24 results about "In vitro transfection" patented technology

The present invention relates to the field of genetic engineering, and specifically relates to a vector and a method for transfecting non-activated T cells in vitro. Disclosed in the present invention are a vector and a method of using the vector to come into contact with non-activated T cells and transfect the non-activated T cells. The surface of the vector comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor binding fragments thereof that specifically bind to endocytic receptors of non-activated T cells; and the vector can carry a nucleic acid encoding a CAR, enters the non-activated T cells by means of endocytosis initiated by the specific binding of the endocytic receptors of the non-activated T cells, and then effectively transfects the non-activated T cells. Therefore, the technical problem whereby it is difficult to effectively transfect non-activated T cells in the process of preparing CAR-T cells in vitro is effectively solved, thereby reducing the cost of preparing CAR-T cells and shortening the preparation period.

The invention provides an application of a mesenchymal stem cell modified by an IGF-1 (Insulin-like Growth Factor 1) gene in treatment of premature ovarian failure. The invention provides a modified IGF-1mRNA (Insulin-like Growth Factor). The modified IGF-1mRNA comprises a 5'end capping structure (Cap) and a 3 '-polyA tailed structure of an RNA (Ribonucleic Acid) sequence, and encapsulating the modified IGF-1 mRNA (messenger Ribonucleic Acid) by using an ionizable lipid. The invention further provides a preparation method of the liposome based on the ionizable lipid, a method for improving the transfection efficiency of the IGF-1 in-vitro transfected mesenchymal stem cells and the IGF-1 transfected mesenchymal stem cells obtained by adopting the method, MSCs are promoted to continuously and stably express IGF-1, the MSCs can stably secrete factors to promote cell proliferation and differentiation, and the granular cell secretion activity is maintained. The IGF-1 transfected mesenchymal stem cells can be used for preparing a medicine for treating premature ovarian failure, the IGF-1 modified MSCs can assist the premature ovarian failure in recovery, and the technical problem that the treatment effect cannot be improved due to the fact that the dosage of the MSCs is limited in the clinical treatment process can be solved.

The present invention provides a lipid nanoparticle (LNP) formulation comprising at least one sialic acid (SA)-containing lipid. The LNP formulation can effectively bind to cell surface Siglec, transfect specific cells in vitro, and target specific cells in vivo. The present invention also provides a method for using the LNP composition described herein for pharmaceutical applications. For example, the LNP provided herein is useful for intracellular delivery of nucleic acid therapeutic agents to a subject.

The invention discloses an in-vitro transcription system of theileria annulata transformed cells and a construction method of the in-vitro transcription system, the in-vitro transcription system comprises IVT-EGFP-mRNA, and the structure of the IVT-EGFP-mRNA is 5 'UTR-Cl-Kozak-EGFP-Pacl-3' UTR-polyA (120). According to the invention, mRNA for coding the EGFP gene is synthesized through in-vitro transcription, through capping, purification and agarose gel electrophoresis analysis, the expression condition of the target gene in TaNM1 cells is finally verified through an in-vitro transfection test and an indirect immunofluorescence technology, and the result shows that the in-vitro transcription system transfected and coded with the EGFP successfully expresses the EGFP, and the expression condition of the target gene in the TaNM1 cells is verified. The invention shows that a system for transfecting the EGFP gene into the theileria annulata transformed cell TaNM1 through an in-vitro transcription method is successfully established, and an important reference value is provided for design and research and development of theileria annulata RNA vaccines.

The present invention provides a lipid nanoparticle (LNP) formulation comprising at least one sialic acid (SA)—containing lipid. The LNP formulation is capable of effectively binding cell surface Siglecs, transfecting certain cells in vitro and targeting certain cells in vivo. The present invention also provides methods of using the LNP compositions described herein for pharmaceutical applications. For example, the LNPs provided herein are useful for the intracellular delivery of a nucleic acid therapeutic to a subject.

The application discloses a multifunctional multi-arm star polymer and a preparation method and application thereof, and belongs to the technical field of biomedical materials.The functional multi-arm star polymer disclosed by the application takes an internal ring polymer as a core molecule, and a functionalized acrylate monomer is used to prepare the multi-arm star polymer through ATRP.The polymer prepared by the application has the characteristics of adjustable terminal reaction site of the core molecule, controllable and narrow molecular weight distribution of the number, length and functionalization unit of the arm molecule, excellent affinity to mRNA, and the like.The complex nanoparticles formed by the polymer and mRNA are uniformly distributed and have good stability.The in-vitro transfection result shows that the polymer can efficiently deliver mRNA in various tissue cell lines, which provides an application basis for further genetic disease, cancer gene therapy and virus vaccine development.

