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BACKGROUND: Adagrasib, a KRASG12C inhibitor, irreversibly and selectively binds KRASG12C, locking it in its inactive state. Adagrasib showed clinical activity and had an acceptable adverse-event profile in the phase 1-1b part of the KRYSTAL-1 phase 1-2 study. METHODS: In a registrational phase 2 cohort, we evaluated adagrasib (600 mg orally twice daily) in patients with KRASG12C -mutated non-small-cell lung cancer (NSCLC) previously treated with platinum-based chemotherapy and anti-programmed death 1 or programmed death ligand 1 therapy. The primary end point was objective response assessed by blinded independent central review. Secondary end points included the duration of response, progression-free survival, overall survival, and safety. RESULTS: As of October 15, 2021, a total of 116 patients with KRASG12C -mutated NSCLC had been treated (median follow-up, 12.9 months); 98.3% had previously received both chemotherapy and immunotherapy. Of 112 patients with measurable disease at baseline, 48 (42.9%) had a confirmed objective response. The median duration of response was 8.5 months (95% confidence interval [CI], 6.2 to 13.8), and the median progression-free survival was 6.5 months (95% CI, 4.7 to 8.4). As of January 15, 2022 (median follow-up, 15.6 months), the median overall survival was 12.6 months (95% CI, 9.2 to 19.2). Among 33 patients with previously treated, stable central nervous system metastases, the intracranial confirmed objective response rate was 33.3% (95% CI, 18.0 to 51.8). Treatment-related adverse events occurred in 97.4% of the patients - grade 1 or 2 in 52.6% and grade 3 or higher in 44.8% (including two grade 5 events) - and resulted in drug discontinuation in 6.9% of patients. CONCLUSIONS: In patients with previously treated KRASG12C -mutated NSCLC, adagrasib showed clinical efficacy without new safety signals. (Funded by Mirati Therapeutics; ClinicalTrials.gov number, NCT03785249.).
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Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Proteínas Proto-Oncogénicas p21(ras) , Acetonitrilos/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/patología , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Mutación , Piperazinas/uso terapéutico , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Pirimidinas/uso terapéuticoRESUMEN
The NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Non-Small Cell Lung Cancer (NSCLC) provide recommendations for the treatment of patients with NSCLC, including diagnosis, primary disease management, surveillance for relapse, and subsequent treatment. The panel has updated the list of recommended targeted therapies based on recent FDA approvals and clinical data. This selection from the NCCN Guidelines for NSCLC focuses on treatment recommendations for advanced or metastatic NSCLC with actionable molecular biomarkers.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/terapia , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/terapia , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Biomarcadores de Tumor/genética , Terapia Molecular Dirigida/métodos , Estadificación de NeoplasiasRESUMEN
Mesothelioma is a rare cancer that originates from the mesothelial surfaces of the pleura and other sites, and is estimated to occur in approximately 3,500 people in the United States annually. Pleural mesothelioma is the most common type and represents approximately 85% of these cases. The NCCN Guidelines for Mesothelioma: Pleural provide recommendations for the diagnosis, evaluation, treatment, and follow-up for patients with pleural mesothelioma. These NCCN Guidelines Insights highlight significant updates to the NCCN Guidelines for Mesothelioma: Pleural, including revised guidance on disease classification and systemic therapy options.
