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Oligodeoxyribonucleotide Uptake in Primary Human Hematopoietic Cells
Is Enhanced by Cationic Lipids and Depends on the Hematopoietic Cell Subset
By Ralf Kronenwett, Ulrich Steidl, Michael Kirsch, Georg Sczakiel, and Rainer Haas
The use of antisense oligodeoxyribonucleotides (ODN) is a
potential method to switch off gene expression. The poor
cellular uptake of ODN in primary cells still is a limiting factor
that may contribute to the lack of functional efficacy. Various
forms of cationic lipids have been developed for efficient
delivery of nucleic acids into different cell types. We examined the two cationic lipids DOTAP and DOSPER to improve
uptake of ODN into primary human hematopoietic cells.
Using a radiolabeled 23-mer, ODN uptake into blood-derived
mononuclear cells could be increased 42- to 93-fold by
DOTAP and 440- to 1,025-fold by DOSPER compared with
application of ODN alone. DOTAP was also effective for
delivery of ODN into leukocytes within whole blood, which
may resemble more closely the in vivo conditions. As assessed by fluorescein isothiocyanate–conjugated ODN both
cationic lipids enhanced cytoplasmic accumulation of ODN
in endosome/lysosome-like structures with a partial shift of
fluorescence to the whole cytoplasm and the nucleus following an incubation of 24 hours. ODN uptake by cationic lipids
into different hematopoietic cell subsets was examined by
dual-color immunofluorescence analysis with subset-specific monoclonal antibodies. We found a cell type–dependent
delivery of ODN with greatest uptake in monocytes and
smallest uptake in T cells. CD341 cells, B cells, and granulocytes took up ODN at an intermediate level. Uptake of ODN
into isolated CD341 cells could be increased 100- to 240-fold
using cationic lipids compared with application of ODN
alone. Stimulation of CD341 cells by interleukin-3 (IL-3), IL-6,
and stem cell factor did not significantly improve cationic
lipid-mediated ODN delivery. Sequence-specific antisense
effects in clonogenic assays could be shown by transfection
of bcr-abl oncogene-directed antisense ODN into primary
cells of patients with chronic myelogenous leukemia using
this established protocol. In conclusion, cationic lipids may
be useful tools for delivery of antisense ODN into primary
hematopoietic cells. These studies provide a basis for clinical
protocols in the treatment of hematopoietic cells in patients
with hematologic malignancies and viral diseases by antisense ODN.
r 1998 by The American Society of Hematology.
A
cytosol due to the limited capacity of the endocytotic process
and the lysosomal degradation. In the light of the ongoing
clinical studies, an increased stability and uptake of ODN into
hematopoietic cells could improve the efficacy of antisense
nucleic acid–based therapies. A variety of cationic lipids with
low toxicity are protective against degradation and can facilitate
the transport of ODN into different cell types mainly by
endocytosis even in the presence of human serum.16-19
In this study, we examined the delivery of phosphorothioatemodified ODN by cationic lipids into primary human hematopoietic cells, including CD341 cells using the cationic lipids
DOTAP and DOSPER under ex vivo culture conditions as well
as under conditions resembling the in vivo situation. For
quantitative analyses the experiments were performed with
32P-labeled ODN followed by liquid scintillation counting and
gel electrophoresis of cellular extracts. In addition, fluoresceinlabeled ODN were used to examine subcellular localization and
cell subset-dependent uptake. For demonstration of functional
effects of cationic lipid-mediated ODN delivery primary cells of
patients with chronic myelogenous leukemia (CML) were
treated by bcr-abl–directed ODN and suppression of clonogenic
growth was examined.
NTISENSE oligodeoxyribonucleotides (ODN) are capable
of downregulating gene expression and are used for the
assessment of gene function and for therapeutic purposes.1-3
Several clinical trials are ongoing with antisense ODN directed
to hematopoietic cells for the treatment of hematologic malignancies and viral diseases. For instance, a human immunodeficiency virus-1 (HIV-1)–directed antisense ODN is used for
systemic in vivo administration in patients with aquired immunodeficiency syndrome to protect normal T lymphocytes and
macrophages.4 In other clinical trials bcr-abl-, c-myb-, or
p53-directed antisense ODN were used for systemic therapy or
for ex vivo treatment of hematopoietic cells in patients with
acute and chronic myelogenous leukemia or advanced myelodysplastic syndrome.5-8 Functional efficacy of ODN requires not
only the selection of an appropriate target sequence9-12 but also
a sufficient intracellular concentration. The latter one depends
on the degree of cellular uptake, the intracellular distribution,
and the rate of degradation of ODN by serum and cytoplasmic
nucleases.1 Anionic ODN cannot diffuse through cell membranes, but are actively taken up by endocytosis.13-15 As a result,
only a small amount of extracellular ODN is available in the
From Klinische Kooperationseinheit Molekulare Hämatologie/
Onkologie and Forschungsschwerpunkt Angewandte Tumorvirologie,
Deutsches Krebsforschungszentrum, Heidelberg, Germany; and Medizinische Klinik und Poliklinik V, Universität Heidelberg, Heidelberg,
Germany.
Submitted May 29, 1997; accepted September 29, 1997.
Address reprint requests to Rainer Haas, MD, Medizinische Klinik
und Poliklinik V, Universität Heidelberg, Hospitalstr. 3, 69115 Heidelberg, Germany.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to indicate
this fact.
r 1998 by The American Society of Hematology.
