159 results about "Lentivirus" patented technology

The invention belongs to the technical field of biological medicine, and particularly relates to an engineered cell microvesicle and a delivery system based on the engineered cell microvesicle, the system realizes efficient preparation of 1-5 [mu] m cell microvesicles, and the cell microvesicles have a large space volume and can be used for preparing the cell microvesicles. The carrier can be used for loading and delivery of target protein, polypeptide and recombinase which are specifically expressed in mother cells. The system transfects mother cells through lentivirus transfection or plasmid transfection to further produce cell microvesicles, and the microvesicles can load more goods and inherit membrane proteins of the mother cells, and can also effectively load intracellular proteins to realize effective delivery. By virtue of good structural stability, high immunogenicity and excellent biocompatibility, the cell microvesicle reduces systematic toxic and side effects of a traditional carrier, is expected to become an effective drug delivery system, and has great application potential in the field of gene therapy.

Among other things, provided herein are systems that replace the natural random integration activity of a retrovirus with site-specific integration machinery. This approach allows for a more precise targeting of a gene of interest into a human genome, e.g., for therapeutic purposes. The system may include integration-deficient retrovirus (e.g., lentivirus) (IDLV), in which the natural integration activity has been reduced (e.g., by mutation to the viral integrase polypeptide). Instead, the system may comprise a site-specific recombinase (e.g., a serine recombinase, e.g., a serine integrase) capable of directing insertion of a template DNA, or portion thereof, into a desired site in the human genome.

The present application relates to an anti-CD3 antibody and its application. The present application develops a specific antibody molecule targeting CD3 epsilon chain, which can activate T cells by combining with CD3 epsilon on T cells. A T cell binding protein containing the CD3 antibody is further designed, which can specifically target and bind to CD3 epsilon on the surface of T cells in vivo and in vitro, so as to achieve moderate activation of T cells. The CAR lentivirus containing the T cell binding protein of the present application can induce the generation of CAR-T cells in vitro and in vivo, and the CAR-T cells have good tumor cell killing efficiency.

The invention discloses an HER2-CAR lentivirus and application thereof, and belongs to the technical field of lentiviruses. The invention provides the CAR with a brand new structure, the expression level, the stability and the targeting property of the CAR protein in immune cells such as T cells and NK cells can be remarkably improved, the survival time of engineered immune cells in vivo can be prolonged, the lasting anti-tumor capacity of the engineered immune cells can be prolonged, and the tumor cell removing capacity can be improved; the lentiviral vector provided by the invention is high in infection efficiency and wide in host range, provides a reliable platform for large-scale preparation of clinical-grade CAR-T / CAR-NK cell products, and has huge development potential and application value in the field of immunotherapy of HER2 positive solid tumors.

The embodiment of the invention discloses a heavy chain variable region and a light chain variable region of an anti-GPC3 monoclonal antibody and application of the heavy chain variable region and the light chain variable region. The high-affinity anti-GPC3 sequence is obtained through screening, the binding specificity of the high-affinity anti-GPC3 sequence and the GPC3 antigen is high, and dissociation is slow. The sequence is used as an extracellular targeting domain to construct GPC3-CAR, after macrophages are transduced through lentivirus, CAR-M can specifically recognize GPC3 positive target cells, activate intracellular signal channels and remarkably improve in-vitro phagocytosis and killing activity, the hepatoma cell lysis effect is better, and technical support is provided for anti-hepatoma application.

The invention provides a chimeric envelope glycoprotein, a preparation method thereof and an envelope plasmid, a packaging method and a kit of the chimeric envelope glycoprotein, and particularly relates to a preparation method of the chimeric envelope glycoprotein, the chimeric envelope glycoprotein and a lentivirus envelope plasmid. The invention discloses a pseudotyped packaging method of lentivirus and a kit. The amino acid sequence of the wild type VSV-G envelope glycoprotein is chimeric with the amino acid sequences of other envelope glycoproteins, and compared with the initial wild type envelope glycoprotein, the obtained chimeric envelope glycoprotein can effectively improve the lentivirus packaging efficiency, biological activity and other properties.

