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(12) United States Patent (10) Patent No.: US 7820,442 B2 Petersen-Mahrt Et Al

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(12) United States Patent (10) Patent No.: US 7820,442 B2 Petersen-Mahrt Et Al USOO7820442B2 (12) United States Patent (10) Patent No.: US 7820,442 B2 Petersen-Mahrt et al. (45) Date of Patent: Oct. 26, 2010 (54) ACTIVATION INDUCED DEAMINASE (AID) Yamanaka et al., Apollipoprotein B mRNA-editing protein induces hepatocellular carcinoma and dysplasia in transgenic animals, Proc. (75) Inventors: Svend K. Petersen-Mahrt, Cambridge Natl. Acad. Sci. USA, Biochemisty, Aug. 1995, vol. 92, pp. 8483 (GB); Reuben Harris, Cambridge (GB); 8487. International Search Report Related To PCT/GB 03/02002. Michael Samuel Neuberger, Cambridge Martin A. et al., Activation-Induced Cytidine Deaminase Turns On (GB); Rupert Christopher Somatic Hypermutation. In Hybridomas, Nature, vol. 415, Feb. 24. Landsdowne Beale, Cambridge (GB) 2002, pp. 802-806. Jarmuz A. et al. An Anthropoid-Specific Locus Of Orphan C To U (73) Assignee: Medical Research Council, London RNA-Editing Enzymes On Chromosome-22, Genomics, vol. 79, No. (GB) 3, Mar. 2002, pp. 285-296. Manis JP, Mechanism And Control OfClass-Switch Recombination, (*) Notice: Subject to any disclaimer, the term of this Trends in Immunology, Elsevier, Cambridge, GB, vol. 23, No. 1, Jan. patent is extended or adjusted under 35 1, 2002, pp. 31-39. U.S.C. 154(b) by 0 days. MacGinnitie A.J. et al., Mutagenesis. Of Apobec-1. The Catalytic Subunit Of The Mammalian Apollipoprotein B mRNA Editing (21) Appl. No.: 10/985,321 Enzyme, Reveals Distinct Domains That Meiate Cytosine Nucleoside Deaminase, RNA Binding, and RNA Editing Activity, The Journal of Biological Chemistry, vol. 270, No. 24. Jun. 16, 1995, (22) Filed: Nov. 10, 2004 pp. 14768-14775. Okazaki IM. et al., The Aid Enzyme Induces Class Switch Recom (65) Prior Publication Data bination. In Fibroblasts, Nature, vol. 416, Mar. 21, 2002, pp.340-345. US 2006/OO19262 A1 Jan. 26, 2006 Yelamos J., et al., Targeting of Non-IG Sequences. In Place Of The V Segment By Somatic Hypermutation, Nature, MacMillan Journals Related U.S. Application Data Ltd. London, GB, vol. 376, No. 6537, 1995, pp. 225-229. Harris RS, Petersen-Mahrt SK. et al., RNA Editing Enzyme (63) Continuation of application No. PCT/GB03/02002, APOBEC1 And Some Of Its Homologs Can Act AS DNA Mutators, filed on May 9, 2003. Molecular Cell, vol. 10, No. 2002, pp. 1247-1253. Petersen-Mahrt SK. et al., Aid Mutates E. coli suggesting a DNA (30) Foreign Application Priority Data Deamination Mechanism for Antibody Diversification, Nature, vol. 418, Jul. 4, 2002, pp. 99-103. May 10, 2002 (GB) ................................. O2 10755.5 Yoshikawa, K. et al., “AID Enzyme-Induced Hypermutation in an Jun. 14, 2002 (GB) ... ... O2137.51.1 Actively Transcribed Gene in Fibroblasts.” Science 296:2033-2036 Jul. 29, 2002 (GB) ................................. O217519.8 (2002). Conticello, S.G. et al., “Evolution of the AID/APOBEC Family of (51) Int. Cl. Polynucleotide (Deoxy)cytidine Deaminases.” Molecular Biology CI2N 15/87 (2006.01) and Evolution 22(2):367-377 (2005). Cumbers, S.J. et al., “Generation and iterative affinity maturation of CI2N IS/00 (2006.01) antibodies in vitro using hypermutating B-cell lines.” Nature GOIN33/554 (2006.01) Biotech. 