RESUMEN
BACKGROUND: RPT193 is an orally administered small molecule antagonist of the human C-C motif chemokine receptor 4 (CCR4) that inhibits the migration and downstream activation of T-helper Type 2 (Th2) cells. We investigated single- and multiple-ascending doses of RPT193 in healthy subjects, and multiple doses of RPT193 in subjects with moderate-to-severe atopic dermatitis (AD). METHODS: This was a first-in-human randomized, placebo-controlled Phase 1a/1b monotherapy study (NCT04271514) to evaluate the safety, tolerability, pharmacokinetics, pharmacodynamics, and CCR4 surface receptor occupancy in eligible healthy subjects and subjects with moderate-to-severe AD. Clinical efficacy and skin biomarker effects of RPT193 monotherapy were assessed as exploratory endpoints in AD subjects. RESULTS: In healthy (n = 72) and AD subjects (n = 31), once-daily RPT193 treatment was generally well tolerated, with no serious adverse events reported and all treatment-emergent adverse events reported as mild/moderate. In AD subjects, numerically greater improvements in clinical efficacy endpoints were observed with RPT193 monotherapy versus placebo up to the end of the treatment period (Day 29), with statistically significant improvement, compared to Day 29 and placebo, observed 2 weeks after the end of treatment (Day 43) on several endpoints (p < .05). Moreover, significant changes in the transcriptional profile were seen in skin biopsies of RPT193-treated versus placebo-treated subjects at Day 29, which were also significantly correlated with improvements in clinical efficacy measures. CONCLUSIONS: To our knowledge, this is the first clinical study with an oral CCR4 antagonist that showed clinical improvement coupled with modulation of the cutaneous transcriptomic profile in an inflammatory skin disease.
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Dermatitis Atópica , Humanos , Dermatitis Atópica/tratamiento farmacológico , Dermatitis Atópica/patología , Piel/patología , Células Th2/patología , Resultado del Tratamiento , Método Doble Ciego , Índice de Severidad de la Enfermedad , Receptores CCR4/uso terapéuticoRESUMEN
General control nonderepressible 2 (GCN2) protein kinase is a cellular stress sensor within the tumor microenvironment (TME), whose signaling cascade has been proposed to contribute to immune escape in tumors. Herein, we report the discovery of cell-potent GCN2 inhibitors with excellent selectivity against its closely related Integrated Stress Response (ISR) family members heme-regulated inhibitor kinase (HRI), protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase (PERK), as well as good kinome-wide selectivity and favorable PK. In mice, compound 39 engages GCN2 at levels ≥80% with an oral dose of 15 mg/kg BID. We also demonstrate the ability of compound 39 to alleviate MDSC-related T cell suppression and restore T cell proliferation, similar to the effect seen in MDSCs from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI) as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced by treatment with compound 39 demonstrating the complementarity of these two mechanisms.
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Células Supresoras de Origen Mieloide , eIF-2 Quinasa , Animales , Hemo , Ratones , Ratones Noqueados , Proteínas Serina-Treonina Quinasas , Linfocitos T/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
The Epstein-Barr Virus (EBV) is involved in the etiology of multiple hematologic and epithelial human cancers. EBV+ tumors employ multiple immune escape mechanisms, including the recruitment of immunosuppressive regulatory T cells (Treg). Here, we show some EBV+ tumor cells express high levels of the chemokines CCL17 and CCL22 both in vitro and in vivo and that this expression mirrors the expression levels of expression of the EBV LMP1 gene in vitro. Patient samples from lymphoblastic (Hodgkin lymphoma) and epithelial (nasopharyngeal carcinoma; NPC) EBV+ tumors revealed CCL17 and CCL22 expression of both tumor cell-intrinsic and -extrinsic origin, depending on tumor type. NPCs grown as mouse xenografts likewise showed both mechanisms of chemokine production. Single cell RNA-sequencing revealed in vivo tumor cell-intrinsic CCL17 and CCL22 expression combined with expression from infiltrating classical resident and migratory dendritic cells in a CT26 colon cancer mouse tumor engineered to express LMP1. These data suggest that EBV-driven tumors employ dual mechanisms for CCL17 and CCL22 production. Importantly, both in vitro and in vivo Treg migration was effectively blocked by a novel, small molecule antagonist of CCR4, CCR4-351. Antagonism of the CCR4 receptor may thus be an effective means of activating the immune response against a wide spectrum of EBV+ tumors.
