Development of sensitive and unbiased integration site analyses to comprehensivly assess biosafety and efficiency of innovative vectors

开发敏感且公正的整合位点分析，以全面评估创新载体的生物安全性和效率

• 批准号: 22811434
• 负责人: Dr. Manfred Schmidt, Ph.D.
• 金额: --
• 依托单位: Abteilung Translationale Medizinische Onkologie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2006
• 资助国家: 德国
• 起止时间: 2005-12-31 至 2012-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/22811434?language=en
• 关键词: Development; sensitive; unbiased; integration; site

Gene therapy with insertional vector systems has been coming of age, achieving unprecedented levels of efficiency, success, but unfortunately also side effects. Our recent preclinical and clinical gene therapy studies have revealed that insertional mutagenesis is not rare, but may lead to frequent gene activation in hematopoiesis, producing insertion-activated expansion of cell clones, and in its worst cases has resulted in oncogenesis. We and others have demonstrated that redesigning integrating vectors might diminish or avoid unwanted insertional gene activation, but even with such vectors, any integration by definition remains mutagenic, and poses a finite biosafety risk. Therefore, the quest for cellular gene delivery has rendered integrase deficient vectors particularly attractive, because they maintain the ease of use, effectiveness and lack of acute toxicity of retrovirus and lentivirus vectors but potentially minimize the likelihood of severe side effects. Transduction by integrase deficient lentiviral vectors has been attributed to episomal DNA circles and was able to correct clinically relevant rodent in vivo disease models. Towards therapeutic applications in quiescent or less frequently dividing cells in retina, heart and muscle, we aim to characterize and quantify nuclear episomal vector forms, and characterize residual integrations by statistically relevant large-scale mapping analysis. Integrase deficient vectors dilute out1 transgene expression in dividing cells, thus becoming an ideal system to study the influence of particular transcription factors such as MDS1/EVI1on the proliferation and differentiation of early repopulating bone marrow cells. To combine non-toxic nucleic acid transfer of lentivirus particles with the preferable insertion and capacity of transposases, we will generate and characterize the potential biological advantages of a stably integrating combined vector system.

插入载体系统的基因治疗已经成熟，取得了前所未有的效率和成功，但不幸的是也有副作用。我们最近的临床前和临床基因治疗研究表明，插入突变并不少见，但可能会导致造血系统中频繁的基因激活，产生插入激活的细胞克隆扩张，在最糟糕的情况下会导致肿瘤发生。我们和其他人已经证明，重新设计整合载体可能会减少或避免不必要的插入基因激活，但即使使用这样的载体，任何整合按照定义仍然是突变的，并构成有限的生物安全风险。因此，对细胞基因传递的探索使得整合酶缺陷载体特别有吸引力，因为它们保持了逆转录病毒和慢病毒载体的易用性、有效性和急性毒性，但潜在地将严重副作用的可能性降至最低。整合酶缺陷慢病毒载体的转导已被归因于DNA环上的外体，并能够纠正临床相关的啮齿动物体内疾病模型。对于视网膜、心脏和肌肉中静止或较少分裂的细胞的治疗应用，我们的目标是表征和量化核上体载体形式，并通过统计相关的大规模图谱分析来表征残留整合。整合酶缺陷载体稀释了分裂细胞中Out1基因的表达，成为研究MDS1/EVI1等特定转录因子对早期再生骨髓细胞增殖和分化影响的理想系统。为了将慢病毒颗粒的无毒核酸转移与转座酶的良好插入和能力相结合，我们将产生并鉴定稳定整合的组合载体系统的潜在生物学优势。