WO1988003558A1 - Eucaryotic expression system - Google Patents

Abstract

Vectors, improved host cells and improved methods for producing a heterologous protein by culturing an improved eucaryotic host cell of this invention transformed or transfected with a vector capable of directing the expression of the heterologous protein. An expression vector containing DNA encoding an anti-sense RNA to GRP78 protein-encoding mRNA is used to increase levels of heterologous proteins.

Description

EUCARYOTIC EXPRESSION SYSTEM

Background of the Invention

This invention relates to improvements in the expression an secretion of heterologous proteins from eucaryotiσ cells.

The information which determines the destiny of a secrete protein is contained in its primary structure, and much of thi information may involve dictating appropriate post-translationa modification and correct conformation. The steps in th exocytotic pathway of the processing and transit of membrane spanning and secretory proteins in mammalian cells have bee described (for reviews see Farquhar, Ann Rev Cell Biol, 1985 ornfeld & Kornf ld, Ann Rev Biochem, 1985) . A large body o work has shown that proteins destined for the cell surface ar first cotranslationally translocated into the lumen of th endoplas ic reticulum (ER) mediated by a signal sequence at o near the a ino terminus of the nascent chain (Blobel Dobberstein, J. Cell Biol, 1975; Walter et al., Cell, 1984) Inside the endoplasmic reticulum the signal sequence is usuall removed and a high mannose oligosaccharide core unit i transferred to asparagine residues located in the sequence Asn-X Ser/Thr where X can be any amino acid, except perhaps proline. This N-linked core glycosylation occurs cotranslationally and i appears that the efficiency of glycosylation is dependent on the presentation of an appropriate conformation of the peptide chain as it enters the endoplasmic reticulum. Potential N-linked glycosylation sites may no longer be accessible after the protein has folded (Kornfeld & Kornfeld) .

Proteins move from the endoplasmic reticulum to the Golgi apparatus where modifications such as sulfation and processing of the high mannose oligosaccharide chain to a complex type occurs and the proteins are directed to their proper destinations (Dunp y & Rothman, Cell, 1985) . The movement from the ER to the

Golgi has been identified as the rate limiting step in intracellular transport (Lodish et al.. Nature, 1983; Fitting &

Kabat, JBC, 1982, and J. Cell Biol, 1985). Few proteins resident in the ER have been extensively studied for their interaction with secretory proteins transiting that compartment.

Environmental stresses such as heat shock induce the synthesis in prokaryotic and eukaryotic cells of a set of highly conserved heat shock proteins. (Schlesinger, J. Cell Biol, 1986). hsp70 is the most abundant of these induced proteins. Proteins related to hsp70 are found in unstressed mammalian cells. There are three main members of the mammalian hsp70-like group of proteins: hsp70, hsc70, and GRP78 (Pelham, Cell, 1986) Following heat shock, synthesis of hsp70 is induced and the protein migrates to the nucleus where it is found in tight association with nucleoli. hsp70 can be released from this association by the addition of ATP in vitro. It has been hypothesized that hsp70 disaggregates heat damaged proteins by an ATP dependent mechanism to facilitate recovery from heat shock (Lewis & Pelham, EMBO J, 1985) . hsc70 is found at high basal levels in growing cells and is only slightly heat inducible (Pelham, Cell, 1986) . hsc70 has recently been identified as "uncoating ATPase", a constitively expressed enzyme that releases clathrin triskelions from coated vesicles in an ATP dependent reaction (Chappell et al. , Cell, 1986, Unge ickell, EMBO J, 1985) .

GRP78 was initially reported to be one of two proteins whose synthesis was induced by glucose starvation in chick fibroblasts (Shiu et al. , PNAS, 1977). Its synthesis can also be induced by inhibitors of N-linked glycosylation such as tunicamycin, glucosamine or 2-deoxygTucose (Olden et al., PNAS, 1979, Pouyssegur et al.- Cell, 1977). GRP78 is not heat inducible and its basal level is high in secreting- cells. Some work on the transcriptional activation of the GRP78 gene has been reported (Lin et al., Mol. Cell Biol., 1986; Resendez et al. , Mol. Ce Biol., 1985; Chang et al. , Proc. Nat ' 1. Acad. Sci., 1987) Recently it has been shown that GRP78 is similar if not identica to immunoglobulin heavy chain binding protein (BiP) (Munro an Pelham, Cell, 1986). GRP78 is therefore also referred t hereinafter as BiP/GRP78 or simply, BiP. BiP was first describe for its association with immunoglobulin heavy chains in pre- cells (Haas and Wable, Nature, 1983) . BiP transiently complexe with immunoglobulin heavy chain in the endoplasmic reticulum o secreting hybridomas. When assembly with light chains occurs Bi dissociates from the complex. In the absence of light chains Bi remains associated with heavy chains and this complex is no transported from the endoplasmic reticulum to the Golgi apparatu (Bole et al. , J. Cell Biol., 1986) . These subcellula fractionation studies showed that BiP is predominantly localize to the endoplasmic reticulum. The heavy chain-BiP complex ca be dissociated in the presence of ATP suggesting a functiona analogy with the hsp70 complex in heat shocked nucleoli. (Munro Pelham, Cell, 1986) .

We believe that BiP/GRP78 may associate in secreting cells wit underglycosylated or improperly folded proteins in th endoplasmic reticulum and help clear them in analogy to the hypo thesized role of hsp70 in the nucleus (Pelham, Cell, 1986) . Suc a function is consistent with the induction of increased level of GRP78 synthesis under conditions which disrupt N-linke glycosylation. Recent studies on abberant proteins which fail t transit out- of the ER have been interpreted to show that Bi binds to them in the ER although the identity of grp78 and BiP was disputed (Gething et al.. Cell, 1986; Sharma et al., EMBO J, 1985) and the extent and degree of such binding was not specif¬ ically characterized. BiP/GRP78 may also associate with partial¬ ly assembled proteins and retain them in the ER until assembly and processing is complete as is the case for the processing of immunoglobulin heavy chain (Bole et al., J Cell Biol, 1986). Independent of the research on BiP mentioned above, we have conducted extensive research on the production of glycoproteins, including Factor VIII, in genetically engineered host cells. In the course of this research we have surprisingly found that a significant proportion of Factor VIII and analogs thereof (also referred to hereinafter, simply as "Factor VIII") and analogs of tPA produced in vitro, e.g. in CHO cells, is not secreted into the cell culture medium. We have now surprisingly found that secretion levels for Factor VIII and other proteins can be decreased by providing higher intracellular levels of BiP and can be increased by reducing the intracellular BiP level.

