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Chang J, Yang X, Zhang T, Sun H, Cheng H, Jia Z, Li Y, Ma S, Sun T, Cao J. High-Throughput Screening to Identify Novel Compounds Affecting the Genome Editing Efficiency of CRISPR System. Molecules 2025; 30:1811. [PMID: 40333840 PMCID: PMC12029788 DOI: 10.3390/molecules30081811] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2025] [Revised: 04/08/2025] [Accepted: 04/12/2025] [Indexed: 05/09/2025] Open
Abstract
Genome editing is a promising therapeutic strategy for genetic disorders by modifying the genome precisely, especially the CRISPR/Cas9 system. However, a major limitation of CRISPR/Cas9 in gene therapy is the biosafety issues caused by off-target effects. Compounds that can modulate the genome editing efficiency of the CRISPR/Cas9 system, especially those reducing the off-target effects, are potentially useful pharmacological tools for improving the effectiveness and safety of genome editing. Here, we performed high-throughput screening in HEK 293FT cells to discover compounds that decrease or increase the genome editing efficiency of the CRISPR/Cas9 system from 9930 compounds. After two rounds of screening, we identified that CP-724714, a ErbB2 (HER2) tyrosine kinase inhibitor, decreased the CRISPR/Cas9 efficiency and reduced the off-target effects by suppressing the efficiency of CRISPR/Cas9, and was thus named a CRISPR decelerator (or inhibitor), while Clofarabine, a DNA synthesis inhibitor, increased the efficiency of CRISPR/Cas9, and was named a CRISPR accelerator. We further identified four compounds (Tranilast, Cerulenin, Rosolic acid and Resveratrol) that affected the efficiency of single-strand annealing (SSA) repair. Among them, Tranilast, Cerulenin and Rosolic acid are potential SSA decelerators, while Resveratrol is a potential SSA accelerator. These identified compounds may be useful in optimizing mammalian genetic manipulation techniques.
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Affiliation(s)
- Jiasong Chang
- Key Laboratory of Cellular Physiology, Shanxi Medical University, Ministry of Education, Taiyuan 030001, China; (J.C.); (X.Y.); (Z.J.); (Y.L.)
- Department of Physiology, Shanxi Medical University, Taiyuan 030001, China
| | - Xiulong Yang
- Key Laboratory of Cellular Physiology, Shanxi Medical University, Ministry of Education, Taiyuan 030001, China; (J.C.); (X.Y.); (Z.J.); (Y.L.)
- Department of Physiology, Shanxi Medical University, Taiyuan 030001, China
| | - Tong Zhang
- Biological Science Research Center, Southwest University, Chongqing 400715, China; (T.Z.); (H.S.); (S.M.)
| | - Hao Sun
- Biological Science Research Center, Southwest University, Chongqing 400715, China; (T.Z.); (H.S.); (S.M.)
| | - Hongying Cheng
- Department of Preschool Education, Lvliang Teachers College, Lvliang 033001, China;
| | - Zhangrong Jia
- Key Laboratory of Cellular Physiology, Shanxi Medical University, Ministry of Education, Taiyuan 030001, China; (J.C.); (X.Y.); (Z.J.); (Y.L.)
- Department of Physiology, Shanxi Medical University, Taiyuan 030001, China
| | - Yiying Li
- Key Laboratory of Cellular Physiology, Shanxi Medical University, Ministry of Education, Taiyuan 030001, China; (J.C.); (X.Y.); (Z.J.); (Y.L.)
- Department of Physiology, Shanxi Medical University, Taiyuan 030001, China
| | - Sanyuan Ma
- Biological Science Research Center, Southwest University, Chongqing 400715, China; (T.Z.); (H.S.); (S.M.)
| | - Teng Sun
- Key Laboratory of Cellular Physiology, Shanxi Medical University, Ministry of Education, Taiyuan 030001, China; (J.C.); (X.Y.); (Z.J.); (Y.L.)
- Department of Physiology, Shanxi Medical University, Taiyuan 030001, China
| | - Jimin Cao
- Key Laboratory of Cellular Physiology, Shanxi Medical University, Ministry of Education, Taiyuan 030001, China; (J.C.); (X.Y.); (Z.J.); (Y.L.)
- Department of Physiology, Shanxi Medical University, Taiyuan 030001, China
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Kim DH, Choi SH, Sung JJ, Kim S, Yi H, Park S, Park CW, Oh YW, Lee J, Kim DS, Kim JH, Park CY, Kim DW. Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs. Exp Mol Med 2025; 57:184-192. [PMID: 39762408 PMCID: PMC11799516 DOI: 10.1038/s12276-024-01375-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2024] [Revised: 09/04/2024] [Accepted: 10/08/2024] [Indexed: 02/07/2025] Open
Abstract
Hemophilia A (HA) is caused by mutations in coagulation factor VIII (FVIII). Genome editing in conjunction with patient-derived induced pluripotent stem cells (iPSCs) is a promising cell therapy strategy, as it replaces dysfunctional proteins resulting from genetic mutations with normal proteins. However, the low expression level and short half-life of FVIII still remain significant limiting factors in the efficacy of these approaches in HA. Here, we constructed a functionally enhanced FVIII variant, F309S/E1984V-mutated B domain-deleted (BDD)-FVIII (FE-FVIII), with increased activity and stability. We inserted FE-FVIII with a human elongation factor-1 alpha (EF1α) promoter into the AAVS1 locus of HA patient-derived iPSCs via CRISPR/Cas9 (D10A) nickase to ensure expression in any cell type. FE-FVIII was expressed not only in undifferentiated FE-FVIII-inserted (FE-KI) iPSCs but also in endothelial cells (ECs) differentiated from them in vitro. Compared with mice transplanted with wild-type BDD-FVIII-containing ECs, immunocompetent HA mice intravenously transplanted with FE-KI ECs presented a 2.12-fold increase in FVIII activity in the blood and an approximately 20% greater survival rate after hemorrhagic tail injury. For sustained efficacy, FE-KI ECs were subcutaneously transplanted into immunodeficient HA mice, resulting in amelioration of the hemophilia phenotype for more than 3 months. This strategy can improve FVIII function and may provide a universal therapeutic approach for treating HA.
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Affiliation(s)
- Do-Hun Kim
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
- S. Biomedics Co., Ltd, 28 Seongsui-ro 26-gil, Seongdong-gu, Seoul, 04797, Korea
| | - Sang-Hwi Choi
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Jin Jea Sung
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Sieun Kim
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
- Brain Korea 21 PLUS Program for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Hanui Yi
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
- Brain Korea 21 PLUS Program for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Sanghyun Park
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Chan Wook Park
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Young Woo Oh
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
- Brain Korea 21 PLUS Program for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Jungil Lee
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
- Brain Korea 21 PLUS Program for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Dae-Sung Kim
- Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Korea
| | - Jong-Hoon Kim
- Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Korea
| | - Chul-Yong Park
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea.
- S. Biomedics Co., Ltd, 28 Seongsui-ro 26-gil, Seongdong-gu, Seoul, 04797, Korea.
| | - Dong-Wook Kim
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea.
- S. Biomedics Co., Ltd, 28 Seongsui-ro 26-gil, Seongdong-gu, Seoul, 04797, Korea.
- Brain Korea 21 PLUS Program for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea.
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Azeez SS, Hamad RS, Hamad BK, Shekha MS, Bergsten P. Advances in CRISPR-Cas technology and its applications: revolutionising precision medicine. Front Genome Ed 2024; 6:1509924. [PMID: 39726634 PMCID: PMC11669675 DOI: 10.3389/fgeed.2024.1509924] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2024] [Accepted: 11/28/2024] [Indexed: 12/28/2024] Open
Abstract
CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated proteins) has undergone marked advancements since its discovery as an adaptive immune system in bacteria and archaea, emerged as a potent gene-editing tool after the successful engineering of its synthetic guide RNA (sgRNA) toward the targeting of specific DNA sequences with high accuracy. Besides its DNA editing ability, further-developed Cas variants can also edit the epigenome, rendering the CRISPR-Cas system a versatile tool for genome and epigenome manipulation and a pioneering force in precision medicine. This review explores the latest advancements in CRISPR-Cas technology and its therapeutic and biomedical applications, highlighting its transformative impact on precision medicine. Moreover, the current status of CRISPR therapeutics in clinical trials is discussed. Finally, we address the persisting challenges and prospects of CRISPR-Cas technology.
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Affiliation(s)
- Sarkar Sardar Azeez
- Department of Medical Laboratory Technology, Soran Technical College, Erbil Polytechnic University, Erbil, Kurdistan Region, Iraq
| | - Rahin Shareef Hamad
- Nursing Department, Soran Technical College, Erbil Polytechnic University, Erbil, Kurdistan Region, Iraq
| | - Bahra Kakamin Hamad
- Department of Medical Laboratory Technology, Erbil Health and Medical Technical College, Erbil Polytechnic University, Erbil, Kurdistan Region, Iraq
| | - Mudhir Sabir Shekha
- Department of Biology, College of Science, Salahaddin University, Erbil, Kurdistan Region, Iraq
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
| | - Peter Bergsten
- Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
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Hui KK, Yamanaka S. iPS cell therapy 2.0: Preparing for next-generation regenerative medicine. Bioessays 2024; 46:e2400072. [PMID: 38922935 DOI: 10.1002/bies.202400072] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2024] [Revised: 06/04/2024] [Accepted: 06/06/2024] [Indexed: 06/28/2024]
Abstract
This year marks the tenth anniversary of the world's first transplantation of tissue generated from induced pluripotent stem cells (iPSCs). There is now a growing number of clinical trials worldwide examining the efficacy and safety of autologous and allogeneic iPSC-derived products for treating various pathologic conditions. As we patiently wait for the results from these and future clinical trials, it is imperative to strategize for the next generation of iPSC-based therapies. This review examines the lessons learned from the development of another advanced cell therapy, chimeric antigen receptor (CAR) T cells, and the possibility of incorporating various new bioengineering technologies in development, from RNA engineering to tissue fabrication, to apply iPSCs not only as a means to achieve personalized medicine but also as designer medical applications.
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Affiliation(s)
- Kelvin K Hui
- Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
| | - Shinya Yamanaka
- Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan
- CiRA Foundation, Kyoto, Japan
- Gladstone Institute of Cardiovascular Disease, San Francisco, California, USA
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Mukhametzyanova L, Schmitt LT, Torres-Rivera J, Rojo-Romanos T, Lansing F, Paszkowski-Rogacz M, Hollak H, Brux M, Augsburg M, Schneider PM, Buchholz F. Activation of recombinases at specific DNA loci by zinc-finger domain insertions. Nat Biotechnol 2024; 42:1844-1854. [PMID: 38297187 PMCID: PMC11631766 DOI: 10.1038/s41587-023-02121-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2023] [Accepted: 12/22/2023] [Indexed: 02/02/2024]
Abstract
Recombinases have several potential advantages as genome editing tools compared to nucleases and other editing enzymes, but the process of engineering them to efficiently recombine predetermined DNA targets demands considerable investment of time and labor. Here we sought to harness zinc-finger DNA-binding domains (ZFDs) to program recombinase binding by developing fusions, in which ZFDs are inserted into recombinase coding sequences. By screening libraries of hybrid proteins, we optimized the insertion site, linker length, spacing and ZFD orientation and generated Cre-type recombinases that remain dormant unless the insertionally fused ZFD binds its target site placed in the vicinity of the recombinase binding site. The developed fusion improved targeted editing efficiencies of recombinases by four-fold and abolished measurable off-target activity in mammalian cells. The ZFD-dependent activity is transferable to a recombinase with relaxed specificity, providing the means for developing fully programmable recombinases. Our engineered recombinases provide improved genome editing tools with increased precision and efficiency.
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Affiliation(s)
- Liliya Mukhametzyanova
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
| | - Lukas Theo Schmitt
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
- Seamless Therapeutics GmbH, Dresden, Germany
| | - Julia Torres-Rivera
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
| | - Teresa Rojo-Romanos
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
- Seamless Therapeutics GmbH, Dresden, Germany
| | - Felix Lansing
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
- Seamless Therapeutics GmbH, Dresden, Germany
| | | | - Heike Hollak
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
- Seamless Therapeutics GmbH, Dresden, Germany
| | - Melanie Brux
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
| | - Martina Augsburg
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
| | - Paul Martin Schneider
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany
- Seamless Therapeutics GmbH, Dresden, Germany
| | - Frank Buchholz
- Medical Systems Biology, Medical Faculty, Technical University Dresden, Dresden, Germany.
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Aslan C, Zolbanin NM, Faraji F, Jafari R. Exosomes for CRISPR-Cas9 Delivery: The Cutting Edge in Genome Editing. Mol Biotechnol 2024; 66:3092-3116. [PMID: 38012525 DOI: 10.1007/s12033-023-00932-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/03/2023] [Accepted: 10/02/2023] [Indexed: 11/29/2023]
Abstract
Gene mutation correction was challenging until the discovery of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas). CRISPR is a new era for genome modification, and this technology has bypassed the limitations of previous methods such as zinc-finger nuclease and transcription activator-like effector nuclease. Currently, this method is becoming the method of choice for gene-editing purposes, especially therapeutic gene editing in diseases such as cardiovascular, neurological, renal, genetic, optical, and stem cell, as well as blood disorders and muscular degeneration. However, finding the optimum delivery system capable of carrying this large complex persists as the main challenge of this technology. Therefore, it would be ideal if the delivery vehicle could direct the introduction of editing functions to specific cells in a multicellular organism. Exosomes are membrane-bound vesicles with high biocompatibility and low immunogenicity; they offer the best and most reliable way to fill the CRISPR/Cas9 system delivery gap. This review presents the current evidence on the molecular mechanisms and challenges of CRISPR/Cas9-mediated genome modification. Also, the role of CRISPR/Cas9 in the development of treatment and diagnosis of numerous disorders, from malignancies to viral infections, has been discussed. Lastly, the focus is on new advances in exosome-delivery technologies that may play a role in CRISPR/Cas9 delivery for future clinical settings.
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Affiliation(s)
- Cynthia Aslan
- Research Center for Integrative Medicine in Aging, Aging Research Institute, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Naime Majidi Zolbanin
- Experimental and Applied Pharmaceutical Sciences Research Center, Urmia University of Medical Sciences, Urmia, Iran
- Department of Pharmacology and Toxicology, School of Pharmacy, Urmia University of Medical Sciences, Urmia, Iran
| | - Fatemeh Faraji
- Hazrat-e Rasool General Hospital, Antimicrobial Resistance Research Center, Institute of Immunology and Infectious Diseases, Iran University of Medical Sciences, Floor 3, Building No. 3, Niyayesh St, Sattar Khan St, Tehran, 1445613131, Iran.
| | - Reza Jafari
- Cellular and Molecular Research Center, Cellular and Molecular Medicine Research Institute, Clinical Research Institute, Urmia University of Medical Sciences, Shafa St., Ershad Blvd., P.O. Box: 1138, Urmia, 57147, Iran.
- Department of Immunology and Genetics, Faculty of Medicine, Urmia University of Medical Sciences, Urmia, Iran.
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Banda A, Impomeni O, Singh A, Baloch AR, Hu W, Jaijyan DK. Precision in Action: The Role of Clustered Regularly Interspaced Short Palindromic Repeats/Cas in Gene Therapies. Vaccines (Basel) 2024; 12:636. [PMID: 38932365 PMCID: PMC11209408 DOI: 10.3390/vaccines12060636] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2024] [Revised: 05/21/2024] [Accepted: 06/04/2024] [Indexed: 06/28/2024] Open
Abstract
Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated enzyme-CAS holds great promise for treating many uncured human diseases and illnesses by precisely correcting harmful point mutations and disrupting disease-causing genes. The recent Food and Drug Association (FDA) approval of the first CRISPR-based gene therapy for sickle cell anemia marks the beginning of a new era in gene editing. However, delivering CRISPR specifically into diseased cells in vivo is a significant challenge and an area of intense research. The identification of new CRISPR/Cas variants, particularly ultra-compact CAS systems with robust gene editing activities, paves the way for the low-capacity delivery vectors to be used in gene therapies. CRISPR/Cas technology has evolved beyond editing DNA to cover a wide spectrum of functionalities, including RNA targeting, disease diagnosis, transcriptional/epigenetic regulation, chromatin imaging, high-throughput screening, and new disease modeling. CRISPR/Cas can be used to engineer B-cells to produce potent antibodies for more effective vaccines and enhance CAR T-cells for the more precise and efficient targeting of tumor cells. However, CRISPR/Cas technology has challenges, including off-target effects, toxicity, immune responses, and inadequate tissue-specific delivery. Overcoming these challenges necessitates the development of a more effective and specific CRISPR/Cas delivery system. This entails strategically utilizing specific gRNAs in conjunction with robust CRISPR/Cas variants to mitigate off-target effects. This review seeks to delve into the intricacies of the CRISPR/Cas mechanism, explore progress in gene therapies, evaluate gene delivery systems, highlight limitations, outline necessary precautions, and scrutinize the ethical considerations associated with its application.
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Affiliation(s)
- Amrutha Banda
- Department of Biology, The College of New Jersey, Ewing Township, NJ 08618, USA
| | - Olivia Impomeni
- Department of Biology, The College of New Jersey, Ewing Township, NJ 08618, USA
| | - Aparana Singh
- Department of Chemistry, National Institute of Technology Agartala, Agartala 799046, India;
| | - Abdul Rasheed Baloch
- Department of Anatomy and Neurobiology, School of Medicine, Virginia Commonwealth University, Richmond, VA 23284, USA;
| | - Wenhui Hu
- Department of Anatomy and Neurobiology, School of Medicine, Virginia Commonwealth University, Richmond, VA 23284, USA;
| | - Dabbu Kumar Jaijyan
- Department of Anatomy and Neurobiology, School of Medicine, Virginia Commonwealth University, Richmond, VA 23284, USA;
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Alayoubi AM, Khawaji ZY, Mohammed MA, Mercier FE. CRISPR-Cas9 system: a novel and promising era of genotherapy for beta-hemoglobinopathies, hematological malignancy, and hemophilia. Ann Hematol 2024; 103:1805-1817. [PMID: 37736806 DOI: 10.1007/s00277-023-05457-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Accepted: 09/15/2023] [Indexed: 09/23/2023]
Abstract
Gene therapy represents a significant potential to revolutionize the field of hematology with applications in correcting genetic mutations, generating cell lines and animal models, and improving the feasibility and efficacy of cancer immunotherapy. Compared to different genetic engineering tools, clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9) emerged as an effective and versatile genetic editor with the ability to precisely modify the genome. The applications of genetic engineering in various hematological disorders have shown encouraging results. Monogenic hematological disorders can conceivably be corrected with single gene modification. Through the use of CRISPR-CAS9, restoration of functional red blood cells and hemostasis factors were successfully attained in sickle cell anemia, beta-thalassemia, and hemophilia disorders. Our understanding of hemato-oncology has been advanced via CRIPSR-CAS9 technology. CRISPR-CAS9 aided to build a platform of mutated genes responsible for cell survival and proliferation in leukemia. Therapeutic application of CRISPR-CAS9 when combined with chimeric antigen receptor (CAR) T cell therapy in multiple myeloma and acute lymphoblastic leukemia was feasible with attenuation of CAR T cell therapy pitfalls. Our review outlines the latest literature on the utilization of CRISPR-Cas9 in the treatment of beta-hemoglobinopathies and hemophilia disorders. We present the strategies that were employed and the findings of preclinical and clinical trials. Also, the review will discuss gene engineering in the field of hemato-oncology as a proper tool to facilitate and overcome the drawbacks of chimeric antigen receptor T cell therapy (CAR-T).
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Affiliation(s)
- Abdulfatah M Alayoubi
- Department of Biochemistry and Molecular Medicine, College of Medicine, Taibah University, Madinah, Saudi Arabia
| | | | | | - François E Mercier
- Divisions of Experimental Medicine & Hematology, Department of Medicine, Faculty of Medicine, McGill University, Montreal, Quebec, Canada
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Zangi AR, Amiri A, Pazooki P, Soltanmohammadi F, Hamishehkar H, Javadzadeh Y. Non-viral and viral delivery systems for hemophilia A therapy: recent development and prospects. Ann Hematol 2024; 103:1493-1511. [PMID: 37951852 DOI: 10.1007/s00277-023-05459-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Accepted: 09/17/2023] [Indexed: 11/14/2023]
Abstract
Recent advancements have focused on enhancing factor VIII half-life and refining its delivery methods, despite the well-established knowledge that factor VIII deficiency is the main clotting protein lacking in hemophilia. Consequently, both viral and non-viral delivery systems play a crucial role in enhancing the quality of life for hemophilia patients. The utilization of viral vectors and the manipulation of non-viral vectors through targeted delivery are significant advancements in the field of cellular and molecular therapies for hemophilia. These developments contribute to the progression of treatment strategies and hold great promise for improving the overall well-being of individuals with hemophilia. This review study comprehensively explores the application of viral and non-viral vectors in cellular (specifically T cell) and molecular therapy approaches, such as RNA, monoclonal antibody (mAb), and CRISPR therapeutics, with the aim of addressing the challenges in hemophilia treatment. By examining these innovative strategies, the study aims to shed light on potential solutions to enhance the efficacy and outcomes of hemophilia therapy.
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Affiliation(s)
- Ali Rajabi Zangi
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
- Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, 5166-15731, Iran
| | - Ala Amiri
- Department of Biotechnology, Faculty of Biological Sciences, Alzahra University, Tehran, Iran
| | - Pouya Pazooki
- Cellular and Molecular Biology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Fatemeh Soltanmohammadi
- Student Research Committee, Tabriz University of Medical Sciences, Tabriz, Iran
- Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, 5166-15731, Iran
| | - Hamed Hamishehkar
- Drug Applied Research Center, Tabriz University of Medical Science, Tabriz, 5166-15731, Iran
| | - Yousef Javadzadeh
- Department of Pharmaceutics, Faculty of Pharmacy, Tabriz University of Medical Sciences, Tabriz, 5166-15731, Iran.
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Xiao R, Chen Y, Hu Z, Tang Q, Wang P, Zhou M, Wu L, Liang D. Identification of the Efficient Enhancer Elements in FVIII-Padua for Gene Therapy Study of Hemophilia A. Int J Mol Sci 2024; 25:3635. [PMID: 38612447 PMCID: PMC11011560 DOI: 10.3390/ijms25073635] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/13/2024] [Revised: 03/13/2024] [Accepted: 03/21/2024] [Indexed: 04/14/2024] Open
Abstract
Hemophilia A (HA) is a common X-linked recessive hereditary bleeding disorder. Coagulation factor VIII (FVIII) is insufficient in patients with HA due to the mutations in the F8 gene. The restoration of plasma levels of FVIII via both recombinant B-domain-deleted FVIII (BDD-FVIII) and B-domain-deleted F8 (BDDF8) transgenes was proven to be helpful. FVIII-Padua is a 23.4 kb tandem repeat mutation in the F8 associated with a high F8 gene expression and thrombogenesis. Here we screened a core enhancer element in FVIII-Padua for improving the F8 expression. In detail, we identified a 400 bp efficient enhancer element, C400, in FVIII-Padua for the first time. The core enhancer C400 extensively improved the transcription of BDDF8 driven by human elongation factor-1 alpha in HepG2, HeLa, HEK-293T and induced pluripotent stem cells (iPSCs) with different genetic backgrounds, as well as iPSCs-derived endothelial progenitor cells (iEPCs) and iPSCs-derived mesenchymal stem cells (iMSCs). The expression of FVIII protein was increased by C400, especially in iEPCs. Our research provides a novel molecular target to enhance expression of FVIII protein, which has scientific value and application prospects in both viral and nonviral HA gene therapy strategies.
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Affiliation(s)
| | | | | | | | | | | | | | - Desheng Liang
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China; (R.X.); (Y.C.); (Z.H.); (M.Z.)
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Lotfi M, Ashouri A, Mojarrad M, Mozaffari-Jovin S, Abbaszadegan MR. Design Principles of a Novel Construct for HBB Gene-Editing and Investigation of Its Gene-Targeting Efficiency in HEK293 Cells. Mol Biotechnol 2024; 66:517-530. [PMID: 37266832 DOI: 10.1007/s12033-023-00739-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2023] [Accepted: 03/27/2023] [Indexed: 06/03/2023]
Abstract
Beta-thalassemia is one of the most common monogenic inherited disorders worldwide caused by different mutations in the hemoglobin subunit beta (HBB) gene. Genome-editing based on clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 system (CRISPR/Cas9) has raised the hope for life-long gene therapy of beta-thalassemia. In a proof-of-concept study, we describe the detailed design and assess the efficacy of a novel homology-directed repair (HDR)-based CRISPR construct for targeting the HBB locus. The selected sgRNAs were designed and cloned into an optimized CRISPR plasmid. The HDR donor templates containing a reporter and a selection marker flanked by the piggyBac Inverted Tandem Repeat (ITRs), the homology arms and the delta thymidine kinase (ΔTK) gene for negative selection were constructed. The efficiency of on-target mutagenesis by the eSpCas9/sgRNAs was assessed by mismatch assays. HDR-positive cells were isolated by treatment with G418 or selection based on truncated Neuron Growth Factor Receptor (tNGFR) expression using the Magnetic Activated Cell Sorting (MACS) method followed by ganciclovir (GCV) treatment to eliminate cells with random genomic integration of the HDR donor template. In-out PCR and sanger sequencing confirmed HDR in the isolated cells. Our data showed ~ 50% efficiency for co-transfection of CRISPR/donor template plasmids in HEK293 cells and following G418 treatment, the HDR efficiency was detected at ~ 37.5%. Moreover, using a clinically-relevant strategy, HDR events were validated after selection for tNGFR+ cells followed by negative selection for ΔTK by GCV treatment. Thus, our HDR-based gene-editing strategy could efficiently target the HBB locus and enrich for HDR-positive cells.
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Affiliation(s)
- Malihe Lotfi
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Atefeh Ashouri
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Majid Mojarrad
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Sina Mozaffari-Jovin
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
| | - Mohammad Reza Abbaszadegan
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
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12
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Nuttle X, Burt ND, Currall B, Moysés-Oliveira M, Mohajeri K, Bhavsar R, Lucente D, Yadav R, Tai DJC, Gusella JF, Talkowski ME. Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries. CELL REPORTS METHODS 2024; 4:100672. [PMID: 38091988 PMCID: PMC10831954 DOI: 10.1016/j.crmeth.2023.100672] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/21/2023] [Revised: 08/14/2023] [Accepted: 11/21/2023] [Indexed: 01/25/2024]
Abstract
New technologies and large-cohort studies have enabled novel variant discovery and association at unprecedented scale, yet functional characterization of these variants remains paramount to deciphering disease mechanisms. Approaches that facilitate parallelized genome editing of cells of interest or induced pluripotent stem cells (iPSCs) have become critical tools toward this goal. Here, we developed an approach that incorporates libraries of CRISPR-Cas9 guide RNAs (gRNAs) together with inducible Cas9 into a piggyBac (PB) transposon system to engineer dozens to hundreds of genomic variants in parallel against isogenic cellular backgrounds. This method empowers loss-of-function (LoF) studies through the introduction of insertions or deletions (indels) and copy-number variants (CNVs), though generating specific nucleotide changes is possible with prime editing. The ability to rapidly establish high-quality mutational models at scale will facilitate the development of isogenic cellular collections and catalyze comparative functional genomic studies investigating the roles of hundreds of genes and mutations in development and disease.
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Affiliation(s)
- Xander Nuttle
- Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Department of Neurology, Harvard Medical School, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA.
| | - Nicholas D Burt
- Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA
| | - Benjamin Currall
- Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA
| | - Mariana Moysés-Oliveira
- Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Department of Neurology, Harvard Medical School, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA
| | - Kiana Mohajeri
- Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA; PhD program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA, USA
| | - Riya Bhavsar
- Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Department of Neurology, Harvard Medical School, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA
| | - Diane Lucente
- Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA
| | - Rachita Yadav
- Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Department of Neurology, Harvard Medical School, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA
| | - Derek J C Tai
- Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Department of Neurology, Harvard Medical School, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA
| | - James F Gusella
- Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA; Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, USA; Harvard Stem Cell Institute, Cambridge, MA, USA
| | - Michael E Talkowski
- Center for Genomic Medicine and Department of Neurology, Massachusetts General Hospital, Boston, MA, USA; Department of Neurology, Harvard Medical School, Boston, MA, USA; Stanley Center for Psychiatric Research, Broad Institute, Cambridge, MA, USA; Program in Medical and Population Genetics, Broad Institute, Cambridge, MA, USA.
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13
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Punetha M, Saini S, Chaudhary S, Yadav PS, Whitworth K, Green J, Kumar D, Kues WA. Induced Pluripotent Stem Cells in the Era of Precise Genome Editing. Curr Stem Cell Res Ther 2024; 19:307-315. [PMID: 36880183 DOI: 10.2174/1574888x18666230307115326] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2022] [Revised: 11/22/2022] [Accepted: 12/06/2022] [Indexed: 03/08/2023]
Abstract
Genome editing has enhanced our ability to understand the role of genetics in a number of diseases by facilitating the development of more precise cellular and animal models to study pathophysiological processes. These advances have shown extraordinary promise in a multitude of areas, from basic research to applied bioengineering and biomedical research. Induced pluripotent stem cells (iPSCs) are known for their high replicative capacity and are excellent targets for genetic manipulation as they can be clonally expanded from a single cell without compromising their pluripotency. Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR/Cas RNA-guided nucleases have rapidly become the method of choice for gene editing due to their high specificity, simplicity, low cost, and versatility. Coupling the cellular versatility of iPSCs differentiation with CRISPR/Cas9-mediated genome editing technology can be an effective experimental technique for providing new insights into the therapeutic use of this technology. However, before using these techniques for gene therapy, their therapeutic safety and efficacy following models need to be assessed. In this review, we cover the remarkable progress that has been made in the use of genome editing tools in iPSCs, their applications in disease research and gene therapy as well as the hurdles that remain in the actual implementation of CRISPR/Cas systems.
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Affiliation(s)
- Meeti Punetha
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, 125001, Haryana, India
| | - Sheetal Saini
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, 125001, Haryana, India
| | - Suman Chaudhary
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, 125001, Haryana, India
| | - Prem Singh Yadav
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, 125001, Haryana, India
| | - Kristin Whitworth
- Division of Animal Sciences, University of Missouri, Columbia, MO, 65211, USA
| | - Jonathan Green
- Division of Animal Sciences, University of Missouri, Columbia, MO, 65211, USA
| | - Dharmendra Kumar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar, 125001, Haryana, India
| | - Wilfried A Kues
- Department of Biotechnology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Höltystr 10, 31535, Neustadt, Germany
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14
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Ragni MV, Chan SY. Innovations in RNA therapy for hemophilia. Blood 2023; 142:1613-1621. [PMID: 37478403 PMCID: PMC10862240 DOI: 10.1182/blood.2022018661] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Revised: 06/05/2023] [Accepted: 06/27/2023] [Indexed: 07/23/2023] Open
Abstract
Given the shortcomings of current factor-, nonfactor-, and adeno-associated virus gene-based therapies, the recent advent of RNA-based therapeutics for hemophilia is changing the fundamental approach to hemophilia management. From small interfering RNA therapeutics that knockdown clot regulators antithrombin, protein S, and heparin cofactor II, to CRISPR/Cas9 gene editing that may personalize treatment, improved technologies have the potential to reduce bleeds and factor use and avoid inhibitor formation. These novel agents, some in preclinical studies and others in early phase trials, have the potential to simplify treatment and improve hemostasis and quality of life. Furthermore, because these therapies arise from manipulation of the coagulation cascade and thrombin generation and its regulation, they will enhance our understanding of hemostasis and thrombosis and ultimately lead to better therapies for children and adults with inherited bleeding disorders. What does the future hold? With the development of novel preclinical technologies at the bench, there will be fewer joint bleeds, debilitating joint disease, orthopedic surgery, and improved physical and mental health, which were not previously possible. In this review, we identify current limitations of treatment and progress in the development of novel RNA therapeutics, including messenger RNA nanoparticle delivery and gene editing for the treatment of hemophilia.
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Affiliation(s)
- Margaret V. Ragni
- Division of Hematology Oncology, Department of Medicine, University of Pittsburgh, Hemophilia Center of Western Pennsylvania, Pittsburgh, PA
| | - Stephen Y. Chan
- Division of Cardiology, Department of Medicine, Vascular Medicine Institute, Pittsburgh, PA
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15
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Lotfi M, Morshedi Rad D, Mashhadi SS, Ashouri A, Mojarrad M, Mozaffari-Jovin S, Farrokhi S, Hashemi M, Lotfi M, Ebrahimi Warkiani M, Abbaszadegan MR. Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells. Stem Cell Rev Rep 2023; 19:2576-2596. [PMID: 37723364 PMCID: PMC10661828 DOI: 10.1007/s12015-023-10585-3] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 06/30/2023] [Indexed: 09/20/2023]
Abstract
Rapid advancement in genome editing technologies has provided new promises for treating neoplasia, cardiovascular, neurodegenerative, and monogenic disorders. Recently, the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has emerged as a powerful gene editing tool offering advantages, including high editing efficiency and low cost over the conventional approaches. Human pluripotent stem cells (hPSCs), with their great proliferation and differentiation potential into different cell types, have been exploited in stem cell-based therapy. The potential of hPSCs and the capabilities of CRISPR/Cas9 genome editing has been paradigm-shifting in medical genetics for over two decades. Since hPSCs are categorized as hard-to-transfect cells, there is a critical demand to develop an appropriate and effective approach for CRISPR/Cas9 delivery into these cells. This review focuses on various strategies for CRISPR/Cas9 delivery in stem cells.
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Affiliation(s)
- Malihe Lotfi
- Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Dorsa Morshedi Rad
- School of Biomedical Engineering, University of Technology Sydney, Sydney, Australia
| | - Samaneh Sharif Mashhadi
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Atefeh Ashouri
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Majid Mojarrad
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Sina Mozaffari-Jovin
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Shima Farrokhi
- Student Research Committee, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Maryam Hashemi
- Nanotechnology Research Center, Pharmaceutical Technology Institute, Mashhad University of Medical Sciences, Mashhad, Iran
- Department of Pharmaceutical Biotechnology, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad, Iran
| | - Marzieh Lotfi
- Department of Medical Genetics, School of Medicine, Shahid Sadoughi University of Medical Sciences and Health Services, Yazd, Iran
| | - Majid Ebrahimi Warkiani
- School of Biomedical Engineering, University of Technology Sydney, Sydney, Australia.
- Institute for Biomedical Materials and Devices (IBMD), Faculty of Science, University of Technology Sydney, Sydney, Australia.
| | - Mohammad Reza Abbaszadegan
- Medical Genetics Research Center, Mashhad University of Medical Sciences, Mashhad, Iran.
- Department of Medical Genetics and Molecular Medicine, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran.
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16
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De Pablo-Moreno JA, Miguel-Batuecas A, Rodríguez-Merchán EC, Liras A. Treatment of congenital coagulopathies, from biologic to biotechnological drugs: The relevance of gene editing (CRISPR/Cas). Thromb Res 2023; 231:99-111. [PMID: 37839151 DOI: 10.1016/j.thromres.2023.10.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2023] [Revised: 09/09/2023] [Accepted: 10/02/2023] [Indexed: 10/17/2023]
Abstract
Congenital coagulopathies have, throughout the history of medicine, been a focus of scientific study and of great interest as they constitute an alteration of one of the most important and conserved pathways of evolution. The first therapeutic strategies developed to address them were aimed at restoring the blood components lost during hemorrhage by administering whole blood or plasma. Later on, the use of cryoprecipitates was a significant breakthrough as it made it possible to decrease the volumes of blood infused. In the 1970' and 80', clotting factor concentrates became the treatment and, from the 1990's to the present day, recombinant factors -with increasingly longer half-lives- have taken over as the treatment of choice for certain coagulopathies in a seamless yet momentous transition from biological to biotechnological drugs. The beginning of this century, however, saw the emergence of new advanced (gene and cell) treatments, which are currently transforming the therapeutic landscape. The possibility to use cells and viruses as well as specific or bispecific antibodies as medicines is likely to spark a revolution in the world of pharmacology where therapies will be individualized and have long-term effects. Specifically, attention is nowadays focused on the development of gene editing strategies, chiefly those based on CRISPR/Cas technology. Rare coagulopathies such as hemophilia A and B, or even ultra-rare ones such as factor V deficiency, could be among those deriving the greatest benefit from these new developments.
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Affiliation(s)
- Juan A De Pablo-Moreno
- Department of Genetic, Physiology and Microbiology, Biology School, Complutense University of Madrid, Spain
| | - Andrea Miguel-Batuecas
- Department of Genetic, Physiology and Microbiology, Biology School, Complutense University of Madrid, Spain
| | - E Carlos Rodríguez-Merchán
- Osteoarticular Surgery Research, Hospital La Paz Institute for Health Research-IdiPAZ (La Paz University Hospital-Autonomous University of Madrid), Spain
| | - Antonio Liras
- Department of Genetic, Physiology and Microbiology, Biology School, Complutense University of Madrid, Spain.
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17
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Xia K, Wang F, Tan Z, Zhang S, Lai X, Ou W, Yang C, Chen H, Peng H, Luo P, Hu A, Tu X, Wang T, Ke Q, Deng C, Xiang AP. Precise Correction of Lhcgr Mutation in Stem Leydig Cells by Prime Editing Rescues Hereditary Primary Hypogonadism in Mice. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2023; 10:e2300993. [PMID: 37697644 PMCID: PMC10582410 DOI: 10.1002/advs.202300993] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/13/2023] [Revised: 07/20/2023] [Indexed: 09/13/2023]
Abstract
Hereditary primary hypogonadism (HPH), caused by gene mutation related to testosterone synthesis in Leydig cells, usually impairs male sexual development and spermatogenesis. Genetically corrected stem Leydig cells (SLCs) transplantation may provide a new approach for treating HPH. Here, a novel nonsense-point-mutation mouse model (LhcgrW495X ) is first generated based on a gene mutation relative to HPH patients. To verify the efficacy and feasibility of SLCs transplantation in treating HPH, wild-type SLCs are transplanted into LhcgrW495X mice, in which SLCs obviously rescue HPH phenotypes. Through comparing several editing strategies, optimized PE2 protein (PEmax) system is identified as an efficient and precise approach to correct the pathogenic point mutation in Lhcgr. Furthermore, delivering intein-split PEmax system via lentivirus successfully corrects the mutation in SLCs from LhcgrW495X mice ex vivo. Gene-corrected SLCs from LhcgrW495X mice exert ability to differentiate into functional Leydig cells in vitro. Notably, the transplantation of gene-corrected SLCs effectively regenerates Leydig cells, recovers testosterone production, restarts sexual development, rescues spermatogenesis, and produces fertile offspring in LhcgrW495X mice. Altogether, these results suggest that PE-based gene editing in SLCs ex vivo is a promising strategy for HPH therapy and is potentially leveraged to address more hereditary diseases in reproductive system.
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Affiliation(s)
- Kai Xia
- Center for Stem Cell Biology and Tissue EngineeringKey Laboratory for Stem Cells and Tissue EngineeringMinistry of Education National‐Local Joint Engineering Research Center for Stem Cells and Regenerative Medicine Zhongshan School of MedicineSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Fulin Wang
- Department of Urology and AndrologyThe First Affiliated HospitalSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Zhipeng Tan
- Center for Stem Cell Biology and Tissue EngineeringKey Laboratory for Stem Cells and Tissue EngineeringMinistry of Education National‐Local Joint Engineering Research Center for Stem Cells and Regenerative Medicine Zhongshan School of MedicineSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Suyuan Zhang
- Center for Stem Cell Biology and Tissue EngineeringKey Laboratory for Stem Cells and Tissue EngineeringMinistry of Education National‐Local Joint Engineering Research Center for Stem Cells and Regenerative Medicine Zhongshan School of MedicineSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Xingqiang Lai
- Cardiovascular DepartmentThe Eighth Affiliated HospitalSun Yat‐sen UniversityShenzhenGuangdong518033China
| | - Wangsheng Ou
- State Key Laboratory of Ophthalmology Zhong Shan Ophthalmic CenterSun Yat‐sen UniversityGuangzhouGuangdong510000China
| | - Cuifeng Yang
- Department of Urology and AndrologyThe First Affiliated HospitalSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Hong Chen
- Center for Stem Cell Biology and Tissue EngineeringKey Laboratory for Stem Cells and Tissue EngineeringMinistry of Education National‐Local Joint Engineering Research Center for Stem Cells and Regenerative Medicine Zhongshan School of MedicineSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Hao Peng
- Center for Stem Cell Biology and Tissue EngineeringKey Laboratory for Stem Cells and Tissue EngineeringMinistry of Education National‐Local Joint Engineering Research Center for Stem Cells and Regenerative Medicine Zhongshan School of MedicineSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Peng Luo
- Department of Urology and AndrologyThe First Affiliated HospitalSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Anqi Hu
- Center for Stem Cell Biology and Tissue EngineeringKey Laboratory for Stem Cells and Tissue EngineeringMinistry of Education National‐Local Joint Engineering Research Center for Stem Cells and Regenerative Medicine Zhongshan School of MedicineSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Xiang'an Tu
- Department of Urology and AndrologyThe First Affiliated HospitalSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Tao Wang
- Center for Stem Cell Biology and Tissue EngineeringKey Laboratory for Stem Cells and Tissue EngineeringMinistry of Education National‐Local Joint Engineering Research Center for Stem Cells and Regenerative Medicine Zhongshan School of MedicineSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Qiong Ke
- Center for Stem Cell Biology and Tissue EngineeringKey Laboratory for Stem Cells and Tissue EngineeringMinistry of Education National‐Local Joint Engineering Research Center for Stem Cells and Regenerative Medicine Zhongshan School of MedicineSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Chunhua Deng
- Department of Urology and AndrologyThe First Affiliated HospitalSun Yat‐sen UniversityGuangzhouGuangdong510080China
| | - Andy Peng Xiang
- Center for Stem Cell Biology and Tissue EngineeringKey Laboratory for Stem Cells and Tissue EngineeringMinistry of Education National‐Local Joint Engineering Research Center for Stem Cells and Regenerative Medicine Zhongshan School of MedicineSun Yat‐sen UniversityGuangzhouGuangdong510080China
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18
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Selin C, Lambert L, Morain S, Nelson JP, Barlevy D, Farooque M, Manley H, Scott CT. Researching the future: scenarios to explore the future of human genome editing. BMC Med Ethics 2023; 24:72. [PMID: 37735670 PMCID: PMC10512597 DOI: 10.1186/s12910-023-00951-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Accepted: 09/04/2023] [Indexed: 09/23/2023] Open
Abstract
BACKGROUND Forward-looking, democratically oriented governance is needed to ensure that human genome editing serves rather than undercuts public values. Scientific, policy, and ethics communities have recognized this necessity but have demonstrated limited understanding of how to fulfill it. The field of bioethics has long attempted to grapple with the unintended consequences of emerging technologies, but too often such foresight has lacked adequate scientific grounding, overemphasized regulation to the exclusion of examining underlying values, and failed to adequately engage the public. METHODS This research investigates the application of scenario planning, a tool developed in the high-stakes, uncertainty-ridden world of corporate strategy, for the equally high-stakes and uncertain world of the governance of emerging technologies. The scenario planning methodology is non-predictive, looking instead at a spread of plausible futures which diverge in their implications for different communities' needs, cares, and desires. RESULTS In this article we share how the scenario development process can further understandings of the complex and dynamic systems which generate and shape new biomedical technologies and provide opportunities to re-examine and re-think questions of governance, ethics and values. We detail the results of a year-long scenario planning study that engaged experts from the biological sciences, bioethics, social sciences, law, policy, private industry, and civic organizations to articulate alternative futures of human genome editing. CONCLUSIONS Through sharing and critiquing our methodological approach and results of this study, we advance understandings of anticipatory methods deployed in bioethics, demonstrating how this approach provides unique insights and helps to derive better research questions and policy strategies.
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Affiliation(s)
- Cynthia Selin
- School for the Future of Innovation in Society at Arizona State University, PO Box 876002, 85287-6002, Tempe, AZ, USA.
| | - Lauren Lambert
- School of Sustainability at Arizona State University, 4th floor, Walton Center for Planetary Health, 85281, Tempe, AZ, USA
| | - Stephanie Morain
- Berman Institute of Bioethics, Johns Hopkins University, 1809 Ashland Ave, 21212, Baltimore, MD, USA
| | - John P Nelson
- School of Public Policy, Georgia Institute of Technology, 685 Cherry St., Suite 107, 30332, Atlanta, GA, USA
| | - Dorit Barlevy
- Center for Medical Ethics and Health Policy, Baylor College of Medicine, One Baylor Plaza, Suite 310D, 77030, Houston, TX, USA
| | - Mahmud Farooque
- Consortium for Science, Policy and Outcomes, Arizona State University, 1800 I Street, 20006, Washington, DC, USA
| | - Haley Manley
- Center for Medical Ethics and Health Policy, Baylor College of Medicine, One Baylor Plaza, Suite 310D, 77030, Houston, TX, USA
| | - Christopher T Scott
- Center for Medical Ethics and Health Policy, Baylor College of Medicine, One Baylor Plaza, Suite 310D, 77030, Houston, TX, USA
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19
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Ebrahimi S, Khosravi MA, Raz A, Karimipoor M, Parvizi P. CRISPR-Cas Technology as a Revolutionary Genome Editing tool: Mechanisms and Biomedical Applications. IRANIAN BIOMEDICAL JOURNAL 2023; 27:219-46. [PMID: 37873636 PMCID: PMC10707817 DOI: 10.61186/ibj.27.5.219] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 02/01/2023] [Accepted: 06/14/2023] [Indexed: 12/17/2023]
Abstract
Programmable nucleases are powerful genomic tools for precise genome editing. These tools precisely recognize, remove, or change DNA at a defined site, thereby, stimulating cellular DNA repair pathways that can cause mutations or accurate replacement or deletion/insertion of a sequence. CRISPR-Cas9 system is the most potent and useful genome editing technique adapted from the defense immune system of certain bacteria and archaea against viruses and phages. In the past decade, this technology made notable progress, and at present, it has largely been used in genome manipulation to make precise gene editing in plants, animals, and human cells. In this review, we aim to explain the basic principle, mechanisms of action, and applications of this system in different areas of medicine, with emphasizing on the detection and treatment of parasitic diseases.
