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(12) Patent Application Publication (10) Pub. No.: US 2006/0248604 A1 Lewin Et Al US 20060248604A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2006/0248604 A1 Lewin et al. (43) Pub. Date: Nov. 2, 2006 (54) ADENO-ASSOCIATED VIRUS-DELIVERED (60) Provisional application No. 60/13 1942, filed on Apr. RBOZYME COMPOSITIONS AND 30, 1999. METHODS OF USE Publication Classification (76) Inventors: Alfred S. Lewin, Gainesville, FL (US); Nicholas Muzyczka, Gainesville, FL (US); William W. Hauswirth, (51) Int. Cl. Gainesville, FL (US); Christian AOIK 67/027 (2006.01) Teschendorf, Essen (DE); Corinna C40B 30/06 (2006.01) Burger, Gainesville, FL (US) (52) U.S. Cl. ...................................... 800/9: 435/6: 800/14 Correspondence Address: HAYNES AND BOONE, LLP (57) ABSTRACT 901 MAIN STREET, SUITE 3100 DALLAS, TX 75202 (US) Provided are methods for the identification of novel genes (21) Appl. No.: 11/256,607 involved in a variety of cellular processes, including retinal degeneration, retinal disease, cancer, memory and learning, (22) Filed: Oct. 21, 2005 amylotropic lateral sclerosis, and methods for the identifi Related U.S. Application Data cation of the function of a variety of genes and gene fragments of unknown function. The genes thus identified, (62) Division of application No. 09/561,498, filed on Apr. as well as the compositions used in the identification meth 28, 2000, now abandoned. ods, are also provided. Patent Application Publication Nov. 2, 2006 Sheet 1 of 28 US 2006/0248604 A1 f1(+) origin TR SMOSOSA Hindi Patent Application Publication Nov. 2, 2006 Sheet 2 of 28 US 2006/0248604 A1 f(t) origin TR f1(+) origin EF1 alpha prom ApR Hindl s pTR-UFCREB23Ohh Ns, 6824 bp Creb23Oh IRES CoE 1 ori GFP (F64L, S65T) TR SV40 poly(A) FIG. 4 Patent Application Publication Nov. 2, 2006 Sheet 3 of 28 US 2006/0248604 A1 f1 (+) origin EF1 alpha promot April PTR-UFCREB288hh . Hi? d 6827 bp . NSi IRES ColE1 ori GFP (F64L S65T) TR SV40 poly(A) FIG. 5 Patent Application Publication Nov. 2, 2006 Sheet 4 of 28 US 2006/0248604 A1 5' N N N N N G U C N N N N N N Target (SEQ ID NO:2) 3' N N N N N C A N N N N N N A C U G A A U G AG C - G SEO D NO:1 G - C U - A C - G G - C G U C U U G G A G U C C GC U GC Target (murine pl6) (SEQ ID NO:4) ' A C C U C A G C G A C G A C U G A. A U G. AG C - G SEQ ID NO:3 G - C U - A C - G G - C G U C U FIG. 7 Patent Application Publication Nov. 2, 2006 Sheet 5 of 28 US 2006/0248604 A1 * CA UGG G U C GC A G G UU Target (murine pl9ARF) (SEQ ID NO:6) G UA C C C A C G UCC A A A C U SEQ ID NO:5 s' U CACAGU C G G A UAU Target (murine p53) (SEQID