Lentiviral Vector Production from a Stable Packaging Cell Line Using a Packed Bed Bioreactor.

Self-inactivating lentiviral vectors (LVVs) are used regularly for genetic modification of cells, including T cells and hematopoietic stem cells for cellular gene therapy. As vector demand grows, scalable and controllable methods are needed for production. LVVs are typically produced in HEK293T cells in suspension bioreactors using serum-free media or adherent cultures with serum. The iCELLis® is a packed-bed bioreactor for adherent or entrained cells with surface areas from 0.53 to 500 m2. Media are pumped through the fixed bed and overflows, creating a thin film that is replenished with oxygen and depleted of CO2 as media return to the reservoir. We describe the optimization and scale-up of the production of GPRTG-EF1α-hγc-OPT LVV using a stable packaging cell line in the iCELLis Nano 2-cm to the 10-cm bed height low compaction bioreactors (0.53 and 2.6 m2 surface area) and compare to the productivity and efficacy of GPRTG-EF1α-hγc-OPT LVV manufactured under current Good Manufacturing Practice (cGMP) using 10-layer cell factories for the treatment of X-linked severe combined immunodeficiency. By optimizing fetal bovine serum (FBS) concentration, pH post-induction, and day of induction, we attain viral yields of more than 2 × 107 transducing units/mL. We compared transduction efficiency between LVVs produced from the iCELLis Nano and cell factories on healthy, purified CD34+ cells and found similar results.

Changes in the proteome of Candida albicans in response to azole, polyene, and echinocandin antifungal agents.

The yeast Candida albicans is an opportunistic human fungal pathogen and the cause of superficial and systemic infections in immunocompromised patients. The classes of antifungal agents most commonly used to treat Candida infections are the azoles, polyenes, and echinocandins. In the present study, we identified changes in C. albicans protein abundance using two-dimensional polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization-time of flight mass spectroscopy following exposure to representatives of the azole (ketoconazole), polyene (amphotericin B), and echinocandin (caspofungin) antifungals in an effort to elucidate the adaptive responses to these classes of antifungal agents. We identified 39 proteins whose abundance changed in response to ketoconazole exposure. Some of these proteins are involved in ergosterol biosynthesis and are associated with azole resistance. Exposure to amphotericin B altered the abundance of 43 proteins, including those associated with oxidative stress and osmotic tolerance. We identified 50 proteins whose abundance changed after exposure to caspofungin, including enzymes involved in cell wall biosynthesis and integrity, as well as the regulator of beta-1,3-glucan synthase activity, Rho1p. Exposure to caspofungin also increased the abundance of the proteins involved in oxidative and osmotic stress. The common adaptive responses shared by all three antifungal agents included proteins involved in carbohydrate metabolism. Some of these antifungal-responsive proteins may represent potential targets for the development of novel therapeutics that could enhance the antifungal activities of these drugs.

Upc2p-associated differential protein expression in Candida albicans.

The gain-of-function mutation G648D in UPC2 causes ERG11 up-regulation and increased fluconazole resistance in Candida albicans. In this study, we performed 2-DE and PMF to identify proteomic alterations in an ERG11-overexpressing fluconazole-resistant C. albicans clinical isolate compared with its fluconazole-susceptible parent strain. We identified 23 differentially expressed proteins, and among them, seven became differentially expressed in a C. albicans wild-type strain after the introduction of a UPC2 allele carrying this mutation. These Upc2p-regulated proteins may contribute to fluconazole resistance in C. albicans.

Proteomic analysis of Mrr1p- and Tac1p-associated differential protein expression in azole-resistant clinical isolates of Candida albicans.

Azole resistance in Candida albicans is frequently caused by the overexpression of multi-drug efflux pump genes MDR1, CDR1, and CDR2 due to gain-of-function mutations in the zinc cluster transcription factors Mrr1p and Tac1p. In this study, we performed a comparative proteomic analysis to identify proteins whose expression level is influenced by these transcription factors. Both 2-DE and PMF were used to examine the expression profiles of six pairs of matched C. albicans isolates carrying gain-of-function mutations in either MRR1 or TAC1 resulting in the overexpression of either MDR1 or CDR1 and CDR2. Using this approach, 17 differentially expressed proteins were identified in the MDR1-overexpressing isolates, while 14 were identified in the isolates that overexpress CDR1 and CDR2. Furthermore, we found that the expression of many of these proteins was increased in a wild-type strain of C. albicans after the introduction of a gain-of-function allele of MRR1 or TAC1. Moreover, disruption of MRR1 and TAC1 in isolates carrying gain-of-function mutations resulted in decreased expression of these proteins, confirming their regulation by Mrr1p or Tac1p. Several proteins involved in heat shock and carbohydrate metabolism were differentially expressed in all clinical isolate sets, but these proteins were not dependent upon either Tac1p or Mrr1p.

Biotransformation of 2,4,6-trinitrotoluene (TNT) by the fungus Fusarium oxysporum.

