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1.
Stem Cells Transl Med ; 8(10): 1107-1122, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31140762

RESUMO

Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T-cell development, leading to immunodeficiency or autoimmunity. These defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene-modified postnatal murine TECs into three-dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline-induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4-expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. Stem Cells Translational Medicine 2019;8:1107-1122.

2.
Stem Cell Reports ; 2018 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-30416049

RESUMO

We generated patient-specific disease-free induced pluripotent stem cells (iPSCs) from peripheral blood CD34+ cells and differentiated them into functional endothelial cells (ECs) secreting factor VIII (FVIII) for gene and cell therapy approaches to cure hemophilia A (HA), an X-linked bleeding disorder caused by F8 mutations. iPSCs were transduced with a lentiviral vector carrying FVIII transgene driven by an endothelial-specific promoter (VEC) and differentiated into bona fide ECs using an optimized protocol. FVIII-expressing ECs were intraportally transplanted in monocrotaline-conditioned non-obese diabetic (NOD) severe combined immune-deficient (scid)-IL2rγ null HA mice generating a chimeric liver with functional human ECs. Transplanted cells engrafted and proliferated in the liver along sinusoids, in the long term showed stable therapeutic FVIII activity (6%). These results demonstrate that the hemophilic phenotype can be rescued by transplantation of ECs derived from HA FVIII-corrected iPSCs, confirming the feasibility of cell-reprogramming strategy in patient-derived cells as an approach for HA gene and cell therapy.

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.

4.
EMBO Mol Med ; 10(2): 254-275, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29242210

RESUMO

Transferring large or multiple genes into primary human stem/progenitor cells is challenged by restrictions in vector capacity, and this hurdle limits the success of gene therapy. A paradigm is Duchenne muscular dystrophy (DMD), an incurable disorder caused by mutations in the largest human gene: dystrophin. The combination of large-capacity vectors, such as human artificial chromosomes (HACs), with stem/progenitor cells may overcome this limitation. We previously reported amelioration of the dystrophic phenotype in mice transplanted with murine muscle progenitors containing a HAC with the entire dystrophin locus (DYS-HAC). However, translation of this strategy to human muscle progenitors requires extension of their proliferative potential to withstand clonal cell expansion after HAC transfer. Here, we show that reversible cell immortalisation mediated by lentivirally delivered excisable hTERT and Bmi1 transgenes extended cell proliferation, enabling transfer of a novel DYS-HAC into DMD satellite cell-derived myoblasts and perivascular cell-derived mesoangioblasts. Genetically corrected cells maintained a stable karyotype, did not undergo tumorigenic transformation and retained their migration ability. Cells remained myogenic in vitro (spontaneously or upon MyoD induction) and engrafted murine skeletal muscle upon transplantation. Finally, we combined the aforementioned functions into a next-generation HAC capable of delivering reversible immortalisation, complete genetic correction, additional dystrophin expression, inducible differentiation and controllable cell death. This work establishes a novel platform for complex gene transfer into clinically relevant human muscle progenitors for DMD gene therapy.

5.
Sci Transl Med ; 9(411)2017 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-29021165

RESUMO

Targeted genome editing in hematopoietic stem/progenitor cells (HSPCs) is an attractive strategy for treating immunohematological diseases. However, the limited efficiency of homology-directed editing in primitive HSPCs constrains the yield of corrected cells and might affect the feasibility and safety of clinical translation. These concerns need to be addressed in stringent preclinical models and overcome by developing more efficient editing methods. We generated a humanized X-linked severe combined immunodeficiency (SCID-X1) mouse model and evaluated the efficacy and safety of hematopoietic reconstitution from limited input of functional HSPCs, establishing thresholds for full correction upon different types of conditioning. Unexpectedly, conditioning before HSPC infusion was required to protect the mice from lymphoma developing when transplanting small numbers of progenitors. We then designed a one-size-fits-all IL2RG (interleukin-2 receptor common γ-chain) gene correction strategy and, using the same reagents suitable for correction of human HSPC, validated the edited human gene in the disease model in vivo, providing evidence of targeted gene editing in mouse HSPCs and demonstrating the functionality of the IL2RG-edited lymphoid progeny. Finally, we optimized editing reagents and protocol for human HSPCs and attained the threshold of IL2RG editing in long-term repopulating cells predicted to safely rescue the disease, using clinically relevant HSPC sources and highly specific zinc finger nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9). Overall, our work establishes the rationale and guiding principles for clinical translation of SCID-X1 gene editing and provides a framework for developing gene correction for other diseases.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes/métodos , Marcação de Genes/métodos , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Camundongos , Camundongos SCID
6.
EMBO Mol Med ; 9(11): 1574-1588, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28899930

