Integrated DNA Technologies

Improved Anti-miRNA (AMOs) and Splice-Switching Oligonucleotides (SSOs)Mark Behlke MD, PhD Chief Scientific Officer

Biopolis, Singapore July 26, 2013

Inhibition of miRNAs by Antisense Oligonucleotides

RISCRNA Induced

Silencing Complex

Target mRNA

Inhibit translation, mRNA cleavage …

AAAAA

Transfect AMO

miRNA

Steric blockingEventual Degradation?

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Role of Chemical Modifications

1. Nuclease Stabilization

2. Increased binding affinitya. Tighter binding greater potencyb. Tighter binding decreased specificity

3. Compatible with invasion of RISC?

4. Assist with delivery?

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Chemical Modifications – Sugar alterations

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Chemical Modifications – Phosphorothioate linkage

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Newer AMO designs

Original “antagomir”

M*M*MMMMMMMMMMMMMMMMMM*M*M*M-Chol

M = 2’OMem = 2’MOEF = 2’FD = DNAL = LNA* = PS bond

Chemistries used in anti-miRNA Oligos (AMOs)

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DLDDLDDLDDLDDLDDLDDLDD

D*D*L*D*D*L*D*D*L*D*D*L*D*D*L*D*D*L*D*D

L*D*L*D*D*L*L*D*D*L*D*L*D*L*L

m*m*F*F*F*F*F*F*F*F*F*F*F*F*F*F*F*F*F*m*m

MMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM MMMMMM MMMMMM

L*L*L*L*L*L*L*L*L

Luc/Luc Assay System

One perfect match miRNA binding site was cloned

Renilla luciferase

Fireflyluciferase

miRNA

+AMO

Translation

Light

miRNA

Renilla Luciferase

miRNA Binding

Site

Transfected into Cells

+

miRNA

Degradation

Renilla Luc

No Light

Cleavage

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Rluc/Fluc assay in HeLa cells (express miR-21)

Direct Comparison of Different AMO Chemistries for miR-21 Knockdown

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Fold

inc

reas

e in

RL/

FL

1nM5nM10nM25nM50nM

DNA 2'OMe 2'OMePS-ends

DNA/LNA PS

2'OMe/LNA

2'OMe/LNA PS

2'FLNAends

2'FLNAends PS

HP+RC+HP2'OMe

DNA/LNA

2'OMePS

DNA PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■ 2’OMe ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ 2’OMe PS-ends ■•■•■•■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■•■•■•■ 2’OMe PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■

DNA/LNA ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ DNA/LNA PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■ 2’OMe/LNA ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ 2’OMe/LNA PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■ 2’F LNAends ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ 2’F LNAends PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■ HP+RC+HP 2’OMe ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■

■ = DNA bases ■ = 2’OMe bases ■ = LNA bases ■ = 2’F bases • = PS linkages

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Unmodified DNA or 2’OMe oligos are rapidly degraded in serum – need PS modification or hairpin

(Note: unmodified 2’OMe is stable in cell extracts, even though it degrades in serum)

Stability in Serum

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Interestingly, the DNA/LNA mixmers also require some PS modification (at least on the ends)

(Note: unmodified are all stable in cell extracts)

Stability in Serum

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Interestingly, the DNA/LNA mixmers also require some PS modification (at least on the ends)

(Note: 2’F without PS rapidly degrades in cell extracts)

Stability in Serum

Degraded in cell extracts

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For More Information:

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Insertion of “ZEN” between bases increases duplex stability

Temperature (oC)

20 30 40 50 60 70 80

% m

elte

d du

plex

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100Unmod DNAInternal ZEN

5’-ATCGTTGCTA-3’3’-TAGCAACGAT-5’

5’-ATCGTzTGCTA-3’3’-TAGCA ACGAT-5’

vs.+ ZEN

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2’OMe RNA is:NaturalSafe / NontoxicLess expensive than LNAs or 2’F

Degraded by exonucleases in serumStable to endonucleases in cell extracts

The new napthyl-azo modification increasesTm (PS decreases Tm), blocks exonuclease action, and is compatible with RISC invasion

MzMMMMMMMMMMMMMMMMMMMMMzM

2’OMe with new napthyl-azo (“ZEN”) modifier between end bases

O P

O

O-

O3'

N

O

N

N

5'

NO2

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miR-21 2’OMe AMOs with internal ZEN insertion

2’OMe ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ 2’OMe 3PSends ■•■•■•■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■•■•■•■ 2’OMe PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■

