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1
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Matuszek Z, Brown BL, Yrigollen CM, Keiser MS, Davidson BL. Current trends in gene therapy to treat inherited disorders of the brain. Mol Ther 2025; 33:1988-2014. [PMID: 40181540 DOI: 10.1016/j.ymthe.2025.03.057] [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: 03/17/2025] [Revised: 03/28/2025] [Accepted: 03/28/2025] [Indexed: 04/05/2025] Open
Abstract
Gene therapy development, re-engineering, and application to patients hold promise to revolutionize medicine, including therapies for disorders of the brain. Advances in delivery modalities, expression regulation, and improving safety profiles are of critical importance. Additionally, each inherited disorder has its own unique characteristics as to regions and cell types impacted and the temporal dynamics of that impact that are essential for the design of therapeutic design strategies. Here, we review the current state of the art in gene therapies for inherited brain disorders, summarizing key considerations for vector delivery, gene addition, gene silencing, gene editing, and epigenetic editing. We provide examples from animal models, human cell lines, and, where possible, clinical trials. This review also highlights the various tools available to researchers for basic research questions and discusses our views on the current limitations in the field.
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Affiliation(s)
- Zaneta Matuszek
- Merkin Institute of Transformative Technologies in Healthcare, Broad Institute of Harvard and MIT, Cambridge, MA 02138, USA; Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA
| | - Brandon L Brown
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Center for Epilepsy and Neurodevelopmental Disorders (ENDD), Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Carolyn M Yrigollen
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA
| | - Megan S Keiser
- Department of Neurological Surgery, The Ohio State Wexner Medical Center, Columbus, OH 43210, USA
| | - Beverly L Davidson
- Raymond G. Perelman Center for Cellular and Molecular Therapeutics, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Center for Epilepsy and Neurodevelopmental Disorders (ENDD), Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA; Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
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2
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Bolideei M, Barzigar R, Gahrouei RB, Mohebbi E, Haider KH, Paul S, Paul MK, Mehran MJ. Applications of Gene Editing and Nanotechnology in Stem Cell-Based Therapies for Human Diseases. Stem Cell Rev Rep 2025:10.1007/s12015-025-10857-0. [PMID: 40014250 DOI: 10.1007/s12015-025-10857-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/23/2025] [Indexed: 02/28/2025]
Abstract
Stem cell research is a dynamic and fast-advancing discipline with great promise for the treatment of diverse human disorders. The incorporation of gene editing technologies, including ZFNs, TALENs, and the CRISPR/Cas system, in conjunction with progress in nanotechnology, is fundamentally transforming stem cell therapy and research. These innovations not only provide a glimmer of optimism for patients and healthcare practitioners but also possess the capacity to radically reshape medical treatment paradigms. Gene editing and nanotechnology synergistically enhance stem cell-based therapies' precision, efficiency, and applicability, offering transformative potential for treating complex diseases and advancing regenerative medicine. Nevertheless, it is important to acknowledge that these technologies also give rise to ethical considerations and possible hazards, such as inadvertent genetic modifications and the development of genetically modified organisms, therefore creating a new age of designer infants. This review emphasizes the crucial significance of gene editing technologies and nanotechnology in the progress of stem cell treatments, particularly for degenerative pathologies and injuries. It emphasizes their capacity to restructure and comprehensively revolutionize medical treatment paradigms, providing fresh hope and optimism for patients and healthcare practitioners.
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Affiliation(s)
- Mansoor Bolideei
- Department of Respiratory and Critical Care Medicine, the Center for Biomedical Research, NHC Key Laboratory of Respiratory Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Sciences and Technology, Wuhan, China
| | - Rambod Barzigar
- Department of Biotechnology, SJCE Technical Campus, JSS Research Foundation, University of Mysore, Mysore, 570006, Karnataka, India
| | - Razieh Bahrami Gahrouei
- Department of Pharmacy PES College, Rajiv Gandhi University of Health Sciences, Bangalore, Karnataka, India
| | - Elham Mohebbi
- Department of Medical Microbiology, Immunology, and Cell Biology, Southern Illinois School of Medicine, Springfield, IL, USA
| | - Khawaja Husnain Haider
- Sulaiman AlRajhi Medical School, Al Bukayriyah, AlQaseem, 52726, Kingdom of Saudi Arabia
| | - Sayan Paul
- Department of Biochemistry & Molecular Biology, University of Texas Medical Branch at Galveston, Galveston, TX, 77555, USA.
| | - Manash K Paul
- Department of Radiation Biology and Toxicology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, 576104, India.
| | - Mohammad Javad Mehran
- Department of Biotechnology, SJCE Technical Campus, JSS Research Foundation, University of Mysore, Mysore, 570006, Karnataka, India.
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3
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Allisha J, Das J, Dunnigan T, Sharfstein ST, Datta P. Stipulations of cell and gene therapy and the ties to biomanufacturing. Biotechnol Prog 2025:e3521. [PMID: 39846483 DOI: 10.1002/btpr.3521] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2024] [Revised: 11/15/2024] [Accepted: 11/20/2024] [Indexed: 01/24/2025]
Abstract
Cell and gene therapy (CGT) products are emerging and innovative biopharmaceuticals that hold promise for treating diseases that are otherwise beyond the scope of conventional medicines. The evolution of CGT from a research idea to a promising therapeutic product is due to the complementary advancements across various scientific disciplines. First, the innovations and advancements in gene editing and delivery technology have provided fundamental tools to manipulate genes and cells for therapeutic pursuits. Second, advancements in applied and translational research, including how clinical trials are designed, performed, evaluated, and analyzed, have transformed the technology into a potential therapeutic product. Third, advancements in scaling up the production of CGT products have been critical in delivering the product for preclinical studies, clinical trials, and approved treatments. In parallel, regulatory requirements have continuously evolved, with lessons learned from translational studies and biomanufacturing. These combined efforts have transformed CGT products from a promising concept into a reality with the potential to treat a wide range of diseases. However, continued R&D and regulatory oversight are crucial to further improve the safety, efficacy, and accessibility of CGT products.
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Affiliation(s)
- Justin Allisha
- Department of Life Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA
| | - Juthika Das
- Department of Life Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA
| | - Thomas Dunnigan
- Department of Life Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA
| | - Susan T Sharfstein
- Department of Nanoscale Science and Engineering and The RNA Institute, University at Albany, State University of New York, Albany, New York, USA
| | - Payel Datta
- Department of Life Sciences, Albany College of Pharmacy and Health Sciences, Albany, New York, USA
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4
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Claiborne DT, Detwiler Z, Docken SS, Borland TD, Cromer D, Simkhovich A, Ophinni Y, Okawa K, Bateson T, Chen T, Hudson W, Trifonova R, Davenport MP, Ho TW, Boutwell CL, Allen TM. High frequency CCR5 editing in human hematopoietic stem progenitor cells protects xenograft mice from HIV infection. Nat Commun 2025; 16:446. [PMID: 39774003 PMCID: PMC11707138 DOI: 10.1038/s41467-025-55873-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/13/2024] [Accepted: 01/02/2025] [Indexed: 01/11/2025] Open
Abstract
The only cure of HIV has been achieved in a small number of people who received a hematopoietic stem cell transplant (HSCT) comprising allogeneic cells carrying a rare, naturally occurring, homozygous deletion in the CCR5 gene. The rarity of the mutation and the significant morbidity and mortality of such allogeneic transplants precludes widespread adoption of this HIV cure. Here, we show the application of CRISPR/Cas9 to achieve >90% CCR5 editing in human, mobilized hematopoietic stem progenitor cells (HSPC), resulting in a transplant that undergoes normal hematopoiesis, produces CCR5 null T cells, and renders xenograft mice refractory to HIV infection. Titration studies transplanting decreasing frequencies of CCR5 edited HSPCs demonstrate that <90% CCR5 editing confers decreasing protective benefit that becomes negligible between 54% and 26%. Our study demonstrates the feasibility of using CRISPR/Cas9/RNP to produce an HSPC transplant with high frequency CCR5 editing that is refractory to HIV replication. These results raise the potential of using CRISPR/Cas9 to produce a curative autologous HSCT and bring us closer to the development of a cure for HIV infection.
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Affiliation(s)
- Daniel T Claiborne
- Vaccine and Immunotherapy Center, The Wistar Institute, Philadelphia, PA, USA
| | | | - Steffen S Docken
- Kirby Institute, University of New South Wales, Sydney, NSW, Australia
| | | | - Deborah Cromer
- Kirby Institute, University of New South Wales, Sydney, NSW, Australia
| | | | | | - Ken Okawa
- Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA
| | | | - Tao Chen
- Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA
| | - Wesley Hudson
- Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA
| | | | - Miles P Davenport
- Kirby Institute, University of New South Wales, Sydney, NSW, Australia
| | - Tony W Ho
- CRISPR Therapeutics, Boston, MA, USA
| | | | - Todd M Allen
- Ragon Institute of MGH, MIT and Harvard, Cambridge, MA, USA
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Hiner CR, Mueller AL, Su H, Goldstein H. Interventions during Early Infection: Opening a Window for an HIV Cure? Viruses 2024; 16:1588. [PMID: 39459922 PMCID: PMC11512236 DOI: 10.3390/v16101588] [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: 09/08/2024] [Revised: 10/05/2024] [Accepted: 10/08/2024] [Indexed: 10/28/2024] Open
Abstract
Although combination antiretroviral therapy (ART) has been a landmark achievement for the treatment of human immunodeficiency virus (HIV), an HIV cure has remained elusive. Elimination of latent HIV reservoirs that persist throughout HIV infection is the most challenging barrier to an HIV cure. The progressive HIV infection is marked by the increasing size and diversity of latent HIV reservoirs until an effective immune response is mobilized, which can control but not eliminate HIV infection. The stalemate between HIV replication and the immune response is manifested by the establishment of a viral set point. ART initiation during the early stage limits HIV reservoir development, preserves immune function, improves the quality of life, and may lead to ART-free viral remission in a few people living with HIV (PLWH). However, for the overwhelming majority of PLWH, early ART initiation alone does not cure HIV, and lifelong ART is needed to sustain viral suppression. A critical area of research is focused on determining whether HIV could be functionally cured if additional treatments are provided alongside early ART. Several HIV interventions including Block and Lock, Shock and Kill, broadly neutralizing antibody (bNAb) therapy, adoptive CD8+ T cell therapy, and gene therapy have demonstrated delayed viral rebound and/or viral remission in animal models and/or some PLWH. Whether or not their application during early infection can improve the success of HIV remission is less studied. Herein, we review the current state of clinical and investigative HIV interventions and discuss their potential to improve the likelihood of post-treatment remission if initiated during early infection.
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Affiliation(s)
- Christopher R. Hiner
- Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; (C.R.H.); (A.L.M.)
| | - April L. Mueller
- Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; (C.R.H.); (A.L.M.)
| | - Hang Su
- Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; (C.R.H.); (A.L.M.)
| | - Harris Goldstein
- Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA; (C.R.H.); (A.L.M.)
- Department of Pediatrics, Albert Einstein College of Medicine, Bronx, NY 10461, USA
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6
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Lindegger DJ. Advanced Therapies for Human Immunodeficiency Virus. Med Sci (Basel) 2024; 12:33. [PMID: 39051379 PMCID: PMC11270269 DOI: 10.3390/medsci12030033] [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: 05/14/2024] [Revised: 06/27/2024] [Accepted: 07/05/2024] [Indexed: 07/27/2024] Open
Abstract
Human Immunodeficiency Virus (HIV) remains a significant global health challenge with approximately 38 million people currently having the virus worldwide. Despite advances in treatment development, the virus persists in the human population and still leads to new infections. The virus has a powerful ability to mutate and hide from the human immune system in reservoirs of the body. Current standard treatment with antiretroviral therapy effectively controls viral replication but requires lifelong adherence and does not eradicate the virus. This review explores the potential of Advanced Therapy Medicinal Products as novel therapeutic approaches to HIV, including cell therapy, immunisation strategies and gene therapy. Cell therapy, particularly chimeric antigen receptor T cell therapy, shows promise in preclinical studies for targeting and eliminating HIV-infected cells. Immunisation therapies, such as broadly neutralising antibodies are being investigated to control viral replication and reduce reservoirs. Despite setbacks in recent trials, vaccines remain a promising avenue for HIV therapy development. Gene therapy using technologies like CRISPR/Cas9 aims to modify cells to resist HIV infection or eliminate infected cells. Challenges such as off-target effects, delivery efficiency and ethical considerations persist in gene therapy for HIV. Future directions require further research to assess the safety and efficacy of emerging therapies in clinical trials. Combined approaches may be necessary to achieve complete elimination of the HIV reservoir. Overall, advanced therapies offer new hope for advancing HIV treatment and moving closer to a cure.
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Affiliation(s)
- Daniel Josef Lindegger
- Independent Researcher, 6000 Lucerne, Switzerland;
- Independent Researcher, London SW1A2JR, UK
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7
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Buck AM, LaFranchi BH, Henrich TJ. Gaining momentum: stem cell therapies for HIV cure. Curr Opin HIV AIDS 2024; 19:194-200. [PMID: 38686850 PMCID: PMC11155292 DOI: 10.1097/coh.0000000000000859] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 05/02/2024]
Abstract
PURPOSE OF REVIEW Durable HIV-1 remission has been reported in a person who received allogeneic stem cell transplants (SCTs) involving CCR5 Δ32/Δ32 donor cells. Much of the reduction in HIV-1 burden following allogeneic SCT with or without donor cells inherently resistant to HIV-1 infection is likely due to cytotoxic graft-versus-host effects on residual recipient immune cells. Nonetheless, there has been growing momentum to develop and implement stem cell therapies that lead to durable long-term antiretroviral therapy (ART)-free remission without the need for SCT. RECENT FINDINGS Most current research leverages gene editing techniques to modify hematopoietic stem cells which differentiate into immune cells capable of harboring HIV-1. Approaches include targeting genes that encode HIV-1 co-receptors using Zinc Finger Nucleases (ZFN) or CRISPR-Cas-9 to render a pool of adult or progenitor cells resistant to de-novo infection. Other strategies involve harnessing multipotent mesenchymal stromal cells to foster immune environments that can more efficiently recognize and target HIV-1 while promoting tissue homeostasis. SUMMARY Many of these strategies are currently in a state of infancy or adolescence; nonetheless, promising preclinical and first-in-human studies have been performed, providing further rationale to focus resources on stem cell therapies.
