RESUMEN
Broadly neutralizing antibodies (bNAbs) directed to HIV-1 have shown promise at suppressing viremia in animal models. However, the use of bNAbs for the central nervous system (CNS) infection is confounded by poor penetration of the blood brain barrier (BBB). Typically, antibody concentrations in the CNS are extremely low; with levels in cerebrospinal fluid (CSF) only 0.1% of blood concentrations. Using a novel nanotechnology platform, which we term nanocapsules, we show effective transportation of the human bNAb PGT121 across the BBB in infant rhesus macaques upon systemic administration up to 1.6% of plasma concentration. We demonstrate that a single dose of PGT121 encased in nanocapsules when delivered at 48h post-infection delays early acute infection with SHIVSF162P3 in infants, with one of four animals demonstrating viral clearance. Importantly, the nanocapsule delivery of PGT121 improves suppression of SHIV infection in the CNS relative to controls.
Asunto(s)
Anticuerpos Antivirales/administración & dosificación , Encéfalo/virología , Anticuerpos ampliamente neutralizantes/administración & dosificación , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Carga Viral/efectos de los fármacos , Animales , Animales Recién Nacidos , Barrera Hematoencefálica , Encéfalo/efectos de los fármacos , Humanos , Macaca mulatta , Nanocápsulas , Virus de la Inmunodeficiencia de los SimiosRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1009404.].
RESUMEN
Due to the durability and persistence of reservoirs of HIV-1-infected cells, combination antiretroviral therapy (ART) is insufficient in eradicating infection. Achieving HIV-1 cure or sustained remission without ART treatment will require the enhanced and persistent effective antiviral immune responses. Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy and show promise in treating HIV-1 infection. Persistence, trafficking, and maintenance of function remain to be a challenge in many of these approaches, which are based on peripheral T cell modification. To overcome many of these issues, we have previously demonstrated successful long-term engraftment and production of anti-HIV CAR T cells in modified hematopoietic stem cells (HSCs) in vivo. Here we report the development and in vivo testing of second generation CD4-based CARs (CD4CAR) against HIV-1 infection using a HSCs-based approach. We found that a modified, truncated CD4-based CAR (D1D2CAR) allows better CAR-T cell differentiation from gene modified HSCs, and maintains similar CTL activity as compared to the full length CD4-based CAR. In addition, D1D2CAR does not mediate HIV infection or stimulation mediated by IL-16, suggesting lower risk of off-target effects. Interestingly, stimulatory domains of 4-1BB but not CD28 allowed successful hematopoietic differentiation and improved anti-viral function of CAR T cells from CAR modified HSCs. Addition of 4-1BB to CD4 based CARs led to faster suppression of viremia during early untreated HIV-1 infection. D1D2CAR 4-1BB mice had faster viral suppression in combination with ART and better persistence of CAR T cells during ART. In summary, our data indicate that the D1D2CAR-41BB is a superior CAR, showing better HSC differentiation, viral suppression and persistence, and less deleterious functions compared to the original CD4CAR, and should continue to be pursued as a candidate for clinical study.
Asunto(s)
Infecciones por VIH/virología , Células Madre Hematopoyéticas/citología , Activación de Linfocitos , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Quiméricos de Antígenos/inmunología , Animales , Infecciones por VIH/inmunología , VIH-1/inmunología , Células Madre Hematopoyéticas/inmunología , Humanos , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Ratones , Receptores de Antígenos de Linfocitos T/genética , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/uso terapéuticoRESUMEN
[This corrects the article DOI: 10.1371/journal.ppat.1006753.].