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Quimiocina CCL17/inmunología , Quimiocina CCL22/inmunología , Infecciones por Virus de Epstein-Barr/inmunología , Neoplasias/inmunología , Neoplasias/virología , Linfocitos T Reguladores/inmunología , Animales , Infecciones por Virus de Epstein-Barr/complicaciones , Herpesvirus Humano 4 , Xenoinjertos , Enfermedad de Hodgkin/inmunología , Enfermedad de Hodgkin/virología , Humanos , Ratones , Carcinoma Nasofaríngeo/inmunología , Carcinoma Nasofaríngeo/virología , Neoplasias Nasofaríngeas/inmunología , Neoplasias Nasofaríngeas/virologíaRESUMEN
BACKGROUND: Checkpoint inhibitors (CPIs) such as anti-PD(L)-1 and anti-CTLA-4 antibodies have resulted in unprecedented rates of antitumor responses and extension of survival of patients with a variety of cancers. But some patients fail to respond or initially respond but later relapse as they develop resistance to immune therapy. One of the tumor-extrinsic mechanisms for resistance to immune therapy is the accumulation of regulatory T cells (Treg) in tumors. In preclinical and clinical studies, it has been suggested that tumor trafficking of Treg is mediated by CC chemokine receptor 4 (CCR4). Over 90% of human Treg express CCR4 and migrate toward CCL17 and CCL22, two major CCR4 ligands that are either high at baseline or upregulated in tumors on CPI treatment. Hence, CCR4 antagonism has the potential to be an effective antitumor treatment by reducing the accumulation of Treg into the tumor microenvironment (TME). METHODS: We developed in vitro and in vivo models to assess Treg migration and antitumor efficacy using a potent and selective CCR4 antagonist, CCR4-351. We used two separate tumor models, Pan02 and CT26 mouse tumors, that have high and low CCR4 ligand expression, respectively. Tumor growth inhibition as well as the frequency of tumor-infiltrating Treg and effector T cells was assessed following the treatment with CCR4 antagonist alone or in combination with CPI. RESULTS: Using a selective and highly potent, novel small molecule inhibitor of CCR4, we demonstrate that migration of CCR4+ Treg into the tumor drives tumor progression and resistance to CPI treatment. In tumor models with high baseline levels of CCR4 ligands, blockade of CCR4 reduced the number of Treg and enhanced antitumor immune activity. Notably, in tumor models with low baseline level of CCR4 ligands, treatment with immune CPIs resulted in significant increases of CCR4 ligands and Treg numbers. Inhibition of CCR4 reduced Treg frequency and potentiated the antitumor effects of CPIs. CONCLUSION: Taken together, we demonstrate that CCR4-dependent Treg recruitment into the tumor is an important tumor-extrinsic mechanism for immune resistance. Blockade of CCR4 led to reduced frequency of Treg and resulted in increased antitumor activity, supporting the clinical development of CCR4 inhibitors in combination with CPI for the treatment of cancer. STATEMENT OF SIGNIFICANCE: CPI upregulates CCL17 and CCL22 expression in tumors and increases Treg migration into the TME. Pharmacological antagonism of the CCR4 receptor effectively inhibits Treg recruitment and results in enhanced antitumor efficacy either as single agent in CCR4 ligandhigh tumors or in combination with CPIs in CCR4 ligandlow tumors.
