Retinal cell therapy using iPS cells · Retinal cell therapy using iPS cells Center for...
Transcript of Retinal cell therapy using iPS cells · Retinal cell therapy using iPS cells Center for...
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Retinal cell therapy using iPS cells
Center for Developmental Biology, RIKENLaboratory for Retinal Regeneration
Kobe City Medical Center General HospitalOphthalmology department
Institute of Biomedical Research and Innovation HospitalOphthalmology section
Masayo Takahashi MD, PhD
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embryonic tissueneural stem cell
Regeneration of central nervous system using stem cells
intrinsic neural stem cells
dubious
2. Transplantation
A Replacement therapy
B Trophic effect
1. . Regeneration from intrinsic stem cells
Trophic factorImmature cellSmall amount
Functional cellMature cellEnough amount
Mitotic factorDifferentiation facotr
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Preclinicalresearch
Clinical research(Act on the safety of regenerative medicine) forced on Nov 25, 2014
Regulation for cell therapy in Japan
Clinical trial(revised Pharmaceutical affairs act)
forced on Nov 25, 2014【Regenerative medicine 2014.11.25~ 】
※Earlier Patient Access !
Informed Consent and Post Market Safety Measures
Marketing(further confirmation
of efficacy and safety)
Adaptive Licensing with
conditionClinical
Research
Approval orExpiration ofprovisional
approval
MarketingClinical Trial(confirmation of
probable benefit* and safety**)
Application
* Probable benefit: Confirmation of efficacy with small patient population.** Safety: Earlier detection and evaluation of adverse events.
Enforcement 2015.11.25~
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Retina
Peripheral retina(rod)
fovea(cone)
Optic disc
Retinal pigment epithelium (RPE)
Fundus
Φ2㎜
Macula
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Technology ~retinal cell transplantation~
2017/6/7 5
RPE cells (for AMD)Started clinical study (Safaty)
IssueVariety of cell formsEfficacy
Photoreceptor (for Retinitis pigmentosa)Issue
Structure of photoreceptor cellsPurificationFunctional connection with the host’s neurons
Retinal cell transplantation
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Technology ~retinal cell transplantation~
2017/6/7 Retina Institute Japan 6
RPE cells (for AMD)Started clinical study (Safaty)
IssueVariety of cell formsEfficacy
Photoreceptor (for Retinitis pigmentosa)Issue
Structure of photoreceptor cellsPurificationFunctional connection with the host’s neurons
Retinal cell transplantation
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Dry-type Chronic
AMDDrusenRPE atrophy
Neovascularization retinaRPE Basement
membrane
Choroid
Age-related macular degeneration (AMD)
Wet –typeSubacute
Current treatment(anti-VEGF drug injection)
RPE transplantation
edema / subretinal fluid
exduatehemorrhage
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Treatment for age-related macular degeneration (AMD) with hiPS-RPE cell sheets
Patients with AMD
Pure RPE cells
Skin fibrobrasts
(Reprogramming)
Transplantation
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iPS cells 24 line → 6 line
4 Mo 4 Mo
3 line
2 Mo
Differentiation
RPE cell sheets
Plasmid (Glis1)
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Φ4mm skin incision
無菌試験室
iPS細胞室 皮膚細胞処置室 機器室 分化調製室
着衣室1
エアーロック室1
女子更衣室
男子更衣室
廃棄物保管庫
エントランス
クリーン廊下
準備室2着衣室2
脱衣室2
滅菌室
準備室1
脱衣室1
パスルーム
保管室 QC室
居室
一般廊下
準備室3
脱衣室3
脱衣室4
エアーロック室2
収納庫2
収納庫1
着衣室3
着衣室4
気流方向
Grade AGrade BGrade CGrade D一般空調
清浄度区分
Skin harvesting from the patient
Cell culture in the cell processing facility
Safety cabinet
Incubator
Skin trephine
iPS-derived RPE sheet
Cell Processing Facility (CPF)
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Quality & quantityof hiPSC-RPE cell-sheets
Collagen 4 DAPI Laminin DAPI ZO-1 DAPI
Hiroyuki Kamao
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6w
PEDF
VEGF
apical
basal
Monitoring points : RPE function
PhagocytosisColor
PEDF Growth factors VEGF
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Cluster analysisof gene expression profiles
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RPE & Tumors
• No report of metastatic tumor ever in the history• Even in the familial tumor patients
=various cancer formation with hereditary oncogene (ex. p53) mutations
only hyperplasia of RPE occurs• PEDF (pigment epithelial derived factor)
= strong antitumor factor
• Eye ball is full of Retinoic Acid = antitumor factor
Cells
Environment
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Summary of tumorigenicity test
donor Vector # of animal tumor
1st2010. 11~2011. 9
RPE cellRetrovirus
Sendai virusPlasmid A
57 0
2nd2011. 2~2012 4
RPE cell Plasmid A 11 0
RPE cell:OCT3/4 remnant+ Plasmid A 7 0
3rd2012. 3~10
RPE cell Plasmid B 27 0
4th2012. 12~2013 7
RPE sheet(subcutaneous・subretinal)
Plasmid B 16 0
(Kanemura et al.2014 PlosONE)
Purified hiPS-RPE did not generate tumors
NOD-SCID mice, subcutaneous, 1x10^5 cells in Matrigel
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The most important thing is ….
