Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 40
Filtrar
Mais filtros

Base de dados
Tipo de documento
País de afiliação
Intervalo de ano de publicação
1.
Blood ; 144(9): 977-987, 2024 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-38861668

RESUMO

ABSTRACT: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo. Here, we describe the incorporation of T-cell activation and costimulatory signals onto the surface of VivoVec particles (VVPs) in the form of a multidomain fusion protein and show enhanced in vivo transduction and improved CAR T-cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into nonhuman primates resulted in the robust generation of anti-CD20 CAR T cells and the complete depletion of B cells for >10 weeks. These data validate the VivoVec platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.


Assuntos
Vetores Genéticos , Imunoterapia Adotiva , Lentivirus , Receptores de Antígenos Quiméricos , Linfócitos T , Animais , Lentivirus/genética , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/genética , Linfócitos T/imunologia , Linfócitos T/metabolismo , Humanos , Imunoterapia Adotiva/métodos , Ligantes , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Transdução Genética , Antígenos CD20/imunologia , Antígenos CD20/genética , Ativação Linfocitária
2.
N Engl J Med ; 380(16): 1525-1534, 2019 04 18.
Artigo em Inglês | MEDLINE | ID: mdl-30995372

RESUMO

BACKGROUND: Allogeneic hematopoietic stem-cell transplantation for X-linked severe combined immunodeficiency (SCID-X1) often fails to reconstitute immunity associated with T cells, B cells, and natural killer (NK) cells when matched sibling donors are unavailable unless high-dose chemotherapy is given. In previous studies, autologous gene therapy with γ-retroviral vectors failed to reconstitute B-cell and NK-cell immunity and was complicated by vector-related leukemia. METHODS: We performed a dual-center, phase 1-2 safety and efficacy study of a lentiviral vector to transfer IL2RG complementary DNA to bone marrow stem cells after low-exposure, targeted busulfan conditioning in eight infants with newly diagnosed SCID-X1. RESULTS: Eight infants with SCID-X1 were followed for a median of 16.4 months. Bone marrow harvest, busulfan conditioning, and cell infusion had no unexpected side effects. In seven infants, the numbers of CD3+, CD4+, and naive CD4+ T cells and NK cells normalized by 3 to 4 months after infusion and were accompanied by vector marking in T cells, B cells, NK cells, myeloid cells, and bone marrow progenitors. The eighth infant had an insufficient T-cell count initially, but T cells developed in this infant after a boost of gene-corrected cells without busulfan conditioning. Previous infections cleared in all infants, and all continued to grow normally. IgM levels normalized in seven of the eight infants, of whom four discontinued intravenous immune globulin supplementation; three of these four infants had a response to vaccines. Vector insertion-site analysis was performed in seven infants and showed polyclonal patterns without clonal dominance in all seven. CONCLUSIONS: Lentiviral vector gene therapy combined with low-exposure, targeted busulfan conditioning in infants with newly diagnosed SCID-X1 had low-grade acute toxic effects and resulted in multilineage engraftment of transduced cells, reconstitution of functional T cells and B cells, and normalization of NK-cell counts during a median follow-up of 16 months. (Funded by the American Lebanese Syrian Associated Charities and others; LVXSCID-ND ClinicalTrials.gov number, NCT01512888.).


Assuntos
Bussulfano/administração & dosagem , Terapia Genética , Vetores Genéticos , Subunidade gama Comum de Receptores de Interleucina/genética , Lentivirus , Condicionamento Pré-Transplante , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/terapia , Antígenos de Diferenciação de Linfócitos T/sangue , Linfócitos B/fisiologia , Transplante de Células-Tronco Hematopoéticas , Humanos , Imunoglobulina M/sangue , Lactente , Células Matadoras Naturais , Contagem de Linfócitos , Masculino , Linfócitos T , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/genética , Doenças por Imunodeficiência Combinada Ligada ao Cromossomo X/imunologia
3.
Curr Opin Ophthalmol ; 33(6): 501-506, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36066372

