静脉注射和肌肉注射含有阿尔法疱疹病毒潜伏期相关启动子2的AAV载体一年后,外周组织中转基因的持续表达

IF 2 Q4 VIROLOGY Frontiers in virology Pub Date : 2024-03-18 DOI:10.3389/fviro.2024.1379991
Carola J. Maturana, Esteban A. Engel
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引用次数: 0

摘要

在加强重组腺相关病毒(rAAV)的临床研究方面取得了重大进展。尽管重组腺相关病毒(rAAV)是一种多功能基因递送平台,但其固有的 4.7 kb 封装限制了它所能治疗的疾病范围。在这种情况下,我们发现了一种异常紧凑的长期启动子,与传统启动子相比,它能促进较大基因的表达。这种紧凑型启动子源自阿尔法疱疹病毒伪狂犬病毒的基因组--潜伏期相关启动子 2(LAP2,404 bp)。将驱动 mCherry 报告基因的启动子打包到单链(ss)AAV8 和 AAV9 载体中,通过单次静脉注射或肌肉注射,以 5 x 1011 vg/mouse 的剂量注射到成年 C57BL/6 小鼠体内。为了进行比较,我们同时注射了带有伸长因子-1α启动子(EF1α,1264 bp)的ssAAV8和ssAAV9载体。400 天后,我们将小鼠处死,并检测了小鼠肝脏、肾脏、心脏、肺脏、脾脏、胰腺、骨骼肌和大脑中 mCherry 的表达。我们发现,LAP2 在广泛的细胞和组织中表现出强大的转基因表达能力,可与较大的 EF1α 相媲美。两种给药途径的 AAV8-LAP2 和 AAV9-LAP2 构建体在肝、肾和骨骼肌中都表现出很强的转导和转录能力。然而,在心脏、肺、脾脏、胰腺和大脑中均未检测到表达。我们的研究结果证明了 LAP2 在基因治疗应用中的可行性,因为基因治疗需要在单次病毒载体给药后表达大量或多个治疗基因。
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Persistent transgene expression in peripheral tissues one year post intravenous and intramuscular administration of AAV vectors containing the alphaherpesvirus latency-associated promoter 2

Significant progress has been made in enhancing recombinant adeno-associated virus (rAAV) for clinical investigation. Despite its versatility as a gene delivery platform, the inherent packaging constraint of 4.7 kb imposes restrictions on the range of diseases it can address. In this context, we present findings of an exceptionally compact and long-term promoter that facilitates the expression of larger genes compared to conventional promoters. This compact promoter originated from the genome of the alphaherpesvirus pseudorabies virus, latency-associated promoter 2 (LAP2, 404 bp). Promoter driving an mCherry reporter was packaged into single strand (ss) AAV8 and AAV9 vectors and injected into adult C57BL/6 mice at a dose of 5 x 1011 vg/mouse by single intravenous or intramuscular administration. An ssAAV8 and ssAAV9 vector with elongation factor-1α promoter (EF1α, 1264 bp) was injected side-by-side for comparison. After 400 days, we sacrificed the mice and examined mCherry expression in liver, kidney, heart, lung, spleen, pancreas, skeletal muscle, and brain. We found that LAP2 exhibited robust transgene expression across a wide range of cells and tissues comparable to the larger EF1α, which is currently recognized as a rather potent and ubiquitous promoter. The AAV8-LAP2 and AAV9-LAP2 constructs displayed strong transduction and transcription in liver, kidney, and skeletal muscle on both route of administration. However, no expression was detected in the heart, lung, spleen, pancreas, and brain. The outcomes of our investigation propose the viability of LAP2 for gene therapy applications demanding the expression of large or multiple therapeutic genes following a single viral-vector administration.

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