WO2004016652A2 - Mammaglobin promoter - Google Patents

Abstract

The invention is a mammalian mammaglobin gene promoter. The promoter nucleic acid molecule comprises a sequence 344 bp or greater upstream of the translational start site of the mammaglobin gene. The promoter provides tissue-specific expression, such as breast-specific or tumour-specific expression.

Description

MAMMAGLOBIN PROMOTER

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority from US patent application no. 60/404,125, filed August 19, 2002, which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to promoter sequences for the gene encoding mammaglobin (MGB), a uteroglobin-related protein and the use of the promoter in production of RNA and proteins for therapy and diagnosis in breast, ovary and prostate tissue and other tissues.

BACKGROUND OF THE INVENTION

Some of the most promising cancer gene therapies investigated in the last decade include cyto ine gene transfer that act indirectly by recruiting the patient's immune system to attack the tumor, pro-apoptotic and/or suicide gene therapy that act by directly killing transduced tumor cells, and oncolytic viruses, which induce lysis of infected tumor cells during the last stage of virus replication. Although all of these therapies have shown some degree of efficacy, the damage done to normal tissues by their unrestricted expression limits their therapeutic utility. The use of tissue- or tumor-specific promoters to drive expression of vector-encoded toxic proteins or of replication-inducing proteins in lytic viruses can potentially protect normal tissues from damage without compromising anti-tumor activity.

Several promoters have been identified recently that induce either mammary-specific or tumor-specific expression and thus could be useful for breast cancer (BrCa) gene therapy. The human alpha-lactalbumin (ALA) and bovine beta-lactoglobulin (BLG) promoters are expressed equally in both breast cancer (1) and normal mammary tissue (2, 3), but not in other tissues. Other promoters upregulated in tumor (including mammary carcinoma) cells are the epithelial cell-specific DF3 (4, 5), glycolysis-induced hexokinase type II promoter (6), and the ErbB2 (HER2/r»e-v) promoter (7) that is upregulated in expressed in some normal tissues (9-12). Identification of promoters with a higher degree of specificity or that target a different or larger proportion of human mammary tumors would be desirable. MGB, a uteroglobin-related protein of unknown function, was originally detected by differential RNA expression levels in breast cancer biopsies compared to normal breast tissue (13). MGB protein was detected immunologically in over 80% of the primary and metastatic breast tumors examined in one study (14). In addition, expression of MGB was detected by RT-PCR in 100% of the 57 primary breast tumors examined in two other studies (15, 16), and in normal breast, but no other normal tissues (17). Moreover, over ten-fold higher levels of expression in tumors than patient- matched normal breast tissue were detected by Northern analysis (17). Genbank includes information on MGB at AP003306, NCBI entrez NT_033241 , NCBI MIM entry *605562 and NCBI entry AF015224, but none of this sequence information discloses and characterizes the regulatory elements of the MGB upstream region. Use of the first 1 kb of the MGB promoter was unsuccessful in isolating the DNA sequences responsible for this specificity (18). In that study, a 1 kb fragment of the MGB promoter proximal to the gene induced a low level of reporter gene expression following transfection of MGB(+) breast cancer cells, but expression was also detected in MGB(-) breast cancer and HeLa cell lines. This data was obtained using plasmid transfections. This reference teaches that the promoter is nonspecific and would not be suitable for targeting gene therapy. There remains a need to identify an effective vector and promoter for specific delivery of therapeutic genes to cancer cells in breast and other tissue.

SUMMARY OF THE INVENTION

The present invention relates to regulatory sequences upstream of the mammaglobin (MGB) protein coding sequence. Also provided are nucleic acid constructs and vectors containing the sequences, and uses thereof. The present inventor has identified promoter sequence (SEQ.ID.NO.1) 25 kb upstream of the MGB coding sequence. Accordingly, the invention provides a purified and isolated nucleic acid molecule comprising a mammalian MGB promoter of SEQ.ID.NO.1 or portions thereof. In a preferred embodiment, the minimal promoter region comprises at least the first 300 or 344 bp optionally with additional enhancer elements between 4.4 and 5.5 kb upstream of the translational start site of the MGB gene. Alternatively, the promoter comprises at least 1kb, 1.1 kb, 1.5kb, 2kb, 3kb, 4kb, 4.5kb, 5kb, 5.5kb, 6kb, 10kb, 15kb, 20kb or 25kb upstream of the translational start site (or the transcriptional start site) optionally with additional enhancer elements between 4.4 and 5.5 kb. In another embodiment, the promoter provides tissue-specific expression, preferably breast-specific or tumour-specific. In an optional variation, the invention includes an isolated nucleic acid molecule comprising a mammalian mammaglobin promoter with the provisio that the nucleic acid molecule does not comprise a sequence disclosed in AP003306, NCBI entrez NT_033241 , NCBI MIM entry *605562, NCBI entry AF015224 or Watson et al. (18) (such sequences may optionally still be used in conjunction with a vector of the invention, such as an adenovirus vector). The invention also provides for a recombinant nucleic acid molecule comprising the MGB promoter or a portion thereof operatively linked to a RNA or protein coding sequence, wherein the RNA or protein coding sequence is under the transcriptional control of the promoter region. Preferably, the RNA or protein coding sequence may be selected from the group consisting of suicide genes, proapoptotic genes, or immunomodulatory genes; genes that when expressed in the cells of proliferative cell disorders such as cancer cells, are useful in the treatment of the proliferative cell disorder, such as the cancers described in this application, because they are toxic or detrimental when expressed in cells (usually toxic or detrimental in either normal or cancerous cells). In one embodiment, the recombinant nucleic acid molecule comprising the MGB promoter or a portion thereof is operatively linked to an RNA coding sequence that is an antisense RNA molecule, a small hairpin RNA molecule or a short interfering RNA molecule. In a further embodiment, the antisense RNA molecule, small hairpin RNA molecule or short interfering RNA molecule inactivates a gene or genes or pathways which are useful for the treatment of cell proliferative disorders, such as cancer and which are toxic or detrimental when expressed in such cells. In another embodiment of the invention an antisense RNA molecule, small hairpin RNA molecule or a short interfering RNA molecule, operatively linked to the recombinant nucleic acid molecule comprising the MGB promoter or portion thereof is used in the treatment of a cell proliferative disorder such as cancer. In another embodiment of the invention, the recombinant nucleic acid molecule would be inserted in a vector, preferably an adenovirus vector or a helper-dependent adenovirus vector. In a further embodiment of the invention, a host cell would be transformed with the vector. Preferably the host cell would be a breast cell, breast cancer cell, prostate cancer cell, colon cancer cell, osteosarcoma cell, bladder carcinoma cell or other cancer cell.

The present inventors have shown that vectors carrying expression cassettes driven by MGB promoter fragments demonstrate high levels of expression in several human cancer cell lines including human and murine breast cancer cell lines, prostate cancer cell lines, colon cancer cell lines, osteosarcoma cell lines and bladder cancer cell lines and at greatly reduced levels (to over 1000-fold) in normal cell lines. Accordingly, in another embodiment, the invention provides for a method of treating proliferative cell disorders such as cancer, comprising administering to a mammal having that condition the recombinant nucleic acid molecules of the invention. The recombinant nucleic acid molecules of the invention may be contained in a vector. In another embodiment, the invention provides for a method for gene therapy for a cell proliferative disorder such as cancer, comprising delivering a the recombinant nucleic acid molecule of the invention or the recombinant nucleic acid molecule inserted in a vector, containing an MGB promoter driving expression of a RNA or protein coding sequence to an affected tissue. In a preferred embodiment, the affected tissue would be selected from the group of breast, prostate, colon, bladder, bone or ovarian or breast, prostate, colon, bladder, bone or ovarian derived cancers. In another embodiment, the cancer is metastatic.

Other features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the invention will now be described in relation to the drawings in which:

Figure 1 is a graphical representation of the relative activity of MGB promoter constructs compared to basic plasmid following DNA transfection of normal and breast cancer cell lines.

