Terapia’génica’para’el’estudio’y’tratamiento’de...
Transcript of Terapia’génica’para’el’estudio’y’tratamiento’de...
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Terapia génica para el estudio y tratamiento de enfermedades hepáticas
AEEH
Madrid, 15 Febrero 2017
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Packaging into non-
viral vectors
Direct injection of modified nucleic acids
Packaging into viral vectors
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Packaging into non-
viral vectors
Direct injection of modified nucleic acids
Packaging into viral vectors
Physical methods
NeedleBallistic DNAElectroporationSonoporationPhotoporationMagnetofectionHydroporation
Chemical carriers
Inorganic particlesCalcium phosphateSilicaGoldOther
Natural biodegradableCationic lipidsLipid nano emulsionsSolid lipid nanoparticlesPeptide basedPolymer based
PEIChitosanPLADendrimersPolymethacrylate
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Monogenic disease
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Monogenic disease
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Monogenic disease
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Alpha-1 Antitrypsin Deficiency
Hemophilia A and B
Crigler-Najjar Syndrome
Ornithine transcarbamylase deficiency Glycogen storage disease type Ia Citrullinemia type 1 Phenylketonuria Wilson diseaseHemophilia A and B
Hemophilia A and B
Inherited retinal diseasesNeurodegenerative diseases
MuscleImmune deficits and blood diseases
Hemophilia A
Crigler-Najjar Syndrome
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Editas MedicineAlphaI-Antitrypsin DeficiencyCRISPRCas NHEJ+HDR
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Immuno-oncology: Cancer vaccinesOX40LIL12
Cardiovascular disease: VEGF
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HaploinsufficiencyHNF4a in NASH
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Genetic diseasesHepatic porphirias siALAS1 Ph1Primary hyperoxaluria siGlycolate oxidaseB thalasemia siTMPRSS6 < ironAlpha1 antitrypsin deficiency sia1ATHered ATTR amyloidosis siTTR Ph3Hemophilia siAntithrombin Ph2Complement –mediated disease siC5 Ph1
Hyperchol (Ph2)siPCSK9 (>LDLR)
HyperlipidemiasiApoCIIIsiANGPTL3
InfectionsiPD-L1siHBVsiHDV
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Severe and rareHomozygous Familial Hypercholesterolemia Apo B100 (<col)Familial Chylomicron Syndrome Apo CIII (<TG)Familial Partial Lipodystrophy Apo CIII (>TG)
CardiovascularClotting disorders Factor XI (< thrombosis)High ApoA (< atherosclerosis)Dyslipidemias ANGPTL3 (< cardiovascular risk)
CancerLiver/Brain/Lung/Breast/Bone/MM cancer STAT3Pancreatic/Bladder/Prostate cancer HSP27
LiverHCV miR122HBV viral genomeNASH + type2 diabetes miR103/107NASH DGAT2 (<TG)
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Monogenic disease
- Any monogenic disease that affects the liver
- Described.
- Identified by high-throughput sequencing forpersonalized medicine.
- Use the liver to secrete proteins: hemophilia
Polygenic diseases
Complex diseases that can be treated by expression of a therapeutic gene
One allele Two alleles
Haploinsufficiency
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IGF-I and liver cirrhosis
Chronic injury
AlcoholViruses (HBV, HCV)DrugsToxinsGenetic background
Chronic inflammation (TGFb, TNFa, IL6)
Dead of hepatocytesRegeneration of remaining hepatocytes.
Proliferation as round nodules.Markers of dediferentiation (WT-1)
Profibrogenic factors (TGFb, VEGF, CTGF, PDGF, AR)
Activation of HSCs (with SMA marker)
Collagen deposits = Fibrosis
IGF-I
IGF-I
MalnutritionOsteoporosisHypogonadism
50
100
150
200
CONTROL22 + 1234 + 22
59 + 29
169 + 21
0COMPENSATED
CIRRHOSISCIRRHOSIS WITH ASCITES
UNaV > 10 mmol/24 h. UNaV < 10 mol/24 h
**
* *P < 0.01 vs control
IGF-Ing/ml
AlbuminCoagulation factors
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rIGF-I
Improved Hepatic function
Decreased Oxidative stress
Decreased Fibrosis
Improved Mitochondrial function
Castillo-Cortazar et al., 2000;; Lorenzo-Zuniga et al., 2006
IGF-I and liver cirrhosis
Castilla et al., Gastroenterology, 1997 Lorenzo et al., GUT 2005
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rIGF-I
Improved albumin levels
Tendency to improve energy metabolism
Conchillo et al., 2005
100 µg/kg/day4 months
(7 mg/day for 70 Kg)
IGF-I and liver cirrhosis
Pilot clinical trial
2.5
3
3.5
4
4.5
5
0 120 0 120Placebo IGF-1
days
Albumin (g/dL)
p=0.015
4567891011
0 120 0 120Placebo IGF-1
days
Child-Pugh score
p=0.053
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AAVIGF-I: Cirrhosis treatment.
CCl4 administration8 weeks
> 3,4 x 109 vp/rat
Saline
AAVLuc
AAVIGFI
Serum analysis 16 weeks
4 dpi 2wpi 8wpi 16wpi
Sacrifice
intraarterial
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IGF-I mRNA
HealthyCiCi+AAVLucCi+AAVIGF-I
0
5
10
15
20
25
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtIGF-I)x10
3
IGF-IBP3 mRNA
0
10
20
30
40
50
60
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtIGFBP5)X10
3
AAV1dsa1ATIGF-I expresses active IGF-I in rat liver
Day 4 Week 2 Week 8 Week 16
Liver Total IGF-I
020040060080010001200140016001800
ng/mg of protein
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AAVIGF-I: Restores liver functionality4 dpi 2wpi 8wpi 16wpi
Transaminases
0
100
200
300
400
500
600
700
800
900
AST ALT ALP AST ALT ALP AST ALT ALP
Day 4 Week 2 Week 8
U/l
HealthyCiCi+AAVLucCi+AAVIGF-I
0.00.20.40.60.81.01.21.41.61.8
Day 4 Week 2 Week 8
mg/dl
Bilirubin
0123456
Day 4 Week 2 Week 8
gr/dl
Albumin
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AAVIGF-I: Regresses liver cirrhosis4 dpi 2wpi 8wpi 16wpi
024681012141618
Day 4 Week 2 Week 8 Week 16
Fibrosis%
FibrosisCi+IGF-I
Col I mRNA
01020304050607080
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtCOLI)x10
3
Col IV mRNA
05101520253035404550
Day 4 Week 2 Week 8 Week16
2(CtGADHP-CtCOLIV)x103
HealthyCiCi+AAVLucCi+AAVIGF-I
Healthy Ci Ci+Luc Ci+IGF-I
Sirius red staining
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AAVIGF-I: Regresses liver cirrhosis4 dpi 2wpi 8wpi 16wpi
Ci+IGF-I
024681012141618
Day 4 Week 2 Week 8 Week 16
Fibrosis%
FibrosisCi+IGF-I
Healthy Ci Ci+Luc
Col I mRNA
01020304050607080
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtCOLI)x10
3
Col IV mRNA
05101520253035404550
Day 4 Week 2 Week 8 Week16
2(CtGADHP-CtCOLIV)x103
HealthyCiCi+AAVLucCi+AAVIGF-I
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AAVIGF-I: Induces expression and functionality of several MMPs4 dpi 2wpi 8wpi 16wpi
0
5
10
15
20
25
30
35
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtMMP-1)x103
0
10
20
30
40
50
60
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtMMP-2)x103
0
5
10
15
20
25
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtMMP-9)x106
0
2
4
6
8
10
12
14
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtMMP-14)x10
3
MMP1 mRNA MMP2 mRNA
MMP9 mRNA MMP14 mRNA
HealthyCiCi+AAVLucCi+AAVIGF-I
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AAVIGF-I: Decreases the amount of activated stellate cells.4 dpi 2wpi 8wpi 16wpi
α-SMA
0
20
40
60
80
100
120
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtSMA-1)x103
αSMA mRNA
HealthyCiCi+AAVLucCi+AAVIGF-I
Day 4 Week 2 Week 8
Healthy
Ci
Ci+Luc
Ci+AAVIGF-I
Ci
Ci+IGF-I
Vimentin αSMA
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4 dpi 2wpi 8wpi 16wpi
AAVIGF-I: Decreases the amount of proinflammatory or profibrogenic factors.
