INTRODUCTION:

Gene therapy is a medical field which focuses on the genetic modification of cells to produce a therapeutic effector the treatment of disease by repairing or reconstructing defective genetic material. The first attempt at modifying human DNA was performed in 1980 by Martin Cline, but the first successful nuclear gene transfer in humans, approved by the National Institutes of Health, was performed in May 1989. The first therapeutic use of gene transfer as well as the first direct insertion of human DNA into the nuclear genome was performed by French Anderson in a trial starting in September 1990. It is thought to be able to cure many genetic disorders or treat them over time.

Between 1989 and December 2018, over 2,900 clinical trials were conducted, with more than half of them in phase I. As of 2017, Spark Therapeutics' Luxturna (RPE65 mutation-induced blindness) and Novartis' Kymriah (Chimeric antigen receptor T cell therapy) are the FDA's first approved gene therapies to enter the market. Since that time, drugs such as Novartis' Zolgensma and Alnylam's Patisiran have also received FDA approval, in addition to other companies' gene therapy drugs. Most of these approaches utilize adeno-associated viruses (AAVs) and lentiviruses for performing gene insertions, in vivo and ex vivo, respectively. ASO / siRNA approaches such as those conducted by Alnylam and Ionis Pharmaceuticals require non-viral delivery systems, and utilize alternative mechanisms for trafficking to liver cells by way of GalNAc transporters.

The concept of gene therapy is to fix a genetic problem at its source. If, for instance, a mutation in a certain gene causes the production of a dysfunctional protein resulting (usually recessively) in an inherited disease, gene therapy could be used to deliver a copy of this gene that does not contain the deleterious mutation and thereby produces a functional protein. This strategy is referred to as gene replacement therapy and is employed to treat inherited retinal diseases.

While the concept of gene replacement therapy is mostly suitable for recessive diseases, novel strategies have been suggested that are capable of also treating conditions with a dominant pattern of inheritance.

What is a gene and how does it work:

In the center of every cell in your body is an area called the nucleus. The nucleus contains your DNA (deoxyribose nucleic acid), which is the genetic code that was passed down (inherited) from each of your parents. Genes are made up of anywhere from a few hundred to a few million pieces of DNA . You have 46 chromosomes, which are each made up of thousands of genes. Each person has between 20,000 and 25,000 genes in their body. You have 2 copies of every gene, 1 inherited from your mother and 1 from your father. Genes contain instructions for the cell on how to make proteins. Proteins are needed to perform most life functions. Genes tell cells how to work, control our growth and development, and determine what we look like and how our bodies work. They also play a role in the repair of damaged cells and tissues.¹

Type of Call:

Gene therapy may be classified into two types:

Somatic:

In somatic cell gene therapy (SCGT), the therapeutic genes are transferred into any cell other than a gamete, germ cell, gametocyte, or undifferentiated stem cell. Any such modifications affect the individual patient only, and are not inherited by offspring. Somatic gene therapy represents mainstream basic and clinical research, in which therapeutic DNA (either integrated in the genome or as an external episome or plasmid) is used to treat disease.²

Over 600 clinical trials utilizing SCGT are underwayin the US. Most focus on severe genetic disorders, including immunodeficiencies, haemophilia, thalassaemia, and cystic fibrosis. Such single gene disorders are good candidates for somatic cell therapy. The complete correction of a genetic disorder or the replacement of multiple genes is not yet possible. Only a few of the trials are in the advanced stages.³

Germline:

In germline gene therapy (GGT), germ cells (sperm or egg cells) are modified by the introduction of functional genes into their genomes. Modifying a germ cell causes all the organism's cells to contain the modified gene.

The change is therefore heritable and passed on to later generations. Australia, Canada, Germany, Israel, Switzerland, and the Netherlandsprohibit GGT for application in human beings, for technical and ethical reasons, including insufficient knowledge about possible risks to future generationsand higher risks versus SCGT.The US has no federal controls specifically addressing human genetic modification (beyond FDA regulations for therapies in general).⁴

What is gene therapy:

So, now that we know what genes are, how can they be used to help fight cancer? If a gene becomes damaged, this damage is called a mutation. This can lead to a gene not working properly and a cell growing uncontrollably. When cells grow too quickly or uncontrollably, cancer can form. Keep in mind that developing a cancer is not quite as simple as this, but requires a complex series of multiple mutations⁵. These mutations may be caused by things like smoking, the environment, or they may be inherited (passed down from your parents). It makes sense that if we could repair these mutations, we could possibly stop a cancer from starting. The question is, how do we do this⁶.

