safety of therapeutic oligonucleotides€¦ · presentation outline 2 1. introduction to...

Safety of Therapeutic Oligonucleotides

Patrik Andersson, PhD, ERT

Respiratory & Immunology Safety, Clinical Pharmacology and Safety

Sciences, AstraZeneca R&D Gothenburg, Sweden

OTS Webinar 13 May 2020

Presentation outline

2

1. Introduction to Preclinical Safety Assessment

2. Oligo specific considerations

3. Design, chemistry and sequence

4. Preclinical Safety findings and mechanistic drivers

a. Sequence independent

b. Sequence and hybridisation dependent

c. Sequence, but hybridisation independent

5. Summary

Introduction to Preclinical Safety Assessment

3

Preclinical Safety Assessment of drug candidates - general

4

• Medicines must be both efficacious and safe

• Preclinical Safety Assessment: Identify and

assess potential safety risks for healthy

volunteers and patients

• Risk = outcome x probability

• Benefit:Risk is context dependent

– Severity of disease

– Patient population

– Treatment alternatives

• Main drivers and mitigation of toxicity

A. Target (or “exaggerated pharmacology”)

B. Chemistry

Target Safety Assessment with

experimental verification

Ranking and selecting best compound using in silico/ experimental screening


5

Preclinical DiscoveryPreclinicalDevelop-

mentClinical Development

Life CycleManagement

Candidate

selection

Target

selection

First Time

In HumansRegulatory

Approval?!

Non-GLP studies – ”screening”• In silico

• In vitro

• In vivo: rodent(s)

GLP (Good Laboratory Practice)• In vitro

• In vivo: rodent(s) and non-rodents (mainly NHP)

Preclinical

safety studies

Aim: Document preclinical safety and DMPK

profile to meet regulatory requirements –

which toxicities at what exposure levels?

Aim: Select best compound and

understand potential target

safety concerns

Target

Modality/chemistry

Specific structure or Sequence

Number of

compounds

tested10 to >106 1-2

Target and

chemistry tox

Locked

project

decisions

R&D | Innovative Medicines | Global Safety Assessment

Exposure

Effect

Desired

effectUndesired

effect

Desired

Efficacy

(eg ED90)

Safety margin – efficacy:safety ratio

6

Whether toxicity and size of safety margin are acceptable or not dependends on

the toxicity of concern and the project context, e.g. patient population to be treated

Safety Margin

NOAEL: no observed adverse effect level

NOEL: no observed effect level

LOAEL: lowest observed adverse effect level

Acceptable

finding (eg

NOAEL)

Unacceptable

Toxicity (eg

LOAEL)

• Easy to detect preclinically (short

term, relevant models available)

• Non-vital target organs

• Reversible

• Possible to monitor in the clinic,

i.e. good biomarkers available

• Effect tolerated by patient

population

Less impact More impact

• Difficult/impossible to detect in

preclinical models (e.g. cancer risk)

• Vital organs (e.g. heart, lungs, liver,

kidney, CNS)

• Irreversible

• Difficult/impossible to monitor in

the clinic

• Effect not tolerated by patient

population

Risk = probability x outcome in perspective of the project context

Assessing level of concern for a given toxicity

7

Project context A

• Non-lethal disease

• Alternative treatments available

• Life-long duration

• Both sexes from early adolescence

• Systemic (incl. CNS) exposure needed

• Long half-life anticipated, e.g. mAb

Project context B

• Lethal disease

• No other treatment options

• Short duration

• Only males or females > 70 years of age

• Local /Topical exposure sufficient

• Short half-life anticipated

8

Importance of project context

Same safety profile can result in different assessment depending on project context

Many therapeutic oligo

projects to date against

rare diseases with no

treatment alternatives

Preclinical Safety Assessment – ASO specific

considerations

9

Oligonucleotide therapeutics ≠ small moleculesMany properties differ

10

Size

Uptake mechanism

Mechanism of action

Distributiont1/2

Metabolism

Charge

Same principles, but

• Different focus areas

• Different approaches

• Different screening cascades

Selectivity

Species selectivity

vs.

Modified from Yu et al. (2016) Nucleic Acid Ther 26, 372-380.


11

Preclinical DiscoveryPreclinicalDevelop-

mentClinical Development

Life CycleManagement

Candidate

selection

Target

selection

First Time

In HumansRegulatory

Approval?!

Specifics for oligo therapeutics?

