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Prepublished online as a Blood First Edition Paper on June 21, 2002; DOI 10.1182/blood-2002-04-1245.
GENE THERAPY
From the Laboratory of Genetics, The Salk Institute for
Biological Studies, La Jolla, CA; and the Department of Molecular and
Medical Genetics, Oregon Health Science University, Portland.
Fanconi anemia (FA) is an inherited cancer susceptibility syndrome
caused by mutations in a DNA repair pathway including at least 6 genes
(FANCA, FANCC, FANCD2, FANCE, FANCF, and FANCG). The
clinical course of the disease is dominated by progressive, life-threatening bone marrow failure and high incidence of acute myelogenous leukemia and solid tumors. Allogeneic bone marrow transplantation (BMT) is a therapeutic option but requires HLA-matched donors. Gene therapy holds great promise for FA, but previous attempts
to use retroviral vectors in humans have proven ineffective given the
impaired proliferation potential of human FA hematopoietic progenitors
(HPCs). In this work, we show that using lentiviral vectors efficient
genetic correction can be achieved in quiescent hematopoietic
progenitors from Fanca Fanconi anemia (FA) is a rare autosomal recessive
disorder caused by mutations in a DNA repair pathway that involves at
least 7 genes (FANCA, FANCB/D1, FANCC, FANCD2, FANCE, FANCF,
and FANCG).1-8,31 Fanconi anemia type A and
type C combined account for approximately 80% of all FA
cases.5 Clinical features of FA are considerably heterogeneous and include congenital abnormalities of the skeleton and
other organs (kidney, heart), life-threatening bone marrow failure, and
increased cancer susceptibility. Life expectancy is on average reduced
to 20 years, with patients with serious illness dying before they reach
adulthood and patients with mild forms surviving until the
fifth decade of life. The clinical presentation is usually dominated by
hematological deficiencies derived from bone marrow failure, which can
progressively lead to profound aplastic anemia. If the supportive
treatment for the anemia is successful, patients are at high risk for
acute myelogenous leukemias and solid tumors.9-11
Allogeneic bone marrow transplantation (BMT) is a therapeutic option
but requires HLA-matched donors.12
Gene therapy holds great promise for patients with FA. In spite of
successful retroviral-mediated genetic correction of FA knockout (KO)
mouse hematopoietic progenitors,13 attempts to use
retroviral vectors in patients have proven to be ineffective. This
difference most likely results from the impaired proliferation potential specific to human FA hematopoietic progenitors
(HPCs).14 It must be noted, in fact, that the phenotype of
FA KO mice differs from the clinical syndrome. Their hematopoiesis is
virtually normal under stationary conditions and becomes impaired only
under extreme proliferative stress.15,16 Given that
ex vivo expansion is not an option for FA gene therapy in humans,
transplantation protocols in mice are relevant for clinical
application only if modeled while taking into account the differences
between KO mice and FA patients.
High-efficiency gene delivery into nondividing hematopoietic
progenitors is a major requirement for FA gene therapy in humans because bone marrow cells from FA patients, unlike those from mouse KO
cells, are fragile, grow poorly in vitro, and are therefore refractory
to transduction by retroviral vectors,14 which can transduce only dividing cells. On the other hand, lentiviral vectors can transduce early hematopoietic progenitors capable of in vivo engraftment, and efficient transduction occurs in the absence of
cytokines and other growth factors.17
In this work, we show that using lentiviral vectors, efficient genetic
correction can be achieved in hematopoietic progenitors from
Fanca Animals
Cells, culture media, and reagents
Lentiviral vectors Yamada et al19 described a first-generation HIV-derived lentiviral vector carrying an expression cassette for the FANCC gene. In our work, we chose a self-inactivating vector design and a third-generation, 4-plasmid packaging system.20 Plasmids were cotransfected in 293T cells, and the medium was collected after 48 hours and concentrated by ultracentrifugation at 19 400 rpm in a Beckman SW55 rotor. The virus pellet was resuspended in Hanks balanced salt solution (HBSS), and the content of p24 was determined by enzyme-linked immunosorbent assay (ELISA; NEN, Perkin Elmer Life Sciences, Boston, MA). Vector batches were tested for the absence of replication-competent virus by monitoring p24 antigen expression in the culture medium of transduced SupT1 lymphocytes for at least 3 weeks.Lentiviral transduction of Fancc  /or Fancc/
mice in RPMI supplemented with penicillin/streptomycin. Red cells were depleted in ACK lysis buffer (Bio-Fluids, Rockville, MD). Bone
marrow was resuspended in FANCA or FANCC lentiviral supernatant at
37°C for 24 hours, at a multiplicity of infection (MOI) between 10 and 100.
