WO1993015743A1 - Novel gene therapies employing antisense constructs - Google Patents

Abstract

Modulation of the growth of cells is accomplished by contacting the cells with a vector capable of carrying genetic material into the cells. The genetic material codes for a nucleolar antigen or is antisense to genetic material coding for a nucleolar antigen. Portions of such genetic material, less than the whole, are also effective in certain preferred embodiments. In accordance with preferred embodiments, antisense genetic material, preferably cDNA, to that coding for p120 is used to inhibit growth of human breast carcinoma cells.

Description

NOVEL GENE THERAPIES EMPLOYING ANTISENSE CONSTRUCTS

ACKNOWLEDGEMENTS

Portions of this work may have been supported by the Cancer Research Center, grant PHS-10893, PI, awarded by the National Cancer Institute, Department of Human Services, USPHS.

FIELD OF THE INVENTION This invention is directed to new methods for modulating disease, especially hyperproliferative diseases, through gene therapy employing antisense constructs.

BACKGROUND OF THE INVENTION It has long been known that many diseases in animals are associated with the production of abnormal proteins or by the production of normal proteins in abnormal proportions or amounts. Most pharmaceutical approaches to therapeutics for such diseases are directed at interference with the production of such proteins or interference with their activity. These approaches, while beneficial, have limited applicability. Many diseases are not amenable to such therapeutic approaches, others have been attended by forms of drug resistance, and still others are met by problems of toxicity and intolerance. It has long been desired to modulate the course of disease through the introduction of functioning genetic material into cells to cause modification or alteration of their growth or to effect an alteration of their functioning. Recently, a form of gene therapy has been reported for the treatment of disease linked to an enzyme deficiency. Severe combined immunodeficiency disease due to adenosine deaminase deficiency was treated in human subjects by administering to the subjects autologous lymphocytes transduced with a human adenosine deaminase gene. See, e.g., . French Anderson et al. Human Gene Therapy, 1: 331-362 (1990); and Kantoff et al. Expression of Human Adenosine Deaminase in Non-Human Primates After Retrovirus-Mediated Gene Transfer, J . Exp. Med. 166; 219-234 (1987) . The French Anderson study suggests that diseases which are related to certain protein deficiencies may be treated through gene therapy. There is no evidence that diseases characterized by other etiologies can be treated by such techniques however. In particular, there is no gene therapy for the treatment of diseases characterized by the production of abnormal proteins or of proteins produced in excessive proportions or amounts.

Accordingly, there is a long-felt, but unfulfilled, need for therapeutic methodologies and compositions which operate through novel mechanisms to effect interference with disease states. In particular, it is greatly to be desired to provide compositions and methods which introduce genetic material into cells to cause the production of species which interfere with the synthesis or effect of proteins implicated in disease. It is especially desired to provide such methods and materials which can interfere with hyperproliterative diseases, especially cancers, through the harnessing of cellular apparatus to produce inhibitory proteinaceous species.

OBJECTS OF THE INVENTION It is an object of this invention to provide methods to effect gene therapy for interference with disease states characterized by the production of abnormal proteins or of normal proteins in excessive proportions or amounts.

It is a further object of this invention to provide compositions and materials suitable for the performance of such methods. Yet another object is to provide vectors for introduction into cells, which vectors carry genetic information for transcription in the cells leading to the production of therapeutic peptides. Still another object is to provide therapeutic, diagnostic, and researc reagents for introduction into cells. A further ob_ject of the invention is to interfere with the growth of cells such as cancerous cells, especially human breast carcinoma, through the introduction into such cells of genetic material coding for a nucleolar antigen, its antisense analog, or a portion thereof.

Still other objects should be apparent to persons of ordinary skill in the art from a review of the instant specification and appended claims.

SUMMARY OF THE INVENTION

The present invention provides methods fsr modulating the growth of ^ells comprising contacting the cells with a vector capable of inserting genetic material into the cells. In accordance with the invention, the vector contains material coding for a nucleolar antigen, its antisense analog, or a portion thereof. In accordance with preferred embodiments, the vector is a retroviral vector.

It is preferrec that the genetic material code for an antisense analog to a nucleolar antigen. The resulting incorporation of genetic material produces a product which effects inhibition of cell growth. In ace^* dance with a preferred embodiment, the genetic ma...rial iε NA coding for antisense constructs. In accordance with other preferred embodiments, the methods inhibit the growth of hyperproliferative cells through the insertion of genetic material into the cells coding for antisense product to a nucleolar antigen or a portion thereof. A preferred nucleolar antigen is pl20. When antisense pl20 is coded for by the genetic material to be inserted, the materials and methods of t invention may be particularly directed to the treatment . f cancerous disease, especially human breast carcinoma. In accordance with other embodiments of the present invention, the growth of cells may be enhanced through the introduction into the cells of the vector comprising a portion, less than the whole, of a gene coding for a protein essential to growth of the cells. It is preferred that such proteins be nucleolar antigens, especially pl20.

BRIEF DESCRIPTION OF THE DRAWINGS Figures 1A and IB are drawings depicting pSVX and

PMSG vectors containing pl20 cDNA in its sense or antisense sequences.

Figure 2 is a photograph of a Southern blot of the total DNA from NIH/3T3 clones and estimation of the gene copy number.

Figure 3 is a Northern blot evaluation to determine expression of constructs.

Figure 4 shows the immunofluorescence detection of antigen pl20 in transfected NIH/3T3 cells. Figure 5 is a Western blot of nucleolar antigen pl20 in transfected NIH/3T3 whole cell extracts.

Figure 6 depicts growth of transfected NIH/3T3 clones in complete or serum-free medium.

Figure 7 shows the growth of transfected NIH/3T3 clones on top of the contact inhibited NIH/3T3 monolayer.

Figure 8 depicts growth of transfected NIH/3T3 cells in soft agarose.

Figure 9 shows tumor growth in nude mice. Figure 10 shows the effect of pl20 antisense constructs on transformed NIH/3T3pSVX120 cells.

DETAILED DESCRIPTION OF THE INVENTION

One of the major characteristics of cancer cells is their abnormal, pleomorphic nucleoli. Studies with a monoclonal antibody to the pl20 nucleolar antigen, MAbpl20, have shown positive nucleolar fluorescence in pleomorphic nucleoli of many human cancers but not in most normal resting human cells. Busch, H. The Final Common Pathway of Cancer: Presidential Address, Cancer Res . 50:4830-4838 (1990); Freeman, J. W. , Busch, R. K. , Gyorkey, F. , Gyorkey, P., Ross, B. E., Busch, H. Identification and characterization of a human proliferation-associated nucleolar antigen with a molecular weight of 120,000 expressed in early Gl phase. Cancer Res. 48:1244-1251 (1988). Additional studies have indicated that pl20 is a proliferation-associated nucleolar antigen which is visualized in the early Gl phase of the cell cycle and peaks in S phase. Fonagy, A., Wilson, A., Busch, H. , and Freeman, J.W., Antisense Mediated Specific Inhibition of pl20 Protein Expression and Cell Proliferation, Proc Am. Assoc. Cancer Res. 32:1642 (1991).

A recent clinical study showed that increased survival of breast cancer patients correlated with reduced amounts of the pl20 protein and that the pl20 protein is a prognostic marker in such cases. Busch, H. , et al., supra , (1990); Freeman, J. W. , McGrath, P., Bondada, V., Selliah, N. , Ownby, H. , Maloney, T. , Busch, R. K. , Busch, H. Prognostic Significance of Proliferation Associated Nucleolar Antigen pl20 in Human Breast Carcinoma, Cancer Res. 51:1973-1978 (1991) . It has been found that the pl20 protein was localized to a beaded microfibrillar network of the nucleolus, suggesting that the pl20 antigen is a component of the nucleolar matrix of the highly pleomorphic nucleoli of cancer cells.

Multiple overlapping cDNA clones for pl20 were isolated and sequenced; the genomic DNA sequence was also determined. Busch, H. , et al., supra , (1990); Fonagy, A., Henning, D. , Jhiang, S., Haidar, M. , Busch, R. K. , Larson, R. , Valdez, B. , and Busch, H. , Cloning of the cDNA and Sequence of the Human Proliferating-Cell Nucleolar Protein pl20, Cancer Commun. 1:243-245 (1989); Larson, R. G., Henning, D. , Haidar, M. A., Jhiang, S., Lin, W. L. , Zhang, W. W. , and Busch, H. Genomic Structure of the Human Proliferating Cell Nucleolar Protein pl20, Cancer Commun. 2:63-71 (1990). While much has been learned about pl20 protein, the exact function of the human pl20 protein is not known. Without wishing to be bound by any particular theory, it is possible that it may function as an oncogene.