HP+RC+HP 2’OMe ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ 2’OMe 3’-Z ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■z■ 2’OMe 5’-Z ■z■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ 2’OMe 2-Z ■z■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■z■ 2’OMe 2-Z-PS ■z■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■z■ 2’OMe 3-Z ■z■ ■ ■ ■ ■ ■ ■ ■ ■ ■z■ ■ ■ ■ ■ ■ ■ ■ ■ ■z■ DNA LNA PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■ 2’OMe LNA PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■

■ = DNA bases ■ = 2’OMe bases ■ = LNA bases • = PS linkages z = ZEN mod

Tm 72.7 72.2 68.1 76.3 71.4 74.5 87.3DTm +0.5 -- -4.1 +4.1 -0.8 +2.3 +15.1

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Small DTm can result in large DKa at 37oC

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DNA t c a a c a t c a g t c t g a t a a g c t a 56.3 -16.4 -18.7 1.5 x1013

2 OMe′ U C A A C A U C A G U C U G A U A A G C U A 72.7 - -26.9 9.4 x1018

2 OMe 3PSends′ U*C*A*A C A U C A G U C U G A U A A G*C*U*A 72.2 -0.5 -26.4 4.3 x1018

2 OMe 5 inZEN,3 ZEN′ ′ ′ UzC A A C A U C A G U C U G A U A A G C U Az 76.3 3.6 -30.6 3.8 x1021

K a(37°C) (mol/L)-1Name miR-21 AMO Sequences (5 to 3 )′ ′ T m (°C) ΔT m (°C) ΔG o

37 (kcal/mol)

A 4.1oC increase in Tm between the 2’OMe-PSends AMO and the ZEN-AMO results in an 880-fold increase in the

binding affinity (Ka) at 37oC

Importance of binding affinity for AMO potency

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• It is generally accepted that high binding affinity improves potency for all steric blocking antisense applications (AMO, SSO, mRNA …)

• miRNAs reside in RISC (complexed to protein) and can be stable for weeks. It is critical to be able to invade RISC and inactivate these miRNAs

• miRNAs start as dsRNAs and get reduced to ssRNA form in RISC – thus RISC has machinery that renders the miRNA duplex single-stranded : the AMO must overcome these natural pathways so it does not get treated like a passenger strand• Nuclease “slicer” function in Ago2• Helicase “unwinding” of duplexes in Ago1, Ago2, Ago3, Ago4

• Thus the AMOs need to be nuclease resistant (cannot be cut by Ago2)

• Thus the AMOs need high enough binding affinity to overcome helicase activity• After you reach the “threshold Tm” where helicase can no longer unwind the AMO from

the miRNA guide strand, then increases in binding affinity mostly serve to make cross-reactivity for mismatches worse

miR-21 AMO length walk

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For the miR-21 AMO with ZEN-2’OMe chemistry, the binding affinity threshold to escape helicase unwinding must lie between the 14mer & 15mer

Specificity comparison of AMO chemistries

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Mutant Type

Wildtype MUT 1 MUT 2 MUT 3

a Mutations are notated as blue nucleotides enclosed in red boxes

U C A A C A U C A G U C U G A U A A G C U AU C A A C A U C A G U C A G A U A A G C U AU C A A C C U C A G U C A G A U A A G C U AU C A A C C U C A G U C A G A U A A C C U A

miR-21 AMO Sequences (5′ to 3 )′ a

ZEN-2’OMe DNA/LNA-PS 2’OMe/LNA-PS2’OMe-PSends“Antagomir”

ZEN is non-toxic, whereas PS mod and LNA mod show toxicity

2’OMe 2-Z ■Z■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■Z■ 2’OMe 3PS-ends ■•■•■•■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■•■•■•■ DNA PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■ DNA/LNA PO ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ DNA/LNA PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■ 2’OMe/LNA PO ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ 2’OMe/LNA PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■

■ = DNA bases ■ = 2’OMe bases ■ = LNA bases Z = ZEN mod • = PS linkages

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Se-ries1

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Cytotoxicity Data of "NC1" AMOs transfected into HeLa Cells for 24hrs with INTERFERin™

50nM100nM

% V

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lls

2'OMe 2-Z

Reagent Only

2'OMe3PSends

DNA/LNA PS

PS DNA/LNA PO

2'OMeLNA PS

2'OMe/LNA PO

Stauro-sporine

Pos Cont

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30 nM

100 nM

10 nM

DNA/LNA-PS 2’OMe-ZEN 2’OMe-3PSends 2’OMe2’OMe/LNA-PS

Potency differences do not relate to transfection efficiency

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Different AMOs were transfected at 30 nM.