NO:8) s' A G U G U C A C C U A U A A C U SEQ ID NO:7 FIG. 9 Patent Application Publication Nov. 2, 2006 Sheet 6 of 28 US 2006/0248604 A1 * CA U U AUGU CAAAAGUU Target (murine Rb) (SEQID NO:10) GUAAU A C A U U U U CAA A C U G A U AG SEQ ID NO:9 U FIG. 10A w sees b with UA C C G UC U A G CA UA (nt 2040-2053) (Genbank no. M26391) A U G G C A A U C G U AU A C U G A U AG See ID M/o: 63 Mouse Rb ribozyme 2046 HindII Ribozyme Nsi 1 9. é & B MVO) agct taTGCTACTGATGAGCCGCTTCGGCGGCGAAACGGTAAtgca g6E Ib Mo 6- aTACGATGACTACTCGGCGAAG3CG33GCTTGCCATt (, F.G. 1 OB Patent Application Publication Nov. 2, 2006 Sheet 7 of 28 US 2006/0248604 A1 5"CAGACAGUCCAGGUC 3' Substrate CREB 230 (SEQ ID NO:12) 3'GUCUGUCA GUCCAG 5' A CU A. A U G AG C-G SEQ ID NO: 1 G U C U Oligonucleotides: HindIII sil Oligo 3: 5' agct toACCTGCTGATGAGCCGCTTCGGCGGCGAAACTGTCTGatgca SEQ ID NO: 13 Oligo 4: 3 aCTGGACGACTACTCGGCGAAGCCGCCGCTTT3ACAGACt 5' SEQID NO: 1-4 Catalytic donain Inutant CREB230 Oligo 8: 5' agct toACCTGCTGcTGAcCCGCTTCGGCGGCGAAACTGTCTGatgca SEQID NO: 15 Oligo 9: ' aCTGGACGACgACTgGGCGAAGCCGCCGCTTTGACAGACt 5' SEQID NO:16 FIG. 11 * UCCACAAGUCCAAACAG Substrate CREB 288 (SEQ ID NO:18) AGGUGUUCA GUUUGUC A CU G A d g SEQ ID NO:17 Oligonucleotides: HindIII Nsi Oligo.5 S'agct tcTGTTTGCTGATGAGCCGCTTCGGCGGCGAAACTTGTGGAatgca 3 SEQ ID NO.19 Oligo6 3' aGACAAACGACTACTCGGCGAAGCCGCCGCTTGAACACCTt 5' SEQ ID NO:20 Catalytic domain mutant CREB 288 Oligol O Sagct tcTGTTTGCTGcTGAcCCGCT TCGGCGGCGAAACTTGTGGAatgca 3 SEQ ID No: Oligol l 3' aGACAAACGACgACTgGGCGAAGCCGCCGCT TGAACACCTt 5' SEQID NO:22 FIG. 12 Patent Application Publication Nov. 2, 2006 Sheet 8 of 28 US 2006/0248604 A1 CACAGGAGUCUGUGGAU Substrate CREB 380 (SEQID NO:24) GUGUCCUCA ACACCUA’ A CU G A A U G AG C-G SEQID NO:23 G-C G-C C-G G-C G U C U Oligonucleotides: HindIII Nsi Oligo6 S'agcttATCCACACTGATGAGCCGCTTCGGCGGCGAAACTCCTGTGatgca 3 SEQED NO:25 Oligo7 3 aTAGGTGTGACTACTCGGCGAAGCCGCCGCTTTGAGGACACt 5' SEQ ID NO:26 F.G. 13 Patent Application Publication Nov. 2, 2006 Sheet 9 of 28 US 2006/0248604 A1 WSH --- fixatian in rap frea in primary tissue culture performaldehyd bud nitrogel deterine tumor aC YES types aay for sp represson carry until Fish) cronocoral (McRoostcTION populator southern blot for Northern to for .rcror PCR on serome integrated Proviral expression of the nboryne Na gerome row tre areers tarar rare ?mars was Yr w rewry sw raf te sus one PR fragment irri y -sequence it tru vadu Aires N. aiores We wer rairs rijs 1: irrett S-1ses, re wers were FIG. 14 sequencesu-v are chrkr: try elast search in rura - rperTentary to J3rrprTertury to so war anx or aroWr turner turressor subdismer equence w: rusty of v try archwy's farror stay Posttave ventrol W Cesign iru Yasual Srren of co Arary ave err r sailers rewrite wir t error Raa i specific sequence yed forthon 3f the RNA w posttave core are w nect into rove pitative rurror to Water suppressor grres screer a rers' W: arger rrore tor tructior Patent Application Publication Nov. 2, 2006 Sheet 10 of 28 US 2006/0248604 A1 5 C UAC GUAGC A G A A' (nt 1037-1049) (SEQID NO:30) GAUG CA C G UC UU AP) MoR) we A C U G A U AG pork G SEO D NO:29 A G A FIG. 15 Patent Application Publication Nov. 2, 2006 Sheet 11 of 28 US 2006/0248604 A1 Patent Application Publication Nov. 2, 2006 Sheet 12 of 28 US 2006/0248604 A1 human and rat ABCR (RZ114) eG (b. Muo:32. s' U U c UG G UcuY UGAU C TARGET cuts at position 114 of ABCR AAG ACCA AA CUA G A C U G A U seo d alo 3 AG U FIG. 17A mouse ABCR ge a b wo: 3' S GAA cu Gucu CAA AC TARGET sc U U G A C A A G U U U G A C U G A U AG O 33 S. e 6. W : U FIG. 17B Patent Application Publication Nov. 2, 2006 Sheet 13 of 28 US 2006/0248604 A1 HHRZ35 for mousey-PDE sea b wo:34, 5 Auuc G GucAGCCA C - TARGET y UAA G C C A U C G G UG A C U G A U geo () wo's AG U FIG. 18A Hannerhead HHRZ42 for mouse YPDE sé b is sac Ag AguccAGA AG TARGET nt 73-86 of mouse YPDE U GUC U C A G UC U U C Genbank: NMO12065 A C U A A U. G AG ses to M0.7 S. G - C C - G G U C U F.G. 18B Patent Application Publication Nov. 2, 2006 Sheet 14 of 28 US 2006/0248604 A1 Time course of Cleavage by HHRZ42 - Time 1o 20 30 45 60 120 180 300 600 sec. Na to up as a " gPDE Target Patent Application Publication Nov. 2, 2006 Sheet 15 of 28 US 2006/0248604 A1 (+/+)3SnowAJALCITIAA?LooxzoWOH ?IA?I-T-CHACH-R' Patent Application Publication Nov. 2, 2006 Sheet 16 of 28 US 2006/0248604 A1 SWHOHAAVAAOHGH(CIORI)OLIOLOOS -14u!ZRIH(I)I)odÅLpHAA"I'dSHM9jeºsnou (Ar) on LII a MV Patent Application Publication Nov. 2, 2006 Sheet 17 of 28 US 2006/0248604 A1 IT15-2Rz set wo: to 5’-GGCUGUCAAUCAU-3’ target 3’-CCGAC A UAGUA-5 A U G A A U Seo (b wo. 3 G AG C G w A U FIG. 22 G C G C A G A A IT15-4 * ' ' 5'-ACGGGUCCAAGAU-3 target 3’-UGCCCA gUUCUA-5 U "gA A U Sea. It wo.'? G AG FIG. 23 C G A U. G C G C A G A A Patent Application Publication Nov. 2, 2006 Sheet 18 of 28 US 2006/0248604 A1 IT 15-12 RZ 4ek i? ?o?? 5’-AGAUGUCAGGAUG-3’ target 3'-UCUACA CCUAC-5 A U G A A U SeG (D rv0 3 G^ G FIG. 24 G C G C A G A A IT 15-8 RZ Seo () wo: 5-CAUUGUCUGACAA-3 target 3’-GUAACA AC UGUU-5 A o A A U sco G AS FIG.25 C G A U G C G C A G A A Patent Application Publication Nov. 2, 2006 Sheet 19 of 28 US 2006/0248604 A1 mouse NADH dehydrogenase MWFE subunit int338 S60, 18 AD.
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