Third-Generation Antisense (3GA) Technology: Insights into Mechanism of Action

Reina Improgo, Ph.D.

Idera Pharmaceuticals

Oligonucleotide and Peptide Therapeutics

March 27, 2017

© 2017 Idera

First generation antisense

• Phosphorothioate chemistry was the first chemicalmodification applied to antisense technology

– Increased tissue bioavailability

• Over 2 dozen compounds advanced to clinical developmentand then discontinued

– Unintended immune activation observed; Toll-like Receptors (TLRs)had not yet been discovered

• Discovery of TLR9 explained why the presenceof CG motifs led to immune activation

– Raised questions on whether anti-viral and anti-cancer activity weredue to immune activation rather than antisense mechanism ofaction

Agrawal, S et al., Proc. Natl. Acad. Sci. USA, (1988) 7079-7083; Agrawal, S et al., Proc. Natl. Acad. Sci. USA, (1989) 7790-

7794; Agrawal, S et al., Proc Natl Acad Sci USA. (1991) 7595-9; Galbraith, WM et al., Antisense Res Dev. (1994) 201-206;

Zhang, R et al., Clin Pharmacol Ther. (1995) 44-53; Agrawal, S et al., J Immunol. (2003) 1621.

© 2017 Idera

Chemistry of second generation antisense pioneered by Idera

PS-DNA allowed

RNase H cleavage

Reduced

immune

stimulation

OCH3

OCH3

2’-O-substituted RNA

provided metabolic stability

and binding to RNA

Metelev, V et al., Bioorg. Med. Chem. Lett. (1994) 2929-2934; Zhang, R et al., Biochem Pharmacol. (1995) 545-56.

Agrawal, S et al., Biochem Pharmacol. (1995) 571-6; Yu, D et al., Bioorg Med Chem. (1996) 1685-92. Agrawal, S et al.,

Antisense Nucleic Acid Drug Dev. (1997) 575-84; US Patent # 5,652,355.

© 2017 Idera

Second generation antisense is the most successfulchemistry to date, however, limitations exist

• Clinical proof of concept established against multiple RNA targets

• Limited therapeutic index

– Injection site reactions

– Flu-like symptoms

– Hepatotoxicity

– Thrombocytopenia

– Immunotoxicity

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Nucleic acids and immune receptors

Desmet, et al. Nat Rev Immunol. 2012 Jun 22;12(7):479-91

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Idera’s pipeline of candidates

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Third generation antisense design

First generation

Second generation

Third generation, 3GA

© 2017 Idera

Activation of TLR9 requires 5’-end accessibility

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Linking two antisense sequences via their 5’ ends abrogates immune activation

BCL2 as representative target

Same Bcl2 sequence comparing 3GA vs. antisense dose response

Sequences:

Bcl2 antisense: 5’-TCTCCCAGCGTGCGCCAT-3’

Bcl2 3GA: 3’-TACCGCGTGCGACCCTCT-X-TCTCCCAGCGTGCGCCAT-3’

3GA

© 2017 Idera

Summary of 3GA design

1Bhagat, L et al., J Med Chem (2011) 3027-36. 2Yu, D et al., Bioorg Med Chem Lett (2000) 2585-8; 3Kandimalla, E et al.,

Bioconjugate Chem. (2002) 966-74; 4Yu, D et al., Nucleic Acid Res (2002) 4460-9; 5Putta, M et al., Bioconjugate Chem. 2010

39-45. 6Temsamani, J et al., Ann N Y Acad Sci. (1992) 318-20 U.S. patent #8,431,544 issued to Idera in 2013.

Phosphorothioate backbone

confers stability and

bioavailability

Lack of accessible 5’- ends

abrogates immune

activation1,2,3,4,5

Accessible 3’- ends allows

degradation and clearance6

19- to 21-mer length is optimal

for targeting RNA1

Phosphorothioate backbone

confers stability and

bioavailability

Lack of accessible 5’- ends

abrogates immune

activation1,2,3,4,5

Accessible 3’- ends allows

degradation and clearance6

19- to 21-mer length is optimal

for targeting RNA1

© 2017 Idera

3GA Studies Using PCSK9 as a Target

© 2017 Idera

Structure and chemistry of compounds used in this study

Target Site

Phosphorothioate

Hybrid/Gapmer

5’-5’-Linked

3’-3’-Linked

5’-3’ Tandem Repeat

siRNA Duplex

5’

© 2017 Idera

Comparative gene-silencing activity of 3GA, 1GA, and 3’-3’linked control in cell-based assays

© 2017 Idera

In vivo dose-dependent activity of 3GA targeting PCSK9

% P

CS

K9

Kn

oc

kd

ow

n

Re

lati

ve

to

PB

S

7 0

6 0

5 0

4 0

3 0

2 0

1 0

0

0 3 .5 7 .5 1 5 3 0

3 G A , m g /k g3GA, mg/kg

% P

CS

K9 K

no

ck

do

wn

Rela

tive

to

PB

S1Day 53

PCSK9 3GA, s.c.

