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Blood, Vol. 95 No. 2 (January 15), 2000:
pp. 700-704
RED CELLS
Phenotypic correction of Fanconi anemia group C knockout mice
Kimberly A. Gush,
Kai-Ling Fu,
Markus Grompe, and
Christopher E. Walsh
From the UNC Gene Therapy Center and the Department of Medicine,
University of North Carolina, Chapel Hill, NC; and the
Department of Genetics, Oregon Health Sciences Center, Portland,
OR.
 |
Abstract |
Fanconi anemia (FA) is a genetic disorder characterized by bone
marrow failure, congenital anomalies, and a predisposition to
malignancy. FA cells demonstrate hypersensitivity to DNA cross-linking agents, such as mitomycin C (MMC). Mice with a targeted disruption of
the FANCC gene (fancc  / nullizygous mice)
exhibit many of the characteristic features
of FA and provide a valuable tool for testing novel therapeutic
strategies. We have exploited the inherent hypersensitivity of
fancc / hematopoietic cells to assay for phenotypic
correction following transfer of the FANCC complementary DNA (cDNA)
into bone marrow cells. Murine fancc / bone marrow cells
were transduced with the use of retrovirus carrying the human
fancc cDNA and injected into lethally irradiated recipients.
Mitomycin C (MMC) dosing, known to induce pancytopenia, was used to
challenge the transplanted animals. Phenotypic correction was
determined by assessment of peripheral blood counts. Mice that received
cells transduced with virus carrying the wild-type gene maintained
normal blood counts following MMC administration. All nullizygous
control animals receiving MMC exhibited pancytopenia shortly before
death. Clonogenic assay and polymerase chain reaction analysis
confirmed gene transfer of progenitor cells. These results indicate
that selective pressure promotes in vivo enrichment of fancc-transduced hematopoietic stem/progenitor cells. In
addition, MMC resistance coupled with detection of the transgene in
secondary recipients suggests transduction and phenotypic correction of long-term repopulating stem cells.
(Blood. 2000;95:700-704)
© 2000 by The American Society of Hematology.
| |
Introduction |
Fanconi anemia (FA) is a rare autosomal disorder
characterized by developmental anomalies, bone marrow failure, and
cancer predisposition.1 The hematologic manifestations of
FA predominate, with the majority of patients developing
aplasia, myelodysplasia, or acute leukemia,2 owing to
defective repopulating hematopoietic stem cells. Stem cell
reconstitution via histocompatible matched sibling donor bone marrow
transplantation3 produces a hematologic cure. The potential
of autologous stem cell harvest with subsequent gene transduction
offers an attractive alternative to patients lacking a matched sibling donor.
We have used recombinant viral vectors to transfer fancc
complementary DNA (cDNA) to cells from FA patients. Phenotypic
correction was demonstrated in vitro by resistance to mitomycin C
(MMC), a DNA cross-linker known to induce cell death at nanomolar
concentrations. Transduction of CD34+-enriched progenitors
significantly improved clonogenic growth in both the absence and the
presence of MMC.4,5 On the basis of these results, we
hypothesized that gene-corrected FA stem/progenitor marrow cells
maintain a selective growth advantage in vivo.
The fancc / knockout mouse provides a powerful
tool with which to study the mechanism of FA and the potential benefit
of novel therapeutic strategies in vivo.6,7 These mice
appear hematologically intact; however, upon exposure to such agents as
MMC, fancc / mice develop pancytopenia as a
consequence of marrow aplasia,7,8 recapitulating the
phenotype seen in FA patients. Furthermore, when bone marrow cells are
assayed for clonogenic capacity7 or the ability to
reconstitute themselves, they exhibit significantly
reduced growth potential,9 suggesting that hematopoietic
failure is caused by a primary defect of stem/progenitor cells. Here,
using gene transfer, we demonstrate reconstitution of hematopoiesis in
fancc nullizygous mice.
| |
Materials and methods |
Animals
fancc heterozygous [B6.129Sv-fancc/,
n = 3] mice were inbred by brother-sister mating [F = 5-6] to
obtain homozygous null and wild-type offspring. All animals were
maintained in the animal facility at University of North Carolina,
Chapel Hill, NC, in accordance with Institutional Animal Care & Use
Committee standards in hot-washed, micro-isolator cages supplied with
unlimited mouse chow and water. Lethally irradiated mice
were housed in autoclaved micro-isolator cages, given sterile food and
water, and kept in a pathogen-free room. All mice used in these
experiments were between 2 and 6 months of age.
