Biology Research Report Gene Therapy Sashi Amanda Samarathunga ID No. 00008786 Introduction As the name suggests, Gene Therapy is a class of treatment procedure that utilizes ‘genes’ (DNA segments)- instead of drugs or surgery- to treat or prevent disease. In Gene Therapy, an individual's cells and tissues are inserted with genes that can help treat certain diseases, particularly those that are hereditary. More specifically, a functional allele is inserted over a defective mutant allele but gene targeting is also commonly used; this involves gene amplification (Eugene, 2001). A crucial element in this procedure are proteins as they carry an important role in the cell structure and function; any mutation or defect in them would have an impact on the structure and homeostasis in the cells, triggering diseases. The synthesis and secretion of protein in our cells are guided by genes, hence, by removing and replacing the ‘culprit gene’ with a healthy, functional gene it is possible to acquire more useful and normal proteins (Ermak, 2015). The most popular vectors used in Gene Therapy to transplant healthy genes- or more recently, portions of genes- are viruses, this is because all viruses tend to attack and introduce their genetic material into the host cell as a stage in their process of replication. Scientists recognized that separating the viral DNA and manipulating the virus like a ‘vehicle’ to transport the therapeutic DNA to be a sound and feasible strategy in the procedure of Gene Therapy (Ermak, 2015). The procedure of gene therapy varies depending on the type of cell that is receiving treatment, as of now, there are essentially two types of Gene Therapy; Somatic Gene Therapy and Germline Gene Therapy. The literal definition of the term ‘Somatic’ is pertaining to the body. Therefore Somatic genetic engineering specifically involves making changes to the cells of the body, excepting ova and sperm; however this exception forbids these genetic alterations from being passed on to the next generation. In contrast to Somatic genetic therapy, Germline genetic therapy involves modifications that are assimilated into the genome of future generations (Sheridan, 2011). In general terms, geremline treatment includes alterations to ova and sperm prior to or after fertilisation; which is at the single-cell stage of development. Even though Gene Therapy appears to be a favourable treatment option for certain types of cancer, viral infections, inherited disorders and a variety of other diseases, it is still an experimental technique and has only been tested on diseases that lack a cure. In the future, doctors might be able to treat diseases by inserting a gene into patients’ cells rather than following treatment with drugs or surgery (Rojahn, 2014). But on the bright side, scientists continue to study it in order to confirm it’s safety and performance, and are examining multiple strategies to gene therapy, a few include: ● ● ● Replacing a defective disease-causing gene with a functional copy of the gene. Inactivating, or “knocking out,” a defective gene which is dysfunctional. Introducing the body with a new gene to help fight a disease. ● Procedure As explained, Gene Therapy generally involves changing the genetic format of cells to correct defective genes; this is generally done by replacing or editing a genetic code by adding missing code. Somatic Gene Therapy and Germline Gene Therapy are advanced procedures that intend to alter the genetic code inside a patient's cells to repair, replace or silence a broken gene (Crasto, 2013). In addition to that, they can also institute code that can synthesise a completely new protein. Gene Therapy can be done in two ways, ‘Vivo’ and ‘Ex Vitro’. In vivo therapy, an individual is instituted with a vector (usually a virus) which will then transfer the required therapeutic gene into the patient’s cells. On the other hand, ex vitro introduces a vector that codes for a specific protein (i.e.; chimeric antigen receptor) to an individual’s ‘autologous’ (their own cells) or ‘allogeneic’ (donor cells) cells that are extracted and isolated outside the body which are then modified and instituted back into the patient (refer to figure 01). When comparing the two methods, vivo seems the most convenient as there is no need to gather mitotic cells but ex vitro therapy poses less serious immune risks and is more compatible with the body. Figure: 01 1.1. Somatic Cell Gene Therapy In Somatic cell gene therapy (SCGT), therapeutic genes are placed inside ‘somatic’ cells- any cell (i.e.: bone marrow) other than gamete cells (sperm and ova), these cells do not produce the eggs and sperms that in turn produce the next generation. What this means is that Somatic Cell Gene Therapy aims to cure a disease only in the patient and not in the patient’s descendants; any alteration performed would only have an effect on an individual patient and are not inherited by their offspring. SCGT constitutes conventional clinical and basic research where therapeutic DNA, that is either merged as an external plasmid or episome or into the genome, is utilized to treat disorders (Williams & Orkin, 1986). In the U.S. more than six hundred clinical trials promoting Somatic Cell Gene Therapy have been initiated, most of them focusing on serious hereditary disorders such as haemophilia, cystic fibrosis, thalassaemia and immunodeficiencies which are single gene mutations- when it comes to SCGT, single gene disorders appear to be the best and easiest to work with. SCGT in method encompasses the idea of inserting a corrective, normal gene into the genome of a patient affected by a hereditary disease, thereby permanently fixing the disorder. Figure 02 represents the basic procedure of inserting genetic material into an individual’s genome, utilizing. viruses that hold the human genome required in the place of their own genetic material. Small fat-like molecules, namely ‘liposomes’, can also be used to transfer DNA into cells (Mawillo & Ferrari, 2008). For example, the target cells can be bone marrow cells; bone marrow cells are convenient as they can easily be isolated and re-implanted, they also tend to constantly divide and multiple throughout the span of an individual's life so as to create blood cells- however, this approach is only limited to genes that have a biological role in the blood. In the case of transferring a gene with a different biological role like the liver, lungs, muscle, etc., delivery would have to take place within those target organs. A major problem in multiple instances is accessing the appropriate tissue or multiple tissues (i.e.:. muscles all over the body) and making sure it can be delivered where it is needed. (Mawillo & Ferrari, 2008). Figure: 02 1.2. Germline Cell Gene Therapy Germline Gene Therapy (GGT) involves the modification of germ-cells- more commonly known as gametes (sperm and ova)- by introducing functional and corrective genes through to their genetic structure. Germ cell modification leads to all of the organism’s cells to accommodate this revised gene and thus is heritable, and can be inherited by their offspring and sweeped on to later generations- GGT removes a genetic disorder from a family line forever (refer figure 03). Some advantages of GGT include the fact that germ cells are situated outside the body and hence are easily accessible, this way gene delivery is less of an issue unlike with SCGT. This also connotes the idea that this inserted gene or genes would be present in all of the cells of the patient as it is transferred to progeny cells during growth and development (Hanna, 2006). Although, countries like Canada, Israel, Australia, Germany and the Netherlands forbid GGT for application in humans due to ethical and technical reasons as there is inadequate information and potential safety threats to future generations and include more drawbacks in comparison to Somatic Cell Gene Therapy. Speculating the exact location in the genome a gene would culminate in is impossible, and this suggests risks with normal gene functioning, developing new mutations. Unfortunately, even if these risks were to be overlooked, there is a possibility of developing new ethical problems with regard to serious development of GGT; some examples of these ethical problems are; determining what genetic modifications should be permitted-the main focus would be on correcting detrimental mutations, but others might be considered enhancements, rather than treatments (Hanna, 2006). Figure: 03 Viral And Non-viral Vectors As previously explained, gene therapy mainly focuses on transferring genetic material into cells, there are multiple ways to do this but two prime methods stand out; viral vectors and non-viral vectors. When it comes to viral methods, they utilize viral vectors which are also commonly known as recombinant viruses or biological nanoparticles. Non-viral methods utilize non-viral vectors- DNA complexes or Naked DNA (Sheridan, 2011). 1.1 Viral Methods All viruses tend to attack and insert their genepool into the host cell as a stage in their process of replication. Primary information or ‘instructions’ to replicate viruses are stored in this genetic material and possess the ability to manipulate the host-cell or body to address the requirements of the virus; the host cell is manoeuvred into manufacturing more copies of the virus, inevitably infecting more cells. Most viruses usually do not enter the host cell and instead tend to inject their genetic material towards the host’s cytoplasm, but some other viruses prefer to insinuate themselves into the host cell through the cell membrane by disguising themselves as protein molecules thereby confusing the host cells. Lytic and Lysogenic are two prime virus infections; viruses that follow the lytic cycle break out from their host cell and infect more cells once more viruses have been produced. Lysogenic viruses follow a cycle where they merge their DNA into the genome of the host cells, these viruses will continue to reproduce at the same pace as the cell does causing no harm to the host body until it is triggered (Sheridan, 2011). Retrovirus Retroviruses have their genetic material structured in RNA molecules unlike their host cells who structure is in the form of DNA instead