354 adenoviral eNOS eluting stents result in accelerated re endothelialisation and a reduction in neointima formation in an in vivo rabbiat iliac artery stent model

354 Adenoviral eNOS Eluting Stents Result in Accelerated Re Endothelialisation and a Reduction in Neointima Formation in an In Vivo Rabbiat Iliac Artery Stent Model events including structural stress[.]

events including structural stress to a cell Indeed,we found with

irnmunostainingthat MPS I mice had a marked increase in nuclear

levels ofSTAT3 that isphosphorylatedat tyrosine 705 and at serine

727 Wepropose that massive lysosomal storage exerts stress upon

the cell,which causesactivation ofSTAT3, resultingin upregulation

of the elastase proteins MMP-12 and cathepsin S The second goal

of this study was to determine ifgene therapy could preventdisease

High dose neonatal gene therapy with a retroviral vector resulted in

1000 Ulml of serum IDUA activity and prevented upregulation of

MMP-12 and cathepsin S RNA,and development of aortic disease

in MPS I mice However,animals with <100 units oflDUA activity

after neonatal or adult therapy still developed aortic disease

Simi-larly, MPS I dogs that achieved 500 Ulml of serum IDUA activity

failed to develop aortic disease,while 25 U/ml was not completely

effective Specific inhibitors of one or both of these enzymes may

reduce disease in the aorta and possibly other sites in patients with

MPS I and related disorders

352 Correction of Hypoglycemia Following

Gene Therapy in Dogs Affected with Glycogen

Storage Disease Type la

Carlos R Pinto,ITalmage T Brown,IDaniel M Kozink,IAmanda

K Dcmaster,'Meghan A Kruse; KazuhiroOka.'AndrewBird,'

Mark W.Jackson,4Yuan-TsongChen,' Lawrence Chan.?Dwight

D Kocbcrl.'

IPopulation Health & Pathobiology North Carolina State

Uni-versity College of Veterinary Medicine, Raleigh, NC ; 2 Division

ofDiabetes, E ndocrinology and Metabolism, Baylor College of

Medicine , Durham, NC; JPediatrics /Division ofMedical

Genet-ics, Duke University Medical Center; Durham , NC; 'Faculty0/

Veterinary Medicine, University ofGlasgow, Glasgow, Scotland,

United Kingdom.

The glycogenstorage disease type la (GSO-Ia) described in dogs

closely resembles human GSD-Ia Both GSD-Ia human and canine

patients suffer from complications associated with

glucosc-e-phos-phatase(G6Pase)deficiency(hypoglycemia,hyperlipidemia, growth

retardation,and early death) For the past two decades survival of

human patients that are placed under intensive nutritional

manage-ment with uncooked corn starch has improved; however,long-term

complicationspersist includingrenal failure,nephrolithiasis,hepatic

adenomas, and a high risk for hepatocellular carcinoma Gene

therapy has been pursued as an alternative treatment for GSD-Ia

using a canine model In a previousstudy,we reportedthe successful

delivery of the normal canine G6Pase to the liver of 3 GSD dogs

using an adeno-associated virus type 2 (AAV2) Clinical correction

ofGSO in treateddogs was moderateand extendedsurvival(ranging

from 20 to 86 days of life) was difficult, despite intensive

supple-mental nutritional therapy In the current study, we investigated

the efficacy of an AAV2 vector cross-packaged as AAV8(AAV2/8

vector) (n=3) encoding human glucose-6-phosphatase (lxlOEI3

vector particles/kg), and a helper-dependent adenovirus (I-lOAd)

vector (n=I) encoding canine G6Pase (2xIOE I2 vector particles/

kg) Crossbred carrier females were bred by artificial insemination

to carrier males resulting in the birth of 4 GSD pups At 3 days of

age, 3 ofthe pups received theAAV2/8 vector by intravenous

injec-tion The fourth puppy was injected with the 1·IOAdvector.All4

pups responded positively within the first week to vector injection

with increased blood glucose levels The treated GSO pups require

minimal clinical attention and have not required supplemental

nutritional therapy Biweekly blood glucose levels of treated GSO

pups,after a 2-hour fast,have been within a normal range (73-I16

mg/dL) in contrast to hypoglycemia« 60 mg/dl) that occurs in

fasted untreated GSD pups The increase in body weight of the4

treated pups approaches that ofnon-GSOlitterrnatesin contrast to

the poor growth of untreated GSD pups Survival of these treated

SI34

GSD pups (current age ranges from 2 to 7 months old) is prolonged

in comparisonto untreatedGSO pups that experience88% mortality

by 2 months of age Continuation ofthese studies could provide the basis forjustifying a clinical trialusingAAVand/or HDAd vector-mediated gene therapy in scleeted human cases ofGSD-la CARDIOVASCULAR GENE THERAPY

