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Небесная энциклопедия

Космические корабли и станции, автоматические КА и методы их проектирования, бортовые комплексы управления, системы и средства жизнеобеспечения, особенности технологии производства ракетно-космических систем

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Мониторинг СМИ

Мониторинг СМИ и социальных сетей. Сканирование интернета, новостных сайтов, специализированных контентных площадок на базе мессенджеров. Гибкие настройки фильтров и первоначальных источников.

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Поддерживает ввод нескольких поисковых фраз (по одной на строку). При поиске обеспечивает поддержку морфологии русского и английского языка
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Применить Всего найдено 6360. Отображено 100.
05-01-2012 дата публикации

Immunogenic compositions for inducing an immune response to hiv

Номер: US20120003265A1
Автор: John H. Eldridge, Maninder K. Sidhu, Michael Egan, Zimra Israel
Принадлежит: WYETH LLC

The invention relates to immunogenic compositions for inducing an immune response to HIV comprising combinations of two, three, or four plasmids, where each plasmid is expressing a defined antigen, which may be a single antigen or a fusion of two or three antigens.

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Номер записи: 1
19-01-2012 дата публикации

Dna vaccine for alzheimer's disease

Номер: US20120014987A1
Автор: Yoh Matsumoto
Принадлежит: TOKYO METROPOLITAN INSTITUTE OF MEDICAL SCIENCE

The present invention aims to provide a DNA vaccine for Alzheimer's disease. The present invention provides a recombinant vector which comprises DNA encoding amyloid β and DNA encoding a Th2 cytokine, as well as a DNA vaccine for Alzheimer's disease which comprises this vector.

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Номер записи: 2
16-02-2012 дата публикации

Intergenic regions as insertion sites in the genome of modified vaccinia virus ankara (mva)

Номер: US20120039936A1
Автор: Paul Howley, Sonja Leyrer
Принадлежит: Individual

The present invention relates to novel insertion sites useful for the integration of exogenous sequences into the Modified Vaccinia Ankara (MVA) virus genome. The present invention further provides plasmid vectors to insert exogenous DNA into the genome of MVA. Furthermore, the present invention provides recombinant MVA comprising an exogenous DNA sequence inserted into said new insertion site as medicine or vaccine.

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Номер записи: 3
17-05-2012 дата публикации

Promoters for recombinant viral expression

Номер: US20120121617A1
Автор: Christine Meisinger-Henschel, Eva Felder, Robin Steigerwald
Принадлежит: Bavarian Nordic AS

The invention relates to a promoter selected from a group of nucleic acids consisting of (a) a nucleic acid having the nucleotide sequence of SEQ ID NO:1; (b) a nucleic acid having a nucleotide sequence derived from SEQ ID NO:1, wherein not more than 10 nucleotides have been added, deleted, substituted and/or inverted from the nucleic acid of SEQ ID NO:1; and (c) a nucleic acid sequence having at least 70% identity with the nucleic acid of (a); wherein the promoter has a length of up to and including 27 nucleotides and wherein the promoter according to options (b) and (c) exhibits at least the 70% of the promoter activity of SEQ ID NO:1 as measured by the amount of recombinant protein produced.

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Номер записи: 4
05-07-2012 дата публикации

Reagents and methods for modulating cone photoreceptor activity

Номер: US20120172419A1
Автор: James A. Kuchenbecker, Jay Neitz, Maureen Neitz
Принадлежит: Medical College of Wisconsin Research Foundation Inc, UNIVERSITY OF WASHINGTON

The present invention provides reagents and methods for modulating cone photoreceptor activity, and devices for assessment of cone photoreceptor activity.

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Номер записи: 5
25-10-2012 дата публикации

Identification of rnai targets and use of rnai for rational therapy of chemotherapy-resistant leukemia and other cancers

Номер: US20120272346A1
Автор: Anthony Mazurek, Bruce Stillman, Christopher Vakoc, Johannes Ekkehart Zuber, Katherine McJunkin, Scott W. Lowe
Принадлежит: COLD SPRING HARBOR LABORATORY

Provided is a mosaic mouse model for use in determining the potency of an shRNA in vivo for reducing survival of cancer cells of chemotherapy-resistant leukemia. The syngeneic mouse recipient is transplanted with tet-on competent leukemia cells carrying a bicistronic nucleic acid construct comprising a promoter operably linked to a fusion gene associated with chemotherapy-resistant leukemia, and a sequence encoding a reverse tet-transactivator protein, such that both coding sequences are co-expressed from the promoter. Also provided are methods of treating soft tissue cancers.

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Номер записи: 6
29-11-2012 дата публикации

Use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof

Номер: US20120304321A1
Автор: Emmanuel Lacroix, Frederic Paques, Jean-Charles Epinat, Patrick Chames, Phillippe Duchateau, Sylvain Arnould
Принадлежит: CELLECTIS SA

A monomer of an I-CreI meganuclease variant wherein said monomer when in dimeric form binds and cleaves DNA.

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Номер записи: 7
31-01-2013 дата публикации

Glycine riboswitches, methods for their use, and compositions for use with glycine riboswitches

Номер: US20130029342A1
Автор: Jeffrey Barrick, Maumita Mandal, Ronald R. Breaker
Принадлежит: YALE UNIVERSITY

Riboswitches are structural elements in mRNA that change state when bound by a trigger molecule, and are thus able to regulate gene expression. They can be dissected into two separate domains: one that selectively binds the target (aptamer domain) and another that influences genetic control (expression platform domain). Bacterial glycine riboswitches consist of two tandem aptamer domains which cooperatively bind glycine to regulate the expression of downstream genes. These natural switches are targets for antibiotics and other small molecule therapies. Modified versions of these natural riboswitches can be employed as designer genetic switches that are controlled by specific effector compounds. Disclosed are isolated and recombinant riboswitches, and compositions and methods for selecting and identifying compounds that can activate, inactivate, or block a riboswitch.

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Номер записи: 8
07-02-2013 дата публикации

Compositions, methods and uses for expression of enterobacterium-associated peptides

Номер: US20130034583A1
Автор: Dan T. Stinchcomb, Jorge E. Osorio, Joseph N. Brewoo, Timothy D. Powell
Принадлежит: Inviragen Inc

Embodiments of the present invention generally disclose methods, compositions and uses for generating and expressing enterobacterial-associated peptides. In some embodiments, enterobacterial-associated peptides include, but are not limited to plague-associated peptides. In certain embodiments, methods generally relate to making and using compositions of constructs including, but not limited to, attenuated or modified vaccinia virus vectors expressing enterobacterial-associated peptides. In other embodiments, vaccine compositions are reported of use in a subject.

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Номер записи: 9
18-04-2013 дата публикации

Recombinant production of authentic human proteins using human cell expression systems

Номер: US20130095062A1
Автор: Hui Feng, Ridong Chen, Soon Seog Jeong
Принадлежит: Humanzyme Ltd

The present invention relates to novel expression cassettes and vectors for efficiently producing authentic recombinant human proteins from stable cultures of novel human cell lines, the authentic recombinant proteins produced therefrom, and antibodies raised against those authentic recombinant proteins.

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Номер записи: 10
25-07-2013 дата публикации

Multi plasmid system for the production of influenza virus

Номер: US20130189762A1
Автор: George Kemble, Gregory Duke
Принадлежит: MEDIMMUNE LLC

Vectors and methods for the production of influenza viruses suitable as recombinant influenza vaccines in cell culture are provided. Bi-directional expression vectors for use in a multi-plasmid influenza virus expression system are provided. Additionally, the invention provides methods of producing influenza viruses with enhanced ability to replicate in embryonated chicken eggs and/or cells (e.g., Vero and/or MDCK) and further provides influenza viruses with enhanced replication characteristics. In addition, the present invention includes an improved method of rescue, wherein animal cells (e.g., SF Vero cells) are electroporated with plasmids and vectors of the invention.

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Номер записи: 11
08-08-2013 дата публикации

Use of meganucleases for inducing homologous recombination ex vivo and in toto in vertebrate somatic tissues and application thereof

Номер: US20130203840A1
Автор: Emmanuel Lacroix, Frederic Paques, Jean-Charles Epinat, Patrick Chames, Philippe Duchateau, Sylvain Arnould
Принадлежит: CELLECTIS SA

A single chain homing endonuclease, comprising a first variant of I-CreI having the amino acid sequence of accession number pdb 1g9y and a second variant of I-CreI variant having the amino acid sequence of accession number pdb 1g9y in a single polypeptide.

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Номер записи: 12
29-08-2013 дата публикации

Use of herpes vectors for tumor therapy

Номер: US20130224243A1
Автор: Masahiro Toda, Robert L. Martuza, Samuel D. Rabkin
Принадлежит: Individual

Eliciting a systemic antitumor immune response can be efficacious for a patient who presents with or who is at risk of developing multiple metastatic tumors of a given cell type. To this end a pharmaceutical composition is employed that comprises a defective HSV vector, preferably containing an expressible nucleotide sequence encoding at least one immune modulator.

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Номер записи: 13
07-11-2013 дата публикации

Method for expression of small antiviral rna molecules with reduced cytotoxicity within a cell

Номер: US20130295615A1
Автор: Carlos Lois-Caballe, David Baltimore, Dong Sung An, Irvin S.Y. Chen, Xiao-Feng Qin
Принадлежит: California Institute of Technology CalTech, UNIVERSITY OF CALIFORNIA

In one aspect, the invention provides methods and compositions for the expression of small RNA molecules within a cell using a retroviral vector (FIG. 1 A). Small interfering RNA (siRNA) can be expressed using the methods of the invention within a cell. In a further aspect, the invention provides methods for producing siRNA encoding lentivirus where the siRNA activity may interfere with the lentiviral life cycle. In yet a further aspect, the invention provides methods for expression of a small RNA molecule within a cell, such as an siRNA capable of downregulating CCR5, wherein expression of the small RNA molecule is relatively non-cytotoxic to the cell. The invention also includes small RNA molecules, such as an siRNA capable of downregulating CCR5, that are relatively non-cytotoxic to cells.

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Номер записи: 14
07-11-2013 дата публикации

Synthetic genes and genetic constructs

Номер: US20130298264A1
Автор: Michael Wayne Graham, Robert Norman Rice
Принадлежит: Commonwealth Scientific and Industrial Research Organization CSIRO

The present invention relates generally to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the present invention provides novel synthetic genes and genetic constructs which are capable of repressing delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto.

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Номер записи: 15
21-11-2013 дата публикации

Promoter-regulated differentiation-dependent self-deleting cassette

Номер: US20130312128A1
Автор: David Frendewey, David M. Valenzuela, Guochun Gong, Ka-Man Venus Lai
Принадлежит: Regeneron Pharmaceuticals Inc

Targeting constructs and methods of using them are provided for differentiation-dependent modification of nucleic acid sequences in cells and in non-human animals. Targeting constructs comprising a promoter operably linked to a recombinase are provided, wherein the promoter drives transcription of the recombinase in an differentiated cell but not an undifferentiated cell. Promoters include Blimp1, Prm1, Gata6, Gata4, Igf2, Lhx2, Lhx5, and Pax3. Targeting constructs with a cassette flanked on both sides by recombinase sites can be removed using a recombinase gene operably linked to a 3′-UTR that comprises a recognition site for an miRNA that is transcribed in undifferentiated cells but not in differentiated cells. The constructs may be included in targeting vectors, and can be used to automatically modify or excise a selection cassette from an ES cell, a non-human embryo, or a non-human animal.

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Номер записи: 16
02-01-2014 дата публикации

Chimeric gene constructs for generation of fluorescent transgenic ornamental fish

Номер: US20140007265A1
Автор: Bensheng Ju, Jiangyan He, Tie Yan, Toong Jin Lam, Yanfei Xu, Zhiyuan Gong
Принадлежит: NATIONAL UNIVERSITY OF SINGAPORE

Four zebrafish gene promoters, which are skin specific, muscle specific, skeletal muscle specific and ubiquitously expressed respectively, were isolated and ligated to the 5′ end of the EGFP gene. When the resulting chimeric gene constructs were introduced into zebrafish, the transgenic zebrafish emit green fluorescence under a blue light or ultraviolet light according to the specificity of the promoters used. Thus, new varieties of ornamental fish of different fluorescence patterns, e.g., skin fluorescence, muscle fluorescence, skeletal muscle-specific and/or ubiquitous fluorescence, are developed.

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Номер записи: 17
05-01-2017 дата публикации

Pharmacologically induced transgene ablation system

Номер: US20170000904A1
Автор: Anna P. Tretiakova, James M. Wilson, Jenny A. Sidrane
Принадлежит: University of Pennsylvania Penn

The present invention relates to gene therapy systems designed for the delivery of a therapeutic product to a subject using replication-defective virus composition(s) engineered with a built-in safety mechanism for ablating the therapeutic gene product, either permanently or temporarily, in response to a pharmacological agent—preferably an oral formulation, e.g., a pill. The invention is based, in part, on the applicants' development of an integrated approach, referred to herein as “PITA” (Pharmacologically Induced Transgene Ablation), for ablating a transgene or negatively regulating transgene expression. In this approach, replication-deficient viruses are used to deliver a transgene encoding a therapeutic product (an RNA or a protein) so that it is expressed in the subject, but can be reversibly or irreversibly turned off by administering the pharmacological agent; e.g., by administration of a small molecule that induces expression of an ablator specific for the transgene or its RNA transcript.

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Номер записи: 18
06-01-2022 дата публикации

MULTIPLE VECTOR SYSTEM AND USES THEREOF

Номер: US20220002749A1
Автор: Auricchio Alberto, Colella Pasqualina, Trapani Ivana
Принадлежит:

The present invention relates to constructs, vectors, relative host cells and pharmaceutical compositions which allow an effective gene therapy, in particular of genes larger than 5 Kb. 1. A vector system to express the coding sequence of a gene of interest in a cell , said coding sequence comprising a first portion and a second portion , said vector system comprising: said first portion of said coding sequence (CDS1),', 'a first reconstitution sequence; and, 'a) a first vector comprising said second portion of said coding sequence (CDS2),', 'a second reconstitution sequence,, 'b) a second vector comprisingwherein said first and second reconstitution sequences are selected from the group of:i] the first reconstitution sequence consists of the 3′ end of said first portion of the coding sequence and the second reconstitution sequence consists of the 5′end of said second portion of the coding sequence, said first and second reconstitution sequences being overlapping sequences; orii] the first reconstitution sequence comprises a splicing donor signal (SD) and the second reconstitution sequence comprises a splicing acceptor signal (SA), optionally each one of first and second reconstitution sequence further comprises a recombinogenic sequence,characterized by the fact that either one or both of the first and second vector further comprises a nucleotide sequence of a degradation signal said sequence being located in case of i) at the 3′ end of the CDS1 and/or at the 5′ end of the CDS2 and in case of ii) in 3′ position relative to the SD and/or in 5′ position relative to the SA.2. The vector system according to claim 1 , wherein both of the first and second vector further comprise said nucleotide sequence of a degradation signal claim 1 , wherein the nucleotide sequence of the degradation signal in the first vector is identical to or differs from that in the second vector.3. The vector system according to claim 1 , wherein the first reconstitution sequence comprises a ...

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Номер записи: 19
07-01-2016 дата публикации

Compositions and methods of use thereof for identifying anti-viral agents

Номер: US20160002668A1
Автор: Leor S. WEINBERGER
Принадлежит: J David Gladstone Institutes

The present disclosure provides a recombinant expression vector comprising a nucleotide sequence encoding a herpesvirus transactivator, where the nucleotide sequence is operably linked to a herpesvirus control element. The present disclosure provides cell lines genetically modified to express a herpesvirus transactivator under the control of a herpesvirus control element. The present disclosure provides methods of identifying agents that disrupt feedback regulation of a herpesvirus transcriptional control element by a herpesvirus transactivator.

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Номер записи: 20
05-01-2017 дата публикации

COMPOSITIONS AND METHODS FOR DETECTING AND MODULATING CELL DEATH BY A TRANSLATION REGULATED GENE EXPRESSION SYSTEM

Номер: US20170002377A1
Автор: Carlock Leon, Cypher Maria
Принадлежит:

The technology relates to a nucleic acid expression cassette comprising a TR element encoding an mRNA molecule that is translated in stressed and/or dying cells, and a nucleotide sequence operably linked to the TR element, that is a first open reading frame (ORF) sequence and encodes a polypeptide or a fragment thereof and is co-translated with the TR element. The technology further relates to mammalian cells and a transgenic animal comprising such expression cassette. Further included are kits comprising the expression cassette, and methods for determining toxicity, and killing a target cell. 1122.-. (canceled)123. A nucleic acid expression cassette expressible in mammalian cells , wherein the expression cassette comprises the following elements in a 5′ to 3′ direction:at least one transcriptional effector sequence,{'figref': {'@idref': 'DRAWINGS', 'FIG. 15'}, 'a translational regulatory (TR) element encoding a mammalian mRNA molecule that is at least 80% homologous to a human Proteolipid Protein (plp) gene corresponding to nts 1-831 of a PLP sequence, and is selectively translated in stressed and/or dying cells,'}a nucleotide sequence operably linked to the TR element, that is an open reading frame (ORF) sequence and encodes a polypeptide or a fragment thereof and is co-translated with the TR element, anda polyadenylation sequence,wherein selectively translated in stressed and/or dying cells means that the mRNA translation activity is observed in more than 95% of any cell line transformed with the expression cassette at the peak of translation activity following treatment with an acute toxic agent that induces cell stress and/or death, and that translational levels of the ORF of the expression cassette rise to at least 50% of expression levels of the same ORF when transcribed and translated from the same expression cassette lacking an operably linked TR element following treatment with the acute toxic agent.124. The expression cassette of claim 123 , wherein the ...

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Номер записи: 21
05-01-2017 дата публикации

SYNTHETIC GENES AND GENETIC CONSTRUCTS

Номер: US20170002379A1
Автор: Graham Michael Wayne, Rice Robert Norman
Принадлежит:

The present invention relates generally to synthetic genes for modifying endogenous gene expression in a cell, tissue or organ of a transgenic organism, in particular a transgenic animal or plant. More particularly, the present invention provides novel synthetic genes and genetic constructs which are capable of repressing delaying or otherwise reducing the expression of an endogenous gene or a target gene in an organism when introduced thereto. 133-. (canceled)34. A double-stranded DNA construct comprising a first structural gene sequence comprising 20 consecutive nucleotides identical in sequence to a region of a target gene in an animal cell; a second structural gene sequence comprising 20 consecutive nucleotides identical in sequence to , and in an inverted orientation relative to , the 20 consecutive nucleotides of the first structural gene sequence , such that a repeating sequence which comprises the 20 consecutive nucleotides in length identical to the region of the target gene is present in the DNA construct; a stuffer fragment which consists of nucleotides other than the nucleotides of the repeating sequence and which separates and links the first and second structural gene sequences; a promoter and a transcription termination sequence , wherein the repeating sequence within the DNA construct comprises 20 nucleotides in length , and wherein the first structural gene sequence , the stuffer fragment and the second structural gene sequence are all operably connected to the promoter and the transcription termination sequence.35. The double-stranded DNA construct of claim 34 , wherein the target gene is endogenous to the animal cell.36. The double-stranded DNA construct of claim 34 , wherein the target gene is non-endogenous to the animal cell.37. The double-stranded DNA construct of claim 34 , wherein the region of the target gene is in an exon.38. The double-stranded DNA construct of claim 34 , wherein the target gene is from a lentivirus.39. The double- ...

