Post on 30-Dec-2015
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DNA shuffling Example 2: Antibodies: breaking the natural limit on affinity selection
Natural affinity ceiling for an antibody = lowest Kd of 10-10 M (100 pM):
Kd = off time / on time
Endocytosis rate ~~ 10 min to several hoursSo 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
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J Foote and HN EisenKinetic and Affinity Limits on Antibodies Produced During Immune ResponsesPNAS 1995; 92: 1254-1256
Class 25 last updated 12/6/11 11:00 AM
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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 10-15 M = 5 x 10-14 M = 0.05 pM (compare 100 pM limit for naturally selected Abs)
Slower off-rate than biotin-streptavidin (>5d).
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2x10-6 per second = 5 dayshalf-life
Iterations
Off
tim
e (t
1/2)
Antibodies from yeast scFv selection (Boder et al. and Wittrup, PNAS 2000)
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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)
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Example 4: Enzyme stabilization:
PAI-1, a protease inhibitor (TPA inhibitor)
Error prone DNA shuffling 245X increase in stability ( days)Assay = tPA bindingFound 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.
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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.
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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)
Example 6?: A global version of DNA shuffling -- genome shuffling: A different (unnatural) method of genetic mixing using whole genomes
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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.
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)
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Typical receptor structures
of 217 A-domains
A-domains:(~35-40 AA’s/domain):determine binding specificity of many receptors
as a spacer between domains
(~metaphorically?)
Organization of binding domains in typical mammalian receptors
Degenerate oligos synthesized to code for 35-40 AAs of the A domainsOnly 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 librarySelect one domain at a time, serially, by panning:
LRP = LDL receptor related protein; VLDLR =very low density lipoprotein receptor
2 domains cooperating
Bipartite domain
Dual specificity domain
An A-domian
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Monomer displayedon phage coat
Monomer protein Screened for binding
Build 20 phage dimer poolsfrom 20 best monomers
M13 phage
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”
IL6 = interleukin 6
One domain
Two domains
Three domains
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Finally:Add an IgG-binding domain at the end to prevent rapid clearance(measured half-life of 89 hours in monkeys)
Model structure
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Activity of the anti-IL-6 tetramer C326. (a) AlphaScreen competition analysis comparing ability of C326 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.
LaserReactive Oxygen
Luminescence
Binding measured by a competition assay (“AlphaScreen,” Perkin-Elmer)
Reactive oxygen species can react only over a short distance with an “acceptor” bead
IL6IL6 receptor
Avidin bead:biotinylated IL6 + gp130-Fc:Protein A beadCompetition: IL6 (non-biotinylated) or C326 avimer (10X tighter)
Laser
gp130 = natural IL6 receptor
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More AlphaScreens: effect of combining the 3 domains
20 pM
[So how was M1 selected??]
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Physical assay: Biacore surface plasmon resonance to measure binding kinetics
253 pM0.23 nM
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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
