610 transfer of PAI 1 gene prevents abdominal aortic aneurysm development in mice

610 Transfer of PAI 1 Gene Prevents Abdominal Aortic Aneurysm Development in Mice Molecular Therapy �������� ��� ���� ���������������� �������� ��� ������© ����������� �!����� ����"� �������� S237 ���[.]

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

CARDIOVASCULAR all of the animals examined (gene transfer and controls) EGFP

expression was clearly evident by direct whole mount fluorescent

microscopy in the lung lobes of all gene transferred animals,

demonstrating transgene expression throughout the

post-administration gestational period (2-3 mos post-gene transfer) These

findings are significant because they indicate that (1) direct

administration of a lentiviral vector to the developing lung lobes

during early gestation and (2) subsequent transgene expression

post-administration did not impair lung growth or development in this

nonhuman primate model Further studies with the rhesus monkey

model will be crucial for assessing the safety and efficiency of gene

transfer for the treatment of human congenital lung disease

608 Localized Gene Delivery from

Polyurethane Heart Valve Cusps and Vascular

Implants Using Antibody Tethered Adenovirus

Stanley J Stachelek,1 Cunxian Song,1 Ivan Alferiev,1 Suzanne

DeFelice,1 Xiumin Cui,1 Jeanne M Connolly,1 Richard W

Bianco,2 Robert J Levy.1

1 Cardiology, Children’s Hospital of Philadelphia, Philadelphia,

PA; 2 Experimental Surgery, University of Minnesota, Minneapolis,

MN.

Favorable biocompatibility and durability make polyurethanes

an attractive material for fashioning prosthetic heart valves and

vascular implants We recently reported that bromoalkylated

polyurethane films, modified with covalently attached

bisphosphonate, function as a calcification resistant composite

material for replacement heart valve leaflets We test here the

hypothesis that gene vectors can also be appended to polyurethane

using a variant of our process chemistry, and thereby the prosthetic

leaflet could serve as a gene delivery platform Pendant bromoalkyl

groups on either Carbothane or Tecothane were activated with a

thiol-to-thiol crosslinker

(1,4-di-[3’-(2’-pyridyldithio)-propionamido]butane), and an adenovirus specific antibody

(anti-knob) was covalently linked either directly to the polyurethane film

(Tecothane), or to a collagen buffer layer covalently bound to the

polyurethane (Carbothane) Replication incompetent adenoviral

vectors (type V) containing the gene encoding green fluorescent

protein (AdGFP) or a beta galactosidase reporter molecule (AdLacZ)

were then tethered to the modified polyurethane surface In vitro

analysis of both polyurethane configurations showed that rat arterial

smooth muscle cells (A10), grown on polyurethane with either

tethered AdLac Z or AdGFP, was successfully transduced with the

virus and expressed the appropriate marker gene within 24 hours

In vivo analysis using implantable vascular “buttons” composed of

Delrin, coated on the blood exposed surface with AdGFP appended

to the collagen coated polyurethane matrix, showed GFP gene

expression in 14.3% ± 2.5% of attached cells after 7 days Similarly,

single pulmonary valve leaflet replacement in juvenile sheep showed

that leaflets composed of polyurethane with antibody complexed

AdGFP demonstrated GFP expression in 17.8% ± 1.8% of cells in

the adjacent myocardium, and in 25.1% ± 5.7% of adherent cells In

both in vivo models, FITC autofluorescence was not noted in control

animals, and PCR analyses for the GFP gene noted an absence of the

GFP in the bloodstream and in distal tissues of both experimental

and control animals Cells interacting with the polyurethane surfaces

were immunochemically identified as largely leukocyte common

antigen positive These data represent a marked improvement in

localized transduction efficiency over other vascular gene therapies

We conclude that chemically modified polyurethane vascular

implants are suitable gene delivery devices

609 Delivery of p27 to Rat Carotid Arteries Using Electroporation Protects from Intimal Hyperplasia Following Balloon Injury

Jennifer L Young,1 David A Dean.1

1 Division of Pulmonary and Critical Care Medicine, Northwestern University, Chicago, IL, United States.

Vascular diseases such as atherosclerosis, restenosis and vein graft stenosis affect millions of Americans each year The pathophysiologies of these diseases all consist of unregulated smooth muscle cell proliferation that results in intimal hyperplasia and ultimately vascular occlusion Gene therapy is a promising treatment option for smooth muscle proliferative diseases with several genes identified as potential therapies However, the efficient delivery of these genes to the target cells remains a major limiting factor Our lab recently developed a non-viral delivery method for gene transfer

to the vasculature using electroporation We have demonstrated that plasmid delivery by electroporation results in high level gene expression in rat mesenteric arteries with expression peaking between

