Int J Pharm Pharm Sci, Vol 16, Issue 9, 1-7Review Article

CRISPR-CAS9-MEDIATED EX VIVO GENE EDITING FOR INHERITED HEMATOLOGICAL DISORDERS: ADVANCEMENTS, CHALLENGES, AND CLINICAL POTENTIAL

ATASI RANJAN PANDA1*, SHREEYA DAS2

¹JSS College of Pharmacy, M. Pharm Department of Pharmaceutics, JSS Academy of Higher Education and Research, Mysuru-570015, Karnataka, India. ²Department of Biotechnology, University Institute of Biotechnology, Chandigarh University, Mohali, Punjab, India
*Corresponding author: Atasi Ranjan Panda; Email: atasiranjanoandaniku@gmail.com

Received: 03 Apr 2024, Revised and Accepted: 10 Jul 2024

ABSTRAC

Global healthcare systems have a great challenge in the form of inherited hematological diseases, which necessitates the development of new remedial strategies. By precisely targeting inherited abnormalities, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated ex vivo gene editing has surfaced as a promising approach to treat these diseases. This review offers a comprehensive examination of the advancements, challenges, and clinical eventuality of CRISPR-Cas9-intermediated ex vivo gene editing for inherited hematological diseases. With advancements in CRISPR-Cas9 technology, the eventuality to correct inheritable mutations responsible for inherited hematological diseases is within reach. However, challenges such as off-target effects, immune responses, and ethical considerations need to be addressed for the safe and effective perpetration of this technology. A promising understanding of how CRISPR-Cas9-intermediated gene editing functions in practice is handed by ongoing clinical studies, giving rise to the possibility of advanced remedial approaches and bettered patient issues. By addressing these complications in a human-readable format, this review attempts to provide greater understanding and appreciation for the eventuality of CRISPR-Cas9 technology in revolutionizing the treatment landscape for these challenging disorders and contribute to the ongoing discussion in the field and facilitate further exploration towards effective treatments for these challenging disorders.

Keywords: CRISPR-Cas9, Gene editing, Inherited hematological disorders, Precision medicine, Ex vivo interventions, Genetic mutations, Clinical trials, Off-target effects, Immune responses, Therapeutic advancements

© 2024 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijpps.2024v16i9.51048 Journal homepage: https://innovareacademics.in/journals/index.php/ijpps

INTRODUCTION

In the realm of healthcare, inherited hematological conditions, like sickle cell disease and beta-thalassemia, pose significant health complications globally due to inheritable abnormalities affecting hemoglobin production [1, 2]. An aberrant hemoglobin pattern, which gives red blood cells their distinctive sickle shape and makes them stiff, is reflective of sickle cell diseases. This condition increases the risk of infections, organ damage, and painful occurrences [3]. Conversely, beta-thalassemia results in inadequate erythropoiesis, anemia, and complications such as bone deformities and organ damage due to diminished or nonexistent beta-globin chain synthesis. Traditional treatments focus on symptom management and supportive care, such as blood transfusions and medications, but do not address the underlying genetic defects, necessitating lifelong management [4–6].

Amidst the limitations of traditional treatments, Clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR-associated protein 9 (Cas9) technology, derived from a bacterial immune system has surfaced as a promising frontier for addressing inherited blood diseases like sickle cell disease and beta-thalassemia [5–8]. Its precision in editing DNA sequences offers the potential to correct genetic mutations underlying these conditions, shifting treatment focus from symptom management to addressing the root cause. Understanding the genetic mechanisms driving these disorders, from mutations in the beta-globin gene causing abnormal hemoglobin in sickle cell disease to disruptions in hemoglobin production due to mutations in the Hemoglobin Subunit Beta (HBB) gene in beta-thalassemia, is crucial for guiding targeted interventions [9, 10].

At the forefront of gene editing technology, CRISPR-Cas9 offers unequaled precision, efficiency, and scalability, making it ideally suited for ex vivo gene editing operations in inherited blood disorders. This approach involves editing patients' Hematopoietic Stem Cells (HSCs) outside the body before reintroduction, potentially offering long-term therapeutic benefits and even cures [11–13]. In this review, we delve into the advancements, challenges, and clinical potential of CRISPR-Cas9-mediated ex vivo gene editing for inherited hematological disorders. Our objectives include providing a comprehensive overview of CRISPR-Cas9 technology, examining recent advances in gene editing strategies, and evaluating efficacy and safety through preclinical and ongoing clinical trials. By addressing challenges and limitations, we aim to enhance understanding and pave the way for future innovations in precision medicine, ultimately offering restorative cures and hope for patients' brighter, healthier futures [14-16].

Advancements in crispr-Cas9 technology

Evolution of CRISPR-Cas9: from discovery to application

The journey of CRISPR-Cas9 technology, originating from bacterial vulnerable systems like Streptococcus pyogenes, has been marked by significant mileposts [17]. The first major advance in the evolution of CRISPR-Cas9 gene editing came with the adaption of the system for use in eukaryotic cells, enabling precise targeting of DNA sequences in mammalian cells and revolutionizing genetic research [18]. Subsequent advancements, such as the engineering of high-fidelity Cas9 variants and the introduction of smaller Cas9 orthologs and alternative CRISPR systems like CRISPR from Prevotella and Francisella 1 (CPF1), have expanded the gene-editing toolkit, enhanced precision and effectiveness while minimizing off-target effects [19-21].

Molecular mechanisms of CRISPR-Cas9 editing

At the heart of CRISPR-Cas9 technology lies a significant molecular tool capable of precisely targeting and modifying specific regions of the genome [22, 23]. It consists of two main factors the Cas9 protein and a guide RNA (gRNA). The Cas9 protein acts as a molecular scissor, cutting double-stranded DNA at specific target sequences guided by the gRNA through base pairing. Initially, the gRNA recognizes and binds to a specific DNA sequence adjacent to a Protospacer Adjacent Motif (PAM) sequence. Once bound, Cas9 induces a Double-Stranded Break (DSB) in the DNA, triggering the cell's natural DNA repair mechanisms such as Non-homologous End Joining (NHEJ) and Homology-Directed Repair (HDR) [24–26].

Fig. 1: Schematic diagram illustrating the molecular mechanism of CRISPR-Cas9-mediated gene editing, including guide RNA (gRNA) targeting, Cas9 nuclease activity, and DNA repair mechanisms. (Source: Abdelnour SA, Xie L, Hassanin AA, Zuo E, Lu Y. The potential of CRISPR/Cas9 gene editing as a treatment strategy for inherited diseases. Vol. 9, frontiers in cell and developmental biology. Frontiers media S. A.; 2021) [27]

The DNA repair process following Cas9-mediated cleavage can lead to the introduction of desired genetic modifications, such as gene knockouts, insertions, or replacements. By precisely targeting specific DNA sequences, CRISPR-Cas9 enables researchers to manipulate the genome with unprecedented delicacy and effectiveness [28, 29].

Recent Innovations in CRISPR-Cas9 editing techniques

In the fast-paced world of genetic engineering, recent advancements have expanded the capabilities of CRISPR-Cas9 for precise genome editing. One significant advancement is the development of base editing and prime editing techniques, which enable targeted nucleotide substitutions or precise insertions and deletions without double-stranded breaks, reducing off-target effects [30–32]. Improvements in CRISPR-Cas9 specificity include high-fidelity Cas9 variants and modified gRNAs, enhancing safety and efficacy. Additionally, novel approaches, such as CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa), enable precise regulation of gene expression without altering the underlying DNA sequence [33]. One of the pivotal challenges in employing the full eventuality of CRISPR-Cas9 for remedial purposes lies in delivering the editing ministry to target cells effectively [34, 35]. This has led to the development of various delivery systems, including viral vectors such as Adeno-Associated Viruses (AAVs) and lentiviruses, known for efficiently transducing various cell types, including hematopoietic stem cells. Non-viral delivery methods, such as lipid nanoparticles and electroporation, offer alternative strategies for delivering CRISPR-Cas9 factors, potentially reducing the risk of immune responses and off-target effects [36–39]. Optimization of delivery systems aims to enhance the clinical feasibility of CRISPR-Cas9-mediated ex vivo gene editing for inherited hematological diseases. Continuous innovation and enhancement have driven the evolution of CRISPR-Cas9 technology, leading it to evolve from an introductory exploration tool to a promising therapeutic approach [40, 41].

CRISPR Cas9 ex vivo gene editing strategies for inherited hematological disorders

Inherited hematological disorders encompass a diverse spectrum of genetic conditions affecting the production, structure, or function of blood cells, often resulting in significant morbidity and mortality. Examples include sickle cell disease, thalassemia, hemophilia, and various types of anemia. These diseases are generally caused by mutations in genes involved in hematopoiesis, hemoglobin synthesis, or coagulation pathways. Manifestations can range from mild to severe, with symptoms similar as chronic anemia, pain crises, organ damage, and increased vulnerability to infections or bleeding [42, 43].

Table 1: Summary of genetic mutations associated with inherited blood disorders

S. No. Inherited blood disorder Affected gene Type of mutation Phenotype Reference
1. Sickle Cell Disease HBB Missense Hemolytic Anemia, Vaso-Occlusive Crises, Beta-Thalassemia [44]
2. Beta-Thalassemia HBB Nonsense Microcytic anemia, hepatosplenomegaly [12]
3. Hemophilia A F8 Insertion/Deletion Prolonged bleeding, joint deformities [44]
4. Hemophilia B F9 Missense Prolonged bleeding, spontaneous hemorrhage [44]
5. Fanconi Anemia FANCA Nonsense Bone marrow failure, developmental abnormalities [9]
6. G6PD Deficiency G6PD Deletion Hemolytic anemia, jaundice [45]
7. Alpha-Thalassemia HBA Deletion Hemolytic anemia, skeletal abnormalities [33]
8. Thrombocytopenia MPL Frameshift Low Platelet Count, Easy Bruising [46]
9. Thalassemia Intermedia HBB Insertion Anemia, Fatigue [47]

Fig. 2: EX vivo vs. in vivo strategies for therapeutic CRISPR genome editing (Source: Li R, Wang Q, She K, Lu F, Yang Y. CRISPR/Cas systems usher in a new era of disease treatment and diagnosis. Vol. 3, Molecular Biomedicine. (Springer; 2022) [54]

Inherited blood diseases pose significant challenges for affected individuals and their families, challenging lifelong operation and care. Advances in inheritable testing, prenatal screening, and treatment modalities, including gene remedy and HSCs transplantation, offer hope for improved outcomes. CRISPR-Cas9 technology holds a significant pledge for the treatment of inherited hematological diseases through precise gene editing [48–50]. In the environment of these diseases, similar to sickle cell complaint and beta-thalassemia, CRISPR-Cas9 can be utilized in various ways to correct underlying genetic mutations. One operation involves in vivo gene editing, where CRISPR-Cas9 is directly delivered into the patient's body to target specific cells or tissues affected by the disorder. Another approach is ex vivo gene editing, which entails modifying hematopoietic stem cells or T cells outside the body before reintroduction [51, 52]. While ex vivo strategies offer greater control over editing effectiveness and particularity, in vivo approaches offer the advantage of simplicity and eventuality broader applicability [44, 53].

With a focus on modifying HSCs, ex vivo gene editing, which leverages the potential of CRISPR-Cas9 technology, presents a possible treatment option for hereditary anemia disorders. These HSCs, obtained from the patient's peripheral blood or bone marrow, are targeted due to their ability to generate all blood cell types. CRISPR-Cas9 enables precise modification of specific genomic areas within these HSCs, allowing for the correction of underlying genetic mutations responsible for conditions like sickle cell disease and beta-thalassemia [55-57]. Frequently, mutations in particular genes essential for hematopoiesis and hemoglobin synthesis result in inherited hematological diseases. The HBB gene, encoding the beta-globin subunit of hemoglobin, is a primary target for ex vivo gene editing in conditions like sickle cell disease and beta-thalassemia, where mutations lead to abnormal hemoglobin structure or reduced production [46, 47]. To achieve successful editing of the HBB gene, various strategies are employed. These could involve the insertion of therapeutic transgenes, the removal of disease-causing regions, or the use of CRISPR-Cas9-mediated homology-directed repair. By targeting these key genes and customized editing techniques, scientists aim to restore normal hematopoiesis and alleviate symptoms. With the precision of CRISPR-Cas9 technology, ex vivo gene editing holds promise for individualized and effective treatments for these challenging diseases, offering hope for improved outcomes and a higher quality of life for patients worldwide [58, 59].

Clinical potential and therapeutics applications

Preclinical studies serve as pivotal stepping monuments in assessing the eventuality of CRISPR-Cas9-intermediated gene editing for inherited hematological diseases, such as sickle cell disease and beta-thalassemia. In animal models, such as mice and non-human primates, experimenters have successfully employed CRISPR-Cas9 to precisely target and edit defective genes, restoring normal hematological parameters and alleviating disease symptoms [60]. A significant preclinical study showed that CRISPR-Cas9 could potentially fix the sickle cell mutation in a mouse model of sickle cell disease. In this work, researchers effectively edited the appropriate genetic sequence in hematopoietic stem cells using CRISPR-Cas9, leading to the creation of functional red blood cells with normal hemoglobin levels. These edited cells were then transplanted back into the mice, leading to a significant reduction in sickling and enhancement in overall health. Similarly, in a rat model of beta-thalassemia, CRISPR-Cas9-mediated gene editing increased normal hemoglobin synthesis and reduced symptoms [61, 62]. Another noteworthy preclinical work focused on the successful delivery of CRISPR-Cas9 components into hematopoietic stem cells derived from non-human primates, leading to the production of healthy blood cells and bone marrow replenishment [63, 64]. These preclinical studies emphasize the versatility and efficacity of CRISPR-Cas9-intermediated gene editing across different animal models, pressing its eventuality as a transformative remedial strategy for inherited hematological diseases. Moreover, preclinical studies have played a pivotal part in relating and addressing challenges similar to off-target effects, immune responses, and the need for optimized delivery styles to ensure effective and precise editing, which must be addressed before advancing to clinical trials [65–67].

Clinical trials represent a critical stage in the journey of CRISPR-Cas9 gene editing technology from the laboratory to real-world operations for inherited hematological diseases. These trials aim to estimate the safety, efficacity, and feasibility of CRISPR-Cas9 interventions in mortal cases, furnishing precious perceptivity into their eventuality as remedial strategies [68, 69]. Ongoing and completed clinical trials for conditions like sickle cell disease and beta-thalassemia validate promising preclinical results and restate them into tangible clinical issues. Initial outcomes from these trials have shown varying degrees of success, with some reporting advancements in hematological parameters, reduction of disease-related symptoms, and dragged ages of disease remission in treated patients [70]. For illustration, recent trials for sickle cell disease have demonstrated increased levels of normal hemoglobin and reduced complications in patients treated with CRISPR-Cas9-edited hematopoietic stem cells, offering hope for its potential as a transformative remedy [71, 72].

Table 3: Summary of clinical trials utilizing CRISPR-Cas9 mediated ex vivo gene editing for inherited blood disorders

Clinical trial ID Phase Disease Treatment protocol Outcome Current status References
NCT04925206 Phase 1 Beta-Thalassemia Infusion of CRISPR-Edited CD34+Hematopoietic Stem Cells Increased Hemoglobin Levels, Reduced Transfusion Dependence Recruiting [73]
NCT04205435 Phase 1/2 Beta-thalassemia, Sickle Cell Disease Transplantation of CRISPR-Edited Hematopoietic Stem Cells Improved Hemoglobin Levels, Reduced Disease Complications Active not recruiting [74]
NCT04774536 Phase 1/2 Sickle Cell Disease Transplantation of CRISPR-Edited Hematopoietic Stem Cells Improved Hemoglobin Levels, Reduced Disease Symptoms Ongoing [73]
NCT04293185 Phase 3 Sickle Cell Disease Transplantation of CRISPR-Edited Hematopoietic Stem Cells Increased Hemoglobin Levels, Reduced Transfusion Dependence Recruiting [62]
NCT04592458 Phase 1 Beta-Thalassemia Transplantation of CRISPR-Edited CD34+Hematopoietic Stem Cells Increased Hemoglobin Levels, Reduced Transfusion Dependence Ongoing [62]
NCT03728322 Phase 1 Beta-Thalassemia Transplantation of CRISPR-Edited Hematopoietic Stem Cells Increased Hemoglobin Levels, Reduced Disease Symptoms Recruiting [75]
NCT03745287 Phase 1 Sickle Cell Disease, Beta-Thalassemia Transplantation of CRISPR-Edited CD34+Hematopoietic Stem Cells Improved Hemoglobin Levels, Reduced Disease Symptoms Recruiting [76]
NCT03655678 Phase 1 Beta-Thalassemia Transplantation of CRISPR-Edited CD34+Hematopoietic Stem Cells Improved Hemoglobin Levels, Reduced Transfusion Dependence Recruiting [77]

Despite these promising results, clinical trials using CRISPR-Cas9 for inherited hematological disorders face several challenges, including optimizing delivery methods to target HSCs effectively while mitigating off-target effects and addressing immune responses to edited cells [78–80]. A further obstacle that needs to be overcome is the ethical and regulatory issues that surround the application of gene editing technology in clinical settings. Nonetheless, ongoing trials demonstrate CRISPR-Cas9's potential as a treatment avenue. Looking forward, tailored strategies based on patient genetic profiles hold promise for enhancing therapeutic outcomes and minimizing risks associated with CRISPR-Cas9 interventions [81, 82].

Challenges and limitations

Off-target effects

Understanding the complexities of CRISPR-Cas9-mediated ex vivo gene editing for inherited blood diseases requires addressing significant challenges, such as off-target effects. These effects involve unexpected genetic alterations occurring at sites other than the intended target, potentially disrupting essential genes or activating oncogenes [45, 76, 83]. Despite CRISPR-Cas9's precision, the risk of off-target changes remains, necessitating strategies like protein engineering, precise gRNA design, and bioinformatics tools to mitigate these effects before clinical application. Optimizing delivery methods further enhances targeting accuracy, ensuring the safety and effectiveness of ex vivo gene editing techniques [84–87].

Immunogenicity and host responses

Immunogenicity and Host Responses pose another challenge in CRISPR-Cas9-mediated gene editing, particularly concerning the eventuality of immune responses triggered by the insertion of edited cells into the patient's body [73]. Inserting edited cells using CRISPR-Cas9 may trigger immune responses, leading to cell rejection or inflammation. Addressing immunogenicity involves selecting immunocompatible editing reagents, modulating immune responses through immunosuppressive remedies, and engineering edited cells to evade immune detection. Additionally, advances in gene editing technologies, such as the development of non-viral delivery systems, may help reduce immunological responses and improve the tolerability of ex vivo gene editing interventions [88, 89].

Ethical and regulatory consideration

Exploring ethical considerations surrounding CRISPR-Cas9 gene editing is essential for navigating the complex geography of genetic medicine. Concerns related to germline editing raise profound ethical questions about the eventuality of unintended consequences and the implications for future generations [90]. Equitable access to therapies, consent, privacy, and societal implications must be addressed. Regulatory frameworks must balance scientific innovation with human welfare and ethical principles, necessitating robust oversight and transparent governance structures to ensure responsible and ethical use of CRISPR-Cas9-mediated ex vivo gene editing for inherited hematological disorders [91, 92].

Future directions and opportunities

Looking ahead, the future of CRISPR-Cas9-mediated ex vivo gene editing for inherited hematological disorders is ripe with implicit potential, presenting a horizon of instigative openings and new avenues for exploration. Future endeavors may concentrate on enhancing the precision, efficiency, and safety of gene editing techniques, steering in a new era of targeted therapeutic interventions [93-95]. Furthermore, the integration of CRISPR-Cas9 with other cutting-edge technologies, such as artificial intelligence and machine learning, could enable more precise targeting of genetic mutations and substantiated treatment strategies acclimatized to individual patients. Additionally, solidarity with arising fields like regenerative medicine, synthetic biology, single-cell genomics, bioinformatics, and tissue engineering offers exciting opportunities to explore the complications of inherited hematological disorders at a position of granularity never before imagined. This interdisciplinary approach not only deepens our understanding of disease mechanisms but also illuminates novel therapeutic targets and strategies, offering renewed hope for patients and clinicians alike [96–100]. Likewise, as we navigate the path toward clinical restatement, it's imperative to address the challenges and ethical considerations that accompany the use of CRISPR-Cas9 in mortal cases. Robust regulatory frameworks and transparent communication are essential to ensure responsible use, prioritizing patient safety and autonomy [74, 101]. Exploring combination therapies and integrating gene editing with modalities like gene therapy or small molecule inhibitors offers a holistic approach to target multiple aspects of disease pathology. Additionally, refining disease models will accelerate preclinical research, advancing therapeutic development for inherited hematological disorders [75, 102, 103].

CONCLUSION

The journey of CRISPR-Cas9-intermediated ex vivo gene editing for inherited hematological diseases is both a testament to scientific invention and a reflection of the complications essential in biomedical exploration. Advancements in understanding, refining, and applying CRISPR-Cas9 technology have propelled the field forward, offering unknown openings to address inheritable diseases at their root cause. Preclinical studies have provided insights into efficacy and safety, while ongoing clinical trials offer glimpses of real-world impact. Despite challenges such as off-target effects and ethical considerations, the clinical potential of CRISPR-Cas9 remains promising. Ongoing trials show encouraging results, and as technology evolves and challenges are addressed, new possibilities emerge. Embracing emerging technologies and approaches promises to reshape treatment landscapes, offering hope to affected families. With unwavering commitment and collaboration, we stand poised to realize the clinical eventuality of CRISPR-Cas9, steering in a new era of hope and healing for patients around the globe.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All authors have contributed equally

CONFLICT OF INTERESTS

Declared none

REFERENCES

  1. Lillicrap D. Introduction to a series of reviews on inherited bleeding disorders. Blood. 2015;125(13):2011. doi: 10.1182/blood-2015-01-613588, PMID 25712995.

  2. Weatherall DJ. The inherited diseases of hemoglobin are an emerging global health burden. Blood. 2010;115(22):4331-6. doi: 10.1182/blood-2010-01-251348, PMID 20233970.

  3. Ifeanyi E, Ogechi B, Emmanuel Ifeanyi O, ISSN. Sickle cell anaemia: a review. Scholars Journal of Applied Medical Sciences. 2015;3(6B):2244-52.

  4. Soni S. Gene therapies for transfusion-dependent β-thalassemia: current status and critical criteria for success. Am J Hematol. 2020;95(9):1099-112. doi: 10.1002/ajh.25909, PMID 32562290.

  5. Cao A, Moi P, Galanello R. Recent advances in β-thalassemias. Pediatr Rep. 2011;3(2):e17. doi: 10.4081/pr.2011.e17, PMID 21772954.

  6. Galanello R, Origa R. Open access review biomed central beta-thalassemia. Vol. 5. Orphanet Journal of Rare Diseases; 2010. Available from: http://www.ojrd.com/content/5/1/11 [Last accessed on 16 Aug 2024]

  7. Romito M, Rai R, Thrasher AJ, Cavazza A. Genome editing for blood disorders: state of the art and recent advances. Emerg Top Life Sci. 2019;3(3):289-99. doi: 10.1042/ETLS20180147, PMID 33523137.

  8. Frangoul H, Altshuler D, Cappellini MD, Chen YS, Domm J, Eustace BK. CRISPR-Cas9 gene editing for sickle cell disease and β-thalassemia. N Engl J Med. 2021 Jan 21;384(3):252-60. doi: 10.1056/NEJMoa2031054, PMID 33283989.

  9. Pandey VK, Tripathi A, Bhushan R, Ali A, Dubey PK. Application of CRISPR/Cas9 genome editing in genetic disorders: a systematic review up to date. J Genet Syndr Gene Ther. 2017;08(2). doi: 10.4172/2157-7412.1000321.

  10. Demirci S, Uchida N, Tisdale JF. Gene therapy for sickle cell disease: an update. Cytotherapy. 2018;20(7):899-910. doi: 10.1016/j.jcyt.2018.04.003, PMID 29859773.

  11. Daniel Moreno A, Lamsfus Calle A, Raju J, Antony JS, Handgretinger R, Mezger M. CRISPR/Cas9-modified hematopoietic stem cells present and future perspectives for stem cell transplantation. Bone Marrow Transplant. 2019;54(12):1940-50. doi: 10.1038/s41409-019-0510-8, PMID 30903024.

  12. Cavazzana M, Bushman FD, Miccio A, Andre Schmutz I, Six E. Gene therapy targeting haematopoietic stem cells for inherited diseases: progress and challenges. Nat Rev Drug Discov. 2019;18(6):447-62. doi: 10.1038/s41573-019-0020-9, PMID 30858502.

  13. Walsh RM, Hochedlinger K. A variant CRISPR-Cas9 system adds versatility to genome engineering. Proc Natl Acad Sci USA. 2013;110(39):15514-5. doi: 10.1073/pnas.1314697110, PMID 24014593.

  14. Hille F, Charpentier E. CRISPR-cas: biology mechanisms and relevance. Philos Trans R Soc Lond B Biol Sci. 2016;371(1707). doi: 10.1098/rstb.2015.0496, PMID 27672148.

  15. Chira S, Gulei D, Hajitou A, Zimta AA, Cordelier P, Berindan Neagoe I. CRISPR/Cas9: transcending the reality of genome editing. Mol Ther Nucleic Acids. 2017;7:211-22. doi: 10.1016/j.omtn.2017.04.001, PMID 28624197.

  16. Yamamoto M, Tani K. Current status and recent advances of gene therapy in hematological diseases. Int J Hematol. 2016;104(1):4-5. doi: 10.1007/s12185-016-2036-9, PMID 27250344.

  17. Humbert O, Samuelson C, Kiem HP. CRISPR/Cas9 for the treatment of haematological diseases: a journey from bacteria to the bedside. Br J Haematol. 2021;192(1):33-49. doi: 10.1111/bjh.16807, PMID 32506752.

  18. Ma Y, Zhang L, Huang X. Genome modification by CRISPR/Cas9. FEBS Journal. 2014;281(23):5186-93. doi: 10.1111/febs.13110, PMID 25315507.

  19. Gulei D, Berindan Neagoe I. CRISPR/Cas9: a potential lifesaving tool. What’s next? Mol Ther Nucleic Acids. 2017;9:333-6. doi: 10.1016/j.omtn.2017.10.013, PMID 29246311.

  20. Iyer DN, Schimmer AD, Chang H. Applying CRISPR-Cas9 screens to dissect hematological malignancies. Blood Adv. 2023;7(10):2252-70. doi: 10.1182/bloodadvances.2022008966, PMID 36355853.

  21. Shi H, Jiang M, Wang Z. Comprehensive update on applications of CRISPR/Cas9 for hematological diseases. Int J Clin Exp Med. Vol. 10; 2017. Available from: www.ijcem.com. [Last accessed on 16 Aug 2024]

  22. Singh V, Braddick D, Dhar PK. Exploring the potential of genome editing CRISPR-Cas9 technology. Gene. 2017;599:1-18. doi: 10.1016/j.gene.2016.11.008, PMID 27836667.

  23. Li B, Niu Y, Ji W, Dong Y. Strategies for the CRISPR-based therapeutics. Trends Pharmacol Sci. 2020;41(1):55-65. doi: 10.1016/j.tips.2019.11.006, PMID 31862124.

  24. Gupta D, Bhattacharjee O, Mandal D, Sen MK, Dey D, Dasgupta A. CRISPR-Cas9 system: A new fangled dawn in gene editing. Life Sci. 2019;232:116636. doi: 10.1016/j.lfs.2019.116636, PMID 31295471.

  25. Jiang F, Doudna JA. CRISPR-Cas9 structures and mechanisms; 2017. doi: 10.1146/annurev-biophys.

  26. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8(11):2281-308. doi: 10.1038/nprot.2013.143, PMID 24157548.

  27. Abdelnour SA, Xie L, Hassanin AA, Zuo E, Lu Y. The potential of CRISPR/Cas9 gene editing as a treatment strategy for inherited diseases. Front Cell Dev Biol. 2021;9:699597. doi: 10.3389/fcell.2021.699597, PMID 34977000.

  28. Konishi CT, Long C. Progress and challenges in CRISPR-mediated therapeutic genome editing for monogenic diseases. J Biomed Res. 2020;35(2):148-62. doi: 10.7555/JBR.34.20200105, PMID 33402545.

  29. Bloomer H, Khirallah J, Li Y, Xu Q. CRISPR/Cas9 ribonucleoprotein mediated genome and epigenome editing in mammalian cells. Adv Drug Deliv Rev. 2022 Feb 1;181:114087. doi: 10.1016/j.addr.2021.114087, PMID 34942274.

  30. Nidhi S, Anand U, Oleksak P, Tripathi P, Lal JA, Thomas G. Novel crispr cas systems: an updated review of the current achievements applications and future research perspectives. Int J Mol Sci. 2021;22(7). doi: 10.3390/ijms22073327, PMID 33805113.

  31. Buffa V, Alvarez Vargas JR, Galy A, Spinozzi S, Rocca CJ. Hematopoietic stem and progenitors cells gene editing: beyond blood disorders. Front Genome Ed. 2022;4:997142. doi: 10.3389/fgeed.2022.997142, PMID 36698790.

  32. Rautela I, Uniyal P, Thapliyal P, Chauhan N, Bhushan Sinha V, Dev Sharma M. An extensive review to facilitate understanding of CRISPR technology as a gene editing possibility for enhanced therapeutic applications. Gene. 2021;785:145615. doi: 10.1016/j.gene.2021.145615, PMID 33775851.

  33. Antony JS, Haque AK, Lamsfus Calle A, Daniel Moreno A, Mezger M, Kormann MS. CRISPR/Cas9 system: a promising technology for the treatment of inherited and neoplastic hematological diseases. Adv Cell Gene Ther. 2018 May;1(1):e10. doi: 10.1002/acg2.10.

  34. LaFountaine JS, Fathe K, Smyth HD. Delivery and therapeutic applications of gene editing technologies ZFNs TALENs and CRISPR/Cas9. Int J Pharm. 2015;494(1):180-94. doi: 10.1016/j.ijpharm.2015.08.029, PMID 26278489.

  35. Xu X, Wan T, Xin H, Li D, Pan H, Wu J. Delivery of CRISPR/Cas9 for therapeutic genome editing. J Gene Med. 2019;21(7):e3107. doi: 10.1002/jgm.3107, PMID 31237055.

  36. Wang HX, Li M, Lee CM, Chakraborty S, Kim HW, Bao G. CRISPR/Cas9-based genome editing for disease modeling and therapy: challenges and opportunities for nonviral delivery. Chem Rev. 2017;117(15):9874-906. doi: 10.1021/acs.chemrev.6b00799, PMID 28640612.

  37. Luther DC, Lee YW, Nagaraj H, Scaletti F, Rotello VM. Delivery approaches for CRISPR/Cas9 therapeutics in vivo: advances and challenges. Expert Opin Drug Deliv. 2018;15(9):905-13. doi: 10.1080/17425247.2018.1517746, PMID 30169977.

  38. Qushawy M, Nasr A. Solid lipid nanoparticles (SLNs) as nano drug delivery carriers: preparation characterization and application. Int J App Pharm. 2020;12(1-2):1-9. doi: 10.22159/ijap.2020v12i1.35312.

  39. Nayek S, Venkatachalam A, Choudhury S. Recent nanocochleate drug delivery system for cancer treatment: a review. Int J Curr Pharm Sci. 2019 Nov 15:28-32. doi: 10.22159/ijcpr.2019v11i6.36359.

  40. Liu C, Zhang L, Liu H, Cheng K. Delivery strategies of the CRISPR-Cas9 gene-editing system for therapeutic applications. J Control Release. 2017;266:17-26. doi: 10.1016/j.jconrel.2017.09.012, PMID 28911805.

  41. Karimian A, Azizian K, Parsian H, Rafieian S, Shafiei Irannejad V, Kheyrollah M. CRISPR/Cas9 technology as a potent molecular tool for gene therapy. J Cell Physiol. 2019;234(8):12267-77. doi: 10.1002/jcp.27972, PMID 30697727.

  42. Sankaran VG, Ghazvinian R, Do R, Thiru P, Vergilio JA, Beggs AH. Exome sequencing identifies GATA1 mutations resulting in diamond blackfan anemia. J Clin Invest. 2012 Jul 2;122(7):2439-43. doi: 10.1172/JCI63597, PMID 22706301.

  43. Steinberg MH, Sebastiani P. Genetic modifiers of sickle cell disease. Am J Hematol. 2012;87(8):795-803. doi: 10.1002/ajh.23232, PMID 22641398.

  44. Ben Jehuda R, Shemer Y, Binah O. Genome editing in induced pluripotent stem cells using CRISPR/Cas9. Stem Cell Rev Rep. 2018;14(3):323-36. doi: 10.1007/s12015-018-9811-3, PMID 29623532.

  45. Mohammadian Gol T, Urena Bailen G, Hou Y, Sinn R, Antony JS, Handgretinger R. CRISPR medicine for blood disorders: progress and challenges in delivery. Front Genome Ed. 2022;4:1037290. doi: 10.3389/fgeed.2022.1037290, PMID 36687779.

  46. Huang C, Li Q, Li J. Site specific genome editing in treatment of inherited diseases: possibility progress and perspectives. Med Rev. 2022;2(5):471-500. doi: 10.1515/mr-2022-0029, PMID 37724161.

  47. Mani I. CRISPR-Cas9 for treating hereditary diseases. Prog Mol Biol Transl Sci. 2021;181:165-83. doi: 10.1016/bs.pmbts.2021.01.017, PMID 34127193.

  48. Pellagatti A, Dolatshad H, Yip BH, Valletta S, Boultwood J. Application of genome editing technologies to the study and treatment of hematological disease. Adv Biol Regul. 2016;60:122-34. doi: 10.1016/j.jbior.2015.09.005, PMID 26433620.

  49. Zhang H, McCarty N. CRISPR-Cas9 technology and its application in haematological disorders. Br J Haematol. 2016;175(2):208-25. doi: 10.1111/bjh.14297, PMID 27619566.

  50. Bhattacharjee G, Gohil N, Siruka D, Khambhati K, Maurya R, Ramakrishna S. CRISPR-dCas9 system for epigenetic editing towards therapeutic applications. Prog Mol Biol Transl Sci. 2023 Jan 1;198:15-24. doi: 10.1016/bs.pmbts.2023.02.005, PMID 37225318.

  51. Kim EJ, Kang KH, Ju JH. CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells. Korean J Intern Med. 2017 Jan 1;32(1):42-61. doi: 10.3904/kjim.2016.198, PMID 28049282.

  52. Ho BX, Loh SJ, Chan WK, Soh BS. In vivo genome editing as a therapeutic approach. Int J Mol Sci. 2018;19(9). doi: 10.3390/ijms19092721, PMID 30213032.

  53. Huang K, Zapata D, Tang Y, Teng Y, Li Y. In vivo delivery of CRISPR-Cas9 genome editing components for therapeutic applications. Biomaterials. 2022 Dec 1;291:121876. doi: 10.1016/j.biomaterials.2022.121876, PMID 36334354.

  54. Li R, Wang Q, She K, Lu F, Yang Y. CRISPR/Cas systems usher in a new era of disease treatment and diagnosis. Mol Biomed. 2022;3(1):31. doi: 10.1186/s43556-022-00095-y, PMID 36239875.

  55. Fan Y, Chan JK. Editing the genome ex vivo stem cell therapy. Curr Stem Cell Rep. 2018;4(4):338-45. doi: 10.1007/s40778-018-0148-2.

  56. Al-Saif AM. Gene therapy of hematological disorders: current challenges. Gene Ther. 2019;26(7-8):296-307. doi: 10.1038/s41434-019-0093-4, PMID 31300728.

  57. Koniali L, Lederer CW, Kleanthous M. Therapy development by genome editing of hematopoietic stem cells. Cells. 2021;10(6):1492. doi: 10.3390/cells10061492, PMID 34198536.

  58. Quintana Bustamante O, Fananas Baquero S, Dessy Rodriguez M, Ojeda Perez I, Segovia JC. Gene editing for inherited red blood cell diseases. Front Physiol. 2022;28(13):848261. doi: 10.3389/fphys.2022.848261, PMID 35418876.

  59. Li Y, Glass Z, Huang M, Chen ZY, Xu Q. Ex vivo cell based CRISPR/Cas9 genome editing for therapeutic applications. Biomaterials. 2020;234:119711. doi: 10.1016/j.biomaterials.2019.119711, PMID 31945616.

  60. Bhattacharjee G, Mani I, Gohil N, Khambhati K, Braddick D, Panchasara H. CRISPR technology for genome editing. In: Precision medicine for investigators, practitioners and providers. Elsevier; 2019. p. 59-69.

  61. Gonzalez Romero E, Martinez Valiente C, Garcia Ruiz C, Vazquez Manrique RP, Cervera J, Sanjuan-Pla A. CRISPR to fix bad blood: a new tool in basic and clinical hematology. Haematologica. 2019;104(5):881-93. doi: 10.3324/haematol.2018.211359, PMID 30923099.

  62. Germino Watnick P, Hinds M, Le A, Chu R, Liu X, Uchida N. Hematopoietic stem cell gene addition/editing therapy in sickle cell disease. Cells. 2022;11(11). doi: 10.3390/cells11111843, PMID 35681538.

  63. Xu Y, Li Z. CRISPR cas systems: overview innovations and applications in human disease research and gene therapy. Comput Struct Biotechnol J. 2020;18:2401-15. doi: 10.1016/j.csbj.2020.08.031, PMID 33005303.

  64. CRISPR HX/Cas9 system and its applications in human hematopoietic cells. Blood Cells Mol Dis. 2016;62:6-12. doi: 10.1016/j.bcmd.2016.09.003.

  65. Sharma G, Sharma AR, Bhattacharya M, Lee SS, Chakraborty C. CRISPR-Cas9: a preclinical and clinical perspective for the treatment of human diseases. Mol Ther. 2021;29(2):571-86. doi: 10.1016/j.ymthe.2020.09.028, PMID 33238136.

  66. Foley RA, Sims RA, Duggan EC, Olmedo JK, Ma R, Jonas SJ. Delivering the CRISPR/Cas9 system for engineering gene therapies: recent cargo and delivery approaches for clinical translation. Front Bioeng Biotechnol. 2022;26(10):973326. doi: 10.3389/fbioe.2022.973326, PMID 36225598.

  67. Lino CA, Harper JC, Carney JP, Timlin JA. Delivering crispr: a review of the challenges and approaches. Drug Deliv. 2018;25(1):1234-57. doi: 10.1080/10717544.2018.1474964, PMID 29801422.

  68. Rodriguez Rodriguez DR, Ramirez-Solis R, Garza Elizondo MA, Garza Rodriguez ML, Barrera Saldana HA. Genome editing: a perspective on the application of CRISPR/Cas9 to study human diseases a review. Int J Mol Med. 2019;43(4):1559-74. doi: 10.3892/ijmm.2019.4112, PMID 30816503.

  69. Luthra R, Kaur S, Bhandari K. Applications of CRISPR as a potential therapeutic. Life Sci. 2021;284:119908. doi: 10.1016/j.lfs.2021.119908, PMID 34453943.

  70. Zhang S, Wang Y, Mao D, Wang Y, Zhang H, Pan Y. Current trends of clinical trials involving CRISPR/Cas systems. Front Med (Lausanne). 2023;10(10):1292452. doi: 10.3389/fmed.2023.1292452, PMID 38020120.

  71. Jensen TI, Axelgaard E, Bak RO. Therapeutic gene editing in haematological disorders with CRISPR/Cas9. Br J Haematol. 2019;185(5):821-35. doi: 10.1111/bjh.15851, PMID 30864164.

  72. Rao I, Crisafulli L, Paulis M, Ficara F. Hematopoietic cells from pluripotent stem cells: hope and promise for the treatment of inherited blood disorders. Cells. 2022;11(3)557. doi: 10.3390/cells11030557, PMID 35159366.

  73. Zhou L, Yao S. Recent advances in therapeutic CRISPR-Cas9 genome editing: mechanisms and applications. Mol Biomed. 2023;4(1):10. doi: 10.1186/s43556-023-00115-5, PMID 37027099.

  74. Khoshandam M, Soltaninejad H, Mousazadeh M, Hamidieh AA, Hosseinkhani S. Clinical applications of the CRISPR/Cas9 genome editing system: delivery options and challenges in precision medicine. Genes Dis. 2024;11(1):268-82. doi: 10.1016/j.gendis.2023.02.027, PMID 37588217.

  75. Li H, Yang Y, Hong W, Huang M, Wu M, Zhao X. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms advances and prospects. Signal Transduct Target Ther. 2020;5(1):1. doi: 10.1038/s41392-019-0089-y, PMID 32296011.

  76. Uddin F, Rudin CM, Sen T. CRISPR gene therapy: applications limitations and implications for the future. Front Oncol. 2020;10:1387. doi: 10.3389/fonc.2020.01387, PMID 32850447.

  77. Schacker M, Seimetz D. From fiction to science: clinical potentials and regulatory considerations of gene editing. Clin Transl Med. 2019 Dec;8(1):27. doi: 10.1186/s40169-019-0244-7, PMID 31637541.

  78. Anurogo D, Yuli Prasetyo Budi N, Thi Ngo MH, Huang YH, Pawitan JA. Cell and gene therapy for anemia: hematopoietic stem cells and gene editing. Int J Mol Sci. 2021 Jun 2;22(12):6275. doi: 10.3390/ijms22126275, PMID 34200975.

  79. 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, PMID 33455878.

  80. Bhoopalan SV, Yen JS, Levine RM, Sharma A. Editing human hematopoietic stem cells: advances and challenges. Cytotherapy. 2023 Mar 1;25(3):261-9. doi: 10.1016/j.jcyt.2022.08.003, PMID 36123234.

  81. Hirakawa MP, Krishnakumar R, Timlin JA, Carney JP, Butler KS. Gene editing and CRISPR in the clinic: current and future perspectives. Biosci Rep. 2020;40(4). doi: 10.1042/BSR20200127, PMID 32207531.

  82. Cerci B, Uzay IA, Kara MK, Dincer P. Clinical trials and promising preclinical applications of CRISPR/cas gene editing. Life Sci. 2023 Jan 1;312:121204. doi: 10.1016/j.lfs.2022.121204, PMID 36403643.

  83. Papizan JB, Porter SN, Sharma A, Pruett Miller SM. Therapeutic gene editing strategies using CRISPR-Cas9 for the β-hemoglobinopathies. J Biomed Res. 2021;35(2):115-34. doi: 10.7555/JBR.34.20200096.

  84. Hahn E, Hiemenz M. Therapeutic gene editing with CRISPR: a laboratory medicine perspective. Clin Lab Med. 2020;40(2):205-19. doi: 10.1016/j.cll.2020.02.008, PMID 32439069.

  85. Sun J, Wang J, Zheng D, Hu X. Advances in therapeutic application of CRISPR-Cas9. Brief Funct Genomics. 2020 May 1;19(3):164-74. doi: 10.1093/bfgp/elz031, PMID 31769791.

  86. Sahu S, Poplawska M, Lim SH, Dutta D. CRISPR based precision medicine for hematologic disorders: advancements challenges and prospects. Life Sci. 2023 Nov 15;333:122165. doi: 10.1016/j.lfs.2023.122165, PMID 37832631.

  87. Ates I, Rathbone T, Stuart C, Bridges PH, Cottle RN. Delivery approaches for therapeutic genome editing and challenges. Genes. 2020;11(10):1-32. doi: 10.3390/genes11101113, PMID 32977396.

  88. Burrage LC, Nagamani SC, Campeau PM, Lee BH. Branched chain amino acid metabolism: from rare mendelian diseases to more common disorders. Hum Mol Genet. 2014;23(R1):(R1-8). doi: 10.1093/hmg/ddu123, PMID 24651065.

  89. Sahel DK, Mittal A, Chitkara D. CRISPR/Cas system for genome editing: progress and prospects as a therapeutic tool. J Pharmacol Exp Ther. 2019;370(3):725-35. doi: 10.1124/jpet.119.257287, PMID 31122933.

  90. Doudna JA. The promise and challenge of therapeutic genome editing. Nature. 2020;578(7794):229-36. doi: 10.1038/s41586-020-1978-5, PMID 32051598.

  91. Jacinto FV, Link W, Ferreira BI. CRISPR/Cas9-mediated genome editing: from basic research to translational medicine. J Cell Mol Med. 2020;24(7):3766-78. doi: 10.1111/jcmm.14916, PMID 32096600.

  92. Tavakoli K, Pour Aboughadareh A, Kianersi F, Poczai P, Etminan A, Shooshtari L. Applications of CRISPR-Cas9 as an advanced genome editing system in life sciences. BioTech (Basel). 2021;10(3). doi: 10.3390/biotech10030014, PMID 35822768.

  93. Yip BH. Recent advances in CRISPR/Cas9 delivery strategies. Biomolecules. 2020;10(6). doi: 10.3390/biom10060839, PMID 32486234.

  94. Lu X, Zhang M, Li G, Zhang S, Zhang J, Fu X. Applications and research advances in the delivery of CRISPR/Cas9 systems for the treatment of inherited diseases. Int J Mol Sci. 2023;24(17). doi: 10.3390/ijms241713202, PMID 37686009.

  95. Tay LS, Palmer N, Panwala R, Chew WL, Mali P. Translating CRISPR-cas therapeutics: approaches and challenges. CRISPR J. 2020;3(4):253-75. doi: 10.1089/crispr.2020.0025, PMID 32833535.

  96. Li J, Wu P, Cao Z, Huang G, Lu Z, Yan J. Machine learning-based prediction models to guide the selection of Cas9 variants for efficient gene editing. Cell Rep. 2024 Feb;43(2):113765. doi: 10.1016/j.celrep.2024.113765, PMID 38358884.

  97. Li Y, Zaheri S, Nguyen K, Liu L, Hassanipour F, Pace BS. Machine learning-based approaches for identifying human blood cells harboring CRISPR-mediated fetal chromatin domain ablations. Sci Rep. 2022 Dec 1;12(1):1481. doi: 10.1038/s41598-022-05575-3, PMID 35087158.

  98. Gimeno M, San Jose Eneriz E, Villar S, Agirre X, Prosper F, Rubio A. Explainable artificial intelligence for precision medicine in acute myeloid leukemia. Front Immunol. 2022 Sep 29;13:977358. doi: 10.3389/fimmu.2022.977358, PMID 36248800.

  99. Saxena V, Singh A. An update on bio-potentiation of drugs using natural options. Asian J Pharm Clin Res. 2020 Nov 7;13(11):25-32. doi: 10.22159/ajpcr.2020.v13i11.38889.

  100. Radhika Reddy M, Shiva Gubbiyappa K. A comprehensive review on supersaturable self-nanoemulsifying. Drug Deliv Syst. 2021;14(8). doi: 10.22159/ajpcr.2021v14i8.41987.

  101. Solayappan M, Azlan A, Khor KZ, Yik MY, Khan M, Yusoff NM. Utilization of CRISPR-mediated tools for studying functional genomics in hematological malignancies: an overview on the current perspectives challenges and clinical implications. Front Genet. 2021;12:767298. doi: 10.3389/fgene.2021.767298, PMID 35154242.

  102. Reddy OL, Savani BN, Stroncek DF, Panch SR. Advances in gene therapy for hematologic disease and considerations for transfusion medicine. Semin Hematol. 2020;57(2):83-91. doi: 10.1053/j.seminhematol.2020.07.004, PMID 32892847.

  103. Bhattacharjee G, Gohil N, Khambhati K, Mani I, Maurya R, Karapurkar JK. Current approaches in CRISPR-Cas9 mediated gene editing for biomedical and therapeutic applications. J Control Release. 2022 Mar 1;343:703-23. doi: 10.1016/j.jconrel.2022.02.005, PMID 35149141.