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Construction and application of multiple-reading frame non-integrative lentiviral vector

A lentiviral vector, non-integrating technology, applied in the field of multi-target non-integrating lentiviral vectors and immunotherapy vectors, can solve the problem of heterogeneous population of tumor cells

Active Publication Date: 2016-10-26
李因传
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the mutating characteristics of tumors, heterogeneous populations of tumor cells appear during the growth process, which leads to the limitations of conventional single-target CAR-T therapy.

Method used

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  • Construction and application of multiple-reading frame non-integrative lentiviral vector
  • Construction and application of multiple-reading frame non-integrative lentiviral vector
  • Construction and application of multiple-reading frame non-integrative lentiviral vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Embodiment 1, construction and sequencing identification of vector

[0084] Based on the basic plasmid containing the pBR322 replication origin and the penicillin resistance gene, a linear basic plasmid sequence was constructed by PCR, and the 5'-LTR sequence of the lentivirus, the viral packaging signal sequence, the Rev response element RRE sequence, and the proviral integration element The nuclear entry enhancer sequence cPPT / CTS sequence and a multiple cloning site were integrated into a circular plasmid sequence, and then the lentiviral woodchuck hepatitis virus post-transcriptional regulatory sequence WPRE and 3'-LTR sequence were PCR cloned into the multiple of the plasmid Downstream of the cloning site.

[0085] Based on the plasmid sequence, a series of therapeutic vectors with specific functions are constructed.

[0086] On the basis of the above-mentioned basic plasmid, the S / MAR sequence Seq ID No.19 of human IFN-beta 1 (by PCR method, the 293T cell genome ...

Embodiment 2

[0117] Example 2: In vitro secretion identification of anti-CD47 antibody and in vitro secretion expression identification of SIRPaECD

[0118] 293T cells in DMEM medium (containing 5-10% FBS, containing penicillin streptomycin, mycin) and 5% CO 2Under the conditions, culture at 37°C, use pLV-CMV-MCS-IRES, pLV-CMV-CAR_T_Muc-IRES-anti_CD47 package to infect 293T cells respectively, and lyse the cells after 48 hours of infection.

[0119] The lysate was medium-strength RIPA lysate (50mM Tris-Cl, 150mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1mM MgCl 2 , 5mM EDTA, 5% glycerol, add Roche protease inhibitor cocktail).

[0120] After the protein undergoes SDS-PAGE electrophoresis, the PVDF membrane is transferred to the membrane, after being blocked by skim milk, it is incubated with horseradish peroxidase HRP-labeled goat anti-human IgG1 constant region antibody, the PVDF membrane is treated with a luminescent substrate, and X-ray film Photosensitive, X-ray film is processed w...

Embodiment 3

[0140] Example 3: Cytotoxic effect (ADCC) of anti-CD47 antibody

[0141] The virus (pLV-CMV-CAR_T_Muc-IRES-anti_CD47_IgG1) encoding the CD47 antibody infected 293T cells, and the supernatant was collected after 48 hours and 72 hours, purified through a protein A column (GE Health), eluted, dialyzed, and the resulting antibody The CD47 IgG1 concentrated antibody was quantified, a small amount was used for Western blot detection, and the rest was used for the following ADCC detection.

[0142] Construction of HUVEC target cells: Design primers according to the sequence of human CD47 (GENBANK: NM_001777), use human CD47 plasmid (Sinobiological) as a template to PCR amplify its coding frame sequence, and connect it into our constructed lentiviral vector pLV-CMV-MCS1-IRES -MCS, to obtain HUVEC cells stably transfected with CD47.

[0143] Primer hCD47a-S (XhoI): 5'-GGCCTCGAGATGTGGCCCCTGGTA-3'; hCD47a-R (BamHI): 5'-CGTGGATCCAGTTATTCATCATC-3';

[0144] ADCC was determined by convent...

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Abstract

The invention discloses construction and application of a multiple-reading frame non-integrative lentiviral vector, and relates to the technical field of gene / cell therapy. The lentiviral vector sequentially includes a 5'-LTR sequence, a promoter sequence, a multiple cloning site 1, one or more IRES, a multiple cloning site 2 and a 3'-LTR sequence from upstream to downstream; the vector also comprises a virus packaging signal sequence used for enhancing nuclear entry; and the multiple cloning site 1 and the multiple cloning site 2 are used for loading target exogenous expression gene. Experiments prove that vectors used for cellular immunity therapy of solid tumors, blood tumors, self immune diseases, stem cell therapy, viral diseases and genetic diseases can be effectively constructed on the basis of the lentiviral vector.

Description

technical field [0001] The present invention relates to the fields of gene therapy and cell therapy, in particular to a multi-target non-integrated lentiviral vector and its application in constructing various solid tumors and blood tumors, autoimmune diseases, stem cell therapy, viral diseases and Immunotherapy vector applications for genetic diseases. Background technique [0002] Virus-mediated gene transfer technology is currently a key tool technology for cell therapy and gene targeting operations, and has the advantages of high efficiency and stable integration and expression of the loaded gene. [0003] At present, vector systems such as adenovirus, adeno-associated virus, retrovirus and lentivirus have been mainly developed in the world. Among them, the adenovirus system cannot effectively integrate the genome and is only used for transient expression. Although the retrovirus has high infection and integration efficiency, it is risky. Relatively high sex, adeno-asso...

Claims

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Application Information

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IPC IPC(8): C12N15/867A61K48/00A61K39/395A61P35/00A61P35/02A61P37/02A61P31/12
CPCA61K39/3955A61K39/39558A61K48/005C12N15/86C12N2740/15043A61K39/46447A61K2239/13A61K39/4611A61K39/4631A61K39/0011A61K39/464482C07K14/7051C07K2319/03
Inventor 李因传
Owner 李因传
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