1 A more supervised version of DNA shuffling

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1 A more supervised version of DNA shuffling Multivalent avimer proteins evolved by “exon”

1 A more supervised version of DNA shuffling Multivalent avimer proteins evolved by “exon” shuffling of a family of human receptor domains Nature Biotechnology 23: 1556 (2005) A misnomer; really domain shuffling) Joshua Silverman, et al & Willem Pim C Stemmer Avidia, Inc Strategy: Create therapeutic proteins by combining hundreds of known binding domains from receptor proteins in new random combinations and selecting for binding to a specific target by phage display

2 Organization of binding domains in typical mammalian receptors A-domains: (~35 -40 AA’s/domain): determine

2 Organization of binding domains in typical mammalian receptors A-domains: (~35 -40 AA’s/domain): determine binding speficicity of many receptors Library of Typical receptor structures 217 Adomains (~metaphorically? ) as a spacer between domains 2 domains cooperating Bipartite domain Dual specificity domain Degenerate oligos synthesized to coding for 35 -40 AAs of the A domains Only AA’s naturally found at each position were coded for. Conserved structural AAs were kept constant (blue, yellow). Complexity = 1023. Actually realized = 1010 as phage display particles Select one domain at a time, serially, by panning: LRP = LDL receptor related protein

Isolation of a high affinity binding protein to IL 6 ( interleukin 6 )

Isolation of a high affinity binding protein to IL 6 ( interleukin 6 ) by iterative selection (IL 6 is a target for cancer and inflammation) Phage display (M 13) - IL 6 immobilized on plates. Recovered proteins from first cycle, cloned and tested for IL 6 binding; 20 top binders pursued. Add the domain library to each of the 20 first round winning domains. Again pick best 20 overall. After a third cycle pick the very best binder: = “C 326” IL 6 = interleukin 6 M 13 phage One domain Two domains Monomer displayed Monomer protein Build 20 dimer pools on phage coat Screened for from 20 best monomers binding combined with the same library again Three domains etc. to trimer 3

4 Finally, add an Ig. G-binding domain (like protein A) at the end to

4 Finally, add an Ig. G-binding domain (like protein A) at the end to prevent rapid clearance (measured half-life of 89 hours in monkeys)

Binding measured by a competition assay (“Alpha. Screen”) General scheme Laser Luminescence Reactive Oxygen

Binding measured by a competition assay (“Alpha. Screen”) General scheme Laser Luminescence Reactive Oxygen IL 6 receptor 5 Reactive oxygen species can react only over a short distance with and “acceptor” bead Avidin bead: biotinylated IL 6 : gp 130 -Fc : Protein A bead Competition: IL 6 (nonbiotinylated) or C 326 avimer Activity of the anti-IL-6 tetramer C 326. (a) Alpha. Screen competition analysis comparing ability of C 326 relative to IL-6 itself in inhibiting the interaction of IL-6 with its receptors. An avimer that does not bind to IL -6 is included as a negative control.

6 More Alpha. Screens: effect of combining the 3 domains

6 More Alpha. Screens: effect of combining the 3 domains

7 Physical assay: Biacore surface plasmon resonance to measure binding kinetics

7 Physical assay: Biacore surface plasmon resonance to measure binding kinetics

8 Biological assay: Stimulation of proliferation of TF-1 cells (erythroleukemia line) 16 h of

8 Biological assay: Stimulation of proliferation of TF-1 cells (erythroleukemia line) 16 h of 3 H-Td. R incorporation to measure promotion of DNA synthesis Commercial anti-IL 6 antibodies

9 Table 1 Selected avimer affinities and activitiesa Avimer Target No. of domains Affinity

9 Table 1 Selected avimer affinities and activitiesa Avimer Target No. of domains Affinity (n. M) IC 50 (Biochemical) (n. M) IC 50 (Biological) (n. M) C 426 c. Met 2 <0. 1 b 0. 170 c Actived C 65 CD 40 L 2 <0. 1 e 0. 06 c 0. 1 f C 326 IL-6 3 <0. 2 e 0. 05 c 0. 0008 g C 2810 CD 28 3 0. 1 e n. d. h 0. 6 f C 2 BAFF 3 n. d. h 0. 1 c 0. 4 f

10 Acute phase inflammatory response induced by IL 6 is reversed by avimer C

10 Acute phase inflammatory response induced by IL 6 is reversed by avimer C 326 (in mice) Specific for IL 6 induced inflammation

11 RNA Topics: 1. ) Pre-m. RNA splicing basics 2) Splicing-based therapy 3) RNAi

11 RNA Topics: 1. ) Pre-m. RNA splicing basics 2) Splicing-based therapy 3) RNAi

12 Pre-m. RNA splicing

12 Pre-m. RNA splicing

13 Intron = 80 nts to 100, 000 nts Pre-m. RNA Branch point Phosphotriester

13 Intron = 80 nts to 100, 000 nts Pre-m. RNA Branch point Phosphotriester Lariat m. RNA

14 ATP The spliceosome (5 smalll RNAs + 100 -300 proteins) Intron becomes a

14 ATP The spliceosome (5 smalll RNAs + 100 -300 proteins) Intron becomes a lariat ATP degraded http: //www. swbic. org/education/comp-bio/intron. htm

15 (= “donor” site) (= “acceptor” site)

15 (= “donor” site) (= “acceptor” site)

Finding exons in a sea of introns 16 TTCTCAGTCCTAAACAGGGTAATGGACTGGGGCTGAATCACATGAAGGCAAGGTCAGATTTTTATTATTATGCACATCTAGCTTGAAAATTTTCTGTTAAGTCAATTACAGTGAAAAACCTTACCTGGTATTGAATGCTTGCATTGTATGTCTGGCTATTCTGTGTTTTTAAAATTATAATATCAAAATATTTGTGTTATAAAATATTCTAACTATGGAGGCCATAAACAAGAAGACTAAAGTTCTCTCCTTTC AGCCTTCTGTACACATTTCTTCTCAAGCACTGGCCTATGCATGTATACTATATGCAAAAGTACATATATACATTTATATTTTAACGTATGAGTATAGTTTTAAATGTTATTGGACACTTTTAATATTAGTGTGTCTAGAGCTATCTAATATATTTTAAAGGTTGCATAGCATTCTGTCTTATGGAGATACCATAACTGATTTAACCAGTCCACTATTGATAGACACTATTTTGTTCTTACCGACTGTACTAGAAGAAAC ATTCTTTTACATGTTTGGTACTTGTTCAGCTTTATTCAAGTGGAATTTCTGGGTCAAGGGGAAAGAGTTTATTGAATATTTTGGTATTGCCAAATTTTCCTCTAAGAAGTTGAATCATTTTATACTCCTGATGTTATATGAGAGTACCTTTCTCTTCACAATTTGTCTCTTTTTTTTGAGACAAGGTCTCTGTTGCCCAGGCTGGGGTGCAGCAGAATGATCACAGTTCACTGCAGTCTCAACC TCCTGGGTTCAAGCGATCCTTCCACCTCAGCCTCCTGAGTAGCTGGGACTATAGGTGTGCGCCACCACTCCCAGCTAATATTTTGTAGAAACAGGGTTCGCCATGTTACCCAGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACTGGCCCAGTTTCTACAGTCTCTCTTAATATTGTATATTATCCAGAAAATTTCATTTAATCAGAACCTGCCAGTCTGATAGGTGAAAATGGTATCTTGTTT TTATTTGCATTTAAAATTATGATAGTGGTATGCTTGGTTTTTTTGAAGGTATCAAATTTTTTACCTTATGAAACATGAGGGCAAAGGATGTGATACGTGGAAGATTTAAAATTTTTAATGCATTTTTTTGAGACAAGGTCTTGCTCTATTGTCCAGGCTGGAGTGCAGTGGCACAATCACAGTTCACTCCAGCCTCAACATCCTGCACTAAAGTGATTTTCCCACCTCTCAAGTAGCTGGGAC TACAGGTACATGCTACCATGCCTGGCTAATTTTTTTGCAGGCATGGGGTCTCACTATATTGCCCAGGTTGGTGTGGAAGTTTAATGACTAAGAGGTGTTATAAAGTTTAATGTATGAAACTTTCTATTAAATTCCTGATTTTATTTCTGTAGGACTGAACGTCTTGCTCGAGATGTGATGAAGGAGATGGGAGGCCATCACATTGTAGCCCTCTGTGTGCTCAAGGGGGGCTATAAATTCTTTGC

