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WO1997003195A1 - Nouveaux analogues polypeptidiques de facteur viii:c ayant des sites de protease modifies - Google Patents

Nouveaux analogues polypeptidiques de facteur viii:c ayant des sites de protease modifies Download PDF

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Publication number
WO1997003195A1
WO1997003195A1 PCT/US1996/011444 US9611444W WO9703195A1 WO 1997003195 A1 WO1997003195 A1 WO 1997003195A1 US 9611444 W US9611444 W US 9611444W WO 9703195 A1 WO9703195 A1 WO 9703195A1
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Prior art keywords
residue
factor viii
deletion
pro
analog
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PCT/US1996/011444
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English (en)
Inventor
David T. Hung
Fred E. Cohen
Michael Innis
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Chiron Corporation
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Application filed by Chiron Corporation filed Critical Chiron Corporation
Priority to AU64861/96A priority Critical patent/AU6486196A/en
Publication of WO1997003195A1 publication Critical patent/WO1997003195A1/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 to the discovery that an active Factor VIII.C polypeptide analog can be made that is modified at a site adjacent to an arginine ("Arg") residue, where the Arg is at a site other than at an activating site, creating an arginine-proline (“Arg-Pro”) linkage or a proline-arginine (“Pro-Arg”) linkage.
  • Arg arginine residue
  • Pro-Arg proline-arginine
  • This invention also relates to a Factor V II.C polypeptide analog with a modification that comprises creation of a tripeptide having the formula P 3 -P 2 ⁇ p i wherein P 3 is a residue selected from the group consisting of Phe, Glu, and Pro; P 2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and P is Arg. Additionally, the invention pertains to substitutions at the non-activating Arg residues occurring at positions Arg336, Argl719 and/or Argl721.
  • This invention further relates to an analog complex comprising at least two such analogs, or one such analog and a native Factor VIII.C 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 VIII:C 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 VIII:C.
  • the disease afflicts approximately 1 in 10,000 males.
  • Factor VIII:C 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 VIII:C determined from the cloned cDNA indicates that Factor VIII:C is a heterodimer processed from a larger precursor polypeptide consisting of 2351 amino acids, referred to herein as the precursor or full- length Factor VIII:C molecule, of which the first 19 N- terminal residues comprise the signal sequence. Therefore, the mature Factor VIII.C 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 VIII:C 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 VIII:C molecule comprise a "light chain" with a molecular weight of approximately 80 kD ("the 80 kD subunit") .
  • the heterodimeric mature Factor VIII:C molecule consi ⁇ t ⁇ of the heavy and light chains as ⁇ ociated by a metal ion bridge, and lacking amino acid ⁇ 741-1648 (the B domain) .
  • the mature Factor VIII.C molecule con ⁇ i ⁇ ts 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 NH 2 -A1-A2-B-A3-C1-C2- COOH.
  • Factor VIII.C is known to be activated by plasma proteases such as thrombin.
  • the mature Factor VIII.C 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”)
  • Factor VlllrC Patients suffering from hemophilia A are conventionally treated with purified or substantially purified Factor VlllrC.
  • a difficulty in such treatment is the relatively short half-life of externally administered Factor VIII.C, lasting about 8 to 12 hours.
  • This instability of Factor VIII:C derives in part from its susceptibility to proteolytic cleavage by plasma protease.
  • plasma proteases for example, thrombin, Factor Xa, and activated protein C (“APC”) , inactivate Factor VIII.C by cleaving the molecule at multiple sites. It would be advantageous, therefore, to produce Factor VIII:C polypeptides with improved properties.
  • an active Factor VIII.C polypeptide analog that is substantially the same as a native Factor VIII.C polypeptide, except for modification at a site that is adjacent to a non-activating Arg residue, that is, adjacent to an Arg residue that is not at an activation site, such as Argl689 or Arg372.
  • the modification of the present invention creates an Arg-Pro linkage or a Pro-Arg linkage. Such a modification can be achieved by one or more amino acid substitutions, additions or deletions.
  • non-activating Arg residue is at least one selected from the group consisting of amino acid residues 220, 226, 250, 279, 282, 336, 359, 562, 747, 776, 1310, 1313, 1645, 1648, 1719, and 1721, numbered with respect to the native Factor VIII:C polypeptide sequence, as depicted in Figures 1A-1F.
  • an analog with a modification that comprises creation of a tripeptide having the formula P 3 ⁇ P 2 -p ⁇ # wherein P 3 is a residue selected from the group consisting of Phe, Glu, and Pro; P 2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and P ⁇ is Arg.
  • an active Factor VIII:C polypeptide analog comprising a native Factor VIII.C polypeptide that is modified at at least one non- activating Arg residue selected from the group consisting of Arg336, Argl719 and Argl721, wherein the modification comprises a substitution of any of amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr for Arg336, a substitution of any of amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr for Argl719 and/or a substitution of any of amino acids Glu, Asp, Asn, Gin, Ser and Tyr for Argl721, numbered with respect to the native Factor VIII:C polypeptide sequence.
  • the native Factor VIII.C polypeptide that is modified is selected from the group consisting of (a) a full-length Factor VIII.C molecule comprising a signal peptide and all A, B, and C domains; (b) a native Factor VIII:C molecule comprising all A, B, and C domains and lacking a signal peptide; (c) a truncated Factor VIII.C molecule lacking a signal peptide and at least a portion of the B domain; (d) a cleaved Factor VIII:C molecule containing a light chain subunit of molecular weight of about 80 kD; (e) a cleaved Factor VIII.C molecule containing a heavy chain fragment of molecular weight in a range of about 90 kD to about 200 kD; (f) a cleaved Factor VIII.C molecule comprising a heavy chain fragment of a mo
  • Factor VIII.C molecule containing a heavy chain fragment of molecular weight of about 50 kD (h) a cleaved Factor VIII.C molecule containing a heavy chain fragment of molecular weight of about 43 kD; and (i) a cleaved Factor VIII.C molecule containing a light chain fragment of molecular weight of about 73 kD.
  • an active Factor VIII.C analog complex that contains either at least two Factor VIII.C polypeptide analogs as above, or a Factor VIII.C polypeptide analog and a Factor VIII.C polypeptide, together with a metal ion.
  • the analog complex herein can comprise two Factor VIII.C 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 (f) 73 kD and 43 kD.
  • a method of producing a Factor VIII.C polypeptide analog as above by (a) providing a native Factor VIII.C polypeptide that contains an amino acid sequence, and (b) modifying at least one amino acid re ⁇ idue in the amino acid ⁇ equence to produce an analog as above.
  • nucleic acid molecule that contains a nucleotide sequence that encodes an 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 a nucleic acid molecule or recombinant vector as above.
