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Over 4% of the global population is estimated to live with autoimmune disease, necessitating immunosuppressive treatment that is often chronic, not curative, and carries associated risks. B cells have emerged as key players in disease pathogenesis, as evidenced by partial responsiveness to B cell depletion by antibody-based therapies. However, these treatments often have transient effects due to incomplete depletion of tissue-resident B cells. Chimeric antigen receptor (CAR) T cells targeting B cells have demonstrated efficacy in refractory systemic lupus erythematosus. To this end, we developed an anti-CD19 CAR T cell product candidate, CABA-201, containing a clinically evaluated fully human CD19 binder (IC78) with a 4-1BB costimulatory domain and CD3 zeta stimulation domain for treatment refractory autoimmune disease. Here, we demonstrate specific cytotoxic activity of CABA-201 against CD19+ Nalm6 cells with no off-target effects on primary human cells. Novel examination of CABA-201 generated from primary T cells from multiple patients with autoimmune disease displayed robust CAR surface expression and effective elimination of the intended target autologous CD19+ B cells in vitro. Together, these findings support the tolerability and activity of CABA-201 for clinical development in patients with autoimmune disease.
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Muscle-specific tyrosine kinase myasthenia gravis (MuSK MG) is an autoimmune disease that causes life-threatening muscle weakness due to anti-MuSK autoantibodies that disrupt neuromuscular junction signaling. To avoid chronic immunosuppression from current therapies, we engineered T cells to express a MuSK chimeric autoantibody receptor with CD137-CD3ζ signaling domains (MuSK-CAART) for precision targeting of B cells expressing anti-MuSK autoantibodies. MuSK-CAART demonstrated similar efficacy as anti-CD19 chimeric antigen receptor T cells for depletion of anti-MuSK B cells and retained cytolytic activity in the presence of soluble anti-MuSK antibodies. In an experimental autoimmune MG mouse model, MuSK-CAART reduced anti-MuSK IgG without decreasing B cells or total IgG levels, reflecting MuSK-specific B cell depletion. Specific off-target interactions of MuSK-CAART were not identified in vivo, in primary human cell screens or by high-throughput human membrane proteome array. These data contributed to an investigational new drug application and phase 1 clinical study design for MuSK-CAART for the treatment of MuSK autoantibody-positive MG.
Assuntos
Miastenia Gravis Autoimune Experimental , Receptores Colinérgicos , Humanos , Camundongos , Animais , Receptores Colinérgicos/uso terapêutico , Autoantígenos/uso terapêutico , Miastenia Gravis Autoimune Experimental/tratamento farmacológico , Linfócitos T , Autoanticorpos/uso terapêutico , Imunoglobulina G , Proteínas Tirosina Quinases/uso terapêutico , MúsculosRESUMO
BACKGROUND: Gene-modified autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) reactive against the NY-ESO-1-specific HLA-A*02-restricted peptide SLLMWITQC (NY-ESO-1 SPEAR T-cells; GSK 794), have demonstrated clinical activity in patients with advanced synovial sarcoma (SS). The factors contributing to gene-modified T-cell expansion and the changes within the tumor microenvironment (TME) following T-cell infusion remain unclear. These studies address the immunological mechanisms of response and resistance in patients with SS treated with NY-ESO-1 SPEAR T-cells. METHODS: Four cohorts were included to evaluate antigen expression and preconditioning on efficacy. Clinical responses were assessed by RECIST v1.1. Engineered T-cell persistence was determined by qPCR. Serum cytokines were evaluated by immunoassay. Transcriptomic analyses and immunohistochemistry were performed on tumor biopsies from patients before and after T-cell infusion. Gene-modified T-cells were detected within the TME via an RNAish assay. RESULTS: Responses across cohorts were affected by preconditioning and intra-tumoral NY-ESO-1 expression. Of the 42 patients reported (data cut-off 4June2018), 1 patient had a complete response, 14 