1. Platform approach

2. Patient context

3. Regulatory landscape

4. Toxicities of concern

Safety findings not seen with ASOs

12

hERG

CNS

CV safety

Genotoxicity

Secondary

pharmacology

Small Molecules ASOs

XNot

observed

for ASOs

Reprotox

Most small

molecule in

vitro assays

not relevant

13

Patients and regulatory context

Yesterday Tomorrow

Patient numbers 100’s to 1000’s –

rare/orphan disease

1 to millions – from n=1 to

broad indications

Benefit-Risk balance No/only few alternative

treatments

No alternative treatments to

broad competition

Regulatory experience of

oligo formats

Mainly single stranded,

Gen2.0 ASOs

siRNA, LNA/cEt, anti-miRs,

miR mimics, conjugates

beyond GalNAc

Guidelines No formal, supported by

publications and white

papers

???

n = 1 (milasen)

Potentially millions

Oligonucleotide therapeutics – regulatory environment

14

• In general highly regulated area covered by formal guidelines from ICH, FDA, EMA etc.

• Oligos chemically synthesized – classified as small molecules

• However, no specific formal guidelines exist for oligonucleotide therapeutics

– White papers form OSWG (Oligo Safety Working Group)

• Inhaled oligos

• Hybridisation depedent off-target

• Target safety//Exaggerated pharmacology

• Safety Pharmacology

• Reproductive and developmental toxicity

• Genotoxicity

• Complement activation

• Impurities

• Formulated oligos

• Ongoing working groups: DMPK, Anti-Drug Antibodies, Carcinogenicity, Off-target update, GalNAc conjugates

– Bi-annual DIA/FDA meeting in Washington DC area

– EFPIA (European Federation of Pharmaceutical Industries and Associations)

• Oligo working group

15

Regulatory landscape – will it change?

• Discussions at the DIA/FDA meeting Oct 28-30 2019

– Japanese draft M3-like oligo guideline

– Off-target analysis

– EFPIA survey results

• Most companies are asking for improved guidance

• Different level of experience and views between some authorities

– FDA request for public views on Clinical Pharmacology studies

• DDI – Drug Drug Interactions

• ADA – Anti-drug antibodies

• QT prolongation assessment

• Liver and kidney organ impairment

Preclinical Safety Assessment – design, chemistry and

sequence

16

Nucleotide based drugs

Hybridisation-dependent and independent MOA

• Aptamers

• Immunostimulatory (CpG) oligos

• mRNA

• siRNA (small interfering RNA)

• miR (microRNA)

• ASO (Antisense Oligonucleotides)

- RNase H dependent

- Anti-miR

- Splice modifying oligos

• PGE (Precision Genome Editing)

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

T G C T G C A T C G T A

Hybridisation dependent Hybridisation independent

17

Combining unique mechanisms and great

selectivity - oligos can be applied to

otherwise undruggable targets

Focus on ASOs


• miR (microRNA)


• RNase H dependent gapmer

• Anti-miR

• Splice modifying oligos




Hybridisation-dependent

18

Slow onset of action

Long tissue t1/2 – weeks to months

Long effect duration – weeks to months after last dose

• Double-stranded, few if any PS linkages

• Often requires (lipid) formulation or conjugate (e.g.

GalNAc)

• Single-stranded, often full PS backbone

• Administered in saline

• Conjugate (e.g. GalNAc) enhance potency and reduce dose

Focus on ASOs


• miR (microRNA)


• RNase H dependent gapmer

• Anti-miR

• Splice modifying oligos




Hybridisation-dependent

19

Slow onset of action

Long tissue t1/2 – weeks to months

Long effect duration – weeks to months after last dose

• Double-stranded, few if any PS linkages

• Often requires (lipid) formulation or conjugate (e.g.

GalNAc)

• Single-stranded, often full PS backbone

• Administered in saline

• Conjugate (e.g. GalNAc) enhance potency and reduce dose

Perfect sequence match no guarantee for ASO gapmer activity

All these sequences are perfect

matches to the human transcript

but there is still a variability 0-95%

knockdown

20

Thus, perfect homology not

enough for potent ASO activity

Consequences for species

differences and in silico off-

target analysis

RNase H gapmer screening focused on balance between

sequence dependent potency and tolerability Note: steric blocking ASOs

significantly more restricted

regarding sequence choice

PS-backbone ASOs show uneven distribution

Endogenous uptake mechanism not understood

• Tissue level: many organs not reached by systemic, untargeted ASOs

• Cellular level: only few percent of cellular content active/productive

• In vitro: low endogenous productive uptake and limited predictivity

21

Highest conc: PTC, LSEC, Kupffer

Productive uptake is key parameterProductive uptake = lead to PD

response, i.e. target mRNA knockdown.