Figure 2 is a graphical representation of the activity of the MGB promoter in breast cancer cells lines infected with HDAd vectors carrying large MGB promoter fragments. Figure 3 is a graphical representation of the activity of the 25 kb MGB promoter in normal cell lines and in the T47D breast cancer cell line.

Figure 4 is a graphical representation of the activity of the MGB promoter in other cancer cell lines compared to the activity in normal MRC5 cells Figure 5 a) is a graphical representation of the activity of MGB promoter fragments (estimated multiplicity of infection) after adenovirus transduction.

Figure 5 b) is a graphical representation of the activity of MGB promoter fragments (multiplicity of infection of 50 pfu/cell) after adenovirus transduction.

Figure 6 is a graphical representation of the MGB promoter driven reporter expression in Tumour. Figure 7 is a graphical representation of the MGB promoter driven reporter expression in Liver.

Figure 8 is a graphical representation of the MGB promoter driven reporter expression in Mammary Gland. Figure 9 shows graphical representations of reporter expression driven by different sized MGB promoters.

Figure 10 shows the structure of the MGB promoter fragments used in the analyses. . Dark bars indicate MGB promoter sequences; thin line indicates MGB sequences that have been deleted from the promoter in construction of the corresponding vectors. Promoter fragments analyzed in transfection studies are: pML20 (nt 1 to 3253); pML23 (nt 1 to 4745); pML21 (nt1 to 7270); and pML34 (nt 1 to 8360). Promoter fragments inserted into first generation vectors are: AdML23 (nt 1 to 4745); AdML35 (nt 1 to 2431); and AdML36 (nt 1 to 963). Promoter fragments inserted into HDAd vectors are: HDMP25K (nt 1 to 25373); HDMP17K (nt 1 to 17070); HDMP8K (nt 1 to 8210); HDMP0.9K (nt 1 to 879); HDMP0.34K (nt 1 to 344); HDMP8KS1 (nt 1 to 6944); HDMP8KS2 (nt 1 to 6291); HDMP8KS3 ( nt 1 to 5536); HDMP8KS4 (nt 1 to 344 and 3230 to 8210); HDMP8KS5 (nt 1 to 344 and 3945 to 8210); HDMP8KS6 (nt 1 to 344 and 4425 to 8210); HDMP8KS7 (nt 1 to 344 and 2064 to 8210); HDMP8KS8 (nt 1 to 5158 and 6994 to 8210); and HDMP8KS9 (nt 1 to 4424 and 6994 to 8210).

Figure 11 a) shows (SEQ.ID.NO:1). In a preferred embodiment, the sequence is the 5' upstream region of the MGB gene including the promoter and other regulatory sequences. Sequences used in plasmids & vectors and nucleotide nos. are as follows

* these sequences are not just staffer, but include the Ad sequences comprising the left ITR and packaging signal (nt 2894-3333) and the right ITR (30618-30986); and the plasmid sequences (1-2891) between the Ad ITRs for amplification of the pHD in bacterial prior to transfection of 293 cells for virus rescue.

Figure 12 illustrates a deletion analysis of the region between 5.5 kb and 8 kb upstream of the MGB coding sequence.

Figure 13 illustrates a deletion analysis of the region between 0.34 and 4.5 kb upstream of the MGB coding sequence. Figure 14 investigates the effect of deletion of potential enhancer(s) between 4.4 and 7 kb of the MGB promoter.

Figure 15 is a schematic diagram of the MGB promoter, illustrating the location of the potential minimal MGB promoter between 1 and 344 bp, and a strong enhancer between 4.4 and 5.5 kb upstream of the MGB coding sequence (+64 to -280, and -4.3 to -5.4 kb relative to the transcription start site).

DETAILED DESCRIPTION OF THE INVENTION The invention identifies the regulatory sequences governing expression of the mammaglobin (MGB) gene and a novel vector which allows highly selective expression of genes under the control of the promoter, for example in cancer cells. Additional regulatory sequences were isolated upstream of the 1 kb promoter fragment. Adenovirus (Ad) is a preferred vector used as a delivery vehicle because of the ease of vector construction and propagation, the high titers of virus progeny, and the ability of Ad to infect a wide range of cell types from different species at very high efficiencies (see review (19)). In addition, the cloning capacity of Ad is large, up to 8 kb for first generation vectors and up to about 33 kb for the fully deleted helper-dependent Ad (HDAd) vectors. HDAd vectors carrying expression cassettes driven by MGB promoter fragments of 8-25 kb demonstrated high levels of expression in several human and murine BrCa cell lines and at greatly reduced levels (to over 1000-fold) in normal cell lines.

Nucleic Acid Molecules The present inventors have purified and isolated (SEQ.ID.NO.1) 25 kb upstream of the translational start site of the MGB gene. Accordingly, in one embodiment, the invention provides for a purified and isolated nucleic acid molecule comprising a mammalian MGB gene promoter or portions thereof. In a preferred embodiment, the optimal promoter region comprises 5.5 kb upstream of the translational start site of the MGB gene, with specificity conferred by a minimal promoter within the 344 bp proximal to the translational start site. Preferably, the promoter provides tissue-specific expression, preferably breast-specific or tumour-specific expression.

The invention also provides for fragments of the MGB promoter. Accordingly, the invention provides for isolated nucleic acid molecules, wherein the promoter comprises a portion of a nucleic acid sequence shown in SEQ.ID.NO.1 , wherein the portion is capable of promoting transcription of a gene operatively linked to the promoter. In a preferred embodiment, the fragment comprises nucleotides 1 to 17070, nucleotides 1 to 8360, nucleotides 1 to 8210, nucleotides 1 to 7270, nucleotides 1 to 4745, nucleotides 1 to 3253, nucleotides 1 to 2431 , nucleotides 1 to 963, nucleotides 1 to 879, nucleotides 1 to 344, nucleotides 1 to 6944, nucleotides 1 to 6291 , nucleotides 1 to 5535, nucleotides 3230 to 8210, nucleotides 3945 to 8210, nucleotides 4425 to 8210, nucleotides 2064 to 8210, nucleotides 6994 to 8210, nucleotides 1 to 5158, nucleotides 1 to 4424, or nucleotides 4424 to 5535 of the sequence shown in SEQ.ID.NO.1. The fragment may be 300 to 500 nucleotides, 500 to 1000 nucleotides, 1000-2500 nucleotides, 2500-5000 nucleotides, 5000-10,000 nucleotides or greater than 10,000 nucleotides

In a preferred embodiment, the nucleic acid molecules of this invention comprise:

(a) SEQ ID NO:1 or a portion thereof (such as the specific nucleotide ranges given in this application) or a nucleic acid sequence that is complimentary to SEQ. ID. NO. 1 , or a portion thereof which has MGB promoter activity (MGB promoter activity may be identified by performing an experiment as described in the present application to show that the promoter has at least the same, about the same (e.g. plus or minus 10%) or better activity than the MGB promoter and specificity for cancer cells compared to wild type cells as measured by one or more of the experiments described in this application, such as in the examples), preferably providing tissue specific expression, such as breast-specific or tumor-specific expression;

(b) a nucleic acid sequence that has substantial sequence identity to a nucleic acid sequence of (a); (c) a nucleic acid sequence that is an analog of a nucleic acid sequence of (a) or (b); or

(d) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a), (b) or (c) under stringent hybridization conditions. A sequence has promoter function if it can function as a promoter, i.e. induce transcription as measured by an assay. A promoter is tissue specific if it induces at least 3-fold or more preferably at least 10-fold increased transcription of a downstream gene or cDNA in target tissues or cells relative to nontarget tissues or cells. This could be detected for example, by luciferase assay if the downstream gene encodes luciferase.

