An Ovarian Cancer Tropic Adeno-associated Virus Evolved By DNA Shuffling

As a new technology in combination with modern medicinal and molecular biology, genetherapy has become a new approach for treatment of cancer and hereditary disease. Genetherapy transduce exogenous gene to cells with the help of vectors. Gene therapy vectorsinclude lentivirus(LV), adenovirus(Ad), retrovirus(RV), adeno-associatd virus(AAV),etc.AAV is a nonpathogenic parvovirus. There are over100serotypes of AAV isolated fromand indentified humans and non-human primates so far. Among these, nine naturally occurringserotypes are used commonly. During the infection, various subtypes have different tropism totissues and cells. Compared with other viral vector, AAV has numerous advantages. Firstly,AAV vectors are poorly immunogenic and not associated with disease of human; Secondly,AAV vector can transduce cell efficiently, and AAV vector-mediated gene transfer can expressgene longer, up to several years. Finally, different serotypes of AAV have different tissuetropism. These advantages make AAV an ideal vector. In the past decades, AAV has been usedbroadly as a vector of gene therapy to transduce liver, muscle, retina, etc. with greatachievements.However, the wide host range of AAV results in a problem that AAV lacks the specificityto tissue or cell. Thus, it is crucial to improve the targeting and infection of AAV to cell throughAAV transformation by genetic engineering. The capsid protein coded by the cap gene caninteract with cells by recognizing specific receptor on cell surface. The targeting of AAVdepends on the capsid protein, and the construction of target-based AAV vector has increaseddramatically during these days. Directed evolution of protein provides a good base for thereformation of AAV capsid. We can get highly diversified libraries through directed evolution.Under the selective pressure, in vivo or in vitro, successful and new viruses with enhancedspeciality can be amplified and recovered.DNA shuffling is a homologous recombination technique in vitro. Through modifying single gene or homologous genes with similar function and different sources, this method cancreate new gene which expresses the protein with new function. DNA shuffling has been usedbroadly to evolve the protein in vitro.In this experiment, we used family DNA shuffling technology to transform the cap gene ofAAV1~9serum type (except for AAV5). Through the PCR amplification, we acquired variousparental AAV cap genes. After digesting randomly by DNaseI, we recycled fragments of100~2000bp in size, and used without-primer PCR and with-primer PCR to combine fragmentsinto the full-length DNA. This new cap gene is a stochastic chimera of various serotype capgenes. We connected this cap into the pAdBII vector, constructing a plasmid library.Transfecting HEK293cell, we established the MC-AAV virus library. After5rounds ofselection in vitro, we retrieved a novel qAAV virus that could target and infect ovarian cancercell SK-OV-3f specifically and effectively. A comparative analysis of the gene sequencesrevealed that the chimera is a recombinant of AAV1, AAV2, AAV3and AAV8.We further studied the infectious ability of the qAAV, with parental AAVs as a control.With the same MOI, qAAV was more efficient to infect ovarian cancer cell SK-OV-3f thanother serotypes; Meanwhile, we infected liver cancer cell, lung cancer cell and gastric cancercell with qAAV respectively. The result showed that qAAV was weaker to infect these cancercells, which demonstrated the targeting of qAAV. This mosaic AAV virus library is a solidfoundation for the selection and transformation of AAV.