A more supervised version of DNA shuffling
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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) Joshua Silverman, et al & Willem Pim C Stemmer Avidia, Inc.

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Strategy: Create therapeutic proteins by combining hundreds of known binding

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Strategy create therapeutic proteins by combining hundreds of known binding

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

A misnomer; really domain shuffling)

Strategy:

Create therapeutic proteins by combining hundreds of known binding

domains from receptor proteins in new random combinations and selecting

for binding to a specific target by phage display


Strategy create therapeutic proteins by combining hundreds of known binding

Organization of binding domains in typical mammalian receptors

A-domains:(~35-40 AA’s/domain):

determine binding speficicity of many receptors

Typical receptor structures

Library of 217 A-domains

(~metaphorically?)

as a spacer

between domains

Dual specificity domain

Bipartite domain

2 domains cooperating

Degenerate oligos synthesized to coding for 35-40 AAs of the A domains

Only AA’s naturally found at each position were coded for.

Conserved structural AAs were kept constant (blue, yellow).

Complexity = 1023 .Actually realized = 1010 as phage display particles

Select one domain at a time, serially, by panning:

LRP = LDL receptor related protein


Strategy create therapeutic proteins by combining hundreds of known binding

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 proteins from first cycle, cloned and tested for IL6 binding; 20 top binders pursued.

Add the domain library to each of the 20 first round winning domains. Again pick best 20 overall.

After a third cycle pick the very best binder: = “C326”

IL6 = interleukin 6

One domain

Three domains

Two domains

M13 phage

Monomer displayed

on phage coat

Monomer protein

Screened for

binding

Build 20 dimer pools

from 20 best monomers combined with the same library again

etc. to trimer


Strategy create therapeutic proteins by combining hundreds of known binding

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


Strategy create therapeutic proteins by combining hundreds of known binding

Binding measured by a competition assay (“AlphaScreen”)

General scheme

Reactive oxygen species can react only over a short distance with and “acceptor” bead

Laser

Luminescence

Reactive Oxygen

IL6 receptor

IL6

Avidin bead: biotinylated IL6 : gp130-Fc : Protein A bead

Competition: IL6 (non-biotinylated) or C326 avimer


Strategy create therapeutic proteins by combining hundreds of known binding

More AlphaScreens: effect of combining the 3 domains


Strategy create therapeutic proteins by combining hundreds of known binding

Physical assay: Biacore surface plasmon resonance to measure binding kinetics


Strategy create therapeutic proteins by combining hundreds of known binding

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


Strategy create therapeutic proteins by combining hundreds of known binding

Acute phase inflammatory response induced by IL6 is reversed by avimer C326

(in mice)

Specific for IL6-induced inflammation


Strategy create therapeutic proteins by combining hundreds of known binding

RNA

Topics:

  • 1.) Pre-mRNA splicing basics

  • Splicing-based therapy

  • RNAi


Strategy create therapeutic proteins by combining hundreds of known binding

Pre-mRNA splicing


Strategy create therapeutic proteins by combining hundreds of known binding

Intron = 80 nts to 100,000 nts

Pre-mRNA

Branch point

Phosphotriester

Lariat

mRNA


Strategy create therapeutic proteins by combining hundreds of known binding

ATP

ATP

The spliceosome(5 smalll RNAs + 100-300 proteins)

ATP

Intron becomes a lariat

ATP

degraded

http://www.swbic.org/education/comp-bio/intron.htm


Strategy create therapeutic proteins by combining hundreds of known binding

(= “acceptor” site)

(= “donor” site)


