Improved Anti-miRNA (AMOs) and Splice-Switching Oligonucleotides (SSOs)

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Improved Anti-miRNA (AMOs) and Splice-Switching Oligonucleotides (SSOs), presented by Dr Mark Behlke, Chief Scientific Officer at Integrated DNA Technologies

Transcript of Improved Anti-miRNA (AMOs) and Splice-Switching Oligonucleotides (SSOs)

  • 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 RISC RNA Induced Silencing Complex Target mRNA Inhibit translation, mRNA cleavage AAAAA Transfect AMO miRNA Steric blocking Eventual Degradation? 2
  • Role of Chemical Modifications 1. Nuclease Stabilization 2. Increased binding affinity a. Tighter binding greater potency b. Tighter binding decreased specificity 3. Compatible with invasion of RISC? 4. Assist with delivery? 3
  • Chemical Modifications Sugar alterations 4
  • Chemical Modifications Phosphorothioate linkage 5
  • Newer AMO designs Original antagomir M*M*MMMMMMMMMMMMMMMMMM*M*M*M-Chol M = 2OMe m = 2MOE F = 2F D = DNA L = LNA * = PS bond Chemistries used in anti-miRNA Oligos (AMOs) 6 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 M MMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM M 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 Firefly luciferase miRNA + AMO Translation Light miRNA Renilla Luciferase miRNA Binding Site Transfected into Cells + miRNA Degradation Renilla Luc No Light Cleavage 7
  • Rluc/Fluc assay in HeLa cells (express miR-21) Direct Comparison of Different AMO Chemistries for miR-21 Knockdown 0 10 20 30 40 50 60 70 80 90 100 FoldincreaseinRL/FL 1nM 5nM 10nM 25nM 50nM DNA 2'OMe 2'OMe PS-ends DNA/ LNA PS 2'OMe/ LNA 2'OMe/ LNA PS 2'F LNAends 2'F LNAends PS HP+RC+HP 2'OMe DNA/ LNA 2'OMe PS DNA PS 2OMe 2OMe PS-ends 2OMe PS DNA/LNA DNA/LNA PS 2OMe/LNA 2OMe/LNA PS 2F LNAends 2F LNAends PS HP+RC+HP 2OMe = DNA bases = 2OMe bases = LNA bases = 2F bases = PS linkages 8
  • Unmodified DNA or 2OMe oligos are rapidly degraded in serum need PS modification or hairpin (Note: unmodified 2OMe is stable in cell extracts, even though it degrades in serum) Stability in Serum 9
  • 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 10
  • Interestingly, the DNA/LNA mixmers also require some PS modification (at least on the ends) (Note: 2F without PS rapidly degrades in cell extracts) Stability in Serum Degraded in cell extracts 11
  • For More Information: 12
  • Insertion of ZEN between bases increases duplex stability Temperature (oC) 20 30 40 50 60 70 80 %meltedduplex 0 20 40 60 80 100 Unmod DNA Internal ZEN 5-ATCGTTGCTA-3 3-TAGCAACGAT-5 5-ATCGTzTGCTA-3 3-TAGCA ACGAT-5 vs. + ZEN 13
  • 2OMe RNA is: Natural Safe / Nontoxic Less expensive than LNAs or 2F Degraded by exonucleases in serum Stable to endonucleases in cell extracts The new napthyl-azo modification increases Tm (PS decreases Tm), blocks exonuclease action, and is compatible with RISC invasion MzMMMMMMMMMMMMMMMMMMMMMzM 2OMe with new napthyl-azo (ZEN) modifier between end bases O P O O- O 3' N O N N 5' NO2 14
  • miR-21 2OMe AMOs with internal ZEN insertion 2OMe 2OMe 3PSends 2OMe PS HP+RC+HP 2OMe 2OMe 3-Z z 2OMe 5-Z z 2OMe 2-Z z z 2OMe 2-Z-PS zz 2OMe 3-Z z z z DNA LNA PS 2OMe LNA PS = DNA bases = 2OMe bases = LNA bases = PS linkages z = ZEN mod Tm 72.7 72.2 68.1 76.3 71.4 74.5 87.3 DTm +0.5 -- -4.1 +4.1 -0.8 +2.3 +15.1 15
  • Small DTm can result in large DKa at 37oC 16 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 2OMe 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 2OMe 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 2OMe 5inZEN,3ZEN 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 Ka(37C) (mol/L)-1Name miR-21 AMO Sequences (5 to 3) Tm (C) Tm (C) G o 37 (kcal/mol) A 4.1oC increase in Tm between the 2OMe-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 17 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 18 For the miR-21 AMO with ZEN-2OMe chemistry, the binding affinity threshold to escape helicase unwinding must lie between the 14mer & 15mer
  • Specificity comparison of AMO chemistries 19 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 A U C A A C A U C A G U C A G A U A A G C U A U C A A C C U C A G U C A G A U A A G C U A U 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-2OMe DNA/LNA-PS 2OMe/LNA-PS2OMe-PSends Antagomir
  • ZEN is non-toxic, whereas PS mod and LNA mod show toxicity 2OMe 2-Z Z Z 2OMe 3PS-ends DNA PS DNA/LNA PO DNA/LNA PS 2OMe/LNA PO 2OMe/LNA PS = DNA bases = 2OMe bases = LNA bases Z = ZEN mod = PS linkages 20 0 20 40 60 80 100 120 %ViableCells Cytotoxicity Data of "NC1" AMOs transfected into HeLa Cells for 24hrs with INTERFERin 50nM 100nM 2'OMe 2-Z Reagent Only 2'OMe 3PSends DNA/ LNA PS PS DNA/ LNA PO 2'OMe LNA PS 2'OMe/ LNA PO Stauro- sporine Pos Cont
  • 21 30 nM 100 nM 10 nM DNA/LNA-PS 2OMe-ZEN 2OMe-3PSends 2OMe2OMe/LNA-PS
  • Potency differences do not relate to transfection efficiency 22 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. 23
  • For more information: 24 Molecular Therapy Nucleic Acids, 2013
  • Insulin Regulation in Pancreatic Islets Eran Hornstein Weizmann 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 25
  • Knockout of Dicer leads to reduction in miRNA levels Studied in isolated islets; b-cells comprise ~50-60% of cell mass 26
  • Dicer mutants have elevated glucose, decreased insulin 27
  • 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. 28