WO2008112470A1

WO2008112470A1 - Increased effectiveness of mda-7 with mdr-1 over-expression

Info

Publication number: WO2008112470A1
Application number: PCT/US2008/055877
Authority: WO
Inventors: Paul Fisher; Luni Emdad; Devanand Sarkar; Irina Lebedeva
Original assignee: The Trustees Of Columbia University In The City Of New York
Priority date: 2007-03-09
Filing date: 2008-03-05
Publication date: 2008-09-18

Abstract

The present invention relates to the use of melanoma differentiation associated gene-7, its encoded protein, and related molecules (Collectively, 'Mda-7 Molecules') for improving the response of a subject to chemotherapy and the particular benefit of Mda-7 treatment in a subject resistant to chemotherapy. It is based, at least in part, on the discovery that mda-7 expression increases the response of hitherto resistant cells to chemotherapeutic agent, as well as on the discovery that mda-7 expression is itself enhanced in the context of over- expression of the multidrug resistance gene (mdr-l).

Description

INCREASED EFFECTIVENESS OF MDA-7 WITH MDR-I OVER-EXPRESSION

GRANT INFORMATION

This invention was made with government support under Grant Nos. POl CA104177, ROl CA35675 and ROl CA097318 awarded by the National Institutes of Health/National Cancer Institutes. The government has certain rights in the invention.

PRIORITY BENEFIT This application claims the benefit of priority to United States

Provisional Application Serial No. 60/894,068, filed March 9, 2007, the contents of which is hereby incorporated by reference in its entirety.

1. INTRODUCTION

The present invention relates to the use of melanoma differentiation associated gene-7, its encoded protein, and related molecules ( Collectively, "Mda-7 Molecules") for improving the response of a subject to chemotherapy and the particular benefit of Mda-7 treatment in a subject resistant to chemotherapy. It is based, at least in part, on the discovery that mda-7 expression increases the response of hitherto resistant cells to chemotherapeutic agent, as well as on the discovery that mda-7 expression is itself enhanced in the context of over- expression of the multidrug resistance gene (mdr-l).

2. BACKGROUND OF THE INVENTION

2.1 MDA-7

Melanoma differentiation associated gene-7 (mda-7) (Jiang et al., 1995, Oncogene 11 :2477-2486) is a secreted cytokine belonging to the interleukin (IL)-IO family designated as IL-24 (Pestka et al., 2004, Annu Rev Immunol 22:929- 979). Multiple independent studies demonstrate that delivery of mda-7/IL-24 by a replication incompetent adenovirus, Ad.mda-7, or as a GST-MD A-7 fusion protein selectively kills diverse cancer cells. In contrast to its harmful effects on tumor cells, mda-7/IL-24 does not induce toxicity in normal endothelial and epithelial cells, fibroblasts, melanocytes and astrocytes (Ekmekcioglu et al., 2001, Int J Cancer 94(l):54-59; Ellerhorst et al., 2002, J CHn Oncol 20(4):1069-1074; Fisher, 2005, Cancer Res 65(22):10128-10138; Fisher et al., 2003, Cancer Biol Ther 2 (4 Suppl l):S23-37; Gupta et al., 2006, Pharmacol Ther 111 : 596-628; Huang et al., 2001, Oncogene 20(48):7051-7063; Jiang et al., 1996, Proc Natl Acad Sci U S A

93(17):9160-9165; Lebedeva et al., 2005, MoI Ther 11(1):4-18; Lebedeva et al., 2002, Oncogene 21(5):708-718; Madireddi et al., 2000, Adv Exp Med Biol 465:239-261; Mhashilkar et al., 2001, MoI Med 7(4):271-282; Saeki et al., 2000, Gene Ther 7(23):2051-2057; Saeki et al., 2002, Oncogene 21(29):4558-4566; Sarkar et al., 2006, Anti-Inflammatory & Anti- Allergy Agents in Medicinal Chemistry 5: 259-274; Sarkar et al., 2002, Biotechniques Suppl:30-39; Sarkar et al., 2002, Proc Natl Acad Sci U S A 99(15):10054-10059; Sauane et al., 2003, Cytokine Growth Factor Rev 14(1):35-51; Su et al., 2001, Proc Natl Acad Sci U S A 98(18):10332-10337; Su et al., 1998, Proc Natl Acad Sci U S A 95(24): 14400-14405). In addition, mda-7/IL-24 possesses potent anti-angiogenic, immunostimulatory and bystander activities (Fisher, 2005, Cancer Res 65(22):10128-10138; Fisher et al., 2003, Cancer Biol Ther 2 (4 Suppl l):S23-37; Gupta et al., 2006, Pharmacol Ther 111 : 596-628; Lebedeva et al., 2005, MoI Ther 11(1):4-18). The sum of these attributes makes mda-7/IL-24 a significant candidate for cancer gene therapy (Fisher, 2005, Cancer Res 65(22): 10128- 10138).

Indeed, Ad.mda-7 has been successfully used for a Phase I clinical trial for advanced carcinomas and melanomas and has shown promising results in tumor growth inhibition and induction of cancer apoptosis (Cunningham et al., 2005, MoI Ther 11(1):149-159 ; Fisher, 2005, Cancer Res 65(22):10128-10138; Fisher et al., 2003, Cancer Biol Ther 2 (4 Suppl l):S23-37, ; Lebedeva et al., 2005, MoI Ther

11(1):4-18; Tong et al., 2005, MoI Ther 1 l(l):160-172). Previous studies demonstrate that Ad.mda-7 induces growth suppression and apoptosis in histologically diverse cancer cells containing single or multiple genetic defects, including alterations in p53, pl6/INK4a and/or Rb (Emdad et al., 2006, J Cell Physiol 208(2):298-306; Huang et al., 2001, Oncogene 20(48):7051-7063; Jiang et al., 1996, Proc Natl Acad Sci U S A 93(17):9160-9165; Lebedeva et al., 2002, Oncogene 21(5):708-718; Mhashilkar et al., 2001, MoI Med 7(4):271-282; Saeki et al., 2000, Gene Ther 7(23):2051-2057; Su et al., 2001, Proc Natl Acad Sci U S A 98(18):10332-10337; Su et al., 1998, Proc Natl Acad Sci U S A 95(24):14400-14405). Moreover, Ad.mda-7 is equally effective in inducing apoptosis in breast and lung carcinoma and melanoma cells containing wtp53, mutp53 or which are null for p53 expression (Lebedeva et al., 2002, Lebedeva et al., 2002, Oncogene 21(5):708-718; Madireddi et al., 2000, Adv Exp Med Biol 465:239-261; Saeki et al., 2000, Gene Ther 7(23):2051-2057; Saeki et al., 2002, Oncogene 21(29):4558-4566; Su et al., 1998, Proc Natl Acad Sci U S A 95(24): 14400-14405). A fragment of MDA-7, retaining amino acid residues 104 to 206 of the wild-type sequence, has been found to retain the cancer-specific growth suppressive and apoptosis-inducing properties of the full-length protein (Gupta et al., 2006, Cancer Res. 66(16^:8182-8191).