The term "sequence that has substantial sequence identity" means those nucleic acid sequences which have slight or inconsequential sequence variations from the sequences in (a), i.e., the sequences function in substantially the same manner and promote tissue-specific gene expression. The variations may be attributable to local mutations or structural modifications. Nucleic acid sequences having substantial identity include nucleic acid sequences having at least 65%, more preferably at least: 85%, 90%, 95% (e.g. 90-95%), 98% or 99% identity with the nucleic acid sequence as shown in SEQ.ID.NO.1. Identity is calculated according to methods known in the art. Sequence identity is most preferably assessed by the BLAST version 2.1 program advanced search (preferably using default parameters). BLAST is a series of programs that are available online at http://www.ncbi.nlm.nih.gov/BLAST. The advanced blast search (http://www.ncbi.nlm.nih.gov/blast/bIast.cgi?Jform=1 ) is set to default parameters, (ie Matrix BLOSUM62; Gap existence cost 11 ; Per residue gap cost 1 ; Lambda ratio 0.85 default). References to BLAST searches include: Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403_410; Gish, W. & States, D.J. (1993) "Identification of protein coding regions by database similarity search." Nature Genet. 3:266_272; Madden, T.L., Tatusov, R.L. & Zhang, J. (1996) "Applications of network BLAST server" Meth. Enzymol. 266:131_141 ; Altschul, S.F., Madden, T.L, Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D.J. (1997) "Gapped BLAST and PSI_BLAST: a new generation of protein database search programs." Nucleic Acids Res. 25:3389_3402; Zhang, J. & Madden, T.L. (1997) "PowerBLAST: A new network BLAST application for interactive or automated sequence analysis and annotation." Genome Res. 7:649_656.

The term "sequence that hybridizes" means a nucleic acid sequence that can hybridize to a sequence of (a), (b) or (c) under stringent hybridization conditions. Appropriate "stringent hybridization conditions" which promote DNA hybridization are known to those skilled in the art, or may be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the following may be employed: 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C. The stringency may be selected based on the conditions used in the wash step. For example, the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C. In addition, the temperature in the wash step can be at high stringency conditions, at about 65°C.

The term "a nucleic acid sequence which is an analog" means a nucleic acid sequence which has been modified as compared to the sequence of (a) or (b) wherein the modification does not alter the utility of the sequence (i.e. as promoting tissue-specific expression) as described herein. The modified sequence or analog may have improved properties over the sequence shown in (a) or (b). One example of a modification to prepare an analog is to replace one of the naturally occurring bases (i.e. adenine, guanine, cytosine or thymidine) of the sequence shown in SEQ.ID.NO.1 with one or moremodified bases such as such as xanthine, hypoxanthine, 2- aminoadenine, 6-methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5- halo cytosine, 6-aza uracil, 6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines, 8 amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5- trifluoro cytosine.

Another example of a modification is to include modified phosphorous or oxygen heteroatoms in the phosphate backbone, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages in the nucleic acid molecule shown in SEQ.ID.NO.1. For example, the nucleic acid sequences may contain phosphorothioates, phosphotriesters, methyl phosphonates, and phosphorodithioates.

A further example of an analog of a nucleic acid molecule of the invention is a peptide nucleic acid (PNA) wherein the deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), is replaced with a polyamide backbone which is similar to that found in peptides (P.E. Nielsen, et al Science 1991 , 254, 1497). PNA analogs have been shown to be resistant to degradation by enzymes and to have extended lives in vivo and in vitro. PNAs also bind stronger to a complimentary DNA sequence due to the lack of charge repulsion between the PNA strand and the DNA strand. Other nucleic acid analogs may contain nucleotides containing polymer backbones, cyclic backbones, or acyclic backbones. For example, the nucleotides may have morpholino backbone structures (U.S. Pat. No. 5,034,506). The analogs may also contain groups such as reporter groups, a group for improving the pharmacokinetic or pharmacodynamic properties of nucleic acid sequence.

The present invention also provides recombinant nucleic acid molecules comprising any one of the MGB promoters described herein operatively linked to a protein-coding or RNA-coding sequence (for example, a structural gene), wherein the sequence is under the transcriptional control of the promoter region. Preferably the RNA or protein coding sequence is selected from a group consisting of cancer-specific suicide genes, proapoptotic genes, immunomodulatory genes and other genes which are toxic or detrimental when expressed in normal cells. Examples of such genes include herpes simplex virus thymidine kinase, cytosine deaminase, bax, bak, caspases, interleukin-2, interleukin-12, tumor-necrosis factor-alpha, interferon-alpha, and interferon-beta. The invention also comprises RNA coding sequences selected from the group of short interfering RNAs (siRNAs), small hairpin RNAs (shRNAs), or anti-sense RNAs. SiRNAs, sh RNAs and anti-sense RNAs reduce the abundance and thus the activity of target proteins. Antisense RNA coding sequences are typically obtained from cDNAs and are expressed in the reverse orientation in cells so that the resultant RNA generated is complementary to the endogenous mRNA encoding the target protein. The binding of these two RNAs inhibits the translation of the target mRNA, thereby blocking or reducing the synthesis of the corresponding protein. Patents that describe various uses and modifications of antisense technology include: US Patent Nos. 6,133,246, 6,096,722, 6,040,296, 5,801 ,159 and 5,739,119. Short interfering RNAs (also known as small interfering RNAs) and small hairpin RNAs are double stranded RNA molecules which form secondary structure and have sequence homology to a cellular mRNA target. The former comprises two complementary RNA molecules which hybridize to form a double stranded molecule while the latter forms a double stranded molecule by forming a hairpin structure (ie folding back on itself). Expression of the siRNA or shRNA molecule activates a cellular response which destroys the homologous internal mRNA. This ablates or decreases levels of the mRNA and the protein coded for by the mRNA. U.S. patent applications 20030153519 and 20030148519 describe various uses of RNA interference technologies.

In another embodiment the anti-sense RNAs shRNAs or siRNAs inactivate a gene selected from the group of genes which promote cell proliferation, cell survival, cell division, replication genes, oncogenes, angiogenic factors or other genes whose reduction would be beneficial for the treatment of cell proliferation disorders such as cancer.

In another embodiment, the invention provides a method of expressing a nucleic acid protein- or RNA-coding region in a host cell, the method comprising: (a) introducing the recombinant nucleic acid molecules of the invention into a host cell; and

(b) expressing the protein- or RNA-coding region.

The recombinant nucleic acid molecules may be introduced into tissues or cells using techniques in the art including vectors (retroviral vectors, adenoviral vectors and DNA virus vectors) or physical techniques such as microinjection. The recombinant nucleic acid molecules or vectors containing the molecules may be directly administered in vivo or may be used to transfect cells in vitro which are then administered in vivo. In one embodiment, a vector of the invention is adapted for transfer to a prokaryotic or eukaryotic cell. In another embodiment, the recombinant nucleic acid molecule or vector may be delivered to host cells in a liposome formulation.

In a further embodiment, the recombinant nucleic acid can be introduced into a host cell by electroporation. Examples of vectors that can be used include adeno-associated viruses, lentiviruses, retroviruses, herpes viruses, and pox viruses. The present inventors have shown that the adenovirus gene delivery vehicle permitted specificity and demonstrated a high degree of activity compared to plasmid transfection. Significant specificity was observed with the first generation Ad construct carrying a 4.7 kb fragment of the MGB promoter.

Larger fragments of the promoter in a helper dependent adenovirus vector showed an even greater reduction in non-specific expression, while maintaining a high level of breast cancer-specific expression. Accordingly, in a preferred embodiment, the recombinant nucleic acid molecule may be delivered to host cells in an adenovirus vector or a helper-dependent adenovirus vector.

The invention also provides for a recombinant nucleic acid including two or more non-contiguous nucleic acid fragments of the MGB promoter to be combined in a vector for delivery to host cells. In one embodiment, non- contiguous MGB promoter nucleic acid fragments are delivered by a helper dependent adenovirus vector. Non-coding "stuffer" sequences can be inserted into the helper dependent adenovirus vectors carrying MGB promoter nucleic acid fragments in order to maintain an optimal size for vector encapsidation such as those provided by pC4HSU (Microbix Biosystems Inc.; www.microbix.com) which is referenced in the legend for Figure 11. It should be noted, however that the invention is not limited to the use of particular "stuffer" sequences and any non-coding nucleic acid may be used as "stuffer" sequence.

The invention further provides a host cell transformed with the recombinant nucleic acids or vectors of the invention. Examples of suitable host cells include mammary cells, breast cancer cells and other tumour cells.

