Transcript 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.

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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 10-10 M (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
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J Foote and HN Eisen
Kinetic and Affinity Limits on Antibodies Produced During Immune Responses
PNAS 1995; 92: 1254-1256
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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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Off time (t1/2)
Antibodies from yeast scFv selection (Boder et al. and Wittrup, PNAS 2000)
2x10-6 per
second =
5 days
half-life
Iterations
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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 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.
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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.
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)
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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
Avimers = high affinity ligand binding proteins that are not antibodies,
based on receptor domains.
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.
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Organization of binding domains in typical mammalian receptors
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?)
2 domains
cooperating
Bipartite
domain
Dual
specificity
domain
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
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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
displayed
on phage coat
M13 phage
One
domain
IL6 = interleukin 6
Monomer protein Build 20 phage
Screened for
dimer pools
binding
from 20 best
monomers
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
Binding measured by a competition assay (“AlphaScreen,” Perkin-Elmer)
Laser
Luminescence
Reactive
Oxygen
Laser
IL6
IL6
receptor
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Reactive oxygen
species can react
only over a short
distance with an
“acceptor” bead
Avidin bead:biotinylated IL6 +
gp130-Fc:Protein A bead
Competition:
IL6 (non-biotinylated)
or C326 avimer (10X tighter)
gp130 = natural IL6 receptor
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.
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More AlphaScreens: effect of combining the 3 domains
[So how was
M1 selected??]
20 pM
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Physical assay: Biacore surface plasmon resonance to measure binding kinetics
253 pM
0.23 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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Table 1. Diffedrent selected avimer affinities and activities
Avimer
Target
No. of
domains
Affinity (nM)
IC50
(Biochemical)
(nM)
IC50 (Biological) (nM)
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
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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
RNA
Topics:
1.) Pre-mRNA splicing basics
2) Splicing-based therapy
3) RNAi
Pre-mRNA splicing
Precision
5’
3’
5’
3’
X
X
Orderliness
X
X
Intron = 80 nts to 100,000 nts
Pre-mRNA
Branch point
Phosphotriester
Lariat
mRNA
Sequence of events in splicing
ATP
ATP
The spliceosome
(5 smalll RNAs + 100-300 proteins)
Intron becomes a lariat
ATP
ATP
degraded
http://www.swbic.org/education/comp-bio/intron.htm
5’ss (= “donor” site)
Branch point (BP)
(UACUAAC)
A
(= “acceptor”
site)
3’ss