0006-4971/98/9103-0016$3.00/0
852
MATERIALS AND METHODS
Cells. Human peripheral blood mononuclear cells (MNC) were
obtained from patients with hematologic malignancies or solid tumors
in complete remission. Cells were obtained by leukapheresis using a
Fenwal CS3000 (Baxter Deutschland, Munich, Germany) after cytotoxic chemotherapy supported with recombinant human G-CSF (RmetHuG-CSF; Amgen, Thousands Oaks, CA). Primary cells from
patients with CML were obtained from peripheral blood by vein
puncture. Red blood cells and cell debris were removed by density
centrifugation using the lymphocyte separation medium Lymphoprep
(Nycomed Pharma, Oslo, Norway) as previously described.20 Separation of CD341 cells from leukapheresis products was performed using
the miniMACS immunomagnetic separation system (Miltenyi Biotec,
Blood, Vol 91, No 3 (February 1), 1998: pp 852-862
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OLIGONUCLEOTIDE UPTAKE IN HEMATOPOIETIC CELLS
Bergisch Gladbach, Germany) according to the manufacturer’s instructions as previously described.20 For transfection experiments cells were
cultured in RPMI-1640-medium (CC Pro GmbH, Neustadt/Weinstrasse,
Germany) supplemented with 10% heat-inactivated fetal calf serum
(Biochrom, Berlin, Germany), 100 IU/mL penicillin (Life Technologies, Eggenstein, Germany), 100 µg/mL streptomycin (Life Technologies), 2 mmol/L L-glutamine (Life Technologies). For culture of CD341
cells, interleukin-3 (IL-3) (20 ng/mL), stem cell factor (SCF) (50
ng/mL), and IL-6 (20 ng/mL) were added to the culture medium in some
experiments. Cytokines were obtained from PromoCell (Heidelberg,
Germany).
Oligodeoxyribonucleotide synthesis and labeling. The ODN used
for transfection studies were derived from bcr-abl oncogene-directed
antisense nucleic acids and had the following sequences: antisenseb3a2(A): 58-GCTGAAGGGCTTTTGAACTCTGC-38,11 scrambledb3a2(A): 58-TTATTGAGGGTGATCCGCTAGCC-38, anti-sense-b3a2(B):
58-GCTGAAGGGCTTTTGAACTCTGCTTAAA-38,11 scrambledb3a2(B): 58-AGAGGTCACGCTTTTAGAGATTGCTTCA-38, antisense-b2a2: 58-CGCTGAAGGGCTTCTTCCTTATTGAT-38,21 scrambledb2a2: 58-TGGTCATACAGGCCTATTTCGTCTTG-38. In a data base
research using the softwares of the Heidelberg Unix Sequence Analysis
Resources (HUSAR, version 4.0; DKFZ, Heidelberg, Germany) no
homology of the used scrambled ODN with any human sequence of the
EMBL data bank was found. The ODN were obtained from Interactiva
(Ulm, Germany). They were synthesized using standard phosphoramidate chemistry. The two internucleotide linkages at the 38 and 58 end of
the ODN were phosphorothioates, and the internal deoxyribonucleotides were connected by phosphodiesters. For fluorescent labeling the
last coupling step was performed using fluorescein isothiocyanate
(FITC)-labeled amidite. The ODN were purified by reverse-phase
high-performance liquid chromatography and lyophilized after synthesis by the manufacturer. Before use ODN were resolved in HEPESbuffer (20 mmol/L, pH 7.4). Radioactive labeling of the 5-ends was
performed by phosphorylation as described.22 Briefly, 40 pmol of ODN
was incubated for 45 minutes at 37°C with 70 mmol/L Tris-HCl, pH 7.6,
10 mmol/L MgCl2, 5 mmol/L dithiothreitol, 150 µCi of [g-32P]ATP
(3,000 Ci/mmol; Amersham, Braunschweig, Germany) and 30 U of T4
polynucleotide kinase (New England Biolabs, Schwalbach/Taunus,
Germany) in a final volume of 40 µL. After heating to 68°C for 10
minutes the ODN were purified by gel filtration (Sephadex G-50;
Pharmacia, Freiburg, Germany). After precipitation with ethanol the
ODN were dissolved in TE-buffer (10 mmol/L Tris-HCl, pH 7.6; 1
mmol/L EDTA). Chemicals were obtained from Merck (Darmstadt,
Germany).
Treatment of cells by ODN and cationic lipids. Before transfection,
ODN were incubated with the respective cationic lipid for 15 minutes at
room temperature for formation of ODN/cationic lipid complexes.
Using DOTAP (N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate; Boehringer Mannheim, Mannheim, Germany), 3
µg ODN (corresponding to a final concentration of a 23-mer ODN in
500 µL cell suspension of 0.84 µmol/L) was mixed with 15 µL cationic
lipid (1 µg/µL) and HEPES-buffer (20 mmol/L, pH 7.4) to a final
volume of 75 µL. Using DOSPER (1,3-Di-Oleoyloxy-2-(6-Carboxyspermyl)-propyl-amid; Boehringer Mannheim), 5.4 µg ODN (final
concentration: 1.5 µmol/L) was mixed with 14 µL cationic lipid (1
µg/µL) and HBS-buffer (HEPES 20 mmol/L, pH 7.4; NaCl 150
mmol/L) to a final volume of 100 µL. When using an amount of ODN
differing from that given above, the amount of cationic lipid was altered
correspondingly. For transfection of radioactively labeled ODN, the
specific activity of oligonucleotides was 3 3 109 cpm/µmol. After 15
minutes the ODN/cationic lipid mixture was added dropwise to the cell
suspension which was incubated at 37°C for at least 30 minutes before
transfection. MNC as well as CD341 cells were incubated in a final
volume of 500 µL at a concentration of 1 3 106 cells/mL. Cells were
incubated with the ODN between 30 minutes and 27 hours at 37°C.
