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Researchers analyzed liver tissue from a patient treated with Zolgensma gene therapy and found significant contamination from manufacturing plasmid sequences, specifically REP/CAP genes comprising 0.5-1% of the therapeutic transgene levels. Through detailed sequencing analysis, they identified that these contaminants arise when the AAV P5 promoter is positioned directly downstream of the capsid gene in manufacturing plasmids, allowing Rep-mediated incorporation at specific DNA binding and cutting sites. The findings reveal a specific, preventable mechanism of contamination that depends on plasmid design choices during manufacturing.
Why it matters
This discovery identifies a modifiable factor in gene therapy manufacturing that could improve product purity and reduce unwanted DNA transfer to patients. The findings provide actionable guidance for manufacturers to redesign plasmids and potentially enhance the safety profile of AAV-based gene therapies like Zolgensma and similar products.
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⚠️ Preprint – Noch nicht peer-reviewed
Dieser Artikel wurde noch nicht von unabhängigen Experten begutachtet. Die Ergebnisse sind vorläufig und sollten mit Vorsicht interpretiert werden.
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.