Modified mRNA: Getting to the Heart of Gene Therapy

July 2014

Messenger RNA (mRNA) for clinical use is not a new concept. In 2000, Hans-George Rammensee, Günther Jung and colleagues accidentally discovered that direct injection of messenger RNA elicits an immune response (Hoerr et al., 2000). This discovery led to years of research utilizing mRNA for vaccine development. mRNA naturally has advantages over traditional vaccines: it does not require the use of inactivated virus, it carries negligible risk for insertion into the genome, it can be manipulated to obtain optimal immune response and it can more easily be produced at large scales. Recently, the use of mRNA as a therapeutic agent has been gaining momentum again this time as a tool for gene replacement. mRNA in this context has to be specifically modified to evade the immune system. Recent studies have greatly advanced this field to make mRNA less toxic, more stable and able to translate with greater efficiency through structural nucleic acid modifications (Kariko et al., 2008; Kormann et al., 2011).

Recently, Kenneth Chien’s lab published a pair of research papers utilizing modified mRNA (5-methyl-CTP, 2-thio-UTP and Anti-Reverse Cap Analogue (ARCA)) to regenerate vascular tissue after injury by driving the cell fate of heart progenitor cells in vitro and in vivo. In their first study, published in Cell Research, they examine the developmental stimuli that directs the cell fate of human Isl1+ cardiovascular progenitor cells (Lui et al., 2013). Isl1+ progenitors give rise to three distinct cardiovascular cell lineages: cardiomyocytes, smooth muscle cells and endothelial cells (EC). They hypothesized that by preferentially directing the fate of these progenitors towards the EC lineage using modified mRNA, they may be able to promote vascular regeneration in the heart, an organ that naturally contains a limited capacity for regeneration.

In this study, the authors first identified endothelial growth factor (VEGF-A) as the most abundant angiocrine factor expressed in cardiac endothelial intermediate cells derived from the outflow tract of human fetal hearts. The outflow tract comprises the portion of the left or right ventricle that the blood must pass through before entering the great arteries. Next they utilized modified mRNA to overexpress VEGF-A and they found that they were able to drive the progenitors to an endothelial lineage, both in vitro and in vivo. For the in vivo study, the authors implanted subcutaneously into NOD/SCID mice purified progenitors in Matrigel^® containing either VEGF-A mRNA or vehicle alone as a control. Surprisingly, they found that treatment with VEGF-A not only drove differentiation to ISL1+ ECs in vivo but increased proliferation and reduced apoptosis. Broadly, this suggests that VEGF-A derived from synthetic mRNA may be a suitable therapeutic candidate to induce regeneration after cardiovascular damage.

In the second paper, published in Nature Biotechnology, Zangi et al. utilize modified mRNA to facilitate the conversion of heart progenitor cells to EC after myocardial infarction. In an impressive study utilizing a combination of sophisticated genetic techniques, they compare modified mRNA and plasmid-mediated gene transfer (Zangi et al., 2013). The authors’ initial experiments tested the feasibility of using modified mRNA in their model system. They found that in addition to high transfection efficiency in vitro, they were able to efficiently transfect in vivo through direct injection of modified mRNA mixed with a commercial cationic lipid-based transfection reagent. The rates of transfection were higher in modified mRNA vs plasmid DNA across all cell types.

Next the authors tested delivery and expression of VEGF-A. As previously reported, they found that the transient nature of mRNA after direct myocardial injection (peak expression at 18 hours, return to baseline after 144-150 hours) was essential in maintaining cellular health. Prolonged VEGF-A expression via plasmid delivery rendered the membranes excessively leaky. The modified mRNA also had lower toxicity and immunogenicity than the DNA plasmid as determined by apoptosis and upregulation of RIG-I, INF-α or INF-β.

Finally, after verifying the ability of VEGF-A to direct cell fate towards cardiovascular cell types, the authors found that the modified mRNA derived VEGF-A was effective in producing increased capillary density, reduced infarct size and cell death after myocardial infarction - without the excessive cellular membrane permeability and edema associated with the plasmid derived VEGF-A. Impressively, they found a dramatic increase in survival of mice after myocardial infarction as compared to plasmid transfected and control, likely due to the differing kinetic profiles.

While the safety and efficacy still need to be evaluated in humans, these findings convincingly demonstrate that modified mRNA is a suitable agent for regulation of protein expression in mice and an attractive alternative treatment to traditional gene therapy. The transient nature of mRNA makes it ideal for inducing differentiation and regeneration, thereby allowing the body to naturally repair itself. TriLink is a leader in custom mRNA manufacturing. The recent opening of our therapeutic GMP manufacturing facility allows us to support all phases of your research, from preliminary studies in cell culture through clinical trials. View our list of stocked mRNA for commonly used fluorescent controls, expression factors and genome engineering tools.

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