Application Note:

Use of RJH Transfection Reagents for mRNA Delivery to PBMCs

BACKGROUND

Peripheral blood derived mononuclear cells (PBMCs) are important mediators involved in immune surveillance and are actively explored as the foundation of a wide range of therapies [1]. It is convenient to isolate the cells from patients and re-administer them after desired manipulations. There are various efforts to genetically modify the cells using vectors that permanently integrate their cargo into cellular genome. Transient expression of transgenes, on the other hand, is more desirable to exert some control over the expression kinetics [2]. The messenger RNA (mRNA) has emerged as the reagent of choice for modifying PBMC since it can be processed in cytoplasm and yield therapeutic proteins in large quantities in a short period of time [3]. Non-viral delivery of mRNA minimizes any effects on the host genome and leads to a safer intervention. Transfection reagents developed by RJH Biosciences are particularly suitable for this application since they are highly compatible with human cells and achieve elevated levels of delivery with mRNAs. The transfection reagents are effective in different tissue culture media as well as in high serum concentration (up to 50%), so that they can be effectively used with a range of immune cells. This application note summarizes experience with transfection of PBMC with plasmid DNA (pDNA) and mRNA and using the speciality reagents developed by RJH Biosciences.

MATERIALS and METHODS (general procedures)

  • Complexes of pDNA and mRNA and the transfection reagents were prepared in RPMI without serum. The ratio of the nucleic acid to transfection reagent was typically 1:5 (w/w).
  • The mRNA-Fect was used as the transfection reagent. To a pDNA or mRNA solution in RPMI, mRNAFect solution (undiluted; 1 mg/mL) was added to form complexes. They were incubated for 30 min at room temperature and then added to empty multiwell plates.
  • The PBMCs purified from blood were then added to the wells containing the complexes. Typically, 100,000 PBMCs could be used in a single well of 96-well plate. For comparison, lymphocyte cell lines were also transfected along with the PBMCs in the same way. Typical concentrations for nucleic acids and transfection reagents in cell culture medium were 1 and 5 µg/mL, respectively.
  • It is typically necessary to activate the immune cells with appropriate stimulus before transfection. We found the phorbol myristate acetate/ionomycin (PMA/IO) combination or CD3/CD28 antibody combination to work effectively for activation. In this application note, cells were either not activated at all or activated with PMA/IO or CD3/CD28 antibodies for 2 days before transfection.
  • Transgene expression was determined by fluorescent microscopy and flow cytometry. The mean GFP fluorescence/cells and percentage of cell population positive for GFP are calculated (by normalizing non-treated cells to 1%).

RESULTS and DISCUSSION

To assess efficiency of mRNA-Fect in suspension-growing cells, lymphoblastic K562 cell line derived a female chronic myeloid leukemia patient and monocytic THP-1 cell line derived from a male acute myocytic leukemia patient were initially used. These are common cell lines that are easily propagated in culture for various experimental purposes. Using a GFP-expressing mRNA from a commercial vendor, cells were transfected with a commercial lipofection reagent and mRNA-Fect. GFP expression was investigated after 2 days. As shown in Figure 1, it was typical to obtain 40-60% transfection efficiency in the cell lines, which was higher for mRNA-Fect than a leading commercial lipofection reagent.

mRNA_Transfection_Reagent_PBMC_Cells
Figure 1. Typical transfections seen with lymphocytic cell lines. (Left) Wild type K562 (K562-WT) and THP-1 cells were transfected with a GFP expressing mRNA using a lipofection reagent and mRNA-Fect from RJH Biosciences. The GFP expression was visualized after 2 days with a fluorescence microscope. (Right) The extent of GFP expressing cells was quantified by flow cytometry and summarized as the percentage of cells expressing GFP.

PBMCs from two sources (donors) were transfected without and with CD3/CD28 activation for 24 hours, after which the cells were transfected with pDNA or mRNA complexes formed with mRNA-Fect. Transfection without activation did not lead to significant level of transfection with either mRNA or pDNA complexes. With activation, PBMCs from both sources gave significant GFP expression, although some variation depending on the source was evident.

pDNA_mRNA_Delivery_PBMC_cells_Transfection_reagent_mRNA-Fect

Figure 2. Microscopy images of PBMCs (from two separate donors) transfected with pGFP and mGFP complexes formed with mRNA-Fect. Top row shows the transfection results with CD3/CD28 activated cells, while the bottom row shows the results from inactivated cells. Pictures are overlays of DAPI-stained and GFP expressing cells.

In a separate experiment, PBMCs from another donor were activated with PMA/IO and CD3/CD28 and the extent of transfection was measured after 2 days of transfecting the cells with mGFP complexes of mRNA-Fect. The results are summarized in Figure 3. Similar to the results seen in the microscopy pictures in Figure 2, the inactivated cells did not give any transfection with the mGFP complexes. Both PMA/IO and CD3/CD28 activated cells gave significant transfection, with CD3/CD28 activated cells giving significantly higher levels of GFP expression in the modified cells.

Flow_cytometry_mGFP_mRNA-Fect_PBMC_Cells

Figure 3. Flow cytometry analysis of PBMCs transfected with mGFP complexes formed with mRNA-Fect. Left graph shows the percentage of GFP-positive cells while the right graph shows the extent of GFP fluorescence per cell. The cells were either inactivated, or activated with PMA/IO and CD3/CD28 combination. Note that PMA/IO activated cells gave some fluorescence even in the absence of transfection. The CD3/CD28 activated cells gave much higher GFP fluorescence per cell compared to PMA.IO activated cells.

Benefits of RJH Transfection Reagents

  • High transfection efficiency tailored for specific cell types.
  • Simple protocol that is amenable for automation and scale-up.
  • Minimal toxicity on target cells allowing nucleic acid effects to be manifested without complication.
  • Cost-effective reagent minimizing additional costs in large screens due to transfection reagent
  • Possibility of using the same transfection reagent in animal models, leading to consistent studies.

References

  1. Lee DA. Cellular therapy: Adoptive immunotherapy with expanded natural killer cells. Immunol Rev. 2019, 290(1): 85-99.
  2. Ewart D, Peterson EJ, Steer CJ. A new era of genetic engineering for autoimmune and inflammatory diseases. Semin Arthritis Rheum. 2019, 49: e1-e7.
  3. Uludag H, Ubeda A, Ansari A. At the intersection of biomaterials and gene therapy: Progress in non-viral delivery of nucleic acids. Front Bioeng Biotechnol. 2019, 7: 131.

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