Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs

• Authors: Duo Li, University of Zurich; Erika Schlaepfer, University of Zurich; Annette Audigé, University of Zurich; Mary-Aude Rochat, University of Zurich; Sandra Ivic, University of Zurich; Caitlin N. Knowlton, Emory University; Baek Kim, Emory University; Oliver T. Keppler, University of Frankfurt; Roberto F. Speck, University of Zurich
• Journal Title: Stem Cell Research and Therapy
• Volume: Volume 15, Number 2
• Publisher: BioMed Central | 2015-09-01, Pages 271-280
• Type of Work: Article | Final Publisher PDF
• Publisher DOI: http://doi.org/10.1016/j.scr.2015.06.012
• Subjects: Biology, Cell; Biology, Microbiology; Engineering, Biomedical
• Keywords: Science & Technology; Life Sciences & Biomedicine; Cell & Tissue Engineering; Biotechnology & Applied Microbiology; Cell Biology; SAMHD1; dNTP pools; Transduction; Hematopoietic stem and progenitor cells (HSPCs); Gene engineering; Lentiviral vectors; CD4(+) T-CELLS; GENE-TRANSFER; HIV-1 RESTRICTION; LYMPHOID-TISSUE; IN-VIVO; INFECTION; EXPRESSION; VECTORS; PHOSPHORYLATION; MACROPHAGES

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.