456 Enrichment of Human Hematopoietic Stem/Progenitor Cells Increases Transduction Efficiency for Stem Cell Gene Therapy Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American So[.]
HEMATOLOGIC & IMMUNOLOGIC DISEASES I No SPECT signal in the brain was observed (+/- KI) in mice administered with free 125I- whilst uptake in the thyroid gland was reduced in the presence of KI1 (fig.1a/b) The probe delivered 125Iin the brain with peak uptake (1-5 min) and rapid efflux (>10 min) possibly by iodide efflux systems present at the BBB1-2 (fig 1c/d) Reporter gene function: hNIS cells showed 9-fold higher 125I- uptake over control cells (fig.2a) Pilot study: BLI confirmed that hNIS tumour xenografts can propagate in the mouse brain (fig 2b) Conclusion: In vivo SPECT imaging has demonstrated that an iodopurine probe can cross the BBB and deliver 125I- in the brain The exceptionally low background signal observed after 10 suggests the technique could be highly sensitive at detecting gene expression in animals and humans Studies are currently underway to detemine whether hNIS cells can be detected with SPECT imaging and to quantify the sensitivity of the technique for imaging gene expression in the brain References: Okamura, T Life Sci 2009 Davson, H J Physiol 1973 454 Role and Potential Use of microRNA-29 to Target the Pancreatic Cancer Stoma Jason Kwon,1 Sarah C Nabinger,1 Zachary Vega,1 Smiti S Sahu,1 Janaiah Kota.1 Medical and Molecular Genetics, Indiana University School of Medicine, Indianapolis, IN Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths, with a 5-year survival rate of less than 6% It usually remains undiagnosed until after the cancer has metastasized, and the resulting disseminated tumors are resistant to all forms of existing therapeutic modalities Until recently, therapeutic strategies have mainly focused on targeting the tumor cells themselves However, pancreatic tumors are characterized by a prominent stromal/fibrous reaction around the tumor called desmoplasia, which is a major obstacle for drug delivery to the tumor bed and plays a critical role in pancreatic cancer progression Because of its role in cancer progression and invasion, stroma is an important therapeutic target to improve the efficacy of anti-cancer drugs Although some of the pharmacological based anti-stromal drugs are shown to be therapeutically beneficial in pre-clinical studies, their effects are minimal in the clinic A growing body of evidence suggests that pancreatic stellate cells (PSCs), stromal cells activated in response to autocrine and paracrine pro-inflammatory cytokines/growth factors such as TGF-β1, secrete excessive amounts of extracellular matrix (ECM) proteins, a major component in the stroma formation of PDAC Furthermore, PSCs interact closely with cancer cells to facilitate cancer progression and metastasis miRNAs are endogenously encoded small non-coding RNA molecules that regulate global gene expression and are critical in maintaining cellular homeostasis It is now well established that miRNAs play a critical role in signaling pathways associated with cancer pathogenesis and the fibrotic process of several organs Numerous functional studies have demonstrated the pro- and antitumorigenic/fibrotic activity of specific miRNAs and their therapeutic potential to suppress tumor growth and fibrosis In an effort to understand the role of miRNAs in PSC-mediated PDAC-stromal accumulation, we challenged PSCs with TGF-β1 and estimated miR-29 expression, a known anti-fibrotic miRNA family In our S174 preliminary work, we observed significant loss of miR-29 in TGF- β1 treated PSCs, in correlation with an increase in pro-fibrotic mediator connective tissue growth factor (CTGF) expression and ECM component proteins such as collagen, fibronectin, and laminin Both TGF-β1 and CTGF are known pro-fibrotic inflammatory cytokines/ growth factors, and their up-regulation is well documented in PDAC and chronic pancreatitis, a known risk factor for pancreatic cancer In addition, we observed a loss of miRNA-29 in the pancreata of a PDAC mouse model which