Molecular Therapuetics for Epithelial Disorders

描述(由申请人提供)： 虽然利用逆转录病毒介导的体外基因治疗已经证明了遗传性皮肤病的功能矫正，但这种方法的成本和创伤特点导致了对其他非病毒基因治疗技术的探索。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，该系统在体内将一个表达盒整合到肝细胞的染色体DNA中。稳定的基因转移是通过转座酶基因的瞬时表达实现的，从而导致转座子DNA整合到表达盒的两侧。结果，在血友病B小鼠中实现了人凝血因子IX的终生和治疗性浓度。此外，它没有毒性，如果发生二次转位，也是极其罕见的。该系统的两个主要局限性是有限的基因转移量(-5.0kb)和整合效率，整合效率约为体内转染型肝细胞的5%，并在不同类型的细胞培养中有所不同。虽然这种转座子对血友病等疾病的有效性已经被证明，但在其他疾病中，-dNA插入的效率和大小将需要增加。我们已经取得了进展，我们已经确定了高活性的转座酶突变体，其效率是原始转座酶的8倍，与新的转座子相结合，将整合增加了14倍，并允许至少14kb的DNA分子的有效转座。我们最近已经证明，基于转座子的载体在皮肤模型中工作良好，并计划在基因转移的皮肤模型中测试当前和新获得的高度活跃的转座子。此外，我们将在临床前基因治疗研究中使用我们的新型嵌合转座酶-辅助性腺病毒载体系统。

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.