Compositions and methods for non-targeted activation of endogenous genes Number:6,740,503 from the United States Patent and Trademark Office (PTO) owispatent The present invention is directed generally to activating gene expression or causing over-expression of a gene by recombination methods in situ. The invention also is directed generally to methods for expressing an endogenous gene in a cell at levels higher than those normally found in the cell. In one embodiment of the invention, expression of an endogenous gene is activated or increased following integration into the cell, by non-homologous or illegitimate recombination, of a regulatory sequence that activates expression of the gene. The invention also provides methods for the identification, activation, isolation, and/or expression of genes undiscoverable by current methods since no target sequence is necessary for integration. Thus, by the present invention, endogenous genes, including those associated with human disease and development, may be activated and isolated without prior knowledge of the, sequence, structure, function, or expression profile of the genes.<H Compositions, endogenous, Compositions, an, 6740503.html, Patent, pto, 6740503

Title: Compositions and methods for non-targeted activation of endogenous genes

Abstract: The present invention is directed generally to activating gene expression or causing over-expression of a gene by recombination methods in situ. The invention also is directed generally to methods for expressing an endogenous gene in a cell at levels higher than those normally found in the cell. In one embodiment of the invention, expression of an endogenous gene is activated or increased following integration into the cell, by non-homologous or illegitimate recombination, of a regulatory sequence that activates expression of the gene. The invention also provides methods for the identification, activation, isolation, and/or expression of genes undiscoverable by current methods since no target sequence is necessary for integration. Thus, by the present invention, endogenous genes, including those associated with human disease and development, may be activated and isolated without prior knowledge of the, sequence, structure, function, or expression profile of the genes.

Patent Number: 6,740,503 Issued on 05/25/2004 to Harrington, et al.

Inventors: Harrington; John J. (Mentor, OH), Sherf; Bruce (Spencer, OH), Rundlett; Stephen (Chagrin Falls, OH)
Assignee: Athersys, Inc. (Cleveland, OH)

Appl. No.: 09/484,317

Filed: January 18, 2000

Current U.S. Class: 435/69.1 ; 435/320.1; 435/325; 435/455; 435/69.8

Current International Class: C12N 15/67 (20060101); C12N 15/10 (20060101); C12N 15/63 (20060101); C12N 15/85 (20060101); C12N 15/87 (20060101); C12N 15/90 (20060101); A61K 48/00 (20060101)

Field of Search: 435/320.1,325,69.2,455,69.1,69.8

References Cited [Referenced By]

U.S. Patent Documents


5024939	June 1991	Gorman
5272071	December 1993	Chappel
5547933	August 1996	Lin
5561053	October 1996	Crowley
5578461	November 1996	Sherwin et al.
5580734	December 1996	Treco et al.
5618698	April 1997	Lin
5621080	April 1997	Lin
5641670	June 1997	Treco et al.
5677170	October 1997	Devine et al.
5683905	November 1997	Capon et al.
5707830	January 1998	Calos
5728551	March 1998	Devine et al.
5733761	March 1998	Treco et al.
5750105	May 1998	Newman et al.
5783385	July 1998	Treco et al.
5789215	August 1998	Berns et al.
5830698	November 1998	Reff et al.
5843772	December 1998	Devine et al.
5928867	July 1999	den Dunnen et al.
6136566	October 2000	Sands et al.

Foreign Patent Documents


71893/94	Jun., 1994	AU
0 742 285	Nov., 1996	EP
2 707 091	Jan., 1995	FR
WO 90/14092	Nov., 1990	WO
WO 91/01140	Feb., 1991	WO
WO 91/06667	May., 1991	WO
WO 92/19255	Nov., 1992	WO
WO 92/20808	Nov., 1992	WO
WO 93/09222	May., 1993	WO
WO 94/12650	Jun., 1994	WO
WO 95/31560	Nov., 1995	WO
WO 96/04391	Feb., 1996	WO
WO 96/29411	Sep., 1996	WO
WO 98/14614	Apr., 1998	WO
WO 99/07389	Feb., 1999	WO
WO 99/50426	Oct., 1999	WO