Finding exons in a sea of introns 16 TTCTCAGTCCTAAACAGGGTAATGGACTGGGGCTGAATCACATGAAGGCAAGGTCAGATTTTTATTATTATGCACATCTAGCTTGAAAATTTTCTGTTAAGTCAATTACAGTGAAAAACCTTACCTGGTATTGAATGCTTGCATTGTATGTCTGGCTATTCTGTGTTTTTAAAATTATAATATCAAAATATTTGTGTTATAAAATATTCTAACTATGGAGGCCATAAACAAGAAGACTAAAGTTCTCTCCTTTC AGCCTTCTGTACACATTTCTTCTCAAGCACTGGCCTATGCATGTATACTATATGCAAAAGTACATATATACATTTATATTTTAACGTATGAGTATAGTTTTAAATGTTATTGGACACTTTTAATATTAGTGTGTCTAGAGCTATCTAATATATTTTAAAGGTTGCATAGCATTCTGTCTTATGGAGATACCATAACTGATTTAACCAGTCCACTATTGATAGACACTATTTTGTTCTTACCGACTGTACTAGAAGAAAC ATTCTTTTACATGTTTGGTACTTGTTCAGCTTTATTCAAGTGGAATTTCTGGGTCAAGGGGAAAGAGTTTATTGAATATTTTGGTATTGCCAAATTTTCCTCTAAGAAGTTGAATCATTTTATACTCCTGATGTTATATGAGAGTACCTTTCTCTTCACAATTTGTCTCTTTTTTTTGAGACAAGGTCTCTGTTGCCCAGGCTGGGGTGCAGCAGAATGATCACAGTTCACTGCAGTCTCAACC TCCTGGGTTCAAGCGATCCTTCCACCTCAGCCTCCTGAGTAGCTGGGACTATAGGTGTGCGCCACCACTCCCAGCTAATATTTTGTAGAAACAGGGTTCGCCATGTTACCCAGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACTGGCCCAGTTTCTACAGTCTCTCTTAATATTGTATATTATCCAGAAAATTTCATTTAATCAGAACCTGCCAGTCTGATAGGTGAAAATGGTATCTTGTTT TTATTTGCATTTAAAATTATGATAGTGGTATGCTTGGTTTTTTTGAAGGTATCAAATTTTTTACCTTATGAAACATGAGGGCAAAGGATGTGATACGTGGAAGATTTAAAATTTTTAATGCATTTTTTTGAGACAAGGTCTTGCTCTATTGTCCAGGCTGGAGTGCAGTGGCACAATCACAGTTCACTCCAGCCTCAACATCCTGCACTAAAGTGATTTTCCCACCTCTCAAGTAGCTGGGAC TACAGGTACATGCTACCATGCCTGGCTAATTTTTTTGCAGGCATGGGGTCTCACTATATTGCCCAGGTTGGTGTGGAAGTTTAATGACTAAGAGGTGTTATAAAGTTTAATGTATGAAACTTTCTATTAAATTCCTGATTTTATTTCTGTAGGACTGAACGTCTTGCTCGAGATGTGATGAAGGAGATGGGAGGCCATCACATTGTAGCCCTCTGTGTGCTCAAGGGGGGCTATAAATTCTTTGC TGACCTGCTGGATTACATCAAAGCACTGAATAGAAATAGTGATAGATCCATTCCTATGACTGTAGATTTTATCAGACTGAAGAGCTATTGTGTGAGTATATTTAATATATGATTCTTTTTAGTGGCAACAGTAGGTTTTCTTATATTTTCTTTGAATCTCTGCAAACCATACTTGCTTTCACTTGGTTACAGTGAGATTTTTCTAACATATTCACTAGTACTTTACATCAAAGCCAATACTGTTTTTTTAAAACT AGTCACCTTGGAGGATATATACTTATTTTACAGGTGTGGTTTTTTAAACTCCTTTTAGGAATTGCTGTTGGGACTTGGGATACTTTTTTCACTATACTGGTGACAGATACCCTCTCTTGAGCTACATCGGTTTGTGGGGAGTCAAAAGTCCTTTGGAGCTAGGTTTGACAAATAA GGTGGGTTAACACTTGTTTCCTAGAAAGCACATGGAGAGCTAGAGTATTGGCGAATTGAAGAAATCCC CCTTTTTAACACACTTAAGAAAGGGGACTGCAGGTATACTCAAGAGAGTAAGTCGCACCAGAAACCACTTTTGATCCACAGTCTGCCTGTGTCACACAATTGAAATGCATCACAACATTGACACTGTGGATGAAACAAAATCAGTGTGAATTTTAGTAGTGAATTTCATAATTTGATCGTGCAAACGTTTGATTTTTATTACTTTAGACTATTGTTTCTGATTTTATGTTGGTATTTCCTGTGA GTTACTGTTTTACCTTTAAAATAGGAATTTTTCATACTCTTCAAAGATTAGAACAAATGTCCAGTTTTTGCTGTTTCATGAGTCCTGTCCATCTTTGTAGAAACTCGCCTTATGTTCACATTTTTATTGAGAATAAGACCACTTATCTACATTTAACTATCAACCTCATCCTCTCCATTAATCATCTATTTTAGTGACCCAAGTTTTTGACCTTTTCCATGTTTACATCAATCCTGTAGGTGATTGGGCAGCCAT TTAAGTATTATTATAGACATTTTCACTATCCCATTAAAACCCTTTATGCCCATACATCATAACACTACTTCCTACCCATAAGCTCCTTTTAACTTGTTAAAGTCTTGAATTAAAGACTTGTTTAAACACAAAATTTAGACTTTTACTCAACAAAAGTGATTGATTGATTGATGGTTTACAGTAGGACTTCATTCTAGTCATTATAGCTGCTGGCAGTATAACTGGCCAGCCTTTAATACATTG CTGCTTAGAGTCAAAGCATGTACTTTAGAGTTGGTATGATTTATCTTTTTGGTCTTCTATAGCCTCCTTCCCCATCAGTCTTAATCAGTCTTGTTACGTTATGACTAATCTTTGGGGATTGTGCAGAATGTTATTTTAGATAAGCAAAAACGAGCAAAATAGGGGAGTTTAACTTTAATATTTTCTTTTAAAAAGCATTTCATGTTATAAGATCAATTCTGAGTGGTAGAAAATGCTTTGACATTTTATTTC CATTTTCTACTTTTAGTTTTTTTCCTATTTGTTTAAGATCTTAGAGGATTATTAAGCTGAACTCCTCAACTGATAAAAAGCATGACATCTTAAACATAAGCATATTTTTAGGTTAATTTTCACATAGAAAACAGTTTATGTGAAATTCTATGTAGATATACTATTTTTTTGGTATTGATATGTTTATTTTATTTTATTTTATTTTATTTATTTTTTGAGACA GAGTCTCACTCTGTTGCCCAGGCTGGAGTGCAGTGGCATGATCGTAGCTCACTGCAACCTCCACTCCCGGGTTCAAGCAATTCTTCTGTCTCAGCCTCCCGAGTAGCTGGGACTACAGGTGCCACTATGCCCGGCTAATTTTTGTGTTTTTAGTAGAGATGGGGTTTCACCTTGTTGGTCAGGCTGGTCTCGAACCCCTGACCTCAGGTGATCCACCTCAGCCTCCCAAAGTGCTGGGATTA TAGGCATGAGCCACGTGCCCGGCCGACATGTTAATTTTTTAAAAAAGGCTTTACTGGGGTATATTTTATATAATAATCACATGTTTTAACTATACAATTCCAAGCTTTTTAGTATATTTATAGGGCTATGCAAGGAAGATATACTGTTAAACAGTAGAAATTGAGAAAGCTCTTCTGATAATATCTCTTGATGATGGCTCATGCCTGTAATCTCAGTGCTTTGGAAGGCCAAGACAGCAGAATCACT TGAGGCCAGGGGTTCGAGACCAGCCTGGGCAACACAGCAATACCCTATCTTTACAAATAATAAAAATATCTGTTGATTTGAAGTAAAGTTTTTAAAGACAAGGTCTCATTCTGTCACCCAGGCTGGAATGCAGTAGCAAGATCACAGCTCACTGTGGCCTTGACCTTCTGGGCTCAAGTGATTCTCCCACTTCGGCCTCCCGAGTAGCTGGGACTAACAGGTGTGCACCACCATGGCTAATTTTTATGTTTGTAGAGATTGGGTCTTACTGTGTTGCCCAGGCTGATCCCGAACTCCTGGGCTCAAGCAGTCTTCCTGCCTCAGCCTCTAAAATTGCTGGGATTACAGGCTTGAGTCACCATGCCCAGCCTGAAGTAGCATTTCTACCCTGTTTAATAATTCAGCAGCTTGTCATGTAAGATATTCATATATGCATATAAACATTAGGCAGCTTAATTTGGTAAAACTGTAAAATGGAAATTTTAAATTGTTTGCA GCATCAATAACATTGATGTCAGTATGATTTTTACATGCTGATCTTGACCAATTTGAAACAGTGAGTTAAAATCTGGCTGATCCGTACTAATCCTAAAGAAATATTCTATGAACTATTAAATGTTTCCAGAATATATAAAGAAACATTATGATGTCAACACACCCATCTATTTTTGGAAATAAAAACTCCATTTTTCTTATTAAAGAAAACATGCTTATTAGAAAACATACGGCTGGGTGCAGTGGCACACATGTA ATTCCAGTGCTTTGGGAGATCGAGGTGGGAGAATCACTTGAGGCCAGGAGTTTGAGACCAGCCTAGACAACATAATGAGACCCCCTCTCTACACAAAAAGAATTAGTTGTGCATGGTGGCGTGCACCTGTAGTCCCAGCTACTTGGGAGGCAGGAGCATCCCTTGAGCCTAGGAGTTTGAGACTGCAGGAGTTCGAGACTGAGTGGAATGCAGTGGAACTGCATTCCAGCCTGAGTGACAGAG GGAGACCCTGTCTTAAAAAAATAAGAAAACACAACTGCAGAAAATTATAAAGGATTTAAGTCATTCCAAATATCACTGCCACTTTTTAGAATATTCTAAAGAATTCTCTGTGTACACACATATGCGTACTCTTAATCCAAGTAGCTTGGTAGGATTTTACCTAGTGCCTAGATGGGAAATTG CCTGGGGATTCCAAATACCTATTTCATTAAAGATGTCACTGATTTTAAGAC TTAACACTATTTTTCATACTGCCAAGAAAACACTACCAGTTATAAATGTAAATTGCCATCAATTGTAATACATCAATTTTAGAGCTATTATTAATAAAATGTGCATCTTAGAGCAATGAAATATAGTACTATATATTTGATGACCTTTTCTGCCCTGTGATATTCAGAAAGTTAAATATGGGCTGAGCATGGTGGCTCACACCTGTAATCCCAGTACTTTGGGAAGTCAAGACGGGAGGCTTGAACCCAGGAGTTCAAGACCAGCCTAGGCAATGTAGCGAGACGCCATCTCAAAATATTAAAAATAAGTAAATAAAAAGAAGGTTAAGTATACAAATGTATTTCCTTTGTTGTGAATTTCAATTTTATAGTGATTTTTTGAGACGAAGTCTCACTCTTGTCCCCCAGGCTGGAGTGCGATGGCGTGATCTCAGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCTATACTCCT GCCTTGGCCCCCCGAGTAGCTGGGATTACAGGCGCCTGCTACCATGCCTGGCTAATTTTTGTATTTTTAGTTGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTAGAACTCTTGACCTCTGGTGATCCACCCGCCTCGGACTCCCAAAATGCTGGGATTACAGGCGTGAGCCACCGTGCCTGGCCAGTGGTTTTTTGTTGTTGTTTTGTTTTTGTTTTTGAGAC AGGATCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGTGCCATCTTGGTTCACTGCAACCTCTGCGTGGGCTCAAGCAATCCTCCCACCTCCCTTTCCAGAG TAGCGGGGACCACAGGTGTGTGCCACCACACCTGACTAATTTTTGCATTTTTGTAGAAACAGGGTTTTGCCATGTTGCCCAGGTTGGTCTGAAACT CCTGAGCTCAAACAATCCAACTGCCTTGGCTTCCCTAAGTGAAATTACAG GCATGGGCCACTGTACCCAGTCTAGTGATTTTTTTATTTTTATTTTATTTTTTTACCAAAAAAACAACAAAGCCTCAGGAGGAAAAGTTGATACACAAGTAAATTTTATTGGAAATGTTTTTGTGTGGACCTTAAGCAGAGGGAAAATTAGTCTGCATTATGGTGTATCCAGACTAAATGACTGATATTAAAATGAAATTATTCTTAGGATTTGCAATCTTAGAGAAAACTTTTTCATTTTTAT TTTTTTGAGTTACAAATTATCTTCATTTACATTTGAGAACAGTGAGTCACAGAGGGATTAAGTAACTTACTCAAGATCATACAAGTCTTTGAACCCAATCTTTTAACTCTGCAGAACTCAGAGTCACTCTTATTTGGAAAAACTTTTTAACTGATGTGGATCCTCTAATATGGGCTTCCTATTATTCTCTATTAGTCAGAAGTTTTGCAAGCAGAATTCATTTTGCCAATTACGGGATTTTCCCT CAGTTGCAGTCAAGGTTCATAAAACTATAACTCTTTAATTAGAAATGTTTTTTGAGACAAGGTCTTGCTCTGTTGCCCAGACTGGAATGCAGTGGCATAGTGGCCCATTGCAGCTTTGAACTCCTGGGCTCAAGGGATCCTCTGCCTCAGCCTCCCAAGTATCTGAGACTACAAGTGCCATCACCCATGGCTATTTTAAAAAAATTGTAGAGATAGGGTCTTGCTGTGTTGC CCAGGCTGGTCTCAAACTCCTGGTCTCAAGCAATCCTTCTGCCTTGGTCTCCCAAAGTGCTGAGATTACAGGTGTCAGCCGTTGCACCTGGCCAAAACGATAACTTAAAATACACACACACACAAACACATATGTGTATTTGTGTGTGTGTGTCTCAAAAGGTATCAAAAGAGAATAGCTATAACTTTAGTGTTGATCTTGATAGTGACTTGATTAGGCTCTGTTTAACATC AAAGATGCAAATTAATACTTTGAACATATTAAAAATGCAGAAAATATTGGAGTATTTTAAATTGTATTCTGTATATTTAAGGTATACAACATGATGTTATGGGATACATATAGGTGGTTAAAAGATTACTGCAGTGAAGCAAATTAACGTATCCCTCAACTCACATAGTTACCCATTTTTGTTTTGGTGGCAAGAGGAGCTTAAAATCTCATTTAGTGTGAATCCCAAATACAGCACAATTT TATTACCTATATACTTCATGTTGTACATTATATTTCTAGACTTGTTCATCCTACATATCTGCTACTTTGTATCCTCTGAGCTACATCTCCCCATTTTCTCACTTGCCCCCCAAGTAGTTTCTTAAAGTGTCTCATGTAAGAGGGCAGTAGCTTTCAGCTTAAACTTTTTCTCTGTAGTCGATTTCTTTGAGGTATACTTTTCTCTCCAGAATAGTTAGATGTAGGTATACCACTTTGATGTTGACACTAGTTTAC CTAGAACTTATCTTCTGTAAATCTGTCTCTATTTCCATCTCTGTCTCCATCTTTGTCTCTATCTGTCTATCTATCTATCTATCTATCTATCTATCTAAAGCAAATTCATGCCCTTCTCCTATTGAATCGAGACCATAGACAGGGGTGAGAGAATTTGGCAGGAATGGGGATGTGTATTATCTGTGGCATAAGGAAACTTTACAGAACTAGGTTCAAAA GTATACTTTCTAGTTCTTTCCCATGGCTTTTCACTTTGATGTAGTCCTTATCAGGTAACTGAGGTTTTATATAAGTCCCCTGATTCTTAGAACATGAAGGTGTAGTAGTCAAGGTTGGTCCCTTGAAACCACAAATTTTGTGAAAATTAAGAAAATTTGAATAATTTCCTCAGCAAATACATATTGATCATCTGTTATACAGCCATGAGAAGTGGTTCTGTTGCACACGTTTATCAGATCCTAATCCCA AACCAGGCATAAAATGGAAACCATGAAGATAGGATGAAATAACTTCTGAATGTTTGAAAATAGTGTACTTAAAAATACCAGGTGGTTTTTGTTTTTTGTTTTTTTCTTTTTTTGAGACAGGGTCTCACTCTGTCACCCAGGCTGGAGTGTAGTGGTGCAATCTCATTGCAGTCTTGACCTCCCAGGCTCAGGTTATCTCCCACCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACAT GCCACCACGCCCAGCTAATTTTTTGTAGAGACGGGGTTTCACCCTGTTGCCCAGGCTGGTCTAGAACTCCTGGGCTTAAGCGATCCTCCCACCTCAGCCTCCCAAAGTGCTAGGATTACAGGCATGAGCCACCATGCCTGGCAGAAAATACCAGGTTTTTAAGTATCAGCACTTACTCTTCAATCTTTTCTATTACTATGTTGTGCTAAATGGTATTTTTTAATTAGAGCAATGCTGTTCAA TAGAACTTTGAGGATGGAAATCTTTTATGTTTCTGCTATGTGGTACAGAGCCACTAGTGACATGTGGCTTTTGAGCGCTTGACACATCTTGTGCAACACAGGAACTGAATTTTTAAGTAATTTATATTGCCACATGTGGCTACCGTATGGGACAGTGTAGTACTAGATGATCTGTAAGGGCTGTGCTTCATCAGTGTCGTTTTTTAACTGACAAAAACCTTTAGTTTTTAGTAATGTGTTTAAA AGAATTCATAAAATACAAGTAAACAAATTAACTTGTTACCTGAGCATATGTCCTTTCATACTTATTTTTTCTGCATATTTTGGAAAATGGAATATCTGCCCCTTTTTATCTGAGATACAGTCTACCTCTAAAAATACATGATTCTAACATTCTCACTTTTTGTTGGCATTTGATCAGGGTATAGAAAAACAGTTAAAAGGACAGAGAATGGTTGAGAGATTATGAAGAGAAAATGTGATTGAGTGT GGTAGACTTGGGGCCTGCTTGAATGTTGAGAGAATGACTGTTTTCCGATAAAAAAGTCCATTCTAGGATCCTAAAAGAAGGGTCTGAAGTTCACTGCAGAAAGCTACATAGTACTAAGCCACTAAGGGGACATGGAGCCCTTAGTAATTCCTACCTTAGTAATAGTCTCATCATGCCCTCTTGGGAACCCAGCCTTGTTGATTAGCCTCTCTGCTTTCTCTCCTTATAGTTCAACCTCCCTGTTT GTTCCAAGCAGTTCTTTTCCTGCCCATTTATTATGCATTTCTATACAGCTTTCCTCCTCTTTTTCTATACCATGCTGCAGTTCTTATTGCTACCTAGAGGTTTTCAAAATTCCTAGGGGCGGATAAGTAGGCATAAACAAAGTTCTTCCCTATTATCCTATTTTTTCACCTAGACTGAAGAGGTAGACAAAATAGAAATAAAGACATTAAGGGTATGTGTTTGTAGTCCCAAAGAGCTTCTCTGGCAATTTTG ATGTAGTTGACAGTGACGCTCTGAGTTCAGGACAGATTGGACTCCTTGGCTGAGAGGAGTGAGGAGATAGGACGGTAGAGGGTAGAGCAACTCTGGAGGAAGCTTTCCCCTCACCTTTGCCAGTCCTGTTATCCTAGACTTAACCATAATTAAAGATGAGGCACTCAGTAAAGGGATCTAGTGGGAAGCTTGTTCCAGACAGCCAAGGAGGTTCGCGCAGTTCCTTTGGCCACCCA GGTGGGGTAATTGATCCATGTATGCCATTCATGTACAATGTAGGCACTTATACCTGTATTCCAATGTAGTGAACTATACCATTACTCTTAAATTAATATTCTTTATTAGCTTCCATGGTGGCTATAGGCCAGGCAAGAGAGTTAAGAAAAAATAGCCAGGTATGGTGACTCAAGCCTGTAATCTCGGCACTTTAGGAGGCCGAGGCAGGAGGATAGCTTGAGTCCAGGAGTTCAAGACCAGCCTGAGCA AAATAGTGAGATCCTGTCTCTATTTTT TAAAAAAGCCTTGGGGCAAACAGGAGTATGGAGGTTTGGATGCTAATAGAACAGCAGTGTCTTACTGCTTGGAGTTCTCTTGTTTCTTGTCCTATCACCGTAGCCTTTGGATCACAGCAATTTTTCCATGACTCCATACTTTTCAGTTCTTGAATATTTTTTCCTTTATTCCTCTTGTCTCTGTAAAGACATCAACTGGAGTTGGACTGTAATACCAGGTATCTCCAGA AGATGGCACTATTTAACAGATTTTATAATTTGATGTGAGTCACTGTCATCTGAAGCTTGTTGCCTTTTCTTTCTTCTTTTTTTTCCCCATCAATTCTGTATGTTTGAAATGCTGGGATTTAAGTTAGAATAAGGGATGTCTGTAATTTCCCTAAATTGAGAAGTAATATGCAAAGGTTGATATCAGAAGTCATATGCTCACCTTGCAACACCAAATAATACTGGCCCATTTGTGATTTTTGAA AGTAACACTCCATAATAAATGGATGTATAGAAGCATAACAAAAATAGAAGCACATAAAAGTGAAAAGTCTCATAAACGCCATTGTCACTACTCATGTAATTGCTGTTACAAATTTGTTTAAATGTTGAATAAAAATGGTGTCATAGGCAACACAGTGTTCCACTACTTGGTGTTTTTAATAGCATTATTCTGTCTCAGTGTGCTTTGGATTATCAGGTGCTTTTTAATAGTTGCATGGTATTACATTGTGTAGA TGAACTTGATTAATTTAAATGGTTCCCTGTTAATGGACATGTTGGTTTTTGTGAACAACTGATACAGTGAACATTTTTTAAAAAAAAGAGAGACAGGGTCTTGCTGTGTTTCTCGGGCTGGCCTTGAACTCCTGGGGTCAAGCGATCGTCTTGCCTCCCTGGGATTACAGGCATGAAGCCACCGCACCCGGCCCAGTGAACACTCTTGAATGTATCTTTGTATACTTGTCAAGTGT TTTTGTAGCAATTGATTCCCAGAAGTGGGAATTACATGGAATTAAGTGACATGTTTGCAATTTTAACAGGTATTGCTATGTCATTTTCAAAAGAAGCTATGCCAATTAATACTCTCACCAACAAGAGTGCTTATTTCCCCTCAGCATATTATCAGGCTTAAGTTTTGCCAGTATGGGAGAACAGTAGAATCACATTGTTTTAGTGTTTCTCAGATATAATTTTACACCTTATAACCTTCTCT