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Shrestha A, Pillis DM, Felker S, Chi M, Wagner K, Gbotosho OT, Sieling J, Shadid M, Malik P. Preclinical efficacy of a modified gamma-globin lentivirus gene therapy in Berkeley sickle cell anemia mice and human xenograft models. Mol Ther Methods Clin Dev 2025; 33:101439. [PMID: 40176947 PMCID: PMC11964741 DOI: 10.1016/j.omtm.2025.101439] [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: 07/24/2024] [Accepted: 02/18/2025] [Indexed: 04/05/2025]
Abstract
We previously showed correction of sickle cell anemia (SCA) in mice utilizing a lentiviral vector (LV) expressing human γ-globin. Herein, we made a G16D mutation in the γ-globin gene to generate the G16D mutation (GbGM) LV to increase fetal hemoglobin formation. We also generated an insulated version of this LV, GbGMI, inserting a 36-bp insulator from the Foamy virus in the long terminal repeats of the LV. Preclinical batches of GbGM and GbGMI LV showed both were highly efficacious in correcting SCA in mice, with sustained gene transfer in primary transplanted SCA mice and high hematopoietic stem cell (HSC) transduction in colony-forming unit-spleen in secondary transplanted mice. CRISPR-mediated targeting of the proviruses into the LMO2 proto-oncogene showed remarkably reduced LMO2 activation by both insulated and uninsulated LV, compared to the SFFV γ-RV vector targeted to the same locus. We therefore used the GbGM LV to perform preclinical human CD34+ gene transfer. We assessed gene transfer and engraftment of human HSCs in two immunocompromised mouse models: persistent stable GbGM-transduced cell engraftment was comparable to that of untransduced cells with no detrimental effects on hematopoiesis up to 20 weeks post transplant. These robust preclinical studies in mouse and human HSCs allowed its translation into a clinical trial.
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Affiliation(s)
- Archana Shrestha
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center (CCHMC), Cincinnati, OH 45229, USA
- Aruvant Sciences, New York, NY 10036, USA
| | - Devin M. Pillis
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center (CCHMC), Cincinnati, OH 45229, USA
| | - Sydney Felker
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center (CCHMC), Cincinnati, OH 45229, USA
| | - Mengna Chi
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center (CCHMC), Cincinnati, OH 45229, USA
| | - Kimberly Wagner
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center (CCHMC), Cincinnati, OH 45229, USA
| | - Oluwabukola T. Gbotosho
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center (CCHMC), Cincinnati, OH 45229, USA
| | | | | | - Punam Malik
- Division of Experimental Hematology and Cancer Biology, Cincinnati Children’s Hospital Medical Center (CCHMC), Cincinnati, OH 45229, USA
- Division of Hematology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH 45229, USA
- Department of Pediatrics, University of Cincinnati, Cincinnati, OH 45229, USA
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Handgretinger R, Mezger M. An evaluation of exagamglogene autotemcel for the treatment of sickle cell disease and transfusion-dependent beta-thalassaemia. Expert Opin Biol Ther 2024; 24:883-888. [PMID: 39222044 DOI: 10.1080/14712598.2024.2399134] [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: 06/09/2024] [Accepted: 08/28/2024] [Indexed: 09/04/2024]
Abstract
INTRODUCTION Sickle cell disease is the most common hereditary hemoglobinopathy followed by beta-thalassemia. Until recently, allogeneic stem cell transplantation was the only curative approach. Based on the Crispr-Cas9-technology enabling targeting specific genes of interest, fetal hemoglobin which is normally shut-off after birth can be switched on and sufficient levels can alleviate symptoms in sickle cell disease and avoid transfusions in beta-thalassemia. Two first-in-human clinical studies in sickle cell disease and beta-thalassemia aiming to increase the level of fetal hemoglobin by using Crispr-Cas9 to modify autologous hematopoietic stem cells in patients aged 12-35 years have proved safety and efficacy and have shown promising clinical outcomes. AREAS COVERED The paper summarizes the outcome of the results of the two recently published clinical studies and compares them with the other available curative approaches. EXPERT OPINION Based on the currently available safety and efficacy data of the two published clinical results on gene therapy with Crispr-Cas9 modified autologous stem cells (exagamglogene autotemcel), it can be anticipated that this approach will add significantly to the therapeutic options for patients with sickle cell disease and beta-thalassemia and can be considered for all patients above 12 years of age independent of a suitable allogeneic stem cell donor.
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Affiliation(s)
- Rupert Handgretinger
- Department of Hematology/Oncology, Children's University Hospital, Tübingen, Germany
- Department of Hematology, Abu Dhabi Stem Cell Center and Yas Clinic Khalifa City, Abu Dhabi, UAE
- George and Jennifer Yeo Endowed Chair in Pediatric Oncology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore
| | - Markus Mezger
- Department of Hematology/Oncology, Children's University Hospital, Tübingen, Germany
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Cowan MJ, Yu J, Facchino J, Fraser-Browne C, Sanford U, Kawahara M, Dara J, Long-Boyle J, Oh J, Chan W, Chag S, Broderick L, Chellapandian D, Decaluwe H, Golski C, Hu D, Kuo CY, Miller HK, Petrovic A, Currier R, Hilton JF, Punwani D, Dvorak CC, Malech HL, McIvor RS, Puck JM. Lentiviral Gene Therapy for Artemis-Deficient SCID. N Engl J Med 2022; 387:2344-2355. [PMID: 36546626 PMCID: PMC9884487 DOI: 10.1056/nejmoa2206575] [Citation(s) in RCA: 41] [Impact Index Per Article: 13.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
BACKGROUND The DNA-repair enzyme Artemis is essential for rearrangement of T- and B-cell receptors. Mutations in DCLRE1C, which encodes Artemis, cause Artemis-deficient severe combined immunodeficiency (ART-SCID), which is poorly responsive to allogeneic hematopoietic-cell transplantation. METHODS We carried out a phase 1-2 clinical study of the transfusion of autologous CD34+ cells, transfected with a lentiviral vector containing DCLRE1C, in 10 infants with newly diagnosed ART-SCID. We followed them for a median of 31.2 months. RESULTS Marrow harvest, busulfan conditioning, and lentiviral-transduced CD34+ cell infusion produced the expected grade 3 or 4 adverse events. All the procedures met prespecified criteria for feasibility at 42 days after infusion. Gene-marked T cells were detected at 6 to 16 weeks after infusion in all the patients. Five of 6 patients who were followed for at least 24 months had T-cell immune reconstitution at a median of 12 months. The diversity of T-cell receptor β chains normalized by 6 to 12 months. Four patients who were followed for at least 24 months had sufficient B-cell numbers, IgM concentration, or IgM isohemagglutinin titers to permit discontinuation of IgG infusions. Three of these 4 patients had normal immunization responses, and the fourth has started immunizations. Vector insertion sites showed no evidence of clonal expansion. One patient who presented with cytomegalovirus infection received a second infusion of gene-corrected cells to achieve T-cell immunity sufficient for viral clearance. Autoimmune hemolytic anemia developed in 4 patients 4 to 11 months after infusion; this condition resolved after reconstitution of T-cell immunity. All 10 patients were healthy at the time of this report. CONCLUSIONS Infusion of lentiviral gene-corrected autologous CD34+ cells, preceded by pharmacologically targeted low-exposure busulfan, in infants with newly diagnosed ART-SCID resulted in genetically corrected and functional T and B cells. (Funded by the California Institute for Regenerative Medicine and the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT03538899.).
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Affiliation(s)
- Morton J Cowan
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Jason Yu
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Janelle Facchino
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Carol Fraser-Browne
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Ukina Sanford
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Misako Kawahara
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Jasmeen Dara
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Janel Long-Boyle
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Jess Oh
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Wendy Chan
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Shivali Chag
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Lori Broderick
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Deepak Chellapandian
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Hélène Decaluwe
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Catherine Golski
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Diana Hu
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Caroline Y Kuo
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Holly K Miller
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Aleksandra Petrovic
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Robert Currier
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Joan F Hilton
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Divya Punwani
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Christopher C Dvorak
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Harry L Malech
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - R Scott McIvor
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
| | - Jennifer M Puck
- From the Departments of Pediatrics (M.J.C., J.Y., J.F., C.F.-B., U.S., M.K., J.D., J.L.-B., W.C., S.C., R.C., C.C.D., J.M.P.) and Epidemiology and Biostatistics (J.F.H.), the Smith Cardiovascular Research Institute (M.J.C., J.M.P.), and the School of Pharmacy (J.L.-B.), University of California, San Francisco (UCSF), and UCSF Benioff Children's Hospital (M.J.C., J.F., J.D., J.L.-B., J.O., C.C.D., J.M.P.), San Francisco, the Department of Pediatrics, University of California, San Diego, and Rady Children's Hospital, San Diego (L.B.), and the Department of Pediatrics, UCLA Mattel Children's Hospital, Los Angeles (C.Y.K.) - all in California; the Department of Pediatrics, Johns Hopkins All Children's Hospital, St. Petersburg, FL (D.C.); the Department of Pediatrics, Sainte-Justine University Hospital Center, University of Montreal, Montreal (H.D.); Tuba City Regional Health Care, Tuba City (C.G., D.H.), and Phoenix Children's Hospital, Phoenix (H.K.M.) - both in Arizona; the Department of Pediatrics, University of Washington Seattle Children's Hospital, Seattle (A.P.); Clinical Development, Roche Diagnostics Solutions, Singapore (D.P.); the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD (H.L.M.); and the Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis (R.S.M.)
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4
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Suleman S, Payne A, Bowden J, Haque SA, Zahn M, Fawaz S, Khalifa MS, Jobling S, Hay D, Franco M, Fronza R, Wang W, Strobel-Freidekind O, Deichmann A, Takeuchi Y, Waddington SN, Gil-Farina I, Schmidt M, Themis M. HIV- 1 lentivirus tethering to the genome is associated with transcription factor binding sites found in genes that favour virus survival. Gene Ther 2022; 29:720-729. [PMID: 35513551 PMCID: PMC9750860 DOI: 10.1038/s41434-022-00335-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/07/2021] [Revised: 04/01/2022] [Accepted: 04/06/2022] [Indexed: 01/09/2023]
Abstract
Lentiviral vectors (LV) are attractive for permanent and effective gene therapy. However, integration into the host genome can cause insertional mutagenesis highlighting the importance of understanding of LV integration. Insertion site (IS) tethering is believed to involve cellular proteins such as PSIP1/LEDGF/p75, which binds to the virus pre-integration complexes (PICs) helping to target the virus genome. Transcription factors (TF) that bind both the vector LTR and host genome are also suspected influential to this. To determine the role of TF in the tethering process, we mapped predicted transcription factor binding sites (pTFBS) near to IS chosen by HIV-1 LV using a narrow 20 bp window in infected human induced pluripotent stem cells (iPSCs) and their hepatocyte-like cell (HLC) derivatives. We then aligned the pTFBS with these sequences found in the LTRs of native and self-inactivated LTRs. We found significant enrichment of these sequences for pTFBS essential to HIV-1 life cycle and virus survival. These same sites also appear in HIV-1 patient IS and in mice infected with HIV-1 based LV. This in silco data analysis suggests pTFBS present in the virus LTR and IS sites selected by HIV-1 LV are important to virus survival and propagation.
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Affiliation(s)
- Saqlain Suleman
- Department of Life Sciences, College of Health, Medicine & Life Sciences, Brunel University London, Uxbridge, UK
- Testavec Ltd, Queensgate House, Maidenhead, UK
| | - Annette Payne
- Testavec Ltd, Queensgate House, Maidenhead, UK
- Department of Computer Science, College of Engineering Design and Physical Sciences, Brunel University London, Uxbridge, UK
| | - Johnathan Bowden
- Department of Life Sciences, College of Health, Medicine & Life Sciences, Brunel University London, Uxbridge, UK
| | - Sharmin Al Haque
- Department of Life Sciences, College of Health, Medicine & Life Sciences, Brunel University London, Uxbridge, UK
| | - Marco Zahn
- Genewerk GmbH, Heidelberg, Germany
- University Heidelberg, Medical Faculty, Heidelberg, Germany
| | - Serena Fawaz
- Department of Life Sciences, College of Health, Medicine & Life Sciences, Brunel University London, Uxbridge, UK
| | - Mohammad S Khalifa
- Department of Life Sciences, College of Health, Medicine & Life Sciences, Brunel University London, Uxbridge, UK
| | - Susan Jobling
- Testavec Ltd, Queensgate House, Maidenhead, UK
- Institute of Environment, Health and Societies, College of Business, Arts and Social Sciences, Brunel University London, Uxbridge, UK
| | - David Hay
- Centre for Regenerative Medicine, The University of Edinburgh, Edinburgh, UK
| | | | | | - Wei Wang
- Genewerk GmbH, Heidelberg, Germany
| | | | | | - Yasuhiro Takeuchi
- Division of Infection and Immunity, University College London, London, UK
- Division of Advanced Therapies, National Institute for Biological Standards and Control, Potters Bar, UK
| | - Simon N Waddington
- Gene Transfer Technology, EGA Institute for Women's Health, University College London, London, UK
- MRC Antiviral Gene Therapy Research Unit, Faculty of Health Sciences, University of the Witswatersrand, Johannesburg, South Africa
| | | | - Manfred Schmidt
- Genewerk GmbH, Heidelberg, Germany
- Department of Translational Oncology, NCT and DKFZ, Heidelberg, Germany
| | - Michael Themis
- Department of Life Sciences, College of Health, Medicine & Life Sciences, Brunel University London, Uxbridge, UK.
- Division of Ecology and Evolution, Department of Life Sciences, Imperial College London, London, UK.
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5
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Dhorne-Pollet S, Fitzpatrick C, Da Costa B, Bourgon C, Eléouët JF, Meunier N, Burzio VA, Delmas B, Barrey E. Antisense oligonucleotides targeting ORF1b block replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Front Microbiol 2022; 13:915202. [PMID: 36386681 PMCID: PMC9644129 DOI: 10.3389/fmicb.2022.915202] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2022] [Accepted: 09/29/2022] [Indexed: 10/15/2023] Open
Abstract
The ongoing COVID-19 pandemic continues to pose a need for new and efficient therapeutic strategies. We explored antisense therapy using oligonucleotides targeting the severe acute respiratory syndrome coronavirus (SARS-CoV-2) genome. We predicted in silico four antisense oligonucleotides (ASO gapmers with 100% PTO linkages and LNA modifications at their 5' and 3'ends) targeting viral regions ORF1a, ORF1b, N and the 5'UTR of the SARS-CoV-2 genome. Efficiency of ASOs was tested by transfection in human ACE2-expressing HEK-293T cells and monkey VeroE6/TMPRSS2 cells infected with SARS-CoV-2. The ORF1b-targeting ASO was the most efficient, with a 71% reduction in the number of viral genome copies. N- and 5'UTR-targeting ASOs also significantly reduced viral replication by 55 and 63%, respectively, compared to non-related control ASO (ASO-C). Viral titration revealed a significant decrease in SARS-CoV-2 multiplication both in culture media and in cells. These results show that anti-ORF1b ASO can specifically reduce SARS-CoV-2 genome replication in vitro in two different cell infection models. The present study presents proof-of concept of antisense oligonucleotide technology as a promising therapeutic strategy for COVID-19.
