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WO1997003191A1 - Nouveaux analogues de polypeptides du facteur viii:c comprenant des domaines ou des sous-domaines du facteur v - Google Patents

Nouveaux analogues de polypeptides du facteur viii:c comprenant des domaines ou des sous-domaines du facteur v Download PDF

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Publication number
WO1997003191A1
WO1997003191A1 PCT/US1996/011013 US9611013W WO9703191A1 WO 1997003191 A1 WO1997003191 A1 WO 1997003191A1 US 9611013 W US9611013 W US 9611013W WO 9703191 A1 WO9703191 A1 WO 9703191A1
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Prior art keywords
factor
viilc
factor viilc
analog
polypeptide
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PCT/US1996/011013
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English (en)
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David T. Hung
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Chiron Corporation
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Priority to AU62918/96A priority Critical patent/AU6291896A/en
Publication of WO1997003191A1 publication Critical patent/WO1997003191A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/755—Factors VIII, e.g. factor VIII C (AHF), factor VIII Ag (VWF)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates generally to active Factor VIILC polypeptide analogs having improved properties. More particularly, the invention pertains to Factor VII C polypeptide analogs having modifications such that A and/or C domains of Factor V, or subdomains of Factor V, are present in the molecule.
  • This invention further relates to analog complexes comprising at least two such analogs, or one such analog and a native Factor VIILC polypeptide, nucleic acid molecules encoding such analogs, vectors and host cells comprising the nucleic acid molecules, pharmaceutical compositions comprising the analogs or analog complexes, methods of making the analogs, nucleic acid molecules, vectors and host cells, and methods of prevention or treatment of active Factor VIILC deficiency using the analogs, complexes, and/or nucleic acid molecules, vectors and host cells.
  • Hemophilia A is an X-chromosome-linked inherited bleeding diathesis that results from the deficiency of an active blood clotting factor termed Factor VIILC.
  • the disease afflicts approximately 1 in 10,000 males.
  • Factor VIILC is a large glycoprotein that participates in the blood coagulation cascade that ultimately converts soluble fibrinogen to insoluble fibrin clot, effecting hemostasis.
  • the deduced primary amino acid sequence of human Factor VIILC determined from the cloned cDNA indicates that Factor VIILC is a heterodimer processed from a larger precursor polypeptide consisting of 2351 amino acids, referred to herein as the precursor or full-length Factor VIILC molecule, of which the first 19 N-terminal residues comprise the signal sequence. Therefore, the mature Factor VIILC molecule, starting with Alal, which does not contain the signal peptide sequence, includes a sequence of 2332 amino acids. Amino acids from about 1 to about 1648 of the mature Factor VIILC molecule give rise to "heavy chain" fragments with molecular weights ranging from approximately 90 kD to 200 kD.
  • Amino acids from about 1649 to about 2331 of the mature Factor VIILC molecule comprise a "light chain" with a molecular weight of approximately 80 kD ("the 80 kD subunit").
  • the heterodimeric mature Factor VIILC molecule consists of the heavy and light chains associated by a metal ion bridge.
  • the mature Factor VIILC molecule consists of a triplicated A domain of 330 amino acids, a unique B domain of 980 amino acids, and a duplicated C domain of 150 amino acids with the structure NH2-A1-A2-B-A3-C1- C2-COOH. See, e.g., Kaufman, R.
  • Factor VIILC is known to be activated by plasma proteases such as thrombin. During activation, the mature Factor VIILC polypeptide is cleaved to generate heavy and light chain fragments that are further cleaved.
  • cleavage of the light chain after arginine residue 1689 yields a light chain fragment of about 73 kD ("the 73 kD fragment")
  • cleavage of the heavy chain after arginine residue 372 yields smaller heavy chain fragments of about 50 kD and 43 kD ("the 50 kD and 43 kD fragments," respectively), as described in Eaton et al. (1986), Biochem. 25: 505-512.
  • the complex formed by the 50 kD and 73 kD polypeptides appears to be required for Factor VIILC coagulant activity.
  • the heavy and light chain fragments of Factor VIILC are inactivated by plasma proteases.
  • Factor V is also a plasma clotting factor and is both structurally and functionally similar to Factor VIILC.
  • the two molecules have roughly the same molecular weights, they both consist of triplicated A domains that are 30% homologous to ceruloplasmin (a copper-binding protein), they both possess a B domain which is unnecessary for activity and is largely removed during proteolytic activation of each factor, and both possess duplicated C domains which are homologous to slime mold lectins and are implicated in binding to glycoconjugates and phospholipid surfaces.
  • ceruloplasmin a copper-binding protein
  • both factors consist of heavy and light chains bound together by a metal ion bridge. The heavy and light chains are 40% identical between Factor V and Factor VIILC.
  • Factor Xa binds Factor V and activates prothrombin to thrombin.
  • Factor IXa binds Factor VIILC and activates Factor X to Xa.
  • Factor V has a plasma half-life of about 36 hours
  • Factor VIILC has a plasma half-life of only about 8 to 12 hours.
  • the short half-life of Factor VIILC presents problems in the treatment of hemophilia A.
  • an active Factor VIILC polypeptide analog that is substantially the same as a native Factor VIILC polypeptide, except for the presence of one or more Factor V domains or subdomains selected from the group consisting of a Factor V A domain, a Factor V C domain, a subdomain of the Factor V A domain, and a subdomain of the Factor V C domain.
  • a modification can be achieved by one or more amino acid substitutions, additions or deletions.
  • the native Factor VIILC polypeptide that is modified is selected from the group consisting of (a) a full-length Factor VIILC molecule comprising a signal peptide and all A, B, and C domains; (b) a native Factor VIILC molecule comprising all A, B, and C domains and lacking a signal peptide; (c) a truncated Factor VIILC molecule lacking a signal peptide and at least a portion of the B domain; (d) a cleaved Factor VIILC molecule containing a light chain subunit of molecular weight of about 80 kD; (e) a cleaved Factor VIILC molecule containing a heavy chain fragment of molecular weight in a range of about 90 kD to about 200 kD; (f) a cleaved Factor VIILC molecule comprising a heavy chain fragment of a molecular weight of about 90
  • an active Factor VIILC analog complex that contains either at least two Factor VIILC polypeptide analogs as above, or a Factor VIILC polypeptide analog and a Factor VIILC polypeptide, together with a metal ion.
  • the analog complex herein can comprise two Factor VIILC polypeptide analogs, and the two analogs can be selected from the group consisting of analogs of molecular weights of about (a) 80 kD and 90 kD; (b) 73 kD and 90 kD; (c) 80 kD and 50 kD; (d) 80 kD and 43 kD; (e) 73 kD and 50 kD; and (0 73 kD and 43 kD.
