Chemically Modified mRNA Encoding Cas Endonucleases and Base Editors
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Solution Overview
Problem
Existing CRISPR delivery systems for gene editing in human cells are inefficient and cumbersome, necessitating improved methods for easier and more effective delivery of CRISPR effectors.
Innovation Solution
Development of chemically modified RNA (modRNA) sequences encoding Cas endonucleases or base editors, combined with mutated p53 proteins, to enhance gene editing efficiency, utilizing structures like m7G (5') pppN1pN2p (CAP0) or m7G (5') pppN1mpNp (CAP1) and incorporating UTRs and polyA tails, with optional guide RNAs for targeted editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional plasmid or viral vector systems are used to deliver CRISPR effectors, then delivery can be achieved, but the process is cumbersome and editing efficiency is low
Solution Approach 1:
The patent applies parameter changes by chemically modifying RNA molecules with specific structures (m7G cap, pseudouridine modifications, polyA tail lengths) to optimize delivery efficiency and editing performance. These chemical parameter modifications enable modRNA to overcome the limitations of traditional plasmid and viral vector systems, achieving higher editing efficiency with simpler delivery processes.
2Productivity
If chemically modified RNA sequences are used, then gene editing efficiency is significantly enhanced, but the molecular structure complexity increases
Solution Approach 1:
The patent applies local quality by introducing specific chemical modifications at particular locations within the RNA structure - such as m7G cap at the 5' end, pseudouridine residues at specific positions, and polyA tail at the 3' end. Each modification serves a specific functional purpose and is placed strategically to maximize editing efficiency while maintaining overall structural manageability.
Solution Approach 2:
The modRNA constructs represent composite molecular structures combining multiple chemical modification types (capping groups, base modifications, tail structures) into a single functional RNA molecule. This composite approach integrates various chemical features that collectively enhance stability, delivery, and editing efficiency beyond what single modifications could achieve.
3Ease of operation
If modRNA delivery system is used, then delivery is quicker and easier, but the chemical modification process adds complexity
Solution Approach 1:
The patent applies preliminary action by pre-modifying the RNA molecules with chemically stable structures (m7G capping, pseudouridine incorporation, polyA tailing) before delivery. These preliminary chemical modifications are performed during in vitro transcription, creating ready-to-deliver modRNA molecules that require no further modification at the target site, thereby simplifying the overall delivery process while maintaining manufacturing feasibility.
Data Source
AI summary
Disclosed herein are gene editing systems comprising chemically modified RNAs (modRNAs) encoding Cas endonucleases or base editors. Wherein a gene editing system comprising a first chemically modified RNA (modRNA) comprising a sequence encoding a CRISPRassociated (Cas) endonuclease or a base editor; and a second modRNA comprising a sequence encoding a mutated p53 protein, wherein the mutated p53 protein inhibits a wild type p53 protein from binding to a target domain thereof.