(even with genetic changes)
Safety issue ( risk size) will be different depending on
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1. Final cell types 2. Treatment procedure (i.v. or surgery)3. Host environment (diseases)
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Hazard Level
1. The protocol that sometimes makes tumor in vivo2. Purity: contamination of tumor cells
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↑ Actual hazard difficult to prove negative ↓ Possible hazard easy to find
3. Genetic change: SNV, Indel, CNV、Plasmid remnant4. Epigenetic change …..
Regulatory Science VS Basic Science
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Quality 2: Purity
Quantity: Small amount of cells
Quality 1: function
OCT ; fine examination
Requirements for replacement therapy
Safety: no tumor formation
OCTOCT
Kanemura et al. 2013 Scientific report
Kuroda et al. 2012 PlosONE
Clinical application
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SURGERY ROOM(SEP. 12. 2014)
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iPS-RPE sheet transplantation
iPS-RPE sheet transplantation
CNV removal
vitreous
retina
lensInjectionneedle
injection
subretinalinjectionneedle
refluxfluid
Wet AMD
2mm
1mm
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Fundus photograph after the surgery
3 month 6 month 1 year
1 week 4 week3 day
Animal experiment
rejection
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0.3 0.3
Anti-VEGF injection
Visual acuity before & after the surgery
13 times injection into the eyeball
0.01
0.1
1
-240 -216 -192 -168 -144 -120 -96 -72 -48 -24 0 24 48 72 96 120
preoperative BCVA postoperative BCVA
postoperative weeks
BC
VA
surgery
preoperative weeks
1 year
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NEI VFQ-25 (QOL)
術前W
Pre
The patient, who has remained anonymous, told The Japan Times:
‘I am glad I received the treatment. I feel my eyesight has brightened and widened.’
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B B’3 days 1 year
1 week
1 year
pretreatment
1 year results: OCT (transvers section)
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Fixation tests using MP-1
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March, 2017
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EDITORIALPolar Extremes in the Clinical Use of Stem Cells
George Q. Daley, M.D., Ph.D.N Engl J Med 2017; 376:1075-1077
March 16, 2017
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Preclinicalresearch
Clinical research(Act on the safety of regenerative medicine) forced on Nov 25, 2014
Regulation for cell therapy in Japan
Clinical trial(revised Pharmaceutical affairs act)
forced on Nov 25, 2014【Regenerative medicine 2014.11.25~ 】
※Earlier Patient Access !
Informed Consent and Post Market Safety Measures
Marketing(further confirmation
of efficacy and safety)
Adaptive Licensing with
conditionClinical
Research
Approval orExpiration ofprovisional
approval
MarketingClinical Trial(confirmation of
probable benefit* and safety**)
Application
* Probable benefit: Confirmation of efficacy with small patient population.** Safety: Earlier detection and evaluation of adverse events.
Enforcement 2015.11.25~
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HLA 6 loci homozygous
iPS line
HLA types CiRA
SheetSurgery risk: Big
SuspensionSurgery risk: Small
AutoCost: Big
Auto/Sheet8.2013~(Clinical research)
Auto/SuspensionApplication in preparation
AlloCost : Small
Allo/SheetApplication in preparation
Allo/SuspensionApproved by the ministry
2.3.2017
Types of hiPS-RPE cell transplantation
HLA 6 loci match 17% of Japanese population
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MHC homo iPS-RPE transplantation into hetero matched monkey eye: no immune rejection
MHC Homo (HT-1)
MHC Homo iPS cells
MHC Hetero
MHC Hetero(MHC Matched)
Control
MHC (MHC mismatched)
iPS-RPE cells(MHC Homo:1121A1) Subretinal transplantation w/o immunosuppression
MHC mismatched monkey → rejection
MHC matched monkey → no rejection
Retina intact
Retinal inflammatory findingsOCT 2M
OCT 2M
RPE graft sheet RPE graft sheet
Cystic edemaDisappearance of outer retinal layers
Immune reaction to the MHC matched or unmatched iPS-RPE(in normal monkey )
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The scheme of new clinical study using allograft
A*24:02-B*52:01-C*12:02-DRB1*15:02-DQB1*06:01-DPB1*09:01 Cell suspension
Superdonor blood cells
Frozen → culture → graft
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Japanese man is first to receive 'reprogrammed' stem cells from another person
World-first transplant, used to treat macular degeneration, represents a major step forward in movement to create banks of ready-made stem cells.
