305 Surface Epitopes Involved in Cell Motility and Tumor Progression Are Expressed in Low Density Cultures but Not in Confluent Cultures of Human Multipotent Stromal Cells (hMSCs) treated as well as c[.]
treated as well as control mice The remaining cells were grown in either methycellulose or in 96 well plates Second generation recipient mice were sacrificed 14 days after transplant and the sp and nsp cells isolated and grown in either methycellulose or in 96 well plates Tissue culture grown colonies were stained for donor (LacZ+) cells, expression ofvimentin, endothelin, or F4/80 (macrophages) In the first generation recipient mice, those pre-treated with MnSOO-PL had increased expression ofLacZ+ sp and nsp cells per esophagus (38.4 ± 4.2% and 21.8 ± 5.5% , respectively) compared to the control irradiated mice (21.4 ± 4.4%, p=0.0105 , and 5.2 ± 2.4% , p=0.0277, respectively) Second generation MnSOD treated recipient mice, transplanted with SPcells from MnSOD-PL treated mice had more LacZ+ SP cells per esophagus (47.9 + 3.5%) than that detected in mice injected with NSP cells from the same donors (22.3 ± 2.4%, p < 0.0001), control irradiation only SP cells (22.3 ± 2.0, p = 0.000 I) or control irradiation only NSP (9.6 ± 2.5%, p < 0.000 I) Explanted second generation LacZ+ cell derived colonies were stained with vimentin , endothelin, or F4/80 For all SP and NSP subpopulation, colonies formed in vitro contained viment in and/or endothelin positive with few F4/80 positive cells In both the first and second generation recipient mice , more multi-lineage LacZ+ colony forming cells were recovered from mice pretreated with MnSOO-PL Therefore, esophageal irradiation induced ROS can be reduced by swallowed MnSOO-PL treatment improved engraftment of esophageal stem cells 303 Efficient Gene Transfer into Long-Term Repopulating Hematopoietic Stem Cells from Clinically Relevant Sources Martina Cesani,'« Alessia Capotondo.P Eugenio Montini,' Laura Tononi,' Manfred Schmidt,' Fabrizio Benedicenti,' Anna Zingale; Francesca Santoni de Sio,' Samantha Scaramuzza,' Christof Von Kalle,5 Luigi Naldinj,l ,3 Alessandra BiffiY [San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milano, Italy ; 2Experimental Neurology InstiIIIte, San Raffaele Scientific Institute , Milano, Italy; J/1ta-Salute University, San Raffaele Scientific Institute , Milano , Italy; "Department ofBiomedical Science and Biotechnology University ofBrescia, Brescia, Italy; 5National Center for Tumor Diseases, National Centerfor Tumor Diseases, Heidelberg, Germany, In the perspective ofclinical application oflentiviral vectors (LV) in hematopoietic stem cells (HSC) gene therapy for Metachromatic Leukodystrophy, we optimized LVtransduction ofhuman HSC from clinically relevant sources, such as bone marrow and mobilized peripheral blood (BMIMPB) In order to confirm transduction of long-term repopulating, multi-lineage differentiating hHSC by LV, we monitored the engraftment and differentiation oftransduced cells in RAG2-/-IL2R-gamma chain-/- mice This xenograft model is characterized by a long life-span and the ability to sustain T cell differentiation and thymic maturation oftransplanted human cells We first demonstrated efficient LV transduction of cord-blood derived HSC and reached high transgene expression long-term in the multilineage human graft of transplanted mice We then tested different transduction protocols on MPB/BM cells using LV encoding either the therapeutic arylsulfatasc A (ARSA) or GFP, tuning vector dose, number of transduction hits and total time of in vitro culture We reached efficient and reproducible gene marking of HSC (ranging from 40% to 70% transduction in the various conditions), assayed both in liquid culture and c1onogenic assays , with a mean of 1.5-4 vector copy number (VCN) per genome Sustained transgene expression was observed, MPB HSC, transduced with a single vector hit at multiplicity of infection (MOl) of 100, efficiently repopulated recipient mice long-term upon transplantation We detected human cells engraftmcnt in all hematopoietic organs up to 18 weeks after transplantation, and we observed multi-lineage differentiation of Molecular Therapy Volume 15,Supplement I ~, \ )' 2007 Coprright © The American Society of G ene Th erapy transduced cells Interestingly, thymic maturation of human lymphoid progenitors was documented When isolated from chimeric mice 18 weeks post-transplant, human cells retained transgene expression at comparable levels to the in vitro samples Moreover, LAM PCR demonstrated a polyclonal pattern of LV integrations in both pre-transplant samples and tissues retrieved from repopulated mice, without apparent indication of in vivo skewing Overall these data demonstrate in a stringent in vivo model the capability of LV to efficiently transduce human MPB/BM HSC without impairing their long-term repopulation and differentiation 304 A Transformed Cell Population Derived from Long Term Cultured Mesenchymal Stem Cells Has No Functional Effect after Transplantation into the Injured Heart Nan Ma,l Wenzhong Li; Oario Furlani,' Lee-Lee Ong,' Karola Lutzow,' Christian Klopsch, I Andreas Liebold, I Andreas Lendlein,' Gustav Steinhoff.' [Department ofCardiac Surgery; FKGO, Rostock, Germany; 2GKSS Forschungszentrum