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Mesotelioma Maligno , Mesotelioma , Neoplasias Pleurales , Humanos , Pleura , Mesotelioma/diagnóstico , Mesotelioma/terapia , Neoplasias Pleurales/diagnóstico , Neoplasias Pleurales/terapiaRESUMEN
The retrovirus human immunodeficiency virus-1 (HIV-1) is the causative agent of AIDS. Although treatment of HIV/AIDS with antiretroviral therapy provides suppression of viremia, latent reservoirs of integrated proviruses preclude cure by current antiviral treatments. Understanding the mechanisms of host-viral interactions may elucidate new treatment strategies. Here, we performed a CRISPR/Cas9 transcriptional activation screen using a high-complexity, genome-wide sgRNA library to identify cellular factors that inhibit HIV-1 infection of human CD4+ T cells. MT4 cells were transduced with a CRISPR/Cas9 sgRNA library and infected with nef-deficient HIV-1NL4-3 expressing ganciclovir-sensitive thymidine kinase, thus enabling selection of HIV-1-resistant cells for analysis of enriched sgRNAs. After validation of screen hits, multiple host factors essential for HIV-1 infection were identified, including SET (SET nuclear proto-oncogene) and ANP32A (acidic nuclear phosphoprotein 32A, PP32A), which together form a histone acetylase inhibitor complex. Using multiple human cell lines and peripheral blood mononuclear cells (PBMCs) from healthy donors and HIV-1-infected individuals, we demonstrate that SET depletion increased HIV-1 infectivity by augmenting DNA integration without significantly changing sites of integration. Conversely, SET overexpression decreased HIV-1 integration and infectivity. SET protein expression was significantly reduced in PBMCs from HIV-1-infected individuals and was downregulated by HIV-1 infection of healthy donor cells in vitro. Notably, HIV-1-induced downregulation of SET could be alleviated by inhibition of the protease granzyme A. Altogether, we have identified cellular inhibitors of HIV-1 infection on a genome-wide scale, which affords new insight into host-virus interactions and may provide new strategies for HIV-1 treatment.
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VIH-1 , Humanos , Sistemas CRISPR-Cas , Histona Acetiltransferasas , VIH-1/genética , Leucocitos Mononucleares , Proteínas Nucleares , Proteínas de Unión al ARN , Activación Transcripcional , Integración ViralRESUMEN
The NCCN Guidelines for Non-Small Cell Lung Cancer (NSCLC) provide recommendations for management of disease in patients with NSCLC. These NCCN Guidelines Insights focus on neoadjuvant and adjuvant (also known as perioperative) systemic therapy options for eligible patients with resectable NSCLC.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/terapia , Terapia NeoadyuvanteRESUMEN
Mesothelioma is a rare cancer originating in mesothelial surfaces of the peritoneum, pleura, and other sites. These NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) focus on peritoneal mesothelioma (PeM). The NCCN Guidelines for PeM provide recommendations for workup, diagnosis, and treatment of primary as well as previously treated PeM. The diagnosis of PeM may be delayed because PeM mimics other diseases and conditions and because the disease is so rare. The pathology section was recently updated to include new information about markers used to identify mesothelioma, which is difficult to diagnose. The term "malignant" is no longer used to classify mesotheliomas, because all mesotheliomas are now defined as malignant.
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Mesotelioma Maligno , Mesotelioma , Humanos , Oncología Médica , Mesotelioma/diagnóstico , Mesotelioma/terapia , PeritoneoRESUMEN
NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines) for Non-Small Cell Lung Cancer (NSCLC) provide recommended management for patients with NSCLC, including diagnosis, primary treatment, surveillance for relapse, and subsequent treatment. Patients with metastatic lung cancer who are eligible for targeted therapies or immunotherapies are now surviving longer. This selection from the NCCN Guidelines for NSCLC focuses on targeted therapies for patients with metastatic NSCLC and actionable mutations.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/terapia , Humanos , Inmunoterapia , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Oncología Médica , Recurrencia Local de NeoplasiaRESUMEN
