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1
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Thornburg CD, Pipe SW, Cantore A, Unzu C, Jones M, Miesbach WA. Clinical perspective: Advancing hemophilia treatment through gene therapy approaches. Mol Ther 2025:S1525-0016(25)00297-7. [PMID: 40263938 DOI: 10.1016/j.ymthe.2025.04.023] [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: 12/23/2024] [Revised: 03/21/2025] [Accepted: 04/16/2025] [Indexed: 04/24/2025] Open
Abstract
Hemophilia, a congenital bleeding disorder, can cause arthropathy, impaired mobility, pain, and life-threatening hemorrhage events, significantly impacting quality of life for patients and caregivers. Current therapies, although effective, necessitate costly lifelong treatment, often in specialized settings. However, as a monogenic disorder caused by loss-of-function genetic variants, hemophilia is amenable to gene therapy. In this article, three primary gene therapy approaches at the forefront of clinical development are reviewed. Adeno-associated virus-based gene therapy, having secured approval in the EU, UK, and US after promising phase 3 trial results, demonstrates clear superiority over standard-of-care treatment. Lentivirus-based approaches capable of transducing dividing and nondividing cells may improve the durability of treatment and have low susceptibility to pre-existing neutralizing antibodies to viral vectors. Finally, gene editing techniques such as zinc finger nucleases and CRISPR aim to correct genetic defects directly, holding promise as novel, effective, and highly durable therapeutic strategies in adults and children with hemophilia. This review provides a comprehensive summary of the current status of these gene therapy approaches, highlighting advantages, limitations, and potential future developments.
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Affiliation(s)
- Courtney D Thornburg
- National Institutes of Health, National Heart, Lung, and Blood Institute, Division of Blood Diseases and Resources, Bethesda, MD, USA
| | - Steve W Pipe
- Pediatric Hematology-Oncology, University of Michigan, Ann Arbor, MI, USA
| | - Alessio Cantore
- San Raffaele Telethon Institute for Gene Therapy, IRCCS San Raffaele Scientific Institute, Milan, Italy; Vita-Salute San Raffaele University, Milan, Italy
| | - Carmen Unzu
- DNA and RNA Medicine Division, CIMA Universidad de Navarra, Pamplona, Spain
| | | | - Wolfgang A Miesbach
- Department of Haemostaseology University Hospital Frankfurt, Frankfurt, Germany.
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Kim DH, Choi SH, Sung JJ, Kim S, Yi H, Park S, Park CW, Oh YW, Lee J, Kim DS, Kim JH, Park CY, Kim DW. Long-term correction of hemophilia A via integration of a functionally enhanced FVIII gene into the AAVS1 locus by nickase in patient-derived iPSCs. Exp Mol Med 2025; 57:184-192. [PMID: 39762408 PMCID: PMC11799516 DOI: 10.1038/s12276-024-01375-z] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2024] [Revised: 09/04/2024] [Accepted: 10/08/2024] [Indexed: 02/07/2025] Open
Abstract
Hemophilia A (HA) is caused by mutations in coagulation factor VIII (FVIII). Genome editing in conjunction with patient-derived induced pluripotent stem cells (iPSCs) is a promising cell therapy strategy, as it replaces dysfunctional proteins resulting from genetic mutations with normal proteins. However, the low expression level and short half-life of FVIII still remain significant limiting factors in the efficacy of these approaches in HA. Here, we constructed a functionally enhanced FVIII variant, F309S/E1984V-mutated B domain-deleted (BDD)-FVIII (FE-FVIII), with increased activity and stability. We inserted FE-FVIII with a human elongation factor-1 alpha (EF1α) promoter into the AAVS1 locus of HA patient-derived iPSCs via CRISPR/Cas9 (D10A) nickase to ensure expression in any cell type. FE-FVIII was expressed not only in undifferentiated FE-FVIII-inserted (FE-KI) iPSCs but also in endothelial cells (ECs) differentiated from them in vitro. Compared with mice transplanted with wild-type BDD-FVIII-containing ECs, immunocompetent HA mice intravenously transplanted with FE-KI ECs presented a 2.12-fold increase in FVIII activity in the blood and an approximately 20% greater survival rate after hemorrhagic tail injury. For sustained efficacy, FE-KI ECs were subcutaneously transplanted into immunodeficient HA mice, resulting in amelioration of the hemophilia phenotype for more than 3 months. This strategy can improve FVIII function and may provide a universal therapeutic approach for treating HA.
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Affiliation(s)
- Do-Hun Kim
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
- S. Biomedics Co., Ltd, 28 Seongsui-ro 26-gil, Seongdong-gu, Seoul, 04797, Korea
| | - Sang-Hwi Choi
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Jin Jea Sung
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Sieun Kim
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
- Brain Korea 21 PLUS Program for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Hanui Yi
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
- Brain Korea 21 PLUS Program for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Sanghyun Park
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Chan Wook Park
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Young Woo Oh
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
- Brain Korea 21 PLUS Program for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Jungil Lee
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
- Brain Korea 21 PLUS Program for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea
| | - Dae-Sung Kim
- Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Korea
| | - Jong-Hoon Kim
- Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, 02841, Korea
| | - Chul-Yong Park
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea.
- S. Biomedics Co., Ltd, 28 Seongsui-ro 26-gil, Seongdong-gu, Seoul, 04797, Korea.
| | - Dong-Wook Kim
- Department of Physiology, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea.
- S. Biomedics Co., Ltd, 28 Seongsui-ro 26-gil, Seongdong-gu, Seoul, 04797, Korea.
- Brain Korea 21 PLUS Program for Medical Science, Yonsei University College of Medicine, 50-1 Yonsei-ro, Seodaemun-gu, Seoul, 03722, Korea.
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Jeddou H, Tzedakis S, Chaouch MA, Sulpice L, Samson M, Boudjema K. Viability Assessment During Normothermic Machine Liver Perfusion: A Literature Review. Liver Int 2025; 45:e16244. [PMID: 39821671 PMCID: PMC11740183 DOI: 10.1111/liv.16244] [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: 06/30/2024] [Revised: 12/25/2024] [Accepted: 01/03/2025] [Indexed: 01/19/2025]
Abstract
BACKGROUND AND OBJECTIVE The discrepancy between donor organ availability and demand leads to a significant waiting-list dropout rate and mortality. Although quantitative tools such as the Donor Risk Index (DRI) help assess organ suitability, many potentially viable organs are still discarded due to the lack of universally accepted markers to predict post-transplant outcomes. Normothermic machine perfusion (NMP) offers a platform to assess viability before transplantation. Thus, livers considered unsuitable for transplantation based on the DRI can be evaluated and potentially transplanted. During NMP, various viability criteria have been proposed. These criteria are neither homogeneous nor consensual. In this review, we aimed to describe the viability criteria during NMP and evaluate their ability to predict hepatic graft function following transplantation. We conducted a PubMed search using the terms 'liver transplantation', 'normothermic machine perfusion' and 'assessment', including only English publications up to February 2024. Viability assessment during NMP includes multiple hepatocellular and cholangiocellular criteria. Lactate clearance and bile production are commonly used indicators, but their ability to predict post-transplant outcomes varies significantly. The predictive value of cholangiocellular criteria such as bile pH, bicarbonate and glucose levels remains under investigation. Novel markers, such as microRNAs and proteomic profiles, offer the potential to enhance graft evaluation accuracy and provide insights into the molecular mechanisms underlying liver viability. Combining perfusion parameters with biomarkers may improve the prediction of long-term graft survival. Future research should focus on standardising viability assessment protocols and exploring real-time biomarker evaluations, which could enhance transplantation outcomes and expand the donor pool.
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Affiliation(s)
- Heithem Jeddou
- Department of Hepatobiliary and Digestive SurgeryUniversity Hospital, Rennes 1 UniversityRennesFrance
- Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail)‐UMR_S 1085, Université de RennesRennesFrance
| | - Stylianos Tzedakis
- Department of Hepato‐Biliary, Digestive and Endocrine SurgeryCochin Hospital, APHPParisFrance
- Université Paris CitéParisFrance
| | - Mohamed Ali Chaouch
- Department of Visceral and Digestive SurgeryMonastir University HospitalMonastirTunisia
| | - Laurent Sulpice
- Department of Hepatobiliary and Digestive SurgeryUniversity Hospital, Rennes 1 UniversityRennesFrance
- INSERM OSS U1242, University Hospital, Rennes 1 UniversityRennesFrance
| | - Michel Samson
- Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail)‐UMR_S 1085, Université de RennesRennesFrance
| | - Karim Boudjema
- Department of Hepatobiliary and Digestive SurgeryUniversity Hospital, Rennes 1 UniversityRennesFrance
- Inserm, EHESP, Irset (Institut de recherche en santé, environnement et travail)‐UMR_S 1085, Université de RennesRennesFrance
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Kim JJ, Kurial SNT, Choksi PK, Nunez M, Lunow-Luke T, Bartel J, Driscoll J, Her CL, Dhillon S, Yue W, Murti A, Mao T, Ramos JN, Tiyaboonchai A, Grompe M, Mattis AN, Syed SM, Wang BM, Maher JJ, Roll GR, Willenbring H. AAV capsid prioritization in normal and steatotic human livers maintained by machine perfusion. Nat Biotechnol 2025:10.1038/s41587-024-02523-6. [PMID: 39881029 DOI: 10.1038/s41587-024-02523-6] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/17/2023] [Accepted: 12/02/2024] [Indexed: 01/31/2025]
Abstract
Therapeutic efficacy and safety of adeno-associated virus (AAV) liver gene therapy depend on capsid choice. To predict AAV capsid performance under near-clinical conditions, we established side-by-side comparison at single-cell resolution in human livers maintained by normothermic machine perfusion. AAV-LK03 transduced hepatocytes much more efficiently and specifically than AAV5, AAV8 and AAV6, which are most commonly used clinically, and AAV-NP59, which is better at transducing human hepatocytes engrafted in immune-deficient mice. AAV-LK03 preferentially transduced periportal hepatocytes in normal liver, whereas AAV5 targeted pericentral hepatocytes in steatotic liver. AAV5 and AAV8 transduced liver sinusoidal endothelial cells as efficiently as hepatocytes. AAV capsid and steatosis influenced vector episome formation, which determines gene therapy durability, with AAV5 delaying concatemerization. Our findings inform capsid choice in clinical AAV liver gene therapy, including consideration of disease-relevant hepatocyte zonation and effects of steatosis, and facilitate the development of AAV capsids that transduce hepatocytes or other therapeutically relevant cell types in the human liver with maximum efficiency and specificity.
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Affiliation(s)
- Jae-Jun Kim
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA
| | - Simone N T Kurial
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA
- Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA
| | - Pervinder K Choksi
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA
- Tetrad Graduate Program, University of California, San Francisco, San Francisco, CA, USA
| | - Miguel Nunez
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
| | - Tyler Lunow-Luke
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
- School of Medicine, University of California, Irvine, Irvine, CA, USA
| | - Jan Bartel
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA
| | - Julia Driscoll
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA
| | - Chris L Her
- Department of Medicine, University of California, San Francisco, San Francisco, CA, USA
- Liver Center, University of California, San Francisco, San Francisco, CA, USA
- Pliant Therapeutics, South San Francisco, CA, USA
| | - Simaron Dhillon
- Department of Medicine, University of California, San Francisco, San Francisco, CA, USA
- Liver Center, University of California, San Francisco, San Francisco, CA, USA
- Stone Research Foundation, San Francisco, CA, USA
| | - William Yue
- Department of Medicine, University of California, San Francisco, San Francisco, CA, USA
| | - Abhishek Murti
- Department of Medicine, University of California, San Francisco, San Francisco, CA, USA
| | - Tin Mao
- Ambys Medicines, South San Francisco, CA, USA
- Genentech, South San Francisco, CA, USA
| | - Julian N Ramos
- Ambys Medicines, South San Francisco, CA, USA
- Adverum Biotechnologies, Redwood City, CA, USA
| | - Amita Tiyaboonchai
- Oregon Stem Cell Center, Oregon Health & Science University, Portland, OR, USA
- Department of Pediatrics, Oregon Health & Science University, Portland, OR, USA
| | - Markus Grompe
- Oregon Stem Cell Center, Oregon Health & Science University, Portland, OR, USA
- Department of Pediatrics, Oregon Health & Science University, Portland, OR, USA
- Department of Molecular and Medical Genetics, Oregon Health & Science University, Portland, OR, USA
| | - Aras N Mattis
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA
- Liver Center, University of California, San Francisco, San Francisco, CA, USA
- Department of Pathology, University of California, San Francisco, San Francisco, CA, USA
| | - Shareef M Syed
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
| | - Bruce M Wang
- Department of Medicine, University of California, San Francisco, San Francisco, CA, USA
- Liver Center, University of California, San Francisco, San Francisco, CA, USA
| | - Jacquelyn J Maher
- Department of Medicine, University of California, San Francisco, San Francisco, CA, USA
- Liver Center, University of California, San Francisco, San Francisco, CA, USA
| | - Garrett R Roll
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA
| | - Holger Willenbring
- Department of Surgery, University of California, San Francisco, San Francisco, CA, USA.
- Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, San Francisco, CA, USA.
- Liver Center, University of California, San Francisco, San Francisco, CA, USA.
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5
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Mitani S, Hosoda C, Onodera Y, Takabayashi Y, Sakata A, Shima M, Tatsumi K. Efficient generation of liver sinusoidal endothelial-like cells secreting coagulation factor VIII from human induced pluripotent stem cells. Mol Ther Methods Clin Dev 2024; 32:101355. [PMID: 39559558 PMCID: PMC11570519 DOI: 10.1016/j.omtm.2024.101355] [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: 12/28/2023] [Accepted: 10/15/2024] [Indexed: 11/20/2024]
Abstract
Liver sinusoidal endothelial cells (LSECs) and LSEC progenitor cells (LPCs) derived from human pluripotent stem cells (PSCs) are expected as valuable cell sources for the development of cell therapy for hemophilia A, a congenital deficiency of coagulation factor VIII (FVIII), as LSECs are responsible for FVIII production. However, there is room for improvement in the efficiency of LSEC and LPC differentiation from human PSCs. In this study, we sought to optimize the method of mesoderm differentiation induction, the initial step of LSEC differentiation from human PSCs, to efficiently induce LSEC-like cells capable of secreting FVIII from human induced pluripotent stem cells (iPSCs). Following optimization of the concentration and stimulation period of CHIR99021 (glycogen synthase kinase 3β inhibitor), bone morphogenetic protein 4, fibroblast growth factor 2, and Activin A in the mesoderm induction step, approximately 65% and 54% of cells differentiated into LPCs and LSEC-like cells, respectively. Furthermore, we observed substantial FVIII protein secretion from LSEC-like cells in vitro. In conclusion, we established an efficient method for obtaining LPCs and functional LSEC-like cells from human iPSCs in vitro.
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Affiliation(s)
- Seiji Mitani
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Chihiro Hosoda
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Yu Onodera
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Yoko Takabayashi
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Asuka Sakata
- Medicinal Biology of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Midori Shima
- Medicinal Biology of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Kohei Tatsumi
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
- Medicinal Biology of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
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Vir P, Gunasekera D, Dorjbal B, McDaniel D, Agrawal A, Merricks EP, Ragni MV, Leissinger CA, Stering AI, Lieuw K, Nichols TC, Pratt KP. Lack of factor VIII detection in humans and dogs with an intron 22 inversion challenges hypothesis regarding inhibitor risk. J Thromb Haemost 2024; 22:3415-3430. [PMID: 39233012 DOI: 10.1016/j.jtha.2024.08.007] [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: 06/30/2024] [Revised: 08/03/2024] [Accepted: 08/05/2024] [Indexed: 09/06/2024]
Abstract
BACKGROUND Almost half of severe hemophilia A (HA) cases are caused by an intron 22 inversion (Int22Inv) mutation, which truncates the 26-exon F8 messenger RNA (mRNA) after exon 22. Another F8 transcript, F8B, is initiated from within F8-intron-22. F8B mRNA consists of a short exon spliced to exons 23 to 26 and is expressed in multiple human cell types. It has been hypothesized that Int22Inv patients have self-tolerance to partial factor (F)VIII proteins expressed from these 2 transcripts. FVIII is expressed in endothelial cells, primarily in the liver and lungs. Several studies have reported FVIII expression in other cell types, although this has been controversial. OBJECTIVES To determine if partial FVIII proteins are expressed from intron 22-inverted and/or F8B mRNA and if FVIII is expressed in nonendothelial cells. METHODS A panel of FVIII-specific antibodies was validated and employed to label FVIII in cells and tissues and for immunoprecipitation followed by western blots and mass spectrometry proteomics analysis. RESULTS Immunofluorescent staining localized FVIII to endothelial cells in liver sections from non-HA but not HA-Int22Inv dogs. Neither FVIII nor FVIIIB was detected in human peripheral blood mononuclear cells, B cell or T cell lines, or cell lines expanded from peripheral blood mononuclear cells, whereas FVIII antigen and activity were readily detected in primary nonhemophilic liver sinusoidal endothelial cells. CONCLUSION If FVIII is expressed in nonendothelial cells or if partial FVIII proteins are expressed in HA-Int22Inv, the concentrations are below the detection limits of these sensitive assays. Our results argue against promotion of immune tolerance through expression of partial FVIII proteins in Int-22Inv patients.
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Affiliation(s)
- Pooja Vir
- Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA; The Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland, USA
| | - Devi Gunasekera
- Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA; The Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland, USA
| | - Batsukh Dorjbal
- Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA; The Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland, USA
| | - Dennis McDaniel
- Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA; Biological Instrumentation Center, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
| | - Atul Agrawal
- Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA; The Henry M. Jackson Foundation for the Advancement of Military Medicine, Bethesda, Maryland, USA
| | - Elizabeth P Merricks
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | | | - Cindy A Leissinger
- Department of Medicine, Louisiana Center for Bleeding and Clotting Disorders, Tulane University School of Medicine, New Orleans, Louisiana, USA
| | - Allen I Stering
- Walter Reed National Military Medical Center, Bethesda, Maryland, USA
| | - Kenneth Lieuw
- Walter Reed National Military Medical Center, Bethesda, Maryland, USA; Department of Pediatrics, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA
| | - Timothy C Nichols
- Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA
| | - Kathleen P Pratt
- Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland, USA.
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7
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Grigore A, Dragomir M, Călugăru OT, Jardan D, Jardan C, Brînză M, Bălănescu P, Coriu D. Mutational Profile in Romanian Patients with Hemophilia A. Int J Mol Sci 2024; 25:8366. [PMID: 39125936 PMCID: PMC11312815 DOI: 10.3390/ijms25158366] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2024] [Revised: 07/25/2024] [Accepted: 07/30/2024] [Indexed: 08/12/2024] Open
Abstract
Hemophilia A (HA) is an X-linked recessive bleeding disorder caused by mutations in the F8 gene, resulting in deficient or dysfunctional factor VIII (FVIII). This study aimed to characterize the mutational profile of HA in Romanian patients using next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA). A total of 107 patients were analyzed, revealing pathogenic or likely pathogenic variants in 96.3% of cases. The identified mutations included missense (30.5%), nonsense (9.1%), small deletions (6.4%), small insertions (2.1%), splice-site variants (4.3%), large deletions (1.6%), and large duplications (1.1%). Large intron inversion was previously found in 37.5% of the patients. Novel variants accounted for 21.5% of identified mutations, expanding the spectrum of F8 variants in this population. This study underscores the genetic heterogeneity of HA and provides insights into genotype-phenotype correlations, aiding in clinical management and prenatal diagnosis.
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Affiliation(s)
- Andra Grigore
- Hematology (Clinic and Laboratory) Discipline-Fundeni Clinical Institute, “Carol Davila” University of Medicine and Pharmacy, 020021 Bucharest, Romania
- Department of Hematology and Bone Marrow Transplant, Fundeni Clinical Institute, 022328 Bucharest, Romania
| | - Mihaela Dragomir
- Department of Hematology and Bone Marrow Transplant, Fundeni Clinical Institute, 022328 Bucharest, Romania
| | - Onda-Tabita Călugăru
- Department of Hematology and Bone Marrow Transplant, Fundeni Clinical Institute, 022328 Bucharest, Romania
| | - Dumitru Jardan
- Molecular Biology Laboratory, Medlife, 010093 Bucharest, Romania
| | - Cerasela Jardan
- Department of Hematology and Bone Marrow Transplant, Fundeni Clinical Institute, 022328 Bucharest, Romania
- Pediatrics Discipline-Fundeni Clinical Institute, “Carol Davila” University of Medicine and Pharmacy, 020021 Bucharest, Romania
| | - Melen Brînză
- Department of Hematology and Bone Marrow Transplant, Fundeni Clinical Institute, 022328 Bucharest, Romania
| | - Paul Bălănescu
- Internal Medicine Discipline-Colentina Clinical Hospital, “Carol Davila” University of Medicine and Pharmacy, 020021 Bucharest, Romania
| | - Daniel Coriu
- Hematology (Clinic and Laboratory) Discipline-Fundeni Clinical Institute, “Carol Davila” University of Medicine and Pharmacy, 020021 Bucharest, Romania
- Department of Hematology and Bone Marrow Transplant, Fundeni Clinical Institute, 022328 Bucharest, Romania
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8
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Tavares V, Savva-Bordalo J, Rei M, Liz-Pimenta J, Assis J, Pereira D, Medeiros R. Haemostatic Gene Expression in Cancer-Related Immunothrombosis: Contribution for Venous Thromboembolism and Ovarian Tumour Behaviour. Cancers (Basel) 2024; 16:2356. [PMID: 39001418 PMCID: PMC11240748 DOI: 10.3390/cancers16132356] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/08/2024] [Revised: 06/24/2024] [Accepted: 06/26/2024] [Indexed: 07/16/2024] Open
Abstract
Ovarian cancer (OC) is the deadliest gynaecological malignancy. Identifying new prognostic biomarkers is an important research field. Haemostatic components together with leukocytes can drive cancer progression while increasing the susceptibility to venous thromboembolism (VTE) through immunothrombosis. Unravelling the underlying complex interactions offers the prospect of uncovering relevant OC prognostic biomarkers, predictors of cancer-associated thrombosis (CAT), and even potential targets for cancer therapy. Thus, this study evaluated the expression of F3, F5, F8, F13A1, TFPI1, and THBD in peripheral blood cells (PBCs) of 52 OC patients. Those with VTE after tumour diagnosis had a worse overall survival (OS) compared to their counterparts (mean OS of 13.8 ± 4.1 months and 47.9 ± 5.7 months, respectively; log-rank test, p = 0.001). Low pre-chemotherapy F3 and F8 expression levels were associated with a higher susceptibility for OC-related VTE after tumour diagnosis (χ2, p < 0.05). Regardless of thrombogenesis, patients with low baseline F8 expression had a shorter progression-free survival (PFS) than their counterparts (adjusted hazard ratio (aHR) = 2.54; p = 0.021). Among those who were not under platelet anti-aggregation therapy, low F8 levels were also associated with a shorter OS (aHR = 6.16; p = 0.006). Moving forward, efforts should focus on external validation in larger cohorts.
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Affiliation(s)
- Valéria Tavares
- Molecular Oncology and Viral Pathology Group, Research Center of IPO Porto (CI-IPOP)/Pathology and Laboratory Medicine Dep., Clinical Pathology SV/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre (Porto. CCC), 4200-072 Porto, Portugal;
- ICBAS—Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal
- Faculty of Medicine of the University of Porto (FMUP), 4200-072 Porto, Portugal;
| | - Joana Savva-Bordalo
- Department of Medical Oncology, Portuguese Institute of Oncology of Porto (IPO Porto), 4200-072 Porto, Portugal; (J.S.-B.); (D.P.)
| | - Mariana Rei
- Department of Gynaecology, Portuguese Institute of Oncology of Porto (IPO Porto), 4200-072 Porto, Portugal;
| | - Joana Liz-Pimenta
- Faculty of Medicine of the University of Porto (FMUP), 4200-072 Porto, Portugal;
- Department of Medical Oncology, Centro Hospitalar de Trás-os-Montes e Alto Douro (CHTMAD), 5000-508 Vila Real, Portugal
| | - Joana Assis
- Clinical Research Unit, Research Center of IPO Porto (CI-IPOP)/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Center (Porto. CCC), 4200-072 Porto, Portugal;
| | - Deolinda Pereira
- Department of Medical Oncology, Portuguese Institute of Oncology of Porto (IPO Porto), 4200-072 Porto, Portugal; (J.S.-B.); (D.P.)
| | - Rui Medeiros
- Molecular Oncology and Viral Pathology Group, Research Center of IPO Porto (CI-IPOP)/Pathology and Laboratory Medicine Dep., Clinical Pathology SV/RISE@CI-IPOP (Health Research Network), Portuguese Oncology Institute of Porto (IPO Porto)/Porto Comprehensive Cancer Centre (Porto. CCC), 4200-072 Porto, Portugal;
- ICBAS—Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4050-313 Porto, Portugal
- Faculty of Medicine of the University of Porto (FMUP), 4200-072 Porto, Portugal;
- Faculty of Health Sciences, Fernando Pessoa University, 4200-150 Porto, Portugal
- Research Department, Portuguese League Against Cancer (NRNorte), 4200-172 Porto, Portugal
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9
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Tian SP, Ge JY, Song YM, Yu XQ, Chen WH, Chen YY, Ye D, Zheng YW. A novel efficient strategy to generate liver sinusoidal endothelial cells from human pluripotent stem cells. Sci Rep 2024; 14:13831. [PMID: 38879647 PMCID: PMC11180100 DOI: 10.1038/s41598-024-64195-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2024] [Accepted: 06/06/2024] [Indexed: 06/19/2024] Open
Abstract
Liver sinusoidal endothelial cells (LSECs) are highly specialized endothelial cells (ECs) that play an important role in liver development and regeneration. Additionally, it is involved in various pathological processes, including steatosis, inflammation, fibrosis and hepatocellular carcinoma. However, the rapid dedifferentiation of LSECs after culture greatly limits their use in vitro modeling for biomedical applications. In this study, we developed a highly efficient protocol to induce LSEC-like cells from human induced pluripotent stem cells (hiPSCs) in only 8 days. Using single-cell transcriptomic analysis, we identified several novel LSEC-specific markers, such as EPAS1, LIFR, and NID1, as well as several previously revealed markers, such as CLEC4M, CLEC1B, CRHBP and FCN3. These LSEC markers are specifically expressed in our LSEC-like cells. Furthermore, hiPSC-derived cells expressed LSEC-specific proteins and exhibited LSEC-related functions, such as the uptake of acetylated low density lipoprotein (ac-LDL) and immune complex endocytosis. Overall, this study confirmed that our novel protocol allowed hiPSCs to rapidly acquire an LSEC-like phenotype and function in vitro. The ability to generate LSECs efficiently and rapidly may help to more precisely mimic liver development and disease progression in a liver-specific multicellular microenvironment, offering new insights into the development of novel therapeutic strategies.
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Affiliation(s)
- Shang-Ping Tian
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, and South China Institute of Large Animal Models for Biomedicine, School of Pharmacy and Food Engineering, Wuyi University, Jiangmen, Guangdong, China
| | - Jian-Yun Ge
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, and South China Institute of Large Animal Models for Biomedicine, School of Pharmacy and Food Engineering, Wuyi University, Jiangmen, Guangdong, China
| | - Yu-Mu Song
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, and South China Institute of Large Animal Models for Biomedicine, School of Pharmacy and Food Engineering, Wuyi University, Jiangmen, Guangdong, China
| | - Xiao-Qing Yu
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, and South China Institute of Large Animal Models for Biomedicine, School of Pharmacy and Food Engineering, Wuyi University, Jiangmen, Guangdong, China
| | - Wen-Hao Chen
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, and South China Institute of Large Animal Models for Biomedicine, School of Pharmacy and Food Engineering, Wuyi University, Jiangmen, Guangdong, China
| | - Yu-Ying Chen
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, and South China Institute of Large Animal Models for Biomedicine, School of Pharmacy and Food Engineering, Wuyi University, Jiangmen, Guangdong, China
- State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, China
| | - Di Ye
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, and South China Institute of Large Animal Models for Biomedicine, School of Pharmacy and Food Engineering, Wuyi University, Jiangmen, Guangdong, China
| | - Yun-Wen Zheng
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, and South China Institute of Large Animal Models for Biomedicine, School of Pharmacy and Food Engineering, Wuyi University, Jiangmen, Guangdong, China.
- Institute of Regenerative Medicine, and Department of Dermatology, Affiliated Hospital of Jiangsu University, Jiangsu University, Zhenjiang, Jiangsu, China.
- Department of Medical and Life Sciences, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba, Japan.
- Institute of Medical Science, Center for Stem Cell Biology and Regenerative Medicine, The University of Tokyo, Tokyo, Japan.
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10
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Alayoubi AM, Khawaji ZY, Mohammed MA, Mercier FE. CRISPR-Cas9 system: a novel and promising era of genotherapy for beta-hemoglobinopathies, hematological malignancy, and hemophilia. Ann Hematol 2024; 103:1805-1817. [PMID: 37736806 DOI: 10.1007/s00277-023-05457-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Accepted: 09/15/2023] [Indexed: 09/23/2023]
Abstract
Gene therapy represents a significant potential to revolutionize the field of hematology with applications in correcting genetic mutations, generating cell lines and animal models, and improving the feasibility and efficacy of cancer immunotherapy. Compared to different genetic engineering tools, clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated protein 9 (Cas9) emerged as an effective and versatile genetic editor with the ability to precisely modify the genome. The applications of genetic engineering in various hematological disorders have shown encouraging results. Monogenic hematological disorders can conceivably be corrected with single gene modification. Through the use of CRISPR-CAS9, restoration of functional red blood cells and hemostasis factors were successfully attained in sickle cell anemia, beta-thalassemia, and hemophilia disorders. Our understanding of hemato-oncology has been advanced via CRIPSR-CAS9 technology. CRISPR-CAS9 aided to build a platform of mutated genes responsible for cell survival and proliferation in leukemia. Therapeutic application of CRISPR-CAS9 when combined with chimeric antigen receptor (CAR) T cell therapy in multiple myeloma and acute lymphoblastic leukemia was feasible with attenuation of CAR T cell therapy pitfalls. Our review outlines the latest literature on the utilization of CRISPR-Cas9 in the treatment of beta-hemoglobinopathies and hemophilia disorders. We present the strategies that were employed and the findings of preclinical and clinical trials. Also, the review will discuss gene engineering in the field of hemato-oncology as a proper tool to facilitate and overcome the drawbacks of chimeric antigen receptor T cell therapy (CAR-T).
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Affiliation(s)
- Abdulfatah M Alayoubi
- Department of Biochemistry and Molecular Medicine, College of Medicine, Taibah University, Madinah, Saudi Arabia
| | | | | | - François E Mercier
- Divisions of Experimental Medicine & Hematology, Department of Medicine, Faculty of Medicine, McGill University, Montreal, Quebec, Canada
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11
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Yap KK, Schröder J, Gerrand YW, Dobric A, Kong AM, Fox AM, Knowles B, Banting SW, Elefanty AG, Stanley EG, Yeoh GC, Lockwood GP, Cogger VC, Morrison WA, Polo JM, Mitchell GM. Liver specification of human iPSC-derived endothelial cells transplanted into mouse liver. JHEP Rep 2024; 6:101023. [PMID: 38681862 PMCID: PMC11046210 DOI: 10.1016/j.jhepr.2024.101023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/28/2023] [Revised: 01/19/2024] [Accepted: 01/23/2024] [Indexed: 05/01/2024] Open
Abstract
Background & Aims Liver sinusoidal endothelial cells (LSECs) are important in liver development, regeneration, and pathophysiology, but the differentiation process underlying their tissue-specific phenotype is poorly understood and difficult to study because primary human cells are scarce. The aim of this study was to use human induced pluripotent stem cell (hiPSC)-derived LSEC-like cells to investigate the differentiation process of LSECs. Methods hiPSC-derived endothelial cells (iECs) were transplanted into the livers of Fah-/-/Rag2-/-/Il2rg-/- mice and assessed over a 12-week period. Lineage tracing, immunofluorescence, flow cytometry, plasma human factor VIII measurement, and bulk and single cell transcriptomic analysis were used to assess the molecular and functional changes that occurred following transplantation. Results Progressive and long-term repopulation of the liver vasculature occurred as iECs expanded along the sinusoids between hepatocytes and increasingly produced human factor VIII, indicating differentiation into LSEC-like cells. To chart the developmental profile associated with LSEC specification, the bulk transcriptomes of transplanted cells between 1 and 12 weeks after transplantation were compared against primary human adult LSECs. This demonstrated a chronological increase in LSEC markers, LSEC differentiation pathways, and zonation. Bulk transcriptome analysis suggested that the transcription factors NOTCH1, GATA4, and FOS have a central role in LSEC specification, interacting with a network of 27 transcription factors. Novel markers associated with this process included EMCN and CLEC14A. Additionally, single cell transcriptomic analysis demonstrated that transplanted iECs at 4 weeks contained zonal subpopulations with a region-specific phenotype. Conclusions Collectively, this study confirms that hiPSCs can adopt LSEC-like features and provides insight into LSEC specification. This humanised xenograft system can be applied to further interrogate LSEC developmental biology and pathophysiology, bypassing current logistical obstacles associated with primary human LSECs. Impact and implications Liver sinusoidal endothelial cells (LSECs) are important cells for liver biology, but better model systems are required to study them. We present a pluripotent stem cell xenografting model that produces human LSEC-like cells. A detailed and longitudinal transcriptomic analysis of the development of LSEC-like cells is included, which will guide future studies to interrogate LSEC biology and produce LSEC-like cells that could be used for regenerative medicine.
