The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a versatile and effective approach that has been explored in the context of multiple genetic disorders, including sickle cell disease (SCD). This system has proven to be efficient in correcting the sickle mutation in the β-globin (HBB) gene and a single delivery of CRISPR/Cas9 can accomplish a permanent gene modification.

According to So Hyun Park and Gang Bao, researchers from Rice University, Houston, Texas, “With the advancement of CRISPR/Cas9 technology, autologous transplant of gene-edited hematopoietic stem cells could potentially provide a cure for most patients with SCD.”

CRISPR/Cas9-Based SCD Therapy in Clinical Trials

As of early 2021, there were 2 trials (NCT03745287 and NCT04443907) investigating the potential of CRISPR-Cas9-modified human hematopoietic stem cells (HSCs) in SCD.

One of the studies had just published preliminary data of the first participant with severe SCD treated with CRISPR-Cas9 editing of the BCL11A enhancer to reactivate the expression of fetal hemoglobin (HbF) in adult red blood cells (RBCs). “Short-term follow-up for the first two patients [ie, one with transfusion-dependent β-thalassemia and another one with severe SCD] infused with edited autologous [hematopoietic stem and progenitor cells] revealed pancellular, elevated, and stable HbF expression providing transfusion independence and elimination of vaso-occlusive episodes,” DeMirci et al wrote in a review article.

Recently, the US Food and Drug Administration (FDA) approved another CRISPR/Cas9-based experimental treatment for SCD for clinical trial (NCT04774536). The study, led by Mark Walters, MD, director of the Pediatric Blood and Marrow Transplant Program at UCSF Benioff Children’s Hospital Oakland in California aims to administer a single infusion of sickle allele modified CD34+ HSPCs in subjects with severe SCD, thereby producing healthy RBCs. “Based on our experience with bone marrow transplants, we predict that correcting 20% of the genes should be sufficient to out-compete the native sickle cells and have a strong clinical benefit,” Dr. Walters explained.

Donald Kohn, MD, professor at the David Geffen School of Medicine at UCLA and a member of the UCLA Broad Stem Cell Research Center in Los Angeles, California.
(Credit: UCLA)

Current evidence suggests that this technique is safer than stem cell transplantation from a bone marrow donor, as noted by Donald Kohn, MD, professor at the David Geffen School of Medicine at UCLA and member of the UCLA Broad Stem Cell Research Center in Los Angeles, California: “Gene therapy and gene editing allow each patient to serve as their own stem cell donor.” This is an enormous advantage as it does not require the usually intense search for a matched stem cell donor.

However, there are many challenges that still limit the translation of gene editing-based strategies into clinical practice, in particular the risk of off-target mutations.

Risks of CRISPR/Cas9-Induced Off-Target Mutations

One of the major concerns regarding the use of the CRISPR/Cas9 system for genome editing is its potential off-target activity. “The off-target activity of Cas9 nuclease can cause disruption of normal gene function and genome instability via large chromosomal rearrangements, which is of serious concern in human gene therapies, potentially leading to difficult-to-predict side effects,” Park and Bao explained.

Evidence suggests that the long-term expression of Cas9 nuclease in transfected cells might lead to the accumulation of off-target cleavages over time, which would be deleterious for cells. Therefore, researchers have been trying to incorporate improvements in the system to address this issue. Among the strategies are the delivery of guide RNA (gRNA) and Cas9 as a pre-complexed ribonucleoprotein, which was shown to be well-tolerated in CD34+ HSPCs, despite eliciting a DNA damage response (DDR), and the use of high-fidelity Cas9. However, these approaches were not sufficient to eliminate the risk of aberrant events.

The detection and quantification of such aberrant events are also challenging. Currently, Cas9 off-target activity is commonly quantified by polymerase chain reaction (PCR) amplification, followed by next-generation sequencing. However, it is now known that this approach misses many off-target sites due to a detection limit of 0.1 % by deep sequencing for accurate indel identification.

“Although the large deletions/insertions at the on- and off-target sites and the large chromosomal rearrangements between on- and off-target sites typically have low occurrence, they pose a significant safety concern since even a very small number of HSCs harboring these detrimental events could cause hematological malignancies after HSCT,” Park and Bao said.

How Far Are We From a Cure?

There are additional issues that preoccupy researchers. For instance, the editing protocols currently adopted on SCD clinical trials use electroporation to deliver the system into cells. This method showed superior efficiency in terms of editing and fast kinetics, however, it was also associated with high toxicity. This is because cell permeabilization requires high-voltage shock, which decreases the number of viable HSCs and might be a problem for patients who are poor HSC mobilizers.

Moreover, currently available data is not sufficient to determine if the ability of the CRISPR/Cas9 system to reactivate HbF is sustained over the lifetime and what the long-term consequences of gene-edited stem cells are, if any.

Ultimately, “the simplest, most efficient, and, importantly, cost-effective editing strategy for therapeutic levels of HbF induction in adult RBCs is yet to be defined but offers a potentially curative therapy for millions living with SCD,” Demirci et al wrote in a review article published in the journal Molecular Therapy – Methods & Clinical Development.

Reference

Park SH, Bao G. CRISPR/Cas9 gene editing for curing sickle cell disease. Transfus Apher Sci. 2021;60(1):103060. doi:10.1016/j.transci.2021.103060

Demirci S, Leonard A, Essawi K, Tisdale JF. CRISPR-Cas9 to induce fetal hemoglobin for the treatment of sickle cell disease. Mol Ther – Methods Clin Dev. 2021;23:276-285. doi:10.1016/j.omtm.2021.09.010

FDA approves first test of CRISPR to correct genetic defect causing sickle cell disease. News release. Berkeley News; March 30, 2021.