Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In trying to develop methods of gene therapy for Gaucher disease that will avoid the morbidity and mortality associated with bone marrow (BM) ablation, we transplanted BM stem cells transduced with a retroviral vector containing the human glucocerebrosidase cDNA into normal, nonablated, syngeneic mice. Donor BM from untreated male mice or treated with 5-fluorouracil (5-FU) was transduced ex vivo using a standard 4-day transduction protocol. Recipient female mice were injected one time only or once daily for 5 consecutive days or once a week for 5 consecutive weeks using 2 x 10(7) (untreated BM) or 2 x 10(6) (5-FU-treated BM) cells per injection. Initial transduction efficiency into colony-forming unit-spleen (CFU-S) was 80% to 100%. Recipient analysis was performed at least 6 months after the last transplantation. The best engraftment of donor stem cells, up to 5% by secondary CFU-S analysis, was obtained with multiple injections of transduced BM not previously treated with 5-FU. Polymerase chain reaction (PCR) amplification for both the transgene and the Y chromosome identified the progeny of transduced stem cells in various hematopoietic and non-hematopoietic organs. The copy number of the transgene in stem cells was 0.13 to 2.8. Transgene expression was shown by reverse transcriptase-PCR, in situ hybridization, and immunohistochemistry. No serious side effects of the procedure were noted. We conclude that multiple transplants of retrovirally transduced BM cells into nonablated recipients may be a safe and effective therapeutic modality for a number of genetic hematopoietic disorders.
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PMID:Transfer of the human glucocerebrosidase gene into hematopoietic stem cells of nonablated recipients: successful engraftment and long-term expression of the transgene. 762 Jan 75

In an attempt to improve our gene transfer efficiency into hematopoietic stem cells and to evaluate the capacity of immunoselected CD34+Thy-1+(CDw90) cells to reconstitute hematopoiesis following myeloablation, bone marrow (BM) transplantation was performed using autologous, immunoselected CD34+Thy-1+ cells in rhesus macaques. BM samples were positively selected for cells that express CD34, further subdivided using high gradient immunomagnetic selection for cells that express Thy-1, and transduced using a 7-day supernatant transduction protocol with a replication-defective retroviral vector that contained the human glucocerebrosidase (GC) gene. Circulating leukocytes were evaluated using a semiquantitative polymerase chain reaction (PCR) assay for the human GC gene, with the longest surviving animal evaluated at day 558. Provirus was detected at all time points in both CD20+ B cells and CD2+ dim T cells, but long-term gene transfer was not observed in the granulocyte population. The CD2+ dim population was phenotypically identified as being CD8+ natural killer cells. By day 302 and day 330, both the CD2+ bright and dim cell populations and sorted CD4+ and CD8+ cells had detectable provirus. Vector-derived GC mRNA was detected by reverse transcriptase (RT)-PCR analysis as far out as day 588. Thus, CD34+Thy-1+ cells isolated using high gradient magnetic separation techniques can engraft, be transduced with a replication-defective retroviral vector, and contribute to CD20+ B lymphocytes, CD8+ T lymphocytes, and CD4+ T lymphocytes; making them a suitable cell population to target for gene therapies involving lymphocytes.
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PMID:Transplantation and gene transfer of the human glucocerebrosidase gene into immunoselected primate CD34+Thy-1+ cells. 894 51