91 results about "Donor cell" patented technology

Disclosed are compositions and methods for preventing graft versus host disease (GVHD) in subjects receiving donor cells. In particular, chimeric antigen receptor (CAR) polypeptides are disclosed that can be used with adoptive cell transfer suppress alloreactive donor cells. Also disclosed are immune effector cells, such as T cells or Natural Killer (NK) cells, that are engineered to express these CARs. Therefore, also disclosed are methods of suppressing alloreactive donor cells in a subject receiving transplant donor cells that involves adoptive transfer of the disclosed immune effector cells engineered to express the disclosed CARs.

Genetically modified cells that are compatible with multiple subjects, e.g., universal donor cells, and methods of generating said genetic modified cells are provided herein. The universal donor cells comprise at least one genetic modification within or near at least one gene that encodes a survival factor, wherein the genetic modification comprises an insertion of a polynucleotide encoding a tolerogenic factor. The universal donor cells may further comprise at least one genetic modification within or near a gene that encodes one or more MHC-I or MHC-II human leukocyte antigens or a component or a transcriptional regulator of a MHC-I or MHC-II complex, wherein said genetic modification comprises an insertion of a polynucleotide encoding a second tolerogenic factor.

Several embodiments disclosed herein relate to methods and compositions for enhanced expansion of NK cells in culture, through the repeated co-culturing of NK cells with feeder cells and the selective use of stimulatory interleukins. In several embodiments, the methods utilize one or more soluble interleukins as culture media supplements at one or more time points during expansion of the NK cell, or other immune cell, which results in a highly expanded and highly cytotoxic population of cells, for use in, for example allogeneic cellular immunotherapy.

The invention discloses a conjugational transfer-assisted orthogonal targeted mutation evolution system, which comprises a donor strain and a receptor strain, a donor strain intracellular muter performs targeted mutation on a target sequence to obtain a mutation sequence, a non-self-transferable conjugational transfer functional plasmid is introduced into the donor strain, and a non-self-transferable conjugational transfer functional plasmid is introduced into the receptor strain. The mutant sequence is unidirectionally and horizontally transferred from a donor strain cell to a receptor strain cell, and the complete orthogonality of the mutants in the initial donor strain and the mutant sequence in the receptor strain is realized through the orthogonality between the donor strain and the receptor strain. The results of a sacB-sucrose reverse screening experiment show that the mutation frequency of an ortho-MutaT7 system reaches 2.96 * 10 <-2 > at 96 hours, which is 1840.9 times of the natural mutation frequency of the sacB in donor cells, and the ortho-MutaT7 system generates mutation of 12 basic group change types in a target sequence. The Ortho-MutaT7 system developed by the invention realizes mutation diversity and also has orthogonality, leakage mutation and off-target mutation of mutants in host cells are fully avoided, and a direct relationship between a mutation sequence and a phenotype is established.

Genetically modified cells that are compatible with multiple subjects, e.g., universal donor cells, and methods of generating said genetic modified cells are provided herein. The universal donor cells comprise at least one genetic modification within or near at least one gene that encodes a survival factor, wherein the genetic modification comprises an insertion of a polynucleotide encoding a tolerogenic factor. The universal donor cells may further comprise at least one genetic modification within or near a gene that encodes one or more MHC-I or MHC-II human leukocyte antigens or a component or a transcriptional regulator of a MHC-I or MHC-II complex, wherein said genetic modification comprises an insertion of a polynucleotide encoding a second tolerogenic factor.

The application discloses a gene editing system for constructing a three-gene (GHR gene, IGF1 gene and IGF2 gene) combined mutation small pig nuclear transfer donor cell and application thereof. The application provides a kit comprising GHR-E5-gRNA2 shown in SEQ ID NO: 36, GHR-E5-gRNA3 shown in SEQ ID NO: 37, IGF1-E4-gRNA1 shown in SEQ ID NO: 38, IGF1-E4-gRNA2 shown in SEQ ID NO: 39, IGF2-E4-gRNA2 shown in SEQ ID NO: 40, IGF2-E4-gRNA7 shown in SEQ ID NO: 41 and NCN protein. The NCN protein is a Cas9 protein or a fusion protein with the Cas9 protein. The application adopts CRISPR / Cas9 technology combined with double gRNA editing to perform combined knockout of the GHR gene, the IGF1 gene and the IGF2 gene, and obtains a single cell clone with three-gene combined knockout, thereby laying a foundation for breeding small pigs and growth and development disorder or retardation model pigs through somatic cell nuclear transfer animal cloning technology in the later stage.

The present disclosure describes systems, devices, and methods for high-throughput electroporation of a donor cell. The systems can include a perforated membrane having one or more layers, each comprising a plurality of channels disposed through each layer. The systems can also include spacer arrays in contact with a perforated membrane to create a plurality of cell electroporation chambers for high-throughput cell electroporation.

The application discloses a gene editing system for constructing a muscle weakness disease model pig nuclear transfer donor cell with a DOK7 gene mutation and application thereof. The application provides application of DOK7-gRNA2 shown in SEQ ID NO: 16, DOK7-gRNA4 shown in SEQ ID NO: 17 and NCN protein in preparation of a kit. The application further provides a method for preparing a recombinant cell, comprising the following steps: co-transfecting a pig cell with DOK7-gRNA2, DOK7-gRNA4 and NCN protein to obtain a recombinant cell. The recombinant cell is a recombinant cell with a mutated DOK7 gene. The use of any of the above kits is to prepare a recombinant cell, to prepare a muscle weakness model pig, to prepare a muscle weakness cell model, a muscle weakness tissue model or a muscle weakness organ model. The application has great application value for research and development of muscle weakness disease drugs and revealing the pathogenesis of the disease.

The present disclosure provides compositions and methods for cell transplantation therapy based on forced expression of an exogenous HLA-F protein in donor cells to be transplanted into a subject. In some embodiments, the donor cells express an exogenous chimeric HLA-F protein comprising an extracellular region comprising an HLA-F alpha 1 domain, an HLA alpha 2 domain, an HLA-F alpha 3 domain, a linker and a β2m protein.

To provide genetically modified cells that are compatible with multiple subjects, e.g., universal donor cells, and to provide methods of generating the genetic modified cells.SOLUTION: The universal donor cells comprise at least one genetic modification within or near at least one gene encoding a survival factor, the genetic modification comprising the insertion of a polypeptide encoding a tolerogenic factor. The universal donor cell may further comprise at least one genetic modification within or near a gene encoding one or more MHC-I or MHC-II human leukocyte antigens or components or transcriptional regulators of the MHC-I or MHC-II complexes, the genetic modification comprising the insertion of a polypeptide encoding a second tolerogenic factor.SELECTED DRAWING: Figure 1

The invention discloses a line establishment method and a culture system of bovine trophoblast stem cells. The stem cell line is obtained by separation and induction from bovine blastocyst. The embryonic stem cell line has stability. The method can be applied to various life science and medical fields such as bovine animal breeding and breeding, gene editing models, animal cloned donor cells, medical musculoskeletal injury experiments, drug development carriers, vaccine production cell banks, embryo quality analysis and the like, and can be applied to large-scale production and application.

Provided are methods for rapid multiplex HLA genotyping and transcript quantitation. In some embodiments, the presently disclosed methods include providing a. sample from the cell, tissue, or organ that has mRNA derived from an HLA gene product; reverse transcribing the mRNA to produce a. pool of cDNAs; amplifying the cDNAs that result from reverse transcription of HLA-specific mRNAs to produce a pool of HLA-specific cDNAs; and determining the sequence of each HLA-specific cDNAs, whereby the HLA status of the cell, tissue, organ, or subject is determined. Also provided are methods for genotyping donor cells, tissues, and / or organs meant for transplantation into recipients. In some embodiments, the methods can be used to identify the presence of absence of each of HLA-A, -B, -C, -DRB 1 / 3 / 4 / 5, -DQA1, and -DQB1 in a biological sample.

The present disclosure provides bovine animals and methods to create recipient bovine animals for spermatogenic stem cell transplantation / complementation through modulation of the NANOS2 gene. In one embodiment genome editing was used to create bovine animals with insertions or deletions (indels) that inactivate or otherwise modulate NANOS2 gene activity so that resulting male bovines lack functional germ cells yet retain functional testicular somatic cells, and bovine females are fertile. These bovine males can then be transplanted or complemented with donor cells capable of giving rise to cells of the spermatogenic lineage, or spermatogenic stem cells and used for breeding.

Disclosed are compositions and methods for preventing graft versus host disease (GVHD) or allograft rejection in subjects receiving donor cells. Also disclosed are methods enhancing regulatory T (Treg) cells for use in preventing GVHD. Also disclosed are methods of suppressing alloreactive donor cells in a subject receiving transplant donor cells that involves adoptive transfer of the treated Treg cells. Also disclosed are enhanced Treg cells produced by the disclosed methods that have been engineered to express chimeric antigen receptor (CAR) polypeptide cells.