Geesthacht GmbH, Institut Fiir Polymerforschung, Teltow, Germany Mesenchymal stem cells are characterized by their self renewal and differentiation potential They have been evaluated widely both as a cell source tor various therapeutic applications and as a platform for ex vivo genetic manipulation In this study, mesenchymal stem cells were isolated from normal Lewis rat bone marrow underwent spontaneous transformation following long term (3-4 months) in vitro culture The cells were noted to appear distinctly different from typical MSCs They were morphologically spherical and cuboidal to short spindle in shape In terms ofcell surface characteristics, the cells were C045', C029'0", C09010" and CO 117' They exhibited accelerated proliferation and lost of contact inhibition Six weeks after transplantation into the rat infarcted myocardium, encapsulated structure and calcifications were found in the infarcted area or/and border zone Furthermore, there was no significant improvement on cardiac function by pressure-volume loop measurement after cell transplantation Our study highlights the need for thorough biosafety investigations of long-term cultivation ofmesenchymal stem cell to achieve the full clinical therapeutic benefits 305 Surface Epitopes Involved in Cell Motility and Tumor Progression Are Expressed in Low Density Cultures but Not in Confluent Cultures of Human Multipotent Stromal Cells (hMSCs) Ryang Hwa Lee*, Min Jeong Seo", Andrey A Pulin, Carl Gregory, Joni Ylostalo , Darwin J Prockop 'Center for Gene Therapy, Tulane University Health Sciences Center; New Orleans, LA *R.H.L and MJ.$ contributed equally to the work Human rnultipotent stromal cells (hMSCs) from bone marrow can be cloned as single-cell derived colonies, but during proliferation the cells undergo phenotypic changes In cultures ofhMSCs that were plated at low density, the changes include progression from small, rapidly proliferating cells to large, nat cells that are poised for differentiation In parallel with these changes, there are dramatic alterations in the profiles of expressed genes Because of these phenotypic changes as the cells expand , it has been difficult to obtain reliable markers to characterize hMSCs by surface epitopes Here, we have identified a series of surface epitopes that early passage hMSCs express in low density cultures but not in confluent cultures: hepatocyte growth factor receptor (HGFR), podocalyxin-Iike protein (POOXL), integrin-a6, integrin-a4, the receptor for stem cell derived factor-I (CXCR4) and the receptor for fractal kine (CX3RI) Interestingly, siRNA-mediated downregulated POOXL cause the cells to aggreSI15 gate into immobile clusters The results suggest that the epitopes are useful to assay the quality ofhMSCs cultures for use in animal models and clinical trials CELL PROCESSI NG VECTOR PRODUCTION 306 Testing Large-Scale Lentiviral Vector Preparations for Replication Competent Lentivirus Lisa Duffy,' Sue Koop ,' ling Yao,' Robert Getty,' Scott Cross,' Lakshmi Sastry, Kenneth Cornctta.' 'Medical and Molecular Genetics/National Gene Vector Laborato/yo Indiana University School ofMedicine, Indianapolis IN As lentiviral vectors move into the clinical arena, the need to screen for replication competent lentivirus (RCL) will be an im- portant step in certifying vector supernatant We have previously reportedan analysis ofRCL testing methods (Sastryet al Molecular Therapy 8:830-9, 2003) and now report our initial experience with RCL screening In brief, the two-phase assay utilizes an amplification phase where the T cell line C8166 is exposed to the test article After hour incubation the cells are passaged for weeks and cell free media is used to inoculate naive C8166 cells which are then passagedfor I week (indicator phase), RCL is presentwhen p24-gag I-IIV-I antigen (by ELISA)and/or psi-gag recombinant(by PCR)are detected in indicator phase cells.An attenuated HIV-I virus (R8.71) is used as a positive control The assay was validated by performing infection of C8166 cells according to the protocol using R8.71 at the TCID50 (0.5 infectious unit/culture).Three cultures were tested per assay and three independent assays were performed Seven of nine cultures tested positive at the indicator phase for both p24 antigen and psi-gag recombinants, which validated the assay and confirmed the limits of detection was approximately I infectious unit Since validation, the assay has been uscd to test large scale vectorpreparationsand cells in independent assays For each assay, three positive controls were inoculated at the estimated TCID50 at the start of the amplification phase and were subsequently carried through the indicatorphase In addition, fiveculturesof naive C8166 were inoculated with virus at the TCID50 at the start ofthe indicator phase In reviewing the positive control portion ofthe amplification phase, 24 of 27 cultures at thc end of the amplification phase had elevatedp24 antigen levelsand after passageintothe indicatorphase, 27 of27 had detectable virus by p24 and by psi-gag recombination PCR For the positive controls set up at the start of the indicator phase, 41 of 45 were positive for p24 and 36 of 45 were positive for psi-gag recombinants at the end of the week of culture These findings suggest the