020 | Developing a Neuro-Specific Tool for RNAi silencing: from nice ideas to real challenges

Cellular and Molecular Neurobiology

Author: Tomas Eidelman | Email: tomas.eidelman@gmail.com

Tomas Eidelman , Hernán Ezequiel Hauché Pedernera , Oscar Gershanik , Maria Elena Avale , Melina Paula Bordone , Juan Esteban Ferrario

1° aboratorio de Neurobiología de la Enfermedad de Parkinson, IB3, UBA-CONICET Ciudad Autónoma de Buenos Aires, Argentina
2° aboratorio de Terapéutica Experimental en Procesos Neurodegenerativos ,INGEBI-CONICET Ciudad Autónoma de Buenos Aires, Argentina
3° Universidad Favaloro, Ciudad Autónoma de Buenos Aires

The use of RNAi technology to control gene expression holds great promise as a potential therapeutic strategy.RNA-based medicines have gained clinical approval, and others are in various stages of research(Zhu et al 2022).We’ve developed a RNAi targeting Fyn tyrosine kinase’s mRNA, which combined with lentiviral particles delivered into the striatum, reduced dyskinesia in a pre-clinical mice model of Parkinson’s disease (Bordone et al 2021).Although viral transduction is restricted to the infected areas, Fyn expression is ubiquitous throughout the different striatal cells, mediating different roles.Thus our plan involves enhancing the accuracy of gene knock-down within specific neuronal subgroups.Our aim is to design a molecular scalpel to provide a fine therapeutic option that shall direct therapeutic effect and reduce side effects.To reach this goal we’ve designed a strategy using a modified Cre-LoxP system to restrict expression of RNA molecules into dopamine D1R-expressing neurons.We’ve cloned the synapsin promoter inverted between lox71/lox66 sequences upstream a EGFP reporter that should only express in the presence of the recombinase Cre.We cloned and tested this construct in the N2A cell line and in this poster we will discuss our strategy, present in vitro experiments results and discuss this and other strategies towards reaching our goal.The next step is to go forward with the already validated miRNA against Fyn and test its efficacy in a mouse model of LID.