Selective Immunoaffinity-Based Enrichment of CD34⁺Cells Transduced with Retroviral Vectors Containing an Intracytoplasmatically Truncated Version of the Human Low-Affinity Nerve Growth Factor Receptor (ΔLNGFR) Gene

Abstract

Human hematopoietic stem cells remain one of the most promising target cells for gene therapeutic approaches to treat malignant and nonmalignant diseases. To rapidly characterize transduced cells and to isolate these from residual nontransduced, but biologically equivalent, cells, we have used a Moloney murine leukemia virus (Mo-MuLV)-based retroviral vector containing the intracytoplasmatically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene. Supernatant transduction of CD34+ cells (mean purity 97%) in fibronectin-coated tissue culture flasks resulted in 5.5-45% (mean 26%) transduced cells expressing deltaLNGFR (LNGFR+ cells). After transduction, more than 65% of the transduced cells remained CD34+. Compared with control (mock- and nontransduced) CD34+ cells, transduction did not decrease the cloning efficiency of CD34+ cells. Immunomagnetic selection of the transduced cells with a monoclonal anti-LNGFR antibody resulted in >90% LNGFR+ cells. Further phenotypic characterization of these highly enriched LNGFR+ cells indicated that the majority co-expressed the CD34 and CD38 antigens. These results show that transduced cells expressing an ectopic cell-surface protein can be rapidly and conveniently quantitated and characterized by fluorescence-activated cell sorting (FACS) analysis and fast and efficiently enriched by immunoadhesion using magnetic beads. The use of cell-surface reporters should facilitate optimization of methods of gene transfer into more primitive hematopoietic progenitors.

Persisting multilineage transgene expression in the clonal progeny of a hematopoietic stem cell

“…Such a procedure is relatively well tolerated, avoids the use of mutagenic agents, and is compatible with clinically approved systems for cell processing. 15,30,31 Although the present study revealed an intact hematopoiesis after transplantation of immunoselected transgenic HSCs in secondary recipients, the clinical utility will be dependent on the demonstration of polyclonal reconstitution following selection prior to primary BMT. Since CD34 is expressed in clinically used HSC populations, human tCD34 cannot currently be proposed for clinical use, except when introduced into subpopulations that are CD34-negative (such as T cells).…”

Establishment of an optimised gene transfer protocol for human primary T lymphocytes according to clinical requirements

“…Cells were infected at high densities (1 × 10 6 /ml) with retroviral vectors encoding as a gene transfer marker a truncated human low-affinity nerve growth factor receptor (⌬LNGFR) lacking the intracytoplasmatic domain. 6,12 The use of surface proteins as gene transfer markers allows rapid and convenient determination of gene transfer efficiencies by flow cytometry. 13 The vectors were produced by a PG13 producer cell line, 14 which utilises the Gibbon ape leukaemia virus (GALV) env protein.…”

FMEV vectors: both retroviral long terminal repeat and leader are important for high expression in transduced hematopoietic cells

“…These were the cytoplasmic marker enhanced green fluorescent protein (EGFP, or simply ␥ in our vector nomenclature), 12 and the cell surface marker, low affinity nerve growth factor receptor lacking the cytoplasmic signal transduction domain (⌬LNGFR, or simply ␦). 13 Expression of these genes allows preparative sorting of transduced cells by flow cytometry (EGFP) 14 or, which may be of greater utility for clinical settings, also by immunoaffinity (⌬LNGFR). 13 However, potential drawbacks such as immunogenicity (EGFP) or interference with signalling pathways (⌬LNGFR) still remain to be elucidated.…”

“…13 Expression of these genes allows preparative sorting of transduced cells by flow cytometry (EGFP) 14 or, which may be of greater utility for clinical settings, also by immunoaffinity (⌬LNGFR). 13 However, potential drawbacks such as immunogenicity (EGFP) or interference with signalling pathways (⌬LNGFR) still remain to be elucidated. 15 In the present study, we preferentially addressed the utility of these genes as reporters for vector expression.…”