The University of Hong KongLi Ka Shing Faculty of Medicine香港大學李嘉誠醫學院

Press ConferenceApril 18, 2018

HKU Discovers the Application of Human Induced Pluripotent Stem Cells in Precision

Medicine for Hereditary Diseases

Professor David Siu Chung-wahClinical Professor of Department of Medicine

Speaker

“An emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person.”*

– Enables physicians to tailor medical treatment for each patient

– Supports the development of molecularly targeted drugs based on biologic pathways

– Identifies at-risk populations for targeted prevention prior to disease onset

*NIH - https://ghr.nlm.nih.gov/primer/precisionmedicine/definition

What is Precision Medicine?

In precision medicine, the patients' disease risks, prognoses, and treatment responses can be predicted based on the behaviors of their hiPSC derivatives in cell culture

Patient-specific hiPSC derivatives recapitulate the phenotypes of their in vivo counterparts

Differences in patients' clinical phenotypes are the result of their unique DNA sequences

Differential gene expression patterns cause different cellular and tissue phenotypes

Role of Human Induced Pluripotent Stem Cell (hiPSC) Technology in Precision Medicine

Human Induced Pluripotent Stem Cell (hiPSC)-Based Platforms for Drug Development

28

Skin biopsy and dermal fibroblast

expansion

bFGF supplement

2-10 7 14 21

Emergence of ES cell-like colonies

Manual picking and subculturing of iPS cell

colonies

ROCK kinase inhibitorLentiviral Transduction

0

Switch to MatrigelTM coated

plate

1

Day

Reprogramming genes ROCK kinase inhibitor

Skin cells

Micro-dissection & clonalexpansion

hiPSCs

Lai WH, ... Tse HF, Siu CW: Cell Reprogram. 2010 Dec;12(6):641-53.

Animal Product-Free hiPSC Production

Embryoid bodies Smooth muscle cells Skin and nerve cells

Liver cells Heart cells Blood vessel cells

Lai WH, ... Tse HF, Siu CW: Cell Reprogram. 2010 Dec;12(6):641-53.

Branching into various cell types of the body

Beating Heart Muscle Generated from hiPSC

• Alternative splicing of LMNA gene generates lamin A and C,the intermediate filament protein of nuclear lamina.

• Serve as a matrix to maintain chromosome and genomeintegrity

• Mutations in LMNA referred to as “laminopathies, whichcause Hutchinson Gilford Progeria (premature agingsyndrome) and muscular dystrophy, to familial dilatedcardiomyopathy (DCM).

Laminopathies

Hutchinson-Gilford Progeria Syndrome In HGPS patients the cell nucleus has dramatically aberrant morphology (Scaffidi et al., 2005).

Laminopathies are rare human degenerative disorders with a wide spectrum of clinical phenotypes.

Laminopathies

0-20 years

Atrial fibrillation 30-60 years

Cardiomyopathy VT/VF 40-60 years

2nd to 3rd AV nodal block 20-40 years

Asymptomatic Conduction block 1st degree AV block•Sick Sinus Rhythm

SIU CW Aging (2011)

LAMIN A/C Related Cardiomyopathy

Pedigree of LMNAR225X/WT probands with two females (II-5 & II-9) and one male (II-7)

WT:GCACAGAACACCTGGGGC

MT:GCACAGAACACTGGGGC

heterozygous

frameshift mutation

T518fs

Deletion of ‘c’

Frameshift

WT: ATGCAGCAGCAG

MT: ATGCAGTAGCAG

heterozygous

nonsense mutation

Q354X

C to T

Q

stop

TAG premature stop

WT: CGTCATGAGACCCGACTGGTGG

MT: CGTCATGAGACCTGACTGGTGG

heterozygous

nonsense mutation

R225X

C to T

stop

TGA premature stop

Schematic diagram of LMNA/C structure presenting mutation sites of our enrolled patients

Head Coil 1A Coil 1B Coil 2 Tail

1 240 244 350

35 70 82

644

1 240 244 350

35 70 82

572

Lamin A

Lamin C

1 225

35 70 82

R225X

1 240 244 354

35 70 82

Q354X

1 240 244 350

35 70 82

T518fs

2 3 4 5 6 7 8 9 10 11 121

c.675C>T c.1062C>T c.1554delC

LMNA

Lamin A HeadHead

Coil1A Coil 1B Coil 2 Tail

NLS

Tru

nca

ted

Lam

inA

518

Schematic Diagram Illustrating the LMNAMutations Involved in Current Study: Nonsense

and Frame-Shift Mutations in LMNA

Table 1. Cardiac manifestations in affected subjects bearing LMNA mutation in present studyAbbreviations: 1 HB: first degree heart block; 2 HB: second degree heart block; 3 HB: third degree heart block; AF: Atrialfibrillation; AICD: automatic implantable cardioverter defibrillator; PPB: permanent pacemaker; AV block: atrio-ventricular block;CHB: complete heart block; PR: P-R interval; VT: ventricular tachyarrhythmia, DCM: dilated cardiomyopathy.* Three probands were come from the same family.

