RESUMEN
Delivery of siRNA is a key hurdle to realizing the therapeutic promise of RNAi. By targeting internalizing cell surface antigens, antibody-siRNA complexes provide a possible solution. However, initial reports of antibody-siRNA complexes relied on non-specific charged interactions and have not been broadly applicable. To assess and improve this delivery method, we built on an industrial platform of therapeutic antibodies called THIOMABs, engineered to enable precise covalent coupling of siRNAs. We report that such coupling generates monomeric antibody-siRNA conjugates (ARCs) that retain antibody and siRNA activities. To broadly assess this technology, we generated a battery of THIOMABs against seven targets that use multiple internalization routes, enabling systematic manipulation of multiple parameters that impact delivery. We identify ARCs that induce targeted silencing in vitro and extend tests to target prostate carcinoma cells following systemic administration in mouse models. However, optimal silencing was restricted to specific conditions and only observed using a subset of ARCs. Trafficking studies point to ARC entrapment in endocytic compartments as a limiting factor, independent of the route of antigen internalization. Our broad characterization of multiple parameters using therapeutic-grade conjugate technology provides a thorough assessment of this delivery technology, highlighting both examples of success as well as remaining challenges.
Asunto(s)
Anticuerpos , ARN Interferente Pequeño/administración & dosificación , Animales , Anticuerpos/genética , Anticuerpos/inmunología , Anticuerpos/metabolismo , Línea Celular , Endosomas/metabolismo , Ratones , Neoplasias/genética , Ingeniería de Proteínas , Interferencia de ARN , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismoRESUMEN
RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3'-CH2-CO-NH-5' amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typical A-form duplex. The amide carbonyl group points into the major groove and assumes an orientation that is similar to the P-OP2 bond in the phosphate linkage. Amide linkages are well hydrated by tandem waters linking the carbonyl group and adjacent phosphate oxygens. Amides are tolerated at internal positions of both the guide and passenger strand of siRNAs and may increase the silencing activity when placed near the 5'-end of the passenger strand. As a result, an siRNA containing eight amide linkages is more active than the unmodified control. The results suggest that RNAi may tolerate even more extensive amide modification, which may be useful for optimization of siRNAs for in vivo applications.
Asunto(s)
Amidas/química , Interferencia de ARN , ARN Interferente Pequeño/química , Células HeLa , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , Fosfatos/química , ARN Interferente Pequeño/síntesis químicaRESUMEN
To provide a systematic analysis of genes that regulate epithelial cell migration, we performed a high throughput wound healing screen with MCF-10A breast epithelial cells, using siRNAs targeting 1,081 human genes encoding phosphatases, kinases and proteins predicted to influence cell migration and adhesion. The primary screen identified three categories of hits: those that accelerate, those that inhibit and those that impair migration with associated effects on cell proliferation or metabolism. Extensive validation of all the hits yielded 66 high confidence genes that, when downregulated, either accelerated or impaired migration; 42 of these high confidence genes have not been previously associated with motility or adhesion. Time-lapse video microscopy revealed a broad spectrum of phenotypic changes involving alterations in the extent and nature of disruption of cell-cell adhesion, directionality of motility, cell polarity and shape, and protrusion dynamics. Informatics analysis highlighted three major signalling nodes, beta-catenin, beta1-integrin and actin, and a large proportion of the genes that accelerated migration impaired cell-cell adhesion.
Asunto(s)
Movimiento Celular/genética , Células Epiteliales/citología , Células Epiteliales/metabolismo , Pruebas Genéticas , ARN Interferente Pequeño/genética , Actinas/genética , Actinas/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Redes Reguladoras de Genes , Humanos , Integrina beta1/genética , Integrina beta1/metabolismo , Fenotipo , Reproducibilidad de los Resultados , Transducción de Señal , Factores de Tiempo , Transfección , Cicatrización de Heridas , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
RNA sequences having up to three consecutive internal amide linkages were synthesized and studied using UV and NMR spectroscopy. The amide modifications did not interfere with normal base-pairing and A-type RNA conformation. Three consecutive amides were well tolerated in the passenger strand of siRNA and caused little change in RNAi activity.
