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The origin of replication (also called the replication origin) is a particular sequence in a genome at which replication is initiated. Propagation of the genetic material between generations requires timely and accurate duplication of DNA by semiconservative replication prior to cell division to ensure each daughter cell receives the full complement of chromosomes. This can either involve the replication of DNA in living organisms such as prokaryotes and eukaryotes, or that of DNA or RNA in viruses, such as double-stranded RNA viruses. Synthesis of daughter strands starts at discrete sites, termed replication origins, and proceeds in a bidirectional manner until all genomic DNA is replicated. Despite the fundamental nature of these events, organisms have evolved surprisingly divergent strategies that control replication onset. Although the specific replication origin organization structure and recognition varies from species to species, some common characteristics are shared.
Reduced immunogenic gene therapy protocols using circular, extended cruciform containing non-viral vectors
PCT designated stage expiredWO2025090786A1Nucleic acid vectorImmunological disordersRepetitive SequencesOrigin of replication
Provided herein are non-immunogenic, circular, non-integrating, non-viral DNA vectors, and methods of using such DNA vectors, which are capable of forming one or more specialized secondary structures, for example extended cruciform structures, and express one or more therapeutic genes. These DNA vectors capable of forming one or more specialized secondary structures, for example extended cruciform structures, provide for increased nucleus entry, episomal stability, and long-term transgene expression, and have unique and highly unexpectedly reduced immunogenicity. The one or more specialized secondary structures, for example extended cruciform structures, formed by the circular DNA vectors provided herein are stabilized through the interaction of two specifically oriented repeating sequences, for example AAV-derived inverted terminal repeat (ITR) sequences, separated by a small, non-repeating sequence, for example a non-repeating sequence comprising a small bacterial origin of replication (Ori).
Owner:RAMPART BIOSCIENCE INC
Non-immunogenic circular, non-viral DNA vectors
The present disclosure relates to circular, non-viral DNA vectors, compositions including one or more of the disclosed vectors, and methods for delivering and / or expressing one or more therapeutic genes (e.g., proteins) in mammals, e.g., human patients. In some embodiments, the present disclosure is directed to circular, non-viral DNA vectors, such as circular non-viral DNA vectors including at least two inverted repeat sequences, where the at least two inverted repeat sequences are separated by a non-repeated nucleotide sequence which is not part of the at least two inverted repeat sequences. In some embodiments, the disclosed circular, non-viral DNA vectors do not include a “DD element.” In some embodiments, the disclosed circular, non-viral DNA vectors do not include a “DD element,” but include at least a portion of a bacterial origin of replication.
Owner:RAMPART BIOSCIENCE INC
Non-immunogenic circular, non-viral DNA vectors
The present disclosure relates to circular, non-viral DNA vectors, compositions including one or more of the disclosed vectors, and methods for delivering and / or expressing one or more therapeutic genes (e.g., proteins) in mammals, e.g., human patients. In some embodiments, the present disclosure is directed to circular, non-viral DNA vectors, such as circular non-viral DNA vectors including at least two inverted repeat sequences, where the at least two inverted repeat sequences are separated by a non-repeated nucleotide sequence which is not part of the at least two inverted repeat sequences. In some embodiments, the disclosed circular, non-viral DNA vectors do not include a “DD element.” In some embodiments, the disclosed circular, non-viral DNA vectors do not include a “DD element,” but include at least a portion of a bacterial origin of replication.
Owner:RAMPART BIOSCIENCE INC
Non-immunogenic circular, non-viral DNA vectors
The present disclosure relates to circular, non-viral DNA vectors, compositions including one or more of the disclosed vectors, and methods for delivering and / or expressing one or more therapeutic genes (e.g., proteins) in mammals, e.g., human patients. In some embodiments, the present disclosure is directed to circular, non-viral DNA vectors, such as circular non-viral DNA vectors including at least two inverted repeat sequences, where the at least two inverted repeat sequences are separated by a non-repeated nucleotide sequence which is not part of the at least two inverted repeat sequences. In some embodiments, the disclosed circular, non-viral DNA vectors do not include a “DD element.” In some embodiments, the disclosed circular, non-viral DNA vectors do not include a “DD element,” but include at least a portion of a bacterial origin of replication.