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Affiliation(s)
- Kiryu K. Yap
- O’Brien Department of St Vincent’s Institute, Fitzroy, VIC, Australia
- University of Melbourne Department of Surgery, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia
| | - Jan Schröder
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia
- Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
- Australian Regenerative Medicine Institute, Clayton, VIC, Australia
- Doherty Institute & University of Melbourne Department of Microbiology and Immunology, Parkville, VIC, Australia
| | - Yi-Wen Gerrand
- O’Brien Department of St Vincent’s Institute, Fitzroy, VIC, Australia
| | - Aleksandar Dobric
- O’Brien Department of St Vincent’s Institute, Fitzroy, VIC, Australia
| | - Anne M. Kong
- O’Brien Department of St Vincent’s Institute, Fitzroy, VIC, Australia
| | - Adrian M. Fox
- University of Melbourne Department of Surgery, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia
- Hepatobiliary Surgery Unit, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia
| | - Brett Knowles
- University of Melbourne Department of Surgery, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia
- Hepatobiliary Surgery Unit, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia
| | - Simon W. Banting
- University of Melbourne Department of Surgery, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia
- Hepatobiliary Surgery Unit, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia
| | - Andrew G. Elefanty
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC, Australia
- Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC, Australia
| | - Eduoard G. Stanley
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia
- Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC, Australia
- Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC, Australia
| | - George C. Yeoh
- Harry Perkins Institute of Medical Research and Centre for Medical Research, University of Western Australia, Perth, WA, Australia
| | - Glen P. Lockwood
- ANZAC Research Institute and University of Sydney, Concord, NSW, Australia
| | - Victoria C. Cogger
- ANZAC Research Institute and University of Sydney, Concord, NSW, Australia
| | - Wayne A. Morrison
- O’Brien Department of St Vincent’s Institute, Fitzroy, VIC, Australia
- University of Melbourne Department of Surgery, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia
- Australian Catholic University, Fitzroy, VIC, Australia
| | - Jose M. Polo
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia
- Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia
- Australian Regenerative Medicine Institute, Clayton, VIC, Australia
- Adelaide Centre for Epigenetics, South Australian Immunogenomics Cancer Institute, University of Adelaide, Adelaide, SA, Australia
| | - Geraldine M. Mitchell
- O’Brien Department of St Vincent’s Institute, Fitzroy, VIC, Australia
- University of Melbourne Department of Surgery, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia
- Australian Catholic University, Fitzroy, VIC, Australia
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12
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Pierce GF, Fong S, Long BR, Kaczmarek R. Deciphering conundrums of adeno-associated virus liver-directed gene therapy: focus on hemophilia. J Thromb Haemost 2024; 22:1263-1289. [PMID: 38103734 DOI: 10.1016/j.jtha.2023.12.005] [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: 09/15/2023] [Revised: 11/07/2023] [Accepted: 12/01/2023] [Indexed: 12/19/2023]
Abstract
Adeno-associated virus gene therapy has been the subject of intensive investigation for monogenic disease gene addition therapy for more than 25 years, yet few therapies have been approved by regulatory agencies. Most have not progressed beyond phase 1/2 due to toxicity, lack of efficacy, or both. The liver is a natural target for adeno-associated virus since most serotypes have a high degree of tropism for hepatocytes due to cell surface receptors for the virus and the unique liver sinusoidal geometry facilitating high volumes of blood contact with hepatocyte cell surfaces. Recessive monogenic diseases such as hemophilia represent promising targets since the defective proteins are often synthesized in the liver and secreted into the circulation, making them easy to measure, and many do not require precise regulation. Yet, despite initiation of many disease-specific clinical trials, therapeutic windows are often nonexistent, resulting in excess toxicity and insufficient efficacy. Iterative progress built on these attempts is best illustrated by hemophilia, with the first regulatory approvals for factor IX and factor VIII gene therapies eventually achieved 25 years after the first gene therapy studies in humans. Although successful gene transfer may result in the production of sufficient transgenic protein to modify the disease, many emerging questions on durability, predictability, reliability, and variability of response have not been answered. The underlying biology accounting for these heterogeneous responses and the interplay between host and virus is the subject of intense investigation and the subject of this review.
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Affiliation(s)
- Glenn F Pierce
- World Federation of Hemophilia, Montreal, Quebec, Canada.
| | - Sylvia Fong
- BioMarin Pharmaceutical Inc, Research and Early Development, Novato, California, USA
| | - Brian R Long
- BioMarin Pharmaceutical Inc, Research and Early Development, Novato, California, USA
| | - Radoslaw Kaczmarek
- Department of Pediatrics, Indiana University School of Medicine, Wells Center for Pediatric Research, Indiana, USA; Laboratory of Glycobiology, Hirszfeld Institute of Immunology and Experimental Therapy, Wroclaw, Poland
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13
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Wang JH, Gessler DJ, Zhan W, Gallagher TL, Gao G. Adeno-associated virus as a delivery vector for gene therapy of human diseases. Signal Transduct Target Ther 2024; 9:78. [PMID: 38565561 PMCID: PMC10987683 DOI: 10.1038/s41392-024-01780-w] [Citation(s) in RCA: 180] [Impact Index Per Article: 180.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/05/2023] [Revised: 02/08/2024] [Accepted: 02/19/2024] [Indexed: 04/04/2024] Open
Abstract
Adeno-associated virus (AAV) has emerged as a pivotal delivery tool in clinical gene therapy owing to its minimal pathogenicity and ability to establish long-term gene expression in different tissues. Recombinant AAV (rAAV) has been engineered for enhanced specificity and developed as a tool for treating various diseases. However, as rAAV is being more widely used as a therapy, the increased demand has created challenges for the existing manufacturing methods. Seven rAAV-based gene therapy products have received regulatory approval, but there continue to be concerns about safely using high-dose viral therapies in humans, including immune responses and adverse effects such as genotoxicity, hepatotoxicity, thrombotic microangiopathy, and neurotoxicity. In this review, we explore AAV biology with an emphasis on current vector engineering strategies and manufacturing technologies. We discuss how rAAVs are being employed in ongoing clinical trials for ocular, neurological, metabolic, hematological, neuromuscular, and cardiovascular diseases as well as cancers. We outline immune responses triggered by rAAV, address associated side effects, and discuss strategies to mitigate these reactions. We hope that discussing recent advancements and current challenges in the field will be a helpful guide for researchers and clinicians navigating the ever-evolving landscape of rAAV-based gene therapy.
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Affiliation(s)
- Jiang-Hui Wang
- Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
- Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
- Centre for Eye Research Australia, Royal Victorian Eye and Ear Hospital, East Melbourne, VIC, 3002, Australia
- Ophthalmology, Department of Surgery, University of Melbourne, East Melbourne, VIC, 3002, Australia
| | - Dominic J Gessler
- Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
- Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
- Department of Neurological Surgery, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
- Department of Neurosurgery, University of Minnesota, Minneapolis, MN, 55455, USA
| | - Wei Zhan
- Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
- Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
- Department of Medicine, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
- Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
| | - Thomas L Gallagher
- Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA
| | - Guangping Gao
- Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA.
- Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA.
- Li Weibo Institute for Rare Diseases Research, University of Massachusetts Chan Medical School, Worcester, MA, 01605, USA.
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Lawton SM, Manson MA, Fan MN, Chao TY, Chen CY, Kim P, Campbell C, Cai X, Vander Kooi A, Miao CH. Ultrasound-mediated gene delivery specifically targets liver sinusoidal endothelial cells for sustained FVIII expression in hemophilia A mice. Mol Ther 2024; 32:969-981. [PMID: 38341614 PMCID: PMC11163219 DOI: 10.1016/j.ymthe.2024.02.010] [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: 09/11/2023] [Revised: 12/30/2023] [Accepted: 02/07/2024] [Indexed: 02/12/2024] Open
Abstract
The ability to target the native production site of factor VIII (FVIII)-liver sinusoidal endothelial cells (LSECs)-can improve the outcome of hemophilia A (HA) gene therapy. By testing a matrix of ultrasound-mediated gene delivery (UMGD) parameters for delivering a GFP plasmid into the livers of HA mice, we were able to define specific conditions for targeted gene delivery to different cell types in the liver. Subsequently, two conditions were selected for experiments to treat HA mice via UMGD of an endothelial-specific human FVIII plasmid: low energy (LE; 50 W/cm2, 150 μs pulse duration) to predominantly target endothelial cells or high energy (HE; 110 W/cm2, 150 μs pulse duration) to predominantly target hepatocytes. Both groups of UMGD-treated mice achieved persistent FVIII activity levels of ∼10% over 84 days post treatment; however, half of the HE-treated mice developed low-titer inhibitors while none of the LE mice did. Plasma transaminase levels and histological liver examinations revealed minimal transient liver damage that was lower in the LE group than in the HE group. These results indicate that UMGD can safely target LSECs with a lower-energy condition to achieve persistent FVIII gene expression, demonstrating that this novel technology is highly promising for therapeutic correction of HA.
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Affiliation(s)
| | | | - Meng-Ni Fan
- Seattle Children's Research Institute, Seattle, WA, USA
| | - Ting-Yen Chao
- Seattle Children's Research Institute, Seattle, WA, USA
| | - Chun-Yu Chen
- Seattle Children's Research Institute, Seattle, WA, USA
| | - Peter Kim
- Seattle Children's Research Institute, Seattle, WA, USA
| | | | - Xiaohe Cai
- Seattle Children's Research Institute, Seattle, WA, USA
| | | | - Carol H Miao
- Seattle Children's Research Institute, Seattle, WA, USA; University of Washington, Seattle, WA, USA.
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Romano L, Schütte L, van Hest R, Meijer K, Laros-van Gorkom B, Nieuwenhuizen L, Eikenboom J, Heubel-Moenen F, Uitslager N, Coppens M, Fijnvandraat K, Driessens M, Polinder S, Cnossen M, Leebeek F, Mathôt R, Kruip M, DAVID and SYMPHONY Consortium. Tachyphylaxis and reproducibility of desmopressin response in perioperative persons with nonsevere hemophilia A: implications for clinical practice. Res Pract Thromb Haemost 2024; 8:102367. [PMID: 38660455 PMCID: PMC11039391 DOI: 10.1016/j.rpth.2024.102367] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2023] [Revised: 01/25/2024] [Accepted: 02/11/2024] [Indexed: 04/26/2024] Open
Abstract
Background Desmopressin is frequently used perioperatively in persons with nonsevere hemophilia A. However, increase in factor (F)VIII:C after desmopressin use is interindividually highly variable. Tachyphylaxis has only been reported in test setting for persons with hemophilia A, with a remaining response of approximately 70% after a second dose compared with that after a first dose. Objectives To study tachyphylaxis of FVIII:C response after multiple administration(s) of desmopressin in perioperative persons with nonsevere hemophilia A. Methods We studied FVIII:C levels after desmopressin before (day 0 [D0]) and on days 1 (D1) and 2 (D2) after surgery in 26 patients of the DAVID and Little DAVID studies. We studied tachyphylaxis by comparing the responses at D1 and D2 with that at D0. We also assessed the reproducibility of the D0 response in comparison to an earlier performed desmopressin test. Results The median absolute FVIII:C increase was 0.50 IU/mL (0.35-0.74; n = 23) at D0, 0.21 IU/mL (0.14-0.28; n = 17) at D1, and 0.23 IU/mL (0.16-0.30; n = 11) at D2. The median percentage of FVIII increase after the second administration (D1) compared with the first (D0) was 42.9% (29.2%-52.5%; n = 17) and that of the third (D2) compared with the first (D0) was 36.4% (23.7%-46.9%; n = 11). The FVIII:C desmopressin response at D0 was comparable with the desmopressin test response in 74% of the patients. Conclusion Tachyphylaxis in the surgical setting was considerably more pronounced than previously reported, with FVIII:C at D1 and D2 of 36% to 43% of the initial response. Our results may have important implications for monitoring repeated desmopressin treatment when used perioperatively.
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Affiliation(s)
- L.G.R. Romano
- Department of Hematology, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - L.M. Schütte
- Department of Hematology, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - R.M. van Hest
- Department of Hospital Pharmacy and Clinical Pharmacology, Amsterdam University Medical Centers - University of Amsterdam, Amsterdam, The Netherlands
| | - K. Meijer
- Department of Hematology, University Medical Center Groningen, Groningen, The Netherlands
| | | | - L. Nieuwenhuizen
- Department of Hematology, Máxima Medical Center, Veldhoven, The Netherlands
| | - J. Eikenboom
- Department of Internal Medicine, Division of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands
| | - F.C.J.I. Heubel-Moenen
- Department of Hematology, Maastricht University Medical Center+, Maastricht, The Netherlands
| | - N. Uitslager
- Van Creveldkliniek, University Medical Center Utrecht, Utrecht, The Netherlands
| | - M. Coppens
- Department of Hematology, Amsterdam University Medical Centers - University of Amsterdam, Amsterdam, The Netherlands
- Pulmonary Hypertension & Thrombosis, Amsterdam Cardiovascular Sciences, Amsterdam, The Netherlands
| | - K. Fijnvandraat
- Department of Paediatric Hematology, Amsterdam University Medical Centers - University of Amsterdam, Emma Children's Hospital, Amsterdam, The Netherlands
- Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands
| | - M.H.E. Driessens
- Netherlands Hemophilia Patient Society, Nijkerk, The Netherlands
| | - S. Polinder
- Department of Public Health, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - M.H. Cnossen
- Department of Pediatric Hematology, Erasmus MC - Sophia Children's Hospital, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - F.W.G. Leebeek
- Department of Hematology, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - R.A.A. Mathôt
- Department of Hospital Pharmacy and Clinical Pharmacology, Amsterdam University Medical Centers - University of Amsterdam, Amsterdam, The Netherlands
| | - M.J.H.A. Kruip
- Department of Hematology, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - DAVID and SYMPHONY Consortium
- Department of Hematology, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
- Department of Hospital Pharmacy and Clinical Pharmacology, Amsterdam University Medical Centers - University of Amsterdam, Amsterdam, The Netherlands
- Department of Hematology, University Medical Center Groningen, Groningen, The Netherlands
- Department of Hematology, Radboud University Medical Center, Nijmegen, The Netherlands
- Department of Hematology, Máxima Medical Center, Veldhoven, The Netherlands
- Department of Internal Medicine, Division of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands
- Department of Hematology, Maastricht University Medical Center+, Maastricht, The Netherlands
- Van Creveldkliniek, University Medical Center Utrecht, Utrecht, The Netherlands
- Department of Hematology, Amsterdam University Medical Centers - University of Amsterdam, Amsterdam, The Netherlands
- Pulmonary Hypertension & Thrombosis, Amsterdam Cardiovascular Sciences, Amsterdam, The Netherlands
- Department of Paediatric Hematology, Amsterdam University Medical Centers - University of Amsterdam, Emma Children's Hospital, Amsterdam, The Netherlands
- Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands
- Netherlands Hemophilia Patient Society, Nijkerk, The Netherlands
- Department of Public Health, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
- Department of Pediatric Hematology, Erasmus MC - Sophia Children's Hospital, Erasmus University Medical Center, Rotterdam, The Netherlands
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Romano LGR, Schütte LM, van Hest RM, Meijer K, Laros-van Gorkom BAP, Nieuwenhuizen L, Eikenboom J, Heubel-Moenen FCJI, Uitslager N, Coppens M, Fijnvandraat K, Driessens MHE, Polinder S, Cnossen MH, Leebeek FWG, Mathôt RAA, Kruip MJHA. Peri-operative desmopressin combined with pharmacokinetic-guided factor VIII concentrate in non-severe haemophilia A patients. Haemophilia 2024; 30:355-366. [PMID: 38343113 DOI: 10.1111/hae.14946] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2023] [Revised: 12/10/2023] [Accepted: 01/13/2024] [Indexed: 03/14/2024]
Abstract
INTRODUCTION Non-severe haemophilia A patient can be treated with desmopressin or factor VIII (FVIII) concentrate. Combining both may reduce factor consumption, but its feasibility and safety has never been investigated. AIM We assessed the feasibility and safety of combination treatment in nonsevere haemophilia A patients. METHODS Non-severe, desmopressin responsive, haemophilia A patients were included in one of two studies investigating peri-operative combination treatment. In the single-arm DAVID study intravenous desmopressin (0.3 μg/kg) once-a-day was, after sampling, immediately followed by PK-guided FVIII concentrate, for maximally three consecutive days. The Little DAVID study was a randomized trial in patients undergoing a minor medical procedure, whom received either PK-guided combination treatment (intervention arm) or PK-guided FVIII concentrate only (standard arm) up to 2 days. Dose predictions were considered accurate if the absolute difference between predicted and measured FVIII:C was ≤0.2 IU/mL. RESULTS In total 32 patients (33 procedures) were included. In the DAVID study (n = 21), of the FVIII:C trough levels 73.7% (14/19) were predicted accurately on day 1 (D1), 76.5% (13/17) on D2. On D0, 61.9% (13/21) of peak FVIII:C levels predictions were accurate. In the Little DAVID study (n = 12), on D0 83.3% (5/6) FVIII:C peak levels for both study arms were predicted accurately. Combination treatment reduced preoperative FVIII concentrate use by 47% versus FVIII monotherapy. Desmopressin side effects were mild and transient. Two bleeds occurred, both despite FVIII:C > 1.00 IU/mL. CONCLUSION Peri-operative combination treatment with desmopressin and PK-guided FVIII concentrate dosing in nonsevere haemophilia A is feasible, safe and reduces FVIII consumption.
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Affiliation(s)
- Lorenzo G R Romano
- Department of Hematology, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Lisette M Schütte
- Department of Hematology, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Reinier M van Hest
- Department of Hospital Pharmacy and Clinical Pharmacology, Amsterdam University Medical Centers-University of Amsterdam, Amsterdam, The Netherlands
| | - Karina Meijer
- Department of Hematology, University Medical Center Groningen, Groningen, The Netherlands
| | | | | | - Jeroen Eikenboom
- Department of Internal Medicine, Division of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands
| | | | - Nanda Uitslager
- Van Creveldkliniek, University Medical Center Utrecht, Utrecht, The Netherlands
| | - Michiel Coppens
- Department of Hematology, Amsterdam University Medical Centers-University of Amsterdam, Amsterdam, The Netherlands
- Pulmonary Hypertension & Thrombosis, Amsterdam Cardiovascular Sciences, Amsterdam, The Netherlands
| | - Karin Fijnvandraat
- Department of Pediatric Hematology, Amsterdam University Medical Centers-University of Amsterdam, Emma Children's Hospital, Amsterdam, The Netherlands
- Department of Plasma Proteins, Sanquin Research, Amsterdam, The Netherlands
| | | | - Suzanne Polinder
- Department of Public Health, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Marjon H Cnossen
- Department of Pediatric Hematology and Oncology, Erasmus MC, Sophia Children's Hospital, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Frank W G Leebeek
- Department of Hematology, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Ron A A Mathôt
- Department of Hospital Pharmacy and Clinical Pharmacology, Amsterdam University Medical Centers-University of Amsterdam, Amsterdam, The Netherlands
| | - Marieke J H A Kruip
- Department of Hematology, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
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17
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Jamil MA, Al-Rifai R, Nuesgen N, Altmüller J, Oldenburg J, El-Maarri O. The role of microRNAs in defining LSECs cellular identity and in regulating F8 gene expression. Front Genet 2024; 15:1302685. [PMID: 38440189 PMCID: PMC10910020 DOI: 10.3389/fgene.2024.1302685] [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: 09/26/2023] [Accepted: 01/03/2024] [Indexed: 03/06/2024] Open
Abstract
Introduction: Coagulation Factor VIII (FVIII) plays a pivotal role in the coagulation cascade, and deficiencies in its levels, as seen in Hemophilia A, can lead to significant health implications. Liver sinusoidal endothelial cells (LSECs) are the main producers and contributors of FVIII in blood, a fact we have previously elucidated through mRNA expression profiling when comparing these cells to other endothelial cell types. Methods: Our current investigation focuses on small microRNAs, analyzing their distinct expression patterns across various endothelial cells and hepatocytes. Results: The outcome of this exploration underscores the discernible microRNAs expression differences that set LSECs apart from both hepatocytes (193 microRNAs at p < 0.05) and other endothelial cells (72 microRNAs at p < 0.05). Notably, the 134 and 35 overexpressed microRNAs in LSECs compared to hepatocytes and other endothelial cells, respectively, shed light on the unique functions of LSECs in the liver. Discussion: Our investigation identified a panel of 10 microRNAs (miR-429, miR-200b-3p, miR-200a-3p, miR-216b-5p, miR-1185-5p, miR-19b-3p, miR-192-5p, miR-122-5p, miR-30c-2-3p, and miR-30a-5p) that distinctly define LSEC identity. Furthermore, our scrutiny extended to microRNAs implicated in F8 regulation, revealing a subset (miR-122-5p, miR-214-3p, miR-204-3p, and miR-2682-5p) whose expression intricately correlates with F8 expression within LSECs. This microRNA cohort emerges as a crucial modulator of F8, both directly through suppression and indirect effects on established F8-related transcription factors. The above microRNAs emerged as potential targets for innovative therapies in Hemophilia A patients.
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Affiliation(s)
- Muhammad Ahmer Jamil
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
| | - Rawya Al-Rifai
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
| | - Nicole Nuesgen
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
| | - Janine Altmüller
- Cologne Center for Genomics (CCG), Faculty of Medicine, University Hospital Cologne, University of Cologne, Cologne, Germany
| | - Johannes Oldenburg
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
| | - Osman El-Maarri
- Institute of Experimental Hematology and Transfusion Medicine, University Hospital Bonn, Bonn, Germany
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18
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Onodera Y, Kobayashi J, Mitani S, Hosoda C, Banno K, Horie K, Okano T, Shimizu T, Shima M, Tatsumi K. Terminus-Selective Covalent Immobilization of Heparin on a Thermoresponsive Surface Using Click Chemistry for Efficient Binding of Basic Fibroblast Growth Factor. Macromol Biosci 2024; 24:e2300307. [PMID: 37774391 DOI: 10.1002/mabi.202300307] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/02/2023] [Revised: 09/02/2023] [Indexed: 10/01/2023]
Abstract
Cell therapy using endothelial cells (ECs) has great potential for the treatment of congenital disorders, such as hemophilia A. Cell sheet technology utilizing a thermoresponsive culture dish is a promising approach to efficiently transplant donor cells. In this study, a new method to prepare terminus-selective heparin-immobilized thermoresponsive culture surfaces is developed to facilitate the preparation of EC sheets. Alkynes are introduced to the reducing terminus of heparin via reductive amination. Cu-catalyzed azide-alkyne cycloaddition (CuAAC) facilitates efficient immobilization of the terminus of heparin on a thermoresponsive surface, resulting in a higher amount of immobilized heparin while preserving its function. Heparin-immobilized thermoresponsive surfaces prepared using CuAAC exhibit good adhesion to human endothelial colony-forming cells (ECFCs). In addition, upon further binding to basic fibroblast growth factor (bFGF) on heparin-immobilized surfaces, increased proliferation of ECFCs on the surface is observed. The confluent ECFC monolayer cultured on bFGF-bound heparin-immobilized thermoresponsive surfaces exhibits relatively high fibronectin accumulation and cell number and detaches at 22 °C while maintaining the sheet-like structure. Because heparin has an affinity for several types of bioactive molecules, the proposed method can be applied to facilitate efficient cultures and sheet formations of various cell types.
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Affiliation(s)
- Yu Onodera
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, 840 Shijocho, Kashihara, Nara, 634-8521, Japan
| | - Jun Kobayashi
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns, 8-1 Kawadacho, Shinjuku-ku, Tokyo, 162-8666, Japan
| | - Seiji Mitani
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, 840 Shijocho, Kashihara, Nara, 634-8521, Japan
| | - Chihiro Hosoda
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, 840 Shijocho, Kashihara, Nara, 634-8521, Japan
| | - Kimihiko Banno
- Department of Physiology II, Nara Medical University, 840 Shijocho, Kashihara, Nara, 634-8521, Japan
| | - Kyoji Horie
- Department of Physiology II, Nara Medical University, 840 Shijocho, Kashihara, Nara, 634-8521, Japan
| | - Teruo Okano
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns, 8-1 Kawadacho, Shinjuku-ku, Tokyo, 162-8666, Japan
| | - Tatsuya Shimizu
- Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, TWIns, 8-1 Kawadacho, Shinjuku-ku, Tokyo, 162-8666, Japan
| | - Midori Shima
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, 840 Shijocho, Kashihara, Nara, 634-8521, Japan
| | - Kohei Tatsumi
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, 840 Shijocho, Kashihara, Nara, 634-8521, Japan
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Mücke MM, Fong S, Foster GR, Lillicrap D, Miesbach W, Zeuzem S. Adeno-associated viruses for gene therapy - clinical implications and liver-related complications, a guide for hepatologists. J Hepatol 2024; 80:352-361. [PMID: 37890721 DOI: 10.1016/j.jhep.2023.10.029] [Citation(s) in RCA: 20] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/09/2023] [Revised: 10/13/2023] [Accepted: 10/17/2023] [Indexed: 10/29/2023]
Abstract
Gene therapy has garnered increasing interest over recent decades. Several therapies employing gene transfer mechanisms have been developed, and, of these, adeno-associated virus (AAV) vectors have demonstrated viability for use with in vivo gene therapy. Several AAV-based therapeutics have received regulatory approval in the last few years including those for retinal disease, spinal muscular atrophy or aromatic L-amino acid decarboxylase deficiency. Lately, with the introduction of novel liver-directed AAV vector-based therapeutics for the treatment of haemophilia A and B, gene therapy has attracted significant attention in the hepatology community, with the liver increasingly recognised as a target for gene therapy. However, the introduction of foreign DNA into hepatocytes is associated with a risk of hepatic reactions, with raised ALT (alanine aminotransferase) and AST (aspartate aminotransferase) being - so far - the most commonly reported side effects. The complete mechanisms underlying the ALT flairs remain to be determined and the long-term risks associated with these new treatments is not yet known. The liver community is increasingly being asked to support liver-directed gene therapy to mitigate potential liver associated harm. In this review, we focus on AAV vector-based gene therapy, shedding light on this promising technique and its remarkable success in haemophilia, with a special focus on hepatic complications and their management in daily clinical practice.
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Affiliation(s)
- Marcus Maximilian Mücke
- Department of Internal Medicine I, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany
| | - Sylvia Fong
- Research and Early Development, BioMarin Pharmaceutical. Inc, San Rafael, United States
| | - Graham R Foster
- Barts Liver Centre, Blizard Institute, QMUL, London, United Kingdom.
| | - David Lillicrap
- Department of Pathology and Molecular Medicine, Queen's University, Kingston, Canada
| | - Wolfgang Miesbach
- Department of Internal Medicine II, Haemostaseology and Haemophilia Centre, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany
| | - Stefan Zeuzem
- Department of Internal Medicine I, University Hospital Frankfurt, Goethe University, Frankfurt am Main, Germany
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20
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Ay C, Frenzel L, Pinachyan K, Le Quellec S. Gene therapy for haemophilia A and B, from basic principles to clinical implementation: An illustrated review. Haemophilia 2024; 30:5-15. [PMID: 38111029 DOI: 10.1111/hae.14907] [Citation(s) in RCA: 6] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2023] [Revised: 11/21/2023] [Accepted: 11/28/2023] [Indexed: 12/20/2023]
Abstract
INTRODUCTION With recent approval of the first two gene therapies for haemophilia A and B, educational materials about AAV-based gene therapy are needed by the haemophilia community for a better understanding of this novel therapeutic approach and helping healthcare providers and patients making personalized choices amongst an increasing array of therapeutic options. AIM To provide a comprehensive summary of the whole process of AAV-based gene therapy from basic principles to clinical implementation through an illustrated review. METHODS The authors, with expertise in and knowledge about gene therapy for haemophilia A and B, reviewed relevant articles from PubMed database and translated them into illustrations. RESULTS The review is divided into eight illustrated sections providing an overview of gene therapy for haemophilia A and B from haemophilia basics and current treatment landscape, principles of the AAV-based liver-directed gene therapy, through exploring the efficacy and safety results of published phase III clinical trials, current and future challenges, to implementation in clinical practice, including the hub and spoke models and the patient journey. CONCLUSION This illustrated review educates healthcare professionals on AAV-based gene therapy for haemophilia A and B enabling them to further educate their peers and their patients.
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Affiliation(s)
- Cihan Ay
- Department of Medicine I, Clinical Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria
| | - Laurent Frenzel
- Laboratory of Cellular and Molecular Mechanisms of Hematological Disorders and Therapeutical Implications, Labex GR-Ex, Imagine Institute, Inserm, Paris Descartes - Sorbonne Paris Cité University, Paris, France
- Hematology unit care, Hemophilia Center, Necker Hospital, Paris, France
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21
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Westwood LJ, Le Couteur DG, Hunt NJ, Cogger VC. Strategies to target and genetically modify the liver sinusoid. SINUSOIDAL CELLS IN LIVER DISEASES 2024:161-189. [DOI: 10.1016/b978-0-323-95262-0.00008-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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22
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Mitani S, Onodera Y, Hosoda C, Takabayashi Y, Sakata A, Shima M, Tatsumi K. Generation of functional liver sinusoidal endothelial-like cells from human bone marrow-derived mesenchymal stem cells. Regen Ther 2023; 24:274-281. [PMID: 37575681 PMCID: PMC10412721 DOI: 10.1016/j.reth.2023.07.006] [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: 06/26/2023] [Revised: 07/15/2023] [Accepted: 07/24/2023] [Indexed: 08/15/2023] Open
Abstract
Introduction Liver sinusoidal endothelial cells (LSECs) are specialized vascular endothelial cells that play an important role in the maintenance of biological homeostasis. However, the lack of versatile human LSECs has hindered research on LSECs and development of medical technologies for liver diseases including hemophilia A. In this study, we developed a technique to induce LSEC differentiation from human bone marrow-derived mesenchymal stem cells (BM-MSCs). Methods To induce LSECs from human BM-MSCs, cytokines and chemical compounds associated with signaling implicated in LSEC differentiation and liver development were screened. Then LSEC-related genes and proteins expression in the differentiated cells were analyzed by qPCR and flow cytometry analysis, respectively. LSEC-related functions of the differentiated cells were also examined. Results We found that the gene expression of LSEC markers, such as LYVE1, was considerably increased by culturing human BM-MSCs with bone morphogenetic protein 4, fibroblast growth factor 8b, transforming growth factor-β signal inhibitor, and cyclic AMP. Furthermore, the differentiated cells expressed LSEC marker proteins and clearly demonstrated LSEC-specific functions, such as the uptake of hyaluronic acid. Conclusions Our result indicate that the functional LSEC-like cells were successfully generated from human BM-MSCs using our established protocol.
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Affiliation(s)
- Seiji Mitani
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Yu Onodera
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Chihiro Hosoda
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Yoko Takabayashi
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Asuka Sakata
- Medicinal Biology of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Midori Shima
- Medicinal Biology of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
| | - Kohei Tatsumi
- Advanced Medical Science of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
- Medicinal Biology of Thrombosis and Hemostasis, Nara Medical University, Kashihara, Nara 634-8521, Japan
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23
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Randi AM, Jones D, Peghaire C, Arachchillage DJ. Mechanisms regulating heterogeneity of hemostatic gene expression in endothelial cells. J Thromb Haemost 2023; 21:3056-3066. [PMID: 37393001 DOI: 10.1016/j.jtha.2023.06.024] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/17/2023] [Revised: 05/30/2023] [Accepted: 06/20/2023] [Indexed: 07/03/2023]
Abstract
The hemostatic system involves an array of circulating coagulation factors that work in concert with platelets and the vascular endothelium to promote clotting in a space- and time-defined manner. Despite equal systemic exposure to circulating factors, bleeding and thrombotic diseases tend to prefer specific sites, suggesting an important role for local factors. This may be provided by endothelial heterogeneity. Endothelial cells differ not only between arteries, veins, and capillaries but also between microvascular beds from different organs, which present unique organotypic morphology and functional and molecular profiles. Accordingly, regulators of hemostasis are not uniformly distributed in the vasculature. The establishment and maintenance of endothelial diversity are orchestrated at the transcriptional level. Recent transcriptomic and epigenomic studies have provided a global picture of endothelial cell heterogeneity. In this review, we discuss the organotypic differences in the hemostatic profile of endothelial cells; we focus on 2 major endothelial regulators of hemostasis, namely von Willebrand factor and thrombomodulin, to provide examples of transcriptional mechanisms that control heterogeneity; finally, we consider some of the methodological challenges and opportunities for future studies.
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Affiliation(s)
- Anna M Randi
- National Heart and Lung Institute, Imperial College London, London, UK.
| | - Daisy Jones
- National Heart and Lung Institute, Imperial College London, London, UK
| | - Claire Peghaire
- University of Bordeaux, Unité Mixte de Recherche-1034 INSERM, Biology of Cardiovascular Diseases, Pessac, France
| | - Deepa J Arachchillage
- Centre for Haematology, Department of Immunology and Inflammation, Imperial College London, London, UK; Department of Haematology, Imperial College Healthcare NHS Trust, London, UK. https://twitter.com/DeepaArachchil1
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24
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De Wolf D, Singh K, Chuah MK, VandenDriessche T. Hemophilia Gene Therapy: The End of the Beginning? Hum Gene Ther 2023; 34:782-792. [PMID: 37672530 DOI: 10.1089/hum.2023.112] [Citation(s) in RCA: 21] [Impact Index Per Article: 10.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/08/2023] Open
Abstract
Extensive preclinical research over the past 30 years has culminated in the recent regulatory approval of several gene therapy products for hemophilia. Based on the efficacy and safety data in a recently conducted phase III clinical trial, Roctavian® (valoctocogene roxaparvovec), an adeno-associated viral (AAV5) vector expressing a B domain deleted factor VIII (FVIII) complementary DNA, was approved by the European Commission and Food and Drug Administration (FDA) for the treatment of patients with severe hemophilia A. In addition, Hemgenix® (etranacogene dezaparvovec) was also recently approved by the European Medicines Agency and the FDA for the treatment of patients with severe hemophilia B. This product is based on an AAV5 vector expressing a hyper-active factor IX (FIX) transgene (FIX-Padua) transgene. All AAV-based phase III clinical trials to date show a significant increase in FVIII or FIX levels in the majority of treated patients, consistent with a substantial decrease in bleeding episodes and a concomitant reduction in factor usage obviating the need for factor prophylaxis in most patients. However, significant interpatient variability remains that is not fully understood. Moreover, most patients encountered short-term asymptomatic liver inflammation that was treated by immune suppression with corticosteroids or other immune suppressants. In all phase III trials to date, FIX expression has appeared relatively more stable than FVIII, though individual patients also had prolonged FVIII expression. Whether lifelong expression of clotting factors can be realized after gene therapy requires longer follow-up studies. Further preclinical development of next-generation gene editing technologies offers new prospects for the development of a sustained cure for hemophilia, not only in adults, but ultimately in children with hemophilia too.
