51 results about "Inverted Terminal Repeat" patented technology

The invention relates to the field of biological medicine, and particularly discloses a regulatory element combination for driving nucleic acid expression based on ITR-enhancer and application of the regulatory element combination. The regulatory element combination for driving nucleic acid expression based on an ITR-enhancer comprises at least one inverted terminal repeat (ITR) and at least one enhancer, the regulatory element combination is delivered through an AAV vector. The invention also provides a recombinant nucleic acid molecule for improving specific delivery of the target gene. The recombinant nucleic acid molecule comprises the regulatory element combination, an exogenous target gene and polyadenylic acid. The AAV vector provided by the invention can be used for driving the expression of a target gene without the action of a traditional promoter, and has the advantages of enhancer tissue specificity, short sequence (50-100bp), pathological microenvironment response and the like, thereby breaking through the capacity, targeting and dynamic limitation of the traditional AAV vector, improving the DNA (Deoxyribose Nucleic Acid) carrying capacity of the AAV vector, and simultaneously improving the expression of the target gene. The tissue and spatio-temporal expression specificity of a target gene is realized.

The invention belongs to the technical field of medicines, and discloses application of an adeno-associated virus vector in preparation of medicines for preventing and treating RSV infection and / or diseases caused by RSV. According to the invention, a recombinant adeno-associated virus vector is adopted for the first time to construct a medicine for preventing and treating RSV infection or diseases caused by RSV infection, and based on this, the invention provides a nucleic acid molecule which comprises: (a1) a first region which comprises a first adeno-associated virus inverted terminal repeat sequence, and (a2) a second region which comprises a second adeno-associated virus inverted terminal repeat sequence; and (a2) a second region comprising a gene encoding a respiratory syncytial virus antigen; the recombinant adeno-associated virus containing the nucleic acid molecule can generate better humoral immune effect and cellular immune effect, has a certain serum neutralization effect on strains of A and B subtypes of RSV, and particularly, when the antigen is a fusion protein of the respiratory syncytial virus or a fusion protein mutant of the respiratory syncytial virus, the recombinant adeno-associated virus has a certain serum neutralization effect on the strains of A and B subtypes of the RSV. The recombinant adenovirus vector containing the nucleic acid molecule can successfully realize the expression of the F protein before RSV fusion.

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.

Herein is reported a method for producing a recombinant adeno-associated viral particle preparation (rAAVp), the method comprising the steps of culturing a mammalian cell comprising an expression cassette directed against: a non-adeno-associated viral gene interposed between two AAV inverted terminal repeats (ITRs), and thereby producing the rAAVp; an adeno-associated virus rep gene; an adeno-associated virus cap gene; an adeno-associated virus E1A gene; an adeno-associated virus E1B gene; an adeno-associated virus E2A gene; an adeno-associated virus E4orf6; and an adeno-associated virus VA RNA gene, wherein the culturing is carried out at a pH value in the range of pH 7.4 to pH 7.6 and including a terminal value. The yield of the rAAVp produced by the culture performed at a pH value in the range of pH 7.4 to pH 7.6 and including a terminal value is higher than the yield of the rAAVp produced by the culture performed at a pH value in the range of pH 7.0 to pH 7.2 and including a terminal value, and the yield of the rAAVp produced by the culture performed at a pH value in the range of pH 7.4 to pH 7.6 and including a terminal value is lower than the yield of the rAAVp produced by the culture performed at a pH value in the range of pH 7.0 to pH 7.2. And the rAAVp produced by the culture at a pH value in the range of pH 7.4 to pH 7.6 and including an endvalue has a higher percentage of intact particles than the rAAVp produced by the culture at a pH value in the range of pH 7.0 to pH 7.2 and including an endvalue.

Compositions and methods for the treatment of retinal degeneration are provided. In one aspect, provided herein is adeno-associated virus (AAV) vector comprising an AAV capsid having encapsidated therein a vector genome comprising AAV inverted terminal repeat (ITR) sequences, a human protein kinase B (AKT) coding sequence, and expression control sequences that direct expression of AKT in a host cell.

The present disclosure provides methods for assessing the integrity of inverted terminal repeat sequences in AAV plasmids by restriction digestion followed by capillary electrophoresis.