Cardiac Manifestations (age of diagnosis in years)

Affectedsubjects

Sex

heartblock AF VT/VF Cardiomyopat

hyAICD/Pacemaker Age of death

SF5* (II-7)LMNAR225X/WT M CHB (49) + (49) + (50) - PPM (49);AICD

(52) (57)

SF29*(II-9)LMNAR225X/WT F CHB (48) + (48) - - PPM (49);AICD

(53) -

SF36*(II-5)LMNAR225X/WT F CHB (51) + (52) + (60) + DCM (60) ICD (60) -

SF11LMNAframeshift/

WTM 3 HB (46) + (49) - - Pacemaker (50) -

SF26LMNAE422X/WT M CHB (50) + (54) + (54) + DCM (57) ICD (?) (64)

SF30LMNAQ534X/WT M CHB (50) + (50) + (56) + DCM (50) PPM: AICD (50) (56)

SF39LMNAT918fs/WT M CHB (43) + (43) + (47)) + DCM (47) AICD (47) -

ActininLMNA

LMNAwt/wt

ActininLMNA

LMNAR225X/wt

ActininLMNA

LMNAwt/wt +GFP

LMNAwt/wt + shLMNA

ActininLMNA

ActininLMNA

ActininLMNA

ActininLMNA

LMNAwt/wt

LMNAR225X/wt

ActininLMNA

ActininLMNA

LMNAwt/wt +GFP

LMNAwt/wt + shLMNA

基線 電刺激

hiPSC Heart muscle cells from patients with Cardiac Laminopathy exhibit nuclear blebbing

upon electrical stimulation

SIU CW Aging (2011)

PTC 124 Chemical Structure

• PTC124 (Ataluren) is a novel small molecular CFTR-G542X nonsense allele inhibitor.• In safety pharmacology studies in rats and dogs, oral administration of PTC124 (Ataluren) induces

no adverse neurological, pulmonary, or cardiovascular effects at doses through 1500 mg/kg.• In toxicology studies in rats and dogs at oral doses through 1500 mg/kg for 28 days, PTC124

(Ataluren) has shown good tolerability.• FDA approved a new indication for orphan drug PTC 124 (made by PTC Therapeutics, Inc.),

allowing its use in the treatment of Duchenne muscular dystrophy (MD) caused by a nonsensemutation in the dystrophin gene.

(Nature, Vol 447 2007)

PTC124 (Ataluren)

X

50S

30S

F-MET

ARGGLY SER PRO THR

50S

30S

F-MET

ARGGLY SER PRO

A U G C G C G G A U C C C C C A C C U G A

5’ 3’Codon 1(f-MET/Start)

Codon 2(ARG)

Codon 3(GLY)

Codon 4(SER)

Codon 5(PRO)

Codon 6(THR)

Codon 7(STOP)

X X X X X X X X X

THR

A U G C G C G G A U C C C C C A C C U G A

5’ 3’Codon 1(f-MET/Start)

Codon 2(ARG)

Codon 3(GLY)

Codon 4(SER)

Codon 5(PRO)

Codon 6(THR)

Codon 7(STOP)

X X X X X X X X X

PTC124

X X XX

PTC124 reverses or alleviates nonsense mutation

Lamin A/C

ACTB

Lamin A/C

ACTB

Lamin A/C

ACTB

Lamin A/C

ACTB

Lamin A/C

ACTB

Lamin A/C

ACTB

Lamin A/C

ACTB

Skin Fibroblast iPSC-CMC

PTC (µM)0 50

PTC (µM)0 50

LMNAR225X/wt

LMNAT518fs/wt

LMNAwt/wt

LMNAQ354X/wt

Lamin A/C

ACTB

Effects of PTC124 on Expression of Lamin A/C Proteins in Dermal Fibroblasts and hiPSC-

Derived Cardiomyocytes

LMNAWT/WT

CT

RE

lect

ric

ally

st

ress

edE

lect

ric

ally

str

ess

ed+

PT

C12

4

100 µm

100 µm

100 µm

LMNAR225X/WT

100 µm

100 µm

100 µm

LMNAQ354X/WT

100 µm

100 µm

100 µm

LMNAT518fs/WT

100 µm

100 µm

100 µm

Nuclear blebbing in the hiPSC-derived cardiomyocytes

DNA content (PI)

