dCas9 Fusion Proteins for Frataxin Expression Modulation

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Solution Overview

Problem

Current treatments for Friedreich's Ataxia primarily focus on symptom management rather than addressing the fundamental etiology and disease mechanism, as they fail to effectively modulate the reduced activity and dysregulation of the frataxin (FXN) gene expression.

Innovation Solution

The development of DNA-targeting systems, including fusion proteins and guide RNAs, that bind to and target the frataxin locus to modulate its expression, specifically using deactivated Cas9-transcriptional activator fusion proteins and guide RNAs targeting promoter and enhancer regions to increase FXN expression.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If conventional treatments are used for Friedreich's Ataxia, then symptom management is provided, but the fundamental etiology and disease mechanism are not addressed

Engineering Contradiction:

Improveeffectiveness of treatmentVSAvoidcomplexity of therapeutic approach

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The patent uses dCas9 (deactivated Cas9) as an intermediary molecule that binds to regulatory DNA elements of the FXN gene without causing DNA breaks. This intermediary enables targeted transcriptional activation through fusion with activator domains (VP64, p65, p300), thereby increasing frataxin expression to address the fundamental etiology of Friedreich's Ataxia while avoiding the harmful effects of traditional gene editing approaches

Inventive Principle:

Principle #24Intermediary (Mediator)

2Quantity of substance

If DNA-targeting systems with effector domains are used to increase FXN transcription, then frataxin expression is increased, but the risk of genetic disruption or DNA breaks increases

Engineering Contradiction:

Improvefrataxin expression levelVSAvoidgenetic disruption or DNA break

Core Design Contradiction:

Quantity of substanceVSObject-affected harmful factors

Solution Approach 1:

The patent converts the potentially harmful nuclease activity of Cas9 into a beneficial non-destructive binding mechanism by using deactivated Cas9 (dCas9). This allows the system to target regulatory DNA elements and increase frataxin transcription through effector domains (VP64, p65, p300) without causing DNA breaks or genetic disruption, thereby safely achieving therapeutic frataxin expression levels

Inventive Principle:

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

3Power

If multiple effector domains are combined for synergistic activation, then transcription activation is enhanced, but the device complexity increases

Engineering Contradiction:

Improvetranscription activation strengthVSAvoidfusion protein structure

Core Design Contradiction:

PowerVSDevice complexity

Solution Approach 1:

The patent merges multiple effector domains (VP64, p65, p300) into single fusion proteins with dCas9 to achieve synergistic transcription activation. These combined effector domains work together to strongly activate frataxin transcription from targeted regulatory elements, providing enhanced therapeutic effect while maintaining a manageable protein structure through systematic domain fusion

Inventive Principle:

Principle #5Merging (Combining)

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

These systems effectively increase frataxin expression in cells from patients with Friedreich's Ataxia, both at the mRNA and protein levels, offering a potential therapeutic approach by targeting regulatory DNA elements for controlled and synergistic activation, thereby addressing the disease's underlying cause.

Implementation Method 1

a guide RNA (gRNA) that binds a target site in a regulatory DNA element of a frataxin (FXN) locus

Methodology Applied

Scientific EffectHybridization:

Implementation Method 2

a deactivated Cas9 (dCas9) protein fused to one or more effector domains, wherein the gRNA binds a target site

Methodology Applied

Scientific EffectProtein-RNA binding:

Implementation Method 3

at least one effector domain that increases transcription of the FXN locus

Methodology Applied

Scientific EffectTranscriptional activation:

Data Source

PatentUS20240254483A1Compositions and methods for modulating expression of frataxin (FXN)

Publication Date: 2024.08.01 TUNE THERAPEUTICS INC

AI summary

Provided in some aspects are compositions, such as DNA-targeting systems, fusion proteins, guide RNAs (gRNAs), and pluralities and combinations thereof, that bind to or target a frataxin (FXN) locus. In particular, the present disclosure relates to the modulation of expression of the FXN gene. In some aspects, the present disclosure also relates to poly nucleotides, vectors, cells and pluralities and combinations thereof, that encode or comprise the DNA-targeting systems, fusion proteins, gRNAs or pluralities or combinations thereof, and methods and uses related to the provided compositions, for example, in modulating the expression of FXN, and/or in the treatment or therapy of diseases or disorders that involve the activity, function or expression of FXN, such as Friedreich's Ataxia (FA).