The UAB NF Research Program is dedicated to the discovery and development of new, life-changing therapies for people with all forms of NF. To advance this mission, UAB maintains a robust and internationally recognized research program that includes a range of preclinical research and clinical trials for all forms of NF. The primary focus of our program is the development of genome-guided therapeutics with the goal of restoring function to the mutated NF1 gene or gene product. We have been pioneers in this effort for NF1 and have developed resources to enable this research through many funding sources, including the Gilbert Family Foundation, a private foundation established for the purpose of developing effective therapies for NF1.
This month’s blog features insight from two genetic scientists collaborating on preclinical research projects focused on genome-guided therapeutics. Deeann Wallis, Ph.D., UAB Associate Professor of Genetics, and Robert Kesterson, Ph.D., Professor of Cancer Precision Medicine and Director of Genetically Engineered Models facility at the Pennington Biomedical Research Center at Louisiana State University (and formerly Professor of Genetics, UAB), give an overview of promising preclinical research initiatives and discuss the strengths of the UAB NF Program in accelerating the development of genome-guided therapeutics with the potential to transform the treatment landscape for NF.
What are some of the most promising preclinical initiatives focused on genome-guided therapeutics in your laboratory?
Dr. Wallis: I am working on a project, in conjunction with researchers from Teesside University in Middlesbrough, England, that focuses on correcting NF mutations in cell and mouse model systems using a technique called exon skipping. This process causes cells to skip over the region bearing mutations in the genetic code while still producing a functional protein. A gene is encoded in segments, called exons, which code for the amino acids of a protein, separated by introns, which are intervening sequences. This project is focused on identifying exons within the NF1 gene that can be skipped while still maintaining function of the gene, allowing these mutations to be bypassed.
We have made good progress with exon skipping in our laboratory. We have defined several exons we can skip and have also developed antisense oligonucleotides (ASOs) that can be used to target different exons. ASOs are chemically modified chains of nucleotides that can be used to target a specific gene product of interest. At this point, we have demonstrated exon skipping in animal models, although we have not yet introduced the therapy into animals to treat before development of NF features. A limitation of exon skipping is that it is not available for all patients because it targets specific mutations that are not shared by all individuals with NF1.
Dr. Kesterson: While Dr. Wallis and I collaborate on several projects funded by eight separate grants from the Gilbert Family Foundation, her work focuses primarily on developing cell culture models and much of my work involves developing animal model systems of specific human NF1 mutations for use in testing genome-guided therapeutics.
We have a gene replacement project, in partnership with Yale University, that has moved to Phase B and involves replacing the NF1 gene using nanoparticles as the delivery system. Gene replacement therapy for other diseases has previously used adenoviruses to deliver the corrected gene product into cells. This delivery process is more challenging with NF1 due to the large size of the NF1 gene exceeding the normal size capacity to be delivered by viral vectors. However, we have evidence that gene replacement therapy using nanoparticle delivery is working in our animal models.
Also, as Dr. Wallis explained, we are using antisense oligonucleotides (ASOs) for exon skipping. A German foundation is supporting my research to study the recurring Y489C mutation found in a subset of patients in which a “cryptic” splice site causes aberrant skipping of an exon region. We have developed cell lines and mouse models with the mutation to study plexiform and cutaneous neurofibromas. Using the antisense oligos, it is possible to repress the mutated splice site to restore gene function. We can essentially force the gene to splice correctly using this technology.
Which genome-guided therapeutic approaches are the closest, and most distant, from reaching the human clinical trial stage?
Dr. Kesterson: In conjunction with researchers at Yale University, we are using the CRISPR/Cas9 gene editing system to perform gene editing in NF1 animal models with mutations found in human patients. While this technology is advancing at a remarkable pace, there are serious limitations to overcome for its successful application to NF1. To perform gene editing at the DNA level and convert it to a normal sequence, the process must be 100% efficient with no mistakes. For diseases related to blood-borne or marrow-related genetic disorders, stem cells can be isolated to correct the gene more easily. This is not an option for NF1 because tumors are comprised of a mass of cells that are more difficult to target. While gene editing with CRISPR/Cas9 is still a hopeful technology, the complications in using this technology for NF1 make it a more distant technology for clinical trials.
Dr. Wallis: A genome-guided therapeutic approach that is closer to clinical trials is based on research conducted by UAB genetic scientist David Bedwell, Ph.D., on premature stop, or nonsense mutations, which affect about 20% of people with NF1. This type of mutation inserts a signal that tells the protein production machinery in the cell to stop production of neurofibromin before the complete protein is made, resulting in a truncated or nonfunctioning protein. Drug compounds have been identified that show promise in overcoming the effects of premature stop mutations, and some of these have been tested successfully in animal models for NF1. These drug compounds are being tested for other genetic diseases as well, which accelerates the process of moving this research to clinical trials.
What specific strengths of the UAB NF Research Program have helped to advance the pace of research focused on genome-guided therapeutics?
Dr. Kesterson: The collaborative interactions and expertise of the faculty, based on the team that Dr. Korf has built, has provided the framework for pioneering research in this area. We have the most diverse animal models of any program in the country, and we routinely receive requests from researchers around the world interested in gaining access to our models to study NF1. Several years ago, Dr. Korf asked me to develop more animal models for NF1, and now these models are in demand worldwide and have served as the foundation of leading-edge research in genome-guided therapeutics. For example, one of the first animal models I developed was for the premature stop, or nonsense, mutation. Dr. Bedwell in our program used this model as the basis of his research to prove that nonsense suppression is a viable therapeutic approach.
Dr. Wallis: A strength of our research program is the UAB Medical Genomics Laboratory, the world’s gold standard for NF genetic testing and a leader in identifying new NF mutations and maintaining the world’s largest repository of NF mutations.
Our projects are also synergistic and address key areas that collectively are more informative than any one project alone, including the amount of functional neurofibromin needed to restore gene function, the mechanism of drug delivery that is most effective for specific cell types, and the types of NF1 mutations affecting gene function. Because we take this collective approach, we are answering many questions within in a single project that help to advance our colleagues’ research and accelerate the pace of developing genome-guided therapeutics for NF1.