High-titer modular retroviral vectors enabled by an antisense cassette design preventing dsRNA formation during virus production.

Retroviral vectors have demonstrated durable safety and therapeutic benefit in the context of cell and gene therapies, but achieving modular, independently controlled gene expression remains a major design challenge. Although antisense cassette architectures enable such regulation, yet they are limited by sharply reduced viral titers caused by double-stranded RNA (dsRNA) formation from convergent transcription during virus production. We developed an antisense cassette strategy that prevents dsRNA generation and preserves high titers. The design incorporates a sense-oriented intron containing an antisense polyadenylation signal into the transfer plasmid, enforcing premature termination of antisense transcription. This intron is subsequently spliced out of the packaged genomic RNA, thereby preventing inverted-promoter-driven dsRNA formation. The approach is compatible with both γ-retroviral and lentiviral vectors, supports diverse promoter combinations, and accommodates external introns as well as microRNA-based short hairpin RNAs for concurrent gene knockdown. As proof of concept, we engineered a dual-cassette vector containing an activation-inducible module paired with a constitutive transgene cassette, achieving robust modular expression alongside high titers. These findings establish a versatile platform enabling independent regulation of multiple genes in retroviral vectors and support the advancement of both basic research and therapeutic applications.