120 proper timing of VEGF expression is required for the formation of stable and mature blood vessels

120 Proper Timing of VEGF Expression Is Required for the Formation of Stable and Mature Blood Vessels disease However, SVGs have limited durability Furthermore, there are no effective treatments to pr[.]

disease However, SVGs have limited durability Furthermore, there

are no effective treatments to prevent SVG failure.Gene transfer to

SVGs at the time of implantation has been introduced as a method

to modify the degenerative process and prevent vein graft failure

The most common viral vector employed has been recombinant

adenovirus and therefore,the duration of experimental transgene

expression has been relatively short compared to the anticipated

clinical life of the grafts It is unelear whether early and transient

transgene expression will correlate with late freedom from graft

failure In contrast to adenoviral vectors,adeno-associated viral

(AAV) vectors have been shown to provide stable long-term

trans-gene expression in vivo and are more clinically applicable since they

induce less inflammatory response and are not associated with human

pathogenic conditions.Very little is known regarding transduction of

AAV vectors in veins of higher mammals, ineluding humans

Suc-cessful treatment ofSVGs in patients with AAV vectors requires a

more comprehensive understanding ofthe transduction process I-Iere

we define the optimalconditions for AAV mediated transduction of

canine and human saphenous vein utilizing an in vitro model

Hu-man saphenous vein segments were acquired after coronary bypass

surgery Canine saphenous vein specimens were obtained from

Class A and Class B mongrels after euthanasia These specimens

were treated intraluminally with a suspension of either Adenoviral

or AAV luciferase vectors Parameters which were tested included:

physical (dose, duration of exposure,temperature,distension

pres-sure, oncotic pressure); chemical (proteosome inhibitors, heparin,

pluorinic gel); and genetic (capsid optimization) Veinsegments were

maintained in culture for 2 days, after which they were assayed for

viability and luciferase activity.Interestingly, of the AAV caps ids

examined,a genetically modified AAV3b variant (termed SASTG)

yielded substantially greater luciferase activity than eitherAAV I or

AAV2;expression levels approached,in some p instances,that ofthe

adenoviral luciferase vector Luciferase activity was dose dependent

with Ix I012tvp demonstrating highest transduction efficiency The

transduction efficiency ofAAV-SASTG luciferase was not affected

by oncotic pressure, however, it was increased at higher luminal

distention pressures This study demonstrates that AAV is an

effec-tive vector for transgene delivery to canine and human saphenous

vein grafts Moreover, the AAV-SASTG capsid modification

pro-vides superior transduction efficiency in this model as compared to

AAVI and AAV2 SASTG has transduction efficiency that in some

instances approaches adenovirus This combined with preliminary

data that show SASTG'simproved function in the setting ofheparin,

makes SASTG the ideal capsid type for gene therapy for coronary

artery bypass grafting Preliminary studies arc currently underway

examining this improved transduction efficiency in a canine vein

graft model These data will also be reported

Vector Inhibits Neointimal Hyperplasia in a Rabbit

Vein Graft Model

Qing-HaiMcng,' Scott A Irvine; Jean R.Mcliwan,'Stephen L

Hart.'

IMolecular Immunology Unit, UCL Institute ofChild Health,

London , United Kingdom; 'Cenne for Cardiovascular Medicine

and Biology, University College London, London, United

King-dom

Vein graft failure is a main subject of vascular investigations

Efforts have been focused on preventing neointimal hyperplasia

which is the major cause of long term failure Proliferation and

mi-gration of vascular smooth muscle cells and adventitial fibroblasts

contribute significantly to neointima formation and have become

the target of gene therapy However,the lack of safe and efficient

vectors has significantly hindered the progress in this field We have

developed a synthetic vector system termed a Receptor-Targeted

Molecular Therapy Volume1 5 SupplementI ~ br 2007

C opyright © T he Am erican So ci ety o t Gen e Therapy

Nanocomplex (RTN) which comprises a cationic lipid, a cationic targeting peptide and plasmid DNA,to elicit synergistic effects

on gene delivery.The RTN system has demonstrated efficient and consistent gene transfer in many cell types and animal models In this study, we aim to transfer therapeutic genes, TIMP-I (tissue inhibitor of metalloprotcinasc-l ) and iNOS (inducible nitric oxide synthase), to the vessel wall in a rabbit vein graft model to evaluate the efficacy ofthe RTN vector in vascular gene therapy Rabbit vein graft surgery with cuffed anastomosis was performed to position a fragment of jugular vein to carotid artery Ex vivo gene delivery was carried out, adventitially with ends of vein graft clipped, by immersing vein graft for 20 minutes in a saline solution of the RTN vector Efficiency of gene delivery was firstly evaluated with