RESUMO

PURPOSE OF REVIEW: The current article will update and review the clinical and radiological manifestations and management of rhino-orbital mucormycosis (ROM). RECENT FINDINGS: There has been an increase in cases of ROM worldwide, especially in India. Immunosuppression (especially diabetes mellitus) is a known predisposing risk factor for ROM. Delayed diagnosis and treatment of ROM can be vision or life-threatening. This article reviews the clinical and radiologic features, treatment, and prognosis of ROM with special emphasis on new and emerging therapies. SUMMARY: ROM is an angioinvasive fungal infection that affects the sinuses and orbits and may present to ophthalmologists. Clinicians should have a high index of suspicion for ROM, especially in patients with poorly controlled diabetes mellitus or other immunosuppression. Corticosteroid treatment (including the recent COVID-19 pandemic) may be a predisposing risk factor for ROM.


Assuntos
COVID-19 , Oftalmopatias , Mucormicose , Doenças Orbitárias , Humanos , Mucormicose/diagnóstico , Mucormicose/epidemiologia , Mucormicose/terapia , Doenças Orbitárias/diagnóstico , Doenças Orbitárias/terapia , Pandemias
4.
Blood ; 134(16): 1298-1311, 2019 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-31416800

RESUMO

Therapeutic gene delivery to hematopoietic stem cells (HSCs) holds great potential as a life-saving treatment of monogenic, oncologic, and infectious diseases. However, clinical gene therapy is severely limited by intrinsic HSC resistance to modification with lentiviral vectors (LVs), thus requiring high doses or repeat LV administration to achieve therapeutic gene correction. Here we show that temporary coapplication of the cyclic resveratrol trimer caraphenol A enhances LV gene delivery efficiency to human and nonhuman primate hematopoietic stem and progenitor cells with integrating and nonintegrating LVs. Although significant ex vivo, this effect was most dramatically observed in human lineages derived from HSCs transplanted into immunodeficient mice. We further show that caraphenol A relieves restriction of LV transduction by altering the levels of interferon-induced transmembrane (IFITM) proteins IFITM2 and IFITM3 and their association with late endosomes, thus augmenting LV core endosomal escape. Caraphenol A-mediated IFITM downregulation did not alter the LV integration pattern or bias lineage differentiation. Taken together, these findings compellingly demonstrate that the pharmacologic modification of intrinsic immune restriction factors is a promising and nontoxic approach for improving LV-mediated gene therapy.


Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/virologia , Proteínas de Membrana/efeitos dos fármacos , Resveratrol/farmacologia , Transdução Genética/métodos , Animais , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Vetores Genéticos , Xenoenxertos , Humanos , Lentivirus , Proteínas de Membrana/metabolismo , Camundongos , Transporte Proteico/efeitos dos fármacos
5.
Gene Ther ; 27(12): 545-556, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32341484

RESUMO

Autologous gene therapy using lentiviral vectors (LVs) holds promise for treating monogenetic blood diseases. However, clinical applications can be limited by suboptimal hematopoietic stem cell (HSC) transduction and insufficient quantities of available vector. We recently reported gene therapy for X-linked severe combined immunodeficiency using a protocol in which patient CD34+ cells were incubated with two successive transductions. Here we describe an improved protocol for LV delivery to CD34+ cells that simplifies product manipulation, reduces vector consumption, and achieves greater vector copy number (VCN) of repopulating HSCs in mouse xenotransplantation assays. Notable findings include the following: (1) the VCN of CD34+ cells measured shortly after transduction did not always correlate with the VCN of repopulating HSCs after xenotransplantation; (2) single-step transduction at higher CD34+ cell concentrations (2-4 × 106/ml) conserved LV without compromising HSC VCN; (3) poloxamer F108 (LentiBOOST) increased HSC VCN by two- to threefold (average from three donors); (4) although LentiBOOST + prostaglandin E2 combination further increased VCN in vitro, the VCN observed in vivo were similar to LentiBOOST alone; (5) cyclosporine H increased the HSC VCN to a similar or greater extent with LentiBOOST in vivo. Our findings delineate an improved protocol to increase the VCN of HSCs after CD34+ cell transduction with clinically relevant LVs.