TCTATAAATTGTCTATTTGTGTTCATTCTCCATTTTCCTATGGGTTCTTATTGTTGGAGCCCAATATATAAAAGGGGGTATTTGTTACAGAACCTCTTCAGTTTTGGTTCATGCCTGGGTTTTTACCCTTTCTACGGATGTTAAAATTCTCTATTTTCTTCCAGTCCACTTATGGCTTTATTTTTTACATTTAGATTTTAATCCGTCTGGAATTTTTGTGTATGCTGTGAGGTAGGGACCATACTT TTATTTTTTCCCAAATGGGTTACTAGTTGGCCAAACATCATTTATTGAATAATTCATCTTTTCCCTACTGACTCGAAATACCATCTTTATTGTATACTAAATCCTCATATAGTTCTGGGTCTGTTTCTGGGCTCTACTTTGTTCATTTACTGTGCTGGTACTGCACCGTTGTAATTGCTGTGGCTTTGTGGTATGGCTTGCTCTCTGCTAGGGCAAGTCGAAGCTCTTTTGTTCACCTGCTCTTTCACCCAA ATTTTCTGTCCTGAATCCAGCACAGCCAAATTATGGTCATTGTCACCACCAACTACAGTGGGTGTTGAGCATTTCCCATTGAATCTCCTGTAAGGGTTTTATTGGATTCTGTGATAGCAGTAAAATGGGAGCCTAAGAGGTATTCCTTAAAGGACTACTAATCAGACCTGGTTTCCCAGATGATGCTGAAGATGACGGGGCCTGGGCTAGACTTTTGAGGGACATATCCTTGGGGTTGGGTGTGATATAGACC AGCCCTTACAATTTGCTTGACTCATGGGAATCGTACAGGGCCAGAACCAGACACCTGTCATGCTAATAACTTCCCTCACAATTCAGAAATCACTGTGATTGAAGATGGGTGGCTGTTATAATACTACCCACTTAAAAATGGATGTAACCCATTTTTTAGGACTCTTAAAAACATCAAATCAGTAATGGCCGATTAGGACTTTTTAATTTTTACTAATCTCTACTTGAAAGTTTTCTAGTCATTTCAGGAAACC TAATTCTTATAATTCATATCATTTAGAATATCATAATGCTATGGATATTAGCTAACTTCTCAAATCTTCTAGTTCTCATTTAATTTGAAGTTTGTGTACATAAGGATATACATATGTGTAGATATATATAGTTTTTTTAACTAGAATGACCAGTCAACAGGGGACATAAAAGTAATTGGTGGAGATGATCTCTCAACTTTAACTGGAAAGGTATCTTGAAAGGGAA GAAAAGCACTTCATACCGAGTCAATTAGTAACAGTGTGCTTTCAATCACTAAGAGATAATTTACATAGTATAACTAAATGGGTTATTTAACCCTTGGAAGCAGTCTAGGTTAATTATCGTTCCCTAGGTCATGTAGTAAAAAGACAGTAGAATCCAACATTAACCTTAAATGTCCATATTGTCAAGTACTGCTGTCTGCCTCTGTGGGACTCTAATTTGGGATCCTTCAAAAAACATTGATGGGGGAAA AGATAGCCTTTAAAAAAAACAAACCTATGTGAGTCTATGTGAGGTAGACTCACATAGTTTCCTAAAAGATAGCAAAGCAGTATTATGTAGTGGCTGAAAGTGTGAGTTCCGGAGCCTGACAACTGATTCAAAGCATGGCTTAGTACTTCCTAACTCTGACCTTGGGCAAGTTACTTAACCTCTCTGTGTCCCATATGTGATTAGGGTGAGGTTGATAATAGCAGCCATAGAGTTAAGAGGATTAAGT GCTATAATGCAAGTAGAGCTCTTACAACAGTTTCTGGTAAATCACTCAATAAATTCAGACATACTATTATTTTAAGAAATCTCAAAGAGTTTTCTTGTACCTTAAAATTCTCCTAGTGTGAACCATTGGTTTTGGTATATTGTGCTTCCATGTAGTTTAATATCAAGATGTTTTTAGATTTCCCTTTTAATTTGTTGACCCATTGGTTGTTCAGGAGCATGCTGTTTACCTGAAAATAATGGAGATATTAAGGTA TTTGAATATTTATCTTCTAGTACATTGAAAAACTTTTTGAGAGTAACCAATAATAAATGATGGAATGCTACTGCTTTTTTGAAGCTGCCAGTTATTGTTTACACTATGCCAAATATAAAGGCATTAATCTCATAAAAGTTTCACAACAATCCTGTGAGGGAGACGATATCCCCATTTTACAAATCAGGAAATTAAGACTTAATAAGGTTAAAAGACTTGCCCCAAAGTCACAGAACCAGTAAGTGGTAG AGCTTGAATACAGACCTGACTCTAAAGCTCTTTCTTTAGATTTTAGTGTTCATTGCTTACTTGAATGAGTATCTATAAGAAAACTTTAACATGTAAAACTTCTGTGAAATTATCTTGTCCCATATCAGGGTCATGTCAAACTAATGTCCTCCTCAGCATCTTTGGAAAACTTCAGAGGAGAAATGAGCTTTGCCCCTCCTGTTCATTTCCTATTCCACTAG GAGACCTGTCCTTCCCTTTCAGCAT GCTTTGTCCATATTTAGAAGCTGTTGAAGCCATTACTTGTCTGGTCAGTTTTTAGTGCTGGAATGGACCTAGCCTTTTAGGCCTTCTGAGATTTAGTTTGATCTCGTCTTTCCCACCTAATGGCTC TGTTCTACTACATAGATTTGATCTGAAACAGTTCTCTGTTTCTAAAATAACTTTTCATGATAGTCACAGTAAAGTACATTTATTATGGAAAAATCAATAAGTATAACGAGTGAAAGTTATTTCTTGGT GGTAAGATTATGGGATTATTTGAACTTTCTGTTTCATTGTATTTATTTATTTTTGTGATGGAGTCTCACTCTGCTGCCCAGGCTGGAGTGCAGTAGTACGATCTTGGCTCACTGCAACCTCCCCTTCCCAGTTCAAGTGATTCTCCTGCCTCAGACTCCCAAGTAGCTGGGATTACAGGCGCACGCCACCATGCCTGGCTAATTTTTTTATCTTTAGTAGAGACAGGGTTTCACCATGTTGACCAG GCTGATCTCCAACTCCTGATCTCAGGTATCCACCTGCCTCAGCCTCCCAAAGTACCGGGATTACGGGTGTGAGCCACCCTGGCCTCATTTTGTCTTTTGGGGGTATTTTTGTGTGCAGATATGTATATAAATATTTTTCCCTCTTTTCCCCAGTTAGTATTTGAGCAGATGAACTTTGGACCCGAATACCTGTATTCAAGTCTCTAATACCACTTCTTGGCTATTTTCATTTTATCAAATGGCCTCTT ATCCTCGTTTTTCTCATTTATTAAGTAGAGATGTAACTACTTGATATAATTCAAAAACTCAATAATGGCATTCTTTTGTTTTTTAGACTCTAGTGTCTGTACTCCTTGTACCATGCTGGGATTCATTTGAACAATTGCATGGCTTTTTTAGTGTATTATTAAATTTGCAGTTTACTTAGAATTTACTGGGACCTCATACAAATGGGAAAAAAACATAACTGTGTTACTCATTTGCTGTGTGCCTTTGGATTGACCCTAT TTTTTGTATTCATTTTCTCCCCATGTCCTGAGTTCCACTTTGAATAAAAAAGTAATTTTTTTCCTGTAAAATAGGCTACCAATAGGCTGCAGTTGTCTATAGTAGCTGCTTCACTGAGGAGAGCTCAGCATGAGAGAAATAGTATGAATTGCCACAAGTTATGGGCTAGCCTTACTTCATTCTGTACTTGGACCTGTTTAGGCTTCTAAGAGATCTTACCTCCAACAATAAACTGCTTTGAGACATGA AAAGGTGGAAGCTTTACTTGGTTATAACTTTTAATACCTAGAACAGTGAGTCTTCAAACTTGTATTTGCATGCCCAATTTATAAAAAGTTTCCTGAGCATTTACCCCTAATATATGCATTTTAAATTATATATGATTTATGGTAATAATAATATGTTACAAAATACAAAAATATAGATTAAACAAGGTGAGGTTAAAAAATTTAAAAGTTCTAATCTTGCAAACCAGTGGATCTTTTGTGCCTT ACTCTGGTAAACACTGTCTTAGAAGAATATATAGAACATTAAAATCTTAATGCTATAGTTATATGACAGAGTATGATGAGAGCTACAGATAAACAACACATCATGAATCTTCTTGTGGCAGTGTTTATAACCATTATGTGAAATGCTGCCTCATTCTTATAACTAGCATAAGAACAGATAGGACTTTCTCGATTTTGAGGGGTAATTATTAGATGGTATTTTCTGTTAAGGACTCTTCCAGCTATAAAATTCTTAAAT GTAGAAAGCGAAGTGAGGGTTTATGGTGAGAGGAAGCATTGGTATCATGTTTTAGTGTAGTCCAAGAATATGGACACATCCAGAAAATGCAGATCAAGTTTAGCCTAATGAGAAAATATATTTTGGAGTCCATATGGTAAATTATGTGATTTTTGAGTTATTGTACAAATATAATTCTTAGAATGTTAGAGTCAGGAGACTATAAGAGACCAACTGCTTCAAGTTTCATTTAACACATGGGAAACTAAGGC GAGAGAAATTTCAAGACTTGCCCAAGATTAGACCTCTTGTTAAGTAATGAAAGTGTTTTAAAAACAGGTGGGTCAAATTCTGTTTTTAAAATTTCCATTATGATGAAAATTTCAGTATTACAGGCTTCCAAATCCCAGCAGATGGGCCACTTGTTTAAAGGAGAGTTTGATATAATAAAGCATCTAAAAACAAGAGTTTGGATAATTCCTTAGGGTTGTTATGATGTGATTTGACTTATAATTGGAAATACCGTTTTA TTCATTGTACTGATTTTCATTTCTCTTTTTCTTCTAGAATGTCTTGATTGTGGAAGTTCACATTTACTTTTAATATAACATTTATGACTTTTCTAACTTAGTATGCACCATCCTAAAGGTAAGCCAGGGAGAGAAATTCCTCTGCATCAGTTTTAATGGTGGGCTTGTGTTCTAAAGGAGTGAGATTGGTTTTTTGTAAAGACTACTTAGTAATTTGTTTTTACCAATAATGGTATACTTCCTACCTCTC TTTTTTTAGTTTGAAGTATTTTCTAAACATAACTCTCTATTTATCTATAATATATACATATCTTATATTTTATGTATATATATCTTGCTTAGATTTTGTCTTATGTAATATTTGGTACATAAAAAATAATATTTATAGACTATTTTCCATGTGTTATTATGTGCTAAAGTATTTTGTATCTTAGCACCGAGAGGCTAAGCAGTTTCCTAGGGTTACCAGCTAGTAAACTAAG GGAAACCTTTACTTCCTTTAGCTCAGTGGTTCTCAAAATGTGGTTCCCTAGACCAAAAGTATTAATATCAGACAAGAACCTACCGAATCAAAATATCTGTGATGAGGCCCAGCAAGCTATGCTTTAACAAGTTTCCGAGTGATTCTGATGCTAAGGTTTAGGATCCCTTGTTTTTACTCATAAGTCACTTTCTCATTAAGGCCTTCCCTGGCCATCCTATATAAAATCTCATGTTTTCACACCGTCAACTTC