patients had partial responses, 24 patients had stable disease, and 3 patients had progressive disease. The magnitude of gene-modified T-cell expansion shortly after infusion was associated with response in patients with high intra-tumoral NY-ESO-1 expression. Patients receiving a fludarabine-containing conditioning regimen experienced increases in serum IL-7 and IL-15. Prior to infusion, the TME exhibited minimal leukocyte infiltration; CD163+ tumor-associated macrophages (TAMs) were the dominant population. Modest increases in intra-tumoral leukocytes (≤5%) were observed in a subset of subjects at approximately 8 weeks. Beyond 8 weeks post infusion, the TME was minimally infiltrated with a TAM-dominant leukocyte infiltrate. Tumor-associated antigens and antigen presentation did not significantly change within the tumor post-T-cell infusion. Finally, NY-ESO-1 SPEAR T cells trafficked to the TME and maintained cytotoxicity in a subset of patients. CONCLUSIONS: Our studies elucidate some factors that underpin response and resistance to NY-ESO-1 SPEAR T-cell therapy. From these data, we conclude that a lymphodepletion regimen containing high doses of fludarabine and cyclophosphamide is necessary for SPEAR T-cell persistence and efficacy. Furthermore, these data demonstrate that non-T-cell inflamed tumors, which are resistant to PD-1/PD-L1 inhibitors, can be treated with adoptive T-cell based immunotherapy. TRIAL REGISTRATION: ClinicalTrials.gov, NCT01343043 , Registered 27 April 2011.
Assuntos
Antígenos de Neoplasias/imunologia , Imunoterapia Adotiva , Proteínas de Membrana/imunologia , Sarcoma Sinovial/imunologia , Sarcoma Sinovial/terapia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Biomarcadores , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Citocinas/metabolismo , Citotoxicidade Imunológica , Antígenos HLA-A/imunologia , Humanos , Imuno-Histoquímica , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/imunologia , Linfócitos do Interstício Tumoral/metabolismo , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/genética , Receptores de Antígenos Quiméricos/metabolismo , Sarcoma Sinovial/patologia , Especificidade do Receptor de Antígeno de Linfócitos T , Resultado do Tratamento , Microambiente Tumoral/imunologiaRESUMO
This study in patients with relapsed, refractory, or high-risk multiple myeloma (MM) evaluated the safety and activity of autologous T cells engineered to express an affinity-enhanced T-cell receptor (TCR) that recognizes a peptide shared by cancer antigens New York esophageal squamous cell carcinoma-1 (NY-ESO-1) and L-antigen family member 1 (LAGE-1) and presented by HLA-A*02:01. T cells collected from 25 HLA-A*02:01-positive patients with MM expressing NY-ESO-1 and/or LAGE-1 were activated, transduced with self-inactivating lentiviral vector encoding the NY-ESO-1c259TCR, and expanded in culture. After myeloablation and autologous stem cell transplant (ASCT), all 25 patients received an infusion of up to 1 × 1010 NY-ESO-1 specific peptide enhanced affinity receptor (SPEAR) T cells. Objective response rate (International Myeloma Working Group consensus criteria) was 80% at day 42 (95% confidence interval [CI], 0.59-0.93), 76% at day 100 (95% CI, 0.55-0.91), and 44% at 1 year (95% CI, 0.24-0.65). At year 1, 13/25 patients were disease progression-free (52%); 11 were responders (1 stringent complete response, 1 complete response, 8 very good partial response, 1 partial response). Three patients remained disease progression-free at 38.6, 59.2, and 60.6 months post-NY-ESO-1 SPEAR T-cell infusion. Median progression-free survival was 13.5 months (range, 3.2-60.6 months); median overall survival was 35.1 months (range, 6.4-66.7 months). Infusions were well tolerated; cytokine release syndrome was not reported. No fatal serious adverse events occurred during study conduct. NY-ESO-1 SPEAR T cells expanded in vivo, trafficked to bone marrow, demonstrated persistence, and exhibited tumor antigen-directed functionality. In this MM patient population, NY-ESO-1 SPEAR T-cell therapy in the context of ASCT was associated with antitumor activity. This trial was registered at www.clinicaltrials.gov as #NCT01352286.
Assuntos
Antígenos de Neoplasias/imunologia , Imunoterapia Adotiva , Proteínas de Membrana/imunologia , Mieloma Múltiplo/imunologia , Mieloma Múltiplo/terapia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Terapia Combinada , Citocinas/metabolismo , Feminino , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante de Células-Tronco Hematopoéticas/métodos , Humanos , Imunoterapia Adotiva/efeitos adversos , Imunoterapia Adotiva/métodos , Masculino , Proteínas de Membrana/antagonistas & inibidores , Pessoa de Meia-Idade , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos Quiméricos/genética , Transplante Autólogo , Resultado do Tratamento , Adulto JovemRESUMO
We evaluated the safety and activity of autologous T cells expressing NY-ESO-1c259, an affinity-enhanced T-cell receptor (TCR) recognizing an HLA-A2-restricted NY-ESO-1/LAGE1a-derived peptide, in patients with metastatic synovial sarcoma (NY-ESO-1c259T cells). Confirmed antitumor responses occurred in 50% of patients (6/12) and were characterized by tumor shrinkage over several months. Circulating NY-ESO-1c259T cells were present postinfusion in all patients and persisted for at least 6 months in all responders. Most of the infused NY-ESO-1c259T cells exhibited an effector memory phenotype following ex vivo expansion, but the persisting pools comprised largely central memory and stem-cell memory subsets, which remained polyfunctional and showed no evidence of T-cell exhaustion despite persistent tumor burdens. Next-generation sequencing of endogenous TCRs in CD8+ NY-ESO-1c259T cells revealed clonal diversity without contraction over time. These data suggest that regenerative pools of NY-ESO-1c259T cells produced a continuing supply of effector cells to mediate sustained, clinically meaningful antitumor effects.Significance: Metastatic synovial sarcoma is incurable with standard therapy. We employed engineered T cells targeting NY-ESO-1, and the data suggest that robust, self-regenerating pools of CD8+ NY-ESO-1c259T cells produce a continuing supply of effector cells over several months that mediate clinically meaningful antitumor effects despite prolonged exposure to antigen. Cancer Discov; 8(8); 944-57. ©2018 AACR.See related commentary by Keung and Tawbi, p. 914This article is highlighted in the In This Issue feature, p. 899.
Assuntos
Antígenos de Neoplasias/imunologia , Proteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Sarcoma Sinovial/terapia , Linfócitos T/transplante , Transferência Adotiva , Adulto , Linfócitos T CD8-Positivos/metabolismo , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Metástase Neoplásica , Projetos Piloto , Sarcoma Sinovial/imunologia , Linfócitos T/imunologia , Resultado do Tratamento , Adulto JovemRESUMO
Adoptive T-cell therapy, incorporating engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs), target tumor antigens with high affinity and specificity. To increase the potency of adoptively transferred T cells, patients are conditioned with lymphodepleting chemotherapy regimens prior to adoptive T-cell transfer (ACT), and data suggest that fludarabine is an important component of an effective regimen. In a recent clinical trial using CAR-T cells engineered to target the CD19 B-cell antigen to treat acute lymphoblastic leukemia, JCAR-015 (NCT02535364), two patient deaths due to cerebral edema led to trial suspension. The lymphodepleting agent fludarabine was suggested as the causative agent, in part due to its known association with neurotoxicity and its ability to induce greater potency. In a similar CAR-T study also incorporating fludarabine in the preconditioning regimen, ZUMA-1 (NCT02348216), one patient died of cerebral edema. However, subsequent deaths in the JCAR-015 study after removal of fludarabine and improved understanding behind the mechanisms of CAR-T-related encephalopathy syndrome (CRES) indicate that fludarabine is not the primary causative agent of cerebral edema and that it can be safely incorporated into the preconditioning regimen for ACT. Since entering clinical use in the late 1980s as a chemotherapy agent, fludarabine and similar analogs have been associated with lethal neurological toxicity, yet the manifestation and timing of symptoms are distinct to those observed recently in ACT. Herein, we review the history of fludarabine development as a chemotherapeutic agent, and discuss the safety of its continued use in preconditioning regimens for ACT.
