Cystic fibrosis (CF) is an autosomal recessive genetic disorder that affects all parts of the body, but mainly the lungs. It causes lung infections and limits breathing, lowering quality of life and, without proper treatment, survival rate for patients. CF is quite rare as only approximately 70,000 people worldwide have CF and, although it affects every racial and ethnic group, it is mainly found in Caucasians. Despite the rarity of CF as a genetic disorder, there has been much research regarding its genetic basis and possible treatments that involve biotechnology. It is well-established that CF is caused by a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) protein. CFTR normally acts as an ion channel, facilitating the transport of Cl⁻ from the cytoplasm to the extracellular matrix, attracting water to the cell surface and allowing cilia to sweep excess mucus out of airways. In CF patients, CFTR is dysfunctional, so Cl⁻ is not transported to the matrix, so water doesn’t hydrate the cell surface, causing mucus to clog up airways. Since CF is caused by a single gene, CRISPR is a viable option for treating CF as it can replace the mutated CFTR gene with a functional one, allowing lung cells to produce functional CFTR that attenuates CF symptoms. I first researched the basis of CF to understand the causes, effects, and diagnosis of the disease. Then, I researched the genetic and proteomic basis of CF to understand how biotechnology can treat it. Finally, I read several journal articles to gain a better understanding of how CRISPR has been used to treat CF, and what further research is needed to make this a viable treatment plan for patients.
Basics of the CFTR Protein. Cystic Fibrosis Foundation. (n.d.). Retrieved January 18, 2022, from https://www.cff.org/research-clinical-trials/basics-cftr-protein{:target="_blank"}
This article explained the purpose of functional CFTR (cystic fibrosis transmembrane conductance regulator), and how its mutation causes CF. CFTR is a protein made of approximately 1,480 amino acids that acts as an ion channel, facilitating the transport of Cl⁻ from the cytoplasm to the extracellular matrix. This extracellular Cl⁻ attracts water to the cell surface, allowing cilia (hairs on the surface of lung cells) to sweep excess mucus out of airways. In CF patients, CFTR is dysfunctional, so Cl⁻ is not transported to the matrix, so water doesn’t hydrate the cell surface. This causes mucus covering cells to become thick and sticky, clogging the airways.
CF Genetics: The Basics. Cystic Fibrosis Foundation. (n.d.). Retrieved January 18, 2022, from https://www.cff.org/intro-cf/cf-genetics-basics{:target="_blank"}
This article was helpful in summarizing the genetic basis of CF. CF is an autosomal recessive disease, which means that if a person inherits 2 mutated copies of CFTR, they have CF, but if they inherit only 1 mutated copy of CFTR, they are a carrier that doesn’t have CF, but can pass it on to their children. Approximately 10,000,000 Americans are CF carriers, while approximately 30,000 Americans have CF: it makes logical sense that there are more carriers than patients since it is less likely to inherit 2 than 1 mutated gene.
Types of CFTR Mutations. Cystic Fibrosis Foundation. (n.d.). Retrieved January 18, 2022, from https://www.cff.org/research-clinical-trials/types-cftr-mutations{:target="_blank"}
This article provided an in-depth view of the 5 types of CF-causing mutations. Protein production mutations—such as nonsense and slice mutations—results in dysfunctional CFTR being produced. In protein processing mutations—such as missense mutations and F508del, the most common CF-causing mutation—normal CFTR is produced, but it is made dysfunctional by the time it reaches the cell membrane or never reaches the membrane at all. Gating mutations cause CFRT to be permanently closed as an ion channel. Conduction mutations cause Cl⁻ to flow through CFRT slower. Insufficient protein means that simply not enough CFTR is produced to deal with all the Cl⁻.