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Affiliation(s)
- Sahar Ebrahimi
- Molecular Systematics Laboratory, Parasitology Department, Pasteur Institute of Iran, Tehran, Iran
- Molecular Medicine Department, Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran
| | - Mohammad Ali Khosravi
- Molecular Medicine Department, Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran
| | - Abbasali Raz
- Malaria and Vector Research Group (MVRG), Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran
| | - Morteza Karimipoor
- Molecular Medicine Department, Biotechnology Research Center (BRC), Pasteur Institute of Iran, Tehran, Iran
| | - Parviz Parvizi
- Molecular Systematics Laboratory, Parasitology Department, Pasteur Institute of Iran, Tehran, Iran
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20
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Adlat S, Vázquez Salgado AM, Lee M, Yin D, Wangensteen KJ. Emerging and potential use of CRISPR in human liver disease. Hepatology 2023:01515467-990000000-00538. [PMID: 37607734 PMCID: PMC10881897 DOI: 10.1097/hep.0000000000000578] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/17/2023] [Accepted: 08/13/2023] [Indexed: 08/24/2023]
Abstract
CRISPR is a gene editing tool adapted from naturally occurring defense systems from bacteria. It is a technology that is revolutionizing the interrogation of gene functions in driving liver disease, especially through genetic screens and by facilitating animal knockout and knockin models. It is being used in models of liver disease to identify which genes are critical for liver pathology, especially in genetic liver disease, hepatitis, and in cancer initiation and progression. It holds tremendous promise in treating human diseases directly by editing DNA. It could disable gene function in the case of expression of a maladaptive protein, such as blocking transthyretin as a therapy for amyloidosis, or to correct gene defects, such as restoring the normal functions of liver enzymes fumarylacetoacetate hydrolase or alpha-1 antitrypsin. It is also being studied for treatment of hepatitis B infection. CRISPR is an exciting, evolving technology that is facilitating gene characterization and discovery in liver disease and holds the potential to treat liver diseases safely and permanently.
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Affiliation(s)
- Salah Adlat
- Division of Gastroenterology and Hepatology, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA
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21
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Elhawary NA, AlJahdali IA, Abumansour IS, Azher ZA, Falemban AH, Madani WM, Alosaimi W, Alghamdi G, Sindi IA. Phenotypic variability to medication management: an update on fragile X syndrome. Hum Genomics 2023; 17:60. [PMID: 37420260 PMCID: PMC10329374 DOI: 10.1186/s40246-023-00507-2] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2023] [Accepted: 07/03/2023] [Indexed: 07/09/2023] Open
Abstract
This review discusses the discovery, epidemiology, pathophysiology, genetic etiology, molecular diagnosis, and medication-based management of fragile X syndrome (FXS). It also highlights the syndrome's variable expressivity and common comorbid and overlapping conditions. FXS is an X-linked dominant disorder associated with a wide spectrum of clinical features, including but not limited to intellectual disability, autism spectrum disorder, language deficits, macroorchidism, seizures, and anxiety. Its prevalence in the general population is approximately 1 in 5000-7000 men and 1 in 4000-6000 women worldwide. FXS is associated with the fragile X messenger ribonucleoprotein 1 (FMR1) gene located at locus Xq27.3 and encodes the fragile X messenger ribonucleoprotein (FMRP). Most individuals with FXS have an FMR1 allele with > 200 CGG repeats (full mutation) and hypermethylation of the CpG island proximal to the repeats, which silences the gene's promoter. Some individuals have mosaicism in the size of the CGG repeats or in hypermethylation of the CpG island, both produce some FMRP and give rise to milder cognitive and behavioral deficits than in non-mosaic individuals with FXS. As in several monogenic disorders, modifier genes influence the penetrance of FMR1 mutations and FXS's variable expressivity by regulating the pathophysiological mechanisms related to the syndrome's behavioral features. Although there is no cure for FXS, prenatal molecular diagnostic testing is recommended to facilitate early diagnosis. Pharmacologic agents can reduce some behavioral features of FXS, and researchers are investigating whether gene editing can be used to demethylate the FMR1 promoter region to improve patient outcomes. Moreover, clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 and developed nuclease defective Cas9 (dCas9) strategies have promised options of genome editing in gain-of-function mutations to rewrite new genetic information into a specified DNA site, are also being studied.
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Affiliation(s)
- Nasser A. Elhawary
- Department of Medical Genetics, College of Medicine, Umm Al-Qura University, Mecca, 21955 Saudi Arabia
| | - Imad A. AlJahdali
- Department of Community Medicine, College of Medicine, Umm Al-Qura University, Mecca, Saudi Arabia
| | - Iman S. Abumansour
- Department of Medical Genetics, College of Medicine, Umm Al-Qura University, Mecca, 21955 Saudi Arabia
| | - Zohor A. Azher
- Department of Medical Genetics, College of Medicine, Umm Al-Qura University, Mecca, 21955 Saudi Arabia
| | - Alaa H. Falemban
- Department of Pharmacology and Toxicology, College of Medicine, Umm Al-Qura University, Mecca, 24382 Saudi Arabia
| | - Wefaq M. Madani
- Department of Hematology and Immunology, Faculty of Medicine, Umm Al-Qura University, Mecca, Saudi Arabia
| | - Wafaa Alosaimi
- Department of Hematology, Maternity and Children Hospital, Mecca, Saudi Arabia
| | - Ghydda Alghamdi
- Department of Medical Genetics, College of Medicine, Umm Al-Qura University, Mecca, 21955 Saudi Arabia
| | - Ikhlas A. Sindi
- Department of Biology, Faculty of Science, King Abdulaziz University, Jeddah, 21589 Saudi Arabia
- Preparatory Year Program, Batterjee Medical College, Jeddah, 21442 Saudi Arabia
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22
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Lučanský V, Holubeková V, Kolková Z, Halašová E, Samec M, Golubnitschaja O. Multi-faceted CRISPR/Cas technological innovation aspects in the framework of 3P medicine. EPMA J 2023; 14:201-217. [PMID: 37275547 PMCID: PMC10201107 DOI: 10.1007/s13167-023-00324-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Accepted: 05/10/2023] [Indexed: 06/07/2023]
Abstract
Since 2009, the European Association for Predictive, Preventive and Personalised Medicine (EPMA, Brussels) promotes the paradigm change from reactive approach to predictive, preventive, and personalized medicine (PPPM/3PM) to protect individuals in sub-optimal health conditions from the health-to-disease transition, to increase life-quality of the affected patient cohorts improving, therefore, ethical standards and cost-efficacy of healthcare to great benefits of the society at large. The gene-editing technology utilizing CRISPR/Cas gene-editing approach has demonstrated its enormous value as a powerful tool in a broad spectrum of bio/medical research areas. Further, CRISPR/Cas gene-editing system is considered applicable to primary and secondary healthcare, in order to prevent disease spread and to treat clinically manifested disorders, involving diagnostics of SARS-Cov-2 infection and experimental treatment of COVID-19. Although the principle of the proposed gene editing is simple and elegant, there are a lot of technological challenges and ethical considerations to be solved prior to its broadly scaled clinical implementation. This article highlights technological innovation beyond the state of the art, exemplifies current achievements, discusses unsolved technological and ethical problems, and provides clinically relevant outlook in the framework of 3PM.
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Affiliation(s)
- Vincent Lučanský
- Jessenius Faculty of Medicine in Martin (JFMED CU), Biomedical Center, Comenius University in Bratislava, Martin, Slovakia
| | - Veronika Holubeková
- Jessenius Faculty of Medicine in Martin (JFMED CU), Biomedical Center, Comenius University in Bratislava, Martin, Slovakia
| | - Zuzana Kolková
- Jessenius Faculty of Medicine in Martin (JFMED CU), Biomedical Center, Comenius University in Bratislava, Martin, Slovakia
| | - Erika Halašová
- Jessenius Faculty of Medicine in Martin (JFMED CU), Biomedical Center, Comenius University in Bratislava, Martin, Slovakia
| | - Marek Samec
- Department of Pathophysiology, Jessenius Faculty of Medicine, Comenius University in Bratislava, Martin, Slovakia
| | - Olga Golubnitschaja
- Predictive, Preventive, Personalised (3P) Medicine, Department of Radiation Oncology, University Hospital Bonn, Rheinische Friedrich-Wilhelms-Universität Bonn, 53127 Bonn, Germany
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Tang Q, Hu Z, Zhao J, Zhou T, Tang S, Wang P, Xiao R, Chen Y, Wu L, Zhou M, Liang D. CRISPR-Mediated In Situ Introduction or Integration of F9-Padua in Human iPSCs for Gene Therapy of Hemophilia B. Int J Mol Sci 2023; 24:ijms24109013. [PMID: 37240366 DOI: 10.3390/ijms24109013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2023] [Revised: 05/10/2023] [Accepted: 05/17/2023] [Indexed: 05/28/2023] Open
Abstract
Hemophilia B (HB) is an X-linked recessive disease caused by F9 gene mutation and functional coagulation factor IX (FIX) deficiency. Patients suffer from chronic arthritis and death threats owing to excessive bleeding. Compared with traditional treatments, gene therapy for HB has obvious advantages, especially when the hyperactive FIX mutant (FIX-Padua) is used. However, the mechanism by which FIX-Padua works remains ambiguous due to a lack of research models. Here, in situ introduction of F9-Padua mutation was performed in human induced pluripotent stem cells (hiPSCs) via CRISPR/Cas9 and single-stranded oligodeoxynucleotides (ssODNs). The hyperactivity of FIX-Padua was confirmed to be 364% of the normal level in edited hiPSCs-derived hepatocytes, providing a reliable model for exploring the mechanism of the hyperactivity of FIX-Padua. Moreover, the F9 cDNA containing F9-Padua was integrated before the F9 initiation codon by CRISPR/Cas9 in iPSCs from an HB patient (HB-hiPSCs). Integrated HB-hiPSCs after off-target screening were differentiated into hepatocytes. The FIX activity in the supernatant of integrated hepatocytes showed a 4.2-fold increase and reached 63.64% of the normal level, suggesting a universal treatment for HB patients with various mutations in F9 exons. Overall, our study provides new approaches for the exploration and development of cell-based gene therapy for HB.
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Affiliation(s)
- Qiyu Tang
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China
| | - Zhiqing Hu
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China
| | - Junya Zhao
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China
| | - Tao Zhou
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China
| | - Shuqing Tang
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China
| | - Peiyun Wang
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China
| | - Rou Xiao
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China
| | - Yan Chen
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China
| | - Lingqian Wu
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China
| | - Miaojin Zhou
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China
| | - Desheng Liang
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha 410078, China
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Bhatia S, Pooja, Yadav SK. CRISPR-Cas for genome editing: Classification, mechanism, designing and applications. Int J Biol Macromol 2023; 238:124054. [PMID: 36933595 DOI: 10.1016/j.ijbiomac.2023.124054] [Citation(s) in RCA: 44] [Impact Index Per Article: 22.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Revised: 02/24/2023] [Accepted: 03/12/2023] [Indexed: 03/18/2023]
Abstract
Clustered regularly interspersed short pallindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system (CRISPR-Cas) came into light as prokaryotic defence mechanism for adaptive immune response. CRISPR-Cas works by integrating short sequences of the target genome (spacers) into the CRISPR locus. The locus containing spacers interspersed repeats is further expressed into small guide CRISPR RNA (crRNA) which is then deployed by the Cas proteins to evade the target genome. Based on the Cas proteins CRISPR-Cas is classified according to polythetic system of classification. The characteristic of the CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new arenas due to which today CRISPR-Cas has evolved as cutting end technique in the field of genome editing. Here, we discuss about the evolution of CRISPR, its classification and various Cas systems including the designing and molecular mechanism of CRISPR-Cas. Applications of CRISPR-Cas as a genome editing tools are also highlighted in the areas such as agriculture, and anticancer therapy. Briefly discuss the role of CRISPR and its Cas systems in the diagnosis of COVID-19 and its possible preventive measures. The challenges in existing CRISP-Cas technologies and their potential solutions are also discussed briefly.
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Affiliation(s)
- Simran Bhatia
- Center of Innovative and applied Bioprocessing, Sector-81, Knowledge City, Mohali, India; Regional Center for Biotechnology, Faridabad, India
| | - Pooja
- Center of Innovative and applied Bioprocessing, Sector-81, Knowledge City, Mohali, India
| | - Sudesh Kumar Yadav
- Center of Innovative and applied Bioprocessing, Sector-81, Knowledge City, Mohali, India; Regional Center for Biotechnology, Faridabad, India.
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Hu Z, Wu Y, Xiao R, Zhao J, Chen Y, Wu L, Zhou M, Liang D. Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing. Front Genet 2023; 14:1115831. [PMID: 36968612 PMCID: PMC10033665 DOI: 10.3389/fgene.2023.1115831] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2022] [Accepted: 02/27/2023] [Indexed: 03/11/2023] Open
Abstract
Introduction: Hemophilia A (HA) is the most common genetic bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). As the second predominant pathogenic mutation in hemophilia A severe patients, F8 Intron one inversion (Inv1) completely splits the F8 gene into two parts and disrupts the F8 transcription, resulting in no FVIII protein production. The part which contains exon 2-exon 26 covers 98% of F8 coding region.Methods: We hypothesized that in situ genetic manipulation of F8 to add a promoter and exon one before the exon two could restore the F8 expression. The donor plasmid included human alpha 1-antitrypsin (hAAT) promoter, exon one and splicing donor site (SD) based on homology-mediated end joining (HMEJ) strategy was targeted addition in hemophilia A patient-derived induced pluripotent stem cell (HA-iPSCs) using CRISPR/Cas9. The iPSCs were differentiated into hepatocyte-like cells (HPLCs).Results: The hAAT promoter and exon one were targeted addition in HA-iPSCs with a high efficiency of 10.19% via HMEJ. The FVIII expression, secretion, and activity were detected in HPLCs derived from gene-targeted iPSCs.Discussion: Thus, we firstly rescued the 140 kb reversion mutation by gene addition of a 975 bp fragment in the HA-iPSCs with Inv1 mutation, providing a promising gene correction strategy for genetic disease with large sequence variants.
