FDA Presentation, April 2008


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"Safety Considerations for the Clinical
Application of Human Embryonic Stem Cells"


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FDA Presentation, April 2008

  1. 1. Safety Considerations for the ClinicalApplication of Human Embryonic Stem CellsApril 2008
  2. 2. General Considerations
  3. 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 initialoptimism 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.3
  4. 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 immunerejection of transplanted cells.• Confirm results with the human cells transplanted to immunodeficient/immunosuppressedrecipients.• Appropriate identification methods should be established so that the end dispositionof 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-repairmechanisms 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 andwhether the cells get trapped in the lungs, heart, liver, spleen or brain.4
  5. 5. Stimulation of Endogenous Repair Activities• Cells do not survive long-term.• Highly dependent on immune reaction.• Immune reaction varies depending on species combinationbeing studied.• Should be tested in multiple species combinations to safelymake sure cells do not survive.• Effects more likely to be due to mechanisms independent ofthe cell type used.• Benefits should be demonstrated in comparison to control cells.• There should be benefits unique to the therapeutic cell beingused.• 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 togrowth (e. g. cerebral spinal space)5
  6. 6. Graft Dependent Repair• Graft can be analyzed in detail.• Benefit vs presence of undesired cell types can beassessed.• 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 betterassessed.• Fewer unknowns when cells survive.• Site of administration and injection method moreimportant.6
  7. 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 fusionartifact.• Cell survival needs to be sufficient to account for functional benefit orbenefit needs to be dependent on cell type used.• Transplant of human cells to experimental animal species allows forprecise assessment of cell survival.7
  8. 8. Cross Species Transplants• Immuno-permissive Model• Systemic immunosuppression (Cyclosporin, FK-506, etc).• Immuno-deficient recipient.• Immunodeficient animal models vary depending on cellsbeing transplanted and the location of the transplant, sothe correct model needs to be selected.• Nude Mouse and Rat models acceptable for transplant toan immunoprivileged site.• NOD, SCID, ß2 knockout mouse strain or equivalentrequired for most cells when not transplanted to animmunoprivileged site (must eliminate B-cell, T-cell and NKcell activity).8
  9. 9. Cell Sourcing• The cells to be used clinically should be derived from thesame source as the cells tested pre-clinically.– Human cells behave differently than those from otherspecies.– Require use of immune suppression or immune deficientrecipients.• Cell shipping and storage conditions should be modeledafter what will be used clinically.– Shipping and storage can effect cell function and survival.– Thawing of cryopreserved cells at clinical site introducesrisk and sterility issues.– A specially trained team needs to be utilized if cells are tobe shipped frozen and thawed before administration.9
  10. 10. Cell Manufacturing• Cells used in pre-clinical studies should be produced byprocess 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.10
  11. 11. Mode of Delivery• Injection device to be used clinically should be tested forbiocompatibility.• Look at short term effects on cell viability.• Look at long term effects on cell function.• Cells can reveal deficiencies in the injection device notrevealed by passing media through the device.• Mass effect created by the cells in suspension can dragdebris 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 testsafety for human injection.11
  12. 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.12
  13. 13. Specific Example: hES-derived RetinalPigmented Epithelial (RPE) Cells
  14. 14. 14RPE Cell FunctionRPELayerDeterioratesin Patientswith AMDand otherRetinalDegenerativeDiseases Pigmented cells Allow for direct selection Adhere to Bruchs membrane Phagocytose shedphotoreceptor segments Produce trophic factorshES derived RPE cells
  15. 15. RPE Cell Production157 – 14 days 2 months 1 month
  16. 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.16
  17. 17. RPE Survival and Integration• Human to rat transplant• Immunopriviliged location• Cyclosporin Immunosuppression• Cells survive and provide long-term benefit, >100days.• No evidence for cell rejection• Cells integrate and function to preventphotoreceptor loss• Dose can be estimated for larger eye of a human.• Primate studies undertaken to confirm capabilityto inject projected cell dose.17
  18. 18. 18RPE Rescue in the RCS RatRCS retina at P100 withH9-RPE injection:A: low power view of retinashowing extensive rescue.B: high power of (b)showing rescuedphotoreceptors.C: high power of (c)showing non-rescue area.PhotoreceptorRescue in RCS Ratby transplantedH9-RPE cells
  19. 19. Clinical Considerations for the use of Retinal• 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 oreliminating need for immunosuppression.• Eye is a self-contained space, limiting issues ofcell migration.• The target location in the eye can be easilyvisualized and monitored.19