Strategy create therapeutic proteins by combining hundreds of known binding

Finding exons in a sea of introns

TTCTCAGTCCTAAACAGGGTAATGGACTGGGGCTGAATCACATGAAGGCAAGGTCAGATTTTTATTATTATGCACATCTAGCTTGAAAATTTTCTGTTAAGTCAATTACAGTGAAAAACCTTACCTGGTATTGAATGCTTGCATTGTATGTCTGGCTATTCTGTGTTTTTATTTTAAAATTATAATATCAAAATATTTGTGTTATAAAATATTCTAACTATGGAGGCCATAAACAAGAAGACTAAAGTTCTCTCCTTTCAGCCTTCTGTACACATTTCTTCTCAAGCACTGGCCTATGCATGTATACTATATGCAAAAGTACATATATACATTTATATTTTAACGTATGAGTATAGTTTTAAATGTTATTGGACACTTTTAATATTAGTGTGTCTAGAGCTATCTAATATATTTTAAAGGTTGCATAGCATTCTGTCTTATGGAGATACCATAACTGATTTAACCAGTCCACTATTGATAGACACTATTTTGTTCTTACCGACTGTACTAGAAGAAACATTCTTTTACATGTTTGGTACTTGTTCAGCTTTATTCAAGTGGAATTTCTGGGTCAAGGGGAAAGAGTTTATTGAATATTTTGGTATTGCCAAATTTTCCTCTAAGAAGTTGAATCATTTTATACTCCTGATGTTATATGAGAGTACCTTTCTCTTCACAATTTGTCTCTTTTTTTTTTTTTTTTGAGACAAGGTCTCTGTTGCCCAGGCTGGGGTGCAGTGCAGCAGAATGATCACAGTTCACTGCAGTCTCAACCTCCTGGGTTCAAGCGATCCTTCCACCTCAGCCTCCTGAGTAGCTGGGACTATAGGTGTGCGCCACCACTCCCAGCTAATATTTTTATTTTGTAGAAACAGGGTTCGCCATGTTACCCAGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCACTGGCCCAGTTTCTACAGTCTCTCTTAATATTGTATATTATCCAGAAAATTTCATTTAATCAGAACCTGCCAGTCTGATAGGTGAAAATGGTATCTTGTTTTTATTTGCATTTAAAAAAAATTATGATAGTGGTATGCTTGGTTTTTTTGAAGGTATCAAATTTTTTACCTTATGAAACATGAGGGCAAAGGATGTGATACGTGGAAGATTTAAAAAAAATTTTTAATGCATTTTTTTGAGACAAGGTCTTGCTCTATTGTCCAGGCTGGAGTGCAGTGGCACAATCACAGTTCACTCCAGCCTCAACATCCTGCACTAAAGTGATTTTCCCACCTCACCTCTCAAGTAGCTGGGACTACAGGTACATGCTACCATGCCTGGCTAATTTTTTTTTTTTTGCAGGCATGGGGTCTCACTATATTGCCCAGGTTGGTGTGGAAGTTTAATGACTAAGAGGTGTTTGTTATAAAGTTTAATGTATGAAACTTTCTATTAAATTCCTGATTTTATTTCTGTAGGACTGAACGTCTTGCTCGAGATGTGATGAAGGAGATGGGAGGCCATCACATTGTAGCCCTCTGTGTGCTCAAGGGGGGCTATAAATTCTTTGCTGACCTGCTGGATTACATCAAAGCACTGAATAGAAATAGTGATAGATCCATTCCTATGACTGTAGATTTTATCAGACTGAAGAGCTATTGTGTGAGTATATTTAATATATGATTCTTTTTAGTGGCAACAGTAGGTTTTCTTATATTTTCTTTGAATCTCTGCAAACCATACTTGCTTTCATTTCACTTGGTTACAGTGAGATTTTTCTAACATATTCACTAGTACTTTACATCAAAGCCAATACTGTTTTTTTAAAACTAGTCACCTTGGAGGATATATACTTATTTTACAGGTGTGTGTGGTTTTTTAAATAAACTCCTTTTAGGAATTGCTGTTGGGACTTGGGATACTTTTTTCACTATACATACTGGTGACAGATACCCTCTCTTGAGCTACATCGGTTTGTGGGGAGTCAAAAGTCCTTTGGAGCTAGGTTTGACAAATAAGGTGGGTTAACACTTGTTTCCTAGAAAGCACATGGAGAGCTAGAGTATTGGCGAATTGAAGAAATCCCCCTTTTTTTTTAACACACTTAAGAAAGGGGACTGCAGGTATACTCAAGAGAGTAAGTCGCACCAGAAACCACTTTTGATCCACAGTCTGCCTGTGTCACACAATTGAAATGCATCACAACATTGACACTGTGGATGAAACAAAATCAGTGTGAATTTTAGTAGTGAATTTCATTCATAATTTGATCGTGCAAACGTTTGATTTTTATTACTTTAGACTATTGTTTCTGATTTTATGTTGGGTTGGTATTTCCTGTGAGTTACTGTTTTACCTTTAAAATAGGAATTTTTCATACTCTTCAAAGATTAGAACAAATGTCCAGTTTTTGCTGTTTCATGAATGAGTCCTGTCCATCTTTGTAGAAACTCGCCTTATGTTCACATTTTTATTGAGAATAAGACCACTTATCTACATTTAACTATCAACCTCATCCTCTCCATTAATCATCTATTTTAGTGACCCAAGTTTTTGACCTTTTCCATGTTTACATCAATCCTGTAGGTGATTGGGCAGCCATTTAAGTATTATTATAGACATTTTCACTATCCCATTAAAACCCTTTATGCCCATACATCATAACACTACTTCCTACCCATAAGCTCCTTTTAACTTGTTAAAGTCTTGCTTGAATTAAAGACTTGTTTAAACACAAAATTTAGACTTTTACTCAACAAAAGTGATTGATTGATTGATTGATTGATTGATGGTTTACAGTAGGACTTCATTCTAGTCATTATAGCTGCTGGCAGTATAACTGGCCAGCCTTTAATACATTGCTGCTTAGAGTCAAAGCATGTACTTTAGAGTTGGTATGATTTATCTTTTTGGTCTTCTATAGCCTCCTTCCCCATCCCCATCAGTCTTAATCAGTCTTGTTACGTTATGACTAATCTTTGGGGATTGTGCAGAATGTTATTTTAGATAAGCAAAAACGAGCAAAATAGGGGAGTTTAACTTTAATATTTTCTTTTAAAAAGCATTTCATGTTATAAGATCAATTCTGAGTGGTAGAAAATGCTTTGACATTTTATTTCCATTTTCTACTTTTAGTTTTTTTCCTATTTGTTTAAGATCTTAGAGGATTATTAAGCTGAACTCCTCAACTGATAAAAAGCATGACATCTTAAACATAAGCAAAGCATATTTTTAGGTTAATTTTCACATAGAAAACAGTTTATTTTATGTGAAATTCTATGTAGATATACTATTTTTTTGGTATTTATTGATATGTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTTATTTATTTATTTTTTTTTTTGAGACAGAGTCTCACTCTGTTGCCCAGGCTGGAGTGCAGTGGCATGATCGTAGCTCACTGCAACCTCCACTCCCGGGTTCAAGCAATTCTTCTGTCTCAGCCTCCCGAGTAGCTGGGACTACAGGTGCCTGCCACTATGCCCGGCTAATTTTTGTGTTTTTAGTAGAGATGGGGTTTCACCTTGTTGGTCAGGCTGGTCTCGAACCCCTGACCTCAGGTGATCCACCCACCTCAGCCTCCCAAAGTGCTGGGATTATAGGCATGAGCCACGTGCCCGGCCGACATGTTAATTTTTTAAAAAAGGCTTTACTGGGGTATATTTTATATAATATAATAATCACATGTTTTAACTATACAATTCCAAGCTTTTTAGTATATTTATAGGGCTATGCAAGGAAGATATACTGTTAAACAGTAGAAATTGAGAAAGCTCTTCTGATAATATCTCTTGATTTGATGATGGCTCATGCCTGTAATCTCAGTGCTTTGGAAGGCCAAGACAGCAGAATCACTTGAGGCCAGGGGTTCGAGACCAGCCTGGGCAACACAGCAATACCCTATCTTTACAAATAATAAAAATATCTGTTGATTTGAAGTAAAGTTTTTTTTTAAAGACAAGGTCTCATTCTGTCACCCAGGCTGGAATGCAGTAGCAAGATCACAGCTCACTGTGGCCTTGACCTTCTGGGCTCAAGTGATTCTCCCACTTCGGCCTCCCGAGTAGCTGGGACTAACAGGTGTGCACCACCATGGCTGGCTAATTTTTTTTTATGTTTGTAGAGATTGGGTCTTACTGTGTTGCCCAGGCTGATCCCGAACTCCTGGGCTCAAGCAGTCTTCCTGCCTCAGCCTCTAAAATTGCTGGGATTACAGGCTTGAGTCACCATGCCCAGCCTGAAGTAGCATTTCTACCCTGTTTAATAATTCAGCAGCTTGTCATGTAAGATATTCATATATGCATATAAACATTAGGCAGCTTAATTTGGTAAAACTGTAAAATGGAAATTTTAAATTGTTTGCAGCATCAATAACATTGATGTCAGTATGATTTTTACATGCTGATCTTGACCAATTTGAAACAGTGAGTTAAAATCTGGCTGATCCGTACTAATCCTAAAGAAATATTCTATGAACTATTAAATGTTTCCAGAATATATAAAGAAACATTATGATGTCAACACACCCATCTATTTTTTTTTGGAAATAAAAACTCCATTTTTCTTATTAAAGAAAACATGCTTATTAGAAAACATACGGCTGGGTGCAGTGGCACACATGTAATTCCAGTGCTTTGGGAGATCGAGGTGGGAGAATCACTTGAGGCCAGGAGTTTGAGACCAGCCTAGACAACATAATGAGACCCCCTCTCTACACAAAAAGAATTAGTTGTGCATGGTGGCGTGCACCTGTAGTCCCAGCTACTTGGGAGGCAGAGGCAGGAGCATCCCTTGAGCCTAGGAGTTTGAGACTGCAGGAGTTCGAGACTGAGTGGAATGCAGTGGAACTGCATTCCAGCCTGAGTGACAGAGGGAGACCCTGTCTTAAAAAAATAAGAAAGAAAACACAACTGCAGAAAATTATAAAGGATTTAAGTCATTCCAAATATCACTGCCACTTTTTATTTAGAATATTCTAAAGAATTCTCTCTCTGTGTACACACACACATATGCGTACTCTTAATCCAAGTAGCTTGGTAGGATTTTATTTACCTAGTGCCTAGATGGGAAATTGCCTGGGGATTCCAAATACCTATTTCATTAAATTAAAGATGTCACTGATTTTAAGACTTAACACTATTTTTCATACTGCCAAGAAAGAAAACACTACCAGTTATAAATGTAAATTGCCATCAATTGTAATACATCAATTTTAGAGCTATTATTAATAAAATGTGAATGTGCATCTTAGAGCAATGAAATATAGTACTATATATTTGATGACCTTTTCTGCCCTGTGATATTCAGAAAGTGAAAGTTAAATATGGGCTGAGCATGGTGGCTCACACCTGTAATCCCAGTACTTTGGGAAGTCAAGACGGGAGGCTGGCTTGAACCCAGGAGTTCAAGACCAGCCTAGGCAATGTAGCGAGACGCCATCTCAAAATATTAAAAATAAGTAAATAAGTAAATAAAAAGAAGGTTAAGTATACAAATGTATTTCCTTTGTTGTGAATTTATTTCAATTTTATAGTGATTTTTTTTTTTTGAGACGAAGTCTCACTCTTGTCCCCCAGGCTGGAGTGCGATGGCGTGATCTCAGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCTATACTCCTGCCTTGGCCCCCCGAGTAGCTGGGATTACAGGCGCCTGCTACCATGCCTGGCTAATTTTTGTATTTTTAGTTGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTAGAACTCTTGACCTCTGGTGATCCACCCGCCTCGGACTCCCAAAATGCTGGGATTACAGGCGTGAGCCACCGTGCCTGGCCAGTGGTTTTTTGTTGTTGTTGTTGTTGTTTTGTTTTGTTTTTGTTTTTGTTTTTGTTTTGAGACAGGATCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGTGCCATCTTGGTTCACTGCAACCTCTGCGTGGGCTCAAGCAATCCTCCCACCTCCCTTTCCAGAGTAGCGGGGACCACAGGTGTGTGCCACCACACCTGACTAATTTTTGCATTTTTTTTTGTAGAAACAGGGTTTTGCCATGTTGCCCAGGTTGGTCTGAAACTCCTGAGCTCAAACAATCCAACTGCCTTGGCTTCCCTAAGTGAAATTACAGGCATGGGCCACTGTACCCAGTCTAGTGATTTTTTTATTTTTATTTTTATTTTATTTTATTTTATTTTTTTACCAAAAAAACAACAAAGCCTCAGGAGGAAAAGTTGATACACAAGTAAATTTTATTGGAAATGTTTTTGTGTGGACCTTAAGCAGAGGGAAAATTAGTCTGCATTATGGTGTATCCAGACTAAATGACTGATATTAAAATGAAATTATTCTTAGGATTTGCAATCTTAGAGAAAACTTTTTCATTTTTATTTTTTTGAGTTACAAATTATCTTCATTTACATTTGAGAACAGTGAGTCACAGAGGGATTAAGTAACTTACTCAAGATCATACAAGTCTTTGATTTGAACCCAATCTTTTAACTCTGCAGAACTCAGAGTCACTCTTATTTGGAAAAACTTTTTAACTGATGTGGATCCTCTAATATGGGCTTCCTATTATTCATTCTCTATTAGTCAGAAGTTTTGCAAGCAGACAGAATTCATTTTGCCAATTACGGGATTTTCCCTCAGTTGCAGTCAAGGTTCATAAAACTATAACTCTTTATCTTTAATTAGAAATGTTTTTTTTTTTGAGACAAGGTCTTGCTCTGTTGCCCAGACTGGAATGCAGTGGCATAGTGGCCCATTGCAGCTTTGAACTCCTGGGCTCAAGGGATCCTCTGCCTCAGCCTCCCAAGTATCTGAGACTACAAGTGCGTGCCATCACCCATGGCTATTTTAAAAAAAAAAAAAATTGTAGAGATAGGGTCTTGCTGTGTTGCCCAGGCTGGTCTCAAACTCCTGGTCTCAAGCAATCCTTCTGCCTTGGTCTCCCAAAGTGCTGAGATTACAGGTGTCAGCCGTTGCACCTGGCCAAAACGATAACTTAAAATACACACACACACACACACACACAAACACATATGTGTATTTGTGTGTGTGTGTGTGTGTGTGTGTCTCAAAAGGTATCAAAAGAGAATAGCTATAACTTTAGTGTTGATCTTGATAGTGACTTGATTAGGCTCTGTTTAACATCAAAGATGCAAATTAATACTTTCTTTGAACATATTAAAAATGCAGAAAATATTGGAGTATTTTATTTTAAATAAATTGTATTCTGTATATTTAAGGTATACAACATGATGTTATGGGATACATATAGGTGGTTAAAAGATTACTGCAGTGAAGCAAATTAACGTATCCCTCAACTCACATAGTTACCCATTTTTTTTTTGTTTTGGTGGCAAGAGGAGCTTAAAATCTCATTTAGTGTGAATCCCAAATACAGCACAATTTTATTACCTATATACTTCATGTTGTACATTATATTTCTAGACTTGTTCATCCTACATATCTGCTACTTTGTATCCTCTGAGCTACATCTCCCCATTTTCTCACTTGCCCCCCAAGTAGTTTCTTAAAGTGTCTCATGTAAGAGGGCAGTAGCTTTCAGCTTAAACTTTTTCTCTGTATGTAGTCGATTTCTTTGAGGTATACTTTTCTCTCCAGAATAGTTAGATGTAGGTATACCACTTTGATGTTGACACTAGTTTACCTAGAACTTATCTTCTGTAAATCTGTCTCTATTTCCATCTCTGTCTCCATCTTTGTCTCTATCTCTATCTGTCTATCTCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTATCTAAAGCAAATTCATGCCCTTCTCCTATTTATTGAATCGAGACCATAGACAGGGGTGAGAGAAAGAATTTGGCAGGAATGGGGATGTGTATTATCTGTGGCATAAGGAAACTTTACAGAACTAGGTTCAAAAGTATACTTTCTAGTTCTTTCCCATGGCTTTTCACTTTGATGTAGTCCTTATCAGGTAACTGAGGTTTTATATAAGTCCCCTGATTCTTAGAACATGAAGGTGTAGTAGTCAAGGTTGGTCCCTTGAAACCACAAATTTTGTGAAAAAAAATTAAGAAAATTTGAATAATTTCCTCAGCAAATACATATTGATCATCTGTTATACAGCCATGAGAAGTGGTTCTGTTGCACACGTTTATTTTATCAGATCCTAATCCCAAACCAGGCATAAAATGGAAACCATGAAGATAGGATGAAATAACTTCTGAATGTTTGAATGTTTGAAAATAGTGTACTTAAAAATACCAGGTGGTTTTTGTTTGTTTTTTGTTTTTTTCTTTTTTTGAGACAGGGTCTCACTCTGTCACCCAGGCTGGAGTGTAGTGGTGCAATCTCATCTCATTGCAGTCTTGACCTCCCAGGCTCAGGTTATCTCCCACCTCAGCCTCCCAAGTAGCTGGGACTACAGGCACATGCCACCACGCCCAGCTAATTTTTTGTATTTTTTGTAGAGACGGGGTTTCACCCTGTTGCCCAGGCTGGTCTAGAACTCCTGGGCTTAAGCGATCCTCCCACCTCAGCCTCCCAAAGTGCTAGGATTACAGGCATGAGCCACCATGCCTGGCAGAAAATACCAGGTTTTTAAGTATCAGCACTTACTCTTCAATCTTTTCTATTACTATGTTGTGCTAAATGGTATTTTTTATTTAATTAGAGCAATGCTGTTCAATAGAACTTTCTTTGAGGATGGAAATCTTTTATGTTTCTGCTATGTGGTACAGAGCCACTAGTGACATGTGGCTTTTGAGCGCTTGACACATCTTGTGCAACACAGGAACTGAATTTTTAAGTAATTTATATTGCCACATGTGGCTACCGTATGGGACAGTGTAGTACTAGATGATCTGTAAGGGCTGTGCTTCATCAGTGTCGTTTTTTAACTGACAAAAACCTTTAGTTTTTTTTTTAGTAATGTGTTTATTTAAAAGAATTCATAAAATACAAGTAAACAAATTAACTTGTTACCTGAGCATATGTCCTTTCATACTTATTTTTTCTGCATACATATTTTGGAAAATGGAATATCTGCCCCTTTTTTTTTATCTGAGATACAGTCTACCTCTAAAAATACATGATTCTAACATTCTCACTTTTTGTTGGCATTTGATCAGGGTATAGAAAAACAGTTAAAAGGACAGAGAATGGTTGAGAGATTATGATATGAAGAGAAAATGTGATTGAGTGTGGTAGACTTGGGGCCTGCTTGAATGTTGAGAGAATGACTGTTTTCCGATAAAAAAAAAAAGTCCATTCTAGGATCCTAAAAGAAGGGTCTGAAGTTCACTGCAGAAAGCAAGCTACATAGTACTAAGCCACTAAGGGGACATGGAGCCCTTAGTAATTCCTACCTTAGTAATAGTCTCATCATGCCCTCTTGGGAACCCAGCCTTGTTGATTAGCCTCTCTGCTTTCTCTCCTTATAGTTCAACCTCCCTGTTTGTTCCAAGCAGTTCTTTTCCTGCCCATTTATTATGCATTTCTATACAGCTTTCCTCCTCTTTTTCTATACCATGCTGCAGTTCTTATTGCTACCTAGAGGTTTTCAAAATTCCTAGGGGCGGATAAGTAGGCATAAACAAAGTTCTTCCCTATTATCCTTCCTATTTTTTCACCTAGACTGAAGAGGTAGACAAAATAGAAATAAAGACATTAAGGGTATGTGTTTGTAGTCCCAAAGAGCTTCTCTGGCAATTTTGATGTAGTTGACAGTGACGCTCTGAGTTCAGGACAGATTGGACTCCTTGGCTGAGAGGAGTGAGGAGATAGGACGGTAGAGGAGAGGGTAGAGCAACTCTGGAGGAAGCTTTCCCCTCACCTTTGCCAGTCCTGTTATCCTAGACTTAACCATAATTAAAGATGAGGGAGGCACTCAGTAAAGGGATCTAGTGGGAAGCTTGTTCCAGACAGCCAAGGAGGGAGGTTCGCGCAGTTCCTTTGGCCACCCAGGTGGGGTAATTGATCCATGTATGCCATTCATGTACAATGTAGGCACTTATACCTGTATTCCAATGTAGTGAACTATACCATTACTCTTAAATTAATATTCTTTATTAGCTTCCATGGTGGCTATAGGCCAGGCAAGAGAGTTAAGAAAAAATAAATAGCCAGGTATGGTGACTCAAGCCTGTAATCTCGGCACTTTAGGAGGCCGAGGCAGGAGGATAGCTTGAGTCCAGGAGTTCAAGACCAGCCTGAGCAAAATAGTGAGATCCTGTCTCTATTTTTTAAAAAAGCCTTGGGGCAAACAGGAGTATGGAGGTTTGGATGCTAATAGAACAGCAGTGTCTTACTGCTTGGAGTTCTCTTGTTTCTTGTCCTATCACCGTAGCCTTTGGATCACAGCAATTTTTCCATGACTCCATACTTTTCAGTTCTTGAATATTTTTTCCTTTATTCCTCTTGTCTCTGTAAAGACATCAACTGGAGTTGGACTGTAATACCAGGTATCTCCAGAAGATGGCACTATTTAACAGATTTTATAAATAATTTGATGTGAGTCACTGTCATCTGAAGCTTGTTGCCTTTTCTTTCTTTCTTCTTTCTTTTTTTTCCCCATCAATTCTGTATGTTTGAAATGCTGGGATTTAAGTTAGTTAGAATAAGGGATGTCTGTAATTTCCCTAAATTGAGAAGTAATATGCAAAGGTTGATATCAGAAGTCATATGCTCACCTTGCAACACCAAATAATACTGGCCCATTTGTGATTTTTGAAAGTAACACTCCATAATAAATGGATGTATATATAGAAGCATAACAAAAATAGAAGCACATAAAAGTGAAAAGTCTCATAAACGCCATTGTCACTACTCATGTAATTGCTGTTACAAATTTGTTTAAATGTTGAATAAAAATGGTGTCATAGGCAACACAGTGTTCCACTACTTGGTGTTTTTAATAGCATTATTCTGTCTCAGTGTGCTTTGGATTATCAGGTGCTTTTTAATAGTTGCATGGTATTACATTGTGTAGATGAACTTGATTAATTTAAATGGTTCCCTGTTAATGGACATGTTGGTTTGTTTTTGTGAACAACTGATACAGTGAACATTTATTTTTTAAATAAAAAAAAGAGAGACAGGGTCTTGCTGTGTTTCTCGGGCTGGCCTTGAACTCCTGGGGTCAAGCGATCGTCTTGCCTCTGCCTCCCTGGGATTACAGGCATGAAGCCACCGCACCCGGCCCAGTGAACACTCTTGAATGTATCTTTGTATACTTGTCAAGTGTTTTTGTAGCAATTGATTCCCAGAAGTGGGAATTACATGGAATTAAGTGACATGCATGTTTGCAATTTTAACAGGTATTGCTATGTCATTTTCAAAAGAAGCTATGCCAATTAATACTCTCACCAACAAGAGTGCTTATTTCCCCTCAGCATATTATCAGGCTTAAGTTTTGCCAGTATGGGTGGGAGAACAGTAGAATCACATTGTTTTAGTGTTTGTTTCTCAGATAGATATAATTTTACACCTTATAACCTTCTCTTCTATAAATTGTCTATTTGTGTTCATTCTCCATTTTCCTATGGGTTCTTATTGTTGGAGCCCAATATATAAAAGGGGGTATTTGTTACAGAACCTCTTCAGTTTTGGTTCATGTCATGCCTGGGTTTTTACCCTTTCTACGGATGTTAAAAAAAATTCTCTATTTTCTTCCAGTCCACTTATGGCTTTATTTTTTACATTTAGATTTTAATCCGTCTGGAATTTATTTTTGTGTATGCTGTGAGGTAGGGACCATACTTTTATTTTTTCCCAAATGGGTTACTAGTTGGCCAAACATCATTTATTGAATAATTCATCTTTTCCCTACTGACTCGAAATACCATCTTTATTGTATACTAAATCCTCATATAGTTCTGGGTCTGTTTCTGGGCTCTACTTTGTTCATTTACTGTGCTGGTACTGCACCGTTGTAATTGCTGTGGCTTTGTGGTATGGTATGGCTTGCTCTCTGCTAGGGCAAGTCGAAGCTCTTTTGTTCACCTGCTCTTTCACCCAAATTTTCTGTCCTGAATCCAGCACAGCCAAATTATGGTCATTGTCACCACCAACTACAGTGGGTGTTGAGCATTTCCCATTGAATCTCCTGTAAGGGTTTTATTGGATTCTGTGATAGCAGTAAAATGGGAGCCTAAGAGGTATTCCTTAAAGGACTACTAATCAGACCTGGTTTCCCAGATGATGCTGAAGATGACGGGGCCTGGGCTAGACTTTTGAGGGACATATCCTTGGGGTTGGGTGTGATATAGACCAGCCCTTACAATTTGCTTGACTCATGGGAATCGTACAGGGCCAGAACCAGACACCTGTCATGCTAATAACTTCCCTCACAATTCAGAAATCACTGTGATTGAAGATGGGTGGCTGTTATAATACTACCCACTTAAAAATGGATGTAACCCATTTTTTAGGACTCTTAAAAACATCAAATCAGTAATGGCCGATTAGGACTTTTTAATTTTTACTAATCTCTACTTGAAAGTTTTCTAGTCATTCATTTCAGGAAACCTAATTCTTATAATTCATATCATTTAGAATATCATAATGCTATGGATATTAGCTAGCTAACTTCTCAAATCTTCTAGTTCTCATTTAATTTGAAGTTTGTGTGTGTACATAAGGATATACATATACATATGTGTGTGTAGATATATATATATATAGTTTTTTTTTTTTTAACTAGAATGACCAGTCAACAGGGGACATAAAAGTAATTGGTGGAGATGATCTCTCAACTTTAACTGGAAAGGTATGTATCTTGAAAGGGAAGAAAAAAAAGCACTTCATACCGAGTCAATTAGTAACAGTGTGCTTTCAATCAATCACTAAGAGATAATTTACATAGTATAACTAAATGGGTTATTTAACCCTTGGAAGCAGTCTAGGTTAATTATCGTTCCCTAGGTCATGTAGTAAAAAGACAGTAGAATCCAACATTAACCTTAAATGTCCATATTGTCAAGTACTGCTGTCTGCCTCTGTGGGACTCTAATTTGGGATCCTTCAAAAAACATTGATGGGGGAAAAGATAGCCTTTAAAAAAAAAAAAAAAACAAACCTATGTGAGTCTATGTGAGGTAGACTCACATAGTTTCCTAAAAGATAGCAAAGCAGTATTATGTAGTGGCTGAAAGTGTGAGTTCCGGAGCCTGACAACTGATTCAAAGCATGGCTTAGTACTTCCTAACTCTGACCTTGGGCAAGTTACTTAACCTCTCTGTGTCCCATATGTGATTAGGGTGAGGTTGATAATAGCAGCCATAGAGTTAAGAGGATTAAGTGCTATAATGCAAGTAGAGCTCTTACAACAGTTTCTGGTAAATCACTCAATAAATTCAGACATACTATTATTTTAAGAAATCTCAAAGAGTTTTCTTGTACCTTAAAATTCTCCTAGTGTGAACCATTGGTTTTGGTATATTGTGCTTCCATGTAGTTTAATATCAAGATGTTTTTAGATTTCCCTTTTAATTTATTTGTTGACCCATTGGTTGTTCAGGAGCATGCTGTTTACCTGAAAATAATGGAGATATTAAGGTATTTGAATATTTATCTTCTAGTACATTGAAAAACTTTTTGAGAGTAACCAATAATAAATGATGGAATGCTACTGCTTTTTTTTTTTGAAGCTGCCAGTTATTGTTTACTTACACTATGCCAAATATAAAGGCATTAATCTCATAAAAGTTTCACAACAATCCTGTGAGGGAGACGATATCCCCATTTTACAAATCAGGAAATTAAGACTTAATAAGGTTAAAAGACTTGCCCCAAAGTCACAGAACCAGTAAGTGGTAGAGCTTGAATTTGAATACAGACCTGACTCTAAAGCTCTTTTCTTTCTTTAGATTTTAGTGTTCATTGCTTACTTGAATGAGTATCTATAAGAAAACTTTAACATGTAAAACTTCTGTGAAATTATCTTGTCCCATATCAGGGTCATGTCAAACTAATGTCCTCCTCAGCATCTTTGGAAAACTTCAGAGGAGAAATGAGCTTTGCCCCTCCTGTTCATTTCCTATTCCACTAGGAGACCTGTCCTTCCCTTTCAGCATGCTTTGTCCATATTTAGAAGCTGTTGAAGCCATTACTTGTCTGGTCAGTTTTTAGTGCTGGAATGGACCTAGCCTTTTAGGCCTTCTGAGATTTAGTTTGATCTCGTCTTTCCCACCTAATGGCTCTGTTCTACTACATAGATTTGATCTGAAACAGTTCTCTGTTTCTAAAATAACTTTCTTTTCATGATAGTCACAGTAAAGTACATTTATTATGGAAAAATCAATAAGTATAACGAGTGAAAGTTATTTCTTGGTGGTAAGATTATGGGATTATTTGAACTTTCTGTTTCATTGTATTTTATTTATTTATTTATTTTTGTGATGGAGTCTCACTCTGCTGCCCAGGCTGGAGTGCAGTAGTACGATCTTGGCTCACTGCAACCTCCCCTTCCCAGTTCAAGTGATTCTCCTGCCTCAGACTCCCAAGTAGCTGGGATTACAGGCGCACGCCACCATGCCTGGCTAATTTTTTTATCTTTAGTAGAGACAGGGTTTCACCATGTTGACCAGGCTGATCTCCAACTCCTGATCTCAGGTATCCACCTGCCTCAGCCTCCCAAAGTACCGGGATTACGGGTGTGAGCCACCCTGCCTGGCCTCATTTTGTCTTTTGGGGGTATTTTTGTGTGCAGATATATATGTATATAAATATTTTTCCCTCTTTTCCCCAGTTAGTATTTGAGCAGATGAACTTTGGACCCGAATACCTGTATTCAAGTCTCTAATACCACTTCTTGGCTATTTTCATTTTATCAAATGGCCTCTTATCCTCGTTTTTCTCATTTATTAAGTAGAGATGTAACTACTTGATATAATTCAAAAACTCAATAATGGCATTCTTTTGTTTTTTAGACTCTAGTGTCTGTACTCCTTGTACCATGCTGGGATTCATTTGAACAATTGCATGGCTTTTTTAGTGTATTATTAAATTTGCAGTTTACTTAGAATTTACTGGGACCTCATACAAATGGGAAAAAAACATAACTGTGTTACTCATTTGCTGTGTGCCTTTGGATTGACCCTATTTTTTGTATTCATTTTCTCCCCATGTCCTGAGTTCCACTTTGAATAAAAAAGTAATTTTTTTCCTGCCTGTAAAATAGGCTACCAATAGGCTGCAGTTGTCTATAGTAGCTGCTTCACTGAGGAGAGCTCAGCATGAGAGAAATAGTATGAATTGCTTGCCACAAGTTATGGGCTAGCCTTACTTCATTCTGTACTTGGACCTGTTTAGGCTTCTAAGAGATCTTACCTCCAACAATAAACTGCTTTGAGACATGAAAAGGTGGAAGCTTTACTTGGTTATAACTTTACTTTTAATACCTAGAACAGTGAGTCTTCAAACTTGTATTTGCATGCCCAATTTATAAAAAGTTTCCTGAGCATTTACCCCTAATATATGCATTTTAAATTATATATGATTTATGGTAATAATAATATATATGTTACAAAATACATACAAAAATATAGATTAAACAAGGTGAGGTTAAAAAATTTAAAAGTTCTAATCTTTCTTGCAAACCAGTGGATCTTTTGTGCCTTACTCTGGTAAACACTGTCTTAGAAGAATATATAGAACATTAAAATCTTAATGCTATAGTTATATGACAGAGTATGATGAGAGCTACAGATAAACAACACATCATGAATCTTCTTGTGGCAGTGTTTATAACCATTATGTGAAATGCTGCCTCATTCTTATAACTAGCATAAGAACAGATAGGACTTTCTCGATTTTGAGGGGTAATTATTAGATGGTATTTTCTGTTAAGGACTCTTCCAGCTATAAAATTCTTAAATGTAGAAAGCGAAGTGAGGGTTTATGGTGAGAGGAAGCATTGGTATCATGTTTTAGTGTAGTCCAAGAATATGGACACATCCAGAAAATGCAGATCAAGTTTAGCCTAATGAGAAAATATATTTTGGAGTCCATATGGTAAATTAAATTATGTGATTTTTGAGTTATTGTACAAATATAATTCTTAGAATGTTAGAGTCAGGAGACTATAAGAGACCAACTGCTTCAAGTTTCATTTAACACATGGGAAACTAAGGCGAGAGAAATTTCAAGACTTGCCCAAGATTAGACCTCTTGTTAAGTAATGAAAGTGTTTTAAAAACAGGTGGGTCAAATTCTGTTTTTAAAATTTCCATTATGATGAAAATTTCAGTATTACAGGCTTCCAAATCCCAGCAGATGGGCCACTTGTTTAAAGGAGAGTTTGATATAATAAAGCATCTAAAAACAAGAGTTTGGATAATTCCTTAGGGTTGTTATGATGTGATTTGACTTATAATTGGAAATACCGTTTTATTCATTGTACTGATTTTCATTTCTCTTTTTCTTCTAGAATGTCTTGATTGTGGAAGTAAGTTCACATTTACTTTTAATATAACATTTATGACTTTTCTAACTTAGTATGCACCATCCTAAAGGTAAGCCAGGGAGAGAAATTCCTCTGCATCAGTTTTAATGGTGGGCTTGTGTTCTAAAGGAGTGAGATTGGTTTTTTGTAAAGACTACTTAGTAATTTGTTTTTACCAATAATGGAATGGTATACTTCCTACCTCTCTTTTTTTAGTTTGAAGTATTTTCTTTCTAAACATAACTCTCTCTCTCTATTTATCTATATATAATATATACATATATATCTTATATTTTATGTATATATATATATATCTTGCTTAGATTTTGTCTTATGTAATATTTGGTACATAAAAAATAATATTTATAATTTATAGACTATTTTCCATGTGTTATTATGTGCTAAAGTATTTTGTATCTTAGCACCGAGAGGCTAAGCAGTTTCCTAGGGTTACCAGCTAGTAAACTAAGGGAAACCTTTACTTCCTTTAGCTCAGTGGTTCTCAAAATGTGGTTCCCTAGACCAAAAGTATTAATATCAGACAAGAACCTACCGAATCAAAATATCTGTGATGAGGCCCAGCAAGCTATGCTTTAACAAGTTTCCGAGTGATTCTGATGCATGCTAAGGTTTAGGATCCCTTGTTTTTACTCATAAGTCACTTTCTCATTAAGGCCTTCCCTGGCCATCCTATATAAAATCTCATGTTTTCACACCGTCAACTTCGTATTCCTCCTCAATACTTTTATTTTCCTGATCACTTATCACTAACAGCCTCTCTCTCTCTCTCTCTCTCTCTCTATGTATATATATATATATATCACTTATCACTGTCTAACAGCCTCTCTTTATATATATATAATCTATAGATTATATATATATGCAGCATTGTGCAATCATTATCACGCTCAATTTTAAAACATTTTCATTTCCCCACAAAGAAACCCAATCCCCTTAGCCATCACTCCCAATTTTCCCTTCCCCCAGCACCTAGCAAACTGATCATCTACCTACTTGCTGTCTATAAGATTTGCCTATTCTGGACATTTTGTATAAATAGAATCATACAATATGTGGCCTTTTGTATCTGGCTTCTCTCACTTAATGTTTTCAAGGTTCATTCATGTTGTGGAGTATATCTGCACTCATTTCCTTTTTATTGCCAAATTGTATGGATAGACAGGTGTTCCTCAACTGTGTCCTGATAAACCCATCTGAAGTTGAAAATATCATAAGTTGAAAATGGATTTACTACTTTGATAAATCTATCCTAAAGTCAGAAAAATCTCATGTTGGAACCATCGTAAGTTGGATACCATCTGAATTACATTTTTGTTATCCATTCACTGGTTGACAGACGTTAGGTTGTTTCCACTGATGCTCCTTATTTCTCGTACCTGAAATGTCCTTATTCCCTCCCTTCTTATCCCATGTTTAAGTCATTTAAGACCCAGCTCAAACGTCACCTCCACAAAACCTTCCTTGATACCCCTTTCCTCTTCAATTCACTTGGACCTTTTGCATTTAATTTTAATTTTTATTTTTTTTAAGACAGAGTCTCACTCTGTCACCAGGCTGGAGTGCAGTGGTATGATCTCAGCTCACTAACTACTCTGCCTCCCAGGTTCAAGCAATTCTCATGTCTCAGCCTCCCAAGTAGCTGGGACTACAGGTGTGCGCCACCATGCCTGGCTAATTGTGTGTGTGTGTGTGTGTATGTATGTATGTATATATGTGTGTGTGTGTATATATATATATACACAAACATATATAAATATATATACATATATATATATACACACATATATAAATATATATACATATATATATATACACACACACACACATATATATATATATAGTTTTTTTTTTTTTAAGTAGAGATGGGGTTTTGCCATGTTGGCCAGGCTGGTCTGGCCTCAAGCCATCCTCCCACCTCGGCCTCGCAAAGTGCTGGTATTATAGGCATGAGCCACTGTGCCTGGCCTGCATTTCATTTTAATTATAAAATATTTTGAACTCAGAAAAAAGGGTATGCTGAATACCTACGTACCCACAAAAGTATTAACATTTTGCCATATTTGCTTCTGATCTTATTTTTTTTGAGAAATTAAAGATCATAATACAACTAAAGCCCCATTTCTTTCCCTTCATTCCCAGAAGTATGACAATTATCCTTAAAGTTGATATATATCATTCCCATGCATGTTTTTTATACTTCCCTAGTACAAGTTAGCTGTATCCTCTGCTCAGGGGCTCATCAAGCTGAATCAAGGGACTCATGATCCTCTTCAAAGTTCCTTCAGGTTGTTGGCAGAATTTAGTTCCTTGTGATTGTAGGACTGAGGGCCCGTTTTCTCACTGGCTGCTGGCCAGGGGTTGCTCCCAGATATTTAAAGGCTCATGCCCTAGCCCATGACAGTCTCACAACATGGCAGCTGACTTCTTCAAAACCAGCAGGAGAATCTTGCTCTAGTCTACCACATAACCTAATCACAGGAGCGGCTATCCCGTTATTTTCACAGATCCTGGTCACATTCAAGGGGAGGGAACCCTTCTGTGTGTGTACACCAGGAGGCAGGAATTTTTTTTTTCTTTTTCTTTTTTGTTAAAAAGTCTTAAAGTCTTTTATCCCTAAAGGAGGCAGGAATTTTGAGAGCCATCAGAATTCTGCCTACCACAGCCCAGAAATCTGCATTTTTCACAAGTCTCCAGCCATGATGTTTCTGATGGCTCACACTGCTTTATTCCATTTTTAAAGAGTATTTTTATTGAAAAGCATTAGGGTTATGGTTTAAAAAATATTTTCCCTAACAAAGATGGGTTTGTTTAGAGTCCTACTTTTGACTAAATAGCTGAGATTCACTTTTATGTAAAGTTCATTTTATAGCGTTATTAATTTGGGTGCCTTTAAAAATAGTATAAAGCATGTTTCTCGAGTGTAGTCTGTTAGCCACCTATATTGGAGAGTTGGGAGGAGAGAGTCTCTATCTTGAATTTATGGGAAAAATTCTAAAATACTTTTTATAATGAAGGACAACATCATAACTCCCTAATAAAATGTGCATGTATATATTCAAATTTGCTGTCATTGATCCTGCACCTACAAAATCCAGTCCTGGGGGCTGGCATTCTTACTGCTTGCTGAGGGCCAGATGATATAGATTCCAGAATATCTCCATGTAGATTTTGGTGAGAATTACTGTGCTGAAAAGAATGACAGTATTGCAGTTATACATGGGGGTTTTGGTACTTTATATTGTGACTCTGAATTTAAAGCTATGCAATGTCTTCTTTTTTGAAAGGATATAATTGACACTGGCAAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAAAGATGGTCAAGGTCGCAAGGTATGTATGACATTTTGACACAGAATATTTTCCTCATTTGAAGGGGGATTAAGTGATTGCTTCTTTTTAAGGATAAATGTTTTCAACTGTCATTTTATCTTCGAAAAGTAATGTAATCTCATATAAGACTTAAGATATAATCCTTTTAAATAATTTTGTCATGTGTTAATAAAGCTCATAATTACAGTCACTTCCTTGCTAATATTAACATTTGGTTTTCAGCATGCTAATTATATCAGTTTGTCCTGAATAGCATGGCAGAGGATTTTGGGCCCCCTTGCAAAATTAAGAATAAGGATTCCAAAGCGGGTGAGGAAGTGATAGGAAGGGGTGGGCCCTGAAGATCTGGACCTCCTGGAATTGAGTGATGAATGCTGCATCTTCTTTGTGTCTGTAGTGAAATTTTATAATGCCTGCTTCCTTTTTTATTAAGTCGGCCTCACCTCCTCACCTTACCTATGCTGTTTTACTTTTGCTTTTATAGTTCTACCTGTGTTTATTTCTCATTTTCGTTTCATCTCTCAACAACTCTGGGGTGGCATTATTATTCCCACTTTTCAGATAAGGTTACTGAGGCATAGGGAATTGTCCAAAGGTACAGAGCTAGTCCGCTATAGAGATGAGATTTGAACCCAGGGAACCTGGCTCACAGTTTATGCTTTTGCCTACCTTAAGTTTTTAATAGAGTGACATCAAACAAACATTTAAGAATATGTTTTTCTTTTCCTTTTATAATTTCATTAAAAACATTAAGTCTCTGATCAGTCTGCAGTTTTTATGTAGGGGTCAGGTAATGTTCTAACTTCTGCTTTTTCCTAAGTGATTAACAGGTTTTTATAAGCCCTTTTGAAAAAATCACGGTATCTGTCGAGCATCTTTGAATCAGAGTAAGCCTTCTAGTGAGTCATATGTCAGCAGTTTGACTGTATGGGCTTTTCTAATATCCAGTTCAAGTGTTTATCAGTGAGTTTTTCTTTTAAATAGATTTGGGACAGGTACTATGAGAGTATATAAGTGATACGTTATAGGACACTAACTAGTATCCTATGAAATGGCAAAAACTGCAATCACTTTTGCACCAACCAAATAGAAACTAATCAGTGCACTTGCTTATTTTTCTACATGCTCTTTAGGGTTTTAAATGTCAACCTACTGTGGCATAGACTTTAATCCTCTGGGTATTCTTTTGTTGTTCTTTCCTGGTATATGCTGTGGAATTGAGATAGACTGGTTCGTGAGCGAGAGATTTTGTGTTGCCACAGGTAGGACATGCTCAAACAATACTTGGGTCATTTCTTGACCCAAGTCATCTATTCACCATAGTTTTGTAGCACCGATCTTGCATACATTTCATGTATCTTCTTTGAACCCCACGTCAGTGCTGCTTATATGATACTCAGAAATTAAACACTAAGGAATAAGATTTTCAGGTAGGATTGAGTTTTGGAGGGTCACAAATCTTGTAATGTCTAATATTTCCACTCTCCCTGCTGAGAATTAGTTTTGGCTTCCTTGGAGGTGATATCGCCTCTGTTGAGTATAAGTGGCCTACTGTGATCACACCACTGCACTCCAGCCTGGGTGACAGAGTGAGACCCTGTCTCAGAAAAAAAAAAAAAAAAAAAGAATGCATGGCCTAGATGACTTCTAAGGTTTTTCCCACCCAGTTCCAGTTTTCATGTTCTAGGCAGAGCAGTAAAGTGAGAAACACATGGACTTGGGAGTTTAGTCTCGCATTTCACTGCCACTTAATCTGAGCGACTATTCCATATTTAATCTCTCTGAATGTATTTACTCATCTTTAAAGGGGAATGATTATTAACATCTTTTTCTCAGGGAAACTATATGAGTCAAGGAGATAATATATTTGAAAATCTTTTTAACTGCAAAGCGCTGTTTCACTGTTGGTTATAATGTGATTGATCTCATTGTAGTGAGCAGCTGCTTAATTGCGTTTTAGAATGTAGGGAAGATAGTAATATTTTTCACATTATATATGTAGCTGGTTCTGGAACTGTAAACATACTCCTTTTTTATGGAGATCTGAGTCACGTACCATAAAATTCACTCTTTTAAAGTTGTACAATCCAGTGGTTTTTGATATATTCAGAGTTGTGCATCTGCTACCACTATTTCATTTTGGAACCCAAAGAAACCTTGTACCCATTAGCAGTCATTCTCCCTTCTCCCAGCCCCTGGCAACTACTAATCTACTTTCTACAGAAAGTCCGTACAGATTTGTGTATTATGGACATTCCATATAAATGGACTCATGCAATATCCTGTCTTCTTTCACTTAGCATAGTGTTTTCAAGGTTCATCTAGGTTGGGGCATGTATCAGTACTTCATCCCTTGTTTTGGCTGAATAATATTTCATTGTACAAATATATCACATTTTGCTTATCCATCTGTTGGTGAACATTTGAGTTTCTACCTGTTGGCTTTTATGAATAATGTTGATTTGAATGTTTGTGTACAAGTATGAATACCTGTTTTCAGGTCTCTTGAGTATATAGTTGCTAGGTCATATAGTAACTCTGTGTTTAACATTTTGAGGAATTGCCCGACTATTTAACAAGGTATATGTACTGTTTTACACCAGTAACATATGAGGGTTCCAATATCTCCACATCCTTGACAACACTTGTTACTGTCCTTTTTATTGTAGCCATCCTAGTGGCTATGATGTGGTATCTCATTGTGGTTTTGATTTGTGTTTCTCTGATGCTGATGATGTTGAACATGTTTTCATCTGCTTATTGGCCATTTACATATATCTTCTTAAGAACGGTTACCCATTTACAGTATGGAAAATGCTTCAGATGCAACTCTAGTCATGCCTTAGAGATGGAGCTTTATTAAACATTCAGATCTCTAGGCATATGAAGTGCTGAGTTCTCTTGAACTCCTAATACAGATTGCACTGAGTTTAGTGATACCTTTTCTGGAGCATTCCTGAGTTCAGGTAGGGAGAAGGGTTTTTGCTGTGATTGGCTTGTTATGTTCTTTCTAAATGGAAATAGAATTGAAGTGTCTCCTCTCTCCATTTA


