terapia’génica’para’el’estudio’y’tratamiento’de...

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