|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 92 No. 10 (November 15), 1998:
pp. 3537-3545
RAPID COMMUNICATION
Gene Delivery to Human B-Precursor Acute Lymphoblastic Leukemia Cells
By
Leo Mascarenhas,
Renata Stripecke,
Scott S. Case,
Dakun Xu,
Kenneth I. Weinberg, and
Donald B. Kohn
From the Divisions of Research Immunology/Bone Marrow Transplantation
and Hematology/ Oncology, Department of Pediatrics, University of
Southern California School of Medicine, Childrens Hospital Los Angeles,
Los Angeles, CA.
 |
ABSTRACT |
Autologous leukemia cells engineered to express immune-stimulating
molecules may be used to elicit antileukemia immune responses. Gene
delivery to human B-precursor acute lymphoblastic leukemia (ALL) cells
was investigated using the enhanced green fluorescent protein (EGFP) as
a reporter gene, measured by flow cytometry. Transfection of the Nalm-6
and Reh B-precursor ALL leukemia cell lines with an expression plasmid
was investigated using lipofection, electroporation, and a polycationic
compound. Only the liposomal compound Cellfectin showed significant
gene transfer (3.9% to 12% for Nalm-6 cells and 3.1% to 5% for Reh
cells). Transduction with gibbon-ape leukemia virus pseudotyped Moloney
murine leukemia virus (MoMuLV)-based retrovirus vectors was
investigated in various settings. Cocultivation of ALL cell lines with
packaging cell lines showed the highest transduction efficiency for
retroviral gene transfer (40.1% to 87.5% for Nalm-6 cells and 0.3%
to 9% for Reh cells), followed by transduction with viral supernatant on the recombinant fibronectin fragment CH-296 (13% to 35.5% for Nalm-6 cells and 0.4% to 6% Reh cells), transduction on human bone
marrow stroma monolayers (3.2% to 13.3% for Nalm-6 cells and 0% to
0.2% Reh cells), and in suspension with protamine sulfate (0.7% to
3.1% for Nalm-6 cells and 0% for Reh cells). Transduction of both
Nalm-6 and Reh cells with human immunodeficiency virus-type 1 (HIV-1)-based lentiviral vectors pseudotyped with the vesicular stomatitis virus-G envelope produced the best gene transfer efficiency, transducing greater than 90% of both cell lines. Gene delivery into
primary human B-precursor ALL cells from patients was then investigated
using MoMuLV-based retrovirus vectors and HIV-1-based lentivirus
vectors. Both vectors transduced the primary B-precursor ALL cells with
high efficiencies. These studies may be applied for investigating gene
delivery into primary human B-precursor ALL cells to be used for
immunotherapy.
© 1998 by The American Society of Hematology.
| |
INTRODUCTION |
ACUTE LYMPHOBLASTIC leukemia (ALL),
usually of B-precursor lymphoblastic origin, is the most frequently
occurring cancer in childhood. The overall event-free survival at 5 years varies between 70% and 80%.1 However, the prognosis
of patients with certain high-risk forms of ALL or patients who relapse
after conventional chemotherapy is poor. This group of patients
accounts for a significant number of children with cancer.
B-precursor ALL cells are inefficient antigen-presenting cells and
thereby evade the body's immune surveillance system.2 Based on previous preclinical studies in solid tumor
therapy,3,4 the expression of immunomodulatory factors by
B-precursor ALL cells may facilitate the presentation of
tumor-associated antigens to the immune system and cause an immune
response against the leukemia. The possibility of introducing various
immunomodulatory genes into neoplastic cells has been successfully
documented.4-6 Recently, disabled single-cycle
virus-herpes simplex virus (DISC-HSV) vectors have been shown to
transduce human malignant hematopoietic cells transiently.7
However, there has been no systematic comparison of different methods
of gene transfer to ALL cells reported in the scientific literature.
Gene therapy for ALL will require efficient gene delivery procedures.
We investigated gene delivery to the human B-precursor ALL
cell lines Nalm-6 and Reh. The enhanced green fluorescent protein
(EGFP) was used as a reporter gene to measure gene transfer efficiency.
Methods evaluated included direct DNA delivery of an expression plasmid
using electroporation, lipofection, and the polycationic compound
SuperFect. Gene delivery using Moloney murine leukemia virus
(MoMuLV)-based retroviral vectors and human immunodeficiency
virus-type 1 (HIV-1)-based lentiviral vectors was also investigated.