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Affiliation(s)
- Amanda M Buck
- Division of Experimental Medicine, University of California San Francisco, San Francisco, California, USA
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8
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Vasconcelos Komninakis S, Domingues W, Saeed Sanabani S, Angelo Folgosi V, Neves Barbosa I, Casseb J. CRISPR/CAS as a Powerful Tool for Human Immunodeficiency Virus Cure: A Review. AIDS Res Hum Retroviruses 2024; 40:363-375. [PMID: 38164106 DOI: 10.1089/aid.2022.0148] [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] [Indexed: 01/03/2024] Open
Abstract
Despite care and the availability of effective antiretroviral treatment, some human immunodeficiency virus (HIV)-infected individuals suffer from neurocognitive disorders associated with HIV (HAND) that significantly affect their quality of life. The different types of HAND can be divided into asymptomatic neurocognitive impairment, mild neurocognitive disorder, and the most severe form known as HIV-associated dementia. Little is known about the mechanisms of HAND, but it is thought to be related to infection of astrocytes, microglial cells, and macrophages in the human brain. The formation of a viral reservoir that lies dormant as a provirus in resting CD4+ T lymphocytes and in refuge tissues such as the brain contributes significantly to HIV eradication. In recent years, a new set of tools have emerged: the gene editing based on the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system, which can alter genome segments by insertion, deletion, and replacement and has great therapeutic potential. This technology has been used in research to treat HIV and appears to offer hope for a possible cure for HIV infection and perhaps prevention of HAND. This approach has the potential to directly impact the quality of life of HIV-infected individuals, which is a very important topic to be known and discussed.
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Affiliation(s)
- Shirley Vasconcelos Komninakis
- Laboratory of Medical Investigation (LIM56) of the School of Medicine/Institute de Tropical Medicine, Department of Dermatology, São Paulo University, São Paulo, São Paulo, Brazil
| | - Wilson Domingues
- Laboratory of Medical Investigation (LIM56) of the School of Medicine/Institute de Tropical Medicine, Department of Dermatology, São Paulo University, São Paulo, São Paulo, Brazil
| | - Sabri Saeed Sanabani
- Laboratory of Medical Investigation (LIM56) of the School of Medicine/Institute de Tropical Medicine, Department of Dermatology, São Paulo University, São Paulo, São Paulo, Brazil
| | - Victor Angelo Folgosi
- Laboratory of Medical Investigation (LIM56) of the School of Medicine/Institute de Tropical Medicine, Department of Dermatology, São Paulo University, São Paulo, São Paulo, Brazil
| | - Igor Neves Barbosa
- Institute of Genetic Biology at the Biological Institute of São Paulo University, São Paulo, São Paulo, Brazil
| | - Jorge Casseb
- Laboratory of Medical Investigation (LIM56) of the School of Medicine/Institute de Tropical Medicine, Department of Dermatology, São Paulo University, São Paulo, São Paulo, Brazil
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Dudek AM, Feist WN, Sasu EJ, Luna SE, Ben-Efraim K, Bak RO, Cepika AM, Porteus MH. A simultaneous knockout knockin genome editing strategy in HSPCs potently inhibits CCR5- and CXCR4-tropic HIV-1 infection. Cell Stem Cell 2024; 31:499-518.e6. [PMID: 38579682 PMCID: PMC11212398 DOI: 10.1016/j.stem.2024.03.002] [Citation(s) in RCA: 3] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2023] [Revised: 12/29/2023] [Accepted: 03/06/2024] [Indexed: 04/07/2024]
Abstract
Allogeneic hematopoietic stem and progenitor cell transplant (HSCT) of CCR5 null (CCR5Δ32) cells can be curative for HIV-1-infected patients. However, because allogeneic HSCT poses significant risk, CCR5Δ32 matched bone marrow donors are rare, and CCR5Δ32 transplant does not confer resistance to the CXCR4-tropic virus, it is not a viable option for most patients. We describe a targeted Cas9/AAV6-based genome editing strategy for autologous HSCT resulting in both CCR5- and CXCR4-tropic HIV-1 resistance. Edited human hematopoietic stem and progenitor cells (HSPCs) maintain multi-lineage repopulation capacity in vivo, and edited primary human T cells potently inhibit infection by both CCR5-tropic and CXCR4-tropic HIV-1. Modification rates facilitated complete loss of CCR5-tropic replication and up to a 2,000-fold decrease in CXCR4-tropic replication without CXCR4 locus disruption. This multi-factor editing strategy in HSPCs could provide a broad approach for autologous HSCT as a functional cure for both CCR5-tropic and CXCR4-tropic HIV-1 infections.
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Affiliation(s)
- Amanda M Dudek
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - William N Feist
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Elena J Sasu
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Sofia E Luna
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Kaya Ben-Efraim
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Rasmus O Bak
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Biomedicine, Aarhus University, 8000 Aarhus, Denmark; Aarhus Institute of Advanced Studies (AIAS), Aarhus University, 8000 Aarhus, Denmark
| | - Alma-Martina Cepika
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA
| | - Matthew H Porteus
- Department of Pediatrics, Stanford University School of Medicine, Stanford, CA 94305, USA; Institute for Stem Cell Biology & Regenerative Medicine, Stanford University School of Medicine, Stanford, CA 94305, USA.
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10
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Li K, Zhang Q. Eliminating the HIV tissue reservoir: current strategies and challenges. Infect Dis (Lond) 2024; 56:165-182. [PMID: 38149977 DOI: 10.1080/23744235.2023.2298450] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/09/2023] [Accepted: 12/16/2023] [Indexed: 12/28/2023] Open
Abstract
BACKGROUND Acquired immunodeficiency syndrome (AIDS) is still one of the most widespread and harmful infectious diseases in the world. The presence of reservoirs housing the human immunodeficiency virus (HIV) represents a significant impediment to the development of clinically applicable treatments on a large scale. The viral load in the blood can be effectively reduced to undetectable levels through antiretroviral therapy (ART), and a higher concentration of HIV is sequestered in various tissues throughout the body, forming the tissue reservoir - the source of viremia after interruption treatment. METHODS We take the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) as a guideline for this review. In June 2023, we used the Pubmed, Embase, and Scopus databases to search the relevant literature published in the last decade. RESULTS Here we review the current strategies and treatments for eliminating the HIV tissue reservoirs: early and intensive therapy, gene therapy (including ribozyme, RNA interference, RNA aptamer, zinc finger enzyme, transcriptional activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/associated nuclease 9 (CRISPR/Cas9)), 'Shock and Kill', 'Block and lock', immunotherapy (including therapeutic vaccines, broadly neutralising antibodies (bNAbs), chimeric antigen receptor T-cell immunotherapy (CAR-T)), and haematopoietic stem cell transplantation (HSCT). CONCLUSION The existence of an HIV reservoir is the main obstacle to the complete cure of AIDS. Choosing the appropriate strategy to deplete the HIV reservoir and achieve a functional cure for AIDS is the focus and difficulty of current research. So far, there has been a lot of research and progress in reducing the HIV reservoir, but in general, the current research is still very preliminary. Much research is still needed to properly assess the reliability, effectiveness, and necessity of these strategies.
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Affiliation(s)
- Kangpeng Li
- National Center for Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China
| | - Qiang Zhang
- National Center for Infectious Diseases, Beijing Ditan Hospital, Capital Medical University, Beijing, China
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11
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Volodina OV, Fabrichnikova AR, Anuchina AA, Mishina OS, Lavrov AV, Smirnikhina SA. Evolution of Prime Editing Systems: Move Forward to the Treatment of Hereditary Diseases. Curr Gene Ther 2024; 25:46-61. [PMID: 38623982 DOI: 10.2174/0115665232295117240405070809] [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: 01/04/2024] [Revised: 03/19/2024] [Accepted: 03/20/2024] [Indexed: 04/17/2024]
Abstract
The development of gene therapy using genome editing tools recently became relevant. With the invention of programmable nucleases, it became possible to treat hereditary diseases due to introducing targeted double strand break in the genome followed by homology directed repair (HDR) or non-homologous end-joining (NHEJ) reparation. CRISPR-Cas9 is more efficient and easier to use in comparison with other programmable nucleases. To improve the efficiency and safety of this gene editing tool, various modifications CRISPR-Cas9 basis were created in recent years, such as prime editing - in this system, Cas9 nickase is fused with reverse transcriptase and guide RNA, which contains a desired correction. Prime editing demonstrates equal or higher correction efficiency as HDR-mediated editing and much less off-target effect due to inducing nick. There are several studies in which prime editing is used to correct mutations in which researchers reported little or no evidence of off-target effects. The system can also be used to functionally characterize disease variants. However, prime editing still has several limitations that could be further improved. The effectiveness of the method is not yet high enough to apply it in clinical trials. Delivery of prime editors is also a big challenge due to their size. In the present article, we observe the development of the platform, and discuss the candidate proteins for efficiency enhancing, main delivery methods and current applications of prime editing.
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Affiliation(s)
- Olga V Volodina
- Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522, Moscow, Russia
| | | | - Arina A Anuchina
- Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522, Moscow, Russia
| | - Olesya S Mishina
- Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522, Moscow, Russia
| | - Alexander V Lavrov
- Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522, Moscow, Russia
| | - Svetlana A Smirnikhina
- Laboratory of Genome Editing, Research Centre for Medical Genetics, 115522, Moscow, Russia
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12
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Williams L, Larsen J. Nanoparticle-mediated delivery of non-viral gene editing technology to the brain. Prog Neurobiol 2024; 232:102547. [PMID: 38042249 PMCID: PMC10872436 DOI: 10.1016/j.pneurobio.2023.102547] [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: 07/18/2023] [Revised: 11/01/2023] [Accepted: 11/26/2023] [Indexed: 12/04/2023]
Abstract
Neurological disorders pose a significant burden on individuals and society, affecting millions worldwide. These disorders, including but not limited to Alzheimer's disease, Parkinson's disease, and Huntington's disease, often have limited treatment options and can lead to progressive degeneration and disability. Gene editing technologies, including Zinc Finger Nucleases (ZFN), Transcription Activator-Like Effector Nucleases (TALEN), and Clustered Regularly Interspaced Short Palindromic Repeats-associated Protein 9 (CRISPR-Cas9), offer a promising avenue for potential cures by targeting and correcting the underlying genetic mutations responsible for neurologic disorders. However, efficient delivery methods are crucial for the successful application of gene editing technologies in the context of neurological disorders. The central nervous system presents unique challenges to treatment development due to the blood-brain barrier, which restricts the entry of large molecules. While viral vectors are traditionally used for gene delivery, nonviral delivery methods, such as nanoparticle-mediated delivery, offer safer alternatives that can efficiently transport gene editing components. Herein we aim to introduce the three main gene editing nucleases as nonviral treatments for neurologic disorders, the delivery barriers associated with brain targeting, and the current nonviral techniques used for brain-specific delivery. We highlight the challenges and opportunities for future research in this exciting and growing field that could lead to blood-brain barrier bypassing therapeutic gene editing.
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Affiliation(s)
- Lucian Williams
- Department of Bioengineering, Clemson University, Clemson, SC 29631, USA
| | - Jessica Larsen
- Department of Bioengineering, Clemson University, Clemson, SC 29631, USA; Department of Chemical Engineering, Clemson University, Clemson, SC 29631, USA.
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Au TY, Arudkumar J, Assavarittirong C, Benjamin S. Killing two birds with one stone: CRISPR/Cas9 CCR5 knockout hematopoietic stem cells transplantation to treat patients with HIV infection and hematological malignancies concurrently. Clin Exp Med 2023; 23:4163-4175. [PMID: 37500934 DOI: 10.1007/s10238-023-01129-7] [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: 02/07/2023] [Accepted: 06/23/2023] [Indexed: 07/29/2023]
Abstract
Human immunodeficiency virus (HIV) is known to cause hematological malignancy. Hematopoietic stem cell transplantation (HPSCT) is an advanced treatment for that. Currently, there are three successful HIV-eliminated cases, and two received HPSCT from CCR5-absent donors. It is well established that the CCR5 protein on the cell surface assists human immunodeficiency virus entry. Preliminary studies have revealed that knocking out CCR5 and/or CXCR4 may inhibit the viral entry of HIV, which may prove promising in the further development of HIV treatment options. Herein, we suggest performing autologous or allogeneic HSCT with CCR5 KO hematopoietic stem cells in patients who suffer from complicated HIV conditions, particularly drug-resistant HIV or a concurrent diagnosis of HIV with lymphoma/leukemia, to achieve complete HIV remission. Nevertheless, at the clinical forefront of CRISPR-HIV technology, more efforts should be directed to advance nonhuman primate (NHP) models for studies of HIV pathogenesis and off-target assessments within this system. CRISPR-Cas9 knock out of host HSCT-expressing CCR5 or CXCR4 may confer HIV-resistance, which when applied to bedside therapeutics in an allogeneic or autologous manner can warrant a permanent and effective treatment outcome.
