RESUMEN
Regulators and industry are actively seeking improvements and alternatives to current models and approaches to evaluate potential carcinogenicity of gene therapies (GTs). A meeting of invited experts was organized by NC3Rs/UKEMS (London, March 2023) to discuss this topic. This article describes the consensus reached among delegates on the definition of vector genotoxicity, sources of uncertainty, suitable toxicological endpoints for genotoxic assessment of GTs, and future research needs. The collected recommendations should inform the further development of regulatory guidelines for the nonclinical toxicological assessment of GT products.
Asunto(s)
Terapia Genética , Terapia Genética/efectos adversos , Terapia Genética/métodos , Humanos , Factores de Riesgo , Animales , Vectores Genéticos/efectos adversos , Consenso , Neoplasias/terapia , Neoplasias/genética , Medición de RiesgoRESUMEN
Methods to engineer the genomes of human cells for therapeutic intervention continue to advance at a remarkable pace. Chimeric antigen receptor-engineered T lymphocytes have pioneered the way for these therapies, initially beginning with insertions of chimeric antigen receptor transgenes into T-cell genomes using classical gene therapy vectors. The broad use of clustered regularly interspaced short palindromic repeats (CRISPR)-based technologies to edit endogenous genes has now opened the door to a new era of precision medicine. To add complexity, many engineered cellular therapies under development integrate gene therapy with genome editing to introduce novel biological functions and enhance therapeutic efficacy. Here, we review the current state of scientific, translational, and regulatory oversight of gene-edited cell products.
RESUMEN
Recurrent glioblastoma (rGBM) remains a major unmet medical need, with a median overall survival of less than 1 year. Here we report the first six patients with rGBM treated in a phase 1 trial of intrathecally delivered bivalent chimeric antigen receptor (CAR) T cells targeting epidermal growth factor receptor (EGFR) and interleukin-13 receptor alpha 2 (IL13Rα2). The study's primary endpoints were safety and determination of the maximum tolerated dose. Secondary endpoints reported in this interim analysis include the frequency of manufacturing failures and objective radiographic response (ORR) according to modified Response Assessment in Neuro-Oncology criteria. All six patients had progressive, multifocal disease at the time of treatment. In both dose level 1 (1 ×107 cells; n = 3) and dose level 2 (2.5 × 107 cells; n = 3), administration of CART-EGFR-IL13Rα2 cells was associated with early-onset neurotoxicity, most consistent with immune effector cell-associated neurotoxicity syndrome (ICANS), and managed with high-dose dexamethasone and anakinra (anti-IL1R). One patient in dose level 2 experienced a dose-limiting toxicity (grade 3 anorexia, generalized muscle weakness and fatigue). Reductions in enhancement and tumor size at early magnetic resonance imaging timepoints were observed in all six patients; however, none met criteria for ORR. In exploratory endpoint analyses, substantial CAR T cell abundance and cytokine release in the cerebrospinal fluid were detected in all six patients. Taken together, these first-in-human data demonstrate the preliminary safety and bioactivity of CART-EGFR-IL13Rα2 cells in rGBM. An encouraging early efficacy signal was also detected and requires confirmation with additional patients and longer follow-up time. ClinicalTrials.gov identifier: NCT05168423 .
