Gene therapy strategies and prospects for neurofibromatosis type 1 / 中国修复重建外科杂志

OBJECTIVE@#To summarize the gene therapy strategies for neurofibromatosis type 1 (NF1) and related research progress.@*METHODS@#The recent literature on gene therapy for NF1 at home and abroad was reviewed. The structure and function of the NF1 gene and its mutations were analyzed, and the current status as well as future prospects of the transgenic therapy and gene editing strategies were summarized.@*RESULTS@#NF1 is an autosomal dominantly inherited tumor predisposition syndrome caused by mutations in the NF1 tumor suppressor gene, which impair the function of the neurofibromin and lead to the disease. It has complex clinical manifestations and is not yet curable. Gene therapy strategies for NF1 are still in the research and development stage. Existing studies on the transgenic therapy for NF1 have mainly focused on the construction and expression of the GTPase-activating protein-related domain in cells that lack of functional neurofibromin, confirming the feasibility of the transgenic therapy for NF1. Future research may focus on split adeno-associated virus (AAV) gene delivery, oversized AAV gene delivery, and the development of new vectors for targeted delivery of full-length NF1 cDNA. In addition, the gene editing tools of the new generation have great potential to treat monogenic genetic diseases such as NF1, but need to be further validated in terms of efficiency and safety.@*CONCLUSION@#Gene therapy, including both the transgenic therapy and gene editing, is expected to become an important new therapeutic approach for NF1 patients.

Research on the Ethical Supervision of Human Gene Editing Technology: Based on the Perspective of Game Theory / 中国医学伦理学

In view of the current norms and demands of human gene editing technology at home and abroad, the paper explained that the regulatory difficulties faced by human gene editing technology were due to the conflict between economic interests and moral bottom line by constructing a game model in a hypothetical way. On this basis, the ideas of the supervision mode of human gene editing technology were put forward: establish unified international standards based on the country as the main body, enact more stringent and effective laws, to jointly deal with the behavior of genetic manipulation of human gametes, zygotes and embryos for the purpose of reproductive, and ensure the normalization and legalization of gene editing technology to avoid technology abuse.

Advances in corneal stromal regeneration and repair / 国际眼科杂志(Guoji Yanke Zazhi)

Corneal stroma is a significant part of the cornea and plays a significant role in the eye's refractive system. Although corneal transplantation is now the most effective treatment for corneal stromal disease, its advancement has been constrained by a shortage of donors, the need for prolonged immunosuppressive medicine to prevent rejection, and low graft survival rates. An alternate strategy is to use the corneal stroma's natural capacity for regeneration to create the ideal conditions for the collagenous extracellular matrix of the stroma to self-renew. However, it is challenging to replicate the intricate ultrastructure of the corneal stroma in vitro. Regenerative medicine has so been used to address these issues. These approaches refer to numerous disciplines, including stem cell-induced differentiation, tissue engineering and gene editing. This article provides potential directions for the future clinical applications of corneal stromal regeneration and repair while summarizing pertinent techniques, research progress, and issues.

Logic-gated tumor-microenvironment nanoamplifier enables targeted delivery of CRISPR/Cas9 for multimodal cancer therapy

Recent innovations in nanomaterials inspire abundant novel tumor-targeting CRISPR-based gene therapies. However, the therapeutic efficiency of traditional targeted nanotherapeutic strategies is limited by that the biomarkers vary in a spatiotemporal-dependent manner with tumor progression. Here, we propose a self-amplifying logic-gated gene editing strategy for gene/H2O2-mediated/starvation multimodal cancer therapy. In this approach, a hypoxia-degradable covalent-organic framework (COF) is synthesized to coat a-ZIF-8 in which glucose oxidase (GOx) and CRISPR system are packaged. To intensify intracellular redox dyshomeostasis, DNAzymes which can cleave catalase mRNA are loaded as well. When the nanosystem gets into the tumor, the weakly acidic and hypoxic microenvironment degrades the ZIF-8@COF to activate GOx, which amplifies intracellular H+ and hypoxia, accelerating the nanocarrier degradation to guarantee available CRISPR plasmid and GOx release in target cells. These tandem reactions deplete glucose and oxygen, leading to logic-gated-triggered gene editing as well as synergistic gene/H2O2-mediated/starvation therapy. Overall, this approach highlights the biocomputing-based CRISPR delivery and underscores the great potential of precise cancer therapy.