Carrier Testing for Cystic Fibrosis. Cystic Fibrosis Foundation. (n.d.). Retrieved January 18, 2022, from https://www.cff.org/intro-cf/carrier-testing-cystic-fibrosis{:target="_blank"}
This article helped explain how people with a family history of CF can determine their chances of having a child with CF. Since CF is an autosomal recessive disease, a person can have 1 copy of the mutated CFTR gene: while they do not express symptoms, as a carrier, they can pass it onto their children. If 2 carriers have a child, there is a ¼ probability that child will have CF, a ½ probability that child will be a carrier, and a ¼ probability that they will be normal. If a carrier and a patient have a child, there is a ½ probability that child will have CF and a ½ probability that child will be a carrier.
CFTR-Related Metabolic Syndrome (CRMS). Cystic Fibrosis Foundation. (n.d.). Retrieved January 18, 2022, from https://www.cff.org/intro-cf/cftr-related-metabolic-syndrome-crms{:target="_blank"}
This article revealed that patients with intermediate symptoms of CF have CFTR-related metabolic syndrome (CRMS, also known as CF Screen Positive, Inconclusive Diagnosis (CFSPID)). A patient is diagnosed as having CRMS if they have a sweat Cl⁻ value less than 30 mmol/L and 2 CFTR mutations (at least 1 of which has no physical symptoms) or a sweat Cl⁻ value between 30 and 59 mmol/L and 0 or 1 CF-causing mutations. The future of patients with CRMS is unclear as, while they are not as afflicted as CF patients, they have an increased risk of airways, intestines, pancreas, or reproductive problems.
Find Out More About Your Mutations. Cystic Fibrosis Foundation. (n.d.). Retrieved January 18, 2022, from https://www.cff.org/intro-cf/find-out-more-about-your-mutations{:target="_blank"}
This article revealed the CFTR2 database, which provides information about different CF mutations and associated symptoms. This is particularly useful for patients that have had their CFTR gene sequenced because, once they know their specific mutation, they can develop a treatment plan specialized to them. For example, they can choose modulators that target the exact mutation to better deal with the source of their illness. This proves that DNA sequencing plays a major role in helping CF patients develop treatment plans.
Newborn Screening for CF. Cystic Fibrosis Foundation. (n.d.). Retrieved January 18, 2022, from https://www.cff.org/intro-cf/newborn-screening-cf{:target="_blank"}
This article provided invaluable information about the major method of detecting CF, newborn screening (NBS). NBS is a national program to check if babies have certain health conditions, including CF. Within the first few days of a baby’s life, a few blood drops from a heel prick are put on a Guthrie card, which is sent to be tested for CF. Doctors check levels of immunoreactive trypsinogen (IRT), a pancreatic protein, in the blood: babies with CF have high IRT levels, but that can also be caused by premature or stressful delivery, etc. If the results find that the baby had high levels of blood IRT, they may have CF, so a sweat test is performed.
Sweat test. Cystic Fibrosis Foundation. (n.d.). Retrieved January 18, 2022, from https://www.cff.org/intro-cf/sweat-test{:target="_blank"}
This article helped explain the sweat test, the most realizable method of diagnosing CF. This painless 1-hour test involves measuring the amount of Cl⁻ in the sweat, as high levels of Cl⁻ in sweat is associated with CF. A colorless, odorless chemical (pilocarpine) and electrical stimulation are applied to the arm or leg to stimulate the sweat glands, producing sweat which is then collected and sent for testing. Results return the same day: if the Cl⁻ sweat concentration is less than 29 mmol/L, it is unlikely that the person has CF; if it is between 30 and 59 mmol/L, additional testing is needed; if it is greater than 60 mmol/L, they likely have CF.
Theratyping. Cystic Fibrosis Foundation. (n.d.). Retrieved January 18, 2022, from https://www.cff.org/research-clinical-trials/theratyping{:target="_blank"}
This article explained the process of theratyping, which matches therapies to specific types of mutations to identify which modulators work on which mutations. The necessity of theratyping derives from the fact that it is difficult for companies to produce modulators for patients with rare mutations since there are not enough patients available for a clinical trial. Researchers are testing modulators on cell lines with rare CFTR mutations to see if it improves protein function. This effectively replaces the need for clinical trials for patients with rare mutations and allows researchers to match modulators to rare mutations, saving time, money, and lives.
