Expression of active human factor IX in mammary tissue and of milk non human transgenic mammals

Recombinant Factor IX characterized by a high percentage of active protein can be obtained in the milk of transgenic animals that incorporate chimeric DNA molecules according to the present invention. Transgenic animals of the present invention are produced by introducing into developing embryos DNA that encodes Factor IX, such that the foreign DNA is stably incorporated in the DNA of germ line cells of the mature animal. Particularly efficient expression was accomplished using a chimeric construct comprising a mammary gland specific promoter, Factor IX cDNA that lacked the complete or any portion of the 5\u27-untranslated and 3\u27-untranslated region, which is substituted with a 5\u27- and 3\u27-end of the mouse whey acidic protein gene. In vitro cell cultures of cells explanted from the transgenic mammal of the invention and methods of producing Factor IX from such said culture and methods of treating hemophilia B are also described

TREATMENT OF HEMOPHILIA WITH HUMAN FACTORIX PRODUCED IN MAMIMARY TISSUE OF TRANSGENIC MAMMALS

Recombinant Factor IX characterized by a high percentage of active protein can be obtained in the milk of transgenic animals that incorporate chimeric DNA molecules according to the present invention. Transgenic animals of the present invention are produced by introducing into developing embryos DNA that encodes Factor IX, such that the foreign DNA is stably incorporated in the DNA of germ line cells of the mature animal. Particularly efficient expression was accomplished using a chimeric construct comprising a mammary gland specific promoter, Factor IX cDNA that lacked the complete or any portion of the 5\u27-untranslated and 3\u27-un-translated region, which is substituted with a 5- and 3\u27-end of the mouse whey acidic protein gene. In vitro cell cultures of cells explanted from the transgenic mammal of the invention and methods of producing Factor IX from such said culture and methods of treating hemophilia B are also described

TRANSGENIC NONHUMAN MAMMALS PRODUCING FIBRINOGEN IN MILKAND METHODS OF PRODUCING FIBRIN

A transgenic, non-human mammalian animal is capable of expressing a heterologous gene for human or other recombinant physiologically functional fibrinogen holoprotein or individual subunit chain polypeptides thereofora modified or fusion fibrinogen in mammary glands of the animals and secreting the expressed product into a body fluid. Methodol ogy employing such a mammal yields recombinant physiologically functional fibrinogens, subunit chain polypeptides thereof, and modified or fusion fibrinogens

EXPRESSION OF HUMAN PROTEIN C IN MAMMARY TISSUE OF TRANSGENIC MAMMALS

Recombinant protein C characterized by a high percentage of active protein can be obtained in the milk of transgenic mammals that incorporate DNAs according to the present invention. Transgenic mammals of the present invention are produced by introducing into developing embryos DNA that encodes protein C, such that the DNA is stably incorporated in the DNA of germ line cells of the mature mammals and inherited in normal, mendelian fashion

TREATMENT OF HEMOPHILIA WITH HUMAN FACTORIX PRODUCED IN MAMIMARY TISSUE OF TRANSGENIC MAMMALS

Recombinant Factor IX characterized by a high percentage of active protein can be obtained in the milk of transgenic animals that incorporate chimeric DNA molecules according to the present invention. Transgenic animals of the present invention are produced by introducing into developing embryos DNA that encodes Factor IX, such that the foreign DNA is stably incorporated in the DNA of germ line cells of the mature animal. Particularly efficient expression was accomplished using a chimeric construct comprising a mammary gland specific promoter, Factor IX cDNA that lacked the complete or any portion of the 5\u27-untranslated and 3\u27-un-translated region, which is substituted with a 5- and 3\u27-end of the mouse whey acidic protein gene. In vitro cell cultures of cells explanted from the transgenic mammal of the invention and methods of producing Factor IX from such said culture and methods of treating hemophilia B are also described

Compositions, Methods And Kits Relating To Thrombin Degradation Resistant Fibroblast Growth Factor-1

The invention relates to novel degradation resistant FGF-1, and methods for producing and using the same. More specifically, the invention relates to identification of a thrombin degradation resistant FGF-1, an a nucleic acid encoding the same. The thrombin degradation resistant FGF-1 can elicit responses that are otherwise typically impeded by degradation of FGF-1 by thrombin. Thrombin degradation resistant FGF-1 is an important molecule for effecting an FGF-1 response that would be otherwise inhibited by thrombin. Thus, the present invention provides a powerful therapeutic for diseases or disorders wherein an FGF-1 response can mediate a reduction in the frequency or intensity of a symptom of the disease or disorder but for degradation of FGF-1 before it can effect the response

