It’s estimated that there are over ten thousand diseases that afflict humans (Smith et al, 2022). Only about five hundred of those diseases have treatments, and out of those, most treatments are not curative but rather manage the symptoms and allow a patient to have a relatively decent quality of life with the disease (Kessler, 2016).
Wouldn’t it be wonderful if we had a versatile tool that could cure thousands of different diseases? With the recent approval of CRISPR gene editing medicines, we may be witnessing the starting point of a new wave of genetic medicines.
The regulatory approval of CRISPR therapies for Sickle Cell Disease and Transfusion Dependent b-Thalassemia provides a new and seemingly curative option for patients with these diseases. These approvals are a landmark for the first approved CRISPR therapies, with many more to come across a broad range of diseases.
CRISPR is a genetic modification tool first discovered in bacteria that now has widespread industrial applications, including in human health. Compared to previous genome editing tools, CRISPR has a couple of very important improvements.
First, it is very precise, meaning it only edits the intended genomic target and not at other random sites in your DNA which could lead to problems.
Second, CRISPR is flexible, meaning it is relatively easy to make a new CRISPR that targets a different gene. In comparison, it was expensive and took a lot of time to optimize previous gene editing tools to target different genes.
Together, these advantages make CRISPR a viable approach to treat a wide range of human diseases.
In this article, we will cover the rapid rise of CRISPR from initial discovery to the clinic, discuss the CRISPR therapies approved for Sickle Cell Anemia and Transfusion Dependent b-Thalassemia, and survey the horizon for the next series of CRISPR therapies that are currently in development.
Article Table of Contents
Regulatory Approval of First CRISPR Therapies
Transfusion Dependent b-Thalassemia
Next CRISPR Medicines on the Horizon
Alternative Therapeutic Approaches for SCD and TDT
The Rapid Rise of CRISPR
CRISPR is an acronym for clustered regularly interspaced short palindromic repeats. What a mouthful – I bet you can see why everyone says CRISPR instead! These molecular systems were originally discovered in bacteria, and you can think of them as bacteria’s immune system (Jansen et al, 2002).
CRISPR, it turns out, is a primary tool many bacteria use to recognize and fend off bacteriophage invaders. Bacteriophages are viruses that infect and replicate in bacteria. Multicellular organisms like humans have specialized immune systems that help us deal with invaders like viruses. But a bacterium, which is only a single cell, has a more compact molecular defense system.
At its most basic, CRISPR has two essential components: one component that localizes CRISPR to a specific DNA sequence, and another component that cuts or otherwise modifies DNA. The localization component is an RNA molecule termed a guide RNA that binds to specific DNA sequence (Figure 1). This piece is why CRISPR can easily be adapted to target new genes as you just change the guide RNA to bind to a new DNA sequence. The components that cut DNA are CRISPR associated proteins, such as Cas9 and Cas 12 which we’ll discuss more in the Next “CRISPR Medicines” section.
Figure 1. Essential CRISPR pieces include a guide RNA
(magenta) that binds to DNA (light green) in a sequence specific manner and
directs the CRISPR complex to a particular place in the genome (light and dark
green), and a nuclease (plum) such as Cas9 or Cas12 that cuts DNA on both
strands.
Regulatory Approval of the First CRISPR Therapies
Sickle Cell Disease (SCD) and Transfusion Dependent b-Thalassemia (TDT) are both anemias involving red blood cell dysfunction and deficiencies in oxygen transport throughout the body. Each disease has a defect in hemoglobin, which is the protein complex that binds to oxygen and distributes oxygen from our lungs to the rest of our body. The defect in hemoglobin is different in each disease – but the mechanism used to overcome these defects with CRISPR therapies is the same.
Sickle Cell Disease
Hemoglobin is normally a tetramer formed by two a-hemoglobin and two b-hemoglobin proteins (Figure 2). SCD patients have a mutation in b-hemoglobin that causes hemoglobin to switch from a tetramer to a very long rod-shaped oligomer. These oligomers are so long that they disrupt the normal, round red blood cells into a sickle shape (Figure 3).
Figure 2. Normal hemoglobin forms a tetramer that
binds to oxygen (left) (PDB: 2DHB). A mutation in the b subunit of hemoglobin causes the tetramers to oligomerize into
long rod-shaped structures (right).
The sickle shape causes these red blood cells to easily rupture and to get caught and cause blockages in blood vessels leading to vasculo-occlusive crises. These blockages cause a lack of oxygen and severe pain to the organs with disrupted blood flow and are a common cause of hospitalization for SCD patients. When the blockages occur in the lungs or the brain, they are immediately life threatening. Over the long run, the cumulative stress of reduced oxygen supply and vasculo-occlusive crises leads to organ damage and a lifespan for SCD patients that, on average, is over twenty years shorter than the general population (U.S. Centers for Disease Control and Prevention, 2024).
