Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.1.21 (thymidine kinase)
 7,561 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Retroviral gene transfer has been used successfully to correct the glucocerebrosidase (GCase) deficiency in primary hematopoietic cells from patients with Gaucher disease. For this model of somatic gene therapy, we developed a high-titer, amphotropic retroviral vector designated NTG in which the human GCase gene was driven by the mutant polyoma virus enhancer/herpesvirus thymidine kinase gene (tk) promoter (Py+/Htk). NTG normalized GCase activity in transduced Gaucher fibroblasts and efficiently infected human monocytic and erythroleukemic cell lines. RNA blot-hybridization (Northern blot) analysis of these hematopoietic cell lines showed unexpectedly high-level expression from the Moloney murine leukemia virus long terminal repeat (Mo-MLV LTR) and levels of Py+/Htk enhancer/promoter-initiated human GCase RNA that approximated endogenous GCase RNA levels. Furthermore, NTG efficiently infected human hematopoietic progenitor cells. Detection (by means of the polymerase chain reaction) of the provirus in approximately one-third of NTG-infected progenitor colonies that had not been selected in G418-containing medium indicates that relative resistance to G418 underestimated the actual gene transfer efficiency. Northern blot analysis of NTG-infected, progenitor-derived cells showed expression from both the Mo-MLV LTR and the Py+/Htk enhancer/promoter. NTG-transduced hematopoietic progenitor cells from patients with Gaucher disease generated progeny in which GCase activity had been normalized.
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PMID:Correction of glucocerebrosidase deficiency after retroviral-mediated gene transfer into hematopoietic progenitor cells from patients with Gaucher disease. 231 24

The human glucocerebrosidase (GC) gene has been transferred efficiently into spleen colony-forming unit (CFU-S) multipotential hematopoietic progenitor cells, and production of human GC RNA and protein has been achieved in transduced CFU-S colonies. High-titer retroviral vectors containing the human GC cDNA were constructed. Mouse bone marrow cells were stimulated with hematopoietic growth factors, infected by coculture with producer cells, and injected into lethally irradiated animals. Four vectors were compared with respect to gene-transfer efficiency into CFU-S progenitors. One vector (G vector) required high concentrations of interleukins 3 and 6 during stimulation and coculture for efficient transduction of CFU-S progenitors. The remaining three vectors (NTG, GTN, and GI vectors) transduced these progenitors at infection frequencies approaching 100% using low concentrations of hematopoietic growth factors to stimulate cell division prior to and during the infection. Vectors using the viral long terminal repeat enhancer/promoter to drive the human GC cDNA produced high levels of human GC RNA in the progeny of CFU-S progenitors after gene transfer. When an internal herpes simplex thymidine kinase promoter assisted by a mutant polyoma enhancer was used to drive the human GC cDNA (NTG vector), little or no human GC RNA was detected in transduced CFU-S colonies. All three vectors producing human GC RNA in CFU-S colonies can generate human GC as detected by immunochemical analysis of CFU-S colonies. NTG vector-infected bone marrow cells were transplanted into W/Wv recipients to generate long-term reconstituted mice. The capacity of the viral long terminal repeat and the internal thymidine kinase promoter to direct synthesis of RNA in transduced bone marrow and spleen cells 5 months after bone marrow transplantation reflected the performance of these promoters in NTG-transduced CFU-S colonies.
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PMID:Production of human glucocerebrosidase in mice after retroviral gene transfer into multipotential hematopoietic progenitor cells. 257 69

In this study, we describe the efficiency of second gene translation in bicistronic constructs containing either a short (36bp) synthetic intercistron or known internal ribosomal entry sites (IRES). Experiments were performed using two different gene combinations: Herpes simplex virus-thymidine kinase (HSV-TK) and neomycine (NEO) or human glucocerebrosidase (hGC) and a methotrexate (MTX) resistant mutant dihydrofolate reductase (DHFR). We demonstrate that upon transfection, second gene translation is efficient using either an IRES or a 36-bp intercistron. Infection with retrovirus carrying the TK and NEO genes linked via a 36-bp intercistron resulted in both G418R (NEO expression) and gancyclovir (GCV) sensitivity (TK expression), indicating that both genes were expressed and thus that the genomic DNA and RNA of this bicistronic construct were intact. Likewise, retrovirus carrying the hGC and mutant DHFR gene separated by a short intercistron was harvested from MTXR murine PsiCRE cells. However, infection of PA317 cells with this virus supernatant did not result in the presence of hGC enzyme activity in these murine cells. Proviral DNA and RNA analyses indicated that the hGC coding region was lost from the original construct in the infected PA317 cells. In contrast, retrovirus carrying the hGC and DHFR cDNAs was linked via an IRES functioned as expected. Based on these results, we conclude that the efficiency of second gene translation using short synthetic intercistrons might prove useful in bicistronic constructs, depending on the gene combination used.
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PMID:Second gene expression in bicistronic constructs using short synthetic intercistrons and viral IRES sequences. 983 67