Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production
Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, rep...
| Main Authors: | , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2024-03-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/87730 |
| _version_ | 1827304759386701824 |
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| author | Nina K Jain Pierce J Ogden George M Church |
| author_facet | Nina K Jain Pierce J Ogden George M Church |
| author_sort | Nina K Jain |
| collection | DOAJ |
| description | Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single-codon mutations to the AAV2 rep gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2 rep mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large-scale gene therapy production. |
| first_indexed | 2024-04-24T17:44:34Z |
| format | Article |
| id | doaj.art-fc3d27322d8842f3ac88f95d8b811992 |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-24T17:44:34Z |
| publishDate | 2024-03-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-fc3d27322d8842f3ac88f95d8b8119922024-03-27T17:22:47ZengeLife Sciences Publications LtdeLife2050-084X2024-03-011210.7554/eLife.87730Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV productionNina K Jain0https://orcid.org/0000-0003-3020-4162Pierce J Ogden1https://orcid.org/0000-0003-3444-6214George M Church2https://orcid.org/0000-0001-6232-9969Wyss Institute for Biologically Inspired Engineering, Boston, United States; Department of Genetics, Harvard Medical School, Boston, United StatesWyss Institute for Biologically Inspired Engineering, Boston, United States; Department of Genetics, Harvard Medical School, Boston, United StatesWyss Institute for Biologically Inspired Engineering, Boston, United States; Department of Genetics, Harvard Medical School, Boston, United StatesRecombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single-codon mutations to the AAV2 rep gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2 rep mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large-scale gene therapy production.https://elifesciences.org/articles/87730adeno-associated virusgene therapyrep |
| spellingShingle | Nina K Jain Pierce J Ogden George M Church Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production eLife adeno-associated virus gene therapy rep |
| title | Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production |
| title_full | Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production |
| title_fullStr | Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production |
| title_full_unstemmed | Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production |
| title_short | Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 rep gene on AAV production |
| title_sort | comprehensive mutagenesis maps the effect of all single codon mutations in the aav2 rep gene on aav production |
| topic | adeno-associated virus gene therapy rep |
| url | https://elifesciences.org/articles/87730 |
| work_keys_str_mv | AT ninakjain comprehensivemutagenesismapstheeffectofallsinglecodonmutationsintheaav2repgeneonaavproduction AT piercejogden comprehensivemutagenesismapstheeffectofallsinglecodonmutationsintheaav2repgeneonaavproduction AT georgemchurch comprehensivemutagenesismapstheeffectofallsinglecodonmutationsintheaav2repgeneonaavproduction |