CpG-Modified AAV Vectors for Lower TLR9 Immunogenicity

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Solution Overview

Problem

Unmethylated CpG dinucleotides in adeno-associated virus (AAV) vectors trigger immune responses and toxicity due to recognition by the Toll-like receptor 9 pathway, posing challenges in recombinant AAV vector production and delivery.

Innovation Solution

Modifying AAV vectors by reducing CpG dinucleotides and increasing their methylation, using CRISPR-associated endonucleases and RNA methyltransferases to target CpG dinucleotides for methylation, and employing helper polynucleotides with reduced or increased CpG dinucleotides to minimize immune response triggers.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Ease of manufacture

If AAV vectors contain unmethylated CpG dinucleotides, then the vectors can be produced using standard methods, but they trigger immune responses and toxicity through Toll-like receptor 9 pathway recognition

Engineering Contradiction:

Improveease of manufactureVSAvoidimmune response triggers

Core Design Contradiction:

Ease of manufactureVSObject-affected harmful factors

Solution Approach 1:

The patent changes the chemical state of CpG dinucleotides by increasing methylation levels. Helper polynucleotides are engineered with enhanced CpG methylation, transforming unmethylated immunostimulatory sequences into methylated forms that do not activate Toll-like receptor 9, thereby reducing immune response while maintaining manufacturability

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent extracts and removes immunostimulatory CpG dinucleotides from helper polynucleotides. By designing helper polynucleotides with reduced CpG content or completely eliminating unmethylated CpG sequences, the vectors produce fewer immune response triggers while maintaining essential viral functions

Inventive Principle:

Principle #2Taking out (Extraction)

2Object-affected harmful factors

If helper polynucleotides are modified to reduce CpG dinucleotides or increase methylation, then immune response triggers are reduced, but the complexity of vector production increases

Engineering Contradiction:

Improveimmune response triggersVSAvoidcomplexity of vector production

Core Design Contradiction:

Object-affected harmful factorsVSDevice complexity

Solution Approach 1:

The patent applies preliminary action by pre-engineering helper plasmids with modified CpG characteristics (reduced content or increased methylation) before viral production. This upfront modification of helper polynucleotides ensures that the resulting viral vectors inherently possess reduced immunogenicity without requiring complex post-production modifications or additional processing steps

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The patent uses specially designed helper polynucleotides as intermediaries to transfer the desired low-immunogenicity characteristics to the final AAV vector product. These modified helper polynucleotides serve as mediators that enable production of therapeutic vectors with reduced CpG-induced immune responses through standard viral production workflows

Inventive Principle:

Principle #24Intermediary (Mediator)

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

Reduces immune response triggers, enhancing the safety and efficacy of AAV vectors by minimizing CpG-induced toxicity and immunogenicity, thereby improving therapeutic delivery.

Implementation Method 1

increasing their methylation, using CRISPR-associated endonucleases and RNA methyltransferases to target CpG dinucleotides for methylation

Methodology Applied

Scientific EffectMethylation:

Data Source

PatentUS12514933B2Modified CPG dinucleotides for recombinant viral vector production

Publication Date: 2026.01.06 SIREN BIOTECHNOLOGY INC

AI summary

The disclosure relates generally to methods of producing recombinant viral vectors with reduced immunogenicity.