422 development of HIV 1 based lentiviral vectors expressing pre trans splicing molecules (PTMs) for liver directed therapies

422 Development of HIV 1 Based Lentiviral Vectors Expressing Pre Trans Splicing Molecules (PTMs) for Liver Directed Therapies Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The Americ[.]

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S162

GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY II

418 Increased Expression of Human

Glucocerebrosidase Activity in Bone Marrow of

Gaucher Mice Following Macrophage Mediated

Gene Therapy

Arlene T Lim,1 Edward I Ginns.1

1 Program in Medical Genetics, Clinical Pathology, University of

Massachusetts Medical School, Worcester, MA.

Gaucher disease, caused by beta-glucocerebrosidase (GBA)

deficiency, is the most common lysosomal storage disorder

Accumulation of glucocerebroside in the bone marrow predisposes

patients with Gaucher disease to skeletal complications, which

are often the disorder’s most disabling manifestations Frequently,

bisphosphonates are administered as bone resorption inhibitors

along with enzyme replacement therapy, and although there is

skeletal improvement, bone density often remains below normal, and

bisphosphonates can be accompanied by adverse side effects A novel

approach to increasing GBA activity in bone marrow utilizes yeast

glucan particles (YGP) to deliver DNA nanoplexes encoding human

GBA (YGP-GBA DNA) to macrophage Following administration to

Gaucher mice by either oral gavage or intraperitoneal (IP) injection,

the YGP-GBA DNA formulations are taken up by macrophage

through a dectin-1 receptor mediated mechanism and carried to a

wide range of tissues, including bone In small pilot experiments,

5 and 7 weeks of daily oral administration of YGP-GBA DNA to

Gaucher mice resulted in 7% and 39% increase in GBA bone marrow

activity, respectively Intraperitoneal administration of YGP-GBA

DNA increased bone marrow GBA activity to 24% and 77% after

2 and 7 weeks of treatment, respectively The environment in the

gastrointestinal tract could contribute to DNA degradation and may

in part explain the increased ef cacy of GBA expression from IP

administration These initial results demonstrate that administration

of YGP-GBA DNA formulations increase GBA activity in Gaucher

mouse bone marrow, and that this therapeutic strategy has the

potential to address the current limitations in delivery of GBA to

bone Additional studies to characterize more details of the increase in

GBA activity in bone marrow of treated Gaucher mice are in progress

and include immunohistology, Western blot analyses, PCR analyses

and  ow cytometry to determine the percentage of macrophage and

other bone marrow cells expressing human GBA

419 Cell Sheet Engineering toward

Bioengineering of Functional Neo-Islets

Takahiro Saito,1,2 Kazuo Ohashi,2 Hirofumi Shimizu,1 Rie Utoh,2

Kazuya Ise,1 Masayuki Yamato,2 Teruo Okano,2 Mitsukazu Gotoh.1

1 Department of Surgery, Fukushima Medical University,

Fukushima, Japan; 2 Institute of Advanced Biomedical Engineering

and Science, Tokyo Women’s Medical University, Shinjyuku, Tokyo,

Japan.

[Background] Cell-based therapy using pancreatic islets has

been established as a promising new approach for treating

insulin-dependent diabetes mellitus (DM) Clinical trails and experimental

researches have been conducted based on the transplantation of islet, a

cluster of approx 3,000 cells To advance the islet-based therapies for

DM, it is clear that further improvements are needed to optimize the

conditions to maximize the longevity of the transplanted cell systems

Creation of functional islet tissues made of dissociated islet cells have

rarely been attempted The present study was designed to establish

a novel tissue engineering approach for diabetes mellitus (DM) by

fabricating a monolayered tissue sheet composed of dissociated single

pancreatic islet cells for the creation of functional and transplantable

neo-islet construct

[Experimental methods] Temperature-responsive culture dishes

speci c for islet cell culturing was prepared by covalent immobilization

of the temperature-responsive polymer poly(N-isopropylacrylamide)

