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...

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Bibliographic Details
Published in:eLife 2024-03, Vol.12
Main Authors: Jain, Nina K, Ogden, Pierce J, Church, George M
Format: Article
Language:English
Subjects:
adeno-associated virus
Amino acids
Bar codes
Binding sites
Capsid
Capsid Proteins - genetics
Cytomegalovirus
Dependovirus - genetics
Gene mapping
Gene therapy
Genes
Genetic Vectors - genetics
Genetics and Genomics
Genomes
Mutagenesis
Mutation
Packaging
Proteins
rep
Rep gene
Rep protein
Serotypes
Viruses
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Summary: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 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 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.
ISSN:2050-084X
2050-084X
DOI:10.7554/eLife.87730