E-StimControl

Ap

op

toti

c c

ell

(TU

NE

L p

osi

tive

: B

rdU

-FIT

C)

FITC

Log

LMN

AR22

5X/w

t LM

NAT5

18f

s/w

t

LMN

Awt

/wt

E-Stim+ PTC

LMN

AQ35

4X/w

t

6.32 ± 1.72

33.78 ± 1.72

28.20 ± 13.70

59.85 ± 13.79

7.48 ± 3.94

69.96 ± 7.49

17.38 ± 7.23

43.78 ± 15.30

8.78 ± 3.38

20.6 ± 3.91

26.13 ± 5.75

54.58 ± 16.28

Evaluation of TUNEL-Positive Apoptotic Cell in Electrically Stressed and PTC124-Treated

Cardiomyocytes

Co

ntr

actil

ity(%

)

D

S

DL

Ds: Duration of shorteningDL: Duration of Lengthening

Cell shortening: Video based edge detection used to record cell shortening

Peak High

Baseline

Field electrical pacing at 40 Vat the frequency of 0.5 Hz, 1 Hz, 1.5 Hz and 2 Hz

LMNAWT/WT

PTC124CTR

0.0 0.5 1.0 1.5 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca2

+i(n

M)

40

45

50

55

60

65

Cel

l le

ng

th (

uM

)

0.0 0.5 1.0 1.5 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca2

+i(n

M)

40

45

50

55

60

65

Cel

l le

ng

th (

uM

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca

2+

i(nM

)

40

45

50

55

60

65

Ce

ll le

ng

th (

uM

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca2

+i(n

M)

40

45

50

55

60

65

Cel

l len

gth

(u

M)

LMNAR225X/WT

PTC124CTR

0.0 0.5 1.0 1.5 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca2

+

i(nM

)

20

30

40

50

60

Cel

l le

ng

th (

uM

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca2

+

i(nM

)

20

30

40

50

60

Cel

l len

gth

(u

M)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca2

+

i(nM

)

20

30

40

50

60

Ce

ll le

ng

th (

uM

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca2

+

i(nM

)

20

30

40

50

60

Ce

ll le

ng

th (

uM

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca2

+i(n

M)

20

25

30

35

40

45

50

55

60

Cel

l le

ng

th (

uM

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca2

+i(n

M)

20

25

30

35

40

45

50

55

60

Cel

l le

ng

th (

uM

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca

2+ i(n

M)

20

25

30

35

40

45

50

55

60

Cel

l le

ng

th (

uM

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca

2+ i(n

M)

40

41

42

43

44

45

Cel

l le

ng

th (

uM

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca2

+

i(nM

)

20

25

30

35

40

45

50

55

60

Cel

l le

ng

th (

uM

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca

2+

i(nM

)

20

25

30

35

40

45

50

55

60

Ce

ll le

ng

th (

uM

)

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

Time (s)

Ca2

+i(n

M)

40

45

50

55

60

65

70

Cel

l len

gth

(u

M)

0.0 0.5 1.0 1.5 2.0

20

40

60

80

100

120

140

160

180

200

Time (s)

Ca2

+

i(nM

)

40

45

50

55

60

Cel

l len

gth

(u

M)

0.5

Hz

1 H

z1.

5 H

z2

HzDiastolic Ca2+

concentration

Ca2+ Transient: the calcium indicator,Fura-2 AM was loaded into cells

Am

pli

tud

e

Simultaneous Recording of Cardiac Cell Contractile Force and Calcium Transients of

Single Cardiomyocytes

• Precision medicine is a new trend in modern medicine. It aimsto allow tailing disease treatment and prevention according toindividual variability in genes, environment, and lifestyle foreach person.

• hiPSC technology provides an unique platform for cliniciansand scientists to study the underlying mechanisms of variousdiseases.

• More importantly, while the clinical manifestations are verysimilar in patients with the same disease, hiPSC technologyallow better prediction to clinical responses prior to realclinical trials. This is particularly important for rare diseases,whose number of patients often too small for meaningfulclinical trials.

Conclusions

• In fact, since 2008, we have generated more than 20 disease-specific hiPSC line. This hiPSC bank is a unique platform forinnovative biomedical research and drug development inHong Kong and Mainland China.

• The present work demonstrate the feasibility of hiPSCtechnology in precision medicine for rare disease,representing a step forwards to its clinical applications.

Conclusions