1' RNAi

1
RNA Topics

• 1.) RNAi
• Pre-mRNA splicing basics
• Splicing-based therapy

2
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
3
siRNA short interfering RNA
miRNA microRNA naturally occurring
2 nt overhangs
(RNA-induced silencing complex)
Protect against viral RNA, repetitive sequence
transcripts
4
Introduction of long DS RNA into mammalian cells
will trigger the interferon response Cessation
of protein synthesis via activation of PKR
(protein kinase RNA), and phosphorylation of
eIF2 Global degradation of mRNA (without any
sequence specificity, RNase L activation) Spread
to neighboring cells (induction and secrtion of
interferon) Most small DS RNAs do not trigger
this response(lt30 bp)
5
Generation of siRNA in vitro
Chemical synthesis, annealing of 22-mers
(bypasses Dicer)
Introduce long DS RNA into cells, let Dicer make
siRNA
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
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Limitations of siRNA silencing in mammalian cells
Transient nature of the response (3 days)
Transfection problems (cell type, refractoriness)
Non-renewable nature of siRNAs ()
7
Generation of siRNA in vivo (can give permanent
knockdown)
Not good for interferon- response cells
Trans-association (TTTTT acts as a terminator)
Most common, using U6 or H1 promoter
miRNA
8
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)
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Some recent 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 ? 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)
10
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
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. So far 1700 genes examined for
phenotypes (e.g., fat metabolism)
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Systemic RNAi worms, plants, mammals In
plants, get permanent post-transcriptional gene
silencing (PTGS, transcriptional level) Worms
effect can last though several generations Amplifi
ed by reverse transcriptase Influx/efflux via a
specific transmembrane protein (in worms)
Raison detre? 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, RNAi inhibits action of SS mRNA
12
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) Note 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.
13
(No Transcript)
14
Target GFP- lacZ fusion
Mag. of b
lt 1 cell/animal
Nuclear GFP
Mitochondrial GFP
Both GFP mRNAs knocked out (or down) (for mitoch
localization and for nuclear localization).
Works targeting the lacZ mRNA sequences as well
15
Works at a distance from the siteof injection
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In situ hybridizations
No RNA injected
No probe
SS antisense RNA
DS RNA
Transcript disappears (RNA degraded)
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Therapeutic targets of RNA interference
localized target
ss RNA virus target
Dominant allele target
18
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.
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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
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Plasma ApoB
Plasma cholesterol
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Pre-mRNA splicing
22
ATP
ATP
ATP
ATP
http//www.swbic.org/education/comp-bio/intron.htm
23
(No Transcript)
24
Finding exons in a sea of introns
TTCTCAGTCCTAAACAGGGTAATGGACTGGGGCTGAATCACATGAAGGCA
AGGTCAGATTTTTATTATTATGCACATCTAGCTTGAAAATTTTCTGTTAA
GTCAATTACAGTGAAAAACCTTACCTGGTATTGAATGCTTGCATTGTATG
TCTGGCTATTCTGTGTTTTTATTTTAAAATTATAATATCAAAATATTTGT
GTTATAAAATATTCTAACTATGGAGGCCATAAACAAGAAGACTAAAGTTC
TCTCCTTTCAGCCTTCTGTACACATTTCTTCTCAAGCACTGGCCTATGCA
TGTATACTATATGCAAAAGTACATATATACATTTATATTTTAACGTATGA
GTATAGTTTTAAATGTTATTGGACACTTTTAATATTAGTGTGTCTAGAGC
TATCTAATATATTTTAAAGGTTGCATAGCATTCTGTCTTATGGAGATACC
ATAACTGATTTAACCAGTCCACTATTGATAGACACTATTTTGTTCTTACC
GACTGTACTAGAAGAAACATTCTTTTACATGTTTGGTACTTGTTCAGCTT
