Background: Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are aggressive sarcomas that can arise in Neurofibromatosis type 1 (NF1) patients. MPNST prognosis is dismal, and no therapy is known, making the identification of actionable targets an urgent need. MPNST malignancy is shaped by changes in metabolic and survival pathways. MPNST cells express high levels of the isoform 2 of the glycolytic enzyme hexokinase (HK2), which is known to enhance both glucose metabolism and resistance to death stimuli. Hence, HK2 is a potential target for MPNST therapeutic approaches.

Methods: We have assessed the efficacy in inducing MPNST cell death of a HK2-targeting peptide (HK2pep) that dislodges HK2 from mitochondria-endoplasmic reticulum contact sites called MAMs (mitochondria-associated membranes). Moreover, we have evaluated the in vitro and in vivo effect of HK2pep versions that are activatable by matrix metalloproteases (MMPs) found in the MPNST microenvironment.

Results:
We have observed that HK2pep rapidly induces a massive, Ca2+-dependent death of MPNST cells. Moreover, various MMP-activatable HK2pep forms inhibit in vitro tumorigenicity of MPNST cells. The HK2pep version activatable by MMP2/9 is highly effective in hampering MPNST growth in vivo, both after xenografting cells in nude mice, and on a mouse model of MPNST induction that we have generated, the Nf1f/f; p53f/f; PLP-CreERT2 mouse.

Conclusion:
Our data indicate that detaching HK2 from MAMs could constitute a novel anti-MPNST therapeutic strategy, which could be flexibly adapted to the protease expression pattern of the tumor microenvironment.

Background:
Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder primarily characterized by café-au-lait macules and development of nerve sheath tumors. Individuals with NF1 inherit one mutated and one wild-type NF1 allele, and loss of the remaining wild-type copy drives uncontrolled Schwann cell proliferation and tumorigenesis. Affected individuals may develop cutaneous neurofibromas, plexiform neurofibromas (PNFs), and malignant peripheral nerve sheath tumors (MPNSTs).
Gene therapy has been hindered by the large NF1 coding sequence, which exceeds the capacity of most viral vectors. Foamy viral (FV) vectors, with their larger cargo capacity and favorable integration profile, provide a promising alternative.
Methods:
We generated a codon optimized NF1 isoform 2 (coNF1) and produced high titer foamy viral (FV) stocks using a four-plasmid system. FV-coNF1 efficiently transduced multiple cell lines for NF1 and Ras pathway analyses. In vivo, MPNST xenografts in NOD/SCID mice received intratumoral FV-coNF1, and tumors were monitored for 77 days to assess growth response.
Results:
We generated a foamy viral vector encoding coNF1 with strong expression and efficient plasmid propagation. FV-coNF1 drove robust neurofibromin production, reduced proliferation in tumor cells, and lowered Ras activity in multiple NF1 deficient cell lines. In vivo, intratumoral FV-coNF1 caused marked regression of ST88-14 and NF90-8 xenografts, with most tumors eradicated by day 77.
Conclusion:
We generated a full-length, codon-optimized NF1 foamy viral vector that produced high titer stocks and drove strong, dose-dependent neurofibromin expression. FV-coNF1 reduced proliferation, suppressed Ras activity, and caused marked xenograft regression, supporting its potential as a therapeutic for MPNSTs and other NF1-related tumors.

Background: Neurofibromatosis type 1 (NF1) is caused by loss-of-function mutations in NF1. Patients develop peripheral nerve sheath tumours of Schwann-cell origin, with current treatments only modifying symptoms, without addressing the underlying genetic defect. Gene replacement could address this directly, but the large NF1 sequence exceeds adeno-associated viral vector capacity, and anti-vector immunity constrains the repeat dosing likely needed for durable benefit. Non-viral lipid nanoparticles (LNPs) accommodate large cargo and permit redosing, yet concentrating their effect in the tumour, not the whole body, remains challenging.
Methods: We engineered a codon-optimised full-length NF1 expression cassette and tested it in NF1 loss-of-function cells, assessing transgene expression and functional rescue. To evaluate in vivo delivery, we encapsulated a mRNA reporter in LNPs and compared locoregional intravascular delivery with intravenous administration in wild-type mice and an orthotopic xenograft of immortalised neurofibroma-derived human Schwann-cells.
Results: The codon-optimised cassette drove approximately two-fold higher transgene expression than a previously published full-length NF1 construct and restored neurofibromin function. In vivo, in wild-type mice, locoregional intravascular administration increased reporter expression by up to 70-fold compared with systemic delivery. Critically, this advantage carried over to a disease-relevant model: in the xenograft, locoregional delivery produced a pronounced increase in intratumoural reporter expression versus systemic administration, confirming tumour-preferential expression.
Conclusion: Locoregional intravascular delivery of LNPs substantially enhances targeted transgene expression relative to systemic administration in both wild-type and tumourbearing NF1 mice. Combined with a codon-optimised NF1 cassette capable of functional rescue, this strategy supports the feasibility of a non-viral, Schwann cell-directed gene-replacement therapy for NF1.

Background: In cells of neurofibromatosis type 1 patients one NF1 allele is mutated. The remaining wild-type NF1 allele does not produce enough neurofibromin causing haplo-insufficiency. It was hypothesized that transcriptional stimulation of the wild-type NF1 gene would alleviate or overcome the haploinsufficiency phenotype.
Methods: A firefly luciferase-reporter was generated driven by the NF1 promoter and used to select gRNAs capable of activating transcription in conjunction with dCAS9 linked to a transcriptional activator (dCAS9-VP64). Transient transfection experiments examined the induction of NF1 expression at the transcriptional and protein level in Hek293T cells by qPCR and Western blotting. A high-throughput re-purposing drug screen, involving 1540 drugs, was performed to identify drugs that induce the NF1 promoter using a GFP-reporter construct.
Results: Twelve guide RNAs were designed and tested using the NF1 promoter driven luciferase reporter and dCAS9-VP64. Three guide RNAs, guiding dCAS9-VP64 to specific locations on the NF1 promoter, were selected and activated luciferase expression 5 – 10-fold. Transient expression experiments in HEK293T cells revealed a 1.3 – 2-fold induction of endogenous NF1 expression at the transcriptional and protein level. A high-throughput drug screen was performed revealing ten drugs that were capable of inducing the NF1 promoter. The most promising drug candidates are currently investigated to determine their effects on endogenous neurofibromin expression in HEK293T cells.
Conclusion: NF1 gene expression can be enhanced by dCAS9-VP64 binding to specific sites on the NF1 promoter as well as by re-purposed drugs. This study highlights new therapeutic avenues for the management of neurofibromatosis type 1.