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CN-121975746-A - Application of chikungunya virus structural protein in improving stability of pseudoviruses, construction method, vaccine, antibody evaluation and in-vivo gene therapy product

CN121975746ACN 121975746 ACN121975746 ACN 121975746ACN-121975746-A

Abstract

The invention belongs to the field of genetic engineering, and in particular relates to application and a construction method of chikungunya virus structural protein in improving the stability of pseudoviruses, and vaccine and antibody evaluation and in-vivo gene therapy products. Constructing human codon optimized Asian strain CHIKV-E3+E2+6K+E1 structural protein eukaryotic vector, designing 2 kinds of target T cell binding expression plasmids, co-transferring the vector and psPAX kinds of auxiliary plasmid containing SFFV promoter and ZsGreen reporter gene (or CAR therapeutic gene) lentivirus destination vector into 293T cell, culturing, filtering and centrifuging to prepare lentivirus. The lentivirus can efficiently infect Jurkat cells and activate human T cells, remarkably reduces the infection efficiency of 293T cells to reduce the off-target effect, has excellent stability in human serum, and provides a safe and efficient gene delivery tool for in-vivo CAR-T therapy.

Inventors

  • LIU JUN
  • ZHOU CHUNYAN
  • LI LIPING
  • JIN LI
  • XIE WEIJIAN

Assignees

  • 复百澳(苏州)生物医药科技有限公司

Dates

Publication Date
20260505
Application Date
20251215

Claims (12)

  1. 1. The application of the chikungunya virus structural protein in construction of pseudoviruses is characterized in that the nucleic acid sequence of the chikungunya virus structural protein is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.
  2. 2. The use according to claim 1, wherein the chikungunya virus is asian strain chikungunya virus.
  3. 3. The use according to claim 1, wherein the pseudoviral vector system is selected from at least one of a lentiviral vector system, a retroviral vector system, an adenoviral vector system or an adeno-associated viral vector system.
  4. 4. The use according to claim 1, wherein the nucleic acid sequence of the chikungunya virus structural protein is shown in SEQ ID No. 1.
  5. 5. A method for constructing a pseudovirus is characterized by comprising the steps of constructing a eukaryotic vector for expressing a chikungunya virus structural protein, co-transfecting a host cell with an auxiliary plasmid and a target vector containing a defective genome by the eukaryotic vector, culturing the host cell, and then collecting and preparing the pseudovirus, wherein the nucleic acid sequence of the chikungunya virus structural protein is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.
  6. 6. The method of claim 5, wherein the pseudoviral vector system is selected from at least one of a lentiviral vector system, a retroviral vector system, an adenoviral vector system, or an adeno-associated viral vector system.
  7. 7. The method according to claim 5, wherein the objective vector comprises a promoter selected from at least one of SFFV promoter, CMV promoter or EF 1. Alpha. Promoter and a functional gene selected from at least one of reporter gene or therapeutic gene.
  8. 8. The method of claim 7, wherein the reporter gene is at least one of a ZsGreen gene or a GFP gene, and the therapeutic gene is at least one selected from a CAR gene, a gene editing system encoding gene, or a cytokine encoding gene.
  9. 9. The method of claim 5, further comprising constructing a targeting molecule expression plasmid, wherein the targeting molecule in the targeting molecule expression plasmid is selected from at least one of an antibody, nanobody, ligand, or aptamer, and wherein the target of the targeting molecule is selected from at least one of CD3, CD7, BCMA, or CD 19.
  10. 10. The targeting virus vector is characterized by comprising a chikungunya virus structural protein, wherein the nucleic acid sequence of the chikungunya virus structural protein is SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.
  11. 11. Use of the targeted viral vector of claim 10 for the preparation of a product for gene therapy of cells in vivo.
  12. 12. The use of claim 11, wherein the in vivo cellular gene therapy comprises in vivo CAR-T cell therapy.

