One goal of gene therapy is to deliver genes to somatic tissue in a manner that provides correction of inborn or acquired deficiencies and imbalances. Gene-based drug delivery offers a number of advantages over administration of recombinant proteins. These advantages include: conservation of native protein structure; improved biological activity; prolonged exposure to protein in the therapeutic range; prolonged availability of protein from each administration; avoidance of systemic toxicities; and avoidance of infectious and toxic impurities.
Efforts to develop gene-based delivery of therapeutic proteins such as, for example, erythropoietin (“EPO”) for treatment of anemias of various etiologies have been underway for almost a decade. EPO is produced primarily in the kidney in adults and is responsible for stimulating the production of red blood cells from progenitor cells. In patients with renal insufficiency, compromised EPO production results in anemia. Low serum EPO levels may also be seen in anemic patients with cancer, as well as those with rheumatoid arthritis, HIV infection, ulcerative colitis, sickle cell anemia, and in anemia of prematurity. However, certain proteins such as erythropoietin may have adverse effects if administration is not carefully controlled. For example, unregulated exposure to erythropoietin may result in life-threatening erythroid hyperplasia. In humans suffering from polycythemia, or a high red-blood cell count, prophylactic phlebotomy or blood removal is employed to maintain a hematocrit level below 45%. In animal models of erythropoietin-gene therapy, unregulated expression systems routinely result in hematocrit levels in the range of 60-85%. (Savino, R., et al., International Patent Publication No. WO0009713, “Adenoviral Vectors Encoding Erythropoietin and Their Use in Gene Therapy”; Podsakoff, G., et al., U.S. Pat. No. 5,846,528; Svensson, E., et al., Hum Gene Ther 8(15):1797 (1997); Lemieux, P., et al., Gene Therapy 7:986 (2000)). Periodic blood removal may be necessitated to avoid stroke and other severe polycythemic pathologies. (Zhou, S., et al., Gene Therapy 5, 665 (1998)).
In unregulated viral-vector-based erythropoietin-gene-delivery systems, the resulting hematocrit has been found to be viral dose dependent. Attempted adjustment of the hematocrit has been through empirical titration of the administered viral dose. (Kessler, et al., Proc. Natl. Acid. Sci. 93:14082 (1996)). Control of adverse and life threatening side effects through viral-dose titration, however, does not provide a satisfactory margin of therapeutic safety. What is needed for expression of proteins such as erythropoietin is the ability to closely regulate expression of the introduced gene across a range of administration dosages.
Several regulated gene-expression systems for erythropoietin have been explored. For example, Rizzuto, G., et al., Proc. Natl. Acad. Sci. 96:6417 (1999), utilized a tetracycline-inducible promoter to drive expression of a mouse EPO gene from plasmid DNA administered in saline with electroporation. As a consequence of the high basal level of expression in this system, however, the amount of plasmid DNA that could be administered for controlled expression had to be empirically titrated to a sufficiently low delivery amount.
Regulated viral vector systems for EPO delivery have also been described. For example, Rendahl, K. et al., Nature Biotechnology 16:757 (1998), reported a regulated two-viral vector system in which administration of tetracycline is designed to down regulate EPO production through the interaction of the two-vector gene products. However, control of EPO production was dependent on administered viral dose with gradual uncontrolled rise in hematocrit levels at higher viral doses. Ye, X., et al., Science 283:88 (1999) also reported the use of a two-viral vector system designed to be regulated by rapamycin. Although plasma EPO levels could be regulated by rapamycin, the hematocrit level could not, thereby indicating a basal level of EPO expression sufficient to stimulate a maximal increase in hematocrit.
In addition, the use of viral vectors is complicated by the generation of immune responses to the vector in immunocompetent hosts. As a consequence, viral vectors are considered to have limited readministration potential. Manning, W. et al., International Published Application WO09906562, “Method Enabling Readministration of AAV Vector Via Immunosuppression of Host”, described efforts to control this phenomena through the use of transient immunosuppression at the time of vector delivery. But immunosuppression, in general, may lead to undesirable side effects.
The use of a viral vector for ex vivo transformation of fibroblasts to provide a mutated steroid-hormone-regulated system of erythropoietin gene expression has also been reported. (Serguera, C. et al., Human Gene Therapy 10:375 (1999)). However, induction of gene expression by mifepristone resulted in polycythemia that was not reversible upon cessation of mifepristone treatment. What is needed is an improved regulated system where increases in hematocrit are not obtained in the absence of specific induction.
Recombinant interferon alpha “IFN-alpha” is the primary treatment for chronic hepatitis C virus infection. The current best treatment regimen (interferon with ribavirin) has a relatively low response rate that is attributed in part to the short half-life of interferon alpha in the circulation. Emerging therapies are interferons with covalently attached polyethylene glycol moieties (peginterferon) that are shown to have a longer half-life, sustained absorption and a slower rate of clearance. Clinical trials have indicated that use of peginterferon given once weekly is more effective than using non-modified interferon three times weekly. However, all of the routinely injected IFN-alpha protein therapies are associated with substantial side effects that result in part from the high levels of interferon that are obtained by bolus injection. What is needed is a long term continuous and consistent expression of low circulating levels of INF-alpha such that a sufficient anti-viral level is obtained without toxic peak levels. A potential method of achieving this goal with a minimum number of treatments is gene therapy. An adenoviral delivery system for expression of interferon alpha from the liver has been reported to provide protection of the liver from a hepatitis virus infection in a mouse model. Aurisicchio et al., J Virol 2000 May; 74(10):4816-23. However, because of potential adverse effects with uncontrolled interferon expression, the ability to regulate expression of the interferon may be required. What is needed in this context is a tightly regulated gene expression system for interferon alpha whereby induction can be obtained through administration of a non-toxic small molecule inducer.
Furthermore, the etiologies of many disease states are characterized by expression of a mutated protein or lack of protein expression due to a defect in one or more genes. Current treatment regimens include administration of human-derived protein or recombinant protein products to supplement the loss of endogenously produced protein. These proteins when administered are often viewed by the host as foreign, leading to the generation of antibodies to the administered protein that renders the treatment regimen ineffective. One example of a class of diseases that are due to a genetic absence of functional protein is hemophilia. Hemophilia A and B are caused by functional deficiencies in Factor VIII and Factor IX respectively. Hemophilic patients have a high incidence of developing inhibitors to replacement factors and much effort is focused on how to avoid this complication.
It has been demonstrated that expression of foreign proteins using a gene therapy approach can result in an immune response against the foreign protein. This response can be cellular, humoral or both and can result in rapid loss of vector-bearing cells (Fields et al., Mol Ther 1(3):225 (2000); Song et al. Hum Gene Ther 8(10):1207 (1997); Michou, et al., Gene Ther 4(5):473 (1997); Dai et al., Proc Natl Acad Sci USA 92(5):1401 (1995)). As an example of the effects of foreign transgene expression, when recombinant human EPO (“hEPO”) transgenes are delivered to mice, immune responses to the foreign transgene product can neutralize elevations in hematocrit level, and antibody cross-reactivity to endogenous EPO can result in erythroid hypoplasia that may lead to fatal anemia. (Tripathy, et al. Nat Med. 2:545 (1996); (Kessler, et al., Proc. Natl. Acid. Sci. 93:14082 (1996)).
Hence, what is needed is an improved, regulated gene expression system having extremely low levels of basal expression while retaining high inducibility. What is further needed is a system for minimizing the potential for developing of an immune response to therapeutic gene products.