267 image guided, liver targeted hydrodynamic gene delivery in dogs preclinical assessment of effectiveness and safety of the procedure

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 ©[.]

Molecular Therapy Volume 23, Supplement 1, May 2015 Copyright © The American Society of Gene & Cell Therapy

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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

1 Department of Genetics, University of North Carolina-Chapel

Hill, Chapel Hill, NC; 2 Gene 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 4 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

1 Louisiana State University, Baton Rouge; 2 The 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

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

1 Division of Gastroenterology and Hepatology, Graduate School

of Medical and Dental Sciences, Niigata University, Niigata, Japan; 2 Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA; 3 Institute 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 image-guided 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 7 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 1 week after hydrodynamic gene delivery and sustained for 4 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

of the oxygen saturation of the Hb in the blood of the injected lobe

from 30 to 0 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 4 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,

liver-targeted 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,1 Misty L Noble,1 Keith R Loeb,2 James Harrang,1

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

1 Pediatrics, Seattle Children’s Reserach Institute and University

of Washington, Seattle, WA; 2 Fred Hutchinson Cancer Research

Center, Seattle, WA; 3 Benaroya Research Institute, Seattle, WA;

4 Sonic Concepts, Inc., Seattle, WA; 5 University of North Carolina,

Chapel Hill, NC.

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 2 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 3

ml of Definity® MBs in 8 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 4 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

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

1 Centre de Biophysique Moléculaire-CNRS UPR4301, CNRS and University of Orléans, Orléans, France; 2 Unité de Technologies Chimiques et Biologiques pour la Santé – CNRS UMR8258 - Inserm U1022, CNRS & INSERM, Pris, France; 3 CEMCA, 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

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