APLP2 Safe Harbor Knock-In Using Intronic CRISPR Targeting

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Solution Overview

Problem

Existing methods for modifying the Amyloid Beta Precursor Like Protein 2 (APLP2) gene often disrupt its expression or function, leading to undesirable consequences.

Innovation Solution

A method using a CRISPR system to introduce a composition comprising a CRISPR nuclease and an RNA molecule comprising a guide sequence portion having 17-50 nucleotides, which affects a double strand break in the APLP2 gene, allowing for targeted insertion of a donor molecule under the control of the APLP2 promoter, enabling expression of a desired sequence without knocking out the gene.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Manufacturing precision

If traditional gene modification methods are used to introduce a desired sequence, then the sequence can be inserted into the genome, but the APLP2 gene expression is disrupted or knocked out

Engineering Contradiction:

Improvetargeted sequence insertionVSAvoidAPLP2 gene expression

Core Design Contradiction:

Manufacturing precisionVSReliability

Solution Approach 1:

The patent uses a CRISPR nuclease system as an intermediary to create a controlled double-strand break at a specific location within the APLP2 gene (intron 1 or intron 2), which then enables precise donor molecule integration through homology-directed repair. This intermediary mechanism allows targeted insertion while preserving overall gene structure and expression, resolving the contradiction between precise modification and gene functionality.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The invention applies local quality by targeting specific intronic regions (intron 1 or intron 2) of the APLP2 gene rather than random genomic locations. By confining the modification to these specific local regions that do not disrupt critical coding sequences or regulatory elements, the patent achieves precise sequence insertion while maintaining the integrity and expression of the APLP2 gene.

Inventive Principle:

Principle #3Local quality

2Manufacturing precision

If a double strand break is induced in the APLP2 gene to enable donor molecule insertion, then targeted knock-in can be achieved, but there is risk of disrupting gene expression

Engineering Contradiction:

Improveknock-in at specific locusVSAvoidgene expression disruption

Core Design Contradiction:

Manufacturing precisionVSObject-affected harmful factors

Solution Approach 1:

The patent employs preliminary action by using a CRISPR nuclease guided by a specifically designed RNA molecule to create a double-strand break at a predetermined location (intron 1 or intron 2 of APLP2) before donor molecule introduction. This pre-positioned break ensures that subsequent homology-directed repair will integrate the donor sequence at the exact desired location, achieving precise knock-in while avoiding disruption of critical gene regions.

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The invention converts the potentially harmful double-strand break into a beneficial tool for precise gene modification. By intentionally creating a controlled break at a safe intronic location and providing a donor molecule with homology arms, the system harnesses the cell's natural homology-directed repair mechanism to achieve precise knock-in. The harmful break is transformed into a precise editing event that preserves gene expression.

Inventive Principle:

Principle #22Blessing in disguise (Convert harm into benefit)

3Adaptability or versatility

If existing gene modification strategies are employed, then genetic changes can be made, but deleterious disruptions to the APLP2 gene occur

Engineering Contradiction:

Improvegene modification capabilityVSAvoidAPLP2 gene function

Core Design Contradiction:

Adaptability or versatilityVSReliability

Solution Approach 1:

The patent applies parameter changes by modifying the guide RNA sequence parameters to target specific intronic regions of the APLP2 gene. By adjusting the nucleotide sequence of the guide RNA to match intron 1 or intron 2 regions, the CRISPR system can be precisely directed to these locations, enabling gene modification capability while preserving the critical coding and regulatory sequences that maintain APLP2 gene function.

Inventive Principle:

Principle #35Parameter changes

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

Enables the expression and secretion of a protein of interest in desired body tissues without disrupting APLP2 gene expression, facilitating precise genetic modifications.

Implementation Method 1

a complex of the CRISPR nuclease and the RNA molecule affects a double strand break in at least one allele of the APLP2 gene

Methodology Applied

Scientific EffectCRISPR-mediated DNA cleavage:

Data Source

PatentUS20260021141A1Strategies For Knock-Ins At APLP2 Safe Harbor Sites

Publication Date: 2026.01.22 EMENDOBIO INC

AI summary

RNA molecules comprising a guide sequence portion having 17-50 contiguous nucleotides in the sequence set forth in any one of SEQ ID NOs: 1-159641 and compositions, methods, and uses thereof. Further, wherein a method for modifying in a cell at least one allele of the Amyloid Beta Precursor Like Protein 2 (APLP2) gene is disclosed.