Major bottlenecks in development of therapeutic post transcriptional gene silencing (PTGS) agents (e.g. ribozymes, RNA interference, antisense) include the challenge of mapping rare accessible regions of the mRNA target that are open for annealing and cleavage, testing and optimization of agents in human cells to identify lead agents, testing for cellular toxicity, and preclinical evaluation in appropriate animal models of disease. Methods for rapid and reliable cellular testing of PTGS agents are needed to identify potent lead candidates for optimization. Our goal was to develop a means of rapid assessment of many RNA agents to identify a lead candidate for a given mRNA associated with a disease state. We developed a rapid human cell-based screening platform to test efficacy of hammerhead ribozyme (hhRz) or RNA interference (RNAi) constructs, using a model retinal degeneration target, human rod opsin (RHO) mRNA. The focus is on RNA Drug Discovery for diverse retinal degeneration targets. To validate the approach, candidate hhRzs were tested against NUH↓ cleavage sites (N = G,C,A,U; H = C,A,U) within the target mRNA of secreted alkaline phosphatase (SEAP), a model gene expression reporter, based upon in silico predictions of mRNA accessibility. HhRzs were embedded in a larger stable adenoviral VAI RNA scaffold for high cellular expression, cytoplasmic trafficking, and stability. Most hhRz expression plasmids exerted statistically significant knockdown of extracellular SEAP enzyme activity when readily assayed by a fluorescence enzyme assay intended for high throughput screening (HTS). Kinetics of PTGS knockdown of cellular targets is measureable in live cells with the SEAP reporter. The validated SEAP HTS platform was transposed to identify lead PTGS agents against a model hereditary retinal degeneration target, RHO mRNA. Two approaches were used to physically fuse the model retinal gene target mRNA to the SEAP reporter mRNA. The most expedient way to evaluate a large set of potential VAI-hhRz expression plasmids against diverse NUH↓ cleavage sites uses cultured human HEK293S cells stably expressing a dicistronic Target-IRES-SEAP target fusion mRNA. Broad utility of this rational RNA drug discovery approach is feasible for any ophthalmological disease-relevant mRNA targets and any disease mRNA targets in general. The approach will permit rank ordering of PTGS agents based on potency to identify a lead therapeutic compound for further optimization.
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Terapia Genética/métodos , ARN Catalítico/uso terapéutico , ARN Mensajero/genética , Tratamiento con ARN de Interferencia/métodos , Degeneración Retiniana/genética , Células Cultivadas , Biología Computacional/métodos , Técnicas de Silenciamiento del Gen/métodos , Vectores Genéticos/genética , Humanos , Degeneración Retiniana/diagnóstico , Degeneración Retiniana/terapia , Epitelio Pigmentado de la Retina/patologíaRESUMEN
BACKGROUND: The ADAURA trial confirmed adjuvant Osimertinib's efficacy in EGFR-mutated Non-small-cell lung cancer (NSCLC), yet the limited mature overall survival (OS) data at approval poses a challenge. This study explores patient preferences in the absence of complete OS information, hypothesizing that disease-free survival (DFS) benefit alone may influence adjuvant Osimertinib pursuit. METHODS: At Roswell Park Comprehensive Cancer Center (Jan-Dec 2021), patients assessed for adjuvant therapy received a survey probing OS and DFS preferences. Scenarios were (a) minimum OS justifying Osimertinib, (b) minimum DFS improvement justifying 3-years of adjuvant Osimertinib, (c) minimum 5-year DFS percent change, and (d) minimum OS justifying copay changes. Results were analyzed. RESULTS: Of 524 NSCLC patients, 51 participated. Scenario 1 saw 56% requiring a 12-month OS benefit for Osimertinib justification. In scenario 2, 72% deemed a 12-month DFS benefit sufficient. Scenario 3 revealed 31% opting out despite a 10% OS increase. Scenario 4 showed varied willingness to pay, with 33% unwilling to any shoulder copayment even with a 10-year OS benefit. CONCLUSION: This study explores patient preferences without complete OS data, revealing diverse thresholds. Factors include employment, education, and willingness to pay. Findings underscore shared decision-making importance. Limitations include sample size, potential biases, and regional focus; larger cohorts are needed for validation.
