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Blood, Vol. 95 No. 10 (May 15), 2000:
pp. 3078-3084
GENE THERAPY
Limiting numbers of G156A
O6-methylguanine-DNA methyltransferase-transduced marrow
progenitors repopulate nonmyeloablated mice after drug selection
Brian M. Davis,
Omer N. Koç, and
Stanton L. Gerson
From the Division of Hematology/Oncology, Case Western Reserve
University, the Molecular Virology Training Program, and the Ireland
Cancer Center, University Hospitals of Cleveland, Cleveland, OH.
 |
Abstract |
The limited efficacy of hematopoietic gene therapy can be improved
by in vivo selection for transduced long-term repopulating cells
(LTRC). We selected for G156A MGMT ( MGMT) transduced LTRC present in
5 × 104 to 100 × 104 marrow cells
infused into nonmyeloablated mice by the administration of
O6-benzylguanine (BG) and BCNU every 3 to 4 weeks. To
facilitate engraftment, mice were given a nonablative dose of BG and
BCNU before infusion. Without selection, MGMT was not detected in any hematopoietic colony-forming units (CFU) 24 to 30 weeks after infusion. After BG and BCNU, MGMT+ CFU were frequently
detected, and their proportions increased with each treatment cycle.
After 2 to 3 cycles of BG and BCNU, many mice were stably reconstituted
with 75% to 100% MGMT+ CFU for at least 6 months,
representing up to 940-fold enrichment. Thus, BG and BCNU stem cell
toxicity allows MGMT-transduced LTRC to repopulate the bone marrow.
This degree of selection pressure in nonmyeloablated mice is far
greater than that observed in previous drug-resistance gene transfer
studies. These data support our approved clinical trial to select for
drug-resistant, transduced hematopoietic cells, potentially decreasing
cumulative drug-induced myelosuppression in patients with cancer. These
data also suggest that MGMT may be a potent, dominant, selectable
marker for use in dual gene therapy.
(Blood. 2000;95:3078-3084)
© 2000 by The American Society of Hematology.
| |
Introduction |
Successful clinical gene transfer into hematopoietic
cells requires the replacement of endogenous marrow with transduced
cells. This necessitates either myeloablation and reconstitution with transplanted cells or the ability to select strongly for the transduced population. These strategies are limited by the low rate of
transduction into long-term repopulating cells (LTRC) and, in a
nonmyeloablative setting, infusion of small numbers of cells relative
to a patient's total marrow, after which the infused cells must
compete for engraftment. A number of hematopoietic gene therapy
clinical trials have generated poor results, with genetic modification
in less than 0.1% of cells.1-4 The use of a
drug-resistance gene to enable selection for transduced hematopoietic
stem cells (HSC) has been explored as a strategy to improve the outcome
of these trials. Examples of drug-resistance gene transfer and
selection include MDR-1,5,6 DHFR,7,8 and
wtMGMT.9 In these studies, marrow from drug-treated mice yielded greater numbers of transduced, drug-resistant progenitors and
increased tolerance to chemotherapeutic agents than did untreated controls. However, none of these studies have efficiently selected for
LTRC without prior myeloablation.
Here, we report dramatic in vivo selection for LTRC transduced with a
mutant form of MGMT ( MGMT) in nonmyeloablated mice. MGMT encodes the
protein O6-alkylguanine-DNA-alkyltransferase (AGT), which
repairs BCNU lesions on DNA by direct transfer onto its active
site.10 The mutant AGT ( AGT) contains a single amino
acid substitution from glycine to alanine at amino acid 156, which
blocks inactivation by the AGT inhibitor, O6-benzylguanine
(BG).11 BCNU forms a chloroethyl lesion on O6 of guanine, which, if not repaired by AGT, is converted to a permanent, covalent, interstrand DNA cross-link between the modified guanine and
the opposite strand cytosine in 8 to 12 hours.12 This
distinguishes BCNU from other chemotherapeutic agents used to mediate
in vivo selection; BCNU is a stem cell toxin because cross-links formed in quiescent early hematopoietic cells are cytotoxic regardless of the
time lag between drug exposure and cell division. BG depletes wtAGT in
bone marrow cells, sensitizing both early and late progenitors to
BCNU.13 However, CD34+ cells that overexpress
MGMT efficiently repair DNA damage formed by BCNU even in the
presence of BG and are selectively protected from
cytotoxicity.14 In previous myeloablation/transplantation studies, we found that MGMT-transduced stem cells were
protected and enriched after BG and BCNU treatment.15 This
led us to test whether MGMT transduction and drug
selection would allow small numbers of LTRC to repopulate the
hematopoietic compartment of nonmyeloablated mice with drug-resistant
transduced cells.