Researchers are testing several ways of using gene therapy in the treatment of cancer:

Replace missing or non-functioning (non-working) genes. For example, p53 is a gene called a "tumor suppressor gene." Its job is just that: to suppress (prevent or stop) tumors from forming. Cells that are missing this gene, or have a non-functioning copy because of a mutation, may be "fixed" by adding functioning copies of p53 to the cell.

· Oncogenes are mutated genes that can cause either a new cancer or the spread of an existing cancer (metastasis). By stopping the function of these genes, the cancer and/or its spread may be stopped.

· Use the body's own immune system by inserting genes into cancer cells that then trigger the body to attack the cancer cells as foreign invaders.

· Insert genes into cancer cells so that chemotherapy, radiation therapy, or hormone therapies can attack the cancer cells more easily.

· Create "suicide genes" that can enter cancer cells and cause them to self-destruct.

· Cancers need a blood supply to grow and survive, and they form their own blood vessels to do so. Genes can be used to prevent these blood vessels from forming, which starves the tumor to death (also called anti-angiogenesis).

· Use genes to protect healthy cells from the side effects of therapy, allowing higher doses of chemotherapy and radiation to be given.^7,8

How is gene therapy given:

Gene delivery is one of the biggest challenges of gene therapy. You can imagine it would be hard to actually inject these genes into the tiny cells, so a carrier, or a "vector," is used to do this. Typically, viruses are used as the vectors. The virus vector must be genetically changed to carry human DNA. These viruses are like those that cause the common cold, only they are "deactivated" (turned off) so that they will not cause the patient to actually get the cold.⁹

In some cases, some cells are taken from the patient and the virus is exposed to the cells in the laboratory. The virus with the desired gene attached finds its way into the cells. These cells are allowed to grow in the laboratory. The grown cells are then given back to the patient by intravenous (IV) infusion or are injected into a body cavity (i.e. the lung) or a tumor. ¹⁰

In other cases, the vector with the attached gene is directly inserted into the patient by intravenous infusion or is injected into a body cavity or a tumor. Once the gene has reached the cell, it must go to the cell's nucleus (where the DNA, or genetic code is) and combine with the human genetic material. Then it needs to be "turned on," to produce the protein product of that gene. For gene delivery to be successful, the protein that is produced must work properly. As you can see, that’s a number of steps that need to go right for the therapy to work.¹¹

Methods of gene therapy:

Gene therapy implies an approach that aims to modify, delete, or replace abnormal gene(s) at a target cell. Such target cells may be malignant primary or metastatic nodules, circulating tumor cells or dormant stem cells, and specific cells such as T-cell lymphocytes or dendritic cells. With the presence of over 20,000 active genes in human cells, exposed to numerous factors whether hereditary, environmental, infectious or spontaneous, unlimited possibilities for gene mutation, aberration, dysfunction or deletion have been expected, leading to clinical presentation of various medical disorders, including cancer. Furthermore, genomics of cancer evolve between primary and metastases¹². For example, estrogen receptor gene (ESR J) mutations in breast cancer were found in proportion of metastases but not in primary tumors. Whole-exome sequencing of metastatic samples reported among the top 17 mutated genes, only five were mutated in primary tumors. The evolution from minority clone to lethal metastases follows branched evolution. Thus, tumors with high a level of intratumor heterogenicity and genomic instability could be more likely to escape from targeted therapies such as gene therapy, unless such a branched evolution is taken into consideration¹³. Hence, gene therapy is somewhat difficult to achieve, with limited success. Presently, most approaches are for monogenic gene therapy, tackling one or more critical gene defects. Selection of the appropriate mode of gene therapy is based on the assessment of the immune status, and determination of the molecular nature of a patient’s disease. With the recent increases in knowledge of molecular biology of various medical disorders, a more advanced and comprehensive gene therapy approach will ultimately become available, with anticipated improved results.¹⁴

