Gene therapy with drug resistance genes

Zaboikin, Michail; Srinivasakumar, Narasimhachar; Schuening, Friedrich

doi:10.1038/sj.cgt.7700912

Cited by 25 publications

(18 citation statements)

References 93 publications

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“…Likewise, pemetrexed was barely active in HMI11 (IC 50 Ͼ 50 M) but highly active in CMM (IC 50 ϭ 18 nM). Methotrexate and pemetrexed are both known to inhibit DHFR, resulting in inhibition of folate metabolism and subsequent biosynthesis of thymidine nucleotides (37). Hence, the lower activity observed in HMI11 is likely due to rescue by the high concentrations of folate in HMI11 medium compared to CMM.…”

Section: Resultsmentioning

confidence: 98%

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Creek

Nijagal

Kim

et al. 2013

Antimicrob Agents Chemother

134

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dIn vitro culture methods underpin many experimental approaches to biology and drug discovery. The modification of established cell culture methods to make them more biologically relevant or to optimize growth is traditionally a laborious task. Emerging metabolomic technology enables the rapid evaluation of intra-and extracellular metabolites and can be applied to the rational development of cell culture media. In this study, untargeted semiquantitative and targeted quantitative metabolomic analyses of fresh and spent media revealed the major nutritional requirements for the growth of bloodstream form Trypanosoma brucei. The standard culture medium (HMI11) contained unnecessarily high concentrations of 32 nutrients that were subsequently removed to make the concentrations more closely resemble those normally found in blood. Our new medium, Creek's minimal medium (CMM), supports in vitro growth equivalent to that in HMI11 and causes no significant perturbation of metabolite levels for 94% of the detected metabolome (<3-fold change; ␣ ‫؍‬ 0.05). Importantly, improved sensitivity was observed for drug activity studies in whole-cell phenotypic screenings and in the metabolomic mode of action assays. Four-hundred-fold 50% inhibitory concentration decreases were observed for pentamidine and methotrexate, suggesting inhibition of activity by nutrients present in HMI11. CMM is suitable for routine cell culture and offers important advantages for metabolomic studies and drug activity screening.T rypanosoma brucei is the kinetoplastid parasite responsible for human African trypanosomiasis (HAT), a potentially fatal infection of sub-Saharan Africa. Current treatments for HAT are inadequate because of high toxicity and complex administration regimens, and new drugs are urgently required to ensure ongoing control of the disease in the face of emerging reports of drug resistance (1). Newly invigorated efforts to bring novel compounds forward as drugs have led to several promising candidates emerging through screening against parasites in culture (2-5). In vitro continuous culture methods enable the routine screening of compounds for trypanocidal activity against bloodstream form trypanosomes, for example, with resazurin fluorescence-based assays (5-7).Culture methods for bloodstream form T. brucei were first developed in the 1970s and required feeder layers to support continuous growth (8). This requirement could be overcome by regular supplementation with cysteine (9) and supplementation with mammalian serum (10). Continuous culture was successfully maintained by the addition of reducing and chelating agents (2-mercaptoethanol and bathocuproine sulfonate) to modified Iscove medium with 10% fetal bovine serum (HMI11) (11). HMI11 is now widely used for routine cell culture of laboratory strains of T. brucei and underpins many of the approaches employed in the search for new trypanocidal compounds. Nevertheless, HMI11 is a very rich medium compared to the natural in vivo environment of bloodstream form T. brucei, which may give...

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Section: Resultsmentioning

confidence: 98%

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Creek

Nijagal

Kim

et al. 2013

Antimicrob Agents Chemother

134

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“…In addition to the widely publicized malignant side effects in the trials involving correction of X-linked severe combined immunodeficiency, 8 inefficient transduction, poor long-term expression, and engraftment failure of ex vivo manipulated cells have slowed the practical advancement of gene therapy. 2,[9][10][11] Newer, self-inactivating (SIN), HIV-based lentiviral vectors have recently been explored as a means to avoid some of the pitfalls noted in the previous paragraph. 12 Lentiviruses do not require cells to be in cycle, 13,14 thus allowing for transduction during relatively short-term ex vivo culture.…”

Section: Introductionmentioning

confidence: 99%

“…[9][10][11]19 Several different approaches to programmed drug resistance have been evaluated: ectopic expression of the multidrug resistance 1 gene product (MDR1), 20 cytidine deaminase (CD), 21 glutathione S-transferase (GST), 22 or cytosolic 5Ј-nucleotidase I (cN-I) 23 ; or expression of mutated forms of dihydrofolate reductase (⌬-DHFR) 24 or O 6 -methylguanine-DNA-methyltransferase (⌬-MGMT). 25 Concerns with these strategies remain, though, because of a lack of effectiveness in selecting hematopoietic stem cells (HSCs) in vivo (⌬-DHFR, cN-I), potential development of multiple-drug resistant leukemia (MDR1), 26 or the use of highly genotoxic, carcinogenic drugs for selection (GST, MDR1, ⌬-MGMT).…”

Section: Introductionmentioning

confidence: 99%

Interfering RNA-mediated purine analog resistance for in vitro and in vivo cell selection

Porter

DeGregori

2008

Blood

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The advancement of gene therapy has been slowed, in part, by inefficient transduction of targeted cells and poor longterm engraftment of genetically modified cells. Thus, the ability to select for a desired population of cells within a recipient would be of great benefit for improving gene therapy. Proposed strategies for in vivo cell selection using drug resistance genes have had disappointing outcomes and/or require highly genotoxic medications to be effective. We hypothesized that resistance to purine analogs, a well-tolerated, relatively low-toxicity class of medications, could be provided to cells using interfering RNA against hypoxanthine phosphoribosyl transferase. Using a lentiviral vector, we found that interfering RNA-mediated purine analog resistance (iPAR) provided relative resistance to 6-thioguanine (

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“…58,59 For this purpose, genes involved in the resistance to the drugs have been used as transgenes to protect myeloid stem cells.…”

Section: Myeloprotection With Cytidine Deaminasementioning

confidence: 99%

Development of gene therapy in association with clinically used cytotoxic deoxynucleoside analogues

2009

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The clinical use of cytotoxic deoxynucleoside analogues is often limited by resistance mechanisms due to enzymatic deficiency, or high toxicity in nontumor tissues. To improve the use of these drugs, gene therapy approaches have been proposed and studied, associating clinically used deoxynucleoside analogues such as araC and gemcitabine and suicide genes or myeloprotective genes. In this review, we provide an update of recent results in this area, with particular emphasis on human deoxycytidine kinase, the deoxyribonucleoside kinase from Drosophila melanogaster, purine nucleoside phosphorylase from Escherichia coli, and human cytidine deaminase. Data from literature clearly show the feasibility of these systems, and clinical trials are warranted to conclude on their use in the treatment of cancer patients.

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Gene therapy with drug resistance genes

Cited by 25 publications

References 93 publications

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Metabolomics Guides Rational Development of a Simplified Cell Culture Medium for Drug Screening against Trypanosoma brucei

Interfering RNA-mediated purine analog resistance for in vitro and in vivo cell selection

Development of gene therapy in association with clinically used cytotoxic deoxynucleoside analogues

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