136 results about "Inverted Terminal Repeat" patented technology

Provided herein are transposon / transposase systems for transposing and selecting of transposed cells where the system includes a transposase or polynucleotide encoding a transposase; and an exogenous nucleic acid sequence comprising: a transposon comprising: a 5' inverted terminal repeat (ITR), a first polynucleotide encoding a gene of interest; and a 3' inverted terminal repeat (ITR); and (ii) a polynucleotide encoding an at least first selection marker located outside of the 5' ITR and 3' ITR, where upon introducing (a) and (b) into a cell the transposon is transposed into the cell's genome and the polynucleotide encoding the selection marker is episomal.

The present disclosure provides nucleic acid molecules comprising a first inverted terminal repeat (ITR), a second ITR, and a genetic cassette encoding a miRNA and / or a therapeutic protein. In certain embodiments, the therapeutic protein comprises a clotting factor, e.g., a FVIII polypeptide, a FIX polypeptide, or a fragment thereof. In some embodiments, the first ITR and / or the second ITR is an ITR of a non-adeno-associated virus (AAV). The present disclosure also provides methods of treating bleeding disorders such as hemophilia comprising administering to the subject the nucleic acid molecule or a polypeptide encoded thereby.

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).

Provided herein is a recombinant AAV (rAAV) comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises an AAV 5' inverted terminal repeat (ITR), an expression cassette comprising engineered nucleic acid sequence encoding human Tafazzin and operably linked thereto regulatory sequences, and an AAV 3' ITR. Also provided is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer, and a method of treatment of Barth Syndrome or a disease associated with a mutation in a Tafazzin (TAZ) gene.

The disclosure relates to nanoparticles comprising a surface-exposed immune cell binding moiety, a transposable element comprising a gene sequence flanked by inverted terminal repeats (ITRs), a nucleic acid encoding a transposase with specificity for the ITRs, and an mRNA encoding a first chimeric antigen receptor (CAR) or T-cell receptor (TCR). Methods are also described for treating a disease or disorder by administering such nanoparticles to a subject in need thereof.

Provided herein are composition comprising a) a first recombinant adeno-associated viral (rAAV) vector genome comprising a first expression cassette comprising a promoter operably linked to nucleic acid sequence comprising a 5' portion of an otoferlin gene, wherein the first expression cassette is flanked by inverted terminal repeats (ITRs); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising a 3' portion of an otoferlin gene, wherein the second expression cassette is flanked by ITRs, and wherein the composition can comprise about 4E10-9E13 vg / mL, optionally about about 4.1E10- 4.1E12 total vg or about 8.1E10-8.1E12 total vg and the use of these compositions to treat hearing loss in a subject.

In some aspects, cardiac-specific expression cassettes are provided herein. In some aspects, provided herein is an expression cassette comprising a polynucleotide sequence encoding a gene product for therapy of a heart disease, wherein the polynucleotide sequence is operably linked to promoter (e.g., a cardiac-specific promoter), and optionally an enhancer (e.g., a cardiac-specific enhancer). In some aspects, the disclosure provides recombinant adeno-associated virus (rAAV) virions, comprising a capsid protein and a viral genome comprising an expression cassette comprising a polynucleotide sequence encoding a therapeutic gene product, e.g., dwarf open reading frame (DWORF) polypeptide, operably linked to a promoter, the expression cassette flanked by inverted terminal repeats. The disclosure further provides pharmaceutical compositions and methods of treating or preventing heart disease.

Disclosed are data pipelines for string extraction, clustering, and comparison. A method may include extracting, from each plasmid genome sequence sequenced from genomes of plasmids, based on the presence of fixed flanking sequence markers (FFSMs) in the plasmid genome sequence, sequence regions. Each sequence region is within the FFSMs and includes a candidate inverted terminal repeat (ITR) sequence. The example method further includes clustering, based on perfect sequence identity, two or more sequence regions of the sequence regions to generate a clusters, merging, based on an alignment between their corresponding sequence regions, two or more clusters of the clusters; when a single cluster remains, identifying, based on a local alignment, a genotype of a candidate ITR sequence of the single cluster, and manufacturing, based on the genotype of the candidate ITR sequence, a plurality of AAV vectors using plasmids having ITR sequences with the genotype of the candidate ITR sequence.

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.

The present disclosure includes a composition including a) one or more nucleic acid constructs, wherein the one or more nucleic acid constructs encode: at least one of Replicase 68 (Rep68) and Replicase 78 (Rep78); and at least one protein of viral origin having nucleic acid replication and / or transgene amplification promotion activity, and b) nucleic acid construct comprising a coding region encoding at least one gene of interest (GOI) and at least one inverted terminal repeat (ITR) sequence on each side of the GOI, and methods of making and using the composition.

