Molecular design of controllable recombinant adeno-associated virus (AAV) expression systems for enhanced vector production.

ABSTRACT

Recombinant adeno-associated virus (rAAV) is the leading vector for the delivery of gene therapies. However, low viral genome (VG) titers are common and the proportion of "full" capsids containing the therapeutic gene payload can be highly variable. The coordinated molecular design of plasmids encoding viral components and Helper functions remains a major challenge for rAAV manufacturing. Here we present the design of improved Rep/Cap and Helper plasmids for rAAV2/8 production, (i) a Rep/Cap expression vector harboring independently controllable rep and cap genes and (ii) an improved Helper plasmid harboring E4 gene deletion variants. First, an optimized Rep/Cap vector utilized a truncated p5 promoter, a p5 cis-regulatory element at the 3' end in combination with a heterologous promoter to drive Cap expression and an additional copy of the rep52/40 gene to overexpress short Rep proteins. We demonstrate that Rep78 is essential for efficient rAAV2/8 production in HEK293 cells, and a higher ratio of short Rep to long Rep proteins enhances genome packaging. Second, we identified regulators and open reading frames within the Helper plasmid that contribute to increased rAAV2/8 production. While L4-33k/22k is integral to optimal production, the use of E4orf6-6/7 subset significantly enhanced VG titer. Together, an optimal combination of engineered Rep/Cap and Helper plasmid variants increased VG titer by 3.1-fold. This study demonstrates that configuring and controlling the expression of the different AAV genetic elements contributes toward high rAAV production and product quality (full/empty capsid ratio).