Functionalization of virus-like particles for improved in vivo immune cell engineering

To address the limitations of conventional chimeric antigen receptor (CAR) T cell manufacturing, we developed a novel in vivo CAR engineering platform based on functionalized virus-like particles (VLPs) that self-assemble with a minimal viral genome to efficiently package and deliver mRNA payloads. Our initial studies compared the level and stability of transgene (GFP) expression in primary human T cells transduced with VSV-G pseudotyped lentivirus (LV) with those transduced with our mRNA-delivering VLPs and circular DNA-forming non-integrating LV (NILV). In these studies, our VLPs transduce over 20% more cells at an equivalent titer than both LV and NILV. As expected, GFP expression from VLP-treated and NILV-treated T cells declined 72 hours after VLP removal, consistent with transient transgene expression. To further improve the platform, we modified the VLP envelope in two ways. First, we added an anti-CD3-targeting single-chain variable fragment (scFv) fused to a low-density lipid (LDL) receptor-blind mutant of the VSV-G fusogen protein (VSVg mut). This construct promotes T cell-specific targeting without non-specific cell binding. In a co-culture model of T cells and Raji B cells, we demonstrated that our scFv-coated VLPs can transduce up to 15% of T cells while less than 1% of B cells are transduced. Because the targeting molecule is derived from blinatumomab, anti-CD3 scFv-coated VLPs also induced upregulation of activation markers CD69 and CD25 in primary human T cells relative to a non-functionalized VLP control. Second, we engineered our VLPs to express an immune-enhancing molecule to both improve mRNA delivery and augment the cytotoxic activity of engineered T cells. While scFv-coated VLPs delivering an anti-CD19 CAR transduced less than 5% of rested primary T cells, enhancer-coated VLPs transduced about 35% of cells and dual-functionalized VLPs (scFv + enhancer) transduced approximately 24% of cells. Next, we used in vitro live cell imaging assays (Incucyte) to quantify killing of CD19+ Nalm6 cells with functionalized VLP-treated rested T cells over 72 hours. While the cells treated with only anti-CD3 scFv-coated VLPs did not elicit a significant anti-tumor activity (demonstrated by continued growth of the tumor cells), targeting T cells using just the enhancer-coated VLPs lead to ~75% decrease in tumor cells compared to control targeted T cells. Notably, T cells treated with dual functionalized VLPs (anti-CD3 scFv plus enhancer) led to almost complete tumor cell killing (>95%). In conclusion, our novel VLP system with using dual immune cell targeting via anti-CD3 and functionalized envelope enhances mRNA delivery and anti-CD19 CAR-T cell-mediated cytotoxicity in vitro . In vivo studies using humanized mouse models are underway to validate this in vivo engineering and anti-tumor efficacy.