溶瘤病毒（OV）通过其复制作用和随后肿瘤细胞的溶解促进抗肿瘤免疫反应，从而触发免疫感知途径的激活。在表达促进免疫细胞招募和激活的转基因的情况下，武装OV是增强其治疗效益的一种有吸引力的策略。对于具有临床经验的一类OV——小肠病毒科病毒，由于基因组物理包装的限制、高重组率以及病毒介导的转基因分泌抑制，转基因的表达受到了多方面的限制。在这里，我们评估了武装塞内卡谷病毒（SVV）具有相关免疫调节转基因的策略。具体而言，在武装SVV的情况下，我们评估了转基因的最大尺寸和稳定性、转基因的分泌以及转基因包含对病毒健康状况的影响。我们发现SVV无法表达分泌的有效负载，其转基因包装能力仅为病毒基因组大小的约10%。为了实现转基因的表达，我们开发了SVV复制体，具有更大的转基因尺寸容量和分泌能力。SVV复制体可以通过共感染细胞中的病毒在转录中被包装，在周围细胞中表达免疫调节转基因，从而提供了一种增强这种治疗增加抗肿瘤免疫反应潜力的手段。© 2023 The Author(s).

Oncolytic viruses (OVs) promote the anti-tumor immune response as their replication, and the subsequent lysis of tumor cells, triggers the activation of immune-sensing pathways. Arming OVs by expressing transgenes with the potential to promote immune cell recruitment and activation is an attractive strategy to enhance OVs' therapeutic benefit. For picornaviruses, a family of OVs with clinical experience, the expression of a transgene is limited by multiple factors: genome physical packaging limits, high rates of recombination, and viral-mediated inhibition of transgene secretion. Here, we evaluated strategies for arming Seneca Valley virus (SVV) with relevant immunomodulatory transgenes. Specificially in the contex of arming SVV, we evaluated transgene maximum size and stabiltity, transgene secretion, and the impact of transgene inclusion on viral fitness. We find that SVV is not capable of expressing secreted payloads and has a transgene packaging capacity of ∼10% of viral genome size. To enable transgene expression, we developed SVV replicons with greater transgene size capacity and secretion capabilities. SVV replicons can be packaged in trans by virus in co-infected cells to express immunomodulatory transgenes in surrounding cells, thus providing a means to enhance the potential of this therapeutic to augment the anti-tumor immune response.© 2023 The Author(s).