The efficiency of the MoMuLV vectors and the HIV-1-based vectors in
transducing leukemia cells was evaluated with or without the use of the
recombinant fibronectin fragment CH-296 as a support
matrix.8
Gene transfer to Nalm-6 and Reh cells by electroporation was minimal
and variable and there was significant cell mortality. There was no
measurable gene transfer with SuperFect. Of the cationic lipids
evaluated, Cellfectin alone produced reproducible, low levels of gene
transfer. MoMuLV-based retroviral vectors transduced Nalm-6 cells more
efficiently than Reh cells. High levels of stable gene transduction
were achieved in Nalm-6 cells by cocultivation with murine packaging
cells and by using cell-free virus supernatant on either human bone
marrow stroma or CH-296 as a support matrix. The highest levels of gene
transfer in both Nalm-6 and Reh cells were achieved using lentivirus
vectors pseudotyped with the vesicular stomatitis virus-G (VSV-G)
envelope. Primary B-precursor ALL cells from patients were efficiently
transduced by both MoMuLV-based and HIV-1-based vectors pseudotyped
with the VSV-G envelope.
| |
MATERIALS AND METHODS |
Cells.
The human ALL cell lines used in this study were Nalm-6 (provided by Dr
Dario Campana, St Jude Children's Research Hospital, Memphis, TN) and
Reh (obtained from American Type Culture Collection, Rockville, MD),
which have been previously characterized as B-precursor ALL
cells.9,10 Both cells express the early B-lymphoid antigen CD19 on greater than 95% of cells. The cells were cultured in RPMI
1640 medium with L-glutamine (BioWittaker, Walkersville, MD), 10%
fetal bovine serum (FBS), and penicillin/streptomycin (50 U/mL)
(leukemia medium) at 37°C, 5% CO2. Experiments were performed when the cells were in the exponential growth phase. PG13
vector producing fibroblasts and 293T, 293A, and HT-29 cells were
cultured in Dulbecco's modified Eagle's medium (DMEM; BioWittaker, Walkersville, MD), 10% FBS, L-glutamine (2 mmol/L), and
penicillin/streptomycin (50 U/mL) at 37°C and 5% CO2.
Primary B-precursor ALL cells were obtained from cryopreserved cells
remaining from diagnostic bone marrow aspirates taken from pediatric
ALL patients with marrows that were more than 90% replaced with ALL
blasts. The cells were cryopreserved in 10% dimethyl sulfoxide (DMSO)
and 90% FBS. These cells were thawed at 37°C using a thawing
medium containing AIM-V serum-free medium (GIBCO/BRL,
Grand Island, NY), 30% FBS, 20 U/mL heparin, and 0.2 U/mL
DNAse. Primary ALL cells were cultured in AIM-V serum-free medium
containing 0.5 to 1.5 µg/mL of CD40L (Immunex, Seattle, WA) on
monolayers of irradiated (3,000 cGy) allogeneic primary human bone
marrow stroma (2 × 104 cells per well) at a cell
density of 1 × 105 cells per well in 48-well
flat-bottom plates at 37°C, 5% CO2 on the day before
transduction.
Reporter constructs.
The reporter gene EGFP (Clontech Laboratories, Palo Alto, CA) was used
for the detection of transfected/transduced leukemia cells by
fluorescence-activated cell sorting (FACS) analysis. To make the
expression plasmid VR-EGFP, EGFP was inserted into the expression
plasmid VR-1012 under transcriptional control of the CMV I/E promoter
(provided by Dr Robert H. Zougg, Vical Inc, San Diego, CA;
Fig 1). Plasmids for transfection were
prepared using Qiagen Endotoxin free Plasmid Maxi kit (Qiagen,
Valencia, CA).
View larger version (22K):
[in this window]
[in a new window]
| Fig 1.
Diagrams of the plasmid and vectors expressing EGFP.
Cartoons of the expression plasmid VR-EGFP, the MoMuLV-based vector
encoding EGFP used in the packaging cell line PG-13, and the reporter
gene vectors pHR -CMV-EGFP and MLV-CMV-EGFP used in the transient
packaging system. VR-EGFP contains a CMV promoter/enhancer (CMV
prom/enh) and intron from CMV, the EGFP gene and bovine growth hormone
polyadenylation signal (BGH term) in a plasmid-encoding kanamycin
resistance (Kan). L-EGFP-SN is an MoMuLV-based vector containing the
5LTR and 3LTR from MoMuLV, the EGFP gene, an SV40 early
promoter, and the bacterial neomycin phosphotransferase gene (NEO).
pHR-CMV-EGFP is a lentiviral vector containing HIV-1-derived
elements, including the 5LTR and 3LTR, the splice donor
(SD) and splice acceptor (SA), the packaging signal ( ), a portion of
the gag encoding region (GA), a CMV prom/enh (CMV), and the EGFP gene.
MLV-CMV-EGFP is another MoMuLV-based vector containing the 5LTR
and 3LTR of MoMuLV, the bacterial neomycin phosphotransferase
gene (NEO), the CMV prom/enh (CMV), and the EGFP gene.
|
|
Retroviral vectors.