Assuntos
Receptores de Antígenos de Linfócitos T/uso terapêutico , Vidarabina/análogos & derivados , Antígenos CD19/imunologia , Humanos , Imunoterapia Adotiva/métodos , Síndromes Neurotóxicas/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Vidarabina/efeitos adversos , Vidarabina/farmacologia , Vidarabina/uso terapêuticoRESUMO
Persistence of T cells engineered with chimeric antigen receptors (CARs) has been a major barrier to use of these cells for molecularly targeted adoptive immunotherapy. To address this issue, we created a series of CARs that contain the T cell receptor-zeta (TCR-zeta) signal transduction domain with the CD28 and/or CD137 (4-1BB) intracellular domains in tandem. After short-term expansion, primary human T cells were subjected to lentiviral gene transfer, resulting in large numbers of cells with >85% CAR expression. In an immunodeficient mouse xenograft model of primary human pre-B-cell acute lymphoblastic leukemia, human T cells expressing anti-CD19 CARs containing CD137 exhibited the greatest antileukemic efficacy and prolonged (>6 months) survival in vivo, and were significantly more effective than cells expressing CARs containing TCR-zeta alone or CD28-zeta signaling receptors. We uncovered a previously unrecognized, antigen-independent effect of CARs expressing the CD137 cytoplasmic domain that likely contributes to the enhanced antileukemic efficacy and survival in tumor bearing mice. Furthermore, our studies revealed significant discrepancies between in vitro and in vivo surrogate measures of CAR efficacy. Together these results suggest that incorporation of the CD137 signaling domain in CARs should improve the persistence of CARs in the hematologic malignancies and hence maximize their antitumor activity.
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Leucemia/terapia , Receptores de Antígenos de Linfócitos T/fisiologia , Transdução de Sinais/fisiologia , Linfócitos T/imunologia , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/imunologia , Animais , Antígenos CD28/genética , Antígenos CD28/imunologia , Sobrevivência Celular , Células Cultivadas , Vetores Genéticos/genética , Humanos , Imunoterapia Adotiva/métodos , Lentivirus/genética , Leucemia/genética , Leucemia/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Reação em Cadeia da Polimerase , Receptores de Antígenos de Linfócitos T/genética , Proteínas Recombinantes/genética , Transdução de Sinais/genética , Linfócitos T/citologia , Linfócitos T/metabolismo , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Lentiviral vector-based gene therapy has been used to target the human immunodeficiency virus (HIV) using an antisense env payload. We have analyzed lentiviral-vector integration sites from three treated individuals. We compared integration sites from the ex vivo vector-transduced CD4+ cell products to sites from cells recovered at several times after infusion. Integration sites were analyzed using 454 pyrosequencing, yielding a total of 7,782 unique integration sites from the ex vivo product and 237 unique sites from cells recovered after infusion. Integrated vector copies in both data sets were found to be strongly enriched within active genes and near epigenetic marks associated with active transcription units. Analysis of integration relative to nucleosome structure on target DNA indicated favoring of integration in outward facing DNA major grooves on the nucleosome surface. There was no indication that growth of transduced cells after infusion resulted in enrichment for integration sites near proto-oncogene 5'-ends or within tumor suppressor genes. Thus, this first look at the longitudinal evolution of cells transduced with a lentiviral vector after infusion of gene modified CD4+ cells provided no evidence for abnormal expansions of cells due to vector-mediated insertional activation of proto-oncogenes.
