35 results about "RNA transfection" patented technology

A method of mRNA production for use in transfection is provided, that involves in vitro transcription of PCR generated templates with specially designed primers, followed by polyA addition, to produce a construct containing 3′ and 5′ untranslated sequence (“UTR”), a 5′ cap and / or Internal Ribosome Entry Site (IRES), the gene to be expressed, and a polyA tail, typically 50-2000 bases in length. This RNA can efficiently transfect different kinds of cells. This approach results in increased efficiency (fidelity and productivity) of mRNA synthesis and is less time consuming because it does not require cloning, and also consequently eliminates the unwanted errors and effects related to RNA made on DNA templates obtained with cloning techniques. The results of transfection of RNAs demonstrate that RNA transfection can be very effective in cells that are exceedingly difficult to transfect efficiently with DNA constructs. Further, the levels of gene expression following mRNA transfection are consistent from cell to cell in an experiment and these levels can be controlled over a wide range simply by changing the amount of mRNA that is transfected, and without obvious cytotoxic effects due to the levels of RNA per se. Due to high efficiency the cells can be simultaneously transfected with multiple genetic constructs. The method can be used to deliver genes into cells not- or only poorly transfectable for DNA, in vitro and in vivo.

RNA prepared by in vitro transcription using a polymerase chain reaction (PCR)-generated template can be introduced into a cell to modulate cell activity. This method is useful in de-differentiating somatic cells to pluripotent, multipotent, or unipotent cells; re-differentiating stem cells into differentiated cells; or reprogramming of somatic cells to modulate cell activities such as metabolism. Cells can also be transfected with inhibitory RNAs, such as small interfering RNA (siRNA) or micro RNA (miRNA), or combinations thereof to induce reprogramming of somatic cells. For example, target cells are isolated from a donor, contacted with one or more RNA's causing the cells to be de-differentiated, re-differentiated, or reprogrammed in vitro, and administered to a patient in need thereof. The resulting cells are useful for treating one or more symptoms of a variety of diseases and disorders, for organ regeneration, and for restoration of the immune system.

The present invention provides reagents and methods for RNA transfection and protein expression

Compositions and methods of making cells using RNA, and cells made using the disclosed compositions and methods are also provided. In exemplary embodiments, RNA is transfected into cells to effect a molecular, biological, physiological, or histological change in the cells. In preferred embodiments, the RNA is prepared in vitro, more preferably using a DNA template according to the provided compositions and methods. Methods for treating or inhibiting a disorder or disease such cancer are also provided. The methods can include, for example, locally or systemically administering to the host an effective amount of one or more RNAs; or an effective amount of population of cells isolated from the subject or a syngeneic or histocompatible subject, contacted ex vivo with one or RNAs, and optionally expanded. The cells can be, for example, immune cells or stem cells.

The requirement of T cell activation for efficient expression of genes after messenger ribonucleic acid (mRNA) transfection is leveraged to identify and enrich antigen-specific T cells responding to antigen-pulsed dendritic cells (DCs). RNA transfection of marker genes is used for the selection and enrichment of antigen-specific T cells for use in adoptive immunotherapy. RNA-modified T cells are also used for the generation of enhanced effector populations for use in adoptive immunotherapy. Genes whose transient expression may significantly enhance the in vivo function of T cells (i.e., migratory receptors, anti-apoptotic genes or cytokines enhancing T cell proliferation / differentiation) are used in this modality.

This invention relates to cationic lipids. More particularly the invention relates to biodegradable cationic lipids having a plurality of cationic headgroups and one or more lipophilic tail groups. The lipids are of utility in various applications, and in particular in permitting transfection of molecules, and in particular DNA and RNA, into cells. As such the lipids have specific utility in the field of gene therapy as well as other applications such as delivery of small molecules into cells, detergents, and metal ion complexation for medical or industrial applications.