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Affiliation(s)
| | - Christopher Fitzpatrick
- INRAE, AgroParisTech, GABI, Université Paris-Saclay, Jouy-en-Josas, France
- Universidad Andrés Bello, Santiago, Chile
| | - Bruno Da Costa
- INRAE, UMR VIM, Université Paris-Saclay, Jouy-en-Josas, France
| | - Clara Bourgon
- INRAE, UMR VIM, Université Paris-Saclay, Jouy-en-Josas, France
| | | | - Nicolas Meunier
- INRAE, UMR VIM, Université Paris-Saclay, Jouy-en-Josas, France
| | - Verónica A. Burzio
- Universidad Andrés Bello, Santiago, Chile
- Centro Científico y Tecnológico de Excelencia Ciencia, Vida/Andes Biotechnologies SpA, Santiago, Chile
| | - Bernard Delmas
- INRAE, UMR VIM, Université Paris-Saclay, Jouy-en-Josas, France
| | - Eric Barrey
- INRAE, AgroParisTech, GABI, Université Paris-Saclay, Jouy-en-Josas, France
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6
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Pancreatic Transdifferentiation Using β-Cell Transcription Factors for Type 1 Diabetes Treatment. Cells 2022; 11:cells11142145. [PMID: 35883588 PMCID: PMC9315695 DOI: 10.3390/cells11142145] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/16/2022] [Revised: 07/01/2022] [Accepted: 07/06/2022] [Indexed: 01/25/2023] Open
Abstract
Type 1 diabetes is a chronic illness in which the native beta (β)-cell population responsible for insulin release has been the subject of autoimmune destruction. This condition requires patients to frequently measure their blood glucose concentration and administer multiple daily exogenous insulin injections accordingly. Current treatments fail to effectively treat the disease without significant side effects, and this has led to the exploration of different approaches for its treatment. Gene therapy and the use of viral vectors has been explored extensively and has been successful in treating a range of diseases. The use of viral vectors to deliver β-cell transcription factors has been researched in the context of type 1 diabetes to induce the pancreatic transdifferentiation of cells to replace the β-cell population destroyed in patients. Studies have used various combinations of pancreatic and β-cell transcription factors in order to induce pancreatic transdifferentiation and have achieved varying levels of success. This review will outline why pancreatic transcription factors have been utilised and how their application can allow the development of insulin-producing cells from non β-cells and potentially act as a cure for type 1 diabetes.
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Magrin E, Semeraro M, Hebert N, Joseph L, Magnani A, Chalumeau A, Gabrion A, Roudaut C, Marouene J, Lefrere F, Diana JS, Denis A, Neven B, Funck-Brentano I, Negre O, Renolleau S, Brousse V, Kiger L, Touzot F, Poirot C, Bourget P, El Nemer W, Blanche S, Tréluyer JM, Asmal M, Walls C, Beuzard Y, Schmidt M, Hacein-Bey-Abina S, Asnafi V, Guichard I, Poirée M, Monpoux F, Touraine P, Brouzes C, de Montalembert M, Payen E, Six E, Ribeil JA, Miccio A, Bartolucci P, Leboulch P, Cavazzana M. Long-term outcomes of lentiviral gene therapy for the β-hemoglobinopathies: the HGB-205 trial. Nat Med 2022; 28:81-88. [PMID: 35075288 DOI: 10.1038/s41591-021-01650-w] [Citation(s) in RCA: 60] [Impact Index Per Article: 20.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/08/2020] [Accepted: 11/30/2021] [Indexed: 01/19/2023]
Abstract
Sickle cell disease (SCD) and transfusion-dependent β-thalassemia (TDT) are the most prevalent monogenic disorders worldwide. Trial HGB-205 ( NCT02151526 ) aimed at evaluating gene therapy by autologous CD34+ cells transduced ex vivo with lentiviral vector BB305 that encodes the anti-sickling βA-T87Q-globin expressed in the erythroid lineage. HGB-205 is a phase 1/2, open-label, single-arm, non-randomized interventional study of 2-year duration at a single center, followed by observation in long-term follow-up studies LTF-303 ( NCT02633943 ) and LTF-307 ( NCT04628585 ) for TDT and SCD, respectively. Inclusion and exclusion criteria were similar to those for allogeneic transplantation but restricted to patients lacking geno-identical, histocompatible donors. Four patients with TDT and three patients with SCD, ages 13-21 years, were treated after busulfan myeloablation 4.6-7.9 years ago, with a median follow-up of 4.5 years. Key primary endpoints included mortality, engraftment, replication-competent lentivirus and clonal dominance. No adverse events related to the drug product were observed. Clinical remission and remediation of biological hallmarks of the disease have been sustained in two of the three patients with SCD, and frequency of transfusions was reduced in the third. The patients with TDT are all transfusion free with improvement of dyserythropoiesis and iron overload.
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Affiliation(s)
- Elisa Magrin
- Biotherapy Department, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France.,Centre d'Investigation Clinique-Biothérapie, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Michaela Semeraro
- Centre d'Investigation Clinique-Unité de Recherche Clinique, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France.,Université de Paris, Paris, France
| | - Nicolas Hebert
- Univ Paris Est Creteil, INSERM, EFS, IMRB, Créteil, France.,Hôpital Henri Mondor, Assistance Publique-Hôpitaux de Paris, Université Paris-Est Créteil, Créteil, France
| | - Laure Joseph
- Biotherapy Department, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Alessandra Magnani
- Biotherapy Department, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France.,Centre d'Investigation Clinique-Biothérapie, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Anne Chalumeau
- IMAGINE Institute, Université de Paris, Sorbonne Paris Cité, Paris, France
| | - Aurélie Gabrion
- Biotherapy Department, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France.,Centre d'Investigation Clinique-Biothérapie, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Cécile Roudaut
- Biotherapy Department, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France.,Centre d'Investigation Clinique-Biothérapie, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Jouda Marouene
- Centre d'Investigation Clinique-Unité de Recherche Clinique, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Francois Lefrere
- Biotherapy Department, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Jean-Sebastien Diana
- Biotherapy Department, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Adeline Denis
- IMAGINE Institute, Université de Paris, Sorbonne Paris Cité, Paris, France
| | - Bénédicte Neven
- Pediatric Immunology and Hematology Department, Hôpital Necker Enfants-Malades, Paris, France
| | - Isabelle Funck-Brentano
- Pediatric Immunology and Hematology Department, Hôpital Necker Enfants-Malades, Paris, France
| | - Olivier Negre
- CEA, INSERM, Université Paris-Saclay, Division of Innovative Therapies, Institut François Jacob, Fontenay aux Roses, France.,Bluebird Bio, Inc., Cambridge, MA, USA
| | - Sylvain Renolleau
- Pediatric Intensive Care Unit, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Valentine Brousse
- Department of General Pediatrics and Pediatric Infectious Diseases, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Laurent Kiger
- Univ Paris Est Creteil, INSERM, EFS, IMRB, Créteil, France
| | - Fabien Touzot
- Biotherapy Department, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France.,Centre d'Investigation Clinique-Biothérapie, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Catherine Poirot
- Department of Hematology, Fertility Preservation, Hôpital Saint Louis, Paris, France.,Sorbonne Université, Paris, France
| | - Philippe Bourget
- Pharmacy Department, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Wassim El Nemer
- Institut National de la Transfusion Sanguine (INTS), Paris, France
| | - Stéphane Blanche
- Pediatric Immunology and Hematology Department, Hôpital Necker Enfants-Malades, Paris, France
| | - Jean-Marc Tréluyer
- Centre d'Investigation Clinique-Unité de Recherche Clinique, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France.,Université de Paris, Paris, France
| | | | | | - Yves Beuzard
- Univ Paris Est Creteil, INSERM, EFS, IMRB, Créteil, France.,CEA, INSERM, Université Paris-Saclay, Division of Innovative Therapies, Institut François Jacob, Fontenay aux Roses, France
| | | | - Salima Hacein-Bey-Abina
- Biotherapy Department, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France.,Centre d'Investigation Clinique-Biothérapie, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Vahid Asnafi
- Université de Paris, Institut Necker-Enfants Malades, INSERM U1151, Assistance Publique-Hôpitaux de Paris, Hôpital Necker Enfants-Malades, Paris, France
| | - Isabelle Guichard
- Service de Médecine Interne, Hôpital Nord, CHU de Saint-Étienne, Saint-Étienne, Paris, France
| | - Maryline Poirée
- Department of Pediatric Hematology-Oncology, Centre Hospitalier Universitaire Lenval, Nice, France
| | - Fabrice Monpoux
- Unité d'Hémato-Oncologie Infantile. Hôpital de l'Archet 2, Nice, France
| | - Philippe Touraine
- Department of Endocrinology and Reproductive Medicine, Assistance Publique-Hopitaux de Paris, La Pitié-Salpêtrière, and Sorbonne University, Pierre et Marie Curie School of Medicine, Paris, France
| | - Chantal Brouzes
- Laboratory of Onco-hematology, Hôpital Necker-Enfants Malades, Paris, France
| | - Mariane de Montalembert
- Department of General Pediatrics and Pediatric Infectious Diseases, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France
| | - Emmanuel Payen
- CEA, INSERM, Université Paris-Saclay, Division of Innovative Therapies, Institut François Jacob, Fontenay aux Roses, France
| | - Emmanuelle Six
- IMAGINE Institute, Université de Paris, Sorbonne Paris Cité, Paris, France
| | - Jean-Antoine Ribeil
- Biotherapy Department, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France.,Centre d'Investigation Clinique-Biothérapie, Hôpital Universitaire Necker Enfants-Malades, GH Paris Centre, Paris, France.,Bluebird Bio, Inc., Cambridge, MA, USA
| | - Annarita Miccio
- IMAGINE Institute, Université de Paris, Sorbonne Paris Cité, Paris, France
| | - Pablo Bartolucci
- Univ Paris Est Creteil, INSERM, EFS, IMRB, Créteil, France.,Hôpital Henri Mondor, Assistance Publique-Hôpitaux de Paris, Université Paris-Est Créteil, Créteil, France
| | - Philippe Leboulch
- CEA, INSERM, Université Paris-Saclay, Division of Innovative Therapies, Institut François Jacob, Fontenay aux Roses, France. .,Genetics Division, Department of Medicine, Brigham & Women's Hospital and Harvard Medical School, Boston, MA, USA.
| | - Marina Cavazzana
- Université de Paris, Paris, France. .,IMAGINE Institute, Université de Paris, Sorbonne Paris Cité, Paris, France. .,Biotherapy Department and Clinical Investigation Center, Assistance Publique Hopitaux de Paris, INSERM, Paris, France.
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8
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Thuret I, Ruggeri A, Angelucci E, Chabannon C. OUP accepted manuscript. Stem Cells Transl Med 2022; 11:407-414. [PMID: 35267028 PMCID: PMC9052404 DOI: 10.1093/stcltm/szac007] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/04/2021] [Accepted: 12/13/2021] [Indexed: 01/19/2023] Open
Abstract
Beta-thalassemia is one of the most common monogenic disorders. Standard treatment of the most severe forms, i.e., transfusion-dependent thalassemia (TDT) with long-term transfusion and iron chelation, represents a considerable medical, psychological, and economic burden. Allogeneic hematopoietic stem cell transplantation from an HLA-identical donor is a curative treatment with excellent results in children. Recently, several gene therapy approaches were evaluated in academia or industry-sponsored clinical trials as alternative curative options for children and young adults without an HLA-identical donor. Gene therapy by addition of a functional beta-globin gene using self-inactivating lentiviral vectors in autologous stem cells resulted in transfusion independence for a majority of TDT patients across different age groups and genotypes, with a current follow-up of multiple years. More recently, promising results were reported in TDT patients treated with autologous hematopoietic stem cells edited with the clustered regularly interspaced short palindromic repeats-Cas9 technology targeting erythroid BCL11A expression, a key regulator of the normal switch from fetal to adult globin production. Patients achieved high levels of fetal hemoglobin allowing for discontinuation of transfusions. Despite remarkable clinical efficacy, 2 major hurdles to gene therapy access for TDT patients materialized in 2021: (1) a risk of secondary hematological malignancies that is complex and multifactorial in origin and not limited to the risk of insertional mutagenesis, (2) the cost—even in high-income countries—is leading to the arrest of commercialization in Europe of the first gene therapy medicinal product indicated for TDT despite conditional approval by the European Medicines Agency.