  • a method of producing an active Factor VIII.C 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 a nucleic acid molecule as above comprising: (a) providing a nucleic acid molecule that encodes a native Factor VIII:C polypeptide as above and (b) modifying at least one codon to provide the analog.
  • a method of producing a recombinant vector that contains a 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 a recombinant vector.
  • composition that contains an active Factor VIII:C polypeptide analog or analog complex as above, and a pharmaceutically acceptable excipient.
  • methods for prevention or treatment of active Factor VIII:C deficiency in a mammal comprising administering thereto a therapeutically effective amount of (a) an active Factor VIII:C polypeptide analog as above, or (b) an active Factor VIII:C polypeptide analog complex as above, or (c) a nucleic acid molecule as above, or (d) a recombinant vector as above, or (e) a nucleic acid molecule as above together with and an active Factor VIII:C polypeptide analog, or (f) a recombinant vector as above together with an active Factor VIII:C polypeptide analog.
  • Factor VIII:C polypeptide analogs can be made that have improved properties. These analogs include one or more amino acid residues that are modified from the native structure. The modification can be at least one amino acid substitution, addition or deletion, at an amino acid residue adjacent to an Arg residue so as to generate, for example, an Arg-Pro linkage or a Pro-Arg linkage.
  • the Factor VIII:C polypeptide analog can also include a modification that comprises creation of a tripeptide having the formula P 3 _ P 2 ⁇ p ⁇ , wherein P 3 is a residue selected from the group consisting of Phe, Glu, and Pro; P 2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and P 2 is Arg.
  • P 3 is a residue selected from the group consisting of Phe, Glu, and Pro
  • P 2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720
  • P 2 is Arg.
  • the above modifications do not occur at a site of Factor VIII:C activation.
  • non-activating Arg residues, Arg336 and Argl719 can be substituted with amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr and/or the non-activating Arg residue Argl721 can be substituted with Glu, Asp, Asn, Gin, Ser and Tyr, to impart a Factor VIII:C analog with improved properties.
  • Fractor VIII:C 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 sub ⁇ tituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring.
  • a Factor VIII:C polypeptide includes but is not limited to, for example, the following Factor VIII:C polypeptides: (a) a full-length Factor VIII:C molecule comprising a signal peptide and all A, B, and C domains; (b) a mature Factor VIII:C molecule comprising all A, B, and C domains and lacking the signal peptide; (c) a truncated Factor VIII:C molecule lacking the signal peptide and at least a portion of the B domain; (d) a cleaved Factor VIII:C molecule comprising a light chain subunit of about 80 kD; (e) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of about 90 kD; (f) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of about 50 kD; (g) a cleaved Factor VIII:C molecule comprising a heavy chain fragment of about 43 k
  • Factor VIII:C 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/Trp/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 VIII:C 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 VIII:C molecule.
  • a "nucleic acid molecule” as used herein, 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 viru ⁇ or viral vector or from a linear or circular plasmid.
  • regulatory control refers to control of expres ⁇ ion of a polynucleotide ⁇ equence by a regulatory element to which the polynucleotide sequence is operably linked.
  • the nature of such regulatory control differs depending upon the host organism.
  • prokaryotes such regulatory control is effected by regulatory sequences which generally include, for example, a promoter, and/or a transcription termination sequence.
  • eukaryotes generally, such regulatory sequences include, for example, a promoter and/or a transcription termination sequence.
  • 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 VIII:C analogs of the invention may be determined by means known in the art, for example, by using the commercially available Coate ⁇ t assay.
  • the effective amount is ⁇ ufficient to bring about prevention of further deterioration or treatment to improve coagulation, that is, to enhance coagulation properties such that hemostasis is achieved.
  • 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 ⁇ ubstances 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, polysaccharide ⁇ , 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 i ⁇ available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 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 VIII:C polypeptide analogs with improved properties.
  • the analogs include Pro-Arg bonds or Arg-Pro bonds at non- activating Arg residues.
  • the modification will not introduce an Arg-Ala, Arg-Met, Arg-Gly, Arg-Ser or Arg-Thr bond at the modified site.
  • the modification can comprise creation of a tripeptide having the formula 3 _ p 2 ⁇ p i' wherein P 3 is a residue selected from the group consisting of Phe, Glu, and Pro; P 2 is any amino acid residue except Ser and P2 is not Leu335 and is not Asnl720; and P-L is Arg.
  • Arg residue when the non-activating Arg residue is Arg220, representative modification ⁇ can compri ⁇ e an insertion of at least one Pro residue between Asp219 and Arg 220; an insertion of at least one Pro residue between Arg220 and Asp221; a deletion comprising residues Asp221, Ala222, Ala223, Ser224, Ala225, Arg226, Ala227, and Trp228; an insertion of at least one Phe residue between Gln218 and Asp219; an insertion of at least one Glu residue between Gln218 and Asp219; an insertion of at least one Pro residue between Gln218 and Asp219; a deletion comprising residues Thr212, Lys213, Asn214, Ser215, Leu216, Met217, and Gln218; a ⁇ ub ⁇ titution of at least one Phe residue at Gln218; a substitution of at least one Glu residue at Gln218; and/
  • representative modifications can comprise an insertion of at least one Pro residue between Ala225 and Arg226; an insertion of at least one Pro residue between Arg226 and Ala227; a deletion comprising residues Ala227 and Trp228; a deletion comprising residues Ala227 and Trp228 and insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Ala227, Trp228, Pro229, and Lys230, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Phe residue between Ser224 and Ala225; an insertion of at least one Glu residue between Ser224 and Ala225; an insertion of at least one Pro residue between Ser224 and Ala225; (d) a substitution of at least one Phe residue at Ser224; a substitution of at least one Glu residue at Ser224; and/or a substitution of at least one Pro residue at Ser224.
