This invention relates to gene therapy. More specifically, the present invention relates to delivery of DNA encoding secreted proteins such as interferon proteins in humans and animals.
Interferons (also referred to as xe2x80x9cIFNxe2x80x9d or xe2x80x9cIFNsxe2x80x9d) are proteins having a variety of biological activities, some of which are antiviral, immunomodulating and antiproliferative. They are relatively small, species-specific, single chain polypeptides, produced by mammalian cells in response to exposure to a variety of inducers such as viruses, polypeptides, mitogens and the like. Interferons protect animal tissues and cells against viral attack and are an important host defense mechanism. In most cases, interferons provide better protection to tissues and cells of the kind from which they have been produced than to other types of tissues and cells, indicating that human-derived interferon could be more efficacious in treating human diseases than interferons from other species.
There are several distinct types of human interferons, generally classified as leukocyte (interferon-alpha [xcex1]), fibroblast (interferon-beta [xcex2]) and immune (interferon-gamma [xcex3]), and a large number of variants thereof. General discussions of interferons can be found in various texts and monographs including: The Interferon System (W. E. Stewart, II, Springer-Verlag, N.Y. 1979); and Interferon Therapy (World Health Organization Technical Reports Series 676, World Health Organization, Geneva 1982), incorporated herein by reference.
Interferons have potential in the treatment of a large number of human cancers since these molecules have anti-cancer activity which acts at multiple levels. First, interferon proteins can directly inhibit the proliferation of human tumor cells. The anti-proliferative activity is also synergistic with a variety of approved chemotherapeutic agents such as cis-platin, 5FU and taxol. Secondly, the immunomodulatory activity of interferon proteins can lead to the induction of an anti-tumor immune response. This response includes activation of NK cells, stimulation of macrophage activity and induction of MHC class I surface expression leading to the induction of anti-tumor cytotoxic T lymphocyte activity. Moreover, some studies further indicate that IFN-xcex2 protein may have anti-angiogenic activity. Angiogenesis, new blood vessel formation, is critical for the growth of solid tumors. Evidence indicates that IFN-xcex2 may inhibit angiogenesis by inhibiting the expression of pro-angiogenic factors such as bFGF and VEGF. Lastly, interferon proteins may inhibit tumor invasiveness by affecting the expression of enzymes such as collagenase and elastase which are important in tissue remodeling.
Interferons also appear to have antiviral activities that are based on two different mechanisms. For instance, type I interferon proteins (xcex1 and xcex2) can directly inhibit the replication of human hepatitis B virus (xe2x80x9cHBVxe2x80x9d) and hepatitis C virus (xe2x80x9cHCVxe2x80x9d), but can also stimulate an immune response which attacks cells infected with these viruses.
Specifically, and despite its potential therapeutic value, interferon proteins have only had limited clinical success against viral hepatitis and solid tumors. IFN-xcex1 has been approved for the treatment of both HBV and HCV; however, the response rate in both cases is only approximately 20%. While interferon proteins have been approved for the treatment of some cancers such as lymphomas, leukemias, melanoma and renal cell carcinoma, the majority of clinical trials in which interferons are used alone or in combination with conventional chemotherapeutic agents in the treatment of solid tumors have been unsuccessful.
The method of administering interferon is an important factor in the clinical application of this important therapeutic agent. Systemic administration of interferon protein by either intravenous, intramuscular or subcutaneous injection has been most frequently used with some success in treating disorders such as hairy cell leukemia, Acquired Immune Deficiency Syndrome (AIDS) and related Kaposi""s sarcoma. It is known, however, that proteins in their purified form are especially susceptible to degradation. In particular, for interferon-beta, the primary mechanism(s) of interferon degradation in solution are aggregation and deamidation. The lack of interferon stability in solutions and other products has heretofore limited its utility. Furthermore, following parenteral interferon protein administration (intramuscular, subcutaneous or intravenous) the clearance rate of interferon protein is very rapid. Therefore, parenteral protein administration may not allow the localization of sufficient interferon at the active site (the solid tumor, or, in the case of hepatitis, the liver). The amount of interferon that can be given parenterally in patients is limited by the side-effects observed at high interferon doses. A more effective therapy is clearly needed.
This application is directed toward eliminating the problems associated with delivering a secreted protein such as interferon protein as a therapeutic. The present invention is directed to a method of interferon therapy in which the gene encoding the secreted protein rather than the protein itself, is delivered.
Accordingly, it is one object of the instant invention to provide a method of gene therapy based on the use of genetically engineered cells and to the use thereof for delivering a secreted protein such as an interferon to a mammalian recipient. The instant invention satisfies these and other objects by providing methods for forming a cell expression system, the expression system produced thereby and pharmaceutical compositions containing the same. The cell expression system expresses a gene encoding one or more secreted proteins and is useful as a vehicle for delivering the gene product to the mammalian recipient in situ. In a preferred embodiment, the mammalian recipient is a human.
In one embodiment of the invention, a cell expression system is described for expressing in a cell of a mammalian recipient in vivo, an interferon protein for treating a condition. The expression system comprises a cell of the same species as the mammalian recipient and an expression vector contained therein for expressing the interferon protein. Preferably, the mammalian recipient is a human and the expression vector comprises a viral vector.