2.2 MDR-I

The mdr-1 gene encodes a multi-drug resistant transporter protein, MDR-1/P-glycoprotein (also referred to herein as "P-gp"), a 170 kDa glycosylated membrane protein which is a member of the ATP-binding cassette superfamily of membrane transporters (Meijer et al., 1999, J. Clin. Pathol. 52:450-454; Sakeada et al., 2002, Biol. Pharma Cull. 25:1391-1400). MDR-1/P-glycoprotein acts as a molecular pump which can expel chemotherapeutic agents from tumor cells. The phenomenon of multi-drug resistance arises in a wide variety of cancers, and creates serious obstacles to successful cancer treatment (Mignona et al., 2006, BMC Cancer, 6:293). Mdr-1 over-expression has been linked to a worse clinical prognosis which is both dependent as well as independent of multi-drug resistance (Mignona et al., 2006, BMC Cancer 6:293; Hille et al., 2006, Anticancer Drugs 17(9 V.1041 -1044: Matthews et al., 2006, Leuk. Lymphoma 470i}:2308-2313).

In addition to its obstructive role in cancer therapy, over-expression of mdr-1 gene and protein also interferes with chemotherapy of diseases other than cancer. MDR-1 is believed to function in the transport and metabolism of antiretroviral agents, and variants of MDR-1 have been associated with different levels of therapeutic response in human immunodeficiency virus infection (Saitoh et al., 2005, AIDS Jj9{4}:371-380). Hepatitis B virus has been shown to transactivate mdr-1 gene expression (Doong et al., 1998, J. Hepatol. 29(6): 872-878). Further, Mdr- 1 over-expression has been associated with various autoimmune diseases (Levy et al., 2002, Br. J. Haematol. 118(3):836-838: Tsujimura et al., 2004, Genes Cells 9(121:1265-1273).

3. SUMMARY OF THE INVENTION

The present invention relates to a method for improving the therapeutic response of a subject to chemotherapy, where the subject had previously been resistant to chemotherapy and/or where the mdr-1 gene and/or MDR-I protein are shown to be over-expressed, comprising administering, to the subject, an effective amount of a Mda-7 molecule (mda-7 nucleic acid, MDA-7 protein, M4 nucleic acid or M4 protein, defined below) in conjunction with one or more chemotherapeutic agent. It is based, at least in part, on the discovery that expression of mda-7 in drug-resistant cancer cells resulted in increased accumulation of, and sensitivity to, the drug.

In addition, in alternative embodiments, the present invention provides for a method of treating a subject suffering from a cancer found to be resistant to chemotherapy and/or where the mdr-1 gene and/or MDR-I protein are shown to be over-expressed comprising administering, to the subject, an effective amount of a Mda-7 molecule. This aspect is based, at least in part, on the discovery that the anticancer effect of the melanoma differentiation associated gene -7 (mda-7) was increased in the context of over-expression of the multidrug resistance (MDR-I) gene.

4. BRIEF DESCRIPTION OF THE FIGURES FIGURE IA-C. A. Western blot analysis of endogenous P-gp levels in SW620/WT and SW620/DOX cells. B. Effect of different doses of doxorubicin (Dox) on cell growth in SW620/WT and SW620/DOX cells. Graphs show the average ± SD of results of 3 independent experiments. C. Effect of Dox on apoptosis induction in SW620/WT and SW620/DOX cells by Annexin V binding assay. Results are the average ± SD from replicate studies.

FIGURE 2A-C. A. SW620/WT (also referred to herein as "SW620") and SW620/DOX cells were infected with either Ad.vec or Ad.mda7 (25 pfu/cell) and then treated with the indicated concentrations of Dox for 48 h. Percentage of apoptotic cells were determined by Annexin V binding assay and flow cytometry. B. Cells were infected with either Ad.vec or Ad.mda7 (25 pfu/cell) and then treated with the indicated concentrations of Dox for 24 h. Total proteins were extracted and Western blot analysis was performed with the indicated antibodies. C. Effect of Ad.mda7 on the accumulation of Dox in SW620/WT and SW620/DOX cells. Upper panel, representative flow cytometric image of Dox accumulation. Lower panel, graphical presentation of Dox accumulation. Values are the average ± SD of three independent experiments. FIGURE 3 A-B. Effect of Ad.mda7 on the efflux of Dox from

SW620/WT and SW620/DOX cells. A. Representative flow cytometric image of Dox retention. B. Graphical presentation of Dox retention. Values are the average ± SD of three independent experiments.

FIGURE 4A-C. A. SW620/WT and SW620/DOX cells were infected with either Ad.vec or Ad.mda7 with the indicated MOI and cell viability was determined by MTT assay 5 days after infection. Values are the average ± SD of three independent experiments. B. Effect of Ad.mda7 infection on apoptosis induction in SW620/WT and SW620/DOX cells by Annexin V binding assay. C. MDA-7/IL-24 protein expression in SW620/WT and SW620/DOX cells following infection with Ad.mda-7 determined by Western blot analysis. FIGURE 5A-D. A. SW620/WT cells were transiently transfected with either empty vector (pCDNA3.1) or MDR and then infected with either Ad.vec or Ad.mda7 at the indicated MOI. Apoptosis induction was evaluated by Annexin V binding assay 48 h after infection. B. Effect of MDR over-expression on MDA-7/IL- 24 protein expression. Western blot Analysis was performed with the cell lysates harvested at 24 h after infection. C. Colony formation in soft agar as a function of Ad.vec or Ad.mda-7 in the presence or absence of MDR. Data represents mean ± SD of triplicate plates in three independent experiments. D. Graph shows cloning efficiency. FIGURE 6. Effect of down-regulation of MDR (by siRNA) on

Ad.mda7-induced apoptosis in SW620/DOX cells.

FIGURE 7A-B. K-ras localization in (A) SW620/WT and (B) SW620/DOX cells. Confocal images of SW620/WT and SW620/DOX cells immunofluorescently stained with anti-k-Ras antibody.

