DNA methylation and histone modifications are the molecular lock in lentivirally transduced hematopoietic progenitor cells.

Siew Ching Ngai,Rozita Rosli,Akram Al Abbar,Syahril Abdullah

doi:10.1155/2015/346134

Abstract

Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV.

Highlights

Gene therapy is the introduction of therapeutic genes into target cells to treat a disease or medical disorder [1]
Studies on epigenetic effects on transgene expression have been performed on different cell types [4,5,6,7], none have been conducted on Hematopoietic progenitor cells (HPCs) transduced by lentiviral vector (LV)
We believe that the transgene silencing was caused by certain silencing machineries and not due to the loss of proviral DNA. This is consistent with the results shown by He et al [4], in which the decrease of transgene expression by LV at the very early stage after gene transfer was due to transcriptional silencing in murine embryonic carcinoma P19 cells and not due to the deletion of the transgene

Summary

Introduction

Gene therapy is the introduction of therapeutic genes into target cells to treat a disease or medical disorder [1]. Successful gene therapy requires specific, efficient, stable, and high levels of gene transfer into the target cells in order to achieve the therapeutic effects. Lentiviral vectors (LVs) are promising tools in gene therapy for hematological diseases because they can efficiently transduce HPCs, which are quiescent and difficult to target [3]. Epigenetic effects were found as the confounding factor for the transgene expression decline in several cell types [4,5,6,7]. Studies on epigenetic effects on transgene expression have been performed on different cell types [4,5,6,7], none have been conducted on HPCs transduced by LV

Methods

Results

Conclusion