Human gene targeting by viral vectors

Abstract

Stable transduction of mammalian cells typically involves random integration of viral vectors by non-homologous recombination. Here we report that vectors based on adeno-associated virus (AAV) can efficiently modify homologous human chromosomal target sequences. Both integrated neomycin phosphotransferase genes and the hypoxanthine phosphoribosyltransferase gene were targeted by AAV vectors. Site-specific genetic modifications could be introduced into approximately 1% of cells, with the highest targeting rates occurring in normal human fibroblasts. These results suggest that AAV vectors could be used to introduce specific genetic changes into the genomic DNA of a wide variety of mammalian cells, including therapeutic gene targeting applications.

Fig. 1

Chromosomal neo gene correction by AAV vectors, a, Map of the AAV vector genomes used. AAV-SNori is the parent vector containing a functional neo gene. AAV-SNO39 and AAV-SNO648 are identical to AAV-SNori but contain the nt-39 or nt-648 mutations (sequence differences shown), respectively. The locations of the AAV terminal repeats (TR), promoters with transcription start sites (arrows), neo gene, p15A replication origin, polyadenylation site (pA), SalI and fused BamHI-BglII restriction sites, and the probe used in Southern analysis are indicated. b, Southern blot of genomic DNA from untransduced HSNO39 cells and 11 G418-resistant clones of HSNO39 cells transduced with AAV-SNO648, digested with BamHI and probed for neo sequences. The sizes of fragments hybridizing to neo in the parental HSNO39 line are indicated on the left. The positions of size standards are shown on the right. c, Southern analysis as in a but digested with BamHI and SalI. The corrected 2.7-kb fragments are indicated.

Fig. 2

Targeting of the human HPRT locus by AAV vectors, a. Structure of the AAV vectors used and the human locus. The locations of the AAV terminal repeats (TR), exons 2 and 3 of the HPRTgene, Alu repeats O, P, Q and R, and the probe used in Southern analysis are shown. The sequence differences between the wild-type AAV-HPe2/3 and mutant AAV-HPe2/3X vectors are indicated. The positions of relevant restriction sites are depicted in the chromosomal locus. The AAV-HPe2/3X mutation introduces a PvuI or BspCI site, b, Southern blot of genomic DNA from HT-1080 cells and 13 6TG-resistant clones derived from HT-1080 cells transduced with AAV-HPe2/3X, digested with HindIII and probed for HPRT sequences. The positions of size standards are indicated on the right. c, Southern analysis of genomic DNA as in b, but digested with HindIII and PvuI. The mark at 2.2 kb indicates the position of fragments containing the predicted HPRT modification. d. Southern blot of genomic DNA from MHF2 normal fibroblast clones digested with HindIII or HindIII plus BspCI and probed as in b. Two untransduced (MHF2-A and MHF2-B) and four 6TG-resistant clones transduced by AAV-HPe2/3X were analysed.

Fig. 3

Gene targeting is dependent on vector dose. HSNO39 (filled circles) or MHF2 (open squares) cells were infected with AAV-SNO648 and AAV-HPe2/3X, respectively at the indicated MOI (vector genomes per cell), and the percentage of cells with modified neo or HPRT genes was determined (as in Tables 1 and 2). The results of several experiments are combined with each symbol representing a single result.

Fig. 4

Gene targeting by transduction and transfection. HSNO39 cells were either transduced with the AAV-SNO648 vector at a MOI of 4 × 10⁵ vector genomes per cell or transfected with pASNO648 or pASNori2 plasmids, and the fraction of G418-resistant CFU was determined. Transfections were performed by plating 4 × 10⁶ cells per 15-cm dish on day 1, changing the media and transfecting with 20 µg of the indicated plasmid as a calcium-phosphate precipitate on day 2, treating with trypsin and replating dilutions of cells on day 3, and selecting with G418 on day 4 (as in the transduction experiments). pASNO648 was linearized by digestion with SacII before transfection. Mean values with standard errors are plotted.