Yoshiji Fujita, Ph.D.President & CEO

CytoPathfinder Inc.(http://www.cytopathfinder.com)

(3rd March, 2006; Tokyo)

「事業化迫るRNA工学・RNA医薬、その先端と課題」

RNA医薬の新潮流

Current Status of RNAi MedicinesNeuronal Diseases 11Virus Infections 22Cancer 7Opthalmology 4Infectious Diseases 1Immunodeficiency 5GI & Metabolic Diseases 4

Total 54

Pre-clinical 51 Phase – I 3

As of 2004 ; Some are now in P-II and/or P-II Completed

AlnylamAlzeheimer’s Autoimmune Disease

Obesity, Lipidosis, Hepatitis

Neuropathic Pain

Asthma, Chronic Obstacle Pheumonia

Discovery Pre-Clinical Phase-I

AMD (VEGF-siRNA)

Terminated (Aug. 2005)

Merck (Hybridon)

✓Huntington’s α-SynucleinMayo Clinic

Cancer, InflammationMerck, IsisWO OI/36646 2001

Genesis R&D Allergy (IND in 2005)

Atugen Diabetes (IND in ‘05/’06)

Sirna AMD (VEGFR-1)Phase I (〜 ‘04)

Lilly

Cancer (VEGFR-1)Lilly

CytRx Diabetes, Obesity

Acuity Phama. AMD (Cand5 (VEGF) )Phase I (〜 ‘04)

ALS (IND ’05 scheduled)

Nucleonics HBV (Novosom)HCV (Novosom)

IC-Vec HBV, HCV

Many companies are targeting viral infection diseases

Phase II (’06)

Impacts of RNAi Technologies on Drug Discovery1. Target Identification & Validation Process

Central Dogma Changed – Direct Forward

2. Quick Move to Clinical Trial when Target Validated

* Elimination of Redundant Works !* Cost / Time Saving !

Less than 2 Years

* High Success Rate !* Cost / Time Saving !* Estimated To Shortening ca. 4 Years Till Launching Products

3. RNAi Medicines = Assumed to be ChemicalsNo Need Troublesome QC Like AntibodyCombination of 4 Nucleotides Fits For Every Therapeutic NCEs* Production ; Simple & Multi-Purpose Use

4. Pharmacogenomics = Simple (Focused Target)

Barriers to Tackle RNAi Medicine

1. Cost of Goods To be Resolved in Future

2. Very Unstable in Serum / Plasma Need DDS* Half-Life Controllable from Minutes to Hours / Days

* Need Cell / Tissue-Specific Delivery

3. Complicated IP Situation* Who Owns / Wins What ? What Claims Granted ?

* Need More Methods

* Applicable Therapeutic Area vs. Reimbursement

How Changed ?

Target Identification & Validation Process

Magnitude of Impact ?

Multiple Phenotypic Readouts

Cellular Functions

Validated Target

Target molecules

Conventional Approach

Disease

Hard to prioritize !

Series of experiments of cDNAcloning, protein expression and establishment of assay (e.g. Enzyme, Receptor Assay), followed by HTS.

Apoptosis / NecrosisProliferation / ShapeAdhesion / AggregationSignaling Profile Cytokine ProfileSecretion

High Throughput RNAi Transfection

Speed, Efficiency, Reproducibility ↑↑

3/14

Hits

RNAi MedicineLow Molecular Medicine

Significant Improvement in

TargetID

TargetVal

Lead Opt

Pre-Clinical

Phase III

Approval & Launch

Phase II

Phase I

HTS & Lead IDDisease

Drug Discovery Process

Project Number 57 36 / 22 14 8 / 4.5 / 2 1

siRNA High Success RateTime (M) 24 24 24 73 8

siRNA < 24 Months

Power of TFA SystemEvidences !

