Enhanced AAV production via rational design of a novel pHelper vector integrated with HSV-1 helper genes

Adeno-associated virus (AAV) vectors are widely used in gene therapy owing to their safety, stability, and broad tissue tropism. However, current plasmid-based AAV manufacturing platforms suffer from low yield and high manufacturing cost, limiting their scalability for clinical and commercial applications. Rational engineering of pHelper vector offers an effective strategy to enhance AAV production. In this study, we engineered a novel helper vector (UL12-ICP22-miniHelper) by integrating <i>UL12</i> and <i>ICP22</i> genes from herpes simplex virus type 1 (HSV-1) into a size-reduced pHelper backbone (mini-pHelper) with partial deletion of <i>E2a</i> and <i>E4</i> regions. In a triple-plasmid transfection system, UL12-ICP22-miniHelper increased AAV5 vector yield from 1.35 × 10¹¹ to 2.85 × 10¹¹ vg/mL (2.11-fold) without altering the proportion of full capsids. Enhanced productivity was also observed across multiple serotypes, with increases of 2.24-fold for AAV1, 1.54-fold for AAV2, 1.88-fold for AAV6, and 2.03-fold for AAV9, while maintaining transduction efficiency. Mechanistic analysis indicated that the improved productivity was associated with elevated viral genome replication and increased Rep/Cap protein expression. Collectively, these results demonstrate that the novel UL12-ICP22-miniHelper provides a broadly applicable and cost-effective strategy for improving AAV vector manufacturing in both clinical and industrial applications.