Study Hgb-205: Outcomes of Gene Therapy for Hemoglobinopathies Via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex Vivo with a Lentiviral βΑ-T87Q-Globin Vector (LentiGlobin® BB305 Drug Product)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4797-4797 ◽  
Author(s):  
Marina Cavazzana ◽  
Jean-Antoine Ribeil ◽  
Emmanuel Payen ◽  
Felipe Suarez ◽  
Yves Beuzard ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adverse Events ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Drug Product ◽  
Thalassemia Major ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Equity Ownership

Abstract Background: In patients with β-thalassemia major, hematopoietic stem cell (HSC) gene therapy has the potential to induce production of β-globin, γ-globin or modified β-globin in the red blood cell lineage and reduce or stop the need for blood transfusions. We have previously presented early results for 2 subjects with β0/βE -thalassemia major that suggested that transplantation with autologous CD34+ cells transduced with a replication-defective, self-inactivating LentiGlobin BB305 lentiviral vector containing an engineered β-globin gene (βA-T87Q) resulted in near-normal levels of total hemoglobin (Hb) early after HSC infusion. Herein, we provide additional follow-up data on these two subjects. Subjects and Methods: After obtaining informed consent, subjects with β-thalassemia major underwent HSC collection via peripheral blood apheresis and CD34+ cells were selected. Estimation of the mean ex- vivo vector copy number (VCN) was obtained by quantitative PCR performed on pooled colony-forming progenitors. Subjects underwent myeloablation with intravenous busulfan, followed by infusion of transduced CD34+ cells. Subjects were monitored for hematological engraftment, βA-T87Q-globin expression (by high performance liquid chromatography) and transfusion requirements. Integration site analysis (ISA, by linear amplification-mediated PCR and high-throughput sequencing on nucleated cells) and replication-competent lentivirus (RCL) assays were performed. Results: As of 31 July 2014, two subjects with β0/βE thalassemia major (Subjects 1201 and 1202) have undergone infusion with drug product. The outcome of these two subjects to date is shown in Table 1. The initial safety profile is consistent with myeloablation, without serious adverse events or drug product-related adverse events. Both subjects remain transfusion independent. ISA analyses in both the subjects at 3 months shows polyclonal reconstitution. An additional 2 subjects have been enrolled in this study but have not yet undergone drug product infusion. Conclusion: In the first two subjects, early transfusion independence was achieved and has been maintained as of 31 July 2014. Further follow up data on these two subjects and additional data on subjects who have undergone drug product infusion in this study will be presented. Gene therapy using autologous HSC transduced with LentiGlobin BB305 lentiviral vector is a promising approach for the treatment of patients with β-thalassemia major. Abstract 4797. Table 1. Preliminary Results of Dosing Parameters and Transplantation Outcomes Subject Age (years) and gender Genotype BB305 Drug Product Day of Neutrophil Engraftment Drug Product-related Adverse Events Day of last pRBC transfusion Day of last follow up βA-T87Q-Hb at last follow-up visit /Total Hb (g/dL) VCNa CD34+ cell dose (x106 per kg) 1201 19 F β0/βE 1.5 8.9 Day +13 None Day +10 Day +180 7.2/10.2 1202 16 M β0/βE 2.1 13.6 Day +15 None Day +12 Day +90 6.8/11.0 As of 31 July 2014 a VCN, mean vector copy number Disclosures Payen: bluebird bio, Inc: Consultancy. Beuzard:bluebird bio, Inc: Consultancy, Equity Ownership. Sandler:bluebird bio, Inc: Employment, Equity Ownership. Soni:bluebird bio, Inc.: Employment, Equity Ownership. De Montalembert:Novartis : Speakers Bureau. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Initial Results from the Northstar Study (HGB-204): A Phase 1/2 Study of Gene Therapy for β-Thalassemia Major Via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex Vivo with a Lentiviral βΑ-T87Q -Globin Vector (LentiGlobin BB305 Drug Product)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 549-549 ◽  
Author(s):  
Alexis A. Thompson ◽  
John E Rasko ◽  
Suradej Hongeng ◽  
Janet L. Kwiatkowski ◽  
Gary Schiller ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Adverse Events ◽  
Research Funding ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Drug Product ◽  
Thalassemia Major ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Background: Hematopoietic stem cell (HSC) gene therapy has the potential to induce globin production and mitigate the need for blood transfusions in β-thalassemia major. Promising early results for 2 subjects with β0/βE -thalassemia major in the ongoing HGB-205 study suggested that transplantation with autologous CD34+ cells transduced with a replication-defective, self-inactivating LentiGlobin BB305 lentiviral vector containing an engineered β-globin gene (βA-T87Q) can be safe and yield robust production of βA-T87Qglobin resulting in rapid transfusion independence. The Northstar study (HGB-204), which uses the same lentivirus vector and analogous study design as study HGB-205, is multi-center and multi-national, and centralizes drug product manufacturing. Herein, we provide the initial data on subjects enrolled and treated in this study. Subjects and Methods: Transfusion-dependent subjects with β-thalassemia major undergo HSC collection via mobilized peripheral blood apheresis and CD34+ cells are selected. Estimation of the mean ex-vivo vector copy number (VCN) is obtained by quantitative PCR performed on pooled colony-forming progenitors. Subjects undergo myeloablation with intravenous busulfan, followed by infusion of transduced CD34+ cells. Subjects are monitored for hematologic engraftment, βA-T87Q -globin expression (by high performance liquid chromatography) and transfusion requirements. Integration site analysis (ISA, by linear amplification-mediated PCR and high-throughput sequencing on nucleated cells) and replication-competent lentivirus (RCL) assays are performed for safety monitoring. Results: As of 31 July 2014, 3 subjects have undergone HSC collection and ex-vivo LentiGlobin BB305 gene transfer. One subject (Subject 1102) has undergone myeloablation and drug product infusion. Outcomes data are shown in Table 1. The initial safety profile is consistent with myeloablation, without serious adverse events or gene therapy-related adverse events. This subject has increasing production of βA-T87Q-globin: the proportion of βA-T87Qglobin was 1.5%, 10.9% and 19.5% of total Hb at 1, 2 and 3 months post-infusion, respectively. This subject received pRBCs on Day +14 following drug product infusion and required no further transfusions until a single unit of pRBC was transfused on Day +96 for a Hb of 8.6 g/dL and fatigue. Two additional subjects have undergone drug product manufacture and are awaiting transplantation. Safety data related to ISA and RCL assays are pending. Abstract 549. Table 1 Preliminary results of dosing parameters and transplantation outcomes Subject Age (years) and Gender Genotype BB305 Drug Product Day of Neutrophil Engraftment Drug Product- related Adverse Events βA-T87Q-Hb at last follow-up visit /Total Hb (g/dL) VCN CD34+ cell dose (x106 per kg) 1102 18 F β0/βE 1.0/1.1a 6.5 Day +17 None 1.77/8.6 1104 21 F β0/βE 0.7/0.7a 5.4 P P P 1106 20 F β0/β0 1.5 12.3 P P P As of 31 July 2014; P, pending a If more than one drug product were manufactured, the VCN of each drug product lot is presented. Conclusion: The first subject treated on the Northstar study has safely undergone drug product infusion with autologous HSCs transduced with LentiGlobin BB305 lentiviral vector and is producing steadily increasing amounts of βA-T87Q-globin. Additional follow-up of this subject plus data on additional subjects who undergo drug product infusion will be presented at the meeting. Ex-vivo gene transfer of βA-T87Q-globin to autologous HSCs is a promising approach for the treatment of patients with β-thalassemia major. Disclosures Thompson: ApoPharma: Consultancy; Novartis: Consultancy, Research Funding; Amgen: Research Funding; Glaxo Smith Kline: Research Funding; Mast: Research Funding; Eli Lilly: Research Funding. Kwiatkowski:Shire Pharmaceuticals and Sideris Pharmaceuticals: Consultancy. Schiller:Sunesis, Amgen, Pfizer, Bristol Myers Squibb: Research Funding. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Petrusich:bluebird bio, Inc.: Employment, Equity Ownership. Soni:bluebird bio, Inc.: Employment. Walters:Via Cord and AllCells, Inc.: Medical Director Other.

