Protein Engineering, Vol. 13, No. 9, 611-615,
September 2000
© 2000 Oxford University Press
Communication |
Functional EGFP–dystrophin fusion proteins for gene therapy vector development
Unité de Recherche en Génétique Humaine, Centre Hospitalier de l'Université Laval, CHUQ, Faculté de Médecine, Université Laval, Sainte-Foy, Québec, G1V 4G2, Canada and 1 INSERM U 523, Institut de Myologie, Groupe Hospitalier Pitié-Salpêtrière, Paris, France
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Abstract |
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Transfection and transduction studies involving the use of the full-length dystrophin (11 kb) or the truncated mini-gene (6 kb) cDNAs are hampered by the large size of the resulting viral or non-viral expression vectors. This usually results in very low yields of transgene-expressing cells. Moreover, the detection of the few transgene-expressing cells is often tedious and costly. For these reasons, expression vectors containing the enhanced green fluorescent protein (EGFP) fused with the N-termini of mini- and full-length human dystrophin were constructed. These constructs were tested by transfection of Phoenix cells with Effectene, resulting after 48 h in a green fluorescent signal in 20% of cells. Analysis of the cell extracts by immunoblotting with the use of a monoclonal antibody specific to the dystrophin C-terminus confirmed the expression of EGFP–mini- (240 kDa) and EGFP–full-length human dystrophin (450 kDa) fusion proteins. Moreover, following the in vivo electroporation of the plasmids containing the EGFP–mini- and full-length dystrophin in mouse muscles, both fluorescent proteins were observed in cryostat sections in their normal location under the plasma membrane. This indicates that the fusion of EGFP to dystrophin or mini-dystrophin did not interfere with the normal localization of the protein. In conclusion, the fusion of EGFP provides a good tool for the search of the best methods to introduce mini- or full-length dystrophin cDNA in the cells (in vitro) or muscle fibers (in vivo) for the establishment of a treatment by gene therapy of Duchenne muscular dystrophy patients.
Keywords: cell transfection/Duchenne muscular dystrophy/EGFP/EGFP-dystrophin fusion proteins/electroporation/gene therapy/human dystrophin
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Duchenne muscular dystrophy (DMD), which affects one in 3500 males, is one of the most prevalent types of muscular dystrophy and is characterized by the rapid progression of muscle degeneration which occurs early in life. The mutated gene in DMD, found on the X chromosome, normally encodes a large protein named dystrophin (427 kDa) which is required inside muscle cells for structural support (Acsadi et al., 1996
; Koenig et al., 1988). This protein includes 3685 encoded amino acids separated in four domains. These domains consist of a 240 amino acid N-terminal domain (similar to the actin binding domain of 
-actinin), a rod-shaped domain formed by 25 triple-helical segments (similar to the repeat domains of spectrin), a cysteine-rich (domain similar in part to the entire COOH domain of the Dictyostelium -actinin) and a C-terminal domain (420 amino acids C-terminal) which is unique to dystrophin. Dystrophin is a cytoskeletal protein possessing similar characteristics to spectrin -actinin leading its large structure to adopt a rod shape ~150 nm in length. Mini-dystrophin (210 kDa) has been described (England et al., 1990) as lacking the spectrin-like domain (exon 17–48 of the full-length dystrophin) and possesses binding properties similar to the full gene since the -actinin and the C-terminal domains were conserved. Gene therapy is a possible therapeutic option to treat DMD. The therapeutic gene may be introduced either directly in vivo using viral or non-viral vectors or ex vivo by transfecting or infecting the muscle precursor cells (myoblasts) (Karpati et al, 1997; McHowell, 1999). To date, vectors and transduction methods still need to be developed to transfer dystrophin efficiently to target cells in a clinically feasible protocol.
Recently, green fluorescent protein (GFP) has been fused with many proteins at either the N- or C-terminal end, providing an interesting marker system for many research applications such as microscopy detection and flow cytometry (Cheng et al., 1996). To study the best delivery systems for mini-dystrophin and full-length dystrophin cDNAs, we have constructed fusion proteins consisting of `enhanced' GFP (EGFP) linked to the N-terminus of dystrophin and mini-dystrophin. In the present study, dystrophin expression cassettes were designed to be accommodated in viral or non-viral vectors in which the promoter can be removed and replaced by another without affecting the fusion protein. Also, the evaluation of the functionality of both constructs in vivo was further assessed by electroporation (Aihara and Miyazaki, 1998) of dystrophin constructs in mouse skeletal muscles. Since dystrophin is mainly expressed as a subsarcolemmal protein in skeletal muscle fibers, we investigated whether the constructs could be expressed and would accumulate at relevant localizations in vivo in muscle tissue. In this paper, we report that the dystrophin fused to EGFP could be useful in a rapid and quantitative evaluation of the expression of recombinant dystrophin, both in vitro and in vivo.
