Considerations for RNAi Therapeutic Design

Life Science Technology Symposium

Iselin, NJ

16 September 2009

Dr. Paul Bauerformer Director of Target Biology, Pfizer RNAi Therapeutic Unit

Director of Biology, Forma Therapeutics

Pfizer RNAi Therapeutic Unit

structural modifications expanding IP space screening and SAR development targeting and formulation RNAi mechanism and processing disease biology safety and therapeutic index PK/PD

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The RNAi Therapeutic Unit, as part of the Biotherapeutic & Bioinnovation Center, will develop a cutting edge nucleic acid-based

drug discovery platform and deliver Phase II clinical successes in Oncology and Metabolic Disease

Strategic approach has 3 components:Strategic approach has 3 components:Design PlatformDelivery Platform

Therapeutic Programs

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Delivery (including activity at site) Potency (sub nM) Safety (RNA and formulations) Cost of goods (unmodified GLP RNA >$10K/year) Immune stimulation Off-target effects Stability and duration Intellectual property

Primary Issues for RNAi Therapy

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RNAi Therapeutic Opportunities

Selection Criteria• clinical product concept• market assessment• knowledge of target and disease, particularly clinical• genetic linkage of target or pathway in human samples• validation of target through chemical or RNAi inhibition, overexpression, or genetically modified mouse phenotype• feasibility for RNAi sequence design and assays• feasibility for selective delivery and possible targeting ligands• availability of cell and animal models of disease• assessment of potential safety issues• applicability of target to other diseases

Current RNAi Clinical Programs

RNAi Therapeutic Critical Path

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Sequence Design& Synthesis

Delivery Design& Synthesis

Pre-clinicalGLP

DeliveryAssays

In VitroADME

In VitroToxicology

Cell-basedAssays

In VivoEfficacy

In VivoToxicology

In VivoADME

DiseaseIdentification

Scale-upGMP

BiochemicalAssays

ClinicalIND

BiomarkerDevelopment

FormulationSynthesis

In VivoDelivery

TargetSelection

RNAi Therapeutic Critical Path

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Sequence Design& Synthesis

Delivery Design& Synthesis

Pre-clinicalGLP

DeliveryAssays

In VitroADME

In VitroToxicology

Cell-basedAssays

In VivoEfficacy

In VivoToxicology

In VivoADME

DiseaseIdentification

Scale-upGMP

BiochemicalAssays

ClinicalIND

BiomarkerDevelopment

FormulationSynthesis

In VivoDelivery

TargetSelection

in silico sequence design

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Algorithm considerations• sequence specificity• splice regions/SNPs• off-target effects• thermodynamic stability• immune or toxic motifs

Recommended approach for cell-based screening:• identify desired cell line and function• test a variety of transfection agents for efficacy and toxicity using a control gene• use in silico algorithms to develop ~5 sequences against gene of interest• optimize conditions (concentration, duration, etc.)• develop ~2 mismatch control sequences• screen for functional activity

Cenix algorithm

Kinase

mRNA knockdown screening

platedsiRNA

• Mon (20 plates)choose format (1pt triplicate, 3pt triplicate, 10pt IC50) and top conc. (10nM, 3nM)

transfection

• Tues/Wed (20 plates)

storage

SelecTcells

bDNAdetection

• Thurs/Fri (40 plates w/controls)

dataanalysis

• Mon/Tues after (in database)

• Mon (20 Plates & 3 Flasks)

storage

DesignTeams

DiseaseTeams

chemicalsynthesis

storage

new

retest

prioritization

mRNA

LE

CE BL Panomics bDNA assay 8

R _ start

0

20

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71 366 611 792 969 1089 1180 1275 1350 1402 1528 1779 1852 1943 2140 2364 2411 2687

% k

nock

dow

n @

0.4n

MGene Walk Cell Line A

9position along gene

Scatter Plot

-20

0

20

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0 10 20 30 40 50 60 70 80

Compare Cell Lines A & B

10

% k

nock

dow

n @

0.4n

MC

ell L

ine

A

% knockdown @0.4nMCell Line B

Common modifications and designs

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modifications designs

5’

5’3’

3’

5’

5’3’

3’

RISC substrates

Dicer substrates

RNA

DNA

modified base

5’

5’

5’

5’

5’

5’

5’

5’

3’

3’

3’

3’

3’

3’

3’

3’

