How CRISPR Is Being Used to Cure Disease

Gene therapy aims to modify genes for therapeutic benefit, either by correcting, disrupting, or replacing mutated sequences that are causing disease. In 2012, Doudna and Charpentier showed the utility of CRISPR for altering genetic sequences in vitro, paving the way for the development of CRISPR into a versatile, robust, and convenient tool that has revolutionized gene editing.¹ CRISPR can now be used both in vivo and ex vivo to both repair mutations or improve cellular function and is providing exciting opportunities for gene therapy to treat disease.

Historically the application of gene editing in the clinic had been limited to Zinc Finger Nucleases (ZFNs) and TALENs, but in 2016, the first clinical trial using CRISPR used PD-1 knockout T cells for non-small cell lung cancer.² Now, there are trials underway in several areas, including blood disorders, inherited eye disease, protein-folding disorders, cancer, diabetes, as well as inflammatory and infectious disease. This article looks at some of the current clinical trials using CRISPR and how it could be used to treat human disease.

Ex vivo editing to treat blood disorders

Sickle-cell disease (SCD) and beta thalassemia are genetic blood disorders resulting from mutations to the HBB gene encoding the protein required for hemoglobin in red blood cells. Clinical Therapeutics and Vertex Pharmaceuticals ran the first clinical trial to treat the disease—but rather than directly correcting the gene variant causing disease, they used CRISPR to modify hematopoietic stem cells (HSCs) ex vivo to express high levels of fetal hemoglobin, which is not affected by the mutation.³ Patients in the trial have made remarkable recoveries and the continued presence of edited cells in the bone marrow of treated patients has beem seen more than a year after treatment.⁴

More recent trials to treat blood disorders are underway, including EDIT-301 from Editas Medicine, which uses Cas12a to upregulate fetal hemoglobin by modifying HBG1 and HBG2 gene promoters.⁵ Another approach, utilized by the GPH-101 therapy CEDAR trial from Graphite Bio, is to directly correct the single nucleotide polymorphism (SNP) responsible for the faulty HBB gene.⁶ Two other trials from Beam Therapeutics are utilizing base editing, a development that uses CRISPR machinery but without the introduction of a double-strand break to DNA—potentially providing improved safety for patients.⁷

CRISPR knockout to treat hereditary diseases

There are several disorders that arise from pathogenic mutations that cause the production of abnormal or excessive amounts of protein. But rather than attempting to fix the mutation, CRISPR knockout can be used to prevent the faulty gene from being expressed altogether.

Hereditary transthyretin amyloidosis (hATTR) is a progressive disease caused by a single letter DNA mutation in the TTR gene causing aggregation of misfolded TTR proteins that form amyloid fibrils. Intellia Therapeutics in collaboration with Regeneron are trialing the NTLA-2001 gene therapy to knockout TTR in patients to reduce amyloidosis, which interferes with cellular function leading to death.⁸ Similarly, Intellia Therapeutics is in trials with their NTLA-2002 therapy that uses CRISPR knockout to treat Hereditary Angioedema (HAE).⁹ HAE arises due to a faulty C1 inhibitor protein that regulates inflammation—a lack of C1 inhibitor protein leads to an accumulation of bradykinin in the blood, causing attacks of inflammation that lead to painful and debilitating swelling, which can be fatal. But by using CRISPR to knock out kallikrein B1 (KLKB1), a precursor protein to bradykinin, the treatment aims to reduce bradykinin levels to prevent HAE attacks. These two trials are some of the first to deliver the CRISPR components by lipid nanoparticles, and to deliver systemically rather than to specific cells or tissues. TTR and kallikrein B1 are primarily expressed in the liver, and lipid nanoparticles tend to accumulate there, so the treatments are taking advantage of this.

CRISPR trial to cure blindness

The first in vivo CRISPR clinical trial is currently underway as a potential treatment for Leber Congenital Amaurosis (LCA), the most common inherited childhood blindness.¹⁰ The BRILLIANCE trial by Editas Medicine involves removal of the mutation in the LCA10 photoreceptor gene with a direct injection of the Cas9 and single-guide RNA (sgRNA) straight into the eye. The eye is ideal for in vivo editing, as it is a contained organ with low immunoreactivity, and the adeno-associated virus (AAV) is designed to only be active within photoreceptor cells.

