Neurofibromatosis type 1 (NF1) is caused by a mutation or deletion of the gene neurofibromin 1. This gene controls production of the protein neurofibromin, responsible for regulating cell division and tumor growth. Over the past two decades, researchers have made significant therapeutic developments towards treating many of the various manifestations of NF1. This progress has largely been driven by greater understanding of the natural history of the disease and the functional role of neurofibromin in normal cellular processes, leading to improved biomarkers and therapeutic targets.[1] 

Clinical trials for these therapies have demonstrated benefit to NF1 patients, particularly for progressive low-grade gliomas and plexiform neurofibromas[2]. However, some researchers believe targeting the mutated gene itself could yield a more effective, comprehensive response for a greater range of patients. “In contrast to medication-based treatment, gene editing has the potential of being a true ‘cure’ for NF1,” states Dr. Bruce Korf, Associate Dean for Genomic Medicine at the University of Alabama at Birmingham (UAB) School of Medicine. Dr. Korf is a collaborator on several projects within the Gilbert Family Foundation Gene Therapy Initiative (GTI). “If we could replace the copy of the gene damaged by mutation with a repaired copy, we would hope to be able to prevent, and perhaps even reverse, the many complications of NF1.  We have a long way to go to effectively target and treat the right cells, but we are pleased to see some of the first steps taking place along this journey.”

GTI seeks to explore and develop gene-targeting therapeutic strategies for NF1, including gene replacement and gene editing methods, as well as necessary reagents and in vivo systems. Gene replacement therapies deliver a working copy of the gene to the cell to override the faulty gene, while gene editing therapies revise the mutated portion of a gene at the DNA level.  In both approaches, the expected result is an increase in the expression of functional neurofibromin protein and decrease in disease state.

“Restoration of normal NF1 expression is likely to be more effective [than small drugs] as this complex regulatory protein is once again present in the cell where it can properly mediate all of its regulatory functions,” confirms Dr. Miguel Sena-Esteves, Associate Professor of Neurology at the University of Massachusetts Medical School and one of the Principal Investigators (PIs) in GTI.  “There is evidence suggesting that restoration of NF1 expression in malignant tumor cells triggers cell death or curtails their growth dramatically,” he continues.

Despite this promising evidence, gene therapy development comes with a few key challenges. For gene editing therapies, researchers must ensure that the editing only affects the mutation and avoids off-target effects. Dr. Bob Kesterson, Professor of Genetics at UAB and GTI Project Co-PI, describes, “for gene editing to work, the correction of the mutant gene copy must be extremely efficient and not have unintended consequences such as introducing genetic changes elsewhere or introducing further errors in the NF1 gene. With the discovery of CRISPR-Cas9 and related systems, gene editing technologies are now evolving at an extremely rapid pace, and issues with efficiency and specificity are improving steadily.”

With this in mind, Dr. Charles Gersbach, Associate Professor of Biomedical Engineering at Duke University and Co-PI of a GTI project, aims to develop a new CRISPR-based genome editing tool that restores function to the mutated NF1 gene without modifying off-targets.  He and his team are building on their previous experience developing gene editing to correct other genetic diseases.

Effective delivery to the appropriate cell target is essential to both gene editing and gene replacement strategies, particularly the identification and refinement of the vehicle for gene delivery. Beyond general concerns over the long-term immune effects of using a viral vector, as adeno-associated viruses (AAV) is one of the most common method of gene delivery[3], the NF1 gene itself poses a unique challenge due to its large size.

“For gene replacement, the most commonly used vehicle (or vector) to deliver genes in humans are the adeno-associated viral vectors; however, these have limitations in the size of the ‘cargo’ that can be carried, and the large NF1 gene is problematic,” says Kesterson. “We must use a vehicle that can deliver the gene editing reagents to the intended target and can reach cells throughout the body.”

With this in mind, GTI researchers are exploring new vehicles for gene therapy delivery.  Kesterson and team aim to establish nanoparticle delivery systems for gene replacement and gene editing reagents to correct common mutations on the NF1 gene. Similarly, Gersbach and team are engineering new viruses that specifically target cells in the peripheral nervous system, while Sena-Esteves and team are developing new AAV vectors, zinc finger protein, and antisense oligonucleotides for NF1 delivery and expression restoration.

“The AAV vectors we have designed to delivery full length NF1, or mini-NF1 genes look very promising as we have shown NF1 expression in the brain and various other peripheral tissues after systemic delivery,” describes Sena-Esteves. “Proof-of concept studies are starting soon to demonstrate the feasibility of the new AAV-NF1 vectors in correcting neurological phenotypes and prevent tumor formation in two NF1 mouse models.  Assuming the results are positive and demonstrate a clear benefit in gene therapy treated NF1 mice, our goal is to move this therapy to a first-in-human AAV gene therapy clinical trial.”

In contrast, Kesterson stated, “as an alternative, ‘nanoparticles’ have the capacity to carry much larger cargos, and we have managed to produce full-length NF1 that can replace all domains of the neurofibromin protein.  Nanoparticles also have some additional advantages being able to be modified in ways to selectively target delivery of the cargo to specific cell types including both normal cells and cancerous cells.”

Lastly, developing relevant preclinical models to improve understanding of NF1 and test efficacy of developed therapies is an important step towards bringing NF1 gene therapies to clinical trial.  Kesterson and Zhou’s project includes the development and characterization of an NF1 rat model that carries patient mutations. However, improving the broad availability of both animal models and cell lines still needs to be addressed.

Despite these challenges, GTI aims to pave the way for gene therapies to treat NF1 through a set of innovative and exciting projects. “There are many things that have to be accomplished to make this feasible, but if these challenges can be overcome, some form of gene editing or replacement might eventually become the principal treatment for NF1,” says Korf. “I believe we are taking the initial steps towards what will eventually be a method of treatment that will benefit the NF1 patient community broadly.”