Suppressor tRNA Prime Editing for Premature Stop Codon Readthrough
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Solution Overview
Problem
Nonsense mutations in genomic DNA lead to premature termination codons (PTCs), impeding the translation of full-length proteins and causing pathogenic effects, with existing therapies like adeno-associated viral vectors requiring repeated administration for suppressor tRNA expression, which is challenging.
Innovation Solution
Prime editing is used to convert endogenous tRNAs into suppressor tRNAs by editing their anticodon sequences or inserting suppressor tRNA genes into safe harbor sites in the genome, enabling permanent expression and readthrough of PTCs.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If adeno-associated viral vectors are used to deliver suppressor tRNA, then suppressor tRNA can be expressed to enable stop codon readthrough, but permanent expression is difficult to achieve and repeated administration is required
Solution Approach 1:
The patent uses prime editing to pre-modify endogenous tRNA genes in the genome, converting them into suppressor tRNA genes before they are transcribed. This preliminary genetic modification ensures that the suppressor tRNA will be permanently expressed from the edited genomic DNA, eliminating the need for repeated vector administrations.
Solution Approach 2:
The patent leverages the cell's own endogenous tRNA genes as the substrate for suppression. By editing these native genes using prime editing, the cell's transcriptional machinery automatically produces suppressor tRNA without requiring external continuous supply. The system serves itself by converting its own components into therapeutic agents.
2Reliability
If suppressor tRNA is delivered via AAV vector, then stop codon readthrough can be achieved, but the therapy requires repeated administration which increases treatment complexity
Solution Approach 1:
The prime editing of endogenous tRNA genes is performed as a one-time preliminary action that permanently installs the suppressor function into the genome. This upfront modification simplifies the treatment protocol by eliminating the need for repeated administrations, reducing overall treatment complexity despite the sophistication of the prime editing technology itself.
3Duration of action of stationary object
If endogenous tRNAs are edited into suppressor tRNAs using prime editing, then permanent expression is achieved with minimal perturbation, but the editing process requires precise modification of DNA sequences
Solution Approach 1:
The patent uses prime editing as an intermediary technology that bridges the gap between CRISPR-Cas9 and precise DNA modification. The prime editor system, consisting of a fusion protein of Cas9 and reverse transcriptase along with a pegRNA, serves as a mediator that can install precise nucleotide changes without requiring double-strand breaks, thereby achieving high precision editing of endogenous tRNA genes.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach allows for persistent and safe rescue of protein expression by converting endogenous tRNAs into suppressor tRNAs, tolerable in human cells, with minimal perturbation, and offers a platform for treating genetic diseases caused by PTCs.
Implementation Method 1
Prime editing allows for precise editing of the genomic DNA encoding tRNAs and may provide a platform for the treatment of diseases associated with PTCs
Implementation Method 2
many bacteria and viruses utilize suppressor tRNAs to enable translational stop codon readthrough (e.g., the ribosome goes past the stop codon and continues translating the mRNA into protein)
Data Source
AI summary
Aspects of the disclosure relate to methods, compositions, and systems for editing an endogenous tRNA into a suppressor tRNA, or alternatively, replacing said endogenous tRNA with a suppressor tRNA with using prime editing. Additional aspects relate to compositions comprising the prime editing machinery, pegRNAs, and/or complexes comprising the prime editor and pegRNA that are capable of editing and/or replacing an endogenous tRNA to yield a suppressor tRNA. In some aspects, the disclosure further relates to polynucleotides encoding one or more nucleic acid sequences encoding the prime editor and/or pegRNA, cells comprising the polynucleotides and complexes comprising the prime editor and pegRNA, kits comprising any one of the compositions, complexes, polynucleotides, vectors, and/or cells disclosed herein, and/or delivery systems for administering any one of the compositions, complexes, polynucleotides, vectors to a subject in need thereof. Additional aspects relate to methods for inserting a new suppressor tRNA gene into a target site in a genome (e.g., a safe harbor locus site) using prime editing.