Figure 3. Normal hemoglobin is small and fits into normal red blood cells without perturbing the cells shapes (left). The oligomerized, rod-shaped hemoglobin perturbs red blood cells into a sickle shape in Sickle Cell Disease (right).
The CRISPR therapy CasgevyTM works by activating the expression of fetal hemoglobin (U.S. Food & Drug Administration, 2023). Normally, fetal hemoglobin is expressed in utero, whereas after birth, a- and b-hemoglobin are expressed (Henderson, 2024). CasgevyTM targets and deactivates the BCL11A gene in bone marrow stem cells, where red blood cells are made. Inactivation of BCL11A results in fetal hemoglobin expression in red blood cells derived from the modified bone marrow stem cells (Reardon, 2023). In turn, fetal hemoglobin competes with mutant b-hemoglobin for binding with a-hemoglobin, and thereby limits hemoglobin rod formation and the resulting sickling of red blood cells (Figure 4).
Figure 4. Oligomerized, rod-shaped hemoglobin perturbs red blood cells into a sickle shape in Sickle Cell Disease (left). Fetal hemoglobin forms normal tetramers with a-hemoglobin and limits rod formation driven by mutant b-hemoglobin in Sickle Cell Disease (right).
Transfusion Dependent b-Thalassemia
Transfusion Dependent B-Thalassemia is an anemia that requires patients to undergo blood transfusions as often as every 3-4 weeks. Like SCD, TDT is also caused by a defect in hemoglobin, albeit via a slightly different issue.
Hemoglobin is not mutated in TDT, there is just a lot less of it. For a variety of reasons, hemoglobin protein levels are very low in TDT patients, leading to anemia and need for frequent blood transfusions.
Since the causative problem in TDT is not enough hemoglobin, CasgevyTM also works for TDT to increase fetal hemoglobin expression in red blood cells, as described above.
Therapeutic Administration
The administration of CasgevyTM follows the same protocol for both SCD and TDT patients (US Food and Drug Administration, 2023 & 2024). The patients’ hematopoietic (blood) stem cells are collected from their bone marrow and modified by CRISPR gene editing. Cells that are successfully edited are transplanted back into the patient. Patients undergo high-dose chemotherapy to remove their remaining hematopoietic stem cells before adding the genetically modified cells back in.
The primary outcome for the SCD CasgevyTM clinical trial was freedom from severe vasculo-occlusive crises for at least 12 consecutive months following treatment. 29 of the 31 SCD patients (93.5%) met this outcome. For reference, patients were selected for the trial based on having had at least 2 severe vasculo-occlusive crises in the 2 years prior to treatment.
25 out of 27 TDT patients treated with CasgevyTM are no longer transfusion dependent following the treatment. Some patients have gone over 3 years without transfusion. The other two patients have had an 80% and 96% reduction in transfusion frequency following treatment.
For both SCD and TDT, it is expected that this is a one-time treatment, as the CRISPR-edited hematopoietic blood cells will remain in the patient and continuously generate new red blood cells expressing fetal hemoglobin.
There were a couple of reasons that SCD and TDT anemias made sense as the first diseases to target with CRISPR. One is that since they are anemias, gene editing hematopoietic stem cells can be done using a patient’s own cells and outside of the body. Since the patient’s cells were used, there was no rejection or complication of the transfusion which is a common occurrence in bone marrow transplants from other individuals.
Performing the gene editing of hematopoietic stem cells outside of the patient’s body allows for the cells to be screened before transplanting them back in to ensure that both the correct modification to turn on and fetal hemoglobin expression occurred, and no other off-target gene modifications took place.
Next CRISPR Medicines on the Horizon
CasgevyTM’s approval as the first CRISPR therapy on the market is a breakthrough in genetic medicine. Yet, a lot of questions remain about both the best technological approach for CRISPR therapeutics and the range of diseases that they can treat.
Alternative Therapeutic Approaches for SCD and TDT
As we discussed above, one of the key pieces of CRISPR is a nuclease protein that cuts DNA (Figure 1). CasgevyTM uses the protein Cas9. Another company is currently trying the protein Cas12 as the nuclease instead to treat SCD and TDT patients. They are currently in Phase 1/2 clinical trials, and so far, have results in SCD and TDT patients that are comparable to CasgevyTM (Henderson, 2024). Cas12 has a fundamentally different DNA cleavage mechanism compared to Cas9 (Pickar-Olivar & Gersbach, 2019), so it may be the better nuclease for some CRISPR applications.