Owner:RAMPART BIOSCIENCE INC
Large vectors and methods for high-yield production
Provided herein are methods for the production of a vector with a size of at least 16 kb from bacterial cells comprising the consecutive steps of a) obtaining bacterial cells comprising a vector with a size of at least 16 kb, comprising an inducible origin of replication, b) inoculating culture medium with the bacterial cells comprising the vector, c) culturing the bacterial cells in the culture medium, d) adding one or more inducers of said inducible origin of replication to the culture medium when the bacterial culture has reached an optical density at 600 nm (OD600) of at least 20, e) further culturing the bacterial cells in the culture medium, f) optionally separating the bacterial cells from the culture medium, and g) recovering the plasmid from the bacterial cells. Also provided herein are vectors with a size of at least 16 kb comprising an inducible origin of replication for use in such methods.
Owner:KATHOLIEKE UNIV LEUVEN
Non-integrating viral delivery system and methods related thereto
A non-integrating viral delivery system is disclosed. The system includes a viral carrier, wherein the viral carrier contains a defective integrase gene; a heterologous viral episomal origin of replication; a sequence encoding at least one initiator protein specific for the heterologous viral episomal origin of replication, wherein expression of the sequence encoding the at least one initiator protein specific for the heterologous viral episomal origin of DNA replication is inducible; and at least one gene, gene product, shRNA, siRNA, miRNA, or other RNA of interest.
Owner:AMERICAN GENE TECHNOLOGIES INTERNATIONAL INC
A specific targeted method to eliminate bladder without leaving scars. KPC-2 Gene and IncL plasmid sgRNA sequences, CRISPR / Cas9 system and applications
This invention belongs to the field of bioengineering technology and discloses a method for specific targeting and scarless simultaneous elimination blue KPC‑2 Gene and IncL plasmid sgRNA sequences, CRISPR / Cas9 vector system and applications. This system targets the sgRNA of the IncL plasmid. blue KPC‑2 The IncL-type plasmid of the gene was designed with an sgRNA that specifically recognizes the conserved sequence at its origin of replication, thus eliminating the drug resistance gene while disrupting the plasmid's replication ability. Conjugation transfer to recipient bacteria significantly improved vector delivery efficiency. An arabinose-inducible promoter was used to control Cas9 gene expression, improving system stability and editing success rate. The vector carries a sucrose-inducible self-eliminating gene, enabling controlled vector self-elimination. This system is not a simple superposition but rather constitutes a time-controlled operational flow: efficient delivery via conjugation, controlled cleavage via induction, and finally, induced self-elimination.
Owner:KUNMING UNIV OF SCI & TECH
Improved generation of viral and nonviral nanoplasmid vectors
PendingJP2026116424AOrigin of replicationInverted Repeat Sequences
A method for improving the replication of covalent closed circular plasmids is provided. [Solution] The method comprises the step of preparing a covalent closed circular plasmid having a Pol I-dependent origin of replication and an insert containing a structured DNA sequence selected from the group consisting of a reverse repeat sequence, a directional repeat sequence, a homopolymer repeat sequence, a eukaryotic origin of replication, or a eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication. The method also comprises the step of modifying the covalent closed circular recombinant molecule so that the Pol I-dependent origin of replication is replaced with a Pol III-dependent origin of replication, thereby improving the replication of the covalent closed circular plasmid with the resulting Pol III-dependent origin of replication. A covalent closed circular recombinant DNA molecule without antibiotic markers is also provided.
Owner:ALDEVRON LLC
Vector for gene transfer and gene copy proliferation
The invention relates to a circular vector for plant transformation comprising: a first tethering nucleic acid and a second tethering nucleic acid; a nucleic acid encoding a polynucleotide of interest (POI); a nucleic acid comprising an origin of replication; and two or more nucleic acids encoding replicon proteins and methods for using the same. Also provided are plants produced by the methods of the invention and products produced from the plant.