Update of Results from the Northstar Study (HGB-204): A Phase 1/2 Study of Gene Therapy for Beta-Thalassemia Major Via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex-Vivo with a Lentiviral Beta AT87Q-Globin Vector (LentiGlobin BB305 Drug Product)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 201-201 ◽  
Author(s):  
Mark C. Walters ◽  
John Rasko ◽  
Suradej Hongeng ◽  
Janet Kwiatkowski ◽  
Gary J Schiller ◽  
...  
Keyword(s):  
Adverse Events ◽  
Research Funding ◽  
Lentiviral Vector ◽  
Drug Product ◽  
Thalassemia Major ◽  
Hematopoietic Stem ◽  
Transfusion Independence ◽  
Cd34 Cells ◽  
Post Infusion ◽  
Equity Ownership

Abstract Background: Hematopoietic stem cell (HSC) gene transfer has the potential to induce globin production and mitigate or eliminate blood transfusions in patients with β-thalassemia major. Previously reported early results in subjects with β-thalassemia major participating in the ongoing HGB-205 and HGB-204 (Northstar) studies suggest that transplantation with autologous CD34+ cells transduced with a replication-defective, self-inactivating LentiGlobin BB305 lentiviral vector containing an engineered βA-T87Q-globin gene (LentiGlobin BB305 Drug Product) has a positive safety profile and leads to βA-T87Q globin production and can lead to transfusion independence. Here we provide an update on subjects treated with the LentiGlobin BB305 Drug Product in the Northstar study. Subjects and Methods: HSCs from transfusion-dependent β-thalassemia major subjects are mobilized by a combination of G-CSF and plerixafor, collected via apheresis, and CD34+ cells are selected and transduced with LentiGlobin BB305 lentiviral vector to produce drug product. Subjects undergo myeloablation with busulfan before LentiGlobin BB305 Drug Product infusion. Subjects are monitored for hematologic recovery, vector copy number, βA-T87Q-globinexpression, adverse events, and transfusion requirements after drug product infusion. Integration site analysis (ISA) and replication-competent lentivirus (RCL) assays are performed as part of the safety monitoring. Results: As of 31 July 2015, 10 subjects with transfusion-dependent β-thalassemia (β0/β0 [n=5], β0/βE [n=3], β0/β+ [n=1], and 1 heterozygous β0 genotype) have been infused with drug product. Before enrollment, subjects had received a median of 170 ml/kg/year (range: 137 to 233 ml/kg/year) of red blood cell (RBC) transfusions. The median age of the subjects was 26 years (range: 18 to 35 years), with 8 females and 2 males, and subjects received a median of 7.9 x 106 CD34+ cells/kg (range: 5.3 to 15.0 x106/kg) with median vector copy number of 0.8 (range: 0.3 to 1.5 copies/diploid genome). All subjects engrafted after drug product infusion; median time to engraftment was Day +17 (range +13 to +29) for neutrophils and Day +30 (range: +17 to +35) for platelets. The toxicity profile observed was consistent with autologous transplantation. To date, no ≥ Grade 3 drug-product-related adverse events have been observed, and there is no evidence of clonal dominance or replication competent lentivirus after a median follow-up of 198 days (range: 65 to 492 days) post-infusion. All subjects have detectable vector-derived HbAT87Q with a median peak level of 5.4 g/dL (range: 2.4 to 8.9 g/dL) ≥ 3 months post-infusion. The 7 subjects (3 β0/β0, 2 β0/βE, 1 β0/β+ and 1 heterozygous β0 genotype) monitored for at least 6 months post-infusion are making a median of 5.2 g/dL (range: 1.9 to 8.2 g/dL) of HbAT87Q with total Hb ranging from 8.5 to 11.1 g/dL at their last visit. Of these 7 subjects, 2 β0/β0 subjects have received a single RBC transfusion post-discharge, 1 β0/β0 subject remains transfusion dependent, and all 4 non-β0/β0 subjects have been RBC transfusion-free for ≥ 90 days (median 287 days of transfusion independence, range 171 to 396 days). Conclusion: Ten subjects with β-thalassemia major in the Northstar study have been infused with LentiGlobin BB305 Drug Product without ≥ Grade 3 drug product-related adverse events or evidence of clonal dominance. To date, LentiGlobin derived HbAT87Q is detectable in all infused subjects leading to transfusion independence or reduction in transfusion needs in almost all subjects. Gene therapy with the LentiGlobin BB305 is a promising modality for the treatment of patients with β-thalassemia major. Disclosures Walters: ViaCord and AllCells, Inc: Other: Medical director. Kwiatkowski:ISIS: Membership on an entity's Board of Directors or advisory committees; Shire Pharmaceuticals and Sideris Pharmaceuticals: Consultancy; Sideris Pharmaceuticals: Consultancy; Novartis: Research Funding. Schiller:Sunesis: Honoraria, Research Funding. von Kalle:bluebird bio, Inc.: Consultancy. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Petrusich:bluebird bio, Inc.: Employment, Equity Ownership. Soni:bluebird bio, Inc.: Employment, Equity Ownership. Thompson:bluebird bio, Inc.: Consultancy, Research Funding.