carries oncogenic KrasG12D, a frequent mutation found in >90% PDAC cases Based on these observations, we are currently evaluating the anti-stromal properties of miR-29 in vitro and in vivo to target the pancreatic cancer stroma using gene therapy based approaches 455 Lentiviral Delivered shRNA Inhibition of NANOGP8 or NANOG Activates the Intrinsic Pathway of Apoptosis In Vivo and In Vitro Abid R Mattoo,1 Nikolay Korokhov,1 J M Jessup.1 Laboratory of Experimental Carcinogenesis, National Cancer Institute, Bethesda, MD NANOG and its retrogene NANOGP8 regulate pluripotency and self-renewal in human carcinomas, sarcomas and leukemias while inhibition of NANOGP8 or NANOG decreases spheroid formation by human colorectal carcinomas (CRC) in serum-free medium We postulated that inhibition of NANOGP8 and NANOG by shRNA would increase caspase-dependent apoptosis as CRC undergo 3-D growth in vivo and in vitro Intratumoral injection of lentivirusdelivered (LV) shRNA to NANOGP8 (shNp8-1) or NANOG (shNG-1) at low 1-3 Multiplicity of Infection (MOI) in subcutaneous CX-1 tumors inhibited growth in vivo by 30% compared to tumors injected with a control LV shRNA (shNEG) or left untreated In vitro studies were performed with suspension culture of human CRC cells in serum-free medium in ultra low attachment plates to analyze mechanism of action CRC cells were cultured for 24 hr and then transduced with LV shNp8-1, shNG-1 or shNEG at a MOI of 5-10 and then analyzed days later LV shNp8-1 and shNG-1 reduced total NANOG transcripts with 40-70% of cells transduced and inhibited CRC suspension culture growth by 45- 50% within days, but shNEG did not Growth reduction was secondary to activation of caspase and because LV transduction with shNp8-1 or shNG-1 increased Caspase and activity in single cells and caspase or inhibitors stimulated tumor growth after LV shNp8-1 or shNG-1 transduction while a caspase inhibitor did not CRC cell regrowth after transduction in suspension with LV shNp8-1 or shNG-1 was significantly decreased in a subsequent monolayer colony forming assay Inhibition of NANOGP8 or NANOG activates the intrinsic pathway of caspase-dependent apoptosis in 3-D growth in vitro and in vivo and may enhance the efficacy of treatments that depend on caspase-dependent apoptosis Hematologic & Immunologic Diseases I 456 Enrichment of Human Hematopoietic Stem/ Progenitor Cells Increases Transduction Efficiency for Stem Cell Gene Therapy Kismet Baldwin,1 Fabrizia Urbinati,2 Zulema Romero,2 Michael Kaufman,2 Beatrice Campo-Fernandez,2 Sabine Geiger,2 Donald B Kohn.1,2 Pediatrics, University of California, Los Angeles, Los Angeles, CA; 2Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA Sickle cell disease (SCD) is a multisystem disorder, associated with significant morbidity and mortality The only definitive cure is allogeneic hematopoietic stem cell transplant (HSCT) Autologous Molecular Therapy Volume 22, Supplement 1, May 2014 Copyright © The American Society of Gene & Cell Therapy HEMATOLOGIC & IMMUNOLOGIC DISEASES I stem cell gene therapy for SCD has the potential to treat this illness without the major immunological complications associated with allogeneic HSCT Initial evidence for the efficacy of the modification of human SCD BM CD34+ cells with the bAS3-FB lentiviral (LV) vector for gene therapy of sickle cell disease has been demonstrated (Romero J Clin Invest 123:3317-30, 2013) However, transduction efficiency by b-globin lentiviral vectors using CD34-enriched cell populations is sub-optimal and large vector production batches would be needed to supply clinical trials Isolating a more HSPC enriched population (CD34+/CD38- cells ~ 1% of all CD34+ cells) to transduce could greatly reduce vector needs and, potentially, increase transduction efficiency CD34+/38- cells were isolated from healthy cord blood (CB) CD34+ cells by fluorescence activated cell sorting (FACS) and transduced with the CCL-bAS3-FB LV vector Isolated CD34+/CD38- cells were able to generate progeny over an extended period of long term culture (LTC) compared to the CD34+ cells (p