Closed Linear DNA Production Using Primase-Polymerase Amplification
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Solution Overview
Problem
Current non-viral gene delivery systems, such as plasmid DNA vectors, face challenges including adverse immune responses, low transfection efficiency, and the need for high production yields, while existing processes for producing closed linear DNA (clDNA) are inadequate for large-scale, safe, and cost-effective therapeutic use.
Innovation Solution
A process using a primase/polymerase enzyme for amplifying a DNA template followed by processing to generate clDNA, ensuring high sequence fidelity and efficiency, allowing large-scale production without microorganisms, and enabling the use of diverse templates, including those without primase recognition sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional plasmid DNA vectors are used for gene therapy, then the gene delivery system is easier to store and has greater transgene capacity, but adverse immune responses occur due to bacterial sequences and bioavailability is compromised due to large molecular size
Solution Approach 1:
The invention extracts and removes the bacterial backbone sequences from plasmid DNA vectors, retaining only the essential therapeutic DNA sequence. This is achieved through enzymatic digestion with restriction endonucleases that cleave bacterial DNA sequences while preserving the gene of interest, thereby eliminating immunogenic elements while maintaining transgene capacity
Solution Approach 2:
The plasmid DNA vector is segmented into distinct functional components: the bacterial backbone (removed) and the therapeutic DNA sequence (retained). This segmentation allows separation of the immunogenic bacterial sequences from the functional therapeutic sequence, enabling production of linear DNA that maintains gene delivery capacity without triggering immune responses
2Productivity
If conventional amplification processes are used for clDNA production, then production yield can be achieved, but sequence fidelity is reduced leading to mutated proteins and unspecific products
Solution Approach 1:
The invention introduces a two-stage amplification process using different polymerases as intermediaries. First, a high-fidelity polymerase amplifies the DNA template with maximum accuracy. Second, a different polymerase performs bulk amplification to achieve high production yields. This intermediary high-fidelity amplification step ensures sequence fidelity is maintained even when producing large quantities of clDNA
Solution Approach 2:
The invention performs preliminary high-fidelity amplification before the main production amplification. By first creating a pool of high-quality DNA copies with a high-fidelity polymerase, subsequent amplification steps can achieve high yields without propagating mutations, as the template for bulk amplification already possesses high sequence fidelity
3Productivity
If microorganisms are used in clDNA production processes, then production can be scaled up, but bacterial remains and antibiotic resistance sequences contaminate the final product
Solution Approach 1:
The invention replaces biological production systems (microorganisms) with an in vitro enzymatic system. Restriction endonucleases and DNA polymerases are used in cell-free reactions to amplify and process DNA templates, eliminating the need for microbial cultivation. This substitution produces clDNA without bacterial contamination while maintaining scalability through controlled enzymatic reactions
Solution Approach 2:
The invention uses purified enzymes as intermediaries to perform DNA amplification and processing without microorganisms. Restriction endonucleases serve as intermediaries to cleave bacterial sequences, and DNA polymerases serve as intermediaries to amplify the therapeutic sequence. These enzymatic intermediaries enable large-scale production while avoiding bacterial contamination
4Productivity
If existing clDNA production processes are used, then some production capability is achieved, but the processes are inadequate for large-scale, safe, and cost-effective therapeutic use
Solution Approach 1:
The invention performs preliminary purification and quality control steps before final clDNA production. The process includes initial enzymatic digestion to remove bacterial sequences, followed by selective amplification of the therapeutic sequence. This preliminary action ensures that only high-quality, contamination-free DNA proceeds to large-scale production, thereby ensuring safety for therapeutic use while maintaining productivity
Solution Approach 2:
The invention replaces microbial production systems with in vitro enzymatic systems, eliminating the source of bacterial contamination. By using purified enzymes in controlled cell-free reactions, the process achieves both large-scale production capability and high safety standards required for therapeutic applications, without the risks associated with microbial cultivation
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 process achieves high-quality clDNA production suitable for therapeutic use, with improved transfection efficiency and safety, facilitating efficient gene expression in mammalian cells.
Implementation Method 1
amplifying a DNA template with a primase/polymerase
Data Source
AI summary
The present invention provides a process for the production of a closed linear DNA comprising the steps of (a) providing a DNA template comprising a DNA sequence of interest; (b) amplifying DNA from the DNA template of step (a) wherein the amplification is primed with a primase/polymerase enzyme; (c) generating a closed linear DNA with the amplified DNA produced in step (b); and (d) purifying the closed linear DNA produced in step (c). The invention also provides a closed linear DNA obtainable according to the process of the invention, a pharmaceutical composition comprising a therapeutically effective amount of the closed linear DNA of the invention, and a concatameric DNA comprising repeats of a DNA sequence of interest.