Gene transfer delivery system:

Several methods have been developed to facilitate the entry of genetic materials (transgenes) into target cells, using various vectors. They are broadly divided into two major categories: viral (or bacterial) and non-viral vectors. Viruses usually bind to target cells and introduce their genetic materials into the host cell as part of their replication process. As they enter target cells, they can carry a load of other genetic material called “transgenes¹⁵”. For non-viral vectors, different approaches have been utilized, using physical, chemical, as well as other modes of genetic transfer. Transferring genetic material directly into cells is referred to as “transfection”, while moving them into cells carried by a viral or bacterial vector is termed “transduction”. Non-viral approaches have the advantage of safety and easy modifiability, but have a lower transfection efficiency compared to viral vectors.¹⁶

Physical mediated gene transfer:

DNA genetic material that is coated with nanoparticles from gold or other minerals, and with their kinetic energy supplemented by compressed air or fluid (gene gun), or using ultrasound, can force the genetic material into the target cell, followed by the release of DNA into its nucleus. They are best suited for gene delivery into tissue or in case of gene vaccination¹⁷. The electroporation gene therapy approach aims to achieve cellular membrane disruption with high-voltage electrical pulses, resulting in the formation of nanopores through which naked DNA, foreign genetic materials, and even chemotherapeutic agents can enter cells. This approach is best suited for plasmid DNA-based gene transfer therapy with the advantage of effectiveness in a vast array of cell types, ease of its administration, lack of genome integration with the risk of malignancy, as well as the low potential for unwanted immunogenicity. Electroporation is presently being tested in several clinical trials, especially on patients with malignant melanoma, prostate cancer, colorectal cancer, and leukemia.¹⁸

Chemical mediated gene transfer:

Cationic liposomes are microscopic vesicles of synthetic phospholipids and cholesterol that can enter into cells by endocytosis with the capability of carrying a variety of molecules such as drugs, nucleotides, proteins, plasmids and large genes. Their advantage is selectivity to endothelial cells, a relatively high rate of gene transfer efficiency, a broad application as carriers for many genes, and the lack of severe side effects. When combined with small interfering RNA (siRNA), cationic liposomes may lead to the inhibition of tumor proliferation, inducement of apoptosis, and enhancement of radiosensitivity to tumor cells. Synthetic viruses have been developed to exploit the efficiency of viral vectors and the advantage of liposomes. Once they enter the target cell, DNA is released from the endosome. This method has shown promising results in preclinical studies. Transposons can also transport genetic material inside the cell as well as into the nucleus¹⁹.

Bacterial mediated gene transfer:

Some bacteria have the capability of specifically targeting tumor cells, leading to RNA interference (RNAi) and gene silencing with blockage of RNA functions, including cellular metabolism and protein synthesis. Examples include Escherichia coli, Salmonella typhimurium, Clostridium, and Listeria. Bacterial vectors can deliver pro-drug-converting enzymes and cytotoxic agents into tumor cells, and can mediate the host immune response²⁰. They can be engineered to carry magnetic or fluorescent material to enhance the utility of diagnostic approaches in tumor localization, such as with magnetic resonance imaging (MRI) , and even in the development of cancer vaccines . However, the outcome has been far less pronounced compared to other RNA interference silencing techniques. Overall, genetically engineered bacteria acting as vectors for RNA interference are relatively safe, effective, practical and cheaper to manufacture compared to viral vectors. They selectively colonize and grow within the tumor. They can also be administered orally, hence their use in the management of gastrointestinal disorders.²¹

Viral mediated gene transfer:

