CRISPR-Cas9 Genomic Editing as an Innovation in the Management of Sickle Cell Disease: A Systematic Review

Genomic editing is a group of technologies that scientists have used to alter an organism’s DNA. Of the several genomic editing techniques, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) is well known. The CRISPR-Cas9 system is faster, cheaper, more accurate, more efficient than other genomic editing methods, and it is an adaptation from bacteria’s immune mechanism. Sickle cell diseases (SCDs) are a group of monogenic diseases, and despite their high prevalence and chronic debilitating nature, they continue to have few therapeutic options available. The aim of this study is to review existing literature and current clinical trials on CRISPR-Cas9 genomic editing as an innovation in the management of sickle cell disease (SCD), as well as the current state of treatment for SCD. For this systematic review, PubMed, Google Scholar, African Journals Online (AJOL), and Clinicaltrial.gov articles published up to 6th October, 2022 were searched. Searches for current clinical trials using CRISPR-Cas9 as intervention were conducted by using the search terms such as sickle cell disease, genomic editing, genetics, novel treatments, hematopoietic stem cell transplantation, gene therapy, and CRISPR-Ca9. Studies cited include meta-analyses, original research, prospective clinical trials, online abstracts, literature reviews, retrospective studies, case series, and scientific meetings. The primary search obtained 27,678 articles. Following a review of titles and abstracts, a total of 32 publications and 6 ongoing clinical trials were included in this systematic review based on the recent evidence-based management of SCD. CRISPR-Cas9 genomic editing stands out as a novel, innovative technology which has the potential to cure SCD in children and adults with minimal side effects. Six clinical trials are ongoing with a huge potential for scaling up to Phases 3 and 4.


INTRODUCTION
Genetic medicine is a newer terminology for medical genetics and incorporates areas such as gene therapy. Recent advances in medical genetics are revealing etiologies for morphologic, endocrine, cardiovascular, pulmonary, ophthalmologic, renal, psychiatric, and dermatologic conditions (Ikwuka, 2023a). Sickle cell disease (SCD) encompasses a group of blood disorders resulting from inheriting two mutated copies of the β-globin gene (HBB) from both parents (Frangoul, 2021). HBB, located on chromosome 11p15.5, encodes the β-chain of hemoglobin (Onda, 2005). In normal circumstances, humans have three types of hemoglobin: hemoglobin A (consisting of 2α and 2β chains), hemoglobin A2 (made up of 2α and 2δ chains), and hemoglobin F (HbF) composed of 2α and 2γ chains (Hall, 2020). In sickle cell anemia, the most prevalent form of SCD, red blood cells (RBCs) contain an abnormal hemoglobin variant called hemoglobin S, wherein each of the two β-chains has a specific mutation (an amino acid valine with codon GTG substitutes glutamic acid with codon GAG at position 6) (Hall, 2020). Thus, the genetics of SCD (a hemoglobinopathy) is due to the substitution of valine for glutamic acid at position 6 of both β-globin polypeptide chains. Hemoglobin F persists until about 6 weeks of age. Thereafter, hemoglobin A persists throughout life.
The mode of inheritance of SCD is autosomal recessive (Ikwuka, 2023b). Hemoglobin, a protein responsible for oxygen transport in red blood cells (RBCs), is synthesized during erythropoiesis (initiated in polychromatophil erythroblasts and continuing through the reticulocyte stage) (Hall, 2020). Erythropoiesis is stimulated and enhanced by erythropoietin, a substance synthesized by the kidneys. Results from different studies have shown that high levels of blood pressure, glucose and lipid metabolic disorders, asymptomatic hyperuricemia, activation of systemic immune inflammation and fibrogenesis, contribute to kidney damage (Ikwuka, 2015;Ikwuka, 2017a;Ikwuka, 2017c;Ikwuka, 2017d;Ikwuka, 2017e;Ikwuka, 2018d;Ikwuka, 2019a;Ikwuka, 2019c;Ikwuka, 2022;Ikwuka, 2023d;Ikwuka, 2023e;Virstyuk, 2016;Virstyuk, 2017a;Virstyuk, 2018a;Virstyuk, 2021a;Virstyuk, 2021b), which contributes to anemia on account of disturbed erythropoietin production. However, Dapagliflozin which is a Sodium-Glucose Linked Transporter 2 (SGLT-2) inhibitor and Liraglutide which is a Glucagon-like Peptide 1 Receptor Agonist (GLP-1 RA) have been found to increase the effectiveness of treatment and improve the clinical course of disease in patients with such comorbidities (Ikwuka, 2017b;Ikwuka, 2018a;Ikwuka, 2018b;Ikwuka, 2018c;Ikwuka, 2019b;Virstyuk, 2018b;Virstyuk, 2018c;Virstyuk, 2017b), https://journals.e-palli.com/home/index.php/ajmsi Am. J. Med. Sci. Innov. 2(2) 36-48, 2023 thereby reducing kidney damage, improve kidney functions, and reduce anemia. When hemoglobin S encounters low oxygen levels, it forms long crystals (sometimes 15 micrometers in length) within RBCs, impeding their flow through narrow capillaries. The pointed ends of these crystals can rupture the plasmalemma leading to sickle cell anemia (Hall, 2020). Major free radicals that are of physiological significance are superoxide anion, hydroxyl radical, and hydroperoxyl radical, while non-radical is hydrogen peroxide (Ikwuka, 2023c). Rauwolfia vomitoria has a neuroprotective ability at it elevates antioxidants and suppresses lipid peroxidation (Ekechi, 2023). It is noteworthy that nearly two-thirds of infants worldwide with HbSS or SCD are born in Nigeria, the Republic of Congo, or India, where the childhood mortality rate associated with SCD remains alarmingly high (Piel, 2013). Symptoms and complications of SCD typically manifest around 5 to 6 months of age when fetal hemoglobin (HbF) synthesis significantly declines. These symptoms include severe anemia, episodes of pain (referred to as sickle cell crisis), swelling in the hands and feet, and potential complications such as bacterial infections and stroke (Frangoul, 2021;Hall, 2020). Long-term pain can develop as individuals grow older, and the average life expectancy in developed countries ranges from 40 to 60 years (National Heart, Lung, and Blood Institute, 2015). Newborn screening is the common diagnostic approach for identifying HbSS, and treatment options include penicillin (essential for children under five years with immature immune systems), folic acid supplementation, blood transfusions, vaccinations against encapsulated organisms, transcranial Doppler (TCD) screening to identify stroke risk in children (followed by blood transfusions, if necessary), pain management, hydroxyurea, and intensive hospital-based care (Adams, 1998;Gaston, 1986;Thornburg, 2012;WHO, 2011). Other diagnostic tests for SCD include sickling of the red blood cells on a blood film which is induced by the addition of sodium metabisulfite; and another test referred to as hemoglobin electrophoresis which detects abnormal hemoglobin forms (Ikwuka, 2023b). However, despite the significant need for effective treatment options for SCD patients, current treatments both traditional and newly developed, only ameliorate acute and chronic SCD manifestations without addressing the underlying cause. Hydroxyurea and long-term blood transfusions aim to prevent and treat complications associated with SCD. The recently approved crizanlizumab (Ataga, 2017) has shown a reduced incidence of cellular adhesion and vaso-occlusive crisis in SCD patients, but it does not target the root cause of the disease or fully alleviate its manifestations (Frangoul, 2021). Allogeneic hematopoietic stem cell transplantation (HSCT) remains the sole curative option for SCD, yet less than 20% of eligible patients have a suitable HLA-matched donor (Baronciani, 2016;Eapen, 2019;Gluckman, 2017). Further advances in the understanding of the pathophysiology of SCD contributed to the development of the exciting and novel "Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (CRISPR-Cas9)" genomic editing therapy to cure the disease and its complications. Bacteriophages have the ability to infect bacteria by implanting its genetic material into the bacterial genome (Chaudhary, 2020). Thereafter, bacteria have a natural defense mechanism against bacteriophages, whereby on the first exposure to a bacteriophage they produce CRISPR sequence as a form of genetic memory. CRISPR sequence is always found in association with the Cas9, a nuclease that can cleave the DNA. In subsequent exposure to a similar bacteriophage, the bacteria form guideRNA from the transcription of the CRISPR sequence. The guideRNA finds its target in the bacteriophage DNA, while the Cas9 cleaves the DNA (Chaudhary, 2020). The advantage of this system is that once the CRISPR system has cleaved the DNA, a DNA template carrying the desired sequence can join the cleaved end, thereby facilitating recombination and replacement of the original sequence with the new version. The CRISPR-Cas9 nuclease system can be employed in cultured cells, including stem cells, as well as in fertilized eggs, enabling the generation of transgenic animals with targeted mutations. This genomic editing technique has been extensively studied in various organisms such as yeast, Drosophila, Zebrafish, plants, monkeys, and pigs, in addition to the bacteria from which the technique was originally derived from (Wen, 2017). In the case of SCD, the CRISPR-Cas9 nuclease system is applied to hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of the BCL11A locus on chromosome 2 (Uda, 2008). Normally, BCL11A encodes a transcription factor that inhibits HbF synthesis. The CRISPR-Cas9 nuclease system effectively suppresses BCL11A expression in erythroid-lineage cells, thereby restoring γ-globin synthesis and reactivating HbF production (Canver, 2015;Wu, 2019). Unlike previous genomic editing methods, CRISPR-Cas9 has the capacity to target multiple genes simultaneously, enabling the treatment of not only diseases with point mutations but also those with polygenic mutations. Researchers have recently realized that this system can be engineered to cleave DNA at precisely chosen loci, extending beyond viral DNA to any desired DNA sequence, simply by modifying the guideRNA to match the target (Chaudhary, 2020). In this systematic review, a comprehensive analysis of the current state of research on CRISPR-Cas9 for the treatment of sickle cell disease (SCD) was conducted. By gathering information from multiple sources, evaluation of the progress made with this innovative technology is determined, identified knowledge gaps for further research are checked, and the technology's potential challenges and limitations are discussed.

METHODOLOGY Search Strategy and Selection Criteria
This systematic review aimed to study all available literature on CRISPR-Cas9 genomic editing and its potential in the management of SCD. It also sought to shed more light on Am. J. Med. Sci. Innov. 2(2) 36-48, 2023 the subject matter (considering the fact this technology is novel) and its stage of development is still in the clinical trials. A similar method of literature search as described by Suwito, et al, 2023was used (Suwito, 2023. The literature search was done from the following databases over a period of 2 weeks: PubMed (mostly used), Google Scholar, and African Journals Online (AJOL) using the following terms: CRISPR-Cas9, genomic editing, gene editing, sickle cell disease, hemoglobinopathies, sickle cell anemia, genetic therapy, systematic review, new therapy/novel intervention for sickle cell disease cure/treatment.

Data Sources and Search Engines
A literature search was done from the following databases: PubMed (mostly used), Google Scholar, and African Journals Online (AJOL). While the clinical trials search was done on ClinicalTrials.gov.

Inclusion and Exclusion Criteria
Included in this study were studies that investigated the use of CRISPR-Cas9 genomic editing as a treatment for sickle cell disease, studies that included human participants or human cells/tissues, studies that provided data on the efficacy and/or safety of CRISPR-Cas9 genomic editing for sickle cell disease, and studies that were published in English language within the past 10 years on gene therapy use in SCD, specifically CRISPR-Cas9. Excluded articles were studies not related to CRISPR-Cas9 genomic editing or sickle cell disease, studies that used animal or plant models only, studies not published in English, studies that did not provide data on the efficacy and/or safety of CRISPR-Cas9 genomic editing for sickle cell disease, and studies that had poor methodological quality or a high risk of bias.