GTATTCCTCCTCAATACTTTTATTTTCCTGATCACTTATCACTAACAGCCTCTCTCTCTCTCTATGTATATATATCACTTATCACTGTCTAACAGCCTCTCTTTATATAATCTATAGATTATATATGCAGCATTGTGCAATCATTATCACGCTCAATTTTAAAACATTTTCATTTCCCCACAAAGAAACCCAATCCCCTTAGCCATCACTCCCAATTTTCCCCCAG CACCTAGCAAACTGATCATCTACTTGCTGTCTATAAGATTTGCCTATTCTGGACATTTTGTATAAATAGAATCATACAATATGTGGCCTTTTGTATCTGGCTTCTCTCACTTAATGTTTTCAAGGTTCATGTTGTGGAGTATATCTGCACTCATTTCCTTTTTATTGCCAAATTGTATGGATAGACAGGTGTTCCTCAACTGTGTCCTGATAAACCCATCTGAAGTTGAAAATATCATAAGTTGAAAATG GATTTACTACTTTGATAAATCTATCCTAAAGTCAGAAAAATCTCATGTTGGAACCATCGTAAGTTGGATACCATCTGAATTACATTTTTGTTATCCATTCACTGGTTGACAGACGTTAGGTTGTTTCCACTGATGCTCCTTATTTCTCGTACCTGAAATGTCCTTATTCCCTTCTTATCCCATGTTTAAGTCATTTAAGACCCAGCTCAAACGTCACCTCCACAAAACCTTGATACCCCTTTCCTCTTCA ATTCACTTGGACCTTTTGCATTTAATTTTTATTTTAAGACAGAGTCTCACTCTGTCACCAGGCTGGAGTGCAGTGGTATGATCTCAGCTCACTACTCTGCCTCCCAGGTTCAAGCAATTCTCATGTCTCAGCCTCCCAAGTAGCTGGGACTACAGGTGTGCGCCACCATGCCTGGCTAATTGTGTGTGTGTATGTATATATGTGTGTATATAT ACACAAACATATATAAATATATATACATATATATATATACACACACACATATATATAGTTTTTTTAAGTAGAGATGGGGTTTTGCCATGTTGGCCAGGCTGGTCTGGCCTCAAGCCATCCTCCCACCTCGGCCTCGCAAAGTGCTGGTATTATAGGCATGAGCCACTGTGCCTGCATTTTAATTATAAAATATTTTG AACTCAGAAAAAAGGGTATGCTGAATACCTACGTACCCACAAAAGTATTAACATTTTGCCATATTTGCTTCTGATCTTATTTTGAGAAATTAAAGATCATAATACAACTAAAGCCCCATTTCCCTTCATTCCCAGAAGTATGACAATTATCCTTAAAGTTGATATCATTCCCATGTTTTTTATACTTCCCTAGTACAAGTTAGCTGTATCCTCTGCTCAGGGGCTCATCAAGCTGAATCAAG GGACTCATGATCCTCTTCAAAGTTCCTTCAGGTTGTTGGCAGAATTTAGTTCCTTGTGATTGTAGGACTGAGGGCCCGTTTTCTCACTGGCTGCTGGCCAGGGGTTGCTCCCAGATATTTAAAGGCTCATGCCCTAGCCCATGACAGTCTCACAACATGGCAGCTGACTTCTTCAAAACCAGCAGGAGAATCTTGCTCTAGTCTACCACATAACCTAATCACAGGAGCGGCTATCCCGTTATTTTCACAGATC CTGGTCACATTCAAGGGGAACCCTTCTGTGTACACCAGGAGGCAGGAATTTTTCTTTTTTGTTAAAAAGTCTTTTATCCCTAAAGGAGGCAGGAATTTTGAGAGCCATCAGAATTCTGCCTACCACAGCCCAGAAATCTGCATTTTTCACAAGTCTCCAGCCATGATGTTTCTGATGGCTCACACTGCTTTATTCCATTTTTAAAGAGTATTTTTATTGAAAAGCATTA GGGTTATGGTTTAAAAAATATTTTCCCTAACAAAGATGGGTTTAGAGTCCTACTTTTGACTAAATAGCTGAGATTCACTTTTATGTAAAGTTCATTTTATAGCGTTATTAATTTGGGTGCCTTTAAAAATAGTATAAAGCATGTTTCTCGAGTGTAGTCTGTTAGCCACCTATATTGGAGAGTTGGGAGGAGAGAGTCTCTATCTTGAATTTATGGGAAAAATTCTAAAATACTTTTTATAATGAAGGACAACA TCATAACTCCCTAATAAAATGTGCATGTATATATTCAAATTTGCTGTCATTGATCCTGCACCTACAAAATCCAGTCCTGGGGGCTGGCATTCTTACTGCTGAGGGCCAGATGATATAGATTCCAGAATATCTCCATGTAGATTTTGGTGAGAATTACTGTGCTGAAAAGAATGACAGTATTGCAGTTATACATGGGGGTTTTGGTACTTTATATTGTGACTCTGAATTTAAAGCTATGCAATGTCTTCTTTT TTGAAAGGATATAATTGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAAAGATGGTCAAGGTCGCAAGGTATGACATTTTGACACAGAATATTTTCCTCATTTGAAGGGGGATTAAGTGATTGCTTCTTTTTAAGGATAAATGTTTTCAACTGTCATTTTATCTTCGAAAAGTAATCTCATATAAGACTTAAGATATAATCCTTTTAAATAATTTTGTCATGTGTTA ATAAAGCTCATAATTACAGTCACTTCCTTGCTAATATTAACATTTGGTTTTCAGCATGCTAATTATATCAGTTTGTCCTGAATAGCATGGCAGAGGATTTTGGGCCCCCTTGCAAAATTAAGAATAAGGATTCCAAAGCGGGTGAGGAAGTGATAGGAAGGGGTGGGCCCTGAAGATCTGGACCTCCTGGAATTGAGTGATGAATGCTGCATCTTCTTTGTGTCTGTAGTGAAATTTTATAATGCCTGCTTCCT TTTTTATTAAGTCGGCCTCACCTTACCTATGCTGTTTTACTTTTGCTTTTATAGTTCTACCTGTGTTTATTTCTCATTTTCGTTTCATCTCTCAACAACTCTGGGGTGGCATTATTATTCCCACTTTTCAGATAAGGTTACTGAGGCATAGGGAATTGTCCAAAGGTACAGAGCTAGTCCGCTATAGAGATTTGAACCCAGGGAACCTGGCTCACAGTTTATGCTTTTGCCTACCTTAAGTTTT TAATAGAGTGACATCAAACATTTAAGAATATGTTTTTCCTTTTATAATTTCATTAAAAACATTAAGTCTCTGATCAGTCTGCAGTTTTTATGTAGGGGTCAGGTAATGTTCTAACTTCTGCTTTTTCCTAAGTGATTAACAGGTTTTTATAAGCCCTTTTGAAAAAATCACGGTATCTGTCGAGCATCTTTGAATCAGAGTAAGCCTTCTAGTGAGTCATATGTCAGCAGTTTGACTGTATGGGCTTTT CTAATATCCAGTTCAAGTGTTTATCAGTGAGTTTTTCTTTTAAATAGATTTGGGACAGGTACTATGAGAGTATATAAGTGATACGTTATAGGACACTAGTATCCTATGAAATGGCAAAAACTGCAATCACTTTTGCACCAAATAGAAACTAATCAGTGCACTTGCTTATTTTTCTACATGCTCTTTAGGGTTTTAAATGTCAACCTACTGTGGCATAGACTTTAATCCTCTGGGTATTCTTTTGTTGTTC TTTCCTGGTATATGCTGTGGAATTGAGATAGACTGGTTCGTGAGCGAGAGATTTTGTGTTGCCACAGGTAGGACATGCTCAAACAATACTTGGGTCATTTCTTGACCCAAGTCATCTATTCACCATAGTTTTGTAGCACCGATCTTGCATACATTTCATGTATCTTCTTTGAACCCCACGTCAGTGCTGCTTATATGATACTCAGAAATTAAACACTAAGGAATAAGATTTTCAGGTAGGATTGAGTTTTGGAGG GTCACAAATCTTGTAATGTCTAATATTTCCACTCTCCCTGCTGAGAATTAGTTTTGGCTTCCTTGGAGGTGATATCGCCTCTGTTGAGTATAAGTGGCCTACTGTGATCACACCACTGCACTCCAGCCTGGGTGACAGAGTGAGACCCTGTCTCAGAAAAAAAAAAGAATGCATGGCCTAGATGACTTCTAAGGTTTTTCCCAGTTCCAGTTTTCATGTTCTAGGCAGAGCAGTAAAGTGA GAAACACATGGACTTGGGAGTTTAGTCTCGCATTTCACTGCCACTTAATCTGAGCGACTATTCCATATTTAATCTCTCTGAATGTATTTACTCATCTTTAAAGGGGAATGATTATTAACATCTTTTTCTCAGGGAAACTATATGAGTCAAGGAGATAATATATTTGAAAATCTTTTTAACTGCAAAGCGCTGTTTCACTGTTGGTTATAATGTGATCTCATTGTAGTGAGCAGCTGCTTAATTGCGTTTTAGAA TGTAGGGAAGATAGTAATATTTTTCACATTATATATGTAGCTGGTTCTGGAACTGTAAACATACTCCTTTTTTATGGAGATCTGAGTCACGTACCATAAAATTCACTCTTTTAAAGTTGTACAATCCAGTGGTTTTTGATATATTCAGAGTTGTGCATCTGCTACCACTATTTCATTTTGGAACCCAAAGAAACCTTGTACCCATTAGCAGTCATTCTCCCAGCCCCTGGCAACTACTAATCTACTTTCTA CAGAAAGTCCGTACAGATTTGTGTATTATGGACATTCCATATAAATGGACTCATGCAATATCCTGTCTTCTTTCACTTAGCATAGTGTTTTCAAGGTTCATCTAGGTTGGGGCATGTATCAGTACTTCATCCCTTGTTTTGGCTGAATAATATTTCATTGTACAAATATATCACATTTTGCTTATCCATCTGTTGGTGAACATTTGAGTTTCTACCTGTTGGCTTTTATGAATAATGTTGATTTGAATGTTTGTGTAC AAGTATGAATACCTGTTTTCAGGTCTCTTGAGTATATAGTTGCTAGGTCATATAGTAACTCTGTGTTTAACATTTTGAGGAATTGCCCGACTATTTAACAAGGTATATGTACTGTTTTACACCAGTAACATATGAGGGTTCCAATATCTCCACATCCTTGACAACACTTGTTACTGTCCTTTTTATTGTAGCCATCCTAGTGGCTATGATGTGGTATCTCATTGTGGTTTTGATTTGTGTTTCTCTGATGA TGTTGAACATGTTTTCATCTGCTTATTGGCCATTTACATATATCTTCTTAAGAACGGTTACCCATTTACAGTATGGAAAATGCTTCAGATGCAACTCTAGTCATGCCTTAGAGATGGAGCTTTATTAAACATTCAGATCTCTAGGCATATGAAGTGCTGAGTTCTCTTGAACTCCTAATACAGATTGCACTGAGTTTAGTGATACCTTTTCTGGAGCATTCCTGAGTTCAGGTAGGGAGAAGGGTTTTTGCTGTGA TTGGCTTGTTATGTTCTAAATGGAAATAGAATTGAAGTGTCTCCTCTCTCCATTTA