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Materials and methods |
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Plasmids, pCR3.1 mini-dystrophin and primers
Eukaryotic TA cloning kit containing the plasmid pCR3.1 expression vector was purchased from Invitrogen (Carlsbad, CA). EGFP cDNA was amplified by the polymerase chain reaction (PCR) of the gene contained in plasmid pIRES-EGFP (Clontech, Palo Alto, CA) using the primers C and D shown in Table I. Plasmid pRSVDMD.1 containing human full-length dystrophin cDNA was kindly provided by G.Karpati (Montreal Neurological Institute, McGill University, Canada). Dystrophin mini-gene (lacking exons 17–48 of full-length dystrophin) was obtained by PCR amplification of DNA extracted from cells infected with an adenovirus containing the mini-dystrophin gene (Acsadi et al. 1996; Moisset et al., 1998). Primers A and B (Table I) served for the amplification of mini-dystrophin cDNA with Expand High Fidelity DNA polymerase (Boehringer Mannheim, Montreal, QC, Canada) giving a final product of ~6 kb. The amplified mini-gene fragment was cloned directly in pCR3.1 vector (pCR3.1 mini-dystrophin). A series of six oligonucleotide primers were chemically synthesized using an ABI 394 synthesizer (Perkin-Elmer, Foster City, CA). These primers carrying specific restriction sites are listed in Table I.
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Generation of pCR3.1 EGFP
We amplified with Taq DNA polymerase (Pharmacia Biotech, Montreal, QC, Canada) the EGFP cDNA (0.7 kb) from pIRES-EGFP using oligonucleotides C and D (Table I
). The amplified product was cloned directly in pCR3.1 vector. Sense orientation of the amplified product (EGFP) in pCR3.1 was confirmed by restriction analysis and the construct was tested by transfection in Phoenix cells giving a fluorescent signal. The restriction sites NheI, KpnI and BamHI in the polylinker of the new construct were then deleted by cutting with restriction enzymes flanking these sites, blunting with Klenow enzyme (Pharmacia Biotech) and self-ligating with T4 DNA ligase (Pharmacia Biotech) resulting in pCR3.1 EGFP (Figure 1).
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Generation of pCR3.1 EGFP linked to 5' mini- or full-length dystrophin (pCR3.1 EGFP–5'Dys)
A DNA linker fragment (200 bp) joining EGFP to the 5' portion of the dystrophin cDNA containing the first BamHI site of dystrophin was obtained by PCR amplification of plasmid pRSV DMD.1 with primers E and F (Table I) and cloned in pCR3.1 vector (pCR3.1 link). This recombinant was sequenced (T7 sequencing kit, Pharmacia Biotech) for confirmation of the exact nucleotide sequence of the link between EGFP and dystrophin. The link DNA fragment was then obtained by a double digestion of pCR3.1-link with SalI and XhoI and cloned in the same sites found in pCR3.1 EGFP, making a recombinant pCR3.1 EGFP linked to the 5' portion of the dystrophin cDNA in which the stop codon carried by EGFP in pCR3.1 EGFP was eliminated (pCR3.1 EGFP–5'Dys).
Generation of pCR3.1 EGFP–mini-dystrophin (pMDysE)
We cloned in three steps the other DNA fragments obtained from the following digestions of pCR3.1 mini-dystrophin (pMDys) by directed cloning:
- Step 1:BamHI/NheI digestions of pMDys produced a DNA fragment (~1.1 kb) which was cloned in the same restriction sites of plasmid pCR3.1 EGFP–5'Dys giving an intermediate construct named A.
- Step 2:NheI/KpnI digestions of pMDys produced a DNA fragment (1 kb) which was cloned in the same restriction sites of construct A making a second intermediate construct named B.
- Step 3:KpnI/ApaI digestions of pMDys produced a DNA fragment (~3.6 kb) which was cloned in the same restriction sites of construct B making a final construct for EGFP mini-dystrophin named pCR3.1 EGFP–mini-dystrophin (pMDysE).