Quality Assessment of Included Studies
The articles from the database search were reviewed to tailor them to the inclusion criteria. The abstracts of the articles that met the inclusion criteria were reviewed for relevant keywords. The abstracts and the free complete articles i.e. manuscripts for the selected articles were then read, reviewed, and the information on each of the key areas were summarized. This systematic review was carried out independently by four persons, to minimize errors. The summarized data were later compiled, reviewed, and discussed.

Data Extraction, Synthesis, and Results
The following keywords were used to extract articles from database searches: • "CRISPR-Cas9" • "Genomic editing" • "Sickle cell disease, hemoglobinopathies, and sickle cell anemia" • "Genetic therapy and gene editing" • "Systematic literature review or systematic review" • "New therapy/novel intervention for sickle cell".

Study Selection and Characteristics
The search for articles and abstracts was done using

Figure 1:
Steps of article selection keywords on the three major search engines (PubMed, Google Scholar, and AJOL). However, the mostly used search engine was PubMed due to its advanced features and its large repository of articles. Study selection was based on articles, abstracts, or literature reviews which meet the inclusion criteria. Articles that were found under exclusion criteria were discarded. The diagram below illustrates how articles were selected.

RESULTS
The findings in this study are outlined in Tables 1 and 2.

DISCUSSION
This systematic literature review provides an overview of CRISPR-Cas9 genomic editing and its application to sickle cell disease (SCD), while also addressing the ethical implications associated with this technology in SCD management. Despite the wide use of CRISPR-Cas9 as a mature genomic editing tool, therapeutic applications still face challenges such as off-target effects, complex in vivo Cas9 protein delivery, low gene editing efficiency, and packaging issues. To become an ideal delivery method for therapeutics, CRISPR-Cas9 strategies should exhibit high delivery efficiency, precise targeting ability, and ease of mass production. However, current approaches in this field are far from achieving this desired level of performance (Guo, 2022). While there is significant literature on why SCD is a suitable candidate for CRISPR-Cas9, less attention has been given to the ethical implications of including SCD in CRISPR-Cas9 research. In addition, the implications of CRISPR-Cas9 for sickle cell disease have significant consequences for clinical practice and policy. The following points highlight some of the potential implications:

Improved Outcomes
CRISPR-Cas9 holds the potential to cure SCD by correcting the underlying genetic mutation. This breakthrough could lead to improved outcomes for patients, including reduced pain, enhanced quality of life, and increased lifespan.

Ethical Considerations
The use of CRISPR-Cas9 in humans raises ethical concerns regarding safety and the possibility of unintended consequences. The development of policies is necessary to ensure the ethical and reliable application of CRISPR-Cas9.

Access to Treatment
Issues related to access to CRISPR-Cas9 treatment for SCD patients may arise, particularly in low-and middleincome countries. Policies should be developed to ensure equitable access to the benefits of this technology for all patients in need.

CONCLUSION
After analyzing the available evidence on the use of CRISPR-Cas9 for the management of SCD, it can be concluded that this technology is novel and shows promise as a potential therapeutic option for the condition. Studies have demonstrated the successful correction of the genetic mutation responsible for SCD in clinical settings. One of the main challenges of this technology is the delivery of the CRISPR-Cas9 system to the bone marrow, where the hematopoietic stem cells reside. The off-target effects of the CRISPR-Cas9 system also need to be further studied and minimized to ensure the safety of the treatment. Despite these challenges, the potential benefits of CRISPR-Cas9 for SCD cannot be neglected. The ability to correct the genetic mutation responsible for the condition offers a potentially curative approach to the disease. Overall, further research and clinical trials are necessary to fully evaluate the safety and efficacy of CRISPR-Cas9 as a therapeutic option for SCD. Nevertheless, the current evidence suggests that CRISPR-Cas9 has the potential to revolutionize the treatment of this debilitating disease by offering a curative option.