(PIPAAm) to the tissue culture plastic followed by coating with laminin-5 Pancreatic islets were isolated from Lewis rats using a stationary collagenase digestion protocol To obtain single islet cells, the islets were digested with trypsin-EDTA The dissociated islet cells were then plated onto the laminin-5-PIPAAm dishes After the cells reached con uency, cultured islet cells were harvested as a uniformly connected tissue sheet by lowering the culture temperature from 37°C

to 20°C for 20 min The functionality of the harvested islet cell sheet was assessed by histological examination, cell culture conditions, and

functional activity following transplantation in vivo

[Results] Histological examination showed that the harvested cell sheet was monolayered 2-D structure that had retained

cell-to-cell interactive connections Immunohistological staining revealed that the islet cell sheet predominantly comprised of insulin- (76%) and glucagon- (22%) staining positive cells, respectively Upon re-plating of the tissue sheet into new culture dishes, we detected

a positive response of the tissue sheet to a glucose-challenge test

In vivo functionality of the islet cell sheet was con rmed following transplantation into the subcutaneous space for a period of 7 days Therapeutic values of the islet cell sheets were observed in the transplantation study to the Streptozotocin-induced diabetic

immunode cient mice

[Discussion] The present study describes a new proof-of-concept approach to generate functional monolayered neo-islets in vitro

These neo-islets were able to produce insulin in vivo and to provide therapeutic effects on the diabetic individuals In all, the present studies serve as the foundation for the creation of islet cell-based therapy for DM to provide patients an alternative method for de novo

production of insulin

420 Liver-Speci c Over-Expression of PGC-1Į Does Not Induce a Diabetic Phenotype

Luke Roode, Chunping Qiao, Ruhang Tang, Jiangjang Jiang, Juan

Li, Xiao Xiao

Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina-Chapel Hill, Chapel HIll,

NC.

Peroxisome Proliferator Activated Receptor γ Coactivator 1 (PGC-1α) has been implicated in the pathogenesis of type II diabetes In

the liver, PGC-1α has been demonstrated to activate transcription

of phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-6-phosphatase (G6Pase), two key enzymes involved in the  rst and

last steps of the gluconeogenic pathway: conversion of pyruvate to phosphoenolpyruvate and hydrolyzing the phosphate group to give free glucose, respectively It is hypothesized that PGC-1α activates gluconeogenesis in the liver, resulting in elevated blood glucose measurements However, this hypothesis is based mostly upon in vitro cell culture experimentation The only in vivo evidence of this effect was demonstrated by adenoviral expression of a

CMV-PGC-1α construct in Wistar rats The blood glucose levels of the PGC-CMV-PGC-1α treated animals were higher in both the fed and fasted state, 5 days post administration of 1x10^12 pfu, with PGC-1α expression levels

260% of control

Seeking to further test this hypothesis, we wanted to examine the effect of liver-speci c expression of PGC-1α in vivo We chose to use adeno-associated virus (AAV) as our gene delivery vehicle AAV serotype 9 gives the most ef cient liver transduction out of the known natural serotypes However, this vector also transduces myocardium and skeletal muscle very ef ciently so we constructed a PGC-1α expression cassette driven by liver-speci c alpha-1-antitrypsin (AAT) promoter Additionally, we also wanted to investigate if there was a dose-dependent relationship of PGC-1α expression by injecting 3 different doses of AAV9-AAT-PGC-1α vectors into normal C57/BL6 mice of about 8 weeks of age We found that liver-speci c PGC-1α expression did not induce diabetes in any dose group, as evidenced

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S163

GENETIC AND METABOLIC DISEASES GENE & CELL THERAPY II

by fasting blood glucose levels at 3, 6, and 10 weeks following AAV administration Moreover, a glucose tolerance test at 8 months post administration also showed no effect of PGC-1α expression Western blotting indicates that PGC-1α was increased about 2-3 fold in the high dose group versus control animals RT-PCR of DNA isolated from the liver of the AAV-treated animals indicate that our gene was delivered in a dose-dependent fashion and liver histology will also be examined for any signs of immune response to transgene expression or other abnormalities These results suggest that PGC-1α over-expression in the liver may not result in disregulation of gluconeogenesis and a diabetic phenotype