Other References

US 5,733,746, 3/1998, Treco et al. (withdrawn) .
JL Yates et al., Nature, "Stable replication of plasmids derived from Epstein-Barr virus in various mammalian cells," Feb. 1985, vol. 313, pp. 812-815.* .
Molecular Cloning(2.sup.nd edition)[published by Cold Spring Harbor Laboratory Press, Box 100 Cold Spring Harbor, New York, by Sambrook et al.](1989) pp. 16.3-16.23.* .
Hay, Bruce A., et al., "P Element Insertion-Dependent Gene Activation In The Drosophila Eye," Proc. Natl. Acad. Sci. USA, 94:5195-5200 (May 1997). .
Yoshida, M., et al., "A New Strategy of Gene Trapping in ES Cells Using 3' RACE," Transgenic Research 4:277-287 (1995). .
Ashe, M.P., et al., "Poly(A) site selection in the HIV-1 provirus: inhibition of promoter-proximal polyadenylation by the downstream major splice donor site," Genes & Develop. 9:3008-3025 (1995). .
Auch, D., and Reth, M., "Exon trap cloning: using PCR to rapidly detect and clone exons from genomic DNA fragments," Nucl. Acids Res. 18:6743-6744 (1990). .
Avoustin, P., et al., "Non-homologous recombination within the Major Histocompatability Complex creates a transcribed hybrid sequence," Mammalian Genome 5(12):771-776 (1994). .
Barker, C.S., et al., "Activation of the Prolactin Receptor Gene by Promoter Insertion in a Moloney Murine Leukemia Virus-Induced Rat Thymoma," J. Virol. 66(11):6763-6768 (1992). .
Black, A.C., et al., "HTLV-II Rex Binding and Activity Requires an Intact Splice Donor Site and a Specific RNA Secondary Structure," AIDS Res. & Human Retroviruses 8:870 (1992). .
Brikun, I., et al., "Analysis of CRP-CytR Interactions at the Escherichia coli udp Promoter," J. Bacteriology 178(6):1614-1622 (Mar. 1996). .
Buckler, A.J., et al., "Exon amplification: A strategy to isolate mammalian genes based on RNA splicing," Proc. Natl. Acad. Sci. USA 88:4005-4009 (1991). .
Butturini, A., et al., "Oncogenes in Human Leukemias," Acta Haematologica 78(suppl. 1):2-10 (1987). .
Butturini, A., and Gale, R.P., "Oncogenes and Human Leukemias," Intl. J. Cell Cloning 6(1):2-24 (1988). .
Chakraborty, A.K., et al., "Transforming function of proto-ras genes depends on heterologous promoters and is enhanced by specific point mutations," Proc. Natl. Acad. Sci. USA 88:2217-2221 (1991). .
Chang, W., et al. "Enrichment or Insertional Mutants Following Retrovirus Gene Trap Selection," Virol. 193:737-747 (1993). .
Chow, W.-Y. and Berg, D.E., "In5tac1, a derivative of transposon In5 that generates conditional mutations," Proc. Natl. Acad. Sci. USa 85:6468-6472 (1988). .
Church, D.M., et al., "Isolation of genes from comples sources of mammalian genomic DNA using exon amplification," Nature Genetics, 6:98-105 (1994). .
Crabb, B.S., and Cowman, A.F., "Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum," Proc. Natl. Acad. Sci. USA 93: 7289-7294 (Jul. 1996). .
Datson, N.A., et al., "Specification isolation of 3'-terminal exons of human genes by exon trapping" Nucl. Acids Res. 22(20): 4148-4153 (1994). .
Datson, N.A., et al., "Scanning for genes in large genomic regions: cosmid-based exon trapping of multiple exons in a single product," Nucl. Acids Res. 24:1105-1111 (Mar. 1996). .
De Benedetti, A., and Rhoads, R.E., "A novel BK virus-based episomal vector for expression of foreign genes in mammalian cells," Nucl. Acids Res. 19:1925-1931 (1991). .
Dickson, C., et al., "Tumorigenesis by Mouse Mammary Tumor Virus: Proviral Activation of a Cellular Gene in the Common Integration Region int-2," Cell 37:529-536 (1984). .
Dostatni, N., et al, "Use of Retroviral Vectors for Mapping of Splice Sites in Cottontail Rabbit Papillomavirus," J. Gen. Virol. 69:3093-3100 (1988). .
Duesberg, P.N., et al., "Cancer Genes by Non-Homologous Recombination," In: Boundaries between Promotion and Progression during Carcinogenesis, Sudilovsky, O., et al., eds., Plenum Press, New York, NY, pp. 197-211 (1991). .
Duyk, G.M., et al., "Exon trapping: A genetic screen to identify candidate transcribed sequences in cloned mammalian genomic DNA," Proc. Natl. Acad. Sci. USA 87:8995-8999 (1990). .
Frankel, W., et al., "Retroviral insertional mutagenesis of a target allele in a heterozygous murine cell line," Proc. Natl. Acad. Sci. USA 82:6600-6604 (1985). .
Fujisawa, J.-I., et al., "Functional activation of the long terminal repeat of human T-cell leukemia virus type I by a trans-acting factor," Proc. Natl. Acad. Sci. USA 82:2277-2281 (1985). .
Fung, Y.-K.T., et al., "Activation of the Cellular Oncogene c-erbB by LTR Insertion: Molecular Basis for Induction of Erythroblastosis by Avian Leukosis Virus," Cell 33:357-368 (1983). .
Goff, S.P., "Gene Isolation by Retroviral Tagging," Meth. Enzymology 152:469-481 (1987). .
Hayward, W.S., et al., "Activation of a cellular onc gene by promoter insertion in ALV-induced lymphoid leukosis," Nature 290:475-480 (1981). .
Jackson, I.J., "A reappraisal of non-consensus mRNA splice sites," Nucl. Acids Res. 19(14):3795-3798 (1991). .
Joshi, S., "A Putative Approach for Cloning `Silent` Genes Using Retroviral Vectors," Med. Hypoth. 36:242-245 (1991). .
Joyner, A.L., "Gene Targeting and Gene Trap Screens Using Embryonic Stem Cells: New Approaches to Mammalian Development," BioEssays 13:649-656 (1991). .
Junejo, F., et al., "Sequence analysis of the herpes simplex virus type 1 strain 17 variants 1704, 1705 and 1706 with respect to their origin and effect on the latency-associated transcript sequence," J. Gen. Virol. 72:2311-2315 (1991). .
King, W., et al., "Insertion Mutagenesis of Embryonal Carcinoma Cells by Retroviruses," Science 228:554-558 (1985). .
Kreissig, S., et al., "Expression of peptides encoded by exons in cloned mammalian DNA," Nucl. Acids Res. 24:4358-4359 (Nov. 1996). .
Lemay, G., and Jolicoeur, P., "Rearrangement of a DNA sequence homologous to a cell-virus junction fragment in several Moloney murine leukemia virus-induced rat thymomas," Proc. Natl. Acad. Sci. USA 81:38-42 (1984). .
Liang, T., et al., "Effects of Alternate RNA Splicing on Glucokinase Isoform Activities in the Pancreatic Islet, Liver, and Pituitary," J. Biol. Chem. 266(11):6999-7007 (1991). .
Lih, C.-J., et al., "Rapid identification and isolation of transcriptionally active regions from mouse genomes," Gene 164:289-294 (1995). .
