Transgenic Non-Human Animals Expressing Human Blood Clotting Factors and Uses Thereof

Abstract

The present invention relates, in general, to development of non-human transgenic animals expressing a human blood clotting factor, such as Factor VIII, Factor VII, Factor IX and von Willebrand factor. The invention further provides methods of detecting immunogenic events against human blood clotting factor using the transgenic animals described.

1 . A transgenic non-human animal having a genome comprising a human transgene polynucleotide sequence encoding a human blood clotting factor selected from the group consisting of Factor VIII (FVIII), Factor VII (FVII), Factor IX (FIX), von Willebrand Factor (vWF), Factor II (FII), Factor V (FV), Factor X (FX), Factor XI (FXI), Factor XII (FXII), and Factor XIII (FXIII). 
     
     
         2 . The transgenic animal of  claim 1 , wherein the animal does not express all or part of a polynucleotide encoding an endogenous blood clotting factor corresponding to the human transgene. 
     
     
         3 . The transgenic animal of  claim 1 , wherein the polynucleotide sequence is operably linked to a promoter polynucleotide sequence. 
     
     
         4 . (canceled) 
     
     
         5 . The transgenic animal of  claim 1 , wherein the polynucleotide sequence comprises a poly A polynucleotide sequence. 
     
     
         6 - 10 . (canceled) 
     
     
         11 . The transgenic animal of  claim 1 , wherein the animal also expresses a human major histocompatibility class II gene in place of a major histocompatibility class II gene endogenous to the transgenic animal. 
     
     
         12 . A non-human transgenic animal comprising a polynucleotide encoding a human blood clotting factor selected from the group consisting of Factor VIII, Factor VII, Factor IX, von Willebrand Factor, Factor II, Factor V, Factor X, Factor XI, Factor XII, and Factor XIII, said human blood clotting factor having physiological activity of the human blood clotting factor, said transgenic mammal having in its genome an exogenous transgene construct comprising:
 (a) transcriptional regulatory polynucleotide sequences,   (b) DNA encoding said human blood clotting factor, and   (c) a polyadenylation signal,   wherein (A), (B) and (C) are operably linked in said exogenous gene construct to obtain production of said human blood clotting factor or fragment thereof in said transgenic animal.   
     
     
         13 . The transgenic animal of  claim 12 , wherein the transcriptional regulatory polynucleotide sequences are selected from the group consisting of 5′ transcriptional regulatory polynucleotide sequences, 3′ transcriptional regulatory polynucleotide sequences, internal transcriptional regulatory polynucleotide sequences, and combinations thereof. 
     
     
         14 . The transgenic animal of  claim 13 , wherein the 5′ regulatory sequence is a promoter, optionally comprising an enhancer region. 
     
     
         15 . (canceled) 
     
     
         16 . The transgenic animal of  claim 12 , wherein the animal does not express all or part of a polynucleotide encoding an endogenous blood clotting factor corresponding to the human transgene. 
     
     
         17 - 20 . (canceled) 
     
     
         21 . The transgenic animal of  claim 12 , wherein the transgenic animal also comprises a polynucleotide encoding a human major histocompatibility class II gene in place of the a major histocompatibility class II gene endogenous to the transgenic animal. 
     
     
         22 . A method of producing a transgenic non-human animal comprising a polynucleotide transgene encoding a human blood clotting factor selected from the group consisting of Factor VIII, Factor VII, Factor IX, von Willebrand Factor, Factor II, Factor V, Factor X, and Factor XI, Factor XII, and Factor XIII comprising:
 introducing a polynucleotide sequence encoding said human blood clotting factor into the genomic DNA of the non-human animal to provide a the transgenic non-human animal comprising a polynucleotide encoding the human blood clotting factor and not a corresponding blood clotting factor endogenous to the transgenic animal.   
     
     
         23 . The method of  claim 22 , wherein the introducing is carried out by microinjection or using a viral vector. 
     
     
         24 - 28 . (canceled) 
     
     
         29 . The method of  claim 22 , wherein the polynucleotide sequence is operably linked to a promoter polynucleotide sequence. 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 22 , wherein the polynucleotide sequence comprises a poly A sequence. 
     
