23 results about "Bicistronic mrna" patented technology

The invention discloses a pichia pastoris high-secretion strain screening system and a use method thereof. According to the system, pichia pastoris is used as a host, and the core structure of the system is as follows: an AOX1 promoter, an alpha-factor signal peptide, a target protein-P2A peptide, an alpha-factor signal peptide, a Gamilus fluorescent protein-G4S connecting peptide-Gcw21 anchoring sequence-AOX1 terminator. The use method comprises the steps of vector construction, conversion of enzyme digestion recovery products, clone strain selection and activation, fermentation, inducible expression and screening, and product collection to finally obtain the strain with high yield of target protein. Through integration of bicistronic design, P2A peptide self-cleavage and cell surface display technologies, efficient screening of target protein high-secretion type strains is realized.

Aspects of the disclosure relate to bicistronic AAV nucleic acid constructs comprising a transgene encoding hexosaminidase A (HEXA) and hexosaminidase (HEXB) proteins. In some embodiments, the disclosure provides methods for treating or preventing lysosomal storage disorders, such as Tay-Sachs disease and Sandhoff disease, using bicistronic nucleic acid constructs described by the disclosure.

Provided herein are compositions and methods of using a bicistronic vector for treating or preventing a lysosomal storage disorder (LSD) in a subject. The disclosed compositions comprise a bicistronic vector comprising a promoter, an Internal Ribosome Entry Site (IRES), a polynucleotide encoding a lysosomal enzyme and a polynucleotide encoding a modified GlcNAc-1 phosphotransferase (GlcNAc-1 PTase). The present methods comprise administering to the subject a pharmaceutical composition comprising the bicistronic vector as disclosed herein.

Chimeric antigen receptors (CARs) and bicistronic chimeric antigen receptors (BiCisCARs) that target fibroblast growth factor receptor 4 (FGFR4), CD276, or both are disclosed. The CARs and BiCisCARs include a hinge and transmembrane domain from either CD28 or CD8 and a co-stimulatory domain from either CD28 or 4-1BB. The CARs and BiCisCARs can further include amino acid substitutions in one or more immunoreceptor tyrosine-based activation motifs (ITAMs) of a CD3ζ intracellular signaling domain. Cells expressing the CARs or BiCisCARs can be used for the treatment of cancers that express one or both of FGFR4 and CD276.

This invention discloses a recombinant vector, recombinant bacteria, and fermentation method for high-yield GHK production, belonging to the field of biomedical technology. The recombinant expression vector of this application comprises a bicistronic expression cassette controlled by a T7 / lac heterozygous promoter, sequentially containing the encoding (GHK). n The vector encodes the first cistron of the polypeptide and the second cistron, where n is an integer from 2 to 200. When this vector is transformed into *E. coli* BL21, the resulting recombinant bacteria can simultaneously express (GHK) after IPTG induction. n Polypeptides and trypsinogen. During fermentation, trypsinogen is activated and enables the processing of (GHK). n In situ enzymatic cleavage of peptides efficiently releases GHK. This invention integrates the traditional multi-step process into a "one-step fermentation," simplifying the production process and reducing purification difficulty. Examples show that the GHK yield can reach up to 3.9 g / L, providing an efficient and convenient new strategy for the large-scale biomanufacturing of GHK.

This disclosure relates to bicistronic polypeptide constructs for use in allogeneic gene therapy, such as CAR-T cell therapy. A bicistronic construct comprises a first polynucleotide encoding a therapeutic molecule (e.g., CAR-T or antibody) and a second polynucleotide encoding an immune surveillance masking molecule (ISMM). The ISMM includes a non-functional version of a protein knockout, such as a human leukocyte antigen E genetically fused to a fragment, by insertion of a bicistronic construct, such as beta-2 microglobulin or B2M. Also provided are vectors comprising bicistronic constructs, cells (e.g., CAR-T cells), and methods of use. Kits and manufacturing articles are also provided. This disclosure also provides four novel insertion sites that may be used to insert expression constructs into the B2M gene.

The present disclosure relates to bicistronic polypeptide constructs for use in allogeneic gene therapy, such as CAR-T cell therapy. The bicistronic constructs comprise a first polynucleotide encoding a therapeutic molecule (e.g., CAR-T or an antibody) and a second polynucleotide encoding an immune surveillance masking molecule (ISMM). The ISMs comprise a human leukocyte antigen-E genetically fused to a non-functional form, such as a fragment, of a protein, such as beta-2 microglobulin or B2M, which is knocked out by insertion of the bicistronic construct. Also provided are vectors, cells (e.g., CAR-T cells) comprising the bicistronic constructs and methods of use. Kits and articles of manufacture are also provided. The present disclosure also provides four new insertion sites that can be used to insert an expression construct into the B2M gene.

Chimeric antigen receptors (CARs) and bicistronic chimeric antigen receptors (BiCisCARs) that target fibroblast growth factor receptor 4 (FGFR4), CD276, or both are disclosed. The CARs and BiCisCARs include a hinge and transmembrane domain from either CD28 or CD8 and a co-stimulatory domain from either CD28 or 4-1BB. The CARs and BiCisCARs can further include amino acid substitutions in one or more immunoreceptor tyrosine-based activation motifs (ITAMs) of a CD3ζ intracellular signaling domain. Cells expressing the CARs or BiCisCARs can be used for the treatment of cancers that express one or both of FGFR4 and CD276.

This invention relates to a mouse model of Parkinson's disease and its applications. The construction method includes the following steps: 1) in mice... Snack An exogenous fragment containing the human SNCA G51D coding sequence was knocked into the region upstream of the gene stop codon via homologous recombination, causing the human SNCA G51D coding sequence to interact with the mouse endogenous gene. Snack The gene forms a bicistronic expression structure, and the coding sequence of the human SNCA G51D is shown in SEQ ID No:1; 2) Positive F0 generation mice are backcrossed with wild-type mice, and genotypes are identified and screened to obtain a stable SNCA G51D endogenous knock-in Parkinson's disease mouse model. The gene editing strategy of the Parkinson's disease mouse model preserves the mouse's... Snack Simultaneous endogenous expression of α-synuclein G51D in the gene can mimic two pathogenic forms, exhibiting high physiological relevance and genetic authenticity.

The present disclosure provides nucleic acid molecules (e.g., a plasmid or vector) comprising a nucleic acid sequence encoding a bicistronic or multicistronic LAMP Construct comprising specific fragments of the LAMP luminal domain and an antigenic domain heterologous to the LAMP protein to provide at least one antigen for priming an immune response, wherein the antigen expressed by the Construct is optionally processed and presented to MHC class II molecules, and also a nucleic acid sequence encoding an immune response enhancing polypeptide that is optionally secreted from a host cell. The nucleic acid molecules can be used, for example, for the treatment of disease and in particular, allergies, infectious disease, diabetes, hyperproliferative disorders and / or cancer.

This invention provides a bicistronic CAR-T cell structure that simultaneously targets CD19 and CD72 and its application. The nucleic acid molecule encoding the corresponding chimeric antigen receptors includes at least nucleotide sequences encoding a first chimeric antigen receptor, a second chimeric antigen receptor, and a cleavage peptide. The nucleotide sequence encoding the cleavage peptide is located between the nucleotide sequences encoding the first and second chimeric antigen receptors. The first antigen-binding domain is specific for CD19, and the second antigen-binding domain is specific for CD72. This nucleic acid molecule is a bicistronic structure capable of independently expressing both CD19 and CD72, achieving balanced co-expression of these two independent CAR-T molecules (CD19 CAR and CD72 CAR), thus providing an effective treatment option for relapsed / refractory patients.

The present disclosure provides nucleic acid molecules (e.g., a plasmid or vector) comprising a nucleic acid sequence encoding a bicistronic or multicistronic LAMP Construct comprising specific fragments of the LAMP luminal domain and an antigenic domain heterologous to the LAMP protein to provide at least one antigen for priming an immune response, wherein the antigen expressed by the Construct is optionally processed and presented to MHC class II molecules, and also a nucleic acid sequence encoding an immune response enhancing polypeptide that is optionally secreted from a host cell. The nucleic acid molecules can be used, for example, for the treatment of disease and in particular, allergies, infectious disease, diabetes, hyperproliferative disorders and / or cancer.

This specification provides methods for detecting the expression of bicistronically expressed polypeptides, for example, in a population of immune cells, such as a population of CAR T cells, that have been manipulated into chimeric cytokine receptors. In one aspect, this disclosure provides an in vitro method for detecting bicistronically expressed polypeptides in chimeric antigen receptor (CAR) T cells.

Provided herein arc polynucleotides comprising at least two coding sequences for expression of coding sequences of interest in a cell. Also provided are vectors, recombinant viral genomes, and recombinant virus compositions comprising said polynucleotides, as well as associated methods.

According to the bicistronic DNA vaccine for edwardsiella tarda disease of paralichthys olivaceus, provided by the invention, the bicistronic DNA vaccine can remarkably induce Th1 type immune bias, enhance immune response of fish cells and improve the resistance to intracellular bacterial infection, and has the advantages of being safe, efficient, easy to prepare, high in field popularization adaptability and the like. A fusion fragment of an immunologic adjuvant IL-12 is used in the bicistronic DNA vaccine, and the bicistronic DNA vaccine is constructed by connecting a nucleic acid fragment of a subunit 12p35 of which the coding amino acid sequence is SEQ ID NO: 1 and a nucleic acid fragment of a subunit 12p40 of which the coding amino acid sequence is SEQ ID NO: 3 through a flexible connecting peptide joint. According to the bicistronic DNA vaccine disclosed by the invention, the synergistic expression of the OmpC antigen and the IL-12 adjuvant is realized, the immunogenicity is enhanced, and the problem of insufficient immune effect of the traditional DNA vaccine is solved; and the vaccine has no obvious toxic or side effect on the fish body, does not depend on antibiotics, is green and environment-friendly, and has a good industrialization prospect.