The invention belongs to the technical field of biological medicines, and particularly relates to a membrane-coated oncolytic virus as well as a preparation method and application thereof. The oncolytic virus contains a GSDMD N-terminal expression cassette regulated and controlled by a heated shock promoter, and pyroptosis can be induced under exogenous mild thermal stimulation, so that the virus is promoted to be quickly released, and reinfection and amplification of adjacent tumor cells are realized. A bionic nano-vesicle (iNV) formed by a donor cell membrane modified by genetic engineering is used as an outer layer to coat a genetically engineered oncolytic virus (GOV) to obtain a membrane-coated oncolytic virus iNV-GOV, so that the membrane-coated oncolytic virus iNV-GOV simultaneously has immunocompatibility and active tumor recognition capability, is suitable for solid tumors such as pancreatic ductal adenocarcinoma (PDAC), hepatocellular carcinoma (HCC), intrahepatic cholangiocarcinoma (IHCC) and the like, and can be used for preparing an anti-tumor drug. And the tumor volume can be obviously reduced in mouse in-vivo model experiments.

This invention discloses a gene editing system for constructing a porcine nuclear transplantation donor cell model of atherosclerosis with double gene mutations in AF and its applications. The invention provides a kit comprising plasmid pKG-U6gRNA (APOE-E2-gRNA2), a target sequence binding region as shown in nucleotides 3-22 of SEQ ID NO: 25 (FBN1-gRNA4), a target sequence binding region as shown in nucleotides 3-22 of SEQ ID NO: 26 (FBN1-gRNA6), and FBN1-mutant-ss163 as shown in SEQ ID NO: 27. The kit is used for: preparing recombinant cells; preparing porcine atherosclerosis models; preparing atherosclerosis cell models, atherosclerosis tissue models, or atherosclerosis organ models. The plasmid pKG-U6gRNA (APOE-E2-gRNA2) is transcribed to obtain sgRNA with a target sequence binding region as shown in nucleotides 1-20 of SEQ ID NO: 11. APOE‑E2‑gRNA2 This invention lays the foundation for developing pig models of atherosclerosis through somatic cell nuclear transfer animal cloning technology.

This invention discloses a gene editing system for constructing porcine nuclear transplantation donor cells for cataract disease models with MIP gene mutations and its applications. This invention provides the application of MIP-gRNA2 (SEQ ID NO: 18), MIP-gRNA3 (SEQ ID NO: 19), and NCN protein in a preparation kit. This invention also provides a method for preparing recombinant cells, comprising the following steps: co-transfecting porcine cells with MIP-gRNA2, MIP-gRNA3, and NCN protein to obtain recombinant cells. The recombinant cells are recombinant cells with a mutated MIP gene. The kits described above are used for: preparing recombinant cells; preparing porcine cataract models; preparing cataract cell models, cataract tissue models, or cataract organ models. This invention has significant application value for the development of cataract drugs and for elucidating the pathogenesis of this disease.

Immunotherapeutic Monoclonal Antibody Complexes or Conjugates (IMAC) comprising readily accessible antibodies designed and approved for clinical use are provided using a one-step method that combines killing of existing cancer cells in parallel with induction of long-lasting anti-cancer vaccination. Methods for their use, alone or in combination with cancer killer cells including intentionally mismatched donor T cells, NK cells concomitantly with additional anti-cancer or immune activating agents, or activation of patient's own immune system for personalized treatment of cancer and elimination of undesirable non-malignant cells are also provided. In addition, treatment method based on IMAC can be applied for in vivo vaccination against cancer using an existing malignant lesion as internal anti-cancer vaccine by engagement of patients antigen presenting cells for induction of long-lasting anti-cancer vaccination in situ against residual or recurrent disease.

The technology includes a system to reallocate user experience resources. The system detects network performance in a set of cell areas. The system determines low-performing cell areas and high-performing cell areas by using a network performance model. The system determines common subscribers between the low-performing cell areas and the high-performing cell areas. Based on the common subscribers, the system identifies a recipient cell area and a donor cell area. The system determines a reallocation of user experience resources by using the network performance model to determine scores based on changes to cell site parameters of the donor cell area and the recipient cell area. The system generates an indication that the reallocation will improve user experiences for subscribers of the recipient cell area, without diminishing user experiences for subscribers of the donor cell area.

