Hematology Branch, National Heart, Lung and Blood Institute, Bldg 10/Rm 7C218, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892-1652, USA1
Author for correspondence: Atsushi Handa. Fax +1 301 496 8396. e-mail handaa{at}nih.gov
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Abstract |
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Introduction |
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Much current interest in AAVs stems from their potential use as vectors for gene therapy; virtually all studies have employed recombinant (r) AAV-2. Although AAV-2 undergoes a lytic infection in the presence of helper virus, in the absence of helper virus, wild-type virus can integrate into the host cell genomic DNA. In addition, viral particles are heat stable and resistant to solvents and detergent, withstand a wide range of pH and temperature change and can easily be concentrated (Arella et al., 1990 ). AAV-2-based vectors have been shown to transduce both dividing and non-dividing cells (Podsakoff et al., 1994
) and cells transduced by AAV-2-based vectors can express functional proteins for many months after a single treatment (Monahan et al., 1998
). However, while AAV-2 has a broad host range, not all cells can be infected with AAV-2, possibly due to the absence of the AAV-2 cellular receptor (Mizukami et al., 1996
; Ponnazhagan et al., 1996
). We have shown previously that AAV-2 and AAV-3 do not compete with each other in cell-binding studies (Mizukami et al., 1996
) and we hypothesized that they bind to different cellular receptors and should have different cell tropisms. We therefore constructed rAAV-2 and rAAV-3 vectors expressing green fluorescent protein (GFP) and determined their relative ability to transduce haematopoietic cells. In addition, heparan sulphate and virus-overlay binding studies were performed to study further the differences between rAAV-2 and rAAV-3 vectors.
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Methods |
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CD34-positive cells were obtained from normal bone marrow by a proprietary avidinbiotin immunoaffinity process (Ceprate LC kit, CellPro); purity was checked by flow cytometry and was >90%. Cells were cultured in RPMI 1640 with 10% FCS and cytokines (IL-3, 50 ng/ml; IL-6, 50 ng/ml; and stem cell factor, 100 ng/ml). Lymphocytes were obtained from peripheral blood mononuclear cells and cultured in the presence of IL-2 (500 IU/ml) for 3 days prior to infection with rAAV.
Plasmids.
In order to facilitate subcloning, an EcoRI site was introduced into pAAV3 (the infectious AAV-3 plasmid) by PCR mutagenesis (QuikChange, Stratagene) between the inverted terminal repeat and the Rep coding region (nt 182187). Similarly, an XbaI site was introduced at the 3' end of the genome, before the inverted terminal repeat (nt 45124517). The complete coding sequence, excluding terminal repeats, was excised with EcoRI and XbaI and subcloned into pBluescript to produce the rAAV-3 packaging vector (pAAV3-pac).
A pAAV3-lacZ plasmid was produced by insertion of the human cytomegalovirus (CMV) promoter, a -galactosidase gene and the simian virus 40 (SV40) poly(A) sequence (nt 2641 and 8334506; pCMVbeta, Clontech) into the EcoRI/XbaI site of the mutated pAAV3 vector (Fig. 1a
). The pAAV3-GFP-neo plasmid was then constructed by replacing the
-galactosidase gene in pAAV3-lacZ with the GFP gene and the neomycin resistance gene. A 1·8 kb fragment was deleted from pAAV3-lacZ by digestion with EcoRI and EcoRV followed by blunt-ending with Klenow enzyme and self-ligation. The SalI site in the backbone sequence of the plasmid was deleted and a new SalI site was introduced at the 3' region of the SV40 poly(A) sequence by PCR mutagenesis, changing GGCTAC to GTCGA (pAAV3-lacZ/Sal). The 736 bp fragment that contains the GFP gene was obtained from phGFP-S65T (Clontech) by digestion with HindIII and XbaI and subcloned into pcDNA3 (Invitrogen). The resulting plasmid (pcDNA-GFP) was then digested with MluI and SalI to release a 3·7 kb fragment that contained the GFP gene with the bovine growth hormone poly(A) signal driven by the CMV promoter and the neomycin resistance gene with an SV40 poly(A) signal driven by the SV40 promoter (GFP cassette). This GFP cassette was cloned into previously produced pAAV3-lacZ/SalI so as to replace the
-galactosidase gene.
