Self-immolative plasmid backbone
Inventors
Engels, Bart Marinus • Verhaert, Raymond Michael Dimphena • van der Heijden, Maurice Wilhelmus
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Publication Number
US-11959069-B2
Publication Date
2024-04-16
Expiration Date
Abstract
The invention relates to a method for separating a polynucleotide insert from a polynucleotide vector backbone. The backbone has a plurality of cleavage sites distributed such that the backbone is converted into fragments when the sites are cleaved. This allows straightforward separation of the insert from the backbone. The invention also relates to backbones for use in such a method, and to plasmids and kits comprising such backbones.
Core Innovation
A method is provided for separating a polynucleotide insert from a polynucleotide vector backbone by using a recombinant polynucleotide vector in which the vector backbone comprises a first plurality of cleavage sites that divide the vector backbone into fragments. The fragments are selected from a length of at most 1000 bp and at least 40 bp. The approach is carried out by contacting the recombinant vector with cleavage means capable of specifically cleaving the first plurality of cleavage sites to produce backbone fragments and the insert.
The method includes an optional separation step of separating the insert from the backbone fragments produced by the cleavage. The backbone fragments are generated by cleavage of distributed, insert-free cleavage sites in the polynucleotide vector backbone. In this way, the insert is released while the backbone is reduced to small fragments within the claimed length range.
The disclosure further provides embodiments in which the cleavage sites include restriction sites and the cleavage means may include restriction enzymes, RNA-guided DNA endonucleases, and other sequence-specific nucleases, including sequence-specific ultrasonication, and sequence-specific oxidative or hydrolyzing small molecules. Backbone design features are described to avoid triggering cleavage sites in the insert, while providing a selectable functional selection marker and a multiple cloning site that does not trigger cleavage.
In related embodiments, the isolated sequence is used to enhance transcription in eukaryotic cells by genome integration to create transgenic cells. The disclosure also describes a workflow context including recombinant polynucleotide vectors, amplifying or producing vectors, cleaving to release the nucleotide sequence, separating the nucleotide sequence from backbone fragments, and culturing transgenic cells for expression.
Claims Coverage
The document centers on one independent claim covering a backbone-shredding separation method with an optional separation step, and it is supported by dependent claims that add constraints on fragment lengths, define cleavage site and cleavage means categories, specify exemplary separation techniques, and extend into transcription enhancement via genome integration. The inventive coverage is organized around multiple inventive features present in the claim set: size-limited backbone fragments, cleavage means specific to the backbone cleavage sites, and optional insert/backbone separation, with downstream integration and expression.
Size-limited backbone fragmentation
A recombinant polynucleotide vector comprising the insert and the vector backbone wherein the vector backbone comprises a first plurality of cleavage sites that divide the vector backbone into fragments, wherein all fragments are selected from a length of at most 1000 bp and at least 40 bp.
Backbone-specific cleavage to produce insert and backbone fragments
Contacting the recombinant vector with cleavage means capable of specifically cleaving the first plurality of cleavage sites to produce backbone fragments and the insert.
Optional separation of insert from backbone fragments
Optionally separating the insert from the backbone fragments of the cleavage step.
Restriction sites cleavage and defined cleavage means categories
The method in which the cleavage sites from the first plurality of cleavage sites are restriction sites and the cleavage means are selected from restriction enzymes, RNA-guided DNA endonucleases, sequence-specific nucleases, and sequence-specific ultrasonication, including sequence-specific oxidative or hydrolyzing small molecules.
Separation step using spin column, size exclusion column, or SPRI
Performing separation of the insert from the backbone fragments using one of a spin column, a size exclusion column, or solid phase reversible immobilization (SPRI).
Transcription enhancement via genome integration of the released sequence
Enhancing transcription of a nucleotide sequence of interest in a eukaryotic cell by building a recombinant polynucleotide vector, amplifying it in a microorganism, isolating the amplified vector, cleaving it to release the nucleotide sequence, separating the sequence from backbone fragments, integrating the sequence into the genome to create a transgenic cell, and culturing the transgenic cell to express the sequence.
Overall, the claims cover a method that uses a recombinant polynucleotide vector backbone with a first plurality of cleavage sites to generate small backbone fragments within a defined length range upon backbone-specific cleavage, optionally followed by separating the released insert from the backbone fragments. Dependent claims narrow fragment size limits, specify restriction sites and categories of cleavage means, provide example separation mechanisms, and extend the approach to transcription enhancement via genome integration and expression in eukaryotic transgenic cells.
Stated Advantages
Efficient insert purification with reported purity greater than 99.9%.
Improved contaminant/endotoxin profiles versus gel extraction.
Comparable SeAP expression relative to non-backbone-shredded linearized plasmid.
Improved performance relative to non-backbone-shredded linearized plasmid.
Documented Applications
Purification of an intact polynucleotide insert released from a polynucleotide vector backbone generated by backbone fragmentation, including use of spin column, size-exclusion column, or SPRI.
Enhancing transcription of a nucleotide sequence of interest in a eukaryotic cell by isolating the released nucleotide sequence, integrating it into the genome, creating a transgenic cell, and culturing the transgenic cell for expression.
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