Engineering stem cells with CRISPR for diabetes treatment

Replacing pancreatic cells from healthy donors into individuals with type 1 diabetes has long been considered a potential treatment strategy—however recipients of pancreatic cell transplantation must then take immunosuppressant drugs to maintain transplanted cells, which comes with increased risk of infection. CRISPR Therapeutics and ViaCyte have developed the VCTX210 gene-edited cell therapy to treat type 1 and insulin-dependent type 2 diabetes with the first patient receiving treatment in a phase 1 trial in early 2022.¹¹ Donor-derived cells are edited with CRISPR so that they can evade recognition by the host immune system, providing replacement beta cells without the need for immunosuppression. If successful, this could potentially be a scalable off-the-shelf solution, whereby donor cells can be obtained from any healthy individual rather than requiring a matched donor or cells derived from the patient themselves.

Cancer therapeutics with CRISPR

In 2016, the first clinical trial using CRISPR involved editing a patient’s T cells to prevent expression of the PD-1 gene, therefore improving recognition and targeting of cancer cells¹² and in 2017, CAR-T therapy using CRISPR was approved for use to treat blood cancers. Although treatment of blood-based cancers using CRISPR technology is more advanced, CRISPR is also being used to target solid tumors. Posedia Therapeutics’ P-MUC1C-ALLO1 therapy, which involves allogenic T cells that have been edited to recognize the MUC1-1 antigen expressed on the surface of cancer cells, could be used to treat breast, pancreatic, and gastric cancers.¹³

Treating infectious disease with CRISPR

As well as genetic diseases and cancer, CRISPR can also be used to treat infectious diseases—by targeting bacterial cells directly. In early 2022, a clinical trial by SNIPR Biome dosed the first patients with SNIPR-001 therapy, an orally administered antibiotic that targets specific strains of bacteria without affecting beneficial bacteria.¹⁴ This approach could be utilized in cancer patients to target E. coli strains that could cause sepsis in the bloodstream.

An alternative is to use live bacteriophage to target bacteria and so potentially reduce the reliance on antibiotics and the associated increase in antibiotic resistance. Locus Biosciences is using the CRISPR-Cas3 machinery to target and destroy bacterial genomes. The company completed the first controlled clinical trial for CRISPR-based bacteriophage therapy, where live bacteriophages combined with CRISPR-Cas3 was administered to patients with chronic urinary tract infections (UTIs). Unlike CRISPR-Cas9 that introduces a double-strand break at the target site, CRISPR-Cas9 shreds the DNA resulting in bacterial cell death. A phase 2/3 trial is now ongoing but initial results have shown a decrease in levels of E. coli in the bladder of patient volunteers.¹⁵

Using CRISPR to fight HIV/AIDS

One of the main challenges in treating HIV is eliminating the ‘HIV reservoir’, which contains integrated provirus within host cellular DNA. Despite not actively replicating, it is resistant to antiretroviral treatment and clearance by the host immune system, leading to new rounds of infection if treatment is stopped. However, a potential and exciting therapy was approved for phase1/2 clinical trials in 2021. Excision Biotherapeutics has developed the EBT-101 in vivo therapy, which uses Cas9 from S. aureus (SaCas9) along with two sgRNAs to excise the integrated HIV from the human genome, and potentially offer a cure for the disease.¹⁶

CRISPR in the clinic—the future

Above we have discussed examples where CRISPR is already proceeding through clinical trials. However, there are numerous examples of potential therapies that have demonstrated proof of concept and could be positioned to enter clinical trials soon. For example, PCSK9 is a regulator of blood cholesterol levels and driver of coronary heart disease risk—and there is a growing body of evidence using gene editing to knock-out or knock-down the gene that highlights its potential as a therapeutic target.¹⁷

Just as CRISPR has revolutionized gene editing, there has now been significant progress in developing CRISPR-based therapeutics to treat a wide variety of diseases allowing precision medicine. The development of universal allogenic cell therapies using CRISPR will enable large-scale production of off-the-shelf cells and the use of new CRISPR technologies, such as base editing and prime editing that do not involve the introduction of a double-strand break, will improve patient safety. CRISPR gene editing technology exploded into the biological space—and it looks like the same can be said for its therapeutic use in the clinic.

