Patent Application: US-72361007-A

Abstract:
a baculovirus - based expression system under control of cr 5 promoter improves expression of transgenic nucleic acid molecules in mammalian cells . it also provides a platform for regulated expression of transgenic nucleic acid molecules in mammalian cells .

Description:
cell culture : cho cells and the stable cho cell lines ( cho - cymr - rcta / 10 - 35 and cho - rcta / 227 ) were cultured in cd - cho medium ( gibco , grand island , n . y .) supplemented with 4 mm l - glutamine ( gibco ), 1 × ht supplement ( 0 . 1 mm sodium hypoxanthine and 0 . 016 mm thymidine ) ( gibco ), and 1 × dextran . the cells were maintained in a humidified incubator at 37 ° c . with 5 % co 2 . sf9 cells ( spodoptera frugiperda ) were maintained at 27 ° c . in shaker flasks , agitated at 110 - 120 rpm . sf9 cells were grown in sf900 - ii medium ( gibco , invitrogen life technologies , carlsbad , calif .). all fine chemicals were from sigma aldrich , st . louis , mo . plasmid construction : to construct transfer vectors for generating recombinant baculovirus with a mammalian promoter , the baculovirus polyhedrin ( polh ) promoter in pvl - 1393 was replaced with cr5 promoter ( pvl - 3 - cr5 - gfp , pvl - 3 - cr5 - egfr and pvl - 3 - cr5 - rep ) or cmv5 promoter ( pvl - 3 - cmv5 - gfp , pvl - 3 - cmv5 - rcta ). pvl - 3 - cr5 - gfp , pvl - 3 - cr5 - egfr and pvl - 3 - cr5 - rep were constructed in two steps . first , plasmid padcr5 - gfp ( mullick et al , 2006 ) was digested by restriction enzyme aflii , filled in by klenow fragments , and digested again by bamhi to obtain the dna fragment containing the cr5 promoter . meanwhile , the baculovirus plasmid pvl - 1393 bearing the baculovirus polyhedrin ( polh ) promoter was digested by ecorv and bamhi to remove the polh promoter . the cr5 promoter was then subcloned into the digested vector pvl - 1393 to form the desired bacmam plasmid pvl - 3 - cr5 . the second step was to insert the coding sequences for gfp , egfr and rep downstream of cr5 in the plasmid pvl - 3 - cr5 . the gfp cdna fragment , flanked by bamhi and noti restriction sites , was obtained by pcr amplification from plasmid padcr5 - gfp . the egfr cdna was a kind gift from dr . maria jaramillo . the rep cdna fragment was released from paav - rc ( stratagene ), and then subcloned into a bglii - digested pvl - 3 - cr5 plasmid . construction of bacmam plasmids pvl - 3 - cmv5 - gfp and pvl - 3 - cmv5 - rcta was also carried out in two stages , first to get an empty vector and second to subclone the desired genes into it . in order to obtain a baculovirus vector without any promoter , the plasmid pvl - 1393 was cut by bamhi , filled in by klenow fragments and cut again by ecorv to remove the virus polyhedrin promoter . the resultant vector with two blunt ends was then self - ligated and re - digested by either bglii ( pvl - 3 - cmv5 - gfp ) or smai for the next subcloning . a cdna fragment containing cmv5 - gfp sequences was released from padcmv5 - gfp ( massie et al , 1998 ) by bglii digestion and cloned into bglii - digested vector to generate pvl - 3 - cmv5 - gfp . similarly , a cdna fragment containing cmv5 - rcta sequences was released from pad3 - 4c ( xu et al , 2004 ), by digestion with aflii and pmei and cloned into a smai - digested vector to generate pvl - 3 - cmv5 - rcta . transfer vector pvl - b43 under the control of the cr5 promoter coding for an antibody igg was created by cloning the entire sequence of b43 ( the heavy and light chains ) into the baculovirus . baculovirus construction : recombinant baculoviruses were generated in sf - 9 insect cells using homologous recombination with the linearized baculovirus genome baculogold ( pharmingen ). approximately 10 6 cells were plated onto each well of a 6 - well plate . the transfection mixture comprised 1 μg of the transfer vector dna ( pvlcmv5 - gfp , pvl - cr5 - gfp , pcmv5 - rcta , pvl - cr5 - egfr , pvl - cr5 - rep or pvl - cr5 - b43 ), 0 . 2 μg of the baculovirus dna and the transfection reagent cellfectin ™ ( invitrogen life technologies , carlsbad , calif .) in ipl - 41 basal medium . this was incubated for 45 minutes prior to addition to the cells . the transfection mixture was allowed to incubate with the cells for 5 h at 27 ° c ., followed by replacement of the mixture with fresh medium ( sf900 ii ). these cultures were further incubated at 27 ° c ., supernatants were collected at 96 h post transfection , centrifuged ( 4 ° c ., 1000 g , 10 min ), filtered through a 0 . 22 μm and stored at 4 ° c . these were designated as passage one virus stock . larger volumes of virus stock were made by amplification in shake flask cultures of sf9 cells . briefly this was done as follows . sf9 cells were grown to mid - exponential phase and diluted to 2 − 2 . 5 × 10 6 cells / ml with fresh medium . the cultures were infected with passage one virus stock at a moi of 0 . 1 - 1 . the total and viable cell densities and cell size were measured ( using the automated trypan blue exclusion method , cedex , innovatis , bielfeld , germany ) during the infection process and the supernatant was harvested by centrifugation when viability was less the 80 - 90 %. baculovirus quantification : baculovirus stocks were quantified using a dna staining method previously described by shen et . al , 2002 . briefly , the virus particles were permealized and the viral dna was labeled with the sybr green fluorescent dye ( molecular probes , eugene , oreg .). the viral particles were estimated by counting the labeled dna using a facs ( epics xl - mcl , beckman coulter , miami , fla .) at 550 long pass dichroic and a 525 nm band pass filter . calibration was performed using standard fluorospheres . transduction of cho cells with baculovirus : cho , chocymr - rcta / 10 - 35 and chorcta / 227 cells were grown to 2 × 10 6 cells / ml . at this point , the growth was arrested by decreasing the temperature from 37 ° c . to 30 ° c . the cells were maintained at this temperature for 10 - 16 hr . transduction was initiated by the addition of the baculovirus at the desired moi . sixteen hours later the cells were induced with 40 μg / ml of cumate . analytical methods : total and viable cell densities and cell size were measured using the automated trypan blue exclusion method ( cedex , innovatis , bielfield , germany ). concentrations of glucose , glutamine , lactate and ammonia were measured using the bioanalytical system ( ysi 7100 mbs , yellow springs , ohio ). the proteins produced ( rep and egfr ) were analyzed by sds - page and western blot methods . western blot analysis : cell pellets were extracted in lysis buffer ( 50 mm hepes ph 7 . 4 , 150 mm nacl , 1 % thesit and 0 . 5 % deoxycholate ). following the addition of the lysis buffer , the samples were incubated on ice and periodically vortexed over a 30 minute period . the soluble fraction was collected by centrifugation ( 4 ° c ., 10 - minute , 10000 × g ) and was mixed with sample buffer containing dtt and was heated at 95 ° c . for 5 minutes . samples were run in mops buffer on a 4 - 12 % nu - page gradient gel ( invitrogen ). protein was transferred onto nitrocellulose for 1 hr period . the blots were probed with rabbit anti - human egfr ( santacruz biotechnologies , santa cruz , calif .) or anti - phosphotyrosine 4g - 10 ( upstate , charlottesville , va . usa ) to detect egfr expression and its autophosphorylation activity . antibody binding was detected using the chemiluminescence kit ( boehringer mannheim , mannheim , germany ) and visualized with the kodak imager system . facs - based assay for cell surface egfr expression : 1 . 5 × 10 6 cells per well ( in 6 - well plates ) of chocymr - rcta / 10 - 35 and cho - rcta / 227 cells were infected by baculovirus pvl - cr5 - egfr at an moi of 100 and incubated at 30 ° c . in 5 % co 2 for 5 hours . expression of egfr was induced by the addition of 30 μg / ml cumate . after 48 h the cells were harvested and the cell pellet was re - suspended in 100 μl of 1 × pbs containing 1 μg of primary antibody against egfr ( egfr mab , clone 225 , purified from hybridoma ( atcc )) and incubated for 60 minutes at room temperature . the cells were then washed three times and re - suspended in pbs to obtain a final volume of 100 μl . 