Overview of HSV-Thymidine Kinase
Cancer is a disease that seriously endangers human health. Currently, commonly used treatment methods such as surgery, chemotherapy, and radiotherapy have certain limitations and side effects. An oncolytic virus is a kind of virus that can selectively infect and kill tumor cells and has broad clinical application prospects. Herpes simplex virus type 1 (HSV-1) oncolytic virus is an anticancer drug with the characteristics of high efficiency, specificity, safety, and immune regulation. It has been clinically tested in various solid tumors and achieved certain results. HSV-1 TK gene is an important suicide gene, which can convert non-toxic nucleoside analogs into toxic metabolites, leading to tumor cell death. HSV-1 TK gene can also induce an immune response and enhance the oncolytic effect. The HSV-1 TK gene can be modified by genetic engineering technology to improve its tumor specificity and sensitivity, or it can be used in combination with other genes or drugs to achieve synergistic effects.
Structure Of HSV-Thymidine Kinase
HSV-Thymidine Kinase is an enzyme encoded by herpes simplex virus type 1 (HSV-1) that catalyzes the phosphorylation of thymidine and some nucleosides. The structure of HSV-thymidine kinase is a dimer composed of two identical subunits. Each subunit has about 300 amino acid residues and a molecular weight of about 35 kDa. The structure of HSV-thymidine kinase has been solved by X-ray crystallography, and the crystal structure of the complex formed with its natural substrate thymidine (dT) or the antiviral drug guanosine (GCV) has also been reported. The structure of HSV-thymidine kinase can be divided into two main domains: a larger N-terminal domain and a smaller C-terminal domain, with a deep cleft between them that forms the substrate-binding site. The N-terminal domain contains a nucleoside-binding pocket with a conserved P-loop sequence, which is involved in the binding of the phosphate group of ATP or dTMP. The C-terminal domain contains a base-binding pocket in which there is a conserved HxGH sequence that is involved in the binding of the base portion of thymine or guanidine. Upon substrate binding, relative motion occurs between the two domains, narrowing the cleft and forming a closed active site. In the active site, a nucleoside transfer reaction occurs between substrates, producing nucleoside diphosphates and inorganic phosphates.
Principles and Advantages of HSV-TK Gene Therapy
The principle of HSV-TK gene therapy is to use the herpes simplex virus thymidine kinase (HSV-TK) gene to transfect tumor cells so that it can convert the non-toxic prodrug GCV (Ganciclovir) into a cytotoxic metabolite, thereby Inhibit the proliferation and survival of tumor cells. The advantage of HSV-TK gene therapy is that it has high tumor specificity, does not affect normal cells, and can produce a "bystander effect", that is, a small number of transfected cells can kill surrounding untransfected cells and enhance the therapeutic effect. In addition, HSV-TK gene therapy can also be used in combination with other therapeutic methods, such as anti-angiogenesis, RNA interference, immunotherapy, etc., to achieve the purpose of increasing efficiency and reducing side effects.
Mechanism of HSV-TK Gene Therapy
HSV-TK works synergistically with antiviral drugs
HSV-TK is a thymidine kinase of herpes simplex virus, which can phosphorylate non-toxic antiviral drugs such as GCV or ACV to generate toxic nucleoside triphosphates, thereby inhibiting the growth of tumor cells. DNA synthesis and replication, leading to tumor cell death.
HSV-TK induces tumor cell apoptosis and autophagy
In addition to directly killing tumor cells, the HSV-TK/GCV system can also induce the programmed death of tumor cells by activating the signaling pathways of apoptosis and autophagy. For example, the HSV-TK/GCV system can activate the caspase-3 pathway, causing tumor cell apoptosis characteristics such as DNA fragmentation, chromosomal degeneration, and cytoplasmic shrinkage. The HSV-TK/GCV system can also induce the autophagy of tumor cells, make tumor cells produce autophagosomes, lysosomes and autolysosomes, and other structures, and promote the self-degradation of tumor cells.
HSV-TK activates anti-tumor immune response
The HSV-TK/GCV system not only has a killing effect on transfected tumor cells but can also produce a "bystander effect" on surrounding untransfected tumor cells. That is, the HSV-TK/GCV system can also cause toxic effects on untransfected tumor cells through gap junctions or nucleoside triphosphates released by dissolution. In addition, the HSV-TK/GCV system can also activate the body's immune system, causing the killed tumor cells to release antigens to stimulate immune effector cells such as dendritic cells, macrophages, and T lymphocytes, forming specific and nonspecific anti-tumor immune responses. Some HSV-TK vectors can also overexpress immune stimulatory factors such as GM-CSF to further enhance the immune response.
