16 results about "Genetic transduction" patented technology

The invention belongs to the technical fields of immunotherapy, bioengineering, gene therapy and cell therapy, and provides a preparation method of an enhanced CAR-T cell for overexpressing LYAR, and the enhanced CAR-T cell and application thereof in order to solve the problems that the killing function of the CAR-T cell in solid tumor treatment is limited, the durability is poor, and function depletion is likely to occur. CDNA of the LYAR gene is cloned to an overexpression plasmid vector and packaged into lentivirus, T cells are jointly infected by the lentivirus and the CAR expression virus, the LYAR gene is forced to express under the driving of an EF-1alpha promoter, and stable overexpression of the LYAR in the CAR-T cells is achieved; and the CAR is a CAR targeting EGFR (epidermal growth factor receptor) and CD19. According to the present invention, the gene coding LYAR and the CAR gene are simultaneously transferred into the T cell through the lentivirus infection technology, and are compulsively expressed under the action of the EF1 alpha promoter, such that the anti-tumor function of the CAR-T cell can be significantly enhanced, and the activation and killing functions of the immune cell can be improved;

The invention discloses an enamel secretion prolonging and thickness increasing method based on KLK4 alpha protein, and relates to the technical field of biomedicine and dental materials.According to the method, expression of the KLK4 alpha protein is specifically regulated and controlled, forward intervention in the secretion period in the enamel development process is achieved, synthesis and accumulation of enamel matrix protein are directly promoted, and the effect of prolonging the secretion period of the enamel is achieved. A recombinant lentiviral vector mediated gene transduction technology is utilized to specifically introduce the KLK4alpha gene into a dental embryo glazing cell and realize overexpression, and the expressed KLK4alpha protein remarkably delays the process of transformation from a secretion period to a mature period by regulating the secretion activity of the glazing cell, so that the expression level of the KLK4alpha protein is improved, and the expression level of the KLK4alpha protein is improved. The time sequence intervention effectively prolongs the secretion window period of key organic matrix components such as glaze protein, glaze protein and the like, so that before mineralization is started, the total amount of extracellular matrix net racks is substantially increased, a space is provided for nucleation of hydroxyapatite crystals, and finally, the thickness of a mineral phase is remarkably increased; and the protection performance of the dental crown structure is enhanced.

The present invention provides, among other things, a transgenic chicken. In some embodiments, the transgenic chicken comprises a B cell having an endogenous immunoglobulin heavy chain locus comprising: (a) a functional immunoglobulin heavy chain gene comprising a nucleic acid encoding a heavy chain variable domain having a length of CDR3 in the range of 30 to 60 amino acids and containing at least two cysteine ​​residues; and (b) a plurality of pseudogenes operably linked to the functional immunoglobulin heavy chain gene and, by gene conversion, providing a nucleotide sequence to the nucleic acid encoding the heavy chain variable domain of (a), wherein the pseudogenes are located upstream or downstream of the functional immunoglobulin heavy chain gene.

The present invention provides a single-plasmid inducible expression system and its preparation method and application, which belong to the field of genetic engineering technology. The single-plasmid inducible expression system comprises a single plasmid, and the nucleic acid sequence of the plasmid is shown in SEQ ID No. 1. The single-plasmid inducible expression system constructed by the present invention overcomes the complex operation of twice-establishing the system of traditional induced expression. Only one gene transduction is required to screen and obtain the expected transgenic cell line, which simplifies the operation process and improves efficiency. Puromycin can be used for clone screening after gene transduction, and the tetracycline antibiotic doxycycline is used to induce target gene expression. Compared with the lentiviral system, it is simple to operate, saves costs, can insert a larger exogenous gene length, and has a larger vector capacity; the background expression is lower, the induced expression multiple is higher, and efficient gene expression can be achieved while having lower background leakage.

The invention discloses a dynamic culture amplification method of NK cells or engineered derivatives thereof, and belongs to the technical field of biology. The method comprises the following steps: sorting peripheral blood to obtain NK cells; mixing the culture medium with K562 feeder cells according to a first proportion, and carrying out first co-culture activation and amplification; optionally, carrying out CAR (chimeric antigen receptor) gene transduction; mixing the amplified NK cells or the transduced cells with K562 feeder cells according to a second proportion, and carrying out secondary co-culture activation and continuous amplification under a dynamic culture condition; and finally, the NK cells or the CAR-NK cells are obtained. The method breaks through the limitation of traditional standing co-culture, realizes efficient and stable amplification of NK / CAR-NK cells under dynamic conditions, has the advantages of high amplification efficiency, high cell viability and purity, simplified operation and easy large-scale amplification, and provides a new technical scheme for preparation of immune cell therapy products.

The invention relates to the technical field of cell culture, and discloses a cell culture method based on a car-t therapy, which comprises a channel conversion valve, a peristaltic pump, a dynamic culture unit, a static culture unit, a centrifugal transduction mechanism, an electric pinch valve, a material bag, a flow meter, a magnetic controller, a magnetic bead cleaning unit and a waste liquid collection unit. According to the cell culture method based on the car-t therapy, the cell culture efficiency is improved, the clinical transformation period is shortened, the time loss is reduced through automatic material transmission, the culture environment is accurately controlled, the cell activity is optimized, the gene transduction efficiency is improved through a centrifugal transduction mechanism, exogenous pollution is avoided through a totally-closed process, the process safety is enhanced, and the pollution risk is reduced; the whole process is automatic, personal errors are reduced, the process management and control capability is improved in traceability, automation and intelligence are achieved, labor dependence is reduced, cultivation cost is reduced, technology popularization is promoted, materials are efficiently utilized, waste is reduced, and labor cost is reduced through automation.

[Problem] There is no gene transduction method with high efficiency and high specificity for skin, subcutaneous tissue, skin ulcer surfaces, and their constituent cells. More generally, achieving high gene transduction efficiency in vivo is challenging. Additionally, it is difficult to morphologically and functionally regenerate skin appendages and to regenerate lost peripheral tissues of the upper and lower limbs (upper arm, forearm, hand, fingers, thigh, lower leg, foot, toes).[Solution] This invention provides solutions to these challenges through a novel AAV (adeno-associated virus) capsid with high gene transduction efficiency and high specificity for skin, subcutaneous tissue, and skin ulcer surface cells, as well as a novel AAV capsid with high gene transduction efficiency and high specificity for epidermal tissue and its constituent keratinocytes. Additionally, the invention provides a novel tissue-specific promoter with high gene expression efficiency and high specificity for epidermal tissue and its constituent keratinocytes, and a pharmaceutical composition that enhances gene transduction efficiency by co-administering steroids alongside in vivo gene transduction. The invention further includes a pharmaceutical composition that promotes the regeneration, neogenesis, and functional enhancement of skin appendages through in vivo gene transduction, thereby compensating for functional impairments of skin appendages. Additionally, it provides a research and development platform for developing methods to regenerate peripheral and missing tissues of the upper and lower limbs, as well as a pharmaceutical composition for achieving tissue regeneration. Thus, the present invention provides methods for overcoming these challenges.

The invention provides a method for changing a cell strain HLA (human leukocyte antigen) to present peptide, a cell strain with changed HLA to present peptide and application of the cell strain. The method for changing the cell strain HLA to present peptide comprises the following steps. The HLA gene is transduced to a K562 cell strain by lentivirus, so that the cell strain expresses an HLA molecule. Then, the cell strain expressing the HLA molecules is transduced by lentivirus with the AIRE gene, and a gene transcription map and a protein map expressed by the cell strain are changed through the over-expressed AIRE effect, so that the variety of peptides presented by the HLA molecules is changed.

The present invention relates to a dual-gene-transduced T cell using a CTLA-4 variant and a chimeric antigen receptor and a composition for anti-cancer immunotherapy comprising the same, and according to an aspect, the dual-gene-transduced T cell using the CTLA-4 variant and the chimeric antigen receptor and the composition for anti-cancer immunotherapy comprising the same, by introducing the CTLA-4 variant, the anti-cancer immunotherapy can be obtained by inducing the CTLA-4 variant and the chimeric antigen receptor. According to the present invention, it is possible to induce T cell function enhancement without a precursor that causes side effects such as autoimmune diseases, and to inject a small amount of T cells to exhibit an improved tumor treatment efficiency and an effect of suppressing tumor recurrence, and thus it can be usefully used in immune cell therapy for cancer treatment.