Outcomes of Gene Therapy for Severe Sickle Disease and Beta-Thalassemia Major Via Transplantation of Autologous Hematopoietic Stem Cells Transduced Ex Vivo with a Lentiviral Beta AT87Q-Globin Vector

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 202-202 ◽  
Author(s):  
Marina Cavazzana ◽  
Jean-Antoine Ribeil ◽  
Emmanuel Payen ◽  
Felipe Suarez ◽  
Yves Beuzard ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Lentiviral Vector ◽  
Drug Product ◽  
Thalassemia Major ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Post Infusion ◽  
The Subject ◽  
Equity Ownership

Abstract Background: In patients with hemoglobinopathies, hematopoietic stem cell (HSC) gene therapy has the potential to induce production of functional β-globin in the red blood cell lineage with the aim of reducing or eliminating the symptoms of disease. Previous results from 1 subject with severe sickle cell disease (SCD; 6 months follow-up) and 2 subjects with β0/βE-thalassemia major (up to 15 months follow-up) treated in clinical study HGB-205 suggested that transplantation with autologous CD34+ cells transduced with the LentiGlobin BB305 lentiviral vector containing an engineered βA-T87Q-globin gene (LentiGlobin BB305 Drug Product) resulted in near-normal levels of total hemoglobin (Hb) and rapid clinical improvement. Here we provide data on a new subject enrolled and additional follow-up data on the 3 subjects previously presented in Study HGB-205. Subjects and Methods: Subjects with severe SCD underwent HSC collection via bone marrow harvest, while subjects with β-thalassemia major underwent HSC collection via peripheral blood apheresis following mobilization. CD34+ cells were selected and transduced with LentiGlobin BB305 lentiviral vector to produce the drug product. Subjects underwent myeloablation with intravenous busulfan, followed by infusion of drug product. Subjects were monitored for hematological engraftment, vector copy number, βA-T87Q-globin expression, adverse events and transfusion requirements. Integration site analysis (ISA) and replication-competent lentivirus (RCL) assays were performed. Prophylactic RBC transfusions were continued in subjects with SCD who were on chronic transfusion pre-transplant to maintain HbS <30%, followed by gradual taper over time. Results: As of 31 July 2015, 1 subject with severe SCD (Subject 1204, βS/βS with multiple vaso-occlusive crises, silent infarct, acute chest syndrome, and on prophylactic transfusions) and 3 subjects withβ-thalassemia major (Subjects 1201, 1202 and 1203) have been infused with the LentiGlobin BB305 Drug Product. The outcome of these subjects to date is shown in Table 1. No subject has experienced a drug product-related adverse event, and ISA analyses demonstrate highly polyclonal reconstitution without clonal dominance. The subject with severe SCD is producing approximately 51.5% of anti-sickling hemoglobin (48% HbAT87Q, 1.8% HbF, 1.7% HbA2) at 9 months post-infusion. This subject has not had a post-infusion hospitalization for a SCD-related event despite stopping chronic transfusions at Day +88. Both subjects with β0/βE-thalassemia major have remained transfusion-free for at least 15 months post-infusion, with a consistent expression of βA-T87Q-globin; the subject with β0/β0-thalassemia major has only had 1 month follow-up post-drug product infusion to date. Conclusion: The subject with severe SCD is producing approximately 51.5% anti-sickling globins with HbS of 48.5% and remains free of SCD-related events despite stopping chronic transfusion therapy. Two subjects with β0/βE-thalassemia major remain transfusion-free for at least 15 months post infusion of LentiGlobin BB305 Drug Product. Gene therapy using autologous HSC transduced with LentiGlobin BB305 lentiviral vector is a promising approach for the treatment of patients with hemoglobinopathies. Table 1. Demographics and Transplantation Outcomes Subject Age (years)/ Sex (M/F) Genotype BB305 Drug Product Day of Neutrophil Engraftment Drug Product- related Adverse Events Day of Last pRBC Transfusion Last Study Visit (Months) Hb at Last Visit (g/dL) VCNa CD34+ cell dose (x106 per kg) Subject with severe sickle cell disease HbAT87Q/HbF/ HbS/Total Hb 1204 13/ M βS/βS 1.2 / 1.0 5.6 Day +37 None Day +88 9M 5.5/0.2/5.5/11.4 Subjects with β-thalassemia major Hb AT87Q/ Total Hb 1201 18/ F β0/βE 1.5 8.9 Day +13 None Day +10 18M 7.8/10.7 1202 16/ M β0/βE 2.1 13.6 Day +15 None Day +12 15M 9.7/12.8 1203 19/ M β0/β0 0.8 8.8 Day +28 None Day +15 1M Pending/9.2 As of 31 July 2015 aVCN, vector copy number; F=female; M= Male for gender, and months for day of last follow-up Disclosures Payen: bluebrid bio: Consultancy. Beuzard:bluebird bio Inc: Consultancy, Equity Ownership. von Kalle:bluebird bio, Inc.: Consultancy. Sandler:bluebird bio, Inc.: Employment, Equity Ownership. Soni:bluebird bio, Inc.: Employment, Equity Ownership. De Montalembert:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Leboulch:bluebird bio: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

