Mastering the Molecular Scissors: Sickle Cell Gene Editing Unleashed

By: Blair Wagner

The more we delve into the intricacies of DNA and our genetic composition, the more gene technologies are being developed. In order to combat bloodborne illnesses, scientists have developed new gene editing technologies. One bloodborne disease that affects millions of people globally and can be treated by this form of gene editing is select forms of Sickle cell disease. In this report, the focus will be on examining the effects and applications of CRISPR/Cas9, a revolutionary gene editing technique. Additionally, this report will specifically delve into the specific implementation of a technique known as Casgevy. The progress of gene technology has revolutionized medicine, allowing for the creation of innovative technologies such as CRISPR/Cas9. However, it does come with ethical concerns. By conducting a more thorough examination of this topic, we can analyze the various economic, ethical, global, and engineering aspects that impact the progress and implementation of CRISPR/Cas9 as a potential remedy for sickle cell anemia and beta thalassemia-induced SCD.  

SCD is a genetic disorder caused by a mutation in the β-globin gene called sickle hemoglobin. The term “sickle cell” refers to the shape of blood cells found within a person who is a carrier for the disease. Sickle cells inhibit the ability of hemoglobin in red blood cells to carry oxygen, causing the cells to appear sickle-shaped. More specifically, amounts of fetal hemoglobin are lower in sickle cell patients than normal, which results in an imbalance in oxygen present within blood. These cells can sometimes clump together leading to small blood vessel blockages. There are over 400 different variations of the β-globin gene each with different severity levels. Some severe forms of SCD are beta thalassemia and sickle cell anemia. The difference between beta thalassemia and sickle cell anemia is that sickle cell anemia is caused by a very specific gene mutation and beta thalassemia can be caused by multiple gene mutations. While their symptoms are similar, beta thalassemia does not cause red blood cells to sickle. Persons with sickle cell have significant morbidity and mortality rates; some symptoms include chronic anemia, acute chest syndrome, strokes, renal dysfunction, vaso-occlusive pain crisis, and increased susceptibility to bacterial infections. More than 50,000 individuals are affected with SCD, making it one of the most prevent genetic disorders in the US  Koch et al. (2000).

Figure 1. Birth Prevalence of β-globin gene variants in California newborn population taken from Koch et al. (2000). 

Sickle Cell affects about 1 in 100,000 people in the United States, 1 in 365 African American babies, and 1 out of every 1300 non-white Hispanic babies (CDC, 2023). The mortality rate of SCD has decreased dramatically during the past 25 years with the mortality rate dropping 42% for children ages 4 and younger from 1999 to 2002 due to increased rates of vaccination and development of treatments for SCD (CDC, 2023). While there is no definite cure for any SCD variant there are different treatment options for it some being bone marrow and blood transplants. Other treatment options include Voxelotor a blood cell sickling prevention treatment for people 4 and under, Hydroxyurea a complication prevention treatment, penicillin a infection prevention treatment, and a new gene editing treatment called Casgevy. 

A genome is a set of all the DNA instructions found within a cell. The human genome is contained in 23 pairs of chromosomes.  In order to combat harmful mutations present within or genomes, scientists developed gene editing technologies. Gene editing technology was first developed in the 1970s where molecular biologists gained the ability to manipulate molecules of DNA making it possible to generate novel biotechnology (Hsu et al. 2014). Researchers can directly edit or regulate functions of DNA and their sequences, shedding light on the functional and organizational systems as well as genetic variations in eukaryotic cells. Eukaryotic genomes contain billions of difficult-to-manipulate DNA bases, but scientists have been able to successfully manipulate a genome through homologous recombination. This technique requires high amounts of precision as desired results occur typically only within every 1 in 10^6 - 10^9 cells (Hoban et al. 2016). Homologous recombination is a process in which nucleotides are exchanged between two similar or identical molecules of DNA. Homologous recombination allows cells to access and copy intact DNA sequence information and repair targeted parts of DNA strands.  Gene editing integrates exogenous repair templates that contain sequence homology to the donor site, leading to the successful editing of a gene. One form of gene editing is CRISPR/Cas9 which specifically acts as a pair of “molecular scissors” and cuts out parts of a genome and adds in a part depending on what part of a genome they are editing (Hsu et al. 2014).  

Figure 2. Gene editing Technologies Repair Sequence taken from Yoshino et al. (2019).  Machinery taken from Hsu et al. (2014). 

Scheme of knock-in of a marker sequence into an endogenous target gene by gene editing.  

(a) Illustration of the knock-in.   

(b) Structure of the donor vector containing 3xFLAG sequence between the homology arms for the ACTB gene.  

The target sequence of the gRNA, highlighted in yellow, is placed on the antisense strand around the stop codon (highlighted by red color) of ACTB. The DSB is created 10 bp upstream from the stop codon and 3 bp downstream of the PAM sequence (indicated by the scissors). The donor vector contains 5′ and 3′ arms and a marker sequence, highlighted in blue, just upstream of the stop codon. 

Figure 3. Phenotypic classification of Hemochromatosis associated with hepcidin deficiency is proposed (National Library Of Medicine, 2018).

