Principles of Gene Therapy
Gene Manipulation Strategies
The five different approaches to gene manipulation, including the current uses & potential disease targets of each strategy
Additional CAR-T Cell and CRISPR video resources
Transcription of Therapeutic Gene Manipulation Strategies
[The transcription below is of the video at the top of this page.]
In this chapter we will provide information on the various gene manipulation strategies. We will discuss the different ways in which genetic mutations can cause disease, examine the 5 different approaches to gene manipulation and how they can be applied to treat those diseases.
Gene therapy can be used to treat monogenic and complex diseases, with the nature of the disease determining the appropriate approach1. There are five approaches to gene manipulation: Gene addition, gene inhibition, gene replacement, gene editing, and genetic modulation of immune cells1,2. Each approach has its strengths and weaknesses and is selected based on the nature of the disease1,2.
We will start by looking at the first of these strategies: Gene addition.
Here you can see a complex disorder, caused by defects in multiple genes as well as environmental factors that result in the disease1.
Gene addition works by introducing a new gene into the body to target a specific aspect of the disease pathway1.
Once the therapeutic gene is added, it provides factors that are then protective, resulting in easing of the disease.
An application of the gene addition approach is in genetic calcium deficits that can cause heart failure1. These deficits can be addressed by the addition of a gene that elevates calcium levels. Another good use is the addition of a gene to promote antibody production, a very useful option in combating infectious diseases1.
Next, we will look at the second gene manipulation strategy: Gene inhibition.
The second form of genetic manipulation is gene inhibition1.
Some genetic diseases are caused by a mutated gene that codes for production of mutant proteins, or ‘toxic overexpression’ of required proteins. With these gain-of-function mutations it’s necessary to turn off the mutated gene.
In this example, you can see the mutant gene coding for incorrect messenger RNA, which results in the disease-causing protein.
Gene inhibition involves introducing a small interfering RNA that either triggers RNA degradation, or binds to and prevents the messenger RNA being translated into a protein. This stops production of the disease-causing protein, diminishing its toxic effect.
The inhibition approach is particularly suited for the treatment of diseases that are caused by gain-of-function mutations – those that lead to this toxic overexpression or mutant forms of proteins1.
For example, a mutant, misfolded form of the enzyme superoxide dismutase I is responsible for amyotrophic lateral sclerosis2, and the mutant huntingtin protein is responsible for Huntington’s disease1.
Now we move on to the third gene manipulation strategy: Gene replacement.
Some diseases are caused by a mutation in a single gene that results in a lack of a particular protein.
Such diseases are well suited to treatment with the gene replacement approach1.
The mutation shown here prevents the gene’s essential product from being coded.
The mutant gene is not cut out and replaced in gene replacement therapy as it does not need to be.
Instead, a normal functional copy of the gene is inserted – this corrects the lack of activity of the mutant gene, resulting in the missing protein being produced.
Gene replacement therapy has great potential to treat a number of diseases1,2.
A gene replacement therapy product, voretigene neparvovec-rzyl, has already been approved for the treatment of inherited retinal disease3.
Other monogenic disease targets suited to this form of gene replacement include spinal muscular atrophy, lipoprotein lipase deficiency, cystic fibrosis, Leber congenital amaurosis, alpha-1 antitrypsin deficiency, and hemophilia1,4.
However, rigorous testing will be needed to address possible off-target effects. We will now move on to the fourth gene manipulation strategy: Gene editing.
The fourth form of gene manipulation is gene editing.
Here you see a disease caused by a mutant gene. In gene editing, nucleases ‘edit’ the mutant gene that is causing disease in order to correct the mutation1-3.
This approach has traditionally involved cutting out a mutant disease-causing section of DNA from the genome and replacing it with a new section of DNA using the cell’s naturally occurring DNA repair mechanisms1. However, more recently, tools have been developed that allow for base editing without the need for DNA cleavage2,3. Such base editors can produce C to T conversions (and vice versa) via deamination reactions2,3.
Gene editing is particularly suited to the treatment of infectious diseases by modifying the protein targets that allow a microbe to infect the cell. For example, by modifying a particular gene that encodes a T-cell receptor, it can render the T cell resistant to HIV infection1.
Gene editing is a relatively new technology with additional trials expected to be performed in the next decade2.
The use of the CRISPR-Cas9 system has revolutionized genome engineering, and further variants of this system show promise for gene editing3.
Finally, we will look at the fifth gene manipulation strategy: Genetic modulation of immune cells.
In a complex disorder driven by genetic and environmental factors1, the genetic modulation of immune cells is the fifth approach that can be used for gene manipulation2. It involves the introduction of genes that promote a stronger immune response, thus killing the diseased cells.
There are different ways in which genetic modulation can be used to incite immune cells to target and kill diseased cells. A gene encoding a foreign antigen could be introduced into diseased cells, triggering an immune response2. A cytokine gene could be introduced into diseased cells or non-diseased cells, particularly immune cells, to prompt an enhanced immune response2. Or, a gene could be added to immune cells to enable them to target diseased cells for destruction1. An example of this is a receptor that has been engineered to bind to a certain protein in the diseased cells.
These gene additions enhance the immune response to diseased cells, which leads to their subsequent death and alleviation of the disease.
The ‘genetic modulation of immune cells’ approach can be used in cancer to introduce a gene that promotes the death of targeted cancer cells1.
For example, T cells can be engineered to express antigen receptors that promote targeting of cells that express tumor-associated antigens2. This technique is known as CAR T-cell therapy, or CAR-T. Tisagenlecleucel3 and axicabtagene ciloleucel4 are approved CAR T-cell therapies.
In summary, gene therapy can be used to treat monogenic and complex diseases, with the nature of the disease determining the appropriate approach.
As we have seen, there are five approaches to gene manipulation: Gene addition, gene inhibition, gene replacement, gene editing, and genetic modulation of immune cells.
Each approach is suited to particular types of diseases.
More information on some of the topics discussed can be seen in the video resources available in this chapter.
- Wang D, Gao G. Discov Med 2014;18:151–161.
- Strachan T, Read AP. Genetic Approaches to Treating Disease, in Human Molecular Genetics. Florida: CRC Press, 1999; pp. 696–699.
- Furukawa Y, et al. J Biol Chem 2016;291:4144–4155.
- Gambello MJ, Li H. J Genet Genomics 2018;45:61–70.
- U.S. FDA. News Release. December 19, 2017. FDA approves novel gene therapy to treat patients with a rare form of inherited vision loss. Available at: https://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm589467.htm. Accessed January 23, 2019.
- Eid A, et al. Biochem J 2018;475:1955–1964.
- Gaudelli NM, et al. Nature 2017;551:464–471.
- Dunbar CE, et al. Science 2018;359:175.
- U.S. FDA. Approved Drugs. May 1, 2018. FDA approves tisagenlecleucel for adults with relapsed or refractory large B-cell lymphoma. Available at: https://www.fda.gov/Drugs/InformationOnDrugs/ApprovedDrugs/ucm606540.htm. Accessed January 10, 2019.
- U.S. FDA. Approved Products. YESCARTA (axicabtagene ciloleucel). Available at: https://www.fda.gov/biologicsbloodvaccines/cellulargenetherapyproducts/approvedproducts/ucm581222.htm. Accessed January 10, 2019.