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Acrilamidas , Compuestos de Anilina , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Prioridad del Paciente , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/cirugía , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/cirugía , Acrilamidas/uso terapéutico , Compuestos de Anilina/uso terapéutico , Femenino , Masculino , Persona de Mediana Edad , Anciano , Quimioterapia Adyuvante/métodos , Adulto , Antineoplásicos/uso terapéutico , Anciano de 80 o más Años , Indoles , PirimidinasRESUMEN
Neoadjuvant chemoimmunotherapy improves pathologic complete response rate and event-free survival in patients with resectable non-small cell lung cancer (NSCLC) versus chemotherapy alone. NeoCOAST was the first randomized, multidrug platform trial to examine novel neoadjuvant immuno-oncology combinations for patients with resectable NSCLC, using major pathologic response (MPR) rate as the primary endpoint. Eighty-three patients received a single cycle of treatment: 26 received durvalumab (anti-PD-L1) monotherapy, 21 received durvalumab plus oleclumab (anti-CD73), 20 received durvalumab plus monalizumab (anti-NKG2A), and 16 received durvalumab plus danvatirsen (anti-STAT3 antisense oligonucleotide). MPR rates were higher for patients in the combination arms versus durvalumab alone. Safety profiles for the combinations were similar to those of durvalumab alone. Multiplatform immune profiling suggested that improved MPR rates in the durvalumab plus oleclumab and durvalumab plus monalizumab arms were associated with enhanced effector immune infiltration of tumors, interferon responses and markers of tertiary lymphoid structure formation, and systemic functional immune cell activation. SIGNIFICANCE: A neoadjuvant platform trial can rapidly generate clinical and translational data using candidate surrogate endpoints like MPR. In NeoCOAST, patients with resectable NSCLC had improved MPR rates after durvalumab plus oleclumab or monalizumab versus durvalumab alone and tumoral transcriptomic signatures indicative of augmented immune cell activation and function. See related commentary by Cooper and Yu, p. 2306. This article is featured in Selected Articles from This Issue, p. 2293.
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Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Anticuerpos Monoclonales/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Terapia NeoadyuvanteRESUMEN
Breast magnetic resonance imaging (MRI) is routinely used as a problem-solving tool, but its benefit for this indication remains unclear. The records of 3001 consecutive breast MR examinations between January 1, 2003 and June 6, 2007 were reviewed to identify all those performed for the clinical indication of problem solving. Details of clinical presentation, mammography and ultrasound (US) findings, follow-up recommendations, and pathology outcomes were recorded. Benign versus malignant outcomes were determined by biopsy or 12 months of follow-up imaging and linkage with the regional tumor registry. Problem solving was the clinical indication for 204 of 3001 (7%) of all examinations. Forty-two of 204 examinations (21%) had suspicious or highly suspicious MRI assessments with recommendation for biopsy and 62 of 204 (79%) examinations were assessed as negative, benign, or probably benign. Thirty-six biopsies were performed based on MRI findings and 14 cancers were diagnosed. Biopsy was indicated for 11 of 14 (79%) cancers based on suspicious mammographic or US findings identified prior to MRI. One incidental cancer was detected by MRI alone in a patient at high risk for breast cancer, and two cancers were detected in patients with suspicious nipple discharge and negative mammogram and US. A single false-negative MRI occurred in a patient whose evaluation for a palpable lump prompted biopsy. Problem-solving breast MRI rarely identifies otherwise occult cancer and can be falsely negative in patients with suspicious findings on mammogram and US. Until the benefits and risks of problem-solving MRI are clarified, it should be used judiciously.
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Neoplasias de la Mama/diagnóstico , Imagen por Resonancia Magnética , Diagnóstico Diferencial , Femenino , Humanos , Persona de Mediana Edad , Valor Predictivo de las Pruebas , Solución de Problemas , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
We perform a CRISPR-Cas9 genome-wide screen in glioblastoma stem cells and identify integrin αvß5 as an internalization factor for Zika virus (ZIKV). Expression of αvß5 is correlated with ZIKV susceptibility in various cells and tropism in developing human cerebral cortex. A blocking antibody against integrin αvß5, but not αvß3, efficiently inhibits ZIKV infection. ZIKV binds to cells but fails to internalize when treated with integrin αvß5-blocking antibody. αvß5 directly binds to ZIKV virions and activates focal adhesion kinase, which is required for ZIKV infection. Finally, αvß5 blocking antibody or two inhibitors, SB273005 and cilengitide, reduces ZIKV infection and alleviates ZIKV-induced pathology in human neural stem cells and in mouse brain. Altogether, our findings identify integrin αvß5 as an internalization factor for ZIKV, providing a promising therapeutic target, as well as two drug candidates for prophylactic use or treatments for ZIKV infections.
