Abstracts

Rationale: BRAF mutations leading to aberrant MAPK/ERK signaling are one of the major driver mutations in low-grade pediatric brain tumors (e.g., pilocytic astrocytoma, ganglioglioma, dysembryoplastic neuroepithelial tumor, and diffuse astrocytoma). These variants are highly associated with poor prognosis and intractable epilepsy. We previously identified that BRAF mutations originate from neural stem cells during embryonic brain development and lead to epilepsy-associated pediatric brain tumors (Koh et al, Nat Med 2018). Existing BRAF inhibitors such as Vemurafenib and Dabrafenib are specific to BRAF V600E protein and have limited penetrance into the blood-brain barrier (BBB). Antisense oligonucleotide (ASO) therapy via intrathecal injection bypasses BBB and degrades the transcript of the target gene in the brain. Thus, ASO therapy is a promising and effective therapeutic tool in neurological disorders with genetically and molecularly identified targets.

Methods: We developed the pre-clinical mouse model of Braf-derived pediatric brain tumors with intractable epilepsy byintroducing Braf p.V600E mutation into neural stem cells during embryonic brain development. These mice show a gradual increase in tumor size and epileptic seizures and a decrease in survival rate over time, thereby recapitulating BRAF-derived pediatric brain tumors with intractable epilepsy. We administrated the most effective ASO inhibiting mouse Braf RNA selected by in silico and in vitro screening. ASO was delivered by single bolus intracerebroventricular (i.c.v.) injection at 10 weeks after pre-monitoring of behavior seizure and tumor size. _x000D_
Results: Oral administration of existing BRAF inhibitors could not significantly reduce seizures and tumor size in BRAF pediatric tumor models. Single ICV injection of Braf ASO successfully reduced the transcript and encoded protein in a dose-dependent manner and its knock-down effect lasted at least 3 months. Injected ASOs are well distributed to CD34-positive regions and various cell types in Braf-derived neuro-glial tumors. The behavioral and electrographic seizure frequency is remarkably reduced after treatment of the ASO. The size of tumors and enlarged dysmorphic neurons are significantly reduced. The survival rate was significantly increased. Also, the bodyweight was rescued in Braf ASO treated group, compared to the vehicle-treated control group. No behavioral and molecular toxicity was found at two months after i.c.v. administration of ASO.

Conclusions: In our study, ASO therapy targeting BRAF shows strong therapeutic effects on various aspects of BRAF-derived pediatric brain tumors, including the tumor size, epileptic seizures, survival rate, and dysmorphic neurons. Therefore, ASO therapy targeting BRAF will be a promising and effect treatment in BRAF-derived pediatric brain tumors with intractable epilepsy.

Funding: National Research Foundation of Korea (NRF), Korea government, Ministry of Science and ICT. Grant Number: 2019R1A3B2066619 / 2020R1A6A3A13076897; Suh Kyungbae Foundation; SoVarGen