Assuntos
Transplante de Células-Tronco Hematopoéticas , Lentivirus , Animais , Antígenos CD34 , Terapia Genética , Vetores Genéticos/genética , Células-Tronco Hematopoéticas , Humanos , Lentivirus/genética , Camundongos , Transdução Genética
6.
Cytotherapy ; 20(10): 1278-1287, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30249524

RESUMO

BACKGROUND AIMS: γ-globin expression can be induced by various gene modification strategies, which could be beneficial for hemoglobin (Hb) disorders. To translate promising ideas into clinics, large animal models have proven valuable to evaluate safety and efficacy of the approaches; however, in vitro erythroid differentiation methods have not been established to determine whether they can be modeled in nonhuman primates. METHODS: We optimized erythroid differentiation culture to produce high-level adult Hb from rhesus hematopoietic progenitor cells by using low (LC) or high cytokine concentration (HC) protocols with or without feeder cells. In addition, we established rhesus globin protein analysis using reverse-phase high performance liquid chromatography and mass spectrometry. RESULTS: Robust adult Hb production at protein levels was observed in the LC protocol when feeder cells were used, whereas the HC protocol resulted in higher baseline fetal Hb levels (P < 0.01). We then compared lentiviral transduction of rhesus cells between serum-containing LC media and serum-free StemSpan-based differentiation media, revealing 100-fold more efficient transduction in serum-free differentiation media (P < 0.01). Finally, rhesus CD34+ cells were transduced with lentiviral vectors encoding artificial zinc finger proteins (ZF-Ldb1), which can reactivate γ-globin expression via tethering the transcriptional co-regulator Ldb1 to γ-globin promoters, and were differentiated in the optimized erythroid differentiation method. This resulted in marked increases of γ-globin levels compared with control groups (P < 0.01). DISCUSSION: In conclusion, we developed an efficient rhesus erythroid differentiation protocol from hematopoietic progenitor cells with low fetal and high adult Hb production. Further studies are warranted to optimize gene modification and transplantation of rhesus hematopoietic progenitor cells.


Assuntos
Técnicas de Cultura de Células/métodos , Terapia Genética/métodos , Células-Tronco Hematopoéticas/citologia , gama-Globinas/genética , Animais , Diferenciação Celular , Cromatografia Líquida de Alta Pressão/métodos , Proteínas de Ligação a DNA/genética , Células-Tronco Hematopoéticas/metabolismo , Hemoglobinopatias/terapia , Hemoglobinas/análise , Humanos , Proteínas com Domínio LIM/genética , Macaca mulatta , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Espectrometria de Massas em Tandem , Fatores de Transcrição/genética , Transdução Genética , Dedos de Zinco/genética , gama-Globinas/análise
8.
Surg Endosc ; 30(11): 4968-4975, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-26983434

RESUMO

BACKGROUND: Transumbilical single-port laparoscopic appendectomy (SPLA) is a promising procedure that features less pain, faster recovery of postoperative bowel function and superior cosmetic results. We performed a retrospective comparative analysis of SPLA versus conventional laparoscopic surgery (CLA) to evaluate the safety and efficacy in acute appendicitis. METHODS: From December 2008 to November 2013, laparoscopic surgery was performed on 636 patients with acute appendicitis at the Department of Surgery, Chuncheon Sacred Heart Hospital. Under approval of Institutional Review Board, data concerning baseline characteristics, operative outcomes, postoperative complications and postoperative functional recovery were compared between both procedures. RESULTS: After exclusion of 18 patients, 618 patients treated for acute appendicitis were included. SPLA was performed in 375 patients and CLA in 243 patients. Complicated appendicitis was more prevalent in the CLA group (26.3 %) than in the SPLA group (17.1 %) (p = 0.005). There was no difference between groups in operation time (p = 0.235), postoperative duration of hospital stay (p = 0.672) and readmission rate (p = 0.688). The rate of postoperative complications was similar in both groups (10.7 % in SPLA vs. 11.1 % in CLA, p = 0.862). In subgroup analysis of complicated appendicitis, more patients needed conversion to open surgery in the SPLA group (15.6 vs. 1.6 %, p = 0.005). CONCLUSION: In uncomplicated appendicitis, SPLA can be performed safely and efficiently. However, more selective indication for SPLA should be applied in cases of complicated appendicitis because of the greater risk of open conversion.