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Inmunoterapia/métodos , Neoplasias/inmunología , Neoplasias/terapia , Receptores CCR4/inmunología , Linfocitos T Reguladores/inmunología , Animales , Femenino , Humanos , Ratones , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The deubiquitinase USP7 regulates the levels of multiple proteins with roles in cancer progression and immune response. Thus, USP7 inhibition may decrease oncogene function, increase tumor suppressor function, and sensitize tumors to DNA-damaging agents. We have discovered a novel chemical series that potently and selectively inhibits USP7 in biochemical and cellular assays. Our inhibitors reduce the viability of multiple TP53 wild-type cell lines, including several hematologic cancer and MYCN-amplified neuroblastoma cell lines, as well as a subset of TP53-mutant cell lines in vitro Our work suggests that USP7 inhibitors upregulate transcription of genes normally silenced by the epigenetic repressor complex, polycomb repressive complex 2 (PRC2), and potentiate the activity of PIM and PI3K inhibitors as well as DNA-damaging agents. Furthermore, oral administration of USP7 inhibitors inhibits MM.1S (multiple myeloma; TP53 wild type) and H526 (small cell lung cancer; TP53 mutant) tumor growth in vivo Our work confirms that USP7 is a promising, pharmacologically tractable target for the treatment of cancer.
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Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Animales , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Femenino , Humanos , Ratones , Modelos MolecularesRESUMEN
The C-C chemokine receptor 4 (CCR4) is broadly expressed on regulatory T cells (Treg) as well as other circulating and tissue-resident T cells. Treg can be recruited to the tumor microenvironment (TME) through the C-C chemokines CCL17 and CCL22. Treg accumulation in the TME has been shown to dampen the antitumor immune response and is thought to be an important driver in tumor immune evasion. Preclinical and clinical data suggest that reducing the Treg population in the TME can potentiate the antitumor immune response of checkpoint inhibitors. We have developed small-molecule antagonists of CCR4, featuring a novel piperidinyl-azetidine motif, that inhibit the recruitment of Treg into the TME and elicit antitumor responses as a single agent or in combination with an immune checkpoint blockade. The discovery of these potent, selective, and orally bioavailable CCR4 antagonists, and their activity in in vitro and in vivo models, is described herein.
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Antineoplásicos/química , Antineoplásicos/farmacología , Azetidinas/química , Azetidinas/farmacología , Receptores CCR4/antagonistas & inhibidores , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Azetidinas/farmacocinética , Azetidinas/uso terapéutico , Línea Celular Tumoral , Perros , Humanos , Macaca fascicularis , Neoplasias/tratamiento farmacológico , Neoplasias/inmunología , Piperidinas/química , Piperidinas/farmacocinética , Piperidinas/farmacología , Piperidinas/uso terapéutico , Receptores CCR4/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Linfocitos T Reguladores/inmunologíaRESUMEN
USP7 is a promising target for cancer therapy as its inhibition is expected to decrease function of oncogenes, increase tumor suppressor function, and enhance immune function. Using a structure-based drug design strategy, a new class of reversible USP7 inhibitors has been identified that is highly potent in biochemical and cellular assays and extremely selective for USP7 over other deubiquitinases. The succinimide was identified as a key potency-driving motif, forming two strong hydrogen bonds to the allosteric pocket of USP7. Redesign of an initial benzofuran-amide scaffold yielded a simplified ether series of inhibitors, utilizing acyclic conformational control to achieve proper amine placement. Further improvements were realized upon replacing the ether-linked amines with carbon-linked morpholines, a modification motivated by free energy perturbation (FEP+) calculations. This led to the discovery of compound 41, a highly potent, selective, and orally bioavailable USP7 inhibitor. In xenograft studies, compound 41 demonstrated tumor growth inhibition in both p53 wildtype and p53 mutant cancer cell lines, demonstrating that USP7 inhibitors can suppress tumor growth through multiple different pathways.