5. DETAILED DESCRIPTION OF THE INVENTION For clarity of presentation, and not by way of limitation, the detailed description of the invention is divided into the following subsections:

(i) Mda-7 molecules; (ii) methods of administering Mda-7 molecules;

(iii) use of Mda-7 to improve response to chemotherapy; and (iv) use of Mda-7 therapy in MDRl -expressing subjects. 5.1 MDA-7 MOLECULES

This application uses a convention whereby a nucleic acid is referred to by lower case letters (e.g., mda-7 or mdr-1), a protein is referred to by capital letters (e.g., MDA-7 or MDR-I) and both are generically referred to by an initial capital letter followed by lower case (e.g. Mda-7 or Mdr-1).

A "mda-7 nucleic acid" as defined herein is a nucleic acid which may be:

(i) a nucleic acid having a nucleotide sequence comprising SEQ ID NO:1 (nucleotides 275 to 895 of GenBank Accession No. U 16261; Jiang et al., 1995, Oncogene 11 -.2477-2486), which encodes wild-type human MDA-7 protein;

(ii) a nucleic acid which encodes an MDA-7 protein having an amino acid sequence comprising SEQ ID NO:2 (GenBank Accession No. Q13007; Jiang et al., 1995, Oncogene 11 :2477-2486; the human wild-type MDA-7 amino acid sequence); (iii) a nucleic acid having a nucleotide sequence comprising SEQ ID NO:3, which encodes a version of wild-type human MDA-7 protein lacking the signal sequence;

(iv) a nucleic acid which encodes a protein (lacking a signal peptide) having an amino acid sequence comprising SEQ ID NO:4 (the human wild-type MDA-7 amino acid sequence lacking the signal peptide);

(v) a nucleic acid having a nucleotide sequence which is at least 90 percent or at least 95 percent homologous to SEQ ID NO: 1 ;

(vi) a nucleic acid having a nucleotide sequence which is at least 90 percent or at least 95 percent homologous to SEQ ID NO:3; (vii) a nucleic acid which encodes a protein having an amino acid sequence which is at least 90 percent or at least 95 percent homologous to SEQ ID NO:2; or (viii) a nucleic acid which encodes a protein having an amino acid sequence which is at least 90 percent or at least 95 percent homologous to SEQ ID NO:4 (where homology may be determined, for example, using standard software such as BLAST or FASTA).

A MDA-7 protein, as defined herein is a protein which may be: (i) a protein having an amino acid sequence comprising SEQ ID NO:2 (the human wild-type MDA-7 amino acid sequence); (ii) a protein (lacking a signal peptide) having an amino acid sequence comprising SEQ ID NO: 4 (the human wild-type MDA-7 amino acid sequence lacking the signal peptide);

(iii) a protein having an amino acid sequence which is at least 90 percent or at least 95 percent or at least 98 percent homologous to SEQ ID NO:2; or (iv) _ a protein having an amino acid sequence which is at least 90 percent or at least 95 percent or at least 98 percent homologous to SEQ ID NO:4 (where homology may be determined, for example, using standard software such as BLAST or FASTA).

In further non-limiting embodiments of the invention, as an alternative to using an mda-7 nucleic acid or a MDA-7 protein as defined above, the M4 fragment of wild-type MDA-7 may be used, having the sequence of residues 104 to 206 of SEQ ID NO:2, which is identified herein as SEQ ID NO:5, or a protein which is at least 90 percent, at least 95 percent, or at least 98 percent homologous thereto (all of which are defined herein to be "M4 proteins"). A nucleic acid encoding a M4 protein is referred to as a "M4 nucleic acid" herein, and includes, but is not limited to, a nucleic acid, the sequence of which is SEQ ID NO:6.

"Mda-7 molecules" is defined herein as the class of molecules including mda-7 nucleic acids, MDA-7 proteins, M4 nucleic acids, and M4 proteins. The present invention provides for mda-7 nucleic acids or M4 nucleic acids operably linked to a promoter element and optionally comprised in an expression vector. For example, the mda-7 nucleic acid or M4 nucleic acid may be operably linked to a suitable promoter element, such as, but not limited to, the cytomegalovirus immediate early (CMV) promoter, the Rous sarcoma virus (RSV) long terminal repeat promoter, the human elongation factor 1 α promoter, the human ubiquitin c promoter, etc. It may be desirable, in certain embodiments of the invention, to use an inducible promoter. Non-limiting examples of inducible promoters include the murine mammary tumor virus promoter (inducible with dexamethasone), commercially-available tetracycline-responsive or ecdysone- responsive promoters, etc. It may also be desirable to utilize a promoter which is selectively active in the cancer cell to be treated, for example the PEG-3 gene promoter (U.S. No. 6,472,520). Examples of tissue- and cancer cell-specific promoters are well known to those of ordinary skill in the art.

Other elements that may be included in an mda-7 or M4 nucleic acid - bearing vector include transcription start sites, stop sites, polyadenylation sites, ribosomal binding sites, etc.

Suitable expression vectors include virus-based vectors and non- virus based DNA or RNA delivery systems. Examples of appropriate virus-based vectors include, but are not limited to, those derived from retroviruses, for example Moloney murine leukemia-virus based vectors such as LX, LNSX, LNCX or LXSN (Miller and Rosman, 1989, Biotechniques 7:980-989); lentiviruses, for example human immunodeficiency virus ("HIV"), feline leukemia virus ("FIV") or equine infectious anemia virus (ΕIAV)-based vectors (Case et al, 1999, Proc. Natl. Acad. Sci. U.S.A. 96: 22988-2993; Curran et al, 2000, Molecular Ther. 1:31-38; Olsen, 1998, Gene Ther. 5:1481-1487; United States Patent Nos. 6,255,071 and 6,025, 192); adenoviruses (Zhang, 1999, Cancer Gene Ther. 6(2):113-138; Connelly, 1999, Curr. Opin. MoL Ther. i{5):565-572; Stratford-Perricaudet, 1990, Human Gene Ther. 1:241-256; Rosenfeld, 1991, Science 252:431-434; Wang et al, 1991, Adv. Exp. Med. Biol. 309:61-66; Jaffe et al, 1992, Nat. Gen. 1:372-378; Quantin et al, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:2581-2584; Rosenfeld et al, 1992, Cell 68:143-155;

