Elsevier

Stem Cell Research

Volume 15, Issue 2, September 2015, Pages 271-280
Stem Cell Research

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

https://doi.org/10.1016/j.scr.2015.06.012Get rights and content
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Highlights

  • SAMHD1 is highly expressed in HSPCs already at 2 h culturing in a medium enriched with cytokines conventionally used for transduction of HSPCs.

  • Expression levels of SAMHD1 in cultured HSPCs are comparable to those found in MDMs.

  • HSPCs exposed to HR-GFP-Vpx + showed significant increase of GFP+ cells which was associated with a decrease in SAMHD1 expression.

  • GFP+ cells were detected mostly within the population of cells containing low amounts of SAMHD1.

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.

Abbreviations

HSPCs
hematopoietic stem and progenitor cells
VLPs
viral like particles
IL-2R
interleukin-2 receptor
SIN
self-inactivating
SAMHD1
sterile alpha motif domain and HD domain-containing protein 1
dGTP
deoxyguanosine-triphosphate
dNTPs
deoxynucleoside triphosphates
dNs
deoxynucleosides
DCs
dendritic cells
PBMCs
peripheral blood mononuclear cells
MDMs
monocyte-derived macrophages
SCF
stem cell factor
Flt3L
Fms-related tyrosine kinase 3 ligand
IL-3
interleukin-3
TPO
thrombopoietin
PEI
polyethylenimine
GAPDH
glyceraldehyde 3-phosphate dehydrogenase
MNE
mean normalized gene expression
EVF
efavirenz
RTV
raltegravir
TGV
total gray values
MFI
mean fluorescence intensity

Keywords

SAMHD1
dNTP pools
Transduction
Hematopoietic stem and progenitor cells (HSPCs)
Gene engineering
Lentiviral vectors

Cited by (0)

Author contribution: DL designed, conducted and analyzed all experiments. ES, AA, M-A.R, SI, C-N.K, BK, O-T.K assisted in some experiment. DL, AA and RS wrote the paper.