terapia’génica’para’el’estudio’y’tratamiento’de...
TRANSCRIPT
Terapia génica para el estudio y tratamiento de enfermedades hepáticas
AEEH
Madrid, 15 Febrero 2017
Packaging into non-
viral vectors
Direct injection of modified nucleic acids
Packaging into viral vectors
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
Monogenic disease
Monogenic disease
Monogenic disease
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
Editas MedicineAlphaI-Antitrypsin DeficiencyCRISPRCas NHEJ+HDR
Immuno-oncology: Cancer vaccinesOX40LIL12
Cardiovascular disease: VEGF
HaploinsufficiencyHNF4a in NASH
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
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)
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
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
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
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
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
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
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
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
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
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
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
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
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
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
AAVIGF-I: Requirement of inducible expression systems.
Tet-inducible system
Cirrhosis-inducible system
TGFb TNFa CTGF HNF4a
AdHNF4aAAVHNF4a
LentiHNF4aLentiFoxa3LentiHNF1A
Miofibroblasts in vitro
AdHNF4a
AAVHNF4a
HNF4a: Liver cirrhosis reversion by transdiferentiation of myofibroblasts into hepatocytes
LentiHNF4aLentiFoxa3LentiHNF1AMiofibroblasts in vitro
AAV6HNF4aFoxa1, Foxa2, Foxa3, Gata4,
Hnf1a
AdHNF4aAdFoxa3AdHNF1AMiofibroblasts in vivo
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
Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
AAVIGF-I
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
AAVIGF-I
IGF-I
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
AAVIGF-I
IGF-I
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
AAVIGF-I
IGF-I
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
AAVIGF-I
IGF-I
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
Hepatocytes
HSCs
Kupffer
TGFβ TGFβ TGFβTNFα
TNFα TNFα
Collagen
AAVIGF-I
IGF-I
VEGFCTGF
Cirrhotic liver
CTGF
WT-1
Hepatocytes
HSCs
Kupffer
TGFβTNFα
Collagen
AAVIGF-I
IGF-I
Cirrhotic liver
CTGF
WT-1
Hepatocytes
HSCs
Kupffer
TGFβTNFα
Collagen
AAVIGF-I
IGF-I
Cirrhotic liver
HGF
HGF
HGF
HGF
HGF CTGF
WT-1
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
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
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
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
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
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
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
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
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