Multicellular living organisms and unmodified parts thereof and – Nonhuman animal – Transgenic nonhuman animal
Reexamination Certificate
1999-03-04
2001-07-03
Crouch, Deborah (Department: 1632)
Multicellular living organisms and unmodified parts thereof and
Nonhuman animal
Transgenic nonhuman animal
C800S015000, C800S016000, C800S017000, C800S018000, C800S007000, C800S025000, C435S069600
Reexamination Certificate
active
06255554
ABSTRACT:
BACKGROUND OF THE INVENTION
This invention relates generally to transgenic animals and their use as bioreactors for the production of clinically useful quantities of proteins. More specifically, this invention relates to a non-human transgenic animal genetically engineered to express recombinant human Factor VIII protein or von Willebrand Factor protein and to secrete newly expressed protein into a body fluid from which the protein can readily be isolated.
Factor VIII (“F8”) is a blood plasma glycoprotein of about 260 kDa molecular mass produced in the liver of mammals. It is a critical component of the cascade of coagulation reactions that lead to blood clotting. Within this cascade is a step in which Factor IXa, in conjunction with F8, converts Factor X to an activated form, Factor Xa. F8 acts as a cofactor at this step, being required with calcium ions and phospholipid for the activity of Factor IXa. The two most common hemophilic disorders are caused by a deficiency of functional F8 (Hemophilia A, about 80% of all cases) or functional Factor IXa (Hemophilia B or Christmas Factor disease).
Until recently, the standard treatment of Hemophilia A involved frequent infusions of preparations of F8 concentrates derived from the plasmas of human donors. While this replacement therapy is generally effective, such treatment puts patients at risk for virus-transmissible diseases such as hepatitis and AIDS. Although this risk has been reduced by further purification of F8 from plasma by immunopurification using monoclonal antibodies, and by inactivating viruses by treatment with either an organic solvent or heat, such preparations have greatly increased the cost of treatment, and are not without risk. For these reasons, patients have been treated episodically, rather than prophylactically. A further complication is that about 15% of patients develop inhibitory antibodies to thus-prepared F8.
An important advance in the treatment of Hemophilia A has been the isolation of cDNA clones encoding the complete 2,351 amino acid sequence of human F8 (see, Wood et al,
Nature
, 312: 330 (1984) and U.S. Pat. No. 4,757,006, Jul. 12, 1988) and the provision of the human F8 gene DNA sequence and recombinant methods for its production).
Analysis of the deduced primary amino acid sequence of human F8 determined from the cloned cDNA indicates that it is a heterodimer processed from a larger precursor polypeptide. The heterodimer consists of a C-terminal light chain of about 80 kDa in a metal ion-dependent association with an about 210 kDa N-terminal heavy chain fragment. See review by Kaufman,
Transfusion Med. Revs
., 6: 235 (1992). Physiological activation of the heterodimer occurs through proteolytic cleavage of the protein chains by thrombin (Factor IIa). Thrombin cleaves the heavy chain to a 90 kDa protein, and thence to 54 kDa and 44 kDa fragments. Thrombin also cleaves the 80 kDa light chain to a 72 kDa protein. It is the latter protein, and the two heavy chain fragments (54 kDa and 44 kDa above), held together by calcium ions, that constitute active F8. Inactivation occurs when the 72 kDa and 54 kDa proteins are further cleaved by thrombin, activated protein C or Factor Xa. In plasma, this F8 complex is stabilized by association with a 50-fold excess of von Willebrand Factor protein (“vWF”), which appears to inhibit proteolytic destruction of F8.
The amino acid sequence of F8 is organized into three structural domains: a triplicated A domain of 330 amino acids, a single B domain of 980 amino acids, and a duplicated C domain of 150 amino acids. The B domain has no homology to other proteins and provides 18 of the 25 potential asparagine (N)-linked glycosylation sites of this protein. Although porcine and human F8 show striking divergence in their B domains, the porcine protein can be used to treat Hemophilia A in humans. This suggests either that the B chain is not critical to the biological activity of the holomolecule, or that multiple versions of this domain are similarly effective. Porcine and human F8 show 80-85% homology in two of the three A domains.
Although the hepatocyte is likely to be the cell type that produces F8 in vivo, to date there are no known natural cell lines that express this protein. Kaufman, 1992, above, and Wood et al. 1984, above transfected transformed hamster kidney cells with a vector containing a gene that encodes F8, and expressed this protein. Kaufman,
Nature
342: 207 (1989), expressed recombinant F8 in transformed CHO cells, but production and secretion of newly synthesized protein into the conditioned growth medium was very low. This was said to have been due to three factors: a requirement for the presence of the vWF in the conditioned medium used in these culture systems in order to protect newly secreted F8 from proteolytic destruction (in the absence of vWF, Factor VIII was secreted as incomplete chains that were subsequently degraded); incomplete secretion of newly synthesized F8 from the cells (most remained in the endoplasmic reticulum); and, a low level of F8 mRNA as a result of a post-translational event. Stable recombinant F8 was secreted by CHO cells only when the gene for vWF was concurrently expressed. Additional drawbacks to the use of mammalian tissue culture systems for the production of F8 in clinically useful quantities are the expense of growth media and the limited production capacity of mammalian tissue culture systems.
An important need remains for an efficient and relatively inexpensive means of producing large quantities of infectious particle-free, human F8 protein suitable for clinical use. The transgenic animal system described below that produces human F8 recombinantly satisfies this need.
It has been estimated, for example, by Paleyanda et al, in RECOMBINANT TECHNOLOGY IN HEMOSTASIS AND THROMBOSIS eds., Hoyer et al., (Plenum Press, NY 1991), that the U.S. market for F8 is about 600,000,000 units per year. At a specific activity of 5,000 U/mg, about 120 g a year are required. Assuming an achievable expression level of 50 mg/L in the milk of a transgenic animal of the invention and a 50% loss of the protein during purification, it has been estimated that about 1 cow (producing 6,000 L of milk yearly), 10 goats, sheep or pigs (producing 500 L of milk yearly), or 5,333 rabbits (producing 0.9 L of milk yearly) would be more than sufficient to supply all of this country's needs for F8 (Paleyanda et al., above).
SUMMARY OF THE INVENTION
It is therefore an object of this invention to provide a non-human, transgenic female mammalian animal that produces F8.
It is another object of the invention to provide a process for producing transgenic mammals with the aforementioned characteristic.
It is still another object of the invention to provide a method for producing F8 in commercially valuable amounts, by inducing a non-human transgenic female mammal to secrete expressed F8 into its milk, and isolating this protein from the milk.
It is yet another object to provide a transgenic animal that has had stably integrated into its genome DNAs encoding both F8 and vWF, such that both proteins are expressed and secreted into the animal's milk.
These objects have been realized by the production of a non-human transgenic female mammalian animal into whose genome is stably integrated an exogenous recombinant double stranded DNA sequence comprising a protein promoter specifically active in a secretory gland operably linked to a DNA sequence encoding F8 or vWF or both proteins and a signal peptide effective in directing secretion of newly expressed F8 protein into a body fluid of the animal. More particularly, the protein promoter is for a milk protein and the signal peptide directs the expressed F8 into the animal's milk.
These objects are also realized by the provision of double stranded DNA constructs comprising a tissue-specific active protein promoter operably linked to a DNA sequence encoding F8 or vWF and a DNA sequence encoding a signal peptide that directs newly expressed F8 or vWF into the host animal's body fluid, which con
Drohan William N.
Lubon Henryk
Velander William H.
American National Red Cross
Crouch Deborah
Foley & Lardner
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