In another embodiment, the expression system comprises a plurality of cells of the same species as the mammalian recipient and an expression vector contained therein for expressing the secreted protein. The expression vector is contained within only a portion of the plurality of cells. Preferably, at least 0.3% by number of the cells contain the vector. The preferred secreted protein is an interferon and the most preferred interferons are alpha, beta, gamma and consensus interferon, with beta interferon being the most preferred.
In other embodiments, the cell expression system comprises a plurality of cancer cells and at least a portion of the cancer cells contain an adenoviral vector having an isolated polynucleotide encoding, upon expression, an interferon. In this cell expression system, the adenoviral vectors are selected from the group consisting of: (a) an adenoviral vector having a deletion and/or mutation in its E1 gene; (b) an adenoviral vector having a deletion and/or mutation in its E2a gene, said vector expressing human interferon-beta; (c) an adenoviral vector having a deletion and/or mutation in both its E1 and E4 genes, and (d) an adenoviral vector having a deletion of all of its genes; said vector expressing human interferon-beta.
A pharmaceutical composition for delivery of a secreted protein to a site of a mammalian recipient, is also encompassed within the invention. The composition comprises a carrier and a plurality of genetically modified cells of the same species as the mammalian recipient and at least a portion of the cells contain an expression vector for expressing an effective amount of the secreted protein. The preferred secreted protein is an interferon. The composition encompasses compositions for both in vivo and ex vivo delivery.
A method for making an ex vivo gene therapy pharmaceutical preparation for administration to a mammalian recipient is another embodiment. The method includes the steps of: (a) forming a plurality of cells of the same species as the mammalian recipient, (b) introducing an expression vector for expressing a secreted protein into at least one cell of the plurality to form at least one genetically modified cell and (c) placing the at least one genetically modified cell in a pharmaceutically acceptable carrier to obtain a pharmaceutical preparation that is suitable for administration to a site of the mammalian recipient.
Another embodiment of the invention is a method for gene therapy, which comprises genetically modifying at least one cell of a mammalian recipient via the steps of (a) introducing an expression vector for expressing a secreted protein into at least one cell to form at least one genetically modified cell and (b) allowing the genetically modified cell to contact a site of the mammalian recipient. This method may further comprise the step of removing at least one cell from the mammalian recipient prior to the step of introducing the expression vector. In yet another embodiment, the step of introducing the vector comprises introducing a vector to only a portion of the plurality of cells.
A method of ex vivo gene therapy is encompassed by the invention and includes the steps of removing a plurality of cells from a subject; administering a recombinant adenovirus to at least one cell of the plurality of cells, such that there exists an excess of cells not containing the adenovirus. The adenovirus can have a deletion in its E1 gene and includes an isolated polynucleotide encoding a secreted protein. The plurality of cells is reintroduced back into the subject.
In a method of in vivo gene therapy, the steps include administering an adenoviral vector that includes an isolated polynucleotide encoding human interferon-beta (xcex2) protein directly into a cell of a subject without first removing said cell from the subject. The in vivo and ex vivo methods allow for topical, intraocular, parenteral, intranasal, intratracheal, intrabronchial, intramuscular, subcutaneous intravenous, intramuscular, and intraperitoneal administration.
The present invention has several advantages. Interferon gene therapy may allow for very high local interferon concentrations with low systemic levels. This could result in greater efficacy with lower side-effects. Also, interferons delivered by gene therapy would be present at a fairly constant level, unlike the situation observed in interferon protein therapy in which very high interferon xe2x80x9cburstsxe2x80x9d or peaks in protein concentration (which could lead to toxicity) that occur after protein injection, are followed by very low levels in which the interferon concentration is likely to be too low to be effective.
Patient convenience is also a critical factor. While frequent injections of interferon protein are necessary, a single administration, or a few infrequent administrations, of a vector expressing the interferon gene could provide long-term stable production of the protein. Gene therapy could allow the delivery of interferons in a controlled manner to a distinct target organ or tumor. An autocrine system can be established in which the same cells express, secrete and take up the interferon. Thus, very high local doses can be achieved. These high doses cannot be achieved by parenteral protein administration due to toxicity problems.
Since interferon proteins are secreted out of cells, every cell in a tumor or in the hepatitis-infected liver need not be transduced by the interferon gene. Those cells which do not take up the gene will be affected by neighboring cells (the so-called xe2x80x9cbystander effectxe2x80x9d) which have the gene and secrete the interferon protein. This is a significant finding and is likely to have dramatic effects on treatment regimens since not every cell in a tumor mass or in an organ need contain an expression vector.
Lastly, parenteral interferon administration has been shown to lead to the generation of anti-interferon antibodies. It may be that this potentially neutralizing antibody response can be lessened following introduction of the interferon gene to a distinct local region. Besides the local expression, the interferon expressed will be produced by endogenous human cells and, therefore, will be more natural in its structure and glycosylation and, possibly, less immunogenic than interferon protein produced in bacteria, yeast or Chinese hamster ovary cells and then purified and injected parenterally.