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Materials and methods |
MFG-MGMT vector and producer cells
The MGMT cDNA was cloned from VACO 6 colon cancer
cells.16 VACO 6 MGMT cDNA differed slightly from the
published sequence17 (accession code NM002412), encoding a
Leu at codon 67 (Phe in published sequence) and a Phe at codon 73 (Leu
in published sequence); the sequence diversity did not affect the
activity or BG resistance of AGT. The VACO 6 sequence was confirmed in
MGMT transgenic mice.16 The G156A substitution was
introduced into MGMT as described elsewhere.14 The MGMT
cDNA was subcloned into the MFG retroviral vector (kindly provided by
Dr P Robbins, University of Pittsburgh, Pittsburgh, PA), and GP + E86
and GP + envAm12 producer cell lines (kindly provided by Dr
A. Bank, Columbia University, New York, NY) were established as
described elsewhere.14 The titer of the retroviral producer
cells was 5 × 105 infectious particles/mL.
Transduction protocol
Bone marrow progenitors were obtained from the femur and tibia of 6- to 8-week-old male C3H/HeNCrlBR mice (Charles River, Wilmington, MA) 48 hours after treatment with 150 mg/kg 5-fluorouracil (5-FU; Pharmacia,
Kalamazoo, MI). The cells were transduced as previously
described.15 The producers were rendered replication defective by treatment with 10 µg/mL of mitomycin C (Bedford
Laboratories, Bedford, OH). Untransduced cells were exposed to the same
conditions as were transduced cells. Transduced and untransduced cells
were mixed at 1:4 and 1:20 ratios at a final concentration of
5 × 106 cells/mL and were used for transplantation.
Then 3 × 106 cells from each mix were treated with
20 µmol/L BG and 0 to 40 µmol/L BCNU and were plated
in methylcellulose.
Cell infusion and drug administration
Recipient 7- to 9-week-old male C3H/HeNCrlBR mice received 30 mg/kg
BG and 10 mg/kg BCNU 48 hours before infusion with 1 × 106
bone marrow cells by tail vein injection. Mice were kept in
microisolator cages and given water supplemented with bacitracin and neomycin.
BG and BCNU were solubilized and injected intraperitoneally as
previously described.15 BCNU and Sentry Grade Union Carbide PEG-400 were obtained from the Developmental Therapeutics Branch, National Cancer Institute (Bethesda, MD). BG was synthesized by Dr
Robert Moschel at the Frederick Cancer Research Institute (Frederick, MD). Mice were treated with 30 mg/kg BG and 10 mg/kg BCNU or
were left untreated every 3 to 4 weeks after infusion, and they were killed 24 to 30 weeks after infusion.
In vitro drug treatment and colony-forming unit assay
Bone marrow cells were incubated with 0 or 20 µmol/L BG for 1 hour
followed by 0 to 80 µmol/L BCNU for 2 hours. Cells were washed free
of drug and plated in triplicate in methylcellulose containing 100 ng/mL rSCF (Amgen, Thousand Oaks, CA), 100 U/mL mIL-3 (Genzyme,
Cambridge, MA), 7.5 U/mL hEPO (Amgen), 40 µL pokeweed mitogen spleen
cell-conditioned medium, and 0.1 mmol/L hemin and were cultured for 7 days at 37°C and 5% CO2. Colony-forming unit (CFU)
colonies larger than 50 cells were enumerated.
Polymerase chain reaction for provirus detection
Polymerase chain reaction (PCR) analysis of genomic DNA was
performed as previously described.15 Previously described
proviral specific primers were used to amplify a 443-bp fragment, and
mouse  -globin primers were used to amplify a 400-bp
fragment.15 The PCR conditions were 40 cycles of 94°C
for 45 seconds, 60°C for 45 seconds, and 72°C for 1.5 minutes.
