39 results about "Viral dna" patented technology

Provided herein are lipids having the Formula I or Formula Ia:and pharmaceutically acceptable salts thereof, wherein R′, R1, R2, R3, R4, R5, R6a, R6b, X1, X2, and n are as defined herein for Formula I and Formula Ia, respectively. Also provided herein are lipid nanoparticle (LNP) compositions comprising lipid having the Formula I or Ia and a capsid-free, non-viral vector (e.g., ceDNA). In one aspect of any of the aspects or embodiments herein, these LNPs can be used to deliver a capsid-free, non-viral DNA vector to a target site of interest (e.g., cell, tissue, organ, and the like).

The application describes ceDNA vectors having linear and continuous structure for delivery and expression of a transgene. ceDNA vectors comprise an expression cassette flanked by two ITR sequences, where the expression cassette encodes a transgene encoding FVIII protein. Some ceDNA vectors further comprise cis-regulatory elements, including regulatory switches. Further provided herein are methods and cell lines for reliable gene expression of FVIII protein in vitro, ex vivo and in vivo using the ceDNA vectors. Provided herein are method and compositions comprising ceDNA vectors useful for the expression of FVIII protein in a cell, tissue or subject, and methods of treatment of diseases with said ceDNA vectors expressing FVIII protein. Such FVIII protein can be expressed for treating disease, e.g., hemophilia A.

The invention discloses a quadruple real-time fluorescent quantitative PCR (Polymerase Chain Reaction) primer probe combination for detecting African swine fever virus and application. The primer probe combination comprises a first primer probe combination, a second primer probe combination, a third primer probe combination and a fourth first primer probe combination, the primer probe is capable of specifically targeting ASFV p72 gene, CD2v-I gene, CD2v-II gene and I177L gene, and the detection kit developed based on the primer probe combination can rapidly diagnose African swine fever virus and identify CD2v gene I and II type and CD2v and I177L gene deleted strains. The kit has extremely high sensitivity, the lowest detection limit can reach 8 copies / mu L, it is ensured that trace virus DNA can be detected, and missing detection is avoided.

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.

Disclosed herein are DNA amplification methods for quantifying DNA fragments of a target DNA in a sample by size. This can be used, for example, to detect tumor-derived viral DNA in blood sample and distinguish it from larger viral DNA from non-tumor sources. In particular, disclosed herein are methods of detecting, monitoring or treating a human papilloma virus (HPV)-associated malignancy in a subject that involves detecting a presence or absence of at least one circulating tumor-derived HPV DNA in a sample from the subject. Kits for accomplishing the same are also provided.

Provided herein are circular, non-integrating, non-viral DNA vectors which express a TNALP polypeptide in an improved expression cassette, wherein the DNA vectors are capable of forming one or more specialized secondary structures, for example an extended cruciform structure, and methods of using such DNA vectors. These DNA vectors having improved expression cassettes provide for increased potency and long-term TNALP transgene expression.

Provided herein are improved DNA vectors (e.g., circular DNA vectors and / or non-viral DNA vectors) that can provide features including enhanced expression, persistence, safety, and manufacturability. Also provided herein are methods of producing such DNA vectors, host cells (e.g., engineered bacterial cells useful for producing such DNA vectors) containing such DNA vectors, methods of using such DNA vectors (e.g., engineered bacterial cells useful for producing such DNA vectors), and methods of using such DNA vectors. Methods of expressing therapeutic sequences in target cells by administering such DNA vectors and methods of treating diseases or conditions by administering such DNA vectors), as well as pharmaceutical compositions containing such DNA vectors.

Lipids having Formula (I): (SEE FORMULA) and pharmaceutically acceptable salts thereof are provided herein, wherein R1, R2, a and b are as defined herein. Lipid nanoparticle (LNP) compositions comprising lipids having Formula (I) and a capsid-free, non-viral vector (e.g., eDNA) are also provided herein. In any aspect or embodiment hereof, these LNPs may be used to deliver a capsid-free, non-viral DNA vector to a target site of interest (e.g., cell, tissue, organ, and the like).

A recombinant lentiviral vector comprising a coding sequence for an OR protein fused to a coding sequence for a fluorescent protein or a subunit of a fluorescent protein, and a promoter active in human cells operatively linked to the coding sequences. A recombinant lentivirus comprising a recombinant genome comprising an RNA that generates an ANCH sequence upon retrotranscription. A recombinant eukaryotic cell comprising a genomically integrated DNA copy of the recombinant lentiviral vector. A method of observing lentiviral DNA in a eukaryotic cell, comprising: providing a recombinant eukaryotic cell that produces a fusion protein comprising an OR protein, fused to a fluorescent protein or a subunit of a fluorescent protein; infecting the recombinant eukaryotic cell with a recombinant lentivirus comprising a recombinant genome comprising an RNA that generates an ANCH sequence upon retro-transcription, under conditions sufficient for reverse transcription of the recombinant lentiviral genome comprising an ANCH sequence; allowing the OR protein to bind to the ANCH sequence; and detecting the fluorescent protein or subunit of the fluorescent protein to thereby observe the lentiviral DNA in the eukaryotic cell. This tool can be suitable also for in vivo applications (e.g. humanized mice) as well as for screening of new antiretroviral compounds. The HIV-1 ANCHOR system can be extended to the study of other viruses or for the screening of antiviral compounds, e.g. against SARS-CoV2.

