Pharmaceutical composition targeting kuptake cells for treating cancer

A composition and drug technology, applied in the field of drug delivery, can solve the problems of low specificity of targeting Kupffer cells, complex mechanism of action in vivo, cumbersome operation procedures, etc., and achieve the treatment of liver metastases with low cost and strong operability Effect

Pending Publication Date: 2022-05-10
UNIV OF SCI & TECH OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these solutions generally have high preparation costs, cumbersome operation procedures, and large-scale industrial production are difficult; especially important is the complex mechanism of action in vivo, and the specificity of targeting Kupffer cells is not high

Method used

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  • Pharmaceutical composition targeting kuptake cells for treating cancer
  • Pharmaceutical composition targeting kuptake cells for treating cancer
  • Pharmaceutical composition targeting kuptake cells for treating cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Detection of the expression of bacteria-delivered DNA plasmids in living liver Kupffer cells

[0061] 1. Construction of pX459-zsGreen plasmid

[0062] Using the pX459 vector (addgene, #78623) as a blueprint, cut the vector with restriction endonuclease AgeI (NEB, #R0552S) to linearize the vector; then, use the Escherichia coli recombinase Exnase II (Vazyme, #C214) The zsGreen1 coding DNA sequence (as shown in SEQ ID NO:39) containing the start codon ATG and the stop codon TGA is recombined into the vector to obtain the pX459-zsGreen plasmid expressing by zsGreen1 instead of Cas9, so that zsGreen1 can be used as a reporter gene To explore whether the pX459 vector and its encoded protein can be expressed in Kupffer cells. The principle is: the pX459-zsGreen plasmid is driven by a eukaryotic promoter and will not be expressed in bacteria. By injecting mice with bacteria containing the plasmid, if the plasmid DNA is successfully delivered into Kupffer cells and...

Embodiment 2

[0065] Embodiment 2: Contrast the safety of using common escherichia coli (Top10) and low toxicity escherichia coli (Clearcoli)

[0066] 1. Comparing the 7-day survival rate of blood injection of low-toxic E. coli and common E. coli

[0067] In order to reduce the immune response caused by bacterial injection, we selected LPS (lipopolysaccharide, bacterial lipopolysaccharide) mutant low-toxic Escherichia coli instead of common Escherichia coli as the delivery vector of plasmid DNA. Through the tail vein of the mouse, respectively, 1×10 9 CFU of Escherichia coli Top10 or Clearcoli (Lucigen, #60810-1) (or known as BL21(DE3)) was injected into the blood of mice, and the survival of mice within seven days was observed and recorded, such as figure 2 Shown in A: the seven-day survival rate of mice injected with Top10 tail vein is about 80%, while the seven-day survival rate of mice injected with Clearcoli tail vein is increased to 100%.

[0068] 2. Comparing the expression of i...

Embodiment 3

[0074] Example 3: Construction of pX459-2U6-SapI-BsaI-CRE and pPP441-2U6-SapI-BsaI-CRE vectors

[0075] 1. pX459 (CRISPR / Ca9 system) transformation:

[0076]Using pX459 (addgene, #78623) as the blueprint, first double-enzyme the vector with restriction enzymes BsiWI (NEB, R0553S) and BsaI (NEB, R3733S) (two sites on the puromycin resistance gene) cutting, cutting out the puromycin resistance gene, and then using Escherichia coli recombinase Exnase II (Vazyme, C214) to convert the CRE enzyme (to eliminate the redundant BsaI restriction site on the vector, its sequence is shown in SEQ ID NO: 40 ) was recombined into the vector to obtain a vector in which puromycin was replaced by CRE enzyme; then, between the restriction endonuclease sites XbaI (NEB, R0145S) and KpnI (NEB, R0142S) in front of the CMV enhancer Insert T4 DNA ligase (TIANGEN, NG201) into the multiple cloning site (HindIII / XhoI / BamHI / SalI, whose sequence is shown in SEQ ID NO: 41), and then insert U6-BsaI between X...

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Abstract

The invention provides a pharmaceutical composition for treating cancer, especially cancer liver metastasis, the pharmaceutical composition comprises Escherichia coli and a pharmaceutically acceptable excipient, the Escherichia coli comprises a recombinant vector, and the recombinant vector is connected with a nucleotide sequence of sgRNA of a target gene and Cas9 protein/Casphi protein. A CRISPR/Cas gene editing system coded by plasmid DNA is delivered into Kuptake cells through the recombinant vector, and in-situ gene editing and modification can be carried out on the Kuptake cells. By knocking out c-Maf and MafB gene expressions of Kuphog cells through the method, the lethality of Kuphog cells to metastatic liver tumors can be remarkably enhanced, and the purpose of treating the liver metastatic cancer is achieved.

Description

technical field [0001] The invention belongs to the field of drug delivery, and in particular relates to a drug composition targeting Kupffer cells for treating cancer, especially liver metastases of cancer. Background technique [0002] The liver's unique dual blood supply and immune tolerance characteristics make the liver the main site of tumor metastasis. Liver metastatic cancer is not only prone to refractory disease, but also poorly responds to current T cell-based tumor immunotherapy, so it is urgent to develop new treatment methods. [0003] Kupffer cells (Kupffer cells), as liver-resident macrophages, account for 15% of the total number of liver cells, are the largest group of tissue macrophages in the body, and play a key role in regulating the immune tolerance microenvironment of the liver . In the early stage of tumor liver metastasis, Kupffer cells can phagocytize a large number of circulating tumor cells to perform immune surveillance function, but in the lat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00A61K38/46A61P35/00C12N15/113C12N15/70C12N15/55C12N1/21C12R1/19
CPCA61K48/0008A61K48/005A61K38/465A61P35/00C12N15/1135C12N9/22C12N15/70C12N2310/20C12N2800/101
Inventor 曾筑天刘维张清李璐姚奇
Owner UNIV OF SCI & TECH OF CHINA
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