Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer

Fulvio Mavilio, Giuliana Ferrari, Silvano Rossini, Nadia Nobili, Chiara Bonini, Giulia Casorati, Catia Traversari, Claudio Bordignon

Research output: Contribution to journalArticle

217 Citations (Scopus)

Abstract

Peripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low- affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cells lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T-cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector- mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.

Original languageEnglish
Pages (from-to)1988-1997
Number of pages10
JournalBlood
Volume83
Issue number7
Publication statusPublished - Apr 1 1994

Fingerprint

Gene transfer
Lymphocytes
Blood
T-cells
Nerve Growth Factor Receptor
Genes
Gene expression
Cells
T-Lymphocytes
Gene Expression
Gene therapy
Proviruses
Cell Line
Cell- and Tissue-Based Therapy
Fluorescence
Reporter Genes
Genetic Therapy
RNA
Population
Fluorescent Antibody Technique

ASJC Scopus subject areas

  • Hematology

Cite this

Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer. / Mavilio, Fulvio; Ferrari, Giuliana; Rossini, Silvano; Nobili, Nadia; Bonini, Chiara; Casorati, Giulia; Traversari, Catia; Bordignon, Claudio.

In: Blood, Vol. 83, No. 7, 01.04.1994, p. 1988-1997.

Research output: Contribution to journalArticle

Mavilio, F, Ferrari, G, Rossini, S, Nobili, N, Bonini, C, Casorati, G, Traversari, C & Bordignon, C 1994, 'Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer', Blood, vol. 83, no. 7, pp. 1988-1997.
Mavilio, Fulvio ; Ferrari, Giuliana ; Rossini, Silvano ; Nobili, Nadia ; Bonini, Chiara ; Casorati, Giulia ; Traversari, Catia ; Bordignon, Claudio. / Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer. In: Blood. 1994 ; Vol. 83, No. 7. pp. 1988-1997.
@article{32c8b84eec7742c8b210195613aad723,
title = "Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer",
abstract = "Peripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low- affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cells lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T-cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector- mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100{\%} gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.",
author = "Fulvio Mavilio and Giuliana Ferrari and Silvano Rossini and Nadia Nobili and Chiara Bonini and Giulia Casorati and Catia Traversari and Claudio Bordignon",
year = "1994",
month = "4",
day = "1",
language = "English",
volume = "83",
pages = "1988--1997",
journal = "Blood",
issn = "0006-4971",
publisher = "American Society of Hematology",
number = "7",

}

TY - JOUR

T1 - Peripheral blood lymphocytes as target cells of retroviral vector-mediated gene transfer

AU - Mavilio, Fulvio

AU - Ferrari, Giuliana

AU - Rossini, Silvano

AU - Nobili, Nadia

AU - Bonini, Chiara

AU - Casorati, Giulia

AU - Traversari, Catia

AU - Bordignon, Claudio

PY - 1994/4/1

Y1 - 1994/4/1

N2 - Peripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low- affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cells lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T-cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector- mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.

AB - Peripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders. We have systematically compared four retroviral vectors, designed according to different strategies, for their efficiency in transfer and expression in human PBLs of the same reporter gene. The receptor gene used in the study codes for the human low- affinity nerve growth factor receptor (LNGFR), and is not expressed on the majority of human hematopoietic cells, thus allowing quantitative analysis of the transduced gene expression by immunofluorescence, with single cell resolution. Peripheral blood mononuclear cells (PBMCs), as well as human hematopoietic cells lines of myeloid and lymphoid origin, were transduced with the four vectors and analyzed for efficiency of gene transfer, integration and stability of vector proviruses, and LNGFR expression at both RNA and protein level. Fluorescence-activated cell sorter analysis of coexpression of LNGFR and lineage-specific cell surface markers was performed in transduced cell lines, PBLs, and T-cell clones to study gene expression on specific cell subpopulations. Although crucial differences were observed among different constructs, all retroviral vectors could transduce, under appropriate infection conditions, T-cell populations representative of the normal immune repertoire. Gene transfer and expression could be demonstrated also in circulating progenitors of mature T cells. Expression of the transduced gene was heterogeneous among cell populations infected with the different vectors, with optimal results obtained by two of the four constructs. Finally, we have devised a simple protocol based on vector- mediated gene transfer and positive immunoselection of the transduced cells that produces virtually 100% gene-modified cells. This may represent a crucial improvement in the way of designing efficacious protocols involving the use of gene-modified T lymphocytes in clinical studies.

UR - http://www.scopus.com/inward/record.url?scp=0028220643&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0028220643&partnerID=8YFLogxK

M3 - Article

C2 - 8142665

AN - SCOPUS:0028220643

VL - 83

SP - 1988

EP - 1997

JO - Blood

JF - Blood

SN - 0006-4971

IS - 7

ER -