Many diseases of the human body are the result of mutations in certain key genes that are responsible for the proper development, anatomy, and function of human homeostasis. Thus, unless a medication or treatment option can be found that specifically targets the consequences of a mutation - for example, a diet that avoids phenylalanine to counteract effects from phenylketonuria (PKU) - curing or even mitigating the disorder is often not easily possible, and patients have to live with their illness, often for an entire lifetime (Blau, 2016). The problem is that while mutations are easily introduced into the human genome and consequently get inherited over several generations, there is no way to repair the mutations and restore the genes to their original, 'healthy' status.
Recent studies, however, have seen the development of several techniques that allow the exchange of bases in the DNA with pinpoint precision. One example that has seen the most promise and is slowly moving into clinical trials is the so-called clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9, or in short, CRISPR/Cas9 (Tian et al., 2019). Using this technology, DNA sequences can be specifically targeted and 'repaired', thus potentially curing genetic conditions. Importantly, not all genetic conditions can be easily healed even with the technology to repair specific mutations, as not all parts of the body are easily accessible, and some mutations cause problems during development that cannot be easily reversed later on. However, genetic conditions that involve the immune system are likely more easily accessible for genetic therapies, and therefore, CRISPR/Cas9 has been discussed in the treatment of allergies and immunological conditions. Importantly, recent research has identified several mutations that underly airway inflammation and asthma, suggesting ways to treat these conditions using CRISPR/Cas9 (Goodman et al., 2017).
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The goals of this paper are to (1.) provide an overview of the CRISPR/Cas9 system, including the latest developments and practical considerations; (2.) take a look at the current research and clinical treatments pertaining to asthma and similar immunological conditions; and (3.) point out ways in which CRISPR/Cas9 can help cure patients afflicted with asthma, or at least mitigate their symptoms.
The CRISPR/Cas9 methodology - from bench to bedside
The CRISPR/Cas9 system has its origins in prokaryotes and likely functions as a primitive immune system. Incoming alien DNA is recognized by the bacterium, attacked, and cut. Prokaryotes usually have a large and diverse repertoire of defense mechanisms, which include restriction endonucleases that have been long used in basic research for their exquisite specificity in cutting DNA sequences. The special feature of the CRISPR/Cas9 system, however, is its 'programmability', as it consists of two main components: the Cas9 endonuclease and a 'guiding' single-strand RNA fragment, which binds to the complementary DNA strand and thus demarcates the sequence at which Cas9 can cut that strand. Thus, in the lab, the CRISPR/Cas9 system can be specifically targeted to cut in the vicinity of any DNA sequence, as long as there is a guidance RNA (gRNA) molecule present that is complementary to that sequence (Knott & Doudna, 2018). Importantly, the Cas9 endonuclease will only cut a strand open at a specific stretch of sequence; if at the same time DNA nucleotides are present, the cell-internal DNA repair machinery will insert those bases, and it will do so while making mistakes, allowing for the introduction of mutations - or their repair, when the DNA sequence is restored to the wildtype allele.
It is important to note that the CRISPR/Cas9 system is not without flaws. For example, it can induce off-target effects (OTEs), that is, attack sequences that are not in the desired sequence or gene to be mutated. In basic research studies in the lab, OTEs can be controlled through an appropriate experimental design. However, for clinical applications, OTEs need to be eliminated as much as possible. First, the gRNA target sequence needs to be specific enough. If the base pair needs to be changed adjacent to a repetitive sequence, the chances are increased that the system will target other regions within the genome. In addition, local single nucleotide polymorphisms (SNPs) can reduce the affinity of the gRNA fragment to the target DNA, which also increases the likelihood of OTEs (Kimberland et al., 2018).
There are several ways to introduce the CRISPR/Cas9 system into the human body for clinical trials and therapies. First, the system can be delivered via an adenoviral vector; DNA for Cas9 and the guidance RNA are inserted into the adenoviral DNA sequence. The virus is then injected close to the target tissue, upon which it enters the target cells and allows for the CRISPR/Cas9 system to become active. The system can also be attached to artificial vectors, such as synthetic liposomes, injected and subsequently delivered into the target tissue. Third, cells - for example, hematopoietic stem cells (HPSCs) - can be taken from the patient, modified using the CRISPR/Cas9 system ex vivo in the lab, and then reinserted into the patient. The 'repaired' stem cells can then proliferate, giving rise to 'healthy progenitors'. Importantly, the mutation is not eliminated across every tissue in the whole body; however, as many genes are only required in specific tissues, it is enough to abolish the mutation in stem cells that belong to the respective tissue (Gulei & Berindan-Neagoe, 2017).
Asthma and the immune system
Asthma is a chronic lung disease that leaves the airways inflamed. As a consequence, the muscles lining the airways tighten, and the epithelial cells secret a mucus-like substance that narrows the passage of air. The result is troubled breathing, which can become life-threatening in severe cases. In most cases, there is no clear underlying reason for the airway inflammations, which periodically flare up; as a consequence, asthma treatment regimens have emphasized symptom management and mitigation instead of focusing on an actual cure.
