A team led by Johns Hopkins and Kennedy Kreiger Institute researchers has developed a new gene therapy strategy that may overcome longstanding barriers to treating neurofibromatosis type 1 (NF1) as well as other conditions related to a single gene alteration.

The experimental therapy combines a miniaturized version of the NF1 gene with a uniquely engineered adeno-associated virus (AAV) vector that efficiently targets tumor tissue while limiting uptake in the liver and other healthy organs. This is an important development, as nontargeted uptake is a significant limitation of naturally occurring (nonengineered) AAV products.

Published Sept. 29 in Nature Communications, the study demonstrates that when the vector — named AAV-NF (K55) — is paired with the payload GRD-C24, it suppresses tumor growth in xenograft mouse models of NF1-related cancers. The findings establish a foundation for advancing toward larger-animal safety studies, leading to a first-in-human clinical trial.

NF1 is one of the most common single gene disorders worldwide. It stems from mutations in the NF1 gene that produce neurofibromin, a protein that normally regulates RAS and its signaling pathway. When the gene is disrupted, the pathway becomes overactive, driving tumor formation throughout the body. Up to 15% of these tumors turn into treatment-resistant and aggressive cancers, such as the sarcoma called malignant peripheral nerve sheath tumor and brain cancers like glioblastoma. Because NF1 tumors are driven by a single genetic defect, researchers see a unique opportunity for gene-based therapy to address them at their source.

“When the NF1 gene mutates, it leaves the RAS pathway hyperactivated, and cells — especially Schwann cells — proliferate without control,” says Renyuan Bai, associate professor of neurological surgery at the Johns Hopkins University School of Medicine. “That’s what leads to the formation of neurofibromas, which can progress to the dangerous sarcoma, malignant peripheral nerve sheath tumors.”

The NF1 gene is more than twice the size an AAV can carry. To overcome that logistical hurdle, the team created a “mini-NF1” construct, retaining the core enzyme region responsible for turning off RAS hyperactivity. They fused it with a short cell membrane-binding sequence from RAS so the hybrid protein could locate precisely where growth control occurs inside the cell.

Using capsid evolution, the team engineered a tumor-targeted AAV vector, AAV-NF. Libraries of modified capsids were injected into mice bearing human NF1 tumors, and variants that most effectively reached tumor cells were enriched. After multiple rounds, AAV-K55 emerged, delivering the mini-NF1 payload efficiently while minimizing liver uptake. In animal models, it significantly slowed growth of NF1 tumors, including MPNST, supporting translation to human studies.

This work occurred through the Johns Hopkins Neurofibromatosis Therapeutic Acceleration Program (NTAP) NF1 Gene Replacement Initiative. Researchers say the data reported in this manuscript, as well as two patents, a pipeline to clinical translation and commercial development, and multiple collaborations exemplify NTAP’s mission.

“The results of this study reflect excellent collaboration among experts in gene therapy development and NF1, and were supported by guidance from leaders across medicine and biotechnology,” says study co-author Jaishri Blakeley, director of the Johns Hopkins Comprehensive Neurofibromatosis Center and NTAP. “This work shows that bold, technically challenging problems can be transformed into therapeutic opportunities.”

The team is conducting dose-escalation and safety studies in mice, and is working with Johns Hopkins Technology Ventures to advance the research into nonhuman-primate testing and ultimately first-in-human trials. The next step is to validate safety and efficiency in higher models to move toward trials for cancers in need of effective therapies, including NF1-driven sarcoma and glioma.

Other contributors include Christine Pratilas of the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center, and collaborators from the Kennedy Krieger Institute, Indiana University School of Medicine and the Germans Trias i Pujol Research Institute in Spain.

The study also honors the late Verena Staedtke, whose daring vision and leadership was central to the project’s success.

A patent application on the new AAV vectors for NF1 gene replacement therapy with Renyuan Bai and Verena Staedtke as co-inventors has been provisionally filed by The Johns Hopkins University (63/697,752). Other authors declare no competing interests.

The NTAP Gene Therapy Replacement Initiative is made possible by funding from Bloomberg Philanthropies.