Chemically Modified sgRNA for CRISPR Delivery
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Solution Overview
Problem
Current genome editing delivery systems, such as CRISPR/Cas9, face challenges in achieving efficient and safe systemic delivery for therapeutic genome editing, particularly due to off-target effects and immunogenicity concerns with viral vectors, and lack of fully non-viral, systemic Cas9 genome editing systems for in vivo gene modification.
Innovation Solution
Development of a nucleic acid sequence comprising a DNA-binding domain, a Cas protein-binding domain, and a transcription terminator domain, with specific modifications like phosphorothioate bonds and fluorinated nucleotides, to enhance the stability and specificity of CRISPR/Cas9 delivery using lipid nanoparticles for systemic gene editing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If viral vectors (AAV) are used for CRISPR delivery, then long-term CRISPR expression is achieved, but off-target effects increase and immunogenicity risk increases
Solution Approach 1:
The patent extracts the CRISPR expression function from the viral vector delivery system. Instead of using AAV to deliver and express Cas9 long-term, the invention delivers Cas9 as pre-formed RNP complexes that exert their effect immediately and are transient, while using a separate non-viral mechanism for sgRNA delivery. This separation eliminates the need for long-term viral expression while maintaining editing functionality.
Solution Approach 2:
The patent employs transient, disposable RNP complexes instead of persistent viral vectors. The Cas9 protein delivered as RNP performs its genome editing function and is then degraded by cellular mechanisms, providing a short-lived, non-immunogenic alternative to AAV that avoids long-term presence in human tissue.
2Productivity
If Cas9 is stably expressed by AAV delivery, then efficient genome editing is achieved, but T cell responses to AAV capsid limit repeat dosing
Solution Approach 1:
The invention extracts the genome editing efficiency function from stable viral expression. By delivering Cas9 as pre-assembled RNP complexes with chemically modified sgRNAs, the system achieves efficient editing in a single transient delivery event, eliminating the need for stable AAV expression and thereby avoiding capsid-induced T cell responses that would prevent repeat dosing.
Solution Approach 2:
The patent introduces chemically modified nucleic acids (phosphorothioate bonds, fluorinated nucleotides) as intermediaries that enhance the stability and activity of the sgRNA-Cas9 complex. These modifications allow the RNP to resist nucleases and maintain functionality without requiring persistent viral expression, enabling efficient single-dose delivery that avoids immunogenicity issues.
3Object-affected harmful factors
If non-viral delivery system is used, then immunogenicity risk is reduced, but systemic delivery efficiency for major organs is insufficient
Solution Approach 1:
The patent applies parameter changes to the nucleic acid chemistry itself. By incorporating phosphorothioate bonds at critical positions and fluorinated nucleotides in the sgRNA sequence, the invention fundamentally alters the chemical properties of the RNA to enhance its stability against nucleases and improve its binding affinity to Cas9. These parameter changes enable the non-viral RNP complex to achieve systemic delivery efficiency comparable to viral methods while maintaining low immunogenicity.
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 modified nucleic acid sequence enables efficient and specific genome editing with reduced off-target effects and immunogenicity, facilitating systemic delivery and gene modification in major organs like the liver while minimizing side effects.
Implementation Method 1
the bonds between the first position through fourth position nucleotides are phosphorothioate bonds and the bond between the sixth through 11th position of nucleotides are phosphorothioate bonds
Implementation Method 2
with specific modifications like phosphorothioate bonds and fluorinated nucleotides
Data Source
AI summary
The disclosure relates to compositions comprising and methods for chemical modification of single guide RNA (sgRNA), tracrRNA and/or crRNA used individually or in combination with one another or Cas system components. Compositions comprising modified ribonucleic acids have been designed with chemical modification for even higher efficiency as unmodified native strand of sgRNA. Administration of modified ribonucleic acids will allow decreased immune response when administered to a subject, increased stability, increased editing efficiency and facilitated in vivo delivery of sgRNA via various delivery platforms. The disclosure also relates to methods of decreasing off-target effect of CRISPR and a CRISPR complex.