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. 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(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. 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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? 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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.