Poster CC7 - CRISPR/Cas9 genome editing delivered by BV system

Introduction: Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 technology holds immense promise for cancer therapy owing to its capacity to selectively target and edit genomic DNA. CRISPR/Cas9 offers the potential to correct or disrupt genes implicated in tumor growth or drug resistance, and to modify immune cells to target and eradicate cancerous cells more effectively. However, the lack of a reliable delivery method poses a significant obstacle to the clinical translation of in vivo genome editing. Currently, lipid nanoparticles, hydrodynamic injection, and viral vectors such as lentivirus and adeno-associated viruses, represent the most common methods for in vivo gene delivery. Yet, these methods are plagued by low delivery efficiency and the risk of viral gene integration, thereby limiting their applicability in CRISPR/Cas9 delivery. Baculovirus vectors (BV), derived from an insect virus, harbor a single molecule of circular supercoiled double-stranded DNA ranging from 80 to 180 kb, encoding 100-200 proteins. Notably, BVs exhibit high efficiency in infecting various types of mammalian cells, and no integration into the genome has been reported. CpG sequences abundant within the baculovirus genome, eliciting host-cell activation through Toll-like receptor 9 (TLR9), thereby contributing to the induction of both innate and adaptive immune responses. Previous study has demonstrated that combination of magnetic nanoparticle and a magnet field enhanced BV transduction efficiency in vivo. In this study, we developed baculoviral vectors for the delivery of CRISPR/Cas9 into tumor cells and investigated the impact of BV-pdl1 transfection in MC38 cells.

Materials and

Methods: BV-eGFP, BV-SpCas9, and BV-pdl1 vectors were constructed using the Bac-to-Bac baculovirus expression system and extracted with the ZR BAC DNA Miniprep Kit. The recombinant bacmids were separately transduced into SF-9 cells using Cellfectin II. BV of passage 3 was utilized in the purification study. The titration of BV stock was determined by BacPAK baculovirus rapid titer kit (Takara). MC38 cells were obtained from ATCC and cultured in DMEM-F12 medium supplemented with 10% FBS. To assess BV transfection efficiency in MC38 cells, different multiplicities of infection (MOI) of BV were added to the medium and co-cultured with MC38 for 24 hours before analysis by flow cytometry. PD-L1 knockout efficiency was evaluated 48 hours after BV transfection in MC38 cells. Cell cytotoxicity of BV towards MC38 cells were determined with CCK8 after 24 hours and 48 hours of transfection. To assess immune responses caused by BV-transfected MC38, mouse splenocytes were isolated and co-cultured with BV transfected MC38 cells for 24 hours before analysis with CCK8. Gene expression levels of Granzyme B, IFN-γ, IL-2, IL-10, IL-12 in splenocytes after 24 hours co-culture with BV-transfected MC38 cells were determined by RT-qPCR. Additionally, RNA was isolated from MC38 cells and BV-pdl1-transfected MC38 cells and analyzed using the nCounter tumor signaling 360 panel chip to investigate changes in signaling pathways.

Results, Conclusions, and Discussions: A standard curve for BV titer detection was constructed using the BacPAK baculovirus rapid titer kit (Fig.1A). Among six sgRNAs predicted by VBC-Score to target the mouse Pdl1 gene, the sgRNA with the highest editing efficiency was determined using the T7E1 assay (Fig.1B). MC38 cells infected with BV packaging Cas9 nuclease and the sgRNA exhibited a lower PD-L1 expression (45.5%) compared to untreated cells (Fig.1C). At MOI of 5 and 20, BV-SpCas9 and BV-pdl1 transfections showed little impact on MC38 cell growth (Fig.1D). However, at higher MOIs such as 100 and 500, BV transfection led to MC38 growth inhibition, particularly notable in BV-pdl1-treated MC38 cells, which exhibited significantly higher cytotoxicity than BV-SpCas9-treated MC38 cells (Fig.1E). Protein expression increased with increasing MOIs of both BV-eGFP and BV-pdl1, while PD-L1 expression showed a different trend (Fig.1F & 1G). Consistent with previous studies, the BV we produced demonstrated transient expression property (Fig.1H). Furthermore, splenocytes co-cultured with BV-pdl1-transfected MC38 cells exhibited higher cell cytotoxicity compared to untreated MC38 cells (37.3% vs. 28.7%) (Fig.2A). Gene expressions of Granzyme B, IFN-γ, IL-2, IL-10, and IL-12 further supported the notion that BV transfection stimulated splenocytes to secrete higher amounts of cytokines for tumor cell killing and lower IL-10 production (Fig.2B). nCounter tumor signaling 360 panel analysis of the whole RNA isolated from MC38 and BV-pdl1-transfected MC38 revealed that among the 506 genes analyzed, 26 genes were significantly downregulated (p-Adj < 0.05, fold change ≤ -1.5), and 81 genes were significantly upregulated (p-Adj < 0.05, fold change ≥ -1.5) (Fig.2C). BV-pdl1 transfection upregulated the expressions of 31 signaling pathways and downregulated the expressions of 7 signaling pathways in MC38 cells (Fig.2D). BV-pdl1 showed high efficacy in Pdl1 gene disruption in tumor cell and BV itself as a delivery vehicle proved synergy effect with genome editing to stimulate strong immune response against tumor.

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