Gene Therapy for the Hemophilias

血友病的基因治疗

• 批准号: 6605732
• 负责人: Christopher E Walsh
• 金额: $ 33.9万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6605732
• 关键词: SCID mouse; adeno associated virus group; biotechnology; coagulation factor VIII; dogs; gene delivery system; gene therapy; genetically modified animals; hemophilia As; laboratory mouse; nonhuman therapy evaluation; recombinant virus; transfection /expression vector

DESCRIPTION (provided by applicant): Effective gene therapy will revolutionize the treatment of the hemophilias. Recombinant adeno-associated virus (rAAV) vectors are considered among the most promising viral vectors for hemophilia gene therapy. The non-pathogenic nature of AAV, the ability to transduce mitotic and post-mitotic cells, and the capacity for stable persistence of rAAV/transgene sequences are unique among all viral vectors. A major obstacle in the application of rAAV in gene therapy for hemophilia A (factor VIII deficiency) is the conflict of the limited packaging capacity of rAAV and the large size of the human FVIII gene. The major rate-limiting aspect of this delivery system has always been the small packaging capacity (5kb) of rAAV. Factor VIII with its large cDNA (7.0 Kb) is an excellent model to test a variety of new approaches for AAV-mediated gene transfer. Here we present compelling evidence supporting the use of AAV vectors for the expression human factor VIII gene therapy. We developed several different novel approaches for the expression of functional factor VIII. First, we developed rAAV vectors carrying a truncated version of the full-length FVIII cDNA. Removal of the B-domain sequence of factor VIII (~4.0 Kb) results in a fully functional protein (termed B-domain deleted, BDD FVIII which express therapeutic levels of functional FVIII in vivo. Despite truncation of the FVIII sequence, the use of small (<250 bp) enhancer/promoter elements is still required for AAV packaging. Further truncation of the FVIII sequence and modification of the transcriptional elements are proposed. Second, AAV dimerization can be used to overcome vector-packaging limitations. AAV proviral DNA is characterized by head-to-tail concatamers. Here, the FVIII gene is divided and packaged into two individual AAV vectors. Dimerization dramatically increased by amplifying the conversion of single to double-strand intermediates. Third, a totally novel RNA repair strategy relies on the use of spliceosome-mediated trans-splicing. Here two independent pre-messenger RNA transcripts are spliced together via the native cellular splicing machinery. We present molecular, protein and functional data demonstrating correction of the FVIII knockout mouse phenotype using this method. Fourth, AAV type 2 is the predominant serotype used for gene transfer studies. We propose that alternate AAV serotypes differ in terms of their cellular tropism. We demonstrate that non-type 2 AAV serotypes effect significantly higher levels of factor IX expression and will be used to test factor VIII expression. Each method will be optimized and in AAV vectors for FVIII production and tested in vivo using immunodeficient and FVIII knockout mice and hemophilic A canines.

描述(申请人提供)：有效的基因治疗将彻底改变血友病的治疗方法。重组腺相关病毒(RAAV)载体被认为是血友病基因治疗最有前景的病毒载体之一。AAV的非致病性、转导有丝分裂细胞和有丝分裂后细胞的能力以及稳定保持rAAV/转基因序列的能力在所有病毒载体中都是独一无二的。RAAV在血友病A(凝血因子VIII缺乏症)基因治疗中应用的一个主要障碍是rAAV有限的包装容量与人类FVIII基因的大小之间的矛盾。这种递送系统的主要限速方面一直是rAAV的小包装容量(5kb)。因子具有较大的cDNA7.0kb，是检测AAV介导的多种基因转移新途径的极佳模型。在这里，我们提出了令人信服的证据，支持使用AAV载体进行人类因子VIII的表达基因治疗。我们开发了几种不同的新方法来表达功能因子VIII。首先，我们开发了携带全长FVIII基因的截短版本的rAAV载体。去除因子VIII的B结构域序列(~4.0kb)会导致一个全功能蛋白(称为B结构域缺失，BDD FVIII)，它在体内表达功能性FVIII的治疗水平。尽管FVIII序列被截短，但AAV包装仍然需要使用小的(&lt；250个碱基)增强子/启动子元件。建议进一步截短FVIII序列和修改转录元件。其次，AAV二聚化可以用来克服载体包装的局限性。AAV前病毒DNA的特征是头到尾的串联。在这里，FVIII基因被分割并包装成两个单独的AAV载体。通过放大单链到双链中间体的转化，二聚化大大增加。第三，一种全新的RNA修复策略依赖于剪接体介导的反式剪接的使用。在这里，两个独立的前信使RNA转录本通过天然的细胞剪接机制被剪接在一起。我们提供了分子、蛋白质和功能数据，证明了使用这种方法对FVIII基因敲除小鼠表型的纠正。第四，AAV2型是用于基因转移研究的主要血清型。我们认为，不同的AAV血清型具有不同的细胞嗜性。我们证明，非2型AAV血清型显著影响因子IX的表达水平，并将用于测试因子VIII的表达。每种方法都将进行优化，并在AAV载体中生产FVIII，并在体内使用免疫缺陷和FVIII基因敲除小鼠和血友病A犬进行测试。