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C426 cMet 2 <0.1 0.170 Active
C65 CD40L 2 <0.1 0.06 0.1
C326 IL-6 3 <0.2 0.05 0.0008
C2810 CD28 3 0.1 n.d. 0.6
C2 BAFF 3 n.d. 0.1 0.4
Table 1. Diffedrent selected avimer affinities and activities
Avimer TargetNo. of
domainsAffinity (nM)
IC50
(Biochemical) (nM)
IC50 (Biological) (nM)
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Acute phase inflammatory response induced by IL6 is reversed by avimer C326(in mice)
Specific for IL6-induced inflammation
hIL1 – human interleukin 1
1.) Pre-mRNA splicing basics
2) Splicing-based therapy
3) RNAi
RNA
Topics:
Pre-mRNA splicing
5’ 3’
Pre c ision
XX
5’ 3’
X
X
Orderliness
Branch point
Phosphotriester
Lariat
Intron = 80 nts to 100,000 nts
mRNA
Pre-mRNA
ATP
ATP
ATP
ATP
http://www.swbic.org/education/comp-bio/intron.htm
The spliceosome(5 smalll RNAs + 100-300 proteins)
Intron becomes a lariat
degraded
Sequence of events in splicing
5’ss 3’ssA
(UACUAAC)
Bra
nc
h p
oin
t (BP
)
(= “donor” site)(= “acceptor”
site)
TTCTCAGTCCTAAACAGGGTAATGGACTGGGGCTGAATCACATGAAGGCAAGGTCAGATTTTTATTATTATGCACATCTAGCTTGAAAATTTTCTGTTAAGTCAATTACAGTGAAAAACCTTACCTGGTATTGAATGCTTGCATTGTATGTCTGGCTATTCTGTGTTTTTATTTTAAAATTATAATATCAAAATATTTGTGTTATAAAATATTCTAACTATGGAGGCCATAAACAAGAAGACTAAAGTTCTCTCCTTTCAGCCTTCTGTACACATTTCTTCTCAAGCACTGGCCTATGCATGTATACTATATGCAAAAGTACATATATACATTTATATTTTAACGTATGAGTATAGTTTTAAATGTTATTGGACACTTTTAATATTAGTGTGTCTAGAGCTATCTAATATATTTTAAAGGTTGCATAGCATTCTGTCTTATGGAGATACCATAACTGATTTAACCAGTCCACTATTGATAGACACTATTTTGTTCTTACCGACTGTACTAGAAGAAACATTCTTTTACATGTTTGGTACTTGTTCAGCTTTATTCAAGTGGAATTTCTGGGTCAAGGGGAAAGAGTTTATTGAATATTTTGGTATTGCCAAATTTTCCTCTAAGAAGTTGAATCATTTTATACTCCTGATGTTATATGAGAGTACCTTTCTCTTCACAATTTGTCTCTTTTTTTTTTTTTTTTGAGACAAGGTCTCTGTTGCCCAGGCTGGGGTGCAGTGCAGCAGAATGATCACAGTTCACTGCAGTCTCAACCTCCTGGGTTCAAGCGATCCTTCCACCTCAGCCTCCTGAGTAGCTGGGACTATAGGTGTGCGCCACCACTCCCAGCTAATATTTTTATTTTGTAGAAACAGGGTTCGCCATGTTACCCAGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACTGGCCCAGTTTCTACAGTCTCTCTTAATATTGTATATTATCCAGAAAATTTCATTTAATCAGAACCTGCCAGTCTGATAGGTGAAAATGGTATCTTGTTTTTATTTGCATTTAAAAAAAATTATGATAGTGGTATGCTTGGTTTTTTTGAAGGTATCAAATTTTTTACCTTATGAAACATGAGGGCAAAGGATGTGATACGTGGAAGATTTAAAAAAAATTTTTAATGCATTTTTTTGAGACAAGGTCTTGCTCTATTGTCCAGGCTGGAGTGCAGTGGCACAATCACAGTTCACTCCAGCCTCAACATCCTGCACTAAAGTGATTTTCCCACCTCACCTCTCAAGTAGCTGGGACTACAGGTACATGCTACCATGCCTGGCTAATTTTTTTTTTTTTGCAGGCATGGGGTCTCACTATATTGCCCAGGTTGGTGTGGAAGTTTAATGACTAAGAGGTGTTTGTTATAAAGTTTAATGTATGAAACTTTCTATTAAATTCCTGATTTTATTTCTGTAGGACTGAACGTCTTGCTCGAGATGTGATGAAGGAGATGGGAGGCCATCACATTGTAGCCCTCTGTGTGCTCAAGGGGGGCTATAAATTCTTTGCTGACCTGCTGGATTACATCAAAGCACTGAATAGAAATAGTGATAGATCCATTCCTATGACTGTAGATTTTATCAGACTGAAGAGCTATTGTGTGAGTATATTTAATATATGATTCTTTTTAGTGGCAACAGTAGGTTTTCTTATATTTTCTTTGAATCTCTGCAAACCATACTTGCTTTCATTTCACTTGGTTACAGTGAGATTTTTCTAACATATTCACTAGTACTTTACATCAAAGCCAATACTGTTTTTTTAAAACTAGTCACCTTGGAGGATATATACTTATTTTACAGGTGTGTGTGGTTTTTTAAATAAACTCCTTTTAGGAATTGCTGTTGGGACTTGGGATACTTTTTTCACTATACATACTGGTGACAGATACCCTCTCTTGAGCTACATCGGTTTGTGGGGAGTCAAAAGTCCTTTGGAGCTAGGTTTGACAAATAAGGTGGGTTAACACTTGTTTCCTAGAAAGCACATGGAGAGCTAGAGTATTGGCGAATTGAAGAAATCCCCCTTTTTTTTTAACACACTTAAGAAAGGGGACTGCAGGTATACTCAAGAGAGTAAGTCGCACCAGAAACCACTTTTGATCCACAGTCTGCCTGTGTCACACAATTGAAATGCATCACAACATTGACACTGTGGATGAAACAAAATCAGTGTGAATTTTAGTAGTGAATTTCATTCATAATTTGATCGTGCAAACGTTTGATTTTTATTACTTTAGACTATTGTTTCTGATTTTATGTTGGGTTGGTATTTCCTGTGAGTTACTGTTTTACCTTTAAAATAGGAATTTTTCATACTCTTCAAAGATTAGAACAAATGTCCAGTTTTTGCTGTTTCATGAATGAGTCCTGTCCATCTTTGTAGAAACTCGCCTTATGTTCACATTTTTATTGAGAATAAGACCACTTATCTACATTTAACTATCAACCTCATCCTCTCCATTAATCATCTATTTTAGTGACCCAAGTTTTTGACCTTTTCCATGTTTACATCAATCCTGTAGGTGATTGGGCAGCCATTTAAGTATTATTATAGACATTTTCACTATCCCATTAAAACCCTTTATGCCCATACATCATAACACTACTTCCTACCCATAAGCTCCTTTTAACTTGTTAAAGTCTTGCTTGAATTAAAGACTTGTTTAAACACAAAATTTAGACTTTTACTCAACAAAAGTGATTGATTGATTGATTGATTGATTGATGGTTTACAGTAGGACTTCATTCTAGTCATTATAGCTGCTGGCAGTATAACTGGCCAGCCTTTAATACATTGCTGCTTAGAGTCAAAGCATGTACTTTAGAGTTGGTATGATTTATCTTTTTGGTCTTCTATAGCCTCCTTCCCCATCCCCATCAGTCTTAATCAGTCTTGTTACGTTATGACTAATCTTTGGGGATTGTGCAGAATGTTATTTTAGATAAGCAAAAACGAGCAAAATAGGGGAGTTTAACTTTAATATTTTCTTTTAAAAAGCATTTCATGTTATAAGATCAATTCTGAGTGGTAGAAAATGCTTTGACATTTTATTTCCATTTTCTACTTTTAGTTTTTTTCCTATTTGTTTAAGATCTTAGAGGATTATTAAGCTGAACTCCTCAACTGATAAAAAGCATGACATCTTAAACATAAGCAAAGCATATTTTTAGGTTAATTTTCACATAGAAAACAGTTTATTTTATGTGAAATTCTATGTAGATATACTATTTTTTTGGTATTTATTGATATGTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTATTTATTTTTTTTTTTGAGACAGAGTCTCACTCTGTTGCCCAGGCTGGAGTGCAGTGGCATGATCGTAGCTCACTGCAACCTCCACTCCCGGGTTCAAGCAATTCTTCTGTCTCAGCCTCCCGAGTAGCTGGGACTACAGGTGCCTGCCACTATGCCCGGCTAATTTTTGTGTTTTTAGTAGAGATGGGGTTTCACCTTGTTGGTCAGGCTGGTCTCGAACCCCTGACCTCAGGTGATCCACCCACCTCAGCCTCCCAAAGTGCTGGGATTATAGGCATGAGCCACGTGCCCGGCCGACATGTTAATTTTTTAAAAAAGGCTTTACTGGGGTATATTTTATATAATATAATAATCACATGTTTTAACTATACAATTCCAAGCTTTTTAGTATATTTATAGGGCTATGCAAGGAAGATATACTGTTAAACAGTAGAAATTGAGAAAGCTCTTCTGATAATATCTCTTGATTTGATGATGGCTCATGCCTGTAATCTCAGTGCTTTGGAAGGCCAAGACAGCAGAATCACTTGAGGCCAGGGGTTCGAGACCAGCCTGGGCAACACAGCAATACCCTATCTTTACAAATAATAAAAATATCTGTTGATTTGAAGTAAAGTTTTTTTTTAAAGACAAGGTCTCATTCTGTCACCCAGGCTGGAATGCAGTAGCAAGATCACAGCTCACTGTGGCCTTGACCTTCTGGGCTCAAGTGATTCTCCCACTTCGGCCTCCCGAGTAGCTGGGACTAACAGGTGTGCACCACCATGGCTGGCTAATTTTTTTTTATGTTTGTAGAGATTGGGTCTTACTGTGTTGCCCAGGCTGATCCCGAACTCCTGGGCTCAAGCAGTCTTCCTGCCTCAGCCTCTAAAATTGCTGGGATTACAGGCTTGAGTCACCATGCCCAGCCTGAAGTAGCATTTCTACCCTGTTTAATAATTCAGCAGCTTGTCATGTAAGATATTCATATATGCATATAAACATTAGGCAGCTTAATTTGGTAAAACTGTAAAATGGAAATTTTAAATTGTTTGCAGCATCAATAACATTGATGTCAGTATGATTTTTACATGCTGATCTTGACCAATTTGAAACAGTGAGTTAAAATCTGGCTGATCCGTACTAATCCTAAAGAAATATTCTATGAACTATTAAATGTTTCCAGAATATATAAAGAAACATTATGATGTCAACACACCCATCTATTTTTTTTTGGAAATAAAAACTCCATTTTTCTTATTAAAGAAAACATGCTTATTAGAAAACATACGGCTGGGTGCAGTGGCACACATGTAATTCCAGTGCTTTGGGAGATCGAGGTGGGAGAATCACTTGAGGCCAGGAGTTTGAGACCAGCCTAGACAACATAATGAGACCCCCTCTCTACACAAAAAGAATTAGTTGTGCATGGTGGCGTGCACCTGTAGTCCCAGCTACTTGGGAGGCAGAGGCAGGAGCATCCCTTGAGCCTAGGAGTTTGAGACTGCAGGAGTTCGAGACTGAGTGGAATGCAGTGGAACTGCATTCCAGCCTGAGTGACAGAGGGAGACCCTGTCTTAAAAAAATAAGAAAGAAAACACAACTGCAGAAAATTATAAAGGATTTAAGTCATTCCAAATATCACTGCCACTTTTTATTTAGAATATTCTAAAGAATTCTCTCTCTGTGTACACACACACATATGCGTACTCTTAATCCAAGTAGCTTGGTAGGATTTTATTTACCTAGTGCCTAGATGGGAAATTGCCTGGGGATTCCAAATACCTATTTCATTAAATTAAAGATGTCACTGATTTTAAGACTTAACACTATTTTTCATACTGCCAAGAAAGAAAACACTACCAGTTATAAATGTAAATTGCCATCAATTGTAATACATCAATTTTAGAGCTATTATTAATAAAATGTGAATGTGCATCTTAGAGCAATGAAATATAGTACTATATATTTGATGACCTTTTCTGCCCTGTGATATTCAGAAAGTGAAAGTTAAATATGGGCTGAGCATGGTGGCTCACACCTGTAATCCCAGTACTTTGGGAAGTCAAGACGGGAGGCTGGCTTGAACCCAGGAGTTCAAGACCAGCCTAGGCAATGTAGCGAGACGCCATCTCAAAATATTAAAAATAAGTAAATAAGTAAATAAAAAGAAGGTTAAGTATACAAATGTATTTCCTTTGTTGTGAATTTATTTCAATTTTATAGTGATTTTTTTTTTTTGAGACGAAGTCTCACTCTTGTCCCCCAGGCTGGAGTGCGATGGCGTGATCTCAGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCTATACTCCTGCCTTGGCCCCCCGAGTAGCTGGGATTACAGGCGCCTGCTACCATGCCTGGCTAATTTTTGTATTTTTAGTTGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTAGAACTCTTGACCTCTGGTGATCCACCCGCCTCGGACTCCCAAAATGCTGGGATTACAGGCGTGAGCCACCGTGCCTGGCCAGTGGTTTTTTGTTGTTGTTGTTGTTGTTTTGTTTTGTTTTTGTTTTTGTTTTTGTTTTGAGACAGGATCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGTGCCATCTTGGTTCACTGCAACCTCTGCGTGGGCTCAAGCAATCCTCCCACCTCCCTTTCCAGAGTAGCGGGGACCACAGGTGTGTGCCACCACACCTGACTAATTTTTGCATTTTTTTTTGTAGAAACAGGGTTTTGCCATGTTGCCCAGGTTGGTCTGAAACTCCTGAGCTCAAACAATCCAACTGCCTTGGCTTCCCTAAGTGAAATTACAGGCATGGGCCACTGTACCCAGTCTAGTGATTTTTTTATTTTTATTTTTATTTTATTTTATTTTATTTTTTTACCAAAAAAACAACAAAGCCTCAGGAGGAAAAGTTGATACACAAGTAAATTTTATTGGAAATGTTTTTGTGTGGACCTTAAGCAGAGGGAAAATTAGTCTGCATTATGGTGTATCCAGACTAAATGACTGATATTAAAATGAAATTATTCTTAGGATTTGCAATCTTAGAGAAAACTTTTTCATTTTTATTTTTTTGAGTTACAAATTATCTTCATTTACATTTGAGAACAGTGAGTCACAGAGGGATTAAGTAACTTACTCAAGATCATACAAGTCTTTGATTTGAACCCAATCTTTTAACTCTGCAGAACTCAGAGTCACTCTTATTTGGAAAAACTTTTTAACTGATGTGGATCCTCTAATATGGGCTTCCTATTATTCATTCTCTATTAGTCAGAAGTTTTGCAAGCAGACAGAATTCATTTTGCCAATTACGGGATTTTCCCTCAGTTGCAGTCAAGGTTCATAAAACTATAACTCTTTATCTTTAATTAGAAATGTTTTTTTTTTTGAGACAAGGTCTTGCTCTGTTGCCCAGACTGGAATGCAGTGGCATAGTGGCCCATTGCAGCTTTGAACTCCTGGGCTCAAGGGATCCTCTGCCTCAGCCTCCCAAGTATCTGAGACTACAAGTGCGTGCCATCACCCATGGCTATTTTAAAAAAAAAAAAAATTGTAGAGATAGGGTCTTGCTGTGTTGCCCAGGCTGGTCTCAAACTCCTGGTCTCAAGCAATCCTTCTGCCTTGGTCTCCCAAAGTGCTGAGATTACAGGTGTCAGCCGTTGCACCTGGCCAAAACGATAACTTAAAATACACACACACACACACACACACAAACACATATGTGTATTTGTGTGTGTGTGTGTGTGTGTGTGTCTCAAAAGGTATCAAAAGAGAATAGCTATAACTTTAGTGTTGATCTTGATAGTGACTTGATTAGGCTCTGTTTAACATCAAAGATGCAAATTAATACTTTCTTTGAACATATTAAAAATGCAGAAAATATTGGAGTATTTTATTTTAAATAAATTGTATTCTGTATATTTAAGGTATACAACATGATGTTATGGGATACATATAGGTGGTTAAAAGATTACTGCAGTGAAGCAAATTAACGTATCCCTCAACTCACATAGTTACCCATTTTTTTTTTGTTTTGGTGGCAAGAGGAGCTTAAAATCTCATTTAGTGTGAATCCCAAATACAGCACAATTTTATTACCTATATACTTCATGTTGTACATTATATTTCTAGACTTGTTCATCCTACATATCTGCTACTTTGTATCCTCTGAGCTACATCTCCCCATTTTCTCACTTGCCCCCCAAGTAGTTTCTTAAAGTGTCTCATGTAAGAGGGCAGTAGCTTTCAGCTTAAACTTTTTCTCTGTATGTAGTCGATTTCTTTGAGGTATACTTTTCTCTCCAGAATAGTTAGATGTAGGTATACCACTTTGATGTTGACACTAGTTTACCTAGAACTTATCTTCTGTAAATCTGTCTCTATTTCCATCTCTGTCTCCATCTTTGTCTCTATCTCTATCTGTCTATCTCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTAAAGCAAATTCATGCCCTTCTCCTATTTATTGAATCGAGACCATAGACAGGGGTGAGAGAAAGAATTTGGCAGGAATGGGGATGTGTATTATCTGTGGCATAAGGAAACTTTACAGAACTAGGTTCAAAAGTATACTTTCTAGTTCTTTCCCATGGCTTTTCACTTTGATGTAGTCCTTATCAGGTAACTGAGGTTTTATATAAGTCCCCTGATTCTTAGAACATGAAGGTGTAGTAGTCAAGGTTGGTCCCTTGAAACCACAAATTTTGTGAAAAAAAATTAAGAAAATTTGAATAATTTCCTCAGCAAATACATATTGATCATCTGTTATACAGCCATGAGAAGTGGTTCTGTTGCACACGTTTATTTTATCAGATCCTAATCCCAAACCAGGCATAAAATGGAAACCATGAAGATAGGATGAAATAACTTCTGAATGTTTGAATGTTTGAAAATAGTGTACTTAAAAATACCAGGTGGTTTTTGTTTGTTTTTTGTTTTTTTCTTTTTTTGAGACAGGGTCTCACTCTGTCACCCAGGCTGGAGTGTAGTGGTGCAATCTCATCTCATTGCAGTCTTGACCTCCCAGGCTCAGGTTATCTCCCACCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACATGCCACCACGCCCAGCTAATTTTTTGTATTTTTTGTAGAGACGGGGTTTCACCCTGTTGCCCAGGCTGGTCTAGAACTCCTGGGCTTAAGCGATCCTCCCACCTCAGCCTCCCAAAGTGCTAGGATTACAGGCATGAGCCACCATGCCTGGCAGAAAATACCAGGTTTTTAAGTATCAGCACTTACTCTTCAATCTTTTCTATTACTATGTTGTGCTAAATGGTATTTTTTATTTAATTAGAGCAATGCTGTTCAATAGAACTTTCTTTGAGGATGGAAATCTTTTATGTTTCTGCTATGTGGTACAGAGCCACTAGTGACATGTGGCTTTTGAGCGCTTGACACATCTTGTGCAACACAGGAACTGAATTTTTAAGTAATTTATATTGCCACATGTGGCTACCGTATGGGACAGTGTAGTACTAGATGATCTGTAAGGGCTGTGCTTCATCAGTGTCGTTTTTTAACTGACAAAAACCTTTAGTTTTTTTTTTAGTAATGTGTTTATTTAAAAGAATTCATAAAATACAAGTAAACAAATTAACTTGTTACCTGAGCATATGTCCTTTCATACTTATTTTTTCTGCATACATATTTTGGAAAATGGAATATCTGCCCCTTTTTTTTTATCTGAGATACAGTCTACCTCTAAAAATACATGATTCTAACATTCTCACTTTTTGTTGGCATTTGATCAGGGTATAGAAAAACAGTTAAAAGGACAGAGAATGGTTGAGAGATTATGATATGAAGAGAAAATGTGATTGAGTGTGGTAGACTTGGGGCCTGCTTGAATGTTGAGAGAATGACTGTTTTCCGATAAAAAAAAAAAGTCCATTCTAGGATCCTAAAAGAAGGGTCTGAAGTTCACTGCAGAAAGCAAGCTACATAGTACTAAGCCACTAAGGGGACATGGAGCCCTTAGTAATTCCTACCTTAGTAATAGTCTCATCATGCCCTCTTGGGAACCCAGCCTTGTTGATTAGCCTCTCTGCTTTCTCTCCTTATAGTTCAACCTCCCTGTTTGTTCCAAGCAGTTCTTTTCCTGCCCATTTATTATGCATTTCTATACAGCTTTCCTCCTCTTTTTCTATACCATGCTGCAGTTCTTATTGCTACCTAGAGGTTTTCAAAATTCCTAGGGGCGGATAAGTAGGCATAAACAAAGTTCTTCCCTATTATCCTTCCTATTTTTTCACCTAGACTGAAGAGGTAGACAAAATAGAAATAAAGACATTAAGGGTATGTGTTTGTAGTCCCAAAGAGCTTCTCTGGCAATTTTGATGTAGTTGACAGTGACGCTCTGAGTTCAGGACAGATTGGACTCCTTGGCTGAGAGGAGTGAGGAGATAGGACGGTAGAGGAGAGGGTAGAGCAACTCTGGAGGAAGCTTTCCCCTCACCTTTGCCAGTCCTGTTATCCTAGACTTAACCATAATTAAAGATGAGGGAGGCACTCAGTAAAGGGATCTAGTGGGAAGCTTGTTCCAGACAGCCAAGGAGGGAGGTTCGCGCAGTTCCTTTGGCCACCCAGGTGGGGTAATTGATCCATGTATGCCATTCATGTACAATGTAGGCACTTATACCTGTATTCCAATGTAGTGAACTATACCATTACTCTTAAATTAATATTCTTTATTAGCTTCCATGGTGGCTATAGGCCAGGCAAGAGAGTTAAGAAAAAATAAATAGCCAGGTATGGTGACTCAAGCCTGTAATCTCGGCACTTTAGGAGGCCGAGGCAGGAGGATAGCTTGAGTCCAGGAGTTCAAGACCAGCCTGAGCAAAATAGTGAGATCCTGTCTCTATTTTTTAAAAAAGCCTTGGGGCAAACAGGAGTATGGAGGTTTGGATGCTAATAGAACAGCAGTGTCTTACTGCTTGGAGTTCTCTTGTTTCTTGTCCTATCACCGTAGCCTTTGGATCACAGCAATTTTTCCATGACTCCATACTTTTCAGTTCTTGAATATTTTTTCCTTTATTCCTCTTGTCTCTGTAAAGACATCAACTGGAGTTGGACTGTAATACCAGGTATCTCCAGAAGATGGCACTATTTAACAGATTTTATAAATAATTTGATGTGAGTCACTGTCATCTGAAGCTTGTTGCCTTTTCTTTCTTTCTTCTTTCTTTTTTTTCCCCATCAATTCTGTATGTTTGAAATGCTGGGATTTAAGTTAGTTAGAATAAGGGATGTCTGTAATTTCCCTAAATTGAGAAGTAATATGCAAAGGTTGATATCAGAAGTCATATGCTCACCTTGCAACACCAAATAATACTGGCCCATTTGTGATTTTTGAAAGTAACACTCCATAATAAATGGATGTATATATAGAAGCATAACAAAAATAGAAGCACATAAAAGTGAAAAGTCTCATAAACGCCATTGTCACTACTCATGTAATTGCTGTTACAAATTTGTTTAAATGTTGAATAAAAATGGTGTCATAGGCAACACAGTGTTCCACTACTTGGTGTTTTTAATAGCATTATTCTGTCTCAGTGTGCTTTGGATTATCAGGTGCTTTTTAATAGTTGCATGGTATTACATTGTGTAGATGAACTTGATTAATTTAAATGGTTCCCTGTTAATGGACATGTTGGTTTGTTTTTGTGAACAACTGATACAGTGAACATTTATTTTTTAAATAAAAAAAAGAGAGACAGGGTCTTGCTGTGTTTCTCGGGCTGGCCTTGAACTCCTGGGGTCAAGCGATCGTCTTGCCTCTGCCTCCCTGGGATTACAGGCATGAAGCCACCGCACCCGGCCCAGTGAACACTCTTGAATGTATCTTTGTATACTTGTCAAGTGTTTTTGTAGCAATTGATTCCCAGAAGTGGGAATTACATGGAATTAAGTGACATGCATGTTTGCAATTTTAACAGGTATTGCTATGTCATTTTCAAAAGAAGCTATGCCAATTAATACTCTCACCAACAAGAGTGCTTATTTCCCCTCAGCATATTATCAGGCTTAAGTTTTGCCAGTATGGGTGGGAGAACAGTAGAATCACATTGTTTTAGTGTTTGTTTCTCAGATAGATATAATTTTACACCTTATAACCTTCTCTTCTATAAATTGTCTATTTGTGTTCATTCTCCATTTTCCTATGGGTTCTTATTGTTGGAGCCCAATATATAAAAGGGGGTATTTGTTACAGAACCTCTTCAGTTTTGGTTCATGTCATGCCTGGGTTTTTACCCTTTCTACGGATGTTAAAAAAAATTCTCTATTTTCTTCCAGTCCACTTATGGCTTTATTTTTTACATTTAGATTTTAATCCGTCTGGAATTTATTTTTGTGTATGCTGTGAGGTAGGGACCATACTTTTATTTTTTCCCAAATGGGTTACTAGTTGGCCAAACATCATTTATTGAATAATTCATCTTTTCCCTACTGACTCGAAATACCATCTTTATTGTATACTAAATCCTCATATAGTTCTGGGTCTGTTTCTGGGCTCTACTTTGTTCATTTACTGTGCTGGTACTGCACCGTTGTAATTGCTGTGGCTTTGTGGTATGGTATGGCTTGCTCTCTGCTAGGGCAAGTCGAAGCTCTTTTGTTCACCTGCTCTTTCACCCAAATTTTCTGTCCTGAATCCAGCACAGCCAAATTATGGTCATTGTCACCACCAACTACAGTGGGTGTTGAGCATTTCCCATTGAATCTCCTGTAAGGGTTTTATTGGATTCTGTGATAGCAGTAAAATGGGAGCCTAAGAGGTATTCCTTAAAGGACTACTAATCAGACCTGGTTTCCCAGATGATGCTGAAGATGACGGGGCCTGGGCTAGACTTTTGAGGGACATATCCTTGGGGTTGGGTGTGATATAGACCAGCCCTTACAATTTGCTTGACTCATGGGAATCGTACAGGGCCAGAACCAGACACCTGTCATGCTAATAACTTCCCTCACAATTCAGAAATCACTGTGATTGAAGATGGGTGGCTGTTATAATACTACCCACTTAAAAATGGATGTAACCCATTTTTTAGGACTCTTAAAAACATCAAATCAGTAATGGCCGATTAGGACTTTTTAATTTTTACTAATCTCTACTTGAAAGTTTTCTAGTCATTCATTTCAGGAAACCTAATTCTTATAATTCATATCATTTAGAATATCATAATGCTATGGATATTAGCTAGCTAACTTCTCAAATCTTCTAGTTCTCATTTAATTTGAAGTTTGTGTGTGTACATAAGGATATACATATACATATGTGTGTGTAGATATATATATATATAGTTTTTTTTTTTTTAACTAGAATGACCAGTCAACAGGGGACATAAAAGTAATTGGTGGAGATGATCTCTCAACTTTAACTGGAAAGGTATGTATCTTGAAAGGGAAGAAAAAAAAGCACTTCATACCGAGTCAATTAGTAACAGTGTGCTTTCAATCAATCACTAAGAGATAATTTACATAGTATAACTAAATGGGTTATTTAACCCTTGGAAGCAGTCTAGGTTAATTATCGTTCCCTAGGTCATGTAGTAAAAAGACAGTAGAATCCAACATTAACCTTAAATGTCCATATTGTCAAGTACTGCTGTCTGCCTCTGTGGGACTCTAATTTGGGATCCTTCAAAAAACATTGATGGGGGAAAAGATAGCCTTTAAAAAAAAAAAAAAAACAAACCTATGTGAGTCTATGTGAGGTAGACTCACATAGTTTCCTAAAAGATAGCAAAGCAGTATTATGTAGTGGCTGAAAGTGTGAGTTCCGGAGCCTGACAACTGATTCAAAGCATGGCTTAGTACTTCCTAACTCTGACCTTGGGCAAGTTACTTAACCTCTCTGTGTCCCATATGTGATTAGGGTGAGGTTGATAATAGCAGCCATAGAGTTAAGAGGATTAAGTGCTATAATGCAAGTAGAGCTCTTACAACAGTTTCTGGTAAATCACTCAATAAATTCAGACATACTATTATTTTAAGAAATCTCAAAGAGTTTTCTTGTACCTTAAAATTCTCCTAGTGTGAACCATTGGTTTTGGTATATTGTGCTTCCATGTAGTTTAATATCAAGATGTTTTTAGATTTCCCTTTTAATTTATTTGTTGACCCATTGGTTGTTCAGGAGCATGCTGTTTACCTGAAAATAATGGAGATATTAAGGTATTTGAATATTTATCTTCTAGTACATTGAAAAACTTTTTGAGAGTAACCAATAATAAATGATGGAATGCTACTGCTTTTTTTTTTTGAAGCTGCCAGTTATTGTTTACTTACACTATGCCAAATATAAAGGCATTAATCTCATAAAAGTTTCACAACAATCCTGTGAGGGAGACGATATCCCCATTTTACAAATCAGGAAATTAAGACTTAATAAGGTTAAAAGACTTGCCCCAAAGTCACAGAACCAGTAAGTGGTAGAGCTTGAATTTGAATACAGACCTGACTCTAAAGCTCTTTTCTTTCTTTAGATTTTAGTGTTCATTGCTTACTTGAATGAGTATCTATAAGAAAACTTTAACATGTAAAACTTCTGTGAAATTATCTTGTCCCATATCAGGGTCATGTCAAACTAATGTCCTCCTCAGCATCTTTGGAAAACTTCAGAGGAGAAATGAGCTTTGCCCCTCCTGTTCATTTCCTATTCCACTAGGAGACCTGTCCTTCCCTTTCAGCATGCTTTGTCCATATTTAGAAGCTGTTGAAGCCATTACTTGTCTGGTCAGTTTTTAGTGCTGGAATGGACCTAGCCTTTTAGGCCTTCTGAGATTTAGTTTGATCTCGTCTTTCCCACCTAATGGCTCTGTTCTACTACATAGATTTGATCTGAAACAGTTCTCTGTTTCTAAAATAACTTTCTTTTCATGATAGTCACAGTAAAGTACATTTATTATGGAAAAATCAATAAGTATAACGAGTGAAAGTTATTTCTTGGTGGTAAGATTATGGGATTATTTGAACTTTCTGTTTCATTGTATTTTATTTATTTATTTATTTTTGTGATGGAGTCTCACTCTGCTGCCCAGGCTGGAGTGCAGTAGTACGATCTTGGCTCACTGCAACCTCCCCTTCCCAGTTCAAGTGATTCTCCTGCCTCAGACTCCCAAGTAGCTGGGATTACAGGCGCACGCCACCATGCCTGGCTAATTTTTTTATCTTTAGTAGAGACAGGGTTTCACCATGTTGACCAGGCTGATCTCCAACTCCTGATCTCAGGTATCCACCTGCCTCAGCCTCCCAAAGTACCGGGATTACGGGTGTGAGCCACCCTGCCTGGCCTCATTTTGTCTTTTGGGGGTATTTTTGTGTGCAGATATATATGTATATAAATATTTTTCCCTCTTTTCCCCAGTTAGTATTTGAGCAGATGAACTTTGGACCCGAATACCTGTATTCAAGTCTCTAATACCACTTCTTGGCTATTTTCATTTTATCAAATGGCCTCTTATCCTCGTTTTTCTCATTTATTAAGTAGAGATGTAACTACTTGATATAATTCAAAAACTCAATAATGGCATTCTTTTGTTTTTTAGACTCTAGTGTCTGTACTCCTTGTACCATGCTGGGATTCATTTGAACAATTGCATGGCTTTTTTAGTGTATTATTAAATTTGCAGTTTACTTAGAATTTACTGGGACCTCATACAAATGGGAAAAAAACATAACTGTGTTACTCATTTGCTGTGTGCCTTTGGATTGACCCTATTTTTTGTATTCATTTTCTCCCCATGTCCTGAGTTCCACTTTGAATAAAAAAGTAATTTTTTTCCTGCCTGTAAAATAGGCTACCAATAGGCTGCAGTTGTCTATAGTAGCTGCTTCACTGAGGAGAGCTCAGCATGAGAGAAATAGTATGAATTGCTTGCCACAAGTTATGGGCTAGCCTTACTTCATTCTGTACTTGGACCTGTTTAGGCTTCTAAGAGATCTTACCTCCAACAATAAACTGCTTTGAGACATGAAAAGGTGGAAGCTTTACTTGGTTATAACTTTACTTTTAATACCTAGAACAGTGAGTCTTCAAACTTGTATTTGCATGCCCAATTTATAAAAAGTTTCCTGAGCATTTACCCCTAATATATGCATTTTAAATTATATATGATTTATGGTAATAATAATATATATGTTACAAAATACATACAAAAATATAGATTAAACAAGGTGAGGTTAAAAAATTTAAAAGTTCTAATCTTTCTTGCAAACCAGTGGATCTTTTGTGCCTTACTCTGGTAAACACTGTCTTAGAAGAATATATAGAACATTAAAATCTTAATGCTATAGTTATATGACAGAGTATGATGAGAGCTACAGATAAACAACACATCATGAATCTTCTTGTGGCAGTGTTTATAACCATTATGTGAAATGCTGCCTCATTCTTATAACTAGCATAAGAACAGATAGGACTTTCTCGATTTTGAGGGGTAATTATTAGATGGTATTTTCTGTTAAGGACTCTTCCAGCTATAAAATTCTTAAATGTAGAAAGCGAAGTGAGGGTTTATGGTGAGAGGAAGCATTGGTATCATGTTTTAGTGTAGTCCAAGAATATGGACACATCCAGAAAATGCAGATCAAGTTTAGCCTAATGAGAAAATATATTTTGGAGTCCATATGGTAAATTAAATTATGTGATTTTTGAGTTATTGTACAAATATAATTCTTAGAATGTTAGAGTCAGGAGACTATAAGAGACCAACTGCTTCAAGTTTCATTTAACACATGGGAAACTAAGGCGAGAGAAATTTCAAGACTTGCCCAAGATTAGACCTCTTGTTAAGTAATGAAAGTGTTTTAAAAACAGGTGGGTCAAATTCTGTTTTTAAAATTTCCATTATGATGAAAATTTCAGTATTACAGGCTTCCAAATCCCAGCAGATGGGCCACTTGTTTAAAGGAGAGTTTGATATAATAAAGCATCTAAAAACAAGAGTTTG
GATAATTCCTTAGGGTTGTTATGATGTGATTTGACTTATAATTGGAAATACCGTTTTATTCATTGTACTGATTTTCATTTCTCTTTTTCTTCTAGAATGTCTTGATTGTGGAAGTAAGTTCACATTTACTTTTAATATAACATTTATGACTTTTCTAACTTAGTATGCACCATCCTAAAGGTAAGCCAGGGAGAGAAATTCCTCTGCATCAGTTTTAATGGTGGGCTTGTGTTCTAAAGGAGTGAGATTGGTTTTTTGTAAAGACTACTTAGTAATTTGTTTTTACCAATAATGGAATGGTATACTTCCTACCTCTCTTTTTTTAGTTTGAAGTATTTTCTTTCTAAACATAACTCTCTCTCTCTATTTATCTATATATAATATATACATATATATCTTATATTTTATGTATATATATATATATCTTGCTTAGATTTTGTCTTATGTAATATTTGGTACATAAAAAATAATATTTATAATTTATAGACTATTTTCCATGTGTTATTATGTGCTAAAGTATTTTGTATCTTAGCACCGAGAGGCTAAGCAGTTTCCTAGGGTTACCAGCTAGTAAACTAAGGGAAACCTTTACTTCCTTTAGCTCAGTGGTTCTCAAAATGTGGTTCCCTAGACCAAAAGTATTAATATCAGACAAGAACCTACCGAATCAAAATATCTGTGATGAGGCCCAGCAAGCTATGCTTTAACAAGTTTCCGAGTGATTCTGATGCATGCTAAGGTTTAGGATCCCTTGTTTTTACTCATAAGTCACTTTCTCATTAAGGCCTTCCCTGGCCATCCTATATAAAATCTCATGTTTTCACACCGTCAACTTCGTATTCCTCCTCAATACTTTTATTTTCCTGATCACTTATCACTAACAGCCTCTCTCTCTCTCTCTCTCTCTCTCTATGTATATATATATATATATCACTTATCACTGTCTAACAGCCTCTCTTTATATATATATAATCTATAGATTATATATATATGCAGCATTGTGCAATCATTATCACGCTCAATTTTAAAACATTTTCATTTCCCCACAAAGAAACCCAATCCCCTTAGCCATCACTCCCAATTTTCCCTTCCCCCAGCACCTAGCAAACTGATCATCTACCTACTTGCTGTCTATAAGATTTGCCTATTCTGGACATTTTGTATAAATAGAATCATACAATATGTGGCCTTTTGTATCTGGCTTCTCTCACTTAATGTTTTCAAGGTTCATTCATGTTGTGGAGTATATCTGCACTCATTTCCTTTTTATTGCCAAATTGTATGGATAGACAGGTGTTCCTCAACTGTGTCCTGATAAACCCATCTGAAGTTGAAAATATCATAAGTTGAAAATGGATTTACTACTTTGATAAATCTATCCTAAAGTCAGAAAAATCTCATGTTGGAACCATCGTAAGTTGGATACCATCTGAATTACATTTTTGTTATCCATTCACTGGTTGACAGACGTTAGGTTGTTTCCACTGATGCTCCTTATTTCTCGTACCTGAAATGTCCTTATTCCCTCCCTTCTTATCCCATGTTTAAGTCATTTAAGACCCAGCTCAAACGTCACCTCCACAAAACCTTCCTTGATACCCCTTTCCTCTTCAATTCACTTGGACCTTTTGCATTTAATTTTAATTTTTATTTTTTTTAAGACAGAGTCTCACTCTGTCACCAGGCTGGAGTGCAGTGGTATGATCTCAGCTCACTAACTACTCTGCCTCCCAGGTTCAAGCAATTCTCATGTCTCAGCCTCCCAAGTAGCTGGGACTACAGGTGTGCGCCACCATGCCTGGCTAATTGTGTGTGTGTGTGTGTGTATGTATGTATGTATATATGTGTGTGTGTGTATATATATATATACACAAACATATATAAATATATATACATATATATATATACACACATATATAAATATATATACATATATATATATACACACACACACACATATATATATATATAGTTTTTTTTTTTTTAAGTAGAGATGGGGTTTTGCCATGTTGGCCAGGCTGGTCTGGCCTCAAGCCATCCTCCCACCTCGGCCTCGCAAAGTGCTGGTATTATAGGCATGAGCCACTGTGCCTGGCCTGCATTTCATTTTAATTATAAAATATTTTGAACTCAGAAAAAAGGGTATGCTGAATACCTACGTACCCACAAAAGTATTAACATTTTGCCATATTTGCTTCTGATCTTATTTTTTTTGAGAAATTAAAGATCATAATACAACTAAAGCCCCATTTCTTTCCCTTCATTCCCAGAAGTATGACAATTATCCTTAAAGTTGATATATATCATTCCCATGCATGTTTTTTATACTTCCCTAGTACAAGTTAGCTGTATCCTCTGCTCAGGGGCTCATCAAGCTGAATCAAGGGACTCATGATCCTCTTCAAAGTTCCTTCAGGTTGTTGGCAGAATTTAGTTCCTTGTGATTGTAGGACTGAGGGCCCGTTTTCTCACTGGCTGCTGGCCAGGGGTTGCTCCCAGATATTTAAAGGCTCATGCCCTAGCCCATGACAGTCTCACAACATGGCAGCTGACTTCTTCAAAACCAGCAGGAGAATCTTGCTCTAGTCTACCACATAACCTAATCACAGGAGCGGCTATCCCGTTATTTTCACAGATCCTGGTCACATTCAAGGGGAGGGAACCCTTCTGTGTGTGTACACCAGGAGGCAGGAATTTTTTTTTTCTTTTTCTTTTTTGTTAAAAAGTCTTAAAGTCTTTTATCCCTAAAGGAGGCAGGAATTTTGAGAGCCATCAGAATTCTGCCTACCACAGCCCAGAAATCTGCATTTTTCACAAGTCTCCAGCCATGATGTTTCTGATGGCTCACACTGCTTTATTCCATTTTTAAAGAGTATTTTTATTGAAAAGCATTAGGGTTATGGTTTAAAAAATATTTTCCCTAACAAAGATGGGTTTGTTTAGAGTCCTACTTTTGACTAAATAGCTGAGATTCACTTTTATGTAAAGTTCATTTTATAGCGTTATTAATTTGGGTGCCTTTAAAAATAGTATAAAGCATGTTTCTCGAGTGTAGTCTGTTAGCCACCTATATTGGAGAGTTGGGAGGAGAGAGTCTCTATCTTGAATTTATGGGAAAAATTCTAAAATACTTTTTATAATGAAGGACAACATCATAACTCCCTAATAAAATGTGCATGTATATATTCAAATTTGCTGTCATTGATCCTGCACCTACAAAATCCAGTCCTGGGGGCTGGCATTCTTACTGCTTGCTGAGGGCCAGATGATATAGATTCCAGAATATCTCCATGTAGATTTTGGTGAGAATTACTGTGCTGAAAAGAATGACAGTATTGCAGTTATACATGGGGGTTTTGGTACTTTATATTGTGACTCTGAATTTAAAGCTATGCAATGTCTTCTTTTTTGAAAGGATATAATTGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAAAGATGGTCAAGGTCGCAAGGTATGTATGACATTTTGACACAGAATATTTTCCTCATTTGAAGGGGGATTAAGTGATTGCTTCTTTTTAAGGATAAATGTTTTCAACTGTCATTTTATCTTCGAAAAGTAATGTAATCTCATATAAGACTTAAGATATAATCCTTTTAAATAATTTTGTCATGTGTTAATAAAGCTCATAATTACAGTCACTTCCTTGCTAATATTAACATTTGGTTTTCAGCATGCTAATTATATCAGTTTGTCCTGAATAGCATGGCAGAGGATTTTGGGCCCCCTTGCAAAATTAAGAATAAGGATTCCAAAGCGGGTGAGGAAGTGATAGGAAGGGGTGGGCCCTGAAGATCTGGACCTCCTGGAATTGAGTGATGAATGCTGCATCTTCTTTGTGTCTGTAGTGAAATTTTATAATGCCTGCTTCCTTTTTTATTAAGTCGGCCTCACCTCCTCACCTTACCTATGCTGTTTTACTTTTGCTTTTATAGTTCTACCTGTGTTTATTTCTCATTTTCGTTTCATCTCTCAACAACTCTGGGGTGGCATTATTATTCCCACTTTTCAGATAAGGTTACTGAGGCATAGGGAATTGTCCAAAGGTACAGAGCTAGTCCGCTATAGAGATGAGATTTGAACCCAGGGAACCTGGCTCACAGTTTATGCTTTTGCCTACCTTAAGTTTTTAATAGAGTGACATCAAACAAACATTTAAGAATATGTTTTTCTTTTCCTTTTATAATTTCATTAAAAACATTAAGTCTCTGATCAGTCTGCAGTTTTTATGTAGGGGTCAGGTAATGTTCTAACTTCTGCTTTTTCCTAAGTGATTAACAGGTTTTTATAAGCCCTTTTGAAAAAATCACGGTATCTGTCGAGCATCTTTGAATCAGAGTAAGCCTTCTAGTGAGTCATATGTCAGCAGTTTGACTGTATGGGCTTTTCTAATATCCAGTTCAAGTGTTTATCAGTGAGTTTTTCTTTTAAATAGATTTGGGACAGGTACTATGAGAGTATATAAGTGATACGTTATAGGACACTAACTAGTATCCTATGAAATGGCAAAAACTGCAATCACTTTTGCACCAACCAAATAGAAACTAATCAGTGCACTTGCTTATTTTTCTACATGCTCTTTAGGGTTTTAAATGTCAACCTACTGTGGCATAGACTTTAATCCTCTGGGTATTCTTTTGTTGTTCTTTCCTGGTATATGCTGTGGAATTGAGATAGACTGGTTCGTGAGCGAGAGATTTTGTGTTGCCACAGGTAGGACATGCTCAAACAATACTTGGGTCATTTCTTGACCCAAGTCATCTATTCACCATAGTTTTGTAGCACCGATCTTGCATACATTTCATGTATCTTCTTTGAACCCCACGTCAGTGCTGCTTATATGATACTCAGAAATTAAACACTAAGGAATAAGATTTTCAGGTAGGATTGAGTTTTGGAGGGTCACAAATCTTGTAATGTCTAATATTTCCACTCTCCCTGCTGAGAATTAGTTTTGGCTTCCTTGGAGGTGATATCGCCTCTGTTGAGTATAAGTGGCCTACTGTGATCACACCACTGCACTCCAGCCTGGGTGACAGAGTGAGACCCTGTCTCAGAAAAAAAAAAAAAAAAAAAGAATGCATGGCCTAGATGACTTCTAAGGTTTTTCCCACCCAGTTCCAGTTTTCATGTTCTAGGCAGAGCAGTAAAGTGAGAAACACATGGACTTGGGAGTTTAGTCTCGCATTTCACTGCCACTTAATCTGAGCGACTATTCCATATTTAATCTCTCTGAATGTATTTACTCATCTTTAAAGGGGAATGATTATTAACATCTTTTTCTCAGGGAAACTATATGAGTCAAGGAGATAATATATTTGAAAATCTTTTTAACTGCAAAGCGCTGTTTCACTGTTGGTTATAATGTGATTGATCTCATTGTAGTGAGCAGCTGCTTAATTGCGTTTTAGAATGTAGGGAAGATAGTAATATTTTTCACATTATATATGTAGCTGGTTCTGGAACTGTAAACATACTCCTTTTTTATGGAGATCTGAGTCACGTACCATAAAATTCACTCTTTTAAAGTTGTACAATCCAGTGGTTTTTGATATATTCAGAGTTGTGCATCTGCTACCACTATTTCATTTTGGAACCCAAAGAAACCTTGTACCCATTAGCAGTCATTCTCCCTTCTCCCAGCCCCTGGCAACTACTAATCTACTTTCTACAGAAAGTCCGTACAGATTTGTGTATTATGGACATTCCATATAAATGGACTCATGCAATATCCTGTCTTCTTTCACTTAGCATAGTGTTTTCAAGGTTCATCTAGGTTGGGGCATGTATCAGTACTTCATCCCTTGTTTTGGCTGAATAATATTTCATTGTACAAATATATCACATTTTGCTTATCCATCTGTTGGTGAACATTTGAGTTTCTACCTGTTGGCTTTTATGAATAATGTTGATTTGAATGTTTGTGTACAAGTATGAATACCTGTTTTCAGGTCTCTTGAGTATATAGTTGCTAGGTCATATAGTAACTCTGTGTTTAACATTTTGAGGAATTGCCCGACTATTTAACAAGGTATATGTACTGTTTTACACCAGTAACATATGAGGGTTCCAATATCTCCACATCCTTGACAACACTTGTTACTGTCCTTTTTATTGTAGCCATCCTAGTGGCTATGATGTGGTATCTCATTGTGGTTTTGATTTGTGTTTCTCTGATGCTGATGATGTTGAACATGTTTTCATCTGCTTATTGGCCATTTACATATATCTTCTTAAGAACGGTTACCCATTTACAGTATGGAAAATGCTTCAGATGCAACTCTAGTCATGCCTTAGAGATGGAGCTTTATTAAACATTCAGATCTCTAGGCATATGAAGTGCTGAGTTCTCTTGAACTCCTAATACAGATTGCACTGAGTTTAGTGATACCTTTTCTGGAGCATTCCTGAGTTCAGGTAGGGAGAAGGGTTTTTGCTGTGATTGGCTTGTTATGTTCTTTCTAAATGGAAATAGAATTGAAGTGTCTCCTCTCTCCATTTA
