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CN-122005865-A - Application of adeno-associated virus vector in preparation of medicine for treating ovarian cancer

CN122005865ACN 122005865 ACN122005865 ACN 122005865ACN-122005865-A

Abstract

The invention relates to the technical field of biological medicine, and in particular discloses application of an adeno-associated virus vector in preparation of a medicine for treating ovarian cancer. The adeno-associated virus vector comprises an engineered AAV2 capsid and a polynucleotide loaded therein and carrying a therapeutic gene, wherein the amino terminus of VP2 protein of the AAV2 capsid is inserted with an EC1 targeting peptide, and the VP1 protein has R585A and R588A mutations. Compared with the conventional viral vector, the vector can specifically target ovarian cancer cells, realize efficient infection, remarkably reduce the infection capability of normal tissues such as livers and the like, and has advantages in the aspect of safety. In vivo experiments show that the tail vein injection of the adeno-associated virus vector carrying HSV-TK combined with ganciclovir can obviously inhibit tumor proliferation of mice with ovarian cancer transplantation tumor, and has no obvious damage to tissues and organs such as liver and kidney of the mice.

Inventors

  • XIONG YANQIANG
  • LV YAFENG
  • Zhou mou

Assignees

  • 武汉儿童医院

Dates

Publication Date
20260512
Application Date
20260206

Claims (10)

  1. 1. The application of the adeno-associated virus vector in preparing the medicine for treating the ovarian cancer is characterized in that the adeno-associated virus vector comprises an engineered AAV2 capsid and a polynucleotide carrying a therapeutic gene loaded in the adeno-associated virus vector, wherein the amino terminal of VP2 protein of the AAV2 capsid is inserted with an EC1 targeting peptide, and the VP1 protein has R585A and R588A mutation.
  2. 2. The method of claim 1, wherein the EC1 targeting peptide is a targeting peptide for EpCAM, the amino acid sequence of which is shown in SEQ ID NO. 1, and the nucleotide sequence of DNA encoding the EC1 targeting peptide is shown in SEQ ID NO. 2.
  3. 3. The method of claim 1, further comprising the step of preparing an adeno-associated viral vector, said step of preparing comprising: (1) Taking AAV2 capsid plasmid pRC as a template, mutating threonine at 138 th site of VP1 protein of pRC plasmid into alanine to obtain pRVP/3 plasmid for expressing VP1 and VP3, wherein the DNA sequence of pRVP1/3 plasmid is shown as SEQ ID NO. 3; (2) Taking AAV2 capsid plasmid pRC as a template, and mutating methionine at 1 st and 203 st positions of VP2 protein of pRC plasmid into alanine to obtain a pVP2 plasmid for expressing VP2, wherein the DNA sequence of the pVP2 plasmid is shown as SEQ ID NO. 4; (3) The pHelper plasmid, pAAV plasmid, pRVP/3 plasmid obtained in step (1) and pVP2 plasmid obtained in step (2) are mixed and packaged into AAV virus.
  4. 4. The method of claim 1, wherein the therapeutic gene is one or more of a suicide gene, an endogenously targeted interference/correction gene, an anti-angiogenic gene, and an immune related gene.
  5. 5. The method according to claim 4, wherein the therapeutic gene is a suicide gene.
  6. 6. The method according to claim 5, wherein the suicide gene is the HSV-TK gene.
  7. 7. The method of claim 6, wherein the pharmaceutical composition further comprises ganciclovir.
  8. 8. The method of claim 1, wherein the ovarian cancer is an ovarian cancer that is EpCAM positive.
  9. 9. The use according to claim 1, wherein the medicament has one or more of the following effects: (1) The targeting agent has high ovarian cancer targeting; (2) Inhibit growth of ovarian cancer tumor.
  10. 10. The method according to claim 1, wherein the pharmaceutical composition is in the form of an injection, and the injection is in the form of an injection and/or a freeze-dried powder injection.

