Human Embryonic Stem Cells: Considerations for

Therapeutic Development

Jane Lebkowski Ph.D.Geron Corporation

Cell, Tissue and Gene Therapy Advisory Committee MeetingApril 10, 2008

Human Embryonic Stem Cells

Blastocyst

Human Embryonic Stem Cells

Neural Cells

Spinal CordInjury

Parkinson’sDisease

Cardiomyocytes

Heart Failure

Islets

Diabetes

Osteoblasts

OsteoporosisAnd BoneFractures

Chondrocytes

Arthritis

Hepatocytes

Drug DiscoveryLiver Failure

Dendritic Cells

Tolerance InductionCancer Immunotherapy

Large Characterized cGMP Banks

Therapeutic Cells

hESCs: New Cell Type for Potential Broad Therapeutic Application

Simplify, Standardize, and Scale hESC Growth

Develop Reproducible Methods to Selectively Differentiate Cells

Develop Parameters to Characterize Differentiated Cell Populations

Define Efficacy, Potency, & Safety of Cell

Populations

Develop Methods to Deliver Cells to Target Tissue

Define Need for Immunosuppression

Demonstrate Safety & Efficacy in Clinical Trials

Develop Scaled Low Cost Production Methods

Necessary Technological Developments

Spinal Cord Injury: GRNOPC1 Transplantation

Differentiation Process

• Characterization of Materials• Starting Material• Adventitious Agents

• Characterization of Unit Operations• Cell Density• Culture Format• Timing of Induction

• Storage

• Release Testing

GRNOPC1 Manufacturing Process Flow Diagram: Differentiation

ST

AG

E I

I: D

IFF

ER

EN

TIA

TIO

N

CommentsAnalytical Methods

Unit OperationsMaterials

Cell Culture Vessel

uhESC Expansion

Passaging Enzyme

Glial Progenitor Medium (GPM)

Matrix Coated Vessels

Passaging Enzyme

Cell Count Enumeration

STAGE III: Harvest,

Formulation,Fill and Finish

Medium

Growth Factor

Supplement

Medium

Matrix Coated Vessels

GPM

GPM

GPM

D-PBS

Growth Factor

Growth Factor

Growth Factor

Growth Factor

Growth Factor

Growth Factor

GPM

Growth Factor

GPM

Growth Factor

= Analytical Method

Key = Off Page Reference

= Process FlowUnit Operation

Raw Material

= Manufacturing Stage

1 = Process Step

Prepare Embryoid Bodies8

Culture Embryoid Bodies in

Suspension9

Plate Embryoid Bodies10

Culture Cells11

Passage Cells12

Culture Cells13

Day Number Specified

Day Number Specified

Day Number Specified

Day Number Specified

GRNOPC1 Contains Oligodendroglial Progenitors

Lineage Marker

Neural Progenitors Nestin

Oligodendroglial Progenitors Olig 1

Oligodendroglial Progenitors NG2

Oligodendroglial Progenitors PDGFRa

Early Ectoderm Pax 6

Early Ectoderm Sox 10

Neurons TubIII

Astrocytes GFAP

Early Endoderm HNF3

Endoderm AFP

Early Mesoderm GATA4

Mesoderm MSA

Undifferentiated hESCs OCT4

Undifferentiated hESCs Tra-1-60

Phenotype Testing of Over 75 Lots

• Multiple Markers Required

• Lineage Specific Markers

• Specific Antibodies

• Detection and Quantitation Limits of Assays

• Potency Assays

Challenges

IdentityPurity

StrengthPotency

Considerations for Nonclinical Studies for hESC-Based Therapeutics

Final Product: What is the Product Designed to Do?What is the Target Site for Activity?

Final Product Production Scale:Does Scale Effect Composition?

Formulation:Cryopreserved Format?Selective Cell Survival?

Clinical Administration:Site of Administration?Effects on Performance and Potential Adverse Events?Need for Immunosuppression?Dose Required?

Pharmacology Studies9 mos GRNOPC1

hNucEC

1mm

100 m

9 mos vehiclehNucEC

1mm

100 m

In Vitro Activity• Protein and Gene Expression• Factor Production• Structural/ Metabolic Activity

In Vivo Survival• Survive Delivery• Immune Responses

Phenotype• In Vitro and In Vivo• Maturation Over Time• Proliferative Capacity

Activity in Disease Models• Clinical Efficacy• Histological Efficacy• Dose• Human Equivalent Dose• Delivery Site and Volume• Timing of Treatment

What Is the Activity of the Cells?