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Affiliation(s)
- Zhiqing Hu
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
| | - Yong Wu
- Shenzhen Baoan Women’s and Children’s Hospital, Jinan University, Shenzhen, China
| | - Rou Xiao
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
| | - Junya Zhao
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
| | - Yan Chen
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
| | - Lingqian Wu
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
| | - Miaojin Zhou
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
- *Correspondence: Miaojin Zhou, ; Desheng Liang,
| | - Desheng Liang
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
- *Correspondence: Miaojin Zhou, ; Desheng Liang,
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26
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Applying the CRISPR/Cas9 for treating human and animal diseases: a comprehensive review. ANNALS OF ANIMAL SCIENCE 2023. [DOI: 10.2478/aoas-2023-0009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 03/03/2023]
Abstract
Abstract
Recently, genome editing tools have been extensively used in many biomedical sciences. The gene editing system is applied to modify the DNA sequences in the cellular system to comprehend their physiological response. A developing genome editing technology like clustered regularly short palindromic repeats (CRISPR) is widely expended in medical sciences. CRISPR and CRISPR-associated protein 9 (CRISPR/Cas9) system is being exploited to edit any DNA mutations related to inherited ailments to investigate in animals (in vivo) and cell lines (in vitro). Remarkably, CRISPR/Cas9 could be employed to examine treatments of many human genetic diseases such as Cystic fibrosis, Tyrosinemia, Phenylketonuria, Muscular dystrophy, Parkinson’s disease, Retinoschisis, Hemophilia, β-Thalassemia and Atherosclerosis. Moreover, CRISPR/Cas9 was used for disease resistance such as Tuberculosis, Johne’s diseases, chronic enteritis, and Brucellosis in animals. Finally, this review discusses existing progress in treating hereditary diseases using CRISPR/Cas9 technology and the high points accompanying obstacles.
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Kim HJ, Kim G, Chi KY, Kim H, Jang YJ, Jo S, Lee J, Lee Y, Woo DH, Han C, Kim SK, Park HJ, Kim JH. Generation of multilineage liver organoids with luminal vasculature and bile ducts from human pluripotent stem cells via modulation of Notch signaling. Stem Cell Res Ther 2023; 14:19. [PMID: 36737811 PMCID: PMC9898924 DOI: 10.1186/s13287-023-03235-5] [Citation(s) in RCA: 16] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2022] [Accepted: 01/03/2023] [Indexed: 02/05/2023] Open
Abstract
BACKGROUND The generation of liver organoids recapitulating parenchymal and non-parenchymal cell interplay is essential for the precise in vitro modeling of liver diseases. Although different types of multilineage liver organoids (mLOs) have been generated from human pluripotent stem cells (hPSCs), the assembly and concurrent differentiation of multiple cell types in individual mLOs remain a major challenge. Particularly, most studies focused on the vascularization of mLOs in host tissue after transplantation in vivo. However, relatively little information is available on the in vitro formation of luminal vasculature in mLOs themselves. METHODS The mLOs with luminal blood vessels and bile ducts were generated by assembling hepatic endoderm, hepatic stellate cell-like cells (HscLCs), and endothelial cells derived entirely from hPSCs using 96-well ultra-low attachment plates. We analyzed the effect of HscLC incorporation and Notch signaling modulation on the formation of both bile ducts and vasculature in mLOs using immunofluorescence staining, qRT-PCR, ELISA, and live-perfusion imaging. The potential use of the mLOs in fibrosis modeling was evaluated by histological and gene expression analyses after treatment with pro-fibrotic cytokines. RESULTS We found that hPSC-derived HscLCs are crucial for generating functional microvasculature in mLOs. HscLC incorporation and subsequent vascularization substantially reduced apoptotic cell death and promoted the survival and growth of mLOs with microvessels. In particular, precise modulation of Notch signaling during a specific time window in organoid differentiation was critical for generating both bile ducts and vasculature. Live-cell imaging, a series of confocal scans, and electron microscopy demonstrated that blood vessels were well distributed inside mLOs and had perfusable lumens in vitro. In addition, exposure of mLOs to pro-fibrotic cytokines induced early fibrosis-associated events, including upregulation of genes associated with fibrotic induction and endothelial cell activation (i.e., collagen I, α-SMA, and ICAM) together with destruction of tissue architecture and organoid shrinkage. CONCLUSION Our results demonstrate that mLOs can reproduce parenchymal and non-parenchymal cell interactions and suggest that their application can advance the precise modeling of liver diseases in vitro.
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Affiliation(s)
- Hyo Jin Kim
- grid.222754.40000 0001 0840 2678Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul, 02841 South Korea
| | - Gyeongmin Kim
- grid.222754.40000 0001 0840 2678Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul, 02841 South Korea
| | - Kyun Yoo Chi
- grid.222754.40000 0001 0840 2678Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul, 02841 South Korea
| | - Hyemin Kim
- grid.418982.e0000 0004 5345 5340Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon, 34114 South Korea
| | - Yu Jin Jang
- grid.89336.370000 0004 1936 9924Department of Molecular Biosciences, The University of Texas at Austin, Austin, TX 78712 USA
| | - Seongyea Jo
- grid.222754.40000 0001 0840 2678Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul, 02841 South Korea ,grid.418982.e0000 0004 5345 5340Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon, 34114 South Korea
| | - Jihun Lee
- grid.222754.40000 0001 0840 2678Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul, 02841 South Korea
| | - Youngseok Lee
- grid.222754.40000 0001 0840 2678Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul, 02841 South Korea
| | - Dong-Hun Woo
- Department of Stem Cell Biology, NEXEL Co., Ltd, Seoul, 07802 South Korea
| | - Choongseong Han
- Department of Stem Cell Biology, NEXEL Co., Ltd, Seoul, 07802 South Korea
| | - Sang Kyum Kim
- grid.254230.20000 0001 0722 6377College of Pharmacy, Chungnam National University, Daejeon, 34134 South Korea
| | - Han-Jin Park
- grid.418982.e0000 0004 5345 5340Department of Predictive Toxicology, Korea Institute of Toxicology, Daejeon, 34114 South Korea
| | - Jong-Hoon Kim
- Laboratory of Stem Cells and Tissue Regeneration, Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-Ro, Seongbuk-Gu, Seoul, 02841, South Korea.
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Methods for CRISPR-Cas as Ribonucleoprotein Complex Delivery In Vivo. Mol Biotechnol 2023; 65:181-195. [PMID: 35322386 DOI: 10.1007/s12033-022-00479-z] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2021] [Accepted: 03/14/2022] [Indexed: 01/18/2023]
Abstract
The efficient delivery of CRISPR-Cas components is still a key and unsolved problem. CRISPR-Cas delivery in the form of a Cas protein+sgRNA (ribonucleoprotein complex, RNP complex), has proven to be extremely effective, since it allows to increase on-target activity, while reducing nonspecific activity. The key point for in vivo genome editing is the direct delivery of artificial nucleases and donor DNA molecules into the somatic cells of an adult organism. At the same time, control of the dose of artificial nucleases is impossible, which affects the efficiency of genome editing in the affected cells. Poor delivery efficiency and low editing efficacy reduce the overall potency of the in vivo genome editing process. Here we review how this problem is currently being solved in scientific works and what types of in vivo delivery methods of Cas9/sgRNA RNPs have been developed.
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29
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Ramamurthy RM, Rodriguez M, Ainsworth HC, Shields J, Meares D, Bishop C, Farland A, Langefeld CD, Atala A, Doering CB, Spencer HT, Porada CD, Almeida-Porada G. Comparison of different gene addition strategies to modify placental derived-mesenchymal stromal cells to produce FVIII. Front Immunol 2022; 13:954984. [PMID: 36591257 PMCID: PMC9800010 DOI: 10.3389/fimmu.2022.954984] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2022] [Accepted: 11/28/2022] [Indexed: 12/23/2022] Open
Abstract
Introduction Placenta-derived mesenchymal cells (PLCs) endogenously produce FVIII, which makes them ideally suited for cell-based fVIII gene delivery. We have previously reported that human PLCs can be efficiently modified with a lentiviral vector encoding a bioengineered, expression/secretion-optimized fVIII transgene (ET3) and durably produce clinically relevant levels of functionally active FVIII. The objective of the present study was to investigate whether CRISPR/Cas9 can be used to achieve location-specific insertion of a fVIII transgene into a genomic safe harbor, thereby eliminating the potential risks arising from the semi-random genomic integration inherent to lentiviral vectors. We hypothesized this approach would improve the safety of the PLC-based gene delivery platform and might also enhance the therapeutic effect by eliminating chromatin-related transgene silencing. Methods We used CRISPR/Cas9 to attempt to insert the bioengineered fVIII transgene "lcoET3" into the AAVS1 site of PLCs (CRISPR-lcoET3) and determined their subsequent levels of FVIII production, comparing results with this approach to those achieved using lentivector transduction (LV-lcoET3) and plasmid transfection (Plasmid-lcoET3). In addition, since liver-derived sinusoidal endothelial cells (LSECs) are the native site of FVIII production in the body, we also performed parallel studies in human (h)LSECs). Results PLCs and hLSECs can both be transduced (LV-lcoET3) with very high efficiency and produce high levels of biologically active FVIII. Surprisingly, both cell types were largely refractory to CRISPR/Cas9-mediated knockin of the lcoET3 fVIII transgene in the AAVS1 genome locus. However, successful insertion of an RFP reporter into this locus using an identical procedure suggests the failure to achieve knockin of the lcoET3 expression cassette at this site is likely a function of its large size. Importantly, using plasmids, alone or to introduce the CRISPR/Cas9 "machinery", resulted in dramatic upregulation of TLR 3, TLR 7, and BiP in PLCs, compromising their unique immune-inertness. Discussion Although we did not achieve our primary objective, our results validate the utility of both PLCs and hLSECs as cell-based delivery vehicles for a fVIII transgene, and they highlight the hurdles that remain to be overcome before primary human cells can be gene-edited with sufficient efficiency for use in cell-based gene therapy to treat HA.
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Affiliation(s)
- Ritu M. Ramamurthy
- Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, United States
| | - Martin Rodriguez
- Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, United States
| | - Hannah C. Ainsworth
- Department of Biostatistics and Data Sciences Wake Forest School of Medicine, Winston Salem, NC, United States
| | - Jordan Shields
- Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Emory University, Atlanta, GA, United States
| | - Diane Meares
- Department of Medicine, Hematology and Oncology, Wake Forest School of Medicine, Winston Salem, NC, United States
| | - Colin Bishop
- Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, United States
| | - Andrew Farland
- Department of Medicine, Hematology and Oncology, Wake Forest School of Medicine, Winston Salem, NC, United States
| | - Carl D. Langefeld
- Department of Biostatistics and Data Sciences Wake Forest School of Medicine, Winston Salem, NC, United States
| | - Anthony Atala
- Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, United States
| | - Christopher B. Doering
- Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Emory University, Atlanta, GA, United States
| | - H. Trent Spencer
- Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Emory University, Atlanta, GA, United States
| | - Christopher D. Porada
- Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, United States
| | - Graça Almeida-Porada
- Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, United States
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Chen X, Niu X, Liu Y, Zheng R, Yang L, Lu J, Yin S, Wei Y, Pan J, Sayed A, Ma X, Liu M, Jing F, Liu M, Hu J, Wang L, Li D. Long-term correction of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration. J Genet Genomics 2022; 49:1114-1126. [PMID: 35691554 DOI: 10.1016/j.jgg.2022.06.001] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2022] [Revised: 05/29/2022] [Accepted: 06/01/2022] [Indexed: 01/14/2023]
Abstract
CRISPR/Cas9-mediated site-specific insertion of exogenous genes holds potential for clinical applications. However, it is still infeasible because homologous recombination (HR) is inefficient, especially for non-dividing cells. To overcome the challenge, we report that a homology-independent targeted integration (HITI) strategy is used for permanent integration of high-specificity-activity Factor IX variant (F9 Padua, R338L) at the albumin (Alb) locus in a novel hemophilia B (HB) rat model. The knock-in efficiency reaches 3.66%, as determined by droplet digital PCR (ddPCR). The clotting time is reduced to a normal level four weeks after treatment, and the circulating factor IX (FIX) level is gradually increased up to 52% of the normal level over nine months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through primer-extension-mediated sequencing (PEM-seq), no significant off-target effect is detected. This study not only provides a novel model for HB but also identifies a promising therapeutic approach for rare inherited diseases.
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Affiliation(s)
- Xi Chen
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Xuran Niu
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Yang Liu
- The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China
| | - Rui Zheng
- Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
| | - Lei Yang
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Jian Lu
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Shuming Yin
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Yu Wei
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Jiahao Pan
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Ahmed Sayed
- Biochemistry Laboratory, Chemistry Department, Faculty of Science, Assiut University, Assiut 71516, Egypt
| | - Xueyun Ma
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Meizhen Liu
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | | | - Mingyao Liu
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China
| | - Jiazhi Hu
- The MOE Key Laboratory of Cell Proliferation and Differentiation, Genome Editing Research Center, School of Life Sciences, Peking-Tsinghua Center for Life Sciences, Peking University, Beijing 100871, China.
| | - Liren Wang
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
| | - Dali Li
- Shanghai Frontiers Science Center of Genome Editing and Cell Therapy, Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai 200241, China.
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31
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Tang LV, Tao Y, Feng Y, Ma J, Lin W, Zhang Y, Zhang Y, Wu T, Cai Y, Lu H, Wei J, Corral J, Hu Y. Gene editing of human iPSCs rescues thrombophilia in hereditary antithrombin deficiency in mice. Sci Transl Med 2022; 14:eabq3202. [PMID: 36449603 DOI: 10.1126/scitranslmed.abq3202] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/03/2022]
Abstract
Hereditary antithrombin deficiency is caused by SERPINC1 gene mutations and predisposes to recurrent venous thromboembolism that can be life-threatening. Therefore, lifelong anticoagulation is required, which has side effects and may not be effective. In this study, peripheral blood mononuclear cells from a patient with severe antithrombin deficiency were reprogrammed into induced pluripotent stem cells (iPSCs). The mutation was corrected using CRISPR-Cas9 and Cre/LoxP genome editing. iPSCs were differentiated into hepatocytes, which were injected into the spleen of antithrombin knockout mice to restore the activity of antithrombin and reduce the thrombophilic state. Human iPSC-differentiated hepatocytes colonized mice and secreted antithrombin stably, normalizing antithrombin in plasma (activity: from 46.8 ± 5.7% to 88.6 ± 7.6%, P < 0.0001; antigen: from 146.9 ± 19.5 nanograms per milliliter to 390.7 ± 16.1 nanograms per milliliter, P < 0.0001). In venous thrombosis model, the rate of thrombosis in mice treated with edited hepatocytes, parental hepatocytes, and wild-type mice were 60, 90, and 70%, respectively. The thrombus weight was much lighter in mice treated with edited hepatocytes compared with parental hepatocytes (7.25 ± 2.00 milligrams versus 15.32 ± 2.87 milligrams, P = 0.0025) and showed no notable difference compared with that in wild-type mice (10.41 ± 2.91 milligrams). The activity and concentration of antithrombin remained high for 3 weeks after injection. The liver and kidney function markers showed no obvious abnormality during the observation period. This study provides a proof of principle for correction of mutations in patient-derived iPSCs and potential therapeutic applications for hereditary thrombophilia.