RESUMO

Gene targeting constitutes a new step in the development of gene therapy for inherited diseases. Although previous studies have shown the feasibility of editing fibroblasts from Fanconi anemia (FA) patients, here we aimed at conducting therapeutic gene editing in clinically relevant cells, such as hematopoietic stem cells (HSCs). In our first experiments, we showed that zinc finger nuclease (ZFN)-mediated insertion of a non-therapeutic EGFP-reporter donor in the AAVS1 "safe harbor" locus of FA-A lymphoblastic cell lines (LCLs), indicating that FANCA is not essential for the editing of human cells. When the same approach was conducted with therapeutic FANCA donors, an efficient phenotypic correction of FA-A LCLs was obtained. Using primary cord blood CD34+ cells from healthy donors, gene targeting was confirmed not only in in vitro cultured cells, but also in hematopoietic precursors responsible for the repopulation of primary and secondary immunodeficient mice. Moreover, when similar experiments were conducted with mobilized peripheral blood CD34+ cells from FA-A patients, we could demonstrate for the first time that gene targeting in primary hematopoietic precursors from FA patients is feasible and compatible with the phenotypic correction of these clinically relevant cells.


Assuntos
Antígenos CD34/metabolismo , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Anemia de Fanconi/patologia , Edição de Genes/métodos , Células-Tronco Hematopoéticas/metabolismo , Animais , Sequência de Bases , Células Cultivadas , Dependovirus/genética , Anemia de Fanconi/metabolismo , Proteína do Grupo de Complementação A da Anemia de Fanconi/metabolismo , Sangue Fetal/citologia , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Espécies Reativas de Oxigênio/metabolismo , Nucleases de Dedos de Zinco/genética , Nucleases de Dedos de Zinco/metabolismo
7.
EMBO Mol Med ; 9(11): 1558-1573, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28835507

RESUMO

Lentiviral vectors (LV) are powerful and versatile vehicles for gene therapy. However, their complex biological composition challenges large-scale manufacturing and raises concerns for in vivo applications, because particle components and contaminants may trigger immune responses. Here, we show that producer cell-derived polymorphic class-I major histocompatibility complexes (MHC-I) are incorporated into the LV surface and trigger allogeneic T-cell responses. By disrupting the beta-2 microglobulin gene in producer cells, we obtained MHC-free LV with substantially reduced immunogenicity. We introduce this targeted editing into a novel stable LV packaging cell line, carrying single-copy inducible vector components, which can be reproducibly converted into high-yield LV producers upon site-specific integration of the LV genome of interest. These LV efficiently transfer genes into relevant targets and are more resistant to complement-mediated inactivation, because of reduced content of the vesicular stomatitis virus envelope glycoprotein G compared to vectors produced by transient transfection. Altogether, these advances support scalable manufacturing of alloantigen-free LV with higher purity and increased complement resistance that are better suited for in vivo gene therapy.


Assuntos
Edição de Genes/métodos , Vetores Genéticos/metabolismo , Lentivirus/genética , Animais , Antígenos CD55/metabolismo , Linhagem Celular , Fator IX/genética , Fator IX/metabolismo , Terapia Genética , Vetores Genéticos/genética , Células HEK293 , Hemofilia B/terapia , Humanos , Isoantígenos/imunologia , Proteína Cofatora de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Complemento 3b/metabolismo , Transfecção
8.
Mol Ther ; 25(10): 2254-2269, 2017 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-28807569