Summary of the Invention

This invention provides an anti-sense expression vector capable of directing the transcription of mRNA complementary to mRNA encoding- GRP78 protein (BiP) . The anti-sense expression vector thus directs the transcription of "anti-sense" mRNA which is capable of hybridizing to part or all of the endogenous GRP78/BiP-encoding mRNA of a eucaryotic host cell, thereby preventing or decreasing, preferably significantly decreasing, the level of translation of GRP78/BiP mRNA and thus the level of BiP/GRP78 protein in a host cell transformed or transfected with the anti-sense expression vector of this invention. The anti- sense expression Vector comprises a DNA sequence encoding part or all of a GRP78 protein or an expression control sequence thereof, operatively linked in reverse orientation to an expression control sequence permitting transcription of the anti-sense mRNA. The expression control sequence comprises a promoter and optionally an enhancer to which the promoter is responsive, as well as other optional genetic elements, all as are well known in the art. The anti-sense mRNA, and thus the corresponding DNA in the anti-sense expression vector, (i) need not be full-length, i.e. may contain fewer bases or base pairs than the host cell's BiP-encoding mRNA or DNA, and/or (ii) may be mutagenized or otherwise contain a number of substituted bases or base pai for naturally occurring ones, so long as the anti-sense mR hybridizes to a sufficient portion of the host cell's GRP78/B mRNA to prevent or decrease, preferably significantly, the lev of GRP78/BiP mRNA translation. Prevention or reduction o translation can be conveniently measured by methods describ hereinafter. The anti-sense expression vector may also contai one or more a plifiable markers permitting the amplification o gene copy number by conventional techniques, one or mor selectable markers, and other elements heretofore generally know in the art to be useful in expression vectors, as disclosed i greater detail below.

Suitable anti-sense expression vectors, as are described i greater detail herein, may be synthesized by techniques wel known in the art. The components of the vectors such a bacterial replicons, selection genes, amplifiable markers enhancers, promoters, and the like may be obtained from natura sources or synthesized by known procedures. See Kaufman et al. 1982, J.Mol. Biol. ,159;601-621; Kaufman, 1985, Proc. Natl. Acad Sci. 8.: 689-693. The DNA sequence encoding the BiP anti-sens mRNA may be obtained or synthesized as described hereinafter.

This invention further encompasses an improved eucaryotic hos cell for expressing a heterologous protein such as Factor VIII; t-PA; von Willebrand Factor (VWF) ; erythropoietin; lymphokine such as GM-CSF, other CSFs, 11-2, 11-3; etc., or analogs thereof. Factor VIII analogs are described, e.g. i International Applications PCT/US87/01299 and PCT/US87/00033 an in U.S. Serial No. 068,865 (filed July 2, 1987), the contents of which are hereby incorporated by reference. t-PA analogs are described e.g. in U.S. Serial Nos. 861,699; 853,781; 825,104 and 882,051 and in PCT/US87/00257, the contents of which are hereby incorporated by reference. The improved host cell of this invention comprises a host cell transformed or transfected with an anti-sense expression vector of this invention, or the progeny thereof. The improved host cell may be a bacterial, yeast, fungal, plant, insect or mammalian cell or cell line, and is preferably a mammalian cell or cell line.

Established cell lines, including transformed cell lines, are suitable as hosts. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants (including relatively undifferentiated cells such as hematopoetic stem cells) are also suitable. Candidate cells need not be genotypically deficient in a selection gene so long as the selection gene is dominantly acting.

The host cells preferably will be established mammalian cell lines. For stable integration of vector DNA into chromosomal DNA, and for subsequent amplification of the integrated vector DNA, both by conventional methods, CHO (Chinese Hamster Ovary) cells are presently preferred. Alternatively, vector DNA may include all or part of the bovine papilloma virus genome (Lusky et al., 1984, Cell 2_6:391-401) and be carried in cell lines such as C127 mouse cells as a stable episomal element. Other usable mammalian cell lines include HeLa, COS-1 monkey cells, melanoma cell lines such as Bowes cells, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HAK hamster cell lines and the like.

The improved host cell, or the progeny thereof, may further be transformed or transfected with one or more expression vectors capable of directing the expression of the desired protein. This may be accomplished directly, i.e., by transforming or transfecting the host cell or its progeny with an expression vector encoding the desired protein, prior or subsequent to transformation or transfection of the host cell with the anti- sense vector of this invention. Alternatively, this may be accomplished "indirectly", i.e., by fusion of cells transformed or transfected with the anti-sense vector or their progeny wi cells transformed or transfected with the vector encoding t desired protein. Suitable vectors for the expression of a lar number of diverse proteins are known in the art and are eith publicly available or may be synthesized by purely convention techniques. Vectors containing DNA encoding the followin proteins, for example, have been deposited with the American Typ Culture Collection (ATCC) of Rockville, MD: Factor VIII (pSP64 VIII, ATCC No. 39812); a Factor VIII analog,"LA", lacking 58 amino acids (pDGR-2, ATCC No. 53100) ;t-PA and analogs thereo (see e.g. International Application WO 87/04722, published 1 August 1987); VWF (pMT2-VWF, ATCC No. 67122); EPO (pRKl-4, ATC No.39940; pdBPVMMTneo 342-12 (BPV-type vector, ATCC No.37224) and GM-CSF (pCSF-1, ATCC No. 39754) .

An improved method is thus provided for producing a heterologou protein, e.g. Factor VIII, etc. The method comprises culturing eucaryotic host cell transformed with a vector capable o directing the expression of the heterologous protein, or th pregeny thereof, the host cell or its progeny being additionall transformed or transfected with (a) an anti-sense GRP78/Bi vector of this invention; (b) a vector, preferably amplified t multi-copy number, containing a DNA sequence substantially th same as the following heterologous DNA sequence, or a portio thereof so long as it is capable of reducing or preventing Bi induction under conditions which otherwise typically induce Bi expression:

(5 ' )

S a I

CGGGGGCCCA ACGTGAGGGG AGGACCTGGA CGGTTACCGG CGGAAACTGG TTTCCAGGTG

Pvu I AGAGGTCACC CGAGGGACAG GCAGCTGCTC AACCAATAGG ACCAGCTCTC AGGGCGGATG

CGCCTCTCAT TGGCGGTCCG CTAAGAATGA CCAGTAGCCA ATGAGTTCGG CTGGGGGGCG

Rsa I

CGTACCAGTG ACGTGAGTTG CGGAGGAGGC CGCTTCGAAT CGGCAGCGGC CAGCGTTGGT

Stu I GGCATGAACC AACCAGCGGC CTCCAACGAG TAGCGAGTTC ACCAATCGGA GGCCTCCACG

Bssh I ACGGGGCTGC GGGGAGGATA TATAAGCCGA GTCGGCGACC GGCGCGCTCG AATAACCCGG

(3^») (single, coding strand shown alone for simplicity) or a DNA sequence at least about 70% homologous thereto; or (c) both (a) and (b) .

