Discovery Biologics, Merck & Co., Inc., South San Francisco, California, USA.
Discovery Oncology, Merck & Co., Inc., South San Francisco, California, USA.
J Virol. 2023 May 31;97(5):e0030923. doi: 10.1128/jvi.00309-23. Epub 2023 Apr 18.
Coxsackievirus A21 (CVA21) is a naturally occurring RNA virus that, in preclinical studies and clinical trials, has demonstrated promising potential in treating a range of malignancies. Other oncolytic viruses, such as adenovirus, vesicular stomatitis virus, herpesvirus, and vaccinia virus, all can be engineered to carry one or more transgenes for various purposes, including immune modulation, virus attenuation, and induction of apoptosis of tumor cells. However, it remained unknown whether CVA21 can express therapeutic or immunomodulatory payloads due to its small size and high mutation rate. Using reverse genetics techniques, we demonstrated that a transgene encoding a truncated green fluorescent protein (GFP) of up to 141 amino acids (aa) can be successfully carried in the 5' end of the coding region. Furthermore, a chimeric virus carrying an eel fluorescent protein, UnaG (139 aa), was also made and shown to be stable, and it maintained efficient tumor cell-killing activity. Similar to other oncolytic viruses, the likelihood of delivering CVA21 by the intravenous route is low due to issues like blood absorption, neutralizing antibodies, and liver clearance. To address this problem, we designed the CVA21 cDNA under the control of a weak RNA polymerase II promoter, and subsequently, a stable cell pool in 293T cells was made by integrating the resulting CVA21 cDNA into the cell genome. We showed that the cells are viable and able to persistently generate rCVA21 . The carrier cell approach described here may pave the way to designing new cell therapy strategies by arming with oncolytic viruses. As a naturally occurring virus, coxsackievirus A21 is a promising oncolytic virotherapy modality. In this study, we first used reverse genetics to determine whether A21 can stably carry transgenes and found that it could express up to 141 amino acids of foreign GFP. The chimeric virus carrying another fluorescent eel protein UnaG (139 amino acids) gene also appeared to be stable over at least 7 passages. Our results provided guidance on how to select and engineer therapeutic payloads for future A21 anticancer research. Second, the challenges of delivering oncolytic viruses by the intravenous route hamper the broader use of oncolytic viruses in the clinic. Here, we used A21 to show that cells could be engineered to stably carry and persistently release the virus by harboring the viral cDNA in the genome. The approach we presented here may pave a new way for oncolytic virus administration using cells as carriers.
柯萨奇病毒 A21（CVA21）是一种天然存在的 RNA 病毒，在临床前研究和临床试验中，已显示出在治疗多种恶性肿瘤方面有很大的潜力。其他溶瘤病毒，如腺病毒、水疱性口炎病毒、疱疹病毒和牛痘病毒，都可以被工程改造以携带一个或多个用于各种目的的转基因，包括免疫调节、病毒减毒和诱导肿瘤细胞凋亡。然而，由于其体积小、突变率高，人们并不知道 CVA21 是否可以表达治疗或免疫调节有效载荷。我们使用反向遗传学技术证明，长达 141 个氨基酸（aa）的截断绿色荧光蛋白（GFP）的转基因可以成功地携带在编码区的 5'端。此外，还构建了携带鳗鲡荧光蛋白 UnaG（139aa）的嵌合病毒，该病毒也很稳定，并保持了有效的杀伤肿瘤细胞的活性。与其他溶瘤病毒一样，由于血液吸收、中和抗体和肝脏清除等问题，静脉内途径传递 CVA21 的可能性很低。为了解决这个问题，我们设计了在弱 RNA 聚合酶 II 启动子控制下的 CVA21 cDNA，随后通过将所得的 CVA21 cDNA 整合到细胞基因组中，在 293T 细胞中制造了一个稳定的细胞池。我们表明细胞是存活的，并能够持续产生 rCVA21。本文所述的载体细胞方法可能为通过武装溶瘤病毒来设计新的细胞治疗策略铺平道路。作为一种天然存在的病毒，柯萨奇病毒 A21 是一种很有前途的溶瘤病毒治疗模式。在这项研究中，我们首先使用反向遗传学来确定 A21 是否能够稳定地携带转基因，并发现它可以表达多达 141 个氨基酸的外源 GFP。携带另一种荧光鳗鲡蛋白 UnaG（139 个氨基酸）基因的嵌合病毒至少经过 7 次传代也似乎很稳定。我们的结果为如何选择和设计用于未来 A21 抗癌研究的治疗性有效载荷提供了指导。其次，通过静脉途径传递溶瘤病毒的挑战阻碍了溶瘤病毒在临床上的更广泛应用。在这里，我们使用 A21 表明，通过将病毒 cDNA 携带在基因组中，细胞可以被工程改造为稳定携带并持续释放病毒。我们提出的方法可能为使用细胞作为载体进行溶瘤病毒给药开辟了一条新途径。
