Molecular Therapuetics for Epithelial Disorders

上皮疾病的分子治疗

• 批准号: 6908586
• 负责人: PAUL KHAVARI
• 金额: $ 70.98万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6908586
• 关键词: Molecular; Therapuetics; Epithelial; Disorders

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.

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基因皮肤病的功能性纠正，但该过程的成本和创伤性特征导致人们寻找用于基因治疗的其他非病毒技术。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续水平的基因表达的困扰。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。稳定的基因转移是通过转座酶基因的瞬时表达实现的，从而导致表达盒侧翼的转座子DNA整合。结果，B 型血友病小鼠体内的人因子 IX 达到了终生治疗浓度。而且，没有毒性，二次转座发生的情况也极为罕见。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，整合效率约为转染的5％：体内肝细胞，并且在培养物中的不同细胞类型之间存在差异。虽然这种转座子的有效性已被证明对血友病等疾病有效，但在其他疾病中，-DNA 插入片段的效率和大小仍需要提高。我们取得的进展是，我们已经鉴定出高活性转座酶突变体，其效率比原始转座酶高 8 倍以上，与新转座子组合可将整合提高 14 倍，并允许有效转座至少 14 kb 的 DNA 分子。我们最近表明，基于转座子的载体在皮肤模型中效果良好，并计划在基因转移的皮肤模型中测试当前和新获得的高活性转座子。此外，我们将在临床前基因治疗研究中使用我们新的嵌合转座酶辅助依赖性腺病毒载体系统。