Aplicaciones clínicas de la herramienta CRISPR-Cas / Clinical Applications of the CRISPR-Cas Tool

Resumen El desarrollo de tecnologías para la edición del genoma ha abierto la posibilidad de apuntar directamente y modificar secuencias genómicas en casi todo tipo de células eucariotas. La edición del genoma ha ampliado nuestra capacidad para dilucidar la contribución de la genética a las enfermedades al promover la creación de modelos celulares y animales más precisos de procesos patológicos y ha comenzado a mostrar su potencial en una variedad de campos, que van desde la investigación básica hasta la biotecnología aplicada y biomédica. Entre estas tecnologías, el uso de las repeticiones palindrómicas cortas agrupadas regularmente espaciadas ha acelerado, en gran medida, el progreso de la edición de genes desde el concepto hasta la práctica clínica, generando, además, interés debido, no solo a su precisión y eficiencia, sino también a la rapidez y a los costos necesarios para su implementación en comparación con otras tecnologías de edición genómica. En esta revisión se presenta información recabada de publicaciones indexadas en la base de datos PubMed que se encontraron mediante el uso de palabras claves asociadas con la tecnología y que se filtraron para retener solo aquellas con evidencias de avances clínicamente relevantes y que permiten demostrar algunas de las aplicaciones que tiene esta tecnología en la investigación, pronóstico y tratamiento de enfermedades genéticas, cardiovasculares, virales, entre otras; esto con el objetivo de dar a conocer la situación actual de los avances en aplicaciones clínicas de la herramienta CRISPR-Cas y fomentar aún más la investigación en esta tecnología, la cual, tal como se evidencia a lo largo de esta revisión, posee una gran versatilidad y un amplio rango de aplicaciones, lo que ofrece una enorme oportunidad en el campo de la medicina genómica, pero que, a su vez, requiere un mayor fomento en su investigación para mejorar la tecnología y acercarla aún más a consolidar aplicaciones clínicas de uso seguro, confiable y consistente.

Abstract The development of genome editing technologies has opened up the possibility of directly targeting and modifying genomic sequences in almost all types of eukaryotic cells. Genome editing has expanded our ability to elucidate the contribution of genetics to disease by promoting the creation of more precise cellular and animal models of disease processes and has begun to show its potential in a variety of fields, ranging from basic research to applied and biomedical biotechnology. Among these technologies, the use of clustered regularly spaced short palindromic repeats have greatly accelerated the progress of gene editing from concept to clinical practice, further generating interest due not only to its precision and efficiency, but also to the speed and costs required for its implementation compared to other genomic editing methods. This review presents information collected from indexed publications in the PubMed database that were found by using keywords associated with the technology and filtered to retain only those with evidence of clinically relevant advances that demonstrate some of the applications that this technology has in research, prognosis, and treatment of genetic, cardiovascular, and viral diseases, among others; this with the aim of show the current situation of advances in clinical applications of the CRISPR-Cas tool and further encourage research in this technology, which, as evidenced throughout this review, has a great versatility and a wide range of applications, which offers an enormous opportunity in the field of genomic medicine but which, in turn, requires greater support in its research to improve the technology and bring it even closer to consolidating clinical applications of safe, reliable and consistent use.

CRISPR/Cas9 genome editing approaches for psychiatric research

Currently, genome editing technologies, such as clustered regularly interspaced short palindromic repeats (CRISPR/Cas9), are predominantly used to model genetic diseases. This genome editing system can correct point or frameshift mutations in risk genes. Here, we analyze and discuss the advantages of genome editing, its current applications, and the feasibility of the CRISPR/Cas9 system in research on psychiatric disorders. These disorders produce cognitive and behavioral alterations and their etiology is associated with polygenetic and environmental factors. CRISPR/Cas9 may reveal the biological mechanisms of psychiatric disorders at a basic research level, translating a suitable clinical approach for use in the diagnosis and treatment of psychiatric disorders. Genetic diagnosis and treatment for these disorders have not yet been fully established in psychiatry due to the limited understanding of their heterogeneity and polygenicity. We discuss the challenges and ethical issues in using CRISPR/Cas9 as a tool for diagnosis or gene therapy.