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Antivirales/uso terapéutico , Células-Madre Neurales/metabolismo , Receptores de Vitronectina/genética , Infección por el Virus Zika/virología , Virus Zika/patogenicidad , Antivirales/farmacología , Humanos , Receptores de Vitronectina/metabolismoRESUMEN
Importance: Nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, are commonly prescribed medications with anti-inflammatory and antiplatelet properties used long term to decrease the risk of cardiovascular events. A recent study showed that aspirin was associated with improved survival in patients with head and neck squamous cell carcinoma (HNSCC) who were treated with surgery. Objective: To examine whether use of NSAIDs during definitive chemoradiation therapy (CRT) was associated with improved outcomes in patients with HNSCC. Design, Setting, and Participants: This cohort study analyzed patients with HNSCC who were treated with CRT at a single institution between January 1, 2005, and August 1, 2017. Patient and tumor characteristics included age, race/ethnicity, smoking status, alcohol use, comorbidities (respiratory, cardiovascular, immune, renal, endocrine), disease stage, human papillomavirus status, and treatment duration. Data were analyzed from May 1, 2019, to March 17, 2020. Exposures: Patients were dichotomized by NSAID use during treatment. Main Outcomes and Measures: The association of NSAID use with patterns of failure, disease-specific survival (DSS), and overall survival (OS) was examined using multivariate Cox proportional hazard regression models. Survival estimates for OS and DSS were generated using Kaplan-Meier survival curves. Results: A total of 460 patients (median [interquartile range] age, 60 [53.9-65.6] years; 377 [82.0%] men) were included in the analysis. Among these patients, 201 (43.7%) were taking NSAIDs during treatment. On univariate analysis, NSAID use (hazard ratio [HR], 0.63; 95% CI, 0.43-0.92; P = .02) was associated with better OS. On Cox regression analysis, after backward selection adjustment for potentially confounding factors (age, smoking status, primary tumor site, human papillomavirus status, diabetes, stroke, hyperlipidemia), NSAID use remained significantly associated with better OS (HR, 0.59; 95% CI, 0.38-0.90; P = .02). NSAID use was associated with significantly better OS at 5 years compared with patients who did not take concurrent NSAIDs (63.6% [56 of 88 patients]; 95% CI, 58%-73% vs 56.1% [83 of 148 patients]; 95% CI, 50%-63%; P = .03). NSAID use was not associated with better DSS in univariate (HR, 0.82; 95% CI, 0.48-1.41; P = .47) or multivariate (HR, 0.98; 95% CI, 0.57-1.70; P = .44) analysis. NSAID use was not associated with better response to treatment (HR, 1.44; 95% CI, 0.91-2.27; P = .12) or distant failure (HR, 1.12; 95% CI, 0.68-1.84; P = .65). Change in local control with NSAID use was not statistically significant (HR, 0.59; 95% CI, 0.31-1.10; P = .10). Conclusions and Relevance: This cohort study suggests a possible OS advantage for patients taking NSAIDs during chemoradiation for HNSCC. Further studies examining this association are warranted.