Asunto(s)
Amidas/química , Interferencia de ARN , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Secuencia de Bases , Células HeLa , Humanos , Modelos Moleculares , Conformación de Ácido Nucleico , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/síntesis químicaRESUMEN
Influenza virus encodes only 11 viral proteins but replicates in a broad range of avian and mammalian species by exploiting host cell functions. Genome-wide RNA interference (RNAi) has proven to be a powerful tool for identifying the host molecules that participate in each step of virus replication. Meta-analysis of findings from genome-wide RNAi screens has shown influenza virus to be dependent on functional nodes in host cell pathways, requiring a wide variety of molecules and cellular proteins for replication. Because rapid evolution of the influenza A viruses persistently complicates the effectiveness of vaccines and therapeutics, a further understanding of the complex host cell pathways coopted by influenza virus for replication may provide new targets and strategies for antiviral therapy. RNAi genome screening technologies together with bioinformatics can provide the ability to rapidly identify specific host factors involved in resistance and susceptibility to influenza virus, allowing for novel disease intervention strategies.
Asunto(s)
Ensayos Analíticos de Alto Rendimiento/métodos , Virus de la Influenza A/genética , Gripe Humana/terapia , Interferencia de ARN , Proteínas Virales/genética , Animales , Humanos , Metaanálisis como Asunto , MicroARNs/genética , MicroARNs/metabolismo , FN-kappa B/metabolismo , Proteína Quinasa C/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/fisiologíaRESUMEN
Metastatic prostate cancer (PCa) is one of the leading causes of death from cancer in men. The molecular mechanisms underlying the transition from localized tumor to hormone-refractory metastatic PCa remain largely unknown, and their identification is key for predicting prognosis and targeted therapy. Here we demonstrated that increased expression of a splice variant of the Kruppel-like factor 6 (KLF6) tumor suppressor gene, known as KLF6-SV1, in tumors from men after prostatectomy predicted markedly poorer survival and disease recurrence profiles. Analysis of tumor samples revealed that KLF6-SV1 levels were specifically upregulated in hormone-refractory metastatic PCa. In 2 complementary mouse models of metastatic PCa, KLF6-SV1-overexpressing PCa cells were shown by in vivo and ex vivo bioluminescent imaging to metastasize more rapidly and to disseminate to lymph nodes, bone, and brain more often. Interestingly, while KLF6-SV1 overexpression increased metastasis, it did not affect localized tumor growth. KLF6-SV1 inhibition using RNAi induced spontaneous apoptosis in cultured PCa cell lines and suppressed tumor growth in mice. Together, these findings demonstrate that KLF6-SV1 expression levels in PCa tumors at the time of diagnosis can predict the metastatic behavior of the tumor; thus, KLF-SV1 may represent a novel therapeutic target.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/metabolismo , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas/metabolismo , Animales , Apoptosis/genética , Línea Celular Tumoral , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Regulación Neoplásica de la Expresión Génica/fisiología , Humanos , Inmunohistoquímica , Factor 6 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/antagonistas & inhibidores , Factores de Transcripción de Tipo Kruppel/genética , Metástasis Linfática , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones SCID , Metástasis de la Neoplasia , Procesos Neoplásicos , Prostatectomía , Neoplasias de la Próstata/cirugía , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Interferencia de ARN , Factores de Tiempo , Trasplante HeterólogoRESUMEN
The tumour microenvironment can be a potent carcinogen, not only by facilitating cancer progression and activating dormant cancer cells, but also by stimulating tumour formation. We have previously investigated stromelysin-1/matrix metalloproteinase-3 (MMP-3), a stromal enzyme upregulated in many breast tumours, and found that MMP-3 can cause epithelial-mesenchymal transition (EMT) and malignant transformation in cultured cells, and genomically unstable mammary carcinomas in transgenic mice. Here we explain the molecular pathways by which MMP-3 exerts these effects: exposure of mouse mammary epithelial cells to MMP-3 induces the expression of an alternatively spliced form of Rac1, which causes an increase in cellular reactive oxygen species (ROS). The ROS stimulate the expression of the transcription factor Snail and EMT, and cause oxidative damage to DNA and genomic instability. These findings identify a previously undescribed pathway in which a component of the breast tumour microenvironment alters cellular structure in culture and tissue structure in vivo, leading to malignant transformation.
