In vitro NIH3T3 mouse embryonic fibroblast cell model does not predict AAV2 or AAVdj-mediated cell transformation.

IF 3.3 3区医学 Q2 PHARMACOLOGY & PHARMACY

Toxicology and applied pharmacology Pub Date : 2025-01-12 DOI:10.1016/j.taap.2025.117229

Luping Qiu, Steven W Kumpf, Elias M Oziolor, Mark Sheehan, James E Finley, David M Rubitski, Jessie Qian, Mark M Gosink, Anna K Kopec, Thomas A Lanz, Andrew D Burdick

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引用次数: 0

Abstract

One of the potential risk factors of recombinant adeno-associated virus (rAAV)-based gene therapy is insertional mutagenesis, which has been associated with the development of hepatocellular carcinoma (HCC) in rAAV-treated neonatal mice. The objective of this study was to investigate if well-established in vitro cell transformation assays (CTA) in mouse cell lines can detect AAV2 or AAVdj-mediated cell transformation. Since AAV integration at the Rian locus in neonatal mice has been implicated in AAV-mediated HCC, an rAAV vector specifically targeting the mouse Rian locus and an additional rAAV vector previously shown to cause HCC in neonatal mice were both tested for the induction of cell transformation in NIH3T3 cells. To increase the frequency of AAV DNA integration at the Rian locus in the genome of NIH3T3 cells, double-strand breaks in Rian locus of NIH3T3 cells were created by CRISPR-Cas9 to increase the homologous crossover between viral DNA and the cell genome. When transduced cells were assayed in CTA, the transformation frequency observed in AAV-transduced NIH3T3 cells was not significantly different from that of untreated vehicle cells. The finding that rAAV is unable to transform the NIH3T3 in vitro indicates that either the transformation rate is less than the spontaneous rate of NIH3T3 cellular transformation, or in vitro CTA are not predictive of rAAV-induced HCC in mice.

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体外NIH3T3小鼠胚胎成纤维细胞模型不能预测AAV2或aavdj介导的细胞转化。

重组腺相关病毒（rAAV）基因治疗的潜在危险因素之一是插入性突变，这与rAAV治疗的新生小鼠肝细胞癌（HCC）的发生有关。本研究的目的是研究在小鼠细胞系中建立的体外细胞转化试验（CTA）是否可以检测AAV2或aavj介导的细胞转化。由于AAV在新生小鼠Rian基因座的整合与AAV介导的HCC有关，因此，专门针对小鼠Rian基因座的rAAV载体和先前显示在新生小鼠中引起HCC的另一个rAAV载体都被用于诱导NIH3T3细胞的细胞转化。为了提高NIH3T3细胞基因组Rian位点上AAV DNA整合的频率，利用CRISPR-Cas9在NIH3T3细胞的Rian位点上制造双链断裂，增加病毒DNA与细胞基因组的同源交叉。当在CTA中检测转导细胞时，观察到aav转导的NIH3T3细胞的转化频率与未处理的载体细胞无显著差异。rAAV在体外不能转化NIH3T3，说明其转化速率小于NIH3T3细胞自发转化速率，或者体外CTA不能预测rAAV诱导小鼠肝癌的发生。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Toxicology and applied pharmacology 医学-毒理学

CiteScore

6.80

自引率

2.60%

发文量

309

审稿时长

32 days

期刊介绍： Toxicology and Applied Pharmacology publishes original scientific research of relevance to animals or humans pertaining to the action of chemicals, drugs, or chemically-defined natural products. Regular articles address mechanistic approaches to physiological, pharmacologic, biochemical, cellular, or molecular understanding of toxicologic/pathologic lesions and to methods used to describe these responses. Safety Science articles address outstanding state-of-the-art preclinical and human translational characterization of drug and chemical safety employing cutting-edge science. Highly significant Regulatory Safety Science articles will also be considered in this category. Papers concerned with alternatives to the use of experimental animals are encouraged. Short articles report on high impact studies of broad interest to readers of TAAP that would benefit from rapid publication. These articles should contain no more than a combined total of four figures and tables. Authors should include in their cover letter the justification for consideration of their manuscript as a short article.