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A Beginner's Guide to Gene Therapy

For a few years now, gene therapy has been in the spotlight of scientific research. This innovative approach has its roots in the 1970s, when Roger Stanfield suggested that “good DNA” could be used to restore defective DNA in patients with genetic disorders (Rogers, 1971). Fifty years later, dozens of gene therapies have been approved and more and more gene therapy drugs are on clinical trials. According to the FDA, gene therapy aims to modify or manipulate a patient’s DNA in order to treat or cure a disorder (Center for Biologics Evaluation and Research). The correct version of a gene is packed into a vector. The vector is delivered into the patient’s cells. Cellular machinery translates the correct version of the gene and thus produces the desired protein. Gene therapy targets the core of the disease, aiming at correcting the genetic mistake itself, rather than just fighting the symptoms. This revolutionary approach has endless applications, it is flexible and constitutes a new era in therapeutics. However, genetic manipulation comes with practical challenges and ethical considerations that still need to be overcome.

Who runs the world? Proteins!

Before discovering the insights of gene therapy, it is important to understand the background of gene expression. Mammalian DNA consists of nucleobases: Adenine (A), Thymine (T), Cytosine (C), Guanine (G). Genetic information is saved in the DNA in the form of the genetic code. The genetic code consists of 64 combinations of the nucleobases in triads. The first step of gene expression is the transformation of DNA to RNA, by the process of transcription. Afterwards, the RNA is translated into amino acids. Finally, cellular machinery organises the amino acids into proteins (Costa dos Santos, Renovato-Martins, & de Brito, 2021).

Mutations in the DNA can lead into the creation of defective proteins. A mutation is a change in the DNA sequence. These changes can be alterations in the nucleobase sequence (substitution, deletion, insertion), chromosomal mutations concerning whole regions of chromosomes or copy number variations. Somatic mutations arise spontaneously after errors in the replication of DNA or due to exposure to mutagens such as the UV radiation or chemicals. Mutations in the DNA of the eggs or sperm are called germline mutations and are passed on to next generations.

Figure 1: DNA mutation (Newman, 2018)

Current gene therapy approaches

The two main types of gene therapy are gene addition and gene editing. Gene editing aims to either silence a gene (gene silencing) or to correct it (gene correction). Gene addition techniques deliver to the cell the correct form of a malfunctioning gene. Once the correct genetic material is in the cell, gene expression machinery will transcribe and translate the “healthy” gene, providing the cell and thus the organism with the correct form of the desired protein (Tang & Xu, 2020). Gene editing is a more complicated approach; its main goal is to correct the mutation. The most popular gene editing techniques are Zinc Finger Nucleases (ZFNs), Transcription activator-like effector nucleases (TALENs) and the Nobel-awarded CRISPR. With this editing techniques, cells are administered the editing machinery, which recognise and modify the desired part of the DNA (Janik et al., 2020).

Both types of gene therapies can be conducted as ex vivo, in vivo and in situ. In ex vivo gene therapies, patient’s cells are collected and provided with the gene therapy reagents outside the body. The engineered cells are then reintroduced into the patient. This kind of approach is usually preferable for treating blood disorders, as blood cells can be easily removed and reintroduced into the organism. For the in vivo gene therapy, the gene therapy reagents are injected into the patient’s body, usually intravenously. Finally, in situ therapies are directly delivered, usually by injection, into the specific diseased organ, such as the eye or the liver (Kaufmann, Büning, Galy, Schambach, & Grez, 2013).

But how does DNA enter the cell? There are surprisingly many ways. DNA is delivered into the cell by a vector. A vector can either be viral or non-viral. Viral vectors are viruses whose pathogenic action has been artificially removed. Examples of viral vectors are the Adeno-associated virus (AAV), retrovirus and lentivirus (Ghosh, Brown, Jenkins, & Campbell, 2020). The desired genetic material is integrated into the viral genome along with other regulatory elements that allow gene expression. Consequently, once the virus is in the human cells, depending on the type of virus, it is either integrating into the human genome or it remains in the cytoplasm. Gene expression machinery of the cell transcribe and translate the new genetic information (Bulcha, Wang, Ma, Tai, & Gao, 2021).

Figure 2: Viral Gene Therapy (Bulcha, Wang, Ma, Tai, & Gao, 2021)

Non-viral vectors are either chemical or physical. Chemical vectors, such as liposomes and polymers, can carry the target genes, preventing them from degradation, and are able to enter the cell, usually by a natural process called endocytosis (Gehl, 2003). Physical methods for gene delivery aim at a higher gene transfection rate than viral methods. One of the most well-known methods is electroporation, that uses short-high voltage pulses to enable the DNA to surpass the cell membrane and enter the cell. Microinjection is another efficient technique that delivers the genetic material to the cell. A micropipette is used for the injection. This technique has a very low cytotoxicity and a high efficiency of transduction (Zhang & Yu, 2008).

Diving into the world of gene therapies

On September 1990, the first approved gene therapy was performed on a 4-year-old girl suffering from severe combined immunodeficiency (SCID). The little girl, Ashanti DeSilva, had a specific form of SCID, called Adenosine Deaminase Deficiency (ADA), which is characterised by the absence of the enzyme adenosine deaminase. Anderson and his colleagues extracted Ashanti’s blood cells, delivered the adenosine deaminase gene and then re-injected the engineered blood cells into the girl. The treatment was successful and offered long-term results (Anderson, 1990).

