Rational design for controlled release of Dicer-substrate siRNA harbored in phi29 pRNA-based nanoparticles

Daniel W Binzel,Songchuan Guo,Hongran Yin,Tae Jin Lee,Shujun Liu,Dan Shu,Peixuan Guo

doi:10.1016/j.omtn.2021.07.021

Abstract

Small interfering RNA (siRNA) for silencing genes and treating disease has been a dream since ranking as a top Breakthrough of the Year in 2002 by Science. With the recent FDA approval of four siRNA-based drugs, the potential of RNA therapeutics to become the third milestone in pharmaceutical drug development has become a reality. However, the field of RNA interference (RNAi) therapeutics still faces challenges such as specificity in targeting, intracellular processing, and endosome trapping after targeted delivery. Dicer-substrate siRNAs included onto RNA nanoparticles may be able to overcome these challenges. Here, we show that pRNA-based nanoparticles can be designed to efficiently harbor the Dicer-substrate siRNAs in vitro and in vivo to the cytosol of tumor cells and release the siRNA. The structure optimization and chemical modification for controlled release of Dicer-substrate siRNAs in tumor cells were also evaluated through molecular beacon analysis. Studies on the length requirement of the overhanging siRNA revealed that at least 23 nucleotides at the dweller’s arm were needed for dicer processing. The above sequence parameters and structure optimization were confirmed in recent studies demonstrating the release of functional Survivin siRNA from the pRNA-based nanoparticles for cancer inhibition in non-small-cell lung, breast, and prostate cancer animal models.

Highlights

RNA interference (RNAi) is a post-transcriptional gene regulation pathway used by different classes of small RNAs.[1]
RNA nanoparticles harboring Small interfering RNA (siRNA) were processed by Dicer into functional siRNA resulting in cleavage of target gene RNA nanoparticles based on the phi[29] packaging RNA (pRNA) were constructed to include Lamin[25] siRNA extended on the pRNA 50/30 terminal end
Lamin A/C was chosen as a model gene to Molecular Therapy: Nucleic Acids demonstrate the specific cleavage of messenger RNA (mRNA) by a 50 rapid amplification of cDNA ends (RACE) technique to prove that the gene-silencing activity of pRNA nanoparticles is mediated by RNA interference.[73] pRNA-Lamin[25] was designed similar to that described by Zhang et al.,[74] with 25-bp siRNA linked to a pRNA vector by a double uracil (-UU-) linker (Figure 1A)