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(12) United States Patent (10) Patent No.: US 6,790,941 B2 Barbas, III Et Al

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(12) United States Patent (10) Patent No.: US 6,790,941 B2 Barbas, III Et Al USOO6790941B2 (12) United States Patent (10) Patent No.: US 6,790,941 B2 Barbas, III et al. (45) Date of Patent: *Sep. 14, 2004 (54) ZINC FINGER PROTEIN DERIVATIVES AND 5,702.914. A 12/1997 Evans et al. METHODS THEREFOR 5,789,538 A * 8/1998 Rebar et al. ................ 530/324 5,792,640 A 8/1998 Chandrasegaran (75) Inventors: Carlos F. Barbas, III, San Diego, CA 5,869,618 A 2/1999 Lippman et al. (US); Joel M. Gottesfeld, Del Mar, CA 5,871,902 A 2/1999 Weininger et al. (US); Peter E. Wright, La Jolla, CA 5,871,907 A 2/1999 Winter et al. s s s 5,916,794. A 6/1999 Chandrasegaran (US) 5,939,538 A 8/1999 Leavitt et al. (73) Assignee: The Scripps Research Institute, La 6,001.885. A 12/1999 Vega et al. Jolla, CA (US) 6,007,988 A 12/1999 Choo et al. s 6,013,453 A 1/2000 Choo et al. ( c: ) Notice: Subject to any disclaimer, the term of this 6.242,568 B1 6/2001 Barbas et al. ............... 530/350 patent is extended or adjusted under 35 2002/0081.614 A1 6/2002 Case et al. U.S.C. 154(b) by 0 days. FOREIGN PATENT DOCUMENTS This patent is Subject to a terminal dis- WOEP WO95/19431875567 11/1998y: claimer. WO WO 96/06110 2/1996 WO WO 96/06166 2/1996 (21) Appl. No.: 09/500,700 WO WO 96/11267 4/1996 22) Filled: Feb. 9,9 2000 WO WO 96/20951 7/1996 (65) Prior Publication Data W W o: 19. US 2003/0059767 A1 Mar. 27, 2003 W W 35 i.FC Related U.S. Application Data WO WO 98/53058 11/1998 WO WO 98/53059 11/1998 (63) Continuation of application No. 08/863,813, filed on May WO WO 98/53060 11/1998 27, 1997, now Pat. No. 6,140,466, which is a continuation- WO WO 98/54311 12/1998 in-part of application No. 08/676,318, filed as application No. PCT/US95/00829 on Jan. 18, 1995, now Pat. No. OTHER PUBLICATIONS 6.242,568, which is a continuation-in-part of application No. 66 08/312,604, filed on Sep. 28, 1994, now abandoned, which Celenza, J. L., et al., Science, vol. 233, "A yeast gene that is a continuation-in-part of application No. 08/183,119, filed is essential for release from glucose repression encodes a on Jan. 18, 1994, now abandoned. protein kinase”, pp. 1175-1180, 1986.* (51) Int. Cl.'7 .................................................. co7K 100 Kinzler,nzler, K.W.,K. W., et al., Nature, Vol.vol. 332,s TheC GL1 geneCC. S a (52) U.S. Cl 530/400, 530/350, 530/358; member of the Kruppel family of Zinc finger proteins”, pp. Oa - 1 - O - - - - - 53633,53632.435.60?.- - - - - - - - - - - - - - - - - - s s 435,607.s 371-374, 1988. c: 435/455; 435/471; 435/235.1; 435/320.1; (List continued on next page.) 435/325:s 435/252.3 Primaryy Examiner-JerryE. -G LeffCS (58) Field of Search ................................. 530/350, 358, (74) Attorney, Agent, or Firm-Gray Cary Ware & 530/400; 536/23.1, 23.2; 435/69.1, 69.7, Friedenrich, LLP 6,455, 471,235.1, 320.1, 325, 252.3 (57) ABSTRACT (56) References Cited Zinc finger proteins of the Cys-His type represent a class of U.S. PATENT DOCUMENTS malleable DNA binding proteins which may be selected to 4,990,607 A * 2/1991 Katagiri et al. ............ 536/23.6 bind diverse Sequences. Typically, Zinc finger proteins con 5,096,814 A 3/1992 Aivasidis et al. taining three Zinc finger domains, like the murine transcrip 5,096,815 A * 3/1992 Ladner et al. ............. 435/69.1 tion factor Zif268 and the human transcription factor Sp1, 5,198.346 A 3/1993 Ladner et al. ............. 435/69.1 5,223,409 A 6/1993 Ladner et al. bind nine contiguous base pairs (bp). To create a class of 5.243,041 A * 9/1993 Fernandez-Pol ........... 536/23.5 proteins which would be generally applicable to target 5,302,519 A 4/1994 Blackwood et al. unique sites within complex genomes, the present invention 5,324.638 A 6/1994 Tao et al. .................. 435/69.1 provides a polypeptide linker that fuses two three-finger 5,324.818 A 6/1994 Nabel et al. ................ 530/350 proteins. Two Six-fingered proteins were created and dem 5,324.819 A 6/1994 Oppermann et al. onstrated to bind 18 contiguous bp of DNA in a Sequence 5,340,739 A * 8/1994 Stevens et al. .......... 435/240.1 Specific fashion. Expression of these proteins as fusions to 5,348.864 A 9/1994 Barbacid activation or repression domains allows transcription to be 5,350,840 A * 9/1994 Call et al................... 536/23.1 specifically up or down modulated within cells. Polydactyl 5,356.802 A 10/1994 Chandrasegaran Zinc finger proteins are broadly applicable as genome 5,376,530 A * 12/1994 De The et al................... 435/6 5,403,484 A 4/1995 Ladner et al. ........... 