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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 Molecular T[.]
CELL PROCESSING AND VECTOR PRODUCTION 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 capsid or a self-complementary genome conguration 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 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 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 Children’s GMP, LLC, Memphis, TN; 2Hematology, St Jude Children’s Research Hospital, Memphis, TN; 3Surgery, St Jude Children’s Research Hospital, Memphis, TN; 4UCL Cancer Institute, University College, London, United Kingdom We utilized a 293T-based 2-plasmid transient transfection system coupled with a column chromatography purication process to produce a high quality self-complementary AAV2/8 hFIX clinicalgrade vector for treatment of Hemophilia B Yields of >2x1012/10stack 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 mircouidized lysates generated from pellets of transfected cells were puried by group separation on sepharose beads followed by anion exchange chromatography The virus-containing fractions were further processed through gel ltration and ultraltration 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 successive rounds of amplication indicated the quantity of the rcAAV contaminant is less than or equal to 103 vg per 2.25x1010 vg of vector, which equates to ≤ rcAAV in 2.25x107 vg Additional studies have conrmed 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 Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy 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 efcient, 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 Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; 2Institute 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 S261 CELL PROCESSING AND VECTOR PRODUCTION quantitatively evaluate the distribution and survival of transplanted cells, NIH/3T3 cells, a mouse broblast cell line, were genetically labeled with rey luciferase to obtain 3T3/Luc cells Two 8-mm full-thickness excisional skin wounds were made on the dorsum of ICR mice after hair removal Then, 3T3/Luc cells pretreated with or without adhesamine were intradermally transplanted around the wounds, and the survival of cells and the size of the wounds were periodically measured 3T3/Luc cells rapidly disappeared from the administration site, but thosetransplanted with adhesamine were detected at the site for a much longer period of time Such changes were not obtained with cells transplanted with collagen or bronectin In accordance with the cell survival, 3T3/Luc cells with adhesamine healed the wound signicantly faster than untreated cells Similar results were obtained using primary bone marrow derived cells not only in ICR mice but in diabetic db/db mice, in the latter of which wound healing is impaired compared with normal mice These results strongly suggest that adhesamine can be used to increase the therapeutic effects of transplanted cells for wound healing by increasing their survival time after transplantation 671 Characterization of Positioning Effect of pol III-shRNA Transcription Unit in scAAV Vector Genome on the Packaging Efciency and Functionality of shRNA Silencing Jun Xie,1 Ran He,1 Qin Su,1 Guangping Gao.1 Gene Therapy Center and Viral Vector Core, UMass Medical School, Worcester, MA Recombinant adeno-associated virus (rAAV) can efciently deliver shRNAs to different animal models and accomplish stable knockdown of target genes Small sizes of Pol III-shRNA expression cassettes make them more suitable for self-complementary rAAV (scrAAV) design which is more efcient than single stranded vector However, one of the challenges in production of rAAV-shRNA, particularly, scrAAV-shRNA, is the significant decrease in vector yields as compared to the vectors without shRNA inserts We compared vector yields of scrAAV preparations of different serotypes expressing different shRNAs with those of scrAAVs without a shRNA cassette We found that the average yield for 17 lots of scrAAV-shRNA vectors was only 25% of that for 55 lots of regular scrAAVs while all vector lots were produced in 1x10e9 of 293 cells by the conventional triple transfection and CsCl purication method In addition, EM analysis suggested that the vectors carrying shRNAs had signicant higher rations of empty to full virions Further analysis indicated that, in most of those scrAAV-shRNA vectors, shRNA expression cassettes were cloned into downstream of a Pol II promoter-driven EGFP reporter gene cassette and at a position immediately adjacent to the 3’ inverted terminal repeat (ITR) We hypothesized that negative impact of shRNA inserts on the vector yield was due to the close vicinity between the two strong hairpin structures (i.e replication competent 3’ ITR of the scrAAV and shRNA insert), which hinders vector genome rescue, replication and/or packaging To test this hypothesis and characterize possible positioning effect of shRNA in scrAAVs, we constructed a panel of six scrAAV genomes in which a U6 -shRNA cassette targeting re y luciferase (FFLuc) gene was cloned either immediately after 5’ crippled ITR or 3’ intact ITR or in an intron between