Vpx-Independent Lentiviral Transduction and shRNA-Mediated Protein Knock-Down in Monocyte-Derived Dendritic Cells

Abstract

The function of dendritic cells (DCs) in the immune system is based on their ability to sense and present foreign antigens. Powerful tools to research DC function and to apply in cell-based immunotherapy are either silencing or overexpression of genes achieved by lentiviral transduction. To date, efficient lentiviral transduction of DCs or their monocyte derived counterparts (MDDCs) required high multiplicity of infection (MOI) or the exposure to the HIV-2/SIV protein Vpx to degrade viral restriction factor SAM domain and HD domain-containing protein 1 (SAMHD1). Here we present a Vpx-independent method for efficient (>95%) transduction of MDDCs at lower MOI. The protocol can be used both for ectopic gene expression and knock-down. Introducing shRNA targeting viral entry receptor CD4 and restriction factor SAMHD1 into MDDCs resulted in down-regulation of targeted proteins and, consequently, expected impact on HIV infection. This protocol for MDDCs transduction is robust and free of the potential risk arising from the use of Vpx which creates a virus infection-prone environment, potentially dangerous in clinical setting.

Cas9-mediated knockout of Ndrg2 enhances the regenerative potential of dendritic cells for wound healing

“…Hence, previous studies have reported relatively low transduction efficiencies (<40%) or required very high vector doses, which can induce undesired DC maturation or even cell toxicity 65 . Approaches to induce proteasomal degradation of SAMHD1 using Vpx or shRNA-mediated down-regulating of SAMHD1 to improve transduction efficiency have been described; however, these techniques are not suitable for the development of clinical-grade DC therapies since they increase the risk for cell infection with viruses and induce a cytotoxic T-cell response 66 , 67 .…”

Cas9-Mediated Knockout of Ndrg2 Enhances the Regenerative Potential of Dendritic Cells for Wound Healing

“…60 Approaches to induce proteasomal degradation of SAMHD1 using Vpx or shRNAmediated down-regulating of SAMHD1 to improve transduction efficiency have been described; however these techniques are not suitable for the development of clinical-grade DC therapies, since they increase the risk for cell infection with viruses and induce a cytotoxic T cell response. 61,62 Recently, Cas9 RNP-based gene editing of CAR T cells have been implicated for enhanced solid tumor killing. 22 Furthermore, despite being early in the clinical test stage, Cas9 RNPediting of murine primary DCs has been demonstrated to be an effective approach leading to high KO rates for several target genes, while preserving cell viability and in vitro functional characteristics.…”

“…60 Approaches to induce proteasomal degradation of SAMHD1 using Vpx or shRNA-mediated down-regulating of SAMHD1 to improve transduction efficiency have been described; however these techniques are not suitable for the development of clinical-grade DC therapies, since they increase the risk for cell infection with viruses and induce a cytotoxic T cell response. 61,62…”