Human immunodeficiency virus type 1 vectors efficiently transduce human hematopoietic stem cells - PubMed (original) (raw)
Human immunodeficiency virus type 1 vectors efficiently transduce human hematopoietic stem cells
R E Sutton et al. J Virol. 1998 Jul.
Abstract
Lentiviruses are potentially advantageous compared to oncoretroviruses as gene transfer agents because they can infect nondividing cells. We demonstrate here that human immunodeficiency virus type 1 (HIV-1)-based vectors were highly efficient in transducing purified human hematopoietic stem cells. Transduction rates, measured by marker gene expression or by PCR of the integrated provirus, exceeded 50%, and transduction appeared to be independent of mitosis. Derivatives of HIV-1 were constructed to optimize the vector, and a deletion of most of Vif and Vpr was required to ensure the long-term persistence of transduced cells with relatively stable expression of the marker gene product. These results extend the utility of this lentivirus vector system.
Figures
FIG. 1
(A) HIV vector constructs. The parent construct was HIV-AP (reference and data not shown), which is based upon NL4-3 (top). Deletions are indicated by delimited bars, and each frameshift mutation is indicated by x. In pHIV-PV, CMV designates the immediate-early cytomegalovirus promoter and polyA represents a cellular polyadenylation addition site. Genes are not precisely to scale. (B) HIV vectors with the 5′ LTR _Bsp_EI site removed (for PCR analysis) and with the marker human CD4 (for FACS analysis). See the text for details.
FIG. 2
Transduction increases if HSC are exposed to cytokines for 2 days. CD34+ or CD34+ Thy1+ cells were placed into cytokine-containing medium, and at 0, 24, and 48 h ultrafiltered HIV-APΔenv(VSV G) was added for an overnight incubation. For the bald virus, the VSV G expression plasmid was omitted from the original transfection. To inhibit reverse transcriptase, zidovudine (AZT) was added to a final concentration of 0.5 mM. All cell samples were fixed at 96 h, developed with BCIP/NBT, and scored visually for a brownish-purple color change. At least 100 cells were counted for each determination.
FIG. 3
Aphidocolin does not inhibit transduction. Increasing amounts of aphidocolin (Sigma) were added to CD34+ Thy1+ cells 1 day before overnight transduction with concentrated HIV-APΔenv(VSV G) and left in the medium until fixation was carried out. Transduction was scored by BCIP/NBT or Vector Red staining as specified by the manufacturer. Vector Red caused positive cells to be bright red, and they were easily visible by light microscopy.
FIG. 4
Transduced cells have a DNA content profile similar to that of untransduced cells. CD34+ cells were transduced overnight with ultracentrifuge-concentrated bald HIV-CD4 (left) or HIV-CD4(VSV G) (right). Two days later, the cells were analyzed by flow cytometry on a FACStar equipped with a UV laser. For CD4 measurements, the primary antibody was mouse anti-CD4–PE used at a 1:10 dilution. For DNA content measurement, the cells were incubated with 0.75 mM Hoechst dye 33342 (Molecular Probes) for 1 h at 37°C. Only live cells were quantified; they were gated by exclusion of the dye propidium iodide. N.D., not determined.
FIG. 5
Transduction is independent of the S phase. CD34+ cells were transduced overnight with ultracentrifuge-concentrated bald HIV-CD4 (mock) or HIV-CD4(VSV G) and at the same time incubated in the presence or absence of 30 μg of BrdU per ml. Two days later, the cells were fixed and incubated with mouse anti-BrdU followed by FITC-IgG. After further washing, the cells were incubated with mouse anti-CD4–PE (1:10 dilution) and analyzed by flow cytometry on a FACScan. Similar results were obtained in a 4-h experiment. BrdU-positive cells represent those that were in the S phase.