scholarly journals Extensive Metabolic Correction of Hurler Disease By Hematopoietic Stem Cell-Based Gene Therapy: Preliminary Results from a Phase I/II Trial

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 607-607 ◽  
Author(s):  
Bernhard Gentner ◽  
Maria Ester Bernardo ◽  
Francesca Tucci ◽  
Erika Zonari ◽  
Francesca Fumagalli ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Phase I ◽  
Board Of Directors ◽  
Financial Support ◽  
Joint Venture ◽  
Ex Vivo ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Equity Ownership

Allogeneic hematopoietic stem cell transplantation (HSCT) performed early in life is the current standard of care for patients with severe type 1 mucopolysaccharidosis (Hurler disease), a metabolic disorder caused by mutations in the alpha-L-iduronidase (IDUA) gene, leading to impaired breakdown of glycosaminoglycans (GAG). Secretion of IDUA by donor-derived hematopoietic cells may cross-correct non-hematopoietic cells, slowing progression of tissue damage and cognitive decline. Nevertheless, Hurler patients undergoing HSCT manifest substantial residual disease burden, e.g. on the skeleton and central nervous system (CNS). We conducted a phase I/II clinical study (NCT03488394) to test whether infusion of autologous CD34+ hematopoietic stem and progenitor cells (HSPC) transduced ex vivo with a lentiviral vector coding for the IDUA gene was feasible, safe and capable of restoring enzymatic activity in the patients' blood and tissues, up to supraphysiologic levels. The trial originally planned to enroll 6 Hurler patients with preserved neurocognitive function (DQ/IQ&gt;70) that had no access to a suitable allogeneic donor. Sample size has recently been increased to 8 patients. By July 2019, six patients have been treated at a median age of 24 months (range: 14-34), with a median follow up of 4 months (range: 1-13). In all patients, we collected a high number of autologous HSPC by leukapheresis following mobilization with lenograstim and plerixafor, resulting in drug products with a median of 21 million CD34+ cells/kg (range: 13-29). Transduction efficiency was high with a median above 80% and a vector copy number (VCN) of 1.7 (range: 1.0-5.2), employing a shortened, 2 day transduction protocol that included prostaglandin E2. All patients showed rapid hematopoietic recovery following myeloablative conditioning with busulfan (targeted to an AUC of 80mg*h/L), fludarabine (160mg/sqm) and rituximab (375mg/sqm). Median duration of grade 4 neutropenia associated with conditioning was 15.5 days (range: 13-19). Also associated with conditioning, Grade 3 thrombocytopenia lasted 4 days, while only 2 out of 6 patients experienced a platelet drop below 20,000/mcL on a single day, in the absence of transfusion support. Adverse events were mild and compatible with myeloablative conditioning, with the exception of patient 3 who experienced an anaphylactic reaction on day+12, which promptly responded to antihistamines, IV fluids and steroids. All evaluable patients showed sustained, supraphysiologic blood IDUA activity (dried blood spot), which was on average 3 fold above the upper limit of normal (evaluable patients: n=5 at 1 month, n=4 at 2 months, n=3 at 3 months). Notably, in n=4 Hurler patients treated with allogeneic HSCT, we detected IDUA activity that ranged within the lowest quartile of normal in spite of full donor chimerism, suggesting substantial gain achieved by overexpressing IDUA in ex vivo genetically-modified autologous HSPC. Urinary GAG excretion fell to normal levels within 3-6 months. IDUA activity was also detected in the cerebrospinal fluid (CSF) of treated patients, accompanied by a logfold reduction in CSF GAGs in the 2 patients with longest follow up. This suggests that gene therapy accomplishes full metabolic correction of tissues, including the CNS. Gene therapy did not induce antibodies against the IDUA protein, while pre-existing antibodies induced by enzyme replacement therapy before gene therapy rapidly disappeared. Patient 1 who reached the 1-year follow-up demonstrated a stable cognitive score, improved findings on brain and spine MRI, resumed growth velocity and an improvement of his skeletal phenotype. The preliminary results from our phase I/II study compare favorably with the standard of care in terms of safety and efficacy, and highlight the potential of genetic engineering of HSPC grafts for therapeutic gain-of-function. Disclosures Gentner: Genenta Science: Consultancy, Equity Ownership, Research Funding. Parini:Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; BioMarin: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Ultragenyx: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; SOBI: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Orphan Europe: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support; Sanofi-Genzyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Financial Support. Naldini:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was then licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; Genenta Science: Consultancy, Equity Ownership; Magenta Therapeutics: Equity Ownership. Aiuti:San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Wiskott-Aldrich Syndrome (WAS) gene therapy was licensed to GlaxoSmithKline (GSK) in 2014. It was than licensed to Orchard Therapeutics (OTL) in April 2018. OTL is the current sponsor of the clinical trial.; San Raffaele Telethon Institute for Gene Therapy (SR-TIGET), a joint venture between Fondazione Telethon and Ospedale San Raffaele (OSR): Other: Study PI.