Strategy create therapeutic proteins by combining hundreds of known binding

Some types of alternative splicing

Alt. 3’ SS

Alt. 5’ SS

Exon skipping

|pA

|pA

Different termination


Strategy create therapeutic proteins by combining hundreds of known binding

Alternative splicing:

Occurs in almost all of the 25,000 human gene transcripts


Alternative splicing in the alpha tropomyosin gene 7 isoforms

Alternative splicing in the alpha-tropomyosin gene (7 isoforms)

Similar proteins but subtly different to suit different tissues


Strategy create therapeutic proteins by combining hundreds of known binding

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


Strategy create therapeutic proteins by combining hundreds of known binding

A cautionary note:

95% of human genes show evidence of alternative splicing

Low levels could be simply mistakes.

Or genes trying out new exons to see if they are useful, or give them a chancew to become useful (through mutation, evolution)

But there are still a very large number documented cases so there is no doubt that alternative splicing greatly increases the complexity of the mamalian proteome.


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)

Many human genetic diseases are caused by mutations causing missplicing


Strategy create therapeutic proteins by combining hundreds of known binding

Therapeutic intervention at the level of pre-mRNA splicing

Alternative splicing

Unwanted alternative = included

Use antisense  skipped

Bias alternative splicing

Against an unwanted isoform (e.g., Bcl-X alt. spl.:

Bcl-XS = promotes apoptosis;

Bcl-XL = inhibits apoptosis and promotes cell growth, cancer)

Pseudo exon activated  disease

Antisense = block and skip

unwanted pseudo exon

Alternative 5’ splicing

Unwanted = longer exon

Antisense  shorter isoform


Strategy create therapeutic proteins by combining hundreds of known binding

Nonsense mutation

Antisense-induced skipping

d

x

Expendable exon (e.g., protein with many repeated domains)

Exon must be multiple of 3 in length to maintain reading frame after skipping


Strategy create therapeutic proteins by combining hundreds of known binding

Therapeutic intervention at the level of pre-mRNA splicing

  • Interfere with improper splicing caused by splice site creation or activation

  • E.g., beta-thalassemia (R. Kole) in which a splice site has been created by a mutation

  • 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


Strategy create therapeutic proteins by combining hundreds of known binding

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


Strategy create therapeutic proteins by combining hundreds of known binding

Sazani P, et al. and Kole R.

Systemically delivered antisense oligomers upregulate gene expression in mouse tissues

Nat Biotechnol. 2002 Dec;20(12):1228-33.

EGFP: Enhanced green fluorescent protein = model system

Antisense “RNA” injected into tail vein, RNA was modified for stability

Mutant globin intron has activated splice sites

Actin promoter, universally expressed. Exon skipping yields green fluorescence


Strategy create therapeutic proteins by combining hundreds of known binding

RNA modification for stabilization

Instead of deoxyribose or ribose

Modified phosphate

Still base pairs OK


Strategy create therapeutic proteins by combining hundreds of known binding

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)


Strategy create therapeutic proteins by combining hundreds of known binding

RNA modification

Also can add 2’ MOE

-O-CH2-CH2-O-CH3

MOE = methoxyethyl -

Phosphorothioate deoxyoligonucleotides


Strategy create therapeutic proteins by combining hundreds of known binding

No antisense:

Antisense treatment in

cell cultures (ex vivo) from themouse with the mutant EGFP gene

Control oligo (C)(50 nt downstream)was ineffective.

Max. effect = 40%


Strategy create therapeutic proteins by combining hundreds of known binding

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)

Deliver antisense to ends of exon with the nonsense mutation in mdx mice (model for Duchenne’s) to promote the skipping of the nonsense-bearing exon and so avoid truncation of the protein .

Use AAV (adeno-associated virus) to deliver the antisense gene

Measure:

mRNA with skipped exon

dystrophin protein

muscle histochemistry for dystrophin


Strategy create therapeutic proteins by combining hundreds of known binding

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


Strategy create therapeutic proteins by combining hundreds of known binding

U7 promoter

Consensus binding site

for Sm proteins (to target to pre-mRNA)

compl. to splice donor site

compl. to branch

Double target synergistic (loop?) (Kole)

U7: normally hybridizes with seq. at 3’ end of histone mRNAs to effect cleavage;

Binds 2 Sm proteins; in coiled (Cajal) bodies (RNA processing centers?); low concentrations (1000’s of molecules per cell)

U7OPT: Change Sm binding site to consensus for all snRNAs (spliceosomal, for delivery there);

high copy no.; no longer in coiled bodies;

Now include anti-splice site segments as well. In permanent transfectants can effect > 50% inactivation of a globin cryptic site.

Gorman, L., Suter, D., Emerick, V., Schumperli, D. & Kole, R.. Proc Natl Acad Sci U S A95, 4929-34 (1998).


Strategy create therapeutic proteins by combining hundreds of known binding

Expression of U7

antisense construct

RT-PCR

U7OPT-A.S.

Endog. U7

(slow onset =conclude slow mRNA turnover)

0 2 4 6 13 weeks

Splicing assay (RT-PCR)

Skip exon 23, after 2-4 wks.

0 2 4 6 8 13 weeks

normal

Dystrophin protein

(Western)


Strategy create therapeutic proteins by combining hundreds of known binding

dystrophin

Muscle immuno-histochemistry

dystrophin-associated antigens

Normal

Untreated mdx

Treated mdx

Top,

middle ,and

bottom


Strategy create therapeutic proteins by combining hundreds of known binding

RNAi = RNA interference

Short double stranded RNA molecules trigger the

degradation of the complementary sequence in the cell,

and can inhibit translation of the targeted mRNA

Their introduction into a cell can greatly reduce any protein whose mRNA

is targeted.

Inhibition is usually incomplete in mammalian cells

Thus “gene knockdown” as opposed to knock-out

Alternative technologies:

Antisense RNA: block translation or splicing

Ribozymes: RNAs that cleave other RNAs, sequence specifically


Strategy create therapeutic proteins by combining hundreds of known binding

siRNA = short interfering RNA

miRNA = microRNA

naturally occurring siRNA

(Primary transcript)

2 nt overhangs

(RNA-induced

silencing complex)

Single-stranded RNA

More common

No cleavage if imperfectly complementary, but translation inhibition

Cleavage if perfectly complementary

Protect against viral RNA, repetitive sequence transcripts


Strategy create therapeutic proteins by combining hundreds of known binding

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

Cessation of protein synthesis via activation of PKR (protein kinase RNA-activated), and phosphorylation of eIF2

Global degradation of mRNA (without any sequence specificity, RNase L activation)

Spread to neighboring cells (induction and secretion of interferon)

Most small DS RNAs do not trigger this response(<30 bp)


Strategy create therapeutic proteins by combining hundreds of known binding

Generation of siRNA in vitro

Chemical synthesis, annealing of 22-mers (bypasses dicing by Dicer)

T7-mediated in vitro transcription of each complementary strand. Anneal to make long DS RNA and transfer to cells. Let Dicer make siRNA in the cell

Also, can use controlled RNase to generatefragments (cheaper)

Introduce perfect hairpin RNA into cells,

let Dicer make siRNA

Introduce imperfect hairpin RNA into cells(based on mRNA sequence) and

let Dicer make miRNA


Strategy create therapeutic proteins by combining hundreds of known binding

Limitations of siRNA silencing in mammalian cells

Transient nature of the response (~3 days)

Transfection problems (cell type, refractoriness)

Non-renewable nature of siRNAs ($$)


Strategy create therapeutic proteins by combining hundreds of known binding

Generation of siRNA in vivo (can give permanent knockdown)

Not good for interferon-

responsive cells

Allow trans-association

(TTTTT acts as a terminator)

Most common,

using U6 or H1 promoter

U6 = small nuclear RNA used for splicing.H1 = RNA element of RNase P, used in tRNA processing.