853
After incubation, the cells were washed at 4°C once with PBS-buffer
(KCl 0.2 g/L; KH2PO4 0.2 g/L; NaCl 8.00 g/L; Na2HPO4 1.15 g/L) and
with PBS-buffer containing 1% bovine serum albumin, respectively. To
remove ODN bound to the cell membrane the acid-salt elution method
was used as described.23 Briefly, the cell pellet was suspended in an
ice-cold solution containing 0.5 mol/L NaCl and 0.2 mol/L acetic acid
(pH 2.5), incubated at 4°C for 10 minutes and centrifuged at 1,000g for
5 minutes. After washing and acid-salt elution, the cells were either
treated with proteinase K lysis buffer (10 mmol/L Tris-HCl, pH 8.3; 50
mmol/L KCl; 1.5 mmol/L MgCl2; 1.5% Tween-20; 1 µg/mL Proteinase
K) for measurement of cell-associated radioactivity or washed with
PBS-buffer for flow cytometry.
For assessment of cellular uptake of the FITC-molecule cells were
incubated for 2 and 24 hours with 0.3 µmol/L FITC (Aldrich, Steinheim,
Germany) in the presence or absence of cationic lipids.
For transfection of ODN into cells within whole blood, 500 µL of
blood anticoagulated by EDTA was incubated in the presence or
absence of cationic lipids. The incubation was performed using a rotator
to avoid sedimentation of blood cells. After incubation, 100 µL of
samples was mixed with 1.9 mL Becton Dickinson 1 3 FACS lysing
solution (Becton Dickinson, Heidelberg, Germany) for 5 minutes at
room temperature for lysis of erythrocytes. The cells were washed,
resuspended in proteinase K lysis buffer, and analyzed by scintillation
counting.
Quantification of cell-associated ODN. To determine cell-associated radioactivity cell pellets were incubated with 40 µL of the
proteinase K lysis buffer for 1 hour at 56°C. Twenty microliters was
analyzed by counting in a liquid scintillation counter (Beckman
Instruments, Fullerton, CA) with 1 mL of liquid scintillation cocktail
(Ready Safe; Beckman Instruments). Cellular ODN uptake was expressed as pmol/106 cells. Lysates were examined by polyacrylamide
gel electrophoresis under denaturing conditions (12% polyacrylamide
gel containing 7 mol/L urea in 89 mmol/L Tris-borate buffer, pH 8.3) to
determine the proportion of degraded ODN as well as of removed 58
phosphate residues. Gels were dried and exposed to x-ray film. Gels
were scanned with a PhosphorImager (Molecular Dynamics, Krefeld,
Germany) and band intensities were measured using the IMAGE
QUANT software (Molecular Dynamics).
Time-dependent decrease of cell-associated full-length ODN was
computer-fitted by nonlinear regression curves with single- and doubleexponential decays. Best fit was achieved using a double exponential
function. The curves were compatible to an at least two-phase or
compartment model which can be described by a function of the form:
C(t) 5 Ae-k1t 1 Be-k2t. C(t) is the amount of full-length ODN at time t,
while A and B indicate the amount of full-length ODN at t 5 0 in each
phase or compartment. The coefficients k1 and k2 are the rate constants
for the decay of full-length ODN from the respective compartment.24
The half-life of ODN in each compartment was obtained from the
equation: t1/2 5 ln2 / k.25 The overall half-life of intracellular full-length
ODN was calculated from the double-exponential function assuming
that C(t1/2) 5 C(0)/2.
Immunofluorescence staining and flow cytometry. After washing
1 3 106 transfected cells were stained with the phycoerytherin
(PE)-conjugated monoclonal antibodies (MoAbs) CD7 (clone 3A1;
Sigma, Deisenhofen, Germany), CD13 (clone L138; Becton Dickinson,
Heidelberg, Germany), CD15 (clone 80H5; Immunotech, Marseille,
France), CD19 (clone 467; Becton Dickinson), or CD34 (clone 8612;
Becton Dickinson) in 500 µL PBS at 4°C for 30 minutes. Isotypeidentical MoAbs served as control (IgG1-PE, Becton Dickinson;
IgG2a-PE, Becton Dickinson; IgM-PE, Immunotech). After antibody
staining, cells were washed and suspended in PBS-buffer. For determination of the proportion of viable cells, propidium iodide (Sigma) was
added at a final concentration of 10 µg/mL before analysis. The cells
were analyzed using a Becton Dickinson FACScan with a 2-W argon
ion laser. Fluorescence was measured using 530/15 nm (FITC) and
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854
KRONENWETT ET AL
575/36 nm (PE) band pass filter. Data were analyzed using the Becton
Dickinson Lysis II software after gating on viable cells.
For fluorescence microscopy, FITC-ODN transfected cells were
transferred onto slides by centrifugation (1,000 rpm, 5 minutes) with a
Shandon Cytospin3 (Life Sciences International, Frankfurt am Main,
Germany). In some experiments cells were fixed using 2% paraformaldehyde for 20 minutes. For staining of nuclei, slides were washed once
for 5 minutes with 2 3 SSC (20 3 stock solution: 3 mol/L NaCl; 0.3
mol/L sodium citrate, pH 7.0), once for 10 minutes with 2 3 SSC
containing 0.2 µg/mL 48,68-Diamidino-2-Phenylindole (DAPI; Sigma),
and once for 5 minutes with 2 3 SSC containing 0.05% Tween 20. After
staining, cells were embedded under cover slips in Vecta-Shield
mounting medium (Vector Laboratories, Burlingame, CA). Analysis
was performed using a fluorescent microscope (Zeiss Axioskop, Jena,
Germany).
Clonogenic assay of primary CML cells transfected by ODN.
Mononuclear cells from peripheral blood from patients with CML were
incubated two times with an interval of 16 hours by ODN (final
concentrations: 1 µmol/L in first and 0.5 µmol/L in second incubation)
using the cationic lipid DOTAP as described above. After transfection
the cells were seeded in methylcellulose growth medium (MethoCult
H4433; StemCell Technology, Vancouver, Canada) at a density between
5 3 104 and 2 3 105 cells/mL.21,26 The colony numbers (colonyforming unit granulocyte/macrophage [CFU-GM], burst-forming unit
erythrocyte [BFU-E]) were counted after 2 weeks. The type of bcr-abl
fusion point in each sample was determined by polymerase chain
reaction following reverse transcription as described.27
RESULTS
The use of radioactively labeled ODN provides a suitable
method to study ODN uptake into cells in vitro, as it permits to
measure the amount and the proportion of internalized fulllength ODN.28 Still, it cannot be distinguished between uptake
into living and dead cells or specific uptake into the different
cell types of blood-derived MNC. FACS analysis permits the
assessment of uptake of FITC-labeled ODN on a single cell
level, while the intracellular localization and time-dependent
distribution can be examined by fluorescence microscopy.16,29-31
In this study, these methods were used to address quantitative as
well as qualitative aspects of ODN uptake into primary human
hematopoietic cells.