Bar C hart

R _ start

-20

0

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1390 1403 1419 1479 1553 1688 1770 1785 1796 1827 1853 1906 1937 1948 1988 2090

Gene Walk Designs #1, #2, and #3

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position along gene

% k

nock

dow

n @

0.4n

M

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-10 0 10 20 30 40 50 60 70 80

siRNA Designs #1, #2, and #3

Design #1 vs. Design #2

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0

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-20 0 20 40 60

Design #1 vs. Design #3

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RNAi mechanism of action

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purity of siRNAstability to nucleases

interaction with delivery vehicle“Formulation”

entry into cell

entry into cytosol

“Internalization”

association with RISC complex

activity of Dicer

“Association”

accessibility of mRNA “Accessibility”

duration of active complex

activity of complex“Activity”

“Variation”level of RISC components

level of Dicer

kinetics of mRNA

Formulation

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• purity• stability• interaction with delivery vehicle• common delivery options for siRNA

fromDharmacon

Nanocarriers Liposome Nanosphere Nanocapsule

Micelle

ConjugatesPolymer Dendrimer

Cationic polymer and/or lipid

+ +

+

Complexes

S

R

R

R

ROH

S

R

R

R

ROH

Macromolecule

Internalization

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• fluorescently labeled siRNA visualized by confocal microscopy• majority of siRNA lipoplexes internalize via endocytosis• recent data suggests that functional siRNA may not enter this way

Engelke & Rossi Meth. Enzymol. 392:120 (2005)

Lu, et al. Mol. Pharmaceutics 6:763-771 (2009)

Association

170 10 20 30 40 50 60 70 80 90 100 110

0

10

20

30

40

50

60

70

KDspec. = 3.9 0.5 nM

[PAZ] (nM)

Inte

nsi

ty (

RU

)

0 25 50 75 100 125 150 175 200 225 250 275 300 325 3500

10

20

30

KDspec. = 12.6 2.5 nM

[Ds 21-mer siRNA] (nM)

Inte

nsi

ty (

RU

)

Ago2 PAZ Domain Binding to Immobilized Biotinylated ds 21-mer siRNA ds 21-mer siRNA Binding to Immobilized Ago2 PAZ Domain

-10

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-30 0 30 60 90 120 150 180 210 240 270 300

Fc=2 Spot=1-r

Ago2 PAZ100 - 1.5625 nM1/2 dilution

Inte

nsi

ty (

RU

)

Time (s)

-5

5

15

25

35

45

-30 0 30 60 90 120 150 180 210 240 270 300

Fc=2 Spot=1-r

ds 21-mer RNA1000 - 1.37 nM1/3 dilution

Inte

nsi

ty (

RU

)

Time (s)

Variation

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• Dicer and Ago2 levels vary between cell lines and disease states

• mRNA synthesis rates, steady state levels, and degradation rates of target genes also vary• target sequence can differ between species used for efficacy, safety, pre-clinical, and clinical models

Merritt, et al. NEJM 359:2641-50 (2009)

Accessibility

19Zhang, et al. Nucl. Acids Res. 31:e72 (2003)

• mRNA accessibility site tagging• microarray profiling• reverse transcriptase priming

Activity in vitro

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position along gene

Assay Conditions:[mRNA substrate] = 5 nM[siRNA] = 50 nM75 g/mL S100 fraction of cell lysate30 min reaction

Readout: siRNA-dependent RNA substrate degradation by qPCR

Considerations for RNAiTherapeutic Design

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Part of Mechanism

Target Issue?

Cell Issue?

Design Issue?

Estimated Importance

Formulation no no yes low

Internalization no yes yes high

Association no yes yes unknown

Variation yes yes no medium

Accessibility yes yes no low

Activity yes yes yes high

Higher order cell-based assays• Primary human cells (normal and disease)• Functional assays (target, pathway and phenotypic)• Tissue engineered models (single and multiple cell types)• Mechanistic assays (biomarkers, toxicity, metabolism, etc.)• In vivo models (delivery, ADME, function)

Acknowledgements

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• Rob Stanton, Qing Cao, Lingling Shen, Jason Hughes, Jeremy Little• Xu Xu, Larry Drew, Meta Foster, Yiding Yan, Tracy Chen, Yuxin Wang• Joe Wu, Gayatri Deshmuch, Mark Bernard, Suzanne Jacques-O’Hagan, Meg Mabuchi• Zhigang Wang, Liann Wang, Xiao-Qin Ren• Eugen Uhlmann (Düsseldorf)