421 Promoter Selection for AAV Vector-Mediated Factor IX Expression in Skeletal Muscle

Akihiro Kume,1 Hiroya Yagi,1 Hiroaki Mizukami,1 Masashi Urabe,1 Tomonori Tsukahara,1 Akira Ishiwata,2 Jun Mimuro,2 Seiji Madoiwa,2 Tsukasa Ohmori,2 Yoichi Sakata,2 Keiya Ozawa.1

1 Genetic Therapeutics, Jichi Medical University, Shimotsuke, Tochigi, Japan; 2 Cell and Molecular Medicine, Jichi Medical University, Shimotsuke, Tochigi, Japan.

Hemophilia is an X-linked bleeding diathesis caused by a de ciency of either coagulation factor VIII (FVIII; hemophilia A)

or factor IX (FIX; hemophilia B) While re ned management with coagulation factor products has signi cantly improved the prognosis, regular supplementation of FVIII or FIX would be preferable for prophylaxis Gene therapy is an ideal modality for this purpose, and adeno-associated virus (AAV) vectors are promising vehicles for gene delivery to target tissues such as liver and muscle In developing AAV vectors for hemophilia, ef cient promoters are required to achieve therapeutic levels of FVIII or FIX, while tissue-speci c expression is desirable for safety reasons These tasks are particularly challenging with AAV vectors, because the size of their genome is strictly limited Considering the accessibility and safety, skeletal muscle

is a superb target tissue for hemophilia gene therapy Therefore we investigated the ef cacy of several small-sized promoters including cytomegalovirus promoter (CMV), truncated form of elongation factor-1 promoter (EFS), muscle creatine kinase minimal promoter linked with one (MCK) or two (dMCK) enhancer core elements In our reporter constructs, these promoters were placed to drive the human FIX (hFIX) gene Human embryonic kidney 293 cells, Huh7 human hepatoma cells, C2C12 mouse myoblasts were transfected with the expression plasmids, and the hFIX in the culture medium was examined by ELISA and immunoblot As expected, CMV promoter was strongest in 293, Huh7 and undifferentiated C2C12 cells while MCK and dMCK promoters had trace activity During C2C12 differentiation into myotubes, however, MCK and dMCK promoters demonstrated greater activity than other promoters Based on this result, we constructed serotype 1-pseudotyped self-complementary AAV vectors with CMV-hFIX (AAV1/CMV-hFIX) and dMCK-hFIX (AAV1/dMCK-hFIX) for in vivo evaluation 1 × 1011 genome copies

of the AAV vectors were injected to the hindlimb muscle of C57BL/6 mice and plasma hFIX was measured by ELISA The hFIX levels were gradually increased and reached to 4.9 ± 3.7 % with AAV1/

CMV-hFIX (n = 4) and 2.1 ± 2.9 % with AAV1/dMCK-hFIX (n = 4)

at 11 weeks postinjection The hFIX expression was maintained at these levels up to 25 weeks The reason for the discrepancy between

in vitro and in vivo hFIX expression, i.e CMV promoter worked better than dMCK in vivo, is currently under investigation Since the plasma hFIX level was generally low and signi cantly deviated even

in each cohort, intramuscular gene delivery might not be successful

The animals were sacri ced and tissue DNA analysis is ongoing

Much more potent promoter may be required for in vivo expression following muscle-directed gene transfer using AAV vectors.α

422 Development of HIV-1 Based Lentiviral Vectors Expressing Pre-Trans-Splicing Molecules (PTMs) for Liver-Directed Therapies

Madaiah Puttaraju,1 S Gary Mans eld,1 Jun Wang,1 Nikolay Korokhov,1 Jenice D’Costa,1 Ziping C Chen,1 Katherine Radd,1