Provided herein are circular, non-integrating, non-viral DNA vectors which express a TNALP polypeptide in an improved expression cassette, wherein the DNA vectors are capable of forming one or more specialized secondary structures, for example an extended cruciform structure, and methods of using such DNA vectors. These DNA vectors having improved expression cassettes provide for increased potency and long-term TNALP transgene expression.

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.

Provided herein are lipids having the Formula (I):and pharmaceutically acceptable salts thereof, wherein R1, R2, a, and b are as defined herein. Also provided herein are lipid nanoparticle (LNP) compositions comprising lipid having the Formula (I) and a capsid-free, non-viral vector (e.g., ceDNA). In one aspect of any of the aspects or embodiments herein, these LNPs can be used to deliver a capsid-free, non-viral DNA vector to a target site of interest (e.g., cell, tissue, organ, and the like).

The invention relates to the technical field of real-time fluorescent PCR (polymerase chain reaction) detection and analysis, in particular to a method for detecting a rat parvovirus KRV strain through real-time fluorescent PCR and application of the method. The method comprises the following steps: (1) sample pretreatment: taking rat visceral organ tissues, cecum contents or excrement, blood, swabs and cell cultures as experimental animal detection samples; adding normal saline with the same volume to prepare homogenate, centrifuging, and collecting supernate for detection; blood and ascites are directly used for detection; (2) virus extraction: taking 200 [mu] L of the treated sample, carrying out virus extraction by adopting a FastPure Viral DNA / RNA Mini Kit (RC311-01) kit of the Norazan company, and taking a proper amount of final product sample virus as a subsequent template; and (3) carrying out real-time fluorescent quantitative PCR amplification by using the specific primer designed by the invention and a TaqMan probe, collecting a fluorescent signal, and carrying out analysis and judgment.

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.

The present disclosure provides methods and compositions of modified oligonucleotide primer and probe combinations, structurally modified with locked nucleic acids, quenchers, and dyes, effective to detect tumor-derived Human Papilloma Virus (HPV) and tumor-derived Epstein-Barr virus (EBV) and, especially, to distinguish viral DNA derived from tumors from viral DNA derived from infectious viral particles.

The present disclosure relates generally to compositions and methods comprising recombinant integration deficient retroviral vectors where the expression of cargo(s) of interest is enhanced by the presence of elements that inhibit the silencing of viral DNA.

To provide a non-viral DNA vector for expressing a FVIII therapeutic agent and its use.SOLUTION: The present application describes ceDNA vectors having a linear and continuous structure for transgene delivery and expression. ceDNA vectors comprise an expression cassette flanked by two ITR sequences, wherein the expression cassette encodes a transgene encoding a FVIII protein. Some ceDNA vectors further comprise a cis-regulatory element comprising a regulatory switch. Further provided herein are methods and cell lines for reliable gene expression of FVIII proteins in vitro, ex vivo, and in vivo using ceDNA vectors.SELECTED DRAWING: None

Detection of viruses present in cells The present invention provides 1. A method for identifying viral DNA integrated into the genome of at least one mammalian test cell, comprising: (a) determining the presence or absence of viral DNA in a DNA sample obtained from a mammalian test cell; (b) determining the methylation status of at least one CpG site in the viral DNA of a DNA sample obtained from a mammalian test cell; and the presence of viral DNA after step (a) and a hypermethylated state in step (b) indicates that the viral DNA has been integrated into the mammalian test cell and that the virus is inactive, respectively; the presence of viral DNA after step (a) and a baseline methylation state or a hypomethylated state in step (b) indicates that the viral DNA has integrated into the mammalian test cell and that the virus is active, respectively; the absence of viral DNA after step (a) indicates that the viral DNA has not integrated into the mammalian test cell; Methods performed on arrays Regarding.

Compositions and methods for treating a viral condition. The composition is administered to the subject in a therapeutic amount. The composition includes a first medicament effective for up-regulating disulfide bond donation and exchange, and a second medicament comprising a DNA polymerase inhibitor, a protease inhibitor, a DNA / RNA inhibitor, an intron splicer, or combinations thereof. The first medicament is effective for causing viruses to dissociate from integration sites and actively replicate with availability of disulfide bonds. The second medicament is effective for supporting or improving degradation, slicing, and / or inhibition of viral RNA, viral DNA, and / or viral proteins. The virus is Coronavirus, Epstein Barr virus, human papillomavirus, or herpes simplex virus.

The present disclosure provides methods and compositions of modified oligonucleotide primer and probe combinations, structurally modified with locked nucleic acids, quenchers, and dyes, that are effective for detecting tumor-derived human papillomavirus (HPV) and tumor-derived Epstein-Barr virus (EBV), and in particular, that are effective for distinguishing tumor-derived viral DNA from viral DNA derived from infectious viral particles.