Retroviral vector
The amphotropic, Murine Moloney Leukemia Virus-based,
retroviral vector containing human FANCC cDNA was collected from
supernatant of a stable producer cell line 52-19 as previously
described.4 Briefly, 1 to 2 × 106
producer 52-19 cells were plated in 10 cm dishes and grown to 75%
confluence. Fresh medium was added, and supernatant
containing virus was harvested 24 hours later. Viral supernatant,
harvested from producer cell line #17,5
carrying the Fanconi anemia group A
(fanca) cDNA served as negative control. Mock transduction of wild-type cells served as a positive control.
Retroviral mediated bone marrow transduction
fancc/ mice were euthanized by CO2
asphyxiation, and bone marrow cells were harvested from both femurs and
tibias. Red cells were depleted in ACK lysing buffer
(Bio-Fluids, Rockville, MD). The bone marrow cells were then
subjected to a 48-hour prestimulation period in Dulbecco's
Modified Eagle Medium (GibcoBRL, Gaithersburg, MD), 20% fetal bovine
serum (HyClone, Logan, UT), and penicillin/streptomycin, which was
supplemented with rmIL-3 (25 ng/mL) (R&D Systems, Minneapolis, MN),
rhIL-6 (50 ng/mL) (gift of Dr Robert Donahue, National Institutes of
Health), and rmSCF (25 ng/mL) (gift from Genetics Institute, Cambridge,
MA). The cells were pelleted and resuspended in viral supernatant with protamine sulfate (5 µg/mL) and
cytokines for 24 hours. Transduction was repeated for an additional 24 hours before injection of cells into recipient animals.
Bone marrow transplantation
Recipient wild-type female mice received whole body  -irradiation
in a single dose of 10 Gy, by means of a 137Cs
source 4 to 6 hours before transplantation.
Anesthetized recipient mice received injection of
1 × 106 transduced bone marrow cells through the
retro-orbital plexus. At week 16 postinjection, MMC
(CalBioChem, La Jolla, CA) was administered by means of intraperitoneal
injection at a dose of 0.3 mg/kg weekly for 6 weeks.
Hematological assay
Peripheral blood (20 to 50 µL) was collected by
means of tail venipuncture into microcapillary tubes precoated with
ethylenediaminetetraacetic acid. The samples were analyzed at the UNC
Animal Clinic Laboratory on an ABC Vet automated blood counter (ABX
Hematology Inc, Garden Grove, CA).
DNA isolation and analysis
Peripheral blood (50 to 100 µL) was collected by
means of tail venipuncture into heparin-treated microcapillary tubes,
and mature red blood cells were depleted by suspension in ACK lysing buffer (Bio-Fluids, Rockville, MD). DNA from nucleated cells was isolated with the use of the QIAamp Blood Kit (Qiagen, Valencia, CA)
and the manufacturer's protocol. DNA polymerase chain reaction (PCR)
(Perkin-Elmer Cetus, Norwalk, CT) incorporating
32P-deoxycytidine triphosphate was performed
with the use of 100 ng DNA and primers specific for the vector
(upstream primer 5'-ACAGATGGAA-TCGTCTTGGC; downstream primer
5'-CCTGTCTCTTGATCAGATCGG). Amplification conditions were as
follows: 95°C for 2 minutes (1 cycle), 95°C for 1 minute, 55°C for 1 minute, and 72°C for 2 minutes (35 cycles), followed by extension at 72°C for 8 minutes. Samples were electrophoresed on
5% polyacrylamide gels, and autoradiography was performed. Semiquantitative PCR, used to estimate the percentage of transduced cells within the graft, was based on comparison of band intensity, which was performed with the use of DNA isolated from a known vector
copy-control (cell line 52-19).
In vitro clonogenic assays
Bone marrow cells were harvested from recipients; red cells were
depleted as above; and 2 × 104 cells each were
incubated in methylcellulose media (Methocult M3530-Stem Cell
Technologies, Vancouver, BC, Canada) in both the presence and absence
of 10 nM MMC (CalBioChem). The cultures were incubated with high
humidity at 37°C and 5% CO2 for 15 days, and colonies
were enumerated.
In vivo repopulation assays
Unfractionated bone marrow cells isolated from primary recipient
mice were pooled, and 3 to 4 × 106 cells were
injected into secondary lethally irradiated wild-type female mice
following the same protocol described above. The secondary recipients
received weekly doses of MMC (0.3mg/kg) for 7 weeks.
Statistical analyses
Two-tailed t tests were performed for comparison of mean
colony numbers between corrected, wild-type, and null cells.
