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

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 cell 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.

[1]  E. Gilboa,et al.  Transfer of the ADA gene into bone marrow cells and peripheral blood lymphocytes for the treatment of patients affected by ADA-deficient SCID. , 1993, Human gene therapy.

[2]  R. Giavazzi,et al.  Transfer of the ADA gene into human ADA-deficient T lymphocytes reconstitutes specific immune functions. , 1992, Blood.

[3]  A. Miller,et al.  Human gene therapy comes of age , 1992, Nature.

[4]  E. Gilboa,et al.  An in vivo model of somatic cell gene therapy for human severe combined immunodeficiency. , 1991, Science.

[5]  Eli Gilboa,et al.  Overexpression of TAR sequences renders cells resistant to human immunodeficiency virus replication , 1990, Cell.

[6]  E. Gilboa,et al.  Retroviral vector-mediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADA-deficient marrow cells. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[7]  B. Sullenger,et al.  Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[8]  D. J. Kaplan,et al.  Complete sequence and structure of the gene for human adenosine deaminase. , 1986, Biochemistry.

[9]  M. Chao,et al.  Expression and structure of the human NGF receptor , 1986, Cell.

[10]  A. Miller,et al.  Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production , 1986, Molecular and cellular biology.

[11]  G. Keller,et al.  Expression of a foreign gene in myeloid and lymphoid cells derived from multipotent haematopoietic precursors , 1985, Nature.

[12]  Tak W. Mak,et al.  A human T cell-specific cDNA clone encodes a protein having extensive homology to immunoglobulin chains , 1984, Nature.

[13]  D. Baltimore,et al.  Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus , 1983, Cell.

[14]  P. Thomas,et al.  Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[15]  W. Rutter,et al.  Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. , 1979, Biochemistry.

[16]  M Vapalahti,et al.  [Human gene therapy]. , 1996, Duodecim; laaketieteellinen aikakauskirja.

[17]  T. Lee,et al.  Overexpression of RRE-derived sequences inhibits HIV-1 replication in CEM cells. , 1992, The New biologist.

[18]  K. Cornetta,et al.  Human gene transfer: characterization of human tumor-infiltrating lymphocytes as vehicles for retroviral-mediated gene transfer in man. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[19]  A. Miller,et al.  Improved retroviral vectors for gene transfer and expression. , 1989, BioTechniques.

[20]  E. Gilboa,et al.  Transfer and expression of cloned genes using retroviral vectors , 1986 .

[21]  P Berg,et al.  Transformation of mammalian cells to antibiotic resistance with a bacterial gene under control of the SV40 early region promoter. , 1982, Journal of molecular and applied genetics.