Another approach is to try to fix the mutation with b-hemoglobin in SCD patients, rather than turn on fetal hemoglobin. A University of California consortium is currently the only non-profit organization in this space, and they are planning to enroll patients for a phase 1 clinical trial with a fix-it approach starting in early 2025 (Fernandes, 2021).
Other strategies are simply fine-tuning the process already in place with CasgevyTM treatment. One optimization in this regard is to try to find a lower dose of chemotherapy for ablating a patient’s hematopoietic stem cells before transplanting the gene-edited stem cells back in. The chemotherapy can have toxic effects on other body parts, so finding a lower dose that maintains HSC ablation but minimizes other damage would be an improvement.
Another approach would be to deliver the CRISPR gene editor into the patient rather than editing the stem cells outside of the body. The current protocol allows for scientists to check for the desired gene edits as well as look for off target edits. It also limits any off-target effects to only the hematopoietic stem cells since those are the ones being treated. However, so far off-target DNA cuts do not seem to be an issue.
Editing in a patient would enable CRISPR targeting of a wider variety of cell types, would be faster, and may be cheaper because it would be less labor intensive and wouldn’t rely on a few highly-specialized labs. However, targeting CRISPR to the cell type that is desired to be edited may be an issue as systemic delivery of CRISPR would expose many cell types to these genome editors, most of which you wouldn’t want editing of any kind to occur.
Additional Disease Targets
In addition to sickle cell and b-thalassemia, there are CRISPR-based therapies for a wide range of diseases currently being tested in clinical trials, including:
- cancers
- inflammatory disease
- chronic bacterial infection
Cancers
There are many clinical trials currently being conducted on different types of leukemias and lymphomas. Many of these trials are using CRISPR to support CAR T Cell approaches. CAR stands for chimeric antigen receptor, and this is a special type of membrane bound protein that is engineered into a patient’s own T Cells to help them recognize and eliminate cancer cells (Figure 5).
Figure 5. A Car T cell (green) has an engineered
receptor (green and gray) that binds to a cancer cell (orange) antigen (light
orange and gray).
CAR T therapies typically have a strong impact initially, but eventually the cancer returns (De Marco et al, 2023). Additionally, the therapy is quite expensive, costing hundreds of thousands of dollars.
Researchers are using CRISPR to try to improve current CAR T therapies. One way to do this is by modifying the T cells to better recognize cancer cells. Sometimes this means removing a protein such that the cancer’s own defense mechanisms can’t ward off the engineered T cells.
Another role for CRISPR in CAR T therapy is to enable allogenic T cell sources. Currently, each patient’s CAR T therapies are engineered from their own T cells. The advantage of this approach is that the engineered T cells are well tolerated and aren’t flagged by their immune system as being foreign invaders. The downside is that this approach is very expensive and takes several weeks to engineer the cells – sometimes patients die before the CAR T cells are ready.
Currently researchers are using CRISPR to modify T cells to enable CAR T therapies to be made from a healthy donor. These CAR T therapies would be “off the shelf” ready – patients could start them immediately, and they would be less expensive as new cells wouldn’t have to be made for every patient. The problem is that the immune system will flag foreign T cells and mount an immune response, but researchers are genetically modifying donor T cells with CRISPR to make them more tolerable in allogenic transplants (Henderson, 2024).
These therapies are being tested in a wide range of leukemias and lymphomas including acute myeloid leukemia, B-cell lymphoma, multiple myeloma, B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, and T-cell lymphoma (CRISPR Medicine News, 2024).
CRISPR clinical trials are also being conducted on many solid tumor cancers including ovarian cancer, gastro-intestinal cancer, non-small cell lung cancer, and renal cell carcinoma (CRISPR Medicine News, 2024). Similar to the leukemia and lymphoma space, most of these trials are focused on improving CAR T therapies for the treatment of solid tumors.
Inflammatory Disease
Clinical trials are currently underway for hereditary angioedema (HAE), a severe inflammatory response. HAE patients have severe attacks of inflammation and swelling in their extremities, face, intestine, and airway. The attacks occur every couple of weeks, last for a few days, and can be life threatening.
A CRISPR-Cas9 treatment that reduces the level of an inflammatory protein called C-1 inhibitor protein is currently being tested. 10 patients have had treatment administered, and so far, the results are very promising. Since treatment attacks have been reduced 95% in HAE patients, including in 6 patients who have had no attacks and have discontinued other HAE preventative measures (Longhurst et al, 2024). These results suggest that the CRISPR HAE therapy may be curative.
