932 results about "Lentivirus" patented technology

The invention belongs to the field of pharmaceutical and biological engineering, and relates to a CRISPR/Cas9 recombinant lentiviral vector for human immunodeficiency virus gene therapy and a lentivirus of the CRISPR/Cas9 recombinant lentiviral vector. The recombinant lentiviral vector is prepared by carrying out enzyme digestion on a lentiviral vectorlentiCRISPR by BsmBI and connecting into a BsmBI cohesive end-containing CXCR4 specific target sequence to recombine; the obtained CRISPR/Cas9 recombinant lentiviral vector is capable of mutating gene sequences at four different loci of ahuman immunodeficiency virusco-receptor CXCR4 and themutatuin rate is high and up to 25-75%. The cells transformed by the recombinant lentiviral vector cannot be infected by the human immunodeficiency virus. Compared with theRNAi-Knockdown, ZFN and TALEN technologies, the method has higher efficiency of suppressing the human immunodeficiency virus replication; the system is rapid to construct, simple and low in cost, is capable of preventing the invasion of the human immunodeficiency virus and is suitable for human immunodeficiency virus gene therapy.

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.

Lentiviral vectors modified at the 5′ LTR or both the 5′ and 3′ LTR are useful in the production of recombinant lentivirus vectors (See the Figure). Such vectors can be produced in the absence of a functional tat gene. Multiple transformation of the host cell with the vector carrying the transgene enhances virus production. The vectors can contain inducible or conditional promoters.

The invention is directed to peptides having antimicrobial activity (antimicrobial peptides). The antimicrobial peptides of the present invention are analogs of the Lentivirus Lytic Peptide 1 (LLP1) amino acid sequence. The invention is further directed to peptides referred to as the Lytic Base Unit (LBU) peptides derived from the LLP1 analogs, also having antimicrobial activity. In addition, the present invention is also directed to methods of using the peptides in a variety of contexts, including the treatment or prevention of infectious diseases. The antimicrobial LLP1 analog peptides and the LBU peptides (collectively eLLPs) may be highly active under high salt conditions and in biologic fluids. In addition, the eLLPs are effective when presented either in soluble form, or when attached to a solid surface. Furthermore, the peptides of the present invention are selectively active against a wide variety of bacterial pathogens and exhibit minimal toxicity to eukaryotic cells in vitro and in vivo.

The invention belongs to the field of gene engineering, and particularly relates to a method for specifically knocking out CCR5 genes of human bodies through CRISPR/SaCas9 and sgRNA used for specifically targeting CCR5 genes. The invention provides the method for specifically knocking out the CCR5 genes of the human bodies through CRISPR/SaCas9 and sgRNA used for specifically targeting the CCR5 genes and relates to a CRISPR/SaCas9 recombinant lentivirus and adeno-associated virus vector system and application. CRISPR/SaCas9 recombinant lentivirus and adeno-associated virus vectors can express SaCas9-RNA aiming at the CCR5 target spot of one same gene of the human bodies and rhesus monkeys, therefore, the application range is wider, and the efficiency is higher. The preparation method is easy to construct, the targeting efficiency is high, and a novel technology is provided for the gene therapy of HIV.

A modified polypeptide corresponding to an envelope glycoprotein of a primate lentivirus is described. The polypeptide has been modified from the wild-type structure so that it has at least two of the glycosylation sites proximal to the CD4 binding site or chemokine receptor site altered so that the alteration prevents glycosylation at that site or where glycosylation sites distal to these sites have been derivatized with a molecular adjuvant, while retaining the overall 3-dimensional structure of a discontinuous conserved epitope of the wild-type protein. Preferably, the polypeptide has both changes. Preferably, the primate lentivirus is HIV, and the protein is HIV-1 gp 120.

The invention provides a humanized PD-L1 tumor cell line MC-38-hPD-L1, a builtanimal tumor model with the same and a method for constructing the humanized PD-L1 tumor cell line. The method particularly includes knocking out animal-origin PD-L1 by the aid of CRISPR-CAS9; carrying out amplification and cultivation to obtain knocked-out cell banks; extracting DNA (deoxyribonucleic acid) and carrying out PCR (polymerase chain reaction) amplification; recycling and cloning amplification products; carrying out over-expression on human-origin PD-L1 in MC-38 cell lines of mPD-L1 KO by the aid of lentivirus systems; packaging lentivirus and screening Puromycin to obtain the humanized MC-38 cell line of PD-L1. The humanized PD-L1 tumor cell line, the animal tumor model and the method have the advantages that as shown by results, high killing efficiency and multiplication capacity are obviously presented by tumor infiltration CD8 T lymphocytes after antibody treatment is carried out, tumor infiltration Treg cells can be obviously inhibited after antibody treatment is carried out, and accordingly the method is proved to be effective and feasible from the aspect of molecular mechanisms.

