1

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

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 10-15 M = 5 x 10-14 M = 0.05 pM (compare 100 pM limit for naturally selected Abs)

Slower off-rate than biotin-streptavidin (>5d).

3

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)

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

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.

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

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

8

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)

9

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

10

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

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

     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

13

More AlphaScreens: effect of combining the 3 domains

20 pM

[So how was M1 selected??]

14

Physical assay: Biacore surface plasmon resonance to measure binding kinetics

253 pM0.23 nM

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

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