Some types of alternative splicing Alt. 3’ SS Alt. 5’ SS Exon skipping |p.

Some types of alternative splicing Alt. 3’ SS Alt. 5’ SS Exon skipping |p. A Different termination |p. A 17

18 Alternative splicing: Occurs in almost all of the 25, 000 human gene transcripts

18 Alternative splicing: Occurs in almost all of the 25, 000 human gene transcripts

19 Alternative splicing in the alpha-tropomyosin gene (7 isoforms) Similar proteins but subtly different

19 Alternative splicing in the alpha-tropomyosin gene (7 isoforms) Similar proteins but subtly different to suit different tissues

20 The alternative splicing champion Dscam transcript alternative splicing (Drosophila) 12 38 33 2

20 The alternative splicing champion Dscam transcript alternative splicing (Drosophila) 12 38 33 2 Each isoform has one exon 4, mutually exclusively Exon choice within each class is mutually exclusive. Codes for axon guidance proteins as well as function in the fly’s immune response 60 kb gene 115 total exons/gene 38, 016 combinations

21 A cautionary note: 95% of human genes show evidence of alternative splicing Low

21 A cautionary note: 95% of human genes show evidence of alternative splicing Low levels could be simply mistakes. Or genes trying out new exons to see if they are useful, or give them a chancew to become useful (through mutation, evolution) But there are still a very large number documented cases so there is no doubt that alternative splicing greatly increases the complexity of the mamalian proteome.