Data pipelines for string extraction, clustering, and comparison are disclosed. A method can include extracting sequence regions from each plasmid genome sequence sequenced from a genome of a plasmid based on presence of a fixed flanking sequence marker (FFSM) in the plasmid genome sequence. Each sequence region is within the FFSM and includes a candidate inverted terminal repeat (ITR) sequence. An example method further includes clustering two or more of the sequence regions based on perfect sequence identity to generate clusters; merging two or more of the clusters based on alignments between corresponding sequence regions of the two or more clusters; when a single cluster remains, identifying a genotype of a candidate ITR sequence of the single cluster based on local alignments; and using a plasmid having an ITR sequence with the genotype of the candidate ITR sequence to manufacture a plurality of AAV vectors based on the genotype of the candidate ITR sequence.

To provide methods for treating Huntington's disease.SOLUTION: Aspects of the present disclosure relate to compositions and methods useful for treating Huntington's disease. In some embodiments, the disclosure provides interfering nucleic acids (for example, artificial miRNAs) targeting a huntingtin gene (HTT) and methods for treating Huntington's disease using the same. Accordingly, in some aspects, the disclosure provides an isolated nucleic acid comprising or encoding a sequence set forth in any one of SEQ ID NOs: 1-22. In one aspect, an isolated nucleic acid is disclosed herein, comprising: (a) a first region comprising a first adeno-associated virus (AAV) inverted terminal repeat (ITR) or a variant thereof; and (b) a second region comprising an introduced gene encoding one or more miRNAs, each miRNA comprising a seed sequence complementary to SEQ ID NO: 25.SELECTED DRAWING: None

Disclosed herein is a recombinant viral vector comprising an AAV 5′ inverted terminal repeat (ITR) sequence, an AAV 3′ ITR sequence, and an expression cassette flanked by the AAV 5′ ITR and 3′ ITR sequences. According to the embodiments of the present disclosure, the expression cassette comprises a first and a second coding sequences respectively encoding a human neuraminidase 1 (NEU1) and a human protective protein / cathepsin A (PPCA). Also disclosed herein are a recombinant adeno-associated virus (AAV) comprising the recombinant viral vector, a pharmaceutical composition comprising the recombinant AAV, and uses thereof in treating sialidosis.

The invention relates to circular DNA molecule for rescuing recombinant adenoviruses comprising a recombinant adenoviral genome with two inverted terminal repeats (ITRs) flanking the genome ends, wherein at least one of the ITRs is associated with a target sequence adjacent to a PAM sequence, wherein the target sequence is configured for generating an RNA-guided DNA endonuclease-mediated DNA double strand break at the external end of or in close proximity outside the external end of the respective ITR, preferably within less than about 15 nucleotides. The invention also relates to a kit and a method for rescuing recombinant adenoviruses comprising or using a circular DNA molecule as described herein.

A method for producing a nucleic acid-encapsulated adeno-associated virus (AAV) hollow particle, comprising the following steps: (1) preparing a linear nucleic acid fragment comprising a sequence for A region and a sequence for D′ region in an AAV inverted terminal repeat (ITR) (i.e., an AD sequence) or a sequence complementary to the AD sequence and a target gene sequence; (2) introducing the nucleic acid fragment prepared in step (1) into a cell capable of producing an AAV hollow particle; and (3) culturing the cell obtained in step (2).

Described herein is an adeno-associated virus (AAV) complex platform including an asymmetrically modified inverted terminal repeat (ITR). The AAV complex has advantages of increased productivity and expression efficiency of a transgene, and decreased genotoxicity, by having an asymmetric ITR in which any one of two ITRs is modified. Also, described herein is a composition comprising the adeno-associated virus complex and a method of gene therapy.

The disclosure provides adeno-associated virus (AAV) helper plasmid variants with improved safety profiles. In certain aspects, an AAV helper plasmid variant of the disclosure comprises one or more inactivating mutations in at least one of the following adenovirus genes or genome sequences: (a) adenoviral fiber gene; (b) adenoviral precursor terminal protein gene; (c) adenoviral L1-52K gene; (d) adenoviral 100K gene; (e) adenoviral PVIII gene; (f) adenoviral E4 region open reding frame; (g) adenoviral inverted terminal repeat (ITR) sequence; (h) L3-23K region gene; (i) hexon-assembly gene; or (j) a combination of any of (a)-(i).

