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Functional Correction of Fanconi Anemia Group C Hematopoietic Cells by the Use of a Novel Lentiviral Vector
doi:10.1006/mthe.2001.0287, available online at http://www.idealibrary.com on IDEAL
ARTICLE
Functional Correction of Fanconi Anemia Group C Hematopoietic Cells by the Use of a Novel Lentiviral Vector Kaoru Yamada,* John C. Olsen,† Manij Patel,† Kathleen W. Rao,‡ and Christopher E. Walsh*,§,1 *UNC Gene Therapy Center, †Cystic Fibrosis/Pulmonary Research and Treatment Center, ‡Department of Pathology, and § Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Received for publication December 4, 2000; accepted in revised form February 9, 2001; published online April 16, 2001
Lentiviral vectors transduce nondividing hematopoietic cells more efficiently than other currently available vector systems. Here we report the results of human hematopoietic cell gene transfer using lentiviral vectors based upon human immunodeficiency virus (HIV-1) and equine infectious anemia virus (EIAV). EIAV is a nonprimate lentivirus and is severely restricted in its host range to horses and closely related equines. We employed the EIAV vector system to test for gene transfer to human Fanconi anemia (FA) hematopoietic cells by comparison with HIV-1- and Moloney murine leukemia virus-based systems. Fanconi anemia is characterized by bone marrow failure secondary to stem cell dysfunction. Fanconi anemia group C EBV-transformed lymphoblasts were transduced with all three viral vectors. Phenotypic correction of FA cells, as measured by mitomycin C drug resistance, was observed with a similar efficiency in all vector systems. This is the first description of lentiviral correction of FA cells and suggests that lentiviral vectors may be useful for FA gene transfer. Key Words: EIAV vector; lentivirus; Fanconi anemia group C (FANCC).
INTRODUCTION Oncoretroviruses such as Moloney murine leukemia virus (MoMLV) have provided the backbone for most retroviral gene transduction vectors. However, the inefficiency of gene transfer to nondividing cells is a major limitation of MoMLV-based vectors (1–3). Lentivirus vectors derived from human immunodeficiency virus type 1 (HIV-1) infect and integrate into quiescent cells (4, 5). Pseudotyped HIV employing the vesicular stomatitis virus-glycoprotein (VSV-G) envelope (6) broadens the host range of the vector (4, 7–11). Efficient HIV viral replication requires four accessory proteins (Vif, Vpr, Vpu, and Nef) that play a role in HIV pathogenesis (12). To improve the safety of the HIV-1 vector system genes encoding HIV accessory proteins can be removed to prevent production of wild-type HIV (9, 11). As an alternative to HIV-1-based vectors, the nonprimate lentiviral counterparts, feline immunodeficiency virus (13, 14) and equine infectious anemia virus (EIAV) (15, 16), are under development. EIAV has a relatively
1 To whom correspondence and reprint requests should be addressed at Room 7101, Thurston Building, CB#7352, Chapel Hill, NC 27599. Fax: (919) 966-0907. E-mail: [email protected].
MOLECULAR THERAPY Vol. 3, No. 4, April 2001 Copyright © The American Society of Gene Therapy 1525-0016/01 $35.00
simple genome organization, having only three accessory proteins. Alternate splicing derives accessory genes Rev, S2, and Tat. EIAV Rev functions for nuclear export of nonspliced or incompletely spliced RNA and Tat functions to increase the steady-state levels of viral RNA. The function of S2 is not completely understood, but is not required for replication of wild-type virus in culture or for gene transfer by recombinant EIAV vectors (17). S2 may have a role in progression of infectious anemia in its natural host (18). Wild-type EIAV has never been reported as an agent transmitted to humans (19). Previous studies have established the feasibility of generating recombinant EIAV vectors at titers sufficient for use in gene transfer experiments (15, 16). Recombinant EIAV vectors are produced following transient transfection of a transgene plasmid and a plasmid carrying the VSV-G envelope into a producer cell line carrying the EIAV gag, pol, and rev ((15) and Patel and Olsen, unpublished data). EIAV vectors stably integrate reporter genes into both transformed and normal cells from humans and rodents. Nondividing cells can be transduced in culture and in vivo by EIAV vectors (16). These results suggest that EIAV vectors can be used to transfer genes to human hematopoietic cells. Recently we demonstrated the retroviral gene comple-
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FIG. 1. Schematic diagram of FANCC vector plasmid constructs. EIAV and HIV vectors contain the CMV immediate early promoter as internal promoter. The EIAV vector also contains a fusion of the CMV promoter and the R/U5 region of the 5⬘ LTR sequence. EIAV Rev responsive element is located downstream of the FANCC gene. Transcription of the FANCC gene is driven from the LTR in MoMLV vector. CMV, human cytomegalovirus; SV, SV40 promoter; neo, neomycin resistance gene.
mentation of Fanconi anemia hematopoietic cells (20). In this study, we chose hematopoietic cells derived from patients with Fanconi anemia to test gene transduction by EIAV- and HIV-1-based vectors. Fanconi anemia (FANC) is an autosomal recessive disease characterized by bone marrow failure due to stem/progenitor cell dysfunction. FANC cells are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC). Exposure of FANC cells to MMC leads to cell cycle arrest, cell apoptosis, and an increased frequency of chromosomal breakage thought to be the result of defective DNA repair (21, 22). Seven FANC subgroups, defined as FANCA–G have been described by complementation analysis (23). Genetic complementation of FANC cells with the appropriate FANC cDNA leads to normalized cell cycle kinetics, regular cell growth, and absence of chromosomal damage in the presence of drug. Here we describe phenotypic correction of FANCC group cells using lentiviral vectors.
MATERIALS
AND
METHODS
Cell Lines/Cell Culture Human 293T cells, an amphotropic MoMLV vector-producing cell line (clone 52-19 (20)), and B241 cells, a 293-derived cell line stably expressing EIAV gag, pol, and rev genes (Patel and Olsen, unpublished), were cultured in Dulbecco’s modified Eagle’s medium with high glucose (DMEM; Gibco BRL, Gaithersburg, MD) and 10% fetal bovine serum. Epstein–Barr virustransformed B cell lines, HSC536N and PD4L cells, established from FANCC patients carrying different mutations (20), were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum.
Construction of Transgene Plasmids To construct FANCC gene transfer vectors, the 1.9-kb FANCC gene cDNA from pG1FASVna (20) plasmid was digested by NotI restriction enzyme and ligated into an EIAV vector (pECG3CR, Patel and Olsen, unpublished). The EGFP gene in HIV-LL-CG plasmid (kindly provided by Dr. Kafri, UNC) was replaced by FANCC cDNA (Fig. 1).