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Affiliation(s)
- Tsz Yuen Au
- Center for Medical Education in English, Poznan University of Medical Sciences, Poznan, Poland
| | - Jayshen Arudkumar
- South Australian Health and Medical Research Institute, Adelaide, SA, Australia.
- The University of Adelaide, Adelaide, SA, Australia.
| | - Chanika Assavarittirong
- Center for Medical Education in English, Poznan University of Medical Sciences, Poznan, Poland
| | - Shamiram Benjamin
- Center for Medical Education in English, Poznan University of Medical Sciences, Poznan, Poland
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14
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Murugesan R, Karuppusamy KV, Marepally S, Thangavel S. Current approaches and potential challenges in the delivery of gene editing cargos into hematopoietic stem and progenitor cells. Front Genome Ed 2023; 5:1148693. [PMID: 37780116 PMCID: PMC10540692 DOI: 10.3389/fgeed.2023.1148693] [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: 01/20/2023] [Accepted: 08/17/2023] [Indexed: 10/03/2023] Open
Abstract
Advancements in gene delivery and editing have expanded the applications of autologous hematopoietic stem and progenitor cells (HSPCs) for the treatment of monogenic and acquired diseases. The gene editing toolbox is growing, and the ability to achieve gene editing with mRNA or protein delivered intracellularly by vehicles, such as electroporation and nanoparticles, has highlighted the potential of gene editing in HSPCs. Ongoing phase I/II clinical trials with gene-edited HSPCs for β-hemoglobinopathies provide hope for treating monogenic diseases. The development of safe and efficient gene editing reagents and their delivery into hard-to-transfect HSPCs have been critical drivers in the rapid translation of HSPC gene editing into clinical studies. This review article summarizes the available payloads and delivery vehicles for gene editing HSPCs and their potential impact on therapeutic applications.
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Affiliation(s)
- Ramya Murugesan
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, India
- Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Karthik V. Karuppusamy
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, India
- Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Srujan Marepally
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, India
| | - Saravanabhavan Thangavel
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Christian Medical College Campus, Vellore, Tamil Nadu, India
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15
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Zheng R, Zhang L, Parvin R, Su L, Chi J, Shi K, Ye F, Huang X. Progress and Perspective of CRISPR-Cas9 Technology in Translational Medicine. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2023; 10:e2300195. [PMID: 37356052 PMCID: PMC10477906 DOI: 10.1002/advs.202300195] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/09/2023] [Revised: 03/29/2023] [Indexed: 06/27/2023]
Abstract
Translational medicine aims to improve human health by exploring potential treatment methods developed during basic scientific research and applying them to the treatment of patients in clinical settings. The advanced perceptions of gene functions have remarkably revolutionized clinical treatment strategies for target agents. However, the progress in gene editing therapy has been hindered due to the severe off-target effects and limited editing sites. Fortunately, the development in the clustered regularly interspaced short palindromic repeats associated protein 9 (CRISPR-Cas9) system has renewed hope for gene therapy field. The CRISPR-Cas9 system can fulfill various simple or complex purposes, including gene knockout, knock-in, activation, interference, base editing, and sequence detection. Accordingly, the CRISPR-Cas9 system is adaptable to translational medicine, which calls for the alteration of genomic sequences. This review aims to present the latest CRISPR-Cas9 technology achievements and prospect to translational medicine advances. The principle and characterization of the CRISPR-Cas9 system are firstly introduced. The authors then focus on recent pre-clinical and clinical research directions, including the construction of disease models, disease-related gene screening and regulation, and disease treatment and diagnosis for multiple refractory diseases. Finally, some clinical challenges including off-target effects, in vivo vectors, and ethical problems, and future perspective are also discussed.
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Affiliation(s)
- Ruixuan Zheng
- Joint Centre of Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
- Division of Pulmonary MedicineThe First Affiliated HospitalWenzhou Medical UniversityWenzhouZhejiang325000P. R. China
- Wenzhou Key Laboratory of Interdiscipline and Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
| | - Lexiang Zhang
- Joint Centre of Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
- Wenzhou Key Laboratory of Interdiscipline and Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
- Oujiang Laboratory (Zhejiang Lab for Regenerative MedicineVision and Brain Health); Wenzhou InstituteUniversity of Chinese Academy of SciencesWenzhouZhejiang325000P. R. China
| | - Rokshana Parvin
- Oujiang Laboratory (Zhejiang Lab for Regenerative MedicineVision and Brain Health); Wenzhou InstituteUniversity of Chinese Academy of SciencesWenzhouZhejiang325000P. R. China
| | - Lihuang Su
- Joint Centre of Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
- Division of Pulmonary MedicineThe First Affiliated HospitalWenzhou Medical UniversityWenzhouZhejiang325000P. R. China
- Wenzhou Key Laboratory of Interdiscipline and Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
| | - Junjie Chi
- Joint Centre of Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
- Wenzhou Key Laboratory of Interdiscipline and Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
| | - Keqing Shi
- Joint Centre of Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
- Wenzhou Key Laboratory of Interdiscipline and Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
| | - Fangfu Ye
- Joint Centre of Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
- Oujiang Laboratory (Zhejiang Lab for Regenerative MedicineVision and Brain Health); Wenzhou InstituteUniversity of Chinese Academy of SciencesWenzhouZhejiang325000P. R. China
- Beijing National Laboratory for Condensed Matter PhysicsInstitute of PhysicsChinese Academy of SciencesBeijing100190P. R. China
| | - Xiaoying Huang
- Joint Centre of Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
- Division of Pulmonary MedicineThe First Affiliated HospitalWenzhou Medical UniversityWenzhouZhejiang325000P. R. China
- Wenzhou Key Laboratory of Interdiscipline and Translational MedicineThe First Affiliated Hospital of Wenzhou Medical UniversityWenzhouZhejiang325000P. R. China
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16
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Rojo-Romanos T, Karpinski J, Millen S, Beschorner N, Simon F, Paszkowski-Rogacz M, Lansing F, Schneider PM, Sonntag J, Hauber J, Thoma-Kress AK, Buchholz F. Precise excision of HTLV-1 provirus with a designer-recombinase. Mol Ther 2023; 31:2266-2285. [PMID: 36934299 PMCID: PMC10362392 DOI: 10.1016/j.ymthe.2023.03.014] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2022] [Revised: 02/06/2023] [Accepted: 03/12/2023] [Indexed: 03/19/2023] Open
Abstract
The human T cell leukemia virus type 1 (HTLV-1) is a pathogenic retrovirus that persists as a provirus in the genome of infected cells and can lead to adult T cell leukemia (ATL). Worldwide, more than 10 million people are infected and approximately 5% of these individuals will develop ATL, a highly aggressive cancer that is currently incurable. In the last years, genome editing tools have emerged as promising antiviral agents. In this proof-of-concept study, we use substrate-linked directed evolution (SLiDE) to engineer Cre-derived site-specific recombinases to excise the HTLV-1 proviral genome from infected cells. We identified a conserved loxP-like sequence (loxHTLV) present in the long terminal repeats of the majority of virus isolates. After 181 cycles of SLiDE, we isolated a designer-recombinase (designated RecHTLV), which efficiently recombines the loxHTLV sequence in bacteria and human cells with high specificity. Expression of RecHTLV in human Jurkat T cells resulted in antiviral activity when challenged with an HTLV-1 infection. Moreover, expression of RecHTLV in chronically infected SP cells led to the excision of HTLV-1 proviral DNA. Our data suggest that recombinase-mediated excision of the HTLV-1 provirus represents a promising approach to reduce proviral load in HTLV-1-infected individuals, potentially preventing the development of HTLV-1-associated diseases.
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Affiliation(s)
- Teresa Rojo-Romanos
- Medical Systems Biology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technical University Dresden, 01307 Dresden, Germany
| | - Janet Karpinski
- Medical Systems Biology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technical University Dresden, 01307 Dresden, Germany
| | - Sebastian Millen
- Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany
| | - Niklas Beschorner
- PROVIREX Genome Editing Therapies GmbH, Luruper Hauptstrasse 1, 22547 Hamburg, Germany
| | - Florian Simon
- Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany
| | - Maciej Paszkowski-Rogacz
- Medical Systems Biology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technical University Dresden, 01307 Dresden, Germany
| | - Felix Lansing
- Medical Systems Biology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technical University Dresden, 01307 Dresden, Germany
| | - Paul Martin Schneider
- Medical Systems Biology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technical University Dresden, 01307 Dresden, Germany
| | - Jan Sonntag
- Medical Systems Biology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technical University Dresden, 01307 Dresden, Germany
| | - Joachim Hauber
- PROVIREX Genome Editing Therapies GmbH, Luruper Hauptstrasse 1, 22547 Hamburg, Germany
| | - Andrea K Thoma-Kress
- Institute of Clinical and Molecular Virology, Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 91054 Erlangen, Germany
| | - Frank Buchholz
- Medical Systems Biology, Faculty of Medicine and University Hospital Carl Gustav Carus, Technical University Dresden, 01307 Dresden, Germany.
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17
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Baroncini L, Bredl S, Nicole KP, Speck RF. The Humanized Mouse Model: What Added Value Does It Offer for HIV Research? Pathogens 2023; 12:pathogens12040608. [PMID: 37111494 PMCID: PMC10142098 DOI: 10.3390/pathogens12040608] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2023] [Revised: 04/13/2023] [Accepted: 04/14/2023] [Indexed: 04/29/2023] Open
Abstract
In the early 2000s, novel humanized mouse models based on the transplantation of human hematopoietic stem and progenitor cells (HSPCs) into immunocompromised mice were introduced (hu mice). The human HSPCs gave rise to a lymphoid system of human origin. The HIV research community has greatly benefitted from these hu mice. Since human immunodeficiency virus (HIV) type 1 infection results in a high-titer disseminated HIV infection, hu mice have been of great value for all types of HIV research from pathogenesis to novel therapies. Since the first description of this new generation of hu mice, great efforts have been expended to improve humanization by creating other immunodeficient mouse models or supplementing mice with human transgenes to improve human engraftment. Many labs have their own customized hu mouse models, making comparisons quite difficult. Here, we discuss the different hu mouse models in the context of specific research questions in order to define which characteristics should be considered when determining which hu mouse model is appropriate for the question posed. We strongly believe that researchers must first define their research question and then determine whether a hu mouse model exists, allowing the research question to be studied.
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Affiliation(s)
- Luca Baroncini
- Department of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, 8091 Zurich, Switzerland
| | - Simon Bredl
- Department of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, 8091 Zurich, Switzerland
| | - Kadzioch P Nicole
- Department of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, 8091 Zurich, Switzerland
| | - Roberto F Speck
- Department of Infectious Diseases and Hospital Epidemiology, University Hospital of Zurich, University of Zurich, 8091 Zurich, Switzerland
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18
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Vamva E, Ozog S, Leaman DP, Yu-Hong Cheng R, Irons NJ, Ott A, Stoffers C, Khan I, Goebrecht GK, Gardner MR, Farzan M, Rawlings DJ, Zwick MB, James RG, Torbett BE. A lentiviral vector B cell gene therapy platform for the delivery of the anti-HIV-1 eCD4-Ig-knob-in-hole-reversed immunoadhesin. Mol Ther Methods Clin Dev 2023; 28:366-384. [PMID: 36879849 PMCID: PMC9984920 DOI: 10.1016/j.omtm.2023.02.004] [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: 03/01/2022] [Accepted: 02/08/2023] [Indexed: 02/12/2023]
Abstract
Barriers to effective gene therapy for many diseases include the number of modified target cells required to achieve therapeutic outcomes and host immune responses to expressed therapeutic proteins. As long-lived cells specialized for protein secretion, antibody-secreting B cells are an attractive target for foreign protein expression in blood and tissue. To neutralize HIV-1, we developed a lentiviral vector (LV) gene therapy platform for delivery of the anti-HIV-1 immunoadhesin, eCD4-Ig, to B cells. The EμB29 enhancer/promoter in the LV limited gene expression in non-B cell lineages. By engineering a knob-in-hole-reversed (KiHR) modification in the CH3-Fc eCD4-Ig domain, we reduced interactions between eCD4-Ig and endogenous B cell immunoglobulin G proteins, which improved HIV-1 neutralization potency. Unlike previous approaches in non-lymphoid cells, eCD4-Ig-KiHR produced in B cells promoted HIV-1 neutralizing protection without requiring exogenous TPST2, a tyrosine sulfation enzyme required for eCD4-Ig-KiHR function. This finding indicated that B cell machinery is well suited to produce therapeutic proteins. Lastly, to overcome the inefficient transduction efficiency associated with VSV-G LV delivery to primary B cells, an optimized measles pseudotyped LV packaging methodology achieved up to 75% transduction efficiency. Overall, our findings support the utility of B cell gene therapy platforms for therapeutic protein delivery.
