34 results about "Inverted Repeat Sequences" patented technology

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.

The present invention relates to a method for propagating plasmids containing at least one inverted repeat, wherein said method comprises the step of cultivating the Escherichia coli bacteria at a temperature of at least 39°C. Moreover, the invention relates to a plasmid encoding for a cargo nucleic acid including at least one inverted repeat from a method of the present invention. The invention further relates to a cargo nucleic acid including at least one inverted repeat obtained from a method of the present invention.

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.

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.

This disclosure generally relates to super minimal transposon inverted repeat sequence (ITR) polynucleotides, compositions comprising the polynucleotides and methods of using compositions comprising the polynucleotides for the ex vivo and in vivo delivery of nucleic acids to cells, in particular, in vivo delivery of therapeutic genes to treat genetic disorders or diseases.

The application belongs to the technical field of biology, and discloses a kind of Myzus persicae densovirus, amplification and connector method and its application, Myzus persicae densovirus name is Myzus persicae densovirus 2, abbreviated as MpDV2, preserved in China typical culture preservation center, and the preservation number is V202281.The Myzus persicae densovirus MpDV2 provided in the application is a virus strain that has not been isolated and identified before and has pathogenicity to Myzus persicae, and the full-length sequence of the genome of the Myzus persicae densovirus MpDV2 (including the complete terminal inverted repeat sequence) is disclosed for the first time, and the influence of the virus infection on the growth and development of Myzus persicae and the population dynamics / growth.The Myzus persicae densovirus MpDV2 of the application significantly reduces the intrinsic growth rate (r), net reproduction rate (R0) and week-long growth rate (lambda) of Myzus persicae population, and prolongs the average generation time (T) of the population, and the MpDV2 infection has a significant influence on the growth and development of Myzus persicae and population growth.

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.

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.

To provide nucleic acids (including AAV expression cassettes), AAV vectors, and compositions for use in methods of treating and / or delaying the onset of diseases associated with mutations in mecp2 genes, such as Rett Syndrome.SOLUTION: Also provided herein are methods for treating a brain-derived neurotrophic factor (BDNF) - associated disease and / or delaying the onset of a brain-derived neurotrophic factor (BDNF) - associated disease. The present disclosure provides a nucleic acid comprising an adeno-associated virus (AAV) expression cassette, wherein the AAV expression cassette comprises, in a 5' to 3' direction, a 5' inverted terminal repeat (ITR), a synthetic activity-dependent promoter, a Rett syndrome-associated gene, and a 3' ITR.SELECTED DRAWING: None

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.

Provided are a plasmid system for recombinant adeno-associated virus packaging and the use thereof. Provided is a plasmid system for rAAV packaging. The plasmid system comprises: a plasmid containing two terminal inverted repeat sequences, an adenovirus helper plasmid and an rAAV recombinant packaging plasmid, wherein a site sequence recognized by a recombinase and / or a recombinase sequence of the sequence is inserted into at least one of the plasmid containing two terminal inverted repeat sequences, the adenovirus helper plasmid, and the rAAV recombinant packaging plasmid. During the rAAV production process, the ratio of non-rAAV-related sequences being incorrectly packaged into the AAV viral capsid can be effectively reduced, and the purity of the rAAV product is increased, so that the druggability of the rAAV product is greatly improved.

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.

The invention belongs to the technical field of biological medicine, and particularly relates to the field of gene therapy and pain therapy. The invention firstly provides a new application of the ASIC3 gene in anti-inflammation. The invention further provides a sgRNA and AAV-CRISPRi virus system for targeted inhibition of the ASIC3 gene, the AAV-CRISPRi virus system is a CRISPRi system based on AAV delivery, and the core functional structure sequence of the AAV-CRISPRi virus system is a 5 '-ITR inverted repeat sequence, an EF1 alpha core promoter, a ZIM3 transcription inhibition structural domain, a linker, a sadCas9-NLS nuclear localization signal, an SV40 poly (A) termination signal, a U6 promoter, a sgRNA-3'-ITR inverted repeat sequence. The AAV-CRISPRi virus system can realize efficient and long-acting transcriptional inhibition on the ASIC3 gene in dorsal root ganglion neurons, and provides a novel treatment strategy with tissue targeting, lasting effect and no addiction risk for relieving inflammatory and neuropathic chronic pain.

The invention discloses a construction method and application of an AAV single-molecule sequencing library and a kit, and belongs to the technical field of biological medicine detection. In order to solve the technical problem that ligase steric hindrance is caused by a secondary structure of an AAV inverted repeat (ITR), a Cas9-RNP compound specifically targeting an ITR stem region (Stem Region) is used for carrying out directional cutting on a single-chain genome, a closed-loop hairpin structure is removed, and a blunt-end double-chain DNA handle of 10-50 bp is reserved at the tail end. In cooperation with a forced dissociation step of the enzyme, the method eliminates connection steric hindrance and significantly improves the linker connection efficiency. According to the method, double-end cyclization is not needed, sequencing can be achieved only through single-end connection, and therefore a complete genome, a truncated body and a covalent head-to-tail concatemer can be captured without bias, and the method can be used for accurately measuring the physical packaging capacity limit of the AAV carrier.