The application discloses a method for detecting residual fragment distribution of recombinant lentivirus host cell DNA, which uses real-time fluorescent quantitative PCR to accurately detect HEK293T host cell DNA residual in 122bp, 244bp and 562bp three fragment length distribution in a recombinant lentivirus sample. The method of the application adopts a novel prepGEM enzyme to crack the rLVV virus, and the virus is cracked within 5-15 minutes, the enzyme is inactivated within 2-10 minutes, the virus sample amount is small, and the virus cracking solution can be directly applied to a real-time fluorescent quantitative PCR reaction. Compared with the magnetic bead method, the prepGEM enzyme cracking method is more stable and reasonable, and the variance and CV are smaller.

The application provides a quality control for detecting respiratory pathogen nucleic acid, which comprises any one or more of the following respiratory pathogens: coronavirus, influenza virus, adenovirus, Mycoplasma pneumoniae, Streptococcus pneumoniae, rhinovirus or / and Legionella pneumophila. The full-length genome sequence of the coronavirus is divided into 6 target fragments, each with a length of 4000-5500 bp. The quality control of the application covers all detection target sequences (or target points) of the coronavirus, has a wide coverage, comprehensively detects target points, and does not have the phenomenon of missed detection. The quality control of the application contains main respiratory pathogen infections, has a wide range, and uses real virus samples and lentivirus samples as raw materials, and has more accurate detection target points. The preparation of the coronavirus quality control of the application integrates the target gene sequence into the host genome through the lentivirus vector, knocks out the self-replication gene in the preparation process, and has a "self-inactivation" ability, so that the recombinant lentivirus cannot replicate in the target cells and infect other cells.

The invention belongs to the technical field of tumor immunotherapy, and relates to an anti-Claudin18.2 single-domain antibody, a multifunctional fusion protein, a recombinant natural killer cell (CT-CAR-NK), and preparation and application thereof. Through alpaca immunization and phage display library construction and panning, the single-domain antibody VHH-C18.2-1 specifically combined with Claudin18.2 is obtained, and the amino acid sequence of the single-domain antibody VHH-C18.2-1 is SEQ ID NO: 1. The amino acid sequence of the designed fusion protein is SEQ ID NO: 3, the fusion protein sequentially comprises a VHH-C18.2-1, a flexible Linker, a TGF-beta RII extracellular domain, a CD8alpha hinge region, a CD8alpha transmembrane region, a 4-1BB intracellular domain and a CD3zeta intracellular domain from the N end to the C end, and the fusion protein has the functions of targeting, resisting TGF-beta inhibition and activating signals. The fusion protein gene transfects human peripheral blood CD56 + CD3-NK cells through lentivirus to obtain CT-CAR-NK, in-vitro verification shows that the CT-CAR-NK still keeps efficient killing in an immunosuppression environment, tumor growth can be remarkably inhibited in vivo, the lifetime can be prolonged, and a safe and efficient scheme is provided for Claudin18.2 positive solid tumor treatment.

The present application relates to a kind of based on logic gate double-target point chimeric antigen receptor modified TIL cell, lentivirus expression vector and application, belong to tumor immunotherapy and gene editing technical field.The TIL cell based on logic gate double-target point chimeric antigen receptor modified in the application, double-target point chimeric antigen receptor includes chimeric antigen receptor EGFR and chimeric antigen receptor GD2;Chimeric antigen receptor EGFR is composed of CD8 alpha signal peptide, anti-EGFR single-chain antibody, CD8 alpha transmembrane region, 4-1BB costimulatory domain and CD3 zeta intracellular signal domain in series;Chimeric antigen receptor GD2 is composed of CD8 alpha signal peptide, anti-GD2 single-chain antibody, CD28 transmembrane region, CD27 costimulatory domain and CD3 zeta intracellular signal domain in series.The present application solves the defects that lentivirus transduction targeting is poor in prior art, CAR signal activation specificity is insufficient, TIL cell is easily exhausted, has the advantages that gene integration is accurate, signal transduction is controllable, in-vivo survival time is long, can be efficiently used for the immunotherapy of double-antigen co-expression solid tumor.

