CRISPR/Cas9-Mediated Gene Editing Ameliorates Neurotoxicity (Article Sample)

CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease
Thaha Ahmed Choudhury
CUNY College
Professor
DATE
“CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease” by Yang et al., 2017.
In their research paper “CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease”, Yang et al. (2017) investigates the possible gene therapy applications of the CRISPR-Cas9 system to treat Huntington’s disease in mice. The Huntington’s disease is one of the CAG trinucleotide repeat disorder of the nerve system caused by the expansions of these glutamine repeats within the genes. These trinucleotides expansions occurs in the amino terminal regions of the huntingtin (HTT) gene. The cytotoxic mHTT (mutant huntingtin) proteins are responsible for neural degradations in the Huntington’s disease. Several gene therapy strategies including siRNA, CRISPR/Cas9 and antisense oligonucleotides have been successfully applied to selectively inhibit the mHTT expressions in mice using recombinant mutant huntingtin genes. Studies have shown that knock-out of the HTT in the brains of adult mice had no effects in their neuronal viabilities as well as the survival and growth of the animals. Besides, knockin (KI) mice expressing mutant huntingtin proteins have been shown to successfully develop well in early embryonic stages. Put together, the researchers argued that the elimination of the amino terminal polyglutamine repeats could potentially be used to manage the Huntington’s disease. In this research, Yang and colleagues investigated the effects of mHTT suppression in HD140Q-KI mice striatum using CRISPR-Cas9.
The researchers started by testing whether the CRISPR-Cas9 can edit the polyglutamine (polyQ) repeats in the mutant huntingtin (mHTT) gene in mice. This experiment was necessary in validating their previous hypothesis that claimed that elimination of the amino terminal polyglutamine repeats could possibly reduce the severity of the Huntington’s disease. They designed four gRNAs (guide RNAs) targeting the regions of the DNA on both sides of the human HTT exon 1 CAG trinucleotide repeats and marked them from T1, T2, T3 and T4 depending on their regions of action. Next, Yang and colleagues then added each of the designed gRNAs to HEK293 cells expressing stable human HTT exon 1 consisting of 120 CAG trinucleotides (polyQ) repeats. When they performed western blotting analysis, they observed a steep decline of mHTT in the transfected cells. To further support their findings, the team investigated the efficiency of using two HTT guide RNAs on HEK293 cells. They observed that T1 and T3 pairs of the HTT guide RNAs were the most efficient and therefore chosen for further researches in this paper.
The researchers then investigated the consequences of removing HTT in the in HD140Q-KI mice model. This was done since numerous age-associated deficits in motor functions and nuclear buildup of mHTT have been linked to HD140Q-KI mice. The team expressed the two guide RNAs (T1 and T3) under Uc6 promoter in an AAV vector with RFP reporter system and an AAV-CMV-Cas9. They then mixed the two viruses before performing stereotaxic injection into the striatum of the mouse at three weeks. Their Western blotting analysis revealed confirmed predominant expression of Cas9 and RFP reporter in the injected striatum. Additionally, when they further experimented the injection of AAV-CMV-Cas9 and AAV-HTT-gRNA when the homozygous HD140Q-KI mice were three or nine months old at either side of the striatum. They also injected the contralateral striatum with either AAV-CMV-Cas9 or with AAV-HTT-gRNA. Their results showed that AAVs mainly transduced cortical and hippocampus many striatal and needle pathways in the third week after injection. Similarly, a substantial decline of mHTT in the striatum was caused by HTT-gRNA as reported in the western blot at nine months. The decline was further reported by both immuno-stain and the double immunofluorescence staining which showed a reduced nuclear accumulation of the mHTT and confirmed that the decline in mHTT staining is influenced by the HTT-gRNA expression respectively.
Yang and colleagues further investigated the effects of HTT-gRNA/Cas9 transduction on the protein compositions of different regions of the brain. This was important to understand possible correlations of the Huntington’s disease with proteins like neuronal marker-NeuN, apoptosis marker-caspase 3, p62 autophagy marker and glial fibrillary acidic protein (GFAP). They compared the protein compositions of the hippocampus, striatum and cortex regions of the HD140Q-KI mice injected with AAV-CMV-Cas9 and AAV-HTT-gRNA or control guide RNA via Western blotting. Their results indicated that while there was no change in the cellular expression of NeuN, p62, and caspase 3, conversely, amplified GFAP expression seen. They concluded that mHTT knockdown was responsible for the increased expression of the glial fibrillary acidic protein due, a phenomenon that they postulated might be due to the increased numbers of the reactive astrocytes.
The team then investigated the possibility of using CRISPR-Cas9 system to therapeutically manage the Huntington’s disease in heterozygous HD140Q-KI mice model. Understanding this was important since most patients with Huntington’s disease have heterozygous genes for Huntington’s disease, the gene mutation arises are responsible for such allelic disparities. The researchers used AAV-HTT-gRNAs (T1 and T3) together with AAVCas9 that had been expressed under the neuronal Mecp2 (methyl-CpG –binding protein) promoter. They then injected the pre-mixed viruses into striatum of HD140Q-KI mice aged nine-months. The team then did an immune-stain of the of the transfected striatum. Their results showed the availabilities of the Red Flourescent proteins in the dopamine. Additionally, the cAMP-regulated DARPP-32 (neuronal phosphoprotein) as well as neuronal phosphoprotein were also found to be present in their results. These observations indicated that the striatal transduction of the medium spiny neurons had taken place upon injection of AAVs, through the CRISPR-Cas9 system.
The researchers next investigated if the neuronal mHTT depletion through CRISPR-Cas9 system had effects on the HD140Q KI mice motor functions. This