(Pol III)

(Pol II)

miRNA


Strategy create therapeutic proteins by combining hundreds of known binding

Potential determinants of efficient siRNA-directed gene silencing

siRNA

Incorporation into the RNA-inducing silencing complex (RISC); stability in RISC.

Base-pairing with mRNA.

Cleavage of mRNA.

mRNA

Base-pairing with siRNA.

The position of the siRNA-binding target region.

Secondary and tertiary structures in mRNA.

Binding of mRNA-associated proteins.

The rate of mRNA translation.

The number of polysomes that are associated with translating mRNA.

The abundance and half-life of mRNA.

The subcellular location of mRNA.

Delivery

Transfection (lipofection, electroporation, hydrodynamic injection (mouse))

Virus infection (esp. lentivirus (e.g., retrovirus like HIV that can integrate into non-dividing cells)


Strategy create therapeutic proteins by combining hundreds of known binding

Some applications:

Target oncogene Ras V12 (G12V) – silenced mutant ras without silencing the WT allele. Reduced the oncogenic phenotype (soft agar growth, tumor formation in nude mice)

T-lymphocytes infected with anti-CCR5 RNA

lower levels of this HIV receptor, and lower levels of infection (5-7X)

Target an enzyme in mouse ES cells with a hairpin vector, Isolate a knockdown, make a mouse.

Mouse shows same knockdown phenotype in its cells.

So can target the whole mammalian organism,

Just inject a GFP silencer gene into single cell embryos of a GFP mouse:

Can find a chimeric GFP mouse with reduced GFP

Progeny carry it in the germ line,

Get a complete knockdown mouse, without ES cells (easier)


Strategy create therapeutic proteins by combining hundreds of known binding

Delivery inan intact organism

Hydrodynamic injection (sudden large volume) of straight siRNA (no vector) into the tail vein of a newborn mouse

Get silencing of co-injected luciferase vector in a variety of tissues

High throughput siRNA for gene discovery

C.elegans, 19,000 genes

Make a library 17,000 siRNA genes in plasmids in E. Coli.

Feed the clones of E. coli to the worms.

Look for phenotypes.

1700 genes examined for phenotypes (as of 2005)

(e.g., fat metabolism phenotypes found)


Strategy create therapeutic proteins by combining hundreds of known binding

Systemic RNAi: worms, plants, mammals

In plants, get permanent post-transcriptional gene silencing (PTGS, transcriptional level)

Worms: effect can last though several generations

Amplified by reverse transcriptase

Influx/efflux via a specific transmembrane protein (in worms)

Raisons d’etre?

Infection, many viruses go through a DS RNA phase.

Repeat element silencing? (1 million Alus, + others  half the human genome)

Transcribed in either direction, so could form DS RNA, then RNAi inhibits action of SS ‘mRNA”


Strategy create therapeutic proteins by combining hundreds of known binding

Discovery of RNA interference using double-stranded RNA

Nature (1998) 391: 806

Discovered RNAi as they tracked down the effective agent in antisense experiments

(DS RNA contaminating their SS antisense preparations had all the inhibitory activity)

Paper characterized by nice controls and variations:

Several genes, whole animal phenotype, protein product (GFP), RNA level (in situs)

Phenotype of null mutant is specifically mimicked.

Introns and promoter sequences ineffective.

DS RNA from a different sequence + SS antisense RNA vs. the target: ineffective

DS RNA linked (chimeric molecule) to a single stranded portion vs, the target: ineffective

Transport of DS RNA between cells and amplification implied.


Strategy create therapeutic proteins by combining hundreds of known binding

In situ hybridizations

No probe

No RNA injected

SS

antisense

RNA

DS RNA

Transcript disappears (RNA degraded)


Strategy create therapeutic proteins by combining hundreds of known binding

p. 173

Alnylam Pharmaceuticals Inc.

Target : Apolipoprotein B, involved in binding cholesterol to low density lipoproteins (LDLs)

Made in liver and jejunum.

An important factor for high serum cholesterol and atherosclerosis.

Tested ~80 siRNAs for reduction of ApoB mRNA in a hepatoma cell line in culture.

Used 2 best.

Stabilized the siRNA by:

1) Substituted sulfur for a hydroxyl oxygen at the 3’ end linkage (phosphorothioate)

2) Added some methyl groups to the sugars of the last 2 nucleotides

3) Conjugated cholesterol to the 3’ end. This dramatically improved serum half-life and efficacy.

[Promoted entry into cells as well?]

Injected into tail veins of mice.


Strategy create therapeutic proteins by combining hundreds of known binding

Recovery of siRNA from injected mice.

RPA = RNase protection assay

cholesterol-conjugated siRNA

non-conjugated siRNA

Control RNAs

Inject once a day for 3 days,

measure 24 h after last injection

60-70% reduction in

Apolipoprotein B mRNA


Strategy create therapeutic proteins by combining hundreds of known binding

Plasma ApoB

Plasma cholesterol


Strategy create therapeutic proteins by combining hundreds of known binding

Some therapeutic targets of RNA interference

localized target

ss RNA virus target

Dominant allele target

(can be made allele-specific)


Strategy create therapeutic proteins by combining hundreds of known binding

Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol. Sunilkumar et al. and Keerti S. Rathore. PNAS (2006) 103: 18054

Cotton: 20 million cotton farmers, in Asia and Africa.

For every 1 kg of fiber, plant  1.65 kg seed = 21% oil, 23% protein.

BUT:

Seed contains the terpenoid gossypol:

Which protects the plant from infections,

But which is:

cardiotoxic and hepatotoxic

Oil is OK, but protein is contaminated with gossypol

44 million metric tons of cottonseed produced each year  9.4 million tons of protein. Enough to satisfy the protein requirement of 500 million people.

Terpenoid-negative cotton mutants are susceptible to infection and so are not commercially viable.


Strategy create therapeutic proteins by combining hundreds of known binding

Delta-cadinene synthase

Target the mRNA specifying the first step in gossypol synthesis


Strategy create therapeutic proteins by combining hundreds of known binding

Recombinant plasmid T1 grows in the bacteria Agrobacterium tumefaciens

which can be used as a vector for plant transfection

shRNA

neo gene

terminator

 alpha-globulin promoter, active only in seeds

The T-DNA region of the binary vector pAGP-iHP-dCS. Arrows indicate the primers used in the PCR analyses.

RB-right T-DNA border

tOCS:octopine synthase terminator

dCS: 604-bp d-cadinene synthase sequence

pAGP: cotton a-globulin promoter (seed specific)

pNOS: nopaline synthase promoter

nptII:neomycin phosphotransferase II

tNOS: nopaline synthase terminator

LB: left T-DNA border.

dCS = delta-cadinene synthase


Strategy create therapeutic proteins by combining hundreds of known binding

NEO-RESISTANT TRANSFECTANT PLANTS

Ten seeds from two transgenic plants from F1 of selfed matings

Gossypol level

0.1 ug/mg

PCR for transgene

Note transgene-null segregants have normal gossypol levels


Strategy create therapeutic proteins by combining hundreds of known binding

HPLC (high performance liquid chromatography)

Null segregant

Spots on seed indicate terpenoid glands


Strategy create therapeutic proteins by combining hundreds of known binding

RT-PCR assay for the mRNA for the enzyme delta-cadinene synthase:

Low to undetectable levels in the siRNA knocked-down plants

PCR of DNA for transgene


Strategy create therapeutic proteins by combining hundreds of known binding

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

WT Transfectant1 Transfectant 2


Strategy create therapeutic proteins by combining hundreds of known binding

Low gossypol level analyzed through two generations of one homozygous plant were stable at 0.19 ug/mg +/- 0.013 (SEM, 50 seeds).

WHO limit for human consumption is 0.6 ug/mg, so OK.

Other plants could be similarly targeted:

Lathyrus sativus, grass pea, a hardy tropical/subtropical legume plant (neurotoxin = beta-N-oxalylamino-L-alanine)

Fava beans, cassava beans: toxins = cyanogenic and contains fava glycosides (toxic to people with low levels of the enzymes glucose-6-phosphate dehydrogenease (G6PD), which is common.

fava bean

cassava bean


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