David Cyranoski 3/28/2017
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Technology ~retinal cell transplantation~
2017/6/7 Retina Institute Japan 32
RPE cells (for AMD)Started clinical study (Safaty)
IssueVariety of cell formsEfficacy
Photoreceptor (for Retinitis pigmentosa)Issue
Structure of photoreceptor cellsPurificationFunctional connection with the host’s neurons
Retinal cell transplantation
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厚生労働科学研究費科学研究費補助金 難治性疾患克服研究事業網脈絡膜・視神経萎縮症に関する研究 平成17年度総括・分担研究報告書 p.p.263-267 (2006)Health Labour Sciences Research Grant for Research on Measures for Intractable Diseases
Causative diseases of visual impairment in Japan (2005)
Graph1
glaucoma
diabetic retinopathy
retinitis pigmentosa
AMD
high myopia
cataract
others
causative disease of defect in vision
AMD; 9%
cataract; 3%
others; 27%
20.7
19
13.7
9.1
7.8
3.2
26.5
Sheet1
causative disease of defect in vision
glaucoma20.7
diabetic retinopathy19
retinitis pigmentosa13.7
AMD9.1
high myopia7.8
cataract3.2
others26.5
グラフのデータ範囲の大きさを変更するには、範囲の右下隅をドラッグしてください。
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Photoreceptor degeneration
Retinitis pigmentosa•More than 30000 patients in Japan•No treatment
After birth(normal)
photoreceptor
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Self-organizing optic-cup morphogenesis in three-dimensional culture
Mototsugu Eiraku, Nozomu Takata, Hiroki Ishibashi, Masako Kawada,Eriko Sakakura, Satoru Okuda, Kiyotoshi Sekiguchi, Taiji Adachi, Yoshiki Sasai (CDB, RIKEN)
April 7, 2011
http://www.nature.com/nature/journal/v472/n7341/full/nature09941.html
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Retinal tissue derived from mouse ES cells in vitroSensory retina RPE
緑:神経網膜
OS
Green:photoreceptor
IS
Nucleus
Dr. Eiraku
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Outer segment
Host retina
Donor sheet
RPE
Outer segment
Photoreceptor
Michiko MandaiMD, Phd
Mouse retinal sheet transplantation
AssawachananontMD, Phd
Stem Cell Reports 2014
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Graft
Host retina
Host-graft synaptic contact in direct contact pattern
Mouse retinal sheet transplantation
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MED probe (MED-P5155)1.2mm
Electrode size: 50x50µm 1.2mm
Evalutation of Function after transplantation
Grafted cell inSubretinal space
Glass Micropipette
GCL
INL
ONL
Dim light stimulation
MED64 Amp
MED probe (64 channels)
Graft
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Light intensity:750cds/m2Stimulus duration:10ms1Hz low cut filter
X-axis:Time (500ms/DIV)Y-axis:Amplitude(0.05mV/DIV)
microERG
光刺激のタイミング
on
Response to the light stimuli after transplantation
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stimuli
ElectricalShock
5s
Training with light & sound
Within 2w (avoidance ratio:70%
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Host with good transplants(post OCT selection)
Light only wild type rd1
After transplantation….
Rd1-2J
wt
light
Light +beep
Graft-
beep
light
Electric shock
5 sec
light
wt
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Donor and host photoreceptors engage in material transfer following transplantation of post-mitotic photoreceptor
precursors R.A. Pearson1,*, A. Gonzalez-Cordero1, E.L. West1,**, J.R. Ribeiro1,**, N. Aghaizu1,**, D. Goh1, R.D. Sampson1, A. Georgiadis1, P.V. Waldron1, Y. Duran1, A. Naeem1, M. Kloc1, E. Cristante1, K.