In another embodiment, the invention provides for a non-human transgenic mammal carrying the recombinant nucleic acids or vectors of the invention. In another embodiment, the transgenic mammal is carrying the recombinant nucleic acids or vectors of the invention in a specific tissue or organ or tumor derived from a specific tissue or organ. In another embodiment, the tissue or organ or tumor derived from a tissue or organ, includes, but is not limited to, the prostate, breast, colon, bladder or bone. In another embodiment, the transgenic mammal is carrying the recombinant nucleic acids or vectors of the invention in mammary tissue or tumours derived from mammary tissue. In a preferred embodiment, the non-human mammal is a rodent, more preferably mouse. In a further embodiment the non-human transgenic animal carrying the recombinant nucleic acids or vectors of the invention is used to screen for or test the efficacy of RNA or protein coding sequences that may be useful in the treatment of cancer. Therapeutic Methods

The term "effective amount" as used herein means an amount effective at dosages and for periods of time necessary to enhance the level of nucleic acid of interest.

The term "treatment or treating" as used herein means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treating" can also mean prolonging survival as compared to expected survival if not receiving treatment.

Since an animal suffering from disease, disorder or abnormal physical state can be treated by administering a vector including all or part of the MGB promoter and a therapeutic gene, the methods and compositions of the invention are useful to modify the development/progression of cell proliferation disorders such as cancer. The invention also includes a transformed cell containing the vector and the recombinant nucleic acid molecule sequences.

The present inventors have demonstrated the specificity of the MGB promoter to direct expression in cancerous cells. Accordingly, the invention provides for a method of reducing cell proliferation, comprising administering to a mammal having that condition the recombinant nucleic acid molecule or vector containing all or part of the MGB promoter driving expression of a protein or RNA-coding gene. The invention also provides for a method of treating cancer, comprising administering to a mammal having that condition the recombinant nucleic acid molecule or vector containing all or part of the MGB promoter driving expression of a protein or RNA-coding gene (useful MGB fragments are those described herein). The vectors of the invention are used to reduce proliferation of cells that exhibit symptoms of a cell proliferation disorder, such as dividing for longer or persisting longer than wild type cells. These symptoms are typically shown by changes compared to wild type cells, such as changes in cell cycle time, reduced death rate, percentage of cells in different stages of cellular development (growth or S-phase fractionation) or number of receptors, such as estrogen or progesterone receptors. The invention also provides for a method for gene therapy for reducing cell proliferation and/or cancer, comprising the step of delivering a recombinant nucleic acid molecule or vector to affected tissue. In a preferred embodiment, the cancer is breast, prostate, colon, bladder, bone or ovarian cancer. In another embodiment, the cancer is metastatic. Preferably the RNA or protein coding sequence is selected from a group consisting of suicide genes, proapoptotic genes, immunomodulatory genes, siRNA sequences, shRNA sequences or anti-sense RNA sequences and other genes which are toxic or detrimental when expressed in normal or diseased cells (such as cancer cells). Examples of such genes include herpes simplex virus thymidine kinase, cytosine deaminase, bax, bak, caspases, interleukin-2, interleukin-12, tumor necrosis factor-alpha, interferon-alpha, and interferon- beta, preferably the human genes or fragments, homologs or derivatives thereof, all of which are are readily apparent to those in the art.

In a further embodiment, the invention also provides for pharmaceutical compositions containing the recombinant nucleic acid or vector. Such pharmaceutical compositions can be for intralesional, intravenous, topical, rectal, parenteral, local, inhalant or subcutaneous, intradermal, intramuscular, intrathecal, transperitoneal, oral, and intracerebral use. The composition can be in liquid, solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, gels, membranes, tubelets, solutions or suspensions. The recombinant nucleic acid molecule or vector is preferably injected in a saline solution either intravenously, intraperitoneally or subcutaneously.

The pharmaceutical compositions of the invention can be intended for administration to humans or animals. Dosages to be administered depend on individual needs, on the desired effect and on the chosen route of administration.

The pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the pharmaceutical compositions include, albeit not exclusively, the active compound or substance in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids. The pharmaceutical compositions may additionally contain other anti- tumour agents.

A pharmaceutical composition comprising the nucleic acid molecules of the invention may be used in gene therapy to modulate the growth of cell proliferative disorders such as cancer growth. Recombinant molecules comprising a nucleic acid sequence comprising an MGB promoter or fragment thereof operatively linked to an RNA or protein coding sequence, which may be directly introduced into cells or tissues in vivo using delivery vehicles such as retroviral vectors, adenoviral vectors and DNA virus vectors. They are optionally introduced into cells in vivo using physical techniques such as microinjection and electroporation or chemical methods such as coprecipitation and incorporation of DNA into iiposomes. Recombinant molecules are optionally delivered in the form of an aerosol or by lavage. The nucleic acid molecules of the invention are optionally applied extracellularly such as by direct injection into cells. Tumor-specific promoters in cancer therapy may be used in the development of oncolytic viruses. These are lytic viruses, for example adenovirus, modified to replicate only in tumor cells. Lytic viruses could be generated by placing genes responsible for virus replication (generally the early gene E1A and E1 B in the case of adenovirus) under control of tumor- specific promoters. Thus the virus replicates in and lyses only tumor cells. For these cancer therapeutic agents it is critical that the promoter is inactive in normal cells. Accordingly, in another embodiment, the invention provides an adenovirus vector, or any other lytic virus, such as (but not limited to) herpes viruses or pox viruses, wherein the replication genes are under the control of the mammaglobin promoter or portions thereof thus producing a conditionally replicating virus. Preferably, the specific genes controlled by the mammaglobin promoter in an adenovirus vector would be adenovirus early region 1A and 1 B genes, E1A or E1 B cDNAs, or derivatives of the E1A and E1 B genes (i.e., truncated or mutant versions of these genes that still function in tumor cells). Oncolytic viruses could also carry therapeutic genes like suicide genes, proapoptotic genes, immunomodulatory genes and other genes, which are toxic or detrimental when expressed in normal cells. Examples of such genes include herpes simplex virus thymidine kinase, cytosine deaminase, bax, bak, caspases, interleukin-2, interleukin-12, tumor necrosis factor-alpha, interferon-alpha, and interferon-beta.

In a further embodiment, the invention provides a method of treating cancer in a mammal comprising administering to the mammal an effective amount of this oncolytic vector.

Experimental Models

The present invention also includes methods and experimental models for studying the function of the MGB promoter protein. Cells, tissues and non- human animals are created by a recombinant expression vector including all or part of the MGB promoter and a nucleic acid of interest. Thus, another aspect of the invention relates to a transgenic non-human mammal carrying the vector including all or a portion of the MGB promoter, such as an adenovirus vector. The vector is optionally in one or more tissues or tumors derived from one or more tissues or organism, such as mammary tissue or a tumor derived from mammary tissue, prostate tissue or a tumor derived from prostate tissue, colon tissue or a tumor derived from colon tissue, bone tissue or a tumor derived from bone tissue, bladder tissue or a tumor derived from bladder tissue. The transgenic non-human mammal is optionally a rodent, such as a mouse. The transgenic non-human mammal is useful in screening for RNA or protein coding genes useful in the treatment of cancer. For example, the vector may be administered to the transgenic non-human mammal, such as a mammal having breast, prostate, bone, colon or other cancer, to determine whether the therapeutic nucleic acid under the control of the promoter reduces the cancer, for example by reducing tumour size, metastasis or other properties compared to a control mammal not receiving the therapeutic nucleic acid.

