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Targeting RNA

challenges for the rational discovery of oligonucleotide drugs.
by

Morten Lindow

on 12 February 2014

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Transcript of Targeting RNA

RNAseH
inactive compartment
RISC
splicing
endosome -> lysosome
e.g escape from lysomomes
sequestering
RNAseH
target
degradation
Nucleus
Cell
intravenous
subcutanous
via lymph to venous blood

Life cycle of an oligonucleotide
cellular uptake
Targeting RNA: challenges for the rational discovery of oligonucleotide drugs
Morten Lindow
Santaris Pharma and University of Copenhagen
mol@santaris.com
Oligos bind to proteins
PS backbone enhances protein binding
Protein binding reduces filtration
tinyLNA structure
2' - 4' lock
some oligos cause liver injuries at low doses
...some don't
Bioinformatics challenge:
predict likelihood of liver toxicity
RNAseH
export of spliced mRNAs
RNAseH is active in the nucleus
RISC is probably not
pinocytosis, endocytosis?
Challenge:
which features improve uptake?
Challenge:
which features improve endosomal escape?
Challenges:
dynamic model could improve thinking and understanding
what is relevant targetable sequence
specificity
Challenges:
measuring and predicting
specifity
target
accessibility
local structure, RNA-binding proteins
simulate
reactions
Oligo meets target(s)
Knockdown and specificity
Targetable sequence?
(patho)physiology
The promise of oligonucleotides
as drugs

Generic
Fast and cheap drug discovery
small molecules target
tertiary
or
quaternary
structure of
proteins
oligonucleotides target
primary
structure of
RNA
Rational drug design for RNA targets
Rational drug design for protein targets
Time to develop HTS assay for
protein
target: 5.1 month
RNA
target: <1 month
(Drug Discovery World 2010)
Number of compounds to screen:
protein
target: millions
RNA
target: hundreds
235 active drug programs
in 2011
Just get the RefSeq
Introns, Tss, transcript variants, expression patterns
RefSeq only has one variant
- nice...
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
Andreas Petri
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!
Problem:
some oligos are
too toxic
For most common dosing regime (75 mg/kg):
evaluate performance
using cross-validation
Dose regime
used for training
at lower doses
we over-predict tox
at higher doses
we underpredict tox
RNA accessibility
Oligos in the kidney
Sakari Kauppinen
Susanna
Obad
Anders Krogh
Jeppe Vinther
Factors influencing knockdown
Nothing beats experiments
Learning from experiments
Specificity prediction from sequence
iteration
Affinity to target site
Prediction: Vienna RNA
Measurement: Tm
Local structure of target site
Prediction: Vienna RNA, sfold, etc etc
Measurement: FragSeq, SHAPE-seq,
COAT
Local protein binding to target region
Motifs?
Measurement:
COAT
Oligo dimerization
Prediction: Exiqon, Vienna RNA, VectorNTI
Measurement: melting curves
Oligo self-folding
Prediction: Exiqon, Vienna RNA, VectorNTI
Oligo protein binding
Known motifs?
Measurement:
COAT
-proteomics
Binding to (off)target
RNAseH recruitment
RNAseH expression level
Oligo sequence motifs
Target region motifs and folds
Target regions and oligo protein binding
Reach target cells
ADME
Plasma protein binding
reduce glomerular filtration
Properties of the tissue
Vascularization of tissue
Fenestration of capillaries
Cellular uptake
Escape endosomes
Cell type
Oligo protein binding
measurememt: COAT
Protein binding
Acid stability?
Q: Is a long oligo more specific than short?
Q: Also if the hybridization
stringency is constant?
"The longer the oligo, the
less likely
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
RNAseH or
RISC
enzyme
mediated cleavage
Requirements for binding?
requirements for cleavage?
RISC
target is a microRNA
target is a long RNA
steric block
Chemistry of oligonucleotides
Acknowledgements
Predicting drug properties
Types of target RNAs and oligos
Oligo drug discovery workflow
Development candidate(s)
in silico design and filtering
antimiR design space and criteria
TargetSurveyor
Choose a target
Peter Hagedorn
Santaris Pharma
pha@santaris.com
protein coding transcripts
long ncRNA
miRNA
mixmer ASOs
gapmer ASOs
siRNAs, shRNAs, miR-mimics
splice alteration
Three main mechanisms for oligonucleotides
Direct nucleolytic enzymes to complementary RNA
Steric block of sites on complementary RNA
Aptamers

Chemical synthesis
in vitro potency screen
in vivo efficacy and safety screen
Detailed characterization of lead candidates
two stages:
rough screen at one or two concentrations
Full dose response curves for a subset
Typically:
5 mice receive 5 times 15 mg/kg i.v. over 16 days
Readouts
macroscopic evaluation
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
4
Wienholds et al, Science (2005)
miR-122 is abundantly expressed in the liver
miR-122 and hepatitis C virus
AAAAA
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
Virus accumulation
Jopling et al, Science 2005
HCV genome
Converging on the liver
Drug discovery
Drug development
Discovering miravirsen: efficacy ‘screen’ of 10 LNA-oligonucleotides
Elmen&Lindow et al., Nature 2008
SPC3649 / miravirsen
Sylvia Schultz
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
Miravirsen
Animal Pharmacology Study in Chimpanzees
Miravirsen produced long-lasting suppression of HCV viremia
16
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.
Individual patients
Cooper et al, J Hepat, 2009
Lanford et al, Science 2010
LNA-antimiR targeting the host factor
miR-122
Individual chimps
Non-clinical Safety Studies
20
Good toxicology profile from GLP toxicology
Phase 1a trial in healthy human volunteers
SPC3649-202
Miravirsen
Clinical Trials Overview
22
DDI drug-drug interaction; CSR clinical study report
Miravirsen
Number of subjects exposed to dosing
23
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.
Study Outline
SPC3649-203
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
Dose Selection
Weekly doses expected to reach target hepatic exposure required for anti-viral activity
26
MIRAVIRSEN
4 weeks
Wk 0
Dose 1
Wk 1
Dose 2
Wk 2
Dose 3
Wk 3
Dose 4
Wk 4
Dose 5
14 weeks
FOLLOW-UP PERIOD
Wk 7
P&R cohort 1
Wk 10
P&R cohort 2&3
Wk 18
END
HCV RNA Decline (not tainted by SOC)
SPC3649-203 All Cohorts
27
5 dosing events
Follow-up
Smoothed HCV decline
Phase 2 trial
of miravirsen
Individual HCV RNA Levels (before SOC)
28
Safety, General
SPC3649-203
Adverse events
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

Liver tests
AST, ALT and GGT tend to decrease with miravirsen treatment in all dose groups

Injection-site events
Injection site reactions are uncommon (2 patients with local erythema and itching in cohort of 7 mg/kg)
Generic drug discovery
Full transcript