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1.
N Engl J Med ; 390(18): 1649-1662, 2024 May 09.
Article in English | MEDLINE | ID: mdl-38661449

ABSTRACT

BACKGROUND: Exagamglogene autotemcel (exa-cel) is a nonviral cell therapy designed to reactivate fetal hemoglobin synthesis by means of ex vivo clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 gene editing of autologous CD34+ hematopoietic stem and progenitor cells (HSPCs) at the erythroid-specific enhancer region of BCL11A. METHODS: We conducted a phase 3, single-group, open-label study of exa-cel in patients 12 to 35 years of age with sickle cell disease who had had at least two severe vaso-occlusive crises in each of the 2 years before screening. CD34+ HSPCs were edited with the use of CRISPR-Cas9. Before the exa-cel infusion, patients underwent myeloablative conditioning with pharmacokinetically dose-adjusted busulfan. The primary end point was freedom from severe vaso-occlusive crises for at least 12 consecutive months. A key secondary end point was freedom from inpatient hospitalization for severe vaso-occlusive crises for at least 12 consecutive months. The safety of exa-cel was also assessed. RESULTS: A total of 44 patients received exa-cel, and the median follow-up was 19.3 months (range, 0.8 to 48.1). Neutrophils and platelets engrafted in each patient. Of the 30 patients who had sufficient follow-up to be evaluated, 29 (97%; 95% confidence interval [CI], 83 to 100) were free from vaso-occlusive crises for at least 12 consecutive months, and all 30 (100%; 95% CI, 88 to 100) were free from hospitalizations for vaso-occlusive crises for at least 12 consecutive months (P<0.001 for both comparisons against the null hypothesis of a 50% response). The safety profile of exa-cel was generally consistent with that of myeloablative busulfan conditioning and autologous HSPC transplantation. No cancers occurred. CONCLUSIONS: Treatment with exa-cel eliminated vaso-occlusive crises in 97% of patients with sickle cell disease for a period of 12 months or more. (CLIMB SCD-121; ClinicalTrials.gov number, NCT03745287.).


Subject(s)
Anemia, Sickle Cell , Fetal Hemoglobin , Hematopoietic Stem Cell Transplantation , Adolescent , Adult , Child , Female , Humans , Male , Young Adult , Anemia, Sickle Cell/complications , Anemia, Sickle Cell/genetics , Anemia, Sickle Cell/therapy , Antigens, CD34 , Busulfan/therapeutic use , CRISPR-Cas Systems , Fetal Hemoglobin/biosynthesis , Fetal Hemoglobin/genetics , Gene Editing , Hematopoietic Stem Cells , Repressor Proteins , Transplantation Conditioning , Cell- and Tissue-Based Therapy/methods , Myeloablative Agonists/therapeutic use , Europe , North America
2.
Mol Ther ; 32(5): 1202-1218, 2024 May 01.
Article in English | MEDLINE | ID: mdl-38454604

ABSTRACT

In recent years, a growing number of clinical trials have been initiated to evaluate gene therapy approaches for the treatment of patients with transfusion-dependent ß-thalassemia and sickle cell disease (SCD). Therapeutic modalities being assessed in these trials utilize different molecular techniques, including lentiviral vectors to add functional copies of the gene encoding the hemoglobin ß subunit in defective cells and CRISPR-Cas9, transcription activator-like effector protein nuclease, and zinc finger nuclease gene editing strategies to either directly address the underlying genetic cause of disease or induce fetal hemoglobin production by gene disruption. Here, we review the mechanisms of action of these various gene addition and gene editing approaches and describe the status of clinical trials designed to evaluate the potentially for these approaches to provide one-time functional cures to patients with transfusion-dependent ß-thalassemia and SCD.