RESUMO

T regulatory cells (Tregs) play a key role in modulating T cell responses. Clinical trials showed that Tregs modulate graft-versus-host disease (GvHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, their ability to mediate anti-leukemic activity (graft-versus-leukemia [GvL]) is largely unknown. Enforced interleukin-10 (IL-10) expression converts human CD4+ T cells into T regulatory type 1 (Tr1)-like (CD4IL-10) cells that suppress effector T cells in vitro and xenoGvHD in humanized mouse models. In the present study, we show that CD4IL-10 cells mediate anti-leukemic effects in vitro and in vivo in a human leukocyte antigen (HLA) class I-dependent but antigen-independent manner. The cytotoxicity mediated by CD4IL-10 cells is granzyme B (GzB) dependent, is specific for CD13+ target cells, and requires CD54 and CD112 expression on primary leukemic target blasts. CD4IL-10 cells adoptively transferred in humanized mouse models directly mediate anti-tumor and anti-leukemic effects. In addition, when co-transferred with peripheral blood mononuclear cells (PBMCs), CD4IL-10 cells contribute to the GvL activity but suppress xenoGvHD mediated by the PBMCs. These findings provide for the first time a strong rationale for CD4IL-10 cell immunotherapy to prevent GvHD and promote GvL in allo-HSCT for myeloid malignancies.


Assuntos
Interleucina-10/metabolismo , Leucemia Mieloide/terapia , Leucócitos Mononucleares/metabolismo , Linfócitos T Reguladores/metabolismo , Linfócitos T CD4-Positivos , Humanos , Imunoterapia , Leucemia Mieloide/imunologia , Leucemia Mieloide/metabolismo , Modelos Biológicos
9.
Blood ; 130(5): 606-618, 2017 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-28637663

RESUMO

Transfer of T-cell receptors (TCRs) specific for tumor-associated antigens is a promising approach for cancer immunotherapy. We developed the TCR gene editing technology that is based on the knockout of the endogenous TCR α and ß genes, followed by the introduction of tumor-specific TCR genes, and that proved safer and more effective than conventional TCR gene transfer. Although successful, complete editing requires extensive cell manipulation and 4 transduction procedures. Here we propose a novel and clinically feasible TCR "single editing" (SE) approach, based on the disruption of the endogenous TCR α chain only, followed by the transfer of genes encoding for a tumor-specific TCR. We validated SE with the clinical grade HLA-A2 restricted NY-ESO-1157-165-specific TCR. SE allowed the rapid production of high numbers of tumor-specific T cells, with optimal TCR expression and preferential stem memory and central memory phenotype. Similarly to unedited T cells redirected by TCR gene transfer (TCR transferred [TR]), SE T cells efficiently killed NY-ESO-1pos targets; however, although TR cells proved highly alloreactive, SE cells showed a favorable safety profile. Accordingly, when infused in NSG mice previously engrafted with myeloma, SE cells mediated tumor rejection without inducing xenogeneic graft-versus-host disease, thus resulting in significantly higher survival than that observed in mice treated with TR cells. Overall, single TCR gene editing represents a clinically feasible approach that is able to increase the safety and efficacy of cancer adoptive immunotherapy.


Assuntos
Transferência Adotiva , Edição de Genes/métodos , Memória Imunológica , Mieloma Múltiplo , Proteínas de Neoplasias , Fragmentos de Peptídeos , Receptores de Antígenos de Linfócitos T , Linfócitos T , Animais , Linhagem Celular Tumoral , Feminino , Técnicas de Transferência de Genes , Doença Enxerto-Hospedeiro , Camundongos , Mieloma Múltiplo/genética , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Linfócitos T/transplante , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Cell ; 167(1): 219-232.e14, 2016 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-27662090

RESUMO

Gene silencing is instrumental to interrogate gene function and holds promise for therapeutic applications. Here, we repurpose the endogenous retroviruses' silencing machinery of embryonic stem cells to stably silence three highly expressed genes in somatic cells by epigenetics. This was achieved by transiently expressing combinations of engineered transcriptional repressors that bind to and synergize at the target locus to instruct repressive histone marks and de novo DNA methylation, thus ensuring long-term memory of the repressive epigenetic state. Silencing was highly specific, as shown by genome-wide analyses, sharply confined to the targeted locus without spreading to nearby genes, resistant to activation induced by cytokine stimulation, and relieved only by targeted DNA demethylation. We demonstrate the portability of this technology by multiplex gene silencing, adopting different DNA binding platforms and interrogating thousands of genomic loci in different cell types, including primary T lymphocytes. Targeted epigenome editing might have broad application in research and medicine.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Edição de Genes/métodos , Inativação Gênica , Marcação de Genes/métodos , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Repressoras/metabolismo , Domínio Catalítico , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Células-Tronco Embrionárias/metabolismo , Engenharia Genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Proteínas Repressoras/genética , Linfócitos T/metabolismo
11.
Oncoimmunology ; 5(5): e1122860, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-27467932

