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Transcript of Targeting RNA
endosome -> lysosome
e.g escape from lysomomes
via lymph to venous blood
Life cycle of an oligonucleotide
Targeting RNA: challenges for the rational discovery of oligonucleotide drugs
Santaris Pharma and University of Copenhagen
Oligos bind to proteins
PS backbone enhances protein binding
Protein binding reduces filtration
2' - 4' lock
some oligos cause liver injuries at low doses
predict likelihood of liver toxicity
export of spliced mRNAs
RNAseH is active in the nucleus
RISC is probably not
which features improve uptake?
which features improve endosomal escape?
dynamic model could improve thinking and understanding
what is relevant targetable sequence
measuring and predicting
local structure, RNA-binding proteins
Oligo meets target(s)
Knockdown and specificity
The promise of oligonucleotides
Fast and cheap drug discovery
small molecules target
Rational drug design for RNA targets
Rational drug design for protein targets
Time to develop HTS assay for
target: 5.1 month
target: <1 month
(Drug Discovery World 2010)
Number of compounds to screen:
235 active drug programs
Just get the RefSeq
Introns, Tss, transcript variants, expression patterns
RefSeq only has one variant
Ensembl has four...hmm
ESTs from many different tissues
* any from liver?
thanks...only one polyA site
RNAseq could complement the ESTs
Three different data sets...
Not much expression in
the liver cell line??
Avoid common SNPs
Pragmatic data integration, data reduction and visualization
Label drug candidates according to experiments
causes liver toxicity?
causes kidney toxicty?
makes mice drowsy?
Oligonucleotides are sequences :-)
Train two Markov chains:
1. the 'toxic'
2. the 'safe'
Months of work to organize data to allow meaningful categorization
No they are not!
some oligos are
For most common dosing regime (75 mg/kg):
used for training
at lower doses
we over-predict tox
at higher doses
we underpredict tox
Oligos in the kidney
Factors influencing knockdown
Nothing beats experiments
Learning from experiments
Specificity prediction from sequence
Affinity to target site
Prediction: Vienna RNA
Local structure of target site
Prediction: Vienna RNA, sfold, etc etc
Measurement: FragSeq, SHAPE-seq,
Local protein binding to target region
Prediction: Exiqon, Vienna RNA, VectorNTI
Measurement: melting curves
Prediction: Exiqon, Vienna RNA, VectorNTI
Oligo protein binding
Binding to (off)target
RNAseH expression level
Oligo sequence motifs
Target region motifs and folds
Target regions and oligo protein binding
Reach target cells
Plasma protein binding
reduce glomerular filtration
Properties of the tissue
Vascularization of tissue
Fenestration of capillaries
Oligo protein binding
Q: Is a long oligo more specific than short?
Q: Also if the hybridization
stringency is constant?
"The longer the oligo, the
it is to find a target even allowing for a couple of mismatches"
"The longer the oligo,
the more likely
it is to be able to find imperfect basepairing sites with sufficiently low free energy of binding"
The PCR fallacy
Lots of data from a few oligonucleotides
A bit of data from many oligonucleotides
Systematic collection and analysis of data from drug discovery screening programs
'number of off-targets'
Length of oligonucleotide
Requirements for binding?
requirements for cleavage?
target is a microRNA
target is a long RNA
Chemistry of oligonucleotides
Predicting drug properties
Types of target RNAs and oligos
Oligo drug discovery workflow
in silico design and filtering
antimiR design space and criteria
Choose a target
protein coding transcripts
siRNAs, shRNAs, miR-mimics
Three main mechanisms for oligonucleotides
Direct nucleolytic enzymes to complementary RNA
Steric block of sites on complementary RNA
in vitro potency screen
in vivo efficacy and safety screen
Detailed characterization of lead candidates
rough screen at one or two concentrations
Full dose response curves for a subset
5 mice receive 5 times 15 mg/kg i.v. over 16 days
efficacy biomarker (if available)
target knock-down in relevant tissue
ALT and AST
(kidney injury markers)
iterate when necessary
Depends on project
(experimental specificity determination)
(medicinal chemistry lead optimizations)
Miravirsen - World's first antimiRNA medicine
Biodistribution of LNA-phosphorothioate oligonucleotides
Wienholds et al, Science (2005)
miR-122 is abundantly expressed in the liver
miR-122 and hepatitis C virus
HCV is a single stranded RNA virus
HCV genome resembles an mRNA
170 million infected worldwide
Current treatment often ineffective and
with serious side effects
2x miR-122 binding
sites in 5’NTR
Jopling et al, Science 2005
Converging on the liver
Discovering miravirsen: efficacy ‘screen’ of 10 LNA-oligonucleotides
Elmen&Lindow et al., Nature 2008
SPC3649 / miravirsen
Antagonism of miR-122 leads to reduced plasma cholesterol
Elmen&Lindow et al, Nature 2008
Single i.v. injection of
miravirsen in mice
Three i.v. injections of miravirsen
in African green monkeys
Esau et al, Cell Metab 2006
Kreutzfeldt et al. Nature 2005
Animal Pharmacology Study in Chimpanzees
Miravirsen produced long-lasting suppression of HCV viremia
Lanford Science 2010
Rebound of HCV at D175 (wk 25) same time as re-emergence of free miRNA-122
Anti-viral activity in vivo
Can HCV mutate to escape treatment?
Small molecule inhibitor of viral polymerase.
Cooper et al, J Hepat, 2009
Lanford et al, Science 2010
LNA-antimiR targeting the host factor
Non-clinical Safety Studies
Good toxicology profile from GLP toxicology
Phase 1a trial in healthy human volunteers
Clinical Trials Overview
DDI drug-drug interaction; CSR clinical study report
Number of subjects exposed to dosing
MIR was safe and well tolerated in 77 healthy volunteers in single and multiple dose (x5 weekly doses) phase 1 trials. No dose limiting toxicities were identified.
4 weeks miravirsen s.c., simple saline solution
3 dosing cohorts : 3, 5 and 7 mg/kg x weekly doses
36 patients : 12 patients per dosing cohort (9 active and 3 placebo)
14 weeks of follow-up with regular visits
3 mg/kg cohort : pegIFN/RBV was allowed 3 weeks after last dose
5 & 7 mg/kg cohort : pegIFN/RBV was allowed 6 weeks after last dose
Weekly doses expected to reach target hepatic exposure required for anti-viral activity
P&R cohort 1
P&R cohort 2&3
HCV RNA Decline (not tainted by SOC)
SPC3649-203 All Cohorts
5 dosing events
Smoothed HCV decline
Phase 2 trial
Individual HCV RNA Levels (before SOC)
No dose-limiting toxicities
AEs mostly mild; no AE resulted in discontinuation
1 SAE (unrelated to treatment) in a patient who received 7 mg/kg
AST, ALT and GGT tend to decrease with miravirsen treatment in all dose groups
Injection site reactions are uncommon (2 patients with local erythema and itching in cohort of 7 mg/kg)
Generic drug discovery