Soluble Hook Fusion Protein for Reversible ER Retention

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Solution Overview

Problem

Existing molecular systems for controlling intracellular trafficking of target proteins face limitations such as large hook sizes impairing lentivirus production, impaired gene expression due to internal ribosome entry sites (IRES), and irreversible ER retention that prevents protein retrieval from the membrane.

Innovation Solution

Development of a soluble hook fusion protein with cytoplasmic ER retention signals that can work in a 'trans' configuration, allowing reversible control of membrane protein expression and retrieval from the Golgi and later compartments, and inclusion of an endocytosis signal for ER retrograde transport.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If traditional hooks (STIM1-NN, Ii, KDEL) are used for ER retention, then the target protein can be retained in the ER, but the hook size becomes large (above 4 kb) which impairs lentivirus production

Engineering Contradiction:

ImproveER retention capabilityVSAvoidlentivirus production efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent extracts and utilizes only the essential ER retention signal sequences (KKXX or K(X)KXX at the C-terminus, or RKR/RKX at the N-terminus) from the larger hook proteins, separating the retention function from the bulky structural components. This extracted signal sequence approach achieves ER retention with a minimal 5-10 amino acid tag, dramatically reducing the insert size from above 4 kb to well below 1 kb, thereby restoring lentivirus production efficiency while maintaining reliable ER retention capability.

Inventive Principle:

Principle #2Taking out (Extraction)

2Adaptability or versatility

If IRES is used for bicistronic or multicistronic vector construction, then multiple proteins can be expressed from a single vector, but gene expression is impaired

Engineering Contradiction:

Improvemulti-protein expression capabilityVSAvoidgene expression efficiency

Core Design Contradiction:

Adaptability or versatilityVSProductivity

Solution Approach 1:

The patent introduces the 2A peptide sequence as an intermediary self-cleaving element between the hook protein coding sequence and the reporter gene. This 2A peptide acts as a molecular mediator that enables simultaneous translation of both proteins from a single mRNA transcript while allowing their physical separation. The result is restored high-level expression of both the hook protein and reporter, eliminating the expression impairment caused by direct IRES linkage while maintaining the versatility of multi-protein expression from one vector.

Inventive Principle:

Principle #24Intermediary (Mediator)

3Reliability

If hooks are stably anchored in the ER membrane, then ER retention is achieved, but the system becomes irreversible and target proteins cannot be retrieved from the membrane

Engineering Contradiction:

ImproveER retention stabilityVSAvoidreversibility and protein retrieval capability

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The patent transforms the static, irreversible ER retention system into a dynamic, reversible one by using soluble hook proteins that can freely diffuse in the cytoplasm rather than being stably anchored in the ER membrane. The soluble hook proteins dynamically associate with target proteins containing SBP tags, forming reversible complexes that can be controlled by adding biotin to displace the hook proteins. This dynamic system maintains reliable ER retention when needed while enabling complete reversibility and protein retrieval from the membrane by simply removing the soluble hook proteins through biotin competition.

Inventive Principle:

Principle #15Dynamics

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 efficient and reversible control of target protein localization, reducing the size of hook constructs, improving lentivirus production, and preventing protein leakage to the plasma membrane, while allowing retrieval from the Golgi and later compartments.

Implementation Method 1

a soluble hook fusion protein that binds to a target membrane protein in the cytosol

Methodology Applied

Scientific EffectProtein-protein interaction:

Implementation Method 2

at least one cytoplasmic carboxy terminal endoplasmic reticulum (ER) retention signal and/or at least one cytoplasmic amino terminal endoplasmic reticulum (ER) retention signal

Methodology Applied

Scientific EffectSignal-mediated protein targeting:

Implementation Method 3

inclusion of an endocytosis signal for ER retrograde transport

Methodology Applied

Scientific EffectEndocytosis:

Data Source

PatentUS12252517B2Hook fusion protein for regulating the cellular trafficking of a target protein

Publication Date: 2025.03.18 INSTITUT CURIE

AI summary

A hook fusion protein, which includes a hook domain and at least one cytoplasmic carboxyl endoplasmic reticulum (ER) retention signal and/or at least one cytoplasmic amino terminal endoplasmic reticulum (ER) retention signal; wherein the hook fusion protein is a soluble protein that localizes in the cytoplasm. Also, a nucleic acid system for intracellular targeting control including a nucleic acid encoding a target fusion protein including a hook fusion protein, and a nucleic acid encoding a target fusion protein including a hook-binding domain; wherein the target fusion protein is a membrane protein; and wherein the hook fusion protein localizes in the ER when bound to the target fusion protein. Additionally, a vector system, viral particle system, host cell and kit include these nucleic acids. Further, the vector system, viral particle system, host cell or kit for use as a medicament, in particular for immunotherapy.