title: Lentivirus vector driven by polybiquitin C promoter without woodchuck posttranscriptional regulatory element and central polypurine tract generates low level and short-lived reporter gene expression.
abstract: Lentivirus (LV) encoding woodchuck posttranscriptional regulatory element (WPRE) and central polypurine tract (cPPT) driven by CMV promoter have been proven to act synergistically to increase both transduction efficiency and gene expression. However, the inclusion of WPRE and cPPT in a lentiviral construct may pose safety risks when administered to human. A simple lentiviral construct driven by an alternative promoter with proven extended duration of gene expression without the two regulatory elements would be free from the risks. In a non-viral gene delivery context, gene expression driven by human polybiquitin C (UbC) promoter resulted in higher and more persistent expression in mouse as compared to cytomegalovirus (CMV) promoter. In this study, we measured the efficiency and persistency of green fluorescent protein (GFP) reporter gene expression in cells transduced with LV driven by UbC (LV/UbC/GFP) devoid of the WPRE and cPPT in comparison to the established LV construct encoding WPRE and cPPT, driven by CMV promoter (LV/CMV/GFP). However, we found that LV/UbC/GFP was inferior to LV/CMV/GFP in many aspects: (i) the titer of virus produced; (ii) the levels of reporter gene expression when MOI value was standardized; and (iii) the transduction efficiency in different cell types. The duration of reporter gene expression in selected cell lines was also determined. While the GFP expression in cells transduced with LV/CMV/GFP persisted throughout the experimental period of 14 days, expression in cells transduced with LV/UbC/GFP declined by day 2 post-transduction. In summary, the LV driven by the UbC promoter without the WPRE and cPPT does not exhibit enhanced or durable transgene expression.
authors.affiliation: Medical Genetics Laboratory, Clinical Genetics Unit, Faculty of Medicine & Health Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. siewching_ngai@imu.edu.my
authors.forename: Siew Ching
authors.initials: SC
authors.lastname: Ngai
authors.forename: Rozita
authors.initials: R
authors.lastname: Rosli
authors.forename: Norshariza
authors.initials: N
authors.lastname: Nordin
authors.forename: Abhi
authors.initials: A
authors.lastname: Veerakumarasivam
authors.forename: Syahril
authors.initials: S
authors.lastname: Abdullah
mesh_terms: D000818:Animals; D002460:Cell Line / Q000821:virology; D003587:Cytomegalovirus / Q000235:genetics; D015870:Gene Expression; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D025821:Polyubiquitin / Q000235:genetics; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D013997:Time Factors; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes
chemical_list: D025821:Polyubiquitin; D049452:Green Fluorescent Proteins