Editing the Human Genome with CRISPR/Cas: A Review of its Molecular Basis, Current Clinical Applications, and Bioethical Implications

ABSTRACT CRISPR/Cas genes evolved in prokaryotic organisms as a mechanism of defense designed to identify and destroy genetic material from threatening viruses. A breakthrough discovery is that CRISPR/Cas system can be used in eukaryotic cells to edit almost any desired gene. This comprehensive review addresses the most relevant work in the CRISPR/Cas field, including its history, molecular biology, gene editing capability, ongoing clinical trials, and bioethics. Although the science involved is complex, we intended to describe it in a concise manner that could be of interest to diverse readers, including anyone dedicated to the treatment of patients who could potentially benefit from gene editing, molecular biologists, and bioethicists. CRISPR/Cas has the potential to correct inherited diseases caused by single point mutations, to knock-in the promoter of a gene whose expression is highly desirable or knockout the gene coding for a deleterious protein. CRISPR/Cas technique can also be used to edit ex vivo immune cells and reinsert them in patients, improving their efficiency in attacking malignant cells, limiting the infectious potential of viruses or modulating xenotransplant rejection. Very important bioethical considerations on this topic include the need to internationally regulate its use by ad hoc expert committees and to limit its use until safety and bioethical issues are satisfactorily resolved.

Biological Basis of Five-phase Evolution of Malignant Tumors from Perspective of "Immune Editing" / 中国实验方剂学杂志

Immune escape is one of the ten hallmarks of tumors， which plays an important role in the occurrence and development of tumors. Immune escape refers to a process where tumor cells remodel and edit the immune system through the model of immune clearance， immune balance， and immune escape to "transform" the immune cells into immunosuppressive cells in the tumor microenvironment， so as to support immune escape. The five-stage evolution is the summary of tumor pathogenesis by professor LI Jie. He believes that the gradual development of tumors follows the core pathogenesis of "deficiency-cold-toxin-obstruction-collapse"， in which "depression" runs through the whole process， and cancer toxin is the key. Based on immune editing， this paper combined phenotypic characteristics of tumor cells with the core pathogenesis of the five-stage evolution of professor LI to reveal the biological basis of malignant tumor five-stage evolution. The results indicate that the prominent change from deficiency to cold is the reduction of immune surveillance and the prominent change from toxin to obstruction is immune escape. The final stage of collapse is the outcome of immune failure. Depression is the booster of tumor immune editing. Therefore， the method of reinforcing the healthy Qi and removing toxins was proposed to regulate the immune editing and cut off the five-stage evolution of tumors. Supplementing Qi and warming Yang can reinforce the healthy Qi and restore immune surveillance. Removing toxins and dredging can reverse toxins and immune escape. The harmonizing method can maintain the dynamic balance of immune cells/immunosuppressive cells. Resolving depression can truncate tumor immune editing. Those methods can provide a certain reference for the treatment based on microscopic syndrome differentiation in traditional Chinese medicine （TCM）. In future studies， it is necessary to further explore the specific mechanism of the regulation of immune editing with the methods of supplementing Qi and warming Yang， removing toxins and dredging， their combination， and resolving depression， so as to find out specific Chinese medicines and targets and provide more sufficient evidence for the regulation of tumor immune editing by TCM.

Research progress on clustered regularly interspaced short palindromic repeats-associated 9 system for the treatment of HSV / 中华检验医学杂志

Herpes simplex virus (HSV) is a double-stranded DNA enveloped virus that causes severe effects on the human body by infecting the skin and nerve tissues. Because of latency and reactivation, the rapid detection and eradication of HSV are great challenges for clinical treatments. In recent years, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system has developed rapidly in the field of gene editing and detection due to its simple design and high targeting efficiency.