RESUMEN
Chimeric Antigen Receptor (CAR) T-cells have emerged as a powerful immunotherapy for various forms of cancer and show promise in treating HIV-1 infection. However, significant limitations are persistence and whether peripheral T cell-based products can respond to malignant or infected cells that may reappear months or years after treatment remains unclear. Hematopoietic Stem/Progenitor Cells (HSPCs) are capable of long-term engraftment and have the potential to overcome these limitations. Here, we report the use of a protective CD4 chimeric antigen receptor (C46CD4CAR) to redirect HSPC-derived T-cells against simian/human immunodeficiency virus (SHIV) infection in pigtail macaques. CAR-containing cells persisted for more than 2 years without any measurable toxicity and were capable of multilineage engraftment. Combination antiretroviral therapy (cART) treatment followed by cART withdrawal resulted in lower viral rebound in CAR animals relative to controls, and demonstrated an immune memory-like response. We found CAR-expressing cells in multiple lymphoid tissues, decreased tissue-associated SHIV RNA levels, and substantially higher CD4/CD8 ratios in the gut as compared to controls. These results show that HSPC-derived CAR T-cells are capable of long-term engraftment and immune surveillance. This study demonstrates for the first time the safety and feasibility of HSPC-based CAR therapy in a large animal preclinical model.
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Linfocitos T CD4-Positivos/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/terapia , Células Madre Hematopoyéticas/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/terapia , Animales , Linfocitos T CD4-Positivos/trasplante , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Modelos Animales de Enfermedad , Terapia Genética/métodos , Infecciones por VIH/virología , Trasplante de Células Madre Hematopoyéticas/métodos , Inmunoterapia/métodos , Macaca nemestrina , Masculino , Receptores de Antígenos de Linfocitos T/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/metabolismo , Síndrome de Inmunodeficiencia Adquirida del Simio/virologíaRESUMEN
In a recent clone-tracking experiment, millions of uniquely tagged hematopoietic stem cells (HSCs) and progenitor cells were autologously transplanted into rhesus macaques and peripheral blood containing thousands of tags were sampled and sequenced over 14 years to quantify the abundance of hundreds to thousands of tags or "clones." Two major puzzles of the data have been observed: consistent differences and massive temporal fluctuations of clone populations. The large sample-to-sample variability can lead clones to occasionally go "extinct" but "resurrect" themselves in subsequent samples. Although heterogeneity in HSC differentiation rates, potentially due to tagging, and random sampling of the animals' blood and cellular demographic stochasticity might be invoked to explain these features, we show that random sampling cannot explain the magnitude of the temporal fluctuations. Moreover, we show through simpler neutral mechanistic and statistical models of hematopoiesis of tagged cells that a broad distribution in clone sizes can arise from stochastic HSC self-renewal instead of tag-induced heterogeneity. The very large clone population fluctuations that often lead to extinctions and resurrections can be naturally explained by a generation-limited proliferation constraint on the progenitor cells. This constraint leads to bursty cell population dynamics underlying the large temporal fluctuations. We analyzed experimental clone abundance data using a new statistic that counts clonal disappearances and provided least-squares estimates of two key model parameters in our model, the total HSC differentiation rate and the maximum number of progenitor-cell divisions.
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Hematopoyesis/fisiología , Células Madre Hematopoyéticas , Modelos Biológicos , Animales , Diferenciación Celular/fisiología , Rastreo Celular , Células Clonales/citología , Células Clonales/fisiología , Biología Computacional , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Macaca mulattaRESUMEN
UNLABELLED: Although the use of chimeric antigen receptors (CARs) based on single-chain antibodies for gene immunotherapy of cancers is increasing due to promising recent results, the earliest CAR therapeutic trials were done for HIV-1 infection in the late 1990s. This approach utilized a CAR based on human CD4 as a binding domain and was abandoned for a lack of efficacy. The growing number of HIV-1 broadly neutralizing antibodies (BNAbs) offers the opportunity to generate novel CARs that may be more active and revisit this modality for HIV-1 immunotherapy. We used sequences from seven well-defined BNAbs varying in binding sites and generated single-chain-antibody-based CARs. These CARs included 10E8, 3BNC117, PG9, PGT126, PGT128, VRC01, and X5. Each novel CAR exhibited conformationally relevant expression on the surface of transduced cells, mediated specific