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.

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.

An adeno-associated virus (AAV) complex platform is disclosed that includes an asymmetrically modified terminal inverted repeat (ITR). The AAV complex has an asymmetric ITR in which any one of two ITRs is modified, and thus has the advantages of increased productivity and transgene expression efficiency, and reduced genetic toxicity. Also disclosed are compositions comprising the adeno-associated virus complexes and gene therapy methods.

A method for producing recombinant adeno-associated virus particle preparations (rAAVp) is reported herein, comprising the steps of culturing mammalian cells containing expression cassettes for non-adeno-associated virus genes, adeno-associated virus rep genes, adeno-associated virus cap genes, adeno-associated virus E1A genes, adeno-associated virus E1B genes, adeno-associated virus E2A genes, adeno-associated virus E4orf6, and adeno-associated virus VA RNA genes located between two AAV terminal inverted repeat sequences (ITRs), and producing rAAVp therefrom, wherein the culturing is performed at a pH value between pH 7.4 and pH 7.6. The yield of rAAVp produced by culturing at a pH value between pH 7.4 and pH 7.6 is higher than that of rAAVp produced by culturing at a pH value between pH 7.0 and pH 7.2, and the percentage of perfect particles is higher for rAAVp produced by culturing at a pH value between pH 7.4 and pH 7.6 than for rAAVp produced by culturing at a pH value between pH 7.0 and pH 7.2.

This invention discloses an RNAi recombinant vector resistant to both whitefly and tomato yellow leaf curl virus (TYLCV), its construction method, and its applications. The method involves constructing a DNAβ promoter associated with a geminivirus expressed in the phloem into the RNAi vector pCambia1391, obtaining a 1391::DNAβpro vector backbone. RT-PCR is used to amplify a partial fragment of the whitefly key gene BtACTB, a key region of TYLCV (including the intergenic spacer region for viral replication initiation, some coat proteins, and replication-related proteins), and its inverted repeat sequences. This constructs a recombinant vector with an inverted hairpin structure driven by the phloem promoter, simultaneously targeting key sequences of both whitefly and TYLCV. The constructed recombinant vector is then introduced into tomatoes via Agrobacterium-mediated transformation. Screening yields tomato plants that reduce whitefly survival and inhibit TYLCV infection.

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.

This invention belongs to the field of biotechnology and discloses a high-yield rAAV plasmid system and its applications. The high-yield rAAV plasmid system of this invention includes a transgenic plasmid containing two terminal inverted repeat sequences, a packaging plasmid, a helper plasmid, and an shRNA expression cassette; the shRNA expression cassette includes a sequence capable of targeting the target gene in the transgenic plasmid. In the rAAV production process, the plasmid system of this invention inhibits the transcriptional expression of the target gene by expressing shRNA to target the coding region or target sequence of the target gene on the transgenic plasmid. The carefully designed target sequence, unlike coding regions of human and mammalian genes, can be broadly used in various transgenic plasmids, thereby increasing rAAV yield and significantly improving the rAAV production efficiency of mammalian cells, achieving the technical effects of improved rAAV druggability and reduced production costs.

The invention discloses insecticidal dsRNA synthesized according to KCC as well as a synthesis method and application of the dsRNA. The gene sequence of the dsRNA is shown as SEQ ID NO: 1 or at least comprises a gene sequence with 90% of identity of the sequence shown as SEQ ID NO: 1. After the compound is introduced into the body of a test insect through a micro-injection method, the accumulated death rate can reach 98.33%, and the lethal effect is remarkable. According to the invention, a recombinant expression vector containing an inverted repeat sequence of the gene is constructed, and high-efficiency expression and accumulation of dsRNA are realized through IPTG induction in escherichia coli. After the dsRNA-containing engineering bacteria are mixed with flour, test insects are fed, and the accumulative fatality rate can reach 58.33%. A metal organic framework (MOF) material is used for loading the obtained dsRNA, the stability and delivery efficiency of the dsRNA can be further improved, the final death rate can reach 65.56% after a test insect compound is fed, and an effective means is provided for application of nucleic acid biopesticide in prevention and control of stored grain pests.

The present invention relates to a method for propagating plasmids containing at least one inverted repeat, the method comprising the step of culturing the Escherichia coli bacteria of step (i) for at least 16 hours at a temperature of at least 39°C. Furthermore, the invention relates to a plasmid encoding a cargo nucleic acid comprising at least one inverted repeat from a method of the present invention. The invention further relates to a cargo nucleic acid comprising at least one inverted repeat obtained by a method of the present invention.

