Oligonucleòtids: Aplicacions a la Biotecnologia...
Transcript of Oligonucleòtids: Aplicacions a la Biotecnologia...
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Ramon EritjaIQAC-CSIC, Barcelona
CIBER-BBN
Oligonucleòtids: Aplicacions a la
Biotecnologia Farmacèutica
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Nucleic Acids Chemistry Group
DNA nanotechnologyDNA origami
DNA structure: G-quadruplex, Triplex, Aptamers
siRNAantisense
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Sintetitzados automàtics de DNA i RNA
tetrazol
+
(CH2)2(CH2)2
R
R
R
R
R
R
RDCC, DMAP
Ac2ODMAP
I2
Detritilación
Acoplamiento
Acetilación
Oxidación
(n - 1)
ODMTO B
O C
O
C
O
OH
ODMTO B
O C
O
C
O
NH
OHO B
O C
OO
DMTO B'
O
PCEO N i Pr2
O
C
B
O
O
HO
AcOO
B
O C
O
OB
O C
O
OB
O C
O
ODMTO B'
O
PCEO O
ODMTO B'
O
PCEO
O
O
H2N
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ElsEls ààcidscids nucleicsnucleics, medicaments dels , medicaments dels futurfutur ??
• Antisense
• Ribozims
• Triplex, pinces (diana dsDNA)
• Aptamers (diana proteïnes)
• RNA de interferencia, siRNA
• Antigomirs, microRNAmimetics (diana miRNA)
• Esquè (Decoys, diana proteïnesd’unió a DNA)
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ANTISENSE strategy :
Oligonucleotides of 15-25 nucleotides complementary to a specific mRNA
Pros: Specificity is given by the sequence. Universal and simple design
Cons: degradation by nucleases, poor cellular uptake, immunostimulation
Nucleic Acids
cell
membrane
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APTAMERS: Nucleic Acids that have the property of bindingto a specific protein. They are obtained by selection (SELEX)
SELEX
MACUGEN: treatment of macular degeneration
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RNA interference
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1990. RNA interference observed in Petunias by Jorgensen1998. Discovery of RNA interference in C. elegansby Fire & Mello1999. siRNA as posttranscriptional gene silencing by Baulcombe2001. Use of synthetic siRNA by Tuschl2006. Nobel Prize for Physiology or Medicine to Fire & Mello
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Development of modified siRNA as potential drugs
• Design of nuclease-resistant siRNA
• Development of formulations for local and intravenous administration
• Improvement of cellular uptake and delivery
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CHEMICAL MODIFICATION OF siRNA/ antisense
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Inhibition of TNF-αααα expressionby chemically modified siRNAs
Why TNF-α ?• Because overexpresssion of this gene is found a large number of diseases:
cancer, autoimmune diseases: rheumatoid arthritis, ankylosing spondylitis, Crohn’s disease, psoriasis, refractory asthma…..
• These disorders are treated with TNF inhibitors such monoclonal antibodies• There are siRNA that have good inhibitory activity (Sioudet al.)• Possibility of external adminitration• Inconvenient: Innate immune response
Synthesis (IQAC-CSIC, IRB Barcelona):Clara Caminal, Anna Aviñó,Santiago Grijalvo, Alvaro S omozaAnalysis (Facultat de Medicina. Universitat de Barcelona, Bellvitge)Sandra M. Ocampo, Francesc X. Blasco, José C. Perale sAnimal model (Universitat Autónoma de Barcelona, Bellatera)Ester Fernández, Carolina Romero, Joan Burgueño
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LNAPhosphorothioate 2’-OMe Propanediol
OHOP
O-
O
O
Modified siRNA (modifications on sense strand)
5’-GUGCCUAUGUCUCAGCCUC-dT-dT-(CH2)3-OH-3’PropSense-Propanediol
5’-GUGCCUAUGUCUCAGCCUC-T-T-(CH2)3-OH-3’LNASense LNA-Propanediol
5’-GUGCCUAUGUCUCAGCCUC-dT*dT*(CH2)3-OH-3’PSSense PS-Propanediol
5’-guGCCUAUGUCUCAGCCUC-dT-dT-(CH2)3-OH-3’OMe-PropSense OMe-Propanediol
5’-GUGCCUAUGUCUCAGCCUC-dT-dT-3’SSense
5’-GAGGCUGAGACAUAGGCAC-dT-dT-3’AsAntisense
SecuenciasiRNAModificación
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Nuclease resistance (sera)
0
1
4
24
Unm
.
