Evaluation of the loading capacity and patterns of packaged DNA in AAV genomes of different sizes using long-read sequencing
The loading capacity of adeno-associated virus (AAV) vectors is reportedly 4.7–5.0 kb, which limits the size of genes that can be treated with gene therapy. However, the effects of oversized genomes on the integrity of packaged AAV genomes are poorly understood. Herein, nanopore long-read sequencing...
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Elsevier
2025-06-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050125000695 |
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| author | Mitsuki Kosaka Ai Fujita Sajiki Kosuke Fujita Kazuhisa Yamada Kenichi Kawano Kiichi Hirazawa Tsuyoshi Matsuno Masahiko Takada Nobuhiro Shimozawa Ken-ichi Inoue Kenya Yuki Koji M. Nishiguchi |
| author_facet | Mitsuki Kosaka Ai Fujita Sajiki Kosuke Fujita Kazuhisa Yamada Kenichi Kawano Kiichi Hirazawa Tsuyoshi Matsuno Masahiko Takada Nobuhiro Shimozawa Ken-ichi Inoue Kenya Yuki Koji M. Nishiguchi |
| author_sort | Mitsuki Kosaka |
| collection | DOAJ |
| description | The loading capacity of adeno-associated virus (AAV) vectors is reportedly 4.7–5.0 kb, which limits the size of genes that can be treated with gene therapy. However, the effects of oversized genomes on the integrity of packaged AAV genomes are poorly understood. Herein, nanopore long-read sequencing was used to evaluate genomic integrity in AAV vectors harboring genomes of various sizes. AAV had a reduced proportion of full-length genomes at a vector length of 4.9 kb, which declined rapidly between 4.9 and 5.0 kb. This was mainly attributable to defects in genome packaging rather than genome synthesis. Furthermore, the pattern of packaged DNA was unique to the arrangement of the components of the oversized genome. However, an 86.3% reduction in the proportion of full-length genomes (4.7 vs. 5.0 kb) was not consistent with the retained expression of the reporter gene in the mouse retina. This discrepancy might be partially attributable to the preferential inclusion of the region containing the reporter gene. These results highlight the utility of long-read sequencing in assessing the genomic integrity and design of AAV vectors, as the pattern of packaged genomes appears to be unique to each vector, particularly for oversized AAV genomes. |
| format | Article |
| id | doaj-art-28a6c7e3728d451c98502fffb67ac8bc |
| institution | Kabale University |
| issn | 2329-0501 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj-art-28a6c7e3728d451c98502fffb67ac8bc2025-08-20T03:52:19ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012025-06-0133210147410.1016/j.omtm.2025.101474Evaluation of the loading capacity and patterns of packaged DNA in AAV genomes of different sizes using long-read sequencingMitsuki Kosaka0Ai Fujita Sajiki1Kosuke Fujita2Kazuhisa Yamada3Kenichi Kawano4Kiichi Hirazawa5Tsuyoshi Matsuno6Masahiko Takada7Nobuhiro Shimozawa8Ken-ichi Inoue9Kenya Yuki10Koji M. Nishiguchi11Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, JapanDepartment of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan; Division for Advanced Medical Research, Center for Research of Laboratory Animals and Medical Research Engineering, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan; Corresponding author: Ai Fujita Sajiki, MD, PhD, Department of Ophthalmology, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya, Aichi 466-8550, Japan.Department of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, JapanDepartment of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, JapanDepartment of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, Japan; Department of Ophthalmology, Yokkaichi Municipal Hospital, Yokkaichi, Mie 510-8567, JapanDepartment of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, JapanDepartment of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, JapanSystems Neuroscience Section, Center for the Evolutionary Origins of Human Behavior, Kyoto University, Inuyama, Aichi 484-8506, JapanTsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition, Tsukuba, Ibaraki 305-0843, JapanSystems Neuroscience Section, Center for the Evolutionary Origins of Human Behavior, Kyoto University, Inuyama, Aichi 484-8506, JapanDepartment of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, JapanDepartment of Ophthalmology, Nagoya University Graduate School of Medicine, Nagoya, Aichi 466-8550, JapanThe loading capacity of adeno-associated virus (AAV) vectors is reportedly 4.7–5.0 kb, which limits the size of genes that can be treated with gene therapy. However, the effects of oversized genomes on the integrity of packaged AAV genomes are poorly understood. Herein, nanopore long-read sequencing was used to evaluate genomic integrity in AAV vectors harboring genomes of various sizes. AAV had a reduced proportion of full-length genomes at a vector length of 4.9 kb, which declined rapidly between 4.9 and 5.0 kb. This was mainly attributable to defects in genome packaging rather than genome synthesis. Furthermore, the pattern of packaged DNA was unique to the arrangement of the components of the oversized genome. However, an 86.3% reduction in the proportion of full-length genomes (4.7 vs. 5.0 kb) was not consistent with the retained expression of the reporter gene in the mouse retina. This discrepancy might be partially attributable to the preferential inclusion of the region containing the reporter gene. These results highlight the utility of long-read sequencing in assessing the genomic integrity and design of AAV vectors, as the pattern of packaged genomes appears to be unique to each vector, particularly for oversized AAV genomes.http://www.sciencedirect.com/science/article/pii/S2329050125000695adeno-associated virusAAV vectorgene therapyAAV genome loading capacityAAV genome packaging patternlong-read sequencing |
| spellingShingle | Mitsuki Kosaka Ai Fujita Sajiki Kosuke Fujita Kazuhisa Yamada Kenichi Kawano Kiichi Hirazawa Tsuyoshi Matsuno Masahiko Takada Nobuhiro Shimozawa Ken-ichi Inoue Kenya Yuki Koji M. Nishiguchi Evaluation of the loading capacity and patterns of packaged DNA in AAV genomes of different sizes using long-read sequencing Molecular Therapy: Methods & Clinical Development adeno-associated virus AAV vector gene therapy AAV genome loading capacity AAV genome packaging pattern long-read sequencing |
| title | Evaluation of the loading capacity and patterns of packaged DNA in AAV genomes of different sizes using long-read sequencing |
| title_full | Evaluation of the loading capacity and patterns of packaged DNA in AAV genomes of different sizes using long-read sequencing |
| title_fullStr | Evaluation of the loading capacity and patterns of packaged DNA in AAV genomes of different sizes using long-read sequencing |
| title_full_unstemmed | Evaluation of the loading capacity and patterns of packaged DNA in AAV genomes of different sizes using long-read sequencing |
| title_short | Evaluation of the loading capacity and patterns of packaged DNA in AAV genomes of different sizes using long-read sequencing |
| title_sort | evaluation of the loading capacity and patterns of packaged dna in aav genomes of different sizes using long read sequencing |
| topic | adeno-associated virus AAV vector gene therapy AAV genome loading capacity AAV genome packaging pattern long-read sequencing |
| url | http://www.sciencedirect.com/science/article/pii/S2329050125000695 |
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