Each resulting immunogenic neoantigen sequence from this extensive screening process will then be incorporated into a single gene with the fascin promoter and edited into the genome of differentiated dendritic cells via CRISPR (Population 1). The fascin gene promoter is highly active in mature dendritic cells, making it an ideal element of the researchers' neoantigen gene construct (Bros et al. 2003). A second gene construct identical to the first but including a signal sequence directed to the endoplasmic reticulum (ER) will be edited via CRISPR into a second population (Population 2) of differentiated dendritic cells. These two populations will be kept together in vitro and all dendritic cells will be treated in solution with Poly(I:C), a toll-like receptor 3 (TLR3) agonist shown to induce activation of dendritic cells (Garzon-Muvdi et al. 2018). Since the fascin gene promoter is active in mature dendritic cells, expression of these genes in both dendritic cell populations will result in expression of both endogenous and exogenous neoantigen peptides. In population 1, the expressed endogenous neoantigen peptides will be processed by the proteasome and subsequently move to the ER, bind to MHC class I, and display on the plasma membrane. In population 2, the addition of the signal sequence to the ER will result in the secretion of neoantigen peptides, which then can be recognized by population 1 dendritic cells as “exogenous.” While displaying these same neoantigens on MHC class I, these dendritic cells will also phagocytose the neoantigens secreted by population 2. Since these neoantigens are exogenous relative to population 1, they will be displayed via MHC class II on the plasma membrane.
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