  • a method of producing a Factor VIILC polypeptide analog as above by (a) providing a native Factor VIILC polypeptide that contains an amino acid sequence, and (b) modifying at least one amino acid residue in the amino acid sequence to produce an analog as above.
  • nucleic acid molecule that contains a nucleotide sequence that encodes the analog as above.
  • a recombinant vector that contains the nucleic acid molecule as above and a regulatory element, where the nucleic acid molecule is placed under regulatory control of the regulatory element.
  • a recombinant host cell that contains the nucleic acid molecule or recombinant vector as above.
  • a method of producing an active Factor VIILC polypeptide analog as above comprising: (a) providing the recombinant host cell as above, and (b) allowing the recombinant host cell to express the analog.
  • a method of producing the nucleic acid molecule as above comprising: (a) providing a nucleic acid molecule that encodes a native Factor VIILC polypeptide as above and (b) modifying at least one codon to provide the analog.
  • a method of producing the recombinant vector that contains the nucleic acid molecule as above comprising linking a regulatory element to the nucleic acid molecule.
  • a method of producing a recombinant host cell that comprises a nucleic acid molecule as above, comprising transforming a host cell with the nucleic acid molecule, or transforming a host cell with the recombinant vector.
  • composition that contains the active Factor VIILC polypeptide analog or analog complex as above, and a pharmaceutically acceptable excipient.
  • methods for prevention or treatment of active Factor VIILC deficiency in a mammal comprising administering thereto a therapeutically effective amount of (a) an active Factor VIILC polypeptide analog as above, or (b) an active Factor VIILC polypeptide analog complex as above, or (c) the nucleic acid molecule as above, or (d) the recombinant vector as above, or (e) the nucleic acid molecule as above together with and an active Factor VIILC polypeptide analog, or (f) the recombinant vector as above together with an active Factor VIILC polypeptide analog.
  • Factor VIILC polypeptide analogs can be made that have improved properties. These analogs carry one or more amino acid residues that are modified from the native structure such that A and/or C domains of Factor V, or subdomains of Factor V, are present in the molecule.
  • the modification can be at least one amino acid substitution, addition or deletion, to any A and/or C domain or subdomain of the native Factor VIILC molecule, so long as Factor VIILC activity is not destroyed.
  • Fractor VIILC polypeptide refers to a polymer of amino acids and does not refer to a specific length of the product.
  • peptides, oligopeptides, and proteins are included within the definition of polypeptide. This term also does not exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations and the like. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid, including, for example, unnatural amino acids, polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • a Factor VIILC polypeptide includes but is not limited to, for example, the following Factor VIILC polypeptides: (a) a full-length Factor VIILC molecule comprising a signal peptide and all A, B, and C domains; (b) a mature Factor VIILC molecule comprising all A, B, and C domains and lacking the signal peptide; (c) a truncated Factor VIILC molecule lacking the signal peptide and at least a portion of the B domain; (d) a cleaved Factor VIILC molecule comprising a light chain subunit of about 80 kD; (e) a cleaved Factor VIILC molecule comprising a heavy chain fragment of about 90 kD; (f) a cleaved Factor VIILC molecule comprising a heavy chain fragment of about 50 kD; (g) a cleaved Factor VIILC molecule comprising a heavy chain fragment of about 43 kD; and (h) a
  • Factor VIILC polypeptides also include muteins or derivatives of the polypeptides with conservative amino acid changes that do not alter the biological activity of the polypeptide from which the mutein or derivative is made.
  • Such muteins or derivatives may have, for example, amino acid insertions, deletions, or substitutions in the relevant molecule that do not substantially affect its properties.
  • the mutein or derivative can include conservative amino acid substitutions, such as substitutions which preserve the general charge, hydrophobicity/hydrophilicity, and/or stearic bulk of the amino acid substituted, for example Gly/ Ala; Val/Ile/Leu; Asp/Glu; Lys/ Arg; Asn/Gln; and Phe/T ⁇ /Tyr.
  • the mutein or derivative should exhibit the same general structure as the native polypeptide, and may also include polypeptides having one or more peptide mimics or peptoids.
  • active in reference to the polypeptide analogs herein refers to biological activity, such as coagulation or pro-coagulation activity. Such activity is measured by using standard assays for blood plasma samples, such as, for example, the Coatest assay or the activated partial thromboplastin time test (APTT).
  • An "active" Factor VIILC polypeptide analog will have at least about 50% of the coagulation or pro-coagulation activity displayed by the native molecule, preferably at least about 60% to 80% , and more preferably at least about 90% or more of the coagulation or procoagulation activity displayed by the native Factor VIILC molecule.
  • nucleic acid molecule refers to either RNA or DNA or its complementary strands thereof, that contains a nucleotide sequence.
  • regulatory element refers to an expression control sequence that is conventionally used to effect expression of a gene.
  • a regulatory element includes one or more components that affect transcription or translation, including transcription and translation signals.
  • Such a sequence can be derived from a natural source or synthetically made, as in hybrid promoters and includes, for example, one or more of a promoter sequence, an enhancer sequence, a combination promoter/enhancer sequence, an upstream activation sequence, a downstream termination sequence, a polyadenylation sequence, an optimal 5' leader sequence to optimize initiation of translation, and a Shine-Dalgarno sequence.
  • the expression control sequence that is appropriate for expression of the present polypeptide differs depending upon the host system in which the polypeptide is to be expressed.
  • such a sequence in prokaryotes, can include one or more of a promoter sequence, a ribosomal binding site, and a transcription termination sequence.
  • such a sequence in eukaryotes, for example, can include one or more of a promoter sequence, and a transcription termination sequence.
  • a component that is necessary for transcription or translation is lacking in the nucleic acid molecule of the present invention, such a component can be supplied by a vector.
  • Regulatory elements suitable for use herein may be derived from a prokaryotic source, an eukaryotic source, a virus or viral vector or from a linear or circular plasmid.
  • regulatory control refers to control of expression of a polynucleotide sequence by a regulatory element to which the polynucleotide sequence is operably linked. The nature of such regulatory control differs depending upon the host organism. In prokaryotes, such regulatory control is effected by regulatory sequences which generally include, for example, a promoter, and/or a transcription termination sequence. In eukaryotes, generally, such regulatory sequences include, for example, a promoter and/or a transcription termination sequence. Additionally, other components which control of expression, for example, a signal peptide sequence or a secretory leader sequence for secretion of the polypeptide, and a terminator for transcriptional termination, may be attached thereto to facilitate regulatory control of expression.
  • terapéuticaally effective amount is meant an amount of analog that will improve the blood coagulation properties as compared to coagulation in the absence of the analog.
  • a “therapeutically effective amount” will fall within a relatively broad range that can be determined through routine trials.
  • the activity of the Factor VIILC analogs of the invention may be determined by means known in the art, for example, by using the commercially available Coatest assay.