At a given cellular concentration this

varies between cell types and in vivo-in

vitro

Important: except very

high tissue concentrations

achieved at high dose tox

studies – ASO

accumulation is not toxic

per se

Preclinical safety assessment: PS ASO-specific considerations

• Within ASO class, safety spectrum (”class effects”)

relatively well understood

- Focus screening and optimization on fewer endpoints

- Adverse findings for one sequence can have

perceptional impact on entire class

• Compared to small molecules, limited availability of

predictive in vitro models

- Screening in vivo rather than in vitro

- For long time insufficient data to build predictive in

silico models

• In theory, ”all” potential hybridization dependent

off-targets can be identified and assessed (in

contrast to small molecules)

- Potential consequences of off-target hits can be

assessed by designing rodent active ASOs against

• RNase H gapmers often poor species cross-

reactivity – use of (rodent active) surrogates of

same chemistry and design

• Where is target expressed and what level of

activity is needed for pharmacological effect?

- Will dictate doses required and activity in other

organs (especially liver and kidney)

- Restricted number of tissues/cell types of true

target safety concern

• Long effect duration for both desired and

undesired pharmacology

22

Target Safety/Exaggerated Pharmacology Chemistry/Compound safety

Chemical modifications – PS backbone and 2’ribose

2’MOE

2’OMe

2’F

2’cEt

2’LNA

5’GalNAc conjugation

3’GalNAc conjugation

PS backbone

Improved nuclease

resistance and PK

properties but

decreased affinity

to target RNA

Further

improved

nuclease

resistance

and increased

affinity to

target RNA

Main driver for

ADME properties

and sequence-

independent

toxicities

23

From: Wan and Seth (2016) J Med Chem 59, 9645-9667.


Exposure

Effect

Desired

effect

Undesired

effect

Safety margins - conjugates

24

Conjugates can

increase safety

margin if toxicity in

other tissue


Exposure

Effect

Desired

effect

Undesired

effect

Safety margins - conjugates

25

Or lead to no

change if

toxicity in

same target

cell

Oligo chemistries

PS backbone and ribose modifications

26

2’O-ME-RNA

• Naturally occuring

post-transcriptional

modification

From: Khvorova, A., and Watts, J.K. (2017). Nat Biotechnol 35, 238-248.

Colour legend

2’-O-MOE RNA

• ”Generation 2” chemistry

• Adds nuclease resistance

• Used in large number of

clinical trials

2’4’ bridged nucleic acids LNA and cEt,

”Gen 2.5”

• LNA = Locked Nucleic Acid

• cEt = Constrained Ethyl

• High affinity modification

• Shorter ASOs

• Higher potency on-target, but also

increased off-target

2’-F-RNA

• High affinity modification

• Does not add nuclease

resistance

PS

backbone

Stereochemistry

https://www.ncbi.nlm.nih.gov/pubmed/28244990

Preclinical Safety findings

27

Safety findings observed with single stranded PS ASOs

28

• Activation of alternative

complement (NHP)

• Coagulation prolongation

• Oligo accumulation effects: e.g.

renal tubular epithelium, histiocytes

in multiple tissue

Sequence-independentSequence dependent

• Off-target (ASO:RNA)

• Proinflammatory effects

– Flu like symptoms

– Injection site reactions

– Anti-drug antibodies

– Thrombocytopenia

• Liver tox

• Kidney tox

PS backbone, mainly plasma Cmax driven

– mitigated by low dose/ dosing regimenLow doses + extensive and stringent

safety screening

High conc. tissues

Sequence dependent

Hybridization dependent

On-target

Off-target

Liver

Hybridization independent

Immune stimulatory

Pro-inflammatory

ADAThrombo-cytopenia

Sequence independent

Hybridization independent

Coagulation prolongation

Complement activation

Accumulation related tox

Liver and kidney

Local injection

ASO safety - sequence dependence or not

29

A. Sequence independent

(PS backbone driven)B. Sequence and (RNA)

hybridization dependent

C. Sequence, but not RNA

hybridization dependent

30

Three categories of safety concerns for oligos

• Coagulation time

prolongation

• Complement activation (but

sequence differences

observed)

• ASO accumulation in liver

and kidney

• Liver toxicity

• Kidney toxicity

• Proinflammatory

– Injection site reaction

– Flu-like symptoms

– Thrombocytopenia

– ADA

• On-target safety

• Off-target (RNA)

Mitigated by Target Safety

Assessment and in

silico/RNA seq analysis

Mitigated by keeping

doses lowMitigated by in vitro and in vivo

screening

A. Sequence independent

31

Chemistry driving toxicity – complement activation

Henry 2016, Nucleic Acid

Ther 26, 210-215.

