DNA shuffling Example 2: Antibodies: breaking the natural limit on affinity selection

1. 1 Class 25 last updated 12/6/11 11:00 AM DNA shuffling Example 2: Antibodies: breaking the natural limit on affinity selection Natural affinity ceiling for an antibody = lowest Kd of 10-10M (100 pM): Kd = off time / on time Endocytosis rate ~~ 10 min to several hours So no selection for off time (t1/2) longer than ~<3 h (104 sec) Diffusion-limited on rate ~ 106 M-1s-1 Selection limit of affinity of natural antibody evolution: Off rate 1/off time; On rate 1/on time Kd = off rate / on rate = (1/10+4) / (10+6)= 10-10 = Kd --------------------------------------------------------------------------------------------------- J Foote and HN EisenKinetic and Affinity Limits on Antibodies Produced During Immune ResponsesPNAS 1995; 92: 1254-1256

2. 2 Directed evolution of antibody fragments with monovalent femtomolar antigen-binding affinity. Eric T. Boder, Katarina S. Midelfort, and K. Dane Wittrup. Proc Natl Acad Sci U S A. 2000; 97(20): 10701–10705. Used the FACS to selected single chain anti-fluorescein antibodies displayed on the surface of yeast cells. Competed with free fluorescein. DNA shuffled. 4 cycles. Selected for slow off times. Achieved 50 fM affinities. That’s femtomolar, 50 x 10-15M = 5 x 10-14M = 0.05 pM (compare 100 pM limit for naturally selected Abs) Slower off-rate than biotin-streptavidin (>5d).

3. 3 Antibodies from yeast scFv selection (Boder et al. and Wittrup, PNAS 2000) Off time (t1/2) 2x10-6 per second = 5 days half-life Iterations

4. 4 • Example 3. Selections for antibody characteristics other than Ag binding: • scMAb + DNA shuffling + ribosome display + selection in DTT (disulfide reducing agent). • Ab folding without need for disulfide bond (-SH oxidation to –S-S- not needed), • as well as high ligand affinity. • Lutz Jermutus, Annemarie Honegger, Falk Schwesinger, Jozef Hanes, and Andreas Pluckthun, PNAS 98:75-80 (2001)

5. 5 Example 4: Enzyme stabilization: PAI-1, a protease inhibitor (TPA inhibitor) Error prone DNA shuffling  245X increase in stability ( days) Assay = tPA binding Found 11 aa changes, presumably affecting protein folding Back-cross to remove non-contributory mutations: DNA shuffle best clone with original WT DNA. Maintain selective pressure. Analyze “progeny”: see 2 of the 11 aa changes lost, not needed, replaced by WT sequence. J Mol Biol. 2001 Jan 26;305(4):773-83. Different structural requirements for plasminogen activator inhibitor 1 (PAI-1) during latency transition and proteinase inhibition as evidenced by phage-displayed hypermutated PAI-1 libraries. Stoop AA, Eldering E, Dafforn TR, Read RJ, Pannekoek H.

6. 6 • Example 5: Viral tropism: murine leukemia virus, a retrovirus • MLV, 6 strains, all  poor infection of CHO cells. • DNA shuffled envelope gene of the 6 strains • chimeric virus that can infect CHO cells And selected incidentally for resistance to inactivation under conditions of laboratory manipulation (100X centrifugation-resistant) Nat Genet. 2000 Aug;25(4):436-9. Molecular breeding of viruses. Soong NW, Nomura L, Pekrun K, Reed M, Sheppard L, Dawes G, Stemmer WP.

7. 7 Example 6?: A global version of DNA shuffling -- genome shuffling: A different (unnatural) method of genetic mixing using whole genomes Zhang et al., Nature 415, 644 (2002) Can mix Streptomyces genomes by protoplast fusion  effectively diploid bacteria The fused cells will generate recombinant haploid spores. Target: tylosin production (an antibiotic) Mutagenize a culture, collect 22,000 survivors. Screen all 22,000 for tylosin synthesis, pick the top 11. Protoplast fuse all top 11 with each other. Collect 1000 progeny. Screen 100 for tylosin, collect the best 7. Protoplast fuse again. Collect 1000 again. Screen 100 for tylosin again. Characterize the best 2: Tylosin production is up 9-fold. So productivity is up 9-fold, without a lot but not tremendous amount of work (22000 screen max)

8. 8 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) Joshua Silverman, et al & Willem Pim C Stemmer Avidia, Inc Avimers = high affinity ligand binding proteins that are not antibodies, based on receptor domains. A misnomer; really domain shuffling) Strategy: Create therapeutic proteins by combining hundreds of known binding domains from receptor proteins in new mutated random combinations and selecting for binding to a specific target by phage display.

9. Organization of binding domains in typical mammalian receptors 9 A-domains:(~35-40 AA’s/domain): determine binding specificity of many receptors Typical receptor structures An A-domian of 217 A-domains as a spacer between domains (~metaphorically?) Dual specificity domain Bipartite domain 2 domains cooperating Degenerate oligos synthesized to code 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 (6 cys and 4 Ca binders + 2 others). Complexity = 1023 .Actually realized = 1010 as phage display particles = domain library Select one domain at a time, serially, by panning: LRP = LDL receptor related protein; VLDLR =very low density lipoprotein receptor

10. 10 Isolation of a high affinity binding protein to IL6 ( interleukin 6 ) by iterative selection (IL6 is a target for cancer and inflammation). Phage display (M13). IL6 immobilized on plates. Recovered phage from first cycle, cloned and tested for IL6 binding: 20 top binders pursued. Added the same 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: = “C326” Monomer protein Screened for binding Build 20 phage dimer pools from 20 best monomers Monomer displayed on phage coat One domain Two domains Three domains M13 phage IL6 = interleukin 6

11. 11 Finally: Add an IgG-binding domain at the end to prevent rapid clearance (measured half-life of 89 hours in monkeys) Model structure

12. 12 Binding measured by a competition assay (“AlphaScreen,” Perkin-Elmer) Luminescence Reactive oxygen species can react only over a short distance with an “acceptor” bead Laser Laser Reactive Oxygen IL6 receptor IL6 Avidin bead:biotinylated IL6 + gp130-Fc:Protein A bead Competition: IL6 (non-biotinylated) or C326 avimer (10X tighter) gp130 = natural IL6 receptor

13. 13 More AlphaScreens: effect of combining the 3 domains [So how was M1 selected??] 20 pM

14. 14 Physical assay: Biacore surface plasmon resonance to measure binding kinetics 253 pM 0.23 nM

15. 15 Biological assay: Stimulation of proliferation of TF-1 cells (erythroleukemia line) 16 h of 3H-TdR incorporation to measure promotion of DNA synthesis Commercial anti-IL6 antibodies

16. 16

17. 17 Acute phase inflammatory response induced by IL6 is reversed by avimer C326 (in mice) Specific for IL6-induced inflammation hIL1 – human interleukin 1

18. RNA Topics: • 1.) Pre-mRNA splicing basics • Splicing-based therapy • RNAi

19. Pre-mRNA splicing

20. Intron = 80 nts to 100,000 nts Pre-mRNA Branch point Phosphotriester Lariat mRNA

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

22. (= “acceptor” site) (= “donor” site)

