Gene Therapy in Coronary Heart Disease

Slide 1 © 2003 By Default!
Gene Therapy in
Coronary Heart Disease
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 Research into gene transfer techniques as potential therapeutic strategies

for cardiovascular disease began in the early 1980s.
 One of the first reports of successful gene transfer to vascular cells was by
Nabel et al in 1989.
 First human clinical trial in vascular gene therapy was started in 1994 by
Professor Isner.
 Therapeutic Angiogenesis –
A strategy for treating myocardial ischemia by inducing
neovascularization of the heart by use of “angiogens,” mediators that
induce the formation of blood vessels.
 Rationale –
In the adult heart, the genes coding for angiogens and their receptors
are expressed in insufficient levels in most individuals to provide robust
formation of collaterals in response to chronic ischemia.

Pro-angiogenic molecules
 Angiopoietin
 Del-1
 Fibroblast growth factors (FGF)
 Granulocyte-macrophage colony-stimulating factor (GM-CSF)
 Granulocyte colony-stimulating factor (G-CSF)
 Hepatocyte growth factor (HGF)
 Hypoxia inducible factor (HIF)
 Monocyte chemoattractant protein (MCP)-1
 Nitric oxide (NO), nitric oxide synthases (NOS)
 Platelet-derived growth factor (PDGF)
 Vascular endothelial growth factors (VEGF)
Angiogens
 VEGF
VEGF serves as a major angiogen in normal cardiac development
Exclusive to endothelial cells
High specificity for receptors
Initiates a cascade of events - endothelial cell migration, proliferation,

aggregation into tubelike structures, and networking of the arterial
and venous systems
Activates of many other genes involved in angiogenic response
The VEGF gene has 4 forms - VEGF121 and VEGF165 important.
Involved in the first human trial in vascular gene therapy started in

1994
treating PVD, and for therapeutic angiogenesis since 1997.

 FGF
Associated with tumor angiogenesis
Not as specific as VEGF, stimulates the proliferation of endothelial

cells, smooth muscle cells and fibroblasts
Not only stimulates VEGF production but also increases the density of
PDGF receptors.
The increased cellularity of the FGF-stimulated vessels produces more

mature vessels.
FGF, but not VEGF, was shown to participate in arteriogenesis.
Recent studies demonstrate cardioprotection action.
23 structurally similar forms of FGF. FGF-1, -2, -4 and -5 have been

used in angiogenic studies with no differences in efficacy.
Choosing a gene therapy vector
 Plasmid DNA –
Can transfer large size DNA
Low efficacy and low specificity
Efficiency increased somewhat by addition of liposomes
Highly safe
 Genetically modified viruses –
Adenovirus,
adeno-associated virus,
baculovirus,
herpes simplex virus,
lentivirus,
retrovirus
Sendai virus
Retroviruses Adenovirus serotype 5

Stable integration into the host genome Lack of genome integration
long-lasting gene-transfer effect transient expression
Inability to infect non-dividing cells High-level transfer
Low titres Highly efficient
New more efficient pseudotypes High conc produced
Stable in storage
 Mixed
Sendai virus liposome
Efficient
Safety ?
Adenovirus liposome
Efficacy lower than adenovirus
 Most clinical trials in gene therapy so far have used a retrovirus,

DNA/liposome combination or adenovirus


Choosing a delivery method
 Epicardial -
Requires thoracotomy (during CABG or separately).
Surgical complications
Vector can be specifically injected near ischaemic areas
 Percutaneous endocardial injection –
Easier for the patient
Finding the ischemic sites can be challenging
Electromechanical (NOGA) mapping to localize ischemic areas,
requires specialized catheters, instrumentation and skills.
Direct injection may also cause complications and restrict vector
distribution within the myocardium.
 Intracoronary infusion –
Infuses the vector throughout the coronary vascular tree
Less risk of complications than a myocardial injection
Systemically dissemination, some toxicity depending on the dose.
Other possible modes of administration include infusion during

stenting or retrograde infusion into the coronary veins.
Slide 10
Phase II clinical trials testing angiogenic © 2003 By Default!
factors to treat CHD

