Integration profile of retroviral vector in gene therapy treated patients is cell‐specific according to gene expression and chromatin conformation of target cell

Luca Biasco,Clelia Di Serio,Christof Von Kalle,Alessandro Ambrosi,Maria Grazia Roncarolo,Danilo Pellin,Manfred Schmidt,Alessandro Aiuti,Cynthia Bartholomae,Immacolata Brigida

doi:10.1002/emmm.201000108

Abstract

The analysis of genomic distribution of retroviral vectors is a powerful tool to monitor ‘vector-on-host’ effects in gene therapy (GT) trials but also provides crucial information about ‘host-on-vector’ influences based on the target cell genetic and epigenetic state. We had the unique occasion to compare the insertional profile of the same therapeutic moloney murine leukemia virus (MLV) vector in the context of the adenosine deaminase-severe combined immunodeficiency (ADA-SCID) genetic background in two GT trials based on infusions of transduced mature lymphocytes (peripheral blood lymphocytes, PBL) or a single infusion of haematopoietic stem/progenitor cells (HSC). We found that vector insertions are cell-specific according to the differential expression profile of target cells, favouring, in PBL-GT, genes involved in immune system and T-cell functions/pathways as well as T-cell DNase hypersensitive sites, differently from HSC-GT. Chromatin conformations and histone modifications influenced integration preferences but we discovered that only H3K27me3 was cell-specifically disfavoured, thus representing a key epigenetic determinant of cell-type dependent insertion distribution. Our study shows that MLV vector insertional profile is cell-specific according to the genetic/chromatin state of the target cell both in vitro and in vivo in patients several years after GT.

Highlights

Retroviral vectors have been extensively used in gene therapy (GT) applications as an effective tool to permanently integrate a therapeutic gene of interest into a target cell, conferring in most cases stable and efficient transgene expression over time (Kohn (1) The San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milano, Italy.(2) Universita Vita-Salute San Raffaele, Milano, Italy. (3) CUSSB, Universita Vita-Salute, Milan, Italy. (4) National Center for Tumor Diseases (NCT), German Cancer Research& Candotti, 2009; Naldini, 2009)
Distribution of vector integrations from PBL-GT and haematopoietic stem/progenitor cells (HSC)-GT To compare the insertional profile of murine leukemia virus (MLV) vector in PBL-GT and haematopoietic stem cell gene therapy (HSC-GT), we identified a total of 4157 unique insertions from ADA-SCID patients (2198 from PBL-GT and 1959 from HSC-GT) by the combination of shotgun cloning-generated insertion libraries of ligation mediated PCR (LM-PCR) products and pyrosequencing of linear amplification mediated PCR (LAMPCR) products (Table 1)
From HSC-GT patients, we retrieved retroviral integration sites (RIS) from both CD3þ T-cells and CD15þ granulocytes in vivo, whereas only T-cells were analysed in the PBL-GT group since in these patients the vector is undetectable in other lineages

Summary

Introduction

The analyses of integration sites from adenosine deaminase deficient SCID (ADA-SCID), SCID-X1, and chronic granulomatous disease (CGD) trials (Aiuti et al, 2007; Deichmann et al, 2007; Ott et al, 2006) show the presence of specific regions with recurrent integrations (common integration sites, CIS), it remains undefined to what extent the presence of CIS is the result of positive clonal selection after cell infusion or instead derives from preferential targeting for integration at the time of transduction (Cattoglio et al, 2007). Insertion site selection during in vitro transduction could be driven by tethering of transcription factors (TF) to specific regions according to TF binding sites location (Felice et al, 2009) and seems dependent on cellular determinants as well as on vector design (Lewinski et al, 2006)

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Conclusion