This invention discloses a method for screening and validating personalized neoantigens for liver cancer and its application. DNA and RNA are extracted from tumor and adjacent normal tissue samples from patients and subjected to high-throughput sequencing. Mutant peptides are screened, and the patient's HLA typing is determined. The binding affinity of the mutant peptides to HLA alleles is then evaluated, and antigen presentation ability is predicted. Candidate neoantigen peptides are comprehensively screened. The candidate neoantigens undergo coding sequence sequencing, cloning, PCR amplification, in vitro transcription, capping and tailing modifications, and encapsulation to prepare an mRNA-LNP vaccine. The obtained mRNA-LNP vaccine is then transfected into 293T cells in vitro to verify antigen expression efficiency, completing the functional quality control of the mRNA-LNP vaccine. This invention provides a highly efficient, closed-loop system for screening personalized neoantigens for liver cancer and validating vaccines, an integrated approach that directly guides mRNA vaccine construction and in vivo immunogenicity evaluation from multi-omics data screening.

The utility model discloses a micro-fluidic chip for preparing LNP, an LNP synthesis module and an automatic preparation system of mRNA vaccine and medicine. The micro-fluidic chip is provided with a bent sample introduction channel and a mixing flow channel consisting of transverse flow channels and vertical flow channels which are alternately communicated, so that a water phase and an organic phase can be automatically and fully mixed, and LNP can be prepared in a high-flux manner. According to the micro-fluidic chip, an mRNA vaccine and medicine preparation system is introduced through an LNP synthesis module, the system is also integrated with various peripheral equipment and a pipetting workstation, and complete-flow, high-throughput, rapid, integrated and automatic preparation of mRNA preparation and purification, LNP encapsulation, in-vitro transfection and cellular level characterization in mRNA vaccine and medicine research is completed for the first time; and a foundation is laid for realizing standardization of a whole mRNA preparation process.

The present invention provides a lipid nanoparticle (LNP) formulation comprising at least one sialic acid (SA)-containing lipid. The LNP formulation is capable of effectively binding cell surface Siglecs, transfecting certain cells in vitro and targeting certain cells in vivo. The present invention also provides methods of using the LNP compositions described herein for pharmaceutical applications. For example, the LNPs provided herein are useful for the intracellular delivery of a nucleic acid therapeutic to a subject.

The invention relates to the field of gene engineering, in particular to a vector and a method for transfecting non-activated T cells in vitro. The invention discloses a vector and a method for contacting a non-activated T cell and transfecting the non-activated T cell by using the vector. The surface of the vector comprises one or more antibodies or antigen-binding fragments thereof and / or ligands or receptor-binding fragments thereof, wherein the antibodies or the antigen-binding fragments thereof and / or the ligands or the receptor-binding fragments thereof are specifically bound with an endocytosis receptor of the non-activated T cell; the vector can carry nucleic acid for coding the CAR, and enters and effectively transfects the non-activated T cells through the endocytosis caused by the endocytosis receptor specifically combined with the non-activated T cells, so that the technical problem that the non-activated T cells are difficult to effectively transfect in the process of preparing the CAR-T cells in vitro is effectively solved, and the CAR-T cells can be used for preparing the CAR-T cells. Further, the cost for preparing the CAR-T cells is reduced, and the preparation period is shortened.

The present invention provides a lipid nanoparticle (LNP) formulation, the lipid nanoparticle (LNP) formulation comprising at least one sialic acid (SA)-containing lipid. The LNP preparation can effectively bind Siglecs on the surface of a cell, transfect certain cells in vitro, and target certain cells in vivo. The present invention also provides methods of using the LNP compositions described herein for pharmaceutical applications. For example, the LNPs provided by the invention can be used for delivering nucleic acid therapeutic agents into cells of a subject.

The present invention relates to a cancer cell membrane-encapsulated lipid nanoparticle for nucleic acid drug delivery, a preparation method therefor and the use thereof. The solution comprises encapsulating the outer surface of LNP with a layer of a cancer cell membrane to endow same with a biological activity similar to that of tumor cells, so that the LNP can effectively reside at a lesion for a long time after being injected into the tumor tissue of a patient. Moreover, experimental data shows that CLNPs encapsulated with the cell membrane of cancer cell HepG2 have a generally improved transfection efficiency compared with that of LNPs, and for three different liposome formulations (LNP1, LNP2 and LNP3), the transfection efficiency is improved to varying degrees in three different cell lines. In an in vitro transfection experiment in MC38, the maximum increase of 7.5-fold is achieved.