An MoMuLV vector encoding EGFP (L-EGFP-SN) was constructed by inserting
the Bgl II-Not I fragments containing the EGFP gene into the Hpa I site in the polylinker of LXSN11
(Fig 1). L-EGFP-SN was packaged by transfecting the vector plasmid into
PG13 cells, which produce virus particles pseudotyped with the
gibbon-ape leukemia virus (GALV) envelope.12 Transfected
cells were selected in G418 and high-titer clones were selected by
sorting EGFP-positive single-cell clones using a flow
cytometry-assisted automatic cell deposition unit. The titer of these
clones was assessed by transduction of HT-29 cells and subsequent G418
selection. Viral supernatant from high-titer clones was collected after
incubation of confluent plates for 48 hours at 32°C,
5%CO2. Cell-free supernatant from a high-titer clone
(106 infectious units/mL) was used in the transduction
experiments.
Virus particles containing an MoMuLV-based vector encoding EGFP under
control of an internal CMV promoter (MLV-CMV-EGFP; Fig 1)13
and pseudotyped with the VSV-G envelope were produced by cotransfecting
293T cells with the vector plasmid, packaging plasmid pHIT
60,14 and the VSV-G protein encoding plasmid
pMDG13 using the calcium phosphate method and collecting
viral supernatant for a period of 72 hours, starting 24 hours after
transfection. Viral supernatants were concentrated by
ultracentrifugation at 19,000 rpm for 2 hours and 20 minutes at
22°C and were titered by assessing expression of EGFP in transduced
293A cells by flow cytometry. Briefly, 1 × 105 293A
cells were plated in each well of a 6-well plate. The following day,
cells from three representative wells were trypsinized and counted, and
the average number of cells was determined. Three dilutions (1/500,
1/5,000, and 1/50,000) of concentrated viral supernatant were used to
infect the 293A cells. Polybrene (10 µg/mL) was used as a
transduction adjuvant. Medium was changed after 24 hours and cells were
analyzed for the percentage of cells expressing EGFP 48 hours after
transduction. Titer was calculated using the formula: percentage of
cells expressing EGFP × dilution factor × average number of
cells per well/100.15 Multiplicities of infection (MOI) for
leukemia cells were calculated as infectious units as determined on
293A cells divided by the number of leukemia cells exposed to the
vector.
Lentiviral vectors.
The HIV-1-based lentiviral vectors used were those described by
Naldini et al (kindly provided by Drs Didier Trono and Romain Zuffrey
[University of Geneva, Geneva, Switzerland] and Dr Luigi Naldini [Cell Genesys, Foster City,
CA]).12,16 A vector containing the EGFP gene
under control of the CMV promoter (pHR-CMV-EGFP) was produced by
transient cotransfection of 293T cells with plasmids encoding the
vector; the HIV-1 gag, pol, tat, and rev genes ( r 8.91); and the
VSV-G envelope (pMDG; Fig 1) using the calcium phosphate
method.13 The viral supernatants were concentrated and
titered as described above. Viral supernatant was used to infect cells
in transduction experiments.
Gene transfer methods.
For direct DNA delivery, two methods were used. (1) Electroporation of
leukemia cells was performed using the Gene Pulser II RF module from
Bio-Rad (Hercules, CA). Leukemia cells were washed once
with Hank's Balanced Salt Solution (HBSS) and resuspended at a
concentration of 107/mL in HEPES buffered media (15 mmol/L
potassium phosphate buffer, pH 7.2, 1 mmol/L magnesium chloride, 2 mmol/L HEPES, 250 mmol/L mannitol) with 12 µg/mL of VR-EGFP plasmid
DNA. Four-hundred-microliter samples were placed in cuvettes and
experiments were performed by varying the voltage (50, 100, and 150 V)
and burst interval (0.2, 0.5, and 1 second) while keeping the
percentage of modulation (100%), radio frequency (40 KHz), burst
duration (2 milliseconds), and the number of bursts (5) constant.
Cuvettes were placed on ice immediately after electroporation for 5 minutes. The electroporated cells were then placed in leukemia medium
and cultured for 48 hours before analysis of transfection. (2) The
cationic lipids used for transfection were Lipofectin, Lipofectamine,
Cellfectin, and Dimrie/C (GIBCO/BRL). Transfection was performed
following the manufacturer's instructions initially and then optimized
in later experiments. Briefly, Lipofectin (10 µL), Lipofectamine (10 µL), Cellfectin (15 µL), and Dimrie/C (5 µL) were added to 250 µL of AIM-V serum-free medium (GIBCO/BRL). These samples were added
to the first column of a 24-well plate that had 250 µL of AIM-V in
every well and incubated at room temperature for 30 minutes. Two
hundred fifty microliters of VR-EGFP plasmid DNA at a concentration of
12.8 µg/mL of AIM-V was added to the first well in each column and 5 twofold dilutions were performed. Leukemia cells were washed twice with
AIM-V and resuspended to obtain a cell density of 8 × 106/mL. Fifty microliters of cells was added to each well
containing the lipid-DNA complexes and incubated for 4 hours at
37°C, 5% CO2. Fresh AIM-V medium (0.6 mL) was then
added to each well and incubated for 48 hours in the same conditions.
