Gene-silencing oligonucleotides: Innovative design of oligonucleotides with enhanced efficacy and RNAilike mechanism of action

Transcription

1 Gene-silencing oligonucleotides: Innovative design of oligonucleotides with enhanced efficacy and RNAilike mechanism of action Nicola La Monica, Ekambar R. Kandimalla, Sudhir Agrawal Idera Pharmaceuticals, Inc. 167 Sidney Street, Cambridge MA 2139 USA EuroTIDES 211 Berlin, November 211

2 Nucleic Acids Therapeutics: Over Three Decades of research 2

3 Gene Silencing Approaches Antisense (1978) sirna (1992) Synthetic RNA or DNA Synthetic RNA No length-dependent activity Backbone modifications permissible Naked delivery mer optimal for activity Limited modifications permissible Lipid-based delivery RNase H mediated Ago/Dicer mediated Immune activation and off- target effects Immune activation and off- target effects 3

4 Gene Silencing Approaches Antisense (1978) sirna (1992) Synthetic RNA or DNA Synthetic RNA No length-dependent activity Backbone modifications permissible Naked delivery mer optimal for activity Limited modifications permissible Lipid-based delivery RNase H mediated Ago/Dicer mediated Immune activation and off- target effects Immune activation and off- target effects 4

5 Design of Gene-Silencing Oligo (GSO) Synthetic DNA or RNA TLRmediated immune response is mitigated due to 5 end inaccessibility Phosphorothioate modification provides stability Systemic delivery without lipid formulation 19 and 21-mer show optimal gene-silencing activity 5

6 Chemistry of GSO GSO Structure Linker Synthetic DNA or RNA PO or PS backbone O - S O OH S - P O O P O O 6

7 GSO has Optimal Activity with 19-mer Arms Antisense 5 GSO MyD88 RNA (R.Q.).8.4 MyD88 RNA (R.Q.).8.4. PBS 15 - mer 17 - mer 19 - mer 21 - mer 23 - mer 25 - mer Length of Oligo. PBS 15-mer 17-mer 19-mer 21-mer 23-mer 25-mer Length of Oligo J774 cell culture, 48 hr incubation 7

8 5 RLM-RACE Analysis: Comparison of sirna and GSO cleavage products 5 RLM-RACE Assay Harvest RNA 48hr post transfection Predicted sirna cleavage site Perform RNA-Linker-Mediated Rapid Amplification of cdna ends (RLM-RACE) 5 GSO or sirna RNA linker RNA ligation Sequence amplified cleavage products 5 cdna synthesis 1 st PCR 2 nd PCR 8

9 sirna and RNA GSO have similar cleavage products MyD88 Target sequence 5 -GCAGGCTGCTA GAGCTGCTGGCCTT..- Expected Cleavage Site Target sequence sirna cleavage product 5 -GCAGGCTGCTA GAGCTGCTGGCCTT AGTAGAAA GAGCTGCTGGCCTT..- RNA GSO cleavage product 2 -O-Me GSO 5 - AGTAGAAA GAGCTGCTGGCCTT..- No Cleavage Product AGTAGAAA - Linker 9

10 DNA GSOs induce cleavage of target mrna at multiple sites MyD88 Target sequence 5 -GCAGGCTGCTA GAGCTGCTGGCCTT..- Expected Cleavage Site Target sequence 19-mer sirna 5 -GCAGGCTGCTA GAGCTGCTGGCCTT AGTAGAAA GAGCTGCTGGCCTT AGTAGAAA GGAGTACTGGCCTT mer DNA GSO Control DNA with --linkage 5 - GTAGAAA AGAGCTGCTGGCCTT GGAAGAAAGAAGGAGTAAAGGCCTT..- No Cleavage Product AGTAGAAA - Linker GGA - Unmatched/ambiguous sequence 1

11 Insights into Structure-Activity Relationship Studies of GSOs Progress to Date: Comparative Activity of GSO and Antisense Affinity of GSO and Antisense to the target RNA Requirement of 19-mer arms of GSOs Effect of mis-matches in each arm of GSO Do the 19-mer arms need to be the same Effect of presence of 5 -phosphate on the activity of GSO Effect of linker on the activity of GSO 11

12 GSO has greater gene silencing activity than Antisense Oligonucleotide (ASO) GSO GSO ASO 5 Antisense oligonucleotide (ASO) % Inhibition of MyD88 RNA µg/ml 1 µg/ml J774 cell culture, 48 hr incubation 12

13 GSO and ASO have similar affinity to target mrna 7 T m, o C 6 5 GSO ASO 4 15-mer 17-mer 19-mer 21-mer 23-mer 25-mer Length of Oligo T m with target RNA 13

14 GSOs require Two 19mer Arms for Optimal Activity mer mer mer mer mer % Inhibition of Protein mer J774 cell culture, 3 µg/ml, 48 hr incubation 14

15 Requirement of 19-mer arms of GSOs in vivo mer mer % Inhibition of induced IL mer mer mer mer GSO, s.c. 24 hr 2 hr TLR Agonist, s.c. Serum IL-12 Analysis 15

16 Mismatches in GSO sequence result in loss of activity 25 GSO 1XmmGSO 2XmmGSO GSO X X X X X 1Xmm GSO % Inhibition of Protein X X X X X X X X X X 2Xmm GSO -1 MyD88 p38 PBS GSO 1Xmm 2Xmm GSO GSO X = mismatch nucleotide J774 cells: 1 µg/ml, 48 hr incubation 16