Subject(s)
Genetic Therapy , Hemoglobinopathies , Animals , Humans , Anemia, Sickle Cell/therapy , Anemia, Sickle Cell/genetics , beta-Thalassemia/therapy , beta-Thalassemia/genetics , Clinical Trials as Topic , CRISPR-Cas Systems , Gene Editing/methods , Genetic Therapy/methods , Genetic Vectors/genetics , Genetic Vectors/administration & dosage , Hemoglobinopathies/therapy , Hemoglobinopathies/genetics , Lentivirus/genetics
3.
J Assist Reprod Genet ; 41(4): 1027-1034, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38358434

ABSTRACT

PURPOSE: To describe the experience of performing ovarian tissue cryopreservation (OTC) before hematopoietic stem cell transplantation (HSCT), among girls/women with severe sickle cell disease (SCD)(SS or S/ß0-thalassemia) who are, besides the usual surgical risk, at risk of SCD-related complications during the fertility preservation procedure for improving their counseling and management. METHODS: This retrospective study included 75 patients (girls/women) with SCD who have had OTC before myeloablative conditioning regimen (MAC) for HSCT. Characteristics of patients and data on OTC, ovarian status follow-up, and results of ovarian tissue transplantation (OTT) were collected in medical records. RESULTS: At OTC, the median (IQR 25-75; range) age of the patients was 9.6 (6.9-14.1; 3.6-28.3) years, 56/75 were prepubertal, and no SCD or surgery-related complications occurred. The median follow-up post-HSCT was > 9 years. At the last follow-up, among prepubertal patients at HSCT, 26/56 were ≥ 15 years old and presented with a premature ovarian insufficiency (POI), except 2, including the patient who had received an OTT to induce puberty. Eight were 13-15 years old and presented for POI. The remaining 22 patients were under 13. Among the 19 patients who were menarche at HSCT, 2 died 6 months post-HSCT and we do not have ovarian function follow-up for the other 2 patients. All the remaining patients (n = 15) had POI. Five patients had OTT. All had a return of ovarian function. One patient gave birth to a healthy baby. CONCLUSION: OTC is a safe fertility preservation technique and could be offered before MAC independent of the patient's age.


Subject(s)
Anemia, Sickle Cell , Cryopreservation , Fertility Preservation , Hematopoietic Stem Cell Transplantation , Ovary , Primary Ovarian Insufficiency , Humans , Female , Fertility Preservation/methods , Hematopoietic Stem Cell Transplantation/methods , Hematopoietic Stem Cell Transplantation/adverse effects , Cryopreservation/methods , Anemia, Sickle Cell/therapy , Ovary/transplantation , Child , Adolescent , Adult , Follow-Up Studies , Young Adult , Child, Preschool , Retrospective Studies , Transplantation Conditioning/methods , Transplantation Conditioning/adverse effects , Pregnancy
5.
Cell Stem Cell ; 31(4): 435-436, 2024 Apr 04.
Article in English | MEDLINE | ID: mdl-38579680

ABSTRACT

Lee et al.1 analyzed the impacts of lentiviral vector transduction and CRISPR-Cas9/homology-directed repair editing on hematopoietic stem and progenitor cell (HSPC) engraftment and clonal dynamics. The study suggests that relative to lentiviral-vector-mediated gene addition, homology-directed repair editing is inefficient in vivo and might impair the engraftment and differentiation of HSPCs.


Subject(s)
Gene Editing , Hematopoietic Stem Cell Transplantation , Animals , Humans , Hematopoietic Stem Cells/metabolism , Models, Animal , CRISPR-Cas Systems/genetics
6.
Biomedicines ; 12(7)2024 Jul 05.
Article in English | MEDLINE | ID: mdl-39062069

ABSTRACT

Recombinase-activating gene (RAG)-deficient SCID patients lack B and T lymphocytes due to the inability to rearrange immunoglobulin and T cell receptor genes. The two RAG genes act as a required dimer to initiate gene recombination. Gene therapy is a valid treatment alternative for RAG-SCID patients who lack a suitable bone marrow donor, but developing such therapy for RAG1/2 has proven challenging. Using a clinically approved lentiviral vector with a codon-optimized RAG1 gene, we report here preclinical studies using CD34+ cells from four RAG1-SCID patients. We used in vitro T cell developmental assays and in vivo assays in xenografted NSG mice. The RAG1-SCID patient CD34+ cells transduced with the RAG1 vector and transplanted into NSG mice led to restored human B and T cell development. Together with favorable safety data on integration sites, these results substantiate an ongoing phase I/II clinical trial for RAG1-SCID.

