Abstract：
Recombinant adeno-associated virus (rAAV) vector gene delivery systems have demonstrated great promise in clinical trials but continue to face durability and dose-related challenges. Unlike rAAV gene therapy, integrating gene addition approaches can provide curative expression in mitotically active cells and pediatric populations. We explored a novel in vivo delivery approach based on an engineered transposase, Sleeping Beauty (SB100X), delivered as an mRNA within a lipid nanoparticle (LNP), in combination with an rAAV-delivered transposable transgene. This combinatorial approach achieved correction of ornithine transcarbamylase deficiency in the neonatal Spfash mouse model following a single delivery to dividing hepatocytes in the newborn liver. Correction remained stable into adulthood, while a conventional rAAV approach resulted in a return to the disease state. In non-human primates, integration by transposition, mediated by this technology, improved gene expression 10-fold over conventional rAAV-mediated gene transfer while requiring 5-fold less vector. Additionally, integration site analysis confirmed a random profile while specifically targeting TA dinucleotides across the genome. Together, these findings demonstrate that transposable elements can improve rAAV-delivered therapies by lowering the vector dose requirement and associated toxicity while expanding target cell types.
摘要：
重组腺相关病毒(rAAV)载体基因递送系统已在临床试验中显示出巨大的前景,但继续面临耐久性和剂量相关的挑战。与rAAV基因治疗不同,整合基因添加方法可以在有丝分裂活性细胞和儿科人群中提供治愈性表达。我们探索了一种基于工程化转座酶的新型体内递送方法，睡美人(SB100X)在脂质纳米颗粒（LNP）内作为mRNA递送，与rAAV递送的可转座转基因组合。这种组合方法在单次递送至新生肝脏中的分裂肝细胞后,在新生Spfash小鼠模型中实现了鸟氨酸转碳淀粉酶缺乏的校正。矫正一直稳定到成年，而传统的rAAV方法导致疾病状态的恢复。在非人灵长类动物中，通过换位整合，通过这项技术，与常规rAAV介导的基因转移相比，基因表达提高了10倍，同时需要5倍的载体。此外,整合位点分析证实了随机谱,同时特异性靶向跨基因组的TA二核苷酸.一起,这些发现表明,转座因子可以通过降低载体剂量需求和相关毒性,同时扩增靶细胞类型来改善rAAV递送疗法.
