201results about How to "High transduction efficiency" patented technology

Decreasing the expression of genes in a mammalian subject has multiple applications ranging from cancer therapy to anti-infective therapy or treatment of autosomal dominant genetic disorders. Yet, there is still a lack of efficient technologies to achieve that goal in mammalian subjects in vivo. The present invention relates to methods for decreasing gene expression by administering to a mammalian subject a recombinant adeno-associated viral vector in vivo with said vector comprising an RNA interference (RNAi) expression cassette whose RNA expression products directly or indirectly lead to a decrease in expression of the corresponding RNAi target gene. Upon successful transduction with the recombinant adeno-associated viral vector, the RNA expression products of the RNAi expression cassette will decrease the cellular concentration of the mRNA transcripts of the RNAi target gene, thus resulting in decreased concentration of the protein encoded by the RNAi target gene.

Disclosed are water-soluble gel-based compositions for the delivery of recombinant adeno-associated virus (rAAV) vectors that express nucleic acid segments encoding therapeutic constructs including peptides, polypeptides, ribozymes, and catalytic RNA molecules, to selected cells and tissues of vertebrate animals. Also disclosed are gel-based rAAV compositions are useful in the treatment of mammalian, and in particular, human diseases, including for example, cardiac disease or dysfunction, and musculoskeletal disorders and congenital myopathies, including, for example, muscular dystrophy, acid maltase deficiency (Pompe's disease), and the like. In illustrative embodiments, the invention provides rAAV vectors comprised within a biocompatible gel composition for enhanced viral delivery/transfection to mammalian tissues, and in particular to vertebrate muscle tissues such as a human heart or diaphragm tissue.

The invention discloses a CAR-T transgene vector based on replication defective recombinant lentivirus. The CAR-T transgene vector comprises an original nuclear replicon pUCOri sequence, a resistance gene AmpR sequence containing ampicillin, a virus replicon SV40 Ori sequence, a lentivirus packaging cis element, ZsGreen1 green fluorescent protein, an IRES ribosome binding sequence, a human EF1 alpha promoter , a chimeric antigen receptor of second-generation CAR or third-generation CAR and a regulating element, wherein the original nuclear replicon pUCOri sequence is used for plasmid replication; the resistance gene AmpR sequence is used for massively proliferating target strains; the virus replicon SV40 Ori sequence is used for enhancing replication in eukaryocyte; the lentivirus packaging cis element is used for lentivirus packaging; the ZsGreen1 green fluorescent protein is used for expressing green fluorescent for eukaryocyte; the IRES ribosome binding sequence is used for jointly transcribing and expressing protein; the human EF1 alpha promoter is used for conducting eukaryotic transcription on antigen receptor genes; the chimeric antigen receptor is used for forming the second-generation CAR or the third-generation CAR integrating recognition, transfer and start; the regulating element is used for enhancing expression efficiency of transgenes and used after eWPRE-enhanced type woodchuck hepatitis b virus is transcribed. Besides, the invention further discloses a construction method and application of the vector. By means of the CAR-T transgene vector and the construction method and application of the vector, secretion of cell factors and an in vitro killing effect of CAR-T cells can be remarkably improved, and the clinical treatment effect is remarkable.

The present invention demonstrates that VSV-G-pseudotyped lentivirus vectors efficiently transduce AEC in primary culture and in vivo with transduction favored by virus application from the apical side. Transduction efficiency in AEC increased with increasing MOI and greatly exceeded that achieved with a similarly pseudotyped MLV retrovirus vector. The present invention also demonstrates the successful in vivo transfer of genes through lentivirus vector transduction. Mammals injected with lentivirus vector via the trachea expressed the reporter protein in alveolar epithelial cells within 48 to 72 hours after infection.

A modified adeno-associated virus (AAV) capsid protein comprising at least one non-native amino acid that confers to the modified AAV particles new properties, such as increased transduction efficiency and reduced immunogenicity. These modified AAV proteins and particles are particularly useful for gene therapy and the treatment of various diseases and conditions.