Finding exons in a sea of introns
Alt. 3’ SS
Alt. 5’ SS
Exon skipping
Some types of alternative splicing
|pA
Different termination
|pA
Alternative splicing: Occurs in almost all of the 25,000 human gene transcripts
Alternative splicing in the alpha-tropomyosin gene (7 isoforms)
Similar proteins
but subtly different to
suit different tissues
Exon choice within each class is mutually exclusive.Codes for axon guidance proteins as well as function in the fly’s immune response60 kb gene115 total exons/gene38,016 combinations
Each isoform has one exon 4, mutually exclusively
Dscam transcript alternative splicing (Drosophila)The alternative splicing champion
12 38 33 2
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.
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)
Alternative splicingUnwanted alternative = includedUse antisense skippedBias alternative splicingAgainst an unwanted isoform (e.g., Bcl-X alt. spl.:Bcl-XS = promotes apoptosis;Bcl-XL = inhibits apoptosis and promotes cell growth, cancer)
Alternative 5’ splicingUnwanted = longer exonAntisense shorter isoform
Pseudo exon activated diseaseAntisense = block and skip unwanted pseudo exon
Anti-sense RNA:A therapeutic intervention at the level of pre-mRNA splicing
Got this far
x
Nonsense mutation
Expendable exon (e.g., protein with many repeated domains)Exon must be multiple of 3 in length to maintain reading frame after skipping
Antisense-induced skippingd
Therapeutic intervention at the level of pre-mRNA 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 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
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
Instead of deoxyribose or ribose
Modified phosphate
RNA modification for stabilization
Still base pairs OK!
ase
ase
Attached 1 to 4 lysines here
PNA = peptide nucleic acid
Amide bonds,No ribose
B = a nucleic acid baseRNA modification
Even more extreme and more stable: peptide nucleic acids (PNAs)
Base pairs even better than natural nucleic acids (higher melting temperatures)
Also can add 2’ MOE
-O-CH2-CH2-O-CH3
Phosphorothioate deoxyoligonucleotides
MOE = methoxyethyl -
RNA modification
Sazani P, et al. and Kole R.Systemically delivered antisense oligomers upregulate gene expression in mouse tissuesNat Biotechnol. 2002 Dec;20(12):1228-33.
EGFP: Enhanced green fluorescent protein = model system
Actin promoter, universally expressed. Induced exon skipping yields green fluorescence
Mutant globin intron has activated splice sites
Antisense “RNA” injected into tail vein, RNA was modified for stability
Antisense treatment incell cultures (ex vivo) from themouse with the mutant EGFP gene
Control oligo (C)(50 nt downstream)was ineffective.
Max. effect = 40%
No antisense:
Dystrophin gene 2400 kb, mRNA = 14 kb, 79 exons: a giant geneProtein maintains muscle cell membrane integrityMutation: Duchenne’s muscular dystrophySome 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 exondystrophin proteinmuscle histochemistry for dystrophin
= 3 X 71
Branch site (consensus = YNYTRAY)
protein
mRNA
79
Use antisense RNA to target the branch point upstream of the offending exon 23 and the donor splice site downstream of the exon.
BP = branch point; SD = splice donor
Sequences targeted by antisense
U7 promoter
compl. to splice donor site compl. to branchConsensus binding site for Sm proteins (to target to pre-mRNA)
Double target synergistic (loop?) (Kole)
ITR = inverted terminal repeat, characteristic of AAV
0 2 4 6 13 weeks
Expression of U7 antisense construct
Splicing assay (RT-PCR)
Dystrophin protein (Western)
Endog. U7 U7SmOPT-A.S.
RT-PCR
Skip exon 23, after 2-4 wks.
(slow onset =conclude slow mRNA turnover)
normal0 2 4 6 8 13 weeks
transgenic U7
included
Top, middle ,and bottom
Normal
Untreated mdx
Treated mdx
dystrophin dystrophin-associated antigensMuscle immuno-histochemistry
intriguing