20:1129-1134 (2002). GOIN33/569 (2006.01) Storb, U. et al., “Somatic hypermutation of immunoglobulin and CI2P I/O (2006.01) non-immunoglobulin genes.” Phil. Trans. R. Soc. Lond. B356:13-19 CI2P39/00 (2006.01) (2001). CI2P2/06 (2006.01) Wang, L. et al., “Evolution of new nonantibody proteins via iterative CI2P 2L/04 (2006.01) somatic hypermutation.” PNAS USA 101(48): 16745-16749 (2004). C7H 2L/02 (2006.01) Kawai et al., “Isolation and Analysis of Lipase-Overproducing C7H 2L/04 (2006.01) Mutants of Serratia marcescens,” Journal of Bioscience and Bioengineering, 91(4): 409-415 (2001). (52) U.S. Cl. ........................ 435/463; 435/7.32:435/41; Stefan et al., “Directed Evolution of B-Galactosidase from 435/42:435/69.1; 435/69.7:435/.440,536/23.1; Escherichia coli by Mutator Strains Defective in the 3'-5' 536/23.2:536/23.4 Exonuclease Activity of DNA polymerase III.” FEBS Letters, 493: (58) Field of Classification Search ..................... 435/6, 139-143 (2001). 435/7.1, 325, 326,455, 7.32, 41, 42, 69.1, 435/69.7, 440, 463: 536/23.1, 23.2, 23.4 (Continued) See application file for complete search history. Primary Examiner Thaian N Ton (56) References Cited (74) Attorney, Agent, or Firm Leydig, Voit & Mayer, Ltd. U.S. PATENT DOCUMENTS (57) ABSTRACT 2005/0054073 A1* 3/2005 Honjo et al. ................ 435/232 2005/0095712 A1* 5/2005 Martin et al. ............... 435/455 The present invention identifies that the expression of Acti Vation Induced Deaminase (AID) or its homologues in cells FOREIGN PATENT DOCUMENTS confers a mutator phenotype and thus provides a method for AU T16 748 B 10, 1996 generating diversity in a gene or gene product as well as cell EP 1174 509 A1 1, 2002 lines capable of generating diversity in defined gene products. WO WOO3,061363 T 2003 The invention also provides methods of modulating a mutator phenotype by modulating AID expression or activity. OTHER PUBLICATIONS Siddiquiet al. Exp. Cell Research, 252: 154-164, 1999.* 22 Claims, 15 Drawing Sheets US 7,820.442 B2 Page 2 OTHER PUBLICATIONS and Somatic Hypermutation.” J. Exp. Med., 195: 529-534 (Feb. 2002). Harris et al., AID is essential for Immunoglobulin V. Gene Conver Peterson-Mahrt et al. In Vitro Deamination of Cytosine to Uracil in sion in a Cultured B Cell Line, Current Biology, 12(5): 435-438 (Mar. Single-Stranded DNA by Apollipoprotein B Editing Complex Cata 5, 2002). lytic Subunit 1 (APOBEC1), The Journal of Biological Chemistry, Martin et al., “Somatic Hypermutation of the AID Transgene in Band 278(22): 19583-19586 (May 30, 2003). Non-B Cells.” PNAS, 99(9): 12304-12308 (2002). Pham et al., “Processive