Disclosed are capsid-modified rAAV expression vectors, as well as infectious virions, compositions, and pharmaceutical formulations that include them. Also disclosed are methods of preparing and using novel capsid-protein-mutated rAAV vector constructs in a variety of diagnostic and therapeutic applications including, inter alia, as delivery agents for diagnosis, treatment, or amelioration of one or more symptoms of disease or abnormal conditions via in situ and/or ex vivo mammalian gene therapy methods. Also disclosed are large-scale production methods for capsid-modified rAAV expression vectors, viral particles, and infectious virions having improved transduction efficiencies over those of the corresponding, un-modified, rAAV vectors, as well as use of the disclosed compositions in the manufacture of medicaments for a variety of in vitro and/or in vivo applications.

The invention discloses a nucleic acid for coding a chimeric antigen receptor protein expressed on the surface of the human T lymphocyte. The chimeric antigen receptor protein comprises an extracellular binding domain, a transmembrane domain and an intracellular signal domain, and the extracellular binding domain, the transmembrane domain and the intracellular signal domain are orderly connected. The extracellular binding domain comprises a single-chain antibody scFv (epidermal growth factor receptor EGFR) for specific recognition of 287th-302th amino acid epitopes of EGFR.

The invention provides a CD19 targeted CAR (chimeric antigen receptor)-T cell, a preparation method and an application and relates to the field of immune cells. The activation capacity of T cells in CAR-T cell therapy can be improved, the problem of insufficient transfection efficiency can be solved, and the CAR-T cell has a high killing capacity for CD19. The CD19 targeted CAR-T cell expresses a CD19 targeted CAR gene on surface, and the CD19 targeted CAR gene is formed by connecting a single-chain antibody CD19ScFv of CD19, a hinge region and a transmembrane region of CD8, an intracellular signal structure of CD28, an intracellular signal structure of 4-1BB and an intracellular signal structural domain of CD3zeta in series, and the base sequence of the CD19 targeted CAR gene is shown as SEQ ID NO:2.

The present invention provides methods and compositions for improving the efficacy of viral transduction of cells. More particularly, the present invention provides methods and materials useful for safely and reliably improving the efficiency of methods for transducing cells, such as human hematopoietic stem cells (HSC), with viruses and/or viral vectors. The compositions and methods are useful for therapeutic indications amenable to treatment with hematopoietic stem cell gene therapies.

Compositions and methods are provided for preparation of concentrated stock solutions of AAV virions without aggregation. Formulations for AAV preparation and storage are high ionic strength solutions (e.g. μ˜500 mM) that are nonetheless isotonic with the intended target tissue. This combination of high ionic strength and modest osmolarity is achieved using salts of high valency, such as sodium citrate. AAV stock solutions up to 6.4×10¹³ vg/mL are possible using the formulations of the invention, with no aggregation being observed even after ten freeze-thaw cycles. The surfactant Pluronic® F68 may be added at 0.001% to prevent losses of virions to surfaces during handling. Virion preparations can also be treated with nucleases to eliminate small nucleic acid strands on virions surfaces that exacerbate aggregation.

The invention discloses a method for jointly preparing CAR-NK (chimeric antigen receptor-natural killer) cells and CAR-NKT (natural killer T) cells. The method includes steps of 1), adding peripheral blood mononuclear cells into cell culture media, pre-activating NK cells and NKT cells in the peripheral blood mononuclear cells and selectively activating and amplifying the NK cells and the NKT cells in an in-vitro manner; 2), transducing mixed cells obtained at the step 1) by the aid of lentiviral vectors with gene sequences of chimeric antigen receptors. The method has the advantages that the CAR-NK cells and the CAR-NKT cells can be prepared from NKs and NKTs of healthy donors in a standardized and batched manner; the purity of the NK cells and the NKT cells can respectively reach 60% and 30% at least without purification, and accordingly preliminary purification can be omitted; the in-vivo activation and amplification degrees and the in-vivo existence time of the CAR-NK cells and the CAR-NKT cells which are jointly prepared by the aid of the method can be controlled by means of clinical medication.