To make the gene editing process less difficult, researchers have developed an RNA-guided endonuclease called Cas9, which stems from microbial adaptive immune systems aka CRISPR (Clustered Regularly Interspersed Palindromic Repeats). This technology, known as Casgevy (exagamglogene anatomical), has been modified by scientists to treat various genetic disorders, including sickle cell, frequent vaso-occlusive crisis, anemia and beta thalassemia, in individuals aged 12 and above (Hsu et al. 2020). Casgevy is a treatment option that primarily targets genetic diseases like sickle cell anemia and beta thalassemia. It has been proven to have a success rate of 93.5%, according to the FDA. This treatment was first approved on December 8th by the FDA (Food and Drug Administration) after a successful trial run titled CTX001. CTX001 was the first clinical trial application of CRISPR/Cas9 in the treatment of the two forms of SCD. During the procedure, stem cells were extracted from the participant and sent to a lab in order to stimulate the production of fetal hemoglobin instead of adult hemoglobin in the body. After blood stem cells are sent to a laboratory, scientists focus on the BCL11A gene to remove the mutation and replace it with instructions that prompt your body to produce higher levels of adult hemoglobin and lower levels of fetal hemoglobin. Scientists will modify it to have the opposite effect since fetal hemoglobin acts as a genetic regulator that prevents the formation of sickle hemoglobin polymers. Once the gene alteration is finished, the patient will receive the blood treated with Casgevy and will be closely monitored for 4-6 weeks to determine if they have been cured (Porteus et al. 2020). 

Figure 3. Process of Casgevy Treatment from a Sickle Cell Disease patient to cell reinfusion taken from Porteus et al. (2020).

Genetic editing, particularly techniques like CRISPR-Cas9, raises ethical considerations due to its potential to modify the DNA of humans. As genetic editing introduces permanent changes to a genome, there is a risk of introducing unwanted genetic mutations to your DNA. Another consequence is that gene editing can lead to people using this technology improperly to enhance their own physical traits, which could widen the healthcare disparity gap because of the high treatment cost. Due to a lack of understanding about the long term potential harm that gene editing can cause, it can be difficult to obtain informed consent from patients because of gene editing's ability to affect future generations. The potential health and safety risks that gene editing may pose to germline cells (sperm, eggs, and embryos) are significant concerns when it comes to the effects on future generations (Ranish et al. 2022). Due to its high cost, genetic editing technology is not widely available and may contribute to the emergence of social and economic inequities. Therefore, there are worries about the possibility of using gene editing for objectives other than enhancement, even as it shows promise for treating genetic problems. This calls into question issues of equity, fairness, and the effect on cultural norms and values. Due to the high expense of treatment, genetic editing is one way that healthcare disparities may develop. On average, genetic editing involving CRISPR/Cas9 can cost upwards of 450,000 to 2.1 million dollars for a single treatment (NIH, 2023), which may not be affordable to the vast majority of people. SCD impacts communities all across the nation regardless of sex, gender, or race.  

Even though multiple impacts are seen within communities, there are disparities in sickle cell treatment and trait carriers of SCD. Approximately 80% of sickle cell diagnosis are people of African or Mediterranean descent (CDC, 2019). The prevalence of SCD contributes to health disparities among African Americans particularly due to socioeconomic factors. Seeing a doctor is cost prohibitive for a lot of these individuals. We can especially see healthcare disparities in African Americans in terms of healthcare coverage. According to the CDC, 13.4% of African Americans aged 18-64 are living without healthcare insurance and 18.8% of adults aged 18 and older are found to live in fair or poor health. Thus individuals with SCD may face challenges such as limited access to quality healthcare leading to differences in health outcomes. Although the reasoning behind higher prevalence of SCD in African Americans is still unknown, there are some theories that attempt to explain this phenomenon.There is a widely accepted theory claiming that SCD developed as a body defense against malaria, which explains why SCD is more common in African Americans than in other racial groups (CDC, 2019). Malaria is primarily present in Sub-Saharan Africa along with other regions with unfavorable environmental conditions. It is a disease carried by mosquito bites. Ninety-four percent of deaths from malaria occur in Sub-Saharan Africa alone (World Health Organization, 2023). Given that malaria has been a problem for hundreds of years, people of African descent may be more likely to have experienced genetic adaptations that have increased the prevalence of sickle cell disease. Additionally, we can see how widespread sickle cell disease has become throughout the African diaspora and how common it is among African Americans and other Africans. Given that SCD is believed to be a hereditary characteristic, the African diaspora provides insight into the disease's prevalence among African Americans. 

By utilizing CRISPR as a gene therapy for SCD, we can specifically address the genetic makeup of our DNA, potentially eradicating genetic blood disorders such as SCD. This condition can impact people of all ages and currently has limited treatment methods available.. Unlike conventional disease treatments, gene therapy has the ability to alter your genetic composition, which was previously considered impossible even with medication. The application of this revolutionary gene therapy could enable humanity to surpass the once unattainable physical boundaries of the human body. This treatment shows potential in promoting global change and aiding in the fight against blood-borne diseases, although there is still significant work to be done before fully implementing and expanding its availability. Actions aimed at tackling the effects of sickle cell disease encompass advancements in research, enhancements in healthcare accessibility, and active involvement of the community to ensure fair treatment and assistance for individuals impacted by this hereditary condition. 