proliferation and killing in response to HIV-1-infected cells, and conferred potent antiviral activity (reduction of viral replication in log10 units) to transduced CD8(+) T lymphocytes. The antiviral activity of these CARs was reproducible but varied according to the strain of virus. These findings indicated that BNAbs are excellent candidates for developing novel CARs to consider for the immunotherapeutic treatment of HIV-1. IMPORTANCE: While chimeric antigen receptors (CARs) using single-chain antibodies as binding domains are growing in popularity for gene immunotherapy of cancers, the earliest human trials of CARs were done for HIV-1 infection. However, those trials failed, and the approach was abandoned for HIV-1. The only tested CAR against HIV-1 was based on the use of CD4 as the binding domain. The growing availability of HIV-1 broadly neutralizing antibodies (BNAbs) affords the opportunity to revisit gene immunotherapy for HIV-1 using novel CARs based on single-chain antibodies. Here we construct and test a panel of seven novel CARs based on diverse BNAb types and show that all these CARs are functional against HIV-1.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Receptores de Antígenos/inmunología , Receptores del VIH/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales/inmunología , Infecciones por VIH/inmunología , Infecciones por VIH/virología , Humanos , Células Jurkat , Homología de Secuencia de Aminoácido , Anticuerpos de Cadena Única/inmunologíaRESUMEN
BACKGROUND: How a potentially diverse population of hematopoietic stem cells (HSCs) differentiates and proliferates to supply more than 10(11) mature blood cells every day in humans remains a key biological question. We investigated this process by quantitatively analyzing the clonal structure of peripheral blood that is generated by a population of transplanted lentivirus-marked HSCs in myeloablated rhesus macaques. Each transplanted HSC generates a clonal lineage of cells in the peripheral blood that is then detected and quantified through deep sequencing of the viral vector integration sites (VIS) common within each lineage. This approach allowed us to observe, over a period of 4-12 years, hundreds of distinct clonal lineages. RESULTS: While the distinct clone sizes varied by three orders of magnitude, we found that collectively, they form a steady-state clone size-distribution with a distinctive shape. Steady-state solutions of our model show that the predicted clone size-distribution is sensitive to only two combinations of parameters. By fitting the measured clone size-distributions to our mechanistic model, we estimate both the effective HSC differentiation rate and the number of active HSCs. CONCLUSIONS: Our concise mathematical model shows how slow HSC differentiation followed by fast progenitor growth can be responsible for the observed broad clone size-distribution. Although all cells are assumed to be statistically identical, analogous to a neutral theory for the different clone lineages, our mathematical approach captures the intrinsic variability in the times to HSC differentiation after transplantation.
Asunto(s)
Células Sanguíneas/fisiología , Diferenciación Celular , Linaje de la Célula , Células Madre Hematopoyéticas/fisiología , Homeostasis , Macaca mulatta/sangre , Animales , Células Sanguíneas/citología , Células Clonales/citología , Células Clonales/metabolismo , Células Madre Hematopoyéticas/citología , Modelos BiológicosRESUMEN
Chimeric antigen receptors (CARs) are artificially engineered receptors that confer a desired specificity to immune effector T cells. As an HIV-1-specific CAR, CD4ζ CAR has been extensively tested in vitro as well as in clinical trials. T cells modified with this CAR mediated highly potent anti-HIV-1 activities in vitro and were well-tolerated in vivo, but exerted limited effects on viral load and reservoir size due to poor survival and/or functionality of the transduced cells in patients. We hypothesize that ectopic expression of CD4ζ on CD8(+) T cells renders them susceptible to HIV-1 infection, resulting in poor survival of those cells. To test this possibility, highly purified CD8(+) T cells were genetically modified with a CD4ζ-encoding lentiviral vector and infected with HIV-1. CD8(+) T cells were vulnerable to HIV-1 infection upon expression of CD4ζ as evidenced by elevated levels of p24(Gag) in cells and culture supernatants. Concurrently, the number of CD4ζ-modified CD8(+) T cells was reduced relative to control cells upon HIV-1 infection. To protect these cells from HIV-1 infection, we co-expressed two anti-HIV-1 shRNAs previously developed by our group together with CD4ζ. This combination vector was able to suppress HIV-1 infection without impairing HIV-1-dependent effector activities of CD4ζ. In addition, the number of CD4ζ-modified CD8(+) T cells maintained similar levels to that of the control even under HIV-1 infection. These results suggest that protecting CD4ζ-modified CD8(+) T cells from HIV-1 infection is required for prolonged HIV-1-specific immune surveillance.