It is now believed possible to modulate the growth of cells by contacting the cells with a vector capable of inserting genetic material into the cells. In accordance with certain embodiments of the invention, the genetic material contained within the vector, which will subsequently be expressed by the cells codes for a nucleolar antigen. In accordance with other embodiments, an antisense analog or construct corresponding to genetic material coding for a nucleolar antigen, or a portion thereof is used.

While a number of types of cells may benefit from practice of the present invention, it is preferred that the cells be hyperproliferative. The family of hyperproliferative cells and the conditions or diseases associated therewith are well-known to persons of ordinary skill in the art. Noteworthy among such diseases are various forms of cancer. It is believed to be particularly useful to employ the compositions and methods of the present invention in the treatment of cancerous disease, especially carcinomas. In accordance with preferred embodiments, the methods and materials of the present invention are directed to human breast carcinoma to effect interference with its development or spread. It has been found that in predictive animal models, the growth of human breast carcinoma can be halted. The present invention is not so limited, however, and a wide variety of cancers and other hyperproliferative diseases may be treated through practice of the present invention. It is also possible to employ the materials and methods of the present invention to encourage the growth of cells. Thus, it has been found that when cells are contacted by vectors containing genetic material coding for nucleolar antigens, that the development of such cells can be encouraged. This has been found to be true for human breast carcinoma in predictive animal models. Accordingly, the present invention is not limited to the interference with cellular proliferation, since such development may, in the alternative, be encouraged.

The vectors which are to be used in accordance with the present invention are best defined by their functionality. The term vector is well understood by persons of ordinary skill in the art to be a cellular or sub-cellular moiety which is, at once, capable of being transfected into cells while carrying genetic material within itself which is foreign to the cells to be transfected. It is understood that not all vectors are capable of effective transfection into all cell lines or to all species of animal. THUS, persons of ordinary skill in the art will know to select vectors which are capable of transfection into the cells of interest while carrying the foreign genetic material with the result that the transfected cells are capable of expressing the foreign genetic material. Vectors which are useful in practicing the processes of the present invention are, therefore, best defined by their function. Such vectors are said to be capable of inserting genetic material into the cells whose growth is to be modulated or interfered with in a fashion such that the genetic material can be expressed. At present, a number of vectors are viewed as being exemplary and preferred for practice of embodiments of this invention.

A number of vector types and vectors are known for use in the introduction of genes into mammalian cells, especially human cells. See in this regard Eglitis, M.A. and French Anderson, W. Retroviral Vectors for Introduction of Genes into Mammalian Cells, Bio Techniques , 6:608-613 (1988). The foregoing reference of Eglitis and French Anderson is directed to retroviral vectors for such use. Indeed, retroviral vectors are generally preferred for performance of preferred embodiments of the present invention, although other forms of vectors may also be useful. Among the retroviral vectors which may be so used is N2 and vectors derived from N2, which vectors have been used by French Anderson in the therapy described hereinabove. It is to be understood that the basic vector into which genetic material is to be introduced for subsequent transfection into the cells whose activity is to be modulated is a matter of choice to be exercised by persons of ordinary skill in the art. At present, preferred vectors include pSVX, N2, pLNCX, pLXSN, pMSG, pMAM, pCEP4, pMEP4 and pREP4. It is preferred that the vector be pSVX or an N2-derived vector.

Genetic material to be incorporated into the vector for subsequent transfection into cells is incorporated generally in accordance with standard procedures. Thus, the methodology set forth in Eglitis and French Anderson, Supra . for augmenting the genetic material of the basic vectors with foreign genetic material for subsequent insertion into mammalian cells may be employed and adapted by persons of ordinary skill in the art by practicing the present invention. Other methodologies for incorporating genetic material into vectors may be used as well.

In accordance with the invention, genetic material is included within the vectors, which genetic material is incorporated into the cells when the cells are transfected with the vector. Subsequent transcription of the genetic material modulates the growth of the cells. The genetic material for inclusion in the vectors is genetic material which codes for nucleolar antigen, its antisense analog or counterpart, or a portion thereof. Nucleolar antigens are known per se to be immunogenic proteins found in the nucleolus.

It is believed that when genetic material, especially cDNA 3T3 cells, coding for other nucleolar antigens is incorporated into cells, proliferation can be enhanced. A principal object of the present invention is to interfere with the growth of cells, however. Accordingly, it has been found that when antisense cDNA of the pl20 genome is incorporated into a vector and human breast carcinoma cells transfected with the vector, that the proliferation of the cells is significantly interfered with; therapeutic effect upon the carcinoma is demonstrated. It has also been found that portions of the genetic material coding for a nucleolar antigen or of the antisense counterpart or analog thereto, less than the whole, may also be useful for incorporation into vectors for modulation of the growth of cells. This effect is entirely unexpected. Thus, genetic material, such as antisense cDNA for portions of the gene for nucleolar antigen pl20, has been found to interfere with proliferation of human breast carcinoma.

The term "antisense" as applied to genetic material is known to persons of ordinary skill in the art to relate to an oligonucleotide which is Watson-Crick complementary to a given gene sequence. Accordingly, for cDNA, an antisense analog to a genetic strand would substitute Watson- rick counterpart nucleotides for each original nucleotide in the sequence. As applied to the present invention, the antisense analog to geneti: material coding for nucleolar antigen is constructed in just that way. Such construction may be accomplished conveniently using automated, solid state synthetic techniques using equipment which is commercially available such as that which is supplied by the Applied Biosystems Corporation of California.

In accordance with practice of preferred embodiments of the present invention, it is preferred that all or a portion of the antisense counterpart to genetic material coding for a nucleolar antigen, especially pl20, be incorporated into vectors for use in the transfection of cells to inhibit their growth. Sequence ID Listing No. 1 hereto sets forth the sequence listinr of the complementary DNA, cDNA, coding for nucleolar antigen pl20. Further details of this structure are set forth in Larson et al.. Supra. (1990). Sequence ID Listing No. 2 is the cDNA sequence of the antisense counterpart to the pl20 genome of Sequence ID Listing No. 1 with the 5' untranslated region. Sequence ID Listing No. 3 sets forth a portion of the antisense cDNA counterpart of Sequence ID Listing No. 2 at the 3¹ untranslated region, which portion has been found to be useful for incorporation into vectors in accordance with the process of the present invention to effect inhibition of the growth of hyperproliferative cells, specifically human breast carcinoma cells. Sequence ID Listing No. 4 is antisense to the B region of pl20, while Sequence ID Listing No. 5 is antisense at the 5' untranslated region of the gene. Other portions of the antisense counterpart to the pl20 cDNA may also be useful, especially those which contain the 3 ' or 5' untranslated portion or the "B" region.

It is to be understood that any portion which is effective in inhibiting the growth of cells in accordance with the teachings of the present invention may be useful for its practice. It is contemplated that portions of the gene coding for pl20, other than the ones identified to date, will also find utility in the practice of one or more embodiments of this invention. It is also believed that antisense constructs and portions thereof corresponding to the genetic material coding for other nucleolar antigens will be similarly useful for inhibiting the proliferation, development or growth of cells. It is anticipated that a wide variety of hyperproliferative cells and diseases may be inhibited by employing such antisense constructs and effective portions.

While the genetic material which is preferred for use in accordance with the present invention is cDNA, other genetic forms including native DNA, RNA and artificial oligonucleotides which are similar in structure and effect to "wild type" nucleic acids may be so employed. The effective requirement is that the genetic material be capable of being incorporated into vectors which, in turn, may be effectively transfected into mammalian cells and that such cells be capable of expressing the genetic material thus introduced.

Persons of ordinary skill in the art will have no difficulty in determining appropriate conditions for accomplishing the incorporation of the selected genetic material into the vectors selected for use with the particular mammalian cells to be treated. Indeed, many such systems have been reported. Similarly, selection of appropriate conditions for transfection of those cells with the vectors is within the routine abilities of persons of ordinary skill in the art. For example, the protocol set forth in French Anderson in Human Gene Therapy (1990) Supra, for clinical use of gene therapy may be adapted for application to the present invention. Analogous protocols for other mammalian species may also be employed.

While the present methods and materials are believed to be general in scope, applicability of those methods and materials have been demonstrated by reference to a particular nucleolar antigen, pl20 and a disease which is clearly related to expression of pl20, human breast carcinoma.

Several constructs were prepared using the pSVX vector for transfection into NIH/3T3 cells; these constructs contained the complete pl20 cDNA in sense (forward; pSVX120) and antisense (reverse; pSVX021) orientations with respect to the LTR. Following electroporation into NIH/3T3 cells and selection of the clones, 1 - 2 copies of the plasmids were present per cell. Northern blots using labeled pl20 riboprobes indicated that the sense pl20 and the antisense pl20 transcripts were produced. The presence of pl20 and p021 mRNA was further confirmed by RNAse protection assay (data not shown) . Cells containing the cDNA in the sense orientation produced human pl20 which localized to the nucleolus as shown by indirect immunofluorescence; the pl20 protein was also shown by Western blot analysis to be present in whole-cell extracts.

