Prime editing-installed suppressor tRNAs for disease-agnostic genome editing

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• Precise genome editing tools like base and prime editing can fix gene variants but require mutation-specific agents.
• Nonsense mutations causing premature stop codons are targets for suppressor tRNAs (sup-tRNAs), but current methods need lifelong treatment or cause toxicity.
• Existing sup-tRNA therapies face limitations in potency and require continuous administration.

Purpose of the Study:

• To develop a novel, disease-agnostic genome-editing strategy for rescuing nonsense mutations.
• To engineer an optimized, endogenous suppressor tRNA (sup-tRNA) using prime editing.
• To evaluate the therapeutic potential of prime editing-mediated readthrough of premature termination codons (PERT) in genetic disease models.

Main Methods:

• Utilized prime editing to permanently convert a dispensable endogenous tRNA into an optimized sup-tRNA.
• Screened thousands of human tRNA variants to identify those with the highest sup-tRNA potential.
• Optimized prime editing agents for single-locus installation of engineered sup-tRNAs without overexpression.
• Tested PERT in human cell models of Batten disease, Tay-Sachs disease, and cystic fibrosis.
• Administered a prime editor in vivo to convert a mouse tRNA into a sup-tRNA in a Hurler syndrome model.

Main Results:

• Achieved efficient readthrough of premature termination codons and protein rescue in cell models.
• Demonstrated extensive rescue of disease pathology in a mouse model of Hurler syndrome following in vivo PERT delivery.
• Confirmed PERT did not induce readthrough of natural stop codons.
• Observed no significant transcriptomic or proteomic alterations.
• Identified specific tRNA variants with potent sup-tRNA capabilities.

Conclusions:

• PERT is a potent, disease-agnostic genome-editing strategy for treating genetic diseases caused by nonsense mutations.
• This approach enables permanent correction via endogenous sup-tRNA generation, avoiding lifelong administration or overexpression toxicity.
• PERT shows promise for a single therapeutic agent to address diverse genetic disorders by targeting premature stop codons.
• The findings support the development of novel therapeutic genome-editing approaches for a broad range of genetic conditions.