title: Long-term reproducible expression in human fetal liver hematopoietic stem cells with a UCOE-based lentiviral vector.
abstract: Hematopoietic Stem Cell (HSC) targeted gene transfer is an attractive treatment option for a number of hematopoietic disorders caused by single gene defects. However, extensive methylation of promoter sequences results in silencing of therapeutic gene expression. The choice of an appropriate promoter is therefore crucial for reproducible, stable and long-term transgene expression in clinical gene therapy. Recent studies suggest efficient and stable expression of transgenes from the ubiquitous chromatin opening element (UCOE) derived from the human HNRPA2B1-CBX3 locus can be achieved in murine HSC. Here, we compared the use of HNRPA2B1-CBX3 UCOE (A2UCOE)-mediated transgene regulation to two other frequently used promoters namely EF1α and PGK in human fetal liver-derived HSC (hflHSC). Efficient transduction of hflHSC with a lentiviral vector containing an HNRPA2B1-CBX3 UCOE-eGFP (A2UCOE-eGFP) cassette was achieved at higher levels than that obtained with umbilical cord blood derived HSC (3.1x; p<0.001). While hflHSC were readily transduced with all three test vectors (A2UCOE-eGFP, PGK-eGFP and EF1α-eGFP), only the A2-UCOE construct demonstrated sustained transgene expression in vitro over 24 days (p<0.001). In contrast, within 10 days in culture a rapid decline in transgene expression in both PGK-eGFP and EF1α-eGFP transduced hflHSC was seen. Subsequently, injection of transduced cells into immunodeficient mice (NOD/SCID/Il2rg-/-) demonstrated sustained eGFP expression for the A2UCOE-eGFP group up to 10 months post transplantation whereas PGK-eGFP and EF1α-eGFP transduced hflHSC showed a 5.1 and 22.2 fold reduction respectively over the same time period. We conclude that the A2UCOE allows a more efficient and stable expression in hflHSC to be achieved than either the PGK or EF1α promoters and at lower vector copy number per cell.
authors.affiliation: Experimental Fetal Medicine Group, Department of Obstetrics and Gynecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Niraja
authors.initials: N
authors.lastname: Dighe
authors.affiliation: Interdisciplinary Research Group in Infectious Diseases, Singapore-Massachusetts Institute of Technology Alliance for Research and Technology, Singapore, Singapore.
authors.forename: Maroun
authors.initials: M
authors.lastname: Khoury
authors.affiliation: Experimental Fetal Medicine Group, Department of Obstetrics and Gynecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Citra
authors.initials: C
authors.lastname: Mattar
authors.affiliation: Experimental Fetal Medicine Group, Department of Obstetrics and Gynecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; Division of Bioengineering, School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore.
authors.forename: Mark
authors.initials: M
authors.lastname: Chong
authors.affiliation: Experimental Fetal Medicine Group, Department of Obstetrics and Gynecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
authors.forename: Mahesh
authors.initials: M
authors.lastname: Choolani
authors.affiliation: Interdisciplinary Research Group in Infectious Diseases, Singapore-Massachusetts Institute of Technology Alliance for Research and Technology, Singapore, Singapore; Koch Institute for Integrative Cancer Research and Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts, United States of America.
authors.forename: Jianzhu
authors.initials: J
authors.lastname: Chen
authors.affiliation: Department of Medical and Molecular Genetics, King's College London School of Medicine, Guys Hospital, London, United Kingdom.
authors.forename: Michael N
authors.initials: MN
authors.lastname: Antoniou
authors.affiliation: Experimental Fetal Medicine Group, Department of Obstetrics and Gynecology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; Department of Reproductive Medicine, KK Women's and Children's Hospital, Singapore, Singapore; Cancer and Stem Cell Program, Duke-NUS Graduate Medical School, Singapore, Singapore.
authors.forename: Jerry K Y
authors.initials: JK
authors.lastname: Chan
mesh_terms: D000818:Animals; D019175:DNA Methylation / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008099:Liver / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D058990:Molecular Targeted Therapy / Q000379:methods*; D010735:Phosphoglycerate Kinase / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D019076:Transgenes / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; C439768:hnRNP A2; D010735:Phosphoglycerate Kinase