Engineering inter-promoter spacing in baculovirus dual-expression systems enhances transcription and reduces rAAV2...

Global delivery of gene therapies demands scalable manufacturing, yet the field is limited by the genome-deficient empty capsids. While the baculovirus expression vector system (BEVS) serves as a leading platform for industrial scale-up, its packaging efficiency is frequently compromised. This failure is largely driven by an imbalanced expression of Rep and Cap proteins. Here, we showed that the conventional back-to-back arrangement of p10 and polh promoters induced transcriptional interference, which suppressed viral protein stoichiometry and limited genome encapsulation. Using systematic dual-fluorescence reporter system, we discover an unexpected non-linear response, in which specific spacer lengths (133-161 bp) paradoxically enhance bidirectional transcription rather than simply relieving transcription repression. Nucleotide-resolution mapping identified a sharp structural optimum at 153 bp that triggered transcriptional enhancement beyond separated promoter controls. Integrating this 153 bp configuration into functional rAAV2 packaging systems restored Rep and Cap expression. Consequently, genome replication efficiency tripled, and the accumulation of empty capsids dropped precipitously from 84.9% to 20.1%, while transduction efficiency in mammalian cells remained uncompromised. These findings define a design rule for dual-promoter expression vector that is readily implementable in rAAV manufacturing. This work establishes a translatable engineering strategy to enhance both product quality and manufacturing efficiency for rAAV-based gene therapies.