Generation of pCR3.1 EGFP full-length dystrophin (pDysE)
We cloned in two consecutive steps the other DNA fragments obtained by digestion of pRSVDMD.I and by digestion of pMDysE.
- Step 2':NheI/KpnI digestions of pRSVDMD.I produced a DNA fragment (6.1 kb) which was cloned in the same restriction sites of construct A (see above for pMDysE) giving an intermediate construct named C.
- Step 3':KpnI/ApaI digestions of pMDysE produced a DNA fragment (~3.6 kb) which was cloned in the same restriction sites of construct B' making a final construct for EGFP–full-length dystrophin pCR3.1 (pDysE).
Transfection of EGFP–dystrophin constructs into cultured cells
Phoenix cells lines (made by G.Nolan and provided by ATCC) were maintained in Dulbecco's modified Eagle's medium high glucose (DMEM; GIBCO/BRL, Burlington, ON, Canada) at 37°C under 5% CO2. Culture medium was supplemented with 10% fetal bovine serum (GIBCO/BRL), penicillin (10 000 IU/ml, GIBCO/BRL), streptomycin (10 µg/ml, GIBCO/BRL), sodium pyruvate (1 mM) (Sigma, St. Louis, MO) and glutamine (2 mM) (Sigma). Transfections of pMDysE and pDysE were performed with the Effectene transfection reagent (Qiagen, Mississauga, ON, Canada) according to the manufacturer's indications. The fluorescence signal was observed by microscopy after 24 and 48 h.
Protein extraction and Western blot analysis
For protein extraction, cells were rinsed twice with phosphate-buffered saline (PBS), lysed in 200 µl of 10 mM Tris–HCl pH 7.5, 10% SDS, 1 mM dithiothreitol (DTT), 1 mM PMSF and treated by a procedure described by Wessel and Flügge (1984). Pellets resulting from the extraction procedure were partially dissolved in electrophoresis loading buffer (200 µl) containing 10% SDS and centrifuged before loading on the gel. Approximately 10 µg of protein (10 µl) were electrophoresed on 6% SDS–PAGE gel and transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked with 5% non-fat milk in PBS–Tween-20 (0.05%) (PBS–T). The primary antibody was NCL-Dys 2 (Novocastra Laboratories, Newcastle upon Tyne, UK), a monoclonal antibody directed against the carboxy-terminal 17 amino acids of dystrophin, diluted 1:300 in PBS–T with 1% non-fat milk. The secondary antibody was a goat anti-mouse antibody conjugated to horseradish peroxidase (Jackson Immunoresearch Laboratories) diluted 1:10 000 in PBS–T with 3% non-fat milk. The chemiluminescent signal was analyzed by a 2 min exposure to autoradiography film after detection with Renaissance reagent (NEN, Boston, MA).
Electroporation of mouse muscles with pMdysE or pDysE
Two-month-old C57BL6J mice were used for this study. Three experimental groups included three mice each and both Tibialis anterior muscles received the naked DNA and were electroporated. Each of the three groups received one of the following constructions: plasmid containing only EGFP, pMdysE and pDysE. Gene transfer was achieved using the ECM830 electroporator (Genetronics, San Diego, CA) according to the settings described by Mir et al. (1999). Briefly, 20 µl of naked DNA in solution in clinical-grade saline buffer (1 mg/ml) were injected into both Tibialis anterior muscles. An electric field was applied using plate electrodes. Given the potential immunogenicity of EGFP protein (Stripecke et al., 1999), animals were immunosuppressed with FK506 (2.5 mg/kg.day) starting on the day of electrotransfer (Kinoshita et al., 1994).
Muscle examination
Five days after gene transfer, the animals were killed and the muscles were snap-frozen in isopentane cooled in liquid nitrogen. Serial cryostat sections were prepared throughout the whole muscle length, mounted in phosphate buffer–glycerol (1:1) and observed under UV illumination (Zeiss microscope).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
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Figure 1
illustrates the schematic procedure for obtaining the final constructs giving EGFP fused to human dystrophin mini- or full-length cDNA following the strategy described in Materials and methods. For both constructs, the PmeI and NotI restriction sites flanking the transgene allow excision of the fusion gene from its original vector pCR3.1 and subcloning it in a viral or a non-viral vector, making an ideal tool for the rapid evaluation of the efficiency of transduction or transfection. Moreover, the CMV promoter and the EGFP DNA fragment can be deleted separately by the use of single cutting restriction enzymes in both constructs (Figure 1) giving different options to replace the CMV promoter or to remove the EGFP once the transfection or transduction conditions are well established.