Key Takeways

• CRISPR-Cas9 is a gene editing technology developed from endogenous bacterial immune system and is comprised of two components—a short guide RNA (sgRNA) that guides the Cas9 endonuclease to the target site
• Once at the target site, the Cas9 will introduce a double-strand break (DSB), activating the non-homologous end joining (NHEJ) endogenous DNA repair pathway, resulting in a frameshift mutation and subsequent gene knockout
• However, if an exogenous template is present alongside the CRISPR machinery, the DSB will trigger the Homologous Repair (HR) pathway, which allows for sequences to be replaced or modified
• The development of CRISPR-based therapeutics for the clinic will need GMP-grade sgRNA. Synthego, a genome engineering company that provides GMP-like and GMP-grade sgRNA, has recently broken ground on a new facility that will enable expedited CRISPR-based cell and gene therapy development, accelerating the path to clinic for new therapies

References

1. Jinek, Martin et al. “A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Science (New York, N.Y.) vol. 337,6096 (2012): 816-21. doi:10.1126/science.1225829

2. Liu, Qian. “World-First Phase I Clinical Trial for CRISPR-Cas9 PD-1-Edited T-Cells in Advanced Nonsmall Cell Lung Cancer.” Global medical genetics vol. 7,3 (2020): 73-74. doi:10.1055/s-0040-1721451

3. Frangoul. N., et al. “CRISPR-Cas9 Gene Editing for Sickle Cell Disease and β-Thalassemia”. N Engl J Med 2021;384:252-60.(2021) doi:10.1056/NEJMoa2031054

4. Vertex and CRISPR Therapeutics Present New Data in 22 Patients With Greater Than 3 Months Follow-Up Post-Treatment With Investigational CRISPR/Cas9 Gene-Editing Therapy, CTX001™ at European Hematology Association Annual Meeting—Press release from CRISPR Therapeutics  May 2021

5. EDIT-301 for Autologous HSCT in Subjects with Severe Sickle Cell Disease. Last updated posted 12 October, 2022

6. Gene Correction in Autologous CD34+ Hematopoietic Stem Cells (HbS to HbA) to Treat Severe Sickle Cell Disease (CEDAR). Last updated 7 October 2022

7. https://beamtx.com/pipeline/ (29 Nov 22)

8. Study to Evaluate Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of NTLA-2001 in Patients with Hereditary Transthyretin Amyloidosis With Polyneuropathy (ATTRv-PN) and Patients With Transthyretin Amyloidosis-Related Cardiomyopathy (ATTR-CM). Last updated 29 December 2021

9. NTLA-2002 in Adults with Hereditary Angioedema (HAE) (NTLA-2002). Last updated 20 July 2022

10. Editas Medicine Announces Positive Initial Clinical Data From Ongoing Phase 1/2 Brilliance Clinical Trial Of EDIT-101 For LCA10—Press release from Editas Medicine, 29 September 2021

11. First CRISPR Therapy for Type 1 Diabetes Set to Enter Clinical Trial. CRISPR Medicine News, 17 Nov 2021

12. Liu, Qian. “World-First Phase I Clinical Trial for CRISPR-Cas9 PD-1-Edited T-Cells in Advanced Nonsmall Cell Lung Cancer.” Global medical genetics vol. 7,3 (2020): 73-74. doi:10.1055/s-0040-1721451

13. P-MUC1C-ALLO1 Allogeneic CAR-T Cells in the Treatment of Subjects with Advanced or Metastatic Solid Tumors. Last updated 28 October 2022

14. A Study Investigating the Safety, Recovery, and Pharmacodynamics of Multiple Oral Administrations of SNIPR001 in Healthy Subjects. Last updated 7 November 2022

15. Locus Biosciences completes first-of-its-kind controlled clinical trial for CRISPR-enhanced bacteriophage therapy. Press release. 24 February 2021

16. Study of EBT-101 in Aviremic HIV-1 Infected Adults on Stable ART. Last updated 19 October 2022

17. Musunuru, Kiran. “Moving toward genome-editing therapies for cardiovascular diseases.” The Journal of Clinical investigation vol. 132,1 (2022): e148555. doi:10.1172/JCI148555