Finding exons in a sea of introns
TTCTCAGTCCTAAACAGGGTAATGGACTGGGGCTGAATCACATGAAGGCAAGGTCAGATTTTTATTATTATGCACATCTAGCTTGAAAATTTTCTGTTAAGTCAATTACAGTGAAAAACCTTACC TGGTATTGAATGCTTGCATTGTATGTCTGGCTATTCTGTGTTTTTATTTTAAAATTATAATATCAAAATATTTGTGTTATAAAATATTCTAACTATGGAGGCCATAAACAAGAAGACTAAAGTTCTCT CCTTT
CAGCCTTCTGTACACATTTCTTCTCAAGCACTGGCCTATGCATGTATACTATATGCAAAAGTACATATATACATTTATATTTTAACGTATGAGTATAGTTTTAAATGTTATTGGACACTTTTAAT ATTAGTGTGTCTAGAGCTATCTAATATATTTTAAAGGTTGCATAGCATTCTGTCTTATGGAGATACCATAACTGATTTAACCAGTCCACTATTGATAGACACTATTTTGTTCTTACCGACTGTACTAG AAGAAA
CATTCTTTTACATGTTTGGTACTTGTTCAGCTTTATTCAAGTGGAATTTCTGGGTCAAGGGGAAAGAGTTTATTGAATATTTTGGTATTGCCAAATTTTCCTCTAAGAAGTTGAATCATTTTATACTCCTGATGTTATATGAGAGTACCTTTCTCTTCACAATTTGTCTCTTTTTTTTTTTTTTTTGAGACAAGGTCTCTGTTGCCCAGGCTGGGGTGCAGTGCAGCAGAATGATCACAGTTCACTGCAGTCT CAAC
CTCCTGGGTTCAAGCGATCCTTCCACCTCAGCCTCCTGAGTAGCTGGGACTATAGGTGTGCGCCACCACTCCCAGCTAATATTTTTATTTTGTAGAAACAGGGTTCGCCATGTTACCCAGCCTCC CAAAGTGCTGGGATTACAGGCATGAGCCACTGGCCCAGTTTCTACAGTCTCTCTTAATATTGTATATTATCCAGAAAATTTCATTTAATCAGAACCTGCCAGTCTGATAGGTGAAAATGGTATCTTGT T
TTTATTTGCATTTAAAAAAAATTATGATAGTGGTATGCTTGGTTTTTTTGAAGGTATCAAATTTTTTACCTTATGAAACATGAGGGCAAAGGATGTGATACGTGGAAGATTTAAAAAAAATTTTT AATGCATTTTTTTGAGACAAGGTCTTGCTCTATTGTCCAGGCTGGAGTGCAGTGGCACAATCACAGTTCACTCCAGCCTCAACATCCTGCACTAAAGTGATTTTCCCACCTCACCTCTCAAGTAGCTG GGA
CTACAGGTACATGCTACCATGCCTGGCTAATTTTTTTTTTTTTGCAGGCATGGGGTCTCACTATATTGCCCAGGTTGGTGTGGAAGTTTAATGACTAAGAGGTGTTTGTTATAAAGTTTAATGTA TGAAACTTTCTATTAAATTCCTGATTTTATTTCTGTAGGACTGAACGTCTTGCTCGAGATGTGATGAAGGAGATGGGAGGCCATCACATTGTAGCCCTCTGTGTGCTCAAGGGGGGCTATAAATTCTT TG
CTGACCTGCTGGATTACATCAAAGCACTGAATAGAAATAGTGATAGATCCATTCCTATGACTGTAGATTTTATCAGACTGAAGAGCTATTGTGTGAGTATATTTAATATATGATTCTTTTTAGTG GCAACAGTAGGTTTTCTTATATTTTCTTTGAATCTCTGCAAACCATACTTGCTTTCATTTCACTTGGTTACAGTGAGATTTTTCTAACATATTCACTAGTACTTTACATCAAAGCCAATACTGTTTTT TTAAAA
CTAGTCACCTTGGAGGATATATACTTATTTTACAGGTGTGTGTGGTTTTTTAAATAAACTCCTTTTAGGAATTGCTGTTGGGACTTGGGATACTTTTTTCACTATACATACTGGTGACAGATACC CTCTCTTGAGCTACATCGGTTTGTGGGGAGTCAAAAGTCCTTTGGAGCTAGGTTTGACAAATAAGGTGGGTTAACACTTGTTTCCTAGAAAGCACATGGAGAGCTAGAGTATTGGCGAATTGAAGAAATC
CCCCTTTTTTTTTAACACACTTAAGAAAGGGGACTGCAGGTATACTCAAGAGAGTAAGTCGCACCAGAAACCACTTTTGATCCACAGTCTGCCTGTGTCACACAATTGAAATGCATCACAACATTGACACTGTGGATGAAACAAAATCAGTGTGAATTTTAGTAGTGAATTTCATTCATAATTTGATCGTGCAAACGTTTGATTTTTATTACTTTAGACTATTGTTTCTGATTTTATGTTGGGTTGGTATTTCCTGT
GAGTTACTGTTTTACCTTTAAAATAGGAATTTTTCATACTCTTCAAAGATTAGAACAAATGTCCAGTTTTTGCTGTTTCATGAATGAGTCCTGTCCATCTTTGTAGAAACTCGCCTTATGTTCAC ATTTTTATTGAGAATAAGACCACTTATCTACATTTAACTATCAACCTCATCCTCTCCATTAATCATCTATTTTAGTGACCCAAGTTTTTGACCTTTTCCATGTTTACATCAATCCTGTAGGTGATTGG GCAGCC
ATTTAAGTATTATTATAGACATTTTCACTATCCCATTAAAACCCTTTATGCCCATACATCATAACACTACTTCCTACCCATAAGCTCCTTTTAACTTGTTAAAGTCTTGCTTGAATTAAAGACTTGTTTAAACACAAAATTTAGACTTTTACTCAACAAAAGTGATTGATTGATTGATTGATTGATTGATGGTTTACAGTAGGACTTCATTCTAGTCATTATAGCTGCTGGCAGTATAACTGGCCAGCCTTTAATACATT
GCTGCTTAGAGTCAAAGCATGTACTTTAGAGTTGGTATGATTTATCTTTTTGGTCTTCTATAGCCTCCTTCCCCATCCCCATCAGTCTTAATCAGTCTTGTTACGTTATGACTAATCTTTGGGGA TTGTGCAGAATGTTATTTTAGATAAGCAAAAACGAGCAAAATAGGGGAGTTTAACTTTAATATTTTCTTTTAAAAAGCATTTCATGTTATAAGATCAATTCTGAGTGGTAGAAAATGCTTTGACATTT TATTT
CCATTTTCTACTTTTAGTTTTTTTCCTATTTGTTTAAGATCTTAGAGGATTATTAAGCTGAACTCCTCAACTGATAAAAAGCATGACATCTTAAACATAAGCAAAGCATATTTTTAGGTTAATTTTCACATAGAAAACAGTTTATTTTATGTGAAATTCTATGTAGATATACTATTTTTTTGGTATTTATTGATATGTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTATTTATTTT TTTTTTTGAGAC
AGAGTCTCACTCTGTTGCCCAGGCTGGAGTGCAGTGGCATGATCGTAGCTCACTGCAACCTCCACTCCCGGGTTCAAGCAATTCTTCTGTCTCAGCCTCCCGAGTAGCTGGGACTACAGGTGCCT GCCACTATGCCCGGCTAATTTTTGTGTTTTTAGTAGAGATGGGGTTTCACCTTGTTGGTCAGGCTGGTCTCGAACCCCTGACCTCAGGTGATCCACCCACCTCAGCCTCCCAAAGTGCTGGGATT