About Cystic Fibrosis. Cystic Fibrosis Foundation. (n.d.). Retrieved January 18, 2022, from https://www.cff.org/intro-cf/about-cystic-fibrosis{:target="_blank"}
This article gave a general overview of the symptoms and treatments of CF, as well as particularly helpful statistics on the prevalence of CF. Most patients with CF have salty-tasting skin, chronic coughing, and frequent infections, and they are diagnosed either by newborn screening or a sweat test. Treatments are personalized to each CF treatment, but generally include air clearance methods, inhaled medicines, and pancreatic enzymes. Across all racial and ethnic groups, approximately 30,000 Americans out of 70,000 people worldwide have CF, and about 1,000 new cases are diagnosed annually.
Da Silva Sanchez, A., Paunovska, K., Cristian, A., & Dahlman, J. E. (2020). Treating Cystic Fibrosis with mRNA and CRISPR. Human gene therapy, 31(17-18), 940–955. https://doi.org/10.1089/hum.2020.137{:target="_blank"}
This peer-reviewed journal article describes past studies that used mRNA or CRISPR to treat CF. It helpfully explained that RNA therapies (such as small interfering RNA, siRNAs, or antisense oligonucleotides, ASOs) use RNA to either under- or over-express a particular gene, while mRNA therapies use mRNA to express a protein that is normally, nonexistent, underexpressed, or dysfunctional. Since CF is caused by a single mutation in the CFTR gene, but is present in multiple cell types, mRNA is a passable method of treating CF. There are 2 main methods of using mRNA to treat CF: patients can take a drug containing mRNA that codes for functional CFTR; patients can take a drug containing mRNA that codes for CRISPR-based gene editors and a guide RNA, specific to their mutation, that can fix the mutation. While several past studies have demonstrated the feasibility of these methods to treat CF, there are drawbacks to each, such as ensuring the drug reaches the lungs. The article also provided a helpful diagram, Figure 2, of the 6 classes of CF-causing mutations, and listed various mRNA drugs used to treat them. This source was unique from the other journal articles about potential CF treatments as it considered the uses of RNA-based drugs, rather than gene editing through CRISPR.
Geurts, M. H., de Poel, E., Amatngalim, G. D., Oka, R., Meijers, F. M., Kruisselbrink, E., van Mourik, P., Berkers, G., de Winter-de Groot, K. M., Michel, S., Muilwijk, D., Aalbers, B. L., Mullenders, J., Boj, S. F., Suen, S., Brunsveld, J. E., Janssens, H. M., Mall, M. A., Graeber, S. Y., van Boxtel, R., ... Clevers, H. (2020). CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank. Cell stem cell, 26(4), 503–510.e7. https://doi.org/10.1016/j.stem.2020.01.019{:target="_blank"}
This article recounts a recent successful study in using CRISPR to treat CF. The researchers used adenine base editing (ABE), a biotechnological process that converts A-T pairs to G-C pairs in DNA. ABE is a form of CRISPR, involving the SpCas9-ABE and xCas9-ABE enzyme instead of the typical Cas9; it is less controversial, and somewhat more efficient as it does not break then repair the DNA, only replacing certain base pairs. They collected 664 intestinal organoid samples of patients with CF and applied ABE-based CRISPR to 4 samples. In all 4 samples, the organoid’s genome was altered to have the correct CFTR gene and produced functional CFTR, suggesting that CRISPR can treat CR at the source. This source included a similar study as that of Schwank, et al is it demonstrated the use of CRISPR in editing the CFTR gene.