Fig.1 The HSV-TK suicide gene therapy system. (Izmirli, 2016)
Clinical Application of HSV-TK Gene Therapy
HSV-TK gene therapy for glioma
The mechanism of HSV-TK gene therapy for glioma is to transfect the HSV-TK gene into tumor cells and then administer the non-toxic nucleoside analog ganciclovir (GCV), which is induced by HSV-TK enzymes and converted into toxic metabolites, thereby inhibiting DNA synthesis and proliferation of tumor cells and inducing apoptosis. In addition, the HSV-TK/GCV system can also induce a bystander effect and an anti-tumor immune response, enhancing its therapeutic effect. The HSV-TK/GCV system has been evaluated in several clinical trials, showing certain levels of safety and efficacy. For example, it has been found that the injection of adenovirus-mediated HSV-TK/GCV into mice with gliomas can significantly inhibit the growth of tumors and reduce the lung metastasis rate of tumors by about 40%.
HSV-TK gene therapy for prostate cancer
The mechanism of HSV-TK gene therapy for prostate cancer is similar to the treatment of gliomas. It also uses the HSV-TK enzyme to convert GCV into toxic metabolites, kill tumor cells, and induce bystander effects and antitumor immune responses. The HSV-TK/GCV system has also been evaluated in clinical trials for prostate cancer, showing some safety and efficacy. For example, this method has also achieved good clinical results in the treatment of prostate cancer patients who have failed treatment or metastasized.
HSV-TK gene therapy for other types of cancer
In addition to glioma and prostate cancer, the HSV-TK/GCV system has also been used in gene therapy for other types of cancer, such as breast cancer, ovarian cancer, liver cancer, pancreatic cancer, colorectal cancer, head and neck cancer, etc. In these cancers, the HSV-TK/GCV system also exhibits certain inhibitory effects on tumor growth and metastasis and can activate the immune system against tumors. However, the clinical application of the HSV-TK/GCV system in other types of cancer needs further exploration and optimization.
Challenges and Prospects of HSV-TK Gene Therapy
Currently, HSV-TK gene transfection methods mainly include viral and non-viral vectors, each with its own advantages and disadvantages. Viral vectors have high efficiency and stability, but there are also problems such as safety, immune response, and tissue specificity. Non-viral vectors are relatively safe and have low immunity, but they have low transfection efficiency and are easily degraded by enzymes in vivo. Therefore, it is necessary to develop more efficient, specific, and safer HSV-TK gene transfection methods, such as the use of new vectors or technologies such as stem cells, nanoparticles, and gene guns. Although HSV-TK gene therapy can kill surrounding tumor cells through the bystander effect, it may also damage normal cells, especially hematopoietic stem cells and liver cells with high proliferation abilities. In addition, the dose of GCV also needs to be controlled within a reasonable range to avoid systemic side effects such as bone marrow suppression and renal function damage.
In order to improve the safety and tolerability of HSV-TK gene therapy, the following strategies can be adopted:
- use mutant or improved HSV-TK enzymes, such as A168H mutant, to reduce the toxicity to normal cells;
- use tissue-specific or Inducible promoters to control the expression of HSV-TK genes, such as the tumor-specific promoter hTERT2 or promoters regulated by the tumor microenvironment;
- the dose and time of GCV can be adjusted by combining or staging drug use to reduce adverse reactions.
Currently, there are several approaches to optimizing HSV-TK gene therapy regimens in combination with other anticancer strategies. First, it is used in combination with radiation therapy or chemotherapy to increase the sensitivity of tumor cells or reduce the damage to normal cells by means of synergistic enhancement or complementary antagonistic mechanisms. Second, in combination with immunotherapy, the antigens released by tumor cell apoptosis induced by HSV-TK gene therapy can be used to stimulate the body to produce a specific immune response, or immune cells can be used as carriers of HSV-TK genes to enhance tumor-targeting sex and lethality. Third, it is used in combination with other suicide gene therapy, using different enzyme-precursor systems or the same enzyme-different precursor systems to achieve multiple killing or cross-killing and increase the death rate of tumor cells.
In summary, HSV-1 oncolytic virus is a promising anticancer strategy, and the HSV-1 TK gene is one of its core components, which has broad application potential. In the future, it is necessary to further optimize the expression regulation, administration route, and dosage of the HSV-1 TK gene to improve its clinical transformation rate and cure rate.
Reference
- Izmirli, et al. (2016). The war against cancer: Suicide gene therapy. Advances in Modern Oncology Research. 2. 10.18282/amor.v2.i3.103.