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Linfócitos T CD4-Positivos/metabolismo , Vetores Genéticos/genética , Infecções por HIV/genética , Infecções por HIV/terapia , Lentivirus/genética , Integração Viral/genética , Ensaios Clínicos Fase I como Assunto , Ensaios Clínicos Fase II como Assunto , Biologia Computacional , Humanos , Proto-Oncogene Mas , Análise de Sequência de DNARESUMO
We report findings from a clinical evaluation of lentiviral vectors in a phase I open-label nonrandomized clinical trial for HIV. This trial evaluated the safety of a conditionally replicating HIV-1-derived vector expressing an antisense gene against the HIV envelope. Five subjects with chronic HIV infection who had failed to respond to at least two antiviral regimens were enrolled. A single i.v. infusion of gene-modified autologous CD4 T cells was well tolerated in all patients. Viral loads were stable, and one subject exhibited a sustained decrease in viral load. CD4 counts remained steady or increased in four subjects, and sustained gene transfer was observed. Self-limiting mobilization of the vector was observed in four of five patients. There is no evidence for insertional mutagenesis after 21-36 months of observation. Immune function improved in four subjects. Lentiviral vectors appear promising for gene transfer to humans.
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Vetores Genéticos/genética , Lentivirus/fisiologia , Replicação Viral/genética , Adulto , Técnicas de Transferência de Genes , HIV-1/genética , HIV-1/imunologia , HIV-1/metabolismo , Humanos , Pessoa de Meia-Idade , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismoRESUMO
We examined the ability of a HIV-1-based vector (VRX494) encoding a 937-bp antisense HIV-1 envelope sequence to inhibit the replication of chimeric SIV/HIV-1 viruses encoding the HIV-1 envelope. Challenge of VRX494-transduced CEMx174 cells resulted in potent inhibition of HIV-1 and several SHIV strains. To evaluate the potential efficacy of the VRX494 vector for stem cell gene therapy, rhesus CD34(+) bone marrow cells were transduced with VRX494 and then cultured on thymus stroma to induce T cell differentiation. Transduction conditions for CD34(+) cells were optimized to yield high transduction efficiency with minimal effective multiplicity of infection. Purified CD4(+) GFP(+) T cells derived from VRX494-transduced CD34(+) cells strongly inhibited SHIV HXBC2P 3.2 and SHIV 89.6P replication compared to controls. Southern blot analysis of VRX494-transduced T cell clones revealed a subset of cells with multiple proviral copies per cell. Expression of GFP and the antisense inhibitor in VRX494-transduced cells was upregulated by Tat. Analysis of HIV-1 envelope sequences in VRX494-transduced cells revealed modifications consistent with those mediated by double-stranded RNA-dependent adenosine deaminase. These results indicate that the macaque/SHIV model should serve as a useful preclinical model to evaluate this lentiviral vector expressing an HIV-1 antisense inhibitor for stem cell gene therapy for AIDS.
Assuntos
Antígenos CD34/biossíntese , Linfócitos T CD4-Positivos/imunologia , HIV-1/genética , Células-Tronco Hematopoéticas/metabolismo , Lentivirus/genética , Vírus da Imunodeficiência Símia/genética , Replicação Viral , Adenosina Desaminase/metabolismo , Animais , Antígenos CD34/genética , Southern Blotting , Células da Medula Óssea/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Linhagem Celular , Relação Dose-Resposta a Droga , Citometria de Fluxo , Produtos do Gene env/metabolismo , Produtos do Gene rev/metabolismo , Produtos do Gene tat/metabolismo , Terapia Genética/métodos , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Hematopoéticas/virologia , Humanos , Leucócitos Mononucleares/metabolismo , Macaca mulatta , Modelos Genéticos , Oligonucleotídeos Antissenso/química , RNA/química , Retroviridae/genética , Células-Tronco/metabolismo , Linfócitos T/metabolismo , Regulação para Cima , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
