355 results about "Transfection" 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.

The invention discloses a nucleic acid transfection system and method based on automatic control, and relates to the technical field of genetic engineering.The method comprises the steps that after target cells are intelligently cultured to be in a suitable state, an automatic system selects a transfection reagent and prepares a compound according to cell types; the cells and the compound are mixed through low shear force and then incubated; multi-modal monitoring equipment is used for tracking nucleic acid distribution and cell states in real time, and incubation conditions are dynamically adjusted; analyzing the monitoring data based on a machine learning algorithm and optimizing transfection parameters; after transfection, culture and multi-dimensional analysis are automatically executed. The system correspondingly comprises a cell culture module, a reagent preparation module, a mixed incubation module, a real-time monitoring module, a dynamic optimization module and a subsequent analysis module. Through closed-loop automatic control and intelligent optimization, the transfection efficiency and stability are remarkably improved, the cytotoxicity is reduced, manual intervention is reduced, and a standardized solution is provided for recombinant gene expression.

Provided herein are formulations for oral delivery or administration of therapeutic nucleic acids. An oral formulation can include: a nucleic acid; at least one casein protein; and a chitosan. In some embodiments, the formulations are liposome-free formulations having no cationic lipids typically used as transfection reagents in conventional nucleic acid formulations for oral delivery. The formulations provided herein find use in treating a condition associated with inflammation and / or fibrosis, or a cardiometabolic disorder by oral administration.

The invention relates to an shRNA (short hairpin Ribonucleic Acid) interference sequence of a targeted silence PCSK9 gene and a construction method and lipid-lowering application of a recombinant adeno-associated virus vector of the shRNA interference sequence. Hyperlipidaemia is a metabolic disease characterized by abnormal rising of cholesterol and triglyceride levels in blood, and the design of lipid-lowering drugs is the focus of attention to improvement of hyperlipidaemia. Proprotein convertase subtilisin / kexin type 9 (PCSK9) can be combined with a low-density lipoprotein receptor (LDL-R) and degrade the LDL-R, so that accumulation of LDL-C in blood is further promoted, and hyperlipidemia is caused. Aiming at the key target PCSK9, a specific shRNA interference sequence is designed, and a recombinant adeno-associated virus vector (rAAV) carrying the sequence is constructed by an enzyme digestion-connection method. In-vitro experiments prove that the vector can remarkably reduce the expression level of PCSK9 protein, so that the cyclic utilization of a low-density lipoprotein receptor (LDL-R) is promoted, and the concentration of low-density lipoprotein cholesterol (LDL-C) in plasma is reduced. The rAAV vector provided by the invention has the characteristics of low production cost, high transfection efficiency, lasting action time and the like, and provides a new thought for gene therapy of hyperlipidemia.

The invention discloses application of fluorinated ionizable lipid as a gene drug delivery carrier, and belongs to the technical field of biological medicine. The gene drug delivery carrier provided by the invention is prepared from fluorinated ionizable lipids, cationic lipids, auxiliary lipids, structural lipids and PEG lipids, wherein the molar percentage of the fluorinated ionizable lipids in the gene drug delivery carrier is 1%-10%. According to the invention, the molar percentage of the fluorinated ionizable lipid in the total lipid is optimized to 1-10%, so that the cytotoxicity is obviously reduced, and more excellent nucleic acid transfection efficiency is realized in vivo and in vitro; various nucleic acid drugs including DNA, mRNA, siRNA and CRISPR-Cas9 ribonucleoprotein can be efficiently delivered, action targets of the nucleic acid drugs are expanded to cell nucleuses and cytoplasm, and the nucleic acid drugs are more accordant with the action mechanism of modern gene therapy and editing.

This invention discloses a hepatocyte model with silencing or overexpression of LCMT1 and its construction method, comprising: passage and seeding cells in logarithmic growth phase into well plates; diluting plasmid siRNA1 or siRNA2 or an overexpression plasmid to an appropriate concentration, and then mixing it with transfection reagent dilution to obtain a transfection mixture; washing the cells in each well of the plate and adding another culture medium, then gently dripping the transfection mixture into the well plate, shaking well, and culturing in a cell culture incubator for a period of time; replacing the culture medium with complete culture medium without antibiotics to obtain the constructed hepatocyte model with silencing or overexpression of LCMT1. This invention, by constructing a hepatocyte model with silencing or overexpression of LCMT1 in the liver, allows for validation experiments to be conducted to study the development mechanism of liver fibrosis. It is suitable for widespread application in various laboratories and provides an experimental model basis for research on liver fibrosis and drug trials.

