Gene therapy in oncology

Gene therapy against cancer

Inna Burkova, "Biomolecule"Thanks to the rapid development of medicine, innovative techniques, medicines, and equipment aimed at treating complex diseases such as cancer are being created.

Recently, much attention has been paid to gene therapy as a promising method of treating cancer, which in the future will become a particularly important tool for preventing and reducing cancer mortality.

This article briefly discusses the ways of disease development, as well as the use of innovative gene therapy techniques in oncology.

Deadly ClawsHumanity has faced this mysterious disease since before our era.

Learned men in various parts of the world tried to understand and treat it: in Ancient Egypt – Ebers, in India – Sushruta, Greece – Hippocrates. All of them and many other doctors were fighting a dangerous and serious enemy – cancer. And although this battle continues to this day, it is difficult to determine whether there are chances of a complete and final victory. After all, the more we study the disease, the more questions arise – is it possible to completely cure cancer? How to avoid illness? Is it possible to make treatment fast, affordable and inexpensive?

Thanks to Hippocrates and his observation (it was he who saw the similarity of the tumor and the tentacles of cancer), the term carcinoma (Greek carcinos) or cancer (Latin cancer) appeared in ancient medical treatises. In medical practice, malignant neoplasms are classified differently: carcinomas (from epithelial tissues), sarcomas (from connective, muscular tissues), leukemia (in blood and bone marrow), lymphomas (in the lymphatic system) and others (develop in other types of cells, for example, glioma – brain cancer). But in everyday life, the term "cancer" is more popular, which implies any malignant tumor.

Mutations: to die or to live forever?Numerous genetic studies have revealed that the appearance of cancer cells is the result of genetic changes.

Errors in replication (copying) and repair (error correction) DNA leads to changes in genes, including those controlling cell division. The main factors that contribute to the damage of the genome, and in the future – the acquisition of mutations, are endogenous (attack of free radicals formed during metabolism, chemical instability of some DNA bases) and exogenous (ionizing and UV radiation, chemical carcinogens). When mutations are fixed in the genome, they contribute to the transformation of normal cells into cancer cells. Such mutations mainly occur in proto-oncogenes, which normally stimulate cell division. As a result, a gene can be permanently "switched on", and mitosis (division) does not stop, which, in fact, means malignant degeneration. If inactivating mutations occur in genes that normally inhibit proliferation (tumor suppressor genes), control over division is lost, and the cell becomes "immortal" (Fig. 1).

Figure 1. Genetic model of cancer: colon cancer. The first step is the loss or inactivation of two alleles of the ARS gene on the fifth chromosome. In the case of familial cancer (familiar adenomatous polyposis, FAP), one mutation of the APC gene is inherited. The loss of both alleles leads to the formation of benign adenomas. Subsequent mutations of genes on chromosomes 12, 17, 18 of a benign adenoma can lead to transformation into a malignant tumor. Source: [1].Obviously, the development of certain types of cancer involves a change in most or even all of these genes and can take place in various ways.

It follows from this that each tumor should be considered as a biologically unique object. To date, there are special genetic information bases on cancer containing data on 1.2 million mutations from 8,207 tissue samples belonging to 20 types of tumors: Cancer Genome Atlas and Catalog of Somatic Mutations in Cancer (Catalogue of Somatic Mutations in Cancer, COSMIC) [2].

The result of a gene malfunction is uncontrolled cell division, and in subsequent stages – metastasis to various organs and parts of the body through blood and lymphatic vessels. This is a rather complex and active process, which consists of several stages. Individual cancer cells are separated from the primary focus and spread with blood throughout the body. Then, using special receptors, they attach to endothelial cells and express proteinases that break down matrix proteins and form pores in the basement membrane. Having destroyed the extracellular matrix, cancer cells migrate deep into healthy tissue. Due to autocrine stimulation, they divide, forming a node (1-2 mm in diameter). With a lack of nutrition, some of the cells in the node die, and such "dormant" micrometastases can remain latent in the tissues of the organ for a long time. Under favorable conditions, the node grows, the gene for vascular endothelial growth factor (VEGF) and fibroblast growth factor (FGFb) are activated in the cells, and angiogenesis (formation of blood vessels) is initiated (Fig. 2).

