Therapeutic adenine base editor with minimized off-target effects

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• Adenine base editors (ABEs) are powerful tools for precise genome editing.
• ABE8e, a highly efficient ABE, exhibits significant genome-wide off-target effects.
• Off-target mutations pose a safety concern for clinical applications of ABEs.

Purpose of the Study:

• To identify and develop adenine base editor variants with improved specificity.
• To assess and mitigate the off-target effects of ABE8e.
• To evaluate the therapeutic potential of a novel, high-fidelity ABE variant.

Main Methods:

• Saturation mutagenesis was performed on eight amino acid sites of the TadA8e deaminase domain.
• Genome-wide single nucleotide variants (SNVs) were analyzed using two-cell embryo injection (GOTI) assays.
• The developed ABE variant was tested for its ability to correct a disease-causing mutation in a mouse model of hereditary tyrosinemia type I.

Main Results:

• ABE8e demonstrated a ~30-fold increase in SNVs compared to controls, indicating prevalent off-target effects.
• A novel variant, ABE8eY149V, was engineered, exhibiting high editing efficiency with no detectable off-target mutations.
• ABE8eY149V editing of the Hpd gene rescued lethality in hereditary tyrosinemia type I mice.

Conclusions:

• ABE8eY149V significantly enhances the safety profile of adenine base editing by eliminating off-target effects.
• The engineered ABE8eY149V variant demonstrates broad applicability by fusing with various Cas homologs.
• ABE8eY149V holds substantial promise for safe and effective ABE-based gene therapies.