Provided herein is a recombinant AAV (rAAV) comprising an AAV capsid and a vector genome packaged therein, wherein the vector genome comprises an AAV 5′ inverted terminal repeat (ITR), an engineered nucleic acid sequence encoding a functional hSGSH, optionally comprising stabilizing amino acid changes, a regulatory sequence which direct expression of hSGSH in a target cell, and an AAV 3′ ITR. Also provided is a pharmaceutical composition comprising a rAAV as described herein in a formulation buffer, and a method of treating a human subject diagnosed with MPS IIIA.

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 invention relates to a rep plasmid comprising at least one adeno-associated virus replication protein coding sequence encoding at least one functional rep protein, at least one polyadenylation signal sequence downstream the adeno-associated virus replication protein coding sequence, and a minimal element comprising one or more rep-binding elements downstream the polyadenylation signal sequence. In another aspect, the invention provides a transgene plasmid comprising a CMVie promotor, a transgene, SV40 PolyA, a 5' and a 3' inverted terminal repeats derived from J01901.1 AAV2; wherein the transgene plasmid is selected from one of the following: a conventional single-stranded genome recombinant adeno-associated virus, or a self-complementary genome recombinant adeno-associated virus.

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.

The invention belongs to the technical field of biological medicine, and particularly relates to a rabies virus G protein carrier, a rabies virus vaccine and a preparation method and application of the rabies virus G protein carrier and the rabies virus vaccine. The rabies virus G protein vector pscAAV-G is obtained by inserting a coding gene of rabies virus G protein into a coding region between inverted terminal repetitive sequences at two ends of a double-chain adeno-associated virus vector, so that the problem of expression delay of single-chain AAV (ssAAV) is solved, and rapid and efficient expression of the rabies virus G protein is realized; furthermore, an endogenous nucleic acid adjuvant is embedded into a non-coding region of the pscAAV-G, so that the rabies virus G protein carrier pscAAV-G-Adj is prepared, and Th1 type immune response is remarkably improved. According to a rabies virus vaccine prepared by using the rabies virus G protein carrier, the antigen expression efficiency and immunogenicity of an AAV carrier vaccine are remarkably improved, long-acting protection of single inoculation is realized, and the defects that an existing rabies vaccine is short in immune cycle and needs to be inoculated for multiple times are overcome.

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

Provided herein are compositions comprising a) a first recombinant adeno-associated virus (rAAV) vector genome (vg) comprising a first expression cassette comprising a promoter operably linked to a nucleic acid sequence comprising a 5'part of an orthodontic protein gene wherein the expression cassette is flanked by an inverted terminal repeat (ITR); and b) a second rAAV vector genome comprising a second expression cassette comprising a nucleic acid sequence comprising a 3'portion of an orthodontic protein gene wherein the expression cassette is flanked by an ITR wherein the compositions comprise about 4.1 E10-4.1 E12 total vg or about 8.1 E10-8.1 E12 total vg, and the use of these compositions to treat hearing loss in a subject.

An isolated linear nucleic acid molecule comprising in this order: a first adeno-associated virus (AAV) inverted terminal repeat (ITR), a nucleotide sequence of interest and a second AAV ITR, wherein said nucleic acid molecule is devoid of AAV capsid protein coding sequences. The said nucleic acid molecule can be applied to a host at repetition without eliciting an immune response. Methods for producing and purifying this nucleic acid molecule, and use of the same for therapeutic purposes are also provided.

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 the technical field of crossing of molecular biology, nucleic acid detection and bioinformatics, and discloses an in-vivo detection method for synchronously detecting CRISPR off-target sites and viral vector integration sites. According to the in-vivo detection method for synchronously detecting the off-target site of the CRISPR and the integration site of the virus vector, the reverse terminal repeat (ITR) of the AAV vector is used as a tag sequence for the first time, and a universal PCR (Polymerase Chain Reaction) amplification primer is designed and a unique molecular identifier (UMI) technology is introduced to detect the off-target site of the CRISPR and the integration site of the virus vector. And meanwhile, an off-target event and an AAV carrier integration event in an in-vivo CRISPR editing process are captured and accurately recognized. Compared with the existing schemes such as GUIDE-seq, DISCOVER-Seq +, OliTag-seq, U-DiTaS and the like, the in-vivo detection method for synchronously detecting the CRISPR off-target site and the virus vector integration site, provided by the invention, has the advantage that remarkable breakthrough is realized in the aspects of in-vivo applicability, detection depth, operation economy and industrialization standardization.

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.

To provide a method for producing mammalian cells containing a lentiviral packaging vector.SOLUTION: A method of producing a lentiviral packaging vector-containing mammalian cells, comprising: a. transfecting a mammalian cell with a packaging vector comprising an expression cassette encoding: 1. a lentiviral regulator of expression of virion proteins (REV) gene under control of a first promoter; 2. a lentiviral envelope gene under control of a second promoter; and 3. a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene both under control of a third promoter where the expression cassette is flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITR); b. culturing the transfected mammalian cell; and c. isolating the lentiviral packaging vector-containing mammalian cell.SELECTED DRAWING: Figure 1

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).

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

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.

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.