title: Comparison of HIV-1 and EIAV-based lentiviral vectors in corneal transduction.
abstract: In this study we compare the ability of self-inactivating Human Immunodeficiency Virus 1 (HIV-1) and Equine Infectious Anaemia Virus (EIAV)-based vectors to mediate gene transfer to rabbit and human corneas and to a murine corneal endothelial cell line. Both vectors were pseudotyped with vesicular stomatitis virus-G (VSV-G) envelope and contained marker transgenes under the control of an internal CMV promoter. For specificity of action, the heterologous promoter in the EIAV-vector was exchanged for an inducible E-Selectin promoter, previously shown to regulate gene-expression in a plasmid system. We show that EIAV is more efficient than HIV in transducing human and rabbit corneal endothelial cells. Rabbit corneal endothelial cells are transduced in higher quantity than human corneal endothelial cells. In the inducible system, however, we detected impairment between the vector and its internal E-Selectin promoter. Instead of controlled transgene expression or silencing of promoter activity, the U3-modified long-terminal-repeats (LTR) impaired the conditional activity of the E-Selectin promoter. Significant transgene expression was seen without stimulation of the inducible promoter. We show efficient transduction by lentiviruses of a corneal endothelial cell line and of full thickness corneas from different species, confirming that those vectors would be appropriate tools for gene therapy of selected corneal diseases. However, the modification within the U3-LTR did not adequately allow regulated transgene expression. These findings have important implications for vector design for diagnostic or therapeutic opportunities.
authors.affiliation: Jefferiss Research Trust Laboratories, Wright-Fleming Institute, Faculty of Medicine, Division of Medicine, Department of GU Medicine, St Mary's Campus Medicine, Imperial College London, Norfolk Place, London W2 1PG, UK. s.beutelspacher@imperial.ac.uk
authors.forename: Sven Christoph
authors.initials: SC
authors.lastname: Beutelspacher
authors.forename: Navid
authors.initials: N
authors.lastname: Ardjomand
authors.forename: Peng Hong
authors.initials: PH
authors.lastname: Tan
authors.forename: Gillian Sarah
authors.initials: GS
authors.lastname: Patton
authors.forename: D Frank P
authors.initials: DF
authors.lastname: Larkin
authors.forename: Andrew J T
authors.initials: AJ
authors.lastname: George
authors.forename: Myra O
authors.initials: MO
authors.lastname: McClure
mesh_terms: D000818:Animals; D002460:Cell Line; D003315:Cornea / Q000502:physiology*; D019040:E-Selectin / Q000235:genetics; D004728:Endothelium, Corneal / Q000502:physiology; D005434:Flow Cytometry / Q000379:methods; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006801:Humans; D004860:Infectious Anemia Virus, Equine / Q000235:genetics*; D008081:Liposomes; D051379:Mice; D008807:Mice, Inbred BALB C; D011401:Promoter Regions, Genetic / Q000235:genetics; D011817:Rabbits; D014161:Transduction, Genetic / Q000379:methods; D014162:Transfection / Q000379:methods; D019076:Transgenes / Q000235:genetics
chemical_list: D019040:E-Selectin; D008081:Liposomes; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins