Industrial scale production of plasmid DNA for vaccine and gene therapy: plasmid design, production, and purification

The past several years have witnessed a rapidly increasing number of reports on utilizing plasmid DNA as a vector for the introduction of genes into mammalian cells for use in both gene therapy and vaccine applications. “Naked DNA vaccines” allow the foreign genes to be transiently expressed in transfected cells, mimicking intracellular pathogenic infection and triggering both the humoral and cellular immune responses. While considerable attention has been paid to the potential of such vaccines to mitigate a number of infections, substantially less consideration has been given to the practical challenges of producing large amounts of plasmid DNA for therapeutic use in humans, for both clinical studies and, ultimately, full-scale manufacturing. Doses of naked DNA vaccines are on the order of milligrams, while typical small-scale Escherichia coli fermentations may routinely yield only a few mg/l of plasmid DNA. There have been many investigations towards optimizing production of heterologous proteins over the past three decades, but in these cases, the plasmid DNA was not the final product of interest. This review addresses the current state-of-the-art means for the production of plasmid DNA at large scale in compliance with existing regulatory guidelines. The impact of the nature of the plasmid vector on the choice of fermentation protocols is presented, along with the effect of varying cultivation conditions on final plasmid content. Practical considerations for the large-scale purification of plasmid DNA are also discussed.     

Subtype-selective nicotinic receptor antagonists: potential as tobacco use cessation agents

N-n-Alkylpicolinium and N,N'-alkyl-bis-picolinium analogues were assessed in nicotinic receptor (nAChR) assays. The most potent and subtype-selective analogue, N,N'-dodecyl-bis-picolinium bromide (bPiDDB), inhibited nAChRs mediating nicotine-evoked [(3)H]dopamine release (IC(50)=5 nM; I(max) of 60%), and did not interact with alpha4beta2* or alpha7* nAChRs. bPiDDB represents the current lead compound for development as a tobacco use cessation agent.     

Photosynthesis and nutrient composition of spinach and fenugreek grown under elevated carbon dioxide concentration

The effect of elevated carbon dioxide concentration on the changes in the biomass, photosynthesis and nutrient composition was investigated in two leafy vegetables. Spinach (Spinacia oleracea L.) and fenugreek (Trigonella foenum-graecum L.) plants were grown in open top chambers under either ambient (ACO₂, 350 ± 50 μmol mol⁻¹) or elevated (ECO₂, 600 ± 50 μmol mol⁻¹) CO₂ concentration and analyzed 40, 60 and 80 days after exposure. The plants grown in ECO₂ had higher net photosynthetic rate and lower stomatal conductance when compared with the plants grown in ACO₂. ECO₂ also changed the nutrient composition: a lower N, Mg and Fe contents and higher C and Ca contents were observed in the leaves of plants exposed to ECO₂ than in those grown at ACO₂.     

The effects of apolipoprotein F deficiency on high density lipoprotein cholesterol metabolism in mice

Apolipoprotein F (apoF) is 29 kilodalton secreted sialoglycoprotein that resides on the HDL and LDL fractions of human plasma. Human ApoF is also known as Lipid Transfer Inhibitor protein (LTIP) based on its ability to inhibit cholesteryl ester transfer protein (CETP)-mediated transfer events between lipoproteins. In contrast to other apolipoproteins, ApoF is predicted to lack strong amphipathic alpha helices and its true physiological function remains unknown. We previously showed that overexpression of Apolipoprotein F in mice reduced HDL cholesterol levels by 20-25% by accelerating clearance from the circulation. In order to investigate the effect of physiological levels of ApoF expression on HDL cholesterol metabolism, we generated ApoF deficient mice. Unexpectedly, deletion of ApoF had no substantial impact on plasma lipid concentrations, HDL size, lipid or protein composition. Sex-specific differences were observed in hepatic cholesterol content as well as serum cholesterol efflux capacity. Female ApoF KO mice had increased liver cholesteryl ester content relative to wild type controls on a chow diet (KO: 3.4+/-0.9 mg/dl vs. WT: 1.2+/-0.3 mg/dl, p<0.05). No differences were observed in ABCG1-mediated cholesterol efflux capacity in either sex. Interestingly, ApoB-depleted serum from male KO mice was less effective at promoting ABCA1-mediated cholesterol efflux from J774 macrophages relative to WT controls.     

In silico characterization and molecular dynamics simulation of Pfcyc-1, a cyclin homolog of Plasmodium falciparum

Malaria is still one of the deadly diseases resulting in deaths of millions of people worldwide and situation has become worse due to alarming rise in anti-malarial drug resistance. Genome sequence availability of Plasmodium falciparum, the main causal organism of severe malaria in humans, has enabled identification of various parasite cell cycle regulators like several cyclins and cyclin dependent kinases or CDKs which are promising novel drug targets for Malaria. Here, we present in silico characterization of tertiary structure of Pfcyc-1, a P. falciparum cyclin homolog, which enables identification of key structural elements that contribute to its tertiary structure and function. We have investigated the structure and dynamics of Pfcyc-1 structural model by performing 10?ns molecular dynamics (MD) simulation. Our study indicates that despite poor sequence similarities with cyclin H and A, the characteristic structural cyclin domains are conserved in Pfcyc-1 too. The Pfcyc-1 model reveals a cyclin box, consisting of two tandemly repeating five-helix bundles separated by a linker hinge peptide. Furthermore, the amino acid residues in other known cyclins mediating cyclin-CDK interactions are conserved in Pfcyc-1. The model and its MD simulation offer a first ever structural annotation of any plasmodium cyclin, which along with sequence comparisons, helps in identification of important functional residues mediating the Pfcyc-1-CDK like interactions.     