23. Finding exons in a sea of introns TTCTCAGTCCTAAACAGGGTAATGGACTGGGGCTGAATCACATGAAGGCAAGGTCAGATTTTTATTATTATGCACATCTAGCTTGAAAATTTTCTGTTAAGTCAATTACAGTGAAAAACCTTACCTGGTATTGAATGCTTGCATTGTATGTCTGGCTATTCTGTGTTTTTATTTTAAAATTATAATATCAAAATATTTGTGTTATAAAATATTCTAACTATGGAGGCCATAAACAAGAAGACTAAAGTTCTCTCCTTTCAGCCTTCTGTACACATTTCTTCTCAAGCACTGGCCTATGCATGTATACTATATGCAAAAGTACATATATACATTTATATTTTAACGTATGAGTATAGTTTTAAATGTTATTGGACACTTTTAATATTAGTGTGTCTAGAGCTATCTAATATATTTTAAAGGTTGCATAGCATTCTGTCTTATGGAGATACCATAACTGATTTAACCAGTCCACTATTGATAGACACTATTTTGTTCTTACCGACTGTACTAGAAGAAACATTCTTTTACATGTTTGGTACTTGTTCAGCTTTATTCAAGTGGAATTTCTGGGTCAAGGGGAAAGAGTTTATTGAATATTTTGGTATTGCCAAATTTTCCTCTAAGAAGTTGAATCATTTTATACTCCTGATGTTATATGAGAGTACCTTTCTCTTCACAATTTGTCTCTTTTTTTTTTTTTTTTGAGACAAGGTCTCTGTTGCCCAGGCTGGGGTGCAGTGCAGCAGAATGATCACAGTTCACTGCAGTCTCAACCTCCTGGGTTCAAGCGATCCTTCCACCTCAGCCTCCTGAGTAGCTGGGACTATAGGTGTGCGCCACCACTCCCAGCTAATATTTTTATTTTGTAGAAACAGGGTTCGCCATGTTACCCAGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACTGGCCCAGTTTCTACAGTCTCTCTTAATATTGTATATTATCCAGAAAATTTCATTTAATCAGAACCTGCCAGTCTGATAGGTGAAAATGGTATCTTGTTTTTATTTGCATTTAAAAAAAATTATGATAGTGGTATGCTTGGTTTTTTTGAAGGTATCAAATTTTTTACCTTATGAAACATGAGGGCAAAGGATGTGATACGTGGAAGATTTAAAAAAAATTTTTAATGCATTTTTTTGAGACAAGGTCTTGCTCTATTGTCCAGGCTGGAGTGCAGTGGCACAATCACAGTTCACTCCAGCCTCAACATCCTGCACTAAAGTGATTTTCCCACCTCACCTCTCAAGTAGCTGGGACTACAGGTACATGCTACCATGCCTGGCTAATTTTTTTTTTTTTGCAGGCATGGGGTCTCACTATATTGCCCAGGTTGGTGTGGAAGTTTAATGACTAAGAGGTGTTTGTTATAAAGTTTAATGTATGAAACTTTCTATTAAATTCCTGATTTTATTTCTGTAGGACTGAACGTCTTGCTCGAGATGTGATGAAGGAGATGGGAGGCCATCACATTGTAGCCCTCTGTGTGCTCAAGGGGGGCTATAAATTCTTTGCTGACCTGCTGGATTACATCAAAGCACTGAATAGAAATAGTGATAGATCCATTCCTATGACTGTAGATTTTATCAGACTGAAGAGCTATTGTGTGAGTATATTTAATATATGATTCTTTTTAGTGGCAACAGTAGGTTTTCTTATATTTTCTTTGAATCTCTGCAAACCATACTTGCTTTCATTTCACTTGGTTACAGTGAGATTTTTCTAACATATTCACTAGTACTTTACATCAAAGCCAATACTGTTTTTTTAAAACTAGTCACCTTGGAGGATATATACTTATTTTACAGGTGTGTGTGGTTTTTTAAATAAACTCCTTTTAGGAATTGCTGTTGGGACTTGGGATACTTTTTTCACTATACATACTGGTGACAGATACCCTCTCTTGAGCTACATCGGTTTGTGGGGAGTCAAAAGTCCTTTGGAGCTAGGTTTGACAAATAAGGTGGGTTAACACTTGTTTCCTAGAAAGCACATGGAGAGCTAGAGTATTGGCGAATTGAAGAAATCCCCCTTTTTTTTTAACACACTTAAGAAAGGGGACTGCAGGTATACTCAAGAGAGTAAGTCGCACCAGAAACCACTTTTGATCCACAGTCTGCCTGTGTCACACAATTGAAATGCATCACAACATTGACACTGTGGATGAAACAAAATCAGTGTGAATTTTAGTAGTGAATTTCATTCATAATTTGATCGTGCAAACGTTTGATTTTTATTACTTTAGACTATTGTTTCTGATTTTATGTTGGGTTGGTATTTCCTGTGAGTTACTGTTTTACCTTTAAAATAGGAATTTTTCATACTCTTCAAAGATTAGAACAAATGTCCAGTTTTTGCTGTTTCATGAATGAGTCCTGTCCATCTTTGTAGAAACTCGCCTTATGTTCACATTTTTATTGAGAATAAGACCACTTATCTACATTTAACTATCAACCTCATCCTCTCCATTAATCATCTATTTTAGTGACCCAAGTTTTTGACCTTTTCCATGTTTACATCAATCCTGTAGGTGATTGGGCAGCCATTTAAGTATTATTATAGACATTTTCACTATCCCATTAAAACCCTTTATGCCCATACATCATAACACTACTTCCTACCCATAAGCTCCTTTTAACTTGTTAAAGTCTTGCTTGAATTAAAGACTTGTTTAAACACAAAATTTAGACTTTTACTCAACAAAAGTGATTGATTGATTGATTGATTGATTGATGGTTTACAGTAGGACTTCATTCTAGTCATTATAGCTGCTGGCAGTATAACTGGCCAGCCTTTAATACATTGCTGCTTAGAGTCAAAGCATGTACTTTAGAGTTGGTATGATTTATCTTTTTGGTCTTCTATAGCCTCCTTCCCCATCCCCATCAGTCTTAATCAGTCTTGTTACGTTATGACTAATCTTTGGGGATTGTGCAGAATGTTATTTTAGATAAGCAAAAACGAGCAAAATAGGGGAGTTTAACTTTAATATTTTCTTTTAAAAAGCATTTCATGTTATAAGATCAATTCTGAGTGGTAGAAAATGCTTTGACATTTTATTTCCATTTTCTACTTTTAGTTTTTTTCCTATTTGTTTAAGATCTTAGAGGATTATTAAGCTGAACTCCTCAACTGATAAAAAGCATGACATCTTAAACATAAGCAAAGCATATTTTTAGGTTAATTTTCACATAGAAAACAGTTTATTTTATGTGAAATTCTATGTAGATATACTATTTTTTTGGTATTTATTGATATGTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTATTTATTTTTTTTTTTGAGACAGAGTCTCACTCTGTTGCCCAGGCTGGAGTGCAGTGGCATGATCGTAGCTCACTGCAACCTCCACTCCCGGGTTCAAGCAATTCTTCTGTCTCAGCCTCCCGAGTAGCTGGGACTACAGGTGCCTGCCACTATGCCCGGCTAATTTTTGTGTTTTTAGTAGAGATGGGGTTTCACCTTGTTGGTCAGGCTGGTCTCGAACCCCTGACCTCAGGTGATCCACCCACCTCAGCCTCCCAAAGTGCTGGGATTATAGGCATGAGCCACGTGCCCGGCCGACATGTTAATTTTTTAAAAAAGGCTTTACTGGGGTATATTTTATATAATATAATAATCACATGTTTTAACTATACAATTCCAAGCTTTTTAGTATATTTATAGGGCTATGCAAGGAAGATATACTGTTAAACAGTAGAAATTGAGAAAGCTCTTCTGATAATATCTCTTGATTTGATGATGGCTCATGCCTGTAATCTCAGTGCTTTGGAAGGCCAAGACAGCAGAATCACTTGAGGCCAGGGGTTCGAGACCAGCCTGGGCAACACAGCAATACCCTATCTTTACAAATAATAAAAATATCTGTTGATTTGAAGTAAAGTTTTTTTTTAAAGACAAGGTCTCATTCTGTCACCCAGGCTGGAATGCAGTAGCAAGATCACAGCTCACTGTGGCCTTGACCTTCTGGGCTCAAGTGATTCTCCCACTTCGGCCTCCCGAGTAGCTGGGACTAACAGGTGTGCACCACCATGGCTGGCTAATTTTTTTTTATGTTTGTAGAGATTGGGTCTTACTGTGTTGCCCAGGCTGATCCCGAACTCCTGGGCTCAAGCAGTCTTCCTGCCTCAGCCTCTAAAATTGCTGGGATTACAGGCTTGAGTCACCATGCCCAGCCTGAAGTAGCATTTCTACCCTGTTTAATAATTCAGCAGCTTGTCATGTAAGATATTCATATATGCATATAAACATTAGGCAGCTTAATTTGGTAAAACTGTAAAATGGAAATTTTAAATTGTTTGCAGCATCAATAACATTGATGTCAGTATGATTTTTACATGCTGATCTTGACCAATTTGAAACAGTGAGTTAAAATCTGGCTGATCCGTACTAATCCTAAAGAAATATTCTATGAACTATTAAATGTTTCCAGAATATATAAAGAAACATTATGATGTCAACACACCCATCTATTTTTTTTTGGAAATAAAAACTCCATTTTTCTTATTAAAGAAAACATGCTTATTAGAAAACATACGGCTGGGTGCAGTGGCACACATGTAATTCCAGTGCTTTGGGAGATCGAGGTGGGAGAATCACTTGAGGCCAGGAGTTTGAGACCAGCCTAGACAACATAATGAGACCCCCTCTCTACACAAAAAGAATTAGTTGTGCATGGTGGCGTGCACCTGTAGTCCCAGCTACTTGGGAGGCAGAGGCAGGAGCATCCCTTGAGCCTAGGAGTTTGAGACTGCAGGAGTTCGAGACTGAGTGGAATGCAGTGGAACTGCATTCCAGCCTGAGTGACAGAGGGAGACCCTGTCTTAAAAAAATAAGAAAGAAAACACAACTGCAGAAAATTATAAAGGATTTAAGTCATTCCAAATATCACTGCCACTTTTTATTTAGAATATTCTAAAGAATTCTCTCTCTGTGTACACACACACATATGCGTACTCTTAATCCAAGTAGCTTGGTAGGATTTTATTTACCTAGTGCCTAGATGGGAAATTGCCTGGGGATTCCAAATACCTATTTCATTAAATTAAAGATGTCACTGATTTTAAGACTTAACACTATTTTTCATACTGCCAAGAAAGAAAACACTACCAGTTATAAATGTAAATTGCCATCAATTGTAATACATCAATTTTAGAGCTATTATTAATAAAATGTGAATGTGCATCTTAGAGCAATGAAATATAGTACTATATATTTGATGACCTTTTCTGCCCTGTGATATTCAGAAAGTGAAAGTTAAATATGGGCTGAGCATGGTGGCTCACACCTGTAATCCCAGTACTTTGGGAAGTCAAGACGGGAGGCTGGCTTGAACCCAGGAGTTCAAGACCAGCCTAGGCAATGTAGCGAGACGCCATCTCAAAATATTAAAAATAAGTAAATAAGTAAATAAAAAGAAGGTTAAGTATACAAATGTATTTCCTTTGTTGTGAATTTATTTCAATTTTATAGTGATTTTTTTTTTTTGAGACGAAGTCTCACTCTTGTCCCCCAGGCTGGAGTGCGATGGCGTGATCTCAGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCTATACTCCTGCCTTGGCCCCCCGAGTAGCTGGGATTACAGGCGCCTGCTACCATGCCTGGCTAATTTTTGTATTTTTAGTTGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTAGAACTCTTGACCTCTGGTGATCCACCCGCCTCGGACTCCCAAAATGCTGGGATTACAGGCGTGAGCCACCGTGCCTGGCCAGTGGTTTTTTGTTGTTGTTGTTGTTGTTTTGTTTTGTTTTTGTTTTTGTTTTTGTTTTGAGACAGGATCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGTGCCATCTTGGTTCACTGCAACCTCTGCGTGGGCTCAAGCAATCCTCCCACCTCCCTTTCCAGAGTAGCGGGGACCACAGGTGTGTGCCACCACACCTGACTAATTTTTGCATTTTTTTTTGTAGAAACAGGGTTTTGCCATGTTGCCCAGGTTGGTCTGAAACTCCTGAGCTCAAACAATCCAACTGCCTTGGCTTCCCTAAGTGAAATTACAGGCATGGGCCACTGTACCCAGTCTAGTGATTTTTTTATTTTTATTTTTATTTTATTTTATTTTATTTTTTTACCAAAAAAACAACAAAGCCTCAGGAGGAAAAGTTGATACACAAGTAAATTTTATTGGAAATGTTTTTGTGTGGACCTTAAGCAGAGGGAAAATTAGTCTGCATTATGGTGTATCCAGACTAAATGACTGATATTAAAATGAAATTATTCTTAGGATTTGCAATCTTAGAGAAAACTTTTTCATTTTTATTTTTTTGAGTTACAAATTATCTTCATTTACATTTGAGAACAGTGAGTCACAGAGGGATTAAGTAACTTACTCAAGATCATACAAGTCTTTGATTTGAACCCAATCTTTTAACTCTGCAGAACTCAGAGTCACTCTTATTTGGAAAAACTTTTTAACTGATGTGGATCCTCTAATATGGGCTTCCTATTATTCATTCTCTATTAGTCAGAAGTTTTGCAAGCAGACAGAATTCATTTTGCCAATTACGGGATTTTCCCTCAGTTGCAGTCAAGGTTCATAAAACTATAACTCTTTATCTTTAATTAGAAATGTTTTTTTTTTTGAGACAAGGTCTTGCTCTGTTGCCCAGACTGGAATGCAGTGGCATAGTGGCCCATTGCAGCTTTGAACTCCTGGGCTCAAGGGATCCTCTGCCTCAGCCTCCCAAGTATCTGAGACTACAAGTGCGTGCCATCACCCATGGCTATTTTAAAAAAAAAAAAAATTGTAGAGATAGGGTCTTGCTGTGTTGCCCAGGCTGGTCTCAAACTCCTGGTCTCAAGCAATCCTTCTGCCTTGGTCTCCCAAAGTGCTGAGATTACAGGTGTCAGCCGTTGCACCTGGCCAAAACGATAACTTAAAATACACACACACACACACACACACAAACACATATGTGTATTTGTGTGTGTGTGTGTGTGTGTGTGTCTCAAAAGGTATCAAAAGAGAATAGCTATAACTTTAGTGTTGATCTTGATAGTGACTTGATTAGGCTCTGTTTAACATCAAAGATGCAAATTAATACTTTCTTTGAACATATTAAAAATGCAGAAAATATTGGAGTATTTTATTTTAAATAAATTGTATTCTGTATATTTAAGGTATACAACATGATGTTATGGGATACATATAGGTGGTTAAAAGATTACTGCAGTGAAGCAAATTAACGTATCCCTCAACTCACATAGTTACCCATTTTTTTTTTGTTTTGGTGGCAAGAGGAGCTTAAAATCTCATTTAGTGTGAATCCCAAATACAGCACAATTTTATTACCTATATACTTCATGTTGTACATTATATTTCTAGACTTGTTCATCCTACATATCTGCTACTTTGTATCCTCTGAGCTACATCTCCCCATTTTCTCACTTGCCCCCCAAGTAGTTTCTTAAAGTGTCTCATGTAAGAGGGCAGTAGCTTTCAGCTTAAACTTTTTCTCTGTATGTAGTCGATTTCTTTGAGGTATACTTTTCTCTCCAGAATAGTTAGATGTAGGTATACCACTTTGATGTTGACACTAGTTTACCTAGAACTTATCTTCTGTAAATCTGTCTCTATTTCCATCTCTGTCTCCATCTTTGTCTCTATCTCTATCTGTCTATCTCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTAAAGCAAATTCATGCCCTTCTCCTATTTATTGAATCGAGACCATAGACAGGGGTGAGAGAAAGAATTTGGCAGGAATGGGGATGTGTATTATCTGTGGCATAAGGAAACTTTACAGAACTAGGTTCAAAAGTATACTTTCTAGTTCTTTCCCATGGCTTTTCACTTTGATGTAGTCCTTATCAGGTAACTGAGGTTTTATATAAGTCCCCTGATTCTTAGAACATGAAGGTGTAGTAGTCAAGGTTGGTCCCTTGAAACCACAAATTTTGTGAAAAAAAATTAAGAAAATTTGAATAATTTCCTCAGCAAATACATATTGATCATCTGTTATACAGCCATGAGAAGTGGTTCTGTTGCACACGTTTATTTTATCAGATCCTAATCCCAAACCAGGCATAAAATGGAAACCATGAAGATAGGATGAAATAACTTCTGAATGTTTGAATGTTTGAAAATAGTGTACTTAAAAATACCAGGTGGTTTTTGTTTGTTTTTTGTTTTTTTCTTTTTTTGAGACAGGGTCTCACTCTGTCACCCAGGCTGGAGTGTAGTGGTGCAATCTCATCTCATTGCAGTCTTGACCTCCCAGGCTCAGGTTATCTCCCACCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACATGCCACCACGCCCAGCTAATTTTTTGTATTTTTTGTAGAGACGGGGTTTCACCCTGTTGCCCAGGCTGGTCTAGAACTCCTGGGCTTAAGCGATCCTCCCACCTCAGCCTCCCAAAGTGCTAGGATTACAGGCATGAGCCACCATGCCTGGCAGAAAATACCAGGTTTTTAAGTATCAGCACTTACTCTTCAATCTTTTCTATTACTATGTTGTGCTAAATGGTATTTTTTATTTAATTAGAGCAATGCTGTTCAATAGAACTTTCTTTGAGGATGGAAATCTTTTATGTTTCTGCTATGTGGTACAGAGCCACTAGTGACATGTGGCTTTTGAGCGCTTGACACATCTTGTGCAACACAGGAACTGAATTTTTAAGTAATTTATATTGCCACATGTGGCTACCGTATGGGACAGTGTAGTACTAGATGATCTGTAAGGGCTGTGCTTCATCAGTGTCGTTTTTTAACTGACAAAAACCTTTAGTTTTTTTTTTAGTAATGTGTTTATTTAAAAGAATTCATAAAATACAAGTAAACAAATTAACTTGTTACCTGAGCATATGTCCTTTCATACTTATTTTTTCTGCATACATATTTTGGAAAATGGAATATCTGCCCCTTTTTTTTTATCTGAGATACAGTCTACCTCTAAAAATACATGATTCTAACATTCTCACTTTTTGTTGGCATTTGATCAGGGTATAGAAAAACAGTTAAAAGGACAGAGAATGGTTGAGAGATTATGATATGAAGAGAAAATGTGATTGAGTGTGGTAGACTTGGGGCCTGCTTGAATGTTGAGAGAATGACTGTTTTCCGATAAAAAAAAAAAGTCCATTCTAGGATCCTAAAAGAAGGGTCTGAAGTTCACTGCAGAAAGCAAGCTACATAGTACTAAGCCACTAAGGGGACATGGAGCCCTTAGTAATTCCTACCTTAGTAATAGTCTCATCATGCCCTCTTGGGAACCCAGCCTTGTTGATTAGCCTCTCTGCTTTCTCTCCTTATAGTTCAACCTCCCTGTTTGTTCCAAGCAGTTCTTTTCCTGCCCATTTATTATGCATTTCTATACAGCTTTCCTCCTCTTTTTCTATACCATGCTGCAGTTCTTATTGCTACCTAGAGGTTTTCAAAATTCCTAGGGGCGGATAAGTAGGCATAAACAAAGTTCTTCCCTATTATCCTTCCTATTTTTTCACCTAGACTGAAGAGGTAGACAAAATAGAAATAAAGACATTAAGGGTATGTGTTTGTAGTCCCAAAGAGCTTCTCTGGCAATTTTGATGTAGTTGACAGTGACGCTCTGAGTTCAGGACAGATTGGACTCCTTGGCTGAGAGGAGTGAGGAGATAGGACGGTAGAGGAGAGGGTAGAGCAACTCTGGAGGAAGCTTTCCCCTCACCTTTGCCAGTCCTGTTATCCTAGACTTAACCATAATTAAAGATGAGGGAGGCACTCAGTAAAGGGATCTAGTGGGAAGCTTGTTCCAGACAGCCAAGGAGGGAGGTTCGCGCAGTTCCTTTGGCCACCCAGGTGGGGTAATTGATCCATGTATGCCATTCATGTACAATGTAGGCACTTATACCTGTATTCCAATGTAGTGAACTATACCATTACTCTTAAATTAATATTCTTTATTAGCTTCCATGGTGGCTATAGGCCAGGCAAGAGAGTTAAGAAAAAATAAATAGCCAGGTATGGTGACTCAAGCCTGTAATCTCGGCACTTTAGGAGGCCGAGGCAGGAGGATAGCTTGAGTCCAGGAGTTCAAGACCAGCCTGAGCAAAATAGTGAGATCCTGTCTCTATTTTTTAAAAAAGCCTTGGGGCAAACAGGAGTATGGAGGTTTGGATGCTAATAGAACAGCAGTGTCTTACTGCTTGGAGTTCTCTTGTTTCTTGTCCTATCACCGTAGCCTTTGGATCACAGCAATTTTTCCATGACTCCATACTTTTCAGTTCTTGAATATTTTTTCCTTTATTCCTCTTGTCTCTGTAAAGACATCAACTGGAGTTGGACTGTAATACCAGGTATCTCCAGAAGATGGCACTATTTAACAGATTTTATAAATAATTTGATGTGAGTCACTGTCATCTGAAGCTTGTTGCCTTTTCTTTCTTTCTTCTTTCTTTTTTTTCCCCATCAATTCTGTATGTTTGAAATGCTGGGATTTAAGTTAGTTAGAATAAGGGATGTCTGTAATTTCCCTAAATTGAGAAGTAATATGCAAAGGTTGATATCAGAAGTCATATGCTCACCTTGCAACACCAAATAATACTGGCCCATTTGTGATTTTTGAAAGTAACACTCCATAATAAATGGATGTATATATAGAAGCATAACAAAAATAGAAGCACATAAAAGTGAAAAGTCTCATAAACGCCATTGTCACTACTCATGTAATTGCTGTTACAAATTTGTTTAAATGTTGAATAAAAATGGTGTCATAGGCAACACAGTGTTCCACTACTTGGTGTTTTTAATAGCATTATTCTGTCTCAGTGTGCTTTGGATTATCAGGTGCTTTTTAATAGTTGCATGGTATTACATTGTGTAGATGAACTTGATTAATTTAAATGGTTCCCTGTTAATGGACATGTTGGTTTGTTTTTGTGAACAACTGATACAGTGAACATTTATTTTTTAAATAAAAAAAAGAGAGACAGGGTCTTGCTGTGTTTCTCGGGCTGGCCTTGAACTCCTGGGGTCAAGCGATCGTCTTGCCTCTGCCTCCCTGGGATTACAGGCATGAAGCCACCGCACCCGGCCCAGTGAACACTCTTGAATGTATCTTTGTATACTTGTCAAGTGTTTTTGTAGCAATTGATTCCCAGAAGTGGGAATTACATGGAATTAAGTGACATGCATGTTTGCAATTTTAACAGGTATTGCTATGTCATTTTCAAAAGAAGCTATGCCAATTAATACTCTCACCAACAAGAGTGCTTATTTCCCCTCAGCATATTATCAGGCTTAAGTTTTGCCAGTATGGGTGGGAGAACAGTAGAATCACATTGTTTTAGTGTTTGTTTCTCAGATAGATATAATTTTACACCTTATAACCTTCTCTTCTATAAATTGTCTATTTGTGTTCATTCTCCATTTTCCTATGGGTTCTTATTGTTGGAGCCCAATATATAAAAGGGGGTATTTGTTACAGAACCTCTTCAGTTTTGGTTCATGTCATGCCTGGGTTTTTACCCTTTCTACGGATGTTAAAAAAAATTCTCTATTTTCTTCCAGTCCACTTATGGCTTTATTTTTTACATTTAGATTTTAATCCGTCTGGAATTTATTTTTGTGTATGCTGTGAGGTAGGGACCATACTTTTATTTTTTCCCAAATGGGTTACTAGTTGGCCAAACATCATTTATTGAATAATTCATCTTTTCCCTACTGACTCGAAATACCATCTTTATTGTATACTAAATCCTCATATAGTTCTGGGTCTGTTTCTGGGCTCTACTTTGTTCATTTACTGTGCTGGTACTGCACCGTTGTAATTGCTGTGGCTTTGTGGTATGGTATGGCTTGCTCTCTGCTAGGGCAAGTCGAAGCTCTTTTGTTCACCTGCTCTTTCACCCAAATTTTCTGTCCTGAATCCAGCACAGCCAAATTATGGTCATTGTCACCACCAACTACAGTGGGTGTTGAGCATTTCCCATTGAATCTCCTGTAAGGGTTTTATTGGATTCTGTGATAGCAGTAAAATGGGAGCCTAAGAGGTATTCCTTAAAGGACTACTAATCAGACCTGGTTTCCCAGATGATGCTGAAGATGACGGGGCCTGGGCTAGACTTTTGAGGGACATATCCTTGGGGTTGGGTGTGATATAGACCAGCCCTTACAATTTGCTTGACTCATGGGAATCGTACAGGGCCAGAACCAGACACCTGTCATGCTAATAACTTCCCTCACAATTCAGAAATCACTGTGATTGAAGATGGGTGGCTGTTATAATACTACCCACTTAAAAATGGATGTAACCCATTTTTTAGGACTCTTAAAAACATCAAATCAGTAATGGCCGATTAGGACTTTTTAATTTTTACTAATCTCTACTTGAAAGTTTTCTAGTCATTCATTTCAGGAAACCTAATTCTTATAATTCATATCATTTAGAATATCATAATGCTATGGATATTAGCTAGCTAACTTCTCAAATCTTCTAGTTCTCATTTAATTTGAAGTTTGTGTGTGTACATAAGGATATACATATACATATGTGTGTGTAGATATATATATATATAGTTTTTTTTTTTTTAACTAGAATGACCAGTCAACAGGGGACATAAAAGTAATTGGTGGAGATGATCTCTCAACTTTAACTGGAAAGGTATGTATCTTGAAAGGGAAGAAAAAAAAGCACTTCATACCGAGTCAATTAGTAACAGTGTGCTTTCAATCAATCACTAAGAGATAATTTACATAGTATAACTAAATGGGTTATTTAACCCTTGGAAGCAGTCTAGGTTAATTATCGTTCCCTAGGTCATGTAGTAAAAAGACAGTAGAATCCAACATTAACCTTAAATGTCCATATTGTCAAGTACTGCTGTCTGCCTCTGTGGGACTCTAATTTGGGATCCTTCAAAAAACATTGATGGGGGAAAAGATAGCCTTTAAAAAAAAAAAAAAAACAAACCTATGTGAGTCTATGTGAGGTAGACTCACATAGTTTCCTAAAAGATAGCAAAGCAGTATTATGTAGTGGCTGAAAGTGTGAGTTCCGGAGCCTGACAACTGATTCAAAGCATGGCTTAGTACTTCCTAACTCTGACCTTGGGCAAGTTACTTAACCTCTCTGTGTCCCATATGTGATTAGGGTGAGGTTGATAATAGCAGCCATAGAGTTAAGAGGATTAAGTGCTATAATGCAAGTAGAGCTCTTACAACAGTTTCTGGTAAATCACTCAATAAATTCAGACATACTATTATTTTAAGAAATCTCAAAGAGTTTTCTTGTACCTTAAAATTCTCCTAGTGTGAACCATTGGTTTTGGTATATTGTGCTTCCATGTAGTTTAATATCAAGATGTTTTTAGATTTCCCTTTTAATTTATTTGTTGACCCATTGGTTGTTCAGGAGCATGCTGTTTACCTGAAAATAATGGAGATATTAAGGTATTTGAATATTTATCTTCTAGTACATTGAAAAACTTTTTGAGAGTAACCAATAATAAATGATGGAATGCTACTGCTTTTTTTTTTTGAAGCTGCCAGTTATTGTTTACTTACACTATGCCAAATATAAAGGCATTAATCTCATAAAAGTTTCACAACAATCCTGTGAGGGAGACGATATCCCCATTTTACAAATCAGGAAATTAAGACTTAATAAGGTTAAAAGACTTGCCCCAAAGTCACAGAACCAGTAAGTGGTAGAGCTTGAATTTGAATACAGACCTGACTCTAAAGCTCTTTTCTTTCTTTAGATTTTAGTGTTCATTGCTTACTTGAATGAGTATCTATAAGAAAACTTTAACATGTAAAACTTCTGTGAAATTATCTTGTCCCATATCAGGGTCATGTCAAACTAATGTCCTCCTCAGCATCTTTGGAAAACTTCAGAGGAGAAATGAGCTTTGCCCCTCCTGTTCATTTCCTATTCCACTAGGAGACCTGTCCTTCCCTTTCAGCATGCTTTGTCCATATTTAGAAGCTGTTGAAGCCATTACTTGTCTGGTCAGTTTTTAGTGCTGGAATGGACCTAGCCTTTTAGGCCTTCTGAGATTTAGTTTGATCTCGTCTTTCCCACCTAATGGCTCTGTTCTACTACATAGATTTGATCTGAAACAGTTCTCTGTTTCTAAAATAACTTTCTTTTCATGATAGTCACAGTAAAGTACATTTATTATGGAAAAATCAATAAGTATAACGAGTGAAAGTTATTTCTTGGTGGTAAGATTATGGGATTATTTGAACTTTCTGTTTCATTGTATTTTATTTATTTATTTATTTTTGTGATGGAGTCTCACTCTGCTGCCCAGGCTGGAGTGCAGTAGTACGATCTTGGCTCACTGCAACCTCCCCTTCCCAGTTCAAGTGATTCTCCTGCCTCAGACTCCCAAGTAGCTGGGATTACAGGCGCACGCCACCATGCCTGGCTAATTTTTTTATCTTTAGTAGAGACAGGGTTTCACCATGTTGACCAGGCTGATCTCCAACTCCTGATCTCAGGTATCCACCTGCCTCAGCCTCCCAAAGTACCGGGATTACGGGTGTGAGCCACCCTGCCTGGCCTCATTTTGTCTTTTGGGGGTATTTTTGTGTGCAGATATATATGTATATAAATATTTTTCCCTCTTTTCCCCAGTTAGTATTTGAGCAGATGAACTTTGGACCCGAATACCTGTATTCAAGTCTCTAATACCACTTCTTGGCTATTTTCATTTTATCAAATGGCCTCTTATCCTCGTTTTTCTCATTTATTAAGTAGAGATGTAACTACTTGATATAATTCAAAAACTCAATAATGGCATTCTTTTGTTTTTTAGACTCTAGTGTCTGTACTCCTTGTACCATGCTGGGATTCATTTGAACAATTGCATGGCTTTTTTAGTGTATTATTAAATTTGCAGTTTACTTAGAATTTACTGGGACCTCATACAAATGGGAAAAAAACATAACTGTGTTACTCATTTGCTGTGTGCCTTTGGATTGACCCTATTTTTTGTATTCATTTTCTCCCCATGTCCTGAGTTCCACTTTGAATAAAAAAGTAATTTTTTTCCTGCCTGTAAAATAGGCTACCAATAGGCTGCAGTTGTCTATAGTAGCTGCTTCACTGAGGAGAGCTCAGCATGAGAGAAATAGTATGAATTGCTTGCCACAAGTTATGGGCTAGCCTTACTTCATTCTGTACTTGGACCTGTTTAGGCTTCTAAGAGATCTTACCTCCAACAATAAACTGCTTTGAGACATGAAAAGGTGGAAGCTTTACTTGGTTATAACTTTACTTTTAATACCTAGAACAGTGAGTCTTCAAACTTGTATTTGCATGCCCAATTTATAAAAAGTTTCCTGAGCATTTACCCCTAATATATGCATTTTAAATTATATATGATTTATGGTAATAATAATATATATGTTACAAAATACATACAAAAATATAGATTAAACAAGGTGAGGTTAAAAAATTTAAAAGTTCTAATCTTTCTTGCAAACCAGTGGATCTTTTGTGCCTTACTCTGGTAAACACTGTCTTAGAAGAATATATAGAACATTAAAATCTTAATGCTATAGTTATATGACAGAGTATGATGAGAGCTACAGATAAACAACACATCATGAATCTTCTTGTGGCAGTGTTTATAACCATTATGTGAAATGCTGCCTCATTCTTATAACTAGCATAAGAACAGATAGGACTTTCTCGATTTTGAGGGGTAATTATTAGATGGTATTTTCTGTTAAGGACTCTTCCAGCTATAAAATTCTTAAATGTAGAAAGCGAAGTGAGGGTTTATGGTGAGAGGAAGCATTGGTATCATGTTTTAGTGTAGTCCAAGAATATGGACACATCCAGAAAATGCAGATCAAGTTTAGCCTAATGAGAAAATATATTTTGGAGTCCATATGGTAAATTAAATTATGTGATTTTTGAGTTATTGTACAAATATAATTCTTAGAATGTTAGAGTCAGGAGACTATAAGAGACCAACTGCTTCAAGTTTCATTTAACACATGGGAAACTAAGGCGAGAGAAATTTCAAGACTTGCCCAAGATTAGACCTCTTGTTAAGTAATGAAAGTGTTTTAAAAACAGGTGGGTCAAATTCTGTTTTTAAAATTTCCATTATGATGAAAATTTCAGTATTACAGGCTTCCAAATCCCAGCAGATGGGCCACTTGTTTAAAGGAGAGTTTGATATAATAAAGCATCTAAAAACAAGAGTTTGGATAATTCCTTAGGGTTGTTATGATGTGATTTGACTTATAATTGGAAATACCGTTTTATTCATTGTACTGATTTTCATTTCTCTTTTTCTTCTAGAATGTCTTGATTGTGGAAGTAAGTTCACATTTACTTTTAATATAACATTTATGACTTTTCTAACTTAGTATGCACCATCCTAAAGGTAAGCCAGGGAGAGAAATTCCTCTGCATCAGTTTTAATGGTGGGCTTGTGTTCTAAAGGAGTGAGATTGGTTTTTTGTAAAGACTACTTAGTAATTTGTTTTTACCAATAATGGAATGGTATACTTCCTACCTCTCTTTTTTTAGTTTGAAGTATTTTCTTTCTAAACATAACTCTCTCTCTCTATTTATCTATATATAATATATACATATATATCTTATATTTTATGTATATATATATATATCTTGCTTAGATTTTGTCTTATGTAATATTTGGTACATAAAAAATAATATTTATAATTTATAGACTATTTTCCATGTGTTATTATGTGCTAAAGTATTTTGTATCTTAGCACCGAGAGGCTAAGCAGTTTCCTAGGGTTACCAGCTAGTAAACTAAGGGAAACCTTTACTTCCTTTAGCTCAGTGGTTCTCAAAATGTGGTTCCCTAGACCAAAAGTATTAATATCAGACAAGAACCTACCGAATCAAAATATCTGTGATGAGGCCCAGCAAGCTATGCTTTAACAAGTTTCCGAGTGATTCTGATGCATGCTAAGGTTTAGGATCCCTTGTTTTTACTCATAAGTCACTTTCTCATTAAGGCCTTCCCTGGCCATCCTATATAAAATCTCATGTTTTCACACCGTCAACTTCGTATTCCTCCTCAATACTTTTATTTTCCTGATCACTTATCACTAACAGCCTCTCTCTCTCTCTCTCTCTCTCTCTATGTATATATATATATATATCACTTATCACTGTCTAACAGCCTCTCTTTATATATATATAATCTATAGATTATATATATATGCAGCATTGTGCAATCATTATCACGCTCAATTTTAAAACATTTTCATTTCCCCACAAAGAAACCCAATCCCCTTAGCCATCACTCCCAATTTTCCCTTCCCCCAGCACCTAGCAAACTGATCATCTACCTACTTGCTGTCTATAAGATTTGCCTATTCTGGACATTTTGTATAAATAGAATCATACAATATGTGGCCTTTTGTATCTGGCTTCTCTCACTTAATGTTTTCAAGGTTCATTCATGTTGTGGAGTATATCTGCACTCATTTCCTTTTTATTGCCAAATTGTATGGATAGACAGGTGTTCCTCAACTGTGTCCTGATAAACCCATCTGAAGTTGAAAATATCATAAGTTGAAAATGGATTTACTACTTTGATAAATCTATCCTAAAGTCAGAAAAATCTCATGTTGGAACCATCGTAAGTTGGATACCATCTGAATTACATTTTTGTTATCCATTCACTGGTTGACAGACGTTAGGTTGTTTCCACTGATGCTCCTTATTTCTCGTACCTGAAATGTCCTTATTCCCTCCCTTCTTATCCCATGTTTAAGTCATTTAAGACCCAGCTCAAACGTCACCTCCACAAAACCTTCCTTGATACCCCTTTCCTCTTCAATTCACTTGGACCTTTTGCATTTAATTTTAATTTTTATTTTTTTTAAGACAGAGTCTCACTCTGTCACCAGGCTGGAGTGCAGTGGTATGATCTCAGCTCACTAACTACTCTGCCTCCCAGGTTCAAGCAATTCTCATGTCTCAGCCTCCCAAGTAGCTGGGACTACAGGTGTGCGCCACCATGCCTGGCTAATTGTGTGTGTGTGTGTGTGTATGTATGTATGTATATATGTGTGTGTGTGTATATATATATATACACAAACATATATAAATATATATACATATATATATATACACACATATATAAATATATATACATATATATATATACACACACACACACATATATATATATATAGTTTTTTTTTTTTTAAGTAGAGATGGGGTTTTGCCATGTTGGCCAGGCTGGTCTGGCCTCAAGCCATCCTCCCACCTCGGCCTCGCAAAGTGCTGGTATTATAGGCATGAGCCACTGTGCCTGGCCTGCATTTCATTTTAATTATAAAATATTTTGAACTCAGAAAAAAGGGTATGCTGAATACCTACGTACCCACAAAAGTATTAACATTTTGCCATATTTGCTTCTGATCTTATTTTTTTTGAGAAATTAAAGATCATAATACAACTAAAGCCCCATTTCTTTCCCTTCATTCCCAGAAGTATGACAATTATCCTTAAAGTTGATATATATCATTCCCATGCATGTTTTTTATACTTCCCTAGTACAAGTTAGCTGTATCCTCTGCTCAGGGGCTCATCAAGCTGAATCAAGGGACTCATGATCCTCTTCAAAGTTCCTTCAGGTTGTTGGCAGAATTTAGTTCCTTGTGATTGTAGGACTGAGGGCCCGTTTTCTCACTGGCTGCTGGCCAGGGGTTGCTCCCAGATATTTAAAGGCTCATGCCCTAGCCCATGACAGTCTCACAACATGGCAGCTGACTTCTTCAAAACCAGCAGGAGAATCTTGCTCTAGTCTACCACATAACCTAATCACAGGAGCGGCTATCCCGTTATTTTCACAGATCCTGGTCACATTCAAGGGGAGGGAACCCTTCTGTGTGTGTACACCAGGAGGCAGGAATTTTTTTTTTCTTTTTCTTTTTTGTTAAAAAGTCTTAAAGTCTTTTATCCCTAAAGGAGGCAGGAATTTTGAGAGCCATCAGAATTCTGCCTACCACAGCCCAGAAATCTGCATTTTTCACAAGTCTCCAGCCATGATGTTTCTGATGGCTCACACTGCTTTATTCCATTTTTAAAGAGTATTTTTATTGAAAAGCATTAGGGTTATGGTTTAAAAAATATTTTCCCTAACAAAGATGGGTTTGTTTAGAGTCCTACTTTTGACTAAATAGCTGAGATTCACTTTTATGTAAAGTTCATTTTATAGCGTTATTAATTTGGGTGCCTTTAAAAATAGTATAAAGCATGTTTCTCGAGTGTAGTCTGTTAGCCACCTATATTGGAGAGTTGGGAGGAGAGAGTCTCTATCTTGAATTTATGGGAAAAATTCTAAAATACTTTTTATAATGAAGGACAACATCATAACTCCCTAATAAAATGTGCATGTATATATTCAAATTTGCTGTCATTGATCCTGCACCTACAAAATCCAGTCCTGGGGGCTGGCATTCTTACTGCTTGCTGAGGGCCAGATGATATAGATTCCAGAATATCTCCATGTAGATTTTGGTGAGAATTACTGTGCTGAAAAGAATGACAGTATTGCAGTTATACATGGGGGTTTTGGTACTTTATATTGTGACTCTGAATTTAAAGCTATGCAATGTCTTCTTTTTTGAAAGGATATAATTGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAAAGATGGTCAAGGTCGCAAGGTATGTATGACATTTTGACACAGAATATTTTCCTCATTTGAAGGGGGATTAAGTGATTGCTTCTTTTTAAGGATAAATGTTTTCAACTGTCATTTTATCTTCGAAAAGTAATGTAATCTCATATAAGACTTAAGATATAATCCTTTTAAATAATTTTGTCATGTGTTAATAAAGCTCATAATTACAGTCACTTCCTTGCTAATATTAACATTTGGTTTTCAGCATGCTAATTATATCAGTTTGTCCTGAATAGCATGGCAGAGGATTTTGGGCCCCCTTGCAAAATTAAGAATAAGGATTCCAAAGCGGGTGAGGAAGTGATAGGAAGGGGTGGGCCCTGAAGATCTGGACCTCCTGGAATTGAGTGATGAATGCTGCATCTTCTTTGTGTCTGTAGTGAAATTTTATAATGCCTGCTTCCTTTTTTATTAAGTCGGCCTCACCTCCTCACCTTACCTATGCTGTTTTACTTTTGCTTTTATAGTTCTACCTGTGTTTATTTCTCATTTTCGTTTCATCTCTCAACAACTCTGGGGTGGCATTATTATTCCCACTTTTCAGATAAGGTTACTGAGGCATAGGGAATTGTCCAAAGGTACAGAGCTAGTCCGCTATAGAGATGAGATTTGAACCCAGGGAACCTGGCTCACAGTTTATGCTTTTGCCTACCTTAAGTTTTTAATAGAGTGACATCAAACAAACATTTAAGAATATGTTTTTCTTTTCCTTTTATAATTTCATTAAAAACATTAAGTCTCTGATCAGTCTGCAGTTTTTATGTAGGGGTCAGGTAATGTTCTAACTTCTGCTTTTTCCTAAGTGATTAACAGGTTTTTATAAGCCCTTTTGAAAAAATCACGGTATCTGTCGAGCATCTTTGAATCAGAGTAAGCCTTCTAGTGAGTCATATGTCAGCAGTTTGACTGTATGGGCTTTTCTAATATCCAGTTCAAGTGTTTATCAGTGAGTTTTTCTTTTAAATAGATTTGGGACAGGTACTATGAGAGTATATAAGTGATACGTTATAGGACACTAACTAGTATCCTATGAAATGGCAAAAACTGCAATCACTTTTGCACCAACCAAATAGAAACTAATCAGTGCACTTGCTTATTTTTCTACATGCTCTTTAGGGTTTTAAATGTCAACCTACTGTGGCATAGACTTTAATCCTCTGGGTATTCTTTTGTTGTTCTTTCCTGGTATATGCTGTGGAATTGAGATAGACTGGTTCGTGAGCGAGAGATTTTGTGTTGCCACAGGTAGGACATGCTCAAACAATACTTGGGTCATTTCTTGACCCAAGTCATCTATTCACCATAGTTTTGTAGCACCGATCTTGCATACATTTCATGTATCTTCTTTGAACCCCACGTCAGTGCTGCTTATATGATACTCAGAAATTAAACACTAAGGAATAAGATTTTCAGGTAGGATTGAGTTTTGGAGGGTCACAAATCTTGTAATGTCTAATATTTCCACTCTCCCTGCTGAGAATTAGTTTTGGCTTCCTTGGAGGTGATATCGCCTCTGTTGAGTATAAGTGGCCTACTGTGATCACACCACTGCACTCCAGCCTGGGTGACAGAGTGAGACCCTGTCTCAGAAAAAAAAAAAAAAAAAAAGAATGCATGGCCTAGATGACTTCTAAGGTTTTTCCCACCCAGTTCCAGTTTTCATGTTCTAGGCAGAGCAGTAAAGTGAGAAACACATGGACTTGGGAGTTTAGTCTCGCATTTCACTGCCACTTAATCTGAGCGACTATTCCATATTTAATCTCTCTGAATGTATTTACTCATCTTTAAAGGGGAATGATTATTAACATCTTTTTCTCAGGGAAACTATATGAGTCAAGGAGATAATATATTTGAAAATCTTTTTAACTGCAAAGCGCTGTTTCACTGTTGGTTATAATGTGATTGATCTCATTGTAGTGAGCAGCTGCTTAATTGCGTTTTAGAATGTAGGGAAGATAGTAATATTTTTCACATTATATATGTAGCTGGTTCTGGAACTGTAAACATACTCCTTTTTTATGGAGATCTGAGTCACGTACCATAAAATTCACTCTTTTAAAGTTGTACAATCCAGTGGTTTTTGATATATTCAGAGTTGTGCATCTGCTACCACTATTTCATTTTGGAACCCAAAGAAACCTTGTACCCATTAGCAGTCATTCTCCCTTCTCCCAGCCCCTGGCAACTACTAATCTACTTTCTACAGAAAGTCCGTACAGATTTGTGTATTATGGACATTCCATATAAATGGACTCATGCAATATCCTGTCTTCTTTCACTTAGCATAGTGTTTTCAAGGTTCATCTAGGTTGGGGCATGTATCAGTACTTCATCCCTTGTTTTGGCTGAATAATATTTCATTGTACAAATATATCACATTTTGCTTATCCATCTGTTGGTGAACATTTGAGTTTCTACCTGTTGGCTTTTATGAATAATGTTGATTTGAATGTTTGTGTACAAGTATGAATACCTGTTTTCAGGTCTCTTGAGTATATAGTTGCTAGGTCATATAGTAACTCTGTGTTTAACATTTTGAGGAATTGCCCGACTATTTAACAAGGTATATGTACTGTTTTACACCAGTAACATATGAGGGTTCCAATATCTCCACATCCTTGACAACACTTGTTACTGTCCTTTTTATTGTAGCCATCCTAGTGGCTATGATGTGGTATCTCATTGTGGTTTTGATTTGTGTTTCTCTGATGCTGATGATGTTGAACATGTTTTCATCTGCTTATTGGCCATTTACATATATCTTCTTAAGAACGGTTACCCATTTACAGTATGGAAAATGCTTCAGATGCAACTCTAGTCATGCCTTAGAGATGGAGCTTTATTAAACATTCAGATCTCTAGGCATATGAAGTGCTGAGTTCTCTTGAACTCCTAATACAGATTGCACTGAGTTTAGTGATACCTTTTCTGGAGCATTCCTGAGTTCAGGTAGGGAGAAGGGTTTTTGCTGTGATTGGCTTGTTATGTTCTTTCTAAATGGAAATAGAATTGAAGTGTCTCCTCTCTCCATTTA

