593 A phase IIb Double Blind Placebo Controlled Trial of Non Viral Gene Transfer for Cystic Fibrosis Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell[.]
CARDIOVASCULAR AND PULMONARY DISEASES II serum levels of low-density lipoproteins (LDL-C) and an early onset of cardiovascular disease Adeno-associated viral (AAV) vectors expressing human LDLR (AAVhLDLR) have been demonstrated to correct the FH phenotype in mouse models of the disease However, factors that negatively regulate LDLR transgene expression can dampen AAVLDLR expression Among these, proprotein convertase subtilisin kexin (PCSK9) and inducible degrader of LDLR (IDOL) are known to lower LDLR levels within cells and as such are the targets of next generation cholesterol lowering drugs PCSK9 levels are higher in FH patients due to lack of endogenous LDLR expression and can reduce vector efficacy In the present study, we evaluated novel “hyperactive” AAVhLDLR variants that avoid degradation by both PCSK9 and IDOL First, amino acid substitutions were introduced into the coding sequence of hLDLR to ablate interaction with human PCSK9 which were screened in the presence of hPCSK9 using an in vitro assay Next, the best performing construct was evaluated in DKO mice; a mouse model of FH lacking endogenous mouse LDLR and APOBEC expression Administration of AAV vectors encoding wild type hLDLR to DKO mice stably expressing hPCSK9 led to significantly diminished LDLR activity; however, LDLR constructs carrying the L318D amino acid substitution in the EGF-A domain (LDLRL318D) could overcome hPCSK9 inhibition and led to stable correction of cholesterol levels Similarly, a LDLR-K809R\C818A construct with mutations K809R/C818A that are known to confer resistance to hIDOL were indeed unaffected in mice stably expressing hIDOL Finally, the AAVLDLR-L318D\K809R\C818A hyperactive vector, that carried all three amino acid substitutions, was less impacted than wild type mice when administered to DKO mice expressing hPCSK9 or hIDOL Regulatory factors are expected to be higher in the absence of endogenous gene expression to remove them and as such can reduce the efficacy of AAV transgene expression We present here a novel approach to engineer a hyperactive transgene that increases activity by overcoming regulatory pathways 592 An In Vivo, AAV-Based Selection Strategy for the Identification of Novel Cytokines Inducing Cardiac Retention of Mesenchymal Stem Cells After Ischemic Damage Francesca Bortolotti,1 Giulia Ruozi,1 Antonella Falcione,1 Laura Ukovich,1 Lorena Zentilin,1 Serena Zacchigna,1 Mauro Giacca.1 Molecular Medicine, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy There is much evidence indicating that mesenchymal stem cells (MSCs), once injected into the heart, sustain and improve myocardial function after an ischemic insult Mounting information indicates that the benefits of MSC therapy are attributable to paracrine effects, leading to enhanced cardiomyocyte resistance to apoptotic signals and improved cardiac function The therapeutic potential of MSCs, therefore, strictly depends on survival and retention of these cells at the site of injury We developed an innovative Functional Selection (FunSel) procedure aimed at the direct, in vivo selection of cytokines mediating survival or retention of bone marrow (BM)-derived MSCs after intracardiac injection We generated an arrayed collection of different murine cytokines (including all known interleukins and chemokines) cloned into an AAV2 vector plasmid backbone along with an individual 10-bp long barcode specifically identifying each factor Two pooled AAV2 vectors were then generated (40 factors per pool) and used for the batched, ex vivo transduction of BM-MSCs; these cells were then implanted into infarcted or normal murine hearts Vector DNA was recovered from the hearts three weeks after Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy implantation, a time at which most of the MSCs have disappeared, and the relative barcode abundance was determined by next generation sequencing Through this approach, we identified a series of cytokines promoting cardiac retention of the transduced MSCs Interestingly, two of the selected factors, Ccl2 (MCP-1) and Ccl7 (MCP-3) were already associated to MSC homing and survival Cardiotrophin1 (Ctf1), a well known cardiomyocyte hypertrophic factor and another still poorly characterized cytokine were also found highly enriched in both ischemic and non-ischemic conditions The candidate genes identified by FunSel were subsequently individually validated; the results obtained indicated that transduction with AAV vectors coding for Ctf1, CCl7 and the newly identified cytokine conferred a marked survival and retention advantage to MSCs in vivo The mechanism of action of the identified cytokines is currently under investigation 593 A phase IIb Double-Blind PlaceboControlled Trial of Non-Viral Gene Transfer for Cystic Fibrosis Ian A Pringle,1 Eric W Alton,1 Chris A Boyd,1 Seng H Cheng,2 S Cunningham,1 Deborah R Gill,1 Uta Griesenbach,1 Tracy E Higgins,1 Stephen C Hyde,1 Alastair J Inness,1 David D Porteous,1 Ronald K Scheule.2 UK Cystic Fibrosis