Gerard J McGarrity,1 Laurent M Humeau.1

1 VIRxSYS Corporation, Gaithersburg, MD.

Spliceosome mediated RNA trans-splicing (SMaRT) is one of the few RNA-based technologies that can potentially restrict the production of a protein of therapeutic interest to a speci c cell type or organ We combined this RNA trans-splicing technology with the company’s proven lentiviral vector (LVs) delivery system

to maximize PTM delivery to target cells and achieve persistent gene expression We generated VSV-G pseudotyped HIV-1 based LVs encoding a PTM that trans-splices human apolipoprotein A-I (hapoA-I) into highly abundant human albumin pre-mRNAs in hepatocytes to increase blood levels of this protein and ultimately correct familial apolipoprotein A-I de ciency This PTM cassette contains a binding domain targeted to intron 1 of human albumin, splice acceptor sequences and hapoA-I coding sequence Initial testing of these LV-hapoA-I PTMs were performed in HepG2, and human primary hepatocytes These cell types produce and process albumin and apoA-I, and therefore are ideal cell models for pre-testing clinical candidate LV-hapoA-I PTMs The low conservation of intron sequences among species precludes the use of most animal models for pre-testing our clinical candidate human PTMs HepG2 cells and hepatocytes were transduced ef ciently by LV-PTMs and analysis of RNA from these cells by RT-PCR for transcripts containing albumin exon 1 fused to hapoA-I coding sequences con rmed accurate trans-splicing into human albumin pre-mRNAs We also quanti ed levels

of hapoA-I PTM RNAs, albumin pre-mRNAs, trans-spliced mRNA, and LV copies per cell by qRT-PCR and qPCR, respectively; all four parameters showed clear dose response relationships Increased MOIs produced a linear increase in the number of LV copies per cell, and cell samples with higher copy values had higher levels of unspliced PTM RNA Similarly, analysis of trans-splicing level showed a close linear relationship with PTM RNA (Figure) and produced in the range

of 5-20 copies of trans-spliced chimeric mRNA per cell in HepG2 and 1-5 copies in human primary hepatocytes In all samples analyzed

a higher level of PTM RNA or target pre-mRNA resulted in higher levels of trans-splicing These results demonstrate: 1 successful integration of the company’s LV delivery platform with SMaRT technology, 2 trans-splicing to a true endogenous pre-mRNA target in human primary hepatocytes, and 3 con rm and extend our previous observations of the intimate relationship between PTM RNA, target pre-mRNA and trans-splicing Currently, experiments are in progress

to assess the therapeutic potential of these LV-PTMs in engineered animal models Results from these studies will serve as a foundation for the future development of liver directed RNA-based therapies

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S164

CARDIOVASCULAR GENE & CELL THERAPY

Cardiovascular Gene & Cell Therapy

423 AAV-Mediated Intramyocardial VEGF-B167

Expression Exerts a Potent Cardioprotective Effect

in Small and Large Animal Models of Myocardial

Infarction and Heart Failure

Lorena Zentilin,1 Vincenzo Lionetti,2 Uday Puligadda,1 Silvia

Moimas,1 Serena Zacchigna,1 Martino Pepe,3 Mohammed

Mamdani,3 Khaled Qanud,3 Xiabin Xu,3 Thomas H Hintze,3 Fabio

A Recchia,3 Mauro Giacca.1

1 International Centre for Genetic Engineering and Biotechnology

(ICGEB), Trieste, Italy; 2 Scuola Superiore Sant’Anna, Pisa, Italy;