week amplification does increase sensitivity compared to a week amplification In terms of test articles, of the RCLassays were used to screen large scale vector productions that had a combined pre-concentration volume of over 100 liters, of which 5% of the total final product was tested In addition, 10' post-productioncells were also screened from each of the productions and assays were performed on cells transduced with clinical grade vector To date, no RCL has been detected in any of the test articles submitted for analysis This analysis provides encouraging news for lentiviral vector development, although continued refinement of the RCL assay is warranted to maximize sensitivity while streamlining the methodology S1I6 307 Generation of Lentivirus Vectors Using Recombinant Baculoviruses Hanna P Lesch.!' Sanna Turpeinen,':' Einari A Niskanen,' Anssi Mahonen,':' Kari Airenne, I Seppo Yla-Herttuala.v-' I Department ofBiotechnology and Molecular Medicine A.I Vlrtanen Institute University ofKuopio, Kuopio, Finland; 2Department ofMedicine and Gene Therapy Unit A./ Virtanen Institute University ofKuopio, Kuopio, Finland; j Kuopio University Hospital Kuopio University Hospital Kuopto, Finland; "Department ofBiological and Environmental Science NanoScience Center; University of'Jyvaskyla Jyviiskyld, Finland; sArk Therapeutics qg Ark Therap eutics qg Kuopio, Finland The production ofreplication defective lentiviral vectors in clinical scale is challenging The four plasmid method by conventional transienttransfectionto producethird generation lentiviralvectors is tedious, time consuming and suffers from batch-to-batch variation Some inducible stable production cell lines are available but the toxicity of several viral proteins prohibits constitutive expression Baculovirus technology offers an attractive possibility to a scalable virus production as a result of ease production and concentration of baculoviruses, efficient transduction of suspension mammalian cells in serum free conditions and safety of the baculoviruses, As a firststep towards scalable lentiviralproductionsystem we have constructed four recombinant baculoviruscs, BAC-transfer, BAC-gagpol, BAC-VSVg and BAC-rcv,expressing all c1cmcnts required for safe lentivirus vectorgeneration After 293T cell transduction with recombinant baculoviruses functional Ientiviruses were produced Different baculovirus concentrationswere used to findoptimal baculovirusconcentrationfor lentivirusproduction The un-concentrated lentiviral titers in cell culture mediums were on avarege 1,21 x 106 TUlml which are comparable to titers ofthe lentivirus produced by the conventional four plasmid method Lentivirus transduced Hela cells were grown for three months without loosing the GFP exprcssion Our results show for the first time that baculoviruses can be used for the production of lcntiviruscs in mammalian cells 308 Titer and Long Term Stability of Retroviral Titers Produced in Murine Based Producer Cell Lines Lisa Duffy,' Sue Koop,' ling Yao,' Kenneth Cornctta.' 'Medical and Molecular Genetics/National Gene l-ector Laboratory Indiana University School ofMedicine Indianapolis IN While the spectrum of integratingvectors under considerationfor clinical use continues to expand, vectors based on gamma retroviruses continue to be utilized and improved To better understand factors involved with vector production, we studied the impact of genome size, temperature, harvest timing, and long-term storage on vector titer.Tostudy genomesize we prospectively designed a series of deletions in the transgene portion of the GcSAM vector (kindly provided by Richard Morgan, NIH) that contains non-expressing neomycin phosphotransferase (neo) and beta galactosidases genes Eight constructs were evaluated with genome sizes ranging from 3258 to 6970 bascpairs Titer was assessed using real-time PCR for detection of vector RNA using a probe and primer sct designed within the packaging sequence Titer for vector genornes between 4559 and 6346 basepairs had similar titers, with a decrease outside this range The effect of temperature and harvest intervals was also evaluated Data from 27 PG13and GP+envAM12derived Master Cell Banks(MCB)generatedin our facilitywascompiled, evaluating titer of vector produced at 32 or 37°C and harvest intervals of 8, 12 or 24 hours Thc majority of PG 13 based ccll Iincs (13/27) had the highest titers whcn harvested at 24 hours and 32°C, although 6/27 wcre optimal at 37°C, 12 hour harvest; 4/27 wcrc optimal at 37°C, 24 hour harvest; and 4/27 were optimal at 32 DC, 12 hour Molecular The 4lpy Volume 15.Supplement t• •\ br 2007 Co pyright © "111e American SOI;ic;ty o f Gene Thcrapj- ... Department ofBiotechnology and Molecular Medicine A.I Vlrtanen Institute University ofKuopio, Kuopio, Finland; 2Department ofMedicine and Gene Therapy Unit A./ Virtanen Institute University ofKuopio,... Gene l-ector Laboratory Indiana University School ofMedicine Indianapolis IN While the spectrum of integratingvectors under considerationfor clinical use continues to expand, vectors based on gamma... cells. An attenuated HIV-I virus (R8.71) is used as a positive control The assay was validated by performing infection of C8166 cells according to the protocol using R8.71 at the TCID50 (0.5 infectious