20 μl of a secondary antibody ( goat anti - mouse igg alexa - green ) was added to each tube . after 30 minutes of incubation at 4 ° c ., the stained cells were washed with pbs , re - suspended in 500 μl of 1 % paraformaldehyde , and analyzed by facs for egfr expression . pvl - cmv5 - gfp and pvl - cr5 - gfp baculovirus vectors were constructed and recombinant baculoviruses were generated and amplified using the sf - 9 insect cells . transient gene expression using the baculoviruses was examined in chinese hamster ovary ( cho ) cell lines that stably express the cumate - modulated transactivator , rcta . these cell lines are the chocymr - rcta / 10 - 35 cell line ( xu et al , 2004 ) in which expression is induced by cumate addition , and the cho - rcta / 227 cell line in which rcta expression is constitutively high resulting in cumate - independent reporter gene expression . both cell lines are adapted for growth in suspension , in serum free medium . in addition to the reporter gene gfp ( for green fluorescent protein ), the system was used successfully to express significant amounts of a kinase - competent human epidermal growth factor receptor ( egfr ), and a rep aav protein that is toxic to most mammalian cells . the work described herein with baculovirus ( bacmam ) containing the cr5 promoter is aimed at enhancing baculovirus - mediated transient transgene expression in cho cells . high level of protein expression obtained with the cr5 promoter offers the benefits of using the baculovirus at lower mois and thus offers a significant improvement for large scale protein production in cho cells . as opposed to protein production , where one cell type can be used to produce several proteins , for functional analysis it is important to use a relevant cell type for functional analysis of each protein . making stable cell lines expressing the transactivator in each cell type of interest is feasible , but too cumbersome to be effective . it is therefore desirable to deliver both elements of the regulatory expression system , the reporter cr5 - gfp and the activator rcta , by baculovirus - mediated transduction . a baculovirus expressing rcta was therefore generated under the control of a strong constitutive promoter , cmv5 ( pvl - cmv5 - rcta ). strong expression and tight regulation in cho cell lines using co - infection with the two baculoviruses was found . the results herein show that the baculovirus expression system of the present invention , wherein transgene expression is regulated by a cumate - switch , offers a new and powerful tool for the expression of genes in mammalian cells . given the strong activity of the cr5 promoter , lower mois can be used , thus decreasing the demand for very high titer stocks . protein expression in mammalian cells may therefore be facilitated , be it for high - level production or functional analysis of the protein in question . the cho cell line chocymr - rcta / 10 - 35 was transduced with bacmam - cmv5 - gfp and bacmam - cr5 - gfp baculoviruses . fig1 shows the facs data for gfp expression , with fig1 a showing the profile of the transduced cell population with respect to gfp expression and fig1 b quantifying the results . as expected , low gfp expression was observed in the cells transduced with bacmam - cr5 - gfp in the absence of cumate ( fig1 a : gray line ; fig1 b : fluorescence index ( f . i . )=% of gfp positive cells × mean fluorescence intensity = 212 . 5 ). of note , however , is that this level ( f . i .= 212 . 5 ) is comparable to the fluorescence observed with cmv5 - gfp ( fig1 a : gray line ; fig1 b : f . i .= 244 ). moreover , upon addition of cumate , gfp expression from bacmam - cr5 - gfp increased 40 - fold ( fig1 a : black line ; fig1 b : f . i .= 9037 ). treatment with butyrate further increased gfp expression 2 . 5 - fold ( fig1 a : filled area ; fig1 b : f . i .= 24 , 000 ). expression from the cmv5 promoter was not significantly affected by cumate ( fig1 a : solid line ; fig1 b : f . i .= 298 ), but was enhanced 3 - fold by butyrate treatment ( fig1 a : filled area ; fig1 b : f . i .