Protein replacement therapy for patients with hemophilia or other inherited protein deficiencies is often complicated by pathogenic antibody responses, including antibodies that neutralize the therapeutic protein or that predispose to potentially life-threatening anaphylactic reactions by formation of IgE. Using murine hemophilia B as a model, we have developed a prophylactic protocol against such responses that is non-invasive and does not include immune suppression or genetic manipulation of the patient's cells. Oral delivery of coagulation factor IX (F. IX) expressed in chloroplasts, bioencapsulated in plant cells, effectively blocked formation of inhibitory antibodies in protein replacement therapy. Inhibitor titers were mostly undetectable and up to 100-fold lower in treated mice when compared to controls. Moreover, this treatment eliminated fatal anaphylactic reactions that occurred after 4 to 6 exposures to intravenous F. IX protein. While only 20-25% of control animals survived after 6-8 F. IX doses, 90-95% of tolerized mice survived 12 injections without signs of allergy or anaphylaxis. This high-responder strain of hemophilia B mice represents the first hemophilic animal model to study anaphylactic reactions. The plant material was effective over a range of oral antigen doses (equivalent to 5-80 μg recombinant F.IX/kg), and controlled inhibitor formation and anaphylaxis long-term, up to 7 months. Oral antigen administration caused a deviant immune response that suppressed formation of IgE and inhibitory antibodies. This cost-effective and efficient approach to oral delivery of protein antigens to the gut should be applicable to several genetic diseases that are prone to pathogenic antibody responses during treatment.

Disclosed herein are methods and compositions for insertion of transgene sequences encoding proteins involved in clotting into the genome of a cell for treating conditions including hemophilias.

The present invention provides codon optimized Factor IX sequences, vectors and host cells comprising codon optimized Factor IX sequences, polypeptides encoded by codon optimized Factor IX sequences, and methods of producing such polypeptides. The present invention also provides methods of treating bleeding disorders such as hemophilia comprising administering to the subject a codon optimized Factor IX nucleic acid sequence or the polypeptide encoded thereby.

Disclosed herein are compositions and methods for modulating the production of a protein in a target cell. The compositions and methods disclosed herein are capable of ameliorating diseases associated with protein or enzyme deficiencies.

Fusion proteins comprising a protein expression enhancing polypeptide linked to a target protein binding domain and nucleic acid molecules encoding such fusion proteins are described for use in enhancing expression and/or location of a targeted protein of interest, for restoring lost functions in cells, and for treating disease. Additional fusion proteins comprising a target protein of interest modified with a fusion partner comprising a protein expression enhancing polypeptide are also disclosed.

The present invention relates to variants of factor IX (F.IX) or activated factor IX (F.IXa), wherein the variant is characterized in that it has clotting activity in absence of its cofactor. The present invention furthermore relates to variants of factor IX (F.IX) or activated factor IX (F.IXa), wherein the variant is characterized in that it has increased F.IX clotting activity compared to wildtype. The present invention furthermore relates to the use of these variants for the treatment and/or prophylaxis of bleeding disorders, in particular hemophilia A and/or hemophilia B or hemophilia caused or complicated by inhibitory antibodies to F.VIII. The present invention also relates to further variants of factor IX (F.IX) which have desired properties and can, thus be tailored for respective specific therapeutic applications.

In various embodiments, disclosed herein are methods of assessing therapeutic efficacy of an anticoagulant. Preferably, the method comprising providing a blood sample; perfusing the blood sample over a surface coated with collagen or immobilized rTF; measuring platelet aggregation and fibrin deposition on the surface coated with collagen or immobilized rTF; and assessing therapeutic efficacy of the anticoagulant based on the volume of platelet aggregates and/or deposited fibrin.

The present invention relates to modified Factor IX polypeptides comprising a mutation at a position corresponding to position 347 of wild type immature (precursor) Factor IX, polynucleotides encoding the polypeptides, and treatments utilising the polypeptides or polynucleotides.

Provided are formulations, compositions and methods for delivering biological moieties such as modified nucleic acids into cells to modulate protein expression. Such compositions and methods include the delivery of biological moieties, and are useful for production of proteins.

The present invention relates to a nucleic acid molecule encoding human albumin for increasing the levels and/or activity of a protein or polypeptide encoded by a transgene, comprising a sequence defined by SEQ ID NO: 14 or a sequence having at least 80% sequence identity to said sequence, its use in nucleic acid expression cassettes and vectors containing liver-specific regulatory elements and codon-optimized factor IX, factor VIII, factor VII or factor VIIa transgenes, methods employing these expression cassettes and vectors and uses thereof. The present invention is particularly useful for applications using liver-directed gene therapy, in particular for the treatment of hemophilia A, hemophilia B or factor VII deficiency.