The invention provides a DNA library screening method and application of an intracellular mRNA sensor switch. The DNA library screening method comprises the following steps: designing a DNA library containing a switch sequence of a degenerate basic group; preparing a circular expression vector library; preparing a minimum linear expression vector library; performing seamless cloning; carrying out RCA rolling circle amplification; carrying out telomerase digestion; carrying out cell transfection; extracting RNA (Ribonucleic Acid) and carrying out reverse transcription; carrying out library building of next-generation sequencing by taking the cDNA as a template; carrying out next-generation sequencing; analyzing data and comparing to obtain the DNA library of the mRNA sensor switch. According to the DNA library screening method provided by the invention, more sensor switch sequences can be provided for a single site on mRNA in cells, and more selection possibilities are further provided for subsequent targeted therapy.

The invention discloses application of beta-nicotinamide mononucleotide in regulation and control of gene editing efficiency, belongs to the field of gene editing treatment, and finds that the beta-nicotinamide mononucleotide (NMN) can efficiently inhibit the activity of CRISPR-Cas9, CRISPR-Cas12 and CRISPR-Cas13 systems in a broad-spectrum manner for the first time. The application comprises emergency blocking of off-target effect in gene editing clinical treatment, biological safety prevention and control of a virus vector gene editing system, and CRISPR activity regulation and control of in-vitro non-diagnostic purpose. Experiments show that NMN can inhibit CRISPR-mediated gene damage and cell death in a cell model, the inhibition efficiency in an in-vitro enzyme digestion system reaches 68.7%, and cell growth or transfection efficiency is not affected. The invention provides an innovative solution for safe application of CRISPR (clustered regularly interspaced short palindromic repeats) technology, and the NMN is approved to be taken orally as a health care product, so that the NMN has extremely strong clinical application potential. Compared with the existing CRISPR (clustered regularly interspaced short palindromic repeats)-resistant protein or synthetic small-molecule inhibitor, the NMN has the advantages of endogenous property, high biocompatibility, good oral safety and the like.

The invention relates to an amino acid derivative carrier as well as a preparation method and application thereof, and belongs to the technical field of biology, and the preparation method of the amino acid derivative carrier comprises the following steps: providing a cationic amino acid monomer and a neutral-polyanion block polymer template; a cationic amino acid monomer, a neutral-polyanion block polymer template, a cross-linking agent and a photoinitiator are mixed and react under the irradiation of ultraviolet light to obtain an amino acid derivative carrier, and the amino acid derivative carrier is cationic nanogel. The amino acid derivative carrier prepared by the preparation method disclosed by the invention can be used as a nucleic acid drug delivery carrier, and has the advantages of strong nucleic acid loading capacity, high transfection efficiency, safety and the like.

The invention relates to screening methods. In particular, it relates to a method of identifying a functional TCR from a library of particles, which displays a plurality of different T cell receptors (TCRs). The method may comprise a) exposing the library of particles to a target antigen to identify TCRs that bind to the target antigen, b) transfecting a plurality of T cells with nucleic acid encoding TCRs identified in step (a), c) exposing the plurality of transfected cells to the target antigen, and d) selecting cells having TCR activity in the presence of the target antigen. Transfection of the T cells is such that each T cell comprises a nucleic acid encoding a single TCR from the library of particles, the nucleic acid is integrated into the genome of the T cell at a single identical pre-defined locus, and the TCR is in a single chain format and comprises an alpha chain variable domain, a beta cain variable domain and a constant domain. The T cells do not express endogenous TCR. A T-cell comprising a recombinase mediated landing pad, wherein the T cell constitutively expresses TCR constant domain and does not express endogenous TCR, is also claimed.