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Affiliation(s)
- Eirini Vamva
- Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, USA
| | - Stosh Ozog
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA
- Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA
| | - Daniel P. Leaman
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA
| | - Rene Yu-Hong Cheng
- Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, USA
| | - Nicholas J. Irons
- Department of Statistics, University of Washington, Seattle, WA, USA
| | - Andee Ott
- Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, USA
| | - Claire Stoffers
- Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, USA
| | - Iram Khan
- Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, USA
| | | | - Matthew R. Gardner
- Department of Infectious Diseases, The Scripps Research Institute, Jupiter, FL, USA
| | - Michael Farzan
- Department of Infectious Diseases, The Scripps Research Institute, Jupiter, FL, USA
| | - David J. Rawlings
- Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, USA
- Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA
- Department of Immunology, University of Washington School of Medicine, Seattle, WA, USA
| | - Michael B. Zwick
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA
| | - Richard G. James
- Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, USA
- Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA
- Department of Pharmacology, University of Washington School of Medicine, Seattle, WA, USA
| | - Bruce E. Torbett
- Center for Immunity and Immunotherapies, Seattle Children’s Research Institute, Seattle, WA, USA
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, USA
- Department of Pediatrics, University of Washington School of Medicine, Seattle, WA, USA
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA
- Institute for Stem Cell and Regenerative Medicine, Seattle, WA, USA
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19
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Third patient free of HIV after receiving virus-resistant cells. Nature 2023; 615:13-14. [PMID: 36807347 DOI: 10.1038/d41586-023-00479-2] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/22/2023]
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20
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Phan HTL, Kim K, Lee H, Seong JK. Progress in and Prospects of Genome Editing Tools for Human Disease Model Development and Therapeutic Applications. Genes (Basel) 2023; 14:483. [PMID: 36833410 PMCID: PMC9957140 DOI: 10.3390/genes14020483] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2022] [Revised: 02/09/2023] [Accepted: 02/10/2023] [Indexed: 02/17/2023] Open
Abstract
Programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas, are widely accepted because of their diversity and enormous potential for targeted genomic modifications in eukaryotes and other animals. Moreover, rapid advances in genome editing tools have accelerated the ability to produce various genetically modified animal models for studying human diseases. Given the advances in gene editing tools, these animal models are gradually evolving toward mimicking human diseases through the introduction of human pathogenic mutations in their genome rather than the conventional gene knockout. In the present review, we summarize the current progress in and discuss the prospects for developing mouse models of human diseases and their therapeutic applications based on advances in the study of programmable nucleases.
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Affiliation(s)
- Hong Thi Lam Phan
- Department of Physiology, Korea University College of Medicine, Seoul 02841, Republic of Korea
| | - Kyoungmi Kim
- Department of Physiology, Korea University College of Medicine, Seoul 02841, Republic of Korea
- Department of Biomedical Sciences, Korea University College of Medicine, Seoul 02841, Republic of Korea
| | - Ho Lee
- Graduate School of Cancer Science and Policy, National Cancer Center, Goyang 10408, Republic of Korea
| | - Je Kyung Seong
- Korea Mouse Phenotyping Center, Seoul National University, Seoul 08826, Republic of Korea
- Laboratory of Developmental Biology and Genomics, BK21 PLUS Program for Creative Veterinary Science Research, Research Institute for Veterinary Science, College of Veterinary Medicine, Seoul National University, Seoul 08826, Republic of Korea
- Interdisciplinary Program for Bioinformatics, Program for Cancer Biology, BIO-MAX/N-Bio Institute, Seoul National University, Seoul 08826, Republic of Korea
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21
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Colas C, Volodina O, Béland K, Pham TNQ, Li Y, Dallaire F, Soulard C, Lemieux W, Colamartino ABL, Tremblay-Laganière C, Dicaire R, Guimond J, Vobecky S, Poirier N, Patey N, Cohen ÉA, Haddad E. Generation of functional human T cell development in NOD/SCID/IL2rγ null humanized mice without using fetal tissue: Application as a model of HIV infection and persistence. Stem Cell Reports 2023; 18:597-612. [PMID: 36736326 PMCID: PMC9969074 DOI: 10.1016/j.stemcr.2023.01.003] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/31/2022] [Revised: 01/04/2023] [Accepted: 01/05/2023] [Indexed: 02/05/2023] Open
Abstract
Humanization of mice with functional T cells currently relies on co-implantation of hematopoietic stem cells from fetal liver and autologous fetal thymic tissue (so-called BLT mouse model). Here, we show that NOD/SCID/IL2rγnull mice humanized with cord blood- derived CD34+ cells and implanted with allogeneic pediatric thymic tissues excised during cardiac surgeries (CCST) represent an alternative to BLT mice. CCST mice displayed a strong immune reconstitution, with functional T cells originating from CD34+ progenitor cells. They were equally susceptible to mucosal or intraperitoneal HIV infection and had significantly higher HIV-specific T cell responses. Antiretroviral therapy (ART) robustly suppressed viremia and reduced the frequencies of cells carrying integrated HIV DNA. As in BLT mice, we observed a complete viral rebound following ART interruption, suggesting the presence of HIV reservoirs. In conclusion, CCST mice represent a practical alternative to BLT mice, broadening the use of humanized mice for research.
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Affiliation(s)
- Chloé Colas
- Department of Microbiology and Immunology, Université de Montréal, Montreal, QC H3T 1J4, Canada; CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada
| | - Olga Volodina
- Montreal Clinical Research Institute (IRCM), Montreal, QC H2W 1R7, Canada
| | - Kathie Béland
- CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada
| | - Tram N Q Pham
- Department of Microbiology and Immunology, Université de Montréal, Montreal, QC H3T 1J4, Canada; Montreal Clinical Research Institute (IRCM), Montreal, QC H2W 1R7, Canada
| | - Yuanyi Li
- CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada
| | - Frédéric Dallaire
- Montreal Clinical Research Institute (IRCM), Montreal, QC H2W 1R7, Canada
| | - Clara Soulard
- Department of Microbiology and Immunology, Université de Montréal, Montreal, QC H3T 1J4, Canada; CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada
| | - William Lemieux
- Department of Microbiology and Immunology, Université de Montréal, Montreal, QC H3T 1J4, Canada; CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada
| | - Aurélien B L Colamartino
- Department of Microbiology and Immunology, Université de Montréal, Montreal, QC H3T 1J4, Canada; CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada
| | - Camille Tremblay-Laganière
- Department of Microbiology and Immunology, Université de Montréal, Montreal, QC H3T 1J4, Canada; CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada
| | - Renée Dicaire
- CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada
| | | | - Suzanne Vobecky
- Department of Cardiac Surgery, CHU Sainte-Justine, Montreal, QC H3T 1C5, Canada
| | - Nancy Poirier
- Department of Cardiac Surgery, CHU Sainte-Justine, Montreal, QC H3T 1C5, Canada
| | - Natasha Patey
- CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada; Department of Pathology, CHU Sainte-Justine, Université de Montréal, Montreal, QC H3T 1C5, Canada
| | - Éric A Cohen
- Department of Microbiology and Immunology, Université de Montréal, Montreal, QC H3T 1J4, Canada; Montreal Clinical Research Institute (IRCM), Montreal, QC H2W 1R7, Canada.
| | - Elie Haddad
- Department of Microbiology and Immunology, Université de Montréal, Montreal, QC H3T 1J4, Canada; CHU Sainte-Justine Research Center, Montreal, QC H3T 1C5, Canada; Department of Pediatrics, Université de Montréal, Montreal, QC H3T 1C5, Canada.
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22
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Danaeifar M. Recent advances in gene therapy: genetic bullets to the root of the problem. Clin Exp Med 2022:10.1007/s10238-022-00925-x. [PMID: 36284069 DOI: 10.1007/s10238-022-00925-x] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2022] [Accepted: 10/14/2022] [Indexed: 12/28/2022]
Abstract
Genetics and molecular genetic techniques have changed many perspectives and paradigms in medicine. Using genetic methods, many diseases have been cured or alleviated. Gene therapy, in its simplest definition, is application of genetic materials and related techniques to treat various human diseases. Evaluation of the trends in the field of medicine and therapeutics clarifies that gene therapy has attracted a lot of attention due to its powerful potential to treat a number of diseases. There are various genetic materials that can be used in gene therapy such as DNA, single- and double-stranded RNA, siRNA and shRNA. The main gene editing techniques used for in vitro and in vivo gene modification are ZNF, TALEN and CRISPR-Cas9. The latter has increased hopes for more precise and efficient gene targeting as it requires two separate recognition sites which makes it more specific and can also cause rapid and sufficient cleavage within the target sequence. There must be carriers for delivering genes to the target tissue. The most commonly used carriers for this purpose are viral vectors such as adenoviruses, adeno-associated viruses and lentiviruses. Non-viral vectors consist of bacterial vectors, liposomes, dendrimers and nanoparticles.
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23
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Advances in CRISPR/Cas9. BIOMED RESEARCH INTERNATIONAL 2022; 2022:9978571. [PMID: 36193328 PMCID: PMC9525763 DOI: 10.1155/2022/9978571] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 08/09/2022] [Accepted: 08/22/2022] [Indexed: 11/30/2022]
Abstract
CRISPR/Cas9 technology has become the most examined gene editing technology in recent years due to its simple design, yet low cost, high efficiency, and simple operation, which can also achieve simultaneous editing of multiple loci. It can also be carried out without using plasmids, saving lots of troubles caused by plasmids. CRISPR/Cas9 has shown great potential in the study of genes or genomic functions in microorganisms, plants, animals, and human beings. In this review, we will examine the history, structure, and basic mechanisms of the CRISPR/Cas9 system, describe its great value in precision medicine and sgRNA library screening, and dig its great potential in a new field: DNA information storage.
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Abana CZY, Lamptey H, Bonney EY, Kyei GB. HIV cure strategies: which ones are appropriate for Africa? Cell Mol Life Sci 2022; 79:400. [PMID: 35794316 PMCID: PMC9259540 DOI: 10.1007/s00018-022-04421-z] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2022] [Revised: 06/09/2022] [Accepted: 06/10/2022] [Indexed: 11/10/2022]
Abstract
Although combination antiretroviral therapy (ART) has reduced mortality and improved lifespan for people living with HIV, it does not provide a cure. Patients must be on ART for the rest of their lives and contend with side effects, unsustainable costs, and the development of drug resistance. A cure for HIV is, therefore, warranted to avoid the limitations of the current therapy and restore full health. However, this cure is difficult to find due to the persistence of latently infected HIV cellular reservoirs during suppressive ART. Approaches to HIV cure being investigated include boosting the host immune system, genetic approaches to disable co-receptors and the viral genome, purging cells harboring latent HIV with latency-reversing latency agents (LRAs) (shock and kill), intensifying ART as a cure, preventing replication of latent proviruses (block and lock) and boosting T cell turnover to reduce HIV-1 reservoirs (rinse and replace). Since most people living with HIV are in Africa, methods being developed for a cure must be amenable to clinical trials and deployment on the continent. This review discusses the current approaches to HIV cure and comments on their appropriateness for Africa.
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Affiliation(s)
- Christopher Zaab-Yen Abana
- Department of Virology, College of Health Sciences, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana
| | - Helena Lamptey
- Department of Immunology, College of Health Sciences, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana
| | - Evelyn Y Bonney
- Department of Virology, College of Health Sciences, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana
| | - George B Kyei
- Department of Virology, College of Health Sciences, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra, Ghana.
- Departments of Medicine and Molecular Microbiology, Washington University in St. Louis, 660 S. Euclid Ave, St. Louis, MO, USA.
- Medical and Scientific Research Center, University of Ghana Medical Centre, Accra, Ghana.
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Maslennikova A, Mazurov D. Application of CRISPR/Cas Genomic Editing Tools for HIV Therapy: Toward Precise Modifications and Multilevel Protection. Front Cell Infect Microbiol 2022; 12:880030. [PMID: 35694537 PMCID: PMC9177041 DOI: 10.3389/fcimb.2022.880030] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/20/2022] [Accepted: 04/25/2022] [Indexed: 11/18/2022] Open
Abstract
Although highly active antiretroviral therapy (HAART) can robustly control human immunodeficiency virus (HIV) infection, the existence of latent HIV in a form of proviral DNA integrated into the host genome makes the virus insensitive to HAART. This requires patients to adhere to HAART for a lifetime, often leading to drug toxicity or viral resistance to therapy. Current genome-editing technologies offer different strategies to reduce the latent HIV reservoir in the body. In this review, we systematize the research on CRISPR/Cas-based anti-HIV therapeutic methods, discuss problems related to viral escape and gene editing, and try to focus on the technologies that effectively and precisely introduce genetic modifications and confer strong resistance to HIV infection. Particularly, knock-in (KI) approaches, such as mature B cells engineered to produce broadly neutralizing antibodies, T cells expressing fusion inhibitory peptides in the context of inactivated viral coreceptors, or provirus excision using base editors, look very promising. Current and future advancements in the precision of CRISPR/Cas editing and its delivery will help extend its applicability to clinical HIV therapy.
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Affiliation(s)
- Alexandra Maslennikova
- Cell and Gene Technology Group, Institute of Gene Biology of Russian Academy of Science, Moscow, Russia
| | - Dmitriy Mazurov
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Institute of Gene Biology of Russian Academy of Science, Moscow, Russia
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26
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Wu Y, Gu H, Bao Y, Lin T, Wang Z, Gu D, Shen H, Xian H, Fan Y, Mao R. USP7 sustains an active epigenetic program via stabilizing MLL2 and WDR5 in diffuse large B-cell lymphoma. Cell Biochem Funct 2022; 40:379-390. [PMID: 35411950 DOI: 10.1002/cbf.3702] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2022] [Revised: 03/25/2022] [Accepted: 03/30/2022] [Indexed: 12/24/2022]
Abstract
Activated B-cell-like (ABC)-diffuse large B-cell lymphoma (ABC-DLBCL) is a common subtype of non-Hodgkin's lymphoma with poor prognosis. The survival of ABC-DLBCL relies on constitutive activation of BCR signaling, but the underlying molecular mechanism is not fully addressed. By mining The Cancer Genome Atlas database, we found that the expression of ubiquitin-specific protease 7 (USP7) is significantly elevated in three cancer types including DLBCL. Interestingly, unlike germinal center B-cell-like (GCB)-DLBCL, ABC-DLBCL shows upregulated expression of USP7. Inhibiting the enzymatic activity of USP7 (P22077) has a drastic effect on ABC-DLBCL, but not GCB-DLBCL cells. Compared to GCB-DLBCL, ABC-DLBCL cells show transcriptional upregulation of multiple components of BCR-signaling. USP7 inhibition significantly reduces the expression of upregulated components of BCR signaling. Mechanistically, USP7 inhibition greatly reduces the methylation of histone 3 on lysine 4 (H3K4me2), which is an epigenetic marker for active enhancers. USP7 inhibition greatly reduces the protein level of WDR5 and MLL2, key components of lysine-specific methyltransferase complex (complex of proteins associated with Set1 [COMPASS]). In ABC-DLBCL cells, USP7 stabilizes WDR5 and MLL2. In patients, the expression of USP7 is significantly associated with components of BCR signaling (LYN, SYK, BTK, PLCG2, PRKCB, MALT1, BCL10, and CARD11) and targets of BCR signaling (MYC and IRF4). In summary, we demonstrated an essential role of USP7 in ABC-DLBCL by organizing an oncogenic epigenetic program via stabilization of WDR5 and MLL2. Targeting USP7 might be a novel and efficient approach to treat patients with ABC-DLBCL and it might be better than targeting individual components such as BTK in BCR signaling.