At Research Institute of Cell Engineering / AIST-Amagasaki

(TFA = Transfection Array)

1. Compound Profiling of Unknown Target (Cancer)

2. Identify Molecules Responsible For Cellular Events (Neurite Extension)

3. Pathway Analysis

4. Sequential Analysis Of Cellular Events

Inkjet printingInkjet printingRNAiRNAi x Reporterx Reporter Pour cellPour cell suspension suspension into dishinto dish

CultivationCultivation

ObserObservvationation

PathwayPathway analysanalysiiss

Co-transfection array① siRNA and reporter are co-transfected

Putting different kinds of siRNA on a spot allowssimultaneous knock-down of multiple targets

② High screeningefficiency usingcellular functionalassay

③ Unique analysis algorithm

About Transfection Array System

The principle of acceleration of solid-phase transfection

Nuclear

Narrow space

Cell membrane

1) The accelerator activates phagocytosisof DNA/polymers.

2) DNA may easily access to the nuclear because the space between cell membrane and nuclear membrane is closed very much.

DNA

Cell membrane

Nuclearmembrane

cell

Nu

Accelerator DNA/polymer

Glass slide

H2OH2O

H2O H2O H2OH2O

Merit ２：Solid-phase TransfectionHeighten throughput, lower in cost of RNAi

8/14

Pre-incubationOf cells

24 hr（critical）

Inoculation of cells

RNAi＋transfection reagents

Added after 5 min.

Exchangemedium

After 1 hr（critical）,change medium

48 – 72 hr incubation

Inoculation of cells RNAi layer

48 – 72 hr incubation

【Solid-phase transfection】【Conventional transfection】

Both transfection and incubation are preformed simultaneously.

Functional AssayFluorescence reporterChromogenic reporter Immuno-stainingDNA micro-arrayProtein-chip

All kinds of wells (96-384) and arrays are available.

High ThroughputMinimizing cost and maximizing throughput of RNAi by solid-phase transfection

Advantages over Conventional MethodCurrent problems of RNAi technology and our solutions

① Transfection Efficiency 10-20 times increase

Average in optimal conditions = 20%; Only 1-2% for difficult cell types

Average = 60-80%; 40% in case of difficult cell types

② Throughput 100 times increase

Manual process due to multiple-step and complicated operation

Accelerator

Solid-phase Transfection Complete automation by

single-step operation（Solution volume: milliliter nano-liter (1/1000,000)

③ Analysis of Cellular-

Limited to specific analysis per function, induced per stimuli (RNAi, drug candidate, etc.)

Parallel and Real-time Monitoring Analysis of various intra-cellular functions

AlgorithmEvent InformationFirst in the world to perform high-level functional analysis

0

GFP

inte

nsity

/mm

2 10

8

5.0

2.5

10

7.5

HEK293 HeLa NIH3T3 HepG2 hMSCs

easy difficult

Conventional MedthodCytoPathfinder’s Method

Improvement in Transfection efficiency using CytoPathfinder’sproprietary promoters

Human Cells

Origin TransfectionEfficiency (%)

hMSC Mesenchymal 40Stem Cell

HepG2 Hepatic Cancer 80

MCF7 Breast Cancer 80

U251 Glioma 80

SHSY5Y Neuroblastoma 60

NTERA-2 Embryonic 80Carcinoma

High Transfection Efficiency

Practical Approach Toward RNAi Medicine And Conventional Low Molecular Medicine

siRNA TFACell

Target Molecules

Representatives

siRNAs➀ ➁ ③ ④ ⑤

20〜30 siRNAs/Gene

Prioritize the most effective sequences

siRNA ③DDS

In vitro, in vivo

Drugs

SNPs (Allele, Frequency, Ethnics…) Pharmacogenomics

Pipelines

Efficacy; Responder / Non-Responder

Proteins (Biological Functions) Membrane Protein Crystallization

Collaborations- Academia- Pharmaceuticals Comp.