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Antiinflamatorios no Esteroideos , Neoplasias de Cabeza y Cuello , Carcinoma de Células Escamosas de Cabeza y Cuello , Anciano , Aspirina , Quimioradioterapia , Femenino , Neoplasias de Cabeza y Cuello/epidemiología , Neoplasias de Cabeza y Cuello/mortalidad , Neoplasias de Cabeza y Cuello/terapia , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Modelos de Riesgos Proporcionales , Estudios Retrospectivos , Carcinoma de Células Escamosas de Cabeza y Cuello/epidemiología , Carcinoma de Células Escamosas de Cabeza y Cuello/mortalidad , Carcinoma de Células Escamosas de Cabeza y Cuello/terapiaRESUMEN
To identify lead candidate allele-independent hammerhead ribozymes (hhRz) for the treatment of autosomal dominant mutations in the human rod opsin (RHO) gene, we tested a series of hhRzs for potential to significantly knockdown human RHO gene expression in a human cell expression system. Multiple computational criteria were used to select target mRNA regions likely to be single stranded and accessible to hhRz annealing and cleavage. Target regions are tested for accessibility in a human cell culture expression system where the hhRz RNA and target mRNA and protein are coexpressed. The hhRz RNA is embedded in an adenoviral VAI RNA chimeric RNA of established structure and properties which are critical to the experimental paradigm. The chimeric hhRz-VAI RNA is abundantly transcribed so that the hhRzs are expected to be in great excess over substrate mRNA. HhRz-VAI traffics predominantly to the cytoplasm to colocalize with the RHO mRNA target. Colocalization is essential for second-order annealing reactions. The VAI chimera protects the hhRz RNA from degradation and provides for a long half-life. With cell lines chosen for high transfection efficiency and a molar excess of hhRz plasmid over target plasmid, the conditions of this experimental paradigm are specifically designed to evaluate for regions of accessibility of the target mRNA in cellulo. Western analysis was used to measure the impact of hhRz expression on RHO protein expression. Three lead candidate hhRz designs were identified that significantly knockdown target protein expression relative to control (p<0.05). Successful lead candidates (hhRz CUC [see in text downward arrow] 266, hhRz CUC [see in text downward arrow] 1411, hhRz AUA [see in text downward arrow] 1414) targeted regions of human RHO mRNA that were predicted to be accessible by a bioinformatics approach, whereas regions predicted to be inaccessible supported no knockdown. The maximum opsin protein level knockdown is approximately 30% over a 48h paradigm of testing. These results validate a rigorous computational bioinformatics approach to detect accessible regions of target mRNAs in cellulo. The opsin knockdown effect could prove to be clinically significant when integrated over longer periods in photoreceptors. Further optimization and animal testing are the next step in this stratified RNA drug discovery program. A recently developed novel and efficient screening assay based upon expression of a dicistronic mRNA (RHO-IRES-SEAP) containing both RHO and reporter (SEAP) cDNAs was used to compare the hhRz 266 lead candidate to another agent (Rz525/hhRz485) already known to partially rescue retinal degeneration in a rodent model. Lead hhRz 266 CUC [see in text downward arrow] proved more efficacious than Rz525/hhRz485 which infers viability for rescue of retinal degeneration in appropriate preclinical models of disease.
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Técnicas de Silenciamiento del Gen/métodos , Terapia Genética/métodos , ARN Catalítico/metabolismo , Degeneración Retiniana/terapia , Opsinas de Bastones/genética , Adenoviridae/genética , Algoritmos , Células Cultivadas , Biología Computacional/métodos , Vectores Genéticos/genética , Humanos , Mutación , ARN Catalítico/genética , ARN Mensajero/genética , Degeneración Retiniana/genética , TransfecciónRESUMEN