8

Measurement of PCSK9 mRNA in liver

Gene Silencing Effect

Treatment Period

C57/BL6

30157.53.50

© 2017 Idera

In vivo activity of 3GA is sustained up to 12 days post-treatment

% P

CS

K9

Kn

oc

kd

ow

n

Re

lati

ve

to

PB

S

4 Days Post-Treatment

1 G A 2 G A 3 G A

% P

CS

K9

K

no

ck

do

wn

Re

lativ

e to

PB

S

4 D a y s P o s t-T re a tm e n t

1 0 0

9 0

8 0

7 0

6 0

5 0

4 0

3 0

2 0

1 0

0

1 G A 2 G A 3 G A

% P

CS

K9

K

no

ck

do

wn

Re

lativ

e to

PB

S

1 2 D a y s P o s t-T r e a tm e n t

1 0 0

9 0

8 0

6 0

7 0

5 0

4 0

3 0

2 0

1 0

0

1GA 2GA 3GA

% P

CS

K9

Kn

oc

kd

ow

n

Re

lati

ve

to

PB

S

12 Days Post-Treatment

1 G A 2 G A 3 G A

% P

CS

K9

K

no

ck

do

wn

Re

lativ

e to

PB

S

4 D a y s P o s t-T re a tm e n t

1 0 0

9 0

8 0

7 0

6 0

5 0

4 0

3 0

2 0

1 0

0

1 G A 2 G A 3 G A

% P

CS

K9

K

no

ck

do

wn

Re

lativ

e to

PB

S

1 2 D a y s P o s t-T r e a tm e n t

1 0 0

9 0

8 0

6 0

7 0

5 0

4 0

3 0

2 0

1 0

0

1GA 2GA 3GA

Day

15 mg/kg, s.c.

Measurement of PCSK9 mRNA in liver

Gene Silencing EffectTreatment Period

1 2 43 9 175

Treatment Period

C57/BL6

© 2017 Idera

Sustained activity of 3GA in vivo is not associated with increased tissue accumulation

Liver AccumulationIn

tac

t O

lig

os

in

Liv

er

(mg

/g)

1GA 2GA 3GA1 G A 2 G A 3 G A

0

1 0 0

2 0 0

3 0 0

L iv e r A c c c u m u la tio n

Day

15 mg/kg, s.c.

Measurement of intact oligos by HPLC

Tissue Accumulation in Liver

Treatment Period

1 2 43 95

Treatment Period

C57/BL6

© 2017 Idera

Insights into the Mechanism of Action of 3GA

© 2017 Idera

Two major mechanisms of gene silencing

Antisense RNAi

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Elucidating the mechanism of action of 3GA by analysis of targeted RNA cleavage products

Analysis of

Cleavage Products

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RLM-RACE

Cleaved RNA

Ligation of RACE Adapter to 5’ End of Cleaved RNA

cDNA Synthesis

1st Round PCR using 5’ RACE Outer Primer and 3’ Gene-Specific Outer Primer

2nd Round PCR using 5’ RACE Inner Primer and 3’ Gene-Specific Inner Primer

AAAAm7G

AAAAAdapter

AAAAAdapter

Adapter

Adapter

Gel Analysis, Cloning, and Sequencing

Sequence Analysis

© 2017 Idera

Sites of RLM-RACE target and primers

661 uguggugcug auggaggaga cccagaggcu acagauugaa caaacugccc accgccugca

721 gacccgggcu gcccgccggg gcuaugucau caagguucua cauaucuuuu augaccucuu

781 cccuggcuuc uuggugaaga ugagcaguga ccuguugggc cuggcccuga aguugcccca

841 uguggaguac auugaggaag acuccuuugu cuucgcccag agcaucccau ggaaccugga

901 gcgaauuauc ccagcauggc accagacaga ggaagaccgc uccccugaug gaagcagcca

961 gguggaggug uaucucuuag auaccagcau ccagggugcc caucgggaga uugagggcag

Mouse PCSK9 mRNA

© 2017 Idera

Various antisense compounds used for RLM-RACE analysis

% P

CS

K9

Kn

oc

kd

ow

n

Re

lati

ve

to

Un

tre

ate

d C

on

tro

l

0

- 1 0

- 2 0

- 3 0

- 4 0

- 5 0

- 6 0

- 7 0

- 8 0

- 9 0

-1 0 0

A n tis e n s e C o m p o u n d

% P

CS

K9 K

no

ck

do

wn

Re

lati

ve

to

Un

tre

ate

d C

on

tro

l

Hepa1-6 cells were transfected with 50 nM oligos using Lipofectamine 2000. After 16 hours, RNA was

isolated and PCSK9 expression was measured via qPCR using TagMan probes (ThermoFisher).

