Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1997-12-30
2004-11-16
Guzo, David (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S235100, C435S069100, C435S069600, C530S350000, C530S384000, C536S023100, C536S023500
Reexamination Certificate
active
06818439
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods of administration of recombinant gene transfer vehicles for the treatment of hemophilia, thrombosis, and other diseases. The present invention also relates generally to recombinant retroviruses, and more specifically, to high titer recombinant retroviral particle preparations suitable for a variety of applications.
BACKGROUND OF THE INVENTION
A variety of human disorders can be treated by the methods described herein. For example, hemophilia is a genetic disease characterized by a severe blood clotting deficiency. As such, it will be amenable to treatment by gene therapy. In hemophilia A, an X-chromosome linked genetic defect disrupts the gene encoding factor VIII, a trace plasma glycoprotein which acts as a cofactor in conjunction with factor IXa in the activation of factor X. In humans, the factor VIII gene codes for 2,351 amino acids. The protein has six domains, designated from amino to carboxy terminus as A1, A2, B, A3, C1, and C2, respectively (Wood et al., 1984,
Nature
312:330; Vehar et al., 1984,
Nature
312:337; and Toole et al., 1984,
Nature
312:342) with a deduced molecular weight of about 280 kilo Daltons (kD). The 980 amino acid B domain is deleted in the activated procoagulant form of the protein. Additionally, in the native protein two polypeptide chains, the heavy and light chain, flanking the B domain, are bound to a divalent calcium cation.
The genetic defect causing hemophilia A affects about one in every 10,000 males. Due to the resultant clotting deficiency, those afflicted with the disease suffer severe bleeding episodes due to small injuries, internal bleeding, and joint hemorrhage, which leads to arthropathy, the major cause of morbidity in hemophilia. Normal levels of factor VIII average between 50 to 200 ng/ml of blood plasma (Mannucci, P. M. in
Practical Laboratory Hematology,
ed. Koepke, J. A., Churchill Livingstone, N.Y., pp:347-371, 1990); however, patients suffering from mild to moderate hemophilia A typically have plasma levels well below 2-60 ng/ml, while levels below about 2 ng/mL result in severe hemophilia.
Previously, therapy for hemophilia A involved repeated administration off human factor VIII purified from blood products pooled in lots from over 1000 donors. However, because of the low levels of circulating factor VIII, resulting pharmaceutical products using the natural protein typically were highly impure, with an estimated purity by weight (factor VIII to total protein) of approximately 0.04%. Due to the frequency of administration and inability to remove various human pathogens from such preparations, more than 90% of those suffering from hemophilia A were infected in the 1980s with the human immunodeficiency virus (HIV) as a result of their therapy. Many of these HIV infected patients and other HIV negative hemophiliacs have also been infected by Hepatitis B in the same way. Fortunately, recent advances in genetic engineering have lead to the commercial availability of a recombinant form of the protein free from contamination with human pathogens, except for those potentially derived from tissue culture origin of the proteins, or from human serum albumin used in formulation of the recombinant protein. However, this form of therapy is expensive and chronic, and a large proportion of hemophiliacs continue to rely on plasma-derived products due to expense and or shortages of the recombinant product. In addition, most hemophilia A patients in the Unites States do not presently receive factor VIII maintenance therapy, but instead only receive the polypeptide prior to activities or events which might cause bleeding, such as surgery, or to treat spontaneous bleeding. Interestingly, this is despite evidence showing that hemophilic arthropathy can be prevented by administering from an early age prophylactic amounts of factor VIII, typically 24-40 IU per kilogram bodyweight, three times a week. Such therapy kept factor VIII concentrations from falling below 1% of normal (Nillson, et al.,
J. Internal Med.
232:23, 1992). For these reasons, a genetic therapy affording continuous, long term therapeutically effective expression levels or amounts of factor VIII, i.e., to decrease the severity of or eliminate the clotting disorder associated with hemophilia A, would be of great benefit.
A condition clinically indistinguishable from Hemophilia A is Hemophilia B, resulting from the deficiency of clotting factor IX. The incidence of this condition is about 5-fold lower than that of hemophilia A, and presents many of the same therapeutic challenges and difficulties. For similar reasons, it would be of great benefit to provide a gene therapy to these patients.
Factor X deficiency results in a rare but serious bleeding disorder affecting 1 in 500,000 known as Stuart disease. Le et al., 1997,
Blood
89:1254-9, describes therapeutic levels of functional human factor X in rats after retroviral mediated hepatic gene therapy. As in the case of hemophilia A and B, a genetic therapy affording continuous, long term therapeutically effective expression levels or amounts of factor X, i.e., to decrease the severity of or eliminate the clotting disorder associated with hemophilia B, would be of great benefit.
The present invention also provides for gene therapy delivery of other clotting factors for treatment or prophalaxis of thrombosis. Venous thromboembolism has an annual incidence of 1/1000 in the general population (Dahlback, 1995,
Blood
85:607). Precipitating factors can include hemostatic challenges such as surgery, fractures, inflammation, immobilization, pregnancy, oral contraceptive use, trauma, cancer, etc. Thrombosis is often familial, and a number of genetic risk factors have been identified. The clinical condition in which recurrent thrombosis occurs has been dubbed thrombophilia. The natural defenses against thrombosis involve two major systems: serpin inhibitors of thrombin, e.g., antithrombin III, the major pathway by which heparin exerts its clinical antithrombin effect, and the protein C system. Gene therapy for thrombosis disorders is needed and is addressed by the instant invention.
The present invention also provides methods for treatment of diseases such as viral hepatitis. Currently, the only approved treatment for chronic hepatitis B, C and D infections is the use of alpha interferon 2a and 2b. However, for patients with hepatitis B infections only about 35% of patients infected as adults responded to such treatment, and in perinatal infectees only about 10% responded to treatment (Perrillo et al., 1990,
New Eng. J. Med.
323:295-301). For hepatitis C infections, despite apparent short term success utilizing such therapy, six months after termination of treatment half of the patients who responded to therapy had relapsed. (Davis et al.,
New Eng. J. Med.
321:1501-1506). In pilot studies for hepatitis D infections, 25-60% of patients responded to alpha interferon therapy. Sustained responses were rare; 85-90% of patients who responded had relapsed. (di Bisceglie, A. M. D.,
Viral Hepatitis A to F: An Update,
1994). In addition, a further difficulty with alpha interferon therapy is that the composition frequently has toxic side effects which require reduced dosages for sensitive patients. Thus, improved methods for treatment of viral hepatitis are needed and are addressed by the present invention.
The instant invention also relates to the production and use of high titer recombinant retroviruses. Since the discovery of DNA in the 1940s and continuing through the most recent era of recombinant DNA technology, substantial research has been undertaken in order to realize the possibility that the course of disease may be affected through interaction with the nucleic acids of living organisms. Most recently, a wide variety of methods have been described for altering or affecting genes, including for example, viral vectors derived from retroviruses, adenoviruses, vaccinia viruses, herpes viruses, and adeno-associated viruses (see Jolly, 1994,
Cancer Gene Therapy
1(1):5
Chang Stephen
DePolo Nicholas J.
Greengard Judith
Hsu David Chi-Tang
Ibanez Carlos E.
Blackburn Robert P.
Chiron Corporation
Cullman Louis C.
Guzo David
Harbin Alisa A.
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