Owner:CLEMSON UNIVERSITY
Non-immunogenic circular, non-viral DNA vectors
The present disclosure relates to circular, non-viral DNA vectors, compositions including one or more of the disclosed vectors, and methods for delivering and / or expressing one or more therapeutic genes (e.g., proteins) in mammals, e.g., human patients. In some embodiments, the present disclosure is directed to circular, non-viral DNA vectors, such as circular non-viral DNA vectors including at least two inverted repeat sequences, where the at least two inverted repeat sequences are separated by a non-repeated nucleotide sequence which is not part of the at least two inverted repeat sequences. In some embodiments, the disclosed circular, non-viral DNA vectors do not include a “DD element.” In some embodiments, the disclosed circular, non-viral DNA vectors do not include a “DD element,” but include at least a portion of a bacterial origin of replication.
Owner:RAMPART BIOSCIENCE INC
Optimized lentiviral transfer vectors and uses thereof
PendingUS20260062712A1Polypeptide with localisation/targeting motifImmunoglobulin superfamilyHeterologousOrigin of replication
The invention features lentiviral transfer vectors that include heterologous nucleic acids to be introduced into a cell. The lentiviral transfer vector may be characterized by the following features: (a) including a cytomegalovirus (CMV) promoter; (b) including a polynucleotide encoding a partial gag protein that includes a mutated INS1 inhibitory sequence that reduces restriction of nuclear export of RNA; (c) not including a polynucleotide encoding the INS2, INS3, and INS4 inhibitory sequences of gag; (d) not including an SV40 origin of replication and / or an f1 origin of replication; (e) including a cPPT sequence that contains splice site; (f) including an EF1alpha promoter with intact splice donor and acceptor sites; and (g) including hepatitis B PRE with mutation in start codon of X protein ORF.
Owner:NOVARTIS AG +1
CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR / Cas9) system for specific targeted traceless elimination of blaNDM-1 gene and IncN plasmid and application
PendingCN121950810AOvercome the bottleneck of conversion difficultiesImprove delivery efficiencyHydrolasesMicroorganism based processesGene deliveryOrigin of replication
The invention belongs to the technical field of biological genetic engineering, and discloses a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / associated protein 9) system for specific targeted traceless elimination of a blaNDM-1 gene and an IncN plasmid and application of the CRISPR / Cas9 system. According to the system, sgRNA of an original point conserved sequence is copied by designing an IncN type plasmid carrying a blaNDM-1 gene in a targeting manner, so that the replication capability of the plasmid is destroyed while a drug-resistant gene is eliminated; the carrier delivery efficiency is greatly improved by conjugational transfer to recipient bacteria; the expression of the Cas9 gene is controlled by adopting an arabinose inducible promoter, so that the constitutive toxicity of Cas9 protein is reduced, and the system stability and the editing success rate are improved; the sucrose-induced carrier self-elimination gene carried by the carrier realizes controllable self-elimination of the carrier. The system is not simply superposed, but forms an operation process with a controllable time sequence: efficient delivery is firstly carried out through joint, then cutting is controlled through induction, and finally self-elimination is induced.
Owner:KUNMING UNIV OF SCI & TECH
DNA production methods
PCT designated stageWO2026102108A1Genetic material ingredientsFermentationOrigin of replicationDouble strand
The disclosure provides, for example, a method of making a composition comprising purified DNA molecules. In some embodiments, the method comprises contacting a solution comprising a plurality of circular, double-stranded DNA molecules to a hydrophobic interaction chromatography matrix. In some embodiments, the circular, double-stranded DNA molecules lack one or both of a bacterial origin of replication and a selectable marker.
Owner:FLAGSHIP PIONEERING INNOVATIONS VII LLC
Generating improved viral and non-viral nanoplasmid vectors
PendingCN120249340AMicroorganism based processesNucleic acid vectorOrigin of replicationInverted Repeat Sequences
The present invention provides for the production of improved viral and non-viral nanoplasmid vectors. Specifically, the invention provides an antibiotic-label-free covalent closed circular recombinant DNA molecule comprising: a. An antibiotic-label-free insert comprising a structured DNA sequence selected from the group consisting of a reverse repeat, a forward repeat, a homopolymerized repeat and an eukaryotic origin of replication; b. A Pol III-dependent origin of replication comprising an R6K [gamma] origin of replication having at least 95% sequence identity to a sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 18; and c. An RNA-OUT RNA selection marker, which comprises an RNA-IN modulating RNA-OUT RNA having at least 95% sequence identity with SEQ ID NO: 6, and a method for detecting the RNA-OUT RNA.
