Screening The Adeno-associated Virus For Targeting And Infecting Of Nasopharyngeal Carcinoma Cells By Establishing DNA Shuffling Library

With the development of science and technology, gene therapy is becoming the trend ofcancer therapy in the future. The so-called tumor gene therapy is through a variety of vectorsintroducing therapeutic genes into tumor or other somatic cells by gene transfer technology inorder to correct the excessive activation of genes or compensate for defective genes. Thus, thetarget cells can get the new features to kill or inhibit tumor cell and achieve the purpose oftreating tumors. In this process, introducing the therapeutic gene into target cells by thecorresponding vector is a critical step. Therefore, looking for an ideal gene vector become animportant aspect of tumor gene therapy.Adeno-associated virus (AAV) belongs to the Parvoviridae family. Due to its remarkablecharacteristics, AAV had become one of very potential virus vector for many years:1) nohuman disease have been found related with AAV;2) AAV has the ability of infecting mitoticcells and non-mitotic cells;3) the range of its host in the human body is wide, such as heart,muscle, liver, etc; In addition, AAV can mediate long-term expression of the target gene intarget cells. But AAV can cause non-specific infections because of its wide host range and thenon-specific expression of carried target gene can lead to off target or immune response,which limits its usage in the gene therapy. Thus, improving the property of AAV targeting andinfection is a key to improve its function as a vector.The AAV’s targeting is determined by the AAV capsid, which contains antigen epitopes,and the latter can direct contact with the host surface and generate an immune response whenAAV invades target cells. Therefore, changing AAV capsid through appropriate methods canyield new types of AAV with efficient targeting and infection on target cells. Currently,Scientists have discovered more than100kinds of AAV serotypes. These different AAVserotypes have high homology, and the AAV2research is the most thoroughly studied of them.Hence this research was based on the archetype AAV2/1, AAV2/2, AAV2/3, AAV2/4, AAV2/7,AAV2/8, AAV2/9which are preserved in our laboratory. AAV2rep gene was used as universalrep gene which provided a basis for universal primer design in the back. This subject used DNA shuffling technology to recombinate the sequences of AAV capsid,and established chimeric AAV library. The library was then used for targeted screen againstthe nasopharyngeal carcinoma cells. The concrete steps are as follows：(1) Establish pAdB-rc vector molecular clone libraryFirst, got AAV capsid sequences by nested PCR, named cap sequence fragments, andrecovered them by gel extraction. Second, digested cap sequence fragments by theDnaseⅠand recovered200-1000bp fragments by gel extraction. Third, using differentenzymes to recombinate the restriction fragments by PCR without primers. Fourth, under theconditions of different enzymes, we amplified recombinant sequences by PCR with primerscontaining restriction sites to obtain a large number of chimeric cap sequence fragments. Bycomparing the DNA shuffling results yielded under a variety of conditions, we screened outthe optimum condition of DNA shuffling, and then repeated DNA shuffling with the optimumcondition to amplify the chimeric cap sequence fragments. Finally, we connected the obtainedsequences with PADB II vector by restriction enzyme digestion to construct pAdB-rc vectors,and transformed them into the competence of SURE bacteria to establish pAdB-rc vectorclone library.(2) Screening of the chimeric AAVOn the basis of the clone library, clones were picked out and the plasmids were extracted,The virus was packaged in HEK293cells by calcium phosphate transfection method to obtainchimeric AAV library. And the obtained chimeric AAV was concentrated and its titer need tobe measured. Then added the virus to the nasopharyngeal carcinoma cells CNE-3by a certainMOI value. By four rounds of screening, we obtained the chimeric AAV virus which can targetand infect CNE-3cells.(3) Detection of Chimeric AAV’s infectious and targeting propertyUsing PCR to isolated the chimeric AAV and the chimeric cap sequence fragment wascombinated with pAdB II by Restriction Enzyme ligation method, sequenced, choose the highproportion of clone by analysis of sequencing results, named cAAV. After obtained cAAV, wepackaged recombinant AAV use cAAV and pAAV with EGFP gene by double-plasmid calciumphosphate transfection method in HEK293, and named cAAV-EGFP. Meanwhile, we used the archetype AAV and pAAV packaged recombinant AAV by the same method, and namedAAV1-EGFP, AAV2-EGFP, AAV3-EGFP, AAV4-EGF, AAV7-EGFP, AAV8-EGFP, AAV9-EGFP.Then we used these eight kinds of recombinant AAVs to infect CNE-3cells respectively andobserved expression of the green fluorescent, proving that the cAAV-EGFP had efficientinfectious to CNE-3cells. At the same time, we used the eight kinds of recombinant AAVs toinfect other cancer cell, and observed expression of the green fluorescent, proving that thecAAV-EGFP had the ability of targeting CNE-3.As a summary, based on the various exploration of DNA shuffling, we found that used PfuDNA polymerase to recombinate by PCR without primer, then used KOD-Plus-DNApolymerase to amplify, could get best result. After that the gained chimeric AAV library waspackaged by the calcium phosphate method, and used the virus library to targeted screen CNE-3nasopharyngeal carcinoma cells. Through the four rounds of screening, we got chimeric AAV(cAAV) preliminary. From sequencing results, the chimeric AAV named as cAAV wascomposed of AAV1, AAV2, AAV3and AAV8. Finally through the detection of the cAAV’sinfectious and targeting property, this study found that the cAAV has higher infectivity to CNE-3and its targeting property to CNE-3was more exclusive compared with its archetype. In brief,the research makes a basic exploration for building chimeric AAV library reasonably as well astargeted screening of therapeutic genes.