A major challenge in finding a cure for HIV-1/AIDS is the difficulty in identifying and eradicating persistent reservoirs of replication-competent provirus. Long noncoding RNAs (lncRNAs, >200 nucleotides) are increasingly recognized to play important roles in pathophysiology. Here, we report the first genome-wide expression analysis of lncRNAs in HIV-1-infected primary monocyte-derived macrophages (MDMs). We identified an lncRNA, which we named HIV-1-enhanced lncRNA (HEAL), that is upregulated by HIV-1 infection of MDMs, microglia, and T lymphocytes. Peripheral blood mononuclear cells of HIV-1-infected individuals show elevated levels of HEAL Importantly, HEAL is a broad enhancer of multiple HIV-1 strains because depletion of HEAL inhibited X4, R5, and dual-tropic HIV replications and the inhibition was rescued by HEAL overexpression. HEAL forms a complex with the RNA-binding protein FUS, which facilitates HIV replication through at least two mechanisms: (i) HEAL-FUS complex binds the HIV promoter and enhances recruitment of the histone acetyltransferase p300, which positively regulates HIV transcription by increasing histone H3K27 acetylation and P-TEFb enrichment on the HIV promoter, and (ii) HEAL-FUS complex is enriched at the promoter of the cyclin-dependent kinase 2 gene, CDK2, to enhance CDK2 expression. Notably, HEAL knockdown and knockout mediated by RNA interference (RNAi) and CRISPR-Cas9, respectively, prevent HIV-1 recrudescence in T cells and microglia upon cessation of azidothymidine treatment in vitro Our results suggest that silencing of HEAL or perturbation of the HEAL-FUS ribonucleoprotein complex could provide a new epigenetic silencing strategy to eradicate viral reservoirs and effect a cure for HIV-1/AIDS.IMPORTANCE Despite our increased understanding of the functions of lncRNAs, their potential to develop HIV/AIDS cure strategies remains unexplored. A genome-wide analysis of lncRNAs in HIV-1-infected primary monocyte-derived macrophages (MDMs) was performed, and 1,145 differentially expressed lncRNAs were identified. An lncRNA named HIV-1-enhanced lncRNA (HEAL) is upregulated by HIV-1 infection and promotes HIV replication in T cells and macrophages. HEAL forms a complex with the RNA-binding protein FUS to enhance transcriptional coactivator p300 recruitment to the HIV promoter. Furthermore, HEAL knockdown and knockout prevent HIV-1 recrudescence in T cells and microglia upon cessation of azidothymidine treatment, suggesting HEAL as a potential therapeutic target to cure HIV-1/AIDS.
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Epigénesis Genética , Regulación Viral de la Expresión Génica/fisiología , Infecciones por VIH/fisiopatología , VIH-1/fisiología , Regiones Promotoras Genéticas/fisiología , ARN Largo no Codificante/fisiología , Replicación Viral/fisiología , HumanosRESUMEN
PURPOSE: To systematically evaluate human rod opsin (hRHO) mRNA for potential target sites sensitive to posttranscriptional gene silencing (PTGS) by hammerhead ribozyme (hhRz) or RNA interference (RNAi) in human cells. To develop a comprehensive strategy to identify and optimize lead candidate agents for PTGS gene therapeutics. METHODS: In multidisciplinary RNA drug discovery, computational mRNA accessibility and in vitro experimental methods using reverse transcription-polymerase chain reaction (RT-PCR) were used to map accessibility in full-length hRHO transcripts. HhRzs targeted predicted accessible and inaccessible sites and were screened for cellular knockdown using a bicistronic reporter construct. Lead hhRz and RNAi PTGS agents were rationally optimized for target knockdown in human cells. RESULTS: Systematic screening of hRHO mRNA targeting agents resulted in lead candidate identification of a novel hhRz embedded in an RNA scaffold. Rational optimization strategies identified a minimal 725 hhRz as the most active agent. Recently identified tertiary accessory elements did not enhance activity. A 725-short-hairpin RNA (shRNA) agent exerts log-order knockdown. Silent modulation of the 725-hhRz target site in hRHO mRNA resulted in resistance to knockdown. CONCLUSIONS: Combining rational RNA drug design with cell-based screening allowed rapid identification of lead agents targeting hRHO. Optimization strategies identified the agent with highest intracellular activity. These agents have therapeutic potential in a mutation-independent strategy for adRP, or other degenerations where hRHO is a target. This approach can be broadly applied to any validated target mRNA, regardless of the disease. TRANSLATIONAL RELEVANCE: This work establishes a platform approach to develop RNA biologicals for the treatment of human disease.