AMO transfection efficiency was assessed using ISH (in situ hybridization).

Cells were fixed and hybridized with a Dig-probe and detected with an A647-anti-Dig antibody

Final design rules

MzMMMMMMMMMMMMMMMMMMMMz (N-1)-length

MzMMMMMMMMMMMMMMMMMMMMzM Full-length

Either during transfection (in serum) or exposure to cytoplasmic nucleases, the first AMO gets degraded to the second AMO. Positioning the ZEN at the

3’-end removes most of the Nearest Neighbor effects and avoids any degradation. Making the final AMO be 1 base shorter than the miRNA target

can also slightly increase potency.

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For more information:

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Molecular Therapy Nucleic Acids, 2013

Insulin Regulation in Pancreatic Islets

Eran HornsteinWeizmann Institute

• Investigated a possible role for miRNAs in the regulation of insulin secretion

• Dicer1 conditional KO using tamoxifen inducible Cre recombinase with rat insulin promoter

• Examine changes in insulin levels and glucose regulation with miRNA production disrupted

• Identified SOX6 and Bhlhe22 as negative regulators of insulin secretion, with miRNAs 22, 24, 148, and 182 regulating expression of these genes, thereby indirectly regulating insulin secretion

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Knockout of Dicer leads to reduction in miRNA levels

Studied in isolated islets; b-cells comprise ~50-60% of cell mass

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Dicer mutants have elevated glucose, decreased insulin

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Up-regulation of transcription repressors in Dicer mutants

Sox6 and Bhlhe22 are repressors of insulin transcription. Increased levels of repressors lower insulin and raise glucose levels.

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miRNA regulation of Sox6 and Bhlhe22?

• miRNA expression in pancreatic islets was examined using microarrays

• Potential binding sites for several highly expressed miRNAs were found in the 3’-UTRs of Sox6 and Bhlhe22

• AMOs were synthesized to specifically suppress these miRNAs to investigate the role of these species in normal cells (not Dicer mutants)

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AMOs increase SOX6 and Bhlhe22 levels, lowering glucose

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miRNA regulatory pathway for Insulin secretion

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For more information:

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Regulation of Cystic Fibrosis (CFTR)

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• Investigated role of miRNAs in regulation of CFTR expression and found a major role for miR-138

• miR-138 does not directly regulate CFTR but instead acts as a suppressor of SIN3A, which is a suppressor of CFTR transcription

• miR-138 AMO increases SIN3A levels which lowers CFTR levels

• miR-138 mimic lowers SIN3A levels which raises CFTR levels (same effect is seen using anti-SIN3A siRNA)

• Not just transcription/translation effect – salvages D508 mutants!

Paul McCrayUniversity of Iowa

Effect of miR-138 AMO vs. miR-mimic on WT CFTR

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SIN3A knockdown rescues CFTR D508 expression on cell surface

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The D508 mutant retains Cl- channel activity, but is degraded in the EPR and never reaches the cell surface; SIN3A knockdown not only increases

CFTR expression, it alters processing and permits the semi-functional mutant CFTR protein to reach the cell surface.

miR-138 mimic restores Cl- conductance in CF airway cells

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A new target for CF therapy?

For more information, see:

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Use of the ZEN modification: Splice Switching Oligos (SSOs)

Another use for this kind of antisense technology: SSOs • Steric blocking mechanism of action

• 2’OMe RNA, LNA, PMO, PNA

• Bypass stop codon or other errors present by causing splicing to shift and deleting the affected mutation. Many diseases exist which could be treated by this mechanism, including DMD, SMA, b-Thalassemia, and many more

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Duchenne Muscular Dystrophy (DMD)

Genetics – An X-linked recessive disorder affecting approx 1 in 3500 boys

Cause - An absence of dystrophin, a protein that helps keep muscle cells intact.

Onset - Early childhood - about 2 to 6 years.

Symptoms - Generalized weakness and muscle wasting first affecting the muscles of the hips, pelvic area, thighs and shoulders. Loss of ambulation, development of respiratory problems (diaphragm), cardiomyopathy and death in 20’s/30’s.

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Collaboration with the Wood lab to study SSO in “mdx” mouse

C57BL/10ScSn-Dmdmdx/J mouse• Stop codon in Exon 23• Develops DMD phenotype• Salvage with exon skipping SSOs = skip exon 23 and

you get an in-frame semi-functional dystrophin protein• PCR assay:

• Full length = 1kb product• Skip exon 23 = 700 bp product• Skip exons 22+23 = 550 bp product

Samir EL AndaloussiSuzan HammondGraham McCloreyMatthew Wood

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H2k (myoblast) cell culture

• H2k cells were grown at 33°C in 10% CO2 atmosphere using DMEM media supplemented with 20% FBS and 0.5% chick embryo serum; grown on gelatinised plates.