Description

Application of adeno-associated virus vector in preparation of medicine for treating ovarian cancer Technical Field The invention relates to the technical field of biological medicine, in particular to application of an adeno-associated virus vector in preparation of a medicine for treating ovarian cancer. Background Ovarian cancer is one of the most common malignant tumors of female reproductive system, and has poor clinical treatment effect and prognosis due to low early diagnosis rate, high recurrence rate and easy chemotherapy resistance, and the mortality rate is the first position of gynecological tumors, and the total survival rate is only about 30% in 5 years. Although surgery and adjuvant chemotherapy have improved survival rates of ovarian cancer patients to some extent in recent years, the problems of chemotherapy resistance and recurrence of ovarian cancer all bring great challenges to clinical diagnosis and treatment. Adeno-associated virus (AAV) vector as a gene therapy vector has good application potential in the field of ovarian cancer therapy research. However, when the existing AAV vector is administered systemically, a large amount of viral particles are taken up by normal tissues such as liver, which not only reduces the effective drug dose at the tumor site, but also may induce side effects such as hepatotoxicity. Disclosure of Invention In order to solve the problem of insufficient targeting of an AAV vector in the ovarian cancer treatment process, the invention provides application of an adeno-associated virus vector in preparation of a medicine for treating ovarian cancer. The carrier shows remarkable targeting enrichment capability in ovarian cancer transplantation tumor after injection, and remarkably reduces the infection capability to normal tissues such as liver, muscle and the like. The tumor-bearing mice are further treated by tail vein injection of the virus vector loaded with suicide gene HSV-TK, and the result shows that compared with a wild AAV2 virus vector, the virus vector loaded with HSV-TK provided by the invention can obviously inhibit the growth of ovarian cancer tumors of the mice after being combined with ganciclovir. Based on the findings, the invention provides the following technical scheme: The invention provides application of an adeno-associated virus vector in preparing a medicament for treating ovarian cancer. The virus vector comprises an engineered AAV2 capsid and a therapeutic gene-carrying polynucleotide loaded therein, wherein the VP2 protein amino-terminus of the AAV2 capsid is inserted with an EC1 targeting peptide, and the VP1 protein has R585A and R588A mutations. In some embodiments of the invention, the EC1 targeting peptide is a targeting peptide for EpCAM, the amino acid sequence of which is shown in SEQ ID NO. 1, and the nucleotide sequence of the DNA encoding the EC1 targeting peptide is shown in SEQ ID NO. 2. In some embodiments of the invention, the method further comprises the step of preparing an adeno-associated viral vector: (1) Taking AAV2 capsid plasmid pRC as a template, mutating threonine at 138 th site of VP1 protein of pRC plasmid into alanine to obtain pRVP/3 plasmid for expressing VP1 and VP3, wherein the DNA sequence of pRVP1/3 plasmid is shown as SEQ ID NO. 3; (2) Taking AAV2 capsid plasmid pRC as a template, and mutating methionine at 1 st and 203 st positions of VP2 protein of pRC plasmid into alanine to obtain a pVP2 plasmid for expressing VP2, wherein the DNA sequence of the pVP2 plasmid is shown as SEQ ID NO. 4; (3) The pHelper plasmid, pAAV plasmid, pRVP/3 plasmid obtained in step (1) and pVP2 plasmid obtained in step (2) are mixed and packaged into AAV virus. In some embodiments of the invention, the therapeutic gene is one or more of a suicide gene, an endogenously targeted interference/correction gene, an anti-angiogenic gene, an immune related gene. In some embodiments of the invention, the therapeutic gene is a suicide gene. In some embodiments of the invention, the suicide gene is the HSV-TK gene. In some embodiments of the invention, the medicament further comprises ganciclovir. In some embodiments of the invention, the ovarian cancer is EpCAM positive expressed ovarian cancer. In some embodiments of the invention, the therapeutic agent has one or more of the following effects: (1) The targeting agent has high ovarian cancer targeting; (2) Inhibit growth of ovarian cancer tumor. In some embodiments of the invention, the medicament is in the form of injection, and the injection is injection and/or freeze-dried powder injection. Compared with the prior art, the invention has at least the following beneficial effects: The invention provides an application of an adeno-associated virus vector in preparing a medicament for treating ovarian cancer. The mouse living body imaging result shows that compared with a wild AAV2 virus vector, the AAV2M EC1 virus vector provided by the invention is obviously enriched in ovarian