© 2017 Idera

Cleavage sites observed with 1GA, 2GA, and 3GA

90%

3’-CCACGACTACCTCCTCUGG-5’

3’-CCACGACTACCTCCTCTGG-5’

3’-CCACGACTACCTCCTCTGG-X

3/10 4/10

1/2725/27

1/102/10 4/10 3/10

2/101/10

1/27

97%

90%

1GA

3GA

2GA

659 – AUUGUGGUGCUGAUGGAGGAGACCCAGAG - 687

659 – AUUGUGGUGCUGAUGGAGGAGACCCAGAG - 687

659 – AUUGUGGUGCUGAUGGAGGAGACCCAGAG - 687

Numerator indicates number of sequences cleaved at the indicated site (arrowhead).

Denominator indicates total number of sequences cleaved within the target site (underlined).

© 2017 Idera

3GA cleavage sites are different from those of controlcompounds

X-CCACGACTACCTCCTCTGG

3’-CCACGACTACCTCCTCTGG

1/1

1/1

3’-CCACGACTACCTCCTCTGG-X

25/271/27 1/27

97%

3GA

Control 1

Control 2

659 – AUUGUGGUGCUGAUGGAGGAGACCCAGAG - 687

659 – AUUGUGGUGCUGAUGGAGGAGACCCAGAG - 687

659 – AUUGUGGUGCUGAUGGAGGAGACCCAGAG - 687

Numerator indicates number of sequences cleaved at the indicated site (arrowhead).

Denominator indicates total number of sequences cleaved within the target site (underlined).

© 2017 Idera

Cleavage sites observed with 3GA are in a similar region as observed with siRNA

3’-dTdTCCACGACUACCUCCUCUGG-5’

3’-CCACGACTACCTCCTCTGG-X

25/271/27 1/27

1/301/30

17/3011/30

97%

100%

3GA

siRNA

659 – AUUGUGGUGCUGAUGGAGGAGACCCAGAG - 687

659 – AUUGUGGUGCUGAUGGAGGAGACCCAGAG - 687

Numerator indicates number of sequences cleaved at the indicated site (arrowhead).

Denominator indicates total number of sequences cleaved within the target site (underlined).

© 2017 Idera

Hypothesis: central region of 3GA is involved in cleavage

© 2017 Idera

3’-CCACGACTACCTCCTCTGG-X-GGTCTCCTCCATCAGCACC-3’3GA

3’-CCACGACTACATCCTCTGG-X-GGTCTCCTACATCAGCACC-3’MM9

3’-CCACGACTAACTCCTCTGG-X-GGTCTCCTCAATCAGCACC-3’MM10

MM11 3’-CCACGACTTACTCCTCTGG-X-GGTCTCCTCCTTCAGCACC-3’

Insertion of a single mismatch in the central region of 3GA

© 2017 Idera

3GA activity is impacted by the insertion of mismatches at the 9th, 10th, and 11th positions

Sites of mismatches in 3GA

Sites of mismatches in 1GA

1GA MM9 MM10 MM11

- 5’

© 2017 Idera

In vivo activity of 3GA is impacted by the insertion of site-specific mismatches at 9th, 10th, and 11th positionsPCSK9

Day

15 mg/kg 3GA, s.c.

Measurement of PCSK9 mRNA in liver

Gene Silencing EffectTreatment Period

1 2 43 95

Treatment Period

C57/BL6

© 2017 Idera

In vivo activity of 3GA is significantly impacted by the insertion of site-specific mismatches at 9th, 10th, and 11th positions

ApoB

Day

15 mg/kg 3GA, s.c.

Measurement of ApoB mRNA in liver

Gene Silencing EffectTreatment Period

1 2 43 95

Treatment Period

C57/BL6

Compounds were targeted against mApoB nucleotides 2709 - 2727.

© 2017 Idera

RLM-RACE for mPCSK9 3GA with a single mismatch at the 10th position in vivo

© 2017 Idera

Specificity of 3GA allows targeting of point mutations

© 2017 Idera

WT 3GA but Not V600E 3GA Silences WT Expression

HCT116 Colorectal Carcinoma (Homozygous WT)

HCT116 cells were treated with WT 3GA or V600E 3GA using Lipofectamine 2000. After 16 hours, cell lysate was prepared and RNA was isolated.

