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

使用插入载体系统的基因治疗已经成熟，达到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，插入突变并不罕见，但可能导致造血过程中频繁的基因激活，产生插入激活的细胞克隆扩增，在最坏的情况下导致肿瘤发生。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，任何整合根据定义仍然是诱变的，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具有吸引力，因为它们保持了逆转录病毒和慢病毒载体的易用性、有效性和缺乏急性毒性，但潜在地将严重副作用的可能性降到最低。整合酶缺陷慢病毒载体的转导已归因于外体DNA环，并能够纠正临床相关的啮齿动物体内疾病模型。为了在视网膜、心脏和肌肉的静止或不太频繁分裂的细胞中的治疗应用，我们的目标是表征和量化核附着体载体形式，并通过统计相关的大规模制图分析表征残留整合。整合酶缺陷载体稀释了分裂细胞中1的转基因表达，因此成为研究特定转录因子如MDS1/ evi1对早期再填充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结合起来，我们将产生并表征一个稳定整合的组合载体系统的潜在生物学优势。

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.