The present disclosure provides virus-like particles (VLPs) comprising: i) a CRISPR-Cas effector polypeptide; ii) a recombinant lentivirus comprising a nucleotide sequence encoding a therapeutic polypeptide having a length of from about 250 amino acids to about 3,000 amino acids, where the VLP comprises a pseudotyping viral glycoprotein and / or a polypeptide that provides for binding to a target cell. The present disclosure provides systems for producing a VLP. The present disclosure provides methods of delivering a therapeutic protein, using a VLP of the present disclosure.

The invention discloses a method for low-density amplification of T cells and an obtained product, and belongs to the technical field of cell culture. The method comprises the following steps: activating a cell population containing T cells, and co-culturing with a lentiviral vector carrying a target gene for transduction; the method comprises the following steps: transduction is carried out on cells, then the transduction cells are inoculated into a shake flask at an inoculation density of not higher than 2 * 10 < 5 > cells / mL, shake culture is carried out to realize amplification, and amplification of 30-120 times can be realized. According to the method, a low-density cell microenvironment is optimized through dynamic oscillation, the problems of signal insufficiency, factor dilution and metabolism imbalance are solved, the efficiency bottleneck of traditional low-density amplification is broken through, and the method is suitable for large-scale production. The method has the beneficial effects of simplicity in operation, high cell product viability, high purity, low cost, wide scale adaptability and the like. The invention also provides a T cell product prepared by the method.

The invention discloses an affinity peptide ligand for separation and purification of a VSV-G pseudotype lentiviral vector and application of the affinity peptide ligand. The affinity peptide ligand contains an amino acid sequence combined with a VSV-G pseudotype lentiviral vector envelope protein; the amino acid sequence is any one of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8. A biological raw material solution containing VSV-G pseudotype lentivirus vector components flows into the affinity chromatography medium, and after adsorption and cleaning, a target vector can be collected through a mild elution step; the recovery rate of virus vector particles is 78% or above, the removal rate of Vero host cell impure protein is 90% or above, the double-stranded DNA residue is 48% or below, and the separation effect is very remarkable.

The invention provides an organoid for simulating a dormancy and recurrence process of breast cancer cells as well as a construction method and application of the organoid. The construction method comprises the following steps: culturing breast cancer cells, and carrying out flow sorting on surface markers CD44 and ITGB4 to obtain an ITGB4 low-expression (ITGB4low) cell population; packaging a lentiviral vector, infecting a cell population with low expression of ITGB4, and carrying out flow sorting to obtain a stably transfected cell population; and carrying out induction treatment on the stably transfected cell population and culturing to obtain the organoid. The organoid provided by the invention provides powerful support for prevention and / or treatment of metastatic breast cancer, and shows extraordinary application prospects in accurate and efficient screening of drugs for targeting dormancy-recurrence process.

The invention discloses an application of an RGS1 gene inhibitor in preparation of a medicine for treating obesity-related metabolic diseases, relates to the technical field of biological medicines, and particularly relates to an application of the RGS1 gene inhibitor in preparation of a medicine for treating obesity-related metabolic diseases. The inhibitor of the RGS1 gene is used for preparing a medicine for treating obesity-related metabolic inflammatory diseases, and the medicine is used for activating a PINK1 / Parkin mediated mitochondrial autophagy pathway in macrophages; the inhibitor of the RGS1 gene is a shRNA (short hairpin Ribonucleic Acid) lentiviral expression vector capable of realizing targeted silencing of RGS1 gene expression; the application of the RGS1 gene inhibitor in preparation of drugs for treating obesity-related metabolic diseases has significant beneficial effects; the drug can accurately activate a PINK1 / Parkin mediated mitochondrial autophagy pathway in macrophages, has high targeting property, and improves the safety and effectiveness of the drug.

