TnpB Nucleobase Editing via LNP Delivery for In Vivo Precision
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Solution Overview
Problem
Existing genome-editing technologies like CRISPR/Cas9 have limitations in efficiency, precision, and scalability, particularly in vivo applications, and there is a need for improved systems that utilize innovative systems to address these challenges.
Innovation Solution
The use of TnpB-based systems comprising a TnpB polypeptide and a recombinant TnpB ncRNA, which are engineered to enhance the efficacy of the efficacy of the system.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If CRISPR/Cas9 genome-editing technologies are used, then sequence-specific modifications can be introduced into genomes, but editing efficiency and precision are limited
Solution Approach 1:
The patent uses a synthetic mRNA template that copies and encodes the TnpB protein sequence, allowing expression of the editing enzyme without introducing it directly into the cell. This template-based approach enables precise control over protein production and improves editing consistency
Solution Approach 2:
The patent modifies the TnpB system by changing parameters such as using synthetic mRNA instead of direct protein delivery, optimizing the mRNA sequence for efficient translation, and adjusting delivery parameters to achieve improved editing precision and efficiency
2Reliability
If existing genome-editing systems are used, then gene editing can be performed, but deliverability in vivo is insufficient
Solution Approach 1:
The patent replaces direct mechanical delivery of protein or DNA with an mRNA-based system that leverages the cell's natural translation machinery. The mRNA is delivered via lipid nanoparticles, and the cell's ribosomes translate it into TnpB protein, improving deliverability and reducing immunogenicity
Solution Approach 2:
The patent introduces mRNA as an intermediary carrier between the delivery vehicle and the functional protein. This intermediary allows the editing system to be delivered in a stable, non-immunogenic form that can be translated into active enzyme within the cell
3Manufacturing precision
If TnpB-based systems are engineered with recombinant ncRNA, then precision and efficiency are enhanced, but system complexity increases
Solution Approach 1:
The patent merges the guide RNA and scaffold RNA functions into a single recombinant ncRNA molecule that simultaneously guides the TnpB protein to the target site and provides structural stability. This consolidation reduces the number of separate components and simplifies delivery
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
The TnpB-based systems provide enhanced precision and efficiency in gene editing, with improved deliverability and scalability, making them suitable for treating genetic disorders and complex diseases.
Implementation Method 1
a recombinant TnpB ncRNA... which is capable of associating with the TnpB polypeptide to form a complex such that the complex localizes to a target nucleic acid sequence... and binds thereto
Implementation Method 2
The TnpB protein has a nuclease activity which results in the cutting of one or both strands of DNA
Data Source
AI summary
The disclosure provides nucleic acid-containing lipid nanoparticle (LNP) compositions and methods relating to the delivery of TnpB nucleobase editing systems comprising TnpB polypeptides, engineered TnpB ncRNAs, and optionally one or more additional accessory functionalities (e.g., a deaminase, reverse transcriptase, recombinase, nuclease, a donor template, or combinations thereof) for use in applications such as precision gene editing.