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Номер записи: 22
04-01-2018 дата публикации

MODIFIED POLYNUCLEOTIDES FOR THE PRODUCTION OF ONCOLOGY-RELATED PROTEINS AND PEPTIDES

Номер: US20180002393A1
Автор: Bancel Stephane, Chakraborty Tirtha, de Fougerolles Antonin, Ejebe Kenechi, Elbashir Sayda M., Ellsworth Jeff Lynn, Guild Justin, Hatala Paul, John Matthias, Roy Atanu, Schrum Jason P., Whoriskey Susan, Wood Kristy M.
Принадлежит:

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of oncology-related polynucleotides, oncology-related primary transcripts and oncology-related mmRNA molecules. 1. An mRNA encoding a polypeptide of interest selected from the group consisting of SEQ ID NOs 4704-9203 , wherein said mRNA comprises a coding region selected from the group consisting of SEQ ID NOs: 9204-33882.2. The mRNA of claim 1 , wherein the mRNA comprises at least one untranslated region 5′ relative to the coding region and at least one untranslated region 3′ relative to the coding region.3. The mRNA of claim 2 , wherein the 5′ untranslated region is heterologous to the coding region of the mRNA.4. The mRNA of claim 2 , wherein the 3′ untranslated region is heterologous to the coding region of the mRNA.5. The mRNA of claim 2 , wherein the 5′ untranslated region and the 3′ untranslated region are heterologous to the coding region of the mRNA.6. The mRNA of claim 2 , wherein the mRNA comprises at least two stop codons.7. A pharmaceutical composition comprising the mRNA of and a pharmaceutically acceptable excipient.8. The pharmaceutical composition of claim 7 , wherein the pharmaceutically acceptable excipient is selected from a solvent claim 7 , aqueous solvent claim 7 , non-aqueous solvent claim 7 , dispersion media claim 7 , diluent claim 7 , dispersion claim 7 , suspension aid claim 7 , surface active agent claim 7 , isotonic agent claim 7 , thickening or emulsifying agent claim 7 , preservative claim 7 , lipid claim 7 , lipidoids liposome claim 7 , lipid nanoparticle claim 7 , core-shell nanoparticles claim 7 , polymer claim 7 , lipoplex claim 7 , peptide claim 7 , protein claim 7 , cell claim 7 , hyaluronidase claim 7 , and mixtures thereof.9. The pharmaceutical composition of claim 8 , where the pharmaceutical composition comprises a lipid and wherein said lipid is selected from DLin-DMA claim 8 , DLin-K-DMA claim 8 , DLin-KC2-DMA claim 8 , ...

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Номер записи: 23
04-01-2018 дата публикации

USE OF ADENOVIRUS AND NUCLEIC ACIDS CODING THEREFOR

Номер: US20180002674A1
Автор: Holm Per Sonne
Принадлежит:

This invention relates to the use of an adenovirus to treat cancer, for example. The adenovirus may be replication deficient in cells that lack Y box binding protein. The adenovirus may encode an oncogene or an oncogene product, which may transactivate at least one viral gene. 155.-. (canceled)56. A pharmaceutical composition comprising an adenovirus , wherein the adenovirus is AdΔ24 and is replication deficient in cells that lack Y box binding protein 1 (“YB-1”) in the nucleus , and wherein the pharmaceutical composition further comprises a pharmaceutically active compound selected from the group consisting of a cytokine , a metalloproteinase inhibitor , an angiogenesis inhibitor , a cytostatic , and a cell cycle inhibitor.57. The composition of claim 56 , wherein the adenovirus is capable of replicating in cells that contain YB-1 in the nucleus.58. The composition of claim 56 , wherein the virus codes for YB-1.59. The composition of claim 58 , wherein the YB-1 is under the control of a tissue and/or tumor specific promoter.60. The composition of claim 56 , wherein the adenovirus is E1B 19 K-minus.61. A method for the treatment of cancer claim 56 , comprising administering to a subject in need thereof an adenovirus claim 56 , wherein the adenovirus is AdΔ24 and is replication deficient in cells that lack Y box binding protein 1 (“YB-1”) in the nucleus.62. The method of claim 61 , wherein the tumor expresses YB-1.63. The method of claim 62 , wherein the tumor has YB-1 in the nucleus.64. The method of claim 63 , wherein the tumor has YB-1 in the nucleus independent of the cell cycle.65. The method of claim 61 , wherein the cancer is formed from a tumor claim 61 , or part thereof claim 61 , which is resistant to pharmacologically effective agents.66. The method of claim 65 , wherein the cells that form the tumor claim 65 , or part thereof claim 65 , overexpress membrane-bound transport protein P glycoprotein.67. A method for the treatment of cancer claim 56 , ...

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Номер записи: 24
04-01-2018 дата публикации

Vectors Conditionally Expressing Therapeutic Proteins, Host Cells Comprising the Vectors, and Uses Thereof

Номер: US20180002719A1
Автор: Brandon Cuthbertson, Charles C. Reed, Jeremiah F. Roeth, Sunil Chada, William E. Fogler
Принадлежит: Intrexon Corp

This invention relates to the field of therapeutics. Most specifically, the invention provides methods of generating conditionally expressing vectors for one or more immuunomodulators under the control of a gene expression modulation system in the presence of activating ligand and uses for therapeutic purposes in animals. These vector may be provided to treat a variety of disorders, e.g., neoplastic disorders, through direct injection or through in vitro engineered cells, such as dendritic cells.

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Номер записи: 25
07-01-2021 дата публикации

RNA-GUIDED GENE EDITING AND GENE REGULATION

Номер: US20210002665A1
Автор: Black Joshua B., Gersbach Charles A., Hilton Isaac B., Kabadi Ami M., Ousterout David G., Perez-Pinera Pablo, Thakore Pratiksha I.
Принадлежит:

Disclosed herein are Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) 9-based system related compositions and methods of using said CRISPR/Cas9-based system related compositions for altering gene expression and genome engineering. Also disclosed herein are compositions and methods of using said compositions for altering gene expression and genome engineering in muscle, such as skeletal muscle and cardiac muscle. 1. A DNA targeting system for deleting exon 51 of a dystrophin gene , the system comprising Cas9 and at least one guide RNA (gRNA) , wherein the at least one gRNA binds and targets a polynucleotide sequence comprising SEQ ID NO: 67 , SEQ ID NO: 70 , SEQ ID NO: 100 , or SEQ ID NO: 680.2. An isolated polynucleotide encoding the DNA targeting system of .3. A vector comprising the isolated polynucleotide of .4. A cell comprising the isolated polynucleotide of .5. A method of treating a subject in need thereof having a mutant dystrophin gene claim 1 , the method comprising administering to the subject the DNA targeting system of .6. The method of claim 5 , wherein the subject is suffering from Duchenne muscular dystrophy.7. A method of correcting a mutant dystrophin gene in a cell claim 1 , the method comprising administering to a cell containing a mutant dystrophin gene the DNA targeting system of .8. A composition for genome editing in a muscle of a subject claim 1 , the composition comprising a modified adeno-associated virus (AAV) vector and a nucleotide sequence encoding the DNA targeting system of claim 1 , wherein the muscle is skeletal muscle or cardiac muscle.9. The composition of claim 8 , wherein the modified AAV vector has enhanced cardiac and skeletal muscle tissue tropism. This application is a continuation of U.S. patent application Ser. No. 14/895,316, filed Dec. 2, 2015, which is the national stage filing under 35 U.S.C. 371 of International Patent Application No. PCT/US2014/041190, filed Jun. 5, 2014, ...

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Номер записи: 26
07-01-2021 дата публикации

TARGETED INTEGRATION OF NUCLEIC ACIDS

Номер: US20210002669A1
Автор: Crawford Yongping Guo, Gao Albert Eric, MISAGHI Shahram, Ng Chi Kin Domingos, SHEN Amy, Snedecor Bradley R., Zhou Meixia
Принадлежит: Genentech, Inc.

The presently disclosed subject matter relates to targeted integration (TI) host cells suitable for the expression of recombinant proteins, as well as methods of producing and using said TI host cells. 177-. (canceled)78. A targeted integration (TI) host cell comprising an exogenous nucleotide sequence integrated at an integration site within a specific locus of the genome of the host cell , wherein the locus is at least about 90% homologous to a sequence selected from: contig NW_006874047.1; contig NW_006884592.1 , contig NW_006881296.1 , contig NW_003616412.1 , contig NW_003615063.1 , contig NW_006882936.1 , and contig NW_003615411.1.79. The TI host cell of claim 78 , wherein the nucleotide sequence immediately 5′ of the integrated exogenous nucleotide sequence is selected from the group consisting of(a) sequences at least about 90% homologous to nucleotides 41190-45269 of NW_006874047.1, nucleotides 63590-207911 of NW_006884592.1, nucleotides 253831-491909 of NW_006881296.1, nucleotides 69303-79768 of NW_003616412.1, nucleotides 293481-315265 of NW_003615063.1, nucleotides 2650443-2662054 of NW_006882936.1, and nucleotides 82214-97705 of NW_003615411.1; or(b) sequences at least 15 base pairs, at least 20 base pairs, at least 30 base pairs, at least 40 base pairs, at least 50 base pairs, at least 75 base pairs, at least 100 base pairs, at least 150 base pairs, at least 200 base pairs, at least 300 base pairs, at least 400 base pairs, at least 500 base pairs, at least 1,000 base pairs, at least 1,500 base pairs, at least 2,000 base pairs, at least 3,000 base pairs from nucleotide 45269 of NW_006874047.1, nucleotide 207911 of NW_006884592.1, nucleotide 491909 of NW_006881296.1, nucleotide 79768 of NW_003616412.1, nucleotide 315265 of NW_003615063.1, nucleotide 2662054 of NW_006882936.1, and nucleotide 97705 of NW_003615411.1.80. The TI host cell of claim 78 , wherein the nucleotide sequence immediately 3′ of the integrated exogenous nucleotide sequence is selected ...

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Номер записи: 27
03-01-2019 дата публикации

METHOD FOR EXPRESSION OF SMALL ANTIVIRAL RNA MOLECULES WITH REDUCED CYTOTOXICITY WITHIN A CELL

Номер: US20190002883A1
Автор: An Dong Sung, Baltimore David, Chen Irvin S.Y., Lois-Caballe Carlos, Qin Xiao-Feng
Принадлежит:

In one aspect, the invention provides methods and compositions for the expression of small RNA molecules within a cell using a retroviral vector (FIG. A). Small interfering RNA (siRNA) can be expressed using the methods of the invention within a cell. In a further aspect, the invention provides methods for producing siRNA encoding lentivirus where the siRNA activity may interfere with the lentiviral life cycle. In yet a further aspect, the invention provides methods for expression of a small RNA molecule within a cell, such as an siRNA capable of downregulating CCR5, wherein expression of the small RNA molecule is relatively non-cytotoxic to the cell. The invention also includes small RNA molecules, such as an siRNA capable of downregulating CCR5, that are relatively non-cytotoxic to cells. 120.-. (canceled)21. A retroviral construct for expressing an RNA within a cell , comprising:a nucleic acid having the R and U5 sequences from a 5′ lentiviral long terminal repeat (LTR);a self-inactivating lentiviral 3′ LTR; anda first promoter configured to be operably linked to a first RNA coding region encoding a first RNA,wherein the first promoter is located between the 5′ LTR and the 3′ LTR, and wherein the first RNA comprises a RNA duplex.22. The retroviral construct of claim 21 , wherein the first RNA comprises a sequence that is at least about 90% complementary to a target region of a pathogenic virus genome or genome transcript.23. The retroviral construct of claim 22 , wherein the pathogenic virus is human immunodeficiency virus (HIV) claim 22 , hepatitis A virus (HAV) claim 22 , hepatitis B virus (HBV) claim 22 , hepatitis C virus (HCV) claim 22 , cytomegalovirus (CMV) claim 22 , herpes simplex virus (HSV) claim 22 , influenza virus claim 22 , adeno virus claim 22 , human papillomavirus claim 22 , Coxsackieviruses claim 22 , Measles virus claim 22 , or poliovirus.24. The retroviral construct of claim 23 , wherein the pathogenic virus is HIV-1 or HIV-2.25. The ...

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Номер записи: 28
03-01-2019 дата публикации

HYBRID DUAL RECOMBINANT AAV VECTOR SYSTEMS FOR GENE THERAPY

Номер: US20190002916A1
Автор: Francois Achille, KALATZIS Vasiliki
Принадлежит:

The invention relates to constructs, vectors, relative host cells and pharmaceutical compositions which allow an effective gene therapy, in particular of genes larger than 5Kb by using an improved hybrid dual recombinant AAV vector system. 121-. (canceled)22. A dual construct composed a pair of nucleic acid sequences: the 5′ end portion of a nucleic acid sequence of a synthetic intron comprising a nucleic acid sequence of a splicing donor (SD) signal (SEQ ID NO: 1), and', 'a nucleic acid sequence of a recombinogenic region; and, '(a) a first nucleic acid sequence comprising a nucleic acid sequence of a recombinogenic region, and', 'the 3′ end portion of a nucleic acid sequence of a synthetic intron comprising a branch site and a polypyrimidine tract and a nucleic acid sequence of a splicing acceptor (SA) signal (SEQ ID NO: 2)., '(b) a second nucleic acid sequence comprising23. The dual construct of claim 22 , wherein the recombinogenic region is a polynucleotide sequence derived or originating from alkaline phosphatase (AP) or from bacteriophage F1 (AK) claim 22 , or other polynucleotide sequences known as a homologous recombination hotspot.24. The dual construct of claim 22 , wherein the recombinogenic region AP comprises SEQ ID NO: 3 claim 22 , a fragment thereof or a derived codon-modified (mAP) (SEQ ID NO: 6) claim 22 , into which all ATG codons on both DNA strands were removed.25. A hybrid dual construct system suitable for expressing the coding sequence of a gene of interest in an host cell claim 22 , comprising: a 5′-inverted terminal repeat (5′-ITR) sequence;', 'a promoter sequence;', 'the 5′ end portion of said coding sequence, said 5′ end portion being operably linked to and under control of said promoter;', 'the 5′ end portion of a sequence of a synthetic intron comprising a nucleic acid sequence of a splicing donor (SD) signal (SEQ ID NO: 1);', 'a nucleic acid sequence of a recombinogenic region; and', 'a 3′-inverted terminal repeat (3′-ITR) sequence;, ' ...

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Номер записи: 29
20-01-2022 дата публикации

MULTICISTRONIC VECTOR FOR SURFACE ENGINEERING LENTIVIRAL PARTICLES

Номер: US20220017920A1
Автор: BEITZ Laurie, Scharenberg Andrew
Принадлежит:

The disclosure relates generally to nucleic acid vectors and packaging cell lines for in vivo expansion of T-cells. More particularly, the disclosure relates to intravenous or intratumoral injection of a lentiviral particle adapted for transduction and expansion of tumor-infiltrating lymphocytes in vivo. 1. A multicistronic vector for surface-engineering lentiviral particles , comprising a polynucleotide operatively linked to a promoter , 'wherein the plurality of polypeptides comprise a fusion glycoprotein or functional variant thereof and one or more non-viral proteins capable of viral surface display.', 'wherein the polynucleotide encodes a plurality of polypeptides joined by linkers comprising peptides capable of inducing ribosome skipping or self-cleavage, and'}2. The multicistronic vector of claim 1 , wherein the linkers comprise 2A peptides each independently selected from the group consisting of P2A (SEQ ID NO: 14) claim 1 , T2A (SEQ ID NO: 15) claim 1 , E2A (SEQ ID NO: 16) claim 1 , and F2A (SEQ ID NO: 17).3. The multicistronic vector of or claim 1 , where one or more of the linkers comprises a sequence encoding the residues Gly-Ser-Gly.4. The multicistronic vector of any one of to claim 1 , wherein the the plurality of polypeptides comprises 2 claim 1 , 3 claim 1 , 4 claim 1 , 5 claim 1 , 6 claim 1 , 7 claim 1 , 8 claim 1 , 9 claim 1 , 10 claim 1 , 11 claim 1 , 12 claim 1 , 13 claim 1 , 14 claim 1 , 15 claim 1 , 16 claim 1 , 17 claim 1 , 18 claim 1 , 19 or 20 proteins capable of viral surface display.5. The multicistronic vector of any one of to claim 1 , wherein the fusion glycoprotein or functional variant thereof is a viral fusion glycoprotein or a functional variant thereof.6. The multicistronic vector of claim 5 , wherein the viral fusion glycoprotein or functional variant thereof is Cocal virus G (COCVG) protein or a functional variant thereof.7. The multicistronic vector of any one of to claim 5 , wherein the non-viral proteins capable of viral ...

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Номер записи: 30
20-01-2022 дата публикации

IMPROVED VECTOR SYSTEMS FOR CAS PROTEIN AND SGRNA DELIVERY, AND USES THEREFOR

Номер: US20220017921A1
Автор: Doench John, Dubrot Juan, Haining William Nicholas, Manguso Robert, Yates Kathleen
Принадлежит:

The present disclosure provides vectors, methods and kits for for delivery and stable expression of CRISPR/Cas components capable of inducing genetic modification of cells, followed by recombinase-mediated excision of some or all of these components after the cells have been successfully genetically modified. The disclosed vectors and methods provide for reduced immunogenic effects arising from one or more CRISPR/Cas components. The disclosed vectors comprise coding sequences that encode a Cas protein, detectable markers and a guide RNA. The disclosed vectors provide for the subsequent genomic excision of the CRISPR/Cas components after successful genetic modification, as mediated by recombinase recognition of recombination sites flanking one or more of the disclosed coding sequences. The present disclosure further provides methods of generating a population of genetically modified tumor cells for screening a candidate target gene for cancer immunotherapy. 1. A method of producing a population of genetically modified cells , comprising:(i) providing a population of cells; wherein the first integration vector is a replication defective retroviral vector derived from a primate lentivirus,', 'wherein the first integration vector comprises a first nucleic acid sequence comprising a first promoter operably linked to a Cas protein coding sequence encoding a Cas protein; and at least a first 3′ site-specific recombination site located 3′ to the Cas coding sequence, and', 'wherein the first integrating vector is capable of integration into the genomes of at least a portion of the population of cells;, '(ii) introducing a first integration vector into at least a portion of the population of cells,'}(iii) introducing an sgRNA into at least a portion of the population of cells, wherein the sgRNA is capable of guiding the Cas protein to a target site in the genomes of at least a portion of the population of cells, and wherein the Cas protein is capable of double-stranded DNA ...

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Номер записи: 31
12-01-2017 дата публикации

BIOCONTROL

Номер: US20170009253A1
Автор: Alphey Luke
Принадлежит: Oxitec Limited

Provided is an arthropod male germline gene expression system suitable for conditional expression of an effector gene in an Arthropod male germline. The system comprises a first expression unit comprising an effector gene and a promoter therefor operably linked thereto; and a second expression unit. Said second unit comprises a coding sequence for a transcription factor and an upstream regulatory element operably linked thereto, the transcription factor being capable of acting upon the promoter in the first expression unit to drive expression of the effector gene. The upstream regulatory element includes a promoter for the transcription factor; and a 5′ UTR adjacent a start site for the transcription factor coding sequence. The upstream regulatory element driving sufficient expression of the transcription factor such that the transcription factor protein in turn drives transcription of the effector gene before meiosis. Also provided are uses of the system for instance in methods of biocontrol and quality control. 2) A gene expression system according to claim 1 , wherein the transcription factor is a transcriptional activator claim 1 , such as tTA claim 1 , GAL4 or their variants.3) A gene expression system according to any preceding claim claim 1 , wherein the effector is endonuclease claim 1 , most preferably a 3-Zn finger nuclease.4) A gene expression system according to any preceding claim claim 1 , wherein the promoter of the first expression unit is a minimal promoter.5) A gene expression system according to any preceding claim claim 1 , wherein the promoter of the upstream regulatory element in the second expression unit is most from topi claim 1 , aly or Beta-2 Tubulin (B2T) or homologues thereof.6) A gene expression system according to any preceding claim claim 1 , wherein the 5′ UTR in the upstream regulatory element of the second expression unit is that from hsp83 claim 1 , preferably from Medfly or homologues of hsp83 claim 1 , particularly the homologue ...