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Affiliation(s)
- Yuanyuan Wu
- Laboratory of Basic Medicine, Medical College, Nantong University, Nantong, Jiangsu, China
| | - Hongyan Gu
- Department of Respiratory Medicine, Nantong Sixth People's Hospital, Nantong, Jiangsu, China
| | - Yuhua Bao
- Department of Emergency Medicine, The Affiliated Hospital of Nantong University, Nantong University, Nantong, Jiangsu, China
| | - Ting Lin
- Laboratory of Basic Medicine, Medical College, Nantong University, Nantong, Jiangsu, China.,Department of Pathophysiology, Nantong University, Nantong, Jiangsu, China
| | - Zhenyu Wang
- Laboratory of Basic Medicine, Medical College, Nantong University, Nantong, Jiangsu, China.,Department of Pediatric Surgery, Affiliated Hospital of Nantong University, Nantong University, Nantong, Jiangsu, China
| | - Donghua Gu
- The Department of Urology, The Second Affiliated Hospital of Nantong University, Nantong University, Nantong, Jiangsu, China
| | - Haoliang Shen
- Department of Critical Care Medicine, Affiliated Hospital of Nantong University, Nantong University, Nantong, Jiangsu, China
| | - Hua Xian
- Department of Pediatric Surgery, Affiliated Hospital of Nantong University, Nantong University, Nantong, Jiangsu, China
| | - Yihui Fan
- Laboratory of Basic Medicine, Medical College, Nantong University, Nantong, Jiangsu, China.,Department of Pathogenic Biology, Medical College, Nantong University, Nantong, Jiangsu, PR China
| | - Renfang Mao
- Laboratory of Basic Medicine, Medical College, Nantong University, Nantong, Jiangsu, China.,Department of Pathophysiology, Nantong University, Nantong, Jiangsu, China
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27
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Scheller SH, Rashad Y, Saleh FM, Willingham KA, Reilich A, Lin D, Izadpanah R, Alt EU, Braun SE. Biallelic, Selectable, Knock-in Targeting of CCR5 via CRISPR-Cas9 Mediated Homology Directed Repair Inhibits HIV-1 Replication. Front Immunol 2022; 13:821190. [PMID: 35386712 PMCID: PMC8978527 DOI: 10.3389/fimmu.2022.821190] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/23/2021] [Accepted: 02/22/2022] [Indexed: 01/17/2023] Open
Abstract
Transplanting HIV-1 positive patients with hematopoietic stem cells homozygous for a 32 bp deletion in the chemokine receptor type 5 (CCR5) gene resulted in a loss of detectable HIV-1, suggesting genetically disrupting CCR5 is a promising approach for HIV-1 cure. Targeting the CCR5-locus with CRISPR-Cas9 was shown to decrease the amount of CCR5 expression and HIV-1 susceptibility in vitro as well as in vivo. Still, only the individuals homozygous for the CCR5-Δ32 frameshift mutation confer complete resistance to HIV-1 infection. In this study we introduce a mechanism to target CCR5 and efficiently select for cells with biallelic frameshift insertion, using CRISPR-Cas9 mediated homology directed repair (HDR). We hypothesized that cells harboring two different selectable markers (double positive), each in one allele of the CCR5 locus, would carry a frameshift mutation in both alleles, lack CCR5 expression and resist HIV-1 infection. Inducing double-stranded breaks (DSB) via CRISPR-Cas9 leads to HDR and integration of a donor plasmid. Double-positive cells were selected via fluorescence-activated cell sorting (FACS), and CCR5 was analyzed genetically, phenotypically, and functionally. Targeted and selected populations showed a very high frequency of mutations and a drastic reduction in CCR5 surface expression. Most importantly, double-positive cells displayed potent inhibition to HIV-1 infection. Taken together, we show that targeting cells via CRISPR-Cas9 mediated HDR enables efficient selection of mutant cells that are deficient for CCR5 and highly resistant to HIV-1 infection.
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Affiliation(s)
- Stefan H Scheller
- Applied Stem Cell Laboratory, Medicine/Heart and Vascular Institute, Tulane University Health Sciences Center, New Orleans, LA, United States.,Department of Cardiology and Angiology, Faculty of Medicine, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany
| | - Yasmine Rashad
- Applied Stem Cell Laboratory, Medicine/Heart and Vascular Institute, Tulane University Health Sciences Center, New Orleans, LA, United States
| | - Fayez M Saleh
- Division of Immunology, Tulane National Primate Research Center, Tulane University School of Medicine, Covington, LA, United States.,Department of Medical Microbiology, Faculty of Medicine, University of Tabuk, Tabuk, Saudi Arabia
| | - Kurtis A Willingham
- Applied Stem Cell Laboratory, Medicine/Heart and Vascular Institute, Tulane University Health Sciences Center, New Orleans, LA, United States
| | - Antonia Reilich
- Applied Stem Cell Laboratory, Medicine/Heart and Vascular Institute, Tulane University Health Sciences Center, New Orleans, LA, United States
| | - Dong Lin
- Applied Stem Cell Laboratory, Medicine/Heart and Vascular Institute, Tulane University Health Sciences Center, New Orleans, LA, United States.,Department of Surgery, Tulane University Health Science Center, New Orleans, LA, United States
| | - Reza Izadpanah
- Applied Stem Cell Laboratory, Medicine/Heart and Vascular Institute, Tulane University Health Sciences Center, New Orleans, LA, United States.,Department of Surgery, Tulane University Health Science Center, New Orleans, LA, United States
| | - Eckhard U Alt
- Applied Stem Cell Laboratory, Medicine/Heart and Vascular Institute, Tulane University Health Sciences Center, New Orleans, LA, United States.,Isar Klinikum Munich, Munich, Germany
| | - Stephen E Braun
- Applied Stem Cell Laboratory, Medicine/Heart and Vascular Institute, Tulane University Health Sciences Center, New Orleans, LA, United States.,Division of Immunology, Tulane National Primate Research Center, Tulane University School of Medicine, Covington, LA, United States.,Department of Pharmacology, Tulane University Health Science Center, New Orleans, LA, United States
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Karuppusamy KV, Demosthenes JP, Venkatesan V, Christopher AC, Babu P, Azhagiri MK, Jacob A, Ramalingam VV, Rangaraj S, Murugesan MK, Marepally SK, Varghese GM, Srivastava A, Kannangai R, Thangavel S. The CCR5 Gene Edited CD34+CD90+ Hematopoietic Stem Cell Population Serves as an Optimal Graft Source for HIV Gene Therapy. Front Immunol 2022; 13:792684. [PMID: 35359982 PMCID: PMC8963924 DOI: 10.3389/fimmu.2022.792684] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2021] [Accepted: 02/14/2022] [Indexed: 11/13/2022] Open
Abstract
Transplantation of allogenic hematopoietic stem and progenitor cells (HSPCs) with C-C chemokine receptor type 5 (CCR5) Δ32 genotype generates HIV-1 resistant immune cells. CCR5 gene edited autologous HSPCs can be a potential alternative to hematopoietic stem cell transplantation (HSCT) from HLA-matched CCR5 null donor. However, the clinical application of gene edited autologous HSPCs is critically limited by the quality of the graft, as HIV also infects the HSPCs. In this study, by using mobilized HSPCs from healthy donors, we show that the CD34+CD90+ hematopoietic stem cells (HSCs) express 7-fold lower CD4/CCR5 HIV receptors, higher levels of SAMHD1 anti-viral restriction factor, and possess lower susceptibility to HIV infection than the CD34+CD90- hematopoietic progenitor cells. Further, the treatment with small molecule cocktail of Resveratrol, UM729 and SR1(RUS) improved the in vivo engraftment potential of CD34+CD90+ HSCs. To demonstrate that CD34+CD90+ HSC population as an ideal graft for HIV gene therapy, we sort purified CD34+CD90+ HSCs, treated with RUS and then gene edited the CCR5 with single sgRNA. On transplantation, 100,000 CD34+CD90+ HSCs were sufficient for long-term repopulation of the entire bone marrow of NBSGW mice. Importantly, the gene editing efficiency of ~90% in the infused product was maintained in vivo, facilitating the generation of CCR5 null immune cells, resistant to HIV infection. Altogether, CCR5 gene editing of CD34+CD90+ HSCs provide an ideal gene manipulation strategy for autologous HSCT based gene therapy for HIV infection.
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Affiliation(s)
- Karthik V. Karuppusamy
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Vellore, India
- Manipal Academy of Higher Education, Manipal, India
| | | | - Vigneshwaran Venkatesan
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Vellore, India
- Manipal Academy of Higher Education, Manipal, India
| | - Abisha Crystal Christopher
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Vellore, India
- Thiruvalluvar University, Vellore, India
| | - Prathibha Babu
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Vellore, India
- Manipal Academy of Higher Education, Manipal, India
| | - Manojkumar K. Azhagiri
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Vellore, India
- Manipal Academy of Higher Education, Manipal, India
| | - Annlin Jacob
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Vellore, India
- Manipal Academy of Higher Education, Manipal, India
| | | | - Sumathi Rangaraj
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Vellore, India
| | | | | | - George M. Varghese
- Department of Infectious Diseases, Christian Medical College, Vellore, India
| | - Alok Srivastava
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Vellore, India
- Department of Hematology, Christian Medical College, Vellore, India
| | - Rajesh Kannangai
- Department of Clinical Virology, Christian Medical College, Vellore, India
| | - Saravanabhavan Thangavel
- Centre for Stem Cell Research (CSCR), A Unit of InStem Bengaluru, Vellore, India
- *Correspondence: Saravanabhavan Thangavel,
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29
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Peterson CW, Venkataraman R, Reddy SS, Pande D, Enstrom MR, Radtke S, Humbert O, Kiem HP. Intracellular RNase activity dampens zinc finger nuclease-mediated gene editing in hematopoietic stem and progenitor cells. Mol Ther Methods Clin Dev 2022; 24:30-39. [PMID: 34977270 PMCID: PMC8671732 DOI: 10.1016/j.omtm.2021.11.010] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2021] [Accepted: 11/19/2021] [Indexed: 12/21/2022]
Abstract
Over the past decade, numerous gene-editing platforms which alter host DNA in a highly specific and targeted fashion have been described. Two notable examples are zinc finger nucleases (ZFNs), the first gene-editing platform to be tested in clinical trials, and more recently, CRISPR/Cas9. Although CRISPR/Cas9 approaches have become arguably the most popular platform in the field, the therapeutic advantages and disadvantages of each strategy are only beginning to emerge. We have established a nonhuman primate (NHP) model that serves as a strong predictor of successful gene therapy and gene-editing approaches in humans; our recent work shows that ZFN-edited hematopoietic stem and progenitor cells (HSPCs) engraft at lower levels than CRISPR/Cas9-edited cells. Here, we investigate the mechanisms underlying this difference. We show that optimized culture conditions, including defined serum-free media, augment engraftment of gene-edited NHP HSPCs in a mouse xenograft model. Furthermore, we identify intracellular RNases as major barriers for mRNA-encoded nucleases relative to preformed enzymatically active CRISPR/Cas9 ribonucleoprotein (RNP) complexes. We conclude that CRISPR/Cas9 RNP gene editing is more stable and efficient than ZFN mRNA-based delivery and identify co-delivered RNase inhibitors as a strategy to enhance the expression of gene-editing proteins from mRNA intermediates.
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Affiliation(s)
- Christopher W. Peterson
- Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
- Department of Medicine, University of Washington, Seattle, WA 98195, USA
| | - Rasika Venkataraman
- Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
| | - Sowmya S. Reddy
- Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
| | - Dnyanada Pande
- Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
| | - Mark R. Enstrom
- Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
| | - Stefan Radtke
- Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
| | - Olivier Humbert
- Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
| | - Hans-Peter Kiem
- Stem Cell and Gene Therapy Program, Fred Hutchinson Cancer Research Center, Seattle, WA 98109-1024, USA
- Department of Medicine, University of Washington, Seattle, WA 98195, USA
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30
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Nanoparticle-based strategies to target HIV-infected cells. Colloids Surf B Biointerfaces 2022; 213:112405. [PMID: 35255375 DOI: 10.1016/j.colsurfb.2022.112405] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2021] [Revised: 01/31/2022] [Accepted: 02/07/2022] [Indexed: 02/06/2023]
Abstract
Antiretroviral drugs employed for the treatment of human immunodeficiency virus (HIV) infections have remained largely ineffective due to their poor bioavailability, numerous adverse effects, modest uptake in infected cells, undesirable drug-drug interactions, the necessity for long-term drug therapy, and lack of access to tissues and reservoirs. Nanotechnology-based interventions could serve to overcome several of these disadvantages and thereby improve the therapeutic efficacy of antiretrovirals while reducing the morbidity and mortality due to the disease. However, attempts to use nanocarriers for the delivery of anti-retroviral drugs have started gaining momentum only in the past decade. This review explores in-depth the various nanocarriers that have been employed for the treatment of HIV infections highlighting their merits and possible demerits.