TATTCAAGTGGAATTTCTGGGTCAAGGGGAAAGAGTTTATTGAATATTTT
GGTATTGCCAAATTTTCCTCTAAGAAGTTGAATCATTTTATACTCCTGAT
GTTATATGAGAGTACCTTTCTCTTCACAATTTGTCTCTTTTTTTTTTTTT
TTTGAGACAAGGTCTCTGTTGCCCAGGCTGGGGTGCAGTGCAGCAGAATG
ATCACAGTTCACTGCAGTCTCAACCTCCTGGGTTCAAGCGATCCTTCCAC
CTCAGCCTCCTGAGTAGCTGGGACTATAGGTGTGCGCCACCACTCCCAGC
TAATATTTTTATTTTGTAGAAACAGGGTTCGCCATGTTACCCAGCCTCCC
AAAGTGCTGGGATTACAGGCATGAGCCACTGGCCCAGTTTCTACAGTCTC
TCTTAATATTGTATATTATCCAGAAAATTTCATTTAATCAGAACCTGCCA
GTCTGATAGGTGAAAATGGTATCTTGTTTTTATTTGCATTTAAAAAAAAT
TATGATAGTGGTATGCTTGGTTTTTTTGAAGGTATCAAATTTTTTACCTT
ATGAAACATGAGGGCAAAGGATGTGATACGTGGAAGATTTAAAAAAAATT
TTTAATGCATTTTTTTGAGACAAGGTCTTGCTCTATTGTCCAGGCTGGAG
TGCAGTGGCACAATCACAGTTCACTCCAGCCTCAACATCCTGCACTAAAG
TGATTTTCCCACCTCACCTCTCAAGTAGCTGGGACTACAGGTACATGCTA
CCATGCCTGGCTAATTTTTTTTTTTTTGCAGGCATGGGGTCTCACTATAT
TGCCCAGGTTGGTGTGGAAGTTTAATGACTAAGAGGTGTTTGTTATAAAG
TTTAATGTATGAAACTTTCTATTAAATTCCTGATTTTATTTCTGTAGGAC
TGAACGTCTTGCTCGAGATGTGATGAAGGAGATGGGAGGCCATCACATTG
TAGCCCTCTGTGTGCTCAAGGGGGGCTATAAATTCTTTGCTGACCTGCTG
GATTACATCAAAGCACTGAATAGAAATAGTGATAGATCCATTCCTATGAC
TGTAGATTTTATCAGACTGAAGAGCTATTGTGTGAGTATATTTAATATAT
GATTCTTTTTAGTGGCAACAGTAGGTTTTCTTATATTTTCTTTGAATCTC
TGCAAACCATACTTGCTTTCATTTCACTTGGTTACAGTGAGATTTTTCTA
ACATATTCACTAGTACTTTACATCAAAGCCAATACTGTTTTTTTAAAACT
AGTCACCTTGGAGGATATATACTTATTTTACAGGTGTGTGTGGTTTTTTA
AATAAACTCCTTTTAGGAATTGCTGTTGGGACTTGGGATACTTTTTTCAC
TATACATACTGGTGACAGATACCCTCTCTTGAGCTACATCGGTTTGTGGG
GAGTCAAAAGTCCTTTGGAGCTAGGTTTGACAAATAAGGTGGGTTAACAC
TTGTTTCCTAGAAAGCACATGGAGAGCTAGAGTATTGGCGAATTGAAGAA
ATCCCCCTTTTTTTTTAACACACTTAAGAAAGGGGACTGCAGGTATACTC
AAGAGAGTAAGTCGCACCAGAAACCACTTTTGATCCACAGTCTGCCTGTG
TCACACAATTGAAATGCATCACAACATTGACACTGTGGATGAAACAAAAT
CAGTGTGAATTTTAGTAGTGAATTTCATTCATAATTTGATCGTGCAAACG
TTTGATTTTTATTACTTTAGACTATTGTTTCTGATTTTATGTTGGGTTGG
TATTTCCTGTGAGTTACTGTTTTACCTTTAAAATAGGAATTTTTCATACT
CTTCAAAGATTAGAACAAATGTCCAGTTTTTGCTGTTTCATGAATGAGTC
CTGTCCATCTTTGTAGAAACTCGCCTTATGTTCACATTTTTATTGAGAAT
AAGACCACTTATCTACATTTAACTATCAACCTCATCCTCTCCATTAATCA
TCTATTTTAGTGACCCAAGTTTTTGACCTTTTCCATGTTTACATCAATCC
TGTAGGTGATTGGGCAGCCATTTAAGTATTATTATAGACATTTTCACTAT
CCCATTAAAACCCTTTATGCCCATACATCATAACACTACTTCCTACCCAT
AAGCTCCTTTTAACTTGTTAAAGTCTTGCTTGAATTAAAGACTTGTTTAA
ACACAAAATTTAGACTTTTACTCAACAAAAGTGATTGATTGATTGATTGA
TTGATTGATGGTTTACAGTAGGACTTCATTCTAGTCATTATAGCTGCTGG
CAGTATAACTGGCCAGCCTTTAATACATTGCTGCTTAGAGTCAAAGCATG
TACTTTAGAGTTGGTATGATTTATCTTTTTGGTCTTCTATAGCCTCCTTC
CCCATCCCCATCAGTCTTAATCAGTCTTGTTACGTTATGACTAATCTTTG
GGGATTGTGCAGAATGTTATTTTAGATAAGCAAAAACGAGCAAAATAGGG
GAGTTTAACTTTAATATTTTCTTTTAAAAAGCATTTCATGTTATAAGATC
AATTCTGAGTGGTAGAAAATGCTTTGACATTTTATTTCCATTTTCTACTT
TTAGTTTTTTTCCTATTTGTTTAAGATCTTAGAGGATTATTAAGCTGAAC
TCCTCAACTGATAAAAAGCATGACATCTTAAACATAAGCAAAGCATATTT
TTAGGTTAATTTTCACATAGAAAACAGTTTATTTTATGTGAAATTCTATG
TAGATATACTATTTTTTTGGTATTTATTGATATGTTTATTTTATTTTATT
TTATTTTATTTTATTTTATTTTATTTTATTTATTTATTTTTTTTTTTGAG
ACAGAGTCTCACTCTGTTGCCCAGGCTGGAGTGCAGTGGCATGATCGTAG
CTCACTGCAACCTCCACTCCCGGGTTCAAGCAATTCTTCTGTCTCAGCCT
CCCGAGTAGCTGGGACTACAGGTGCCTGCCACTATGCCCGGCTAATTTTT
GTGTTTTTAGTAGAGATGGGGTTTCACCTTGTTGGTCAGGCTGGTCTCGA
ACCCCTGACCTCAGGTGATCCACCCACCTCAGCCTCCCAAAGTGCTGGGA
TTATAGGCATGAGCCACGTGCCCGGCCGACATGTTAATTTTTTAAAAAAG
GCTTTACTGGGGTATATTTTATATAATATAATAATCACATGTTTTAACTA
TACAATTCCAAGCTTTTTAGTATATTTATAGGGCTATGCAAGGAAGATAT
ACTGTTAAACAGTAGAAATTGAGAAAGCTCTTCTGATAATATCTCTTGAT
TTGATGATGGCTCATGCCTGTAATCTCAGTGCTTTGGAAGGCCAAGACAG
CAGAATCACTTGAGGCCAGGGGTTCGAGACCAGCCTGGGCAACACAGCAA
TACCCTATCTTTACAAATAATAAAAATATCTGTTGATTTGAAGTAAAGTT
TTTTTTTAAAGACAAGGTCTCATTCTGTCACCCAGGCTGGAATGCAGTAG
CAAGATCACAGCTCACTGTGGCCTTGACCTTCTGGGCTCAAGTGATTCTC
CCACTTCGGCCTCCCGAGTAGCTGGGACTAACAGGTGTGCACCACCATGG
CTGGCTAATTTTTTTTTATGTTTGTAGAGATTGGGTCTTACTGTGTTGCC
CAGGCTGATCCCGAACTCCTGGGCTCAAGCAGTCTTCCTGCCTCAGCCTC
TAAAATTGCTGGGATTACAGGCTTGAGTCACCATGCCCAGCCTGAAGTAG
CATTTCTACCCTGTTTAATAATTCAGCAGCTTGTCATGTAAGATATTCAT
ATATGCATATAAACATTAGGCAGCTTAATTTGGTAAAACTGTAAAATGGA
AATTTTAAATTGTTTGCAGCATCAATAACATTGATGTCAGTATGATTTTT
