High expression of a B-domain deleted factor VIII gene in a human hepatic cell line

Journal of Biotechnology61(1998)165–173High expression of a B-domain deleted factor VIII gene in ahuman hepatic cell lineSabine E.Herlitschka,Uwe Schlokat,Falko G.Falkner*,Friedrich Dorner Hyland-IMMUNO AG,Biomedical Research Center,Uferstraße15,A-2304Orth,Donau,AustriaReceived27January1997;received in revised form14January1998;accepted16January1998AbstractThe expression of a modified human coagulation factor VIII cDNA in a human liver-derived cell line is described.A B-domain deleted FVIII(rVIIIdB928)cDNA controlled by a strong viral promoter/enhancer was linked to a dominant selection-/amplification marker and transfected into the human hepatic cell line SK-HEP-1.By means of this system,up to3.5U rFVIIIdB928/106cells×24h could be detected immediately after selection without gene amplification.This level is orders of magnitude higher than that obtained in Chinese hamster ovary(CHO)cells under the same conditions.Efficient expression of rFVIIIdB928in SK-HEP-1cells was temperature dependent,a4-fold higher level of activity was achieved in culture supernatants at decreased incubation temperatures of28o C.This system allows the production of high amounts of recombinant rFVIIIdB928without time and labour consuming gene amplification procedures.©1998Elsevier Science B.V.All rights reserved.Keywords:Recombinant factor VIII;SK-HEP-1hepatic cells;New vector host system1.IntroductionBlood clotting factor VIII(FVIII)is an essen-tial co-factor for factor IXa dependent activation of factor X.FVIII can be activated proteolytically by a variety of coagulation proteins,including thrombin and factor Xa.The plasma concentra-tion of FVIII is low,approximately100ng ml−1(Hoyer,1987).Analysis of the cloned cDNA for human FVIII(Gitschier et al.,1984;Toole et al., 1984;Vehar et al.,1984;Wood et al.,1984;Truett et al.,1985)has provided more information about its gene and protein structure.The full length FVIII cDNA(FVIIIfl)contains an open reading frame coding for a polypeptide of2351amino acids.This primary translation product is orga-nized in structural domains that occur in the order A1-A2-B-A3-C1-C2(Toole et al.,1984). The FVIII single polypeptide chain has a molecu-*Corresponding author.Tel.:+431201004232;fax:+43 122122716;e-mail:falknef@0168-1656/98/$19.00©1998Elsevier Science B.V.All rights reserved. PII S0168-1656(98)00035-2S.E.Herlitschka et al./Journal of Biotechnology61(1998)165–173 166lar mass of approximately300kDa and is pro-cessed into an amino terminal heavy chain of 90–210kDa and a carboxy terminal light chain of 80kDa,which form a heterodimer linked by divalent metal ions(Hoyer,1987).The B-domain, located on the heavy chain of FVIII,is encoded by a single large exon and is highly glycosylated, harbouring19of the25N-linked glycosylation sites.The B-domain is released upon co-factor activation;its functional significance,however,is still unknown.The liver is most likely one of the major sites of FVIII biosynthesis(Wion et al., 1985;Lewis et al.,1985;Hellman et al.,1989). Moreover,the hepatic cell line HepG2naturally expresses small amounts of FVIII(Ingerslev et al., 1988).Deficiency or abnormalities in the FVIII gene lead to the X-chromosomally linked,reces-sive bleeding disorder haemophilia A(Antonar-akis et al.,1987),a condition that affects10–20 per100000males.Generally,plasma-derived FVIII products have been used for the treatment of haemophilia A. Advances in molecular biology during the last decade have opened a new way towards an alter-native supply of FVIII.Cloned FVIII cDNA expressed in various eukaryotic expression sys-tems have allowed the production of recombinant FVIII(Kaufman,1989).However,these ap-proaches demonstrated that FVIII is not effi-ciently expressed in eukaryotic cells,unless the FVIII cDNA is amplified.Removal of the B-do-main resulted in improved expression levels (Eaton et al.,1986a,b;Toole et al.,1986;Pittman et al.,1993;Mertens et al.,1993).However,high level FVIII expressing CHO cell clones were ob-tained only after extensive amplification via methotrexate(MTX)and co-expression