Important note: Information in this article was accurate in 1995. The state of the art may have changed since the publication date.
Retroviral vector-mediated gene transfer into peripheral blood lymphocytes: a target cell type for gene therapy of congenital and acquired disorders (Meeting abstract).
Abstract:
Peripheral blood lymphocytes (PBLs) are key target cells for gene therapy of a number of inherited and acquired blood disorders We have systematically analyzed the crucial variables involved in high efficiency gene transfer with specific attention to in vitro and in vivo functional properties associated with cell manipulation required for cell activation and division, and retroviral vector-mediated gene transfer. For this purpose, we have developed a number of vectors, designed according to different strategies, for efficient transfer and expression in human PBLs of the same reporter gene. The receptor gene utilized 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. FACS analysis of co-expression of LNGFR and lineage-specific cell surface markers was carried out 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. Additionally, 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. An additional advantage of this strategy is related to the possibility of PBL infection during antigen stimulation of effector cells. Since retroviral vectors infect almost exclusively dividing cells, positive selection of transduced cells allow a significant enrichment of antigen-specific lymphocytes. Finally, culture conditions necessary for in vitro high efficiency of gene transfer and in vivo long term survival include: (1)short term in vitro culture, and (2)low levels of in vitro stimulation and activation. This general strategy may represent a crucial improvement on the way to design efficacious protocols involving the use of gene-modified T-lymphocytes in clinical studies and is now in application in a number of clinical and preclinical disease models including: (1) human somatic cell gene therapy in patients affected by ADA deficient SCID; (2) Transfer of the HSV-tK gene into donor PBL for in vivo immunomodulation of donor antitumor immunity after allo-BMT; (3) intracellular intervention against HIV. The first 2 studies are in clinical application. (1) Deficiency of the enzyme adenosine deaminase (ADA) resulting in a variant of severe combined immunodeficiency (SCID). Following the administration of transduced PBLs, we observed: (a) in vivo, long term survival of transduced cells; (b) in vector-transduced T-cell clones vector-derived ADA production was confirmed by TLC analysis; (c) immune reconstitution, including antigen-specific proliferation and Ig production. (2) The infusion of donors lymphocytes after allogeneic BMT is a potentially useful therapeutic tool for relapse of hematologic malignancies and for other complications related to the aggressive immunosuppression regimens. However, severe graft vs host disease (GvHD) may result from this therapeutic approach. In order to circumvent this complication we constructed and tested at preclinical and clinical levels retroviral vectors for transfer of the HSV-tk gene. After infusion, the transduced lymphocytes could be detected in the blood of recipients by FACS and PCR analyses. A progressive increase of the infused marked lymphocytes was observed in one patient affected by a EBV-BLPD and was accompanied by a complete clinical response, and in one patient with CML. In the two cases, the administration of ganciclovir resulted in elimination of marked donor PBL, and near resolution of all clinical and biochemical signs of acute GvHD.
Keywords: Adenosine Deaminase/BIOSYNTHESIS/DEFICIENCY/GENETICS Bone Marrow Transplantation Chromatography, Thin Layer Flow Cytometry *Gene Therapy *Gene Transfer Genetic Vectors Graft vs Host Disease/PREVENTION & CONTROL Hereditary Diseases/*THERAPY Human *Lymphocytes Polymerase Chain Reaction Receptors, Nerve Growth Factor/GENETICS Retroviridae/*GENETICS Severe Combined Immunodeficiency/THERAPY Transplantation, Homologous ABSTRACT
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Important note: Information in this article was accurate in 1995. The state of the art may have changed since the publication date.