BACKGROUND: A stable packaging cell line facilitates large-scale lentivirus vector manufacture. However, it has been difficult to produce clinical-scale HIV-1-based lentiviral vectors using a packaging cell line, in part due to toxicity of packaging genes, and gene silencing that occurs during the long culture period necessary for sequential addition of packaging constructs. METHODS: To avoid these problems, we developed a three-level cascade gene regulation system designed to remove tetracycline transactivator (tTA) from cytomegalovirus immediate early promoter (CMV)-controlled expression to reduce cytotoxicity from constitutive expression of tTA and leaky expression of packaging genes. We also performed a one-step integration of the three packaging plasmids to shorten the culture time for clonal selection. RESULTS: Although leaky expression of p24 and vector production still occurred despite the three-level regulation system, little cytotoxicity was observed and producer cells could be expanded for large-scale production. Producer cells yielded remarkably stable vector production over a period greater than 11 days with the highest titer 3.5 x 10(7) transducing units (TU)/ml and p24 300 ng/ml, yielding 2.2 x 10(11) TU and 1.8 milligram (mg) p24 from one cell factory. No replication-competent lentivirus (RCL) was detected. Long-term analysis demonstrated that, although the cells are genetically stable, partial gene silencing occurs after 2-3 months in culture; however, the one-step construct integration allowed prolonged vector production before significant gene silencing. Concentrated vector resulted in 90% transduction in CD4+ lymphocytes at 20 TU per cell. CD34+ progenitor cells were transduced at 41-46% efficiency, and long-term initiating culture (LTC-IC) was transduced at 45-51%. CONCLUSIONS: These results demonstrate for the first time HIV-1-based lentiviral vector production on the large scale using a packaging cell line.
Assuntos
Vetores Genéticos/genética , HIV-1/genética , Lentivirus/genética , Sequência de Bases , Linhagem Celular , Células Clonais , Clonagem Molecular , Códon , Ensaio de Imunoadsorção Enzimática , Proteínas de Fusão gag-pol/química , Proteínas de Fusão gag-pol/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Produtos do Gene rev/química , Produtos do Gene rev/genética , Produtos do Gene tat/química , Produtos do Gene tat/genética , Engenharia Genética , Vetores Genéticos/biossíntese , Células HeLa , Humanos , Cinética , Glicoproteínas de Membrana/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Plasmídeos , Tetraciclina/farmacologia , Transdução Genética , Transfecção , Proteínas do Envelope Viral/metabolismo , Vírion/metabolismo , Replicação Viral , Produtos do Gene rev do Vírus da Imunodeficiência Humana , Produtos do Gene tat do Vírus da Imunodeficiência HumanaRESUMO
This review is intended to exemplify the roles and responsibilities of the two agencies under the Department of Health and Human Services, the National Institutes of Health and the Food and Drug Administration, that have oversight for human gene transfer clinical protocols, as seen through our experience of bringing a first-in-its-class lentiviral vector to clinical trials. In response to the changing circumstances in gene therapy research between 1999 and 2002, the concerns of these agencies regarding gene therapy have been evolving. This review provides an overview of the major safety concerns regarding insertional oncogenesis, the generation of a replication- competent lentivirus (RCL), and vector mobilization thought to be related to lentiviral vectors, which had to be addressed during the regulatory review process before initiating the clinical trial. Specific monitoring assays to address these concerns were established to test for RCL generation, vector mobilization, persistence of vector-modified cells, and abnormal clonal expansion of modified cells. We hope to provide a basic understanding and appreciation of the regulatory process and major safety concerns, toward providing useful insight to those presently embarking on the development of clinical application of lentiviral vectors.