Owner:ALDEVRON LLC
Double-strand circular DNA vector
The present invention addresses the problem of providing a new replication initiation region capable of increasing the yield of plasmid DNA. Provided is a double-strand circular DNA vector having a replication initiation region in which at least the base corresponding to position 22 in the base sequence represented by SEQ ID NO: 1 is an adenine base.
Owner:KANEKA CORP
Novel shuttle vector
The present invention addresses the problem of providing a novel vector using a replication initiation point derived from Thermus thermophilus. The problem is solved by providing a shuttle vector that includes a specific first replication initiation point derived from Thermus thermophilus and a second replication initiation point derived from a biological species other than the Thermus thermophilus.
Owner:OSAKA UNIVERSITY
Proteus mirabilis homologous recombination system expression plasmid, expression system, application and gene knockout / knockin method
PendingCN122012561AMicroorganism based processesStable introduction of DNAHeterologousOrigin of replication
The invention belongs to the field of biology, and discloses a proteus mirabilis homologous recombination system expression plasmid which comprises a pBR322 replication starting point, a resistance gene, an arabinose inducible promoter and a proteus mirabilis-derived homologous recombination operon. The homologous recombination operon is used for coding a TPM66325 protein, an EPM66325 protein, a single-chain binding protein SSB and a fake release protein. The expression plasmid has relatively high recombination efficiency in proteus mirabilis GDMCC 66325 and can also play a recombination role in a proteus mirabilis standard strain ATCC35659, and the expression plasmid is obtained by knocking out a related gene bcsB formed by a hemolysin related gene hpmA and a biological membrane of the proteus mirabilis GDMCC 66325 and directly knocking in a heterologous gene after a related gene cluster is adjusted by flagellum. The effectiveness of the expression plasmid in recombinant operation of proteus mirabilis is verified; meanwhile, the invention further discloses a proteus mirabilis homologous recombination system, application and a gene knockout / knockin method.
Owner:SOUTH CHINA AGRICULTURAL UNIVERSITY
Non-immunogenic cyclic non-viral DNA vectors
PendingCN119866375APeptide/protein ingredientsHydrolasesOrigin of replicationInverted Repeat Sequences
The present disclosure relates to cyclic non-viral DNA vectors, compositions comprising one or more of the disclosed vectors, and methods of delivering and / or expressing one or more therapeutic genes (e.g., proteins) in mammals, e.g., human patients. In some embodiments, the present disclosure relates to cyclic non-viral DNA vectors, such as cyclic non-viral DNA vectors comprising at least two inverted repeats, where the at least two inverted repeats are separated by a non-repeating nucleotide sequence that is not part of the at least two inverted repeats. In some embodiments, the disclosed cyclic non-viral DNA vectors do not include "DD elements". In some embodiments, the disclosed cyclic non-viral DNA vectors do not include a "DD element", but include at least a portion of a bacterial origin of replication.
Owner:RAMPART BIOSCIENCE INC
Biosynthesis method
The present invention relates generally to methods for producing products by cells, in particular to the field of industrial biotechnology or microbiology, in which useful chemicals and products, such as biological products such as proteins, are produced using living cells. The present invention relates to a method of producing a product by a cell, the method comprising: excising one or more nucleic acid sequences from a chromosome in the cell wherein the one or more nucleic acid sequences comprise a gene of interest and a starting point of replication; forming a DNA molecule outside the chromosome, wherein the DNA molecule outside the chromosome comprises a resected target gene and a replication starting point which are resected; and inducing expression of the target gene from the DNA molecule outside the chromosome. The invention also relates to cells for producing the product, polynucleotide vectors and uses thereof.
Owner:GEARBOX BIOSCIENCES EUROPE
A shuttle vector for expression in Escherichia coli and Bacillus
The present invention belongs to the field of molecular biology and provides a shuttle vector for expression in Escherichia coli (E. coli) and Bacilli, said shuttle vector comprising a high-copy replication origin functional in E. coli, a low to medium-copy ORI functional in Bacilli, and a synthetic constitutive regulatory nucleic acid that confers reduced constitutive expression compared to the corresponding initiation regulatory nucleic acid molecule in bacterial cells.