Liu, Z., et al., "The E6 Gene of Human Papillomavirus Type 16 Is Sufficient for Transformation of Baby Rat Kidney Cells in Cotransfection with Activated Ha-ras," Virology 201:388-396 (1994). .
Molders, H., et al., "Integration of transfected LTR sequences into the c-raf proto-oncogene: activation by promoter insertion," EMBO J. 4(3):693-698 (1985). .
Moore, R., et al., "Sequence, topography and protein coding potential of mouse int-2: a putative oncogene activated by mouse mammary tumour virus," EMBO J. 5(5):919-924 (1986). .
Muller, R., and Muller, D., "Co-transfection of normal NIH/3T3 DNA and retroval LTR sequences: a novel strategy for the detection of potential c-onc genes," EMBO J. 3(5):1121-1127 (1984). .
Muth, K., et al., "Disruption of Genes Regulated During Hematopoietic Differentiation of Mouse Embryonic Stem Cells," Develop. Dynamics 212:277-283 (Jun. 1998). .
Neel, B.G., et al., "Molecular Analysis of the c-myc Locus in Normal Tissue and in Avian Leukosis Virus-Induced Lymphomas," J. Virol. 44(1):158-166 (1982). .
Nicolas, J.-F., and Rubenstein, J.L.R., "Retroviral Vectors," In: Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Rodriguez. R.L., et al., eds., Butterworths Publs., Stoneham, MA, pp. 493-513 (1988). .
Nilsen, T.W., et al., "c-erbB Activation in ALV-Induced Erythroblastosis: Novel RNA Processing and Promoter Insertion Result in Expression of an Amino-Truncated EGF Receptor," Cell 41:719-726 (1985). .
Niwa, H., et al., "An Efficient Gene-Trap Method Using Poly A Trap Vectors and Characterization of Gene-Trap Events," J. Biochem. 113:343-349 (1993). .
Nottenburg, C., and Varmus, H.E., "Features of the Chicken c-myc Gene That Influence the Structure of c-myc RNA in Normal Cells and Bursal Lymphomas," Mol. Cell. Biol. 6(8):2800-2806 (1986). .
Nouvel, P., "The mammalian genome shaping activity of reverse transcriptase," Genetica 93:191-201 (1994). .
Nusse, R., "The activation of cellular oncogenes by retroviral insertion," Trends in Genetics 2:244-247 (1986). .
Nusse, R., and Berns, A., "Cellular Oncogene Activation by Insertion of Retroviral DNA; Genes Identified by Provirus Tagging," In: Cellular Oncogenes Activation, Klein, G., ed., Marcel Dekker, Inc., New York, NY, pp. 95-119 (1988). .
Payne, G.S., et al., "Multiple arrangements of viral DNA and an activated host oncogene in bursal lymphomas," Nature 295:209-215 (1982). .
Peters, G., et al., "Tumorigenesis by Mouse Mammary Tumor Virus: Evidence for a Common Region for Provirus Integration in Mammary Tumors," Cell 33:369-377 (1983). .
Petitclerc, D., et al., "The effect of various introns and transcription terminators on the efficiency of expression vectors in various cultured cell lines and in the mammary gland of transgenic mice," J. Biotechnol. 40:169-178 (1995). .
Reinhart, T.A., et al., "RNA Splice Site Utilization by Simian Immunodeficiency Viruses Derived from Sooty Mangabey Monkeys," Virology 224:338-344 (Oct. 1, 1996). .
Robinson, W.S., "Molecular Events in the Pathogenesis of Hepadnavirus-Associated Hepatocellular Carcinoma," Annu. Rev. Med. 45:297-323 (1994). .
Salminen, M., et al., "Efficient Poly A Trap Approach Allows the Capture of Genes Specifically Active in Differentiated Embryonic Stem Cells and in Mouse Embryos," Develop. Dynamics 212:326-333 (Jun. 1998). .
Segal, D., et al., "Genetic Transformation of Drosophila Cells in Culture by P Element-Mediated Transposition," Somatic Cell and Mol. Genetics 22(2):159-165 (Mar. 1996). .
Shen-Ong, G.L.C., et al., "Activation of the c-myb Locus by Viral Insertional Mutagenesis in Plasmacytoid Lymphosarcomas," Science 226:1077-1080 (1984). .
Shen-Ong, G.L.C., et al., "Two Modes of c-myb Activation in Virus-Induced Mouse Myeloid Tumors," Mol. Cell. Biol. 6(2):380-392 (1986). .
Shin, S., and Steffen, D.L., "Frequent activation of the lck gene by promoter insertion and aberrant splicing in murine leukemia virus-induced rat lymphomas," Oncogene 8:141-149 (1993). .
Simpson, K., et al., "Stable Episomal Maintenance of Yeast Artificial Chromosomes in Human Cells," Mol. Cell. Biol. 16:5117-5126 (Sep. 1996). .
Stary, A., and Sarasin, A., "Simian virus 40 (SV40) large T antigen-dependent amplification of an Epstein-Barr virus-SV40 hybrid shuttle vector integrated into the human HeLa cell genome," J. Gen. Virol. 73:1679-1685 (1992). .
Steffen, D., "Proviruses are adjacent to c-myc in some murine leukemia virus-induced lymphomas," Proc. Natl. Acad. Sci. USA 81:2097-2101 (1984). .
Sun, T.-Q., "Human artificial episomal chromosomes for cloning large DNA fragments in human cells," Nature Genetics 8:33-41 (1994). .
Takami, Y., et al., "Cloning and Characterization of Human Papillomavirus Type 52 From Cervical Carcinomas in Indonesia," Int. J. Cancer 48:516-522 (1991). .
Tashiro, S., et al, "Identification of illegitimate recombination hot spot of the retinoic acid receptor alpha gene involved in 15;17 chromosomal translocation of acute promyelocytic leukemia," Oncogene 9:1939-1945 (1994). .
Tsichlis, P.N., et al., "A common region for proviral DNA integration in MoMuLV-induced rat thymic lymphomas," Nature 302:445-449 (1983). .
Unger, R.E., et al., "Simian Immunodeficiency Virus (SIVmac) Exhibits Complex Splicing for tat. rev. and env mRNA," Virology 182:177-185 (1991). .
van Ooyen, A., and Nusse, R., "Structure and Nucleotide Sequence of the Putative Mammary Oncogene int-1; Proviral Insertions Leave the Protein-Encoding Domain Intact," Cell 39:233-240 (1984). .
Voss, A.K., et al., "Efficiency Assessment of the Gene Trap Approach," Develop. Dynamics 212:171-180 (Jun. 1998). .
Westphal, E.M., et al., "A System for Shuttling 200-kb BAC/PAC Clones into Human Cells: Stable Extrachromosomal Persistence and Long-Term Ectopic Gene Activation," Human Gene Ther. 9:1863-1873 (Sep. 1998). .
Xiong, J.-W., et al., "Large-Scale Screening for Developmental Genes in Embryonic Stem Cells and Embryoid Bodies Using Retroviral Entrapment Vectors," Develop. Dynamics 212:181-197 (Jun. 1998). .
Zhou, H., and Duesberg, P.H., "A retroviral promoter is sufficient to convert proto-src to a transforming gene that is distinct from the src gene of Rous sarcoma virus," Proc. Natl. Acad. Sci. USA 87:9128-9132 (1990). .
Dialog File 351, Derwent WPI English language abstract for FR 2 707 091, 1995. .
Drug Discovery News, "Lexicon Genetics, Inc.," Genetic Engin. News 19:35 (Mar. 1999)..

