Strategy:

1
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, Qiang Liu, Alice
Bakker, Wayne To, Amy Duguay, Ben M Alba, Richard
Smith, Alberto Rivas, Peng Li, Hon Le, Erik
Whitehorn, Kevin W Moore, Candace Swimmer, Victor
Perlroth, Martin Vogt, Joost Kolkman  Willem Pim
C Stemmer Avidia, Inc., 2450 Bayshore Parkway,
Mountain View, California 94043
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
2
Organization of binding domains in typical
mammalian receptors
A-domains(35-40 AAs/domain) determine binding
speficicity of many receptors
Typical receptor structures
of 217 A-domains
(metaphorically?)
as a spacer between domains
2 domains cooperating
Bipartite domain
Dual specificity domain
Degenerate oligos synthesized to coding for 35-40
AAs of the A domains Only AAs naturally found at
each position were coded for. Conserved
structural AAs were kept constant. Complexity
1023 . Actually realized 1010 as phage display
particles Select one domain at a time, serially,
by panning
LRP LDL receptor related protein
3
Isolation of a high affinity binding protein to
IL6 ( interleukin 6 ) by iterative selection
(IL6 is a target for cancer and
inflammation) IL6 immobilized on
plates. Recovered proteins from first cycle
cloned and tested for IL6 binding 20 top binders
pursued. Add the domian library to each of the
20 first round winning dominas. Again pick best
20 overall. After a third cycle pick the very
best binder C326
Monomer protein Screened for binding
Monomer displayed on phage coat
Build 20 dimers pools from 20 best monomers
M13 phage
IL6
4
Add an IgG-binding domain at the end to prevent
rapid clearance (measured half-life of 89 hours
in monkeys)
5
Binding measured by a competition assay
(AlphaScreen)
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
6
More AlphaScreens effect of combining the 3
domains
7
Physical assay Biacore surface plasmon
resonance to measure binding kinetics
8
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
9
(No Transcript)
10
Acute phase inflammatory response induced by IL6
is reversed by avimer C326 (in mice)
Specific for IL6-induced inflammation
11
RNA Topics

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

12
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
13
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
14
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(lt30
bp)
15
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
16
Limitations of siRNA silencing in mammalian cells
Transient nature of the response (3 days)
Transfection problems (cell type, refractoriness)
Non-renewable nature of siRNAs ()
17
Generation of siRNA in vivo (can give permanent
knockdown)
Not good for interferon- responsive cells
Allow trans-association (TTTTT acts as a
terminator)
(Pol III)
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 II)
miRNA
18
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)
19
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)
20
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)
21
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)
Raisons 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, then RNAi inhibits action of
SS mRNA
22
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.
23
In situ hybridizations
No RNA injected
No probe
SS antisense RNA
DS RNA
Transcript disappears (RNA degraded)
24
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.
25
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
26
Plasma ApoB
Plasma cholesterol
27
Some therapeutic targets of RNA interference
localized target
ss RNA virus target
Dominant allele target (can be made
allele-specific)
28
Engineering cottonseed for use in human nutrition
by tissue-specific reduction of toxic gossypol.
Ganesan Sunilkumar, LeAnne M. Campbell,
Lorraine Puckhaber, Robert D. Stipanovic, 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.
29
Delta-cadinene synthase
Target the mRNA specifying the first step in
gossypol synthesis
30
Recombinant plasmid T1 grows in the bacteria
Agrobacterium tumefaciens which can be used as a
vector for plant transfection
dCS delta-cadinene synthase
shRNA
neo gene
terminator
? alpha-globulin promoter
a-globulin promoteris 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 tOCSoctopine synthase terminator dCS
604-bp d-cadinene synthase sequence pAGP cotton
a-globulin promoter (seed specific) pNOS
nopaline synthase promoter nptIIneomycin
phosphotransferase II tNOS nopaline synthase
terminator LB left T-DNA border.
31
NEO-RESISTANT TRANSFECTANT PLANTS
Ten seeds from two transgenic plants from F1 of
selfed matings
0.1 ug/mg
PCR for transgene
Note transgene-null segregants have normal
gossypol levels
32
HPLC (high performance liquid chromatography)
Null segregant
Spots on seed indicate terpenoid glands
33
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
34
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 an for roots.
35
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. -------------------------------------------
--------------------------------------------------
---------------------------------------- Other
plants could be similarly targeted Lathyrus
sativus, 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
36
Pre-mRNA splicing
37
Intron 80 nts to 100,000 nts
Branch point
Phosphotriester
Lariat
38
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
39
( acceptror site)
( donor site)
40
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
41
Some types of alternative splicing
Alt. 3 SS
Alt. 5 SS
Exon skipping
pA
Different termination
42
Alternative splicing Occurs in more than half
of the 30,000 human gene transcripts
43
Alternative splicing in the alpha-tropomyosin
gene (7 isoforms)
Similar proteins but subtley different to suit
different tissues
44
The alternative splicing champion
Dscam transcript alternative splicing
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 flys immune response 60
kb gene 115 total exons/gene 38,016 combinations
45
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 even only one EST is found
among several homologous sequences that contain
an extra exon, the gene is classified as an
alternative splicer Could be simply mistakes.
Or genes trying out new exons to see if they
are useful, or 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.
46
Got this far
47
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.,
  ß-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)

48
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
d
Antisense-induced skipping
Nonsense mutation
x
Expendable exon (e.g., protein with many repeated
domains) Exon must be multiple of 3 in length to
maintain reading frame afterskipping
49
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
  (dont want mRNA destroyed here, want to correct
  its splicing

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
50
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
Antisense RNA injected into tail vein, RNA was
modified for stability
Actin promoter, universally expressed.
Exon skipping yields green fluorescence
51
RNA modification for stabilization
Instead of deoxyribose or ribose
Modified phosphate
Still base pairs OK
52
Even more extreme and more stable peptide
nucleic acids
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)
53
RNA modification
Also can add 2 MOE
-O-CH2-CH2-O-CH3
methoxyethyl -
Phosphorothioate deoxyoligonucleotides
54
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
55
Dystrophin gene 2400 kb, mRNA 14 kb, 79 exons
a giant gene 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 mice (model for
Duchennes). Use AAV (adeno-associated virus) to
deliver the antisense gene Measure mRNA with
skipped exon Dystrophin protein muscle
histochemistry for dystrophin
56
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 targets by antisense
57
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 (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
segments 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).
58
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)
59
dystrophin
dystrophin-associated antigens
Muscle immuno-histochemistry
Normal
Untreated mdx
Treated mdx
Top, middle ,and bottom