Griesenbach, U., Davies, J. C., & Alton, E. (2016). Cystic fibrosis gene therapy: a mutation-independent treatment. Current opinion in pulmonary medicine, 22(6), 602–609. https://doi.org/10.1097/MCP.0000000000000327{:target="_blank"}
This somewhat recent journal article reviews past developments in gene therapy to treat CF. It notes that past research has emphasized how complex it would be to change the genotype of lung cells to have a functional CFTR gene, as it is quite difficult to target a drug to the lungs and have the drug pass the mucus protective coating on the lungs. Past research has also shown that nonviral gene transfer has the opportunity to produce functional CFTR, thus attenuating the effects of CF temporarily; this research seems promising in treating CF, so further studies will be conducted. Viral gene transfer, specifically through lentiviral vectors, has also proven to be a plausible method of treating CF as these viruses can be altered to make them more able to enter the lung cells that need to express CFTR correctly. Simply, this article helpfully summarized recent findings on the possibility of using both nonviral and viral gene therapy to treat CF.
Maule, G., Arosio, D., & Cereseto, A. (2020). Gene Therapy for Cystic Fibrosis: Progress and Challenges of Genome Editing. International journal of molecular sciences, 21(11), 3903. https://doi.org/10.3390/ijms21113903{:target="_blank"}
This recent journal article reviews methods of gene therapy that have proven viable in treating CF. It first explains that since CF is caused by a single recessive mutation, gene complementation is a viable method of treatment. The targeted integration of a functional CFTR gene into the lung's genome through nucleases, such as Cas9, can also treat CF. The mutation of CFTR can also be corrected either through homology directed repair or non-homologous end joining. Additionally, base editors can correct point mutations in the CFTR gene to help the protein become functional. Simply, this article outlined various methods of gene therapy that have the potential to treat CF.
Schwank, G., Koo, B. K., Sasselli, V., Dekkers, J. F., Heo, I., Demircan, T., Sasaki, N., Boymans, S., Cuppen, E., van der Ent, C. K., Nieuwenhuis, E. E., Beekman, J. M., & Clevers, H. (2013). Functional repair of CFTR by CRISPR/Cas9 in intestinal stem cell organoids of cystic fibrosis patients. Cell stem cell, 13(6), 653–658. https://doi.org/10.1016/j.stem.2013.11.002{:target="_blank"}
This journal article describes a study that demonstrates the plausibility of using homologous recombination in primary adult stem cells to treat CF. First, the researchers created epithelial organoids out of human intestinal stem cells. In some human intestinal stem cells, they used CRISPR to edit the CFTR gene through homologous recombination, in which they introduce the correct CFTR gene through a plasmid vector to replace the mutated CFTR gene in the CF patient. These edited stem cells then produced organoids that did not express CF conditions: instead, the edited stem cell produced an organism composed of cells with the correct CFTR gene, which expressed the functional protein. This suggests that CRISPR-based homologous recombination of intestinal stem cells in CF patients is a viable method to treat CF. This source built upon the CRISPR treatment description included in the study by Geurts, et al. as it considered the genetic editing of stem cells, not differentiated cells.
Kenigsberg, B. (2022, January 20). ‘Salt in My Soul’ Review: Living, Even Thriving, With Illness. New York Times. Retrieved January 23, 2022, from https://www.nytimes.com/2022/01/20/movies/salt-in-my-soul-review.html?referringSource=articleShare{:target="_blank"}
This article describes a documentary entitled “Salt in my Soul” which narrates the life of Mallory Smith, a CF patient who died in 2017. It was particularly helpful in considering the mental aspects of living with such a pervasive illness: even though CF affected every aspect of Mallory’s life, she resolved to stay positive in the face of hardship. The documentary is composed of audio and visual recordings that Mallory kept in a diary throughout her life to journal her experiences with CF. In her will, she revealed the password for her diary, so that her experiences could be publicized to help the public understand what life with CF is truly like.
Purcell, J. (2022, January 10). Living with an Invisible Illness. Cystic Fibrosis Foundation. Retrieved January 18, 2022, from https://www.cff.org/community-posts/2022-01/living-invisible-illness?sf158274053=1&fbclid=IwAR1hO46zMasPP7HUIqpyvZzIHUVUxLDJCwxPwSNqzu_06qaZqxUaPqA9Dcc{:target="_blank"}
This article was particularly helpful in examining the mental effects of CF on patients. As a CF patient herself, the author, Julia Purcell provides a unique perspective on the mental effects of CF. As an “invisible” illness, it is not apparent that a patient is sick just from looking at them, even though they require various treatments, so others often do not understand their requirements. As Purcell explains, “I wish people knew that even if I don’t look sick, I still struggle with the mental and physical aspects of cystic fibrosis.” This emphasizes that CF affects every aspect of the patient’s life, including mental health.