Frequently, transformed cells have lower serum dependence than their normal counterparts. Their properties are associated with in vitro transformation and are related to changes in growth characteristics, genetic properties and neoplastic properties. The anchorage-independent growth of NIH/3T3pSVX120 cells and their cytomorphological changes are characteristic of a transformed phenotype, suggesting that the pl20 constructs might function like an oncogene. Similar anchorage-independent and serum-independent growth has been found in NIH/3T3 cells transformed with the ras oncogene or

• the hhcM oncogene derived from a human hepatocellular carcinoma. See Sugimoto, et al., Thy-1 as a Negative Growth Regulator in ras-Transformed Mouse Fibroblasts, Cancer Res . , 51: 99-104 (1991) and Yang, et al. , A Human Hepatocellular Carcinoma 3.0 Kilobases DNA Sequence Transforms Both Rat Liver Cells and NIH3T3 Fibroblasts and Encodes a 52 KiloDalton Protein, Cancer Res. 50(Suppl.): 5658s-5667s (1990).

The transfected pl20 cDNA in the sense orientation resulted in loss of contact inhibition in monolayers and colony formation in soft agarose. Neither the control, pSVX vector nor the antisense, pSVX021 produced these effects. In vivo studies on Hsd:Athymic Nude-nu male mice showed that the cells transfected with pl20 in the sense orientation produced rapidly growing solid tumors. These tumors were visible one week following the s.c. transplantation. Non-transfected NIH/3T3 cells or transfected NIH/3T3 cells with the vector alone produced tumors that grew more slowly.

The presence of the antisense, pSVX021 construct in NIH/3T3 cells markedly delayed tumor growth when compared with the vector alone and with the pl20 in the sense orientation. The slower growth of the antisense pl20 containing NIH/3T3 cells (NIH/3T3pSVX021) (Figs. 6 and 9) may result from effects on the NIH/3T3 mouse pl20 mRNA. It has been estimated that there is a 77 per cent nucleotide similarity between the human and mouse pl20 cDNA. Although there have been many reports on the use of antisense molecules to affect gene expression, it has now been shown that the whole antisense construct reduced the growth rate of these cells in vivo . The growth of pl20-containing cells was markedly inhibited by transfection of the antisense pl20 construct (pMSG021) and was inhibited even more by dexamethasone stimulation.

The mechanism of the increased growth rate of the tumors and cells transformed by the pl20 sense construct has not yet been shown with certainty. Without wishing to be bound by any particular theory, it may be that the overproduction of the pl20 protein activates other genes or accelerates other cellular growth events. The growth inhibitory effect of the antisense construct appears to be particularly significant, suggesting the use of such antisense oligonucleotides in cancer treatment.

Oligonucleotides designed to hybridize to specific mRNA sequences have been utilized to inhibit the expression of specific proteins. Antisense oligonucleotides have been used successfully to inhibit oncogenes such as c-myc or c-myb. Wickstrom, E.L, Sandgren, E. , Bacon, T. , Wickstrom, E., Werking, C, Brinster, R. , Antisense DNA Methyl phosphonate Inhibition of c-myc Gene Expression in Transgenic Mice, Proc Am. Assoc. Cancer Res. 32:2550 (1991); Melani, C. , Rivoltini, L. , Parmiani, G. , Calabretta, B. , and Colombo, M.P., Inhibition of Proliferation by c-myb Antisense Oligodeoxynucleotides in Colon Adenocarcinoma Cell Lines That Express c-myb. Cancer Res. 51:2897-2901 (1991). Fonagy et al. in Fonagy, A. , Wilson, A. , Busch, H. , and Freeman, J.W. , Antisense Mediated Specific Inhibition of pl20 Protein Expression and Cell Proliferation, Proc Am. Assoc. Cancer Res. 32:1642 (1991) demonstrated inhibition of pl20 protein expression and cell proliferation with an antisense oligonucleotide in a human lymphocyte system in vitro .

Since the sense pl20 protein increased cell proliferation and malignant transformation of normal NIH/3T3 cells, and the antisense pl20 inhibited the increased cell growth and returned the pl20-transformed cell line to its normal phenotype after dexamethasone induction, antisense pl20 oligonucleotide molecules are believed to be useful as therapeutic anticancer agents.

The invention is further pointed out by reference to the following, non-limiting examples.

EXAMPLE 1

Cloning of pl20 cDNA Into an Expression Vector:

The plasmid pET120, which contained the pl20 cDNA prepared. in accordance with Valdez, B. C. , Busch, R. K. , and Busch, H. Phosphorylation of the human cell proliferation- associated nucleolar protein pl20, Biochem. Biophys. Res. Commun. 173:423-430 (1990) was cut with Ncol and Sspl, and then treated with Klenow DNA polymerase I. The two fragments were separated on 1% agarose gel and the 3.0 kb pl20 cDNA excised and purified using the Geneclean kit of Bio 101, Inc.. An expression vector, pSVX, that contains the neomycin resistance gene (Cepko, C. L. , Roberts, B. E. , and Mulligan, R. C. Construction and applications of a highly transmissible murine retrovirus shuttle vector. Cell 37:1053-1062 (1984)), was linearized with BamHI and blunt ended with Klenow fragment. The purified pl20 cDNA fragment was ligated with the linearized pSVX. Insertion of the pl20 cDNA in the BamHI site of pSVX increases pl20 expression under the control of Moloney murine leukemia virus LTR. Figure la is a schematic description of the pSVX vector depicting the insertion site for the cDNA into the BamHI site. The orientation of the insert was determined by Hindlll digestion. Clones containing the pl20 cDNA in the sense direction are referred to as pSVXl20. The "antisense" clones contain the reverse orientation of the pl20 cDNA (pSVX021) . The orientation is with respect to the upstream LTR of the pSVX vector.

Another construct was made using the pMSG vector. The full-length pl20 cDNA in reverse orientation was cloned downstream of the MMTV-LTR and was designated as pMSG021. Figure lb is a schematic showing a pMSG vector having the full-length pl20 cDNA in reverse orientation cloned downstream of the MMTV-LTR, designated pMSG021. These pMSG constructs have dexamethasone-inducible promoters and gpt-selection genes.

EXAMPLE 2

Transfection by Electroporation

Logarithmic-phase growth cells were harvested with trypsin/EDTA, centrifuged at 800 rpm for 5 min in a Fischer Centrific centrifuge, and washed in PBS (lOmM phosphate, 150 mM NaCl, pH 7.2). 3x106 cells/ml were resuspended in lx HeBS (20 mM Hepes pH 7.05, 137 mM NaCl, 0.5 mM KC1, 0.7 mM Na₂HP0₄, 6 mM dextrose) containing 500 μg/ l of sonicated salmon testis DNA (Sigma) . The pSVX or MSG plasmid constructs (20 μg/ml DNA) were then added. The cells were exposed to a single voltage pulse (220 V, 960 μF, Gene-Pulser, BIO-RAD) at room temperature, allowed to remain in the buffer for 10 min and then plated onto 10 cm cell culture dishes (Falcon) . The optimal parameters of electroporation (220 V, 960 uF, single pulse) were determined previously for NIH/3T3 cells by colony-forming assays (cell killing) and MAbpl20 immunostaining (gene transfer) . The sense pl20 (pSVXl20) , the antisense pl20 (pSVX021) or the pSVX vector alone were electroporated into NIH/3T3 cells.

EXAMPLE 3 Selecting Media The pSVX plasmid and the pSVX120 or the pSVX021 constructs contained a Neo gene. Geneticin^® (G418 sulfate) containing D-MEM medium was used for cell selection. The Geneticin^® concentration of 600 μg/ml active at 10^" surviving fraction was determined by colony formation. The pMSG plasmid and the antisense pl20 pMSG constructs (pMSG021) contained the gpt-selection gene. The D-MEM-gpt selecting medium contained 250 μg/ml xanthine, 25 μg/ml mycophenolic acid, 2 μg/ml aminopterin 10 μg/ml thymidine, and 15 μg/ml hypoxanthine.

EXAMPLE 4

DNA Isolation and Analysis

Total DNA was extracted from monolayer cells in accordance with the procedure set forth in Sambrook, J. , Fritsch, E.F., and Maniatis, T. Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, 2nd Ed. pp.9.16-9.19 (1989) using an end-sealed and U-shaped Pasteur pipette to spool the DNA. The DNA was resuspended in TE buffer (lOmM Tris-HCl pH 8.0, ImM EDTA) and incubated with 20 μg/ml RNase at 37°C for 2 hours. The sample was made to 0.5% SDS and treated with 100 μg/ml proteinase K at 50°C for 3 hours. The solution was extracted with phenol equilibrated with 0.5 M Tris-HCl, pH 8.0. The purified DNA was precipitated by addition of 0.1 volume of 3.5 M sodium acetate and 2.5 volumes of ethanol. DNA was digested with restriction enzymes according to reaction conditions recommended by BRL-GIBCO. The DNA fragments were separated on 0.8% agarose gels and transferred to Zeta-Probe blotting membrane (BIO-RAD) . Blotting, prehybridization, hybridization and washing of filters were carried out according to the manufacturer.