OMe-P OMe Prop LNA PS
Tim
e (h
rs)
Ocampo et al. 2013
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W.M I.A Chol PS LNA OMe Pep Scr0
10
20
30
40100
200
** **
**** **
** **
pg/m
l TN
F-a
W.M I.A Chol PS LNA OMe Pep Scr0
10
20
30
40
** * *
pg/m
l TNF
-a
Effect of the modifications in the sense strand
Hela cells Macrophages
s.m OMe Prop OMe-Prop Scr0
50
100800
900
1000***
TN
F-αα αα
(pg/
ml)
s.m OMe-Prop OMe Scr 0
20
40
60***
*##
pg/m
l TN
F-a
Ocampo et al. 2013
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IN VIVO: siTNF78(OMe-Prop) AMELIORATES MURINE DSS COLITIS
DISEASE ACTIVITY INDEX
HEALTHY siTNF siTNF-OMe siTNF78 siScr-OMe
0
2
4
6
8
10
*** P<0.001, Kruskal-Wallis.
* P <0.05 vs. HEALTHY CONTROL. (Dunn's post-test).
**
ns
ns
5%DSS
DA
I
IMPROVES CLINICAL & BIOCHEMICAL INDEXES
MYELOPEROXIDASE ACTIVITY
HEALTHY siTNF siTNF-OMe siTNF78 siScr-OMe
0.00
0.25
0.50
0.75
1.00
1.25
1.50
1.75
*
***+
***P<0.001, 1 Way ANOVA.*P<0.05 and ***P<0.001 vs. Healthy Control.
+P<0.05 siScr-OMe vs. siTNF78. (Bonferroni's post-test).
5%DSS
MP
O a
ctiv
ity
(U
/mg
we
t ti
ssu
e)
Ester Fernandez et al.
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HISTOLOGICAL SCORE
HEALTHY siTNF siTNF-OMe siTNF78 siScr-OMe
0
5
10
15
20
25
***P<0.001, Kruskal-Wallis.
* P<0.05, *** P<0.001, vs. HEALTHY CONTROL.
+ P<0.05 siScr-OMe vs. siTNF78. (Dunn's post-test).
*
***
*
+
ns
5% DSS
HS
HEALTHY siTNF-OMesiTNF siTNF 78 siScr-OMe
IN VIVO: siTNF78 (OMe-Prop) AMELIORATES MURINE DSS COLIT IS
IMPROVES CLINICAL & BIOCHEMICAL INDEXES
IMPROVES CLINICAL & HISTOLOGICAL SCORE
HEALTHY
SiTNF78
SIScr-OMe
Ester Fernandez et al.
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days
healthy
Scr
unmodified
OMe
OMe-Prop
Survival curves
Ester Fernandez et al.
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Healthy siTNF78 siTNF siTNF-OMe siScr-OMe
siTNF78
siTNF-OMesiScr-OMe
75
60
23 500
MICROARRAY ANALYSIS (25000 genes)
Increased expression of
genes related to tissue
repair and normal colon
function
Decreased expression of
genes related to
inflammation and innate
immunity
Ester Fernandez et al.
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Off-target effects. Estimulation of innate immune respon se
Several modified RNA have been demonstrated that are able toreduce immune response
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Beneficial effects of OMe-Prop siRNA
• Increase stability to nucleases (modification at both 5’and 3’ ends)
• The modification on the 5’-end directs the guide strandto RISC (through phosphorylation of guide strand).
