Gene Therapy Research Unit, Children's Medical Research Institute, Faculty of Medicine and Health, The University of Sydney and Sydney Children's Hospitals Network, Westmead, NSW, Australia.
Tessera Therapeutics, Inc., Somerville, MA, USA.
J Gene Med. 2024 Aug;26(8):e3726. doi: 10.1002/jgm.3726.
Conventional adeno-associated viral (AAV) vectors, while highly effective in quiescent cells such as hepatocytes in the adult liver, confer less durable transgene expression in proliferating cells owing to episome loss. Sustained therapeutic success is therefore less likely in liver disorders requiring early intervention. We have previously developed a hybrid, dual virion approach, recombinant AAV (rAAV)/piggyBac transposon system capable of achieving stable gene transfer in proliferating hepatocytes at levels many fold above conventional AAV vectors. An alternative transposon system, Sleeping Beauty, has been widely used for ex vivo gene delivery; however liver-targeted delivery using a hybrid rAAV/Sleeping Beauty approach remains relatively unexplored.
We investigated the capacity of a Sleeping Beauty (SB)-based dual rAAV virion approach to achieve stable and efficient gene transfer to the newborn murine liver using transposable therapeutic cassettes encoding coagulation factor IX or ornithine transcarbamylase (OTC).
At equivalent doses, rAAV/SB100X transduced hepatocytes with high efficiency, achieving stable expression into adulthood. Compared with conventional AAV, the proportion of hepatocytes transduced, and factor IX and OTC activity levels, were both markedly increased. The proportion of hepatocytes stably transduced increased 4- to 8-fold from <5%, and activity levels increased correspondingly, with markedly increased survival and stable urinary orotate levels in the OTC-deficient Spf mouse following elimination of residual endogenous murine OTC.
The present study demonstrates the first in vivo utility of a hybrid rAAV/SB100X transposon system to achieve stable long-term therapeutic gene expression following delivery to the highly proliferative newborn mouse liver. These results have relevance to the treatment of genetic metabolic liver diseases with neonatal onset.
传统的腺相关病毒（AAV）载体在静止细胞（如成年肝脏中的肝细胞）中非常有效，但由于外显子丢失，在增殖细胞中赋予的转基因表达持续时间较短。因此，在需要早期干预的肝脏疾病中，持续的治疗成功可能性较小。我们之前开发了一种混合的、双病毒体方法，即重组腺相关病毒（rAAV）/piggyBac 转座子系统，该系统能够在增殖的肝细胞中实现稳定的基因转移，其水平比传统的 AAV 载体高出许多倍。另一种转座子系统，Sleeping Beauty，已被广泛用于体外基因传递；然而，使用混合 rAAV/Sleeping Beauty 方法进行肝靶向传递仍相对未被探索。
我们研究了基于 Sleeping Beauty（SB）的双 rAAV 病毒体方法，使用编码凝血因子 IX 或鸟氨酸转氨甲酰酶（OTC）的可转座治疗盒，实现对新生小鼠肝脏的稳定和高效基因转移的能力。
在等效剂量下，rAAV/SB100X 以高效率转导肝细胞，实现成年后的稳定表达。与传统的 AAV 相比，转导的肝细胞比例以及因子 IX 和 OTC 活性水平都显著增加。稳定转导的肝细胞比例从<5%增加了 4-8 倍，活性水平相应增加，在消除残余内源性小鼠 OTC 后，OTC 缺陷 Spf 小鼠的生存能力和稳定的尿尿酸水平得到显著提高。
本研究首次证明了混合 rAAV/SB100X 转座子系统在向高度增殖的新生小鼠肝脏递送后实现稳定的长期治疗性基因表达的体内应用。这些结果与新生儿发病的遗传代谢性肝脏疾病的治疗有关。
