Preprint links the P5 region in DNA used to produce rAAV to Rep and Cap gene fragments in a liver biopsy after Zolgensma treatment

Preprint links the P5 region in DNA used to produce rAAV to Rep and Cap gene fragments in a liver biopsy after Zolgensma treatment

In a preprint dated September 14, researchers reanalyzed DNA from a liver biopsy of a child who received Zolgensma to treat spinal muscular atrophy. The authors reconstructed a possible sequence of events through which a plasmid used to manufacture the treatment could have left fragments of the Rep and Cap genes, which are needed to assemble the viral vector, in the final product.

Zolgensma delivers a therapeutic gene into cells using a recombinant adeno-associated virus, or rAAV. During rAAV production, cells receive plasmids, which are circular DNA molecules. One plasmid carries the therapeutic gene. Another carries the Rep and Cap genes: Rep helps replicate and package DNA, while Cap encodes the viral capsid protein.

A clinical study had already identified fragments of all three production plasmids in this child's biopsy. The authors of the new preprint reconstructed fragments of one of those plasmids from the biopsy data. One end of a contiguously assembled REP/CAP fragment fell within P5, near the site where Rep cuts DNA. In long sequencing reads from the same biopsy, P5 was the breakpoint in five of the 16 fragments that matched the assembled REP/CAP sequence.

P5 regulates the production of the large forms of Rep needed to produce the vector. P5 contains a Rep binding site and an adjacent DNA cleavage site. In the authors' reconstruction of the plasmid map, P5 lay immediately after Cap, with REP/CAP upstream of P5. When Rep cuts DNA near P5, a fragment upstream of that site can be packaged into a separate rAAV particle. In this biopsy, the copy number of REP/CAP sequences was 0,5–1% of the therapeutic gene copy number.

In a peer-reviewed study published in 2026, researchers modified the part of P5 involved in DNA cleavage and restored the start codon, the site where synthesis of the large forms of Rep begins. In cell systems used to produce rAAV, this combination reduced detectable P5-associated impurities by approximately 50-fold while maintaining a high vector yield.