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GENE THERAPY
From the Hematopoietic Gene Therapy Program,
Centro de Investigaciones Energéticas,
Medioambientales y Tecnológicas (CIEMAT)/Marcelino
Botín Foundation, Madrid, Spain; the Department of Pediatric
Hematology and Oncology, Children's Hospital Heinrich Heine
University, Düsseldorf, Germany; and the Department of Clinical
Genetics and Human Genetics, Free University Medical Center, Amsterdam,
The Netherlands.
Fanconi anemia (FA) is a rare autosomal recessive disease,
characterized by bone marrow failure and cancer predisposition. So far, 8 complementation groups have been identified, although mutations in FANCA account for the disease in the majority
of FA patients. In this study we characterized the hematopoietic phenotype of a Fanca knockout mouse model and corrected the
main phenotypic characteristics of the bone marrow (BM)
progenitors using retroviral vectors. The hematopoiesis of these
animals was characterized by a modest though significant
thrombocytopenia, consistent with reduced numbers of BM megakaryocyte
progenitors. As observed in other FA models, the hematopoietic
progenitors from Fanca Fanconi anemia (FA) is a rare autosomal recessive
disease characterized by developmental abnormalities, bone marrow (BM)
failure, and predisposition to cancer, predominantly acute myeloid
leukemia.1,2 To date, 8 complementation groups have been
identified (FA-A, C, E, D1, D2, E, F, and G), and 6 FA genes have
already been cloned: FANCA,3
FANCC,4 FANCD2,5
FANCE,6 FANCF,7 and
FANCG.8 Mutations in the FANCA gene
account for the disease in about 60% to 70% of all FA
patients.1,2
Although the physiological role of FA proteins is still not well
understood, protein interaction studies have shown that FANCA, C, E, F,
and G form a functional complex.9 Interestingly, recent studies have shown that this complex is involved in the ubiquitination of FANCD2, which then interacts with the breast cancer susceptibility protein BRCA1,10 thus indicating a link between the FA
protein complex and the BRCA1 repair machinery.
To understand the pathogenesis of FA and to facilitate the development
of therapeutic approaches for FA, knockout mice with a targeted
disruption in 3 FA genes (Fancc, Fanca, and
Fancg) were generated.11-15 These animals reproduced
the chromosomal instability to DNA cross-linking agents and compromised
gametogenesis observed in human FA patients, but only mild
hematopoietic defects were observed in these animal
models11-17 (also reviewed in Wong and Buchwald18).
In this study we describe a characteristic FA phenotype in the
hematopoietic system of Fanca-deficient mice and show for
the first time a genetic correction in the phenotype of
Fanca Animals
Flow cytometry
Lin  Sca-1+
cells, BM samples were subjected to red blood cell lysis and then
sorted by using the MultiSort Kit (Miltenyi Biotec, Gladbach, Germany)
following manufacturer's recommendations. Briefly, cells were stained
with anti-Sca-1-FITC for 30 minutes at 4°C, washed with
purification buffer (PB; PBS 1x + 0.5% BSA), and
stained with anti-FITC MultiSort MicroBeads for 15 minutes at 4°C.
Cells were subjected to a positive immunomagnetic selection using an
MS column type (Miltenyi Biotec). MultiSort MicroBeads were
removed by incubation with the MultiSort release reagent for 10 minutes
at 6°C to 12°C and then passed through a second magnetic column to
remove unattached magnetic beads. Sca-1+ cells were then
stained with a Lin cocktail with biotinylated antibodies
against CD3, B220, TER119, GR-1, and Mac-1 for 30 minutes at 4°C, and
washed and incubated again with streptavidin MicroBeads for 15 minutes
at 6°C to 12°C. A third column was finally applied to remove the
Lin+ cells. LinSca-1+ cells were
washed with PB and resuspended in IMDM. On average 89%-pure
populations of LinSca-1+ were obtained, the
recovery being 30% to 60% of the input number of
LinSca-1+cells.