Asunto(s)
Antígenos CD4/inmunología , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Infecciones por VIH/terapia , VIH-1/inmunología , Inmunoterapia , ARN Interferente Pequeño/inmunología , Antígenos CD4/genética , Ingeniería Celular , Proliferación Celular , Células Cultivadas , Expresión Génica , Infecciones por VIH/inmunología , VIH-1/genética , Humanos , ARN Interferente Pequeño/genéticaRESUMEN
UNLABELLED: We recently demonstrated that a soluble protein, Gas6, can facilitate viral entry by bridging viral envelope phosphatidylserine to Axl, a receptor tyrosine kinase expressed on target cells. The interaction between phosphatidylserine, Gas6, and Axl was originally shown to be a molecular mechanism through which phagocytes recognize phosphatidylserine exposed on dead cells. Since our initial report, several groups have confirmed that Axl/Gas6, as well as other phosphatidylserine receptors, facilitate entry of dengue, West Nile, and Ebola viruses. Virus binding by viral envelope phosphatidylserine is now a viral entry mechanism generalized to many families of viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most of the known human phosphatidylserine-recognizing molecules, including MFG-E8, TIM-1, -3, and -4, CD300a, BAI1, and stabilin-1 and -2, for their abilities to facilitate virus binding and infection. Using pseudotyped lentiviral vectors, we found that a soluble phosphatidylserine-binding protein, MFG-E8, enhances transduction. Cell surface receptors TIM-1 and -4 also enhance virus binding/transduction. The extent of enhancement by these molecules varies, depending on the type of pseudotyping envelope proteins. Mutated MFG-E8, which binds viral envelope phosphatidylserine without bridging virus to cells, but, surprisingly, not annexin V, which has been used to block phagocytosis of dead cells by concealing phosphatidylserine, efficiently blocks these phosphatidylserine-dependent viral entry mechanisms. These results provide insight into understanding the role of viral envelope phosphatidylserine in viral infection. IMPORTANCE: Envelope phosphatidylserine has previously been shown to be important for replication of various envelope viruses, but details of this mechanism(s) were unclear. We were the first to report that a bifunctional serum protein, Gas6, bridges envelope phosphatidylserine to a cell surface receptor, Axl. Recent studies demonstrated that many envelope viruses, including vaccinia, dengue, West Nile, and Ebola viruses, utilize Axl/Gas6 to facilitate their entry, suggesting that the phosphatidylserine-mediated viral entry mechanism can be shared by various enveloped viruses. In addition to Axl/Gas6, various molecules are known to recognize phosphatidylserine; however, the effects of these molecules on virus binding and entry have not been comprehensively evaluated and compared. In this study, we examined most human phosphatidylserine-recognizing molecules for their abilities to facilitate viral infection. The results provide insights into the role(s) of envelope phosphatidylserine in viral infection, which can be applicable to the development of novel antiviral reagents that block phosphatidylserine-mediated viral entry.
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Receptores de Superficie Celular/metabolismo , Receptores Virales/metabolismo , Fenómenos Fisiológicos de los Virus , Línea Celular , Humanos , Fagocitosis , Fosfatidilserinas/metabolismo , Receptores de Superficie Celular/genética , Receptores Virales/genética , Acoplamiento Viral , Virosis/genética , Virosis/metabolismo , Virosis/virología , Internalización del Virus , Virus/genéticaRESUMEN
BACKGROUND: We previously developed an antibody-avidin fusion protein (ch128.1Av) specific for the human transferrin receptor 1 (TfR1; CD71) to be used as a delivery vector for cancer therapy and showed that ch128.1Av delivers the biotinylated plant toxin saporin-6 into malignant B cells. However, as a result of widespread expression of TfR1, delivery of the toxin to normal cells is a concern. Therefore, we explored the potential of a dual targeted lentiviral-mediated gene therapy strategy to restrict gene expression to malignant B cells. Targeting occurs through the use of ch128.1Av or its parental antibody without avidin (ch128.1) and through transcriptional regulation using an immunoglobulin promoter. METHODS: Flow cytometry was used to detect the expression of enhanced green fluorescent protein (EGFP) in a panel of cell lines. Cell viability after specific delivery of the therapeutic gene FCU1, a chimeric enzyme consisting of cytosine deaminase genetically fused to uracil phosphoribosyltransferse that converts the 5-fluorocytosine (5-FC) prodrug into toxic metabolites, was monitored using the MTS or WST-1 viability assay. RESULTS: We found that EGFP was specifically expressed in a panel of human malignant B-cell lines, but not in human malignant T-cell lines. EGFP expression was observed in all cell lines when a ubiquitous promoter was used. Furthermore, we show the decrease of cell viability in malignant plasma cells in the presence of 5-FC and the FCU1 gene. CONCLUSIONS: The present study demonstrates that gene expression can be restricted to malignant B cells and suggests that this dual targeted gene therapy strategy may help to circumvent the potential side effects of certain TfR1-targeted protein delivery approaches.
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Antígenos CD/genética , Antígenos CD/inmunología , Linfocitos B/citología , Lentivirus/genética , Receptores de Transferrina/genética , Receptores de Transferrina/inmunología , Proteínas Recombinantes de Fusión/genética , Anticuerpos/genética , Anticuerpos/inmunología , Antígenos CD/biosíntesis , Linfocitos B/metabolismo , Línea Celular Tumoral , Citosina Desaminasa/genética , Flucitosina/metabolismo , Terapia Genética/efectos adversos , Vectores Genéticos/genética , Vectores Genéticos/uso terapéutico , Proteínas Fluorescentes Verdes , Células HEK293 , Humanos , Células Jurkat , Neoplasias/genética , Neoplasias/terapia , Pentosiltransferasa/genética , Profármacos/efectos adversos , Profármacos/uso terapéutico , Regiones Promotoras Genéticas , Receptores de Transferrina/biosíntesis , Linfocitos T/citología , Linfocitos T/metabolismo , Transducción Genética , Virus de la Estomatitis Vesicular Indiana/genéticaRESUMEN
Selective T-cell depletion prior to cell or organ transplantation is considered a preconditioning regimen to induce tolerance and immunosuppression. An immunotoxin consisting of a recombinant anti-CD3 antibody conjugated with diphtheria toxin was used to eliminate T-cells. It showed significant T-cell depletion activity in the peripheral blood and lymph nodes in animal models used in previous studies. To date, a comprehensive evaluation of T-cell depletion and CD3 proliferation for all lymphoid tissues has not been conducted. Here, two rhesus macaques were administered A-dmDT390-SCFBdb (CD3-IT) intravenously at 25 µg/kg twice daily for four days. Samples were collected one day prior to and four days post administration. Flow cytometry and immunofluorescence staining were used to evaluate treatment efficiency accurately. Our preliminary results suggest that CD3-IT treatment may induce higher depletion of CD3 and CD4 T-cells in the lymph nodes and spleen, but is ineffective in the colon and thymus. The data showed a better elimination tendency of CD4 T-cells in the B-cell zone relative to the germinal center in the lymph nodes. Further, CD3-IT treatment may lead to a reduction in germinal center T follicular helper CD4 cells in the lymph nodes compared to healthy controls. The number of proliferating CD3 T-cell indicated that repopulation in different lymphoid tissues may occur four days post treatment. Our results provide insights into the differential efficacy of CD3-IT treatment and T-cell proliferation post treatment in different lymphoid tissues. Overall, CD3-IT treatment shows potential efficacy in depleting T-cells in the periphery, lymph nodes, and spleen, making it a viable preconditioning regimen for cell or organ transplantation. Our pilot study provides critical descriptive statistics and can contribute to the design of larger future studies.