ATAGGCATGAGCCACGTGCCCGGCCGACATGTTAATTTTTTAAAAAAGGCTTTACTGGGGTATATTTTATATAATATAATAATCACATGTTTTAACTATACAATTCCAAGCTTTTTAGTATATTT ATAGGGCTATGCAAGGAAGATATACTGTTAAACAGTAGAAATTGAGAAAGCTCTTCTGATAATATCTCTTGATTTGATGATGGCTCATGCCTGTAATCTCAGTGCTTTGGAAGGCCAAGACAGCAGAATCAC
TTGAGGCCAGGGGTTCGAGACCAGCCTGGGCAACACAGCAATACCCTATCTTTACAAATAATAAAAATATCTGTTGATTTGAAGTAAAGTTTTTTTTTAAAGACAAGGTCTCATTCTGTCACCCA GGCTGGAATGCAGTAGCAAGATCACAGCTCACTGTGGCCTTGACCTTCTGGGCTCAAGTGATTCTCCCACTTCGGCCTCCCGAGTAGCTGGGACTAACAGGTGTGCACCACCATGGCTGGCTAATTTT
TTTTTATGTTTGTAGAGATTGGGTCTTACTGTGTTGCCCAGGCTGATCCCGAACTCCTGGGCTCAAGCAGTCTTCCTGCCTCAGCCTCTAAAATTGCTGGGATTACAGGCTTGAGTCACCATGCC CAGCCTGAAGTAGCATTTCTACCCTGTTTAATAATTCAGCAGCTTGTCATGTAAGATATTCATATATGCATATAAACATTAGGCAGCTTAATTTGGTAAAACTGTAAAATGGAAATTTTAAATTGTTT GCA
GCATCAATAACATTGATGTCAGTATGATTTTTACATGCTGATCTTGACCAATTTGAAACAGTGAGTTAAAATCTGGCTGATCCGTACTAATCCTAAAGAAATATTCTATGAACTATTAAATGTTT CCAGAATATATAAAGAAACATTATGATGTCAACACACCCATCTATTTTTTTTTGGAAATAAAAACTCCATTTTTCTTATTAAAGAAAACATGCTTATTAGAAAACATACGGCTGGGTGCAGTGGCACACATGTA
ATTCCAGTGCTTTGGGAGATCGAGGTGGGAGAATCACTTGAGGCCAGGAGTTTGAGACCAGCCTAGACAACATAATGAGACCCCCTCTCTACACAAAAAGAATTAGTTGTGCATGGTGGCGTGCA CCTGTAGTCCCAGCTACTTGGGAGGCAGAGGCAGGAGCATCCCTTGAGCCTAGGAGTTTGAGACTGCAGGAGTTCGAGACTGAGTGGAATGCAGTGGAACTGCATTCCAGCCTGAGTGACAGAG
GGAGACCCTGTCTTAAAAAAATAAGAAAGAAAACACAACTGCAGAAAATTATAAAGGATTTAAGTCATTCCAAATATCACTGCCACTTTTTATTTAGAATATTCTAAAGAATTCTCTCTCTGTGTACACACACACATATGCGTACTCTTAATCCAAGTAGCTTGGTAGGATTTTATTTACCTAGTGCCTAGATGGGAAATTGCCTGGGGATTCCAAATACCTATTTCATTAAATTAAAGATGTCACTGATTTTAAGAC
TTAACACTATTTTTCATACTGCCAAGAAAGAAAACACTACCAGTTATAAATGTAAATTGCCATCAATTGTAATACATCAATTTTAGAGCTATTATTAATAAAATGTGAATGTGCATCTTAGAGCAATGAAATATAGTACTATATATTTGATGACCTTTTCTGCCCTGTGATATTCAGAAAGTGAAAGTTAAATATGGGCTGAGCATGGTGGCTCACACCTGTAATCCCAGTACTTTGGGAAGTCAAGACGGGA GGCT
GGCTTGAACCCAGGAGTTCAAGACCAGCCTAGGCAATGTAGCGAGACGCCATCTCAAAATATTAAAAATAAGTAAATAAGTAAATAAAAAGAAGGTTAAGTATACAAATGTATTTCCTTTGTTGT GAATTTATTTCAATTTTATAGTGATTTTTTTTTTTTGAGACGAAGTCTCACTCTTGTCCCCCAGGCTGGAGTGCGATGGCGTGATCTCAGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCTATACT CC
TGCCTTGGCCCCCCGAGTAGCTGGGATTACAGGCGCCTGCTACCATGCCTGGCTAATTTTTGTATTTTTAGTTGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTAGAACTCTTGACCTCTG GTGATCCACCCGCCTCGGACTCCCAAAATGCTGGGATTACAGGCGTGAGCCACCGTGCCTGGCCAGTGGTTTTTTGTTGTTGTTGTTGTTGTTTTGTTTTGTTTTTGTTTTTGTTTTTGTTTTGAGA
CAGGATCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGTGCCATCTTGGTTCACTGCAACCTCTGCGTGGGCTCAAGCAATCCTCCCACCTCCCTTTCCAGAG TAGCGGGGACCACAGGTGTGTGCCACCACACCTGACTAATTTTTGCATTTTTTTTTGTAGAAACAGGGTTTTGCCATGTTGCCCAGGTTGGTCTGAAACT CCTGAGCTCAAACAATCCAACTGCCTTGGCTTCCCTAAGTGAAATTACA
GGCATGGGCCACTGTACCCAGTCTAGTGATTTTTTTATTTTTATTTTTATTTTATTTTATTTTATTTTTTTACCAAAAAAACAACAAAGCCTCAGGAGGAAAAGTTGATACACAAGTAAATTTTA TTGGAAATGTTTTTGTGTGGACCTTAAGCAGAGGGAAAATTAGTCTGCATTATGGTGTATCCAGACTAAATGACTGATATTAAAATGAAATTATTCTTAGGATTTGCAATCTTAGAGAAAACTTTTTC ATTTTTA
TTTTTTTGAGTTACAAATTATCTTCATTTACATTTGAGAACAGTGAGTCACAGAGGGATTAAGTAACTTACTCAAGATCATACAAGTCTTTGATTTGAACCCAATCTTTTAACTCTGCAGAACTCAGAGTCACTCTTATTTGGAAAAACTTTTTAACTGATGTGGATCCTCTAATATGGGCTTCCTATTATTCATTCTCTATTAGTCAGAAGTTTTGCAAGCAGACAGAATTCATTTTGCCAATTACGGGATT TTCCC
TCAGTTGCAGTCAAGGTTCATAAAACTATAACTCTTTATCTTTAATTAGAAATGTTTTTTTTTTTGAGACAAGGTCTTGCTCTGTTGCCCAGACTGGAATGCAGTGGCATAGTGGCCCATTGCAG CTTTGAACTCCTGGGCTCAAGGGATCCTCTGCCTCAGCCTCCCAAGTATCTGAGACTACAAGTGCGTGCCATCACCCATGGCTATTTTAAAAAAAAAAAAAATTGTAGAGATAGGGTCTTGCTGTGTT G