Asunto(s)
Diferenciación Celular , Células Epiteliales/metabolismo , Inestabilidad Genómica/genética , Metaloproteinasa 3 de la Matriz/metabolismo , Mesodermo/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Proteína de Unión al GTP rac1/metabolismo , Empalme Alternativo/genética , Animales , Línea Celular , Transformación Celular Neoplásica , Daño del ADN , Células Epiteliales/citología , Células Epiteliales/enzimología , Humanos , Metaloproteinasa 3 de la Matriz/genética , Mesodermo/citología , Mesodermo/enzimología , Ratones , Ratones Transgénicos , Mitocondrias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteína de Unión al GTP rac1/genética , Proteínas de Unión al GTP rho/metabolismoRESUMEN
Despite rapid progress in the development of potent and selective small interfering RNA (siRNA) agents for skin disorders, translation to the clinic has been hampered by the lack of effective, patient-friendly delivery technologies. The stratum corneum poses a formidable barrier to efficient delivery of large and/or charged macromolecules including siRNAs. Intradermal siRNA injection results in effective knockdown of targeted gene expression but is painful and the effects are localized to the injection site. The use of microneedle arrays represents a less painful delivery method and may have utility for the delivery of nucleic acids, including siRNAs. For this purpose, we developed a loadable, dissolvable protrusion array device (PAD) that allows skin barrier penetration. The PAD tips dissolve upon insertion, forming a gel-like plug that releases functional cargo. PAD-mediated delivery of siRNA (modified for enhanced stability and cellular uptake) resulted in effective silencing of reporter gene expression in a transgenic reporter mouse model. PAD delivery of luciferase reporter plasmids resulted in expression in cells of the ear, back, and footpad skin as assayed by intravital bioluminescence imaging. These results support the use of PADs for delivery of functional nucleic acids to cells in the skin with an efficiency that may support clinical translation.
Asunto(s)
Portadores de Fármacos , Genes Reporteros/genética , Plásmidos/genética , ARN Interferente Pequeño/fisiología , Piel/metabolismo , Animales , Femenino , Pie , Silenciador del Gen/fisiología , Ratones , Microscopía Fluorescente , ARN Interferente Pequeño/genéticaRESUMEN
Pairing between the hexamer seed region of a small interfering RNA (siRNA) guide strand (nucleotides 2-7) and complementary sequences in the 3' UTR of mature transcripts has been implicated as an important element in off-target gene regulation and false positive phenotypes. To better understand the association between seed sequences and off-target profiles we performed an analysis of all possible (4096) hexamers and identified a nonuniform distribution of hexamer frequencies across the 3' UTR transcriptome. Subsequent microarray analysis of cells transfected with siRNAs having seeds with low, medium, or high seed complement frequencies (SCFs) revealed that duplexes with low SCFs generally induced fewer off-targets and off-target phenotypes than molecules with more abundant 3' UTR complements. These findings provide the first experimentally validated strategy for designing siRNAs with enhanced specificity and allow for more accurate interpretation of high throughput screening data generated with existing siRNA/shRNA collections.