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31
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Engineered Zinc Finger Protein Targeting 2LTR Inhibits HIV Integration in Hematopoietic Stem and Progenitor Cell-Derived Macrophages: In Vitro Study. Int J Mol Sci 2022; 23:ijms23042331. [PMID: 35216446 PMCID: PMC8875109 DOI: 10.3390/ijms23042331] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2022] [Revised: 02/15/2022] [Accepted: 02/16/2022] [Indexed: 12/22/2022] Open
Abstract
Human hematopoietic stem/progenitor cell (HSPC)-based gene therapy is a promising direction for curing HIV-1-infected individuals. The zinc finger protein (2LTRZFP) designed to target the 2-LTR-circle junction of HIV-1 cDNA was previously reported as an intracellular antiviral molecular scaffold that prevents HIV integration. Here, we elucidate the efficacy and safety of using 2LTRZFP in human CD34+ HSPCs. We transduced 2LTRZFP which has the mCherry tag (2LTRZFPmCherry) into human CD34+ HSPCs using a lentiviral vector. The 2LTRZFPmCherry-transduced HSPCs were subsequently differentiated into macrophages. The expression levels of pro-apoptotic proteins of the 2LTRZFPmCherry-transduced HSPCs showed no significant difference from those of the non-transduced control. Furthermore, the 2LTRZFPmCherry-transduced HSPCs were successfully differentiated into mature macrophages, which had normal phagocytic function. The cytokine secretion assay demonstrated that 2LTRZFPmCherry-transduced CD34+ derived macrophages promoted the polarization towards classically activated (M1) subtypes. More importantly, the 2LTRZFPmCherry transduced cells significantly exhibited resistance to HIV-1 integration in vitro. Our findings demonstrate that the 2LTRZFPmCherry-transduced macrophages were found to be functionally and phenotypically normal, with no adverse effects of the anti-HIV-1 scaffold. Our data suggest that the anti-HIV-1 integrase scaffold is a promising antiviral molecule that could be applied to human CD34+ HSPC-based gene therapy for AIDS patients.
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Boutin J, Cappellen D, Rosier J, Amintas S, Dabernat S, Bedel A, Moreau-Gaudry F. ON-target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected. CRISPR J 2022; 5:19-30. [DOI: 10.1089/crispr.2021.0120] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Affiliation(s)
- Julian Boutin
- Bordeaux University, Bordeaux, France
- INSERM U1035, Biotherapy of Genetic Diseases, Inflammatory Disorders and Cancers, Bordeaux, France
- Biochemistry Laboratory, University Hospital Bordeaux, Bordeaux, France
| | - David Cappellen
- Bordeaux University, Bordeaux, France
- INSERM U1035, Biotherapy of Genetic Diseases, Inflammatory Disorders and Cancers, Bordeaux, France
- Tumor Biology and Tumor Bank Laboratory, University Hospital Bordeaux, Bordeaux, France
| | - Juliette Rosier
- Bordeaux University, Bordeaux, France
- INSERM U1035, Biotherapy of Genetic Diseases, Inflammatory Disorders and Cancers, Bordeaux, France
| | - Samuel Amintas
- Bordeaux University, Bordeaux, France
- INSERM U1035, Biotherapy of Genetic Diseases, Inflammatory Disorders and Cancers, Bordeaux, France
- Tumor Biology and Tumor Bank Laboratory, University Hospital Bordeaux, Bordeaux, France
| | - Sandrine Dabernat
- Bordeaux University, Bordeaux, France
- INSERM U1035, Biotherapy of Genetic Diseases, Inflammatory Disorders and Cancers, Bordeaux, France
- Biochemistry Laboratory, University Hospital Bordeaux, Bordeaux, France
| | - Aurélie Bedel
- Bordeaux University, Bordeaux, France
- INSERM U1035, Biotherapy of Genetic Diseases, Inflammatory Disorders and Cancers, Bordeaux, France
- Biochemistry Laboratory, University Hospital Bordeaux, Bordeaux, France
| | - François Moreau-Gaudry
- Bordeaux University, Bordeaux, France
- INSERM U1035, Biotherapy of Genetic Diseases, Inflammatory Disorders and Cancers, Bordeaux, France
- Biochemistry Laboratory, University Hospital Bordeaux, Bordeaux, France
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33
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Jasinska AJ, Pandrea I, Apetrei C. CCR5 as a Coreceptor for Human Immunodeficiency Virus and Simian Immunodeficiency Viruses: A Prototypic Love-Hate Affair. Front Immunol 2022; 13:835994. [PMID: 35154162 PMCID: PMC8829453 DOI: 10.3389/fimmu.2022.835994] [Citation(s) in RCA: 26] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/15/2021] [Accepted: 01/10/2022] [Indexed: 12/14/2022] Open
Abstract
CCR5, a chemokine receptor central for orchestrating lymphocyte/cell migration to the sites of inflammation and to the immunosurveillance, is involved in the pathogenesis of a wide spectrum of health conditions, including inflammatory diseases, viral infections, cancers and autoimmune diseases. CCR5 is also the primary coreceptor for the human immunodeficiency viruses (HIVs), supporting its entry into CD4+ T lymphocytes upon transmission and in the early stages of infection in humans. A natural loss-of-function mutation CCR5-Δ32, preventing the mutated protein expression on the cell surface, renders homozygous carriers of the null allele resistant to HIV-1 infection. This phenomenon was leveraged in the development of therapies and cure strategies for AIDS. Meanwhile, over 40 African nonhuman primate species are long-term hosts of simian immunodeficiency virus (SIV), an ancestral family of viruses that give rise to the pandemic CCR5 (R5)-tropic HIV-1. Many natural hosts typically do not progress to immunodeficiency upon the SIV infection. They have developed various strategies to minimize the SIV-related pathogenesis and disease progression, including an array of mechanisms employing modulation of the CCR5 receptor activity: (i) deletion mutations abrogating the CCR5 surface expression and conferring resistance to infection in null homozygotes; (ii) downregulation of CCR5 expression on CD4+ T cells, particularly memory cells and cells at the mucosal sites, preventing SIV from infecting and killing cells important for the maintenance of immune homeostasis, (iii) delayed onset of CCR5 expression on the CD4+ T cells during ontogenetic development that protects the offspring from vertical transmission of the virus. These host adaptations, aimed at lowering the availability of target CCR5+ CD4+ T cells through CCR5 downregulation, were countered by SIV, which evolved to alter the entry coreceptor usage toward infecting different CD4+ T-cell subpopulations that support viral replication yet without disruption of host immune homeostasis. These natural strategies against SIV/HIV-1 infection, involving control of CCR5 function, inspired therapeutic approaches against HIV-1 disease, employing CCR5 coreceptor blocking as well as gene editing and silencing of CCR5. Given the pleiotropic role of CCR5 in health beyond immune disease, the precision as well as costs and benefits of such interventions needs to be carefully considered.
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Affiliation(s)
- Anna J. Jasinska
- Division of Infectious Diseases, Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States
- Department of Molecular Genetics, Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
- Eye on Primates, Los Angeles, CA, United States
| | - Ivona Pandrea
- Department of Pathology, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States
- Department of Infectious Diseases and Immunology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, United States
| | - Cristian Apetrei
- Division of Infectious Diseases, Department of Medicine, School of Medicine, University of Pittsburgh, Pittsburgh, PA, United States
- Department of Infectious Diseases and Immunology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, United States
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34
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Mohamed H, Gurrola T, Berman R, Collins M, Sariyer IK, Nonnemacher MR, Wigdahl B. Targeting CCR5 as a Component of an HIV-1 Therapeutic Strategy. Front Immunol 2022; 12:816515. [PMID: 35126374 PMCID: PMC8811197 DOI: 10.3389/fimmu.2021.816515] [Citation(s) in RCA: 33] [Impact Index Per Article: 11.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2021] [Accepted: 12/13/2021] [Indexed: 12/26/2022] Open
Abstract
Globally, human immunodeficiency virus type 1 (HIV-1) infection is a major health burden for which successful therapeutic options are still being investigated. Challenges facing current drugs that are part of the established life-long antiretroviral therapy (ART) include toxicity, development of drug resistant HIV-1 strains, the cost of treatment, and the inability to eradicate the provirus from infected cells. For these reasons, novel anti-HIV-1 therapeutics that can prevent or eliminate disease progression including the onset of the acquired immunodeficiency syndrome (AIDS) are needed. While development of HIV-1 vaccination has also been challenging, recent advancements demonstrate that infection of HIV-1-susceptible cells can be prevented in individuals living with HIV-1, by targeting C-C chemokine receptor type 5 (CCR5). CCR5 serves many functions in the human immune response and is a co-receptor utilized by HIV-1 for entry into immune cells. Therapeutics targeting CCR5 generally involve gene editing techniques including CRISPR, CCR5 blockade using antibodies or antagonists, or combinations of both. Here we review the efficacy of these approaches and discuss the potential of their use in the clinic as novel ART-independent therapies for HIV-1 infection.
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Affiliation(s)
- Hager Mohamed
- Department of Microbiology and Immunology, Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
| | - Theodore Gurrola
- Department of Microbiology and Immunology, Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
| | - Rachel Berman
- Department of Microbiology and Immunology, Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
| | - Mackenzie Collins
- Department of Microbiology and Immunology, Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
| | - Ilker K. Sariyer
- Department of Microbiology, Immunology, and Inflammation, Center for Neurovirology and Gene Editing, School of Medicine, Temple University, Philadelphia, PA, United States
| | - Michael R. Nonnemacher
- Department of Microbiology and Immunology, Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
| | - Brian Wigdahl
- Department of Microbiology and Immunology, Center for Molecular Virology and Translational Neuroscience, Institute for Molecular Medicine and Infectious Disease, Drexel University College of Medicine, Philadelphia, PA, United States
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35
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Disruption of HIV-1 co-receptors CCR5 and CXCR4 in primary human T cells and hematopoietic stem and progenitor cells using base editing. Mol Ther 2022; 30:130-144. [PMID: 34737067 PMCID: PMC8753564 DOI: 10.1016/j.ymthe.2021.10.026] [Citation(s) in RCA: 18] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/25/2021] [Revised: 08/31/2021] [Accepted: 10/27/2021] [Indexed: 01/07/2023] Open
Abstract
Disruption of CCR5 or CXCR4, the main human immunodeficiency virus type 1 (HIV-1) co-receptors, has been shown to protect primary human CD4+ T cells from HIV-1 infection. Base editing can install targeted point mutations in cellular genomes, and can thus efficiently inactivate genes by introducing stop codons or eliminating start codons without double-stranded DNA break formation. Here, we applied base editors for individual and simultaneous disruption of both co-receptors in primary human CD4+ T cells. Using cytosine base editors we observed premature stop codon introduction in up to 89% of sequenced CCR5 or CXCR4 alleles. Using adenine base editors we eliminated the start codon in CCR5 in up to 95% of primary human CD4+ T cell and up to 88% of CD34+ hematopoietic stem and progenitor cell target alleles. Genome-wide specificity analysis revealed low numbers of off-target mutations that were introduced by base editing, located predominantly in intergenic or intronic regions. We show that our editing strategies prevent transduction with CCR5-tropic and CXCR4-tropic viral vectors in up to 79% and 88% of human CD4+ T cells, respectively. The engineered T cells maintained functionality and overall our results demonstrate the effectiveness of base-editing strategies for efficient and specific ablation of HIV co-receptors in clinically relevant cell types.
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36
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Xun J, Zhang X, Guo S, Lu H, Chen J. Editing out HIV: application of gene editing technology to achieve functional cure. Retrovirology 2021; 18:39. [PMID: 34922576 PMCID: PMC8684261 DOI: 10.1186/s12977-021-00581-1] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2021] [Accepted: 11/05/2021] [Indexed: 03/01/2023] Open
Abstract
Highly active antiretroviral therapy (HAART) successfully suppresses human immunodeficiency virus (HIV) replication and improves the quality of life of patients living with HIV. However, current HAART does not eradicate HIV infection because an HIV reservoir is established in latently infected cells and is not recognized by the immune system. The successful curative treatment of the Berlin and London patients following bone marrow transplantation inspired researchers to identify an approach for the functional cure of HIV. As a promising technology, gene editing-based strategies have attracted considerable attention and sparked much debate. Herein, we discuss the development of different gene editing strategies in the functional cure of HIV and highlight the potential for clinical applications prospects. ![]()
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Affiliation(s)
- Jingna Xun
- Scientific Research Center, Shanghai Public Health Clinical Center, Fudan University, 2901 Caolang Road, Shanghai, 201508, China.,State Key Laboratory of Genetic Engineering, School of Life Sciences, Fudan University, Shanghai, China
| | - Xinyu Zhang
- Scientific Research Center, Shanghai Public Health Clinical Center, Fudan University, 2901 Caolang Road, Shanghai, 201508, China
| | - Shuyan Guo
- Shanghai Foreign Language School, Shanghai International Studies University, Shanghai, China
| | - Hongzhou Lu
- Department of Infectious Diseases and Immunology, Shanghai Public Health Clinical Center, Fudan University, 2901 Caolang Road, Shanghai, 201508, China
| | - Jun Chen
- Department of Infectious Diseases and Immunology, Shanghai Public Health Clinical Center, Fudan University, 2901 Caolang Road, Shanghai, 201508, China.