Asunto(s)
Receptores ErbB , Glioblastoma , Inmunoterapia Adoptiva , Subunidad alfa2 del Receptor de Interleucina-13 , Receptores Quiméricos de Antígenos , Humanos , Glioblastoma/terapia , Glioblastoma/inmunología , Glioblastoma/diagnóstico por imagen , Glioblastoma/patología , Subunidad alfa2 del Receptor de Interleucina-13/inmunología , Persona de Mediana Edad , Masculino , Receptores Quiméricos de Antígenos/inmunología , Femenino , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/métodos , Recurrencia Local de Neoplasia/inmunología , Recurrencia Local de Neoplasia/patología , Adulto , Anciano , Neoplasias Encefálicas/inmunología , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/patología , Inyecciones Espinales , Dosis Máxima ToleradaRESUMEN
The use of lentiviral vectors in cell and gene therapy is steadily increasing, both in commercial and investigational therapies. Although existing data increasingly support the usefulness and safety of clinical-grade lentiviral vectors used in cell manufacturing, comprehensive studies specifically addressing their long-term stability are currently lacking. This is significant considering the high cost of producing and testing GMP-grade vectors, the limited number of production facilities, and lengthy queue for production slots. Therefore, an extended shelf life is a critical attribute to justify the investment in large vector lots for investigational cell therapies. This study offers a thorough examination of essential stability attributes, including vector titer, transduction efficiency, and potency for a series of clinical-grade vector lots, each assessed at a minimum of 36 months following their date of manufacture. The 13 vector lots included in this study were used for cell product manufacturing in 16 different clinical trials, and at the time of the analysis had a maximum storage time at -80°C of up to 8 years. The results emphasize the long-term durability and efficacy of GMP-grade lentiviral vectors for use in ex vivo cell therapy manufacturing.
RESUMEN
Chimeric antigen receptor (CAR) T cells demonstrate remarkable success in treating hematological malignancies, but their effectiveness in non-hematopoietic cancers remains limited. This study proposes enhancing CAR T cell function and localization in solid tumors by modifying the epigenome governing tissue-residency adaptation and early memory differentiation. We identify that a key factor in human tissue-resident memory CAR T cell (CAR-TRM) formation is activation in the presence of the pleotropic cytokine, transforming growth factor ß (TGF-ß), which enforces a core program of both "stemness" and sustained tissue residency by mediating chromatin remodeling and concurrent transcriptional changes. This approach leads to a practical and clinically actionable in vitro production method for engineering peripheral blood T cells into a large number of "stem-like" CAR-TRM cells resistant to tumor-associated dysfunction, possessing an enhanced ability to accumulate in situ and rapidly eliminate cancer cells for more effective immunotherapy.
Asunto(s)
Neoplasias , Receptores Quiméricos de Antígenos , Humanos , Receptores Quiméricos de Antígenos/metabolismo , Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Citocinas/metabolismo , InmunoterapiaRESUMEN
Chimeric antigen receptor (CAR) T cells have not induced meaningful clinical responses in solid tumors. Loss of T cell stemness, poor expansion capacity, and exhaustion during prolonged tumor antigen exposure are major causes of CAR T cell therapeutic resistance. Single-cell RNA-sequencing analysis of CAR T cells from a first-in-human trial in metastatic prostate cancer identified two independently validated cell states associated with antitumor potency or lack of efficacy. Low expression of PRDM1, encoding the BLIMP1 transcription factor, defined highly potent TCF7 [encoding T cell factor 1 (TCF1)]-expressing CD8+ CAR T cells, whereas enrichment of HAVCR2 [encoding T cell immunoglobulin and mucin-domain containing-3 (TIM-3)]-expressing CD8+ T cells with elevated PRDM1 was associated with poor outcomes. PRDM1 knockout promoted TCF7-dependent CAR T cell stemness and proliferation, resulting in marginally enhanced leukemia control in mice. However, in the setting of PRDM1 deficiency, a negative epigenetic feedback program of nuclear factor of activated T cells (NFAT)-driven T cell dysfunction was identified. This program was characterized by compensatory up-regulation of NR4A3 and other genes encoding exhaustion-related transcription factors that hampered T cell effector function in solid tumors. Dual knockout of PRDM1 and NR4A3 skewed CAR T cell phenotypes away from TIM-3+CD8+ and toward TCF1+CD8+ to counter exhaustion of tumor-infiltrating CAR T cells and improve antitumor responses, effects that were not achieved with PRDM1 and NR4A3 single knockout alone. These data underscore dual targeting of PRDM1 and NR4A3 as a promising approach to advance adoptive cell immuno-oncotherapy.