Improvement of cellular uptake of ODN by DOTAP and
DOSPER. The radioactively labeled phosphorothioate-modified 23-mer scrambled-b3a2(A) ODN was used for quantitative
analysis of ODN uptake into blood-derived MNC by means of
DOTAP and DOSPER. The following parameters were evaluated: (1) the weight ratio of ODN to cationic lipid,32 (2) the
dependency on ODN concentration, and (3) the time course of
cationic lipid-mediated ODN uptake.
Cells were incubated for 2 hours with 0.3 µmol/L radioactively labeled ODN complexed to different amounts of DOTAP
resulting in lipid/ODN ratios (µg/µg) between 1:2 and 10:1.
Before analysis, cells were washed according to the acid-salt
method to remove extracellularly membrane-bound ODN.23
The cell-associated radioactivity varied between 3 and 30
pmol/106 cells with a maximum when the DOTAP/ODN ratio
was between 5:1 and 7:1 (µg:µg) (data not shown). This
corresponds to a charge ratio between 2:1 and 3:1 (1/2).
Having defined the best DOTAP/ODN ratio, cells were incubated with increasing amounts of ODN/DOTAP complexes up
to the toxicity limit of 30 µg DOTAP/mL (Fig 1A). The
Fig 1. Effect of cationic lipids on cell-associated ODN. Bloodderived mononuclear cells were incubated for 2 hours with increasing
amounts of radiolabeled ODN in the absence (X) or presence (W) of
DOTAP (A) or DOSPER (B). The amount of cell-associated ODN was
measured by liquid scintillation counting of cellular extracts. (C)
Analysis of extracts from cells incubated with ODN/DOSPER complexes by denaturing 12% polyacrylamide gel electrophoresis. Fulllength 23-mer ODN are indicated.
cell-associated radioactivity was dose-dependent without reaching a plateau. The peak value of 120 pmol/106 cells was
observed at 0.84 µmol/L ODN which corresponds to 7.2 3 107
ODN molecules per cell. The increase of DOTAP-mediated
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OLIGONUCLEOTIDE UPTAKE IN HEMATOPOIETIC CELLS
ODN uptake varied from 42- to 93-fold (mean, 70-fold;
standard deviation [SD], 26) in comparison with the application
of ODN alone. DNA extracts of transfected cells were analyzed
by polyacrylamide gel electrophoresis to measure the proportion of full-length ODN (Fig 1C). As shown by x-ray films and
PhosphorImager analysis, the proportion of full-length ODN to
whole cell–associated radioactivity was more than 80%.
For the assessment of the time course of ODN uptake, cells
were incubated between 30 minutes and 27 hours with 0.3
µmol/L ODN complexed to DOTAP (Fig 2). Approximatly two
thirds of the maximum cell-associated radioactivity was observed within the first 2 hours of incubation. During the
following 22 hours intracellular ODN accumulated at a significantly lower rate, suggesting that an incubation time between 2
and 8 hours is sufficient for delivery of ODN by DOTAP into
blood-derived MNC.
Using DOSPER the uptake of ODN in MNC could also be
improved significantly, but some parameters were different
from the results obtained with DOTAP. The maximum of
cell-associated radioactivity was found at a DOSPER/ODN
ratio of 2.6:1 (µg:µg) which corresponds to a charge ratio of 3:1
(1/2). There were 800 pmol ODN associated with 106 cells
(4.8 3 108 molecules/cell) when the ODN concentration was
1.5 µmol/L (Fig 1B), and the DOSPER-mediated increase of
uptake varied between 440- and 1,025-fold (mean, 690-fold;
SD, 220) compared with the application of ODN alone. The
time course of ODN uptake by DOSPER was also different in
comparison with DOTAP, as the cell-associated radioactivity
reached a maximum after 2 hours of incubation (Fig 2) followed
by a decrease as a function of time. In addition, uptake of the
radioactively labeled 26-mer scrambled-b2a2 as well as the
28-mer scrambled-b3a2(B) ODN with different base compositions was measured to look for a sequence dependency of ODN
delivery. No differences in cationic lipid-mediated uptake were
Fig 2. Time course of cationic lipid-mediated ODN uptake in
blood-derived mononuclear cells. Cells were incubated with 0.3
mmol/L radiolabeled ODN complexed to DOTAP (X) or DOSPER (W)
for the indicated times. Means and standard deviations of three
independent experiments are presented.
855
observed between the 23-mer, the 26-mer, and the 28-mer ODN
(data not shown).
Subcellular localization of ODN after transfection with
cationic lipids. The functional activity of antisense ODN
depends on their access to the cellular compartment of the
biological target. Therefore, the subcellular localization and
intracellular trafficking of ODN was evaluated by fluorescence
microscopy using FITC-labeled 23-mer scrambled-b3a2(A)
ODN (Fig 3). Incubation of blood-derived MNC with ODN/
DOTAP complexes resulted in a faint cytoplasmic stain of
lymphoid cells at a circumscript perinuclear area on one side of
the cell (Fig 3A). In contrast, approximately 90% of cells with
monocyte appearance showed a bright fluorescent staining with
a spotted distribution within the cytosol (Fig 3B). Although
there was no nuclear fluorescence found after 2 hours of
incubation, a homogenous nuclear stain with a pronounced
accumulation in the nucleoli was observed after 24 hours (Fig
3C). In approximately 80% of cells the cytoplasmic stain
persisted in spotted endosome/lysosome-like structures after 24
hours (Fig 3D), whereas the other cells developed a homogenous cytoplasmic fluorescence (Fig 3E). Similar intracellular
distributions of ODN were obtained with DOSPER except for
the difference that the proportion of cells with a homogenous
cytoplasmic fluorescence after 24 hours reached 50% (data not
shown). There was no difference between fixed and unfixed
cells, indicating that fixation does not influence subcellular
localization of ODN.