Positive and negative controls included genomic DNA from previously
demonstrated MGMT+ CFU and untransduced CFU,
respectively, and a water control to ensure the lack of contaminating
MGMT sequences in reagents.
Blood analyses
Blood was obtained from the tail vein of mice and mixed with 0.5 mol/L EDTA to prevent clotting. Counts were performed using a Sysmex
K-100 (Baxter, Deerfield, IL) and normalized for the blood-to-EDTA ratio.
FACS analysis for AGT expression
Spleens and thymi were minced into small pieces then grated through
a fine mesh to generate single-cell suspension. Blood was obtained by
cardiac puncture, and mononuclear cells were isolated by
Ficoll-Hypaque separation. Cells were prepared for flow cytometry as
previously described.15 Flow cytometry was performed using a Becton Dickinson FACScan, and 20,000 events were analyzed.
| |
Results |
Preinfusion treatment of mice with 30 mg/kg BG and 10 mg/kg BCNU is
not myeloablative
Myelosuppression produced by BG and BCNU was measured in mice
treated with 10 mg/kg BG 1 hour before 25 to 50 mg/kg BCNU or with 30 mg/kg BG before 10 to 50 mg/kg BCNU. The LD50 of the drug combination was 10 mg/kg BG plus 40 mg/kg BCNU or 30 mg/kg BG plus 30 mg/kg BCNU. In subsequent experiments, we administered 30 mg/kg BG and
10 mg/kg BCNU, which produced moderate myelosuppression. In
mice killed 48 hours after treatment, hind limb marrow cellularity decreased 57% to 10.25 ± 2.5 × 106 cells in
BG- and BCNU-treated mice (n = 4) compared with
23.8 ± 3.4 × 106 cells in controls (n = 3).
The concentration of committed progenitors per
1 × 105 marrow cells decreased 77%, from
189 ± 34 to 43 ± 12; BFU-E were reduced by 77%,
161 ± 28 versus 36 ± 10; CFU-GM were reduced by 79%,
25 ± 6 versus 5.4 ± 1.9; and CFU-GEMM were reduced by 63%, 2.5 ± 1.9 versus 0.9 ± 0.9. Blood counts were significantly
reduced (P < .05; n = 3) 7 days after drug treatment for
WBC (5.0 ± 0.6 vs. 1.3 ± 0.4 [×103/µL]),
RBC (3.8 ± 0.7 vs. 2.7 ± 0.5 [×106/µL]),
and platelets (564 ± 297 vs. 169 ± 45
[×103/µL]), but they recovered by day 12. Nineteen
additional mice received 30 mg/kg BG and 10 mg/kg BCNU; they were
followed up for more than 6 months and had normal blood counts,
indicating that this dose was myelosuppressive but not myeloablative.
Transduction efficiency to colony-forming units
An MFG-MGMT retroviral vector was used to transduce
5-FU-enriched mouse bone marrow-derived progenitors.14
After a 48-hour transduction, 75% (72 of 96) of CFU were
MGMT+ by PCR. Transduced cells were mixed with
untransduced cells such that the transduced cell population constituted
5%, 25%, or 100% of the mixture. To assess the relative protection
from BG and BCNU after MGMT transduction, cells were treated with 20 µmol/L BG and increasing doses of BCNU. The BCNU IC50
plus 20 µmol/L BG increased from 3 µmol/L to 19.5 µmol/L and 9.5 µmol/L in mixtures of 100% and 25%
MGMT-transduced cells, respectively (Figure
1).
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| Fig 1.
BG and BCNU resistance in transduced CFU.
After transduction, bone marrow was mixed with untransduced cells at
final ratios of 1:4 and 1:20. The cells were treated with 20 µmol/L
BG and 0 to 40 µmol/L BCNU then plated in methylcellulose, and CFU
growth was scored. Error bars represent mean ± SD.  , MGMT
transduced;  , 25% MGMT transduced;  , 5% MGMT transduced;
 , untransduced.