Primary Examiner: Shukla; Ram R.
Attorney, Agent or Firm: Brown; Anne Athersys, Inc.

Parent Case Text

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser. No. 09/276,820, filed Mar. 26, 1999, entitled "COMPOSITIONS AND METHODS FOR NON-TARGETED ACTIVATION OF ENDOGENOUS GENES" which is a continuation-in-part of U.S. application Ser. No. 09/263,814 of John J. Harrington, Bruce Sherf, and Stephen Rundlett, entitled "Compositions and Methods for Non-targeted Activation of Endogenous Genes," filed Mar. 8, 1999 now abandoned, which is a continuation-in-part of U.S. application Ser. No. 09/253,022, filed Feb. 19, 1999 now abandoned, which is a continuation-in-part of U.S. application Ser. No. 09/159,643, filed Sep. 24, 1998 now abandoned, which is a continuation-in-part of U.S. application Ser. No. 08/941,223, filed Sep. 26, 1997 now abandoned, the disclosures of all of which are incorporated herein by reference in their entireties.

Claims

What is claimed is:

1. A method for expression of a protein encoded by a genomic DNA fragment, said method comprising: (A) transfecting a host cell in vitro with a vector comprising: (a) a promoter operably linked to: (i) an exon; and (ii) a splice donor that defines the 3' end of the exon; and (b) one or more selectable markers; and (c) a genomic DNA fragment, wherein said vector is not integrated into the genome of the host cell or, when integrated into the genome of the host cell, does not integrate by homologous recombination into or around the gene that is expressed on the genomic DNA fragment, wherein said promoter, exon and splice donor are operably linked to a gene or portion thereof in said genomic DNA fragment, said gene or portion thereof encoding a protein and comprising one or more splice acceptor sequences, wherein said exon and/or promoter is heterologous to said gene or portion thereof on the genomic DNA fragment, wherein said exon lacks a functional translation start codon and wherein said splice donor in a transcript containing said exon is spliced to said splice acceptor on the genomic DNA fragment, wherein a spliced transcript is produced comprising said exon and said gene or portion thereof encoding a protein, and wherein said transcript is translated in said cell.

2. The method of claim 1, wherein said vector further comprises a viral origin of replication.

3. The method of claim 2, wherein said viral origin of replication is Epstein Barr Virus oriP.

4. A vector comprising: (a) a promoter operably linked to: (i) an exon; and (ii) a splice donor site that define the 3' end of the exon; (b) one or more selectable markers; (c) one or more viral origins of replication; and (d) a genomic DNA fragment, wherein said promoter, exon and splice donor are operably linked to a gene or portion thereof in said genomic DNA fragment, said gene or portion thereof encoding a protein and comprising one or more splice acceptor sequences, wherein said exon and/or promoter is heterologous to said gene or portion thereof on the genomic DNA fragment, wherein said exon either lacks a functional translation start codon or encodes a functional translation start codon and an open reading frame that is not terminated by a stop codon, and wherein when said vector and genomic DNA fragment are introduced into a host cell that is capable of expressing protein, said splice donor in a transcript containing said exon is spliced to said splice acceptor on the genomic DNA fragment, wherein a spliced transcript is produced comprising said exon and said gene or portion thereof encoding a protein, and wherein said transcript is translated in said cell.

5. The vector of claim 1 or 4, wherein said selectable marker lacks a polyadenylation signal.

6. The vector of claim 4, further comprising one or more genes encoding one or more viral replication proteins.

7. The vector of claim 1 or 4, further comprising a sequence encoding an amplifiable marker.

8. A host cell in vitro comprising the vector and genomic DNA fragment of claim 4.

9. The host cell of claim 8, wherein said cell is an isolated cell.

10. A host cell in vitro comprising the vector of claim 5.

11. A host cell in vitro comprising the vector of claim 6.

12. A host cell in vitro comprising the vector of claim 7.

13. A host cell in vitro stably expressing a protein encoded by a genomic DNA fragment, wherein said host cell comprises a vector comprising: (a) a promoter operably linked to an exon defined at the 3' end by a splice donor, (b) a selectable marker, and (c) a genomic DNA fragment, wherein said vector is not integrated into the genome of the host cell or, when integrated into the genome of the host cell, does not integrate by homologous recombination into or around the gene that is expressed on the genomic DNA fragment, wherein said promoter, exon and splice donor are operably linked to a gene or portion thereof in said genomic DNA fragment, said gene or portion thereof encoding a protein and comprising one or more splice acceptor sequences, wherein said promoter and/or said exon is heterologous to said gene or portion thereof on said genomic DNA fragment, and wherein said splice donor in a transcript containing said exon is spliced to said splice acceptor on the genomic DNA fragment, wherein a spliced transcript is produced comprising said exon and said gene or portion thereof encoding a protein, and wherein said transcript is translated in said cell.

14. The host cell of claim 13, wherein said vector and genomic DNA fragment are integrated into the genome of said cell.

15. The host cell of claim 13, wherein said vector further comprises a viral origin of replication and is maintained within said host cell as an episome.

16. The host cell of claim 14, wherein said vector further comprises one or more additional selectable markers.

17. The host cell of claim 15, wherein said viral origin of replication is Epstein Barr Virus oriP.

18. A method for expression of a protein encoded by a genomic DNA fragment, said method comprising: (A) transfecting a host cell in vitro with a vector comprising (a) a promoter operably linked to: (i) an exon; and (ii) a splice donor site that defines the 3' end of the exon; (b) one or more selectable markers; (c) one or more viral origins of replication; and (d) a genomic DNA fragment, wherein said promoter, exon and splice donor are operably linked to a gene or portion thereof in a genomic DNA fragment, said gene or portion thereof encoding a protein and comprising one or more splice acceptor sequences, wherein said exon and/or said promoter is heterologous to said gene or portion thereof on the genomic DNA fragment, wherein said exon encodes a functional translation start codon and an open reading frame that is not terminated by a stop codon and wherein said splice donor in a transcript containing said exon is spliced to said splice acceptor on the genomic DNA fragment, wherein a spliced transcript is produced comprising said exon and said gene or portion thereof encoding a protein, and wherein said transcript is translated in said cell.