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Production and purification of rAAV-2 and rAAV-3.
Vector plasmids (10 µg) containing GFP and the appropriate packaging plasmid (2 µg) were co-transfected into COS-7 cells (106 cells in 400 µl) by electroporation (170 eV, 500 F; Gene Pulser, Bio-Rad). The cells were infected with Ad5 (m.o.i.=5) 24 h later, and the cells were harvested after 40 h. The monolayer was washed and adherent cells were dislodged by scraping, resuspended in PBS and lysed by six cycles of freezing and thawing. After ultrasonication, sodium deoxycholate was added to a final concentration of 0·5% and allowed to incubate for 30 min at 37 °C. CsCl was added to a density of 1·4 g/cm3 and rAAV and adenovirus were separated by ultracentrifugation at 35000 g for 40 h. The visible rAAV band was removed and a second CsCl purification was performed. To remove the CsCl, rAAV was dialysed against PBS for 24 h and the virus was stored at -80 °C until use.
Particle titres were measured for both rAAV-2 and rAAV-3 by DNA dot-blot hybridization. Filters were prepared by applying GFP-containing plasmids, rAAV-2 and rAAV-3 that had been denatured in 10 M NaOH, onto nylon membranes, baking for 2 h in a vacuum oven and hybridizing with a random-primed 32P-labelled probe (GFP segment obtained from double restriction enzyme digestion of the GFP/Neo plasmid with HindIII and XbaI). Genomic titres were calculated by comparison with dilutions of the GFP/Neo plasmid.
All preparations were tested for contamination by wild-type AAV-2 and AAV-3 by PCR amplification (data not shown) and were routinely negative. The biological infectious titre of preparations was calculated by using COS-7 cells: cells were infected with different concentrations of rAAV and the number of GFP-positive cells detected by fluorescence microscope (Nikon) was assessed 48 h post-infection. An m.o.i. of 1 was calculated as one AAV infectious particle per cell.
Virus titration and infection of cell lines.
Cells (104) were cultured in medium with 2% FCS to avoid overgrowth. After 6 h culture, the medium was removed from adherent cell lines and the monolayers were overlaid with virus-containing medium (m.o.i. of 5 or 100). For suspension lines, cells were pelleted by centrifugation and resuspended in virus-containing medium. After incubation with virus for 2 h at 4 °C, the cells were transferred to a 37 °C incubator for 15 min, fresh medium (100 µl) was added to the cells and the cells were cultured for 7 days in 96 well culture plates (Costar). The plates were examined for GFP-positive cells by fluorescence microscopy and the number of positive cells was calculated.
Inhibition of transduction by heparin.
To test the ability of heparin to compete with cell binding, COS-7 cells were grown in RPMI 1640 and rAAV-2 and rAAV-3 (m.o.i.=5) were added directly with different concentrations of heparin (Sigma). After 7 days incubation, the number of GFP-positive cells was estimated and inhibition was calculated.
Virus-overlay binding assay.
Virus-overlay assays with iodinated AAV-2 and AAV-3 were performed as described previously (Mizukami et al., 1996 ). Briefly, membrane proteins derived from HeLa S3 cells were separated by SDSPAGE and transferred to nitrocellulose and non-specific binding sites were blocked with 5% BSA in PBSTween prior to incubation with 125I-labelled AAV-2 and AAV-3 (1011 particles/ml). After extensive washing, virus binding was visualized by exposure to a phosphorimager screen (Molecular Dynamics) or X-ray film.