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MGMT-transduced cell infusion and mouse survival after BG and
BCNU treatment
Recipient mice were pretreated with 30 mg/kg BG and 10 mg/kg BCNU to
induce myelosuppression and improve engraftment. Cohorts of mice were
infused 48 hours later with 1 × 106 progenitors
from the 5%, 25%, or 100% MGMT-transduced cell mixtures, such
that the number of cells infused from the transduced cell culture was
5, 25, or 100 ( × 104) cells (approximately 4, 20, or 75 [×104] transduced cells). Noninfused control mice
did not receive MGMT-transduced cells. Mice received 0, 1, 2, or 3 postinfusion cycles of BG and BCNU administered every 3 to 4 weeks.
Only 1 of 6 control mice survived 4 cycles of BG and BCNU. In contrast,
66% of mice from the 5 × 104 cohort, 91% from the
25 × 104 cohort, and 100% from the
100 × 104 cohort survived 3 or 4 cycles of BG and
BCNU treatment (P < .005; Table
1), indicating a striking survival
advantage for mice receiving MGMT-transduced cells.
Selection for transduced progenitors
Mice were killed 24 to 30 weeks after cell infusion to test for the
presence of MGMT-transduced LTRC at a time when infused, short-term,
repopulating progenitors had been exhausted. There was no difference in
mean bone marrow cellularity between drug-treated cohorts infused with
MGMT-transduced cells (30.7 × 106 ± 5.8
cells/mouse) and mice that did not receive postinfusion BG and BCNU
(33.3 × 106 ± 3.9 cells/mouse). The
proportion of CFU that contained the MGMT proviral sequence was
determined by proviral-specific PCR on individual colonies (Figure
2). The MGMT proviral sequence was
undetectable in any of the 168 marrow-derived CFU tested from 9 mice
that did not receive postinfusion BG and BCNU. PCR performed on whole
bone marrow preparations from these mice also did not amplify the
MGMT sequence. We expected a limit of detection of 1%. Furthermore,
there was no detectable AGT expression in the bone marrow of these
mice by Western blot. The inability to detect genetically modified
cells suggests that transduced, infused progenitors are at a
competitive disadvantage to endogenous stem cells in contributing to
long-term hematopoiesis. This is consistent with previous reports
suggesting that 5-FU-enriched, cytokine-stimulated cells engraft
poorly to nonmyeloablated mice.18-20
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| Fig 2.
In vivo selection for MGMT-transduced CFU.
Representative PCR on genomic DNA obtained from individual bone
marrow-derived CFU from mice in the 25 × 104 cohort
given (a) 0 cycles (b) 1 cycle, (c) 2 cycles, or (d) 3 cycles of BG and
BCNU; (e) mouse from 100 × 104 cohort given 3 cycles of BG and BCNU. +, positive control;  , negative control;
H20, water control (no genomic DNA).
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In contrast, at least some CFU from each mouse in the
100 × 104 MGMT+ (n = 9) and the
25 × 104 MGMT+ (n = 9) cohorts and
9 of 10 of the mice in the 5 × 104
MGMT+ cohort treated with 2 or 3 cycles of BG and BCNU
contained the MGMT provirus. The proportion of CFU with provirus
correlated with the number of cycles of BG and BCNU received and the
number of MGMT-transduced cells infused (Table
2). Every CFU tested in 4 of 5 mice from
the 100 × 104 cohort, which received 3 cycles of BG
and BCNU, were MGMT+, and 14 of 16 CFU were
MGMT+ in the fifth mouse. Furthermore, every CFU tested
from 1 mouse from the 100 × 104 cohort treated
twice and from 1 mouse from the
25 × 104 cohort treated with 3 cycles of BG and
BCNU was MGMT+, whereas 75% or more of the CFU from the
other 6 mice in these cohorts contained MGMT. One dose of BG and
BCNU was sufficient to mediate selection for MGMT-transduced LTRC at
the 6-month time point because CFU from 3 of 4 mice from the
25 × 104 and the 100 × 104
cohorts and 2 of 4 mice from the 5 × 104 cohort
contained the MGMT provirus. This implies that 1 cycle of drug
exerted remarkable selection pressure, resulting in the survival of
MGMT+ LTRC among endogenous HSC 6 months after infusion
and drug treatment.
