Targeted Genome Insertion Using nrRT and Modified Uridine RNA
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Solution Overview
Problem
Current methods for inserting DNA into host cell genomes face issues such as immune response, mutagenicity, and non-specific integration, especially in post-mitotic cells like neurons, and existing gene therapy technologies have limitations in safety and efficiency.
Innovation Solution
The use of a two-RNA system comprising an mRNA encoding a non-LTR retrotransposon reverse transcriptase protein (nrRT) and a template RNA with modified uridines to facilitate site-specific integration of heterologous polynucleotides into eukaryotic genomes, avoiding the need for viral vectors and minimizing cellular toxicity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If DNA transgenes are introduced into the cytoplasm of a cell, then gene integration can be achieved, but an immune response is induced that can be harmful to cells or the organism
Solution Approach 1:
The patent changes the chemical parameters of the nucleosides in the template RNA by incorporating modified uridines (such as N1-methyl-pseudouridine, pseudouridine, 5-methyluridine, or 5-methyoxyuridine) instead of standard uridines. This parameter change reduces the immunogenicity of the RNA while maintaining its functionality as a template for reverse transcription and integration into the host genome.
2Reliability
If homologous recombination is used for DNA integration at a target site, then integration can occur, but a potentially mutagenic double-strand break must be introduced in the genomic DNA
Solution Approach 1:
The patent replaces the mechanical process of creating double-strand breaks (as done in CRISPR-Cas9 or TALEN systems) with a biochemical process using reverse transcription. The nrRT protein catalyzes the conversion of template RNA into cDNA that is then integrated into the host genome through target-primed reverse transcription, avoiding the need for mutagenic double-strand breaks.
3Productivity
If homologous recombination is used for DNA integration, then integration efficiency is improved in dividing cells, but integration at non-specific locations occurs in post-mitotic cells such as neurons
Solution Approach 1:
The patent introduces an RNA template with modified uridines as an intermediary carrier that directs the nrRT protein to specific target sites. The modified uridines serve as a mediator that enhances the stability and specificity of the RNA-protein complex, enabling precise targeting in post-mitotic cells without relying on cell division.
4Ease of manufacture
If standard uridines are used in template RNA, then the RNA can be cleaved by ribozymes for processing, but insertion efficiency into the eukaryotic genome is reduced
Solution Approach 1:
The patent changes the chemical structure of uridines in the template RNA to modified versions (N1-methyl-pseudouridine, pseudouridine, 5-methyluridine, or 5-methyoxyuridine). This parameter change prevents ribozyme cleavage while simultaneously enhancing the insertion efficiency of the payload sequence into the eukaryotic genome, likely due to increased RNA stability and improved reverse transcription efficiency.
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 enhances the efficiency and fidelity of gene insertion, allows for targeted integration in non-dividing cells, and reduces immunogenicity and mutagenesis, providing long-term expression and safety in gene therapy applications.
Implementation Method 1
an RNA encoding a non-LTR retrotransposon reverse transcriptase protein (nrRT) comprising a reverse transcriptase domain and an endonuclease domain; and a template RNA... the nrRT is expressed in the cell and catalyzes insertion of a double stranded heterologous polynucleotide comprising the payload sequence at the target site in the eukaryotic genome
Implementation Method 2
the template RNA comprises one or more modified uridine (U) nucleosides selected from the group consisting of N1-methyl-pseudouridine (N1mΨU), pseudouridine (ΨU), 5-methyluridine (5meU), 5-methyoxyuridine (5moU), and mixtures thereof... the template RNA comprising modified U increases the insertion efficiency of the payload sequence into the eukaryotic genome
Data Source
AI summary
The present disclosure provides compositions and methods for inserting heterologous payload sequences into a target-site in a host cell genome. The compositions and methods use non-LTR retrotransposon reverse transcriptase proteins that bind template RNAs comprising a payload sequence that encodes a protein or regulatory RNA. The template RNA can comprise modified uridines that are not cleavable by a ribozyme. The incorporation of modified uridines increases the efficiency of integration and expression of the payload sequence and decreases cellular toxicity.