of von Willebrand factor(Kaufman et al.,1989).In the present study,we describe the expression of the B-domain deleted FVIII variant FVIII-del(741–1668),also called FVIIIdB928.Within this cDNA,the entire B-domain was deleted by fusion of the codon for Arg740with that of Ser1669. (Leyte et al.,1991).This FVIIIdB928fusion protein was expressed at low levels in C127cells (Mertens et al.,1993;Donath et al.,in press). Here,we show that the human hepatic cell line SK-HEP-1is an excellent cell line to obtain high levels of FVIII expression without gene amplifica-tion at decreased temperatures.In addition,the recombinant factor VIII obtained in this system has been characterized.2.Materials and methods2.1.ChemicalsOligonucleotides were synthesized with an auto-mated DNA synthesizer(model394,Applied Biosystems,USA).Culture media and antibiotics were purchased from Gibco(Breda,Netherlands) and Seromed(Berlin,Germany).Restriction en-zymes and DNA-modifying enzymes were pur-chased from New England Biolabs(USA)and Boehringer Mannheim(Germany).2.2.Cell lines and plasmidsCell lines were obtained from the American Type Culture Collection.The plasmids p11k-ATA18-FVIII and p11k-ATA18-FVIIIdB928and the anti FVIII antibodies CLB Cag9,Cag A and Cag69were provided by Dr J.A.van Mourik from the department of Blood Coagulation,Cen-tral Laboratory of the Netherlands Red Cross Blood Transfusion Service,Amsterdam, Netherlands.2.3.Plasmid constructions2.3.1.pCMVFVIIIdB928def/EDHProThe FVIIIdB928def cDNA,derived from the plasmid p11k-ATA18-FVIIIdB928,contains a deletion of the B-domain between the amino acids 741–1668(Leyte et al.,1991).The cDNA was isolated as Sal I-Hin dIII fragment,the cohesive ends werefilled in with DNA polymerase I (Klenow fragment)and ligated with the Sma I digested plasmid pCMV/EDHPro(Herlitschka, 1994).The resulting plasmid was designated pCMVFVIIIdB928def/EDHPro.2.3.2.pUC18/FVIIIdefIn plasmid pCMVFVIIIdB928def/EDHPro,the FVIII cDNA contains an insertion of six nucleo-S.E.Herlitschka et al./Journal of Biotechnology61(1998)165–173167 Fig.1.Structure of the FVIII expression unit.The transcription unit,containing the human cytomegalovirus(CMV)promotor/en-hancer,the gene of interest(rFVIIIdB928cDNA),an internal ribosomal entry site(EMCV IRES),the selection marker,the SV40 intron and polyadenylation site is shown.The marker is a chimeric construct consisting of the wild-type dihydrofolate reductase cDNA and the hygromycin phosphotransferase gene fused in frame.tides coding for the two additional amino acids alanine and lysine located between position2044 and2045with respect to the mature full length FVIII protein of2332amino acids.In order to restore the original FVIII sequence,the insertion was removed via a multi-step process taking ad-vantage of the Apa I site adjacent to the insertion. First,the Bam HI-Xba I FVIIIdB928def fragment from plasmid pB2/FVIIIdB928(Herlitschka, 1994)was subcloned into Bam HI-Xba I digested pUC18,generating the plasmid pUC18/FVIIIdef.2.3.3.pUC18/rFVIIIpUC18/FVIIIdef was digested with Esp I and Apa I,the300bp fragment containing the inser-tion was removed and replaced by a new Esp I-Apa I fragment,resulting in plasmid pUC18/rFVIII.This new Esp I-Apa I fragment was generated by PCR using plasmid pUC18/ FVIIIdef as template and primers c2443,5%GAT GGT ATC TGC TCA GCA TGG GCA ATG-3% and c2346,5%-GGC CAG CTT TGG GGC CCA CTG TCC ATA TTG TCC TGA AGC TGT A-3%,which anneal at the Esp I and the Apa I site,respectively.As primer c2346has the origi-nal FVIII sequence,the six base pair insertion was thus eliminated.Sequencing of the FVIII Esp I-Apa I fragment confirmed the correct sequence at position2044of the complete FVIII open reading frame.2.3.4.pB2/rFVIIdB928Plasmid pUC18/rFVIII was digested with Bam HI and Xba I and the isolated FVIII frag-ment was reinserted into pB2/FVIIIdB928,also treated with Bam HI and Xba I,resulting in plas-mid pB2/rFVIIIdB928.2.3.5.pCMVrFVIIIdB928/EDHProThe repaired FVIIIdB928Bgl II-Sfu I fragment was exchanged for the FVIIIdB928Bgl II-Sfu I fragment containing the two codon insertion in plasmid pCMVFVIIIdB928def/EDHPro,generat-ing the expression