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BACKGROUND: Resistance to immune checkpoint inhibitors (ICIs) has been linked to local immunosuppression independent of major ICI targets (e.g., PD-1). Clinical experience with response prediction based on PD-L1 expression suggests that other factors influence sensitivity to ICIs in non-small cell lung cancer (NSCLC) patients. METHODS: Tumor specimens from 120 NSCLC patients from 10 institutions were evaluated for PD-L1 expression by immunohistochemistry, and global proliferative profile by targeted RNA-seq. RESULTS: Cell proliferation, derived from the mean expression of 10 proliferation-associated genes (namely BUB1, CCNB2, CDK1, CDKN3, FOXM1, KIAA0101, MAD2L1, MELK, MKI67, and TOP2A), was identified as a marker of response to ICIs in NSCLC. Poorly, moderately, and highly proliferative tumors were somewhat equally represented in NSCLC, with tumors with the highest PD-L1 expression being more frequently moderately proliferative as compared to lesser levels of PD-L1 expression. Proliferation status had an impact on survival in patients with both PD-L1 positive and negative tumors. There was a significant survival advantage for moderately proliferative tumors compared to their combined highly/poorly counterparts (p = 0.021). Moderately proliferative PD-L1 positive tumors had a median survival of 14.6 months that was almost twice that of PD-L1 negative highly/poorly proliferative at 7.6 months (p = 0.028). Median survival in moderately proliferative PD-L1 negative tumors at 12.6 months was comparable to that of highly/poorly proliferative PD-L1 positive tumors at 11.5 months, but in both instances less than that of moderately proliferative PD-L1 positive tumors. Similar to survival, proliferation status has impact on disease control (DC) in patients with both PD-L1 positive and negative tumors. Patients with moderately versus those with poorly or highly proliferative tumors have a superior DC rate when combined with any classification schema used to score PD-L1 as a positive result (i.e., TPS ≥ 50% or ≥ 1%), and best displayed by a DC rate for moderately proliferative tumors of no less than 40% for any classification of PD-L1 as a negative result. While there is an over representation of moderately proliferative tumors as PD-L1 expression increases this does not account for the improved survival or higher disease control rates seen in PD-L1 negative tumors. CONCLUSIONS: Cell proliferation is potentially a new biomarker of response to ICIs in NSCLC and is applicable to PD-L1 negative tumors.
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Antineoplásicos Inmunológicos/uso terapéutico , Antígeno B7-H1/antagonistas & inhibidores , Carcinoma de Pulmón de Células no Pequeñas , Proliferación Celular/genética , Neoplasias Pulmonares , Adulto , Anciano , Anciano de 80 o más Años , Antígeno B7-H1/metabolismo , Secuencia de Bases , Biomarcadores , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Resistencia a Antineoplásicos , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Masculino , Persona de Mediana Edad , Análisis de SupervivenciaRESUMEN
Development of post-transcriptional gene silencing (PTGS) agents for therapeutic purposes is an immense challenge in modern biology. Established technologies used to knockdown a specific target RNA and its cognate protein: antisense, ribozyme, RNAi, all conditionally depend upon an initial, critical annealing event of the PTGS ligand to a target RNA. In this review we address the nature of the bottlenecks, emphasizing the biocomplexity of target RNA structure, that currently limit PTGS therapeutic development. We briefly review existing and emerging technologies designed to release these constraints to realize the potential of PTGS agents in gene based therapies.
Asunto(s)
Terapia Genética/métodos , Interferencia de ARN , Enfermedades de la Retina/terapia , Marcación de Gen/métodos , Terapia Genética/tendencias , Humanos , Procesamiento Postranscripcional del ARN/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Opsinas de Bastones/genéticaRESUMEN
Genome-wide functional genomic screens utilizing the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system have proven to be a powerful tool for systematic genomic perturbation in mammalian cells and provide an alternative to previous screens utilizing RNA interference technology. The wide availability of these libraries through public plasmid repositories as well as the decreasing cost and speed in quantifying these screens using high-throughput next-generation sequencing (NGS) allows for the adoption of the technology in a variety of laboratories interested in diverse biologic questions. Here, we describe the protocol to generate next-generation sequencing libraries from genome-wide CRISPR genomic screens.