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Therapeutic Gene Editing for Sickle Cell Disease

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“It is heartbreaking to see my child go through this crisis. When my son, [Eric*], was five years old, he had a sickle cell crisis and the doctors told me that he might have a 50/50 chance of surviving. My heart just broke in half,” said the mother of an eight-year-old boy with sickle cell disease (SCD).1 Sickle cell crises are also referred to as pain crises, as this is how they manifest. The pain can occur anywhere in the body, last from hours to a week, and range from mild to excruciating. Pain is the clinical hallmark of SCD, being so particular to the disease that in some languages, the disease name refers to the pain. Pain crisis is the most common cause of hospitalization for patients with SCD, with many frequently spending days, weeks or even months in hospital.

Sickle cell disease is the most common inherited blood disorder in the US, affecting 70,000 to 80,000 Americans
. Worldwide, there are an estimated 300,000 babies born each year with SCD. It is a group of autosomal recessive genetic disorders that causes atypical hemoglobin molecules, which distorts red blood cells into a sickle, or crescent, shape. This characteristically leads to anemia, pain crises, repeated infections, and other complications. The pain is caused by vaso-occlusion: when sickled erythrocytes, which are stiff and inflexible, clump together and get stuck in capillaries. These episodes deprive tissues and organs of oxygen-rich blood and can lead to organ damage, especially in the lungs, kidneys, spleen, and brain. A particularly serious complication is pulmonary hypertension, which occurs in about a third of adults with SCD and can lead to heart failure. SCD is caused by a point mutation in both alleles of the human beta-globin gene (HBB).

The only curative treatment is hematopoietic stem cell (HSC) transplantation, which has over 90% event-free survival when a matched donor is used. However, treatment-related complications – such as the potential for graft rejection, infections, prolonged immunosuppression, acute or chronic graft-vs-host disease, and disease relapses – limit its broad acceptability and applicability. Moreover, the availability of matched donors is limited. More than 80% of patients do not have a human leukocyte antigen-identical sibling donor. However, we may soon have a better cure. There is real hope, for Eric and thousands of others who are born with this debilitating disease, that CRISPR gene editing can correct the mistake in their genes that is responsible for SCD.

Refining gene editing for clinical use

Our work at Integrated DNA Technologies (IDT) involves the development of the highest quality tools to support and better facilitate the research conducted by biomedical scientists. One such development is that of an improved enzyme for gene editing using CRISPR/Cas9. As CRISPR/Cas9 technology directly edits genes, it may be used to develop an SCD therapy based on ex vivo gene editing of HSCs for autologous transplants, applicable for most if not all patients, as sibling donors would not be needed.

The most commonly used gene editing system, CRISPR/Cas9, co-opts the ability of bacterial nucleases, like Cas9, coupled to target-specific guide ribonucleic acid oligomers (gRNAs), to selectively cut DNA, enabling the insertion, deletion or substitution of disease-causing mutations to produce healthier versions of the DNA sequence. However, there is concern regarding the hypothetical risks of off-target gene editing, potentially resulting in effects such as the activation of oncogenes or deactivation of tumor suppressors. To address this concern, many scientists have developed Cas9 variants with the aim of improving its selectivity, compared with the wild-type (WT) Cas9. Increased selectivity has however come at the cost of nuclease activity.

The delivery method can also improve selectivity. The Cas9 nuclease and gRNA can be delivered as DNA expression cassettes in plasmids or viral vectors, as purified RNAs with Cas9 mRNA, or as a Cas9–gRNA ribonucleoprotein (RNP) complex. Methods resulting in higher, more sustained levels of Cas9 and gRNA in the cells tend to yield both greater cutting efficiency and off-target effects (OTEs). Delivery using RNPs leads to a “fast on, fast off” effect, with an initially high level of genome editing machinery followed by rapid decay. This results in highly efficient gene editing, while OTEs are minimized. That said, OTEs still occur.

To tackle this problem, we at IDT devised an unbiased bacterial screen to isolate RNP-delivered Cas9 variants that provide highly specific cleavage with minimal OTEs, and nuclease activity comparable with WT Cas9. Thus, we selected a high-fidelity (HiFi) variant that, compared with other high-fidelity Cas9 nucleases, demonstrates substantially better specificity and fewer OTEs, while not comprising on WT-level nuclease activity. The results of this work and clinical applications were published in Nature Medicine in 2018 (Vakulskas, et al. 2018).2 IDT have partnered with Aldevron to supply a Good Manufacturing Practice (GMP) grade of the enzyme for clinical use.

Editing genes to treat sickle cell disease

Dr Matthew Porteus, from Stanford Medicine, is leading a proposed Phase I SCD clinical trial to evaluate a gene editing therapy that corrects the actual disease-causing HBB mutation, potentially offering full restoration of normal hemoglobin function. The HiFi Cas9 enzyme was used to show that the sickle HBB allele could be efficiently corrected, while simultaneously reducing problematic off-target editing from ~30% to less than 1%, in the preclinical studies. The Aldevron GMP form of the enzyme has shown similar results and will ultimately be used in the clinical trial. Knowing that the clinical trial will employ effectively the same nuclease used in the preclinical studies can build confidence with regulatory authorities when considering the proposed trial.

Alongside patients with SCD, our partners at Aldevron, and the trial investigators, we at IDT hope to see great clinical trial results, which will realize the promise of gene editing to cure SCD.

* Not patient’s real name


1. eMedicine Health. Sickle cell crisis. Available at: https://comments.emedicinehealth.com/sickle_cell_crisis/viewer-comments_em-139.htm (accessed July 2019).

2. Vakulskas CA, Dever DP, Rettig GR, Turk R, et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat Med. 2018;24:1216–24.

Author Biography

Dr Christopher Vakulskas is a senior staff scientist in the molecular genetics research group at IDT. Dr Vakulskas earned his PhD in microbiology at the University of Iowa, where he studied genetic regulatory circuits in pathogenic bacterial species. After earning his PhD, he became an NIH Postdoctoral Fellow at the University of Florida, where he studied RNA binding proteins and posttranscriptional gene regulation. At IDT, Dr Vakulskas has managed contract research projects, led process development for CRISPR protein purification, and developed novel CRISPR proteins, including Alt-R S.p. HiFi Cas9 Nuclease 3NLS and Alt-R A.s. Cas12a (Cpf1) Ultra.