EXAMPLE 5

Neomycin Phosphotransferase II (NPT II) , ELISA Assay

The presence of the pSVX constructs in the NIH/3T3 cells was further analyzed by the expression of neomycin phosphotransferase II (NPT II) . NPT II was detected in the transfected cells with an enzyme-linked immunosorbant assay (ELISA) kit (5 Prime - 3 Prime, Inc.). The various transfected cell lines that grew on 10 cm cell culture dishes were scraped and transferred to a conical tube. The pellets were suspended in 200 μl of PBS and were subjected to three freeze (at -70°C) and thaw (at 37°C) cycles, of 10-15 minutes each. The supernatants were collected, stored at -70°C and analyzed for NPT II.

EXAMPLE 6 Protein Blots

The whole cells from transfected and non-transfected NIH/3T3 cells were solubilized in Laemmli buffer and heated at 100°C for 5 minutes. The extracts were loaded on an SDS (0.1%) polyacrylamide (7.5%) gel and electrophoresed for 1 hour at 200V on a BIORAD minigel apparatus. Proteins were transferred to nitrocellulose membrane by the method of Towbin et al.; Towbin, H. , Stahelin, T. , and Gordan, J., Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets procedure and some applications, Proc. Natl . Acad. Sci . USA, 76:4350-4354 (1979). The available binding sites were treated with blocking buffer (10 mM Tris- HC1, pH 7.5, 3% BSA, 150 mM NaCl, 10% chicken serum) . The M >pl20 was added at a 1:400 dilution of ascites in TEST buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.05% Tween-20) , and incubated for 2 hr at room temperature. The second antibody, a phosphatase conjugated goat anti-mouse (Promega) was added at a 1:5000 dilution; incubation was for 1 hr. The band was developed in substrate containing buffer (Promega) ; the reaction was terminated with a 20 mM Tris-HCl, pH 8.0, 2 mM EDTA buffer.

EXAMPLE 7

SNA Preparation and Analysis

Poly(A)+ RNAs were prepared using Fast TrackTM mRNA isolation Kit (Invitrogen Co., San Diego, CA) . Equal amounts of poly(A)+ RNA were denatured and fractionated on a 1.2% agarose gel containing formaldehyde in accordance with Sambrook, J. , Fritsch, E.F., and Maniatis, T. , Molecular Cloning. A Laboratory Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, 2nd Ed. pp.7.43-7.45 (1989), and transferred to Zeta-probe blotting membrane (BIO-RAD) . Sense and antisense hybridization probes were synthetized using an RNA transcription kit (Stratagene) and pBS120 template (pl20 cDNA in the Bluescript vector (Stratagene) ) . Prehybridization and hybridization were done as recommended by the supplier.

EXAMPLE 8

Indirect Immunofluorescence

Asynchronous cells in logarithmic growth-phase were used for immunostaining. The cells were grown on slides, air dried, and fixed in formaldehyde/PBS for 20 minutes. The slides were washed in PBS and the cells permeabilized in acetone at -20 °C for 4 minutes. Anti-pl20 monoclonal (MAbpl20) or polyclonal (PAbpl20) antibodies (1:50 or 1:20 dilution) were added and incubated in a moist chamber at 37°C for 60 minutes according to the method of Freeman, J. W. , Busch, R. K. , Gyorkey, F. , Gyorkey, P., Ross, B. E., Busch, H. Identification and Characterization of a Human Proliferation-Associated Nucleolar Antigen with a Molecular Weight of 120,000 Expressed in Early Gl Phase, Cancer Res . 48:1244-1251 (1988) . The sample was washed three times in PBS for 20 min/wash. The primary antibody was detected with fluorescein-conjugated, goat anti-mouse immunoglobulin (Cappel, dilution: 1:20 in PBS) at 37°C for 35 minutes. The slides were washed three times in PBS and covered with n-propyl gallate containing glycerol-PBS as described in Giloh, H. , and Sedat, J. W. , Fluorescence microscopy: Reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate, Science , 217:1252-1255 (1982).

EXAMPLE 9 Cell Culture

NIH/3T3 (ATCC CRL 1658, contact-inhibited NIH Swiss mouse embryo) cells were cultured in Dulbecco's Modified Eagle Medium (D-MEM) (GIBCO) , supplemented with 10% fetal bovine serum (FBS) (GIBCO) and 1% penicillin-streptomycin liquid (10000 IU/ml penicillin G sodium, 10 mg/ml streptomycin sulphate in 0.85% saline) (GIBCO) . Based on the doubling time and on the three-day plating schedule, 6.5 x 10 exponentially growing cells were serially plated into T-75 cell culture flasks (Falcon) . All cell lines were negative for mycoplasma infection as determined by a DNA stain.

EXAMPLE 10

Colony Formation in Soft Agarose

The NIH/3T3pSVX, NIH/3T3pSVX021, and NIH/3T3pSVX120 cultured clones were trypsinized and 2.5x10 - l.Oxlo'¹ viable cells per dish were suspended in a final agarose concentration of 0.4% and pipetted onto the top of the prepared 0.8% agarose base. The triplicate plates were incubated in a humidified incubator at 37°C for 3-6 weeks. The plates were stained with INT (p-Iodonitrotetrazolium violet, Sigma) and the colonies with a diameter greater than 0.2 mm were counted under a 7x measuring magnifier.

EXAMPLE 11 In Vivo Studies in Nude Mice

The exponentially growing NIH/3T3pSVX120 and control cells (NIH/3T3, NIH/3T3pSVX, NIH3T3pSVX021) were washed and resuspended in serum-free D-MEM medium. 2xl0⁶ viable cells (determined by trypan blue exclusion) , in 0.2 ml D-MEM were injected s.c. into the homozygous mutant, Hsd:Athymic Nude-nu male mice according to the method of Shin, S., Use of nude mice for tumorigenicity testing and mass propagation in Jakoby, W.B. and Pastan, I.H. (eds.). Methods in Enzymology, Vol. LVIII: Cell Culture, pp. 370-379. San Diego, CA: Academic Press, Inc. (1979) . Tumor growth was followed by daily measurement of the three orthogonal diameters (L, W and H) , and volume (V) was calculated by the equation V = π/6 x (L x W x H) . All animal experimentation followed the guidelines of the Baylor College of Medicine and New York Academy of Sciences.

EXAMPLE 12

Presence of the pSVX Recombinant Plasmid in Transfected NIH/3T3 Cells

To determine „_. t the Geneticin^® -resistant NIH/3T3 clones contained the pSVX recombinant plasmids, Southern blot analysis, dot blot hybridization and neomycin phospho¬ transferase II assays vere performed. For the Southern blot analysis, ten micrograms of total DNA were digested to completion with Hindlll (A) or EcoRI (B) , electrophoresed on 0.8% agarose gels, and transferred to Zeta-Probe nylon membranes (BIO-RAD) . The filters were hybridized to randomly-pri »med 32P-labeled pSVX120 whole plasmi■d and washed according to the supplier of the membrane (BIO-RAD) . Lanes for panels A and B, (1) nontransfected NIH/3T3; (2) NIH/3T3pSVX (vector alone transfected) ; (3) NIH/3T3pSVX021 (antisense pl20 construct transfected) ; (4) NIH/3T3pSVX120 (sense pl20 construct transfected) ; (5) 90 picograms pSVX120 plasmid. Size of molecular weight markers (kb) is shown on the left side. Panel C, The gene copy number, was estimated by dot blot hybridization. Each standard pSVX120 plasmid, corresponding to 1-5 copies, was mixed with 10 μg of NIH/3T3 total DNA. Copy number was determined using 10 μg total DNA of each clone and probed with 32P-labeled pSVX120 plasmi.d. The filter was cut and the radioactivity of each dot was determined using a liquid scintillation counter.

The analysis of total DNA from the transfected NIH/3T3 clones digested with restriction enzymes showed the presence of bands that hybri •di■zed wi.th the 32P-labeled pSVXl20 probe as shown in Figures 2A and 2B) . The hybridizing bands from the NIH/3T3pSVX120 clone digested with Hindlll (Fig. 2A, lane 4) or EcoRI (Fig. 2B, lane 4) showed patterns similar to the pSVX120 plasmid digested with the same enzymes (Fig. 2, A and B, lane 5) . This result implies that the plasmid was not integrated into the NIH/3T3 genome. Despite the low gene copy number (Fig. 2C) , the plasmids were retained by the cells after 10 passages. The presence of the SV40 origin of replication (SVori) in the constructs shown in Figure 1A may enable them to replicate as episomes.

