AAV-Mediated Gene Targeting

  • Daniel G. MillerEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 807)


The precise alteration of sequences by homologous recombination is an important strategy for gene therapies as well as investigating gene function and cellular DNA repair pathways. Inefficient delivery of template DNA to the nucleus using transfection or electroporation methods is one limitation of the frequency of homologous recombination in primary cells. AAV vectors can be used to efficiently deliver single stranded DNA recombination templates to the nucleus of primary cells and the AAV genome structure with single DNA strands stabilized by inverted terminal repeat sequences is likely one reason for the increase in recombination frequencies observed. Thus, an AAV-mediated gene targeting approach allows cells from normal or disease-affected individuals to be modified for careful study. When clones of primary cells can be expanded, autologous transplantation of phenotypically corrected cells is also feasible. Here we describe a basic approach to gene targeting using an AAV-mediated strategy. Vector design strategies are discussed, and protocols for altering expressed and non-expressed loci in primary somatic cells, and stem cells are reviewed.

Key words

Homologous recombination Gene targeting Gene editing Genome engineering Targeted repair Targeted gene disruption AAV-mediated 


  1. 1.
    Gao, G., Vandenberghe, L.H., Alvira, M.R., Lu, Y., Calcedo, R., Zhou, X. and Wilson, J. M. (2004) Clades of adeno-associated viruses are widely disseminated in human tissues. J. Virol. 78, 6381–6388.PubMedCrossRefGoogle Scholar
  2. 2.
    Gao, G., Alvira, M.R., Wang, L., Calcedo, R., Johnston, J. and Wilson, J. M. (2002) Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy. Proc. Natl. Acad. Sci. U.S.A. 99, 11854–11859.PubMedCrossRefGoogle Scholar
  3. 3.
    Nakai, H., Storm, T. and Kay, M. (2000) Recruitment of single-stranded recombinant adeno-associated virus vector genomes and intermolecular recombination are responsible for stable transduction of liver in vivo. J. Virol. 74, 9451–9463.PubMedCrossRefGoogle Scholar
  4. 4.
    Ferrari, F., Samulski, T., Shenk, T. and Samulski, R. (1996) Second-strand synthesis is a rate-limiting step for efficient transduction by recombinant adeno-associated virus vectors. J. Virol. 70, 3227–3234.PubMedGoogle Scholar
  5. 5.
    McCarty, D. M. (2008) Self-complementary aav vectors; advances and applications. Mol. Ther. 16, 1648–1656.PubMedCrossRefGoogle Scholar
  6. 6.
    McCarty, D. M., Fu, H., Monahan, P.E., Toulson, C.E., Naik, P. and Samulski, R. J. (2003) Adeno-associated virus terminal repeat (tr) mutant generates self-complementary vectors to overcome the rate-limiting step to transduction in vivo. Gene Ther. 10, 2112–2118.PubMedCrossRefGoogle Scholar
  7. 7.
    Hirata, R. and Russell, D. (2000) Design and packaging of adeno-associated virus gene targeting vectors. J. Virol. 74, 4612–4620.PubMedCrossRefGoogle Scholar
  8. 8.
    Chen, Z., Yant, S., He, C., Meuse, L., Shen, S. and Kay, M. (2001) Linear dnas concatemerize in vivo and result in sustained transgene expression in mouse liver. Mol. Ther. 3, 403–410.PubMedCrossRefGoogle Scholar
  9. 9.
    Nakai, H., Yant, S., Storm, T., Fuess, S., Meuse, L. and Kay, M. (2001) Extrachromosomal recombinant adeno-associated virus vector genomes are primarily responsible for stable liver transduction in vivo. J. Virol. 75, 6969–6976.PubMedCrossRefGoogle Scholar
  10. 10.
