Abstracts

Rationale: STXBP1-encephalopathy is a broad neurological disorder caused by mutations in the pre-synaptic gene STXBP1, crucially involved in neurotransmitter release at the synapse. The core symptoms of this disorder include intellectual disability, epilepsy, movement disorders, and autism spectrum disorder1. Nowadays, the therapeutic approach to this neurodevelopmental encephalopathy is mainly based on symptomatic treatment of seizures, which are often drug-resistant, and occupational and physical therapy to maximize the developmental potential. We propose a new targeted therapeutic approach in the wake of precision medicine. SINEUPs are a class of natural and synthetic non-coding RNA composed of a Binding Domain (BD) specifically binding an mRNA’s ATG and an Effector Domain (ED) which increases the translation by 1,5-5 fold2. These compounds can be used to specifically increase the protein levels in case of haploinsufficiency due to loss-of-function mutations, in vivo, in site-specific manner, and without risking genetic off-target effects. Here we present preliminary results of the use of SINEUPs to increase STXBP1 protein levels in a human cell line.References:1. Stamberger H, et al. STXBP1 encephalopathy: A neurodevelopmental disorder including epilepsy. Neurology.2016;86:954-62.2. Zucchelli S, et al. SINEUPs are modular antisense long non-coding RNAs that increase synthesis of target proteins in cells. Front Cell Neurosci. 2015b; 9:174. Methods: After bioinformatic analysis of the promoter region of interest, SINEUPs are designed with 3 different BD for STXBP1. The constructs are cloned in plasmid vectors and transfected into SKNBE(2) cells using Lipofectamine. The proteins are extracted by RIPA buffer at 48-hours post-transfection, and the protein levels assessed by Western Blot. Target mRNA quantification and SINEUPs expression is assessed by qRT-PCR. SINEUPs with the only ED and with a Scrambled-BD are used as negative controls (NC). eGFPpdual and SINEUP-eGFP are used as positive controls of transfection.  Results: We designed three SINEUPs: -40/+32, -40/+4, -14/+4 from the ATG of STXBP1, and cloned them into plasmid vectors. SKNBE(2) cells/well were transfected with 1 ug of the plasmids using Lipofectamine. Transfection control experiments (n=3) with GFP and SINEUP-GFP showed a mean 4-fold increase in GFP levels at Western blot at 48h (Fig.1). The pilot transfections with 3 STXBP1-SINEUPs at 48h showed a 1.2 to 1.8 fold increase in STXBP1 levels (normalized per GAPDH and related to the NCs), when compared to the NCs and the Not-Treated samples. RNA levels of STXBP1 after the administration of SINEUPs remained stable as expected.  Conclusions: Preliminary results prove the SINEUPs’ potential to specifically increase the protein levels without off-target effects on the genome. We are going to perform several tests at different time-points to select the most performant construct. The best STXBP1-SINEUP will be tested in mutated patients’ neurons differentiated in our Lab from induced pluripotent stem cells to evaluate the functional rescue potential of SINEUPs on the synaptic vesicles release and recycling. This is an extremely flexible approach to target many developmental and epileptic encephalopathies caused by haploinsufficiency and therefore to address these diseases in a more tailored and radical way.  Funding: No funding