Total DNA from NIH/3T3pSVX and NIH/3T3pSVX021 digested with Hindlll or EcoRI had a number of bands that hybridized with the P-labeled pSVX120 probe (Fig. 2, A and B, lanes 2 and 3) . The clones contained 1 - 2 copies of the construct per cell (Fig. 2C) . The presence of the pSVX vector in the clones was further confirmed by the expression of neomycin phosphotransferase II (125-355 pg NPT II / mg total protein) from the neomycin resistance gene.

EXAMPLE 13

Expression of pSVXl20 mRNA in NIH/3T3 Cells: Northern Blots To determine whether the transfected sense or antisense pl20 constructs were expressed in NIH/3T3 cells, poly(A)+ enriched RNA was prepared from pSVX, pSVX021, and pSVX120 clones for Northern blotting. Equal amounts of poly(A)+ enriched RNA were fractionated on agarose gel and hybri •di•zed to a 32P-labeled pl20 ri•boprobe as shown in Fi«gure 3. The sense transcript, 7.5 kb, the length between the two LTRs containing pl20 cDNA, was detected in the RNA from pSVX120-transfected NIH/3T3 cells (Fig. 3B, lane 3), but not in the RNA from pSVX- or pSVX021-transfected NIH/3T3 cells (Fig. 3B, lanes 1 and 2) . Antisense transcripts were detected in the pSVX021 clone (Fig. 3A, lane 2) but not in the pSVX- or pSVX120-transfected NIH/3T3 cells. The shorter transcript (6.5 kb) probably represents a spliced transcript. The 2.8 kb band detected by the pl20 antisense riboprobe in the 3 samples (Fig. 3B) probably represents mouse pl20 mRNA, which is similar in size to the pl20 mRNA from HeLa cells.

EXAMPLE 14

Im unochemical Detection of Antigen pl20

Cells grown on slides were fixed, permeabilized, and analyzed by indirect immunofluorescence using MAbpl20. The NIH/3T3pSVX120 clones exhibited bright nucleolar fluorescence as shown in Figure 4B, indicating the presence of the human pl20 protein. There was no detectable fluorescence in the non-transfeeted NIH/3T3; NIH/3T3pSVX; NIH/3T3pSVX021 clones as shown in Figure 4A, because the MAbpl20 is human specific and does not immunoreact with mouse nucleolar proteins.

Fig. 5 shows the results from Western blot analysis using specific MAbpl20 on a 7.5% Laemmli gel containing whole cell extracts from the different clones. The blot was developed with MAbpl20 and the Promega phosphatase reagents. Lane 1 shows a positive control with HeLa nucleoli; pl20 is the major band. Lane 2 contains the prestained molecular weight markers; the 116 kD marker was juxtaposed to the pl20 band in the HeLa extract. Lane 3 and 4, which did not contain pl20, were whole cell extracts from the NIH/3T3pSVX clone and the NIH/3T3pSVX021 clone, respectively. Lanes 5 and 6 contained whole cell extracts from the NIH/3T3pSVX120 clone. The pl20 bands were clearly seen (arrow) .

EXAMPLE 15 Growth in Complete or Serum-Free Medium

In complete medium, the NIH/3T3, NIH/3T3pSVX or NIH3T3pSVX120 cells grew at similar rates; the population doubling times (PDT) were not significantly different. Approximately 24 hours was required for confluency. The NIH/3T3 and NIH/3T3pSVX cells were contact inhibited by the sixth day after plating. The NIH/3T3pSVX120 transfected clone started to form multiple layers, overgrew from the fifth day after plating, and formed rapidly growing foci. The population doubling time (PDT) for this multiple layered overgrowing phase was 106 hours. The antisense pl20 construct containing, NIH/3T3pSVX021 cells were contact inhibited by day 12, and grew slower than the control; the PDT was 40 hours. See Figure 6A. Although the NIH/3T3pSVX120 cells did not require serum for growth, their growth in serum-free medium was slower than in serum containing medium; the PDT was 115 hours, which is similar to the PDT of NIH/3T3pSVX120 in the overgrowing phase in complete medium. See Figure 6B. The NIH/3T3pSVX and NIH/3T3pSVX021 clones divided only once or twice but no further in the serum-free medium. The NIH/3T3 cells without serum died during the two week period. For the analysis, the cells were trypsinized, stained with Trypan blue and counted. Three parallel dishes were used for each datum point. Panel A, Growth in completed medium. The NIH/3T3pSVX120 clones overgrew and formed multiple layers. NIH/3T3, and NIH/3T3pSVX cells were contact inhibited the sixth day after plating. The NIH/3T3pSVX021 cells were also contact inhibited, however, these cells grew slower. Panel B, Growth in serum-free medium; NIH/3T3pSVXl20 cells did not require serum, however, the growth in serum-free medium was slower.

EXAMPLE 16

Growth on confluent Monolayers Colony formation was observed when the NIH/3T3- pSVX120 transformed cells were plated on top of the contact inhibited NIH/3T3 monolayer as shown in Figure 7B. The colony-forming efficiency was 20 %. The NIH/3T3, NIH/3T3pSVX, or NIH/3T3pSVX021 cells showed no colony formation above the confluent NIH/3T3 monolayer. See Figure 7A which indicates their requirement for anchorage-dependent growth. Figures 7A and 7B are at 90X magnification.

EXAMPLE 17 Growth in Soft Agarose

Transfected NIH/3T3 cells were plated in soft agarose. In three repetitive experiments, 1000, 5000, and 10,000 cells were seeded into three parallel wells. Only the NIH/3T3pSVX120 transformed cells formed colonies that grew progressively to larger than 0.2 mm diameter in soft agarose as shown in Figure 8B. The colony forming efficiency (PE) of the pl20-containing NIH/3T3 cells was 5.6 %. The NIH/3T3, NIH/3T3pSVX, and NIH/3T3pSVX021 cells did not form colonies in semisolid medium (colony forming efficiency, 0.05%), See Figure 8A.

EXAMPLE 18

Tumor Growth in Nude Mice

Studies were designed to assess the in vivo growth characteristics of the three transfected cell lines. In these studies, transfected NIH/3T3 cells were transplanted s.c. into nude mice. Experiments were done in triplicate, using three different batches of each transfected cell line. In each experiment, three or six animals were used in a group. Figure 9 shows that the NIH/3T3pSVX120 cells induced tumors which grew more rapidly than the NIH/3T3pSVX induced tumors. The

NIH/3T3pSVX021 (antisense) induced tumors which grew very slowly; their growth delay was much longer than the NIH/

3T3pSVX- or NIH/3T3pSVX120-induced tumors. The parameters of tumor growth (Table 1) show that the tumor growth delay (TGD) was 12.3 days for the cells containing the antisense construct, and 6.0 - 6.5 days for the pSVX or pSVX120 construct-containing tumors. The tumor growth time (TGT) for the antisense pSVX021 containing tumor was 4 - 6 times greater than for the pSVX or the pSVX120 tumors. The difference in growth was significant (p less than 0.01) . The other reported tests are tumor volume doubling time, TVDT-1, calculated from the first phase of log/linear plot of growth curves; TVDT-2, calculated from the second phase of log/linear plot of growth curves; TG-1, tumor growth rate of the first phase of tumor growth and TG-2, tumor growth rate of the second phase of tumor growth.

TABLE 1

TEST 3T3pSVX

TGD (days) 6.5

TVDT-1 (h) 40

TVDT-2 (h) 90

TGT (days) 16.5

TG-1 (h^"1; X10^"2) 1.7 TG-2 (h^'1; xio^"3) 7.7

EXAMPLE 19

Effect of Antisense pl20 Constructs on Transformed

NIH/3T3pSVX120 Cells. To learn more about the effect of the antisense pl20, thepreviously-characterized, transformedNIH/3T3pSVX120 cells producing human pl20 were transfected by electroporation with the antisense, pMSG021 construct. Figure 10 shows the growth characteristics of the transformed sense NIH/3T3pSVX120 cells transfected with the pMSG vector alone (circles) and the transformed clones after transfection with the antisense pl20 construct (triangles) . For induction, 1 μM dexamethasone in D-MEM medium was used for 48 hours (filled symbols, dashed lines) . The cells were trypsinized, stained with Trypan blue and counted. Three parallel dishes were used for each datum point. The growth rate (G=ln2/DT) of the sense NIH/3T3pSVX120 transfected with the antisense, pMSG021 (open triangles, solid line, G= l.OxlO^-2 hr^-1) , was reduced by 52% compared to the sense 3T3pSVX120 transfected with the pMSG vector alone (open circles, solid line, G=2.1xlθ^"2 hr^"1) . After stimulation with dexamethasone, the growth rate was reduced by 62% (dashed line with filled triangles, G=7.7xlθ^"3 hr^"1) . Northern blot analysis showed a decreased level of pl20 mRNA, the immunofluorescence was also markedly reduced, and the cells returned to their original untransformed phenotype (data not shown) .

SEQUENCE LISTING (1) GENERAL INFORMATION:

(i) APPLICANT: Harris Busch, et al.

(ii) TITLE OF INVENTION: NOVEL GENE THERAPIES EMPLOY ANTISENSE CONSTRUCTS

(iii) NUMBER OF SEQUENCES: 4

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Woodcock Washburn Kurtz Mackiewicz & Nor

(B) STREET: One Liberty Place - 46th Floor

(C) CITY: Philadelphia

(D) STATE: PA

(E) COUNTRY: USA

(F) ZIP: 19103

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: DISKETTE, 3.5 INCH, 1.44 Mb STORAGE

(B) COMPUTER: IBM PS/2

(C) OPERATING SYSTEM: PC-DOS

(D) SOFTWARE: WORDPERFECT 5.0/5.1 (Vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: n/a

(B) FILING DATE: herewith

(C) CLASSIFICATION: (vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE:

(Viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: John W. Caldwell

(B) REGISTRATION NUMBER: 28,937

(C) REFERENCE/DOCKET NUMBER: BAY-0003 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (215) 568-3100

(B) TELEFAX: (215) 568-3439 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2984

(B) TYPE: nucleic acid

(C) ^?VRANDEDNESS: single

(D) !'. POLOGY: linear (iv) i sTTI-SENSE: no

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

CATTTCTGCC TGCCACAGTA CCATGGGGCG CAAGTTGGAC CCTACGAAGG 50

AGAAGCGGGG GCCAGGCCGA AAGGCCCGGA AGCAGAAGGG TGCCGAGACA 100

GAACTCGTCA GATTCTTGCC TGCAGTAAGT GACGAAAATT CCAAGAGGCT 150

GTCTAGTCGT GCTCGAAAGA GGGCAGCCAA GAGGAGATTG GGCTCTGTTG 200

AAGCCCCTAA GACAAATAAG TCTCCTGAGG CCAAACCATC GCCTGGAAAG 250

CTACCAAAAG GGATCTCTGC AGGAGCTGTC CAGACAGCTG GTAAGAAGGG 300

ACCCCAGTCC CTATTTAATG CTCCTCGAGG CAAGAAGCGC CCAGCACCTG 350

GCAGTGATGA GGAAGAGGAG GAGGAAGACT CTGAAGAAGA TGGTATGGTG 400

AACCACGGGG ACCTCTGGGG CTCCGAGGAC GATGCTGATA CGGTAGATGA 450

CTATGGAGCT GACTCCAACT CTGAGGATGA GGAGGAAGGT GAAGCGTTGC 500

TGCCCATTGA AAGAGCTGCT CGGAAGCAGA AGGCCCGGGA AGCTGCTGCT 550

GGGATCCAGT GGAGTGAAGA GGAGACCGAG GACGAGGAGG AAGAGAAAGA 600

AGTGACCCCT GAGTCAGGCC CCCCAAAGGT GGAAGAGGCA GATGGGGGCC 650

TG_.CAGATCAA TGTGGATGAG GAACCATTTG TGCTGCCCCC TGCTGGGGAG 700

ATGGAGCAGG ATGCCCAGGC TCCAGACCTG CAACGAGTTC ACAAGCGGAT 750

CCAGGATATT GTGGGAATTC TGCGTGATTT TGGGGCTCAG CGGGAGGAAG 800

GGCGGTCTCG TTCTGAATAC CTGAACCGGC TCAAGAAGGA TCTGGCCATT 850 TACTACTCCT ATGGAGACTT CCTGCTTGGC AAGCTCATGG ACCTCTTCCC 900

TCTGTCTGAG CTGGTGGAGT TCTTAGAAGC TAATGAGGTG CCTCGGCCCG 950

TCACCCTCCG GACCAATACC TTGAAAACCC GACGCCGAGA CCTTGCACAG 1000

GCTCTAATCA ATCGTGGGGT TAACCTGGAT CCCCTGGGCA AGTGGTCAAA 1050

GACTGGACTA GTGGTGTATG ATTCTTCTGT GCCCATTGGT GCTACCCCCG 1100

AGTACCTGGC TGGGCACTAC ATGCTGCAGG GAGCCTCCAG CATGTTGCCC 1150

GTCATGGCCT TGGCACCCCA GGAACATGAG CGGATCCTGG ACATGTGTTG 1200

TGCCCCTGGA GGAAAGACCA GCTACATGGC CCAGCTGATG AAGAACACGG 1250

GTGTGATCCT TGCCAATGAC GCCAATGCTG AGCGGCTCAA GAGTGTTGTG 1300

GGCAACTTGC ATCGGCTGGG AGTCACCAAC ACCATTATCA GCCACTATGA 1350

TGGGCGCCAG TTCCCCAAGG TGGTGGGGGG CTTTGACCGA GTACTGCTGG 1400

ATGCTCCCTG CAGTGGCACT GGGGTCATCT CCAAGGATCC AGCCGTGAAG 1450

ACTAACAAGG ATGAGAAGGA CATCCTGCGC TGTGCTCACC TCCAGAAGGA 1500

GTTGCTCCTG AGTGCTATTG ACTCTGTCAA TGCGACCTCC AAGACAGGAG 1550

GCTACCTGGT TTACTGCACC TGTTCTATCA CAGTAGAAGA GAATGAGTGG 1600

GTGGTAGACT ATGCTCTGAA AAAGAGGAAT GTGCGACTGG TGCCCACGGG 1650

CCTAGACTTT GGCCAGGAAG GTTTTACCCG CTTTCGAGAA AGGCGCTTCC 1700

ACCCCAGTCT GCGTTCTACC CGACGCTTCT ACCCTCATAC CCACAATATG 1750

GATGGGTTCT TCATTGCCAA GTTCAAGAAA TTTTCCAATT CTATCCCTCA 1800

GTCCCAGACA GGAAATTCTG AAACAGCCAC ACCTACAAAT GTAGACTTGC 1850

CTCAGGTCAT CCCCAAGTCT GAGAACAGCA GCCAGCCAGC CAAGAAAGCC 1900

AAGGGGGCTG CAAAGACAAA GCAGCAGCTG CAGAAACAGC AACATCCCAA 1950

GAAGGCCTCC TTCCAGAAGC TGAATGGCAT CTCCAAAGGG GCAGACTCAG 2000

AATTGTCCAC TGTACCTTCT GTCACAAAGA CCCAAGCTTC CTCCAGCTTC 2050

CAGGATAGCA GTCAGCCAGC TGGAAAAGCC GAAGGGATCA GGGAGCCAAA 2100

GGTGACTGGG AAGCTAAAGC AACGATCACC TAAATTACAG TCCTCCAAGA 2150

AAGTTGCTTT CCTCAGGCAG AATGCCCCTC CCAAGGGCAC AGACACACAA 2200

ACACCGGCTG TGTTATCCCC ATCCAAGACT CAGGCCACCC TGAAACCTAA 2250 GGACCATCAT CAGCCCCTTG GAAGGGCCAA GGGGGTTGAG AAGCAGCAGT 2300

TCGCAGAGCA GCCTTTTGAG AAAGCTGCCT TCCAGAAACA GAATGATACC 2350

CCCAAGGGCC TCAGCCTCCC ACTGTGTCTC CCATCCGTTC CAGCCGCCCC 2400

CCACCAGCAA AGAGGAAGAA ATCTCAGTCC AGGGGCAACA GCCAGCTGCT 2450

GCTATCTTAG ATGGTTGAAA ACTAGACGGG TGGCTCACTG CCATTGTCAC 2500

CAGGTTGGAA CTCTTGCCTC TGTGAGGATG CCTTCTCTAC TGTGCATACC 2550

CATGAAATTT AATACACATT TTAAAACCTC TGGCCACTGA GTATTTTTGA 2600

GGGAACTGGG TGCTGCCTTT GCCGCTTTAA GGGTAAAGAT GGGAGGTGGG 2650

ACTGGGATCT TCTGAGGAGA GGGAATGGTT GGGTGCTACT GAGAAATAAG 2700

GTCAGTTTTA GGCTCCTGCA TCCTTGGGTT TTCCCAGGAA GGGAAAGCCC 2750

TGGAGTCAGA GCTAGGGGTG ATTGAACTCC GGGGAGAGGA GGGCAATAGA 2800

AGTGTAGGGG AAGGCGGGGC ACCTAGGCTC ATGCCTGTGA ATCCCAGCAC 2850

TTTCTGGGAG GCCAAGGCGG GTGGATCACC TGAGGTCAGG TGTTCGAGAC 2900

CAGCCTGGCT AACATGGCGA AACCTCGTCT CTACTAAAAA TACAAAAAAT 2950

CAGCCGGGTG TGGTGGCGGG TGCCTGTAAT CCCA 2984

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) _ENGTH: 2984 (B^* TYPE: nucleic acid (C STRANDEDNESS: single (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