Description

Application of chikungunya virus structural protein in improving stability of pseudoviruses, construction method, vaccine, antibody evaluation and in-vivo gene therapy product Technical Field The invention belongs to the field of genetic engineering, and in particular relates to application and a construction method of chikungunya virus structural protein in improving the stability of pseudoviruses, and vaccine and antibody evaluation and in-vivo gene therapy products. Background In Vivo chimeric antigen receptor T cell (In Vivo CAR-T) therapy is taken as an important development direction of the cell therapy field, T cells are directly reprogrammed into CAR-T cells In Vivo through an innovative gene delivery technology, and complex processes such as In vitro separation, modification and amplification are not needed, so that the treatment link is simplified, the personalized preparation cost is reduced, potential safety hazards such as T cell exhaustion possibly brought by In vitro gene modification can be avoided, and the method becomes a core technical path for promoting the transformation of CAR-T therapy to the "instant use" and expanding the clinical application range. The current gene delivery technology of In Vivo CAR-T (In Vivo CAR-T) therapy mainly focuses on two major directions of viral vector transformation and non-viral vector innovation, wherein a lentiviral vector becomes one of the mainstream technology choices due to the advantages of high gene integration efficiency, strong continuous expression capability and the like. The existing lentiviral vector is modified to be mostly unfolded around vesicular stomatitis virus G protein (VSV-G), on one hand, the combination of the vesicular stomatitis virus G protein and a natural receptor low density lipoprotein receptor (LDL-R) is blocked through amino acid mutation, the risk of non-target cell infection is reduced, on the other hand, the resistance of the vector to a serum complement system is improved through mutation, the in vivo circulation time is prolonged, and meanwhile, targeting molecules such as anti-CD 3 single-chain variable fragments (scFv) and the like are coupled on the surface of the virus, so that the accurate delivery of T cells is realized. In addition, other membrane fusion proteins such as the Cocal glycoprotein are also studied to construct lentiviral vectors, or the performance of the vectors is further optimized through engineering nanobodies and costimulatory ligand modification, but the VSV-G mutant is still used as a core fusion protein in the whole technical route. However, the existing lentiviral vector based on VSV-G mutant still faces a key bottleneck in clinical application, namely, firstly, the serum stability is insufficient, even though the vector is subjected to mutation transformation, the vector is still easy to recognize and clear by a complement system after entering human blood, so that the effective circulation time is short, and the target cells are difficult to reach efficiently, secondly, the off-target risk is not completely solved, and part of VSV-G mutant can still infect non-target cells such as 293T cells through a non-specific way while reducing the binding capacity with LDL-R, so that the potential safety hazard of treatment is increased, thirdly, the synergic property of the fusion protein function and the targeting property is difficult to balance, and the coupling of targeting molecules (such as scFv) can interfere with the pH-dependent membrane fusion function of the VSV-G mutant, or influence the structural stability of virus particles, so that the gene delivery efficiency is reduced. These problems limit the application of lentiviral vectors in CAR-T therapy in vivo, and there is a need to develop a completely new fusion protein system to break through the bottleneck of the prior art. Chikungunya virus (Chikungunya Virus, CHIKV) is an enveloped virus of the genus alphavirus of the family togaviridae, providing a new technical idea for solving the above-mentioned problems. The virus has the capability of stable survival in human blood, can avoid complement system clearance after entering into host blood through aedes bite, and has the structural proteins (E1, E2, E3 and 6K) playing key roles in the virus infection process, namely, the E2 protein is responsible for combining with host cell receptors, the E1 protein mediates fusion of virus envelope and cell membrane, and the E3 and 6K proteins assist structural protein folding, transportation and viral particle assembly budding, so that a high-efficiency and stable infection mechanism is formed. The unique structure and functional characteristics make the novel lentivirus fusion protein have natural advantages in the aspect of developing the novel lentivirus fusion protein, not only can realize high-efficiency membrane fusion through the structural protein, but also is hopeful to improve the stability of the carrier by virtue of the natural s