Bone marrow transplantation After lentiviral transduction, BM cells were washed and transplanted in Fanca/ or
Fancc/ recipients (n = 6 per group). Each
animal received 1 × 106 cells through the retro-orbital
plexus, without any preconditioning. One week after transplantation,
they were given a single dose of 40 mg/kg cyclophosphamide (CPA)
intraperitoneally. Mice that underwent transplantation were
bled every 4 weeks for analysis of the integrated vector. The same
protocol was used for secondary transplantation of bone marrow
harvested from reconstituted primary recipients.
Semiquantitative PCR for the detection of the FANCA/FANCC provirus The proviral copy number in peripheral blood DNA of lenti-FANCC animals that underwent transplantation was measured as described21 using a 4-primer PCR, including one primer pair specific for the proviral DNA (human FANCC cDNA) and a second pair specific for an unrelated autosomal locus (murine Fanca gene). PCR products were analyzed by Southern blotting.21 Hybridization signals were visualized using a Molecular Dynamics PhosphorImager SI (Molecular Dynamics, Sunnyvale, CA) and quantitated using the software IPLab Gel (Signal Analytics, Vienna, VA).Semiquantitation of the FANCA provirus was performed similarly to that of the FANCC provirus. Briefly, murine 3T3 cells transduced or nontransduced with the FANCA lentivirus were used as the standards for the determination of the copy number of the provirus from the peripheral blood of mice that underwent transplantation with lentiviral-transduced BM. Southern blot analysis verified that the transduced cell line used as the standard contained only a single copy of the FANCA provirus, and a standard curve was generated as described previously.21 Primers specific for human FANCA were 5'-GCAGTTTGCCAGCGATTTCC-3' and 5'-CTTCCTGATGTTTTCTTCCCTGA-3', resulting in a 120-bp product. Primers 5'-TTCCTTCAAAGCTGCTGGGG-3' and 5'-CAGTGACATCTTCCTTCCTAACTCC-3' were designed to amplify a 942-bp region of the fanca gene as an internal control; 5'-TTGCGATTCAACAAGTCAGGG-3' was used as probe for the lenti-FANCA provirus, and 5'-AGACACAATCATCTGTAGTGATTCT-3' was used as a probe for Fanca. The percentage contribution of each allele was determined as explained elsewhere.21 Twelve-day spleen colony-forming unit assay Forty thousand unfractionated BM cells from the animals reconstituted with lenti-FANCA/C-transduced HSCs were transplanted into lethally irradiated wild-type recipients killed on day 12 after transplantation. Genomic DNA was prepared from spleen colonies,22 and 4-primer PCR was performed as described above.Southern blot analysis was performed on each colony to determine the proviral copy number. For FANCC, 5 µg DNA from each spleen colony-forming unit (CFU-S) DNA was SpeI-digested and probed with a NotI-XbaI fragment (596 bp) from the human FANCC cDNA. Southern blot analysis for FANCA provirus was performed in the same way, but a XhoI-XhoI fragment (2100 bp) from the human FANCA cDNA was used as a probe. FANCA and FANCC protein expression FANCA and FANCC protein expression were analyzed in the BM of the recipients of FANCC/FANCA lentiviral-transduced bone marrow by Western immunoblotting, using anti-FANCA and anti-FANCC polyclonal antisera, kindly provided by Drs Alan D'Andrea and Grover Bagby.Hematological protection against MMC toxicity Untreated Fanca/ and
Fancc/ mice (n = 6 each), control
wild-type mice (n = 6), and secondary recipients of FANCC
or FANCA lentiviral-transduced bone marrow (n = 6
each) were exposed to MMC (0.3 mg/kg body weight, single
intraperitoneal injection per week for 6 weeks). Peripheral blood was
collected before MMC treatment and every 2 weeks thereafter. Samples
were analyzed at Antech Diagnostics (Portland, OR) on an ABC Vet
automated blood counter.
