The evolving landscape of recombinant adeno-associated virus (rAAV) manufacturing: Engineering vector plasmids for upstream production

Adeno-associated virus (AAV) vectors have emerged as one of the most versatile and clinically validated platforms for in vivo gene delivery, owing to their safety profile, broad tissue tropism, and capacity for long-term transgene expression. However, the high vector doses required for systemic administration, together with the rapid expansion of clinical pipelines, have outpaced current manufacturing capacities, exposing a critical bottleneck in upstream recombinant AAV (rAAV) production. This challenge is particularly acute for the field's gold-standard platform, the HEK293 transient transfection system; despite its dominance, its inherent stoichiometric and scalability limitations underscore the need for a deeper molecular and genetic engineering-driven understanding of the system. To address this, the present review examines the core molecular-level engineering principles essential to transcending these productivity limits within the upstream bioprocess framework. We discuss advances in the molecular design of production plasmids, including rep, cap, ITR, and helper gene optimization; the evolution of plasmid architecture through consolidation of multi-plasmid systems (from three to dual and single configurations); and recent progress in developing inducible, site-specifically integrated, and suspension-adapted packaging and producer cell lines. Together, these genetic and cell-line engineering strategies illustrate how genetic elements, plasmid system structure, and cell-line platform design collectively shape vector productivity, quality attributes, and scalability. By integrating these dimensions into a coherent upstream manufacturing perspective, the review highlights persistent technical challenges and outlines strategic directions for next-generation, high-yield, industrial-scale rAAV production platforms.