Transfection of ODN by cationic lipids using whole peripheral blood. The data presented are helpful for the design of ex
vivo transfection protocols of ODN into MNC. On the other
hand, antisense ODN can also be used for systemic intravenous
administration. Therefore, we measured uptake of radioactively
labeled ODN in cells of whole blood which may resemble more
closely the in vivo conditions. The peripheral blood was
anticoagulated by EDTA, because heparin is a negatively
charged macromolecule that may interfere with cationic lipids.33,34 In comparison with the use of ODN alone, the uptake
into leukocytes was approximately 30-fold and 20-fold greater
using DOTAP and DOSPER, respectively (Fig 4). Polyacrylamide gel analyses of DNA extracted from transfected cells
were performed to exclude that the radioactive label was
displaced from the ODN by serum phosphatases and nucleases.
PhosphorImager analyses of gels showed that the proportion of
full-length ODN to whole cell–associated radioactivity was
more than 80% in each sample (data not shown). As shown by
FACS analysis with FITC-labeled ODN complexed to cationic
lipids, the fluorescence staining was only found in leukocytes,
whereas the erythrocytes were negative (data not shown).
Cell subset-dependent ODN uptake. Data obtained from
uptake studies using radioactively labeled ODN provide mean
values of cell-associated ODN of all cell subsets in the MNC
fraction. Therefore, DOTAP-mediated uptake of FITC-labeled
ODN was examined by dual-color immunofluorescence analysis with subset specific PE-conjugated MoAbs. The results of
one representative experiment of three experiments are shown
in Figs 5 and 6. As assessed by propidium iodide staining, the
proportion of dead cells was less than 5%, reflecting the low
toxicity of the transfection. The mean FITC-fluorescence of all
cells was approximately 35-fold greater compared with ODN
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856
KRONENWETT ET AL
Fig 3. Subcellular localization of FITC-labeled ODN. After transfection of 0.3 mmol/L FITC-labeled ODN using DOTAP, cells were transferred on
slides by centrifugation, stained by DAPI, and analyzed by fluorescence microscopy. The upper row shows the DAPI stained nuclei, the lower row
the intracellular distribution of FITC-labeled ODN. (A and B) Representative cells after incubation with ODN/DOTAP complexes for 2 hours. (C
through E) Representative cells after an incubation time of 24 hours.
treatment alone (Fig 5). As assessed by sideward scatter gating,
greatest uptake of ODN into monocytes could be seen in both
samples, with and without DOTAP. This was confirmed by the
antibody staining of lineage-specific antigens (Fig 6). Monocytes, as assessed by gating on CD131 and CD152 cells, had the
Fig 4. Effect of DOTAP and DOSPER on ODN uptake into leukocytes using whole blood. Cells were incubated for 2 hours with 0.3
mmol/L radiolabeled ODN complexed to cationic lipids. After lysis of
erythrocytes, radioactivity associated with white blood cells was
measured by liquid scintillation counting. The results from two
experiments are indicated.
greatest cell-associated fluorescence intensity. The smallest
uptake was observed in CD71 T cells, while the uptake was
intermediate in CD151 myeloid cells, in CD191 B cells, as well
as in CD341 hematopoietic progenitor and stem cells.
After a 24-hour incubation of cells with ODN/DOTAP
complexes, the mean FITC-fluorescence further increased approximately 4-fold in CD131 and CD151 cells and approximately 2.5-fold in CD341 cells. In contrast, uptake of ODN into
CD71 and CD191 lymphocytes was not enhanced following the
longer incubation time (data not shown). Small and large cells
were gated within each subpopulation to exclude that the
different efficiency of uptake among the subsets was related to
cell volume. The proportion of cell subsets was 78% for CD131
cells, 52% for CD151 cells, 24% for CD191 cells, 20% for
CD71 cells, and 1.5% for CD341 cells. We found no correlation
between the proportion of each cell subset and ODN uptake.
The same subset-specific ODN uptake was seen using mononuclear cells from patients with CML in chronic phase as well
as using DOSPER instead of DOTAP (data not shown).
Incubation of cells with FITC in the presence or absence of
cationic lipids showed no increased cellular fluorescence after 2
hours as well as after 24 hours in comparison with untreated
cells.
ODN uptake in CD341 cells. For application of antisense
ODN in ex vivo treatment protocols uptake into enriched
CD341 hematopoietic progenitor cells is of special interest.
Therefore, delivery of ODN by cationic lipids into isolated
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OLIGONUCLEOTIDE UPTAKE IN HEMATOPOIETIC CELLS
857
Fig 5. Analysis of ODN uptake into blood-derived mononuclear cells by flow cytometry.
Cells were incubated for 2 hours
with 0.3 mmol/L FITC-labeled
ODN in the absence (B) or presence (C) of DOTAP. (A) Background fluorescence of cells
without ODN incubation. The
relative mean fluorescence intensities (Ym) are indicated.
blood-derived CD341 cells was examined using radioactively
labeled ODN. After an incubation of CD341 cells with the
ODN/cationic lipid complexes for 2 hours, the cell-associated
radioactivity was increased 100-fold (SD, 6) using DOTAP and
240-fold (SD, 32) using DOSPER compared with application of
ODN alone, respectively (data not shown). Isolated CD341
cells were also cultured in the presence of IL-3, IL-6, and SCF
for 48 hours before transfection to examine whether ODN
uptake is greater after cellular activation. The cytokines were
chosen, as they stimulate proliferation of CD341 cells without
loss of long-term culture potential.35 Still, stimulation with
growth factors did not significantly improve the cationic
lipid-mediated ODN uptake in comparison with unstimulated
CD341 cells (Fig 7). As assessed by trypan blue staining and a
colony-forming assay in semisolid culture medium, DOTAP
and DOSPER had no toxic effects on CD341 cells after an
incubation time of 10 hours with the cationic lipid/ODN
complexes (data not shown).