3 New York Medical College, Valhalla, NY.

VEGF-B, a member of the vascular endothelial growth factor

family, is emerging as a major cardiovascular protecting factor

We have recently observed, in a rat model of acute myocardial

infarction, that the intramyocardial injection of AAV2-VEGF-B167,

which selectively binds VEGFR-1, signi cantly improves both

regional and global cardiac contractility and maintains favorable

tissue remodeling over time in the absence of a signi cant angiogenic

response (Zentilin et al 2010 FASEB J., in press) We found that

cardiomyocytes expressed VEGFR-1, VEGFR-2 and Neuropilin-1

and that, in particular, VEGFR-1 was speci cally upregulated upon

exposure of cardiomyocytes to hypoxia/reoxygenation or cardiotoxic

drugs Histological and molecular analysis highlighted a marked

antiapoptotic effect exerted both in vitro on isolated neonatal

cardiomyocytes and in vivo after induction of ischemia

We now show that VEGF-B is capable to elicit a compensatory,

hypertrophic response mediated by the up-regulation of genes

involved in intracellular calcium transients such as ryanodyne

receptors and SERCA2a, as well as of PGC-1a, a powerful regulator

of mitochondrial metabolism and cardiac energetics Consistent with

this conclusion, we found that VEGF-B exerts a similarly striking,

angiogenesis-unrelated cardioprotective effect in a pacing-induced

model of non-ischemic heart failure in chronically instrumented dogs

Control animals subjected to 4 weeks of continuous pacing suffered

from an overt congestive heart failure; on the contrary, at the same

time point, intramyocardial AAV9-mediated VEGF-B expression

maintained heart contractility and prevented LV wall thinning Also

in this model, apoptosis, revealed as a number of TUNEL positive

cardiomyocytes and cleaved caspase 3, was signi cantly reduced

in myocardium expressing VEGF-B In a consistent manner,

phosphorylation of Akt, a major negative regulator of apoptosis,

was superphysiological in AAV-VEGF-B treated tissue, while the

inhibitory phosphorylation of the pro-apoptotic intracellular mediators

GSK-3β and FoxO3a, was reduced in the control and normalized

in the AAV-VEGF-B animals Cardiac VEGFR-1 expression was reduced four-fold in all paced dogs, suggesting that exogenous VEGF-B exerted a compensatory receptor stimulation

Together, these results point toward VEGFR-1 signalling in the heart as an important mediator of cardiomyocyte function, with clear therapeutic implications

424 Osteopontin Enhances the Angiogenic Potential of Endothelial Progenitor Cells

Erin E Vaughan,1 Angela Duffy,2 Timothy O’Brien.1

1 Regenerative Medicine Institute, National University of Ireland, Galway, Ireland; 2 Medtronic, Galway, Ireland.

Patients with Type 1 Diabetes Mellitus (T1DM) often demonstrate

an inability to generate an appropriate angiogenic response to ischemia One possible reason for this may be due to a decrease in both the number and function of endothelial progenitor cells (EPCs)

We have previously shown that EPCs cultured from patients with T1DM are impaired in their ability to adhere to activated endothelial cells as well as their ability to induce tubule formation in a matrigel assay Further, we observed a down- regulation of the angiogenic protein osteopontin (OPN) in diabetic EPCs We hypothesized that the down regulation of OPN may contribute to the impaired function of diabetic EPCs Interestingly, when diabetic EPCs were pre-incubated with recombinant OPN for 24 hours their ability to induce tubule formation was restored to levels observed from healthly control EPCs In vitro, this suggests that OPN plays an important role in EPC-mediated angiogenesis To further explore the role of OPN in EPC mediated angiogenesis, we isolated EPCs from OPN knockout mice and found they too were impaired in their ability to bind activated endothelial cells and form tubules in a matrigel tubule assay Further, this impairment was reversed upon exposure to OPN We went on to determine that conditioned media (CM) from wild-type (WT) EPCs was suf cient to enhance tubule formation, suggesting that secreted factors are responsible for the EPC-mediated angiogenic response

Interestingly, we found that incubation of the CM with an osteopontin neutralizing antibody did not abrogate tubule formation Thus, it does not appear that OPN acts directly on the endothelial cells to induce tubule formation Therefore, we hypothesized that OPN may

be acting in an autocrine manner on EPCs to induce the secretion

of additional angiogenic proteins Indeed, using a chemi-array, we found that exposure of KO EPCs to OPN induced the release of IL-6, TGF alpha, and FGF alpha to levels secreted by WT cells To assess the role of OPN in vivo, a murine model of hind-limb ischemia was used Ischemia was induced in the left limb of OPN KO animals and laser doppler blood  ow measurements were obtained pre- and post-operatively as well as at days 7 and 14 post surgery At the time of surgery an intramuscular injection of: diluent (control);1 million OPN KO EPCs; or 1 million KO EPCs pre-incubated with recombinant OPN was performed At day 7, signi cant improvement was noted in the mice injected with KO EPCs pre-incubated with OPN and this improvement was enhanced at day 14 There was no signi cant improvement noted with the injection of KO EPCs alone