Cells were then washed and analyzed for transfection. SuperFect
(Qiagen) transfection reagent was also used following the
manufacturer's instructions. Leukemia cells were washed once with HBSS
and then plated at a concentration of 2.5 × 106 cells in 2.5 mL of leukemia medium in each well of a
6-well plate. VR-EGFP plasmid DNA (2.5 µg) was diluted in 75 µL of
AIM-V and 10 µL of SuperFect reagent was added to the DNA solution.
The mixture was vortexed for 10 seconds and then incubated at room temperature for 10 minutes to allow complex formation. Transfection complexes were then added dropwise to each well while gently swirling the plate to ensure uniform distribution of the complexes to the cells.
Retroviral- and lentiviral-mediated gene transfer.
Retroviral- and lentiviral-mediated gene transduction of leukemia cells
was evaluated using allogeneic human bone marrow or the recombinant
fibronectin fragment CH-296 (RetroNectin; Takara Shuzo Co, Ltd, Otsu,
Shiga, Japan) as a support matrix, with bovine serum albumin (BSA) as a
control. Six-well plates were coated with 2 mL/well of 50 µg/mL
CH-296 for 2 hours at room temperature and then blocked for 30 minutes with 2% BSA in sterile water. Coated wells were
then washed with HBSS (BioWittaker) containing 2.5 mmol/L vol/vol
HEPES. Control wells were coated with 2% BSA for 30 minutes and then
washed with the HEPES/Hank's solution. Leukemia cells (1 × 106) were plated in each well in 1 mL of leukemia medium.
Viral supernatant was diluted to give the calculated MOI, added to each
well, and then incubated at 37°C, 5% CO2 for 24 hours.
Leukemia cells were mobilized using enzyme-free cell dissociation
buffer (GIBCO/BRL), washed twice with leukemia medium, and then
incubated for 24 to 48 hours at 37°C, 5% CO2 before
FACS analysis. Transduction experiments on primary human B-precursor
ALL cells were performed under culture conditions described earlier.
Retroviral and lentiviral vector supernatants were added at the
indicated MOI and the cells were cultured for 48 hours at 37°C, 5%
CO2. Two days after addition of the vector, leukemia cells
were harvested for analysis of transduction.
FACS analysis.
For analysis of EGFP expression, cells were washed once with
phosphate-buffered saline, resuspended at 107/mL, and then
fixed with 1% paraformaldehyde. Detection of EGFP expression was
accomplished with a FACScan cytometer equipped with a 488-nm argon
laser for excitation of the reporter protein and a 530/30-nm bandpass
filter for monitoring the fluorescent emissions. To establish
background for fluorescence and to set gates for data acquisition,
sham-transduced or sham-transfected cells were used. Primary ALL cells
were stained with a monoclonal antibody to the B-precursor cell antigen
CD19 and conjugated to phycoerthrin (CD19-PE; Becton Dickinson, San
Jose, CA), followed by fixation in 1% paraformaldehyde
and FACS analysis. Care was taken to analyze cells that fell in the
lymphocyte gate based on forward and side scatter characteristics and
that were positive for the expression of CD19. Mean fluorescence
intensity was used to calculate levels of EGFP expression.
| |
RESULTS |
Stability of EGFP expression.
Leukemia cells transduced with L-EGFP-SN by cocultivation on vector
producing fibroblasts or on CH-296 were sorted by FACS for those
expressing EGFP and then placed in culture. Leukemia cells were split
1:10 every 3 to 4 days. Cell aliquots were then cryopreserved every
week. At the completion of 2 months of culture, leukemia cells frozen
at various time points were thawed and placed in culture for 24 hours
and then simultaneously analyzed by flow cytometry. The level of
expression of EGFP remained between 82.54% and 94.53% for leukemia
cells transduced with the L-EGFP-SN vector for Nalm-6 and Reh cells
(Fig 2A and B). Leukemia cells transduced with the lentiviral vectors but not sorted by FACS also showed stable
expression of EGFP for at least 3 weeks in culture (Fig 2A and B). The
cells grew well in culture, suggesting that EGFP was a stable and
nontoxic reporter gene in these cells. Leukemia cells transiently
transfected with the expression plasmid VR-EGFP using Cellfectin showed
relatively stable expression of EGFP for approximately 1 week in
culture before falling to undetectable levels (Fig 2A and B). These
cells were not FACS sorted.
View larger version (21K):
[in this window]
[in a new window]
View larger version (21K):
[in this window]
[in a new window]
| Fig 2.
Stability of EGFP expression in leukemia cells in
culture. Stability of EGFP expression in leukemia cells in culture
after transfection with the expression plasmid ( ) VR-EGFP and
transduction by the retroviral vector ( ) L-EGFP-SN and the
lentiviral vector ( ) pHR-CMV-EGFP. The transfected cells and
the lentiviral-transduced cells were not FACS sorted before culture,
but the retroviral-transduced cells were. Day 0 is 48 hours after
transfection or transduction. (A) Nalm-6 cells; (B) Reh cells.
|
|
Direct DNA delivery.
