267 Image Guided, Liver Targeted Hydrodynamic Gene Delivery in Dogs Preclinical Assessment of Effectiveness and Safety of the Procedure Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright ©[.]
Physical Methods of Delivery and Chemical/Molecular Conjugates Together, these data support a vector dose dependency threshold for capsid antigen presentation on hepatocyte MHC I molecules and demonstrates that the novel AAV2-LiA and LiC capsids avoid detection by capsid specific CD8 T cells 265 Polysialic Acid as a Novel Regulator of AAV Tropism in the Developing Brain Giridhar Murlidharan,1,2 Aravind Asokan.1,2 Department of Genetics, University of North Carolina-Chapel Hill, Chapel Hill, NC; 2Gene Therapy Center, University of North Carolina-Chapel Hill, Chapel Hill, NC Recombinant AAV vectors are currently being exploited in a number of clinical trials that aim to correct neurological diseases In many cases, it is important that therapeutic intervention occurs in the infantile stage of life Thus, it is important to understand how AAV vectors interact with the constantly changing environment in the brain during neurodevelopment Recombinant AAV is thought to spread through the central nervous system (CNS) by exploiting cerebrospinal fluid (CSF) flux and hijacking axonal transport pathways The role of host receptors that mediate these processes in the developing brain are not well understood In the current study, we utilized AAV serotype as a model to evaluate whether ubiquitously expressed 2,3-linked sialic acid and the developmentally regulated marker, 2,8-linked polysialic acid (PSA) regulate viral transport and tropism in the neonatal brain Modulation of the levels of SA and PSA in cell culture studies using specific neuraminidases revealed possibly opposing roles of the two glycans on AAV4 transduction Interestingly, upon intracranial injection into lateral ventricles of the neonatal mouse brain, a low affinity AAV4 mutant displayed a striking shift in cellular tropism from 2,3-linked SA+ ependymal lining to 2,8-linked PSA+ migrating progenitors in the rostral migratory stream and olfactory bulb In addition, this gain-of-function phenotype correlated with robust CNS spread of the mutant through paravascular transport pathways Consistent with these observations, altering glycan dynamics within the brain by co-administering SA and PSA specific neuraminidases resulted in striking changes to the cellular tropisms and transduction efficiencies of both parental and mutant vectors We postulate that glycan signatures associated with host development can be exploited to redirect novel AAV vectors to specific cell types in the brain 266 G-Force Loading of Virus Vectors into Vesicles for Enhanced Gene Therapy Vehicles Zachary Fitzpatrick,1 Andreas Maus,2 Bence Gyorgy,2 Dakai Mu,2 Krisztian Csomos,2 Jolan E Walter,2 Casey A Maguire.2 Louisiana State University, Baton Rouge; 2The Massachusetts General Hospital, Charlestown, MA Adeno-associated virus (AAV) vectors have shown remarkable efficiency in a number of preclinical models of disease in several organs including the eye, brain, muscle, heart, and liver A major limitation to long term transduction, especially via the systemic route, is pre-existing immunity (humoral and cell-mediated) as well as the high vector load required to achieve sufficient levels of gene expression in the desired organ Natural nanoparticles released from all cells, called extracellular vesicles (EVs), may have utility in creating better AAV vectors for human gene therapy We have previously shown that harvesting AAV vectors from the media of 293T producer cells contains AAV vectors endogenously associated with EVs (called ev-AAV, aka vexosomes) We have gone on to demonstrate that 293T-derived ev-AAV can evade neutralizing anti-AAV antibodies and greatly increase transduction in mice An alternative strategy would be to load purified AAV vectors into separately purified EVs This would have the benefit of using EVs S106 from a variety of cellular sources with desired biological activity and also to use donor-derived autologous EVs Here we demonstrate a simple method for loading EVs with AAV vectors using high speed centrifugation forces Iodixanol density gradient-purified AAV was mixed with conditioned media containing EVs from 293T cells and also primary human peripheral blood mononuclear cells (PBMCs) and then ultracentrifuged to co-pellet EVs and AAV The pellet was resuspended in media and used in transduction assays and anti-AAV antibody neutralization assays Strikingly we found that this process bestowed greatly enhanced transduction of cells in culture (36-fold) as well as resistance to neutralizing anti-AAV antibodies To demonstrate that EVs were essential to this enhancement, we mixed AAV with plain media (no EVs) or depleted conditioned media of EVs before addition of AAV using either Triton-X-100 EV lysis or ultracentrifugation In all cases there was a large decrease in the transduction efficiency and antibody evasion compared to conditioned EV media mixed with AAV Together these data suggest that g-force loading of AAV into EVs represents a promising method to increase performance of the vector We will continue to optimize the protocol and