Asunto(s)
ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Regiones no Traducidas 3' , Secuencia de Bases , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Células HeLa , Humanos , Internet , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Complejo Silenciador Inducido por ARN/metabolismo , TransfecciónRESUMEN
Hepatitis C virus (HCV) is a leading cause of liver cirrhosis and hepatocellular carcinoma worldwide. Currently available treatment options are of limited efficacy, and there is an urgent need for development of alternative therapies. RNA interference (RNAi) is a natural mechanism by which small interfering RNA (siRNA) or short hairpin RNA (shRNA) can mediate degradation of a target RNA molecule in a sequence-specific manner. In this study, we screened in vitro-transcribed 25-bp shRNAs targeting the internal ribosome entry site (IRES) of HCV for the ability to inhibit IRES-driven gene expression in cultured cells. We identified a 44-nt region at the 3'-end of the IRES within which all shRNAs efficiently inhibited expression of an IRES-linked reporter gene. Subsequent scans within this region with 19-bp shRNAs identified even more potent molecules, providing effective inhibition at concentrations of 0.1 nM. Experiments varying features of the shRNA design showed that, for 25-bp shRNAs, neither the size of the loop (4-10 nt) nor the sequence or pairing status of the ends affects activity, whereas in the case of 19-bp shRNAs, larger loops and the presence of a 3'-UU overhang increase efficacy. A comparison of shRNAs and siRNAs targeting the same sequence revealed that shRNAs were of comparable or greater potency than the corresponding siRNAs. Anti-HCV activity was confirmed with HCV subgenomic replicons in a human hepatocyte line. The results indicate that shRNAs, which can be prepared by either transcription or chemical synthesis, may be effective agents for the control of HCV.
Asunto(s)
Hepacivirus/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Regiones no Traducidas 5' , Secuencia de Bases , Línea Celular , Expresión Génica , Vectores Genéticos , Hepacivirus/metabolismo , Humanos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , ARN Viral/genética , TransfecciónRESUMEN
Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi). RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi. To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3'-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.
Asunto(s)
Ingeniería Genética/métodos , Interferencia de ARN , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , Alineación de Secuencia/métodos , Análisis de Secuencia de ARN , Animales , Secuencia de Bases , Ciclofilinas/genética , Ciclofilinas/metabolismo , Dípteros , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Datos de Secuencia Molecular , Isomerasa de Peptidilprolil , ARN Interferente Pequeño/análisisRESUMEN
The ability of small hairpin RNAs (shRNAs) to inhibit hepatitis C virus internal ribosome entry site (HCV IRES)-dependent gene expression was investigated in cultured cells and a mouse model. The results indicate that shRNAs, delivered as naked RNA or expressed from vectors, may be effective agents for the control of HCV and related viruses.
Asunto(s)
Antivirales , Hepatitis C/terapia , ARN Interferente Pequeño/farmacología , Animales , Células Cultivadas , Expresión Génica , Terapia Genética , Hepatitis C/genética , Hepatocitos , Humanos , Ratones , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/uso terapéuticoRESUMEN
Many applications in molecular biology can benefit from improved PCR amplification of DNA segments containing a wide range of GC content. Conventional PCR amplification of DNA sequences with regions of GC less than 30%, or higher than 70%, is complex due to secondary structures that block the DNA polymerase as well as mispriming and mis-annealing of the DNA. This complexity will often generate incomplete or nonspecific products that hamper downstream applications. In this study, we address multiplexed PCR amplification of DNA segments containing a wide range of GC content. In order to mitigate amplification complications due to high or low GC regions, we tested a combination of different PCR cycling conditions and chemical additives. To assess the fate of specific oligonucleotide (oligo) species with varying GC content in a multiplexed PCR, we developed a novel method of sequence analysis. Here we show that subcycling during the amplification process significantly improved amplification of short template pools (~200 bp), particularly when the template contained a low percent of GC. Furthermore, the combination of subcycling and 7-deaza-dGTP achieved efficient amplification of short templates ranging from 10-90% GC composition. Moreover, we found that 7-deaza-dGTP improved the amplification of longer products (~1000 bp). These methods provide an updated approach for PCR amplification of DNA segments containing a broad range of GC content.
Asunto(s)
Composición de Base , Islas de CpG , ADN/química , ADN/síntesis química , Guanina/análogos & derivados , Reacción en Cadena de la Polimerasa/métodos , Guanina/químicaRESUMEN
The fields of antibody engineering, enzyme optimization and pathway construction rely increasingly on screening complex variant DNA libraries. These highly diverse libraries allow researchers to sample a maximized sequence space; and therefore, more rapidly identify proteins with significantly improved activity. The current state of the art in synthetic biology allows for libraries with billions of variants, pushing the limits of researchers' ability to qualify libraries for screening by measuring the traditional quality metrics of fidelity and diversity of variants. Instead, when screening variant libraries, researchers typically use a generic, and often insufficient, oversampling rate based on a common rule-of-thumb. We have developed methods to calculate a library-specific oversampling metric, based on fidelity, diversity, and representation of variants, which informs researchers, prior to screening the library, of the amount of oversampling required to ensure that the desired fraction of variant molecules will be sampled. To derive this oversampling metric, we developed a novel alignment tool to efficiently measure frequency counts of individual nucleotide variant positions using next-generation sequencing data. Next, we apply a method based on the "coupon collector" probability theory to construct a curve of upper bound estimates of the sampling size required for any desired variant coverage. The calculated oversampling metric will guide researchers to maximize their efficiency in using highly variant libraries.