RESUMO

The factors that determine whether disseminated transformed cells in vivo yield neoplastic lesions have only been partially identified. We established an ad hoc model of peritoneal carcinomatosis by injecting colon carcinoma cells in mice. Tumor cells recruit inflammatory leukocytes, mostly macrophages, and generate neoplastic peritoneal lesions. Phagocyte depletion via clodronate treatment reduces neoplastic growth. Colon carcinoma cells release a prototypic damage-associated molecular pattern (DAMP)/alarmin, High Mobility Group Box1 (HMGB1), which attracts leukocytes. Exogenous HMGB1 accelerates leukocyte recruitment, macrophage infiltration, tumor growth and vascularization. Lentiviral-based HMGB1 knockdown or pharmacological interference with its extracellular impair macrophage recruitment and tumor growth. Our findings provide a preclinical proof of principle that strategies based on preventing HMGB1-driven recruitment of leukocytes could be used for treating peritoneal carcinomatosis.

12.
Stem Cell Reports ; 5(4): 558-68, 2015 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-26344905

RESUMO

Autosomal recessive osteopetrosis is a human bone disease mainly caused by TCIRG1 gene mutations that prevent osteoclasts resorbing activity, recapitulated by the oc/oc mouse model. Bone marrow transplantation is the only available treatment, limited by the need for a matched donor. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of autologous cells to generate gene corrected osteoclasts might represent a powerful alternative. We generated iPSCs from oc/oc mice, corrected the mutation using a BAC carrying the entire Tcirg1 gene locus as a template for homologous recombination, and induced hematopoietic differentiation. Similarly to physiologic fetal hematopoiesis, iPSC-derived CD41(+) cells gradually gave rise to CD45(+) cells, which comprised both mature myeloid cells and high proliferative potential colony-forming cells. Finally, we differentiated the gene corrected iPSC-derived myeloid cells into osteoclasts with rescued bone resorbing activity. These results are promising for a future translation into the human clinical setting.


Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Osteoclastos/citologia , Osteopetrose/terapia , Reparo Gênico Alvo-Dirigido/métodos , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Diferenciação Celular , Linhagem Celular , Hematopoese , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Células Mieloides/citologia , Células Mieloides/metabolismo , Osteoclastos/metabolismo , Osteopetrose/genética
14.
Genome Res ; 24(8): 1251-9, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24879558

RESUMO

Endogenous retroelements (EREs) account for about half of the mouse or human genome, and their potential as insertional mutagens and transcriptional perturbators is suppressed by early embryonic epigenetic silencing. Here, we asked how ERE control is maintained during the generation of induced pluripotent stem cells (iPSCs), as this procedure involves profound epigenetic remodeling. We found that all EREs tested were markedly up-regulated during the reprogramming of either mouse embryonic fibroblasts, human CD34(+) cells, or human primary hepatocytes. At the iPSC stage, EREs of some classes were repressed, whereas others remained highly expressed, yielding a pattern somewhat reminiscent of that recorded in embryonic stem cells. However, variability persisted between individual iPSC clones in the control of specific ERE integrants. Both during reprogramming and in iPS cells, the up-regulation of specific EREs significantly impacted on the transcription of nearby cellular genes. While transcription triggered by specific ERE integrants at highly precise developmental stages may be an essential step toward obtaining pluripotent cells, the broad and unspecific unleashing of the repetitive genome observed here may contribute to the inefficiency of the reprogramming process and to the phenotypic heterogeneity of iPSCs.


