Combinatorial engineering of a gene therapy vector: directed evolution of adeno-associated virus.
BACKGROUND
Viruses are being exploited as vectors to deliver therapeutic genetic information into target cells. The success of this approach will depend on the ability to overcome current limitations, especially in terms of safety and efficiency, through molecular engineering of the viral particles.


METHODS
Here we show that in vitro directed evolution can be successfully performed to randomize the viral capsid by error prone PCR and to obtain mutants with improved phenotype.


RESULTS
To demonstrate the potential of this technology we selected several adeno-associated virus (AAV) capsid variants that are less efficiently neutralized by human antibodies. These mutations can be used to generate novel vectors for the treatment of patients with pre-existing immunity to AAV.


CONCLUSIONS
Our results demonstrate that combinatorial engineering overcomes the limitations of rational design approaches posed by incomplete understanding of the infectious process and at the same time offers a powerful tool to dissect basic viral biology by reverse genetics.
Perabo
Luca
L
Gene Center, Ludwig-Maximilians-University of Munich, Germany. perabo@lmb.uni-muenchen.de
Endell
Jan
J
King
Susan
S
Lux
Kerstin
K
Goldnau
Daniela
D
Hallek
Michael
M
Büning
Hildegard
H
D000595:Amino Acid Sequence; D000229:Dependovirus / Q000235:genetics* / Q000276:immunology; D019020:Directed Molecular Evolution; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000276:immunology; D006367:HeLa Cells; D006801:Humans; D007108:Immune Tolerance; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction