1 Introduction

Sickle Cell Anemia (SCA) is a debilitating hereditary blood disorder that affects millions of individuals worldwide. The advent of gene editing technologies has opened new avenues for potential curative treatments for this condition. Sickle Cell Anemia is caused by a single-point mutation in the β-globin gene (HBB), leading to the production of abnormal hemoglobin S (HbS) (DeWitt et al., 2016; Romero et al., 2018). This mutation results in the deformation of red blood cells into a sickle shape, causing vaso-occlusion, severe pain, and progressive organ damage (DeWitt et al., 2016; Park et al., 2019). The disease is most prevalent in regions where malaria is endemic, as the sickle cell trait provides some protection against malaria (Romero et al., 2018). Current treatments, such as hydroxyurea and blood transfusions, provide symptomatic relief but do not offer a cure. Allogeneic hematopoietic stem cell transplantation (HSCT) is the only curative option available, but it is limited by donor availability and the risk of graft-versus-host disease (Park et al., 2019; Zarghamian et al., 2023).

Gene editing technologies, particularly CRISPR/Cas9, base editors, and homology-directed repair (HDR), have shown promising results in correcting the genetic mutation responsible for SCA (Hossain and Bungert, 2017; Newby et al., 2021; Rosanwo and Bauer, 2021). CRISPR/Cas9 has been used to disrupt regulatory elements that inhibit fetal hemoglobin (HbF) production, thereby compensating for defective adult hemoglobin (Rosanwo and Bauer, 2021; Zarghamian et al., 2023). Base editors, such as adenine base editors (ABE), have been employed to convert the sickle cell allele into a non-pathogenic variant, showing durable gene editing in preclinical models (Zeng et al., 2020; Newby et al., 2021). These technologies offer the potential for a one-time, autologous treatment that could eliminate the pathogenic HbS and generate benign hemoglobin variants (Newby et al., 2021; Germino-Watnick et al., 2022).

The study is to provide a comprehensive overview of the current advancements in gene editing technologies for the treatment of Sickle Cell Anemia. We discuss the various strategies employed, their efficacy and safety profiles, and the challenges that need to be addressed for clinical translation. By synthesizing findings from recent studies, this study seeks to highlight the transformative potential of gene editing as a universal curative option for SCA.

2 Overview of Sickle Cell Anemia

2.1 Genetic and molecular basis

Sickle cell anemia (SCA) is a hereditary blood disorder caused by a single nucleotide mutation in the beta-globin gene (HBB), resulting in the production of abnormal hemoglobin known as hemoglobin S (HbS) (DeWitt et al., 2016; Lin et al., 2017; Cisneros and Thein, 2020; Quagliano et al., 2022). This mutation leads to the polymerization of HbS under low oxygen conditions, causing red blood cells to deform into a sickle shape. These sickle-shaped cells are less flexible and can obstruct blood flow, leading to various complications (Figure 1) (DeWitt et al., 2016; Newby et al., 2021; Wilkinson et al., 2021). The discovery of the BCL11A gene, a major repressor of the γ-globin gene, has been pivotal in understanding the switch from fetal hemoglobin (HbF) to adult hemoglobin, providing new avenues for therapeutic intervention (Cisneros and Thein, 2020; Frangoul et al., 2020; Quagliano et al., 2022).

2.2 Pathophysiology and symptoms

The pathophysiology of SCA is primarily driven by the sickling of red blood cells, which leads to vaso-occlusion, hemolysis, and chronic inflammation (DeWitt et al., 2016; Frangoul et al., 2020). These processes result in severe pain crises, known as vaso-occlusive episodes, and progressive organ damage. Common symptoms include anemia, episodes of pain, swelling in the hands and feet, frequent infections, and delayed growth in children (Cisneros and Thein, 2020; Frangoul et al., 2020). The chronic hemolysis also leads to complications such as jaundice, gallstones, and an increased risk of stroke (Randolph and Zhao, 2015; DeWitt et al., 2016).

2.3 Epidemiology and global impact

Sickle cell anemia predominantly affects individuals of African, Mediterranean, Middle Eastern, and Indian ancestry, with the highest prevalence in sub-Saharan Africa (Romero et al., 2018; Frangoul et al., 2020). It is estimated that millions of people worldwide are affected by SCA, with approximately 300,000 infants born with the condition each year (Randolph and Zhao, 2015; Frangoul et al., 2020). The disease poses a significant public health challenge, particularly in low-resource settings where access to comprehensive care and curative treatments like hematopoietic stem cell transplantation (HSCT) is limited (Romero et al., 2018; Lin et al., 2019). The global burden of SCA includes high morbidity and mortality rates, with many patients experiencing a reduced quality of life and life expectancy (Romero et al., 2018; Frangoul et al., 2020).

3 Fundamentals of Gene Editing Technologies

Gene editing technologies have revolutionized the field of genetic research and therapy, offering unprecedented precision in modifying the genome. These technologies are particularly promising for treating genetic disorders such as sickle cell anemia (SCA), which is caused by a single point mutation in the beta-globin gene.

3.1 CRISPR-Cas9 technology

CRISPR-Cas9 is a groundbreaking gene editing technology that has transformed genetic research and therapeutic applications. The system consists of two key components: the Cas9 enzyme, which acts as molecular scissors to cut DNA, and a guide RNA (gRNA) that directs Cas9 to the specific location in the genome to be edited. This technology allows for precise modifications by creating double-strand breaks (DSBs) at targeted sites, which are then repaired by the cell's natural repair mechanisms, often leading to insertions or deletions (indels) (Ji, 2020; Uchida et al., 2021).

CRISPR-Cas9 has shown significant potential in treating sickle cell anemia by targeting the BCL11A gene, which represses fetal hemoglobin (HbF) expression. By disrupting this gene, researchers have successfully increased HbF levels, ameliorating the symptoms of SCA (Zeng et al., 2020; Uchida et al., 2021). However, the technology is not without its challenges, including off-target effects and the risk of unintended mutations (Ji, 2020; Newby et al., 2021).

3.2 Other Gene editing tools

In addition to CRISPR-Cas9, several other gene editing tools have been developed, each with unique mechanisms and applications. Transcription Activator-Like Effector Nucleases (TALENs) and Zinc Finger Nucleases (ZFNs) are two such technologies that predate CRISPR-Cas9. Both TALENs and ZFNs use engineered proteins to create DSBs at specific genomic locations, which are then repaired by the cell's natural mechanisms (Wilkinson et al., 2021).

While TALENs and ZFNs offer high specificity, they are more complex and time-consuming to design compared to CRISPR-Cas9. Nonetheless, they have been successfully used to correct the sickle cell mutation in hematopoietic stem cells, demonstrating their potential in treating genetic disorders (Wilkinson et al., 2021).

3.3 Base and prime editing

Base editing and prime editing are recent advancements in the field of gene editing that offer more precise and efficient modifications without creating DSBs. Base editors are fusion proteins that combine a deaminase enzyme with a CRISPR-Cas9 variant, allowing for the direct conversion of one DNA base to another. There are two main types of base editors: cytosine base editors (CBEs) and adenine base editors (ABEs), which enable C-to-T and A-to-G conversions, respectively (Kantor et al., 2020; Chu et al., 2021).

Prime editing is a more versatile tool that can introduce all twelve possible base-to-base conversions, as well as small insertions and deletions. Prime editors use a modified Cas9 enzyme fused to a reverse transcriptase, guided by a prime editing guide RNA (pegRNA) to specify the target site and the desired edit (Kantor et al., 2020). This technologyhas shown promise in correcting the sickle cell mutation with high precision and minimal off-target effects (George et al., 2022).

Both base and prime editing hold significant potential for treating sickle cell anemia by directly correcting the point mutation responsible for the disease. These technologies offer a safer and more efficient alternative to traditional gene editing methods, paving the way for new therapeutic strategies (Kantor et al., 2020; George et al., 2022). The advancements in gene editing technologies, particularly CRISPR-Cas9, base editing, and prime editing, have opened new avenues for the treatment of sickle cell anemia. These tools offer precise and efficient methods for correcting genetic mutations, bringing us closer to potential cures for this debilitating disease.

4 Applications of Gene Editing in Sickle Cell Anemia

4.1 Gene editing in hematopoietic stem cells

Gene editing in hematopoietic stem cells (HSCs) has emerged as a promising approach to treat sickle cell anemia (SCA). Various gene editing technologies, including CRISPR/Cas9 and base editors, have been employed to correct the mutation in the HBB gene responsible for SCA. For instance, CRISPR/Cas9 has been used to correct the sickle cell mutation in human HSCs, which maintained the edits in vivo and produced enough normal hemoglobin to potentially benefit patients with SCA (Zeng et al., 2020). Additionally, base editing has shown potential in converting the SCD allele into a non-pathogenic variant, resulting in significant reduction of sickling in erythroid progeny (Zeng et al., 2020; Newby et al., 2021). These approaches have demonstrated the feasibility of achieving therapeutic-level gene correction in HSCs, which can be engrafted and maintained over time in animal models (Zeng et al., 2020; Uchida et al., 2021).

4.2 Animal models of sickle cell anemia

Animal models, particularly mice, play a crucial role in the preclinical evaluation of gene editing strategies for SCA. Humanized sickle mouse models, which carry the human sickle hemoglobin gene, have been extensively used to study the efficacy of gene editing techniques. For example, a study using a humanized globin-cluster SCD mouse model demonstrated that CRISPR/Cas9-mediated HBB correction in HSCs resulted in stable hemoglobin-A production and normalized red blood cell features following autologous transplantation (Bak et al., 2018). These models allow researchers to assess the long-term effects of gene editing and the potential for clinical translation.

4.3 Successes in preclinical trials

Preclinical trials have shown promising results in correcting the HBB mutation in animal models. For instance, CRISPR/Cas9-mediated gene correction in SCD CD34+ cells achieved therapeutic-level gene correction at both DNA and protein levels, with corrected cells contributing to normal hemoglobin production in xenograft mouse models (Figure 2) (Uchida et al., 2021). Similarly, base editing of HSCs from SCD patients resulted in high-frequency conversion of the sickle allele to a non-pathogenic variant, significantly reducing hypoxia-induced sickling in mice (Bak et al., 2018).

Gene editing has also led to improvements in phenotype and survival rates in animal models of SCA. Studies have shown that gene-corrected HSCs can ameliorate the sickle phenotype, resulting in near-normal hematological parameters and reduced organ pathology. For example, lentiviral delivery of a human gamma-globin gene in a humanized sickle mouse model corrected the sickle phenotype and improved survival rates (Zeng et al., 2020). Additionally, CRISPR/Cas9-edited HSCs demonstrated long-term engraftment and persistence, contributing to improved erythropoiesis and reduced sickling in vivo (Bak et al., 2018; Wilkinson et al., 2021). Gene editing technologies have shown significant potential in correcting the HBB mutation and improving the phenotype and survival rates in animal models of SCA. These preclinical successes pave the way for future clinical trials and the potential for curative therapies for patients with sickle cell anemia.

5 Clinical Trials and Advances

5.1 Current clinical trials

Recent advancements in gene editing technologies have paved the way for promising clinical trials aimed at treating sickle cell anemia (SCA). One notable approach involves the use of viral transduction to introduce anti-sickling β-like globin genes into hematopoietic stem/progenitor cells (HSPCs). Early-phase clinical studies have shown promising preliminary results, indicating the potential of this method to provide a curative treatment for SCA (Zarghamian et al., 2023).

Another significant development is the use of CRISPR-Cas nucleases and base editors to reactivate γ-globin expression, which can replace the faulty β-globin chain. This approach has demonstrated efficacy in both preclinical animal models and clinical trials, showing promising results in terms of safety and effectiveness (Lin et al., 2019). Additionally, the use of zinc finger nucleases (ZFNs) to disrupt the BCL11A erythroid enhancer has been shown to increase fetal hemoglobin (HbF) levels in patients with SCA, with ongoing phase 1/2a clinical trials further evaluating this method (DeWitt et al., 2016; Rosanwo and Bauer, 2021) (Figure 3). Moreover, the investigational gene-edited autologous hematopoietic stem cell medicine, EDIT-301, which employs AsCas12a to edit the HBG1/2 promoters, has shown rapid and sustained normalization of hemoglobin levels and increased HbF production in patients with severe SCA and transfusion-dependent beta-thalassemia (TDT). These clinical trials highlight the potential of gene editing technologies to provide a curative treatment for SCA (Figure 3) (Rahimmanesh et al., 2022).

5.2 Case studies

Several case studies have demonstrated the potential of gene editing technologies in treating SCA, For instance, a study by DeWitt et al. (2016) utilized CRISPR/Cas9 to edit hematopoietic stem cells from SCA patients, resulting in successful engraftment in a mouse model and the production of normal hemoglobin, indicating potential clinical benefits (Ikawa et al., 2019; Newby et al., 2021). Another case study involved the use of a custom adenine base editor (ABE8e-NRCH) to convert the SCD allele into a non-pathogenic variant, resulting in significant reduction of hypoxia-induced sickling and near-normal hematological parameters in mice (Cisneros and Thein, 2020). A study on the use of base editing to upregulate fetal hemoglobin (HbF) in CD34+ HSPCs showed that editing at key regulatory motifs within the HBG1 and HBG2 promoters resulted in significant increases in HbF levels, providing protection to the majority of SCA patients (Moran et al., 2018). These case studies underscore the transformative potential of gene editing technologies in providing a curative treatment for SCA (Figure 4) (Cisneros and Thein, 2020). The ongoing clinical trials and case studies highlight the significant advancements in gene editing technologies for the treatment of sickle cell anemia. These approaches offer promising prospects for providing a universal curative option for patients with SCA, with ongoing research and clinical trials continuing to refine and optimize these treatments (DeWitt et al., 2016; Moran et al., 2018; Lin et al., 2019; Cisneros and Thein, 2020; Newby et al., 2021; Hanna et al., 2023; Zarghamian et al., 2023).

6 Challenges and Barriers to Implementation

6.1 Technical challenges

Gene editing technologies, particularly CRISPR-Cas9, have shown promise in treating sickle cell anemia (SCA) by targeting specific genetic mutations. However, several technical challenges remain. One significant issue is the efficiency and specificity of gene modification. Achieving high editing efficiency without off-target effects is crucial for clinical success (Lin et al., 2017; Ikawa et al., 2019; Frangoul et al., 2020). Additionally, the delivery of gene-editing tools to hematopoietic stem cells (HSCs) and ensuring their robust and non-toxic engraftment pose significant hurdles (Rosanwo and Bauer, 2021). The optimization of these processes is essential to maximize therapeutic benefits while minimizing potential risks (Ebens et al., 2019; Lin et al., 2019).

6.2 Ethical and regulatory issues

The ethical implications of gene editing are profound, particularly concerning germline modifications that could be inherited by future generations. While current therapies focus on somatic cells, the potential for germline editing raises concerns about unintended consequences and long-term effects (Romero et al., 2018; Rahimmanesh et al., 2022). Regulatory frameworks must evolve to address these ethical considerations, ensuring that gene editing technologies are applied responsibly and ethically. Moreover, the approval process for new gene therapies is complex and requires rigorous evaluation to ensure safety and efficacy (Urnov, 2017).

6.3 Socioeconomic barriers

The high cost of gene editing therapies presents a significant barrier to widespread implementation. Current ex vivo HSC gene therapy platforms are expensive, limiting access to treatment, especially in regions where SCA is most prevalent (Lin et al., 2019). Ensuring equitable access to these advanced therapies requires addressing the economic disparities that exist in healthcare systems globally. Additionally, the infrastructure needed to support gene therapy, including specialized facilities and trained personnel, is not universally available, further exacerbating access issues (Romero et al., 2018).

6.4 Regulatory and safety evaluations

Regulatory and safety evaluations are critical to the successful implementation of gene editing technologies. Ensuring that edited cells do not cause adverse effects, such as insertional mutagenesis or off-target gene disruptions, is paramount (DeWitt et al., 2016; Lin et al., 2017). Comprehensive in vivo safety and toxicology studies are necessary to evaluate the long-term effects of gene editing (Ebens et al., 2019). Regulatory bodies must establish clear guidelines and robust monitoring systems to oversee the clinical application of these therapies, ensuring that they meet stringent safety and efficacy standards (Urnov, 2017). While gene editing technologies hold great promise for treating sickle cell anemia, addressing the technical, ethical, socioeconomic, and regulatory challenges is essential for their successful and equitable implementation. Continued research and collaboration among scientists, ethicists, policymakers, and healthcare providers are crucial to overcoming these barriers and realizing the full potential of gene editing in curing SCA.

7 Future Perspectives

7.1 Technological advancements

The future of gene editing technologies in treating sickle cell anemia (SCA) is promising, with several advancements on the horizon. CRISPR/Cas9 has shown high efficiency in editing human primary hematopoietic stem and progenitor cells (HSPCs) to upregulate fetal hemoglobin (HbF), which can ameliorate the symptoms of SCA (Lin et al., 2017; Frangoul et al., 2020). Additionally, base editing technologies, such as cytosine and adenine base editors, offer precise genome modifications without introducing double-strand breaks, which can further enhance the safety and efficacy of gene therapies (Lin et al., 2019; Zeng et al., 2020). These advancements are crucial for achieving high editing efficiencies and ensuring the long-term viability and functionality of edited cells (Lin et al., 2017; Zeng et al., 2020).

7.2 Combining therapies

Combining gene editing with other therapeutic approaches holds significant potential for treating SCA. For instance, integrating gene editing with viral transduction techniques can enhance the overall therapeutic outcome by ensuring robust and sustained expression of therapeutic genes (Romero et al., 2018; Quintana-Bustamante et al., 2022). Moreover, the combination of base editing and CRISPR/Cas9 technologies can target multiple genetic loci simultaneously, thereby increasing the therapeutic efficacy and reducing the disease burden in patients (Lin et al., 2019; Zeng et al., 2020). This multi-faceted approach can address the limitations of single-modality treatments and provide a more comprehensive solution for SCA (Hossain and Bungert, 2017; Romero et al., 2018).

7.3 Long-term vision

The long-term vision for gene editing in SCA involves not only curing the disease but also ensuring the accessibility and affordability of these therapies. As gene editing technologies advance, it is essential to optimize the delivery methods, improve the specificity and efficiency of gene modifications, and ensure the safety of edited cells (Hossain and Bungert, 2017). Additionally, addressing the challenges of implementing these therapies in regions where SCA is endemic is crucial for making these treatments universally available (Romero et al., 2018). Ultimately, the goal is to develop a universal curative option for SCA that can be safely and effectively administered to all patients, regardless of their geographic or socioeconomic status (Romero et al., 2018).

8 Concluding Remarks

Gene editing technologies have shown significant promise in the treatment of sickle cell anemia (SCA). Various approaches, including CRISPR/Cas9, base editing, and gene addition/editing, have demonstrated the potential to correct the genetic mutation responsible for SCA or to induce the expression of fetal hemoglobin (HbF) to compensate for defective adult hemoglobin. Studies have reported successful ex vivo editing of hematopoietic stem/progenitor cells (HSPCs) and their subsequent engraftment in animal models, leading to reduced sickling of red blood cells and improved hematological parameters.

Future research should focus on optimizing the efficiency and specificity of gene editing techniques to minimize off-target effects and ensure long-term safety and efficacy. Additionally, addressing the challenges of delivering gene-edited cells to patients, particularly in regions where SCA is endemic, is crucial. Further studies are needed to refine the protocols for clinical-scale production of gene-edited HSPCs and to evaluate the long-term outcomes of these therapies in clinical trials.

The advancements in gene editing technologies offer a transformative potential for the treatment of SCA. The ability to correct the underlying genetic defect or to induce HbF expression provides a promising curative approach. The durability and effectiveness of these treatments in preclinical models and early-phase clinical trials are encouraging, suggesting that gene editing could become a universal curative option for SCA in the near future. Continued research and clinical development will be essential to bring these therapies to patients worldwide, offering hope for a definitive cure for SCA.

Acknowledgments

Thank you to the two anonymous peer reviewers for their feedback on the manuscript.

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.