What is gene therapy
Gene therapy is described as a being an up-and-coming branch of medicine which uses the patient’s genomic information in order to improve the clinical care they receive, for example during their diagnosis or when choosing their treatment plan (NHGRI, 2018). Gene therapy is a method in which functioning genes can be delivered to particular host cells where that gene is faulty. This functioning gene can be transfected, transformed or transduced. Transfection is the process of integrating nucleic acids using non-viral delivery into eukaryotic cells. Transformation is described as non-viral DNA transfer into bacteria, non-animal eukaryotic cells and plant cells. Transduction, on the other hand, is a term used to describe the viral delivery of DNA into targeted host cells. This new nucleic acid is then able to replicate itself within its new host cells which would undergo cell division, thus replicating cells that now contain the new functioning gene.
Why gene therapies are used
The genetic makeup of every individual differs, this variation results in different immune responses, drug metabolism and different responses to the same drug. It is evident that most drug dosages do not affect every patient in the same manner, this is due to each individual’s genetic factors accounting for approximately 20% – 95% of the variation seen in drug responses (Ahmed et al. 2016). However, majority of therapies are “one size fits all” meaning that they are not designed to accommodate for different individuals’ needs, and it is apparent that this is not always suitable as every individual has different genes. An example of this is trastuzumab which is a drug that is only beneficial to women with breast cancer who are positive in the Human Epidermal growth factor Receptor 2 (HER2) gene (Goddard et al. 2011). Gene therapy is an option that enables this matter to be managed. DNA of patients can be altered in order to regulate a specific gene, stop gene expression, or to add a gene in order to rectify hereditary diseases and to prevent disease by removing or substituting abnormal, faulty genes with functioning genes. Gene therapy is advantageous because the therapy only works on that specific patient and depending on the type of gene therapy, in theory, it could offer a permanent change and potentially a cure for their condition. Gene therapy links to personalised medicine because it is specifically tailored to the patient and the therapy offers a more targeted treatment for their symptoms.
Achieving gene therapy
In order for gene therapy to work, vectors are required to deliver the gene into the target cells and specific primers are used to control the location and the way the gene gets expressed. A functional copy of the faulty gene is introduced into a vector in preparation for delivery into the target cell. The type and location of the target cells are factors that should be considered when deciding which vector to use. Figure 1 shows how the wanted gene is delivered to the host cell and is replicated in order to produce that specific encoded protein in those targeted cells. In this particular example, the vector used is an adenovirus.
Figure 1: The use of an adenovirus to deliver the new, functioning gene into a target cell (Lumen Learning)
Types of gene delivery methods
Viral vectors are used for efficient gene delivery because they transduce their own genetic information effectively into host cells for replication. The viral genes are disabled so that the vector loses its ability to cause disease and the desired, functional genes are inserted for transduction. Gag, Pol and Env are genes that are required to make new viral particles, the infected host cell would be deficient in these particular genes so that they would be unable to produce infectious particles. This means that only the patient receives the vector which carries the therapy and it cannot be transmitted to others. Many of the successful gene therapies are conducted using viral vectors, for example the treatment of cystic fibrosis using Adeno-Associated Virus (AAV).
Cystic fibrosis is a genetic disorder that is caused by the mutated Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene which codes for chloride channels that are involved in the upkeep of a thin layer of fluid that surrounds the mucosal membranes of multiple organs and ducts including the liver, pancreas and the airways. A faulty CFTR gene results in the absence of this layer of fluid which causes an accumulation of sticky mucus. This can lead to numerous problems including inflammation and chronic lung infection (Guggino and Cebotaru 2017). The main cause of death in patients with cystic fibrosis is lung disease, making cystic fibrosis an ideal condition to develop a gene therapy for. This is because the lungs are organs that can be easily accessed by vectors used in gene therapy. As a result, there have been many clinical trials which have targeted the delivery of packaged CFTR cDNA in AAV into the airways of cystic fibrosis patients for a direct pathway into the lungs (Stoltz, Meyerholz and Welsh 2015).
