Linear DNA with NLS and End Adaptors for Nuclear Delivery
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Solution Overview
Problem
Existing gene therapy and gene editing technologies face challenges in efficiently delivering DNA to the nucleus of both dividing and non-dividing cells, particularly due to inefficient trafficking and the presence of unwanted genetic material and susceptibility to nuclease digestion.
Innovation Solution
A linear DNA product is developed with a double-stranded region and adaptor molecules at each end, enhanced with nuclease resistance and appended with a nuclear localization signal (NLS) for improved nuclear delivery.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If plasmids are used for gene therapy delivery, then DNA can be delivered to cells, but the DNA contains unwanted genetic material (e.g., antibiotic resistance genes) and is susceptible to nuclease digestion
Solution Approach 1:
The plasmid DNA is segmented into a linear DNA product containing only the essential therapeutic gene cassette, separated from the bacterial backbone and unwanted genetic material. This segmentation allows delivery of pure therapeutic DNA without antibiotic resistance genes or other harmful elements.
Solution Approach 2:
The unwanted genetic material (bacterial backbone, antibiotic resistance genes, origin of replication) is extracted and removed from the plasmid structure. Only the essential eukaryotic expression cassette is retained, creating a purified therapeutic DNA product.
2Productivity
If naked DNA is delivered to non-dividing cells, then nuclear entry is possible, but trafficking to the nucleus is inefficient
Solution Approach 1:
A nuclear localization signal (NLS) peptide is introduced as an intermediary component that mediates nuclear import of the linear DNA product. The NLS binds to importin proteins which facilitate active transport through nuclear pore complexes, enabling efficient nuclear delivery in both dividing and non-dividing cells.
Solution Approach 2:
The DNA structure is changed from circular plasmid to linear configuration with specific terminal adaptations. This parameter change enables recognition by nuclear import machinery and facilitates active transport into the nucleus, overcoming the inefficiency of naked DNA delivery.
3Object-generated harmful factors
If linear DNA is used instead of plasmids, then unwanted genetic material is removed, but the linear DNA is susceptible to exonuclease digestion
Solution Approach 1:
The linear DNA product is constructed as a composite structure with a eukaryotic expression cassette protected by terminal adaptations. The 5' and 3' ends are modified with structures (such as hairpins or protein binders) that confer exonuclease resistance, creating a stable therapeutic DNA product that combines purity with durability.
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 linear DNA product effectively traverses the nuclear envelope, enhancing nuclear uptake and resistance to exonuclease digestion, suitable for both gene therapy and gene editing applications.
Implementation Method 1
a nuclear localisation signal (NLS) molecule is appended to the linear DNA product
Implementation Method 2
the linear DNA product is resistant to nuclease digestion
Data Source
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AI summary
The invention relates to a linear DNA product that is resistant to nuclease (e.g. exonuclease) digestion, and which is suitable for delivery to the nucleus of a cell. The invention also relates to related methods of production and uses of the linear DNA product.