Taken together this data demonstrates that the pre-treatment of OPN

KO EPCs with recombinant OPN results in an improved response

to induced hind limb ischemia Our data suggests this may be due

to the induced secretion of angiogenic proteins Further, we have shown that the treatment of diabetic EPCs with OPN improves their angiogenic potential in vitro Although further research is needed, this is a promising step for the future of autologus cell therapy in the treatment of ischemia related conditions

Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S165

CARDIOVASCULAR GENE & CELL THERAPY

425 Arginine and Tetrahydrobiopterin Synergistically Potentiate the Antirestenotic Effect

of Vascular Gene Therapy with Inducible Nitric Oxide Synthase

Ilia Fishbein,1 Peter Sobolewki,1 Richard F Adamo,1 Ivan S

Alferiev,1 Ariel Fishbein,1 Marina Bakay,1 Michael Chorny,1 Robert

J Levy.1

1 Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA.

Background: Gene therapy with nitric oxide synthase (NOS)

isoforms in order to increase production of NO in the vasculature has been recognized as a potential therapeutic approach for the prevention

of restenosis However, the optimal activity of NOS was shown to depend on the availability of arginine (Arg) and tetrahydrobiopterin (BH4) via the prevention of NOS decoupling and reactive oxygen species (ROS) formation Here we investigated the impact of Arg and BH4 supplementation on NO production, ROS formation, SMC proliferation and neointimal thickening in balloon-injured rat carotid arteries transduced with adenovirus encoding inducible NOS (AdiNOS)

Methods: Rat carotid arteries were balloon-injured and delivered

intralumenally with 1.6x109 pfu of AdLacZ (control, n=6) or AdiNOS (n=24) The latter were then randomized into 4 groups of 1) no additional co-treatment; 2) Arg only (500 mg/kg/d); 3) BH4 only (10 mg/kg/d); or 4) Arg+BH4 Six hours before the sacri ce at day

14 the rats were injected with 40 mg/kg BrdU In similarly treated animals (n=4 per group), which were euthanized at day 3, the direct effects of iNOS overexpression +/- Arg/BH4 supplementation on NO and ROS production were determined using DAF  uorometry and DHE staining respectively

Results: In comparison with the AdLacZ control, decreases of

20%, 27%, 36% and 71% in neointimal area and 26%, 39%, 36%

and 70% of BrdU labeling were found in the Ad-iNOS-treated rats receiving none, Arg only, BH4 only and Arg+BH4 supplementation, respectively Compared to non-injured carotid arteries, the arteries of rats locally treated with AdLacZ, AdiNOS and AdiNOS+Arg/BH4 exhibited 3.7-, 3.8-, and 1.3-fold increases in ROS production and 0.4-, 1.1- and 2.2-fold changes in NO levels, respectively

Conclusions: Combined Arg and BH4 supplementation potentially

augments the therapeutic effectiveness of AdiNOS vascular gene transfer, probably via normalization of ROS/NO balance secondary

to the recoupling of iNOS

426 Intra-Myocardial Administration of

ACRX-100 Promotes Cardiac Bene t and Improves Angiogenesis in a Porcine Model of Heart Failure

Joseph M Pastore,1 Timothy J Miller,1 Rahul A Aras,1 Marc S

Penn.2

1 Juventas Therapeutics, Inc., Cleveland, OH; 2 Stem Cell Biology and Regenerative Medicine, Cleveland Clinic, Cleveland, OH.

ACRX-100 comprises a non-viral DNA plasmid engineered to transiently express human Stromal-cell Derived Factor 1 (SDF-1)

SDF-1 triggers a number of protective molecular cascades that are both anti-in ammatory and anti-apoptotic Furthermore, SDF-1

is a strong chemoattractant of stem cells and progenitor cells that promote tissue preservation and blood vessel development Previous studies have demonstrated that SDF-1 expression is increased in the myocardium after a myocardial infarction, but expression lasts for less than a week, and therefore the induced stem cell homing response quickly fades This short duration of SDF-1 expression reduces the potential for tissue repair and suggests that therapeutic interventions which prolong the ability of SDF-1 to stimulate the stem cell homing process may be bene cial for patients that have damaged heart tissue A 57-pig safety and ef cacy study evaluated

cardiac functional response, toxicity and bio-distribution in a porcine model of heart failure after treatment with escalating doses of