scholarly journals Flipping the Switch: Initial Results of Genetic Targeting of the Fetal to Adult Globin Switch in Sickle Cell Patients

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1023-1023 ◽  
Author(s):  
Erica B. Esrick ◽  
Christian Brendel ◽  
John P Manis ◽  
Myriam A Armant ◽  
Helene Negre ◽  
...  
Keyword(s):  
Adverse Events ◽  
Board Of Directors ◽  
Research Funding ◽  
Lentiviral Vector ◽  
Employment Equity ◽  
Advisory Committees ◽  
Cd34 Cells ◽  
Equity Ownership ◽  
Initial Results

Abstract Autologous gene therapy (GT) for beta-hemoglobinopathies has demonstrated encouraging early safety and efficacy using addition of a sickling-resistant globin gene to stem cells. Another therapeutic strategy for sickle cell disease (SCD) is erythroid-specific inactivation of BCL11A, which is a validated repressor of gamma globin expression (Sankaran et al. Science 2011). This approach has the distinct advantage of simultaneously inducing fetal hemoglobin (HbF) while coordinately decreasing sickle hemoglobin (HbS). Since hemoglobin (Hb) polymerization in sickle red cells is highly dependent on the intracellular concentration of HbS and is strongly inhibited by HbF, effective BCL11A repression should prevent the sickling phenotype within red cells. We have shown that erythroid-specific expression of microRNA-adapted shRNAs (shRNAmiR) targeting BCL11A effectively induces HbF in human erythroid cells derived from transduced HSCs, largely attenuating the hematologic effects of SCD in a murine model while avoiding negative effects in HSCs and B lymphocytes (Brendel et al. JCI 2016). Here we report the initial results of a pilot clinical study utilizing a shRNAmiR lentiviral vector (LVV) targeting BCL11A for autologous GT in SCD patients. Transduction of hematopoietic cells with GMP lentiviral vector (BCH_BB-LCRshRNA(miR)) expressing the shRNAmiR for BCL11A in an erythroid-specific fashion showed no toxicity in engraftment and genotoxicity assays, efficient transduction rates of 80-95% of CD34+-derived erythroid colony forming cells from healthy donors and SCD patients, and >95% of transduced erythroid colonies demonstrating HbF levels of 50-95% of total Hb. Transduction at clinical scale with plerixafor mobilized CD34+ cells from three SCD donors yielded vector copies of 3.7 - 5.2/cell. Patients with severe SCD were screened for eligibility according to an IND enabled, IRB-approved investigator-initiated protocol. The first cohort included patients ≥ 18 years old. After at least 3 months of protocol-required transfusions, autologous CD34+ cells were collected by plerixafor mobilization and apheresis, and then transduced under GMP conditions with the BCH_BB-LCRshRNA(miR) vector. As of July 28, 2018, 3 patients representing the adult cohort had undergone a total of 3 (n=2) or 4 (n=1) days of mobilization. Mean single-day apheresis yields were 3.2 (range 1.5 - 6.8) x 106 CD34+ cells/kg. No Grade 3 or 4 AEs were attributed to mobilization and collection, although one subject developed an incidentally-discovered line-associated atrial clot and pulmonary embolism. Transduced cell products for these 3 patients have cell doses of 3.3 - 6.7 x 106 CD34+ cells/kg, VCN of 3.3 - 5.1 copies per cell and >95% vector-positive CD34+-derived colonies. One subject (BCL002), who had been regularly transfused for 17 years, has undergone infusion of gene-modified cells after myeloablative busulfan conditioning and achieved neutrophil engraftment after 22 days. Post-infusion follow-up is 78 days. At the time of the last analysis 76 days after GT and 64 days after last RBC transfusion (Table 1) subject BCL002 had a sustained Hb of >10 g/dL and, compared to pre-GT, there was a notable absence of irreversibly sickled cells on peripheral smear and a persistently low absolute reticulocyte count consistent with markedly reduced hemolysis. Hb electrophoresis showed 23.3% HbF, 51.8% HbS and 22.3% HbA (from residual transfused cells) with a HbF/(HbF+HbS) ratio of 29.7%. At day 76, the number of F cells had risen to 59.7% with 12pg HbF/F cell. In flow-sorted immature erythroid cells γ-globin mRNA was >80% of total β-like globins in the graft-derived population and BCL11A protein was reduced by ~90%. Adverse events observed from the start of conditioning until latest follow-up were consistent with myeloablative conditioning, and there have been no product-related adverse events and no SCD-related complications. These early results show: (1) feasibility of enrollment, cell procurement, and GMP manufacturing of gene modified CD34+ cells in 3 adult SCD patients; (2) the first proof of principle demonstrating shRNAmiR-based gene knockdown in humans, and (3) successful rapid induction of HbF in the first patient infused, with marked attenuation of hemolysis in the early phase of autologous reconstitution. Based on the trajectory of increasing HbF/(HbF+HbS), near full suppression of the SCD phenotype is expected. Disclosures Esrick: Bluebird Bio: Honoraria. Negre:bluebird bio: Other: Spouse employed by bluebird Bio. Dansereau:Bluebird Bio: Consultancy. Braunewell:Bluebird Bio: Employment, Equity Ownership. Christiansen:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Nikiforow:Kite Pharma: Consultancy. Achebe:Luitpold Pharmaceutical: Consultancy; AMAG Pharmaceuticals, Inc.: Membership on an entity's Board of Directors or advisory committees; Syros pharmaceuticals: Consultancy. Negre:Bluebird Bio: Employment, Equity Ownership, Other: Salary. Heeney:Sancilio Pharmaceuticals: Consultancy, Research Funding; Ironwood: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees; Vertex/Crisper: Other: Data Monitoring Committee; Pfizer: Research Funding; Astra Zeneca: Consultancy, Research Funding. Williams:Bluebird Bio: Research Funding.