Asunto(s)
Neoplasias , Receptores de Esteroides , Masculino , Humanos , Ratones , Animales , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Linfocitos T CD8-positivos , Inmunoterapia Adoptiva/métodos , Receptor 2 Celular del Virus de la Hepatitis A/genética , Receptor 2 Celular del Virus de la Hepatitis A/metabolismo , Neoplasias/patología , Línea Celular Tumoral , Proteínas de Unión al ADN/metabolismo , Receptores de Esteroides/metabolismo , Receptores de Hormona Tiroidea/metabolismo , Proteínas del Tejido Nervioso/metabolismoRESUMEN
Chimeric antigen receptor (CAR) T cells have induced remarkable antitumor responses in B cell malignancies. Some patients do not respond because of T cell deficiencies that hamper the expansion, persistence, and effector function of these cells. We used longitudinal immune profiling to identify phenotypic and pharmacodynamic changes in CD19-directed CAR T cells in patients with chronic lymphocytic leukemia (CLL). CAR expression maintenance was also investigated because this can affect response durability. CAR T cell failure was accompanied by preexisting T cell-intrinsic defects or dysfunction acquired after infusion. In a small subset of patients, CAR silencing was observed coincident with leukemia relapse. Using a small molecule inhibitor, we demonstrated that the bromodomain and extra-terminal (BET) family of chromatin adapters plays a role in downregulating CAR expression. BET protein blockade also ameliorated CAR T cell exhaustion as manifested by inhibitory receptor reduction, enhanced metabolic fitness, increased proliferative capacity, and enriched transcriptomic signatures of T cell reinvigoration. BET inhibition decreased levels of the TET2 methylcytosine dioxygenase, and forced expression of the TET2 catalytic domain eliminated the potency-enhancing effects of BET protein targeting in CAR T cells, providing a mechanism linking BET proteins and T cell dysfunction. Thus, modulating BET epigenetic readers may improve the efficacy of cell-based immunotherapies.
Asunto(s)
Inmunoterapia Adoptiva , Leucemia Linfocítica Crónica de Células B/inmunología , Leucemia Linfocítica Crónica de Células B/terapia , Proteínas/antagonistas & inhibidores , Proteínas/inmunología , Receptores Quiméricos de Antígenos/inmunología , Linfocitos T/inmunología , Antígenos CD19/inmunología , Azepinas/farmacología , Epigénesis Genética , Glucólisis/efectos de los fármacos , Humanos , Tolerancia Inmunológica , Memoria Inmunológica , Leucemia Linfocítica Crónica de Células B/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Receptores Quiméricos de Antígenos/genética , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Triazoles/farmacologíaRESUMEN
BackgroundWe conducted a phase I clinical trial that infused CCR5 gene-edited CD4+ T cells to determine how these T cells can better enable HIV cure strategies.MethodsThe aim of trial was to develop RNA-based approaches to deliver zinc finger nuclease (ZFN), evaluate the effect of CCR5 gene-edited CD4+ T cells on the HIV-specific T cell response, test the ability of infused CCR5 gene-edited T cells to delay viral rebound during analytical treatment interruption, and determine whether individuals heterozygous for CCR5 Δ32 preferentially benefit. We enrolled 14 individuals living with HIV whose viral load was well controlled by antiretroviral therapy (ART). We measured the time to viral rebound after ART withdrawal, the persistence of CCR5-edited CD4+ T cells, and whether infusion of 10 billion CCR5-edited CD4+ T cells augmented the HIV-specific immune response.ResultsInfusion of the CD4+ T cells was well tolerated, with no serious adverse events. We observed a modest delay in the time to viral rebound relative to historical controls; however, 3 of the 14 individuals, 2 of whom were heterozygous for CCR5 Δ32, showed post-viral rebound control of viremia, before ultimately losing control of viral replication. Interestingly, only these individuals had substantial restoration of HIV-specific CD8+ T cell responses. We observed immune escape for 1 of these reinvigorated responses at viral recrudescence, illustrating a direct link between viral control and enhanced CD8+ T cell responses.ConclusionThese findings demonstrate how CCR5 gene-edited CD4+ T cell infusion could aid HIV cure strategies by augmenting preexisting HIV-specific immune responses.REGISTRATIONClinicalTrials.gov NCT02388594.FundingNIH funding (R01AI104400, UM1AI126620, U19AI149680, T32AI007632) was provided by the National Institute of Allergy and Infectious Diseases (NIAID), the National Institute on Drug Abuse (NIDA), the National Institute of Mental Health (NIMH), and the National Institute of Neurological Disorders and Stroke (NINDS). Sangamo Therapeutics also provided funding for these studies.
