Abstract
<p class="MsoBodyText" style="text-align:justify"><span style="font-size:12pt"><span style="line-height:200%"><span style="font-family:"Times New Roman",serif"><b>Background: </b> Insect baculovirus is a promising vector for gene delivery into mammalian cells. We developed a Cre/loxP-based hybrid baculovirus comprising two viruses: one expressing Cre recombinase and the other harboring the transgene flanked by two loxP sites. Co-transduction of mammalian cells confers Cre expression, which excises the loxP-flanking cassette off baculovirus genome and catalyzes DNA minicircle formation, thereby prolonging transgene expression. Two separate baculoviruses avoid undesirable recombination and loss of transgene during virus production process, but reduces the gene delivery efficiency and complicates applications.</span></span></span></p><p class="MsoBodyText" style="text-align:justify"><span style="font-size:12pt"><span style="line-height:200%"><span style="font-family:"Times New Roman",serif"><b>Methods: </b>To develop an all-in-one baculovirus harboring both the<i> cre</i> gene and loxP-flanking transgene cassette, we exploited synthetic biology strategy to control Cre expression. We controlled <i>cre </i>transcription with a mammalian EF-1<span style="font-family:Symbol">a</span> promoter, and regulated <i>cre</i> translation with an intron inserted within the coding region and bantam miRNA binding site at the 3’UTR.</span></span></span></p><p class="MsoBodyText" style="text-align:justify"><span style="font-size:12pt"><span style="line-height:200%"><span style="font-family:"Times New Roman",serif"><b>Significant findings: </b> The all-in-one baculovirus vector selectively conferred Cre/loxP-mediated recombination in mammalian cells, but not in <i>E. coli</i> and Sf-9 cells, hence circumventing transgene excision from the virus genome during gene cloning in <i>E. coli</i> and virus amplification in insect cells.<a name="_Hlk88551060"><span style="color:red"> The all-in-on vector enabled formation of DNA minicircle in mammalian cells at efficiencies exceeding 80% and implicated its potential for gene delivery.</span> </a> Our design may be expanded to other gene delivery systems that require two separate vectors and is beneficial to gene therapy applications.</span></span></span></p>