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 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.

A reversible functional defect of CD8+ T lymphocytes involving loss of tetramer labeling

“…Murine P1.aza r cells, derived from P815 mouse mastocytoma cell line, were cultured in DMEM (Life Technologies) supplemented with 10% FCS and AAG [38]. EBV-B.MAGE-1 cells were obtained by transduction with the retroviral vector M1-CSM, which encodes both the full-length MAGE-1 protein and a truncated form of the human low-affinity nerve growth factor receptor, according to the procedure described previously [39]. Human recombinant IL-2 was purchased from Eurocetus (Amsterdam, The Netherlands).…”

Hematopoietic stem cell gene therapy: towards clinically significant gene transfer efficiency

“…Coding sequences for the cytoplasmic transducing domains have been deleted. Vectors containing this transgene transduce lymphocytes efficiently, and transduced cells can be selected using FACS or magnetic anti-NGFR-coated beads (44). However, there are concerns regarding the ectopic expression of a potential growth-altering receptor protein.…”

Specific pharmacological dimerization of KDR in lentivirally transduced human hematopoietic cells activates antiapoptotic and proliferative mechanisms

“…The chimeric receptor was constructed within a shuttle vector and contained a truncated version of the low affinity growth factor receptor (tLNGFR) fused in frame with one modified copy of FKBP12 (F36V), which allows for binding of the CID AP20187, and the cytoplasmic portion of human KDR. tLNGFR, kindly provided by Dr. Warren Pear (University of Pennsylvania, Philadelphia, PA), encodes the extracellular and transmembrane domains of this molecule and targets the chimera to the cell membrane (33). FKBP12 (F36V) was excised from pC4Fv1E (kindly provided by Ariad Pharmaceuticals, Cambridge, MA) and ligated downstream of tLNGFR.…”