Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.5.4.4 (adenosine deaminase)
 5,136 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reports of 1- to 2-log higher gene transfer levels in purified CD34+ cells or marrow CFU compared with levels in mature circulating blood cells after transplantation of retrovirally transduced primitive human hematopoietic cells have resulted in concern that transduced progenitors do not contribute proportionally to ongoing hematopoiesis (Kohn et al., 1995; Brenner, 1996). To study the issue in a relevant large animal, we analyzed samples of mature blood cells, marrow CD34-enriched cells and marrow CD34-depleted cells, and marrow CFU from a cohort of 11 rhesus transplanted with retrovirally transduced cells and followed for up to 5.5 years. They were transplanted with CD34-enriched bone marrow (BM) or G-CSF/SCF-mobilized peripheral blood (PB) cells transduced with vectors containing either neo, human glucocerebrosidase, or murine adenosine deaminase genes. There were no significant differences between the levels of vector sequences found in BM CD34+ cells, BM CD34- cells, PB granulocytes, or PB mononuclear cells (MNCs) in any animal. In four animals transplanted with SCF/G-CSF-primed BM cells and analyzed 3-6 months posttransplantation, the percentage of CFU containing the neo vector appeared to be 1 log higher than the representation of marked cells in the PB of these animals, but this discrepancy did not persist at time points greater than 6 months posttransplantation. The level of CFU marking was no higher than PB granulocyte or MNC marking at any time points in the other animals. Low levels of mature gene-modified cells probably reflect poor transduction of repopulating stem cells, not a block in differentiation or specific immune rejection of mature cells. This study represents the longest follow-up of primates transplanted with transduced hematopoietic cells, and it is encouraging that the levels of vector-containing cells appear stable for up to 5 years.
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PMID:No discrepancy between in vivo gene marking efficiency assessed in peripheral blood populations compared with bone marrow progenitors or CD34+ cells. 1009 6

Human lymphocytes remain among the most promising target cells for gene therapy. Gene-modified lymphocytes have been used successfully to treat adenosine deaminase (ADA)-deficient patients and to control GvHD after allogeneic BMT. Because activation and proliferation of T cells are necessary for efficient retrovirus-mediated gene transfer and subsequent selection of transduced cells, mononuclear cells (MNC) from steady-state and G-CSF-stimulated peripheral blood were activated by short exposure to the mitogen PHA, the anti-CD3 antibody OKT3, or both in the presence of different concentrations of recombinant IL-2. Using OKT3 (10 or 30 ng/ml) and IL-2 (100 U/ml), T cells expanded efficiently during a 14-day culture period. Cell expansion was similar under serum-free conditions. The immunophenotypic profile over time showed a marked increase in CD8+ cells, leading to a reversed CD4/CD8 ratio of 1:2 and a slight increase in CD56+ cells. Supernatant-based centrifugal transduction of primary human T lymphocytes was compared with supernatant transduction on the extracellular matrix protein fibronectin. Transduction with cell-free retrovirus-containing supernatant in tissue culture flasks coated with human plasma fibronectin led to significantly higher transduction efficiencies (20% +/- 7.5%) than centrifugal transduction in uncoated culture flasks (13.6% +/- 5.1%)(p = 0.041). To both rapidly characterize transduced cells and isolate these from residual nontransduced but biologically equivalent cells, an amphotropic Moloney murine leukemia virus (MoMuLV)-based retroviral vector containing the intracytoplasmically truncated human low-affinity nerve growth factor receptor (deltaLNGFR) cDNA as a marker gene was used. FACS sorting of T cells after transduction resulted in >90% LNGFR+ cells and was much faster than enrichment of transduced cells through growth in G418-selection medium. These results show that supernatant-based retroviral gene transfer into primary human T lymphocytes can be enhanced by fibronectin. Ectopic expression of a cell surface protein can be used to rapidly and conveniently quantitate transduction efficiency through FACS analysis and to efficiently enrich transduced cells through FACS sorting.
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PMID:Expansion and fibronectin-enhanced retroviral transduction of primary human T lymphocytes for adoptive immunotherapy. 1063 78