353 Ad2-Mediated Expression of a Constitutively Stable Hypoxic Inducible Factor-1 ex Enhances Collateral Development and Reduces Vascular Leakage in a Diabetic Rat Model of Hindlimb Ischemia

Adam J Belanger; HidetoshiKajiwara,'ZhengyuLuo,' Akihiro Urabc.'Seng H.Cheng,'SeibuMochlzuki.'CanwenJiang.'

'Applied Discovery Research, Genzyme , Corp , Framingham, MA; 2Dept ofInternal Medicine, Cardiovascular Division , Jikei University School ofMedicne, Tokyo, Japan.

TypeII diabetes is a common co-morbidity of peripheral artery disease Hypoxia-inducible factor-l (HIF-I), a master regulator

of the expression ofangiogenic growth factors.has been shown to stimulate angiogenesis in various preclinical studies In this study

we investigatedthe effectsofadenoviral vectorsexpressingvascular endothelial growth factor (Ad/2VEGF) or a hybrid hypoxia-induc-ible factor-I alpha (Ad2/H1F-IalVPI6) on collateral development and vascularleakinessin diabetic rats Theright femoral artery of 8-week-oldZucker lean (ZL) or Zuckerdiabetic fatty(ZDF) ratswas removed to induce hindlimb ischemia Three days aftersurgery,the mRNA levels of endogenous VEGF in the hindlimb muscle were elevated in ZL rats but not in their ZOF counterparts Seven days aftersurgery, however, this elevation diminished to comparable levels of that of ZDF rats Thirty-five days after surgery, endog-enous collateraldevelopment,as determined angiographically,was significantly less inZDF rats,comparedto ZL animals These results suggest that the endogenous angiogenic response in ZDF rats was retarded,possibly due to decreased VEGF expression In separate groups of animals, seven days after surgery, Ad2/1-IIF-IalVPI6, (Ad/2VEGF),or Ad2/CMVEV (a control vector expressing no transgene) was injected into the thigh muscles Both Ad2/HIF-IaI

VPI6 and (Ad/2VEGF) increasedcollateraldevelopment.Vascular leakiness,as determined by tissue Evans-blue dye content, was significantly higher in ischemic limbs of ZDF rats.compared to ZL rats Ad2/HIF-1alVP I 6 but not Ad2IVEGF reduced the vascular leakiness These results suggest that although Ad2/HIF-1 alVPI6 and Ad2IVEGFare capable ofpromoting collateral development in the diabetic ischemiclimb,Ad2/HIF-IalVP I6 also reducedvascular leakiness These results suggest that angiogenic gene therapy for diabetes using hybrid HIF-Ia may have an advantage over VEGF gene therapy

354 Adenoviral eNOS-Eluting Stents Result

in Accelerated Re-Endothelialisation and a Reduction in Neointima Formation in anIn Vivo

Rabbiat Iliac Artery Stent Model Faisal Sharif, Sean O Hynes,Ronan Cooney,Linda Howard,Jill McMahon, Kieran Daly, James Crowley, FrankBarry,Timothy O'Brien

'Department a/Med icine, Regenerative Medicine Institute, Na-tional University ofIreland Galway, Galway, Ireland.

Introduction: Localdeliveryof anti-proliferative drugs by drug elutingstentsfor coronary artery disease results in inhibition of smooth muscle cell and endothelialcell proliferation.Impairmentof endothelial repair increasesthe risk of'stent thrombosis In the

pres-Molecular Therapy Yofum e 15 S upplement I, \b y 2007

Co pyright © '111C Am erican Socie tyo f G ene TI ICr.lpr

ent study we attempted to achieve an enhanced rc-cndothclialization

of deployed stents while simultaneously inhibiting intimal

We have previously shown that over expression of eNOS

follow-ing vascular injury have beneficial effects on vascular remodelfollow-ing

Therefore deliveryof eNOS to the blood vessel wall using

delivery,re-endothelialization and intimal hyperplasiawere assessed

in iliacarteries from46 normal diet and

16hypercholesteremicrab-bits Successful gene delivery of eNOS was confirmed by RT-PCR