TRIAL AGENT ENDPOINT RESULT
VIVA IC VEGF ETT 60d -
FIRST IC FGF2 ETT 90d -
FGF2 with FGF2 during CABG Angina class +
CABG
AGENT IC Adeno FGF4 ETT 4wks +
AGENT2 “ SPECT +
KAT VEGF after PCI Perfusion 6mths +
REVASC IC Adeno-VEGF ETT 6mths +
Euroinject one Percardial Adeno- Perfusion 3mths -

VEGF
VEGF2 Endocardial ETT, Angina class +

Plasmid VEGF

Ad5FGF-4 to treat chronic myocardial
ischaemia
 FGF Initiating RevaScularization Trial (FIRST)
Design - double-blinded, randomized, controlled trial
337 patients with stable angina.
Result - The frequency of angina was decreased at 90 days

the difference compared with placebo was not
sustained at 180 days.
ET was not increased at any time point.
 Injection of a sustained-release preparation of rFGF-2 directly

into the heart muscle during CABG
A small study
Perfusion defects were smaller in patients treated with
rFGF-2 and followed for 32 months.
More freedom from angina
2 late deaths (pancreatic cancer and undetermined cause)
The AGENT study programme
STUDY PHASE PATIENTS OBJECTIVES STATUS
AGENT II 79 Safety, selection of

dose, exercise
Complete
12-mth FU
duration complete;
telephone
contact every
6 mths
AGENT2 II 52 Safety, effect on

myocardial
“
perfusion
AGENT3 II/III 450 Change in ETT Stopped

Jan 2004
Safety and efficacy
AGENT4 II/III 450 “ “

AGENT (Angiogenic GENe Therapy) trials

 first randomized, double blind, placebo controlled trial of gene therapy
 AIM To test the clinical efficacy and safety of Ad5FGF-4
 DESIGN
patients with chronic stable angina (Class II or III)

randomized to receive either Ad5FGF-4 or placebo
Ad5FGF-4 was administered via 90-s intracoronary infusions.
Blood tests and physical examinations, which were performed at

baseline and at 1, 2, 4, 8 and 12 weeks, and at 6 and 12 months.
 EVALUATION
viral infectivity assay on pulmonary artery blood, venous blood and

urine; an assessment of FGF-4 levels in serum, adenoviral neutralizing
antibody titres; and semen analysis using PCR for adenoviral DNA
Tolerability and safety Ad5FGF-4

 FGF-4 protein was not detectable at any time in the plasma samples.
 No virus could be detected in the urine.
 Semen testing at 8 weeks showed no detectable adenoviral DNA.
 In a long-term mean follow-up time of 311 days
No evidence of myocarditis, new heart failure and reduction in LVEF
No cases of retinal neovascularization.
14 patients hospitalized for worsening angina(10 from active t/t group)
Two patients experiencing transient elevated SGPT and bilirubin
 Two patients were diagnosed with malignancies (metastatic colon cancer
and brain tumour (glioblastoma multiforme) ,both tumours were negative for
Ad5FGF-4 DNA.
 One patient had a skin lesion removed, diagnosed as a squamous cell Ca.
This patient had several skin lesions removed previously, all thought to be
pre-malignant.
Slide 15
The VIVA Trial © 2003 By Default!
Vascular Endothelial Growth Factor in Ischemia for Vascular