Asunto(s)
Biblioteca de Genes , Variación Genética , Variación Genética/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Modelos Teóricos , Probabilidad , Alineación de SecuenciaRESUMEN
RNA interference is widely recognized for its utility as a functional genomics tool. In the absence of reliable target site selection tools, however, the impact of RNA interference (RNAi) may be diminished. The primary determinants of silencing are influenced by highly coordinated RNA-protein interactions that occur throughout the RNAi process, including short interfering RNA (siRNA) binding and unwinding followed by target recognition, cleavage, and subsequent product release. Recently developed strategies for identification of functional siRNAs reveal that thermodynamic and siRNA sequence-specific properties are crucial to predict functional duplexes (Khvorova et al., 2003; Reynolds et al., 2004; Schwarz et al., 2003). Additional assessments of siRNA specificity reveal that more sophisticated sequence comparison tools are also required to minimize potential off-target effects (Jackson et al., 2003; Semizarov et al., 2003). This chapter reviews the biological basis for current computational design tools and how best to utilize and assess their predictive capabilities for selecting functional and specific siRNAs.
Asunto(s)
Interferencia de ARN , Algoritmos , Animales , Secuencia de Bases , Línea Celular , Humanos , MicroARNs/genética , Datos de Secuencia Molecular , TermodinámicaRESUMEN
As scientists make strides toward the goal of developing a form of biological engineering that's as predictive and reliable as chemical engineering is for chemistry, one technology component has become absolutely critical: gene synthesis. Gene synthesis is the process of building stretches of deoxyribonucleic acid (DNA) to order--some stretches based on DNA that exists already in nature, some based on novel designs intended to accomplish new functions. This process is the foundation of synthetic biology, which is rapidly becoming the engineering counterpart to biology.
Asunto(s)
Bioingeniería/métodos , Biología Sintética/métodos , BiocombustiblesRESUMEN
Screening of RNA interference (RNAi) libraries in primary T cells is labor-intensive and technically challenging because these cells are hard to transfect. Chemically modified, self-delivering small interfering RNAs (siRNAs) offer a solution to this problem, because they enter hard-to-transfect cell types without needing a delivery reagent and are available in library format for RNAi screening. In this study, we have screened a library of chemically modified, self-delivering siRNAs targeting the expression of 72 distinct genes in conjunction with an image-based high-content-analysis platform as a proof-of-principle strategy to identify genes involved in lymphocyte function-associated antigen-1 (LFA-1)-mediated migration in primary human T cells. Our library-screening strategy identified the small GTPase RhoA as being crucial for T cell polarization and migration in response to LFA-1 stimulation and other migratory ligands. We also demonstrate that multiple downstream assays can be performed within an individual RNAi screen and have used the remainder of the cells for additional assays, including cell viability and adhesion to ICAM-1 (the physiological ligand for LFA-1) in the absence or presence of the chemokine SDF-1α. This study therefore demonstrates the ease and benefits of conducting siRNA library screens in primary human T cells using self-delivering, chemically modified siRNAs, and it emphasizes the feasibility and potential of this approach for elucidating the signaling pathways that regulate T cell function.