Electroporation resulted in massive cell destruction (70% cell death).
Of the viable cells recovered, gene transfer was negligible as assessed
by expression of EGFP by flow cytometry. In one experiment, there were
approximately 0.5% to 1% of the cells transfected (data not shown).
SuperFect, a polycationic compound, also had no efficacy in
transfecting the leukemia cell lines.
Four lipofection compounds (Lipofectin, Lipofectamine, Cellfectin, and
Dimrie/C) were screened for transfection of the leukemia cell lines.
Only Cellfectin produced significant results. At the optimal dilution,
Cellfectin produced 7.2% transfection of Nalm-6 cells
(Fig 3A) and 2.2% of Reh cells (Fig 3B).
View larger version (23K):
[in this window]
[in a new window]
View larger version (31K):
[in this window]
[in a new window]
| Fig 3.
Transfection of leukemia cells using different
lipofectants. Transfection of leukemia cells with () Lipofectin,
( ) Lipofectamine, () Cellfectin, and ( ) Dimrie-C. 0 dilution
corresponds to 10 µL Lipofectin, 10 µL Lipofectamine, 15 µL
Cellfectin, and 5 µL Dimrie/C. (A) Nalm-6 cells; (B) Reh cells.
|
|
Experiments were then designed using Cellfectin at various
concentrations (15, 7.5, 3.25, and 1.625 µL) together with varied amounts of plasmid DNA (1.6, 3.2, and 6.4 µg). Cellfectin at 3.25 µL and 1.6 µg of plasmid DNA per 100,000 leukemia cells resulted in
transfection of 22% of the Nalm-6 cells and 11% of the Reh cells
(Fig 4). The intensity of EGFP fluorescence
was brighter for Nalm-6 cells than for REH cells (data not shown).
View larger version (19K):
[in this window]
[in a new window]
| Fig 4.
Transfection of leukemia cell lines using various
quantities of VR-EGFP plasmid DNA. Transfection of () Nalm-6 and
() Reh cells with cellfectin using different amounts of VR-EGFP
plasmid DNA.
|
|
MoMuLV-based retroviral gene transfer.
Cocultivation of Nalm-6 cells with the fibroblast packaging cell line
PG13 L-EGFP-SN resulted in gene transfer efficiency of 41.5%. Reh
cells were transduced less well under similar conditions, resulting in
a gene transfer efficiency of 0.71% (Table
1). Varying concentrations of protamine sulfate (1, 2, 4, and 8 µg/mL) were used as transduction adjuvants together with cell-free
retroviral supernatants. Protamine sulfate at 2 µg/mL produced the
most efficient gene transfer in both Nalm-6 and Reh cells (Table 1).
Transduction was next evaluated using the recombinant fibronectin
fragment CH-296 (50 µg/mL) as a support matrix or BSA (2%) as a
control. The results are shown in Table 1. A total of
43.5% of Nalm-6 cells were transduced on fibronectin compared with
13.3% on BSA. A total of 2.4% of Reh cells were transduced on
fibronectin compared with 0.2% on BSA. Three rounds of transduction on
3 consecutive days was more efficient than three rounds of transduction
on a single day (Table 2).
In another experiment, transduction of Nalm-6 and Reh cells was also
evaluated comparing cell-free viral supernatant alone, viral
supernatant with protamine sulfate, viral supernatant with protamine
sulfate on allogeneic human bone marrow stroma, and viral supernatant
on recombinant CH-296 or on BSA. The results are shown in
Table 3. Recombinant CH-296 provided the
best support matrix and produced the highest gene transfer in both cell
lines. Varying concentrations of fibronectin (25, 50, 75, and 96 µg/mL) used to coat the culture dishes were also evaluated for the
effect on gene transfer efficiency. No significant differences were
noted (data not shown).
HIV-1-based lentiviral gene transfer.
Lentiviral vectors produced very efficient gene transfer into both
Nalm-6 and Reh cells. A total of 90.16% of Nalm-6 cells and 99.77% of
Reh cells were transduced at an MOI of 30 (Fig 5A). Transduction of the leukemia cell
lines with GALV-pseudotyped MoMuLV and lentiviral vectors was compared
on fibronectin, with BSA as a control. Gene transfer was enhanced on
recombinant CH-296 when compared with BSA for the GALV-pseudotyped
MoMuLV vector but not for the lentiviral vector (Fig 5B).
View larger version (32K):
[in this window]
[in a new window]
View larger version (29K):
[in this window]
[in a new window]
| Fig 5.