22 Many human genetic diseases are caused by mutations causing missplicing • 1) Frank

22 Many human genetic diseases are caused by mutations causing missplicing • 1) Frank splicing mutations loss of an exon loss of a gene product or of an isoform (e. g. , β-thalassemia, loss of a hemoglobin) • 2) More rarely, but on the increase (in terms of discovery), activation of a false exon (e. g. , muscular dystrophy, cystic fibrosis: protein function disrupted or protein terminated prematurely) • 3) Theoretically, loss of a splicing factor (? ) (lower organisms)

23 Therapeutic intervention at the level of pre-m. RNA splicing Pseudo exon activated disease

23 Therapeutic intervention at the level of pre-m. RNA splicing Pseudo exon activated disease Antisense = block and skip unwanted pseudo exon Alternative splicing Unwanted alternative = included Use antisense skipped Bias alternative splicing Against an unwanted isoform (e. g. , Bcl-X alt. spl. : Bcl-XS = promotes apoptosis; Bcl-XL = inhibits apoptosis and promotes cell growth, cancer) Alternative 5’ splicing Unwanted = longer exon Antisense shorter isoform

24 Nonsense mutation d Antisense-induced skipping x Expendable exon (e. g. , protein with

24 Nonsense mutation d Antisense-induced skipping x Expendable exon (e. g. , protein with many repeated domains) Exon must be multiple of 3 in length to maintain reading frame after skipping

25 Therapeutic intervention at the level of pre-m. RNA splicing A. Interfere with improper

25 Therapeutic intervention at the level of pre-m. RNA splicing A. Interfere with improper splicing caused by splice site creation or activation E. g. , beta-thalassemia (R. Kole) in which a splice site has been created by a mutation Use complementary DNA (antisense) Rapidly degraded: Use modified bases, sugars: PNA, morpholino, 2’ OMe, Normally, DNA-RNA hybrids + endogenous RNase H type activity RNA destruction Modified antisense DNA circumvents this problem (don’t want m. RNA destroyed here, want to correct its splicing

26 B. Bias alternative splicing ratios Target the unwanted isoform exon-intron joint. e. g.

26 B. Bias alternative splicing ratios Target the unwanted isoform exon-intron joint. e. g. , BCL-2 isoforms, one is pro-apoptotic, one anti-apoptotic. The latter increased in many cancers Target the anti-apoptotic isoform in cancer cells. e. g. , GABA-a-gamma-2 receptor (GABA = gamma amino butyric acid, a neurotransmitter) Long and short forms. Long form associated with mental illness. C. Skip offensive exons e. g. , nonsense truncations in dystrophin

27 Sazani P, et al. and Kole R. Systemically delivered antisense oligomers upregulate gene

27 Sazani P, et al. and Kole R. Systemically delivered antisense oligomers upregulate gene expression in mouse tissues Nat Biotechnol. 2002 Dec; 20(12): 1228 -33. EGFP: Enhanced green fluorescent protein = model system Antisense “RNA” injected into tail vein, RNA was modified for stability Mutant globin intron has activated splice sites Actin promoter, universally expressed. Exon skipping yields green fluorescence

28 RNA modification for stabilization Instead of deoxyribose or ribose Modified phosphate Still base

28 RNA modification for stabilization Instead of deoxyribose or ribose Modified phosphate Still base pairs OK

Even more extreme and more stable: peptide nucleic acids (PNAs) RNA modification B =

Even more extreme and more stable: peptide nucleic acids (PNAs) RNA modification B = a nucleic acid base Amide bonds, No ribose PNA = peptide nucleic acid Attached 1 to 4 lysines here Base pairs even better than natural nucleic acids (higher melting temperatures) 29

30 RNA modification Also can add 2’ MOE -O-CH 2 -O-CH 3 MOE =

30 RNA modification Also can add 2’ MOE -O-CH 2 -O-CH 3 MOE = methoxyethyl - Phosphorothioate deoxyoligonucleotides

31 No antisense: Antisense treatment in cell cultures (ex vivo) from the mouse with

31 No antisense: Antisense treatment in cell cultures (ex vivo) from the mouse with the mutant EGFP gene Control oligo (C) (50 nt downstream) was ineffective. Max. effect = 40%

32 Dystrophin gene 2400 kb, m. RNA = 14 kb, 79 exons: a giant

32 Dystrophin gene 2400 kb, m. RNA = 14 kb, 79 exons: a giant gene Protein maintains muscle cell membrane integrity Mutation: Duchenne’s muscular dystrophy Some cases (~half) are due to stop codons (nonsense) in a repetitious exon (spectrin -like repeat) Deliver antisense to ends of exon with the nonsense mutation in mdx mice (model for Duchenne’s) to promote the skipping of the nonsense-bearing exon and so avoid truncation of the protein. Use AAV (adeno-associated virus) to deliver the antisense gene Measure: m. RNA with skipped exon dystrophin protein muscle histochemistry for dystrophin

33 Use antisense RNA to target the branch point upstream of the offending exon

33 Use antisense RNA to target the branch point upstream of the offending exon 23 and the donor splice site downstream of the exon. protein m. RNA = 3 X 71 79 BP = branch point; SD = splice donor Branch site (consensus = YNYTRAY) Sequences targeted by antisense

34 U 7 promoter compl. to splice donor site compl. to branch Double target

34 U 7 promoter compl. to splice donor site compl. to branch Double target synergistic (loop? ) (Kole) Consensus binding site for Sm proteins (to target to pre-m. RNA) U 7: normally hybridizes with seq. at 3’ end of histone m. RNAs to effect cleavage; Binds 2 Sm proteins; in coiled (Cajal) bodies (RNA processing centers? ); low concentrations (1000’s of molecules per cell) U 7 OPT: Change Sm binding site to consensus for all sn. RNAs (spliceosomal, for delivery there); high copy no. ; no longer in coiled bodies; Now include anti-splice site segments as well. In permanent transfectants can effect > 50% inactivation of a globin cryptic site. Gorman, L. , Suter, D. , Emerick, V. , Schumperli, D. & Kole, R. . Proc Natl Acad Sci U S A 95, 4929 -34 (1998).

35 Expression of U 7 antisense construct RT-PCR U 7 OPT-A. S. Endog. U

35 Expression of U 7 antisense construct RT-PCR U 7 OPT-A. S. Endog. U 7 0 2 4 6 13 weeks (slow onset = conclude slow m. RNA turnover) Splicing assay (RT-PCR) Skip exon 23, after 2 -4 wks. normal 0 2 4 6 8 13 weeks Dystrophin protein (Western)

Muscle immuno -histochemistry Normal Untreated mdx Top, middle , and bottom dystrophin-associated antigens 36

Muscle immuno -histochemistry Normal Untreated mdx Top, middle , and bottom dystrophin-associated antigens 36

37 RNAi = RNA interference Short double stranded RNA molecules trigger the degradation of

37 RNAi = RNA interference Short double stranded RNA molecules trigger the degradation of the complementary sequence in the cell, and can inhibit translation of the targeted m. RNA Their introduction into a cell can greatly reduce any protein whose m. RNA is targeted. Inhibition is usually incomplete in mammalian cells Thus “gene knockdown” as opposed to knock-out Alternative technologies: Antisense RNA: block translation or splicing Ribozymes: RNAs that cleave other RNAs, sequence specifically

38 si. RNA = short interfering RNA mi. RNA = micro. RNA naturally occurring

38 si. RNA = short interfering RNA mi. RNA = micro. RNA naturally occurring si. RNA (Primary transcript) 2 nt overhangs (RNA-induced silencing complex) Single-stranded RNA More common Protect against viral RNA, repetitive sequence transcripts Cleavage if perfectly complementary No cleavage if imperfectly complementary, but translation inhibition

39 Introduction of long DS RNA into mammalian cells will trigger the “interferon response:

39 Introduction of long DS RNA into mammalian cells will trigger the “interferon response: Cessation of protein synthesis via activation of PKR (protein kinase RNAactivated), and phosphorylation of e. IF 2 Global degradation of m. RNA (without any sequence specificity, RNase L activation) Spread to neighboring cells (induction and secretion of interferon) Most small DS RNAs do not trigger this response(<30 bp)

40 Generation of si. RNA in vitro Chemical synthesis, annealing of 22 -mers (bypasses

40 Generation of si. RNA in vitro Chemical synthesis, annealing of 22 -mers (bypasses dicing by Dicer) T 7 -mediated in vitro transcription of each complementary strand. Anneal to make long DS RNA and transfer to cells. Let Dicer make si. RNA in the cell Also, can use controlled RNase to generate fragments (cheaper) Introduce perfect hairpin RNA into cells, let Dicer make si. RNA Introduce imperfect hairpin RNA into cells (based on m. RNA sequence) and let Dicer make mi. RNA

41 Limitations of si. RNA silencing in mammalian cells Transient nature of the response

41 Limitations of si. RNA silencing in mammalian cells Transient nature of the response (~3 days) Transfection problems (cell type, refractoriness) Non-renewable nature of si. RNAs ($$)

