Gene Therapy for the Hemophilias

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，转导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都是独一无二的。rAAV在血友病A（因子VIII缺乏症）基因治疗中应用的主要障碍是rAAV有限的包装能力与人FVIII基因大尺寸的冲突。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VIII具有较大的cDNA (7.0 Kb)，是测试各种aav介导的基因转移新方法的良好模型。在这里，我们提出了令人信服的证据，支持使用AAV载体表达人因子VIII基因治疗。我们开发了几种不同的表达功能因子VIII的新方法。首先，我们开发了rAAV载体，携带全长FVIII cDNA的截断版本。去除因子VIII的b结构域序列（~4.0 Kb）会产生完全功能的蛋白（称为b结构域删除，BDD FVIII），其在体内表达治疗水平的功能性FVIII。尽管截断了FVIII序列，但AAV包装仍然需要使用小的（<250 bp）增强子/启动子元件。建议进一步截断FVIII序列并修改转录元件。其次，AAV二聚化可以用来克服载体封装的限制。AAV原病毒DNA的特征是头尾相连。在这里，FVIII基因被分离并包装成两个单独的AAV载体。二聚化通过放大单链到双链中间体的转化而显著增加。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。在这里，两个独立的前信使RNA转录物通过原生细胞剪接机制拼接在一起。我们提出了分子、蛋白质和功能数据，证明使用这种方法可以纠正FVIII基因敲除小鼠的表型。第四，AAV 2型是用于基因转移研究的主要血清型。我们认为不同的AAV血清型在细胞趋向性方面有所不同。我们证明非2型AAV血清型对因子IX表达水平有显著影响，并将用于检测因子VIII表达。每种方法都将在AAV载体上进行优化，用于生产FVIII，并在免疫缺陷小鼠和FVIII敲除小鼠以及血友病A犬的体内进行测试。

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.