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Affiliation(s)
- Dries De Wolf
- Department of Gene Therapy and Regenerative Medicine, Vrije Universiteit Brussel, Brussels, Belgium
| | - Kshitiz Singh
- Department of Gene Therapy and Regenerative Medicine, Vrije Universiteit Brussel, Brussels, Belgium
| | - Marinee K Chuah
- Department of Gene Therapy and Regenerative Medicine, Vrije Universiteit Brussel, Brussels, Belgium
- Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium
| | - Thierry VandenDriessche
- Department of Gene Therapy and Regenerative Medicine, Vrije Universiteit Brussel, Brussels, Belgium
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Giuli L, Pallozzi M, Venturini G, Gasbarrini A, Ponziani FR, Santopaolo F. Molecular Mechanisms Underlying Vascular Liver Diseases: Focus on Thrombosis. Int J Mol Sci 2023; 24:12754. [PMID: 37628933 PMCID: PMC10454315 DOI: 10.3390/ijms241612754] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2023] [Revised: 08/07/2023] [Accepted: 08/11/2023] [Indexed: 08/27/2023] Open
Abstract
Vascular liver disorders (VLDs) comprise a wide spectrum of clinical-pathological entities that primarily affect the hepatic vascular system of both cirrhotic and non-cirrhotic patients. VLDs more frequently involve the portal and the hepatic veins, as well as liver sinusoids, resulting in an imbalance of liver homeostasis with serious consequences, such as the development of portal hypertension and liver fibrosis. Surprisingly, many VLDs are characterized by a prothrombotic phenotype. The molecular mechanisms that cause thrombosis in VLD are only partially explained by the alteration in the Virchow's triad (hypercoagulability, blood stasis, and endothelial damage) and nowadays their pathogenesis is incompletely described and understood. Studies about this topic have been hampered by the low incidence of VLDs in the general population and by the absence of suitable animal models. Recently, the role of coagulation imbalance in liver disease has been postulated as one of the main mechanisms linked to fibrogenesis, so a novel interest in vascular alterations of the liver has been renewed. This review provides a detailed analysis of the current knowledge of molecular mechanisms of VLD. We also focus on the promising role of anticoagulation as a strategy to prevent liver complications and to improve the outcome of these patients.
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Affiliation(s)
- Lucia Giuli
- Hepatology Unit, CEMAD Centro Malattie Dell’Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (L.G.); (M.P.); (G.V.); (F.R.P.); (F.S.)
| | - Maria Pallozzi
- Hepatology Unit, CEMAD Centro Malattie Dell’Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (L.G.); (M.P.); (G.V.); (F.R.P.); (F.S.)
| | - Giulia Venturini
- Hepatology Unit, CEMAD Centro Malattie Dell’Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (L.G.); (M.P.); (G.V.); (F.R.P.); (F.S.)
| | - Antonio Gasbarrini
- Hepatology Unit, CEMAD Centro Malattie Dell’Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (L.G.); (M.P.); (G.V.); (F.R.P.); (F.S.)
- Dipartimento di Medicina e Chirurgia Traslazionale, Università Cattolica del Sacro Cuore, 00168 Rome, Italy
| | - Francesca Romana Ponziani
- Hepatology Unit, CEMAD Centro Malattie Dell’Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (L.G.); (M.P.); (G.V.); (F.R.P.); (F.S.)
| | - Francesco Santopaolo
- Hepatology Unit, CEMAD Centro Malattie Dell’Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (L.G.); (M.P.); (G.V.); (F.R.P.); (F.S.)
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26
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Romano LG, van Vulpen LF, den Exter PL, Heubel-Moenen FC, Hooijmeijer HL, Coppens M, Fijnvandraat K, Schols SE, Ypma PF, Smit C, Driessens MH, Rosendaal FR, van der Bom JG, Gouw SC, Kruip MJ. Desmopressin in nonsevere hemophilia A: patient perspectives on use and efficacy. Res Pract Thromb Haemost 2023; 7:100281. [PMID: 37601028 PMCID: PMC10439392 DOI: 10.1016/j.rpth.2023.100281] [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: 01/10/2023] [Revised: 05/22/2023] [Accepted: 06/01/2023] [Indexed: 08/22/2023] Open
Abstract
Background Desmopressin increases plasma factor VIII and von Willebrand factor levels in persons with nonsevere hemophilia A. Patients' perspectives on desmopressin are relevant to increase and optimize its suboptimal use. However, patients' views on desmopressin are not reported. Objectives To evaluate the perspectives of persons with nonsevere hemophilia A on desmopressin use, barriers for its use, side effects, and their knowledge about desmopressin's efficacy and side effects. Methods Persons with nonsevere hemophilia A were included in a cross-sectional, national, multicenter study. Questionnaires were filled out by adult patients and children aged ≥12 years themselves. Caretakers filled out questionnaires for children aged <12 years. Results In total, 706 persons with nonsevere hemophilia A were included (544 mild, 162 moderate, [age range, 0-88 years]). Of 508 patients, 234 (50%) patients reported previous desmopressin use. Desmopressin was considered as at least moderately effective in 171 of 187 (90%) patients. Intranasal administration was the modality of choice for 138 of 182 (76%) patients. Flushing was the most reported side effect in 54 of 206 (26%) adults and 7 of 22 (32%) children. The most frequently reported advantage and disadvantage were the convenience of intranasal, out-of-hospital administration by 56% (126/227) and side effects in 18% (41/227), respectively. Patients' self-perceived knowledge was unsatisfactory or unknown in 28% (63/225). Conclusion Overall, desmopressin was most often used intranasally and considered effective, with flushing as the most common side effect. The most mentioned advantage was the convenience of intranasal administration and disadvantage was side effects. More information and education on desmopressin could answer unmet needs in patients with current or future desmopressin treatment.
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Affiliation(s)
- Lorenzo G.R. Romano
- Department of Hematology, Hemophilia Treatment Center, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
| | - Lize F.D. van Vulpen
- Center for Benign Hematology, Thrombosis and Hemostasis, Van Creveldkliniek, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands
| | - Paul L. den Exter
- Department of Internal Medicine, Division of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, The Netherlands
| | | | - Helene L. Hooijmeijer
- Department of Pediatrics, University Medical Center Groningen, Groningen, The Netherlands
| | - Michiel Coppens
- Department of Vascular Medicine, Amsterdam University Medical Center, Amsterdam, The Netherlands
- Pulmonary Hypertension & Thrombosis, Amsterdam Cardiovascular Sciences, Amsterdam, The Netherlands
| | - Karin Fijnvandraat
- Department of Pediatric Hematology, Amsterdam University Medical Center - Emma Children’s Hospital, University of Amsterdam, Amsterdam, The Netherlands
- Department of Molecular Cellular Hemostasis, Sanquin Research and Landsteiner Laboratory, Amsterdam, The Netherlands
| | - Saskia E.M. Schols
- Department of Hematology, Radboud University Medical Center and Hemophilia Treatment Center, Nijmegen-Eindhoven-Maastricht, Nijmegen, The Netherlands
| | - Paula F. Ypma
- Department of Hematology, Haga Hospital, The Hague, The Netherlands
| | - Cees Smit
- Netherlands Hemophilia Patient Society (NVHP), Nijkerk, The Netherlands
| | | | - Frits R. Rosendaal
- Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands
| | - Johanna G. van der Bom
- Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands
| | - Samantha C. Gouw
- Department of Pediatric Hematology, Amsterdam University Medical Center - Emma Children’s Hospital, University of Amsterdam, Amsterdam, The Netherlands
- Department of Clinical Epidemiology, Leiden University Medical Center, Leiden, The Netherlands
| | - Marieke J.H.A. Kruip
- Department of Hematology, Hemophilia Treatment Center, Erasmus MC, Erasmus University Medical Center, Rotterdam, The Netherlands
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Airola C, Pallozzi M, Cerrito L, Santopaolo F, Stella L, Gasbarrini A, Ponziani FR. Microvascular Thrombosis and Liver Fibrosis Progression: Mechanisms and Clinical Applications. Cells 2023; 12:1712. [PMID: 37443746 PMCID: PMC10341358 DOI: 10.3390/cells12131712] [Citation(s) in RCA: 6] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2023] [Revised: 06/14/2023] [Accepted: 06/16/2023] [Indexed: 07/15/2023] Open
Abstract
Fibrosis is an unavoidable consequence of chronic inflammation. Extracellular matrix deposition by fibroblasts, stimulated by multiple pathways, is the first step in the onset of chronic liver disease, and its propagation promotes liver dysfunction. At the same time, chronic liver disease is characterized by alterations in primary and secondary hemostasis but unlike previously thought, these changes are not associated with an increased risk of bleeding complications. In recent years, the role of coagulation imbalance has been postulated as one of the main mechanisms promoting hepatic fibrogenesis. In this review, we aim to investigate the function of microvascular thrombosis in the progression of liver disease and highlight the molecular and cellular networks linking hemostasis to fibrosis in this context. We analyze the predictive and prognostic role of coagulation products as biomarkers of liver decompensation (ascites, variceal hemorrhage, and hepatic encephalopathy) and liver-related mortality. Finally, we evaluate the current evidence on the application of antiplatelet and anticoagulant therapies for prophylaxis of hepatic decompensation or prevention of the progression of liver fibrosis.
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Affiliation(s)
- Carlo Airola
- Hepatology Unit, CEMAD Centro Malattie dell'Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (C.A.); (M.P.); (L.C.); (F.S.); (L.S.); (A.G.)
| | - Maria Pallozzi
- Hepatology Unit, CEMAD Centro Malattie dell'Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (C.A.); (M.P.); (L.C.); (F.S.); (L.S.); (A.G.)
| | - Lucia Cerrito
- Hepatology Unit, CEMAD Centro Malattie dell'Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (C.A.); (M.P.); (L.C.); (F.S.); (L.S.); (A.G.)
| | - Francesco Santopaolo
- Hepatology Unit, CEMAD Centro Malattie dell'Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (C.A.); (M.P.); (L.C.); (F.S.); (L.S.); (A.G.)
| | - Leonardo Stella
- Hepatology Unit, CEMAD Centro Malattie dell'Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (C.A.); (M.P.); (L.C.); (F.S.); (L.S.); (A.G.)
| | - Antonio Gasbarrini
- Hepatology Unit, CEMAD Centro Malattie dell'Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (C.A.); (M.P.); (L.C.); (F.S.); (L.S.); (A.G.)
- Dipartimento di Medicina e Chirurgia Traslazionale, Università Cattolica del Sacro Cuore, 00168 Rome, Italy
| | - Francesca Romana Ponziani
- Hepatology Unit, CEMAD Centro Malattie dell'Apparato Digerente, Medicina Interna e Gastroenterologia, Fondazione Policlinico Universitario Gemelli IRCCS, 00168 Rome, Italy; (C.A.); (M.P.); (L.C.); (F.S.); (L.S.); (A.G.)
- Dipartimento di Medicina e Chirurgia Traslazionale, Università Cattolica del Sacro Cuore, 00168 Rome, Italy
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Gong J, Yang R, Zhou M, Chang LJ. Improved intravenous lentiviral gene therapy based on endothelial-specific promoter-driven factor VIII expression for hemophilia A. Mol Med 2023; 29:74. [PMID: 37308845 DOI: 10.1186/s10020-023-00680-z] [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: 02/28/2023] [Accepted: 06/06/2023] [Indexed: 06/14/2023] Open
Abstract
BACKGROUND Hemophilia A (HA) is an X-linked monogenic disorder caused by deficiency of the factor VIII (FVIII) gene in the intrinsic coagulation cascade. The current protein replacement therapy (PRT) of HA has many limitations including short term effectiveness, high cost, and life-time treatment requirement. Gene therapy has become a promising treatment for HA. Orthotopic functional FVIII biosynthesis is critical to its coagulation activities. METHODS To investigate targeted FVIII expression, we developed a series of advanced lentiviral vectors (LVs) carrying either a universal promoter (EF1α) or a variety of tissue-specific promoters, including endothelial-specific (VEC), endothelial and epithelial-specific (KDR), and megakaryocyte-specific (Gp and ITGA) promoters. RESULTS To examine tissue specificity, the expression of a B-domain deleted human F8 (F8BDD) gene was tested in human endothelial and megakaryocytic cell lines. Functional assays demonstrated FVIII activities of LV-VEC-F8BDD and LV-ITGA-F8BDD in the therapeutic range in transduced endothelial and megakaryocytic cells, respectively. In F8 knockout mice (F8 KO mice, F8null mice), intravenous (iv) injection of LVs illustrated different degrees of phenotypic correction as well as anti-FVIII immune response for the different vectors. The iv delivery of LV-VEC-F8BDD and LV-Gp-F8BDD achieved 80% and 15% therapeutic FVIII activities over 180 days, respectively. Different from the other LV constructs, the LV-VEC-F8BDD displayed a low FVIII inhibitory response in the treated F8null mice. CONCLUSIONS The LV-VEC-F8BDD exhibited high LV packaging and delivery efficiencies, with endothelial specificity and low immunogenicity in the F8null mice, thus has a great potential for clinical applications.
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Affiliation(s)
- Jie Gong
- Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 611731, China
- School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610054, China
| | - Rui Yang
- School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610054, China
| | - Min Zhou
- Chengdu Women's and Children's Central Hospital, School of Medicine, University of Electronic Science and Technology of China, Chengdu, 611731, China
| | - Lung-Ji Chang
- School of Medicine, University of Electronic Science and Technology of China, Chengdu, 610054, China.
- Shenzhen Geno-Immune Medical Institute, 6 Yuexing 2nd Rd., 2nd Floor, Nanshan Dist., Shenzhen, 518057, Guangdong Province, China.
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29
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Tanaka KA, Terada R, Butt AL, Mazzeffi MA, McNeil JS. Factor VIII: A Dynamic Modulator of Hemostasis and Thrombosis in Trauma. Anesth Analg 2023; 136:894-904. [PMID: 37058725 DOI: 10.1213/ane.0000000000006356] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 04/16/2023]
Abstract
A trace amount of thrombin cleaves factor VIII (FVIII) into an active form (FVIIIa), which catalyzes FIXa-mediated activation of FX on the activated platelet surface. FVIII rapidly binds to von Willebrand factor (VWF) after secretion and becomes highly concentrated via VWF-platelet interaction at a site of endothelial inflammation or injury. Circulating levels of FVIII and VWF are influenced by age, blood type (nontype O > type O), and metabolic syndromes. In the latter, hypercoagulability is associated with chronic inflammation (known as thrombo-inflammation). In acute stress including trauma, releasable pools of FVIII/VWF are secreted from the Weibel-Palade bodies in the endothelium and then augment local platelet accumulation, thrombin generation, and leukocyte recruitment. Early systemic increases of FVIII/VWF (>200% of normal) levels in trauma result in a lower sensitivity of contact-activated clotting time (activated partial thromboplastin time [aPTT] or viscoelastic coagulation test [VCT]). However, in severely injured patients, multiple serine proteases (FXa plasmin and activated protein C [APC]) are locally activated and may be systemically released. Severity of traumatic injury correlates with prolonged aPTT and elevated activation markers of FXa, plasmin, and APC, culminating in a poor prognosis. In a subset of acute trauma patients, cryoprecipitate that contains fibrinogen, FVIII/VWF, and FXIII is theoretically advantageous over purified fibrinogen concentrate to promote stable clot formation, but comparative efficacy data are lacking. In chronic inflammation or subacute phase of trauma, elevated FVIII/VWF contributes to the pathogenesis of venous thrombosis by enhancing not only thrombin generation but also augmenting inflammatory functions. Future developments in coagulation monitoring specific to trauma patients, and targeted to enhancement or inhibition of FVIII/VWF, are likely to help clinicians gain better control of hemostasis and thromboprophylaxis. The main goal of this narrative is to review the physiological functions and regulations of FVIII and implications of FVIII in coagulation monitoring and thromboembolic complications in major trauma patients.
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Affiliation(s)
- Kenichi A Tanaka
- From the Department of Anesthesiology, University of Oklahoma College of Medicine, Oklahoma City, Oklahoma
| | - Rui Terada
- From the Department of Anesthesiology, University of Oklahoma College of Medicine, Oklahoma City, Oklahoma
| | - Amir L Butt
- From the Department of Anesthesiology, University of Oklahoma College of Medicine, Oklahoma City, Oklahoma
| | - Michael A Mazzeffi
- Department of Anesthesiology, University of Virginia School of Medicine, Charlottesville, Virginia
| | - John S McNeil
- Department of Anesthesiology, University of Virginia School of Medicine, Charlottesville, Virginia
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30
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Li J, Lu H, Zhang J, Li Y, Zhao Q. Comprehensive Approach to Assessment of Liver Viability During Normothermic Machine Perfusion. J Clin Transl Hepatol 2023; 11:466-479. [PMID: 36643041 PMCID: PMC9817053 DOI: 10.14218/jcth.2022.00130] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/20/2022] [Revised: 06/14/2022] [Accepted: 08/10/2022] [Indexed: 01/18/2023] Open
Abstract
Liver transplantation is the most effective treatment of advanced liver disease, and the use of extended criteria donor organs has broadened the source of available livers. Although normothermic machine perfusion (NMP) has become a useful tool in liver transplantation, there are no consistent criteria that can be used to evaluate the viability of livers during NMP. This review summarizes the criteria, indicators, and methods used to evaluate liver viability during NMP. The shape, appearance, and hemodynamics of the liver can be analyzed at a macroscopic level, while markers of liver injury, indicators of liver and bile duct function, and other relevant indicators can be evaluated by biochemical analysis. The liver can also be assessed by tissue biopsy at the microscopic level. Novel methods for assessment of liver viability are introduced. The limitations of evaluating liver viability during NMP are discussed and suggestions for future clinical practice are provided.
