Single Donor Repair Template for Multiplex Genome Editing

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Solution Overview

Problem

Existing genome editing technologies struggle to efficiently and simultaneously edit multiple genetic loci using DNA repair templates, particularly with adeno-associated virus (AAV) templates, which are not suitable for targeting multiple genetic loci, and require multiple nucleases and templates, making them ineffective for complex cell-based therapies.

Innovation Solution

A single DNA donor repair template is designed with pairs of homology arms targeting multiple sites, including engineered nuclease cleavage sites in immune system genes, and transgenes like immunopotency enhancers or antigen receptors, delivered via recombinant AAV vectors, enabling multiplex genome editing in immune effector cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Adaptability or versatility

If multiple nucleases and multiple DNA donor repair templates are used to target multiple genetic loci, then the ability to edit multiple sites is achieved, but the device complexity and manufacturing difficulty increase significantly

Engineering Contradiction:

Improveability to target multiple genetic lociVSAvoidnumber of nucleases and templates required

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The patent merges multiple DNA donor repair templates into a single multiplexed template that contains multiple AAV replication origins and multiple pairs of homology arms. This single template can simultaneously target multiple genetic loci, eliminating the need for multiple separate templates and reducing overall system complexity while maintaining the ability to edit multiple sites

Inventive Principle:

Principle #5Merging (Combining)

Solution Approach 2:

The single DNA donor repair template is designed with universal functionality to perform multiple editing tasks simultaneously. It contains multiple AAV replication origins that enable independent replication of different AAV minigene cassettes, and multiple pairs of homology arms that can target different genetic loci, making one template serve the function of multiple templates

Inventive Principle:

Principle #6Universality (Multi-functionality)

2Reliability

If adeno-associated virus (AAV) DNA repair templates are used for single locus editing, then low immunogenicity and efficient homology-directed recombination are achieved, but the template cannot simultaneously target multiple genetic loci

Engineering Contradiction:

Improveefficiency of homology-directed recombinationVSAvoidability to target multiple genetic loci

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The DNA donor repair template is segmented into multiple AAV minigene cassettes, each containing a pair of homology arms and an AAV replication origin. These segmented cassettes are arranged in tandem repeats within a single plasmid, allowing each cassette to independently function for homology-directed recombination at different genetic loci while maintaining the low immunogenicity characteristics of AAV

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Multiple AAV minigene cassettes are nested within a single DNA donor repair template structure. Each cassette is self-contained with its own homology arms and replication origin, nested sequentially within the larger plasmid framework, allowing the template to deliver multiple editing functions through a single vector

Inventive Principle:

Principle #7Nested doll (Nesting)

Data Source

PatentUS12559742B2Donor repair templates multiplex genome editing

Publication Date: 2026.02.24 NOVO NORDISK AS

AI summary

The present disclosure provides improved multiplex genome editing compositions and methods. The disclosure further provides genome edited cells for the prevention, treatment, or amelioration of at least one symptom of a hemoglobinopathy, a cancer, an infectious disease, an autoimmune disease, an inflammatory disease, or an immunodeficiency.