Single or double? Uncovering the packaging of pesky DNA impurities in rAAV
New insights into the packaging of DNA impurities may help improve rAAV purification.
In a recent study, researchers from the Graduate School of Science, Technology and Innovation at Kobe University (Kobe, Japan) revealed insights into the nature and origin of DNA impurities packaged into recombinant adeno-associated virus (rAAV) capsids for gene therapy products. Their findings offer a better understanding of how DNA impurities are packaged into rAAV capsids and may help improve rAAV purification methods, ultimately enhancing the safety and efficacy of gene therapy products.
rAAV vectors are extensively used in gene therapy to deliver therapeutic genes into target cells. Typically, rAAVs are produced by transfecting HEK293 cells with two or three plasmids: a packaging plasmid, a transfer plasmid, and, in some cases, a helper plasmid. However, DNA impurities can be co-packaged into rAAV capsids during production, which can compromise the quality, safety and efficacy of the final product. These impurities include unwanted DNA mistakenly packaged into AAV capsids, known as non-vector genome DNA. Common sources of these impurities are plasmid DNA and host cell DNA (e.g., HEK293 cell DNA).
In this study, the researchers aimed to deepen their understanding of DNA impurities derived from plasmid and host cell sources in rAAV vectors, in particular, the strand form of these impurities. They also examined whether the type and quantity of impurities varied across different rAAV serotypes and how consistently these contaminants are packaged into capsids.
To investigate this, they produced and purified four rAAV serotypes (rAAV1, rAAv2, rAAV5 and rAAV6), all carrying a ZsGreen1 reporter gene. Before analysis, the samples were treated with DNase I, ensuring that only encapsulated DNA was analyzed. The encapsulated material was then extracted and analyzed using ddPCR with primers targeting the three plasmids used in the vector production, as well as HEK293T host cell DNA.
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The team found that 0.69–3.27% of the DNA inside purified rAAV capsids was derived from plasmids, with the vast majority (over 98%) originating from the backbone of the transfer plasmid, pAAV-ZsGreen1. Impurities from the packaging and helper plasmids (pRC and pHelper) were present at significantly lower levels.
To determine whether the encapsulated impurities were single- or double-stranded, the extracted DNA was treated with DNases nuclease P1 and lambda exonuclease. Nuclease P1, which degrades single-stranded DNA, resulted in substantial degradation of the extracted DNA impurities, while lambda exonuclease, which specifically targets double-stranded DNA had no observable effect, indicating that the impurities were primarily single-stranded. To verify these results, the researchers included control experiments using known single- and double-stranded DNA, which validated the nuclease digestion results.
The presence of single-stranded DNA impurities within the vector capsid suggests that these fragments may be packaged into the rAAV capsids in a manner similar to the intended therapeutic genome. This finding may prove important for improving rAAV purification methods, which could ultimately enhance the safety and efficacy of gene therapy products.
The researchers also noted that the adverse effects of single-stranded DNA impurities in gene therapy may differ from those of double-stranded impurities. As discussed in the paper, the researchers believe, “it may be necessary to reconsider or evaluate the effects of single-strand form of DNA impurities independently.”
Thomas Gallagher, Managing Editor of Human Gene Therapy at the University of Massachusetts Chan Medical School (MA, USA), emphasized the importance of studies like this in understanding the sources of DNA impurities during rAAV production:
“Studies like this are incredibly important to better understand how DNA impurities are generated and what sources they come from during the production process in order to improve the efficiency of manufacturing and purity of the final rAAV product, which will help make the therapy more effective and reduce adverse events for patients.”
