584 regenerative cell therapy for liver diseases with skin adipose tissues derived stem cells

584 Regenerative Cell Therapy for Liver Diseases with Skin Adipose Tissues Derived Stem Cells Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therap[.]

STEM CELL THERAPIES II 582 Ultrasound Gene Delivery to Mesenchymal Stem Cells: Targeting Tumor Therapy Tom Haber,1 Marcelle Machluf.1 Biotechnology and Food Engineering, Technion – Israel Institute of Technology, Haifa, Israel INTRODUCTION: Gene therapy of stem cells such as mesenchymal stem cells, is an exciting therapeutic concept that offers the promise of therapy for an array of disorders and malignancies MSC have been demonstrated to migrate to tumors and ischemic tissue thus can be used as a carrier of therapeutic DNAs Ultrasound (US) is a non-viral approach used to deliver genes into cells and tissue Non-viral vectors are emerging as substitutes to the viral ones since they are considered safer, easier to prepare and lack immunogenic response introduced Ultrasound Contrast Agents (USCA) are gasfilled microbubbles that are used for medical applications The assumption that cavitation is the major bioeffect attributed to gene delivery, suggested the addition of USCA to the medium to decrease the threshold for cavitation, thus increase transfections In the present study, we aim to design and characterize a gene delivery system based on Therapeutic US (TUS) waves for the transfection of MSC The transfected cells will be then used as gene delivery vehicles for the therapy of cancer EXPERIMENTAL METHODS: In-vitro transfection: Mesenchymal stem cells were seeded in 6-well plates Plasmid DNS (pDNA) encoding for Luciferase or GFP was added to the cells and cells were exposed to TUS different parameters with and without USCA The transfection level was detected days post transfection using illuminometer, and transfection efficiency was measured days post transfection using FACS The effect of TUS on the viability of the cells was measured using AlamarBlue assay Effect of TUS on Cells’ surface markers analysis: rMSCs (Ps 7) were exposed to TUS of different parameters and analyzed for different cell surface markers such as: CD31, CD34, CD90 and CD44 The cells were then analyzed for the expression by using FACS Cells not exposed to TUS served as control Effect of TUS on cells’ morphology-Microscope analyses: Cells were cultured, exposed to TUS application, fixated and stained with Phalloidin-FITC for actin filaments, with DAPI for nuclei and with Dil for membrane stain Effect of TUS on cells’ membrane permeability and integrity: Fluorescent conjugated dextran was added to the cells before and in different time points after the TUS application The cells were exposed to TUS different parameters with and without USCA The transfection efficiency was measured using FACS in order to understand the kinetics of membrane permeability in different TUS parameters RESULTS AND DISCUSSION: TUS of 2W/cm2, 30% DC operated for 20 or 30 led to the transfection of MSC, when using 10 or 12 mg/ml of cDNA Adding USCA resulted in higher transfection efficiency TUS did not affect significantly the viability of the cells and did not alter the stemness of the cells When dextran was added at different time points post TUS, transfection was negligible and lower compared to adding the dextran before the TUS In-situ Time-laps imaging revealed that during TUS application the nucleus undergoes reversible morphological changes CONCLUSION: Our study demonstrates that TUS can efficiently transfect MSC while maintaining their viability These results suggest that transfected MSC may be used as a carrier of therapeutic DNAs in order to migrate to tumors and ischemic tissue 583 Universal Stem Cell Gene Therapy Platform: Combining Ex Vivo Strategies and Injectable Tissue/Organ-Specific Extracellular Matrix (ECM) Hydrogels Roger Bertolotti.1 CNRS, Gene Therapy and Regulation Research, Faculty of Medicine, University of Nice - Sophia Antipolis, Nice, France Tissue/organ-specific extracellular matrix (ECM) hydrogels are emerging promising natural scaffolds for regenerative medicine Non-invasive catheter/syringe-based delivery of solubilized ECM relies on an adjustment of the gelation properties/kinetics restricting the liquid-solid phase transition to the final location within the recipient tissue/organ Such an injectable scaffold recapitulates the native ECM environment, thereby providing relevant cells with their tissue-specific matrix interactions These ECM hydrogels are currently successfully used to recruit endogenous repopulating cells in animal models and will be soon experimented for the delivery of transplanted cells for basic regenerative medicine (e.g myocardium regeneration, Singelyn & Christman, 2010) Such exciting data prompted us to devise a strategy for ex vivo gene therapy whereby therapeutic homing of engineered stem cells is achieved by tissue/ organ-specific ECM hydrogel vehicules Organs like liver are very attractive targets since in vivo gelation of injected ECM hydrogel will entrap engineered hepatic stem cells in regenerative structures that will compete with the original mutated tissue during the involution phase of the enlarged organ Apoptotic involution will most