The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression

BACKGROUND: Efficient delivery and expression of plasmids (pDNA) is a major concern in gene therapy and DNA vaccination using non-viral vectors. Besides the use of adjuvants, the pDNA vector itself can be designed to maximize survival in nuclease-rich environments. Homopurine-rich tracts in polyadenylation sequences have been previously shown to be especially important in pDNA resistance. METHODOLOGY: The effect of modifications in the poly A sequence of a model pDNA vector (pVAX1GFP) on nuclease resistance and transgene expression was investigated. Four poly A sequences were studied: bovine growth hormone (BGH), mutant BGH, SV40 and a synthetic poly A. Plasmid resistance (half-life) was assessed through in vitro incubations with mammalian nucleases. The impact in transgene expression was studied by quantifying pDNA, mRNA, and GFP expression in CHO, hybridoma and HeLa cells. RESULTS AND CONCLUSIONS: In vitro and cell culture studies indicate that plasmids containing the SV40 and the synthetic poly A sequences present significant improvements in nuclease resistance (up to two-fold increase in half-life). However, RT-PCR analysis demonstrated that significant reduction in mRNA steady-state levels were responsible for a decrease in transgene expression and detected transfection level of CHO and hybridoma cells when using the more resistant plasmids. Interestingly, transfection of HeLa cells demonstrated that both poly A efficiency and plasmid resistance interfere significantly in transgene expression. The results strongly suggest that the choice of the poly A is important, not only for mRNA maturation/stability, but also for pDNA resistance, and should thus be taken into consideration in the design and evaluation of pDNA vectors.     

Integrated detection of intrinsic fluorophores in live microbial cells using an array of thin film amorphous silicon photodetectors

Two-dimensional fluorescence spectroscopy (2D FS) provides a non-invasive means to assess cell condition without the introduction of changes to the cell environment. The method relies on the measurement of the excitation-emission fluorescence intensity matrix of key intrinsic fluorophores, like aromatic amino acids, enzyme cofactors, and vitamins. Commonly used detection systems are complex, with multiple bandpass filters, and are hard to miniaturize. Here, an amorphous silicon photodetector array system integrated with amorphous silicon-carbon alloy filters designed to detect three key fluorophores - tryptophan (Trp), reduced nicotine adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) - is demonstrated. These intrinsic fluorophores were detected in pure solutions and also in suspended yeast cells. The array system was used to monitor changes in intrinsic fluorophore concentration when a yeast cell solution was subject to a thermal shock stress.     

Recombination frequency in plasmid DNA containing direct repeats--predictive correlation with repeat and intervening sequence length

In this study, a simple non-linear mathematical function is proposed to accurately predict recombination frequencies in bacterial plasmid DNA harbouring directly repeated sequences. The mathematical function, which was developed on the basis of published data on deletion-formation in multicopy plasmids containing direct-repeats (14-856 bp) and intervening sequences (0-3872 bp), also accounts for the strain genotype in terms of its recA function. A bootstrap resampling technique was used to estimate confidence intervals for the correlation parameters. More than 92% of the predicted values were found to be within a pre-established +/-5-fold interval of deviation from experimental data. The correlation does not only provide a way to predict, with good accuracy, the recombination frequency, but also opens the way to improve insight into these processes.     

Development of process flow sheets for the purification of supercoiled plasmids for gene therapy applications.

Human clinical trial of gene therapy with nonviral vectors demands large amounts of pharmaceutical-grade plasmid DNA. Since standard molecular biology methods cannot be used for this purpose, there is a need for the development of processing methodologies for the large-scale production and purification of plasmids. This work describes several studies that were undertaken during the development of process flow-sheets for the downstream processing of supercoiled plasmids. Anion-exchange HPLC was used as a routine technique for monitoring plasmid purity in process streams. The use of RNase or high temperatures during alkaline lysis was proved unnecessary. Instead, RNA could be completely removed by performing sequentially clarification with a chaotropic salt, concentration with PEG, and ion-exchange and size-exclusion chromatography. Also, clarification of streams by precipitation was independent of the chaotropic salt used. Furthermore, by proceeding directly from cell lysis to chromatography it was possible to obtain plasmid with purity/quality identical to that of the one obtained when clarification and concentration were included in the process. This strategy has the advantage of increasing the overall process yield to 38%. The plasmid thus purified was depleted of RNA, chromosomal DNA, and proteins. Additionally, no animal-derived enzymes, alcohols, or toxic solvents were used, rendering validation potentially easier. The results described in this report also indicate that downstream processing times and costs can be considerably reduced without affecting plasmid purity.     

Translational features of human alpha 2b interferon production in Escherichia coli

The yield of human alpha 2b interferon in Escherichia coli was optimized by replacement of low-usage arginine codons located in the mRNA 5' end. The differences observed among the various gene variants suggest that codon usage, Shine-Dalgarno-like sequences, and mRNA secondary structure contribute to the performance of E. coli translation machinery.     

Tempol Moderately Extends Survival in a hSOD1G93A ALS Rat Model by Inhibiting Neuronal Cell Loss,Oxidative Damage and Levels of Non-Native hSOD1G93A Forms

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive dysfunction and death of motor neurons by mechanisms that remain unclear. Evidence indicates that oxidative mechanisms contribute to ALS pathology, but classical antioxidants have not performed well in clinical trials. Cyclic nitroxides are an alternative worth exploring because they are multifunctional antioxidants that display low toxicity in vivo. Here, we examine the effects of the cyclic nitroxide tempol (4-hydroxy-2,2,6,6-tetramethyl piperidine-1-oxyl) on ALS onset and progression in transgenic female rats over-expressing the mutant hSOD1^G93A . Starting at 7 weeks of age, a high dose of tempol (155 mg/day/rat) in the rat´s drinking water had marginal effects on the disease onset but decelerated disease progression and extended survival by 9 days. In addition, tempol protected spinal cord tissues as monitored by the number of neuronal cells, and the reducing capability and levels of carbonylated proteins and non-native hSOD1 forms in spinal cord homogenates. Intraperitoneal tempol (26 mg/rat, 3 times/week) extended survival by 17 days. This group of rats, however, diverted to a decelerated disease progression. Therefore, it was inconclusive whether the higher protective effect of the lower i.p. dose was due to higher tempol bioavailability, decelerated disease development or both. Collectively, the results show that tempol moderately extends the survival of ALS rats while protecting their cellular and molecular structures against damage. Thus, the results provide proof that cyclic nitroxides are alternatives worth to be further tested in animal models of ALS.     

Quantitation of non-amplified genomic DNA by bead-based hybridization and template mediated extension coupled to alkaline phosphatase signal amplification

Klenow I polymerase activity was combined with solid phase DNA hybridization to detect non-amplified genomic DNA (gDNA) sequences from Escherichia coli. Aminopropyl-controlled pore glass surface-bound oligonucleotides were hybridized to fragmented gDNA. The template-mediated extension at the 3′-terminus of the immobilized probe was then promoted in the presence of Klenow I polymerase and digoxigenin-labeled nucleotides. Detection of the extended probes was accomplished with an anti-digoxigenin alkaline phosphatase conjugate protocol coupled to colorimetric or fluorescent detection. Using the colorimetric protocol, the proof-of-concept was established. The fluorescence-based methodology, on the other hand, provided the basis for a quantitative interpretation of the data, affording a detection limit of 5 pM gDNA.     

Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$