scholarly journals Advances in the Gene Therapy of Patients with Fanconi Anemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1022-1022 ◽  
Author(s):  
Juan A. Bueren ◽  
Susana Navarro ◽  
Wei Wang ◽  
Rebeca Sanchez-Dominguez ◽  
Eva Merino ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Fanconi Anemia ◽  
Research Funding ◽  
Bone Marrow Failure ◽  
Insertion Site ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Equity Ownership

Abstract Fanconi anemia (FA) is a DNA repair syndrome characterized by bone marrow failure, congenital abnormalities and cancer predisposition. Based on previous experimental results showing the in vivo proliferative advantage of gene corrected FA patients' hematopoietic stem cells (HSCs; Rio, Navarro et al. Blood 2017) a gene therapy trial in non-conditioned FA-A patients was initiated in 2016. Six patients have been treated to-date using fresh and cryopreserved CD34+ cells mobilized to peripheral blood with G-CSF and plerixafor, and transduced with the PGK-FANCA.Wpre* lentiviral vector. Cell doses infused in four patients with a follow-up of at least 12 months varied from 0.6 to 1.4 million CD34+ cells/kg. Transduction efficacies of these samples, determined as vector copies per cell, ranged from 0.17 to 0.53 copies/cell. Despite the absence of patients' conditioning, a marked in vivo expansion of gene-corrected cells was observed in all hematopoietic cell lineages analyzed in BM and PB. Significantly, up to 44% of corrected cells were determined in total PB cells at the most recent follow-up visit (24 month) in the first treated patient. Insertion site analyses in PB cells showed an oligoclonal pattern of hematopoietic reconstitution, and revealed engraftment of multipotent corrected HSCs and no evidence of insertion-site mediated clonal expansion. Functional studies showed significant increases in the resistance of BM progenitors to mitomycin C in all treated patients. Additionally, patients with higher levels of corrected cells also showed significant increases in the chromosomal stability of T cells exposed to diepoxybutane. Finally, analyses discriminating the presence of corrected and uncorrected PB cells in these patients showed marked increases in the total number of corrected leukocytes, contrasting to progressive decreases of uncorrected cells. Our studies demonstrate for the first time that lentiviral-mediated gene therapy results in progressive engraftment and phenotypic correction of HSCs in non-conditioned FA patients, suggesting that this gene therapy approach may constitute a low-toxicity option for the treatment and prevention of BMF in patients with FA. Disclosures Bueren: Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Navarro:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Casado:Rocket Pharmaceuticals Inc: Patents & Royalties. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Schmidt:GeneWerk GmbH: Employment; German Cancer Research Center: Employment; bluebird bio: Consultancy. Rio:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Sevilla:Rocket Pharmaceuticals Inc: Honoraria, Patents & Royalties.

Hematopoietic Stem Cell Gene Therapy Trial with Lentiviral Vector in X-Linked Adrenoleukodystrophy

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 821-821 ◽  
Author(s):  
Marina Cavazzana-Calvo ◽  
Nathalie Cartier ◽  
Salima Hacein-Bey Abina ◽  
Gabor Veres ◽  
Manfred Schmidt ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Cd34 Cells ◽  
Hsc Transplantation

Abstract We report preliminary results in 3 children with cerebral X-linked adrenoleukodystrophy (ALD) who received in September 2006, January 2007 and June 2008 lentiviral vector transduced autologous hematopoietic stem cell (HSC). We have previously demonstrated that cerebral demyelination associated with cerebral ALD can be stopped or reversed within 12–18 months by allogeneic HSC transplantation. The long term beneficial effects of HCT transplantation in ALD are due to the progressive turn-over of brain macrophages (microglia) derived from bone-marrow cells. For the current HSC gene therapy procedure, we used mobilized peripheral blood CD34+ cells that were transduced ex vivo for 18 hours with a non-replicative HIV1-derived lentiviral vector (CG1711 hALD) at MOI25 and expressing the ALD cDNA under the control of the MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted) promoter, and in the presence of 4 human recombinant cytokines (Il- 3, Stem Cell Factor [SCF], Flt3-ligand and Megakaryocyte Growth and Differentiation Factor [MGDF]) and CH-296 retronectine. Transduced cells were frozen to perform the required (RCL) safety tests. After thawing and prior to reinjection, 50%, 30% and 40% of transduced CD34+ cells expressed the ALD protein with a mean of 0.7, 0.6 and 0.65 copies of integrated provirus per cell. Transduced CD34+ cells were infused to ALD patients after a conditioning regimen including full doses of cyclophosphamide and busulfan. Hematopoietic recovery occured at day 13–15 post-transplant and the procedure was uneventful. In patient P1 and P2, the percentage of lymphocytes and monocytes expressing the ALD protein declined from day 60 to 6 months after gene therapy (GT) and remained stable up to 16 months post-GT. In P1, 9 to 13% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein 16 months post-transplant. In P2 and at the same time-point after transplant, 10 to 18% of CD14+, CD3+, CD19+ and CD15+ cells expressed ALD protein. ALD protein was expressed in 18–20% of bone marrow CD34+ cells from patients P1 and P2, 12 months post-transplant. In patient P3, 20 to 23% of CD3+, CD14+ and CD15+ cells expressed ALD protein 2 months after transplant. Tests assessing vector-derived RCL and vector mobilization were negative up to the last followups in the 3 patients. Integration of the vector was polyclonal and studies of integration sites arein progress. At 16 months post-transplant, HSC gene therapy resulted in neurological effects comparable with allogeneic HSC transplantation in patient P1 and P2. These results support that: ex-vivo HSC gene therapy using HIV1-derived lentiviral vector is not associated with the emergence of RCL and vector mobilization; a high percentage of hematopoietic progenitors were transduced expressing ALD protein in long term; no early evidence of selective advantage of the transduced ALD cells nor clonal expansion were observed. (This clinical trial is sponsored by Institut National de la Santé et de la Recherche Médicale and was conducted in part under a R&D collaboration with Cell Genesys, Inc., South San Francisco, CA)

