Benjamin Kleinstiver's team at Harvard Medical School and Massachusetts General Hospital published a research paper titled "Optimization of base editors for the functional correction of SMN2 as a treatment for spinal muscular atrophy" in Nature Biomedical Engineering.
This study used an adenine base editor based on the SpCas9 variant SpRY (ABE8e-SpRY) to reverse C6T of exon 7 of the SMN2 gene in spinal muscular atrophy (SMA) cells and mouse models via an A•T-to-G•C edit. This restored SMN protein levels and improved the body weight, exercise capacity and survival rate of the mouse model. These results suggest that using base editing or other CRISPR-based gene editing technologies to target specific sites in the SMN2 gene and restore SMN protein levels may be helpful in the treatment of SMA patients.
The leading cause of SMA cases is loss-of-function mutations in the SMN1 gene, but SMN1 is not the only player. The copy number of the SMN2 gene is an important factor affecting the severity of SMA. This C-to-T polymorphism in the sixth nucleotide of SMN2 exon 7 (referred to as C6T) causes the skipping of exon 7 in most SMN2 transcripts due to alternative splicing, resulting in non-functional SMN protein. Therefore, the research team tried to use base editing technology to directly edit the SMN2 gene to restore the SMN protein.
Figure 1. Schematic of SMN1 and SMN2 in unaffected individuals and in patients with SMA.
As early as 2020, Benjamin Kleinstiver's team published a paper in the Science journal and developed a variant of SpCas9, SpRY, which no longer needs to rely on specific PAM sequences and therefore can recognize and edit almost any site on the genome.
The research team developed a SpRY-based adenine base editor (ABE8e-SpRY) to reverse C6T (i.e., A-to-G editing) of exon 7 of the SMN2 gene. They applied this editing strategy to fibroblasts extracted from SMA patients and significantly improved the retention of exon 7 in SMN2's mRNA, thereby increasing SMN protein expression levels, and did not find any unexpected off-target editing.
Then, they further used dual-vector adeno-associated virus (AAV) to deliver gRNA and AB into the SMA mouse model. In the tissues of primary interest, A-to-G editing was detected at an average of ~6% (brain) and ~4% (spinal cord), which was able to significantly increase SMN transcript levels by 2-4-fold. The study also showed that longer follow-up resulted in higher levels of base editing. Furthermore, additional intravenous injections increased base editing in liver and heart tissue compared with intracerebroventricular injection alone. Moreover, over time, the body weight, motor function, and survival of these treated mouse models improved significantly. This illustrates that the restoration of peripheral SMN protein is crucial for the phenotypic recovery of SMA mice and can enhance its phenotypic recovery.
Figure 2. AAV-mediated delivery of base editors for in vivo SMN2 C6T editing.
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All these experimental results indicate that base editing-mediated precise editing of C6T in exon 7 of the SMN2 gene can significantly increase functional SMN protein levels, thus providing a new therapeutic strategy for the treatment of SMA. More broadly, this work demonstrates that genes can be precisely modified using adaptable CRISPR nucleases such as SpRY, opening new doors to using this approach to treat other diseases.
Reference
Alves C R R, et al. Optimization of base editors for the functional correction of SMN2 as a treatment for spinal muscular atrophy. Nature Biomedical Engineering, 2023: 1-14.