Virginia Tech Intellectual Properties, Inc.

Other

American Red Cross

National Institutes of Health, a Component of the US Dept. of Health & Human Services

The University of North Carolina at Chapel Hill

Center for Innovative Technology

American National Red Cross

Minnesota Mining and Manufacturing Company

Progenetics LLC

Virginia Intellectual Property Division

William Hugold Velander

William N Drohan

Henryk Lubon

John L Johnson

Polly Matzinger

Tirumalai Kamala

Oral Alpan

Dean S Milbrath

Tracy Dale Wilkins

Wolfgang G Glasser

Julian D Cooper

Lothar Hennighausen

Tanya K O'Sickey

Raymond L Page

Roger Van Tassell

Kevin E Van Cott

Mary Ann H Johnson, Legal Representative

Anuradha Subramanian

Carolyn Orthner

Jeffrey A Kaster

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 FIX protein without the benefit of the presence of a complete milk gene sequence for gene rescue, and a signal sequence is active in directing newly expressed Factor IX into the milk of the animal at levels in an unactivated form that is suitable for subsequent processing for therapeutic applications in treating Hemophilia B. The transgenic mammals are preferably pigs, cows, sheep, goats and rabbits. The applications include milk derivatives for oral delivery and oral tolerization in the treatment of Hemophilia B.

Production of high levels of transgenic factor IX without gene rescue, and its therapeutic uses

Disclosed herein is a simple method for the treatment of antigen-deficiency diseases, by orally administering to a subject a therapeutically effective amount of the deficient antigen, wherein the antigen is not present in a liposome. In one embodiment, the method increases hemostasis in a subject having hemophilia A or B, by orally administering to the hemophiliac a therapeutically effective amount of the appropriate clotting factor other than in a liposome, sufficient to induce oral tolerance and supply exogenous clotting factor to the subject.

Induction of tolerance by oral administration of factor VIII and treatment of hemophilia

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 thereof or a modified or fusion fibrinogen in mammary glands of the animals and secreting the expressed product into a body fluid. Methodology employing such a mammal yields recombinant physiologically functional fibrinogens, subunit chain polypeptides thereof, and modified or fusion fibrinogens.

Transgenic nonhuman mammals producing fibrinogen in milk and methods of producing fibrin

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'-untranslated and 3&#x2032;-untranslated region, which is substituted with a 5-&#x2032; and 3&#x2032;-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 Factor IX produced in mammary tissue of transgenic mammals

Oral treatment of hemophilia

Transgenic non-human mammals producing fibrinogen in their milk

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's Factor into milk may be used to stabilize newly-secreted Factor VIII.

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

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&prime;-untranslated and 3&prime;-untranslated region, which is substituted with a 5&prime;- and 3&prime;-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.

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

An isolated DNA sequence which regulates the expression of a heterologous gene composed of a mouse whey acidic protein promoter having a length of greater than about 2.4 kb extending upstream from the unique KpnI site in the mouse whey acidic protein gene is disclosed. Specifically a mouse whey acidic protein promoter of about 4.1-4.2 kb in length extending upstream from the unique KpnI site is preferred. This mouse whey acid protein promoter is operably linked to a DNA sequence encoding a heterologous polypeptide and used to prepare transgenic non-human mammals expressing the heterologous polypeptide in their milk. Particularly efficient expression of both cDNAs and genomic DNAs encoding heterologous polypeptides was obtained in transgenic non-human mammals using this promoter, known as the long whey acidic protein promoter.

Expression of a heterologous protein C in mammary tissue of transgenic animals using a long whey acidic protein promoter

The antitope of an antibody is masked with a masking agent, followed by immobilization on a support. The masking agent is then eluted to produce an improved immunosorbent, which is capable of binding more than double the amount of an antigen than existing immunosorbents having the same antibody bound at the same density. Preferably, the masking agent is an antigen or other compound having an epitope for which the antitope of the bound antibody has an avidity. In a preferred embodiment, greater than 30% of the bound antibodies maintain the same vicinity as when unbound for specific antigen or hapten molecules. Preferably, the support is formed of any conventional immunosorbent support material which allows the bound and unbound antibody to maintain an avidity for the same compounds or antigens. In a preferred embodiment, the support is formed of agarose, cellulose, piezo crystals, microtiter plates, or other materials which will not reduce the avidity of a bound antibody for an antigen in comparison to the avidity of the unbound antibody for the same antigen. Preferably, the masked antibodies are bound to supports which are activated with a reagent, such as, but not limited to, cyanogen bromide, carbonyldiimidazole, N-hydroxysuccinimide, hydrazine, hydrazide, and toluene sulfonylchloride. In another preferred embodiment, masked antibody complexes are immobilized through affinity complexing to proteins bound to a support.

Immunosorbents with developed binding efficiency and method of manufacture

Relating to chromatographic processes and ion-exchange and affinity matrices, a spatial installation method for a bifunctional reagent that crosslinks and/or activates a polymer matrix is disclosed, with inside-outside installation of a bifunctional reagent on and within a polymer matrix. The polymer matrix is cellulose, agarose, or chitosan particles. The installation may be followed by inside-outside ligand attachment, by further reacting the matrix with a ligand or ionic group so that a higher concentration of ligand or ionic moiety occurs on the intra-particle volume than the outer matrix surface.

Inside-out crosslinked and commercial-scale hydrogels, and

A method is disclosed for coupling a ligand within a porous support. The method involves mixing ligand and porous support under conditions sufficient to suppress coupling conditions of the ligand to the porous support while enhancing the relative rate of diffusion, to the rate of reaction, of the ligand into and within the porous support, and then altering the conditions to enhance rapid coupling of the ligand within the porous support. The alteration from diffusion conditions to coupling conditions involves a change in the reaction solution of pH, ionic strength, temperature, coupling competitor, such that a relatively lower Thiele Modulus during diffusion conditions changes to a relatively higher Thiele Modulus during coupling conditions. Derivatized porous supports produced according to the method are also disclosed. The derivatized porous support has enhanced functional efficiency. Derivatized porous supports prepared from azlactone-functional porous supports are also disclosed.

Method of coupling ligands onto supports and products produced therefrom

Transgenic mammals expressing human coagulation factor VIII

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.

Expression of human protein C in mammary tissue of transgenic mammals

Growing community of inventors

Blacksburg, VA, United States of America

William Hugold Velander