The application discloses a kind of elastin-like polypeptide V 20 K 40 L, mRNA vaccine and preparation method and application in preparation tumor prevention, mitigation or treating drug.The elastin-like polypeptide carrier V 20 K 40 L provided by the application has good biocompatibility, and has no adverse reaction in vivo.The elastin-like polypeptide V 20 K 40 L provided by the application has amphiphilic structure, can be loaded Melan-A mRNA by hydrophobic self-assembly and electrostatic adsorption, and is in the state of shrinkage when higher than Tt temperature, which can prevent the uncontrollable leakage of mRNA.Furthermore, V 20 K 40 L contains lysosome escape peptide, which can further improve the transfection efficiency of mRNA by promoting lysosome escape.The elastin-like polypeptide V 20 K 40 L in the application can deliver Melan-A mRNA efficiently and safely, and realize the prevention and treatment of melanoma.

The invention discloses a method for screening and identifying ribosome entry sites in a short chain, which comprises the following steps of: (1) screening a short chain IRES sequence with the length of less than 100nt from an IRES database, constructing a screening library and synthesizing; (2) constructing a screening vector containing double reporter genes, cloning the short-chain IRES into a linearized screening vector, and generating circular RNA which only depends on the short-chain IRES sequence to recruit ribosome expression of the reporter genes in cells by the screening vector cloned with the short-chain IRES sequence; and (3) carrying out cell transfection on the screening vector cloned with the short-chain IRES sequence, screening out cells expressing reporter genes through cell sorting, and sequencing through a ribosome-newborn peptide chain compound to obtain the short-chain IRES sequence capable of recruiting ribosome to start circular RNA translation in the cells. According to the invention, a short-chain IRES sequence library is constructed, and short IRES capable of starting circular RNA translation is screened, so that transfection limitation caused by overlarge molecular weight of the current IRES sequence is overcome.

The invention provides a method for introducing an exogenous substance into a cell, the method comprising exposing the cell to a transient pressure reduction in the presence of the exogenous substance. Apparatus for performing the method of the invention is also provided.

This invention relates to a self-adjuvanted biomimetic lipid nanoparticle, its microfluidic assembly method, and its anti-tumor applications. The self-adjuvanted biomimetic lipid nanoparticle has a structure in which an activated dendritic cell membrane (ADCM) is coated on the surface of a lipid nanoparticle (LNP) loaded with mRNA. The advancements of this invention compared to traditional LNPs are: the self-adjuvanted biomimetic lipid nanoparticle (ADCM-LNP) exhibits a faster cellular uptake rate and higher transfection efficiency, thereby inducing a strong Th1-type immune response and a potent CTL-mediated tumor-killing effect. In vivo biodistribution studies have shown that this system exhibits significant spleen-targeting (splenic tropism) and demonstrates excellent therapeutic efficacy in a HER2-positive breast cancer model.

The application provides a group of small interfering RNAs (siRNAs) targeting pyroptosis-related inflammatory reaction genes and application thereof. The pyroptosis-related inflammatory reaction genes include IL1A, IL1B, IL6, HMGB1, S100A8, S100A9 and BACH1. The application designs and synthesizes specific siRNA sequences for the above genes, and verifies that the siRNAs can efficiently and specifically inhibit the mRNA expression level of the corresponding genes through cell transfection and real-time fluorescent quantitative PCR. The application also provides a composition containing the siRNAs, a pharmaceutical composition and application thereof in the preparation of a drug for treating diseases mediated by pyroptosis-related inflammatory reaction genes (especially inflammatory diseases). The siRNAs and the composition thereof provide a new effective strategy for treating diseases related to excessive activation of pyroptosis-related inflammatory reactions, and have a wide application prospect.

In one aspect, a method of treating an NK cell to induce a gene edit. The method may include contacting an NK cell with a transfection composition including poly-L-glutamic acid (y- PGA), a single-stranded oligo deoxynucleotide (ssODN) enhancer, or a combination thereof; contacting the NK cell with an editing composition including: a DNA-PK inhibitor, and an HD AC inhibitor; electroporating the NK cell; and contacting the NK cell with a recovery composition including DNase I. In another aspect, a method of preparing an engineered NK cell comprising a fluorescent reporter at an endogenous locus. In another aspect, a method of treating an NK cell to introduce an exogenous nucleic acid.

The present invention relates to novel cell culture and transfection methods using a wildtype Caf1 ("Caf1-WT") polymer or a variant thereof. Use of a Caf1-WT polymer or a variant thereof to increase cell viability, cell proliferation, cell migration and / or cell clustering; promote the formation of a 3D cell structure; and / or promote suspension cell culture of cells that are normally adherent are also provided herein.

