通过基因衣壳标记对溶瘤腺病毒进行体内动态监测。

Dynamic monitoring of oncolytic adenovirus in vivo by genetic capsid labeling.

作者信息

Le Long P, Le Helen N, Dmitriev Igor P, Davydova Julia G, Gavrikova Tatyana, Yamamoto Seiji, Curiel David T, Yamamoto Masato

机构信息

Division of Human Gene Therapy, Department of Medicine, University of Alabama, Birmingham, AL, USA.

出版信息

J Natl Cancer Inst. 2006 Feb 1;98(3):203-14. doi: 10.1093/jnci/djj022.

DOI:10.1093/jnci/djj022

PMID:16449680

Abstract

BACKGROUND

Conditionally replicative adenoviruses represent a promising strategy to address the limited efficacy and safety issues associated with conventional cancer treatment. Despite rapid translation into human clinical trials and demonstrated safety, the fundamental properties of oncolytic adenovirus replication and spread and host-vector interactions in vivo have not been completely evaluated.

METHODS

We developed a noninvasive dynamic monitoring system to detect adenovirus replication. We constructed capsid-labeled E1/E3-deleted and wild-type adenoviruses (Ad-wt) by fusing the minor capsid protein IX with red fluorescent proteins mRFP1 and tdimer2(12), resulting in Ad-IX-mRFP1, Ad-IX-tdimer2(12), and Ad-wt-IX-mRFP1. Virus DNA replication, encapsidation, cytopathic effect, thermostability, and binding to primary receptor (coxsackie adenovirus receptor) were analyzed using real-time quantitative polymerase chain reaction, cell viability (MTS) assay, and fluorescence microscopy. Athymic mice (n = 4) carrying xenograft tumors that were derived from A549 lung adenocarcinoma cells were intratumorally inoculated with Ad-wt-IX-mRFP1, and adenovirus replication was dynamically monitored with a fluorescence noninvasive imaging system. Correlations between fluorescence signal intensity and viral DNA synthesis and replication were calculated using Pearson's correlation coefficient (r).

RESULTS

The red fluorescence label had little effect on viral DNA replication, encapsidation, cytopathic effect, thermostability, and coxsackie adenovirus receptor binding. The fluorescent signal correlated with viral DNA synthesis and infectious progeny production both in vitro and in vivo (in A549 cells, r = .99 and r = .65; in tumors, r = .93 and r = .92, respectively). The replication efficiency of Ad-wt-IX-mRFP1 in vivo was variable, and replication and viral spreading and persistence were limited, consistent with clinical observations.

CONCLUSIONS

Genetic capsid labeling provides a promising approach for the dynamic assessment of oncolytic adenovirus function in vivo.

摘要

背景

条件性复制腺病毒是一种很有前景的策略，可解决与传统癌症治疗相关的疗效有限和安全性问题。尽管已迅速进入人体临床试验并证明了安全性，但溶瘤腺病毒在体内的复制、传播以及宿主与载体相互作用的基本特性尚未得到全面评估。

方法

我们开发了一种用于检测腺病毒复制的非侵入性动态监测系统。通过将次要衣壳蛋白IX与红色荧光蛋白mRFP1和tdimer2(12)融合，构建了衣壳标记的E1/E3缺失型和野生型腺病毒（Ad-wt），分别得到Ad-IX-mRFP1、Ad-IX-tdimer2(12)和Ad-wt-IX-mRFP1。使用实时定量聚合酶链反应、细胞活力（MTS）测定法和荧光显微镜分析病毒DNA复制、衣壳化、细胞病变效应、热稳定性以及与主要受体（柯萨奇腺病毒受体）的结合。对携带源自A549肺腺癌细胞的异种移植肿瘤的无胸腺小鼠（n = 4）进行瘤内接种Ad-wt-IX-mRFP1，并使用荧光非侵入性成像系统动态监测腺病毒复制。使用Pearson相关系数（r）计算荧光信号强度与病毒DNA合成及复制之间的相关性。

结果

红色荧光标记对病毒DNA复制、衣壳化、细胞病变效应、热稳定性以及柯萨奇腺病毒受体结合的影响很小。荧光信号在体外和体内（在A549细胞中，r分别为0.99和0.65；在肿瘤中，r分别为0.93和0.92）均与病毒DNA合成和感染性子代产生相关。Ad-wt-IX-mRFP1在体内的复制效率各不相同，复制以及病毒传播和持久性均有限，这与临床观察结果一致。