Affects of CF on the Body. https://www.nhlbi.nih.gov/sites/default/files/inline-images/cystic-fibrosis-body.jpg{:target="_blank"}
CFTR Protein. https://ars.els-cdn.com/content/image/1-s2.0-S0092867417302398-fx1.jpg{:target="_blank"}
CRISPR-Cas9. https://www.pearson.com/us/content/dam/one-dot-com/one-dot-com/us/en/higher-ed/en/custom-product/urry-campbell-biology-11e/images/kf-keep-current-scientific-advances.png{:target="_blank"}
Embryonic Development. https://cdn1.byjus.com/wp-content/uploads/2020/09/Embryo-Development.png{:target="_blank"}
Nebulizer. https://cysticfibrosisnewstoday.com/wp-content/uploads/2019/07/shutterstock_621845186.jpg{:target="_blank"}
Pancreatic Enzyme Supplement Capsules. https://www.creon.com/dist/images/cr30-pills.jpg{:target="_blank"}
Percussion Vest. https://bronchiectasis.com.au/wp-content/uploads/2015/09/The-Vest.jpg{:target="_blank"}
Person Graphic. https://upload.wikimedia.org/wikipedia/commons/thumb/0/03/Protein_CFTR_PDB_1xmi.png/800px-Protein_CFTR_PDB_1xmi.png{:target="_blank"}
Prognosis over Time. https://cystic-fibrosis.com/wp-content/uploads/2021/06/CF-life-expectancy-IAI.png{:target="_blank"}
Splice Site Mutation. https://upload.wikimedia.org/wikipedia/commons/thumb/1/15/SplicingSlide.png/375px-SplicingSlide.png{:target="_blank"}
Stem Cell Diagram. https://slideplayer.com/slide/16704027/96/images/14/Cell+division+White+blood+cells.jpg{:target="_blank"}
Sweat Test. https://www.cff.org/sites/default/files/styles/image_card/public/webimage-A65FA1B9-6634-431A-9BA6E23A07E9D43B.jpg?itok=rAg3Lga-{:target="_blank"}
Trikafta Diagram. https://www.trikafta.com/sites/default/files/moa-graphic-2%403x.png{:target="_blank"}
Trikafta Packaging. https://www.trikafta.com/sites/default/files/6-11-packaging-mockup-option-1-1%403x_060321.png{:target="_blank"}
Types of Mutations. https://medlineplus.gov/genetics/understanding/mutationsanddisorders/possiblemutations/{:target="_blank"}
From my research, I have learned that biotechnology will be the future of CF treatment as it targets the genetic source of the disorder, rather than its symptoms, to improve patients’ lives. CRISPR is especially important in the treatment of CF as it can replace the dysfunctional CFTR gene with a functional one, essentially curing the disease by producing the CFTR proteins the lungs need to maintain the proper water-salt balance. While CRISPR has been successfully used in various studies to genetically edit patient-derived organoids to produce functional CFTR, in live patients, it would be more difficult to get the CRISPR-based drugs into lung cells, where it is needed to function. Not only is it generally difficult to apply CRISPR to live cells, but lung cells are especially difficult targets as their mucus layer prevents foreign bodies from entering the cell. Despite this, researchers have made various strides in applying CRISPR to cultured human epithelial cells in order to emulate the difficulty of applying drugs to mucus-covered lung cells in live patients. Research in using biotechnology to treat CF is ongoing, and I was surprised to learn that these studies are so well-funded and readily available, considering that CF is a relatively rare genetic disease. It seems that, even though CF affects few patients, since its effects have a truly devastating effect on patients’ lives, much research has been done to help them.