Aptamers

Compound Profiling

Rationale Drug CombinationMechanism Existing Drugs

Low Molecular Drug

Drug Delivery System is The KeyTo Succeed In RNAi Medicine

* Liposome / Cubosome

* Virus Vector

* Chemical Modification

- PEG- Cholesterol

NIH Sets Aside $5.6 Million to Fund RNAi Research Programs in 2006 8/12/2005

The cash, which is open to US-based institutions only, is aimed at helping researchers improve their understanding of the uptake and processing of RNAi compounds by target tissues; assess stability, half-life, and off-target effects of these agents in target tissues; and determine optimal delivery methods for RNAi molecule uptake by the target tissues.

CytoPathfinder’s Endeavor For CubosomeType II Cubic Liquid Crystal

Water ChannelΦ〜5 nm

Φ〜100 nm

Proprietary Lipid ; 55〜65%Water ; 45〜35%

Characteristics compared with Liposome;1. Superior strength against mechanical stress to preserve structure

2. Larger lipid/water-surface per unit volume (~500m2/g lipid )Incorporating larger amount of chemicals (e.g. proteins of ca. 500KDa)

3. Controlled release / reactions through water channel size

Molecular Shape and Liquid Crystals

Water Continuous

Alkyl-ChainContinuous

Hexagonal Liquid Crystal (HI)

Lamellar Liquid Crystal (La)

Cubic Liquid Crystal

Revrs. Hexagonal Liquid Crystal (HII)

キュービック液晶

他の相領域間の狭い濃度範囲に存在光学的に透明等方性で複屈折性を示さない結晶類似の構造、機械的に強い

Complicated IP SituationDespite New Patents on the Horizon, RNAiIP Field Still Muddy; Is Licensing Always an Option? [1/26/2006]

In 2005, RNAi Science Advances and Big Pharma Takes Notice[1/5/2006] ・・・・・ But,

Both Alnylam Pharmaceuticals and Sirna Therapeutics said this week that they have gotten word from the US Patent and Trademark Office that certain of their IP would be issued as patents — yet questions about the RNAi IP landscape remain.

Qiagen

Contents Suppliers Market Size = $ Ca. 500 MGrowth = Ca. 10% / YearConsumables market ; sample prep, RNAi, microRNA, gene expression, array products

Sigma-Aldrich

- Optimizing G/C Content

- Off-target silencing effects- Contamination

- Whole Genome (> 20K siRNAs)

Ambion (ABI)Dharmacon(Fisher Scientific)

Invitrogen

Validation ! Cost ! Reproducibility !

Cenix GenOway

Alnylam Acuity CytRxSirnaPharmaceuticals Therapeutics Corp.Pharmaceutical

Pharma. Co.

Bayer, Schering AGNovartis, Merck Allergan, Lilly

Phase II / DB macular edemaCompleted phase II / age-related macular degeneration

AstraZeneca Alcon

?

Non-Profit Consortium

RNAi Therapeutic Users

Three IP Barriers for ＲＮＡｉ Drug Discovery

Research Tool- Target Identify- Target Validation- Pathway Analysis- Compound Profiling- Multi Well / Array (TFA)

siRNA, miRNA, shRNA…

低分子医薬

DDS- Liposome / Cubosome- Chem. Modification(Chol. PEG etc.)

- Viral Vector

Therapeutic UseAlnylam (2002-226; 30 IPs)

Sirna (213 IP-Hits)

CytRx (56 IP-Hits)(18〜23 Nucleotides)

EP1144623 (〜25 Nucleotides)EP1214945 (15〜49 Nucleotides)

核酸医薬

Optimal Nuclear Sequence

Discovery Target

RNAi technology is truly “Destructive” to force changes in whole drug discovery process ;

CONCLUSION

RNAi medicine is direct forward outcomes but is just one exit !Low molecular, existing & under-development drugs can be re-profiled for better and safer uses (Alternative indication as well)

Limitation by cell-lines ; not reflecting “real world”“Increasing demands on primary & stem Cells “

Cutting-Edge Cell Sorting Technology For

Phenotypic Study

Future Need !