Calculations were performed with statistical analysis software (Prism,
GraphPad Software Inc.)
| |
Results |
Bone marrow cells were harvested from femurs and
tibias of fancc / mice, and unfractionated cells
were infected under conditions that promote efficient
transduction.10,11 Supernatant containing recombinant
retrovirus carrying either the human fancc cDNA4 or
an irrelevant cDNA5 was used. Wild-type recipients were subjected to gamma-irradiation (10 Gy) and received either
1 × 106 unfractionated marrow cells transduced with
rFANCC virus (n = 6), 1 × 106 cells transduced
with rFANCA virus (n = 6), 1 × 106 mock infected
wild-type (n = 3) cells, or no cells (n = 3). As expected, all
animals not receiving cells died within 8 days of irradiation. Control
animals receiving cells transduced with an irrelevent vector failed to
engraft with the same efficiency as those transduced with fancc
virus, and only 50% survived the 12-week engraftment
period. At 13 weeks posttransplant, peripheral blood counts were
performed and found to be normal in all remaining mice, indicating that
engraftment was satisfactory (Table).
To test whether phenotypic correction of the defective fancc
/ bone marrow cells occurred, we challenged all mice with
weekly intraperitoneal doses of MMC (0.3 mg/kg/wk for 6 weeks). This dosing regimen produces pancytopenia and bone marrow aplasia in untreated fancc / mice.8 Peripheral
blood counts were performed to monitor the effectiveness of MMC. As
expected, a marked decrease in all peripheral blood counts from
nullizygous untreated mice was observed (Table). Bone marrow histology
confirmed severe marrow hypocellularity (less than 5%). The 3 remaining animals that received cells transduced with irrelevant vector
(negative control) demonstrated normalized peripheral blood counts
before the MMC regimen; all of these animals, however, developed severe
pancytopenia following MMC exposure and died. In marked contrast, all
mice receiving FANCC-transduced cells demonstrated normal
peripheral blood counts after completing the MMC regimen (Table). No
evidence of leukemia or solid tumor was observed in any of the animals studied.
To verify gene transfer, we used DNA isolated from peripheral blood for
semiquantitative PCR. Primers were designed to generate a PCR product
spanning the vector and the transgene. We detected the transgene using
samples from FANCC-transduced mice prior to MMC administration
at levels averaging 0.01 vector genome copies/cell. Following MMC
exposure, detection of the transgene increased roughly tenfold in all
mice, to levels averaging 0.1 genome copies copies/cell (Figure 2A).
To demonstrate the transduction of primitive progenitor cells, we
performed in vitro clonogenic assays. The animals were killed, and
their marrow was harvested and plated in methylcellulose in both the
absence and the presence of MMC. No significant colony growth of
/cells is observed at 10 nM MMC, whereas
clonogenic growth of +/+ cells is not impaired.12 Animals
receiving either FANCC-transduced or wild-type (+/+) bone
marrow produced similar clonogenic results at 10 nM MMC,
(Figure 1). When wild-type or FANCC-corrected cells are compared with FANCC / cells, a
significant difference in clonogenic capacity is observed
(P = .0002, 2-tailed t test). To determine if
phenotypic correction of progenitor cells correlated with vector
transduction, PCR of individual colonies was performed. A positive
vector-specific PCR product was detected in more than 70% of all
colonies (in both the absence and presence of MMC) sampled (Figure 2B).
Marrow was harvested from secondary recipients, and clonogenic assays
were performed (data not shown). Results were comparable to those shown
in Figure 1 for the primary recipients.
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| Fig 1.
Clonogenic assay of FANCC gene-transduced bone
marrow.
Marrow was harvested from primary recipients, and
2 × 104 cells were plated in methylcellulose in the
presence or absence of 10 nM MMC. Duplicate cultures were
established from each animal. Colonies were enumerated at day 15 and
total number of colonies shown. (BFU, CFU-GM, and
CFU-mix colonies represented 20%, 70%, and 10%,
respectively.) Bone marrow from untreated knockout
(/), +/+, and FANCC-transduced recipients
is presented as the mean colony number ± SEM.  , wild-type;  ,
corrected;  , null.
|
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View larger version (61K):
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| Fig 2.
Vector-specific PCR.
(A) PCR was performed with the use of DNA isolated from peripheral
blood samples taken from primary recipients before and after MMC
administration. The standard curve was generated with the use of serial
dilution of a known vector-copy cell line, 52-19. (B) Methylcellulose
cultures were performed with the use of bone marrow isolated from
animals receiving FANCC-transduced cells. Day-15 bone
marrow colonies were enumerated and isolated for PCR analysis. (C) PCR
was performed with the use of DNA isolated from peripheral blood
samples taken from secondary recipients before and after MMC
administration. Positive control DNA was derived from cell line 52-19 and negative control DNA was isolated from cell line 17.