FIG. 6
Transduction by HIV-APΔenvΔR1(VSV G) results in cytotoxicity. CD34+ Thy1+ cells were transduced overnight with ultrafiltered bald or HIV-APΔenvΔR1(VSV G). Three days later, the cells were plated into methylcellulose. Colony types and AP staining were determined 2 weeks later. GM, granulocyte-macrophage; E, erythroid; mixed, mixed cell types. Only a few AP+ colonies were observed, and these were small compared to AP− colonies. This experiment was repeated twice more with CD34+ cells, and similar results were obtained.
FIG. 7
Expression of AP marker declines over time. CD34+ cells were transduced in triplicate overnight with ultracentrifuge-concentrated VSV G pseudotyped HIV vectors (Table 1). Note that the viruses HIV-AP G-P-E-F-V-, HIV-AP E-F-V-T-, HIV-AP E-F-V-R-, and HIV-AP E-F-V-R-T- were produced by cotransfection with pHIV-PV (Fig. 1A) and pME VSV G. After transduction, the cells were either plated out into methylcellulose and 3 weeks later stained for AP with BCIP/NBT or maintained in bulk culture in the presence of IL-3, IL-6, and stem cell factor and periodically stained for AP. No staining was observed for cells infected with bald HIV-APΔenvΔVifΔVpr (data not shown). For each construct, the percentage of AP+ colonies observed in methylcellulose was similar to what was seen in bulk culture. ∗, P < 0.0001 compared to HIV-APΔenvΔVifΔVpr; ∗∗, _P_ > 0.05 compared to HIV-AP E-F-V-R- but P < 0.0001 compared to HIV-APΔenvΔVifΔVpr; §, P < 0.0001 for both HIV-APΔR1 and HIV-AP E-F-V-R-T- compared to HIV-APΔenvΔ VifΔ Vpr (all results obtained by two-way balanced analysis of variance). Error bars have been omitted for clarity.
FIG. 8
PCR assays for vector proviruses in MC colonies. CD34+ cells were transduced overnight with ultracentrifuge-concentrated HIV-APΔenvΔVifΔVpr-BspE1(VSV G) or the bald control virus stock, treated with DNase 1 at 100 μg/ml in the presence of 5 mM MgCl2 for 24 h, and then plated into methylcellulose. (A) Colonies contain fully replicated forms of HIV provirus. In this experiment, 42% of the cells initially stained positive for AP. Three weeks later, DNA was prepared from individual colonies and PCRs were carried out with primers NL43-171U and NL43-831L as described in Materials and Methods. At that time, only 24% of the colonies stained positive for AP. A portion of each PCR product was digested with _Bsp_EI, size fractionated on a 1.2% agarose gel, transferred to Hybond N+ (Amersham), and hybridized to a 32P-labelled DNA probe encompassing the HIV-1 LTR. The filter was washed with 0.2× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate)–0.1% sodium dodecyl sulfate at 65°C for a total of 30 min and exposed to X-ray film for 20 min. First 12 lanes, bald virus; last 12 lanes, HIV-APΔenvΔVifΔVpr-BspE1(VSV G). The expected product size prior to _Bsp_EI digestion is 660 bp; after digestion, it is 523 bp. None of the first 12 and 10 of the last 12 reactions were judged to be positive. When β-globin control primers were used, only one sample was negative. (B) Colonies contain integrated forms of HIV provirus. In this experiment, the initial transduction rate was 65% and at 3 weeks 24% of the colonies stained positive for AP. DNA was prepared from individual colonies, and all 24 were positive when the β-globin control primers were used. None of the mock-transduced and all 12 of the HIV-APΔenvΔVifΔVpr-BspE1(VSV G)-transduced colonies were positive by PCR with the primers used in panel A. Samples were then subjected to Alu-PCR as described previously (4), and the products were size fractionated on a 1.5% agarose gel prestained with ethidium bromide. None of the mock-transduced and 10 of 12 HIV-APΔenvΔVifΔVpr-BspE1(VSV G)-transduced colonies gave the expected size of product, as indicated.
References
- Chatterjee S, Lu D, Podsakoff G, Wong K K. Strategies for efficient gene transfer into hematopoietic cells: the use of adeno-associated virus vectors in gene therapy. Ann N Y Acad Sci. 1995;770:79–90. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Medical