Figure 2. Spread of metastases. Drawing from the website pharmaceuticalintelligence.com .

However, cells are armed with special mechanisms that protect against the development of tumors:

• imprinting is a mechanism of epigenetic modifications that controls the normal growth and proper development of the body. Any abnormalities in the methylation of certain genes can contribute to the occurrence of cancer. For example, studies have found that the loss of imprinting after inactivation of the maternal allele of the IgF2 gene increases the risk of developing rectal cancer by 3-5 times [3];
• DNA repair (for example, single-nucleotide excision repair protects DNA from mutations caused by carcinogenic agents) [4];
• Cell cycle checkpoints – use specific messenger proteins such as ATM, ATR and the RAD17-RFC complex to search for damage in DNA molecules. Signaling proteins activate p53 and inactivate cyclin-dependent kinases, which, in turn, inhibits the cell cycle from G1 to S (G1/S restriction point), DNA replication in the S-phase and G2-phase (G2/M-restriction point) [5];
• programmed cell death – apoptosis and associated regulatory genes have a huge impact on the occurrence of a malignant phenotype. Some oncogenic mutations disrupt apoptosis, which leads to the initiation of carcinogenesis and metastasis [6];
• immune system – activation of natural killer cells (NK – natural killer cells), macrophages, neutrophils, eosinophils and specific T-cytotoxic cells; synthesis of cytokines and specific antibodies [7].

Traditional methods and their disadvantagesIf the body's defense systems failed, and the tumor still began to develop, only the intervention of doctors can save.

For a long period, doctors have used three main "classical" therapies:

• surgical (complete removal of the tumor). It is used when the tumor is small and well localized. They also remove part of the tissues that come into contact with the malignant neoplasm. The method is not used in the presence of metastases;
• radiation – irradiation of a tumor with radioactive particles to stop and prevent the division of cancer cells. Healthy cells are also sensitive to this radiation and often die;
• chemotherapy – drugs that inhibit the growth of rapidly dividing cells are used. Medications also have a negative effect on normal cells.

The above approaches may not always save the patient from cancer. Often, single cancer cells remain during surgical treatment, and the tumor can relapse, and with chemotherapy and radiation therapy, side effects occur (decreased immunity, anemia, hair loss, etc.), which lead to serious consequences, and often to the death of the patient. Nevertheless, traditional methods of treatment that can defeat cancer are improving every year and new ones are emerging, such as biological therapy, hormone therapy, the use of stem cells, bone marrow transplantation, as well as various supportive therapies. Gene therapy is considered the most promising, since it is aimed at the root cause of cancer – compensation for the malfunction of certain genes.

Gene therapy as a perspectiveAccording to PubMed, interest in gene therapy (GT) of cancers is growing rapidly, and today GT combines a number of techniques that operate with cancer cells both in the body (in vivo) and outside it (ex vivo) (Fig. 3).

Figure 3. Two main strategies of gene therapy. Ex vivo genetic material using vectors is transferred to cells grown in culture (transduction), and then the transgenic cells are administered to the recipient; in vivo introduction of the vector with the desired gene in a specific tissue or organ. Picture from [8].

Gene therapy, in vivo gene transfer involves the introduction of genetic constructs in cancer cells or in tissues that surround the tumor [9]. Gene therapy ex vivo consists of isolation of stem cells from blood or bone marrow of the patient, the embedding of the therapeutic gene in their genome and the introduction of transducerwith cells back into the patient. For these purposes, use special vectors created by genetic engineering. As a rule, the viruses that detect and destroy cancer cells, while remaining harmless to healthy tissue of the body, or non-viral vectors.