Previous reports have shown various levels of transfection in different cells in order to evaluate gene transfer of full-length or mini-dystrophin (Clemens et al., 1995; Acsadi et al., 1996; Yanagihara et al., 1996; McCaster et al., 1997; Floyd et al., 1998). However, a separate ß-galactosidase gene was usually used as a reporter gene in most cases. In the present study, transfection of pDysE or pMdysE constructs in Phoenix cells resulted in a good fluorescent signal in about 20% of the cells (Figure 2). This observation was confirmed by Western blot analysis of equal amounts of Phoenix cell proteins (Figure 3) showing bands of the expected size of 450 kDa for pDysE (Figure 3, lane 2) and 240 kDa for pMDysE (Figure 3, lane 3). Overall, these results show the suitability of EGFP fused to mini- or full-length dystrophin as a tool to determine rapidly the transfection efficiency in different cells instead of ß-galactosidase or EGFP alone.
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We performed electroporation of pMdysE and pDysE in mouse muscle in order to verify whether a normal localization of dystrophin fused to EGFP was preserved. Our results illustrated in Figure 4
indicated that the chimeric EGFP–dystrophin proteins produced by pDysE were directed to the subsarcolemmal compartment in the muscle fibers where fluorescence activity was dominant. Moreover, a similar observation was also made for pMdysE whereas the plasmid containing only EGFP expressed a cytoplasmic activity with great fluorescence intensity (results not shown). The percentages of fibers transduced by pMdysE or pDysE ranged from 5 to almost 30% 5 days after electroporation. These results suggest that at least some part of the biological activity of each component of the fusion proteins is preserved in the present constructs. The EGFP part is responsible for the fluorescence activity and the dystrophin part targets the whole protein to the relevant cellular compartment.
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Finally, this is the first demonstration of the ability to evaluate directly by fluorescence the expression level of dystrophin following transfection (in vitro) and electroporation (in vivo). It is known that the main target cells for ex vivo gene therapy in DMD are myoblasts which are the vehicles able to bring the dystrophin gene to muscle fibers lacking this protein (Mendell et al., 1995
; Partridge and Davies, 1995). The current constructs thus enable us to test different transfection methods such as lipofection or adenofection to deliver efficiently EGFP–dystrophin constructs to DMD myoblasts in primary culture (Campeau et al., 2000). Moreover, recombinant adenovirus vectors which are promising for delivering dystrophin gene (ex or in vivo) (Floyd et al., 1998; Moisset et al, 1998) could contain EGFP–mini- or full-length dystrophin cassettes with muscle-specific promoters, therefore improving the specificity of dystrophin expression. Similar applications can be made for retroviral vectors with EGFP human dystrophin mini-gene. Many hurdles remain in the application of gene therapy as a treatment of DMD, but rapid methods to test the function of constructs to deliver dystrophin ex or in vivo are necessary to establish the goal of effective gene therapy for DMD. The present study must be considered as the first step in setting up a strategy to permit the dystrophin gene to enter the myoblasts with a view to transplanting them in dystrophic muscles using mdx mice as a model. Moreover, viral vectors such as adenovirus-adeno-associated virus or Herpes amplicon can also be used to transfer in or ex vivo the cassette EGFP–dystrophin. The current constructs will certainly trigger an immune response following in vivo gene therapy since EGFP, the corrective gene or other elements in the vector are immunogenic. For this reason, the constructs used for this study should be modified once the best transfection method has been established. We have nevertheless established an important construct to improve the transfection and detection of the recombinant dystrophin.
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Notes |
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2 To whom correspondence should be addressed.E-mail: jacques-p.tremblay{at}crchul.ulaval.ca
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Acknowledgments |
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This work was supported by the Association Franciaise contre les Myopathies (AFM) and the Muscular Dystrophy Association (MDA).The authors thank Franciois Tardif for his technical assistance with the preparation of the figures. Pierre-Alain Moisset was supported by an FRSQ–FCAR studentship.
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Received March 25, 2000; revised July 1, 2000; accepted July 14, 2000.
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