    Nakai, H., Wu, X., Fuess, S., Storm, T., Munroe, D., Montini, E., Burgess, S., Grompe, M. and Kay, M. (2005) Large-scale molecular characterization of adeno-associated virus vector integration in mouse liver. J. Virol. 79, 3606–3614.PubMedCrossRefGoogle Scholar
  11. 11.
    Nakai, H., Montini, E., Fuess, S., Storm, T., Grompe, M. and Kay, M. (2003) Aav serotype 2 vectors preferentially integrate into active genes in mice. Nat. Genet. 34, 297–302.PubMedCrossRefGoogle Scholar
  12. 12.
    Miller, D., Trobridge, G., Petek, L., Jacobs, M., Kaul, R. and Russell, D. (2005) Large-scale analysis of adeno-associated virus vector ­integration sites in normal human cells. J. Virol. 79, 11434–11442.PubMedCrossRefGoogle Scholar
  13. 13.
    Miller, D., Rutledge, E. and Russell, D. (2002) Chromosomal effects of adeno-associated virus vector integration. Nat. Genet. 30, 147–148.PubMedCrossRefGoogle Scholar
  14. 14.
    Miller, D., Petek, L. and Russell, D. (2004) Adeno-associated virus vectors integrate at chromosome breakage sites. Nat. Genet. 36, 767–773.PubMedCrossRefGoogle Scholar
  15. 15.
    Hirata, R., Chamberlain, J., Dong, R. and Russell, D. (2002) Targeted transgene insertion into human chromosomes by adeno-associated virus vectors. Nat. Biotechnol. 20, 735–738.PubMedCrossRefGoogle Scholar
  16. 16.
    Inoue, N., Dong, R., Hirata, R. and Russell, D. (2001) Introduction of single base substitutions at homologous chromosomal sequences by adeno-associated virus vectors. Mol. Ther. 3, 526–530.PubMedCrossRefGoogle Scholar
  17. 17.
    Russell, D. and Hirata, R. (1998) Human gene targeting by viral vectors. Nat. Genet. 18, 325–330.PubMedCrossRefGoogle Scholar
  18. 18.
    Kohli, M., Rago, C., Lengauer, C., Kinzler, K.W. and Vogelstein, B. (2004) Facile methods for generating human somatic cell gene knockouts using recombinant adeno-associated viruses. Nucleic Acids Res. 32, e3.PubMedCrossRefGoogle Scholar
  19. 19.
    Chamberlain, J., Schwarze, U., Wang, P., Hirata, R., Hankenson, K., Pace, J., Underwood, R., Song, K., Sussman, M., Byers, P. and Russell, D. (2004) Gene targeting in stem cells from individuals with osteogenesis imperfecta. Science 303, 1198–1201.PubMedCrossRefGoogle Scholar
  20. 20.
    Miller, D., Petek, L. and Russell, D. (2003) Human gene targeting by adeno-associated virus vectors is enhanced by dna double-strand breaks. Mol. Cell. Biol. 23, 3550–3557.PubMedCrossRefGoogle Scholar
  21. 21.
    Porteus, M., Cathomen, T., Weitzman, M. and Baltimore, D. (2003) Efficient gene targeting mediated by adeno-associated virus and dna double-strand breaks. Mol. Cell. Biol. 23, 3558–3565.PubMedCrossRefGoogle Scholar
  22. 22.
    Porteus, M. H. and Carroll, D. (2005) Gene targeting using zinc finger nucleases. Nat. Biotechnol. 23, 967–973.PubMedCrossRefGoogle Scholar
  23. 23.
    Durai, S., Mani, M., Kandavelou, K., Wu, J., Porteus, M.H. and Chandrasegaran, S. (2005) Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells. Nucleic Acids Res. 33, 5978–5990.PubMedCrossRefGoogle Scholar
  24. 24.
    Bibikova, M., Beumer, K., Trautman, J.K. and Carroll, D. (2003) Enhancing gene targeting with designed zinc finger nucleases. Science 300, 764.PubMedCrossRefGoogle Scholar
  25. 25.
    Deng, C. and Capecchi, M. R. (1992) Reexamination of gene targeting frequency as a function of the extent of homology between the targeting vector and the target locus. Mol. Cell. Biol. 12, 3365–3371.PubMedGoogle Scholar
  26. 26.
    Thomas, K. R., Folger, K.R. and Capecchi, M. R. (1986) High frequency targeting of genes to specific sites in the mammalian genome. Cell 44, 419–428.PubMedCrossRefGoogle Scholar
  27. 27.
