Use of RJH Transfection Reagents in Co-Delivery of Plasmid DNA and short interfering RNA

Application Note:

Use of RJH Transfection Reagents in Co-Delivery of Plasmid DNA and short interfering RNA

BACKGROUND

The convergence genetic bioinformatics and nucleic acid (DNA and RNA) chemistry with sophisticated delivery systems has significantly enhanced therapeutic application of nucleic acids. The promising outcomes of nucleic acids have been reflected in many clinical trails (Nat. Rev. Drug Discov. 14, 843-856, 2015; J Gene Med. 20, e3015, 2018). A variety of transfection reagents has been used to deliver plasmid DNA (pDNA), microRNA, short interfering RNA (siRNA) and antisense oligonucleotides (ASOs) to human cells. However, co-delivery of different nucleic acids packaged with a single transfection agent has not been well explored. Disease conditions where multiple signaling pathways are involved in abnormal physiology will benefit from the co-delivery approach (J. Control. Rel. 256, 153-169, 2017). For example, co-delivery of pDNA and siRNA can enable simultaneous knockdown of undesirable proteins with siRNA and forced expression of desirable proteins with pDNA. To deliver pDNA and siRNA with the same reagent will be desirable if one wishes to ensure co-delivery to individual cells. This is not always guaranteed if one were to formulate the pDNA and siRNA complexes separately.

Co-delivering pDNA and siRNA with a single transfection reagent is challenging since the two molecules are structurally different; functional pDNAs are long (>3000 base pairs) flexible molecules while siRNAs are short (<30 base pairs) rigid molecules. complexation and condensation of such different nucleic acids will be ideally undertaken by reagents. one the transfection reagents developed by RJH Biosciences has been identified for effective co-delivery of pDNA and siRNA. This application note summarizes the technical experience for pDNA and siRNA co-delivery in breast cancer cells.

PROCEDURE

Human breast cancer cells (MDA-MB-231) were seeded in 24-well plates. The cells were allowed to attach and proliferate for 24 hours prior to nucleic acid treatment.

  • Complexes of nucleic acids and transfection reagent were prepared in DMEM without serum. The ratio of the total nucleic acid to transfection reagent was 1:5 (w/w).
  • ALL-Fect was used as the transfection reagent. To an ALL-Fect solution in DMEM, a mixture of pDNA (TRAIL plasmid and specific siRNA (against SOD1) solution (1:4 w/w ratio between pDNA and siRNA) were added to form ternary complexes of transfection reagent/pDNA/siRNA. The complexes were incubated for 30 min at room temperature and then added directly to the cells.
  • The investigate uptake of nucleic acid complexes with confocal microscopy, cells were seeded on coverslip (15 mm diameter) inserted into 24 well plate and allowed to attach and proliferate for 24 hours prior to treatment. Cy3-labelled pDNA and FAM-labelled siRNA were used to formulate the complexes. The cells were fixed 24 hours after complex addition for analysis with confocal microscopy (Figure 1).
  • The efficiency of transgene expression by pDNA was determined by TRAIL secretion (2 days posttransfection). A non-specific and a SOD1-specific siRNA was used to formulate the complexes along with the pDNA (Figure 2A). An ELISA was used to determine protein concentration in cell culture supernatants.
  • The efficiency of gene silencing by siRNA was determination by RT-PCR (2 days post-transfection) and using a SOD1-specific siRNA. The same complexes as above were used to treat the cells (Figure 2B).

RESULTS

The uptake of the complexes from confocal microscopy analysis is shown in Figure 1. The transgene expression and gene silencing by the complexes are shown in Figure 2.

Figure 1. Confocal micrographs (scale bars: 20 µm) of MDA-MB-231 cells after co-delivery of pDNA and siRNA. DAPI-stained cell nucleus is visualized as blue. FAM-labelled siRNA in complexes is visualized as green dots. Cy3- labelled pDNA in complexes is visualized as red dots. The merged image shows distinct particles that are mostly orange that is indicative of entrapment of both siRNA and pDNA in the complexes. One predominant green (siRNA) particle is also evident. The particles are distributed in the cytosolic region of the cells as well as being associated with the nucleus.

Figure 2.(A) Transfection of a TRAIL expressing pDNA in MDA-MB-231 cells in the presence of a non-specific and specific siRNA. An effective transfection is obtained at 1:5 ratio of nucleic acid:ALL-Fect reagent, with abundant secretion of the transgene protein. The presence of specific siRNA did not affect the protein secretion. (B). The same complexes were used to transfect MDA-MB-231 cells but this time investigating the silencing of the specific target (SOD1). Formulating the complexes with pDNA and non-specific siRNA did not lead to any silencing, but addition of a specific siRNA significantly down-regulated the mRNA levels of SOD1. The presence of pDNA did not affect silencing activity.

ACKNOWLEDGEMENT

Data courtesy of Ms. Bindu Thapa, graduate student at the Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Canada.

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