Assuntos
Apendicectomia/métodos , Apendicite/cirurgia , Laparoscopia/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Conversão para Cirurgia Aberta/estatística & dados numéricos , Feminino , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , Estudos Retrospectivos , Adulto Jovem
9.
Nat Methods ; 9(10): 973-5, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22941364

RESUMO

Targeted DNA double-strand breaks introduced by rare-cleaving designer endonucleases can be harnessed for gene disruption applications by engaging mutagenic nonhomologous end-joining DNA repair pathways. However, endonuclease-mediated DNA breaks are often subject to precise repair, which limits the efficiency of targeted genome editing. To address this issue, we coupled designer endonucleases to DNA end-processing enzymes to drive mutagenic break resolution, achieving up to 25-fold enhancements in gene disruption rates.


Assuntos
Quebras de DNA de Cadeia Dupla , Endonucleases/fisiologia , Animais , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Exodesoxirribonucleases/fisiologia , Células HEK293 , Humanos , Camundongos , Fosfoproteínas/fisiologia , Receptores CCR5/fisiologia
10.
Nat Methods ; 8(8): 671-6, 2011 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-21743461

RESUMO

Site-specific genome engineering technologies are increasingly important tools in the postgenomic era, where biotechnological objectives often require organisms with precisely modified genomes. Rare-cutting endonucleases, through their capacity to create a targeted DNA strand break, are one of the most promising of these technologies. However, realizing the full potential of nuclease-induced genome engineering requires a detailed understanding of the variables that influence resolution of nuclease-induced DNA breaks. Here we present a genome engineering reporter system, designated 'traffic light', that supports rapid flow-cytometric analysis of repair pathway choice at individual DNA breaks, quantitative tracking of nuclease expression and donor template delivery, and high-throughput screens for factors that bias the engineering outcome. We applied the traffic light system to evaluate the efficiency and outcome of nuclease-induced genome engineering in human cell lines and identified strategies to facilitate isolation of cells in which a desired engineering outcome has occurred.


Assuntos
Dano ao DNA/genética , Reparo do DNA/genética , Genes Reporter/genética , Engenharia Genética/métodos , Genoma/genética
11.
Blood ; 119(19): 4395-407, 2012 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-22431569

RESUMO

The immunodeficiency disorder Wiskott-Aldrich syndrome (WAS) leads to life-threatening hematopoietic cell dysfunction. We used WAS protein (WASp)-deficient mice to analyze the in vivo efficacy of lentiviral (LV) vectors using either a viral-derived promoter, MND, or the human proximal WAS promoter (WS1.6) for human WASp expression. Transplantation of stem cells transduced with MND-huWASp LV resulted in sustained, endogenous levels of WASp in all hematopoietic lineages, progressive selection for WASp+ T, natural killer T and B cells, rescue of T-cell proliferation and cytokine production, and substantial restoration of marginal zone (MZ) B cells. In contrast, WS1.6-huWASp LV recipients exhibited subendogenous WASp expression in all cell types with only partial selection of WASp+ T cells and limited correction in MZ B-cell numbers. In parallel, WS1.6-huWASp LV recipients exhibited an altered B-cell compartment, including higher numbers of λ-light-chain+ naive B cells, development of self-reactive CD11c+FAS+ B cells, and evidence for spontaneous germinal center (GC) responses. These observations correlated with B-cell hyperactivity and increased titers of immunoglobulin (Ig)G2c autoantibodies, suggesting that partial gene correction may predispose toward autoimmunity. Our findings identify the advantages and disadvantages associated with each vector and suggest further clinical development of the MND-huWASp LV for a future clinical trial for WAS.


