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1.
Lancet Haematol ; 6(5): e239-e253, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30981783

RESUMO

BACKGROUND: Wiskott-Aldrich syndrome is a rare, life-threatening, X-linked primary immunodeficiency characterised by microthrombocytopenia, infections, eczema, autoimmunity, and malignant disease. Lentiviral vector-mediated haemopoietic stem/progenitor cell (HSPC) gene therapy is a potentially curative treatment that represents an alternative to allogeneic HSPC transplantation. Here, we report safety and efficacy data from an interim analysis of patients with severe Wiskott-Aldrich syndrome who received lentiviral vector-derived gene therapy. METHODS: We did a non-randomised, open-label, phase 1/2 clinical study in paediatric patients with severe Wiskott-Aldrich syndrome, defined by either WAS gene mutation or absent Wiskott-Aldrich syndrome protein (WASP) expression or a Zhu clinical score of 3 or higher. We included patients who had no HLA-identical sibling donor available or, for children younger than 5 years of age, no suitable 10/10 matched unrelated donor or 6/6 unrelated cord blood donor. After treatment with rituximab and a reduced-intensity conditioning regimen of busulfan and fludarabine, patients received one intravenous infusion of autologous CD34+ cells genetically modified with a lentiviral vector encoding for human WAS cDNA. The primary safety endpoints were safety of the conditioning regimen and safety of lentiviral gene transfer into HSPCs. The primary efficacy endpoints were overall survival, sustained engraftment of genetically corrected HSPCs, expression of vector-derived WASP, improved T-cell function, antigen-specific responses to vaccinations, and improved platelet count and mean platelet volume normalisation. This interim analysis was done when the first six patients treated had completed at least 3 years of follow-up. The planned analyses are presented for the intention-to-treat population. This trial is registered with ClinicalTrials.gov (number NCT01515462) and EudraCT (number 2009-017346-32). FINDINGS: Between April 20, 2010, and Feb 26, 2015, nine patients (all male) were enrolled of whom one was excluded after screening; the age range of the eight treated children was 1·1-12·4 years. At the time of the interim analysis (data cutoff April 29, 2016), median follow-up was 3·6 years (range 0·5-5·6). Overall survival was 100%. Engraftment of genetically corrected HSPCs was successful and sustained in all patients. The fraction of WASP-positive lymphocytes increased from a median of 3·9% (range 1·8-35·6) before gene therapy to 66·7% (55·7-98·6) at 12 months after gene therapy, whereas WASP-positive platelets increased from 19·1% (range 4·1-31·0) to 76·6% (53·1-98·4). Improvement of immune function was shown by normalisation of in-vitro T-cell function and successful discontinuation of immunoglobulin supplementation in seven patients with follow-up longer than 1 year, followed by positive antigen-specific response to vaccination. Severe infections fell from 2·38 (95% CI 1·44-3·72) per patient-year of observation (PYO) in the year before gene therapy to 0·31 (0·04-1·11) per PYO in the second year after gene therapy and 0·17 (0·00-0·93) per PYO in the third year after gene therapy. Before gene therapy, platelet counts were lower than 20 × 109 per L in seven of eight patients. At the last follow-up visit, the platelet count had increased to 20-50 × 109 per L in one patient, 50-100 × 109 per L in five patients, and more than 100 × 109 per L in two patients, which resulted in independence from platelet transfusions and absence of severe bleeding events. 27 serious adverse events in six patients occurred after gene therapy, 23 (85%) of which were infectious (pyrexia [five events in three patients], device-related infections, including one case of sepsis [four events in three patients], and gastroenteritis, including one case due to rotavirus [three events in two patients]); these occurred mainly in the first 6 months of follow-up. No adverse reactions to the investigational drug product and no abnormal clonal proliferation or leukaemia were reported after gene therapy. INTERPRETATION: Data from this study show that gene therapy provides a valuable treatment option for patients with severe Wiskott-Aldrich syndrome, particularly for those who do not have a suitable HSPC donor available. FUNDING: Italian Telethon Foundation, GlaxoSmithKline, and Orchard Therapeutics.