likely equally affect both resident and transplanted cells, thereby securing an efficient engraftment of therapeutic cells even if they not have a selective advantage over their mutant cognates Our tissue/organspecific ECM hydrogel strategy is thus discussed in terms of 1) hydrogel-based delivery and entrapment of ex vivo engineered stem cells into regenerative structures in which they can generate wild-type tissue with the revascularizating support of infiltrating endogenous vascular cells, 2) of reconstitution of tissue/organ-specific niches for engraftment of stable pools of therapeutic stem cells in order to secure long-term wild-type cell turn-over, and 3) of substitution in appropriate cases of ECM-linked growth/differentiative factors for transient topical regenerative gene therapy Such an ECM hydrogel strategy stands thus as a perfect complement to basic stem cell gene therapy which is driven by the homing ability of many tissue-specific stem cells (e.g engraftment of hematopoietic stem cells into their bone marrow niche, homing of endothelial progenitor cells into ischemic foci or tumor-tropism of neural stem cells) Of note, our proposed universal platform for autologous stem cell gene therapy has a transient regenerative gene therapy arm aimed at magnifying the homing ability of therapeutic stem cells Synergistic combinations based on the use of ECM hydrogels are thus discussed in light of the main features of our proposed universal platform which relies on 1) long-term gene therapy mediated by endonuclease-boosted gene targeting (gene repair/alteration or targeted transgene integration) and cybridization (mitochondrial DNA transfer), 2) on transient gene therapy aimed at optimizing stem cell regenerative ability (see above) or at epigenetic gene control (e.g potential genesis of fully pluripotent transgene-free iPS cells) and 3) on patient-specific iPS cells for most ex vivo applications 584 Regenerative Cell Therapy for Liver Diseases with Skin Adipose Tissues-Derived Stem Cells Sin-Ting Wang,1 Chi-Chen Lin.1 Institute of Biomedical Science, Taichung, Taiwan Liver diseases have long been the major threats to the health of people in Taiwan for decades Unfortunately, current therapeutic options for many acute and chronic liver diseases are still very Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S223 CELL PROCESSING AND VECTOR PRODUCTION limited and the outcome is usually unsatisfactory Stem cell-based therapy, due to its regenerative and reparative natures, may serve as a promising therapeutic strategy for many liver diseases in the future In the previous publication of our collaborators, it has been demonstrated that AT-MSCs may be further differentiated into hepatocyte-like cells; more importantly, it has also been shown that, human AT-MSC-derived hepatocytoid cells or even the original ATMSCs, when injected into nude mice with acute liver injury via tail vein, may migrate to the damaged liver tissues and integrate with the recipient liver tissues and the recipient mice even showed improved liver function Although this previous study of our collaborators has demonstrated the possibility of using AT-MSCs as a candidate stem cell-based therapy for liver diseases, there are still many important questions need to be addressed First of all, it is still unclear about the mechanisms governing the process of homing and migration of these AT-MSC cells to liver tissues Since chemokine receptor has been reported as an important mediator for stem cell homing, we hypothesize that it may also play an important role in the AT-MSCs homing to liver Secondly, it is important to determine whether AT-MSCs (without further differentiation efforts) can be use directly in cell therapy for liver diseases We then conducted several series of experiments and have found the following results: (1) chemokine receptor CCR4 is highly expressed by AT-MSCs at mRNA and protein level, (2) CCR4 is functional in AT-MSCs measured by chemotaxis assay, (3) induced by injection of galactosamine (an inhibitor of hepatic RNA synthesis) and bacterial lipopolysaccharide, the liver tissue in mouse showed increased expression of CCR4 ligands CCL22 and CCL17 Cell Processing and Vector Production 585 Scalable Manufacturing of Recombinant Adeno-Associated Virus Using Suspension HEK293 Cells Joshua C Grieger,1 Stephen M Soltys,1 Joy R Burleyson,1 Richard J Samulski.1,2 Gene Therapy Center, University of North Carolina, Chapel Hill, NC; 2Pharmacology, University of North Carolina, Chapel Hill, NC Adeno-associated virus (AAV) has shown great promise as a gene therapy vector in multiple aspects of pre-clinical and clinical applications Many developments including new serotypes as well as self complementary vectors are now entering the clinic With these ongoing vector developments, continued effort has been focused on scalable manufacturing processes that can efficiently generate high titer, highly pure and potent quantities of AAV vectors Utilizing the relatively simple and efficient transfection system of HEK293 cells, originally developed in our lab, as a starting point, we have successfully adapted an adherent HEK293 cell line from a qualified clinical master cell bank to grow in serum-free suspension conditions in