Deletion of the LTR Enhancer/Promoter Has No Impact on the Integration Profile of MLV Vectors in Human Hematopoietic Progenitors

Arianna Moiani,Fulvio Mavilio,Julia Debora Suerth,Annarita Miccio,Danilo Pellin,Gianluca De Bellis,Christopher Baum,Ermanno Rizzi,Marco Severgnini,Yasuhiro Ikeda

doi:10.1371/journal.pone.0055721

Abstract

Moloney murine leukemia virus (MLV)-derived gamma-retroviral vectors integrate preferentially near transcriptional regulatory regions in the human genome, and are associated with a significant risk of insertional gene deregulation. Self-inactivating (SIN) vectors carry a deletion of the U3 enhancer and promoter in the long terminal repeat (LTR), and show reduced genotoxicity in pre-clinical assays. We report a high-definition analysis of the integration preferences of a SIN MLV vector compared to a wild-type-LTR MLV vector in the genome of CD34+ human hematopoietic stem/progenitor cells (HSPCs). We sequenced 13,011 unique SIN-MLV integration sites and compared them to 32,574 previously generated MLV sites in human HSPCs. The SIN-MLV vector recapitulates the integration pattern observed for MLV, with the characteristic clustering of integrations around enhancer and promoter regions associated to H3K4me3 and H3K4me1 histone modifications, specialized chromatin configurations (presence of the H2A.Z histone variant) and binding of RNA Pol II. SIN-MLV and MLV integration clusters and hot spots overlap in most cases and are generated at a comparable frequency, indicating that the reduced genotoxicity of SIN-MLV vectors in hematopoietic cells is not due to a modified integration profile.

Highlights

Retroviral integration is a non-random process, whereby the viral RNA genome, reverse transcribed into double-stranded DNA and assembled in pre-integration complexes (PICs), associates with the host cell chromatin and integrates through the activity of the viral integrase [1]
To identify differences in the integration preferences of murine leukemia virus (MLV) and SIN-MLV in hematopoietic stem/progenitor cells (HSPCs), we first analyzed the relationship between integration sites and Known Genes (UCSC definition) in the human genome: integration were annotated as TSS-proximal when occurring in an interval of 62.5 kb from the TSS of any Known Gene, intragenic when occurring inside a Known Gene .2.5 kb from the TSS, and intergenic in all other cases
Retroviruses select their target integration sites by tethering their PICs to the host cell chromatin through protein-protein interactions that appear to be specific for each retrovirus type [2]

Summary

Introduction

Retroviral integration is a non-random process, whereby the viral RNA genome, reverse transcribed into double-stranded DNA and assembled in pre-integration complexes (PICs), associates with the host cell chromatin and integrates through the activity of the viral integrase [1]. Recent clinical studies have shown that transplantation of stem cells genetically modified by retroviral vectors may cure severe genetic diseases such as immunodeficiencies [6,7,8], skin adhesion defects [9] and lysosomal storage disorders [10]. Some of these studies showed the genotoxic consequences of retroviral gene transfer technology. The MLV U3 enhancer contains repeated binding sites for cellular transcription factors (TF), which may play a role in tethering retroviral pre-integration complexes to transcriptionally active regulatory regions and contribute to the MLV genotoxic characteristics [20]

Methods

Results

Conclusion