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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 94 No. 10 (November 15), 1999:
pp. 3349-3357
Improved Expression in Hematopoietic and Lymphoid Cells in Mice After
Transplantation of Bone Marrow Transduced With a Modified Retroviral
Vector
By
Stephanie Halene,
Lijun Wang,
Robert M. Cooper,
David C. Bockstoce,
Paul B. Robbins, and
Donald B. Kohn
From the Division of Research Immunology/Bone Marrow Transplantation,
Childrens Hospital Los Angeles, Los Angeles, CA; and the Department of
Pediatrics and Microbiology, University of Southern California School
of Medicine, Los Angeles, CA.
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ABSTRACT |
Retroviral vectors based on the Moloney murine leukemia virus
(MoMuLV) are currently the most commonly used vehicles for stable gene
transfer into mammalian hematopoietic cells. But, even with reasonable
transduction efficiency, expression only occurs in a low percentage of
transduced cells and decreases to undetectable levels over time. We
have previously reported the modified MND LTR
(myeloproliferative sarcoma virus enhancer,
negative control region deleted, dl587rev
primer-binding site substituted) to show increased expression frequency
and decreased methylation in transduced murine embryonic stem cells and
hematopoietic stem cells. We have now compared expression of the
enhanced green fluorescent protein (eGFP) from a vector using the
MoMuLV LTR (LeGFPSN) with that from the modified vector (MNDeGFPSN) in
mature hematopoietic and lymphoid cells in the mouse bone marrow
transplant (BMT) model. In primary BMT recipients, we observed a higher
frequency of expression from the MND LTR (20% to 80%) in
hematopoietic cells of all lineages in spleen, bone marrow, thymus, and
blood compared with expression from the MoMuLV LTR (5% to 10%).
Expression from the MND LTR reached 88% in thymic T lymphocytes and
54% in splenic B lymphocytes for up to 8 months after BMT. The mean
fluorescence intensity of the individual cells, indicating the amount
of protein synthesized, was 6- to 10-fold higher in cells expressing
MNDeGFPSN compared with cells expressing LeGFPSN. Transduction
efficiencies determined by DNA polymerase chain reaction of vector copy
number were comparable for the 2 vectors. Therefore, the MND vector
offers an improved vehicle for reliable gene expression in
hematopoietic cells.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
EFFECTIVE GENE TRANSFER and expression in
pluripotent hematopoietic stem cells (HSC) could provide new therapies
for inherited disorders of myeloid and lymphoid cells (such as
hemoglobinopathies, storage disorders, and immune deficiencies), for
infectious diseases (such as acquired immunodeficiency syndrome
[AIDS]), and for acquired disorders (such as cardiovascular
diseases).1,2 Efficient transduction, stable integration,
and persistent expression of the relevant gene are prerequisites in
gene therapy targeting HSC. The Moloney murine leukemia virus (MoMuLV),
an endogenous murine retrovirus, is the most commonly used retroviral
vector for gene transfer studies in vitro and in vivo. It has been
widely used in clinical trials, for marking studies of repopulating
stem cells or malignant cells, for replacement of the defective gene in
patients with genetic disorders, and for insertion of anti-human immunodeficiency virus (HIV) genes into hematopoietic cells of HIV-infected patients.3
Cocultivation of murine bone marrow on vector-producing fibroblasts or
transduction on recombinant fibronectin results in high transduction
efficiency (70% to 100%) and stable integration into the genome of
murine HSC and their progeny. However, expression of the desired gene
driven by the MoMuLV LTR is often subject to silencing in vitro and in
vivo.4-6 We have previously shown that a modified vector,
MND (myeloproliferative sarcoma virus enhancer,
negative control region deleted, dl587rev
primer-binding site substituted), derived from the MoMuLV, shows a
significantly higher frequency of expression than the standard MoMuLV
vector in embryonic carcinoma (F9) and embryonic stem cells (CCE) in vitro7,8 and in murine HSC and their progeny in
vivo.9 Although the analysis of secondary spleen foci
(2° colony-forming units-spleen [CFU-S]) as
performed by Robbins et al9 is a stringent assay for
transduction of and expression in HSC and their progeny, expression in
hematopoietic and lymphoid cells of primary transplant recipients is
more relevant to clinical applications and therefore warrants detailed studies.