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Affiliation(s)
- Isabelle Thuret
- Department of Pediatric Onco-Hematology, Center for Hemoglobinopathies, La Timone Hospital, Marseille University, Marseille, France
| | - Annalisa Ruggeri
- Hematology and Bone Marrow Transplant Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy
- Eurocord, Hopital Saint Louis, Paris, France
- EBMT Cellular Therapy and Immunobiology Working Party, Leiden, the Netherlands
| | - Emanuele Angelucci
- Hematology and Cellular Therapy, IRCCS Ospedale Policlinico San Martino, Genova, Italy
| | - Christian Chabannon
- Corresponding author: Christian Chabannon, MD, PhD, Institut Paoli-Calmettes, Aix-Marseille Université, Marseille, France. Tel: +33 491 223 441;
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9
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Cherqui S. Hematopoietic Stem Cell Gene Therapy for Cystinosis: From Bench-to-Bedside. Cells 2021; 10:3273. [PMID: 34943781 PMCID: PMC8699556 DOI: 10.3390/cells10123273] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/02/2021] [Revised: 11/17/2021] [Accepted: 11/19/2021] [Indexed: 12/31/2022] Open
Abstract
Cystinosis is an autosomal recessive metabolic disease that belongs to the family of lysosomal storage disorders. The gene involved is the CTNS gene that encodes cystinosin, a seven-transmembrane domain lysosomal protein, which is a proton-driven cystine transporter. Cystinosis is characterized by the lysosomal accumulation of cystine, a dimer of cysteine, in all the cells of the body leading to multi-organ failure, including the failure of the kidney, eye, thyroid, muscle, and pancreas, and eventually causing premature death in early adulthood. The current treatment is the drug cysteamine, which is onerous and expensive, and only delays the progression of the disease. Employing the mouse model of cystinosis, using Ctns-/- mice, we first showed that the transplantation of syngeneic wild-type murine hematopoietic stem and progenitor cells (HSPCs) led to abundant tissue integration of bone marrow-derived cells, a significant decrease in tissue cystine accumulation, and long-term kidney, eye and thyroid preservation. To translate this result to a potential human therapeutic treatment, given the risks of mortality and morbidity associated with allogeneic HSPC transplantation, we developed an autologous transplantation approach of HSPCs modified ex vivo using a self-inactivated lentiviral vector to introduce a functional version of the CTNS cDNA, pCCL-CTNS, and showed its efficacy in Ctns-/- mice. Based on these promising results, we held a pre-IND meeting with the Food and Drug Administration (FDA) to carry out the FDA agreed-upon pharmacological and toxicological studies for our therapeutic candidate, manufacturing development, production of the GMP lentiviral vector, design Phase 1/2 of the clinical trial, and filing of an IND application. Our IND was cleared by the FDA on 19 December 2018, to proceed to the clinical trial using CD34+ HSPCs from the G-CSF/plerixafor-mobilized peripheral blood stem cells of patients with cystinosis, modified by ex vivo transduction using the pCCL-CTNS vector (investigational product name: CTNS-RD-04). The clinical trial evaluated the safety and efficacy of CTNS-RD-04 and takes place at the University of California, San Diego (UCSD) and will include up to six patients affected with cystinosis. Following leukapheresis and cell manufacturing, the subjects undergo myeloablation before HSPC infusion. Patients also undergo comprehensive assessments before and after treatment to evaluate the impact of CTNS-RD-04 on the clinical outcomes and cystine and cystine crystal levels in the blood and tissues for 2 years. If successful, this treatment could be a one-time therapy that may eliminate or reduce renal deterioration as well as the long-term complications associated with cystinosis. In this review, we will describe the long path from bench-to-bedside for autologous HSPC gene therapy used to treat cystinosis.
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Affiliation(s)
- Stephanie Cherqui
- Department of Pediatrics, Division of Genetics, University of California, La Jolla, San Diego, CA 92093, USA
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10
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Rattananon P, Anurathapan U, Bhukhai K, Hongeng S. The Future of Gene Therapy for Transfusion-Dependent Beta-Thalassemia: The Power of the Lentiviral Vector for Genetically Modified Hematopoietic Stem Cells. Front Pharmacol 2021; 12:730873. [PMID: 34658870 PMCID: PMC8517149 DOI: 10.3389/fphar.2021.730873] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2021] [Accepted: 09/09/2021] [Indexed: 01/15/2023] Open
Abstract
β-thalassemia, a disease that results from defects in β-globin synthesis, leads to an imbalance of β- and α-globin chains and an excess of α chains. Defective erythroid maturation, ineffective erythropoiesis, and shortened red blood cell survival are commonly observed in most β-thalassemia patients. In severe cases, blood transfusion is considered as a mainstay therapy; however, regular blood transfusions result in chronic iron overload with life-threatening complications, e.g., endocrine dysfunction, cardiomyopathy, liver disease, and ultimately premature death. Therefore, transplantation of healthy hematopoietic stem cells (HSCs) is considered an alternative treatment. Patients with a compatible human leukocyte antigen (HLA) matched donor can be cured by allogeneic HSC transplantation. However, some recipients faced a high risk of morbidity/mortality due to graft versus host disease or graft failure, while a majority of patients do not have such HLA match-related donors. Currently, the infusion of autologous HSCs modified with a lentiviral vector expressing the β-globin gene into the erythroid progenitors of the patient is a promising approach to completely cure β-thalassemia. Here, we discuss a history of β-thalassemia treatments and limitations, in particular the development of β-globin lentiviral vectors, with emphasis on clinical applications and future perspectives in a new era of medicine.
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Affiliation(s)
- Parin Rattananon
- Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Ratchathewi, Thailand
| | - Usanarat Anurathapan
- Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Ratchathewi, Thailand
| | - Kanit Bhukhai
- Department of Physiology, Faculty of Science, Mahidol University, Ratchathewi, Thailand
| | - Suradej Hongeng
- Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Ratchathewi, Thailand
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11
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De Simone G, Quattrocchi A, Mancini B, di Masi A, Nervi C, Ascenzi P. Thalassemias: From gene to therapy. Mol Aspects Med 2021; 84:101028. [PMID: 34649720 DOI: 10.1016/j.mam.2021.101028] [Citation(s) in RCA: 32] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/05/2021] [Accepted: 09/19/2021] [Indexed: 12/26/2022]
Abstract
Thalassemias (α, β, γ, δ, δβ, and εγδβ) are the most common genetic disorders worldwide and constitute a heterogeneous group of hereditary diseases characterized by the deficient synthesis of one or more hemoglobin (Hb) chain(s). This leads to the accumulation of unstable non-thalassemic Hb chains, which precipitate and cause intramedullary destruction of erythroid precursors and premature lysis of red blood cells (RBC) in the peripheral blood. Non-thalassemic Hbs display high oxygen affinity and no cooperativity. Thalassemias result from many different genetic and molecular defects leading to either severe or clinically silent hematologic phenotypes. Thalassemias α and β are particularly diffused in the regions spanning from the Mediterranean basin through the Middle East, Indian subcontinent, Burma, Southeast Asia, Melanesia, and the Pacific Islands, whereas δβ-thalassemia is prevalent in some Mediterranean regions including Italy, Greece, and Turkey. Although in the world thalassemia and malaria areas overlap apparently, the RBC protection against malaria parasites is openly debated. Here, we provide an overview of the historical, geographic, genetic, structural, and molecular pathophysiological aspects of thalassemias. Moreover, attention has been paid to molecular and epigenetic pathways regulating globin gene expression and globin switching. Challenges of conventional standard treatments, including RBC transfusions and iron chelation therapy, splenectomy and hematopoietic stem cell transplantation from normal donors are reported. Finally, the progress made by rapidly evolving fields of gene therapy and gene editing strategies, already in pre-clinical and clinical evaluation, and future challenges as novel curative treatments for thalassemia are discussed.
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Affiliation(s)
- Giovanna De Simone
- Dipartimento di Scienze, Università Roma Tre, Viale Guglielmo Marconi 446, 00146, Roma, Italy
| | - Alberto Quattrocchi
- Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche, Facoltà di Farmacia e Medicina, "Sapienza" Università di Roma, Corso della Repubblica, 79, 04100, Latina, Italy
| | - Benedetta Mancini
- Dipartimento di Scienze, Università Roma Tre, Viale Guglielmo Marconi 446, 00146, Roma, Italy
| | - Alessandra di Masi
- Dipartimento di Scienze, Università Roma Tre, Viale Guglielmo Marconi 446, 00146, Roma, Italy
| | - Clara Nervi
- Dipartimento di Scienze e Biotecnologie Medico-Chirurgiche, Facoltà di Farmacia e Medicina, "Sapienza" Università di Roma, Corso della Repubblica, 79, 04100, Latina, Italy.
| | - Paolo Ascenzi
- Dipartimento di Scienze, Università Roma Tre, Viale Guglielmo Marconi 446, 00146, Roma, Italy; Accademia Nazionale dei Lincei, Via della Lungara 10, 00165, Roma, Italy.
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12
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Gene Therapies for Transfusion-Dependent β-Thalassemia. Indian Pediatr 2021. [DOI: 10.1007/s13312-021-2263-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/20/2022]
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13
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Bonner MA, Morales-Hernández A, Zhou S, Ma Z, Condori J, Wang YD, Fatima S, Palmer LE, Janke LJ, Fowler S, Sorrentino BP, McKinney-Freeman S. 3' UTR-truncated HMGA2 overexpression induces non-malignant in vivo expansion of hematopoietic stem cells in non-human primates. Mol Ther Methods Clin Dev 2021; 21:693-701. [PMID: 34141824 PMCID: PMC8181581 DOI: 10.1016/j.omtm.2021.04.013] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2021] [Accepted: 04/22/2021] [Indexed: 12/16/2022]
Abstract
Vector-mediated mutagenesis remains a major safety concern for many gene therapy clinical protocols. Indeed, lentiviral-based gene therapy treatments of hematologic disease can result in oligoclonal blood reconstitution in the transduced cell graft. Specifically, clonal expansion of hematopoietic stem cells (HSCs) highly expressing HMGA2, a chromatin architectural factor found in many human cancers, is reported in patients undergoing gene therapy for hematologic diseases, raising concerns about the safety of these integrations. Here, we show for the first time in vivo multilineage and multiclonal expansion of non-human primate HSCs expressing a 3' UTR-truncated version of HMGA2 without evidence of any hematologic malignancy >7 years post-transplantation, which is significantly longer than most non-human gene therapy pre-clinical studies. This expansion is accompanied by an increase in HSC survival, cell cycle activation of downstream progenitors, and changes in gene expression led by the upregulation of IGF2BP2, a mRNA binding regulator of survival and proliferation. Thus, we conclude that prolonged ectopic expression of HMGA2 in hematopoietic progenitors is not sufficient to drive hematologic malignancy and is not an acute safety concern in lentiviral-based gene therapy clinical protocols.
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Affiliation(s)
- Melissa A. Bonner
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | | | - Sheng Zhou
- Experimental Cell Therapeutics Lab, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Zhijun Ma
- Department of Bone Marrow Transplant and Cell Therapy, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Jose Condori
- Experimental Cell Therapeutics Lab, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Yong-Dong Wang
- Department of Cell and Molecular Biology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Soghra Fatima
- Immunology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Lance E. Palmer
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Laura J. Janke
- Veterinary Pathology Core, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Stephanie Fowler
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
| | - Brian P. Sorrentino
- Department of Hematology, St. Jude Children’s Research Hospital, Memphis, TN 38105, USA
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14
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Nualkaew T, Sii-Felice K, Giorgi M, McColl B, Gouzil J, Glaser A, Voon HPJ, Tee HY, Grigoriadis G, Svasti S, Fucharoen S, Hongeng S, Leboulch P, Payen E, Vadolas J. Coordinated β-globin expression and α2-globin reduction in a multiplex lentiviral gene therapy vector for β-thalassemia. Mol Ther 2021; 29:2841-2853. [PMID: 33940155 PMCID: PMC8417505 DOI: 10.1016/j.ymthe.2021.04.037] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2020] [Revised: 03/08/2021] [Accepted: 04/27/2021] [Indexed: 01/30/2023] Open
Abstract
A primary challenge in lentiviral gene therapy of β-hemoglobinopathies is to maintain low vector copy numbers to avoid genotoxicity while being reliably therapeutic for all genotypes. We designed a high-titer lentiviral vector, LVβ-shα2, that allows coordinated expression of the therapeutic βA-T87Q-globin gene and of an intron-embedded miR-30-based short hairpin RNA (shRNA) selectively targeting the α2-globin mRNA. Our approach was guided by the knowledge that moderate reduction of α-globin chain synthesis ameliorates disease severity in β-thalassemia. We demonstrate that LVβ-shα2 reduces α2-globin mRNA expression in erythroid cells while keeping α1-globin mRNA levels unchanged and βA-T87Q-globin gene expression identical to the parent vector. Compared with the first βA-T87Q-globin lentiviral vector that has received conditional marketing authorization, BB305, LVβ-shα2 shows 1.7-fold greater potency to improve α/β ratios. It may thus result in greater therapeutic efficacy and reliability for the most severe types of β-thalassemia and provide an improved benefit/risk ratio regardless of the β-thalassemia genotype.
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Affiliation(s)
- Tiwaporn Nualkaew
- Hudson Institute of Medical Research, Clayton, Melbourne, VIC 3168, Australia; Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom 73170, Thailand; Murdoch Children's Research Institute, Parkville, Melbourne, VIC 3052, Australia
| | - Karine Sii-Felice
- Division of Innovative Therapies, CEA François Jacob Biology Institute, 18 route du Panorama, 92260, Fontenay-aux-Roses, France; Paris-Saclay University, CEA, INSERM, Center for Immunology of Viral, Auto-immune, Hematological and Bacterial Diseases (IMVA-HB/IDMIT), 18 route du Panorama, 92260 Fontenay-aux-Roses & Le Kremlin Bicêtre, France
| | - Marie Giorgi
- Division of Innovative Therapies, CEA François Jacob Biology Institute, 18 route du Panorama, 92260, Fontenay-aux-Roses, France
| | - Bradley McColl
- Murdoch Children's Research Institute, Parkville, Melbourne, VIC 3052, Australia
| | - Julie Gouzil
- Division of Innovative Therapies, CEA François Jacob Biology Institute, 18 route du Panorama, 92260, Fontenay-aux-Roses, France
| | - Astrid Glaser
- Murdoch Children's Research Institute, Parkville, Melbourne, VIC 3052, Australia
| | - Hsiao P J Voon
- Department of Biochemistry and Molecular Biology, Cancer Program, Biomedicine Discovery Institute, Monash University, Clayton, VIC 3800, Australia
| | - Hsin Y Tee
- Hudson Institute of Medical Research, Clayton, Melbourne, VIC 3168, Australia
| | - George Grigoriadis
- Hudson Institute of Medical Research, Clayton, Melbourne, VIC 3168, Australia
| | - Saovaros Svasti
- Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom 73170, Thailand; Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
| | - Suthat Fucharoen
- Thalassemia Research Center, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom 73170, Thailand
| | - Suradej Hongeng
- Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand
| | - Philippe Leboulch
- Division of Innovative Therapies, CEA François Jacob Biology Institute, 18 route du Panorama, 92260, Fontenay-aux-Roses, France; Genetics Division, Brigham & Women's Hospital and Harvard Medical School, Boston, MA 02115, USA.
| | - Emmanuel Payen
- Division of Innovative Therapies, CEA François Jacob Biology Institute, 18 route du Panorama, 92260, Fontenay-aux-Roses, France; Paris-Saclay University, CEA, INSERM, Center for Immunology of Viral, Auto-immune, Hematological and Bacterial Diseases (IMVA-HB/IDMIT), 18 route du Panorama, 92260 Fontenay-aux-Roses & Le Kremlin Bicêtre, France.
| | - Jim Vadolas
- Hudson Institute of Medical Research, Clayton, Melbourne, VIC 3168, Australia; Murdoch Children's Research Institute, Parkville, Melbourne, VIC 3052, Australia.
