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WO1993015743A1 - Nouvelles therapies geniques mettant en ×uvre des structures non codantes - Google Patents

Nouvelles therapies geniques mettant en ×uvre des structures non codantes Download PDF

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Publication number
WO1993015743A1
WO1993015743A1 PCT/US1993/000893 US9300893W WO9315743A1 WO 1993015743 A1 WO1993015743 A1 WO 1993015743A1 US 9300893 W US9300893 W US 9300893W WO 9315743 A1 WO9315743 A1 WO 9315743A1
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genetic material
cells
vector
material comprises
antisense
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PCT/US1993/000893
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English (en)
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Harris Busch
Ben Valdez
Laszlo Perlaky
Yasuo Saijo
Rose Busch
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Baylor College Of Medicine
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Publication of WO1993015743A1 publication Critical patent/WO1993015743A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • 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.
  • 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.
  • 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.
  • compositions and materials suitable for the performance of such methods are provided.
  • 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 j ect 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.
  • 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.
  • the vector contains material coding for a nucleolar antigen, its antisense analog, or a portion thereof.
  • the vector is a retroviral vector.
  • 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.
  • the genetic ma...rial i ⁇ 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.
  • 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.
  • 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.
  • Figures 1A and IB are drawings depicting pSVX and
  • PMSG vectors containing pl20 cDNA in its sense or antisense sequences are included in 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.
  • 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).
  • the genetic material contained within the vector which will subsequently be expressed by the cells codes for a nucleolar antigen.
  • an antisense analog or construct corresponding to genetic material coding for a nucleolar antigen, or a portion thereof is used.
  • 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.
  • 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.
  • the materials and methods of the present invention to encourage the growth of cells.
  • the present invention is not limited to the interference with cellular proliferation, since such development may, in the alternative, be encouraged.
  • 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.
  • 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).
  • Eglitis and French Anderson is directed to retroviral vectors for such use.
  • retroviral vectors are generally preferred for performance of preferred embodiments of the present invention, although other forms of vectors may also be useful.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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.
  • antisense molecules 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. , Purani, G. , Calabretta, B.
  • antisense pl20 oligonucleotide molecules are believed to be useful as therapeutic anticancer agents.
  • 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.
  • 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.
  • FIG. 1 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.
  • 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 Na 2 HP0 4 , 6 mM dextrose) containing 500 ⁇ g/ l of sonicated salmon testis DNA (Sigma) . The pSVX or MSG plasmid constructs (20 ⁇ g/ml DNA) were then added.
  • the cells were exposed to a single voltage pulse (220 V, 960 ⁇ F, 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
  • D-MEM medium was used for cell selection.
  • the Geneticin ® concentration of 600 ⁇ g/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 ⁇ g/ml xanthine, 25 ⁇ g/ml mycophenolic acid, 2 ⁇ g/ml aminopterin 10 ⁇ g/ml thymidine, and 15 ⁇ g/ml hypoxanthine.
  • 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 ⁇ g/ml RNase at 37°C for 2 hours.
  • the sample was made to 0.5% SDS and treated with 100 ⁇ g/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.
  • Neomycin Phosphotransferase II NPT II
  • NPT II neomycin phosphotransferase II
  • ELISA enzyme-linked immunosorbant assay
  • 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.
  • 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.
  • 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.
  • NIH/3T3 ATCC CRL 1658, contact-inhibited NIH Swiss mouse embryo
  • D-MEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • penicillin-streptomycin liquid 10000 IU/ml penicillin G sodium, 10 mg/ml streptomycin sulphate in 0.85% saline
  • 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.
  • 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.
  • NIH/3T3pSVX120 and control cells were washed and resuspended in serum-free D-MEM medium.
  • 2xl0 6 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.
  • 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.
  • 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.
  • 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.
  • 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 probably represents mouse pl20 mRNA, which is similar in size to the pl20 mRNA from HeLa cells.
  • 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) .
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • PE colony forming efficiency
  • 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.
  • FIG. 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) .
  • 1 ⁇ M dexamethasone in D-MEM medium was used for 48 hours (filled symbols, dashed lines) . The cells were trypsinized, stained with Trypan blue and counted.
  • was reduced by 52% compared to the sense 3T3pSVX120 transfected with the pMSG vector alone (open circles, solid line, G 2.1xl ⁇ "2 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) .
  • GAACTCGTCA GATTCTTGCC TGCAGTAAGT GACGAAAATT CCAAGAGGCT 150

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Abstract

L'invention concerne la modulation de la croissance cellulaire qui consiste à mettre en contact les cellules avec un vecteur capable de transporter la matière génétique dans les cellules. La matière génétique code pour un antigène nucléolaire ou est non codante par rapport à la matière génétique codant pour un antigène nucléolaire. Des parties de cette matière génétique, représentant moins de la totalité de cette matière, sont également efficaces dans certains modes de réalisation préférés. Conformément à des modes de réalisation préférés, la matière génétique non codante, de préférence l'ADNc, par rapport à celle codant pour la p120 est utilisée pour empêcher la croissance des cellules cancéreuses du sein chez la femme.
PCT/US1993/000893 1992-02-18 1993-01-28 Nouvelles therapies geniques mettant en ×uvre des structures non codantes WO1993015743A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995023855A3 (fr) * 1994-03-02 1995-09-28 Univ Illinois PROTEINES INHIBITRICES PROPRES AU DOMAINE DE KUNITZ DERIVEES DE L'INHIBITEUR DU PRECURSEUR DE LA β-PROTEINE AMYLOIDE DE LA MALADIE D'ALZHEIMER
EP0672178A4 (fr) * 1992-02-19 1996-03-20 Baylor College Medicine Modulation de la croissance cellulaire au moyen d'oligonucleotides.
JP3484198B2 (ja) 1994-10-14 2004-01-06 大鵬薬品工業株式会社 オリゴヌクレオチド及びこれを有効成分とする制癌剤

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CANCER RESEARCH, Volume 50, issued 15 August 1990, H. BUSCH, "The Final Common Pathway of Cancer: Presidential Address", pages 4830-4838. *
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, Volume 86, issued May 1989, G. ANFOSSI et al., "An Oligomer Complementary to C-Myb-Encoded mRNA Inhibits Proliferation of Human Myeloid Leukemia Cell Lines", pages 3379-3383. *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0672178A4 (fr) * 1992-02-19 1996-03-20 Baylor College Medicine Modulation de la croissance cellulaire au moyen d'oligonucleotides.
US5656743A (en) * 1992-02-19 1997-08-12 Baylor College Of Medicine Oligonucleotide modulation of cell growth
WO1995023855A3 (fr) * 1994-03-02 1995-09-28 Univ Illinois PROTEINES INHIBITRICES PROPRES AU DOMAINE DE KUNITZ DERIVEES DE L'INHIBITEUR DU PRECURSEUR DE LA β-PROTEINE AMYLOIDE DE LA MALADIE D'ALZHEIMER
JP3484198B2 (ja) 1994-10-14 2004-01-06 大鵬薬品工業株式会社 オリゴヌクレオチド及びこれを有効成分とする制癌剤

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