Viral Vector Production EIAV and HIV vectors were produced by transient transfection using CaPO4-mediated methods. For EIAV vector generation, B241 cells were plated at 1 ⫻ 107 cells per collagen- (Vitrogen, Cohesion, Palo Alto, CA) coated 10-cm tissue culture dish 24 h before transfection. Then cells were
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transfected with 10 g each of the EIAV/FANCC vector and VSV-G (pCIVSV-G) plasmids. After overnight transfection, fresh medium containing 10 mM sodium butyrate (Sigma) was added (24). Recombinant HIV vector was produced using a similar cotransfection method. Ten micrograms each of three vector plasmids, HIV/FANCC vector, HIV virus proteinexpressing vector (CMV⌬R8.2 (25)), and VSV-G vector (pMDG) was transfected into 2.5 ⫻ 106 293T cells that were plated 24 h prior onto poly-Llysine- (Sigma) coated 10-cm tissue culture dishes. Following 2 days of culture after transfection, HIV and EIAV vector supernatants were harvested and filtered through 0.45-m filters and virus was concentrated about 100-fold by ultracentrifugation at 50,000g for 2.5 h at 4°C. One-day culture supernatants of amphotropic retrovirus vector 52-19 (20) producer cells were harvested and then filtered. All supernatants containing virus were frozen at ⫺80°C until use.
Virus Titer Nonconcentrated virus was subjected to RNA dot blot analysis as an index of virus particle number. Virus was precipitated from 1 ml of culture medium by the addition of 0.5 ml solution of 6% polyethylene glycol (MW 8000) (Sigma) and 0.3 M NaCl and then centrifuged at 14, 000 rpm for 20 min at 4°C. RNA was extracted from the virions with phenol– chloroform and digested with 10 U of RNase free DNase I (Promega Biotec, Madison, WI) in the presence of 200 U RNasin ribonuclease inhibitor (Promega Biotec) for 30 min at 37°C to eliminate contamination of plasmid DNA and then reextracted with phenol– chloroform. Viral RNA from 1 ml of culture medium was dot-blotted onto nylon membrane (Hybond N⫹; Amersham Corp., Arlington Heights, IL) and then hybridized using a 32 P-labeled random-primed 1.9-kb size FANCC cDNA as a probe. Standard curves were made by serial dilution of each vector plasmid. Titers of EIAV/FANCC and HIV/FANCC vectors were estimated by comparing genome content with vectors of known infectivity encoding surrogate markers (LacZ gene in pEC-LacZ plasmid and GFP gene in HIV-LL-CG plasmid). To determine infectivity, 293T cells were plated at 3 ⫻ 105 cells per well in six-well plates with 3 ml final volume. The day after plating, cells were transduced at 37°C overnight with serial dilution of EIAV/LacZ and HIV/ GFP virus vectors in the presence of 5 g/ml protamine sulfate. For determination of X-gal staining titer for EIAV vector, at 48 h after transduction, cells were fixed with 1 ml 0.5% glutaraldehyde in PBS containing 1 mM MgCl2. Cells were stained in X-gal solution for 2 h at 37°C and the number of blue-stained colonies was counted under microscope. Virus titer was determined by multiplying with the dilution factor. For determination of GFP titer for HIV vector, at 48 h after transduction, cells were harvested and fixed with 4% paraformaldehyde and then analyzed with a flow cytometer for GFP fluorescence. Virus titer was determined by multiplying the percentage of GFP-positive cells by the dilution factor. The titer of the MoMLV vector was estimated by determining the number of neomycinresistant colonies using NIH 3T3 cells.
Gene Transfer to FANC Hematopoietic Cells HSC536N and PD4L lymphoblasts were incubated with vector preparations containing protamine sulfate (5 g/ml) at a m.o.i. of 1. After overnight transduction, cells were harvested, washed with PBS twice, and cultured further for 2 days in DMEM. Cells were maintained in medium containing 10 nM MMC for 4 weeks to select gene-complemented cells. Mock-transduced lymphoblasts cultured under the same conditions yielded no viable cells after 4 weeks.
Molecular Analysis of FANCC-Transduced Cells Southern blot analysis. Genomic DNA was isolated from transduced lymphoblasts using the DNeasy Tissue Kit (Qiagen, Valencia, CA). Ten micrograms of DNA was digested with the particular restriction enzymes whose restriction sites are located within each LTR: StuI for EIAV, BspEI for HIV, and KpnI for MoMLV. DNA was hybridized using a 32P-labeled 1.9-kb-size FANCC cDNA probe. Mock-transduced HSC536N cells served as a source for negative control DNA. Standard curves were made by digestion of the mock-transduced HSC536N DNA with serial copy numbers of each vector plasmid. The expected band sizes were 4.9 kb for EIAV, 4.7 kb for HIV, and 4.6 kb for MoMLV. MOLECULAR THERAPY Vol. 3, No. 4, April 2001 Copyright © The American Society of Gene Therapy
ARTICLE FANCC, we assumed the functional titer of FANCC vectors was also 1:100.
TABLE 1 Virus Titer Virus vector/ transgene EIAV/LacZ EIAV/FANCC HIV/GFP HIV/FANCC MoMLV/FANCC⫹Neo a
Target cells
Infectious particle unit/ml
Virus particle number/mla
293T
6 ⫻ 104
293T
5 ⫻ 10
NIH 3T3
5 ⫻ 106
1 ⫻ 106 1 ⫻ 106 1 ⫻ 107 1 ⫻ 107 1 ⫻ 108
5
Analyzed by dot blot with transgene cDNA as the probes.
Northern blot analysis. Total RNA was extracted from selected HSC536N cells by the AGPC method (26) using TRIzol Reagent (Gibco BRL). Polyadenylated RNA was further purified by Oligo(dT) Cellulose Spin Column Kit (Eppendorf–5 Prime, Inc., Boulder, CO). Two micrograms of polyadenylated RNA was subjected to electrophoresis in a 1% agarose–formaldehyde gel. The estimated size of RNA transcripts was calculated by comparison with the mobility of ribosomal RNA from total RNA run in the same gel (not shown). After RNA blotting onto nylon membranes, hybridization was performed using a 32P-labeled FANCC cDNA probe. The intensity of each band was quantified following phosphorimage analysis.
Functional Assessment of Transduced Cells Cell cycle analysis. Transduced lymphoblasts were incubated at 1 ⫻ 105/ml with 100 nM MMC for 2 days. Cells were harvested and fixed with ethanol and then incubated with 2 mg/ml RNase (Sigma) at 37°C followed by staining with 0.05 mg/ml propidium iodide (Sigma) for 20 min in the dark. DNA content data were acquired by FACSCalibur (Becton–Dickinson, San Jose, CA) using a CellQuest software program (Becton–Dickinson). Cell cycle analysis was performed using ModFit LT (Verity Software House, Inc., Topsham, ME). MMC sensitivity assay. Transduced or control lymphoblasts were plated at 1 ⫻ 105/ml in 12-well plates and then cultured with various concentrations of MMC (0, 1, 5, 10, 50, and 100 nM) for 14 days. Cellular viability was assayed by trypan blue exclusion. Samples were performed in duplicate. Cytogenetic breakage. Lymphoblast cultures were analyzed for cytogenetic breakage and exchange figures (radial formation) by exposure to 100 nM MMC for 2 days. Cultures were harvested after a 90-min exposure to 0.50 g/ml colcemid and 10 g/ml ethidium bromide. After a 10-min treatment with 0.075 M KCl, the cells were fixed with a 3:1 mixture of methanol:acetic acid. Slides were prepared using wet slides, air dried, and stained with Wright’s stain. Fifty metaphase figures from each culture were scored for obvious breaks, gaps larger than a chromatid width, and exchange figures.