  • Arg250 When the non-activating Arg residue is Arg250, representative modifications can comprise an insertion of at least one Pro residue between His249 and Arg250; an insertion of at least one Pro residue between Arg250 and Lys251; a deletion comprising residues Lys251 and Ser252 and insertion of at least one Pro residue to replace the deleted residues; a substitution of Lys251 with at lea ⁇ t one Pro residue; a deletion comprising residues Gly244, Leu245, Ile246, Gly247, Cys248, and His249; a deletion comprising residues Arg240, Ser241, Leu242, Pro243, Gly244, Leu245, Ile246, Gly247, Cys248, and Hi ⁇ 249 and an in ⁇ ertion of at least one Pro residue to replace the deleted residue ⁇ ; an in ⁇ ertion of at lea ⁇ t one Phe re ⁇ idue between Cy ⁇ 248 and His249; an insertion of at least one Glu residue between Cys248 and Hi ⁇
  • Arg279 When the non-activating Arg residue is Arg279, representative modifications can comprise an insertion of at least one Pro residue between Val278 and Arg279; an insertion of at least one Pro residue between Arg279 and Asn280; a deletion comprising residues Asn280, His281, and Arg282 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising Asn280, His281, Arg282, Gln283, Ala284, and Ser285, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at lea ⁇ t one Phe re ⁇ idue between Leu277 and Val278; an in ⁇ ertion of at least one Glu residue between Leu277 and Val278; an insertion of at least one Pro residue between Leu277 and Val278; a deletion comprising residue Leu277; a deletion comprising residue Val278; a deletion comprising residues Gly273, His274, Thr275, Phe27
  • Arg282 When the non-activating Arg residue is Arg282, representative modifications can comprise an insertion of at least one Pro residue between His281 and Arg282; an insertion of at least one Pro residue between Arg282 and Gln283; a deletion comprising residues Arg279, Asn280, and His281 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Gln283, Ala284, and Ser285, and an insertion of at least one Pro residue to replace the deleted residue ⁇ ; a deletion comprising residues Gln283, Ala284, Ser285, Leu286, Glu287, Ile288, and Ser289; an insertion of at least one Phe residue between Asn280 and His281; an insertion of at least one Glu residue between Asn280 and His281; an in ⁇ ertion of at least one Pro residue between Asn280 and His281; a deletion comprising residues Leu277, Val278, Arg279, and Asn
  • Arg336 When the non-activating Arg re ⁇ idue is Arg336, representative modifications can compri ⁇ e an insertion of at least one Pro residue between Leu335 and Arg336; a deletion comprising residues Gln334, and Leu335; an insertion of at least one Pro residue between Arg336 and Met337; a deletion comprising residues Met337, and Lys338 and insertion of at least one Pro residue in place of the deleted residues; a deletion comprising residue Leu335 and an in ⁇ ertion of at least one Phe residue between Pro333 and Gln334; a deletion comprising re ⁇ idue Leu335 and an in ⁇ ertion of at least one Glu residue between Pro333 and Gln334; a deletion comprising residue Leu335 and an insertion of at least one Pro residue between
  • Pro333 and Gln334 a deletion comprising residue Leu335; a deletion comprising residue ⁇ Gln334, and Leu335; a deletion comprising residues Pro333, Gln334, and Leu335; a deletion comprising residues Glu332, Pro333, Gln334, and Leu335; a deletion comprising residue Leu335 and a sub ⁇ titution of at least one Phe residue at Pro333; and/or a deletion comprising residue Leu335 and a substitution of at least one Glu residue at Pro333.
  • Arg359 When the non-activating Arg residue is Arg359, representative modifications can comprise an insertion of at least one Pro residue between Val358 and Arg359; an insertion of at least one Pro residue between Arg359 and Phe360; a deletion comprising residues Phe360, Asp361, A ⁇ p362, A ⁇ p363, A ⁇ n364, and Ser365; a deletion compri ⁇ ing residues Phe360, Asp361, A ⁇ p362, A ⁇ p363, Asn364, Ser365, Pro366, and Ser367, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Phe residue between Val357 and Val358; an insertion of at least one Glu residue between Val357 and Val358; an insertion of at least one Pro residue between Val357 and Val358; a deletion comprising residues Met355, Asp356, and Val357; a substitution of at least one Phe residue at Val357; a substitution of at least one Glu
  • Arg562 When the non-activating Arg residue is Arg562, representative modifications can comprise an insertion of at least one Pro residue between Gln561 and Arg562; an insertion of at least one Pro residue between Arg562 and Gly563; a deletion comprising residues Ser558, Val559, Asp560, Gln561, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Lys556, Glu557, Ser558, Val559, Asp560, Gln561, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Gly563, Asn564, Gln565, Ile566, Met567, and Ser568, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Gly563, Asn564, Gln565, Ile566, Met567, Ser568, Asp569, Lys570, and Arg571, and an insertion of at
  • representative modifications can comprise an insertion of at least one Pro residue between Ser746 and Arg747; a deletion comprising residue Ser746 and an insertion of at least one Pro residue to replace the deleted residue; a deletion comprising residue His748; a deletion comprising residue His748 and an insertion of at least one Pro residue to replace the deleted residue; an insertion of at least one Pro residue between Arg747 and His748; a deletion comprising residues His748, Pro749, and Ser750 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising Ser743, Gln744, Asn745, Ser746; a deletion comprising His748, Pro749, Ser750, Thr751, Arg752, Gln753, and Lys754, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residue Ser746 and an insertion of at least one Phe residue between Gln744 and Asn745;
  • Arg776 When the non-activating Arg residue is Arg776, representative modifications can comprise an insertion of at least one Pro residue between His775 and Arg776; an insertion of at least one Pro residue between Arg776 and Thr777; a deletion compri ⁇ ing re ⁇ idue Thr777; a deletion compri ⁇ ing re ⁇ idue Thr777 and an insertion of at least one Pro residue to replace the deleted residue; a deletion comprising residues Trp772, Phe773, Ala774, and His775; a deletion comprising residues Trp772, Phe773, Ala774, and His775, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Lys768, Thr769, Asp770, Pro771,
  • Argl310 When the non-activating Arg residue is Argl310, representative modifications can comprise an insertion of at least one Pro residue between Glnl309 and Argl310; an insertion of at least one Pro residue between Argl310 and Serl311; a deletion comprising residue Serl311 and an insertion of at least one Pro residue to replace the residue; a deletion comprising residues Ser1311, Lysl312, and Argl313 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Serl311, Lysl312, Argl313, Alal314, Leul315, and Lysl316, and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Serl311, Lysl312, Argl313, Alal314, Leul315, Lysl316, Glnl317, Phel318, Argl319, and Leul320; a deletion comprising residues Serl311, Lysl312,
  • Argl313 When the non-activating Arg residue is Argl313, representative modifications can comprise an insertion of at least one Pro residue between Lysl312 and Argl313; a deletion comprising residue Lysl312 and an insertion of at least one Pro residue to replace the deleted residue; a deletion compri ⁇ ing residues Argl310, Serl311, and Lysl312, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at lea ⁇ t one Pro re ⁇ idue between Argl313 and Alal314; a deletion comprising residues Alal314, Leul315, and Lysl316, and an in ⁇ ertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Alal314, Leul315, Lysl316, Glnl317, Phel318, and Argl319 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Alal314, Leul315,