19. A method for detecting expression of a protein encoded by a genomic DNA fragment, said method comprising: (A) transfecting a host cell in vitro with a vector comprising (a) a promoter operably linked to: (i) an exon; and (ii) a splice donor site that defines the 3' end of the exon; and (b) one or more selectable markers; and (c) a genomic DNA fragment, wherein said vector is not integrated into the genome of the host cell or, when integrated into the genome of the host cell, does not integrate by homologous recombination into or around the gene that is expressed on the genomic DNA fragment, wherein said promoter, exon and splice donor are operably linked to a gene or portion thereof in a genomic DNA fragment, said gene or portion thereof encoding a protein and comprising one or more splice acceptor sequences, wherein said exon and/or said promoter is heterologous to said gene or portion thereof on the genomic DNA fragment, wherein said exon encodes a functional translation start codon and an open reading frame that is not terminated by a stop codon and wherein said splice donor in a transcript containing said exon is spliced to said splice acceptor on the genomic DNA fragment, wherein a spliced transcript is produced comprising said exon and said gene or portion thereof encoding a protein, and wherein said transcript is translated in said cell; and (B) screening said cell for expression of a desired protein.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention is in the fields of molecular biology and cellular biology. The invention is directed generally to activation of gene expression or causing over-expression of a gene by recombination methods in situ. More specifically, the invention is directed to activation of endogenous genes by non-targeted integration of specialized activation vectors, which are provided by the invention, into the genome of a host cell. The invention also is directed to methods for the identification, activation, and isolation of genes that were heretofore undiscoverable, and to host cells and vectors comprising such isolated genes. The invention also is directed to isolated genes, gene products, nucleic acid molecules, and compositions comprising such genes, gene products and nucleic acid molecules, that may be used in a variety of therapeutic and diagnostic applications. Thus, by the present invention, endogenous genes, including those associated with human disease and development, may be identified, activated, and isolated without prior knowledge of the sequence, structure, function, or expression profile of the genes.

2. Related Art

Identification and over-expression of novel genes associated with human disease is an important step towards developing new therapeutic drugs. Current approaches to creating libraries of cells for protein over-expression are based on the production and cloning of cDNA. Thus, in order to identify a new gene using this approach, the gene must be expressed in the cells that were used to make the library. The gene also must be expressed at sufficient levels to be adequately represented in the library. This is problematic because many genes are expressed only in very low quantities, in a rare population of cells, or during short developmental periods.

Furthermore, because of the large size of some mRNAs, it is difficult or impossible to produce full length cDNA molecules capable of expressing the biologically active protein. Lack of full-length cDNA molecules has also been observed for small mRNAs and is thought to be related to sequences in the message that are difficult to produce by reverse transcription or that are unstable during propagation in bacteria. As a result, even the most complete cDNA libraries express only a fraction of the entire set of possible genes.

Finally, many cDNA libraries are produced in bacterial vectors. Use of these vectors to express biologically active mammalian proteins is severely limited since most mammalian proteins do not fold correctly and/or are improperly glycosylated in bacteria.

Therefore, a method for creating a more representative library for protein expression, capable of facilitating faithful expression of biologically active proteins, would be extremely valuable.

Current methods for over-expressing proteins involve cloning the gene of interest and placing it, in a construct, next to a suitable promoter/enhancer, polyadenylation signal, and splice site, and introducing the construct into an appropriate host cell.

An alternative approach involves the use of homologous recombination to activate gene expression by targeting a strong promoter or other regulatory sequence to a previously identified gene.

WO 90/14092 describes in situ modification of genes, in mammalian cells, encoding proteins of interest. This application describes single-stranded oligonucleotides for site-directed modification of genes encoding proteins of interest. A marker may also be included. However, the methods are limited to providing an oligonucleotide sequence substantially homologous to a target site. Thus, the method requires knowledge of the site required for activation by site-directed modification and homologous recombination. Novel genes are not discoverable by such methods.

WO 91/06667 describes methods for expressing a mammalian gene in situ. With this method, an amplifiable gene is introduced next to a target gene by homologous recombination. When the cell is then grown in the appropriate medium, both the amplifiable gene and the target gene are amplified and there is enhanced expression of the target gene. As above, methods of introducing the amplifiable gene are limited to homologous recombination, and are not useful for activating novel genes whose sequence (or existence) is unknown.

WO 91/01140 describes the inactivation of endogenous genes by modification of cells by homologous recombination. By these methods, homologous recombination is used to modify and inactivate genes and to produce cells which can serve as donors in gene therapy.

WO 92/20808 describes methods for modifying genomic target sites in situ. The modifications are described as being small, for example, changing single bases in DNA. The method relies upon genomic modification using homologous DNA for targeting.

WO 92/19255 describes a method for enhancing the expression of a target gene, achieved by homologous recombination in which a DNA sequence is integrated into the genome or large genomic fragment. This modified sequence can then be transferred to a secondary host for expression. An amplifiable gene can be integrated next to the target gene so that the target region can be amplified for enhanced expression. Homologous recombination is necessary to this targeted approach.

WO 93/09222 describes methods of making proteins by activating an endogenous gene encoding a desired product. A regulatory region is targeted by homologous recombination and replacing or disabling the region normally associated with the gene whose expression is desired. This disabling or replacement causes the gene to be expressed at levels higher than normal.

WO 94/12650 describes a method for activating expression of and amplifying an endogenous gene in situ in a cell, which gene is not expressed or is not expressed at desired levels in the cell. The cell is transfected with exogenous DNA sequences which repair, alter, delete, or replace a sequence present in the cell or which are regulatory sequences not normally functionally linked to the endogenous gene in the cell. In order to do this, DNA sequences homologous to genomic DNA sequences at a preselected site are used to target the endogenous gene. In addition, amplifiable DNA encoding a selectable marker can be included. By culturing the homologously recombinant cells under conditions that select for amplification, both the endogenous gene and the amplifiable marker are co-amplified and expression of the gene increased.

