235 LncRNA Saf Interacts with SPF45 To Promote Fas Receptor Alternative Splicing in Maturing Erythroid Cells Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gen[.]
GENE REGULATION cohort at the highest safe and tolerated dose Safety and signs of clinical benefit will be assessed throughout these trials The doseescalation phase of the RetinoStat® trial is complete whilst this phase is in progress for StarGen™ and UshStat® Subretinal administration of all three products has been safe and well tolerated, causing no ocular inflammation or immune responses in any patients and there have been no SAEs related to the products In the RetinoStat® trial, the secreted nature of the endostatin and angiostatin transgenes has meant that expression of these proteins could be quantified over time in each patient from aqueous tap samples Transduction of the retina following subretinal injection of RetinoStat® produced both proteins that were detectable in the aqueous humour at a dose dependent level that increased over time in each patient In summary, ocular gene therapies based on the LentiVector® platform continue to show good safety following subretinal delivery into patients for three different ocular indications The platform has proved to be a highly effective delivery system for relatively large genes into target retinal cells, resulting in stable and long-term expression The overexpression of Rtl1 induced the upregulation of Ppargc1a and oxidative phosphorylation genes in HCCs The overexpression of Rtl1 in murine embryo fibroblasts recapitulated the upregulation of Ppargc1a Since Ppargc1a is a target of microRNA 136, our data suggest that Rtl1 regulates cancer cell metabolism by squelching the microRNAs encoded within anti-Rtl1, thus acting as a competing endogenous RNA Overall, by LV-based insertional mutagenesis we identified new liver cancer genes that represent novel prognostic markers and therapeutic targets for human HCCs Studying HCCs induced by single traceable events, we found that different oncogenes converge towards the regulation of the Dlk1-Dio3 region, that may represent an important hub in the regulation of cancer cell metabolism Gene Regulation Zhaohui Wang,1 Donald D Rao,1 Phillip B Maples,1 Neil Senzer,1,2 John Nemunaitis.1,2,3,4 Gradalis, Inc, Dallas; 2Mary Crowley Cancer Researh Centers, Dallas; 3Texas Oncology, PA, Dallas; 4Medical City HCA, Dallas 233 Lentiviral Vector-Based Insertional Mutagenesis Identifies New Oncogenes and Molecular Networks That Control Hepatocyte Transformation and Metabolism Marco Ranzani,1 Daniela Cesana,1 Cynthia Bartholomae,2 Francesca Sanvito,3 Stefano Annunziato,1 Michela Riba,5 Fabrizio Benedicenti,1 Pierangela Gallina,1 Claudio Doglioni,3 Christof von Kalle,2 Yoon Jun Kim,6 Elia Stupka,5 Manfred Schmidt,2 Giovanni Tonon,7 Luigi Naldini,1 Eugenio Montini.1 HSR-TIGET, Milan, Italy; 2NCT, Heidelberg, Germany; HSR Department of Pathology, Milan, Italy; 4HSR-Center for Translational Genomics and Bioinformatics, Milan, Italy; 5Seoul National University College of Medicine, Seoul, Korea; 6HSRFunctional Genomic of Cancer Unit, Milan, Italy We developed a forward genetics approach based on a new lentiviral vector (LV)-based insertional mutagen by which we could efficiently induce hepatocellular carcinoma (HCC) in different mouse models From 30 LV-induced HCCs we retrieved LV integrations and identified Rtl1, Braf, Fign, and Sos1 as candidate cancer loci We validated in vivo the causative role in HCCs of all the genes by forced expression in the mouse liver We found that the newly identified cancer genes are overexpressed and amplified or deleted in human HCCs and that their expression or the gene expression signature induced by their upregulation can predict the survival of HCC patients By whole-transcriptome gene expression analysis of the LV-induced HCCs, we found that Fign and Braf specifically caused the upregulation of several Maternally Expressed Genes and MicroRNAs (MEGM) encoded within the imprinted Dlk1Dio3 region This pattern of MEGM induction was recapitulated in murine hepatocytes in vivo before overt cell transformation Accordingly, we found that human HCCs and other solid tumors display the concurrent upregulation of FIGN or BRAF with MEGMs Overexpression of truncated BRAF in human primary hepatocytes induced the upregulation of MEGMs and the downregulation of PPARGC1A (a master regulator of oxidative phosphorylation), its targets and genes involved in oxidative phosphorylation We validated PPARGC1A as a direct target of microRNA 136 (encoded within the DLK1-DIO3 region), thus providing a mechanistic framework to explain how the DLK1-DIO3 region is involved in the regulation of cancer metabolism On the other hand, LV integrations targeting the Dlk1-Dio3 region in HCCs induced the upregulation of the paternally expressed gene Rtl1, which is transcribed in the opposite orientation of and complementary to anti-Rtl1, a maternally expressed transcript encoding for microRNAs, including microRNA 136 S90 234 Detection of RNA Interference (RNAi) Mediated mRNA Cleavage in Fresh Injected Tumor Tissue from Patients a Phase I Trial of an Intratumoral pbi-shRNA™ Lipoplex Targeting Stathmin-1 We developed a novel “bifunctional” shRNA technology to knockdown the expression of target genes through both cleavagedependent mRNA degradation and the cleavage-independent mRNA degradation, sequestration and translational suppression A bifunctional shRNA