Team:Hong Kong HKU/Gene Delivery
Gene Delivery
mRNA knockdown
Artificial miRNA Silencing cassette
After plasmid gene delivery by Salmonella by endocytosis pathway and endosomal escape, plasmid enters the nucleus and replicates. Artificial miRNA construct (eSIBR cassette) would be produced from transcription, and processed with innate miRNA pathways (Figure).
The production of miRNA would lead to down-regulation of target genes. In our case, it leads to decreased stemness and drug-resistance.
Immunofluorescence microscopy
The NDC is labelled with Cy5. In the immunofluorescence microscopy photo above, we could see the specific binding of NDC to specific cells in spheroid even with repeated washing with mild detergent (PBST). The specific binding of NDC showed the specific targeting and binding of NDC to certain spheroid cells.
Salmonella Aptamer further increasing specific delivery
As suggested by experts, in order to further increase delivery efficiency and safety of the system, a Salmonella aptamer were incorporated onto the NDC. The photo shows the more saturated Cy5 signal, indicating Salmonella facilitated delivery of NDC. The NDC location is more spread out which resembles the Salmonella distribution pattern in spheroid.
amiRNA casette expression
The GFP reporter protein reports the expression of eSIBR cassette, which expresses artificial miRNA constructs. The photo shows the expression of GFP starts as soon as Salmonella invades the spheroid. Intense GFP signals were observed at day 7 (5 days after bacto-infection), showing that Salmonella invasion, phagosomal escape, plasmid delivery to nucleus and also amplification inside mammalian nucleus takes time. It also takes time for amiRNA expression and mRNA downregulation. However, the actual mRNA down-regulation time was not investigated.
Control has been established so to proof that GFP signal is not induced by Salmonella itself, and also there is no leakage of GFP expression with CMV promoter as proven by E. Coli carrying the construct.
Spheroid cytotoxicity assay
In monitoring of spheroid size, the construct alone does not produce a difference in growth of spheroid, without combining with chemotherapy drugs. The cytotoxicity assay with chemotherapy drug (doxorubicin) using MTT was performed, however not shown here due to interference with bacterial signal.
A point to note is that flagella overexpression increases the cytotoxicity of the bacteria, due to higher accumulation of bacteria. However, the flagella overexpression also decreases the escape of bacteria to external environment, which increases the safety level of bacteria therapeutics for clinical application.
Overview of the gene delivery scheme for mRNA knockdown