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Номер записи: 32
08-01-2015 дата публикации

Synthetic Transcriptional Control Elements and Methods of Generating and Using Such Elements

Номер: US20150011007A1
Автор: Arkin Adam P., Liu Chang, Qi Lei S.
Принадлежит:

Provided herein are nucleic acid constructs that contain a synthetic control element that includes a cis-regulator of translation, and an adapter translation-coupled regulator of transcription. Further provided herein are nucleic acid constructs that contain nucleic acid sequences under the control of the synthetic control elements. Also provided are compositions and methods related to the nucleic acid constructs. 1. A nucleic acid construct comprising a synthetic TCRT (translation-coupled regulator of transcription) comprising in a 5′ to 3′ order:(a) a cis-regulator of translation; and(b) an adapter TCRT,wherein (a) and (b) are operably linked so that the transcription of sequences that are 3′ of and operably linked to the synthetic TCRT is regulated by (a).2. The nucleic acid construct of claim 1 , further comprising a nucleic acid sequence of interest that is positioned 3′ of the synthetic TCRT and is operably linked to the synthetic TCRT such that the synthetic TCRT regulates transcription of the nucleic acid sequence of interest.3. The nucleic acid construct of claim 2 , wherein the nucleic acid sequence of interest comprises a sequence selected from the group consisting of: an insertion site claim 2 , a sequence encoding a polypeptide claim 2 , a sequence encoding a sense RNA claim 2 , a sequence encoding an antisense RNA claim 2 , a sequence encoding a ribonucleic acid aptamer responsive to an environmental cue claim 2 , and a sequence encoding an RNA that is responsive to an intracellular or environmental cue.4. The nucleic acid construct of claim 1 , wherein the cis-regulator of translation is selected from the group consisting of: a riboswitch that senses small molecules claim 1 , a ribosomal binding site-including sequence that is repressed or activated through antisense molecules claim 1 , a nucleic acid aptamer responsive to an environmental cue claim 1 , an RNA sequence responsive to an intracellular or environmental cue claim 1 , and a tuned ribosome ...

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Номер записи: 33
11-01-2018 дата публикации

REGULATION OF GENE EXPRESSION BY APTAMER-MEDIATED MODULATION OF ALTERNATIVE SPLICING

Номер: US20180010146A1
Автор: Boyne Alex K., Danos Olivier F., Guo Xuecui, Volles Michael J.
Принадлежит:

The invention provides a platform and methods of using the platform for the regulation of the expression of a target gene using exposure to an aptamer ligand (for example, a small molecule). The platform features a polynucleotide gene regulation cassette that is placed in the target gene and includes a synthetic riboswitch positioned in the context of a 5′ intron-alternative exon-3′ intron. The riboswitch comprises an effector region and a sensor region (e.g., an aptamer that binds a small molecule ligand) such that the alternative exon is spliced into the target gene mRNA when the ligand is not present thereby preventing expression of the target gene. When the ligand is present, the alternative exon is not spliced into the target gene mRNA thereby providing expression of the target gene. 1. A polynucleotide cassette for the regulation of the expression of a target gene comprisinga. a riboswitchb. an alternatively-spliced exon, flanked by a 5′ intron and a 3′ intron,wherein the riboswitch comprises (i) an effector region comprising a stem that includes the 5′ splice site of the 3′ intron, and (ii) an aptamer,wherein the alternatively-spliced exon comprises a stop codon that is in-frame with the target gene when the alternatively-spliced exon is spliced into the target gene mRNA.2. The polynucleotide cassette of claim 1 , wherein the aptamer binds a small molecule ligand.3. The polynucleotide cassette of claim 1 , wherein the 5′ and 3′ introns are derived from an endogenous intron from the target gene.4. The polynucleotide cassette of claim 1 , wherein the 5′ and 3′ introns are exogenous to the target gene.5. The polynucleotide cassette of claim 1 , wherein the 5′ and 3′ introns are derived from intron 2 of the human β-globin gene.6. The polynucleotide cassette of claim 1 , wherein the 5′ intron comprises a stop codon in-frame with the target gene.7. The polynucleotide cassette of claim 1 , wherein the 5′ and 3′ introns are each independently from about 50 to about ...

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Номер записи: 34
14-01-2021 дата публикации

RECOMBINANT HVT VECTORS EXPRESSING MULTIPLE ANTIGENS OF AVIAN PATHOGENS AND USES THEREOF

Номер: US20210010033A1
Автор: Bublot Michel, Kassa Aemro, Linz Perry, Mebatsion Teshome, Pritchard Joyce
Принадлежит: Boehringer Ingelheim Animal Health USA Inc.

The present invention provides recombinant herpesvirus of turkeys (HVT) vectors that contain and express antigens of avian pathogens, compositions comprising the recombinant HVT vectors and polyvalent vaccines comprising the recombinant HVT vectors. The present invention further provides methods of vaccination against a variety of avian pathogens and method of producing the recombinant HVT vectors. 1. A recombinant herpesvirus of turkeys (HVT) vector comprising:a first heterologous polynucleotide encoding a first avian pathogen antigen; anda second heterologous polynucleotide encoding a second avian pathogen antigen,wherein the first and the second heterologous polynucleotides are linked by IRES or P2A.2. The vector of claim 1 , wherein the first and second avian pathogen antigens are each claim 1 , independently claim 1 , an Infectious Bursal Disease Virus (IBDV) VP2 antigen claim 1 , an Infectious Laryngotracheitis Virus (ILTV) glycoprotein D (gD) antigen claim 1 , or a Newcastle Disease Virus F (NDV-F) antigen.3. The vector of claim 1 , wherein the first and second avian pathogen antigens are each claim 1 , independently claim 1 , an IBDV VP2 antigen having at least 80% sequence identity to SEQ ID NO: 2 claim 1 , an ILTV gD antigen having at least 80% sequence identity to SEQ ID NO: 17 claim 1 , or a NDV-F antigen having at least 80% sequence identity to SEQ ID NOs: 5 and/or 22.4. The vector of claim 1 , wherein the first and second heterologous polynucleotides each comprise claim 1 , independently claim 1 , a nucleotide sequence encoding an IBDV VP2 antigen that has at least 70% sequence identity to SEQ ID NO: 1 claim 1 , a nucleotide sequence encoding an ILTV gD antigen that has at least 70% sequence identity to SEQ ID NO: 16 claim 1 , or a nucleotide sequence encoding a NDV-F antigen that has at least 70% sequence identity to SEQ ID NOs: 3 claim 1 , 4 or 215. The vector of claim 1 , wherein the first heterologous polynucleotide is operably linked to a mCMV IE ...

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Номер записи: 35
10-01-2019 дата публикации

DNA VECTORS, TRANSPOSONS AND TRANSPOSASES FOR EUKARYOTIC GENOME MODIFICATION

Номер: US20190010505A1
Автор: Caves Kate, Govindrajan Sridhar, Lee Maggie, Minshull Jeremy, Ness Jon, WELCH Mark
Принадлежит:

The present invention provides polynucleotide vectors for high expression of heterologous genes. Some vectors further comprise novel transposons and transposases that further improve expression. Further disclosed are vectors that can be used in a gene transfer system for stably introducing nucleic acids into the DNA of a cell. The gene transfer systems can be used in methods, for example, gene expression, bioprocessing, gene therapy, insertional mutagenesis, or gene discovery. 119-. (canceled)20XenopusXenopus. A method of creating a transgenic cell comprising introducing into a eukaryotic cell a transposon comprising the sequence 5′-CCYTTTBMCTGCCA-3′ (SEQ ID NO:19) inverted in orientation in the two transposon ends , and a transposase , wherein the transposase integrates the transposon into the genome of the cell , wherein either{'i': 'Xenopus', 'i. the transposase comprises an amino acid sequence at least 90% identical to SEQ ID NO: 48 and which differs from SEQ ID NO:48 by at least one amino acid shown in Table 4 column C or D, and wherein the transposase, when fused to a heterologous nuclear localization signal, can excise a transposon from a polynucleotide of the nucleotide sequence of SEQ ID NO: 44 with increased activity relative to the transposase of the amino acid sequence of SEQ ID NO:48 fused to a heterologous nuclear localization signal, or'}{'i': 'Xenopus', 'ii. the transposase comprises the amino acid sequence of SEQ ID NO:48 or 49 fused to a heterologous nuclear localization sequence.'}21Xenopus. The method of wherein the transposase comprises at least one amino acid substitution relative to the amino acid sequence of SEQ ID NO:48 at one of the following positions (numbered according to SEQ ID NO: 48): 6 claim 20 , 7 claim 20 , 16 claim 20 , 19 claim 20 , 20 claim 20 , 21 claim 20 , 22 claim 20 , 23 claim 20 , 24 claim 20 , 26 claim 20 , 28 claim 20 , 31 claim 20 , 34 claim 20 , 67 claim 20 , 73 claim 20 , 76 claim 20 , 77 claim 20 , 88 claim 20 , 91 ...

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Номер записи: 36
09-01-2020 дата публикации

Enhanced Production of Recombinant Proteins By Transient Transfection of Suspension-Growing Mammalian Cells

Номер: US20200010808A1
Автор: Durocher Yves, Kamen Amine, Perret Sylvie, Pham Phuong
Принадлежит: NATIONAL RESEARCH COUNCIL OF CANADA

Disclosed is a new process for the production of recombinant proteins, by transient transfection of suspension-grown human embryonic kidney cells (293 cell line and its genetic variants) with an expression vector, using polyethylenimine (PEI) as a transfection reagent. In a preferred embodiment, the process uses 293E cells expressing the Epstein-Barr virus (EBV) EBNA 1 protein, in combination with an oriP-based episomal expression vector having an improved cytomegalovirus expression cassette comprising the CMV5 promoter. The process combines in a single step the cell growth, transfection and protein expression, is carried out without changing the culture medium, and allows to achieve high expression levels in a short period of time. The process may be carried out in a serum-free, low-protein culture medium, is easily scalable, compatible with continuous production processes, and fully adapted to high-throughput production of milligram quantities of recombinant proteins. 150-. (canceled)52. The expression vector of claim 51 , wherein the size of the expression vector is from about 4185 base pairs to about 5925 base pairs.53. The expression vector of claim 51 , wherein the fragment of SEQ ID NO: 1 consists of a BxtXI-EcoRI FR fragment consisting of nucleotides 5 to 299 of SEQ ID NO: 1.54. The expression vector of claim 51 , wherein the fragment of SEQ ID NO: 2 consists of a BxtXI FR fragment consisting of nucleotides 300 to 595 of SEQ ID NO: 1.55. The expression vector of claim 51 , further comprising an antibiotic resistance gene and a bacterial origin of replication claim 51 , wherein the antibiotic resistance gene and the bacterial origin of replication are located between SEQ ID NO: 1 or the fragment thereof and the 5′ end of the CMV5 promoter.56. The expression vector of claim 55 , wherein the bacterial origin of replication is pMB1 and/or the antibiotic resistance gene is an ampicillin resistance gene.57. The expression vector of claim 51 , further comprising a ...

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Номер записи: 37
21-01-2016 дата публикации

REAGENTS AND METHODS FOR MODULATING CONE PHOTORECEPTOR ACTIVITY

Номер: US20160015288A1
Автор: Hauswirth William W., Kuchenbecker James A., Neitz Jay, Neitz Maureen
Принадлежит:

The present invention provides reagents and methods for modulating cone photoreceptor activity, and devices for assessment of cone photoreceptor activity.

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Номер записи: 38
03-02-2022 дата публикации

EXPRESSION VECTORS FOR EUKARYOTIC EXPRESSION SYSTEMS

Номер: US20220033845A1
Автор: DU Zhimei, JIANG Bo, TANG Xiaoyan
Принадлежит: Merck Sharp & Dohme Corp.

The invention provides expression vectors for expressing recombinant proteins (e.g., biologics) in mammalian cells. Also provided are host cells comprising the expression vectors, methods of producing the recombinant proteins, and methods of propagating the expression vectors. 171-. (canceled)72. An expression vector comprising:(a) a first expression cassette comprising the following elements in the order of upstream to downstream: a promoter operably linked to an insertion site for a gene of interest (GOI), an internal ribosome entry site (IRES), a polynucleotide encoding a eukaryotic selectable marker, and a polyadenylation (polyA) signal;(b) a second expression cassette comprising a polynucleotide encoding a bacterial selectable marker; and(c) a bacterial plasmid origin of replication;wherein optionally the first expression cassette further comprises one or more regulatory element; andwherein optionally the regulatory element is an enhancer, an insulator, a locus control region (LCR), a matrix attachment region (MAR), a scaffold attachment region (SAR), an expression augmenting sequence element (EASE), an adenovirus tripartite leader (TPL), or a ubiquitous chromatin opening element (UCOE).73. The expression vector of claim 72 , further comprising two inverted terminal repeat (ITR) sequences flanking the first expression cassette; wherein optionally the ITR is piggyBac ITR.74. The expression vector of claim 73 , wherein(a) the IRES comprises a polynucleotide sequence of SEQ ID NO:1, 2, 3, 23, 24, or 25;(b) the eukaryotic selectable marker is a neomycin phosphotransferase, a histidinol dehydrogenase, a hygromycin B phosphotransferase, a xanthine-guanine phosphoribosyltransferase, a dihydrofolate reductase, a tryptophan synthetase, a puromycin N-acetyl-transferase, a thymidine kinase, an adenine phosphoribosyl transferase, a glutamine synthetase, an adenosine deaminase, or metallothionein-1; wherein optionally the eukaryotic selectable marker is a glutamine ...

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Номер записи: 39
21-01-2016 дата публикации

Constructs containing multiple expression cassettes for cancer therapy

Номер: US20160015834A1
Автор: Avraham Hochberg, Doron Amit
Принадлежит: Yissum Research Development Co of Hebrew University of Jerusalem

The present invention relates to the field of cancer treatment, particularly to a novel constructs useful for treating tumors expressing H19 and/or IGF-II. More specifically, the invention provides compositions and methods utilizing a nucleic acid construct enabling expression of a cytotoxic gene product directed by more than one tumor specific promoter.

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Номер записи: 40
19-01-2017 дата публикации

Chimeric nucleic acid molecules with non-aug translation initiation sequences and uses thereof

Номер: US20170016026A1
Автор: Robert Z. Florkiewicz
Принадлежит: Tapimmune Inc USA

The present disclosure relates to nucleic acid vaccine compositions and methods for preventing or treating pathological conditions, such as cancer or infectious disease. Further, the disclosure provides methods for more efficient production of antigens via mRNA containing one or more non-conventional start codons to promote multiplex initiation of translation in eukaryotic cells.

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Номер записи: 41
21-01-2016 дата публикации

STEM CELL GENE TARGETING

Номер: US20160017371A1
Автор: Fehling Hans J., Gadue Paul, Irion Stefan, KELLER Gordon, Luche Herve
Принадлежит:

The invention provides a method for generating a transgenic eukaryotic cell population having a modified human Rosa26 locus, which method includes introducing a functional DNA sequence into the human Rosa26 locus of starting eukaryotic cells. Also provided are targeting vectors useful in the method, as well as a cell population and a transgenic non-human animal comprising a modified human Rosa26 locus. Finally, the invention provides an isolated DNA sequence corresponding to the human Rosa26 locus. 120-. (canceled)21. A method for modifying a human Rosa26 gene in an isolated human stem cell , which method comprises introducing exogenous DNA into the human Rosa26 gene in an isolated human embryonic stem cell , wherein said exogenous DNA is a gene expression cassette comprising an expressible gene of interest operatively linked to a heterologous promoter , or wherein said exogenous DNA is a gene expression cassette comprising an expressible gene of interest which is introduced into the Rosa26 gene by homologous recombination such that expression of the DNA sequence is under the control of the endogenous Rosa26 promoter.22. The method of claim 21 , wherein the exogenous DNA is introduced into the human embryonic stem cell by homologous recombination with a targeting vector comprising said exogenous DNA flanked by DNA sequences homologous to the human Rosa26 gene.23. The method of claim 21 , wherein the human stem cell is selected from the group consisting of primary cells and immortalized cells.24. The method of claim 21 , wherein the gene of interest is selected from the group consisting of a gene encoding a recombinase claim 21 , a reporter and mutations and combinations thereof.25. The method of claim 21 , wherein the heterologous promoter is selected from the group consisting of a constitutive ubiquitous promoter claim 21 , a constitutive tissue specific promoter claim 21 , an inducible ubiquitous promoter and an inducible tissue specific promoter.26. The method of ...

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Номер записи: 42
16-01-2020 дата публикации

Stable gene transfer to proliferating cells

Номер: US20200016278A1
Автор: Andras Nagy, Ian Alexander, Sharon CUNNINGHAM
Принадлежит: Childrens Medical Research Institute, Mount Sinai Hospital Corp, Sydney Childrens Hospitals Network Randwick and Westmead

Provided herein are methods for facilitating or inducing stable transgene integration and expression in a proliferating cell, comprising administering to the cell (i) a recombinant AAV (rAAV) vector comprising the transgene flanked by transposon-derived inverted terminal repeat sequences, which sequences are in turn flanked by AAV-derived inverted terminal repeat regions, and (ii) a source of a transposase that recognises said transposon-derived inverted terminal repeat sequences and directs the genomic integration of the transgene into the genome of the proliferating cell. Also provide are methods and transgene delivery systems for the treatment or prevention of diseases affecting, associated with or characterised by proliferating cells.

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Номер записи: 43
21-01-2021 дата публикации

METHODS FOR DIRECTED DIFFERENTIATION OF PLURIPOTENT STEM CELLS TO IMMUNE CELLS

Номер: US20210017494A1
Автор: BRANDL Andrew J., BURTON Sarah A., MUNN Christie, RAJESH Deepika, SWANSON Bradley, VODYANYK Maksym A., WANG Wen Bo, Zhang Xin
Принадлежит: FUJIFILM Cellular Dynamics, Inc.

Provided herein are methods for the efficient in vitro differentiation of somatic cell-derived pluripotent stem cells to hematopoietic precursor cells, and the further differentiation of the hematopoietic precursor cells into immune cells of various myeloid or lymphoid lineages, particularly T cells, NK cells, and dendritic cells. The pluripotent cells may be maintained and differentiated under defined conditions; thus, the use of mouse feeder cells or serum is not required in certain embodiments for the differentiation of the hematopoietic precursor cells. 1. A method of producing immune cells comprising:(a) obtaining pluripotent stem cells (PSCs), wherein the PSCs are reprogrammed from a population of somatic cells;(b) differentiating the PSCs to hematopoietic precursor cells (HPCs); and(c) culturing the HPCs under conditions to promote immune cell differentiation, thereby producing immune cells.2. The method of claim 1 , wherein the PSCs are induced pluripotent stem cells (iPSCs).3. The method of claim 1 , wherein the PSCs are embryonic stem cells (ECSs).4. The method of claim 1 , wherein the immune cells are lymphoid cells.5. The method of claim 4 , wherein the lymphoid cells are T cells claim 4 , B cells claim 4 , and/or NK cells.6. The method of claim 1 , wherein the immune cells are myeloid cells.7. The method of claim 6 , wherein the myeloid cells are dendritic cells.8. The method of claim 1 , wherein the population of somatic cells are a population of blood cells or a population of skin cells.9. The method of claim 8 , wherein the population of blood cells comprises T cells claim 8 , B cells claim 8 , and/or NK cells.10. The method of claim 8 , wherein the population of blood cells is further defined as progenitor blood cells claim 8 , peripheral blood mononuclear cells claim 8 , or lymphoblastoid cells.11. The method of claim 1 , wherein the population of somatic cells is mammalian12. The method of claim 1 , wherein the population of somatic cells is ...