Viral vectors

As the use of viral vectors retroviruses, adenoviruses, AAV viruses, lentiviruses, herpes viruses, and others. These viruses differ in the efficiency of transduction, interaction with cells (recognition and infection) and DNA. The main criterion is safety and no risk of uncontrolled spread of the virus DNA: if the genes are inserted in the wrong place of the human genome, they can cause harmful mutations and initiate tumor development. It is also important to consider the level of expression of the transferred genes to prevent inflammatory or immune response of the body when hyperintense target proteins (table 1).

Table 1. Viral vectors [10].

Non-viral vectors

For transfer of transgenic DNA is also used non-viral vectors. Polymer-carriers of drugs are structures of nanoparticles used for delivery of drugs with low molecular weight, e.g., oligonucleotides, peptides, siRNA. Due to its small size, nanoparticles are absorbed by cells and can penetrate into the capillaries, which is very convenient to deliver "healing" of the molecules in the most remote places in the body. This technique is often used for the inhibition of tumor angiogenesis. But there is a risk of accumulation of particles in other organs such as the bone marrow, which can lead to unpredictable consequences [11]. The most popular methods of non-viral DNA delivery include liposomes and electroporation.

Synthetic cationic liposomes are currently recognized as a promising method of delivery of functional genes. The positive charge on the surface of the particles provides the fusion with negatively charged cell membranes. Cationic liposomes neutralize the negative charge of the DNA chain, making it more compact spatial structure and contribute to the efficient condensation. Plasmid-liposome complex has several important advantages: can accommodate a genetic design of virtually unlimited size, there is no risk of replication or recombination, is virtually immune response in the host organism. The disadvantage of this system is a low duration of therapeutic effect, and the re-entry can appear side-effects [12].

Electroporation is a popular method of non-viral DNA delivery, quite simple and not causing an immune response. By using electric pulses induced on the surface of cells formed pores, and plasmid DNA can easily penetrate into the intracellular space [13]. Gene therapy in vivo using electroporation has proven its effectiveness in a number of experiments on murine tumors. It is possible to carry any genes, e.g., genes of cytokines (IL-12) and cytotoxic genes (TRAIL), which contributes to the development of a wide spectrum of therapeutic strategies. In addition, this approach can be effective for the treatment of metastatic and primary tumors [14].

The choice of technique

Depending on the type of tumor and its progression for a patient gets the most effective treatment method. Currently, we have developed a promising new technique for gene therapy against cancer, among which oncolytic viral GT, GT Pro (prodrug therapy), immunotherapy, GT, using stem cells.

Oncolytic viral gene therapy

For this technique is used by viruses that use special genetic manipulation become oncolytic – cease to reproduce healthy cells and affect only the tumor. A good example of this therapy is to ONYX-015 – modified adenovirus that expresses a protein not Е1В. In the absence of this protein, the virus cannot replicate in cells with normal p53 [15]. Two vectors are constructed on the base of the herpes simplex virus (HSV-1) – G207 and NV1020 also carry mutations of several genes that replicate only in cancer cells [16]. A great advantage of this technique is that when performing intravenous injection of oncolytic viruses are carried by the blood throughout the body and can fight with metastases. The main problems that arise when working with viruses is a potential risk of immune response in the recipient's body, as well as uncontrolled integration of genetic constructs into the genome of healthy cells, and as a result, the occurrence of cancer.

Kinooperatora a Pro-enzymatic therapy

Based on the introduction of tumor tissue "suicide" genes, in which the cancer cells die. These transgenes encode enzymes that activate intracellular cytotoxic agents, TNF receptors, and other important components for the activation of apoptosis. Suicidal combination of genes prodrug should ideally meet the following requirements [17]: controlled gene expression; correct transformation of the selected prodrug into an active anti-cancer agent; full activation of prodrug without additional endogenous enzymes.

Minus the therapy is that the tumors are all defense mechanisms typical of healthy cells, and they gradually adapt to the damaging factors and the prodrug. The adaptation process promotes the expression of cytokines (autocrine regulation), of the factors regulating the cell cycle (selection of the most resistant cancer clones), MDR-gene (responsible for susceptibility to some drugs).