Asunto(s)
Antirretrovirales/administración & dosificación , Linfocitos T CD4-Positivos , Edición Génica , Infecciones por VIH , VIH-1/fisiología , Transfusión de Linfocitos , Receptores CCR5 , Replicación Viral/inmunología , Adulto , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/trasplante , Linfocitos T CD8-positivos/inmunología , Femenino , Infecciones por VIH/genética , Infecciones por VIH/inmunología , Infecciones por VIH/terapia , Humanos , Masculino , Persona de Mediana Edad , Receptores CCR5/genética , Receptores CCR5/inmunología , Carga Viral/genética , Carga Viral/inmunología , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
MazF is an Escherichia coli-derived endoribonuclease that selectively cleaves ACA sequences of mRNA prevalent in HIV. We administered a single infusion of autologous CD4 T lymphocytes modified to express a Tat-dependent MazF transgene to 10 HIV-infected individuals (six remaining on antiretroviral therapy [ART]; four undergoing treatment interruption post-infusion) in order to provide a population of HIV-resistant immune cells. In participants who remained on ART, increases in CD4 and CD8 T cell counts of ~200 cells/mm3 each occurred within 2 weeks of infusion and persisted for at least 6 months. Modified cells were detectable for several months in the blood and trafficked to gastrointestinal lymph tissue. HIV-1 Tat introduced ex vivo to the modified CD4+ T cells induced MazF expression in both pre- and post-infusion samples, and MazF expression was detected in vivo post-viral-rebound during ATI. One participant experienced mild cytokine release syndrome. In sum, this study of a single infusion of MazF-modified CD4 T lymphocytes demonstrated safety of these cells, distribution to lymph tissue and maintenance of Tat-inducible MazF endoribonuclease activity, as well as sustained elevation of blood CD4 and CD8 T cell counts. Future studies to assess effects on viremia and latent proviral reservoir are warranted.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Endorribonucleasas/genética , Infecciones por VIH/inmunología , Infecciones por VIH/virología , VIH-1/fisiología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endorribonucleasas/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Terapia Genética , Infecciones por VIH/metabolismo , Infecciones por VIH/terapia , Carga Viral , Replicación ViralRESUMEN
Chimeric antigen receptor (CAR) T cell therapy has come of age, offering a potentially curative option for patients who are refractory to standard anti-cancer treatments. The success of CAR T cell therapy in the setting of acute lymphoblastic leukemia and specific types of B cell lymphoma led to rapid regulatory approvals of CD19-directed CAR T cells, first in the United States and subsequently across the globe. Despite these major milestones in the field of immuno-oncology, growing experience with CAR T cells has also highlighted the major limitations of this strategy, namely challenges associated with manufacturing a bespoke patient-specific product, intrinsic immune cell defects leading to poor CAR T cell function as well as persistence, and/or tumor cell resistance resulting from loss or modulation of the targeted antigen. In addition, both on- and off-tumor immunotoxicities and the financial burden inherent in conventional cellular biomanufacturing often hamper the success of CAR T cell-based treatment approaches. Herein, we provide an overview of the opportunities and challenges related to the first form of gene transfer therapy to gain commercial approval in the United States. Ongoing advances in the areas of genetic engineering, precision genome editing, toxicity mitigation methods and cell manufacturing will improve the efficacy and safety of CAR T cells for hematologic malignancies and expand the use of this novel class of therapeutics to reach solid tumors.