test the gene delivery functions in vivo Physical Methods of Delivery and Chemical/ Molecular Conjugates 267 Image-Guided, Liver-Targeted Hydrodynamic Gene Delivery in Dogs - Preclinical Assessment of Effectiveness and Safety of the Procedure Kenya Kamimura,1 Hiroyuki Abe,1 Takeshi Yokoo,1 Tsutomu Kanefuji,1 Takeshi Suda,1 Yuji Kobayashi,1 Guisheng Zhang,2 Masafumi Oda,3 Yutaka Aoyagi,1 Shuji Terai,1 Dexi Liu.2 Division of Gastroenterology and Hepatology, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan; 2Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA; 3Institute for Research Collaboration and Promotion, Niigata University, Niigata, Japan Image-guided, liver-targeted hydrodynamic gene delivery was evaluated in dogs to assess the safety and effectiveness of the procedure for human clinical trial The procedure involves imageguided catheter insertion to hepatic veins in the dogs followed by computer-controlled hydrodynamic gene delivery using newly developed injector for clinical use Optimum parameters for the procedure, including the location of the balloon catheter in the hepatic vein, intravascular pressure upon injection, injection volume, and sequential injections to multiple lobes of the injected liver, were employed as previously we reported Hydrodynamic injection of human factor IX expressing plasmid (pBS-hFIX) was performed in dogs (female, 20 kg) and short- and long-term effects of the procedure were assessed We demonstrate that plasma level of human factor IX and coagulation activity in dogs increased from the background level at ~20 ng/ml and ∼8% to 21768.1 ng/ml and 36.8%, respectively in week after hydrodynamic gene delivery and sustained for weeks Transgene expression in the hepatocytes was also confirmed by immunohistochemical stainings For safety assessment, we demonstrated that the impacts of image-guided liver-targeted hydrodynamic gene delivery are localized in the site of injection in the liver Histological analysis showed significant expansion of sinusoids at the injected site to 190% of their original size Hepatic microcirculation analysis of liver-targeted hydrodynamic injection using reflectance spectrophotometry showed significant decrease Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy Physical Methods of Delivery and Chemical/Molecular Conjugates of the oxygen saturation of the Hb in the blood of the injected lobe from 30 to unit upon the injection and recovered smoothly after the injection Serum analysis showed a transient, 10- to 20-fold increase in hepatobiliary enzymes, including aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase after hydrodynamic injections, which recovered within days We also explored for the first time, the levels of the cytokines following the hydrodynamic injections to the large animals There was no increase in the systemic inflammatory cytokines of IFN-γ, IL-8, IL-18, and IL-4, although an increase in serum levels of TNF-α, IL-10, MCP-1, canine KC, and IL-6, which were related to vascular stretching representing the sinusoidal expansion was observed No impacts on their respiratory, cardiovascular conditions, or long-term body weight changes were observed throughout the study These results of preclinical assessments support the clinical applications of image-guided, livertargeted hydrodynamic gene delivery This work was supported in part by NIH grants RO1EB002946 and RO1HL075542, and by Japanese Society for the Promotion of Science Grants 22890064, 23790595, and 26860354 268 Enhanced Gene Expression of Factor VIII by Ultrasound-Mediated Gene Delivery in Dogs Carol H Miao, Misty L Noble, Keith R Loeb, James Harrang, Christian S Kuhr,3 Scott S Graves,2 Kyle P Morrison,4 Timothy Nichols,5 Bryn Smith,1 Shuxian Song,1 Rainer F Storb,2 George W Keilman.4 Pediatrics, Seattle Children’s Reserach Institute and University of Washington, Seattle, WA; 2Fred Hutchinson Cancer Research Center, Seattle, WA; 3Benaroya Research Institute, Seattle, WA; Sonic Concepts, Inc., Seattle, WA; 5University of North Carolina, Chapel Hill, NC 1 Previously we demonstrated that ultrasound (US)-mediated gene delivery (UMGD) can significantly enhance reporter gene transfer into the mouse and rat livers This nonviral gene transfer strategy can bypass many obstacles encountered by viral gene therapy Most significantly, we have achieved therapeutic levels of FVIII following UMGD into hemophilia A mice Recently we have successfully developed prototype US systems including several unfocused and semi-focused transducers to treat large tissue volumes in canine and swine In order to facilitate the translation of this technology to treat hemophilia, we have recently treated normal dogs with UMGD of FVIII plasmids using an open surgery procedure Four mg of a high-expressing, liver-specific pBS-HCRHPI-FVIIIA plasmid and ml of Definity® MBs in ml total PBS solution were injected via the segmental portal vein branch with simultaneous exposure of the target liver lobe to therapeutic US (1.1 MHz frequency, 20 cycle pulses, 50 Hz pulse repetition frequency) for minutes using the large diameter transducers A sham-treated dog received an equivalent pGL4/MB dose, but was not exposed to tUS We used an apodized dual element unfocused