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Affiliation(s)
| | | | | | | | - Qiang Zhao
- Correspondence to: Qiang Zhao, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, Guangdong, China. ORCID: https://orcid.org/0000-0002-6369-1393. Tel: +86-15989196835, E-mail:
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31
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van Dievoet MA, Stephenne X, Rousseaux M, Lisman T, Hermans C, Deneys V. The use of prothrombin complex concentrate in chronic liver disease: A review of the literature. Transfus Med 2023. [PMID: 36941801 DOI: 10.1111/tme.12969] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2022] [Revised: 02/07/2023] [Accepted: 03/08/2023] [Indexed: 03/23/2023]
Abstract
Patients with chronic liver disease (CLD) and cirrhosis present a rebalanced hemostatic system in the three phases of haemostasis. This balance is however unstable and can easily tip towards bleeding or thrombosis. Management of both spontaneous bleeding and bleeding during invasive procedures remains a challenge in this patient population. Transfusion of blood products can result in circulatory overload and thereby worsen portal hypertension. As an alternative to fresh frozen plasma (FFP), prothrombin complex concentrates (PCC) may have merit in patients with liver disease because of their low volume. The impact of PCC in in-vitro spiking experiments of cirrhotic plasma is promising, but also warrants cautious use in light of thromboembolic risk. The majority of existing studies carried-out in CLD patients are retrospective or do not have an adequate control arm. A prospective study (the PROTON trial) was set up in 2013 to investigate the utility of PCC in patients undergoing liver transplantation. However, the study has never recruited the planned number of patients. Robust data on PCC safety in CLD is also required. The limited existing evidence does not seem to indicate an excessive thromboembolic risk. Currently, the utilisation of PCC in CLD cannot be routinely recommended but can provide an option for carefully selected cases in which other measures were not sufficient to control bleeding and after delicately weighing risks and benefits.
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Affiliation(s)
- Marie-Astrid van Dievoet
- Laboratory Department, Cliniques Universitaires Saint-Luc, Brussels, Belgium
- Pediatric Hepatology and Cellular Therapy Laboratory, Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain, Brussels, 1200, Belgium
| | - Xavier Stephenne
- Pediatric Hepatology and Cellular Therapy Laboratory, Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain, Brussels, 1200, Belgium
- Pediatric Gastroenterology and Hepatology Division, Cliniques universitaires Saint-Luc, Brussels, Belgium
| | - Madeleine Rousseaux
- Laboratory Department, Cliniques Universitaires Saint-Luc, Brussels, Belgium
| | - Ton Lisman
- Surgical Research Laboratory and Section of Hepatobiliary Surgery and Liver Transplantation, Department of Surgery, University of Groningen, University Medical Center Groningen, Groningen, The Netherlands
| | - Cedric Hermans
- Haemostasis and Thrombosis Unit/Haemophilia Treatment Centre/Division of Haematology, Cliniques Universitaires Saint-Luc, Brussels, Belgium
| | - Véronique Deneys
- Laboratory Department, Cliniques Universitaires Saint-Luc, Brussels, Belgium
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32
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Hu Z, Wu Y, Xiao R, Zhao J, Chen Y, Wu L, Zhou M, Liang D. Correction of F8 intron 1 inversion in hemophilia A patient-specific iPSCs by CRISPR/Cas9 mediated gene editing. Front Genet 2023; 14:1115831. [PMID: 36968612 PMCID: PMC10033665 DOI: 10.3389/fgene.2023.1115831] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2022] [Accepted: 02/27/2023] [Indexed: 03/11/2023] Open
Abstract
Introduction: Hemophilia A (HA) is the most common genetic bleeding disorder caused by mutations in the F8 gene encoding coagulation factor VIII (FVIII). As the second predominant pathogenic mutation in hemophilia A severe patients, F8 Intron one inversion (Inv1) completely splits the F8 gene into two parts and disrupts the F8 transcription, resulting in no FVIII protein production. The part which contains exon 2-exon 26 covers 98% of F8 coding region.Methods: We hypothesized that in situ genetic manipulation of F8 to add a promoter and exon one before the exon two could restore the F8 expression. The donor plasmid included human alpha 1-antitrypsin (hAAT) promoter, exon one and splicing donor site (SD) based on homology-mediated end joining (HMEJ) strategy was targeted addition in hemophilia A patient-derived induced pluripotent stem cell (HA-iPSCs) using CRISPR/Cas9. The iPSCs were differentiated into hepatocyte-like cells (HPLCs).Results: The hAAT promoter and exon one were targeted addition in HA-iPSCs with a high efficiency of 10.19% via HMEJ. The FVIII expression, secretion, and activity were detected in HPLCs derived from gene-targeted iPSCs.Discussion: Thus, we firstly rescued the 140 kb reversion mutation by gene addition of a 975 bp fragment in the HA-iPSCs with Inv1 mutation, providing a promising gene correction strategy for genetic disease with large sequence variants.
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Affiliation(s)
- Zhiqing Hu
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
| | - Yong Wu
- Shenzhen Baoan Women’s and Children’s Hospital, Jinan University, Shenzhen, China
| | - Rou Xiao
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
| | - Junya Zhao
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
| | - Yan Chen
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
| | - Lingqian Wu
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
| | - Miaojin Zhou
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
- *Correspondence: Miaojin Zhou, ; Desheng Liang,
| | - Desheng Liang
- Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, China
- *Correspondence: Miaojin Zhou, ; Desheng Liang,
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Di Minno G, Castaman G, De Cristofaro R, Brunetti-Pierri N, Pastore L, Castaldo G, Trama U, Di Minno M. Progress, and prospects in the therapeutic armamentarium of persons with congenital hemophilia. Defining the place for liver-directed gene therapy. Blood Rev 2023; 58:101011. [PMID: 36031462 DOI: 10.1016/j.blre.2022.101011] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2022] [Revised: 08/12/2022] [Accepted: 08/16/2022] [Indexed: 02/07/2023]
Abstract
In persons with congenital severe hemophilia A (HA) living in high-income countries, twice weekly intravenous infusions of extended half-life (EHL) factor VIII (FVIII) products, or weekly/biweekly/monthly subcutaneous injections of emicizumab are the gold standard home treatments to grant days without hurdles and limitations. Once weekly/twice monthly infusions of EHL Factor IX (FIX) products achieve the same target in severe hemophilia B (HB). Gene therapy, which is likely to be licensed for clinical use within 1-2 years, embodies a shift beyond these standards. At an individual patient level, a single functional gene transfer leads to a > 10-yr almost full correction of the hemostatic defect in HB and to a sustained (3-6-yrs) expression of FVIII sufficient to discontinue exogenous clotting factor administrations. At the doses employed, the limited liver toxicity of systemically infused recombinant adeno-associated virus (rAAV) vectors is documented by long-term (12-15 yrs) follow-ups, and pre-existing high-titer neutralizing antibodies to the AAV5 vector are no longer an exclusion criterion for effective transgene expression with this vector. A safe durable treatment that converts a challenging illness to a phenotypically curable disease, allows persons to feel virtually free from the fears and the obligations of hemophilia for years/decades. Along with patient organizations and health care professionals, communicating to government authorities and reimbursement agencies the liberating potential of this substantial innovation, and disseminating across the Centers updated information on benefits and risks of this strategy, will align expectations of different stakeholders and establish the notion of a potentially lifelong cure of hemophilia.
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Affiliation(s)
- Giovanni Di Minno
- Hub Center for Hemorrhagic and Thrombotic Disorders, Dep. of Clinical Medicine and Surgery, School of Medicine, Federico II University, Naples, Italy.
| | - Giancarlo Castaman
- Center for Bleeding Disorders and Coagulation, Careggi University Hospital, Florence, Italy.
| | - Raimondo De Cristofaro
- Center for Hemorrhagic and Thrombotic Diseases, Foundation University Hospital A. Gemelli IRCCS, Catholic University of the Sacred Heart, Rome, Italy.
| | - Nicola Brunetti-Pierri
- Telethon Institute of Genetics and Medicine, Pozzuoli, Italy; Dept of Translational Medicine, School of Medicine, Università degli Studi di Napoli "Federico II", Italy.
| | - Lucio Pastore
- CEINGE-Biotecnologie Avanzate, and Department of Molecular Medicine and Medical Biotechnology, School of Medicine, University of Naples Federico II, 80131 Naples, Italy.
| | - Giuseppe Castaldo
- CEINGE-Biotecnologie Avanzate, and Department of Molecular Medicine and Medical Biotechnology, School of Medicine, University of Naples Federico II, 80131 Naples, Italy.
| | - Ugo Trama
- Coordination of the Regional Health System, General Directorate for Health Protection, Naples, Italy.
| | - Matteo Di Minno
- Hub Center for Hemorrhagic and Thrombotic Disorders, Dep. of Clinical Medicine and Surgery, School of Medicine, Federico II University, Naples, Italy.
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Dietrich CG, Geier A, Merle U. Non-alcoholic fatty liver disease and COVID-19: Harmless companions or disease intensifier? World J Gastroenterol 2023; 29:367-377. [PMID: 36687116 PMCID: PMC9846932 DOI: 10.3748/wjg.v29.i2.367] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/01/2022] [Revised: 11/09/2022] [Accepted: 12/21/2022] [Indexed: 01/06/2023] Open
Abstract
The pandemics of coronavirus disease 2019 (COVID-19) and non-alcoholic fatty liver disease (NAFLD) coexist. Elevated liver function tests are frequent in COVID-19 and may influence liver damage in NAFLD, while preexisting liver damage from NAFLD may influence the course of COVID-19. However, the prognostic relevance of this interaction, though, is unclear. Obesity is a risk factor for the presence of NAFLD as well as a severe course of COVID-19. Cohort studies reveal conflicting results regarding the influence of NAFLD presence on COVID-19 illness severity. Striking molecular similarities of cytokine pathways in both diseases, including postacute sequelae of COVID-19, suggest common pathways for chronic low-activity inflammation. This review will summarize existing data regarding the interaction of both diseases and discuss possible mechanisms of the influence of one disease on the other.
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Affiliation(s)
| | - Andreas Geier
- Division of Hepatology, Department of Medicine II, University Hospital Wuerzburg, Wuerzburg 97080, Germany
| | - Uta Merle
- Department of Gastroenterology and Hepatology, University Hospital Heidelberg, Heidelberg 69120, Germany
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35
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Pipe SW, Arruda VR, Lange C, Kitchen S, Eichler H, Wadsworth S. Characteristics of BAY 2599023 in the Current Treatment Landscape of Hemophilia A Gene Therapy. Curr Gene Ther 2023; 23:81-95. [PMID: 36111754 DOI: 10.2174/1566523222666220914105729] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2022] [Revised: 08/12/2022] [Accepted: 08/13/2022] [Indexed: 11/22/2022]
Abstract
Hemophilia A, a single gene disorder leading to deficient Factor VIII (FVIII), is a suitable candidate for gene therapy. The aspiration is for single administration of a genetic therapy that would allow the production of endogenous FVIII sufficient to restore hemostasis and other biological processes. This would potentially result in reliable protection from bleeding and its associated physical and emotional impacts. Gene therapy offers the possibility of a clinically relevant improvement in disease phenotype and transformational improvement in quality of life, including an opportunity to engage in physical activities more confidently. Gene therapy products for hemophilia A in advanced clinical development use adeno-associated viral (AAV) vectors and a codon-optimized B-domain deleted FVIII transgene. However, the different AAV-based gene therapies have distinct design features, such as choice of vector capsid, enhancer and promoter regions, FVIII transgene sequence and manufacturing processes. These, in turn, impact patient eligibility, safety and efficacy. Ideally, gene therapy technology for hemophilia A should offer bleed protection, durable FVIII expression, broad eligibility and limited response variability between patients, and long-term safety. However, several limitations and challenges must be overcome. Here, we introduce the characteristics of the BAY 2599023 (AAVhu37.hFVIIIco, DTX 201) gene therapy product, including the low prevalence in the general population of anti-AAV-hu37 antibodies, as well as other gene therapy AAV products and approaches. We will examine how these can potentially meet the challenges of gene therapy, with the ultimate aim of improving the lives of patients with hemophilia A.
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Affiliation(s)
- Steven W Pipe
- Departments of Pediatrics and Pathology, University of Michigan, Ann Arbor, MI, USA
| | - Valder R Arruda
- Division of Hematology, Department of Pediatrics, Center for Cell and Molecular Therapeutics at Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
| | | | - Stephen Kitchen
- Sheffield Haemophilia and Thrombosis Centre, Sheffield Teaching Hospitals, Sheffield, UK
| | - Hermann Eichler
- Institute of Clinical Hemostaseology and Transfusion Medicine, Saarland University and University Hospital, Homburg/Saar, Germany
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Ramamurthy RM, Rodriguez M, Ainsworth HC, Shields J, Meares D, Bishop C, Farland A, Langefeld CD, Atala A, Doering CB, Spencer HT, Porada CD, Almeida-Porada G. Comparison of different gene addition strategies to modify placental derived-mesenchymal stromal cells to produce FVIII. Front Immunol 2022; 13:954984. [PMID: 36591257 PMCID: PMC9800010 DOI: 10.3389/fimmu.2022.954984] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/27/2022] [Accepted: 11/28/2022] [Indexed: 12/23/2022] Open
Abstract
Introduction Placenta-derived mesenchymal cells (PLCs) endogenously produce FVIII, which makes them ideally suited for cell-based fVIII gene delivery. We have previously reported that human PLCs can be efficiently modified with a lentiviral vector encoding a bioengineered, expression/secretion-optimized fVIII transgene (ET3) and durably produce clinically relevant levels of functionally active FVIII. The objective of the present study was to investigate whether CRISPR/Cas9 can be used to achieve location-specific insertion of a fVIII transgene into a genomic safe harbor, thereby eliminating the potential risks arising from the semi-random genomic integration inherent to lentiviral vectors. We hypothesized this approach would improve the safety of the PLC-based gene delivery platform and might also enhance the therapeutic effect by eliminating chromatin-related transgene silencing. Methods We used CRISPR/Cas9 to attempt to insert the bioengineered fVIII transgene "lcoET3" into the AAVS1 site of PLCs (CRISPR-lcoET3) and determined their subsequent levels of FVIII production, comparing results with this approach to those achieved using lentivector transduction (LV-lcoET3) and plasmid transfection (Plasmid-lcoET3). In addition, since liver-derived sinusoidal endothelial cells (LSECs) are the native site of FVIII production in the body, we also performed parallel studies in human (h)LSECs). Results PLCs and hLSECs can both be transduced (LV-lcoET3) with very high efficiency and produce high levels of biologically active FVIII. Surprisingly, both cell types were largely refractory to CRISPR/Cas9-mediated knockin of the lcoET3 fVIII transgene in the AAVS1 genome locus. However, successful insertion of an RFP reporter into this locus using an identical procedure suggests the failure to achieve knockin of the lcoET3 expression cassette at this site is likely a function of its large size. Importantly, using plasmids, alone or to introduce the CRISPR/Cas9 "machinery", resulted in dramatic upregulation of TLR 3, TLR 7, and BiP in PLCs, compromising their unique immune-inertness. Discussion Although we did not achieve our primary objective, our results validate the utility of both PLCs and hLSECs as cell-based delivery vehicles for a fVIII transgene, and they highlight the hurdles that remain to be overcome before primary human cells can be gene-edited with sufficient efficiency for use in cell-based gene therapy to treat HA.
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Affiliation(s)
- Ritu M. Ramamurthy
- Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, United States
| | - Martin Rodriguez
- Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, United States
| | - Hannah C. Ainsworth
- Department of Biostatistics and Data Sciences Wake Forest School of Medicine, Winston Salem, NC, United States
| | - Jordan Shields
- Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Emory University, Atlanta, GA, United States
| | - Diane Meares
- Department of Medicine, Hematology and Oncology, Wake Forest School of Medicine, Winston Salem, NC, United States
| | - Colin Bishop
- Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, United States
| | - Andrew Farland
- Department of Medicine, Hematology and Oncology, Wake Forest School of Medicine, Winston Salem, NC, United States
| | - Carl D. Langefeld
- Department of Biostatistics and Data Sciences Wake Forest School of Medicine, Winston Salem, NC, United States
| | - Anthony Atala
- Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, United States
| | - Christopher B. Doering
- Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Emory University, Atlanta, GA, United States
| | - H. Trent Spencer
- Department of Pediatrics, Aflac Cancer and Blood Disorders Center, Children’s Healthcare of Atlanta, Emory University, Atlanta, GA, United States
| | - Christopher D. Porada
- Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, United States
| | - Graça Almeida-Porada
- Fetal Research and Therapy Program, Wake Forest Institute for Regenerative Medicine, Winston Salem, NC, United States
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Hough C, Notley C, Mo A, Videl B, Lillicrap D. Heterogeneity and reciprocity of FVIII and VWF expression, and the response to shear stress in cultured human endothelial cells. J Thromb Haemost 2022; 20:2507-2518. [PMID: 35950488 PMCID: PMC9850489 DOI: 10.1111/jth.15841] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2022] [Revised: 07/06/2022] [Accepted: 07/18/2022] [Indexed: 01/25/2023]
Abstract
BACKGROUND Substantial phenotypic heterogeneity exists in endothelial cells and while much of this heterogeneity results from local microenvironments, epigenetic modifications also contribute. METHODS Cultured human umbilical vein endothelial cells, human pulmonary microvascular endothelial cells, human hepatic sinusoidal endothelial cells, human lymphatic endothelial cells (hLECs), and two different isolations of endothelial colony forming cells (ECFCs) were assessed for levels of factor VIII (FVIII) and von Willebrand factor (VWF) RNA and protein. The intracellular location and co-localization of both proteins was evaluated with immunofluorescence microscopy and stimulated release toof FVIII and VWF from Weibel-Palade bodies (WPBs) was evaluated. Changes in expression of FVIII and VWF RNA after hLECs and ECFCs were exposed to 2 or 15 dynes/cm2 of laminar shear stress were also assessed. RESULTS We observed considerable heterogeneity in FVIII and VWF expression among the endothelial cells. With the exception of hLECs, FVIII RNA and protein were barely detectable in any of the endothelial cells and a reciprocal relationship between levels of FVIII and VWF appears to exist. When FVIII and VWF are co-expressed, they do not consistently co-localize in the cytoplasm. However, in hLECs where significantly higher levels of FVIII are expressed, FVIII and VWF co-localize in WPBs and are released together when stimulated. Expression of both FVIII and VWF is markedly reduced when hLECs are exposed to higher or lower levels of laminar shear stress, while in ECFCs there is a minimal response for both proteins. CONCLUSIONS Variable levels of FVIII and VWF RNA and protein exist in a subset of cultured human endothelial cells. Higher levels of FVIII present in hLECs co-localize with VWF and are released together when exposed to a secretagogue.
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Affiliation(s)
- Christine Hough
- Department of Pathology and Molecular Medicine, Richardson Laboratory, Queen's University, Kingston, Ontario, Canada
| | - Colleen Notley
- Department of Pathology and Molecular Medicine, Richardson Laboratory, Queen's University, Kingston, Ontario, Canada
| | - Aomei Mo
- Department of Pathology and Molecular Medicine, Richardson Laboratory, Queen's University, Kingston, Ontario, Canada
| | - Barbara Videl
- Department of Pathology and Molecular Medicine, Richardson Laboratory, Queen's University, Kingston, Ontario, Canada
| | - David Lillicrap
- Department of Pathology and Molecular Medicine, Richardson Laboratory, Queen's University, Kingston, Ontario, Canada
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Castaman G, Di Minno G, De Cristofaro R, Peyvandi F. The Arrival of Gene Therapy for Patients with Hemophilia A. Int J Mol Sci 2022; 23:10228. [PMID: 36142153 PMCID: PMC9499514 DOI: 10.3390/ijms231810228] [Citation(s) in RCA: 16] [Impact Index Per Article: 5.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/25/2022] [Revised: 09/01/2022] [Accepted: 09/03/2022] [Indexed: 11/16/2022] Open
Abstract
Historically, the standard of care for hemophilia A has been intravenous administration of exogenous factor VIII (FVIII), either as prophylaxis or episodically. The development of emicizumab, a humanized bispecific monoclonal antibody mimicking activated FVIII, was a subsequent advance in treatment. However, both exogenous FVIII and emicizumab require repeated and lifelong administration, negatively impacting patient quality of life. A recent breakthrough has been the development of gene therapy. This allows a single intravenous treatment that could result in long-term expression of FVIII, maintenance of steady-state plasma concentrations, and minimization (or possibly elimination) of bleeding episodes for the recipient's lifetime. Several gene therapies have been assessed in clinical trials, with positive outcomes. Valoctocogene roxaparvovec (an adeno-associated viral 5-based therapy encoding human B domain-deleted FVIII) is expected to be the first approved gene therapy in European countries, including Italy, in 2022. Some novel challenges exist including refining patient selection criteria, managing patient expectations, further elucidation of the durability and variability of transgene expression and long-term safety, and the development of standardized 'hub and spoke' centers to optimize and monitor this innovative treatment. Gene therapy represents a paradigm shift, and may become a new reference standard for treating patients with hemophilia A.
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Affiliation(s)
- Giancarlo Castaman
- Center for Bleeding Disorders, Department of Oncology, Careggi University Hospital, Largo Brambilla 3, 50134 Firenze, Italy
| | - Giovanni Di Minno
- Regional Reference Centre for Hemo-Coagulation Diseases, Federico II University, Via S. Pansini 5, 80131 Naples, Italy
| | - Raimondo De Cristofaro
- Servizio Malattie Emorragiche e Trombotiche, Dipartimento di Medicina e Chirurgia Traslazionale, Fondazione Policlinico Universitraio “A. Gemelli” IRCCS, Università Cattolica S. Cuore Roma, Largo Francesco Vito, 1, 00168 Rome, Italy
| | - Flora Peyvandi
- Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, Fondazione Luigi Villa, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, 20122 Milan, Italy
- Department of Pathophysiology and Transplantation, Università degli Studi di Milano, Via Pace 9, 20122 Milan, Italy
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Pablo-Moreno JAD, Serrano LJ, Revuelta L, Sánchez MJ, Liras A. The Vascular Endothelium and Coagulation: Homeostasis, Disease, and Treatment, with a Focus on the Von Willebrand Factor and Factors VIII and V. Int J Mol Sci 2022; 23:ijms23158283. [PMID: 35955419 PMCID: PMC9425441 DOI: 10.3390/ijms23158283] [Citation(s) in RCA: 35] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2022] [Revised: 07/22/2022] [Accepted: 07/23/2022] [Indexed: 11/27/2022] Open
Abstract
The vascular endothelium has several important functions, including hemostasis. The homeostasis of hemostasis is based on a fine balance between procoagulant and anticoagulant proteins and between fibrinolytic and antifibrinolytic ones. Coagulopathies are characterized by a mutation-induced alteration of the function of certain coagulation factors or by a disturbed balance between the mechanisms responsible for regulating coagulation. Homeostatic therapies consist in replacement and nonreplacement treatments or in the administration of antifibrinolytic agents. Rebalancing products reestablish hemostasis by inhibiting natural anticoagulant pathways. These agents include monoclonal antibodies, such as concizumab and marstacimab, which target the tissue factor pathway inhibitor; interfering RNA therapies, such as fitusiran, which targets antithrombin III; and protease inhibitors, such as serpinPC, which targets active protein C. In cases of thrombophilia (deficiency of protein C, protein S, or factor V Leiden), treatment may consist in direct oral anticoagulants, replacement therapy (plasma or recombinant ADAMTS13) in cases of a congenital deficiency of ADAMTS13, or immunomodulators (prednisone) if the thrombophilia is autoimmune. Monoclonal-antibody-based anti-vWF immunotherapy (caplacizumab) is used in the context of severe thrombophilia, regardless of the cause of the disorder. In cases of disseminated intravascular coagulation, the treatment of choice consists in administration of antifibrinolytics, all-trans-retinoic acid, and recombinant soluble human thrombomodulin.
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Affiliation(s)
- Juan A. De Pablo-Moreno
- Department of Genetics, Physiology and Microbiology, School of Biology, Complutense University, 28040 Madrid, Spain; (J.A.D.P.-M.); (L.J.S.)
| | - Luis Javier Serrano
- Department of Genetics, Physiology and Microbiology, School of Biology, Complutense University, 28040 Madrid, Spain; (J.A.D.P.-M.); (L.J.S.)
| | - Luis Revuelta
- Department of Physiology, School of Veterinary Medicine, Complutense University of Madrid, 28040 Madrid, Spain;
| | - María José Sánchez
- Centro Andaluz de Biología del Desarrollo (CABD), Consejo Superior de Investigaciones Científicas (CSIC), Junta de Andalucía, Pablo de Olavide University, 41013 Sevilla, Spain;
| | - Antonio Liras
- Department of Genetics, Physiology and Microbiology, School of Biology, Complutense University, 28040 Madrid, Spain; (J.A.D.P.-M.); (L.J.S.)
- Correspondence:
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Barbon S, Stocco E, Rajendran S, Zardo L, Macchi V, Grandi C, Tagariello G, Porzionato A, Radossi P, De Caro R, Parnigotto PP. In Vitro Conditioning of Adipose-Derived Mesenchymal Stem Cells by the Endothelial Microenvironment: Modeling Cell Responsiveness towards Non-Genetic Correction of Haemophilia A. Int J Mol Sci 2022; 23:ijms23137282. [PMID: 35806285 PMCID: PMC9266329 DOI: 10.3390/ijms23137282] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2022] [Revised: 06/22/2022] [Accepted: 06/27/2022] [Indexed: 11/24/2022] Open
Abstract
In recent decades, the use of adult multipotent stem cells has paved the way for the identification of new therapeutic approaches for the treatment of monogenic diseases such as Haemophilia A. Being already studied for regenerative purposes, adipose-derived mesenchymal stem cells (Ad-MSCs) are still poorly considered for Haemophilia A cell therapy and their capacity to produce coagulation factor VIII (FVIII) after proper stimulation and without resorting to gene transfection. In this work, Ad-MSCs were in vitro conditioned towards the endothelial lineage, considered to be responsible for coagulation factor production. The cells were cultured in an inductive medium enriched with endothelial growth factors for up to 21 days. In addition to significantly responding to the chemotactic endothelial stimuli, the cell populations started to form capillary-like structures and up-regulated the expression of specific endothelial markers (CD34, PDGFRα, VEGFR2, VE-cadherin, CD31, and vWF). A dot blot protein study detected the presence of FVIII in culture media collected from both unstimulated and stimulated Ad-MSCs. Remarkably, the activated partial thromboplastin time test demonstrated that the clot formation was accelerated, and FVIII activity was enhanced when FVIII deficient plasma was mixed with culture media from the untreated/stimulated Ad-MSCs. Overall, the collected evidence supported a possible Ad-MSC contribution to HA correction via specific stimulation by the endothelial microenvironment and without any need for gene transfection.
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Affiliation(s)
- Silvia Barbon
- Section of Human Anatomy, Department of Neuroscience, University of Padova, 35121 Padova, Italy; (S.B.); (E.S.); (V.M.); (A.P.); (R.D.C.)
- Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling—TES, Onlus, 35030 Padova, Italy; (C.G.); (P.P.P.)
| | - Elena Stocco
- Section of Human Anatomy, Department of Neuroscience, University of Padova, 35121 Padova, Italy; (S.B.); (E.S.); (V.M.); (A.P.); (R.D.C.)
- Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling—TES, Onlus, 35030 Padova, Italy; (C.G.); (P.P.P.)
| | - Senthilkumar Rajendran
- Department of Surgery Oncology and Gastroenterology, University of Padova, 35124 Padova, Italy;
| | - Lorena Zardo
- Haematology and Haemophilia Centre, Castelfranco Veneto Hospital, 31033 Castelfranco Veneto, Italy; (L.Z.); (G.T.)
| | - Veronica Macchi
- Section of Human Anatomy, Department of Neuroscience, University of Padova, 35121 Padova, Italy; (S.B.); (E.S.); (V.M.); (A.P.); (R.D.C.)
| | - Claudio Grandi
- Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling—TES, Onlus, 35030 Padova, Italy; (C.G.); (P.P.P.)
| | - Giuseppe Tagariello
- Haematology and Haemophilia Centre, Castelfranco Veneto Hospital, 31033 Castelfranco Veneto, Italy; (L.Z.); (G.T.)
| | - Andrea Porzionato
- Section of Human Anatomy, Department of Neuroscience, University of Padova, 35121 Padova, Italy; (S.B.); (E.S.); (V.M.); (A.P.); (R.D.C.)
- Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling—TES, Onlus, 35030 Padova, Italy; (C.G.); (P.P.P.)
| | - Paolo Radossi
- Haematology and Haemophilia Centre, Castelfranco Veneto Hospital, 31033 Castelfranco Veneto, Italy; (L.Z.); (G.T.)
- Correspondence:
| | - Raffaele De Caro
- Section of Human Anatomy, Department of Neuroscience, University of Padova, 35121 Padova, Italy; (S.B.); (E.S.); (V.M.); (A.P.); (R.D.C.)
- Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling—TES, Onlus, 35030 Padova, Italy; (C.G.); (P.P.P.)
| | - Pier Paolo Parnigotto
- Foundation for Biology and Regenerative Medicine, Tissue Engineering and Signaling—TES, Onlus, 35030 Padova, Italy; (C.G.); (P.P.P.)
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Kaczmarek R. Gene therapy - are we ready now? Haemophilia 2022; 28 Suppl 4:35-43. [PMID: 35521736 PMCID: PMC9325484 DOI: 10.1111/hae.14530] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2021] [Revised: 02/20/2022] [Accepted: 02/21/2022] [Indexed: 01/19/2023]
Abstract
Introduction Haemophilia therapy has evolved from rudimentary transfusion‐based approaches to an unprecedented level of innovation with glimmers of functional cure brought by gene therapy. After decades of misfires, gene therapy has normalized factor (F)VIII and factor (F)IX levels in some individuals in the long term. Several clinical programmes testing adeno‐associated viral (AAV) vector gene therapy are approaching completion with imminent regulatory approvals. Discussion Phase 3 studies along with multiyear follow‐up in earlier phase investigations raised questions about efficacy as well as short‐ and long‐term safety, prompting a reappraisal of AAV vector gene therapy. Liver toxicities, albeit mostly low‐grade, occur in the first year in at least some individuals in all haemophilia A and B trials and are poorly understood. Extreme variability and unpredictability of outcome, as well as a slow decline in factor expression (seemingly unique to FVIII gene therapy), are vexing because immune responses to AAV vectors preclude repeat dosing, which could increase suboptimal or restore declining expression, while overexpression may result in phenotoxicity. The long‐term safety will need lifelong monitoring because AAV vectors, contrary to conventional wisdom, integrate into chromosomes at the rate that calls for vigilance. Conclusions AAV transduction and transgene expression engage the host immune system, cellular DNA processing, transcription and translation machineries in ways that have been only cursorily studied in the clinic. Delineating those mechanisms will be key to finding mitigants and solutions to the remaining problems, and including individuals who cannot avail of gene therapy at this time.
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Affiliation(s)
- Radoslaw Kaczmarek
- Coagulation Products Safety Supply and Access Committee, World Federation of Hemophilia, Montreal, Quebec, Canada.,Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, Indiana, USA
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Gage BK, Merlin S, Olgasi C, Follenzi A, Keller GM. Therapeutic correction of hemophilia A by transplantation of hPSC-derived liver sinusoidal endothelial cell progenitors. Cell Rep 2022; 39:110621. [PMID: 35385743 DOI: 10.1016/j.celrep.2022.110621] [Citation(s) in RCA: 10] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2021] [Revised: 01/27/2022] [Accepted: 03/14/2022] [Indexed: 01/19/2023] Open
Abstract
Liver sinusoidal endothelial cells (LSECs) form the predominant microvasculature in the liver where they carry out many functions including the secretion of coagulation factor VIII (FVIII). To investigate the early origins of this lineage, we develop an efficient and scalable protocol to produce human pluripotent stem cell (hPSC)-derived LSEC progenitors characterized as venous endothelial cells (VECs) from different mesoderm subpopulations. Using a sensitive and quantitative vascular competitive transplantation assay, we demonstrate that VECs generated from BMP4 and activin A-induced KDR+CD235a/b+ mesoderm are 50-fold more efficient at LSEC engraftment than venous cells from BMP4 and WNT-induced KDR+CD235a/b- mesoderm. When transplanted into immunocompromised hemophilia A mice (NSG-HA), these VECs engraft the liver, proliferate, and mature to functional LSECs that secrete bioactive FVIII capable of correcting the bleeding phenotype. Together, these findings highlight the importance of appropriate mesoderm induction for generating hPSC-derived LSECs capable of functioning in a preclinical model of hemophilia A.
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Affiliation(s)
- Blair K Gage
- McEwen Stem Cell Institute, University Health Network, Toronto, ON M5G1L7, Canada.
| | - Simone Merlin
- Department of Health Sciences, School of Medicine, University of Piemonte Orientale, 28100 Novara, Italy
| | - Cristina Olgasi
- Department of Health Sciences, School of Medicine, University of Piemonte Orientale, 28100 Novara, Italy
| | - Antonia Follenzi
- Department of Health Sciences, School of Medicine, University of Piemonte Orientale, 28100 Novara, Italy
| | - Gordon M Keller
- McEwen Stem Cell Institute, University Health Network, Toronto, ON M5G1L7, Canada; Department of Medical Biophysics, University of Toronto, Toronto, ON M5G1L7, Canada.
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Coagulation Factors Accumulate During Normothermic Liver Machine Perfusion Regardless of Donor Type and Severity of Ischemic Injury. Transplantation 2022; 106:510-518. [PMID: 33756546 DOI: 10.1097/tp.0000000000003763] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/25/2022]
Abstract
BACKGROUND Coagulation factors may inform on liver function during normothermic machine perfusion (NMP). We investigated whether graft ischemic injury impairs the accumulation of anticoagulation factors during NMP of porcine and human livers. METHODS Dynamics of FV, FVII, FVIII, FIX, and FX during NMP and their correlation with graft injury was investigated in porcine livers with minimal (no warm ischemia, n = 5) or severe injury (60 min warm ischemia, n = 5). Next, FV, FVIII, FIX, fibrinogen, and antithrombin were measured in 35 matched human liver NMPs from the COPE trial. Correlation of these factors with outcomes was explored. Livers were categorized in to 4 groups depending on donor type and posttransplant peak aspartate aminotransferase (AST) as surrogate of minimal (peak < 500 IU/L) or moderate injury (peak > 1000 IU/L). RESULTS Factor concentrations increased significantly during NMP regardless of severity of injury. In porcine livers, factor concentrations were 2- to 6-fold lower in severely injured grafts (all P < 0.05). All factors negatively correlated with AST (coefficient range: from -0.50 to -0.93; all P < 0.05) and lactate (range: from -0.51 to -0.67; all P < 0.05). In human livers, no difference in factor accumulation rates and no correlation with other markers were observed. One graft with primary nonfunction had low rate of factor accumulation. CONCLUSIONS Anticoagulation factors accumulate during NMP regardless of donor type and severity of injury. In pigs, severe ischemic injury resulted in significantly lower factor concentrations. In human livers with life-sustaining function, they do not correlate with hepatic injury. Whether low concentrations predict nonfunction in high-risk livers with severe injury requires further investigation.
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Son JS, Park CY, Lee G, Park JY, Kim HJ, Kim G, Chi KY, Woo DH, Han C, Kim SK, Park HJ, Kim DW, Kim JH. Therapeutic correction of hemophilia A using 2D endothelial cells and multicellular 3D organoids derived from CRISPR/Cas9-engineered patient iPSCs. Biomaterials 2022; 283:121429. [DOI: 10.1016/j.biomaterials.2022.121429] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/29/2021] [Revised: 01/26/2022] [Accepted: 02/17/2022] [Indexed: 01/19/2023]
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Tricot T, Verfaillie CM, Kumar M. Current Status and Challenges of Human Induced Pluripotent Stem Cell-Derived Liver Models in Drug Discovery. Cells 2022; 11:442. [PMID: 35159250 PMCID: PMC8834601 DOI: 10.3390/cells11030442] [Citation(s) in RCA: 11] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/06/2021] [Revised: 01/13/2022] [Accepted: 01/24/2022] [Indexed: 02/08/2023] Open
Abstract
The pharmaceutical industry is in high need of efficient and relevant in vitro liver models, which can be incorporated in their drug discovery pipelines to identify potential drugs and their toxicity profiles. Current liver models often rely on cancer cell lines or primary cells, which both have major limitations. However, the development of human induced pluripotent stem cells (hiPSCs) has created a new opportunity for liver disease modeling, drug discovery and liver toxicity research. hiPSCs can be differentiated to any cell of interest, which makes them good candidates for disease modeling and drug discovery. Moreover, hiPSCs, unlike primary cells, can be easily genome-edited, allowing the creation of reporter lines or isogenic controls for patient-derived hiPSCs. Unfortunately, even though liver progeny from hiPSCs has characteristics similar to their in vivo counterparts, the differentiation of iPSCs to fully mature progeny remains highly challenging and is a major obstacle for the full exploitation of these models by pharmaceutical industries. In this review, we discuss current liver-cell differentiation protocols and in vitro iPSC-based liver models that could be used for disease modeling and drug discovery. Furthermore, we will discuss the challenges that still need to be overcome to allow for the successful implementation of these models into pharmaceutical drug discovery platforms.
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Affiliation(s)
| | | | - Manoj Kumar
- Stem Cell Institute, KU Leuven, 3000 Leuven, Belgium; (T.T.); (C.M.V.)
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Gifre-Renom L, Daems M, Luttun A, Jones EAV. Organ-Specific Endothelial Cell Differentiation and Impact of Microenvironmental Cues on Endothelial Heterogeneity. Int J Mol Sci 2022; 23:ijms23031477. [PMID: 35163400 PMCID: PMC8836165 DOI: 10.3390/ijms23031477] [Citation(s) in RCA: 36] [Impact Index Per Article: 12.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/31/2021] [Revised: 01/14/2022] [Accepted: 01/19/2022] [Indexed: 02/04/2023] Open
Abstract
Endothelial cells throughout the body are heterogeneous, and this is tightly linked to the specific functions of organs and tissues. Heterogeneity is already determined from development onwards and ranges from arterial/venous specification to microvascular fate determination in organ-specific differentiation. Acknowledging the different phenotypes of endothelial cells and the implications of this diversity is key for the development of more specialized tissue engineering and vascular repair approaches. However, although novel technologies in transcriptomics and proteomics are facilitating the unraveling of vascular bed-specific endothelial cell signatures, still much research is based on the use of insufficiently specialized endothelial cells. Endothelial cells are not only heterogeneous, but their specialized phenotypes are also dynamic and adapt to changes in their microenvironment. During the last decades, strong collaborations between molecular biology, mechanobiology, and computational disciplines have led to a better understanding of how endothelial cells are modulated by their mechanical and biochemical contexts. Yet, because of the use of insufficiently specialized endothelial cells, there is still a huge lack of knowledge in how tissue-specific biomechanical factors determine organ-specific phenotypes. With this review, we want to put the focus on how organ-specific endothelial cell signatures are determined from development onwards and conditioned by their microenvironments during adulthood. We discuss the latest research performed on endothelial cells, pointing out the important implications of mimicking tissue-specific biomechanical cues in culture.
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Affiliation(s)
- Laia Gifre-Renom
- Centre for Molecular and Vascular Biology, Department of Cardiovascular Sciences, Katholieke Universiteit Leuven (KU Leuven), BE-3000 Leuven, Belgium; (L.G.-R.); (M.D.); (A.L.)
| | - Margo Daems
- Centre for Molecular and Vascular Biology, Department of Cardiovascular Sciences, Katholieke Universiteit Leuven (KU Leuven), BE-3000 Leuven, Belgium; (L.G.-R.); (M.D.); (A.L.)
| | - Aernout Luttun
- Centre for Molecular and Vascular Biology, Department of Cardiovascular Sciences, Katholieke Universiteit Leuven (KU Leuven), BE-3000 Leuven, Belgium; (L.G.-R.); (M.D.); (A.L.)
| | - Elizabeth A. V. Jones
- Centre for Molecular and Vascular Biology, Department of Cardiovascular Sciences, Katholieke Universiteit Leuven (KU Leuven), BE-3000 Leuven, Belgium; (L.G.-R.); (M.D.); (A.L.)
- Department of Cardiology, CARIM School for Cardiovascular Diseases, Maastricht University, 6229 ER Maastricht, The Netherlands
- Correspondence:
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Aoki H, Ogiwara K, Hasegawa M, Nogami K. Hemostatic rebalance in neonatal intrahepatic cholestasis with citrin deficiency. Pediatr Int 2022; 64:e14741. [PMID: 33851467 DOI: 10.1111/ped.14741] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/30/2021] [Revised: 03/16/2021] [Accepted: 04/09/2021] [Indexed: 01/10/2023]
Abstract
BACKGROUND Neonatal intrahepatic cholestasis with citrin deficiency (NICCD) results in coagulopathy due to decreased levels of vitamin (V)K-dependent clotting factors, similar to biliary atresia (BA). However, the involvement of VK-independent coagulant and anticoagulant factor(s) remains unknown. We examined relationships between coagulant and anticoagulant potential before and after nutritional treatment in NICCD. METHODS Three cases (aged 12, 21, and 45 days) with NICCD-associated coagulopathy were evaluated with standard coagulation/anticoagulation tests and comprehensive coagulation assays, rotational thromboelastometry, and protein C/protein S (PC/PS) pathway function assay (ThromboPath® ), before and after nutritional treatment. RESULTS In all cases, activated partial thromboplastin time and prothrombin time were significantly prolonged, which is associated with very low levels of VK-independent fibrinogen and antithrombin. The initiation of nutritional treatment of medium-chain triglycerides oil improved these levels within the normal range, although low levels of other clotting factors were modestly increased. Whole blood- rotational thromboelastometry analysis revealed near-normal coagulation potential, even before treatment, comparable to healthy adults, and supportive of their non-bleeding symptoms. The introduction of nutritional treatment had further improved comprehensive coagulation potential. The global PC/PS-pathway function assay demonstrated the absence of the features of this function associated with the pathogenesis of NICCD. Compared to BA, the plasma levels of fibrinogen and antithrombin in all cases were markedly low, whilst those after treatment improved, especially to similar level of BA. CONCLUSIONS Neonatal intrahepatic cholestasis with citrin deficiency has the characteristic of rebalancing hemostatic mechanisms associated with coagulant and anticoagulant potential involving low levels of fibrinogen and antithrombin, suggesting a pathophysiological coagulopathy distinct from BA.
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Affiliation(s)
- Hirosato Aoki
- Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
| | - Kenichi Ogiwara
- Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
| | - Mari Hasegawa
- Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
| | - Keiji Nogami
- Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan
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Koui Y, Himeno M, Mori Y, Nakano Y, Saijou E, Tanimizu N, Kamiya Y, Anzai H, Maeda N, Wang L, Yamada T, Sakai Y, Nakato R, Miyajima A, Kido T. Development of human iPSC-derived quiescent hepatic stellate cell-like cells for drug discovery and in vitro disease modeling. Stem Cell Reports 2021; 16:3050-3063. [PMID: 34861166 PMCID: PMC8693663 DOI: 10.1016/j.stemcr.2021.11.002] [Citation(s) in RCA: 17] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/22/2020] [Revised: 11/01/2021] [Accepted: 11/02/2021] [Indexed: 02/07/2023] Open
Abstract
Hepatic stellate cells (HSCs) play a central role in the progression of liver fibrosis by producing extracellular matrices. The development of drugs to suppress liver fibrosis has been hampered by the lack of human quiescent HSCs (qHSCs) and an appropriate in vitro model that faithfully recapitulates HSC activation. In the present study, we developed a culture system to generate qHSC-like cells from human-induced pluripotent stem cells (hiPSCs) that can be converted into activated HSCs in culture. To monitor the activation process, a red fluorescent protein (RFP) gene was inserted in hiPSCs downstream of the activation marker gene actin alpha 2 smooth muscle (ACTA2). Using qHSC-like cells derived from RFP reporter iPSCs, we screened a repurposing chemical library and identified therapeutic candidates that prevent liver fibrosis. Hence, hiPSC-derived qHSC-like cells will be a useful tool to study the mechanism of HSC activation and to identify therapeutic agents.
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Affiliation(s)
- Yuta Koui
- Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
| | - Misao Himeno
- Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
| | - Yusuke Mori
- Bio Science & Engineering Laboratory, Research & Development Management Headquarters, FUJIFILM Corporation, 577 Ushijima, Kaisei-machi, Ashigarakami-gun, Kanagawa 258-8577, Japan
| | - Yasuhiro Nakano
- Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
| | - Eiko Saijou
- Laboratory of Computational Genomics, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
| | - Naoki Tanimizu
- Department of Tissue Development and Regeneration, Research Institute for Frontier Medicine, Sapporo Medical University School of Medicine, S-1, W-17, Chuo-ku, Sapporo 060-8556, Japan
| | - Yoshiko Kamiya
- Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
| | - Hiroko Anzai
- Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
| | - Natsuki Maeda
- Department of Bioengineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
| | - Luyao Wang
- Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
| | - Tadanori Yamada
- Bio Science & Engineering Laboratory, Research & Development Management Headquarters, FUJIFILM Corporation, 577 Ushijima, Kaisei-machi, Ashigarakami-gun, Kanagawa 258-8577, Japan
| | - Yasuyuki Sakai
- Department of Chemical System Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan
| | - Ryuichiro Nakato
- Laboratory of Computational Genomics, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
| | - Atsushi Miyajima
- Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan
| | - Taketomo Kido
- Laboratory of Cell Growth and Differentiation, Institute for Quantitative Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan.
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Olgasi C, Borsotti C, Merlin S, Bergmann T, Bittorf P, Adewoye AB, Wragg N, Patterson K, Calabria A, Benedicenti F, Cucci A, Borchiellini A, Pollio B, Montini E, Mazzuca DM, Zierau M, Stolzing A, Toleikis P, Braspenning J, Follenzi A. Efficient and safe correction of hemophilia A by lentiviral vector-transduced BOECs in an implantable device. Mol Ther Methods Clin Dev 2021; 23:551-566. [PMID: 34853801 PMCID: PMC8606349 DOI: 10.1016/j.omtm.2021.10.015] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2021] [Revised: 10/06/2021] [Accepted: 10/29/2021] [Indexed: 11/18/2022]
Abstract
Hemophilia A (HA) is a rare bleeding disorder caused by deficiency/dysfunction of the FVIII protein. As current therapies based on frequent FVIII infusions are not a definitive cure, long-term expression of FVIII in endothelial cells through lentiviral vector (LV)-mediated gene transfer holds the promise of a one-time treatment. Thus, here we sought to determine whether LV-corrected blood outgrowth endothelial cells (BOECs) implanted through a prevascularized medical device (Cell Pouch) would rescue the bleeding phenotype of HA mice. To this end, BOECs from HA patients and healthy donors were isolated, expanded, and transduced with an LV carrying FVIII driven by an endothelial-specific promoter employing GMP-like procedures. FVIII-corrected HA BOECs were either directly transplanted into the peritoneal cavity or injected into a Cell Pouch implanted subcutaneously in NSG-HA mice. In both cases, FVIII secretion was sufficient to improve the mouse bleeding phenotype. Indeed, FVIII-corrected HA BOECs reached a relatively short-term clinically relevant engraftment being detected up to 16 weeks after transplantation, and their genomic integration profile did not show enrichment for oncogenes, confirming the process safety. Overall, this is the first preclinical study showing the safety and feasibility of transplantation of GMP-like produced LV-corrected BOECs within an implantable device for the long-term treatment of HA.
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Affiliation(s)
- Cristina Olgasi
- Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy
| | - Chiara Borsotti
- Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy
| | - Simone Merlin
- Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy
| | - Thorsten Bergmann
- Department of Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, 97082 Würzburg, Germany
| | - Patrick Bittorf
- Department of Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, 97082 Würzburg, Germany
| | - Adeolu Badi Adewoye
- Institute of Inflammation and Ageing, College of Medical and Dental Sciences, University of Birmingham, B15 2TT Birmingham, UK
| | - Nicholas Wragg
- Guy Hilton Research Centre, School of Pharmacy and Bioengineering, Keele University, Staffordshire, ST47QB Stoke-on-Trent, UK
| | | | | | | | - Alessia Cucci
- Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy
| | - Alessandra Borchiellini
- Haematology Unit Regional Center for Hemorrhagic and Thrombotic Diseases, City of Health and Science University Hospital of Molinette, 10126 Turin, Italy
| | - Berardino Pollio
- Immune-Haematology and Transfusion Medicine, Regina Margherita Children Hospital, City of Health and Science University Hospital of Molinette, 10126 Turin, Italy
| | | | | | - Martin Zierau
- IMS Integrierte Management Systeme e. K., 64646 Heppenheim, Germany
| | - Alexandra Stolzing
- Centre for Biological Engineering, School of Mechanical, Electrical and Manufacturing Engineering, Loughborough University, LE113TU Loughborough, UK
- SENS Research Foundation, Mountain View, CA 94041, USA
| | | | - Joris Braspenning
- Department of Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, 97082 Würzburg, Germany
| | - Antonia Follenzi
- Department of Health Sciences, University of Piemonte Orientale, 28100 Novara, Italy
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George LA. Hemophilia gene therapy: ushering in a new treatment paradigm? HEMATOLOGY. AMERICAN SOCIETY OF HEMATOLOGY. EDUCATION PROGRAM 2021; 2021:226-233. [PMID: 34889378 PMCID: PMC8877054 DOI: 10.1182/hematology.2021000254] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/04/2023]
Abstract
After 3 decades of clinical trials, repeated proof-of-concept success has now been demonstrated in hemophilia A and B gene therapy. Current clinical hemophilia gene therapy efforts are largely focused on the use of systemically administered recombinant adeno-associated viral (rAAV) vectors for F8 or F9 gene addition. With multiple ongoing trials, including licensing studies in hemophilia A and B, many are cautiously optimistic that the first AAV vectors will obtain regulatory approval within approximately 1 year. While supported optimism suggests that the goal of gene therapy to alter the paradigm of hemophilia care may soon be realized, a number of outstanding questions have emerged from clinical trial that are in need of answers to harness the full potential of gene therapy for hemophilia patients. This article reviews the use of AAV vector gene addition approaches for hemophilia A and B, focusing specifically on information to review in the process of obtaining informed consent for hemophilia patients prior to clinical trial enrollment or administering a licensed AAV vector.
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Affiliation(s)
- Lindsey A. George
- Correspondence Lindsey A. George, University of Pennsylvania School of Medicine, Colket Translational Research Bldg, Rm 5016, 3501 Civic Center Blvd, Philadelphia, PA 19104; e-mail:
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