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 1. Exon skipping in myoblast cells using RJH
transfection reagents. Three different RJH reagents (1:
Leu-Fect A, 2 and 3: 2 proprietary formulations) were used
for ASO delivery to myotubes cells. The transfection
reagents were formulated at ASO:carrier ratios of 1:5 and
1:10 (w/w) using exon-51 specific and mock ASO. The
positive responses of myotubes to ASO-complexes are
indicated by the appearance of exon-51 skipped band.
Approximately 70-80% exon51 skipping was obtained in
this experiment, based on RT-PCR and densitometric
analysis of the bands.