Role of arginine-304 in the diphosphate-triggered active site closure mechanism of trichodiene synthase

The X-ray crystal structures of R304K trichodiene synthase and its complexes with inorganic pyrophosphate (PP(i)) and aza analogues of the bisabolyl carbocation intermediate are reported. The R304K substitution does not cause large changes in the overall structure in comparison with the wild-type enzyme. The complexes with (R)- and (S)-azabisabolenes and PP(i) bind three Mg2+ ions, and each undergoes a diphosphate-triggered conformational change that caps the active site cavity. This conformational change is only slightly attenuated compared to that of the wild-type enzyme complexed with Mg2+(3)-PP(i), in which R304 donates hydrogen bonds to PP(i) and D101. In R304K trichodiene synthase, K304 does not engage in any hydrogen bond interactions in the unliganded state and it donates a hydrogen bond to only PP(i) in the complex with (R)-azabisabolene; K304 makes no hydrogen bond contacts in its complex with PP(i) and (S)-azabisabolene. Thus, although the R304-D101 hydrogen bond interaction stabilizes diphosphate-triggered active site closure, it is not required for Mg2+(3)-PP(i) binding. Nevertheless, since R304K trichodiene synthase generates aberrant cyclic terpenoids with a 5000-fold reduction in kcat/KM, it is clear that a properly formed R304-D101 hydrogen bond is required in the enzyme-substrate complex to stabilize the proper active site contour, which in turn facilitates cyclization of farnesyl diphosphate for the exclusive formation of trichodiene. Structural analysis of the R304K mutant and comparison with the monoterpene cyclase (+)-bornyl diphosphate synthase suggest that the significant loss in activity results from compromised activation of the PP(i) leaving group.     

Monitoring of RNA clearance in a novel plasmid DNA purification process

As the field of plasmid DNA-based vaccines and therapeutics matures, improved methods for impurity clearance monitoring are increasingly valuable for process development and scale-up. Residual host-cell RNA is a major impurity in current large-scale separation processes for the production of clinical-grade plasmid DNA. Current RNA detection technologies include quantitative rtPCR, HPLC, and fluorescent dye-based assays. However, these methodologies are difficult to employ as in-process tests primarily as a result of impurity and buffer interferences. To address the need for a method of measuring RNA levels in various process intermediates, a sample pretreatment strategy has been developed that utilizes spermidine affinity precipitation to eliminate a majority of solution impurities, followed by a quantitative precipitation with alcohol to concentrate RNA and allow detection at lower concentrations. RNA concentrations as low as 80 ng/mL have been measured using detection with gel electrophoresis and 20 ng/mL if microplate-based detection with Ribogreen fluorescent dye is used. The assay procedure has been utilized to troubleshoot RNA clearance issues encountered during scale-up of a novel, non-chromatographic purification process for plasmid DNA. Assay results identified residual liquor removal inadequacies as the source of elevated RNA levels, enabling process modifications in a timely fashion.     

The effect of the etched (et) mutation on the amylolytic enzyme activities in germinating kernels and seedlings of Zea mays

Summary The etched (et) mutation in maize causes distinct depressions and structural gaps in the endosperm and also gives rise to virescent seedlings, - and -Amylase activities were observed to be higher in et ⁺ et ⁺ kernels and seedlings as compared to that of the et et mutant. The total amylase and -amylase trends during germination also differed between normal and mutant kernels and seedlings (it increases in the wildtype and decreases in et et). On the contrary, the overall -amylase trend was found to be similar in both genotypes (slight decrease during germination). The native gel electrophoresis of crude enzyme extracts did not reveal any qualitative differences in and amylases during germination. The germinating et et kernels initially showed lower levels of starch compared with the wild type kernels, whereas no such difference was found at later stages of germination. It is concluded that et gene associated endosperm lesions lead to an impairment of starch degradation in germinating kernels resulting in virescent seedlings.     

Genetic microheterogeneity and phenotypic variation of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> arginase in clinical isolates

Background  

Clinical isolates of the gastric pathogen Helicobacter pylori display a high level of genetic macro- and microheterogeneity, featuring a panmictic, rather than clonal structure. The ability of H. pylori to survive the stomach acid is due, in part, to the arginase-urease enzyme system. Arginase (RocF) hydrolyzes L-arginine to L-ornithine and urea, and urease hydrolyzes urea to carbon dioxide and ammonium, which can neutralize acid.