The enhanced green fluorescent protein (eGFP) gene has
been shown previously to be useful as a reporter gene in hematopoietic cells in vitro and in vivo.10-13 Analysis of the expression
of the eGFP reporter gene by fluorescence-activated cell sorting (FACS)
allows assessment of expression in individual cells of each
hematopoietic lineage from various tissues in vivo.
We therefore compared expression of eGFP in primary re- cipients of
bone marrow, transduced with either the modified vector, MNDeGFPSN, or
the MoMuLV-based vector, LeGFPSN. The time course and
distribution of expression were monitored over 8 months to allow for
hematopoietic cells derived from transduced, mature progenitors to be
replaced by cells derived from transduced, primitive precursors and
HSC. Secondary recipients were transplanted with marrow harvested from
primary recipients 2 to 6 months after bone marrow transplantation
(BMT) and 2°CFU-S were analyzed at 12 to 14 days after BMT.
Semiquantitative polymerase chain reaction (PCR) was performed to
assess vector copy number in cell populations. Our results show that,
with equal vector copy numbers, the MND vector is expressed in a
significantly higher percentage of hematopoietic and lymphoid cells
than the MoMuLV vector.
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MATERIALS AND METHODS |
Retroviral vectors.
The LN vector, in which the MoMuLV LTR drives expression of the
neomycin resistance gene, was constructed by A. Dusty Miller (Fred
Hutchinson Cancer Center, Seattle, WA).14 The
LeGFPSN vector was constructed by insertion of the Bgl
II-Not I fragment containing the eGFP gene (Clontech
Laboratories, Palo Alto, CA) into the Hpa I site in the
polylinker of L-X-SN. The MNDeGFPSN vector was constructed as
previously described by Robbins et al8 (Fig 1).
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| Fig 1.
Vector constructs. Vectors were constructed so that the
LTR drives expression of the reporter gene for eGFP and the SV40
promoter drives expression of the gene for neomycin resistance. The
5'LTR of LeGFPSN is the MoMuLV LTR. The MND LTR is based on the
MoMuLV LTR, but was modified by replacing the MoMuLV enhancer by the
MPSV enhancer (M), deleting the negative control region (N) and
replacing the primer binding site (PBS) of the MoMuLV by the PBS of the
endogenous murine retrovirus dl587rev (D). Vectors are otherwise
identical.
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Packaging of retroviral vectors.
The PA317 cell line, derived from NIH 3T3 fibroblasts, was obtained
from the American Type Culture Collection (Rockville, MD).15 The GP+E-86 ecotropic packaging
cells16 were a generous gift from Arthur Bank (Columbia
University, New York, NY). LN was packaged in a high titer clone of
PA317 packaging cells in A. Dusty Miller's laboratory. The vectors
LeGFPSN and MNDeGFPSN were packaged in the GP+E-86 packaging cells.
High titer clones were generated for LN (1 × 106/mL),
LeGFPSN (2 × 106/mL), and MNDeGFPSN (1 × 106/mL), as measured by G418 resistance on 3T3 fibroblasts.
BMT.
Bone marrow of C57BL/6 (Charles River Laboratories, Wilmington, MA)
male donor mice was transplanted into lethally (10 Gy) irradiated
female C57BL/6 recipients after transduction. Donor bone marrow cells
were transduced by cocultivation on irradiated vector-producing
fibroblasts in the presence of murine interleukin-3 (IL-3), human IL-6,
and murine stem cell factor (SCF) as described by Robbins
et al.8 Animals were injected intravenously with 3 to 5 × 106 nucleated cells transduced with either LN,
LeGFPSN, or MNDeGFPSN retroviral vectors. Five separate BMTs were
performed with each set, including all 3 vectors. Bone marrow of some
primary recipients was used to reconstitute a second generation of
lethally irradiated female recipients. Twelve to 14 days after the
secondary transplants, 2°CFU-S were analyzed by PCR for the
presence of the vector and by FACS for eGFP expression. All procedures
involving animals were approved by the Animal Care Committee at
Childrens Hospital Los Angeles (Los Angeles, CA).
Tissue processing.
Animals were killed at varying time points after BMT. Immediately after
lethal CO2 inhalation, spleen, thymus, bone marrow, and
blood were harvested on ice. Single-cell suspensions were prepared for
FACS analysis and DNA preparation.
DNA analysis.
A semiquantitative PCR assay was established to measure the vector copy
number in samples of murine tissue. We used a GP+E-86 clone, containing
5 copies of the MNDeGFPSN vector, to generate a standard curve. DNA of
this clone was diluted with DNA of nontransduced GP+E-86 cells, such
that the template input was maintained at 40 ng per reaction. The
standard curve was based on samples diluted to represent 5, 3.75, 2.5, 1.25, 0.625, and 0 copies/cell. Semiquantitative PCR was performed with
primers to eGFP (forward primer, 5'-TAC GGC AAG CTG ACC CTG AAG
TTC-3'; reverse primer, 5'-CGT CCT TGA AGA AGA TGG TGC
G-3'), yielding a product of 193 bp. Reactions were performed in
a 50 µL reaction, with PCR buffer (10× buffer II; Perkin
Elmer-Cetus, Norwalk, CT), 1.5 mmol/L MgCl2, primers at 1 µmol/L, and dNTP at 0.2 nmol/L using the Perkin Elmer Gene Amp PCR
System 9600. The reaction was conducted for 20 cycles with denaturation
at 94°C for 30 seconds, annealing at 58°C for 1 minute, and
extension at 72°C for 1 minute. In parallel, an aliquot of each DNA
sample was subjected to PCR with primers to  -actin to control for
DNA content. -Actin primers were previously described by Tanaka et
al.17 PCR was conducted at the same conditions as used for
eGFP amplification. The -actin primers yield a PCR product of 226 bp. Reaction products were electrophoresed and transferred to nylon
membranes. Southern blots were performed with a probe labeled with
32P by random priming (Stratagene, La Jolla, CA) specific
for eGFP or -actin accordingly, generated with the above-mentioned
primers. Kodak x-ray film (Eastman Kodak, Rochester, NY) was exposed to blots for 2 to 5 hours at room temperature, and the band intensity was
quantitated by densitometry using a SciScan 5000 (US Biochemical, Cleveland, OH).
FACS analysis.
Nonspecific antibody binding was blocked with 10 µL/1 million cells
of murine  -globulin (IgG; 1 mg/mL; Sigma, St Louis, MO) and
incubated for 30 minutes at 4°C. For analysis of lineage-specific eGFP expression, cells were stained with phycoerythrin (PE)-conjugated antibodies against murine CD4, CD8, B220, Mac-1, Gr-1, and Ter-119 (Pharmingen, San Diego, CA). Excess antibody was removed by washing twice, once with red cell lysis buffer (Ortho-mune TM; Ortho Diagnostic Systems Inc, Raritan, NJ) and once with Dulbecco's phosphate-buffered saline (DPBS; Bio Whittaker, Walkersville, MD). Detection
of eGFP expression and PE staining was accomplished on a FACScan
cytometer equipped with a 488 nm argon laser for excitation and 530/30
nm (eGFP) and 575/28 nm (PE) bandpass filters for monitoring the fluorescent emissions.
Statistical analysis.
Statistical analysis was performed to compare frequency of expression,
vector copy numbers, and mean fluorescence intensities between
MNDeGFPSN and LeGFPSN. Two-sided tests were performed at  = .05 level of significance. The t-test was used when normality and
equal variances were present. The Aspin-Welch Test was used when
normality of the data was fulfilled, but equal variances were rejected.
Either the Mann-Whitney U or the Wilcoxon Rank-Sum Test for Difference
in Medians was applied when normality and equal variances were
rejected, depending on the presence of ties.
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RESULTS |
Gene transfer/BMT.
Bone marrow from male C57BL/6 mice was collected, transduced with
either LeGFPSN, MNDeGFPSN, or LN, and transplanted into irradiated
female C57BL/6 recipient mice. At specific times after gene
transfer/BMT, expression of eGFP was assessed by FACS analysis in cells
of the myeloid, erythroid, and lymphoid lineages from the bone marrow,
spleen, thymus, and blood of recipients. Tissues of recipients
transplanted with bone marrow transduced by the LN vector, which does
not contain eGFP, were used as negative controls. At each time-point, a
set of mice transplanted with cells transduced by each of the 3 vectors
was analyzed simultaneously. A total of 35 sets of mice were studied.