plasmid pCMVrFVIIIdB928/ EDHPro.The schematic structure of the transcription unit is shown in Fig.1.For transfor-mation of the plasmids,the bacterial strain E.coli HB101was used.2.4.Cell cultureSK-HEP-1(ATCC HTB52),293(ATCC CRL 1573)and Chinese hamster ovary(CHO)cells, deficient in dihydrofolate reductase activity (Urlaub and Chasin,1980)were grown in basal medium supplemented with10%fetal calf serum (FCS).Basal medium contains DMEM:HAMs F12(1:1)including penicillin(100U ml−1),strep-tomycin(100v g ml−1)and L-glutamine(2mM). The medium for CHO cells was additionally sup-plemented with10v g ml−1adenosine,thymidine and deoxyadenosine.Expression plasmid pCMVrFVIIIdB928/EDH-Pro was introduced into the cells by using the calcium-phosphate method(Graham and van der Eb,1973)or by electroporation(Neumann et al., 1982).A total of48h post-transfection the cells were subcultured1:20in hygromycin B selective medium(basal medium including200v g hy-gromycin B).Transfected CHO cells were subcul-tured in dhfr selective medium(basal medium without thymidine,glycine and hypoxanthine sup-plemented with10%dialyzed fetal calf serum). After2–3weeks,the resulting colonies were iso-lated,grown to confluence and tested for FVIIIS.E.Herlitschka et al./Journal of Biotechnology61(1998)165–173 168expression.Positive clones were further subcloned and again tested for FVIII expression.2.5.Quantification of FVIIIFor analysis of FVIII procoagulant activity,log phase cells were rinsed and fed with serum free DMEM:HAMs F12containing2.5v g recombi-nant von Willebrand factor(vWF).The cells were incubated for24h at28°C.Conditioned medium samples were harvested and FVIII activity was determined using a chromogenic assay(COAT-TEST VIII:C/4)that measures the FVIII depen-dent generation of FXa from FX(Chromogenix AB,Sweden).FVIII concentrations were ex-pressed in U ml−1;1U is approximately100ng FVIII.Cell numbers were determined with a cell counter(Technicon HI,Bayer,Germany).2.6.Western blotsThe blots were performed according to Towbin et al.(1979).The anti FVIII antibody CLB Cag9 (Leyte et al.,1989),which recognizes the heavy chain epitope of amino acids713–740,was di-luted1:500,as were the anti-light chain antibodies CLB Cag A and69(Leyte et al.,1991),recogniz-ing the C2domain of FVIII.To detect GRP78, the monoclonal antibody anti-GRP78(Stress Gene)was used in a1:1000dilution and incu-bated for36h.The second antibody was the alkaline phosphatase conjugated anti-mouse anti-body(Biorad),which was used in a1:7500dilu-tion for4h.2.7.Thrombin treatmentPlasma-derived FVIII(Monoclate,Armour, USA),recombinant full length FVIII(CHO,Hy-land/Baxter;BHK,Bayer/Cutter)and recombi-nant SK-HEP-1cell derived B-domain deleted FVIII were used at a concentration of25U ml−1 FVIII.Thrombin treatment was performed with 0.25U ml−1thrombin(Sigma,USA)per unit FVIII.Samples were incubated with phospholipid and50mM CaCl2at37°C for5min,followed by the addition of thrombin.After2,5,15,25,35 and60min of incubation the reactions were stopped with SDS-gel loading buffer.3.Results3.1.Structural features of the genetic constructs The FVIII cDNA lacking the B-domain (FVIII-dB928)was cloned into the expression plasmid pCMV/EDHPro(Herlitschka,1994).As the FVIII-dB928cDNA contained an insertion of six nucleotides not found in any FVIII cDNAs sequenced thus far,the cDNA was repaired using a PCR-based mutation procedure described in Section2.The resulting expression plasmid was designated pCMVrFVIIIdB928/EDHPro.Within this plasmid,expression of the cDNA is driven by the human cytomegalovirus(CMV)immediate early gene promotor and enhancer(Boshart et al., 1985).The plasmid also contains a simian virus40 (SV40)intron and polyadenylation site,an ampi-cillin resistance gene and pUC19sequences.For selection and amplification purposes,a selection-/ amplification marker consisting of the wild-type (wt)dihydrofolate reductase(dhfr)cDNA fused in frame with the hygromycin phosphotransferase (hph)gene was used(Herlitschka,1994).The rFVIIIdB928cDNA was linked with the marker cassette via the encephalomyocarditis virus5%un-translated region(EMCV5%UTR),which acts as an