CCCAGGCTGGTCTCAAACTCCTGGTCTCAAGCAATCCTTCTGCCTTGGTCTCCCAAAGTGCTGAGATTACAGGTGTCAGCCGTTGCACCTGGCCAAAACGATAACTTAAAATACACACACACACA CACACACACAAACACATATGTGTATTTGTGTGTGTGTGTGTGTGTGTGTGTCTCAAAAGGTATCAAAAGAGAATAGCTATAACTTTAGTGTTGATCTTGATAGTGACTTGATTAGGCTCTGTTTAACA T
CAAAGATGCAAATTAATACTTTCTTTGAACATATTAAAAATGCAGAAAATATTGGAGTATTTTATTTTAAATAAATTGTATTCTGTATATTTAAGGTATACAACATGATGTTATGGGATACATAT AGGTGGTTAAAAGATTACTGCAGTGAAGCAAATTAACGTATCCCTCAACTCACATAGTTACCCATTTTTTTTTTGTTTTGGTGGCAAGAGGAGCTTAAAATCTCATTTAGTGTGAATCCCAAATACAG CACAAT
TTTATTACCTATATACTTCATGTTGTACATTATATTTCTAGACTTGTTCATCCTACATATCTGCTACTTTGTATCCTCTGAGCTACATCTCCCCATTTTCTCACTTGCCCCCCAAGTAGTTTCTT AAAGTGTCTCATGTAAGAGGGCAGTAGCTTTCAGCTTAAACTTTTTCTCTGTATGTAGTCGATTTCTTTGAGGTATACTTTTCTCTCCAGAATAGTTAGATGTAGGTATACCACTTTGATGTTGACAC TAGTTT
ACCTAGAACTTATCTTCTGTAAATCTGTCTCTATTTCCATCTCTGTCTCCATCTTTGTCTCTATCTCTATCTGTCTATCTCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATC TATCTAAAGCAAATTCATGCCCTTCTCCTATTTATTGAATCGAGACCATAGACAGGGGTGAGAGAAAGAATTTGGCAGGAATGGGGATGTGTATTATCTGTGGCATAAGGAAACTTTACAGAACTAGG TTCAA
AAGTATACTTTCTAGTTCTTTCCCATGGCTTTTCACTTTGATGTAGTCCTTATCAGGTAACTGAGGTTTTATATAAGTCCCCTGATTCTTAGAACATGAAGGTGTAGTAGTCAAGGTTGGTCCCT TGAAACCACAAATTTTGTGAAAAAAAATTAAGAAAATTTGAATAATTTCCTCAGCAAATACATATTGATCATCTGTTATACAGCCATGAGAAGTGGTTCTGTTGCACACGTTTATTTTATCAGATCCTAATCC
CAAACCAGGCATAAAATGGAAACCATGAAGATAGGATGAAATAACTTCTGAATGTTTGAATGTTTGAAAATAGTGTACTTAAAAATACCAGGTGGTTTTTGTTTGTTTTTTGTTTTTTTCTTTTT TTGAGACAGGGTCTCACTCTGTCACCCAGGCTGGAGTGTAGTGGTGCAATCTCATCTCATTGCAGTCTTGACCTCCCAGGCTCAGGTTATCTCCCACCTCAGCCTCCCAAGTAGCTGGGACTACAGGC AC
ATGCCACCACGCCCAGCTAATTTTTTGTATTTTTTGTAGAGACGGGGTTTCACCCTGTTGCCCAGGCTGGTCTAGAACTCCTGGGCTTAAGCGATCCTCCCACCTCAGCCTCCCAAAGTGCTAGG ATTACAGGCATGAGCCACCATGCCTGGCAGAAAATACCAGGTTTTTAAGTATCAGCACTTACTCTTCAATCTTTTCTATTACTATGTTGTGCTAAATGGTATTTTTTATTTAATTAGAGCAATGCTGT TC
AATAGAACTTTCTTTGAGGATGGAAATCTTTTATGTTTCTGCTATGTGGTACAGAGCCACTAGTGACATGTGGCTTTTGAGCGCTTGACACATCTTGTGCAACACAGGAACTGAATTTTTAAGTA ATTTATATTGCCACATGTGGCTACCGTATGGGACAGTGTAGTACTAGATGATCTGTAAGGGCTGTGCTTCATCAGTGTCGTTTTTTAACTGACAAAAACCTTTAGTTTTTTTTTTAGTAATGTGTTTA TTTA
AAAGAATTCATAAAATACAAGTAAACAAATTAACTTGTTACCTGAGCATATGTCCTTTCATACTTATTTTTTCTGCATACATATTTTGGAAAATGGAATATCTGCCCCTTTTTTTTTATCTGAGATACAGTCTACCTCTAAAAATACATGATTCTAACATTCTCACTTTTTGTTGGCATTTGATCAGGGTATAGAAAAACAGTTAAAAGGACAGAGAATGGTTGAGAGATTATGATATGAAGAGAAAATGTGATTGAGT
GTGGTAGACTTGGGGCCTGCTTGAATGTTGAGAGAATGACTGTTTTCCGATAAAAAAAAAAAGTCCATTCTAGGATCCTAAAAGAAGGGTCTGAAGTTCACTGCAGAAAGCAAGCTACATAGTAC TAAGCCACTAAGGGGACATGGAGCCCTTAGTAATTCCTACCTTAGTAATAGTCTCATCATGCCCTCTTGGGAACCCAGCCTTGTTGATTAGCCTCTCTGCTTTCTCTCCTTATAGTTCAACCTCCCTG T
TTGTTCCAAGCAGTTCTTTTCCTGCCCATTTATTATGCATTTCTATACAGCTTTCCTCCTCTTTTTCTATACCATGCTGCAGTTCTTATTGCTACCTAGAGGTTTTCAAAATTCCTAGGGGCGGA TAAGTAGGCATAAACAAAGTTCTTCCCTATTATCCTTCCTATTTTTTCACCTAGACTGAAGAGGTAGACAAAATAGAAATAAAGACATTAAGGGTATGTGTTTGTAGTCCCAAAGAGCTTCTCTGGCAATTT
TGATGTAGTTGACAGTGACGCTCTGAGTTCAGGACAGATTGGACTCCTTGGCTGAGAGGAGTGAGGAGATAGGACGGTAGAGGAGAGGGTAGAGCAACTCTGGAGGAAGCTTTCCCCTCACCTTT GCCAGTCCTGTTATCCTAGACTTAACCATAATTAAAGATGAGGGAGGCACTCAGTAAAGGGATCTAGTGGGAAGCTTGTTCCAGACAGCCAAGGAGGGAGGTTCGCGCAGTTCCTTTGGCCACC
CAGGTGGGGTAATTGATCCATGTATGCCATTCATGTACAATGTAGGCACTTATACCTGTATTCCAATGTAGTGAACTATACCATTACTCTTAAATTAATATTCTTTATTAGCTTCCATGGTGGCT ATAGGCCAGGCAAGAGAGTTAAGAAAAAATAAATAGCCAGGTATGGTGACTCAAGCCTGTAATCTCGGCACTTTAGGAGGCCGAGGCAGGAGGATAGCTTGAGTCCAGGAGTTCAAGACCAGCCTGAG
CAAAATAGTGAGATCCTGTCTCTATTTTTTAAAAAAGCCTTGGGGCAAACAGGAGTATGGAGGTTTGGATGCTAATAGAACAGCAGTGTCTTACTGCTTGGAGTTCTCTTGTTTCTTGTCCTATCACCGTAGCCTTTGGATCACAGCAATTTTTCCATGACTCCATACTTTTCAGTTC TTGAATATTTTTTCCTTTATTCCTCTTGTCTCTGTAAAGACATCAACTGGAGTTGGACTGTAATACCAGGTATCTCCA