Cells were stained with PE-conjugated antibodies against specific
surface antigens on granulocytes (Gr-1), monocytes/macrophages (Mac-1),
T lymphocytes (CD4 and CD8), B lymphocytes (B220), and the erythroid
lineage (Ter-119).
Representative FACS dot blots and histogram plots of a set of mice
analyzed 4.5 months after BMT are shown in
Fig 2A and B. These dot blots
show a higher percentage of cells expressing eGFP with the MNDeGFPSN
vector than with the LeGFPSN vector.
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| Fig 2.
FACS analysis of eGFP expression. FACS dot blots and
histogram blots from a representative pair of mice are shown. (A and B)
Cells from bone marrow, spleen, thymus, and blood from recipients of
LeGFPSN-transduced, MNDeGFPSN-transduced, and LN-transduced (data not
shown) marrow were harvested and analyzed by FACS for
eGFP expression (x axis) in granulocytes (Gr-1), monocytes/macrophages
(MAC-1), B cells (B220), T cells (CD4, CD8), and RBC (Ter-119).
Lineage-specific staining is shown on the y-axis. Quadrant statistics
give the percentage of gated events. (C) Distribution of fluorescence intensity is
shown in histogram plots for LeGFPSN (dark line) and MNDeGFPSN (solid
area).
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eGFP as a reporter also allows measurement of the level of expression
as the fluorescence intensity of expressing cells. Mean fluorescence
intensities were analyzed by gating only on the eGFP expressing cell
populations to avoid skewing of the data by differences in the absolute
numbers of expressing and nonexpressing cells.
In addition to the higher number of eGFP(+) cells, the level of
expression was on average 6- to 10-fold higher in eGFP(+) cells of
recipients of MNDeGFPSN-transduced marrow than in eGFP(+) cells of
recipients of LeGFPSN-transduced marrow, as shown in representative
histograms in Fig 2C and in Table 1.
Copy number/cell by semiquantitative PCR.
The higher frequency and level of eGFP expression from MNDeGFPSN could
be due to a higher gene transfer by MNDeGFPSN than by LeGFPSN, leading
to differences in copy number/cell, rather than reflecting differences
in LTR-driven expression. To assess the copy number/cell,
semiquantitative PCR was performed on DNA samples derived from bone
marrow, spleen, blood, and thymus or from cells of specific lineages
sorted by FACS. A standard curve was generated based on a murine
fibroblast vector-producer clone containing 5 copies/cell of an
eGFP-containing vector. PCR with primers specific for murine -actin
served as an internal standard to control for DNA loading. DNA of
tissues from recipients transplanted with the control vector LN were
used as negative controls.
Results from all samples analyzed are shown in
Table 2. The data on the percentages of
eGFP-expressing cells in the table were derived only from those animals
for which the vector copy number/cell was assessed. Differences in the
percentages of expressing cells were significantly higher for MNDeGFPSN
compared with LeGFPSN for this subset, whereas the copy numbers per
cell were not statistically different between MNDeGFPSN and LeGFPSN for
all tissues and cell lineages analyzed. Overall, the percentage of
eGFP-expressing cells when normalized for a single vector copy/cell was
3.8 to 7.1 times higher for MNDeGFPSN than for LeGFPSN.
eGFP expression in tissues.
Mice were killed and tissues were analyzed by FACS for eGFP expression
at 2 weeks, 8 weeks, 4 to 5 months, and 6 to 8 months after BMT. Two
weeks after BMT, eGFP expression was detectable in 40% to 55% of
cells from bone marrow, spleen, and blood from recipients of
MNDeGFPSN-transduced marrow and in 5% to 25% of cells from recipients
of LeGFPSN-transduced marrow (P = not significant; Fig 3A). Six to 8 months after transplant,
expression levels were 25% to 35% of cells from recipients of
MNDeGFPSN-transduced marrow, but were less than 5% in cells from
recipients of LeGFPSN-transduced marrow (P < .05).
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| Fig 3.
eGFP expression in tissues. eGFP expression was assessed
in bone marrow, spleen, blood (A), and thymus (B) from recipients of
LeGFPSN-transduced, MNDeGFPSN-transduced, and
LN-transduced (data not shown) bone marrow 2 weeks, 8 to
12 weeks, 4 to 5 months, and 6 to 8 months after BMT.