Compositions comprising a mixed population of recombinant adeno-associated virus (rAAV) and methods of reducing deamidation in the AAV capsid of rAAV are provided.SOLUTION: Provided is a composition comprising a mixed population of recombinant adeno-associated viruses (rAAVs), wherein each of the rAAVs comprises (a) an AAV capsid comprising about 60 capsid vp1, vp2, and vp3 proteins, and (b) a vector genome in the AAV capsid, wherein the vector genome comprises AAV inverted terminal repeat sequences and a non-AAV nucleotide sequence encoding a product operably linked to sequences directing expression of the product in host cells.SELECTED DRAWING: Figure 1-1

Provided is a method for producing a protein / nucleic acid complex which comprises a capsid protein derived from an adeno-associated virus and a genomic nucleic acid having at least one inverted terminal repeat sequence and has infectiousness to mammals. The genomic nucleic acid further includes a target nucleic acid that encodes a target protein. The method comprises: (a-1) a step for providing an isolated first nucleic acid that comprises a linear single-stranded nucleic acid, a linear double-stranded nucleic acid, or a circular single-stranded nucleic acid and includes at least one inverted terminal repeat sequence and a target nucleic acid that encodes a target protein; (a-2) a step for providing an isolated first nucleic acid that includes at least one inverted terminal repeat sequence and a target nucleic acid that encodes a target protein; and (b) a step for bringing a first protein into contact with the first nucleic acid in a solvent to form the protein / nucleic acid complex.

Provided are methods for treating an ocular neovascular disease in an individual by reducing Auction of central subfoveal thickness (CST) or central retinal thickness (CRT), comprising administering a unit dose of recombinant adeno-associated virus (rAAV) particles to an eye of the individual, wherein the rAAV particles comprise: a) a nucleic acid encoding a polypeptide comprising an amino acid sequence with at least about 95% identity to the amino acid sequence of SEQ ID NO: 35 and Ranked by AAV2 inverted terminal repeats (ITRs), and b) an AAV2 capsid protein comprising an amino acid sequence LGETTRP (SEQ ID NO: 14) inserted between positions 587 and 588 of the capsid protein, wherein the amino acid residue numbering corresponds to an AAV2 VP1 capsid protein.

A recombinant AAV (rAAV) suitable for use in treating adult onset neurodegeneration caused by granulin (GRN) haploinsufficiency, such as progranulin (PGRN)-related frontotemporal dementia (FTD), is provided. The rAAV comprises (a) an adeno-associated virus 1 capsid, and (b) a vector genome packaged in the AAV capsid, said vector genome comprising AAV inverted terminal repeats, a coding sequence for human progranulin, and regulatory sequences which direct expression of the progranulin. Also provided are a method for treating a human patient with PGRN-FTD and other adult onset neurodegeneration caused by granulin (GRN) haploinsufficiencies, comprising delivering to the central nervous system (CNS) a recombinant adeno-associated virus (rAAV) having an adeno-associated virus 1 (AAV1) capsid, said rAAV further comprising a vector genome packaged in the AAV capsid, said vector genome comprising AAV inverted terminal repeats, a coding sequence for human progranulin, and regulatory sequences which direct expression of the progranulin.

The application discloses a method for simultaneously producing multiple recombinant adeno-associated viruses, and belongs to the field of gene therapy. The method comprises the following steps: stably integrating multiple target genes into a host cell genome or stably integrating multiple serotype capsid protein Cap genes into the host cell genome; screening a single clone cell which stably expresses the target genes or the serotype capsid protein Cap genes; co-transfecting a transfer plasmid containing AAV inverted terminal repeat sequences, a Rep gene plasmid and an adenovirus auxiliary gene plasmid into the single clone cell which stably expresses the target genes or the serotype capsid protein Cap genes; and harvesting and purifying recombinant AAV viruses to obtain an AAV virus mixture with a specific ratio of target genes or a hybrid AAV virus with a specific ratio of capsid proteins.

