CN-121975747-A - Double-targeting recombinant herpes simplex virus and application thereof in detection of circulating tumor cells

Abstract

The invention relates to biotechnology and medical diagnosis field, disclose a double targeting recombinant herpes simplex virus and its application in the detection of circulating tumor cells, this virus has been through systematic genetic modification, have deleted neurovirulence gene ICP34.5 and immune escape gene ICP47 in order to improve the security; the ICP4 gene essential for virus replication is placed under the control of a tumor specific hTERT promoter, and green fluorescent protein is expressed in a coupling way, so that the virus can only replicate in tumor cells and fluoresce, meanwhile, the critical invasion protein gD on the surface of the virus is modified, and the virus is redirected by inserting the structural domain of human lectin CD301 so as to be preferentially combined with a specific glycosylation marker on the surface of the tumor cells. The double targeting mechanism enables the recombinant virus to be used as a high-sensitivity and high-specificity living probe, specifically traces the living CTC in a complex biological sample (such as blood), and provides a brand-new and functional detection tool for liquid biopsy of cancers.

Inventors

• ZHANG WEN
• WEN ZHANG
• CHEN SIYU

Assignees

• 重庆臻伽亦生物科技有限公司

Dates

Publication Date

20260505

Application Date

20260409

Claims (10)

1. 1. A double-targeting recombinant herpes simplex virus is characterized in that a virus genome is obtained by modifying a template by taking a bacterial artificial chromosome containing an HSV-1 full-length genome as a starting point, and the modifying process comprises the following steps: a) The ICP34.5 gene and ICP47 gene were deleted; b) The endogenous ICP4 expression cassette was deleted; c) At the deletion position of the endogenous ICP4 expression cassette, an expression cassette encoding ICP4 protein and a reporter protein driven by a human telomerase reverse transcriptase promoter is inserted; d) The gene encoding the surface glycoprotein gD was engineered, the sequence encoding the amino acid at the N-terminal portion thereof was deleted, and a sequence encoding a fusion protein comprising at least two lectin domains of human lectin CD301 linked by a linker peptide was inserted at the deletion position.
2. 2. The double-targeted recombinant herpes simplex virus of claim 1, wherein in step c), the reporter protein is a fluorescent protein or a luminescent protein, and the fluorescent protein comprises at least one of a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, or any variant thereof.
3. 3. A double-targeted recombinant herpes simplex virus according to claim 1, wherein in step c) the expression cassette achieves co-expression of ICP4 protein and reporter protein by a member selected from the group consisting of 2A self-cleaving peptide comprising at least one of T2A, P2A, E2A, F A or internal ribosome entry site.
4. 4. A double-targeted recombinant herpes simplex virus according to claim 1, wherein in step d) the gene encoding the surface glycoprotein gD is engineered with the deletion of the sequence encoding amino acids 6-38.
5. 5. The double-targeted recombinant herpes simplex virus of claim 1, wherein in step d), the linker peptide is a flexible linker peptide or a rigid linker peptide.
6. 6. The double-targeted recombinant herpes simplex virus of claim 5, wherein the flexible linker peptide comprises at least one of (Gly 4 Ser) n (n≥1), (Gly) 8, EGKSSGSGSESKST, GSAGSAAGSGEF, and the rigid linker peptide comprises at least one of A (EAAAK) nA (n≥2) and hCG beta CTP.
7. 7. The use of a double-targeting recombinant herpes simplex virus according to any one of claims 1-6 for the preparation of a product for detecting circulating tumor cells.
8. 8. The use of a double-targeted recombinant herpes simplex virus according to claim 7 for preparing a circulating tumor cell detection product, wherein the sample is contacted with the recombinant herpes simplex virus and the expression of the reporter protein is detected.
9. 9. The use of a double-targeted recombinant herpes simplex virus according to claim 8 for preparing a circulating tumor cell detection product, wherein the sample is a blood sample.
10. 10. The use of a double-targeting recombinant herpes simplex virus according to claim 9 in the preparation of a circulating tumor cell detection product, wherein the circulating tumor cell detection product is a diagnostic reagent or a kit, and the diagnostic reagent or the kit comprises the recombinant herpes simplex virus according to any one of claims 1-6.