  • representative modifications can comprise a deletion comprising residues Vall642, Leul643, and Lysl644; a deleteion compri ⁇ ing residue Lysl644 and an in ⁇ ertion of at lea ⁇ t one Pro residue to replace the deleted residue; an insertion of at least one Pro residue between Argl645 and Lysl644; an insertion of at least one Pro re ⁇ idue between Argl645 and Hisl646; a deletion comprising residues Hisl646, Glnl647, and Argl648 and an in ⁇ ertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Hisl646, Glnl647, Argl648, Glul649, Ilel650, Thrl651, and Argl652, and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Phe residue between Leul643 and Ly ⁇
  • Argl648 When the non-activating Arg residue is Argl648, representative modifications include a deletion comprising residues Vall642, Leul643, Lysl644, Argl645, Hisl646, and Glnl647; an insertion of at least one Pro residue between Glnl647 and Argl648; a deletion comprising residues Lysl644, Argl645, Hisl646, and Glnl647 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Glul649, Ilel650, Thrl651, and Argl652 and an insertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Pro residue between Argl648 and Glul649; an insertion of at least one Phe residue between Hisl646 and Glnl647; an insertion of at least one Glu residue between Hisl646 and Glnl647; an in ⁇ ertion of at least one Pro residue between His
  • Argl719 When the non-activating Arg residue is Argl719, representative modifications a deletion comprising residues Hisl716, Vall717, and Leul718; an in ⁇ ertion of at least one Pro residue between Leul718 and Argl719; a deletion comprising residues Asnl720, and Argl721 and an in ⁇ ertion of at least one Pro residue to replace the deleted residues; an insertion of at least one Pro residue between Argl719 and Asnl720; an insertion of at least one Phe residue between Vall717 and Leul718; an insertion of at least one Glu residue between Vall717 and Leul718; an insertion of at least one Pro residue between Vall717 and Leul718; a deletion comprising residue ⁇
  • Argl721 representative modifications include a deletion comprising residues Hisl716, Vall717, Leul718, Argl719, and Asnl720; an insertion of at least one Pro residue between A ⁇ nl720 and Argl721; an insertion of at least one Pro residue between Argl721 and Alal722; a deletion comprising residues Alal722, Glnl723, and Serl724 and an insertion of at least one Pro residue to replace the deleted residues; a deletion comprising residues Alal722, Glnl723, Serl724, Glyl725, Serl726, and Vall727; a deletion comprising residues Alal722, Glnl723, Serl724, Glyl725, and Serl726, and an
  • polypeptide analogs contemplated by the present invention are tho ⁇ e analogs including substitutions of the non-activating Arg residues found at positions Arg336, Argl719 and Argl721, numbered with respect to the native Factor VIII:C polypeptide ⁇ equence.
  • tho ⁇ e analogs including substitutions of the non-activating Arg residues found at positions Arg336, Argl719 and Argl721, numbered with respect to the native Factor VIII:C polypeptide ⁇ equence.
  • any of amino acids Pro, Glu, Asp, Asn, Gin, Ser and Tyr can be substituted at position Arg336 and/or position Argl719, to generate a Factor VIII:C polypeptide analog with improved propertie ⁇ .
  • any of amino acids Glu, Asp, Asn, Gin, Ser and Tyr can be substituted at position Argl721.
  • the Factor VIII:C polypeptide analog of this embodiment will include a substitution of Arg336 with Pro, a substitution of Arg 1719 with Pro and/or a substitution of Argl721 with Glu. Nucleic acid molecules encoding the present
  • Factor VIII:C polypeptide analogs can be made by modifying the native nucleic acid sequences that encode the Factor VIII:C polypeptide or cDNA sequences that encode the Factor VIII:C polypeptides. Such modification can be done by conventional techniques such as site- directed mutagenesis. For example, the M13 method for site directed mutagene ⁇ is is known, as described in Zoller and Smith, Nucleic Acids Re ⁇ . (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 .
  • one or more of the codons encoding a residue adjacent to an arginine, preferably, at the carboxy side can be mutated by substitution, deletion or addition, to a codon encoding proline, thereby creating polypeptides with either Pro-Arg bonds or Arg-Pro bonds where none existed before, provided that the modification does not affect an activation cleavage site, such as Arg372 and Arg740.
  • the codons encoding proline include CCU, CCT, CCG, CCA, and CCC.
  • the nucleic acid molecule ⁇ of the pre ⁇ ent invention can al ⁇ o be made ⁇ ynthetically by piecing together nucleic acid molecules encoding heavy and light chain fragments derived from cDNA clones or genomic clones containing Factor VIII:C coding sequences, preferably cDNA clones, using known linker sequences. Alternatively, the entire sequence or portions of nucleic acid sequence ⁇ encoding analogs described above may be prepared by synthetic methods (e.g. using DNA synthe ⁇ i ⁇ 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.
  • DNA cloning and in posses ⁇ ion of the DNA encoding native Factor VIII:C polypeptide will be able to prepare suitable DNA molecules for production of the present analogs using known cloning procedure ⁇ (e.g. restriction enzyme digestion, exonuclease digestion, ligation, and other appropriate procedures) outlined in any of the following: Sambrook, et al , MOLECULAR CLONING: A LABORATORY MANUAL 2nd ed. (Cold Spring Harbor Laboratory Press, 1989) ; DNA CLONING, Vol. I and II, D.N. Glover ed. (IRL Pres ⁇ , 1985); OLIGONUCLEOTIDE SYNTHESIS, M.J. Gait ed.
  • cloning procedure e.g. restriction enzyme digestion, exonuclease digestion, ligation, and other appropriate procedures
  • 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 site ⁇ 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 epi ⁇ omal existence or of integration into the host cell genome.
  • replication systems typically derived from viruses that infect mammalian host cells.
  • Illustrative replication sy ⁇ tems include the replication systems from Simian virus 40, adenoviru ⁇ , bovine papilloma viru ⁇ , polyoma viru ⁇ , Ep ⁇ tein Barr viru ⁇ , 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 cell ⁇ , including bacterial, yeast, insect and mammalian expression sy ⁇ tems.
  • 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
  • an eukaryotic host cell can be used for expression of the Factor VIII:C polypeptide analogs.
  • the expression cassettes are introduced into the host cell by conventional methods, depending on the expression system used, as de ⁇ cribed 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.
  • Coexpres ⁇ ion of more than one Factor VIII:C polypeptide analog may be de ⁇ ired. For example, it may be de ⁇ irable to expre ⁇ the light and heavy chain ⁇ u ⁇ ing ⁇ eparate constructs. In this regard, either or both of the light and heavy chains may include modifications as described above.
  • Coexpression refers to the expression of two or more Factor VIII:C polypeptides in a ⁇ ingle ho ⁇ t cell. Thus, for example, the expression of the 90 kD specie ⁇ 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 VIII:C polypeptides would be con ⁇ idered "coexpressed" for purposes of the present definition as would the expression of two genes as a dicistronic construct employing an internal ribosome entry site.
  • proteins expres ⁇ ed from the same vector but driven by separate regulatory elements would also be considered “coexpressed.”
  • the term also refers to the expres ⁇ ion 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 purposes of the present invention.
