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The Increasingly Important Role of Lentivirus Packaging


Abstract: Recently, studies indicate that many diseases like tumors, are closely related to genetic abnormalities. As a result, gene therapy has become a crucial way of clinical treatment of diseases. In order to achieve efficient transfer or transfection of genes, finding suitable gene therapy vectors is the focus of current research. At present, commonly used vectors include eukaryotic vectors and viral vectors, among which viral vectors have attracted attention in recent years. The more frequently used groups include adenovirus, retrovirus, lentivirus and the like. Lentivirus is a gene therapy vector developed on the basis of human immunodeficiency type I virus (HIV-1) with the advantages of large capacity of carrying gene fragments, high transfection efficiency, wide host range, long-term stable expression, etc. It is an ideal vector for transferring genes of interest and used for clinical treatment. The lentivirus vector has been gradually improved and developed to the third generation of the four-plasmid system. The lentivirus gene expression system is a widely used genetic manipulation tool. The plasmid is a genetic unit capable of autonomous replication outside the chromosome, a vector capable of amplifying and expressing a foreign gene to guide bacteria, and a main tool for genetic engineering. The expression system consists of a plasmid of interest for expressing particular gene and a packaging plasmid for the components gag/pol, Rev and the like. The packaging plasmid provides the structural proteins, polymerases and envelope proteins necessary for the packaging of viral genomic mRNA into intact virions. Obtaining high titers of the virus is the key to the later research on gene function. In practice, researchers mostly use commercial lentivirus packaging products, or simply mix individual packaging plasmids, and it is often difficult to obtain optimized viral packaging efficiency for the genes of interest. When the target gene is large, it is possible to obtain a highly efficient virus virion by selecting the optimized virus packaging system. However, research on the components of viral packaging systems to obtain perfect viruses is still lacking. This article reviews the research status of lentivirus plasmid extraction, lentivirus packaging, and lentivirus purification to improve lentivirus titer.

Keywords: lentivirus packaging, lentivirus purification, titer

u Optimization of lentivirus plasmid extraction
In modern biological experiments, it is often necessary to prepare a large number of high-quality plasmids. The extraction efficiency and purity of plasmid DNA are essential for subsequent steps. Common methods to extract plasmid DNA are:

SDS alkaline lysis

Alkali cracking combined with phenol chloroform method

Boiling method

Kit method

The cost of the kit method is so high that most laboratories often use boiling method and classical alkali lysis method. According to relevant literature reports, although the boiling method is short in time and simple in operation, the reaction is too intense during the experiment, which is apt to cause plasmid breakage and low recovery rate. High yield as the alkali lysis method could bring about, it is easy to retain phenol chloroform, and difficult to remove protein impurities. These results are directly related to subsequent molecular biology experiments.

Some researchers have combined the improved alkaline lysis method with hollow fiber ultrafiltration, molecular exclusion chromatography and other purification methods for large-scale production of lentivirus, and successfully obtained a large number of plasmid DNA with high purity and concentration. In addition, the increase in capsid protein, reverse transcriptase and envelope protein in viral packaging is mainly achieved by adjusting the ratio of packaging plasmids, which greatly affects the efficiency of plasmid transfection and lentivirus packaging.

u Lentivirus packaging methods
Viral packaging principally contains two methods of transient transfection and construction of stable packaging cell lines. Currently, lentiviruses used in clinical trials of gene therapy mainly adopt transient transfection method.

Selection of transiently transfected cell lines

In order to increase the virus titer, the researchers used 293T cells with high viral packaging capacity, which can express SV40 large T antigen, and the plasmid containing the SV40 origin of replication can be replicated. The virus titer produced by 293T cells under the same experimental conditions is four times that of HEK293 cells, and 293T cells can be domesticated into serum-free suspension cells, which is conducive to the expansion of virus packaging.

Transient transfection method

Commonly used transient transfection methods are calcium phosphate transfection, lipofection, polyethyleneimine (PEI), and cell electroporation.



Calcium phosphate precipitation method is safe, cheap, and applicable in large-scale production. While, the cell culture needs to add serum to reduce the toxicity of calcium phosphate to cells, and the method is greatly affected by the pH of the medium. In the experiment, the preparation of the solution and the experimental operation is strictly demanded.

The lipofection method has high virus titer yield and low required cell volume, but it is expensive and difficult to be used for large-scale promotion of industry.

The virus yield obtained by PEI transfection method is higher than that of calcium phosphate transfection method and dispensable to change the medium in cell culture. The transfection efficiency is less affected by the pH of the medium, but the PEI-plasmid complex is also toxic to cells.

Electroporation is more suitable for the transfection of suspension cells. In addition, the density and growth state of the cells to be transfected are also important factors affecting the transfection efficiency. Generally, cells with low passage times and vigorous growth stages are selected.



u Lentivirus purification
Laboratory lentiviruses, usually crude viruses obtained by ultracentrifugation, contain more impurities and cannot be used for gene therapy or for the large-scale production of lentivirus purification. To achieve the goal of human gene therapy, lentivirus must remove protein impurities, DNA impurities, endotoxin, etc. from the product to ensure the quality, safety and effectiveness of the product.

Centrifugation and membrane separation techniques

Centrifugation and membrane separation techniques are used for the initial separation and concentration of the desired product, which are suitable for the separation and concentration of heat sensitive substances such as viruses, and has the characteristics of high throughput and low resolution.

Nuclease digestion

The lentivirus packaging process is contaminated with plasmid DNA and host cell nucleic acid with nucleases adopted to remove impurities. And the final viral product is tested to ensure no nuclease residue.

Chromatography

Chromatography is widely used in large-scale purification of biomolecules, featuring fast, high resolution and recyclability. Lentivirus is negatively charged at physiological pH. Ion exchange chromatography is the most efficient chromatographic method for lentivirus purification that can efficiently remove heteroproteins and DNA and at the same time concentrate.

u Prospects and outlooks
With increasing application of lentivirus in clinical trials, researchers are inevitable to study the improvement of lentivirus packaging yield from various aspects. Subsequent analysis can be through orthogonal design and statistical analysis to optimize the various influencing factors of virus packaging & production in order to obtain more effective solutions, while improving the quality and stability of the product. In addition, the construction of safe and effective lentivirus stable packaging cell lines and quality control standards is also the focus of future research. The current lentivirus production is still mainly transfected with adherent cells, and the development of lentivirus packaging cell lines will promote large-scale suspension cells. The development of cultivating technology will also significantly increase the production capacity of lentiviruses.

References
[1] Journal of Yangtze University (Nat Sci Edit) Mar.2014, Vo1.11 No.9

[2] Segura MM, Mangion M, Gaillet B, et al, New developments in lentiviral vector design, production and purification [J]. Expert Opin Biol Ther, 2013, 13 (7):987-1012
publi le vendredi 02 aot 14:39

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