The fungus Fusarium oxysporum was isolated and identified from the aquatic plant M. aquaticum. The capability of this fungus to transform 2,4,6-trinitrotoluene (TNT) in liquid cultures was investigated TNT was added to shake flask cultures and transformed into 2-amino-4,6-dinitrotoluene (2-A-DNT), 4-amino-2,6-dinitrotoluene (4-A-DNT), and 2,4-diamino-6-nitrotoluene (2,4-DAT) via 2- and 4-hydroxylamino-dinitrotoluene derivatives, which could be detected as intermediate metabolites. Transformation of TNT, 2-A-DNT, and 4-A-DNT was observed by whole cultures and with isolated mycelium. Cell-free protein extracts from the extracellular, soluble, and membrane-bound fractions were prepared from this fungus and tested for TNT-reducing activity. The concentrated extracellular culture medium was unable to transform TNT; however, low levels of TNT transformation were observed by the membrane fraction in the presence of nicotinamide adenine dinucleotide phosphate in an argon atmosphere. A concentrated extract of soluble enzymes also transformed TNT, but to a lesser extent. When TNT toxicity was studied with this fungus, a 50% decrease in the growth of F. oxysporum mycelium was observed when exposed to 20 mg/L TNT.

Differences in the biotransformation of 2,4,6-trinitrotoluene (TNT) between wild and axenically grown isolates of Myriophyllum aquaticum.

The aim of this study was to demonstrate the potential for aquatic plants and their associated microbes to bioremediate wetland sites contaminated with 2,4,6-trinitrotoluene (TNT). The transformation of TNT was studied using both wild and axenically grown isolates of Myriophyllum aquaticum (parrot feather). Differences in TNT transformation rates and nitroaromatic metabolites were observed between different plants. The wild isolates, containing a consortium of associated microorganisms, transformed TNT into 2-amino-4,6-dinitrotoluene (2-A-DNT) and 4-amino-2,6-dinitrotoluene (4-A-DNT) via 2- and 4-hydroxylamino-dinitrotoluene, which were detected as intermediates. The wild M. aquaticum also converted the metabolites, 2-A-DNT and 4-A-DNT, into low levels of 2,4-diaminotoluene (2,4-DAT). The axenically grown plants, containing no cultureable microorganisms, also transformed TNT into 2-A-DNT and 4-A-DNT, but at a much lower rate than that observed for the wild isolates. Unlike the wild plants, axenically grown M. aquaticum could not transform either 2-A-DNT or 4-A-DNT into 2,4-DAT over the incubation period. The differences in the performance between these plants could indicate that plant-associated microorganisms assisted in the overall transformation of TNT. For each plant, unidentifiable metabolites were observed and the soluble monoamino-derivatives present in the wild and axenic medium accounted for 14 and 7% of the initial TNT concentration, respectively. Thus, the majority of nitroaromatic derivatives remained associated with the plant tissues. Furthermore, only 7 and 3% of the initial TNT concentration were extracted as monoamino-derivatives from the tissues of the wild and axenically grown plants, respectively.

Purification and partial characterization of an acid phosphatase from Spirodela oligorrhiza and its affinity for selected organophosphate pesticides.

An acid phosphatase from the aquatic plant Spirodela oligorrhiza (duckweed) was isolated by fast protein liquid chromatography and partially characterized. The enzyme was purified 1871-fold with a total yield of 40%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the pure acid phosphatase resolved a single protein band that migrated to approximately 60 kDa. Nondenaturing SDS-PAGE electrophoresis revealed a single protein band around 120 kDa after staining with Coomassie Brilliant blue. Quantitative gel filtration chromatography estimated a native molecular mass of this enzyme to be 120 kDa. Thus, this acid phosphatase likely functions as a homodimer, consisting of two similar 60 kDa subunits. An electrophoretic technique using the flourogenic substrate 4-methylumbelliferyl phosphate enabled visualization of an acid phosphatase activity that corresponded to the protein band at 120 kDa on a nondenaturing PAGE gel. It was determined that the acid phosphatase had a pH optimum of 6.0 at 25 degrees C. The enzyme activity appeared to be stable over a broad range of temperatures (10-40 degrees C) and in the presence of the metals Zn2+, Mn2+, and Mg2+ as well as the chelating agents ethylenedinitrilotetraacetic acid and ethylene glycol tetraacetic acid. It was shown that this acid phosphatase could hydrolyze a variety of physiological organophosphate compounds including beta-glycerophosphate, phosphoserine, adenosine triphosphate, adenosine diphosphate, adenosine monphosphate, and pyrophosphate. Furthermore, analysis using capillary electrophoresis demonstrated that this hydrolytic enzyme could transform a wide array of organophosphate pesticides including S-2-ethylthioethyl O,O-dimethylphosphorothioate (demeton-S-methyl); S-1,2-bis(ethoxycarbonyl)ethyl O,O-dimethylphosphorodithioate (malathion); O,O-dimethyl O-4-nitrophenyl (paraoxon); O,O,O,O-tetraethyldithiopyrophosphate (sulfatep); O-2-chloro-4-nitrophenyl O,O-dimethylphosphorothioate (dicapthon); and 2,2-dichlorovinyl dimethylphosphate (dichlorvos).