Asunto(s)
Neoplasias Hematológicas/terapia , Inmunoterapia Adoptiva/métodos , Receptores Quiméricos de Antígenos/genética , Ingeniería Genética/métodos , Humanos , Inmunoterapia Adoptiva/efectos adversos , Inmunoterapia Adoptiva/economía , Neoplasias/terapiaRESUMEN
CRISPR-Cas9 gene editing provides a powerful tool to enhance the natural ability of human T cells to fight cancer. We report a first-in-human phase 1 clinical trial to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. Two genes encoding the endogenous T cell receptor (TCR) chains, TCRα (TRAC) and TCRß (TRBC), were deleted in T cells to reduce TCR mispairing and to enhance the expression of a synthetic, cancer-specific TCR transgene (NY-ESO-1). Removal of a third gene encoding programmed cell death protein 1 (PD-1; PDCD1), was performed to improve antitumor immunity. Adoptive transfer of engineered T cells into patients resulted in durable engraftment with edits at all three genomic loci. Although chromosomal translocations were detected, the frequency decreased over time. Modified T cells persisted for up to 9 months, suggesting that immunogenicity is minimal under these conditions and demonstrating the feasibility of CRISPR gene editing for cancer immunotherapy.
Asunto(s)
Traslado Adoptivo , Sistemas CRISPR-Cas , Edición Génica , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Linfocitos T/inmunología , Linfocitos T/trasplante , Anciano , Proteína 9 Asociada a CRISPR , Ingeniería Celular , Femenino , Humanos , Masculino , Persona de Mediana Edad , Receptor de Muerte Celular Programada 1/genética , TransgenesRESUMEN
The advent of engineered T cells as a form of immunotherapy marks the beginning of a new era in medicine, providing a transformative way to combat complex diseases such as cancer. Following FDA approval of CAR T cells directed against the CD19 protein for the treatment of acute lymphoblastic leukemia and diffuse large B cell lymphoma, CAR T cells are poised to enter mainstream oncology. Despite this success, a number of patients are unable to receive this therapy due to inadequate T cell numbers or rapid disease progression. Furthermore, lack of response to CAR T cell treatment is due in some cases to intrinsic autologous T cell defects and/or the inability of these cells to function optimally in a strongly immunosuppressive tumor microenvironment. We describe recent efforts to overcome these limitations using CRISPR/Cas9 technology, with the goal of enhancing potency and increasing the availability of CAR-based therapies. We further discuss issues related to the efficiency/scalability of CRISPR/Cas9-mediated genome editing in CAR T cells and safety considerations. By combining the tools of synthetic biology such as CARs and CRISPR/Cas9, we have an unprecedented opportunity to optimally program T cells and improve adoptive immunotherapy for most, if not all future patients.
Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Inmunoterapia Adoptiva , Receptores Quiméricos de Antígenos/uso terapéutico , Antígenos CD19 , Humanos , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Linfocitos T/inmunologíaRESUMEN
Replication-competent retrovirus/lentivirus (RCR/L) and insertional oncogenesis are potential safety risks with integrating viruses in gene-modified cell therapies. As such, the Food and Drug Administration guidances outline RCR/L-monitoring methods throughout the entire gene therapy treatment cycle. We present data for 17 vector lots, 375 manufactured T cell products, and 308 patients post-infusion across both HIV and oncology indications, showing no evidence of RCR/L. Given our data, a Poisson probability model estimates that a single patient, or a group of patients, would need to be followed for at least 52.8 years to observe one positive RCR/L event, highlighting the unlikelihood of RCR/L development. Additionally, we estimate the median time for lentivirus-modified T cell products to fall below the 1% vector sequence threshold in peripheral or whole blood that would trigger vector integration site analysis. These estimated times are 1.4 months in hematologic malignancies, 0.66 month in solid tumors, and 0.92 month in HIV. Based on these considerable safety data in HIV and oncology and recent Biologics License Applications filed for lentiviral-modified T cell products for hematologic malignancies, this may be an opportune time to re-evaluate the current guidelines for T cell gene therapy product testing and long-term patient monitoring.