The DNA insert in the vector in (b) is believed to contain a regulatory element required for induction of transcription of the GRP78 gene, perhaps by binding with an endogenous trans-acting transcriptional regulatory factor which is produced by the host cell in response to BiP-inducing conditions. It is believed that the factor normally binds to an endogenous transcription control element linked to the endogenous BiP gene and thereby causes increased BiP mRNA levels under BiP-inducing conditions. The presence of the vector in (b) , preferably amplified to multi¬ copy number, is believed to compete with the endogenous transcription control sequence for binding to the transcription initiation factor, and thus to block the BiP induction pathway.

This method permits the production at increased levels of heterologous proteins using eucaryotic, preferably mammalian, host cells which contain reduced levels of BiP. By "reduced levels of BiP", as the phrase is used herein, we mean levels of BiP below, preferably at least 20% below, more preferably least 50% below, and even more preferably at least 75% below, t level of BiP in CHO cells under BiP-inducing conditions such glucose deprivation. Factor VIII overexpression, treatment wi tunicamycin or A23187, etc. BiP levels may be convenientl measured by standard procedures such as immunological assa using antibodies directed to BiP or to BiP-heterologous protei complex. Alternatively, BiP levels may be measured indirectly b measuring endogenous levels of RNA encoding BiP using standar procedures such as Northern or Southern blotting.

This method is believed to produce higher recoverable yields o heterologous proteins, which may otherwise be intractably boun within the ER, owing to decreased ER levels of BiP resulting fro decreased levels of translation of the host cell's endogenou BiP-encoding mRNA, in the case of (a) ; decreased levels o induction of the host cell's endogenous BiP gene, in the case o (b) ; and decreased levels of BiP induction and translation, i the case of (c) . Other methods for achieving reduced endogenou BiP levels which will occur to those skilled in this art ar expected to be operative to a greater or lesser extent than th specific methods disclosed herein, and should thus be viewed a equivalent methods.

In the practice of this invention stable transformants prepare for use in the improved method or their progeny are screened fo decreased expression of BiP and/or expression of the heterologou protein by standard immunological or enzymatic assays. Th presence of anti-sense mRNA or mRNA encoding the heterologou protein and/or DNA encoding the heterologous protein or a DN sequence present in the vectors of (a) and/or (b) , above, may be detected by standard procedures such as Northern or Southern blotting respectively. Transient expression of the anti-sense vector and/or the DNA encoding the heterologous protein during the several days after introduction of the respective vector(s) into COS-1 monkey cells is measured without selection by activity or immunological assay of the proteins in the culture medium.

Following expression of the DNA encoding the heterologous protein, the protein so produced may be recovered, purified, and/or characterized, all by known methods.

This invention also encompasses a "sense" GRP78/BiP expression vector which comprises a DNA sequence encoding GRP78/BiP operatively linked to an expression control sequence. Construction of an exemplary GRP78/BiP expression vector is described in detail below, but it should be understood that other BiP expression vectors may be readily prepared by purely conventional techniques using a GRP78/BiP-encoding DNA sequence and readily available or synthesizable components, as is true for other exemplary vectors of this invention described hereinafter.

This invention also encompasses a eucaryotic host cell, as described above, but which is transformed or transfected with a GRP78/BiP expression vector. Such an engineered host cell or its progeny may be further transformed with an expression vector capable of directing the transcription of a heterologous protein, also as described above. The resultant cell or its progeny may then be cultured in an improved method for producing the heterologous protein, wherein improperly glycosylated or folded heterologous protein is not secreted into the culture medium, but is instead retained within the host cell by virtue of association with intracellular BiP.

Detailed Description of the Invention

We have examined the role of BiP/GRP78 in the processing and secretion of a variety of proteins, including human factor VIII (FVIII) and analogs thereof, human tissue plasminogen activator (tPA) and analogs thereof and human von Willebrand Factor (vWF) in eucaryotic host cells such as stable Chinese hamster ova (CHO) cell lines. FVIII is synthesized as a single chai precursor of about 250 kd and subsequently processed to a "hea chain" of about 200 kd and a "light chain" doublet of about 8 kd. FVIII has a plethora of potential N-linked glycosylatio sites. Twenty of the twenty-five sites are located within th middle one-third of the molecule which has been defined as the domain. (Toole et al.. Nature, 1984) Deletion of this domai to produce the "LA" variant of FVIII results in higher levels o FVIII secretion (Toole et al., PNAS, 1986). tPA has a molecula weight of about 68,000 kd and contains four potential N-linke glycosylation sites of which three are typically occupied (Poh et al., Biochem, 1984). vWF is a large glycoprotein synthesize as an approximately 260,000 kd precursor which forms di ers i the endoplasmic reticulum and is subsequently processed in Golg and post-Golgi compartments to approximately 100,000 and 220,00 kd forms (Bonthron et al.. Nature, 1986). These processed form undergo interdimer disulfide bond formation to form hig molecular weight multimers (Wagner & Marder, J. Cell Biol. 1984) .

Our results indicate that the occupancy of N-linked glycosylatio sites on a protein plays a role in the extent of BiP association. Underglycosylation of a protein results in increased Bi association and retention inside the cell. This block t secretion may be dependent on expression level. We believe tha BiP/GRP78 plays a major role in the processing and transport o secreted glycoproteins.

Results

Association of FVIII and Deleted Form LA with BiP

In order to qualitatively assess the role of BiP/GRP78 in the secretory pathway we examined a variety of stable CHO cell lines by pulse and chase experiments. The time course of association of FVIII and BiP was analyzed by comparing the amount of FVIII which was detected by immunoprecipitation with a monoclonal antibody specific for BiP against that precipitated with a monoclonal specific for FVIII. Following a 1 hour pulse with ³⁵S ethionine roughly 85% of wild-type (wt) FVIII was detected in a complex with BiP as indicated by the amount of FVIII seen in the anti-BiP immunoprecipitation compared to that precipitated by the anti-FVIII monoclonal. Only the 250kD single chain form was found to be associated with BiP. No processed 80kD form was precipitated by the anti-Bip monoclonal although it is present in the cells at this time. BiP was observed to migrate slightly faster than the 80kD doublet.

