Marius Wernig, MD, PhDInstitute for Stem Cell Biology and Regenerative

Medicineand Department of Pathology

Stanford University School of Medicine

Potential therapeutic applications of pluripotent stem cells

mid stage embryo

early embryo

fertilized egg

Ectoderm Mesoderm Endoderm Germ cells

Nuclear Transfer

adult tissues:

nucleus

skin cells

egg

early embryo

Dolly

Dolly‘s “mother“

“reprogramming”

nucleus

skin cells

egg

embryonic stem cells

differentiated donor cells

early embryo

?

“Therapeutic” cloning

transplantation

nucleus

adult cells

enucleated oocyte

iPS cells

differentiated donor cells

transplantation

blastocyst

?

Induced pluripotent stem (iPS) cells

Oct4Oct4Sox2Sox2Klf4Klf4

c-Mycc-Myc

adult cells

repaired iPS cells

differentiated donor cells

transplantation

Gene repair in iPS cells

Oct4Oct4Sox2Sox2Klf4Klf4

c-Mycc-MyciPS cells

gene targeting

Correction of sickle cell anemia (hS/hS)

Correction of the sickle allele by gene targeting

hS

hA

hA/hS

#3 #25#10

#11

0

2

4

6

8

10

12

14

16

18

Hb (g/dl) RBC (x10^3/µl)

hA/hShS/hStreated

**

Jacob Hanna, Rudolf Jaenisch

DEBDystrophic

(scarring)

Epidermolysis(destruction of skin)

Bullosa (blistering)

DEBRDEB

Autosomal Recessive

DDEB

Autosomal Dominant

RDEBBlisters 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)

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

RDEB: widespread blisters causing wounds that never

heal

Complications of RDEB

Mitten hand deformitySquamous cell

carcinoma

Pulkkinen L., Uitto J. Matrix Biology 18., 1999., p. 29-42

Cutaneous basement membrane zoneCutaneous basement membrane zone

Human type VII Collagen (Col7A1)

R

DE

Bm

uta

tion

sR

DE

B/D

DE

Bm

uta

tion

s

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 Gly-X-YGly-X-YNC1 NC

2

Ex Vivo genetic correction of RDEB Preclinical Data for FDA

+ Skin BMZ analyses

Epidermal SheetProduction

RDEBKeratinocytesFrom skin biopsy

Viral transferLZRSE-Col7A1

MouseGrafting

6-12 days6-12 days 3-7 days3-7 days 1 days1 days 12-60 12-60 daysdays

Pig Dermis

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 10x10xAnti-Dsg3 MabAnti-Dsg3 MabHoechst 33342Hoechst 33342

Anti-Type VII Collagen MabAnti-Type VII Collagen MabHoechst 33342Hoechst 33342

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)

adult cells

repaired iPS cells

differentiated donor cells

iPS cell gene targeting approach

Oct4Oct4Sox2Sox2Klf4Klf4

c-Mycc-MyciPS cells

adult cells

repaired iPS cells

differentiated donor cells

Bottlenecks for translation to human cells

Oct4Oct4Sox2Sox2Klf4Klf4

c-Mycc-MyciPS cells

gene

targ

etin

g

safe reprogramming

differentiation transplantation✓

1. Published iPS cell derivation strategies

a. integration-free

Repeated plasmid transfections (regular, minicircles)Protein transductionAdeno virusesSendai 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)

Derivation of EB-iPS cell lines

11 33

11 1414

11 22

118118

118118

118118

11 11811833

Patient 1

Patient 2

Patient 3

Patient 4

11 1515 118118

11 117117 118118

11 5454 118118

11 8686 118118

Pei Wang, Thomas Leung, Seung Kim

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%

Differentiation of hES cells into engraftable keratinocytes

Guenou et al Lancet 2009

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

Vittorio Sebastiano

Dana Ting Yeo

Bahareh Haddad

Jesse Karmazin

Acknowledgements

Sandra MeloAnthony Oro

Thomas LeungPei WangSeung Kim

Zurab SiprashviliAndrea TichyAl Lane

Funding: California Institute for Regenerative Medicine