Asunto(s)
Terapia Genética , Vectores Genéticos/genética , Infecciones por VIH/genética , Lentivirus/genética , Neoplasias/genética , Retroviridae/genética , Linfocitos T/inmunología , Linfocitos T/metabolismo , Ensayos Clínicos como Asunto , Terapia Genética/métodos , Infecciones por VIH/inmunología , Infecciones por VIH/terapia , Humanos , Inmunoterapia Adoptiva , Neoplasias/inmunología , Neoplasias/mortalidad , Neoplasias/terapia , Receptores Quiméricos de Antígenos/genética , Receptores Quiméricos de Antígenos/metabolismoRESUMEN
The role of the negative elongation factor (NELF) in maintaining HIV latency was investigated following small hairpin RNA (shRNA) knockdown of the NELF-E subunit, a condition that induced high levels of proviral transcription in latently infected Jurkat T cells. Chromatin immunoprecipitation (ChIP) assays showed that latent proviruses accumulate RNA polymerase II (RNAP II) on the 5' long terminal repeat (LTR) but not on the 3' LTR. NELF colocalizes with RNAP II, and its level increases following proviral induction. RNAP II pause sites on the HIV provirus were mapped to high resolution by ChIP with high-throughput sequencing (ChIP-Seq). Like cellular promoters, RNAP II accumulates at around position +30, but HIV also shows additional pausing at +90, which is immediately downstream of a transactivation response (TAR) element and other distal sites on the HIV LTR. Following NELF-E knockdown or tumor necrosis factor alpha (TNF-α) stimulation, promoter-proximal RNAP II levels increase up to 3-fold, and there is a dramatic increase in RNAP II levels within the HIV genome. These data support a kinetic model for proviral transcription based on continuous replacement of paused RNAP II during both latency and productive transcription. In contrast to most cellular genes, HIV is highly activated by the combined effects of NELF-E depletion and activation of initiation by TNF-α, suggesting that opportunities exist to selectively activate latent HIV proviruses.
Asunto(s)
Regulación Viral de la Expresión Génica , VIH-1/fisiología , Provirus/fisiología , ARN Polimerasa II/genética , Factores de Transcripción/fisiología , Latencia del Virus/genética , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Jurkat , Regiones Promotoras Genéticas , Provirus/genética , Interferencia de ARN , ARN Polimerasa II/biosíntesis , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño , Análisis de Secuencia de ADN , Factores de Transcripción/genética , Transcripción Genética , Factor de Necrosis Tumoral alfa/metabolismo , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Human ß-defensin 3 (hBD-3) activates antigen-presenting cells through Toll-like receptors (TLRs) 1/2. Several TLR1/2 agonists have been identified but little is known about how they might differentially affect cellular activation. We compared the effects of hBD-3 with those of another TLR1/2 agonist, Pam(3) CSK(4) , in human monocytes. Monocytes incubated with hBD-3 or Pam(3) CSK(4) produced interleukin-6 (IL-6), IL-8 and IL-1ß, but only Pam(3) CSK(4) induced IL-10. The IL-10 induction by Pam(3) CSK(4) caused down-modulation of the co-stimulatory molecule, CD86, whereas CD86 expression was increased in monocytes exposed to hBD-3. Assessment of signalling pathways linked to IL-10 induction indicated that mitogen-activated protein kinases were activated similarly by hBD-3 or Pam(3) CSK(4) , whereas the non-canonical nuclear factor-κB pathway was only induced by Pam(3) CSK(4) . Our data suggest that the lack of non-canonical nuclear factor-κB signalling by hBD-3 could contribute to the failure of this TLR agonist to induce production of the anti-inflammatory cytokine, IL-10, in human monocytes.