A novel lentiviral vector targets gene transfer into human hematopoietic stem cells in marrow from patients with bone marrow failure syndrome and in vivo in humanized mice

Blood ◽  
2012 ◽  
Vol 119 (5) ◽  
pp. 1139-1150 ◽  
Author(s):  
Cecilia Frecha ◽  
Caroline Costa ◽  
Didier Nègre ◽  
Fouzia Amirache ◽  
Didier Trono ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Bone Marrow Failure ◽  
Humanized Mice ◽  
Hematopoietic Stem ◽  
Cd34 Cells

AbstractIn vivo lentiviral vector (LV)–mediated gene delivery would represent a great step forward in the field of gene therapy. Therefore, we have engineered a novel LV displaying SCF and a mutant cat endogenous retroviral glycoprotein, RDTR. These RDTR/SCF-LVs outperformed RDTR-LVs for transduction of human CD34+ cells (hCD34+). For in vivo gene therapy, these novel RDTR/SCF-displaying LVs can distinguish between the target hCD34+ cells of interest and nontarget cells. Indeed, they selectively targeted transduction to 30%-40% of the hCD34+ cells in cord blood mononuclear cells and in the unfractionated BM of healthy and Fanconi anemia donors, resulting in the correction of CD34+ cells in the patients. Moreover, RDTR/SCF-LVs targeted transduction to CD34+ cells with 95-fold selectivity compared with T cells in total cord blood. Remarkably, in vivo injection of the RDTR/SCF-LVs into the BM cavity of humanized mice resulted in the highly selective transduction of candidate hCD34+Lin− HSCs. In conclusion, this new LV will facilitate HSC-based gene therapy by directly targeting these primitive cells in BM aspirates or total cord blood. Most importantly, in the future, RDTR/SCF-LVs might completely obviate ex vivo handling and simplify gene therapy for many hematopoietic defects because of their applicability to direct in vivo inoculation.

scholarly journals Lentiviral Vector-Mediated Gene Transfer Restores CD18 Expression in Long-Term Repopulating Hematopoietic Stem and Progenitor Cells Isolated from a Patient with Leukocyte Adhesion Deficiency Type 1

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 1859-1859
Author(s):  
Richard H. Smith ◽  
Daisuke Araki ◽  
Andre Larochelle
Keyword(s):  
Gene Therapy ◽  
Lentiviral Vector ◽  
Ex Vivo ◽  
Leukocyte Adhesion ◽  
Myeloid Cells ◽  
Cd11b Expression ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Ex Vivo Gene Therapy ◽  
Deficiency Type