Kinetic analyses of cell- and medium-associated radioactivity were performed to determine the intracellular half-life of
ODN in CD341 cells and the efflux rate of cell-associated
radioactivity. Radioactively labeled ODN/DOSPER complexes
were added to CD341 cells for 2 hours, the medium was
removed and the cells were incubated with fresh medium
devoid of ODN. The time courses of cell-associated as well as
medium-associated radioactivity were measured. The cell-
associated radioactivity decreased, while the extracellular radioactivity increased as a function of time (Fig 8A). As shown by
PhosphorImager analysis of polyacrylamide gels of cellular
DNA extracts, the cell-associated ODN degraded during incubation. The proportion of full-length ODN to whole cellassociated radioactivity decreased from 80% to 55% after 24
hours (Fig 8B). Degradation products were more than 95%
mononucleotides or phosphate residues without successively
shortened ODN (data not shown). These results indicate a very
fast degradation after removal of the 58 or 38 cap-phosphorothioates. Multiplication of the functions of cell-associated radioactivity and of proportion of full-length ODN resulted in a curve
reflecting the kinetics of full-length ODN (Fig 8B), which is a
relevant parameter with regard to the efficacy of antisense
ODN. The kinetics best fit to a bi-exponential function in which
the exponential terms represent at least two compartments or
phases.24,25 The ODN half-lives of the individual phases or
compartments were calculated from the computer fit. The first
phase was rapid with a half-life of approximately 2 hours,
whereas the second phase was slow, reflected by a half-life of
approximately 30 hours. This resulted in an overall half-life of
full-length ODN in CD341 cells of about 10 hours.
Using DOTAP a similar bi-exponential curve with the same
overall half-life of full-length ODN was found (data not
shown).
Fig 6. Subset dependent ODN
uptake into primary blood-derived mononuclear cells. Cells
were incubated for 2 hours with
0.3 mmol/L FITC-labeled ODN
complexed to DOTAP and stained
with the indicated PE-conjugated
lineage-specific antibodies before FACS-analysis. Cells were
analyzed after gating on the lineage-specific antibody staining.
The background fluorescence
with an isotype-specific control
antibody is shown in the first dot
blot. The relative mean FITC fluorescence intensities (Ym) are indicated.
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
858
KRONENWETT ET AL
target mRNA. Colony formation of bcr-abl2 cells was not
influenced after transfection of ODN by DOTAP. The transfection procedure alone had also no effect on cell proliferation.
DISCUSSION
In this report we show that ODN uptake into human
blood-derived MNC can be greatly improved by cationic lipids.
Because primary hematopoietic cells are target cells for the
treatment of patients with hematologic diseases and viral
infections by antisense ODN,4-8 an efficient delivery of nucleic
acids is required to obtain functional effects. We found an
increase of ODN uptake by the cationic lipids DOTAP and
DOSPER between 30- and 800-fold in comparison to the values
achieved with ODN alone. This enhancement is significantly
greater than that observed for other cell types. For instance,
Fig 7. Influence of cellular activation of CD341 cells on cationic
lipid-mediated ODN uptake. Cells were incubated for 2 hours with 0.3
mmol/L radiolabeled ODN complexed to DOTAP (N) or DOSPER (M)
with or without a 48-hour preculture in IL-3, IL-6, and SCF supplemented medium. The amount of cell-associated ODN was measured
by liquid scintillation counting. The results of two experiments are
indicated.
Functional effects of DOTAP-mediated ODN delivery. ODN
used in the experiments presented were derived from antisense
sequences directed against the CML-related bcr-abl oncogene.
The CML can serve as a model for antisense ODN-mediated
inhibition of leukemic cell growth, because the bcr-abl rearrangement of the Philadelphia-chromosome (Ph1) that results from a
reciprocal translocation between chromosomes 9 and 22 leads
to the expression of a pathological p210 bcr-abl fusion protein
in more than 90% of patients. Depending on whether the second
exon of the abl gene fuses with bcr exon 2 or 3, two different
types of bcr-abl mRNA (b2a2 and b3a2) are found in most cases
of CML.36
The functional effects of ODN transfected by DOTAP was
examined using primary hematopoietic cells from 9 patients
with CML. Seven patients were in chronic phase without
previous treatment, 1 patient was in accelerated phase, and 1 in
blast crisis. Mononuclear cells were obtained from peripheral
blood and the type of bcr-abl fusion point was determined by
polymerase chain reaction following reverse transcription of
cellular RNA. The b3a2 fusion point-directed antisenseb3a2(B) ODN and the b2a2-directed antisense-b2a2 ODN as
well as the respective control ODN with the same nucleotide
compositions in scrambled sequence (scrambled-b3a2(B),
scrambled-b2a2) were used to assess the antiproliferative
activity on the patients’ cells in clonogenic assays. ODN were
transfected two times with an interval of 16 hours at concentrations of 1 µmol/L and 0.5 µmol/L, respectively. The b3a2
antisense sequence was chosen based on kinetic in vitro
selection to achieve specific hybridization with the b3a2 bcr-abl
fusion sequences while sparing the wild-type sequences bcr or
abl.11 In 3 of 9 cases, fusion point-specific inhibition of colony
formation was observed ranging between 39% and 72% at a low
variation within the experiments performed in duplicates (Table
1). In none of the 9 cases, any inhibitory effects were observed
with scrambled control sequences or alternative junctional
antisense ODN; ie, inhibition was only measured with bcr-abl
junction-specific antisense ODN in cells with the appropriate
Fig 8. (A) Time course of extracellular (X) and cell-associated (W)
radioacivity in CD341 cells. After a 2-hour incubation with radiolabeled ODN/DOSPER complexes the medium was removed and cells
were resuspended in fresh medium. Radioactivity was measured at
indicated time points by liquid scintillation counting. (B) Time course
of cell-associated full-length ODN (X). The proportion of full-length
ODN to the whole cell-associated radioactivity as determined by
PhosphorImager analysis of a polyacrylamide gel is shown as a
function of time by (n). Multiplication of this function with the
function of cell-associated radioactivity (W) resulted in the curve
indicating the time course of cell-associated full-length ODN.