Assuntos
Retrovirus Endógenos/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Transcriptoma , Animais , Células Cultivadas , Reprogramação Celular , Inativação Gênica , Humanos , Camundongos , Regulação para Cima
15.
EMBO Mol Med ; 6(6): 835-48, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24859981

RESUMO

Gene targeting is progressively becoming a realistic therapeutic alternative in clinics. It is unknown, however, whether this technology will be suitable for the treatment of DNA repair deficiency syndromes such as Fanconi anemia (FA), with defects in homology-directed DNA repair. In this study, we used zinc finger nucleases and integrase-defective lentiviral vectors to demonstrate for the first time that FANCA can be efficiently and specifically targeted into the AAVS1 safe harbor locus in fibroblasts from FA-A patients. Strikingly, up to 40% of FA fibroblasts showed gene targeting 42 days after gene editing. Given the low number of hematopoietic precursors in the bone marrow of FA patients, gene-edited FA fibroblasts were then reprogrammed and re-differentiated toward the hematopoietic lineage. Analyses of gene-edited FA-iPSCs confirmed the specific integration of FANCA in the AAVS1 locus in all tested clones. Moreover, the hematopoietic differentiation of these iPSCs efficiently generated disease-free hematopoietic progenitors. Taken together, our results demonstrate for the first time the feasibility of correcting the phenotype of a DNA repair deficiency syndrome using gene-targeting and cell reprogramming strategies.


Assuntos
Reprogramação Celular , Proteína do Grupo de Complementação A da Anemia de Fanconi/genética , Anemia de Fanconi/genética , Anemia de Fanconi/terapia , Marcação de Genes , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Terapia Genética/métodos , Hematopoese , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo
16.
Nature ; 510(7504): 235-240, 2014 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-24870228

RESUMO

Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases.


Assuntos
Marcação de Genes/métodos , Genoma Humano/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Reparo Gênico Alvo-Dirigido/métodos , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Animais , Antígenos CD34/metabolismo , DNA Complementar/genética , Endonucleases/metabolismo , Sangue Fetal/citologia , Sangue Fetal/metabolismo , Sangue Fetal/transplante , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas , Humanos , Subunidade gama Comum de Receptores de Interleucina/genética , Masculino , Camundongos , Mutação/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia
17.
Nat Commun ; 4: 2597, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24169527

RESUMO

The possibility of generating neural stem/precursor cells (NPCs) from induced pluripotent stem cells (iPSCs) has opened a new avenue of research that might nurture bench-to-bedside translation of cell transplantation protocols in central nervous system myelin disorders. Here we show that mouse iPSC-derived NPCs (miPSC-NPCs)-when intrathecally transplanted after disease onset-ameliorate clinical and pathological features of experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Transplanted miPSC-NPCs exert the neuroprotective effect not through cell replacement, but through the secretion of leukaemia inhibitory factor that promotes survival, differentiation and the remyelination capacity of both endogenous oligodendrocyte precursors and mature oligodendrocytes. The early preservation of tissue integrity limits blood-brain barrier damage and central nervous system infiltration of blood-borne encephalitogenic leukocytes, ultimately responsible for demyelination and axonal damage. While proposing a novel mechanism of action, our results further expand the therapeutic potential of NPCs derived from iPSCs in myelin disorders.


Assuntos
Terapia Baseada em Transplante de Células e Tecidos/métodos , Doenças Desmielinizantes/terapia , Encefalomielite Autoimune Experimental/terapia , Células-Tronco Pluripotentes Induzidas/fisiologia , Fator Inibidor de Leucemia/genética , Esclerose Múltipla/terapia , Células-Tronco Neurais/fisiologia , Animais , Biomarcadores/metabolismo , Diferenciação Celular , Movimento Celular , Doenças Desmielinizantes/imunologia , Doenças Desmielinizantes/patologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/patologia , Feminino , Expressão Gênica , Células-Tronco Pluripotentes Induzidas/citologia , Injeções Espinhais , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Fator Inibidor de Leucemia/metabolismo , Camundongos , Camundongos Transgênicos , Esclerose Múltipla/imunologia , Esclerose Múltipla/patologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/transplante , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Oligodendroglia/citologia , Oligodendroglia/fisiologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo
18.
Mol Ther ; 21(9): 1695-704, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23760447

RESUMO

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.