Kruczek1, K. Warre-Cornish1,w, J.C. Sowden2, A.J. Smith1 & R.R. Ali1,3,*
Retinal transplantation of photoreceptors results in donor–host cytoplasmic exchange
Tiago Santos-Ferreira1,*, S ́ılvia Llonch1,*, Oliver Borsch1,*, Kai Postel1, Jochen Haas1 & Marius Ader1
Published 4 Oct 2016 It was cytoplasmic exchange !!
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Graft
Host retina
Host-graft synaptic contact in direct contact pattern
Mouse retinal sheet transplantation
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Effect of Regenerative medicine
1. Donor cells
2. Host environment
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Glial scar
Rod degeneration Cone degeneration Bipolar degenration
Iba1
ONL
Red: Iba1Microglia
ONLINL
INL
ONL
INLONL
3w
4w2w 3w
Immflamaion
Red: GFAPMuller glia
4w2w 7w 12w
Time window
Host environment and transplantation(rd mice:rapid photoreceptor degeneration model mice)
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Retinitis Pigmentosa
ONL
ONL (Photoreceptor cell layer) +
Transplantation
In human patients, various stages exist in one eye
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Patient-derived iPSCs studies
Photoreceptor
RPE
Retina
RGC
Photoreceptor dysfunction/degeneration
RGC dysfunction/degenerationLeber hereditary optic neuropathy
Normal tension glaucoma
・1 in 4000~8000 individuals・~50 causal genes identified・~60% are sporadic/unknown
(Mofiried from Nat Rev Neurosci.)
Retinitis Pigmentosa
⑨ MT-ND4 ComplexI
・1 in ~50000 individuals ・80% of Japanese LHON patients
・70~90% in Japanese patients・Causal genes characterized (MYOC WDR36 OPTN) ⑩ NTG (uncharacterized)
① RHO outer segment② RP1 clila③ PRPH2 outer segment④ RP9 splicing factor⑤ PRPH2 outer segment⑥ RP2 cilia⑦ EYS outer segment/cilia⑧ PRPF31 splicing factor/cilia
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Preclinicalresearch
Clinical research(Act on the safety of regenerative medicine) forced on Nov 25, 2014
Regulation for cell therapy in Japan
Clinical trial(revised Pharmaceutical affairs act)
forced on Nov 25, 2014【Regenerative medicine 2014.11.25~ 】
※Earlier Patient Access !
Informed Consent and Post Market Safety Measures
Marketing(further confirmation
of efficacy and safety)
Adaptive Licensing with
conditionClinical
Research
Approval orExpiration ofprovisional
approval
MarketingClinical Trial(confirmation of
probable benefit* and safety**)
Application
* Probable benefit: Confirmation of efficacy with small patient population.** Safety: Earlier detection and evaluation of adverse events.
Enforcement 2015.11.25~
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Lab Member
Project LeaderMasayo Takahashi
Deputy Project Leader
Michiko Mandai
Sunao Sugita
Research ScientistAkishi Onishi
Akiko Maeda
Genshiro Sunagawa
Ojos Matsuyama
Yuki Arai
Akiko Yoshida
Student TraineeShiniciro ItoSatoshi IrahaKaori UedaSo GotoKenichi MakabeWataru KobayashiCody KimeAnouk GeorgesRyutaro Akiba
CPC teamHiroyuki KitajimaNoriko SakaiYumiko ShibataMotoki TeragdaMitsuhiro Nishida
Retinal cell therapy using iPS cellsスライド番号 2スライド番号 3Retina Technology ~retinal cell transplantation~Technology ~retinal cell transplantation~スライド番号 7 Treatment for age-related macular degeneration (AMD) with hiPS-RPE cell sheetsスライド番号 9Quality & quantity� of hiPSC-RPE cell-sheetsMonitoring points : RPE functionCluster analysis �of gene expression profiles RPE & Tumors Summary of tumorigenicity testスライド番号 15Hazard Levelスライド番号 17スライド番号 18iPS-RPE sheet transplantationFundus photograph �after the surgeryVisual acuity before & after the surgeryNEI VFQ-25 (QOL)スライド番号 23スライド番号 24スライド番号 25スライド番号 26スライド番号 27スライド番号 28スライド番号 29The scheme of new clinical study using allograftスライド番号 31Technology ~retinal cell transplantation~スライド番号 33スライド番号 34Self-organizing optic-cup morphogenesis �in three-dimensional cultureスライド番号 36スライド番号 37スライド番号 38スライド番号 39スライド番号 40スライド番号 41スライド番号 42スライド番号 43スライド番号 44スライド番号 45スライド番号 46スライド番号 47スライド番号 48スライド番号 49スライド番号 50