  • the effective amount is sufficient to bring about prevention of further deterioration or treatment to improve coagulation, that is, to enhance coagulation properties such that hemostasis is achieved.
  • the exact amount necessary will vary depending on the subject being treated; the age and general condition of the individual to be treated; the functionality of the endogenous Factor VIILC gene present in the individual; and the mode of administration, among other factors.
  • An appropriate effective amount can be readily determined by one of skill in the art. For example, depending on the severity of active Factor VIILC polypeptide deficiency, up to about 1000 to about 3000 U of Factor VIILC polypeptide analog can be given to an average person such as a 70 kg male patient. Alternatively, sufficient Factor VIILC polypeptide analog or analog complex can be given to establish a plasma level of about 0.5 to about 2 U/ml of Factor VIILC analog or combination analog and native polypeptide. See U.S. Patent Nos. 3,631,018; 3,652,530, and 4,069,216 for methods of administration and amounts.
  • pharmaceutically acceptable excipient refers to an excipient for administration of a therapeutic agent, in vivo, and refers to any pharmaceutical agent that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol.
  • Pharmaceutically acceptable salts can be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • Suitable carriers may also be present and are generally large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive virus particles. Such carriers are well known to those of ordinary skill in the art. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ. 1991).
  • the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • the present invention provides Factor VIILC polypeptide analogs with improved properties.
  • the analogs include modifications to the native Factor VIILC amino acid sequence which comprise amino acid substitutions, deletions or additions. Modifications can be made to the native Factor VIILC sequence by the addition to, or substitution of, any of the A and/or C domains or subdomains in Factor VIILC, with co ⁇ esponding A and/or C domains or subdomains derived from Factor V. Furthermore, additional subdomains of Factor VIILC which are unnecessary for activity, can also be deleted.
  • domain is meant any of the A or C domains found in the Factor VIILC and Factor V molecules. See, e.g. , Kaufman, R.
  • subdomain any portion of an A or C domain found in the Factor VIILC and Factor V molecules, respectively, regardless of size.
  • a subdomain can comprise nearly the whole domain or as little as a single amino acid.
  • modifications which include the replacement of one or more selected A and/or C domains or subdomains of Factor VIILC by one or more co ⁇ esponding domains, or subdomains of Factor V, using, e.g., site-directed mutagenesis.
  • co ⁇ esponding is meant a domain or subdomain in Factor V which occurs in the co ⁇ esponding position to that found in Factor VIILC.
  • Al , A2, A3, Cl and C2 domains of Factor V co ⁇ espond to the Al, A2, A3, Cl and C2 domains, respectively, found in Factor VIILC.
  • substitutions result in an analog with improved qualities, such as enhanced stability, increased plasma half-lives, increased antigenicity and the like.
  • the present modifications can include substitutions of any of the triplicated A domains, duplicated C domains, portions of these domains, or even point mutations within these domains, that result in a molecule displaying Factor VIILC activity and which has improved characteristics.
  • Particularly useful modifications include the substitution of Phe270, Phe652 and Tyr 1786, of Factor VIILC, with a His residue that is found in Factor V at the corresponding sites (His242, His597 and His 1643, respectively).
  • Nucleic acid molecules encoding the present Factor VIILC polypeptide analogs can be made by modifying the native nucleic acid sequences that encode the Factor VIILC polypeptide or cDNA sequences that encode the Factor VIILC polypeptides.
  • the M13 method for site directed mutagenesis is known, as described in Zoller and Smith, Nucleic Acids Res. (1982) 10: 6487- 6500, Methods Enzymol. (1983) 100: 468-500, and DNA (1984) 3: 479-488, using single stranded DNA, and the method of Morinaga et al. Bio /technol. 636-639 (July 1984), using heteroduplexed DNA.
  • one or more of the codons in the selected domain of Factor VIILC can be mutated by substitution, deletion or addition, to one or more codons encoding a domain or subdomain of Factor V, provided that the modification does not destroy Factor VIILC activity.
  • the codon encoding the native amino acid at this position will be substituted with a codon encoding His, such as CAT and CAC.
  • nucleic acid molecules of the present invention can also be made synthetically by piecing together nucleic acid molecules encoding heavy and light chain fragments derived from cDNA clones or genomic clones containing Factor VIILC coding sequences, preferably cDNA clones, using known linker sequences. Alternatively, the entire sequence or portions of nucleic acid sequences encoding analogs described above may be prepared by synthetic methods (e.g. using DNA synthesis machines). Once made, the nucleic acid molecules can be inserted in vectors for production of recombinant vectors for transcription and translation of the nucleic acid molecules.
  • PCR PROTOCOLS A GUIDE TO METHODS AND APPLICATIONS, Innis, Gelfand, Sninsky, and White (eds.) (Academic Press, 1990).
  • a vector suitable for use herein for the production of a recombinant vector comprises a nucleic acid sequence with one or more restriction enzyme recognition sites into which the present nucleic acid molecule of the invention can be inserted.
  • This vector also typically contains a selection marker for detection of the presence of the vector in the host cell.
  • the vector can also provide, if desired, one or more regulatory elements or control sequences for expression of the nucleic acid molecule.
  • the present vector can be derived from a plasmid, a virus, a cosmid, or a bacteriophage. This vector is typically capable of behaving as an autonomous unit of replication when introduced into a host cell.
  • the vector may be one that is capable of episomal existence or of integration into the host cell genome.
  • a wide variety of replication systems are available, typically derived from viruses that infect mammalian host cells.
  • Illustrative replication systems include the replication systems from Simian virus 40, adenovirus, bovine papilloma virus, polyoma virus, Epstein Ban virus, and the like.
  • the nucleic acid molecule of the present invention can be inserted at an appropriate restriction site in the vector so as to be placed under the control of one or more regulatory elements in the vector to form a recombinant vector that can be used for transfection or transformation of a host cell.
  • the host cells of the invention can be, for example, prokaryotic or eukaryotic host cells, including bacterial, yeast, insect and mammalian expression systems.
  • the analogs of the present invention are expressed in mammalian host cell systems.
  • the regulatory elements to be used in the vector depend on the host system that is to be utilized.
  • a prokaryotic host cell can be used for amplification of the nucleic acid molecule of the present invention, while an eukaryotic host cell can be used for expression of the Factor VIILC polypeptide analogs.
  • the expression cassettes are introduced into the host cell by conventional methods, depending on the expression system used, as described further below. Where viruses are involved, transfection or transduction may be employed.
  • the particular manner in which the host cell is transformed is not critical to this invention, depending substantially upon whether the expression cassettes are joined to a replication system and the nature of the replication system and associated genes.
  • Coexpression of more than one Factor VIILC polypeptide analog may be desired.
  • either or both of the light and heavy chains may include modifications as described above.