1 and 3 days post transfer Based on our results in the mesenteric arteries, we reasoned that electroporation could be used to deliver genes to other vascular beds including larger arteries Using a square wave electroporator (200V/cm, 8 pulses of 10msec duration), we delivered a CMV driven luciferase plasmid to the common carotid arteries of rats In animals receiving DNA plus electroporation, we obtained an average of 56.38 pg/mg total protein with values as high

as 180 pg/mg protein at 2 days post-transfer In contrast, luciferase expression was undetected in arteries receiving DNA but no electroporation These levels are similar to those achieved in the mesenteric vessels, thus providing evidence that our previously described gene delivery method for the vasculature can be successfully applied to multiple vascular beds To extend these findings to a vascular disease model, we have focused on a rat model for restenosis Other labs have demonstrated that early expression

of the small cell cycle inhibitor p27 can inhibit intimal hyperplasia following vascular injury Therefore we hypothesized that delivering

a p27 expressing plasmid to the common carotid arteries of rats by electroporation immediately following balloon injury would decrease the degree of intimal thickening after injury, providing protection from intimal hyperplasia and vessel occlusion Indeed, we found that delivering as little as 50 μg of plasmid expressing p27 from the CMV immediate early promoter was sufficient to decrease the intimal to medial area ratio four-fold compared to untreated carotid arteries, (balloon injury only) at 14 days post-injury Further, injured arteries treated with the p27 plasmid without electroporation had the same degree of intimal hyperplasia as control balloon-injured arteries Taken together, these results demonstrate that electroporation is an efficient method for gene transfer and expression

in rat carotid arteries and can be effectively used to deliver therapeutic genes for the treatment of smooth muscle proliferative disease in vivo

610 Transfer of PAI-1 Gene Prevents Abdominal Aortic Aneurysm Development in Mice

Hu Sheng Qian,1 Ana Freay,2 Perry Liu,1 Katalin Kauser,2 Linda Cashion,3 Jon Morser,2 Ron Vergona,1 William P Dole,2 Mark E Sullivan,1 Gary G Deng.2

1 Department of Pharmacology, Berlex Biosciences, Inc, Richmond,

CA, United States; 2 Department of Cardiovascular Research, Berlex Biosciences, Inc, Richmond, CA, United States;

3 Department of Gene Therapy, Berlex Biosciences, Inc, Richomnd,

CA, United States.

Vessel wall matrix destruction is a key event in abdominal aortic aneurysm (AAA) formation and rupture We have previously shown that urokinase plasminogen activator (uPA) is highly expressed in

Molecular Therapy Vol 7, No 5, May 2003, Part 2 of 2 Parts

Copyright © The American Society of Gene Therapy

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CARDIOVASCULAR

AAA and is critical for its development In this study, we examined

the effects of overexpression of a natural inhibitor of uPA,

plasminogen activator inhibitor-1 (PAI-1), through gene delivery on

the development of AAA Methods: Angiotensin II (Ang II) (1.44

mg/kg/day) was administered to 6-month old male mice deficient in

containing either the human PAI-1 gene (Ad-PAI-1) or the luciferase

gene (Ad-Luc) by localized delivery through intra-advential injection

or systemic delivery through tail vein injection In vivo gene

expression and biodistribution of luciferase was monitored by a

cooled charge coupled device (CCCD) camera imaging system in

living mice The expression of human PAI-1 was determined by

both ELISA and immunohistology Abdominal aortas were examined

after 30 days and the tissues were harvested for histology and protein

or enzyme activity measurement Results: After 30 days of Ang II

treatment, there were no significant differences in general appearance

and cholesterol levels between the PAI-1 treated and the

Ad-Luc treated groups through localized or systemic gene delivery

Systemic delivery of the PAI-1 gene did not affect AAA incidence

(78% vs 90% in Ad-Luc controls, p=0.125) In contrast, AAA

development was completely prevented in the localized delivery

group (0% in Ad.PAI-1 vs 55.6% in Ad-Luc controls, p<0.05)