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15
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Raes L, De Smedt SC, Raemdonck K, Braeckmans K. Non-viral transfection technologies for next-generation therapeutic T cell engineering. Biotechnol Adv 2021; 49:107760. [PMID: 33932532 DOI: 10.1016/j.biotechadv.2021.107760] [Citation(s) in RCA: 41] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2020] [Revised: 04/24/2021] [Accepted: 04/24/2021] [Indexed: 12/24/2022]
Abstract
Genetically engineered T cells have sparked interest in advanced cancer treatment, reaching a milestone in 2017 with two FDA-approvals for CD19-directed chimeric antigen receptor (CAR) T cell therapeutics. It is becoming clear that the next generation of CAR T cell therapies will demand more complex engineering strategies and combinations thereof, including the use of revolutionary gene editing approaches. To date, manufacturing of CAR T cells mostly relies on γ-retroviral or lentiviral vectors, but their use is associated with several drawbacks, including safety issues, high manufacturing cost and vector capacity constraints. Non-viral approaches, including membrane permeabilization and carrier-based techniques, have therefore gained a lot of interest to replace viral transductions in the manufacturing of T cell therapeutics. This review provides an in-depth discussion on the avid search for alternatives to viral vectors, discusses key considerations for T cell engineering technologies, and provides an overview of the emerging spectrum of non-viral transfection technologies for T cells. Strengths and weaknesses of each technology will be discussed in relation to T cell engineering. Altogether, this work emphasizes the potential of non-viral transfection approaches to advance the next-generation of genetically engineered T cells.
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Affiliation(s)
- Laurens Raes
- Laboratory of General Biochemistry & Physical Pharmacy, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium
| | - Stefaan C De Smedt
- Laboratory of General Biochemistry & Physical Pharmacy, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium
| | - Koen Raemdonck
- Laboratory of General Biochemistry & Physical Pharmacy, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium
| | - Kevin Braeckmans
- Laboratory of General Biochemistry & Physical Pharmacy, Ghent University, Ottergemsesteenweg 460, 9000 Ghent, Belgium.
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16
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Kim HS, Hwang GH, Lee HK, Bae T, Park SH, Kim YJ, Lee S, Park JH, Bae S, Hur JK. CReVIS-Seq: A highly accurate and multiplexable method for genome-wide mapping of lentiviral integration sites. MOLECULAR THERAPY-METHODS & CLINICAL DEVELOPMENT 2021; 20:792-800. [PMID: 33768124 PMCID: PMC7961857 DOI: 10.1016/j.omtm.2020.10.012] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 04/06/2020] [Accepted: 10/17/2020] [Indexed: 12/16/2022]
Abstract
Lentiviruses have been widely used as a means of transferring exogenous DNAs into human cells to treat various genetic diseases. Lentiviral vectors are fundamentally integrated into the host genome, but their integration sites are generally unpredictable, which may increase the uncertainty for their use in therapeutics. To determine the viral integration sites in the host genome, several PCR-based methods have been developed. However, the sensitivities of the PCR-based methods are highly dependent on the primer sequences, and optimized primer design is required for individual target sites. In order to address this issue, we developed an alternative method for genome-wide mapping of viral insertion sites, named CReVIS-seq (CRISPR-enhanced Viral Integration Site Sequencing). The method is based on the sequential steps: fragmentation of genomic DNAs, in vitro circularization, cleavage of target sequence in a CRISPR guide RNA-specific manner, high-throughput sequencing of the linearized DNA fragments in an unbiased manner, and identification of viral insertion sites via sequence analysis. By design, CReVIS-seq is not affected by biases that could be introduced during the target enrichment step via PCR amplification using site specific primers. Furthermore, we found that multiplexed CReVIS-seq, using collections of different single-guide RNAs (sgRNAs), enables simultaneous identification of multiple target sites and structural variations (i.e., circularized viral genome), in both single cell clones and heterogeneous cell populations.
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Affiliation(s)
- Heon Seok Kim
- Division of Oncology, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA
| | - Gue-Ho Hwang
- Department of Chemistry, Hanyang University, Seoul 04763, South Korea.,Research Institute for Natural Sciences, Hanyang University, Seoul 04763, South Korea
| | - Hyomin K Lee
- Department of Medicine, Graduate School, Hanyang University, Seoul 04763, South Korea
| | - Taegeun Bae
- Department of Medicine, Graduate School, Kyung Hee University, Seoul 02447, South Korea
| | - Seong-Ho Park
- Department of Medicine, Graduate School, Hanyang University, Seoul 04763, South Korea
| | - Yong Jun Kim
- Department of Pathology, College of Medicine, Kyung Hee University, Seoul 02447, South Korea.,Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul 02447, South Korea
| | - Sun Lee
- Department of Pathology, College of Medicine, Kyung Hee University, Seoul 02447, South Korea.,Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul 02447, South Korea
| | - Jae-Hoon Park
- Department of Pathology, College of Medicine, Kyung Hee University, Seoul 02447, South Korea.,Department of Biomedical Science, Graduate School, Kyung Hee University, Seoul 02447, South Korea
| | - Sangsu Bae
- Department of Chemistry, Hanyang University, Seoul 04763, South Korea.,Research Institute for Natural Sciences, Hanyang University, Seoul 04763, South Korea
| | - Junho K Hur
- Department of Genetics, College of Medicine, Hanyang University, Seoul 04763, South Korea.,Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul 04763, South Korea
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17
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High-level protein production in erythroid cells derived from in vivo transduced hematopoietic stem cells. Blood Adv 2020; 3:2883-2894. [PMID: 31585952 PMCID: PMC6784527 DOI: 10.1182/bloodadvances.2019000706] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2019] [Accepted: 08/18/2019] [Indexed: 12/12/2022] Open
Abstract
We developed an in vivo hematopoietic stem cell (HSC) transduction approach that involves HSC mobilization from the bone marrow into the peripheral bloodstream and the IV injection of an integrating, helper-dependent adenovirus (HDAd5/35++) vector system. HDAd5/35++ vectors target human CD46, a receptor that is abundantly expressed on primitive HSCs. Transgene integration is achieved by a hyperactive Sleeping Beauty transposase (SB100x) and transgene marking in peripheral blood cells can be increased by in vivo selection. Here we directed transgene expression to HSC-derived erythroid cells using β-globin regulatory elements. We hypothesized that the abundance and systemic distribution of erythroid cells can be harnessed for high-level production of therapeutic proteins. We first demonstrated that our approach allowed for sustained, erythroid-lineage specific GFP expression and accumulation of GFP protein in erythrocytes. Furthermore, after in vivo HSC transduction/selection in hCD46-transgenic mice, we demonstrated stable supraphysiological plasma concentrations of a bioengineered human factor VIII, termed ET3. High-level ET3 production in erythroid cells did not affect erythropoiesis. A phenotypic correction of bleeding was observed after in vivo HSC transduction of hCD46+/+/F8-/- hemophilia A mice despite high plasma anti-ET3 antibody titers. This suggests that ET3 levels were high enough to provide sufficient noninhibited ET3 systemically and/or locally (in blood clots) to control bleeding. In addition to its relevance for hemophilia A gene therapy, our approach has implications for the therapy of other inherited or acquired diseases that require high levels of therapeutic proteins in the blood circulation.
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18
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19
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Soni S. Gene therapies for transfusion dependent β-thalassemia: Current status and critical criteria for success. Am J Hematol 2020; 95:1099-1112. [PMID: 32562290 DOI: 10.1002/ajh.25909] [Citation(s) in RCA: 16] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2020] [Revised: 05/30/2020] [Accepted: 06/16/2020] [Indexed: 01/19/2023]
Abstract
Thalassemia is one of the most prevalent monogenic diseases usually caused by quantitative defects in the production of β-globin leading to severe anemia. Technological advances in genome sequencing, stem cell selection, viral vector development, transduction and gene editing strategies now allow for efficient exvivo genetic manipulation of human stem cells that can lead to production of hemoglobin, leading to a meaningful clinical benefit in thalassemia patients. In this review, the status of the gene-therapy approaches available for transfusion dependent thalassemia are discussed, along with the critical criteria that affect efficacy and lessons that have been learned from the early phase clinical trials. Salient steps necessary for the clinical development, manufacturing, and regulatory approvals of gene therapies for thalassemia are also highlighted, so that the potential of these therapies can be realized. It is highly anticipated that gene therapies will soon become a treatment option for patients lacking compatible donors for hematopoietic stem cell transplant and will offer an alternative for definitive treatment of β-thalassemia.
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Affiliation(s)
- Sandeep Soni
- Division of Pediatric Stem Cell Transplant and RM Lucile Packard Children's Hospital, Stanford University Palo Alto California
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20
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Lamsfus-Calle A, Daniel-Moreno A, Antony JS, Epting T, Heumos L, Baskaran P, Admard J, Casadei N, Latifi N, Siegmund DM, Kormann MSD, Handgretinger R, Mezger M. Comparative targeting analysis of KLF1, BCL11A, and HBG1/2 in CD34 + HSPCs by CRISPR/Cas9 for the induction of fetal hemoglobin. Sci Rep 2020; 10:10133. [PMID: 32576837 PMCID: PMC7311455 DOI: 10.1038/s41598-020-66309-x] [Citation(s) in RCA: 40] [Impact Index Per Article: 8.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2019] [Accepted: 05/19/2020] [Indexed: 12/22/2022] Open
Abstract
β-hemoglobinopathies are caused by abnormal or absent production of hemoglobin in the blood due to mutations in the β-globin gene (HBB). Imbalanced expression of adult hemoglobin (HbA) induces strong anemia in patients suffering from the disease. However, individuals with natural-occurring mutations in the HBB cluster or related genes, compensate this disparity through γ-globin expression and subsequent fetal hemoglobin (HbF) production. Several preclinical and clinical studies have been performed in order to induce HbF by knocking-down genes involved in HbF repression (KLF1 and BCL11A) or disrupting the binding sites of several transcription factors in the γ-globin gene (HBG1/2). In this study, we thoroughly compared the different CRISPR/Cas9 gene-disruption strategies by gene editing analysis and assessed their safety profile by RNA-seq and GUIDE-seq. All approaches reached therapeutic levels of HbF after gene editing and showed similar gene expression to the control sample, while no significant off-targets were detected by GUIDE-seq. Likewise, all three gene editing platforms were established in the GMP-grade CliniMACS Prodigy, achieving similar outcome to preclinical devices. Based on this gene editing comparative analysis, we concluded that BCL11A is the most clinically relevant approach while HBG1/2 could represent a promising alternative for the treatment of β-hemoglobinopathies.
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Affiliation(s)
- Andrés Lamsfus-Calle
- University Children's Hospital. Department of Pediatrics I, Hematology and Oncology, University of Tübingen, Tübingen, Germany
| | - Alberto Daniel-Moreno
- University Children's Hospital. Department of Pediatrics I, Hematology and Oncology, University of Tübingen, Tübingen, Germany
| | - Justin S Antony
- University Children's Hospital. Department of Pediatrics I, Hematology and Oncology, University of Tübingen, Tübingen, Germany
| | - Thomas Epting
- Institute for Clinical Chemistry and Laboratory Medicine, Medical Center, Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Lukas Heumos
- Quantitative Biology Center (QBiC), University of Tübingen, Tübingen, Germany
| | - Praveen Baskaran
- Quantitative Biology Center (QBiC), University of Tübingen, Tübingen, Germany
| | - Jakob Admard
- Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany
| | - Nicolas Casadei
- Institute of Medical Genetics and Applied Genomics, University of Tübingen, Tübingen, Germany
| | - Ngadhnjim Latifi
- Interfaculty Institute of Biochemistry, University of Tübingen, Tübingen, Germany
| | - Darina M Siegmund
- University Hospital Freiburg. Department of Hematology, Oncology, and Stem-Cell Transplantation, Medical Center, University of Freiburg, Freiburg, Germany
| | - Michael S D Kormann
- University Children's Hospital. Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tübingen, Tübingen, Germany
| | - Rupert Handgretinger
- University Children's Hospital. Department of Pediatrics I, Hematology and Oncology, University of Tübingen, Tübingen, Germany
| | - Markus Mezger
- University Children's Hospital. Department of Pediatrics I, Hematology and Oncology, University of Tübingen, Tübingen, Germany.
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21
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Udry F, Decembrini S, Gamm DM, Déglon N, Kostic C, Arsenijevic Y. Lentiviral mediated RPE65 gene transfer in healthy hiPSCs-derived retinal pigment epithelial cells markedly increased RPE65 mRNA, but modestly protein level. Sci Rep 2020; 10:8890. [PMID: 32483256 PMCID: PMC7264209 DOI: 10.1038/s41598-020-65657-y] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2019] [Accepted: 05/08/2020] [Indexed: 12/15/2022] Open
Abstract
The retinal pigment epithelium (RPE) is a monolayer of cobblestone-like epithelial cells that accomplishes critical functions for the retina. Several protocols have been published to differentiate pluripotent stem cells into RPE cells suitable for disease modelling and therapy development. In our study, the RPE identity of human induced pluripotent stem cell (hiPSC)-derived RPE (iRPE) was extensively characterized, and then used to test a lentiviral-mediated RPE65 gene augmentation therapy. A dose study of the lentiviral vector revealed a dose-dependent effect of the vector on RPE65 mRNA levels. A marked increase of the RPE65 mRNA was also observed in the iRPE (100-fold) as well as in an experimental set with RPE derived from another hiPSC source and from foetal human RPE. Although iRPE displayed features close to bona fide RPE, no or a modest increase of the RPE65 protein level was observed depending on the protein detection method. Similar results were observed with the two other cell lines. The mechanism of RPE65 protein regulation remains to be elucidated, but the current work suggests that high vector expression will not produce an excess of the normal RPE65 protein level.
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Affiliation(s)
- Florian Udry
- Department of ophthalmology, Unit of Retinal Degeneration and Regeneration, University of Lausanne, Hôpital ophtalmique Jules-Gonin, 1004, Lausanne, Switzerland
| | - Sarah Decembrini
- Department of ophthalmology, Unit of Retinal Degeneration and Regeneration, University of Lausanne, Hôpital ophtalmique Jules-Gonin, 1004, Lausanne, Switzerland
- Department of Biomedicine, University Hospital Basel & University Basel, Hebelstr. 20, 4031, Basel, Switzerland
| | - David M Gamm
- McPherson Eye Research Institute, Waisman Center and Department of Ophthalmology and Visual Sciences, and University of Wisconsin-Madison, Madison, USA
| | - Nicole Déglon
- Neuroscience Research Center, Laboratory of Neurotherapies and Neuromodulation, Lausanne University Hospital and University of Lausanne, Lausanne, Switzerland
| | - Corinne Kostic
- Department of ophthalmology, Unit of Retinal Degeneration and Regeneration, University of Lausanne, Hôpital ophtalmique Jules-Gonin, 1004, Lausanne, Switzerland
| | - Yvan Arsenijevic
- Department of ophthalmology, Unit of Retinal Degeneration and Regeneration, University of Lausanne, Hôpital ophtalmique Jules-Gonin, 1004, Lausanne, Switzerland.