Assuntos
Dependovirus/genética , Endonucleases/genética , Marcação de Genes , Recombinação Homóloga , Queratinócitos/metabolismo , Células-Tronco/metabolismo , Integração Viral , Animais , Linhagem Celular , Transplante de Células , Células Cultivadas , Dependovirus/metabolismo , Endonucleases/metabolismo , Vetores Genéticos , Humanos , Camundongos , Transdução Genética , Dedos de Zinco
19.
Nat Med ; 18(5): 807-815, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22466705

RESUMO

The transfer of high-avidity T cell receptor (TCR) genes isolated from rare tumor-specific lymphocytes into polyclonal T cells is an attractive cancer immunotherapy strategy. However, TCR gene transfer results in competition for surface expression and inappropriate pairing between the exogenous and endogenous TCR chains, resulting in suboptimal activity and potentially harmful unpredicted antigen specificities of the resultant TCRs. We designed zinc-finger nucleases (ZFNs) that promoted the disruption of endogenous TCR ß- and α-chain genes. Lymphocytes treated with ZFNs lacked surface expression of CD3-TCR and expanded with the addition of interleukin-7 (IL-7) and IL-15. After lentiviral transfer of a TCR specific for the Wilms tumor 1 (WT1) antigen, these TCR-edited cells expressed the new TCR at high levels, were easily expanded to near purity and were superior at specific antigen recognition compared to donor-matched, unedited TCR-transferred cells. In contrast to unedited TCR-transferred cells, the TCR-edited lymphocytes did not mediate off-target reactivity while maintaining their anti-tumor activity in vivo, thus showing that complete editing of T cell specificity generates tumor-specific lymphocytes with improved biosafety profiles.


Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II/farmacologia , Lentivirus/genética , Leucemia/imunologia , Receptores de Antígenos de Linfócitos T/genética , Especificidade do Receptor de Antígeno de Linfócitos T , Dedos de Zinco , Sequência de Bases , Técnicas de Transferência de Genes , Humanos , Células Jurkat , Dados de Sequência Molecular , Linfócitos T/imunologia , Proteínas WT1/genética
20.
Neurobiol Dis ; 46(1): 41-51, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22405424

RESUMO

Neuronal disorders, like Huntington's disease (HD), are difficult to study, due to limited cell accessibility, late onset manifestations, and low availability of material. The establishment of an in vitro model that recapitulates features of the disease may help understanding the cellular and molecular events that trigger disease manifestations. Here, we describe the generation and characterization of a series of induced pluripotent stem (iPS) cells derived from patients with HD, including two rare homozygous genotypes and one heterozygous genotype. We used lentiviral technology to transfer key genes for inducing reprogramming. To confirm pluripotency and differentiation of iPS cells, we used PCR amplification and immunocytochemistry to measure the expression of marker genes in embryoid bodies and neurons. We also analyzed teratomas that formed in iPS cell-injected mice. We found that the length of the pathological CAG repeat did not increase during reprogramming, after long term growth in vitro, and after differentiation into neurons. In addition, we observed no differences between normal and mutant genotypes in reprogramming, growth rate, caspase activation or neuronal differentiation. However, we observed a significant increase in lysosomal activity in HD-iPS cells compared to control iPS cells, both during self-renewal and in iPS-derived neurons. In conclusion, we have established stable HD-iPS cell lines that can be used for investigating disease mechanisms that underlie HD. The CAG stability and lysosomal activity represent novel observations in HD-iPS cells. In the future, these cells may provide the basis for a powerful platform for drug screening and target identification in HD.


Assuntos
Técnicas de Cultura de Células/métodos , Doença de Huntington/genética , Doença de Huntington/metabolismo , Lisossomos/genética , Proteínas do Tecido Nervoso/genética , Células-Tronco Pluripotentes/metabolismo , Animais , Linhagem Celular , Fibroblastos/citologia , Fibroblastos/fisiologia , Heterozigoto , Homozigoto , Humanos , Proteína Huntingtina , Doença de Huntington/patologia , Lisossomos/metabolismo , Camundongos , Camundongos SCID , Mutação , Proteínas do Tecido Nervoso/metabolismo , Fenótipo , Teratoma/genética , Teratoma/metabolismo , Ativação Transcricional/fisiologia
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