Virus Vector-Mediated Gene Transfer and Phenotypic Correction of FANC Lymphoblasts Epstein–Barr virus-transformed lymphoblast cell lines, HSC536N and PD4L, were established from FANCC patients as previously described (27). Spontaneous phenotypic reversion, i.e., resistance to drug, has not occurred in these cell lines, making them well suited to gene complementation studies. HSC536N carries a T to C point mutation while PD4L contains a G deletion causing a frameshift mutation and a truncated protein. Both mutations totally abolish FANC protein function. Transduction of each cell line was performed using EIAV, HIV, or MoMLV/ FANCC at a m.o.i. of 1. After overnight incubation with virus, cells were cultured in 10 nM MMC for 4 weeks (27) to select complemented cells. No viable mock-transduced cells were observed in culture after 4 weeks. Molecular analysis of gene transduction was performed to confirm the presence of proviral DNA. Southern blot analysis of genomic DNA harvested from the transduced HSC536N cells and hybridized with FANCC-specific probe is shown in Fig. 2. We estimated that proviral copy number for each vector was 0.3 copy/cell. Northern blot analysis was performed on MMC-selected HSC536N cells (Fig. 3). A single RNA species hybridized to the probe for both MoMLV- and HIV-transduced samples. The band sizes were 4.5 and 2.8 kb, respectively, consistent with that of the predicted transcript size for each vector. Analysis of EIAV-transduced cells produced two bands, 4.0 and 3.0 kb, consistent with transcription originating from both the LTR and the internal CMV promoters. An estimation of the steady-state levels of vector RNA transcripts yielded a ratio of 3:2:1 (HIV:MoMLV:EIAV). The LTR promoter activity of EIAV in hematopoietic cells might cause promoter interference of the internal CMV promoter, resulting in lower transcription levels in these target cells.
RESULTS Generation of FANCC Gene Expression Vectors VSV-G-pseudotyped EIAV- and HIV-1-based lentiviral vectors were generated by transient transfection. To estimate the titers of recombinant lentiviral vectors, dot blot hybridization analysis was performed and infectious-particle units were measured by the number of GFP- and LacZ-positive cells. Shown in Table 1, we reproducibly observed that the relative infectious-particle number-toviral genome ratio was 1 to 100 for both EIAV and HIV. In order to compare the effectiveness of EIAV and HIV/ MOLECULAR THERAPY Vol. 3, No. 4, April 2001 Copyright © The American Society of Gene Therapy
FIG. 2. Southern blot analysis of gene-transduced HSC536N lymphoblasts. Genomic DNA was isolated from gene-transduced cells and digested with restriction endonucleases that cut uniquely within each LTR. Genomic DNA from each transduced cell line was electrophoresed in an agarose gel, transferred onto nylon membranes, and hybridized using a 32P-labeled 1.9-kb size FANCC fragment as a probe. The standard curve was prepared using serial dilutions of plasmid and parental cell line genomic DNA and run with each sample. The expected 1.9-kb band is indicated (arrow). Data shown are representative of duplicate experiments.
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ARTICLE DISCUSSION
FIG. 3. Northern blot analysis of FANCC-transduced HSC536N lymphoblasts. Total RNA was isolated from transduced cells and mRNA was purified using an oligo(dT) column. 32P-labeled FANCC DNA was used as a probe. The expected sizes of the RNA transcripts are indicated. Each single transcript from HIV and MoMLV vector-transduced cells was detected (2.8 kb, HIV; 4.5 kb, MoMLV). The two bands for EIAV-transduced cells are consistent with the size of transcripts from EIAV LTR (4 kb) and CMV internal promoter (3 kb).
We demonstrated functional complementation of FANCC cell lines using EIAV- and HIV-based vectors. Phenotypic correction was determined by three different functional assays, including MMC sensitivity on cell proliferation, cell cycle kinetics, and cytogenetic assays. Although we found lower FANCC transgene expression in EIAV-transduced cells compared with HIV and MoMLV, there was no difference in terms of FANC correction. These data represent the first example of EIAV-mediated gene transfer using a disease-specific cDNA. Several reports have demonstrated efficient gene transduction of human CD34⫹ cells by HIV vectors in vitro (5, 28 –30). HIV vectors showed the capability to transduce genes into quiescent CD34⫹Lin⫺Thy-1⫹CD38⫺ cells (31), overcoming a major limitation of retrovirus vectors, which need cell mitosis for vector integration. Fully reconstituted NOD/SCID mice with HIV-1 vector-trans-
The hallmark of FANC is cellular hypersensitivity to DNA cross-linking agents. Cell cycle arrest, accelerated apoptosis, and aberrant chromosomal breakage characterize the FANC phenotype. The clastogenic agent, MMC, causes DNA damage and growth arrest in the G2 phase of the cell cycle. We tested whether FANCC gene complementation restored cell cycle arrest. FANCC gene-transduced cells were subjected to cell cycle analysis after culture with 100 nM MMC for 2 days. Cells were stained with propidium iodide and then analyzed with flow cytometry (Fig. 4). The percentage of cells in the G2/M phase increased only in mock-transduced cells treated with MMC. In contrast, no significant change in the fraction of cycling cells was observed in any of the gene-transduced or normal lymphoblasts following MMC treatment (Table 2). Drug sensitivity of each cell line was measured by cell viability with cultured cells in various concentrations of MMC (0, 1, 5, 10, 50, and 100 nM). As expected, both mock-transduced cell lines were highly sensitive to MMC with an effective drug concentration yielding 50% reduction in cell viability (EC50) of 1 to 5 nM MMC (Fig. 5). In contrast, regardless of the vector used, transduced cells exhibited growth equivalent to lymphoblasts established from a normal individual. The EC50 of both normal and corrected lymphoblasts was 30 nM. We then determined the frequency of chromosomal aberrations in both treated and untreated cells. Metaphase preparations of cells were made following cell incubation with 100 nM MMC for 2 days. As expected, the formation of quadriradials was 35% of all metaphases examined, significantly greater than normal. Aberrations in greater than 20% of metaphases are considered diagnostic for FANC. In contrast, cytogenetic results for each vector-transduced cell line were less than 20% and comparable with normal lymphoblasts (Table 3).