Compositions of assembly solutions and stabilization solutions that can achieve kinetic control of the size and composition of polycationic / nucleic acid particles and the optimized process for reproducible and scalable production of concentrated, shelf-stable nanoparticle vehicles for in vitro transfection of cell culture in bioreactors are disclosed.

The invention discloses a nucleic acid delivery carrier based on natural lipids and a preparation method and application thereof, the natural lipids of the nucleic acid delivery carrier comprise coix seed oil, brucea javanica oil and soybean oil, and other components comprise DOTAP, DOPE, glycerin and deionized water. The nucleic acid delivery carrier for delivering the siRNA drug has difference in cellular uptake and transfection efficiencies, and the lysosome escape condition and lipid droplet co-localization result taking the coix seed oil and the brucea javanica oil as lipids are superior to those of soybean oil; and the in-vitro transfection efficiency of the coix seed oil nucleic acid delivery vector on AsPC-1 and human embryo kidney cell strain HEK293T cells has no significant difference with the cell transfection efficiency of a commercially available nucleic acid delivery vector. The constructed natural nucleic acid delivery carrier is prepared into a freeze-dried preparation, so that the stability of the nucleic acid delivery carrier can be improved; the natural nucleic acid delivery carrier which is redissolved after being freeze-dried still keeps relatively good transfection efficiency.

The invention relates to the field of biomedicine, in particular to application of Dicer and miRNA regulated by the Dicer as markers in preparation of products for diagnosing, preventing and / or treating tumor liver metastasis. In-vivo and in-vitro experiments find that the Dicer in the macrophages is knocked down, so that M2 type macrophages can be reprogrammed into M1 type macrophages, and the proliferation of colon cancer cells is inhibited. According to the invention, siDicer1 entrapped M2pep-LNP is subjected to in-vitro transfection, and miRNA sequencing and verification are carried out on the M2pep-LNP, so that the miR-148a-3p and the miR-1981-5p are found to be possibly potential targets of Dicer for regulating and controlling the phenotype of macrophages. The miRNA mimics and the inhibitor are further used for carrying out functional verification on the two kinds of miRNAs, and the result shows that the miR-148a-3p and the miR-1981-5p can be used for reprogramming the M2 type macrophages by regulating and controlling the Akt signal channel.

The present invention provides a lipid nanoparticle (LNP) formulation comprising at least one sialic acid (SA) - containing lipid. The LNP formulation is capable of effectively binding cell surface Siglecs, transfecting certain cells in vitro and targeting certain cells in vivo. The present invention also provides methods of using the LNP compositions described herein for pharmaceutical applications. For example, the LNPs provided herein are useful for the intracellular delivery of a nucleic acid therapeutic to a subject.

Methods for extracorporeal transfection of an immune effector cell are provided. The method includes contacting blood, or a cell-containing fraction thereof, from a subject with a nanoparticle. The nanoparticle includes a surface-exposed immune effector binding moiety. Furthermore, the nanoparticle includes a nucleic acid encoding a chimeric antigen receptor (CAR) or T-cell receptor (TCR).

The invention discloses a multifunctional multi-miktoarm star polymer as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials. According to the functional multi-miktoarm star polymer disclosed by the invention, an internal cyclization polymer is used as a core molecule, and the internal cyclization polymer and a functional acrylate monomer are subjected to ATRP (Atom Transfer Radical Polymerization) to prepare the multi-miktoarm star polymer. The polymer prepared by the invention has the characteristics of adjustable reaction sites at the tail ends of core molecules, controllable number and length of arm molecules and functional units, narrower molecular weight distribution and the like, and has excellent affinity to mRNA, and composite nanoparticles formed by the polymer and the mRNA are more uniform in distribution and good in stability. An in-vitro transfection result shows that mRNA can be efficiently delivered in various tissue cell lines, so that an application basis is provided for further genetic disease and cancer gene therapy and viral vaccine development.

The application discloses a kind of automatic high-throughput preparation mRNA vaccine and medicine method.The method uses following microfluidic chip and preparation system.Microfluidic chip has curved sample channel and mixed flow passage of alternating communication transverse flow channel and vertical flow channel, can be fully mixed and high-throughput preparation LNP to water phase and organic phase.The microfluidic chip is introduced mRNA vaccine and medicine preparation system by LNP synthesis module, the system is also integrated with a variety of peripheral equipment and pipetting workstation, for the first time, from mRNA vaccine and medicine research mRNA preparation and purification, LNP encapsulation, in vitro transfection, cell level characterization The whole process of high-throughput, fast integrated automatic preparation is completed, and foundation is laid for realizing the standardization of mRNA whole preparation process.