Mastrangeli et al, 1993, J. Clin. Invest. 91:225-234; Ragot et al, 1993, Nature 361:647-650; Hayaski et al, 1994, J. Biol. Chem. 269:23872-23875; Bett et al, 1994, Proc. Natl. Acad. Sci. U.S.A. 91:8802-8806), for example Ad5/CMV-based El- deleted vectors (Li et al, 1993, Human Gene Ther. 4:403-409); adeno-associated viruses, for example pSub201 -based AAV2-derived vectors (Walsh et al , 1992, Proc. Natl. Acad. Sci. U.S.A. _.89:7257-7261); herpes simplex viruses, for example vectors based on HSV-I (Geller and Freese, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:1149- 1153); baculoviruses, for example AcMNPV-based vectors (Boyce and Bucher, 1996, Proc. Natl. Acad. Sci. U.S.A. 93:2348-2352); SV40, for example SVluc (Strayer and Milano, 1996, Gene Ther. 2:581-587); Epstein-Barr viruses, for example EBV-based replicon vectors (Hambor et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85:4010-4014); alphaviruses, for example Semliki Forest virus- or Sindbis virus-based vectors (Polo et al, 1999, Proc. Natl. Acad. Sci. U.S.A. 96:4598-4603); vaccinia viruses, for example modified vaccinia virus (MVA)-based vectors (Sutter and Moss, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10847-10851) or any other class of viruses that can efficiently transduce human tumor cells and that can accommodate the nucleic acid sequences required for therapeutic efficacy.

Non-limiting examples of non- virus-based delivery systems which may be used according to the invention include, but are not limited to, so-called naked nucleic acids (Wolff et al. , 1990, Science 247: 1465- 1468), nucleic acids encapsulated in liposomes (Nicolau et al, 1987, Methods in Enzymology 149:157-176), nucleic acid/lipid complexes (Legendre and Szoka, 1992, Pharmaceutical Research 9:1235- 1242), and nucleic acid/protein complexes (Wu and Wu, 1991, Biother. 3:87-95).

In specific, non-limiting embodiments of the invention, the expression vector is an El -deleted human adenovirus vector of serotype 5, although those of ordinary skill in the art would recognize that many of the different naturally-occurring human Ad serotypes or Ad vectors derived from non-human adenoviruses may substitute for human Ad 5 -derived vectors. In a preferred, specific, non-limiting embodiment, a recombinant replication-defective Ad.mda-7 virus for use as an mda-7 vector may be created in two steps as described in Su et al. , 1998, Proc. Natl. Acad. Sci. U.S.A. 95:14400-14405. Specifically, the coding region of the mda-7 gene may be cloned into a modified Ad expression vector pAd.CMV (Falck-Pedersen et al. , 1994, MoI. Pharmacol. 45:684-689). This vector contains, in order, the first 355 bp from the left end of the Ad genome, the CMV promoter, DNA encoding splice donor and acceptor sites, the coding region of the mda-7 cDNA, DNA encoding a polyA signal sequence from the β globin gene, and ~3 kbp of adenovirus sequence extending from within the ElB coding region. This arrangement allows high-level expression of the cloned sequence by the CMV promoter, and appropriate RNA processing. The recombinant virus may be created in vitro in 293 cells (Graham et al, 1977, J. Gen. Virol. 36:59-72) by homologous recombination between an mda-7-containing version of pAd.CMV and plasmid pJM17, which contains the whole of the Ad genome cloned into a modified version of pBR322 (McGrory et al, 1988, Virology 163:614-617). pJM17 gives rise to Ad genomes in vivo, but they are too large to be packaged in mature Ad capsids. This constraint is relieved by recombination with the vector to create a packageable genome {Id.) containing the mda-7 gene. The recombinant virus is replication defective in human cells except 293 cells, which express adenovirus ElA and ElB. Following transfection of the two plasmids, infectious virus may be recovered, and the genomes may be analyzed to confirm the recombinant structure, and then virus may be plaque purified by standard procedures (Volkert and Young, 1983, Virology 125: 175- 193).

In a specific, non-limiting embodiment of the invention, the infectivity of an adenovirus vector carrying an mda-7 gene may be improved by inserting an Arg-Gly-Asp motif into the fiber know (Ad5-Delta24RGD), as described in Lamfers et al, 2002, Cancer Res. 62:5736-5742. MDA-7 or M4 protein may be produced in vivo in an organism or in an isolated cell which may be part of a cell population. For example, MDA-7 or M4 protein may be produced in a recombinant expression and then collected and purified using methods known in the art. Such proteins may be administered comprised in liposomes, nanoparticles, with a carrier, etc. (see below).

5.2 METHODS OF ADMINISTERING MDA-7 MOLECULES

In preferred, non-limiting embodiments, a mda-7 nucleic acid or a M4 nucleic acid may be operably linked to a suitable promoter and introduced into a cell in need of such treatment (a "target cell"), which, as defined herein, is a cell which exhibits multidrug resistance and may have been demonstrated to over express the mdr-1 gene. In preferred embodiments, the mda-7 nucleic acid or the M4 nucleic acid may be contained in a viral vector, operably linked to a promoter element that is either inducible or constitutively active in the target cell. In preferred, non-limiting embodiments, the viral vector is a replication-defective adenovirus (as described above).

In a specific, non-limiting embodiment of the invention, a viral vector containing a nucleic acid encoding a mda-7 nucleic acid or a M4 nucleic acid in expressible form, operably linked to a suitable promoter element, may be administered to a population of target cells at a multiplicity of infection (MOI) ranging from 10- 100 MOI.

In another specific, non-limiting embodiment, the amount of a viral vector administered to a subject, preferably a human subject, may be 1 X 10⁹ pfu to 1 X 10¹² pfu.

In specific, non-limiting embodiments, a mda-7 nucleic acid or a M4- nucleic acid, preferably operably linked to a promoter element, comprised in a vector or otherwise, may be introduced into a cell ex vivo and then the cell may be introduced into a subject. For example, a nucleic acid encoding mda-7 may be introduced into a cell of a subject ex vivo and then the cell containing the nucleic acid may be optionally propagated and then (with its progeny) introduced into the subject. Alternatively, a MDA-7 protein or a M4 protein may be used in protein/peptide therapy of a subject in need of such treatment. As such, the MDA-7 protein or M4 protein may be prepared by chemical synthesis or recombinant DNA techniques, purified by methods known in the art, and then administered to a subject in need of such treatment. MDA-7 protein or M4 protein may be comprised, for example, in solution, in suspension, and/or in a carrier particle such as microparticles, liposomes, or other protein-stabilizing formulations known in the art In a non- limiting specific example, formulations of MDA-7 protein or M4 protein may stabilized by addition of zinc and/or protamine stabilizers as in the case of certain types of insulin formulations. Alternatively, in specific non-limiting embodiments, a MDA-7 protein or a M4 protein may be linked covalently or non-covalently, to a carrier protein which is preferably non-immunogenic.

