Use of RJH Transfection Reagents in Antisense Oligonucleotide Aso Delivery


Use of RJH Transfection Reagents in Antisense Oligonucleotide Delivery


The convergences of sophisticated delivery systems, oligonucleotide chemistry and genetic bioinformatics has significantly enhanced the exploration of antisense oligonucleotides for therapeutic purposes, which has been reflected in many clinical studies (Nat. Rev. Drug Discov. 11, 125–140, 2012; Annu. Rev. Pharmacol. Toxicol. 50, 259–293, 2010). ASO is a single stranded nucleic acid first introduced in 1978 by Stephenson and Zamecnik to silence target genes (PNAS, 75, 285-288, 1978). Since then, it has been developed as a potential tool for diverse application ranging from restoration of protein expression to modification of mutant protein. RNaseH-mediated degradation or the modulation of splicing of complementary mRNA is the fundamental mode of action in ASObased therapy. Studies have explored ASO mediated experimental and therapeutic approaches to modulate biological function at molecular level in both in vitro and in vivo model (Hum. Gene Ther. 26, 475–485, 2015). A variety of transfection reagents have been used to deliver ASO to human cells. Complexes of ASO with polycations (e.g., peptides, cationic polymers and dendrimers) are convenient to prepare and offer safe alternatives for use. The transfection reagents developed by RJH Biosciences have been tested and found suitable the ASO delivery. This note summarizes the technical experience for ASO delivery for ASO-mediated modulation of splicing in muscle cells by an independent researcher.


  • Immortalized human myoblasts were differentiated to myotubes and seeded in 12-wells for transfection.
  • Cells were transfected with ASO skipping DMD exon 51 or random control (mock) ASO complexes by using
  • complexes formed at 1:5 and 1:10 ratios
  • Polymer solutions (100 or 200 µg) were added to ASO solution (20 µg) to get complexes at 1:5 and 1:10 ratios.
  • The complexes were incubated for 30 min at room temperature before addition to the myotubes
  • Transfection medium was replaced after 2 days with medium and cells were incubated for 2 additional days.
  • Cells were harvested 4 days post-transfection (or 8 days post-differentiation). RNA was collected using Trizol
  • cDNA synthesized using SuperScript IV, and RT-PCR done using GoTaq for primer in DMD exon 49/50 and exon
  • 52. PCR products were resolved in 2% agarose gels, and post-stained with GelRed.


Figure 2. CRISPR-Cas9 genome editing efficiency using a commercial kit designed for CD4 editing. Transfection of the cells was performed with and Lipo2000 and 4 transfection reagents from RJH Biosciences that is currently under development. The relative editing efficiency with RJH reagents matched or exceeded the efficiency obtained with the leading lipofection reagent.