GAAGATGGCACTATTTAACAGATTTTATAAATAATTTGATGTGAGTCACTGTCATCTGAAGCTTGTTGCCTTTTCTTTCTTTCTTCTTTCTTTTTTTTCCCCATCAATTCTGTATGTTTGAAATG CTGGGATTTAAGTTAGTTAGAATAAGGGATGTCTGTAATTTCCCTAAATTGAGAAGTAATATGCAAAGGTTGATATCAGAAGTCATATGCTCACCTTGCAACACCAAATAATACTGGCCCATTTGTGATTTTT
GAAAGTAACACTCCATAATAAATGGATGTATATATAGAAGCATAACAAAAATAGAAGCACATAAAAGTGAAAAGTCTCATAAACGCCATTGTCACTACTCATGTAATTGCTGTTACAAATTTGTT TAAATGTTGAATAAAAATGGTGTCATAGGCAACACAGTGTTCCACTACTTGGTGTTTTTAATAGCATTATTCTGTCTCAGTGTGCTTTGGATTATCAGGTGCTTTTTAATAGTTGCATGGTATTACAT TGTGT
AGATGAACTTGATTAATTTAAATGGTTCCCTGTTAATGGACATGTTGGTTTGTTTTTGTGAACAACTGATACAGTGAACATTTATTTTTTAAATAAAAAAAAGAGAGACAGGGTCTTGCTGTGTT TCTCGGGCTGGCCTTGAACTCCTGGGGTCAAGCGATCGTCTTGCCTCTGCCTCCCTGGGATTACAGGCATGAAGCCACCGCACCCGGCCCAGTGAACACTCTTGAATGTATCTTTGTATACTTGTCAA G
TGTTTTTGTAGCAATTGATTCCCAGAAGTGGGAATTACATGGAATTAAGTGACATGCATGTTTGCAATTTTAACAGGTATTGCTATGTCATTTTCAAAAGAAGCTATGCCAATTAATACTCTCAC CAACAAGAGTGCTTATTTCCCCTCAGCATATTATCAGGCTTAAGTTTTGCCAGTATGGGTGGGAGAACAGTAGAATCACATTGTTTTAGTGTTTGTTTCTCAGATAGATATAATTTTACACCTTATAA CCTT
CTCTTCTATAAATTGTCTATTTGTGTTCATTCTCCATTTTCCTATGGGTTCTTATTGTTGGAGCCCAATATATAAAAGGGGGTATTTGTTACAGAACCTCTTCAGTTTTGGTTCATGTCATGCCT GGGTTTTTACCCTTTCTACGGATGTTAAAAAAAATTCTCTATTTTCTTCCAGTCCACTTATGGCTTTATTTTTTACATTTAGATTTTAATCCGTCTGGAATTTATTTTTGTGTATGCTGTGAGGTAGG GACCAT
ACTTTTATTTTTTCCCAAATGGGTTACTAGTTGGCCAAACATCATTTATTGAATAATTCATCTTTTCCCTACTGACTCGAAATACCATCTTTATTGTATACTAAATCCTCATATAGTTCTGGGTC TGTTTCTGGGCTCTACTTTGTTCATTTACTGTGCTGGTACTGCACCGTTGTAATTGCTGTGGCTTTGTGGTATGGTATGGCTTGCTCTCTGCTAGGGCAAGTCGAAGCTCTTTTGTTCACCTGCTCTT TCAC
CCAAATTTTCTGTCCTGAATCCAGCACAGCCAAATTATGGTCATTGTCACCACCAACTACAGTGGGTGTTGAGCATTTCCCATTGAATCTCCTGTAAGGGTTTTATTGGATTCTGTGATAGCAGT AAAATGGGAGCCTAAGAGGTATTCCTTAAAGGACTACTAATCAGACCTGGTTTCCCAGATGATGCTGAAGATGACGGGGCCTGGGCTAGACTTTTGAGGGACATATCCTTGGGGTTGGGTGTGATATA
GACCAGCCCTTACAATTTGCTTGACTCATGGGAATCGTACAGGGCCAGAACCAGACACCTGTCATGCTAATAACTTCCCTCACAATTCAGAAATCACTGTGATTGAAGATGGGTGGCTGTTATAA TACTACCCACTTAAAAATGGATGTAACCCATTTTTTAGGACTCTTAAAAACATCAAATCAGTAATGGCCGATTAGGACTTTTTAATTTTTACTAATCTCTACTTGAAAGTTTTCTAGTCATTCATTTC AGGA
AACCTAATTCTTATAATTCATATCATTTAGAATATCATAATGCTATGGATATTAGCTAGCTAACTTCTCAAATCTTCTAGTTCTCATTTAATTTGAAGTTTGTGTGTGTACATAAGGATATACAT ATACATATGTGTGTGTAGATATATATATATATAGTTTTTTTTTTTTTAACTAGAATGACCAGTCAACAGGGGACATAAAAGTAATTGGTGGAGATGATCTCTCAACTTTAACTGGAAAGGTATGTATC TTGAAAG
GGAAGAAAAAAAAGCACTTCATACCGAGTCAATTAGTAACAGTGTGCTTTCAATCAATCACTAAGAGATAATTTACATAGTATAACTAAATGGGTTATTTAACCCTTGGAAGCAGTCTAGGTTAATTATCGTTCCCTAGGTCATGTAGTAAAAAGACAGTAGAATCCAACATTAACCTTAAATGTCCATATTGTCAAGTACTGCTGTCTGCCTCTGTGGGACTCTAATTTGGGATCCTTCAAAAAACATTGAT GGG
GGAAAAGATAGCCTTTAAAAAAAAAAAAAAAACAAACCTATGTGAGTCTATGTGAGGTAGACTCACATAGTTTCCTAAAAGATAGCAAAGCAGTATTATGTAGTGGCTGAAAGTGTGAGTTCCGGAGCCTGACAACTGATTCAAAGCATGGCTTAGTACTTCCTAACTCTGACCTTGGGCAAGTTACTTAACCTCTCTGTGTCCCATATGTGATTAGGGTGAGGTTGATAATAGCAGCCATAGAGTTAAGAGG A
TTAAGTGCTATAATGCAAGTAGAGCTCTTACAACAGTTTCTGGTAAATCACTCAATAAATTCAGACATACTATTATTTTAAGAAATCTCAAAGAGTTTTCTTGTACCTTAAAATTCTCCTAGTGT GAACCATTGGTTTTGGTATATTGTGCTTCCATGTAGTTTAATATCAAGATGTTTTTAGATTTCCCTTTTAATTTATTTGTTGACCCATTGGTTGTTCAGGAGCATGCTGTTTACCTGAAAATAATGGA GATATT
AAGGTATTTGAATATTTATCTTCTAGTACATTGAAAAACTTTTTGAGAGTAACCAATAATAAATGATGGAATGCTACTGCTTTTTTTTTTTGAAGCTGCCAGTTATTGTTTACTTACACTATGCC AAATATAAAGGCATTAATCTCATAAAAGTTTCACAACAATCCTGTGAGGGAGACGATATCCCCATTTTACAAATCAGGAAATTAAGACTTAATAAGGTTAAAAGACTTGCCCCAAAGTCACAGAACCAGTAAG
TGGTAGAGCTTGAATTTGAATACAGACCTGACTCTAAAGCTCTTTTCTTTCTTTAGATTTTAGTGTTCATTGCTTACTTGAATGAGTATCTATAAGAAAACTTTAACATGTAAAACTTCTGTGAA ATTATCTTGTCCCATATCAGGGTCATGTCAAACTAATGTCCTCCTCAGCATCTTTGGAAAACTTCAGAGGAGAAATGAGCTTTGCCCCTCCTGTTCATTTCCTATTCCACTAG GAGACCTGTCCTTCCCTTT
