美国BioMarin制药公司Sylvia Fong研究组近日取得一项新成果。他们的研究揭示了腺病毒相关基因治疗血友病A后转基因mRNA和蛋白质产生的个体间差异。2022年4月11日出版的《自然-医学》发表了这项成果。

在1/2期临床试验 (NCT02576795) 的子研究中，收集五名参与者进行基因转移后2.6-4.1年的肝活检样本。主要目标是检查valoctocogene roxaparvovec(AAV5-hFVIII-SQ)对肝脏组织病理学的影响，确定转导模式和转导AAV5-hFVIII-SQ基因组的肝细胞百分比，表征和量化载体DNA的游离形式并量化转基因表达（hFVIII-SQ RNA hFVIII-SQ 蛋白）。组织病理学显示在表达hFVIII-SQ蛋白的肝细胞中未发现发育异常、结构变形、纤维化或慢性炎症，并且未检测到内质网应激。肝细胞对载体基因组染色呈阳性，显示出用更高剂量转导更多细胞的趋势。

分子分析表明存在与长期表达相关的全长、倒置末端重复融合、环状游离基因组。尽管都成功进行了基因转导，但仍存在转基因表达的个体间差异，可能受宿主介导的载体转录、hFVIII-SQ蛋白翻译和转导后分泌机制的影响。总体而言，这些结果表明AAV5-hFVIII-SQ在给药后持续存在附加的载体结构，并阐明了介导个体间变异性的潜在机制。

据悉，通过单次静脉输注AAV5-hFVIII-SQ实现细胞因子VIII基因转移，可使严重血友病A患者获得持续5年的临床益处。然而，尚未有临床报道AAV5-hFVIII-SQ衍生FVIII持续表达的分子机制。

附：英文原文

Title: Interindividual variability in transgene mRNA and protein production following adeno-associated virus gene therapy for hemophilia A

Author: Fong, Sylvia, Yates, Bridget, Sihn, Choong-Ryoul, Mattis, Aras N., Mitchell, Nina, Liu, Su, Russell, Chris B., Kim, Benjamin, Lawal, Adebayo, Rangarajan, Savita, Lester, Will, Bunting, Stuart, Pierce, Glenn F., Pasi, K. John, Wong, Wing Yen

Issue&Volume: 2022-04-11

Abstract: Factor VIII gene transfer with a single intravenous infusion of valoctocogene roxaparvovec (AAV5-hFVIII-SQ) has demonstrated clinical benefits lasting 5 years to date in people with severe hemophilia A. Molecular mechanisms underlying sustained AAV5-hFVIII-SQ-derived FVIII expression have not been studied in humans. In a substudy of the phase 1/2 clinical trial (NCT02576795), liver biopsy samples were collected 2.6–4.1 years after gene transfer from five participants. Primary objectives were to examine effects on liver histopathology, determine the transduction pattern and percentage of hepatocytes transduced with AAV5-hFVIII-SQ genomes, characterize and quantify episomal forms of vector DNA and quantify transgene expression (hFVIII-SQ RNA and hFVIII-SQ protein). Histopathology revealed no dysplasia, architectural distortion, fibrosis or chronic inflammation, and no endoplasmic reticulum stress was detected in hepatocytes expressing hFVIII-SQ protein. Hepatocytes stained positive for vector genomes, showing a trend for more cells transduced with higher doses. Molecular analysis demonstrated the presence of full-length, inverted terminal repeat-fused, circular episomal genomes, which are associated with long-term expression. Interindividual differences in transgene expression were noted despite similar successful transduction, possibly influenced by host-mediated post-transduction mechanisms of vector transcription, hFVIII-SQ protein translation and secretion. Overall, these results demonstrate persistent episomal vector structures following AAV5-hFVIII-SQ administration and begin to elucidate potential mechanisms mediating interindividual variability.

DOI: 10.1038/s41591-022-01751-0

Source: https://www.nature.com/articles/s41591-022-01751-0