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Affiliation(s)
- Liang V Tang
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Yanyi Tao
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Yuanzheng Feng
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Jiewen Ma
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Wenyi Lin
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Yuyang Zhang
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Yi Zhang
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Tingting Wu
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Yaohua Cai
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Hui Lu
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
| | - Jun Wei
- iRegene Therapeutics Co. Ltd., Wuhan 430070, PR China
| | - Javier Corral
- Servicio de Hematología y Oncología Médica, Hospital Universitario Morales Meseguer, Centro Regional de Hemodonación, Universidad de Murcia, IMIB-Arrixaca, CIBERER, Ronda de Garay S/N, 30003 Murcia, Spain
| | - Yu Hu
- Institute of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
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Petazzi P, Miquel‐Serra L, Huertas S, González C, Boto N, Muñiz‐Diaz E, Menéndez P, Sevilla A, Nogués N. ABO gene editing for the conversion of blood type A to universal type O in Rh null donor-derived human-induced pluripotent stem cells. Clin Transl Med 2022; 12:e1063. [PMID: 36281739 PMCID: PMC9593258 DOI: 10.1002/ctm2.1063] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2022] [Revised: 08/13/2022] [Accepted: 09/06/2022] [Indexed: 01/28/2023] Open
Abstract
The limited availability of red cells with extremely rare blood group phenotypes is one of the global challenges in transfusion medicine that has prompted the search for alternative self-renewable pluripotent cell sources for the in vitro generation of red cells with rare blood group types. One such phenotype is the Rhnull , which lacks all the Rh antigens on the red cell membrane and represents one of the rarest blood types in the world with only a few active blood donors available worldwide. Rhnull red cells are critical for the transfusion of immunized patients carrying the same phenotype, besides its utility in the diagnosis of Rh alloimmunization when a high-prevalence Rh specificity is suspected in a patient or a pregnant woman. In both scenarios, the potential use of human-induced pluripotent stem cell (hiPSC)-derived Rhnull red cells is also dependent on ABO compatibility. Here, we present a CRISPR/Cas9-mediated ABO gene edition strategy for the conversion of blood type A to universal type O, which we have applied to an Rhnull donor-derived hiPSC line, originally carrying blood group A. This work provides a paradigmatic example of an approach potentially applicable to other hiPSC lines derived from rare blood donors not carrying blood type O.
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Affiliation(s)
- Paolo Petazzi
- Josep Carreras Leukemia Research InstituteBarcelonaSpain
| | - Laia Miquel‐Serra
- Immunohematology LaboratoryBarcelonaSpain
- Transfusional medicine. Vall d'Hebron Research Institute (VHIR)BarcelonaSpain
| | - Sergio Huertas
- Immunohematology LaboratoryBarcelonaSpain
- Transfusional medicine. Vall d'Hebron Research Institute (VHIR)BarcelonaSpain
| | - Cecilia González
- Immunohematology LaboratoryBarcelonaSpain
- Transfusional medicine. Vall d'Hebron Research Institute (VHIR)BarcelonaSpain
| | - Neus Boto
- Immunohematology LaboratoryBarcelonaSpain
| | - Eduardo Muñiz‐Diaz
- Immunohematology LaboratoryBarcelonaSpain
- Transfusional medicine. Vall d'Hebron Research Institute (VHIR)BarcelonaSpain
- Department of MedicineUniversitat Autònoma de Barcelona (UAB)BarcelonaSpain
| | - Pablo Menéndez
- Josep Carreras Leukemia Research InstituteBarcelonaSpain
- Department of Biomedicine, School of MedicineUniversity of BarcelonaBarcelonaSpain
- Centro de Investigación Biomédica en Red de Cáncer‐CIBER‐ONCInstituto de Salud Carlos IIIBarcelonaSpain
- Red Española de Terapias Avanzadas (TERAV)Instituto de Salud Carlos III (RICORS, RD21/0017/0029)
- Institució Catalana de Recerca i Estudis Avançats (ICREA)BarcelonaSpain
| | - Ana Sevilla
- Department of Cell BiologyPhysiology and Immunology, Faculty of Biology, University of BarcelonaBarcelonaSpain
- Institute of Biomedicine of the University of Barcelona (IBUB)BarcelonaSpain
| | - Núria Nogués
- Immunohematology LaboratoryBarcelonaSpain
- Transfusional medicine. Vall d'Hebron Research Institute (VHIR)BarcelonaSpain
- Department of MedicineUniversitat Autònoma de Barcelona (UAB)BarcelonaSpain
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Mapook A, Hyde KD, Hassan K, Kemkuignou BM, Čmoková A, Surup F, Kuhnert E, Paomephan P, Cheng T, de Hoog S, Song Y, Jayawardena RS, Al-Hatmi AMS, Mahmoudi T, Ponts N, Studt-Reinhold L, Richard-Forget F, Chethana KWT, Harishchandra DL, Mortimer PE, Li H, Lumyong S, Aiduang W, Kumla J, Suwannarach N, Bhunjun CS, Yu FM, Zhao Q, Schaefer D, Stadler M. Ten decadal advances in fungal biology leading towards human well-being. FUNGAL DIVERS 2022; 116:547-614. [PMID: 36123995 PMCID: PMC9476466 DOI: 10.1007/s13225-022-00510-3] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2022] [Accepted: 07/28/2022] [Indexed: 11/04/2022]
Abstract
Fungi are an understudied resource possessing huge potential for developing products that can greatly improve human well-being. In the current paper, we highlight some important discoveries and developments in applied mycology and interdisciplinary Life Science research. These examples concern recently introduced drugs for the treatment of infections and neurological diseases; application of -OMICS techniques and genetic tools in medical mycology and the regulation of mycotoxin production; as well as some highlights of mushroom cultivaton in Asia. Examples for new diagnostic tools in medical mycology and the exploitation of new candidates for therapeutic drugs, are also given. In addition, two entries illustrating the latest developments in the use of fungi for biodegradation and fungal biomaterial production are provided. Some other areas where there have been and/or will be significant developments are also included. It is our hope that this paper will help realise the importance of fungi as a potential industrial resource and see the next two decades bring forward many new fungal and fungus-derived products.
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Affiliation(s)
- Ausana Mapook
- Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100 Thailand
| | - Kevin D. Hyde
- Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100 Thailand
- School of Science, Mae Fah Luang University, Chiang Rai, 57100 Thailand
- Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 Yunnan China
- Research Center of Microbial Diversity and Sustainable Utilization, Chiang Mai University, Chiang Mai, 50200 Thailand
- Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200 Thailand
- Innovative Institute of Plant Health, Zhongkai University of Agriculture and Engineering, Haizhu District, Guangzhou, 510225 China
| | - Khadija Hassan
- Department Microbial Drugs, Helmholtz Centre for Infection Research (HZI), and German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Inhoffenstrasse 7, 38124 Brunswick, Germany
| | - Blondelle Matio Kemkuignou
- Department Microbial Drugs, Helmholtz Centre for Infection Research (HZI), and German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Inhoffenstrasse 7, 38124 Brunswick, Germany
| | - Adéla Čmoková
- Laboratory of Fungal Genetics and Metabolism, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic
| | - Frank Surup
- Department Microbial Drugs, Helmholtz Centre for Infection Research (HZI), and German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Inhoffenstrasse 7, 38124 Brunswick, Germany
- Institute of Microbiology, Technische Universität Braunschweig, Spielmannstraße 7, 38106 Brunswick, Germany
| | - Eric Kuhnert
- Centre of Biomolecular Drug Research (BMWZ), Institute for Organic Chemistry, Leibniz University Hannover, Schneiderberg 38, 30167 Hannover, Germany
| | - Pathompong Paomephan
- Department Microbial Drugs, Helmholtz Centre for Infection Research (HZI), and German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Inhoffenstrasse 7, 38124 Brunswick, Germany
- Department of Biotechnology, Faculty of Science, Mahidol University, 272 Rama VI Road, Ratchathewi, Bangkok, 10400 Thailand
| | - Tian Cheng
- Department Microbial Drugs, Helmholtz Centre for Infection Research (HZI), and German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Inhoffenstrasse 7, 38124 Brunswick, Germany
- Laboratory of Fungal Genetics and Metabolism, Institute of Microbiology, Czech Academy of Sciences, Prague, Czech Republic
| | - Sybren de Hoog
- Center of Expertise in Mycology, Radboud University Medical Center / Canisius Wilhelmina Hospital, Nijmegen, The Netherlands
- Key Laboratory of Environmental Pollution Monitoring and Disease Control, Guizhou Medical University, Guiyang, China
- Microbiology, Parasitology and Pathology Graduate Program, Federal University of Paraná, Curitiba, Brazil
| | - Yinggai Song
- Department of Dermatology, Peking University First Hospital, Peking University, Beijing, China
| | - Ruvishika S. Jayawardena
- Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100 Thailand
- School of Science, Mae Fah Luang University, Chiang Rai, 57100 Thailand
| | - Abdullah M. S. Al-Hatmi
- Center of Expertise in Mycology, Radboud University Medical Center / Canisius Wilhelmina Hospital, Nijmegen, The Netherlands
- Natural and Medical Sciences Research Center, University of Nizwa, Nizwa, Oman
| | - Tokameh Mahmoudi
- Department of Biochemistry, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Nadia Ponts
- INRAE, UR1264 Mycology and Food Safety (MycSA), 33882 Villenave d’Ornon, France
| | - Lena Studt-Reinhold
- Department of Applied Genetics and Cell Biology, Institute of Microbial Genetics, University of Natural Resources and Life Sciences, Vienna (BOKU), Tulln an der Donau, Austria
| | | | - K. W. Thilini Chethana
- Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100 Thailand
- School of Science, Mae Fah Luang University, Chiang Rai, 57100 Thailand
| | - Dulanjalee L. Harishchandra
- Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100 Thailand
- School of Science, Mae Fah Luang University, Chiang Rai, 57100 Thailand
- Beijing Key Laboratory of Environment Friendly Management on Fruit Diseases and Pests in North China, Institute of Plant Protection, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097 China
| | - Peter E. Mortimer
- Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 Yunnan China
- Centre for Mountain Futures (CMF), Kunming Institute of Botany, Chinese Academy of Science, Kunming, 650201 Yunnan China
| | - Huili Li
- Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 Yunnan China
- Centre for Mountain Futures (CMF), Kunming Institute of Botany, Chinese Academy of Science, Kunming, 650201 Yunnan China
| | - Saisamorm Lumyong
- Research Center of Microbial Diversity and Sustainable Utilization, Chiang Mai University, Chiang Mai, 50200 Thailand
- Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200 Thailand
- Academy of Science, The Royal Society of Thailand, Bangkok, 10300 Thailand
| | - Worawoot Aiduang
- Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200 Thailand
| | - Jaturong Kumla
- Research Center of Microbial Diversity and Sustainable Utilization, Chiang Mai University, Chiang Mai, 50200 Thailand
- Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200 Thailand
| | - Nakarin Suwannarach
- Research Center of Microbial Diversity and Sustainable Utilization, Chiang Mai University, Chiang Mai, 50200 Thailand
- Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai, 50200 Thailand
| | - Chitrabhanu S. Bhunjun
- Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100 Thailand
- School of Science, Mae Fah Luang University, Chiang Rai, 57100 Thailand
| | - Feng-Ming Yu
- Center of Excellence in Fungal Research, Mae Fah Luang University, Chiang Rai, 57100 Thailand
- School of Science, Mae Fah Luang University, Chiang Rai, 57100 Thailand
- Yunnan Key Laboratory of Fungal Diversity and Green Development, Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 Yunnan China
| | - Qi Zhao
- Yunnan Key Laboratory of Fungal Diversity and Green Development, Key Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming, 650201 Yunnan China
| | - Doug Schaefer
- Centre for Mountain Futures (CMF), Kunming Institute of Botany, Chinese Academy of Science, Kunming, 650201 Yunnan China
| | - Marc Stadler
- Department Microbial Drugs, Helmholtz Centre for Infection Research (HZI), and German Centre for Infection Research (DZIF), Partner Site Hannover-Braunschweig, Inhoffenstrasse 7, 38124 Brunswick, Germany
- Institute of Microbiology, Technische Universität Braunschweig, Spielmannstraße 7, 38106 Brunswick, Germany
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Song G, Ma Y, Gao X, Zhang X, Zhang F, Tian C, Hou J, Liu Z, Zhao Z, Tian Y. CRISPR/Cas9-mediated genetic correction reverses spinocerebellar ataxia 3 disease-associated phenotypes in differentiated cerebellar neurons. LIFE MEDICINE 2022; 1:27-44. [PMID: 39872157 PMCID: PMC11749335 DOI: 10.1093/lifemedi/lnac020] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/11/2022] [Accepted: 06/27/2022] [Indexed: 01/29/2025]
Abstract
The neurodegenerative disease spinocerebellar ataxia type 3 (SCA3; also called Machado-Joseph disease, MJD) is a trinucleotide repeat disorder caused by expansion of the CAG repeats in the ATXN3 gene. Here, we applied a CRISPR/Cas9-mediated approach using homologous recombination to achieve a one-step genetic correction in SCA3-specific induced pluripotent stem cells (iPSCs). The genetic correction reversed disease-associated phenotypes during cerebellar region-specific differentiation. In addition, we observed spontaneous ataxin-3 aggregates specifically in mature cerebellar neurons differentiated from SCA3 iPSCs rather than in SCA3 pan-neurons, SCA3 iPSCs or neural stem cells, suggesting that SCA3 iPSC-derived disease-specific and region-specific cerebellar neurons can provide unique cellular models for studying SCA3 pathogenesis in vitro. Importantly, the genetically corrected cerebellar neurons did not display typical SCA3 aggregates, suggesting that genetic correction can subsequently reverse SCA3 disease progression. Our strategy can be applied to other trinucleotide repeat disorders to facilitate disease modeling, mechanistic studies and drug discovery.
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Affiliation(s)
- Guoxu Song
- Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yuying Ma
- Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Xing Gao
- Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
| | - Xuewen Zhang
- Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
| | - Fei Zhang
- Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Chunhong Tian
- Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jiajia Hou
- Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
| | - Zheng Liu
- Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
| | - Zixin Zhao
- Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
| | - Yong Tian
- Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
- University of Chinese Academy of Sciences, Beijing 100049, China
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35
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Chupradit K, Thongsin N, Tayapiwatana C, Wattanapanitch M. A precise gene delivery approach for human induced pluripotent stem cells using Cas9 RNP complex and recombinant AAV6 donor vectors. PLoS One 2022; 17:e0270963. [PMID: 35797389 PMCID: PMC9262223 DOI: 10.1371/journal.pone.0270963] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Accepted: 06/17/2022] [Indexed: 11/18/2022] Open
Abstract
Genome editing in human induced pluripotent stem cells (hiPSCs) offers a potential tool for studying gene functions in disease models and correcting genetic mutations for cell-based therapy. Precise transgene insertion in hiPSCs represents a significant challenge. In the past decade, viral transduction has been widely used due to its high transduction efficiency; however, it can result in random transgene integration and variable transgene copy numbers. Non-viral-based strategies are generally safer but limited by their low transfection efficiency in hiPSCs. Recently, genome engineering using adeno-associated virus (AAV) vectors has emerged as a promising gene delivery approach due to AAVs’ low immunogenicity, toxicity, and ability to infect a broad range of cells. The following protocol describes the workflow for genome editing in hiPSCs using the CRISPR/Cas9 ribonucleoprotein (RNP) complex combined with the recombinant AAV serotype 6 (AAV6) donor vectors to introduce a gene of interest (GOI) fused with mCherry fluorescent reporter gene into the AAVS1 safe harbor site. This approach leads to efficient transgene insertion and is applicable to precise genome editing of hiPSCs or other types of stem cells for research purposes.