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 Tat-based vaccine composition comprising at least one antigen coupled to at least one immunostimulatory lentivirus trans-activator of transcription (Tat) molecule wherein the antigen is a cancer antigen an infectious disease antigen or a fragment thereof and methods to treat disease by administering the Tat-based vaccine composition. An additional Tat-based vaccine composition comprising immunostimulatory lentivirus Tat is provided.

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 recombinant lentiviral vector containing gRNA sequence specifically targeting CCR5 and application thereof. A lentivirus of the CRISPR/Cas9 recombinant lentiviral vector containing gRNA sequence specifically targeting CCR5 gene Delta 32 region is constructed that the lentivirus can introduce cells into a CRISPR/Cas9 system specific to CCR5, double-chain breakage occurs to a specific site of CCR5 gene, a random mutation is introduced to a breakage site after repairing by means of nonhomogeneous recombinant terminal binding, and the mutation rate reaches 90% and above. As gRNA is a nonhomogeneous region of CCR5 and CCR2, detection shows that the missing efficiency of the two gRNAs is lower than 0.2%. Cells modified via the recombinant lentivirus have significantly decreased efficiency of HIV (human immunodeficiency virus) infection. The system is quick to construct, simple and low in price, and is applicable to gene therapy of acquired immune deficiency syndrome.

The invention discloses a CRISPR-Cas9 targeted and knockout human breast cancer cell SLC30A1 gene and the specific sgRNA of the gene. Firstly, sgRNA for specific targeting of the SLC30A1 gene is obtained, and a base sequence of the sgRNA is shown in SEQ ID NO.1; secondly, the sgRNA of the SLC30A1 gene is constructed to a lentivirus vector system containing Cas9 proteins; finally, CRISPR/Cas9 lentivirus containing the sgRNA is infected with a human breast cancer cell MDA-MB-231 to obtain a cell strain of which the SLC30A1 protein expression level is obviously reduced. The CRISPR-Cas9 targeted and knockout human breast cancer cell SLC30A1 gene and the specific sgRNA of the gene disclosed by the invention have the advantages of simple operation steps, good sgRNA targetability and high cuttingefficiency on the SLC30A1 gene; in addition, the constructed CRISPR/Cas9 lentivirus system has the advantage of high knockout efficiency and can specifically knock out the SLC30A1 gene so as to obtain the human breast cancer cell having the SLC30A1 gene knocked out; therefore, a powerful tool is provided for further researching an action mechanism of the SLC30A1 in breast cancer cells.

The invention discloses a CRISPR/Cas9 targeted knockout human colorectal carcinoma cell DEAF1 gene and specific sgRNA thereof. The CRISPR/Cas9 targeted knockout human colorectal carcinoma cell DEAF1 gene and the specific sgRNA thereof are characterized in that: firstly, sgRNA of a second exon of the specific targeted DEAF1 gene is obtained and the base sequence of the sgRNA is shown as SEQ ID NO.1; secondly, the sgRNA of the DEAF1 gene is constructed into a lentiviral vector system, which contains Cas9 protein; finally, the CRISPR/Cas9 lentivirus containing the sgRNA is infected with human colorectal carcinoma cell HT-29 cell, so that a cell strain of which DEAF1 protein expression level is obviously reduced is obtained. The CRISPR/Cas9 targeted knockout human colorectal carcinoma cell DEAF1 gene disclosed by the invention has the advantages of simple operation steps, good sgRNA target ability and high cutting efficiency for the DEAF1 gene; in addition, the constructed CRISPR/Cas9 lentiviral vector system has the advantage of high knockout efficiency and can specifically knock out the DEAF1 gene to obtain the human colorectal carcinoma cells knocking out the DEAF1 gene, and therebya powerful tool is provided for further studying an action mechanism of DEAF1 in the colorectal carcinoma cells.