Assuntos
Linhagem da Célula/genética , Terapia Genética/métodos , Células-Tronco Hematopoéticas/metabolismo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Animais , Células Cultivadas , Regulação da Expressão Gênica , Vetores Genéticos , Células-Tronco Hematopoéticas/fisiologia , Humanos , Lentivirus/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Camundongos Transgênicos , Resultado do Tratamento , Regulação para Cima/genética , Síndrome de Wiskott-Aldrich/patologia , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína da Síndrome de Wiskott-Aldrich/fisiologia
12.
Nucleic Acids Res ; 40(2): e14, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22110042

RESUMO

Two major limitations to achieve efficient homing endonuclease-stimulated gene correction using retroviral vectors are low frequency of gene targeting and random integration of the targeting vectors. To overcome these issues, we developed a reporter system for quick and facile testing of novel strategies to promote the selection of cells that undergo targeted gene repair and to minimize the persistence of random integrations and non-homologous end-joining events. In this system, the gene target has an I-SceI site upstream of an EGFP reporter; and the repair template includes a non-functional EGFP gene, the positive selection transgene MGMTP140K tagged with mCherry, and the inducible Caspase-9 suicide gene. Using this dual fluorescent reporter system it is possible to detect properly targeted integration. Furthermore, this reporter system provides an efficient approach to enrich for gene correction events and to deplete events produced by random integration. We have also developed a second reporter system containing MGMTP140K in the integrated target locus, which allows for selection of primary cells with the integrated gene target after transplantation. This system is particularly useful for testing repair strategies in primary hematopoietic stem cells. Thus, our reporter systems should allow for more efficient gene correction with less unwanted off target effects.


Assuntos
Endodesoxirribonucleases/metabolismo , Marcação de Genes/métodos , Genes Reporter , Linhagem Celular , Corantes Fluorescentes/análise , Genoma , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Proteínas Metiltransferases/análise , Proteínas Metiltransferases/genética
13.
Eur J Immunol ; 42(5): 1327-36, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22311635

RESUMO

Transitional and naïve mature peripheral B cells respond very differently to B-cell receptor (BCR) cross-linking. While transitional B cells undergo apoptosis upon BCR engagement, mature B cells survive and proliferate. This differential response correlates with the capacity of mature, but not transitional B cells to transcribe genes that promote cell survival and proliferation, including those encoding c-Myc and the Bcl-2 family members Bcl-xL and A1. We recently demonstrated that transitional B cells fail to assemble transcriptional machinery at the promoter region of these target genes despite equivalent cytoplasmic signaling and nuclear translocation of key transcription factors including NF-κB and nuclear factor of activated T cells (NFAT). The transcription factor myocyte enhancer factor-2C (MEF2C) is regulated by both calcineurin and mitogen-activated protein kinase signaling pathways, and is essential for proliferation and survival downstream of BCR engagement in mature B cells. In this work, we demonstrate that transitional B cells have intrinsically low levels of MEF2C protein and DNA-binding activity, and that this developmental difference in MEF2C expression is functionally significant. Forced expression of MEF2C in transitional B cells promoted cell survival, proliferation, and upregulation of pro-survival genes. Thus, low MEF2C expression limits transitional B-cell responsiveness to BCR engagement before these cells reach maturity.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Fatores de Regulação Miogênica/genética , Células Precursoras de Linfócitos B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Animais , Proteínas Reguladoras de Apoptose/genética , Proteína 11 Semelhante a Bcl-2 , Proliferação de Células , Sobrevivência Celular , Fatores de Transcrição MEF2 , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fatores de Regulação Miogênica/análise , Proteínas Proto-Oncogênicas/genética , Regulação para Cima
14.
J Virol ; 86(1): 373-81, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22013043

RESUMO

Here we report a novel viral glycoprotein created by replacing a natural receptor-binding sequence of the ecotropic Moloney murine leukemia virus envelope glycoprotein with the peptide ligand somatostatin. This new chimeric glycoprotein, which has been named the Sst receptor binding site (Sst-RBS), gives targeted transduction based on three criteria: (i) a gain of the use of a new entry receptor not used by any known virus; (ii) targeted entry at levels comparable to gene delivery by wild-type ecotropic Moloney murine leukemia virus and vesicular stomatitis virus (VSV) G glycoproteins; and (iii) a loss of the use of the natural ecotropic virus receptor. Retroviral vectors coated with Sst-RBS gained the ability to bind and transduce human 293 cells expressing somatostatin receptors. Their infection was specific to target somatostatin receptors, since a synthetic somatostatin peptide inhibited infection in a dose-dependent manner and the ability to transduce mouse cells bearing the natural ecotropic receptor was effectively lost. Importantly, vectors coated with the Sst-RBS glycoprotein gave targeted entry of up to 1 × 10(6) transducing U/ml, a level comparable to that seen with infection of vectors coated with the parental wild-type ecotropic Moloney murine leukemia virus glycoprotein through the ecotropic receptor and approaching that of infection of VSV G-coated vectors through the VSV receptor. To our knowledge, this is the first example of a glycoprotein that gives targeted entry of retroviral vectors at levels comparable to the natural capacity of viral envelope glycoproteins.