The progress of the gene editing therapy of inherited retinal diseases based on CRISPR/Cas9 / 中华眼底病杂志

Inherited retinal diseases (IRDs) are the major cause of refractory blinding eye diseases, and gene replacement therapy has already made preliminary progress in the treatment of IRDs. For IRDs that cannot be treated by gene replacement therapy, gene editing provides an alternative therapeutic method. Strategies like disruption of pathogenic variants with or without gene augmentation therapy and precise repair of pathogenic variants can be applied for IRDs with various inheritance patterns and pathogenic variants. In animal models of retinitis pigmentosa, Usher syndrome, Leber congenital amaurosis, cone rod cell dystrophy, and other disorders, CRISPR/Cas9, base editing, and prime editing showed the potential to edit pathogenic variations in vivo, indicating a promising future for gene editing therapy of IRDs.

Recent advances in preclinical research of renal xenotransplantation and selection of potential recipients in the future / 中华器官移植杂志

After continuous efforts from generations of transplant surgeons, kidney transplantation (KT) has become an optimal treatment for end-stage renal disease.However, an imbalance between supply and demand of organs has always restricted the development of KT.For this clinical dilemma, xenotransplantation is expected to become one practical alternative for alleviating organ shortage.This review summarized recent literature reports of kidney xenotransplantation and the latest cases of pig-to-human kidney and heart transplantations.Also clinical transformations and applications of kidney xenotransplantation were discussed.

Construction of human TCRP1 gene knockout chronic myeloid leukemia K562 cell line using CRISPR/Cas9 technology and its biological function detection / 中国医师杂志

Objective:To select human chronic myeloid leukemia (CML) cell line K562 as the experimental object, and use lentivirus mediated CRISPR/Cas9 gene editing technology to construct a stable CML cell line K562/TCRP1-KO that knocks out the tongue cancer resistance related protein 1 (TCRP1) gene; and through functional tests such as cell proliferation, apoptosis, and drug sensitivity, compare the phenotypic differences between K562/TCRP1-KO and control cells (K562/cas9-CTL), and preliminarily explore the possible mechanism of TCRP1 gene involvement in the pathogenesis of CML.Methods:The small guide RNA (sgRNA) targeting TCRP1 was designed at a specific location. After annealing, the oligonucleotide fragments were recombined with the linearized Cas9 expression vector, and the lentivirus packaging system was transfected into 293T cells. The purified virus was collected and infected with K562 cells. Positive polyclons were screened for puromycin pressure, and monoclonal K562/TCRP1-KO was further screened by limited dilution method. Stable cell lines were successfully knocked out by sanger sequencing and Western blot detection; Simultaneously, K562 cells transfected with lentiCRISPR vector were constructed as control cell lines (K562/cas9-CTL); Using cell counting method, cell counting kit 8 (CCK8) method, imatinib (IM) gradient dilution method, and flow cytometry cell proliferation, drug sensitivity, and apoptosis analysis were performed on K562/TCRP1-KO and K562/cas9-CTL, respectively.Results:The sgRNA-Cas9 recombinant plasmid vector for TCRP1 knockout was successfully constructed, and after transfection into 293T cells, TCRP1 knockout monoclonal cell lines were successfully screened using limited dilution method. Compared with K562/cas9-CTL cells, the proliferation ability of K562/TCRP1-KO cells was significantly reduced, IM drug sensitivity was significantly enhanced, and the process of cell apoptosis was significantly accelerated (all P<0.05). Conclusions:A CML cell line with TCRP1 knockout was successfully constructed using CRISPR/Cas9. TCRP1 may act as a cancer related gene to affect the proliferation, IM resistance, and apoptosis process of CML cells.

Research on the application of Media studio pro editing technology based on WeChat platform in radiation therapeutics teaching / 中华医学教育探索杂志

Objective:To explore the effect of constructing WeChat platform and introducing PPT (PowerPoint) optimized by Media studio pro editing technology in the teaching of radiation therapeutics.Methods:Sixty undergraduates of medical imaging technology in Fujian Medical University were randomly divided into experimental group and control group in average. The experimental group set up a WeChat group and acquired the optimized PPT before class; control group received classroom teaching and clinical practice according to the traditional teaching mode. Twenty-four items of MCTQ (Maastricht clinical teaching questionnaire) were selected and translated. A total of 30 teachers majoring in tissue radiation oncology and medical imaging were randomly divided into two groups. The questionnaire was used to evaluate the two teaching models. To examine the academic performance of the two groups of students. SPSS 23.0 software was used for independent sample t-test. Results:By comparing the scores of MCTQ questionnaire between the two groups, it was concluded that the teaching mode of the experimental group had significant advantages in 11 aspects, such as clinical practice, obtaining more learning opportunities and so on ( P < 0.05). The scores of practice [(84.67±7.29) vs. (80.03±8.97)] and final evaluation [(81.53±8.78) vs. (76.77±9.49)] of the students in the experimental group were better than those in the control group ( P < 0.05). Conclusion:The application of optimized PPT by Media studio pro editing technology based on WeChat platform is worth popularizing in the teaching of radiation therapeutics.

