Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: DrugBank:EXPT01586 (G418)
 2,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two recombinant retroviral vectors encoding the cDNA of the human adenosine deaminase (ADA; EC 3.5.4.4) gene and the bacterial neomycin resistance (Neo) gene have been used to transduce bone marrow cells obtained from four patients affected by the ADA-deficient variant of severe combined immunodeficiency. By utilizing the long-term marrow culture system, freshly isolated bone marrow cells were subjected to multiple infection cycles with cell-free supernatants containing high titers of viral vector and then maintained in long-term marrow culture in the absence of any overt selection pressure. By using this experimental protocol, about 30-40% of the hematopoietic progenitors were productively transduced with the viral vector, as judged by the appearance of G418-resistant colonies derived from granulocyte/macrophage and multipotent hematopoietic progenitor cells. The vector-encoded human ADA gene was expressed efficiently in both the myeloid and lymphoid progeny of the cultured bone marrow cells, reaching levels between 15% and 100% as compared to the levels of ADA in normal bone marrow cells. The efficiency of gene transfer and ADA production was proportional to the number of infection cycles. Furthermore, transduction of the ADA vectors into the bone marrow cells derived from an ADA-deficient patient restored the capacity of the cells to respond to phytohemagglutinin and interleukin 2.
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PMID:Retroviral vector-mediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADA-deficient marrow cells. 254 45

Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34(+) cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor, Flt3-ligand, and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells, colony-forming cells (CFC), or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period, and the cells infected under either condition regenerated similar numbers of human CD34(+) (myeloid) CFC and human CD19(+) (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However, the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant, 17% +/- 3% and 17% +/- 8%, G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected, the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% +/- 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM), or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17, respectively), whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a >/= 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols.
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PMID:Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential. 955 9

Adenosine deaminase (ADA) deficiency is the primary cause of severe combined immunodeficiency disease and has become a focus for developing innovative approaches to gene therapy. We previously described successful treatment of a Japanese ADA-deficient patient by periodic infusions of genetically modified autologous T lymphocytes transduced with a retroviral vector containing human ADA cDNA. In order to investigate whether polyclonality was restored by the gene therapy and whether the gene-transduced T lymphocytes persisted in the peripheral blood of the patient, we analyzed the T cell clonotype using a T cell receptor-specific RT-PCR/SSCP method. Oligoclonal T cell expansion was observed in every Vbeta family, and the expanded T cell clones were stable throughout the periodic gene therapy. Some of these T cell clones are likely carrying the vector, since they were identical to the clones selected by G418 resistance. Therefore, although it is uncertain when oligoclonal T cells started to expand and what percentage of the oligoclones carries the vector, the peripheral blood of the patient administered the gene therapy included oligoclonal T cells, some of which were identical to the ADA-gene-transduced clones.
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PMID:Gene-transferred oligoclonal T cells predominantly persist in peripheral blood from an adenosine deaminase-deficient patient during gene therapy. 1116 7

Endothelial cells (ECs) play multiple physiological functions and are central to many pathological processes. Various biological studies as well as cell and gene therapy applications would benefit substantially from a procedure that would result in the expansion in culture of large numbers of highly differentiated human ECs. Here, we report the amplification in vitro of human EC populations, which occurred during the first phase of reversible immortalization resulting from the retroviral transfer of an oncogene that was subsequently excised by Cre-loxP-mediated site-specific recombination. Human umbilical vein endothelial cells (HUVECs) and human liver sinusoidal endothelial cells (HLSECs) were transduced with a retroviral vector that expresses the simian virus 40 large T (SV40T) gene flanked by positive and negative selectable markers and a pair of loxP recombination targets. Transduced HUVECs and HLSECs yielded clones with greatly extended life spans, referred to as HNNT-1 and HNNT-2 cells, respectively. HNNT-1 and HNNT-2 cells showed morphological characteristics of ECs and were maintained in culture up to population doubling level (PDL) 80 for HNNT-1 and PDL 65 for HNNT-2 cells. HNNT-1 and HNNT-2 cells were not tumorigenic when transplanted into severe combined immunodeficiency mice and were sensitive to ganciclovir as well as G418. Both cell clones expressed EC markers, which include factor VIII, VEGF receptors (Flt-1 and KDR/Flk-1), and CD34, and endocytosed acetylated low-density lipoproteins. Formation of capillary-like structures in a Matrigel assay was observed with HNNT-1 and HNNT-2 cells until at least PDL 50. Complete elimination of the transferred SV40T gene was achieved in virtually 100% of HNNT-1 and HNNT-2 cells after infection with a recombinant adenovirus expressing the Cre recombinase fused to a nuclear localization signal and subsequent selection with G418. Reverted cells maintained their differentiated EC phenotype. This study extends the utility of the reversible immortalization procedure and provides a means to expand primary human ECs of various sources for basic studies and possible cell and gene therapies.
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PMID:Controlled expansion of human endothelial cell populations by Cre-loxP-based reversible immortalization. 1181 87

Acute liver failure and metabolic liver disorder animal models have demonstrated that hepatocytes transplanted into the liver or spleen survive and participate in the liver repopulation process, and recent studies have documented the usefulness of hepatocyte transplantation in humans. However, despite the promising cell therapy, there are still many restrictions, such as the shortage of donor human livers and the limited lifespan and the functional insufficiency of primary cultured hepatocytes. The immortalized and highly differentiated human hepatocyte could provide an unlimited supply of transplantable cells. In this study, we established an efficient and highly differentiated immortalized human hepatocyte line for bioartificial liver and hepatocyte transplantation research. Hepatocytes isolated from the liver of a 25 year old, brain dead male were transfected with pcDNA3.1 (-) recombinant plasmid containing the genes encoding simian virus 40 (SV40) large tumor antigen. One of the hepatocyte clones, HepLL, displayed highly differentiated liver functions with immortalized characteristics and was selected with a 700-300 microg/ml of G418 technique in 42 days. To characterize this immortalized cell line for cell therapy in the near future, HepLL cells were studied with immunohistochemistry, reverse transcription-polymerase chain reactions, immunoblotting, and tumorigenicity tests. The results revealed that HepLL cells displayed morphologic characteristics of liver parenchymal cells, secreted albumin, synthesized urea and glycogen, and expressed liver enriched functional markers, but there were no tumorigenic qualities after transplantation into severe combined immunodeficiency mice. Thus this immortalized human hepatocyte line is expected to be a useful tool for studying the functions of differentiated human hepatocyte and a promising strategy to resolve the shortages of donor organs and the limits of primary human hepatocyte for transplantation and bioartificial liver support systems.
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PMID:Establishment of highly differentiated immortalized human hepatocyte line with simian virus 40 large tumor antigen for liver based cell therapy. 1596 57