a Pol II promoter and EGFP cDNA in both directions respectively To examine functionalities of the U6-FFLucShRNA expressed from different locations of the vector genome and the shRNA bearing intron, we transfected these vector and appropriate control plasmids together with a luciferase expression plasmid into 293 cells to compare their efciencies of FFLuc gene silencing and EGFP expression The results demonstrated that the shRNA cassette worked equally well at any of positions and both directions Insertion of shRNA cassette into the intron did not affect EGFP expression We then compared vector yields and packaging efciencies of S262 vectors in side-by-side production runs, leading to the following observations First, among the different shRNA positions, vector yields ranked as Intron ≥ 5’ crippled ITR > 3’ intact ITR Secondly, we noticed additional 80% drop in yield when the hairpin structure of shRNA cassette was closer to 3’ ITR which is involved in vector replication Taken together, our results conrmed the positioning effect of shRNA in scrAAV vectors and support our hypothesis that two adjacent hairpin structures affect vector genome replication and packaging efciency The ndings from this study provided some important considerations in scrAAV-shRNA vector design 672 Rapid Production of High Titer Virus Using a Concentrated Retrovirus Expansion Device Ceidy Sanchez,1 Gianpietro Dotti,1 John Wilson,2 Cliona M Rooney,1 Malcolm K Brenner,1 Juan F Vera.1 Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, The Methodist Hospital, Houston, TX; Wilson Wolf Manufacturing Corporation, New Brighton, MN One impediment to the successful development of human therapies using genetically modied hemopoietic cells is the cost and complexity of vector manufacture, which limits the number of studies that can be performed Retroviral transduction is commonly used for such ex vivo modication and usually requires large volumes of retroviral supernatants, the titers of which are low compared with other viral vectors We now describe the use of a Concentrated Retrovirus Expansion Device (CRED) for vector production The CRED is similar in external appearance and shape to conventional roller bottles, and thus can be accommodated by standard roller racks However an internal dialysis membrane allows virus particles to become concentrated in the cell compartment, while the nutrient compartment is separated but easily accessible and can retain up to 250 ml of media without causing virus dilution The CRED has a surface area of 600cm2 in the cell compartment, which, due to diffusion across the dialysis membrane, requires only 10ml of media for cell survival, a 10-fold lower requirement than a traditional cell factory or roller bottle of equivalent area To test the potency and stability of CRED-produced retrovirus we cultured a clinical grade PG-13 producer cell line that produces SFG-CAR-CD19/CD28/Z, which encodes a chimeric antigen receptor directed to CD19 (CD19CAR) This vector is used in a current clinical protocol to transduce T cells for use in patients with advanced CD19+ B cell malignancies We compared our standard procedure for retrovirus production (using a T175 ask) and 30ml of IMDM against the CRED using 10ml of IMDM Retroviral supernatant was harvested and frozen daily once cell conuence was attained (days 4-5 for the CRED and days 2-3 for the T175) Subsequently, to enumerate viable viral particles, 1x104 HeLa cells were seeded in a well plate and after 24 hrs were retrovirally transduced using volumes ranging from 10-300ml in the presence of 1ul of Polybrene After days cultures were evaluated by ow to assess transduction efciency, using an antibody recognizing the CH2CH3 sequence of CAR-CD19/CD28/Z The transduction efciency of the CRED virus was at least times greater than conventional supernatant; 10ul (7.5%±4.5% vs 2.5%±0.28%), 20ul (11.9%±6.3% vs 4.9%±0.1%), 50ul (25.9%±7.4% vs 7.9%±0.9%), 100ul (45.2%±11.5% vs 9.3%±0.6%), 200ul (75.15%±15.6% vs 16.9%±1.5%), 300ul (85.7%±8.6% vs 21.4±1.8%) (n=4 expts) To ensure vector stability we exposed the harvested supernatant to consecutive freeze-thaw cycles, and compared virus titers We initially obtained an 86.8% transduction efciency of HeLa cells which slowly declined after each cycle (82.4%, then 80.2%, 80%, 68.7%, 60.9% and 55.5%) Hence the CRED can produce large quantities of high titer retrovirus without media change This virus can resist freeze-thaw cycles and occupies fold less storage space Molecular Therapy Volume 18, Supplement 1, May 2010 Copyright © The American Society of Gene & Cell Therapy ... effects of transplanted cells for wound healing by increasing their survival time after transplantation 671 Characterization of Positioning Effect of pol III-shRNA Transcription Unit in scAAV Vector... adhesamine were intradermally transplanted around the wounds, and the survival of cells and the size of the wounds were periodically measured 3T3/Luc cells rapidly disappeared from the administration site,... but in diabetic db/db mice, in the latter of which wound healing is impaired compared with normal mice These results strongly suggest that adhesamine can be used to increase the therapeutic effects