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Номер записи: 44
21-01-2021 дата публикации

Genome Editing without Nucleases

Номер: US20210017539A1
Автор: Barzel Adi, Kay Mark A.
Принадлежит:

Methods and compositions are provided for editing the genome of a cell without the use of an exogenously supplied nuclease. Aspects of the methods include contacting a cell with a targeting vector comprising nucleic acid sequence to be integrated into the target locus, where the cell is not also contacted with a nuclease. In addition, reagents, devices and kits thereof that find use in practicing the subject methods are provided. 1. A method for the targeted integration of a transgene into the genome of a cell in the absence of an exogenously provided nuclease , the method comprising:contacting a cell with a recombinant viral vector, the recombinant viral vector comprising:i. a polynucleotide comprising a first nucleic acid sequence and a second nucleic acid sequence, wherein the first nucleic acid sequence encodes the transgene; and the second nucleic acid sequence is positioned 5′ or 3′ to the first nucleic acid sequence and promotes the production of two independent gene products upon integration into the target integration site in the genome of the cell;ii. a third nucleic acid sequence positioned 5′ to the polynucleotide and comprising sequence that is substantially homologous to genomic sequence 5′ of a target integration site in the genome of the cell; andiii. a fourth nucleic acid sequence positioned 3′ of the polynucleotide and comprising sequence that is substantially homologous to genomic sequence 3′ of a target integration site in the genome of the cell;wherein the cell is not contacted with a nuclease or nucleic acid encoding a nuclease.2. The method according to claim 1 , wherein the cell is a non-dividing cell.3. The method according to claim 1 , wherein the contacting occurs in vivo.4. The method according to claim 3 , wherein the method finds use in treating a medical condition associated with a gene deficiency.5. The method according to claim 4 , wherein the medical condition is selected from the group consisting of hemophilia claim 4 , hemophilia ...

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Номер записи: 45
16-01-2020 дата публикации

COMPOSITIONS AND METHODS FOR IMPROVING PLASTID TRANSFORMATION EFFICIENCY IN HIGHER PLANTS

Номер: US20200017868A1
Автор: Maliga Pal
Принадлежит:

Compositions and methods for improving plastid transformation in difficult to transform plants are disclosed. 1. A method for increasing sensitivity to spectinomycin in plastids of higher plants for increasing plastid transformation efficiency , comprising;a) providing a plant comprising a nonfunctional ACC2 nuclear gene;b) introducing one or more plastid transformation vectors into the plastids in cells from said plant, said one or more vectors comprising an aadA spectinomycin resistance marker sequence and a nucleic acid sequence encoding a protein of interest;c) contacting said cells with spectinomycin and selecting plant cells which are resistant to spectinomycin and accumulate said protein of interest in said plastids; andd) culturing said plant cells under conditions suitable to regenerate a transplastomic plant therefrom.2ArabidopsisBrassicaCamelina. The method of claim 1 , wherein said plant is selected from the group consisting of ssp. claim 1 , ssp. claim 1 , and ssp.3. The method of claim 1 , wherein said protein of interest is green fluorescent protein.4. The method of claim 1 , wherein the plant of step a) is a naturally occurring mutant which encodes non-functional or defective ACC2.5. The method of claim 1 , wherein said ACC2 gene is inactivated in said plant using CRISPR/Cas prior to plastid transformation.6Brassica. The method of claim 5 , wherein said plant is a ssp. plant.7Camelina. The method of claim 5 , wherein said plant is a ssp. plant.8. The method of claim 1 , further comprising excising said aadA spectinomycin resistance marker sequence from said plant.9. The method of claim 5 , wherein said protein of interest is selected from the group consisting of a protein conferring herbicide resistance claim 5 , a protein conferring insect resistance claim 5 , a vaccine claim 5 , an antibody claim 5 , regulatory RNA claim 5 , dsRNA claim 5 , siRNA claim 5 , shRNA and insecticidal proteins.10. A method for seed-specific plastid expression comprising: ...

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Номер записи: 46
17-04-2014 дата публикации

Selection of host cells expressing protein at high levels

Номер: US20140106401A1
Автор: Arie Pieter Otte, Henricus J.M. van Blokland, Richard George Antonius Bernardus Sewalt, Theodorus Hendrikus Jacobus Kwaks
Принадлежит: Chromagenics BV

Described is a DNA molecule comprising an open reading frame sequence encoding a selectable marker polypeptide, wherein the DNA molecule in the coding strand comprises a translation start sequence for the selectable marker polypeptide having a GTG or TTG start codon, and wherein the ORF sequence that encodes the selectable marker protein has been mutated to replace at least half of its CpG dinucleotides as compared to the native ORF sequence that encodes the selectable marker protein. Further provided are such DNA molecules wherein the ORF sequence that encodes a selectable marker polypeptide is part of a multicistronic transcription unit that further comprises an open reading frame sequence encoding a polypeptide of interest. Also described are methods for obtaining host cells expressing a polypeptide of interest, wherein the host cells comprise the DNA molecules described herein. Further provided is the production of polypeptides of interest, comprising culturing host cells comprising the DNA molecules described herein.

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Номер записи: 47
22-01-2015 дата публикации

Alphavirus vectors for respiratory pathogen vaccines

Номер: US20150024002A1
Автор: Catherine Greer, John Polo, Silvia Perri, Yasushi Uematsu
Принадлежит: Novartis Vaccines and Diagnostics Inc

Described herein are compositions and methods for stimulating an immune response to one or more proteins derived from one or more respiratory pathogens. In particular, the invention relates to alphavirus replicons, alphavirus vector constructs, alphavirus replicon particles expressing one or more antigens derived from one or more respiratory pathogens as well as to method of making and using these immunogenic compositions.

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Номер записи: 48
10-02-2022 дата публикации

Compositions and methods for treating non-age-associated hearing impairment in a human subject

Номер: US20220040327A1
Автор: Ellen Reisinger, Emmanuel John Simons, Hanan Al-Moyed, Robert NG, Sebastian KÜGLER
Принадлежит: Akouos Inc

Provided herein are compositions that include at least two different nucleic acid vectors, where each of the at least two different vectors includes a coding sequence that encodes a different portion of an otoferlin protein, and the use of these compositions to treat hearing loss in a subject.

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Номер записи: 49
24-01-2019 дата публикации

PLASMID ADDICTION SYSTEM TO DRIVE DESIRED GENE EXPRESSION

Номер: US20190024097A1
Автор: Clarkson Sonya, Kim Daniel, Mattozzi Matthew de la Pena
Принадлежит: Conagen Inc.

The present invention relates to a Plasmid Addiction System for the stabilization of expression plasmids encoding proteins of interest. The invention uses a succinate cycle optimization to ensure the expression of plasmid(s) of interest. By ensuring that plasmids of interest contain genes necessary in the succinate cycle, the system ensures that the passage of the plasmid to daughters and therefore improves the efficiency of production and expression of genes and/or products of interest. 1. A transformed bacterial host cell containing:i) an extrachromosomal DNA sequence encoding at least one protein of interest, the expression of which is regulated and operably associated with at least one extrachromosomal element; and,ii) a DNA sequence encoding at least one necessary succinate pathway gene that has been removed from said bacterial host cell;iii) where such sequences are complementary to a DNA sequence of interest contained in a plasmid; and,iv) where both DNA sequences are positioned upstream or downstream of the ribosomal binding site of the DNA sequence.2. The bacterial cell of claim 1 , wherein said DNA sequence is foreign to said cell.3. The bacterial cell of claim 1 , wherein said extrachromosomal DNA sequence encodes more than one polypeptide of interest.4. The bacterial cell of claim 1 , wherein said extrachromosomal DNA sequence encodes more than one essential succinate pathway gene each of which is essential to the central metabolism of said cell.5. The bacterial cell of claim 1 , wherein said foreign DNA sequence is under the control of a promoter.6. The bacterial cell of claim 1 , wherein said extrachromosomal element is a plasmid.7. A plasmid of claim 6 , wherein the origin of replication is derived from pBR322 claim 6 , pMB1 claim 6 , ColE1 claim 6 , pSC101 or p15A.8. A method of maintaining two or more plasmids in a transformed microbial host cell comprising the bacterial cell of claim 1 , wherein each plasmid utilized contains at least one of the ...

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Номер записи: 50
24-01-2019 дата публикации

VECTOR

Номер: US20190024116A1
Автор: Kokaia Merab
Принадлежит:

The present invention relates to a recombinant adeno-associated viral (r AAV) vector comprising neuropeptide Y (NPY) coding sequence and neuropeptide Y2 receptor (NPY2R) coding sequence. The invention further relates to a AAV particle comprising said vector, wherein the vector is encapsulated by adeno-associated virus (AAV) capsid proteins. Also, a pharmaceutical composition comprising said AAV particle, for use in the prevention or treatment of a neurological disorder in mammals, such as epilepsy. 1. A recombinant adeno-associated viral (rAAV) vector comprising a neuropeptide Y (NPY) coding sequence and a neuropeptide Y2 receptor (NPY2R) coding sequence.247-. (canceled)48. The vector according to claim 1 , wherein said neuropeptide Y (NPY) coding sequence comprises a sequence corresponding to SEQ ID NO:1 or a sequence having at least 90% sequence identity to SEQ ID NO:1.49. The vector according to claim 1 , wherein said neuropeptide Y2 receptor (NPY2R) coding sequence comprises a sequence corresponding to SEQ ID NO:2 or a sequence having at least 90% sequence to SEQ ID NO:2.50. The vector according to claim 1 , wherein the vector further comprises at least one of the functional elements of AAV2 Inverted Terminal Repeat sequences (ITR) claim 1 , hybrid cytomegalovirus enhancer/chicken beta-actin CAG promoter (CAG) claim 1 , internal ribosome entry site (IRES) claim 1 , woodchuck hepatitis post-translational regulatory element (WPRE) claim 1 , or bovine growth hormone polyadenylation (bGH-polyA) signal sequence.51. The vector according to claim 50 , wherein the vector comprises a hybrid cytomegalovirus enhancer/chicken beta-actin CAG promoter (CAG) and said CAG promoter sequence is located upstream of the coding sequences for NPY and NPY2R.52. The vector according to claim 50 , wherein the vector comprises an internal ribosome entry site (IRES) and said IRES sequence is located between the coding sequences for NPY and NPY2R.53. The vector according to claim 50 , ...

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Номер записи: 51
24-01-2019 дата публикации

TISSUE SELECTIVE TRANSGENE EXPRESSION

Номер: US20190024119A1
Автор: Chen Szu-Ying, OBERKOFLER David, RAMAMOORTHI Kartik, TAGLIATELA Stephanie, YOUNG Andrew
Принадлежит:

Provided herein are compositions and methods for selective expression of a transgene. Compositions and methods for selective expression of a transgene comprise one or more human regulatory elements, which, when operably linked to a transgene, can facilitate selective expression of a transgene (e.g., cell-type selective expression) in a target cell as compared to at least one or more non-target cells. 1. A nucleic acid cassette comprising a regulatory element comprising SEQ ID NO: 8 operably linked to a transgene.2. The nucleic acid cassette of claim 1 , wherein the transgene comprises a nucleic acid sequence encoding a DNA binding protein.3. The nucleic acid cassette of claim 2 , wherein the DNA binding protein is a transcriptional activator that modulates an endogenous SCN1A gene.4. The nucleic acid cassette of claim 1 , wherein the nucleic acid cassette is an adeno-associated virus (AAV) vector.5. The nucleic acid cassette of claim 4 , wherein the AAV vector is AAV9 or scAAV9.6. The nucleic acid cassette of claim 3 , wherein the nucleic acid cassette is an adeno-associated virus (AAV) vector.7. The nucleic acid cassette of claim 6 , wherein the AAV vector is AAV9 or scAAV9.8. The nucleic acid cassette of claim 1 , wherein the regulatory element comprises SEQ ID NO: 32.9. The nucleic acid cassette of claim 8 , wherein the transgene comprises a nucleic acid sequence encoding a DNA binding protein.10. The nucleic acid cassette of claim 9 , wherein the DNA binding protein is a transcriptional activator that modulates an endogenous SCN1A gene.11. The nucleic acid cassette of claim 8 , wherein the nucleic acid cassette is an adeno-associated virus (AAV) vector.12. The nucleic acid cassette of claim 11 , wherein the AAV vector is AAV9 or scAAV9.13. The nucleic acid cassette of claim 10 , wherein the nucleic acid cassette is an adeno-associated virus (AAV) vector.14. The nucleic acid cassette of claim 13 , wherein the AAV vector is AAV9.15. The nucleic acid cassette of ...

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Номер записи: 52
24-01-2019 дата публикации

TISSUE SELECTIVE TRANSGENE EXPRESSION

Номер: US20190024120A1
Автор: Chen Szu-Ying, OBERKOFLER David, RAMAMOORTHI Kartik, TAGLIATELA Stephanie, YOUNG Andrew
Принадлежит:

Provided herein are compositions and methods for selective expression of a transgene. Compositions and methods for selective expression of a transgene comprise one or more human regulatory elements, which, when operably linked to a transgene, can facilitate selective expression of a transgene (e.g., cell-type selective expression) in a target cell as compared to at least one or more non-target cells. 1. A nucleic acid cassette comprising a regulatory element comprising SEQ ID NO: 30 operably linked to a transgene.2. The nucleic acid cassette of claim 1 , wherein the transgene comprises a nucleic acid sequence encoding a DNA binding protein.3. The nucleic acid cassette of claim 2 , wherein the DNA binding protein is a transcriptional modulator of an endogenous gene.4. The nucleic acid cassette of claim 2 , wherein the DNA binding protein comprises a zinc finger.5. The nucleic acid cassette of claim 2 , wherein the DNA binding protein is a nuclease-deactivated zinc finger protein.6. The nucleic acid cassette of claim 2 , wherein the DNA binding protein is linked to a transcriptional activator domain.7. The nucleic acid cassette of claim 2 , wherein the DNA binding protein is linked to a transcriptional repressor domain.8. The nucleic acid cassette of claim 2 , wherein the DNA binding protein is a transcriptional activator that modulates an endogenous SCN1A gene.9. The nucleic acid cassette of claim 1 , wherein the nucleic acid cassette is an adeno-associated virus (AAV) vector.10. The nucleic acid cassette of claim 9 , wherein the AAV vector is AAV9 or scAAV9.11. The nucleic acid cassette of claim 8 , wherein the nucleic acid cassette is an adeno-associated virus (AAV) vector.12. The nucleic acid cassette of claim 11 , wherein the AAV vector is AAV9.13. The nucleic acid cassette of claim 11 , wherein the AAV vector is scAAV9.14. A nucleic acid cassette comprising a regulatory element comprising a sequence with at least 95% sequence identity to SEQ ID NO: 30 operably ...

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Номер записи: 53
24-01-2019 дата публикации

TISSUE SELECTIVE TRANSGENE EXPRESSION

Номер: US20190024121A1
Автор: Chen Szu-Ying, OBERKOFLER David, RAMAMOORTHI Kartik, TAGLIATELA Stephanie, YOUNG Andrew
Принадлежит:

Provided herein are compositions and methods for selective expression of a transgene. Compositions and methods for selective expression of a transgene comprise one or more human regulatory elements, which, when operably linked to a transgene, can facilitate selective expression of a transgene (e.g., cell-type selective expression) in a target cell as compared to at least one or more non-target cells. 1. A viral vector comprising a regulatory element operably linked to a transgene , wherein the regulatory element results in selective expression in the CNS such that at least 40% of CNS cells expressing the transgene are parvalbumin (PV) neurons.2. The viral vector of claim 1 , wherein at least 50% of CNS cells expressing the transgene are PV neurons.3. The viral vector of claim 2 , wherein at least 60% of CNS cells expressing the transgene are PV neurons.4. The viral vector of claim 3 , wherein at least 65% of CNS cells expressing the transgene are PV neurons.5. The viral vector of claim 4 , wherein at least 70% of CNS cells expressing the transgene are PV neurons.6. The viral vector of claim 1 , wherein the transgene comprises a nucleic acid sequence encoding a DNA binding protein.7. The viral vector of claim 6 , wherein the DNA binding protein is a transcriptional modulator of an endogenous gene.8. The viral vector of claim 6 , wherein the DNA binding protein comprises a zinc finger.9. The viral vector of claim 6 , wherein the DNA binding protein is linked to a transcriptional activator domain.10. The viral vector of claim 6 , wherein the DNA binding protein is linked to a transcriptional repressor domain.11. The viral vector of claim 6 , wherein the DNA binding protein is a transcriptional activator that modulates an endogenous SCN1A gene.12. The viral vector of claim 1 , wherein the transgene comprises a gene editing protein.13. The viral vector of claim 12 , wherein the gene editing protein is a Cas protein.14. The viral vector of claim 1 , wherein the regulatory ...

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Номер записи: 54
28-01-2021 дата публикации

EXPRESSION VECTOR ELEMENT COMBINATIONS, NOVEL PRODUCTION CELL GENERATION METHODS AND THEIR USE FOR THE RECOMBINANT PRODUCTION OF POLYPEPTIDES

Номер: US20210024952A1
Автор: Huelsmann Peter Michael, Knoetgen Hendrik
Принадлежит:

Herein is reported that for transient transfections the use of the human elongation factor 1 alpha promoter (with Intron A) provides for an enhanced productivity (in LC-HC-SM organization), the use of the bovine growth hormone polyA signal sequence provides for an enhanced productivity compared to use of the SV40 polyA signal sequence, the addition of the HUT to the bGH PolyA signal sequence results in an increased productivity in vectors containing the hCMV promoter and the vector organization LC(3′-5′)-HC-SM results in improved expression. For stable pools it is reported that pools generated with vectors containing the hEFα promoter show an enhanced productivity in batch analysis, clones generated with vectors containing the hEF1α promoter show a reduced number of low producing clones, and clones generated with vectors containing the hEF1α promoter show a higher stability of IgG expression. For single clones it is reported that the vector organization with downstream position of selection marker (LC-HC-SM) has a positive effect on productivity of single clones and that clones generated with vectors containing the bGH polyA signal sequence and the hGT have higher productivities. 1108-. (canceled)109. A method for selecting a recombinant mammalian cell , comprising the steps of: a first expression cassette comprising in 5′ to 3′ direction a hCMV promoter, a nucleic acid encoding an antibody light chain, a bGH polyA signal sequence, and a hGT terminator sequence,', 'a second expression cassette comprising in 5′ to 3′ direction a hCMV promoter, a nucleic acid encoding an antibody heavy chain, a bGH polyA signal sequence, and a hGT terminator sequence, and', 'and thereby obtaining a multitude of recombinant mammalian cells, and, 'a) transfecting a mammalian cell with an expression vector comprising'}b) selecting from the multitude of recombinant mammalian cells a (single) recombinant mammalian cell.110. The method according to claim 109 , wherein the nucleic acid ...

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Номер записи: 55
02-02-2017 дата публикации

Modified polynucleotides for the production of nuclear proteins

Номер: US20170028085A1
Автор: Antonin De Fougerolles, Atanu Roy, Jason P. SCHRUM, Jeff Lynn Ellsworth, Justin Guild, Kenechi Ejebe, Kristy M. WOOD, Matthias John, Paul Hatala, Ron Weiss, Sayda M. ELBASHIR, Stephane Bancel, Susan Whoriskey, Tirtha Chakraborty
Принадлежит: ModernaTx Inc

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules.

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Номер записи: 56
31-01-2019 дата публикации

Affinity maturated t cell receptors and use thereof

Номер: US20190031733A1
Автор: Esther Tzehoval, Rachel Lea Eisenbach, Yosi GOZLAN
Принадлежит: Yeda Research and Development Co Ltd

The present invention relates to methods and systems for increasing the affinity of a T cell receptor (TCR) to its ligand by subjecting the TCR gene to somatic hypermutation. The present invention further relates to use of affinity maturated TCRs to create T cells reactive against a selected antigen.