The application belongs to the technical field of plant genetic engineering, and discloses a functional gene PbCOB.A.1 and application thereof. The in-vitro expressed recombinant protein PbCOB.A.1 can promote pollen tube growth of Rosaceae fruit trees, improve pollination efficiency, and expand the regulation mechanism of non-S factors participating in self-incompatibility reaction in pears. The mechanism of pear pollen tube elongation is researched by using magnetic transfection technology, so that the labor cost can be greatly reduced, and a theoretical basis is provided for pear breeding work.

The invention discloses a nucleic acid transfection reagent and a preparation method thereof and a nucleic acid delivery composition, the nucleic acid transfection reagent comprises lipid nanoparticles and a buffer solution, the lipid nanoparticles are formed through microfluidic mixing and self-assembly and comprise cationic lipid, auxiliary phospholipid and cholesterol, the particle size of the lipid nanoparticles is 30-200 nm, and the polydispersity index is less than 0.3; the buffer solution is a tromethamine buffer solution or a PBS (Phosphate Buffer Solution). According to the nucleic acid transfection reagent, the preparation process is effectively simplified, the process operation time is shortened, meanwhile, the stability of the nucleic acid transfection reagent is improved, long-term storage is facilitated, and cost reduction is facilitated; in the transfection operation, the lipid nanoparticles can be used without using Opti-MEM incubation, and the lipid nanoparticles and the nucleic acid molecules can be uniformly mixed, so that the transfection operation is effectively simplified, and the stability is improved.

The invention provides a chip system which comprises a cell growth area, the cell growth area comprises a cell layer and a plurality of first scaffolds, the cell layer is used for generating viruses, and each first scaffold comprises a plurality of hydrogel layers; the multiple hydrogel layers are stacked to form a groove, and pores of the multiple hydrogel layers are sequentially reduced in the direction from the interior to the exterior of the groove; the groove is used for accommodating the cell layer, so that viruses generated by the cell layer penetrate through the multi-layer hydrogel layer. The chip system is helpful for reducing the phenomenon of cell shedding in the virus transfection process, and can improve the efficiency of transfecting viruses by cells.

This invention discloses an amphoteric compound or its pharmaceutically acceptable salt, complex and its applications, wherein the amphoteric compound has a general structural formula as shown in formula (I); the amphoteric compound or its pharmaceutically acceptable salt of this invention can be used to prepare lipid nanoparticles for drug delivery, and the lipid nanoparticle delivery system has higher transfection efficiency, better delivery efficiency and better mRNA encapsulation rate compared with commercially available delivery systems.

Relates to a lipid composition with targeting property, the lipid composition comprises an active component, a lipid carrier and a targeting component, and the components of the lipid carrier comprise a positively charged component and phospholipid. An active component is entrapped in a lipid carrier containing a component with positive charges, so that the surface of the lipid carrier has positive charges, then the lipid carrier and a targeting component with negative charges are mixed, and the targeting component is adsorbed on the surface of the drug-loaded lipid carrier with positive charges through an electrostatic adsorption effect, so that the drug-loaded lipid carrier which is high in transfection efficiency and good in transfection effect is prepared. The present invention relates to a lipid composition having a targeting property and a high targeting property. The invention relates to a lipid nanoparticle delivery system with high biological activity, tumor targeting property and good safety. The lipid nanoparticle delivery system comprises an active component, cationic lipid, ionizable lipid and neutral phospholipid. The active component is entrapped in the lipid nanoparticle containing the cationic lipid, the ionizable lipid and the neutral phospholipid, and when the molar ratio of the cationic lipid to the ionizable lipid is in a specific range, the lipid nanoparticle has higher biological activity, tumor targeting property and good safety.

Provided is an accurate guide RNA (gRNA) screening method for base editing of an asialoglycoprotein receptor 1 (ASGR1) gene, including the following steps: (1) gRNA design; (2) primer design; (3) in vitro transcription of gRNA; (4) cell transfection; (5) collection of cells, and extraction and polymerase chain reaction (PCR) of a genome; and (6) Sanger sequencing.