At the 4h chase time point processed heavy chain of 200kD and the 80kD light chain doublet can be detected in the conditioned medium. Immunoprecipation of the conditioned medium detected a slight amount of BiP. However there was no associated FVIII observed. Intracellularly the amount of FVIII associated with BiP had decreased to less than 50% as the molecule transits through the cell. At the 2Oh chase time point the ratio of BiP- associated to unassociated FVIII changed. The single chain FVIII had begun to degrade as indicated by a smearing of the 250kD band as analyzed by gel electrophoresis and roughly all of this FVIII which remained in the cell after a long chase was ound complexed with BiP. The amount of BiP had increased in the conditioned medium over this time course but an association with secreted FVIII can not be detected. It is worth noting that through the 2Oh chase time course the amount of GRP78 inside the cells does not significantly change. It is secreted or released from damaged cells at a low rate and appears to be a stable cellular protein with a half life greater than 2Oh.

We then examined the association of LA with BiP in a similar time course. LA is a deleted form of FVIII which has only 7 potential N-linked sites compared to 25 en wt FVIII. At the lh pulse ti point roughly 60% of single chain LA is associated with Bi Single chain LA appears as a doublet of approximately 150 kd. with wt FVIII no 80kd forms are observed to be complexed wi BiP.

During the 4h chase period the association of LA and Bi significantly decreases compared to the earlier time point Single chain, processed heavy chain which migrates as a smea around 90 kD, and 80kD light chain doublet can be detected in th 4h conditioned medium by immunoprecipitation with anti-FVII monoclonal. Also present in the medium is trace unassociate BiP. At the 2 Oh chase time point a small amount of LA remains i the cell and the proportion of LA associated with BiP is slight These experiments indicated that LA exhibits a transien association with BiP inside the cell and, in contrast to w FVIII, is not retained intracellularly in a complex with BiP This suggested that the complexity of the wt FVIII glycosylatio may influence the degree of BiP association since deletion of th highly glycosylated region in LA produced a protein which wa associated with BiP to a lesser degree than wt FVIII. In thi regard it is noteworthy that GRP78 is induced to high levels i CHO cells placed under conditions which affect N-linke glycosylation such as glucose starvation or tunicamyci treatment .

The Effect of Tunicamycin on the Association of LA and BiP

This observation that a population of wt FVIII molecules remained inside the cell complexed with BiP after long chase while LA displayed a transient association prompted us to test whether disruption of the glycosylation of LA would result in greater association with BiP. To examine this concept, LA producing cells were treated overnight with lOug/ml tunicamycin. This treatment inhibits N-linked glycosylation and has been reported to induce increased levels of GRP78 synthesis (Munro and Pelham, 1986) . Following a lh pulse with ³⁵S methionine the extracts of untreated or treated cells were immunoprecipitated with anti-FVIII monoclonal or anti-Bip monoclonal. In the absence of tunicamycin, only a small amount of single chain LA at 150 kd was associated with BiP. In the presence of tunicamycin the molecular weight of the LA doublet was reduced and roughly all of this unglycosylated LA was now associated with BiP. Thus disruption of the glycosylation of LA under conditions which should induce increased levels of GRP78 results in increased association with BiP compared to normally glycosylated LA. This suggested that improper glycoslylation of FVIII might influence its association with BiP.

Of particular interest is the detection of a protein induced by tunicamycin treatment which comigrates with the protein identified as BiP by immunoprecipitation with the anti-BiP monoclonal. The molecular weight of BiP does not change following tunicamycin treatment indicating it is not normally N- 1inked glycosylated.

Association of vWF with BiP.

It was possible that the CHO cells were deficient in some aspect of the secretory pathway and so could not properly process a complex glycoprotein. To explore this we examined the processing of vWF in a stable CHO line in a pulse and chase experiment. The precursor form of vWF has 17 N-linked glycosylation sites spread along the molecule. At the lh pulse time point the 260Kda VWF precursor protein is observed inside CHO cells. Roughly 10% of this protein is found complexed with BiP. VWF is efficiently and rapidly secreted such that at the 4h chase point approximately 90% of the 260kda precursor is gone from the cell extract and the conditioned medium contains the processed forms of 275 and 220 kD. These processed forms are not observed intracellularly to any' significant degree, consistent with observations that th processing of the 260kda precursor to the 275 and 220 for occurs rapidly late in the pathway of VWF secretion. At bo the 4h chase and 20h chase points most of the VWF has bee secreted from the cells. Some VWF is still associated with Bi at the 4h point but little if any BiP-VWF complex is observed a the 2Oh chase point. Despite the fact that VWF is a comple glycosylated protein its association with BiP is transient an most of the protein is efficiently secreted from CHO cells. Thi is in contrast to the situation with wt FVIII and indicates tha CHO cells are competent to efficiently secrete a comple glyσoprotein.

Association of tPA with BiP

To further analyze the role of glycosylation on protein secretio and Bip association we examined the processing of t-PA i glycosylated and unglycosylated forms in CHO cells. T-PA has potential N-linked glycosylation sites of which 3 are utilized t-PA appears as a doublet or roughly 68 kD due to variability i the utilization of one of the three glycosylation sites. t-PA3 is genetically engineered mutant in which the three normall utilized N-linked glycosylation sites have been abolished by As to Gin codon changes in the canonical recognition site sequences See International Application No. PCT/US87/00257.

Glycosylated unmodified t-PA (i.e. wild type, "t-PAwt") wa efficiently processed and secreted in a high producing CHO cel line, t-PAwt cell line. At the pulse time point t-PAwt exhibite a slight association with BiP. During the lh and 3h chas periods most of the t-PAwt had been secreted into the medium an little if any association with BiP could be detected intracellularly at these times. Thus, at high intracellular concentrations t-PAwt is correctly processed and secreted without extensive detectable association with BiP. We next examined the processing of t-PA3x in a low producing cell line, t-PA3x-4, to determine if the absence of N-linked glycosylation in t-PA3x would prevent its efficient secretion, in analogy to our observations with LA. This unglycosylated form of t-PA displays little association with BiP and is efficiently secreted into the medium. The time course of its transit through the cell is similar to that observed for t-PAwt. The majority of the protein has left the cell by the lh and 3h chase time points indicating that t-PA3x does not experience a block in the secretory pathway. Thus, in the absence of glycosylation t-PA remains in a secretion competent form which displays little association with BiP.