The application describes ceDNA vectors having linear and continuous structure for delivery and expression of a transgene. ceDNA vectors comprise an expression cassette flanked by two ITR sequences, where the expression cassette encodes a transgene encoding PAH protein. Some ceDNA vectors further comprise cis-regulatory elements, including regulatory switches. Further provided herein are methods and cell lines for reliable gene expression of PAH protein in vitro, ex vivo and in vivo using the ceDNA vectors. Provided herein are method and compositions comprising ceDNA vectors useful for the expression of PAH protein in a cell, tissue or subject, and methods of treatment of diseases with said ceDNA vectors expressing PAH protein. Such PAH protein can be expressed for treating disease, e.g., Phenylketonuria (PKU).

To provide a method with sensitivity and / or specificity to detect a tumor at an early stage.SOLUTION: Cell-free DNA molecules in a mixture of a biological sample can be analyzed to detect viral DNA. Methylation of viral DNA molecules at one or more sites in the viral genome can be determined. Mixture methylation level (s) can be measured based on one or more amounts of the plurality of cell-free DNA molecules methylated at a set of site (s) of the particular viral genome. The mixture methylation level (s) can be determined in various ways, e.g., as a density of cell-free DNA molecules that are methylated at a site or across multiple sites or regions. The mixture methylation level (s) can be compared to reference methylation level (s), e.g., determined from at least two cohorts of other subjects. The cohorts can have different classifications (including the first condition) associated with the particular viral genome. A first classification of whether the subject has the first condition can be determined based on the comparing.SELECTED DRAWING: None

The present disclosure provides methods and compositions of modified oligonucleotide primer and probe combinations, structurally modified with locked nucleic acids, quenchers, and dyes, effective to detect tumor-derived Human Papilloma Virus (HPV) and tumor-derived Epstein-Barr virus (EBV) and, especially, to distinguish viral DNA derived from tumors from viral DNA derived from infectious viral particles.

The present disclosure relates generally to compositions and methods comprising recombinant integration deficient retroviral vectors where the expression of cargo(s) of interest is enhanced by the presence of elements that inhibit the silencing of viral DNA.

Improved methods for producing recombinant adeno-associated viral capsids (rAAVs) that results in an increase in the percent of filled AAVs are disclosed, and compositions of cells that contain a higher percentage of filled rAAVs. The method uses at least four constructions to provide gene required for rAAV packaging, and transformation of rAAV producing host cells is performed in two temporally separated stages. The filled rAAVs produced by the disclosed methods contain both the viral DNA and the desired transgene. The disclosed methods also minimize the percentage of empty AAV capsids. The four-vector transfection rAAV production method includes a two stage transfection of host cells, temporally separated by at least 7 hrs. Stage 1 involves transfecting host cells with: (1) a Helper construct (2) a Rep construct; and (3) a third construct containing the gene of interest. Stage 2 involves transfecting the cells from Stage 1 with: (4) a fourth construct, containing Rep and Cap genes.

The application discloses a method for screening antiviral target points by targeting viral DNA, and effective sgRNA sequences targeting key DNA regions of varicella-zoster virus, herpes simplex virus type I and Kaposi's sarcoma-associated herpes virus are obtained respectively; dCas9-sgRNA containing viral DNA plasmid is co-transfected, and DNA of the varicella-zoster virus, the herpes simplex virus type I and the Kaposi's sarcoma-associated herpes virus is immunoprecipitated and enriched respectively to screen proteins regulating viral replication; the proteins are identified by mass spectrometry, and it is verified that the method is also applicable to screening proteins regulating viral replication by targeting HIV DNA, so that the method for screening proteins regulating viral replication by targeting viral DNA is established.

Provided herein are methods and compositions for editing the genome of a cell. In some embodiments, a nucleotide sequence of at least 200 nucleotides in length is inserted into a target region in the genome of a cell.

The present invention relates to a method of identifying integrated viral DNA in the genome of at least one mammalian test cell, the method comprising the steps of: (a) determining the presence or absence of viral DNA in a DNA sample obtained from a mammalian test cell; and (b) determining the methylation status of at least one CpG site of the viral DNA in a DNA sample obtained from the mammalian test cell wherein the presence of the viral DNA after step (a) and the hypermethylation in step (b) respectively indicate that the viral DNA is integrated into the mammalian test cell and the virus is inactive; the existence of the virus DNA after the step (a) and the baseline methylation or hypomethylation in the step (b) respectively indicate that the virus DNA is integrated into the mammalian test cell, and the virus is active; and the absence of the viral DNA after step (a) indicates that the viral DNA is not integrated into the mammalian test cell, respectively; and wherein the method is performed on an array.

The present application discloses methods for synthetic production and cell-free synthesis of single stranded adeno-associated virus (AAV) vectors, for delivery and expression of a transgene in host cells. The present invention also relates to an in vitro process for production of closed-ended DNA vectors and corresponding single stranded AAV DNA vector products synthesized from the closed-ended DNA vectors having nicks.