Rational engineering of the P5 TRS-mimic site and REP78/68 start codon yields promoter variants that improve rAAV purity while maintaining high titers.

During recombinant adeno-associated virus (rAAV) production, certain components of the manufacturing system can be encapsidated as unwanted nucleic acid contaminants. Prior work has established that the p5 promoter is critical for efficient vector production and is responsible for a significant portion of this aberrant cross-packaging. The Rep binding element (RBE) and terminal resolution site (TRS)-mimic loop on p5 putatively facilitate off-target packaging of DNA directly adjacent to p5 into rAAV particles. To prevent this, we replaced AAV2 p5 with homologues from several closely related AAV serotypes. All homologues tested that maintained vector production efficiency continued to package p5-adjacent sequences. However, specific mutations of the TRS-mimic site prevented contaminant incorporation but reduced expression of p5-derived Rep proteins and overall production efficiency. When Rep78/68 isoform expression was restored, these new TRS-modified p5 plasmids enabled rAAV production at comparable titers, with significantly reduced p5-associated contaminants, irrespective of scale and serotype. P5-associated contaminants were also observed in the context of rAAV production using covalently closed linear DNA, for which the TRS-mimic modification also significantly reduced DNA contamination while maintaining vector titers. Our findings have implications for efficiently producing rAAVs with increased purity for gene therapy.