ACATGCTGATCTTGACCAATTTGAAACAGTGAGTTAAAATCTGGCTGATC
CGTACTAATCCTAAAGAAATATTCTATGAACTATTAAATGTTTCCAGAAT
ATATAAAGAAACATTATGATGTCAACACACCCATCTATTTTTTTTTGGAA
ATAAAAACTCCATTTTTCTTATTAAAGAAAACATGCTTATTAGAAAACAT
ACGGCTGGGTGCAGTGGCACACATGTAATTCCAGTGCTTTGGGAGATCGA
GGTGGGAGAATCACTTGAGGCCAGGAGTTTGAGACCAGCCTAGACAACAT
AATGAGACCCCCTCTCTACACAAAAAGAATTAGTTGTGCATGGTGGCGTG
CACCTGTAGTCCCAGCTACTTGGGAGGCAGAGGCAGGAGCATCCCTTGAG
CCTAGGAGTTTGAGACTGCAGGAGTTCGAGACTGAGTGGAATGCAGTGGA
ACTGCATTCCAGCCTGAGTGACAGAGGGAGACCCTGTCTTAAAAAAATAA
GAAAGAAAACACAACTGCAGAAAATTATAAAGGATTTAAGTCATTCCAAA
TATCACTGCCACTTTTTATTTAGAATATTCTAAAGAATTCTCTCTCTGTG
TACACACACACATATGCGTACTCTTAATCCAAGTAGCTTGGTAGGATTTT
ATTTACCTAGTGCCTAGATGGGAAATTGCCTGGGGATTCCAAATACCTAT
TTCATTAAATTAAAGATGTCACTGATTTTAAGACTTAACACTATTTTTCA
TACTGCCAAGAAAGAAAACACTACCAGTTATAAATGTAAATTGCCATCAA
TTGTAATACATCAATTTTAGAGCTATTATTAATAAAATGTGAATGTGCAT
CTTAGAGCAATGAAATATAGTACTATATATTTGATGACCTTTTCTGCCCT
GTGATATTCAGAAAGTGAAAGTTAAATATGGGCTGAGCATGGTGGCTCAC
ACCTGTAATCCCAGTACTTTGGGAAGTCAAGACGGGAGGCTGGCTTGAAC
CCAGGAGTTCAAGACCAGCCTAGGCAATGTAGCGAGACGCCATCTCAAAA
TATTAAAAATAAGTAAATAAGTAAATAAAAAGAAGGTTAAGTATACAAAT
GTATTTCCTTTGTTGTGAATTTATTTCAATTTTATAGTGATTTTTTTTTT
TTGAGACGAAGTCTCACTCTTGTCCCCCAGGCTGGAGTGCGATGGCGTGA
TCTCAGCTCACTGCAACCTCTGCCTCCCAGGTTCAAGCTATACTCCTGCC
TTGGCCCCCCGAGTAGCTGGGATTACAGGCGCCTGCTACCATGCCTGGCT
AATTTTTGTATTTTTAGTTGAGATGGGGTTTCACCATGTTGGCCAGGCTG
GTCTAGAACTCTTGACCTCTGGTGATCCACCCGCCTCGGACTCCCAAAAT
GCTGGGATTACAGGCGTGAGCCACCGTGCCTGGCCAGTGGTTTTTTGTTG
TTGTTGTTGTTGTTTTGTTTTGTTTTTGTTTTTGTTTTTGTTTTGAGACA
GGATCTTGCTCTGTCACCCAGGCTGGAGTGCAGTGGTGCCATCTTGGTTC
ACTGCAACCTCTGCGTGGGCTCAAGCAATCCTCCCACCTCCCTTTCCAGA
GTAGCGGGGACCACAGGTGTGTGCCACCACACCTGACTAATTTTTGCATT
TTTTTTTGTAGAAACAGGGTTTTGCCATGTTGCCCAGGTTGGTCTGAAAC
TCCTGAGCTCAAACAATCCAACTGCCTTGGCTTCCCTAAGTGAAATTACA
GGCATGGGCCACTGTACCCAGTCTAGTGATTTTTTTATTTTTATTTTTAT
TTTATTTTATTTTATTTTTTTACCAAAAAAACAACAAAGCCTCAGGAGGA
AAAGTTGATACACAAGTAAATTTTATTGGAAATGTTTTTGTGTGGACCTT
AAGCAGAGGGAAAATTAGTCTGCATTATGGTGTATCCAGACTAAATGACT
GATATTAAAATGAAATTATTCTTAGGATTTGCAATCTTAGAGAAAACTTT
TTCATTTTTATTTTTTTGAGTTACAAATTATCTTCATTTACATTTGAGAA
CAGTGAGTCACAGAGGGATTAAGTAACTTACTCAAGATCATACAAGTCTT
TGATTTGAACCCAATCTTTTAACTCTGCAGAACTCAGAGTCACTCTTATT
TGGAAAAACTTTTTAACTGATGTGGATCCTCTAATATGGGCTTCCTATTA
TTCATTCTCTATTAGTCAGAAGTTTTGCAAGCAGACAGAATTCATTTTGC
CAATTACGGGATTTTCCCTCAGTTGCAGTCAAGGTTCATAAAACTATAAC
TCTTTATCTTTAATTAGAAATGTTTTTTTTTTTGAGACAAGGTCTTGCTC
TGTTGCCCAGACTGGAATGCAGTGGCATAGTGGCCCATTGCAGCTTTGAA
CTCCTGGGCTCAAGGGATCCTCTGCCTCAGCCTCCCAAGTATCTGAGACT
ACAAGTGCGTGCCATCACCCATGGCTATTTTAAAAAAAAAAAAAATTGTA
GAGATAGGGTCTTGCTGTGTTGCCCAGGCTGGTCTCAAACTCCTGGTCTC
AAGCAATCCTTCTGCCTTGGTCTCCCAAAGTGCTGAGATTACAGGTGTCA
GCCGTTGCACCTGGCCAAAACGATAACTTAAAATACACACACACACACAC
ACACACAAACACATATGTGTATTTGTGTGTGTGTGTGTGTGTGTGTGTCT
CAAAAGGTATCAAAAGAGAATAGCTATAACTTTAGTGTTGATCTTGATAG
TGACTTGATTAGGCTCTGTTTAACATCAAAGATGCAAATTAATACTTTCT
TTGAACATATTAAAAATGCAGAAAATATTGGAGTATTTTATTTTAAATAA
ATTGTATTCTGTATATTTAAGGTATACAACATGATGTTATGGGATACATA
TAGGTGGTTAAAAGATTACTGCAGTGAAGCAAATTAACGTATCCCTCAAC
TCACATAGTTACCCATTTTTTTTTTGTTTTGGTGGCAAGAGGAGCTTAAA
ATCTCATTTAGTGTGAATCCCAAATACAGCACAATTTTATTACCTATATA
CTTCATGTTGTACATTATATTTCTAGACTTGTTCATCCTACATATCTGCT
ACTTTGTATCCTCTGAGCTACATCTCCCCATTTTCTCACTTGCCCCCCAA
GTAGTTTCTTAAAGTGTCTCATGTAAGAGGGCAGTAGCTTTCAGCTTAAA
CTTTTTCTCTGTATGTAGTCGATTTCTTTGAGGTATACTTTTCTCTCCAG
AATAGTTAGATGTAGGTATACCACTTTGATGTTGACACTAGTTTACCTAG
AACTTATCTTCTGTAAATCTGTCTCTATTTCCATCTCTGTCTCCATCTTT
GTCTCTATCTCTATCTGTCTATCTCTATCTATCTATCTATCTATCTATCT
ATCTATCTATCTATCTATCTATCTAAAGCAAATTCATGCCCTTCTCCTAT
TTATTGAATCGAGACCATAGACAGGGGTGAGAGAAAGAATTTGGCAGGAA
TGGGGATGTGTATTATCTGTGGCATAAGGAAACTTTACAGAACTAGGTTC
AAAAGTATACTTTCTAGTTCTTTCCCATGGCTTTTCACTTTGATGTAGTC
CTTATCAGGTAACTGAGGTTTTATATAAGTCCCCTGATTCTTAGAACATG
AAGGTGTAGTAGTCAAGGTTGGTCCCTTGAAACCACAAATTTTGTGAAAA
AAAATTAAGAAAATTTGAATAATTTCCTCAGCAAATACATATTGATCATC
TGTTATACAGCCATGAGAAGTGGTTCTGTTGCACACGTTTATTTTATCAG
ATCCTAATCCCAAACCAGGCATAAAATGGAAACCATGAAGATAGGATGAA
ATAACTTCTGAATGTTTGAATGTTTGAAAATAGTGTACTTAAAAATACCA
GGTGGTTTTTGTTTGTTTTTTGTTTTTTTCTTTTTTTGAGACAGGGTCTC
ACTCTGTCACCCAGGCTGGAGTGTAGTGGTGCAATCTCATCTCATTGCAG
TCTTGACCTCCCAGGCTCAGGTTATCTCCCACCTCAGCCTCCCAAGTAGC
TGGGACTACAGGCACATGCCACCACGCCCAGCTAATTTTTTGTATTTTTT
GTAGAGACGGGGTTTCACCCTGTTGCCCAGGCTGGTCTAGAACTCCTGGG
CTTAAGCGATCCTCCCACCTCAGCCTCCCAAAGTGCTAGGATTACAGGCA
TGAGCCACCATGCCTGGCAGAAAATACCAGGTTTTTAAGTATCAGCACTT
ACTCTTCAATCTTTTCTATTACTATGTTGTGCTAAATGGTATTTTTTATT
TAATTAGAGCAATGCTGTTCAATAGAACTTTCTTTGAGGATGGAAATCTT
TTATGTTTCTGCTATGTGGTACAGAGCCACTAGTGACATGTGGCTTTTGA
GCGCTTGACACATCTTGTGCAACACAGGAACTGAATTTTTAAGTAATTTA
TATTGCCACATGTGGCTACCGTATGGGACAGTGTAGTACTAGATGATCTG
TAAGGGCTGTGCTTCATCAGTGTCGTTTTTTAACTGACAAAAACCTTTAG
TTTTTTTTTTAGTAATGTGTTTATTTAAAAGAATTCATAAAATACAAGTA
AACAAATTAACTTGTTACCTGAGCATATGTCCTTTCATACTTATTTTTTC
TGCATACATATTTTGGAAAATGGAATATCTGCCCCTTTTTTTTTATCTGA
GATACAGTCTACCTCTAAAAATACATGATTCTAACATTCTCACTTTTTGT
TGGCATTTGATCAGGGTATAGAAAAACAGTTAAAAGGACAGAGAATGGTT
GAGAGATTATGATATGAAGAGAAAATGTGATTGAGTGTGGTAGACTTGGG
GCCTGCTTGAATGTTGAGAGAATGACTGTTTTCCGATAAAAAAAAAAAGT
CCATTCTAGGATCCTAAAAGAAGGGTCTGAAGTTCACTGCAGAAAGCAAG
CTACATAGTACTAAGCCACTAAGGGGACATGGAGCCCTTAGTAATTCCTA
CCTTAGTAATAGTCTCATCATGCCCTCTTGGGAACCCAGCCTTGTTGATT
AGCCTCTCTGCTTTCTCTCCTTATAGTTCAACCTCCCTGTTTGTTCCAAG
CAGTTCTTTTCCTGCCCATTTATTATGCATTTCTATACAGCTTTCCTCCT
CTTTTTCTATACCATGCTGCAGTTCTTATTGCTACCTAGAGGTTTTCAAA
ATTCCTAGGGGCGGATAAGTAGGCATAAACAAAGTTCTTCCCTATTATCC
TTCCTATTTTTTCACCTAGACTGAAGAGGTAGACAAAATAGAAATAAAGA
CATTAAGGGTATGTGTTTGTAGTCCCAAAGAGCTTCTCTGGCAATTTTGA
TGTAGTTGACAGTGACGCTCTGAGTTCAGGACAGATTGGACTCCTTGGCT
GAGAGGAGTGAGGAGATAGGACGGTAGAGGAGAGGGTAGAGCAACTCTGG
AGGAAGCTTTCCCCTCACCTTTGCCAGTCCTGTTATCCTAGACTTAACCA
TAATTAAAGATGAGGGAGGCACTCAGTAAAGGGATCTAGTGGGAAGCTTG
TTCCAGACAGCCAAGGAGGGAGGTTCGCGCAGTTCCTTTGGCCACCCAGG
TGGGGTAATTGATCCATGTATGCCATTCATGTACAATGTAGGCACTTATA
CCTGTATTCCAATGTAGTGAACTATACCATTACTCTTAAATTAATATTCT
TTATTAGCTTCCATGGTGGCTATAGGCCAGGCAAGAGAGTTAAGAAAAAA
TAAATAGCCAGGTATGGTGACTCAAGCCTGTAATCTCGGCACTTTAGGAG
GCCGAGGCAGGAGGATAGCTTGAGTCCAGGAGTTCAAGACCAGCCTGAGC
AAAATAGTGAGATCCTGTCTCTATTTTTTAAAAAAGCCTTGGGGCAAACA
GGAGTATGGAGGTTTGGATGCTAATAGAACAGCAGTGTCTTACTGCTTGG
AGTTCTCTTGTTTCTTGTCCTATCACCGTAGCCTTTGGATCACAGCAATT
TTTCCATGACTCCATACTTTTCAGTTCTTGAATATTTTTTCCTTTATTCC
TCTTGTCTCTGTAAAGACATCAACTGGAGTTGGACTGTAATACCAGGTAT
CTCCAGAAGATGGCACTATTTAACAGATTTTATAAATAATTTGATGTGAG
TCACTGTCATCTGAAGCTTGTTGCCTTTTCTTTCTTTCTTCTTTCTTTTT
TTTCCCCATCAATTCTGTATGTTTGAAATGCTGGGATTTAAGTTAGTTAG
AATAAGGGATGTCTGTAATTTCCCTAAATTGAGAAGTAATATGCAAAGGT
TGATATCAGAAGTCATATGCTCACCTTGCAACACCAAATAATACTGGCCC
ATTTGTGATTTTTGAAAGTAACACTCCATAATAAATGGATGTATATATAG
AAGCATAACAAAAATAGAAGCACATAAAAGTGAAAAGTCTCATAAACGCC
ATTGTCACTACTCATGTAATTGCTGTTACAAATTTGTTTAAATGTTGAAT
AAAAATGGTGTCATAGGCAACACAGTGTTCCACTACTTGGTGTTTTTAAT
AGCATTATTCTGTCTCAGTGTGCTTTGGATTATCAGGTGCTTTTTAATAG
TTGCATGGTATTACATTGTGTAGATGAACTTGATTAATTTAAATGGTTCC
CTGTTAATGGACATGTTGGTTTGTTTTTGTGAACAACTGATACAGTGAAC
ATTTATTTTTTAAATAAAAAAAAGAGAGACAGGGTCTTGCTGTGTTTCTC
GGGCTGGCCTTGAACTCCTGGGGTCAAGCGATCGTCTTGCCTCTGCCTCC
CTGGGATTACAGGCATGAAGCCACCGCACCCGGCCCAGTGAACACTCTTG
AATGTATCTTTGTATACTTGTCAAGTGTTTTTGTAGCAATTGATTCCCAG
AAGTGGGAATTACATGGAATTAAGTGACATGCATGTTTGCAATTTTAACA
GGTATTGCTATGTCATTTTCAAAAGAAGCTATGCCAATTAATACTCTCAC
CAACAAGAGTGCTTATTTCCCCTCAGCATATTATCAGGCTTAAGTTTTGC
CAGTATGGGTGGGAGAACAGTAGAATCACATTGTTTTAGTGTTTGTTTCT
CAGATAGATATAATTTTACACCTTATAACCTTCTCTTCTATAAATTGTCT
ATTTGTGTTCATTCTCCATTTTCCTATGGGTTCTTATTGTTGGAGCCCAA
TATATAAAAGGGGGTATTTGTTACAGAACCTCTTCAGTTTTGGTTCATGT
CATGCCTGGGTTTTTACCCTTTCTACGGATGTTAAAAAAAATTCTCTATT
TTCTTCCAGTCCACTTATGGCTTTATTTTTTACATTTAGATTTTAATCCG
TCTGGAATTTATTTTTGTGTATGCTGTGAGGTAGGGACCATACTTTTATT
TTTTCCCAAATGGGTTACTAGTTGGCCAAACATCATTTATTGAATAATTC
ATCTTTTCCCTACTGACTCGAAATACCATCTTTATTGTATACTAAATCCT
CATATAGTTCTGGGTCTGTTTCTGGGCTCTACTTTGTTCATTTACTGTGC
TGGTACTGCACCGTTGTAATTGCTGTGGCTTTGTGGTATGGTATGGCTTG
CTCTCTGCTAGGGCAAGTCGAAGCTCTTTTGTTCACCTGCTCTTTCACCC
AAATTTTCTGTCCTGAATCCAGCACAGCCAAATTATGGTCATTGTCACCA
CCAACTACAGTGGGTGTTGAGCATTTCCCATTGAATCTCCTGTAAGGGTT
TTATTGGATTCTGTGATAGCAGTAAAATGGGAGCCTAAGAGGTATTCCTT
AAAGGACTACTAATCAGACCTGGTTTCCCAGATGATGCTGAAGATGACGG
GGCCTGGGCTAGACTTTTGAGGGACATATCCTTGGGGTTGGGTGTGATAT
AGACCAGCCCTTACAATTTGCTTGACTCATGGGAATCGTACAGGGCCAGA
ACCAGACACCTGTCATGCTAATAACTTCCCTCACAATTCAGAAATCACTG
TGATTGAAGATGGGTGGCTGTTATAATACTACCCACTTAAAAATGGATGT
AACCCATTTTTTAGGACTCTTAAAAACATCAAATCAGTAATGGCCGATTA
GGACTTTTTAATTTTTACTAATCTCTACTTGAAAGTTTTCTAGTCATTCA
TTTCAGGAAACCTAATTCTTATAATTCATATCATTTAGAATATCATAATG
CTATGGATATTAGCTAGCTAACTTCTCAAATCTTCTAGTTCTCATTTAAT
TTGAAGTTTGTGTGTGTACATAAGGATATACATATACATATGTGTGTGTA
GATATATATATATATAGTTTTTTTTTTTTTAACTAGAATGACCAGTCAAC
AGGGGACATAAAAGTAATTGGTGGAGATGATCTCTCAACTTTAACTGGAA
AGGTATGTATCTTGAAAGGGAAGAAAAAAAAGCACTTCATACCGAGTCAA
TTAGTAACAGTGTGCTTTCAATCAATCACTAAGAGATAATTTACATAGTA
TAACTAAATGGGTTATTTAACCCTTGGAAGCAGTCTAGGTTAATTATCGT
TCCCTAGGTCATGTAGTAAAAAGACAGTAGAATCCAACATTAACCTTAAA
TGTCCATATTGTCAAGTACTGCTGTCTGCCTCTGTGGGACTCTAATTTGG
GATCCTTCAAAAAACATTGATGGGGGAAAAGATAGCCTTTAAAAAAAAAA
AAAAAACAAACCTATGTGAGTCTATGTGAGGTAGACTCACATAGTTTCCT
AAAAGATAGCAAAGCAGTATTATGTAGTGGCTGAAAGTGTGAGTTCCGGA
GCCTGACAACTGATTCAAAGCATGGCTTAGTACTTCCTAACTCTGACCTT
GGGCAAGTTACTTAACCTCTCTGTGTCCCATATGTGATTAGGGTGAGGTT
GATAATAGCAGCCATAGAGTTAAGAGGATTAAGTGCTATAATGCAAGTAG
AGCTCTTACAACAGTTTCTGGTAAATCACTCAATAAATTCAGACATACTA
TTATTTTAAGAAATCTCAAAGAGTTTTCTTGTACCTTAAAATTCTCCTAG
TGTGAACCATTGGTTTTGGTATATTGTGCTTCCATGTAGTTTAATATCAA
GATGTTTTTAGATTTCCCTTTTAATTTATTTGTTGACCCATTGGTTGTTC
AGGAGCATGCTGTTTACCTGAAAATAATGGAGATATTAAGGTATTTGAAT
ATTTATCTTCTAGTACATTGAAAAACTTTTTGAGAGTAACCAATAATAAA
TGATGGAATGCTACTGCTTTTTTTTTTTGAAGCTGCCAGTTATTGTTTAC
TTACACTATGCCAAATATAAAGGCATTAATCTCATAAAAGTTTCACAACA
ATCCTGTGAGGGAGACGATATCCCCATTTTACAAATCAGGAAATTAAGAC
TTAATAAGGTTAAAAGACTTGCCCCAAAGTCACAGAACCAGTAAGTGGTA
GAGCTTGAATTTGAATACAGACCTGACTCTAAAGCTCTTTTCTTTCTTTA
GATTTTAGTGTTCATTGCTTACTTGAATGAGTATCTATAAGAAAACTTTA
ACATGTAAAACTTCTGTGAAATTATCTTGTCCCATATCAGGGTCATGTCA
AACTAATGTCCTCCTCAGCATCTTTGGAAAACTTCAGAGGAGAAATGAGC
TTTGCCCCTCCTGTTCATTTCCTATTCCACTAGGAGACCTGTCCTTCCCT
TTCAGCATGCTTTGTCCATATTTAGAAGCTGTTGAAGCCATTACTTGTCT
GGTCAGTTTTTAGTGCTGGAATGGACCTAGCCTTTTAGGCCTTCTGAGAT
TTAGTTTGATCTCGTCTTTCCCACCTAATGGCTCTGTTCTACTACATAGA
TTTGATCTGAAACAGTTCTCTGTTTCTAAAATAACTTTCTTTTCATGATA
GTCACAGTAAAGTACATTTATTATGGAAAAATCAATAAGTATAACGAGTG
AAAGTTATTTCTTGGTGGTAAGATTATGGGATTATTTGAACTTTCTGTTT
CATTGTATTTTATTTATTTATTTATTTTTGTGATGGAGTCTCACTCTGCT
GCCCAGGCTGGAGTGCAGTAGTACGATCTTGGCTCACTGCAACCTCCCCT
TCCCAGTTCAAGTGATTCTCCTGCCTCAGACTCCCAAGTAGCTGGGATTA
CAGGCGCACGCCACCATGCCTGGCTAATTTTTTTATCTTTAGTAGAGACA
GGGTTTCACCATGTTGACCAGGCTGATCTCCAACTCCTGATCTCAGGTAT
CCACCTGCCTCAGCCTCCCAAAGTACCGGGATTACGGGTGTGAGCCACCC
TGCCTGGCCTCATTTTGTCTTTTGGGGGTATTTTTGTGTGCAGATATATA
TGTATATAAATATTTTTCCCTCTTTTCCCCAGTTAGTATTTGAGCAGATG
AACTTTGGACCCGAATACCTGTATTCAAGTCTCTAATACCACTTCTTGGC
TATTTTCATTTTATCAAATGGCCTCTTATCCTCGTTTTTCTCATTTATTA
AGTAGAGATGTAACTACTTGATATAATTCAAAAACTCAATAATGGCATTC
TTTTGTTTTTTAGACTCTAGTGTCTGTACTCCTTGTACCATGCTGGGATT
CATTTGAACAATTGCATGGCTTTTTTAGTGTATTATTAAATTTGCAGTTT
ACTTAGAATTTACTGGGACCTCATACAAATGGGAAAAAAACATAACTGTG
TTACTCATTTGCTGTGTGCCTTTGGATTGACCCTATTTTTTGTATTCATT
TTCTCCCCATGTCCTGAGTTCCACTTTGAATAAAAAAGTAATTTTTTTCC
TGCCTGTAAAATAGGCTACCAATAGGCTGCAGTTGTCTATAGTAGCTGCT
TCACTGAGGAGAGCTCAGCATGAGAGAAATAGTATGAATTGCTTGCCACA
AGTTATGGGCTAGCCTTACTTCATTCTGTACTTGGACCTGTTTAGGCTTC
TAAGAGATCTTACCTCCAACAATAAACTGCTTTGAGACATGAAAAGGTGG
AAGCTTTACTTGGTTATAACTTTACTTTTAATACCTAGAACAGTGAGTCT
TCAAACTTGTATTTGCATGCCCAATTTATAAAAAGTTTCCTGAGCATTTA
CCCCTAATATATGCATTTTAAATTATATATGATTTATGGTAATAATAATA
TATATGTTACAAAATACATACAAAAATATAGATTAAACAAGGTGAGGTTA
AAAAATTTAAAAGTTCTAATCTTTCTTGCAAACCAGTGGATCTTTTGTGC
CTTACTCTGGTAAACACTGTCTTAGAAGAATATATAGAACATTAAAATCT
TAATGCTATAGTTATATGACAGAGTATGATGAGAGCTACAGATAAACAAC
ACATCATGAATCTTCTTGTGGCAGTGTTTATAACCATTATGTGAAATGCT
GCCTCATTCTTATAACTAGCATAAGAACAGATAGGACTTTCTCGATTTTG
AGGGGTAATTATTAGATGGTATTTTCTGTTAAGGACTCTTCCAGCTATAA
AATTCTTAAATGTAGAAAGCGAAGTGAGGGTTTATGGTGAGAGGAAGCAT
TGGTATCATGTTTTAGTGTAGTCCAAGAATATGGACACATCCAGAAAATG
CAGATCAAGTTTAGCCTAATGAGAAAATATATTTTGGAGTCCATATGGTA
AATTAAATTATGTGATTTTTGAGTTATTGTACAAATATAATTCTTAGAAT
GTTAGAGTCAGGAGACTATAAGAGACCAACTGCTTCAAGTTTCATTTAAC
ACATGGGAAACTAAGGCGAGAGAAATTTCAAGACTTGCCCAAGATTAGAC
CTCTTGTTAAGTAATGAAAGTGTTTTAAAAACAGGTGGGTCAAATTCTGT
TTTTAAAATTTCCATTATGATGAAAATTTCAGTATTACAGGCTTCCAAAT
CCCAGCAGATGGGCCACTTGTTTAAAGGAGAGTTTGATATAATAAAGCAT
CTAAAAACAAGAGTTTGGATAATTCCTTAGGGTTGTTATGATGTGATTTG
ACTTATAATTGGAAATACCGTTTTATTCATTGTACTGATTTTCATTTCTC
TTTTTCTTCTAGAATGTCTTGATTGTGGAAGTAAGTTCACATTTACTTTT
AATATAACATTTATGACTTTTCTAACTTAGTATGCACCATCCTAAAGGTA
AGCCAGGGAGAGAAATTCCTCTGCATCAGTTTTAATGGTGGGCTTGTGTT
CTAAAGGAGTGAGATTGGTTTTTTGTAAAGACTACTTAGTAATTTGTTTT
TACCAATAATGGAATGGTATACTTCCTACCTCTCTTTTTTTAGTTTGAAG
TATTTTCTTTCTAAACATAACTCTCTCTCTCTATTTATCTATATATAATA
TATACATATATATCTTATATTTTATGTATATATATATATATCTTGCTTAG
ATTTTGTCTTATGTAATATTTGGTACATAAAAAATAATATTTATAATTTA
TAGACTATTTTCCATGTGTTATTATGTGCTAAAGTATTTTGTATCTTAGC
ACCGAGAGGCTAAGCAGTTTCCTAGGGTTACCAGCTAGTAAACTAAGGGA
AACCTTTACTTCCTTTAGCTCAGTGGTTCTCAAAATGTGGTTCCCTAGAC
CAAAAGTATTAATATCAGACAAGAACCTACCGAATCAAAATATCTGTGAT
GAGGCCCAGCAAGCTATGCTTTAACAAGTTTCCGAGTGATTCTGATGCAT
GCTAAGGTTTAGGATCCCTTGTTTTTACTCATAAGTCACTTTCTCATTAA
GGCCTTCCCTGGCCATCCTATATAAAATCTCATGTTTTCACACCGTCAAC
TTCGTATTCCTCCTCAATACTTTTATTTTCCTGATCACTTATCACTAACA
GCCTCTCTCTCTCTCTCTCTCTCTCTCTATGTATATATATATATATATCA
CTTATCACTGTCTAACAGCCTCTCTTTATATATATATAATCTATAGATTA
TATATATATGCAGCATTGTGCAATCATTATCACGCTCAATTTTAAAACAT
TTTCATTTCCCCACAAAGAAACCCAATCCCCTTAGCCATCACTCCCAATT
TTCCCTTCCCCCAGCACCTAGCAAACTGATCATCTACCTACTTGCTGTCT
ATAAGATTTGCCTATTCTGGACATTTTGTATAAATAGAATCATACAATAT
GTGGCCTTTTGTATCTGGCTTCTCTCACTTAATGTTTTCAAGGTTCATTC
ATGTTGTGGAGTATATCTGCACTCATTTCCTTTTTATTGCCAAATTGTAT
GGATAGACAGGTGTTCCTCAACTGTGTCCTGATAAACCCATCTGAAGTTG
AAAATATCATAAGTTGAAAATGGATTTACTACTTTGATAAATCTATCCTA
AAGTCAGAAAAATCTCATGTTGGAACCATCGTAAGTTGGATACCATCTGA
ATTACATTTTTGTTATCCATTCACTGGTTGACAGACGTTAGGTTGTTTCC
ACTGATGCTCCTTATTTCTCGTACCTGAAATGTCCTTATTCCCTCCCTTC
TTATCCCATGTTTAAGTCATTTAAGACCCAGCTCAAACGTCACCTCCACA
AAACCTTCCTTGATACCCCTTTCCTCTTCAATTCACTTGGACCTTTTGCA
TTTAATTTTAATTTTTATTTTTTTTAAGACAGAGTCTCACTCTGTCACCA
GGCTGGAGTGCAGTGGTATGATCTCAGCTCACTAACTACTCTGCCTCCCA
GGTTCAAGCAATTCTCATGTCTCAGCCTCCCAAGTAGCTGGGACTACAGG
TGTGCGCCACCATGCCTGGCTAATTGTGTGTGTGTGTGTGTGTATGTATG
TATGTATATATGTGTGTGTGTGTATATATATATATACACAAACATATATA
AATATATATACATATATATATATACACACATATATAAATATATATACATA
TATATATATACACACACACACACATATATATATATATAGTTTTTTTTTTT
TTAAGTAGAGATGGGGTTTTGCCATGTTGGCCAGGCTGGTCTGGCCTCAA
GCCATCCTCCCACCTCGGCCTCGCAAAGTGCTGGTATTATAGGCATGAGC
CACTGTGCCTGGCCTGCATTTCATTTTAATTATAAAATATTTTGAACTCA
GAAAAAAGGGTATGCTGAATACCTACGTACCCACAAAAGTATTAACATTT
TGCCATATTTGCTTCTGATCTTATTTTTTTTGAGAAATTAAAGATCATAA
TACAACTAAAGCCCCATTTCTTTCCCTTCATTCCCAGAAGTATGACAATT
ATCCTTAAAGTTGATATATATCATTCCCATGCATGTTTTTTATACTTCCC
TAGTACAAGTTAGCTGTATCCTCTGCTCAGGGGCTCATCAAGCTGAATCA
AGGGACTCATGATCCTCTTCAAAGTTCCTTCAGGTTGTTGGCAGAATTTA
GTTCCTTGTGATTGTAGGACTGAGGGCCCGTTTTCTCACTGGCTGCTGGC
CAGGGGTTGCTCCCAGATATTTAAAGGCTCATGCCCTAGCCCATGACAGT
CTCACAACATGGCAGCTGACTTCTTCAAAACCAGCAGGAGAATCTTGCTC
TAGTCTACCACATAACCTAATCACAGGAGCGGCTATCCCGTTATTTTCAC
AGATCCTGGTCACATTCAAGGGGAGGGAACCCTTCTGTGTGTGTACACCA
GGAGGCAGGAATTTTTTTTTTCTTTTTCTTTTTTGTTAAAAAGTCTTAAA
GTCTTTTATCCCTAAAGGAGGCAGGAATTTTGAGAGCCATCAGAATTCTG
CCTACCACAGCCCAGAAATCTGCATTTTTCACAAGTCTCCAGCCATGATG
TTTCTGATGGCTCACACTGCTTTATTCCATTTTTAAAGAGTATTTTTATT
GAAAAGCATTAGGGTTATGGTTTAAAAAATATTTTCCCTAACAAAGATGG
GTTTGTTTAGAGTCCTACTTTTGACTAAATAGCTGAGATTCACTTTTATG
TAAAGTTCATTTTATAGCGTTATTAATTTGGGTGCCTTTAAAAATAGTAT
AAAGCATGTTTCTCGAGTGTAGTCTGTTAGCCACCTATATTGGAGAGTTG
GGAGGAGAGAGTCTCTATCTTGAATTTATGGGAAAAATTCTAAAATACTT
TTTATAATGAAGGACAACATCATAACTCCCTAATAAAATGTGCATGTATA
TATTCAAATTTGCTGTCATTGATCCTGCACCTACAAAATCCAGTCCTGGG
GGCTGGCATTCTTACTGCTTGCTGAGGGCCAGATGATATAGATTCCAGAA
TATCTCCATGTAGATTTTGGTGAGAATTACTGTGCTGAAAAGAATGACAG
TATTGCAGTTATACATGGGGGTTTTGGTACTTTATATTGTGACTCTGAAT
TTAAAGCTATGCAATGTCTTCTTTTTTGAAAGGATATAATTGACACTGGC
AAAACAATGCAGACTTTGCTTTCCTTGGTCAGGCAGTATAATCCAAAGAT
GGTCAAGGTCGCAAGGTATGTATGACATTTTGACACAGAATATTTTCCTC
ATTTGAAGGGGGATTAAGTGATTGCTTCTTTTTAAGGATAAATGTTTTCA
ACTGTCATTTTATCTTCGAAAAGTAATGTAATCTCATATAAGACTTAAGA
TATAATCCTTTTAAATAATTTTGTCATGTGTTAATAAAGCTCATAATTAC
AGTCACTTCCTTGCTAATATTAACATTTGGTTTTCAGCATGCTAATTATA
TCAGTTTGTCCTGAATAGCATGGCAGAGGATTTTGGGCCCCCTTGCAAAA
TTAAGAATAAGGATTCCAAAGCGGGTGAGGAAGTGATAGGAAGGGGTGGG
CCCTGAAGATCTGGACCTCCTGGAATTGAGTGATGAATGCTGCATCTTCT
TTGTGTCTGTAGTGAAATTTTATAATGCCTGCTTCCTTTTTTATTAAGTC
GGCCTCACCTCCTCACCTTACCTATGCTGTTTTACTTTTGCTTTTATAGT
TCTACCTGTGTTTATTTCTCATTTTCGTTTCATCTCTCAACAACTCTGGG
GTGGCATTATTATTCCCACTTTTCAGATAAGGTTACTGAGGCATAGGGAA
TTGTCCAAAGGTACAGAGCTAGTCCGCTATAGAGATGAGATTTGAACCCA
GGGAACCTGGCTCACAGTTTATGCTTTTGCCTACCTTAAGTTTTTAATAG
AGTGACATCAAACAAACATTTAAGAATATGTTTTTCTTTTCCTTTTATAA
TTTCATTAAAAACATTAAGTCTCTGATCAGTCTGCAGTTTTTATGTAGGG
GTCAGGTAATGTTCTAACTTCTGCTTTTTCCTAAGTGATTAACAGGTTTT
TATAAGCCCTTTTGAAAAAATCACGGTATCTGTCGAGCATCTTTGAATCA
GAGTAAGCCTTCTAGTGAGTCATATGTCAGCAGTTTGACTGTATGGGCTT
TTCTAATATCCAGTTCAAGTGTTTATCAGTGAGTTTTTCTTTTAAATAGA
TTTGGGACAGGTACTATGAGAGTATATAAGTGATACGTTATAGGACACTA
ACTAGTATCCTATGAAATGGCAAAAACTGCAATCACTTTTGCACCAACCA
AATAGAAACTAATCAGTGCACTTGCTTATTTTTCTACATGCTCTTTAGGG
TTTTAAATGTCAACCTACTGTGGCATAGACTTTAATCCTCTGGGTATTCT
TTTGTTGTTCTTTCCTGGTATATGCTGTGGAATTGAGATAGACTGGTTCG
TGAGCGAGAGATTTTGTGTTGCCACAGGTAGGACATGCTCAAACAATACT
TGGGTCATTTCTTGACCCAAGTCATCTATTCACCATAGTTTTGTAGCACC
GATCTTGCATACATTTCATGTATCTTCTTTGAACCCCACGTCAGTGCTGC
TTATATGATACTCAGAAATTAAACACTAAGGAATAAGATTTTCAGGTAGG
ATTGAGTTTTGGAGGGTCACAAATCTTGTAATGTCTAATATTTCCACTCT
CCCTGCTGAGAATTAGTTTTGGCTTCCTTGGAGGTGATATCGCCTCTGTT
GAGTATAAGTGGCCTACTGTGATCACACCACTGCACTCCAGCCTGGGTGA
CAGAGTGAGACCCTGTCTCAGAAAAAAAAAAAAAAAAAAAGAATGCATGG
CCTAGATGACTTCTAAGGTTTTTCCCACCCAGTTCCAGTTTTCATGTTCT
AGGCAGAGCAGTAAAGTGAGAAACACATGGACTTGGGAGTTTAGTCTCGC
ATTTCACTGCCACTTAATCTGAGCGACTATTCCATATTTAATCTCTCTGA
ATGTATTTACTCATCTTTAAAGGGGAATGATTATTAACATCTTTTTCTCA
GGGAAACTATATGAGTCAAGGAGATAATATATTTGAAAATCTTTTTAACT
GCAAAGCGCTGTTTCACTGTTGGTTATAATGTGATTGATCTCATTGTAGT
GAGCAGCTGCTTAATTGCGTTTTAGAATGTAGGGAAGATAGTAATATTTT
TCACATTATATATGTAGCTGGTTCTGGAACTGTAAACATACTCCTTTTTT
ATGGAGATCTGAGTCACGTACCATAAAATTCACTCTTTTAAAGTTGTACA
ATCCAGTGGTTTTTGATATATTCAGAGTTGTGCATCTGCTACCACTATTT
CATTTTGGAACCCAAAGAAACCTTGTACCCATTAGCAGTCATTCTCCCTT
CTCCCAGCCCCTGGCAACTACTAATCTACTTTCTACAGAAAGTCCGTACA
GATTTGTGTATTATGGACATTCCATATAAATGGACTCATGCAATATCCTG
TCTTCTTTCACTTAGCATAGTGTTTTCAAGGTTCATCTAGGTTGGGGCAT
GTATCAGTACTTCATCCCTTGTTTTGGCTGAATAATATTTCATTGTACAA
ATATATCACATTTTGCTTATCCATCTGTTGGTGAACATTTGAGTTTCTAC
CTGTTGGCTTTTATGAATAATGTTGATTTGAATGTTTGTGTACAAGTATG
AATACCTGTTTTCAGGTCTCTTGAGTATATAGTTGCTAGGTCATATAGTA
ACTCTGTGTTTAACATTTTGAGGAATTGCCCGACTATTTAACAAGGTATA
TGTACTGTTTTACACCAGTAACATATGAGGGTTCCAATATCTCCACATCC
TTGACAACACTTGTTACTGTCCTTTTTATTGTAGCCATCCTAGTGGCTAT
GATGTGGTATCTCATTGTGGTTTTGATTTGTGTTTCTCTGATGCTGATGA
TGTTGAACATGTTTTCATCTGCTTATTGGCCATTTACATATATCTTCTTA
AGAACGGTTACCCATTTACAGTATGGAAAATGCTTCAGATGCAACTCTAG
TCATGCCTTAGAGATGGAGCTTTATTAAACATTCAGATCTCTAGGCATAT
GAAGTGCTGAGTTCTCTTGAACTCCTAATACAGATTGCACTGAGTTTAGT
GATACCTTTTCTGGAGCATTCCTGAGTTCAGGTAGGGAGAAGGGTTTTTG
CTGTGATTGGCTTGTTATGTTCTTTCTAAATGGAAATAGAATTGAAGTGT
CTCCTCTCTCCATTTA
25
Some types of alternative splicing
Alt. 3 SS
Alt. 5 SS
Exon skipping
pA
Different termination
26
Alternative splicing Occurs in more than half
of the 30,000 human gene transcripts
27
Alternative splicing in the alpha-tropomyosin
gene (7 isoforms)
28
Dscam transcript alternative splicing
Each isoform has one exon 4, mutually exclusively
The dscam transcript in Drosophila, the
alternative splicing champion. Thought to code
for axon guidance proteins. 60 KB gene 115
exons 38,016 combinations
29
A cautionary note 60 of human genes show
evidence of alternative splicing Much of this
evidence is from EST database analysis EST
expressed sequence tags obtained by randomly
sequencing short stretches (200 nt) of cDNAs