construct (designated as pbi-shSTMN1) targeting human stathmin-1 (STMN1) is being tested in a Phase I clinical trial (BB-IND 14938) Pbi-shSTMN1 is formulated with a proprietary cationic liposome delivery system to form a lipoplex (pbi-shRNA™ LP) As part of this pbi-shSTMN1 LP Phase I study, we designed a RNA Ligase Mediated RACE (RLM-RACE) assay, and employed it to detect sequence specific cleavage of Stathmin1 mRNA as the result of RNAi action via RACE amplification The Phase I study is a dose escalation trial (4 patients per cohort) involving single intratumoral (IT) injection in advanced cancer patients The patients in first dose cohort (n=4) were injected with 0.7mg of pbi-shRNA™ LP per injection The second dose cohort of patients with per injection dose of 1.4 mg (n=4) is currently being enrolled Briefly, a core needle biopsy was done to collect a sample a day before treatment, at either 24 or 48 hours, and at days after IT injection For some patients, tumor was resected days after injection Total RNA was extracted from tumor samples and used for RLM-RACE assay The assay design will be presented in a flow chart The predicted cleavage site of Stathmin-1 mRNA by pbi-shSTMN1 had previously been confirmed in HTC-116 cells transfected with pbi-shSTMN1 In an effort to utilize this assay as part of a GLP process necessary for expanded clinical development, HCT-116 cells transfected with pbi-shSTMN1 were spiked into human tumors to be utilized as positive controls.Results of study cohort samples will be reported 235 LncRNA Saf Interacts with SPF45 To Promote Fas Receptor Alternative Splicing in Maturing Erythroid Cells Olga Villamizar,1 Christopher B Chambers,1 Janice M Riberdy,2 Derek A Persons,2 Andrew Wilber.1 Medical Microbiology, Immunology and Cell Biology, Southern Illinois University School of Medicine, Springfield, IL; Hematology, St Jude Children’s Research Hospital, Memphis, IL Erythropoiesis is tightly controlled to ensure adequate red blood cell production throughout life This process is regulated by multiple physiologic mechanisms, including FasR/FasL-mediated apoptosis FasR pre-mRNA can be alternatively spliced to create a soluble isoform (sFas) that renders cells insensitive to FasL-induced Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy GENE REGULATION apoptosis The long noncoding RNA (lncRNA) Saf was reported to participate in this process LncRNAs are >200 nucleotide transcripts that form complexes with various RNA-binding proteins, including splicing factors, to regulate and diversify gene function Last year, we reported that the 1.5-kb lncRNA Saf was regulated by GATA-1 and KLF1 and induced during maturation of human erythroid cells As the precise mechanism of Saf function is unknown, we extend our findings to include gene expression analysis, cellular localization, and identification of Saf interacting proteins For gene expression studies, two human cell lines, Jurkat T-cells and K562 erythroleukemia cells, were transduced with lentivirus particles encoding for Saf/ GFP or GFP alone Total RNA was isolated from the resulting GFP+ cells and genome wide expression analyses performed on HG-U133_Plus2 cartridges Saf over expression had no effect on global gene transcription, supporting a post-transcriptional role To assess localization, total RNA was extracted from the nuclear and cytoplasmic fractions of MCF7 cells that endogenously express Saf RT-PCR analysis revealed that Saf was localized in the nucleus and fraction purity verified with the nuclear-retained 47S pre-rRNA Saf associated proteins were isolated by RNA pulldown of in vitro transcribed Saf RNAs (-/+ Biotin-UTP) that were mixed with K562 nuclear lysates and recovered using anti-streptavidin beads Mass spectrometry analysis identified eight partners unique to biotinlabeled Saf One protein with highest counts, human splicing factor (SPF45) also called RNA-binding motif protein 17 (RBM17), was confirmed to interact with Saf by RNA co-immunoprecipation using SPF45-specific antibodies in nuclear lysates prepared from K562 cells with stable expression of Saf and RT-PCR Specific interaction was demonstrated by the failure to detect Saf using either control IgG or GATA-1 antibodies SPF45 regulates alternative splicing of FasR pre-mRNA by inducing exon skipping to generate sFas proteins To monitor this molecular event in maturing erythroid cells, we developed a qRT-PCR assay to indirectly measure this isoform (FasRExon6) as a ratio of total FasR mRNA levels and detected a 2.5-fold increase in these sFas-encoding transcripts in late stage adult BM-derived erythroblasts Regulation of apoptosis is essential for erythropoiesis and many other developmental processes Our finding that Saf participates in FasR alternative splicing through direct interaction with SPF45 reveals a novel layer of modulation of the cell death program that is required for the proper generation of mature red blood cells 236 Stable and Selective Production of Therapeutic Protein in the Breastmilk of NonTransgenic Mice Via Early Gestational IntraAmniotic Gene Transfer Jesse D Vrecenak,1 Miroslaw Kozlowski,1 Carlyn A Todorow,1 Antoneta Radu,1 Alan W Flake.1 Center for Fetal Research, Children’s Hospital of Philadelphia, Philadephia, PA Fetal gene transfer offers unique potential to achieve sustained transgene expression within rapidly cycling cell populations through transduction of tissue-specific stem cells Due to its hormonally responsive