a reporter gene,GFP,in cultured rabbit aorta rings Incubation of the rings, for periods as short as 15 minutes,with RTN formulations could elicit efficient transfection with GFP,with peak expression observed on day-5 Deliveryof~-Galactosidase in vivo in the vein graft model led to efficient transfection throughout the vein wall

on day 7 Another 29 New Zealand white rabbits (3.0-3.5kg) were divided into 4 groups of vein graft surgery and gene transfer with RTNs carrying different genes: I) TIMP-I group (n=7); 2) iNOS group (n=7); 3) pCI control group (n=8): vehicle plasmid containing

no functional gene; 4) surgery control group (n=7): no RTN vector added in the salinesolution Vein graft samples were collected on day-28 and morphometry analysis was performed on paraffin sec-tions with Elastin-VG staining Significant inhibition ofneointima formation was observed in iNOS group compared to control groups

in terms ofboth neointimal area (60% reduction compared to surgery group and 77% reduction to pCI group) and thickness (41% reduc-tion compared to surgery group and 46% reducreduc-tion to pCI group, standardized by the ratio of neointima to medial layer) However, TIMP-I gene transfer showed no Inhibitory effect on neointimal hyperplasia In addition, plasmid DNA was detected by peR in vein graft samples but not in organ samples (lung,liver, kidney, and testis) from iNOS transfected rabbits The results of this study demonstrate the efficacy of RTN vectors in vascular gene therapy, which suggests a novel selection of a safe and efficient vector to deliver genes to vascular system In addition,we have confirmed the therapeutic effects of iNOS,but not TIMP-I,gene transfer on neointimal hyperplasia in vein graft disease

Required for the Formation of Stable and Mature Blood Vessels

SabrinaTafuro ,IEduardAyuso,'Serena Zacchigna,ILorena

'Molecular Medicine Laboratory; International Centre for Gen etic Engineering and Biotechnolgoy (ICGEB), Trieste , Italy ; / Centre ofAnimal Biotechnology and Gene Therapy (CBATEG), Universitat Autonoma de Barcelona, Barce/ona, Spain;'Struuura

Complessa di Medicina Nucleare, Az Ospedaliero-Universitaria Ospedali Riuniti, Trieste, Italy.

AAV vectors represent an outstanding tool for pro-angiogenic gene transfer,given the specific tropism ofthese vectors for skeletal and cardiac muscle cells We have previously observed that the injcc-tion ofAAV-VEGF in the mouse skeletal musele induced marked nco-vascularization,persisting for several months To establish the kinetics of the angiogenic response and the duration of the VEGF stimulus required to promote functional vessel formation, we now constructed novel AAV vectors in which the expression ofVEGF165 was regulated by an inducible Tet-On system When the vectors were injected in the mice tibialis anterior muscle,the expression

of the VEGF mRNA was found to be strictly dependent upon the administration ofdoxyclycline in the drinking water To our surprise,

we discovered that VEGP expression for a period of 15 days led to

S47

the formation ofan unstable vasculature, which regressed when gene

expression was turned off In contrast, the continuous expression

ofthe factor for at least 30 days was required for the formation for

stable vessels, that persisted upon withdrawal of the stimulus.To

determine the functional capability of the newly formed vessels,

we performed static scintigraphy using a gamma camera equipped

with pinhole collimator (Siemens Ecam), after 30 days ofVEGF

expression and after withdrawal ofdoxycycline for 15 days.At both

time points functional images of the mice legs were acquired by the

injection of 3.7 mBq of99mTe Tetrofosmin in basal condition and

of37 MBq after IO min of hind-limb muscle contraction, to mimic

physiological exercise We discovered that the vasculature formed

by continuous VEGF expression not only did not increase basal

blood flow, but it even decreased perfusion after muscle exercise

This remarkable finding was probably related to a pathological

increase in the leakiness of the newly formed vasculature and to

the formation of artero-venousshunts that exclude perfusion of the

microvasculature In contrast,the vessels persisting after cessation

ofthe VEGF stimulus were found to maintain functional perfusion

after increased physiological blood flow demand When analyzed

histologically by the injectionof'fluorescentlectins, the vasculature

formedaftercontinuous VEGF expression consisted ofcapillaries or

increased diameter and abnormal shape, which showed remarkably

altered permeability, Together, these results clearly indicate that

functionalblood vessel formation requires an appropriate duration

ofthe VEGF stimulus,casting doubts on the potential ofVEGF gene

therapy trials that exploit systemswith short-term or unregulated

expression of this therapeutic gene

Multi-Functional Echogenic Liposomes for

Image-Guided and Ultrasound-Controlled

Oligonucleotide Delivery

Shaoling Huang,' Robert C.Macbonald,' David D.Mcl'herson.'