FIG. 4. Cell cycle kinetics of gene-corrected lymphoblasts. Virus vectortransduced HSC536N cells were compared to the parental mock-transduced cell lines. Cells were cultured in the presence or absence of 100 nM MMC for 48 h, fixed, stained with propidium iodide, and the DNA content analyzed. The results shown are representative of three separate experiments.
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ARTICLE TABLE 2
TABLE 3
Cell Cycle Analysis of Gene-Transduced Lymphoblast
Cytogenetic Analysis of Gene-Transduced HSC536N Cells
Normal lymphoblast Normal lymphoblast⫹MMC HSC536N Mock Mock⫹MMC EIAV EIAV⫹MMC HIV HIV⫹MMC MoMLV MoMLV⫹MMC PD4L Mock Mock⫹MMC EIAV EIAV⫹MMC HIV HIV⫹MMC MoMLV MoMLV⫹MMC
G0/G1
S
G2/M (%)
43.1 21.8
42.0 51.3
14.9 27.0
49.8 33.9 51.1 35.0 53.6 38.2 54.9 37.8
24.0 28.7 34.7 40.2 32.9 37.1 31.4 35.6
26.3 37.9a 13.9 25.0b 13.6 24.5b 13.8 27.3b
63.1 49.4 75.2 67.3 66.7 57.5 66.8 47.0
25.4 13.4 15.4 17.2 21.0 22.6 21.0 22.9
11.6 36.6a 9.5 15.6b 12.0 20.3b 12.3 20.2b
Note. Cells were incubated in the absence or presence of 100 nM MMC for 48 h. The mean data of two separate experiments are shown. There is a statistical significance with paired t test (P ⬍ 0.002) between mock-transduced (a) and transduced (b) cells.
duced CD34⫹ cells showed stable multilineage gene expression in bone marrow cells (32). In the mouse models, efficient GFP expression and long-term reconstitution of bone marrow using stem cells (33) and therapeutic hemoglobin synthesis in -thalassemic mice have been demonstrated (34). Although these positive results of gene transduction with HIV vectors have been obtained in vitro and in mouse models, one marking study using nonhuman
Normal lymphoblast Mock Total number of chromatid breaks Number of cells with chromatid breaksa Number of chromatid exchangesb
EIAV
HIV MoMLV
18
147
30
14
18
13
42
22
11
11
0
15
4
0
1
Note. Gene-transduced cells were incubated in the presence of 100 nM MMC for 48 h. Fifty cells from each cell line were analyzed for chromatid breaks and exchange figures (radials). a Mean of duplicated experiments. b Greater than 20% radial formation is diagnostic for FANC.
primates did not show efficient HIV vector gene transduction (35). Transduced gene expression in peripheral blood cells of reconstituted animals was not higher than that of MoMLV vectors. Regarding this, some modification of vector design or optimization of transduction protocol to enhance long-term gene expression was suggested. Previously, amphotropic retrovirus vectors have been used for gene transduction of peripheral CD34⫹ cells purified from Fanconi anemia group C patients (36). When using retroviruses such as MoMLV, cytokines are needed to stimulate quiescent target cells to enter the cell cycle. Although cytokine stimulation results in high transduction efficiencies of progenitors, it may cause irreversible differentiation of stem cells required to support long-term hematopoietic reconstitution. Despite repeated administration of transduced cells by MoMLV vectors, stable FANCC gene expression has not been observed. The ability of lentivirus vectors to transduce cells in G0/G1 phase of the cell cycle may enable stem cell transduction with-
FIG. 5. Sensitivity of FANCC gene-transduced lymphoblasts to MMC. Cell viability of gene-transduced or nontransduced lymphoblasts cultured with various concentrations of MMC. All experiments were performed in duplicate. MOLECULAR THERAPY Vol. 3, No. 4, April 2001 Copyright © The American Society of Gene Therapy
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ARTICLE out cytokine prestimulation (Yamada, unpublished data) and simplify the procedure of clinical gene therapy. EIAV has several desirable features as a gene delivery vector. Expression of EIAV proteins in human cells appears to be relatively nontoxic, allowing for generation of producer cell lines (15, 37, 38). Efficient gene transfer into cell cycle-arrested human cells by EIAV vectors was first demonstrated by Olsen (15). Moreover, Mitrophanous et al. demonstrated that EIAV vectors containing marker genes could transduce rat neuron and glial cells in vivo with efficiencies comparable to HIV vectors (16). Limitations of the EIAV vector system include lower titers and reduced transcription levels in hematopoietic cells. In comparison with MoMLV- and HIV-1-based vectors, at equivalent m.o.i. and proviral copy numbers/cell, the transcript levels produced from the EIAV vector were lowest. We speculate that the reduced EIAV transcriptional activity may be due to promoter interference between the EIAV LTR and the internal promoter. This may be unique to hematopoietic cells. The basal transcription level from the LTR in hematopoietic cells may be high even in the absence of Tat. Improved design of EIAV vectors is ongoing, analogous to the recent improvements in HIV-1based vectors (39, 40). The development of self-inactivating vectors would also ensure the safety of EIAV vectors especially when hematopoietic cells are targeted. Our data suggest that EIAV vectors are comparable to current HIV-1-based vectors. Efforts are aimed at improving EIAV vector production and gene expression. Studies are in progress testing EIAV gene transfer using primary human hematopoietic stem cells for eventual gene therapy trials. ACKNOWLEDGMENTS The authors thank Susan Bowyer for technical assistance on cytogenetics assay. This work was supported in part by grants from the American Cancer Society, the Leukemia and Lymphoma Society, and the Fanconi Anemia Research Fund.