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Antineoplásicos/administración & dosificación , Antineoplásicos/química , Descubrimiento de Drogas/métodos , Peptidasa Específica de Ubiquitina 7/antagonistas & inhibidores , Peptidasa Específica de Ubiquitina 7/química , Administración Oral , Animales , Línea Celular Tumoral , Cristalografía por Rayos X/métodos , Humanos , Ratones , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Estructura Terciaria de Proteína , Peptidasa Específica de Ubiquitina 7/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto/métodosRESUMEN
Recruitment of suppressive CD4+ FOXP3+ regulatory T cells (Treg) to the tumor microenvironment (TME) has the potential to weaken the antitumor response in patients receiving treatment with immuno-oncology (IO) agents. Human Treg express CCR4 and can be recruited to the TME through the CC chemokine ligands CCL17 and CCL22. In some cancers, Treg accumulation correlates with poor patient prognosis. Preclinical data suggests that preventing the recruitment of Treg and increasing the population of activated effector T cells (Teff) in the TME can potentiate antitumor immune responses. We developed a novel series of potent, orally bioavailable small molecule antagonists of CCR4. From this series, several compounds exhibited high potency in distinct functional assays in addition to good in vitro and in vivo ADME properties. The design, synthesis, and SAR of this series and confirmation of its in vivo activity are reported.
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Movimiento Celular/efectos de los fármacos , Pirazinas/farmacología , Pirazoles/farmacología , Receptores CCR4/antagonistas & inhibidores , Linfocitos T Reguladores/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Animales , Ciclohexanos/síntesis química , Ciclohexanos/farmacocinética , Ciclohexanos/farmacología , Descubrimiento de Drogas , Humanos , Ratones Transgénicos , Estructura Molecular , Piperazinas/síntesis química , Piperazinas/farmacocinética , Piperazinas/farmacología , Pirazinas/síntesis química , Pirazinas/farmacocinética , Pirazoles/síntesis química , Pirazoles/farmacocinética , Ratas , Relación Estructura-ActividadRESUMEN
Autophagy is the primary process for recycling cellular constituents through lysosomal degradation. In addition to nonselective autophagic engulfment of cytoplasm, autophagosomes can recognize specific cargo by interacting with ubiquitin-binding autophagy receptors such as SQSTM1/p62 (sequestosome 1). This selective form of autophagy is important for degrading aggregation-prone proteins prominent in many neurodegenerative diseases. We carried out a high content image-based siRNA screen (4 to 8 siRNA per gene) for modulators of autophagic flux by monitoring fluorescence of GFP-SQSTM1 as well as colocalization of GFP-SQSTM1 with LAMP2 (lysosomal-associated membrane protein 2)-positive lysosomal vesicles. GFP-SQSTM1 and LAMP2 phenotypes of primary screen hits were confirmed in 2 cell types and profiled with image-based viability and MTOR signaling assays. Common seed analysis guided siRNA selection for these assays to reduce bias toward off-target effects. Confirmed hits were further validated in a live-cell assay to monitor fusion of autophagosomes with lysosomes. Knockdown of 10 targets resulted in phenotypic profiles across multiple assays that were consistent with upregulation of autophagic flux. These hits include modulators of transcription, lysine acetylation, and ubiquitination. Two targets, KAT8 (K[lysine] acetyltransferase 8) and CSNK1A1 (casein kinase 1, α 1), have been implicated in autophagic regulatory feedback loops. We confirmed that CSNK1A1 knockout (KO) cell lines have accelerated turnover of long-lived proteins labeled with (14)C-leucine in a pulse-chase assay as additional validation of our screening assays. Data from this comprehensive autophagy screen point toward novel regulatory pathways that might yield new therapeutic targets for neurodegeneration.
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Autofagia , Ensayos Analíticos de Alto Rendimiento/métodos , Imagenología Tridimensional , ARN Interferente Pequeño/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular , Técnicas de Silenciamiento del Gen , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Reproducibilidad de los ResultadosRESUMEN
Unbiased discovery approaches have the potential to uncover neurobiological insights into CNS disease and lead to the development of therapies. Here, we review lessons learned from imaging-based screening approaches and recent advances in these areas, including powerful new computational tools to synthesize complex data into more useful knowledge that can reliably guide future research and development.
