Biomedical subjects

A S Coroadinha

Publications and source records attributed to A S Coroadinha.

7 recordsLinked to original sources

PubMed2006

Effect of osmotic pressure on the production of retroviral vectors: Enhancement in vector stability.

The use of Moloney murine leukaemia virus (MoMLV) derived retroviral vectors in gene therapy requires the production of high titer preparations. However, obtaining high titers of infective MoMLV retroviral vectors is difficult due to the vector inherent instability. In this work the effect of the cell culture medium osmotic pressure upon the virus stability was studied. The osmolality of standard medium was raised from 335 up to 500 mOsm/kg using either ionic (sodium chloride) or non-ionic osmotic agents (sorbitol and fructose). It was observed that, independently of the osmotic agent used, the infectious vector inactivation rate was inversely correlated with the osmolality used in the production media; therefore, the use of high medium osmolalities enhanced vector stability. For production purposes a balance must be struck between cell yield, cell productivity and retroviral stability. From the conditions tested herein sorbitol addition, ensuring osmolalities between 410 and 450 mOsm/kg, yields the best production conditions; NaCl hampered the viral infectious production while fructose originates lower cell yields. Lipid extractions were performed for cholesterol and phospholipid analyses showing that more stable viral vectors had a 10% reduction in the cholesterol content. A similar reduction in cholesterol was observed in the producer cells. A detailed analysis of the major phospholipids composition, type and fatty acid content, by mass spectrometry did not show significant changes, confirming the decrease in the cholesterol to phospholipids ratio in the viral membrane as the major reason for the increased vector stability.

Cell Line↗

PubMed2006

A new generation of retroviral producer cells: predictable and stable virus production by Flp-mediated site-specific integration of retroviral vectors.

We developed a new strategy that provides well-defined high-titer producer cells for recombinant retroviruses in a minimum amount of time. The strategy involves the targeted integration of the retroviral vector into a chromosomal locus with favorable properties. For proof of concept we established a novel HEK293-based retroviral producer cell line, called Flp293A, with a single-copy retroviral vector integrated at a selected chromosomal locus. The vector was flanked by noninteracting Flp-recombinase recognition sites and was exchanged for different retroviral vectors via Flp-mediated cassette exchange. All analyzed cell clones showed correct integration and identical titers for each of the vectors, confirming that the expression characteristics from the parental cell were preserved. Titers up to 2.5 x 10(7) infectious particles/10(6) cells were obtained. Also, high-titer producer cells for a therapeutic vector that encodes the 8.9-kb collagen VII cDNA in a marker-free cassette were obtained within 3 weeks without screening. Thus, we provide evidence that the precise integration of viral vectors into a favorable chromosomal locus leads to high and predictable virus production. This method is compatible with other retroviral vectors, including self-inactivating vectors and marker-free vectors. Further, it provides a tool for evaluation of different retroviral vector designs.

Animals↗

PubMed2006

Effect of medium sugar source on the production of retroviral vectors for gene therapy.

The production of gene therapy retroviral vectors presents many difficulties, mainly due to vector instability and low cell productivities hampering the attainment of high titers of infectious viral vectors. The objective of this work is to increase the production titers of retroviral vectors by manipulating the sugar carbon sources used in bioreaction. Four sugars were tested (glucose, galactose, sorbitol, and fructose) on an established Moloney murine leukemia virus (MoMLV) producer cell line. Galactose and sorbitol did not support cell growth or vector production. Glucose supplemented at 25 mM supported the highest cell growth; however, the use of glucose or fructose at 83 and 140 mM have shown to improve the infectious vector titer three to fourfold. The reasons for the titer improvements were further analyzed and, although, the cell-specific productivity in viral transgene RNA and reverse transcriptase were augmented 5- and 6-fold for glucose at 140 mM and 14- and 16-fold for fructose at 140 mM, comparing with glucose at 25 mM, these increases did not seem sufficient to account for the 14- (140 mM glucose) and 32- (140 mM fructose) fold increment obtained for the infectious particles-specific productivity. Further accounting the enhancement in the titers was the improvement in the viral stability, the half-life of the vectors was enhanced by 30-60%. This resulted in a product quality with a superior ratio of infectious to total particles, thus reducing the most problematic contaminant in the production of retroviral vectors, non-infectious retroviral particles.

Carbohydrates↗

PubMed2006

Retrovirus producer cell line metabolism: implications on viral productivity.