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To determine if phenotypic correction had occurred at the level of the
repopulating stem cell, marrow was harvested from the experimental
animals and pooled for transfer to secondary recipients. Lethally
irradiated wild-type mice were injected with 3 to
4 × 106 unfractionated bone marrow cells pooled
from animals receiving FANCC-transduced cells. Thirteen
weeks posttransplant, the secondary recipients received weekly doses of
MMC (0.3 mg/kg, intraperitoneally) for 7 weeks. All 5 secondary
recipients survived MMC with normal blood counts (Table).
DNA was isolated from peripheral blood samples taken before and after
MMC, and vector-specific PCR was performed. Vector-specific PCR from
peripheral blood DNA taken prior to MMC dosing detected the presence of
the transgene at a level of 0.01 copies/cell or less. However, a
tenfold to fiftyfold increase in the PCR signal of all secondary
recipient animals was observed following MMC treatment (Figure
2C). Normalization of peripheral blood counts (Table) in these
secondary recipients is consistent with an expansion of stem cells
incorporating the FANCC transgene.
| |
Discussion |
FA is a recessive inherited disorder characterized by a
progressive pancytopenia due to marrow aplasia.13 Patients
are diagnosed within the first decade of life, and without
histocompatible allogeneic bone marrow transplant (BMT), they die as
young adults. Patients undergoing BMT experience severe complications
related to pretransplant conditioning regimens and have an elevated
lifetime risk for developing malignancies.1 Alternative
treatments for FA patients include autologous BMT using gene-transduced
peripheral blood or bone marrow stem/progenitor cells.14
Results of 4 Fanconi Anemia Group C patients receiving retroviral
vector-transduced peripheral blood CD34+
cells demonstrated gene-marked peripheral blood cells and increased clonogenic growth; however, no significant effect on peripheral blood
counts were observed, implying that long-term repopulating cells were
not transduced. Thus, for gene therapy in FA to be successful,
transduction of the rare population of hematopoietic stem cells is
required. Since FA constitutes a disorder of stem cell function, it is
an excellent model for gene therapy of hematopoietic stem cells.
Several investigators14,15 have demonstrated defects within
the hematopoietic stem/progenitor compartment in FA patients. The
deficiency of functional FANC proteins in stem/progenitor cells is
assumed to be involved in the pathophysiology in the hematologic
manifestation of FA. It is experimentally difficult to study
hematopoietic stem cell function in FA patients owing to the lack of a
quantitative in vivo assay and the reduced numbers of cells available
from aplastic donors. The availability of animal models with targeted
disruptions of the FANCC gene6,7 provides a
powerful tool with which to address basic questions related to stem
cell function and an in vivo system to assess the utility of novel
therapeutic strategies.
We previously developed an amphotropic retroviral vector carrying the
cDNA for human FANCC.4 In this study, we use a
murine model of FANCC deficiency7 to determine if our
vector would transduce long-term repopulating cells (LTRC) and whether
these corrected cells would restore normal hematopoietic function to aplastic animals. Our experimental strategy has taken into account the
fact that stem/progenitor cells represent a rare subset of the marrow
population16,17 (perhaps even rarer in FANCC null mice) and that stable transduction of murine marrow cells by an amphotropic retroviral vector is relatively
inefficient.11,18 These limitations are directly analogous
to the situation found in aplastic FA patients, who have fewer cells to
target with a vector known to have a relatively low efficiency of
transduction for human cells.11 Because of these built-in
restrictions, we ensured that transduction of a stem/progenitor cell
followed by expression of the transgene would be an exceedingly rare
event. We then asked whether the few corrected cells would restore and maintain normal hematopoiesis in aplastic (lethally irradiated) animals. Animals with irradiation-induced marrow failure were used in
these experiments in order to more closely mimic the FA patient. We
previously reported similar results using nonablated FANCC
/ animals as recipients.19
We believe this is the first report of phenotypic correction of a stem
cell disorder using retroviral gene transfer. Gene-corrected LTRCs from
FANCC / mice will reconstitute aplastic
recipients and maintain normal peripheral blood counts following
exposure to MMC. It was significant to note that the dose of MMC chosen (0.3 mg/kg, chronic administration) is sufficient to cause marrow failure without affecting other organ systems.8 In order to authenticate phenotypic correction of progenitor cells, we performed in
vitro assays using marrow harvested from the primary recipients and
demonstrated improved clonogenic growth. We further demonstrated phenotypic correction of an LTRC population by performing
reconstitution assays into secondary recipients. Control animals, which
received cells transduced with an irrelevant cDNA, all died, strongly
suggesting that the FANCC gene product is required for
radio-protection, stable engraftment of transplanted marrow, and
maintenance of peripheral blood counts upon exposure to genotoxic agents.