Another example of using a viral vector to treat a disease is the use of retroviral vectors to treat X-linked Severe Combined Immunodeficiency (SCID-X1) which is a hereditary disease that is most commonly caused by a mutated Interleukin 2 Receptor Gamma (IL2RG) gene found on the X-chromosome. This is a disease which affects the immune system and results in the lack of development of T lymphocytes (T cells) and Natural Killer cells (NK cells) so patients are highly susceptible to infections which can be fatal. Using a functioning copy of the IL2RG-cDNA in retroviral vectors to treat SCID-X1 has been an effective therapy for younger patients (Baum et al. 2007).
However, there are also disadvantages to using viral vectors for gene delivery. Issues include triggering immune responses, insertional mutagenesis, and also the potential risk associated with mutant viruses and infections. Viruses are pathogens so they can trigger a host immune response. For most viral vectors, the position of where the vectors get inserted cannot be controlled so the random insertion of the viral genome and the gene of interest can cause mutations and further problems. An example of this was seen with patients who developed leukaemia after receiving the IL2RG gene in a retroviral vector (Baum et al. 2007). The cause of this leukaemogenesis was due to the integration of the vector into or near a LMO2 oncogene which drove cell proliferation to higher levels and caused leukaemia (Hacein-Bey-Abina et al. 2008). The use of viral vectors can also be fatal, for example Jesse Gelsinger was the first patient to have died in a gene therapy research trial conducted on Ornithine Transcarbamoylase (OTC) deficiency (Sibbald, 2001).
Therefore, to avoid the challenges that come with using viral vectors, research into the use of non-viral vectors is becoming more prominent. For example, non-viral vectors have been used for cancer gene therapy as they are a safer option than viral vectors because they are non–immunogenic, however they are less efficient than viral vectors due to their low transfection rate (Wang et al.2013). A common non-viral vector that is used are cationic lipids because they can be packaged into liposomes to deliver the DNA into the target cells. The DNA-cationic lipid complex is delivered to the target cell and the liposome membrane fuses together with the host cell membrane to release its contents into those cells, Figure 2 illustrates this process. The use of cationic lipids has been used in the treatment of Central Nervous System (CNS) disorders, for example Lipofectamine 2000 (LK2) is a cationic liposome that can transfect 20% – 25% of primary neurons. (Jayant et al. 2016) These transfected primary neurons can be used in the treatment of CNS conditions.
Figure 2: The use of cationic lipids to deliver DNA to a target cell (ThermoFisher Scientific)
Another example of non-viral gene therapy is the use of Chimeric Antigen Receptors (CARs) to produce modified T cells (CAT-T cell therapy) in the treatment of cancers. T cells are altered by the CARs which allows the cells to target specific cancer cells more effectively and thus, improve the treatment of certain cancers. For example, CAR-T-19 cells were used to treat B lymphocyte malignancies such as Acute Lymphoblastic Leukaemia (ALL) and Chronic Lymphocytic Leukaemia (CLL) (Perez-Amill et al. 2018). Figure 3 shows how CAR-T cell therapy is achieved.
Figure 3: CAR-T cell therapy process (Dana-Farber Cancer Institute)
Prospects of gene therapy
Gene therapy is continuing to become an increasingly prominent field in science, with clinical trials continuously being researched and conducted globally. It is apparent that gene therapy is a useful tool that can be used in conjunction with medicine for improved diagnostics and therapeutic care plans. By developing new and improved methods of gene delivery and plasmids that are increasingly specific to their target sites, gene therapy has endless potential to treat a myriad of disorders and potentially provide cures to many of these. It is possible to develop a tailored therapy that is specific to a patient’s needs by combining their genomic information with the knowledge of which genes are responsible so that they can be targeted. This idea of personalised medicine can help eradicate the “one size fits all” approach used in drugs and therapies which would be a more beneficial and idea approach. There are currently 16 gene therapy products approved by the Food and Drug Administration (FDA) () and with the constant progress with other clinical trials, hopefully more gene therapies and their products will be added to the list to support personalised medicine.