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(1994). Phenotypic correction of Fanconi anemia in human hematopoietic cells with a recombinant adeno-associated virus vector. J. Clin. Invest. 94: 1440 – 1448. 28 Akkina, R. K., et al. (1996). High-efficiency gene transfer into CD34⫹ cells with a human immunodeficiency virus type 1-based retroviral vectors pseudotyped with vesicular stomatitis virus envelope glycoprotein G. J. Virol. 70: 2581–2585. 29 Case, S. S., et al. (1999). Stable transduction of quiescent CD34⫹CD38⫺ human hematopoietic cells by HIV-1-based lentiviral vectors. Proc. Natl. Acad. Sci. USA 96: 2988 – 2993. 30 Evans, J. T., Kelly, P. F., O’Neil, E., and Garcia, V. (1999). Human cord blood CD34⫹CD38⫺ cell transduction via lentivirus-based gene transfer vectors. Hum. Gene Ther. 10: 1479 –1489. 31 Uchida, N., et al. (1998). HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells. Proc. Natl. Acad. Sci. USA 95: 11939 –11944. 32 Guenechea, G., et al. (2000). Transduction of human CD34⫹CD38⫺ bone marrow and cord blood-derived SCID-repopulating cells with third-generation lentiviral vectors. Mol. Ther. 1: 566 –573. 33 Chen, W. Y., et al. (2000). Lentiviral vector transduction of hematopoietic stem cells that mediate long-term reconstitution of lethally irradiated mice. Stem Cell 18: 352–359. 34 May, C., et al. (2000). Therapeutic haemoglobin synthesis in -thalassaemic mice expressing lentivirus-encoded human -globin. Nature 406: 82– 86. 35 An, D. S., et al. (2000). Marking and gene expression by a lentivirus vector in transplanted human and nonhuman primate CD34⫹ cells. J. Virol. 74: 1286 –1295. 36 Liu, J. M., et al. (1999). Engraftment of hematopoietic progenitor cells transduced with the Fanconi anemia group C gene (FANCC). Hum. Gene Ther. 10: 2337–2346. 37 Klages, N., Zufferey, R., and Trono, D. (2000). A stable system for the high-titer production of multiply attenuated lentiviral vectors. Mol. Ther. 2: 170 –176. 38 Kafri, T., Van Praag, H., Ouyang, L., Gage, F. H., and Verma, I. M. (1999). A packaging cell line for lentivirus vectors. J. Virol. 73: 576 –584. 39 Follenzi, A., Ailles, L., Bakovic, S., Geuna, M., and Naldini, L. (2000). Gene transfer by lentiviral vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat. Genet. 25: 217–222. 40 Ramezani, A., Hawley, T., and Hawley, R. (2000). Lentiviral vectors for enhanced gene expression in human hematopoietic cells. Mol. Ther. 2: 458 – 469.
MOLECULAR THERAPY Vol. 3, No. 4, April 2001 Copyright © The American Society of Gene Therapy
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Functional Correction of Fanconi Anemia Group C Hematopoietic Cells by the Use of a Novel Lentiviral Vector Kaoru Yamada,* John C. Olsen,† Manij Patel,† Kathleen W. Rao,‡ and Christopher E. Walsh*,§,1 *UNC Gene Therapy Center, †Cystic Fibrosis/Pulmonary Research and Treatment Center, ‡Department of Pathology, and § Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599 Received for publication December 4, 2000; accepted in revised form February 9, 2001; published online April 16, 2001
Lentiviral vectors transduce nondividing hematopoietic cells more efficiently than other currently available vector systems. Here we report the results of human hematopoietic cell gene transfer using lentiviral vectors based upon human immunodeficiency virus (HIV-1) and equine infectious anemia virus (EIAV). EIAV is a nonprimate lentivirus and is severely restricted in its host range to horses and closely related equines. We employed the EIAV vector system to test for gene transfer to human Fanconi anemia (FA) hematopoietic cells by comparison with HIV-1- and Moloney murine leukemia virus-based systems. Fanconi anemia is characterized by bone marrow failure secondary to stem cell dysfunction. Fanconi anemia group C EBV-transformed lymphoblasts were transduced with all three viral vectors. Phenotypic correction of FA cells, as measured by mitomycin C drug resistance, was observed with a similar efficiency in all vector systems. This is the first description of lentiviral correction of FA cells and suggests that lentiviral vectors may be useful for FA gene transfer. Key Words: EIAV vector; lentivirus; Fanconi anemia group C (FANCC).
INTRODUCTION Oncoretroviruses such as Moloney murine leukemia virus (MoMLV) have provided the backbone for most retroviral gene transduction vectors. However, the inefficiency of gene transfer to nondividing cells is a major limitation of MoMLV-based vectors (1–3). Lentivirus vectors derived from human immunodeficiency virus type 1 (HIV-1) infect and integrate into quiescent cells (4, 5). Pseudotyped HIV employing the vesicular stomatitis virus-glycoprotein (VSV-G) envelope (6) broadens the host range of the vector (4, 7–11). Efficient HIV viral replication requires four accessory proteins (Vif, Vpr, Vpu, and Nef) that play a role in HIV pathogenesis (12). To improve the safety of the HIV-1 vector system genes encoding HIV accessory proteins can be removed to prevent production of wild-type HIV (9, 11). As an alternative to HIV-1-based vectors, the nonprimate lentiviral counterparts, feline immunodeficiency virus (13, 14) and equine infectious anemia virus (EIAV) (15, 16), are under development. EIAV has a relatively
1 To whom correspondence and reprint requests should be addressed at Room 7101, Thurston Building, CB#7352, Chapel Hill, NC 27599. Fax: (919) 966-0907. E-mail: [email protected].
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simple genome organization, having only three accessory proteins. Alternate splicing derives accessory genes Rev, S2, and Tat. EIAV Rev functions for nuclear export of nonspliced or incompletely spliced RNA and Tat functions to increase the steady-state levels of viral RNA. The function of S2 is not completely understood, but is not required for replication of wild-type virus in culture or for gene transfer by recombinant EIAV vectors (17). S2 may have a role in progression of infectious anemia in its natural host (18). Wild-type EIAV has never been reported as an agent transmitted to humans (19). Previous studies have established the feasibility of generating recombinant EIAV vectors at titers sufficient for use in gene transfer experiments (15, 16). Recombinant EIAV vectors are produced following transient transfection of a transgene plasmid and a plasmid carrying the VSV-G envelope into a producer cell line carrying the EIAV gag, pol, and rev ((15) and Patel and Olsen, unpublished data). EIAV vectors stably integrate reporter genes into both transformed and normal cells from humans and rodents. Nondividing cells can be transduced in culture and in vivo by EIAV vectors (16). These results suggest that EIAV vectors can be used to transfer genes to human hematopoietic cells. Recently we demonstrated the retroviral gene comple-
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FIG. 1. Schematic diagram of FANCC vector plasmid constructs. EIAV and HIV vectors contain the CMV immediate early promoter as internal promoter. The EIAV vector also contains a fusion of the CMV promoter and the R/U5 region of the 5⬘ LTR sequence. EIAV Rev responsive element is located downstream of the FANCC gene. Transcription of the FANCC gene is driven from the LTR in MoMLV vector. CMV, human cytomegalovirus; SV, SV40 promoter; neo, neomycin resistance gene.
mentation of Fanconi anemia hematopoietic cells (20). In this study, we chose hematopoietic cells derived from patients with Fanconi anemia to test gene transduction by EIAV- and HIV-1-based vectors. Fanconi anemia (FANC) is an autosomal recessive disease characterized by bone marrow failure due to stem/progenitor cell dysfunction. FANC cells are hypersensitive to DNA cross-linking agents such as mitomycin C (MMC). Exposure of FANC cells to MMC leads to cell cycle arrest, cell apoptosis, and an increased frequency of chromosomal breakage thought to be the result of defective DNA repair (21, 22). Seven FANC subgroups, defined as FANCA–G have been described by complementation analysis (23). Genetic complementation of FANC cells with the appropriate FANC cDNA leads to normalized cell cycle kinetics, regular cell growth, and absence of chromosomal damage in the presence of drug. Here we describe phenotypic correction of FANCC group cells using lentiviral vectors.