In preferred, non-limiting embodiments, a MDA-7 protein or a M4 protein is administered in an amount which achieves a local concentration in the range of 18 to 50 ng per microliter. For example, a subject may be administered a range of 50- 100 mg of MDA-7 protein or M4 protein per kilogram. For a human subject, the dose range may be between 100-2500 mg/day of MDA-7 protein or M4 protein.

Any of the foregoing molecules may be administered by direct instillation, injection (intravenous, subcutaneous, intramuscular, intraarterial, intrathecal, intrahepatic, intratumoral), or any other method or route known in the art.

5.3 USE OF MDA-7 TO IMPROVE RESPONSE TO CHEMOTHERAPY

The present invention relates to a method for improving the therapeutic response of a subject suffering from a disorder to chemotherapy, where the disorder in the subject had previously been resistant to chemotherapy and/or where the mdr-1 gene and/or MDR-I protein are shown to be over-expressed, comprising administering, to the subject, an effective amount of a mda-7 nucleic acid or a MDA-7 protein or a M4 nucleic acid or a M4 protein, in conjunction with one or more chemotherapeutic agent.

The subject may be a human or a non-human subject, but preferably is a human subject. The subject may be suffering from a disorder which is a malignancy or a non-malignant disorder.

Non-limiting examples of malignant disorders include, but are not limited to, any malignancy that exhibits multi-drug resistance, and that preferably has been demonstrated to over-express mdr-1 gene and/or MDR-I protein (also referred to as "P-glycoprotein" or "P-gp" herein). Such malignancies may include, but not be limited to, breast cancer, ovarian cancer, lung cancer (e.g., adenocarcinoma, small- cell carcinoma, non-small cell carcinoma, mesothelioma), colorectal cancer, renal carcinoma, chronic lymphocytic leukemia, acute lymphocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, neuroblastoma, glioblastoma, prostate cancer, pancreatic cancer, gastric cancer, squamous cell carcinoma (e.g., oral squamous cell carcinoma and squamous cell carcinoma of the head and neck), melanoma, hepatic adenocarcinoma, bladder cancer, testicular cancer, and rhabdosarcoma.

In alternative embodiments, subjects for treatment according to the invention include any subject suffering from a non-malignant disease, where the response of the disease to therapy is negatively impacted by multi-drug resistance, where the subject has preferably been demonstrated to over express mdr-1 gene and/or MDR-1 protein. Such subjects may include, but not be limited to, subjects suffering from a viral infection, such as, but not limited to, human immunodeficiency infection, hepatitis B virus infection, Epstein-Barr virus infection, Herpes simplex virus infection, human papilloma virus infection, or hepatitis C infection. In other embodiments, the subject may be suffering from an autoimmune disease, such as, but not limited to, rheumatoid arthritis, juvenile rheumatoid arthritis, system lupus erythematosis, Sjogren's syndrome, scleroderma, polymyositis, or diabetes mellitus. In another non-limiting embodiment, the subject may be suffering from multiple sclerosis.

The nature of the therapeutic response to be improved depends on the nature of the disorder being treated. Where the disorder is a malignant disorder, an improved therapeutic response may be characterized by, for example and not by way of limitation, one or more of the following: higher rate of survival, longer life expectancy, longer period to relapse, increased comfort, decreased tumor burden, decreased pain, decreased rate or incidence of metastasis. Where the disorder is an infectious disorder, an improved therapeutic response may be characterized by, for example and not by way of limitation, one or more of the following: increased survival, longer life expectancy, decreased viral titer, increased CD4+ T cell count, decreased incidence of secondary infection, decreased incidence of associated carcinoma. Where the disorder is an autoimmune disorder, an improved therapeutic response may be characterized by, for example and not by way of limitation, decreased serologic markers of inflammation such as C-reactive protein and erythrocyte sedimentation rate, decreased clinical inflammation, (for arthritis) increased range of motion, decreased pain, longer disease-free interval, decreased need for pain medication or insulin therapy, increased life expectancy, decreased workplace absenteeism. Where the disorder is multiple sclerosis, an improved therapeutic response may be characterized by, for example and not by way of limitation, decreased incidence of relapse, longer relapse-free intervals, shorter duration of relapse, decreased severity of symptoms at relapse, decreased lesions by imaging studies such as MRI, and improved nerve conduction studies.

"Resistance to chemotherapy" means that the subject had been administered one or more chemotherapeutic agent which did not achieve a satisfactory therapeutic response (examples of types of therapeutic responses being set forth above).

"Over-expression of mdr-1 gene or protein" means an increase in the level of mdr-1 gene transcription and/or MDR-I protein levels. In non-limiting example, a sample of cells afflicted by the disorder may be collected from the subject and then tested for levels of mdr-1 mRNA and/or MDR-1 protein, using techniques known in the art, such as PCR analysis, Northern Blot, Western blot, immunohistochemistry, etc. The sequence of the human MDR-1 gene and protein are known. The Genbank accession numbers for mdr-1 nucleic acid and MDR-1 protein are M 14758 and P08183 respectively. "Over-expressing" means an increase relative to a normal control cell, preferably by at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, or at least about 90 percent.

A subject need not be tested for over-expression of Mdr-1 molecules if they have proved to be clinically refractory to treatment. Conversely, a patient need not have been demonstrated to be refractory to treatment if over-expression of mdr-1 gene or MDR-1 protein is demonstrated.

Administration of a mda-7 nucleic acid or a MD A-7 protein or a M4 nucleic acid or a M4 protein may be performed as set forth in the section above. Concurrent administration of one or more chemotherapeutic agent means that the Mda-7 molecule and the one or more chemotherapeutic agent are administered close enough in time to each other for the clinical effect of the Mda-7 molecule may be exerted on the chemotherapeutic agent. The Mda-7 molecule (mda- 7 nucleic acid, MDA-7 protein, M4 nucleic acid or M4 protein) may be administered simultaneously with, prior to, or after the administration of the one or more chemotherapeutic agent (preferably prior to administration of the one or more chemotherapeutic agent). If a mda-7 nucleic acid or a M4 nucleic acid is administered by gene therapy, the time period between administering it and the chemotherapeutic agent may be quite long, and one administration of gene therapy may suffice for a plurality of subsequent chemotherapeutic treatments. If MDA-7 protein or M4 protein is administered, the interval to administration of the one or more chemotherapeutic agent would be shorter in view of degradation of the protein.