Nine years later, the 18-year-old Jesse Gelsinger entered a clinical trial aiming at developing a gene therapy for his disease, a rare metabolic disorder called Ornithine Transcarbamylase Deficiency Syndrome (OTCD). The patient was injected an adenoviral vector carrying the OTC gene. Four days later, Jesse died from an intense viral inflammation (Somia & Verma, 2000). This was a major setback for gene therapies, which lead to intense studies on the safe use of viral vectors. Twenty-four years later the FDA has approved 27 Cell and Gene Therapy products, targeting diseases such as myeloma, retinal dystrophy and Spinal Muscular Atrophy (Center for Biologics Evaluation and Research).

Figure 3: Ashanti DeSilva, age 6 (Thai, 1993)

Challenges and Considerations

Although the landscape of gene therapy seems more than hopeful, there are still challenges that need to be overcome. One of the most permanent challenges is the incorporation of the gene into the correct cell type. Integration of the gene therapy reagents into a different cell type is not only inefficient, but may also cause cytotoxic reactions. Caution is also needed on the insertion site of the gene on the patient’s DNA. If the virus integrates into existing genes or regulatory elements of transcription, gene expression of unrelated genes could be disturbed and cause a series of other adverse events. Moreover, as discussed in the case of Jesse Gelsinger, immunogenicity issues can also be proved extremely dangerous. Current research is focusing on understanding which elements may cause immunogenic reactions and what alternatives can be used (Colella, Ronzitti, & Mingozzi, 2018). Finally, ethical considerations should also be discussed. Intervention in the human nature, unethical use of this technology, distinguishing between “good” and “bad” human features as well as the often unbearable costs of gene therapies are some of the issues that require strict regulation around the gene therapy by the competent bodies.


Gene therapy has great potential in long-term healing of human disease. It targets the defect at its source and is applicable in a variety of disorders. The associated risks and challenges are being currently researched, but advancements in the technology are seen daily. The ultimate goal is that gene therapy becomes a first-line treatment, accessible for people of all economic and social backgrounds.

Bibliographical References

Anderson, W. F. (1990). September 14, 1990: The beginning. Human Gene Therapy, 1(4), 371–372. doi:10.1089/hum.1990.1.4-371 Bulcha, J. T., Wang, Y., Ma, H., Tai, P. W., & Gao, G. (2021). Viral vector platforms within the gene therapy landscape. Signal Transduction and Targeted Therapy, 6(1). doi:10.1038/s41392-021-00487-6 Center for Biologics Evaluation and Research. (n.d.). What is gene therapy? Retrieved from Center for Biologics Evaluation and Research. (n.d.-b). Retrieved from Colella, P., Ronzitti, G., & Mingozzi, F. (2018). Emerging issues in AAV-mediated in vivo gene therapy. Molecular Therapy - Methods & Clinical Development, 8, 87–104. doi:10.1016/j.omtm.2017.11.007 Costa dos Santos, G., Renovato-Martins, M., & de Brito, N. M. (2021). The remodel of the “Central Dogma”: A metabolomics interaction perspective. Metabolomics, 17(5). doi:10.1007/s11306-021-01800-8 Gehl, J. (2003). Electroporation: Theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiologica Scandinavica, 177(4), 437–447. doi:10.1046/j.1365-201x.2003.01093.x Ghosh, S., Brown, A. M., Jenkins, C., & Campbell, K. (2020). Viral vector systems for gene therapy: A comprehensive literature review of progress and biosafety challenges. Applied Biosafety, 25(1), 7–18. doi:10.1177/1535676019899502 Janik, E., Niemcewicz, M., Ceremuga, M., Krzowski, L., Saluk-Bijak, J., & Bijak, M. (2020). Various aspects of a gene editing system—CRISPR–cas9. International Journal of Molecular Sciences, 21(24), 9604. doi:10.3390/ijms21249604 Kaufmann, K. B., Büning, H., Galy, A., Schambach, A., & Grez, M. (2013). Gene therapy on the move. EMBO Molecular Medicine, 5(11), 1642–1661. doi:10.1002/emmm.201202287 Rogers, S. (1971). Change in the structure of Shope papilloma virus-induced arginase associated with mutation of the virus. Journal of Experimental Medicine, 134(6), 1442–1452. doi:10.1084/jem.134.6.1442

Somia, N., & Verma, I. M. (2000). Gene therapy: Trials and tribulations. Nature Reviews Genetics, 1(2), 91–99. doi:10.1038/35038533

Tang, R., & Xu, Z. (2020). Gene therapy: A double-edged sword with great powers. Molecular and Cellular Biochemistry, 474(1–2), 73–81. doi:10.1007/s11010-020-03834-3

Zhang, Y., & Yu, L.-C. (2008). Microinjection as a tool of mechanical delivery. Current Opinion in Biotechnology, 19(5), 506–510. doi:10.1016/j.copbio.2008.07.005

Visual Sources

Figure 1: Newman T (2018). Genetic mutations in healthy tissue are more common than previously thought. [Image]. MedicalNewsToday.

Figure 2: Bulcha, J. T., Wang, Y., Ma, H., Tai, P. W., & Gao, G. (2021). Viral vector platforms within the gene therapy landscape. [Image]. Nature.

Figure 3: Thai T/The LIFE Picture Collection/Getty Images (1993). Ashanthi DeSilva, age 6, March 1993. [Photograph]. Science History.

Author Photo

Matina Laskou

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