Immunotherapy

Thanks to gene therapy, in recent years began to develop immunotherapy – a new approach for the treatment of cancer with anticancer vaccines. The basic strategy of the method of active immunization of the body against cancer antigens (TAA) using the technology of gene transfer [18].

The main difference of recombinant vaccines against other drugs is that they help the patient's immune system to recognize cancer cells and destroy them. In the first stage, the cancer cells obtained from the body of the recipient (autologous cells) or from a special cell lines (allogeneic cells), and then grow them in vitro. In order for these cells could be recognized by the immune system, introduce one or a few genes, which produce immunostimulatory molecules (cytokines) or proteins with an increased number of antigens. After these modifications, the cells continue to be cultured, then lysis is carried out and a ready-made vaccine is obtained.

A wide variety of viral and non-viral vectors for transgenes allows experimentation on various types of immune cells (for example, cytotoxic T cells and dendritic cells) to inhibit the immune response and regression of cancer cells. In the 1990s, it was suggested that tumor infiltrating lymphocytes (TIL) are a source of cytotoxic T-lymphocytes (CTL) and natural killer cells (NK) for cancer cells [19]. Since TIL can be easily manipulated ex vivo, they became the first genetically modified immune cells to be used for anti-cancer immunotherapy [20]. In T-cells removed from the blood of a cancer patient, the genes that are responsible for the expression of receptors for cancer antigens are changed. It is also possible to add genes for greater survival and effective penetration of modified T cells into the tumor. With the help of such manipulations, highly active "killers" of cancer cells are created [21].

When it was proved that most cancers have specific antigens and are able to induce their defense mechanisms [22], it was hypothesized that blocking the immune system of cancer cells would facilitate tumor rejection. Therefore, for the production of most antitumor vaccines, the patient's tumor cells or special allogeneic cells are used as a source of antigens. The main problems of tumor immunotherapy are the likelihood of autoimmune reactions in the patient's body, the absence of an antitumor response, immunostimulation of tumor growth, and others.

Stem cellsA powerful tool of gene therapy is the use of stem cells as vectors for the transfer of therapeutic agents – immunostimulating cytokines, "suicide" genes, nanoparticles and antiangiogenic proteins [23].

In addition to the ability to self-renew and differentiate, stem cells (SC) have a huge advantage over other transport systems (nanopolymers, viruses): the prodrug is activated directly in tumor tissues, which avoids systemic toxicity (the expression of transgenes contributes to the destruction of cancer cells only). An additional positive quality is the "privileged" state of autologous SC – the used own cells guarantee 100% compatibility and increase the level of safety of the procedure [24]. However, the effectiveness of therapy depends on the correct ex vivo transfer of the modified gene to the SC and the subsequent transfer of the transduced cells into the patient's body. In addition, before applying therapy on a large scale, it is necessary to study in detail all possible ways of transformation of SC into cancer cells and develop safety measures to prevent carcinogenic transformation of SC.

ConclusionTo sum up, we can say with confidence that the era of personalized medicine is coming, when a certain effective therapy will be selected for the treatment of each cancer patient.

Individual treatment programs are already being developed that provide timely and proper care and lead to a significant improvement in the condition of patients. Evolutionary approaches for personalized oncology, such as genomic analysis, production of targeted drugs, cancer gene therapy and molecular diagnostics using biomarkers are already bearing fruit [17].

Gene therapy is a particularly promising method of treating oncological diseases. At the moment, clinical trials are being actively conducted, which often confirm the effectiveness of GT in cases where standard anti–cancer treatment – surgery, radiation therapy and chemotherapy - does not help. The development of innovative GT techniques (immunotherapy, oncolytic virotherapy, "suicide" therapy, etc.) will be able to solve the problem of high mortality from cancer, and, perhaps, in the future, the diagnosis of "cancer" will not sound like a verdict. 

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