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Номер записи: 57
31-01-2019 дата публикации

COMPOSITIONS COMPRISING AAV EXPRESSING DUAL ANTIBODY CONSTRUCTS AND USES THEREOF

Номер: US20190031740A1
Автор: Tretiakova Anna, Wilson James M.
Принадлежит:

A recombinant adeno-associated virus (AAV) having an AAV capsid and packaged therein a heterologous nucleic acid which expresses two functional antibody constructs in a cell is described. Also described are antibodies comprising a heavy chain and a light chain from a heterologous antibody. In one embodiment, the antibodies are co-expressed from a vector containing: a first expression cassette which encodes at least a first open reading frame (ORF) for a first immunoglobulin under the control of regulatory control sequences which direct expression thereof; and a second expression cassette which comprises a second ORF, a linker, and a third ORF under the control of regulatory control sequences which direct expression thereof, wherein the second and third ORF for a second and third immunoglobulin construct. The vector co-expressing these two antibody constructs is in one embodiment an AAV, in which the 5′ and 3′ ITRs flank the expression cassettes and regulatory sequences. 1. A method of delivering at least two functional antibodies to a subject , said method comprising administering a recombinant adeno-associated virus (AAV) having an AAV capsid and packaged therein a heterologous nucleic acid which expresses at least two functional monospecific antibodies in a cell , wherein the recombinant AAV expresses a first monoclonal antibody having a first specificity , a second monoclonal antibody having a specificity different from the first monoclonal antibody , and a bifunctional antibody , and wherein the recombinant AAV comprises:a 5′ AAV inverted terminal repeat (ITR);a first expression cassette which encodes at least a first open reading frame (ORF) for a first immunoglobulin under the control of regulatory control sequences which direct expression thereof;a second expression cassette which comprises a second ORF, a linker, and a third ORF under the control of regulatory control sequences which direct expression thereof, wherein the second and third ORF are for a ...

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Номер записи: 58
30-01-2020 дата публикации

CONDITIONALLY IMMORTALIZED CELLS AND METHODS FOR THEIR PREPARATION

Номер: US20200032213A1
Автор: de Vries Antonius Andrianus Franciscus, Pijnappels Daniel Antonie, Schaly Martin Jan
Принадлежит:

The present disclosure relates to genetically modified conditionally immortalized cells and cell lines, provided with a vector comprising a promoter sequence, at least one immortalization gene operably linked to the promoter sequence, a gene coding for an inducible regulator operably linked to the promoter sequence and a response element for the inducible regulator. The present disclosure further relates to methods for their preparation, and to immortalization and differentiation of such cells. 1. A genetically modified eukaryotic cell provided with a vector , the vector comprising:a promoter;at least one immortalization gene operably linked to the promoter;a gene coding for an inducible regulator operably linked to the promoter; anda response element for the inducible regulator.2. The cell according to wherein the promoter is a cell type- or tissue-specific promoter and/or a promoter that is active in a differentiated form of the cell.3. The cell according to claim 1 , wherein the cell is a primary cell or progeny thereof.4. The cell according to of claim 1 , wherein the at least one immortalization gene comprises a simian virus 40 large T antigen (LT) gene or a variant thereof.5. The cell of claim 1 , wherein the inducible regulator is a tetracycline (Tc)-responsive transcriptional repressor claim 1 , and the response element is a Tc response element (TRE).6. The cell of claim 1 , wherein the cell is a cardiac cell and the promoter is a differentiated cardiac cell-specific promoter.7. The cell of wherein said cell is a cardiac cell.8. The cell claim 1 , wherein the promoter is the chimeric striated muscle-specific MHCK7 promoter.9. A cell line comprising a plurality of cells according to .10. A kit of parts comprising:{'claim-ref': {'@idref': 'CLM-00001', 'claim 1'}, 'the cell of or cell line thereof, and'}an agent capable of activating or repressing the in promoter.11. The kit of parts according to wherein the promoter is a Tc-inducible promoter and the agent is ...

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Номер записи: 59
04-02-2021 дата публикации

MODIFIED 5'-UNTRANSLATED REGION (UTR) SEQUENCES FOR INCREASED PROTEIN PRODUCTION IN BACILLUS

Номер: US20210032639A1
Автор: DE LANGE Dennis, FRISCH Ryan L., Masson Helen Olivia
Принадлежит: DANISCO US INC.

The present disclosure is generally modified strains and host cells thereof capable of producing increased amounts of industrially relevant proteins of interest. Other embodiments of the disclosure are related to isolated polynucleotides comprising modified aprE 5′-untranslated region (5′-UTR) nucleic acid sequences, vectors thereof, DNA (expression) constructs thereof, modified (daughter) cells thereof, and methods of making and using the same. 1Bacillus subtilisBacillus subtilis. An isolated polynucleotide comprising a modified aprE 5′-untranslated region (mod-5′-UTR) nucleic acid sequence derived from a wild-type aprE 5′-untranslated region (WT-5′-UTR) nucleic acid sequence SEQ ID NO: 1.2. The polynucleotide of claim 1 , wherein the mod-5′-UTR comprises SEQ ID NO: 2.3. The polynucleotide of claim 1 , wherein the mod-5′-UTR further comprises an upstream (5′) promoter region nucleic acid sequence 5′ and operably linked to the mod-5′-UTR.4. The polynucleotide of claim 1 , wherein the mod-5′-UTR further comprises a downstream (3′) open reading frame (ORF) nucleic acid sequence encoding a protein of interest claim 1 , wherein the ORF sequence is 3′ and operably linked to the mod-5′-UTR.5. The polynucleotide of claim 1 , comprising Formula (I) in the 5′ to 3′ direction:{'br': None, '[Pro][mod-5′-UTR][ORF]; \u2003\u2003(I){'i': 'B. subtilis', 'wherein [Pro] is a promoter region nucleic acid sequence operable in a Bacillus sp. cell, [mod-5′-UTR] is a modified aprE 5′ untranslated region (mod-5′-UTR) nucleic acid sequence and [ORF] is an open reading frame nucleic acid sequence encoding a protein of interest (POI), wherein the [Pro], [mod-5′-UTR] and [ORF] nucleic acid sequences are operably linked.'}6. A vector comprising the polynucleotide of .7. A DNA expression construct comprising the polynucleotide of .8Bacillus. A sp. cell comprising the polynucleotide of .9BacillusBacillus licheniformis. The sp. cell of claim 8 , wherein the cell is a cell.10BacillusBacillus. An ...

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Номер записи: 60
04-02-2021 дата публикации

RNA-GUIDED GENE EDITING AND GENE REGULATION

Номер: US20210032654A1
Автор: Black Joshua B., Gersbach Charles A., Hilton Isaac B., Kabadi Ami M., Ousterout David G., Perez-Pinera Pablo, Thakore Pratiksha I.
Принадлежит:

Disclosed herein are Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) 9-based system related compositions and methods of using said CRISPR/Cas9-based system related compositions for altering gene expression and genome engineering. Also disclosed herein are compositions and methods of using said compositions for altering gene expression and genome engineering in muscle, such as skeletal muscle and cardiac muscle. 120-. (canceled)21. A fusion protein comprising two heterologous polypeptide domains , wherein the first polypeptide domain comprises a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein , wherein the Cas protein comprises a Cas9 , and wherein the second polypeptide domain comprises a p300 histone acetyltransferase effector domain.22. The fusion protein of claim 21 , wherein the Cas9 comprises at least one amino acid mutation that inactivates the nuclease activity of Cas9.23. The fusion protein of claim 22 , wherein the at least one amino acid mutation is at least one of D10A and H840A.24. The fusion protein of claim 22 , wherein the Cas9 comprises the mutations D10A and H840A.25Streptococcus pyogenes.. The fusion protein of claim 21 , wherein the Cas protein is derived from26Neisseria meningitidis.. The fusion protein of claim 21 , wherein the Cas protein is derived from27. The fusion protein of claim 24 , wherein the Cas protein comprises dCas9 (amino acids 36-1403 of SEQ ID NO: 1).28. The fusion protein of claim 21 , wherein the p300 histone acetyltransferase effector domain comprises a wild-type human p300 protein or a mutant human p300 protein claim 21 , or fragments thereof.29. The fusion protein of claim 21 , wherein the p300 histone acetyltransferase effector domain comprises the core lysine-acetyltranserase domain of the human p300 protein.30Streptococcus pyogenes. The fusion protein of claim 21 , wherein the first polypeptide domain comprises a Cas9 derived from and wherein ...

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Номер записи: 61
05-02-2015 дата публикации

Methods of Modulating Smooth Muscle Cell Proliferation and Differentiation

Номер: US20150037290A1
Автор: Cordes Kimberly R., Srivastava Deepak
Принадлежит: THE J. DAVID GLADSTONE INSTITUTES

The present disclosure provides methods of inducing smooth muscle cell differentiation. The present disclosure provides genetically modified cells comprising exogenous miR-143 and/or miR-145 nucleic acids; and artificial tissues comprising the genetically modified cells. The present disclosure provides methods and compositions for reducing pathological angiogenesis. The present disclosure provides methods of inducing therapeutic angiogenesis. The present disclosure provides methods, compositions, and devices for inhibiting vascular smooth muscle cell proliferation.

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Номер записи: 62
11-02-2016 дата публикации

Dna construct and method for transgene expression

Номер: US20160040186A1
Автор: Xiaoyun Liu
Принадлежит: Individual

This invention relates to a DNA construct that is capable of expressing a desired transgene in a trackable manner. The construct comprises in 5′ to 3′ downstream direction: a promoter; a fluorescent reporter gene positioned within an intron defined by a 5′-donor splice site comprising a splice donor sequence and a 3′-acceptor splice site comprising a splice acceptor sequence; a desired gene and a transcription terminator. A method of producing a transgenic host cell having a desired product is also disclosed.

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Номер записи: 63
09-02-2017 дата публикации

In vivo Gene Engineering with Adenoviral Vectors

Номер: US20170037431A1
Автор: Lieber Andre, PAPAYANNOPOULOU Thalia, Richter Maximilian, SAYDAMINOVA Kamola
Принадлежит:

The present invention provides recombinant nucleic acid expression cassetie and helper dependent adenovirus, where the expression cassettes utilize a miRNA based system for controlling expression of nucleases in helper dependent adenoviral viral producer cells, thus permitting production and use for in in vivo gene editing in CD34+ cells. 1. A recombinant nucleic acid expression cassette , comprising at least one first nucleic acid module comprising(i) a first coding region encoding a nuclease capable of generating a DNA break in a CD34+ cell genomic target of interest; and(ii) a second coding region encoding one or more miRNA target sites located in a 3′ untranslated region of the first coding region and at least 60 nucleotides downstream of a translation al stop codon of the first coding region, wherein miRNAs that bind to the one or more encoded miRNA target sites are highly expressed in virus producer cells but not expressed, or expressed at low levels, in CD34+ cells,wherein the first nucleic acid module is operatively linked to a promoter that is active in CD34+ cells.2. The recombinant nucleic acid expression cassette of claim 1 , further comprising a second nucleic acid module encoding a CD46 binding adenoviral fiber polypeptide.3. The recombinant nucleic acid expression cassette of claim 1 , further comprising an inverted terminal repeat (ITR) at each terminus of the recombinant nucleic acid vector claim 1 , wherein the ITR derived from a CD46-binding adenovirus serotype.4. The recombinant nucleic acid expression cassette of claim 1 , further comprising a packaging signal from a CD46-binding adenovirus serotype.5. The recombinant nucleic acid expression cassette of claim 1 , wherein the one or more the miRNA target site comprise a reverse complement of one claim 1 , two claim 1 , or all three miRNA selected from the group consisting of (a) CACUGGUAGA (SEQ ID NO: 1) (has-miR183-5p core) claim 1 , (b) UGUGCUUGAUCUAA (SEQ ID NO: 2) (has-miR218-5p core); and (c ...

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Номер записи: 64
24-02-2022 дата публикации

Compositions and Methods for Generating a Persisting Population of T Cells Useful for the Treatment of Cancer

Номер: US20220056116A1
Автор: Frigault Matthew J., June Carl H., Scholler John, Zhao Yangbing
Принадлежит:

The present invention provides compositions and methods for generating a genetically modified T cells comprising a chimeric antigen receptor (CAR) having an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a CD3 zeta signaling domain, wherein the T cell exhibits prolonged exponential expansion in culture that is ligand independent and independent of the addition of exogenous cytokines or feeder cells. 1. A nucleic acid comprising a sequence encoding a chimeric antigen receptor (CAR) , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 2 or 3.25.-. (canceled)6. The nucleic acid of claim 1 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 2.7. (canceled)8. The nucleic acid of claim 1 , wherein the CAR comprises the amino acid sequence of SEQ ID NO: 3.9. (canceled)10. A T cell comprising the nucleic acid of .1119.-. (canceled)20. A vector comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) claim 1 , the CAR comprising an antigen binding domain claim 1 , a hinge domain claim 1 , a transmembrane domain claim 1 , a costimulatory signaling region claim 1 , and a CD3 zeta signaling domain claim 1 , and wherein when the vector is transduced into a T cell claim 1 , the CAR expressed by the vector contributes to at least one of: increased antigen-independent activation of the transduced T cell claim 1 , increased mean cell volume (MCV) of the transduced T cell claim 1 , increased cell population expansion of the transduced T cell claim 1 , increased proliferation of the transduced T cell claim 1 , increased numbers of progeny of the transduced T cell claim 1 , increased persistence of the transduced T cell population in vitro claim 1 , or increased persistence of the transduced T cell population in vivo.21. The vector of claim 20 , wherein the hinge domain is an IgG4 hinge domain.22. The vector of claim 20 , wherein the antigen binding domain is an anti-cMet binding domain claim 20 , ...

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Номер записи: 65
24-02-2022 дата публикации

CHIMERIC NUCLEIC ACID MOLECULES WITH NON-AUG TRANSLATION INITIATION SEQUENCES AND USES THEREOF

Номер: US20220056473A1
Автор: Florkiewicz Robert Z.
Принадлежит:

The present disclosure relates to nucleic acid vaccine compositions and methods for preventing or treating pathological conditions, such as cancer or infectious disease. Further, the disclosure provides methods for more efficient production of antigens via mRNA containing one or more non-conventional start codons to promote multiplex initiation of translation in eukaryotic cells. 130.-. (canceled)31. A T cell comprising a chimeric nucleic acid molecule comprising a multiplex translation initiation (MTI) sequence comprising from two to about five translation initiation sites operatively linked in frame to a nucleic acid molecule encoding a polypeptide comprising one or more cytokine , wherein at least one of the MTI translation initiation sites is a non-AUG translation initiation site and the MTI allows the production of more than one mole of polypeptide per mole of mRNA ,wherein the chimeric nucleic acid molecule is an mRNA molecule or an mRNA molecule contained in a vector and operably linked to an expression control sequence.32. The T cell of claim 31 , wherein the MTI comprises one claim 31 , two claim 31 , three claim 31 , or four non-AUG translation initiation sites.33. The T cell of claim 32 , wherein the non-AUG translation initiation sites are CUG translation initiation sites.34. The T cell of claim 33 , wherein the MTI comprises an AUG translation initiation site downstream of the CUG translation initiation sites.35. The T cell of claim 31 , wherein the MTI comprises (a) a nucleic acid molecule encoding one or two nuclear localization domains located downstream of two or three CUG translation initiation sites and upstream of an AUG translation initiation site claim 31 , or (b) two or three CUG translation initiation sites upstream of a nucleic acid molecule encoding one or two nuclear localization domains and no AUG translation initiation site.36. The T cell of claim 31 , wherein the MTI comprises a 5′-portion of a human FGF2 gene claim 31 , wherein the 5′- ...

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Номер записи: 66
06-02-2020 дата публикации

CATECHOLAMINE ENZYME FUSIONS

Номер: US20200040361A1
Автор: Kingsman Alan John, Mitrophanous Kyriacos A., Ralph Scott, STEWART Hannah
Принадлежит:

Provided is a construct comprising (i) a nucleotide sequence which encodes tyrosine hydroxylase (TH), (ii) a nucleotide sequence which encodes GTP-cyclohydrolase I (CH1) and (iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC) wherein the nucleotide sequence encoding TH is linked to the nucleotide sequence encoding CH1 such that they encode a fusion protein TH-CH1. Also provided is a construct comprising (i) a nucleotide sequence which encodes tyrosine hydroxylase (TH), (ii) a nucleotide sequence which encodes GTP-cyclohydrolase I (CH1) and (iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC) wherein the nucleotide sequence encoding AADC is linked to the nucleotide sequence encoding TH such that they encode a fusion protein AADC-TH or TH-AADC. Further provided is a viral vector comprising such nucleotide sequences and its use in the treatment and/or prevention of Parkinson's disease. 124-. (canceled)25. A nucleic acid construct comprising:(i) a nucleotide sequence which encodes tyrosine hydroxylase (TH),(ii) a nucleotide sequence which encodes GTP-cyclohydrolase I (CH1), and(iii) a nucleotide sequence which encodes Aromatic Amino Acid Dopa Decarboxylase (AADC);{'sub': L1', 'L2', 'L1', 'L2, 'wherein the nucleic acid construct comprises AADC--TH--CH1 or TH--AADC--CH1, wherein L1 and L2 are linker-encoding sequences.'}26. The nucleic acid construct according to claim 25 , wherein L1 and L2 have the same nucleic acid sequence.27. The nucleic acid construct according to claim 25 , wherein L1 comprises a nucleic acid sequence as set forth in SEQ ID No. 1 or in SEQ ID No. 3.28. The nucleic acid construct according to claim 25 , wherein L2 a comprises a nucleic acid sequence as set forth in SEQ ID No. 1 or in SEQ ID No. 3.29. The nucleic acid construct according to claim 25 , wherein L1 and L2 encode the same amino acid sequence.30. The nucleic acid construct according to claim 25 , wherein either or both ...

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Номер записи: 67
22-02-2018 дата публикации

MESSENGER UNA MOLECULES AND USES THEREOF

Номер: US20180051262A1
Автор: Chivukula Padmanabh, Payne Joseph E., TACHIKAWA Kiyoshi, WARREN Luigi
Принадлежит:

This invention provides a range of translatable messenger UNA (mUNA) molecules. The mUNA molecules can be translated in vitro and in vivo to provide an active polypeptide or protein, or to provide an immunization agent or vaccine component. The mUNA molecules can be used as an active agent to express an active polypeptide or protein in cells or subjects. Among other things, the mUNA molecules are useful in methods for treating rare diseases. 1. A mUNA molecule , comprising one or more UNA monomers , and comprising nucleic acid monomers , wherein the mUNA molecule is translatable to express a polypeptide or protein.2. The molecule of claim 1 , wherein the molecule comprises from 200 to 12 claim 1 ,000 monomers.3. The molecule of claim 1 , wherein the molecule comprises from 200 to 4 claim 1 ,000 monomers.4. The molecule of claim 1 , wherein the molecule comprises from 1 to 8 claim 1 ,000 UNA monomers.5. The molecule of claim 1 , wherein the molecule comprises from 1 to 100 UNA monomers.6. The molecule of claim 1 , wherein the molecule comprises from 1 to 20 UNA monomers.7. The molecule of claim 1 , wherein the molecule comprises one or more modified nucleic acid nucleotides claim 1 , or one or more chemically-modified nucleic acid nucleotides.8. The molecule of claim 1 , wherein the molecule comprises a 5′ cap claim 1 , a 5′ untranslated region of monomers claim 1 , a coding region of monomers claim 1 , a 3′ untranslated region of monomers claim 1 , and a tail region of monomers.9. The molecule of claim 8 , wherein the molecule comprises a translation enhancer in a 5′ or 3′ untranslated region.10. The molecule of claim 1 , wherein the molecule is translatable in vivo.11. The molecule of claim 1 , wherein the molecule is translatable in vitro.12. The molecule of claim 1 , wherein the molecule is translatable in a mammalian cell.13. The molecule of claim 1 , wherein the molecule is translatable in a human in vivo.14. The molecule of claim 1 , wherein a translation ...

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Номер записи: 68
23-02-2017 дата публикации

EVOLUTION OF HIGH-TITER VIRUS-LIKE VESICLES FOR VACCINE APPLICATIONS

Номер: US20170051307A1
Автор: Rose John, ROSE Nina
Принадлежит:

The present invention relates to the discovery of a high titer hybrid-virus vector that gives rise to high titer virus like vesicles (VLVs) that can be used as a vaccine. The invention includes compositions and methods of generating an evolved hybrid-virus vector vaccine and selecting high titer VLVs, methods of treating and/or preventing or immunizing against, a specific disease or disorder, and methods of inducing a memory T cell and B cell immune response in a subject administered the VLV composition produced thereby. Furthermore, the invention encompasses a pharmaceutical composition for vaccinating a subject as well as a high titer protein expression system. 1. A high titer hybrid-virus vector comprising a DNA sequence comprising a promoter sequence operably linked to a DNA sequence encoding alphavirus non-structural protein nucleotide sequences , operably linked to an alphavirus subgenomic RNA promoter , operably linked to a 2A DNA encoding a 2A peptide , which is in turn operably linked to a vesicular stomatitis virus (VSV) G DNA encoding a VSV G protein , wherein the alphavirus non-structural protein nucleotide sequences comprise at least two of the mutations selected from the group consisting of G-4700-A , A-5424-G , G-5434-A , T-5825-C , T-5930-C , A-6047-G , G-6783-A , G-6963-A , G-7834-A , T-8859-A , T-8864-C , G-9211-A , A-10427-G , G-11560-A , A-11871-G and T-11978-C , wherein the vector lacks functional nucleotide sequences which encode alphavirus structural proteins , further wherein when the vector is propagated in a cell culture , titers of at least 10plaque forming units (pfu) per ml of virus like vesicles (VLVs) are obtained.2. The high titer hybrid-virus vector of claim 1 , wherein the alphavirus is Semliki Forest virus (SFV).3. The high titer hybrid-virus vector of claim 1 , wherein the subgenomic RNA promoter is an Semliki Forest virus (SFV) promoter.4. The high titer hybrid-virus vector of claim 1 , wherein the 2A DNA is Thosea asigna virus ...

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Номер записи: 69
26-02-2015 дата публикации

EXPRESSION CONSTRUCTS AND METHODS FOR EXPRESSING POLYPEPTIDES IN EUKARYOTIC CELLS

Номер: US20150056655A1
Автор: AEBISCHER-GUMY Christel, BERTSCHINGER Martin, MORETTI Pierre
Принадлежит:

The invention relates to an expression construct for the expression of polypeptides in host cells using alternative splicing. The expression construct can be used for the expression of polypeptides such as antibodies, antibody fragments and bispecific antibodies by expressing the gene products required for protein expression at the ratio leading to the highest titres or the best product quality profile. 1. An expression construct comprising in a 5′ to 3′ direction:(i) a promoter;(ii) an optional first splice donor site;(iii) a first flanking intron;(iv) a first splice acceptor site;(v) a first exon encoding a first polypeptide;(vi) an optional second splice donor site;(vii) a second flanking intron;(viii) a second splice acceptor site; and(ix) a second exon encoding a second polypeptide,(x) wherein upon entry into a host cell, transcription of the first exon results in expression of the first polypeptide and/or transcription of the second exon results in expression of the second polypeptide.2. An expression construct according to claim 1 , wherein the first and the second flanking introns are selected from the group consisting of: chicken troponin (cTNT) intron 4 claim 1 , cTNT intron 5 and first intron of the human EF1alpha gene.3. An expression construct according to claim 1 , wherein the first and the second flanking introns have a nucleic acid sequence homology of at least 80% for at least 50 nucleotides.4. (canceled)5. (canceled)6. An expression construct according to claim 1 , further comprising at least one polypyrimidine (poly(Y)) tract upstream or downstream of the first exon.7. (canceled)8. (canceled)9. An expression construct according to claim 6 , wherein the poly(Y) tract comprises less than 30 pyrimidine bases.10. (canceled)11. (canceled)12. An expression construct according to claim 1 , wherein the expression construct comprises a third splice donor site claim 1 , an intron and a third splice acceptor site located downstream of said promoter.13. An ...

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Номер записи: 70
26-02-2015 дата публикации

Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded therapeutic protein

Номер: US20150057340A1
Автор: Andreas Thess, Jochen Probst, Thomas Schlake
Принадлежит: CureVac AG

The present invention relates to a nucleic acid sequence, comprising or coding for a coding region, encoding at least one peptide or protein comprising a therapeutic protein or a fragment, variant or derivative thereof, at least one histone stem-loop and a poly(A) sequence or a polyadenylation signal. Furthermore the present invention provides the use of the nucleic acid for increasing the expression of said encoded peptide or protein, particularly for the use in gene therapy. It also discloses its use for the preparation of a pharmaceutical composition, e.g. for use in gene therapy, particularly in the treatment of diseases which are in need of a treatment with a therapeutic peptide or protein, preferably as defined herein. The present invention further describes a method for increasing the expression of a peptide or protein comprising a therapeutic protein or a fragment, variant or derivative thereof, using the nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal.

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Номер записи: 71
15-05-2014 дата публикации

Multi plasmid system for the production of influenza virus

Номер: US20140134208A1
Автор: Erich Hoffmann, George Kemble, HONG Jin, Zhongying Chen
Принадлежит: MEDIMMUNE LLC

Vectors and methods for the production of influenza viruses suitable as recombinant influenza vaccines in cell culture are provided. Bi-directional expression vectors for use in a multi-plasmid influenza virus expression system are provided. Additionally, the invention provides methods of producing influenza viruses with enhanced ability to replicate in embryonated chicken eggs and/or cells (e.g., Vero and/or MDCK) and further provides influenza viruses with enhanced replication characteristics. A method of producing a cold adapted (ca) influenza virus that replicates efficiently at, e.g., 25° C. (and immunogenic compositions comprising the same) is also provided.

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Номер записи: 72
01-03-2018 дата публикации

NEURONAL SPECIFIC TARGETING OF CAVEOLIN EXPRESSION TO RESTORE SYNAPTIC SIGNALING AND IMPROVE COGNITIVE FUNCTION IN THE NEURODEGENERATIVE BRAIN AND MOTOR FUNCTION IN SPINAL CORD

Номер: US20180055950A1
Автор: Head Brian P., Patel Hemal, Patel Piyush M., Roth David M.
Принадлежит:

The invention provides an expression system for producing Caveolin-1 in neuronal cells or neural stem cells comprising a neuron-specific regulatory element and a nucleic acid sequence encoding Caveolin-1. 1. (canceled)2. (canceled)3. (canceled)4. (canceled)5. (canceled)6. (canceled)7. (canceled)8. (canceled)9. (canceled)10. (canceled)11. (canceled)12. (canceled)13. (canceled)14. (canceled)15. (canceled)16. (canceled)17. (canceled)18. (canceled)19. (canceled)20. (canceled)21. (canceled)22. (canceled)23. (canceled)24. (canceled)25. (canceled)26. (canceled)27. (canceled)28. A method of transferring the caveolin-1 gene into a neural stem cell or neuronal cell comprising introducing an expression system into the neural stem cell or neuronal cell , thereby transferring the caveolin-1 gene into a neural stem cell or neuronal cell , respectively wherein the expression system for producing Caveolin-1 in neuronal cells or neural stem cells comprising:(a) a neuron-specific regulatory element; and(b) a nucleic acid sequence encoding Caveolin-1.29. (canceled)30. (canceled)31. (canceled)32. (canceled)33. (canceled)34. (canceled)35. (canceled)36. (canceled)37. (canceled)39. (canceled)40. (canceled)41. (canceled)42. (canceled)43. (canceled)44. (canceled)45. (canceled)46. (canceled)47. (canceled)48. (canceled)49. (canceled)50. (canceled)51. (canceled)52. A method for promoting neural cell growth comprising administering Caveolin-1 or portion thereof to neural cells in a sufficient amount so as to promote neural cell growth.53. A method of increasing synapse formation and improving synaptic function in neurodegenerative diseases by promoting neural cell growth in a subject by the method of claim 52 , thereby increasing synapse formation and improving synaptic function.54. The method of claim 53 , wherein the neurodegenerative disease is Alzheimer's Disease.55. (canceled)56. (canceled)57. (canceled)58. (canceled)59. (canceled)60. (canceled)61. (canceled)62. (canceled)63. (canceled)64. ...

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Номер записи: 73
04-03-2021 дата публикации

NUCLEIC ACID COMPRISING OR CODING FOR A HISTONE STEM-LOOP AND A POLY(A) SEQUENCE OR A POLYADENYLATION SIGNAL FOR INCREASING THE EXPRESSION OF AN ENCODED THERAPEUTIC PROTEIN

Номер: US20210060181A1
Автор: Probst Jochen, SCHLAKE Thomas, THESS Andreas
Принадлежит: CureVac AG

The present invention relates to a nucleic acid sequence, comprising or coding for a coding region, encoding at least one peptide or protein comprising a therapeutic protein or a fragment, variant or derivative thereof, at least one histone stem-loop and a poly(A) sequence or a polyadenylation signal. Furthermore the present invention provides the use of the nucleic acid for increasing the expression of said encoded peptide or protein, particularly for the use in gene therapy. It also discloses its use for the preparation of a pharmaceutical composition, e.g. for use in gene therapy, particularly in the treatment of diseases which are in need of a treatment with a therapeutic peptide or protein, preferably as defined herein. The present invention further describes a method for increasing the expression of a peptide or protein comprising a therapeutic protein or a fragment, variant or derivative thereof, using the nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal. 2. The composition of claim 1 , wherein the polyadenylation sequence comprises the consensus sequence NN(U/T)ANA claim 1 , AA(U/T)AAA or A(U/T)(U/T)AAA.3. The composition of claim 1 , wherein the nucleic acid is a mRNA.4. The composition of claim 1 , comprising a poly(A) sequence.5. The composition of claim 1 , wherein the G/C content of the polypeptide coding region is increased compared with the G/C content of the wild-type nucleic acid of IL-12.6. The composition of claim 1 , wherein the mRNA comprises a 5′ cap structure.7. The composition of claim 4 , wherein the poly(A) sequence comprises about 25 to about 400 adenosine nucleotides.8. The composition of claim 1 , wherein the nucleic acid molecule additionally comprises a poly(C) sequence of about 10 to about 200 cytosine nucleotides.9. The method of claim 1 , wherein the mRNA further comprises a stabilizing sequence from the alpha globin 3′ UTR.10. The composition of claim 3 , wherein the mRNA ...

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Номер записи: 74
04-03-2021 дата публикации

RNA TRANSCRIPTION VECTOR AND USES THEREOF

Номер: US20210060182A1
Автор: Heirman Carlo, Thielemans Kristiaan
Принадлежит: VRIJE UNIVERSITEIT BRUSSEL

The present invention in general relates to an improved RNA transcription vector, which is very suitable for the production of mRNA for in vivo therapeutic purposes. The improvements in the vector in particular reside in the presence of a transcription enhancer and a nuclear retention element. 116-. (canceled)17. An RNA production vector comprising:a transcribable nucleic acid sequence;a 5′ translation enhancer (TE) sequence comprising a tandem repeat of a 9-nucleotide segment of the mouse Gtx homeodomain protein, interspaced by 9-nucleotide fragments of the human beta globin 5′ UTR; anda 3′ nuclear retention sequence (ENE) comprising the poly-adenylated non-translated RNA (PAN) region of the Kaposi's sarcoma associated Herpes virus (KSHV).18. The RNA production vector according to claim 17 , wherein said TE sequence is a 10× tandem repeat of a wild type 9-nucleotide sequence from a Gtx leader sequence (CCGGCGGGT) (SEQ ID NO: 6) linked by a 9-nucleotide sequence derived from a 5′UTR of the human beta globin (TTCTGACAT) (SEQ ID NO: 7).19. The RNA production vector according to claim 17 , wherein said ENE is a 79-nucleotide sequence of the poly-adenylated non-translated RNA (PAN) region of the Kaposi's sarcoma associated Herpes virus (KSHV) claim 17 , comprising a stem-loop structure with an asymmetric internal U-rich loop.20. The RNA production vector according to claim 17 , wherein said transcribable nucleic acid sequence is selected from the group consisting of mRNA encoding CD40L claim 17 , mRNA encoding CD70 claim 17 , mRNA encoding caTLR4 claim 17 , and antigen/disease specific mRNA.21. A method of increasing stability and/or translation efficiency of in vitro transcribed RNA claim 17 , the method comprising:{'claim-ref': {'@idref': 'CLM-00017', 'claim 17'}, '(i) providing an RNA production vector according to , wherein said transcribable nucleic acid sequence is a transcribable DNA sequence that corresponds to said RNA to be transcribed; and'}(ii) transcribing ...

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Номер записи: 75
02-03-2017 дата публикации

NUCLEIC ACID COMPRISING OR CODING FOR A HISTONE STEM-LOOP AND A POLY(A) SEQUENCE OR A POLYADENYLATION SIGNAL FOR INCREASING THE EXPRESSION OF AN ENCODED THERAPEUTIC PROTEIN

Номер: US20170056529A1
Автор: Probst Jochen, SCHLAKE Thomas, THESS Andreas
Принадлежит: CureVac AG

The present invention relates to a nucleic acid sequence, comprising or coding for a coding region, encoding at least one peptide or protein comprising a therapeutic protein or a fragment, variant or derivative thereof, at least one histone stem-loop and a poly(A) sequence or a polyadenylation signal. Furthermore the present invention provides the use of the nucleic acid for increasing the expression of said encoded peptide or protein, particularly for the use in gene therapy. It also discloses its use for the preparation of a pharmaceutical composition, e.g. for use in gene therapy, particularly in the treatment of diseases which are in need of a treatment with a therapeutic peptide or protein, preferably as defined herein. The present invention further describes a method for increasing the expression of a peptide or protein comprising a therapeutic protein or a fragment, variant or derivative thereof, using the nucleic acid comprising or coding for a histone stem-loop and a poly(A) sequence or a polyadenylation signal. 1. Nucleic acid sequence comprising or coding fora) a coding region, encoding at least one peptide or protein;b) at least one histone stem-loop, andc) a poly(A) sequence or a polyadenylation signal; (i) therapeutic proteins for use in the treatment of metabolic or endocrine disorders,', '(ii) therapeutic proteins for use in the treatment of blood disorders, diseases of the circulatory system, diseases of the respiratory system, cancer or tumour diseases, infectious diseases or immunedeficiencies,', '(iii) therapeutic proteins used for hormone replacement therapy,', '(iv) therapeutic proteins used for reprogramming of somatic cells into pluri- or omnipotent stem cells,', '(v) therapeutic proteins selected from adjuvant or immunostimulating proteins; and', '(vi) antibodies., 'wherein said peptide or protein comprises a therapeutic protein or a fragment, variant or derivative thereof, selected from'}2. The nucleic acid according to claim 1 , wherein ...

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Номер записи: 76
21-02-2019 дата публикации

USE OF ADENOVIRUS AND NUCLEIC ACIDS CODING THEREFOR

Номер: US20190055522A1
Автор: Holm Per Sonne
Принадлежит:

The invention relates to the use of a virus, preferably an adenovirus, for producing a medicament. Said virus is replication-deficient in cells which do not contain YB-1 in the core and codes for an oncogene or ongogene product, especially an oncogene protein, which transactivates at least one viral gene, preferably an adenoviral gene, said gene being selected among the group comprising E1B55kDa, E4orf6, E4orf3, and E3ADP. 155-. (canceled)56. A method for the treatment of cancer , the method comprising:administering to a subject in need thereof an adenovirus, wherein the adenovirus expresses E1B55kDa and is replication deficient in cells that lack Y box binding protein 1 (YB-1) in the nucleus and replication competent in cells that have YB-1 in the nucleus;wherein the virus encodes an oncogene protein that transactivates E1B55kDa, and wherein the adenovirus is dl520 lacking a functional CR3 domain.57. The method of claim 56 , wherein dl520 is E1B 19 K-minus.58. The method of claim 56 , wherein the adenovirus is capable of replicating in cells that are p53-positive or p53-negative.59. The method of claim 56 , wherein the oncogene protein is E1A.60. The method of claim 59 , wherein the E1A does not induce nuclear localization of YB-1.61. The method of claim 56 , wherein the oncogene protein is E1A12S.62. The method of claim 56 , wherein the oncogene protein is encoded by a gene that is under the control of a tissue and/or tumor specific promoter.63. The method of claim 56 , wherein the virus encodes YB-1.64. The method of claim 63 , wherein the YB-1 is under the control of a tissue and/or tumor specific promoter.65. The method of claim 56 , wherein the cancer is formed from a tumor claim 56 , or part thereof claim 56 , which is resistant to pharmacological agents.66. The method of claim 65 , wherein the cells that form the tumor claim 65 , or part thereof claim 65 , overexpress membrane-bound transport protein P glycoprotein.67. The method of claim 65 , wherein the ...

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Номер записи: 77
21-02-2019 дата публикации

Simian (gorilla) adenovirus or adenoviral vectors and methods of use

Номер: US20190055579A1
Автор: Douglas E. Brough, Duncan Mcvey, Jason G.D. Gall
Принадлежит: Genvec Inc

The invention provides an adenovirus or adenoviral vector characterized by comprising one or more particular nucleic acid sequences or one or more particular amino acid sequences, or portions thereof, pertaining to, for example, an adenoviral pIX protein, DNA polymerase protein, penton protein, hexon protein, and/or fiber protein.

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Номер записи: 78
01-03-2018 дата публикации

Compositions for Treating Pathological Calcification Conditions, and Methods Using Same

Номер: US20180057820A1
Автор: Demetrios Braddock, Ronald ALBRIGHT
Принадлежит: YALE UNIVERSITY

The present invention includes compositions and methods for treating diseases or disorders associated with pathological calcification or pathological ossification. In certain embodiments, the diseases or disorders are selected from the group consisting of Generalized Arterial Calcification of Infancy (GACI), Idiopathic Infantile Arterial Calcification (IIAC), Ossification of the Posterior Longitudinal Ligament (OPLL), hypophosphatemic rickets, osteoarthritis, calcification of atherosclerotic plaques, PXE, hereditary and non-hereditary forms of osteoarthritis, ankylosing spondylitis, hardening of the arteries occurring with aging, calciphylaxis resulting from end stage renal disease and progeria.

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Номер записи: 79
01-03-2018 дата публикации

VECTORS AND METHODS FOR TARGETED INTEGRATION IN LOCI COMPRISING CONSTITUTIVELY EXPRESSED GENES

Номер: US20180057842A1
Автор: ELEFANTY Andrew, Elliott David, LABONNE Tatiana, STANLEY Ed
Принадлежит:

The invention relates to a vector comprising: a 5′ nucleic acid that is homologous to a genomic sequence 5′ of a stop codon of a constitutively expressed gene; an exogenous nucleic acid; a 3′ nucleic acid that is homologous to a genomic sequence 3′ of the stop codon of the constitutively expressed gene; a translation interruption-reinitiation signal operably linked to the 5′ nucleic acid and the exogenous Targeting nucleic acid, wherein the translation interruption-reinitiation signal is capable of replacing the stop codon of the constitutively expressed gene. 1. A vector comprising:a 5′ nucleic add that is homologous to a genomic sequence 5′ of a genomic stop codon of a constitutively expressed gene;an exogenous nucleic acid;a 3′ nucleic acid that is homologous to a genomic sequence 3′ of the genomic stop codon of the constitutively expressed gene;a translation interruption-reinitiation signal operably linked to the 5′ nucleic acid and the exogenous nucleic acid,wherein the translation interruption-reinitiation signal is capable of replacing the stop codon of the constitutively expressed gene.2. The vector of claim 1 , wherein the constitutively expressed gene comprises GAPDH claim 1 , ACTB claim 1 , HSP90 claim 1 , B2M claim 1 , HPRTI claim 1 , RPLP1 claim 1 , GUSB claim 1 , LDHA claim 1 , NONO claim 1 , PGK1 claim 1 , PPIH claim 1 , RPLPO claim 1 , or TFRC.3. The vector of claim 1 , wherein the translation interruption-reinitiation signal encodes a 2A peptide.4. The vector of claim 3 , wherein the translation inten p io reinitiation signal comprises SEQ ID NO: 1.5. The vector of claim 1 , wherein the 5′ nucleic add comprises about 25 to 100 bases claim 1 , about 200 to 1000 bases claim 1 , about 1 kb to 5 kb claim 1 , or about 3.5 kb.16. The vector of claim 5 , wherein the 5′ nucleic acid comprises SEQ ID NO: 2.7. The vector of claim 1 , wherein the 3′ nucleic acid comprises about 25 to 100 bases claim 1 , about 200 to 1000 bases claim 1 , about 1 kb to 5 kb ...

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Номер записи: 80
01-03-2018 дата публикации

Virus causing respiratory tract illness in susceptible mammals

Номер: US20180057893A1
Автор: Albertus Dominicus Marcellinus Erasmus Osterhaus, Bernadetta Gerarda Van Den Hoogen, Jan Cornelius De Jong, Jan Groen, Ronaldus Adrianus Maria Fouchier
Принадлежит: ERASMUS UNIVERSITY MEDICAL CENTER

The invention relates to the field of virology. The invention provides an isolated essentially mammalian negative-sense single-stranded RNA virus (MPV) within the subfamily Pneumovirinae of the family Paramyxoviridae and identifiable as phylogenetically corresponding to the genus Metapneumovirus and components thereof.

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Номер записи: 81
01-03-2018 дата публикации

METAPNEUMOVIRUS STRAINS AND THEIR USE IN VACCINE FORMULATIONS AND AS VECTORS FOR EXPRESSION OF ANTIGENIC SEQUENCES

Номер: US20180057894A1
Автор: De Jong Jan Cornelius, Fouchier Ronaldus Adrianus Maria, Groen Jan, Osterhaus Albertus Dominicus Marcellinus Erasmus, Van Den Hoogen Bernadetta Gerarda
Принадлежит: ERASMUS UNIVERSITY MEDICAL CENTER ROTTERDAM

Provided is an isolated mammalian negative strand RNA virus, (MPV), within the sub-family Pneumoviridae, of the family Paramyxoviridae. Also provided are isolated mammalian negative strand RNA viruses identifiable as phylogenetically corresponding or relating to the genus and components thereof. In particular, provided is a mammalian MPV, subgroups and variants thereof. Also provided are genomic nucleotide sequences of different isolates of mammalian MPV, in particular, human MPV. Disclosed is the use of the sequence information of different isolates of mammalian MPV for diagnostic and therapeutic methods. Provided are nucleotide sequences encoding the genome of an MPV or a portion thereof, including both mammalian and avian MPV. Further described are chimeric or recombinant viruses encoded by the nucleotide sequences and chimeric and recombinant mammalian MPV that comprise one or more non-native or heterologous sequences. Also provided are vaccine formulations comprising mammalian or avian MPV, including recombinant and chimeric forms thereof. The vaccine preparations encompass multivalent vaccines, including bivalent and trivalent vaccine preparations. 184.-. (canceled)85metapneumovirus. A kit for determining the presence of (MPV) in a mammalian subject , the kit comprising:a DNA probe of at least 10 nucleotides that specifically hybridizes to a target polynucleotide,{'i': 'pneumovirus', 'wherein the DNA probe does not specifically hybridize to a polynucleotide from avian (APV);'}wherein the probe does not hybridize under stringent conditions to a sequence encoding a polypeptide of SEQ ID NOs:322 and 323 or the complement of the sequence;wherein the stringent conditions include a hybridization buffer comprising 6×SSC and 1 mM EDTA; andwherein the target polynucleotide comprises a first nucleic acid encoding a polypeptide that is at least 90% identical to SEQ ID NO:324 or the complement of the first nucleic acid; orwherein the target polynucleotide comprises a ...

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Номер записи: 82
05-03-2015 дата публикации

MODIFIED POLYNUCLEOTIDES

Номер: US20150064235A1
Автор: Bancel Stephane, Chakraborty Tirtha, de Fougerolles Antonin, Ejebe Kenechi, Elbashir Sayda M., Ellsworth Jeff Lynn, Guild Justin, Hatala Paul, John Matthias, Roy Atanu, Schrum Jason P., Whoriskey Susan, Wood Kristy M.
Принадлежит:

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules. 1. An isolated polynucleotide comprising;(a) a first region of linked nucleosides, said first region encoding a polypeptide of interest, said polypeptide of interest selected from the group consisting of Target Nos 1-917; (i) a 5′ untranslated region (5′UTR); and', '(ii) at least one 5′ terminal cap;, '(b) a first flanking region located at the 5′ terminus of said first region comprising;'} (i′) a 3′ untranslated region (3′UTR); and', '(ii′) a 3′ tailing sequence of linked nucleosides; and, '(c) a second flanking region located at the 3′ terminus of said first region comprising;'}wherein at least one of said linked nucleosides comprises at least one modification as compared to the chemical structure of an A G, U or C ribonucleoside and wherein at least one of said 5′UTR or said 3′UTR is not derived from the native untranslated region of said polypeptide of interest.2. The isolated polynucleotide of wherein the first region of linked nucleosides comprises at least an open reading frame of a nucleic acid sequence claim 1 , wherein the nucleic acid sequence selected from the group consisting of SEQ ID NOs: 195-199.3. The isolated polynucleotide of claim 1 , wherein the 3′tailing sequence of linked nucleosides is selected from the group consisting of a poly-A tail of approximately 160 nucleotides and a polyA-G quartet.4. The isolated polynucleotide of which is purified.5. The isolated polynucleotide of claim 1 , wherein the at least one 5′ terminal cap is selected from the group consisting of Cap0 claim 1 , Cap1 claim 1 , ARCA claim 1 , inosine claim 1 , N1-methyl-guanosine claim 1 , 2′fluoro-guanosine claim 1 , 7-deaza-guanosine claim 1 , 8-oxo-guanosine claim 1 , 2-amino-guanosine claim 1 , LNA-guanosine claim 1 , and 2-azido-guanosine.6. (canceled)7. The isolated polynucleotide of claim 1 , wherein at ...

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Номер записи: 83
08-03-2018 дата публикации

CHIMERIC GENE CONSTRUCTS FOR GENERATION OF FLUORESCENT TRANSGENIC ORNAMENTAL FISH

Номер: US20180064074A1
Автор: Gong Zhiyuan, HE Jiangyan, JU Bensheng, LAM Toong Jin, Xu Yanfei, YAN Tie
Принадлежит: NATIONAL UNIVERSITY OF SINGAPORE

Four zebrafish gene promoters, which are skin specific, muscle specific, skeletal muscle specific and ubiquitously expressed respectively, were isolated and ligated to the 5′ end of the EGFP gene. When the resulting chimeric gene constructs were introduced into zebrafish, the transgenic zebrafish emit green fluorescence under a blue light or ultraviolet light according to the specificity of the promoters used. Thus, new varieties of ornamental fish of different fluorescence patterns, e.g., skin fluorescence, muscle fluorescence, skeletal muscle-specific and/or ubiquitous fluorescence, are developed. 141-. (canceled)42. A transgenic fish comprising a chimeric gene comprising a promoter operably linked to an exogenous gene , wherein said promoter comprises a nucleic acid sequence that is identical to the sequence of SEQ ID NO: 7 , SEQ ID NO: 8 , SEQ ID NO: 9 , or SEQ ID NO: 22 , wherein the transgenic fish contains said promoter in germ cells and/or in somatic cells and is capable of breeding to produce viable and fertile transgenic progeny that express the exogenous gene.43. The transgenic fish of claim 42 , wherein the variant polypeptide or variant polynucleotides comprises at least 75% sequence identity to the naturally occurring sequences or fragments.44. The transgenic fish of claim 42 , wherein the variant polypeptide or variant polynucleotides comprises at least 85% sequence identity to the naturally occurring sequences or fragments.45. The transgenic fish of claim 42 , wherein the variant polypeptide or variant polynucleotides comprises at least 95% sequence identity to the naturally occurring sequences or fragments.46. The transgenic fish of claim 42 , wherein the variant polypeptide or variant polynucleotides comprises at least 98% sequence identity to the naturally occurring sequences or fragments.47. The transgenic fish of claim 42 , further comprising a fluorescent protein gene under control of said promoter.48. The transgenic fish of claim 47 , wherein ...

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Номер записи: 84
27-02-2020 дата публикации

GENE THERAPY FOR OCULAR DISORDERS

Номер: US20200061209A1
Автор: Bennett Jean, Bennicelli Jeannette, Sun Junwei
Принадлежит:

Compositions and methods are provided for treating ocular disorders in a subject are provided. In one aspect, an adeno-associated viral vector is provided which includes a nucleic acid molecule comprising a sequence encoding CNGA3. In another aspect, an adeno-associated viral vector is provided which includes a nucleic acid molecule comprising a sequence encoding CNGB3. In another aspect, an adeno-associated viral vector is provided which includes a nucleic acid molecule comprising a sequence encoding REP-1. In desired embodiments, the subject is human, cat, dog, sheep, or non-human primate. 13-. (canceled)4. An adeno-associated virus (AAV) vector comprising an AAV capsid and a nucleic acid sequence comprising AAV inverted terminal repeat sequences and a nucleic acid sequence encoding human Rab Escort Protein-1 (REP-1) , and expression control sequences that direct expression of the REP-1 in a host cell.5. (canceled)6. The viral vector of claim 4 , wherein the REP-1 sequence encodes the protein sequence of SEQ ID NO: 2.78-. (canceled)9. The viral vector of claim 4 , wherein the REP-1 sequence comprises SEQ ID NO: 1.1012-. (canceled)13. An adeno-associated virus (AAV) vector comprising an AAV capsid and a nucleic acid sequence comprising AAV inverted terminal repeat sequences and a nucleic acid sequence encoding human cyclic nucleotide gated channel alpha 3 (CNGA3) claim 4 , and expression control sequences that direct expression of the CNGA3 in a host cell.14. (canceled)15. The viral vector of claim 13 , wherein the CNGA3 sequence encodes the protein sequence of SEQ ID NO: 10.1617-. (canceled)18. The viral vector of claim 13 , wherein CNGA3 sequence comprises SEQ ID NO: 9 or SEQ ID NO: 11.19. The viral vector of any of claim 4 , wherein the expression control sequences comprise a chicken β-actin (CBA) promoter with cytomegalovirus (CMV) enhancer elements.20. (canceled)21. The viral vector of claim 9 , wherein the promoter is a rhodopsin kinase promoter.22. The viral ...

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Номер записи: 85
28-02-2019 дата публикации

REPLICATION-DEFECTIVE ARENAVIRUS VECTORS

Номер: US20190062784A1
Автор: Bergthaler Andreas, Flatz Lukas, Pinschewer Daniel D., Zinkernagel Rolf
Принадлежит: UNIVERSITAT ZURICH

The invention relates to an infectious arenavirus particle that is engineered to contain a genome with the ability to amplify and express its genetic information in infected cells but unable to produce further infectious progeny particles in normal, not genetically engineered cells. One or more of the four arenavirus open reading frames glycoprotein (GP), nucleoprotein (NP), matrix protein Z and RNA-dependent RNA polymerase L are removed or mutated to prevent replication in normal cells but still allowing gene expression in arenavirus vector-infected cells, and foreign genes coding for an antigen or other protein of interest or nucleic acids modulating host gene expression are expressed under control of the arenavirus promoters, internal ribosome entry sites or under control of regulatory elements that can be read by the viral RNA-dependent RNA polymerase, cellular RNA polymerase I, RNA polymerase II or RNA polymerase III. The modified arenaviruses are useful as vaccines and therapeutic agents for a variety of diseases. 1. An infectious arenavirus particle engineered to contain a genome with the ability to amplify and express its genetic information in infected cells but unable to produce further infectious progeny particles in normal , not genetically engineered cells.2. The arenavirus particle according to comprising additional nucleic acids coding for a protein or peptide of interest.3. The arenavirus particle according to comprising additional nucleic acids modulating host gene expression.4. The arenavirus particle according to comprising a modified genome claim 2 , whereini) one or more of the four arenavirus open reading frames glycoprotein (GP), nucleoprotein (NP), matrix protein Z and RNA-dependent RNA polymerase L are removed or mutated to prevent replication in normal cells but still allowing gene expression in arenavirus vector-infected cells;ii) foreign ribonucleic acids coding for one or more proteins of interest are expressed under control of one or ...

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Номер записи: 86
08-03-2018 дата публикации

Compositions for Treating Pathological Calcification Conditions, and Methods Using Same

Номер: US20180064786A1
Автор: ALBRIGHT Ronald, BRADDOCK Demetrios
Принадлежит:

The present invention includes compositions and methods for treating diseases or disorders associated with pathological calcification or pathological ossification. In certain embodiments, the diseases or disorders are selected from the group consisting of Generalized Arterial Calcification of Infancy (GACI), Idiopathic Infantile Arterial Calcification (IIAC), Ossification of the Posterior Longitudinal Ligament (OPLL), hypophosphatemic rickets, osteoarthritis, calcification of atherosclerotic plaques, PXE, hereditary and non-hereditary forms of osteoarthritis, ankylosing spondylitis, hardening of the arteries occurring with aging, calciphylaxis resulting from end stage renal disease and progeria. 1. An isolated cell comprising ENPP1 precursor polypeptide fusion protein , wherein the ENPP1 precursor polypeptide fusion protein comprises an ENPP2 signal peptide domain and a stability domain , wherein the ENPP1 precursor polypeptide fusion protein is proteolytically processed upon secretion from the cell to yield a soluble ENPP1 polypeptide fusion protein comprising the stability domain.2. The cell of claim 1 , wherein the stability domain is selected from the group consisting of albumin and IgG Fc.3. The cell of claim 1 , wherein the ENPP2 signal peptide domain comprises residues 12-30 of ENPP2 (NCBI accession no. NP_001124335 claim 1 , SEQ ID NO: 2).4. The cell of claim 1 , wherein the soluble ENPP1 polypeptide fusion protein comprises residues 96-925 of human ENPP1 [NCBI# 006199 claim 1 , SEQ ID NO: 1].5. The cell of claim 1 , wherein the ENPP1 precursor polypeptide fusion protein lacks residues 77-98 of human ENPP1 [NCBI# 006199 claim 1 , SEQ ID NO: 1].6. The cell of claim 1 , wherein the ENPP1 precursor polypeptide fusion protein comprises residues 1-76 of ENPP1 (SEQ ID NO: 1) claim 1 , residues 12-30 of ENPP2 (SEQ ID NO: 2) claim 1 , and residues 96-925 of ENPP1 (SEQ ID NO: 1).7. The cell of claim 1 , wherein the soluble ENPP1 polypeptide fusion protein further ...

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Номер записи: 87
27-02-2020 дата публикации

SYSTEM AND METHOD FOR CELL TYPE-SPECIFIC TRANSLATION OF RNA MOLECULES IN EUKARYOTES

Номер: US20200063131A1
Автор: Bohlen Heribert, Hoffmann Bernd
Принадлежит:

The present technology comprises methods and RNA constructs for the targeted translation of a polypeptide of interest in a eukaryotic target cell, and to the use thereof in therapeutic clinical applications. 2. Method according to claim 1 , wherein interaction of the anticodogenic element (a) with the mRNA expressed by the target cell results in degradation or inactivation of the blocking element (b) such that translation of the polypeptide of interest can occur.3. Method according to claim 2 , wherein the degradation of the mRNA on the stabilizing element (c) is stopped.4. Method according to claim 1 , characterized in that the anticodogenic element (a) has one or more of the following features:(i) attachment to an RNA sequence specific for the target cell;(ii) formation of double-stranded RNA hybrids of freely adjustable length;(iii) induction of cellular degradation of the introduced RNA construct in the region of the double-stranded RNA hybrid.5. Method according to claim 1 , characterized in that the blocking element (b) has one or more of the following features:(i) the function of the translation initiator element (d) is suppressed;(ii) the blocking element (b) can be degraded or inactivated under suitable conditions;(iii) the suppressive function on the translation initiator element (d) is reversible after inactivation or degradation of the blocking element;(iv) the blocking element (b) performs its function by base pairing in or near the sequence of the translation initiator element (d), thereby changing the secondary structure thereof;(v) the blocking element (b) performs its function by causing other molecules to directly or indirectly inhibit the translation initiator element (d).6. Method according to claim 1 , characterized in that the stabilizing element (c) has one or more of the following features:(i) nucleotide sequence that prevents the degradation of RNA by exo- and/or endonucleases in the 3′ direction;(ii) nucleotide sequence, characterized in ...

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Номер записи: 88
09-03-2017 дата публикации

NUCLEIC ACID CONSTRUCTS ENCODING REPROGRAMMING FACTORS LINKED BY SELF-CLEAVING PEPTIDES

Номер: US20170067081A1
Автор: Carey Bryce Woodbury, Jaenisch Rudolf
Принадлежит:

The disclosure relates to a method of reprogramming one or more somatic cells, e.g., partially differentiated or fully/terminally differentiated somatic cells, to a less differentiated state, e.g., a pluripotent or multipotent state. In further embodiments the invention also relates to reprogrammed somatic cells produced by methods of the invention, to chimeric animals comprising reprogrammed somatic cells of the invention, to uses of said cells, and to methods for identifying agents useful for reprogramming somatic cells. 1. A nucleic acid construct comprising at least three coding regions , a first coding region encoding a first reprogramming factor , a second coding region encoding a second reprogramming factor , and a third coding region that encodes a third reprogramming factor , wherein the first and second coding regions are linked by a nucleic acid that encodes a first self-cleaving peptide and the second and third coding regions are linked by a nucleic acid that encodes a second self-cleaving peptide so as to form a single open reading frame , wherein expression of the first , second , and third reprogramming factors is sufficient for reprogramming a mammalian somatic cell to pluripotency , andwherein the nucleic acid construct does not comprise Oct 3/4, Sox 2 and Klf4 ligated across the 2A sequence of foot-and-mouth disease virus in the order of Oct 3/4, Klf4 and Sox2.2. The nucleic acid construct of claim 1 , further comprising a fourth coding region that encodes a fourth reprogramming factor.3. The nucleic acid construct of claim 1 , wherein: the first or second self-cleaving peptide is a viral 2A peptide or the first and second self-cleaving peptide are a viral 2A peptide.4. The nucleic acid construct of claim 3 , wherein the viral 2A peptide is an aphthovirus 2A peptide.5. The nucleic acid construct of claim 3 , wherein the viral 2A peptide is selected from the group consisting of: a foot-and-mouth disease virus (FMDV) peptide claim 3 , an equine ...

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Номер записи: 89
17-03-2016 дата публикации

EFFECTIVE DELIVERY OF LARGE GENES BY DUAL AAV VECTORS

Номер: US20160076054A1
Автор: Auricchio Alberto, Colella Pasqualina, Trapani Ivana
Принадлежит:

The present invention relates to constructs, vectors, relative host cells and pharmaceutical compositions which allow an effective gene therapy, in particular of genes larger than 5 Kb. 1. A dual construct system to express the coding sequence of a gene of interest in an host cell , said coding sequence consisting of a 5′end portion and of a 3′end portion , said dual construct system comprising:a) a first plasmid comprising in a 5′-3′ direction:a 5′-inverted terminal repeat (5′-ITR) sequence;a promoter sequence;the 5′ end portion of said coding sequence, said 5′end portion being operably linked to and under control of said promoter;a nucleic acid sequence of a splicing donor signal; anda 3′-inverted terminal repeat (3′-ITR) sequence; andb) a second plasmid comprising in a 5′-3′ direction:a 5′-inverted terminal repeat (5′-ITR) sequence;a nucleic acid sequence of a splicing acceptor signal;the 3′end of said coding sequence;a poly-adenylation signal nucleic acid sequence; anda 3′-inverted terminal repeat (3′-ITR) sequence.2. The dual construct system according to wherein upon introduction of said first plasmid and said second plasmid into the host cell claim 1 , said coding sequence reconstitutes by means of the splicing donor and the splicing acceptor signals.3. The dual construct system according to wherein said first plasmid further comprises a nucleic acid sequence of a recombinogenic region in 5′ position of the 3′ITR of said first plasmid and wherein said second plasmid further comprises a nucleic acid sequence of a recombinogenic region in 3′ position of the 5′-ITR of said second plasmid.4. The dual construct system according to wherein the recombinogenic region is a F1 phage recombinogenic region.6. The dual construct system according to claim 1 , wherein the nucleotide sequence of the ITRs derives from the same AAV serotype or from different AAV serotypes.7. The dual construct system according to claim 6 , wherein the 3′-ITR of the first plasmid and the 5′-ITR ...

