Hemophilia B is a common X-chromosome-linked bleeding disorder which is due to a deficiency of biologically active blood coagulation factor IX in plasma. Current protein replacement therapy for hemophilia B is only effective to temporarily relieve bleeding episodes, but its repeated applications required by hemophilia B patients expose them to the risk of several serious complications, notably infection of pathogenic viruses such as HIV-1 and hepatitis virus. Here, we propose to establish a solid experimental base for a safe, effective, and practical gene therapy for hemophilia B based on primary myoblast-mediated gene transfer. A set of highly optimized new-generation retroviral vectors are constructed with a factor IX minigene, and a vector which can express factor IX at the highest level in mouse primary myoblasts and myotubes in culture is identified. Primary myoblast are transduced with the retroviral vector, and implanted into skeletal muscles of syngeneic mice. The systemic production of recombinant factor IX is analyzed, and maximized to reach a therapeutic level of recombinant factor IX (>5% the normal human plasma level) by optimizing individually and in combinations of various parameters and conditions involved in cell isolation and implantation procedures. Any deleterious effects to animals due to the injected myoblasts, such as tumorigenicity and detrimental effects on the muscle strength, are monitored and examined over a long period of time. After the extensive testing in mice, the optimized approach developed is then applied briefly to normal dogs, followed by the final, exhaustive testing with hemophilia B dogs. In the experiments with hemophilia dogs, a retroviral vector which is constructed with a canine factor IX minigene similar to the optimized human factor IX retroviral vector is used to transduce skeletal myoblasts isolated from hemophilia B dogs. The transduced cells are then implanted back into skeletal muscles of the same animals, permitting us to evaluate the effectiveness and safety to the procedure in a totally homogeneous system for at least several years. Finally, we submit the optimized human factor IX retrovirus to a series of extensive safety testings in culture as well as in monkeys which are required to certify it for its ultimate use in human applications in the next phase of this study. Information obtained from the proposed studies should provide us with a sound experimental base for developing a clinical protocol of durable ex vivo gene therapy for hemophilia B. Establishment of this effective, safe and practical gene therapy for hemophilia B should also open up an exciting avenue for its applications to many other diseases, not only various hematological and metabolic disorders which require an efficient systemic and local delivery of transgene products, but also muscular disorders.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL053713-05
Application #
2771399
Study Section
Special Emphasis Panel (ZHL1-CSR-K (S1))
Project Start
1994-09-30
Project End
2000-01-31
Budget Start
1998-09-30
Budget End
2000-01-31
Support Year
5
Fiscal Year
1998
Total Cost
Indirect Cost
Name
University of Michigan Ann Arbor
Department
Genetics
Type
Schools of Medicine
DUNS #
791277940
City
Ann Arbor
State
MI
Country
United States
Zip Code
48109
Kolka, Jacquelyn A; Vreede, Andrew P; Roessler, Blake J (2005) Lipopolysaccharide recognition protein, MD-2, facilitates cellular uptake of E. coli-derived plasmid DNA in synovium. J Gene Med 7:956-64
Allen, David L; Teitelbaum, Daniel H; Kurachi, Kotoku (2003) Growth factor stimulation of matrix metalloproteinase expression and myoblast migration and invasion in vitro. Am J Physiol Cell Physiol 284:C805-15
Krebsbach, Paul H; Zhang, Kezhong; Malik, Ajay K et al. (2003) Bone marrow stromal cells as a genetic platform for systemic delivery of therapeutic proteins in vivo: human factor IX model. J Gene Med 5:11-7
Malik, A K; Wang, J M; Kurachi, K (2001) Effects of a second intron on recombinant MFG retroviral vector. Arch Virol 146:601-9
Kurachi, K; Kurachi, S (2000) Genetic mechanisms of age regulation of blood coagulation: factor IX model. Arterioscler Thromb Vasc Biol 20:902-6
Livant, D L; Brabec, R K; Pienta, K J et al. (2000) Anti-invasive, antitumorigenic, and antimetastatic activities of the PHSCN sequence in prostate carcinoma. Cancer Res 60:309-20
Kurachi, K; Zhang, K; Ameri, A et al. (2000) Genetic and molecular mechanisms of age regulation (homeostasis) of blood coagulation. IUBMB Life 49:189-96
Malik, A K; Monahan, P E; Allen, D L et al. (2000) Kinetics of recombinant adeno-associated virus-mediated gene transfer. J Virol 74:3555-65
Livant, D L; Brabec, R K; Kurachi, K et al. (2000) The PHSRN sequence induces extracellular matrix invasion and accelerates wound healing in obese diabetic mice. J Clin Invest 105:1537-45
Brooks, M (1999) A review of canine inherited bleeding disorders: biochemical and molecular strategies for disease characterization and carrier detection. J Hered 90:112-8

Showing the most recent 10 out of 20 publications