Safety Considerations for the Clinical Application of Human Embryonic Stem Cells April 2008

1. Safety Considerations for the Clinical Application of Human Embryonic Stem CellsApril 2008

2. General Considerations

3. Concerns • Genetic stability of hES cells and their differentiated derivatives. • Karyotype analysis can miss point mutations or chromosomal exchange. • Cell growth requirements and growth rate are important variables for measuring genetic stability • More rapid growth rate can signal problems. • Reduced stringency for cell growth can signal problems. • Factors that accentuate this risk: • Scale-up to produce the numbers of cells needed for clinical administration. • Culture/purification processes. • Feeder-free culture of hES. • Differentiation Signals are not present in adult tissues to instruct ES cells despite initial optimism that this was the case. • Pre-differentiation of cells will be needed. • Accentuates the need for eliminating undifferentiated ES cells from the population. • Long-term follow-up critical to assess tumorigenic potential.

4. Safety Study Requirements • Proof of principle/efficacy animal studies provide critical safety data. • Should mimic route and site of delivery planned clinically. • Start with syngeneic models if possible as this avoids the issues associated with immune rejection of transplanted cells. • Confirm results with the human cells transplanted to immunodeficient/immunosuppressed recipients. • Appropriate identification methods should be established so that the end disposition of transplanted cells can be determined. • Survival or non-survival of transplanted cells needs to be ascertained to assess safety. • Characterization of graft elements or determination that endogenous self-repair mechanisms are being stimulated. • Route of cell delivery important. • Systemic route of delivery vs. direct injection at the site of repair. • Systemic delivery poses unique risks and requirements for the distribution of cells and whether the cells get trapped in the lungs, heart, liver, spleen or brain.

5. Stimulation of Endogenous Repair Activities • Cells do not survive long-term. • Highly dependent on immune reaction. • Immune reaction varies depending on species combination being studied. • Should be tested in multiple species combinations to safely make sure cells do not survive. • Effects more likely to be due to mechanisms independent of the cell type used. • Benefits should be demonstrated in comparison to control cells. • There should be benefits unique to the therapeutic cell being used. • Ectopic survival still needs to be assessed. • There may be cell survival outside the target tissue. • Can be a problem if cells distribute to a site more conducive to growth (e. g. cerebral spinal space)

6. Graft Dependent Repair • Graft can be analyzed in detail. • Benefit vs presence of undesired cell types can be assessed. • Benefits can be correlated to cell survival. • Efficacy studies give significant safety data. • Graft can be assessed for risk of overgrowth. • Consistency of cell preparations can be better assessed. • Fewer unknowns when cells survive. • Site of administration and injection method more important.

7. Cell Survival Assessments • Cell tracking tools can be problematic. • Cell tags (GFP, BrdU, ß-gal, Iridium, Fe-particles or fluorescent dyes) are all subject to significant artifact. • Toxic to cells • Artifactual fluorescence or enzymatic activity. • Markers can be transferred from cell to cell due to fusion, macrophages take up cellular debris containing tags. • Cell survival vs cell fusion needs to be distinguished. • Many instances of cell survival have been shown to be due to cell fusion artifact. • Cell survival needs to be sufficient to account for functional benefit or benefit needs to be dependent on cell type used. • Transplant of human cells to experimental animal species allows for precise assessment of cell survival.

8. Cross Species Transplants • Immuno-permissive Model • Systemic immunosuppression (Cyclosporin, FK-506, etc). • Immuno-deficient recipient. • Immunodeficient animal models vary depending on cells being transplanted and the location of the transplant, so the correct model needs to be selected. • Nude Mouse and Rat models acceptable for transplant to an immunoprivileged site. • NOD, SCID, ß2 knockout mouse strain or equivalent required for most cells when not transplanted to an immunoprivileged site (must eliminate B-cell, T-cell and NK cell activity).

