Fusion Protein Gene Editing Constructs for Site-Specific Integration
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Solution Overview
Problem
Current gene delivery methods, such as lentiviral vectors, face challenges with low efficiency and lack of specificity, leading to unintended insertion mutagenesis and genotoxicity due to random integration in the host genome.
Innovation Solution
Development of nucleic acid constructs comprising fusion proteins of programmable transposases and integrases, such as hyperactive PiggyBac transposases and modified HIV integrases, which enable site-specific integration of exogenous nucleic acids into the genome, using a combination of DNA binding proteins and linkers to enhance specificity and efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If lentiviral vectors are used for gene delivery, then stable infection of dividing and non-dividing cells is achieved, but random integration occurs leading to insertional mutagenesis
Solution Approach 1:
The patent uses a fusion protein that combines a specific DNA-binding domain (Cas9 or zinc finger protein) with an integrase domain. The DNA-binding domain acts as an intermediary that guides the integrase to a specific genomic target site, preventing random integration while maintaining stable infection capability. This intermediary component directs the integration process to predetermined safe harbor loci.
Solution Approach 2:
The patent merges two functional domains into a single fusion protein: a programmable DNA-binding domain (Cas9 or zinc finger protein) and an integrase domain. This combination allows the protein to simultaneously recognize specific genomic sequences and catalyze integration at those sites, resolving the contradiction between stable integration and random insertion by unifying targeting and integration functions in one molecule.
2Productivity
If viral vectors are used for gene delivery, then genetic material is delivered into the genome, but efficiency and specificity are low
Solution Approach 1:
The DNA-binding domain serves as an intermediary that provides high-specificity targeting to predetermined genomic loci. This intermediary component recognizes specific DNA sequences through programmable base pairing (CRISPR-Cas9) or zinc finger-DNA interactions, thereby achieving high integration specificity while maintaining efficient delivery through the integrase catalytic activity.
Solution Approach 2:
The patent utilizes programmable DNA-binding domains where the target sequence can be changed by modifying guide RNA (in Cas9 system) or zinc finger sequences. This parameter change capability allows precise targeting to different genomic locations, achieving both high efficiency and high specificity by simply changing the binding parameters rather than the core integration mechanism.
3Ease of manufacture
If HIV-1 integrase is used for DNA insertion, then integration is catalyzed, but insertion is random and lacks specificity
Solution Approach 1:
The patent combines the catalytic integrase domain with a specific DNA-binding domain in a fusion protein. This merger allows the integrase to retain its catalytic function for efficient integration while the attached DNA-binding domain provides site-specific targeting, thereby achieving both ease of integration catalysis and precision in integration site selection through a single unified protein structure.
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 allows for controlled, site-specific integration of exogenous nucleic acids into the genome, reducing off-target effects and improving the precision of gene editing, particularly for large genes, thereby enhancing the safety and efficacy of gene therapy.
Implementation Method 1
a first DNA binding protein engineered to bind to a specific genomic DNA sequence in a genome
Implementation Method 2
the fusion protein enables insertion of the exogenous nucleic acid into a specific site of the genome
Data Source
AI summary
A nucleic acid constructs are used in improving site-specific insertion of an exogenous nucleic acid into a genome. In some aspects the nucleic acid construct having a first polynucleotide sequence encoding a DNA binding protein engineered to bind to a specific genomic DNA sequence, a second polynucleotide having a modified integrase or a modified transposase that enables insertion of exogenous nucleic acid into the genome, and a nucleic acid sequence encoding a linker between the two nucleotides. In some aspects, the nucleic acid construct encodes a fusion protein, for example, a fusion protein for delivery to a cell by a lentiviral particle.