24. Some types of alternative splicing Alt. 3’ SS Alt. 5’ SS Exon skipping |pA |pA Different termination

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

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

27. 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

28. 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 chance 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 mammalian proteome.

29. 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) Many human genetic diseases are caused by mutations causing missplicing

30. Anti-sense RNA:A therapeutic intervention at the level of pre-mRNA splicing 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) Pseudo exon activated  disease Antisense = block and skip unwanted pseudo exon Alternative 5’ splicing Unwanted = longer exon Antisense  shorter isoform

31. Got this far

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

33. Therapeutic intervention at the level of pre-mRNA splicing • 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 in a hemoglobin gene • 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 mRNA destroyed here, want to correct its splicing PNA = peptide nucleic acids

34. 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 cancersTarget 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

35. RNA modification for stabilization ase Instead of deoxyribose or ribose Modified phosphate ase Still base pairs OK!

36. 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)

37. RNA modification Also can add 2’ MOE -O-CH2-CH2-O-CH3 MOE = methoxyethyl - Phosphorothioate deoxyoligonucleotides

38. 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. Induced exon skipping yields green fluorescence

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

40. Dystrophin gene 2400 kb, mRNA = 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, length = a multiple of 3) Deliver antisense to the 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: mRNA with skipped exon dystrophin protein muscle histochemistry for dystrophin

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

42. U7 promoter Double target synergistic (loop?) (Kole) compl. to branch compl. to splice donor site Consensus binding site for Sm proteins (to target to pre-mRNA) ITR = inverted terminal repeat, characteristic of AAV

43. Expression of U7 antisense construct RT-PCR transgenic U7 U7SmOPT-A.S. Endog. U7 (slow onset =conclude slow mRNA turnover) 0 2 4 6 13 weeks Splicing assay (RT-PCR) included Skip exon 23, after 2-4 wks. 0 2 4 6 8 13 weeks normal Dystrophin protein (Western)

44. dystrophin-associated antigens dystrophin Muscle immuno-histochemistry intriguing Normal Untreated mdx Treated mdx Top, middle ,and bottom