The invention relates to the field of immune cells. According to the preparation method and application of the novel immune cell, lentivirus containing DNA molecules subjected to gene modification is used for transfecting cells in peripheral blood of mammals, so that the cells can be passaged for multiple times, and the cells have the antigen presenting capacity and the capacity of activating and amplifying natural killer cells. After the cell is further genetically modified, better transmembrane transfer, antigen presentation and natural killer cell activation can be realized. After gene modification, the cell and different cytokines or small molecules jointly activate and amplify mononuclear cells to obtain a larger number of natural killer cells with higher purity, epigenetics are regulated and controlled to change the receptor and ligand expression quantity of the natural killer cells, and then the cytotoxicity of effector cells is improved. The invention can be used for preparing antigen presenting cells and CTL cells aiming at different antigens and an application method. The cells and the using method have wide application prospects in the aspects of prevention and treatment of tumors and infectious diseases.

This invention discloses a nanodecoy receptor that blocks the IL-17 signaling pathway, including IL-17RA-CMVs carrying a fusion sequence of PDGFR-TMD and IL-17RA. This invention also provides a method for preparing the aforementioned nanodecoy receptor that blocks the IL-17 signaling pathway, comprising the following steps: S1. Using genetic engineering, IL-17RA and PDGFR-TMD are fused and designed, and a lentivirus carrying the IL-17RA and PDGFR-TMD fusion sequence is coated onto mammalian cells. A cell line stably expressing the IL-17RA and PDGFR-TMD fusion sequence is established in mammalian cells through lentivirus transfection; S2. The cell line from step S1 is taken, and cell membrane vesicles are prepared by ultrasonic disruption and permeabilization extrusion until the fusion sequence of IL-17RA carrying PDGFR-TMD is displayed at high density and correctly oriented on the outer surface of the cell membrane vesicles, forming IL-17RA-CMVs. The nano-decoy receptor of the present invention can efficiently and broadly block the interaction between IL-17 and IL-17RA, avoid such systemic side effects, and achieve treatment only through local minimally invasive delivery, thus solving the side effects problem caused by existing systemic drug administration.

The present application relates to improvements in lentiviral manufacturing for the production of CAR-T cell drug products wherein the manufacturing method comprises a change in time between transfection and harvest of the host cell, and a new vector ratio for transfection. Presented herein are methods of making lentiviruses at various vector ratios and the time between host cell transfection and harvest, as well as transfection compositions comprising various vector ratios.

The invention provides an oligopeptide, and a lentiviral expression vector and a recombinant cell for expressing the oligopeptide. The invention further provides application of the compound in tumor treatment. After the oligopeptide is delivered to HT1080 cells through a lentivirus system, the intracellular SLC7A11 protein level is reduced, the intracellular Fe < 2 + > and lipid peroxidation level is remarkably increased, and the oligopeptide has a remarkable inhibition effect on cell proliferation and has the tumor treatment potential.

The invention particularly relates to a sebastes schlegeli endogenous retrovirus envelope protein percomORF, a lentiviral vector and application of the sebastes schlegeli endogenous retrovirus envelope protein percomORF and the lentiviral vector, and belongs to the field of genetic breeding of molecular biology, and the envelope protein percomORF has an amino acid sequence as shown in SEQ ID NO.1. The invention also covers a membrane fusion protein particle constructed based on the protein, a system for in-vitro assembly of lentivirus particles, a corresponding transformant and a matched kit. By replacing conventional VSVG protein with percomORF protein from sclerobone fish, the infection and transduction efficiency of lentivirus on sclerobone fish cells can be effectively enhanced, and more efficient gene delivery is realized. Besides, the protein belongs to host endogenous Env protein and is beneficial to avoiding immunological rejection caused by introduction of foreign protein, so that the protein has good potential in in-vivo gene delivery application.