internal ribosome entry site(IRES)to generate dicistronic RNAs(Jang and Wimmer,1990).The schematic structure of the promotor,rFVIII-dB928cDNA and marker,termed EDH,is shown in Fig.1.3.2.Unexpected high le6els of rFVIIIdB928 expression in SK-HEP-1cellsThe construct pCMVrFVIIIdB928/EDHPro, containing the FVIII expression cassette,was transfected into SK-HEP-1,293and CHO cells. Transfection was followed by subculturing SK-HEP-1and293cells in hygromycin B selection medium.CHO cells were propagated in DHFR and hygromyin B selection medium,respectively. Resistant colonies were isolated,grown to conflu-ency,incubated for24h at28o C and tested for FVIII expression(Section2).The results from these transfection experiments are summarized in Table1.Consistent with published data(Kauf-S.E.Herlitschka et al./Journal of Biotechnology61(1998)165–173169man et al.,1988),initially selected CHO cells expressed less than0.1mU of full length FVIII per106cells×24h(Herlitschka,1994).In the case of the B-domain deleted rFVIIIdB928,only 0.1–0.5mU could be detected in CHO cells, indicating that the removal of the B-domain did not dramatically increase the detectable amount of rFVIIIdB928.Further subcloning did not im-prove the rFVIIIdB928expression. Surprisingly,thefirst generation clones of293 and SK-HEP-1cells showed much higher expres-sion levels than the corresponding CHO clones.In the case of293cells,100–200mU rFVIIIdB928 per106cells in24h could be found after selection with hygromyin B.However,SK-HEP-1cells ex-hibited even higher expression levels,up to3.5U rFVIIIdB928per106cells in24h could be de-tected in supernatants offirst generation clones grown at28o C after selection.Non-transfected 293and SK-HEP-1cells did not show any en-dogenous FVIII expression(Table1).Further-more,we investigated the properties of SK-HEP-1 cells with respect to the expression stability;we found stable FVIII expression after three sub-cloning steps under maintenance of selection pres-sure over approximately50cell generations(data not shown).3.3.Temperature dependence of FVIII acti6ity in SK-HEP-1and293cellsBiosynthesis and stability of FVIII in recombi-nant CHO cell lines can be increased at decreased Table2Temperature dependence of FVIII activity in recombinant cell clonesTemperature(°C)Cell line373328293c1963451a,b7807203100SK-HEP-1c19446701500Average expression levels of24h supernatants collected over a period of9days are shown.a,b As Table1.temperatures(Rasmussen and Nordfang,1991); thisfinding could also be confirmed for SK-HEP-1and293cells.The SK-HEP-1clone c1963and the293clone c1944were grown to confluence, the growth medium was substituted with serum-free vWF containing medium(Section2)and cells were grown at three different temperatures over a period of9days.Medium was replaced each24h and factor VIII activity was determined in the24 h supernatants.While the optimal temperature for 293cells was at33o C,SK-HEP-1cells were opti-mally expressing FVIIIdB928at28o C(Table2). The increase in activity levels in SK-HEP-1cell supernatants was about fourfold as compared to levels achieved in37o C supernatants.The78kDa glucose regulated protein GRP78, which belongs to the heat shock protein family (Munro and Pelham,1986),has been shown to transiently associate with nascent secreted proteins such as factor VIII and to influence secretion(Dorner et al.,1990).Induction of FVIII synthesis in CHO cells resulted in a50-fold induc-tion of GRP78expression,preventing efficient FVIII secretion(Dorner et al.,1989).In order to investigate whether this type of interaction is re-sponsible for the different expression levels at high and low temperatures,Western blots to de-tect GRP78in non-transfected and in FVII-IdB928expressing SK-HEP-1cells were performed(Fig.2).GRP78levels were,however, not significantly increased in SK-HEP-1cells at high temperature,neither in wild-type cells(lanes 1and2)nor in FVIII expressing cells(lanes4and 5).Therefore,the temperature dependence cannot be explained by increased levels of GRP78at 37o C.Table1Expression of rFVIIIdB928in CHO,293and SK-HEP-1cells Tissue SpeciesCell line Expression level CHO0.1–0.5a,bOvary