Clonogenic assays To determine the number of granulocyte macrophage colony-forming units (CFU-GMs) progenitors present in total BM or spleen cells and in purified LinSca-1+ cells, samples were plated
in MethoCult GF M3534 culture media (StemCell Technologies, Vancouver,
BC, Canada) at a concentration of 5 × 104 cells/plate
and 1-2 × 103 cells/plate, respectively. Samples were
cultured at 37°C in 5% CO2 and fully humidified air, and
7 days later colonies of at least 50 cells were scored under inverted
microscope.19 For the determination of megakaryocyte
colony-forming unit (CFU-Meg) progenitors, total BM and spleen
cells were plated at 1 × 105 cells/plate using the
MegaCult C medium (StemCell Technologies). Seven days later,
megakaryocytic cells were stained with acetylcholinesterase, and
megakaryocytic colonies were defined as aggregates of more than 3 large
brownish cells.20 To determine the sensitivity of
progenitor cells to mitomycin C (MMC), cells were cultured in MethoCult
and MegaCult media containing increasing concentrations of the drug (up
to 100 nM MMC).
In vitro expansion of BM cells Total BM cells were seeded in IMDM supplemented with 20% FBS and 3 different combinations of growth factors: (1) hrIL-11 and mrSCF (kindly provided by Genetics Institute, Cambridge, MA); (2) mrSCF, mIL-3, and hrIL-6 (kindly provided by Immunex); (3) hrIL-11, mrSCF, and hrTPO (kindly provided by Kirin Brewery, Japan); and ProGP (Progenipoietin; dual hFlt3 and hG-CSF receptor agonist; kindly provided by Monsanto, St Louis, MO). Every 7 days, cells were counted, reseeded at the initial cell density, and CFU-GM progenitors evaluated as indicated above. In some experiments purified LinSca-1+ cells were incubated in IMDM with
20% FBS, hrIL-11, and mrSCF at a concentration of
5 × 103 cells/mL. Every 3 days cells were counted and
diluted to the initial cell density. All hematopoietic growth factors
were used at 100 ng/mL, except thrombopoietin (TPO), which was
used at 300 ng/mL. Under our experimental conditions, adherent layers
were not observed in these cultures.
Apoptosis analysis Apoptotic cells were assessed using Annexin-V-FITC (Pharmingen) or tetramethylrhodamine methyl ester (TMRM; Sigma). Annexin-V labeling was performed following manufacturer's instructions. TMRM was used to show loss of mitochondrial inner transmembrane potential associated with the early stages of apoptosis. Cells were incubated for 15 minutes at 37°C in 0.05 µM TMRM in PBS, washed with cold
PBS, resuspended in PBS, and kept on ice
until analysis in the flow cytometer. Cells with damaged mitochondrial
inner transmembrane potential were observed as a population with
fluorescence lower than 580 nm.21
Retroviral vectors and packaging cell lines Two different vectors based on the recombinant vector MSCV2.122 were used in this study. The vector LFAPEG expresses the open reading frame of the human FANCA gene under the control of the retroviral PCC4-cell-pasaged myeloproliferative sarcoma virus (PCMV) long terminal repeat (LTR), and the enhanced green fluorescent protein (EGFP) gene under the phosphoglycerokinase (PGK) promoters. The LPEG vector contains only the PGK promoter/EGFP expression cassette.23 For the generation of the ecotropic vectors, retrovirus-containing supernatants from stable PG13 packaging cells were used to transduce 293T cell-based ecotropic Phoenix cells (kindly provided by Dr Nolan, Stanford University, CA). EGFP+ eco-Phoenix cells were sorted in the EPICS ELITE ESP flow cytometer (Coulter) and subsequently grown in IMDM supplemented with 10% FBS. Supernatants were harvested 24 hours after confluency, filtered through 0.45 µm, and used fresh. Titers ranged between 1 and 1.5 × 106 infective particles/mL, as deduced from the infection of NIH-3T3 cells with serial dilutions of retrovirus containing supernatants in the presence of 5 µg/mL polybrene (Sigma).24Transduction protocol of Lin Sca-1+ cells were
prestimulated for 48 hours in IMDM supplemented with 20% FBS, hrIL-11,
and mrSCF. Prior to infection, plates were coated for 12 hours with 20 µg/cm2 of CH-296 (Retronectin, Takara Shuzo, Otsu, Japan)
and washed with BSA 2% (wt/vol) in PBS.25 Immobilized
fibronectin fragments were preloaded with retroviral particles as
previously described.26 Finally,
LinSca-1+ cells were resuspended at a density
of 5 × 104 cells/mL in fresh supernatants supplemented
with 20% FBS (final concentration), hrIL-11, mrSCF, 5 µg/mL
polybrene (Sigma), and added to the preloaded wells. A total of 4 infections spaced 12 hours apart were conducted. Cells were harvested 4 hours after the last infection cycle to analyze the transduction
efficiency and also the MMC sensitivity and ex vivo expansion ability
of the transduced cells.