However, examination of a high producing t-PA3x cell line, t- PA3x-13, indicated that the association of t-PA3x with BiP is dependent on the expression level. t-PA3x-13 produces roughly 200-fold higher levels of t-PA3x that t-PA3x-4. At high expression levels t-PA3x displays a significant association with BiP in sharp contrast to that observed for the t-PA3x-4 line. The amount of t-PA3x associated with BiP decreases slightly between the pulse time point and the lh chase point. However, the amount of t-PA3x found in a complex with BiP remains the same between the lh and 3h chase points. Strikingly, the proportion of t-PA3x associated with BiP increased through the time course such that at the 3h chase point most of the t-PA3x which remained in the cell was in a complex with BiP. During the time course of this experiment t-PA3x is secreted from the cell but there exists a population of the molecules which are not competent for efficient secretion and apparently enter a stable complex with BiP. This situation is highly reminiscent of that observed with wt FVIII described above. In the case of t-PA3x the efficient of secretion and the extent of BiP association of the unglycosylated protein was influenced by the expression level. The Effect of Tunicamycin on the Association of t-PA and BiP

Another way to examine unglycosylated forms of t-PA is to inhib N-linked glycosylation by tunicamycin treatment. I mun fluorescence analysis showed that tunicamycin treatment of PAwt-producing cells results in accumulation of t-PA in t endoplasmic reticulum. When the t-PAwt cell line is treated wi 10 ug/ml tunicamycin for 1 hour the association of th unglycosylated t-PA with BiP is significantly increased compare to t-PAwt. t-PA-BiP complex is detected at the chase time poin and there is some inhibition of secretion. Similar treatment o the t-PA3x-13 cell line did not produce an alteration in th amount of t-PA3x associated with BiP compared to untreated cell and the protein is secreted while a fraction of the intracellula t-PA remains associated with BiP. This pattern of protei processing in tunicamycin treated t-PA3x-13 cells looks simila to the untreated time course. This indicated that the influenc of tunicamycin treatment on t-PAwt secretion was due to th absence of glycosylation on the molecule itself rather than a indirect effect of the tunicamycin.

It is striking that the t-PAwt treated with tunicamycin profil looks very much like that of t-PA3x at high expression levels In both cases a similar proportion of the unglycosylate molecules are apparently not competent for efficient secretio and remain in an intracellular complex with BiP. At lowe expression levels t-PA3x shows no significant association wit BiP. t-PAwt at lower expression levels is affected to a lesse degree by tunicamycin treatment than the high producer cell line. Thus the association of unglycosylated t-PA with BiP i influenced by the intracellular levels of t-PA.

Unglycosylated t-PA appears as doublet in these experiments. t PA is synthesized with a 12-15 amino acid long propeptide at the amino terminus of the protein (Pennica et al. Nature 1983) . Most probably the higher molecular weight band represents the uncleaved pro-t-PA precursor form while the lower band represents the mature form which has been processed to remove the amino terminal propeptide. Since propeptide cleavage occurs in Golgi and post-Golgi compartments and BiP has been localized to the endoplasmic reticulum, only the pro-t-PA precursor form should be present in the same compartment as BiP. Consistent with this interpretations is the observation that only the higher molecular weight species of the doublet is found associated with BiP while only the lower molecular weight species is secreted.

CoexT-ression of GRP78 and FVIII or LA in Cos Cells

A cDNA coding sequence for Chinese hamster GRP78 was placed in the expression vector pMT2 which is a derivative of p91023b and this expression vector (pMTGRP78) was σotransfected into COS cells with wt FVIII (pMT2VIII) or LA (pMT2LA) expression vectors to examine the consequences of overexpression of GRP78 on FVIII secretion. The transient expression of FVIII was monitored by assaying the conditioned medium for FVIII activity. Expression of GRP78 was detected by immunoprecipitation with the anti-BiP monoclonal. pMT2 may be obtained from pMT2-vWF (ATCC No. 67122) as described in detail below.

Coexpression of GRP78 and FVIII in COS cells consistently resulted in a 6-10 fold reduction in the levels of FVIII activity in the conditioned medium (Table I, below) . The effect of two different replicating vectors in the same cell is a decrease in the expression of both vectors. To compensate for this phenom¬ enon, FVIII or LA vectors were always cotransfected with pCSF-1. pCSF-1 is an expression vector for GM-CSF which carries similar replication and transcription elements as pMT2 (Wong et al Science 1985) .Coexpression of LA and GRP78 in COS cells resulted in a 2-3 fold reduction the levels of LA activity in the medium. The degree of decrease of activity of LA and wt FVIII a consistent with the degree of association of FVIII and LA wi BiP in CHO cells. The heavily glycosylated wt FVIII is mo affected by GRP78 expression than LA in the transient COS syst and also displays a stronger association with BiP in stable C cell lines. This data indicates that high levels of GRP78 c interfere with the secretion of FVIII and is suggestive that B and GRP78 are functionally and structurally similar.

EXAMPLES

A. Preparation of GRP78 cDNA

The particular GRP78 cDNA used is a matter of choice. F example, one may use a Chinese hamster cDNA clone, p3C5, obtain as described (Lee et al JBC 1983), or the Chinese hamster ,cD described below. Alternatively a rat cDNA clone may be obtaine also as described (Munro & Pelham, Cell, 1986) . Sequenc analysis has shown that both of these clones encode the sa protein identified as GRP78. At the amino acid level the rat an hamster proteins are 99.4% homologous. Cloning of a functiona GRP78 cDNA may be effected using one or more oligonucleotide derived from the published sequence of GRP78 and purel conventional techniques as described by Lee et al. or Munro Pelham, supra. Alternatively, a cloned rat cDNA may be obtaine from Dr. Pelham, MRC Laboratory of Molecular Biology, Hills Road Cambridge CB2 2QH, England. Additionally, a DNA sequenc encoding any desired GRP78 may be synthesized, e.g. usin overlapping oligonucleotides which together span the desire sequence.

