mRNA-Fect Transfection Reagent to Deliver mRNA in Breast Cancer Cells
Messenger RNA (mRNA) therapy has been developed as an alternative to DNA-based therapies as it avoids several limiting issues associated with the DNA delivery. It can efficiently modify hard-to-transfect non-dividing cells since it does not require entry into nuclear compartment, and avoids the risk of permanent integration of foreign nuclear material into the host genome. For therapeutic applications, mRNA delivery could be used for treatment of diseases caused by a deficiency in specific proteins, because mRNA sequences can be promptly translated into proteins once they are delivered to the cytoplasm. In diseases where a surplus protein exits, one can envision to deliver mRNA to code for inhibitory proteins.
Despite the promise of a wide range of therapeutic applications, the critical issue with mRNA delivery is now deployment of effective delivery systems. While manufacturing and stability issues related to mRNA production appears to be resolved, effective delivery is limiting specific therapeutic applications. RJH Biosciences is dedicated in developing safe and effective nucleic acid delivery systems for plasmid DNA (pDNA), microRNA, short interfering RNA (siRNA), messenger RNA (mRNA) and antisense oligonucleotides (ASOs) to human cells. A specific transfection reagent developed by RJH Biosciences (mRNA-Fect) has been optimized for effective mRNA delivery to variety of cell lines and primary cells. This application note summarizes the technical experience for mRNA delivery in several breast cancer cells.
Human breast cancer (MCF-7, MDA-MB-231 and SUM-149) cells were seeded in 48-well plates at their exponential growth phase. The cells were allowed to attach to the tissue culture plastic and proliferate for 24 hr prior to nucleic acid treatment.
mRNA-Fect was used as the transfection reagent while a GFP (GFP-mRNA) and a TRAIL coding mRNA (TRAILmRNA) were used as model mRNAs.
Complexes of nucleic acids and transfection reagent were prepared in serum free DMEM. The ratio of the total nucleic acid to transfection reagent was 1:5 (w/w).
To a mRNA-Fect solution in DMEM, mRNA solution was added to form complexes. The complexes were incubated for 30 min at room temperature and then added directly to the cells in the presence of their normal (serum-containing) medium.
The transgene expression by the mRNA-Fect/GFP-mRNA treated cells was determined by fluorescence microscopy (Fig. 1).
Anti-cancer activity of mRNA-Fect/TRAIL-mRNA complexes was determined by the MTT assay after 72 hr of transfection and the outcome is expressed relative to the non-treatment groups (Fig. 2).
In each study, a plasmid expressing GFP was used to formulate the complexes for comparison purposes.
Analysis of transgene expression from mRNA delivery is shown in Fig. 1. The fluorescence microscopy analysis indicated some variability in GFP expression among the breast cancer cells. The extent of transfection was dependent on the individual cell types, as was previously seen with pDNA transfection. The SUM-149 cells readily transfected throughout the population.
Figure 1. Fluorescence micrographs of breast cancer cells after 72 hr of GFP-mRNA transfection. The images show distinct populations of transfected cells.
The anti-cancer activity of TRAIL-mRNA delivery by the mRNA-Fect complexes is shown in Fig. 2. All three types of cells gave the expected growth inhibition with TRAIL-mRNA but not the GFP-mRNA. As in GFP expression, the TRAIL-mRNA response of the SUM-149 cells were the most significant with almost complete induction of cell death with TRAIL-mRNA transfection.
Figure 2. Anticancer activity of mRNA-Fect/TRAIL-mRNA treatment in breast cancer cells after 72 hr of transfection. An effective growth inhibition is obtained at 1:5 ratio of nucleic acid:mRNA-Fect reagent. The delivery of GFP-mRNA did not generate any growth inhibition.
Data courtesy of Ms. Bindu Thapa, graduate student at the Faculty of Pharmacy & Pharmaceutical Sciences, University of Alberta, Canada.