The production of retroviral vectors by human cell lines is still hampered by low titers making it relatively difficult to produce very large quantities of this vector of high interest for clinical gene therapy applications. Thus, to improve vector production, we studied the influence of different sugars alone or combinations of sugars on cell growth, vector titers, and metabolism of the producer cell. The use of fructose at 140 mM or a mixed medium (with glucose at 25 mM and fructose at 140 mM) improved the virus titer three- to fourfold, respectively, and the producer cell productivity by fivefold. The increase in the cell productivity was due to a 1.5-fold increase in the vector stability, the remaining increase being due to higher cell specific productivity. The increase in the productivity was associated with lower glucose oxidation and an increase in the lactate and alanine yield. In the mixed medium, an increase in fatty acids derived from the glucose was observed in parallel with a reduction of glutamate and glutamine synthesis via the tricarboxylic acid (TCA) cycle acetyl-CoA and alpha-ketoglutarate, respectively. Although the higher productivities were associated with severe changes in the glycolysis, TCA cycle, and glutaminolysis, the cell energetic status monitored by phosphocreatine and adenosine triphosphate levels was not significantly affected. The synthesis of fatty acids and phospholipids were enhanced in the fructose or mixed media and are possibly key parameters in retroviral vector production.

Adenosine Triphosphate↗

PubMed2006

The use of recombinase mediated cassette exchange in retroviral vector producer cell lines: predictability and efficiency by transgene exchange.

Currently, retroviral vector producer cell lines must be established for the production of each gene vector. This is done by transfection of a packaging cell line with the gene of interest. In order to find a high-titer retroviral vector producer clone, exhaustive clone screening is necessary, as the random integration of the transgene gives rise to different expression levels. We established a virus producing packaging cell line, the 293 FLEX, in which the viral vector is flanked by two different FRT sites and a selection trap. Using Flp recombinase mediated cassette exchange; this vector can be replaced by another compatible retroviral vector. The first step was the tagging of 293 cells with a lacZ reporter gene, which allowed screening and choosing a high expressing chromosomal locus. After checking that, a single copy of the construct was integrated, cassette exchangeability was confirmed with a reporter targeting construct. Subsequently gag-pol and GaLV envelope genes were stably transfected. The lacZ transgene was replaced by a GFP transgene and the 293 FLEX producer cell line maintained the titer, thus validating the flexibility and efficacy of this producer cell line. The tagged retroviral producer cell clone should constitute a highly advantageous cell line since it has a predictable titer and can be rapidly used for different therapeutic applications.

Cell Line↗

PubMed2006

Relationship between retroviral vector membrane and vector stability.

The present work studies the physico-chemical properties of retroviral vector membrane, in order to provide some explanation for the inactivation kinetics of these vectors and to devise new ways of improving transduction efficiency. For this purpose, vectors with an amphotropic envelope produced by TE Fly A7 cells at two culture temperatures (37 and 32 degrees C) were characterized by different techniques. Electron paramagnetic resonance (EPR) results showed that vectors produced at 32 degrees C are more rigid than those produced at 37 degrees C. Further characterization of vector membrane composition allowed us to conclude that the vector inactivation rate increases with elevated cholesterol to phospholipid ratio. Differential scanning calorimetry (DSC) showed that production temperature also affects the conformation of the membrane proteins. Transduction studies using HCT116 cells and tri-dimensional organ cultures of mouse skin showed that vectors produced at 37 degrees C have higher stability and thus higher transduction efficiency in gene therapy relevant cells as compared with vectors produced at 32 degrees C. Overall, vectors produced at 37 degrees C show an increased stability at temperatures below 4 degrees C. Since vector membrane physico-chemical properties are affected in response to changes in culture temperature, such changes, along with alterations in medium composition, can be used prospectively to improve the stability and the transduction efficiency of retroviral vectors for therapeutic purposes.

Animals↗

PubMed2004

Quantitation of MLV-based retroviral vectors using real-time RT-PCR.

Murine leukaemia virus-based vectors quantitation is a time consuming process that can take up to five days. In order to reduce this time a real-time RT-PCR was developed. This method quantifies vectors without an RNA extraction step, using AMV reverse transcriptase and LightCycler technology. Besides a low quantitation time, this method has the advantages of using a plasmid DNA standard curve with good reproducibility, and of having a high sensitivity (3 x 10(2) particles/microl) as well as an excellent intra- and inter-assay reproducibility. Although the method described quantifies vector particles with RNA whether these particles are infectious or not, it is possible to use it to determine infectious particles concentration after the establishment of a correlation between particles with RNA and infectious particles, for a given set of conditions. This method can also be used to study vector stability by comparison of infectious particles, total particles and particles with RNA.

Genetic Vectors↗