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Results |
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Transduction of erythroid/megakaryoblastoid cells
rAAV-2 was unable to transduce two erythroid cell lines, HEL and TF-1, and two megakaryoblastoid cell lines, Meg-01 and UT-7/Epo, even at high m.o.i. (Table 1). In contrast, rAAV-3 transduced HEL, TF-1, Meg-01 and UT-7/Epo cells, even at an m.o.i. of 5 (Fig. 3
). Neither vector transduced Mo-7e cells at either virus concentration.
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Discussion |
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The inability to transduce all haematopoietic cells with rAAV-2 led to our investigation of other potential AAV-based vectors (Muramatsu et al., 1996 ). We previously determined the complete nucleotide sequence of AAV-3 and showed not only that AAV-3 was serologically distinct from AAV-2 but that it also differed in transcription control elements, including the lack of a typical promoter sequence at position p40 and the presence of the consensus sequence for adenovirus-related transcription factor E4F binding within the upstream region of the p5 promoter (Muramatsu et al., 1996
). In addition, we have shown previously that binding of AAV-3 to cells was not competed for by excess AAV-2 virions, indicating that the two viruses use different antigens as their host cell receptor (Mizukami et al., 1996
). In the present study, using GFP expression, we confirm that both rAAV-2 and rAAV-3 transduce haematopoietic cells but that there is a difference between the vectors, with rAAV-2 transducing lymphoid cells preferentially and rAAV-3 transducing megakaryoblastoid cells preferentially. In addition, we show that there is a difference in the binding of the two viruses to both heparin and related molecules and to membrane proteins in the virus-binding overlay assay. The nature of the AAV-3-binding 42 kDa protein is currently unknown, but it is not fibroblast growth factor receptor or integrin
5, both described as putative co-receptors for AAV-2 (Qing et al., 1999
; Summerford et al., 1999
).
There is currently much interest in trying to transduce stem cells, including haematopoietic stem cells or CD34 cells, with gene therapy vectors. However, we were unable to transduce several myeloid cell lines, including primary CD34 cells, with either vector. Although some have reported that rAAV-2 can transduce CD34 cells (Fisher-Adams et al., 1996 ; Zhou et al., 1994
; Goodman et al., 1994
; Chatterjee et al., 1999
), we and others have subsequently not been able to confirm these results (Alexander et al., 1997
) and transduction of CD34 cells may be particularly inefficient, requiring high titres of virus. However, although further studies are required, our results suggest that rAAV-3 is unlikely to offer any benefit over rAAV-2 vectors as a vehicle for the delivery of genetic material to human CD34 cells.
In a report concerning a different isolate of AAV-3, non-haematopoietic adherent cell lines were tested with rAAV-3 carrying an alkaline phosphatase marker (Rutledge et al., 1998 ). No marked difference (<1 log) in transduction efficiency was noted between rAAV-2 and rAAV-3, although it was shown that the rAAV-3 vector could be used in the presence of anti-AAV-2 sera, suggesting that AAV-3 may have a role as a vector for readministration of genes in the presence of antibody to AAV-2. This is significant, as a recent study reported that 80% of healthy blood donors had anti-AAV-2 antibody and that 70% of them had the neutralizing antibody to AAV-2 (Erles et al., 1999
). Neutralizing antibody efficiently reduces the transduction rate of AAV-2 (Erles et al., 1999
), and one potential use of rAAV-3 vectors may be to evade this neutralization effect. In addition, our results, especially the finding that rAAV-3 vectors transduce haematopoietic cells that are resistant to standard rAAV-2-based vectors, including myeloid and megakaryocytic cells, potentially broaden the utility of the rAAV vectors.
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Footnotes |
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c Present address: Department of Molecular, Microbiology and Immunology, University of Missouri, Columbia, MO, USA.
d Present address: Division of Genetic Therapeutics, Jichi Medical School, Japan.
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Received 7 January 2000;
accepted 3 April 2000.