nature, efforts at transduction of the mammary epithelium in non-transgenic animals have allowed only short-term expression We have previously demonstrated the ability to selectively transduce a mammary epithelial stem cell population through early gestational intra-amniotic injection of targeted lentiviral vector In addition to the obvious clinical applications of selective gene transfer, this technique offers intriguing potential as a novel method for the production of therapeutic proteins Because our technique allows stable transgene expression in the mammary epithelium throughout the life of the animal, we hypothesized that engineered production of human therapeutic proteins could be achieved in breastmilk following early gestational intra-amniotic injection of lentiviral vector encoding Molecular Therapy Volume 21, Supplement 1, May 2013 Copyright © The American Society of Gene & Cell Therapy for a secreted human protein under the control of the mammaryspecific Mouse Mammary Tumor Virus (MMTV) promoter To test this hypothesis, we used the human factor IX protein to facilitate detection in protein-rich mouse breastmilk A lentiviral vector was constructed using the entire MMTV LTR truncated by 25 bp at the 3’ end[1] (courtesy Philip Leder) encoding for GFP and human factor IX Vector was injected into the amniotic cavity (0.35 uL at 1.2 x 10^8 pfu/mL) of the E8-9 Balb/c fetus under ultrasound guidance Surviving female pups were mated with Balb/c males and manually milked following intraperitoneal oxytocin injection (2 IU) Breastmilk was analyzed for the presence of human factor IX protein using ELISA with no mouse cross-reactivity Based upon our vector screen results, our MMTV promoter shows strong GFP expression in both ducts and glands by fluorescence stereomicroscopy, with very low level and sporadic expression in some cortical elements of the brain and no expression in any other tissue, including those of epithelial lineage ELISA demonstrates significant production of human factor IX protein in the breastmilk of injected animals, on the order of 250 ng/mL through months of age The sustained secretion of a human protein in the breastmilk of injected animals offers a novel method for the production of therapeutic proteins with important biotechnology implications Among the challenges inherent in current methods are the complicated folding patterns necessary for the function of certain proteins For such targets, in vitro methods may not be sufficient to recapitulate the required structure, making in vivo methods far superior Depending on the nature of the target, the development of transgenics may be financially or biologically prohibitive Our method offers a novel alternative to this costly and difficult process, allowing scaleable and sustained production of therapeutic proteins in breastmilk Muller, W.J., et al., Single-step induction of mammary adenocarcinoma in transgenic mice bearing the activated c-neu oncogene Cell, 1988 54(1): p 105-15 237 Improved Engraftment of Lentivirally Transduced Adipose Tissue-Derived Stem Cells for Hemophilia B Therapy Using Bioengineered Cell Sheet Technology Inkyong Shim,1 Kazuo Ohashi,1 Kohei Tatsumi,1 Natsumi Watanabe,1 Yuji Kashiwakura,2 Tsukasa Ohmori,2 Yoichi Sakata,2 Makoto Inoue,3 Mamoru Hasegawa,3 Teruo Okano.1 Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University (TWIns), Tokyo, Japan; 2Research Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical University School of Medicine, Tochigi, Japan; 3DNAVEC Corporation, Ibaraki, Japan Hemophilia B is an inherited bleeding disorder caused by the deficiency of Factor IX (FIX), a functional clotting factor Gene transduced cells are considered to be a useful tool in establishing cell-based gene therapies for diseases with monogenic plasma protein deficiencies, while remaining need of improving survival of transplanted cells in vivo Patient-derived adipose tissue-derived stem/stromal cells (ADSCs) is an attractive cell sources This study explored the potential of ADSCs (mADSCs) for human FIX (hFIX) gene transduction with a self-inactivating lentivirus simian immunodeficiency virus (SIV) vector in vitro and their ability to produce and secrete biologically active hFIX in vivo The optimal SIV-vector dose for mADSCs transduction was determined by their transduction efficiency and the production level of hFIX Although the hFIX production levels into culture medium showed a slight decrease in the first week, sustained production levels were observed for the duration of the study (up to weeks) Clotting assay confirmed that the hFIX produced by the ADSCs was biologically active clotting factor For establishing a tissue engineering-based treatment modality with hFIX-transduced ADSCs, we created a contiguous cell sheet format of the hFIX-transduced ADSCs based on cell sheet engineering S91 ... GATA-1 antibodies SPF45 regulates alternative splicing of FasR pre-mRNA by inducing exon skipping to generate sFas proteins To monitor this molecular event in maturing erythroid cells, we developed... partners unique to biotinlabeled Saf One protein with highest counts, human splicing factor (SPF45) also called RNA-binding motif protein 17 (RBM17), was confirmed to interact with Saf by RNA co-immunoprecipation... apoptosis The long noncoding RNA (lncRNA) Saf was reported to participate in this process LncRNAs are >200 nucleotide transcripts that form complexes with various RNA-binding proteins, including