• After 24 h, cells were moved to 37°C in 5% CO2 and media is replaced with differentiation media (DMEM with 5% horse serum). Myotubes form within 3-5 days. These are very difficult to transfect compared to the undifferentiated myoblasts.

• Cells were transfected with LF2000 at the indicated concentrations or naked SSOs are added at 2-4 µM concentration and cells were incubated for 48-96 h in optiMEM.

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PS linkage is important for function (not just nuclease stability)

80 40 20 10 80 40 20 10 U

PS20 EndPS

nM

Myoblasts, lipid transfection, studied at 48h

SSO-EndPSmG*mG*mC*mC mA mA mA mC mC mU mC mG mG mC mU mU mA*mC*mC*mU SSO-PSmG*mG*mC*mC*mA*mA*mA*mC*mC*mU*mC*mG*mG*mC*mU*mU*mA*mC*mC*mU 42

Unlike AMOs, PS linkage is important for function in ZEN SSOs

Myoblasts, lipid transfection, studied at 48h

SSO-ZENmGzmG mC mC mA mA mA mC mC mU mC mG mG mC mU mU mA mC mCzmU SSO-ZEN-PSmGzmG mC*mC*mA*mA*mA*mC*mC*mU*mC*mG*mG*mC*mU*mU*mA*mC mCzmU

80 40 20 10 80 40 20 10 U

ZEN-PO ZEN-PS

nM

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Pilot study: direct intramuscular injection in “mdx” mice

2’OMePS ZENPS

SSO-PSmG*mG*mC*mC*mA*mA*mA*mC*mC*mU*mC*mG*mG*mC*mU*mU*mA*mC*mC*mU SSO-ZEN-PSmGzmG mC*mC*mA*mA*mA*mC*mC*mU*mC*mG*mG*mC*mU*mU*mA*mC mCzmU

• IM injection of 30 µg SSO

• Harvest muscle at day 14

• Immunohistochemical visualization of dystrophin protein

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Functional testing in “mdx” mice

• Plan: 50 mg/kg IV weekly for 8 weeks

• Reality: 50 mg/kg IV 2x weekly for 4 weeks 1 week off 50 mg/kg IP 2x weekly for 4 weeks Study phenotype for a week, then collect tissue

• 4 cohorts, WT untreated, “mdx” untreated, “mdx” 2’OMe-PS, “mdx” ZEN

• Study animals for functional activity

The monitoring system provides an assessment of the motor activity and behavior of the mice, measuring both anxiety-related behavior and locomotor behavior associated with muscle strength. IR beams of light pass through the cage. When the mouse crosses a beam, the light is broken and this is recorded in the software, Digiscan. The system measures 22 forms of activity including rearing, active time, static time as well as distance travelled.

• Examine muscles for dystrophin protein and mRNA splice forms

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Functional testing in “mdx” mice

• Expt was not optimal – think that the IV phase worked but IP did not

• No splice-shifted mRNA was detected (short half life)

• Dystrophin protein was present (long half life)

• Repeating with a 4 week IV regimen

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New ZEN (napthyl-azo) modifier inserted between the terminal bases of a steric-blocking antisense oligo improves nuclease stability and increases binding affinity

Particularly useful when used with 2’OMe RNA; anti-miRNA (AMO; PO form) and splice switching (SSO; PS form) applications; more?

The designs show excellent mismatch specificity, similar to low potency unmodified 2’OMe AMOs, yet achieve the high level of potency normally associated with use of more toxic DNA/LNA-PS mixmers

Both ZEN-AMOs and ZEN-SSOs are being tested in mice now

Available now “off catalog” – just call IDT Tech Support. Full product line in catalog will be available later this year.

Summary

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Integrated DNA TechnologiesKim LennoxScott RoseRichard OwczarzyYong YouMike MarvinAnton HoletsJess AlexanderJoseph WalderMark Behlke

Weizmann InstituteTal Melkman-ZehaviSharon Kredo-RussoAmitai MandelbaumEran Hornstein

Thanks to all the scientists whose work was discussed today!

University of IowaShyam RamachandramMichael WelchPaul McCrayBev Davidson

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University of OxfordSamir EL AndaloussiSuzan HammondGraham McCloreyMatthew Wood