Transcript Potential therapeutic applications of pluripotent stem cells

Potential therapeutic applications of pluripotent
stem cells
Marius Wernig, MD, PhD
Institute for Stem Cell Biology and Regenerative Medicine
and Department of Pathology
Stanford University School of Medicine
fertilized egg
early embryo
Nuclear Transfer
mid stage embryo
Ectoderm
Mesoderm
adult tissues:
Endoderm
Germ cells
Dolly‘s “mother“
Dolly
early embryo
“reprogramming”
egg
skin cells
nucleus
“Therapeutic” cloning
transplantation
differentiated donor cells
embryonic stem cells
early embryo
egg
?
skin cells
nucleus
Induced pluripotent stem (iPS) cells
transplantation
differentiated donor cells
iPS cells
blastocyst
Oct4
Klf4
Sox2
c-Myc
enucleated oocyte
?
adult cells
nucleus
Gene repair in iPS cells
transplantation
differentiated donor cells
repaired iPS cells
gene targeting
iPS cells
Oct4
Klf4 Sox2
c-Myc
adult cells
Correction of sickle cell anemia (hbS/hbS)
Correction of the sickle allele by
gene targeting
hbS
hbA
18
16
*
14
*
12
hbA/hbS
hbS/hbS
treated
10
8
6
4
2
0
Hb (g/dl)
RBC (x10^3/µl)
Jacob Hanna, Rudolf Jaenisch
DEB
Dystrophic
(scarring)
Epidermolysis
(destruction of skin)
Bullosa
(blistering)
DEB
RDEB
Autosomal Recessive
DDEB
Autosomal Dominant
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)
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 deformity
Squamous cell
carcinoma
Cutaneous basement membrane zone
Pulkkinen L., Uitto J. Matrix Biology 18., 1999., p. 29-42
Gly-X-Y
Gly-X-Y
RDEB/DDEB
mutations
NC1
NC2
RDEB
mutations
Human type VII Collagen (Col7A1)
Col7A1: 31,132bp at 3p21. 118exons (largest number yet), 8.9kb ORF, 2944 AA, ~300kDa prot.
Pulkkinen L., Uitto J. Matrix Biology 18., 1999., p. 29-42
Christiano A.M. et al., Genomics. 21., 1994., p. 169-79
Ex Vivo genetic correction of RDEB
Preclinical Data for FDA
RDEB
Viral transfer
Keratinocytes
LZRSE-Col7A1
From skin biopsy
6-12 days
3-7 days
Epidermal
Sheet
Production
Mouse
Grafting
+
Pig Dermis
1 days
12-60
days
Skin BMZ
analyses
Type VII Collagen expression in corrected
RDEB human skin
Anti-Type VII Collagen Mab
Hoechst 33342
10x
Anti-Dsg3 Mab
Hoechst 33342
10x
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
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)
iPS cell gene targeting approach
differentiated donor cells
repaired iPS cells
iPS cells
Oct4
Klf4 Sox2
c-Myc
adult cells
Bottlenecks for translation to human cells
✓
differentiated donor cells
repaired iPS cells
iPS cells
Oct4
Klf4 Sox2
c-Myc
adult cells
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)
Derivation of EB-iPS cell lines
Patient 1
1
118
3
1
118
15
Patient 2
1
14
118
1
117
118
Patient 3
1
2
118
1
54
118
Patient 4
1
1
3
118
86
118
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 iPSderived keratinocytes
Cell autonomous expression in chimeric skin
Callahan et al. Genes Dev 2004
Progenitor Assay in chimeric skin
Sen et al. Nature 2010
Acknowledgements
Sandra Melo
Anthony Oro
Vittorio Sebastiano
Dana Ting Yeo
Bahareh Haddad
Jesse Karmazin
Thomas Leung
Pei Wang
Seung Kim
Zurab Siprashvili
Andrea Tichy
Al Lane
Funding: California Institute for Regenerative Medicine