(A) Transduction of leukemia cell lines with HIV-1-based
lentiviral vectors. Transduction of Nalm-6 and Reh cells with the
lentiviral vector pHR-CMV-EGFP. (Left panels) untransduced
cells; (right panels) transduced cells; (upper panels) Nalm-6 cells;
(lower panels) Reh cells. (B) Comparison of retroviral- and
lentiviral-mediated gene transfer in leukemia cell lines on ()
fibronectin (50 µg/mL) and () BSA (2%).
|
|
To evaluate whether the difference in gene transfer was due to the
vector backbone or due to the viral envelope, gene transduction experiments were performed with viral supernatants containing MoMuLV
vectors pseudotyped with the GALV envelope, MoMuLV vectors pseudotyped
with the VSV-G envelope, and the HIV-based vector pseudotyped with the
VSV-G envelope. We were consistently unable to produce lentiviral
vectors with a GALV pseudotype (Xu and Kohn, unpublished
observation). A range of MOI was used. As before, transduction of Nalm-6 cells by the GALV-pseudotyped MoMuLV vector was markedly enhanced by the presence of a fibronectin support matrix
(Fig 6A). In contrast, fibronectin only
moderately enhanced transduction by the VSV-G-pseudotyped vectors. No
increase in transduction occurred as the MOI of the GALV-pseudotyped
MoMuLV vector was increased from 4 to 20. Gene transfer was MOI
dependent for the vectors pseudotyped with the VSV-G envelope. The
VSV-G-pseudotyped MoMuLV vector transduced Nalm-6 cells poorly. In
contrast, the VSV-G-pseudotyped lentiviral vector efficiently
transduced the Nalm-6 cells, with a maximal transduction of 57% at the
highest MOI tested (20).
View larger version (22K):
[in this window]
[in a new window]
View larger version (20K):
[in this window]
[in a new window]
| Fig 6.
Comparison of vector backbone and viral envelope in
transducing leukemia cells. Transduction of leukemia cells with
MLV-GALV, MLV-VSVG, and pHR-VSVG vectors encoding EGFP at
different MOI on () fibronectin (50 µg/mL) and () BSA (2%).
(A) Nalm-6 cells; (B) Reh cells.
|
|
Similar, although more striking results were seen with the Reh cells
(Fig 6B). Transduction of Reh cells by the GALV-pseudotyped MoMuLV
vector was very poor, although enhanced by fibronectin. No increase in
transduction occurred as the MOI was increased. The VSV-G-pseudotyped
MoMuLV vector transduced even more poorly. However, the
VSV-G-pseudotyped lentiviral vector transduced 88% of the Reh cells
at an MOI of 20. As shown earlier, increasing the MOI to 30 could
transduce almost 100% of the cells.
Primary human B-precursor ALL cells.
Primary B-precursor ALL cells were studied for transduction, comparing
the VSV-G-pseudotyped MoMuLV vector and the HIV-1 vector. Cells were
transduced by a single exposure to cell-free vector supernatant and
analyzed after 2 days for EGFP expression. Because the cells were
cultured on human allogeneic bone marrow stromal monolayers to preserve
leukemia cell viability,17 we costained cells for the
expression of the B-precursor cell antigen CD19 to exclude any stromal
cells that may have been recovered from the culture with leukemia
cells.
Both MoMuLV-based and HIV-1-based vectors transduced primary
B-precursor ALL cells as measured by expression of EGFP. Five patient
samples were analyzed in four different experiments using various MOIs.
Because of the limited availability of primary ALL cells, transduction
could be repeated in two of the five patient samples. CD40L was used
only in experiments no. 1 and 2. There was a large variation in
transduction with both the MoMuLV vectors (2.81% to 74.05%) and HIV-1
vectors (2.2% to 43.35%; Table 4). HIV-1-based vectors transduced primary human pre-B ALL cells with higher efficiency than the MoMuLV-based vectors at an MOI of 3,000, the
highest MOI tested. ALL cells transduced by the HIV-1-based vector had
a higher mean fluorescence intensity of EGFP expression than those
transduced by the MoMuLV-based vector (Fig
7).
View larger version (17K):
[in this window]
[in a new window]
| Fig 7.
Transduction of patient no. 1 primary B-precursor ALL
cells with MLV-CMV-EGFP and pHR-CMV-EGFP. X-axis, EGFP
fluorescence; Y-axis, anti-CD19-PE. (Left panel) Mock-transduced
cells; (center panel) cells transduced with MLV-CMV-EGFP; (right panel)
cells transduced with pHR-CMV-EGFP.
|
|
| |
DISCUSSION |
For clinical application of immunostimulatory gene therapy of ALL,
there is a need to achieve a balance between high efficiency gene
transfer and survival of primary human ALL cells, which may be fragile
and poorly tolerant of extended culture. Genetically modified leukemia
cells used as vaccines will probably be irradiated to prevent their
growth in vivo and, therefore, will only express introduced gene
products for a few days. Thus, gene transfer methods that lead to
stable or transient gene expression may be adequate for this approach.
In this series of experiments, gene delivery to human B-precursor ALL
cell lines was evaluated using a number of transfection and
transduction protocols.
Electroporation was ineffective in transfecting the leukemia cells
lines and also produced high cell mortality. Primary leukemia cells are
prone to undergo apoptosis in vitro; hence, electroporation will
probably be ineffective as a tool for gene transfer to primary cells.