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OLIGONUCLEOTIDE UPTAKE IN HEMATOPOIETIC CELLS
859
Table 1. Fusion Point-Specific Inhibition of Clonogenic Growth of
Primary Cells From Three Patients With CML by bcr-abl–Directed
Antisense ODN
Colony No. as Percentage of Untreated Control
Phase of
Disease
Fusion
Point
DOTAP
Antisense
b3a2
Scrambled
b3a2
Antisense
b2a2
Scrambled
b2a2
CP
CP
AP
b3a2
b2a2
b2a2
90/83
123/128
109/85
49/61
104/107
117/120
83/78
95/97
117/99
85/95
28/35
61/52
83/83
102/104
129/88
Mononuclear blood cells were seeded in semisolid growth medium
after double transfection of ODN (concentrations in first and second
transfection: 1 µmol/L and 0.5 µmol/L, respectively) by DOTAP. The
mean value of colony numbers (CFU-GM, BFU-E) after 2 weeks
compared with untreated controls are indicated. Significant reduction
of colony numbers is marked by bold letters.
Abbreviations: CP, chronic phase; AP, accelerated phase.
Capaccioli et al17 showed a 25-fold increase of ODN uptake into
a lymphoblastic cell line when DOTAP was added. Other
groups described a 2-to 10-fold greater ODN uptake by cationic
lipids into primary endothelial cells as well as leukemic and
cervical cancer cell lines16,28,37 when compared with an incubation of ODN alone. On the other hand, there was no effect of
cationic lipids on the uptake and inhibitory activity of antisense
ODN in primary keratinocytes.29 Using FITC-labeled ODN, we
found that ODN delivery was dependent on the cell type with
smallest uptake into T cells. Uptake into monocytes was
30-fold, into CD341 cells, B cells, and granulocytes about
2-fold greater compared with T cells, while there was no
measurable uptake into erythrocytes. Still, an exact quantification of FITC-labeled ODN uptake is difficult because the
emission intensity of FITC is pH dependent.24,38 Therefore, in a
cellular compartment with low pH, such as lysosomes, the
amount of intracellular FITC-labeled ODN might be underestimated by FACS analysis.
The great variability of uptake obtained with different cell
types and lipids may be related to two essential steps of ODN
uptake into cells: (1) The interaction of the positively charged
cationic lipid/DNA complex with anionic residues on the cell
surface39 and (2) the endocytosis of the complex by the
cells.18,40 There is some evidence that membrane-associated
sulfated proteoglycans are involved in the transfection by
cationic lipids.41 Proteoglycans consist of a core protein covalently linked to one or more sulfated glycosaminoglycans.42
Hematopoietic cells have a different expression pattern of the
hematopoietic proteoglycan core protein (HpPG) dependent on
the lineage and state of differentiation.43 Monocytes, eosinophils, and basophils express the HpPG at higher levels than
lymphocytes, neutrophils, and immature myeloid cells. Therefore, a variable expression of proteoglycans on different cell
types may influence the susceptibility of cells to transfection.
Assuming that ODN/cationic lipid complexes are taken up by
endocytosis,18,40 the differences observed between the cell
subsets could also be explained by a greater endocytosis activity
of monocytes compared with T or B lymphocytes.44 A similar
cell subset–dependent ODN delivery without the use of transfection reagents was described for human and murine hematopoietic cells.31,45 In contrast to the data of Zhao et al,31 who used
ODN alone, we could not observe an increased cellular uptake
of ODN/cationic lipid complexes into CD341 cells following
incubation with a cocktail of the cytokines IL-3, IL-6, and SCF.
This could indicate that uptake of cationic lipid/ODN complexes in contrast to ODN alone is independent from cellular
activation of CD341 cells. The subset dependent uptake is
important with respect to clinical application of antisense ODN.
DOTAP and DOSPER may be less efficient in delivery of ODN
into HIV-1–infected T cells compared with macrophages and
monocytes. Additionally, our data suggest a significant benefit
of cationic lipids for uptake into silent and activated CD341
cells which is of relevance for ex vivo purging protocols of
hematopoietic stem cells.