Lentivector design and characterization To explore the potential of lentiviral vectors in the management of FA, we designed third-generation,20 self-inactivating,23 HIV-derived lentiviral vectors carrying expression cassettes for the FANCA and FANCC genes, respectively, under the control of the cytomegalovirus promoter/enhancer (Figure 1A). High-titer recombinant lentiviruses were generated by transient transfection and concentrated by ultracentrifugation (more than 109 transduction units per milliliter). Biological activity of the proteins encoded by the lenti-FANCA and lenti-FANCC vectors was first assessed by their ability to phenotypically correct human Epstein-Barr virus-immortalized lymphoblastoid cell lines derived from FA patients. These cells undergo growth arrest, chromosomal abnormalities, and cell death when cultured in the presence of DNA-cross-linking agents such as MMC or diepoxybutane.24,25 HSC99 (FANCA/, kindly provided by Dr Buchwald) and
HSC536 (FANCC/)26 cell lines
were transduced with the appropriate lenti-FA or a control
lenti-GFP (green fluoroscent protein) vector at an MOI between
10 and 50 and were subsequently grown in the presence of increasing
concentrations of MMC. The number of viable cells was assessed after 5 days by flow cytometry analysis with propidium iodide exclusion.
Corrected cells showed a fully restored resistance to DNA damage, with
an increase of the LD50 for MMC of approximately 20-fold
compared with controls (Figure 1B-C). Lentiviral vectors also reverted
cell cycle abnormalities in the presence of MMC, as shown by the sharp
decrease in late S/G2-arrested cells after growth in 100 nM
MMC (Figure 1D).
Correction of committed hematopoietic progenitors from
Fanca /18 and
Fancc/15 mice. Bone marrow
mononuclear cells obtained from mice were transduced with lenti-FANCA
or lenti-FANCC, respectively, at an MOI between 10 and 100. Cells were
then assayed for their ability to form colonies in semisolid media in
the presence of MMC. Under these conditions, the colony growth from
bone marrow of Fanca/ and
Fancc/ mice is strongly impaired, mimicking
the deficient hematopoiesis of patients with FA. Genetic correction by
lentiviral vectors resulted in a significant increase in CFC growth in
MMC (Figure 2A).
Ex vivo correction of stem cells from Fanca / or
Fancc/ mice were infected ex vivo with
lentivectors, in the absence of any growth factor or cytokine
stimulation. Recipient mice (n = 6 for each mutant strain) underwent
transplantation with 1 × 106 lentiviral-transduced
cells, without any preconditioning, and received a single dose of 40 mg/kg cyclophosphamide (CPA) 1 week after transplantation for in vivo
selection of the corrected cells.21 We then used
semiquantitative PCR21 to measure the proviral copy number
in peripheral blood DNA over time (Table
1). Before CPA selection, the proviral
signal could not be detected in peripheral blood DNA of either the
Fanca/ or the Fancc/
recipients, indicating less than 0.05 proviral copies/genome. Four
months after CPA administration, the proviral copy number in
Fancc/ recipients had increased to
1.10 ± 0.24 copies/genome and persisted for the duration of the
experiment (1 year after transplantation). The proviral copy number in
Fanca/ recipients ranged between 0.4 and 0.8 copies/genome (Table 1) and was sustained throughout the experiment.
The relatively worse performance of the lenti-FANCA virus was possibly
attributed to the size of the expression cassette, which may lower
packaging efficiency during vector production. These data show
long-term engraftment by CPA-selected, lentiviral-transduced,
hematopoietic progenitors. Expression of the FANCA and FANCC proteins
was detectable by Western blot on the bone marrow primary and secondary
recipient animals (Figure 2B).
We also determined the proviral copy number in secondary recipients, 2 months after transplantation, using semiquantitative PCR. Consistent
with selection at the HSC level, the proviral copy number remained
stable after secondary transplantation (Table 2).
Twelve-day spleen colony-forming unit analysis of corrected bone marrow Bone marrow was harvested from primary recipient mice and was serially transplanted into lethally irradiated wild-type mice. We isolated genomic DNA from spleen colonies at day 12 and determined the presence of the provirus in each colony using PCR. Of the colonies examined, 49 of 50 (98%) were provirus positive in the Fancc experiment, and 37 of 50 (74%) were provirus positive in the Fanca experiment. Colonies that yielded a sufficient amount of genomic DNA were also analyzed by Southern blotting, showing that most had a single proviral integration site (Figure 3A).
Hematological correction of lentiviral-corrected mice To demonstrate that both the Fanca/
and the Fancc/ mice treated with ex
vivo lentiviral gene therapy and in vivo CPA selection were
phenotypically corrected, we exposed treated mice, untreated mutant
controls, and wild-type mice to weekly doses of MMC (0.3 mg/kg).
Platelet counts (PCs) were analyzed at various time-points (Figure 3B).