Owner:BASF SE
Mutant vectors for improved host transformation
PCT designated stageWO2025255554A1Microbiological testing/measurementVirus peptidesOrigin of replicationReplication initiator protein
The invention provides methods of screening replication initiator protein (Rep) mutants that modulate vector's copy number of different origins of replication within Agrobacterium. The invention also provides compositions comprising such Rep mutants that can improve Agrobacterium-mediated transformation efficiency of a vector, such as a binary vector, to a host cell.
Owner:RGT UNIV OF CALIFORNIA
Viral and non-viral nanoplasmid vectors with improved production
ActiveUS12600984B2Microorganism based processesNucleic acid vectorOrigin of replicationInverted Repeat Sequences
A method for improving the replication of a covalently closed circular plasmid is provided. The method includes providing a covalently closed circular plasmid having a Pol I-dependent origin of replication, and an insert including a structured DNA sequence selected from inverted repeat sequences, direct repeat sequences, homopolymeric repeat sequences, eukaryotic origins of replication or eukaryotic promoter enhancer sequences, wherein the structured DNA sequence is located at a distance of less than 1000 bp from the Pol I-dependent origin of replication in the direction of replication. The method also includes modifying the covalently closed circular recombinant molecule such that the Pol I-dependent origin of replication is replaced with a Pol III-dependent origin of replication, whereby the resultant Pol III-dependent origin of replication covalently closed circular plasmid has improved replication. An antibiotic marker free covalently closed circular recombinant DNA molecule is also provided.
Owner:ALDEVRON LLC
Method for the production of replication-defective recombinant polyomaviral vector particles
PCT designated stageWO2025170466A1Genetic material ingredientsDsDNA virusesOrigin of replicationDNA construct
The present invention relates to a method for the production of replication-defective recombinant polyomaviral vector particles, the method comprising the steps of a) providing a mammalian cell line permissive or non-permissive for the wildtype polyomavirus, b) introducing into said cell line a recombinant polyomaviral first DNA construct comprising recombinant DNA and a polyomaviral intergenic region, wherein said recombinant polyomaviral first DNA construct not encoding functional polyomaviral T antigens, and wherein said intergenic region comprises the early and late promoters, the origin-of-replication and the packaging signal, c) culturing the cells of said cell line in a growth medium under conditions allowing the formation of said vector particles, and d) extracting the vector particles from said growth medium and / or said cells, wherein a gene encoding functional polyomaviral large T antigen is introduced into said cell line via a separate second DNA construct.
Owner:AMARNA HLDG
Non-immunogenic circular non-viral DNA vectors
PendingJP2025525583AMetabolism disorderPeptide/protein ingredientsOrigin of replicationInverted Repeat Sequences
The present disclosure relates to circular non-viral DNA vectors, compositions comprising one or more of the disclosed vectors, and methods for delivering and / or expressing one or more therapeutic genes (e.g., proteins) in a mammal, e.g., a human patient. In some embodiments, the disclosure relates to circular non-viral DNA vectors, such as circular non-viral DNA vectors comprising at least two inverted repeat sequences, wherein the at least two inverted repeat sequences are separated by a non-repetitive nucleotide sequence that is not part of the at least two inverted repeat sequences. In some embodiments, the disclosed circular non-viral DNA vectors do not comprise a "DD element." In some embodiments, the disclosed circular non-viral DNA vectors do not comprise a "DD element," but comprise at least a portion of a bacterial origin of replication.
Owner:RAMPART BIOSCIENCE INC
A cytosine base editor based on cynomolgus monkey APOBEC3A and its mutants
The present invention discloses a cytosine base editor based on cynomolgus monkey APOBEC3A and its mutants, belonging to the field of genetic engineering technology. A series of CBE single-base editors constructed by the present invention mainly include sgRNA, mA3A or its mutants, nCas9 (D10A), red fluorescent protein, uracil DNA glycosylase inhibitor, high copy replication origin and ampicillin resistance screening marker gene. The editing window of the single-base editor constructed by the present invention is the C3-C14 site, the editing efficiency can reach up to 75%, and the product purity can reach up to 95%. The present invention uses APOBEC3A of non-human primate cynomolgus monkey as the cytosine deaminase of the CBE system for the first time, enriching the gene editing toolkit, and providing a good idea and direction for the optimization of cytosine base editors in the future.
