| BackgroundAs the most promising new therapeutics,gene therapy has shown obvious advantages in the treatment of hereditary diseases,degenerative diseases and many rare diseases compared with traditional therapies.Gene therapy vectors can be classified as viral vectors and non-viral vectors which include nanoparticles and liposomes.Adeno-associated virus(AAV)vector has become the most commonly used viral vector in clinical trials of gene therapy with its advantages of low immunogenicity,non-integration,long-lasting therapeutic gene expression and high specific tissue and organ tropism.However,the production of recombinant AAV vector remains the biggest obstacle to regular clinical usage,due to its low production efficiency and high cost.AAV is a replication-defective virus,and its productive infection requires the assistance from other helper viruses such as adenovirus and herpes simplex virus to complete its lifecyle.The most widely used rAAV packaging system is the triple transfection of AAV vector plasmid,AAV helper plasmid and adenvirus helper plasmid into human embryonic kidney 293(HEK293)cells.The AAV vector plasmid contains the gene of interest flanked by two inverted teminal repeats(ITRs)of AAV,The AAV helper plasmid contains AAV Rep and Cap genes which provide the essential proteins for AAV replication and packaging in trans.The adenovirus helper plasmid pHelper harboring the adenovirus E2,E4,and VA RNA genes,along with the adenovirus E1 gene which stably integrated into HEK293genome to form the adenovirus helper system.In 2017,we identified Human Bocavirusl(HBoV1)as a novel helper for AAV replication,and the HBoV1 helper genes were identified as NP1,NS2 and BocaSR.BocaSR is a viral Non-coding RNA which shares similarity with adenovirus VAI RNA in sequence and predicted three-dimensional RNA structures.The function of VAI RNA has been extensively studied as a PKR inhibitor.In contrast to VAI RNA,BocaSR doesn't block PKR activation.More importantly,BocaSR can replace with VAI RNA and help AAV replication along with adenovirus E2 and E4 genes.We speculated BocaSR may utilize an alternative pathway in helping AAV replication.PurposeThe purpose of this study is to explore the role and mechanism of helpervirus non-coding RNA(VAI RNA&BocaSR)in helping AAV replication.This study is helpful to understand the replication mechanism of AAV and provide the theoretical basis for the development of a more efficient recombinant adeno-associated virus(rAAV)packaging system.Methods1.The CRISPR-Cas9 gene editing technology was used to construct the PKR gene knockout monoclonal cell lines and verify the knockout effect2.The replication level of AAV was detected in PKR knockout cells.The AAV infectious clone pIAAV2(including ITR,Rep and Cap)plasmid respectively with pHelper plasmid(including E2,E4 and VAI RNA)during or pHelper?VA plasmid(including E2,E4),pBocaHelper plasmid(including NP1,NS2,BocaSR),pBocaHelper?SR plasmid transfection(including NP1,NS2)for the knockout PKR HEK293 monoclonal cell lines,after 48 h by extracting AAV genome DNA and proteins,qPCR was used to detect the replication levels of progeny viruses and WB was used to detect the expression levels of key viral proteins3.The replication levels of AAV in HEK293 and HEK293T cells were detected.pIAAV2 plasmid respectively with pHelper or pHelper?VA,pBocaHelper,pBocaHelper?SR transfection total HEK293 cells and HEK293T cells,after 48 h by extracting Hirt,AAV genome DNA and proteins,DNA,respectively,with the Southern blot detecting virus genome DNA replication,qPCR detection progeny virions packaging level and WB virus key protein expression level4.The HEK293 cell line expressing SV40T antigen was constructed,and the successful construction was verified by WB.Detection in pHelper respectively,pHelper?VA,pBocaHelper or pBocaHelper?SR assisted AAV replication in the cell level.5.The SV40T antigen in HEK293T cells was knocked out by CRISPR-Cas9 gene editing technology,monoclonal cell lines were screened,and the knockout effect of protein expression was verified by Western blot.Southern blot was used to detect the replication status of the plasmid containing the starting point of SV40 replication,so as to verify the function knockout effect of SV40T antigen protein.Detection of AAV in pHelper,pHelper?VA,pBocaHelper or pBocaHelper?SR assisted in the level of replication in the cells6.Transcriptome sequencing was used to compare the effects of non-coding RNA VAI RNA&BocaSR and SV40T antigen on gene expression in HEK293 cells.The transcriptome sequencing results were analyzed to find host factors involved in AAV replication regulated by non-coding RNA or SV40T antigen.Results1.PKR gene knockout HEK293 monoclonal cell line was successfully constructed.PKR protein expression was not detected in PKR knockout cells,and poly(I:C)stimulation did not effectively activate PKR-mediated phosphorylation of eIF2?,compared with control cells.PKR knockdown does not affect the PERK pathway activated by DTT mediated phosphorylation of eIF2?.2.PKR gene knockout does not effectively restore the reduced AAV replication caused by the absence of VAI RNA or BocaSR.PKR in HEK293 cells gene knock out a certain extent,increased the AAV in pHelper?VA or pBocaHelper?SR level of assisted reproduction,but compared with pHelper or pBocaHelper there is still a large gap.3.The function of non-coding RNA(VAI RNA&BocaSR)in the process of assisting AAV replication can be replaced by SV40T antigen.pHelper?VA and pBocaHelper?SR in HEK293T cells auxiliary AAV copy at the same level with pHelper and pBocaHelper respectively.4.A stable cell line expressing SV40T antigen was successfully constructed in HEK293 cells,named HEK293-SV40T.With the increase of passage times,the stable cell line gradually acquired the ability of non-coding RNA-independent assistant AAV replication,and finally reached the same level as AAV replication in the presence of non-coding RNA.5.A monoclonal cell line with SV40T knockout was successfully constructed in HEK293T cells.Non-coding RNA(VAI RNA&BocaSR)was present in the stable cells with HEK293T knockout of SV40T antigen,and the AAV replication level remained unchanged.6.Transcriptome sequencing results showed that compared with HEK293 cells,the stable cell lines of HEK293 overexpressing SV40T were up-regulated and down-regulated in 90 genes in the cells due to the effect of SV40T antigen.Due to the effect of transient transfection of VAI RNA,the expression of 155 genes in the cells was up-regulated and the expression of 184 genes was down-regulated.Due to the effect of transient transfection of BocaSR,the expression of 139 genes in the cells was up-regulated and the expression of 183 genes was down-regulated.Systematic analysis of the sequencing results showed that there was no intersection between the changes of intracellular differential genes caused by SV40T and those caused by transient transfection of non-coding RNA(VAI RNA&BocaSR).In contrast,13 of the intercellular differential genes caused by VAI RNA and BocaSR were up-regulated and 23 were down-regulated.The host factors involved in AAV replication regulated by non-coding rnas are likely to be present in these common differential genesConclusion1.The co-viral non-coding RNA(VAI RNA&BocaSR)is involved in adenoviral replication by mechanisms other than inhibition of the classical protein kinase PKR signaling pathway.2.The function of auxiliary viral non-coding RNA(VAI RNA&BocaSR)to assist AAV replication can be replaced by SV40T.3.SV40T and non-coding RNA(VAI RNA,BocaSR)may assist AAV replication by regulating different signaling pathways,and ultimately change the cell's microenvironment to make it suitable for AAV replication,but the specific signal transduction process remains to be studied. |
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