TGGGATTACA GGCACCCGCC ACCACACCCG GCTGATTTTT TGTATTTTTA 50

GTAGAGACGA GGTTTCGCCA TGTTAGCCAG GCTGGTCTCG AACACCTGAC 100

CTCAGGTGAT CCACCCGCCT TGGCCTCCCA GAAAGTGCTG GGATTCACAG 150 GCATGAGCCT AGGTGCCCCG CCTTCCCCTA CACTTCTATT GCCCTCCTCT 200

CCCCGGAGTT CAATCACCCC TAGCTCTGAC TCCAGGGCTT TCCCTTCCTG 250

GGAAAACCCA AGGATGCAGG AGCCTAAAAC TGACCTTATT TCTCAGTAGC 300

ACCCAACCAT TCCCTCTCCT CAGAAGATCC CAGTCCCACC TCCCATCTTT 350

ACCCTTAAAG CGGCAAAGGC AGCACCCAGT TCCCTCAAAA ATACTCAGTG 400

GCCAGAGGTT TTAAAATGTG TATTAAATTT CATGGGTATG CACAGTAGAG 450

AAGGCATCCT CACAGAGGCA AGAGTTCCAA CCTGGTGACA ATGGCAGTGA 500

GCCACCCGTC TAGTTTTCAA CCATCTAAGA TAGCAGCAGC TGGCTGTTGC 550

CCCTGGACTG AGATTTCTTC CTCTTTGCTG GTGGGGGGCG GCTGGAACGG 600

ATGGGAGACA CAGTGGGAGG CTGAGGCCCT TGGGGGTATC ATTCTGTTTC 650

TGGAAGGCAG CTTTCTCAAA AGGCTGCTCT GCGAACTGCT GCTTCTCAAC 700

CCCCTTGGCC CTTCCAAGGG GCTGATGATG GTCCTTAGGT TTCAGGGTGG 750

CCTGAGTCTT GGATGGGGAT AACACAGCCG GTGTTTGTGT GTCTGTGCCC 800

TTGGGAGGGG CATTCTGCCT GAGGAAAGCA ACTTTCTTGG AGGACTGTAA 850

TTTAGGTGAT CGTTGCTTTA GCTTCCCAGT CACCTTTGGC TCCCTGATCC 900

CTTCGGCTTT TCCAGCTGGC TGACTGCTAT CCTGGAAGCT GGAGGAAGCT 950

TGGGTCTTTG TGACAGAAGG TACAGTGGAC AATTCTGAGT CTGCCCCTTT 1000

GGAGATGCCA TTCAGCTTCT GGAAGGAGGC CTTCTTGGGA TGTTGCTGTT 1050

TCTGCAGCTG CTGCTTTGTC TTTGCAGCCC CCTTGGCTTT CTTGGCTGGC 1100

TGGCTGCTGT TCTCAGACTT GGGGATGACC TGAGGCAAGT CTACATTTGT 1150

AGGTGTGGCT GTTTCAGAAT TTCCTGTCTG GGACTGAGGG ATAGAATTGG 1200

AAAATTTCTT GAACTTGGCA ATGAAGAACC CATCCATATT GTGGGTATGA 1250

GGGTAGAAGC GTCGGGTAGA ACGCAGACTG GGGTGGAAGC GCCTTTCTCG 1300

AAAGCGGGTA AAACCTTCCT GGCCAAAGTC TAGGCCCGTG GGCACCAGTC 1350

GCACATTCCT CTTTTTCAGA GCATAGTCTA CCACCCACTC ATTCTCTTCT 1400

ACTGTGATAG AACAGGTGCA GTAAACCAGG TAGCCTCCTG TCTTGGAGGT 1450

CGCATTGACA GAGTCAATAG CACTCAGGAG CAACTCCTTC TGGAGGTGAG 1500

CACAGCGCAG GATGTCCTTC TCATCCTTGT TAGTCTTCAC GGCTGGATCC 1550 TTGGAGATGA CCCCAGTGCC ACTGCAGGGA GCATCCAGCA GTACTCGGTC 1600

AAAGCCCCCC ACCACCTTGG GGAACTGGCG CCCATCATAG TGGCTGATAA 1650

TGGTGTTGGT GACTCCCAGC CGATGCAAGT TGCCCACAAC ACTCTTGAGC 1700

GCGTCAGCAT TGGCGTCATT GGCAAGGATC ACACCCGTGT TCTTCATCAG 1750

CTGGGCCATG TAGCTGGTCT TTCCTCCAGG GGCACAACAC ATGTCCAGGA 1800

TCCGCTCATG TTCCTGGGGT GCCAAGGCCA TGACGGGCAA CATGCTGGAG 1850

GCTCCCTGCA GCATGTAGTG CCCAGCCAGG TACTCGGGGG TAGCACCAAT 1900

GGGCACAGAA GAATCATACA CCACTAGTCC AGTCTTTGAC CACTTGCCCA 1950

GGGGATCCAG GTTAACCCCA CGATTGATTA GAGCCTGTGC AAGGTCTCGG 2000

CGTCGGGTTT TCAAGGTATT GGTCCGGAGG GTGACGGGCC GAGGCACCTC 2050

ATTAGCTTCT AAGAACTCCA CCAGCTCAGA CAGAGGGAAG AGGTCCATGA 2100

GCTTGCCAAG CAGGAAGTCT CCATAGGAGT AGTAAATGGC CAGATCCTTC 2150

TTGAGCCGGT TCAGGTATTC AGAACGAGAC CGCCCTTCCT CCCGCTGAGC 2200

CCCAAAATCA CGCAGAATTC CCACAATATC CTGGATCCGC TTGTGAACTC 2250

GTTGCAGGTC TGGAGCCTGG GCATCCTGCT CCATCTCCCC AGCAGGGGGC 2300

AGCACAAATG GTTCCTCATC CACATTGATC TGCAGGCCCC CATCTGCCTC 2350

TTCCACCTTT GGGGGGCCTG ACTCAGGGGT CACTTCTTTC TCTTCCTCCT 2400

CGTCCTCGGT CTCCTCTTCA CTCCACTGGA TCCCAGCAGC AGCTTCCCGG 2450

GCCTTCTGCT TCCGAGCAGC TCTTTCAATG GGCAGCAACG CTTCACCTTC 2500

CTCCTCATCC TCAGAGTTGG AGTCAGCTCC ATAGTCATCT ACCGTATCAG 2550

CATCGTCCTC GGAGCCCCAG AGGTCCCCGT GGTTCACCAT ACCATCTTCT 2600

TCAGAGTCTT CCTCCTCCTC TTCCTCATCA CTGCCAGGTG CTGGGCGCTT 2650

CTTGCCTCGA GGAGCATTAA ATAGGGACTG GGGTCCCTTC TTACCAGCTG 2700

TCTGGACAGC TCCTGCAGAG ATCCCTTTTG GTAGCTTTCC AGGCGATGGT 2750

TTGGCCTCAG GAGACTTATT TGTCTTAGGG GCTTCAACAG AGCCCAATCT 2800

CCTCTTGGCT GCCCTCTTTC GAGCACGACT AGACAGCCTC TTGGAATTTT 2850

CGTCACTTAC TGCAGGCAAG AATCTGACGA GTTCTGTCTC GGCACCCTTC 2900 TGCTTCCGGG CCTTTCGGCC TGGCCCCCGC TTCTCCTTCG TAGGGTCCAA 2950 CTTGCGCCCC ATGGTACTGT GGCAGGCAGA AATG 2984

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 398

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (iv) ANTI-SENSE: yes

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:

