In a recent study published in Gene Therapy, researchers utilize the CRISPR-Cas9 system to induce the synthesis of dopamine (DA) within the brains of a rat model for Parkinson’s disease (PD).

Study: CRISPR/sgRNA-directed synergistic activation mediator (SAM) as a therapeutic tool for Parkinson´s disease. Image Credit: Sergey Nivens / Shutterstock.com

Background

The incidence of PD increases with age and is the second most typical neurological disease. PD is brought on by the degeneration of dopaminergic neurons within the substantia nigra pars compacta, which produces DA to control motor control.

The optimal approach for managing PD involves using levodopa (L-DOPA), a precursor that facilitates the formation and substitution of DA. Importantly, this treatment has a finite duration of effectiveness, which usually spans about five years.

Astrocytes are critical for the immunological response of the brain in PD, as demonstrated by animal models and post-mortem investigations. Tyrosine hydroxylase (Th) is an enzyme found inside astrocytes that’s crucial for the production of DA. Previously, the authors of the present study reported that the implantation of astrocytes expression Th successfully increased DA production within the stratium of each rat and non-human primate models of PD.

Clustered often interspaced short palindromic repeats related to Cas (CRISPR-Cas) is a novel technology that has revolutionized gene therapy. Synergistic activation mediator (SAM) is a second-generation CRISPR system that prompts gene expression using enzymatically inactive Cas9 (dCas9) and co-transcriptional activators.

The current study examines the results of endogenous gene activation in stimulating DA production in astrocytes, followed by the implantation of those cells in a rat model of PD. These findings offer a targeted therapeutic approach that may increase the drug-free period of treatment and may be combined with other therapies in advanced cases of PD.

Concerning the study

The researchers analyzed the rat genome to discover potential th small guide ribonucleic acid (sgRNA) sequences that were highly specific and didn’t align with other areas of the rat genome. This led to the identification of 13 sgRNAs for th gene activation.

 The degrees of Th messenger ribonucleic acid (mRNA) in C6 cells transfected with the chosen Th sgRNAs were assessed using real-time polymerase chain response (RT-PCR).

The 6-hydroxydopamine (6-OHDA) lesion stereotaxic surgery is a widely used rat model to check PD. Herein, 6-OHDA is injected directly into the median forebrain bundle (MFB), wherein it destroys dopamine neurons. Control rats underwent the identical procedure but were injected with a 0.1% ascorbic acid in saline solution.

The 6-OHDA rats were subsequently implanted with 20,000 astrocytes (AST) or astroycytes expression Th (AST-TH) in each the anterior and posterior portions of the striatum. Each before and after surgery, rats underwent several behavioral tests including amphetamine-induced rotations, cylinder, and inclined beam balance tests to find out whether astrocyte implantation improved behavioral characteristics of hemi-Parksonian rats.

Study findings 

Of the 13 th sgRNAs that were identified within the evaluation, the researchers discussed the outcomes of TH4 sgRNA, because it achieved the best levels of Th protein expression. Each C6 and AST-TH cells transfected with TH4 sgRNA continuously released DA into the culture medium, which was not observed of their respective untransfected cells. The expression of TH4 sgRNA was also confirmed through immunofluorescence and Western blot evaluation.

Behavioral studies revealed that rats that received primary astrocytes exhibited significantly greater circling behavior as in comparison with rats that received AST-TH cells. Likewise, rats treated with control astrocytes exhibited significant differences in forelimb placement asymmetry (FPA) within the cylinder test as in comparison with AST-TH transplanted rats.

The inclined beam balance test, which is used to measure tremendous motor control and balance, indicated that AST-TH transplanted rats exhibited similar behavioral characteristics to non-Parkinsonian rats.  

Immunohistochemical evaluation of the rat brains indicated that areas of the striatum that were implanted with AST-TH exhibited increased expression of DA, which is comparable to the dearth of DA observed in lesioned areas of the brain. Moreover, the turnover of DA inside AST-TH transplanted brains remained intact, whereas striatum as treated with control astrocytes didn’t exhibit any turnover of DA. These findings show that AST-TH improves the speed of DA metabolism within the brain.

As in comparison with rat brains that were injected with control astroyctes, those implanted with AST-TH exhibited co-localization of Th and glial fibrillary acidic protein (GFAP), the latter of which is a biomarker of neuroinflammation.