Purification And Biological Characteristics Of Adeno-associated Virus EGFP-AAV9

In recent years,virus-mediated gene therapy has been widely concered.Compared with other viruses,adeno-associated virus(AAV)has been so widely used in gene therapy research mainly because of the unique advantages,such as higher safety,wider host ranges,lower immunogenicity and more stable expression in host cells.It is necessary to establish a simple and efficient method to produce large amount of AAV with higher purity and bioactivities to meet clinical research and gene therapy.Produced commonly from the lysed packaging cells,the present AAV purification is time-consuming and laborious because of the combination with other methods as well as the additional needed processes,such as cell lysis,removing cell debris,host cell DNA and protein,and so on.To obtain greater amounts of enhanced green fluorescent protein adeno-associated virus 9(EGFP-AAV9),firstly,both “the mass ratio between packaging plasmids and polyethylenimine(PEI)transfect reagent”and “the molar ratio among three packaging plasmids ”were optimized.Then EGFP-AAV9 particles were collected from the producer cell medium every two days within the 7-day after transfection,due to its ability which can be mostly released into the producer cell medium posttransfection,to achieve maximum EGFP-AAV9 yields.Next,two simple AAV purification methods: Tangential flow filtration(TFF)ultrafiltration and polyethylene glycol(PEG)8000 precipitation,were chosen to concentrate and purify EGFP-AAV9 after comparing the advantages and disadvantages of various AAV purification methods,then the effects of the two methods on EGFP-AAV9'sbiological characteristics,including the purity,the total number of EGFP-AAV9 genomes and the transduction efficiency in human renal epithelial cell line(293T cells)in vitro,were respectively compared through SDS-PAGE,q PCR and transduction efficiency.After that,to achieve higher purity,they were purified by iodixanol ultracentrifugation,desalted and concentrated later by ultrafiltration centrifuge tube(100 k Da).And then the effects of the two purification methods on EGFP-AAV9 were studied by comparing the purification process and the time consumed during purification,and the effects of the two methods on the quality of EGFP-AAV9 were tested in the same way.Finally,the genomic DNA and total RNA were extracted from the infected cells for PCR amplification and RT-PCR,and the total proteins were extracted for Western Blot(WB)to detect the expression of EGFP in 293T cells.In addition,the viruses were also used to infect chicken fibroblast cell line(DF-1)and lymphoma cell line(DT40)to determine the transduction efficiency of EGFP-AAV9 in chicken cells.Results:(1)The best transfection efficiency could be available when the mass ratio of PEI and plasmid was 1:3 and the molar ratio of three-package plasmid was 2:1:1,with which the maximum amount of EGFP-AAV9 could be produced.(2)The two purification methods consumed almost the same time after the purification of EGFP-AAV9 by TFF-Iodixanol and PE8000-Iodixanol,but the EGFP-AAV9 purified by TFF-Iodixanol could obtain higher titers and higher infection efficiency in 293T cells in vitro.(3)The infection efficiency in 293T cells in vitro could be improved by improving the purity of EGFP-AAV9.(4)EGFP expression was successfully detected by WB in protains level and PCR both in DNA and m RNA levels in EGFP-positive 293T cells.(5)The infection efficiency in chicken cells such as DF-1 and DT40 is extremely low,indicating that it may not be suitable for clinical research of chicken cells in vitro and vivo.Conclusion: In this study,the optimal dosage of three plasmids and PEI was determined for producing a large amount of EGFP-AAV9;the TFF-iodixanol method is much more better in the purification of EGFP-AAV9,and the purified EGFP-AAV9 with it could achieve higher efficiency of 293T cell infection;EGFP-AAV9 has a much lower infection efficiency in chicken cells.