  • "Coexpression” as used herein refers to the expression of two or more Factor VIILC polypeptides in a single host cell.
  • the expression of the 90 kD species and the 80 kD species in a single host cell would constitute "coexpression” as used herein.
  • the polynucleotides encoding for the polypeptides can be harbored in a single vector, either under the control of the same regulatory elements or under the control of separate elements.
  • a fusion protein including active portions of the two or more Factor VIILC polypeptides would be considered “coexpressed” for pu ⁇ oses of the present definition as would the expression of two genes as a dicistronic construct employing an internal ribosome entry site.
  • proteins expressed from the same vector but driven by separate regulatory elements would also be considered “coexpressed.”
  • the term also refers to the expression of two or more proteins from separate constructs.
  • the expression of proteins encoded from genes present on separate vectors in a host cell would also be considered “coexpression" for pu ⁇ oses of the present invention.
  • the transformed/transfected cells are then grown in an appropriate nutrient medium.
  • the product can be obtained as a complex of the two Factor VIILC chains, so that the media or cell lysate may be isolated and the Factor VIILC active complex extracted and purified.
  • the full-length molecule can be isolated and treated under complex-forming conditions, e.g., with the addition of calcium and the appropriate enzymes, to form the active complex.
  • Various means are available for extraction and purification, such as affinity chromatography, ion exchange chromatography, hydrophobic chromatography, electrophoresis, solvent-solvent extraction, selective precipitation, and the like.
  • the particular manner in which the product is isolated is not critical to this invention, and is selected to minimize denaturation or inactivation and maximize the isolation of a high-purity active product.
  • Bacterial expression systems can be used to produce the subject Factor VIILC polypeptide analogs and the nucleic acid molecules encoding such analogs.
  • Control elements for use in bacterial systems include promoters, optionally containing operator sequences, and ribosome binding sites.
  • Useful promoters include sequences derived from sugar metabolizing enzymes, such as galactose, lactose (lac) and maltose. Additional examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp), the ⁇ - lactamase (bla) promoter system, bacteriophage ⁇ PL, and T7.
  • synthetic promoters can be used, such as the tac promoter.
  • the 3-lactamase and lactose promoter systems are described in Chang et al. , Nature (1978) 275: 615, and Goeddel et al. , Nature (1979) 281: 544; the alkaline phosphatase, tryptophan (t ⁇ ) promoter system are described in Goeddel et al. , Nucleic Acids Res. (1980) 8: 4057 and EP 36,776 and hybrid promoters such as the tac promoter is described in U.S. Patent No. 4,551 ,433 and deBoer et ai , Proc. Natl. Acad. Sci. USA (1983) 80: 21-25.
  • promoters useful for expression of eukaryotic proteins are also suitable.
  • a person skilled in the art would be able to operably ligate such promoters to the present Factor VIILC polypeptide analog coding sequences, for example, as described in Siebenlist et al. , Cell (1980) 20: 269, using linkers or adapters to supply any required restriction sites.
  • Promoters for use in bacterial systems also generally will contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the Factor VIILC analog polypeptide.
  • SD Shine-Dalgarno
  • the signal sequence can be substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat stable enterotoxin II leaders.
  • a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, Ipp, or heat stable enterotoxin II leaders.
  • the origin of replication from the plasmid pBR322 is suitable for most Gram- negative bacteria.
  • the foregoing systems are particularly compatible with Escherichia coli.
  • numerous other systems for use in bacterial hosts including Gram- negative or Gram-positive organisms such as Bacillus spp. , Streptococcus spp. , Streptomyces spp. , Pseudomonas species such as P. aeruginosa, Salmonella typhimurium, or Serratia marcescans, among others.
  • Methods for introducing exogenous DNA into these hosts typically include the use of CaC ⁇ or other agents, such as divalent cations and DMSO.
  • DNA can also be introduced into bacterial cells by electroporation, nuclear injection, or protoplast fusion as described generally in Sambrook et al. (1989), cited above.
  • the host cell should secrete minimal amounts of proteolytic enzymes.
  • in vitro methods of cloning e.g., PCR or other nucleic acid polymerase reactions, are suitable.
  • Prokaryotic cells used to produce the Factor VIILC analog polypeptides of this invention are cultured in suitable media, as described generally in Sambrook et al. , cited above.
  • Yeast expression systems can also be used to produce the subject
  • Expression and transformation vectors have been developed for transformation into many yeasts.
  • expression vectors have been developed for, among others, the following yeasts: Saccharomyces cerevisiae ,as described in Hinnen et al , Proc. Natl. Acad. Sci. USA (1978) 75: 1929; Ito et al , J. Bacteriol (1983) 153: 163; Candida albicans as described in Kurtz et al , Mol. Cell. Biol. (1986) 6: 142; Candida maltosa, as described in Kunze et al, J.
  • Control sequences for yeast vectors are known and include promoter regions from genes such as alcohol dehydrogenase (ADH), as described in EP 284,044, enolase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3- phosphate-dehydrogenase (GAP or GAPDH), hexokinase, phosphofructokinase, 3- phosphoglycerate mutase, and pyruvate kinase (PyK), as described in EP 329,203.
  • the yeast PH05 gene, encoding acid phosphatase also provides useful promoter sequences, as described in Myanohara et al , Proc. Natl.
  • promoter sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase, as described in Hitzeman et al. , J. Biol. Chem. (1980) 255: 2073, or other glycolytic enzymes, such as pyruvate decarboxylase, triosephosphate isomerase, and phosphoglucose isomerase, as described in Hess et al , J. Adv. Enzyme Reg. (1968) 7: 149 and Holland et al , Biochemistry (1978J 17: 4900.
  • Inducible yeast promoters having the additional advantage of transcription controlled by growth conditions, include from the list above and others the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization.
  • suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EP 073,657.
  • Yeast enhancers also are advantageously used with yeast promoters.
  • synthetic promoters which do not occur in nature also function as yeast promoters.
  • upstream activating sequences (UAS) of one yeast promoter may be joined with the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter.
  • hybrid promoters include the ADH regulatory sequence linked to the GAP transcription activation region, as described in U.S. Patent Nos. 4,876, 197 and 4,880,734.
  • Other examples of hybrid promoters include promoters which consist of the regulatory sequences of either the ADH2, GAL4, GAL10, or PHOS genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK, as described in EP 164,556.
  • a yeast promoter can include naturally occurring promoters of non-yeast origin that have the ability to bind yeast RNA polymerase and initiate transcription.
  • yeast expression vectors include terminators, for example, from GAPDH and from the enolase gene, as described in Holland et al , J. Biol. Chem. (1981) 256: 1385, and leader sequences which encode signal sequences for secretion.