The present invention discloses a recombinant adeno-associated virus of serotype 13, its preparation method and application. By constructing a recombinant adeno-associated virus serotype packaging plasmid containing the Rep gene of AAV2 wild virus and the Cap gene of AAV13 wild virus, and preparing based on a triple plasmid transfection system, a high-titer recombinant adeno-associated virus rAAV2 / 13 can be obtained efficiently and rapidly. The recombinant adeno-associated virus of the present invention can be used as a gene transfer vector to transduce a target gene into nerve cells. It has high expression ability and a smaller diffusion range than rAAV2. Based on this, the rAAV2 / 13 vector can carry the target gene for precise labeling of small brain nuclei for circuit labeling and manipulation in neuroscience research, and can also be used as a vector for gene therapy.

The application provides an attenuated, replication-controllable forward HSV tracing system and a construction method and application thereof. Specifically disclosed is a recombinant herpes simplex virus type 1 crossing a nerve synapse, which is obtained by knocking out two copies of a nerve virulence factor coding gene gamma 34.5 in a H129 strain genome, then back-supplementing an expression-controllable gamma 34.5 self-degradation recombination gene into the original gene site of gamma 34.5, and the expression-controllable gamma 34.5 self-degradation recombination gene contains the gamma 34.5 gene with a function domain participating in the cell autophagy process knocked out and a degradation sequence PEST. The herpes simplex virus provided by the application can be used as a gene transduction carrier for long-term high expression of a target gene, long-term, anterograde crossing multi-level synapse structure tracing of a nerve circuit, functional nerve circuit analysis and the like, and has wide application value in nervous system targeted gene therapy, oncolytic virus therapy, virus replication and pathogenic mechanism analysis, animal infection model establishment and the like.

The present application provides novel viral envelope glycoproteins for viral vector pseudotyping, novel designs for synthetic antigen receptors (SARs), novel signal chains for constructing SAR, novel antigen binding domains, and novel methods for generating SAR expressing cells. These novel methods and compositions have broad applications in cell therapy.

The invention discloses an SMN gene expression cassette, a virus vector carrying the SMN gene expression cassette and application of the virus vector in treatment of spinal muscular atrophy, and relates to the field of gene therapy. The SMN1 gene in a gene expression cassette is subjected to codon optimization, so that the expression quantity of SMN protein after SMN1 gene transduction is improved, and the clinical dosage is expected to be effectively reduced. Meanwhile, a regulatory element such as a miRNA target sequence is also introduced into the gene expression cassette, so that the immune response of an organism to an adeno-associated virus vector and a transgenic product is relieved, and the safety problem possibly caused by a gene therapy is expected to be reduced.

The present application relates to the technical field of vaccine preparation, in particular to a lentivirus dendritic cell vaccine targeting Claudin-18.2 and a preparation method and application thereof. The preparation method comprises the following steps: obtaining immature dendritic cells; using a recombinant lentivirus particle carrying a nucleotide sequence encoding a Claudin-18.2 protein to perform gene transduction on the immature dendritic cells, so that the immature dendritic cells endogenously express a Claudin-18.2 antigen; applying a maturation-promoting stimulus to the transduced dendritic cells to obtain a dendritic cell vaccine that is mature and expresses the Claudin-18.2 antigen. The vaccine can effectively induce an antigen-specific cytotoxic T lymphocyte response, is suitable for preparing a drug for treating Claudin-18.2-positive gastric cancer, and has the characteristics of strong targeting and significant immune activation effect.

Compositions and methods of the invention provide plasmids and methods for efficient production of recombinant adeno-associated viruses (rAAVs) for use in numerous areas including research and therapeutic applications of gene transduction. Plasmids of the invention can include rep-cap plasmids comprising rep and cap genes or portions thereof derived from any AAV serotype or engineered capsid. In certain embodiments, rep and / or cap genes may be derived from AAV2, AAV9, and AAV5 serotypes.

The present invention relates to: feeder cells of natural killer cells, the feeder cells being genetically engineered to express a lunasin protein; and a method for promoting the proliferation or amplification of natural killer cells or enhancing the transduction efficiency of a foreign gene into natural killer cells, by using same.