Characterization of the lympho-hematopoietic tissues of
Fanca /
mice (750 000 ± 40 000 platelets/µL;
P < .05). Flow cytometry analysis of BM, spleen, and
thymus from Fanca+/+ and
Fanca/ mice revealed no significant
differences in the percentage of mature B220+,
GR1+, MAC+, Ter-119+, and
CD3+ cells (Table 1).
Differences in the content of more primitive BM progenitors
(LinSca-1+ or
LinSca-1+c-kit+) were not
significant when both mouse strains were compared. In the thymus,
similar values of CD3+ cells and
CD4+CD8+, CD4+CD8,
CD4CD8+, and
CD4CD8 cells were apparent between both
strains. In no instance were significant differences in the absolute
number of BM, spleen, and thymus cells observed between
Fanca+/+ and Fanca/
mice (data not shown).
Colony-forming unit content in the hematopoietic organs of
Fanca / mice were cultured in methylcellulose
media, allowing the growth of CFU-GM and CFU-Meg progenitors,
respectively (Table 2). Although BM
progenitors from Fanca/ mice were always
below the corresponding values observed in
Fanca+/+ mice, only in the case of BM CFU-Meg
progenitors did differences reach statistical significance
(P < .05).
Mitomycin C sensitivity of Fanca / mice and their respective controls.
As shown in Figure 1, doses as high as 50 or 100 nM MMC were only modestly toxic to CFU-GM progenitors obtained
from Fanca+/+ or
Fanca+/ mice, regardless of whether BM or
spleen cells were used. In sharp contrast to these observations, a low
dose of 5 nM MMC significantly impaired the growth of CFU-GM
progenitors from Fanca/ mice, and 50 nM MMC
almost completely abrogated the clonogenic growth of CFU-GM
progenitors. Similar conclusions of MMC hypersensitivity were
drawn when CFU-Meg progenitors from
Fanca/ mice were assessed (Figure
1).
Ex vivo expansion ability of Fanca /
progenitors, ex vivo expansion cultures with BM from these animals were established (see "Materials and methods"). Initially, cultures were stimulated with IL-11/stem cell factor (SCF) on the basis of
previous data from our laboratory, showing that this combination of
growth factors promotes a significant expansion of hematopoietic progenitors and preserves the repopulating ability of the
graft.27 As shown in Figure
2A, when BM from
Fanca+/+ mice was subjected to ex vivo
expansion, an exponential cell growth was observed during a period of 2 weeks, implying a 300-fold amplification in the cellularity of the
cultures. As shown in the same figure, the amplification observed in
cultures established with Fanca/ BM was 10 times lower. To investigate whether such a difference was specific to
the stimulatory conditions used in these experiments, the proliferation
response to 2 other cytokine combinations was analyzed. When
IL-3/IL-6/SCF stimulation was used, significant differences between the
cellularity of both cultures were also observed. With a more complex
combination of growth factors TPO/ProGP/IL-11/SCFdifferences were
even more marked: Fanca/ BM cells were
capable of expanding the hematopoietic population for only one week,
while the BM of Fanca+/+ mice proliferated for
the whole 3-week period, which was the length of the
experiment (Figure 2A).
