Serine Integrase Fusion Protein for Precise Large-Cargo Gene Transfer

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Solution Overview

Problem

Existing gene transfer methods, such as CRISPR-Cas and viral vectors, are inefficient for precise integration of large genetic cargo, while transposases like PiggyBac pose risks of malignant transformation due to random integration.

Innovation Solution

A fusion protein comprising a large serine integrase with at least 80% sequence identity to Pa01 and a nuclear localization sequence (NLS) at its C-terminus, enhancing integration efficiency and specificity in human cells.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If transposases like PiggyBac are used to transfer large genetic cargo, then transfer efficiency is improved, but random integration causes harmful effects through insertional mutagenesis

Engineering Contradiction:

Improvegene transfer efficiencyVSAvoidinsertional mutagenesis

Core Design Contradiction:

ProductivityVSObject-affected harmful factors

Solution Approach 1:

The patent uses a large serine integrase enzyme as an intermediary to mediate site-specific recombination between the transgene and a predetermined genomic landing pad. This replaces the random integration mechanism of transposases with a controlled, targetted recombination process, maintaining high transfer efficiency while eliminating insertional mutagenesis risks

Inventive Principle:

Principle #24Intermediary (Mediator)

Solution Approach 2:

The patent changes the integration mechanism parameter from random (transposase) to site-specific (serine integrase). By modifying the enzymatic mechanism and adding nuclear localization signals to enhance nuclear uptake, the system achieves both high efficiency and precision, resolving the contradiction between productivity and safety

Inventive Principle:

Principle #35Parameter changes

2Manufacturing precision

If CRISPR-Cas and viral vectors are used for precise gene integration, then integration precision is improved, but efficiency for large genetic cargo (>5 kb) deteriorates

Engineering Contradiction:

Improveintegration precisionVSAvoidgene transfer efficiency

Core Design Contradiction:

Manufacturing precisionVSProductivity

Solution Approach 1:

The large serine integrase system serves multiple functions: it efficiently integrates large DNA cargos (up to 100 kb), maintains high precision through site-specific recombination at predetermined landing pads, and enables safe therapeutic applications. This multi-functional enzyme system resolves the contradiction by achieving both high precision and high productivity for large cargo transfer

Inventive Principle:

Principle #6Universality (Multi-functionality)

3Reliability

If large serine integrase is used for precise DNA integration, then integration specificity is improved, but integration efficiency in human cells deteriorates due to poor nuclear uptake

Engineering Contradiction:

Improveintegration specificityVSAvoidintegration efficiency

Core Design Contradiction:

ReliabilityVSProductivity

Solution Approach 1:

The patent merges the large serine integrase enzyme with a nuclear localization signal (NLS) to create a fusion protein. This combination preserves the integrase's site-specific recombination activity while adding nuclear targeting capability, thereby resolving the contradiction between specificity and efficiency by enabling both precise genomic integration and efficient nuclear delivery in human cells

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

The fusion protein achieves precise integration of DNA fragments up to 100,000 bp with minimal off-target events, suitable for therapeutic applications in human cells.

Implementation Method 1

Large serine integrases are enzymes derived from bacteriophages, which are viruses that infect bacteria. These integrases mediate site-specific and unidirectional recombination, allowing for the precise insertion or excision of DNA segments at specific sequences (so called attachment sites or 'landing pads') within the genome.

Methodology Applied

Scientific EffectSite-specific recombination: Enzyme

Implementation Method 2

a fusion protein comprising a large serine integrase with at least 80% sequence identity to Pa01 and a nuclear localization sequence (NLS) at its C-terminus

Methodology Applied

Scientific EffectNuclear localization:

Data Source

PatentEP4703469A1Fusion proteins, methods and compositions for efficient serine integrase-mediated gene transfer in human cells

Publication Date: 2026.03.04 CHARITE UNIVSMEDIZIN BERLIN KORPERSCHAFT DES OFFENTLICHEN RECHTS

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AI summary

The present invention relates to the field of gene transfer, in particular, targeted gene transfer and enzymes for use in targeted gene transfer. In particular, the present invention provides a nucleic acid encoding a fusion protein having serine integrase activity, wherein the fusion protein comprises a serine integrase consisting of a protein having at least 80% sequence identity to the large serine integrase Pa01 and a nuclear localization sequence at the C-terminus of the serine integrase. The invention further provides a fusion protein encoded by said nucleic acid as well as methods of preparing an engineered cell using the nucleic acids and/or fusion proteins of the invention. Finally, the present invention provides various pharmaceutical compositions and their use in treating a disease or disorder.