The difference of the transfection efficacy between DOTAP
and DOSPER may be related to the tetravalent cationic
structure of the DOSPER molecule, while DOTAP is a monocationic liposomal reagent. The polycationic lipid may lead to a
better formation of the lipid/ODN complex and improved
interaction between the complex and the negatively charged cell
surface. The initial peak after 2 hours with subsequent decrease
of cell-associated radioactivity using DOSPER in comparison
with continuous increase of ODN uptake using DOTAP may be
related to a lower stability of the DOSPER molecule compared
with DOTAP. Therefore, an efficient uptake can be observed
during the first 2 hours of the transfection procedure, but
afterwards metabolization and excretion of cell-associated
radioactivity may overcome the uptake of extracellular ODN as
a result of destabilization of ODN/DOSPER complexes. This
hypothesis is supported by the reduced efficacy of ODN uptake
by DOSPER compared with DOTAP using whole blood conditions instead of the isolated mononuclear cell fraction. Differences in transfection efficacies depending on the chemical
structure of the cationic lipids have been also described for
other liposomes.46
Nucleic acid/cationic lipid complexes are mainly internalized
via endocytosis.18,40 The mechanism of release of the nucleic
acid from the endosome to the cytoplasm as well as the
intracellular trafficking are poorly understood. Recently, a
model for intracellular release of DNA from cationic liposomes
into the cytoplasm was proposed.33,34 After internalization via
endocytosis the DNA/cationic lipid complex destabilizes the
endosomal membrane as a result of a flip-flop exchange
between anionic and cationic lipids. Thereby, the DNA is
displaced from the complex allowing the ODN to diffuse into
the cytoplasm. Alternatively, full-length ODN could also be
released immediately from endosomes to the extracellular
compartment by exocytosis24,25,47 or the DNA/cationic lipid
complex is transferred to lysosomes, where the nucleic acid is
rapidly degraded by nucleases. Our results on the intracellular
localization of ODN as well as the kinetic data are consistent
with these views. Accumulation of ODN in cytoplasmic granules which presumably represent endosomes and lysosomes
was observed after 2 hours of incubation with FITC-labeled
ODN. The nuclear localization of fluorescence after 24 hours
indicated a significant release of ODN from endocytic vesicles
into the cytosol and subsequent transport to the nucleus,
suggesting that ODN may reach the target RNA. Additionally,
the kinetic data on the efflux of cell-associated radioactivity
from ODN-transfected CD341 cells can be described in a
mathematical model for ODN trafficking with at least two
From www.bloodjournal.org by guest on July 31, 2017. For personal use only.
860
phases or compartments.1,24 The first phase or compartment
with a short half-life of 2 hours may reflect the rapid transfer of
ODN from endosomes to the cell surface or to lysosomes with
subsequent degradation and exocytosis. The second and further
phases with slow turnover may reflect the release of ODN from
endosomes to other cellular compartments followed by metabolization. The overall decrease of cell-associated ODN in CD341
cells resulted in a half-life of full-length ODN of 10 hours.
These kinetic data indicate that for targeting a gene which codes
for a RNA and a protein with long half-lives a single transfection of antisense ODN may not be sufficient and further
transfections after intervals of 10 hours using the half ODN
dose may be useful.
The results obtained in ex vivo experiments may differ from
ODN uptake in vivo. Although DOTAP and DOSPER are
effective in the presence of human serum,17 transfection can be
hampered by charged components of the blood such as heparin.34 Therefore, the efficacy of ODN delivery was evaluated
under conditions mimicking an in vivo administration. Using
DOSPER the enhancement of ODN uptake was significantly
decreased from 800-fold to about 20-fold when whole blood
was used instead of defined ex vivo conditions. The efficacy of
DOTAP was less influenced by components of peripheral blood
and still an 30-fold increase of ODN was observed. This
indicates that DOTAP may be suitable for systemic in vivo
application.
Still, one has to take into account that the FITC label or the
sequence of the ODN may influence uptake and intracellular
trafficking of the nucleic acid.48 However, we did not find
differences in the extent of uptake using a 26-mer and a 28-mer
ODN with various sequences, suggesting that the data obtained
with the 23-mer ODN may be representative for other ODN.
Nevertheless, it is important to ensure for each individual ODN
an efficient cationic lipid-mediated delivery into the respective
cell type before using the ODN in cell culture experiments or
clinical trials.
A great uptake of ODN does not necessarily translate into
biological activity, because antisense ODN may lack an efficient binding with the target RNA or they could remain in
endosomes and lysosomes without reaching their target. Because the uptake studies were performed with ODN derived
from bcr-abl oncogene-directed antisense sequences, primary
cells from patients with CML were used to look for functional
effects of ODN delivered by cationic lipids. Reports on the
inhibitory effects of bcr-abl–directed antisense oligodeoxyribonucleotides on the proliferation of Ph1 primary leukemic cells
are heterogeneous and even contradictory with respect to
sequence specificity and efficacy in some instances.21,49-54 This
heterogeneity may be related to differences in cell-culture
conditions, to the target sequence, as well as to the high ODN
concentrations ranging between 5 µmol/L and 20 µmol/L, which
could result in unspecific effects. We found in one third of the
cases a fusion point-specific inhibition of clonogenic growth by
ex vivo treatment of primary CML cells with bcr-abl antisense
ODN/DOTAP complexes using an ODN concentration of only
1 µmol/L, which indicates a benefit of DOTAP for functional
effects of ODN. The lack of growth inhibition observed with
KRONENWETT ET AL
alternative junctional and scrambled control ODN implies that
the antiproliferative activity is due to a specific antisense effect.
A correlation between inhibition of colony formation in these
cases and any patients’ characteristics such as disease status or
type of fusion point was not found. Thus, the antisense ODN
may only be beneficial for a subset of patients with Ph1 CML.
The lack of efficacy in the other patients remains unclear. There
might be additional genomic alterations or alternative signal
transduction pathways that make the cells independent from
bcr-abl expression.55,56
In conclusion, DOTAP and DOSPER have a significant
advantage over the use of ODN alone for delivery of ODN into
primary hematopoietic cells. Thus, using cationic lipids in
clinical studies the ODN can be applied at a lower dose to
reduce side effects and cytotoxicity. The data obtained in this
study, including those obtained with the primary cells of
patients with CML, can serve as basis for ongoing and future
clinical ex vivo purging protocols of hematopoietic stem cells in
the treatment of hematologic diseases such as CML as well as
for systemic in vivo administration of antisense ODN in
leukemias and viral infections.
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1998 91: 852-862
Oligodeoxyribonucleotide Uptake in Primary Human Hematopoietic Cells Is
Enhanced by Cationic Lipids and Depends on the Hematopoietic Cell
Subset
Ralf Kronenwett, Ulrich Steidl, Michael Kirsch, Georg Sczakiel and Rainer Haas
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