The HTT gene is altered in the Huntington disease due to the expansion of a CAG trinucleotide. The CAG trinucleotide recurs within the first exon of the HTT gene, causing the production of a mutant protein within the gene (Ekman et al., 2019). The altered HTT gene then affects the cells and the individual as a whole. This is seen through the multiplication that occurs in the brain leading to the disruption of vital cellular functions like the nucleocytoplasmic transport (Ekman et al., 2019). Consequently, this results in the loss of certain cortical neurons and medium-sized spiny neurons, which project to the striatum. Once the disease manifests, the individual perishes within ~20 years (Ekman et al., 2019).
The rationale behind using genome editing to treat HD is that reducing or deleting mutant HTT gene manifestations in the affected parts of the brain will stop the development of the disease (Ekman et al., 2019). CRISPR-Cas9 disrupts HTT gene expression through DNA binding, where it presents a DNA double-strand break that will stimulate an error-prone DNA reparation path (NHEJ), to introduce the random base deletions and insertions to frame the mutation of the HTT gene (Ekman et al., 2019).
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The authors initially used a reputable reporter that was able to showcase the first exon of the hominid HTT gene with ninety-four glutamines (Ekman et al., 2019). These glutamines were merged to a variant of cyan fluorescent protein (CFP) that linked the mHTT gene manifestation to CFP fluorescence for easier assessment of the effectiveness of the formulated sgRNAs (Ekman et al., 2019).
Cas9 was delivered to mice through the mode of injections. The researchers ensured that the Cas9 was expressed as neurons by using the hSyn promoter to facilitate the manifestation from the AVV vector (Ekman et al., 2019)
The researchers utilized immunohistochemistry to determine where cas9 was actually represented (Ekman et al., 2019). They analyzed sections of the cortical substance from both untreated and treated samples for the manifestation of SaCas9. They also used an antibody that is efficient in recognizing mutant HTT (Ekman et al., 2019). They also used the main neural cell kind of the striatum by utilizing an antibody that aims at the MSN-specific marker DARPP-32. These revealed ~85 percent of DARPP-32+ cells in the areas of injection had the SaCas9 expressed (Ekman et al., 2019). The Cas9 expression altered the mHTT expression evident with the ~40 percent less mHTT protein annexations in DARPP-32+ and in dual SaCas9+ (Ekman et al., 2019). Mice that were treated to the CRISPR-Cas9 also showed a ~50 percent less total mHTT as compared to control animals through the western blot analysis (Ekman et al., 2019).
The CRISPR-Cas9 treatment provides some therapeutic benefit to the HD mice. This was evident as mice exposed to it had decreased hind limb and improved motor functioning as compared to the 80 percent of the control group animals while also having a ~15 percent mean survival rate as equated to control samples (Ekman et al., 2019). These results, therefore, show that the treatment method disrupts the mHTT, thus increasing survival rates and motor functioning.
The main concern associated with using CRISPR-Cas9 for genome editing is that there could be issues of off-target mutations in the cells that may be prompted by the therapeutic method (Ekman et al., 2019).
Although the use of CRISPR-Cas9 to treat HD has been known to have numerous benefits, here are a few limitations. Key to these is the fact that it is unable to reinstate cells that were lost at the onset of the disease before the treatment method is used (Ekman et al., 2019). This, therefore, means that the individual might still be at a loss even with the treatment in place.
Reference
Ekman, F. K., Ojala, D. S., Adil, M. M., Lopez, P. A., Schaffer, D. V., & Gaj, T. (2019). CRISPR-cas9-mediated genome editing increases lifespan and improves motor deficits in a Huntington’s disease mouse model. Molecular Therapy-Nucleic Acids, 17, 829-839.