CAGCATGCTTTGTCCATATTTAGAAGCTGTTGAAGCCATTACTTGTCTGGTCAGTTTTTAGTGCTGGAATGGACCTAGCCTTTTAGGCCTTCTGAGATTTAGTTTGATCTCGTCTTTCCCACCTA ATGGCTCTGTTCTACTACATAGATTTGATCTGAAACAGTTCTCTGTTTCTAAAATAACTTTCTTTTCATGATAGTCACAGTAAAGTACATTTATTATGGAAAAATCAATAAGTATAACGAGTGAAAGTTATT T
CTTGGTGGTAAGATTATGGGATTATTTGAACTTTCTGTTTCATTGTATTTTATTTATTTATTTATTTTTGTGATGGAGTCTCACTCTGCTGCCCAGGCTGGAGTGCAGTAGTACGATCTTGGCTC ACTGCAACCTCCCCTTCCCAGTTCAAGTGATTCTCCTGCCTCAGACTCCCAAGTAGCTGGGATTACAGGCGCACGCCACCATGCCTGGCTAATTTTTTTATCTTTAGTAGAGACAGGGTTTCACCATG TT
GACCAGGCTGATCTCCAACTCCTGATCTCAGGTATCCACCTGCCTCAGCCTCCCAAAGTACCGGGATTACGGGTGTGAGCCACCCTGCCTGGCCTCATTTTGTCTTTTGGGGGTATTTTTGTGTG CAGATATATATGTATATAAATATTTTTCCCTCTTTTCCCCAGTTAGTATTTGAGCAGATGAACTTTGGACCCGAATACCTGTATTCAAGTCTCTAATACCACTTCTTGGCTATTTTCATTTTATCAAA TG
GCCTCTTATCCTCGTTTTTCTCATTTATTAAGTAGAGATGTAACTACTTGATATAATTCAAAAACTCAATAATGGCATTCTTTTGTTTTTTAGACTCTAGTGTCTGTACTCCTTGTACCATGCTG GGATTCATTTGAACAATTGCATGGCTTTTTTAGTGTATTATTAAATTTGCAGTTTACTTAGAATTTACTGGGACCTCATACAAATGGGAAAAAAACATAACTGTGTTACTCATTTGCTGTGTGCCTTT GGATTG
ACCCTATTTTTTGTATTCATTTTCTCCCCATGTCCTGAGTTCCACTTTGAATAAAAAAGTAATTTTTTTCCTGCCTGTAAAATAGGCTACCAATAGGCTGCAGTTGTCTATAGTAGCTGCTTCAC TGAGGAGAGCTCAGCATGAGAGAAATAGTATGAATTGCTTGCCACAAGTTATGGGCTAGCCTTACTTCATTCTGTACTTGGACCTGTTTAGGCTTCTAAGAGATCTTACCTCCAACAATAAACTGCTT TGA
GACATGAAAAGGTGGAAGCTTTACTTGGTTATAACTTTACTTTTAATACCTAGAACAGTGAGTCTTCAAACTTGTATTTGCATGCCCAATTTATAAAAAGTTTCCTGAGCATTTACCCCTAATAT ATGCATTTTAAATTATATATGATTTATGGTAATAATAATATATATGTTACAAAATACATACAAAAATATAGATTAAACAAGGTGAGGTTAAAAAATTTAAAAGTTCTAATCTTTCTTGCAAACCAGTGGATCTTTT
GTGCCTTACTCTGGTAAACACTGTCTTAGAAGAATATATAGAACATTAAAATCTTAATGCTATAGTTATATGACAGAGTATGATGAGAGCTACAGATAAACAACACATCATGAATCTTCTTGTGG CAGTGTTTATAACCATTATGTGAAATGCTGCCTCATTCTTATAACTAGCATAAGAACAGATAGGACTTTCTCGATTTTGAGGGGTAATTATTAGATGGTATTTTCTGTTAAGGACTCTTCCAGCTATA AAATT
CTTAAATGTAGAAAGCGAAGTGAGGGTTTATGGTGAGAGGAAGCATTGGTATCATGTTTTAGTGTAGTCCAAGAATATGGACACATCCAGAAAATGCAGATCAAGTTTAGCCTAATGAGAAAATATATTTTGGAGTCCATATGGTAAATTAAATTATGTGATTTTTGAGTTATTGTACAAATATAATTCTTAGAATGTTAGAGTCAGGAGACTATAAGAGACCAACTGCTTCAAGTTTCATTTAACACATGGG AAA
CTAAGGCGAGAGAAATTTCAAGACTTGCCCAAGATTAGACCTCTTGTTAAGTAATGAAAGTGTTTTAAAAACAGGTGGGTCAAATTCTGTTTTTAAAATTTCCATTATGATGAAAATTTCAGTAT TACAGGCTTCCAAATCCCAGCAGATGGGCCACTTGTTTAAAGGAGAGTTTGATATAATAAAGCATCTAAAAACAAGAGTTTGGATAATTCCTTAGGGTTGTTATGATGTGATTTGACTTATAATTGGA AATA
CCGTTTTATTCATTGTACTGATTTTCATTTCTCTTTTTCTTCTAGAATGTCTTGATTGTGGAAGTAAGTTCACATTTACTTTTAATATAACATTTATGACTTTTCTAACTTAGTATGCACCATCC TAAAGGTAAGCCAGGGAGAGAAATTCCTCTGCATCAGTTTTAATGGTGGGCTTGTGTTCTAAAGGAGTGAGATTGGTTTTTTGTAAAGACTACTTAGTAATTTGTTTTTACCAATAATGGAATGGTAT ACTTCC
TACCTCTCTTTTTTTAGTTTGAAGTATTTTCTTTCTAAACATAACTCTCTCTCTCTATTTATCTATATATAATATATACATATATATCTTATATTTTATGTATATATATATATATCTTGCTTAGA TTTTGTCTTATGTAATATTTGGTACATAAAAAATAATATTTATAATTTATAGACTATTTTCCATGTGTTATTATGTGCTAAAGTATTTTGTATCTTAGCACCGAGAGGCTAAGCAGTTTCCTAGGGTT ACCAGCTAGT
AAACTAAGGGAAACCTTTACTTCCTTTAGCTCAGTGGTTCTCAAAATGTGGTTCCCTAGACCAAAAGTATTAATATCAGACAAGAACCTACCGAATCAAAATATCTGTGATGAGGCCCAGCAAGC TATGCTTTAACAAGTTTCCGAGTGATTCTGATGCATGCTAAGGTTTAGGATCCCTTGTTTTTACTCATAAGTCACTTTCTCATTAAGGCCTTCCCTGGCCATCCTATATAAAATCTCATGTTTTCACA CCG
TCAACTTCGTATTCCTCCTCAATACTTTTATTTTCCTGATCACTTATCACTAACAGCCTCTCTCTCTCTCTCTCTCTCTCTCTATGTATATATATATATATATCACTTATCACTGTCTAACAGCC TCTCTTTATATATATATAATCTATAGATTATATATATATGCAGCATTGTGCAATCATTATCACGCTCAATTTTAAAACATTTTCATTTCCCCACAAAGAAACCCAATCCCCTTAGCCATCACTCCCAATTTTCCCT