435/235.1 Specific transcriptional Switches in gene therapy Strategies 5,436,150 A 7/1995 Chandrasegaran and the development of novel transgenic plants and animals. 5.487,994. A 1/1996 Chandrasegaran Such proteins are useful for inhibiting, activating or enhanc 5,498,530 A 3/1996 Schatz et al. ing gene expression from a Zinc finger-nucleotide binding 5,578.483. A 11/1996 Evans et al. motif containing promoter or other transcriptional control 5,597.693 A * 1/1997 Evans et al. ................... 435/6 element, as well as a structural gene or RNA sequence. 5,639,592 A 6/1997 Evans et al. 5,674,738 A 10/1997 Abramson et al. 9 Claims, 26 Drawing Sheets US 6,790,941 B2 Page 2 OTHER PUBLICATIONS Crozatier, M., et al., Genetics, Vol. 131, "Single amino acid eXchanges in Separate domains of the Drosophila Serendipity Singh, H., et al., Cell, vol. 52, “Molecular cloning of an delta Zinc finger protein cause embryonic and SeX biased enhancer binding protein: Isolation by Screening of an lethality", pp. 905–916, 1992.* expression library with a recognition Site DNA, pp. Thukral, S. K., et al., Molecular and Cellular Biology, vol. 415-423, 1988.* 12, “Mutations in the zinc fingers of ADR1 that change the Wright, J. J., et al., Science, V I. 248, “Expression f a Specificity of DNA binding and transactivation’, pp. zinc-finger gene in HTLV-I- and HTLV-II-transformed 2784-2792, 1992.* cells”, pp. 588-591, 1990.* Quigley, C. A., et al., Molecular Endocrinology, Vol. 6, Debs, R. J., et al., The Journal of Biological Chemistry, vol. “Complete androgen insensitivity due to deletion of exon C 265, "Regulation of gene expression in Vivo by liposome of the androgen receptor gene highlights the functional mediated delivery of a purified transcription factor', pp. importance of the Second Zinc finger of the androgen recep 10189-10192, 1990.* tor in vivo”, pp. 1103-, 1992.* Bergqvist, A., et al., Nucleic Acids Research, Vol. 18, "LOSS Jacobs, G. H., The EMBO Journal, vol. 11, "Determination of DNA-binding and new transcriptional trans-activation of the base recognition positions of Zinc fingers from function in polyomavirus large T-antigen with mutation of sequence analysis”, pp. 4507-4517, 1992.* Zinc finger motif", pp. 2715–2720, 1990.* Yu, M., et al., Proceedings of the National Academy of Rauscher, F. J., et al., Science, vol. 250, “Binding of the Sciences, U.S.A., vol. 90, “A hairpin ribozyme inhibits Wilms tumor locus zinc finger protein to the EGR-1 expression of diverse Strains of human immunodeficiency consensus sequence', pp. 1259-1262, 1990.* virus type 1", pp. 6340-6344, 1993.* South, T. L., et al., Biochemistry, vol. 29, “The nucleocapsid Rollins, M. B., et al., Molecular and Cellular Biology, vol. protein isolated from HIV-1 particles binds Zinc and forms 13, “Role of TFIIIA zinc fingers in Vivo: Analysis f sin retroviral-type zinc fingers", pp., 7786–7789, 1990.* gle-finger function in developing Xenopus embryos', pp. Kudla, B., et al., The EMBO Journal, vol. 9, “The regulatory gene are A mediating nitrogen metabolite repression in 4776–4783, 1993.* Aspergillus nidulans. Mutations affecting Specificity of gene Julian, N., et al., FEBS Letters, vol. 331, “Replacement of activation after a loop residue of a putative Zinc finger”, pp. His23 by Cys in a zinc finger of HIV-1 NCp7 led to change 1355-1464, 1990.* in 1H NMR-derived 3D structure and to a loss of biological Webster, L. C., et al., Proceedings of the National Academy activity”, pp. 43–48, 1993.* of Sciences, USA, Vol. 88, “Conversion of the E1A Cys4 Jamieson, A., C., et al., Biochemistry, vol. 33, "In vitro Zinc finger to a non-functional His2,CyS2 Zinc finger by a Selection of Zinc fingers with altered DNA-binding Speci single point mutation”, pp. 9989-9993, 1991.* ficity”, pp. 5689–5695, 1994.* Thiesen, H.-J., et al., Biochemical and Biophysical Bellefroid, E., J., et al., The EMBO Journal, vol. 12, Research Communications, vol. 175, “Amino acid Substitu “Clustered organization of homologous KRAB Zinc-finger tions in the SP1 Zinc finger domain alter the DNA binding genes with enhanced expression in human T lymphoid affinity to cognate SP1 target site', pp. 333-338, 1991.* cells”, pp. 1363-1374, 1993.* Ray, A., et al., Proceedings f the National Academy of Saleh, M., et al., American Journal of Human Genetics, Vol. Sciences, U.S.A., vol.
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