Non-limiting specific examples of intervals between administration of an Mda-7 molecule and one or more therapeutic agent include up to -5 years ("-" means prior administration of the Mda-7 molecule), up to -3 years, up to -2 years, up to -1 year, up to -12 months, up to -11 months, up to -10 months, up to -9 months, up to -8 months, up to -7 months, up to -6 months, up to -3 months, up to -1 month, up to - 2 weeks, up to -1 week, up to -6 days, up to -5 days, up to -4 days, up to -3 days, up to -2 days, up to -1 day, up to -12 hours, up to -8 hours, up to -4 hours, up to -2 hours, up to -1 hours, 0 hours, up to +1 hour ("+" means that Mda-7 molecules is administered after chemotherapeutic agent (s)), up to +2 hours, up to +4 hours, up to +8 hours, up to +12 hours, up to +1 day, up to +2 days, up to +3 days, up to +4 days, up to +5 days, up to +6 days, up to +1 week, up to +2 weeks, up to +1 month, up to +3 months, up to +6 months, up to +7 months, up to +8 months, up to +9 months, up to +10 months, up to +11 months, up to +1 year, up to + 3 years, and up to + 5 years.

The one or more chemotherapeutic agent selected is related to the nature of the disorder being treated.

Where the disorder is a malignancy, the one or more chemotherapeutic agent may be, for example and not by way of limitation, an alkylating agent such as busulfan, cisplatin, carboplatin, chlorambucil, cyclophosphamide, ifosfamide, dacarbazine, mechlorethamine (nitrogen mustard), melphalan, and temozolomide; a nitrosourea such as streptozocin, carmustine, and lomustine, an antimetabolite such as 5-fluorouracil, capecitabine, 6-mercaptopurine, methotrexate, gemcitabine, cytarabine, fludarabine, and pemetrexed; an anthracyline-like drug such as daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone; a topoisomerase I inhibitor such as topotecan and irinotecan; a topoisomerase II inhibitor such as etoposide and teniposide; or a mitotic inhibitor such as, for example, a taxane such as paclitaxel or docetaxel, or a vinca alkaloid such as vinblastine, vincristine, or vinorelbine, or a combination of two or more of the above. Where the disorder is human immunodeficiency virus infection, the one or more chemotherapeutic agent may be, for example and not by way of limitation, a reverse transcriptase inhibitor, including nucleoside reverse transcriptase inhibitors such as 3TC (lamivudine), abacavir, AZT (zidovudine), d4T (stavudine), ddC (zalcitabine), ddl (didanosine) and FTC (emtricitabine) and non-nucleoside reverse transcriptase inhibitors such as efavirenz, delavirdine, neviripine, etravirine, rilpirivine ; a protease inhibitor such as saquinivir, nelfinivir, ritonivir, indinivir, amprenavir, lopinavir, atazanavir, fosamprenavir, or tipranavir; a fusion inhibitor such enfuvirtide as or an integrase inhibitor such as Merck's L-000870810 or Japan Tobacco's JTK-303, or combinations thereof. Where the disorder is a herpes virus infection, the one or more chemotherapeutic agent may be, for example and not by way of limitation, aciclovir, cidofovir, docosanol, famiciclovir, fomivirsen, foscarnet, ganciclovir, idoxuridine, penciclovir, trifluridine, tromantidine, valaciclovir, valganciclovir, or vidarabine, or combinations thereof. Where the disorder is hepatitis B infection, the one or more chemotherapeutic agent may be, for example and not by way of limitation, adefovir or lamivudine, or a combination thereof.

Where the disorder is hepatitis C infection, a chemotherapeutic agent may be, for example and not by way of limitation, ribivirin.

Where the disorder is multiple sclerosis, the one or more chemotherapeutic agent may be glatiramir or novantrone or a combination thereof.

5.4 USE OF MDA-7 THERAPY IN MDRl -OVER-EXPRESSING SUBJECTS In addition, in alternative embodiments, the present invention provides for a method of treating a subject suffering from a cancer found to be resistant to chemotherapy and/or where the mdr-1 gene and/or MDR-I protein are shown to be over-expressed comprising administering, to the subject, an effective amount of a Mda-7 molecule, for example mda-7 nucleic acid, MDA-7 protein, M4 nucleic acid or M4 protein. This aspect is based, at least in part, on the discovery that the anti-cancer effect of the melanoma differentiation associated gene -7 (mda-7) was increased in the context of over-expression of the multidrug resistance (MDRI) gene. The scope of the various terms used in this paragraph is as set forth in the preceding section. However, according to this embodiment, the Mda-7 molecule need not be administered in conjunction with one or more chemotherapeutic agent.

In one non-limiting set of embodiments, the present invention provides for a method of treating a subject suffering from a cancer, comprising (i) determining that cancer cells of the subject over express the mdr-1 gene and/or MDR-I protein; and (ii) administering, to the subject, an effective amount of an Mda-7 molecule (as described above). In another non-limiting set of embodiments, the present invention provides for a method of evaluating the likelihood that a subject, suffering from a cancer, would benefit from therapy with a Mda-7 molecule, comprising determining whether or not cancer cells in the subject over-express the mdr-1 gene or MDR-I protein (Mdr-1 molecules), wherein over-expression of mdr-1 gene and/or MDR-I protein is consistent with a greater likelihood that the subject would benefit from

Mda-7 molecule therapy.

6. EXAMPLE

6.1 RESULTS

The experiments described in this section were carried out using two related colorectal carcinoma cell lines, SW620/WT and its doxorubicin (Dox)- resistant counterpart, SW620/DOX. Initial experiments were designed to establish a baseline phenotype for these cell lines. FIGURE IA depicts the results of Western blot analysis of endogenous MDR- 1 ("P-gp") levels in S W620/WT and S W620/DOX cells, and shows that the SW620/DOX cell line over expressed P-gp. FIGURE IB depicts the effect of different doses of Dox on cell viability in SW620/WT and S W620/DOX cells. Cells were cultured in the presence of different concentrations of

Dox (0.25, 1, 4, 16, and 64 μM) for 96 hours, and then the effects on cell growth were

determined by MTT assay. Graphs show the average ± SD of results of 3 independent experiments, and demonstrate that Dox had substantially less effect on the viability of SW620/DOX cells. FIGURE 1C particularly addresses the ability of Dox to induce apoptosis in SW620/WT and SW620/Dox cells, based on the levels of Annexin V-

binding. Cells were treated with 0.25, 1, 4, 16, and 64 μM of Dox and were stained

after 48 h with APC-labeled Annexin V and immediately analyzed by flow cytometry. The percentage of early and late apoptotic cells (only Annexin V stained) was calculated using Flow Jo Version 6.31 software. FIGURE 1C shows that the level of Annexin V binding as a result of Dox treatment was greater in SW620/WT cells, and the effect was dose-dependent. Next, the effect of MDA-7 on the responsiveness of S W620/WT and

