Overview of Suicide Genes

Suicide genes refer to a class of genes that can lead to cell death or apoptosis, and they can be divided into two categories: endogenous suicide genes and exogenous suicide genes. Endogenous suicide genes refer to genes that cells themselves have that can regulate cell life cycle and death, such as p53 and Bcl-2. Exogenous suicide genes refer to genes introduced into cells by genetic engineering techniques that can produce cytotoxic substances or sensitize cells to certain drugs, such as thymidine kinase of herpes simplex virus (HSV-TK) and Escherichia coli cytosine deaminase (CD). The characteristic of suicide genes is that they usually require specific conditions or signals to activate, such as low oxygen, high temperatures, or drugs. This avoids damage to normal cells and only targets abnormal or diseased cells. The function of suicide genes is to eliminate harmful cells, such as tumor cells, infected cells, and immune hyperreactive cells, by inducing cell death or apoptosis.

Suicide genes are important in disease treatment, mainly because they can be used as an effective targeted therapy, improve therapeutic efficacy and safety, and reduce side effects and drug resistance. For example, in tumor therapy, by introducing suicide genes into tumor cells or carrier cells (such as viruses and bacteria) and then giving corresponding inducers (such as GCV and 5-FC), tumor cells can produce a large number of toxic metabolites or Sensitize tumor cells to chemotherapeutic drugs, so as to achieve the purpose of eradicating tumors.

Mechanism of Suicide Gene Therapy

There are two main mechanisms of suicide gene therapy: indirect suicide gene therapy and direct suicide gene therapy.

Indirect suicide gene therapy, also known as gene-directed enzyme/prodrug therapy (GDEPT). It refers to the transfection of some enzyme-encoded genes that can convert non-toxic or low-toxic prodrugs into cytotoxic or sensitizing metabolites into target cells and then administer the corresponding prodrugs to induce target cell death or apoptosis. These enzymes are usually derived from viruses or bacteria, such as HSV-TK, CD, and cyanide synthase (Csyn) of Escherichia coli. There is a high degree of specificity between these enzymes and the corresponding prodrugs, such as HSV-TK and GC, CD and 5-FC, Csyn, and sodium nitroprusside (SNP). When these enzymes are expressed in the target cells, the corresponding prodrugs can be given to the target cells to produce a large number of toxic metabolites or sensitize the target cells to other therapeutic methods, so as to achieve the purpose of treatment. For example, HSV-TK can phosphorylate GCV to GCV-monophosphate (GCV-MP), which is further phosphorylated by intracellular kinases to GCV-triphosphate (GCV-TP), which can inhibit DNA polymerase and be embedded in DNA strands, thereby blocking DNA replication and repair and leading to cell death. Indirect suicide gene therapy also has a "bystander effect", that is, cells transfected with suicide genes can transmit toxic metabolites to untransfected adjacent cells through gap junctions or lytic release, thereby expanding the therapeutic effect.

Direct suicide gene therapy, also known as gene-directed toxin therapy (GDAT). It refers to the transfection of some genes that can directly produce cytotoxic substances or induce apoptosis signaling pathways into target cells, thereby inducing target cell death or apoptosis. These genes usually come from humans or other organisms, such as leukemia inhibitory factor (BAX), galectin (LacZ), tumor necrosis factor (TNF). After these genes are expressed in the target cells, they can directly or indirectly activate the effector enzymes of apoptosis, caspases, which lead to the degradation of the nucleus and organelles, and eventually lead to cell death. For example, BAX can promote the permeability of the outer mitochondrial membrane, release cytochrome C and other apoptosis-inducing factors, and activate caspase-9 and caspase-3, thereby triggering apoptosis. The advantage of direct suicide gene therapy is that it does not need to give prodrugs and does not depend on intracellular enzyme activity, so it can avoid some problems of drug resistance and toxicity. However, direct suicide gene therapy also has some disadvantages, such as lack of specificity, bystander effect, and possible immune response.

Delivery Systems for Suicide Gene Therapy

The carrier of suicide gene therapy refers to the system that wraps or carries the suicide gene and transports it into the target cells, which is a key factor affecting the efficacy and safety of suicide gene therapy. Suicide gene therapy delivery systems can be divided into two categories: viral vectors and non-viral vectors.

Viral vectors use some modified viruses to carry and deliver suicide genes, such as adeno-associated virus (AAV), retrovirus (RV), adenovirus (AdV). Viral vectors have the advantages of high-efficiency transfection ability, stable gene expression, and a wide host range, but there are also some disadvantages, such as limited load capacity, immune response, and gene recombination. Different types of viral vectors have their own characteristics, and they need to be selected according to the size of the suicide gene, the type of target cells, and the transfection method. For example, AAV is a small, non-enveloped, non-pathogenic, single-stranded DNA virus that can integrate suicide genes into the chromosomes of host cells for long-term, stable expression. However, the payload capacity of AAV is only about 4.7 kb, which limits its ability to carry larger suicide genes. RV is an enveloped single-stranded RNA virus that can integrate suicide genes into the chromosomes of host cells to achieve long-term stable expression. However, RV can only infect dividing cells and cannot infect quiescent or differentiated cells, which limits its application in solid tumors. AdV is a medium-sized, non-enveloped, double-stranded DNA virus that epigenetically presents a suicide gene in the nucleus of the host cell to achieve temporary or persistent expression. AdV has a large load capacity (about 36 kb) and can carry multiple suicide genes or regulatory elements. However, AdV is prone to cause a strong immune response and cytotoxicity, resulting in decreased transfection efficiency and uncontrolled gene expression.

