Engineered Nuclease Pairing for Precise Repeat Excision In Vivo

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Solution Overview

Problem

Current methods for treating nucleotide repeat expansion disorders, such as Huntington's disease and Fragile X syndrome, are inefficient due to reliance on homologous recombination, which is not the dominant mechanism of DNA break repair in many cell types, making them unlikely to provide significant clinical benefit.

Innovation Solution

The use of a pair of site-specific endonucleases to introduce double-strand breaks at two loci flanking the nucleotide repeat, promoting precise deletion of the intervening sequence through complementary overhangs, independent of homologous recombination, using engineered nucleases like CRISPR/Cas9, TALENs, or meganucleases.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If homologous recombination is used to delete nucleotide repeats, then the treatment can target the disease-causing sequence, but the efficiency is low because homologous recombination is not the dominant mechanism of DNA repair in many cell types

Engineering Contradiction:

Improvedeletion efficiencyVSAvoidclinical benefit likelihood

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent replaces the homologous recombination mechanism with the non-homologous end joining (NHEJ) pathway by using engineered nucleases to create double-strand breaks. The nucleases (such as zinc finger nucleases, TALENs, or CRISPR-Cas9) introduce precise cuts at specific locations flanking the nucleotide repeat, and the cell's dominant NHEJ repair mechanism is harnessed to delete the intervening sequence, thereby substituting an inefficient repair pathway with a more effective one.

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

Solution Approach 2:

The patent changes the key parameter of DNA repair mechanism from homologous recombination to non-homologous end joining. By designing nucleases that create double-strand breaks with specific characteristics (overhangs or blunt ends), the invention shifts the repair pathway preference, utilizing the cell's natural倾向 toward NHEJ for faster and more efficient deletion of the target sequence.

Inventive Principle:

Principle #35Parameter changes

2Device complexity

If a single engineered nuclease is used to target the nucleotide repeat, then the system is simpler, but the precision and control of deletion are reduced

Engineering Contradiction:

Improvenuclease system complexityVSAvoiddeletion precision

Core Design Contradiction:

Device complexityVSManufacturing precision

Solution Approach 1:

The patent divides the deletion task into two separate nuclease components, each targeting one end of the nucleotide repeat. This segmentation allows independent optimization of each nuclease's specificity and efficiency, while the coordinated action of both nucleases ensures precise deletion boundaries. The two-nuclease system creates double-strand breaks at both flanking regions, enabling controlled excision of the intermediate sequence.

Inventive Principle:

Principle #1Segmentation

3Manufacturing precision

If two engineered nucleases are used to flank the nucleotide repeat, then the deletion precision is improved, but the system complexity increases

Engineering Contradiction:

Improvedeletion precisionVSAvoidnuclease system complexity

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The patent employs nuclease platforms (zinc finger nucleases, TALENs, or CRISPR-Cas9) that can be programmed to recognize different DNA sequences through modular design. This universality allows the same basic nuclease architecture to be adapted for targeting multiple locations by simply changing the DNA-binding domain, thereby managing system complexity through standardized, reusable components rather than requiring entirely different molecular tools for each target.

Inventive Principle:

Principle #6Universality (Multi-functionality)

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

This approach achieves a high percentage of precise deletions of the disease-causing nucleotide repeats, eliminating the expanded sequence and restoring a normal phenotype in treated cells and their progeny, without the need for homologous recombination.

Implementation Method 1

engineered nucleases like CRISPR/Cas9, TALENs, or meganucleases

Methodology Applied

Scientific EffectSequence-specific DNA binding:

Implementation Method 2

introduce double-strand breaks at two loci flanking the nucleotide repeat

Methodology Applied

Scientific EffectDNA cleavage:

Implementation Method 3

promoting precise deletion of the intervening sequence through complementary overhangs

Methodology Applied

Scientific EffectComplementary base pairing:

Implementation Method 4

independent of homologous recombination

Methodology Applied

Scientific EffectDNA repair:

Data Source

PatentEP4015633B1Precise deletion of chromosomal sequences in vivo and treatment of nucleotide repeat expansion disorders using engineered nucleases

Publication Date: 2026.04.22 PRECISION BIOSCIENCES INC

Figure 1A~1E
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Figure 2B

AI summary

The present invention provides a method of treating a nucleotide repeat expansion disorder comprising delivering a pair of engineered nucleases, or genes encoding engineered nucleases, to the cells of a patient such that the two nucleases excise the nucleotide repeat responsible for the disease permanently from the genome. The invention provides a general method for treating nucleotide repeat expansion disorders and engineered nucleases suitable for practicing the method. The invention further provides vectors and techniques for delivering engineered nucleases to patient cells.