hInGeTox: a human-based in vitro platform to evaluate lentivirus/host interactions that contribute to genotoxicity.

IF 4.5 3区 医学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Saqlain Suleman, Sharmin Alhaque, Andrew Guo, Huairen Zhang, Annette Payne, Marco Zahn, Serena Fawaz, Mohammad S Khalifa, Susan Jobling, David Hay, Matteo Franco, Raffaele Fronza, Wei Wang, Olga Strobel-Freidekind, Annette Deichmann, Yasuhiro Takeuchi, Irene Gil-Farina, Jan Klapwijk, Stefany Perera, Manfred Schmidt, Michael Themis
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引用次数: 0

Abstract

Lentivirus vectors are effective for treatment of genetic disease. However, safety associated with vector related genotoxicity is of concern and currently available models are not reliably predictive of safety in humans. We have developed hInGeTox as the first human in vitro platform that uses induced pluripotent stem cells and their hepatocyte like cell derivatives to better understand vector-host interactions that relate vectors to their potential genotoxicity. Using lentiviral vectors carrying the eGFP expression cassette under SFFV promoter activity, that only differ by their LTR and SIN configuration, we characterised vector host interactions potentially implicated in genotoxicity. To do this, lentiviral infected cells were subjected to an array of assays and data from these was used for multi-omics analyses of vector effects on cells at early and late harvest time points. Data on the integration sites of lentiviral vectors in cancer genes and differential expression levels of these genes, showed that both vector configurations are capable of activating cancer genes. Through IS tracking in bulk infected cell populations, we also saw an increase in the viral sequence count in cancer genes present over time which were differentially regulated. RNASeq also showed each vector had potential to generate fusion transcripts with the human genome suggestive of gene splicing or vector mediated readthrough from the internal SFFV promoter. Initially, after infection, both vector configurations were associated with differential expression of genes associated cytokine production, however, after culturing over time there were differences in differential expression in cells infected by each LV. This was marked in particular by the expression of genes involved in the response to DNA damage in cells transduced by the SIN vector, suggesting effects likely to prevent tumour development, in contrast to the expression of genes involved in methylation, characteristic of tumour development, in cells transduced by the LTR vector. Both sets of lentiviral infected cells were also found associated with differential expression of MECOM and LMO2 genes known to be associated with clonal dominance, supporting their potential genotoxicity. Alignment of transcriptomic signatures from iPSC and HLC infected cultures with known cancer gene signatures showed the LTR vector with a higher cancer score than the SIN vector over time in iPSC and also in HLC, which further suggests higher genotoxic potential by the LTR configuration lentivirus. By application of hInGeTox to cells infected with LV at the pre-clinical stage of development, we hope that hInGeTox can act as a useful pre-clinical tool to identify lentivirus-host interactions that may be considered contributory to genotoxicity to improve safer lentiviral vector design for gene therapy.

hInGeTox:一个基于人的体外平台,用于评估慢病毒/宿主相互作用对遗传毒性的影响。
慢病毒载体对治疗遗传病是有效的。然而,与媒介相关的遗传毒性相关的安全性令人担忧,目前可用的模型不能可靠地预测人类的安全性。我们已经开发了hInGeTox作为第一个人类体外平台,使用诱导多能干细胞及其肝细胞样细胞衍生物来更好地了解与载体及其潜在遗传毒性相关的载体-宿主相互作用。利用SFFV启动子活性下携带eGFP表达盒的慢病毒载体,仅区别于它们的LTR和SIN配置,我们表征了可能涉及遗传毒性的载体与宿主的相互作用。为此,对慢病毒感染的细胞进行了一系列检测,并将这些数据用于多组学分析,分析在收获早期和后期的时间点上载体对细胞的影响。慢病毒载体在肿瘤基因中的整合位点和这些基因的差异表达水平的数据表明,两种载体构型都能够激活肿瘤基因。通过对大量感染细胞群的IS跟踪,我们还看到随着时间的推移,存在差异调节的癌症基因中的病毒序列计数有所增加。RNASeq还显示,每个载体都有可能与人类基因组产生融合转录物,这表明基因剪接或载体介导的SFFV内部启动子的读取。最初,在感染后,两种载体构型都与细胞因子产生相关基因的差异表达相关,然而,经过一段时间的培养,每种LV感染的细胞中差异表达存在差异。这在由SIN载体转导的细胞中参与DNA损伤反应的基因表达中得到了特别的标记,这表明,与LTR载体转导的细胞中参与甲基化(肿瘤发展的特征)的基因表达相反,这种作用可能会阻止肿瘤的发展。两组慢病毒感染的细胞也被发现与MECOM和LMO2基因的差异表达相关,这些基因已知与克隆优势相关,支持其潜在的遗传毒性。对iPSC和HLC感染培养物中已知癌症基因特征的转录组特征进行比对显示,随着时间的推移,LTR载体在iPSC和HLC中的癌症评分高于SIN载体,这进一步表明LTR结构慢病毒具有更高的遗传毒性潜力。通过将hInGeTox应用于LV感染细胞的临床前开发阶段,我们希望hInGeTox可以作为一种有用的临床前工具,用于鉴定可能有助于遗传毒性的慢病毒-宿主相互作用,从而改进更安全的慢病毒载体设计,用于基因治疗。
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来源期刊
Gene Therapy
Gene Therapy 医学-生化与分子生物学
CiteScore
9.70
自引率
2.00%
发文量
67
审稿时长
4-8 weeks
期刊介绍: Gene Therapy covers both the research and clinical applications of novel therapeutic techniques based on a genetic component. Over the last few decades, significant advances in technologies ranging from identifying novel genetic targets that cause disease through to clinical studies, which show therapeutic benefit, have elevated this multidisciplinary field to the forefront of modern medicine.
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