Undetectable without selection, strong AGT expression was observed
in bone marrow, spleen, and blood mononuclear cells after BG and BCNU
selection. Although not performed in these experiments, we have
observed strong MFG-MGMT expression in thymocytes after BG and
BCNU.21 The proportion of cells overexpressing AGT
increased with each cycle of BG and BCNU, with overexpression in at
least 48% of cells from all sources after 3 cycles (Figure
3). Furthermore, bone marrow-derived CFU
became increasingly resistant with each cycle of drug received (Figure
4; Table 2). In contrast, CFU from mice
that did not receive postinfusion BG and BCNU were as sensitive to BG
and BCNU as untransduced controls. Therefore, MGMT+
cells had a distinct survival advantage after BG and BCNU treatment because of the expression of AGT.
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| Fig 3.
Flow cytometry on bone marrow, splenocytes and blood
mononuclear cells from mice infused with 100 × 104
MGMT-transduced cells after 1, 2, or 3 cycles of BG and BCNU.
Cells were fixed, permeabilized, and stained with the human
AGT-specific monoclonal antibody mT3.1 and a PE-conjugated secondary
antibody before analysis. There was no cross-reactivity with murine
AGT. The proportion of cells overexpressing AGT was determined by
subtracting the histogram obtained from normal mouse tissue.
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| Fig 4.
CFU become increasingly resistant to BG and BCNU after
repetitive drug administration.
Bone marrow cells were treated with 20 µmol/L BG and 0 to 80 µmol/L
BCNU, then plated in methylcellulose. CFU growth was scored, and
resistance curves were generated. Error bars represent SD of mean
percentage survival. , 3 cycles of BG and BCNU; , 2 cycles; ,
1 cycle; dashed line; open box, 0 cycles; dotted line, normal mice.
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Mice infused with MGMT-transduced cells are protected from
myelosuppression
Blood cell counts were monitored in the 25 × 104
and 100 × 104 cohorts and in noninfused controls
during an 80-day period in which mice received 3 cycles of BG and BCNU
at 4-week intervals (3 mice per group; Figure
5). Mice from all 3 cohorts had a similar level of myelosuppression after the first postinfusion cycle of BG and
BCNU, but after the second treatment, mice in the
100 × 104 cohort had higher WBC counts than control
mice (P = .0522). After the third cycle of drug, mice infused
with MGMT-transduced cells had significantly higher counts than the
control group (P < .05). Cumulative myelosuppression
resulted in the death of 2 of 3 control mice after the second cycle,
whereas all mice infused with MGMT-transduced cells survived. In
addition, mice receiving 2 or 3 doses of BG and BCNU were analyzed for
the maintenance of blood counts 28 weeks after infusion. There was no
significant decrease in WBC or RBC counts in drug-treated mice in any
of the cohorts; however, platelet counts
(× 103/µL) were significantly reduced
(325 ± 85 in the 100 × 104 cohort,
362 ± 97 in the 25 × 104 cohort, and
271 ± 109 in the 5 × 104 cohort) compared
with untreated controls (546 ± 35; P < .05). This
reduction may have been caused by toxicity to untransduced early
progenitors, insufficient numbers of transduced LTRC to maintain normal
platelet counts, or poor proviral expression in the megakaryocyte
lineage. These data demonstrate that enrichment for MGMT-transduced
hematopoietic cells by BG and BCNU protects mice from cumulative
myelosuppression induced by this drug combination and that LTRC
were protected by MGMT cDNA transfer and expression.
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| Fig 5.
Infusion of MGMT-transduced cells protects mice from
cumulative myelosuppression.
Blood counts from BG- and BCNU-treated mice were monitored for 70 to 80 days. The first treatment (dashed line) was performed 3 weeks after the
infusion of bone marrow cells, and subsequent treatments were
administered every 4 weeks. Each value represents the mean of 3 mice,
except *, which was obtained from 1 surviving normal mouse. Error bars
represent SD of mean. , 100 × 104 cohort; ,
25 × 104 cohort; , normal mice.