  • DNA encoding suitable signal sequences can be derived from genes for secreted yeast proteins, such as the yeast invertase gene as described in EP 012,873 and JP 62,096,086 and the ⁇ -factor gene, as described in U.S. Patent Nos. 4,588,684, 4,546,083 and 4,870,008; EP 324,274; and WO 89/02463.
  • leaders of non-yeast origin such as an interferon leader, also provide for secretion in yeast, as described in EP 060,057.
  • Methods of introducing exogenous DNA into yeast hosts are well known in the art, and typically include either the transformation of spheroplasts or of intact yeast cells treated with alkali cations. Transformations into yeast can be carried out according to the method described in Van Solingen et al , J. Bact. (1977) 130: 946 and Hsiao et al , Proc. Natl. Acad. Sci. USA (1979) 76: 3829. However, other methods for introducing DNA into cells such as by nuclear injection, electroporation, or protoplast fusion may also be used as described generally in Sambrook et al. , cited above.
  • the native polypeptide signal sequence may be substituted by the yeast invertase, ⁇ -factor, or acid phosphatase leaders.
  • the origin of replication from the 2 ⁇ plasmid origin is suitable for yeast.
  • a suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid described in Kingsman et al , Gene (1979) 7: 141 or Tschemper et al , Gene (1980) 10: 157.
  • the trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan.
  • Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids bearing the Leu2 Gene.
  • a sequence encoding a yeast protein can be linked to a coding sequence of a Factor VIILC polypeptide analog to produce a fusion protein that can be cleaved intracellularly by the yeast cells upon expression.
  • a yeast leader sequence is the yeast ubiquitin gene.
  • Insect expression systems can also be used to produce the Factor VIILC polypeptide analogs and the nucleic acid sequences encoding the analogs.
  • baculovirus expression vectors are recombinant insect viruses in which the coding sequence for a foreign gene to be expressed is inserted behind a baculovirus promoter in place of a viral gene, e.g., polyhedrin, as described in Smith and Summers, U.S. Pat. No. , 4,745,051.
  • An expression construct herein includes a DNA vector useful as an intermediate for the infection or transformation of an insect cell system, the vector generally containing DNA coding for a baculovirus transcriptional promoter, optionally but preferably, followed downstream by an insect signal DNA sequence capable of directing secretion of a desired protein, and a site for insertion of the foreign gene encoding the foreign protein, the signal DNA sequence and the foreign gene being placed under the transcriptional control of a baculovirus promoter, the foreign gene herein being the coding sequence of a Factor VIILC polypeptide analog of this invention.
  • the promoter for use herein can be a baculovirus transcriptional promoter region derived from any of the over 500 baculoviruses generally infecting insects, such as, for example, the Orders Lepidoptera, Diptera, Orthoptera, Coleoptera and Hymenoptera including, for example, but not limited to the viral DNAs of Autographo californica MNPV, Bombyx mori NPV, rrichoplusia ni MNPV, Rachiplusia ou MNPV or Galleria mellonella MNPV, Aedes aegypti, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.
  • insects such as, for example, the Orders Lepidoptera, Diptera, Orthoptera, Coleoptera and Hymenoptera including, for example, but not limited to the viral DNAs of Autographo californica MNPV, Bombyx mori NPV, rricho
  • the baculovirus transcriptional promoter can be, for example, a baculovirus immediate-early gene IEI or IEN promoter; an immediate-early gene in combination with a baculovirus delayed-early gene promoter region selected from the group consisting of a 39K and a Hmdlll fragment containing a delayed-early gene; or a baculovirus late gene promoter.
  • the immediate-early or delayed-early promoters can be enhanced with transcriptional enhancer elements.
  • Particularly suitable for use herein is the strong polyhedrin promoter of the baculovirus, which directs a high level of expression of a DNA insert, as described in Friesen et al (1986) "The Regulation of Baculovirus Gene Expression” in: THE MOLECULAR BIOLOGY OF BACULOVIRUSES (W.Doerfler, ed.); EP 127,839 and EP 155,476; and the promoter from the gene encoding the plO protein, as described in Vlak et al , J. Gen. Virol. (1988) 69: 765-776.
  • the plasmid for use herein usually also contains the polyhedrin polyadenylation signal, as described in Miller et al , Ann. Rev. Microbiol (1988) 42: 177 and a procaryotic ampicillin-resistance (amp) gene and an origin of replication for selection and propagation in E. coli.
  • polyhedrin polyadenylation signal as described in Miller et al , Ann. Rev. Microbiol (1988) 42: 177 and a procaryotic ampicillin-resistance (amp) gene and an origin of replication for selection and propagation in E. coli.
  • DNA encoding suitable signal sequences can also be included and is generally derived from genes for secreted insect or baculovirus proteins, such as the baculovirus polyhedrin gene, as described in Carbonell et al, Gene (1988) 73: 409, as well as mammalian signal sequences such as those derived from genes encoding human ⁇ -interferon as described in Maeda et al , Nature (1985) 575: 592-594; human gastrin-releasing peptide, as described in Lebacq-Verheyden et al , Mol. Cell. Biol. (1988) 8: 3129; human IL-2, as described in Smith et al, Proc. Natl. Acad. Sci.
  • viral strains are publicly available, e.g. , the L-l variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV. Such viruses may be used as the virus for transfection of host cells such as Spodoptera frugiperda cells.
  • baculovirus genes in addition to the polyhedrin promoter may be used.
  • immediate-early genes include immediate-early (alpha), delayed-early (beta), late (gamma), or very late (delta), according to the phase of the viral infection during which they are expressed.
  • the expression of these genes occurs sequentially, probably as the result of a "cascade" mechanism of transcriptional regulation.
  • the immediate-early genes are 0 expressed immediately after infection, in the absence of other viral functions, and one or more of the resulting gene products induces transcription of the delayed-early genes.
  • Some delayed-early gene products induce transcription of late genes, and finally, the very late genes are expressed under the control of previously expressed gene products from one or more of the earlier 5 classes.
  • IEI Autographo californica nuclear polyhedrosis virus
  • Immediate-early genes as described above can be used in combination with a baculovirus gene promoter region of the delayed-early category. Unlike the immediate-early genes, such delayed-early genes require the presence of other viral genes or gene products such as those of the immediate-early genes.
  • the combination of immediate-early genes can be made with any of several delayed-early gene promoter regions such as 39K or one of the delayed-early gene promoters found on the HmdIII fragment of the baculovirus genome. In the present instance, the 39 K promoter region can be linked to the foreign gene to be expressed such that expression can be further controlled by the presence of IEI, as described in L. A.
  • enhancement of the expression of heterologous genes can be realized by the presence of an enhancer sequence in direct cis linkage with the delayed-early gene promoter region.
  • enhancer sequences are characterized by their enhancement of delayed-early gene expression in situations where the immediate-early gene or its product is limited.