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Affiliation(s)
- Koollawat Chupradit
- Research Department, Siriraj Center for Regenerative Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
- Center of Biomolecular Therapy and Diagnostic, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
| | - Nontaphat Thongsin
- Research Department, Siriraj Center for Regenerative Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
- Department of Immunology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
| | - Chatchai Tayapiwatana
- Center of Biomolecular Therapy and Diagnostic, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
- Division of Clinical Immunology, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
- Center of Innovative Immunodiagnostic Development, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai, Thailand
| | - Methichit Wattanapanitch
- Research Department, Siriraj Center for Regenerative Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand
- * E-mail:
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36
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Shin JH, Lee J, Jung YK, Kim KS, Jeong J, Choi D. Therapeutic applications of gene editing in chronic liver diseases: an update. BMB Rep 2022. [PMID: 35651324 PMCID: PMC9252892 DOI: 10.5483/bmbrep.2022.55.6.033] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/23/2022] Open
Abstract
Innovative genome editing techniques developed in recent decades have revolutionized the biomedical research field. Liver is the most favored target organ for genome editing owing to its ability to regenerate. The regenerative capacity of the liver enables ex vivo gene editing in which the mutated gene in hepatocytes isolated from the animal model of genetic disease is repaired. The edited hepatocytes are injected back into the animal to mitigate the disease. Furthermore, the liver is considered as the easiest target organ for gene editing as it absorbs almost all foreign molecules. The mRNA vaccines, which have been developed to manage the COVID-19 pandemic, have provided a novel gene editing strategy using Cas mRNA. A single injection of gene editing components with Cas mRNA is reported to be efficient in the treatment of patients with genetic liver diseases. In this review, we first discuss previously reported gene editing tools and cases managed using them, as well as liver diseases caused by genetic mutations. Next, we summarize the recent successes of ex vivo and in vivo gene editing approaches in ameliorating liver diseases in animals and humans.
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Affiliation(s)
- Ji Hyun Shin
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea
- HY Indang Institute of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Korea
| | - Jinho Lee
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea
- HY Indang Institute of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Korea
| | - Yun Kyung Jung
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea
- HY Indang Institute of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Korea
| | - Kyeong Sik Kim
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea
| | - Jaemin Jeong
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea
- HY Indang Institute of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Korea
| | - Dongho Choi
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea
- HY Indang Institute of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Korea
- Department of HY-KIST Bio-convergence, Hanyang University, Seoul 04763, Korea
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Shin JH, Lee J, Jung YK, Kim KS, Jeong J, Choi D. Therapeutic applications of gene editing in chronic liver diseases: an update. BMB Rep 2022; 55:251-258. [PMID: 35651324 PMCID: PMC9252892] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2022] [Revised: 03/31/2022] [Accepted: 04/22/2022] [Indexed: 02/21/2025] Open
Abstract
Innovative genome editing techniques developed in recent decades have revolutionized the biomedical research field. Liver is the most favored target organ for genome editing owing to its ability to regenerate. The regenerative capacity of the liver enables ex vivo gene editing in which the mutated gene in hepatocytes isolated from the animal model of genetic disease is repaired. The edited hepatocytes are injected back into the animal to mitigate the disease. Furthermore, the liver is considered as the easiest target organ for gene editing as it absorbs almost all foreign molecules. The mRNA vaccines, which have been developed to manage the COVID-19 pandemic, have provided a novel gene editing strategy using Cas mRNA. A single injection of gene editing components with Cas mRNA is reported to be efficient in the treatment of patients with genetic liver diseases. In this review, we first discuss previously reported gene editing tools and cases managed using them, as well as liver diseases caused by genetic mutations. Next, we summarize the recent successes of ex vivo and in vivo gene editing approaches in ameliorating liver diseases in animals and humans. [BMB Reports 2022; 55(6): 251-258].
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Affiliation(s)
- Ji Hyun Shin
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea
- HY Indang Institute of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Korea
| | - Jinho Lee
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea
- HY Indang Institute of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Korea
| | - Yun Kyung Jung
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea
- HY Indang Institute of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Korea
| | - Kyeong Sik Kim
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea
| | - Jaemin Jeong
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea
- HY Indang Institute of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Korea
| | - Dongho Choi
- Department of Surgery, Hanyang University College of Medicine, Seoul 04763, Korea
- HY Indang Institute of Regenerative Medicine and Stem Cell Research, Hanyang University, Seoul 04763, Korea
- Department of HY-KIST Bio-convergence, Hanyang University, Seoul 04763, Korea
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Shojaei Baghini S, Gardanova ZR, Abadi SAH, Zaman BA, İlhan A, Shomali N, Adili A, Moghaddar R, Yaseri AF. CRISPR/Cas9 application in cancer therapy: a pioneering genome editing tool. Cell Mol Biol Lett 2022; 27:35. [PMID: 35508982 PMCID: PMC9066929 DOI: 10.1186/s11658-022-00336-6] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/17/2022] [Accepted: 04/13/2022] [Indexed: 12/20/2022] Open
Abstract
The progress of genetic engineering in the 1970s brought about a paradigm shift in genome editing technology. The clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system is a flexible means to target and modify particular DNA sequences in the genome. Several applications of CRISPR/Cas9 are presently being studied in cancer biology and oncology to provide vigorous site-specific gene editing to enhance its biological and clinical uses. CRISPR's flexibility and ease of use have enabled the prompt achievement of almost any preferred alteration with greater efficiency and lower cost than preceding modalities. Also, CRISPR/Cas9 technology has recently been applied to improve the safety and efficacy of chimeric antigen receptor (CAR)-T cell therapies and defeat tumor cell resistance to conventional treatments such as chemotherapy and radiotherapy. The current review summarizes the application of CRISPR/Cas9 in cancer therapy. We also discuss the present obstacles and contemplate future possibilities in this context.
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Affiliation(s)
- Sadegh Shojaei Baghini
- Plant Biotechnology Department, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
| | - Zhanna R. Gardanova
- Department of Psychotherapy, Pirogov Russian National Research Medical University, 1 Ostrovityanova St., 117997 Moscow, Russia
| | - Saeme Azizi Hassan Abadi
- Department of Nursery and Midwifery, Faculty of Laboratory Science, Islamic Azad University of Chalous, Mazandaran, Iran
| | - Burhan Abdullah Zaman
- Basic Sciences Department, College of Pharmacy, University of Duhok, Kurdistan Region, Iraq
| | - Ahmet İlhan
- Department of Medical Biochemistry, Faculty of Medicine, Cukurova University, Adana, Turkey
| | - Navid Shomali
- Immunology Research Center (IRC), Tabriz University of Medical Sciences, Tabriz, Iran
| | - Ali Adili
- Department of Oncology, Tabriz University of Medical Sciences, Tabriz, Iran
- Senior Adult Oncology Department, Moffitt Cancer Center, University of South Florida, Tampa, USA
| | - Roozbeh Moghaddar
- Department of Pediatric Hematology and Oncology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
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Chen Y, Wen R, Yang Z, Chen Z. Genome editing using CRISPR/Cas9 to treat hereditary hematological disorders. Gene Ther 2022; 29:207-216. [PMID: 33750926 DOI: 10.1038/s41434-021-00247-9] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/23/2020] [Revised: 02/15/2021] [Accepted: 02/19/2021] [Indexed: 02/07/2023]
Abstract
The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile and convenient genome-editing tool with prospects in gene therapy. This technique is based on customized site-specific nucleases with programmable guiding RNAs that cleave and introduce double-strand breaks (DSBs) at the target locus and achieve precise genome modification by triggering DNA repair mechanisms. Human hematopoietic stem/progenitor cells (HSPCs) are conventional cell targets for gene therapy in hematological diseases and have been widely used in most studies. Induced pluripotent stem cells (iPSCs) can be generated from a variety of somatic cells and hold great promise for personalized cell-based therapies. CRISPR/Cas9-mediated genome editing in autologous HSPCs and iPSCs is an ideal therapeutic solution for treating hereditary hematological disorders. Here, we review and summarize the latest studies about CRISPR/Cas9-mediated genome editing in patient-derived HSPCs and iPSCs to treat hereditary hematological disorders. Current challenges and prospects are also discussed.
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Affiliation(s)
- Yan Chen
- Zhanjiang Institute of Clinical Medicine, Zhanjiang Central Hospital, Guangdong Medical University, Zhanjiang, PR China
| | - Ruiting Wen
- Department of Hematology, Zhanjiang Central Hospital, Guangdong Medical University, Zhanjiang, PR China
| | - Zhigang Yang
- Zhanjiang Institute of Clinical Medicine, Zhanjiang Central Hospital, Guangdong Medical University, Zhanjiang, PR China
- Department of Hematology, Zhanjiang Central Hospital, Guangdong Medical University, Zhanjiang, PR China
| | - Zhanghui Chen
- Zhanjiang Institute of Clinical Medicine, Zhanjiang Central Hospital, Guangdong Medical University, Zhanjiang, PR China.
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40
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Mir TUG, Wani AK, Akhtar N, Shukla S. CRISPR/Cas9: Regulations and challenges for law enforcement to combat its dual-use. Forensic Sci Int 2022; 334:111274. [DOI: 10.1016/j.forsciint.2022.111274] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2022] [Revised: 02/19/2022] [Accepted: 03/13/2022] [Indexed: 12/15/2022]
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Vojnits K, Nakanishi M, Porras D, Kim Y, Feng Z, Golubeva D, Bhatia M. Developing CRISPR/Cas9-Mediated Fluorescent Reporter Human Pluripotent Stem-Cell Lines for High-Content Screening. Molecules 2022; 27:molecules27082434. [PMID: 35458632 PMCID: PMC9025795 DOI: 10.3390/molecules27082434] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/28/2022] [Revised: 04/03/2022] [Accepted: 04/06/2022] [Indexed: 12/22/2022] Open
Abstract
Application of the CRISPR/Cas9 system to knock in fluorescent proteins to endogenous genes of interest in human pluripotent stem cells (hPSCs) has the potential to facilitate hPSC-based disease modeling, drug screening, and optimization of transplantation therapy. To evaluate the capability of fluorescent reporter hPSC lines for high-content screening approaches, we targeted EGFP to the endogenous OCT4 locus. Resulting hPSC–OCT4–EGFP lines generated expressed EGFP coincident with pluripotency markers and could be adapted to multi-well formats for high-content screening (HCS) campaigns. However, after long-term culture, hPSCs transiently lost their EGFP expression. Alternatively, through EGFP knock-in to the AAVS1 locus, we established a stable and consistent EGFP-expressing hPSC–AAVS1–EGFP line that maintained EGFP expression during in vitro hematopoietic and neural differentiation. Thus, hPSC–AAVS1–EGFP-derived sensory neurons could be adapted to a high-content screening platform that can be applied to high-throughput small-molecule screening and drug discovery campaigns. Our observations are consistent with recent findings indicating that high-frequency on-target complexities appear following CRISPR/Cas9 genome editing at the OCT4 locus. In contrast, we demonstrate that the AAVS1 locus is a safe genomic location in hPSCs with high gene expression that does not impact hPSC quality and differentiation. Our findings suggest that the CRISPR/Cas9-integrated AAVS1 system should be applied for generating stable reporter hPSC lines for long-term HCS approaches, and they underscore the importance of careful evaluation and selection of the applied reporter cell lines for HCS purposes.
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The most common disease-causing mutation of factor XIII deficiency is corrected by CRISPR/CAS9 gene editing system. Blood Coagul Fibrinolysis 2022; 33:153-158. [PMID: 35221320 DOI: 10.1097/mbc.0000000000001126] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
Factor XIII (FXIII) deficiency is one of the most severe congenital bleeding disorders, with an estimated incidence of one person per one million. Patients with severe FXIII deficiency present a wide range of clinical manifestations, including umbilical cord bleeding, intracranial haemorrhage and recurrent miscarriages. Due to the high rate of life-threatening bleeding, primary prophylaxis is mandatory from the time of diagnosis. Although replacement therapy is the most common therapeutic choice, gene therapy remains the only curative option. In the present study, we assessed the efficacy of the clustered regularly interspaced short palindromic repeats - CRISPR-associated protein 9 (CRISPR/Cas9) system in the correction of the most common FXIII disease-causing mutation (c.562 T > C). A dermal fibroblast was harvested from the human skin biopsy of a young patient with FXIII deficiency. Sanger sequencing was used to confirm the presence of c.562 T>C mutation in the patient and in the harvested fibroblasts. PX459 vector was digested with BbsI restriction enzyme, and after annealing and ligation of two 20-bp guide-RNAs (g-RNAs) close to the PAM (NGG) sequence, the constructed vectors were amplified in Escherichia coli Top 10. Transfection was performed by a nucleofector device, and DNA extraction was performed after puromycin selection and serial dilution from potentially transfected colonies. A 50-bp template oligonucleotide was used to aid homologous repair for correction of the underlying mutation and synonymous mutation as an internal control. The synonymous mutation (AAT to ACT) near the mutation site was used as internal control. Sanger sequencing was done in order to check the gene correction. The c.562 T > C mutation was detected in homozygote state in the primary fibroblasts of the patient and wild-type alleles were confirmed in the normal individual. Colony PCR and sequencing revealed successful cloning of the designed gRNAs. The detected mutation was corrected from a homozygote mutant state (c.562 T > C) to a homozygote wild type in transfected dermal fibroblasts of the patient. The control mutation, as an internal control, was also corrected in the same fibroblasts in the heterozygote manner. The result of the study shows that the CRISPR/CAS9 gene editing system is an effective tool for correction of point mutations in transfected fibroblasts of patients with congenital FXIII deficiency and represents a new, potentially curative, option.
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Eslami-Mossallam B, Klein M, Smagt CVD, Sanden KVD, Jones SK, Hawkins JA, Finkelstein IJ, Depken M. A kinetic model predicts SpCas9 activity, improves off-target classification, and reveals the physical basis of targeting fidelity. Nat Commun 2022; 13:1367. [PMID: 35292641 PMCID: PMC8924176 DOI: 10.1038/s41467-022-28994-2] [Citation(s) in RCA: 20] [Impact Index Per Article: 6.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2020] [Accepted: 02/11/2022] [Indexed: 12/26/2022] Open
Abstract
The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites based on complementarity to a complexed single-guide RNA (sgRNA). However, SpCas9-sgRNA also binds and cleaves genomic off-targets with only partial complementarity. To date, we lack the ability to predict cleavage and binding activity quantitatively, and rely on binary classification schemes to identify strong off-targets. We report a quantitative kinetic model that captures the SpCas9-mediated strand-replacement reaction in free-energy terms. The model predicts binding and cleavage activity as a function of time, target, and experimental conditions. Trained and validated on high-throughput bulk-biochemical data, our model predicts the intermediate R-loop state recently observed in single-molecule experiments, as well as the associated conversion rates. Finally, we show that our quantitative activity predictor can be reduced to a binary off-target classifier that outperforms the established state-of-the-art. Our approach is extensible, and can characterize any CRISPR-Cas nuclease - benchmarking natural and future high-fidelity variants against SpCas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems.