Non-viral vectors have the advantages of no immune response, no gene recombination, and no load capacity limitation, but there are also some disadvantages, such as low transfection ability, unstable gene expression, and difficulty targeting specific cells. Different types of non-viral vectors have their own characteristics, and they need to be selected according to the characteristics of the suicide gene, the characteristics of the target cell, and the transfection conditions. For example, liposomes, which are tiny vesicles composed of double-layered phospholipid molecules, can encapsulate suicide genes inside or outside, thereby protecting suicide genes from degradation by nucleases. However, liposomes are easily bound by serum proteins or complement in the blood, resulting in reduced stability and transfection efficiency. Nanoparticles are tiny particles made of materials such as metals, polymers, and biomacromolecules and can adsorb or wrap suicide genes on the surface or inside, thereby improving the stability and transfection efficiency of suicide genes. However, nanoparticles may cause toxicity and inflammatory responses in cells or tissues, resulting in reduced safety and biocompatibility. Bacteria are a biological carrier that utilizes its own ability to invade and proliferate to deliver suicide genes, which can selectively infect tumor tissues or immunosuppressive tissues to achieve targeted transfection. However, the bacteria may trigger the host's immune response and rejection, resulting in reduced safety and controllability. Stem cells are undifferentiated cells with self-renewal ability and multi-directional differentiation ability, and they can integrate suicide genes into their own chromosomes to achieve long-term stable expression. However, stem cells may cause the host's immune response and tumor formation, resulting in a decrease in their safety and controllability.

Clinical Research Progress of Suicide Gene Therapy

Suicide gene therapy has been clinically tested in various types of cancer, including glioma, prostate cancer, liver cancer, pancreatic cancer, ovarian cancer, etc.

A phase I/II clinical trial for relapsed or refractory glioma using an AAV-based vector to transfect the HSV-TK gene into tumor cells, followed by administration of GCV treatment. A total of 44 patients were enrolled in the trial, of whom 24 received AAV-HSV-TK/GCV and 20 received placebo. The results showed that the median survival period of the AAV-HSV-TK/GCV treatment group was 14.1 months, while that of the placebo group was 6.2 months, and the difference was statistically significant (P = 0.006). In addition, the 6-month and 12-month survival rates were 79% and 46% in the AAV-HSV-TK/GCV-treated group, respectively, compared with 47% and 15% in the placebo group, the difference was also statistically significant (P = 0.01 and P = 0.003). This trial showed that AAV-HSV-TK/GCV treatment can significantly improve the survival time and survival rate of patients with relapsed or refractory gliomas without serious adverse reactions. The advantage of this test is that it uses a high-efficiency, stable, non-pathogenic vector system, and a suicide gene system with high specificity and a strong bystander effect. The trial was limited by its small sample size, which did not adequately assess differences in response among patients with different subtypes or grades of glioma.

A phase II/III clinical trial in locally advanced or metastatic prostate cancer used a retrovirus (RV)-based vector to transfect the HSV-TK gene into tumor cells, followed by GCV therapy. A total of 248 patients were enrolled in the trial, of whom 123 received RV-HSV-TK/GCV and 125 received placebo. The results showed that the overall response rate of the RV-HSV-TK/GCV treatment group was 33.3%, while that of the placebo group was 17.6%, and the difference was statistically significant (P = 0.003). In addition, the median survival time of the RV-HSV-TK/GCV treatment group was 11.4 months, while that of the placebo group was 10.1 months. The difference was not statistically significant (P = 0.22). This trial showed that RV-HSV-TK/GCV treatment significantly improved response rates in patients with locally advanced or metastatic prostate cancer but had no significant effect on survival. The advantage of this assay is the use of a vector system that integrates into the host genome for long-term expression and a suicide gene system with bystander effects and immunostimulatory effects. The limitation of this test is that the RV vector can only infect dividing cells and cannot cover all tumor cells, and GCV treatment may cause adverse reactions such as myelosuppression.

A phase I/II clinical trial for advanced liver cancer used a liposome-based vector to transfect the CD gene into tumor cells, followed by 5-FC treatment. A total of 14 patients were enrolled in the trial, 8 of whom received liposomal CD/5-FC and 6 received placebo. The results showed that the overall response rate of the liposome-CD/5-FC treatment group was 37.5%, while that of the placebo group was 0%, and the difference was statistically significant (P = 0.04). In addition, the median survival time of the liposome-CD/5-FC treatment group was 6.8 months, while that of the placebo group was 4.6 months. The difference was not statistically significant (P = 0.17). This trial showed that liposome-CD/5-FC treatment can significantly improve the response rate in patients with advanced liver cancer, but the effect on survival is not significant. The advantages of this assay are the use of a vector system with no immune response, no gene recombination, no load capacity limitation, and a suicide gene system with low toxicity, high efficiency, and high sensitivity. The limitations of this test are that the liposome carrier is easily bound by serum protein or complement, resulting in a decrease in its stability and transfection efficiency, and that 5-FC treatment may cause adverse reactions such as abnormal liver function.

Table 1. clinical research progress of suicide gene therapy