Cellfectin was the only transfection reagent that produced some gene
transfer. Nalm-6 cells were better transfected than Reh cells. Even
though the level of gene transfer by Cellfectin was relatively low and
expression was transient, it may modify primary ALL cells for a time
sufficient to elicit an immune response in vivo.
MoMuLV vectors have been used in clinical trials for certain genetic
and neoplastic diseases. The potential for use of MoMuLV-based vectors
in ALL will be dependent on whether a patient's primary ALL cell
sample can be transduced efficiently. MoMuLV-based vectors used in the
presence of recombinant fibronectin fragment CH-296 were moderately
efficient in transducing Nalm-6 cells but were inefficient in
transducing Reh cells. The difference in gene transfer to the cell
lines could possibly reflect the heterogeneity of primary ALL cells.
One possible reason could be the different expression of certain
adhesion molecules on ALL cells. Nalm-6 cells expressed high levels of
both VLA-4 and VLA-5, whereas Reh cells expressed only VLA-4
(unpublished observation). These integrins are required
for the binding of the cells to the recombinant fibronectin fragment,
CH-296, and helps colocalize the cells and retrovirus.8 It
is also possible that the Nalm-6 cells express higher levels of the
GALV receptor than Reh cells, leading to better transduction of the
former with the GALV-pseudotyped MoMuLV vector.
Lentiviral vectors efficiently transduced both Nalm-6 and Reh cells.
These vectors overcame the differences between the cell lines seen with
retroviral transduction, making them possibly the best agents for gene
transfer into primary ALL cells. Because the cell lines are
continuously dividing, the high transduction is probably not secondary
to the ability of lentiviral vectors to transduce quiescent cells. The
better transduction may be related to more efficient reverse
transcription and integration in the human leukemia cell genome.
We have extended these studies in the cell lines to analysis of
transduction or primary B-precursor ALL cells from pediatric patients.
Primary ALL cells are very fragile and under the optimal conditions
there is essentially no increase in cell number, although the cells can
be sustained for a few days. Both MoMuLV-based retroviral and
HIV-1-based lentiviral vectors showed reproducible transduction of
these cells. Even though there was no increase in gene transfer with
increasing the MOI across patient samples, there was an increase in
gene transfer within patient samples. To the best of our knowledge, these experiments for the first time show that primary ALL cells can be
transduced by retroviral and lentiviral vectors. We are currently
studying methods to increase transduction efficiency of primary ALL
cells, examining the efficacy of multiple exposures to vector, the use
of higher MOI, or the use of spinoculation protocols. Better culture
conditions for primary human ALL cells may improve gene transfer with
retroviral and lentiviral vectors and also allow ex vivo expansion of
transduced cells. We will also test the delivery and expression of
candidate therapeutic genes such as CD80 and granulocyte-macrophage
colony-stimulating factor (GM-CSF) in primary B-precursor
ALL cells. If successful, these modified cells could be used in
patients with high-risk disease to stimulate an autologous immune
response against leukemia. Even though lentiviral vectors have so far
been shown to be free of replication competent virus, there are still
some safety concerns before they can be used in clinical trials.
Development of safer lentiviral vectors will aid in making this
approach feasible.
| |
ACKNOWLEDGMENT |
The authors acknowledge the help of Karen A. Pepper of the Vector Core
Facility at CHLA for construction of the vectors, Lora W. Barsky for
help with the FACS analysis, and Tanja A. Gruber and Dianne C. Skelton
for their critique of the manuscript.
| |
FOOTNOTES |
Submitted July 9, 1998;
accepted August 31, 1998.
Supported by a grant from an anonymous donor to the USC Norris Cancer
Center and Childrens Hospital Los Angeles for studies of ALL and by a
Translational Grant from the Leukemia Society of America (6211-98).
R.S. is a recipient of the Childrens Hospital Los Angeles Research
Institute Career Development Fellowship.
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.
Address reprint requests to Donald B. Kohn, MD, Childrens Hospital Los
Angeles, 4650 Sunset Blvd, Mailstop #62, Los Angeles, CA 90027; e-mail:
dkohn{at}chla.usc.edu.
| |
REFERENCES |
1.
Rivera GK, Pinkel D, Simone JV, Hancock ML, Crist WM:
Treatment of acute lymphoblastic leukemia. 30 years experience at St. Jude Children's Research Hospital.
N Engl J Med
329:1289, 1993[Abstract/Free Full Text]
2.
Cardoso AA, Schultze JL, Boussiotis VA, Freeman GJ, Seamon MJ, Laszlo S, Billet A, Sallan SE, Gribben JG, Nadler LM:
Pre-B acute lymphoblastic leukemia cells may induce T-cell anergy to alloantigen.
Blood
88:41, 1996[Abstract/Free Full Text]
3.
Gansbacher B, Zin K, Daniels B:
Interleukin-2 gene transfer into tumor cells abrogates tumorigenicity and induces protective immunity.
J Exp Med
172:1217, 1990[Abstract/Free Full Text]
4.