Gene Therapy Consortium, Oxford, Edinburgh & London, United Kingdom; 2Genzyme Corporation, Framingham, MA The UK CF Gene Therapy Consortium is developing both nonviral and lentivirus-mediated CFTR gene transfer to the lung as a potential treatment for cystic fibrosis (CF) Non-viral CFTR gene transfer, where cationic liposomes are complexed with plasmid DNA, can partially correct the ion-transport defects associated with CF, although the effects to date have been transient In order to offer patients realistic clinical benefit, we further developed our non-viral formulation by: 1) generating the novel hCEFI enhancer/ promoter sequence capable of directing persistent in vivo lung CFTR expression; 2) constructing pGM169, a novel plasmid entirely free of CG dinucleotides (CpGs detected by TLR-9) to minimise inflammatory responses, containing a CFTR cDNA under the control of the hCEFI enhancer/promoter; 3) identifying the optimal nonviral liposomal formulation (GL67A) for lung plasmid delivery; 4) selecting the optimal nebuliser (Trudell AeroEclipseII) for delivery of pGM169/GL67A aerosol to the CF lung In a Phase I/IIa openlabel clinical study, we identified a safe dose of the pGM169/GL67A formulation (5m1 containing 13.25mg pGM169 & 75mg GL67A) which directed changes in nasal and lung potential difference measurements consistent with correction of the CF chloride channel defect and improvement in lung function as determined by lung clearance index Subsequently, a multi-dose toxicology programme in mice has shown a cumulative effect of repeated administration, with 12 pGM169/GL67A doses resulting in human CFTR expression at levels equivalent to ~100% of endogenous Cftr levels These studies supported initiation of a Phase IIb double-blind placebo-controlled trial which began in June 2012 CF patients with 50% £ FEV1 £ 90% receive 12 monthly administrations of either pGM169/GL67A (n³60) or 0.9% saline (n³60) in a 1:1 randomisation; all CF mutation classes are included The primary outcome is change in FEV1 with multiple secondary outcomes for clinical efficacy and safety Twenty-four patients also receive a monthly 2ml nasal dose, to allow sequential measurement of nasal PD, and a further 24 have measurements of bronchial PD pre- and post-treatment (randomisation 2:1 for these mechanistic studies) This subgroup will address whether: a) changes in nasal PD predict those in bronchial PD, and b) whether either measurement predicts any clinical changes following treatment S229 CARDIOVASCULAR AND PULMONARY DISEASES II In total, 166 patients have attended screening visits, of which 136 progressed to dosing 61 patients have completed the study and the remainder have had at least doses All pre-treatment bronchoscopies for bronchial PD measurements have been successfully undertaken, and three independent Data Safety Monitoring Board reviews have not identified any problems The study is on track to be completed in summer 2014 The trial is funded by the National Institute for Health Research’s EME programme Work leading up to the trial was funded by the UK CF Trust 594 Cell-Targeted RNA-Based Therapies for Cardiovascular Disease William H Thiel,1 David D Dickey,1 Jennifer Streeter,1 Schickling Brandon,1 Justin P Dassie,1 Maysam Takapoo,1 Xiuying Liu,1 Francis J Miller, Jr.,1 Paloma H Giangrande.1 Internal Medicine, University of Iowa, Iowa City, IA Despite recent progress towards diagnosis and remedy, cardiovascular diseases (CVDs) remain the leading cause of mortality in many countries There is a clear need for more effective treatment and prevention options Cell-targeted therapy has been proposed as a promising new therapeutic strategy for the treatment of pathological vascular smooth muscle cell (VSMC) remodeling, a primary mechanism responsible for many CVDs, including in-stent restenosis, arteriosclerosis, vein graft disease, and cardiac allograft arteriopathy Ideal therapeutic interventions for these diseases would reduce VSMC migration and proliferation without impairing the beneficial remodeling of endothelial cells (re-endothelialization) following injury This process is critical for restoring anti-atherogenic, anti-thrombotic, and anti-proliferative effects on the vessel wall Current preventive and interventional treatments fail to selectively inhibit the pathological activation of VSMCs Our group has pioneered the development and application of cell-targeted RNA ligands (aptamers) that can be utilized for (1) modulating cellular pathways associated with pathogenesis in target cells and (2) delivering therapeutic biomolecules (e.g RNAi modulators) to these cells in vivo1-8 In previous studies, we developed VSMC-specific, cell-internalizing aptamers as a means of selectively delivering therapeutics to VSMCs8 Here we describe the in vitro and in vivo characterization of these aptamers for inhibiting pathological VSMC remodeling We demonstrate the use of these aptamers for inhibiting VSMC-migration in response to multiple agonists while having no effect on endothelial cell remodeling We also demonstrate that the in vitro selected VSMC-specific aptamers retain specificity for VSMCs in vivo