P5 promoter-mediated incorporation explains REP/CAP manufacturing contaminants in patient liver after rAAV gene therapy.

Sequencing of liver tissue from a patient treated with the rAAV gene therapy Zolgensma for spinal muscular atrophy recently revealed contaminating plasmid sequences derived from rAAV manufacturing within the patient's hepatocytes. In particular, REP/CAP-derived sequences were remarkably abundant, corresponding to 0.5-1% of the therapeutic transgene. We hypothesized that these contaminants originated through Rep-mediated incorporation initiated at the AAV P5 promoter. Through reanalysis of the sequencing data, we inferred that an intact P5 promoter had been placed directly downstream of the rAAV capsid gene in the manufacturing plasmid. De novo assembly revealed a contiguous contaminant sequence spanning the rAAV REP and CAP genes and terminating within P5 at the Rep nicking site, immediately downstream of the Rep-binding element (RBE). This analysis also revealed a distinct vector-plasmid backbone contig consistent with reverse packaging. Among partially aligned REP/CAP reads, nearly 17% were linked to rAAV ITR-derived sequence at heterogeneous junctions. Long-read data independently identified the P5 promoter as the most frequent recombination breakpoint region. Together, these findings identify a defined, avoidable mechanism by which REP/CAP manufacturing contaminants arise. Positioning of the P5 promoter within the manufacturing plasmid is therefore a modifiable determinant of rAAV product purity and the transfer of rAAV DNA contaminants to recipient patients.