667 therapy for vaso occlusion in sickle cell disease includes RNA aptamer

667 Therapy for Vaso Occlusion in Sickle Cell Disease Includes RNA Aptamer Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy S260 OLIGONUCLEOT[.]

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S260

OLIGONUCLEOTIDE & RNAI THERAPEUTICS II

666 Nanoparticles are Effective Vehicles

for Systemic Delivery of 2’OMePS Antisense

Oligonucleotides in Exon Skipping-Mediated

Dystrophin Restoration

Alessandra Ferlini,1 Marina Fabris,1 Elena Bassi,1 So a Falzarano,1

Daniela Perrone,2 Francesca Gualandi,1 Patrizia Sabatelli,1,3

Luciano Merlini,1 Paola Rimessi.1

1 Department of Experimental and Diagnostic Medicine, Section of

Medical Genetics, University of Ferrara, Ferrara, Italy; 2

IGM-C.N.R., Unit of Bologna, IOR, Bologna, Italy; 3 Department of

Chemistry, University of Ferrara, Ferrara, Italy.

We used a novel PMMA/N-isopropil-acrylamide+ (NIPAM)

cationic nanoparticle (ZM2-NPs), found to be non-toxic in vitro,

for systemic delivery of 2’-O-methyl full-length phosphorothioate

AONs in the mdx animal model Six weeks-old mdx mice were

intraperitoneally treated: group 1 received 225 mg of naked M23D

AON and group 2 received of 225 mg of M23D AON conjugated

with 2.5 mg of ZM2 NPs Four non-injected mdx mice were used

as controls The animals were injected weekly for 7 weeks (7.5 mg/

kg/injection) and sacri ced 1 week (4) and 12 weeks (4) after the

 nal administration The total amount of M23D AON received by

each animal was 52.5 mg/kg (1575 mg/animal) In treated animals

sacri ced 1 week after  nal administration we observed restored

dystrophin protein synthesis in both skeletal (quadriceps, diaphragm)

and cardiac muscles, allowing protein localization in up to 40%

of muscle  bers The mdx exon 23 skipping level was up to 20%

Western blotting showed the dystrophin protein in all muscles

studied Furthermore, we veri ed that dystrophin restoration also

occurs in the smooth muscle cells of the dorsal skin arrector pili in

ZM2-AON treated mdx mice only In treated animals sacri ced 12

weeks after  nal administration we still observed dystrophin protein

synthesis in up to 8% of muscle  bers in skeletal muscle (quadriceps,

diaphragm) but not in the heart The mdx exon 23 skipping level was

6% (quadriceps), 8% (heart) and 10% (diaphragm) Western blotting

revealed the presence of dystrophin protein in the diaphragm only

We measured the dystrophin transcript amount in all animal groups

by four exon speci c real time PCR assays (ESRAs) In non-treated

mice, we found that the dystrophin transcript amount ranged from

20 to 30% in all muscles of younger mice (7 weeks of age), whereas

it was lower in skeletal muscles (10%-20%) and higher in the heart

(up to 30%) in older mice (19 weeks of age) In ZM2-AON or naked

AON treated animals sacri ced 1 week after  nal administration, the

dystrophin transcript amount didn’t vary signi cantly Differently,

in 12 weeks/sacri ced ZM2-AON treated animals, the transcript

level was signi cantly higher in skeletal muscles but reduced in the

heart In mice treated with naked AONs the amount of transcript was

higher also in the heart In conclusions, ZM2-AON complexes are

able to induce dystrophin restoration, which still persists in skeletal

muscles, though at low level, 12 weeks after  nal administration

Dystrophin transcript amount is not in uenced by naked AON or

NP-AON therapy in early sacri ced mice, whereas both naked AONs

and ZM2-AONs seem to enhance the dystrophin transcript stability

in late sacri ced mice when the basal transcription level is lower The

basal dystrophin transcription amount in the cardiac muscle seems to

be age-related, having a different behavior during AON therapy

667 Therapy for Vaso-Occlusion in Sickle Cell Disease Includes RNA Aptamer

Angela D Burnette,1 Jun-ichi Nishimura,2 Milena Batchvarova,3

Shahid M Nimjee,1 Rahima Zenadi,3 Marilyn J Telen,3 Bruce A

Sullenger.1

1 Surgery, Duke University Medical Center, Durham, NC;

2 Hematology/Oncology, Osaka University Graduate School of Medicine, Osaka, Japan; 3 Hematology/Oncology, Duke University Medical Center, Durham, NC.

Painful vaso-occlusive episodes are characteristic of sickle cell disease (SCD) and are caused in part by the adhesion of sickle erythrocytes (SS-RBC) to the vascular endothelium As a possible treatment for vaso-occlusion to inhibit SS-RBC adhesion, two adhesion molecules: αvβ3 and P-selectin, were targeted by high-af nity aptamers and selected via SELEX (Systematic Evolution of Ligands through EXponential enrichment) technology An in vitro

 ow chamber was used to test the anti-adhesion activity of aptamer 17.16 that binds to human integrin αvβ3 Using human umbilical vein endothelial cells (HUVEC) pre-treated with thrombin, human SS-RBC were passed across the HUVEC at a constant rate Aptamer 17.16 at 30nM had anti-adhesion activity similar to LM609, an inhibitory antibody to αvβ3 However, a control aptamer did not inhibit adhesion Normalized % inhibition of 30nM aptamer 17.16

at 2 dynes/cm2 shear stress was 68%

The anti-adhesive activity of aptamer PF377, which binds to human P-selectin, was tested using primary HUVEC pre-treated with IL-13, followed by histamine Aptamer PF377 at 60nM had anti-adhesion activity similar to heparin, a known inhibitor of SS-RBC adhesion to P-selectin, and 9E1, an inhibitory antibody to P-selectin Normalized

% inhibition of 60nM aptamer PF377 at 1 dyne/cm2 shear stress was 99% Negative controls, a functional aptamer and AC1.2, a non-inhibitory antibody to P-selectin, did not prevent adhesion

CELL PROCESSING AND VECTOR PRODUCTION

These data show the potential of using aptamers to block adhesion molecules as a viable therapeutic option for preventing vaso-occlusion

in SCD

Cell Processing and Vector Production

668 GMP Production of Self-Complementary Serotype 8 AAV Vector for Treatment of Hemophilia B

James A Allay,1 John S Coleman,1 Arthur W Nienhuis,2 Andrew Davidoff,3 Amit C Nathwani,4 Susan Sleep,1 John T Gray.2

1 Children’s GMP, LLC, Memphis, TN; 2 Hematology, St Jude Children’s Research Hospital, Memphis, TN; 3 Surgery, St Jude Children’s Research Hospital, Memphis, TN; 4 UCL Cancer Institute, University College, London, United Kingdom.

We utilized a 293T-based 2-plasmid transient transfection system coupled with a 3 column chromatography puri cation process to produce a high quality self-complementary AAV2/8 hFIX clinical-grade vector for treatment of Hemophilia B Yields of >2x1012 /10-stack cell factory were obtained and produced ∼3.8x1014 total vector genomes (vg) by QPCR Capsid westerns of all transfected cell lysates allowed continuous monitoring of the transfection productivity Benzonase-treated mirco uidized lysates generated from pellets of transfected cells were puri ed by group separation

on sepharose beads followed by anion exchange chromatography

The virus-containing fractions were further processed through gel

 ltration and ultra ltration using a 100kd membrane The vector was formulated in phosphate-buffered saline plus 0.25% human serum albumin The preparation was free of any adventitious agents