BRAF WT levels were measured via qPCR.

+ WT 3GA + V600E 3GA

W T 3 G A V 6 0 0 E 3 G A

0

% B

RA

F W

T E

xp

re

ss

ion

Re

lati

ve

to

Un

tre

ate

d C

on

tro

l-50

-75

© 2017 Idera

V600E 3GA but Not WT 3GA Silences V600E Expression

MCF10A BRAF V600E CompoZr Breast Epithelial Cells (Homozygous Mutant)

MCF10A V600E cells were treated with WT 3GA or V600E 3GA using Lipofectamine 2000. After 16 hours, cell lysate was prepared and RNA was

isolated. BRAF V600E levels were measured via qPCR.

xx

+ WT 3GA + V600E 3GA

W T 3 G A V 6 0 0 E 3 G A

0

% B

RA

F V

60

0E

Ex

pre

ss

ion

Re

lati

ve

to

Un

tre

ate

d C

on

tro

l

-50

-75

-88

© 2017 Idera

V600E 3GA but Not WT 3GA Silences V600E Expression

MCF10A BRAF V600E CompoZr Breast Epithelial Cells (Homozygous Mutant)

MCF10A V600E cells were treated with WT 3GA or V600E 3GA using Lipofectamine 2000. After 16 hours, cell lysate was prepared and RNA was

isolated. BRAF V600E levels were measured via qPCR.

xx

+ WT 3GA + V600E 3GA

W T 3 G A V 6 0 0 E 3 G A

0

% B

RA

F V

60

0E

Ex

pre

ss

ion

Re

lati

ve

to

Un

tre

ate

d C

on

tro

l-50

-75

-88

W T 3 G A V 6 0 0 E 3 G A

0

5 0

1 0 0

1 5 0

Ca

sp

as

e 3

/7 A

cti

vit

y (

% P

BS

Co

ntr

ol)

© 2017 Idera

BRAF V600E Mutation

U>A – V600 E Mutation

GUG – Wild Type codon

GAG – V600E mutation codon

1781 gucaaucauc cacagagacc ucaagaguaa uaauauauuu cuucaugaag accucacagu

1841 aaaaauaggu gauuuugguc uagcuacagu gaaaucucga uggagugggu cccaucaguu

1901 ugaacaguug ucuggaucca uuuuguggau gtcatcagaa tgcaagataa aaatccatac

Human BRAF mRNA

• BRAF is an oncogene that is mutated in about 15% of cancers

• BRAF V600E is the most common BRAF mutation.

-TCGAGATTTCACTGTAGCT-3’WT BRAF 3GA : 3’-TCGATGTCACTTTAGAGCT-X

- TCGAGATTTCTCTGTAGCT-3’V600E BRAF 3GA: 3’-TCGATGTCTCTTTAGAGCT-X

© 2017 Idera

MYD88 L265P

-GATGGGGATCAGTCGCTTC-3’WT 3GA: 3’-CTTCGCTGACTAGGGGTAG-X

-GATGGGGATCGGTCGCTTC-3’L265P 3GA: 3’-CTTCGCTGGCTAGGGGTAG-X

• MYD88 is an adaptor protein activated downstream of TLR signaling.

• MYD88 L265P is a recurrent mutation in hematologic malignancies.

© 2017 Idera

L265P 3GA Silences L265P But Not WT Expression

OCI-Ly10 Cells (Heterozygous)

x

WT Expression

+ L265P 3GA

0

% M

YD

88

Ex

pre

ss

ion

Re

lati

ve

to

Un

tre

ate

d C

on

tro

l

-50

-75

L265P Expression

OCI-Ly10 cells were treated with WT 3GA or L265P 3GA using Lipofectamine RNAiMax. After 48 hours, cell lysate was prepared and RNA was

isolated. BRAF WT levels were measured via qPCR.

© 2017 Idera

Summary

• 3GA is a novel structure.

• 3GA exerts potent gene-silencing activity in cell-based assays as well as in vivo.

• Sustained gene-silencing activity of 3GA in vivo is not associated with tissue

build-up.

• 3GA generates cleavage sites in the target RNA in a similar region as that

observed with siRNA.

• The mechanism of 3GA renders specificity in targeting RNA.

• Specificity and mechanism of 3GA allows allele-specific targeting.

Acknowledgements

Sudhir AgrawalWayne JiangLakshmi BhagatSharanya IyengarEvren Kocabas ArgonMallikarjuna PuttaDong YuJimmy TangIrek NowakDaqing Wang