transducer H105 at 2.5Mpa peak negative pressure (PNP) in the first dog experiment and observed low levels of hFVIII gene expression in the treated liver lobes The second dog experiment was performed using a cylindrically 6.2 MPa at the focal area One day following treatment, the treated liver lobes were sectioned, and representative sections were fixed and stained for FVIII expression by histochemical staining using a polyclonal anti-FVIII antibody Untreated normal dog liver and human liver were used as negative and positive controls Significant hFVIII gene expression was obtained in both treated liver lobes with the expression levels slightly lower than control human liver lobe Furthermore, fairly homogeneous distribution of FVIII gene expression was observed in hepatocytes Next, we performed Western blot analysis to confirm if the staining is specific to human FVIII protein A heavy chain band specific to hFVIII was observed in treated dog plasma and positive hFVIII control, but not in control normal dog plasma and pre-bleed plasma sample from Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy the dog before treatment Significant enhancement of FVIII-specific clotting activity was also obtained in the second treated dog compared to pre-treatment and the first treated dog In addition, transaminase levels and histology analysis indicated minimal tissue damage in treated dog livers Enhancement of FVIII gene expression by UMGD is currently evaluated in hemophilia dogs Our results sugges that UMGD has great potential for therapeutic treatment of hemophilia A 269 Ultrasound-Targeted Delivery of Chemotherapeutic Drug and Nucleic Acids By Gas-Filled Cationic Liposomes Anthony Delalande, Simona Manta, Luka Horvat, Safia Ezzine, Michel Bessodes, Patrick Midoux, Paul-Alain Jaffres, Nathalie Mignet, Chantal Pichon Centre de Biophysique Moléculaire-CNRS UPR4301, CNRS and University of Orléans, Orléans, France; 2Unité de Technologies Chimiques et Biologiques pour la Santé – CNRS UMR8258 Inserm U1022, CNRS & INSERM, Pris, France; 3CEMCA, CNRS UMR 6521, IFR148 ScInBioS, Université Européenne de Bretagne, Université de Brest, Brest, France Ultrasound is widely used in the medical imaging field for diagnostic purposes Twenty years ago, their use has turned to therapeutic applications for musculoskeletal disorders mainly The invention of gas microbubble ultrasound contrast agent was the trigger of a large number of studies on extra-molecules delivery into cells by coupling ultrasound and gas microbubbles This technique called sonoporation has the advantage to deliver locally drugs near or loaded inside microbubbles Gas microbubble is therefore serves as a drug reservoir that could be triggered by ultrasound In this study, we developed gas microbubbles capable of delivering both a cytotoxic agent as Paclitaxel and siRNA to promote tumour regression Microbubbles were developed with original histidylated cationic lipids which are pH-sensitive In vitro measurements have been done to depict the behaviour of these microbubbles in the presence of tumour cells after ultrasound activation Confocal microscopy and flow cytometry experiments demonstrated that cationic microbubbles were able to complex siRNA Ultrasound parameters were optimized in vitro for siRNA delivery in 4T1 cells stably expressing luciferase (luc) Gas-filled cationic microbubbles complexed with anti-luc siRNA led to specific inhibition of luciferase expression under specific ultrasound parameters A proof of principle has been validated in vivo using 4T1 orthotopic murine mammary tumour model Our siRNA-Paclitaxel formulations were injected either inside the tumour or in the blood circulation via tail vein followed by ultrasound application at tumour site A significant inhibition of tumor growth were observed in mice treated weekly with these formulations repeated at least three times In conclusion, we succeeded to produce original gas-filled liposomes made with histidylated cationic liposomes that were ultrasound-sensitive and able to co-deliver efficiently Paclitaxel and siRNA for tumor treatment 270 Nanoparticle-Mediated Rhodopsin cDNA But Not the Intron-Containing DNA Delivery Causes Transgene Silencing in a Rhodopsin Knockout Model Zongchao Han,1 Min Zheng,1 Rajendra Mitra.1 Department of Ophthalmology, University of North Carolina at Chapel Hill, Chapel Hill, NC Gene expression is moderated by a multitude of patterns The cross-talk between the native promoters and all the non-coding endogenous elements, such as transcription factors, enhancers and introns, are tightly controlled in a precise and real-time manner S107 ... Methods of Delivery and Chemical/Molecular Conjugates of the oxygen saturation of the Hb in the blood of the injected lobe from 30 to unit upon the injection and recovered smoothly after the injection... for the first time, the levels of the cytokines following the hydrodynamic injections to the large animals There was no increase in the systemic inflammatory cytokines of IFN-γ, IL-8, IL-18, and. .. observed throughout the study These results of preclinical assessments support the clinical applications of image- guided, livertargeted hydrodynamic gene delivery This work was supported in part by NIH