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22
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Fiedorowicz K, Rozwadowska N, Zimna A, Malcher A, Tutak K, Szczerbal I, Nowicka-Bauer K, Nowaczyk M, Kolanowski TJ, Łabędź W, Kubaszewski Ł, Kurpisz M. Tissue-specific promoter-based reporter system for monitoring cell differentiation from iPSCs to cardiomyocytes. Sci Rep 2020; 10:1895. [PMID: 32024875 PMCID: PMC7002699 DOI: 10.1038/s41598-020-58050-2] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/17/2019] [Accepted: 11/06/2019] [Indexed: 12/14/2022] Open
Abstract
The possibility of using stem cell-derived cardiomyocytes opens a new platform for modeling cardiac cell differentiation and disease or the development of new drugs. Progress in this field can be accelerated by high-throughput screening (HTS) technology combined with promoter reporter system. The goal of the study was to create and evaluate a responsive promoter reporter system that allows monitoring of iPSC differentiation towards cardiomyocytes. The lentiviral promoter reporter system was based on troponin 2 (TNNT2) and alpha cardiac actin (ACTC) with firefly luciferase and mCherry, respectively. The system was evaluated in two in vitro models. First, system followed the differentiation of TNNT2-luc-T2A-Puro-mCMV-GFP and hACTC-mcherry-WPRE-EF1-Neo from transduced iPSC line towards cardiomyocytes and revealed the significant decrease in both inserts copy number during the prolonged in vitro cell culture (confirmed by I-FISH, ddPCR, qPCR). Second, differentiated and contracting control cardiomyocytes (obtained from control non-reporter transduced iPSCs) were subsequently transduced with TNNT2-luc-T2A-Puro-CMV-GFP and hACTC-mcherry-WPRE-EF1-Neo lentiviruses to observe the functionality of obtained cardiomyocytes. Our results indicated that the reporter modified cell lines can be used for HTS applications, but it is essential to monitor the stability of the reporter sequence during extended cell in vitro culture.
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Affiliation(s)
| | | | - Agnieszka Zimna
- Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland
| | - Agnieszka Malcher
- Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland
| | - Katarzyna Tutak
- Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland
| | - Izabela Szczerbal
- Department of Genetics and Animal Breeding, Poznan University of Life Sciences, Poznan, Poland
| | | | | | | | - Wojciech Łabędź
- Department of Spondyloortopaedics and Biomechanics of the Spine, W. Dega University Hospital, Poznan University of Medical Sciences, Poznan, Poland
| | - Łukasz Kubaszewski
- Department of Spondyloortopaedics and Biomechanics of the Spine, W. Dega University Hospital, Poznan University of Medical Sciences, Poznan, Poland
| | - Maciej Kurpisz
- Institute of Human Genetics, Polish Academy of Sciences, Poznan, Poland.
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23
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Karponi G, Zogas N. Gene Therapy For Beta-Thalassemia: Updated Perspectives. APPLICATION OF CLINICAL GENETICS 2019; 12:167-180. [PMID: 31576160 PMCID: PMC6765258 DOI: 10.2147/tacg.s178546] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/22/2019] [Accepted: 09/11/2019] [Indexed: 12/26/2022]
Abstract
Allogeneic hematopoietic stem cell transplantation was until very recently, the only permanent curative option available for patients suffering from transfusion-dependent beta-thalassemia. Gene therapy, by autologous transplantation of genetically modified hematopoietic stem cells, currently represents a novel therapeutic promise, after many years of extensive preclinical research for the optimization of gene transfer protocols. Nowadays, clinical trials being held on a worldwide setting, have demonstrated that, by re-establishing effective hemoglobin production, patients may be rendered transfusion- and chelation-independent and evade the immunological complications that normally accompany allogeneic hematopoietic stem cell transplantation. The present review will offer a retrospective scope of the long way paved towards successful implementation of gene therapy for beta-thalassemia, and will pinpoint the latest strategies employed to increase globin expression that extend beyond the classic transgene addition perspective. A thorough search was performed using Pubmed in order to identify studies that provide a proof of principle on the aforementioned topic at a preclinical and clinical level. Inclusion criteria also regarded gene transfer technologies of the past two decades, as well as publications outlining the pitfalls that precluded earlier successful implementation of gene therapy for beta-thalassemia. Overall, after decades of research, that included both successes and pitfalls, the path towards a permanent, donor-irrespective cure for beta-thalassemia patients is steadily becoming a realistic approach.
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Affiliation(s)
- Garyfalia Karponi
- Department of Veterinary Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece
| | - Nikolaos Zogas
- Department of Biology, Aristotle University of Thessaloniki, Thessaloniki, Greece
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24
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Antony JS, Latifi N, Haque AKMA, Lamsfus-Calle A, Daniel-Moreno A, Graeter S, Baskaran P, Weinmann P, Mezger M, Handgretinger R, Kormann MSD. Gene correction of HBB mutations in CD34 + hematopoietic stem cells using Cas9 mRNA and ssODN donors. Mol Cell Pediatr 2018; 5:9. [PMID: 30430274 PMCID: PMC6236008 DOI: 10.1186/s40348-018-0086-1] [Citation(s) in RCA: 45] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2018] [Accepted: 10/17/2018] [Indexed: 12/13/2022] Open
Abstract
Background β-Thalassemia is an inherited hematological disorder caused by mutations in the human hemoglobin beta (HBB) gene that reduce or abrogate β-globin expression. Although lentiviral-mediated expression of β-globin and autologous transplantation is a promising therapeutic approach, the risk of insertional mutagenesis or low transgene expression is apparent. However, targeted gene correction of HBB mutations with programmable nucleases such as CRISPR/Cas9, TALENs, and ZFNs with non-viral repair templates ensures a higher safety profile and endogenous expression control. Methods We have compared three different gene-editing tools (CRISPR/Cas9, TALENs, and ZFNs) for their targeting efficiency of the HBB gene locus. As a proof of concept, we studied the personalized gene-correction therapy for a common β-thalassemia splicing variant HBBIVS1–110 using Cas9 mRNA and several optimally designed single-stranded oligonucleotide (ssODN) donors in K562 and CD34+ hematopoietic stem cells (HSCs). Results Our results exhibited that indel frequency of CRISPR/Cas9 was superior to TALENs and ZFNs (P < 0.0001). Our designed sgRNA targeting the site of HBBIVS1–110 mutation showed indels in both K562 cells (up to 77%) and CD34+ hematopoietic stem cells—HSCs (up to 87%). The absolute quantification by next-generation sequencing showed that up to 8% site-specific insertion of the NheI tag was achieved using Cas9 mRNA and a chemically modified ssODN in CD34+ HSCs. Conclusion Our approach provides guidance on non-viral gene correction in CD34+ HSCs using Cas9 mRNA and chemically modified ssODN. However, further optimization is needed to increase the homology directed repair (HDR) to attain a real clinical benefit for β-thalassemia. Electronic supplementary material The online version of this article (10.1186/s40348-018-0086-1) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Justin S Antony
- Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tuebingen, Tuebingen, Germany.,University Children's Hospital, Department of Pediatrics I, University of Tuebingen, Tuebingen, Germany.,Department of Hematology, Oncology, Clinical Immunology, University of Tuebingen, Tuebingen, Germany
| | - Ngadhnjim Latifi
- Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tuebingen, Tuebingen, Germany
| | - A K M Ashiqul Haque
- Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tuebingen, Tuebingen, Germany
| | - Andrés Lamsfus-Calle
- University Children's Hospital, Department of Pediatrics I, University of Tuebingen, Tuebingen, Germany
| | - Alberto Daniel-Moreno
- University Children's Hospital, Department of Pediatrics I, University of Tuebingen, Tuebingen, Germany
| | - Sebastian Graeter
- Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tuebingen, Tuebingen, Germany
| | - Praveen Baskaran
- Center for Quantitative Biology, University of Tuebingen, Tuebingen, Germany
| | - Petra Weinmann
- Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tuebingen, Tuebingen, Germany
| | - Markus Mezger
- University Children's Hospital, Department of Pediatrics I, University of Tuebingen, Tuebingen, Germany
| | - Rupert Handgretinger
- University Children's Hospital, Department of Pediatrics I, University of Tuebingen, Tuebingen, Germany
| | - Michael S D Kormann
- Department of Pediatrics I, Pediatric Infectiology and Immunology, Translational Genomics and Gene Therapy in Pediatrics, University of Tuebingen, Tuebingen, Germany.
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25
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Sii-Felice K, Giorgi M, Leboulch P, Payen E. Hemoglobin disorders: lentiviral gene therapy in the starting blocks to enter clinical practice. Exp Hematol 2018; 64:12-32. [PMID: 29807062 DOI: 10.1016/j.exphem.2018.05.004] [Citation(s) in RCA: 30] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2018] [Revised: 05/18/2018] [Accepted: 05/19/2018] [Indexed: 01/19/2023]
Abstract
The β-hemoglobinopathies, transfusion-dependent β-thalassemia and sickle cell disease, are the most prevalent inherited disorders worldwide and affect millions of people. Many of these patients have a shortened life expectancy and suffer from severe morbidity despite supportive therapies, which impose an enormous financial burden to societies. The only available curative therapy is allogeneic hematopoietic stem cell transplantation, although most patients do not have an HLA-matched sibling donor, and those who do still risk life-threatening complications. Therefore, gene therapy by one-time ex vivo modification of hematopoietic stem cells followed by autologous engraftment is an attractive new therapeutic modality. The first proof-of-principle of conversion to transfusion independence by means of a lentiviral vector expressing a marked and anti-sickling βT87Q-globin gene variant was reported a decade ago in a patient with transfusion-dependent β-thalassemia. In follow-up multicenter Phase II trials with an essentially identical vector (termed LentiGlobin BB305) and protocol, 12 of the 13 patients with a non-β0/β0 genotype, representing more than half of all transfusion-dependent β-thalassemia cases worldwide, stopped red blood cell transfusions with total hemoglobin levels in blood approaching normal values. Correction of biological markers of dyserythropoiesis was achieved in evaluated patients. In nine patients with β0/β0 transfusion-dependent β-thalassemia or equivalent severity (βIVS1-110), median annualized transfusion volume decreased by 73% and red blood cell transfusions were stopped in three patients. Proof-of-principle of therapeutic efficacy in the first patient with sickle cell disease was also reported with LentiGlobin BB305. Encouraging results were presented in children with transfusion-dependent β-thalassemia in another trial with the GLOBE lentiviral vector and several other gene therapy trials are currently open for both transfusion-dependent β-thalassemia and sickle cell disease. Phase III trials are now under way and should help to determine benefit/risk/cost ratios to move gene therapy toward clinical practice.
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Affiliation(s)
- Karine Sii-Felice
- UMR E007, Service of Innovative Therapies, Institute of Biology François Jacob and University Paris Saclay, CEA Paris Saclay, Fontenay-aux-Roses, France
| | - Marie Giorgi
- UMR E007, Service of Innovative Therapies, Institute of Biology François Jacob and University Paris Saclay, CEA Paris Saclay, Fontenay-aux-Roses, France
| | - Philippe Leboulch
- UMR E007, Service of Innovative Therapies, Institute of Biology François Jacob and University Paris Saclay, CEA Paris Saclay, Fontenay-aux-Roses, France; Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA; Ramathibodi Hospital, Mahidol University, Bangkok, Thailand
| | - Emmanuel Payen
- UMR E007, Service of Innovative Therapies, Institute of Biology François Jacob and University Paris Saclay, CEA Paris Saclay, Fontenay-aux-Roses, France; INSERM, Paris, France.
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26
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Novel and innovative approaches for treatment of β-thalassemia. PEDIATRIC HEMATOLOGY ONCOLOGY JOURNAL 2017. [DOI: 10.1016/j.phoj.2017.11.153] [Citation(s) in RCA: 7] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
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27
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Qian W, Wang Y, Li RF, Zhou X, Liu J, Peng DZ. Prolonged Integration Site Selection of a Lentiviral Vector in the Genome of Human Keratinocytes. Med Sci Monit 2017; 23:1116-1122. [PMID: 28255155 PMCID: PMC5347986 DOI: 10.12659/msm.903094] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Background Lentiviral vectors have been successfully used for human skin cell gene transfer studies. Defining the selection of integration sites for retroviral vectors in the host genome is crucial in risk assessment analysis of gene therapy. However, genome-wide analyses of lentiviral integration sites in human keratinocytes, especially after prolonged growth, are poorly understood. Material/Methods In this study, 874 unique lentiviral vector integration sites in human HaCaT keratinocytes after long-term culture were identified and analyzed with the online tool GTSG-QuickMap and SPSS software. Results The data indicated that lentiviral vectors showed integration site preferences for genes and gene-rich regions. Conclusions This study will likely assist in determining the relative risks of the lentiviral vector system and in the design of a safe lentiviral vector system in the gene therapy of skin diseases.
