Lentiviral Transduction of CD34+ Cells Induces Genome-Wide Epigenetic Modifications

Yoshiaki Yamagata,Nizar Touleimat,András Paldi,Guillaume Corre,Céline Besse,Anne Galy,Daniel Stockholm,Jörg Tost,Véronique Parietti,Catherine Poinsignon,Damien Delafoy

doi:10.1371/journal.pone.0048943

Abstract

Epigenetic modifications may occur during in vitro manipulations of stem cells but these effects have remained unexplored in the context of cell and gene therapy protocols. In an experimental model of ex vivo gene modification for hematopoietic gene therapy, human CD34+ cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34+ cells to cytokines increased nuclear expression of epigenetic regulators SIRT1 or DNMT1 and caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34+ cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols.

Highlights

The in vitro manipulation of stem cells or embryos is successfully employed in various fields of experimental biology and medicine
The optimization of the hematopoietic stem cells (HSC) gene modification process is a central issue in gene therapy, the question of epigenetic consequences due to the ex vivo culture including the use of the vector, has never been directly explored
Characterization of the Experimental Groups A model of ex vivo gene therapy has been developed using umbilical cord blood CD34+ cells transduced with a lentiviral vector (LV) encoding GFP

Summary

Introduction

The in vitro manipulation of stem cells or embryos is successfully employed in various fields of experimental biology and medicine. Ex vivo gene-corrected autologous hematopoietic stem cells (HSC) are currently tested in early-phase trials to treat severe genetic diseases (for a review see [6]) This approach consists in stable gene modification of the patient’s autologous HSC during a short ex vivo culture in the presence of early-acting hematopoietic cytokines. To be successful, this process must preserve the biological function of gene-corrected HSC such as their engrafting capacity, long-term self-renewal or multi-lineage potency. It is a basic requirement that the process of HSC gene modification during ex vivo culture protocols should avoid cell differentiation and minimally impact on the normal epigenetic regulation and methylation profile of the cells. The optimization of the HSC gene modification process is a central issue in gene therapy, the question of epigenetic consequences due to the ex vivo culture including the use of the vector, has never been directly explored

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