Importantly, no adverse events have yet been reported. Unlike the approved SCD and TDT treatments, this HAE treatment is administered systemically meaning that the CRISPR editing process occurs inside the patient. If this approach is successful long term, it would increase the different types of diseases that CRISPR can treat and should reduce the labor and cost required for such treatments.
Chronic Bacterial Infection
Most urinary tract infections (UTIs) are simply treated with a brief course of antibiotics. However, chronic UTIs occur when the antibiotics don’t work, or the infection keeps recurring.
A new potential treatment for chronic UTIs uses three bacteriophages that have CRISPR-Cas3 engineered into their genome. Unlike Cas9 and Cas12, which make a single cut in DNA, Cas3 repeatedly cuts DNA, shredding it into little pieces (Figure 6) (Morisaka et al, 2019).
These 3 bacteriophages target 95% of the bacteria that cause chronic UTIs and will kill of the infectious bacteria by shredding their DNA. The treatment is delivered directly to the bladder by catheter, which should help avoid systemic off target effects from the treatment. Phase 1 trials demonstrated the safety of the treatment, and reportedly reduced the level of E. coli in the bladders of treated patients (Kim et al, 2021). Phase 2/3 clinical trials to better determine the effectiveness of this treatment are ongoing.
Figure 6. CRISPR Cas3 (indigo) cuts DNA (light and
dark green) repeatedly shredding it to little pieces, in contrast to Cas9 and
Cas 12 which cut DNA once on each strand (Figure 1).
Of course, not all of these treatments will make it to the market. Some may fail clinical trials due to safety reasons. Others may not proceed for business reasons, as has already been the case for several CRISPR therapies besides CasgevyTM that were also targeting SCD and TDT.
Nevertheless, the fraction of these CRISPR therapies that are successful will represent the vanguard of the next generation of genetic medicine. Indeed, in a few decades it’s possible that genetic medicines will be used to treat nearly every type of human disease.
References
Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T. N., Weissig, H., Shindyalov, I. N., & Bourne, P. E. (2000). The Protein Data Bank. Nucleic acids research, 28(1), 235–242. https://doi.org/10.1093/nar/28.1.235
Berman, H., Henrick, K., & Nakamura, H. (2003). Announcing the worldwide Protein Data Bank. Nature structural biology, 10(12), 980. https://doi.org/10.1038/nsb1203-980
Bolton, W., & Perutz, M. F. (1970). Three dimensional fourier synthesis of horse deoxyhaemoglobin at 2.8 Angstrom units resolution. Nature, 228(5271), 551–552. https://doi.org/10.1038/228551a0
Carroll D. (2017). Genome Editing: Past, Present, and Future. The Yale journal of biology and medicine, 90(4), 653–659.
Cohen, J. The latest round in the CRISPR patent battle has an apparent victor, but the fight continues. September 11, 2020. Accessed July 17, 2024. https://www.science.org/content/article/latest-rou...
Cong, L., Ran, F. A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P. D., Wu, X., Jiang, W., Marraffini, L. A., & Zhang, F. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science (New York, N.Y.), 339(6121), 819–823. https://doi.org/10.1126/science.1231143
CRISPR Medicine News (2024). CRISPR Clinical Trials. Accessed July 26, 2024. https://crisprmedicinenews.com/clinical-trials/
De Marco, R. C., Monzo, H. J., & Ojala, P. M. (2023). CAR T Cell Therapy: A Versatile Living Drug. International journal of molecular sciences, 24(7), 6300. https://doi.org/10.3390/ijms24076300
Fernandes, L. (2021). UC Consortium Launches First Clinical Trial Using CRISPR to Correct Gene Defect That Causes Sickle Cell Disease. UC San Francisco. March 30, 2021. Accessed July 23, 2024. https://www.ucsf.edu/news/2021/03/420137/uc-consor...
Henderson, H. CRISPR Clinical Trials: A 2024 Update. Innovative Genomics Institute. March 13, 2024. Accessed July 17, 2024. https://innovativegenomics.org/news/crispr-clinica...
Jansen, R., Embden, J. D., Gaastra, W., & Schouls, L. M. (2002). Identification of genes that are associated with DNA repeats in prokaryotes. Molecular microbiology, 43(6), 1565–1575. https://doi.org/10.1046/j.1365-2958.2002.02839.x
Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science (New York, N.Y.), 337(6096), 816–821. https://doi.org/10.1126/science.1225829
Kessler, G. The Washington Post. Are there really 10,000 diseases and just 500 ‘cures’? November 17, 2016. Accessed July 31, 2024. https://www.washingtonpost.com/news/fact-checker/w...