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No engraftment or selection was observed without the BG and BCNU
preparatory regimen
Interestingly, mice not given a preparatory regimen of BG and BCNU
48 before cell infusion (n = 12) but given 2 cycles of BG and BCNU 3 and 7 weeks after infusion did not have any MGMT+ CFU 6 months after infusion, despite an initial 70% transduction efficiency
into infused CFU. The CFU of these mice were as sensitive to BG and
BCNU as control mice (data not shown). Although Rao et al22
have shown that prior ablation is not required for engraftment when at
least 12.5 × 106 cells are infused, our data
suggest that a niche may be required to allow engraftment of low
numbers of cells. Without this niche, infused LTRC could not be
sufficiently enriched by BG and BCNU. In contrast to mice that had not
received BG and BCNU before infusion, mice given the preparatory
regimen had a 57% reduction in total marrow cellularity at the time of
infusion. This niche formed in the marrow appears to allow improved
engraftment of the infused cells and subsequent BG and BCNU selection
of LTRC.
| |
Discussion |
These data show that the infusion of a small number of
MGMT-transduced early hematopoietic progenitors into nonmyeloablated mice followed by treatment with BG and BCNU allows virtually complete, selective hematopoietic repopulation with MGMT+ cells.
This approach appears to offer an improvement over standard hematopoietic gene transfer methods, which require myeloablative preparation of the host to achieve similar levels of expression. BG and
BCNU appear to exert strong selective pressure at the stem cell
level because genetically modified cells are still observed 6 months after infusion, in some cases 5 months after the last drug
treatment. Efficient selection at the stem cell level and long-term
detection of genetically modified cells suggest applications of BG and
BCNU selection for MGMT+ cells in cancer therapy and
dual gene transfer.
BG and BCNU selection for MGMT+ LTRC is stronger than
has been achieved in other in vivo selection studies. We defined the selection pressure as the fold-enrichment for CFU after BG and BCNU.
Nonmyeloablated mice that received 100 × 104
transduced progenitors (an estimated 6000 MGMT+ CFU) and
2 cycles of BG and BCNU had nearly 100% MGMT+ CFU
(5.5 × 105 total MGMT+ CFU) 4 months
after the final drug treatment, a 92-fold enrichment for
MGMT+ CFU (Figure 6). After
3 cycles, the marrow remained nearly 100% MGMT+,
but AGT expression increased, as shown in flow cytometry and drug-resistance data. Similarly, mice from the
25 × 104 cohort treated with 3 cycles of drug had
nearly 100% MGMT+ CFU, which calculates to 350-fold
enrichment. Mice in the 5 × 104 cohort treated with
3 cycles of drug had approximately 50% MGMT+ CFU, a
940-fold enrichment. Mice treated with only 1 cycle of BG and BCNU had
approximately 20-, 56-, and 120-fold enrichment for
MGMT+ CFU in the 100, 25, and
5 × 104 cohorts, respectively. The strong selection
pressure after 1 cycle of BG and BCNU might have resulted from
BCNU-induced cross-links in unprotected HSC. These cross-links persist
in quiescent HSC and become cytotoxic as they sequentially expand to
support hematopoiesis. This results in an increasing proportion of
MGMT-transduced LTRC repopulating the marrow over time. Additional
cycles of BG and BCNU increase HSC cross-links in unprotected cells and
increases the demand on HSC to proliferate in response to
myelosuppression. Ultimately, the selection pressure weakens because
fewer BG- and BCNU-sensitive cells remain after each cycle.
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| Fig 6.
BG and BCNU mediates strong selection pressure.
Fold enrichment for MGMT+ CFU was calculated by dividing
the total estimated number of MGMT+ CFU at sacrifice by
the number of MGMT+ CFU infused. Within
1 × 105 bone marrow cells, we obtained
approximately 200 CFU, which increased to 800 after 5-FU treatment.
Because the CFU transduction efficiency was 75%, the total number of
infused MGMT+ CFU was 6000, 1500, and 300 in the 100, 25, and 5 × 104 cell populations, respectively. At
sacrifice, we estimated 6 × 105 CFU present in
300 × 106 total mouse bone marrow cells. ,
5 × 104 cohort; , 25 × 104
cohort; , 100 × 104 cohort.
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In these studies, the number of MGMT-transduced LTRC present in
5 × 104 5-FU enriched cells appears to be near the
threshold level of LTRC necessary to partially repopulate a mouse with
drug-resistant progenitors after drug selection. There are an estimated
25 LTRC in a population of 5 × 104 bone marrow
cells after 5-FU treatment.23 Although the transduction efficiency into CFU was 75% in our experiments, relatively quiescent LTRC are expected to be transduced less efficiently by retroviral vectors.24 Assuming 1 MGMT-transduced LTRC was
sufficient to produce enough progeny cells to protect the mouse from
myelosuppression, the variability after infusion of 25 LTRC implies
that the transduction efficiency into LTRC was at least 4%. Southern
blotting performed on the marrow of a mouse infused with
5 × 104 cells and given 2 cycles of BG and BCNU
confirmed the low number of transduced LTRC. A dominant band
and a possible second weaker band was observed, demonstrating that 1 or
2 transduced LTRC were actively involved in hematopoiesis 6 months
after transplantation.