Chinese ham-ster100–200Human293KidneySK-HEP-1Liver Human100–3500The range of FVIII activities determined in serumfree culture supernatants obtained at28o C in the presence of vWF of48 single cell clones is shown.a Activities are given in mU per106cells and24h.b Chromogenic activity was determined by the COATTEST VIII:C/4(Chromogenix AB,Sweden).S.E.Herlitschka et al./Journal of Biotechnology61(1998)165–173170parati6e analysis of rFVIIIdB928withfull length rFVIII and plasma-deri6ed FVIIIWestern blot analyses were performed to fur-ther characterize the secreted FVIII from SK-HEP-1cells in comparison to commerciallyavailable FVIII preparations derived from humanplasma,CHO and BHK cells.As SK-HEP-1cellderived rFVIIIdB928is lacking the entire B-do-main,the primary translation product has a theo-retical molecular mass of163kDa,while the fulllength FVIII has a molecular mass of approxi-mately300kDa.Fig.3A displays the reactionwith the monoclonal antibody CLB Cag9,whichrecognizes the heavy chain of FVIII.For samplescontaining the full length FVIII(lanes3–5),themonoclonal antibody reacted with the expectedbands in the range of90–210kDa.In SK-HEP-1cells expressing rFVIIIdB928(lane2),a band ofapproximately170kDa,which represents the pri-mary translation product,was observed.Addi-tional minor bands of approximately120kDawere also visible.The reactions with the anti-lightchain antibodies CLB Cag A and69are shown inFig.3B.Due to the direct fusion of the heavy andlight chains in rFVIIIdB928,a single band of170Fig.3.Western blot analysis of FVIII samples with mono-clonal antibodies directed against the heavy and the lightne1,negative control consisting of supernatantsfrom non-transfected SK-HEP-1cells incubated in the pres-ence of von Willebrand factor(SK-HEP-1n.c.+vWF).Lane2,FVIIIdB928derived from SK-HEP-1cell clone c6807(rFVIIIdB-SK);lane3,recombinant full length FVIII derivedfrom BHK cells(rFVIIIfl-BHK);lane4,recombinant fulllength FVIII derived from CHO cells(rFVIIIfl-CHO);lane5,plasma-derived FVIII(FVIIIfl-pd).The blot in panel A wasprobed with the heavy chain(h.c.)antibody CLB Cag9.Inpanel B the light chain(l.c.)antibodies CLB Cag A and69,recognizing the C2domain of FVIII,were used.The numbersat the left indicate the molecular weight in kDa.Fig.2.Western blot analysis of GRP78levels in SK-HEP-1 cells at37and28o C.Total cellular proteins obtained from cells grown at37o C and28o C were separated on12%poly-acrylamide gels and probed with a monoclonal antibody di-rected against the human nes1and2,wild-type SK-HEP-1cells(SK-HEP-1wt)at37o C and28o C,respec-tively;lane3,size marker(M);lanes4and5,SK-HEP-1cell clone c7611at37o C and28o C,respectively.kDa appeared(lane2).In samples containing the full length FVIII(lanes3–5),and80kDa doublet was observed(Donath et al.,in press).Quantifications of the SK-HEP-1cell derived and vWF-stabilized culture supernatants by a FVIII specific ELISA(Immunozym FVIII:Ag, Hyland-IMMUNO)confirmed the presence of about equal amounts of antigen and activity. Without addition of vWF to the cell cultureS.E.Herlitschka et al./Journal of Biotechnology61(1998)165–173171medium,the antigen levels were about fourfold higher,confirming instability of the secreted FVIII.In purified FVIII preparations activity ver-sus antigens ratios of50%were achieved(data not shown).Thus,partially activated factor VIII does not contribute to the observed activity levels in SK-HEP-1cell culture supernatants.parati6e analysis of FVIII preparations after thrombin acti6ationFVIII can be proteotypically activated by sev-eral proteins of the coagulation cascade,including factor Xa and thrombin.FVIII has thrombin recognition sites at amino acid positions336,372, 740and1689.Specific cleavages under physiologi-cal conditions generate factor FVIII polypeptides of73,50and43kDa,respectively(Eaton et al., 1986a,b).To further characterize the SK-HEP-1 produced rFVIIIdB928,the thrombin cleavage patterns of different recombinant and plasma-derived FVIII preparations were analyzed by western blotting.As shown in Fig.4A,the proteins generated after60min of thrombin cleav-age of SK-HEP-1produced rFVIIIdB928(lane3) co-migrated