MATERIALS
AND
METHODS
Cell Lines/Cell Culture Human 293T cells, an amphotropic MoMLV vector-producing cell line (clone 52-19 (20)), and B241 cells, a 293-derived cell line stably expressing EIAV gag, pol, and rev genes (Patel and Olsen, unpublished), were cultured in Dulbecco’s modified Eagle’s medium with high glucose (DMEM; Gibco BRL, Gaithersburg, MD) and 10% fetal bovine serum. Epstein–Barr virustransformed B cell lines, HSC536N and PD4L cells, established from FANCC patients carrying different mutations (20), were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum.
Construction of Transgene Plasmids To construct FANCC gene transfer vectors, the 1.9-kb FANCC gene cDNA from pG1FASVna (20) plasmid was digested by NotI restriction enzyme and ligated into an EIAV vector (pECG3CR, Patel and Olsen, unpublished). The EGFP gene in HIV-LL-CG plasmid (kindly provided by Dr. Kafri, UNC) was replaced by FANCC cDNA (Fig. 1).
Viral Vector Production EIAV and HIV vectors were produced by transient transfection using CaPO4-mediated methods. For EIAV vector generation, B241 cells were plated at 1 ⫻ 107 cells per collagen- (Vitrogen, Cohesion, Palo Alto, CA) coated 10-cm tissue culture dish 24 h before transfection. Then cells were
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transfected with 10 g each of the EIAV/FANCC vector and VSV-G (pCIVSV-G) plasmids. After overnight transfection, fresh medium containing 10 mM sodium butyrate (Sigma) was added (24). Recombinant HIV vector was produced using a similar cotransfection method. Ten micrograms each of three vector plasmids, HIV/FANCC vector, HIV virus proteinexpressing vector (CMV⌬R8.2 (25)), and VSV-G vector (pMDG) was transfected into 2.5 ⫻ 106 293T cells that were plated 24 h prior onto poly-Llysine- (Sigma) coated 10-cm tissue culture dishes. Following 2 days of culture after transfection, HIV and EIAV vector supernatants were harvested and filtered through 0.45-m filters and virus was concentrated about 100-fold by ultracentrifugation at 50,000g for 2.5 h at 4°C. One-day culture supernatants of amphotropic retrovirus vector 52-19 (20) producer cells were harvested and then filtered. All supernatants containing virus were frozen at ⫺80°C until use.
Virus Titer Nonconcentrated virus was subjected to RNA dot blot analysis as an index of virus particle number. Virus was precipitated from 1 ml of culture medium by the addition of 0.5 ml solution of 6% polyethylene glycol (MW 8000) (Sigma) and 0.3 M NaCl and then centrifuged at 14, 000 rpm for 20 min at 4°C. RNA was extracted from the virions with phenol– chloroform and digested with 10 U of RNase free DNase I (Promega Biotec, Madison, WI) in the presence of 200 U RNasin ribonuclease inhibitor (Promega Biotec) for 30 min at 37°C to eliminate contamination of plasmid DNA and then reextracted with phenol– chloroform. Viral RNA from 1 ml of culture medium was dot-blotted onto nylon membrane (Hybond N⫹; Amersham Corp., Arlington Heights, IL) and then hybridized using a 32 P-labeled random-primed 1.9-kb size FANCC cDNA as a probe. Standard curves were made by serial dilution of each vector plasmid. Titers of EIAV/FANCC and HIV/FANCC vectors were estimated by comparing genome content with vectors of known infectivity encoding surrogate markers (LacZ gene in pEC-LacZ plasmid and GFP gene in HIV-LL-CG plasmid). To determine infectivity, 293T cells were plated at 3 ⫻ 105 cells per well in six-well plates with 3 ml final volume. The day after plating, cells were transduced at 37°C overnight with serial dilution of EIAV/LacZ and HIV/ GFP virus vectors in the presence of 5 g/ml protamine sulfate. For determination of X-gal staining titer for EIAV vector, at 48 h after transduction, cells were fixed with 1 ml 0.5% glutaraldehyde in PBS containing 1 mM MgCl2. Cells were stained in X-gal solution for 2 h at 37°C and the number of blue-stained colonies was counted under microscope. Virus titer was determined by multiplying with the dilution factor. For determination of GFP titer for HIV vector, at 48 h after transduction, cells were harvested and fixed with 4% paraformaldehyde and then analyzed with a flow cytometer for GFP fluorescence. Virus titer was determined by multiplying the percentage of GFP-positive cells by the dilution factor. The titer of the MoMLV vector was estimated by determining the number of neomycinresistant colonies using NIH 3T3 cells.
Gene Transfer to FANC Hematopoietic Cells HSC536N and PD4L lymphoblasts were incubated with vector preparations containing protamine sulfate (5 g/ml) at a m.o.i. of 1. After overnight transduction, cells were harvested, washed with PBS twice, and cultured further for 2 days in DMEM. Cells were maintained in medium containing 10 nM MMC for 4 weeks to select gene-complemented cells. Mock-transduced lymphoblasts cultured under the same conditions yielded no viable cells after 4 weeks.
Molecular Analysis of FANCC-Transduced Cells Southern blot analysis. Genomic DNA was isolated from transduced lymphoblasts using the DNeasy Tissue Kit (Qiagen, Valencia, CA). Ten micrograms of DNA was digested with the particular restriction enzymes whose restriction sites are located within each LTR: StuI for EIAV, BspEI for HIV, and KpnI for MoMLV. DNA was hybridized using a 32P-labeled 1.9-kb-size FANCC cDNA probe. Mock-transduced HSC536N cells served as a source for negative control DNA. Standard curves were made by digestion of the mock-transduced HSC536N DNA with serial copy numbers of each vector plasmid. The expected band sizes were 4.9 kb for EIAV, 4.7 kb for HIV, and 4.6 kb for MoMLV. MOLECULAR THERAPY Vol. 3, No. 4, April 2001 Copyright © The American Society of Gene Therapy
ARTICLE FANCC, we assumed the functional titer of FANCC vectors was also 1:100.
TABLE 1 Virus Titer Virus vector/ transgene EIAV/LacZ EIAV/FANCC HIV/GFP HIV/FANCC MoMLV/FANCC⫹Neo a
Target cells
Infectious particle unit/ml
Virus particle number/mla
293T
6 ⫻ 104
293T
5 ⫻ 10
NIH 3T3
5 ⫻ 106
1 ⫻ 106 1 ⫻ 106 1 ⫻ 107 1 ⫻ 107 1 ⫻ 108
5
Analyzed by dot blot with transgene cDNA as the probes.