Viruses are small particles that contain either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and may be single-stranded (ss) or double-stranded (ds). The viral structure consists of a genome surrounded by a protective protein coat (viral capsid) which helps the virus attach to host cell receptors, and prevents viral destruction by cell nuclease enzymes²². Some viruses may also have a lipid bilayer envelope derived from the host cell’s membrane, and an outer layer of viral envelope made of glycoprotein. A complete viral particle (virion) by itself is unable to replicate. For propagation, the virus needs to insert its genetic material into a host cell, in order to acquire metabolic and biosynthetic products for viral transcription and replication. VirusesViruses can be modified genetically to be noninfectious. As they enter the cell, they can carry genetic material for delivery into a target cell’s cytoplasm, and subsequently into the nucleus. In monogenic gene therapy, virus vectors can carry a load of 2–10 kb of relevant genes²³. In high complex gene therapy, other supporting molecules can also be added, such as immune-stimulatory molecules to the virus’s DNA for subsequent release during viral replication. The advantage of viral vectors in gene therapy is the ease of purification into high titers, and prolonged gene expression with minimal side effects. Retroviruses including lentiviruses can integrate themselves into host cell genome at the nucleus, while adenoviruses and adeno-associated viruses predominantly persist as extrachromosomal episomes. RNA viruses comprise about 70% of all viruses, and vary greatly in genomic structures. They usually have a higher mutation rate with increased adaptation to attack different host cells²⁴. Single-stranded RNA viruses may have a viral reverse transcriptase enzyme in their genome, which helps in genetic transcription of the viral genome inside the host nucleus, into double-stranded pro-viral DNA. With viral integrase, the pro-viral DNA then integrates with the host DNA making the subsequent transcription of other parts of the virus possible, in order to give rise to a new retrovirus progeny. The proteins of the mature virion are then rearranged to form the new viral particles. Viral particles subsequently destroy the host cell, and release mature viruses to attack neighboring cells. Double-stranded DNA viruses enter the host cells by endocytosis through interaction between the virus and cell receptor. The virus then enters the nucleus through nuclear pores once they escape cellular endosome. The virus subsequently releases two gene products which bind to the retinoblastoma and p53 tumor suppressor genes, thus allowing viral replication. New viruses then cause cell lysis and the released viruses spread to attack neighboring cells.²⁵

What are the side effects of gene therapy:

Given that gene therapy is so new, we do not know all the side effects it may have, particularly long-term side effects that may happen years after receiving this therapy. After initially receiving a type of gene therapy, the patient's immune system may react to the foreign vector. Symptoms of a reaction may include fever, severe chills (called rigors), drop in blood pressure, nausea, vomiting, and headache. These symptoms typically resolve within 24-48 hours of the infusion. Other side effects depend on the type of vector used and how it is given. For example, if the gene is given into a patient's lung, the side effects may affect the lung²⁶.

Some side effects are theoretical, meaning that it is possible that they could happen, yet they have not actually happened in clinical trials as of yet. There is a fear that the genes could enter healthy cells, causing damage to them, which could then lead to another disease or another cancer. If genes enter reproductive cells, they could possibly cause damage to sperm or eggs. It is feared that this damage could then be passed on to future generations. At this time, researchers are very careful to watch for these unwanted side effects and perform tests in animal studies before the therapy is given to humans²⁷. Gene therapy is a potential approach to the treatment of genetic disorders in humans. This is a technique where the absent or faulty gene is replaced by a working gene, so the body can make the correct enzyme or protein and consequently eliminate the root cause of the disease. Are there long term effects of gene therapy.²⁸

Most gene therapies are designed to achieve permanent or long-lasting effects in the human body, and this inherently increases the risk of delayed adverse events. Delayed adverse events associated with gene therapy have been reported before, such as in clinicaltrials for X-linked severe combined immunodeficiency²⁹.

CONCLUSION:

he conclusions from these trials imply that gene therapy has the potential to cure numerous genetic diseases and that the procedures appear to have minimal risks to the patient, but the efficiency of gene transfer and the expression of the corrective genes in the human patients is still very low. Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient's cells instead of using drugs or surgery. Gene therapy replaces a faulty gene or adds a new gene in an attempt to cure disease or improve your body's ability to fight disease. Gene therapy holds promise for treating a wide range of diseases, such as cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS. All of these trials have used somatic cell gene therapy, which has been very difficult due to the problem of getting the genes into enough cells. Advocates of germ-line gene therapy say that this therapy will be much more successful because the procedure will be "infinitely easier.

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Received on 03.11.2021 Modified on 18.12.2021

Res. J. Pharmacology and Pharmacodynamics.2022;14(1):37-42.

DOI: 10.52711/2321-5836.2022.00006