cardiovascular gene therapy - bakerassets.escardio.org/assets/presentations/other2011/...gene...
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Cardiovascular Gene Therapy
Andrew H. Baker University of Glasgow
• Increased understandin of molecular mechanisms of
Why gene therapy?
• Diseases with no pharmacological therapy. • Diseases with suboptimal drug therapies. • Identification of genetic deficiencies. • Increased understanding of molecular mechanisms ofg diseases.
• Increased identification of candidate therapeutic genes. • Procedures that allow gene delivery at the time of surgery.
• Gene transcription cassettes can easily be engineered. • Many diverse vehicles for gene delivery to the vasculature.
• Clear opportunity to beneficially treat diverse CVDs.
Ongoing Clinical Trials
Gene therapy has suffered from:
A – over hype in the early days B – premature advancement to poorly
controlled clinical trials C –– Safety concerns with viruses Safety concerns with viruses C
corrective geneto ta et the liver and ex ress the
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Injected into the bloodstream to target the liver and express therg p
corrective gene
• Substantial induction of cytokines • DIC • Multiorgan failure • Death
Poor knowledge of virus:host interactions and dosing regimens/patient variability. Not predictable from mouse data
Instent restenosis
� Lipid lowering therapy
� Hypertension
� Postangioplasty restenosis
Therapeutic Strategies – to 2011
� Instent restenosis
� Vein graft failure
� Peripheral ischaemia
� Myocardial ischaemia
� Heart Failure
e t erapeut gene
Building a gene therapy vector
• Th h ic• The therapeutic gene • Vector delivery (virus/DNA) • Transcriptional control • Device/system for delivery?
Delivery Vectors
•• Efficient • Selective for the target cell/organ • Nontoxic • Stable in vivo • Easy to produce • Nonimmunogenic • Minimal dissemination for local applications• Minimal dissemination for local applications • Homing ability for systemic applications • Longevity for the appropriate disease • Cellselective gene expression • Regulatable gene expression
Gene therapy: Choice of disease target and vector are both critical
Non viral Viral • lower efficiency • poor selectivity • poor nuclear targeting • toxicity?
• generally higher efficiency • variable nuclear targeting • toxicity? • Immunogenicity
Regulatory Issues
Efficient in vitro not ar cells .
+
( prim y )
LiposomeMediated Gene Transfer
� Cationic lipids mixed with DNA.
� Wide variety.
� Efficient in vitro (not primary cells).
+
++
+ ++
+ +
+
+
� Inefficient release of DNA into cytoplasm.
� Poor nuclear targeting.
� Low efficiency in vivo.
Applications:
Limited due to inefficiency
C
C
A lipid B
peptide
plasmid DNA
LID complex
nuclear pore
endosome nucleus
In vivo transfection of ballooninjured rat
carotid artery
neointima
media
adventitia
lumen
pCIwith peptide pCI plasmid
YlaHerttuala and Alitalo, Nat Med, 2003
AAddeennoovvirusirus –– aa fflleexibxibllee vveeccttoorr ssyysstteemm ffoorr vvaassccululaarr ggeennee ttrarannssffeerr
Key Issues: Tropism Toxicity Innate Immune Response Route of Delivery defines above
nononcogen c c . retrov ruses c n ca tr a eat
i ( f i ) li i l i l d h
Adenoviruses
Common pathogens
3050% of people Adeno +
50 different serotypes
occular and respiratory problems
nononcogenic (cf. retroviruses)
Highly efficient
infect all types of cells
efficient cell entry mechanism
organised delivery to the nucleus
based on replicationdeficiency
Disadvantages
immunogenic
transient gene expression
some cells noninfective
clinical trial death
AAddeennoovviirruuss TTrraannsdsduuccttiioonn iinn
vviittrroo aanndd iinn vviivvoo –– llooccaall aacccceessss ttoo ttaarrggeett
αvβ3/5
integrins CAR
Nucleus
Endosome
Endosome disruption
Nuclear Pore
Lemarchand et al. Circ Res 1993;72:11321138.
French et al. Circulation 90: 24022413, 1994
U s e d e xt e n s iv e l y f o r l o c a l g e n e d e l iv e r y in t h e v a s c ul a r s ys t e m
Human Vein Ex Vivo
George et al., Hum Gene Ther 9;867877, 1998
Proven therapies?DES late thrombosis
Adenovirus delivery using stents
High restenosis rate Instent restenosis
( ) Proven therapies?
Residual lumen
neointima
Stent strut
adventitia
media
DES (late thrombosis)
Fig. 5. PAA-BP-vector binding agent mediated tethering of Ad to steel surfaces in vivo: reporter (GFP) and Ad-iNOS therapeutic results
Copyright ©2006 by the National Academy of Sciences
Fishbein, Ilia et al. (2006) Proc. Natl. Acad. Sci. USA 103, 159-164
Adenovirus Systemic
10
100
0.01
0.1
1
Heart Lung LiverStomachSpleenKidneyBrain
Organ
Courtesy of J. Johnson, Bristol
• Liver disorders • Factory for secreted genes (e.g. haemophilia)
Cellmediated immunity
Adenovirus is highly immunogenic
Transient gene expression
Innate immunity
Viral gene expression
Activates immune response
Cell death
E4
Adenovirus vectors
E1 a b MLP
late transcription
E2
E3
First generation
E2 E4
Second generation
Third generation
erol
(mg/d
l)Pla
smaChole
Plasm
a Chole
st
400
500
600
700
400
500
600
700 HDAd5gE HDAd2gE HDAd5cE FG Ad5 cE Dialysis Buffer C57wt
Lifetime Correction of Hyperlipidemia ApoE/ Mice by a Single Injection of HDAdApoE
Weeks after Injection
0 4 8
12
16
20
24
28
32
36
40
44
48
52
56
60
64
68
72
76
80
84
88
92
96 100
104
108
112
116
sterol (m
g/dl)
0
100
200
300
0
100
200
300
HDAdApoE Treated and Control ApoE/ Mice At Age 2.5 Years
Control Treated
m e o
• li it d t 4.7 kB
Adenoassociated virus (AAV)
• Parvoviruses so called dependoviruses • Small virus not associated with known human pathogenicity • rep/cap genes removed and replaced with expression cassette
• limited to 4.7 kB • no viral genes • less immunogenic • site specific integration (chr 19) • SAFE
• long term gene expression • no very infective for vascular cells • degraded quickly by the proteasome pathway
AAAAVV22 –– ddiiscscoovveerreedd iinn eeaarrllyy 1980s1980s
FGFR1
HSPGs
avb5
integrins
nucleus • Muscle applications • Cardiac/Vascular
Richter et al., Physiol Genomics 2:117127 (2000)
Endothelium Vessel lumen
Smooth muscle
Adventitia
AAV family is now extensive
local Systemic
Gao et al., PNAS 100:60816086 Gao et al., PNAS 2002 99:1185411859
AAV2
AAV cardiac gene delivery
AAV6/9
Gregorevic, Nat Med 10: 828834
AAV2
AAV9
Inagaki et al. Molecular Therapy 14: 4553, 2006
Circ Res Pacak et al. 99 (4): e3.
Next:
3 papers: Adenovirus and vein grafting AAV and cardiac disease Plasmids and peripheral ischaemia
Journal Club.
Further general gene therapy reading:
FOCUS ON GENEBASED THERAPIES Nature Reviews Genetics, May 2011.