Assuntos
Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Criança , Pré-Escolar , Feminino , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Lactente , Itália , Masculino , Mutação , Linfócitos T/imunologia , Linfócitos T/metabolismo , Condicionamento Pré-Transplante/métodos , Resultado do Tratamento , Síndrome de Wiskott-Aldrich/sangue , Síndrome de Wiskott-Aldrich/diagnóstico , Proteína da Síndrome de Wiskott-Aldrich/genética
2.
Nat Med ; 24(11): 1683-1690, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30275570

RESUMO

Hematopoietic stem and progenitor cells (HSPC) are endowed with the role of generating and maintaining lifelong the extremely diverse pool of blood cells1. Clinically, transplantation of human HSPC from an allogeneic healthy donor or infusion of autologous gene-corrected HSPC can effectively replenish defective blood cell production caused by congenital or acquired disorders2-9. However, due to methodological and ethical constraints that have limited the study of human HSPC primarily to in vitro assays10 or xenotransplantation models11,12, the in vivo activity of HSPC has to date remained relatively unexplored in humans13-16. Here we report a comprehensive study of the frequencies, dynamics and output of seven HSPC subtypes in humans that was performed by tracking 148,093 individual clones in six patients treated with lentiviral gene therapy using autologous HSPC transplantation and followed for up to 5 years. We discovered that primitive multipotent progenitor and hematopoietic stem cell (HSC) populations have distinct roles during the initial reconstitution after transplant, compared with subsequent steady-state phases. Furthermore, we showed that a fraction of in vitro-activated HSC are resilient and undergo a defined delayed activation period upon transplant. Finally, our data support the concept that early lymphoid-biased progenitors might be capable of long-term survival, such that they can be maintained independently of their continuous production from HSC. Overall, this study provides comprehensive data on HSPC dynamics after autologous transplantation and gene therapy in humans.

3.
Blood ; 132(22): 2362-2374, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30254128

RESUMO

ARPC1B is a key factor for the assembly and maintenance of the ARP2/3 complex that is involved in actin branching from an existing filament. Germline biallelic mutations in ARPC1B have been recently described in 6 patients with clinical features of combined immunodeficiency (CID), whose neutrophils and platelets but not T lymphocytes were studied. We hypothesized that ARPC1B deficiency may also lead to cytoskeleton and functional defects in T cells. We have identified biallelic mutations in ARPC1B in 6 unrelated patients with early onset disease characterized by severe infections, autoimmune manifestations, and thrombocytopenia. Immunological features included T-cell lymphopenia, low numbers of naïve T cells, and hyper-immunoglobulin E. Alteration in ARPC1B protein structure led to absent/low expression by flow cytometry and confocal microscopy. This molecular defect was associated with the inability of patient-derived T cells to extend an actin-rich lamellipodia upon T-cell receptor (TCR) stimulation and to assemble an immunological synapse. ARPC1B-deficient T cells additionally displayed impaired TCR-mediated proliferation and SDF1-α-directed migration. Gene transfer of ARPC1B in patients' T cells using a lentiviral vector restored both ARPC1B expression and T-cell proliferation in vitro. In 2 of the patients, in vivo somatic reversion restored ARPC1B expression in a fraction of lymphocytes and was associated with a skewed TCR repertoire. In 1 revertant patient, memory CD8+ T cells expressing normal levels of ARPC1B displayed improved T-cell migration. Inherited ARPC1B deficiency therefore alters T-cell cytoskeletal dynamics and functions, contributing to the clinical features of CID.

5.
Front Immunol ; 9: 113, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29456531

RESUMO

Adenosine deaminase-deficient severe combined immunodeficiency disease (ADA-SCID) is a primary immune deficiency characterized by mutations in the ADA gene resulting in accumulation of toxic compounds affecting multiple districts. Hematopoietic stem cell transplantation (HSCT) from a matched donor and hematopoietic stem cell gene therapy are the preferred options for definitive treatment. Enzyme replacement therapy (ERT) is used to manage the disease in the short term, while a decreased efficacy is reported in the medium-long term. To date, eight cases of lymphomas have been described in ADA-SCID patients. Here we report the first case of plasmablastic lymphoma occurring in a young adult with ADA-SCID on long-term ERT, which turned out to be Epstein-Barr virus associated. The patient previously received infusions of genetically modified T cells. A cumulative analysis of the eight published cases of lymphoma from 1992 to date, and the case here described, reveals a high mortality (89%). The most common form is diffuse large B-cell lymphoma, which predominantly occurs in extra nodal sites. Seven cases occurred in patients on ERT and two after haploidentical HSCT. The significant incidence of immunodeficiency-associated lymphoproliferative disorders and poor survival of patients developing this complication highlight the priority in finding a prompt curative treatment for ADA-SCID.