Application of CRISPR/Cas9 genome editing technology in hereditary retinal diseases / 中华实验眼科杂志

Several mutant genes for inherited retinal diseases have been identified, but effective treatments are still lacking.The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system can edit human genomic DNA by nonhomologous end joining or homology-directed repair, offering more possibilities for the treatment of hereditary retinal diseases.CRISPR/Cas9 not only can genetically correct patient-derived induced pluripotent stem cells (iPSCs) to observe their differentiation into retinal cells thereby, thereby exploring the pathogenesis of the disease and implementing cell therapy, but can also be delivered to the body via vectors and directly act on target cells to achieve in vivo gene editing.CRISPR/Cas9 gene editing technology in hereditary retinal diseases has been mainly used in retinitis pigmentosa, hereditary X-linked juvenile retinoschisis, and Leber congenital amaurosis 10, of which the in vitro application of CRISPR/Cas9 for Leber congenital amaurosis 10 has entered the clinical trial stage.In this paper, we reviewed the mechanism and key advances of CRISPR/Cas9 and provided an overview of gene editing in IRDs.

Research progress in the application of CRISPR/Cas9 technology in the treatment of cervical cancer / 国际生物医学工程杂志

Cervical cancer is the fourth-ranked malignant tumor of female cancer in the world, and it seriously threatens women’s health. The main treatment options for patients with cervical cancer are surgery or concurrent chemoradiotherapy. With the development of medical research, researchers are committed to exploring more effective and specific treatment options in order to increase the treatment options for cervical cancer and improve the treatment effect. Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a method in which the Cas9 protein uses guide RNA (gRNA) to target the target gene and achieve precise editing of the target gene. At present, CRISPR/Cas9 technology has become a promising and powerful gene editing tool, a new and effective targeted therapy that has been applied in the treatment of various tumors. The research progress of CRISPR/Cas9 technology in the treatment of cervical cancer is mainly reviewed in terms of action targets, combination therapy strategies, and related drug resistance gene screening in order to provide new strategies for the treatment of cervical cancer.

Research progress in mitochondrial gene editing technology / 浙江大学学报·医学版

Mitochondrial DNA (mtDNA) mutations result in a variety of genetic diseases. As an emerging therapeutic method, mtDNA editing technology recognizes targets more based on the protein and less on the nucleic acid. Although the protein recognition type mtDNA editing technology represented by zinc finger nuclease technology, transcription activator like effector nuclease technology and base editing technology has made some progress, the disadvantages of complex recognition sequence design hinder further popularization. Gene editing based on nucleic acid recognition by the CRISPR system shows superiority due to the simple structure, easy design and modification. However, the lack of effective means to deliver nucleic acids into mitochondria limits application in the field of mtDNA editing. With the advances in the study of endogenous and exogenous import pathways and the deepening understanding of DNA repair mechanisms, growing evidence shows the feasibility of nucleic acid delivery and the broad application prospects of nucleic acid recognition type mtDNA editing technology. Based on the classification of recognition elements, this article summarizes the current principles and development of mitochondrial gene editing technology, and discusses its application prospects.

