Summary. Genetically modified lymphocytes have been successfully used for correction of ADA deficiency in children and in controlling graft-versus-host disease (GvHD) after allogeneic bone marrow transplantation. Low transduction efficiencies are, however, limiting for gene therapeutic strategies based on lymphocytes. In this study we compared protocols for highly efficient gene transfer into human T cells using retroviral vector-containing supernatant. We showed that infection of both human primary T cells and CD4 þ Jurkat cells is most efficient on the matrix component fibronectin. Transduction was carried out with a retroviral vector encoding both the human intracytoplasmatically truncated low-affinity nerve growth factor receptor (DLNGFR) as a gene transfer marker and the Herpes simplex virus thymidine kinase for negative selection. Based on LNGFR expression genetically modified cells were enriched to near purity by magnetic cell sorting (MACS). Enriched cells could be shown to be highly sensitive to ganciclovir.

Summary Advanced chronic idiopathic myelofibrosis (IMF) with osteosclerosis and increase and thickening of bone trabeculae is typically contrasted by the absence or sparse presence of osteoclasts. Because osteoclast formation can be inhibited by osteoprotegerin (OPG) we investigated OPG expression in IMF with severe fibrosis and osteosclerosis, which expressed significantly higher (up to 71‐fold) OPG mRNA levels when compared with prefibrotic cellular IMF and control cases. The receptor activator of nuclear factor kappaB ligand (RANKL), a positive regulator of osteoclast differentiation and putative antagonist of OPG was overexpressed by up to 34‐fold exclusively in advanced IMF. Case‐specific calculation of the RANKL/OPG ratio in advanced IMF showed a wide range without significant differences when compared with the prefibrotic IMF and non‐neoplastic haematopoiesis. Immunohistochemical detection of OPG protein revealed strong labelling of endothelial cells within proliferating vessels in fibrotic IMF and heterogeneously labelled megakaryocytes, and fibroblasts. Osteosclerosis and impaired osteoclast function in IMF appears to be associated with upregulated endothelial OPG expression but concomitant reduction of the antagonist RANKL could not be demonstrated. We conclude that osteosclerosis in IMF is associated with increased endothelial OPG expression without concomitant RANKL downregulation.

Current gene therapeutic protocols directed towards the vectors encoding a truncated human low-affinity nerve treatment of inherited disorders (eg ADA-SCID) and viral growth factor receptor (⌬LNGFR) as a gene transfer infections (eg AIDS), as well as adoptive immunotherapy marker, we obtained transduction frequencies of more than approaches are based on the use of genetically modified 70% of CD3 + cells after two cycles of infection. Our protolymphocytes. Since only insufficient transduction of T cells col is based on the use of FBS-free media for both the is obtained using existing techniques, the development of production of retrovirus-containing supernatant and the culmore efficient gene transfer protocols into these cells is of tivation of the primary T cells. Since the protocol presented great importance. We present here a protocol for the highly here works just as efficiently under large-scale conditions, efficient transduction of human primary T cells at high denit may be easily adapted to clinical needs and 'good manusities (1 × 10 6 /ml) by retroviral infection. Using retroviral facturing practice' (GMP) standards.

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