  • the hr5 enhancer sequence can be linked directly, in cis, to the delayed-early gene promoter region, 39K, thereby enhancing the expression of the cloned heterologous DNA as described in Guarino and Summers (1986a), (1986b), and Guarino et al. (1986).
  • the polyhedrin gene is classified as a very late gene. Therefore, transcription from the polyhedrin promoter requires the previous expression of an unknown, but probably large number of other viral and cellular gene products. Because of this delayed expression of the polyhedrin promoter, state-of-the-art BEVs, such as the exemplary BEV system described by Smith and Summers in, for example, U.S. Pat. No. , 4,745,051 will express foreign genes only as a result of gene expression from the rest of the viral genome, and only after the viral infection is well underway. This represents a limitation to the use of existing BEVs. The ability of the host cell to process newly synthesized proteins decreases as the baculovirus infection progresses.
  • gene expression from the polyhedrin promoter occurs at a time when the host cell's ability to process newly synthesized proteins is potentially diminished for certain proteins such as human tissue plasminogen activator.
  • the expression of secretory glycoproteins in BEV systems is complicated due to incomplete secretion of the cloned gene product, thereby trapping the cloned gene product within the cell in an incompletely processed form.
  • an insect signal sequence can be used to express a foreign protein that can be cleaved to produce a mature protein
  • the present invention is preferably practiced with a mammalian signal sequence for example the Factor VIII signal sequence.
  • An exemplary insect signal sequence suitable herein is the sequence encoding for a Lepidopteran adipokinetic hormone (AKH) peptide.
  • the AKH family consists of short blocked neuropeptides that regulate energy substrate mobilization and metabolism in insects.
  • a DNA sequence coding for a Lepidopteran Manduca sexta AKH signal peptide can be used.
  • Other insect AKH signal peptides, such as those from the Orthoptera Schistocerca gregaria locus can also be employed to advantage.
  • Another exemplary insect signal sequence is the sequence coding for Drosophila cuticle proteins such as CPI, CP2, CP3 or CP4.
  • the desired DNA sequence can be inserted into the transfer vector, using known techniques.
  • An insect cell host can be cotransformed with the transfer vector containing the inserted desired DNA together with the genomic DNA of wild type baculovirus, usually by cotransfection.
  • the vector and viral genome are allowed to recombine resulting in a recombinant virus that can be easily identified and purified.
  • the packaged recombinant virus can be used to infect insect host cells to express a Factor VIILC polypeptide analog.
  • Mammalian expression systems can also be used to produce the Factor VIILC polypeptide analogs and nucleic acid sequences encoding the analogs.
  • Typical promoters for mammalian cell expression include the SV40 early promoter, the CMV promoter, the mouse mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP), and the he ⁇ es simplex virus promoter, among others.
  • Other non-viral promoters such as a promoter derived from the murine metallothionein gene, will also find use in mammalian constructs.
  • Mammalian expression may be either constitutive or regulated (inducible), depending on the promoter.
  • transcription termination and polyadenylation sequences will also be present, located 3' to the translation stop codon.
  • a sequence for optimization of initiation of translation located 5' to the Factor VIILC polypeptide analog coding sequence, is also present.
  • transcription terminator/polyadenylation signals include those derived from SV40, as described in Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2d edition, (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.).
  • Introns, containing splice donor and acceptor sites may also be designed into the constructs of the present invention.
  • Enhancer elements can also be used herein to increase expression levels of the mammalian constructs. Examples include the SV40 early gene enhancer, as described in Dijkema et al , EMBO J. (1985) 4: 761 and the enhancer/promoter derived from the long terminal repeat (LTR) of the Rous Sarcoma Virus, as described in Gorman et al. , Proc. Natl. Acad. Sci. USA (1982b) 79: 6777 and human cytomegalovirus, as described in Boshart et al , Cell (1985) 47: 521.
  • a leader sequence can also be present which includes a sequence encoding a signal peptide, to provide for the secretion of the foreign protein in mammalian cells. Alternatively, the Factor VIII signal peptide can be used.
  • adenovirus tripartite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
  • expression vectors that provide for the transient expression in mammalian cells of DNA encoding the Factor VIILC analog polypeptides. In general, transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in turn, synthesizes high levels of a desired polypeptide encoded by the expression vector.
  • Transient expression systems comprising a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded by cloned DNAs, as well as for the rapid screening of such polypeptides for desired biological or physiological properties.
  • transient expression systems are particularly useful for pu ⁇ oses of identifying additional polypeptides that have Factor VIILC-like activity.
  • the mammalian expression vectors can be used to transform any of several mammalian cells.
  • Methods for introduction of heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
  • dextran-mediated transfection calcium phosphate precipitation
  • polybrene mediated transfection protoplast fusion
  • electroporation encapsulation of the polynucleotide(s) in liposomes
  • direct microinjection of the DNA into nuclei General aspects of mammalian cell host system transformations have been described by Axel in U.S. 4,399,216.
  • a synthetic lipid particularly useful for polynucleotide transfection is N-[l-(2,3-dioleyloxy)propyl]-N,N,N-tri- methylammonium chloride, which is commercially available under the
  • Lipofectin ® (available from BRL, Gaithersburg, MD), and is described by Feigner et al , Proc. Natl. Acad. Sci. USA (1987) 84:7413.
  • Mammalian cell lines available as hosts for expression are also known and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g. , Hep G2), human embryonic kidney cells, baby hamster kidney cells, mouse sertoli cells, canine kidney cells, buffalo rat liver cells, human lung cells, human liver cells, mouse mammary tumor cells, as well as others.
  • ATCC American Type Culture Collection
  • the mammalian host cells used to produce the target polypeptide of this invention may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM1, Sigma) are suitable for culturing the host cells.
  • WO 90/103430 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors such as insulin, transferrin, or epidermal growth factor, salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as Gentamycin(tm) M drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art.
  • the culture conditions such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.
  • the active Factor VIILC analogs produced according to the invention have a variety of uses.
  • the analogs can be used as immunogens for the production of antibodies.
  • the analogs can also be used for the treatment of hemophiliacs and other hosts having blood clotting disorders.
  • the Factor VIILC analogs may display increased plasma half-life or specific activity.
  • the analogs may allow for lower dosages or alternative modes of administration and may improve hemostasis in hemophiliacs.
  • nucleic acid molecules or vectors comprising polynucleotide sequences encoding the Factor VIILC analogs can be used directly for gene therapy and administered using standard gene delivery protocols.
  • the nucleotide sequences encoding the Factor VIILC analogs can be stably integrated into the host cell genome or maintained on a stable episomal element in the host cell. Methods for gene delivery are known in the art. See, e.g. , U.S. Patent No. 5,399,346.
  • retroviral systems have been developed for gene transfer into mammalian cells.
  • retroviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo.