B. Coexpression of Chinese Hamster GRP78 cDNA in Monkey Kidne COS Cells with F(VIII) or LA.

Chinese hamster GRP78 cDNA was placed into a mammalian expressio vector pMT2. This vector is a derivative of p91023B and may be obtained by EcoRI digestion of pMT2-vWF, which has been deposited with the American Type Culture Collection under ATCC No. 67122. EcoRI digestion excises the cDNA insert present in pMT2-vWF, yielding pMT2 in linear form which can be ligated and used to transform E. coli HB101 or DH5 to ampicillin resistance. Plasmid pMT2 DNA can then be prepared by conventional methods. The 1962 nucleotide open reading frame encoding hamster GRP78 was excised by Pstl and EcoRV digestion. The vector was prepared by EcoRI digestion, the EcoRI ends were filled in using Klenow fragment and then the vector was digested with Pstl.

The fragment from the hamster clone was ligated into the prepared pMT2 vector, although as indicated previously, other eucaryotic expression vectors may also be used. The resultant plasmid pMTGRP78 contains the adenovirus-VA genes, SV40 replication origin including enhancer, adenovirus major late promoter (MLP) including tripartite leader and 5' donor splice site, 3' splice acceptor site, GRP78 cDNA insert in proper orientation relative to the MLP for expression of GRP78, DHFR cDNA insert, SV40 early polyadenylation site and pBR322 sequences.

pMTGRP78 was used to cotransfect COS-1 cells along with the FVIII expression vectors pMT2VIII or pMT2LA (Toole et al., PNAS, 1986) using the DEAE dextran procedure (Kaufman, PNAS, 1985). Conditioned medium was harvested at various times beginning 48 hours post-transfection and assayed for FVIII activity as described (Toole et al., Nature, 1984). The results of these experiments are summarized in Table I. Previous work has shown that cotransfection of two different expression vectors reduces the level of expression compared to transfection of a single vector. To compensate for this phenomenon the FVIII expression vectors were cotransfected with pCSF-1, a previously described vector which expresses GM-CSF (Wong et al Science 1985) . pCSF-1 is available from the American Type Culture Collection in E. coli MC1061 as ATCC 39754. The results in Table I show tha coexpression of GRP78 and FVIII resulted in roughly six-ten fol reduction in the level of secreted FVIII activity an coexpression of GRP78 and LA resulted in a roughly two-three fol reduction compared to coexpression of FVIII or LA with GM-CSF Analysis_, of extracts of COS cells cotransfected with pMTLA an pMTGRP78 or pMT2LA and pCSF-1 and subjected to a pulse/chase ³⁵ methionine label showed that in cells expressing pMTGRP78 more L remained associated with BiP/GRP78 following the chase than wa observed in the pCSF-1 cotransf cted cells. These result indicated that overexpression of GRP78 prevented the secretion o FVIII by the intracellular association of FVIII and GRP78 and th retention of this complex in the cell. This suggested tha otherwise secretion competent FVIII might be trapped inside cell expressing high levels of GRP78 and thus a decrease in GRP7 levels would facilitate the secretion of FVIII and othe secretory proteins.

C. Coexpression of Chinese hamster GRP78 antisense cDNA wit F(VIII) in Monkey Kidney COS cells.

Chinese hamster GRP78 cDNA was placed into pMT2 in the opposit orientation from that in the above-described expression vector. The 1962 nucleotide open reading frame was excised as follows. The hamster GRP78 clone was digested with EcoRv and a Pstl linke was ligated to the blunt EcoRV end. The DNA was then cut wit Pstl to excise the 1962 bp open reading frame. The vector pMT2 was prepared by digestion with Pstl. The fragment from th hamster was ligated into the Pstl site of pMT2. The resultant plasmid pMT B2 was analyzed by extensive restriction digest mapping, and it was determined that the vector carries the GRP78 cDNA sequences such that the 3' end of the GRP78 cDNA was closest to the adenovirus major late promoter.

In this orientation transcripts expressed from the adenovirus major late promoter contain sequence which is the complement of the GRP78 coding sequence. Such a RNA is commonly referred to as an antisense RNA. It has been reported that antisense RNA can interact intracellularly with its complementary sense mRNA and block the synthesis of the encoded protein (Kim and Wold, Cell, 1985) .

pMT2αB2 has been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md 20852 (USA) under accession number ATCC 40387. It should be noted that the cDNA insert may be excised with Pstl digestion and the excised cDNA re-inserted in either orientation into an alternative expression vector, using synthetic linkers, if necessary. The excised cDNA may also be used as a hybridization probe to identify and clone other DNA molecules encoding BiP proteins or (by probing a genomic library) containing transcriptional regulatory nucleotide sequences for BiP proteins, by conventional methods.

pMTαB2 was used to cotransfect COS-1 cells along with the FVIII expression vector pMT2VIII using the DEAE dextran procedure. Conditioned medium was harvested at various times beginning 48 hours post-transfection and assayed for FVIII activity. The results of such an experiment are summarized in Table II. In this experiment coexpression of FVIII and antisense GRP78 sequences resulted in a 50% increase in FVIII activity in the conditioned medium compared to coexpression of FVIII and GM- CSF. This data indicates that the introduction of an antisense vector to decrease the intracellular level of GRP78 can result in increased levels of FVIII secretion.

SUBSTITUTE SHEET TABLE I

Cotransfection of FVIII and GRP78 Expression Vectors in

COS-1 Cells

Chromocrenic Activity (milliunits/ml )

No DNA 0 0 0 0 pMT2VIII/pCSF-l 67 93 30 30 pMT2VIII/pMTGRP78 10 19 0 5 pMT2LA/pCSF-l 290 — 536 436

PMT2LA/PMTGRP78 90 -_-; 2_40 2_17

*Shown are the results of four separate experiments. T plasmids indicated were cotransfected into COS-1 cells and the conditioned medium removed for assay by the Kabi Coatest F(VIII) : C method.

TABLE II

Cotransfection of F(VIII) and Antisense GRP78 Expression Vectors in COS-1 Cells

Chromoqenic Activity (milliunits/ml ) pMTVIII/pCSF-1 90 pMTVIII/pMTαB2 135

No DNA 0

The plasmids indicated were cotransfected into COS-1 cells an conditioned medium removed for an assay by the Kabi Coates F (VIII) : C method.

