About me
Development and characterization of LNP for delivery of immunomodulatory CRISPR activation and interference cargo
Location: West Ballroom
Mentor: Dr. Alice Tomei
Type 1 Diabetes (T1D) is a T-cell-driven autoimmune disease characterized by the destruction of insulin-producing β cells within pancreatic islets, causing hyperglycemia and requiring exogenous insulin therapy. Fibroblastic Reticular Cells (FRCs) are unconventional antigen-presenting cells expressing immune inhibitory PD-L1 molecules and low levels of costimulatory factors. PD-L1 suppresses T-cell activation via the PD-1 receptor, making FRCs promising therapeutic targets in T1D. Upregulation of PD-L1 in FRCs could enhance immune suppression, potentially modulating autoimmune responses.
This study explored lipid nanoparticles (LNPs) as delivery vehicles for CRISPR activation and interference (CRISPRa/i) single guide RNA cargo to modulate PD-L1 expression in FRCs. FRCs from non-obese diabetic (NOD) mice were transduced with lentivectors for stable expression of dCas9-VPR and dCas9-KRAB. RiboGreen assays confirmed RNA-loaded LNP stability for at least one week. Viability assays demonstrated that these RNA-loaded LNP formulations were non-toxic, as FRC viability remained stable regardless of LNP dose. Optimal FRC seeding density (25,000 cells/well) determined via RealTime-Glo™ MT Cell Viability Assay showed consistent linear growth (R² > 0.95) over 72 hours. Preliminary flow cytometry confirmed stable baseline FRC phenotype post-transfection with RNA-loaded LNPs.
Our results indicate RNA-loaded LNPs are stable and biocompatible delivery vehicles for CRISPRa/i applications in FRCs, offering a platform to study immunomodulatory mechanisms and explore their therapeutic potential as a novel nucleic acid-based approach for treating T1D.