higher in normocholesterolemic vessels at day 14 with stents

elut-ingAdeNOS (85.34%±7.38 vs 62.66%±10,49, p<0.05) and also at

28 following stent placement there was a significant increase in the

79.26o/o±20.57, p<0.05) in AdeNOS treated vessels in comparison

with controls in hypercholesteremic arteries as assessed by

mor-phometry and quantitative coronary angiography (15.95%±7.63 vs

56.9o/o±38.6, p<0.05) Conclusion: Localized eNOS gene delivery

using adenovirus gene-eluting stents results in enhanced endothelial

regenerationand a reductionin neointimal formation.Therefore, this

simultaneously reducing the risk of stent thrombosis

355 Novel Gene Transfer Vectors To Modulate

the Renin Angiotensin System

Glasgow, Glasgow, UnitedKingdom.

and adrenal function via regulating fluid/ electrolytes and arterial

c.g, stimulating proliferation of smooth muscle cells and fibroblasts,

oxidative stress, inflammation and cardiac hypertrophy Thus, the

the pathophysiology of cardiovascular disease (CVD) Recent

understanding of the RAS has identified other biologically active

metabolites of Angll, including Ang1-7.Ang1-7 may be important

peptides or sustained release from implanted mini pumps We have

developed adenoviral vectors (RAds) expressing Angll or Ang-I-7

in CVDs, including cardiac hypertrophy and atherosclerosis The

expression cassette encodes a fusion protein for a signal peptide (for

secretion), heavy chain IgG (for molecular mass) linked to Angll or

RAd inducedexpression of the fusion protein,confirmed by western

West-ern analysis of concentrated conditioned cell media demonstrated

human vascular smooth muscle cells (VSMC) were transduced with

RAdAngll or RAdAng1-7 and effects on cell numbers measured

at 48 hours VSMC numbers following RAdAngll transduction

percent-age increase following stimulation of cells with exogenous Angll

(I25.6±6.7% of control) VSMC transduced by RAdAngl-7 and

exposed to Angll stimulation were not significantly different in

number to unstimulated control cells but were 75.2±4.5% of Angil

O;lprrighf <0 Tlu: American $(),it,.1)' of' (i"(,."J1{,: Therapy

from an adenoviral vector counteracts Angll stimulation in VSMC Gene delivery of RAS peptide hormones provides a novel method

to modulate this system either tissue-specifically or systemically to

356 Use of Gene Transfer To Identify Cell and Tissue Specific Splicing Factors That Dictate Alternative Splicing of the Human VEGF Gene

Vascular endothelial growth factor (VEGF) plays multiple roles

in maintenance of vascular structure, permeability, repair and remodeling Alternative splicing of the VEGF gene results in the expression of multiple VEGF isoforms named according to the number of amino acids in the mature VEGF protein Among them, VEGFI21, VEGFI65 and VEGFI89 are the most abundant and

ofVEGFI89 provides a better safety profile than expression ofthe shorter isoforms (Amano H et al, Mol Ther 2005; 12:716) The

patterns for VEGP are dependent on tissue and developmental stage

lung It is the objective ofthis study to usc gene transfer to assess the presence of cell and tissue specific alternative splicing factors that dictate the splicing patterns ofVEGF In initial studies, alternative promoters were used to drive expressionof a hybrid cDNA/genomic

out with adenoviral vectors with VEGP expression driven by the CMV promoter for expression in multiple cells types or the albumin

the albumin promoter reduced the relative amount of VEGFI65

by 9,4±1.2-fold compared to expression from the CMV promoter suggesting that mRNA splicing for this isoform was dependent on

a reporter minigene was constructed (pAcGFP-E6) This contained the coding sequence for GFP whose translation was dependent upon

no evidence of inclusion of exon 6A into the final mRNA This suggested either the presence of exon 6A specific exonic splicing

the context of pAcGFP-E6 plasmid Using transfection of VEGP exon 6A reporter constructs into 293 cells, a candidate cis-acting

identified that induced a switch in splicing detectable by a decrease

in green fluorescence and an increase in the inclusion of exon 6A sequence using RT-PCR The deletion of this element resulted in a

overlapped with the silencer consensus sequence of AAGGGG

splicingand dictate the relative expression levels ofVEGF isoforms

in VEGF related angiogenic gene therapy

S135