Assuntos
Técnicas de Transferência de Genes/instrumentação , Vírus da Leucemia Murina de Moloney/genética , Receptores de Somatostatina/metabolismo , Somatostatina/genética , Proteínas do Envelope Viral/genética , Internalização do Vírus , Animais , Sítios de Ligação , Linhagem Celular , Marcação de Genes/instrumentação , Vetores Genéticos/química , Vetores Genéticos/genética , Vetores Genéticos/fisiologia , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Vírus da Leucemia Murina de Moloney/química , Vírus da Leucemia Murina de Moloney/fisiologia , Ligação Proteica , Engenharia de Proteínas , Receptores de Somatostatina/química , Receptores de Somatostatina/genética , Receptores Virais/genética , Receptores Virais/metabolismo , Somatostatina/química , Somatostatina/metabolismo , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/metabolismo
15.
Nucleic Acids Res ; 39(21): e143, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21911364

RESUMO

A key challenge for the academic and biopharmaceutical communities is the rapid and scalable production of recombinant proteins for supporting downstream applications ranging from therapeutic trials to structural genomics efforts. Here, we describe a novel system for the production of recombinant mammalian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system, often requiring <3 weeks to achieve stable, high-level expression: Daedalus. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression. This system can bypass the tedious and time-consuming steps of conventional protein production methods by employing the secretion pathway of serum-free adapted human suspension cell lines, such as 293 Freestyle. Using optimized lentiviral vectors, yields of 20-100 mg/l of correctly folded and post-translationally modified, endotoxin-free protein of up to ~70 kDa in size, can be achieved in conventional, small-scale (100 ml) culture. At these yields, most proteins can be purified using a single size-exclusion chromatography step, immediately appropriate for use in structural, biophysical or therapeutic applications.


Assuntos
Lentivirus/genética , Proteínas Recombinantes/biossíntese , Proteínas de Fase Aguda/biossíntese , Proteínas de Fase Aguda/química , Proteínas de Fase Aguda/genética , Animais , Linhagem Celular , Cristalografia , Citocinas/genética , Citocinas/metabolismo , Técnicas Genéticas , Vetores Genéticos , Humanos , Lipocalina-2 , Lipocalinas/biossíntese , Lipocalinas/química , Lipocalinas/genética , Camundongos , Proteínas Oncogênicas/biossíntese , Proteínas Oncogênicas/química , Proteínas Oncogênicas/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Transdução Genética
16.
Mol Ther Nucleic Acids ; 31: 452-465, 2023 Mar 14.
Artigo em Inglês | MEDLINE | ID: mdl-36852088

RESUMO

Transcriptional enhancers can be in physical proximity of their target genes via chromatin looping. The enhancer at the ß-globin locus (locus control region [LCR]) contacts the fetal-type (HBG) and adult-type (HBB) ß-globin genes during corresponding developmental stages. We have demonstrated previously that forcing proximity between the LCR and HBG genes in cultured adult-stage erythroid cells can activate HBG transcription. Activation of HBG expression in erythroid cells is of benefit to patients with sickle cell disease. Here, using the ß-globin locus as a model, we provide proof of concept at the organismal level that forced enhancer rewiring might present a strategy to alter gene expression for therapeutic purposes. Hematopoietic stem and progenitor cells (HSPCs) from mice bearing human ß-globin genes were transduced with lentiviral vectors expressing a synthetic transcription factor (ZF-Ldb1) that fosters LCR-HBG contacts. When engrafted into host animals, HSPCs gave rise to adult-type erythroid cells with elevated HBG expression. Vectors containing ZF-Ldb1 were optimized for activity in cultured human and rhesus macaque erythroid cells. Upon transplantation into rhesus macaques, erythroid cells from HSPCs expressing ZF-Ldb1 displayed elevated HBG production. These findings in two animal models suggest that forced redirection of gene-regulatory elements may be used to alter gene expression to treat disease.