Angiogenesis
 Design –
Stable exertional angina and
areas of viable but underperfused myocardium
unsuitable for revascularization.
 Method –
Patients were randomized to receive a 20-minute IC infusion of
placebo, low-dose rhVEGF or high-dose rhVEGF followed by 4-hour
IV infusion on days 3, 6, and 9
 Evaluation –
rest thallium scan and gated rest sestamibi scan followed by a
nongated redistribution thallium scan and a gated stress sestamibi
 Primary end point –
change in ETT time from baseline to day 60
 Result –
rhVEGF offered no improvement in all measurements by day 60.
By day 120, high-dose rhVEGF resulted in significant improvement in
angina and favorable trends in ETT time and angina frequency
KAT Trial
 AIM –
Safety and Feasibility of Catheter-Based Local Intracoronary VEGF
Gene Transfer in the Prevention of Postangioplasty and In-Stent Restenosis
and in the Treatment of Chronic Myocardial Ischemia
 Design –
PTCA was performed with standard methods, followed by gene
transfer
90% of the patients were given stents;
37 patients received VEGF adenovirus, 28 patients received VEGF
plasmid liposome and 38 control patients
 Follow-up time - 6 months
 Result –
The overall clinical restenosis rate was 6%.
The minimal lumen diameter and percent of diameter stenosis did not
significantly differ between the study groups.
Myocardial perfusion showed a significant improvement in the VEGF-
Adv–treated patients after the 6-month follow-up.
Gene transfer was well tolerated
Promising Gene- based therapies

Protection of the myocardium at risk
 rAAV intramyocardial delivery of heme oxygenase-1 (HO-1) in a
rat model results in approximately 80% reduction in infarct size
and reduction in myocardial injury leads to post-infarction
functional recovery and normalization of LV dimensions.
 Comparable findings with extra cellular superoxide dismutase
(ec-SOD) gene transfer.
 Significant protection - Cu/Zn SOD catalase, glutathione
peroxidase and stress-induced heat shock proteins.

Restenosis after Angioplasty.

 REGENT-I Trial, currently in progress, to evaluate the efficacy of catheter-
based iNOS gene delivery to prevent restenosis of coronary arteries treated
by PTCA
 Inhibition of the cell cycle using non-phosphorylated Rb (retinoblastoma)
gene or anti-oncogenes such as p53 and p21 has been reported in several
animal models.
 A human trial using naked VEGF165 plasmid DNA has been started for
restenosis after angioplasty in a peripheral artery. the preliminary results
document successful inhibition of the restenosis.
 Preclinical experiments with overexpression of HGF in balloon-injured
arteries accelerated re-endothelialization, thereby attenuating intimal
hyperplasia, have been also reported.

Atherosclerosis and thrombosis

 The over-expression of apoprotein ApoA-1 in mice by IV
adenoviral gene delivery increases serum HDL levels.
 Blockade of monocyte chemoattractant protein-1 (MCP-1)

receptor retards the onset of atheroma and to limit progression
and destabilization of established atherosclerotic lesions in
ApoE deficient mice.
 Lipid-lowering gene therapy in humans has to date only been

evaluated in a small Phase I clinical trial showed a reduction of
6 – 23% in plasma LDL levels.

Morbidity
 Accelerated Atherosclerosis
Over-expression of VEGF in mouse heart led to intramural angiomas
followed by heart failure and death and that VEGF may accelerate
plaque progression in atheroscleroticvessels.
Human subjects participating in clinical trials of VEGF recombinant
protein have shown no evidence of accelerated atherosclerosis.
 Vascular Malformations
Preclinical and clinical studies to date suggest that therapeutic
angiogenesis transfer are unassociated with hemangioma formation.
 Neoplasms
Over-expression of VEGF may induce angiogenesis of occult
neoplasms Among 85 patients undergoing VEGF gene transfer for
myocardial ischemia, neoplasms were diagnosed in 2 patients.
Among 79 patients undergoing IC gene transfer of Ad/FGF new

neoplasms were identified in two treated patients.
The VIVA trial, among 178 patients 3 patients developed new

evidence of cancer (fatal in two) within the 180-day time period of the
trial; all three patients had been randomized to the control arm.
 Retinopathy
Clinical studies to date, including patients with diabetes and a previous
history of retinopathy, have disclosed no evidence to support the
notion of exacerbation of ocular pathology.
 Edema
VEGF was discovered as a tumor-secreted factor that augments
vascular permeability.
Clinically apparent peripheral edema typically developed within 3
weeks after gene transfer and corresponded temporally to an increase
in circulating levels of VEGF,
Edema was promptly attenuated after the administration of oral
diuretics and resolved completely within 2 to 4 weeks after the initiation
of therapy.
 Hypotension
 Arrhythmias