References

A. Ashley-Koch, Q. Yang, R. S. Olney, Sickle Hemoglobin (Hb S) Allele and Sickle Cell Disease: A HuGE Review, American Journal of Epidemiology, Volume 151, Issue 9, 1 May 2000, Pages 839–845, https://doi.org/10.1093/oxfordjournals.aje.a010288  

Ambroise Wonkam; The future of sickle cell disease therapeutics rests in genomics. Dis Model Mech 1 February 2023; 16 (2): dmm049765.   

Analysis Explores Gene Therapy’s Potential to Be Cost-Effective in SCD. (2024, January 22). AJMC.https://www.ajmc.com/view/analysis-explores-gene-therapy-s-potential-to-be-cost-effective-in-scd#   

Black People and Sickle Cell Anemia: Your Questions Answered. (2023, February 2). Healthline.https://www.healthline.com/health/sickle-cell-anemia-black-people#why-more-common-in-black-people  

Center for Disease Control and Prevention. (2019). FastStats - Health of Black or African American Population. CDC. https://www.cdc.gov/nchs/fastats/black-health.htm   

CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells. Molecular therapy : the journal of the American Society of Gene Therapy, 24(9), 1561–1569. https://doi.org/10.1038/mt.2016.148  

CRISPR/Cas9-Mediated Correction of the Sickle Mutation in Human CD34+ cells DeMartino, P., Haag, M. B., Hersh, A. R., Caughey, A. B., & Roth, J. A. (2021). A Budget Impact Analysis of Gene Therapy for Sickle Cell Disease: The Medicaid Perspective. JAMA Pediatrics, 175(6). https://doi.org/10.1001/jamapediatrics.2020.7140  

Hoban, M. D., Lumaquin, D., Kuo, C. Y., Romero, Z., Long, J., Ho, M., Young, C. S., Mojadidi, M., Fitz-Gibbon, S., Cooper, A. R., Lill, G. R., Urbinati, F., Campo-Fernandez, B., Bjurstrom, C. F., Pellegrini, M., Hollis, R. P., & Kohn, D. B. (2016).  doi:doi: https://doi.org/10.1242/dmm.049765  

Hsu, Patrick D., Lander, Eric S., & Zhang, F. (2014). Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell, 157(6), 1262–1278. https://doi.org/10.1016/j.cell.2014.05.010   https://www.cell.com/fulltext/S0092-8674(14)00604-7   

Khemani, K., Katoch, D., & Krishnamurti, L. (2019). Curative Therapies for Sickle Cell Disease. Ochsner journal, 19(2), 131–137. https://doi.org/10.31486/toj.18.0044  

Li, W., Teng, F., Li, T. et al. Simultaneous generation and germline transmission of multiple gene mutations in rat using CRISPR-Cas systems. Nat Biotechnol 31, 684–686 (2013). https://doi.org/10.1038/nbt.2652   

M., & Ranisch, R. (2022). Beyond safety: mapping the ethical debate on heritable genome editing interventions. Humanities and Social Sciences Communications, 9(1). https://doi.org/10.1057/s41599-022-01147-y  

National Human Genome Research Institute. (2017, August 3). ​What are the Ethical Concerns of Genome editing? National Human Genome Research Institute; National Institutes of Health. https://www.genome.gov/about-genomics/policy-issues/Genome-Editing/ethical 

Salih, K. M. A. (2019). The impact of sickle cell anemia on the quality of life of sicklers at school age. Journal of Family Medicine and Primary Care, 8(2), 468–471. https://doi.org/10.4103/jfmpc.jfmpc_444_18  

Subica A. M. (2023). CRISPR in Public Health: The Health Equity Implications and Role of Community in Gene-Editing Research and Applications. American journal of public health, 113(8), 874–882. https://doi.org/10.2105/AJPH.2023.307315  

Synthego | Full Stack Genome Engineering. (n.d.). Www.synthego.com. https://www.synthego.com/crispr-sickle-cell-disease#sickle-cell-crispr-trias 

WHO. (2023, December 4). Malaria. World Health Organization; WHO. https://www.who.int/news-room/fact-sheets/detail/malaria  

Yoshino, Y., Endo, S., Chen, Z., Qi, H., Watanabe, G., & Chiba, N. (2019). Evaluation of site-specific homologous recombination activity of BRCA1 by direct quantitation of gene editing efficiency. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-018-38311-x  

‌Solovieff, N., Hartley, S. W., Baldwin, C. T., Klings, E. S., Gladwin, M. T., Taylor, J. G., Kato, G. J., Farrer, L. A., Steinberg, M. H., & Sebastiani, P. (2011). Ancestry of African Americans with sickle cell disease. Blood Cells, Molecules, and Diseases, 47(1), 41–45.  https://doi.org/10.1016/j.bcmd.2011.04.002