Evaluation of gene therapies for the treatment of Parkinsons disease using nanostructured vehicles and CRISPR/Cas9

Abstract

Parkinson's disease (PD) causes loss of motor control, cognitive and behavioral functions due to damage to dopaminergic neurons. In astrocytes, the neurodegenerative process is due to the imbalance in oxidative stress, making this cell type a focus of interest in the development of Parkinson's desease. Currently, treatments for PD are not definitive, they are intended to attenuate symptoms, and have side effects such as dyskinesias. Research has developed new ways to address this disease through gene therapies, which consist of modifying the expression of genes of interest. This project aims to evaluate a gene therapy based on CRISPR-nCas9 and a non-viral vehicle to change the expression of the pink1 gene. The cellular effects will be quantified by means of three tests focused on MAO-B activation, mitochondrial membrane potential and ROS production. The information collected is fundamental for the rational design of more effective therapies for PD. To achieve the objective, a mixed culture of astrocytes, neurons and microglia was generated, which assimilate PD through MPTP toxicity. The recovery of the cells thanks to CRISPRa-pink1-MNP, allowed to decrease the amount of MAO-B, ROS, the released JC-1 monomers, and intracellular calcium. In addition, we obtained a restoration of the basal values of the cytokines IFN-gamma, IL-17A, IL-6, IL-1alpha and IL-1beta and a recovery of the EAAT1 antibody. allowing us to conclude that the pink1 gene is of great value to treat and counteract cellular symptoms of Parkinson's together with the vehicle made up of the magnetic nanoparticle.