Mortality
Indication Vector/gene Route Nature of Death
CAD NV/VEGF IMyo Resp & Renal failure
CAD NV/VEGF IMyo Cardiogenic shock
CAD Ad/FGF IC Sudden death

CAD Ad/FGF IC Colon cancer
CAD Ad/VEGF IMyo MI, Pneumonia
CAD Ad/VEGF IMyo Ileocolic abscess
CAD Ad/VEGF IMyo Sudden death
Restenosis VEGF IA Cradiac arrest
Restenosis VEGF IA Lung cancer
Unanswered Questions
 Long term safety
 Safest and most efficacious route
 single administration or multiple treatments
 the optimal timing for administering these genes

in the clinical setting
Future of Gene Therapy
 Combination of various angiogenic factors such as VEGFs and

FGFs and angiopoietins may be possible to mimic physiological
angiogenesis more closely.
 Gene cocktails or sequential administration of treatment genes

may be more effective than a single administration of one
growth factor.
 The future will likely see increased demand for the use of vector
systems with the capability to confer tissue-specific expression
of therapeutic transgenes.

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Slide 1 © 2003 By Default!

A Free sample background from www.powerpointbackgrounds.com

 Research into gene transfer techniques as potential therapeutic strategies

A Free sample background from www.powerpointbackgrounds.com

VEGF serves as a major angiogen in normal cardiac development

Exclusive to endothelial cells

High specificity for receptors

Initiates a cascade of events - endothelial cell migration, proliferation,

Activates of many other genes involved in angiogenic response

The VEGF gene has 4 forms - VEGF121 and VEGF165 important.

Involved in the first human trial in vascular gene therapy started in

A Free sample background from www.powerpointbackgrounds.com

Not as specific as VEGF, stimulates the proliferation of endothelial

The increased cellularity of the FGF-stimulated vessels produces more

FGF, but not VEGF, was shown to participate in arteriogenesis.

Recent studies demonstrate cardioprotection action.

23 structurally similar forms of FGF. FGF-1, -2, -4 and -5 have been

 Genetically modified viruses –

Retroviruses Adenovirus serotype 5

 Most clinical trials in gene therapy so far have used a retrovirus,

A Free sample background from www.powerpointbackgrounds.com

A Free sample background from www.powerpointbackgrounds.com

Other possible modes of administration include infusion during

factors to treat CHD

AGENT IC Adeno FGF4 ETT 4wks +

REVASC IC Adeno-VEGF ETT 6mths +

Euroinject one Percardial Adeno- Perfusion 3mths -

VEGF2 Endocardial ETT, Angina class +

A Free sample background from www.powerpointbackgrounds.com

Result - The frequency of angina was decreased at 90 days

 Injection of a sustained-release preparation of rFGF-2 directly

STUDY PHASE PATIENTS OBJECTIVES STATUS

AGENT II 79 Safety, selection of

AGENT2 II 52 Safety, effect on

AGENT3 II/III 450 Change in ETT Stopped

AGENT4 II/III 450 “ “

A Free sample background from www.powerpointbackgrounds.com

AGENT (Angiogenic GENe Therapy) trials

patients with chronic stable angina (Class II or III)

Ad5FGF-4 was administered via 90-s intracoronary infusions.

Blood tests and physical examinations, which were performed at

viral infectivity assay on pulmonary artery blood, venous blood and

Tolerability and safety Ad5FGF-4

Vascular Endothelial Growth Factor in Ischemia for Vascular

Promising Gene- based therapies

A Free sample background from www.powerpointbackgrounds.com

Restenosis after Angioplasty.

A Free sample background from www.powerpointbackgrounds.com

Atherosclerosis and thrombosis

 Blockade of monocyte chemoattractant protein-1 (MCP-1)

 Lipid-lowering gene therapy in humans has to date only been

A Free sample background from www.powerpointbackgrounds.com

Among 79 patients undergoing IC gene transfer of Ad/FGF new

The VIVA trial, among 178 patients 3 patients developed new

A Free sample background from www.powerpointbackgrounds.com

CAD NV/VEGF IMyo Cardiogenic shock

CAD Ad/FGF IC Sudden death

 Long term safety