Abstract Leukocyte adhesion deficiency type 1 (LAD-1) is an inherited primary immunodeficiency caused by loss-of-function mutation within the ITGB2 gene, which encodes the beta2 integrin subunit CD18. Individuals with LAD-1 experience significant loss of neutrophil-mediated innate cellular immune function, resulting in delayed wound healing, severe periodontitis, and life-long bouts of bacterial infection. LAD-1 is a prime candidate for lentiviral vector-mediated genetic intervention as i) it is an intractable, potentially life-threatening disease with limited treatment options, ii) it is amenable to current ex vivo gene therapy procedures, and iii) partial phenotypic correction would present a high likelihood of significant clinical benefit. Allogeneic stem cell transplant can be curative, but suffers from matched donor availability and the potential for graft-versus-host disease. Autologous ex vivo gene therapy may provide a viable alternative to allogeneic transplant in LAD-1 patients. We have evaluated the ability of a CD18-expressing lentiviral vector (LV-hCD18) to mediate ex vivo transduction of LAD-1 patient-derived CD34+ hematopoietic stem and progenitor cells (HSPCs) and subsequent long-term LAD-1 HSPC engraftment in immunodeficient NOD-scid IL2Rg null (NSG) mice. An open reading frame encoding human CD18 was placed under the transcriptional control of the MND promoter (a modified retroviral promoter associated with high levels of stable transgene expression) and packaged in VSV-G-pseudotyped lentiviral particles. After 1 day of pre-stimulation, LAD-1 HSPCs were transduced with LV-hCD18 (MOI = 10) in the presence or absence of transduction-enhancing adjuvants, poloxamer 407 (P407) and prostaglandin E2 (PGE 2), for 24 hours. Sublethally irradiated NSG mice (7 mice/group) were transplanted with either mock-transduced LAD-1 HSPCs, LAD-1 HSPCs transduced in the absence of adjuvants, or LAD-1 HSPCs transduced in the presence of P407/PGE 2. Bone marrow was harvested at ~5.5 months post-transplant for flow cytometric analyses of engraftment efficiency, transgene marking, and human blood cell lineage reconstitution. Bone marrow from mice that received mock-transduced LAD-1 HSPCs showed an average total of 6.45 ± 2.54% (mean ± SEM) CD45+ human cells. Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 7.99 ± 1.82% CD45+ human cells, whereas mice transplanted with LAD-1 HSPCs transduced in the presence of adjuvants showed 7.33 ± 1.90% CD45+ cells. A Kruskal-Wallis statistical test indicated no significant difference in the level of human cell engraftment among the recipient groups (P=0.72). Consistent with the LAD-1 phenotype, human myeloid cells from mice that received mock-transduced LAD-1 HSPCs displayed only background levels of CD18 marking (0.13 ± 0.06% CD45+CD13+CD18+ cells). Mice that received LAD-1 HSPCs transduced in the absence of adjuvants showed 4.05 ± 0.40% CD18+ human myeloid cells (range 2.19% to 5.50%), whereas mice that received LAD-1 HSPCs transduced in the presence of P407/PGE 2 showed 9.56 ± 0.96% CD18+ human myeloid cells (range 4.63% to 13.10%), thus representing a &gt;2-fold increase in in vivo, vector-mediated transgene marking levels when adjuvant was used. Moreover, vector-mediated expression of CD18 rescued endogenous expression of a major CD18 heterodimerization partner in neutrophils, CD11b. In mock-transduced LAD-1 HSPC recipients, CD13+ human myeloid cells were devoid of cell surface CD11b expression (0.01 ± 0.01% CD45+CD13+CD11b+ cells). In contrast, CD13+ human myeloid cells in mice that received LAD-1 HSPCs transduced in the absence of adjuvant showed detectable levels of CD11b expression (2.62 ± 0.19% of CD18-expressing human myeloid cells), and CD11b levels were increased to 6.90 ± 0.98% in LAD-1 HSPCs transduced in the presence of P407/PGE 2. Multilineage engraftment, as evidenced by the presence of CD3+ T cells and CD20+ B cells, was noted within all groups; however, human myeloid cells represented the most prominent human blood cell compartment observed. Colony-forming-unit assays of transduced cells and non-transduced control cells pre-transplant showed similar clonogenic output and colony diversity. In sum, successful transduction, engraftment, transgene marking, CD11b rescue, and multilineage reconstitution supports further development of lentiviral vector-mediated gene therapy for LAD-1. Disclosures No relevant conflicts of interest to declare.

Ex Vivo Expansion of Retrovirally-Transduced Primate CD34+ Cells Results in Preferential Engraftment and Persistence of Clones with MDS1/EVI1 Insertion Sites.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 203-203
Author(s):  
Theo Gomes ◽  
Stephanie Sellers ◽  
Robert E. Donahue ◽  
Rima Adler ◽  
Andre La Rochelle ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Insertional Mutagenesis ◽  
Ex Vivo ◽  
Safety Issue ◽  
Retroviral Vectors ◽  
Ex Vivo Expansion ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Clinical Gene Therapy

Abstract There is increasing evidence that insertional activation of proto-oncogenes by retroviral vectors is a significant safety issue that must be addressed before clinical gene therapy, particularly targeting hematopoietic stem and progenitor cells, can be further developed. The risk of insertional mutagenesis for replication-incompetent retroviral vectors has been assumed to be low until the occurence of T cell leukemias in children treated with HSC-directed gene therapy for X-SCID, and recent evidence that retroviral integration is more common in the promoter region of transcriptionally-active genes. The occurence of “common integration sites” in a particular gene also suggests a non-random insertion pattern, and/or immortalization or other change in the behavior of a clone harboring an insertion in these particular genes. We have previously reported a highly non-random occurence of 14 unique vector integrations in the first two introns of the MDS1/EVI1 proto-oncogene out of a total of 702 identified from myeloid cells of 9 rhesus macaques at least 6 months post-transplantion of retrovirally-transduced CD34+ cells.(Calmels et al, 2005). This same gene locus was found frequently activated by insertions in murine bone marrow cells immortalized in long-term in vitro culture after transduction with retroviral vectors.(Du et al Blood, 2005) To begin to investigate the factors contributing to this worrisome finding, particularly given the very recent report of a marked over-representation of MDS1/EVI1 insertions in a human clinical gene therapy trial for chronic granulomatous disease, we asked whether continued ex vivo expansion of transduced CD34+ cells prior to transplantation would further select for clones with insertions in MDS1/EVI1 or other proto-oncogenes. Rhesus CD34+ cells were transduced with the G1Na standard retroviral vector, identical to that used in the prior studies, using our standard 96 hour transduction protocol in the presence of Retronectin and SCF, FLT3L and thrombopoietin. At the end of transduction, all cells were continued in culture for an additional 7 days under the same culture conditions, and then reinfused into the donor animal following 1200 rads TBI. At 1 month post-transplant there were no CIS and no MDS1/EVI1 insertions identified. However, at 6 months post-transplantation 5 out of 27 (19%) of the unique insertions identified in granulocytes were within the first two introns of MDS1/EVI1, very significantly higher than the 2% of MDS1/EVI1 insertions (14 of 702) identified in animals that were transplanted with cells not subjected to additional ex vivo expansion.(p<.0001) One MDS1/EVI1 clone constituted 14% of overall sequences identified, and the 5 clones constituted 37% of total sequences identified. This strongly suggests that the over-representation of this locus in engrafting cells is due to a potent immortalizing signal provided by activation of the MDS1/EVI1 gene products by the stonger retroviral promoter/enhancer, and that the need for extended ex vivo culture of target cells may select for insertion events activating this locus. It also suggests that strategies involving prolonged ex vivo expansion or selection of transduced cells could increase the risk of gene therapy utilizing integrating vectors targeting primitive hematopoietic cells.

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