The invention discloses a preparation method of a novel target molecule TRE1-dsRNA capable of preventing and treating plant botrytis cinerea through RNAi, and provides a dsRNA molecule targeting trehalase TRE1, the dsRNA molecule is used for preventing and treating plant botrytis cinerea, trehalase plays a key role in the growth and development and pathogenic process of botrytis cinerea, and the dsRNA molecule can be used for preventing and treating plant botrytis cinerea. Therefore, the dsRNA targeting the TRE1 gene is expected to become an efficient disease control means, an expression vector containing a TRE1 gene inverted repeat sequence is constructed in escherichia coli, efficient transcription and accumulation of TRE1-dsRNA molecules are achieved under induction of IPTG, the method not only simplifies the production process of the dsRNA, but also improves the yield and purity of the dsRNA, and the application prospect is broad. The possibility is provided for large-scale preparation of the RNAi biopesticide.

To provide an adeno-associated virus (AAV) vector genome for altering, e.g., enhancing, expression of frataxin (FXN) in vitro and / or in vivo, including an engineered promoter.SOLUTION: Provided is an adeno-associated virus (AAV) vector genome comprising a 5' inverted terminal repeat (ITR), an engineered promoter, a payload region, and a 3' ITR, wherein the payload region encodes a frataxin (FXN) protein.SELECTED DRAWING: None

The invention discloses an RNAi recombinant vector resistant to both bemisia tabaci and tomato yellow leaf curl virus as well as a construction method and application of the RNAi recombinant vector resistant to both bemisia tabaci and tomato yellow leaf curl virus, a DNA beta promoter accompanied by geminivirus expressed in phloem is constructed to an RNAi vector pCambia 1391 vector, and a 1391:: DNA beta pro vector skeleton is obtained; an RT-PCR (reverse transcription-polymerase chain reaction) method is used for amplifying part of fragments of key genes BtACTB of bemisia tabaci, key segments (including a gene spacer region with virus replication initiation, part of coat protein and replication-related protein) of TYLCV and inverted repeat sequences of the key segments to construct a gene which is driven by a phloem promoter and is capable of expressing the bemisia tabaci gene BtACTB. The recombinant vector is of a reverse hairpin structure and simultaneously targets key sequences of bemisia tabaci and tomato yellow leaf curl virus. The constructed recombinant vector is introduced into tomatoes through an agrobacterium transformation method, and tomato plants capable of reducing the survival rate of bemisia tabaci and inhibiting TYLCV infection are obtained through screening.

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 sequence, direct repeat sequence, homopolymeric repeat sequence, eukaryotic origin of replication or 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 includes modifying the covalently closed circular recombinant molecule such that the Pol I-dependent origin of replication is replaced with a Pol Ill-dependent origin of replication, whereby the resultant Pol Ill-dependent origin of replication covalently closed circular plasmid has improved replication. An antibiotic marker free covalently closed circular recombinant DNA molecule is also provided. Recombinant DNA molecules and methods related thereto which include one or more primosomal sites and / or a selectable marker oritented to transcribe away from a structured DNA sequence are also provided.

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.

The invention belongs to the technical field of biological medicine, and particularly relates to the field of gene therapy and pain therapy. The invention provides an sgRNA and AAV-CRISPRi virus system for targeted inhibition of TRPV1 gene, the AAV-CRISPRi virus system is a CRISPRi system based on AAV delivery, and the core functional structure sequence of the AAV-CRISPRi virus system is a 5 '-ITR inverted repeat sequence, an EF1 alpha core promoter, a ZIM3 transcription inhibition structural domain, a linker, a sadCas9-NLS nuclear localization signal-SV40 poly (A) termination signal-U6 promoter, and an sgRNA-3'-ITR inverted repeat sequence, according to the core functional structure, the miniaturization of a CRISPRi system is realized, and the infection success rate and the target gene inhibition efficiency are improved while the plasmid construction and AAV packaging costs are reduced. The AAV-CRISPRi virus system for targeted inhibition of the TRPV1 gene is used for preparing drugs for chronic pain, and has the characteristics of high efficiency, strong specificity and few side effects.

The present invention relates to the field of bioengineering, particularly to a backbone plasmid, a recombinant plasmid, a monkeypox virus particle, and a use. The backbone plasmid is provided by deleting two inverted terminal repeat sequences of a virus and deleting a L2R gene or both L2R and A30.5L genes, wherein the virus includes monkeypox virus. The recombinant plasmid has simple construction, and a virus is rescued by only ligating a ligated fragment of two inverted terminal repeat sequences with one linearized plasmid in vitro. The backbone plasmid is convenient to modify, and a sequence of interest can be rapidly modified by means of homologous fragment recombination in a bacterium; the detection is convenient, and a fluorescent protein and a luciferase reporter gene are both expressed; and the use is safe, and the key gene L2R essential for virus assembly is deleted or both L2R and A30.5L genes are deleted, so that only a single round of infection can be carried out in a cell free of inverse complementary expression, making it impossible to generate a new virus by means of assembly; thus, viral etiology and application research can be carried out in a laboratory having a relatively low level of biological safety.