A construct may be introduced into a cell, such as an embryonic stem cell, by a technique such as transfection, electroporation, injection etc. Cells including the nucleic acid of interest gene may then be identified, for example by Southern blotting, Northern Blotting or by assaying for nucleic acid

The following non-limiting examples are illustrative of the present invention:

EXAMPLES Example 1

MGB promoter activity following DNA transfection of normal and BrCa cell lines

To determine whether the specific expression of MGB in BrCa cells was determined by sequences upstream of the MGB open reading frame, MGB promoter fragments of various sizes were inserted into luciferase reporter gene expression cassettes. These plasmids were used to transfect the normal human fibroblast cell line MRC5, and the human BrCa cell lines MDA-MB468 and T47D. To control for transfection efficiency, a CMV-beta galactosidase (beta-gal) reporter plasmid was cotransfected with the MGB- luciferase constructs, as described in materials and methods. 48 hours post- transfection, cells were harvested and assayed for luciferase and beta-gal activity. As seen in Fig 1 , all the MGB promoter constructs had higher activity than the promoterless Basic plasmid, but little specificity was demonstrated for BrCa cells over normal MRC5 cells. This is similar to the data obtained by Watson and Fleming in transfections with 1 kb or smaller promoter fragments (18). Attempts to use reporter plasmids containing much larger fragments of the MGB promoter have been unsuccessful, but it was not clear whether this was due to a lack of regulatory sequences in the promoter, or due to inefficient delivery of such very large plasmid DNA molecules to target cells using calcium phosphate precipitation. Example 2

Analysis of activity of large fragments of the MGB promoter in HDAd reporter vectors

The large cloning capacity and high transduction efficiency of HDAd vectors allowed further screening of potential regulatory sequences far upstream of the MGB open reading frame. HDAd reporter vectors were constructed carrying 25 kb, 17 kb, and 8 kb of the MGB promoter. These vectors were used to infect confluent 6-cm dishes of normal (MRC5) or BrCa (T47D) human cell lines in triplicate at an MOI of 2000 particles per cell (this is roughly equivalent 20-50 pfu per cell with a first generation vector). Cells were harvested 48 hours later, and equal volumes of lysates were assayed for luciferase activity. As shown in Fig. 2, the activity of the MGB promoter in the BrCa cell line was about 10,000-fold higher than that observed in normal cells. This difference is not due to a defect in Ad delivery to MRC5 cells, since previous experiments have shown higher expression from an Ad- CMV- luciferase vector in MRC5 cells than in T47D cells. Thus it is clear that specificity of this promoter was revealed through Ad vector screening which would not have been apparent with DNA transfections alone.

No difference in activity was seen when the 25 kb, 17 kb and 8 kb promoter fragments were compared, therefore important regulatory sequences must be within the 8 kb sequence proximal to the MGB reading frame.

Other normal cell lines were tested to ensure that expression was specific. Human embryonic kidney HEK cells, human embryonic retinal HER cells, murine fibroblast NIH3T3 cells and T47D BrCa cells were infected with HDMP25K or with a first generation AdCMV-Luc vector as described above. 48 hours later cells were harvested and assayed for luciferase activity. The results shown in Fig 3 were adjusted for differential infectivity of the cell lines by normalizing to activity of the CMV promoter. As with MRC5 cells, the level of expression from the MGB promoter was over 500-fold higher in T47D BrCa than in all normal cell lines tested.

In a similar manner, expression driven by the MGB promoter in other cancer cell lines were compared to that in normal MRC5 cells (Fig 4). Tested lines included BrCa cell lines, human lines T47D, MDAMB468, and MCF7 and murine MT1A2 cells, human prostate cancer cell lines DU145, LNCaP, and PC3; human colon carcinoma Colol cells; human osteosarcoma R970-5 cells, and human bladder carcinoma J82 cells. Cells were infected with the helper- dependent Ad vector carrying a luciferase expression cassette controlled by a 25kb fragment from the MGB promoter at a multiplicity of infection of 2000 particles per cell (roughly equivalent to 50 pfu per cell). 48 hours post- infection, cells were harvested and the lysates assayed for luciferase activity. To correct for different transduction efficiencies of the various cell lines, parallel infections were carried out with an Ad vector carrying a constitutively active mCMV-luciferase cassette. Activity of the MGB promoter was then normalized to activity of the mCMV promoter. These results demonstrate specificity, although with a large range (up to 50-fold) in the level of expression in BrCa lines and show that the MGB promoter are useful for targeting other tumor types.

Example 3

MGB promoter activity following infection of normal and BrCa cell lines with first generation or helper-dependent Ad vectors

A preliminary experiment was carried out to determine whether the same specificity was maintained in a first generation Ad vector as in the HDAd. Due to the smaller cloning capacity however, the largest promoter fragment that would be useful in first generation vectors is about 4-5 kb. Therefore a 4.7 kb MGB promoter Ad construct was generated. In this experiment, the titers of the vector stocks were estimated since they were only partially purified. Cells were plated into 24 well plates, then 24 hours later, infected in duplicate with first generation or helper-dependent Ads at what we now estimate was an MOI of about 2 (this is much lower than MOIs normally used). Infected cell extracts were prepared 48 hours post-infection. Luciferase assays were performed on different volumes of lysate from each cell line such that an equivalent number of cells were used in each assay. AdMCMV-Luc infections were performed in parallel i.e. not co-infected with the MGB-luciferase viruses. The results shown in Fig 5a indicate that the first generation also maintains specificity, but not to as great an extent as the HDAd vector. Furthermore it is possible that the regulatory region of the MGB promoter lies within the first 5 kb. Figure 5b confirms these results. Human breast cancer (T47D), human normal (MRC5), murine breast cancer (MT1A2), or murine normal (516) cell lines were infected at an MOI of 50 pfu/cell with first generation Ad vectors carrying mammaglobin promoter sequences of various sizes from 1 kb to 4.7 kb. Cells were harvested and assayed for luciferase activity at 72 hours post- infection. Therefore first generation vectors maintain BrCa cell specificity and this specificity lies within the first 1kb of the promoter.

Example 4 The MGB promoter is highly specific for breast cancer in a murine tumor model

To determine in vivo specificity of the MGB promoter, expression of luciferase reporter genes driven by the 4.7 kb MGB promoter, the 25 kb MGB promoter, and the constitutive murine CMV promoter following intratumoral injection was compared in a mouse model for breast cancer. Briefly, tumor cells were explanted from a mammary tumor that had developed in a transgenic mouse (the transgenic mouse line carries the oncogenic polyoma middle T antigen gene under control of the MMTV promoter). After a few days in culture, 1 x 10e6 tumor cells were injected subcutaneously into the hind flanks of immunocompetent syngeneic mice. About 3 weeks later, mice (6 per group) were each injected intratumorally with 2 x 10e10 virus particles (corresponding to approximately 5 x 10e8 pfu) of AdML23 (first generation Ad 4.7 kb MGB promoter-luc), HDMP25K (helper-dependent Ad 25 kb MGB promoter-luc), or AdSJ-4 (first generation Ad mCMV promoter-luc). Three mice from each group were euthanized on day 2 and the remainder euthanized on day 7 post-infection. Animals were dissected and tissues snap frozen in liquid N₂. Frozen tissues were thawed in extract buffer (0.1 M K phosphate, pH 7.8, 1 mM DTT, 20 g/ml aprotinin, 50 M leupeptin, 1 mM pepstatin A), homogenized and sonicated on ice. Samples were centrifuged and the supematants assayed for protein (BioRad Protein assay kit) and luciferase activity (Applied Biosystems Luciferase Assay Kit). Luciferase expression was normalized to protein concentration, and the data generated from analysis of tumor, liver, and mammary gland are shown in Figs 6, 7, and 8 respectively. Each bar represents a different animal. Black bars indicate expression on day 2 and empty bars indicate expression on day 7. Previous experiments have demonstrated that control vector infected or mock infected animals do not express luciferase activity in any of these tissues. Significant levels of reporter activity were detected in the tumor with all three promoters on days 2 and 7 post-infection. On day 2, the CMV promoter expressed the highest activity, about 500X greater than either MGB promoter fragment. Activity driven by the CMV promoter decreased substantially by day 7, such that there was not a great difference in activity between the CMV and the MGB promoters at this later time. In contrast, expression in the liver was high both at days 2 and 7 following intratumoral injection of the CMV vector. In the liver, activity of the 4.7 kb MGB promoter was four logs lower than the CMV, and the activity of the 25 kb MGB promoter in an HD vector was undetectable (i.e., greater than 5 logs lower expression than with the CMV promoter). Expression in the mammary gland was detectable, but very low with all promoters, perhaps due to a lack of Ad receptors on these cells. The promoter specificity for breast cancer, determined by the formula

luc activitv/ug protein in tumor luc activity/μg protein in liver

for the CMV promoter was 1.1 on day 2 and 0.01 on day 7, reflecting the fact that expression in the tumor was reduced at the later time. In contrast, the specificity of the 4.7 kb MGB promoter was about 50 on both days 2 and 7. Specificity of the 25 kb MGB promoter was even greater: about 200 on day 2 and 2000 on day 7. When normalized to CMV expression, the specificity of the MGB promoter fragments appear even greater: 58 on day 2 and 3800 on day 7 for the 4.7 kb fragment; and 200 on day 2 and over 100,000 on day 7 for the 25 kb fragment. However, this latter analysis may be misleading if a specific shut-off of CMV activity in the tumor occurred between days 2 and 7. Clearly both the small and large fragments of the MGB promoter are highly specific for mammary tissue and in particular mammary tumors in vivo.