The application discloses a method for stably producing exosomes of regenerative repair and anti-inflammatory molecules of sheep umbilical cord mesenchymal stem cells and application thereof in skin care products, and comprises the following steps: S1, using healthy sheep umbilical cord as raw material, obtaining primary sheep umbilical cord mesenchymal stem cells through aseptic treatment, Wharton's jelly extraction and tissue block adherent culture; S2, introducing an immortalization gene into the primary sheep umbilical cord mesenchymal stem cells through a lentivirus transduction technology, and obtaining an immortalized sheep umbilical cord mesenchymal stem cell line through resistance screening; S3, collecting supernatant after amplification culture of the immortalized sheep umbilical cord mesenchymal stem cell line, and purifying through multi-stage centrifugal impurity removal and PEG precipitation method to obtain stably produced exosomes containing regenerative repair and anti-inflammatory molecules. The exosomes containing regenerative repair and anti-inflammatory molecules provided by the application exhibit significant advantages in yield and quality, and have outstanding application value in the fields of medical beauty anti-aging and skin care products.

The present application provides siRNA and its application for specifically inhibiting CD36 gene expression. In particular, the present application provides siRNA, shRNA, recombinant vector, recombinant lentivirus, siRNA, host cell for specifically inhibiting CD36 gene expression, and its application in preparing a medicament for inhibiting CD36 gene expression / treating CD36 related diseases.

The application discloses application of a PRDM16 gene overexpression reagent in preparation of a medicine for preventing or treating chronic kidney disease, and realizes filling of a technical blank of the treatment of the chronic kidney disease through regulation of the PRDM16 gene by constructing a PRDM16 lentivirus or a PRDM16 plasmid overexpression reagent, and proposes a new way of treating the chronic kidney disease based on a lentivirus and a plasmid overexpression of the PRDM16 gene, which has a significant meaning for preparation of the medicine for preventing or treating the chronic kidney disease.

The invention relates to the technical field of biological medicine, and discloses a lentiviral vector for specifically targeting a target cell and a construction method and application thereof, the lentiviral vector comprises: (1) an antigen targeting artificial protein receptor, the receptor comprising a target cell surface antigen binding domain and a transmembrane domain; (2) a mutated Moreton vesicular disease virus envelope protein; and (3) an expression cassette, wherein the expression cassette comprises a heterologous transgene. The novel lentiviral vector based on the mutated Moreton vesicular disease virus envelope, provided by the invention, can realize specific infection on target cells in vitro and in vivo, and compared with a vesicular stomatitis virus envelope VSV-G, the lentiviral vector constructed based on mutated Moreton vesicular disease virus envelope protein is better in stability in serum, and has a good application prospect. The efficiency of specifically infecting target cells is higher, and the drug effect in a mouse tumor model is more prominent.

The invention belongs to the field of gene engineering, and particularly relates to application of chikungunya virus structural protein in improvement of pseudovirus stability, a construction method of the chikungunya virus structural protein and vaccines, antibody evaluation and in-vivo gene therapy products. The method comprises the following steps: constructing a human codon optimized Asian strain CHIKV-E3 + E2 + 6K + E1 structural protein eukaryotic vector, and designing two types of binder expression plasmids of targeted T cells; the vector, a psPAX2 helper plasmid and a lentivirus target vector containing an SFFV promoter and a ZsGreen reporter gene (or a CAR therapeutic gene) are co-transformed into a 293T cell, and the lentivirus is prepared through culture, filtration and centrifugation. The lentivirus can efficiently infect Jurkat cells and activate human T cells, obviously reduces the infection efficiency on 293T cells so as to reduce the off-target effect, has excellent stability in human serum, and provides a safe and efficient gene delivery tool for in-vivo CAR-T therapy.

The invention belongs to the technical field of biological pharmacy, and particularly relates to application of KMT2A as a target in preparation or screening of products for preventing or treating diabetic peripheral neuralgia diseases. KMT2A is screened as a key molecule for regulating and controlling diabetic peripheral neuralgia by establishing a diabetic peripheral neuralgia (DPN) model and combining an mRNA (messenger Ribonucleic Acid) sequencing technology. According to the invention, the lentivirus containing the KMT2A gene and a pharmaceutically acceptable carrier for treating diabetic peripheral neuralgia are constructed. The lentivirus system LV-shKMT2A packaging the knock-down KMT2A is injected into a DPN mouse through intrathecal injection, and neuroinflammation and pain sensitization of the DPN mouse can be remarkably improved. The invention provides a novel drug target for prevention and treatment of DPN mice, and has important clinical application value.