To evaluate whether indirect effects related to cell interactions or
secretion of inhibitory or toxic molecules played a role in the
differential ex vivo expansions observed in Figure 2A, cultures were
now established with purified hematopoietic precursors. To this end,
progenitor cells were purified as Lin To investigate more deeply the biologic insights involved in
the differential growth of Fanca+/+ and
Fanca
To determine the existence of potential differences in the proportion
of cells entering into apoptosis along the culture, ex vivo-expanded
samples were tested for apoptotic parameters. Changes in the scatter
properties (Figure 4A), changes in the exposure of phosphoserine residues (Annexin-V; Figure 4B), and loss of
mithochondrial inner transmembrane potential (TMRM; Figure 4C) were
determined in these experiments. As shown in the figure, the proportion
of apoptotic cells was markedly increased in cultures from
Fanca
Genetic correction of the hypersensitivity of
Fanca / progenitors could be reversed by the
expression of the human FANCA gene, retroviral-mediated gene
transfer experiments were conducted. In these assays, BM from
Fanca+/+ and Fanca/
mice was enriched on LS precursors and then transduced with
retroviral vectors encoding the FANCA and/or
EGFP cDNAs.
To investigate potential differences in the transduction
efficiency of Fanca+/+ and
Fanca
Data in Figure 6 show the reversion
of MMC hypersensitivity of Fanca
To confirm that the functional correction of
Fanca Correction of the impaired ex vivo expansion ability of
Fanca /
mice were transduced with LPEG and LFAPEG vectors and then
subjected to ex vivo expansion with IL-11/SCF. As expected, the
cellularity of Fanca+/+ LS cultures was markedly
increased between days 7 and 14 of incubation, regardless of the
vectors used for the transduction. Also as expected, the growth of
Fanca/ LS cells transduced with the LPEG
vector was very poor. However, the transduction of LS cells with the
LFAPEG vector (35%-75% transduction efficiency in these experiments;
not shown) allowed the correction of the limited in vitro growth
properties that characterized the Fanca/ LS
progenitor cells.
The progressive cloning of FA genes is facilitating the development of FA experimental models based on the targeted disruption of these genes.18 To date, knockout mice with disruptions in 3 FA genes have been generated. Three strains with disruptions in the Fancc gene,11,12 one of them having disruptions in both the Fancc and Sod1 genes,28 have been developed. In addition, mice having a targeted disruption in Fanca13 and Fancg/Xrcc914,15 genes also have been generated. Taking into account that FA constitutes the most frequent genetic cause of BM failure29 and given that mutations in the FANCA gene account for about 60% to 70% of all FA patients, this Fanca knockout mouse model constitutes an invaluable tool for conducting studies on hematopoietic stem cell (HSC) diseases of genetic etiology. Although a number of characteristics of the human FA disease, like pancytopenia and leukemia predisposition, have not been observed in these FA models, other signs associated with FA, including the hypersensitivity to DNA cross-linking agents, have been reported in these animals.11,13-15,30 In the hematopoietic system, only modest reductions of BM progenitors,12 and in some instances mild thrombocytopenia, have been observed in mice lacking the functional Fancc gene.28 A more profound hematopoietic phenotype has been observed in mice having disruptions in both the Fancc and the Sod1 genes, suggesting the relevance of an altered redox state in the FA phenotype.28 Regarding the hematological description of Fanca-deficient
mice, we observed only modest thrombocytopenia, consistent with data
observed in the original description of these animals.13 In addition, here we show a significant reduction in BM CFU-Meg progenitors, suggesting that the megakaryocytic lineage is particularly affected in these animals. In contrast to the modest effects observed in the hematopoietic tissues of Fanca Taking together the above observations, it could be proposed that only
after exposure to DNA cross-linking agents could a marked hematopoietic