Example 5

Size of promoter required for activity and specificity

In order to show that promoter fragments smaller than 8 kb retain high activity and specificity, HDAds were generated with promoter fragments of 0.34 kb, 0.9 kb, and 8 kbcontrolling expression of a luciferase reporter gene. These vectors were tested for expression in vitro in the human breast cancer cell line T47D and the normal human cell line MRC5 (Figure 9). All MGB promoter fragments displayed high activity in BrCa cells although there was a 100-fold difference in expression levels between the 0.34K fragment and the 8K fragment. Expression in MRC5 cells was two to four orders of magnitude lower than in T47D cells. Thus even the 344 bp MGB promoter is highly specific for BrCa expression, although the absolute level of expression from this fragment of the promoter is significantly reduced relative to the 8 kbp promoter. Example 6

Identification of potential enhancer elements upstream of the MGB coding sequence

To identify potential enhancer elements near the upstream end of the 8kb promoter fragment, a series of vectors were generated carrying the proximal 5.5 kb, 6.3 kb, or 6.9 kb of the MGB promoter. These vectors were compared to the 8 kb promoter vector for specific expression in T47D breast cancer cells relative to normal MRC5 cells (Fig. 12). The structures of the MGB promoter fragments used in this experiment are shown in the top panel of Fig. 12, and the results of the expression analysis are shown below. Sequences carried by the HDAd vectors are indicated by bold arrows. Luciferase expression was determined in HuBrCa (T47D) (black) or normal human fibroblasts (MRC5) (white) 48 hours post-infection with HDAd vectors encoding the luciferase gene driven by sequences 8 kb (HDMP8K), 6.9 kb (HDMP8KS1), 6.3 kb (HDMP8KS2), or 5.5kb (HDMP8KS3), upstream of the MGB translation start site.

These smaller promoter fragments were only slightly less active (5-fold) than the 8 kb vector, showing at most a weak enhancer element in this region between 6.9 kb and 8 kb upstream of the start site.

Vectors were next generated carrying a series of deletions in the proximal end of the 8 kb MGB promoter leaving the minimal promoter from 1- 344 bp upstream of the MGB coding sequence intact (Fig. 13). The structure of the MGB promoter fragments used in this experiment are shown in the top panel of Fig. 13, and the results of the expression analysis are shown below. MGB sequences carried by the HDAd vectors are indicated by bold arrows. Luciferase expression was determined in HuBrCa (T47D) (black) or normal human fibroblasts (MRC5) (white) 48 hours post-infection with HDAd vectors encoding the luciferase gene driven by the full length 8 kb fragment (HDMP8K), a fragment with a deletion in the 8 kb sequence from 0.34 to 2.1 kb (HDMP8KS7), a fragment deleted between 0.34 and 3.2 kb (HDMP8KS4), a fragment deleted between 0.34 and 3.9 kb (HDMP8KS5), or a fragment deleted between 0.34 and 4.4 kb (HDMP8KS6). There was no large difference between any of these promoter fragments in levels of activity or specificity showing that there were no important enhancer elements in the region between 344 bp and 4.4 kb upstream of the MGB coding sequence.

These data show that important regulatory regions reside in the minimal promoter between 1 and 344 bp (including the first 64 bases of the 5' untranslated sequence), and in potential enhancer(s) between 4.4 kb (as defined by the HDMP8KS6 vector) and 5.5 kb (as defined by HDMP8KS3) 5' to the MGB open reading frame. Consistent with these results, deletion of the sequence from 5.1 to 7 kb upstream did not affect activity, but deletion of a larger fragment from 4.4 to 7 kb reduced activity by over 20-fold (Fig. 14). The structures of the MGB promoter fragments used in this experiment are shown in the top panel, and the results of the expression analysis are shown below. MGB sequences carried by the HDAd vectors are indicated by bold arrows. Luciferase expression was determined in HuBrCa (T47D) (black) or normal human fibroblasts (MRC5) (white) 48 hours post-infection with HDAd vectors encoding the luciferase gene driven by the full length 8 kb fragment (HDMP8K), a fragment with a deletion in the 8 kb sequence from 5.1 to 7 kb (HDMP8KS8), or a fragment deleted between 4.4 and 7 kb (HDMP8KS9). A strong enhancer lies between 4.4 and 5.5 kb upstream of the MGB coding sequence (Fig. 15).

Example 7 An optimal MGB promoter is constructed consisting of nt 1-344 ligated to nt 4500-5500 of the MGB sequence in Fig 11a. This promoter as well as the SV40 polyadenylation (polyA) signal are inserted into the first generation Ad shuttle plasmid pDC312 such that a multicloning site is present between the promoter and the polyA site. The potentially therapeutic gene, murine IL- 12 is inserted at the multicloning site. In another similar construct, human IL-2 is inserted at the multicloning site. Both the IL-12 and IL-2 shuttle plasmids are rescued into first generation adenovirus as described (23). After verification that expression is specific in vitro in murine tumor cell lines, the vectors are injected directly into established murine tumors as described in example 4 above. As controls, mice are also injected with vectors that do not encode any transgene, and also with vectors carrying IL-2 and IL-12 under control of the CMV promoter. Tumors are monitored for regression twice weekly. In addition, mice are assessed for liver damage by assay of alanine aminotransferase (ALT) release in the serum. Tumor regression is approximately equivalent in MGB-driven IL-2 (or IL-12) vs. CMV-driven IL-2

(or IL-12) treated mice, but toxicity is lower in the MGB-driven IL-2 mice, which shows that expression not only has been specific for the tumor, but also that specific expression leads to reduced toxicity as predicted.

As an alternative to the first generation MGB-driven IL-2 or IL-12 constructs, the MGB-IL-2 and MGB-IL-12 expression cassettes are inserted into helper-dependent vectors. These vectors have even lower expression in normal tissues than first generation vectors with the MGB promoter, thus also lower toxicity to normal tissues.

A high ratio of tumor regression to overall toxicity, shows that the first generation or helper-dependent MGB-driven IL-2 or IL-12 constructs vectors are suitable for therapeutic agents. Similar experiments are carried out with other therapeutic genes described in this application and in other tumor models.

In summary, the data show that adenovirus vectors carrying the MGB promoter express specifically in mammary cells, in particular mammary carcinoma cells and other cancer cell lines. Promoter fragments as small as 344 bp were highly specific, although the magnitude of expression was reduced by 100-fold when compared to the "full-length" 8 or 25 kb promoter fragments. Insertion of the MGB promoter-driven expression cassette into a helper-dependent Ad vector resulted in a further reduction of activity in normal cells compared to insertion of the promoter into a first generation Ad vector. Specificity of the MGB promoter was not demonstrated in plasmid transfection studies, possibly due to the inefficiency of transduction by this method. Insertion of the promoter into adenovirus vectors facilitated the clear demonstration of MGB promoter specificity.

