CRISPR/Cas9 ZNF274 Binding Site Modification for Genomic Imprinting

Resolve Bottlenecks,
Find Innovative SolutionsGenerate Solutions

Solution Overview

Problem

Current therapeutic strategies for Prader-Willi Syndrome (PWS) are inadequate in activating silenced maternal RNA transcripts, leading to unmet needs in controlling food intake and managing behavioral and psychiatric abnormalities, with no cure available.

Innovation Solution

A guide RNA (gRNA) molecule and DNA targeting system that binds to specific sequences on chromosome 15, reducing the interaction of ZNF274 protein with its binding site, thereby activating maternal transcripts by using CRISPR/Cas9 technology to modify the ZNF274 binding site and restore gene expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If current therapeutic strategies are used, then some symptomatic relief may be achieved, but the silenced maternal RNA transcripts remain inactive and the underlying cause is not addressed

Engineering Contradiction:

Improveeffectiveness of treatmentVSAvoidability to activate silenced genes

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The patent extracts and removes the ZNF274 protein, which is responsible for silencing maternal transcripts, through CRISPR/Cas9-mediated deletion of the ZNF274 gene. This extraction of the harmful silencing factor directly activates the silenced maternal RNA transcripts, addressing the root cause rather than just treating symptoms.

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent uses CRISPR/Cas9 technology as an intermediary system to achieve gene activation. The guide RNA (gRNA) and Cas9 protein work together as intermediary molecules to precisely target and modify the ZNF274 binding site, enabling controlled removal of the silencing mechanism and subsequent activation of maternal transcripts.

Inventive Principle:

Principle #24Intermediary (Mediator)

2Reliability

If ZNF274 protein interaction with binding site is reduced, then maternal transcript activation is achieved, but requires precise modification of specific DNA sequences

Engineering Contradiction:

Improvegene activation efficiencyVSAvoidprecision of DNA modification

Core Design Contradiction:

ReliabilityVSManufacturing precision

Solution Approach 1:

The CRISPR/Cas9 system serves as a precise intermediary mechanism that guides modifications to the exact ZNF274 binding site. The guide RNA provides sequence-specific targeting, ensuring that only the intended DNA sequences are modified while leaving the rest of the genome unchanged, thus achieving high manufacturing precision.

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent applies local quality modification by specifically altering only the ZNF274 binding site sequences while leaving the rest of the genome intact. This localized modification approach ensures that the precision required is concentrated at the specific target site, achieving high accuracy where needed without unnecessary changes elsewhere.

Inventive Principle:

Principle #3Local quality

3Reliability

If CRISPR/Cas9 technology is used to modify ZNF274 binding site, then maternal gene expression is restored, but requires delivery of genetic components into cells

Engineering Contradiction:

Improvetreatment efficacyVSAvoidcomplexity of delivery system

Core Design Contradiction:

ReliabilityVSDevice complexity

Solution Approach 1:

The CRISPR/Cas9 system provides multi-functionality by combining gene targeting, gene editing, and gene activation capabilities in a single platform. This universal system can deliver multiple genetic components (Cas9 protein, guide RNA, and donor DNA templates) that work together to achieve the therapeutic effect, reducing the need for multiple separate delivery systems.

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

The approach effectively reactivates silenced maternal genes, reducing H3K9me3 marks and increasing expression of key transcripts like SNORD116, SNRPN, and other genes, potentially offering a novel treatment for PWS manifestations.

Implementation Method 1

using CRISPR/Cas9 technology to modify the ZNF274 binding site and restore gene expression

Methodology Applied

Scientific EffectCRISPR/Cas9 gene editing:

Implementation Method 2

reducing the interaction of ZNF274 protein with its binding site, thereby activating maternal transcripts

Methodology Applied

Scientific EffectEpigenetic modification:

Implementation Method 3

The approach effectively reactivates silenced maternal genes, reducing H3K9me3 marks and increasing expression of key transcripts

Methodology Applied

Scientific EffectHistone modification:

Data Source

PatentUS12018255B2Compositions and methods for treating disorders of genomic imprinting

Publication Date: 2024.06.25 UNIV OF CONNECTICUT

AI summary

Disclosed herein are compositions, kits, and methods for treating a disorder of genomic imprinting in a subject. The method may include modifying a zinc-finger protein 274 (ZNF274) binding site on maternal chromosome 15 at position 15q11-q13 of the subject, such that the binding of a ZNF274 protein to the ZNF274 binding site is reduced relative to a control. The ZNF274 binding site comprises a polynucleotide having at least 90% sequence identify to SEQ ID NO: 1 or SEQ ID NO: 42. Further provided are DNA targeting systems that bind to a ZNF274 binding site or to a gene encoding a ZNF274 protein.