Values are given as the mean ± standard error of the mean. n is the
number of animals analyzed for each time point and vector. * and **
mark differences in the percentage of expression between MNDeGFPSN and
LeGFPSN that are statistically significant (*P < .05;
**P < .005).
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In contrast to the relatively stable percentage of cells showing eGFP
expression in bone marrow, spleen, and blood from recipients of
MNDeGPFSN-transduced marrow, there was an increase of expression over
time in thymus (Fig 3B). Two weeks after BMT, eGFP expression occurred
in 0.6% ± 0.4% of cells from the thymus of recipients of
MNDeGFPSN-transduced marrow. Six to 8 months after transplant, frequency of expression reached 36.3% ± 11.3%. In recipients of LeGFPSN-transduced marrow, expression remained less than 10% of cells
throughout the time course and was 0.2% ± 0.1% 6 to 8 months after BMT.
eGFP expression in cells of specific lineages.
Expression in cells of specific lineages was assessed for granulocytes,
monocytes/macrophages, B lymphocytes, and T lymphocytes from bone
marrow, spleen, and blood; for T lymphocytes from thymus; and for red
blood cells (RBC) from blood. The percentage of expression by cells of
a specific lineage was calculated by dividing the number of expressing,
lineage-positive cells (RUQ in Fig 2A and B) by the total number of
lineage-positive cells (LUQ + RUQ in Fig 2A and B). Cells from mice
transplanted with LN-transduced marrow were analyzed in parallel as
negative controls for eGFP expression.
The percentage of eGFP expression in B lymphocytes from recipients of
marrow transduced by MNDeGFPSN remained high (25% to 40%) from 2 weeks to 6 to 8 months after BMT, whereas it was less than 5% in
recipients of marrow transduced by LeGFPSN by 6 to 8 months after BMT
(Fig 4A). Overall, expression occurred in a 2- to 30-fold higher cell number under the control of the modified MND
LTR than under the control of the MoMuLV LTR (P < .05). The time course and distribution of eGFP expression in monocytes, macrophages, and granulocytes and RBC were similar to the eGFP expression in B cells (data not shown).
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| Fig 4.
eGFP expression in lineages. eGFP expression is shown for
B lymphocytes (A) and T lymphocytes (B) in bone marrow, spleen, blood,
and thymus from recipients of MNDeGFPSN-transduced and
LeGFPSN-transduced (data for negative control LN not shown) bone
marrow. Animals were killed and analyzed 2 weeks, 8 to 12 weeks, 4 to 5 months, and 6 to 8 months after BMT. Values for the
percentage of cells showing expression were calculated by dividing the
number of expressing lin+ cells by the total number of
lin+ cells and are given as the mean ± standard error
of the mean. n is the number of animals analyzed for each time point
and vector. * and ** mark differences in the percentage of cells
showing expression between MNDeGFPSN and LeGFPSN that are statistically
significant (*P < .05 and **P < .005).
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As we saw for the analysis of the thymus (Fig 3B), the percentage of
eGFP expressing T lymphocytes increased in bone marrow, spleen, blood,
and thymus over time in recipients of MNDeGFPSN-transduced bone marrow
(Fig 4B). In recipients of LeGFPSN-transduced marrow, expression levels
increased only slightly up to 4 to 4.5 months after BMT, but decreased
to less than 5% by 6 to 8 months after BMT. The increase in the
percentage of eGFP(+) T lymphocytes from recipients of
MNDeGFPSN-transduced marrow was most marked in thymus. Less than 5% of
thymic T lymphocytes expressed eGFP 2 weeks after BMT with either
vector. The percentage of eGFP expressing thymic T lymphocytes from
recipients of MNDeGFPSN-transduced bone marrow increased to 24% ± 9.5% after 8 weeks, 26% ± 10.2% after 4 to 5 months, and 36.3% ± 11.3% after 6 to 8 months. At all time points, when under the
control of the MoMuLV LTR, eGFP expression in thymic T lymphocytes
reached 50% in only 1 of 29 mice and remained less than 5% in 24 of
29 mice. When under the control of the MND LTR, 8 of 33 mice showed
expression in more than 50% of thymic T lymphocytes (5 of 33 in
>85%) and only 12 of 33 mice showed levels less than 5% of thymic T
lymphocytes (data not shown).