Assuntos
Terapia Genética , Vetores Genéticos , Lentivirus/genética , Neoplasias/terapia , Pesquisa Biomédica/legislação & jurisprudência , Ensaios Clínicos como Assunto , Regulamentação Governamental , Experimentação Humana/normas , Humanos , Neoplasias/genéticaRESUMO
We report the design of a unique two-plasmid production system for the first lentiviral vector to be evaluated in humans, VRX496. VRX496 is an optimized VSV-G pseudotyped vector derived from HIV-1 that expresses antisense to the HIV envelope gene. We found that a two-plasmid approach to production resulted in higher vector production titers when compared with a three-plasmid approach, which is particularly important for vector production at the large scale. Therefore, we carefully designed a single packaging construct, VIRPAC, for safety by reducing its homology with VRX496 and by insertion of functionally validated genetic elements designed to reduce the risk of generation of a replication-competent lentivirus (RCL). A native cis-acting ribozyme is used to prevent read through into the envelope gene from the upstream gag-pol genes in the packaging vector, thus preventing RNAs containing gag-pol and env together for comparable safety to a three-plasmid system. We demonstrate that there is no significant in vivo vector mobilization using a primary SCID-hu mouse transplantation model, which correlates with the presence of an anti-HIV payload and suggests that inclusion of antisense may be a useful tool to restrict mobilization in other vector constructs. Gene transfer is achieved using a one-step transduction procedure that is simple and clinically translatable, which reaches stable transduction efficiencies of >99% in CD4+ T lymphocytes within 3 days of culture initiation.
Assuntos
Terapia Genética , Vetores Genéticos/genética , Lentivirus/genética , Plasmídeos/genética , Animais , Elementos Antissenso (Genética)/genética , Células Cultivadas , Produtos do Gene env/genética , Engenharia Genética , Vetores Genéticos/efeitos adversos , Vetores Genéticos/biossíntese , HIV-1/genética , Humanos , Camundongos , Camundongos SCID , RNA Catalítico/genética , RNA Catalítico/fisiologia , RNA Viral/metabolismo , Linfócitos T , Transdução Genética , Proteínas Virais/genéticaRESUMO
We present preclinical studies that demonstrate in vitro the feasibility and efficacy of lentivirus-based vector antisense gene therapy for control of HIV replication in primary T lymphocytes isolated from HIV-infected patients discordant for clinical status. VRX496 is a VSV-G-pseudotyped HIV-based vector that encodes an antisense payload against the HIV envelope gene. The antisense payload is under the control of the native LTR promoter, which is highly transactivated by tat upon HIV infection in the cell. Transfer of autologous CD4(+) T lymphocytes genetically modified with VRX496 (VRX496T) into HIV-infected patients is intended to provide a reservoir of cells capable of controlling HIV, potentially delaying AIDS onset. To determine the patient population likely to respond to VRX496 for optimal efficacy, we examined the ability of our research vector, VRX494, to modify and suppress HIV in vitro in lymphocytes isolated from 20 study subjects discordant for CD4 count and viral load. VRX494 is analogous to the clinical vector VRX496, except that it contains GFP as a marker gene instead of the 186-tag marker in the clinical vector. To transfer VRX494 to target cells we developed a novel scalable two-step transduction procedure that has been translated to the clinic in an ongoing clinical trial. This procedure achieved unprecedented transduction efficiencies of 94 +/- 5% in HIV(+) study subject cells. In addition the vector inhibited HIV replication >/=93% in culture regardless of the viral load or CD4 count of the subject or tropism of the virus strain with which they were infected. These findings demonstrate that VRX496T therapy is expected to be beneficial to patients that differ in their status in term of CD4 count and viral load. The methods described represent significant technical advances facilitating execution of lentivirus vector-mediated gene therapy for treatment of HIV and are currently being employed in the first trial evaluating lentivirus vector safety in humans.