17 Effect of Presence of 5 -phosphate on the Activity of GSO One phosphate One phosphate Two phosphate Two phosphates % Inhibition of MyD88 RNA µg/ml 1 µg/ml J774 cell culture, 48 hr incubation 17

18 Do the 19-mer Arms Need to Be the Same DNA/DNA GSO DNA/DNA GSO RNA/DNA GSO 2'-OMe RNA/ 2'-OMe RNA GSO RNA/DNA GSO DNA RNA DNA % Inhibition of MyD88 RNA OMe RNA/2 -OMe RNA GSO 2 -OMe RNA -1 1 µg/ml J774 cell culture, 48 hr incubation 18

19 Length Dependent In Vivo Activity of GSO Length of MyD88 GSO 17-mer 19-mer 21-mer 23-mer 25-mer % Inhibition of IL-12 induction

20 GSO has greater activity than ASO in Vivo 12 % Inhibition of IL-12 induction Dose, mg/kg 24 hr 2 hr Serum IL-12 Analysis GSO, s.c. TLR Agonist, s.c. 2

21 Comparative Duration of Activity of GSO and ASO in vivo 12 % Inhibition of IL-12 induction Post Administration, Days 24 hr 2 hr Serum IL-12 Analysis GSO, s.c. TLR Agonist, s.c. 21

22 GSOs Mitigate Immune Responses Examples of Sequences Evaluated Bcl2 antisense GSO 5 -TCTCCCAGCGTGCGCCAT- -TACCGCGTGCGACCCTCT-X-TCTCCCAGCGTGCGCCAT- H-ras antisense 5 -TCCGTCATCGCTCCTCAGG- C-raf antisense 5 -TCCCGCCTGTGACATGCAT- PKC-a antisense 5 -GTTCTCGCTGGTGAGTTTC- Jahrsdorfer B et al. 22. J. Leuk. Biol. 72: 83 ; Cunnigham, C.C et al. 21. Cancer, 92: 1265 O Dawyer, P.J. et al. Clin. Cancer Res. 5: 3977; Nemunaitis, J. et al. 1999, J. Clin. Oncol. 17:

23 GSOs Mitigate Immune Responses Cytokine/Chemokine, pg/ml Cytokine/chemokine, pg/ml bcl2 PBS Antisense GSO C-raf Cytokine/chemokine, pg/ml Cytokine/chemokine, pg/ml H-ras PBS Antisense GSO PKC-α IL-1α IL-1β IL-2 IL-4 IL-5 IL-6 IL-12 IP-1 KC MCP-1 MIG MIP-1α TNF-α PBS Antisense GSO PBS Antisense GSO Serum Cytokine Analysis 2 hr 1 mg/kg GSO or Antisense, s.c. 23

24 GSOs Targeted to ApoB GSO 7.5 and 15 mg/kg, BIW, s.c Day Collect blood for plasma cholesterol assessment Sacrifice mice for liver mrna and protein levels, and blood for plasma cholesterol Collect blood for plasma cholesterol recovery assessment LDL-c 24

25 GSOs Targeted to ApoB Suppression of ApoB mrna Suppression of ApoB Protein % Change in liver mrna expression compared to PBS control GSO, mg/kg % 7% P <.5 vs PBS % Reduction of ApoB 1 protein ApoB 1 p38 GSO, mg/kg PBS PBS GSO 7.5 mg/kg 15 mg/kg 25

26 GSO Targeted to ApoB Reduction of Cholesterol PBS 7.5 mg/kg 15 mg/kg Serum TC, mg/dl Normal level of cholesterol Animals on high fat diet Dosing period Day P <.5 vs PBS 26

27 GSO is Specific for ApoB mrna At day 22 % Change in mrna expression compared to PBS control ApoB ABCA1 ABCG1 LXR 7% 15 mg/kg dose group 27

28 Anti-miR-21 GSO specifically silences target micro RNA µg/ml Anti-miR-21 GSO Mismatched GSO RNA GSO 2'-OMe-RNA GSO 2'-OMe-RNA AS % change in mir-21 RNA levels % Inhibition of mir HCT-116 cells 28

29 Design of Gene-Silencing Oligo (GSO) Synthetic DNA or RNA TLRmediated immune response is mitigated due to 5 end inaccessibility Phosphorothioate modification provides stability 19 and 21-mer show optimal gene-silencing activity Similar cleavage product as sirna Systemic delivery without lipid formulation 29

30 Gene Silencing Technologies Antisense GSO sirna Synthetic RNA or DNA Synthetic RNA or DNA Synthetic RNA No length-dependent activity Modification of backbone permissible Lipid carrier not required for delivery RNase H mediated Immune activation/ offtarget effect mer optimal for activity Modification of backbone permissible Lipid carrier not required for delivery Ago/Dicer and RNase H mediated No TLR9-mediated Immune Activation mer optimal for activity Limited modifications permissible Lipid-based delivery Ago/Dicer mediated Immune activation/ offtarget effect 3

31 Acknowledgements Lakshmi Bhagat Daqing Wang Michael J. Reardon Victoria Philbin Melissa Precopio Fugang Zhu Dong Yu Mallik Putta Tao Lan Irek Nowak The Idera Team 31