     
         32 . (canceled) 
     
     
         33 . The transgenic animal of  claim 1 , wherein the animal is homozygous for said transgene. 
     
     
         34 . The transgenic animal of  claim 1 , wherein the animal is heterozygous for said transgene. 
     
     
         35 . The method of  claim 22  further comprising introducing a polynucleotide sequence encoding an human major histocompatibility class II gene into the genomic DNA of the non-human animal to replace all or part of a major histocompatibility class II gene endogenous to the animal. 
     
     
         36 . A method for producing a transgenic non-human animal comprising a polynucleotide encoding a human blood clotting factor comprising:
 a) providing a polynucleotide sequence encoding an human blood clotting factor selected from the group consisting of Factor VIII, Factor VII, Factor IX, von Willebrand Factor, Factor II, Factor V, Factor X, Factor XI, Factor XII, and Factor XIII and a positive selectable marker gene, said marker gene flanked by loxP sites;   b) introducing said polynucleotide sequence into an embryonic stem cell from the same animal species as said non-human animal under conditions such that said polynucleotide sequence is homologously recombined into a genomic locus of said embryonic stem cell to produce an embryonic stem cell containing a polynucleotide encoding a human blood clotting factor selected from the group consisting of Factor VIII, Factor VII, Factor IX, von Willebrand Factor, Factor II, Factor V, Factor X, Factor XI, Factor XII, and Factor XIII and said selectable marker gene;   c) injecting said homologously recombined embryonic stem cell into a blastocyst of said non-human animal;   d) introducing said injected blastocyst into a pseudo-pregnant female non-human animal; and   e) permitting said pseudo-pregnant female animal to deliver one or more transgenic animals containing said homologously recombined DNA sequence, wherein said one or more transgenic mice express said human blood clotting factor selected from the group consisting of Factor VIII, Factor VII, Factor IX, von Willebrand Factor, Factor II, Factor V, Factor X, Factor XI, Factor XII, and Factor XIII.   
     
     
         37 . The method of  claim 36 , wherein the polynucleotide comprises a promoter operably linked to said human blood clotting factor gene. 
     
     
         38 . (canceled) 
     
     
         39 . The method of  claim 36 , wherein the polynucleotide sequence comprises a polyA sequence. 
     
     
         40 . The method of  claim 36 , wherein the selectable marker is a neomycin resistance gene, neo. 
     
     
         41 . The method of  claim 36  wherein the one or more transgenic animals from step (e) are crossed with a Cre-deleter strain of mouse. 
     
     
         42 - 45 . (canceled) 
     
     
         46 . The method of  claim 36  further comprising introducing a polynucleotide sequence encoding a human major histocompatibility class II gene into the genomic DNA of the non-human animal said polynucleotide sequence encoding a human major histocompatibility class II gene replacing all or part of a major histocompatibility class II gene endogenous to the transgenic animal such that the transgenic animal does not express its endogenous major histocompatibility class II gene. 
     
     
         47 . A method for screening for antibodies against a human blood clotting factor in a non-human transgenic animal comprising a polynucleotide transgene expressing a human blood clotting factor selected from the group consisting of Factor VIII, Factor VII, Factor IX, von Willebrand Factor, Factor II, Factor V, Factor X, Factor XI, Factor XII, and Factor XIII the method comprising,
 administering to the animal a composition comprising a human blood clotting factor polypeptide, fragment, analog or variant thereof corresponding to the human blood clotting factor expressed in the animal, and   detecting antibodies specific for said human blood clotting factor in a sample from the transgenic animal.   
     
     
         48 . (canceled) 
     
     
         49 . The method of  claim 47 , wherein the composition comprises a fragment, analog or variant of the human blood clotting factor polypeptide expressed by the transgene in the animal. 
     
     
         50 . The method of  claim 49 , wherein the human blood clotting factor administered in the composition comprises a water soluble polymer. 
     
     
         51 . (canceled) 
     
     
         52 . The method of  claim 49 , wherein the human blood clotting factor administered in the composition comprises a polysialyl moiety. 
     
