The bedside and the bench of STXBP1-DEE in the wake of precision medicine

Introduction: STXBP1-encephalopathy is a broad neurological disorder caused by mutations in the presynaptic gene STXBP1, involved in neurotransmitter release at the synapse. The core symptoms of this disorder include intellectual disability, epilepsy, movement disorders and autism spectrum disorder. The available therapeutic approaches are mainly symptomatic. Recently, a new potential targeted therapeutic approach has been developed in the wake of precision medicine: SINEUPs are a class of natural and synthetic non-coding RNA which increases the translation of the target protein, without affecting mRNA levels. In order to plan the clinical trials of targeted therapeutic approaches, a natural history of STXBP1 related disorders is crucial. Aims: 1. To address the causes of phenotypic variability and draft a natural history of STXBP1-DEE using retrospective data of a cohort of STXBP1 patients. 2. To use specific SINEUPs to target STXBP1 mRNA and restore the STXBP1 protein levels in mutated human neurons derived from induced pluripotent stem cells (IPSCs). Methods: 1. Retrospective data (seizures history, neurological examinations, developmental follow-up) were collected from a cohort of STXBP1 patients. A disease-specific composite developmental score (STXBP1_DevScore) was elaborate through expert consensus. 2. Specific SINEUPs targeting STXBP1 mRNA (STXBP1-SINEUPs) were designed. Human neurons were differentiated from IPSCs with CRISPR-induced STXBP1 mutations. STXBP1-SINEUPs were used to treat the neurons. The STXBP1 protein levels after STXBP1-SINEUPs treatment were assessed by Western blot. Results: 1. We collected longitudinal clinical data of 48 STXBP1 patients (mean follow-up period: 8 years). The epilepsy history of STXBP1 patients show the presence of at least two distinct patterns of evolution. Data suggest that epilepsy impact on neuro-developmental consequences in STXBP1-DEE is limited to age at seizure onset. 2. We treated neuronal lines harbouring three different CRISPR-induced mutations in STXBP1, which recapitulated the haploinsufficiency conditions found in patients. In heterozygous neurons, the STXBP1-SINEUPs reached a 1.7-fold mean increase of STXBP1 protein compared to negative control. Conclusions: 1. The use of STXBP1_DevScore allowed to unravel common trajectories in the evolution of STXBP1-related disorders. Efforts should be made to collect natural history data from a larger cohort, in order to identify effective treatment timepoints and rational clinical endpoints for targeted therapies clinical trials. 2. We found that STXBP1-specific SINEUPs have the ability to increase STXBP1 protein levels in human neurons derived from IPSCs, in haploinsufficiency conditions. These results also demonstrate that IPSCsderived human neurons have great potential as a model for target therapy screening in genetic neurodevelopmental and epileptic disorders.

Introduction: STXBP1-encephalopathy is a broad neurological disorder caused by mutations in the pre-synaptic gene STXBP1, involved in neurotransmitter release at the synapse [1]. The core symptoms of this disorder include intellectual disability, epilepsy, movement disorders and autism spectrum disorder [2]. The available therapeutic approaches are only symptomatic. Recently, a new potential targeted therapeutic approach has been developed in the wake of precision medicine: SINEUPs are a class of natural and synthetic non-coding RNA which increases the translation of the target protein, without affecting mRNA levels [3] [4]. In the view of this novel therapeutic scenario, a natural history of STXBP1-related disorders is crucial. Aims: 1. To draft developmental trajectories and identify prognostic factors in STXBP1-DEE using retrospective data of a cohort of individuals with STXBP1 disease mutations. 2. To use specific SINEUPs to target STXBP1 mRNA and restore the STXBP1 protein levels in mutated human neurons derived from induced pluripotent stem cells (IPSCs). Methods: 1. Retrospective data (seizures history, neurological examinations, developmental follow-up) were collected from a cohort of STXBP1 patients. A disease-specific developmental score (STXBP1_DevScore) was elaborated to quantitatively assess the development across different individuals. 2. Specific SINEUPs targeting STXBP1 mRNA (STXBP1-SINEUPs) were designed. Human neurons were differentiated from IPSCs with CRISPR-induced STXBP1 mutations. STXBP1-SINEUPs were used to treat the neurons. The STXBP1 protein levels after STXBP1-SINEUPs treatment were assessed by Western blot. Results: 1. We collected clinical data of 48 STXBP1 patients (mean follow-up period: 8 years). The epilepsy history of STXBP1 patients show the presence of at least two distinct patterns of evolution. Clinical data suggest that the neuro-developmental consequences are independent from the seizure outcome. 2. We treated neuronal lines harbouring three different CRISPR-induced mutations in STXBP1, which recapitulated the haploinsufficiency conditions found in patients. In heterozygous neurons, the STXBP1-SINEUPs led to a significant increase of STXBP1 protein compared to negative control. Conclusions: 1. The clinical data collected suggest some common trajectories in the evolution of STXBP1-related disorders. Age at seizure onset is the only epilepsy-related feature associated with neurodevelopment outcome. Efforts should be made to collect natural history data from a larger cohort, in order to identify effective treatment timepoints and rational clinical endpoints for targeted therapies clinical trials. 2. STXBP1-specific SINEUPs have the ability to increase STXBP1 protein levels in human neurons derived from IPSCs, in haploinsufficiency conditions. These results also demonstrate that IPSCs-derived human neurons have great potential as a model for target therapy screening in genetic neurodevelopmental and epileptic disorders. References: 1. Kovacevic J, Maroteaux G, Schut D, et al. Protein instability, haploinsufficiency, and cortical hyper-excitability underlie STXBP1 encephalopathy. Brain. 2018 May 1;141(5):1350-1374. doi: 10.1093/brain/awy046. PMID: 29538625; PMCID: PMC5917748. 2. Stamberger H, Nikanorova M, Willemsen MH, et al. STXBP1 encephalopathy: A neurodevelopmental disorder including epilepsy. Neurology. 2016 Mar 8;86(10):954-62. doi: 10.1212/WNL.0000000000002457. Epub 2016 Feb 10. PMID: 26865513. 3. Zucchelli S, Cotella D, Takahashi H, et al. SINEUPs: A new class of natural and synthetic antisense long non-coding RNAs that activate translation. RNA Biol. 2015;12(8):771-9. doi: 10.1080/15476286.2015.1060395. PMID: 26259533; PMCID: PMC4615742. 4. Bon C, Luffarelli R, Russo R, et al. SINEUP non-coding RNAs rescue defective frataxin expression and activity in a cellular model of Friedreich's Ataxia. Nucleic Acids Res. 2019 Nov 18;47(20):10728-10743. doi: 10.1093/nar/gkz798. PMID: 31584077; PMCID: PMC6847766.