  • retroviral systems have been described (U.S. Patent No. 5,219,740; Miller and Rosman, BioTechniques (1989) 7:980-990; Miller, A.D., Human Gene Therapy (1990) 7:5-14; Sca ⁇ a et al , Virology (1991) 780:849-852; Burns et al , Proc. Natl.
  • adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J. Virol. (1986) 57:267-214; Bett et al. , J. Virol. (1993) 57:5911-5921 ; Mittereder et al , Human Gene Therapy (1994) 5:717-729; Seth et al , J. Virol.
  • AAV vector systems have been developed for gene delivery. Such systems can include control sequences, such as promoter and polyadenylation sites, as well as selectable markers or reporter genes, enhancer sequences, and other control elements which allow for the induction of transcription.
  • AAV vectors can be readily constructed using techniques well known in the art. See, e.g., U.S. Patent Nos. 5, 173,414 and 5, 139,941 ; International Publication Nos. WO 92/01070 (published 23 January 1992) and WO 93/03769 (published 4 March 1993); Lebkowski et al , Molec. Cell. Biol.
  • vaccinia virus recombinants expressing the novel Factor VIILC analogs can be constructed as follows. The DNA encoding the particular analog is first inserted into an appropriate vector so that it is adjacent to a vaccinia promoter and flanking vaccinia DNA sequences, such as the sequence encoding thymidine kinase (TK). This vector is then used to transfect cells which are simultaneously infected with vaccinia.
  • TK thymidine kinase
  • Homologous recombination serves to insert the vaccinia promoter plus the gene encoding the instant protein into the viral genome.
  • the resulting TK ' recombinant can be selected by culturing the cells in the presence of 5-bromodeoxyuridine and picking viral plaques resistant thereto.
  • a vaccinia based infection/transfection system can be conveniently used to provide for inducible, transient expression of the Factor VIILC analogs in a host cell.
  • cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase.
  • This polymerase displays extraordinar specificity in that it only transcribes templates bearing T7 promoters.
  • cells are transfected with the polynucleotide of interest, driven by a T7 promoter.
  • the polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery.
  • the method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation products. See, e.g., Elroy-Stein and Moss, Proc. Natl. Acad. Sci. USA (1990) 87:6743-6747; Fuerst et al , Proc. Natl. Acad. Sci. USA (1986) 85:8122-8126.
  • avipox viruses such as the fowlpox and canarypox viruses
  • Recombinant avipox viruses expressing immunogens from mammalian pathogens, are known to confer protective immunity when administered to non-avian species.
  • the use of an avipox vector is particularly desirable in human and other mammalian species since members of the avipox genus can only productively replicate in susceptible avian species and therefore are not infective in mammalian cells.
  • Methods for producing recombinant avipoxviruses are known in the art and employ genetic recombination, as described above with respect to the production of vaccinia viruses. See, e.g., WO 91/12882; WO 89/03429; and WO 92/03545.
  • Molecular conjugate vectors such as the adenovirus chimeric vectors described in Michael et al. , J. Biol. Chem. (1993) 268:6866-6869 and Wagner et al , Proc. Natl. Acad. Sci. USA (1992) 89:6099-6103, can also be used for gene delivery.
  • an amplification system can be used that will lead to high level expression following introduction into host cells.
  • a T7 RNA polymerase promoter preceding the coding region for T7 RNA polymerase can be engineered. Translation of RNA derived from this template will generate T7 RNA polymerase which in turn will transcribe more template. Concomitantly, there will be a cDNA whose expression is under the control of the T7 promoter. Thus, some of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to transcription of the desired gene.
  • T7 RNA polymerase can be introduced into cells along with the template(s) to prime the transcription reaction.
  • the amplification template can be generated by PCR techniques.
  • the use of a plasmid is prefe ⁇ ed. Since high level expression of T7 RNA polymerase appears to be lethal to host cells, the plasmid should be one where expression of T7 RNA polymerase can be controlled.
  • a lac operator can be engineered distal or proximal (or both) to the T7 promoter. The binding of the preexisting lac repressor in the appropriate bacterial strain would interfere with the transcription of the template by blocking access to the promoter by T7 RNA polymerase.
  • a plasmid can be constructed where transcription from a bacterial promoter begins 3' of the T7 gene and continues through the 5' end of the T7 promoter. Such transcription will generate an antisense transcript and reduce or eliminate translation of T7 RNA polymerase RNAs.
  • the second transcription unit consisting of the T7 promoter preceding the gene of interest can be provided by a separate plasmid or can be engineered onto the amplification plasmid. Colocalization of the two transcription units is beneficial for ease of manufacturing and ensures that both transcription units will always be together in the cells into which the plasmid is introduced.
  • the T7 RNA polymerase plasmids may include UTRs which comprise an Internal Ribosome Entry Site (IRES) present in the leader sequences of picornaviruses such as the encephalomyocarditis virus (EMCV) UTR (Jang et al. J. Virol. (1989) 65: 1651-1660).
  • IRS Internal Ribosome Entry Site
  • EMCV encephalomyocarditis virus
  • Vectors encoding the subject Factor VIILC analogs can also be packaged in liposomes prior to delivery to the subject or to cells derived therefrom.
  • Lipid encapsulation is generally accomplished using liposomes which are able to stably bind or entrap and retain nucleic acid.
  • the ratio of condensed DNA to lipid preparation can vary but will generally be around 1 : 1 (mg
  • DNA micromoles lipid
  • liposomes as carriers for delivery of nucleic acids, see, Hug and Sleight, Biochim. Biophys. Acta. (1991) 7097: 1-17; Straubinger et al , in METHODS OF ENZYMOLOGY (1983), Vol. 101 , pp. 512-527.
  • Liposomal preparations for use in the instant invention include cationic (positively charged), anionic (negatively charged) and neutral preparations, with cationic liposomes particularly prefened. Cationic liposomes have been shown to mediate intracellular delivery of plasmid DNA (Feigner et al , Proc. Natl. Acad.
  • Cationic liposomes are readily available.
  • N[l-2,3- dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY. (See, also, Feigner et al , Proc. Natl. Acad. Sci. USA (1987) 84:7413-7416).
  • Other commercially available liposomes include transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger).
  • Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g.
  • DOTAP 1,2- bis(oleoyloxy)-3-(trimethylammonio)propane
  • anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL), or can be easily prepared using readily available materials.
  • Such materials include phosphatidyl choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC), dioleoylphosphatidyl glycerol (DOPG), dioleoylphoshatidyl ethanolamine (DOPE), among others. These materials can also be mixed with the DOTMA and DOTAP starting materials in appropriate ratios. Methods for making liposomes using these materials are well known in the art.
  • the liposomes can comprise multilammelar vesicles (MLVs), small unilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs).