FA be apparent in these knockout models. Nevertheless, data in Figures
2-4 reveal the existence of marked in vitro growth defects in
hematopoietic progenitors lacking the Fanca gene. As deduced
from our experiments in Figure 2, the impaired growth ability of
Fanca The results in Figures 3 and 4 add further information regarding the
cellular mechanisms that could account for the in vitro growth defect
observed in Fanca Regarding our observations of increased apoptosis in
Fanca In the context of the gene therapy of FA, we aimed to mimic a clinical
approach, in which particular care has to be taken regarding the in
vivo pharmacological activation of the HSCs. Therefore, although 5-FU
is generally used to promote the proliferation of mouse
HSCs,44,45 in our experiments untreated BM cells were first purified for primitive hematopoietic progenitors and HSCs (Lin Initially, and given the limited information about the transduction
susceptibility of the hematopoietic progenitors from FA compared to
wild-type mice, we investigated the existence of differences in the
transduction efficiency of Fanca+/+ and
Fanca A second observation derived from our gene transfer experiments relates
to the capacity of retroviral vectors encoding the human FANCA
gene to reverse the MMC sensitivity of Fanca-deficient cells. In this respect, data in Figure 6 show for the first time that
the transduction of mouse Fanca Finally, the experiments summarized in Figure 7 were conducted to
investigate the capacity of FANCA-expressing vectors for normalizing the in vitro growth properties of
Fanca Taken together, our analyses of the hematopoiesis of
Fanca
The authors thank I. Ormán for expert assistance with the flow cytometry and cell sorting.
Submitted September 25, 2001; accepted May 10, 2002.
Supported by grants from the Commission of the European Communities; the Comisión Interministerial de Ciencia y Tecnología; the Forschungsverbund Somatische Gentherapie des Bundesministeriums für Bildung und Forschung (beo2103111661), and the Elternitiavtive Kinderkrebsklinik e.V.
P.R. is a recipient of a fellowship from Formación de Personal Investigador (FPI) program of the Ministerio de Ciencia y Tecnología (MCYT).
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.
Reprints: Juan A. Bueren, Hematopoiesis Project, CIEMAT Avda Complutense, no. 22, 28040 Madrid, Spain; e-mail: juan.bueren{at}ciemat.es.
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© 2002 by The American Society of Hematology.
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 R. Larder, L. Chang, M. Clinton, and P. Brown Gonadotropin-Releasing Hormone Regulates Expression of the DNA Damage Repair Gene, Fanconi anemia A, in Pituitary Gonadotroph Cells Biol Reprod, September 1, 2004; 71(3): 828 - 836. [Abstract] [Full Text] [PDF]
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 J. C.Y. Wong, N. Alon, C. Mckerlie, J. R. Huang, M. S. Meyn, and M. Buchwald Targeted disruption of exons 1 to 6 of the Fanconi Anemia group A gene leads to growth retardation, strain-specific microphthalmia, meiotic defects and primordial germ cell hypoplasia Hum. Mol. Genet., August 15, 2003; 12(16): 2063 - 2076. [Abstract] [Full Text] [PDF]
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 A. D. D'Andrea The Fanconi road to cancer Genes & Dev., August 15, 2003; 17(16): 1933 - 1936. [Full Text] [PDF]
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 M. Bogliolo, O. Cabre, E. Callen, V. Castillo, A. Creus, R. Marcos, and J. Surralles The Fanconi anaemia genome stability and tumour suppressor network Mutagenesis, November 1, 2002; 17(6): 529 - 538. [Abstract] [Full Text] [PDF]
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| Copyright © 2002 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
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Abstract
Introduction
)
Fanca+/
) and Fanca
) to MMC. Each point represents the mean ± SEM
corresponding to 4 experiments involving the culture of CFU-GM
progenitors and to 3 experiments with CFU-Meg cultures.
) and Fanca
) were purified and ex vivo expanded with SCF and IL-11.
Cytometry histograms of bone marrow cells before and after purification
of the Lin
