussion with the FDA (Dr. Stephanie Simek, Deputy Director, Division of Cellular and Gene Therapy) that the use of 'helper' viruses for the production of fd Adenoviral vectors was considered problematic as present purification systems did not deliver preparations with 'helper' virus contamination levels of less than 1-in-3 x 1010 particles. Isogenis has produced a novel fd Adenoviral vector production system that avoids the use of a 'helper' virus and replication-efficient recombinants. Isogenis will use this novel vector architecture to produce clinical gene therapy vectors for the treatment of hemophilia A and ornithine transcarbamylase deficiency (OTCD). The activity of these vectors will be tested in pre-clinical animal models. Second, to overcome any remaining immunogenicity, Isogenis has developed innovative immune inhibitory gene transfer vectors. These employ the highly specific, yet effective immune suppression of the natural "veto" immune inhibitory phenomenon. Briefly, cells expressing CD8 on their surface exert a "veto" on T-cells responding to other antigens on their surface. The activity of these vectors will be tested in pre-clinical animal models. The development of these new Adenoviral vectors is a major step forward in getting gene therapy back on track. PUBLIC HEALTH RELEVANCE: The goal of gene therapy is the cure of the disease by the introduction or repair of the defective gene. In the case of hemophilia A and OTCD, the 'missing' enzyme will be directly delivered to the resident hepatocytes. Gene therapy vectors will have to lead to sustained gene expression in their target tissue to provide a permanent cure. Immune responses directed against vector proteins generally negate any therapeutic effect. Therefore, strategies are being sought that allowed the production of effective gene therapy vectors that did no longer contain vector-derived, i.e. viral, genes. In the case of adenoviral vectors such fully deleted vectors had been designed. There efficient production depended on the use of helper viruses. Yet, the use of a helper virus was considered problematic by the FDA. Present purification systems do not deliver preparations with helper virus contamination levels of less than 1-in-3x1010. In this application, we therefore propose to investigate how w novel set of helper-virus independent fully deleted Adenoviral gene transfer vectors function in vivo.