The invention demonstrates the first isolation of the important regulatory sequences determining the specificity of MGB for breast cancer cells. It also provides an isolated promoter and vector with greater specificity and applicability to a broader range of patients (as predicted by the detection of MGB protein in patient samples) than promoters from other putative breast cancer specific genes. Materials and Methods:

Cell culture

The 293 cell line, derived from human embryonic kidney cells transformed with the Ad5 E1 region (20), and its derivative Cre recombinase- expressing 293Cre4 cell line (21) were maintained in minimal essential medium (MEM)-F11. Human prostate cancer cells LNCaP, PC3, and DU145; human colon carcinoma Colol cells; human osteosarcoma R970-5 cells, and human bladder carcinoma J82 cells were cultured as described (24), Human T47D BrCa cells, human MCF7 BrCa cells, human embryonic kidney HEK and retinal HER cells, murine NIH3T3 and 516 fibroblasts and murine MT1A2 BrCa cells were maintained in Dulbecco's modified Eagle's medium (DMEM). Human MDA-MB468 BrCa cells (ATCC #HTB-132) were maintained in DMEM/F12. Normal human lung MRC5 fibroblasts were maintained in alpha- MEM. All media were supplemented with 10% fetal bovine serum (FBS), 2mM L-glutamine, 0.1 mg/ml penicillin and 100 U/ml streptomycin. All cell culture reagents were obtained from GIBCO/lnvitrogen, except FBS that was from Sigma Chemicals.

MGB Promoter isolation

A 173 kilobase (kb) human genomic Bac clone (703H08) carrying the MGB promoter was obtained from the RPCI-11 human genomic BAC library (22) through the Genome Resource Facility at the Hospital for Sick Children, Toronto, Ontario, Canada. An 11 kb Nhe\ DNA fragment, positive for MGB by Southern analysis, was isolated from this clone and inserted into the Spel site of pBluescriptll KS(-) to generate pML16. To reconstitute the 3' end of the MGB promoter, a roughly 1 kb fragment was PCR amplified from human 293 cell genomic DNA and inserted into a derivative of pML16 to generate pML18 such that pML18 carries the proximal 3.3 kb fragment of the MGB promoter, including the entire 64 bp 5' untranslated region (UTR). This PCR-amplified 293 cell DNA fragment is identical in sequence to the MGB promoter sequence of BAG clone 703H08. The promoter/UTR sequence was isolated from pML18 and inserted into the pGL3 reporter plasmid (Promega) upstream of the luceriferase coding sequence to generate pML20. A 4 kb EcoRI fragment, corresponding to MGB promoter sequences immediately 5' to those in pML20, was isolated from pML16 and inserted at the 5' end of the promoter in pML20 to generate pML21 , the 7.3 kb MGB promoter-reporter plasmid. The plasmid pML34 was constructed by replacing a 5.2 kb fragment at the 5' end of the MGB promoter in pML21 with a slightly larger 6.3 kb fragment of the MGB promoter isolated from pCSMP25K (Fig 10, and described below under helper-dependent virus construction). pML34 thus carries 8.4 kb of the MGB promoter in pGL3-based reporter plasmid.

Construction of first generation adenovirus vectors

Ad vectors were generated using the AdMax kit (Microbix Biosystems

Inc.) as described (23). The shuttle plasmid pML22 was constructed by inserting the entire MGB-luciferase expression cassette from pML20 (3.3 kb promoter) into the empty shuttle plasmid pDC312. The shuttle plasmid pML23 was constructed by replacing 830 bp from the 5' end of the MGB promoter in pML22 with a 2.3 kb DNA fragment from pML21 such that a 4.7 kb contiguous MGB promoter was reconstituted. Shuttle plasmids pML35 and pML36 were constructed in a similar manner by inserting expression cassettes carrying 2.4 or 0.96 kb, respectively, of the mammaglobin promoter into pDC312.

The shuttle plasmids pML23, pML35 and pML36 were rescued into first generation Ad vectors AdML23, AdML35 and AdML36, respectively, with pBHGdelEI , E3loxCre following cotransfection of 293 cells. The virus was amplified and purified as described previously (23). Titers were determined by plaque assay and by particle numbers (24).

Construction of helper-dependent adenovirus vectors

An HDAd vector precursor, pCSMP2.4K, was constructed by replacing the sequence from 4498 to 29184 from the HDAd plasmid pC4HSU with a DNA fragment from pML21 carrying 2.4 kb of the MGB promoter, the luciferase gene and the polyA signal. A DNA fragment of approximately 23 kb, corresponding to sequences immediately upstream of the 2.4 kb MGB promoter, was isolated from Bac clone 703H08 and inserted into pCSMP2.4K to generate the helper-dependent plasmid pCSMP25K. For smaller MGB promoter fragment constructs, pCSMP25K was digested with appropriate restriction enzymes; then in the case of plasmids of the deletion series MP8KS1 through S9, the non-contiguous promoter sequences were ligated together. As a last step with all HDAd precursor plasmids, "stuffer" sequences from pC4HSU were inserted if necessary to maintain an optimal size of HDAd vector for encapsidation. All of the HDAds were rescued and amplified in 293Cre4 cells then purified as described (24, 25). Briefly, 293Cre4 cells were transfected with Pmel-digested linearized HDAd plasmid, then co-infected with an Ad2-based helper virus that supplied all viral functions in trans. The packaging signal of the helper virus was flanked by loxP sites, enabling excision of the packaging signal by the Cre recombinase in 293Cre4 cells, and preventing encapsidation of the helper virus. Serial co- infection of 293Cre4 cells with the HDAd and helper virus preferentially amplified the HDAd. The yields with all HDAds described here were similar to those obtained with conventional first generation Ad vectors. The final purified vector concentration (particles per ml) was determined as described (24).

DNA transfections

Target cells were grown in 24-well plates to near confluence. Cells were transfected in duplicate using Lipofectamine PLUS (Invitrogen) according to the manufacturer's instructions. Each well was transfected with 0.8 micrograms of the indicated MGB-reporter plasmid and 0.01 micrograms of pCMV-beta gal to normalize for transfection efficiency. Additional wells were also transfected with 0.8 micrograms of the promoterless pGL3 plasmid alone. Cells were harvested 48 hours after transfection in 80 microliters of Tropix lysis buffer (Tropix, Applied Biosystems). An equivalent volume of lysate from each cell line was used to assay luciferase and beta- galactosidase activity. Luciferase activity was determined using the Applied Biosystems Luciferase Assay Kit. Beta-galactosidase activity was determined using the GalactoLight chemiluminescent assay system (Applied Biosystems). Both chemiluminescent assays were detected using the Tropix TR717 Microplate luminometer.

Virus infections

Cells were plated in culture dishes as indicated. When confluent, cells were infected with Ad vectors as indicated in duplicate or triplicate. 48 hours (or 72 hours for experiment described in Fig 5b) post-infection, cells were harvested in 80 microliters of Tropix lysis buffer and assayed as described above. Where indicated, parallel infections were performed using a first generation AdMCMV-Luc vector at the same MOI.

While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. REFERENCES

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Claims

WE CLAIM:

1. An isolated nucleic acid molecule comprising a mammalian mammaglobin gene promoter.

2. An isolated nucleic acid molecule according to claim 1 wherein the promoter is a human promoter.

3. An isolated nucleic acid molecule according to claim 1 or 2, wherein the nucleic acid molecule comprises a sequence greater than 1 kb upstream of the translational or transcriptional start site of the mammaglobin gene.

4. An isolated nucleic acid molecule according to any one of claims 1 to 3, wherein the promoter provides tissue-specific expression.

5. An isolated nucleic acid molecule according to claim 4, wherein the tissue- specific expression is breast-specific or tumour-specific.

6. An isolated nucleic acid molecule according to any one of claims 1 to 5 wherein the promoter promotes transcription of a protein or RNA coding sequence operatively linked to the promoter.

7. An isolated nucleic acid molecule according to any one of claims 1 to 6 wherein the promoter comprises a nucleic acid sequence as shown in SEQ.ID.NO.1.

8. An isolated nucleic acid molecule according to any one of claims 1 to 6 wherein the promoter comprises a portion of a nucleic acid sequence shown in SEQ.ID.NO.1 , wherein the portion promotes transcription of a protein or RNA coding sequence operatively linked to the portion.