012345678
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtIL-6)x10
3
IL6mRNA
0
2
4
6
8
10
12
14
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtTNFa)x10
5
TNFαmRNA
0123456789
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtTGFb)X10
4
TGFβmRNA
HealthyCiCi+AAVLucCi+AAVIGF-I
0
10
20
30
40
50
60
Day 4 Week 2 Week 8 Week 16
CTGF mRNA
2(CtGADHP-CtCTGF)x103
0
5
10
15
20
25
Day 4 Week 2 Week 8 Week 16
VEGF mRNA
2(CtGADHP-CtVEGF)x103
0510152025303540
Day 4 Week 2 Week 8 Week 16
PDGF mRNA
2(CtGADHP-CtPDGF)x103
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AAVIGF-I: Induces expression of hepatoprotective factors.4 dpi 2wpi 8wpi 16wpi
0
20
40
60
80
100
120
140
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtHGF)x103 HGF mRNA
0
5
10
15
20
25
30
35
40
Day 4 Week 2 Week 8 Week 16
HNF4αmRNA
2(CtGADHP-CtHNF4α)x10
3
0123456789
Day 4 Week 2 Week 8 Week 16
2(CtGADHP-CtWT-1)x106
WT1 mRNA
HealthyCiCi+AAVLucCi+AAVIGF-I
Wound healing response- inflammation- scar formation
Tissue repair programme- hepatocyte functionality- scar degradation
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0
5
10
15
20
25
2(CtGADHP-CtIGF-I)x10
3
Day 4 Week 2 Week 8 Week16 Year 10
10
20
30
40
50
60
2(CtGADHP-CtIGFBP3)x10
3
Day 4 Week 2 Week 8 Week16 Year 1
0
20
40
60
80
100
120
140
2(CtGADHP-CtHGF)x103
Day 4 Week 2 Week 8 Week16 Year 1
AAVIGF-I: Expresses IGF-I one year after vector inoculation.4 dpi 2wpi 8wpi 16wpi
HGF mRNA
IGF-I mRNA IGF-BP3 mRNA
1ypi
HealthyCiCi+AAVLucCi+AAVIGF-I
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AAVIGF-I: Requirement of inducible expression systems.
Tet-inducible system
Cirrhosis-inducible system
TGFb TNFa CTGF HNF4a
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AdHNF4aAAVHNF4a
LentiHNF4aLentiFoxa3LentiHNF1A
Miofibroblasts in vitro
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AdHNF4a
AAVHNF4a
HNF4a: Liver cirrhosis reversion by transdiferentiation of myofibroblasts into hepatocytes
LentiHNF4aLentiFoxa3LentiHNF1AMiofibroblasts in vitro
AAV6HNF4aFoxa1, Foxa2, Foxa3, Gata4,
Hnf1a
AdHNF4aAdFoxa3AdHNF1AMiofibroblasts in vivo
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Mónica Enguita
Jesús Prieto
Jorge QuirogaMaria Vera Luciano Sobrevals
Gene Therapy of liver cirrhosis with IGF-‐I
Gene Therapy of liver monogenic diseases
Bruno Sangro
LncRNAs in HCC
Juan PabloUnfried Victor Segura Lulu Huang
Gloria González-‐Aseguinolaza Rafael Aldabe Cristian SmerdouRuben HernándezAlcoceba
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Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
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Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
AAVIGF-I
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
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Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
AAVIGF-I
IGF-I
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
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Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
AAVIGF-I
IGF-I
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
![Page 39: Terapia’génica’para’el’estudio’y’tratamiento’de ...ww2.aeeh.es/wp-content/uploads/2016/12/1702AEEHMadrid.pdf · B)thalasemia)siTMPRSS6)](https://reader036.fdocuments.us/reader036/viewer/2022081409/60721f29b4630b3899428bc4/html5/thumbnails/39.jpg)
Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
AAVIGF-I
IGF-I
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
![Page 40: Terapia’génica’para’el’estudio’y’tratamiento’de ...ww2.aeeh.es/wp-content/uploads/2016/12/1702AEEHMadrid.pdf · B)thalasemia)siTMPRSS6)](https://reader036.fdocuments.us/reader036/viewer/2022081409/60721f29b4630b3899428bc4/html5/thumbnails/40.jpg)
Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
AAVIGF-I
IGF-I
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
