Less than 20% of the protein coding genome is thought to be targetable by using small molecules. mRNA therapies are not limited in the same way as in theory, they can silence or edit any gene through encoding CRISPR nucleases, or produce any missing protein. However, not all mRNA therapies are equally likely to succeed. The possibility that RNA therapy will work is influenced by the gene itself. One key feature that differentiates genes is the simplicity of the cell signaling that leads to disease. Monogenic diseases, which are caused by mutations in a single gene, are preferable to diseases driven by mutations in several genes. The first FDA-approved siRNA drug consists of an ionizable lipid nanoparticle (LNP) that delivers siRNA targeting the transthyretin (TTR) gene to hepatocytes. The cell signaling diagram of TTR amyloidosis is ideal for an RNA therapy. Mutant TTR protein can lead to disease, thus, reducing the production of mutant TTR protein with siTTR halts disease.
Figure 1. The complexity of the disease can affect the effect of gene therapy. (Da Silva Sanchez A, et al. 2020)
Recently, in a research report entitled "Treating Cystic Fibrosis with mRNA and CRISPR" published in the international journal Human Gene Therapy, scientists from Georgia Institute of Technology have revealed how to use mRNA therapy and CRISPR technology to treat patients with cystic fibrosis.
Most gene targets are located somewhere on a spectrum between TTR and a genetically unstable cancer. One example is cystic fibrosis (CF), a disease caused by autosomal recessive mutations in the CFTR, located on chromosome 7. There are a number of CFTR mutant genotypes. Some of these mutations can already be treated with FDA-approved small molecules. Other mutations cannot and may thus be more dependent on RNA therapies in the future. This study shows that it is possible to treat cystic fibrosis with mRNA therapy, which does not seem to be associated with pathogenic mutations in the patient's body.
Treating CF by delivering mRNA that encodes CFTR has the potential to work in any CF patient, independent of the underlying mutation. It has been estimated that restoring 5% of wild-type CFTR mRNA in the cytosol is enough to ameliorate the symptoms of CF. The researchers have demonstrated that delivering exogenous CFTR mRNA to mice lacking wild-type CFTR leads to the production of functional ion channels. These treatments resulted in improvements in lung functional parameters, including improved forced expiratory volume (FEV) values, and improved ion conductivity in the nasal epithelia in a manner resembling FDA-approved CF drugs.
Another potential therapeutic approach is to use mRNA encoding nucleases such as CRISPR-Cas9 accompanied by gRNA and using them to edit DNA in target cells. The co-delivery of Cas9 mRNA and guide RNA to facilitate editing can bypass some of the disadvantages of delivering protein, using a plasmid or a viral vector. For example, mRNA is transient, allowing for editing to take place during a specific timeframe instead of long-term. In addition, mRNA and guide RNA can be co-delivered within an LNP, which can be optimized to induce delivery in a specific on-target cell type or tissue. This approach has been used to promote potent on-target editing. Recently, a group delivered Cas9 RNP containing chemically modified sgRNA within AAV6 to upper airway basal stem cells and bronchial epithelial cells with the ΔF508 mutation. They achieved 30-50% editing efficiencies which restored CFTR function equivalent to 20-50% of wild-type controls.
Nevertheless, how to use these methods successfully is still a challenge for scientists. First, researchers need to identify drug delivery systems that can reach lung epithelial cells at low doses. Besides, the duration of CFTR protein also needs to be improved after the administration of CFTR mRNA. In the future, researchers will continue to conduct in-depth research to find a variety of effective treatments for cystic fibrosis.