Effect of pH on Infectivity and Morphology of Ecotropic Moloney Murine Leukemia Virus

Ye, Kaiming; Dhiman, Harpreet Kaur; Suhan, Joseph; Schultz, Jerome S.

doi:10.1021/bp0200705

Cited by 22 publications

(31 citation statements)

References 15 publications

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“…293T cells and the packaging cell line GP293 (BD Clontech, Palo Alto, Calif.) were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% heat-inactivated fetal bovine serum (FBS; American Type Culture Collection, Manassas, Va.). The retrovirus producer cell line RVPC-1 was grown in DMEM with 10% non-heat-inactivated FBS (40).…”

Section: Methodsmentioning

confidence: 99%

“…Retroviral vectors were produced by cotransfection of a viral vector, pCMVEGFP_neo (40), into GP293 cells with an Env expression plasmid by using the PolyFect transfection reagent (QIAGEN, Valencia, Calif.) as described elsewhere (41). The viral supernatants were collected 48 h posttransfection, filtered through a 0.45-m membrane filter, and stored at Ϫ70°C until use.…”

Section: Methodsmentioning

confidence: 99%

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Tagging Retrovirus Vectors with a Metal Binding Peptide and One-Step Purification by Immobilized Metal Affinity Chromatography

Jin²,

Ataai

et al. 2004

J Virol

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Retroviral vectors produced from packaging cells are invariably contaminated by protein, nucleic acid, and other substances introduced in the manufacturing process. Elimination of these contaminants from retroviral vector preparations is helpful to reduce unwanted side effects, and purified vector preparations are desirable to improve reproducibility of therapeutic effect. Here we report a novel approach to engineer a metal binding peptide (MBP)-tagged murine leukemia virus (MuLV), allowing for one-step purification of retroviral vectors by immobilized metal affinity chromatography (IMAC). We inserted a His 6 peptide into an ecotropic envelope protein (Env) by replacing part of its hypervariable region sequence with a sequence encoding the His 6 peptide. Display of the His 6 tag on the surface of Env endowed the vectors with a high affinity for immobilized metal ions, such as nickel. We demonstrated that the His 6 -tagged MuLV could be produced to high titers and could be highly purified by one-step IMAC. The protein and DNA contaminants in the purified vector supernatants were below 7 g/ml and 25 pg/ml, respectively, indicating a 1,229-fold reduction in protein contaminant level and a 6,800-fold reduction in DNA contaminant level. About 56% of the viral vectors were recovered in the IMAC purification. The purified vectors retained their functionality and infectivity. These results establish that an MBP can be functionally displayed on the surface of ecotropic retroviruses without interfering with their integrity, and MBP-tagged retroviral vectors can be highly purified by one-step IMAC.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Tagging Retrovirus Vectors with a Metal Binding Peptide and One-Step Purification by Immobilized Metal Affinity Chromatography

Jin²,

Ataai

et al. 2004

J Virol

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“…62 Similarly, ecotropic moloney murine leukemia virus has a narrow window of infectivity between a pH of 5.5 and 8.0 with irreversible damage outside of this range. 63 Here, the need for acid-mediated cation release for both internal gelation strategies becomes an important consideration for developing a viable encapsulation strategy. While both internal gelation strategies require an acid, the pH of the CaCO 3 -GDL is controlled by setting a stoichiometric ratio of GDL to CaCO 3 .…”

Section: Discussionmentioning

confidence: 99%

Microfluidic generation of alginate microgels for the controlled delivery of lentivectors

et al. 2016

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Microgels fabricated through distinct microfluidic procedures encapsulate and release functioning lentivectors in a controlled manner.Please check this proof carefully. Our staff will not read it in detail after you have returned it.Translation errors between word-processor files and typesetting systems can occur so the whole proof needs to be read. Please pay particular attention to: tabulated material; equations; numerical data; figures and graphics; and references. If you have not already indicated the corresponding author(s) please mark their name(s) with an asterisk. Please e-mail a list of corrections or the PDF with electronic notes attached -do not change the text within the PDF file or send a revised manuscript. Corrections at this stage should be minor and not involve extensive changes. All corrections must be sent at the same time.Please bear in mind that minor layout improvements, e.g. in line breaking, table widths and graphic placement, are routinely applied to the final version.Please note that, in the typefaces we use, an italic vee looks like this: n, and a Greek nu looks like this: n.We will publish articles on the web as soon as possible after receiving your corrections; no late corrections will be made.Please return your final corrections, where possible within 48 hours of receipt, by e-mail to: materialsB@rsc.orgQueries for the attention of the authors However, achieving safe, localized, and sufficient gene expression remain key challenges for effective lentivectoral therapy. Localized and efficient gene expression can be promoted by developing material systems to deliver lentivectors. Here, we address the utility of microgel encapsulation as a strategy for the controlled release of lentivectors. Three distinct routes for ionotropic gelation of alginate were incorporated into microfluidic templating to create lentivector-loaded microgels. Comparisons of the three microgels revealed marked differences in mechanical properties, crosslinking environment, and ultimately lentivector release and functional gene expression in vitro. Gelation with chelated calcium demonstrated low utility for gene delivery due to a loss of lentivector function with acidic gelation conditions. Both calcium carbonate gelation, and calcium chloride gelation, preserved lentivector function with a more sustained transduction and gene expression over 4 days observed with calcium chloride gelated microgels. The validation of these two strategies for lentivector microencapsulation may provide a platform for controlled gene delivery.

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“…The optimal cell culture parameters have been shown to be producer cell line and viral vector dependent. The optimal pH range for retroviral vector production was found to be between 6.8 and 7.2 for FLY RD18 and Te FLY A7; outside this range the cell specific productivities were considerable lower (McTaggart and Al-Rubeai 2000; Merten 2004), while the retroviral vector was observed to be stable between pH of 5.5 and 8.0 in ecotropic pseudotyped vectors (Ye et al 2003). Both retroviral vectors (MLV derived) and lentiviral vectors (HIV-1 derived), VSV-G pseudotyped, were stable at pH 7.…”

Section: Bioreaction Physicochemical Parametersmentioning

confidence: 96%

Production of Retroviral and Lentiviral Gene Therapy Vectors: Challenges in the Manufacturing of Lipid Enveloped Virus

Rodrigues¹,

Alves²,

Coroadinha³

2011

Viral Gene Therapy

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Effect of pH on Infectivity and Morphology of Ecotropic Moloney Murine Leukemia Virus

Cited by 22 publications

References 15 publications

Tagging Retrovirus Vectors with a Metal Binding Peptide and One-Step Purification by Immobilized Metal Affinity Chromatography

Tagging Retrovirus Vectors with a Metal Binding Peptide and One-Step Purification by Immobilized Metal Affinity Chromatography

Microfluidic generation of alginate microgels for the controlled delivery of lentivectors

Production of Retroviral and Lentiviral Gene Therapy Vectors: Challenges in the Manufacturing of Lipid Enveloped Virus

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