  • the transformed/transfected cells are then grown in an appropriate nutrient medium. If separate constructs encoding heavy and light chain analogs have been used for coexpression, the product can be obtained as a complex of the two Factor VIII:C chains, so that the media or cell lysate may be isolated and the Factor
  • VIII:C 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- ⁇ olvent extraction, selective precipitation, and the like.
  • affinity chromatography ion exchange chromatography
  • hydrophobic chromatography hydrophobic chromatography
  • electrophoresis solvent- ⁇ olvent 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 VIII:C polypeptide analog ⁇ and nucleic acid sequences encoding the analogs.
  • Control elements for use in bacterial systems include promoters, optionally containing operator ⁇ equences, 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 ⁇ uch as tryptophan (trp) , the ⁇ - lactamase (Jla) promoter system, bacteriophage ⁇ PL, and T7.
  • synthetic promoters can be used, such as the tac promoter.
  • the ⁇ -lactamase and lactose promoter sy ⁇ tems are described in Chang et al . , Nature (1978) 275 : 615, and Goeddel et al . , Nature (1979) 281 : 544; the alkaline phosphatase, tryptophan (trp) promoter system are described in Goeddel et al . , Nucleic Acid ⁇ Re ⁇ . (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 al . , Proc . Natl . Acad . Sci .
  • 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 E ⁇ cherichia coli .
  • numerous other systems for use in bacterial hosts including Gram- negative or Gram-positive organisms such as Bacillu ⁇ ⁇ pp. , Streptococcus ⁇ pp. , Streptomyce ⁇ ⁇ pp . , Pseudomonas species such as P . aerugino ⁇ a, Salmonella typhimurium, or Serratia marce ⁇ cans, among others.
  • Methods for introducing exogenous DNA into these hosts typically include the use of CaCl 2 or other agents, such as divalent cations and DMSO.
  • DNA can also be introduced into bacterial cell ⁇ by eiectroporation, nuclear injection, or protoplast fusion as described generally in Sambrook et al . (1989), cited above. These examples are illustrative rather than limiting.
  • the host cell ⁇ hould 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 VIII:C 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 Factor VIII:C polypeptide analogs and nucleic acid sequences encoding the analogs.
  • Expression and transformation vectors either extrachromosomal replicons or integrating vectors, have been developed for transformation into many yeasts.
  • expres ⁇ ion vectors have been developed for, among others, the following yeast ⁇ : 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 albican ⁇ as described in Kurtz et al . , Mol .
  • 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 isomera ⁇ e, 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.
  • ⁇ uitable promoter sequences for use with yeast host ⁇ include the promoters for 3-phosphoglycerate kina ⁇ e, a ⁇ 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 . ,
  • 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. In addition, synthetic promoters which do not occur in nature also function a ⁇ 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 PH05 genes, combined with the tran ⁇ criptional 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.
  • Other control elements which may be included in the yeast expression vectors are 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 sequence ⁇ 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 de ⁇ cribed in U.S. Patent Nos.
  • 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 H ⁇ iao et al . , Proc . Natl . Acad . Sci . USA (1979) 76 : 3829. However, other methods for introducing DNA into cells such as by nuclear injection, eiectroporation, or protoplast fusion may also be used as described generally in Sambrook et al . , cited above.
  • the native polypeptide signal sequence may be ⁇ ub ⁇ tituted by the yeast invertase, ⁇ -factor, or acid phosphatase leaders.
  • the origin of replication from the 2 ⁇ plasmid origin is ⁇ uitable for yea ⁇ t.
  • a ⁇ uitable 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 T ⁇ che per et al . , Gene (1980) 10 : 157.
  • the trpl gene provide ⁇ a ⁇ election marker for a mutant ⁇ train of yea ⁇ t lacking the ability to grow in tryptophan.
  • Leu2-deficient yeast strains 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 VIII:C polypeptide analog to produce a fusion protein that can be cleaved intracellularly by the yeast cells upon expression.
  • An example, of such a yeast leader sequence i ⁇ the yeast ubiquitin gene.
  • Insect expression sy ⁇ tems can be used to produce the Factor VIII:C polypeptide analogs and 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 sy ⁇ tem, the vector generally containing DNA coding for a baculoviru ⁇ 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 VIII:C 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, rrichoplu ⁇ ia ni MNPV, Rachlplu ⁇ ia ou MNPV or Galleria mellonella MNPV, Aede ⁇ aegypti, Drosophila melanogaster, Spodoptera frugiperda , and Trichoplusia ni .
  • 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 Hindlll 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 ⁇ uitable for u ⁇ e 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
  • the plasmid for use herein usually also contains the polyhedrin polyadenylation signal, a ⁇ described in Miller et al . , Ann . Rev .
  • Microbiol . (1988) 42 111 and a procaryotic ampicillin- resistance (amp) gene and an origin of replication for selection and propagation in E. coli .
  • DNA encoding suitable signal sequence ⁇ can also be included and is generally derived from gene ⁇ for ⁇ ecreted in ⁇ ect or baculoviru ⁇ 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 .
  • 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 employed to advantage in a baculovirus expression system. These 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. Thus, the immediate-early genes are 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.
  • immediate-early genes are 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.
  • IEI a preferred immediate-early gene of Autographo californica nuclear polyhedrosis virus
  • AcMNPV Autographo californica nuclear polyhedrosis virus
  • IEI is pre ⁇ ed in the absence of other viral functions and encodes a product that stimulates the transcription of several genes of the delayed-early class, including the preferred 39K gene, as de ⁇ cribed in Guarino and Summers, J . Virol . (1986) 57 : 563-571 and J. Virol . (1987) 61 : 2091-2099 as well a ⁇ late gene ⁇ , as described in Guanno and Summers, Virol . (1988) 162 : 444-451.
  • 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 Hindlll fragment of the baculovirus genome. In the present instance, the 39 K promoter region can be linked to the foreign gene to be expres ⁇ ed ⁇ uch that expre ⁇ ion can be further controlled by the pre ⁇ ence of IEI, a ⁇ 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 ⁇ equences 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 de ⁇ cribed in Guarino and Summer ⁇ (1986a), (1986b), and Guarino et al .
  • an insect ⁇ ignal ⁇ equence 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 ⁇ ignal 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 insect ⁇ .
  • 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 in ⁇ erted into the tran ⁇ fer vector, u ⁇ ing known technique ⁇ .
  • An insect cell host can be cotran ⁇ formed with the transfer vector containing the inserted de ⁇ ired DNA together with the genomic DNA of wild type baculoviru ⁇ , u ⁇ ually by cotran ⁇ fection.
  • 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 VIII:C polypeptide analog.
  • Mammalian expression systems can also be used to produce the Factor VIII:C polypeptide analogs and nucleic acid sequences encoding the analogs.
  • Typical promoters for mammalian cell expres ⁇ ion include the SV40 early promoter, the CMV promoter, the mou ⁇ e mammary tumor virus LTR promoter, the adenovirus major late promoter (Ad MLP) , and the herpes 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 con ⁇ tructs.
