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应用于血友病A（因子VIII缺乏症）基因治疗的一个主要障碍是rAAV有限的包装能力与人类FVIII基因的大尺寸的冲突。该递送系统的主要速率限制因素始终是 rAAV 的小包装容量（5kb）。因子 VIII 及其大 cDNA (7.0 Kb) 是测试 AAV 介导的基因转移的各种新方法的绝佳模型。在这里，我们提出了令人信服的证据，支持使用 AAV 载体表达人类因子 VIII 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 cDNA 的截短版本的 rAAV 载体。去除因子 VIII 的 B 结构域序列 (~4.0 Kb) 会产生全功能蛋白（称为 B 结构域删除，BDD FVIII，其在体内表达功能性 FVIII 的治疗水平。尽管截断了 FVIII 序列，但 AAV 包装仍需要使用小的 (<250 bp) 增强子/启动子元件。建议进一步截断 FVIII 序列并修饰转录元件。其次，AAV 二聚化可以用于克服载体包装限制的 AAV 前病毒 DNA 的特点是头尾连接体，通过放大单链到双链中间体的转化，二聚化显着增加。我们提供了分子、蛋白质和功能数据，证明使用此方法校正 FVIII 敲除小鼠表型。第四，AAV 2 型是用于基因转移研究的主要血清型。我们证明，非 2 型 AAV 血清型会显着提高因子 IX 的表达水平，并将用于测试因子 VIII 的表达。使用免疫缺陷和 FVIII 敲除小鼠以及血友病 A 犬进行 FVIII 生产和体内测试。

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.