    Lin, F., Sperle, K. and Sternberg, N. (1985) Recombination in mouse l cells between dna introduced into cells and homologous chromosomal sequences. Proc. Natl. Acad. Sci. U.S.A. 82, 1391–1395.PubMedCrossRefGoogle Scholar
  28. 28.
    Yáñez, R. J. and Porter, A. C. G. (2002) A chromosomal position effect on gene targeting in human cells. Nucleic Acids Res. 30, 4892–4901.PubMedCrossRefGoogle Scholar
  29. 29.
    Inoue, N., Hirata, R. and Russell, D. (1999) High-fidelity correction of mutations at multiple chromosomal positions by adeno-associated virus vectors. J. Virol. 73, 7376–7380.PubMedGoogle Scholar
  30. 30.
    Russell, D. W. and Hirata, R. K. (2008) Human gene targeting favors insertions over deletions. Hum. Gene Ther. 19, 907–914.PubMedCrossRefGoogle Scholar
  31. 31.
    Trobridge, G., Hirata, R. and Russell, D. (2005) Gene targeting by adeno-associated virus vectors is cell-cycle dependent. Hum. Gene Ther. 16, 522–526.PubMedCrossRefGoogle Scholar
  32. 32.
    te Riele, H., Maandag, E.R., Clarke, A., Hooper, M. and Berns, A. (1990) Consecutive inactivation of both alleles of the pim-1 proto-oncogene by homologous recombination in embryonic stem cells. Nature 348, 649–651.CrossRefGoogle Scholar
  33. 33.
    Mortensen, R. M. (1993) Double knockouts. production of mutant cell lines in cardiovascular research. Hypertension 22, 646–651.PubMedGoogle Scholar
  34. 34.
    Grim, J. E., Gustafson, M.P., Hirata, R.K., Hagar, A.C., Swanger, J., Welcker, M., Hwang, H.C., Ericsson, J., Russell, D.W. and Clurman, B. E. (2008) Isoform- and cell cycle-dependent substrate degradation by the fbw7 ubiquitin ligase. J. Cell Biol. 181, 913–920.PubMedCrossRefGoogle Scholar
  35. 35.
    Bunz, F., Fauth, C., Speicher, M.R., Dutriaux, A., Sedivy, J.M., Kinzler, K.W., Vogelstein, B. and Lengauer, C. (2002) Targeted inactivation of p53 in human cells does not result in aneuploidy. Cancer Res. 62, 1129–1133.PubMedGoogle Scholar
  36. 36.
    Petek, L.M., Fleckman, P., Miller, D.G. (2010) Efficient KRT14 Targeting and Functional characterization of transplantal human keratinocytes for the treatment of epidermo­lysis Bullosa simplex Mol. Ther. 18, 1624–1632.Google Scholar
  37. 37.
    Maniatis, T, Fritsch, EF, Sambrook, J. Molecular cloning, a laboratory manual. Cold Spring Harbor, New York, 1982.Google Scholar
  38. 38.
    Wang, P., Anton, M., Graham, F.L. and Bacchetti, S. (1995) High frequency recombination between loxp sites in human chromosomes mediated by an adenovirus vector expressing cre recombinase. Somat. Cell Mol. Genet. 21, 429–441.PubMedCrossRefGoogle Scholar
  39. 39.
    Vargas, J. J., Gusella, G.L., Najfeld, V., Klotman, M.E. and Cara, A. (2004) Novel integrase-defective lentiviral episomal vectors for gene transfer. Hum. Gene Ther. 15, 361–372.PubMedCrossRefGoogle Scholar
  40. 40.
    Grimm, D., Kern, A., Rittner, K. and Kleinschmidt, J. (1998) Novel tools for production and purification of recombinant adenoassociated virus vectors. Hum. Gene Ther. 9, 2745–2760.PubMedCrossRefGoogle Scholar
  41. 41.
    Clark, K., Liu, X., McGrath, J. and Johnson, P. (1999) Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses. Hum. Gene Ther. 10, 1031–1039.PubMedCrossRefGoogle Scholar
  42. 42.
    Kaludov, N., Handelman, B. and Chiorini, J. A. (2002) Scalable purification of adeno-associated virus type 2, 4, or 5 using ion-exchange chromatography. Hum. Gene Ther. 13, 1235–1243.PubMedCrossRefGoogle Scholar
  43. 43.
    Zhou, X. and Muzyczka, N. (1998) In vitro packaging of adeno-associated virus dna. J. Virol. 72, 3241–3247.PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  1. 1.Department of PediatricsUniversity of WashingtonSeattleUSA

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