The invention relates to methods and substances for the targeted alteration of genetic information on an RNA level. The substances are artificially produced guide RNAs, which are capable of recruiting endogenous editing enzymes, such as hADAR enzymes, in particular hADAR2 and hADAR1, in order to introduce targeted point mutations in selected mRNAs. The guide RNA consists of multiple segments and is constructed in such a way that individual nucleotides from different segments pair to form a double helix, and the nucleotides of a determined segment form a hairpin structure within the guide RNA. The invention also relates to the method for directed RNA editing, wherein the guide RNA is transfected into the cells in which the RNA editing is to be carried out. The substances and the method can be used for repairing individual, e.g. disease-relevant point mutations, such as those leading to premature stop signals. An advantage of the invention is that endogenous editing enzymes are also used in order to introduce targeted point mutations into the RNA. Only the short guide RNA, used for recruiting endogenous editing enzymes, must be artificially produced for each specific problem and ectopically expressed.

The invention discloses an application of SNHG5 (small nucleolar RNA host gene 5) in diagnosis, evaluation and efficacy prediction of breast cancer. It is found that lncRNA SNHG5 has significant low expression in breast cancer tissue and breast cancer cell lines, knockout of the lncRNA SNHG5 gene can enhance proliferation and migration of breast cancer cells and inhibit apoptosis, and thus, the expression level of long-chain non-coding RNA SNHG5 is closely related with diagnosis, transfer and relapse of tumor, patient prognosis and neoadjuvant chemotherapy effect are closely related, which indicates that the lncRNA SNHG5 gene can be used as a marker for diagnosis, prognosis evaluation or effect prediction. A breast cancer cell model which has low expression of SNHG5 and is established by transfecting breast cancer cell lines with small interference RNA can be used in a preparation method of a preparation for preventing relapse and transfer of breast cancer.

The present invention relates to enhancing RNA expression in a cell such as a cell transfected with RNA by reducing the activity of RNA-dependent protein kinase (PKR). Thus, the present invention provides methods for expressing RNA in a cell comprising the step of reducing the activity of RNA-dependent protein kinase (PKR) in the cell. Reducing the activity of RNA-dependent protein kinase (PKR) in the cell increases the stability of RNA and / or increases the expression of RNA in the cell.

The invention relates to dendritic cells, the NFκB signaling pathway of which has been manipulated by RNA transfection, to the manufacture thereof and to use thereof.

The invention discloses a method for preparing dendritic cells for effectively submitting gastric cancer antigens, and relates to dendritic cells. The method comprises the following steps of: extraction and identification of total RNA (Ribonucleic Acid) of gastric cancer cells MGC-803; synthesis of SOCS1 antagonist pJAK2 (1001-1013) polypeptide; separation of peripheral blood mononuclear cells; acquisition of mononuclear cells; in-vitro induction of immature dendritic cells; RNA transfection of the immature dendritic cells; pJAK2 (1001-1013) polypeptide supported RNA transfection of the dendritic cells; and mature induction of the immature dendritic cells, thus producing mature dendritic cells. By adopting the method, the antigen submission function and the specific immune response level of the dendritic cells can be improved; and the dendritic cells of total RNA transfection of the gastric cancer cells are modified by adopting the SOCS1 antagonist pJAK2 (1001-1013) polypeptide.

A complex includes RNA and a positively charged modified polysaccharide selected from starch, amylose, amylopectin, galactan, chitosan, or dextrin. The complex can be formed into a pharmaceutical composition. The complex can be used in methods for RNA transfection, gene therapy and treatment of a disease, disorder or condition. The positively charged modified polysaccharide can be used in connection with RNA transfection into cells.

A method is described for transferring expressible foreign genes into multi-cellular organisms by means of RNA uptake by sperm cells, followed by endogenous reverse transcription of RNA into cDNA. cDNA molecules are transferred to embryos through fertilization, and are transmitted to born animals and their progeny. The cDNA sequences are mosaically distributed in the cells and tissue of the organisms, and are preferentially expressed in target tissues.

The invention discloses polypeptide for inhibiting a hepatitis C virus infected cell and an application thereof. The sequence of the polypeptide is shown as SEQ ID NO: 1 or SEQ ID NO: 2. Binding adsorption can be realized for the polypeptide and HCV (hepatitis C virus) expressed E2 protein so as to block invasion or transfection of the HCV for cells. The inhibition of the polypeptides TD1 and TD2for HCV infected cells can be detected through a HCV pseudovirus (HCVpp) and HCV RNA (ribonucleic acid) transfected cell cultivation (HCVcc) system, and results show that TD1 and TD2 have certain inhibitory effects on HCVpp and HCVcc infected cells and can be used as an adsorption inhibitor or blocker for the HCV.