ACRX-100 All enrolled pigs had a left ventricular ejection fraction (EF)

<40% and a left ventricular end systolic volume (LVESV) >56.7

ml as measured by echocardiography 30 days post-infarct at which time they received intra-myocardial injections of Saline (control), or ACRX-100 at doses of 7.5 mg (low), 30 mg (mid) or 100 mg (high)

At 60 days post-therapy, ACRX-100 improved LVEF and LVESV at low and mid doses ACRX-100 promoted vasculogenesis at all doses relative to controls, with signi cant increases at the low and mid doses at 30 days post-injection Importantly, increased vessel density (>200 vessels/ eld) correlated with improved cardiac function The difference in change in LVESV between vessel density groups (<200 vessels/ eld: 5.9±11.5 ml vs >200 vessels/ eld: -8.6±13.4 ml-) was statistically signi cant (p<0.05), and the difference in change in LVEF (<200 vessels/ eld: -0.9±10.2% vs >200 vessels/ eld: 7.5±5.5%) approached statistical signi cance (p=0.052) No ACRX-100 dose was associated with signs of toxicity, adverse effects on clinical pathology or histopathology ACRX-100 was distributed primarily

to the heart with negligible amounts found in non-cardiac tissues The product was essentially cleared from all organs within 90-days

of treatment These  ndings indicate that the no observable adverse effect level (NOAEL) for ACRX-100 in the pig model of ischemic heart failure is100 mg Based on these results, the FDA has allowed

an IND to initiate an open label, 16 subject Phase 1 dose-escalation study to evaluate the initial safety of ACRX-100 to treat heart failure

in subjects with ischemic cardiomyopathy (NYHA Class III with prior myocardial infarction)

427 Strategies for Promoting Survival of Transplanted Mesenchymal Stem Cells in a Myocardial Infarction Model

Lisa McGinley,1 Daniel O’Toole,2 Aoife Duffy,1 Xizhe Chen,1

Alessia Stocca,1 Jill McMahon,3 Timothy O’Brien.1

1 REMEDI, NUI, Galway, Ireland; 2 Department of Anaesthesia, NUI, Galway, Ireland; 3 NCBES, NUI, Galway, Ireland.

Ischaemia is a feature of several cardiovascular diseases including atherosclerosis, myocardial ischaemia reperfusion, cardiomyopathies and myocardial infarction (MI) It is characterised by a restriction of regional blood  ow which causes hypercarbia, tissue hypoxia and glucose deprivation with resultant metabolic acidosis Because of their contractile function, cardiomyocytes have a high energy requirement, and so are extremely sensitive to these combined effects, which lead

to cell death by necrosis and apoptosis resulting in permanent loss of tissue and heart failure Mesenchymal stem cell (MSC) transplantation

is one therapeutic approach which aims to repair damaged tissue

and replace lost cells In the in vivo MI model, we have observed

that MSCs are modest in their therapeutic effects This may be due

to the harsh pro-apoptotic, in ammatory microenvironment of the infarct area which is not conducive to MSC survival and engraftment Therefore, protection of MSCs against apoptosis is critical for successful stem cell therapy During ischaemia, cell survival can

be in uenced by expression of genes that promote glycolysis, limit mitochondrial metabolism (both of which reduce oxygen demand), suppress reactive oxygen species and inhibit pro-apoptotic protein expression We propose that transduced MSCs expressing pro-survival genes may possess an increased resistance to ischaemic conditions, improving survival and engraftment of the MSCs in the injured tissue This, in theory, would allow the MSCs to promote repair in the infarct area, either directly or by paracrine action We have constructed VSV-G pseudotyped HIV-1 based lentiviral vectors

expressing pro-survival genes In vitro experiments consisted of

MSC exposure to conditions of ischaemia and its components which

included oxidative stress, hypoxia and complete glucose knockdown

In vivo MI experiments were performed on female Sprague Dawley