Advances in Lentivirus Purification

Moreira, Ana Sofia; Cavaco, David Guia; Faria, Tiago Q.; Alves, Paula M.; Carrondo, Manuel J.T.; Peixoto, Cristina

doi:10.1002/biot.202000019

Cited by 43 publications

(36 citation statements)

References 99 publications

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“…This highlights limited comparability of different retroviral vectors, even though they have the same envelope protein. The observed loss of infectious lentiviral titer due to exposure to high salt concentrations corresponds to the low recoveries reported for anion exchange chromatography during DSP [27].…”

Section: Virus Stability Studymentioning

confidence: 69%

“…It is, therefore, necessary to investigate which process parameters affect the infectious titer. According to current literature it is known that lentiviral vector is prone to damage and loss of infectious titer by several external factors, including temperature, pH, conductivity and shear stress [27]. In this study we analyzed the impact of several physical conditions on the infectivity of a vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped HIV-1 derived LV (Fig 7).…”

Section: Application Of the Newly Established Infectious Titer Assay Protocol To Investigate Lentiviral Vector Stabilitymentioning

confidence: 99%

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Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach

Labisch

Wiese

Barnes³

et al. 2021

PLoS ONE

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The analysis of the infectious titer of the lentiviral vector samples obtained during upstream and downstream processing is of major importance, however, also the most challenging method to be performed. Currently established methods like flow cytometry or qPCR lack the capability of enabling high throughput sample processing while they require a lot of manual handling. To address this limitation, we developed an immunological real-time imaging method to quantify the infectious titer of anti-CD19 CAR lentiviral vectors with a temporal readout using the Incucyte® S3 live-cell analysis system. The infective titers determined with the Incucyte® approach when compared with the flow cytometry-based assay had a lower standard deviation between replicates and a broader linear range. A major advantage of the method is the ability to obtain titer results in real-time, enabling an optimal readout time. The presented protocol significantly decreased labor and increased throughput. The ability of the assay to process high numbers of lentiviral samples in a high throughput manner was proven by performing a virus stability study, demonstrating the effects of temperature, salt, and shear stress on LV infectivity.

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Section: Virus Stability Studymentioning

confidence: 69%

Section: Application Of the Newly Established Infectious Titer Assay Protocol To Investigate Lentiviral Vector Stabilitymentioning

confidence: 99%

Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach

Labisch

Wiese

Barnes³

et al. 2021

PLoS ONE

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“…The complete downstream processing of LVs is composed of numerous individual unit operations which aim to achieve purification, concentration and stability of the vector material (also reviewed in [180]). Briefly, a typical process, involves sequential purification steps consisting of removing cells and their debris followed by enrichment of vector and the removal of host cell or serum proteins, nucleic acids and lipids.…”

Section: Downstream Processing Of Lentiviral Vectorsmentioning

confidence: 99%

Lentiviral Vector Bioprocessing

Perry

Rayat

2021

Viruses

110

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Lentiviral vectors (LVs) are potent tools for the delivery of genes of interest into mammalian cells and are now commonly utilised within the growing field of cell and gene therapy for the treatment of monogenic diseases and adoptive therapies such as chimeric antigen T-cell (CAR-T) therapy. This is a comprehensive review of the individual bioprocess operations employed in LV production. We highlight the role of envelope proteins in vector design as well as their impact on the bioprocessing of lentiviral vectors. An overview of the current state of these operations provides opportunities for bioprocess discovery and improvement with emphasis on the considerations for optimal and scalable processing of LV during development and clinical production. Upstream culture for LV generation is described with comparisons on the different transfection methods and various bioreactors for suspension and adherent producer cell cultivation. The purification of LV is examined, evaluating different sequences of downstream process operations for both small- and large-scale production requirements. For scalable operations, a key focus is the development in chromatographic purification in addition to an in-depth examination of the application of tangential flow filtration. A summary of vector quantification and characterisation assays is also presented. Finally, the assessment of the whole bioprocess for LV production is discussed to benefit from the broader understanding of potential interactions of the different process options. This review is aimed to assist in the achievement of high quality, high concentration lentiviral vectors from robust and scalable processes.

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“…Lentiviruses belonged to the retroviridae family ( 49 , 50 ). Exogenous genes or shRNAs could be effectively integrated into the genomes of dividing or non-dividing cells to achieve the effect of persistent expression of the target sequence ( 51 ).…”

Section: Viral Vectorsmentioning

confidence: 99%

Viral Gene Therapy for Glioblastoma Multiforme: A Promising Hope for the Current Dilemma

Wang

Jia

et al. 2021

Front. Oncol.

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Glioblastoma multiforme (GBM), as one of the most common malignant brain tumors, was limited in its treatment effectiveness with current options. Its invasive and infiltrative features led to tumor recurrence and poor prognosis. Effective treatment and survival improvement have always been a challenge. With the exploration of genetic mutations and molecular pathways in neuro-oncology, gene therapy is becoming a promising therapeutic approach. Therapeutic genes are delivered into target cells with viral vectors to act specific antitumor effects, which can be used in gene delivery, play an oncolysis effect, and induce host immune response. The application of engineering technology makes the virus vector used in genetics a more prospective future. Recent advances in viral gene therapy offer hope for treating brain tumors. In this review, we discuss the types and designs of viruses as well as their study progress and potential applications in the treatment of GBM. Although still under research, viral gene therapy is promising to be a new therapeutic approach for GBM treatment in the future.

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Advances in Lentivirus Purification

Cited by 43 publications

References 99 publications

Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach

Infectious titer determination of lentiviral vectors using a temporal immunological real-time imaging approach

Lentiviral Vector Bioprocessing

Viral Gene Therapy for Glioblastoma Multiforme: A Promising Hope for the Current Dilemma

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