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Gene Therapy for the Hemophilias

血友病的基因治疗

基本信息

批准号：
7074733
负责人：
Christopher E Walsh
金额：
$ 33.1万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
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/7074733
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

项目摘要

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（由申请人提供）：有效的基因治疗将彻底改变血友病的治疗。重组腺相关病毒（rAAV）载体被认为是血友病基因治疗中最有前途的病毒载体之一。AAV的非致病性性质、抑制有丝分裂和有丝分裂后细胞的能力以及rAAV/转基因序列稳定持续的能力在所有病毒载体中是独特的。rAAV在血友病A（因子VIII缺乏）基因治疗中应用的主要障碍是rAAV的有限包装能力与人FVIII基因的大尺寸的冲突。该递送系统的主要限速方面一直是rAAV的小包装容量（5 kb）。具有大cDNA（7.0Kb）的因子VIII是测试用于AAV介导的基因转移的各种新方法的极好模型。在这里，我们提出了令人信服的证据支持使用AAV载体的表达人因子VIII基因治疗。我们开发了几种不同的新方法表达功能因子VIII。首先，我们开发了携带全长FVIII cDNA的截短版本的rAAV载体。去除因子VIII的B结构域序列（约4.0 Kb）产生全功能蛋白（称为B结构域缺失，BDD FVIII），其在体内表达治疗水平的功能性FVIII。尽管截短了FVIII序列，但AAV包装仍然需要使用小（<250 bp）增强子/启动子元件。建议进一步截短FVIII序列和修饰转录元件。第二，AAV二聚化可用于克服载体包装限制。AAV前病毒DNA的特征在于头-尾多联体。在此，FVIII基因被分割并包装到两个单独的AAV载体中。通过放大单链到双链中间体的转化，二聚化显著增加。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转录本通过天然细胞剪接机制剪接在一起。我们目前的分子，蛋白质和功能的数据证明使用这种方法的FVIII敲除小鼠表型的校正。第四，AAV 2型是用于基因转移研究的主要血清型。我们提出，备用AAV血清型在其细胞嗜性方面不同。我们证明，非2型AAV血清型的效果显着更高的水平因子IX的表达，并将用于测试因子VIII的表达。将优化每种方法，并在AAV载体中进行FVIII生产，并使用免疫缺陷和FVIII敲除小鼠和血友病A犬进行体内测试。