Spectrophotometric analysis suggests ∼20% full particles while only very low quantities of non-viral proteins are visible on silver-stained SDS PAGE Residual 293T DNA and protein were 1.15 pg/109 vg and 0.024 µg/ml, respectively Residual plasmid DNA was 1.1% of the total DNA and residual capsid DNA was 0.013% An infectious assay to measure replicative forms of AAV (rcAAV) that may be generated during production was developed in 293T cells using an E1, E3 deleted adenovirus co-infection to provide the requisite helper function QPCR of capsid sequences after 3 successive rounds of ampli cation indicated the quantity of the rcAAV contaminant is less than or equal to 103 vg per 2.25x1010 vg of vector, which equates to

≤ 1 rcAAV in 2.25x107 vg Additional studies have con rmed the long term stability of the vector at -80°C for at least 30 months and for at least 24 hours formulated in the clinical diluent and stored at room temperature within i.v bags This material has been approved

for use in clinical trials in the US and UK The approved clinical trial protocols are currently open and actively recruiting patients To our knowledge this represents the  rst clinically approved AAV vector preparation using either serotype 8 capsid or a self-complementary genome con guration

669 Production of Lentiviral Vectors

in Suspension Cells Using Recombinant Baculoviruses

Hanna P Lesch, Anna E Laitinen, Lauri M Laitinen, Jere T Pikkarainen, Haritha Samaranayake, Seppo Ylä-Herttuala, Kari J Airenne

Department of Biotechnology and Molecular Medicine, A.I Virtanen Institute, University of Kuopio, Kuopio, Finland; Department of Medicine and Gene Therapy Unit, University of Kuopio, Kuopio, Finland; Kuopio University Hospital, Kuopio, Finland; Ark Therapeutics, Kuopio, Finland.

The known fact is that the production of lentiviral vectors in clinical scale is demanding So far the dominating production method is a transient plasmid transfection system using adherent 293T cells Earlier, we have developed the method for baculovirus-mediated for lentiviral vector production in adherent cells However, the expansion

of the adherent cell production to large volumes is troublesome The most practical method for large scale production would be to use suspension cells which necessitates the use of bioreactors in viral vector production In this study we have adapted the lentivirus production to suspension cells using four recombinant baculoviruses expressing all elements required for a safe lentivirus vector production

Up to 109 TU/ml lentivirus titers were achieved when baculoviruses were used in the transduction of suspension 293T cells and lentiviruses were concentrated by ultracentrifugation The produced lentiviruses were accurately characterized and compared to the viruses produced either in adherent 293T cells by calcium phosphate transfection or

in suspension 293T cells by polyethylenimine transfection The production of baculoviruses is easy and rapid, their use is safe and they have low levels of cytotoxicity; therefore they offer a new alternative way for the production of lentiviral vectors In conclusion, the baculovirus system is ef cient, safe and enables high titer lentivirus production in suspension cells

670 Increased Therapeutic Effect of Cell Transplantation for Wound Healing in Diabetic Mice by Prolonging the Survival of Transplanted Cells Using Adhesamine, a Synthetic Adhesion Molecule

Tetsuya Suehara,1 Makiya Nishikawa,1 Yuki Takahashi,1 Sayumi Yamazoe,2 Motonari Uesugi,2 Yoshinobu Takakura.1

1 Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; 2 Institute for Chemical Research, Kyoto University, Kyoto, Japan.

Technology for cell culture and differentiation has greatly increased the possibility of cell-based therapy for a variety of diseases Spatiotemporal distribution of administered cells is an important factor determining the therapeutic effect of such treatment However, the survival of administered cells has not been evaluated well enough

to conclude whether it affects the therapeutic effects of cell-based therapy This is at least partly due to the lack of technologies to increase the availability and survival of cells Recently, Yamazoe

et al have discovered a dumbbell-shaped synthetic small molecule, adhesamine, which increases cell adhesion to culture dishes In the present study, we examined whether adhesamine prolongs the survival of cells after transplantation and increases the therapeutic effects of cell transplantation in mouse models of wound healing To

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