• Less activation of innate immunity
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Liposomal Formulations for Systemic RNAi
– Multi-component lipid formulation• Cationic lipid• Fusogenic lipid• PEG lipid• Cholesterol
– Highly efficient for liver delivery• Hepatocyte-specific gene
silencing achieved
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SNALP Formulation
DLinDMA:DSPC:mPEG2000-C-DMA:Cholesterol (40:10:2:48 molar percent)
mPEG2000-C-DMA
DSPCCholesterol
NO
PO O
O
O H O
O
O
N OO
HN O
O
OO
O
O
n
HOHH
1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA)
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Synthesis of Lipid-oligonucleotide conjugates
Synthetic strategy for the introduction of lipid at the 3’-end
b
+ R-OH a
1 2a, 2b
6a, b
d
5a (80%)5b (67%)
cb
3a (78%)3b (77%)
4a (72%)4b (70%)
where R =
a
b
+ R-OH a
1 2a, 2b
6a, b
d
5a (80%)5b (67%)
cb
3a (78%)3b (77%)
4a (72%)4b (70%)
where R =
a
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10 (80%)
c
9 (97%)
8 (61%)
ba
7
10 (80%)
c
9 (97%)
8 (61%)
ba
7
Synthesis of Lipid-oligonucleotide conjugates
Synthetic strategy for the introduction of lipid at the 5’-end
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Efficient cellular uptake of lipid modified DNA/RNA
Ugarte-Uribe et al., Biochem. Biophys. Acta 2013
OligonucleotideAlexa
R =
ODN-3'-NH2
Lipid oligonucleotide conjugates modified at 3'-ter mini
Alexa
R =
C28-5'-ODN
Lipid oligonucleotide conjugates modified at 5'-ter mini
Oligonucleotide
ODN-3'-C18
ODN-3'-C14
C14-5'-ODN
OligonucleotideAlexa
R =
ODN-3'-NH2
Lipid oligonucleotide conjugates modified at 3'-ter mini
Alexa
R =
C28-5'-ODN
Lipid oligonucleotide conjugates modified at 5'-ter mini
Oligonucleotide
ODN-3'-C18
ODN-3'-C14
C14-5'-ODN
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30 min
30 min 4 hr
4 hrUgarte-Uribe et al., Biochem. Biophys. Acta 2013
Uptake of C28-lipid-DNA conjugate
Formation of lipid-DNA micelles that facilitates cellular uptake
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Developing transfecting agents /formulations for ocular administration
O NH2
O
O NH
O
O NH
O
R1
O
NH
OR2
Gustavo Puras, José Luis Pedraz, UPV, Vitoria; Eduardo Fernandez, UMH
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In vivo gene expression of EGFP post intravitrealinjection
Puras et al. submitted (2014)
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Conclusions
• Specific chemical modifications prevents siRNA degradation by nucleases in serumwithout affecting inhibitory properties. However, the lengthening of half-life of modified siRNAs does not imply more durable silencing effects.
• Double modification in the sense strand of a siRNAimproves in vivoantiflammatory properties in an IBD mouse model. A combination of nuclease resistance and decrease on the innate stimulation seems to be critical for in vivo therapy.
• Lipid modification seems to be the most interesting modificationfor improving cellular uptake. The double hydrocarbon tail derivative (C28) has interesting properties for transfecting nucleic acids in mammalian cells
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Seeman et al. 2007
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Aldaye et al., Science 2008
DNA bidimensional arrays
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DNA Origami
Rothemund, Nature 2006, 440 (16).