  • MLVs multilammelar vesicles
  • SUVs small unilamellar vesicles
  • LUVs large unilamellar vesicles
  • the various liposome-nucleic acid complexes are prepared using methods known in the art. See, e.g., Straubinger et al , in METHODS OF IMMUNOLOGY (1983), Vol. 101 , pp. 512-527; Szoka et al , Proc. Natl. Acad. Sci. USA (1978) 75:4194-4198; Papahadjopoulos et al , Biochim. Biophys.
  • the recombinant vectors may be administered in pharmaceutical compositions as described above.
  • the pharmaceutical compositions will comprise sufficient genetic material to produce a therapeutically effective amount of the analog or analogs, as described above.
  • an effective dose will be from about 0.05 mg/kg to about 50 mg/kg of the DNA constructs in the individual to which it is administered.
  • the compositions of the invention can be administered directly to the subject or, alternatively, in the case of the vectors described above, delivered ex vivo, to cells derived from the subject. Methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and described in e.g., International Publication No.
  • WO 93/14778 published 5 August 1993.
  • such methods will include dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei, all well known in the art.
  • Direct delivery of the compositions will generally be accomplished by injection, either subcutaneously, intraperitoneally, intravenously or intramuscularly.
  • Other modes of administration include oral and pulmonary administration, suppositories, and transdermal applications.
  • Dosage treatment may be a single dose schedule or a multiple dose schedule.
  • the Factor VIILC polypeptide analogs are made using conventional mutagenesis techniques.
  • mutagenesis of the Factor VIILC nucleotide sequences can be performed utilizing plasmids which include sequences encoding the full-length molecule, plasmids encoding the light and heavy chains and various modifications of these molecules, depending on the Factor VIILC analog desired.
  • plasmids are known and described in, e.g. , U.S. Patent no. 5,045,455.
  • the plasmids are first linearized e.g., by using restriction endonucieases which cleave at unique restriction sites.
  • the plasmids are treated with calf intestine phosphatase and separated on low melting temperature tris-acetate agarose gels. The linearized band is extracted by adso ⁇ tion to silica dioxide and eluted in tris-EDTA. The plasmid is then denatured and the desired phosphorylated mutagenic oligonucleotide is added. The mixture is heated and allowed to slowly cool at room temperature.
  • a heteroduplex oligonucleotide mixture can be used and the reactions made with, e.g., 2 mM MgCl 2 , lmM beta-mercaptoethanol, 400 ⁇ M ATP, 100 M deoxynucleotide triphosphate, 3-4 units/ ⁇ L of Klenow fragment of E. coli DNA polymerase I and 400 units/ ⁇ L of T4 DNA ligase.
  • the reactions are terminated using phenolchloroform extraction and ethanol precipitation.
  • DNA obtained is used to transform bacterial host cells and positive clones selected.
  • DNA from the clones is transfe ⁇ ed to nitrocellulose, filters prepared, and hybridized to screening probes to ensure that the mutagenic oligonucleotide is introduced into the co ⁇ ect fragment.
  • Final mutations are confirmed by DNA sequencing.
  • the DNA can be prepared by banding in CsCl and can be used to transfect COS-1 monkey cells as described in Kaufman, PNAS (1982) 82:689. After transfection, the polypeptide analog is isolated and Factor VIILC activity is assayed by the Kabi Coatest chromagenic assay method for the ability to clot Factor VIII deficient plasma before and after thrombin activation.
  • ⁇ xample 1 Construction of a Factor VIILC Polypeptide Analog His242 Residue 270 of Factor VIILC is mutated to a His using the oligonucleotide GAA GTG CAC TCA ATA CAX CTC GAA GGT CAC ACA.
  • the TTC encoding for Phe which normally occurs at the underlined position is mutated to form the codon for His which can be CAX where X is T or C.
  • the mutagenesis is carried out, the co ⁇ ect sequence is confirmed, and the analog produced and tested for activity, as described above.
  • Residue 1786 of Factor VIILC is mutated to a His using the oligonucleotide CGT CCC TAT TCC TTC CAX TCT AGC CTT ATT TCT.
  • the TAT encoding for Tyr which normally occurs at the underlined position is mutated to form the codon for His which can be CAX where X is T or C.
  • the mutagenesis is carried out and the resulting cDNA prepared and transfected, as described above. The analog is assayed also as described above.
  • Factor VIILC polypeptide analogs can be constructed and assayed as described above for the analogs His270, His652 and His 1786.

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Abstract

L'invention concerne des analogues de polypeptides du facteur VIII:C qui sont des polypeptides natifs du facteur VIII:C contenant des parties modifiées dans lesquelles sont présentes au moins un domaine ou un sous-domaine A et/ou C du facteur V. Elle concerne également des molécules d'acide nucléique codant ces analogues de polypeptides du facteur VIII:C, des vecteurs et des cellules hôtes contenant lesdites molécules d'acide nucléique. Elle concerne encore des complexes d'analogues contenant au moins deux de ces analogues. Elle concerne enfin des procédés de production de l'analogue, du complexe d'analogues, des acides nucléiques, des vecteurs et des cellules hôtes, ainsi que des procédés d'utilisation de ces compositions dans la prévention ou dans le traitement de déficiences en polypeptides du facteur VIII:C.
PCT/US1996/011013 1995-07-11 1996-06-28 Nouveaux analogues de polypeptides du facteur viii:c comprenant des domaines ou des sous-domaines du facteur v WO1997003191A1 (fr)

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US6180371B1 (en) 1996-06-26 2001-01-30 Emory University Modified factor VIII
US6458563B1 (en) 1996-06-26 2002-10-01 Emory University Modified factor VIII
US7560107B2 (en) 1996-06-26 2009-07-14 Emory University Modified factor VIII
US7576181B2 (en) 2004-05-03 2009-08-18 Ipsen Biopharm Limited Method of administering porcine B-domainless fVIII
EP2206785A1 (fr) 1998-12-31 2010-07-14 Novartis Vaccines and Diagnostics, Inc. Expression améliorée de polypeptides HIV et production de particules de type virus
US9150637B2 (en) 2010-11-05 2015-10-06 Baxalta Inc. Variant of antihemophilic factor VIII having increased specific activity

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US8951515B2 (en) 1996-06-26 2015-02-10 Emory University Modified factor VIII
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US7560107B2 (en) 1996-06-26 2009-07-14 Emory University Modified factor VIII
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US8501694B2 (en) 2004-05-03 2013-08-06 Emory University Method of administering porcine B-domainless fVIII
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US9150637B2 (en) 2010-11-05 2015-10-06 Baxalta Inc. Variant of antihemophilic factor VIII having increased specific activity
US10053500B2 (en) 2010-11-05 2018-08-21 Baxalta Incorporated Variant of antihemophilic factor VIII having increased specific activity

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