Potent and reversible lentiviral vector restriction in murine induced pluripotent stem cells

Abstract

BackgroundRetroviral vectors are derived from wild-type retroviruses, can be used to study retrovirus-host interactions and are effective tools in gene and cell therapy. However, numerous cell types are resistant or less permissive to retrovirus infection due to the presence of active defense mechanisms, or the absence of important cellular host co-factors. In contrast to multipotent stem cells, pluripotent stem cells (PSC) have potential to differentiate into all three germ layers. Much remains to be elucidated in the field of anti-viral immunity in stem cells, especially in PSC.ResultsIn this study, we report that transduction with HIV-1-based, lentiviral vectors (LV) is impaired in murine PSC. Analyses of early retroviral events in induced pluripotent stem cells (iPSC) revealed that the restriction is independent of envelope choice and does not affect reverse transcription, but perturbs nuclear entry and proviral integration. Proteasomal inhibition by MG132 could not circumvent the restriction. However, prevention of cyclophilin A (CypA) binding to the HIV-1 capsid via use of either a CypA inhibitor (cyclosporine A) or CypA-independent capsid mutants improved transduction. In addition, application of higher vector doses also increased transduction. Our data revealed a CypA mediated restriction in iPSC, which was acquired during reprogramming, associated with pluripotency and relieved upon subsequent differentiation.ConclusionsWe showed that murine PSC and iPSC are less susceptible to LV. The block observed in iPSC was CypA-dependent and resulted in reduced nuclear entry of viral DNA and proviral integration. Our study helps to improve transduction of murine pluripotent cells with HIV-1-based vectors and contributes to our understanding of retrovirus-host interactions in PSC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-017-0358-1) contains supplementary material, which is available to authorized users.

The Ubiquitin Proteasome System Is a Key Regulator of Pluripotent Stem Cell Survival and Motor Neuron Differentiation

“…Pluripotent stem cells were exquisitely sensitive to proteasome inhibition by MG132, with a TC 50 in the nanomolar range. UPS inhibition has previously been reported to have a cytotoxic effect in mouse embryonic stem cells, with doses of 200 nM inducing cytotoxic effects [52]. Together these data highlight the importance of the UPS in pluripotent stem cell survival.…”

CRISPR/Cas9 Immunoengineering of Hoxb8-Immortalized Progenitor Cells for Revealing CCR7-Mediated Dendritic Cell Signaling and Migration Mechanisms in vivo

“…To test this, we transduced dTom + Ccr7 −/− Cas9-Hoxb8 cells with a lentivirus expressing Cerulean and a sgRNA targeting Cxcr4 . This second round of transduction was done in the presence of cyclosporine A, as this has been demonstrated to enhance the transduction rate by overcoming a restriction against lentiviruses ( 47 , 48 ). Transduced cells as well as control Cas9-Hoxb8 cells were subsequently differentiated into mature DCs.…”

Cyclosporine H Overcomes Innate Immune Restrictions to Improve Lentiviral Transduction and Gene Editing In Human Hematopoietic Stem Cells

“…Our findings are of particular relevance in gene therapy settings in which high levels of gene marking are required but difficult to achieve, such as for hemoglobinopathies, where the large and complex human β-globin gene expression cassette limits clinical-scale LV production (Baldwin et al., 2015). CsH utility may extend to other stem cells populations (Geis et al., 2017, Noser et al., 2006). Moreover, CsH helps the IDLV-based editing platforms compete with other non-integrating methods based on AAV or oligonucleotide-mediated donor DNA delivery (De Ravin et al., 2017, Dever et al., 2016, Schiroli et al., 2017, Wang et al., 2015), in particular for patients harboring pre-existing adaptive immunity against some AAV serotypes that may lead to immune recognition of vector and transduced cells (Boutin et al., 2010).…”