The invention belongs to the technical fields of immunology and tumor therapy, and relates to a preparation method and a use of a TCR gene modified CD8+T memory stem cell. The method comprises the following steps: co-incubating a tumor antigen and an immature dendritic cell to obtain a specific tumor antigen-supported mature dendritic cell, co-culturing the specific tumor antigen-supported maturedendritic cell and a CD8+ naive T cell, and adding a stem cell differentiation inhibitor to promote the generation of a CD8+ stem cell-like memory T lymphocyte; separating the CD8+ stem cell-like memory T lymphocyte; and cloning a T cell receptor gene (TCR) for specifically identifying a specific antigenic epitope, and carrying out lentivirus packaging or retroviral vector cotransfection on an autologous CD8+ Tscm cell to in-vitro prepare a CD8+ TCR-Tscm cell specific for different tumor specific antigens. The CD8+ TCR-Tscm cell prepared by the method has the advantages of overcoming of the tumor heterogeneity, high specificity, few adverse reactions, and highly-efficient and lasting tumor preventing and treating effects.

The present invention discloses CRISPR/Cas9 targeted knockout human intestinal cancer cell RITA gene and specific sgRNA thereof. First, sgRNA specifically targeted to a second exon of the RITA gene isobtained, and the base sequence of the sgRNA is as shown in SEQ ID NO. 1; secondly, a sgRNA lentiviral vector system of the RITA gene is constructed, and the sgRNA lentiviral vector system contains Cas9 protein; and finally human intestinal cancer HT-29 cells are infected with CRISPR/Cas9 lentivirus containing the sgRNA to obtain a cell line which is significantly reduced in RITA protein expression level. The invention has the advantages of simple operation steps, good sgRNA targeting property, and high RITA gene cutting efficiency; furthermore, the constructed CRISPR/Cas9 lentivirus system has the advantage of high knock-out efficiency and can specifically knock out the RITA gene to obtain RITA gene-knockout human intestinal cancer cells, and a powerful tool for the further study of theaction mechanism of RITA in the intestinal cancer cells is provided.

A method of constructing a specific CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats associated) to activate a RSPO2 (R-spondin 2) gene is disclosed in the present invention. The method comprises the following steps: designing a sgRNA (single guide RNA) of a specifically targeted human RSPO2 gene; constructing a CRISPR-Cas9 recombinant lentivirus vector of a specifically activated RSPO2 gene; and lentiviral packaging a CRISPR-Cas9 system of the specifically activated RSPO2 gene. The CRISPR-Cas9 system designed by the present invention activates the RSPO2 target gene expression and promotes the activation of the hepatic stellate cell.

The invention provides the construction for a farnesyl pyrophosphoric acid synthetase RNA (Ribonucleic Acid) interference recombinant lentivirus vector, which comprises the following steps of: sieving the most effective target sequence of an FDS (farnesyl diphosphate synthase) gene RNAi (RNA interference) in a tool cell 293T cell, synthesizing the double-stranded DNA of the most effective target sequence, connecting to a pGCSIL-GFP vector and successfully constructing the recombinant vector through enzyme cutting, sequencing and identification. Researches indicate that the constructed RNA interference vector LV-sh-FDS can downwards modulate the expression of an FDS mRNA (Messenger RNA) level in a neonatal rat cardiac myocyte, simultaneously can downwards modulate the expression of myocardial hypertrophy markers such as cell areas and marker genes beta-MHC (Myosin Heavy Chain) and BNP (Brain Natriuretic Peptide), additionally can effectively inhabit the activity of RhoA while downwards modulating the FDS, can be applied in preparing medicaments for treating myocardial hypertrophy diseases and also can be applied in preparing medicaments for cholesterol metabolic control.

The invention discloses a CRISPR-Cas9 for targeting knockout of a human intestinal cancer cell CNR1 gene, and specific sgRNA sequences thereof. Firstly, an sgRNA specifically targeting CNR1 gene second exon is obtained, and base sequences of the sgRNA are as shown in SEQ ID NO. 1; secondly, the sgRNA of the CNR1 gene is constructed to a lentiviral vector system, and the lentiviral vector system contains Cas9 protein; and finally, a CRISPR/Cas9 lentivirus containing the sgRNA is infected with human intestinal cancer HT-29 cells, and a cell strain with CNR1 protein expression level significantlybeing reduced is obtained. The operation steps are simple, good sgRNA targeting is achieved, and the cutting efficiency to the CNR1 gene is high; and in addition, the constructed CRISPR/Cas9 lentiviral system has the advantage of high knockout efficiency, and can specifically knock out the CNR1 gene, human intestinal cancer cells with the CNR1 gene being knocked out is obtained, and therefore a powerful tool for further study of the mechanism of action of CNR1 in intestinal cancer cells is provided.

The present invention relates to retroviral vectors which will infect and confer efficient gene transfer to non-dividing cells including the cells of the central nervous system. The vector system of the present invention is useful as a gene transfer vehicle for gene therapy, i.e. of the central nervous system.