AID-catalysed cytosine deamination on Muramatsu et al., “Specific Expression of Activation-Induced single-stranded DNA simulates Somatic hypermutation.” Nature, Cytidine Deaminase (AID), a Novel Member of the Rina-Editing 424(3): 103-107 (Jul 2003). Deaminase Family in Germinal Center B Cells,” J. Biol. Chem., 274: Rada et al., “HotSpot Focusing of Somatic Hypermutation in MSH2 18470-18476 (1999). Deficient Mice Suggests Two Stages of Mutational Targeting.” Nagaoka et al., “Activation-Induced Deaminase (AID)-Directed Immunity, 9(1): 135-141 (Jul 1998). Hypermutation in the Immunoglobuling Su Region: Implication of AIDInvolvement in a Common Step of Class Switch Recombination * cited by examiner U.S. Patent Oct. 26, 2010 Sheet 1 of 15 US 7820,442 B2 Pose. Two Somatic Hypermutation A/T-biased Uraci DNA Glycosylase Figure l U.S. Patent Oct. 26, 2010 (Z) ---- ? ? US 7.820.442 B2 G?VJO40€/\·--------···--#-OGOZ~~~~~~~~~-…--~~~~.--· eN?maen.No.©5&.“.”0 <!--: !ar????????????????????????????????????????? zexa6?g Sileo eqewolfSuenW &#)C/IV/Jo?O?Ar??????????????????????????????????????, U.S. Patent Oct. 26, 2010 Sheet 3 of 15 US 7.820.442 B2 Vector t t it t t g t gigt tag at cag city tet Cag titt at gag cag and sac ceg ot tat gag att acrg cac a cyt (gt. 8 8 . s. gen agets ccs gge gyt cts acc cyt genegt gea getta gas gtt a gee gta cag cog G g . v. R. O. W. G s 8 S Ted .. 8 B K R a 510 320 tt t t t t tes gen ete sic oca ga agt atgace cyt gas cyt gea age tte gas get cga gag gta caa cog 8 A. G 1? G G a R A r v w 80 te CB t act caa tar ggt cogee gta tagt Cea ato gain aco Cct gas ggit cog aac atc. gigt etgate aac tect ct G R v c is . is is a s 57 19 19 26 70 f 53 81 Transition on G/C 31% Transition of G/C 79% Figure 3 U.S. Patent Oct. 26, 2010 Sheet 4 of 15 US 7.820.442 B2 Vector n=50 gyra ggit gaC tog gC g gly asp Ser ala val tyr Figure 4 U.S. Patent Oct. 26, 2010 Sheet 5 of 15 US 7.820.442 B2 b C e 3. g g Vector APOBEC1 AD - PTG. -- a. W wt ES3A WSOS C93A hamma- hamma ector - All-A Vector APOBEC1 APOBEC -o- Vector --- APOBEC is 10 15 20 25 Time (hr) Figure 5 U.S. Patent Oct. 26, 2010 Sheet 6 of 15 US 7820,442 B2 s Vector g 22 . t gig st t i a t t t ago s att is titt aty is cag ano a e oat it gag atte Reg Gle ana cgt. att et t s t tie sea ste is can age agt et acs 1 an egt get age tta gas get a gue So as ce. sees act ac tacggit cyo gta tyt cerate gas ico cit gaa gi cog aaa to git atgiana""s: te so APOEEC1 138 t EEE te t t s an tit age cay cit tat cag titt its gases cag and are cey cts tet gag att Reg c ana egt. ? to 153 15so isko tee gea ete gge cea age gst cts ace cgigan egt gea gige tte gas gtt ega gae gt cac ceg 1620 650 f - t act cae tact gigt age gta tyt coate gaa aco cat gala gigt cog as atc. gactgate aaa tet S80 f70 27% Transition at dCldG 100% Transition at dCldG Figure 6 U.S. Patent Oct. 26, 2010 Sheet 7 of 15 US 7.820.442 B2 80 a Vector C APOBEC 60- AD APOBEC3C APOBEC3G 40 s O s 2 8 20 : O a stees - c. * c. c. c. s c.
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