Kim, P., Sanchez, A., Kime, J., & ousterout, D. (2021). 1083. Phase 1b Results of Pharmacokinetics, Pharmacodynamics, and Safety for LBP-EC01, a CRISPR-Cas3 Enhanced Bacteriophage Cocktail Targeting Escherichia coli that Cause Urinary Tract Infections. Open Forum Infectious Diseases, 8(Suppl 1), S633. https://doi.org/10.1093/ofid/ofab466.1277
Laurent, M., Geoffroy, M., Pavani, G., & Guiraud, S. (2024). CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments. Cells, 13(10), 800. https://doi.org/10.3390/cells13100800
Longhurst, H. J., Lindsay, K., Petersen, R. S., Fijen, L. M., Gurugama, P., Maag, D., Butler, J. S., Shah, M. Y., Golden, A., Xu, Y., Boiselle, C., Vogel, J. D., Abdelhady, A. M., Maitland, M. L., McKee, M. D., Seitzer, J., Han, B. W., Soukamneuth, S., Leonard, J., Sepp-Lorenzino, L., … Cohn, D. M. (2024). CRISPR-Cas9 In Vivo Gene Editing of KLKB1 for Hereditary Angioedema. The New England journal of medicine, 390(5), 432–441. https://doi.org/10.1056/NEJMoa2309149
Morisaka, H., Yoshimi, K., Okuzaki, Y., Gee, P., Kunihiro, Y., Sonpho, E., Xu, H., Sasakawa, N., Naito, Y., Nakada, S., Yamamoto, T., Sano, S., Hotta, A., Takeda, J., & Mashimo, T. (2019). CRISPR-Cas3 induces broad and unidirectional genome editing in human cells. Nature communications, 10(1), 5302. https://doi.org/10.1038/s41467-019-13226-x
National Cancer Institute. CAR T Cells: Engineering Patients’ Immune Cells to Treat Their Cancers. Updated March 10, 2022. Accessed July 26, 2024. https://www.cancer.gov/about-cancer/treatment/rese...
Nishio, I., Tanaka, T., Sun, S. T., Imanishi, Y., & Ohnishi, S. T. (1983). Hemoglobin aggregation in single red blood cells of sickle cell anemia. Science (New York, N.Y.), 220(4602), 1173–1175. https://doi.org/10.1126/science.6857241
Pauling, l., & Itano, H. A. (1949). Sickle cell anemia a molecular disease. Science (New York, N.Y.), 110(2865), 543–548. https://doi.org/10.1126/science.110.2865.543
Pickar-Oliver, A., & Gersbach, C. A. (2019). The next generation of CRISPR-Cas technologies and applications. Nature reviews. Molecular cell biology, 20(8), 490–507. https://doi.org/10.1038/s41580-019-0131-5
Reardon, S. (2023). FDA Approves First CRISPR Gene Editing Treatment for Sickle Cell Disease. Scientific American. December 8, 2023. Accessed July 17, 2024. https://www.scientificamerican.com/article/fda-app...
Sheridan, C. (2024). The world’s first CRISPR therapy is approved- who will receive it? Nature Biotechnology 42, 3-4. https://www.nature.com/articles/d41587-023-00016-6
Smith, C. I. E., Bergman, P., & Hagey, D. W. (2022). Estimating the number of diseases - the concept of rare, ultra-rare, and hyper-rare. iScience, 25(8), 104698. https://doi.org/10.1016/j.isci.2022.104698
The Broad Insitute. For journalists: Statements and background on the CRISPR patent process. Updated February 28, 2022. Accessed July 19, 2024. https://www.broadinstitute.org/crispr/journalists-...
The Nobel Foundation. The Nobel Prize in Chemistry, 2020. October 7, 2020. Accessed July 18, 2024. https://www.nobelprize.org/prizes/chemistry/2020/p...
The PyMOL Molecular Graphics System, Version 2.5.2 Schrödinger, LLC.
U.S. Centers for Disease Control and Prevention. Data and Statistics on Sickle Cell Disease. May 15, 2024. Accessed July 31, 2024. https://www.cdc.gov/sickle-cell/data/index.html
U.S. Food & Drug Administration. FDA Approves First Gene Therapies to Treat Patients with Sickle Cell Disease. December 8, 2023. Accessed July 17, 2024. https://www.fda.gov/news-events/press-announcement...
U.S. Food & Drug Administration. FDA Roundup: January 16, 2024. January 16, 2024. Accessed July 22, 2024. https://www.fda.gov/news-events/press-announcement...