A method for analytically detecting and determining the transfection efficiency of fluorinated lipid nanoparticles (FLNPs) by utilizing multi-nuclear magnetic resonance comprises the steps of (1) performing 19F magnetic resonance spectroscopy (19F MRS) on the FLNPs to obtain a fluorine spectrum of a control group; (2) transfecting the FLNPs into cells or living mice and performing 19F MRS to obtain a fluorine spectrum of an experimental group; and (3) predicting FLNPs transfection efficiency by calculating the difference in target fluorine peak areas between the experimental and control groups. The method demonstrates excellent specificity, reproducibility, stability, and linearity, providing a robust technical foundation for large-scale screening of FLNPs-based nanomedicines.

The application discloses a kind of chicken primordial germ cell transfection methods, including the following transfection steps: S1, the separation of chicken primordial germ cell is carried out, and grouping culture in culture medium, S2, according to the proportion of 3.5:1 of pPB-GFP transposon plasmid and transposase plasmid mPB dosage Two are mixed, S3, first dilute plasmid with opti-MEN, then dilute transfection reagent with opti-MEN, plasmid diluent and transfection reagent diluent are mixed to obtain compound, the transfection operation of chicken primordial germ cell is carried out by the culture medium of different serum adding amount and adding time in the application, the influence of different serum adding amount and different serum adding time on chicken primordial germ cell transfection efficiency can be understood, then the increase and decrease of the fluorescence brightness in chicken primordial germ cell after transfection are recorded, the growth of chicken primordial germ cell after transfection is understood, so that the most suitable culture medium can be selected, serum adding condition is optimized, and more efficient chicken primordial germ cell transfection can be realized.

The invention discloses a macrophage-targeting lipid nanoparticle and application thereof in malignant tumor treatment, ID3 mRNA is delivered through the lipid nanoparticle, firstly, an ID3 sequence is subjected to codon optimization to improve the expression efficiency and stability of ID3 protein, and the core treatment effect is enhanced; the lipid composition and the targeting ligand of the nanoparticles are further optimized, and mannose modified PEG lipid material (DSPE-PEG2000-Mannose) is specifically combined with the CD206 receptor on the surface of the macrophage, so that the targeting property and the transfection efficiency of the delivery system are remarkably improved. The ID3 mRNA is delivered to the macrophages by utilizing the lipid nanoparticles of the targeted macrophages, so that the tumor cells can be effectively swallowed and killed, the in-situ treatment of pancreatic cancer is realized, and better tumor inhibition and immune activation effects are achieved.

Provided in the present invention are a steroid-cationic lipid compound having a structure as shown in formula (I) and the use thereof. The compound can be used in the preparation of a lipid nanoparticle (LNP) for delivering a therapeutic agent and / or a preventive agent. The LNP prepared using the steroid-cationic lipid compound of the present invention has relatively good stability and transfection efficiency. The use of the LNP for delivering a nucleic acid, e.g., an mRNA, can realize efficient and stable delivery of biologically active substances to target cells or organs, and can induce a high level of specific antibody responses and cellular immune responses in experimental animals. Moreover, the compound has better safety.

The application discloses a novel double-carrier expression system for protein expression and a construction method and application thereof. The novel double-carrier expression system for protein expression comprises a first carrier containing a nucleotide sequence for coding a first functional domain of glutamine synthetase and a nucleotide sequence of light chain and heavy chain of a protein to be expressed; and a second carrier containing a nucleotide sequence for coding a second functional domain of glutamine synthetase and a nucleotide sequence of a ScFv sequence of the protein to be expressed, and an amino acid sequence of the glutamine synthetase is composed of the first functional domain and the second functional domain. By using the expression system, the transfection efficiency and screening efficiency are greatly improved, and the expression amount of the target protein is obviously increased.

Provided herein is a method for preparing the nanoparticle comprising: (i) combining an aqueous phase comprising the nucleic acid with an organic solvent-lipid mixture comprising lipids, wherein the lipids comprise an ionizable lipid and at least one helper lipid and optionally a hydrophilic-polymer lipid conjugate; wherein the aqueous phase comprises a buffer having a concentration of at least 100 mM and has an aqueous phase pH that is lower than a pKa of the ionizable lipid such that the ionizable lipid is substantially charged; wherein the lipid nanoparticle is formed during or subsequent to the combining; and (ii) exchanging a solution external to the lipid nanoparticle with a higher pH solution, thereby producing the nucleic-acid lipid nanoparticle, wherein the nucleic-acid lipid nanoparticle comprises one or more bleb compartments.