17.
J Immunother Cancer ; 11(3)2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36918221

RESUMO

BACKGROUND: Chimeric antigen receptor (CAR) T-cell therapies have demonstrated transformational outcomes in the treatment of B-cell malignancies, but their widespread use is hindered by technical and logistical challenges associated with ex vivo cell manufacturing. To overcome these challenges, we developed VivoVec, a lentiviral vector-based platform for in vivo engineering of T cells. UB-VV100, a VivoVec clinical candidate for the treatment of B-cell malignancies, displays an anti-CD3 single-chain variable fragment (scFv) on the surface and delivers a genetic payload that encodes a second-generation CD19-targeted CAR along with a rapamycin-activated cytokine receptor (RACR) system designed to overcome the need for lymphodepleting chemotherapy in supporting successful CAR T-cell expansion and persistence. In the presence of exogenous rapamycin, non-transduced immune cells are suppressed, while the RACR system in transduced cells converts rapamycin binding to an interleukin (IL)-2/IL-15 signal to promote proliferation. METHODS: UB-VV100 was administered to peripheral blood mononuclear cells (PBMCs) from healthy donors and from patients with B-cell malignancy without additional stimulation. Cultures were assessed for CAR T-cell transduction and function. Biodistribution was evaluated in CD34-humanized mice and in canines. In vivo efficacy was evaluated against normal B cells in CD34-humanized mice and against systemic tumor xenografts in PBMC-humanized mice. RESULTS: In vitro, administration of UB-VV100 resulted in dose-dependent and anti-CD3 scFv-dependent T-cell activation and CAR T-cell transduction. The resulting CAR T cells exhibited selective expansion in rapamycin and antigen-dependent activity against malignant B-cell targets. In humanized mouse and canine studies, UB-VV100 demonstrated a favorable biodistribution profile, with transduction events limited to the immune compartment after intranodal or intraperitoneal administration. Administration of UB-VV100 to humanized mice engrafted with B-cell tumors resulted in CAR T-cell transduction, expansion, and elimination of systemic malignancy. CONCLUSIONS: These findings demonstrate that UB-VV100 generates functional CAR T cells in vivo, which could expand patient access to CAR T technology in both hematological and solid tumors without the need for ex vivo cell manufacturing.


Assuntos
Receptores de Antígenos Quiméricos , Linfócitos T , Humanos , Animais , Cães , Camundongos , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos de Linfócitos T , Leucócitos Mononucleares , Distribuição Tecidual , Engenharia Celular/métodos
18.
Blood ; 115(11): 2146-55, 2010 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-20093406

RESUMO

The immunodeficiency disorder, X-linked agammaglobulinemia (XLA), results from mutations in the gene encoding Bruton tyrosine kinase (Btk). Btk is required for pre-B cell clonal expansion and B-cell antigen receptor signaling. XLA patients lack mature B cells and immunoglobulin and experience recurrent bacterial infections only partially mitigated by life-long antibody replacement therapy. In pursuit of definitive therapy for XLA, we tested ex vivo gene therapy using a lentiviral vector (LV) containing the immunoglobulin enhancer (Emu) and Igbeta (B29) minimal promoter to drive B lineage-specific human Btk expression in Btk/Tec(-/-) mice, a strain that reproduces the features of human XLA. After transplantation of EmuB29-Btk-LV-transduced stem cells, treated mice showed significant, albeit incomplete, rescue of mature B cells in the bone marrow, peripheral blood, spleen, and peritoneal cavity, and improved responses to T-independent and T-dependent antigens. LV-treated B cells exhibited enhanced B-cell antigen receptor signaling and an in vivo selective advantage in the peripheral versus central B-cell compartment. Secondary transplantation showed sustained Btk expression, viral integration, and partial functional responses, consistent with long-term stem cell marking; and serial transplantation revealed no evidence for cellular or systemic toxicity. These findings strongly support pursuit of B lineage-targeted LV gene therapy in human XLA.


