39 results about "Wild type cell" patented technology

A recombinant microbial cell is capable of producing at least one compound having structural Formula II from a carbon source:In Formula II, n=2-3; R1=H or COCH3 or CH2CH2COX with X=OH or O—; R2=CH3 or CH2CH2COX with X=OH or O— where the cell comprises a genetic modification to increase activity relative to its wild-type cell of E4 where E4 is a desferrioxamine or bisucaberin synthetase (EC 6.3.-.-) (E4i) capable of converting N5-aminopentyl-N-(hydroxy)-succinamic acid to desferrioxamine B or H or at least one other linear desferrioxamine or bisucaberin according to Formula II.

The invention discloses a tetra-heterocyclic compound as well as a preparation method and application thereof, and particularly discloses a compound as shown in a formula I, and a stereoisomer or pharmaceutically acceptable salt thereof. The tetra-heterocyclic compound provided by the invention has good proliferation inhibition activity on at least one of NCI-H358 cells, Capan-1 cells and AsPC-1 cells containing KRAS mutation, has relatively weak proliferation inhibition activity on KRAS wild type PC-9 cells, and is expected to treat and / or prevent KRAS mediated diseases.

The present invention relates to mutant enzymes with enhanced properties and methods of using such enzymes to oxidize organic compound substrates. More specifically, provided herein are mutations in cytochrome P450 enzymes that enhance their peroxygenase activity and / or alter product selectivity as compared to wild-type cytochrome P450 enzymes. Also provided are uses of such mutant cytochrome P450 enzymes, e.g., for biocatalytic oxidation of carbon-hydrogen bonds by a peroxygenase pathway, or for oxidation of organic compound substrates.

The invention discloses a chimeric antigen receptor targeting EGFRvIII and application of the chimeric antigen receptor. The chimeric antigen receptor sequentially comprises an EGFRvIII targeting nucleic acid aptamer, a transmembrane domain and an intracellular signal domain from an N end to a C end, the EGFRvIII targeted nucleic acid aptamer is an EGFRvIII specific DNA aptamer obtained on the basis of SELEX (systematic evolution of ligands by exponential enrichment) screening, can only be combined with an extracellular domain of the EGFRvIII, and is not combined with an EGFR (epidermal growth factor receptor) wild type; the intracellular signal domain comprises a costimulatory signal domain and an activation signal domain, the C end of the costimulatory signal domain is connected with the N end of the activation signal domain, and the EGFRvIII specific nucleic acid aptamer screened based on SELEX is adopted as a targeting domain, can only be specifically combined with the extracellular domain of EGFRvIII positive tumor cells, and is not subjected to cross combination with EGFR wild cells and normal cells, so that the EGFRvIII specific nucleic acid aptamer can be used for detecting EGFRvIII positive tumor cells, and the EGFRvIII specific nucleic acid aptamer can be used for detecting EGFRvIII positive tumor cells. The problem of off-target killing caused by the fact that a common antibody scFv fragment of a traditional chimeric antigen receptor (CAR) is easily combined with a wild type EGFR is fundamentally avoided.

T lymphocytes play a key role in the immune response and their functions are intimately linked to metabolic programs. During immune responses, T cells undergo a metabolic reprogramming notably characterized by an increased aerobic glycolysis. Using a quantitative phosphoproteomic approach, the inventors have identified a new transcription factor called Foxk1 as being highly phosphorylated in T cells upon T Cell Receptor (TCR) engagement. The results also indicate that Foxk1 phosphorylation and nuclear translocation is dependent of the AKT-mTOR kinase activities. Using T-cell specific Foxk1 deficient mice (Foxk1- / -), we demonstrated that Foxk1 is required for full T cell activation. Foxk1-deficient T cells exhibited reduced proliferation and cytokine secretion following TCR stimulation. Furthermore, T cells from Foxk1- / - mice were less prone to acquire an effector like phenotype than wild-type cells when challenged in vivo. Conversely, Foxk1 overexpression in T cells enhanced their effector functions in a TCR-dependent manner. In CD8+ T cells, this effect also results into enhanced cancer cell killing capacity in vitro, and improved tumor rejection in vivo. Altogether, these results indicated that Foxk1 is a major regulator of T cell metabolism and thus, of T cell effector functions. Thus, the present invention relates to host immune cells engineered to overexpress a Foxk1 polypeptide and their use of the treatment of cancer.

