How do mRNA delivery mechanisms work?

Messenger RNA (mRNA) transfection can be performed both in vitro and in vivo using a variety of methods (e.g., cationic lipid-mediated delivery or electroporation). Of note, ionizable lipid nanoparticles (LNPs) have become a more desirable mRNA delivery mechanism in clinical research as LNPs have demonstrated reduced toxicity compared to methods relying on cationic lipids [1].

Regardless of transfection method, mRNA can be translated into protein directly within the cytoplasm and therefore does not require nuclear entry. This feature makes mRNA transfection simpler, faster, and more efficient than DNA plasmid transfection (Figure 1). However, because mRNA remains in the cytoplasm and does not integrate into the host cell genome, it will only be transiently expressed.

The simplicity and transient nature of mRNA makes it optimal for studying short-term genetic effects or for the rapid production of recombinant proteins. In gene editing experiments, the transient transfection of Cas9 mRNA ensures that CRISPR-Cas9 is functional in the cell for only a limited amount of time, which can decrease off-target effects. As a mechanism for vaccine delivery, utilizing transient mRNA molecules eliminates the risk for genomic integration, better ensuring the safety of the patient.

Diagram of a cell showing entry of DNA into the nucleus prior to protein expression and mRNA being translated directly into protein within the cytoplasm

Figure 1. DNA transfection vs. mRNA transfection. Both DNA and mRNA payloads enter the cell via endocytosis and must escape the endosome. However, while DNA must enter the nucleus prior to expression of protein, mRNA is able to skip this step and is translated into protein within the cytoplasm.

The design of mRNA molecules for transfection experiments must be carefully considered to ensure success and efficiency. The design of distinct mRNA regions, including the 5’ cap, 3’ poly(A) tail, and untranslated regions (UTRs), will impact the translation and stability of the mRNA in cells.

NOTE: Extra precautions should be taken when handling mRNA molecules due to their instability compared to DNA. Specifically, the use of RNase-free reagents and tips is key to preventing degradation. Long-term storage at –80°C is recommended, and mRNA samples should be kept on ice when in use.

Learn more about mRNA stability

Benefits of mRNA delivery technology

The impact of mRNA delivery technology goes beyond basic research, with invaluable implications in clinical research as well as for the industrial production of proteins in biotechnological and biopharmaceutical settings.

The lack of nuclear entry step required in mRNA delivery mechanisms increases transfection efficiency, especially in primary and hard-to-transfect cell types, while decreasing the time to expression. Other benefits include homogeneity of expression and elimination of the risk for genomic integration.

mRNA applications

Advancements in mRNA delivery technology are leading to a variety of breakthroughs in clinical research. Years of study into better understanding mRNA delivery technology led to the successful deployment of mRNA vaccines against COVID-19 , which has been instrumental to the recognition of mRNA as a pivotal tool across a wide range of experimental applications, including immunotherapy, protein replacement, and gene editing.

Immunotherapy and cancer therapy

mRNA delivery technology has been thrust into the spotlight of immunotherapy development with the rapid and successful deployment of COVID-19 vaccines. By utilizing mRNA as a vector for vaccinations, antigenic proteins are produced using the patient’s own body, allowing for appropriate protein modifications and trafficking. In addition, mRNA vaccines produce noninfectious particles and eliminate the risk of mutagenesis caused by genomic integration, as can be seen with DNA vaccines.

LNP delivery of mRNA also eliminates the need for viral vectors, so specialized facilities for biosafety laboratories are not required. mRNA sequences can also be redesigned to incorporate protein changes more easily as compared to bioengineering specific proteins [2].

In addition to the success of mRNA delivery technology for COVID-19 prevention, this application is undergoing extensive research for other diseases such as Zika virus, HIV, and even certain cancers [3]. A popular and exciting area of cancer therapy research utilizing mRNA delivery technology is CAR-T cell therapy. In this specialized type of targeted cell therapy, extracted T cells can be engineered to express receptors that are able to target specific antigens on cancer cells in a patient, leading to the destruction of these cells and treatment of the disease [3].

Protein replacement

Given the safety and simplicity of mRNA vectors as well as their ability to ensure rapid and efficient protein production, this technology is also emerging as a promising agent for protein replacement therapy, which involves the generation of functional protein to replace defective molecules.

Research is underway for the treatment of many diseases, including mRNA-based CFTR replacement in cases of cystic fibrosis and the treatment of heart failure via the replacement of VEGFA proteins [3].

Gene editing

mRNA is also useful for the delivery of nucleases in gene editing experiments (e.g., TALENs or CRISPR-Cas9). The transient nature of mRNA eliminates the risk for host integration and in the case of Cas9, ensures it is expressed for only a limited amount of time, decreasing off-target effects.

Thermo Fisher Scientific offers the Invitrogen mMessage mMachine T7 Transcription Kit for in vitro production of large amounts of high-quality mRNA and Lipofectamine MessengerMAX for efficient and effective delivery of mRNA into your cells.

Learn more about transfection of mRNA using Lipofectamine MessengerMAX

Visit Transfection Basics to learn more about performing transfection in your lab.

For Research Use Only. Not for use in diagnostic procedures.