Herein is reported a method for producing recombinant adeno-associated viral particle preparation (rAAVp) comprising the step of cultivating a mammalian cell comprising expression cassettes for a non-adeno-associated viral gene, which is interspaced between two AAV inverted terminal repeats (ITRs), an adeno-associated virus rep gene, an adeno-associated virus cap gene, an adeno-associated virus E1A gene, an adeno-associated virus E1B gene, an adeno-associated virus E2A gene, an adeno-associated virus E4orf6 and an adeno-associated virus VA RNA gene, and thereby producing the rAAVp, wherein the cultivating is at a pH value in the range of and including pH 7.4 to pH 7.6. The yield of the rAAVp produced by the cultivating at a pH value in the range of and including pH 7.4 to pH 7.6 is higher than the yield of a rAAVp produced by a cultivating at a pH value in the range of and including pH 7.0 to pH 7.2 and the rAAVp produced by the cultivating at a pH value in the range of and including pH 7.4 to pH 7.6 has a higher percentage of full particles than a rAAVp produced by a cultivating at a pH value in the range of and including pH 7.0 to pH 7.2.

Disclosed are compositions comprising a first nucleic acid sequence comprising: (a) a first inverted terminal repeat (ITR), (b) a second ITR and (c) an intra-ITR sequence, wherein the intra-ITR sequence comprises a transposon sequence, and a second nucleic acid sequence comprising an inter-ITR sequence, wherein the length of the inter-ITR sequence is between 1 and 600 nucleotides, inclusive of the endpoints. Preferably, the compositions are nanotransposons.

The present invention relates to the production of adeno-associated viral vectors in insect cells. The insect cells therefore comprise a first nucleotide sequence encoding the adeno-associated virus (AAV) capsid proteins, whereby the initiation codon for translation of the AAV VP1 capsid protein is a non-ATG, suboptimal initiation codon and wherein the coding sequence for one or more amino acid residues have been inserted between the suboptimal translation initiation codon and the codon encoding the amino acid residue that corresponds to the amino acid residue at position 2 of the wild type capsid amino acid sequence of which the first amino acid residue is alanine, glycine, valine, aspartic acid or glutamic acid. The insect cell further comprises a second nucleotide sequence comprising at least one AAV inverted terminal repeat (ITR) nucleotide sequence; a third nucleotide sequence comprising a Rep52 or a Rep40 coding sequence operably linked to expression control sequences for expression in an insect cell; and, a fourth nucleotide sequence comprising a Rep78 or a Rep68 coding sequence operably linked to expression control sequences for expression in an insect cell. The invention further relates to adeno-associated viral vectors with an altered ratio of the viral capsid proteins.

This disclosure provides nucleic acid molecules comprising a first inverted terminal repeat (ITR), a second ITR, and a gene cassette encoding a target sequence. In some embodiments, the target sequence encodes a microRNA and / or a therapeutic protein. In certain embodiments, the therapeutic protein comprises a clotting factor, a growth factor, a hormone, a cytokine, an antibody, a fragment thereof, and a combination thereof. In some embodiments, the first ITR and / or the second ITR is an ITR of a non-adeno-associated virus (AAV). This disclosure also provides methods of treating a metabolic liver disorder in a subject comprising administering to the subject the nucleic acid molecule or a polypeptide so encoded.

Provided herein are methods and compositions for treatment of Batten disease. Such compositions include a recombinant adeno-associated virus (rAAV), said rAAV comprising an AAV capsid, and a vector genome packaged therein, said vector genome comprising (a) an AAV 5′ inverted terminal repeat (ITR) sequence; (b) a promoter; (c) a CLN2 coding sequence encoding a human TPP1; (d) an AAV 3′ ITR.

According to various aspects of this disclosure, the present disclosure relates to a method for treating or reducing pulmonary inflammation and / or pulmonary fibrosis in a subject in need thereof comprising intramuscularly administering to the subject a recombinant adeno-associated virus (rAAV) vector. In some aspects, the rAAV vector comprises a vector genome and an AAV capsid (e.g., an AAV1 serotype). In some aspects, the vector genome comprises AAV inverted terminal repeats (ITRs) flanking an expression construct comprising a nucleotide sequence encoding a Fibroblast growth factor 21 (FGF21) or functional fragment thereof operably linked to a ubiquitous promoter.