7.
Transplant Cell Ther ; 30(5): 532.e1-532.e16, 2024 May.
Article in English | MEDLINE | ID: mdl-38452872

ABSTRACT

Hematopoietic cell transplantation (HCT) remains the sole available curative treatment for Fanconi anemia (FA), with particularly favorable outcomes reported after matched sibling donor (MSD) HCT. This study aimed to describe outcomes, with a special focus on late complications, of FA patients who underwent umbilical cord blood transplantation (UCBT). In this retrospective analysis of allogeneic UCBT for FA performed between 1988 and 2021 in European Society for Blood and Marrow Transplantation (EBMT)-affiliated centers, a total of 205 FA patients underwent UCBT (55 related and 150 unrelated) across 77 transplant centers. Indications for UCBT were bone marrow failure in 190 patients and acute leukemia/myelodysplasia in 15 patients. The median age at transplantation was 9 years (range, 1.2 to 43 years), with only 20 patients aged >18 years. Among the donor-recipient pairs, 56% (n = 116) had a 0 to 1/6 HLA mismatch. Limited-field radiotherapy was administered to 28% (n = 58) and 78% (n = 160) received a fludarabine (Flu)-based conditioning regimen. Serotherapy consisted of antithymocyte globulin (n = 159; 78%) or alemtuzumab (n = 12; 6%). The median follow-up was 10 years for related UCBT and 7 years for unrelated UCBT. Excellent outcomes were observed in the setting of related UCBT, including a 60-day cumulative incidence (CuI) of neutrophil recovery of 98.1% (95% confidence interval [CI], 93.9% to 100%), a 100-day CuI of grade II-IV acute graft-versus-host disease (GVHD) of 17.3% (95% CI, 9.5% to 31.6%), and a 5-year CuI of chronic GVHD (cGVHD) of 22.7% (95% CI, 13.3% to 38.7%; 13% extensive). Five-year overall survival (OS) was 88%. In multivariate analysis, none of the factors included in the model predicted a better OS. In unrelated UCBT, the 60-day CuI of neutrophil recovery was 78.7% (95% CI, 71.9% to 86.3%), the 100-day CuI of grade II-IV aGVHD was 31.4% (95% CI, 24.6% to 40.2%), and the 5-year CuI of cGVHD was 24.3% (95% CI, 17.8% to 32.2%; 12% extensive). Five-year OS was 44%. In multivariate analysis, negative recipient cytomegalovirus serology, Flu-based conditioning, age <9 years at UCBT, and 0 to 1/6 HLA mismatch were associated with improved OS. A total of 106 patients, including 5 with acute leukemia/myelodysplasia, survived for >2 years after UCBT. Nine of these patients developed subsequent neoplasms (SNs), including 1 donor-derived acute myelogenous leukemia and 8 solid tumors, at a median of 9.7 years (range, 2.3 to 21.8 years) post-UCBT (1 related and 8 unrelated UCBT). In a subset of 49 patients with available data, late nonmalignant complications affecting various organ systems were observed at a median of 8.7 years (range, 2.7 to 28.8 years) post-UCBT. UCB is a valid source of stem cells for transplantation in patients with FA, with the best results observed after related UCBT. After unrelated UCBT, improved survival was observed in patients who underwent transplantation at a younger age, with Flu-based conditioning, and with better HLA parity. The incidence of organ-specific complications and SNs was relatively low. The incidence of SNs, mostly squamous cell carcinoma, increases with time. Rigorous follow-up and lifelong screening are crucial in survivors of UCBT for FA.


Subject(s)
Cord Blood Stem Cell Transplantation , Fanconi Anemia , Graft vs Host Disease , Transplantation Conditioning , Humans , Fanconi Anemia/therapy , Fanconi Anemia/complications , Female , Male , Adult , Child , Child, Preschool , Adolescent , Retrospective Studies , Infant , Transplantation Conditioning/methods , Graft vs Host Disease/epidemiology , Young Adult
8.
Nat Commun ; 15(1): 4965, 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38862518

ABSTRACT

Sickle cell disease is a devastating blood disorder that originates from a single point mutation in the HBB gene coding for hemoglobin. Here, we develop a GMP-compatible TALEN-mediated gene editing process enabling efficient HBB correction via a DNA repair template while minimizing risks associated with HBB inactivation. Comparing viral versus non-viral DNA repair template delivery in hematopoietic stem and progenitor cells in vitro, both strategies achieve comparable HBB correction and result in over 50% expression of normal adult hemoglobin in red blood cells without inducing ß-thalassemic phenotype. In an immunodeficient female mouse model, transplanted cells edited with the non-viral strategy exhibit higher engraftment and gene correction levels compared to those edited with the viral strategy. Transcriptomic analysis reveals that non-viral DNA repair template delivery mitigates P53-mediated toxicity and preserves high levels of long-term hematopoietic stem cells. This work paves the way for TALEN-based autologous gene therapy for sickle cell disease.