The present invention provides a chimeric antigen receptor targeting TROP2, a drug delivery body and its application, belonging to the technical field of hybrid peptides, wherein the amino acid sequence of the chimeric antigen receptor targeting TROP2 is shown in SEQ ID NO: 1 in the sequence table; the chimeric antigen receptor targeting TROP2 is obtained by sequentially connecting the following modules: CD8 signal peptide, TROP2 single-chain antibody, CD8 hinge region, CD8 transmembrane region, CD28 and CD226 costimulatory region, CD3ζ intracellular region, T2A, optimized RISR-RIAD peptide. The CAR-TROP2-iNEs containing the optimized RISR-RIAD peptide prepared by the present invention can improve the killing efficiency of SK-OV-3 cell line and LOVO cell line after in vitro transfection of T cells, with a killing efficiency of 79.6% for SK-OV-3 cells and 87.3% for LOVO cells.

The present invention discloses an ionizable lipid or a pharmaceutically acceptable salt thereof, a composition, and applications. The structure of the ionizable lipid is shown in Formula I, and is primarily composed of a long carbon chain, biodegradable ester bonds, ether bonds, and other structures. The ionizable lipid or a pharmaceutically acceptable salt thereof and nucleic acids of the present invention can be prepared into lipid nanoparticles with a particle size of approximately 110 nm. These lipid nanoparticles exhibit high in vivo and in vitro transfection efficiency. This invention is of great significance for enriching the variety of ionizable lipids, delivering nucleic acid drugs, and preventing or treating diseases. #imgabs0#

The invention discloses a composite lipid nanoparticle containing a cryoprotectant as well as a preparation method and application of the composite lipid nanoparticle. The composite lipid nanoparticle provided by the invention comprises a lipid nanoparticle (LNP) and a cryoprotectant, the lipid nanoparticle comprises a carrier and encapsulated nucleic acid, the carrier comprises an ionizable lipid, auxiliary phospholipid, a cholesterol substance and a pegylated lipid; the cryoprotectant at least comprises betaine. The cryoprotectant in the prepared composite lipid nanoparticles can protect LNP from being damaged in freezing and rewarming treatment, and a good cryoprotection effect is achieved. Meanwhile, after freezing and rewarming treatment, the cryoprotectant can be integrated into the LNP, and the membrane fusion efficiency of the LNP can be improved. By improving the endosome escape efficiency of the LNP, the in-vivo and in-vitro transfection efficiency of the LNP is improved.

This invention provides an ultrasonic perforation device for in vitro transfection and transformation of eukaryotic and prokaryotic cells, comprising: a well plate with a plurality of receiving wells; a driving mechanism; a water tank for holding deionized water; the bottom of the well plate being immersed in the deionized water; a heating element; at least two ultrasonic transducers of different frequencies, disposed below the well plate with their emitting ends immersed in the deionized water; the driving mechanism driving the well plate to move horizontally, so that the receiving well to be tested is located in the far-field region directly above the corresponding ultrasonic transducer. Because the receiving wells are located in the far-field region of the ultrasonic transducer's sound field, the sound intensity of the ultrasonic transducer's sound field changes little with spatial position during transfection, resulting in stable spatial characteristics of the sound field. This facilitates relatively stable ultrasonic cavitation intensity and ultrasonic perforation effect, and the cell membrane's repairable ultrasonic perforation leads to high cell transfection efficiency and high cell viability. Placing the emitting ends of the ultrasonic transducers in deionized water avoids the risk of contamination from direct contact with the cell suspension.

The application discloses an in vitro transcription system of a transformed cell of a circular theileria and a construction method thereof, and the in vitro transcription system comprises IVT-EGFP-mRNA, and the structure is 5'UTR-Cla1-Kozak-EGFP-PacI-3'UTR-polyA(120). The mRNA coding the EGFP gene is synthesized by in vitro transcription, and is subjected to capping, purification and agarose gel electrophoresis analysis. Finally, the expression of the target gene in the TaNM1 cell is verified by in vitro transfection test and indirect immunofluorescence technology. The result shows that the green fluorescent protein EGFP is successfully expressed by the in vitro transcription system coding the EGFP, which indicates that the system for transfecting the EGFP gene into the transformed cell TaNM1 of the circular theileria by the in vitro transcription method is successfully established, and the system provides important reference value for the design and research and development of the RNA vaccine of theileria annulata.