Together, these data highlight the potential for using these aptamers to develop safe therapies with minimal to no off-target effects Additionally, because the aptamers were selected on rat-derived VSMCs, we show cross-species reactivity with both mouse and human cells and vessel segments We may therefore be able to test the same aptamer in preclinical animal models of vascular injury and in future clinical trials in humans Efficacy and safety studies in animal models of vascular injury are ongoing We are also in the process of determining the cellular targets and mechanism of action of these targeted RNA drugs Finally, the VSMC-aptamers are being evaluated for their ability to deliver therapeutic RNAi modulators to VSMCs in culture and in vivo In conclusion, these studies highlight the feasibility of this RNA-based cell-targeted approach and establish a framework for developing cell-targeted therapies for the treatment of CVD Dassie, J P et al Systemic administration of optimized aptamersiRNA chimeras promotes regression of PSMA-expressing tumors Nature biotechnology 27, 839-849, (2009) Thiel, W H et al Rapid identification of cell-specific, internalizing RNA aptamers with bioinformatics analyses of a cellbased aptamer selection PLoS ONE 7, (2012) S230 595 Gene Electrotransfer of VEGF in Angiogenesis and Myocardial Repair Anna Bulysheva,1 Barbara Hargrave,1,2 Robert Strange,3 Len Murray,4 Cathryn Lundberg,1 Niculina Burcus,1 Richard Heller.1,2 Frank Reidy Research Center for Bioelectrics, Old Dominion University, Norfolk, VA; 2School of Medical Diagnostics and Translational Sciences, Old Dominion University, Norfolk, VA; Naval Medical Center Portsmouth, Portsmouth, VA; 4Sobran, Fairfax, VA INTRODUCTION: Ischemia of the myocardium often causes cardiomyocyte cell death and remodeling of the ischemic zone into non-contractile scar tissue that reduces cardiac output, typically leading to heart failure even when best current treatment approaches are employed In this study, we evaluated a new approach using gene electrotransfer to the ischemic myocardium to potentially prevent heart failure Previously, we established a protocol for gene electrotransfer to the left ventricle, determining electrotransfer parameters for safe and effective gene delivery and increased gene expression of luciferase, GFP and vascular endothelial growth factor A (VEGF) compared to controls In the current study, we tested whether gene delivery of VEGF via gene electrotransfer could induce angiogenesis and myocardial repair in a porcine ischemic model METHODS: Domestic farm pigs were anesthetized and intubated, followed by median sternotomy to expose the heart The left anterior descending coronary artery was partially tied off to induce localized ischemia Plasmid encoding VEGF was delivered via electrotransfer to sites surrounding the ischemic region on the border of viable and ischemic myocardium as guided by SPY perfusion analysis Four sites were treated with a plasmid DNA injection followed by electrotransfer with 20ms and 60V pulses, and the negative control groups either received a saline injection or plasmid DNA injection without electrotransfer At and weeks animals were monitored with echography, and angiography immediately before and after the myocardial infarction and treatment Blood was collected at designated time points for troponin-1, creatine kinase MM, and N-terminal-pro-brain natriuretic peptide level analyses Mechanical function of the ventricle was evaluated with a direct pressure sensor during dobutamine stress at weeks, and then cardiac tissue was collected following humane euthanization RESULTS: Arteriograms suggest new blood vessel formations as early as weeks post treatment, as supported by histological evaluation after weeks Further, TCC staining showed notable reduction in scar tissue remodeling in groups with VEGF electrotransfer Echography showed improved cardiac output in electrotransfer groups over controls, confirmed by intraventricular pressure measurements showing enhanced contractility of the left ventricle CONCLUSIONS: Building on our prior work showing the safety and efficiency of gene delivery in this pre-clinical model, we further validate this approach demonstrating favorable therapeutic outcomes over long term studies These results indicate that in vivo VEGF gene electrotransfer to ischemic myocardium improves the structure and function of damaged myocardium and has the potential to become an effective therapy for cardiac ischemia Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy ... with bioinformatics analyses of a cellbased aptamer selection PLoS ONE 7, (2012) S230 595 Gene Electrotransfer of VEGF in Angiogenesis and Myocardial Repair Anna Bulysheva,1 Barbara Hargrave,1,2... preclinical animal models of vascular injury and in future clinical trials in humans Efficacy and safety studies in animal models of vascular injury are ongoing We are also in the process of determining... the feasibility of this RNA-based cell-targeted approach and establish a framework for developing cell-targeted therapies for the treatment of CVD Dassie, J P et al Systemic administration of optimized