The application discloses a preparation method of an easily-recognized cytoplasm control point wax block. The application provides a method for preparing an easily-recognized cytoplasm control point wax block. The method is used for obtaining a cell column by sampling a donor cell wax block, placing the cell column in colored receptor paraffin which is mixed by dye and melted paraffin, and further preparing the cell column into an identifiable quality control wax block containing a quality control point. In the application, the applicant proves that the performance of the wax block is different under different volume ratios of dye and paraffin, and the most suitable ratio of the two is screened. The method provided by the application not only facilitates the recognition of the cytoplasm control point, but also does not interfere with the IHC staining result in the later stage, and has a wide application prospect in clinical diagnosis.

The application discloses a kit for constructing an ATM gene mutation ataxia-telangiectasia model pig nuclear transfer donor cell. The application provides a kit comprising ATM-gRNA1 shown in SEQ ID NO: 16, ATM-gRNA4 shown in SEQ ID NO: 17 and NCN protein. The application also provides a method for preparing a recombinant cell: co-transfecting a pig cell with ATM-gRNA1, ATM-gRNA4 and NCN protein to obtain a recombinant cell. The recombinant cell is a recombinant cell with ATM gene mutation. The kit is used for: preparing a recombinant cell; preparing an ataxia-telangiectasia model pig; preparing an ataxia-telangiectasia cell model or an ataxia-telangiectasia tissue model or an ataxia-telangiectasia organ model. The application has great application value for research and development of ataxia-telangiectasia drugs and revealing the pathogenesis of the disease.

This invention discloses a gene editing system for constructing porcine nuclear transplantation donor cells for ALS models with SOD1 gene mutations and its applications. This invention also provides a method for preparing recombinant cells: SOD1- g RNA1 (target sequence binding region as shown in nucleotides 3-22 of SEQ ID NO: 18), SOD1- g RNA6 (target sequence binding region as shown in nucleotides 3-22 of SEQ ID NO: 19), SOD1-mutant-ss160 (shown in SEQ ID NO: 20), and NCN protein (Cas9 protein or a fusion protein containing Cas9 protein) were co-transfected into porcine cells to obtain recombinant cells. This invention utilizes CRISPR / Cas9 technology combined with ssODN homologous recombination technology to perform point mutation gene editing of the SOD1 gene, mimicking the natural pathogenesis and genetic characteristics of ALS, and obtaining single-cell clones with precise point mutations in the SOD1 gene. This lays the foundation for later breeding of ALS disease model pigs using somatic cell nuclear transfer animal cloning technology.

To provide a method for producing a large number of therapeutic extracellular vesicles (EVs).SOLUTION: There is provided a method for producing a large number of therapeutic extracellular vesicles (EVs) containing high copies of functional nucleic acids and other biomolecules. The method comprises: laying donor cells on a surface of a chip having a three-dimensional (3D) nanochannel electroporation (NEP) biochip formed thereon; adding various plasmids, other transfection vectors, and combinations thereof to a buffer on the chip; applying a pulsatile electric field across the cells laid on the chip surface and a plasmid / vector buffer solution below the chip surface to strongly stimulate the cells and thereby deliver the plasmids / vectors into the cells by non-endocytic transfection; and collecting the EVs secreted by the transfected cells.SELECTED DRAWING: Figure 1

The invention discloses a rapid breeding method for generating larimichthys crocea sperms based on spermatogonial stem cell transplantation. The rapid breeding method comprises the following steps: step S1, performing whole cryopreservation on larimichthys crocea testis; s2, separating the spermatogonial stem cells of the pseudosciaena crocea; s3, dyeing the spermatogonial stem cells of the pseudosciaena crocea; step S4, treatment of nibea albiflora receptors; and S5, transplanting the dyed donor larimichthys crocea spermatogonial stem cells into the nibea albiflora receptor testis. According to the method, when the nibea albiflora acceptor is treated, the traditional busulfan concentration of 40 mg / kg, twice injection and 35 DEG C high-temperature assistance is optimized into 60 mg / kg, single injection and 22 DEG C normal-temperature culture, optimal parameters induced by busulfan are screened out through experiments, the operation process is simplified under the action of ensuring that endogenous germ cells of the acceptor are reduced and immunological rejection is reduced, and the method is suitable for large-scale popularization and application. The harsh requirement of a high-temperature environment is avoided, and meanwhile the field planting efficiency of donor cells in receptors and the survival rate of the receptors are improved.