  • 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 VIII:C polypeptide analog coding sequence, is also present.
  • tran ⁇ cription 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 ⁇ ite ⁇ may al ⁇ o be designed into the constructs of the present invention.
  • Enhancer elements can also be used herein to increase expres ⁇ ion levels of the mammalian construct ⁇ .
  • Example ⁇ 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 de ⁇ cribed in Bo ⁇ hart et al . , Cell (1985) 41 : 521.
  • LTR long terminal repeat
  • a leader ⁇ equence can also be present which includes a sequence encoding a signal peptide, to provide for the secretion of the foreign protein in mammalian cells.
  • the Factor VIII signal peptide can be used.
  • the adenovirus tripartite leader is an example of a leader sequence that provides for secretion of a foreign protein in mammalian cells.
  • transient expres ⁇ ion 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 expre ⁇ sion 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 polypeptide ⁇ for desired biological or physiological properties.
  • transient expression systems are particularly useful for purposes of identifying additional polypeptides that have Factor VIII:C-like activity.
  • the mammalian expres ⁇ ion vector ⁇ 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 fu ⁇ ion, eiectroporation, encapsulation of the polynucleotide(s) in lipo ⁇ omes, and direct microinjection of the DNA into nuclei.
  • General a ⁇ pect ⁇ of mammalian cell ho ⁇ t system transformations have been described by Axel in U.S. 4,399,216.
  • a ⁇ ynthetic lipid particularly u ⁇ eful for polynucleotide transfection is N-[l-(2,3- dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, which is commercially available under the name
  • Lipofectin® (available from BRL, Gaithersburg, MD) , and is de ⁇ cribed 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 ham ⁇ ter 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.
  • the mammalian ho ⁇ t cell ⁇ used to produce the target polypeptide of this invention may be cultured in a variety of media.
  • any of the media described in Ham and Wallace, Meth . Enz . (1979) 58 : 44, Barnes and Sato, Anal. Biochem . (1980) 102 : 255, U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, or 4,560,655, WO 90/103430, WO 87/00195, and U.S. RE 30,985, may be used as culture media for the host cells.
  • any of these media may be supplemented as neces ⁇ ary with hormones and/or other growth factors such as insulin, transferrin, or epidermal growth factor, salt ⁇ ( ⁇ uch a ⁇ 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 ⁇ killed in the art.
  • hormones and/or other growth factors such as insulin, transferrin, or epidermal growth factor, salt ⁇ ( ⁇ uch a ⁇ sodium chloride, calcium, magnesium, and phosphate) , buffers (such as HEPES) , nucleosides (such as adenosine and thymidine) , antibiotics
  • the culture condition ⁇ such as temperature, pH, and the like, are those previously used with the ho ⁇ t cell selected for expres ⁇ ion, and will be apparent to the ordinarily ⁇ killed arti ⁇ an.
  • the active Factor VIII:C 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 VIII:C 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 VIII:C analogs can be used directly for gene therapy and administered using standard gene delivery protocols.
  • the nucleotide sequences encoding the Factor VIII:C analog ⁇ can be stably integrated into the host cell genome or maintained on a stable episomal element in the ho ⁇ t cell.
  • Methods for gene delivery are known in the art. See, e.g., U.S. Patent No. 5,399,346.
  • retroviral based ⁇ ystems 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 u ⁇ ing 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) 1:5-14; Scarpa et al .
  • adenovirus vectors have also been described. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromo ⁇ omally thu ⁇ minimizing the risks associated with insertional mutagenesis (Haj-Ahmad and Graham, J . Virol . (1986) 57:267-274; Bett 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 ⁇ electable markers or reporter genes, enhancer sequence ⁇ , and other control elements which allow for the induction of transcription.
  • AAV vectors can be readily constructed u ⁇ ing technique ⁇ well known in the art. See, e.g., U.S. Patent No ⁇ . 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 .
  • Additional viral vectors which will find use for delivering the nucleic acid molecules encoding the Factor VIII:C analog polypeptides for gene transfer include those derived from the pox family of viruse ⁇ , including vaccinia virus and avian poxvirus.
  • vaccinia virus recombinants expressing the novel Factor VIII:C 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 VIII:C analogs in a host cell. In this system, cells are first infected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase. This polymerase displays vibrant ⁇ pecificity in that it only tran ⁇ cribe ⁇ templates bearing T7 promoters.
  • RNA RNA
  • 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) 83:8122-8126.
  • avipoxviruses such as the fowlpox and canarypox viruses, can also be used to deliver the Factor VIII:C analog genes.
  • 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 specie ⁇ since members of the avipox genus can only productively replicate in susceptible avian specie ⁇ and therefore are not infective in mammalian cell ⁇ .
  • 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 expres ⁇ ion following introduction into host cells.
  • 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, ⁇ ome of the T7 RNA polymerase generated from translation of the amplification template RNA will lead to tran ⁇ cription of the desired gene. Because some T7 RNA polymerase is required to initiate the amplification, 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. However the use of a plasmid is preferred. 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. For example, a lac operator can be engineered distal or proximal (or both) to the T7 promoter. The binding of the preexisting lac repres ⁇ or in the appropriate bacterial strain would interfere with the transcription of the template by blocking access to the promoter by T7 RNA polymerase. Alternatively, or in combination with the above, 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 tran ⁇ cription unit con ⁇ i ⁇ ting of the T7 promoter preceding the gene of interest can be provided by a separate pla ⁇ mid or can be engineered onto the amplification plasmid. Colocalization of the two transcription units i ⁇ beneficial for ea ⁇ e of manufacturing and en ⁇ ures that both transcription units will always be together in the cells into which the plasmid is introduced.
  • the T7 RNA polymera ⁇ e pla ⁇ mid ⁇ may include UTRs which comprise an Internal Ribosome Entry Site (IRES) present in the leader sequence ⁇ of picornaviru ⁇ e ⁇ ⁇ uch a ⁇ the encephalomyocarditi ⁇ virus (EMCV) UTR (Jang et al. J. Virol . (1989) 63:1651-1660).
  • IRS Internal Ribosome Entry Site
  • EMCV encephalomyocarditi ⁇ virus
  • Vectors encoding the subject Factor VIII:C analogs can also be packaged in liposomes prior to delivery to the subject or to cells derived therefrom.
  • Lipid encap ⁇ ulation 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) , or more of lipid.
  • Liposomal preparations for use in the instant invention include cationic (positively charged) , anionic (negatively charged) and neutral preparations, with cationic liposomes particularly preferred.
  • Cationic liposome ⁇ have been ⁇ hown to mediate intracellular delivery of plasmid DNA (Feigner et al . , Proc . Natl .
  • Cationic liposomes are readily available.