As one of the central factors that underly the development of asthma is the inflammation of airway cells, a large body of research has focused on the role of the immune system in asthma. The connection of asthma with allergies is another reason that the immune system may play a decisive role in this condition. Studies have revealed several immunological biomarkers that are associated with an increased risk for asthma and airway inflammation, such as T-cell phenotypes and associated cytokines, endotypes based on eosinophils and neutrophils, and molecules that are derived from lung epithelium, such as osteopontin and CCL-26 (Zissler et al., 2016). Thus, there is a large repertoire of abnormally regulated immune cells that may underly any form of asthma in individual patients. If those patients can be screened for the presence of particular SNPs that are associated with an increased risk for asthma, technologies such as CRISPR/Cas9 can be used to eliminate those SNPs and restore the immune system to its 'normal' function.
Importantly, as it is difficult to find one clear reason for the development of asthma, simply changing one or two SNPs may not be enough to control the disease. However, by targeting hematopoietic stem cells (HPSCs), several downstream populations of immune cells can be simultaneously targeted. For example, CRISPR could be used to block certain transcription factors that promote the development of T-helper cell 2 (Th2) phenotypes, which play an important role in promoting inflammation within lung tissue. At the same time, the expression of certain transcription factors that induce T-helper cell 1 (Th1) and regulatory T-cells (Treg) phenotypes could be promoted, resulting in an overall suppression of the Th2 pro-inflammatory pathways and induction of the Th1 and Treg response, which limit inflammation. Thus, CRISPR could be used to regulate certain subsets of the immune system, in addition to repairing the defects in several genes. As many conditions are of polygenic origin, treating asthma and allergies with CRISPR/Cas9 technology could be a useful framework for the mitigation or even cure of a host of other diseases.
CRISPR/Cas9 has been used for the treatment of aggressive lung cancer in a single patient in China (Cyranoski, 2016); several clinical trials have been proposed that use CRISPR to modify cells within the immune system and other organ systems within the body. In the Chinese study, CRISPR/Cas9 was used in an ex vivo approach. If it turns out that such approaches can be used for CRISPR-based therapies, treatments for more diseases could be developed.
Conclusion
CRISPR/Cas9 is a novel, powerful technology that has enormous potential in the cure of several genetic diseases based on its ability for pinpoint editing of specific DNA sequences. Cas9 is an endonuclease that cuts DNA sequences that bind to a small fragment of guidance RNA (gRNA). As the gRNA sequence can be freely chosen, the system can be programmed to work with almost any sequence. Therefore, it promises to provide treatment and even cure for a whole host of different conditions, from neuronal problems to cancer and diseases of the immune system, such as HIV. Asthma is a chronic inflammation of the lungs and the airways; accumulation of the resulting mucus can make it harder for patients to breathe. Asthma is the result of a misregulation of several different pathways, most of which are within the immune system. Current efforts investigate ways in which hematopoietic stem cells (HPSCs) can be taken from the patient, treated with the CRISPR/Cas9 system in the lab, and reintroduced into the patient. One way in which such modified HPSCs can function is through the downregulation of pro-inflammatory pathways and the upregulation of anti-inflammatory responses.
References
- Blau, N. (2016). Genetics of phenylketonuria: then and now. Human Mutation, 37(6), 508-515.
- Cyranoski, D. (2016). CRISPR gene-editing tested in a person for the first time. Nature News, 539(7630), 479.
- Goodman, M. A., Moradi Manesh, D., Malik, P., & Rothenberg, M. E. (2017). CRISPR/Cas9 in allergic and immunologic diseases. Expert Review of Clinical Immunology, 13, 1, 5 - 9.
- Gulei, D., & Berindan-Neagoe, I. (2017). CRISPR/Cas9: a potential life-saving tool. What’s next? Molecular Therapy - Nucleic Acids, 9, 333-336.
- Kimberland, M. L., Hou, W., Alfonso-Pecchio, A., Wilson, S., Rao, Y., Zhang, S., & Lu, Q. (2018). 6501. Strategies for controlling CRISPR/Cas9 off-target effects and biological variations in mammalian genome editing experiments. Journal of Biotechnology, 284, 91-101.
- Knott, G. J., & Doudna, J. A. (2018). CRISPR-Cas guides the future of genetic engineering. Science, 361(6405), 866-869.
- Tian, X., Gu, T., Patel, S., Bode, A. M., Lee, M. H., & Dong, Z. (2019). CRISPR/Cas9 - an evolving biological tool kit for cancer biology and oncology. npj Precision Oncology, 3(1), 8.
- Zissler, U. M., Esser‐von Bieren, J., Jakwerth, C. A., Chaker, A. M., & Schmidt‐Weber, C. B. (2016). Current and future biomarkers in allergic asthma. Allergy, 71(4), 475-494.
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