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Manfred Schmidt

Publications and source records attributed to Manfred Schmidt.

41 records · Page 3Linked to original sources
PubMed2002

Polyclonal long-term repopulating stem cell clones in a primate model.

Hematopoietic bone marrow stem cells generate differentiated blood cells and, when transplanted, may contribute to other organs, such as the brain, heart, and liver. An understanding of in vivo clonal behavior of stem cells will have important implications for cellular and gene therapy. For the first time, we have directly demonstrated the derivation of circulating peripheral blood cells from individual stem cell clones. We analyzed the clonal composition of retrovirus-marked peripheral blood leukocyte populations in 2 different primate models by a novel direct genomic sequencing technique allowing the identification of vector insertion sites. More than 80 contributing long-term hematopoietic clones were identified in individual rhesus macaque peripheral blood transplant recipients and more than 25 different clones in a baboon marrow transplant recipient. Up to 5 insertion sequences from each animal were used to trace the long-term contribution of stem cell clones in these primate models. Continuous and mostly pluripotent contributions of peripheral blood leukocytes from each of the traced clones could be detected for the entire follow-up period of 23 to 33 months. Our study provides direct molecular evidence for a polyclonal, multilineage, and sustained contribution of individual stem cells to primate hematopoiesis.

Animals↗
PubMed2002

Pharmacologically regulated in vivo selection in a large animal.

The inefficiency of gene transfer has greatly hindered gene therapy. In vivo selection may increase the frequency of genetically modified cells, thereby circumventing this critical limitation. Here we demonstrate regulated in vivo selection in a large animal. CD34(+) cells from 2 dogs were engineered to express a conditional derivative of the thrombopoietin receptor (F36Vmpl). Activation of the receptor through administration of a dimerizing drug, AP20187, produced reversible, drug-dependent rises in genetically modified red cells, white cells, and platelets in both animals, with minimal side effects. Cell growth switches could greatly enhance the efficacy and applicability of gene and cell therapy.

Animals↗
PubMed2002

Molecular evidence of lentiviral vector-mediated gene transfer into human self-renewing, multi-potent, long-term NOD/SCID repopulating hematopoietic cells.

A major challenge in gene therapy is to achieve efficient transduction of hematopoietic stem cells (HSC). It has previously been shown that lentiviral vectors (LV) transduce efficiently human cord blood-derived NOD/SCID mouse repopulating cells (SRC). Here we studied the effect of cytokines during the short ex vivo incubation with vector. Although SRC transduction was efficient without stimulation, the presence of cytokines significantly improved it. The treatment did not affect the engraftment level or the SRC frequency, but seemed to enhance SRC susceptibility to LV. SRC transduced in both conditions repopulated primary and secondary recipients, maintaining stable multi-lineage transgene expression. Using linear amplification-mediated PCR, we then analyzed vector integration in the bone marrow and CFC of the engrafted mice to monitor the clonal activity of the transduced SRC in vivo. We showed polyclonal engraftment, multi-lineage differentiation, and propagation to secondary recipients of individual SRC. We observed multiple integrations in most clones. These results provide the first formal demonstration that primitive human HSC with self-renewal and multi-lineage repopulation capacities were transduced by LV. Our findings are relevant for the design of clinical protocols that exploit this system to reach significant engraftment by genetically modified HSC in the absence of in vivo selection or strong conditioning regimens.

Animals↗
PubMedDate not supplied

Gene therapy of X-linked severe combined immunodeficiency by use of a pseudotyped gammaretroviral vector.

BACKGROUND: X-linked severe combined immunodeficiency (SCID-X1) is caused by mutations in the common cytokine-receptor gamma chain (gamma(c)), resulting in disruption of development of T lymphocytes and natural-killer cells. B-lymphocyte function is also intrinsically compromised. Allogeneic bone-marrow transplantation is successful if HLA-matched family donors are available, but HLA-mismatched procedures are associated with substantial morbidity and mortality. We investigated the application of somatic gene therapy by use of a gibbon-ape-leukaemia-virus pseudotyped gammaretroviral vector. METHODS: Four children with SCID-X1 were enrolled. Autologous CD34-positive haemopoietic bone-marrow stem cells were transduced ex vivo and returned to the patients without preceding cytoreductive chemotherapy. The patients were monitored for integration and expression of the gamma(c) vector and for functional immunological recovery. FINDINGS: All patients have shown substantial improvements in clinical and immunological features, and prophylactic medication could be withdrawn in two. No serious adverse events have been recorded. T cells responded normally to mitogenic and antigenic stimuli, and the T-cell-receptor (TCR) repertoire was highly diverse. Where assessable, humoral immunity, in terms of antibody production, was also restored and associated with increasing rates of somatic mutation in immunoglobulin genes. INTERPRETATION: Gene therapy for SCID-X1 is a highly effective strategy for restoration of functional cellular and humoral immunity.

Antigens, CD34↗