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Affiliation(s)
- Wei Qian
- Institute of Burn Research, Southwest Hospital and Tissue Engineering Research Unit, State Key Laboratory of Trauma, Burns, and Combined Injury, 3rd Military Medical University, Chongqing, China (mainland)
| | - Yong Wang
- Institute of Burn Research, Southwest Hospital and Tissue Engineering Research Unit, State Key Laboratory of Trauma, Burns, and Combined Injury, 3rd Military Medical University, Chongqing, China (mainland)
| | - Rui-Fu Li
- Institute of Burn Research, Southwest Hospital and Tissue Engineering Research Unit, State Key Laboratory of Trauma, Burns, and Combined Injury, 3rd Military Medical University, Chongqing, China (mainland)
| | - Xin Zhou
- Institute of Burn Research, Southwest Hospital and Tissue Engineering Research Unit, State Key Laboratory of Trauma, Burns, and Combined Injury, 3rd Military Medical University, Chongqing, China (mainland)
| | - Jing Liu
- Institute of Burn Research, Southwest Hospital and Tissue Engineering Research Unit, State Key Laboratory of Trauma, Burns, and Combined Injury, 3rd Military Medical University, Chongqing, China (mainland)
| | - Dai-Zhi Peng
- Institute of Burn Research, Southwest Hospital and Tissue Engineering Research Unit, State Key Laboratory of Trauma, Burns, and Combined Injury, 3rd Military Medical University, Chongqing, China (mainland)
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28
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Negre O, Eggimann AV, Beuzard Y, Ribeil JA, Bourget P, Borwornpinyo S, Hongeng S, Hacein-Bey S, Cavazzana M, Leboulch P, Payen E. Gene Therapy of the β-Hemoglobinopathies by Lentiviral Transfer of the β(A(T87Q))-Globin Gene. Hum Gene Ther 2016; 27:148-65. [PMID: 26886832 PMCID: PMC4779296 DOI: 10.1089/hum.2016.007] [Citation(s) in RCA: 128] [Impact Index Per Article: 14.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
β-globin gene disorders are the most prevalent inherited diseases worldwide and result from abnormal β-globin synthesis or structure. Novel therapeutic approaches are being developed in an effort to move beyond palliative management. Gene therapy, by ex vivo lentiviral transfer of a therapeutic β-globin gene derivative (β(AT87Q)-globin) to hematopoietic stem cells, driven by cis-regulatory elements that confer high, erythroid-specific expression, has been evaluated in human clinical trials over the past 8 years. β(AT87Q)-globin is used both as a strong inhibitor of HbS polymerization and as a biomarker. While long-term studies are underway in multiple centers in Europe and in the United States, proof-of-principle of efficacy and safety has already been obtained in multiple patients with β-thalassemia and sickle cell disease.
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Affiliation(s)
- Olivier Negre
- 1 bluebird bio, Cambridge, Massachusetts.,2 CEA, Institute of Emerging Disease and Innovative Therapies (iMETI) , Fontenay aux Roses, France
| | | | - Yves Beuzard
- 2 CEA, Institute of Emerging Disease and Innovative Therapies (iMETI) , Fontenay aux Roses, France .,3 UMR 007, University of Paris 11 and CEA , CEA-iMETI, Fontenay aux Roses, France
| | | | - Philippe Bourget
- 4 Necker Hospital , Assistance Publique-Hôpitaux de Paris, Paris, France
| | | | | | - Salima Hacein-Bey
- 6 Immunology Laboratory, Groupe Hospitalier Universitaire Paris-Sud , Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Marina Cavazzana
- 4 Necker Hospital , Assistance Publique-Hôpitaux de Paris, Paris, France
| | - Philippe Leboulch
- 2 CEA, Institute of Emerging Disease and Innovative Therapies (iMETI) , Fontenay aux Roses, France .,3 UMR 007, University of Paris 11 and CEA , CEA-iMETI, Fontenay aux Roses, France .,5 Mahidol University , Bangkok, Thailand .,7 Harvard Medical School and Genetics Division, Department of Medicine, Brigham & Women's Hospital , Boston, Massachusetts
| | - Emmanuel Payen
- 2 CEA, Institute of Emerging Disease and Innovative Therapies (iMETI) , Fontenay aux Roses, France .,3 UMR 007, University of Paris 11 and CEA , CEA-iMETI, Fontenay aux Roses, France .,8 INSERM , Paris, France
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29
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Kotterman MA, Chalberg TW, Schaffer DV. Viral Vectors for Gene Therapy: Translational and Clinical Outlook. Annu Rev Biomed Eng 2016; 17:63-89. [PMID: 26643018 DOI: 10.1146/annurev-bioeng-071813-104938] [Citation(s) in RCA: 319] [Impact Index Per Article: 35.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
In a range of human trials, viral vectors have emerged as safe and effective delivery vehicles for clinical gene therapy, particularly for monogenic recessive disorders, but there has also been early work on some idiopathic diseases. These successes have been enabled by research and development efforts focusing on vectors that combine low genotoxicity and immunogenicity with highly efficient delivery, including vehicles based on adeno-associated virus and lentivirus, which are increasingly enabling clinical success. However, numerous delivery challenges must be overcome to extend this success to many diseases; these challenges include developing techniques to evade preexisting immunity, to ensure more efficient transduction of therapeutically relevant cell types, to target delivery, and to ensure genomic maintenance. Fortunately, vector-engineering efforts are demonstrating promise in the development of next-generation gene therapy vectors that can overcome these barriers. This review highlights key historical trends in clinical gene therapy, the recent clinical successes of viral-based gene therapy, and current research that may enable future clinical application.
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Affiliation(s)
| | | | - David V Schaffer
- 4D Molecular Therapeutics, San Francisco, California 94107; .,University of California, Berkeley, California 94720-3220;
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30
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Chira S, Jackson CS, Oprea I, Ozturk F, Pepper MS, Diaconu I, Braicu C, Raduly LZ, Calin GA, Berindan-Neagoe I. Progresses towards safe and efficient gene therapy vectors. Oncotarget 2016; 6:30675-703. [PMID: 26362400 PMCID: PMC4741561 DOI: 10.18632/oncotarget.5169] [Citation(s) in RCA: 134] [Impact Index Per Article: 14.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2015] [Accepted: 08/22/2015] [Indexed: 12/11/2022] Open
Abstract
The emergence of genetic engineering at the beginning of the 1970′s opened the era of biomedical technologies, which aims to improve human health using genetic manipulation techniques in a clinical context. Gene therapy represents an innovating and appealing strategy for treatment of human diseases, which utilizes vehicles or vectors for delivering therapeutic genes into the patients' body. However, a few past unsuccessful events that negatively marked the beginning of gene therapy resulted in the need for further studies regarding the design and biology of gene therapy vectors, so that this innovating treatment approach can successfully move from bench to bedside. In this paper, we review the major gene delivery vectors and recent improvements made in their design meant to overcome the issues that commonly arise with the use of gene therapy vectors. At the end of the manuscript, we summarized the main advantages and disadvantages of common gene therapy vectors and we discuss possible future directions for potential therapeutic vectors.
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Affiliation(s)
- Sergiu Chira
- Research Center for Functional Genomics, Biomedicine and Translational Medicine, University of Medicine and Pharmacy "Iuliu Haţieganu", Cluj Napoca, Romania
| | - Carlo S Jackson
- Department of Immunology and Institute for Cellular and Molecular Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa
| | - Iulian Oprea
- Department of Oncology and Pathology, Cancer Center Karolinska, Karolinska Institutet and Karolinska University Hospital, Stockholm, Sweden
| | - Ferhat Ozturk
- Department of Molecular Biology and Genetics, Canik Başari University, Samsun, Turkey
| | - Michael S Pepper
- Department of Immunology and Institute for Cellular and Molecular Medicine, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa
| | | | - Cornelia Braicu
- Research Center for Functional Genomics, Biomedicine and Translational Medicine, University of Medicine and Pharmacy "Iuliu Haţieganu", Cluj Napoca, Romania
| | - Lajos-Zsolt Raduly
- Research Center for Functional Genomics, Biomedicine and Translational Medicine, University of Medicine and Pharmacy "Iuliu Haţieganu", Cluj Napoca, Romania.,Department of Physiopathology, Faculty of Veterinary Medicine, University of Agricultural Science and Veterinary Medicine, Cluj Napoca, Romania
| | - George A Calin
- Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA.,Center for RNA Interference and Non-Coding RNAs, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
| | - Ioana Berindan-Neagoe
- Research Center for Functional Genomics, Biomedicine and Translational Medicine, University of Medicine and Pharmacy "Iuliu Haţieganu", Cluj Napoca, Romania.,Department of Immunology, University of Medicine and Pharmacy "Iuliu Haţieganu", Cluj Napoca, Romania.,Department of Functional Genomics and Experimental Pathology, Oncological Institute "Prof. Dr. Ion Chiricuţă", Cluj Napoca, Romania.,Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston, TX, USA
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31
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Garcia-Gomez M, Calabria A, Garcia-Bravo M, Benedicenti F, Kosinski P, López-Manzaneda S, Hill C, del Mar Mañu-Pereira M, Martín MA, Orman I, Vives-Corrons JLL, Kung C, Schambach A, Jin S, Bueren JA, Montini E, Navarro S, Segovia JC. Safe and Efficient Gene Therapy for Pyruvate Kinase Deficiency. Mol Ther 2016; 24:1187-1198. [PMID: 27138040 PMCID: PMC5088764 DOI: 10.1038/mt.2016.87] [Citation(s) in RCA: 45] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2015] [Accepted: 03/25/2016] [Indexed: 12/17/2022] Open
Abstract
Pyruvate kinase deficiency (PKD) is a monogenic metabolic disease caused by mutations in the PKLR gene that leads to hemolytic anemia of variable symptomatology and that can be fatal during the neonatal period. PKD recessive inheritance trait and its curative treatment by allogeneic bone marrow transplantation provide an ideal scenario for developing gene therapy approaches. Here, we provide a preclinical gene therapy for PKD based on a lentiviral vector harboring the hPGK eukaryotic promoter that drives the expression of the PKLR cDNA. This therapeutic vector was used to transduce mouse PKD hematopoietic stem cells (HSCs) that were subsequently transplanted into myeloablated PKD mice. Ectopic RPK expression normalized the erythroid compartment correcting the hematological phenotype and reverting organ pathology. Metabolomic studies demonstrated functional correction of the glycolytic pathway in RBCs derived from genetically corrected PKD HSCs, with no metabolic disturbances in leukocytes. The analysis of the lentiviral insertion sites in the genome of transplanted hematopoietic cells demonstrated no evidence of genotoxicity in any of the transplanted animals. Overall, our results underscore the therapeutic potential of the hPGK-coRPK lentiviral vector and provide high expectations toward the gene therapy of PKD and other erythroid metabolic genetic disorders.
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MESH Headings
- Anemia, Hemolytic, Congenital Nonspherocytic/genetics
- Anemia, Hemolytic, Congenital Nonspherocytic/metabolism
- Anemia, Hemolytic, Congenital Nonspherocytic/therapy
- Animals
- Blood Cells/metabolism
- Cell Differentiation
- Disease Models, Animal
- Erythrocytes/cytology
- Erythrocytes/metabolism
- Erythropoiesis
- Genetic Therapy/adverse effects
- Genetic Therapy/methods
- Genetic Vectors/genetics
- Glycolysis
- Hematopoietic Stem Cell Transplantation
- Hematopoietic Stem Cells/cytology
- Hematopoietic Stem Cells/metabolism
- Humans
- Lentivirus/genetics
- Metabolic Networks and Pathways
- Metabolome
- Metabolomics
- Mice
- Mice, Transgenic
- Mutation
- Phenotype
- Pyruvate Kinase/deficiency
- Pyruvate Kinase/genetics
- Pyruvate Kinase/metabolism
- Pyruvate Metabolism, Inborn Errors/genetics
- Pyruvate Metabolism, Inborn Errors/metabolism
- Pyruvate Metabolism, Inborn Errors/therapy
- Transduction, Genetic
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Affiliation(s)
- Maria Garcia-Gomez
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) - Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Advanced Therapies Mixed Unit. Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Madrid, Spain
| | - Andrea Calabria
- San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientific Institute, Milan, Italy
| | - Maria Garcia-Bravo
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) - Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Advanced Therapies Mixed Unit. Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Madrid, Spain
| | - Fabrizio Benedicenti
- San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientific Institute, Milan, Italy
| | | | - Sergio López-Manzaneda
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) - Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Advanced Therapies Mixed Unit. Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Madrid, Spain
| | | | - María del Mar Mañu-Pereira
- Red Cell Pathology Laboratory. Hospital Clínic of Barcelona – Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain
| | - Miguel A Martín
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) - Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Advanced Therapies Mixed Unit. Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Madrid, Spain
| | - Israel Orman
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) - Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Advanced Therapies Mixed Unit. Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Madrid, Spain
| | - Joan-LLuis Vives-Corrons
- Red Cell Pathology Laboratory. Hospital Clínic of Barcelona – Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain
| | | | - Axel Schambach
- Institute of Experimental Hematology at Hannover Medical Hospital, Hannover, Germany
| | | | - Juan A Bueren
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) - Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Advanced Therapies Mixed Unit. Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Madrid, Spain
| | - Eugenio Montini
- San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), San Raffaele Scientific Institute, Milan, Italy
| | - Susana Navarro
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) - Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Advanced Therapies Mixed Unit. Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Madrid, Spain
| | - Jose C Segovia
- Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) - Centro de Investigaciones Biomédicas en Red de Enfermedades Raras (CIBERER), Madrid, Spain
- Advanced Therapies Mixed Unit. Instituto de Investigación Sanitaria-Fundación Jiménez Díaz (IIS-FJD), Madrid, Spain
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32
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de Dreuzy E, Bhukhai K, Leboulch P, Payen E. Current and future alternative therapies for beta-thalassemia major. Biomed J 2016; 39:24-38. [PMID: 27105596 PMCID: PMC6138429 DOI: 10.1016/j.bj.2015.10.001] [Citation(s) in RCA: 52] [Impact Index Per Article: 5.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/11/2015] [Accepted: 10/12/2015] [Indexed: 11/15/2022] Open
Abstract
Beta-thalassemia is a group of frequent genetic disorders resulting in the synthesis of little or no β-globin chains. Novel approaches are being developed to correct the resulting α/β-globin chain imbalance, in an effort to move beyond the palliative management of this disease and the complications of its treatment (e.g. life-long red blood cell transfusion, iron chelation, splenectomy), which impose high costs on healthcare systems. Three approaches are envisaged: fetal globin gene reactivation by pharmacological compounds injected into patients throughout their lives, allogeneic hematopoietic stem cell transplantation (HSCT), and gene therapy. HSCT is currently the only treatment shown to provide an effective, definitive cure for β-thalassemia. However, this procedure remains risky and histocompatible donors are identified for only a small fraction of patients. New pharmacological compounds are being tested, but none has yet made it into common clinical practice for the treatment of beta-thalassemia major. Gene therapy is in the experimental phase. It is emerging as a powerful approach without the immunological complications of HSCT, but with other possible drawbacks. Rapid progress is being made in this field, and long-term efficacy and safety studies are underway.
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Affiliation(s)
- Edouard de Dreuzy
- CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay aux Roses, France; University of Paris 11, CEA-iMETI, 92260 Fontenay aux Roses, France
| | - Kanit Bhukhai
- CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay aux Roses, France; University of Paris 11, CEA-iMETI, 92260 Fontenay aux Roses, France
| | - Philippe Leboulch
- CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay aux Roses, France; University of Paris 11, CEA-iMETI, 92260 Fontenay aux Roses, France; Department of Medicine, Harvard Medical School and Genetics Division, Brigham and Women's Hospital, Boston MA, USA; Mahidol University and Ramathibodi Hospital, Bangkok, Thailand
| | - Emmanuel Payen
- CEA, Institute of Emerging Diseases and Innovative Therapies, Fontenay aux Roses, France; University of Paris 11, CEA-iMETI, 92260 Fontenay aux Roses, France; INSERM, Paris, France.
