Modified RNA: An Efficient and Safe Approach for Gene Delivery In Vitro and In Vivo

Objectives: Therapeutic approaches employing viral delivery of candidate genes are frequently associated with safety concerns. In contrast, non-viral methods mostly show only a rather low efficiency of gene transfer. Therefore, we evaluated the usefulness of modified mRNA (mod RNA) to efficiently deliver target genes into cells in vitro and into whole organs in vivo thereby also circumventing the safety issue.

Methods: In our experiments we used the well-established Rosa26^mT/mG-transgenic mouse. All somatic cells constitutively express Tomato Red (mT). In the presence of Cre recombinase (Cre) mT is excised and affected cells change their fluorescence to green due to the expression of GFP. Primary tail-tip fibroblasts (TTFs) were transfected with a mod RNA coding for Cre recombinase (mod Cre RNA). The appearance of green cells was evaluated by fluorescence microscopy and quantified by flow cytometry. Rosa26^mT/mG transgenic mice received up to six direct injections of mod Cre RNA into the heart and green fluorescence was evaluated 4.5 days after operation by fluorescence microscopy. Cre expression was verified by qRT-PCR.

Results: Primary Rosa26^mT/mG transgenic TTFs were transiently transfected by lipofection using 0.25 µg mod Cre RNA. Green cells were readily detected two days post transfection under a fluorescence microscope. In addition, flow cytometry detected a fraction of ∼25% cells which expressed the GFP reporter. In the next series of experiments we applied graded amounts of mod Cre RNA (25, 50 and 75 µg) in vivo by multiple injections directly into the heart of Rosa26^mT/mG mice. Clearly, 4.5 days post-injection green cells were visible around the original injection sites. In addition, their number correlated to the amount of mod Cre RNA initially injected. In parallel, the expression of Cre was evaluated and this parameter also correlated with the quantity of mod Cre RNA used in the experiments. Of note, in all experiments we never encountered elevated toxicity in the TTF culture or visible lesions around the injection sites in the heart.

Conclusion: Our experiments show that the mod RNA approach effectively delivers genes in vitro into cell cultures and, more importantly, can be used in vivo as a safe tool for gene delivery. Therefore, the application of mod RNAs may provide a versatile method to apply appropriate factors in vivo in regenerative therapeutic regimens.