     
         53 . The method of  claim 47 , wherein the human blood clotting factor in the administered composition is a fusion protein. 
     
     
         54 . (canceled) 
     
     
         55 . The method of  claim 47 , wherein the composition further comprises a second human blood clotting factor selected from the group consisting of Factor VIII, Factor VII, Factor IX, von Willebrands Factor, Factor II, Factor V, Factor X, Factor XI, Factor XII, and Factor XIII. 
     
     
         56 - 60 . (canceled) 
     
     
         61 . A method for screening for immunogenicity of a human blood clotting factor in a non-human transgenic animal comprising a transgene expressing a human blood clotting factor polypeptide selected from the group consisting of Factor VIII, Factor VII, Factor IX, von Willebrand Factor, Factor II, Factor V, Factor X, Factor XI, Factor XII, and Factor XIII, the method comprising,
 administering to said animal a composition comprising a human blood clotting factor corresponding to the human blood clotting factor expressed by the transgene in the animal, and   detecting an immunogenic event in the animal subsequent to the administration of the human blood clotting factor polypeptide.   
     
     
         62 . The method of  claim 61 , wherein the immunogenic event is antibody production. 
     
     
         63 . The method of  claim 61 , wherein the composition comprises a fragment, analog or variant of the human blood clotting factor polypeptide expressed by the transgene in the animal. 
     
     
         64 - 65 . (canceled) 
     
     
         66 . A method for determining the effect of a compound on a human blood clotting factor comprising: administering a test compound to a non-human transgenic animal comprising a transgene expressing a human blood clotting factor selected from the group consisting of Factor VIII (FVIII), Factor VII (FVII), Factor IX (FIX), von Willebrand Factor (VWF), Factor II (FII), Factor V (FV), Factor X (FX), Factor XI (FXI), Factor XII (FXII), and Factor XIII (FXIII), and detecting a change in human blood clotting factor activity in a sample taken from the transgenic animal in the presence of the compound compared to the activity of the human blood clotting factor in a sample in the absence of the compound. 
     
     
         67 . The method of  claim 66 , wherein the human blood clotting activity is selected from the group consisting of expression levels of the blood clotting factor, change in blood clotting activity, and protein binding activity. 
     
     
         68 . (canceled) 
     
     
         69 . An experimental animal model that is a non-human transgenic animal expressing at least one human blood clotting factor, wherein the animal does not generate a significant antibody titer against the human blood clotting factor when the native blood clotting factor is administered in solution. 
     
     
         70 . The animal model of  claim 69 , which is an experimental animal model for acquired hemophilia A or hemophilia B. 
     
     
         71 . (canceled) 
     
     
         72 . The experimental animal model of  claim 69  wherein the blood clotting factor is selected from the group consisting of Factor VIII, Factor VII, Factor IX, von Willebrand Factor, Factor II, Factor V, Factor X, Factor XI, Factor XII and Factor XIII. 
     
     
         73 . A method for identifying an agent which induces a break of tolerance to a blood clotting factor, comprising:
 administering a candidate agent to the transgenic non-human animal of  claim 69  expressing a human blood clotting factor,   administering the human blood clotting factor for which the animal is transgenic; and,   detecting anti-blood clotting factor response in the animal,   wherein the candidate agent is a tolerance-breaking agent if the administration of the candidate agent permits production of an anti-blood clotting factor response.   
     
     
         74 . The method of  claim 73 , wherein the response is selected from the group consisting of production of anti-blood clotting factor inhibitors and an immune response. 
     
     
         75 - 77 . (canceled) 
     
     
         78 . The method of  claim 73 , wherein the blood clotting factor is selected from the group consisting of Factor VIII, Factor VII, Factor IX, von Willebrand Factor, Factor II, Factor V, Factor X, Factor XI, Factor XII and Factor XIII. 
     
     
         79 . The method of  claim 22 , wherein the animal is homozygous for said transgene. 
     
     
         80 . The method of  claim 22 , wherein the animal is heterozygous for said transgene.