9. Cell Sourcing • The cells to be used clinically should be derived from the same source as the cells tested pre-clinically. • Human cells behave differently than those from other species. • Require use of immune suppression or immune deficient recipients. • Cell shipping and storage conditions should be modeled after what will be used clinically. • Shipping and storage can effect cell function and survival. • Thawing of cryopreserved cells at clinical site introduces risk and sterility issues. • A specially trained team needs to be utilized if cells are to be shipped frozen and thawed before administration.

10. Cell Manufacturing • Cells used in pre-clinical studies should be produced by process to be used for clinical production. • Cell Source can affect risk of tumor or teratoma formation. • Undifferentiated vs Differentiated ESC • Purity of cell preparation • Mode of culture can affect risk of tumor or teratoma formation. • Monolayer vs cell clusters. • Undifferentiated ES cells can linger in cell clusters. • Less likely an issue with monolayer culture. • Selection process • Length of the selection process is important. • Whether cells are cultured after selection is important.

11. Mode of Delivery • Injection device to be used clinically should be tested for biocompatibility. • Look at short term effects on cell viability. • Look at long term effects on cell function. • Cells can reveal deficiencies in the injection device not revealed by passing media through the device. • Mass effect created by the cells in suspension can drag debris from the injection device not seen with media alone. • Debris cause foreign body reactions at site of injection. • Specific large animal models may be required to test safety for human injection.

12. Tumorigenisis and Biodistribution Studies • Can ideally be combined with animal efficacy studies. • Use immunodeficient of immunosuppressed host. • Use same route of delivery and target location planned for clinical studies. • Not always feasible • Independent studies • Immunodeficient host • Cells tested using route of delivery and location to be used clinically. • Positive control to show cells can survive in host. • Test cells produced by GMP process • This includes any scale-up processes • Should be done with cells under storage and shipping conditions.

13. Specific Example: hES-derived Retinal Pigmented Epithelial (RPE) Cells

14. RPE Cell Function RPE Layer Deteriorates in Patients with AMD and other Retinal DegenerativeDiseases hES derived RPE cells • Pigmented cells • Allow for direct selection • Adhere to Bruchs membrane • Phagocytose shed photoreceptor segments • Produce trophic factors

15. RPE Cell Production 7 – 14 days 2 months 1 month

16. RPE Cell Characterization • RPE cells derived from hES differentiation. • RPE cells selected based on pigmented appearance. • Karyotype confirmed before and after expansion. • After selection RPE cells are cultured and expanded in monolayer culture. • Cells screened for homogeneity and pigment content. • Cells screened for absence of hES cell markers by: • PCR, Western Blot, Immunohistochemistry (e. g. Oct-4, Nanog, Sox-2, etc.). • Non detectable. • RPE screened for presence RPE markers: • PCR, Western Blot, Immunohistochemistry (RPE65, Bestrophin, PEDF, etc) • >95% • RPE screened for in vitro function: phagocytosis, elastin secretion and PEDF production.

17. RPE Survival and Integration • Human to rat transplant • Immunopriviliged location • Cyclosporin Immunosuppression • Cells survive and provide long-term benefit, >100 days. • No evidence for cell rejection • Cells integrate and function to prevent photoreceptor loss • Dose can be estimated for larger eye of a human. • Primate studies undertaken to confirm capability to inject projected cell dose.

18. RPE Rescue in the RCS Rat Photoreceptor Rescue in RCS Rat by transplantedH9-RPE cells RCS retina at P100 withH9-RPE injection: A: low power view of retina showing extensive rescue. B: high power of (b) showing rescued photoreceptors. C: high power of (c) showing non-rescue area.

19. Clinical Considerations for the use of Retinal Pigmented EpithelialCells • Limitation of Risks • Small dose of cells required in the eye. • Confined space. • Focal degeneration • No special devices required for eye injection. • Eye is an immuno-privileged location reducing or eliminating need for immunosuppression. • Eye is a self-contained space, limiting issues of cell migration. • The target location in the eye can be easily visualized and monitored.