title: Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs.
abstract: Understanding how to achieve efficient transduction of hematopoietic stem and progenitor cells (HSPCs), while preserving their long-term ability to self-reproduce, is key for applying lentiviral-based gene engineering methods. SAMHD1 is an HIV-1 restriction factor in myeloid and resting CD4+ T cells that interferes with reverse transcription by decreasing the nucleotide pools or by its RNase activity. Here we show that SAMHD1 is expressed at high levels in HSPCs cultured in a medium enriched with cytokines. Thus, we hypothesized that degrading SAMHD1 in HSPCs would result in more efficient lentiviral transduction rates. We used viral like particles (VLPs) containing Vpx, shRNA against SAMHD1, or provided an excess of dNTPs or dNs to study this question. Regardless of the method applied, we saw no increase in the lentiviral transduction rate. The result was different when we used viruses (HR-GFP-Vpx+) which carry Vpx and encode GFP. These viruses allow assessment of the effects of Vpx specifically in the transduced cells. Using HR-GFP-Vpx+ viruses, we observed a modest but significant increase in the transduction efficiency. These data suggest that SAMHD1 has some limited efficacy in blocking reverse transcription but the major barrier for efficient lentiviral transduction occurs before reverse transcription.
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, 8091, Switzerland. Electronic address: duo.li@usz.ch.
authors.forename: Duo
authors.initials: D
authors.lastname: Li
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, 8091, Switzerland.
authors.forename: Erika
authors.initials: E
authors.lastname: Schlaepfer
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, 8091, Switzerland.
authors.forename: Annette
authors.initials: A
authors.lastname: Audigé
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, 8091, Switzerland.
authors.forename: Mary-Aude
authors.initials: MA
authors.lastname: Rochat
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, 8091, Switzerland.
authors.forename: Sandra
authors.initials: S
authors.lastname: Ivic
authors.affiliation: Center for Drug Discovery, Department of Pediatrics, Emory University, Atlanta, GA 30322, USA.
authors.forename: Caitlin N
authors.initials: CN
authors.lastname: Knowlton
authors.affiliation: Center for Drug Discovery, Department of Pediatrics, Emory University, Atlanta, GA 30322, USA.
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
authors.affiliation: Institute of Medical Virology, National Reference Center for Retroviruses, University of Frankfurt, Frankfurt am Main 60054, Germany.
authors.forename: Oliver T
authors.initials: OT
authors.lastname: Keppler
authors.affiliation: Division of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, 8091, Switzerland. Electronic address: Roberto.speck@usz.ch.
authors.forename: Roberto F
authors.initials: RF
authors.lastname: Speck
mesh_terms: D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D020559:Monomeric GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D000076106:SAM Domain and HD Domain-Containing Protein 1; D054334:Viral Regulatory and Accessory Proteins / Q000235:genetics
chemical_list: D016207:Cytokines; D034741:RNA, Small Interfering; C074286:VPX protein, Human immunodeficiency virus 2; D054334:Viral Regulatory and Accessory Proteins; D000076106:SAM Domain and HD Domain-Containing Protein 1; C484280:SAMHD1 protein, human; D020559:Monomeric GTP-Binding Proteins
keywords: Gene engineering; Hematopoietic stem and progenitor cells (HSPCs); Lentiviral vectors; SAMHD1; Transduction; dNTP pools