The present invention discloses, in one embodiment, a method of using human induced pluripotent stem cells to generate three-dimensional human organ tissue for therapeutic drug toxicity and discovery⋅. In one embodiment, a high throughput microtiter plate is loaded with both wild type and Rett disease 3D spheroids and exposed to a drug library, and activity is measured and analyzed for disease rescue to wild type cell behavior.

This invention relates to the biotechnological production of ω-functionalized carboxylic acids and their esters. Microbial cells are provided for the production of at least one ω-functionalized carboxylic acid ester from at least one alkane, wherein the cells are genetically modified relative to wild-type cells to increase the expression of: (i) an enzyme E1 capable of converting the alkane to the corresponding 1-alkanol; (ii) an enzyme E2 capable of converting the 1-alkanol of (i) to the corresponding 1-alkanal; (iii) an enzyme E3 capable of converting the 1-alkanal of (ii) to the corresponding alkanoic acid; (iv) an enzyme E4 capable of converting the alkanoic acid of (iii) to the corresponding alkanoic acid ester; and (iv) an enzyme E5 capable of converting the alkanoic acid ester of (iv) to the corresponding ω-hydroxy-alkanoic acid ester, and wherein the cells do not contain an increase relative to wild-type cells of the expression of the following enzymes selected from the group consisting of: 20 -E 24 Genetic modification of expression of at least one of them: -E 20 Acyl-ACP thioesterase, -E 21 Acyl-CoA thioesterase, -E 22 Acyl-CoA:ACP transacylase, -E 23 Polyketide synthase, and -E 24 Hexanoic acid synthase.

This disclosure relates to formulations comprising one or more cells, wherein the cells of the formulation are modified to conditionally express: (i) increased levels of one or more immune checkpoint proteins compared to corresponding wild-type cells; (ii) decreased levels of one or more HLA-I proteins compared to corresponding wild-type cells; or a combination of (i) and (ii). This disclosure further relates to methods and constructs for producing said cell formulations, and methods for administering said cell formulations to subjects in need.

The invention belongs to the field of gene engineering, and particularly relates to construction of an RNF31 gene knockout cell line and application of the RNF31 gene knockout cell line as a foot-and-mouth disease virus vaccine production cell line. The method comprises the following steps: firstly, designing two sgRNA sequences at an RNF31 exon by utilizing a CRISPR / Cas9 technology, and connecting with a pX459-puro vector to construct a recombinant plasmid; the method comprises the following steps: transfecting a PK-15 cell with pX459-RNF31-sgRNA, and screening under the action of puromycin, so as to obtain the RNF31 gene knockout cell line. Compared with a wild type cell, the RNF31 gene knockout cell line remarkably promotes FMDV replication and can be used as a production cell line of a foot and mouth disease virus vaccine; and data support is provided for further researching a mechanism of inhibiting FMDV replication by RNF31.

This invention discloses the application of T cells overexpressing PRDM16 in the preparation of anti-tumor or infectious disease drugs. Through experiments, this invention verifies that overexpression of PRDM16 enhances the in vivo and in vitro activity of T cells. It was found that in vitro overexpression of PRDM16 significantly enhances the killing effect of T cells on target cells. In vivo reinfusion of PRDM16 has stronger anti-tumor and anti-infection functions compared to wild-type T cells. Therefore, it can be used in malignant tumors and infectious diseases to enhance the efficacy of T cell adoptive therapy and solve the defect of T cell adoptive therapy where the killing power of target cells is suppressed due to environmental influences.

The invention belongs to the technical field of medicines, and particularly relates to application of a ciclopirox compound in a medicine for treating cancers induced by KRAS G12D mutation, and the cancers comprise pancreatic ductal adenocarcinoma and colorectal cancer. The compound can obviously inhibit the activity of KRAS mutant pancreatic cancer cells, has weak inhibition on KRAS wild type cells, and shows potential targeted therapy advantages. Animal experiment results show that the ciclopirox compound provided by the invention can effectively inhibit tumor growth and down-regulate phosphorylated STK31 protein expression. The composition is clear in component and simple and convenient to prepare, and provides a new strategy for overcoming the drug resistance of pancreatic cancer.

The invention belongs to the technical field of gene engineering, and particularly relates to application of a TP63 gene in improving the expression quantity of exogenous recombinant protein, and the nucleotide sequence of the TP63 gene is shown as SEQ ID NO.1. According to the invention, the TP63 overexpression vector is creatively applied to construction of a recombinant protein expression system, the TP63 overexpression vector is transfected into CHO cells to construct a CHO cell line for overexpressing TP63, and the CHO cell line can significantly improve the expression level of TP63 protein. Meanwhile, the CHO cell line is used as a host cell for recombinant protein expression, compared with a wild type CHO cell line, the expression quantity of recombinant protein can be increased, and the problem that the expression level of antibody expression quantity in an existing CHO cell expression system is low is effectively solved.