Secondary BMT.
To compare expression by the 2 vectors in cells derived from primitive
HSC, we collected bone marrow from 5 randomly chosen pairs of mice
transplanted with transduced bone marrow and transplanted cells into
secondary recipients to generate 2°CFU-S. One primary pair of mice
transduced with MNDeGFPSN and LeGFPSN was killed at 8 weeks and another
at 4 months after transplant and 3 pairs were killed at 6 months after
transplant. The bone marrow from each of the primary recipients was
transplanted into 2 to 4 irradiated secondary recipients. Twelve to 14 days after the secondary transplant, 3 to 5 of the 2°CFU-S-derived
colonies were harvested from the spleen of each secondary recipient and
analyzed for the presence of the vector DNA by PCR for the eGFP gene
and by FACS for eGFP expression. Recipients of LN-transduced marrow
served as negative controls for PCR and FACS analysis.
DNA PCR analysis of the 2°CFU-S showed a transduction efficiency of
100% (54 of 54 foci) for MNDeGFPSN and 82% (46 of 56 foci) for
LeGFPSN. Expression of eGFP was 100% (54 of 54 foci) in 2°CFU-S containing the MNDeGFPSN vector and 0% (0 of 46 foci) in 2°CFU-S containing the LeGFPSN vector (Table 3).
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DISCUSSION |
In this study, we compared the in vivo expression of the reporter gene
eGFP from a modified vector, MNDeGFPSN, and from the MoMuLV vector,
LeGFPSN, in murine hematopoietic and lymphoid cells. The data show a
significantly higher expression in recipients of MNDeGFPSN-transduced
marrow than in recipients of LeGFPSN-transduced marrow 8 weeks to 8 months after BMT. The frequency of expression on average was 6-fold
higher in recipients of MNDeGFPSN-transduced marrow compared with
recipients of LeGFPSN-transduced marrow. Mean fluorescence intensity,
as a measure of the amount of synthesized protein per cell, was on
average 6- to 10-fold higher in MNDeGFPSN-transduced eGFP(+) cells than
in LeGFPSN-transduced eGFP(+) cells.
The higher frequency of eGFP expression and the higher mean
fluorescence intensity for MNDeGFPSN-transduced cells could be related
to a higher vector copy number per cell. A higher copy number could
result in an increased probability of an expression-favoring insertion
site of the vector and therefore to expression of the reporter gene. It
could also lead to a greater intensity of fluorescence and a higher
number of expressing cells through a summation effect of the reporter
gene transcripts from all the vector copies in the cell. Therefore, we
performed semiquantitative PCR analysis to assess average vector copy
number/cell for the 2 vectors on tissues and sorted cell lineages. The
results showed that the copy numbers were not statistically
significantly different between the 2 vectors, ranging from 1 to 4 copies/cell on average, consistent with the equivalent titers of the
vector-producing cell clones. Therefore, differences in expression were
not due to a difference in copy number but were likely due to a higher
probability of expression from the MND LTR than from the standard
MoMuLV LTR. We have previously reported that embryonic carcinoma and
embryonic stem cells expressed at a higher frequency from the MND LTR
(>95%) than from the MoMuLV LTR (<5%).8
Long-term expression.
Two weeks after BMT, the frequency of expression was not significantly
different between the 2 vectors. Both vectors initially expressed in a
high percentage of hematopoietic cells and B lymphocytes in blood,
spleen, and bone marrow. Over time, the percentage of cells expressing
from the standard MoMuLV LTR decreased to a greater extent than from
the modified MND LTR. The decrease in the number of expressing cells
could be due to repression of expression (silencing) in mature
hematopoietic cells that initially expressed the reporter gene.
Alternatively, it could be due to the turnover of mature cells,
resulting in later cell populations being derived from more primitive
precursors, which either never expressed the reporter gene or in which
it underwent silencing. These processes seem to be significantly less
likely to occur in the modified vector, MNDeGPFSN, than in the
MoMuLV-derived vector, LeGFPSN.
Distinct time courses of eGFP expression for different lineages.
In recipients of MNDeGFPSN-transduced marrow, the frequency of eGFP
expression was relatively stable in cells of the myeloid lineage, B
lymphocytes, and RBC. In contrast, in T lymphocytes there was a marked
increase in eGFP expression over time. The increase in expression
frequency was evident in all tissues, but was most prominent in thymus
and least prominent in blood.