Owner:JIANGNAN UNIV
Engineered bacteriophage particle and method of gene therapy by using same
PCT designated stage expiredWO2024243134A3BacteriaMicroorganism based processesGene deliveryOrigin of replication
Provided is an engineered bacteriophage particle for gene delivery including an engineered phagemid having a restructure f1 origin of replication, an M13 phage capsid encapsulating the engineered phagemid, and a cell-targeting ligand displayed on the M13 phage capsid. Also provided is a vector system for producing the engineered bacteriophage particle and a method of gene therapy by using the engineered bacteriophage particle.
Owner:ACAD SINICA +1
Compositions and methods for nucleic acid replication
PCT designated stageWO2025188249A1Microbiological testing/measurementTransferasesOrigin of replicationTelomerase
This disclosure concerns compositions and methods for replicating DNA containing a T7 origin of replication (T7 or / ). Compositions and methods herein use a set of proteins that includes a DNA polymerase (DNAP), an RNA polymerase (RNAP), a single-strand binding protein (SSBP) and a helicase-primase (HP), along with one or more linearising proteins and linearising protein-binding nucleic acid sequences to maintain the replicated DNA in a linear conformation. In one example, the addition of a protelomerase recognition sequence (TeIRL) as the linearising protein-binding nucleic acid sequence to a T7 or / plasmid, in combination with the use of a protelomerase (TelN) as the linearising protein during DNA replication, enabled linearisation, replication and maintenance of a T7 or / plasmid in E. coli. The DNAP may be a mutagenic polymerase, so that methods herein may also be used for DNA mutagenesis and evolution of target DNA sequences.
Owner:NATIONAL UNIVERSITY OF SINGAPORE
Biosynthetic methods
The present invention generally relates to methods for producing products using cells, and more particularly to the field of industrial biotechnology or industrial microbiology in which living cells are used to produce useful chemical substances and products, such as biological products such as proteins. The present invention relates to a method for producing products using cells, comprising the steps of: excising one or more nucleic acid sequences including a target gene and an origin of replication from a chromosome within a cell; forming an extrachromosomal DNA molecule including the excised target gene and the excised origin of replication; and inducing the expression of the target gene from the extrachromosomal DNA molecule. The present invention also relates to cells, polynucleotide vectors, and their use for producing products.
Owner:オウ ギアボックス バイオサイエンシーズ
Method for Producing Plasmid, and Plasmid
Provided are a method for producing a novel plasmid in heterologous expression of a gene and a plasmid. The method includes preparing a first gene cluster containing the plurality of genes by a DNA assembly method using a plasmid transformation system of Bacillus subtilis; and linking, to the first gene cluster, a second gene cluster containing a replication origin of Bacillus subtilis, a replication origin of Escherichia coli, and an initiation sequence for conjugation to an actinomycete.
Owner:SPIBER INC
Generation of improved viral and non-viral nanoplasmid vectors
ActiveCN112154208BMicroorganism based processesNucleic acid vectorOrigin of replicationInverted Repeat Sequences
A method for improving the replication of a covalently closed circular plasmid is provided. The method includes providing a covalently closed circular plasmid, the plasmid having a Pol I-dependent replication origin and an insert, the insert comprising a structured DNA sequence selected from the group consisting of: an inverted repeat sequence, a forward repeat sequence, a homopolymeric repeat sequence, a eukaryotic replication origin, or a eukaryotic promoter enhancer sequence, wherein the structured DNA sequence is located at a distance of less than 1000bp from the Pol I-dependent replication origin along the replication direction. The method also includes modifying the covalently closed circular recombinant molecule so that the Pol I-dependent replication origin is replaced by the Pol III-dependent replication origin, and the Pol III-dependent replication origin covalently closed circular plasmid thus obtained has improved replication. A covalently closed circular recombinant DNA molecule without an antibiotic marker is also provided.
Owner:ALDEVRON LLC