Targeted Delivery of siRNA and CRISPR-Cas9 Using a Nanotechnology Delivery Platform

Abstract

Small interfering RNAs (siRNA) are repressive molecules that inhibit gene expression by promoting mRNA degradation and are being explored as therapeutic agents. However, RNA cannot freely cross cell membranes and requires an effective delivery system that avoids off-target effects and immune activation. Silver sulfide quantum dots (Ag2SQDs) have previously enabled targeted delivery to liver sinusoidal endothelial cells (LSECs) without immune recognition. This thesis investigates their use for targeted oral siRNA delivery to downregulate gene expression.

siRNA was conjugated to a nanocarrier composed of a quantum dot coated with polymers to guide endocytosis, enable lysosomal escape, and promote cytosolic release. This system reduced GAPDH expression following oral administration. In vitro, LSECs showed reduced GAPDH expression after 4 hours with no toxicity. In vivo, orally delivered nanoformulated siRNA significantly reduced LSEC GAPDH expression, comparable to GalNAc tail vein injection, with no effect in hepatocytes, indicating cell-specific targeting.

In a dextran sulfate sodium (DSS) colitis model, siRNA targeting CD98 unexpectedly increased disease severity. In healthy mice, oral delivery reduced CD98 expression in Peyer’s patches, suggesting uptake in gut-associated lymphoid tissue. In diseased mice, nanoparticle accumulation was observed, likely contributing to worsened colitis, highlighting the need for formulation refinement in inflammatory settings.

For CRISPR-Cas9 delivery, free Cas9-GFP showed strong uptake, while nanoparticle-conjugated Cas9-GFP was poorly internalised, indicating challenges related to size, charge, and aggregation.

Overall, Ag2SQDs enabled effective, cell-specific oral siRNA delivery, though further optimisation is needed for CRISPR-Cas9 delivery and use in inflammatory disease.