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Номер записи: 90
07-03-2019 дата публикации

A TRANSPOSON-BASED TRANSFECTION SYSTEM FOR PRIMARY CELLS

Номер: US20190071484A1
Автор: BUNSE Mario, CLAUSS Julian, Izsvák Zsuzsanna, UCKERT Wolfgang
Принадлежит:

The present invention relates to the field of genetic engineering, in particular, to a transposon-based transfection kit suitable for transfection of primary cells, such as T cells, comprising mRNA encoding a transposase, or reagents for generating mRNA encoding said transposase, as well as minicircle DNA comprising the transposon. The invention also relates to a nucleic acid, preferably, a DNA minicircle, comprising a transposon, wherein the transposon encodes a protein and at least one miRNA, wherein the sequences encoding the miRNA are located in an intron and expression of the protein and the miRNA is regulated by the same promoter. The invention also provides a population of cells obtainable with the method of the invention. Methods of transfection are also provided, as well as medical use, e.g. in immunotherapy, in particular, in adoptive T cell therapy or T cell receptor (TCR) or chimeric antigen receptor (CAR) gene therapy. 1. A kit comprising (i) mRNA encoding said transposase; or', '(ii) DNA encoding said transposase functionally linked to a promoter, wherein the kit optionally further comprises reagents suitable for in vitro transcription, comprising ribonucleotide triphosphates, a buffer suitable for transcription, and a RNA polymerase suitable for transcription; and, 'a) a nucleic acid encoding a transposase capable of mobilizing a transposon, wherein the nucleic acid is selected from the group comprising'}b) minicircle DNA comprising said transposon, wherein the transposon encodes at least one protein and/or at least one miRNA, wherein expression of the protein and/or the miRNA is regulated by a promoter.2. The kit of claim 1 , wherein the nucleic acid encoding the transposase is mRNA.3. The kit of claim 1 , wherein the transposase is selected from the group of class II transposable elements comprising piggyBac claim 1 , Tol2 and Tc1/mariner-type transposons comprising Frog Prince and Sleeping Beauty transposase claim 1 , preferably claim 1 , Sleeping ...

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Номер записи: 91
15-03-2018 дата публикации

D-AMINO ACID-INDUCIBLE GENE EXPRESSION SYSTEM FOR RHODOSPORIDIUM AND RHODOTORULA

Номер: US20180073034A1
Автор: JI Lianghui, KOH Chong Mei John, Liu Yanbin
Принадлежит: TEMASEK LIFE SCIENCES LABORATORY LIMITED

The present invention relates to the field of fungal biotechnology, more particularly to a strong inducible gene expression system in fungal species, such as a species of the genus or the genus. 1. A D-amino acid inducible gene expression system operable in a transgenic fungal cell comprising a nucleic acid construct comprising a D-amino acid inducible promoter operably linked to a heterologous polynucleotide , wherein the promoter is selected from the group consisting of:(a) a promoter having at least 50% identity to the nucleotide sequence set forth in SEQ ID NO:94;(b) a promoter having at least 50% identity to the nucleotide sequence set forth in SEQ ID NO:95;(c) a promoter having at least 50% identity to the nucleotide sequence set forth in SEQ ID NO:96;(d) a promoter having at least 50% identity to the nucleotide sequence set forth in SEQ ID NO:97; and(e) a promoter having at least 50% identity to the nucleotide sequence set forth in SEQ ID NO:5.2RhodsporidiumRodottorula. The expression system of claim 1 , wherein the fungal cell is a transgenic fungal cell of a species of genus or genus.3. The expression system of claim 1 , wherein the transgenic fungal cell comprises a mutant D-amino acid oxidase gene having no or reduced D-amino acid oxidase activity compared to a fungal cell comprising a wild type D-amino acid oxidase gene.4. The expression system of claim 1 , wherein the promoter comprises a nucleotide sequence within the promoter having at least 75% identity to the nucleotide sequence set forth in SEQ ID NO:6 claim 1 , wherein said 75% identity is based on the A and G nucleotides in SEQ ID NO:6.5. The expression system of claim 1 , wherein the heterologous polynucleotide is operably linked to a transcription terminator.6. The expression system of claim 1 , wherein the nucleic acid construct contains a nucleotide sequence having at least 75% identity to the nucleotide sequence set forth in SEQ ID NO:7.7. The expression system of claim 6 , wherein the ...

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Номер записи: 92
24-03-2022 дата публикации

TRANSPOSON-BASED TRANSFECTION SYSTEM FOR PRIMARY CELLS

Номер: US20220089671A1
Автор: BUNSE Mario, CLAUSS Julian, Izsvák Zsuzsanna, UCKERT Wolfgang
Принадлежит:

The present invention relates to the field of genetic engineering, in particular, to a transposon-based transfection kit suitable for transfection of primary cells, such as T cells, comprising mRNA encoding a transposase, or reagents for generating mRNA encoding said transposase, as well as minicircle DNA comprising the transposon. The invention also relates to a nucleic acid, preferably, a DNA minicircle, comprising a transposon, wherein the transposon encodes a protein and at least one miRNA, wherein the sequences encoding the miRNA are located in an intron and expression of the protein and the miRNA is regulated by the same promoter. The invention also provides a population of cells obtainable with the method of the invention. Methods of transfection are also provided, as well as medical use, e.g. in immunotherapy, in particular, in adoptive T cell therapy or T cell receptor (TCR) or chimeric antigen receptor (CAR) gene therapy. 1. A kit comprising{'claim-text': ['(i) mRNA encoding said transposase; or', '(ii) DNA encoding said transposase functionally linked to a promoter, wherein the kit optionally further comprises reagents suitable for in vitro transcription, comprising ribonucleotide triphosphates, a buffer suitable for transcription, and a RNA polymerase suitable for transcription; and'], '#text': 'a) a nucleic acid encoding a transposase capable of mobilizing a transposon, wherein the nucleic acid is selected from the group comprising'}b) minicircle DNA comprising said transposon, wherein the transposon encodes at least one protein and/or at least one miRNA, wherein expression of the protein and/or the miRNA is regulated by a promoter.2. The kit of claim 1 , wherein the nucleic acid encoding the transposase is mRNA.3. The kit of any of the preceding claims claim 1 , wherein the transposase is selected from the group of class II transposable elements comprising piggyBac claim 1 , Tol2 and MI/mariner-type transposons comprising Frog Prince and Sleeping ...

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Номер записи: 93
24-03-2022 дата публикации

METHODS AND COMPOSITIONS FOR CIRCULAR RNA MOLECULES

Номер: US20220090137A1
Автор: Asokan Aravind, Borchardt Erin, Marzluff William F., Meganck Rita
Принадлежит:

This invention is directed to AAV compositions for circular RNA expression and methods of expressing covalently closed, circular RNA. 1. An adeno-associated virus (AAV) genome encoding a circular RNA , wherein the AAV genome comprises , from 5′ to 3′:(a) a first inverted terminal repeat;(b) a first intronic element;(c) a nucleotide sequence of interest;(d) a second intronic element; and(d) a second inverted terminal repeat.2. The AAV genome of claim 1 , wherein the first intronic element and the second intronic element are capable of generating a covalently closed circular RNA.3. The AAV genome of claim 2 , wherein the first intronic element and the second intronic element are capable of facilitating formation of a covalently closed circular RNA following transcription of the AAV genome in a mammalian cell.4. The AAV genome of claim 1 , wherein the first intronic element and the second intronic element are derived from the human ZKSCAN1 claim 1 , HIPK3 claim 1 , EPHB4 claim 1 , or Laccase2 genes.5. The AAV genome of claim 1 , wherein the first intronic element and/or the second intronic element comprises the sequence of any one of SEQ ID NOs:13-24 or 29-32.6. The AAV genome of claim 1 , wherein the nucleotide sequence of interest encodes a non-coding RNA.7. The AAV genome of claim 1 , wherein the nucleotide sequence of interest encodes a translatable mRNA.8. The AAV genome of claim 7 , wherein the translatable mRNA encodes a therapeutic protein.9. The AAV genome of claim 7 , comprising an internal ribosome entry site (IRES) capable of diving translation of the translatable mRNA.10. The AAV genome of claim 9 , wherein the IRES is a viral IRES or a cellular IRES.11TriatomaSolenopsis invictaRhopalosiphum padiPlautia stali, Homalodisca coagulata, Homalodisca coagulataEctropis obliquaDrosophilaDrosophilaDrosophilaDrosophilaDrosophilaS. cerevisiaeS. cerevisiae. The AAV genome of claim 9 , wherein the IRES is from Taura syndrome virus claim 9 , virus claim 9 , Theiler's ...

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Номер записи: 94
24-03-2022 дата публикации

MODIFIED ADENOVIRUSES

Номер: US20220090138A1
Автор: Gitlin Leonid, Jooss Karin, Scallan Ciaran Daniel
Принадлежит:

Disclosed herein are compositions that include modified adenoviruses. Also disclosed are nucleotides, cells, and methods associated with the compositions including their use as vaccines. Also disclosed herein are viral vectors using TET promoter system and methods of producing viruses having the same. 1. An adenovirus vector comprising:an adenoviral backbone comprising one or more genes or regulatory sequences of an adenovirus genome, andwherein the adenoviral backbone comprises a partially deleted E4 gene with reference to the adenovirus genome, wherein the partially deleted E4 gene comprises a deleted or partially-deleted E4orf2 region and a deleted or partially-deleted E4orf3 region, and optionally a deleted or partially-deleted E4orf4 region, and [ a MHC class I epitope,', 'a MHC class II epitope,', 'an epitope capable of stimulating a B cell response, or', 'a combination thereof, and', 'optionally wherein the at least one payload nucleic acid sequence further comprises a 5′ linker sequence and/or a 3′ linker sequence, and optionally wherein;, '(1) at least one payload nucleic acid sequence, optionally wherein the at least one payload nucleic acid sequence encodes a polypeptide, optionally wherein the polypeptide comprises an antigen, optionally wherein the antigen comprises, '(2) at least one promoter sequence operably linked to the at least one payload nucleic acid sequence,', '(3) optionally, at least one universal MHC class II antigen-encoding nucleic acid sequence;', '(4) optionally, at least one GPGPG-encoding linker sequence (SEQ ID NO:56); and', '(5) optionally, at least one polyadenylation sequence., 'optionally, wherein the adenovirus vector further comprises a cassette, the cassette comprising2. A chimpanzee adenovirus vector comprising a modified ChAdV68 sequence , wherein the modified ChAdV68 sequence comprises:(a) a partially deleted E4 gene of the E4 gene sequence shown in SEQ ID NO:1 and that lacks at least nucleotides 34,916 to 35,642 of the ...

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Номер записи: 95
19-03-2015 дата публикации

NON-VIRAL EPISOMAL SUICIDE CONSTRUCT

Номер: US20150079682A1
Автор: Atala Anthony, Lu Baisong, Yoo James, Zhao Qingguo
Принадлежит: Wake Forest University Health Sciences

The invention includes compositions and methods for the selective expression of a target gene in a subset of cells. In certain embodiments, the present invention includes a construct comprising a first nucleic acid sequence comprising an episomal maintenance element and a second nucleic acid sequence comprising a target gene wherein the expression of the episomal maintenance element is regulated by a constitutive promoter and the expression of the target gene is regulated by a non-constitutive promoter. The construct is able to maintain episomal state, no matter whether the target gene is expressed in the cell. 1. A composition comprising a first nucleic acid sequence comprising an episomal maintenance element and a second nucleic acid sequence comprising a target gene , wherein the expression of the episomal maintenance element is regulated by a constitutive promoter and the expression of the target gene is regulated by a non-constitutive promoter.2. The composition of claim 1 , wherein the episomal maintenance element is S/MAR.3. The composition of claim 1 , wherein the non-constitutive promoter is selected from the group consisting of an inducible promoter claim 1 , a cell-type specific promoter claim 1 , and a tumor specific promoter.4. The composition of claim 1 , wherein when the target gene is expressed in a cell claim 1 , the cell is susceptible for cell death.5. The composition of claim 1 , wherein the target gene encodes thymidine kinase (TK).6. The composition of claim 3 , wherein the cell-type specific promoter is an undifferentiated stem cell specific promoter.7. The composition of claim 6 , wherein the undifferentiated stem cell specific promoter is selected from the group consisting of OCT4 claim 6 , hTERT claim 6 , and NANOG.8. The composition of claim 1 , wherein the episomal maintenance element provides long term episomal maintenance of the composition in an entire cell population claim 1 , and wherein the non-constitutive promoter provides ...

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Номер записи: 96
05-03-2020 дата публикации

STEM CELL GENE TARGETING

Номер: US20200071719A1
Автор: Fehling Hans J., Gadue Paul, Irion Stefan, KELLER Gordon, Luche Herve
Принадлежит:

The invention provides a method for generating a transgenic eukaryotic cell population having a modified human Rosa26 locus, which method includes introducing a functional DNA sequence into the human Rosa26 locus of starting eukaryotic cells. Also provided are targeting vectors useful in the method, as well as a cell population and a transgenic non-human animal comprising a modified human Rosa26 locus. Finally, the invention provides an isolated DNA sequence corresponding to the human Rosa26 locus. 114-. (canceled)15. A targeting vector comprising an expression cassette comprising a nucleic acid encoding a protein , wherein said nucleic acid is heterologous to a human Rosa 26 gene , said expression cassette flanked by DNA sequences homologous to the human Rosa26 gene.1620.-. (canceled)21. The targeting vector of wherein the expression cassette further comprises a promoter operably linked to the nucleic acid encoding the protein.22. The targeting vector of wherein the promoter is selected from the group consisting of a constitutive ubiquitous promoter claim 21 , a constitutive tissue specific promoter claim 21 , an inducible ubiquitous promoter and an inducible tissue specific promoter.23. The targeting vector of wherein the promoter is heterologous to the human Rosa26 gene.24. The targeting vector of wherein the promoter is the endogenous human Rosa26 promoter.25. The targeting vector of wherein the sequences homologous to the human Rosa26 gene are derived from the 5′ and 3′ flanking arms of the human Rosa26 gene.26. The targeting vector of further comprising tags for protein detection claim 15 , enhancers claim 15 , selection markers claim 15 , and combinations thereof.27. The targeting vector of wherein the nucleic acid encodes a recombinase or a reporter.28. The targeting vector of wherein the expression cassette further comprises a marker gene claim 15 , one or more recombinase recognition sites claim 15 , a poly A signal claim 15 , an intron claim 15 , or ...

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Номер записи: 97
18-03-2021 дата публикации

ADENO-ASSOCIATED VIRUS VECTOR VARIANTS FOR HIGH EFFICIENCY GENOME EDITING AND METHODS THEREOF

Номер: US20210079426A1
Автор: CHATTERJEE Saswati, Smith Laura Jane, WONG Kamehameha
Принадлежит:

Adeno-associated virus (AAV) Clade F vectors or AAV vector variants (relative to AAV9) for precise editing of the genome of a cell and methods and kits thereof are provided. Targeted genome editing using the AAV Clade F vectors or AAV vector variants provided herein occurred at frequencies that were shown to be 1,000 to 100,000 fold more efficient than has previously been reported. Also provided are methods of treating a disease or disorder in a subject by editing the genome of a cell of the subject via transducing the cell with an AAV Clade F vector or AAV vector variant as described herein and further transplanting the transduced cell into the subject to treat the disease or disorder of the subject. Also provided herein are methods of treating a disease or disorder in a subject by in vivo genome editing by directly administering the AAV Clade F vector or AAV vector variant as described herein to the subject. 1146-. (canceled)147. A replication-defective adeno-associated virus (AAV) comprising:(a) a correction genome comprising (i) an editing element for integration into a target locus of a chromosome in a cell, the editing element comprising a coding sequence of a gene operably linked to an exogenous promoter, (ii) a 5′ homologous arm nucleotide sequence 5′ of the editing element, having homology to a 5′ region of the chromosome relative to the target locus, and (iii) a 3′ homologous arm nucleotide sequence 3′ of the editing element, having homology to a 3′ region of the chromosome relative to the target locus; and(b) an AAV capsid comprising an AAV capsid protein comprising the amino acid sequence of amino acids 203-736 of SEQ ID NO: 16.148. The AAV of claim 147 , wherein the editing element comprises a polyadenylation sequence operably linked to the coding sequence.149. The AAV of claim 147 , wherein each of the 5′ and 3′ homologous arm nucleotide sequences independently has a nucleotide length of about 50 to 2000 nucleotides.150. The AAV of claim 147 , wherein ...

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Номер записи: 98
22-03-2018 дата публикации

INTRACELLULAR TRANSLATION OF CIRCULAR RNA

Номер: US20180080041A1
Автор: KRUSE Robert
Принадлежит: Ribokine LLC

A circular mRNA molecule possessing features resembling native mammalian mRNA demonstrates improved translation, while retaining the properties of an extremely long half-life inside cells. This circular mRNA is functional inside mammalian cells, being able to compete against native cellular mRNAs for the eukaryotic translation initiation machinery. The invention possesses additional RNA elements compared to a previous invention containing only an IRES element for successful in vitro or in vivo translation. 1) A vector for making circular mRNA , said vector comprising the following elements operably connected to each other and arranged in the following sequence:a) an RNA polymerase promoter,b) a self circularizing intron 5′ slice junction,c) an IRES,d) an optional 5′ UTR,e) a multiple cloning insertion site for inserting an ORF into said vector,f) a 3′ UTR,g) an optional polyA tract,h) a self circularizing intron 3′ slice junction, andi) an optional RNA polymerase terminator.2) The vector of claim 1 , wherein the RNA polymerase promoter and terminator are from the T7 virus claim 1 , T6 virus claim 1 , SP6 virus claim 1 , T3 virus claim 1 , or T4 virus.3xenopusxenopus) The vector of claim 1 , wherein the 3′ UTRs are from human beta globin claim 1 , human alpha globin claim 1 , beta globin claim 1 , alpha globin claim 1 , human prolactin claim 1 , human GAP-43 claim 1 , human eEF1a1 claim 1 , human Tau claim 1 , human TNF alpha claim 1 , dengue virus claim 1 , hantavirus small mRNA claim 1 , bunyanavirus small mRNA claim 1 , turnip yellow mosaic virus claim 1 , hepatitis C virus claim 1 , rubella virus claim 1 , tobacco mosaic virus claim 1 , human IL-8 claim 1 , human actin claim 1 , human GAPDH claim 1 , human tubulin claim 1 , hibiscus chlorotic rinsgpot virus claim 1 , woodchuck hepatitis virus post-translational regulated element claim 1 , sindbis virus claim 1 , turnip crinkle virus claim 1 , tobacco etch virus or Venezuelan equine encephalitis virus.4xenopus ...

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Номер записи: 99
31-03-2022 дата публикации

Nucleic acid therapeutics for genetic disorders

Номер: US20220096525A1
Автор: Andrey Jury Zarur, Christian Matranga, James Robbins Abshire, Katherine J. Williams, Kelsey A. Haugh
Принадлежит: Greenlight Biosciences Inc

Provided herein, are compositions based on retroviruses (e.g., lentiviruses) comprising one or more nucleic acid molecules encoding retroviral Pol polyprotein components and a nucleic acid molecule comprising one or more transgene sequences flanked by long terminal repeat sequences, for delivery of the one or more transgenes to a target cell ex vivo or in vivo. The compositions are useful for delivering to a target cell (e.g., hematopoietic stem cells (HSCs), liver cells, ocular cells, muscle cells, epithelial cells, T cells, etc.) and/or stably expressing any transgene (e.g., beta-globin, Factor VIII, RP GTPase regulator (RPGR), dystrophin, cystic fibrosis transmembrane conductance regulator (CFTR), a chimeric antigen receptor, etc.) with a biological effect to treat and/or ameliorate the symptoms associated with any disorder related to gene expression (e.g., sickle cell disease, beta-thalassemia, haemophilia B, retinitis pigmentosa, Duchenne muscular dystrophy, cystic fibrosis, cancer, etc.).

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Номер записи: 100