Northern blot analysis. Total RNA was extracted from selected HSC536N cells by the AGPC method (26) using TRIzol Reagent (Gibco BRL). Polyadenylated RNA was further purified by Oligo(dT) Cellulose Spin Column Kit (Eppendorf–5 Prime, Inc., Boulder, CO). Two micrograms of polyadenylated RNA was subjected to electrophoresis in a 1% agarose–formaldehyde gel. The estimated size of RNA transcripts was calculated by comparison with the mobility of ribosomal RNA from total RNA run in the same gel (not shown). After RNA blotting onto nylon membranes, hybridization was performed using a 32P-labeled FANCC cDNA probe. The intensity of each band was quantified following phosphorimage analysis.
Functional Assessment of Transduced Cells Cell cycle analysis. Transduced lymphoblasts were incubated at 1 ⫻ 105/ml with 100 nM MMC for 2 days. Cells were harvested and fixed with ethanol and then incubated with 2 mg/ml RNase (Sigma) at 37°C followed by staining with 0.05 mg/ml propidium iodide (Sigma) for 20 min in the dark. DNA content data were acquired by FACSCalibur (Becton–Dickinson, San Jose, CA) using a CellQuest software program (Becton–Dickinson). Cell cycle analysis was performed using ModFit LT (Verity Software House, Inc., Topsham, ME). MMC sensitivity assay. Transduced or control lymphoblasts were plated at 1 ⫻ 105/ml in 12-well plates and then cultured with various concentrations of MMC (0, 1, 5, 10, 50, and 100 nM) for 14 days. Cellular viability was assayed by trypan blue exclusion. Samples were performed in duplicate. Cytogenetic breakage. Lymphoblast cultures were analyzed for cytogenetic breakage and exchange figures (radial formation) by exposure to 100 nM MMC for 2 days. Cultures were harvested after a 90-min exposure to 0.50 g/ml colcemid and 10 g/ml ethidium bromide. After a 10-min treatment with 0.075 M KCl, the cells were fixed with a 3:1 mixture of methanol:acetic acid. Slides were prepared using wet slides, air dried, and stained with Wright’s stain. Fifty metaphase figures from each culture were scored for obvious breaks, gaps larger than a chromatid width, and exchange figures.
Virus Vector-Mediated Gene Transfer and Phenotypic Correction of FANC Lymphoblasts Epstein–Barr virus-transformed lymphoblast cell lines, HSC536N and PD4L, were established from FANCC patients as previously described (27). Spontaneous phenotypic reversion, i.e., resistance to drug, has not occurred in these cell lines, making them well suited to gene complementation studies. HSC536N carries a T to C point mutation while PD4L contains a G deletion causing a frameshift mutation and a truncated protein. Both mutations totally abolish FANC protein function. Transduction of each cell line was performed using EIAV, HIV, or MoMLV/ FANCC at a m.o.i. of 1. After overnight incubation with virus, cells were cultured in 10 nM MMC for 4 weeks (27) to select complemented cells. No viable mock-transduced cells were observed in culture after 4 weeks. Molecular analysis of gene transduction was performed to confirm the presence of proviral DNA. Southern blot analysis of genomic DNA harvested from the transduced HSC536N cells and hybridized with FANCC-specific probe is shown in Fig. 2. We estimated that proviral copy number for each vector was 0.3 copy/cell. Northern blot analysis was performed on MMC-selected HSC536N cells (Fig. 3). A single RNA species hybridized to the probe for both MoMLV- and HIV-transduced samples. The band sizes were 4.5 and 2.8 kb, respectively, consistent with that of the predicted transcript size for each vector. Analysis of EIAV-transduced cells produced two bands, 4.0 and 3.0 kb, consistent with transcription originating from both the LTR and the internal CMV promoters. An estimation of the steady-state levels of vector RNA transcripts yielded a ratio of 3:2:1 (HIV:MoMLV:EIAV). The LTR promoter activity of EIAV in hematopoietic cells might cause promoter interference of the internal CMV promoter, resulting in lower transcription levels in these target cells.
RESULTS Generation of FANCC Gene Expression Vectors VSV-G-pseudotyped EIAV- and HIV-1-based lentiviral vectors were generated by transient transfection. To estimate the titers of recombinant lentiviral vectors, dot blot hybridization analysis was performed and infectious-particle units were measured by the number of GFP- and LacZ-positive cells. Shown in Table 1, we reproducibly observed that the relative infectious-particle number-toviral genome ratio was 1 to 100 for both EIAV and HIV. In order to compare the effectiveness of EIAV and HIV/ MOLECULAR THERAPY Vol. 3, No. 4, April 2001 Copyright © The American Society of Gene Therapy
FIG. 2. Southern blot analysis of gene-transduced HSC536N lymphoblasts. Genomic DNA was isolated from gene-transduced cells and digested with restriction endonucleases that cut uniquely within each LTR. Genomic DNA from each transduced cell line was electrophoresed in an agarose gel, transferred onto nylon membranes, and hybridized using a 32P-labeled 1.9-kb size FANCC fragment as a probe. The standard curve was prepared using serial dilutions of plasmid and parental cell line genomic DNA and run with each sample. The expected 1.9-kb band is indicated (arrow). Data shown are representative of duplicate experiments.
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ARTICLE DISCUSSION
FIG. 3. Northern blot analysis of FANCC-transduced HSC536N lymphoblasts. Total RNA was isolated from transduced cells and mRNA was purified using an oligo(dT) column. 32P-labeled FANCC DNA was used as a probe. The expected sizes of the RNA transcripts are indicated. Each single transcript from HIV and MoMLV vector-transduced cells was detected (2.8 kb, HIV; 4.5 kb, MoMLV). The two bands for EIAV-transduced cells are consistent with the size of transcripts from EIAV LTR (4 kb) and CMV internal promoter (3 kb).
We demonstrated functional complementation of FANCC cell lines using EIAV- and HIV-based vectors. Phenotypic correction was determined by three different functional assays, including MMC sensitivity on cell proliferation, cell cycle kinetics, and cytogenetic assays. Although we found lower FANCC transgene expression in EIAV-transduced cells compared with HIV and MoMLV, there was no difference in terms of FANC correction. These data represent the first example of EIAV-mediated gene transfer using a disease-specific cDNA. Several reports have demonstrated efficient gene transduction of human CD34⫹ cells by HIV vectors in vitro (5, 28 –30). HIV vectors showed the capability to transduce genes into quiescent CD34⫹Lin⫺Thy-1⫹CD38⫺ cells (31), overcoming a major limitation of retrovirus vectors, which need cell mitosis for vector integration. Fully reconstituted NOD/SCID mice with HIV-1 vector-trans-
The hallmark of FANC is cellular hypersensitivity to DNA cross-linking agents. Cell cycle arrest, accelerated apoptosis, and aberrant chromosomal breakage characterize the FANC phenotype. The clastogenic agent, MMC, causes DNA damage and growth arrest in the G2 phase of the cell cycle. We tested whether FANCC gene complementation restored cell cycle arrest. FANCC gene-transduced cells were subjected to cell cycle analysis after culture with 100 nM MMC for 2 days. Cells were stained with propidium iodide and then analyzed with flow cytometry (Fig. 4). The percentage of cells in the G2/M phase increased only in mock-transduced cells treated with MMC. In contrast, no significant change in the fraction of cycling cells was observed in any of the gene-transduced or normal lymphoblasts following MMC treatment (Table 2). Drug sensitivity of each cell line was measured by cell viability with cultured cells in various concentrations of MMC (0, 1, 5, 10, 50, and 100 nM). As expected, both mock-transduced cell lines were highly sensitive to MMC with an effective drug concentration yielding 50% reduction in cell viability (EC50) of 1 to 5 nM MMC (Fig. 5). In contrast, regardless of the vector used, transduced cells exhibited growth equivalent to lymphoblasts established from a normal individual. The EC50 of both normal and corrected lymphoblasts was 30 nM. We then determined the frequency of chromosomal aberrations in both treated and untreated cells. Metaphase preparations of cells were made following cell incubation with 100 nM MMC for 2 days. As expected, the formation of quadriradials was 35% of all metaphases examined, significantly greater than normal. Aberrations in greater than 20% of metaphases are considered diagnostic for FANC. In contrast, cytogenetic results for each vector-transduced cell line were less than 20% and comparable with normal lymphoblasts (Table 3).