SW620/DOC cells was explored. Experiments were performed in which SW620 and SW620/Dox cells were infected with either (empty vector) Ad.vec or Ad.mda7 (25 pfu/cell) and then treated with the indicated concentrations of Dox for 48 h. The percentages of apoptotic cells were determined by Annexin V binding assay and flow cytometry. As shown in FIGURE 2A, the S W620/DOX cells infected with Ad.vec remained relatively resistant to Dox, but SW620/DOX cells infected with Ad.mda7 exhibited a sensitivity to Dox that approached that of S W620/WT. When cells were infected with either Ad.vec or Ad.mda7 (25 pfu/cell), treated with Dox for 24 h., and then total proteins were extracted and subjected to Western blot analysis, Ad.mda7 infection was found to down regulate P-gp expression in SW620/Dox cells (FIGURE 2B). As shown in FIGURE 2C, the intracellular accumulation of doxorubicin, determined after cells were infected with either Ad.vec or Ad.mda7 (25 pfu/cell) for 24 h, incubated with 2 or 4 μM of Dox for 60 min., washed twice with ice-cold PBS, resuspended in 400 μl PBS and then analyzed by flow cytometry, was increased in Ad.mda7-infected SW620/Dox cells. Thus, FIGURE 2A-C demonstrates that Ad.mda7 infection induces MDR-I reversal in SW620/Dox cells.

The ability of Ad.mda7 infection to increase Dox accumulation in SW620/DOX cells is further supported by FIGURE 3. Cells were incubated with 10 μM doxorubicin at 37⁰C for 30 min (substrate loading phase) and then washed twice with PBS. Cells were then infected with either Ad.vec or Ad.mda7 at the indicated MOI. At the defined time points, cells were harvested, centrifuged and washed in ice- cold PBS. Cell pellets were then resuspended in 400 μl PBS and used immediately for flow cytometric analysis for intracellular doxorubicin retention.

Experiments were then performed to determine the effects of Ad.mda7 infection on viability and apoptosis induction of S W620/WT and S W620/DOX cells independent of Dox. SW620 and SW620/Dox cells were infected with either Ad.vec or Ad.mda7 at MOIs of either 25 or 50 pfu/cell, and then either viability was determined by MTT assay 5 days after infection or apoptosis induction was evaluated by staining infected cells after 48 h with APC-labeled Annexin V followed immediately by flow cytometry analysis. The percentage of early and late apoptotic cells (only Annexin V stained) was calculated using FlowJo Version 6.31 software. As shown in FIGURE 4A-B, Ad.mda7 infection inhibited cell growth and induced apoptosis preferentially in P-gp-expressing mdr-1 variant SW620/Dox cells. Interestingly, as shown in FIGURE 4C, Western blot analysis showed that MDA- 7/IL-24 protein expression was increased in S W620/DOX relative to S W620/WT.

To explore the role of mdr-1 in the enhanced effectiveness of Ad.mda7 in SW620/DOX cells, SW620/WT cells were transiently transfected with either empty vector (pCDNA3.1) or MDR, infected with either Ad.vec or Ad.mda7, and then apoptosis induction was evaluated by Annexin V binding assay 48 h after infection. The percentage of early and late apoptotic cells (only Annexin V stained) was calculated using FlowJo Version 6.31 software. As shown in FIGURE 5 A. over- expression of MDR was observed to induce increased sensitivity to Ad.mda7 infection in SW620 cells. As shown in FIGURE 5B, Western blot analysis performed on cell lysates harvested at 24 h after infection indicated that over-expression of mdr- 1 /MDR-1 was associated with enhanced expression of MDA-7/IL-24 protein. Further, when SW620/WT cells were transiently transfected either with empty vector (pCDNA3.1) or mdr-1, infected with either Ad.vec or Ad.mda7 and, 24 hours later, 1 x 10⁵ cells were replated in 0.4% agar on 0.8% base agar and then, after two weeks, colonies >50 cells were counted under a dissection microscope, it was found that ectopic expression of mdr-l/MDR-1 in SW620 cells significantly inhibited colony formation in soft agar (FIGURE 5C).

To further explore this phenomenon, the effect of down-regulation of mdr-1 in SW620/DOX cells was evaluated. SW620/DOX cells were transiently transfected with either control siRNA or mdr-1 siRNA using lipofectamine prior to infection with either Ad.vec or Ad.mda7. Forty-eight hours later, apoptosis was measured by Annexin V binding assay using APC-conjugated Annexin V by flow cytometry. The percentage of early and late apoptotic cells (only Annexin V stained) was calculated using FlowJo Version 6.31 software. As shown in FIGURE 6, down regulation of mdr-1 partially protected SW620/DOX cells from Ad.mda7-induced apoptosis.

Finally, K-ras localization was studied in SW620/WT and SW620/DOX cells. FIGURE 7 shows confocal images of SW620/WT and SW620/DOX cells immunofluorescently stained with anti-k-Ras antibody. Note extensive plasma-membrane localization in the SW620/WT cell images (typical pattern) and surface distribution in the SW620/DOX cell images.

6.2 DISCUSSION

Inappropriate expression of the multidrug resistance (mdr-1) gene, encoding the P-glycoprotein (P-gp), is frequently implicated in resistance of cancer cells to diverse chemotherapeutic drugs. However, high toxicity of P-gp inhibitors mandates alternative approaches for modulating mdr-1 and promoting death in drug- resistant cancer cells. In this study, the effect of melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), which exhibits cancer-specific apoptosis- inducing properties, was studied in drug-sensitive (SW620/WT) and drug-resistant (SW620/DOX) colorectal carcinoma cell lines. mda-7/IL-24, when administered by means of a replication-incompetent adenovirus, Ad.mda-7, at a multiplicity of infection of 25-50 pfu/cell, significantly increased the sensitivity of SW620/DOX cells to doxorubicin. However, the cytotoxic effect of doxorubicin in the SW620/WT sensitive cell line was not significantly altered by Ad.mda-7 infection. Annexin V binding assays revealed that Ad.mda-7 infection effectively reversed resistance to doxorubicin-induced apoptosis in SW620/DOX cells. In addition, infection of SW620/DOX cells with Ad.mda-7 increased intracellular accumulation of doxorubicin and significantly decreased P-gp expression. Surprisingly, we observed that P-gp over expressing cells (SW620/DOX) displayed increased apoptosis following Ad.mda-7-infection compared to parental SW620/WT cells. Western blot analysis indicated more MDA-7/IL-24 protein in SW620/DOX than SW620/WT cells following infection with Ad.mda-7, which might explain the increased sensitivity of P-gp over expressing cells to Aά.mda-7 infection. Furthermore, over-expression of mdr-1 in SW620/WT cells increased apoptosis and MDA-7/IL-24 protein following Ad.mda-7 infection, while down modulation of mdr-1 in SW620/DOX cells by siRNA led to decreased apoptosis and MDA-7/IL-24 protein following Ad.mda-7 infection. These findings reveal that mda-7/IL-24 is a potent MDR reversal agent, preferentially causing apoptosis in P-gp over expressing MDR cells, suggesting significant clinical implications for the use of mda-7/IL-24 in treating neoplasms that have failed chemotherapy mediated by the P-gp multidrug resistance mechanism. Various references are cited herein, the contents of which are hereby incorporated by reference in their entireties.

