IJMMS_2024v14n1

International Journal of Molecular Medical Science 2024, Vol.14 http://medscipublisher.com/index.php/ijmms © 2024 MedSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. MedSci Publisher, operated by Sophia Publishing Group (SPG), is an international Open Access publishing platform that publishes scientific journals in the field of life science. Sophia Publishing Group (SPG), founded in British Columbia of Canada, is a multilingual publisher. Publisher MedSci Publisher Editedby Editorial Team of International Journal of Molecular Medical Science Email: edit@ijmms.medscipublisher.com Website: http://medscipublisher.com/index.php/ijmms Address: 11388 Stevenston Hwy, PO Box 96016, Richmond, V7A 5J5, British Columbia Canada International Journal of Molecular Medical Science (ISSN 1927-6656) is an open access, peer reviewed journal published online by MedSci Publisher. The journal publishes scientific articles like original research articles, case reports, review articles, editorials, short communications and correspondence of the high quality pertinent to all aspects of human biology, pathophysiology and molecular medical science, including genomics, transcriptomics, proteomics, metabolomics of disease therapy, clinical pharmacology, clinical biochemistry, vaccines, immunology, microbiology, epidemiology, aging, cancer biology, infectious diseases, neurological diseases and myopathies, stem cells and regenerative medicine, vascular and cardiovascular biology, as well as the important implications for human health and clinical practice research. All the articles published in International Journal of Molecular Medical Science are Open Access, and are distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. MedSci Publisher uses CrossCheck service to identify academic plagiarism through the world’s leading plagiarism prevention tool, iParadigms, and to protect the original authors’ copyrights.

International Journal of Molecular Medical Science (online), 2024, Vol. 14, No. 1 ISSN 1927-6656 http://medscipublisher.com/index.php/ijmms © 2024 MedSci Publisher, registered at the publishing platform that is operated by Sophia Publishing Group, founded in British Columbia of Canada. All Rights Reserved. Latest Content Applications of Gene Editing and Gene Therapy in the Treatment of Genetic Disorders Xiaoping Cai International Journal of Molecular Medical Science, 2024, Vol. 15, No. 1, 1-7 The Application Prospects of CRISPR-Cas9 Technology in Gene Therapys Jianer Wang International Journal of Molecular Medical Science, 2024, Vol. 15, No. 1, 8-15 The Application Prospects of Artificial Intelligence in Molecular Medicines Wei Wang, Qikun Huang International Journal of Molecular Medical Science, 2024, Vol. 15, No. 1, 16-23 Glucocorticoid Receptor Signaling: Intricacies and Therapeutic Opportunities ShudanYan International Journal of Molecular Medical Science, 2024, Vol. 15, No. 1, 24-28 Studying the Dynamic Changes of the Immune System Through Single-cell Omics WeiWang International Journal of Molecular Medical Science, 2024, Vol. 15, No. 1, 29-41 Application and Prospects of Gene Editing Technology in the Treatment of Neurological Disorders JennyWu International Journal of Molecular Medical Science, 2024, Vol. 15, No. 1, 42-47 The Development and Clinical Transformation of Gene Therapy in Cardiovascular Diseases Wei Wang, Lingfei Jin International Journal of Molecular Medical Science, 2024, Vol. 15, No. 1, 48-55 Innovative Antiviral Strategy Targeting PLpro: Discovery of Jun12682 and Analysis of Its Antipandemic Effects JiaXuan International Journal of Molecular Medical Science, 2024, Vol. 15, No. 1, 56-60

International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 1-7 http://medscipublisher.com/index.php/ijmms 1 Research Report Open Access Applications of Gene Editing and Gene Therapy in the Treatment of Genetic Disorders Xiaoping Cai Xiongcheng Jianmin Med. Ltd., Zhuji, 311800, China Corresponding email: 2985757244@qq.com International Journal of Molecular Medical Science, 2024, Vol.14, No.1 doi: 10.5376/ijmms.2024.14.0001 Received: 15 Nov., 2023 Accepted: 20 Dec., 2023 Published: 01 Jan., 2024 Copyright © 2024 Cai, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Cai X.P., 2024, Applications of gene editing and gene therapy in the treatment of genetic disorders, International Journal of Molecular Medical Science, 14(1): 1-7 (doi: 10.5376/ijmms.2024.14.0001) Abstract Gene editing and gene therapy are advanced biotechnologies that have gained significant attention in recent years, holding enormous potential for the treatment of genetic disorders. This review provides an overview of the applications of gene editing and gene therapy in the treatment of genetic diseases. It introduces the concept of gene editing and places a specific emphasis on the application of the CRISPR-Cas9 system in genetic disease research and therapy. Furthermore, the review outlines the principles and methods of gene therapy, including gene replacement, gene correction, and gene silencing, discussing their applications in the treatment of genetic disorders. By comparing the advantages and limitations of both gene editing and gene therapy strategies, including technical constraints, safety concerns, and ethical considerations, this review summarizes the current applications of gene editing and gene therapy in the treatment of genetic diseases and provides insights into future trends. The aim of this review is to offer a reference and guidance for research and clinical applications of gene editing and gene therapy in the field of genetic disease treatment. Keywords Gene editing; Gene therapy; Genetic diseases; CRISPR-Cas9; Gene correction Genetic diseases are diseases caused by abnormal gene or chromosome mutations, which impose a significant burden on both patients and society as a whole. Traditional treatment methods primarily focus on alleviating disease symptoms and managing associated complications, but they rarely have the ability to cure the disease itself. However, with the rapid development of gene editing and gene therapy technologies, new treatment strategies are emerging. Gene editing technology, especially the emergence of the CRISPR-Cas9 system, provides an unprecedented option for the treatment of genetic diseases (Hu, 2022). Gene editing allows for the precise modification of abnormal genes within a patient's body, effectively correcting genetic defects and restoring normal gene function. Compared to traditional gene therapy methods, gene editing offers higher precision, a wider range of applications, and greater treatment efficacy. Furthermore, gene editing can also be used to repair a patient's hematopoietic stem cells ex vivo, offering hope incurable by traditional methods. In addition to gene editing, gene therapy is also an important direction in the field of genetic disease treatment (Yang, 2020). Gene therapy uses delivery vectors to introduce normal genes into patients' bodies to repair or replace abnormal genes. This method has made some important breakthroughs in the treatment of monogenic genetic diseases, such as the cure of some rare genetic diseases through gene replacement therapy. In addition, gene therapy can also regulate gene expression through strategies such as stem cells and gene silencing, effectively controlling the development and progression of hereditary diseases. Although the application of gene editing and gene therapy has shown great potential and hope in the treatment of genetic diseases, it also faces some challenges. Technical limitations, security issues, and ethical and legal considerations are currently key issues that need to be addressed. Therefore, further research and standardized regulation are essential. This review will systematically review the application of gene editing and gene therapy in the treatment of genetic diseases, explore the principles, methods, and key application cases of these emerging technologies, and compare

International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 1-7 http://medscipublisher.com/index.php/ijmms 2 their advantages and current challenges. This review paper can provide comprehensive understanding for researchers and clinical doctors, and point out the direction for future research and practice in the treatment of genetic diseases. 1 Application of Gene Editing in the Treatment of Hereditary Diseases 1.1 Definition of gene editing Gene editing refers to the technique of accurately modifying and adjusting the genome of an organism through deliberate human intervention. The main principle is to introduce specific nucleases or protein tools to interact with the target DNA sequence, achieving operations such as DNA mutation, insertion, or deletion. Among them, the most widely used gene editing technology is the CRISPR-Cas9 system. This system utilizes Cas9 protein binds to specific guiding RNA (gRNA) to form complexes for precise identification and binding of target DNA sequences, thereby triggering the process of genome editing. In addition to the CRISPR-Cas9 system, there are other gene editing techniques such as zinc finger nucleases (ZFNs) and deoxyribonucleases (TALENs) that can also achieve precise genome modifications. Different gene editing techniques differ in terms of target selection, editing efficiency, and specificity. 1.2 Application of gene editing in the treatment of genetic diseases 1.2.1 Application in the treatment of monogenic genetic diseases Monogenic genetic diseases, caused by mutations in a single gene, have found new hope in the application of gene editing technologies. The primary tool for gene editing is the CRISPR-Cas9 system, which allows precise modification of the genome to restore normal function. Gene editing can be employed to correct disease phenotypes by repairing mutated genes. This approach is applicable to specific types of mutations,, such as point mutations, insertions, and deletions. By introducing a repair template, gene editing can restore the normal sequence of genes and repair the functionality of disease-associated genes. For instance, in the context of cystic fibrosis, a common monogenic disease, gene editing techniques have been used to repair the CFTR gene. Researchers use the CRISPR-Cas9 system to precisely cleave the mutated gene sequence and introduce a normal repair template into cells through homologous recombination. This repair method has demonstrated feasibility and effectiveness in cell and animal models, providing a new avenue for gene therapy in cystic fibrosis.t. For gene mutations that cannot be repaired, gene editing technologies can facilitate gene replacement, which involves inserting normal gene sequences into damaged gene sites to restore their normal function. This method is applicable to certain monogenic genetic diseases, such as spinal muscular atrophy. For instance, some studies have utilized gene editing techniques to insert the normal SMN1 gene sequence into the genome of patients with spinal muscular atrophy. This gene replacement approach has been shown to increase the levels of SMN protein in patients, thereby improving the symptoms of the disease. Gene editing technology can also be employed for gene silencing, involving the precise cleavage and disruption of the sequence of detrimental genes to inhibit their function. This method is applicable to certain monogenic genetic diseases, such as fragile X syndrome. For example, fragile X syndrome is caused by an expansion mutation of the FMR1 gene on the X chromosome (Figure 1). In the treatment of fragile X syndrome, researchers have utilized the CRISPR-Cas9 system for gene silencing targeting the FMR1 gene (Liao et al., 2022). By accurately cleaving the sequence of this gene, the production of harmful proteins can be reduced, thereby alleviating the symptoms. 1.2.2 Application in the treatment of polygenic genetic diseases Gene editing technology has demonstrated significant potential in the treatment of polygenic genetic diseases. Polygenic genetic diseases are disorders caused by mutations in multiple genes, such as hereditary heart disease, chromosomal abnormality syndromes and so forth. Gene editing can be utilized to repair mutations in multiple genes, in order to restore the functionality of genes associated with polygenic genetic diseases. By introducing multiple repair templates, it is possible to simultaneously correct mutations in different genes. This approach can be applied in certain polygenic genetic diseases, such as hereditary heart disease (Zhang et al., 2020).

International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 1-7 http://medscipublisher.com/index.php/ijmms 3 Figure 1 Differences between fragile X syndrome X chromosome and normal chromosome Gene editing technology can also restore the normal function of genes associated with polygenic genetic diseases by replacing mutations in multiple genes. By simultaneously inserting multiple normal gene sequences, it is possible to ameliorate the pathological consequences of polygenic mutations. This method is applicable to polygenic genetic diseases such as chromosomal abnormality syndromes. Gene editing technology can also be employed to regulate the expression levels of multiple genes. By precisely editing the regulatory sequences of genes, it is possible to increase or decrease the expression of multiple genes, thereby influencing disease-relevant signaling pathways. This method can be applied in certain polygenic genetic diseases, such as polygenic tumors. 2 Gene Therapy in the Treatment of Genetic Diseases 2.1 Definition of gene therapy Gene therapy is a medical technique aimed at treating or preventing diseases by directly intervening in the human genome. It involves the introduction, modification, or deletion of gene sequences into the cells or tissues of patients to repair or alter abnormal or missing gene functions. The goal of gene therapy is to adjust or repair genes, restoring mutated genes or supplementing missing or abnormal genes to achieve therapeutic effects. The potential of this treatment lies in its ability to fundamentally impact the occurrence and progression of diseases. Gene therapy can take various forms, including gene replacement therapy, gene editing therapy, gene enhancement therapy, gene silencing therapy, and more. 2.2 Application of gene therapy in the treatment of genetic diseases 2.2.1 Application in the treatment of monogenic genetic diseases Gene therapy has made significant strides in the treatment of monogenic genetic diseases (Zhou et al., 2020). Gene replacement therapy aims to replace defective genes with normal gene sequences to restore normal gene function. This method can be achieved by introducing normal genes into the cells or tissues of patients. For instance, for patients with cystic fibrosis (Figure 2), normal CFTR genes can be introduced into lung cells to restore the function of chloride ion channels. Gene editing therapy involves directly modifying the patient's gene sequence to correct mutations, insertions, deletions, and other genetic abnormalities. This method utilizes tools such as CRISPR-Cas9 to perform precise gene editing targeted at specific gene mutations. For example, gene editing therapy can be employed to repair mutated genes in hematopoietic stem cells of patients with blood disorders, thereby correcting abnormalities in hemoglobin.

International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 1-7 http://medscipublisher.com/index.php/ijmms 4 Figure 2 Cystic fibrosis In some monogenic genetic diseases, patients may experience a deficiency or insufficient functionality of normal genes. Gene enhancement therapy aims to increase the expression levels of normal genes to achieve therapeutic effects. This method can be implemented by delivering additional normal genes into the patient's body. For instance, through gene enhancement therapy, it is possible to increase the expression of proteins associated with depression-related genes to improve symptoms. 2.2.2 Application in the treatment of polygenic genetic diseases Gene therapy also holds promise in the treatment of polygenic genetic diseases, but compared to single gene inherited diseases, poses more challenges, and research is still in its early stages. Currently, the applications in the treatment of polygenic genetic diseases primarily involve gene silencing therapy, gene regulation therapy, and combination therapy strategies. Gene silencing therapy aims to reduce or eliminate the adverse effects on health by inhibiting the overexpression or production of harmful genes. This can be achieved through RNA interference techniques, such as siRNA or miRNA. By selectively inhibiting the expression of specific genes, it is possible to intervene in the occurrence and progression of polygenic genetic diseases. Polygenic genetic diseases typically involve aberrant regulation of multiple genes. The goal of gene regulation therapy is to restore a normal gene expression pattern by adjusting the balance of the patient's intracellular gene regulatory network. This may involve the use of gene expression regulatory factors (such as transcription factors or nucleases) to modulate the expression levels of relevant genes. The treatment of polygenic genetic diseases often requires the integration of multiple therapeutic strategies. Gene therapy can be combined with other treatment modalities, such as drug therapy, gene editing, or stem cell transplantation, to achieve enhanced therapeutic outcomes. By combining multiple treatment strategies, a more comprehensive intervention in the development and symptoms of polygenic genetic diseases can be achieved. 3 Comparison between Gene Editing and Gene Therapy 3.1 Advantages and limitations in treating genetic diseases The advantages of gene editing include precision; gene editing technologies (such as CRISPR-Cas9) can precisely modify DNA sequences, accurately correcting genetic mutations in patients and restoring normal gene function. Its therapeutic potential is significant; gene editing can potentially cure genetic diseases by repairing or correcting mutations present in the patient's DNA, restoring normal gene expression and function (Li et al., 2017). In terms of persistence, gene editing technology can induce enduring genetic changes in treated cells, which can be passed on to descendant cells, resulting in a long-term therapeutic effect. The limitations of gene editing include safety concerns. Gene editing technology is still in the developmental stage, and unexpected side effects and safety issues, some of which may be unpredictable, could arise. Delivery

International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 1-7 http://medscipublisher.com/index.php/ijmms 5 challenges exist as the precise delivery of editing tools to specific tissues or organs in patients remains a challenge, necessitating effective delivery systems and strategies. In terms of ethical and moral considerations, gene editing involves the direct manipulation of human genes, sparking discussions on ethical and moral issues such as human genetic modification and genetic editing of embryos. The advantages of gene therapy include deliverability (Li et al., 2019). Gene therapy utilizes vectors (such as viruses or plasmids) to introduce normal genes into the patient's body, ensuring accurate delivery to the cells or tissues requiring treatment. With a variety of treatment strategies, gene therapy can intervene in genetic diseases by enhancing the expression of normal genes, suppressing the expression of harmful genes, or supplementing missing genes. Successful cases already exist; in treating certain genetic diseases, gene therapy has achieved significant success, such as in the treatment of some immunodeficiency disorders and retinal diseases. The limitations of gene therapy include clinical application restrictions. Currently, the clinical application of gene therapy is still subject to certain limitations, such as unstable treatment effects and the need for further validation of sustained efficacy. There are concerns about immune reactions and safety issues, the introduction of exogenous genes and vectors may trigger immune responses and safety issues, leading to treatment failure or adverse reactions. Cost is also a factor, the preparation and delivery costs of gene therapy are relatively high, limiting its feasibility for widespread application. Overall, both gene editing and gene therapy hold promise and face challenges as methods for treating genetic diseases. With continuous technological advancements and in-depth research on their safety and efficacy, these two approaches are expected to offer broader prospects for the treatment of genetic diseases in the future. 3.2 Criteria for selection and evaluation in different genetic diseases In selecting and assessing the application of gene editing and gene therapy methods for different genetic diseases, the following are common criteria. Regarding selection criteria, the first consideration is the characteristics of the disease, understanding the types of genetic mutations, the pathogenic mechanisms, and the severity of the disease. This helps determine whether gene editing or gene therapy can correct or alleviate the pathological processes caused by the genetic mutations. Additionally, considerations must include the accessibility of the target cells or tissues, determining whether gene editing or gene therapy techniques can effectively deliver to the specific cells or tissues requiring treatment. Assessing the therapeutic potential involves evaluating the efficacy and feasibility of gene editing or gene therapy methods in correcting genetic mutations, including theoretical effectiveness and pre-clinical study results. In the selection of competitive treatment methods, other possible treatments such as drug therapy or gene replacement therapy should be considered, evaluating the comparative advantages of gene editing or gene therapy against alternative approaches. In terms of evaluation criteria, the first consideration is efficacy and safety, assessing the method's impact on patients' disease symptoms, survival rates, and quality of life, as well as potential treatment risks and adverse reactions. The second consideration is persistence, examining whether the treatment effects can be sustained and whether repeated treatments are necessary. Feasibility and cost-effectiveness should also be taken into account, considering the feasibility of the treatment method, including the preparation and delivery of the treatment plan, and evaluating its cost-effectiveness compared to other treatment methods. Additionally, there is a need to assess the ethical and moral issues inherent in the treatment method (Wang et al., 2021), such as the uncertainty of potential genetic editing or modification impacts on future generations and the potential negative effects on specific ethnic or social groups. The above criteria are for reference only and are not exhaustive. In specific clinical applications, researchers and physicians need to comprehensively consider factors such as disease characteristics, treatment goals, safety, feasibility, and patient needs to make personalized choices and assessments. Additionally, ongoing clinical and scientific research is crucial for continually refining method selection and evaluation criteria.

International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 1-7 http://medscipublisher.com/index.php/ijmms 6 3.3 Complementarity in treating genetic diseases Gene editing and gene therapy complement each other in treating genetic diseases, offering mutual supplementation and synergistic effects at different levels. In terms of the scope of target diseases, gene editing and gene therapy exhibit complementarity in addressing different types of genetic mutations and disease mechanisms. Gene editing can precisely correct genetic mutations by modifying DNA sequences, particularly applicable to monogenic genetic diseases. On the other hand, gene therapy can intervene in various types of genetic diseases through methods such as delivering normal genes or suppressing the expression of harmful genes. Gene editing and gene therapy complement each other in terms of treatment timing. Gene editing typically requires intervention in the early stages of embryos or stem cells to correct genetic mutations that occur early in the patient's body. In contrast, gene therapy can be performed at any point after the patient's birth, even when the disease has already developed and symptoms have appeared. Gene editing and gene therapy possess complementary tissue and organ integration capabilities. Gene editing can achieve systemic efficacy by selectively editing problematic genes as needed. On the other hand, gene therapy can target specific tissues and organs, achieving targeted efficacy by selectively delivering corrective factors. Gene editing and gene therapy exhibit complementarity in terms of the persistence of treatment effects. Gene editing can maintain enduring genetic changes in cells and descendants, providing long-term therapeutic effects (Zhu et al., 2019). Meanwhile, gene therapy can achieve long-term therapeutic effects through sustained gene expression or maintaining stable levels of corrective factors. In summary, gene editing and gene therapy have complementary advantages in treating genetic diseases. Therefore, in specific clinical applications, a flexible combination of gene editing and gene therapy can be chosen based on factors such as the type of disease, treatment goals, and technical feasibility to maximize treatment effectiveness and provide personalized and comprehensive treatment plans for patients. 4 Summary and Outlook Gene editing and gene therapy represent cutting-edge technologies for the treatment of genetic diseases, holding revolutionary potential in genetic modification and intervention. Gene editing, through direct modification of DNA sequences, can precisely correct genetic mutations, potentially curing genetic diseases. Gene therapy, on the other hand, involves delivering normal genes into the patient's body or suppressing the expression of harmful genes to block or improve the course of the disease. Both technologies have significant and undeniable potential in the treatment of genetic diseases. In terms of clinical applications, gene editing and gene therapy have made significant strides. Clinical trials for some genetic diseases, such as cystic fibrosis and hereditary retinal disorders, have been conducted using gene editing or gene therapy, yielding promising results. These studies have laid the groundwork for further exploration of the clinical applications of gene editing and gene therapy. In the future, the application of gene editing and gene therapy in the treatment of genetic diseases will face critical challenges and directions for development. Firstly, there is a need to enhance the precision, efficiency, and safety of gene editing techniques, reducing non-specific editing and potential side effects. Simultaneously, improvements in gene delivery technologies are required to enhance the effective delivery and persistence of gene therapy. Additionally, a deeper understanding of genetic diseases, including disease mechanisms, pathogenic genes, and key factors in disease progression, is essential. This will contribute to better identifying treatment targets and selecting appropriate therapeutic strategies. In the realm of clinical trials and regulation, it is imperative to conduct more clinical trials to assess the efficacy and safety of gene editing and gene therapy. Simultaneously, the establishment of robust regulatory frameworks is essential to ensure the rationality and feasibility of treatments. Leveraging individual genomic information to achieve personalized treatment for genetic diseases is crucial. By integrating gene editing and gene therapy,

International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 1-7 http://medscipublisher.com/index.php/ijmms 7 tailored treatment plans can be devised for each patient. Strengthening collaboration among scientists, clinicians, ethicists, and legal experts is necessary, Considering fully consideration ethical, legal, and societal issues, and formulating appropriate policies and guidelines. In conclusion, gene editing and gene therapy hold significant promise in the treatment of genetic diseases. With ongoing technological advancements and deeper clinical research, gene editing and gene therapy are poised to play even more substantial roles in the future of treating genetic diseases, further enhancing the quality of life and health outcomes for patients. Acknowledgments Special thanks to Mrs. Xuan Jia and Mrs. Zhang Jie for their guidance and review of the paper. Many of their constructive comments have greatly contributed to the improvement of this research. References Hu S.H., Liu Q.Y., Xie D.C., and Huang J.J., 2022, Clinical research progress of CRISPR/Cas genome editing in the treatment of human genetic disorders, Shengming Kexue (Chinese Bulletin of Life Sciences), 34(10): 1250-1263. Liao Y., Zeng X.L., and Yang J., 2022, Application progress of fmr1 gene in diagnosis and treatment of fragile x syndrome, medical recapitulate, (12): 2476-2481. https://doi.org/10.12677/ACM.2022.12121710 Li C.H., Hu B., Weng Y.H., and Huang Y.Y., 2019, current situation and clinical research progresses of gene therapy,Shengming Kexue Yiqi (Life Science Instruments), 17(4): 3-12. Li X., Chui W.T., and Li K., Research advances on techniques of gene editing and its application, Zhongguo Xumu Shouyi (China Animal Husbandry & Veterinary Medicine), 44(8): 2241-2247. Wang H.Y., Li P.F., Xu L.J., Zhang L.W., He C.H., Fan Y.L., Yu J.R., and Xu Z.H., 2021, Ethical governance of gene editing technology, Zhonguo Kexueyuan Yuankan (Bulletin of Chinese Academy of Sciences), 36(11): 1259-1269. Yang X.Y., Wang D.Y., and Gao X., 2020, The Application of gene editing technology and cell therapy in in vitro gene therapy, Zhongguo Shengwu Huaxue Yu Fenzi Shengwu Xuebao (Chinese Journal of Biochemistry and Molecular Biology), 36(11): 1265-1272. Zhang F., Zong Y., MaW., Xu D., Wu J.Y., Construction of congenital heart disease key gene expression vector by Crispr/Cas9 technology, Lanzhou Daxue Xuebao (Journal of Lanzhou University (Medical Sciences)), 46(1): 72-76. Zhou L.J., Wang J.C., Gao H.B., and Zhao D.P., Gene therapy for monogenetic diseases, Keji Daobao (Science & Technology Review), 38(15): 89-100. Zhu B.y., Li L.X., Wang L.R., and Li D.l., 2019, Recent ex vivo gene therapy through genome editing, Zhonguo Xibao Shengwuxue Xuebao (Chinese Journal of Cell Biology), (4): 573-582.

International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 8-15 http://medscipublisher.com/index.php/ijmms 8 Review and Progress Open Access The Application Prospects of CRISPR-Cas9 Technology in Gene Therapys Jianer Wang Infinite (China) Co., Ltd., Zhuji, 31180, China Corresponding email: 1136425476@qq.com International Journal of Molecular Medical Science, 2024, Vol.14, No.1 doi: 10.5376/ijmms.2024.14.0002 Received: 01 Dec., 2024 Accepted: 07 Jan., 2024 Published: 19 Jan., 2024 Copyright © 2024 Wang, This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Preferred citation for this article: Wang J.E., 2024, The application prospects of CRISPR-Cas9 technology in gene therapy, International Journal of Molecular Medical Science, 14(1): 8-15 (doi: 10.5376/ijmms.2024.14.0002) Abstract The CRISPR-Cas9 technology, as a revolutionary gene-editing tool, has achieved remarkable breakthroughs, significantly impacting the field of gene therapy. This review provides an overview of the fundamental principles and technological developments of CRISPR-Cas9, tracing the progress in gene therapy and extensively discussing its applications in the treatment of monogenic inherited diseases and the prospective role in cancer therapy. Additionally, the review delves into the potential applications of CRISPR-Cas9 technology in organ transplantation and regenerative medicine, elucidating the challenges and ethical considerations it faces. While summarizing the research progress of CRISPR-Cas9 technology, the review also looks ahead to the future prospects of CRISPR-Cas9 in gene therapy. This comprehensive review serves as a valuable reference for the innovation and significance of CRISPR-Cas9 technology in the field of gene therapy, providing insights to propel further development and application in this prominent area. Keywords CRISPR-Cas9 technology; Gene therapy; Genetic diseases; Cancer treatment; Organ transplantation Gene therapy, as a revolutionary medical strategy, is offering new hope for human health. With the continuous development and breakthroughs of gene editing technology, CRISPR-Cas9 technology has attracted widespread attention and research as an emerging gene editing tool (Zhang, 2021). The advent of CRISPR-Cas9 technology has brought about revolutionary changes in gene therapy. Its efficient, precise, and customizable characteristics make it an ideal gene editing tool, opening up novel prospects for gene therapy. Gene therapy aims to treat, prevent, or alleviate genetic and other diseases by directly repairing abnormal genes or inserting normal genes into the patient's body. However, gene therapy has long faced several challenges, such as the selection of effective vectors, the precision and safety of gene editing, among others. Traditional gene editing techniques, such as zinc finger nucleases and TALENs, have made significant progress, but they still have certain limitations in terms of precision, efficiency, and operability. In comparison, CRISPR-Cas9 technology has higher precision and manipulability, making gene therapy more feasible and practical. The CRISPR-Cas9 technology has opened a new era in the field of gene editing by harnessing the CRISPR RNA from the natural immune system of bacteria to guide the Cas9 nuclease in gene cutting and editing. The core of this technology lies in the design and synthesis of a short nucleotide sequence, allowing the CRISPR-Cas9 system to accurately recognize and target specific loci in the genome for editing. Compared to traditional gene editing tools, CRISPR-Cas9 technology offers higher manipulability and efficiency, enabling the editing and repair of target genes within a relatively short timeframe. This review aims to comprehensively explore the prospects of CRISPR-Cas9 technology in gene therapy. Challenges faced by CRISPR-Cas9 technology, such as issues related to technical accuracy, non-specific cleavage, and ethical concerns, are discussed. The paper summarizes current research progress and anticipates the future potential of CRISPR-Cas9 technology in gene therapy. It is believed that CRISPR-Cas9 technology will catalyze a revolution in the field of gene therapy, bringing about significant changes and hope for human health. Through this review, we hope to provide a deeper understanding of the importance and innovativeness of CRISPR-Cas9 technology in the field of gene therapy, serving as a reference to further drive the development and application of CRISPR-Cas9 technology.

International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 8-15 http://medscipublisher.com/index.php/ijmms 9 1 The Basic Principles and Technological Innovations of CRISPR-Cas9 Technology 1.1 Discovery and evolution of CRISPR-Cas9 technology CRISPR-Cas9 technology was initially discovered in bacterial genomes (Zhu et al., 2019). In the 1990s, researchers identified a class of repetitive sequences called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), the function of which remained unknown for a long time. Subsequent studies revealed that CRISPR sequences were highly similar to viral sequences in bacteriophage or plasmid genomes. Further research has found that the CRISPR immune system is a natural defense mechanism of bacteria against viral invasions. The CRISPR-Cas system consists of CRISPR sequences and associated Cas proteins, with Cas proteins being crucial factors capable of recognizing and cleaving foreign DNA. In 2012, Emmanuelle Charpentier, along with a research team from Stanford University, discovered a type II CRISPR-Cas system, which includes a protein called Cas9. The Cas9 protein exhibits highly effective gene editing capabilities. The CRISPR-Cas9 technology has garnered significant attention and widespread application due to its simplified design, convenient operation, precise gene editing, broad applicability, and relatively low cost and time consumption. The further development and improvement of CRISPR-Cas9 technology are ongoing. Scientists through in-depth studies of the structure and function of the Cas9 protein, have developed various Cas9 variants, such as nuclease-dead Cas9 (dCas9) and high-fidelity Cas9. Additionally, the introduction of modified single-guide RNA (sgRNA) and chemically modified primers has further enhanced the efficiency and precision of gene editing. 1.2 Basic principles and working mechanism of CRISPR-Cas9 technology CRISPR sequences consist of a set of repetitive DNA sequences and interspersed viral or plasmid DNA segments derived from bacterial or archaeal genomes. These sequences serve as the immune system of bacteria, used to store previously encountered foreign DNA segments to defend against future viral attacks. Through a series of DNA modifications and RNA processing steps, the CRISPR sequences are transcribed into a type of RNA molecule called CRISPR RNA (crRNA). In the CRISPR-Cas9 system, scientists design and construct an artificially synthesized RNA molecule called single-guide RNA (sgRNA). It is formed by the fusion of crRNA and a transcribed RNA segment known as tracrRNA. This sgRNA can bind to the Cas9 protein, forming a complex with the ability to guide Cas9 to recognize and bind to specific DNA sequences (Wang et al., 2019). The Cas9 protein is a central component of the CRISPR-Cas9 system, possessing DNA binding and cleavage functions. Once the sgRNA binds to the Cas9 protein, the complex can recognize a specific region of the target DNA sequence that pairs with the sgRNA. Upon binding to the specific DNA sequence, the Cas9 protein induces a double-strand break, resulting in alterations to the genomic DNA sequence. Following the formation of the double-strand break, the cell initiates its own DNA repair mechanism, attempting to mend the break. Through different repair mechanisms, such as non-homologous end joining or homologous recombination, precise editing of the target gene can occur, leading to insertions, deletions, or replacements in the genome (Figure 1). 1.3 Comparison between CRISPR-Cas9 and traditional gene editing technologies GCRISPR-Cas9 exhibits significant advantages compared to traditional gene editing technologies. The CRISPR-Cas9 technology allows for highly precise gene editing. Different from traditional methods, Cas9 protein can accurately locate and cleave the target DNA sequence by designing appropriate sgRNA. CRISPR-Cas9 technology is highly flexible, as changing the target gene simply involves replacing the sgRNA sequence, whereas traditional methods often require multiple optimizations. Additionally, CRISPR-Cas9 technology is cost-effective and time-saving, utilizing sgRNA and Cas9 protein as the main tools without the need for expensive recombinant enzymes and reagents. Lastly, CRISPR-Cas9 technology is applicable to a variety of organisms, including cells and whole organisms, while traditional methods are typically limited to specific cell types or species (Cao et al., 2020).

International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 8-15 http://medscipublisher.com/index.php/ijmms 10 Figure 1 Application of CRISPR/Cas9 Despite the numerous advantages of CRISPR-Cas9 technology, it also faces several challenges. For instance, unintended cutting or editing events may occur, leading to unexpected genetic alterations. Additionally, the application of CRISPR-Cas9 technology in specific tissues or cell types poses challenges. However, with ongoing technological improvements, it is foreseeable that CRISPR-Cas9 will become a powerful and widely utilized gene editing tool. In summary, CRISPR-Cas9 offers higher precision, flexibility, cost-effectiveness, and shorter working times compared to traditional gene editing techniques. 2 The History and Development of Gene Therapy 2.1 Overview of traditional gene therapy approaches Traditional gene therapy methods refer to non-CRISPR-related techniques used to repair or regulate human genes. These approaches hold potential in the treatment of certain diseases, particularly monogenic disorders. However, they also face challenges such as low delivery efficiency, cell specificity, immune responses, and long-term stability. Traditional gene therapy methods include gene replacement, gene correction, and gene suppression, among others. Gene replacement involves introducing a normal gene into a patient's body to replace a defective gene. This can be achieved by delivering the normal gene into the patient's cells using vectors, such as viral vectors or plasmid DNA. Once the normal gene is introduced and expressed, it can correct the genetic defect and restore normal function. Gene correction refers to the correction or modification of erroneous gene sequences to rectify genetic defects. This can be achieved by using DNA modifying enzymes such as zinc finger nucleases and transcription active factors (Belete, 2021). Gene suppression involves inhibiting the expression of a defective gene through the use of interfering RNA (siRNA) or antisense RNA. Interfering RNA is a short RNA molecule that can interact with specific gene sequences in patient cells, selectively reducing or inhibiting the expression of the targeted gene. Antisense RNA is an RNA molecule that prevents its translation into a protein by interacting with the mRNA molecule of the target gene. 2.2 Progress and limitations of gene therapy Gene therapy is an emerging therapeutic approach that has made significant strides over the past few decades. In terms of clinical applications, gene therapy has successfully been used for some genetic diseases, such as severe combined immunodeficiency and familial hypercholesterolemia (Godbout and Tremblay, 2023). Furthermore, the genetic engineering of patient T cells to express chimeric antigen receptors (CARs) has been successfully applied

International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 8-15 http://medscipublisher.com/index.php/ijmms 11 in the treatment of leukemia. Gene therapy has also shown improvement in ocular diseases like retinal pigmentosa and hereditary optic neuropathy. However, gene therapy also faces limitations and challenges. Delivery efficiency remains a challenge, and improvements are needed to effectively deliver therapeutic genes to target cells and tissues. Safety is another crucial consideration, with attention required for immune reactions, inflammatory responses, or potential adverse events. Ensuring stable expression and persistence of the therapeutic gene, as well as addressing individual variability, are also issues that need resolution. Additionally, the high cost of gene therapy restricts its widespread application, and accessibility is further limited by technical requirements and healthcare resource constraints. Moreover, gene therapy involves ethical and moral considerations, such as privacy protection and fair distribution. Despite the challenges and limitations, gene therapy is a promising field that provides new options for the treatment of many diseases. With advances in scientific technology and accumulated experience, it is believed that gene therapy will continue to evolve in the future, bringing benefits to more patients. 2.3 Breakthrough of CRISPR-Cas9 technology and comparison with traditional gene therapy approaches CRISPR-Cas9 technology is a revolutionary gene editing tool that offers numerous breakthrough advantages compared to traditional gene therapy methods. CRISPR-Cas9 enables precise gene editing in the genome, allowing for gene knockout, repair, and insertion operations. In contrast, traditional gene therapy typically involves gene addition or replacement. CRISPR-Cas9 is characterized by its flexibility and versatility, making it widely applicable in both research and treatment. This technology achieves targeted editing of specific DNA sequences by designing guide RNA, enhancing treatment specificity-a feature often lacking in traditional gene therapy methods. CRISPR-Cas9 technology can directly perform gene editing in vivo, enabling patients to receive treatment without the need for ex vivo cells or gene delivery vectors, thereby enhancing treatment efficiency and convenience (Chen et al., 2019). While CRISPR-Cas9 technology faces some challenges and safety risks, it is considered to have groundbreaking potential, opening up new possibilities in the field of gene therapy. It provides a more precise, flexible, and efficient approach to gene therapy, offering promising prospects for the treatment of genetic diseases and advancements in biological research. As this technology continues to develop and improve, it is believed to bring forth further innovations and progress in the field of gene therapy. 3 CRISPR-Cas9 Technology in the Prospects of Gene Therapy 3.1 Application in the treatment of monogenic genetic diseases CRISPR-Cas9 technology has shown promising progress in the treatment of monogenic genetic diseases (Xu, 2020). Cystic fibrosis, a common genetic disease, can be corrected using CRISPR-Cas9 technology to rectify mutations in the human CFTR gene, restoring its function, which has been successful in both in vitro and in vivo studies. Similarly, for certain genetic blood disorders such as sickle cell anemia and β-thalassemia, CRISPR-Cas9 technology can be applied to correct patients' hematopoietic stem cells, preventing or alleviating disease symptoms. Additionally, immune deficiency disorders like severe combined immunodeficiency can be addressed by using CRISPR-Cas9 technology for gene editing to restore patients' immune function. For neurological disorders such as Huntington's disease, amyotrophic lateral sclerosis, and hereditary retinal dystrophy (Figure 2), CRISPR-Cas9 is also being researched for potential therapeutic applications. While the application in the treatment of monogenic genetic diseases faces some challenges, CRISPR-Cas9-based therapies are still regarded as a highly promising field, offering the potential for more effective and personalized treatment options for patients

International Journal of Molecular Medical Science, 2024, Vol.14, No.1, 8-15 http://medscipublisher.com/index.php/ijmms 12 . Figure 2 Retinitis Pigmentosa 3.2 Application in cancer treatment CRISPR-Cas9 technology holds potential breakthrough applications in cancer treatment. Among them, It can be used to suppress the growth and division of cancer cells through gene editing, disrupting the survival and proliferation capabilities of malignant tumor cells by precisely editing cancer-related genes. Additionally, CRISPR-Cas9 can enhance immunotherapy by editing and modifying patients' immune cells to improve their anti-tumor effects, including knocking out immune checkpoint genes and enhancing CAR-T cell therapies. Furthermore, CRISPR-Cas9 can be employed to restore or correct inactivated or mutated tumor suppressor genes, impeding the development and proliferation of tumors. While the application in cancer treatment still faces challenges, CRISPR-Cas9 technology is poised to become a more effective and personalized therapeutic strategy, offering improved treatment outcomes and survival opportunities for cancer patients. However, further research and clinical trials are still needed to assess the safety and long-term efficacy of CRISPR-Cas9 in cancer treatment. 3.3 Potential applications in organ transplantation and regenerative medicine CRISPR-Cas9 technology demonstrates significant potential applications in the fields of organ transplantation and regenerative medicine (Yu et al., 2021). It can reduce the risk of rejection during organ transplantation and enhance the success rate by gene editing. The technology can precisely edit specific genes in donor and recipient cells, such as HLA genes, thereby increasing the compatibility of transplanted organs. CRISPR-Cas9 plays a crucial role in promoting organ regeneration and repair. By editing and activating specific genes in stem cells, it can accelerate and improve their ability to differentiate into specific cell types, thus facilitating the process of organ regeneration and repair. Moreover, this technology can be applied to organ engineering and in vitro organ cultivation, producing customized organ substitutes that meet the specific needs of individual patients by editing and modifying donor cells or regenerated stem cells. Despite facing technical and ethical challenges in this field, CRISPR-Cas9 holds tremendous potential. This technology is poised to offer new solutions to alleviate organ shortage issues and provide better treatment options for patients. Future research and clinical experiments will further propel the practical application of CRISPR-Cas9 technology in the fields of organ transplantation and regenerative medicine, assessing its safety and feasibility. 4 Potential Applications and Challenges of CRISPR-Cas9 Technology 4.1 Accuracy issues The accuracy of CRISPR-Cas9 technology in gene editing is a crucial topic of discussion. Despite its widespread application and remarkable achievements, there are challenges related to its accuracy. The specificity of the

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