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Affiliation(s)
- Behrouz Eslami-Mossallam
- Kavli Institute of NanoScience and Department of BionanoScience, Delft University of Technology, Delft, 2629HZ, the Netherlands
- Dept. Building Physics and Systems, TNO Building and Construction Research, Leeghwaterstraat 44, Delft, The Netherlands
| | - Misha Klein
- Kavli Institute of NanoScience and Department of BionanoScience, Delft University of Technology, Delft, 2629HZ, the Netherlands
- Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV, Amsterdam, the Netherlands
| | - Constantijn V D Smagt
- Kavli Institute of NanoScience and Department of BionanoScience, Delft University of Technology, Delft, 2629HZ, the Netherlands
- Department of Physics and Astronomy, and LaserLaB Amsterdam, Vrije Universiteit Amsterdam, De Boelelaan 1081, 1081 HV, Amsterdam, the Netherlands
| | - Koen V D Sanden
- Kavli Institute of NanoScience and Department of BionanoScience, Delft University of Technology, Delft, 2629HZ, the Netherlands
| | - Stephen K Jones
- Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA
- Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, 78712, USA
- Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, 78712, USA
- VU LSC-EMBL Partnership for Genome Editing Technologies, Life Sciences Center, Vilnius University, Vilnius, Lithuania
| | - John A Hawkins
- Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA
- Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, 78712, USA
- Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, 78712, USA
- Oden Institute for Computational Engineering and Science, University of Texas at Austin, Austin, TX, 78712, USA
- European Molecular Biology Laboratory, Genome Biology Department, Heidelberg, Germany
| | - Ilya J Finkelstein
- Department of Molecular Biosciences, University of Texas at Austin, Austin, TX, 78712, USA
- Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, TX, 78712, USA
- Center for Systems and Synthetic Biology, University of Texas at Austin, Austin, TX, 78712, USA
| | - Martin Depken
- Kavli Institute of NanoScience and Department of BionanoScience, Delft University of Technology, Delft, 2629HZ, the Netherlands.
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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:cells11030557. [PMID: 35159366 PMCID: PMC8834203 DOI: 10.3390/cells11030557] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2022] [Revised: 01/28/2022] [Accepted: 02/03/2022] [Indexed: 01/26/2023] Open
Abstract
Inherited blood disorders comprise a large spectrum of diseases due to germline mutations in genes with key function in the hematopoietic system; they include immunodeficiencies, anemia or metabolic diseases. For most of them the only curative treatment is bone marrow transplantation, a procedure associated to severe complications; other therapies include red blood cell and platelet transfusions, which are dependent on donor availability. An alternative option is gene therapy, in which the wild-type form of the mutated gene is delivered into autologous hematopoietic stem cells using viral vectors. A more recent therapeutic perspective is gene correction through CRISPR/Cas9-mediated gene editing, that overcomes safety concerns due to insertional mutagenesis and allows correction of base substitutions in large size genes difficult to incorporate into vectors. However, applying this technique to genomic disorders caused by large gene deletions is challenging. Chromosomal transplantation has been proposed as a solution, using a universal source of wild-type chromosomes as donor, and induced pluripotent stem cells (iPSCs) as acceptor. One of the obstacles to be addressed for translating PSC research into clinical practice is the still unsatisfactory differentiation into transplantable hematopoietic stem or mature cells. We provide an overview of the recent progresses in this field and discuss challenges and potential of iPSC-based therapies for the treatment of inherited blood disorders.
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Affiliation(s)
- Ilaria Rao
- IRCCS Humanitas Research Hospital, Via Manzoni 56, 20089 Rozzano, Italy; (I.R.); (L.C.); (M.P.)
- Department of Biomedical Sciences, Humanitas University, Via Rita Levi Montalcini 4, 20090 Pieve Emanuele, Italy
| | - Laura Crisafulli
- IRCCS Humanitas Research Hospital, Via Manzoni 56, 20089 Rozzano, Italy; (I.R.); (L.C.); (M.P.)
- UOS Milan Unit, Istituto di Ricerca Genetica e Biomedica (IRGB), CNR, 20138 Milan, Italy
| | - Marianna Paulis
- IRCCS Humanitas Research Hospital, Via Manzoni 56, 20089 Rozzano, Italy; (I.R.); (L.C.); (M.P.)
- UOS Milan Unit, Istituto di Ricerca Genetica e Biomedica (IRGB), CNR, 20138 Milan, Italy
| | - Francesca Ficara
- IRCCS Humanitas Research Hospital, Via Manzoni 56, 20089 Rozzano, Italy; (I.R.); (L.C.); (M.P.)
- UOS Milan Unit, Istituto di Ricerca Genetica e Biomedica (IRGB), CNR, 20138 Milan, Italy
- Correspondence:
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45
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Son JS, Park CY, Lee G, Park JY, Kim HJ, Kim G, Chi KY, Woo DH, Han C, Kim SK, Park HJ, Kim DW, Kim JH. Therapeutic correction of hemophilia A using 2D endothelial cells and multicellular 3D organoids derived from CRISPR/Cas9-engineered patient iPSCs. Biomaterials 2022; 283:121429. [DOI: 10.1016/j.biomaterials.2022.121429] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2021] [Revised: 01/26/2022] [Accepted: 02/17/2022] [Indexed: 01/19/2023]
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Rezalotfi A, Fritz L, Förster R, Bošnjak B. Challenges of CRISPR-Based Gene Editing in Primary T Cells. Int J Mol Sci 2022; 23:ijms23031689. [PMID: 35163611 PMCID: PMC8835901 DOI: 10.3390/ijms23031689] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2022] [Accepted: 01/29/2022] [Indexed: 12/30/2022] Open
Abstract
Adaptive T-cell immunotherapy holds great promise for the successful treatment of leukemia, as well as other types of cancers. More recently, it was also shown to be an effective treatment option for chronic virus infections in immunosuppressed patients. Autologous or allogeneic T cells used for immunotherapy are usually genetically modified to express novel T-cell or chimeric antigen receptors. The production of such cells was significantly simplified with the CRISPR/Cas system, allowing for the deletion or insertion of novel genes at specific locations within the genome. In this review, we describe recent methodological breakthroughs that were important for the conduction of these genetic modifications, summarize crucial points to be considered when conducting such experiments, and highlight the potential pitfalls of these approaches.
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Affiliation(s)
- Alaleh Rezalotfi
- Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany; (A.R.); (L.F.); (R.F.)
| | - Lea Fritz
- Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany; (A.R.); (L.F.); (R.F.)
| | - Reinhold Förster
- Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany; (A.R.); (L.F.); (R.F.)
- Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, 30625 Hannover, Germany
- German Centre for Infection Research (DZIF), Partner Site Hannover, 30625 Hannover, Germany
| | - Berislav Bošnjak
- Institute of Immunology, Hannover Medical School, 30625 Hannover, Germany; (A.R.); (L.F.); (R.F.)
- Correspondence: ; Tel.: +49-511-532-9731
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Hoersten J, Ruiz-Gómez G, Lansing F, Rojo-Romanos T, Schmitt L, Sonntag J, Pisabarro M, Buchholz F. Pairing of single mutations yields obligate Cre-type site-specific recombinases. Nucleic Acids Res 2022; 50:1174-1186. [PMID: 34951450 PMCID: PMC8789052 DOI: 10.1093/nar/gkab1240] [Citation(s) in RCA: 7] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2021] [Revised: 11/24/2021] [Accepted: 12/06/2021] [Indexed: 12/28/2022] Open
Abstract
Tyrosine site-specific recombinases (SSRs) represent a versatile genome editing tool with considerable therapeutic potential. Recent developments to engineer and evolve SSRs into heterotetramers to improve target site flexibility signified a critical step towards their broad utility in genome editing. However, SSR monomers can form combinations of different homo- and heterotetramers in cells, increasing their off-target potential. Here, we discover that two paired mutations targeting residues implicated in catalysis lead to simple obligate tyrosine SSR systems, where the presence of all distinct subunits to bind as a heterotetramer is obligatory for catalysis. Therefore, only when the paired mutations are applied as single mutations on each recombinase subunit, the engineered SSRs can efficiently recombine the intended target sequence, while the subunits carrying the point mutations expressed in isolation are inactive. We demonstrate the utility of the obligate SSR system to improve recombination specificity of a designer-recombinase for a therapeutic target in human cells. Furthermore, we show that the mutations render the naturally occurring SSRs, Cre and Vika, obligately heteromeric for catalytic proficiency, providing a straight-forward approach to improve their applied properties. These results facilitate the development of safe and effective therapeutic designer-recombinases and advance our mechanistic understanding of SSR catalysis.
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Affiliation(s)
- Jenna Hoersten
- Medical Faculty and University Hospital Carl Gustav Carus, UCC Section Medical Systems Biology, TU Dresden, 01307 Dresden, Germany
| | - Gloria Ruiz-Gómez
- Structural Bioinformatics, BIOTEC TU Dresden, Tatzberg 47-51, 01307 Dresden, Germany
| | - Felix Lansing
- Medical Faculty and University Hospital Carl Gustav Carus, UCC Section Medical Systems Biology, TU Dresden, 01307 Dresden, Germany
| | - Teresa Rojo-Romanos
- Medical Faculty and University Hospital Carl Gustav Carus, UCC Section Medical Systems Biology, TU Dresden, 01307 Dresden, Germany
| | - Lukas Theo Schmitt
- Medical Faculty and University Hospital Carl Gustav Carus, UCC Section Medical Systems Biology, TU Dresden, 01307 Dresden, Germany
| | - Jan Sonntag
- Medical Faculty and University Hospital Carl Gustav Carus, UCC Section Medical Systems Biology, TU Dresden, 01307 Dresden, Germany
| | - M Teresa Pisabarro
- Structural Bioinformatics, BIOTEC TU Dresden, Tatzberg 47-51, 01307 Dresden, Germany
| | - Frank Buchholz
- Medical Faculty and University Hospital Carl Gustav Carus, UCC Section Medical Systems Biology, TU Dresden, 01307 Dresden, Germany
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48
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Lansing F, Mukhametzyanova L, Rojo-Romanos T, Iwasawa K, Kimura M, Paszkowski-Rogacz M, Karpinski J, Grass T, Sonntag J, Schneider PM, Günes C, Hoersten J, Schmitt LT, Rodriguez-Muela N, Knöfler R, Takebe T, Buchholz F. Correction of a Factor VIII genomic inversion with designer-recombinases. Nat Commun 2022; 13:422. [PMID: 35058465 PMCID: PMC8776779 DOI: 10.1038/s41467-022-28080-7] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/26/2021] [Accepted: 12/22/2021] [Indexed: 01/16/2023] Open
Abstract
Despite advances in nuclease-based genome editing technologies, correcting human disease-causing genomic inversions remains a challenge. Here, we describe the potential use of a recombinase-based system to correct the 140 kb inversion of the F8 gene frequently found in patients diagnosed with severe Hemophilia A. Employing substrate-linked directed molecular evolution, we develop a coupled heterodimeric recombinase system (RecF8) achieving 30% inversion of the target sequence in human tissue culture cells. Transient RecF8 treatment of endothelial cells, differentiated from patient-derived induced pluripotent stem cells (iPSCs) of a hemophilic donor, results in 12% correction of the inversion and restores Factor VIII mRNA expression. In this work, we present designer-recombinases as an efficient and specific means towards treatment of monogenic diseases caused by large gene inversions. Correction of disease-causing large genomic inversions remains challenging. Here, the authors developed a dual designer-recombinase system (RecF8) that efficiently corrects a 140 kb inversion frequently found in patients with severe Hemophilia A.
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49
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Jair Lara-Navarro I, Rebeca Jaloma-Cruz A. Current Therapies in Hemophilia: From Plasma-Derived Factor Modalities to CRISPR/Cas Alternatives. TOHOKU J EXP MED 2022; 256:197-207. [DOI: 10.1620/tjem.256.197] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/05/2023]
Affiliation(s)
- Irving Jair Lara-Navarro
- División de Genética, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social
| | - Ana Rebeca Jaloma-Cruz
- División de Genética, Centro de Investigación Biomédica de Occidente, Instituto Mexicano del Seguro Social
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Olgasi C, Borsotti C, Merlin S, Bergmann T, Bittorf P, Adewoye AB, Wragg N, Patterson K, Calabria A, Benedicenti F, Cucci A, Borchiellini A, Pollio B, Montini E, Mazzuca DM, Zierau M, Stolzing A, Toleikis P, Braspenning J, Follenzi A. Efficient and safe correction of hemophilia A by lentiviral vector-transduced BOECs in an implantable device. Mol Ther Methods Clin Dev 2021; 23:551-566. [PMID: 34853801 PMCID: PMC8606349 DOI: 10.1016/j.omtm.2021.10.015] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Revised: 10/06/2021] [Accepted: 10/29/2021] [Indexed: 11/18/2022]
Abstract
Hemophilia A (HA) is a rare bleeding disorder caused by deficiency/dysfunction of the FVIII protein. As current therapies based on frequent FVIII infusions are not a definitive cure, long-term expression of FVIII in endothelial cells through lentiviral vector (LV)-mediated gene transfer holds the promise of a one-time treatment. Thus, here we sought to determine whether LV-corrected blood outgrowth endothelial cells (BOECs) implanted through a prevascularized medical device (Cell Pouch) would rescue the bleeding phenotype of HA mice. To this end, BOECs from HA patients and healthy donors were isolated, expanded, and transduced with an LV carrying FVIII driven by an endothelial-specific promoter employing GMP-like procedures. FVIII-corrected HA BOECs were either directly transplanted into the peritoneal cavity or injected into a Cell Pouch implanted subcutaneously in NSG-HA mice. In both cases, FVIII secretion was sufficient to improve the mouse bleeding phenotype. Indeed, FVIII-corrected HA BOECs reached a relatively short-term clinically relevant engraftment being detected up to 16 weeks after transplantation, and their genomic integration profile did not show enrichment for oncogenes, confirming the process safety. Overall, this is the first preclinical study showing the safety and feasibility of transplantation of GMP-like produced LV-corrected BOECs within an implantable device for the long-term treatment of HA.
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Affiliation(s)
- Cristina Olgasi
- Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy
| | - Chiara Borsotti
- Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy
| | - Simone Merlin
- Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy
| | - Thorsten Bergmann
- Department of Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, 97082 Würzburg, Germany
| | - Patrick Bittorf
- Department of Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, 97082 Würzburg, Germany
| | - Adeolu Badi Adewoye
- Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, B15 2TT Birmingham, UK
| | - Nicholas Wragg
- Guy Hilton Research Centre, School of Pharmacy and Bioengineering, Keele University, Staffordshire, ST47QB Stoke-on-Trent, UK
| | | | | | | | - Alessia Cucci
- Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy
| | - Alessandra Borchiellini
- Haematology Unit Regional Center for Hemorrhagic and Thrombotic Diseases, City of Health and Science University Hospital of Molinette, 10126 Turin, Italy
| | - Berardino Pollio
- Immune-Haematology and Transfusion Medicine, Regina Margherita Children Hospital, City of Health and Science University Hospital of Molinette, 10126 Turin, Italy
| | | | | | - Martin Zierau
- IMS Integrierte Management Systeme e. K., 64646 Heppenheim, Germany
| | - Alexandra Stolzing
- Centre for Biological Engineering, School of Mechanical, Electrical and Manufacturing Engineering, Loughborough University, LE113TU Loughborough, UK
- SENS Research Foundation, Mountain View, CA 94041, USA
| | | | - Joris Braspenning
- Department of Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, 97082 Würzburg, Germany
| | - Antonia Follenzi
- Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy
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