Ramon EritjaIQAC-CSIC, Barcelona

CIBER-BBN

Oligonucleòtids: Aplicacions a la

Biotecnologia Farmacèutica

Nucleic Acids Chemistry Group

DNA nanotechnologyDNA origami

DNA structure: G-quadruplex, Triplex, Aptamers

siRNAantisense

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

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)

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

APTAMERS: Nucleic Acids that have the property of bindingto a specific protein. They are obtained by selection (SELEX)

SELEX

MACUGEN: treatment of macular degeneration

RNA interference

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

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

CHEMICAL MODIFICATION OF siRNA/ antisense

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

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

Nuclease resistance (sera)

0

1

4

24

Unm

.

OMe-P OMe Prop LNA PS

Tim

e (h

rs)

Ocampo et al. 2013

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

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.

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.

days

healthy

Scr

unmodified

OMe

OMe-Prop

Survival curves

Ester Fernandez et al.

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.

Off-target effects. Estimulation of innate immune respon se

Several modified RNA have been demonstrated that are able toreduce immune response

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

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

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)

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

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

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

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

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

In vivo gene expression of EGFP post intravitrealinjection

Puras et al. submitted (2014)

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

Seeman et al. 2007

Aldaye et al., Science 2008

DNA bidimensional arrays

DNA Origami

Rothemund, Nature 2006, 440 (16).

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)

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

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

TBA 29mer

TBA 15mer

5OMeG-TBA 15mer

Thrombin

Rinker et al. Nature Nanotech. 2008

New design. Introduction of two TBA aptamers (15mer and 29mer)

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

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

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

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 .

AFM characterization of the Thrombin- TBA-modified D NA origamis complexes

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.

hAGT REPAIR OF THE METHYL-TBA-ORIGAMI

200 nm

150 nm

50 nm

200 nm

hAGT REPAIR OF THE METHYL-TBA

hAGT titration

hAGT 0x hAGT 2x hAGT 5x hAGT 10x

hAGT excess

% o

f Occ

upat

ion

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.

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