![Page 41: Terapia’génica’para’el’estudio’y’tratamiento’de ...ww2.aeeh.es/wp-content/uploads/2016/12/1702AEEHMadrid.pdf · B)thalasemia)siTMPRSS6)](https://reader036.fdocuments.us/reader036/viewer/2022081409/60721f29b4630b3899428bc4/html5/thumbnails/41.jpg)
Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
AAVIGF-I
IGF-I
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
![Page 42: Terapia’génica’para’el’estudio’y’tratamiento’de ...ww2.aeeh.es/wp-content/uploads/2016/12/1702AEEHMadrid.pdf · B)thalasemia)siTMPRSS6)](https://reader036.fdocuments.us/reader036/viewer/2022081409/60721f29b4630b3899428bc4/html5/thumbnails/42.jpg)
Hepatocytes
HSCs
Kupffer
TGFβTNFα
Collagen
AAVIGF-I
IGF-I
Cirrhotic liver
CTGF
WT-1
![Page 43: Terapia’génica’para’el’estudio’y’tratamiento’de ...ww2.aeeh.es/wp-content/uploads/2016/12/1702AEEHMadrid.pdf · B)thalasemia)siTMPRSS6)](https://reader036.fdocuments.us/reader036/viewer/2022081409/60721f29b4630b3899428bc4/html5/thumbnails/43.jpg)
Hepatocytes
HSCs
Kupffer
TGFβTNFα
Collagen
AAVIGF-I
IGF-I
Cirrhotic liver
HGF
HGF
HGF
HGF
HGF CTGF
WT-1
![Page 44: Terapia’génica’para’el’estudio’y’tratamiento’de ...ww2.aeeh.es/wp-content/uploads/2016/12/1702AEEHMadrid.pdf · B)thalasemia)siTMPRSS6)](https://reader036.fdocuments.us/reader036/viewer/2022081409/60721f29b4630b3899428bc4/html5/thumbnails/44.jpg)
Hepatocytes
HSCs
Kupffer
Collagen
AAVIGF-I
IGF-I
Cirrhotic liver
HGF
HGF
HGF
HGF
HGF
HNF4α
ATP
ATP
ATP
ATP
ATP
ATP
ATPATP
ATP
ATP
ATP
ATP
ATP
ATPATP
![Page 45: Terapia’génica’para’el’estudio’y’tratamiento’de ...ww2.aeeh.es/wp-content/uploads/2016/12/1702AEEHMadrid.pdf · B)thalasemia)siTMPRSS6)](https://reader036.fdocuments.us/reader036/viewer/2022081409/60721f29b4630b3899428bc4/html5/thumbnails/45.jpg)
Hepatocytes
HSCs
Kupffer
Collagen
AAVIGF-I
IGF-I
Cirrhotic liver
HGF
HGF
HGF
HGF
HGF
HNF4α
ATP
ATP
ATP
ATP
ATP
ATP
ATPATP
ATP
ATP
ATP
ATP
ATP
ATPATP
4 days
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Hepatocytes
HSCs
Kupffer
Collagen
AAVIGF-I
IGF-I
Cirrhotic liver
HGF
HGF
HGF
HGF
HGFMMPs
HNF4α
ATP
ATP
ATP
ATP
ATP
ATP
ATPATP
ATP
ATP
ATP
ATP
ATP
ATPATP
2 weeks
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Hepatocytes
HSCs
Kupffer
Collagen
AAVIGF-I
IGF-I
Cirrhotic liver
HGF
HGF
HGF
HGF
HGFMMPs
HNF4α
ATP
ATP
ATP
ATP
ATP
ATP
ATPATP
ATP
ATP
ATP
ATP
ATP
ATPATP
2 weeks
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Hepatocytes
HSCs
Kupffer
AAVIGF-I
IGF-I
Healthy liver
HGF
HGF
HGF
HGF
HGFMMPs
HNF4α
ATP
ATP
ATP
ATP
ATP
ATP
ATPATP
ATP
ATP
ATP
ATP
ATP
ATPATP
2 weeks
![Page 49: Terapia’génica’para’el’estudio’y’tratamiento’de ...ww2.aeeh.es/wp-content/uploads/2016/12/1702AEEHMadrid.pdf · B)thalasemia)siTMPRSS6)](https://reader036.fdocuments.us/reader036/viewer/2022081409/60721f29b4630b3899428bc4/html5/thumbnails/49.jpg)
Hepatocytes
Kupffer
AAVIGF-I
IGF-I
Healthy liver
HGF
HGF
HGF
HGF
HGF
HNF4α
ATP
ATP
ATP
ATP
ATP
ATP
ATPATP
ATP
ATP
ATP
ATP
ATP
ATPATP
16 weeks
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Hepatocytes
Kupffer
AAVIGF-I
IGF-I
Healthy liver
HGF
HGF
HGF
HGF
HGF
HNF4α
ATP
ATP
ATP
ATP
ATP
ATP
ATPATP
ATP
ATP
ATP
ATP
ATP
ATPATP
SAFETY OF IGF-I TREATMENT
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AAVIGF-I: Decreases proliferation4 dpi 2wpi 8wpi 16wpi
Ki67
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
Day 4 Week 2 Week 8 Week 160
5
10
15
20
25
Day 4 Week 2 Week 8 Week 16
PCNA mRNA
2(CtGADHP-CtPCNA)x103
positive cells / 2.1 x 10
7um
2
HealthyCiCi+AAVLucCi+AAVIGF-I
16wpi1 año
1 año
AAVIGF-I
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Tag
17 kT
tag
VP1
VP3
VP2
pA
LP1
IR PEN A/T 72x2G/C
EP LPCORE ORIGIN ENHANCER
ses1. Factors required for replication are deleted;;2. They infect every cell type that has been tested,
both dividing and quiescent3. They result in long term-expression of the
transgene4. SV40 and AAV have small size (45nm/20nm).
IGF-I
SV40 and AAV as vectors for gene therapy
ITR
pA
LucPBGD ITRm
ITR
pA
IGF-IEhAlbAAT ITRm
Double strand