'Internal Medicine, Division ofCardiology, UniversityofTexas

Health ScienceCenter :Houston , TX; ]BiochemistI J\ Molecular

Biologyand Cell Biology Northwestern University, Evanston, IL.

NF-kappaB decoy is a novel drug for atheroma treatment, and

mechanism to enhance local delivery may have important clinical

implications Echogcnie liposomes that co-encapsulate gas and

NF-kappaB decoy will allow directed ultrasound image-guided

deliveryand controlled release.Liposomes containing air were

prepared by conventional procedures of hydrating the lipid film,

sonication, freezing and thawing A single, but critical,

modifica-tion of this method involved pressurizamodifica-tion of lipid dispersion

with air after sonication to encapsulate air into Iiposomes After

equilibration,the sample was frozen The pressure is reduced to

atmospheric and the suspension thawed This method allows the

echogenic liposome to be made anionic or cationic.For anionic

liposomes,NF-kappaB decoy was encapsulatedin hydration step

For cationic liposomes,NF-kappaB decoy was added after

freeze-thawing The echogenicity ofdrug-loaded Iiposomes was measured

by a 20 MHz intravascular ultrasound catheter Ultrasound-triggered

release was achieved by applying I MHz ultrasound at 2 W/cm2

for lOs This Iiposomal preparation results excellent air content (30

III per 5 mg Iiposomes),high ultrasound refclectivity and sufficient

NF-kappaB decoy loading efficiency When added into anionic

liposomes, 15+1-3%of NF-kappaB decoy was encapsulated in

lipsomes A single ultrasound application triggered 40% of release,

When added into cationic echogenic liposomes,90% of

NF-kap-paB decoy can be associated with liposomes Cationic eehogenic

liposomes containing air and NF-kappaB decoy were incubated

with cultured vascular smooth muscle eells(VSMC) to study their

effects on cell proliferation.Ultrasound enhanced the uptake of

NF-kappaB decoy into VSMC and thus enhanced the biologic

cf-S48

feet ofNF-kappa decoy on the inhibition ofVSMC proliferation by 30% This drug delivery system possesses unique properties for cell targeting, ultrasound detection,drug encapsulation,triggered drug release and ultrasound-enhanced uptake.The method present here also applicable for siRNA delivery

UlJlno und enhllrtced the InhIlltlon eITeet01NF kapp B decoy on l he proflteraUo n of vascular smoot h muscle ceUs

Mea n ~ SO n=3

~ Iu. :"b2~ ~~ O~

",.c: ".c:

i'E c~ "'CIIJ 0

tEa++ +++o~

an Efficient Vector for Vascular Gene Delivery

Ilia Fishbein,'Meizan Lai.' RobertJ.Levy.'

'Pediatric Cardiology, The ChildrensHospital ofPhiladelphia Philadelphia, PA

Background and hypothesis Effective gene therapy of vascular disorders remains an elusive target The main reasons contributing

to the relative inefficacy ofvascular gene therapy are poor transfec-tivity/transducibility of cell types in the arterial wall,and the short duration oftransgene expression Adena-associated virus ofserotype

2 pscudotyped with serotype 9 capsid (AAV2/9) has been previously

demonstrated to achieve higher transduction of heart and skeletal

muscle than the parent AAV2 vector However, no study to date has investigated the capability ofAAV2/9 to transduce vasculature

Thus, we hypothesized that the hybrid AAV2/9 serotype may

pos-sess significant transduction potency toward arterial tissue in vivo Herein we report on an extremely robust reporter gene transduction

in rat carotid arteries achieved by local delivery ofAAV2/9

Meth-ods Subconfluent monolaycrs of rat aortic smooth muscle (AW)

and bovine aortic endothelial (BAEC) cells were transduced with AAV2(cmv)GFP,AAV2/9( cmv )GFP and Ad5( emv)GFP at GCIccI!

and particle/cell ratio of Ix IO~. The resultingtransgene expression was determined by live cell fluorometry To investigate efficacy of respective viral vectors in vivo,50 III volumes or the suspensions comprising 5xl01UAAV2/9 GC or 5xl01Uphysical particles ofAdS (both encoding firefly luciferase under control ofthe CMV promoter) were intravascularly delivered for 3 min to isolated endothelium-de-nuded segments ofrat common carotid arteries The ensuing arterial reporter expression was studied by serial intravital bioluminescence imaging Results In A I0 cells experiments,the peak GFP expression achieved with both AAV serotypes at 7 days post-transduction was

18-fold (AAV2/9) and IS-fold (AAV2) lower,than the expressionat

48 hours following Add-mediated transduction.In BAEC cultures AAV2 transduction resulted in 56-fold lower OFP expression levels,

Molecul ar Therapy Vo lume 15 Supp lement I• \br 2007

C opyright © " 111(: Amcri cm Soc iety o f Gen e Therap y