WO 95/31560 describes DNA constructs for homologous recombination. The constructs include a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site. The targeting is achieved by homologous recombination of the construct with genomic sequences in the cell and allows the production of a protein in vitro or in vivo.

WO 96/29411 describes methods using an exogenous regulatory sequence, an exogenous exon, either coding or non-coding, and a splice donor site introduced into a preselected site in the genome by homologous recombination. In this application, the introduced DNA is positioned so that the transcripts under control of the exogenous regulatory region include both the exogenous exon and endogenous exons present in either the thrombopoietin, DNase I, or .beta.-interferon genes, resulting in transcripts in which the exogenous and exogenous exons are operably linked. The novel transcription units are produced by homologous recombination.

U.S. Pat. No. 5,272,071 describes the transcriptional activation of transcriptionally silent genes in a cell by inserting a DNA regulatory element capable of promoting the expression of a gene normally expressed in that cell. The regulatory element is inserted so that it is operably linked to the normally silent gene. The insertion is accomplished by means of homologous recombination by creating a DNA construct with a segment of the normally silent gene (the target DNA) and the DNA regulatory element used to induce the desired transcription.

U.S. Pat. No. 5, 578,461 discusses activating expression of mammalian target genes by homologous recombination. A DNA sequence is integrated into the genome or a large genomic fragment to enhance the expression of the target gene. The modified construct can then be transferred to a secondary host. An amplifiable gene can be integrated adjacent to the target gene so that the target region is amplified for enhanced expression.

Both of the above approaches (construction of an over-expressing construct by cloning or by homologous recombination in vivo) require the gene to be cloned and sequenced before it can be over-expressed. Furthermore, using homologous recombination, the genomic sequence and structure must also be known.

Unfortunately, many genes have not yet been identified and/or sequenced. Thus, a method for over-expressing a gene of interest, whether or not it has been previously cloned, and whether or not its sequence and structure are known, would be useful.

BRIEF SUMMARY OF THE INVENTION

The invention is, therefore, generally directed to methods for over-expressing an endogenous gene in a cell, comprising introducing a vector containing a transcriptional regulatory sequence into the cell, allowing the vector to integrate into the genome of the cell by non-homologous recombination and allowing over-expression of the endogenous gene in the cell. The method does not require previous knowledge of the sequence of the endogenous gene or even of the existence of the gene. Hence, the invention is directed to non-targeted gene activation, which as used herein means the activation of endogenous genes by non-targeted or non-homologous (as opposed to targeted or homologous) integration of specialized activation vectors into the genome of a host cell.

The invention also encompasses novel vector constructs for activating gene expression or over-expressing a gene through non-homologous recombination. The novel construct lacks homologous targeting sequences. That is, it does not contain nucleotide sequences that target host cell DNA and promote homologous recombination at the target site, causing over-expressing of a cellular gene via the introduced transcriptional regulatory sequence.

Novel vector constructs include a vector containing a transcriptional regulatory sequence operably linked to an unpaired splice donor sequence and further contains one or more amplifiable markers.

Novel vector constructs include constructs with a transcriptional regulatory sequence operably linked to a translational start codon, a signal secretion sequence, and an unpaired splice donor site; constructs with a transcriptional regulatory sequence, operably linked to a translation start codon, an epitope tag, and an unpaired splice donor site; constructs containing a transcriptional regulatory sequence operably linked to a translational start codon, a signal sequence and an epitope tag, and an unpaired splice donor site; constructs containing a transcriptional regulatory sequence operably linked to a translation start codon, a signal secretion sequence, an epitope tag, and a sequence-specific protease site, and an unpaired splice donor site.

The vector construct can contain one or more selectable markers for recombinant host cell selection. Alternatively, selection can be effected by phenotypic selection for a trait provided by the activated endogenous gene product.

These vectors, and indeed any of the vectors disclosed herein, and variants of the vectors that will be readily recognized by one of ordinary skill in the art, can be used in any of the methods described herein to form any of the compositions producible by these methods.

The transcriptional regulatory sequence used in the vector constructs of the invention includes, but is not limited to, a promoter. In preferred embodiments, the promoter is a viral promoter. In highly preferred embodiments, the viral promoter is the cytomegalovirus immediate early promoter. In alternative embodiments, the promoter is a cellular, non-viral promoter or inducible promoter.

The transcriptional regulatory sequence used in the vector construct of the invention may also include, but is not limited to, an enhancer. In preferred embodiments, the enhancer is a viral enhancer. In highly preferred embodiments, the viral enhancer is the cytomegalovirus immediate early enhancer. In alternative embodiments, the enhancer is a cellular non-viral enhancer.

In preferred embodiments of the methods described herein, the vector construct be, or may contain, linear RNA or DNA.

The cell containing the vector may be screened for expression of the gene.

The cell over-expressing the gene can be cultured in vitro under conditions favoring the production, by the cell, of desired amounts of the gene product (also referred to interchangeably herein as the "expression product") of the endogenous gene that has been activated or whose expression has been increased. The expression product can then be isolated and purified to use, for example, in protein therapy or drug discovery.

Alternatively, the cell expressing the desired gene product can be allowed to express the gene product in vivo. In certain such aspects of the invention, the cell containing a vector construct of the invention integrated into its genome may be introduced into a eukaryote (such as a vertebrate, particularly a mammal, more particularly a human) under conditions favoring the overexpression or activation of the gene by the cell in vivo in the eukaryote. In related such aspects of the invention, the cell may be isolated and cloned prior to being introduced into the eukaryote.

The invention is also directed to methods for over-expressing an endogenous gene in a cell, comprising introducing a vector containing a transcriptional regulatory sequence and one or more amplifiable markers into the cell, allowing the vector to integrate into the genome of the cell by non-homologous recombination, and allowing over-expression of the endogenous gene in the cell.

The cell containing the vector may be screened for over-expression of the gene.

The cell over-expressing the gene is cultured such that amplification of the endogenous gene is obtained. The cell can then be cultured in vitro so as to produce desired amounts of the gene product of the amplified endogenous gene that has been activated or whose expression has been increased. The gene product can then be isolated and purified.

Alternatively, following amplification, the cell can be allowed to express the endogenous gene and produce desired amounts of the gene product in vivo.

