Neurofibromatosis type 1 (NF1) is caused by mutations of the NF1 gene that lead to a nonfunctional version of the protein neurofibromin. More than 1000 different mutations of the NF1 gene have been identified to cause the disease (1), of which approximately 20% are classified as nonsense mutations (2).
Nonsense, or stop, mutations are genetic mutations that create a premature termination codon (PTC) in a gene, resulting in the production of a truncated, usually nonfunctional, protein (3). In addition, the PTC activates an endogenous cellular pathway, nonsense-mediated mRNA decay (NMD), that degrades mRNA and thus decreases protein expression (4). In the case of NF1, both of these factors result in a loss of neurofibromin function.
Nonsense mutations are responsible for several other genetic diseases. According to the Human Gene Mutation Database, 11% of all genetic diseases are caused by a single-point mutation that produce PTCs (5). The most common pathologies that occur with nonsense mutations are cystic fibrosis (CF), Duchenne muscular dystrophy (DMD), β-thalassemia, and cancer.
There have been several significant advances in therapies that address nonsense mutations. The treatments can be classified under two main approaches: (1) reducing the efficiency of translation termination, and thus the likelihood of stopping translation at PTCs, often referred to as ‘termination suppression’ or ‘read through’ and (2) replacing premature stop mutations with wild-type sequences using gene repair techniques.
Aminoglycosides, particular gentamicin, have been one of the most investigated types of readthrough therapies, having been clinically tested for DMD and CF. However, there is significant concern that long-term use of aminoglycosides could lead to toxicities such as kidney damage and hearing loss, as well as potential suppression of native stop codons.
Other compounds, such as negamycin and Ataluren, utilize a similar mechanism to aminoglycosides while showing fewer side effects. Ataluren is available under the name Translarna in the European Union for the treatment of DMD. The future of therapy development to address nonsense mutations will require less toxic suppression agents for premature stop mutations as well as consideration of combined therapies that simultaneously inhibit NMD. (6)
The Gilbert Family Foundation’s Gene Therapy Initiative (GTI) is currently exploring and identifying potential therapeutics to address NF1 nonsense mutations through three distinct projects.
The first project is led by Dr. Allan Jacobson, Chair and Professor of Microbiology and Physiological Systems at the University of Massachusetts Medical School. Dr. Jacobson is also the co-founder of PTC Therapeutics, the company that developed Ataluren. This project, ‘Ataluren-promoted Therapeutic Nonsense Suppression for Neurofibromatosis’, aims to evaluate the efficacy of the drug on preclinical NF1 models. Success with this project could lead to Phase 2 proof-of-concept clinical trials in NF1 patients with nonsense mutations.
The second project, ‘Exploring Nonsense Suppression as a Treatment for NF1,’ is a collaborative partnership between Dr. David Bedwell, Professor and Chair of Department of Biochemistry and Molecular Genetics at the University of Alabama at Birmingham (UAB), and Drs. Bruce Korf and Mark Suto, at UAB and Southern Research respectively. They are focused on identifying new compounds capable of suppressing PTCs and/or inhibiting NMD via high throughput screening of 157 previously validated compounds.
“We designed a screening strategy that can identify these dual-acting compounds,” says Bedwell. “If we can identify effective PTC suppression compounds, we may be able to restore expression of enough neurofibromin protein to relieve many of the disease phenotypes observed in NF1 patients.”
The team is utilizing a variety of assays that monitor the ability of PTC suppression compounds to: 1) suppress termination at multiple NF1 nonsense mutations, 2) restore full-length neurofibromin expression, and 3) restore neurofibromin function by monitoring the GTP-Ras signaling pathway, which is normally regulated by neurofibromin. The ultimate goal is to develop one or more lead compounds into safe and effective drugs for clinical use.
Lastly, GTI supports the project ‘Development of Mutation Suppression Therapy in a Swine Model of NF1’ led by Dr. Adrienne Watson, Vice President of Research and Development at Recombinetics, LLC, in partnership with Dr. David Largaespada and Dr. Christopher Moertel at the University of Minnesota. The proposed work includes the development of an NF1 pig model harboring a nonsense mutation that recapitulates many clinical manifestations of the human disease, as well as in vitro and in vivo testing of promising PTC suppressors. “In vitro studies will be used to prioritize drugs that show efficacy in primary cell lines derived from the NF1 minipig. Then, drugs will be moved into preclinical trials in NF1 minipigs to establish the safety and efficacy of nonsense mutation suppression therapies for treating patients with NF1,” describes Watson.
As pigs share similar size, anatomy, physiology, and genetics with humans, they are very useful in assessing potential therapeutic interventions. “Our group aims to not only identify novel classes of drugs that may be effective at treating NF1, but also to establish new and relevant preclinical animal models that can help elucidate the underlying biology of NF1 and guide the treatment of all aspects of NF1 from learning disorders, to skeletal abnormalities, to tumor development,” Watson summarizes.
The therapeutic impact of discovering PTC suppressors is significant and wide-ranging, as nonsense mutations lay at the core of many genetic diseases that affect an estimated 20 million patients worldwide. As for NF1 patients, “this class of drugs is exciting because they have the potential to ameliorate both the malignant and non-malignant features of NF1, by addressing the underlying genetic and biochemical cause,” describes Watson.
GTI is thrilled to be working with these dedicated researchers who are applying the most novel theories and promising candidates of nonsense mutation suppression to NF1. We cannot wait to see where these projects lead.
- “NF1 Gene – Genetics Home Reference – NIH.” U.S. National Library of Medicine, National Institutes of Health, 17 Aug. 2020, ghr.nlm.nih.gov/gene/NF1#conditions.
- Li, K., Turner, A., Chen, M., et al. (2016). Mice with missense and nonsense NF1 mutations display divergent phenotypes compared with human neurofibromatosis type I. Disease Models & Mechanisms. 9, 759-767. doi: 10.1242/dmm.025783.
- Biesecker, Leslie G. “Nonsense Mutation.” Genome.gov, www.genome.gov/genetics-glossary/Nonsense-Mutation#:~:text=A%20nonsense%20mutation%20is%20the,%2C%20and%20likely%20nonfunctional%2C%20protein.
- Brogna, S., Wen, J. (2009). Nonsense-mediated mRNA decay (NMD) mechanisms. Nature Structural & Molecular Biology. 16, 107-11.
- Krawczak M, Ball EV, Fenton I. et al. Human gene mutation database-a biomedical information and research resource. Hum Mutat. 2000;15(1):45–51. http://www.hgmd.cf.ac.uk/ac/stats1.php
- Keeling KM, Du M, Bedwell DM. Therapies of Nonsense-Associated Diseases. In: Madame Curie Bioscience Database [Internet]. Austin (TX): Landes Bioscience; 2000-2013. Available from: https://www.ncbi.nlm.nih.gov/books/NBK6183/?log$=activity.