Generation of si. RNA in vivo (can give permanent knockdown) 42 Not good for

Generation of si. RNA in vivo (can give permanent knockdown) 42 Not good for interferonresponsive cells Allow trans-association (TTTTT acts as a terminator) (Pol III) (Pol II) Most common, using U 6 or H 1 promoter U 6 = small nuclear RNA used for splicing. H 1 = RNA element of RNase P, used in t. RNA processing. mi. RNA

43 Potential determinants of efficient si. RNA-directed gene silencing si. RNA Incorporation into the

43 Potential determinants of efficient si. RNA-directed gene silencing si. RNA Incorporation into the RNA-inducing silencing complex (RISC); stability in RISC. Base-pairing with m. RNA. Cleavage of m. RNA Base-pairing with si. RNA. The position of the si. RNA-binding target region. Secondary and tertiary structures in m. RNA. Binding of m. RNA-associated proteins. The rate of m. RNA translation. The number of polysomes that are associated with translating m. RNA. The abundance and half-life of m. RNA. The subcellular location of m. RNA. Delivery Transfection (lipofection, electroporation, hydrodynamic injection (mouse)) Virus infection (esp. lentivirus (e. g. , retrovirus like HIV that can integrate into non-dividing cells)

44 Some applications: Target oncogene Ras V 12 (G 12 V) – silenced mutant

44 Some applications: Target oncogene Ras V 12 (G 12 V) – silenced mutant ras without silencing the WT allele. Reduced the oncogenic phenotype (soft agar growth, tumor formation in nude mice) T-lymphocytes infected with anti-CCR 5 RNA lower levels of this HIV receptor, and lower levels of infection (5 -7 X) Target an enzyme in mouse ES cells with a hairpin vector, Isolate a knockdown, make a mouse. Mouse shows same knockdown phenotype in its cells. So can target the whole mammalian organism, Just inject a GFP silencer gene into single cell embryos of a GFP mouse: Can find a chimeric GFP mouse with reduced GFP Progeny carry it in the germ line, Get a complete knockdown mouse, without ES cells (easier)

45 Delivery inan intact organism Hydrodynamic injection (sudden large volume) of straight si. RNA

45 Delivery inan intact organism Hydrodynamic injection (sudden large volume) of straight si. RNA (no vector) into the tail vein of a newborn mouse Get silencing of co-injected luciferase vector in a variety of tissues High throughput si. RNA for gene discovery C. elegans, 19, 000 genes Make a library 17, 000 si. RNA genes in plasmids in E. Coli. Feed the clones of E. coli to the worms. Look for phenotypes. 1700 genes examined for phenotypes (as of 2005) (e. g. , fat metabolism phenotypes found)

46 Systemic RNAi: worms, plants, mammals In plants, get permanent post-transcriptional gene silencing (PTGS,

46 Systemic RNAi: worms, plants, mammals In plants, get permanent post-transcriptional gene silencing (PTGS, transcriptional level) Worms: effect can last though several generations Amplified by reverse transcriptase Influx/efflux via a specific transmembrane protein (in worms) Raisons d’etre? Infection, many viruses go through a DS RNA phase. Repeat element silencing? (1 million Alus, + others half the human genome) Transcribed in either direction, so could form DS RNA, then RNAi inhibits action of SS ‘m. RNA”

Discovery of RNA interference using double-stranded RNA Nature (1998) 391: 806 Discovered RNAi as

Discovery of RNA interference using double-stranded RNA Nature (1998) 391: 806 Discovered RNAi as they tracked down the effective agent in antisense experiments (DS RNA contaminating their SS antisense preparations had all the inhibitory activity) Paper characterized by nice controls and variations: Several genes, whole animal phenotype, protein product (GFP), RNA level (in situs) Phenotype of null mutant is specifically mimicked. Introns and promoter sequences ineffective. DS RNA from a different sequence + SS antisense RNA vs. the target: ineffective DS RNA linked (chimeric molecule) to a single stranded portion vs, the target: ineffective Transport of DS RNA between cells and amplification implied. 47

48 In situ hybridizations No probe No RNA injected SS antisense RNA DS RNA

48 In situ hybridizations No probe No RNA injected SS antisense RNA DS RNA Transcript disappears (RNA degraded)

49 p. 173 Alnylam Pharmaceuticals Inc. Target : Apolipoprotein B, involved in binding cholesterol

49 p. 173 Alnylam Pharmaceuticals Inc. Target : Apolipoprotein B, involved in binding cholesterol to low density lipoproteins (LDLs) Made in liver and jejunum. An important factor for high serum cholesterol and atherosclerosis. Tested ~80 si. RNAs for reduction of Apo. B m. RNA in a hepatoma cell line in culture. Used 2 best. Stabilized the si. RNA by: 1) Substituted sulfur for a hydroxyl oxygen at the 3’ end linkage (phosphorothioate) 2) Added some methyl groups to the sugars of the last 2 nucleotides 3) Conjugated cholesterol to the 3’ end. This dramatically improved serum half-life and efficacy. [Promoted entry into cells as well? ] Injected into tail veins of mice.

50 Recovery of si. RNA from injected mice. RPA = RNase protection assay cholesterol-conjugated

50 Recovery of si. RNA from injected mice. RPA = RNase protection assay cholesterol-conjugated si. RNA non-conjugated si. RNA Control RNAs Inject once a day for 3 days, measure 24 h after last injection 60 -70% reduction in Apolipoprotein B m. RNA

51 Plasma Apo. B Plasma cholesterol

51 Plasma Apo. B Plasma cholesterol

52 Some therapeutic targets of RNA interference localized target ss RNA virus target Dominant

52 Some therapeutic targets of RNA interference localized target ss RNA virus target Dominant allele target (can be made allele-specific)

53 Engineering cottonseed for use in human nutrition by tissuespecific reduction of toxic gossypol.

53 Engineering cottonseed for use in human nutrition by tissuespecific reduction of toxic gossypol. Sunilkumar et al. and Keerti S. Rathore. PNAS (2006) 103: 18054 Cotton: 20 million cotton farmers, in Asia and Africa. For every 1 kg of fiber, plant 1. 65 kg seed = 21% oil, 23% protein. BUT: Seed contains the terpenoid gossypol: Which protects the plant from infections, But which is: cardiotoxic and hepatotoxic Oil is OK, but protein is contaminated with gossypol 44 million metric tons of cottonseed produced each year 9. 4 million tons of protein. Enough to satisfy the protein requirement of 500 million people. Terpenoid-negative cotton mutants are susceptible to infection and so are not commercially viable.

Delta-cadinene synthase Target the m. RNA specifying the first step in gossypol synthesis 54

Delta-cadinene synthase Target the m. RNA specifying the first step in gossypol synthesis 54

55 sh. RNA Recombinant plasmid T 1 grows in the bacteria Agrobacterium tumefaciens which

55 sh. RNA Recombinant plasmid T 1 grows in the bacteria Agrobacterium tumefaciens which can be used as a vector for plant transfection neo gene terminator alpha-globulin promoter, active only in seeds The T-DNA region of the binary vector p. AGP-i. HP-d. CS. Arrows indicate the primers used in the PCR analyses. RB-right T-DNA border t. OCS: octopine synthase terminator d. CS: 604 -bp d-cadinene synthase sequence p. AGP: cotton a-globulin promoter (seed specific) p. NOS: nopaline synthase promoter npt. II: neomycin phosphotransferase II t. NOS: nopaline synthase terminator d. CS = delta-cadinene synthase LB: left T-DNA border.

56 NEO-RESISTANT TRANSFECTANT PLANTS Gossypol level Ten seeds from two transgenic plants from F

56 NEO-RESISTANT TRANSFECTANT PLANTS Gossypol level Ten seeds from two transgenic plants from F 1 of selfed matings 0. 1 ug/mg PCR for transgene Note transgene-null segregants have normal gossypol levels

57 HPLC (high performance liquid chromatography) Null segregant Spots on seed indicate terpenoid glands

57 HPLC (high performance liquid chromatography) Null segregant Spots on seed indicate terpenoid glands

58 RT-PCR assay for the m. RNA for the enzyme delta-cadinene synthase: Low to

58 RT-PCR assay for the m. RNA for the enzyme delta-cadinene synthase: Low to undetectable levels in the si. RNA knocked-down plants PCR of DNA for transgene

Gossypol (G) and other terpenoids are NOT reduced in the leaves of transgenic plants

Gossypol (G) and other terpenoids are NOT reduced in the leaves of transgenic plants (so resistance to infections should be normal). The same is true other aerial parts of the plant and for roots. WT Transfectant 1 Transfectant 2 59

Low gossypol level analyzed through two generations of one homozygous plant were stable at

Low gossypol level analyzed through two generations of one homozygous plant were stable at 0. 19 ug/mg +/- 0. 013 (SEM, 50 seeds). WHO limit for human consumption is 0. 6 ug/mg, so OK. Other plants could be similarly targeted: Lathyrus sativus, grass pea, a hardy tropical/subtropical legume plant (neurotoxin = beta-N-oxalylamino-L-alanine) Fava beans, cassava beans: toxins = cyanogenic and contains fava glycosides (toxic to people with low levels of the enzymes glucose-6 phosphate dehydrogenease (G 6 PD), which is common. fava bean cassava bean 60