RESUMEN
Cancer immunotherapy has reshaped the landscape of cancer treatment. However, its efficacy is still limited by tumor immunosuppression associated with the excessive production of lactate by cancer cells. Although extensive efforts have been made to reduce lactate concentrations through inhibition of lactate dehydrogenase, such inhibitors disrupt the metabolism of healthy cells, causing severe nonspecific toxicity. We report herein a nanocapsule enzyme therapeutic based on lactate oxidase, which reduces lactate concentrations and releases immunostimulatory hydrogen peroxide, averting tumor immunosuppression and improving the efficacy of immune checkpoint blockade treatment. As demonstrated in a murine melanoma model and a humanized mouse model of triple-negative breast cancer, this enzyme therapeutic affords an effective tool toward more effective cancer immunotherapy.
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Melanoma , Nanocápsulas , Animales , Ratones , Linfocitos T , Inmunoterapia , Melanoma/terapia , Lactatos , Microambiente TumoralRESUMEN
Synthetic siRNA has been considered as a highly promising therapeutic agent for human diseases. However, clinical use of siRNA has been hampered by instability in the body and inability to deliver sufficient RNA interference compounds to the tissues or cells. To address this challenge, we present here a single siRNA nanocapsule delivery technology, which is achieved by encapsulating a single siRNA molecule within a degradable polymer nanocapsule with a diameter around 20 nm and positive surface charge. As proof-of-concept, since CCR5 is considered a major silencing target of HIV therapy, CCR5-siRNA nanocapsules were delivered into 293T cells and successfully downregulated the CCR5 RNA fused with mCherry reporter RNA. In the absence of human serum, nanocapsules and lipofectamine silenced expression of CCR5-mCherry expression to 8% and 15%, respectively. Such nanocapsules maintain the integrity of siRNA inside even after incubation with ribonuclease and serum for 1 h; under the same conditions, siRNA is degraded in the native form or when formulated with lipofectamine. In the presence of serum, CCR5-siRNA nanocapsules knocked down CCR5-mCherry expression to less than 15% while siRNAs delivered through lipofectamine slightly knocked down the expression to 55%. In summary, this work provides a novel platform for siRNA delivery that can be developed for therapeutic purposes.
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Nanocápsulas/química , Interferencia de ARN , ARN Interferente Pequeño/administración & dosificación , Línea Celular , Humanos , Lípidos/química , Modelos Moleculares , Polimerizacion , ARN Interferente Pequeño/genética , Receptores CCR5/genéticaRESUMEN
Inhibiting the expression of the HIV-1 coreceptor CCR5 holds great promise for controlling HIV-1 infection in patients. Here we report stable knockdown of human CCR5 by a short hairpin RNA (shRNA) in a humanized bone marrow/liver/thymus (BLT) mouse model. We delivered a potent shRNA against CCR5 into human fetal liver-derived CD34(+) hematopoietic progenitor/stem cells (HPSCs) by lentiviral vector transduction. We transplanted vector-transduced HPSCs solidified with Matrigel and a thymus segment under the mouse kidney capsule. Vector-transduced autologous CD34(+) cells were subsequently injected in the irradiated mouse, intended to create systemic reconstitution. CCR5 expression was down-regulated in human T cells and monocytes/macrophages in systemic lymphoid tissues, including gut-associated lymphoid tissue, the major site of HIV-1 replication. The shRNA-mediated CCR5 knockdown had no apparent adverse effects on T-cell development as assessed by polyclonal T-cell receptor Vbeta family development and naive/memory T-cell differentiation. CCR5 knockdown in the secondary transplanted mice suggested the potential of long-term hematopoietic reconstitution by the shRNA-transduced HPSCs. CCR5 tropic HIV-1 infection was effectively inhibited in mouse-derived human splenocytes ex vivo. These results demonstrate that lentiviral vector delivery of shRNA into human HPSCs could stably down-regulate CCR5 in systemic lymphoid organs in vivo.