= 780 ). however , even the butyrate - activated cmv5 promoter is 10 - fold lower than the cumate - activated cr5 promoter and 25 - fold lower than the cumate and butyrate - activated cr5 promoter . this demonstrates a significant and unexpected improvement in transgene expression using the bacmam expression system with the cr5 instead of with the cmv5 promoter . the single stranded dna virus aav uses four proteins for its replication function . these are rep 78 , 68 , 52 and 40 . in addition to their role in aav replication , rep 78 and rep 52 inhibit transcription from adenoviral or helper virus genomes ( casto et al , 1967 ; timp et al , 2006 ). interestingly they do so by interfering with the function of cellular transcription factors that are required for adenoviral gene expression ( costello et al , 1997 ; weger et al , 1999 ; harmonat et al , 1998 ; su et al , 2000 ; prasad et al , 2003 ). the expression of these rep proteins is therefore not very well tolerated by most mammalian cells , making any study requiring rep expression a daunting task . therefore , the baculo - cumate system of the present invention was applied to develop an efficient rep - expression system . fig2 shows the result of a western analysis of chocymr - rcta / 10 - 35 ( with and without cumate treatment ) and cho - rcta / 227 cells infected with bacmam - cr5 - rep . four major bands of the expected sizes are detected in extracts from chorcta / 10 - 35 and 227 . in addition , cumate induction of rep expression is evident in the chocymr - rcta / 10 - 35 cell line . thus , the expression system of the present invention advantageously permits expression of the heretofore difficult to express avv rep proteins in mammalian cells . the epidermal growth factor receptor ( egfr ) is a 170 - kd transmembrane glycoprotein that mediates a range of biological responses of the epidermal growth factor . it is also being heavily investigated as a therapeutic target for several forms of cancer ( johnston et al , 2006 ). fig3 a shows the alexa - green fluorescence ( i , ii , v and vi ) and phase ( iii , iv , vii and viii ) images of chocymr - rcta / 10 - 35 ( i - iv ) or cho - rcta / 227 - 13 ( v - viii ) cells transduced with bacmam - cr5 - egfr , wherein egfr expression was detected by virtue of binding to an alexa - green - labelled antibody . the expression of egfr is clearly detectable in both cell lines treated with cumate ( ii and vi ), but as expected , expression in chocymr - rcta was significantly lower without the addition of cumate ( compare i and ii ). a facs profile of the transduced population is shown in fig3 b . in the case of chocymr - rcta cells , the entire cell population is shifted from the negative profile ( black line vs . gray line ), however this shift is much more significant on cumate addition ( filled peak ). in the case of cho - rcta cells , transduction with bacmam - cr5 - egfr is sufficient to induce near - maximal expression of egfr ( black line versus filled profile ). to test whether the egfr molecule was kinase - competent , western blot analysis of the cell extracts was carried out using antibodies that detected either the total egfr or the phosphorylated form . extracts of egf - stimulated hela cells served as a control for activated egfr detection . fig3 c shows egfr expression in chorcta / 10 - 35 cells transduced with bacmam - cr5 - egfr in the presence of cumate . kinase activity of the egfr is demonstrated in panel b wherein the membrane has been probed with an antibody specific to the phosphorylated form of egfr . if the application for which a protein is being expressed involves the function of the protein , it is important that it be expressed in a cell type relevant for its function . a number of cell types that serve as models of physiological / pathological states are not amenable to transfection . moreover , making a stable cell line expressing the activator is impractical . therefore , an investigation was conducted to determine whether co - transduction with two baculoviruses , one expressing the activator and the other a reporter , could be a viable strategy to deliver the gene of interest in a relevant cell type . fig4 compares gfp fluorescence in cho cells co - transduced with bacmam - cr5 - gfp ( moi = 100 ) and bacmam - cmv5 - rcta ( moi = 100 ) with those in chocymr - rcta cells transduced with bacmam - cr5 - gfp ( moi = 100 ). although both scenarios result in gfp expression in the majority of cells ( 88 % gfp positive for chocymr - rcta and 81 . 