Asunto(s)
Interleucina-10/metabolismo , Lipopéptidos/farmacología , FN-kappa B/metabolismo , Receptor Toll-Like 1/agonistas , Receptor Toll-Like 2/agonistas , beta-Defensinas/farmacología , Antígeno B7-2/inmunología , Humanos , Interleucina-1beta/biosíntesis , Interleucina-6/biosíntesis , Interleucina-8/biosíntesis , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/efectos de los fármacos , Receptor Toll-Like 1/inmunología , Receptor Toll-Like 2/inmunologíaRESUMEN
CC Chemokine Receptor 5 (CCR5) is an important mediator of chemotaxis and the primary coreceptor for HIV-1. A recent report by other researchers suggested that primary T cells harbor pools of intracellular CCR5. With the use of a series of complementary techniques to measure CCR5 expression (antibody labeling, Western blot, quantitative reverse transcription polymerase chain reaction), we established that intracellular pools of CCR5 do not exist and that the results obtained by the other researchers were false-positives that arose because of the generation of irrelevant binding sites for anti-CCR5 antibodies during fixation and permeabilization of cells.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Receptores CCR5/metabolismo , Western Blotting , Linfocitos T CD4-Positivos/química , Linfocitos T CD8-positivos/química , Separación Celular , Citoplasma/química , Citoplasma/metabolismo , Reacciones Falso Positivas , Citometría de Flujo , Humanos , Receptores CCR5/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fijación del TejidoRESUMEN
Transcription of the human immunodeficiency virus type 1 (HIV) requires the interaction of the cyclin T1 (CycT1) subunit of a host cellular factor, the positive transcription elongation factor b (P-TEFb), with the viral Tat protein, at the transactivation response element (TAR) of nascent transcripts. Because of this virus-specific interaction, CycT1 may potentially serve as a target for the development of anti-HIV therapies. Here we report the development of a mutant CycT1 protein, containing three threonine-to-alanine substitutions in the linker region between two of the cyclin boxes, which displays a potent dominant negative effect on HIV transcription. Investigation into the inhibitory mechanism revealed that this mutant CycT1 interacted with Tat and the cyclin-dependent kinase 9 (Cdk9) subunit of P-TEFb, but failed to stimulate the Cdk9 kinase activity critical for elongation. This mutant CycT1 protein may represent a novel class of specific inhibitors of HIV transcription which could lead to development of new antiviral therapies.
Asunto(s)
Ciclinas/genética , VIH/genética , Polimorfismo de Nucleótido Simple , Transcripción Genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , Ciclina T , Quinasa 9 Dependiente de la Ciclina/metabolismo , Ciclinas/química , Ciclinas/metabolismo , Humanos , Modelos Moleculares , Conformación Proteica , Subunidades de Proteína/metabolismo , Activación TranscripcionalRESUMEN
BACKGROUND: The positive transcription elongation factor b (P-TEFb) is an essential cellular co-factor for the transcription of the human immunodeficiency virus type 1 (HIV-1). The cyclin T1 (CycT1) subunit of P-TEFb associates with a viral protein, Tat, at the transactivation response element (TAR). This represents a critical and necessary step for the stimulation of transcriptional elongation. Therefore, CycT1 may serve as a potential target for the development of anti-HIV therapies. RESULTS: To create effective inhibitors of HIV transcription, mutant CycT1 proteins were constructed based upon sequence similarities between CycT1 and other cyclin molecules, as well as the defined crystal structure of CycT1. One of these mutants, termed CycT1-U7, showed a potent dominant negative effect on Tat-dependent HIV transcription despite a remarkably low steady-state expression level. Surprisingly, the expression levels of Tat proteins co-expressed with CycT1-U7 were significantly lower than Tat co-expressed with wild type CycT1. However, the expression levels of CycT1-U7 and Tat were restored by treatment with proteasome inhibitors. Concomitantly, the dominant negative effect of CycT1-U7 was abolished by these inhibitors. CONCLUSION: These results suggest that CycT1-U7 inhibits HIV transcription by promoting a rapid degradation of Tat. These mutant CycT1 proteins represent a novel class of specific inhibitors for HIV transcription that could potentially be used in the design of anti-viral therapy.