Molecular Therapuetics for Epithelial Disorders

描述（由申请人提供）： 虽然使用逆转录病毒介导的离体基因治疗已经证明了遗传性皮肤病的功能矫正，但是该过程的成本和创伤特征已经导致了对用于基因治疗的另外的非病毒技术的研究。非病毒载体基因转移载体一直受到无法在体内获得治疗性和持久性基因表达水平的困扰。我们最近描述了一种基于DNA转座子的非病毒载体系统，该系统将表达盒整合到体内肝细胞的染色体DNA中。通过转座酶基因的瞬时表达实现稳定的基因转移，导致转座子DNA整合在表达盒的侧翼。因此，在血友病B小鼠中已经实现了人因子IX的终身和治疗浓度。此外，没有毒性，如果发生继发性易位也是极其罕见的。该系统的两个主要限制是有限的基因转移能力（~ 5.0 kb）和整合效率，其为体内转染肝细胞的~5%，并且在培养物中的不同细胞类型之间变化。虽然这种转座子的有效性已被证明用于血友病等疾病，但在其他疾病中，-DNA插入物的效率和大小需要增加。我们已经取得了进展，因为我们已经鉴定了比原始转座酶效率高> 8倍的超活性转座酶突变体，其与新的转座子组合使整合增加14倍，并允许至少14 kb的DNA分子的有效转座。我们最近表明，转座子为基础的载体在皮肤模型中工作良好，并计划测试目前和新获得的过度活跃转座子在皮肤模型的基因转移。此外，我们将使用我们的新的嵌合转座酶辅助依赖性腺病毒载体系统在临床前基因治疗研究。

DESCRIPTION (provided by applicant): While functional correction of genodermatoses has been demonstrated using retroviral mediated ex-vivo gene therapy, the cost and traumatic features of this procedure has led to the search for additional non-viral technologies for gene therapy. Non-viral vector gene transfer vectors have been plagued by the inability to get both therapeutic and persistent levels of gene expression in vivo. We have recently described a non-viral vector system based on DNA transposons that integrates an expression cassette into the chromosomal DNA of hepatocytes in vivo. Stable gene transfer is achieved by transient expression from the transposase gene resulting in the integration of transposon DNA flanking an expression cassette. As a result, life-long and therapeutic concentrations of human factor IX have been achieved in hemophilia B mice. Moreover, there is no toxicity and secondary transposition if it occurs is extremely rare. The two major limitations of this system are the limited gene transfer capacity (-5.0 kb), and the efficiency of integration, which is ~5% of transfected: hepatocytes in vivo and varies between different cell types in culture. While the effectiveness of this transposon has been demonstrated for diseases like hemophilia, there are other diseases in which the efficiency and size of the -DN A insert will need to be increased. We have made progress in that we have identified hyperactive transposase mutants that are >8x more efficient than the original transposase that in Combination with a new transposon increases integration by 14x and allows for efficient transposition of DNA molecules of at least 14 kb. We have recently shown that transposon-based vectors work well in skin models and plan to test current and newly obtained hyperactive transposons hi skin models of gene transfer. Moreover, we will use our new chimeric transposase-helper dependent adenoviral vector system in preclinical gene therapy studies.