TCCCCCAGCACCTAGCAAACTGATCATCTACCTACTTGCTGTCTATAAGATTTGCCTATTCTGGACATTTTGTATAAATAGAATCATACAATATGTGGCCTTTTGTATCTGGCTTCTCTCACTTA ATGTTTTCAAGGTTCATTCATGTTGTGGAGTATATCTGCACTCATTTCCTTTTTATTGCCAAATTGTATGGATAGACAGGTGTTCCTCAACTGTGTCCTGATAAACCCATCTGAAGTTGAAAATATCA TAAGT
TGAAAATGGATTTACTACTTTGATAAATCTATCCTAAAGTCAGAAAAATCTCATGTTGGAACCATCGTAAGTTGGATACCATCTGAATTACATTTTTGTTATCCATTCACTGGTTGACAGACGTT AGGTTGTTTCCACTGATGCTCCTTATTTCTCGTACCTGAAATGTCCTTATTCCCTCCCTTCTTATCCCATGTTTAAGTCATTTAAGACCCAGCTCAAACGTCACCTCCACAAAACCTTCCTTGATACC CCTTT
CCTCTTCAATTCACTTGGACCTTTTGCATTTAATTTTAATTTTTATTTTTTTTAAGACAGAGTCTCACTCTGTCACCAGGCTGGAGTGCAGTGGTATGATCTCAGCTCACTAACTACTCTGCCTC CCAGGTTCAAGCAATTCTCATGTCTCAGCCTCCCAAGTAGCTGGGACTACAGGTGTGCGCCACCATGCCTGGCTAATTGTGTGTGTGTGTGTGTGTATGTATGTATGTATATATGTGTGTGTGTGTAT AT
ATATATATACACAAACATATATAAATATATATACATATATATATATACACACATATATAAATATATATACATATATATATATACACACACACACACATATATATATATATAGTTTTTTTTTTTTT AAGTAGAGATGGGGTTTTGCCATGTTGGCCAGGCTGGTCTGGCCTCAAGCCATCCTCCCACCTCGGCCTCGCAAAGTGCTGGTATTATAGGCATGAGCCACTGTGCCTGGCCTGCATTTCATTTTAAT TATAAA
ATATTTTGAACTCAGAAAAAAGGGTATGCTGAATACCTACGTACCCACAAAAGTATTAACATTTTGCCATATTTGCTTCTGATCTTATTTTTTTTGAGAAATTAAAGATCATAATACAACTAAAG CCCCATTTCTTTCCCTTCATTCCCAGAAGTATGACAATTATCCTTAAAGTTGATATATATCATTCCCATGCATGTTTTTTATACTTCCCTAGTACAAGTTAGCTGTATCCTCTGCTCAGGGGCTCATC AAGCTG
AATCAAGGGACTCATGATCCTCTTCAAAGTTCCTTCAGGTTGTTGGCAGAATTTAGTTCCTTGTGATTGTAGGACTGAGGGCCCGTTTTCTCACTGGCTGCTGGCCAGGGGTTGCTCCCAGATAT TTAAAGGCTCATGCCCTAGCCCATGACAGTCTCACAACATGGCAGCTGACTTCTTCAAAACCAGCAGGAGAATCTTGCTCTAGTCTACCACATAACCTAATCACAGGAGCGGCTATCCCGTTATTTTC
ACAGATCCTGGTCACATTCAAGGGGAGGGAACCCTTCTGTGTGTGTACACCAGGAGGCAGGAATTTTTTTTTTCTTTTTCTTTTTTGTTAAAAAGTCTTAAAGTCTTTTATCCCTAAAGGAGGCA GGAATTTTGAGAGCCATCAGAATTCTGCCTACCACAGCCCAGAAATCTGCATTTTTCACAAGTCTCCAGCCATGATGTTTCTGATGGCTCACACTGCTTTATTCCATTTTTAAAGAGTATTTTTATTG AAA
AGCATTAGGGTTATGGTTTAAAAAATATTTTCCCTAACAAAGATGGGTTTGTTTAGAGTCCTACTTTTGACTAAATAGCTGAGATTCACTTTTATGTAAAGTTCATTTTATAGCGTTATTAATTT GGGTGCCTTTAAAAATAGTATAAAGCATGTTTCTCGAGTGTAGTCTGTTAGCCACCTATATTGGAGAGTTGGGAGGAGAGAGTCTCTATCTTGAATTTATGGGAAAAATTCTAAAATACTTTTTATAATGAAG
GACAACATCATAACTCCCTAATAAAATGTGCATGTATATATTCAAATTTGCTGTCATTGATCCTGCACCTACAAAATCCAGTCCTGGGGGCTGGCATTCTTACTGCTTGCTGAGGGCCAGATGAT ATAGATTCCAGAATATCTCCATGTAGATTTTGGTGAGAATTACTGTGCTGAAAAGAATGACAGTATTGCAGTTATACATGGGGGTTTTGGTACTTTATATTGTGACTCTGAATTTAAAGCTATGCAAT GTC
TTCTTTTTTGAAAGGATATAATTGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAAAGATGGTCAAGGTCGCAAGGTATGTATGACATTTTGACACAGAATAT TTTCCTCATTTGAAGGGGGATTAAGTGATTGCTTCTTTTTAAGGATAAATGTTTTCAACTGTCATTTTATCTTCGAAAAGTAATGTAATCTCATATAAGACTTAAGATATAATCCTTTTAAATAATTT TGTCAT
GTGTTAATAAAGCTCATAATTACAGTCACTTCCTTGCTAATATTAACATTTGGTTTTCAGCATGCTAATTATATCAGTTTGTCCTGAATAGCATGGCAGAGGATTTTGGGCCCCCTTGCAAAATT AAGAATAAGGATTCCAAAGCGGGTGAGGAAGTGATAGGAAGGGGTGGGCCCTGAAGATCTGGACCTCCTGGAATTGAGTGATGAATGCTGCATCTTCTTTGTGTCTGTAGTGAAATTTTATAATGCCT G
CTTCCTTTTTTATTAAGTCGGCCTCACCTCCTCACCTTACCTATGCTGTTTTACTTTTGCTTTTATAGTTCTACCTGTGTTTATTTCTCATTTTCGTTTCATCTCTCAACAACTCTGGGGTGGCA TTATTATTCCCACTTTTCAGATAAGGTTACTGAGGCATAGGGAATTGTCCAAAGGTACAGAGCTAGTCCGCTATAGAGATGAGATTTGAACCCAGGGAACCTGGCTCACAGTTTATGCTTTTGCCTAC CTT
AAGTTTTTAATAGAGTGACATCAAACAAACATTTAAGAATATGTTTTTCTTTTCCTTTTATAATTTCATTAAAAACATTAAGTCTCTGATCAGTCTGCAGTTTTTATGTAGGGGTCAGGTAATGT TCTAACTTCTGCTTTTTCCTAAGTGATTAACAGGTTTTTATAAGCCCTTTTGAAAAAATCACGGTATCTGTCGAGCATCTTTGAATCAGAGTAAGCCTTCTAGTGAGTCATATGTCAGCAGTTTGACT GTATG
GGCTTTTCTAATATCCAGTTCAAGTGTTTATCAGTGAGTTTTTCTTTTAAATAGATTTGGGACAGGTACTATGAGAGTATATAAGTGATACGTTATAGGACACTAACTAGTATCCTATGAAATGG CAAAAACTGCAATCACTTTTGCACCAACCAAATAGAAACTAATCAGTGCACTTGCTTATTTTTCTACATGCTCTTTAGGGTTTTAAATGTCAACCTACTGTGGCATAGACTTTAATCCTCTGGGTATT CTTTT