The biochemical function of the FA proteins is currently the subject of
debate.20,21 Our data support the hypothesis that FANCC
plays a key role in maintenance and expansion of the stem cell
population. For these experiments, in vivo administration of a low-dose
genotoxic stressor (MMC) enforced expansion and allowed selection of
the corrected cells; however, preliminary experiments suggest that
gene-corrected FANCC / marrow cells are able to
compete favorably in this model system without drug selection.19 This hypothesis is supported by the
observation of somatic reversion with hematopoietic reconstitution in
an FA patient.22 Further studies are needed to confirm this proposition.
This study is relevant for the treatment of FA patients who do not have
an HLA-matched donor for bone marrow transplant. Gene therapy shows
great promise for sustained and permanent correction of FA; however,
the potential of this approach has yet to be demonstrated, owing to
limitations in transduction efficiency and inability to confirm
successful gene transfer into repopulating stem cells.23 This report demonstrates that the efficiency of transduction is not a
limiting factor in gene therapy for FA. The presence of a few corrected
LTRCs is sufficient to restore normal hematopoiesis to aplastic
animals. Gene-correction of stem/progenitor cells will remain a rare
event, and therefore both corrected and uncorrected cells will be
transferred into the patient. Here we set out to determine if
gene-corrected stem/progenitor cells would exhibit preferential growth
and expansion in vivo. Repopulation by gene-corrected cells was
enhanced by low-dose administration of a genotoxic stressor (MMC). This
produced a reduction of the uncorrected mutant cells while
simultaneously enforcing expansion of gene-corrected stem/progenitor cells. Normalization of blood counts upon exposure to MMC and enhancement of vector signal detection show that this in vivo selection
strategy is able to influence the repopulation kinetics in favor of the
few gene-corrected stem cells. The observation that a few
gene-corrected stem/progenitor cells are able to restore normal
hematopoiesis in a background of defective marrow cells suggests that
sustained correction of FA patients is achievable. This study used MMC
in order to demonstrate phenotypic correction (maintenance of normal
peripheral blood counts) and to apply an exogenous selective pressure.
Concerns of toxicity associated with the use of such agents in FA
patients24-26 may limit the usefulness of this approach.
Whether gene-transduced stem cells from aplastic FA patients will
outcompete defective cells without the systemic administration of
marrow toxic drug is currently under investigation.
| |
Footnotes |
Submitted May 14, 1999; accepted September 23, 1999.
Supported by National Institutes of Health grant #HL048347, Leukemia
Society of America grant LSA# 6229-97, and American Cancer Society
grant #RPG-98-246-01-LBC.
Reprints: Christopher E. Walsh, UNC Gene Therapy Center, Room
7101, Thurston Building, CB#7352, University of North Carolina at
Chapel Hill, Chapel Hill, NC 27599; e-mail: cwalsh{at}med.unc.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.
| |
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L. S. Haneline, X. Li, S. L. M. Ciccone, P. Hong, Y. Yang, H. E. Broxmeyer, S.-H. Lee, A. Orazi, E. F. Srour, and D. W. Clapp
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Blood,
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M D Tischkowitz and S V Hodgson
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M. Bogliolo, O. Cabre, E. Callen, V. Castillo, A. Creus, R. Marcos, and J. Surralles
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F. Galimi, M. Noll, Y. Kanazawa, T. Lax, C. Chen, M. Grompe, and I. M. Verma
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J. B. Wilson, M. A. Johnson, A. P. Stuckert, K. L. Trueman, S. May, P. E. Bryant, R. E. Meyn, A. D. D'Andrea, and N. J. Jones
The Chinese hamster FANCG/XRCC9 mutant NM3 fails to express the monoubiquitinated form of the FANCD2 protein, is hypersensitive to a range of DNA damaging agents and exhibits a normal level of spontaneous sister chromatid exchange
Carcinogenesis,
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Y. Yang, Y. Kuang, R. M. De Oca, T. Hays, L. Moreau, N. Lu, B. Seed, and A. D. D'Andrea
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D. A. Williams, A. W. Nienhuis, R. G. Hawley, and F. O. Smith
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Top
Abstract
Introduction