RECEPTOR MEDIATED GENE THERAPY OF HEMOPHILIAS

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The clinical management of hemophilia has become complicated by viral contamination of pooled plasma and the development of antibodies. Hemophilia A and B are both sex-linked recessive disorders, so that gene augmentation should correct the defects. For successful gene therapy of the hemophilias, it is critical to use non-invasive, efficient methods that deliver normal copies of the gene to the liver. It is also desirable to avoid the use of viral sequences since repeated administration of the candidate gene may be necessary. Functional genes have been introduced into hepatocytes in vitro and in vivo by targeting the asialoglycoprotein receptor of the liver. We propose to treat dogs with either classic hemophilia (Factor VIII deficiency, Hemophilia A) and Christmas disease (Factor IX deficiency, Hemophilia B) using an approach to gene transfer developed in our laboratory that targets the asialoglycoprotein receptor. The major innovation in this technique involves the condensation of DNA into a unimolecular form using poly-L-lysine, which is covalently attached to a ligand, i.e. galactose. The DNA complex injected into a systemic vein of an adult animal will efficiently target the liver without the need for invasive procedures, such as partial hepatectomy. Using this technique, we have delivered a chimeric gene containing the promoter from the P-enolpyruvate carboxykinase (PEPCK) gene (-460 to +73), linked to the structural gene for human Factor IX (hFIX) into the livers of adult rats and found prolonged expression of the transgene for up to 140 days (the duration of the experiment). The transgene remained in the liver as an episome. In addition, transcription of the transgene can be induced by feeding the rats a diet devoid of carbohydrate. We propose to extend these studies by introducing the canine Factor IX (cFIX) gene complexes to galactosylated poly-L-lysine into the livers of dogs with Hemophilia B by injecting the DNA complex into the cephalic vein. The level of cFIX in the blood will be monitored by serial measurements using ELISA and Western analysis; the functional activity will be determined by measuring the relative change in the whole blood clotting time and modified activated partial thromboplastin time. Only after we have demonstrated the success of experiments with cFIX treatment of dogs with Hemophilia B, will we attempt to introduce the human Factor VIII (hFVIII) gene into dogs with Hemophilia A. Finally, we will examine potential improvements in the technique by testing alternative ligands to target the DNA to the liver of rats. Other potential improvements that will be tested include the use of a variety of alternative promoters in an attempt to increase the expression of the transgene in the liver and to alter the packaging system used to condense the DNA. The procedure described in this proposal has promise for the treatment of hemophilias and deserves further consideration as an alternative approach to gene therapy of these diseases.