An alternative way to RNA interference to prevent the expression of disease-causing proteins.
siRNA mediates the inhibition of gene expression and has become a major tool for vitro analysis of protein function. It could selectively degrade target mRNA through RNA interference (RNAi) and thus prevent the expression of certain disease-causing proteins. Many researchers are now looking into the development of siRNA-based therapeutics. However, siRNA is not exactly that easy to work with. Free siRNAs are prone to degradation by ribonucleases and sometimes trigger the immune system to cause unnecessary inflammatory response. Hence, there is a need for an efficient carrier for siRNA delivery at the same time minimises problems such as immune responses.
Viral Vectors are tools commonly used by molecular biologists to deliver genetic materials into cells, they are generally efficient but often results in unnecessary immune responses. An alternatives to viral vectors are non-viral vectors such as liposome, polymers and some other nanoparticles.
Cationic dendrimers such as poly(propyleneimine) (PPI), poly(amidoamine) (PAMAM), poly(L-Lysine) (PLL) and carbosilane are able to form complexes with siRNA through charge interactions and shielding it from RNases. However, they are highly stable and do not undergo rapid biodegradation to be removed from the body. In this case, bis-MPA dendrimers came in handy by being more biocompatible and biodegradable at physiologically relevant pH and temperature.
Amino-functional bis-MPA dendrimers of generation 1 to 4 (G1 to G4) have been investigated as a non-viral vector for siRNA delivery in primary neurons, astrocytes, glioma, and glioblastoma cell lines. G2 to G4 formed charge-interaction complexes with siRNA efficiently with G3 and G4 showing dose-dependent cytotoxicity towards primary neurons while G2 shows no signs of toxicity. Fluorescence microscopy experiment also suggested efficient endo-lysosomal escape with distinct mitochondrial subcellular localisation for all generation dendrimers. Transfection was successful in rat glioma and human glioblastoma cells with a 20% reduction in target protein expression as compared to 60% for commercially available transfection reagents such as Interferin®.
There is no clear explanation for the low percentage of protein inhibition by siRNA. Some hypothetical explanations are such as the premature release of siRNA due to the rapid degradation of bis-MPA in the cells. Alternatively, it might also due to a strong charge-interaction between dendrimer and siRNA which inhibits the release of siRNA.
Since this is the first study on bis-MPA dendrimers as a potential non-viral carrier for siRNA delivery, the results are relatively promising and more studies are required to understand the system better and potentially discover ways to take measures to improve the existing protein knockdown rate.