title: Improvement of De Novo Cholesterol Biosynthesis Efficiently Promotes the Production of Human Immunodeficiency Virus Type 1-Derived Lentiviral Vectors.
abstract: The use of lentiviral vectors (LVs) for gene transfer in research, technological, or clinical applications requires the production of large amounts of vector. Mass production of clinical-grade LVs remains a challenge and limits certain perspectives for therapeutic use. Some improvements in LV production protocols have been possible by acting on multiple steps of the production process. The addition of animal-derived cholesterol to the culture medium of producer cells is known to increase the infectivity of LVs. To avoid the use of this animal-derived product in clinical settings, an alternative approach is to increase de novo the production of cholesterol by overexpressing a crucial cholesterogenic enzyme, namely, 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). This project evaluates the impact of such an approach on the production, infectivity, and stability of LVs. We demonstrated that the overexpression of human HMGCR isoform 1 (hHMGCR1) in LV producer cells efficiently increased de novo cholesterol biosynthesis and enhanced by 2- to 3-fold the physical and infectious titers of LVs. We also observed that LVs produced in hHMGCR1-overexpressing cells were comparable in stability to LVs produced under classical conditions and were capable of transducing human CD34+hematopoietic stem/progenitor cells efficiently. Interestingly, we also showed that LV production in the absence of fetal calf serum (FCS) but under hHMGCR1-overexpressing conditions allowed a viral production yield comparable to that achieved under classical conditions in high FCS content, leading the way to the establishment of new LV production protocols on adherent cells without serum.
authors.affiliation: 1 Généthon , Evry, France .|2 INSERM , UMR_S951, Généthon, Evry, France .|3 Université Evry Val d'Essonne , UMR_S951, Evry, France .
authors.forename: Nathalie
authors.initials: N
authors.lastname: Holic
authors.affiliation: 1 Généthon , Evry, France .|2 INSERM , UMR_S951, Généthon, Evry, France .
authors.forename: Sophie
authors.initials: S
authors.lastname: Frin
authors.affiliation: 1 Généthon , Evry, France .|2 INSERM , UMR_S951, Généthon, Evry, France .
authors.forename: Ababacar K
authors.initials: AK
authors.lastname: Seye
authors.affiliation: 1 Généthon , Evry, France .|2 INSERM , UMR_S951, Généthon, Evry, France .|3 Université Evry Val d'Essonne , UMR_S951, Evry, France .
authors.forename: Anne
authors.initials: A
authors.lastname: Galy
authors.affiliation: 1 Généthon , Evry, France .|2 INSERM , UMR_S951, Généthon, Evry, France .|3 Université Evry Val d'Essonne , UMR_S951, Evry, France .
authors.forename: David
authors.initials: D
authors.lastname: Fenard
mesh_terms: D000214:Acyl Coenzyme A / Q000235:genetics; D000818:Animals; D018952:Antigens, CD34 / Q000235:genetics; D002784:Cholesterol / Q000096:biosynthesis* / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics* / Q000627:therapeutic use; D015497:HIV-1 / Q000235:genetics*; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D014161:Transduction, Genetic
chemical_list: D000214:Acyl Coenzyme A; D018952:Antigens, CD34; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D002784:Cholesterol