  • N[1-2, 3-dioleyloxy)propyl]-N,N,N-triethyl- ammonium (DOTMA) liposomes are available under the trademark Lipofectin, from GIBCO BRL, Grand Island, NY.
  • liposome ⁇ examples include tran ⁇ fectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger) .
  • DOTAP/DOPE tran ⁇ fectace
  • Other cationic liposomes can be prepared from readily available materials using techniques well known in the art. See, e.g., Szoka et al . , Proc . Natl . Acad . Sci . USA (1978) 75:4194-4198; PCT Publication No. WO 90/11092 for a description of the synthe ⁇ i ⁇ of DOTAP (1,2-bis(oleoyloxy) -3-(trimethylammonio)propane) liposomes.
  • anionic and neutral liposomes are readily available, such as from Avanti Polar Lipids (Birmingham, AL) , or can be easily prepared u ⁇ ing readily available material ⁇ .
  • material ⁇ include phosphatidyl choline, cholesterol, pho ⁇ phatidyl ethanolamine, dioleoylphosphatidyl choline (DOPC) , dioleoylphosphatidyl glycerol (DOPG) , dioleoylphoshatidyl ethanolamine (DOPE) , among others.
  • DOPC dioleoylphosphatidyl choline
  • DOPG dioleoylphosphatidyl glycerol
  • DOPE dioleoylphoshatidyl ethanolamine
  • 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 liposome ⁇ can compri ⁇ e multilammelar vesicles (MLVs) , ⁇ mall unilamellar vesicles (SUVs) , or large unilamellar vesicles (LUVs) .
  • MLVs multilammelar vesicles
  • SUVs ⁇ mall 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 .
  • the recombinant vectors may be administered in pharmaceutical compositions as de ⁇ cribed 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, eiectroporation, 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 subcutaneou ⁇ ly, intraperitoneally, intravenously or intramuscularly.
  • Other modes of administration include oral and pulmonary administration, suppositories, and transdermal application ⁇ .
  • Do ⁇ age treatment may be a ⁇ ingle do ⁇ e schedule or a multiple dose ⁇ chedule.
  • the present invention will now be illu ⁇ trated by reference to the following examples which set forth particularly advantageou ⁇ embodiment ⁇ .
  • these embodiments are illustrative and are not to be construed as restricting the invention in any way.
  • the Factor VIII:C polypeptide analogs are made using conventional mutagenesi ⁇ techniques.
  • mutagenesis of the Factor VIII:C nucleotide sequence ⁇ can be performed utilizing plasmids which include sequence ⁇ encoding the full-length molecule, plasmids encoding the light and heavy chains and various modifications of these molecules, depending on the Factor VIII:C analog desired.
  • Such plasmid ⁇ are known and de ⁇ cribed in, e.g., U.S. Patent no.
  • the plasmids are first linearized e.g., by using restriction endonucleases which cleave at unique restriction site ⁇ . Once linearized, the plasmids are treated with calf intestine phosphatase and separated on low melting temperature tris-acetate agarose gels. The linearized band is extracted by adsorption to silica dioxide and eluted in tris-EDTA. The pla ⁇ mid is then denatured and the desired phosphorylated mutagenic oligonucleotide i ⁇ 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 , ImM beta-mercaptoethanol, 400 ⁇ M ATP, 100 ⁇ M deoxynucleotide triphosphate, 3-4 units/ ⁇ L of Klenow fragment of E. coli DNA polymera ⁇ e I and 400 unit ⁇ / ⁇ 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 transferred to nitrocellulo ⁇ e, filters prepared, and hybridized to screening probes to ensure that the mutagenic oligonucleotide is introduced into the correct fragment.
  • Final mutations are confirmed by DNA sequencing.
  • the DNA can be prepared by banding in C ⁇ Cl and can be u ⁇ ed to tran ⁇ fect COS-l monkey cell ⁇ a ⁇ de ⁇ cribed in Kaufman, PNAS (1982) 82:689. After transfection, the polypeptide analog is isolated and Factor VIII:C activity is assayed by the Kabi Coatest chromagenic assay method for the ability to clot Factor VIII deficient plasma before and after thrombin activation.
  • Residue 221 of Factor VIII:C is mutated using the oligonucleotide TTC ATG CAG GAT AGG CCX GCT GCA TCT GCT CGG.
  • the GAT encoding for Asp which normally occurs at the underlined position is mutated to form the codon for Pro which can be CCX where X is A, T, G or C.
  • the mutagenesis i ⁇ carried out, the correct sequence is confirmed, and the analog produced and tested for activity, as described above.
  • Factor VIII:C polypeptide analogs can be constructed and assayed as described above.

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Abstract

La présente invention se rapporte à des analogues polypeptidiques de Facteur VIII:C qui sont des polypeptides natifs de Facteur VIII:C contenant des modifications d'aminoacide au niveau d'un ou plusieurs résidus d'aminoacide adjacents à un résidu Arg, pourvu que le résidu Arg ne soit pas un site d'activation du Facteur VIII:C. De telles modifications créent une ou plusieurs liaisons Arg-Pro ou Pro-Arg. L'invention décrit également des molécules d'acide nucléique codant les analogues polypeptidiques de Facteur VIII:C, des vecteurs et des cellules hôtes contenant de telles molécules d'acide nucléique. Des modifications supplémentaires comprennent la création d'un tripeptide de formule P3-P2-P1, dans laquelle P3 représente un résidu choisi dans le groupe contenant Phe, Glu et Pro; P2 représente un résidu d'aminoacide à l'exception de Ser, et P2 ne représente ni Leu355 ni Asn1720; et P1 représente Arg. D'autres modifications consistent en des substitutions au niveau des résidus non activateurs s'effectuant aux positions Arg336, Arg1719 et/ou Arg1721. En outre, l'invention décrit des complexes d'analogues contenant au moins deux de ces analogues. L'invention décrit également des procédés pour produire les analogues, les complexes d'analogues, les acides nucléiques les codant, les vecteurs et les cellules hôtes, ainsi que des procédés d'utilisation de telles compositions pour la prévention ou le traitement de déficiences en polypeptides actives de Facteur VIII:C.