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Abstract
Basic science advances in cancer immunotherapy have resulted in various treatments that have recently shown success in the clinic. Many of these therapies require the insertion of genes into cells to directly kill them or to redirect the host's cells to induce potent immune responses. Other analogous therapies work by modifying effector cells for improved targeting and enhanced killing of tumor cells. Initial studies done using γ-retroviruses were promising, but safety concerns centered on the potential for insertional mutagenesis have highlighted the desire to develop other options for gene delivery. Lentiviral vectors (LVs) have been identified as potentially more effective and safer alternative delivery vehicles. LVs are now in use in clinical trials for many different types of inherited and acquired disorders, including cancer. This review will discuss current knowledge of LVs and the applications of this viral vector-based delivery vehicle to cancer immunotherapy.
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Affiliation(s)
- Robyn Aa Oldham
- Department of Medical Biophysics, University of Toronto, 27 King's College Circle, Toronto, ON M5S, Canada
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Nagree MS, López-Vásquez L, Medin JA. Towards in vivo amplification: Overcoming hurdles in the use of hematopoietic stem cells in transplantation and gene therapy. World J Stem Cells 2015; 7:1233-1250. [PMID: 26730268 PMCID: PMC4691692 DOI: 10.4252/wjsc.v7.i11.1233] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/08/2015] [Accepted: 11/25/2015] [Indexed: 02/06/2023] Open
Abstract
With the advent of safer and more efficient gene transfer methods, gene therapy has become a viable solution for many inherited and acquired disorders. Hematopoietic stem cells (HSCs) are a prime cell compartment for gene therapy aimed at correcting blood-based disorders, as well as those amenable to metabolic outcomes that can effect cross-correction. While some resounding clinical successes have recently been demonstrated, ample room remains to increase the therapeutic output from HSC-directed gene therapy. In vivo amplification of therapeutic cells is one avenue to achieve enhanced gene product delivery. To date, attempts have been made to provide HSCs with resistance to cytotoxic drugs, to include drug-inducible growth modules specific to HSCs, and to increase the engraftment potential of transduced HSCs. This review aims to summarize amplification strategies that have been developed and tested and to discuss their advantages along with barriers faced towards their clinical adaptation. In addition, next-generation strategies to circumvent current limitations of specific amplification schemas are discussed.
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35
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Serrao E, Engelman AN. Sites of retroviral DNA integration: From basic research to clinical applications. Crit Rev Biochem Mol Biol 2015; 51:26-42. [PMID: 26508664 DOI: 10.3109/10409238.2015.1102859] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022]
Abstract
One of the most crucial steps in the life cycle of a retrovirus is the integration of the viral DNA (vDNA) copy of the RNA genome into the genome of an infected host cell. Integration provides for efficient viral gene expression as well as for the segregation of viral genomes to daughter cells upon cell division. Some integrated viruses are not well expressed, and cells latently infected with human immunodeficiency virus type 1 (HIV-1) can resist the action of potent antiretroviral drugs and remain dormant for decades. Intensive research has been dedicated to understanding the catalytic mechanism of integration, as well as the viral and cellular determinants that influence integration site distribution throughout the host genome. In this review, we summarize the evolution of techniques that have been used to recover and map retroviral integration sites, from the early days that first indicated that integration could occur in multiple cellular DNA locations, to current technologies that map upwards of millions of unique integration sites from single in vitro integration reactions or cell culture infections. We further review important insights gained from the use of such mapping techniques, including the monitoring of cell clonal expansion in patients treated with retrovirus-based gene therapy vectors, or patients with acquired immune deficiency syndrome (AIDS) on suppressive antiretroviral therapy (ART). These insights span from integrase (IN) enzyme sequence preferences within target DNA (tDNA) at the sites of integration, to the roles of host cellular proteins in mediating global integration distribution, to the potential relationship between genomic location of vDNA integration site and retroviral latency.
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Affiliation(s)
- Erik Serrao
- a Department of Cancer Immunology and Virology , Dana-Farber Cancer Institute , Boston , MA , USA
| | - Alan N Engelman
- a Department of Cancer Immunology and Virology , Dana-Farber Cancer Institute , Boston , MA , USA
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36
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Negre O, Bartholomae C, Beuzard Y, Cavazzana M, Christiansen L, Courne C, Deichmann A, Denaro M, de Dreuzy E, Finer M, Fronza R, Gillet-Legrand B, Joubert C, Kutner R, Leboulch P, Maouche L, Paulard A, Pierciey FJ, Rothe M, Ryu B, Schmidt M, von Kalle C, Payen E, Veres G. Preclinical evaluation of efficacy and safety of an improved lentiviral vector for the treatment of β-thalassemia and sickle cell disease. Curr Gene Ther 2015; 15:64-81. [PMID: 25429463 PMCID: PMC4440358 DOI: 10.2174/1566523214666141127095336] [Citation(s) in RCA: 88] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2014] [Revised: 11/05/2014] [Accepted: 11/14/2014] [Indexed: 01/27/2023]
Abstract
A previously published clinical trial demonstrated the benefit of autologous CD34(+) cells transduced with a selfinactivating lentiviral vector (HPV569) containing an engineered β-globin gene (β(A-T87Q)-globin) in a subject with β thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 β-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with β-thalassemia major and sickle cell disease.
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Affiliation(s)
| | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | | - Gabor Veres
- bluebird bio, 150 Second Street, Cambridge, MA 02141, USA.
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37
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Cunningham SC, Siew SM, Hallwirth CV, Bolitho C, Sasaki N, Garg G, Michael IP, Hetherington NA, Carpenter K, de Alencastro G, Nagy A, Alexander IE. Modeling correction of severe urea cycle defects in the growing murine liver using a hybrid recombinant adeno-associated virus/piggyBac transposase gene delivery system. Hepatology 2015; 62:417-28. [PMID: 26011400 DOI: 10.1002/hep.27842] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/28/2015] [Accepted: 04/07/2015] [Indexed: 12/17/2022]
Abstract
UNLABELLED Liver-targeted gene therapy based on recombinant adeno-associated viral vectors (rAAV) shows promising therapeutic efficacy in animal models and adult-focused clinical trials. This promise, however, is not directly translatable to the growing liver, where high rates of hepatocellular proliferation are accompanied by loss of episomal rAAV genomes and subsequently a loss in therapeutic efficacy. We have developed a hybrid rAAV/piggyBac transposon vector system combining the highly efficient liver-targeting properties of rAAV with stable piggyBac-mediated transposition of the transgene into the hepatocyte genome. Transposition efficiency was first tested using an enhanced green fluorescent protein expression cassette following delivery to newborn wild-type mice, with a 20-fold increase in stably gene-modified hepatocytes observed 4 weeks posttreatment compared to traditional rAAV gene delivery. We next modeled the therapeutic potential of the system in the context of severe urea cycle defects. A single treatment in the perinatal period was sufficient to confer robust and stable phenotype correction in the ornithine transcarbamylase-deficient Spf(ash) mouse and the neonatal lethal argininosuccinate synthetase knockout mouse. Finally, transposon integration patterns were analyzed, revealing 127,386 unique integration sites which conformed to previously published piggyBac data. CONCLUSION Using a hybrid rAAV/piggyBac transposon vector system, we achieved stable therapeutic protection in two urea cycle defect mouse models; a clinically conceivable early application of this technology in the management of severe urea cycle defects could be as a bridging therapy while awaiting liver transplantation; further improvement of the system will result from the development of highly human liver-tropic capsids, the use of alternative strategies to achieve transient transposase expression, and engineered refinements in the safety profile of piggyBac transposase-mediated integration.
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Affiliation(s)
- Sharon C Cunningham
- Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.,University of Sydney Medical School, Sydney, New South Wales, Australia
| | - Susan M Siew
- Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia
| | - Claus V Hallwirth
- Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia
| | - Christine Bolitho
- Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia
| | - Natsuki Sasaki
- Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia
| | - Gagan Garg
- Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.,Department of Chemistry and Biomolecular Sciences, Macquarie University, Macquarie Park, New South Wales, Australia
| | - Iacovos P Michael
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada
| | - Nicola A Hetherington
- Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia
| | - Kevin Carpenter
- Biochemical Genetics, The Children's Hospital at Westmead, Westmead, Sydney, New South Wales, Australia
| | - Gustavo de Alencastro
- Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia
| | - Andras Nagy
- Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Ontario, Canada.,Institute of Medical Science and Department of Obstetrics & Gynaecology, University of Toronto, Toronto, Ontario, Canada.,Department of Obstetrics & Gynaecology, University of Toronto, Toronto, Ontario, Canada
| | - Ian E Alexander
- Gene Therapy Research Unit, Children's Medical Research Institute and The Children's Hospital at Westmead, Westmead, New South Wales, Australia.,Discipline of Paediatrics and Child Health, The University of Sydney, Sydney, New South Wales, Australia
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Handorf AM, Sollinger HW, Alam T. Genetic Engineering of Surrogate <i>β</i> Cells for Treatment of Type 1 Diabetes Mellitus. ACTA ACUST UNITED AC 2015. [DOI: 10.4236/jdm.2015.54037] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022]
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A PiggyBac-mediated approach for muscle gene transfer or cell therapy. Stem Cell Res 2014; 13:390-403. [PMID: 25310255 DOI: 10.1016/j.scr.2014.08.007] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/02/2014] [Revised: 08/27/2014] [Accepted: 08/29/2014] [Indexed: 11/22/2022] Open
Abstract
An emerging therapeutic approach for Duchenne muscular dystrophy is the transplantation of autologous myogenic progenitor cells genetically modified to express dystrophin. The use of this approach is challenged by the difficulty in maintaining these cells ex vivo while keeping their myogenic potential, and ensuring sufficient transgene expression following their transplantation and myogenic differentiation in vivo. We investigated the use of the piggyBac transposon system to achieve stable gene expression when transferred to cultured mesoangioblasts and into murine muscles. Without selection, up to 8% of the mesoangioblasts expressed the transgene from 1 to 2 genomic copies of the piggyBac vector. Integration occurred mostly in intergenic genomic DNA and transgene expression was stable in vitro. Intramuscular transplantation of mouse Tibialis anterior muscles with mesoangioblasts containing the transposon led to sustained myofiber GFP expression in vivo. In contrast, the direct electroporation of the transposon-donor plasmids in the mouse Tibialis muscles in vivo did not lead to sustained transgene expression despite molecular evidence of piggyBac transposition in vivo. Together these findings provide a proof-of-principle that piggyBac transposon may be considered for mesoangioblast cell-based therapies of muscular dystrophies.
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40
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McAllister RG, Liu J, Woods MW, Tom SK, Rupar CA, Barr SD. Lentivector integration sites in ependymal cells from a model of metachromatic leukodystrophy: non-B DNA as a new factor influencing integration. MOLECULAR THERAPY. NUCLEIC ACIDS 2014; 3:e187. [PMID: 25158091 PMCID: PMC4221599 DOI: 10.1038/mtna.2014.39] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 02/10/2014] [Accepted: 07/07/2014] [Indexed: 02/07/2023]
Abstract
The blood–brain barrier controls the passage of molecules from the blood into the central nervous system (CNS) and is a major challenge for treatment of neurological diseases. Metachromatic leukodystrophy is a neurodegenerative lysosomal storage disease caused by loss of arylsulfatase A (ARSA) activity. Gene therapy via intraventricular injection of a lentiviral vector is a potential approach to rapidly and permanently deliver therapeutic levels of ARSA to the CNS. We present the distribution of integration sites of a lentiviral vector encoding human ARSA (LV-ARSA) in murine brain choroid plexus and ependymal cells, administered via a single intracranial injection into the CNS. LV-ARSA did not exhibit a strong preference for integration in or near actively transcribed genes, but exhibited a strong preference for integration in or near satellite DNA. We identified several genomic hotspots for LV-ARSA integration and identified a consensus target site sequence characterized by two G-quadruplex-forming motifs flanking the integration site. In addition, our analysis identified several other non-B DNA motifs as new factors that potentially influence lentivirus integration, including human immunodeficiency virus type-1 in human cells. Together, our data demonstrate a clinically favorable integration site profile in the murine brain and identify non-B DNA as a potential new host factor that influences lentiviral integration in murine and human cells.
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Affiliation(s)
- Robert G McAllister
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Center for Human Immunology, Western University, London, Ontario, Canada
| | - Jiahui Liu
- Department of Biochemistry, Western University, London, Ontario, Canada
| | - Matthew W Woods
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Center for Human Immunology, Western University, London, Ontario, Canada
| | - Sean K Tom
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Center for Human Immunology, Western University, London, Ontario, Canada
| | - C Anthony Rupar
- 1] Department of Biochemistry, Western University, London, Ontario, Canada [2] Department of Pathology and Laboratory Medicine, Western University, London, Ontario, Canada [3] Department of Pediatrics, Western University, London, Ontario, Canada [4] Children's Health Research Institute, Western University, London, Ontario, Canada
| | - Stephen D Barr
- Department of Microbiology and Immunology, Schulich School of Medicine and Dentistry, Center for Human Immunology, Western University, London, Ontario, Canada
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41
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Burnight ER, Wiley LA, Drack AV, Braun TA, Anfinson KR, Kaalberg EE, Halder JA, Affatigato LM, Mullins RF, Stone EM, Tucker BA. CEP290 gene transfer rescues Leber congenital amaurosis cellular phenotype. Gene Ther 2014; 21:662-72. [PMID: 24807808 DOI: 10.1038/gt.2014.39] [Citation(s) in RCA: 95] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2013] [Revised: 03/06/2014] [Accepted: 03/20/2014] [Indexed: 12/17/2022]
Abstract
Mutations in CEP290 are the most common cause of Leber congenital amaurosis (LCA), a severe inherited retinal degenerative disease for which there is currently no cure. Autosomal recessive CEP290-associated LCA is a good candidate for gene replacement therapy, and cells derived from affected individuals give researchers the ability to study human disease and therapeutic gene correction in vitro. Here we report the development of lentiviral vectors carrying full-length CEP290 for the purpose of correcting the CEP290 disease-specific phenotype in human cells. A lentiviral vector containing CMV-driven human full-length CEP290 was constructed. Following transduction of patient-specific, iPSC-derived, photoreceptor precursor cells, reverse transcriptase-PCR analysis and western blotting revealed vector-derived expression. As CEP290 is important in ciliogenesis, the ability of fibroblast cultures from CEP290-associated LCA patients to form cilia was investigated. In cultures derived from these patients, fewer cells formed cilia compared with unaffected controls. Cilia that were formed were shorter in patient-derived cells than in cells from unaffected individuals. Importantly, lentiviral delivery of CEP290 rescued the ciliogenesis defect. The successful construction and viral transfer of full-length CEP290 brings us closer to the goal of providing gene- and cell-based therapies for patients affected with this common form of LCA.