Assuntos
Agamaglobulinemia/fisiopatologia , Agamaglobulinemia/terapia , Linfócitos B/fisiologia , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Terapia Genética , Lentivirus/genética , Recuperação de Função Fisiológica/fisiologia , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/citologia , Células da Medula Óssea/citologia , Transplante de Medula Óssea , Antígenos CD79/genética , Linhagem Celular , Linhagem da Célula , Modelos Animais de Doenças , Doenças Genéticas Ligadas ao Cromossomo X/fisiopatologia , Terapia Genética/efeitos adversos , Vetores Genéticos/genética , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos/genética , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/metabolismo , Proteínas Tirosina Quinases/uso terapêutico
19.
Mol Ther ; 19(3): 515-25, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21139568

RESUMO

Sustained, targeted, high-level transgene expression in primary B lymphocytes may be useful for gene therapy in B cell disorders. We developed several candidate B-lineage predominant self-inactivating lentiviral vectors (LV) containing alternative enhancer/promoter elements including: the immunoglobulin ß (Igß) (B29) promoter combined with the immunoglobulin µ enhancer (EµB29); and the endogenous BTK promoter with or without Eµ (EµBtkp or Btkp). LV-driven enhanced green fluorescent protein (eGFP) reporter expression was evaluated in cell lines and primary cells derived from human or murine hematopoietic stem cells (HSC). In murine primary cells, EµB29 and EµBtkp LV-mediated high-level expression in immature and mature B cells compared with all other lineages. Expression increased with B cell maturation and was maintained in peripheral subsets. Expression in T and myeloid cells was much lower in percentage and intensity. Similarly, both EµB29 and EµBtkp LV exhibited high-level activity in human primary B cells. In contrast to EµB29, Btkp and EµBtkp LV also exhibited modest activity in myeloid cells, consistent with the expression profile of endogenous Bruton's tyrosine kinase (Btk). Notably, EµB29 and EµBtkp activity was superior in all expression models to an alternative, B-lineage targeted vector containing the EµS.CD19 enhancer/promoter. In summary, EµB29 and EµBtkp LV comprise efficient delivery platforms for gene expression in B-lineage cells.


Assuntos
Linfócitos B/metabolismo , Linfócitos B/patologia , Terapia Genética , Vetores Genéticos/genética , Lentivirus/genética , Proteínas Tirosina Quinases , Tirosina Quinase da Agamaglobulinemia , Agamaglobulinemia/genética , Agamaglobulinemia/terapia , Animais , Linfócitos B/imunologia , Linhagem Celular , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Ordem dos Genes , Genes Reporter/genética , Doenças Genéticas Ligadas ao Cromossomo X/genética , Doenças Genéticas Ligadas ao Cromossomo X/terapia , Vetores Genéticos/administração & dosagem , Células HEK293 , Transplante de Células-Tronco Hematopoéticas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Camundongos SCID , Células Mieloides/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética
20.
Mol Ther Methods Clin Dev ; 20: 635-651, 2021 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-33718514

RESUMO

X-linked agammaglobulinemia (XLA) is an immune disorder caused by mutations in Bruton's tyrosine kinase (BTK). BTK is expressed in B and myeloid cells, and its deficiency results in a lack of mature B cells and protective antibodies. We previously reported a lentivirus (LV) BTK replacement therapy that restored B cell development and function in Btk and Tec double knockout mice (a phenocopy of human XLA). In this study, with the goal of optimizing both the level and lineage specificity of BTK expression, we generated LV incorporating the proximal human BTK promoter. Hematopoietic stem cells from Btk -/- Tec -/- mice transduced with this vector rescued lineage-specific expression and restored B cell function in Btk -/- Tec -/- recipients. Next, we tested addition of candidate enhancers and/or ubiquitous chromatin opening elements (UCOEs), as well as codon optimization to improve BTK expression. An Eµ enhancer improved B cell rescue, but increased immunoglobulin G (IgG) autoantibodies. Addition of the UCOE avoided autoantibody generation while improving B cell development and function and reducing vector silencing. An optimized vector containing a truncated UCOE upstream of the BTK promoter and codon-optimized BTK cDNA resulted in stable, lineage-regulated BTK expression that mirrored endogenous BTK, making it a strong candidate for XLA therapy.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA