Summary
A medRxiv preprint identifies a possible manufacturing mechanism behind REP/CAP DNA sequences found in the liver of a patient treated with the rAAV gene therapy Zolgensma. The proposed mechanism involves the placement of the AAV P5 promoter in a manufacturing plasmid.
A medRxiv preprint reports a sequence-level explanation for how DNA from recombinant adeno-associated virus (rAAV) manufacturing may have entered liver cells of a patient treated with Zolgensma for spinal muscular atrophy. Reanalysis of the patient's liver-sequencing data points to the AAV P5 promoter as a likely starting point for the incorporation of unwanted REP/CAP sequences.
The study is a retrospective sequence reanalysis of liver tissue from one patient. Its measured outcomes were the identity, abundance and structure of contaminating DNA sequences, rather than a clinical safety or efficacy endpoint.
Sequencing reveals a structured manufacturing contaminant
The researchers found that sequences derived from the rAAV REP and CAP genes accounted for approximately 0.5–1% of the therapeutic transgene in the liver sequencing data. REP and CAP are normally used during rAAV production to provide viral replication and capsid functions; they are not intended to form part of the therapeutic genetic cargo delivered to a patient.
Using de novo assembly, a method that reconstructs longer DNA sequences from overlapping sequencing fragments, the researchers identified a contiguous contaminant sequence spanning the REP and CAP genes. The sequence ended within the P5 promoter at a Rep nicking site, immediately downstream of a Rep-binding element, or RBE.
A separate assembled sequence contained vector-plasmid backbone DNA and was consistent with reverse packaging, in which DNA outside the intended vector genome becomes associated with packaged vector material. Among partially aligned REP/CAP reads, nearly 17% were connected to sequences derived from the rAAV inverted terminal repeats, or ITRs, at varied junctions. Long-read sequencing independently identified the P5 promoter as the most frequent recombination breakpoint region.
How the P5 promoter could drive incorporation
rAAV manufacturing uses plasmids to produce the vector genome and the proteins needed to assemble viral particles. The AAV P5 promoter is a regulatory DNA region associated with Rep expression. Rep proteins can recognise an RBE and cut DNA at a nearby nicking site, creating a molecular configuration that can support recombination or incorporation of adjacent sequences.
The researchers inferred from the sequencing data that an intact P5 promoter had been placed directly downstream of the rAAV capsid gene in the manufacturing plasmid. In that arrangement, Rep activity could have promoted incorporation of neighbouring REP/CAP-derived DNA into vector-related material. The matching sequence structures, the ITR-linked junctions and the long-read breakpoint pattern all support this proposed mechanism.
The authors describe the finding as an avoidable source of manufacturing contamination and identify P5 promoter placement as a modifiable determinant of rAAV product purity and the transfer of rAAV DNA contaminants to patients. That points to plasmid design and manufacturing controls as possible intervention points, although the proposed modification was not tested as a clinical intervention in this study.
The report was posted on medRxiv on September 14, 2026, as a preprint. Because the analysis concerns sequencing data from a single treated patient and an inferred manufacturing-plasmid configuration, its immediate contribution is a molecular explanation tied to that case. The authors also disclose patent and royalty interests involving modifications to the AAV P5 promoter and methods for reducing manufacturing-cell DNA contaminants.