D. Development of CHO cell lines with reduced BJP/GRP78 levels and fusion with FVIII producing cell lines

1) Development of CHO cells with reduced BiP/GRP78 levels

Chinese hamster ovary (CHO) cell lines which are DHFR deficient, CHO" (DUKX-Bll) , were grown in an alpha medium supplemented with lOug/ml each of thymidine, deoxyadenosine and adenosine. Cells were cotransfected with pMTαB2 (20μg) and pSV2Neo (2 g) (ATCC No. 37149) by the calcium phosphate coprecipitation procedure (Kaufman et al JMB 1982) . pSV2Neo codes for resistance to the antibiotic G418 (P. Southern & Berg P. 1982 J. Mol. Appl. Genet. 1 327-341). Forty-eight hours post-transfection the cells were plated in alpha medium supplemented with nuσleosides as above and including Img/ml of G418 in order to select for SV2Neo expression. pMTαB2 contains an intact DHFR coding region in the 3' region of the antisense GRP78 transcript. Thus G418 resistant transformants can be subsequently selected for DHFR expression from this mRNA. Growth in alpha media lacking nucleosides with 10% dialyzed fetal calf serum resulted in DHFR⁺ colonies. Five colonies were pooled to produce the A6B line. This line was then amplified by selection for growth in the presence of the folic acid analogue methotrexate at a concentration of .02uM.

Following approximately 8 passages in .02uM methetrexate the BiP/GRP78 level in A6B was compared to CHO DUKX by immunoprecipitation of radiolabeled cell extracts with anti-BiP monoclonal and analysis by SDS PAGE. Additionally, the level of BiP/GRP78 was measured by Western analysis using anti-BiP monoclonal antibody. A6B showed reduced levels of BiP/GRP78 compared to the original CHO line. In addition the level of antisense GRP78 RNA derived from pMT B2 in these cells was determined by Northern analysis. 2) Fusion of H9 with BiP/GRP78 reduced cell line

The A6B cell line was fused with a FVIII producing cell l ine , H9 , by standard polyethylene glycol procedure following treatment of the A6B cells with DEPC to render them nonviable (WE Wright , Chap 5 , The S election of Heterokaryons and Cell Hybrids Using the Biochemical Inhibitors Iodoaceta ide and Diethylpyrocarbonate in Techniques in Somatic Cell Genetics, Ed. JW Shay, Plenum Press) . Two days following cell fusion the cells were plated in luM methetrexate and lmg/ml G418. H9 grows in luM methetrexate and G4 Ϊ8 selects for the chromosome containing the antisense GRP78 sequences derived from A6B cells . After eleven days of growth twenty-two colonies were pooled to produce the cell line designated H9XA6B-9. Determination to the level of FVIII procoagulant activity secreted into the conditioned medium by H9xA6B-9 showed that this cell line yielded two-fold greater activity than the original H9 line.

3 ) Fusion of tPA-3x cell line with cell line having reduced level of BiP/GRP78

Another cell line was developed as follows . CHO DUKX cells as above were cotransfected with pMT B2 (20 ug) and pSV2AdA (2 ug) by the calcium phosphate coprecipitation procedure. pSV2AdA codes for adenosine deaminase and allows for cell growth in the presence of cytotoxic concentrations of adenosine and the drug deoxycoformycin(dCF) . Forty-eight hours post-transf ection the cells were plated in alpha medium supplemented with deoxyadenos ine , thymidine , uridine (U) , alanosine (A) , adenosine (A) , and . 03 uM dCF. Growth in AAU and dCF selects for ADA expression. Colonies were pooled to produce the C6B line. This line was subsequently amplified by selection for growth in 0.1 uM and luM dCF. The BiP/GRP78 protein levels were measured by immunoprecipitation of radiolabeled cell extracts and by Western analysis using anti-BiP monoclonal. The level of GRP78/BiP RNA was also determined by Northern analysis. C6B showed reduced levels of BiP/GRP78 protein and RNA compared to CHO DUKX.

The C6B line growing in 1 uM dCF was fused with a tPA3x producing cell line tPA3x-9. Two days following cell fusion, cells were plated into alpha medium containing AAU, 1 uM dCF and 0.02 uM methotrexate. tPA3x-9 grows in 0.02 methotrexate and dCF selects for the chromosome containing antisense GRP78 sequences. After 11 days of growth 9 colonies were pooled to produce the laE line. Determination of the level of tPA activity secreted into the conditioned medium showed that this line yielded 2-5 fold greater activity than the original tPA3x-9 line.

E. Preparation and use of vectors containing regulatory sequences for BiP/GRP78 transcription

A DNA sequence containing the BiP/GRP78 regulatory element was removed from pUC291R (Lin et al., Mol. Cell Biol., 1986) by digestion of the plasmid with Sma I and Hindi. The DNA fragment so obtained contains the 291 (Smal/Stul) nucleotide sequence bearing the regulatory element required for induction of the rat GRP78 gene. Alternatively, the 360 nucleotide sequence shown above on page 7, or a portion thereof, may be synthesized by purely conventional methods, e.g. through the synthesis of overlapping oligonucleotides which together span the desired sequence. It should be understood that the corresponding GRP78 regulatory sequence derived from other species should also be usable in this invention, whether isolated from a genomic DNA library or chemically synthesized. It is believed that a trans-acting factor or factors may bind to regulatory sequence(s) within the DNA fragment so obtained and thus mediate induction of GRP78 transcription. It has been reported that such DNA fragments can compete for the hypothesized trans-acting factor(s) n vivo (Lin et al., Mol. Cell Biol., 1986).

Operability of the regulatory fragment to be used may be conveniently assayed by methods such as described by Lin et al., Mol. Cell Biol., 1986.

The regulatory DNA sequence to be used is then inserted into a conventional expression vector for use in eucaryotic cells, preferably mammalian cells, either directly or with synthetic linkers if desired. By way of example, the regulatory sequence may be inserted with synthetic EcoRI linkers into the EcoRI site in expression vector, pMT2. pMT2 may be obtained by EcoRI digestion of pMT2-VWF, which has been deposited (29 May 1986) with the American Type

Culture Collection of Rockville, Maryland (USA) under accession number ATCC 67122. EcoRI digestion excises the cDNA insert present in pMT2-VWF, yielding pMT2 in linear form which can be ligated and used to transform E. coli

HB101 or DH-5 to ampicillin resistance. Plasmid pMT2 DNA can be prepared by conventional methods. Of course, other expression vectors known in the art may be used in place of pMT2, using appropriate linkers as desired or necessary.