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Bases de Datos Factuales , Células Madre Pluripotentes Inducidas/fisiología , Investigación , Animales , Enfermedades del Sistema Nervioso Central/terapia , Simulación por Computador , HumanosRESUMEN
Sphingosine kinases (SPHKs) are enzymes that phosphorylate the lipid sphingosine, leading to the formation of sphingosine-1-phosphate (S1P). In addition to the well established role of extracellular S1P as a mitogen and potent chemoattractant, SPHK activity has been postulated to be an important intracellular regulator of apoptosis. According to the proposed rheostat theory, SPHK activity shifts the intracellular balance from the pro-apoptotic sphingolipids ceramide and sphingosine to the mitogenic S1P, thereby determining the susceptibility of a cell to apoptotic stress. Despite numerous publications with supporting evidence, a clear experimental confirmation of the impact of this mechanism on tumor cell viability in vitro and in vivo has been hampered by the lack of suitable tool reagents. Utilizing a structure based design approach, we developed potent and specific SPHK1/2 inhibitors. These compounds completely inhibited intracellular S1P production in human cells and attenuated vascular permeability in mice, but did not lead to reduced tumor cell growth in vitro or in vivo. In addition, siRNA experiments targeting either SPHK1 or SPHK2 in a large panel of cell lines failed to demonstrate any statistically significant effects on cell viability. These results show that the SPHK rheostat does not play a major role in tumor cell viability, and that SPHKs might not be attractive targets for pharmacological intervention in the area of oncology.
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Neoplasias/enzimología , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Permeabilidad Capilar/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/farmacología , Femenino , Técnicas de Silenciamiento del Gen , Humanos , Concentración 50 Inhibidora , Ratones , Neoplasias/genética , Neoplasias/patología , Fosfotransferasas (Aceptor de Grupo Alcohol)/antagonistas & inhibidores , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Interferencia de ARN , Carga Tumoral/efectos de los fármacos , Ensayo de Tumor de Célula Madre , Factor A de Crecimiento Endotelial Vascular/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Advances in the fields of cancer initiating cells and high-throughput in vivo shRNA screens have highlighted a need to observe the growth of tumor cells in cancer models at the clonal level. While in vivo cancer cell growth heterogeneity in xenografts has been described, it has yet to be measured. Here, we tested an approach to quantify the clonal growth heterogeneity of cancer cells in subcutaneous xenograft mouse models. Using a high-throughput sequencing method, we followed the fate in vitro and in vivo of ten thousand HCT-116 cells individually tagged with a unique barcode delivered by lentiviral transduction. While growth in vitro was less homogeneous than anticipated, we still find that 95% of the final cells derived from 80% of the original cells. In xenografts, however, 95% of the retrieved barcoded cells originated from only 6% of the initially injected cells, an effect we term "clonal dominance". We observed this clonal dominance in two additional xenograft models (MDA-MB-468 and A2780(cis)) and in two different host strains (NSG and Nude). By precisely and reproducibly quantifying clonal cancer cell growth in vivo, we find that a small subset of clones accounts for the vast majority of the descendant cells, even with HCT-116, a cell line reported to lack a tumor-initiating compartment. The stochastic in vivo selection process we describe has important implications for the fields of in vivo shRNA screening and tumor initiating cells.
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Transformación Celular Neoplásica , Lentivirus/genética , Animales , Proliferación Celular , Células Clonales/patología , Femenino , Células HCT116 , Humanos , Ratones , ARN Interferente Pequeño/genética , Transducción GenéticaRESUMEN
Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors.