Asunto(s)
Movimiento Celular/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Linfocitos T/fisiología , Expresión Génica , Biblioteca de Genes , Silenciador del Gen , Estudios de Asociación Genética , Humanos , Antígeno-1 Asociado a Función de Linfocito/genética , Cultivo Primario de Células , Reproducibilidad de los Resultados , Transfección , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Despite the development of potent siRNAs that effectively target genes responsible for skin disorders, translation to the clinic has been hampered by inefficient delivery through the stratum corneum barrier and into the live cells of the epidermis. Although hypodermic needles can be used to transport siRNA through the stratum corneum, this approach is limited by pain caused by the injection and the small volume of tissue that can be accessed by each injection. The use of microneedle arrays is a less painful method for siRNA delivery, but restricted payload capacity limits this approach to highly potent molecules. To address these challenges, a commercially available motorized microneedle array skin delivery device was evaluated. This device combines the positive elements of both hypodermic needles and microneedle array technologies with little or no pain to the patient. Application of fluorescently tagged self-delivery (sd)-siRNA to both human and murine skin resulted in distribution throughout the treated skin. In addition, efficient silencing (78% average reduction) of reporter gene expression was achieved in a transgenic fluorescent reporter mouse skin model. These results indicate that this device effectively delivers functional sd-siRNA with an efficiency that predicts successful clinical translation.Molecular Therapy-Nucleic Acids (2013) 2, e129; doi:10.1038/mtna.2013.56; published online 22 October 2013.
RESUMEN
Treatment of skin disorders with short interfering RNA (siRNA)-based therapeutics requires the development of effective delivery methodologies that reach target cells in affected tissues. Successful delivery of functional siRNA to the epidermis requires (1) crossing the stratum corneum, (2) transfer across the keratinocyte membrane, followed by (3) incorporation into the RNA-induced silencing complex. We have previously demonstrated that treatment with microneedle arrays loaded with self-delivery siRNA (sd-siRNA) can achieve inhibition of reporter gene expression in a transgenic mouse model. Furthermore, treatment of human cultured epidermal equivalents with sd-siRNA resulted in inhibition of target gene expression. Here, we demonstrate inhibition of CD44, a gene that is uniformly expressed throughout the epidermis, by sd-siRNA both in vitro (cultured human epidermal skin equivalents) and in vivo (full-thickness human skin equivalents xenografted on immunocompromised mice). Treatment of human skin equivalents with CD44 sd-siRNA markedly decreased CD44 mRNA levels, which led to a reduction of the target protein as confirmed by immunodetection in epidermal equivalent sections with a CD44-specific antibody. Taken together, these results demonstrate that sd-siRNA, delivered by microneedle arrays, can reduce expression of a targeted endogenous gene in a human skin xenograft model.
Asunto(s)
Receptores de Hialuranos/metabolismo , ARN Interferente Pequeño/administración & dosificación , Piel/metabolismo , Animales , Células Cultivadas , Femenino , Expresión Génica , Genes Reporteros , Humanos , Receptores de Hialuranos/genética , Inmunohistoquímica , Queratinocitos/metabolismo , Ratones , Ratones SCID , Agujas , Polimetil Metacrilato , Alcohol Polivinílico , Solubilidad , Trasplante HeterólogoRESUMEN
RNAi screening using pooled shRNA libraries is a valuable tool for identifying genetic regulators of biological processes. However, for a successful pooled shRNA screen, it is imperative to thoroughly optimize experimental conditions to obtain reproducible data. Here we performed viability screens with a library of â¼10,000 shRNAs at two different fold representations (100- and 500-fold at transduction) and report the reproducibility of shRNA abundance changes between screening replicates determined by microarray and next generation sequencing analyses. We show that the technical reproducibility between PCR replicates from a pooled screen can be drastically improved by ensuring that PCR amplification steps are kept within the exponential phase and by using an amount of genomic DNA input in the reaction that maintains the average template copies per shRNA used during library transduction. Using these optimized PCR conditions, we then show that higher reproducibility of biological replicates is obtained by both microarray and next generation sequencing when screening with higher average shRNA fold representation. shRNAs that change abundance reproducibly in biological replicates (primary hits) are identified from screens performed with both 100- and 500-fold shRNA representation, however a higher percentage of primary hit overlap between screening replicates is obtained from 500-fold shRNA representation screens. While strong hits with larger changes in relative abundance were generally identified in both screens, hits with smaller changes were identified only in the screens performed with the higher shRNA fold representation at transduction.