GTTGTTCTTTCCTGGTATATGCTGTGGAATTGAGATAGACTGGTTCGTGAGCGAGAGATTTTGTGTTGCCACAGGTAGGACATGCTCAAACAATACTTGGGTCATTTCTTGACCCAAGTCATCTA TTCACCATAGTTTTGTAGCACCGATCTTGCATACATTTCATGTATCTTCTTTGAACCCCACGTCAGTGCTGCTTATATGATACTCAGAAATTAAACACTAAGGAATAAGATTTTCAGGTAGGATTGAG TT
TTGGAGGGTCACAAATCTTGTAATGTCTAATATTTCCACTCTCCCTGCTGAGAATTAGTTTTGGCTTCCTTGGAGGTGATATCGCCTCTGTTGAGTATAAGTGGCCTACTGTGATCACACCACTG CACTCCAGCCTGGGTGACAGAGTGAGACCCTGTCTCAGAAAAAAAAAAAAAAAAAAAGAATGCATGGCCTAGATGACTTCTAAGGTTTTTCCCACCCAGTTCCAGTTTTCATGTTCTAGGCAGAGCAG T
AAAGTGAGAAACACATGGACTTGGGAGTTTAGTCTCGCATTTCACTGCCACTTAATCTGAGCGACTATTCCATATTTAATCTCTCTGAATGTATTTACTCATCTTTAAAGGGGAATGATTATTAA CATCTTTTTCTCAGGGAAACTATATGAGTCAAGGAGATAATATATTTGAAAATCTTTTTAACTGCAAAGCGCTGTTTCACTGTTGGTTATAATGTGATTGATCTCATTGTAGTGAGCAGCTGCTTAAT TGCG
TTTTAGAATGTAGGGAAGATAGTAATATTTTTCACATTATATATGTAGCTGGTTCTGGAACTGTAAACATACTCCTTTTTTATGGAGATCTGAGTCACGTACCATAAAATTCACTCTTTTAAAGT TGTACAATCCAGTGGTTTTTGATATATTCAGAGTTGTGCATCTGCTACCACTATTTCATTTTGGAACCCAAAGAAACCTTGTACCCATTAGCAGTCATTCTCCCTTCTCCCAGCCCCTGGCAACTACT AATCT
ACTTTCTACAGAAAGTCCGTACAGATTTGTGTATTATGGACATTCCATATAAATGGACTCATGCAATATCCTGTCTTCTTTCACTTAGCATAGTGTTTTCAAGGTTCATCTAGGTTGGGGCATGT ATCAGTACTTCATCCCTTGTTTTGGCTGAATAATATTTCATTGTACAAATATATCACATTTTGCTTATCCATCTGTTGGTGAACATTTGAGTTTCTACCTGTTGGCTTTTATGAATAATGTTGATTTG AATGT
TTGTGTACAAGTATGAATACCTGTTTTCAGGTCTCTTGAGTATATAGTTGCTAGGTCATATAGTAACTCTGTGTTTAACATTTTGAGGAATTGCCCGACTATTTAACAAGGTATATGTACTGTTT TACACCAGTAACATATGAGGGTTCCAATATCTCCACATCCTTGACAACACTTGTTACTGTCCTTTTTATTGTAGCCATCCTAGTGGCTATGATGTGGTATCTCATTGTGGTTTTGATTTGTGTTTCTC TGAT
GCTGATGATGTTGAACATGTTTTCATCTGCTTATTGGCCATTTACATATATCTTCTTAAGAACGGTTACCCATTTACAGTATGGAAAATGCTTCAGATGCAACTCTAGTCATGCCTTAGAGATGG AGCTTTATTAAACATTCAGATCTCTAGGCATATGAAGTGCTGAGTTCTCTTGAACTCCTAATACAGATTGCACTGAGTTTAGTGATACCTTTTCTGGAGCATTCCTGAGTTCAGGTAGGGAGAAGGGT TTT
TGCTGTGATTGGCTTGTTATGTTCTTTCTAAATGGAAATAGAATTGAAGTGTCTCCTCTCTCCATTTA
Some types of alternative splicing
Alt. 3’ SS
Alt. 5’ SS
Exon skipping
|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
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
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)
Anti-sense RNA:
A therapeutic intervention at the level of pre-mRNA 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
Got this far
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
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 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
RNA modification for stabilization
ase
Instead of deoxyribose or ribose
Modified phosphate
ase
Still base pairs OK!
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)
RNA modification
Also can add 2’ MOE
-O-CH2-CH2-O-CH3
MOE = methoxyethyl -
Phosphorothioate deoxyoligonucleotides
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
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%
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
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
U7 promoter
Double target synergistic (loop?) (Kole)
compl. to splice donor site
compl. to branch
ITR = inverted terminal repeat, characteristic of AAV
Consensus binding site
for Sm proteins (to target to
pre-mRNA)
Expression of U7
antisense construct
RT-PCR
transgenic U7SmOPT-A.S.
U7
Endog. U7
0
2
4
6
(slow onset =
conclude slow
mRNA turnover)
13 weeks
included
Splicing assay
(RT-PCR)
Skip exon 23,
after 2-4 wks.
0normal 2
4
6
8
13 weeks
Dystrophin protein
(Western)
Muscle
immunohistochemistry
Normal
intriguing
Untreated
mdx
Treated
mdx
Top,
middle ,and
bottom
dystrophin
dystrophin-associated antigens