Intelligent nanotherapeutic strategies for the delivery of CRISPR system

CRISPR, as an emerging gene editing technology, has been widely used in multiple fields due to its convenient operation, less cost, high efficiency and precision. This robust and effective device has revolutionized the development of biomedical research at an unexpected speed in recent years. The development of intelligent and precise CRISPR delivery strategies in a controllable and safe manner is the prerequisite for translational clinical medicine in gene therapy field. In this review, the therapeutic application of CRISPR delivery and the translational potential of gene editing was firstly discussed. Critical obstacles for the delivery of CRISPR system in vivo and shortcomings of CRISPR system itself were also analyzed. Given that intelligent nanoparticles have demonstrated great potential on the delivery of CRISPR system, here we mainly focused on stimuli-responsive nanocarriers. We also summarized various strategies for CIRSPR-Cas9 system delivered by intelligent nanocarriers which would respond to different endogenous and exogenous signal stimulus. Moreover, new genome editors mediated by nanotherapeutic vectors for gene therapy were also discussed. Finally, we discussed future prospects of genome editing for existing nanocarriers in clinical settings.

Base editors: development and applications in biomedicine / 医学前沿

Base editor (BE) is a gene-editing tool developed by combining the CRISPR/Cas system with an individual deaminase, enabling precise single-base substitution in DNA or RNA without generating a DNA double-strand break (DSB) or requiring donor DNA templates in living cells. Base editors offer more precise and secure genome-editing effects than other conventional artificial nuclease systems, such as CRISPR/Cas9, as the DSB induced by Cas9 will cause severe damage to the genome. Thus, base editors have important applications in the field of biomedicine, including gene function investigation, directed protein evolution, genetic lineage tracing, disease modeling, and gene therapy. Since the development of the two main base editors, cytosine base editors (CBEs) and adenine base editors (ABEs), scientists have developed more than 100 optimized base editors with improved editing efficiency, precision, specificity, targeting scope, and capacity to be delivered in vivo, greatly enhancing their application potential in biomedicine. Here, we review the recent development of base editors, summarize their applications in the biomedical field, and discuss future perspectives and challenges for therapeutic applications.

Ethical and legal implications of gene editing in plant breeding: a systematic literature review / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Biotechnology policies and regulations must be revised and updated to reflect the most recent advances in plant-breeding technology. New Plant Breeding Techniques (NPBT) such as gene editing have been applied to address the myriad of challenges in plant breeding, while the use of NPBT as emerging biotechnological tools raises legal and ethical concerns. This study aims to highlight how gene editing is operationalized in the existing literature and examine the critical issues of ethical and legal issues of gene editing for plant breeding. We carried out a systematic literature review (SLR) to provide the current states of ethical and legal discourses surrounding this topic. We also identified critical research priority areas and policy gaps that must be addressed when designing the future governance of gene editing in plant breeding.

Gene editing of the human embryo: tensions and controversies among scientists / Edição genética do embrião humano: tensões e controvérsias de posicionamentos entre cientistas

Objective: This study analyzed the relation between the position of scientists on embryo editing and the different types of knowledge involved. Methods: A lexical analysis of 151 scientific articles in the PubMed and Web of Science databases was conducted using the IRAMUTEQ software. Results: The results showed that gene editing in embryos is presented in two argumentative branches: the first refers to the editing technique and its possibilities; the second discusses the impacts of these techniques on the public arena. The results demonstrate a consensus regarding the potential of editing; however, dilemmas about its effectiveness were also highlighted. Conclusion: The presence of ethical conflicts with embryo editing among the specialists was evidenced especially regarding the birth of genetically modified babies. Therefore, gene editing is marked by conflicts that are not limited only to biological contexts, but that encompasses different aspects of social life.

Objetivo: O objetivo deste trabalho foi analisar a relação entre o posicionamento dos cientistas sobre a edição de embriões e os diferentes tipos de conhecimento envolvidos nesses debates. Método: Utilizando o software IRAMUTEQ realizou-se uma análise lexical de 151 artigos científicos nas bases de dados PubMed e Web of Science. Resultados: Os resultados demonstraram que a edição genética de embriões se apresenta em dois blocos argumentativos: o primeiro se refere à técnica de edição e suas possibilidades e o segundo discute os impactos dessas técnicas na arena pública. Os achados demonstram consenso sobre as potencialidades da edição, contudo dilemas sobre a sua eficácia foram também destacados. Conclusão: Evidenciou-se a presença de embates éticos sobre a edição de embriões entre os especialistas em relação ao nascimento de bebês geneticamente modificados. Observou-se que a edição genética é marcada por conflitos que não se limitam apenas a contextos biológicos, mas que tangem diferentes aspectos da vida social.