6.
Blood ; 128(1): 45-54, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27129325

RESUMO

Adenosine deaminase (ADA) deficiency is a rare, autosomal-recessive systemic metabolic disease characterized by severe combined immunodeficiency (SCID). The treatment of choice for ADA-deficient SCID (ADA-SCID) is hematopoietic stem cell transplant from an HLA-matched sibling donor, although <25% of patients have such a donor available. Enzyme replacement therapy (ERT) partially and temporarily relieves immunodeficiency. We investigated the medium-term outcome of gene therapy (GT) in 18 patients with ADA-SCID for whom an HLA-identical family donor was not available; most were not responding well to ERT. Patients were treated with an autologous CD34(+)-enriched cell fraction that contained CD34(+) cells transduced with a retroviral vector encoding the human ADA complementary DNA sequence (GSK2696273) as part of single-arm, open-label studies or compassionate use programs. Overall survival was 100% over 2.3 to 13.4 years (median, 6.9 years). Gene-modified cells were stably present in multiple lineages throughout follow up. GT resulted in a sustained reduction in the severe infection rate from 1.17 events per person-year to 0.17 events per person-year (n = 17, patient 1 data not available). Immune reconstitution was demonstrated by normalization of T-cell subsets (CD3(+), CD4(+), and CD8(+)), evidence of thymopoiesis, and sustained T-cell proliferative capacity. B-cell function was evidenced by immunoglobulin production, decreased intravenous immunoglobulin use, and antibody response after vaccination. All 18 patients reported infections as adverse events; infections of respiratory and gastrointestinal tracts were reported most frequently. No events indicative of leukemic transformation were reported. Trial details were registered at www.clinicaltrials.gov as #NCT00598481.


Assuntos
Adenosina Desaminase/deficiência , Agamaglobulinemia/terapia , Terapia Genética , Recuperação de Função Fisiológica , Retroviridae , Imunodeficiência Combinada Severa/terapia , Adenosina Desaminase/genética , Adenosina Desaminase/imunologia , Agamaglobulinemia/genética , Agamaglobulinemia/imunologia , Agamaglobulinemia/mortalidade , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Lactente , Masculino , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Imunodeficiência Combinada Severa/mortalidade , Taxa de Sobrevida
7.
Cell Stem Cell ; 19(1): 107-19, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27237736

RESUMO

Hematopoietic stem/progenitor cells (HSPCs) are capable of supporting the lifelong production of blood cells exerting a wide spectrum of functions. Lentiviral vector HSPC gene therapy generates a human hematopoietic system stably marked at the clonal level by vector integration sites (ISs). Using IS analysis, we longitudinally tracked >89,000 clones from 15 distinct bone marrow and peripheral blood lineages purified up to 4 years after transplant in four Wiskott-Aldrich syndrome patients treated with HSPC gene therapy. We measured at the clonal level repopulating waves, populations' sizes and dynamics, activity of distinct HSPC subtypes, contribution of various progenitor classes during the early and late post-transplant phases, and hierarchical relationships among lineages. We discovered that in-vitro-manipulated HSPCs retain the ability to return to latency after transplant and can be physiologically reactivated, sustaining a stable hematopoietic output. This study constitutes in vivo comprehensive tracking in humans of hematopoietic clonal dynamics during the early and late post-transplant phases.


Assuntos
Rastreamento de Células , Hematopoese , Antígenos CD34/metabolismo , Engenharia Celular , Linhagem da Célula/genética , Pré-Escolar , Células Clonais , Terapia Genética , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lactente , Masculino , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Mutagênese Insercional/genética , Fatores de Tempo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia
8.
Sci Transl Med ; 7(317): 317ra198, 2015 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-26659572

RESUMO

Long-lasting immune protection from pathogens and cancer requires the generation of memory T cells able to survive long-term. To unravel the immunological requirements for long-term persistence of human memory T cells, we characterized and traced, over several years, T lymphocytes genetically modified to express the thymidine kinase (TK) suicide gene that were infused in 10 patients after haploidentical hematopoietic stem cell transplantation (HSCT). At 2 to 14 years after infusion and in the presence of a broad and resting immune system, we could still detect effectors/effector memory (TEM/EFF), central memory (TCM), and stem memory (TSCM) TK(+) cells, circulating at low but stable levels in all patients. Longitudinal analysis of cytomegalovirus (CMV)- and Flu-specific TK(+) cells indicated that antigen recognition was dominant in driving in vivo expansion and persistence at detectable levels. The amount of infused TSCM cells positively correlated with early expansion and with the absolute counts of long-term persisting gene-marked cells. By combining T cell sorting with sequencing of integration (IS), TCRα and TCRß clonal markers, we showed that T cells retrieved long-term were enriched in clones originally shared in different memory T cell subsets, whereas dominant long-term clonotypes appeared to preferentially originate from infused TSCM and TCM clones. Together, these results indicate that long-term persistence of gene-modified memory T cells after haploidentical HSCT is influenced by antigen exposure and by the original phenotype of infused cells. Cancer adoptive immunotherapy might thus benefit from cellular products enriched in lymphocytes with an early-differentiated phenotype.


Assuntos
Rastreamento de Células , Engenharia Genética , Memória Imunológica , Linfócitos T/imunologia , Adulto , Idoso , Antígenos/imunologia , Proliferação de Células , Células Clonais , Feminino , Genes Transgênicos Suicidas , Terapia Genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Contagem de Linfócitos , Depleção Linfocítica , Masculino , Pessoa de Meia-Idade , Fenótipo , Timidina Quinase/metabolismo , Fatores de Tempo , Doadores de Tecidos , Adulto Jovem
9.
Sci Transl Med ; 7(273): 273ra13, 2015 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-25653219

RESUMO

A definitive understanding of survival and differentiation potential in humans of T cell subpopulations is of paramount importance for the development of effective T cell therapies. In particular, uncovering the dynamics in vivo in humans of the recently described T memory stem cells (TSCM) would be crucial for therapeutic approaches that aim at taking advantage of a stable cellular vehicle with precursor potential. We exploited data derived from two gene therapy clinical trials for an inherited immunodeficiency, using either retrovirally engineered hematopoietic stem cells or mature lymphocytes to trace individual T cell clones directly in vivo in humans. We compared healthy donors and bone marrow-transplanted patients, studied long-term in vivo T cell composition under different clinical conditions, and specifically examined TSCM contribution according to age, conditioning regimen, disease background, cell source, long-term reconstitution, and ex vivo gene correction processing. High-throughput sequencing of retroviral vector integration sites (ISs) allowed tracing the fate of more than 1700 individual T cell clones in gene therapy patients after infusion of gene-corrected hematopoietic stem cells or mature lymphocytes. We shed light on long-term in vivo clonal relationships among different T cell subtypes, and we unveiled that TSCM are able to persist and to preserve their precursor potential in humans for up to 12 years after infusion of gene-corrected lymphocytes. Overall, this work provides high-resolution tracking of T cell fate and activity and validates, in humans, the safe and functional decade-long survival of engineered TSCM, paving the way for their future application in clinical settings.


Assuntos
Rastreamento de Células , Engenharia Genética , Células-Tronco Hematopoéticas/citologia , Memória Imunológica , Linfócitos T/citologia , Linfócitos T/metabolismo , Adulto , Sobrevivência Celular , Criança , Células Clonais , Terapia Genética , Humanos , Interleucina-2/farmacologia , Estudos Longitudinais , Subpopulações de Linfócitos/metabolismo , Fenótipo , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Doadores de Tecidos
10.
Science ; 341(6148): 1233151, 2013 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-23845947

RESUMO

Wiskott-Aldrich syndrome (WAS) is an inherited immunodeficiency caused by mutations in the gene encoding WASP, a protein regulating the cytoskeleton. Hematopoietic stem/progenitor cell (HSPC) transplants can be curative, but, when matched donors are unavailable, infusion of autologous HSPCs modified ex vivo by gene therapy is an alternative approach. We used a lentiviral vector encoding functional WASP to genetically correct HSPCs from three WAS patients and reinfused the cells after a reduced-intensity conditioning regimen. All three patients showed stable engraftment of WASP-expressing cells and improvements in platelet counts, immune functions, and clinical scores. Vector integration analyses revealed highly polyclonal and multilineage haematopoiesis resulting from the gene-corrected HSPCs. Lentiviral gene therapy did not induce selection of integrations near oncogenes, and no aberrant clonal expansion was observed after 20 to 32 months. Although extended clinical observation is required to establish long-term safety, lentiviral gene therapy represents a promising treatment for WAS.


Assuntos
Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Criança , Vetores Genéticos , Humanos , Lentivirus , Masculino , Transdução Genética , Integração Viral
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