PCT/US1996/011444 1995-07-11 1996-07-09 Nouveaux analogues polypeptidiques de facteur viii:c ayant des sites de protease modifies WO1997003195A1 (fr)

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Cited By (25)

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WO2000028021A1 (fr) * 1998-11-10 2000-05-18 Baxter Aktiengesellschaft Polypeptide du facteur viii a activite de facteur viii:c
WO2000071714A2 (fr) 1999-05-24 2000-11-30 The American National Red Cross Procedes de reduction de la clairance du facteur viii et compositions correspondantes
US6221349B1 (en) 1998-10-20 2001-04-24 Avigen, Inc. Adeno-associated vectors for expression of factor VIII by target cells
WO2003087161A1 (fr) * 2002-04-18 2003-10-23 Merck Patent Gmbh Facteur viii modifie
US6838437B2 (en) 1996-04-24 2005-01-04 University Of Michigan Inactivation resistant factor VIII
WO2006027111A1 (fr) * 2004-09-06 2006-03-16 Zlb Behring Gmbh Facteur viii de coagulation modifie presentant une meilleure stabilite
US7211559B2 (en) 2003-10-31 2007-05-01 University Of Maryland, Baltimore Factor VIII compositions and methods
US7351577B2 (en) 1998-10-20 2008-04-01 Genzyme Corporation Adeno-associated vector compositions for expression of Factor VIII
US7615622B2 (en) 2001-01-12 2009-11-10 University Of Maryland, Baltimore Methods and compositions for reducing heparan sulfate proteoglycan-mediated clearance of factor VIII
US7632921B2 (en) 2004-11-12 2009-12-15 Bayer Healthcare Llc Site-directed modification of 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
EP2292779A2 (fr) 2003-09-30 2011-03-09 The Trustees of the University of Pennsylvania Variantes des virus associes aux adenovirus (AAV), sequences, vecteurs les contenant, et leur utilisation
EP2357010A1 (fr) 2005-04-07 2011-08-17 The Trustees of The University of Pennsylvania Procédé d'amélioration de la fonction d'un vecteur AAV
WO2011126808A2 (fr) 2010-03-29 2011-10-13 The Trustees Of The University Of Pennsylvania Système d'ablation de transgène induit pharmacologiquement
US8183344B2 (en) 1996-04-24 2012-05-22 University Of Michigan Inactivation resistant factor VIII
WO2012112832A1 (fr) 2011-02-17 2012-08-23 The Trustees Of The University Of Pennsylvania Compositions et procédés pour modifier une spécificité tissulaire et améliorer le transfert d'un gène induit par aav9
US20120297494A1 (en) * 2009-10-16 2012-11-22 Tommy Eugene Howard Compositions and methods of treatment of black hemophiliac patients
WO2013049493A1 (fr) 2011-09-28 2013-04-04 The Trustees Of The University Of Pennsylvania Système d'ablation de transgène médié par un vecteur viral adéno-associé inductible
WO2016168728A2 (fr) 2015-04-16 2016-10-20 Emory University Promoteurs et vecteurs recombinés pour l'expression de protéines dans le foie et utilisation associée
US9719106B2 (en) 2013-04-29 2017-08-01 The Trustees Of The University Of Pennsylvania Tissue preferential codon modified expression cassettes, vectors containing same, and uses thereof
US10272163B2 (en) 2012-12-07 2019-04-30 The Regents Of The University Of California Factor VIII mutation repair and tolerance induction
US11185573B2 (en) 2004-12-06 2021-11-30 Haplomics, Inc. Allelic variants of human factor VIII
US20220033475A1 (en) * 2018-10-23 2022-02-03 The Children's Hospital Of Philadelphia Compositions and methods for modulating factor viii function
WO2022032140A2 (fr) 2020-08-07 2022-02-10 Amicus Therapeutics, Inc. Protéines de ciblage des vésicules et leurs utilisations
CN114106146A (zh) * 2015-02-06 2022-03-01 北卡罗来纳大学查珀尔希尔分校 优化的人类凝血因子viii基因表达盒及其用途

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US7459534B2 (en) 1996-04-24 2008-12-02 The Regents Of The University Of Michigan Inactivation resistant factor VIII
US6838437B2 (en) 1996-04-24 2005-01-04 University Of Michigan Inactivation resistant factor VIII
US7351577B2 (en) 1998-10-20 2008-04-01 Genzyme Corporation Adeno-associated vector compositions for expression of Factor VIII
US6221349B1 (en) 1998-10-20 2001-04-24 Avigen, Inc. Adeno-associated vectors for expression of factor VIII by target cells
WO2000028021A1 (fr) * 1998-11-10 2000-05-18 Baxter Aktiengesellschaft Polypeptide du facteur viii a activite de facteur viii:c
US7544660B2 (en) 1998-11-10 2009-06-09 Stichting Sanquin Bloedvoorziening Factor VIII polypeptide having factor VIII:C activity
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
WO2000071714A2 (fr) 1999-05-24 2000-11-30 The American National Red Cross Procedes de reduction de la clairance du facteur viii et compositions correspondantes
US7615622B2 (en) 2001-01-12 2009-11-10 University Of Maryland, Baltimore Methods and compositions for reducing heparan sulfate proteoglycan-mediated clearance of factor VIII
WO2003087161A1 (fr) * 2002-04-18 2003-10-23 Merck Patent Gmbh Facteur viii modifie
EP2292779A2 (fr) 2003-09-30 2011-03-09 The Trustees of the University of Pennsylvania Variantes des virus associes aux adenovirus (AAV), sequences, vecteurs les contenant, et leur utilisation
EP2292780A2 (fr) 2003-09-30 2011-03-09 The Trustees of the University of Pennsylvania Variantes des virus associes aux adenovirus (AAV), sequences, vecteurs les contenant, et leur utilisation
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US7211559B2 (en) 2003-10-31 2007-05-01 University Of Maryland, Baltimore Factor VIII compositions and methods
WO2006027111A1 (fr) * 2004-09-06 2006-03-16 Zlb Behring Gmbh Facteur viii de coagulation modifie presentant une meilleure stabilite
US7632921B2 (en) 2004-11-12 2009-12-15 Bayer Healthcare Llc Site-directed modification of FVIII
US9364520B2 (en) 2004-11-12 2016-06-14 Bayer Healthcare Llc Factor VIII conjugates
US9096656B2 (en) 2004-11-12 2015-08-04 Bayer Healthcare Llc Factor VIII conjugates
US11185573B2 (en) 2004-12-06 2021-11-30 Haplomics, Inc. Allelic variants of human factor VIII
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EP2357010A1 (fr) 2005-04-07 2011-08-17 The Trustees of The University of Pennsylvania Procédé d'amélioration de la fonction d'un vecteur AAV
US20120297494A1 (en) * 2009-10-16 2012-11-22 Tommy Eugene Howard Compositions and methods of treatment of black hemophiliac patients
WO2011126808A2 (fr) 2010-03-29 2011-10-13 The Trustees Of The University Of Pennsylvania Système d'ablation de transgène induit pharmacologiquement
WO2012112832A1 (fr) 2011-02-17 2012-08-23 The Trustees Of The University Of Pennsylvania Compositions et procédés pour modifier une spécificité tissulaire et améliorer le transfert d'un gène induit par aav9
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US12252528B2 (en) * 2018-10-23 2025-03-18 The Children's Hospital Of Philadelphia Compositions and methods for modulating Factor VIII function
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