SEQUENCE LISTING

SEQ IDNO:1 nucleotides 275 to 895 ofGenBankAccessionNo. U16261 atgaattttc aacagaggct gcaaagcctg tggactttag ccagaccctt ctgccctcct 60

ttgctggcga cagcctctca aatgcagatg gttgtgctcc cttgcctggg ttttaccctg 120

cttctctgga gccaggtatc aggggcccag ggccaagaat tccactttgg gccctgccaa 180

gtgaaggggg ttgttcccca gaaactgtgg gaagccttct gggctgtgaa agacactatg 240

caagctcagg ataacatcac gagtgcccgg ctgctgcagc aggaggttct gcagaacgtc 300

tcggatgctg agagctgtta ccttgtccac accctgctgg agttctactt gaaaactgtt 360

ttcaaaaact accacaatag aacagttgaa gtcaggactc tgaagtcatt ctctactctg 420

gccaacaact ttgttctcat cgtgtcacaa ctgcaaccca gtcaagaaaa tgagatgttt 480

tccatcagag acagtgcaca caggcggttt ctgctattcc ggagagcatt caaacagttg 540

gacgtagaag cagctctgac caaagccctt ggggaagtgg acattcttct gacctggatg 600 cagaaattct acaagctctg a 621

SEQ ID NO:2 = amino acid sequence of complete wild-type MDA-7 protein (GenBank Accession No. U 16261)

MNFQQRLQSL WTLARPFCPP LLATASQMQM WLPCLGFTL LLWSQVSGAQ GQEFHFGPCQ 60

VKGWPQKLW EAFWAVKDTM QAQDNITSAR LLQQEVLQNV SDAESCYLVH TLLEFYLKTV 120

FKNYHNRTVE VRTLKSFSTL ANNFVLIVSQ LQPSQENEMF SIRDSAHRRF LLFRRAFKQL 180

DVEAALTKAL GEVDILLTWM QKFYKL 206

SEQ ID NO: 3 = nucleic acid sequence encoding the portion of wild-type MDA-7 lacking the signal sequence atgcagggcc aagaattcca ctttgggccc tgccaagtga agggggttgt tccccagaaa 60

ctgtgggaag ccttctgggc tgtgaaagac actatgcaag ctcaggataa catcacgagt 120

gcccggctgc tgcagcagga ggttctgcag aacgtctcgg atgctgagag ctgttacctt 180 gtccacaccc tgctggagtt ctacttgaaa actgttttca aaaactacca caatagaaca 240

gttgaagtca ggactctgaa gtcattctct actctggcca acaactttgt tctcatcgtg 300

tcacaactgc aacccagtca agaaaatgag atgttttcca tcagagacag tgcacacagg 360

cggtttctgc tattccggag agcattcaaa cagttggacg tagaagcagc tctgaccaaa 420

gcccttgggg aagtggacat tcttctgacc tggatgcaga aattctacaa gctc 474

SEQ ID NO:4 = amino acid sequence of wild-type MDA-7 protein lacking the signal sequence

MQEFHFGPCQ VKGWPQKLW EAFWAVKDTM QAQDNITSAR LLQQEVLQNV SDAESCYLVH 60

TLLEFYLKTV FKNYHNRTVE VRTLKSFSTL ANNFVLIVSQ LQPSQENEMF SIRDSAHRRF 120

LLFRRAFKQL DVEAALTKAL GEVDILLTWM QKFYKL 156

SEQ ID NO:5 = amino acid sequence of M4; residues 104 to 206 of SEQ ID NO:2.

MESCYLVHTL LEFYLKTVFK NYHNRTVEVR TLKSFSTLAN NFVLIVSQLQ PSQENEMFSI 60 RDSAHRRFLL FRRAFKQLDV EAALTKALGE VDILLTWMQK FYKL 104

SEQ IDNO:6 =portionofSEQ IDNO:1 encoding M4 atggagagct gttaccttgt ccacaccctg ctggagttct acttgaaaac tgttttcaaa 60

aactaccaca atagaacagt tgaagtcagg actctgaagt cattctctac tctggccaac 120

aactttgttc tcatcgtgtc acaactgcaa cccagtcaag aaaatgagat gttttccatc 180

agagacagtg cacacaggcg gtttctgcta ttccggagag cattcaaaca gttggacgta 240

gaagcagctc tgaccaaagc ccttggggaa gtggacattc ttctgacctg gatgcagaaa 300

ttctacaagc tctga 315

Claims

WHAT IS CLAIMED IS:

1. A method for improving the therapeutic response to chemotherapy of a subject suffering from a disorder, comprising (i) demonstrating that the mdr-1 gene and/or MDR-I protein are over-expressed; and (ii) administering, to the subject, an effective amount of a Mda-7 molecule in conjunction with one or more chemotherapeutic agent.

2. The method of claim 1 wherein the Mda-7 molecule is a mda-7 nucleic acid.

3. The method of claim 1 wherein the Mda-7 molecule is a MDA-7 protein.

4. The method of claim 1 wherein the Mda-7 molecule is a M4 nucleic acid.

5. The method of claim 1 wherein the Mda-7 molecule is a M4 protein.

6. A method of treating a subject suffering from a cancer found to be resistant to chemotherapy comprising (i) demonstrating that the mdr-1 gene and/or MDR-I protein are over-expressed and (ii) administering, to the subject, an effective amount of a Mda-7 molecule.

7. The method of claim 6 wherein the Mda-7 molecule is a mda-7 nucleic acid.

8. The method of claim 6 wherein the Mda-7 molecule is a MDA-7 protein.

9. The method of claim 6 wherein the Mda-7 molecule is a M4 nucleic acid.

10. The method of claim 6 wherein the Mda-7 molecule is a M4 protein.