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  • Review Article
  • Published:

Precise hit: adeno-associated virus in gene targeting

Nature Reviews Microbiology volume 3, pages 837–847 (2005)Cite this article

Key Points

  • Gene targeting of chromosomal sequences by homologous recombination is a powerful method for gene correction that can eliminate dominant mutations causing genetic disease. In mammalian cells, this process is inefficient and much effort is directed towards improving the targeting frequency of conventional plasmid systems, which is only 10−6.

  • The discovery that recombinant adeno-associated virus (rAAV) can introduce precise, site-specific modification in the mammalian genome with a frequency of 1% constituted a major breakthrough in the gene-targeting field.

  • This review discusses the unique features of the rAAV system that are responsible for the high targeting efficiency as well as its successful application in gene therapy.

  • Along with the ability to infect multiple cell types from several different species, rAAV can introduce different types of modifications with high fidelity in both exogenously introduced target genes and endogenous genes.

  • The single-stranded viral genome with inverted terminal repeats is a distinct feature of rAAV that probably mediates the recognition of the genome by appropriate cellular factors and its ability to recombine with homologous sequences in the host genome.

  • On the basis of current knowledge, we propose a mechanism of homologous recombination of the rAAV with the chromosomal target.

  • Understanding the mechanism of rAAV-mediated targeting will improve our ability to manipulate the cellular pathways governing this phenomenon and broaden the scope for practical applications.

Abstract

Vectors based on the adeno-associated virus (AAV) have attracted much attention as potent gene-delivery vehicles, mainly because of the persistence of this non-pathogenic virus in the host cell and its sustainable therapeutic gene expression. However, virus infection can be accompanied by potentially mutagenic random vector integration into the genome. A novel approach to AAV-mediated gene therapy based on gene targeting through homologous recombination allows efficient, high-fidelity, non-mutagenic gene repair in a host cell.

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Figure 1: Biology of wild-type and recombinant adeno-associated virus.
Figure 2: Gene targeting with rAAV vectors.
Figure 3: Molecular mechanism of rAAV-mediated gene targeting.

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Acknowledgements

We thank Dr R. M. Linden for discussion.

Author information

Authors and Affiliations

  1. Department of Gene and Cell Medicine, Mount Sinai School of Medicine, New York, USA

    Ana Vasileva

  2. Institute for Physiological Chemistry, Dresden University of Technology, Dresden, Germany

    Rolf Jessberger

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  1. Ana Vasileva
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Correspondence to Rolf Jessberger.

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Supplementary information

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DATABASES

Entrez

AAV2

COL1A1

MUM

PRNP

Saccharomyces cerevisiae

Swiss-Prot

Artemis

ATM kinase

BRCA2

DNA ligase IV

ERCC1

KU70

KU86

MBS85

MRE11

NBS1

RAD50

RAD51

RAD51C

RAD52

RAD54B

RAD54L

RPA

XPF

XRCC3

XRCC4

CDC Infectious Disease Information

BSE

FURTHER INFORMATION

Rolf Jessberger's homepage

Glossary

VECTORS

Nucleic-acid sequences derived from bacterial plasmids or viruses, used for delivery of exogenous DNA or RNA into cells.

TRANSGENE

Artificially reconstituted gene, which can be expressed either pre- or post-integration into the genome; often encodes a therapeutic or a marker gene.

SILENCING

In this context, complete or partial shut-off of transgene expression owing to factors acting at the locus of vector integration and/or the structure of the locus.

HOMOLOGOUS RECOMBINATION

(HR). Substitution (reciprocal exchange or replacement) of a segment of DNA by another that is identical (homologous). Occurs naturally during meiotic recombination and DNA repair.

NON-HOMOLOGOUS END JOINING

(NHEJ). Process of joining two broken DNA ends on the basis of limited or no homology between two DNA molecules, which can result in incorrect recovery of the sequence of a broken DNA molecule. Also functions to create genetic diversity during immune development.

ISOGENIC

Genetically identical (except for sex).

TRANSDUCTION

In this context, equivalent to infection with a virus used as a gene-therapy vector.

EPISOME

Non-integrated, circular, double-stranded DNA molecule with the capacity to integrate into the chromosome of the host cell.

TRANS-SPLICING

A reconstruction of a larger transcript by joining the 5′ and 3′ portions of a transcript from two independent viruses (co-adminstered to the same tissue) that have formed intermolecular concatemers.

MULTIPLICITY OF INFECTION

Synonym of virus load, the number of viral particles or infectious units per infected cell. Usually represented by the number of viral genomes — gcp/cell (genome-containing particles per cell) or TU/cell (transducing units per cell).

ETOPOSIDE

A chemical agent that blocks topoisomerase and therefore introduces random DNA breaks, which results in S-phase arrest.

HYDROXYUREA

Causes depletion of the dNTP pool by inhibition of ribonucleotide reductase and thereby stalls DNA replication. Removal of hydroxyurea might stimulate viral second-strand synthesis.

TYPE I COLLAGEN

The most abundant protein in vertebrates, and a constituent of the extracellular matrix in the connective tissue of bone, skin, tendon, ligament and dentine. It is produced and secreted mostly by fibroblasts and osteoblasts.

GENE CONVERSION

A non-reciprocal transfer of genetic information.

OSTEOGENESIS IMPERFECTA

The most common and the mildest form of a group of rare disorders affecting the connective tissue, which are characterized by fragile bones that fracture easily (brittle bones).

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Vasileva, A., Jessberger, R. Precise hit: adeno-associated virus in gene targeting. Nat Rev Microbiol 3, 837–847 (2005). https://doi.org/10.1038/nrmicro1266

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