It is to be understood, however, that any vector used in the methods described herein can include one or more amplifiable markers. Thereby, amplification of both the vector and the DNA of interest (i.e., containing the over-expressed gene) occurs in the cell, and further enhanced expression of the endogenous gene is obtained. Accordingly, methods can include a step in which the endogenous gene is amplified.

The invention is also directed to methods for over-expressing an endogenous gene in a cell comprising introducing a vector containing a transcriptional regulatory sequence and an unpaired splice donor sequence into the cell, allowing the vector to integrate into the genome of the cell by non-homologous recombination, and allowing over-expression of the endogenous gene in the cell.

The cell containing the vector may be screened for expression of the gene.

The cell over-expressing the gene can be cultured in vitro so as to produce desirable amounts of the gene product of the endogenous gene whose expression has been activated or increased. The gene product can then be isolated and purified.

Alternatively, the cell can be allowed to express the desired gene product in vivo.

The vector construct can consist essentially of the transcriptional regulatory sequence.

The vector construct can consist essentially of the transcriptional regulatory sequence and one or more amplifiable markers.

The vector construct can consist essentially of the transcriptional regulatory sequence and the splice donor sequence.

Any of the vector constructs of the invention can also include a secretion signal sequence. The secretion signal sequence is arranged in the construct so that it will be operably linked to the activated endogenous protein. Thereby, secretion of the protein of interest occurs in the cell, and purification of that protein is facilitated. Accordingly, methods can include a step in which the protein expression product is secreted from the cell.

The invention also encompasses cells made by any of the above methods. The invention encompasses cells containing the vector constructs, cells in which the vector constructs have integrated into the cellular genome, and cells which are over-expressing desired gene products from an endogenous gene, over-expression being driven by the introduced transcriptional regulatory sequence.

The cells can be isolated and cloned.

The methods can be carried out in any cell of eukaryotic origin, such as fungal, plant or animal. In preferred embodiments, the methods of the invention may be carried out in vertebrate cells, and particularly mammalian cells including but not limited to rat, mouse, bovine, porcine, sheep, goat and human cells, and more particularly in human cells.

A single cell made by the methods described above can over-express a single gene or more than one gene. More than one gene in a cell can be activated by the integration of a single type of construct into multiple locations in the genome. Similarly, more than one gene in a cell can be activated by the integration of multiple constructs (i.e., more than one type of construct) into multiple locations in the genome. Therefore, a cell can contain only one type of vector construct or different types of constructs, each capable of activating an endogenous gene.

The invention is also directed to methods for making the cells described above by one or more of the following: introducing one or more of the vector constructs of the invention into a cell; allowing the introduced construct(s) to integrate into the genome of the cell by non-homologous recombination; allowing over-expression of one or more endogenous genes in the cell; and isolating and cloning the cell. The invention is also directed to cells produced by such methods, which may be isolated cells.

The invention also encompasses methods for using the cells described above to over-express a gene, such as an endogenous cellular gene, that has been characterized (for example, sequenced), uncharacterized (for example, a gene whose function is known but which has not been cloned or sequenced), or a gene whose existence was, prior to over-expression, unknown. The cells can be used to produce desired amounts of an expression product in vitro or in vivo. If desired, this expression product can then be isolated and purified, for example by cell lysis or by isolation from the growth medium (as when the vector contains a secretion signal sequence).

The invention also encompasses libraries of cells made by the above described methods. A library can encompass all of the clones from a single transfection experiment or a subset of clones from a single transfection experiment. The subset can over-express the same gene or more than one gene, for example, a class of genes. The transfection can have been done with a single construct or with more than one construct.

A library can also be formed by combining all of the recombinant cells from two or more transfection experiments, by combining one or more subsets of cells from a single transfection experiment or by combining subsets of cells from separate transfection experiments. The resulting library can express the same gene, or more than one gene, for example, a class of genes. Again, in each of these individual transfections, a unique construct or more than one construct can be used.

Libraries can be formed from the same cell type or different cell types.

The invention is also directed to methods for making libraries by selecting various subsets of cells from the same or different transfection experiments.

The invention is also directed to methods of using the above-described cells or libraries of cells to over-express or activate endogenous genes, or to obtain the gene expression products of such over-expressed or activated genes. According to this aspect of the invention, the cell or library may be screened for the expression of the gene and cells that express the desired gene product may be selected. The cell can then be used to isolate or purify the gene product for subsequent use. Expression in the cell can occur by culturing the cell in vitro, under conditions favoring the production of the expression product of the endogenous gene by the cell, or by allowing the cell to express the gene in vivo.

In preferred embodiments of the invention, the methods include a process wherein the expression product is isolated or purified. In highly preferred embodiments, the cells expressing the endogenous gene product are cultured under conditions favoring production of sufficient amounts of gene product for commercial application, and especially for diagnostic, therapeutic and drug discovery uses.

Any of the methods can further comprise introducing double-strand breaks into the genomic DNA in the cell prior to or simultaneously with vector integration.

The invention also is directed to vector constructs that are useful for activating expression of endogenous genes and for isolating the mRNA and cDNA corresponding to the activated genes.

In one such embodiment, the vector construct may comprise (a) a first transcriptional regulatory sequence operably linked to a first unpaired splice donor sequence; (b) a second transcriptional regulatory sequence operably linked to a second unpaired splice donor sequence; and (c) a linearization site, which may be located between the first and second transcriptional regulatory sequences. According to the invention, when the vector construct is transformed into a host cell and then integrates into the genome of the host cell, the first transcriptional regulatory sequence is preferably in an inverted orientation relative to the orientation of the second transcriptional regulatory sequence. In certain preferred such embodiments, the vector may be rendered linear by cleavage at the linearization site.

In another embodiment, the invention provides a linear vector construct having a 3' end and a 5' end, comprising a transcriptional regulatory sequence operably linked to an unpaired spliced donor site, wherein the transcriptional regulatory sequence is oriented in the linear vector construct in an orientation that directs transcription towards the 3' end or the 5' end of the linear vector construct.

In another embodiment, the invention provides a vector construct comprising, in sequential order, (a) a transcriptional regulatory sequence, (b) an unpaired splice donor site, (c) a rare cutting restriction site, and (d) a linearization Site.