To provide a gene therapy for limb-girdle muscular dystrophy type 2C.SOLUTION: The disclosure relates to gene therapy vectors, such as AAV vectors, comprising a polynucleotide encoding γ-sarcoglycan (SGCG), and to methods of using such gene therapy vectors to treat subjects suffering from a muscular dystrophy, e.g., limb-girdle dystrophy type 2C (LGMD2C). Provided in one aspect is a method of treating γ-sarcoglycanopathy in a subject, the method comprising administering to the subject a therapeutically effective amount of a recombinant adeno-associated virus (AAV) vector, the rAAV vector comprising a gene expression cassette comprising a polynucleotide sequence encoding γ-sarcoglycan under transcriptional control of a promoter, where the cassette is flanked by one or more AAV inverted terminal repeats.SELECTED DRAWING: None

A transposon is provided comprising a nucleic acid sequence traversing the chromatin open element (UCOE) as well as a nucleic acid sequence of a gene of interest or a fragment thereof under the control of a promoter and optionally a 3'polyadenylation sequence, both sides of which are Piggybactransposon-specific inverted terminal repeats PB 5 'ITR and PB 3' ITR.

The invention discloses a linear non-viral DNA (deoxyribonucleic acid) vector closed by an ITR (internal transcriptase) terminal as well as a preparation method and application of the linear non-viral DNA vector. The linear non-viral DNA vector comprises the following elements: an asymmetric reverse terminal repeat ITR sequence and an expression cassette; the asymmetric inverted terminal repeat ITR sequence comprises a first ITR sequence and a second ITR sequence, the first ITR sequence and the second ITR sequence are respectively positioned at two ends of an expression cassette sequence so as to enable an expression cassette to be closed, and the expression cassette comprises a cis-regulatory element; the structures of the first ITR sequence and the second ITR sequence are the same or different. The linear non-viral DNA vector is simple to synthesize and low in immunogenicity, can be expressed for a long time, can be used for stable and reliable expression of target genes in vivo and in vitro, and provides a better expression vector for gene therapy.

The invention discloses a circular single-stranded DNA (deoxyribonucleic acid) molecule containing an inverted repeat terminal sequence and application of the circular single-stranded DNA molecule, and relates to the technical field of biology. The annular single-stranded DNA provided by the invention can comprise two gene expression cassettes, which can be the same gene or different genes. When two same genes are inserted, the problem of wrong packaging or empty packaging is avoided no matter whether the capsid packaging is forward packaging or reverse packaging. When two different genes are inserted, the two genes can be packaged in the same capsid, so that the effect of expressing two proteins by one dose of medicine is realized. Therefore, the traditional AAV three-plasmid system can be simplified, and the wrong packaging rate of the traditional AAV three-plasmid system is reduced, so that the AAV drug yield is improved; meanwhile, pollution of non-target genes can be effectively avoided, and potential viruses of an AAV three-plasmid system are removed; and two exogenous genes can be carried at the same time for gene expression, so that wide application of the AAV vector in the field of gene therapy is promoted.

To provide a method for producing a population of high titer recombinant adeno-associated virus (AAV) lacking prokaryotic sequences.SOLUTION: Culturing a human embryo cell strain in suspension, transfecting the human embryo cell strain with (a) nucleic acids sequences encoding helper proteins sufficient for rAAV replication, (b) nucleic acids sequences encoding AAVrep and AAVcap genes, and (c) a closed ended linear duplex rAAV vector nucleic acids comprising at least one inverted terminal repeats (ITR) sequence and a heterologous transgene operably linked to one or more regulatory elements, incubating the transfected human cell strain for about 40 to 400 hours, and optionally, B. lysing the transfected human cell line to purify the nucleic acid sequence encoding the rAAV, thereby producing the rAAV.SELECTED DRAWING: None

The invention relates to the technical field of microorganisms and application thereof, and discloses a piggyBac transposon system, a recombinant vector, a gene editing method, a recombinant strain and application of the piggyBac transposon system, the piggyBac transposon system contains piggyBac transposase, an inverted terminal repeat sequence, a target load gene sequence and a selective marker gene, the vector gene is a functional marker gene and / or a reporter gene, and the expression of the transposase is controlled through a promoter PtrpC; wherein the amino acid sequence of the piggyBac transposase is as shown in SEQ ID NO. 1; and the piggyBac transposase system also contains a vector plasmid. The gene editing method of the piggyBac transposon system can be suitable for efficiently and accurately inserting exogenous genes into gibberella zeylanica, traceless and residue-free genome modification is realized, the problem of exogenous gene residue is avoided, and the method has relatively good industrial value.