Assuntos
Elementos Antissenso (Genética)/genética , Linfócitos T CD4-Positivos/virologia , Vetores Genéticos/genética , Infecções por HIV/terapia , HIV-1/fisiologia , Replicação Viral , Antígenos de Superfície/análise , Antígenos de Superfície/genética , Elementos Antissenso (Genética)/metabolismo , Contagem de Linfócito CD4 , Regulação para Baixo , Terapia Genética/métodos , Infecções por HIV/imunologia , Infecções por HIV/virologia , Humanos , Transdução Genética , Carga ViralRESUMO
We have constructed a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector expressing a 937-base antisense sequence against the HIV-1 envelope gene. Transduction of CD4(+) T lymphocytes with this vector results in expression of the therapeutic antisense sequence and subsequent inhibition of productive HIV-1 replication. In this report, we examined the effect of antisense-mediated suppression on the potential development of virus escape mutants using a permissive T-cell line cultured under conditions that over serial passages specifically allowed for generation and amplification of mutants selected for by antisense pressure. In the resulting virus clones, we found a significant increase in the number of deletions at the envelope target region (91% compared to 27.5% in wild-type HIV). Deletions were most often greater than 1 kb in length. These data demonstrate for the first time that during antisense-mediated suppression of HIV, mutants develop as a direct result of selective pressure on the HIV genomic RNA. Interestingly, in clones where deletions were not observed, there was a high rate of A-G transitions in mutants at the antisense target region but not outside this region, which is consistent with those mutations that are predicted as a result of antisense-mediated modification of double-stranded RNA by the enzyme double-stranded RNA-specific adenosine deaminase. These clones were not found to be escape mutants, as their replicative ability was severely attenuated, and they did not replicate in the presence of vector.
Assuntos
Vetores Genéticos , HIV-1/fisiologia , Lentivirus/genética , RNA Antissenso/farmacologia , Replicação Viral/efeitos dos fármacos , Sequência de Bases , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Células Cultivadas , Produtos do Gene env/antagonistas & inibidores , Produtos do Gene env/genética , Produtos do Gene env/metabolismo , HIV-1/genética , Humanos , Dados de Sequência Molecular , Mutação , RNA Antissenso/metabolismo , Deleção de Sequência , Transdução GenéticaRESUMO
High gene transfer efficiencies have been difficult to achieve in hematopoietic progenitor cells (HPCs) but are important to therapeutic success of HPC gene therapy. Efficient gene transfer is especially challenging with use of column-purified vector for clinical application, as opposed to centrifuged vector commonly used for research. We investigated novel approaches to increase transduction by using a clinically applicable protocol and quantities of column-purified lentiviral vector. Recognizing the association of adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporters with HPC biology, we investigated the effect of transporter inhibitors on transduction. We found the ABC transporter inhibitor verapamil improved transduction efficiency 2- to 6-fold into CD34(+) cells isolated from mobilized peripheral blood, bone marrow, and cord blood. Verapamil also improved transduction in human SCID (severe combined immunodeficient) repopulating cell (SRC) transduction 3- to 4-fold, resulting in 80% to 90% transduction levels in mice receiving primary and secondary transplants without alterations in multilineage reconstitution. Additional ABC transporter substrate inhibitors like quinidine, diltiazem, and ritonavir also enhanced transduction 2- to 3-fold, although ABC transporter inhibitors that are not substrates did not. Enhanced transduction was not observed in mature hematopoietic cells, neurospheres, mesenchymal stem cells, or hepatocytes. Enhancement of transduction in HPCs was observed with vesicular stomatitis virus-G (VSV-G)-pseudotyped lentiviral vector but not with vector pseudotyped with RD114. Thus, we present a new approach for efficient delivery to primitive HPCs by VSV-G-pseudotyped lentiviral vectors.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Vetores Genéticos , Células-Tronco Hematopoéticas/fisiologia , Lentivirus/genética , Transdução Genética/métodos , Adulto , Animais , Antígenos CD34/análise , Bloqueadores dos Canais de Cálcio/farmacologia , Centrifugação , Diltiazem/farmacologia , Terapia Genética/métodos , Células HeLa , Células-Tronco Hematopoéticas/química , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Quinidina/farmacologia , Verapamil/farmacologiaRESUMO
Immune-mediated clearance of virus from the central nervous system (CNS) differs from that of the other organs. Mechanisms of virus control are largely dependent upon the target cell type. Although cytolytic T lymphocytes may mediate clearance of virus from glial cells, non-cytolytic mechanisms mediated by antibody and cytokines dominate clearance from neurons.