FIG. 4. Cell cycle kinetics of gene-corrected lymphoblasts. Virus vectortransduced HSC536N cells were compared to the parental mock-transduced cell lines. Cells were cultured in the presence or absence of 100 nM MMC for 48 h, fixed, stained with propidium iodide, and the DNA content analyzed. The results shown are representative of three separate experiments.
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ARTICLE TABLE 2
TABLE 3
Cell Cycle Analysis of Gene-Transduced Lymphoblast
Cytogenetic Analysis of Gene-Transduced HSC536N Cells
Normal lymphoblast Normal lymphoblast⫹MMC HSC536N Mock Mock⫹MMC EIAV EIAV⫹MMC HIV HIV⫹MMC MoMLV MoMLV⫹MMC PD4L Mock Mock⫹MMC EIAV EIAV⫹MMC HIV HIV⫹MMC MoMLV MoMLV⫹MMC
G0/G1
S
G2/M (%)
43.1 21.8
42.0 51.3
14.9 27.0
49.8 33.9 51.1 35.0 53.6 38.2 54.9 37.8
24.0 28.7 34.7 40.2 32.9 37.1 31.4 35.6
26.3 37.9a 13.9 25.0b 13.6 24.5b 13.8 27.3b
63.1 49.4 75.2 67.3 66.7 57.5 66.8 47.0
25.4 13.4 15.4 17.2 21.0 22.6 21.0 22.9
11.6 36.6a 9.5 15.6b 12.0 20.3b 12.3 20.2b
Note. Cells were incubated in the absence or presence of 100 nM MMC for 48 h. The mean data of two separate experiments are shown. There is a statistical significance with paired t test (P ⬍ 0.002) between mock-transduced (a) and transduced (b) cells.
duced CD34⫹ cells showed stable multilineage gene expression in bone marrow cells (32). In the mouse models, efficient GFP expression and long-term reconstitution of bone marrow using stem cells (33) and therapeutic hemoglobin synthesis in -thalassemic mice have been demonstrated (34). Although these positive results of gene transduction with HIV vectors have been obtained in vitro and in mouse models, one marking study using nonhuman
Normal lymphoblast Mock Total number of chromatid breaks Number of cells with chromatid breaksa Number of chromatid exchangesb
EIAV
HIV MoMLV
18
147
30
14
18
13
42
22
11
11
0
15
4
0
1
Note. Gene-transduced cells were incubated in the presence of 100 nM MMC for 48 h. Fifty cells from each cell line were analyzed for chromatid breaks and exchange figures (radials). a Mean of duplicated experiments. b Greater than 20% radial formation is diagnostic for FANC.
primates did not show efficient HIV vector gene transduction (35). Transduced gene expression in peripheral blood cells of reconstituted animals was not higher than that of MoMLV vectors. Regarding this, some modification of vector design or optimization of transduction protocol to enhance long-term gene expression was suggested. Previously, amphotropic retrovirus vectors have been used for gene transduction of peripheral CD34⫹ cells purified from Fanconi anemia group C patients (36). When using retroviruses such as MoMLV, cytokines are needed to stimulate quiescent target cells to enter the cell cycle. Although cytokine stimulation results in high transduction efficiencies of progenitors, it may cause irreversible differentiation of stem cells required to support long-term hematopoietic reconstitution. Despite repeated administration of transduced cells by MoMLV vectors, stable FANCC gene expression has not been observed. The ability of lentivirus vectors to transduce cells in G0/G1 phase of the cell cycle may enable stem cell transduction with-
FIG. 5. Sensitivity of FANCC gene-transduced lymphoblasts to MMC. Cell viability of gene-transduced or nontransduced lymphoblasts cultured with various concentrations of MMC. All experiments were performed in duplicate. MOLECULAR THERAPY Vol. 3, No. 4, April 2001 Copyright © The American Society of Gene Therapy
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ARTICLE out cytokine prestimulation (Yamada, unpublished data) and simplify the procedure of clinical gene therapy. EIAV has several desirable features as a gene delivery vector. Expression of EIAV proteins in human cells appears to be relatively nontoxic, allowing for generation of producer cell lines (15, 37, 38). Efficient gene transfer into cell cycle-arrested human cells by EIAV vectors was first demonstrated by Olsen (15). Moreover, Mitrophanous et al. demonstrated that EIAV vectors containing marker genes could transduce rat neuron and glial cells in vivo with efficiencies comparable to HIV vectors (16). Limitations of the EIAV vector system include lower titers and reduced transcription levels in hematopoietic cells. In comparison with MoMLV- and HIV-1-based vectors, at equivalent m.o.i. and proviral copy numbers/cell, the transcript levels produced from the EIAV vector were lowest. We speculate that the reduced EIAV transcriptional activity may be due to promoter interference between the EIAV LTR and the internal promoter. This may be unique to hematopoietic cells. The basal transcription level from the LTR in hematopoietic cells may be high even in the absence of Tat. Improved design of EIAV vectors is ongoing, analogous to the recent improvements in HIV-1based vectors (39, 40). The development of self-inactivating vectors would also ensure the safety of EIAV vectors especially when hematopoietic cells are targeted. Our data suggest that EIAV vectors are comparable to current HIV-1-based vectors. Efforts are aimed at improving EIAV vector production and gene expression. Studies are in progress testing EIAV gene transfer using primary human hematopoietic stem cells for eventual gene therapy trials. ACKNOWLEDGMENTS The authors thank Susan Bowyer for technical assistance on cytogenetics assay. This work was supported in part by grants from the American Cancer Society, the Leukemia and Lymphoma Society, and the Fanconi Anemia Research Fund.
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MOLECULAR THERAPY Vol. 3, No. 4, April 2001 Copyright © The American Society of Gene Therapy