597 coordinated co expression of VEGF164 and PDGF BB prevents aberrant angiogenesis and accelerates vascular stabilization

597 Coordinated Co Expression of VEGF164 and PDGF BB Prevents Aberrant Angiogenesis and Accelerates Vascular Stabilization Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American[.]

CARDIOVASCULAR GENE & CELL THERAPY In contrast to PEI-folR and -TfR targeting, PAMAM-fol polyplexes exhibited greatly enhanced gene delivery activity compared to both untargeted and PAMAM-Tf across a wide range of polymer-DNA and ligand-polymer ratios It is noteworthy that PAMAM-folic acid conjugation did not affect PAMAM-DNA binding as measured by EtBr dye exclusion and DNA gel retardation assays Caveolae and proton-pump inhibiting drugs decreased the efciency of all targeted and untargeted PAMAM polyplexes Our results suggest that unmodified PEI and PAMAM are internalized and trafcked by different mechanisms, beginning at the cell surface The addition of ligands to these polymers alters both uptake and gene delivery properties intrinsic to these vehicles This study enabled us to draw a strong mechanistic understanding of ligand density and its effects on PAMAM uptake and transfection, and correlate polyplex exposure time with transfection to provide insight into the kinetics of ligand/receptor binding and turnover Cardiovascular Gene & Cell Therapy 596 Systemic Delivery of AAV2/9 Vectors Improve Cardiac Function in Pompe Disease 595 Successful Gene Delivery with ReceptorTargeted PAMAM Dendrimers Occurs Via Caveolae-Mediated Pathways Mark E Hwang,1 William R Gunther,2 Daniel W Pack.2 Bioengineering, University of Illinois, Urbana-Champaign, Urbana, IL; 2Chemical and Biomolecular Engineering, University of Illinois, Urbana-Champaign, Urbana, IL Viruses have evolved to efciently deliver DNA, often with high specicity, to target cells in the human body Synthetic gene delivery vehicles fall short of their viral counterparts due to both a lack of specic targeting methods and our incomplete understanding of the steps involved in the gene delivery pathway Cationic polymers polyethylenimine (PEI) and polyamidoamine (PAMAM) dendrimer present an effective means to deliver therapeutic genes to mammalian cells in a manner that is non-immunogenic and targetable Both of these prototypical synthetic gene delivery vehicles are characterized by a high density of primary amines that condense and protect cargo DNA and provide easily modiable moieties for receptor-specic targeting of these delivery vehicles Receptors for folic acid (folR) and transferrin (TfR) are overexpressed in a wide range of tumors and have been studied for cell-specic targeting in gene therapy Most signicantly, these two ligands are internalized through two different pathways: folic acid via caveolae, and transferrin via clathrin coated pits We have previously shown that we are able to modulate DNA uptake and gene delivery efciency across a range of polymer:DNA ratios by conjugating folic acid and transferrin ligands to 25-kD PEI (Gabrielson 2009) Specically, 1) PEI is effectively processed through the caveolar pathway, 2) PEI-TfR targeting enhanced gene delivery across a wide range of ligand:polymer ratios (0.1:10.5:1), and 3) PEI-folR targeting was only effective at suboptimal polymer:DNA and low ligand:polymer ratios In this study we conjugated generation four PAMAM to folic acid and transferrin to assess the effect of receptor-specic targeting on dendrimer gene delivery behavior Both gene delivery efciency and PAMAM-DNA polyplex internalization at the cell-surface were studied using YOYO-1 tagged DNA We used caveolae- and clathrin- inhibiting drugs to determine the mechanism of polymerDNA polyplex uptake, in addition to proton-pump inhibitors and buffering agents to determine the role of the endolysosome in targeted vehicle trafcking Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy Darin J Falk,1,2 Meghan S Soustek,1,2 Cathryn S Mah,1,2 Denise A Cloutier,1,2 Sean A Germain,1,2 Barry J Byrne.1,2 Powell Gene Therapy Center, University of Florida, Gainesville, FL; 2Pediatrics, University of Florida, Gainesville Pompe disease is an autosomal recessive genetic disorder characterized by a deciency of the enzyme (acid α-glucosidase; GAA) responsible for degradation of lysosomal glycogen Cardiac dysfunction is a primary feature of this disorder and we have incorporated an animal model of Pompe disease (Gaa-/- mouse) to characterize this aspect To attenuate the progressive and rapid accumulation of glycogen resulting in cardiac dysfunction, adult Gaa-/- mice were administered a single systemic injection of rAAV2/9DES-hGAA (rAAV2/9) Assessment of cardiac function using a 4.7-T magnetic resonance imaging was measured one and three months post vector administration Gaa-/- animals receiving rAAV2/9 vs saline treated Gaa-/- animals demonstrate improvement in cardiac function (ejection fraction; EF%) and dimension (end diastolic cardiac mass; CM) at one month (66.35 ± 2.06% vs 63.21 ± 1.27%, EF%; 4.46 ± 0.17mg/g vs 4.06 ± 0.25 mg/g; CM) and three months (73.51 ]± 3.13% vs 62.65 ± 1.23%, EF%;, 3.73 ± 0.41 mg/g vs 4.91 ± 0.15mg/g; CM) ECG analysis revealed a minor improvement in PR interval at one month (39.25 ± 0.13ms vs 38.09 ± 0.09ms) and signicant elongation in the PR interval at three months (43.9 ± 1.20ms vs 39.6 ± 0.25ms) in rAAV2/9 animals compared to saline Gaa-/- animals These results indicate that systemic delivery of rAAV2/9 vector can augment cardiac function and morphology as early as one month and signicantly improve cardiac indices at three months post treatment in a murine model of Pompe disease 597 Coordinated Co-Expression of VEGF164 and PDGF-BB Prevents Aberrant Angiogenesis and Accelerates Vascular Stabilization Roberto Gianni-Barrera,1 Silvia Reginato,1 Thomas Wolff,1,2 Lorenz Gürke,2 Michael Heberer,1,2 Andrea Ban.1 Cell and Gene Therapy, Department of Biomedicine, Basel University Hospital, Basel, Switzerland; 2Vascular Surgery, Department of Surgery, Basel University Hospital, Basel, Switzerland We previously found that VEGF can induce the formation of normal or aberrant angiogenesis depending strictly on its level of expression in the microenvironment around each producing cell in vivo and not on its total dose Moreover appropriate VEGF levels require at least weeks of continued expression for vascular stabilization We recently S231 CARDIOVASCULAR GENE & CELL THERAPY found that coordinated co-expression of VEGF and PDGF-BB at xed relative levels from a single bicistronic construct prevented aberrant angiogenesis by heterogeneous VEGF levels and yielded a network of normal and functional capillaries through accelerated pericyte recruitment Here we rigorously determined how PDGF-BB modulates VEGF dose-dependent angiogenesis, and specically whether PDGF-BB co-expression can: 1) increase the efcacy of low VEGF levels; 2) increase the safety of high VEGF levels; 3) accelerate the stabilization of VEGF-induced vessels Primary mouse myoblasts were retrovirally transduced to express either VEGF164 (V), PDGF-BB (P), or both at a xed ratio (VIP, for VEGF-IRES-PDGF) Individual clones were isolated from the populations and matched to compare the effects of specic microenvironmental VEGF levels with or without PDGF-BB weeks after implantation in the ear muscle of SCID mice, low (10 ng/106 cells/day) and medium (50 ng/106 cells/day) VEGF levels induced normal angiogenesis, as expected Both vessel length density (VLD) and morphology were not affected by PDGF-BB However, the aberrant structures induced by high VEGF levels (150 ng/106 cells/ day) were completely prevented by PDGF-BB co-expression, leading instead to a homogeneous capillary network and a large increase in VLD (119.4±2.61 vs 63.1±2.48 mm/mm2, p