Genetically modified cells that are compatible with multiple subjects, e.g., universal donor cells, and methods of generating said genetic modified cells are provided herein. The universal donor cells comprise at least one genetic modification within or near a gene that encodes one or more MHC-I or MHC-II human leukocyte antigens or a component or a transcriptional regulator of a MHC-I or MHC-II complex, wherein genetic modification comprises an insertion of a polynucleotide encoding a tolerogenic factor and / or survival factor. The universal donor cells may further comprise at least one genetic modification within or near a gene that encodes a survival factor, wherein said genetic modification comprises an insertion of a polynucleotide encoding a second tolerogenic factor and / or a different survival factor.

Therapeutic extracellular vesicles (EVs) containing high copies of functional nucleic acids and other biomolecules are produced in large quantities by laying donor cells on a surface of a chip, adding various plasmids, other transfection vectors and their combinations to a buffer on the chip, applying a pulsulatic electric field across the cells laid on top of the chip surface and plasmids / vectors buffer solution below the chip surface, and collecting the EVs secreted by the transfected cells. The chip surface has a three-dimensional (3D) nanochannel electroporation (NEP) biochip formed on it, capable of handling large quantities of the donor cells. The buffer is adapted for receiving plasmids and other transfection vectors.

This invention discloses a method and its application for constructing a sickle cell anemia model pig with HBB gene mutations using nuclear transfer donor cells. This invention provides the application of HBB-gU1, HBB-gD3, HBB-mutant-ss174, and NCN proteins in a preparation kit. The kit is used for: preparing recombinant cells; preparing sickle cell anemia model pigs; preparing sickle cell anemia cell models, sickle cell anemia tissue models, or sickle cell anemia organ models. HBB-gU1 is an sgRNA, and its target sequence binding region is shown as nucleotides 3-22 of SEQ ID NO: 18. HBB-gD3 is an sgRNA, and its target sequence binding region is shown as nucleotides 3-22 of SEQ ID NO: 19. HBB-mutant-ss174 is a single-stranded DNA molecule shown in SEQ ID NO: 20. The NCN protein is a Cas9 protein or a fusion protein containing a Cas9 protein. This invention has significant application value for the development of drugs for sickle cell anemia and for elucidating the pathogenesis of the disease.

Immunotherapeutic Monoclonal Antibody Complexes or Conjugates (IMAC) comprising readily accessible antibodies designed and approved for clinical use are provided using a one-step method that combines killing of existing cancer cells in parallel with induction of long-lasting anti-cancer vaccination. Methods for their use, alone or in combination with cancer killer cells including intentionally mismatched donor T cells, NK cells concomitantly with additional anti-cancer or immune activating agents, or activation of patient's own immune system for personalized treatment of cancer and elimination of undesirable non-malignant cells are also provided. In addition, treatment method based on IMAC can be applied for in vivo vaccination against cancer using an existing malignant lesion as internal anti-cancer vaccine by engagement of patient's antigen presenting cells for induction of long-lasting anti-cancer vaccination in situ against residual or recurrent disease.

The application discloses a gene editing system for constructing a GP130 gene mutation gastric cancer model pig nuclear transfer donor cell and application thereof. The gene editing system comprises a high-efficiency Cas9 protein prepared according to the method of the application, a high-efficiency target gRNA for the GP130 gene screened, and a single-chain Donor DNA containing a GP130 mutation site, and the optimal use amount ratio of the components of the system is optimized, and finally the single-cell clone ratio of the target site point mutation is 22.5%, which is much higher than the conventional point mutation efficiency (<5%).

This invention relates to the application of mitochondrial formulations in the preparation of drugs for treating chronic obstructive pulmonary disease (COPD). The mitochondrial formulations contain active mitochondria isolated from healthy donor cells, with a particle size of 400–1000 nm, possessing normal mitochondrial membrane potential and ATP generation function, and capable of being internalized by mammalian COPD lung epithelial cells. In vitro cell experiments have confirmed that this mitochondrial formulation can repair CSE-induced mitochondrial dysfunction in lung epithelial cells, inhibit mPTP opening, reduce mtDNA leakage into the cytoplasm, thereby blocking the activation of the cGAS-STING signaling pathway and reducing the release of inflammatory factors such as IL-1β and TNF-α. In vivo experiments in COPD mouse models show that tail vein injection of the mitochondrial formulation can effectively accumulate in lung tissue, significantly improving lung pathological changes such as emphysema and collagen fiber deposition in model mice. This invention provides a novel strategy for COPD treatment based on repairing mitochondrial function and intervening in inflammatory signaling pathways.