A nucleic acid encoding a chimeric antigen receptor expressed at surface of a T lymphocyte, said chimeric antigen receptor comprises, connected in the order of, an extracellular binding domain, a transmembrane region, and an intracellular signaling domain, wherein the extracellular binding domain comprises a single chain antibody, scFv(GPC3), which specifically recognizes the C-terminal epitope of GPC3. A genetically modified T lymphocyte having a chimeric antigen receptor expressed at surface thereof, and the chimeric antigen receptor is expressed by the nucleic acid described above.

Agents and methods to alter rAAV transduction are provided.

The present invention provides duplexed parvovirus vector genomes that are capable under appropriate conditions of forming a double-stranded molecule by intrastrand base-pairing. Also provided are duplexed parvovirus particles comprising the vector genome. Further disclosed are templates and methods for producing the duplexed vector genomes and duplexed parvovirus particles of the invention. Methods of administering these reagents to a cell or subject are also described. Preferably, the parvovirus capsid is an AAV capsid. It is further preferred that the vector genome comprises AAV terminal repeat sequences.

The invention discloses an anti-CD20 chimeric antigen receptor, an encoding gene, a recombinant expression vector, a construction method of the recombinant expression vector and an application. The anti-CD20 chimeric antigen receptor comprises CD8 leader chimeric receptor signal peptide, CD20 VL, Optimal Linker C, a CD20 single-chain antibody heavy chain VH, a CD8 Hinge chimeric receptor hinge, a CD8 Transmembrane chimeric receptor transmembrane domain, a CD137 chimeric receptor co-stimulated factor and a TCR chimeric receptor T cell activating domain which are connected in series. Moreover, the invention discloses an encoding gene of the anti-CD20 chimeric antigen receptor, a recombinant expression vector, a construction method of the recombinant expression vector, and an application. Secretion of cell factors and an in-vitro killing effect of CAR-T cells are obviously improved, and the CAR-T cells have an outstanding clinical treatment effect.

The invention discloses an anti-EGFRvIII chimeric antigen receptor, an encoding gene, a recombinant expression vector, a construction method of the recombinant expression vector, and an application. The anti-EGFRvIII chimeric antigen receptor comprises CD8 leader chimeric receptor signal peptide, an EGFRvIII single-chain antibody light chain VL, Optimal Linker C, an EGFRvIII single-chain antibody heavy chain VH, a CD8 Hinge chimeric receptor hinge, a CD8 Transmembrane chimeric receptor transmembrane domain, a CD137 chimeric receptor co-stimulated factor and a TCR chimeric receptor T cell activating domain which are connected in series. Moreover, the invention discloses an encoding gene of the anti-EGFRvIII chimeric antigen receptor, a recombinant expression vector, a construction method of the recombinant expression vector, and an application. Secretion of cell factors and an in-vitro killing effect of CAR-T cells are obviously improved, and the CAR-T cells have an outstanding clinical treatment effect.

The invention discloses a CD 123-targeting replication-defective recombinant lentivirus CAR-T transgenic vector. The CD 123-targeting replication-defective recombinant lentivirus CAR-T transgenic vector comprises a prokaryotic replicor pUC Ori sequence used for plasmid duplication; an amicillin resistance gene AmpR sequence used for amplification of a large number of target strains; a virus replicor SV40 Ori sequence used for enhancing the replication in eukaryocytes; a lentivirus packaging cis element used for lentivirus packaging; ZsGreen 1 green fluorescent protein used for green fluorescence expression of eukaryocytes; an IRES ribosome binding sequence used for co-transcription and co-expression of protein; a human EF1 alpha promoter used for eukaryotic transcription of genes of a chimeric antigen acceptor; the chimeric antigen acceptor used for forming second-generation CAR or third-generation CAR integrating recognition, delivery and promoting; an eWPRE element used for enhancing the expression efficiency of transgenes. In addition, the invention further discloses a construction method and applications of the vector. With the vector, the secretion of the cell factors and the in-vitro lethal effect of the CAR-T cells can be obviously improved, and the effect in treatingacute myelogenous leukemia (AML) clinically is outstanding.