Retroviral Vector Stability: Inactivation Kinetics and Membrane Properties

Cruz, Pedro; Carmo, Marlene; Coroadinha, Ana S.; Bengala, André; Gonçalves, Délia; Teixeira, Miguel; Merten, Otto‐Wilhelm; Gény-Fiamma, C.; Carrondo, Manuel J.T.

doi:10.1007/1-4020-3103-3_60

Cited by 5 publications

(7 citation statements)

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“…The virus presented higher cholesterol content than the cells, as also observed in HIV-1 virus (Aloia et al, 1993), as they assemble and bud from the cells in membrane cholesterol rich microdomains (lipid rafts). A reduction in cholesterol content from 35% to 25% occurred in the viral membrane when osmolality was raised from standard 335 to 450 mOsm/ kg, originating a more fluid vector membrane; this is coherent with the higher vector stability obtained at higher osmolalities and with the results described by Beer et al (2003) and Cruz et al (2005) where the more stable retroviral vectors had a more fluid membrane and a lower cholesterol content.…”

Section: Discussionsupporting

confidence: 80%

“…Despite of these observed increments, the cell yield obtained using fructose medium was always lower than the cell yields obtained at the corresponding osmolalities when glucose was used as the sugar source. This was due to the deficient uptake of fructose that was nevertheless enhanced at increasing concentrations (Coroadinha et al, 2005). At 500 mOsm/kg the enhancement in fructose uptake effect was overcome by the osmotic pressure effect and the cell yield was the lowest obtained.…”

Section: Effect Of Osmolality On the Retroviral Vector Productionmentioning

confidence: 94%

“…Thus, the lipid viral membrane is an important part of the virus composition and the viral membrane fluidity has been shown to be correlated with the retrovirus stability and infectivity (Beer et al, 2003;Cruz et al, 2005;Harada et al, 2005). Therefore, this manuscript analyses the membrane phospholipid and cholesterol composition of the retroviral vectors produced in media with different osmolytes and osmolalities and correlates it with the vector stability.…”

Section: Introductionmentioning

confidence: 99%

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

Coroadinha

Silva

Pires

et al. 2006

Biotech & Bioengineering

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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.

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Section: Discussionsupporting

confidence: 80%

Section: Effect Of Osmolality On the Retroviral Vector Productionmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 99%

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

Coroadinha

Silva

Pires

et al. 2006

Biotech & Bioengineering

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“…However, the advantages of culturing cells at 32°C could not be corroborated by other investigators (Forestell et al 1995;Cruz et al 2000) leading to a more detailed examination of the mechanism of thermal stability in retroviruses. It could be shown that retroviral vectors produced at 37°C were characterized by a higher thermal stability than those produced at 32°C (Cruz et al 2005). It was revealed that virus stability was inversely proportional to the level of cholesterol in the viral membrane; hence, depletion of viral cholesterol resulted in increased thermal stability.…”

Section: Culture Conditionsmentioning

confidence: 99%

“…This effect depends on the vector type: retroviral vectors with the amphotropic envelope protein produced at 32°C by TE FLY cells are more rigid than those produced at 37°C, the opposite being observed for vectors with the GALV envelope protein. The differences with respect to rigidity, production temperature and inactivation kinetics observed for different producer cell lines and different env proteins can be explained by differences in the activation energy for the inactivation of these vectors (Cruz et al 2005). Thus, vector thermal stability can be influenced by controlling the individual lipid components in the particle membrane by adjusting cultivation temperature or by the host cell line.…”

Section: Culture Conditionsmentioning

confidence: 99%

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

2006

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Gene therapy is a promising technology for the treatment of several acquired and inherited diseases. However, for gene therapy to be a commercial and clinical success, scalable cell culture processes must be in place to produce the required amount of viral vectors to meet market demand. Each type of vector has its own distinct characteristics and consequently its own challenges for production. This article reviews the current technology that has been developed for the efficient, large-scale manufacture of retrovirus, lentivirus, adenovirus, adeno-associated virus and herpes simplex virus vectors.

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Viral Vectors for Gene Therapy

Warnock

Al‐Rubeai

2014

Encyclopedia of Industrial Biotechnology

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Gene therapy has faced many challenges over the past 20 years as it has made its way toward clinical realization. In 2012, the EMA approved the first gene therapy for European markets, marking a landmark for the technology. Other gene therapies will inevitably follow with an increasing number now progressing to late‐stage clinical trials. To date, manufacturing processes have been able to produce enough viral vectors to satisfy the demands of these studies. However, current processes need to be scaled up if commercial demand is to be met, especially for therapies that treat diseases with large patient populations.

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Retroviral Vector Stability: Inactivation Kinetics and Membrane Properties

Cited by 5 publications

References 10 publications

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

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

Cell Culture Processes for the Production of Viral Vectors for Gene Therapy Purposes

Viral Vectors for Gene Therapy

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