8 % in the case of cho ), the intensity of expression is at least 3 - fold ( mean fluorescence intensity of 848 versus 268 ) higher in the bacmam - cr5 - gfp and bacmam - cmv5 - rcta co - transduction , than in the chocymr - rcta wherein rcta is stably expressed in the host cell line . the most probable explanation for this difference is that the level of rcta in chocymr - rcta is limiting . the results for the expression of the antibody b43 after transduction with the bacmam - cr5 - b43 are shown in fig5 . the transduction was tested on three clones of the inducible cell line chocymr - rcta / 10 - 35 , both heavy and light chains could be detected by probing with the anti - human igg antibody . the clone # 19 showed higher level of secretion . in addition the results with this clone show that in the absence of the inducer ( cumate ) the promoter activity was not detectable for the light chain but was clearly expressed up on addition of cumate . promoter activity was detected for the heavy chain in both the presence and absence of cumate . the disclosures in the references of the following list are herein incorporated by reference . boyce , f . m . and bucher , n . l . r ., 1996 . baculovirus - mediated gene transfer into mammalian cells . proc . natl . acad . sci . usa 93 : 2348 - 2352 . boyce , f . m ., 1998 . u . s . pat . no . 5 , 731 , 182 , issued mar . 24 , 1998 . casto , b . c ., atchison , r . w ., and hammon , w . m ., 1967 . studies on the relationship between adeno - associated virus type 1 ( aav1 ) and adenoviruses . replication of aav in certain cell cultures and its effect on helper adenoviruses . virology 32 : 52 - 59 . condreay , p . j ., witherspoon , s . m ., clay , w . c and kost , t . a ., 1999 . transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector . proc . natl . acad . sci . usa 96 : 127 - 132 . costello , e ., saudan , p ., winocour , e ., pizer , l ., and beard , p ., 1997 . high mobility group chromosomal protein 1 binds to the adeno - associated virus replication protein ( rep ) and promotes rep - mediated site - specific cleavage of dna , atpase activity and transcriptional repression . embo j . 16 : 5943 - 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mony , i ., lamoureux , l ., couture , f ., paquet , l ., guilbault , c ., dionne , j ., chahla , d . et al , 1998 . new adenovirus vectors for protein production and gene transfer . cytotechnology 28 : 53 - 64 . prasad , c . k ., meyers , c ., zhan , d . j ., you , h ., chiriva - internati , m ., mehta , j . l ., liu , y ., and hermonat , p . l ., 2003 . the adeno - associated virus major regulatory protein rep78 - c - jun - dna motif complex modulates ap - 1 activity . virology 314 : 423 - 431 . ramos , l ., kopec , l . a ., sweitzer , s . m ., formwald , j . a ., zhao , h ., mcallister , p ., mcnulty , d . e ., trill , j . j . and kane , j . f ., 2002 . rapid expression of recombinant proteins in modified cho cells using the baculovirus system . cytotechnology 38 : 37 - 41b . shen , c . f ., meghrous , j ., kamen , a ., 2002 . quantitation of baculovirus particles by flow cytometry . journal of virological methods 105 : 321 - 330 . smith , g . e ., summers , m . d ., fraser , m . j ., 1983 . production of human beta interferon in insect cells infected with a baculovirus expression vector . mol . cell . biol . 3 : 2156 - 65 . su , p . f ., chiang , s . y ., wu , c . w ., and wu , f . y ., 2000 . adeno - associated virus major rep 78 protein disrupts binding of tata - binding protein to the p97 promoter of human papillomavirus type 16 . j . virol . 74 : 2459 - 2465 . timpe , j . m , verrill k . c , trempe , j . p ., 2006 . effects of adeno - associated virus on adenovirus replication and gene expression during coinfection . j . virol . 80 : 7807 - 15 . weger , s ., wendland , m ., kleinschmidt , j . a ., and heilbronn , r ., 1999 . the adeno - associated virus type 2 regulatory proteins rep 78 and rep 68 interact with the transcriptional coactivator pc4 . j . virol . 73 : 260 - 269 . other advantages that are inherent to the structure are obvious to one skilled in the art . the embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed . variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims .