Residual DNA impurities in AAV vectors—nature and transcription
Recombinant adeno-associated viruses (rAAVs) produced by transfecting DNA plasmids into mammalian cells can inadvertently package host cell DNA (hcDNA) and plasmid DNA inside their capsids. Although the percentage of these DNA impurities is low compared to the rAAV genome in vector preparations, it...
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Elsevier
2025-09-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050125000981 |
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| author | Hsin-I Jen Patrick Wilkinson Xiaohui Lu Wei Zhang |
| author_facet | Hsin-I Jen Patrick Wilkinson Xiaohui Lu Wei Zhang |
| author_sort | Hsin-I Jen |
| collection | DOAJ |
| description | Recombinant adeno-associated viruses (rAAVs) produced by transfecting DNA plasmids into mammalian cells can inadvertently package host cell DNA (hcDNA) and plasmid DNA inside their capsids. Although the percentage of these DNA impurities is low compared to the rAAV genome in vector preparations, it is essential to characterize the DNA impurities in gene therapy products due to the theoretical risks associated with unwanted gene expression and potential immunogenicity and oncogenicity in treated patients. We performed long-read sequencing in rAAV vector, with a focus on analyzing residual, non-transgene DNA within the capsids. Although we detected host cell and residual plasmid DNA impurities, they were predominantly incomplete sequences without coding potential. This indicated that while DNA impurities may be present in rAAV preparations, host cell and residual plasmid genes were unlikely to be expressed. This was supported by RNA sequencing (RNA-seq) analyses that showed minimal plasmid RNA transcripts and host cell RNA transcripts in the livers of mice dosed with rAAV. Overall, the results from these studies enable data-based risk assessment of co-packaged DNA impurities and a better understanding of potential adverse effects associated with rAAV gene therapy. |
| format | Article |
| id | doaj-art-27a62717f5ba43549c9474df801d8a73 |
| institution | OA Journals |
| issn | 2329-0501 |
| language | English |
| publishDate | 2025-09-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj-art-27a62717f5ba43549c9474df801d8a732025-08-20T02:36:00ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012025-09-0133310150310.1016/j.omtm.2025.101503Residual DNA impurities in AAV vectors—nature and transcriptionHsin-I Jen0Patrick Wilkinson1Xiaohui Lu2Wei Zhang3Global CMC Development, Ultragenyx Pharmaceutical Inc, Woburn, MA, USAGene Therapy Research, Ultragenyx Pharmaceutical Inc, Somerville, MA, USAGlobal CMC Development, Ultragenyx Pharmaceutical Inc, Woburn, MA, USAGlobal CMC Development, Ultragenyx Pharmaceutical Inc, Woburn, MA, USA; Corresponding author: Wei Zhang, Global CMC Development, Ultragenyx Pharmaceutical Inc, Woburn, MA, USA.Recombinant adeno-associated viruses (rAAVs) produced by transfecting DNA plasmids into mammalian cells can inadvertently package host cell DNA (hcDNA) and plasmid DNA inside their capsids. Although the percentage of these DNA impurities is low compared to the rAAV genome in vector preparations, it is essential to characterize the DNA impurities in gene therapy products due to the theoretical risks associated with unwanted gene expression and potential immunogenicity and oncogenicity in treated patients. We performed long-read sequencing in rAAV vector, with a focus on analyzing residual, non-transgene DNA within the capsids. Although we detected host cell and residual plasmid DNA impurities, they were predominantly incomplete sequences without coding potential. This indicated that while DNA impurities may be present in rAAV preparations, host cell and residual plasmid genes were unlikely to be expressed. This was supported by RNA sequencing (RNA-seq) analyses that showed minimal plasmid RNA transcripts and host cell RNA transcripts in the livers of mice dosed with rAAV. Overall, the results from these studies enable data-based risk assessment of co-packaged DNA impurities and a better understanding of potential adverse effects associated with rAAV gene therapy.http://www.sciencedirect.com/science/article/pii/S2329050125000981rAAVresidual DNAhost cell DNA transcriptionPacBio sequencinggene therapyDNA impurities |
| spellingShingle | Hsin-I Jen Patrick Wilkinson Xiaohui Lu Wei Zhang Residual DNA impurities in AAV vectors—nature and transcription Molecular Therapy: Methods & Clinical Development rAAV residual DNA host cell DNA transcription PacBio sequencing gene therapy DNA impurities |
| title | Residual DNA impurities in AAV vectors—nature and transcription |
| title_full | Residual DNA impurities in AAV vectors—nature and transcription |
| title_fullStr | Residual DNA impurities in AAV vectors—nature and transcription |
| title_full_unstemmed | Residual DNA impurities in AAV vectors—nature and transcription |
| title_short | Residual DNA impurities in AAV vectors—nature and transcription |
| title_sort | residual dna impurities in aav vectors nature and transcription |
| topic | rAAV residual DNA host cell DNA transcription PacBio sequencing gene therapy DNA impurities |
| url | http://www.sciencedirect.com/science/article/pii/S2329050125000981 |
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