on SDS-PAGE with those from BHK (lane1),CHO(lane2)and plasma-derived FVIII (lane4).The single band of approximately43 kDa resulted from the binding specificity of the CLB Cag9antibody,which recognizes the heavy chain.Incubation of thrombin treated FVIII sam-ples with the anti-light chain antibodies CLB Cag A and69displayed the73kDA activation product of the light chain(Fig.4B).Again,identi-cal bands were detected with recombinant full length FVIII(lanes1and2),B-domain deleted SK-HEP-1derived FVIII(lane3)and plasma-derived FVIII(lane4).This experiment indicated that thrombin treatment resulted in cleavage products of identical size in the SK-HEP-1cell derived material and the reference FVIII samples.4.DiscussionOne of the few publications describing the ex-pression of FVIII in liver cell lines(Ingerslev et al.,1988),deals with the endogenously expressed FVIII in HepG2cells.In this case,expression levels were found to be around30mU FVIII per approximately106cells in24h.For the expres-sion of human blood coagulation factor VIII by means of recombinant DNA technology,several expression systems have been employed,including viral systems(Pavirani et al.,1987;Webb et al., 1993)and permanent mammalian cell lines(Kauf-man,1989).In cell lines,expression levels of non amplified clones were generally in the low mU range;FVIII constructs lacking the B-domain, however,produced10–20-fold higher levels than full length FVIII constructs(Kaufman,1991; Fig. 4.Western blot analysis of thrombin cleaved FVIII samples.The FVIII samples were treated with thrombin and probed with monoclonal antibodies to detect the activation products.In panel A the heavy chain of FVIII was detected (arrow)with the antibody CLB Cag9.In panel B the light chain of FVIII was detected(arrow)with the antibodies CLB Cag A ne1,BHK cell-derived recombinant FVIII, lane2,CHO cell-derived recombinant FVIII;lane3,SK-HEP-1cell-derived recombinant FVIIIdB928;lane4,plasma-derived FVIII.The numbers at the left indicate the molecular weight in kDa;for abbreviations see Fig.3.S.E.Herlitschka et al./Journal of Biotechnology61(1998)165–173 172Pittman et al.,1993),probably due to higher mRNA stability and the improved secretion effi-ciency(Dorner et al.,1987,1990).First generation clones of the human kidney cell line293expressed up to200mU rFVIIIdB928/106cells in24h.With the human hepatic cell line SK-HEP-1as the expression vehicle,expression levels up3.5U per 106in24h could be achieved with a FVIII cDNA lacking the B-domain.Expression of FVIII in initially selected SK-HEP-1cell clones is therefore orders of magnitude more efficient than the ex-pression obtained in corresponding CHO cell clones.High expression of B-domain deleted FVIII variants have been obtained only after ex-tensive amplification procedures(Pittman et al., 1993).As shown here,SK-HEP-1cells express high levels of biologically active FVIII levels at high and low temperatures.Since GRP78levels were not increased in SK-HEP-1cells at37°C,the temperature dependence cannot be explained by increased levels of GRP78at high temperatures.It remains to be seen which role transcription rates and mRNA stability plays for elevated FVIII levels in SK-HEP-1cells.Furthermore,compara-tive analysis of the thrombin cleavage patterns of rFVIIIdB928with commercially available recom-binant and plasma-derived FVIII preparations confirmed identical proteolytic activation pat-terns.In summary,a new system for the expres-sion of recombinant FVIII is presented which allows more straight-forward analyses of struc-ture-function relationships of FVIII and its mu-tants.Moreover,although the use of human cells would currently be precluded for producing a human therapeutic protein,hepatic cells may not only allow more efficient production of human plasma proteins but may also have beneficial ef-fects on the quality of the recombinant proteins because of more authentic post-translational modifications.AcknowledgementsWe thank C.Kuhn for expert technical assis-tance,Dr G.Antoine and colleagues for oligonu-cleotide synthesis and sequencing and Dr C.P. 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