The expression vector containing the regulatory sequence is then cotransfected or cotransformed into the desired host cell with one or more selectable, amplifiable markers, as is known in the art, and gene copy number of the heterologous DNA may be amplified as desired using conventional methods. The presence of multiple copies of the regulatory sequence in the cellular genome should compete with the endogenous GRP78 expression control sequences for binding wit the trans-acting factor(s) following induction by conditions such as blockage of N- linked glycosylation, over-expression of FVIII : c or analogs thereof , etc. Such competition for trans-acting factors has been reported for a metalothionein I gene (Seguin et al . , Nature, 1984) and suggested for heat shock proteins (McGarvy and Lindquist , Proc . Natl . Acad. Sci . , 1986) . Induction of transcription of the endogenous BiP gene, and thus the induced levels of BiP protein in the resulting cells and their progeny would thus be significantly reduced. Preferably the ratio of induced BiP transcription levels to normal BiP transcription levels is less than about 20 , more preferably less than about 10 , even more preferably less than about 5 , and especially preferably less than about 1.

Transformants or transf ectants , or the progeny thereof, which contain the vector-borne regulatory sequence may additionally be transformed or trans fected with an expression vector capable of directing the synthesis of a desired heterologous protein. Alternatively, they may be fus ed with other c el ls which had been previously transformed or transfected with an expression vector capable of directing the synthesis of the des ired heterologous protein . Suitable vectors capable of directing the synthesis of heterologous proteins are known in the art and discussed previously.

F. Conclusions of BJP/GRP78 Association study

From experiments including those described above, we draw the following conclusions:

1. wt FVIII is associated with BiP and most of the FVIII which is never secreted remains associated with BiP. 20 out of 25 N-linked glycosylation sites are clustered in middle third of the FVIII protein.

2. LA, a deleted form of FVIII which has 18 of 20 clustered glycosylation sites removed, is more efficiently secreted than wt FVIII exhibits a transient association with BiP.

3. The association of LA with BiP can be significantly increased by treatment of cells with the N-linked glycosylation inhibitor tunicamycin.

4. VWF, a complex glyσoprotein which is efficiently secreted by CHO cells, exhibits only a transient association with BiP. The 17 glycosylation sites on VWF are spaced along the molecule rather than clustered as on wt FVIII.

5. tPA exhibits only a slight transient association with BiP. However inhibition of N-linked glycosylation by tunicamycin results in the intracellular retention of some of the unglycosylated molecules in a complex with BiP.

6. tPA3x, an engineered mutant of t-PA which has had three potential N-linked glycosylation sites abolished by replacement of Asn with Gin exhibits only a slight association with BiP at low expression levels. However, at high expression levels a fraction of the unprocessed protein displays a stable association with BiP and is apparently not secreted effeciently. This behavior is similar th that observed for wt t-PA when glycosylation is inhibited.

7 . Intracellular retention of unglycosylated tPA in a complex with BiP is dependent on expression level. tPA3x at low expression levels is not associated with BiP and is effeciently secreted. At 200-fold higher expression levels a significant proportion of tPA3x is associated with BiP. This intracellular retention is similar to that observed for the high producer wt tPA cell line when N-linked glycosylation is inhibited. In a low producing wt tPA cell l ine , H12 B , the eff ect of inhibition of N-linked glycosylation is less pronounced than in higher-producing cells . This suggests that unglycosylated tPA may aggregate when present at high concentrations in the ER leading to its association with BiP.

8. BiP may associate with improperly glycosylated or folded proteins in the endoplasmic reticulum and prevent the ir secretion. BiP probably functions to clear aggregated proteins from the endoplasmic reticulum in an analogous function to hsp70 in heat shocked nucleoli. The problem of protein aggregation or insolubility in the ER may be exacerbated by the high expression levels now attainable by reσombinant DNA expressioin techniques and for some glycσproteins such as FVIII aggregation and consequent association with BiP may prove a barrier to high level secretion.

9. The 20 clustered glycosylation sites in the middle of wt FVIII may be inefficiently glycosylated resulting in aggregation of improperly folded molecules and stable association with BiP . It is also possible that this heavily glycosylated domain assumes a conformation which BiP recognizes as aberrant even if N-linked glycosylation is appropriate. In this situation secretion competent molecules may be trapped in a complex with BiP and reduced levels of BiP may result in higher levels of secretion.

10. Reduction of BiP levels in FVIII producing cell lines results in increased secretion of FVIII acitvity into the conditioned medium. Thsu CHO cell lines with reduced levels of BiP may be of utility in the expression of certain complex glycoproteins.

31A

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Claims

32 What is claimed is:

1. An anti-sense expression vector comprising a DNA sequence encoding a GRP78 protein or a portion thereof operatively linked in reverse orientation to an expression control sequence such that transcription of the DNA sequence produces an anti-sense mRNA capable of hybridizing to a GRP78-encoding mRNA.

2. An improved eucaryotic host cell for expressing a heterologous protein which comprises a eucaryotic host cell transformed with a vector of claim 1, or progeny thereof.

3. A yeast, fungal insect, plant or mammalian host cell of claim 2.

4. An improved host cell of claim 2 which is further transformed with a vector capable of directing the expression of a heterologous protein, or progeny thereof.

5. An improved host cell of claim 4, wherein the heterologous protein is Factor VIII or an analog thereof.

6. An improved method for producing a heterologous protein which comprises culturing a host cell or progeny thereof wherein the host cell is transformed with a vector capable of directing the expression of the heterologous protein, the transformed host cell being additionally transformed with a vector selected from the group consisting of:

a. a vector of claim 1;

b. a vector containing the DNA sequence: 33 (claim 6 , continued)

(5 ' )

CGGGGGCCCA ACGTGAGGGG AGGACCTGGA CGGTTACCGG CGGAAACTGG TTTCCAGGTG AGAGGTCACC CGAGGGACAG GCAGCTGCTC AACCAATAGG ACCAGCTCTC AGGGCGGATG CGCCTCTCAT TGGCGGTCCG CTAAGAATGA CCAGTAGCCA ATGAGTTCGG CTGGGGGGCG CGTACCAGTG ACGTGAGTTG CGGAGGAGGC CGCTTCGAAT CGGCAGCGGC CAGCGTTGGT GGCATGAACC AACCAGCGGC CTCCAACGAG TAGCGAGTTC ACCAATCGGA GGCCTCCACG ACGGGGCTGC GGGGAGGATA TATAAGCCGA GTCGGCGACC GGCGCGCTCG AATAACCCGG (3')

or a portion thereof;

c. a vector containing a DNA sequence at least 70% homologous to the DNA sequence of (b) ; and,

d. combinations of vectors (a) , (b) , and/or (c) .

7. An improved method of claim 6, wherein the heterologous protein is Factor VIII:c or an analog thereof.