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37
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Ebrahimi A, Ahmadi H, Ghasrodashti ZP, Tanideh N, Shahriarirad R, Erfani A, Ranjbar K, Ashkani-Esfahani S. Therapeutic effects of stem cells in different body systems, a novel method that is yet to gain trust: A comprehensive review. Bosn J Basic Med Sci 2021; 21:672-701. [PMID: 34255619 PMCID: PMC8554700 DOI: 10.17305/bjbms.2021.5508] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/22/2021] [Accepted: 04/25/2021] [Indexed: 11/30/2022] Open
Abstract
Stem cell therapy has been used to treat several types of diseases, and it is expected that its therapeutic uses shall increase as novel lines of evidence begin to appear. Furthermore, stem cells have the potential to make new tissues and organs. Thus, some scientists propose that organ transplantation will significantly rely on stem cell technology and organogenesis in the future. Stem cells and its robust potential to differentiate into specific types of cells and regenerate tissues and body organs, have been investigated by numerous clinician scientists and researchers for their therapeutic effects. Degenerative diseases in different organs have been the main target of stem cell therapy. Neurodegenerative diseases such as Alzheimer's, musculoskeletal diseases such as osteoarthritis, congenital cardiovascular diseases, and blood cell diseases such as leukemia are among the health conditions that have benefited from stem cell therapy advancements. One of the most challenging parts of the process of incorporating stem cells into clinical practice is controlling their division and differentiation potentials. Sometimes, their potential for uncontrolled growth will make these cells tumorigenic. Another caveat in this process is the ability to control the differentiation process. While stem cells can easily differentiate into a wide variety of cells, a paracrine effect controlled activity, being in an appropriate medium will cause abnormal differentiation leading to treatment failure. In this review, we aim to provide an overview of the therapeutic effects of stem cells in diseases of various organ systems. In order to advance this new treatment to its full potential, researchers should focus on establishing methods to control the differentiation process, while policymakers should take an active role in providing adequate facilities and equipment for these projects. Large population clinical trials are a necessary tool that will help build trust in this method. Moreover, improving social awareness about the advantages and adverse effects of stem cell therapy is required to develop a rational demand in the society, and consequently, healthcare systems should consider established stem cell-based therapeutic methods in their treatment algorithms.
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Affiliation(s)
- Alireza Ebrahimi
- Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Hanie Ahmadi
- Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Zahra Pourfraidon Ghasrodashti
- Molecular Pathology and Cytogenetics Laboratory, Department of Pathology, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Nader Tanideh
- Stem Cells Technology Research Center, Department of Pharmacology, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Reza Shahriarirad
- Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran
- Thoracic and Vascular Surgery Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Amirhossein Erfani
- Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Keivan Ranjbar
- Student Research Committee, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Soheil Ashkani-Esfahani
- Department of Orthopaedic Surgery, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts, USA
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38
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Kues WA, Kumar D, Selokar NL, Talluri TR. Applications of genome editing tools in stem cells towards regenerative medicine: An update. Curr Stem Cell Res Ther 2021; 17:267-279. [PMID: 34819011 DOI: 10.2174/1574888x16666211124095527] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2021] [Revised: 09/14/2021] [Accepted: 09/25/2021] [Indexed: 11/22/2022]
Abstract
Precise and site specific genome editing through application of emerging and modern gene engineering techniques, namely zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) have swiftly progressed the application and use of the stem cell technology in the sphere of in-vitro disease modelling and regenerative medicine. Genome editing tools facilitate the manipulating of any gene in various types of cells with target specific nucleases. These tools aid in elucidating the genetics and etiology behind different diseases and have immense promise as novel therapeutics for correcting the genetic mutations, make alterations and cure diseases permanently that are not responding and resistant to traditional therapies. These genome engineering tools have evolved in the field of biomedical research and have also shown to have a significant improvement in clinical trials. However, their widespread use in research revealed potential safety issues, which need to be addressed before implementing such techniques in clinical purposes. Significant and valiant attempts are being made in order to surpass those hurdles. The current review outlines the advancements of several genome engineering tools and describes suitable strategies for their application towards regenerative medicine.
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Affiliation(s)
- Wilfried A Kues
- Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health, Department of Biotechnology, Stem Cell Physiology, Höltystr 10, 31535 Neustadt. Germany
| | - Dharmendra Kumar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar-125001, Haryana. India
| | - Naresh L Selokar
- Animal Physiology and Reproduction Division, ICAR-Central Institute for Research on Buffaloes, Hisar-125001, Haryana. India
| | - Thirumala Rao Talluri
- Equine Production Campus, ICAR- National Research Centre on Equines, Bikaner-334001, Rajasthan. India
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39
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Affiliation(s)
- Fyodor D Urnov
- Professor, Molecular and Cell Biology Department, Scientific Director, Technology and Translation, Innovative Genomics Institute, UC Berkeley, 2151 Berkeley Way, Berkeley, CA 94720, USA.
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40
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Benitez EK, Lomova Kaufman A, Cervantes L, Clark DN, Ayoub PG, Senadheera S, Osborne K, Sanchez JM, Crisostomo RV, Wang X, Reuven N, Shaul Y, Hollis RP, Romero Z, Kohn DB. Global and Local Manipulation of DNA Repair Mechanisms to Alter Site-Specific Gene Editing Outcomes in Hematopoietic Stem Cells. Front Genome Ed 2021; 2:601541. [PMID: 34713224 PMCID: PMC8525354 DOI: 10.3389/fgeed.2020.601541] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/01/2020] [Accepted: 11/16/2020] [Indexed: 12/26/2022] Open
Abstract
Monogenic disorders of the blood system have the potential to be treated by autologous stem cell transplantation of ex vivo genetically modified hematopoietic stem and progenitor cells (HSPCs). The sgRNA/Cas9 system allows for precise modification of the genome at single nucleotide resolution. However, the system is reliant on endogenous cellular DNA repair mechanisms to mend a Cas9-induced double stranded break (DSB), either by the non-homologous end joining (NHEJ) pathway or by the cell-cycle regulated homology-directed repair (HDR) pathway. Here, we describe a panel of ectopically expressed DNA repair factors and Cas9 variants assessed for their ability to promote gene correction by HDR or inhibit gene disruption by NHEJ at the HBB locus. Although transient global overexpression of DNA repair factors did not improve the frequency of gene correction in primary HSPCs, localization of factors to the DSB by fusion to the Cas9 protein did alter repair outcomes toward microhomology-mediated end joining (MMEJ) repair, an HDR event. This strategy may be useful when predictable gene editing outcomes are imperative for therapeutic success.
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Affiliation(s)
- Elizabeth K Benitez
- Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
| | - Anastasia Lomova Kaufman
- Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
| | - Lilibeth Cervantes
- Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
| | - Danielle N Clark
- Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
| | - Paul G Ayoub
- Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
| | - Shantha Senadheera
- Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
| | - Kyle Osborne
- Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
| | - Julie M Sanchez
- Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
| | - Ralph Valentine Crisostomo
- Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
| | - Xiaoyan Wang
- Department of General Internal Medicine and Health Services Research, University of California, Los Angeles, Los Angeles, CA, United States
| | - Nina Reuven
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Yosef Shaul
- Department of Molecular Genetics, Weizmann Institute of Science, Rehovot, Israel
| | - Roger P Hollis
- Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
| | - Zulema Romero
- Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
| | - Donald B Kohn
- Department of Microbiology, Immunology & Molecular Genetics, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, United States
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41
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Dalwadi DA, Calabria A, Tiyaboonchai A, Posey J, Naugler WE, Montini E, Grompe M. AAV integration in human hepatocytes. Mol Ther 2021; 29:2898-2909. [PMID: 34461297 DOI: 10.1016/j.ymthe.2021.08.031] [Citation(s) in RCA: 82] [Impact Index Per Article: 20.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/06/2021] [Revised: 08/01/2021] [Accepted: 08/24/2021] [Indexed: 12/17/2022] Open
Abstract
Recombinant adeno-associated viral (rAAV) vectors are considered promising tools for gene therapy directed at the liver. Whereas rAAV is thought to be an episomal vector, its single-stranded DNA genome is prone to intra- and inter-molecular recombination leading to rearrangements and integration into the host cell genome. Here, we ascertained the integration frequency of rAAV in human hepatocytes transduced either ex vivo or in vivo and subsequently expanded in a mouse model of xenogeneic liver regeneration. Chromosomal rAAV integration events and vector integrity were determined using the capture-PacBio sequencing approach, a long-read next-generation sequencing method that has not previously been used for this purpose. Chromosomal integrations were found at a surprisingly high frequency of 1%-3% both in vitro and in vivo. Importantly, most of the inserted rAAV sequences were heavily rearranged and were accompanied by deletions of the host genomic sequence at the integration site.
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Affiliation(s)
- Dhwanil A Dalwadi
- Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239, USA
| | - Andrea Calabria
- San Raffaele Telethon Institute for Gene Therapy, IRCCS Ospedale San Raffaele Scientific Institute, Milan, Italy
| | - Amita Tiyaboonchai
- Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239, USA
| | - Jeffrey Posey
- Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239, USA
| | - Willscott E Naugler
- Department of Medicine, Division of Gastroenterology and Hepatology, Oregon Health and Science University, Portland, OR 97239, USA
| | - Eugenio Montini
- San Raffaele Telethon Institute for Gene Therapy, IRCCS Ospedale San Raffaele Scientific Institute, Milan, Italy
| | - Markus Grompe
- Papé Family Pediatric Research Institute, Department of Pediatrics, Oregon Health and Science University, Portland, OR 97239, USA.
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Khalid K, Padda J, Wijeratne Fernando R, Mehta KA, Almanie AH, Al Hennawi H, Padda S, Cooper AC, Jean-Charles G. Stem Cell Therapy and Its Significance in HIV Infection. Cureus 2021; 13:e17507. [PMID: 34595076 PMCID: PMC8468364 DOI: 10.7759/cureus.17507] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 08/27/2021] [Indexed: 12/02/2022] Open
Abstract
Human immunodeficiency virus (HIV) infection is a major global public health issue. Despite this, the only treatment available in mainstay is antiretroviral therapy. This treatment is not curative, it needs to be used lifelong, and there are many issues with compliance and side effects. In recent years, stem cell therapy has shown promising results in HIV management, and it can have a major impact on the future of HIV treatment and prevention. The idea behind anti-HIV hematopoietic stem/progenitor cell (HSPC)-directed gene therapy is to genetically engineer patient-derived (autologous) HSPC to acquire an inherent resistance to HIV infection. Multiple stem-cell-based gene therapy strategies have been suggested that may infer HIV resistance including anti-HIV gene reagents and gene combinatorial strategies giving rise to anti-HIV gene-modified HSPCs. Such stem cells can hamper HIV progression in the body by interrupting key stages of HIV proliferation: viral entry, viral integration, HIV gene expression, etc.Hematopoietic stem cells (HSCs) may also protect leukocytes from being infected. Additionally, genetically engineered HSCs have the ability to continuously produce protected immune cells by prolonged self-renewal that can attack the HIV virus. Therefore, a successful treatment strategy has the potential to control the infection at a steady state and eradicate HIV from patients. This will allow for a potential future benefit with stem cell therapy in HIV treatment.
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Affiliation(s)
| | | | | | | | | | | | | | | | - Gutteridge Jean-Charles
- Internal Medicine, JC Medical Center, Orlando, USA.,Internal Medicine, AdventHealth & Orlando Health Hospital, Orlando, USA
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43
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González Castro N, Bjelic J, Malhotra G, Huang C, Alsaffar SH. Comparison of the Feasibility, Efficiency, and Safety of Genome Editing Technologies. Int J Mol Sci 2021; 22:10355. [PMID: 34638696 PMCID: PMC8509008 DOI: 10.3390/ijms221910355] [Citation(s) in RCA: 36] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2021] [Revised: 08/26/2021] [Accepted: 09/24/2021] [Indexed: 12/15/2022] Open
Abstract
Recent advances in programmable nucleases including meganucleases (MNs), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats-Cas (CRISPR-Cas) have propelled genome editing from explorative research to clinical and industrial settings. Each technology, however, features distinct modes of action that unevenly impact their applicability across the entire genome and are often tested under significantly different conditions. While CRISPR-Cas is currently leading the field due to its versatility, quick adoption, and high degree of support, it is not without limitations. Currently, no technology can be regarded as ideal or even applicable to every case as the context dictates the best approach for genetic modification within a target organism. In this review, we implement a four-pillar framework (context, feasibility, efficiency, and safety) to assess the main genome editing platforms, as a basis for rational decision-making by an expanding base of users, regulators, and consumers. Beyond carefully considering their specific use case with the assessment framework proposed here, we urge stakeholders interested in genome editing to independently validate the parameters of their chosen platform prior to commitment. Furthermore, safety across all applications, particularly in clinical settings, is a paramount consideration and comprehensive off-target detection strategies should be incorporated within workflows to address this. Often neglected aspects such as immunogenicity and the inadvertent selection of mutants deficient for DNA repair pathways must also be considered.