32

Longer PS ASO – higher

overall negative charge and

more unspecific interaction –

NHP serum in vitro

Henry (2014) Nucl Acid Ther 24: 326

PS backbone

PS backbone,+

2’MOE

Mixed PS/PO

backbone,+ 2’MOE

2’ MOE and mixed PS/PO

backbone modifications to

same sequence decrease

complement activation –

reduced impact of negatively

charged PS backbone

• Plasma Cmax driven activation of alternative

complement system

• PS backbone ASOs bind to the intrinsic

activation inhibitor Factor H

• NHPs approx 10x more sensitive than

humans - adaptation of doses and

administration regimen in tox studies rather

than clinical problem

B. Sequence and hybridization dependent

33

Compiled Safety related information on

• Target distribution - human, rat, mouse, dog (target organs and toxability)

• Biological role of target - theoretical consequences of pharmacological modulation

− In vitro, in vivo, knockout + transgenic models, human mutations

• Selectivity issues - theoretical consequences of pharm. modulation

• Effects of competitor compounds

→ Target Safety Risk assessment in perspective of the project context:

• Indication

• Treatment duration

• Target population (age, sex, co-morbidities, co-medications etc.)

…is used for doing a…

How to assess hybridisation-dependent on and off target concerns?

34

→ defining an experimental plan

...and…

Restricted

distribution but long

effect duration

Off-target analysis

35

• Mainly regarded as concern for oligos with catalytic MOA, i.e. RNase H gapmer ASOs

and siRNA

• No significant hybridisation dependent off-target concerns with ”medium-affinity”

chemistry like 2’MOE

• Higher affinity 2’-modifications (LNA and cEt) increase potency for on-target

hybridisations but also off-targets

– Combined with shorter gapmer ASOs (20 vs. 12-16 mer), this has become more of a

concern

In silico search ofentire

transcriptome

In vitro verification and

marginassessment

Theoretical Off-Target Safety Assessment

Experimental consequence

analysis



Sequence but hybridization independent

36

Oligo sequence and inflammation

CpG motifs and TLR9 activation, friend or foe?

Mitigation: Methylation of cytosines and avoiding CpG motifs

Krieg, Nature Medicine 9, 831 - 835 (2003) Krieg (2006) Nat Rev Drug Disc 5:471

37


• Reversible moderate/consistent and severe (<50K/µl)

platelet reductions not uncommon observation in NHPs

• In 2016, clinical cases of severe thrombocytopenia (TCP;

(<50K/µl) reported for two different MOE-gapmer ASOs

(volanesorsen/Waylivra and inotersen/Tegsedi) at weekly

doses of 300 mg

• TCP also observed in clinic with splice-modulating

Drisapersen (O’ME, full PS) at 6 mg/kg in young boys

• A number of hypotheses evaluated pointing to immune

related, but no specific mechanism yet identified

• Risk factors include

– high dose

– sequence specific factors

– patient population and genetic factors

• Many ASOs have been dosed >175 mg weekly in clinical

trials with no signs of platelet reductions

Thrombocytopenia reported in clinic for three ASOs

38

From FDA AdComm meeting slides May 10 2018: ”UCM608869.pdf”

39

Role of ribose modifications – LNA reduce PS effect

Sewing et al. 2017 PLoS One 12, e0187574

LNA

Adding LNA chemistry to the PS

sequence abrogated a number of in vitro

induced effects

Controls -/+ PS PS backbone -/+ LNA

IL-6

MCP-1

Controls PS backbone -/+ LNA

PLT activation PAC-1 (GP IIb/IIIa)

PLT activationP-selectin

GPVI binding

BiacorePF4 Ab ELISA

3 310

Liver tox with high affinity ASO gapmer – not primary target

knockdown or liver concentration per se

40

ASO conc µg/g liver

ALT level (liver tox marker)

Target mRNA

Highest ASO conc

Good target KD

No ALT

Medium ASO conc

Best target KD

Highest ALT

Lowest ASO conc

Medium target KD

High ALT

3-10-3 LNA ASOs of

different sequence

against same liver target

Hagedorn (2018). Drug Discov Today 23, 101-114.

Low ASO conc

No target KD

High ALT

Liver toxicity more frequently observed with certain chemistries

41

Swayze 2007 Nucleic Acid Res 35:687-700

LNAMOE

Shen (2018) Nucleic Acids Res 46, 2204-2217.

