AAV Ancestral Reconstruction Library Enables Selection of Broadly Infectious Viral Variants
Open accessAbstract
Adeno-associated virus (AAV) vectors have achieved clinical efficacy in treating several diseases. However, enhanced vectors are required to extend these landmark successes to other indications and protein engineering approaches may provide the necessary vector improvements to address such unmet medical needs. To generate new capsid variants with potentially enhanced infectious properties and to gain insights into AAV's evolutionary history, we computationally designed and experimentally constructed a putative ancestral AAV library. Combinatorial variations at 32 amino acid sites were introduced to account for uncertainty in their identities. We then analyzed the evolutionary flexibility of these residues, the majority of which have not been previously studied, by subjecting the library to iterative selection on a representative cell line panel. The resulting variants exhibited transduction efficiencies comparable to the most efficient extant serotypes and, in general, ancestral libraries were broadly infectious across the cell line panel, indicating that they favored promiscuity over specificity. Interestingly, putative ancestral AAVs were more thermostable than modern serotypes and did not use sialic acids, galactose or heparan sulfate proteoglycans for cellular entry. Finally, variants mediated 19- to 31-fold higher gene expression in the muscle compared with AAV1, a clinically used serotype for muscle delivery, highlighting their promise for gene therapy.
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In silico design approach to reconstruct and discover new capsid was first reported in 2015.
“With technological advancements of computational modeling and machine learning, an in silico design approach to reconstruct and discover new capsid was first reported in 2015 [69, 70].”
From Designing and optimizing AAV-mediated gene therapy for neurodegenerative diseases: from bench to bedside (2024) · cites this paper as [70]
The Introduction describes reconstructing the AAV capsid and selecting new, broadly infectious variants, supporting the core description of the approach. It also describes earlier ancestral reconstructions of viral proteins, so this paper does not establish that such an approach was first reported in 2015. The passages have no page numbers in our copy. (LLM, conf=0.85)
Experimental — accuracy not yet measuredAnother vector selection strategy mines the parvovirus ancestral genome to identify capsid sequences that have coevolved with humans.
“Another vector selection strategy mines the parvovirus ancestral genome to identify capsid sequences that have coevolved with humans [115].”
From Risk mitigation of immunogenicity: a key to personalized retinal gene therapy (2021) · cites this paper as [115]
This strategy produced the Anc80 serotype.
“This strategy produced the Anc80 serotype”
From Risk mitigation of immunogenicity: a key to personalized retinal gene therapy (2021) · cites this paper as [115]
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- Authors
- Santiago-Ortiz, J., Ojala, D. S., Westesson, O., Weinstein, J. R., Wong, S. Y., Steinsapir, A., Kumar, S., Holmes, I., & Schaffer, D. V.
- Affiliations
- University of California, Berkeley (US)
- Corresponding author
- Schaffer, D V
- Venue
- Gene Therapy
- Published
- 2015-07-17
- Volume
- 22
- Issue
- 12
- Pages
- 934-946
- ISSN
- 0969-7128 (ISSN-L)
- 1476-5462
- PMC
- PMC4509550
- OpenAlex
- W866037220
- Cited by
- 83
- References
- 64
- Citations by year
- 2026: 5 · 2025: 4 · 2024: 11 · 2023: 7 · 2022: 3 · 2021: 9 · 2020: 9 · 2019: 5 · 2018: 9 · 2017: 11 · 2016: 9 · 2015: 1
- FWCI
- 6.83
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Gene Therapy (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- University of Florida
- National Human Genome Research Institute (R01HG004483)
- National Eye Institute (PN2EY018241, R01EY022975)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Keywords
- adeno-associated virus, ancestral sequence reconstruction, capsid engineering, viral vector design, combinatorial libraries, iterative selection, transduction efficiency, host range, cell line panel, thermal stability, heparan sulfate proteoglycans, muscle gene expression, AAV1, gene therapy vectors
- MeSH terms
- Dependovirus, Animals, Capsid, Cell Line, Female, Genetic Vectors, Humans, Mice, Inbred BALB C, Phylogeny, Genetic Therapy, Gene Library, Sequence Analysis, DNA, Sequence Analysis, Protein, Cell Line, Tumor, Mice, HEK293 Cells
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