The invention belongs to the technical field of biology, and particularly relates to a VP2 protein mutated O-type foot-and-mouth disease recombinant virus as well as preparation and application thereof. The 88th lysine residue of O-type FMDV-VP2 protein is mutated into arginine residue by using a reverse genetic manipulation technology to successfully rescue the O-type foot-and-mouth disease recombinant virus strain rO-VP2K88R, and compared with an O-type foot-and-mouth disease wild type virus strain rO-WT rescued by using the reverse genetic manipulation technology, the recombinant virus strain provided by the invention has the advantages that the recombinant virus strain has the advantages that the yield is high; the growth replication capacity in wild type PK-15 cells is remarkably reduced, and the pathogenicity to suckling rats is also remarkably reduced. Therefore, the obtained O-type foot-and-mouth disease recombinant virus strain rO-VP2K88R possibly has certain immunogenicity and is expected to become a vaccine candidate strain, and a basic research material is provided for researching the foot-and-mouth disease vaccine candidate strain.

Targeting the ability of T cells to enhance aerobic glycolysis upon TCR stimulation or glucose supplementation represents an attractive strategy for the treatment of autoimmune diseases. Using a quantitative phosphoproteomic approach, the inventors have identified a new transcription factor called Foxk1 as being highly phosphorylated in T cells upon T Cell Receptor (TCR) engagement. The results also indicate that Foxk1 phosphorylation and nuclear translocation is dependent of the AKT-mTOR kinase activities. Using T-cell specific Foxk1 deficient mice (Foxk1- / -), the inventors demonstrated that Foxk1 is required for full T cell activation. Foxk1-deficient T cells exhibited reduced proliferation and cytokine secretion following TCR stimulation. Furthermore, T cells from Foxk1- / - mice were less prone to acquire an effector like phenotype than wild-type cells when challenged in vivo. Altogether, these results indicated that targeting can be envisioned to reduce auto-immune reactions. Thus, the present invention relates to the use of Foxk1 inhibitors for the treatment of inflammatory autoimmune diseases.

Provided are a mesenchymal stem cell (MSC) with an enhanced targeted tumor infiltration ability, and the use thereof. The MSC with an enhanced targeted tumor infiltration ability comprises a polynucleotide encoding an NKG2DL-targeting chimeric antigen receptor. The provided MSC with an enhanced targeted tumor infiltration ability can not only normally overexpress the NKG2DL-targeting chimeric antigen receptor, but also has a better solid tumor targeting property, thus being able to effectively break through solid tumor microenvironments and enter the interior of tumors. Compared to wild-type MSC cells, the MSC has a better retention ability at the tumor site, which enhances the survival ability thereof at the tumor site, thereby enabling the MSC to exert a more long-acting and sustained tumor inhibitory effect at the tumor site and further promoting the use of the MSC in the field of solid tumor treatment.

The invention provides an anti-leukemia small molecule compound TIP-20 targeting FLT3 and application of the anti-leukemia small molecule compound TIP-20, and belongs to the technical field of biological medicine. The invention develops a novel FLT3 kinase inhibitor TIP-20, which can enhance the antigen presentation effect of FLT3-ITD leukemia cells, can be combined with FLT3 protein, stabilizes the structure of FLT3, and further inhibits FLT3 phosphorylation and downstream pathway activation. The TIP-20 has an obvious inhibiting effect on the growth of leukemia cell strains, and can inhibit the growth of leukemia cells with FLT3 mutation by inhibiting phosphorylation of FLT3, promoting cell apoptosis and cycle arrest. Moreover, FLT3 wild type cells have good tolerance to TIP-20, have a wide therapeutic window with FLT3-ITD mutated acute myelogenous leukemia cells, and are expected to bring a better choice for treatment of FLT3 mutated AML patients.

The invention discloses an application of a ZFYVE27 gene or an encoded protein thereof in regulating and controlling ASFV resistance of a host, and belongs to the fields of molecular biology and prevention and treatment of animal infectious diseases. A ZFYVE27 gene knockout or silence cell model is constructed by using a CRISPR / Cas9 gene editing technology and an RNAi technology, the gene knockout cells show significant resistance to ASFV infection, the proliferation speed of ASFV on the gene knockout cells is significantly lower than that of wild cells, and it is indicated that the ZFYVE27 gene plays an important role in regulating and controlling host virus resistance. The antiviral regulation function of the ZFYVE27 gene is identified from the cellular level, it is found for the first time that the gene can be used as a potential target for gene editing cells and animal design to be applied to disease-resistant breeding and drug research and development, and the gene has important significance for reducing drug abuse and avoiding epidemic situations.