The invention discloses a construction method of a GS gene-knockout CHO-K1 cell strain. The construction method comprises the following steps: performing adherent culture on CHO-K1 cells, designing and selecting sgRNA, forming a transfection mixture by the selected sgRNA, EGFP mRNA and Cas9mRNA, and performing co-transfection on CHO-K1 to obtain the GS gene-knockout CHO-K1 cell strain. According to the method, the CHO-K1 cells are co-transfected by using the RNA transfection mixture, the method is a GS knockout method based on RNA, meanwhile, a CRISPR / Cas9 gene editing means and GFP screening are combined, thus genome reforming at the DNA level is avoided, the risk that the genome is polluted is reduced, the GS gene knockout efficiency is improved, and the workload of cell screening is reduced; and verification on the GS gene-knockout CHO-K1 cell strain obtained by the invention shows that the GS gene knockout efficiency meets the expectation.

The present invention provides small interfering RNA for a TRAPPC4 gene target point, wherein a sense strand nucleotide sequence is represented by SEQIDNO:1, and an antisense strand nucleotide sequence is represented by SEQIDNO:2. The present invention further provides applications of the small interfering RNA for the TRAPPC4 gene target point in preparation of drugs for prevention or treatment of colorectal carcinoma. The present invention further provides a targeting molecule transport protein microparticle complex of the siRNA and applications of the targeting molecule transport protein microparticle complex in preparation of drugs for prevention or treatment of colorectal carcinoma. According to the present invention, experiment results show that: after the small interfering RNA for the TRAPPC4 gene target point is adopted to transfect human colorectal carcinoma cells to slice TRAPPC4 expression so as to inhibit ERK-MAPK signaling pathway activation, inhibit colorectal carcinoma proliferation and induce apoptosis, such that the small interfering RNA can be used for colorectal carcinoma treatment.

The invention discloses a polypeptide for inhibiting hepatitis C viruses from infecting cells, and its application. The sequence of the polypeptide is represented by SEQ ID NO:1 or SEQ ID NO:2. The polypeptide can be combined and adsorbed to E2 proteins expressed by the HCVs, so the entry or the transfection of the HCVs to the cells is blocked. Results of experiments that the HCV entering cell inhibition effects of polypeptides TD1 and TD2 are detected through an HCV pseudovirus (HCVpp) and HCV RNA transfected cell culture (HCV cc) system show that the polypeptides TD1 and TD2 have certain inhibition effects on HCVpp and HCVcc infected cells, and can be used as HCV adsorption inhibitors or blockers.

The invention discloses a polypeptide for inhibiting hepatitis C viruses from infecting cells, and its application. The sequence of the polypeptide is represented by SEQ ID NO:1 or SEQ ID NO:2. The polypeptide can be combined and adsorbed to E2 proteins expressed by the HCVs, so the entry or the transfection of the HCVs to the cells is blocked. Results of experiments that the HCV entering cell inhibition effects of polypeptides TD1 and TD2 are detected through an HCV pseudovirus (HCVpp) and HCV RNA transfected cell culture (HCV cc) system show that the polypeptides TD1 and TD2 have certain inhibition effects on HCVpp and HCVcc infected cells, and can be used as HCV adsorption inhibitors or blockers.

An eucaryotic expression system using Giardia virus as gene expression carrier is disclosed, which can be the one using DNA transfection mode and the other using RNA transfection mode. Said Giardia virus can effectively infect its trophont and has lower toxin and more number of copies in gene duplication. Said Giardia is easily cultured. Its advantages are high expression efficiency and no pollution to gene.