Embodiments described herein provide orthohepadnavirus capsid protein (Cp) heterodimers, bicistronic vectors encoding the heterodimers, and methods for producing the heterodimers. The heterodimers can be used to form mosaic virus-like particles. In certain embodiments, the heterodimers can form a hexamer, which in turn can be used to nucleate capsid formation, resulting in a Janus particle-like virus-like particle. The hexamer's can then be removed, leaving holey capsids. The capsids can be loaded with, for example, one or more polypeptides, small molecules, or a combination of polypeptides and small molecules. The holes of the holey capsids can be filled with another orthohepadnavirus heterodimer or a homodimer.

The present invention relates to a screening composition for a therapeutic agent for coronavirus infection, comprising a CoV RdRp expression vector and a bicistronic reporter vector, a screening kit for a therapeutic agent for coronavirus infection, comprising the composition, and a method for screening a therapeutic agent for coronavirus infection using the composition or kit. When the screening composition for a therapeutic agent for coronavirus infection, provided by the present invention, is used, candidate materials that can have direct influences on the activity of CoV RdRp can be screened more quickly and easily, and thus, the composition can be widely used in the development of therapeutic agents for coronavirus infection.

Transgenic T cells and vectors for making transgenic T cells are described. The vectors can include a nucleic acid encoding a membrane-bound anti-IL6 (mb-alL6) single chain variable fragment (scFv), and the transgenic T cells can express mb-alL6. The transgenic T cells are useful for suppressing proliferation of IL-6-dependent cells, reducing IL-6 concentration, or both. In one embodiment, the vector is a bicistronic construct encoding the mb-alL6 and an anti-CD19-41 BB-003ζ chimeric antigen receptor (CAR). In another embodiment, an anti-IL-6 scFv can be linked to a 41 BB and 003ζ domains to form an anti-IL-6 CAR. The transgenic T cells expressing said constructs can reduce the linker risk of cytokine release syndrome (CRS) in cancer patients being treated with CAR T cell or for the treatment of autoimmune diseases and inflammatory diseases in which cytokines are involved in pathogenesis.

Provided herein are polynucleotides comprising at least two coding sequences for expression of coding sequences of interest in a cell and two transcriptional regulatory elements. The transcriptional regulatory elements may be tissue specific. Also provided are vectors, recombinant viral genomes, and recombinant virus compositions comprising said polynucleotides, as well as associated methods.

The present disclosure provides compositions comprising IFNE and methods of using the same to treat cancer and / or enhancing responsiveness to immune checkpoint blockade therapy in a patient in need thereof. Also disclosed herein are compositions including tandem bicistronic expression cassettes, and methods of using the same to generate large genomic deletions and / or knock-in gene alterations.

This invention discloses a bicistronic translation-coupled expression vector. The expression vector is constructed by inserting a molecular chaperone, a novel ribosome binding site, and the target protein gene into the pET-28a(+) plasmid. Furthermore, this invention constructs a genetically engineered bacterium using *E. coli* BL21(DE3) as the expression host. Utilizing the bicistronic translation-coupled expression vector, the co-expression of mussel protein Mgfp-3B with the molecular chaperones SUMO and / or TrxA is achieved, promoting the correct folding and translation of Mgfp-3B for expression in a soluble form. This invention enables a simple and rapid production of bioactive soluble mussel protein in *E. coli*, and the bicistronic expression vector avoids the enzymatic chaperone manipulation required for fusion expression. In addition, this invention, through optimized fermentation medium and fermentation process, achieves a soluble mussel protein yield of 200-300 mg / L, simplifying downstream purification processes.

Aspects of the disclosure relate to bicistronic AAV nucleic acid constructs comprising a transgene encoding hexosaminidase A (HEXA) and hexosaminidase (HEXB) proteins. In some embodiments, the disclosure provides methods for treating or preventing lysosomal storage disorders, such as Tay-Sachs disease and Sandhoff disease, using bicistronic nucleic acid constructs described by the disclosure.

The invention discloses a recombinant vector for high yield of GHK, recombinant bacteria and a fermentation method, and belongs to the technical field of biological medicines. The recombinant expression vector comprises a bicistronic expression cassette controlled by a T7 / lac hybrid promoter, the bicistronic expression cassette sequentially comprises a first cistronic for coding (GHK) n polypeptide and a second cistronic for coding trypsinogen, and n is an integer from 2 to 200. The vector is transferred into Escherichia coli BL21, and the obtained recombinant bacterium can synchronously express the GHK n polypeptide and trypsinogen after being induced by IPTG (isopropyl-beta-d-thiogalactoside). In the fermentation process, trypsinogen is activated and in-situ enzyme digestion of the GHK n polypeptide is realized, so that the GHK is efficiently released. According to the method, a traditional multi-step process is integrated into one-step fermentation, the production process is simplified, the purification difficulty is reduced, the highest GHK yield can reach 3.9 g / L according to the embodiment, and an efficient and convenient new strategy is provided for large-scale biological manufacturing of GHK.