and so these viruses contain special enzymes called reverse transcriptase and integrase to help guide the insertion of their RNA into a host cell. A DNA copy of this RNA molecule is formed through a process called reverse transcription (using enzyme reverse transcriptase) in order to be integrated into the host cell’s genetic material. Next, it utilizes the enzyme integrase to incorporate DNA copy into the genome of the host cell (refer to figure 04). Once the host cells start dividing, all it’s daughter cells will also contain this new genetic makeup (Eugene, 2001). Figure: 04 Adenoviruses Adenoviruses cause infections in the intestines, eyes and respiratory system in humans; they store their genetic material in the form of double-stranded DNA. Once infecting a host cell, these viruses have their genetic material in the form of a DNA molecule free in the host cell’s nucleus instead of incorporating it into the host’s genome. Transcription occurs in this extra new DNA similar to other genes except the fact that they’re not recreated during the host cell's cell division process thereby having no effect on its daughter cells. 1.2 Non-viral methods When compared to viral methods, non-viral methods are more advantageous, for example, they pose less adverse immune responses and can also be produced on a large scale. In the past, non-viral methods constituted lower levels of gene expression and transfection and so presented multiple drawbacks but latest developments in vector technology proffered efficient transfection techniques and molecules akin to viruses. As of now, the easiest method to non-viral vector method is injection of naked DNA (Eugene, 2001). Experiments with intramuscular injection of plasmid containing naked DNA were to some extent successful, but still presented low levels of gene expression when compared to other transfection techniques. Figure: 05 Gene gun The method of gene gun utilizes the idea of particle bombardment and is a physical method of DNA transfection. Gene gun method or biolistic particle delivery system is usually used in biotechnology for genetically modifying plants. With the use of a gene gun, the gene gun method delivers vector DNA or plasmid directly into a host cell’s nucleus. The payload is an elemental particle of a heavy metal coated with DNA. The method is also commonly called particle acceleration or microprojectile bombardment. Sonoporation This technique transfers DNA by utilizing ultrasonic frequencies to deliver DNA into cells. It is believed that this process of acoustic cavitation sanctions DNA to advance into the cell by distorting the cell membrane. Hydrodynamic delivery This method involves injecting vasculature swiftly (i.e.; bile duct and tail vein) with a high volume of solution, rich with molecules like DNA plasmids or siRNA, to create elevated hydrostatic pressure which facilitates cell penetration (Hanna, 2006). Adverse effects When it comes to gene therapy there are a multitude of drawbacks and adverse effects, for example some of the unanswered issues include: the therapeutic DNA delivered into target cells must be functional and the cells holding the therapeutic DNA must be stable before gene therapy may become a permanent treatment for an illness. Long-term success is hampered by issues in integrating therapeutic DNA into the genome and the constantly dividing nature of many cells. Another issue isImmune reaction- when a foreign item enters human tissue, the immune system is activated to fight the intruder. It is possible to stimulate the immune system in a way that decreases the efficacy of gene therapy. The immune system's increased reaction to previously encountered viruses decreases the efficacy of repeated treatments.Further more, problems with viral vectors also pose a rather great threat; viral vectors can cause toxicity, inflammatory reactions, and problems with gene regulation and targeting. There are also disorders caused by several genes, variations in many genes influence certain common illnesses, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, complicating treatment. Finally, cost- in 2013, Alipogene tiparvovec, or Glybera, was claimed to be the world's most costly medicine, with a price tag of $1.6 million per patient ((Mawillo & Ferrari, 2008). Reference List Crasto, AM (2013). Glybera – The Most Expensive Drug in the world & First Approved Gene Therapy in the West. All About Drugs. [Retrieved 2 November 2013]. Ermak, G. (2015). Emerging Medical Technologies. World Scientific. Eugene, H. (2001). Gene and Stem Cell Therapies. JAMA. 285 (5): 545–550. Hanna, K. (2006). Germline Gene Transfer. National Human Genome Research Institute. Mavilio, F., Ferrari, G. (2008). Genetic modification of somatic stem cells. The progress, problems and prospects of a new therapeutic technology. EMBO Reports. 9 Suppl 1: S64–69. Rojahn, S.Y., (2014). Genome Surgery. MIT Technology Review. [Retrieved 17 February 2014]. Sheridan, C. (2011). Gene therapy finds its niche. Nature Biotechnology. 29 (2): 121–128. Williams, D.A., Orkin, S.H. (1986). Somatic gene therapy. 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