In another embodiment, the invention provides a vector construct comprising (a) a first transcriptional regulatory sequence operably linked to a selectable marker lacking a polyadenylation signal; and (b) a second transcriptional regulatory sequence operably linked to an exon-splice donor site complex, wherein the first transcriptional regulatory sequence is in the same orientation in the vector construct as is the second transcriptional regulatory sequence, and wherein the first transcriptional regulatory sequence is upstream of the second transcriptional regulatory sequence in the vector construct.

In additional embodiments, the invention provides vector constructs comprising a transcriptional regulatory sequence operably linked to a selectable marker lacking a polyadenylation signal, and further comprising an unpaired splice donor site.

In another embodiment, the invention provides vector constructs comprising a first transcriptional regulatory sequence operably linked to a selectable marker lacking a polyadenylation signal, and further comprising a second transcriptional regulatory sequence operably linked to an unpaired splice donor site.

According to the invention, the transcriptional regulatory sequence (or first or second transcriptional regulatory sequence, in vector constructs having more than one transcriptional regulatory sequence) may be a promoter, an enhancer, or repressor, and is preferably a promoter, including an animal cell promoter, a plant cell promoter, or a fungal cell promoter, most preferably a promoter selected from the group consisting of a CMV immediate early gene promoter, an SV40 T antigen promoter, and a .beta.-actin promoter. Other promoters of animal, plant, or fungal cell origin that may be used in accordance with the invention are known in the art and will be familiar to one of ordinary skill in view of the teachings herein.

The selectable marker used in the vector constructs of the invention may be any marker or marker gene that, upon integration of a vector containing the selectable marker into the host cell genome, permits the selection of a cell containing or expressing the marker gene. Suitable such selectable markers include, but are not limited to, a neomycin gene, a hypoxanthine phosphribosyl transferase gene, a puromycin gene, a dihydrooratase gene, a glutamine syntlietase gene, a histidine D gene, a carbamyl phosphate synthase gene, a dihydrofolate reductase gene, a multidrug resistance 1 gene, an aspartate transcarbamylase gene, a xanthine-guanine phosphoribosyl transferase gene, an adenosine deaminase gene, and a thyrnidine kinase gene.

In related embodiments, the invention provides vector constructs comprising a positive selectable marker, a negative selectable marker, and an unpaired splice donor site, wherein the positive and negative selectable markers and the splice donor site are oriented in the vector construct in an orientation that results in expression of the positive selectable marker in active form, and either non-expression of said negative selectable marker or expression of the negative selectable marker in inactive form, when the vector construct is integrated into the genome of a eukaryotic host cell and activates an endogenous gene in the genome. In certain preferred such embodiments, either the positive selection marker, the negative selection marker, or both, may lack a polyadenylation signal. The positive selection marker used in these aspects of the invention may be any selection marker that, upon expression, produces a protein capable of facilitating the isolation of cells expressing the marker, including but not limited to a neomycin gene, a hypoxanthine phosphribosyl transferase gene, a puromycin gene, a dihydrooratase gene, a glutamine synthetase gene, a histidine D gene, a carbamyl phosphate synthase gene, a dihydrofolate reductase gene, a multidrug resistance 1 gene, an aspartate transcarbamylase gene, a xanthine-guanine phosphoribosyl transferase gene, or an adenosine deaminase gene. Analogously, the negative selection marker used in these aspects of the invention may be any selection marker that, upon expression, produces a protein capable of facilitating removal of cells expressing the marker, including but not limited to a hypoxanthine phosphribosyl transferase gene, a thymidine kinase gene, or a diphtheria toxin gene.

The invention also is directed to eukaryotic host cells, which may be isolated host cells, comprising one or more of the vector constructs of the invention. Preferred such eukaryotic host cells include, but are not limited to, animal cells (including, but not limited to, mammalian (particularly human) cells, insect cells, avian cells, annelid cells, amphibian cells, reptilian cells, and fish cells), plant cells, and fungal (particularly yeast) cells. In certain such host cells, the vector construct may be integrated into the genome of the host cell.

The invention also is directed to primer molecules comprising a PCR-amplifiable sequence and a degenerate 3' terminus. Primer molecules according to this aspect of the invention preferably have the general structure:

wherein a is a whole number from 1 to 100 (preferably from 10 to 30), X is a PCR-amplifiable sequence consisting of a nucleic acid sequence of about 10-20 nucleotides in length, N is any nucleotide, and b is a whole number from 0 to 6. One preferred such primer has the nucleotide sequence 5'-TTTTTTTTTTTTCGTCAGCGGCCGCATCNNNNTTTATT-3' (SEQ ID NO:10). In related embodiments, the primer molecules according to this aspect of the invention may be biotinylated.

The invention also is directed to methods for first strand cDNA synthesis comprising (a) annealing a first primer of the invention (such as the primer described above) to an RNA template molecule to form an first primer-RNA complex, and (b) treating this first primer-RNA complex with reverse transcriptase and one or more deoxynucleoside triphosphate molecules under conditions favoring the reverse transcription of the first primer-RNA complex to synthesize a first strand cDNA.

The invention also is directed to methods for isolating activated genes, particularly from a host cell genome. These methods of the invention exploit the structure of the mRNA molecules produced using the non-targeted gene activation vectors of the invention. One such method of the invention comprises, for example, (a) introducing a vector construct comprising a transcriptional regulatory sequence and an unpaired splice donor site into a host cell (preferably one of the eukaryotic host cells described above), (b) allowing the vector construct to integrate into the genome of the host cell by non-homologous recombination, under conditions such that the vector activates an endogenous gene comprising an exon in the genome, (c) isolating RNA from the host cell, (d) synthesizing first strand cDNA according to the method of the invention described above, (e) annealing a second primer specific for the vector-encoded exon to the first strand cDNA to create a second primer-first strand cDNA complex, and (f) contacting the second primer-first strand cDNA complex with a DNA polymerase under conditions favoring the production of a second strand cDNA substantially complementary to the first strand cDNA. Methods according to this aspect of the invention may comprise one or more additional steps, such as treating the second strand cDNA with a restriction enzyme that cleaves at a restriction site located on the vector downstream of the unpaired splice donor site, or amplifying the second strand cDNA using a third primer specific for the vector-encoded exon and a fourth primer specific for the second primer. The invention also is directed to isolated genes produced according to these methods, and to vectors (which may be expression vectors) and host ce