Conformational Changes in an Epitope Localized to the NH2-terminal Region of Protein C

Murine monoclonal antibodies, developed following immunization with human protein C, were characterized for their ability tboin d antigen in thpe resence of either CaClz or excess EDTA. Three stablec lones were obtained which produced antiboditehsa t bound to protein C only itnh e presence of EDTA. Allt hree antibodies bound to the light choafi np rotein C on immunoblots and also bound to the homologous proteins factor X and prothrombin in solid-phase radioimmunoassays. One antibody, 7D7B10 was purified and studied further. The binding of 7D7B10 to human protein C was characterized bya KOo f 1.4 nM. In competition studies, it was found that the relative affinityo f the antibody for protein C was 20-40-fold higher than for prothrombin, fragment 1 of prothrombin, or factor X. In contrast, 7D7B10 was unable to bind to factor 1X or bovine protein C. The effect of varying Ca2+ concentration on the interaction of the antibody with protein C was complex. Low concentrations of Ca2+ enhanced the formation of the protein C-antibody complex with half-maximal effect occurring at approximately6 0 PM metal ion. However, higher concentrations of Ca2+ completely inhibited 7D7B10 binding to protein C with a K o .o~f 1.1 mM. Furthermore, millimolar concentrations of Mn2+, Ba2+, or Mg2+ also completely abolished antibody binding to protein C. The location of the epitope was delineatedby immunoblotting and peptide studies andf ound to be present in the NHz-termin1a5l residues of protein C. Although residues corresponding to positions 10-13 of human protein C wernee cessary for maximal binding of the antibody, they were not sufficient. No evidence could be found for involvement of the epitope in metal binding per se. Therefore, the effect of Ca2+ on antibody binding is thought to be due to metal-dependent conformational changes in protein C. It seems likelyth at Ca2+ occupatioonf a high affinity site, shown by others to be located in the epidermal growth factor-like domain, causes a conformational change in the NHz-terminarel gion of protein C which is favorable for antibody interaction, whereas Ca2+ binding to thleo w affinity site($, known to be present in the y-carboxyglutamic acid domain, causes an unfavorable conformational change

Affinity Purification of Biologically Active andInactive Forms of Recombinant Human Protein C Produced in Porcine Mammary Gland

Recombinant human protein C (rhPC) secreted in the milk of transgenic pigs was studied. \u27Ikansgenes having different regulatory elements of the murine milk protein, whey acidic protein, were used with cDNA and genomic human protein C (hPC) DNA sequences to obtain lower and higher expressing animals. The cDNA pigs had a range of expression of about 0.1-0.5 g/l milk. Two different genomic hPC pig lines have expressed 0.3 and 1-2 g/l, respectively. The rhPC was first purified at yields greater than 60 per cent using a monoclonal antibody (mAb) to the activation site on the heavy chain of hPC. Subsequent immunopurification with a calcium-dependent mAb directed to the y-carboxyglutamic acid domain of the light chain of hPC was used to fractionate a population having a higher specific anticoagulant activity in vW. The higher percentages of Ca2+-dependent conformers isolated from the total rhPC by immunopurification correlated well with higher specific activity and lower expression. A rate limitation in y-carboxylation of rhPC was clearly identified for the higher expressing animals. Thus, transgenic animals with high expression levels of complex recombinant proteins produced a lower percentage of biologically active protein

Transgenic non-human mammals expressing human coagulation factor VIII and von Willebrand factor

A non-human transgenic mammalian animal, as described above, contains an exogenous double stranded DNA sequence stably integrated into the genome of the animal, which comprises cis-acting regulatory units operably linked to a DNA sequence encoding human Factor VIII protein and a signal peptide, where the cis-acting regulatory units are active in mammary gland cells and the signal peptide is active in directing newly expressed Factor VIII into the milk of the animal. The promoter may be a milk protein promoter such as for whey acidic protein, casein, lactalbumin, or beta-lactoglobulin promoter. The transgenic mammals are preferably farm animals, for example, cows, goats, sheep, rabbits and pigs. Concurrent expression of a gene for human von Willebrand\u27s Factor into milk may be used to stabilize newly-secreted Factor VIII