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Updated: Feb 28, 2026
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Isolation of Next-Generation Gene Therapy Vectors through Engineering, Barcoding, and Screening of Adeno-Associated Virus AAV Capsid Variants
Published on: October 18, 2022
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High capsid-genome correlation facilitates creation of AAV libraries for directed evolution
Mathieu Nonnenmacher1, Harm van Bakel2, Roger J Hajjar1
1Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, New York, New York, USA.
Summary
Directed evolution of adeno-associated virus (AAV) uses natural genome organization to create diverse capsid libraries. This method ensures high capsid-DNA correlation, minimizing mosaicism for effective variant selection.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Directed evolution is key for isolating improved adeno-associated virus (AAV) variants.
- Current AAV libraries rely on natural genome organization for capsid DNA recovery.
- Minimizing capsid-genome mismatch is crucial for successful directed evolution.
Purpose of the Study:
- To investigate capsid-genome correlation in AAV libraries.
- To assess capsid homogeneity and DNA integrity during AAV production.
- To validate the efficiency of natural AAV genome organization for library generation.
Main Methods:
- Utilized natural AAV genome organization for capsid variant expression.
- Employed bulk transfection with semi-random peptide libraries.
- Applied next-generation sequencing to analyze AAV progeny.
Main Results:
- Observed near-homogeneous capsids with high capsid-DNA correlation.
- Demonstrated strong counter-selection against premature stop codons in capsid variants.
- Confirmed low capsid mosaicism and high capsid-genome correlation in natural AAV production.
Conclusions:
- Natural AAV production yields low mosaicism and high capsid-genome correlation.
- This enables efficient, one-step generation of diverse AAV libraries.
- Phenotype-genotype correlation is well-maintained, facilitating directed evolution.