9. An isolated nucleic acid molecule according to any one of claims 1 to 6 wherein the promoter comprises a nucleic acid sequence selected from the group consisting of nucleotides 1 to 17070, nucleotides 1 to 8360, nucleotides 1 to 8210, nucleotides 1 to 7270, nucleotides 1 to 4745, nucleotides 1 to 3253, nucleotides 1 to 2431 , nucleotides 1 to 963, nucleotides 1 to 879, nucleotides 1 to 344, nucleotides 1 to 6944, nucleotides 1 to 6291 , nucleotides 1 to 5535, nucleotides 3230 to 8210, nucleotides 3945 to 8210, nucleotides 4425 to 8210, nucleotides 2064 to

8210, nucleotides 6994 to 8210, nucleotides 1 to 5158, nucleotides 1 to 4424, or nucleotides 4424 to 5535 as shown in SEQ.ID.NO.1. [.

10. An isolated nucleic acid molecule according to claim 1 to 9 wherein the nucleic acid sequence comprises

(a) all or part of SEQ ID NO:1 or a nucleic acid sequence that is complimentary to SEQ ID NO:1 (b) a nucleic acid sequence that has at least 60% sequence identity to a nucleic acid sequence of (a);

(c) a nucleic acid sequence that is an analog of a nucleic acid sequence of (a) or (b); or

(d) a nucleic acid sequence that hybridizes to a nucleic acid sequence of (a), (b) or (c) under stringent hybridization conditions.

11. A recombinant nucleic acid molecule comprising an isolated nucleic acid of any one of claims 1 to 10, operatively linked to a protein or RNA coding sequence, wherein the protein or RNA coding sequence is under the transcriptional control of the promoter region.

12. The recombinant nucleic acid molecule of claim 11 wherein the protein or RNA coding sequence is selected from the group consisting of suicide genes, proapoptotic genes, immunomodulatory genes, anti-sense RNA genes and other genes which are toxic or detrimental when expressed in normal cells.

13.The recombinant nucleic acid molecule of claim 12 where the immunomodulatory gene is interleukin-2 or interleukin-12.

14. A method of expressing a nucleic acid coding region in a host cell, the method comprising:

(a) introducing the recombinant nucleic acid molecule of claims 11 , 12 or 13 into a host cell; and

(b) expressing the coding region.

15. The method according to claim14wherein the recombinant nucleic acid molecule is dispersed in a pharmaceutical composition.

16. The method according to claim 14 or 15, wherein the recombinant nucleic acid molecule is encapsulated in a liposome or introduced by electroporation.

17. A vector comprising the recombinant nucleic acid molecule of claim 11 , 12 or 13.

18. The vector according to claim 17, wherein the vector is selected from the group consisting of adeno-associated viruses, lentiviruses, retroviruses, herpesviruses, pox viruses.

19. The vector according to claim 17, wherein the vector is an adenovirus vector or a helper-dependent adenovirus vector.

20. The vector according to any one of claims 17 to 19, wherein the vector is adapted for transfer to a eukaryotic host cell.

21.A host cell transformed with the vector of any one of claims 17 to 20.

22. A host cell of claim 21 selected from the group consisting of mammary cells, breast cancer cells, prostate cancer cells, colon cancer cells, osteosarcoma cells, bladder cancer cells and other tumour cells.

23. A method of expressing a nucleic acid coding region in a host cell, the method comprising: (a) introducing the vector of any one of claims 17 to 20 into a host cell; and

(b) maintaining the host cell under conditions effective to allow expression of the coding region.

24. The method according to claim 23, wherein the recombinant vector is dispersed in a pharmaceutical composition.

25. The method according to claim 23 or 24, wherein the recombinant vector is encapsulated in a liposome or introduced by electroporation.

26. The method according to claim 23-25, wherein the vector is selected from the group consisting of adeno-associated viruses, lentiviruses, retroviruses, herpesviruses, pox viruses.

27. The method according to claim 26, wherein the recombinant vector is an adenovirus or helper-dependent adenovirus.

28. A method of treating cancer in a mammal, comprising administering to the mammal an effective amount of the vector according to any one of claims

17-20.

29. The method according to claim 28 wherein the cancer is breast cancer, prostate cancer, colon cancer, an osteosarcoma, bladder cancer or ovarian cancer.

30. A method according to any one of claims 28 to 29 wherein the cancer is metastatic.

31. A method according to any one of claims 28 to 30 wherein the mammal is a human.

32. A method for gene therapy for cancer in a mammal, comprising the step of delivering the mammal an effective amount of a vector according to any one of claims 17 to 20.

33. The method according to claim 32 wherein the cancer is breast cancer, prostate cancer, colon cancer, an osteosarcoma, bladder cancer or ovarian cancer.

34. A method according to claims 32 to 33, wherein the cancer is metastatic.

35. A method of claims 32 to 34, wherein the tissue is human.

36. A method of treating cancer, comprising administering to a mammal having that condition the recombinant nucleic acid according to claims 11 , 12 or 13.

37. The method according to claim 36 wherein the cancer is breast cancer.

38. The method according to claim 36 wherein the cancer is prostate cancer.

39. The method according to claim 36 wherein the cancer is colon cancer.

40. The method according to claim 36 wherein the cancer is an osteosarcoma.

41. The method according to claim 36 wherein the cancer is bladder cancer.

42. The method according to claim 36 wherein the cancer is ovarian cancer.

43. A method according to any one of claims 36 to 42 wherein the cancer is metastatic.

44. A method according to any one of claims 36 to 43 wherein the mammal is a human.

45. A method for gene therapy for cancer, comprising the step of delivering a vector according to claims 17 or 20 to an affected tissue.

46. The method according to claim 45 wherein the cancer is breast cancer, prostate cancer, colon cancer, osteosarcoma, bladder cancer or ovarian cancer.

47. A method according to claims 45 to 46, wherein the cancer is metastatic.

48. A vector comprising replication genes under the control of an isolated nucleic acid of any one of claims 1 to 10.

49. The vector of claim 48 comprising an adenovirus vector wherein the replication genes comprise E1A or E1 B.

50. An adenovirus of claims 48 or 49, further comprising a recombinant nucleic acid according to claims 11 or 12.

51. A method of treating cancer in a mammal, comprising administering to the mammal an effective amount of the vector according to any one of claims

49 to 50.

52. The method according to claim 51 wherein the cancer is breast cancer, prostate cancer, colon cancer, osteosarcoma, bladder cancer or ovarian cancer.

53. A method according to claims 51 or 52 wherein the cancer is metastatic.

54. A transgenic non-human mammal carrying the vector according to claims 17 to 20.

55. The transgenic non-human mammal of claim 54 carrying the vector in one or more tissues or tumors derived from one or more tissues or organs.

56. The transgenic non-human mammal of claim 55 where the tissue is mammary tissue or a tumor derived from mammary tissue.

57. The transgenic non-human mammal of claim 55 where the tissue is prostate tissue or a tumor derived from prostate tissue.

58. The transgenic non-human mammal of claim 55 where the tissue is colon tissue or a tumor derived from colon tissue.

59. The transgenic non-human mammal of claim 55 where the tissue is bone tissue or a tumor derived from bone tissue.

60. The transgenic non-human mammal of claim 55 where the tissue is bladder tissue or a tumor derived from bladder tissue.

61. The non-human mammal of claim 54-60, which is a rodent.

62. The non-human mammal of claim 61 , which is a mouse.

63. The non-human mammal of claims 54-62, for use in screening for RNA or protein coding genes useful in the treatment of cancer.

64. Use of a promoter, recombinant nucleic acid molecule vector according to any of claims 1 to 13, 17 to 20 or 48 to 50 for preparation of a medicament.

65. Use of a promoter or vector according to any of claims 1 to 13, 17 to 20 or 48 to 50 as a pharmaceutical substance.

66. Use of a promoter or vector according to any of claims 1 to 13, 17 to 20 or 48 to 50 for treatment of cancer or reduction of cell proliferation.