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APTAMERS: Nucleic acids that have strong affinity for a protein. Obtained by selection on a library (SELEX)
SELEX
MACUGEN: VEGF, macular degeneration
TBA: Thrombin, anticoagulant
Bock et al. Nature 355, 564 (1992)
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Thrombin-binding aptamer (TBA)
• Sequence 5’-GGTTGGTGTGGTTGG-3’ 15b
• Antiparallel quadruplex
Sequence: 5’-G1G2TTG5G6TGTG10G11TTG14G15-3’Antiparallel quadruplexDiscovered by SELEX
Macaya et al. PNAS 90, 3745 (1993)
Binds to heparine exociteAnticoagulant
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A second thrombin-binding aptamer (29 b) binds to the fibrinogen exosite
Tasset et al. J. Mol. Biol., 1997, 272, 688
Both 15 b and 29b TBAs bind to thrombin in a cooperative way
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TBA 29mer
TBA 15mer
5OMeG-TBA 15mer
Thrombin
Rinker et al. Nature Nanotech. 2008
New design. Introduction of two TBA aptamers (15mer and 29mer)
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5’-FAM 3’-DABSYLMe
5’-FAM3’-DABSYL
5’-FAM 3’-DABSYL
hAGT
Me-hAGT
Development of a fluorescent assay for the analysis of theactivity of alkyl-guanine-alkyltransferase using TBA derivatives
HN
N N
N
O
H2N
N
N N
N
O
H2N
CH3
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220 240 260 280 300 320-20
-10
0
10
20
30
40
ΘΘ ΘΘΜΜ ΜΜ
(deg
cm
2 dmol
-1)
λ λ λ λ (nm)
TBA 5-O6-MeG-TBA 6-O6-MeG-TBA
20 30 40 50 60 70 80
0,875
0,900
0,925
0,950
0,975
1,000
1,025
Abs
295
nm
T(ºC)
MB-TBA MB-5-O6-MeG-TBA
TBA: 5´-GGTTGGTGTGGTTGG-3́5-(O6MeG)-TBA: 5́ -GGTT(MeG)GTGTGGTTGG-3́6-(O6MeG)-TBA: 5́ -GGT TGMeG TGT GGT TGG-3́
Methylation of one G residue prevent G-quadruplex formation
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EMSA ASSAYS:
CONDITIONS:
-TAE Mg+2 2.5x
-Thrombin buffer
-1h incubation
-3 h PAGE at 20ºC
TBA5T
TBA5T-Thr
Complex
5OMeG-TBA5T
O-6-methyl guanine on TBA inhibits both quadruplex formation and thrombin binding
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TBA 29mer
TBA 15mer
5OMeG-TBA 15mer
Thrombin
A single methylation in one of the TBA lines of the d ual system was enough to disrupt the interaction with α-thrombin .
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AFM characterization of the Thrombin- TBA-modified D NA origamis complexes
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Human O6-Alkylguanine-DNA-Methyltransferase (hAGT):
- DNA repair protein.
Daniels et al., EMBO J., 19(7) 1719-1730, 2000 Daniels et al.,NSMB, 11(8) 714-720, 2004
- Overexpressed in tumoral cells.
- Inverse correlation between survival and hAGT levels in patients
with malignant gliomas.
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hAGT REPAIR OF THE METHYL-TBA-ORIGAMI
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200 nm
150 nm
50 nm
200 nm
hAGT REPAIR OF THE METHYL-TBA
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hAGT titration
hAGT 0x hAGT 2x hAGT 5x hAGT 10x
hAGT excess
% o
f Occ
upat
ion
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CONCLUSIONS
�The process of α-thrombin recognition and binding to TBA is a quantitative single-molecule reaction, and can be visualized directly on the DNA origami by means of AFM.
� New methodology to study the DNA repair activity of hAGT.
�It can be further developed to design hAGT activity assays, for the identification of potential inhibitors as chemotherapy enhancers.
� The application of the DNA origami as a platform of single-molecule recognition opens the door for the development of new biosensors for the detection of a variety of complexes and the activity of other proteins.
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Thank you for your attention
• IQAC-CSIC: – Anna Aviñó– Carme Fàbrega– Santiago Grijalvo– Sónia Pérez – Sandra M. Ocampo– Montserrat Terrazas– Rubén Ferreira– Maria Tintoré– Adele Alagia– Clara Caminal
• UMH, Elche:– Eduardo Fernández
UB Bellvitge:José Carlos Perales
UAB:Carolina RomeroJoan F. BurgueñoEster Fernández
UPV, Bilbao:Begoña Uribe-UgarteFélix GoñiEster Fernández
UPV, Vitoria: José Luis PedrazGustavo Puras