Dranoff G, Jaffee E, Lazenby A, Golumbek P, Levitsky H, Brose K, Jackson V, Hamada H, Pardoll D, Mulligan RC:
Vaccination with irradiated tumor cells engineered to secrete murine GM-CSF stimulate potent specific and long lasting anti-tumor immunity.
Proc Natl Acad Sci USA
90:3539, 1993[Abstract/Free Full Text]
5.
Vieweg J, Gilboa E:
Considerations for the use of cytokine-secreting tumor cell preparations for cancer treatment.
Cancer Invest
13:193, 1995[Medline]
[Order article via Infotrieve]
6.
Zier KS, Gansbacher B:
The impact of gene therapy on T cell function in cancer.
Hum Gene Ther
6:1259, 1995[Medline]
[Order article via Infotrieve]
7.
Dilloo D, Rill D, Entwistle C, Boursnell M, Zhong W, Holden W, Holladay M, Inglis S, Brenner M:
A novel herpes vector for the high-efficiency transduction of normal and malignant human hematopoietic cells.
Blood
89:119, 1997[Abstract/Free Full Text]
8.
Hanenberg H, Xiao X-L, Dilloo D, Hashino K, Kato I, Williams DA:
Co-localization of retrovirus and target cells on specific fibronectin fragments increases genetic transduction of mammalian cells.
Nat Med
8:876, 1995
9.
Hara H, Luo Y, Harrita K, Seon BK:
Efficient transplantation of human non T-leukemia cells into nude mice and induction of complete regression of the transplanted leukemia by ricin A-chain conjugates of monoclonal antibodies SN5 and SN6.
Cancer Res
48:2876, 1988[Abstract/Free Full Text]
10.
Greaves M, Janossy G:
Patterns of gene expression and the cellular origins of human leukemias.
Biochim Biophys Acta
516:192, 1978
11.
Robbins PB, Yu X-J, Skelton DC, Pepper KA, Wasserman RM, Zhu L, Kohn DB:
Increased probability of expression from modified retroviral vectors in embryonal stem cells and embryonal carcinoma cells.
J Virol
71:9466, 1997[Abstract]
12.
Miller AD, Garcia JV, von Suhr N, Lynch CM, Eiden MV:
Construction and properties of retrovirus packaging cells based on gibbon ape leukemia virus.
J Virol
65:2220, 1991[Abstract/Free Full Text]
13.
Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, Gage FH, Verma I, Trono D:
In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.
Science
272:263, 1996[Abstract]
14. Soneoka A, Cannon PM, Ramsdale EE, Griffiths JC, Romano G,
Kingsman SM, Kingsman AJ: A transient three-plasmid expression system
for the production of high titer retroviral vectors. Nucleic Acids Res
23:4, 628, 1995
15.
Gallardo HF, Tan C, Ory D, Sadelain M:
Recombinant retroviruses pseudotyped with the vesicular stomatitis virus G glycoprotein mediate both stable gene transfer and pseudotransduction in human peripheral blood lymphocytes.
Blood
90:952, 1997[Abstract/Free Full Text]
16.
Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D:
Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo.
Nat Biotech
15:871, 1997[Medline]
[Order article via Infotrieve]
17.
Nishigaki H, Ito C, Manabe A, Masa-aki K, Coustan-Smith E, Yanishevski Y, Behm FG, Raimondi SC, Pui C-H, Campana D:
Prevalence and growth characteristics of malignant stem cells in B-lineage acute lymphoblastic leukemia.
Blood
89:3735, 1997[Abstract/Free Full Text]
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
Y. Soda, K. Tani, Y. Bai, M. Saiki, M. Chen, K. Izawa, S. Kobayashi, S. Takahashi, K. Uchimaru, T. Kuwabara, et al.
A novel maxizyme vector targeting a bcr-abl fusion gene induced specific cell death in Philadelphia chromosome-positive acute lymphoblastic leukemia
Blood,
July 15, 2004;
104(2):
356 - 363.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Kochling, S. A. Konig-Merediz, R. Stripecke, D. Buchwald, A. Korte, H. G. von Einsiedel, F. Sack, G. Henze, K. Seeger, B. Wittig, et al.
Protection of Mice against Philadelphia Chromosome-positive Acute Lymphoblastic Leukemia by Cell-based Vaccination Using Nonviral, Minimalistic Expression Vectors and Immunomodulatory Oligonucleotides
Clin. Cancer Res.,
August 1, 2003;
9(8):
3142 - 3149.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. Stripecke, A. A. Cardoso, K. A. Pepper, D. C. Skelton, X.-J. Yu, L. Mascarenhas, K. I. Weinberg, L. M. Nadler, and D. B. Kohn
Lentiviral vectors for efficient delivery of CD80 and granulocyte-macrophage- colony-stimulating factor in human acute lymphoblastic leukemia and acute myeloid leukemia cells to induce antileukemic immune responses
Blood,
August 15, 2000;
96(4):
1317 - 1326.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
Top
Abstract
Introduction