Recombinant AAV batch profiling by nanopore sequencing elucidates product-related DNA impurities and vector genome length distribution
During production, recombinant adeno-associated virus (rAAV) capsids are equipped with heterogeneous genetic payloads including undesired DNA impurities as well as truncated vector genomes. Comprehensive analysis of encapsidated DNA by long-read next-generation sequencing is destined to guide platfo...
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| Format: | Article |
| Language: | English |
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Elsevier
2025-03-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050125000129 |
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| author | Florian Dunker-Seidler Kathrin Breunig Magdalena Haubner Florian Sonntag Markus Hörer Rebecca C. Feiner |
| author_facet | Florian Dunker-Seidler Kathrin Breunig Magdalena Haubner Florian Sonntag Markus Hörer Rebecca C. Feiner |
| author_sort | Florian Dunker-Seidler |
| collection | DOAJ |
| description | During production, recombinant adeno-associated virus (rAAV) capsids are equipped with heterogeneous genetic payloads including undesired DNA impurities as well as truncated vector genomes. Comprehensive analysis of encapsidated DNA by long-read next-generation sequencing is destined to guide platform optimization and provide crucial insights into safety of gene therapies. We used nanopore sequencing for in-depth profiling of an rAAV9 batch produced using our proprietary split two-plasmid system in a 50-L bioreactor. We compared three methods for single-strand to double-strand DNA conversion and their impact on the sequencing data. We observed a distinct library size profile but comparable impurity distribution. We contrasted recent nanopore sequencing advancements such as the V14 chemistry and dorado basecalling software with the widespread V9 chemistry and detected a markedly increased read quality. Our data highlight a high vector batch quality with low plasmid-derived and host cell DNA impurities of random origin, critical for mitigating associated safety risks. Finally, we compared nanopore data with orthogonal SMRT sequencing data and observed a higher base quality, but largely similar length and impurity profiles. Taken together, nanopore sequencing is a state-of-the-art method for comprehensive, in-depth rAAV vector batch analysis during all stages of gene therapy development. |
| format | Article |
| id | doaj-art-f8ae1c561ee1479ca68e3316df35d06d |
| institution | Kabale University |
| issn | 2329-0501 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj-art-f8ae1c561ee1479ca68e3316df35d06d2025-02-09T05:00:34ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012025-03-01331101417Recombinant AAV batch profiling by nanopore sequencing elucidates product-related DNA impurities and vector genome length distributionFlorian Dunker-Seidler0Kathrin Breunig1Magdalena Haubner2Florian Sonntag3Markus Hörer4Rebecca C. Feiner5Ascend Advanced Therapies GmbH, Fraunhoferstraße 9b, 82152 Planegg-Martinsried, GermanyAscend Advanced Therapies GmbH, Fraunhoferstraße 9b, 82152 Planegg-Martinsried, GermanyAscend Advanced Therapies GmbH, Fraunhoferstraße 9b, 82152 Planegg-Martinsried, GermanyAscend Advanced Therapies GmbH, Fraunhoferstraße 9b, 82152 Planegg-Martinsried, GermanyAscend Advanced Therapies GmbH, Fraunhoferstraße 9b, 82152 Planegg-Martinsried, GermanyAscend Advanced Therapies GmbH, Fraunhoferstraße 9b, 82152 Planegg-Martinsried, Germany; Corresponding author: Rebecca Feiner, Ascend Advanced Therapies GmbH, Fraunhoferstraße 9b, 82152 Planegg-Martinsried, Germany.During production, recombinant adeno-associated virus (rAAV) capsids are equipped with heterogeneous genetic payloads including undesired DNA impurities as well as truncated vector genomes. Comprehensive analysis of encapsidated DNA by long-read next-generation sequencing is destined to guide platform optimization and provide crucial insights into safety of gene therapies. We used nanopore sequencing for in-depth profiling of an rAAV9 batch produced using our proprietary split two-plasmid system in a 50-L bioreactor. We compared three methods for single-strand to double-strand DNA conversion and their impact on the sequencing data. We observed a distinct library size profile but comparable impurity distribution. We contrasted recent nanopore sequencing advancements such as the V14 chemistry and dorado basecalling software with the widespread V9 chemistry and detected a markedly increased read quality. Our data highlight a high vector batch quality with low plasmid-derived and host cell DNA impurities of random origin, critical for mitigating associated safety risks. Finally, we compared nanopore data with orthogonal SMRT sequencing data and observed a higher base quality, but largely similar length and impurity profiles. Taken together, nanopore sequencing is a state-of-the-art method for comprehensive, in-depth rAAV vector batch analysis during all stages of gene therapy development.http://www.sciencedirect.com/science/article/pii/S2329050125000129nanopore sequencing, gene therapyadeno-associated virus, AAVanalyticsbioinformatics |
| spellingShingle | Florian Dunker-Seidler Kathrin Breunig Magdalena Haubner Florian Sonntag Markus Hörer Rebecca C. Feiner Recombinant AAV batch profiling by nanopore sequencing elucidates product-related DNA impurities and vector genome length distribution Molecular Therapy: Methods & Clinical Development nanopore sequencing, gene therapy adeno-associated virus, AAV analytics bioinformatics |
| title | Recombinant AAV batch profiling by nanopore sequencing elucidates product-related DNA impurities and vector genome length distribution |
| title_full | Recombinant AAV batch profiling by nanopore sequencing elucidates product-related DNA impurities and vector genome length distribution |
| title_fullStr | Recombinant AAV batch profiling by nanopore sequencing elucidates product-related DNA impurities and vector genome length distribution |
| title_full_unstemmed | Recombinant AAV batch profiling by nanopore sequencing elucidates product-related DNA impurities and vector genome length distribution |
| title_short | Recombinant AAV batch profiling by nanopore sequencing elucidates product-related DNA impurities and vector genome length distribution |
| title_sort | recombinant aav batch profiling by nanopore sequencing elucidates product related dna impurities and vector genome length distribution |
| topic | nanopore sequencing, gene therapy adeno-associated virus, AAV analytics bioinformatics |
| url | http://www.sciencedirect.com/science/article/pii/S2329050125000129 |
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