1. Marius Wernig, MD, PhD Institute for Stem Cell Biology and Regenerative Medicine and Department of Pathology Stanford University School of Medicine Potential therapeutic applications of pluripotent stem cells

2. mid stage embryo early embryo fertilized egg EctodermMesodermEndodermGerm cells Nuclear Transfer adult tissues:

3. nucleus skin cells egg early embryo Dolly Dolly‘s “mother“ “reprogramming”

4. nucleus skin cells egg embryonic stem cells differentiated donor cells early embryo ? “Therapeutic” cloning transplantation

5. nucleus adult cells enucleated oocyte iPS cells differentiated donor cells transplantation blastocyst ? Induced pluripotent stem (iPS) cells Oct4 Sox2 Klf4 c-Myc

6. adult cells repaired iPS cells differentiated donor cells transplantation Gene repair in iPS cells Oct4 Sox2 Klf4 c-Myc iPS cells gene targeting

7. Correction of sickle cell anemia (h  S/h  S) Correction of the sickle allele by gene targeting hShS hAhA h  A/h  S #3 #25#10#11 h  A/h  S h  S/h  S treated * * Jacob Hanna, Rudolf Jaenisch

8. DEB Dystrophic (scarring) Epidermolysis (destruction of skin) Bullosa (blistering)

9. DEB RDEB Autosomal Recessive DDEB Autosomal Dominant

10. RDEB Blisters present soon after birth Diagnosis is made by absence of type VII collagen in the upper most dermis at junction of epidermis Many years of painful, expanding wounds that never heal Death from malnutrition, infection or squamous cell carcinoma (SCC)

11. Estimated incidence of RDEB is 1-2 infants per million births USA: 4 million annual births leads to 4-8 RDEB infants a year Worldwide: 75 million annual births leads to 75-150 RDEB infants a year

14. RDEB: widespread blisters causing wounds that never heal

15. Complications of RDEB Mitten hand deformity Squamous cell carcinoma

16. Pulkkinen L., Uitto J. Matrix Biology 18., 1999., p. 29-42 Cutaneous basement membrane zone

17. Human type VII Collagen (Col7A1) RDEB mutations RDEB/DDEB mutations Pulkkinen L., Uitto J. Matrix Biology 18., 1999., p. 29-42 Col7A1: 31,132bp at 3p21. 118exons (largest number yet), 8.9kb ORF, 2944 AA, ~300kDa prot. Christiano A.M. et al., Genomics. 21., 1994., p. 169-79 Gly-X-YGly-X-Y NC1 NC2

18. Ex Vivo genetic correction of RDEB Preclinical Data for FDA + Skin BMZ analyses Epidermal Sheet Production RDEB Keratinocytes From skin biopsy Viral transfer LZRSE-Col7A1 Mouse Grafting 6-12 days 3-7 days 1 days 12-60days Pig Dermis

19. Type VII Collagen expression in corrected RDEB human skin Type VII collagen at dermal-epidermal junction Human origin of the epidermis confirmed by human specific Desmoglein 3 protein staining Cell nuclear counterstain dye Hoechst 33342 10x10x Anti-Dsg3 Mab Hoechst 33342 Anti-Type VII Collagen Mab Hoechst 33342

20. Problems with retroviral gene transfer Safety risk due to random integrations of retroviruses Impossible to treat dominant forms of diseases Uncontrolled expression of COL7A1 (throughout epidermis)

21. adult cells repaired iPS cells differentiated donor cells iPS cell gene targeting approach Oct4 Sox2 Klf4 c-Myc iPS cells

22. adult cells repaired iPS cells differentiated donor cells Bottlenecks for translation to human cells Oct4 Sox2 Klf4 c-Myc iPS cells gene targeting safe reprogramming differentiation transplantation ✓

23. 1. Published iPS cell derivation strategies a. integration-free Repeated plasmid transfections (regular, minicircles) Protein transduction Adeno viruses Sendai virus (RNA-based reproductive cycle) b. integration-based Standard Moloney viruses (retroviral silencing) Dox-inducible lentiviruses Transposase-mediated integration (Piggy-back) Excisable lentivirus (4F cassette)

24. Derivation of EB-iPS cell lines

25. 1 1 3 3 1 1 14 1 1 2 2 118 1 1 3 3 Patient 1 Patient 2 Patient 3 Patient 4 1 1 15 118 1 1 117 118 1 1 54 118 1 1 86 118

26. Pei Wang, Thomas Leung, Seung Kim

27. Zinc Finger Nucleases Two domains: Zinc Finger domain DNA-binding domain DNA-cleavage domain High specificity: 18 bp Xenopous: >95% C. elegans: 15% Drosophyla: 15% Human cells: 50%

28. Differentiation of hES cells into engraftable keratinocytes Guenou et al Lancet 2009

29. Testing function of iPS- derived keratinocytes Cell autonomous expression in chimeric skin Progenitor Assay in chimeric skin Callahan et al. Genes Dev 2004 Sen et al. Nature 2010

30. Vittorio Sebastiano Dana Ting Yeo Bahareh Haddad Jesse Karmazin Acknowledgements Sandra Melo Anthony Oro Thomas Leung Pei Wang Seung Kim Zurab Siprashvili Andrea Tichy Al Lane Funding: California Institute for Regenerative Medicine