Most likely, in the lethally irradiated mice, T lymphocytes were
initially derived from transduced donor primary T lymphocytes and
mature lymphoid progenitors, resulting in high numbers of expressing T
lymphocytes in peripheral blood 2 weeks after BMT. Over time, T
lymphocytes were generated from transduced HSC and early lymphoid
precursors by repopulation of the thymus and extrathymic T-cell
development. Observed differences in expression over time in the
myeloid and B-lymphoid lineages compared with T-lymphoid cells could
result from a distinct pattern of regulation of peripheral hematopoietic and thymic engraftment.18
2°CFU-S.
Serial transplantation of transduced murine bone marrow is a stringent
in vivo assay to test vectors targeting HSC. After reconstitution of a
primary recipient for at least 2 months, cells that give rise to
colonies forming in the spleens of secondary BMT recipients
(2°CFU-S) are derived from cells that were HSC at the time of
transduction.19 We have shown previously that the
modifications contained in the MND LTR led to increased expression of a
neomycin reporter gene in 2°CFU-S compared with the MoMuLV LTR.9 In this study, we observed eGFP expression in all of the MNDeGFPSN-transduced 2°CFU-S and none of the LeGFPSN-transduced 2°CFU-S, confirming that the higher expression frequency in primary recipients of MNDeGFPSN-transduced marrow extends to HSC.
Modification of vector sequences has previously been shown to improve
transduction efficiency and expression in in vitro and in vivo systems
targeting HSC and other primitive cells due to their wide clinical
application and the difficulties to reach efficient transduction and
expression in this entity. Riviere et al20 showed
improvement of expression of the human ADA gene from MFG-derived
vectors that included several modifications, such as replacement of the
MoMuLV enhancer or LTR by the myeloproliferative sarcoma virus (MPSV)
or Friend murine leukemia virus enhancer or LTR, respectively, or
introduction of the B2 mutation in the viral tRNA primer binding site.
Eckert et al21 showed improved expression of the multidrug
resistance gene (mdr1) in 2 vectors based on the spleen focus-forming
virus or the myeloproliferative sarcoma virus for the enhancer and the
murine embryonic stem cell virus for the leader.
Pawliuk et al22 compared expression in a murine transplant
model from 2 vectors, 1 containing the MPSV LTR and the other containing the PCC4 cell-passaged myeloproliferative sarcoma virus (PCMV) LTR and a substitution of the primer binding site
to abrogate binding of the repressor binding protein (murine stem cell
virus [MSCV]). In their BMT model, only FACS-sorted,
vector-expressing bone marrow cells were transplanted. By this method,
MSCV led to a high average percentage of expressing donor-derived cells in bone marrow (62%) and peripheral blood lymphocytes (28%) 6 months
after BMT. In the thymus, expression was 15%, despite the presence of
87% donor-derived cells.22
In our study, whole bone marrow was transplanted without preselection
for reporter gene expression. With the MND LTR, we saw similar values
for expression in BM and peripheral blood, but an average expression
frequency in the thymus of 38%.
In conclusion, these studies suggest that the modified vector, MND,
provides an advantage to the MoMuLV LTR for the expression of genes in
hematopoietic and lymphoid cells and especially in T lymphocytes. It
remains to be shown whether the MND LTR provides an advantage in human
cells, although preliminary studies suggest that it does.23
Studies in primary human T lymphocytes in vitro as well as in immune
deficient mice transplanted with CD34+ cells from human
cord blood are in progress.
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ACKNOWLEDGMENT |
The authors thank Earl H. Leonard for help with the statistical
analysis and Denise A. Carbonaro for reviewing the manuscript.
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FOOTNOTES |
Submitted January 20, 1999; accepted June 29, 1999.
Supported by Grant No. CA59318 from the National Cancer Institute of
the National Institutes of Health. D.B.K. is the recipient of a
Elizabeth Glaser Scientist Award from the Pediatric AIDS Foundation.
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, Mailstop #62, Childrens
Hospital Los Angeles, 4650 Sunset Blvd, Los Angeles, CA 90027; e-mail:
dkohn{at}chla.usc.edu.
| |
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