Adenoviral mediated gene expression peaked at day 7 and lasted up

to 28 days Positive PAI-1 staining was detected in association with

both macrophages and smooth muscle cells by immunohistology

Activities of MMP-2 and –9, measured by tissue zymography,

were significantly increased in AAA tissues Local overexpression

of PAI-1 reduced, but did not completely abolish MMP activity

despite the full protection against AAA development Conclusion:

In agreement with our previous data on the essential role of uPA in

the formation of AAA, inhibition of uPA by PAI-1 completely

blocked development of AAA

611 Screening of Cytoprotective Agent and Its

Action Mechanism in Cardiac Cells

BoHee Shin,1,2 HyunJoung Lim,1,2 SeaHyoung Lee,1 Seung-Hyuk

Choi,1 Yangsoo Jang,1,2,3 Ji Hyung Chung,1,3 Hyun-Young Park.1,3

1 Yonsei University College of Medicine, Yonsei Cardiovascular

Research Institute, Seoul, Korea; 2 Yonsei University College of

Medicine, Brain Korea 21 Project for Medical Science, Seoul,

Korea; 3 Yonsei University, Yonsei Research Institute of Aging

Science, Seoul, Korea.

According to a considerable amount of studies, heat shock proteins

(HSPs) are induced by various stressful stimuli such as heat and

hypoxia/ischemia and play and important role in protecting cells

from such stresses in many different types of cells including

myocardial cells Hsp70, a highly inducible form of HSP, has been

know to regulate myocardial function and reduce the cell damages in

hypoxia/ischemia condition In addition, this cytoprotective protein

is known to be involved in several pathologic pathways

We, therefore, investigated protein expressionpatterns regarding

cell survival or death in the cardiac cells under hypoxia condition

,and then performed drug screening to select out compounds that

could induce Hsp70 under the physiological condition Rat cardiac

cells were incubated with various candidate drugs and then expression

of Hsp70 was determined by immunoblotting Furthermore, the cell

viability after the treatment was evaluated using MTT assay After

the screening, the drugs that induced Hsp70 expression without any

significant cell viability changes were selected Among the candidate

drugs, thiamine and several compounds induced Hsp70 without

losing cell viability In addition, we isolated primary cardiomyocytes

from 2day-old SD rats Cultured primary cardiomyocytes were

treated with thiamine and subjected to a Hypoxic condition The

result shows that thiamine induced Hsp70 and protected against

hypoxia insult in cardiomyocytes.Our results imply that thiamine exerted cytoprotective effects in cardiac cells and it is most likely due to the increased expression of Hsp70

612 Efficient Cardiac Gene Transfer Using Percutaneous Aortic Occlusion Technique in the Rat

Tsuyoshi Tsuji,1 Federica del Monte,1 Motoya Hayase,2 J Luis Guerrero,3 Takehisa Abe,4 Yoshiro Yoshikawa,4 Shuichi Kobayashi,5 Shigeki Taniguchi,4 Miyako Takaki,5 Roger J Hajjar.1

1 Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA, United States; 2 Cardiology Laboratory for Integrative Physiology & Imaging, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States; 3 Cardiac Surgery Research, Massachusetts General Hospital, Harvard Medical School, Boston, MA, United States; 4 Department of Surgery III, Nara Medical University, Kashihara, Nara, Japan; 5 Department of Physiology II, Nara Medical University, Kashihara, Nara, Japan.

We have previously developed a cross-pulmonary technique involving the simultaneous occlusion of the aorta and pulmonary artery while injecting adenoviral solutions in the left ventricle Even though this technique has resulted in effective gene transfer, it is highly invasive and not feasible for sequential multiple surgeries

We have therefore developed a percutaneous technique whereby a Fogarty catheter is introduced into the right carotid artery and advanced by radiographic support retrograde towards the proximal aorta Simultaneously from axillary artery another catheter was introduced to the same proximal aortic area The Fogarty balloon was inflated and adenosine was injected into the proximal aorta to perfuse the coronary arteries and increase permeability and decrease heart rate too Through the infusion catheter which lay distal to the balloon (as shown below) 0.25 ml of Ad.bgal was infused over a 10 second period The balloon was kept inflated for a total of 40 sec Five days post-gene transfer, the rats were sacrificed and the number

of affected myocytes was evaluated In all the sections through the left ventricle, 30-50% of the cardiac myocytes showed b-galactosiadase activity The survival rate depended on the speed of adenoviral injection and the size of balloon inflation Fast injections and large balloon size were associated with high procedural mortality

We describe here a reliable and effective method for cardiac gene transfer in close-chested rats that can be used in many models of cardiovascular diseases in this animal