In contrast to BCNU, which facilitates strong selection by stem cell
toxicity, other drugs commonly used to mediate in vivo selection for
drug-resistance genes, such as paclitaxel for MDR1 selection and
methotrexate for DHFR selection, are cytotoxic to cycling cells but not
stem cells.25-27 Because the stem cell pool continues to
participate in hematopoiesis, there is far less selection for
transduced LTRC. Daily administration of these and similar drugs and
cytokines can stimulate stem cells into cell cycle and may result in
some HSC cytotoxicity.27 Repeated treatment has been used
to mediate selection for L22Y DHFR-transduced LTRC by treatment with
trimetrexate and the thymidine transport inhibitor, nitrobenzylmercaptopurineriboside (NBMPR). In a study by Allay et
al,8 a 1:4 mix of transduced-to-untransduced hematopoietic progenitors was transplanted into lethally irradiated mice, and trimetrexate and NBMPR treatment resulted in a 4- to 6-fold enrichment for transduced cells. In contrast, our approach using nonmyeloablated mice permitted us to observe nearly 1000-fold enrichment over the
course of drug-mediated selection, with persistent expression for at
least 6 months.
Because we have demonstrated that selection for MGMT-transduced LTRC
in nonmyeloablated mice can completely repopulate the marrow and
generate drug-resistant hematopoietic progeny, we have suggested that
these concepts be tested in a clinical trial. The hypothesis is that
the infusion of small numbers of transduced progenitor cells would
allow emergence of drug-resistant marrow, resulting in amelioration of
the cumulative myelosuppression previously observed in phase I trials
with BG and BCNU.28,29 The poor transduction efficiency
into early human LTRC may limit this approach. However, the current
perspective that these cells are rarely transduced may be biased by the
use of nonselectable genes. Further experimentation with in vivo
selectable genes must be performed to resolve this issue. Based on the
results presented in this report and on additional results from our
laboratory,30 the United States Food and Drug Administration has recently approved a phase I trial of MGMT gene
transduction of CD34+ cells in patients undergoing
sequential treatment with BG and BCNU.31 The end points of
this trial include detection of transduced cells after each cycle of
chemotherapy, detection of drug-resistant marrow CFU, monitoring for
evidence of cumulative myelosuppression, and therapeutic response to
the drug combination.
These data suggest that LTRC transduced with bicistronic vectors
containing both MGMT and a nonselectable therapeutic gene may be
enriched by BG and BCNU. The success of this approach will be
determined in experiments under way in our laboratory. Until techniques
that improve transduction of stem cells are developed, in vivo
selection for transduced LTRC would potentially ensure that sufficient
numbers of cells are genetically modified and express protein levels
necessary to modify a deficient phenotype. A drawback is the need to
use cytotoxic drugs as selective agents, but this may be accomplished
at doses low enough to avoid toxicity to nonhematopoietic tissues. If
this is validated, in vivo selection using MGMT should be considered
a viable approach to improve the outcome of a number of hematopoietic
gene therapy protocols.
| |
Acknowledgments |
We thank Keunmyoung Lee for creation of the MFG-MGMT vector, Jane
Reese for production of the high-titer producer clone, and James A. Allay for helpful discussions.
| |
Footnotes |
Submitted April 21, 1999; accepted December 27, 1999.
Supported by Public Health Service grants RO1CA73062, RO1ES06288,
UO1CA75525, and P30CA43703.
Reprints: Stanton L. Gerson, Case Western Reserve University
School of Medicine, 10900 Euclid Avenue, BRB 3-West, Cleveland, OH
44106-4937; e-mail: slg5{at}cwru.edu.
The publication costs of this
article were defrayed in part by
page charge payment. Therefore,
and solely to indicate this fact,
this article is hereby marked
"advertisement"
in accordance with 18 U.S.C.
section 1734.
| |
References |
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Abstract
Introduction