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Affiliation(s)
- Nicolás González Castro
- School of Biosciences, Faculty of Science, University of Melbourne, Parkville 3052, Australia; (N.G.C.); (G.M.); (C.H.); (S.H.A.)
| | - Jan Bjelic
- School of Biosciences, Faculty of Science, University of Melbourne, Parkville 3052, Australia; (N.G.C.); (G.M.); (C.H.); (S.H.A.)
| | - Gunya Malhotra
- School of Biosciences, Faculty of Science, University of Melbourne, Parkville 3052, Australia; (N.G.C.); (G.M.); (C.H.); (S.H.A.)
| | - Cong Huang
- School of Biosciences, Faculty of Science, University of Melbourne, Parkville 3052, Australia; (N.G.C.); (G.M.); (C.H.); (S.H.A.)
| | - Salman Hasan Alsaffar
- School of Biosciences, Faculty of Science, University of Melbourne, Parkville 3052, Australia; (N.G.C.); (G.M.); (C.H.); (S.H.A.)
- Biotechnology Department, Environment and Life Sciences Research Center, Kuwait Institute for Scientific Research, Shuwaikh 13109, Kuwait
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44
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Arnesen JA, Hoof JB, Kildegaard HF, Borodina I. Genome Editing of Eukarya. Metab Eng 2021. [DOI: 10.1002/9783527823468.ch10] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/10/2022]
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45
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Daramola OJ, Osasan S, Ali H, Emeagi P. Hematopoietic stem and progenitor cells directly participate in host immune response. AMERICAN JOURNAL OF STEM CELLS 2021; 10:18-27. [PMID: 34327049 PMCID: PMC8310832] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Received: 08/28/2020] [Accepted: 05/27/2021] [Indexed: 06/13/2023]
Abstract
The properties of hematopoietic stem and progenitor cells (HSPCs), including self-renewal and pluripotency, have been extensively studied. These features have been explored in the management of several haematological disorders and malignancies. Although their role as precursors of innate immune cells is well understood, little is known about their direct participation in host immune response. In this review, we explicate the direct role of HSPCs in the host immune response and highlight therapeutic options for the infectious disease burden that is currently ravaging the world, including COVID-19.
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Affiliation(s)
- Olusola Jumoke Daramola
- Department of Histopathology, University of Wirral Teaching Hospital NHS TrustArrowe Park Wirral CH49 5PE, UK
- Institute of Infection, Veterinary and Ecological Sciences, University of LiverpoolLiverpool L69 7BE, UK
- Haemato-Oncology Diagnostic Service, Royal Liverpool University HospitalLiverpool L7 8XP, UK
| | - Stephen Osasan
- Department of Laboratory Medicine and Pathology, University of Alberta CanadaEdmonton, Canada
| | - Hebah Ali
- Haematological Malignancy Diagnostic Service, Leeds Teaching Hospital NHS TrustLS9 7TF, UK
- Leeds Institute of Cancer and Pathology, Faculty of Medicine and Health, University of Leeds LS2 9JTUK
| | - Perpetua Emeagi
- Liverpool Hope University, Department of Biomedical SciencesHope Park, Liverpool L16 9JD, UK
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46
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Ding J, Liu Y, Lai Y. Knowledge From London and Berlin: Finding Threads to a Functional HIV Cure. Front Immunol 2021; 12:688747. [PMID: 34122453 PMCID: PMC8190402 DOI: 10.3389/fimmu.2021.688747] [Citation(s) in RCA: 14] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2021] [Accepted: 05/04/2021] [Indexed: 01/07/2023] Open
Abstract
Despite the ability of combination antiretroviral therapy (cART) to increase the life expectancy of patients infected with human immunodeficiency virus (HIV), viral reservoirs persist during life-long treatment. Notably, two cases of functional cure for HIV have been reported and are known as the "Berlin Patient" and the "London Patient". Both patients received allogeneic hematopoietic stem cell transplantation from donors with homozygous CCR5 delta32 mutation for an associated hematological malignancy. Therefore, there is growing interest in creating an HIV-resistant immune system through the use of gene-modified autologous hematopoietic stem cells with non-functional CCR5. Moreover, studies in CXCR4-targeted gene therapy for HIV have also shown great promise. Developing a cure for HIV infection remains a high priority. In this review, we discuss the increasing progress of coreceptor-based hematopoietic stem cell gene therapy, cART, milder conditioning regimens, and shock and kill strategies that have important implications for designing potential strategies aiming to achieve a functional cure for the majority of people with HIV.
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Affiliation(s)
- Jingyi Ding
- Hospital of Chengdu University of Traditional Chinese Medicine, Chengdu, China
| | - Yanxi Liu
- University of California, Los Angeles, Los Angeles, CA, United States
| | - Yu Lai
- School of Basic Medicine, Chengdu University of Traditional Chinese Medicine, Chengdu, China,*Correspondence: Yu Lai,
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47
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He X, Urip BA, Zhang Z, Ngan CC, Feng B. Evolving AAV-delivered therapeutics towards ultimate cures. J Mol Med (Berl) 2021; 99:593-617. [PMID: 33594520 PMCID: PMC7885987 DOI: 10.1007/s00109-020-02034-2] [Citation(s) in RCA: 48] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2020] [Revised: 12/18/2020] [Accepted: 12/23/2020] [Indexed: 12/17/2022]
Abstract
Gene therapy has entered a new era after decades-long efforts, where the recombinant adeno-associated virus (AAV) has stood out as the most potent vector for in vivo gene transfer and demonstrated excellent efficacy and safety profiles in numerous preclinical and clinical studies. Since the first AAV-derived therapeutics Glybera was approved by the European Medicines Agency (EMA) in 2012, there is an increasing number of AAV-based gene augmentation therapies that have been developed and tested for treating incurable genetic diseases. In the subsequent years, the United States Food and Drug Administration (FDA) approved two additional AAV gene therapy products, Luxturna and Zolgensma, to be launched into the market. Recent breakthroughs in genome editing tools and the combined use with AAV vectors have introduced new therapeutic modalities using somatic gene editing strategies. The promising outcomes from preclinical studies have prompted the continuous evolution of AAV-delivered therapeutics and broadened the scope of treatment options for untreatable diseases. Here, we describe the clinical updates of AAV gene therapies and the latest development using AAV to deliver the CRISPR components as gene editing therapeutics. We also discuss the major challenges and safety concerns associated with AAV delivery and CRISPR therapeutics, and highlight the recent achievement and toxicity issues reported from clinical applications.
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Affiliation(s)
- Xiangjun He
- School of Biomedical Sciences, Faculty of Medicine; Institute for Tissue Engineering and Regenerative Medicine (iTERM), The Chinese University of Hong Kong, Shatin N.T., Hong Kong SAR, China
| | - Brian Anugerah Urip
- School of Biomedical Sciences, Faculty of Medicine; Institute for Tissue Engineering and Regenerative Medicine (iTERM), The Chinese University of Hong Kong, Shatin N.T., Hong Kong SAR, China
| | - Zhenjie Zhang
- School of Biomedical Sciences, Faculty of Medicine; Institute for Tissue Engineering and Regenerative Medicine (iTERM), The Chinese University of Hong Kong, Shatin N.T., Hong Kong SAR, China
| | - Chun Christopher Ngan
- School of Biomedical Sciences, Faculty of Medicine; Institute for Tissue Engineering and Regenerative Medicine (iTERM), The Chinese University of Hong Kong, Shatin N.T., Hong Kong SAR, China
- Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, Shatin N.T., Hong Kong SAR, China
| | - Bo Feng
- School of Biomedical Sciences, Faculty of Medicine; Institute for Tissue Engineering and Regenerative Medicine (iTERM), The Chinese University of Hong Kong, Shatin N.T., Hong Kong SAR, China.
- Centre for Regenerative Medicine and Health, Hong Kong Institute of Science & Innovation, Chinese Academy of Sciences, Shatin N.T., Hong Kong SAR, China.
- Guangzhou Regenerative Medicine and Health Guangdong Laboratory, Guangzhou, 510320, China.
- Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, China.
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48
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Abeynaike S, Paust S. Humanized Mice for the Evaluation of Novel HIV-1 Therapies. Front Immunol 2021; 12:636775. [PMID: 33868262 PMCID: PMC8047330 DOI: 10.3389/fimmu.2021.636775] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/02/2020] [Accepted: 03/01/2021] [Indexed: 12/13/2022] Open
Abstract
With the discovery of antiretroviral therapy, HIV-1 infection has transitioned into a manageable but chronic illness, which requires lifelong treatment. Nevertheless, complete eradication of the virus has still eluded us. This is partly due to the virus’s ability to remain in a dormant state in tissue reservoirs, ‘hidden’ from the host’s immune system. Also, the high mutation rate of HIV-1 results in escape mutations in response to many therapeutics. Regardless, the development of novel cures for HIV-1 continues to move forward with a range of approaches from immunotherapy to gene editing. However, to evaluate in vivo pathogenesis and the efficacy and safety of therapeutic approaches, a suitable animal model is necessary. To this end, the humanized mouse was developed by McCune in 1988 and has continued to be improved on over the past 30 years. Here, we review the variety of humanized mouse models that have been utilized through the years and describe their specific contribution in translating HIV-1 cure strategies to the clinic.
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Affiliation(s)
- Shawn Abeynaike
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, United States.,The Skaggs Graduate Program in Chemical and Biological Sciences, The Scripps Research Institute, La Jolla, CA, United States
| | - Silke Paust
- Department of Immunology and Microbiology, The Scripps Research Institute, La Jolla, CA, United States.,The Skaggs Graduate Program in Chemical and Biological Sciences, The Scripps Research Institute, La Jolla, CA, United States
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49
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Karuppusamy KV, Babu P, Thangavel S. The Strategies and Challenges of CCR5 Gene Editing in Hematopoietic Stem and Progenitor Cells for the Treatment of HIV. Stem Cell Rev Rep 2021; 17:1607-1618. [PMID: 33788143 DOI: 10.1007/s12015-021-10145-7] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/24/2021] [Indexed: 12/29/2022]
Abstract
HIV infection continues to be a serious health issue with an alarming global spread, owing to the fact that attempts at developing an effective vaccine or a permanent cure remains futile. So far, the only available treatment for the clinical management of HIV is the combined Anti-Retroviral Therapy (cART), but the long-term cART is associated with metabolic changes, organ damages, and development and transmission of drug resistant HIV strains. Thus, there is a need for the development of one-time curative treatment for HIV infection. The allogeneic transplantation with the Hematopoietic Stem and Progenitor cells (HSPCs) having 32 bp deletion in Chemokine receptor 5 gene (CCR5 Δ32) demonstrated successful HIV remission in the Berlin and London patients, and highlighted that transplantation of CCR5 null HSPCs is a promising approach for a long- term HIV remission. The advent of gene editing technologies offers a new choice of generating ex vivo CCR5 ablated allogeneic or autologous HSPCs for stem cell transplantation into HIV patients. Many groups are attempting CCR5 disruption in HSPCs using various gene-editing strategies. At least two such studies, involving CCR5 gene editing in HSPCs have entered the clinical trials. This review aims to outline the strategies taken for CCR5 gene editing and discuss the challenges associated with the development of CCR5 manipulated HSPCs for the gene therapy of HIV infection.
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Affiliation(s)
- Karthik V Karuppusamy
- Centre for Stem Cell Research (A unit of inStem, Bengaluru), Christian Medical College, Vellore, Tamil Nadu, India.,Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Prathibha Babu
- Centre for Stem Cell Research (A unit of inStem, Bengaluru), Christian Medical College, Vellore, Tamil Nadu, India.,Manipal Academy of Higher Education, Manipal, Karnataka, India
| | - Saravanabhavan Thangavel
- Centre for Stem Cell Research (A unit of inStem, Bengaluru), Christian Medical College, Vellore, Tamil Nadu, India. .,Manipal Academy of Higher Education, Manipal, Karnataka, India.
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50
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Sar P, Dalai S. CRISPR/Cas9 in epigenetics studies of health and disease. PROGRESS IN MOLECULAR BIOLOGY AND TRANSLATIONAL SCIENCE 2021; 181:309-343. [PMID: 34127198 DOI: 10.1016/bs.pmbts.2021.01.022] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Epigenetics is the heritable phenotypic changes without altering the genotype. Epigenetic processes are such as histone methylation, acetylation, ubiquitination, sumoylation, phosphorylation, ADP ribosylation, DNA methylation and non-coding RNAs interactions associated with structural changes in chromatin. The change of structure is either open chromatin for "active" state or closed chromatin for "inactive" state, that regulates important biological phenomenon like chromatin condensation, gene expression, DNA repair, cellular development, differentiation and homeostasis, etc. However, dysregulation of epigenetic patterns causes diseases like cancer, diabetes, neurological disorder, infectious diseases, autoimmunity etc. Besides, the most important clinical uses of Epigenetics studies are i. identification of disease biomarkers and ii. development of their therapeutics. Epigenetic therapies include epi-drugs, combinatorial therapy, nanocarriers, plant-derived products that are being used for changing the epigenetic pattern to reverse gene expression. However, the developed epi- drugs cause off-target gene and transposable elements activation; promote mutagenesis and carcinogenesis in normal cells, are the major hurdles regarding their clinical use. Therefore, advanced epigenetic therapeutics are required to develop target-specific epigenetic modifications to reverse gene expression pattern. CRISPR-Cas9 (Clustered Regularly Interspaced Palindrome Repeats-associated protein 9) system-mediated gene activation mechanism paves new methods of target-specific epigenetic therapeutics to cure diseases. In this chapter, we discuss how CRISPR/Cas9 and dCas9 have recently been engineered for epigenome editing. Different strategies have been discussed used for epigenome editing based on their efficacy and complexity. Last but not least we have discussed the limitations, different uses of CRISPR/Cas9 and dCas9 in the area of genetic engineering.
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Affiliation(s)
- Pranati Sar
- Institute of Science, NIRMA University, Ahmedabad, India.
| | - Sarat Dalai
- Institute of Science, NIRMA University, Ahmedabad, India.
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