shaker flasks and wave bioreactors that allows for rapid and scalable AAV production Using the triple transfection method, the suspension HEK293 cell line generates greater than 1x105 vector genome containing particles (vg)/cell or greater than 1x1014 vg/L of cell culture prior to purification To achieve these yields, a number of variables were optimized such as selection of a compatible serum-free suspension media that supports both growth and transfection, selection of a transfection reagent and conditions, cell density, and other components essential for vector production A universal purification strategy based on ion exchange chromatography methods was developed that results in high purity vector preps of AAV serotypes 1-6, 8, and various chimeric capsids tested This production approach also results in high full to empty particle ratios After purification, greater than 1x1013 vg was attained from 1L of cell culture from shaker flasks and wave bioreactor bags S224 586 Use of the Counterflow Centrifugal Elutriation in the Preparation of Apheresis Products for Use in Cell Therapy Applications Chy-Anh T Tran,1 Monica Coronado,1 Agnes Gardner,1 Lijing Li,1 Anitha Rao,1 Lan-Feng Cao,1 Qing Liu,1 John A Zaia,1 David L DiGiusto.1 Virology, City of Hope, Duarte, CA Cell separation by counterflow centrifugal elutriation has been described for the preparation of Monocytes for dendritic cell vaccine applications but its use in other cGMP cell manufacturing operations has been limited We have developed protocols that employ the Elutra™ cell separation device to wash and formulate apheresis products for subsequent ex-vivo cell processing procedures G-CSFmobilized peripheral blood progenitor cell products (HPC-A) were collected from healthy donors (after obtaining informed consent) and processed within 24 hours of collection Five fractions were collected for each product as per manufacturer’s instructions Each fraction was analyzed for cell count, viability and differential The cellular composition of each fraction was also evaluated by flow cytometry using antibodies to CD34, CD3, CD14, CD19, CD15, CD56 Our data demonstrate that >99% of red blood cells and platelets were removed with high recoveries (93.1±10.3%, N=6) of CD34+ cells There was also an enrichment of CD34+ cells in two of five fractions (F4+F5) wihch was combined to form a single CD34+ fraction in our latest method In some tissues, the CD34enriched fractions were formulated, stained with antibody to CD34 and washed using the Elutra device and then transferred directly to a magnetic bead selection device (CliniMACS) for further purification Cell purities from the column were extremely high (98.8±0.6%, N=11) and yields were typical for the device (56±11.7%, N=11) We also observed a selective enrichment of cells with a regulatory T-cell (Treg) phenotype (CD4+/CD25+/CD127lo) in two of the five fractions generated from elutriation of non-mobilized peripheral blood apheresis products Both processes were highly automated and closed from receipt of apheresis product through formulation of target-enriched cell fractions The elutriation profiles can be modified to accommodate a variety of cell targets and applications that require a washed, concentrated leukocyte product for downstream processing Therefore elutriation is a feasible method for the initial manipulations associated with primary blood cell therapy products and supports cGMP/cGTP compliant cell processing 587 Alternative Vector Titration Methods for scAAV Vectors Eliminate Systemic Errors Caused by Template Self-Annealing during qPCR Thermocycling Paolo Fagone,1 Susan Sleep,4 James A Allay,4 J Fraser Wright,5 Amit C Nathwani,3 Andrew M Davidoff,2 Arthur W Nienhuis,1 John T Gray.1 Hematology, St Jude Children’s Research Hospital, Memphis, TN; 2Surgery, St Jude Children’s Research Hospital, Memphis, TN; 3Haematology, University College London, London, United Kingdom; 4Children’s GMP, LLC, Memphis, TN; 5Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA Precise and accurate quantitative titration of clinical gene therapy vector preparations is critical for the proper interpretation of vector potency and efficacy In the process of quality control analysis of our clinical grade self-complementary AAV vector for treatment of Hemophilia B, we observed that some vector quantitation assays generated consistently lower titer values than others amongst multiple independent lots of vector product Specifically, our qPCR genome titration assay typically generated values 5-15 fold lower than those determined by UV spectrophotometry We hypothesized Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy ... AT-MSC -derived hepatocytoid cells or even the original ATMSCs, when injected into nude mice with acute liver injury via tail vein, may migrate to the damaged liver tissues and integrate with the... the AT-MSCs homing to liver Secondly, it is important to determine whether AT-MSCs (without further differentiation efforts) can be use directly in cell therapy for liver diseases We then conducted... process of homing and migration of these AT-MSC cells to liver tissues Since chemokine receptor has been reported as an important mediator for stem cell homing, we hypothesize that it may also play