TGGGATTACA GGCACCCGCC ACCACACCCG GCTGATTTTT TGTATTTTTA 50

GTAGAGACGA GGTTTCGCCA TGTTAGCCAG GCTGGTCTCG AACACCTGAC 100

CTCAGGTGAT CCACCCGCCT TGGCCTCCCA GAAAGTGCTG GGATTCACAG 150

GCATGAGCCT AGGTGCCCCG CCTTCCCCTA CACTTCTATT GCCCTCCTCT 200

CCCCGGAGTT CAATCACCCC TAGCTCTGAC TCCAGGGCTT TCCCTTCCTG 250

GGAAAACCCA AGGATGCAGG AGCCTAAAAC TGACCTTATT TCTCAGTAGC 300

ACCCAACCAT TCCCTCTCCT CAGAAGATCC CAGTCCCACC TCCCATCTTT 350

ACCCTTAAAG CGGCAAAGGC AGCACCCAGT TCCCTCAAAA ATACTCAG 398

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1269

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (iv) ANTI-SENSE: yes

( i) SEQUENCE DESCRIPTION: SEQ ID NO:4: GCTTGGGTCT TTGTGACAGA AGGTACAGTG GACAATTC G AGTCTGCCCC 50

TTTGGAGATG CCATTCAGCT TCTGGAAGGA GGCCTTCTTG GGATGTTGCT 100

GTTTCTGCAG CTGCTGCTTT GTCTTTGCAG CCCCCTTGGC TTTCTTGGCT 150

GGCTGGCTGC TGTTCTCAGA CTTGGGGATG ACCTGAGGCA AGTCTACATT 200

TGTAGGTGTG GCTGTTTCAG AATTTCCTGT CTGGGACTGA GGGATAGAAT 250

TGGAAAATTT CTTGAACTTG GCAATGAAGA ACCCATCCAT ATTGTGGGTA 300

TGAGGGTAGA AGCGTCGGGT AGAACGCAGA CTGGGGTGGA AGCGCCTTTC 350

TCGAAAGCGG GTAAAACCTT CCTGGCCAAA GTCTAGGCCC GTGGGCACCA 400

GTCGCACATT CCTCTTTTTC AGAGCATAGT CTACCACCCA CTCATTCTCT 450

TCTACTGTGA TAGAACAGGT GCAGTAAACC AGGTAGCCTC CTGTCTTGGA 500

GGTCGCATTG ACAGAGTCAA TAGCACTCAG GAGCAACTCC TTCTGGAGGT 550

GAGCACAGCG CAGGATGTCC TTCTCATCCT TGTTAGTCTT CACGGCTGGA 600

TCCTTGGAGA TGACCCCAGT GCCACTGCAG GGAGCATCCA GCAGTACTCG 650

GTCAAAGCCC CCCACCACCT TGGGGAACTG GCGCCCATCA TAGTGGCTGA 700

TAATGGTGTT GGTGACTCCC AGCCGATGCA AGTTGCCCAC AACACTCTTG 750

AGCCGCTCAG CATTGGCGTC ATTGGCAAGG ATCACACCCG TGTTCTTCAT 800

CAGCTGGGCC ATGTAGCTGG TCTTTCCTCC AGGGGCACAA CACATGTCCA 850

GGATCCGCTC ATGTTCCTGG GGTGCCAAGG CCATGACGGG CAACATGCTG 900

GAGGCTCCCT GCAGCATGTA GTGCCCAGCC AGGTACTCGG GGGTAGCACC 950

AATGGGCACA GAAGAATCAT ACACCACTAG TCCAGTCTTT GACCACTTGC 1000

CCAGGGGATC CAGGTTAACC CCACGATTGA TTAGAGCCTG TGCAAGGTCT 105

CGGCGTCGGG TTTTCAAGGT ATTGGTCCGG AGGGTGACGG GCCGAGGCAC 1100

CTCATTAGCT TCTAAGAACT CCACCAGCTC AGACAGAGGG AAGAGGTCCA 1150

TGAGCTTGCC AAGCAGGAAG TCTCCATAGG AGTAGTAAAT GGCCAGATCC 1200

^• TTCTTGAGCC GGTTCAGGTA TTCAGAACGA GACCGCCCTT CCTCCCGCTG 1250

AGCCCCAAAA TCACGCAGA 1269 (2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 32

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (iv) ANTI-SENSE: yes

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TGCGCCCCAT GGTACTGTGG CAGGCAGAAA TG 32

Claims

WHAT IS CLAIMED IS:

1. A method for modulating the growth of cells comprising contacting the cells with a vector capable of inserting genetic material into the cells, the vector containing genetic material coding for a nucleolar antigen, an antisense counterpart to such genetic material, or a portion thereof.

2. The method of claim 1 wherein the vector is a retroviral vector.

3. The method of claim 1 wherein the vector is selected from the group consisting of pSVX, N2, pLNCX, pLXSN, pMSG, pMAM, pCEP4, pMEP4 and pREP4.

4. The method of claim 1 wherein the vector is pSVX or an N2-derived vector.

5. The method of claim 1 wherein said genetic material codes for a nucleolar antigen implicated in the growth of hyperproliferative cells.

6. The method of claim 5 wherein the cell growth is inhibited.

7. The method of claim l wherein the genetic material is cDNA.

8. A method for inhibiting the growth of hyperproliferative cells comprising contacting the cells with a vector capable of inserting genetic material into the cells, the vector containing genetic material coding for an antisense counterpart to genetic material coding for a nucleolar antigen, or a portion thereof.

9. The method of claim 8 wherein the nucleolar antigen is pl20.

10. The method of claim 8 wherein the vector is a retroviral vector.

11. The method of claim 8 wherein the vector is selected from the group consisting of pSVX, N2, pLNCX, pLXSN, pMSG, pMAM, pCEP4, pMEP4 and pREP4.

12. The method of claim 8 wherein the vector is pSVX or an N2-derived vector.

13. The method of claim 8 wherein the genetic material is cDNA.

14. The method of claim 8 wherein the genetic material comprises Sequence ID No. 2.

15. The method of claim 8 wherein the genetic material comprises Sequence ID No. 3.

16. The method of claim 8 wherein the genetic material comprises Sequence ID No. 4.

17. The method of claim 8 wherein the genetic material comprises Sequence ID No. 5.

18. A method for inhibiting the growth of a human breast carcinoma comprising contacting cells of the carcinoma with a vector capable of inserting genetic material into the cells, the vector containing at least a portion of an antisense counterpart to genetic material coding for a nucleolar antigen.

19. The method of claim 18 wherein the nucleolar antigen is pl20.

20. The method of claim 18 wherein the vector is a retroviral vector.

21. The method of claim 18 wherein the vector is selected from the group consisting of pSVX, N2, pLNCX, pLXSN, pMSG, pMAM, pCEP4, pMEP4 and pREP4.

22. The method of claim 18 wherein the vector is pSVX or an N2-derived vector.

23. The method of claim 18 wherein the genetic material comprises cDNA.

24. The method of claim 18 wherein the genetic material comprises Sequence ID No. 2.

25. The method of claim 18 wherein the genetic material comprises Sequence ID No. 3.

26. The method of claim 18 wherein the genetic material comprises Sequence ID No. 4.

27. The method of claim 18 wherein the genetic material comprises Sequence ID No. 5.

28. A vector capable of inserting genetic material into cells, said vector containing an antisense counterpart to genetic material coding for a nucleolar antigen, or a portion thereof.

29. The vector of claim 28 comprising a retroviral vector.

30. The vector of claim 28 selected from the group consisting of pSVX, N2, pLNCX, pLXSN, pMSG, pMAM, pCEP4, pMEP4 and pREP4.

31. The method of claim 28 wherein the vector is pSVX or an N2-derived vector.

32. A vector capable of inserting genetic material into cells, said vector containing genetic material coding for at least a portion of an antisense counterpart to genetic material coding for nucleolar antigen pl20.

33. The vector of claim 32 comprising a retroviral vector.

34. The vector of claim 32 selected from the group consisting Of pSVX, N2, pLNCX, pLXSN, pMSG, pMAM, pCEP4, pMEP4 and pREP4.

35. The method of claim 32 wherein the vector is pSVX or an N2-derived vector.

36. The method of claim 32 wherein the genetic material comprises Sequence ID No. 2.

37. The method of claim 32 wherein the genetic material comprises Sequence ID No. 3.

38. The method of claim 32 wherein the genetic material comprises Sequence ID No. 4.

39. The method of claim 32 wherein the genetic material comprises Sequence ID No. 5.

40. A method for altering the growth of c-slls comprising contacting the cells with a vector capable of inserting genetic material into the cells, the vector containing a portion of an antisense counterpart to a gene coding for a protein essential to growth of the cells.

41. The method of claim 40 wherein said protein is a nucleolar antigen.

42. The method of claim 40 wherein said protein is pl20.

43. The method of claim 40 wherein the genetic material comprises Sequence ID No. 3.

44. The method of claim 40 wherein the genetic material comprises Sequence ID No. 4.

45. The method of claim 40 wherein the genetic material comprises Sequence ID No. 5.

46. An oligonucleotide antisense to at least a portion of a gene coding for a nucleolar antigen.

47. The oligonucleotide of claim 46 wherein the nucleolar antigen is pl20.

48. A method of treating human breast carcinoma comprising contacting cells of the carcinoma with a vector capable of inserting genetic material into the cells, the vector containing genetic material antisense to at least a portion of a gene coding for pl20.

49. The method of claim 48 wherein the genetic material comprises an effective portion of Sequence ID No. 2.

50. The method of claim 48 wherein the genetic material comprises an effective portion of Sequence ID No. 3.

51. The method of claim 48 wherein the genetic material comprises an effective portion of Sequence ID No.4.

52. The method of claim 48 wherein the genetic material comprises an effective portion of the 5¹ untranslated region of said gene.

53. The method of claim 48 wherein the genetic material comprises an effective portion of the B region of said gene.