Normal mice were resistant to MMC and did not develop thrombocytopenia,
whereas Fanca/ and
Fancc/ mice rapidly developed PC
abnormalities, leading most (4 of 6) of them to death within 6 weeks.
All mutant mice treated by lentiviral gene therapy and subsequent CPA
selection were protected from the thrombocytopenia and death induced
by MMC.
In many respects, Fanconi anemia is a suitable candidate disease for ex vivo gene therapy. First, the syndrome has a serious clinical outcome, and the only available therapeutic option is allogeneic bone marrow transplantation, associated with considerable morbidity and mortality. Second, corrected cells can have a selective advantage in vivo over the defective ones, as suggested by the high incidence of mosaicism in patients.27 Third, the deficiency in the Fanconi DNA repair pathway specifically affects the hematopoietic stem cell compartment. Although the exact mechanisms accounting for this tissue-specific damage are unclear, the condition provides a valuable model disease for gene transfer into hematopoietic stem cells. Genetic correction of human FA progenitors has proven to be a formidable challenge. Clinical attempts performed so far have relied on oncoretroviral vectors, with overall disappointing results.28 The main reason for this can be found in the fragility of human FA hematopoietic progenitors, in their inability to efficiently grow in vitro, and in their consequent resistance to retroviral transduction. The protocols for ex vivo cytokine expansion of hematopoietic progenitors, refined over the years to improve the efficiency of retroviral vectors with sometimes excellent results,29,30 have failed on human FA tissue. FA knockout mice provide a valuable model for investigating the pathophysiology of FA and for establishing innovative therapeutic strategies. Although the expected DNA repair defect can be documented in cells derived from these animals, the phenotype of FA-deficient mice does not fully reproduce the clinical syndrome. In particular, FA mouse progenitors can be expanded in vitro and transduced efficiently by retroviral vectors.13 FA KO mouse hematopoiesis is virtually normal, and bone marrow failure develops only under extreme proliferative stress.15,16 The selective advantage of corrected progenitors over FA-defective ones in mice is limited under normal conditions,16 whereas it is known to be significant in humans.27 Because transduction protocols based on ex vivo cytokine expansion of
HSCs are not applicable to human FA gene therapy, we explored the
efficiency of lentiviral-derived vectors on totally quiescent
progenitors obtained from FA KO animals. The data presented here show
that lentiviral vectors were remarkably efficient in transducing early
hematopoietic progenitors in Fanca The protocol of transduction we used in this work, including no cytokine prestimulation, low viral dose, and overall minimal ex vivo manipulation followed by in vivo cyclophosphamide selection of corrected stem cells, is particularly relevant for future clinical applications to humans. The next step toward the design of clinical trials for lentiviral FA gene therapy will be to evaluate the efficiency of lentiviral vectors on human primary hematopoietic progenitors obtained from FA patients. These studies are under way. To date, lentiviral vectors have not been approved for clinical use; however, we believe clinical trials for the correction of the FA defect using lentiviral vectors will offer an excellent opportunity because transduction is performed ex vivo, therefore posing fewer safety concerns than with systemic delivery, and correction of FA will be an impetus to attempt a genetic cure of other hematological diseases such as sickle cell anemia, thalassemia, and many immunodeficiencies.
The genes for both FANCB and FANCD1 have recently been shown to be BRCA2.31
We thank Dr Neeltje Kootstra for critical reading of the manuscript and Nien Hoong for excellent technical assistance. We thank Drs Alan D'Andrea, Grover Bagby, and Manuel Buchwald for providing FA cell lines and anti-FANCA/C antisera. F.G.'s permanent address is Department of Biomedical Sciences, University of Sassari Medical School, Sassari, Italy.
Submitted April 4, 2002; accepted June 1, 2002.
Prepublished online as Blood First Edition Paper, June 21, 2002; DOI 10.1182/blood-2002-04-1245.
Supported by the National Institutes of Health (I.M.V., M.G.), Fanconi Anemia Research Fund (I.M.V., M.G.), Jose Carreras International Leukemia Foundation (F.G.), Japan Research Foundation for Clinical Pharmacology (Y.K.), March of Dimes (I.M.V.), Lebensfeld Foundation (I.M.V.), Wayne and Gladys Valley Foundation (I.M.V.), and H.N. and Frances C. Berger Foundation (I.M.V.).
F.G., M.N., and Y.K. contributed equally to this study.
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: Inder M. Verma, Laboratory of Genetics, The Salk Institute for Biological Studies, 10010 N Torrey Pines Rd, La Jolla, CA 92037; e-mail: verma{at}salk.edu.
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Abstract
Introduction