Description

Double-targeting recombinant herpes simplex virus and application thereof in detection of circulating tumor cells Technical Field The invention relates to the field of biotechnology and medical diagnosis, in particular to a double-targeting recombinant herpes simplex virus and application thereof in detection of circulating tumor cells. Background Circulating Tumor Cells (CTCs) are tumor cells that shed from a primary tumor or metastatic tumor and enter the peripheral blood circulation. The detection and analysis of CTCs is of critical clinical significance for early diagnosis, prognosis, efficacy assessment, and personalized treatment regimen formulation of cancer. However, CTCs are very rare in blood, often only a few or even none per milliliter of blood, which makes their detection a great challenge. Current CTC detection techniques are largely divided into physical and biological methods. Physical methods rely on physical properties such as cell size, density, charge, etc., for separation, but have poor specificity. Biological methods are mainly based on capturing and identifying specific antigens on the surface of tumor cells (e.g. EpCAM). There are significant limitations to this CTC detection technique. On the one hand, this method fails to detect a more aggressive subset of CTCs that down-regulate or lack EpCAM expression due to undergoing epithelial-to-mesenchymal transition (EMT), and on the other hand, its sensitivity is limited when the number of CTCs is extremely low. Furthermore, the prior art generally requires complex cell enrichment and immobilization steps, can damage cells, and makes functional analysis of live CTCs difficult. The use of viruses as reporter vectors is an emerging direction to increase detection sensitivity. After infection of cells by the virus, the expression of the reporter gene is a natural signal amplification process. However, existing viral vectors often face challenges with insufficient specificity. Viruses, if they retain their natural cellular affinity, infect large numbers of normal cells, producing a background signal that is indistinguishable. Therefore, how to construct a virus detection tool which can target a wider CTC population and thoroughly eliminate off-target effects is a technical problem to be solved in the field. Disclosure of Invention Aiming at the problem of insufficient specificity of a viral vector in the prior art, the invention provides a double-targeting recombinant herpes simplex virus and application thereof in detection of circulating tumor cells, and aims to solve the problem of missed detection caused by target limitation and the problem of false positive caused by insufficient specificity in the existing CTC detection technology. In order to achieve the aim, the invention adopts the following technical scheme that a double-targeting recombinant herpes simplex virus is obtained by taking a bacterial artificial chromosome containing HSV-1 full-length genome as a starting template for transformation, and the transformation process comprises the following steps: a) The ICP34.5 gene and ICP47 gene were deleted; b) The endogenous ICP4 expression cassette was deleted; c) At the deletion position of the endogenous ICP4 expression cassette, an expression cassette encoding ICP4 protein and a reporter protein driven by a human telomerase reverse transcriptase promoter is inserted; d) The gene encoding the surface glycoprotein gD was engineered, the sequence encoding the amino acid at the N-terminal portion thereof was deleted, and a sequence encoding a fusion protein comprising at least two lectin domains of human lectin CD301 linked by a linker peptide was inserted at the deletion position. Preferably, in step c), the reporter protein is a fluorescent protein or a luminescent protein, and the fluorescent protein comprises at least one of a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, or any variant thereof. Preferably, in step c) the expression cassette achieves co-expression of the ICP4 protein and the reporter protein by means of an element selected from the group consisting of 2A self-cleaving peptides comprising at least one of T2A, P2A, E2A, F A or internal ribosome entry sites. Preferably, in step d) the gene encoding the surface glycoprotein gD is modified by deleting the sequence encoding amino acids 6-38. Preferably, in a modification, in step d), the linker peptide is a flexible linker peptide or a rigid linker peptide. Preferably, as an improvement, the flexible linker peptide comprises at least one of (Gly 4 Ser) n (n≥1), (Gly) 8, EGKSSGSGSESKST, GSAGSAAGSGEF, and the rigid linker peptide comprises at least one of A (EAAAK) nA (n≥2) and hCG beta CTP. Preferably, as an improvement, the use of a double-targeted recombinant herpes simplex virus for the preparation of a product for the detection of circulating tumor cells. Preferably, as a modification, the sample is contacted with a recombinant herp