Converting cells into drug synthesizers
A recently published study from the Koch Institute for Integrative Cancer Research at MIT gets glowing results, literally. Using a messenger RNA (mRNA) transcript that encodes for the bioluminescent, light-emitting protein, called luciferase, MIT researchers were able to track the expression of the aerosolized polymer-bound mRNA that had been delivered to mice epithelial cells via a nebulizer. The findings hold hope for more effective treatments of lung diseases.
Tackling drug delivery
Many believe that RNA drugs will be the next major achievement in drug development technology. These types of drugs either target RNA or use RNA directly as a therapy. Other types of commonly used drugs are small chemical treatments and protein drugs. Chemical drugs are generally non-specific and diffuse throughout the body. They are often not naturally degraded and can accrue to toxic levels–sometimes producing considerable side effects. The development of protein drugs addressed many of these limitations. Protein drugs are well-known, well-established, and well-funded, with the 2016 global market for protein drugs estimated at 172.5 billion USD. However, protein drugs also have drawbacks—the largest arguably being that proteins are sometimes immunogenic, meaning that they are prone to induce an immune response from the body.
However, DNA has received recent attention as a potential therapy. An inhalable lipid-DNA complex that contains the gene Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), which can cause cystic fibrosis when mutated, is currently going through clinical trials. By delivering the correct copy of the CFTR gene, researchers had noted some improvement in patients’ conditions. While researchers are working to optimize the delivery system and enhance the delivery system of the DNA treatment, using DNA poses specific challenges. “One issue with DNA delivery is that a lot of the DNA vectors, or molecules used to carry DNA, are viral,” reports Padmini Pillai, a current postdoctoral fellow doing immunoengineering research at MIT after receiving her PhD in immunobiology from Yale. “This is less favorable since it can induce an immune response. Secondly, DNA needs to enter the nucleus, which is a big ask,” Pillai said. These challenges have led MIT researchers and others to pioneer the investigation of using RNA instead of DNA in aerosolized treatments.
The promise of RNA drugs
Despite a deep understanding of how RNA can regulate gene expression, researchers discounted RNA as a viable therapeutic primarily due to how readily it is degraded. The experiments conducted in the MIT study employ the natural protein-building machinery of the cell. By introducing messenger RNA into the cell, the cell will use its own synthetic pathways to translate this mRNA into a functioning protein. Previous studies have examined the possibility of using nebulized nucleic acids to treat diseases and disorders in the lungs, while others have investigated the use of systemic delivery of mRNA to combat tumors. Both lines of study have yielded positive results. Injections of mRNA packaged in lipid nanoparticles, fat blobs designed to protect RNA from degradation, have shown potential in treating cancer in a mouse model of melanoma. An mRNA-based vaccine that would target tumor cells is currently in clinical trial.
The mRNA transcript does not require delivery inside a viral vector like DNA. Furthermore, mRNA is more easily transported and only temporarily active as it is quickly degraded after it has been translated into protein, enabling researchers to control mRNA expression with precision through readministration. Additionally, mRNA only has to enter the cell membrane, rather than both the cell membrane and the nucleus. One limitation of most current forms of RNA delivery is that they are non-specific. For example, even if only certain cell-types in the lung are diseased or cancerous, an injection of mRNA transcript, and by extension the protein that it will encode, will result in mRNA expression systemically.
The research team investigated two questions: Could aerosolized mRNA be administered effectively through a nebulizer, and what type of nanoparticle would better facilitate mRNA delivery? The inhalation of aerosolized nanoparticles is a noninvasive treatment that has enabled researchers to target the epithelial cells in the lungs with precision. Systemic drugs, which are injected or absorbed into the bloodstream, are expressed throughout the entire body. “The benefit of the nebulized delivery system of treatment is that it facilitates targeted delivery that is isolated to the lung. Further, the nebulizer is completely noninvasive; no needles or sharps [sharp objects] are needed,” Pillai said. This technique initially showed that RNA was able to enter one-quarter of the target epithelial cells in mice, evenly distributed throughout all chambers of the lungs.
The MIT team further engineered a polymer that more efficiently delivered nebulized mRNA to the epithelial cells of the lung. To deliver mRNA via inhalation through a nebulizer, researchers needed to first bind the mRNA to nanoparticles that would help facilitate delivery and protect the mRNA from degradation before they enter cells. The polymer the MIT research team used as their nanoparticle is hPbAE, which is positively charged, biodegradable, hyperbranched polymer. The positive charge on the polymer facilitates stronger binding between the nanoparticle and negatively charged mRNA. Additionally, the positively charged nanoparticle will more easily penetrate the negatively charged cell membrane. Studies cited by the MIT team indicate that a hyperbranched polymer is better suited to aerosolization than a linear polymer. Pillai explains that the branched polymers terminated with polar groups are more efficient due to their increased solubility. The increased solubility results in a nanoparticle that can be easily encased by water molecules and therefore nebulized into water breathable gaseous water particles more easily. Further, the nanoparticle needs to be biodegradable, as previous studies have indicated that non-biodegradable polymers can accumulate in toxic levels.
An RNA future
“While previous studies have used another biomaterial, PEI, to deliver nucleic acids, it is not biodegradable, and its accumulation in the body can lead to side effects,” Pillai said. Pillai is optimistic of the aerosolized-mRNA delivery method due to its ability to facilitate targeted delivery of mRNA to a specific cell type, the lung epithelial cells. She further projects that the aerosolized delivery of mRNA could have a variety of therapeutic applications, not only in cystic fibrosis, but also to a wide range of diseases affecting the epithelium of the lungs, perhaps even viral infections. The field of RNA research is developing therapeutics for a broad range of medical conditions, including cancer. Whatever the condition, this new potential treatment, breaking through a sea of criticism and doubt, is sure to provide a breath of fresh RNA.