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Médula Ósea/metabolismo , Infecciones por VIH/metabolismo , VIH-1 , Trasplante de Células Madre Hematopoyéticas , Hígado/metabolismo , Receptores CCR5/biosíntesis , Timo/metabolismo , Animales , Diferenciación Celular/genética , Modelos Animales de Enfermedad , Regulación hacia Abajo , Técnicas de Silenciamiento del Gen , Infecciones por VIH/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Memoria Inmunológica/genética , Lentivirus , Ratones , Ratones Endogámicos NOD , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores CCR5/genética , Linfocitos T/metabolismo , Transducción Genética , Trasplante HeterólogoRESUMEN
Targeted gene transduction to specific tissues and organs through intravenous injection would be the ultimate preferred method of gene delivery. Here, we report successful targeting in a living animal through intravenous injection of a lentiviral vector pseudotyped with a modified chimeric Sindbis virus envelope (termed m168). m168 pseudotypes have high titer and high targeting specificity and, unlike other retroviral pseudotypes, have low nonspecific infectivity in liver and spleen. A mouse cancer model of metastatic melanoma was used to test intravenous targeting with m168. Human P-glycoprotein was ectopically expressed on the surface of melanoma cells and targeted by the m168 pseudotyped lentiviral vector conjugated with antibody specific for P-glycoprotein. m168 pseudotypes successfully targeted metastatic melanoma cells growing in the lung after systemic administration by tail vein injection. Further development of this targeting technology should result in applications not only for cancers but also for genetic, infectious and immune diseases.
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Marcación de Gen/métodos , Melanoma Experimental/terapia , Virus Sindbis/genética , Animales , Terapia Genética/métodos , Vectores Genéticos , Lentivirus/genética , Luciferasas/biosíntesis , Melanoma Experimental/secundario , Ratones , Proteínas del Envoltorio Viral/genéticaRESUMEN
Chimeric-antigen receptor (CAR) T-cell immunotherapy employs autologous-T cells modified with an antigen-specific CAR. Current CAR-T manufacturing processes tend to yield products dominated by effector T cells and relatively small proportions of long-lived memory T cells. Those few cells are a so-called stem cell memory T (TSCM) subset, which express naïve T-cell markers and are capable of self-renewal and oligopotent differentiation into effector phenotypes. Increasing the proportion of this subset may lead to more effective therapies by improving CAR-T persistence; however, there is currently no standardized protocol for the effective generation of CAR-TSCM cells. Here we present a simplified protocol enabling efficient derivation of gene-modified TSCM cells: Stimulation of naïve CD8+ T cells with only soluble anti-CD3 antibody and culture with IL-7 and IL-15 was sufficient for derivation of CD8+ T cells harboring TSCM phenotypes and oligopotent capabilities. These in-vitro expanded TSCM cells were engineered with CARs targeting the HIV-1 envelope protein as well as the CD19 molecule and demonstrated effector activity both in vitro and in a xenograft mouse model. This simple protocol for the derivation of CAR-TSCM cells may facilitate improved adoptive immunotherapy.
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Receptores Quiméricos de Antígenos , Animales , Antígenos CD19/metabolismo , Linfocitos T CD8-positivos , Humanos , Inmunoterapia Adoptiva/métodos , Ratones , Receptores de Antígenos de Linfocitos T , Receptores Quiméricos de Antígenos/genéticaRESUMEN
Targeting HER-2 over-expressing breast cancer cells with trastuzumab has resulted in significant improvements in both disease-free and overall survival rates. However, despite a favorable initial response, some cancer cells become resistant and develop into fatal metastatic disease. Here we report that we can specifically target HER-2 over-expressing and trastuzumab-resistant breast cancer cells by using an engineered lentivirus which has trastuzumab bound to its envelope. In vitro, this lentiviral construct mediated both the expression of reporter genes, such as enhanced green fluorescent protein (EGFP) and firefly luciferase, as well as the therapeutic gene, herpes thymidine kinase (hTK), in HER-2 over-expressing cells. Subsequent application of the pro-drug ganciclovir selectively killed breast cancer cells in which lentivirus mediated expression of hTK. In vivo, we successfully targeted the expression of firefly luciferase to trastuzumab-resistant breast cancer tumors established in nude mice. Furthermore, we found that systemic administration of trastuzumab-bound lentivirus led to expression of EGFP in circulating trastuzumab-resistant breast cancer cells. In conclusion, HER-2 over-expressing breast cancer cells resistant to trastuzumab can be targeted for selective gene expression and destruction by viruses with envelope-proteins engineered to bind to this antibody.