Scientists find new tools for gene therapy

On June 16, the team of Li Dalian, a professor at the School of Life Sciences of East China Normal University, published a paper in Nature Biotechnology, reporting a series of new adenine switching editing tools (AXBEs and ACBEs), and proved the high efficiency and accuracy of ACBEs in different cell lines and mouse embryos, in which the mouse disease model allele mutation was as high as 100%. This provides new tools for diversified genetic manipulation and gene therapy for the second largest class of single nucleotide variants (SNVs) in humans.

Adenine base reversal schematic. Photo courtesy of East China Normal University

Genetic diseases in humans are mainly caused by genetic mutations, and about 58% are single-base mutations. Currently, single-base editors, which do not rely on DNA double-strand breaks and template participation, are powerful gene editing tools for the treatment of genetic diseases. Existing base editing techniques (CBEs, CGBEs, GBEs, and ABEs) enable cytosine conversion/reversal editing as well as adenine conversion mutations. However, 25% of human single-base mutant genetic diseases still require precise adenine reversal editing (A-to-C or A-to-T) to correct, so it is important to develop efficient and accurate adenine base editors.

Base switching can be achieved by base deamination, while base switching relies on the creation of purine-free pyrimidine-free (AP) sites followed by a base excision repair pathway. Given the inefficient inosine excision repair ability of endogenous glycosidases, the researchers wanted to look for other enzymes that could use inosine as a catalytic substrate. The researchers tried to fuse 9 enzymes with potential inosine excision activity with adenosine deaminase in vitro, and found that mice derived alkyl adenosine DNA glycosidase (mAAG) could achieve 8.7% base switching, and rats, Bacillus subtilis-derived fusion constructs also observed certain adenine reversal mutations.

After a large number of endogenous target evaluation and in vitro enzyme activity experiments, it was found that AXBE has a certain base background sequence preference. Through dependent/undependent off-target assessment at the DNA level and off-target detection at the transcriptome level, AXBE was significantly lower off-target than traditional systems, especially at the RNA level by 90%.

In order to increase the efficiency of adenine switching editing and expand the target range, the researchers identified two key mutations based on structure-oriented rational design and screening, which greatly improved the activity of inosine excision of its substrate and effectively improved the sequence background selectivity of reversal editing. In addition, the mutation can be widely targeted to the genome and expand the scope of application.

“The study found that the adenine swap editor of the mice derived alkyl adenosine DNA glycosidase by screening can achieve adenine reversal in a specific sequence, and further molecular evolution allows editing to be up to 73%. AXBEs bring rich codons and amino acid changes, and will be more suitable for applications such as molecular evolution, genetic screening, and lineage tracing in the future. “This suggests the therapeutic promise of correcting the second largest class of pathogenic SNVs in humans, and ACBEs can also serve as a unique tool to explore site-specific repair mechanisms as opposed to thymine.” (Source: China Science News, Zhang Shuanghu, Huang Xin)

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