Subject(s)
Anemia, Sickle Cell , Gene Editing , Genetic Therapy , Hematopoietic Stem Cells , Transcription Activator-Like Effector Nucleases , Anemia, Sickle Cell/therapy , Anemia, Sickle Cell/genetics , Gene Editing/methods , Animals , Hematopoietic Stem Cells/metabolism , Humans , Female , Mice , Genetic Therapy/methods , Transcription Activator-Like Effector Nucleases/metabolism , Transcription Activator-Like Effector Nucleases/genetics , Hematopoietic Stem Cell Transplantation , beta-Globins/genetics , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , DNA Repair , Mutation , beta-Thalassemia/therapy , beta-Thalassemia/genetics , Disease Models, Animal , Gene Transfer Techniques
9.
Nat Commun ; 15(1): 3662, 2024 Apr 30.
Article in English | MEDLINE | ID: mdl-38688902

ABSTRACT

Hematopoietic stem cell gene therapy (GT) using a γ-retroviral vector (γ-RV) is an effective treatment for Severe Combined Immunodeficiency due to Adenosine Deaminase deficiency. Here, we describe a case of GT-related T-cell acute lymphoblastic leukemia (T-ALL) that developed 4.7 years after treatment. The patient underwent chemotherapy and haploidentical transplantation and is currently in remission. Blast cells contain a single vector insertion activating the LIM-only protein 2 (LMO2) proto-oncogene, confirmed by physical interaction, and low Adenosine Deaminase (ADA) activity resulting from methylation of viral promoter. The insertion is detected years before T-ALL in multiple lineages, suggesting that further hits occurred in a thymic progenitor. Blast cells contain known and novel somatic mutations as well as germline mutations which may have contributed to transformation. Before T-ALL onset, the insertion profile is similar to those of other ADA-deficient patients. The limited incidence of vector-related adverse events in ADA-deficiency compared to other γ-RV GT trials could be explained by differences in transgenes, background disease and patient's specific factors.


Subject(s)
Adenosine Deaminase , Agammaglobulinemia , Genetic Therapy , Genetic Vectors , Hematopoietic Stem Cell Transplantation , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma , Proto-Oncogene Mas , Severe Combined Immunodeficiency , Humans , Adenosine Deaminase/deficiency , Adenosine Deaminase/genetics , Genetic Therapy/methods , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/therapy , Precursor T-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Severe Combined Immunodeficiency/therapy , Severe Combined Immunodeficiency/genetics , Genetic Vectors/genetics , Agammaglobulinemia/therapy , Agammaglobulinemia/genetics , Male , Retroviridae/genetics
10.
Mol Ther Methods Clin Dev ; 31: 101133, 2023 Dec 14.
Article in English | MEDLINE | ID: mdl-38152700

ABSTRACT

Gain-of-function mutations in the PIK3CD gene result in activated phosphoinositide 3-kinase δ syndrome type 1 (APDS1). This syndrome is a life-threatening combined immunodeficiency and today there are neither optimal nor long-term therapeutic solutions for APDS1 patients. Thus, new alternative treatments are highly needed. The aim of the present study is to explore one therapeutic avenue that consists of the correction of the PIK3CD gene through gene editing. Our proof-of-concept shows that TALEN-mediated gene correction of the mutated PIK3CD gene in APDS1 T cells results in normalized phospho-AKT levels in basal and activated conditions. Normalization of PI3K signaling was correlated to restored cytotoxic functions of edited CD8+ T cells. At the transcriptomic level, single-cell RNA sequencing revealed corrected signatures of CD8+ effector memory and CD8+ proliferating T cells. This proof-of-concept study paves the way for the future development of a gene therapy candidate to cure activated phosphoinositide 3-kinase δ syndrome type 1.

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