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Neoplasias de la Mama/terapia , Resistencia a Antineoplásicos , Terapia Genética/métodos , Lentivirus/metabolismo , Receptor ErbB-2/metabolismo , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados , Antineoplásicos/metabolismo , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Supervivencia Celular , Relación Dosis-Respuesta a Droga , Femenino , Ganciclovir/metabolismo , Ganciclovir/farmacología , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lentivirus/genética , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Ratones Desnudos , Células Neoplásicas Circulantes/metabolismo , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/inmunología , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , TrastuzumabRESUMEN
Redirecting the tropism of viral vectors enables specific transduction of selected cells by direct administration of vectors. We previously developed targeting lentiviral vectors by pseudotyping with modified Sindbis virus envelope proteins. These modified Sindbis virus envelope proteins have mutations in their original receptor-binding regions to eliminate their natural tropisms, and they are conjugated with targeting proteins, including antibodies and peptides, to confer their tropisms on target cells. We investigated whether our targeting vectors interact with DC-SIGN, which traps many types of viruses and gene therapy vectors by binding to the N-glycans of their envelope proteins. We found that these vectors do not interact with DC-SIGN. When these vectors were produced in the presence of deoxymannojirimycin, which alters the structures of N-glycans from complex to high mannose, these vectors used DC-SIGN as their receptor. Genetic analysis demonstrated that the N-glycans at E2 amino acid (aa) 196 and E1 aa 139 mediate binding to DC-SIGN, which supports the results of a previous report of cryoelectron microscopy analysis. In addition, we investigated whether modification of the N-glycan structures could activate serum complement activity, possibly by the lectin pathway of complement activation. DC-SIGN-targeted transduction occurs in the presence of human serum complement, demonstrating that high-mannose structure N-glycans of the envelope proteins do not activate human serum complement. These results indicate that the strategy of redirecting viral vectors according to alterations of their N-glycan structures would enable the vectors to target specific cells types expressing particular types of lectins.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Vectores Genéticos , Lectinas Tipo C/metabolismo , Lentivirus , Polisacáridos/metabolismo , Receptores de Superficie Celular/metabolismo , Virus Sindbis/metabolismo , Proteínas del Envoltorio Viral , Animales , Conformación de Carbohidratos , Secuencia de Carbohidratos , Moléculas de Adhesión Celular/genética , Línea Celular , Proteínas del Sistema Complemento/metabolismo , Células Dendríticas/citología , Células Dendríticas/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Humanos , Lectinas Tipo C/genética , Lentivirus/genética , Lentivirus/metabolismo , Datos de Secuencia Molecular , Polisacáridos/química , Polisacáridos/genética , Receptores de Superficie Celular/genética , Virus Sindbis/genética , Transducción Genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Retroviral vector-mediated gene therapy has been successfully used to correct genetic diseases. However, a number of studies have shown a subsequent risk of cancer development or aberrant clonal growths due to vector insertion near or within proto-oncogenes. Recent advances in the sequencing technology enable high-throughput clonality analysis via vector integration site (VIS) sequencing, which is particularly useful for studying complex polyclonal hematopoietic progenitor/stem cell (HPSC) repopulation. However, clonal repopulation analysis using the current methods is typically semiquantitative. Here, we present a novel system and standards for accurate clonality analysis using 454 pyrosequencing. We developed a bidirectional VIS PCR method to improve VIS detection by concurrently analyzing both the 5' and the 3' vector-host junctions and optimized the conditions for the quantitative VIS sequencing. The assay was validated by quantifying the relative frequencies of hundreds of repopulating HPSC clones in a nonhuman primate. The reliability and sensitivity of the assay were assessed using clone-specific real-time PCR. The majority of tested clones showed a strong correlation between the two methods. This assay permits high-throughput and sensitive assessment of clonal populations and hence will be useful for a broad range of gene therapy, stem cell, and cancer research applications.