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Additional experiments designed to restore neurofibromin with temporal control in specific cell lineages in
genetically engineered mouse models or human induced pluripotent stem cell-derived organoids will be
critical. Such experiments may help identify which NF1 manifestations or lesions are most likely to benefit
from gene-targeted therapy, ensuring measurable and durable improvements. Additionally, one must
determine the quantity of neurofibromin to be restored for therapeutic efficacy, considering factors such as
target cell type, NF1 heterozygosity, and mutational landscape. Identifying the specific cell types that must
be targeted-such as Schwann cells, stem cell niches, and cells surrounding neurofibromas-and developing
strategies for effectively targeting these cells will be essential for successful gene therapy approaches.

Development of enabling technology and infrastructure
Developing assays to measure functional neurofibromin and its downstream effectors
A critical area of focus to enable NF1 research is assay development. Developing more sensitive and
reproducible assays with a broader dynamic range to measure functional neurofibromin, Ras activation, and
its downstream effector levels is critical for developing successful gene therapy for NF1. Today, most studies
still depend on semi-quantitative Western blots that measure neurofibromin itself and, at best, a few
downstream Ras effectors. In addition to not necessarily distinguishing between active functional
neurofibromin and inactive full-length neurofibromin, this approach suffers from limited sensitivity,
narrow dynamic range, and poor reproducibility across laboratories. Baseline neurofibromin levels and the
thresholds to reverse or halt specific NF1 manifestations vary and remain unknown; new assays must detect
small yet biologically meaningful changes over several orders of magnitude and must do so quickly enough
to support high-throughput screening and iterative optimization cycles. Cutting-edge technologies such as
[10]
single-cell proteomics can provide high-resolution quantitative data . However, these approaches
currently face limitations related to cost, instrument accessibility, and throughput, restricting their routine
use in large-scale or inter-laboratory applications. A standardized, high-throughput platform that can
simultaneously quantify functional neurofibromin and a multiplexed panel of Ras-pathway downstream
signaling nodes would allow different labs to directly compare interventions, accelerating preclinical
development and providing a quantitative assessment tool for gene- or RNA-based strategies that aim to
restore neurofibromin across diverse cell and tissue types. By providing precise, reproducible readouts of
neurofibromin levels and Ras signaling across multiple cell and tissue contexts and throughout
developmental stages and differentiation states, the same assay will advance NF1 research forward by
enabling the characterization of patient-derived organoid models. These demanding performance criteria
can be satisfied by a high-sensitivity, multiplexed electrochemiluminescence immunoassay platform, which
combines ultrasensitive detection, broad dynamic range, and throughput suitable for screening and
cross-lab standardization. This assay platform is currently being developed with support from the Gilbert
Family Foundation and will be made broadly available to the NF research community. Building on this
foundation, the discovery of biomarkers and the development of relevant assays are crucial for advancing
NF1 gene and other novel therapies to the clinic. The ideal assay would provide predictive value for patient
benefit and patient stratification and support regulatory approval processes. The advancement of such
diagnostic and predictive tools may pave the way for the development of early preventative interventions in
the future, capable of halting or completely preventing the cellular and developmental cascades triggered by
the loss of NF1.

NF1 preclinical model development
While there are several preclinical models currently in use to study various NF1 manifestations, the
development, characterization, and optimization of in vivo and in vitro preclinical models that better
recapitulate the complexity of NF1 biology and symptoms are necessary to enhance the translational
potential of gene therapy. While no single preclinical model may fully capture the full breadth of NF1
manifestations, several established models have yielded valuable insights into specific aspects of the disease.