from a variety of sources. Millions have been
accumulated. If one EST is found among several
homologous sequences that contain is an extra
exon, the gene is classified as an alternative
splicer Could be simply mistakes. But there
are still a very large number documented cases.
30
Many human genetic diseases are caused by
mutations causing missplicing

• 1) Frank splicing mutations, loss of an exon or
  an isoform (thalassemia)
• 2) More rarely, but on the increase (in terms of
  discovery), activation of a false exon (muscular
  dystrophy, cystic fibrosis)
• 3) Theoretically, loss of a splicing factor (?)
  (lower organisms)

31
Therapeutic intervention at the level of pre-mRNA
splicing
Alternative splicing Unwanted alternative
included Antisense ? skipped Bias alternative
splicing Against an unwanted isoform
Pseudo exon activated Antisense block and
skip unwanted pseudo exon
Alternative splicing Unwanted longer
exon Antisense ? shorter isoform
d
Antisense-induced skipping
Nonsense mutation
x
Expendable exon (e.g., with many repeated domains)
32
Therapeutic intervention at the level of pre-mRNA
splicing
A. Interfere with improper splicing caused by
splice site creation or activation 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 (dont
want mRNA destroyed here, want to correct its
splicing E.g., beta-thalassemia (Kole)
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 (GABA gamma amino butyric acid,
a neurotransmitter) receptor Long and short
forms. Long form associated with mental illness.

C. Skip offensive exons e.g., nonsense
truncations in dystrophin
33
Sazani P, Gemignani F, Kang SH, Maier MA,
Manoharan M, Persmark M, Bortner D, Kole
R. Systemically delivered antisense oligomers
upregulate gene expression in mouse tissues. Nat
Biotechnol. 2002 Dec20(12)1228-33.
EGFP Enhanced green fluorescent protein model
system

Mutant globin intron has activated splice sites
Actin promoter, universally expressed.
Exon skipping yields green fluorescence
34
Instead of deoxyribose or ribose
Modified phospahte
35
Amide bonds
PNA peptide nucleic acid
Attached 1 to 4 lysines here
36
Also 2 MOE
-O-CH2-CH2-O-CH3
methoxyethyl -
Phosphorothioate deoxyoligonucleotides
37
Mutant with cryptic splicing activated
Wild type
intron-specific primers,mutant to see mut.
pre-mRNA (accumulates)
intron-specific primers, WT to see WT
pre-mRNA (less)
38
(No Transcript)
39
Antisense treatment in cell cultures (ex vivo)
from themouse with the mutant EGFP gene
Control oligo (C)(50 nt downstream)was
ineffective.
Max. effect 40
40
In vivo antisense treatment, intraperitoneally,
1-4 days, 50 mg/kg.
Mock (background)
mock
41
Brighter exposure
42
RT-PCR of tissuesconfirmsGFP results
43
Dystrophin gene 2400 kb, mRNA 14 kb, 79
exons Protein maintains muscle cell membrane
integrity Mutation Duchennes muscular
dystrophy Some (half) due to stop codon
(nonsense) in a repetitious exon (spectrin-like
repeat) Deliver antisense to ends of exon with
the nonsense mutation in mdx (model for
Duchennes) mice Use AAV (adeno-associated virus)
to deliver the antisense gene Measure mRNA with
skipped expn, dystrophin, and muscle
histochemistry
44
protein
mRNA
3 X 71
Branch site (consensus YNYTRAY)
45
U7 promoter
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 bodies low concentrations
(1000s 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 seqments as well. In permanent
transfectants can effect gt 50 inactivation of a
globin cryptic site. Gorman, L., Suter, D.,
Emerick, V., Schumperli, D. Kole, R.. Proc Natl
Acad Sci U S A 95, 4929-34 (1998).
46
Expression of U7 antisense construct
RT-PCR
U7OPT-A.S.
Endog. U7
(conclude slow mRNA turnover)
0 2 4 6 13 weeks
Splicing assay (RT-PCR)
Skip exon 23
0 2 4 6 8
13 weeks
normal
Dystrophin protein (Western)
47
untreated
normal
48
dystrophin-associated antigens
dystrophin
Normal
Untreated mdx
Treated mdx
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