The invention discloses an eluent for improving the anion exchange chromatography recovery rate of lentivirus and application of the eluent. The eluent is prepared from a basic buffer solution, 50 to 500 mM of NaCl and 0.2 to 2 M of L-arginine; the pH (Potential of Hydrogen) value of the eluent is 6.5 to 8.0. In order to solve the problems that in the prior art, high-salt elution is adopted, so that the lentivirus activity damage is large, and the recovery rate is low, the concentration of a main elution component NaCl in an eluent is reduced, then the main elution component NaCl is compounded with L-arginine, the lentivirus can be effectively eluted, meanwhile, the damage of a high-salt environment to a virus envelope structure is avoided, and the yield of the lentivirus is improved. And the elution efficiency is improved by utilizing the characteristic that L-arginine inhibits protein interaction. When the eluent is used for chromatographic purification, the recovery rate and activity of the lentivirus can be remarkably improved, impurities such as host cell protein and the like are effectively removed, and a key purification solution is provided for preparing a high-purity and high-activity lentiviral vector for gene therapy.

The invention provides application of overexpressed CDCA5 in preparation of a medicine for enhancing the anti-tumor function of T cells, and relates to the technical field of cellular immunotherapy. According to the method for overexpressing the CDCA5, a CDCA5 overexpression agent is used, the CDCA5 overexpression agent is a carrier for overexpressing the CDCA5, and the carrier for overexpressing the CDCA5 is any one of lentivirus and retrovirus. The invention overcomes the defects of the prior art, verifies that the CAR-T overexpressing CDCA5 obviously enhances the anti-tumor function in vivo and in vitro, and provides a new technical direction for anti-tumor treatment.

The application discloses an application of a nuclear receptor target gene in preparation of a biological preparation for blocking activation of diapause larvae, and relates to the field of biotechnology and parasite prevention and control. According to two nuclear receptor targets of Haemonchus contortus HCON_00101910 (regulating activity of worms) and HCON_00023750 (regulating development of larvae), the two nuclear receptor targets are combined with host progesterone and estradiol and are activated, constitute a key signal path in the spring activation process of diapause larvae, and have no homologous genes in mammals, and the safety is excellent. By constructing specific shRNA lentivirus vectors (the silencing efficiency is all greater than or equal to 60%) targeting the two genes, single target or double target synergistic intervention is realized, and the activity-development double key links of worms in the host body can be specifically blocked. The application provides a new paradigm of preventive prevention and control with high specificity and green safety, and provides core technical support for solving the seasonal epidemic problem of blood fluke disease.

The invention discloses a virus aerosol experiment system based on pseudovirus. The virus aerosol experiment system comprises the following steps: preparing a lentiviral vector for expressing green fluorescent protein; adding the lentiviral vector expressing the green fluorescent protein into a PBS solution to obtain a virus aerosol generation solution, adding the virus aerosol generation solution into an aerosol generator, taking a DMEM culture medium containing 1% of fetal calf serum as an aerosol collection solution, putting the aerosol collection solution into a liquid impact type aerosol collector for treatment, culturing HEK293T cells, inoculating the aerosol collection solution with the HEK293T cells, and culturing the HEK293T cells to obtain the lentiviral vector expressing the green fluorescent protein. Culturing the inoculated cells for 72 hours; and after culture is completed, measuring the proportion of cells expressing fluorescence, and calculating the number of active virus particles in the virus aerosol. According to the invention, a set of virus aerosol experiment system is established, the system adopts a pseudovirus expressing fluorescent protein as a model virus to carry out a virus aerosol active particle detection experiment, and the system can be applied to a method and a technology for evaluating detection and disinfection of virus aerosol active particles in air.