2’MOE

2’cEt

2’F

2’LNA

3 310High affinity 2’ modifications increase on-target

potency but also risk for liver toxicity

Transaminases ALT and AST = plasma

markers of hepatocyte toxicity

Role of RNase H in high affinity gapmer liver tox

42

3T3 cells

Apoptosis observed in vitro suppressed

by RNase H knockdown

Dieckmann (2018) Mol Ther Nucleic Acids 10, 45-54.Burel et al. (2016) Nucleic Acids Res 44, 2093-2109.

3 310

KD of RNase H

blunts liver

toxicity for most

sequences

Role of RNase H in high affinity gapmer liver tox

Dieckmann (2018) Mol Ther Nucleic Acids 10, 45-54.

3 310

2’OMe in gap reduces

caspase activation....

….but also knockdown potency

Caspase activation correlates with higher Tm

44

Oligo protein binding - role of chemistry

Crooke et al. (2017) Nat Biotechnol 35, 230-237.

2’-modifications alter

hydrophobicity with impact

on binding affinity to certain

proteins

3 310

45

Oligo protein binding - role of sequence

Vickers and PLoS ONE 11 (8): e0161930. doi:10.1371/journal.pone.0161930

3-10-3 cEt gapmers

with different

sequence show

highly variable

binding to the

P54nrb protein

Crooke et al. (2017) Nat Biotechnol 35, 230-237.

3 310

46

Sequence and chemistry dependent liver toxicity

• Proteins in DHBS family (e.g. PSF, p54nrb)

involved in maintaining DNA integrity

• Liver toxic 2’F sequences show induced DNA

repair, apoptosis

• This was associated with decrease levels of PSF

and p54nrb – not observed with safe sequences

Shen (2018) Nucleic Acids Res 46, 2204-2217.

Toxic 2’F sequences Safe 2’F sequences

3 310

47

Shen (2019) sequence and chemistry dependent liver toxicity

Shen et al. (2019). Nat Biotechnol.

.

3 310

Ribose modification of gap

position 2 converts liver

toxic ASO sequence to a

non-toxic one

Preclinical safety of ASOs – summary

48

• Assessment principles follow small molecule approach

• Consistent safety pattern within oligo class – know what to screen for

• Toxicity mechanisms involve both chemistry and sequence

– Recent improvement in mechanistic understanding very promising –

fun to be a toxicologist!

Questions?

Selected additional references

49

Reviews

• Andersson, P., and Den Besten, C. (2019). Preclinical and Clinical Drug-metabolism, Pharmacokinetics and

Safety of Therapeutic Oligonucleotides. In Advances in Nucleic Acid Therapeutics, S. Agrawal, and M.J.

Gait, eds. (Royal Society of Chemistry), pp. 474-531.

• Frazier, K.S. (2015). Antisense oligonucleotide therapies: the promise and the challenges from a toxicologic

pathologist's perspective. Toxicol Pathol 43, 78-89.

• Henry, S.P., Kim, T.-W., Kramer-Stickland, K., Zanardi, T.A., Fey, R.A., and Levin, A.A. (2008). Toxicologic

properties of 2'-methoxyethyl chimeric antisense inhibitors in animals and man. In Antisense Drug

Technology: Principles, Strategies and Applications, S.T. Crooke, ed. (Boca Raton, FL, USA: CRC Press,

Taylor and Francis group), pp. 327-363.

PS backbone and protein binding

• Crooke, S.T., Vickers, T.A., and Liang, X.H. (2020). Phosphorothioate modified oligonucleotide-protein

interactions. Nucleic Acids Res. (in press)

siRNA and off-target

• Janas, M.M., Schlegel, M.K., Harbison, C.E., Yilmaz, V.O., Jiang, Y., Parmar, R., Zlatev, I., Castoreno, A.,

Xu, H., Shulga-Morskaya, S., et al. (2018). Selection of GalNAc-conjugated siRNAs with limited off-target-

driven rat hepatotoxicity. Nat Commun 9, 723.

Confidentiality Notice

This file is private and may contain confidential and proprietary information. If you have received this file in error, please notify us and remove

it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the

contents of this file is not permitted and may be unlawful. AstraZeneca PLC, 1 Francis Crick Avenue, Cambridge Biomedical Campus,

Cambridge, CB2 0AA, UK, T: +44(0)203 749 5000, www.astrazeneca.com

50

safety of therapeutic oligonucleotides€¦ · presentation outline 2 1. introduction to...

Documents