Where Do The Cells Go?

GRNOPC1 Migration within the Spinal Cord

0

2

4

6

8

10

12

14

16

18

20

2 day 2.4x10^5GRNOPC1

2 day 2.4x10^6GRNOPC1

14 day 2.4x10^5GRNOPC1

14 day 2.4x10^6GRNOPC1

180 day 2.4x10^5GRNOPC1

180 day 2.4x10^6GRNOPC1

Time Point and Dose

Ce

ll D

istr

ibu

tio

n (

mil

lim

ete

rs)

2 day 2.4x10^5 GRNOPC1

2 day 2.4x10^6 GRNOPC1

14 day 2.4x10^5 GRNOPC1

14 day 2.4x10^6 GRNOPC1

180 day 2.4x10^5 GRNOPC1

180 day 2.4x10^6 GRNOPC1

Safety and Efficacy Implications

• Sensitivity of Assay

• Site for Intended Activity

• Sufficient Cells at Site

• Distribution Outside Target Site

• Migration at Local Site

• Over Extended Time

Toxicology Studies

Toxicity of Delivery

• Doses of Product

• Tox Model

• Feasibility of Model

• Duration of Studies

• Duration of Human Cell Survival

GRNOPC1

• Toxicity of Delivery

• Animal Survival

• Clinical Observations

• Systemic Toxicity

• Hematological

• Coagulation Parameters

• Clinical Chemistries

• Macropathology

• Micropathology

• Allodynia

Considerations

Tumorigenicity Studies

• Teratomas

• Ectopic Tissues

• Local Injection Site

• Distal Sites

Challenges

• Human Dose

• Long-Term Cell Survival

• Large Numbers of Animals

• Mimic Human Setting

• Large Animals?

• Homologous ESC Systems?

GRNOPC1 Deliberately Spiked with hESCs

Tumorigenicity Studies: Lessons Learned

Important Factors In Teratoma Formation

• hESCs Cell Number

• Site of Implantation

• Cell Aggregation State

2 x 106 CellsIntraspinal Cord Injection

Assessment 12 mos.

Allogenicity Studies• Immunosuppression Required?

• Duration of Immunosuppression?

Challenges

• hESC-Based Products are Xenografts in All Animal Models

• Allogenicity of Maturing Cells In Vivo

• Humanized Models Challenging

• In Vitro Analyses

• Allogenicity In Vitro

• Inflammatory Site

• Remove Immunosuppression?

• Monitor Engraftment?

• Monitor Rejection?

• Effects of Rejection

Approaches

Cell Delivery in the Clinic

Considerations

• Local Delivery

• Delivery Site

• Intraoperative Delivery

• Rate of Delivery

• Skill Set Required for Delivery

• Effect of Delivery Device on Cells

• Training

Design of Clinical TrialsKey Consideration: Patient Safety

• Protocol

• Delivery

• Logistics of Trial

• Minimize Potential Risks

• Balance Risk with Potential Efficacy

• Define Adverse Events

• Monitor for Adverse Events

• Monitor Cell Survival

• Assess Outcome Measures

• Short and Long-Term Follow-up

• Steering Committee

• DMC

• Outcomes Committee

• Neurosurgeons

• Radiologists

• Investigators

• ESCRO Committee

Interactions with:

Phase 1 Multi-Center Trial

• Open Label Trial

• Subacute, Functionally Complete T3-T12 Lesions

• Transplant 7-14 Days Post Injury

• Temporary Immunosuppression

• Primary Endpoints: Safety Assessments

• Secondary Endpoints: Multiple Outcome Measurements

• Frequent Short and Long-Term MRIs

• Immunological Monitoring

Conclusions

• Numerous Considerations in Developing Cell Therapies

• Some Questions Common To All Cell Therapies

• Some Questions More Specific to hESC-Based Therapies

• Specific Nonclinical Study Designs Based on Clinical Considerations

• Clinical Trial Designs Require Interdisciplinary Input

• Frequent Monitoring Required