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Neoplasias/enzimología , Neoplasias/patología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas ras/genética , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Neoplasias/genética , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas p21(ras) , Interferencia de ARNRESUMEN
Screening small interfering RNA (siRNA) libraries holds the potential to elucidate gene function as well as discover new targets for the therapeutic treatment of disease. Since the inception of siRNA as a discovery tool, there have been progressive improvements in siRNA design algorithms, the transfection reagents used to deliver them, and the assay formats used to monitor phenotypic changes. These changes have helped to improve the quality of the data emerging from siRNA screens. One variable that introduces inconsistency into high-throughput screening (HTS) of siRNA libraries is the state of the cells used in the assays. Multiple factors can contribute to differences in transfection efficiency as well as the basic cell biology, which can lead to differences in the genes identified in siRNA screens. The authors have developed a system using frozen cell aliquots to use in siRNA HTS, so that a major source of variability introduced into cell-based screens can be standardized. In addition, by transiently transfecting plasmids into cell lines and then freezing these cells down to use in siRNA transfection experiments, they have used this same technology to create new cell lines. This process of using aliquots of frozen cells is logistically advantageous in an HTS setting, as it reduces the time spent maintaining cell lines, as well as reducing possible downtime in screening due to lack of cells or poor cell health.
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Criopreservación , Biblioteca Genómica , Ensayos Analíticos de Alto Rendimiento/métodos , ARN Interferente Pequeño/metabolismo , Animales , Línea Celular , Supervivencia Celular , Humanos , ARN Interferente Pequeño/genéticaRESUMEN
RNA interference (RNAi) mediated loss-of-function screens have the potential to delineate biological functions of genes and the proteins they encode. RNAi has proven to be a promising technology for identification and validation of new targets for the pharmacological treatment of many diseases including cancer. Here we review the use of high-throughput RNAi screens, examine the types of targets pursued for oncology indications, and discuss the integration of diverse datasets in both target discovery and drug discovery programs.
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Sistemas de Liberación de Medicamentos , Neoplasias/genética , Interferencia de ARN , Antineoplásicos/farmacología , Diseño de Fármacos , Silenciador del Gen , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/fisiopatologíaRESUMEN
High throughput technologies have the potential to affect all aspects of drug discovery. Considerable attention is paid to high throughput screening (HTS) for small molecule lead compounds. The identification of the targets that enter those HTS campaigns had been driven by basic research until the advent of genomics level data acquisition such as sequencing and gene expression microarrays. Large-scale profiling approaches (e.g., microarrays, protein analysis by mass spectrometry, and metabolite profiling) can yield vast quantities of data and important information. However, these approaches usually require painstaking in silico analysis and low-throughput basic wet-lab research to identify the function of a gene and validate the gene product as a potential therapeutic drug target. Functional genomic screening offers the promise of direct identification of genes involved in phenotypes of interest. In this review, RNA interference (RNAi) mediated loss-of-function screens will be discussed and as well as their utility in target identification. Some of the genes identified in these screens should produce similar phenotypes if their gene products are antagonized with drugs. With a carefully chosen phenotype, an understanding of the biology of RNAi and appreciation of the limitations of RNAi screening, there is great potential for the discovery of new drug targets.
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Evaluación Preclínica de Medicamentos/métodos , Interferencia de ARN , Bibliotecas de Moléculas Pequeñas , Animales , Vectores Genéticos/genética , Humanos , ARN Bicatenario/genética , Reproducibilidad de los Resultados , Análisis de Matrices Tisulares/métodos , TransfecciónRESUMEN
The contribution to genetic diversity of genomic segmental copy number variations (CNVs) is less well understood than that of single-nucleotide polymorphisms (SNPs). While less frequent than SNPs, CNVs have greater potential to affect phenotype. In this study, we have performed the most comprehensive survey to date of CNVs in mice, analyzing the genomes of 42 Mouse Phenome Consortium priority strains. This microarray comparative genomic hybridization (CGH)-based analysis has identified 2094 putative CNVs, with an average of 10 Mb of DNA in 51 CNVs when individual mouse strains were compared to the reference strain C57BL/6J. This amount of variation results in gene content that can differ by hundreds of genes between strains. These genes include members of large families such as the major histocompatibility and pheromone receptor genes, but there are also many singleton genes including genes with expected phenotypic consequences from their deletion or amplification. Using a whole-genome association analysis, we demonstrate that complex multigenic phenotypes, such as food intake, can be associated with specific copy number changes.