Provided herein are genetically modified T cells that exhibit increased proliferation compared to wild-type T cells when stimulated, methods of generating such T cells, and methods of using the T cells for the treatment of a disease such as cancer.

The invention provides mesenchymal stem cells capable of enhancing tumor targeted infiltration capacity and application of the mesenchymal stem cells. The mesenchymal stem cell capable of enhancing the tumor targeted infiltration capacity comprises polynucleotide of a chimeric antigen receptor for coding targeted NKG2DL. The mesenchymal stem cell capable of enhancing the tumor targeting infiltration capacity not only can normally overexpress the NKG2DL targeting chimeric antigen receptor, but also has better solid tumor targeting property, and can effectively break through the solid tumor microenvironment and enter the tumor. Compared with wild type MSC cells, the MSC cells have better retention ability in tumor sites, and the survival ability of the MSC cells in the tumor sites is enhanced, so that the mesenchymal stem cells can play a more long-acting continuous tumor inhibition role in the tumor sites, and the application of the MSC cells in the field of solid tumor treatment is further promoted.

The invention discloses an application of T cells overexpressing PRDM16 in preparation of drugs for resisting tumors or treating infectious diseases, experiments prove that overexpressing PRDM16 can enhance in-vivo and in-vitro activity of the T cells, and discovers that in-vitro overexpressing PRDM16 enables the killing effect of the T cells on target cells to be remarkably enhanced; compared with wild type T cells, in-vivo feedback has stronger anti-tumor and anti-infection functions, so that the T cells can be applied to malignant tumors and infectious diseases, the curative effect of T cell adoptive therapy is enhanced, and the defect that killing power is inhibited due to the fact that target cells are affected by the environment by the T cell adoptive therapy is overcome.

Disclosed herein are single chain dimeric cytokines having improved physiological activity over wild-type cytokine monomers. The single chain dimeric cytokines of the disclosure may be useful in the treatment of various diseases or disorders relating to inflammatory conditions or proliferative conditions. Also disclosed are delivery vectors or agents for delivering the single chain dimeric cytokines to a subject. Such delivery vectors or agents include cells, bacteria and bacteriophages.

The application provides a CRISPR gene editing structural variation detection method, taking the CRISPR edited cell genome DNA as an experimental group, taking the homologous wild type cell genome DNA as a control group, respectively performing Long-range PCR amplification, and using high-throughput double-end sequencing data of the amplification product for bioinformatics analysis, the bioinformatics analysis comprising: (1) using fastp software to perform quality control on the sequencing data; (2) screening target sequencing read pairs of large fragment deletion based on a local alignment algorithm combining k-mer hash table alignment and longest increasing subsequence identification; (3) screening large fragment deletion candidate sequencing read pairs crossing the cleavage site; (4) excluding false positive large fragment deletion based on a k-means clustering algorithm; (5) screening effective large fragment deletion structural variation events. The application can more efficiently and accurately detect large fragment deletion at a CRISPR gene editing target point.

The invention discloses a construction method of a hepatocyte line with high albumin yield, a plasmid and a hepatocyte line. According to the invention, a human serum albumin gene and a YAP gene are inserted into a genome of HepG2 human liver tumor cells so as to obtain a liver cell line with high albumin yield; the nucleotide sequence of the human serum albumin gene is as shown in SEQ ID No: 1, and the nucleotide sequence of the YAP gene is as shown in SEQ ID No: 2. According to the HepcellPro-H cell line constructed by the invention, under a specific culture condition, the concentration of albumin in a cell culture supernatant detected by ELISA (Enzyme-Linked Immunosorbent Assay) is obviously increased compared with that of an unmodified wild type HepG2 cell, the production efficiency is obviously improved, the large-scale extraction period of albumin is shortened, and the potential tumorigenic risk of a viral vector is avoided.

A peptide variant and a fusion protein and use thereof are provided. Specifically, provided are a wild-type B-cell maturation antigen CRD (cysteine-rich domain), a mutation sequence thereof, and a fusion protein comprising the peptide and use thereof.