The present invention provides a novel method for the transduction and / or transfection of plant cells. Cell-penetrating peptides (CPPs) have been successfully employed as nanocarriers to deliver proteins and oligonucleotides to single plant cell microspores as well as multi-cellular zygotic embryos. The efficiency of CPP internalization and further delivery of a macromolecular cargo comprising a protein and / or an oligonucleotide can be enhanced by permeabilization of the zygotic embryos.

The present invention relates to a gene derived from a novel fulminant hepatitis C virus strain, an HCV replicon RNA with a high replication efficiency obtained using the gene, and an HCV replicon-replicating cell transfected with the replicon RNA. When the HCV replicon RNA and the HCV replicon-replicating cell of the present invention are used, HCV proteins can be continuously produced in a large amount.

The invention discloses a traditional Chinese medicine granule for treating viral hepatitis. The traditional Chinese medicine granule is prepared from the following raw material in parts by weight: 16-26 parts of herba artemisiae scopariae, 12-26 parts of radix scutellariae, 12-20 parts of Melilotus officinalis, 10-20 parts of fructus gardeniae, 8-16 parts of herba lysimachiae, 12-16 parts of herba hedyotidis diffusae, 6-18 parts of herba lophatheri and 3-9 parts of radix glycyrrhizae. The traditional Chinese medicine composition has the effects of clearing and activating channels and collaterals, promoting diuresis to alleviate edema, strengthening liver and harmonizing stomach, tonifying kidney and spleen, nourishing yin and producing sperm, improving body immunity, clearing away heat toxin, dispelling dampness and cold and removing RNA transfection caused by virus, and routes of transmission of the virus are blocked from the root; the traditional Chinese medicine composition is suitable for treating hepatitis B from HBsAg,HBeAg and HBcAb test positive to HBsAg, HBeAb and HBcAb test positive and from HBsAg, HBeAb and HBcAb test positive to negative, and also has very good curative effects on hepatitis A, hepatitis C and hepatitis E.

The present invention relates to a gene derived from a novel fulminant hepatitis C virus strain, an HCV replicon RNA with a high replication efficiency obtained using the gene, and an HCV replicon-replicating cell transfected with the replicon RNA. When the HCV replicon RNA and the HCV replicon-replicating cell of the present invention are used, HCV proteins can be continuously produced in a large amount.

The invention belongs to the field of stem cells, and in particular relates to telomeres capable of selectively prolonging adult stem cells in individuals. Individual aging is caused by shortening ofthe telomeres of the multipotent adult stem cells, therefore, only if the telomeres of the multipotent adult stem cells are enabled to extend to the level of young people, the function of the aging tissues and organs in the individuals can be inverted to the level of the young people. For the telomeres, an RNA transfection reagent in the body of an animal is mixed with hTERTmRNA, and the mixture enters the human body through intravenous injection, so that the telomeres of the various adult stem cells in the individuals are prolonged.

The invention discloses a method for preparing dendritic cells for effectively submitting gastric cancer antigens, and relates to dendritic cells. The method comprises the following steps of: extraction and identification of total RNA (Ribonucleic Acid) of gastric cancer cells MGC-803; synthesis of SOCS1 antagonist pJAK2 (1001-1013) polypeptide; separation of peripheral blood mononuclear cells; acquisition of mononuclear cells; in-vitro induction of immature dendritic cells; RNA transfection of the immature dendritic cells; pJAK2 (1001-1013) polypeptide supported RNA transfection of the dendritic cells; and mature induction of the immature dendritic cells, thus producing mature dendritic cells. By adopting the method, the antigen submission function and the specific immune response level of the dendritic cells can be improved; and the dendritic cells of total RNA transfection of the gastric cancer cells are modified by adopting the SOCS1 antagonist pJAK2 (1001-1013) polypeptide.

A method, system, and apparatus for treating a patient with HIV. A vector can be modified from a thymidine kinase gene. The modified vector is expressed in the presence of tat RNA. The modified vector is then package and delivered to HIV-infected cells. The replication of HIV is inhibited by eliminating infected cells in the presence of Ganciclovir. Modified cells are then selected utilizing transient tat RNA transfection and GFP expression. Vector-modified stem cells are then selected for transplantation back into the patient, thereby producing a normal immune system in the patient when the modified vector remains dormant in the absence of HIV tat.