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Affiliation(s)
- E R Burnight
- Department of Opthalmology and Visual Sciences, Stephen A Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - L A Wiley
- Department of Opthalmology and Visual Sciences, Stephen A Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - A V Drack
- Department of Opthalmology and Visual Sciences, Stephen A Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - T A Braun
- 1] Department of Opthalmology and Visual Sciences, Stephen A Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA [2] Biomedical Engineering, University of Iowa, Iowa City, IA, USA
| | - K R Anfinson
- Department of Opthalmology and Visual Sciences, Stephen A Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - E E Kaalberg
- Department of Opthalmology and Visual Sciences, Stephen A Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - J A Halder
- Department of Opthalmology and Visual Sciences, Stephen A Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - L M Affatigato
- Department of Opthalmology and Visual Sciences, Stephen A Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - R F Mullins
- Department of Opthalmology and Visual Sciences, Stephen A Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
| | - E M Stone
- 1] Department of Opthalmology and Visual Sciences, Stephen A Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA [2] Howard Hughes Medical Institute, University of Iowa, Iowa City, IA, USA
| | - B A Tucker
- Department of Opthalmology and Visual Sciences, Stephen A Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Iowa City, IA, USA
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Tubsuwan A, Abed S, Deichmann A, Kardel MD, Bartholomä C, Cheung A, Negre O, Kadri Z, Fucharoen S, von Kalle C, Payen E, Chrétien S, Schmidt M, Eaves CJ, Leboulch P, Maouche-Chrétien L. Parallel assessment of globin lentiviral transfer in induced pluripotent stem cells and adult hematopoietic stem cells derived from the same transplanted β-thalassemia patient. Stem Cells 2014; 31:1785-94. [PMID: 23712774 DOI: 10.1002/stem.1436] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2012] [Revised: 04/21/2013] [Accepted: 05/02/2013] [Indexed: 02/03/2023]
Abstract
A patient with β(E)/β(0) -thalassemia major was converted to transfusion-independence 4.5 years ago by lentiviral gene transfer in hematopoietic stem cells while showing a myeloid-biased cell clone. Induced pluripotent stem cells (iPSCs) are a potential alternative source of hematopoietic stem cells. If fetal to adult globin class, switching does not occur in vivo in iPSC-derived erythroid cells, β-globin gene transfer would be unnecessary. To investigate both vector integration skewing and the potential use of iPSCs for the treatment of thalassemia, we derived iPSCs from the thalassemia gene therapy patient and compared iPSC-derived hematopoietic cells to their natural isogenic somatic counterparts. In NSG immunodeficient mice, embryonic to fetal and a partial fetal to adult globin class switching were observed, indicating that the gene transfer is likely necessary for iPSC-based therapy of the β-hemoglobinopathies. Lentivector integration occurred in regions of low and high genotoxicity. Surprisingly, common integration sites (CIS) were identified across those iPSCs and cells retrieved from isogenic and nonisogenic gene therapy patients with β-thalassemia and adrenoleukodystrophy, respectively. This suggests that CIS observed in the absence of overt tumorigenesis result from nonrandom lentiviral integration rather than oncogenic in vivo selection. These findings bring the use of iPSCs closer to practicality and further clarify our interpretation of genome-wide lentivector integration.
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Affiliation(s)
- Alisa Tubsuwan
- CEA, Institute of Emerging Diseases and Innovative Therapies (iMETI), Fontenay aux Roses, France; INSERM U962 and University Paris Sud 11; Thalassemia Research Centre, Institute of Molecular Biosciences, Mahidol University, Nakornpathom, Thailand; Department of Biochemistry, Faculty of Medicine, Siriraj Hospital, Mahidol University, Nakornpathom, Thailand
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Beard BC, Adair JE, Trobridge GD, Kiem HP. High-throughput genomic mapping of vector integration sites in gene therapy studies. Methods Mol Biol 2014; 1185:321-44. [PMID: 25062639 DOI: 10.1007/978-1-4939-1133-2_22] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Abstract
Gene therapy has enormous potential to treat a variety of infectious and genetic diseases. To date hundreds of patients worldwide have received hematopoietic cell products that have been gene-modified with retrovirus vectors carrying therapeutic transgenes, and many patients have been cured or demonstrated disease stabilization as a result (Adair et al., Sci Transl Med 4:133ra57, 2012; Biffi et al., Science 341:1233158, 2013; Aiuti et al., Science 341:1233151, 2013; Fischer et al., Gene 525:170-173, 2013). Unfortunately, for some patients the provirus integration dysregulated the expression of nearby genes leading to clonal outgrowth and, in some cases, cancer. Thus, the unwanted side effect of insertional mutagenesis has become a major concern for retrovirus gene therapy. The careful study of retrovirus integration sites (RIS) and the contribution of individual gene-modified clones to hematopoietic repopulating cells is of crucial importance for all gene therapy studies. Supporting this, the US Food and Drug Administration (FDA) has mandated the careful monitoring of RIS in all clinical trials of gene therapy. An invaluable method was developed: linear amplification mediated-polymerase chain reaction (LAM-PCR) capable of analyzing in vitro and complex in vivo samples, capturing valuable genomic information directly flanking the site of provirus integration. Linking this method and similar methods to high-throughput sequencing has now made possible an unprecedented understanding of the integration profile of various retrovirus vectors, and allows for sensitive monitoring of their safety. It also allows for a detailed comparison of improved safety-enhanced gene therapy vectors. An important readout of safety is the relative contribution of individual gene-modified repopulating clones. One limitation of LAM-PCR is that the ability to capture the relative contribution of individual clones is compromised because of the initial linear PCR common to all current methods. Here, we describe an improved protocol developed for efficient capture, sequencing, and analysis of RIS that preserves gene-modified clonal contribution information. We also discuss methods to assess dominant/overrepresented gene-modified clones in preclinical and clinical models.
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Affiliation(s)
- Brian C Beard
- Fred Hutchinson Cancer Research Center, University of Washington School of Medicine, Seattle, WA, USA
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44
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Breda L, Rivella S, Zuccato C, Gambari R. Combining gene therapy and fetal hemoglobin induction for treatment of β-thalassemia. Expert Rev Hematol 2013; 6:255-64. [PMID: 23782080 DOI: 10.1586/ehm.13.24] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
β-thalassemias are caused by nearly 300 mutations of the β-globin gene, leading to a low or absent production of adult hemoglobin (HbA). Two major therapeutic approaches have recently been proposed: gene therapy and induction of fetal hemoglobin (HbF) with the objective of achieving clinically relevant levels of Hbs. The objective of this article is to describe the development of therapeutic strategies based on a combination of gene therapy and induction of HbFs. An increase of β-globin gene expression in β-thalassemia cells can be achieved by gene therapy, although de novo production of clinically relevant levels of adult Hb may be difficult to obtain. On the other hand, an increased production of HbF is beneficial in β-thalassemia. The combination of gene therapy and HbF induction appears to be a pertinent strategy to achieve clinically relevant results.
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Affiliation(s)
- Laura Breda
- Department of Pediatrics, Division of Hematology-Oncology, Weill Cornell Medical College, New York, NY, USA.
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45
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Recent progresses in gene delivery-based bone tissue engineering. Biotechnol Adv 2013; 31:1695-706. [DOI: 10.1016/j.biotechadv.2013.08.015] [Citation(s) in RCA: 86] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/07/2013] [Revised: 07/24/2013] [Accepted: 08/19/2013] [Indexed: 12/18/2022]
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46
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Dismuke D, Samulski RJ. Hepatic gene therapy using lentiviral vectors: has safety been established? Hepatology 2013; 58:13-4. [PMID: 23695955 DOI: 10.1002/hep.26460] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/28/2013] [Accepted: 04/11/2013] [Indexed: 01/08/2023]
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47
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Deng Y, Zhou H, Zou D, Xie Q, Bi X, Gu P, Fan X. The role of miR-31-modified adipose tissue-derived stem cells in repairing rat critical-sized calvarial defects. Biomaterials 2013; 34:6717-28. [PMID: 23768901 DOI: 10.1016/j.biomaterials.2013.05.042] [Citation(s) in RCA: 103] [Impact Index Per Article: 8.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/19/2013] [Accepted: 05/22/2013] [Indexed: 12/20/2022]
Abstract
With the increasing application of microRNAs (miRNAs) in the treatment and monitoring of different diseases, miRNAs have become an important tool in biological and medical research. Recent studies have proven that miRNAs are involved in the osteogenic differentiation of stem cells. However, few studies have reported the use of miRNA-modified adult stem cells to repair critical-sized defects (CSDs) using tissue engineering technology. It is known that miR-31 is a pleiotropically acting miRNA that inhibits cancer metastasis and targets special AT-rich sequence-binding protein 2 (Satb2) in fibroblasts. However, it is not clear whether the function of miR-31 is to enhance adipose tissue-derived stem cell (ASC) osteogenesis, along with its association with Satb2, during osteogenic differentiation and bone regeneration. In this study, we systematically evaluated the function of miR-31 in enhancing ASC osteogenesis and the therapeutic potential of miR-31-modified ASCs in a rat CSD model with β-tricalcium phosphate (β-TCP) scaffolds. ASCs were treated with lentivirus (Lenti)-miR-31, Lenti-as-miR-31 (antisense) or Lenti-NC (negative control). These genetically modified ASCs were then combined with β-TCP scaffolds to repair CSDs in rats. The results showed that in cultured ASCs in vitro, Lenti-as-miR-31 significantly enhanced osteogenic mRNA and protein expression when compared with the Lenti-NC group. Moreover, we firstly found that a Runt-related transcription factor 2 (Runx2), Satb2 and miR-31 regulatory loop triggered by bone morphogenetic protein-2 (BMP-2) plays an important role in ASCs' osteogenic differentiation and bone regeneration. More importantly, we found that miR-31-knockdown ASCs dramatically improved the repair of CSDs, including increased bone volume, increased bone mineral density (BMD) and decreased scaffold residue in vivo. These data confirm the essential role of miR-31-modified ASCs in osteogenesis in vitro and in vivo.
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Affiliation(s)
- Yuan Deng
- Department of Ophthalmology, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
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48
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Deyle DR, Khan IF, Ren G, Russell DW. Lack of genotoxicity due to foamy virus vector integration in human iPSCs. Gene Ther 2013; 20:868-73. [PMID: 23388702 PMCID: PMC3655141 DOI: 10.1038/gt.2013.6] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/28/2012] [Revised: 12/13/2012] [Accepted: 12/27/2012] [Indexed: 12/17/2022]
Abstract
Integrating vectors can lead to the dysregulation of nearby chromosomal genes, with important consequences for clinical trials and cellular engineering. This includes the retroviral and lentiviral vectors commonly used for deriving induced pluripotent stem cells (iPSCs). We previously used integrating foamy virus (FV) vectors expressing OCT4, SOX2, MYC, and KLF4 to reprogram osteogenesis imperfecta mesenchymal stem cells (MSCs). Here we have studied the effects of 10 FV vector proviruses on neighboring gene expression in four iPSC lines and their corresponding iPSC-derived mesenchymal stem cells (iMSCs). Gene expression profiles in these iPSC lines showed that none of the 38 genes within 300 kb up- or downstream of integrated proviruses had a significant difference in mRNA levels, including 5 genes with proviruses in their transcription units. In the iMSCs derived from these iPSCs, the same type of analysis showed a single dysregulated transcript out of 46 genes found near proviruses. This frequency of dysregulation was similar to that of genes lacking nearby proviruses, so it may have been due to interclonal variation and/or measurement inaccuracies. While the number of integration sites examined in this paper is limited, our results suggest that integrated FV proviruses do not impact the expression of chromosomal genes in pluripotent human stem cells or their differentiated derivatives. This interpretation is consistent with previous reports that FV vectors have minimal genotoxicity, even when integrating near or within genes.
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Affiliation(s)
- D R Deyle
- Departments of Medicine, University of Washington, Seattle, WA 98195, USA
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Long-term in vivo monitoring of mouse and human hematopoietic stem cell engraftment with a human positron emission tomography reporter gene. Proc Natl Acad Sci U S A 2013; 110:1857-62. [PMID: 23319634 DOI: 10.1073/pnas.1221840110] [Citation(s) in RCA: 36] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
Positron emission tomography (PET) reporter genes allow noninvasive whole-body imaging of transplanted cells by detection with radiolabeled probes. We used a human deoxycytidine kinase containing three amino acid substitutions within the active site (hdCK3mut) as a reporter gene in combination with the PET probe [(18)F]-L-FMAU (1-(2-deoxy-2-(18)fluoro-β-L-arabinofuranosyl)-5-methyluracil) to monitor models of mouse and human hematopoietic stem cell (HSC) transplantation. These mutations in hdCK3mut expanded the substrate capacity allowing for reporter-specific detection with a thymidine analog probe. Measurements of long-term engrafted cells (up to 32 wk) demonstrated that hdCK3mut expression is maintained in vivo with no counter selection against reporter-labeled cells. Reporter cells retained equivalent engraftment and differentiation capacity being detected in all major hematopoietic lineages and tissues. This reporter gene and probe should be applicable to noninvasively monitor therapeutic cell transplants in multiple tissues.
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50
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Abstract
Abstract
High-level production of β-globin, γ-globin, or therapeutic mutant globins in the RBC lineage by hematopoietic stem cell gene therapy ameliorates or cures the hemoglobinopathies sickle cell disease and beta thalassemia, which are major causes of morbidity and mortality worldwide. Considerable efforts have been made in the last 2 decades in devising suitable gene-transfer vectors and protocols to achieve this goal. Five years ago, the first βE/β0-thalassemia major (transfusion-dependent) patient was treated by globin lentiviral gene therapy without injection of backup cells. This patient has become completely transfusion independent for the past 4 years and has global amelioration of the thalassemic phenotype. Partial clonal dominance for an intragenic site (HMGA2) of chromosomal integration of the vector was observed in this patient without a loss of hematopoietic homeostasis. Other patients are now receiving transplantations while researchers are carefully weighing the benefit/risk ratio and continuing the development of further modified vectors and protocols to improve outcomes further with respect to safety and efficacy.
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