The invention relates to the technical field of gene therapy, in particular to a method for knocking out a Bax gene on the basis of an HEK293 cell line by utilizing a CRISPR / Cas9 technology, constructing a stable cell line expressed by foreign protein and improving the AAV packaging efficiency. The invention discloses a Bax2.6 cell strain. A pro-apoptosis factor Bax gene is knocked out from an HEK293T cell. Compared with a wild type HEK293T cell, the Bax2.6 cell strain provided by the invention shows higher cell activity under various stress conditions, the cell survival rate of the Bax2.6 cell strain is improved by at least 20% under the treatment conditions of small molecule inhibitors ZV-FMK, Nec-1, puromycin (Puro), cis-platinum (CDDP) and rotenone, and the Bax2.6 cell strain shows excellent environmental tolerance and production robustness. When the Bax2.6 cell strain provided by the invention is used for transient transfection and packaging of an adeno-associated virus (AAV) vector, the copy number of the obtained AAV virus can be up to 2 * 10 copies / mL, and the AAV packaging efficiency is improved by more than 10 times compared with that of a conventional HEK293T cell line.

The present application relates to an engineered cell, an engineered protomyostatin protein, compositions comprising the same, nucleic acids encoding the same, and methods of production thereof. The engineered cells comprise a myostatin gene that is artificially modified. In some embodiments, the engineered cell expresses less myostatin mRNA as compared to a wild type cell. In addition, the engineered cell can express a mature myostatin protein having the same amino acid sequence as a wild-type animal.

The invention provides an engineered cell as well as a preparation method and application thereof. Relative to a wild type cell, the engineered cell overexpresses a CD47 molecule; based on the density of the CD47 molecules on the cell surface, the distribution density of the CD47 molecules on the surface of the engineered cell is not less than 2700 molecules / mu m < 2 >. The engineered cell has good ultra-low immunogenicity, can avoid killing of T cells, PBMC cells and NK cells at the same time, remarkably prolongs the in-vivo survival time of transplanted cells, is expected to generate universal cell products, and solves the problem of immunological rejection.

This invention belongs to the field of molecular biology, specifically relating to the application of SND1-overexpressing cells in promoting small RNA virus replication. The invention first constructs an SND1 gene overexpression vector, transfects it into BHK-21 cells, and then selects a stable cell line. Infection of the cell line with foot-and-mouth disease virus (FMDV) and Seneca virus (SVV) revealed that the titers of FMDV and SVV, as well as the expression levels of viral RNA and proteins, were significantly higher than in wild-type cells, indicating that SND1 promotes FMDV and SVV replication. The constructed SND1-overexpressing cells can efficiently proliferate FMDV and SVV and can be used as cell lines for FMDV and Seneca virus vaccine production. This provides a new tool for FMDV and SVV mechanism research, vaccine preparation, and the development of detection reagents, and has significant scientific research and application value.

This invention presents the CVOM-2 Gram-negative bacterial strain of Caulobacter crescentus, which has a point mutation in the ompA2 gene resulting in the substitution of arginine 351 for alanine, and a null mutation in the rsaA gene that produces outer membrane vesicles (EMVs) with greater efficiency, quantified as the amount of protein / ml of original culture. These EMVs produce a lower inflammatory response than similar preparations obtained from wild-type Escherichia coli cells or cells modified to reduce this response, or even than preparations obtained from a Caulobacter crescentus strain with a wild-type cell envelope.Genetic methods are also presented that allow the modification of these membrane-bound extracellular matrices (MBs), resulting in the presence of a heterologous protein or antigen in soluble form within the vesicle lumen or associated with the vesicle membrane as part of an integral membrane protein. The antigen may be exposed to the lumen or the vesicle surface, or anchored to the outer membrane via lipids. These MBs induce the generation of antibodies against a heterologous protein present within them. MBs also have an adjuvant effect, inducing antibody generation in response to antigen concentrations that would not otherwise elicit this response.

The invention discloses a construction method of a liver cell line with high blood coagulation factor yield, a plasmid and a cell line. According to the invention, the F10 gene and the YAP gene are inserted into the genome of HepG2 human liver tumor cells to obtain the liver cell line with high blood coagulation factor yield; the nucleotide sequence of the F10 gene is as shown in SEQ ID No: 1, and the nucleotide sequence of the YAP gene is as shown in SEQ ID No: 2. According to the HepcellPro-F cell line constructed by the invention, under specific culture conditions, the concentration of the blood coagulation factors in cell culture supernatant detected by ELISA is obviously increased compared with that of unmodified wild type HepG2 cells, so that the production efficiency is obviously improved, the large-scale extraction period of the blood coagulation factors is shortened, and the potential tumorigenic risk of a viral vector is avoided.