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Effective Transduction of Brain Neurons with Lentiviral Vectors Purified via Ion-Exchange Chromatography

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Abstract

The development of methods to purify viral vectors for gene therapy is one of the most important and urgent problems of modern biology and medicine. Drugs that carry cerebral neurotrophic factors have also recently become increasingly popular. However, viral drugs for gene therapy should meet certain requirements, including a high titer and applicability for in vivo studies. At the same time, the creation of such vectors requires cost-effective, inexpensive, and affordable methods for standard laboratories. This study compares various methods for the purification of lentiviral vectors encoding the brain-derived neurotrophic factor, BDNF. The highest titer (1.12 × 109/mL) was obtained via PEG 6000 precipitation followed by anion-exchange chromatography on two columns of sorbents containing quaternary ammonium groups. Abnormal aggregates of transduced neurons were detected after the injection into the brain of a newborn rat pup of lentiviruses that were purified only via PEG precipitation. This fact confirms the necessity of the proposed additional chromatographic purification stage.

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Funding

This work was financially supported by budgetary funding project no. 0259-2019-0003-C-01.

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  1. Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences (ICG SB), 630090, Novosibirsk, Russia

    E. V. Shaburova & D. A. Lanshakov

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  1. E. V. Shaburova
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  2. D. A. Lanshakov
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Correspondence to D. A. Lanshakov.

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Statement on the welfare of animals. All manipulations with animals in this work were carried out in consent with the International European Standards on Bioethics (86/609-EEC), the Russian Rules for Working with Laboratory Animals (no. 267 June 19, 2003), and the Permission of the Bioethics Committee of the Institute of Cytology and Genetics of the Siberian Branch of the Russian Academy of Sciences.

This article does not contain any studies involving human participants performed by any of the authors.

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Translated by I. Gordon

Abbreviations: BDNF—brain-derived neurotrophic factor; CNS—central nervous system; dNTP—deoxyribonucleotide triphosphate; DTT—dithiotreitol; PBS—phosphate-buffered saline; PEG—polyethylene glycol; PFC—prefrontal cortex; PCR—polymerase chain reaction; qPCR—real-time PCR; RT-PCR—reverse-transcription PCR; TBS—Tris-buffered saline; v.p.—virus particles; VSV-G—protein G of the vesicular stomatitis virus; WPRE—WHP posttranscriptional response element.

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Shaburova, E.V., Lanshakov, D.A. Effective Transduction of Brain Neurons with Lentiviral Vectors Purified via Ion-Exchange Chromatography. Appl Biochem Microbiol 57, 890–898 (2021). https://doi.org/10.1134/S0003683821080044

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