Directed evolution of designer vectors

Access through your institution Buy or subscribe Recombinant adeno-associated viral vectors (rAAV), which are non-pathogenic and can establish persistent transgene expression in various cell

types, are at the forefront of vector design efforts yet present a number of therapeutic challenges. Problems associated with rAAV delivery stem from the properties of the outer shell of

the virus, the capsid, which is a major focus of engineering strategies. In past studies function-enhancing peptide sequences were inserted into specific sites in the capsid gene, providing

improved cell-specific targeting. However, modifying more complex vector activities by this approach would require a greater understanding of the relationship between capsid structure and

specific properties. This new study bypasses this issue by exploiting an approach that has been successful in protein engineering: directed evolution. The authors generated more than 106

AAV2 variants with random mutations throughout the capsid gene. The resulting library was screened for variants with desirable functional properties followed by further rounds of mutagenesis

and screening to enrich the selection. Proof of principle was demonstrated by applying the technique to two clinically relevant problems. One issue is that binding to heparin sulphate can

limit rAAV2 dispersal. Affinity chromatography was used to identify variants with low heparin-binding affinity, additionally confirming the library's high functional diversity. Another

major therapeutic limitation is that patients previously exposed to wild-type AAV2 carry antibodies that bind to and inactivate the vector before it can reach its target. Several variants

were identified that could avoid antibody neutralization _in vitro_. These mutants were used to produce a vector with better delivery efficacy and less antibody neutralization than wild-type

vector _in vivo_. This is a preview of subscription content, access via your institution ACCESS OPTIONS Access through your institution Subscribe to this journal Receive 12 print issues and

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which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions * Read our FAQs * Contact customer support REFERENCES ORIGINAL RESEARCH

PAPER * Maheshri, N. et al. Directed evolution of adeno-associated virus yields enhanced gene delivery vectors. _Nature Biotechnol._ 10.1038/nbt1182 (2006) FURTHER READING * Neylon, C.

Chemical and biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution. _Nucleic Acids Res._ 32, 1448–1459 (2004).

Article  CAS  Google Scholar  Download references Authors * Katherine Whalley View author publications You can also search for this author inPubMed Google Scholar RIGHTS AND PERMISSIONS

Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Whalley, K. Directed evolution of designer vectors. _Nat Rev Drug Discov_ 5, 188 (2006). https://doi.org/10.1038/nrd1999

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