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Acute myeloid leukemia (AML) cells resist differentiation stimuli despite high expression of innate immune receptors, such as Toll-like receptor 9 (TLR9). We previously demonstrated that targeting Signal Transducer and Activator of Transcription 3 (STAT3) using TLR9-targeted decoy oligodeoxynucleotide (CpG-STAT3d) increases immunogenicity of human and mouse AML cells. Here, we elucidated molecular mechanisms of inv(16) AML reprogramming driven by STAT3-inhibition/TLR9-activation in vivo. At the transcriptional levels, AML cells isolated from mice after intravenous administration of CpG-STAT3d or leukemia-targeted Stat3 silencing and TLR9 co-stimulation, displayed similar upregulation of myeloid cell differentiation (Irf8, Cebpa, Itgam) and antigen-presentation (Ciita, Il12a, B2m)-related genes with concomitant reduction of leukemia-promoting Runx1. Single-cell transcriptomics revealed that CpG-STAT3d induced multilineage differentiation of AML cells into monocytes/macrophages, erythroblastic and B cell subsets. As shown by an inducible Irf8 silencing in vivo, IRF8 upregulation was critical for monocyte-macrophage differentiation of leukemic cells. TLR9-driven AML cell reprogramming was likely enabled by downregulation of STAT3-controlled methylation regulators, such as DNMT1 and DNMT3. In fact, the combination of DNA methyl transferase (DNMT) inhibition using azacitidine with CpG oligonucleotides alone mimicked CpG-STAT3d effects, resulting in AML cell differentiation, T cell activation, and systemic leukemia regression. These findings highlight immunotherapeutic potential of bi-functional oligonucleotides to unleash TLR9-driven differentiation of leukemic cells by concurrent STAT3 and/or DNMT inhibition.
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In the search for novel, effective inhibitors of High-Mobility Group Box1 (HMGB1)-a protein involved in various inflammatory and autoimmune diseases as well as in cancer-we herein discovered a set of anti-HMGB1 G-quadruplex(G4)-forming aptamers by using an in vitro selection procedure applied to a doped library of guanine-rich oligonucleotides. The selected DNA sequences were then studied in a pseudo-physiological buffer mimicking the extracellular medium, where HMGB1 exerts its pathological activity, using spectroscopic, electrophoretic, and chromatographic techniques. All the oligonucleotides proved to fold into monomeric G4s and in some cases also dimeric species, stable at physiological temperature. Remarkably, the protein preferentially recognized the sequences forming dimeric parallel G4 structures, as evidenced by a properly designed chemiluminescent binding assay which also highlighted a good selectivity of these aptamers for HMGB1. Moreover, all aptamers showed anti-HMGB1 activity, inhibiting protein-induced cell migration. The acquired data allowed identifying L12 as the best anti-HMGB1 aptamer, featured by high thermal and enzymatic stability, no toxicity at least up to 5â µM concentration on healthy cells, along with potent anti-HMGB1 activity (IC50 ca. 28â nM) and good binding affinity for the protein, thus indicating it as a very promising lead candidate for in vivo studies.
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Aptámeros de Nucleótidos , G-Cuádruplex , Proteína HMGB1 , Aptámeros de Nucleótidos/farmacología , Aptámeros de Nucleótidos/químicaRESUMEN
The identification of new effective therapeutic options for non-small-cell lung cancer (NSCLC) represents a crucial challenge in oncology. Recent studies indicate that cancer-associated fibroblasts (CAFs) participate in tumor progression by establishing a favorable microenvironment that promotes cancer progression. Therefore, the development of strategies inhibiting the interplay between CAFs and cancer cells is considered a winning approach for the development of effective anti-cancer drugs. Among other factors, the signal transducer and activator of transcription-3 (STAT3) has been reported as a key mediator of CAF oncogenic actions, representing a promising therapeutic target. Here, we applied an aptamer-based conjugate (named Gint4.T-STAT3), containing a STAT3 siRNA linked to an aptamer binding and inhibiting the platelet-derived growth factor receptor (PDGFR)ß, to obtain STAT3-specific silencing and interfere with CAF pro-tumorigenic functions. We demonstrated that this molecule effectively delivers the STAT3 siRNA in NSCLC cells, and blocks CAF-induced cancer cell growth and migration and reduced spheroid dimension. In addition, we found that Gint4.T-STAT3 alters CAF phenotype, thus functioning as a double-acting molecule able to inhibit the entire tumor bulk. Our data provide a proof of principle for the targeting of CAF pro-tumor functions through an aptamer-based drug, and can open innovative horizons in NSCLC therapy.
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Non-small-cell lung cancer (NSCLC) is the second most diagnosed type of malignancy and the first cause of cancer death worldwide. Despite recent advances, the treatment of choice for NSCLC patients remains to be chemotherapy, often showing very limited effectiveness with the frequent occurrence of drug-resistant phenotype and the lack of selectivity for tumor cells. Therefore, new effective and targeted therapeutics are needed. In this context, short RNA-based therapeutics, including Antisense Oligonucleotides (ASOs), microRNAs (miRNAs), short interfering (siRNA) and aptamers, represent a promising class of molecules. ASOs, miRNAs and siRNAs act by targeting and inhibiting specific mRNAs, thus showing an improved specificity compared to traditional anti-cancer drugs. Nucleic acid aptamers target and inhibit specific cancer-associated proteins, such as "nucleic acid antibodies". Aptamers are also able of receptor-mediated cell internalization, and therefore, they can be used as carriers of secondary agents giving the possibility of producing very highly specific and effective therapeutics. This review provides an overview of the proposed applications of small RNAs for NSCLC treatment, highlighting their advantageous features and recent advancements in the field.
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Aptámeros de Nucleótidos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , MicroARNs , Humanos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , ARN Interferente Pequeño/genética , Oligonucleótidos/uso terapéutico , Oligonucleótidos Antisentido , MicroARNs/genética , ARN Mensajero , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/uso terapéutico , Aptámeros de Nucleótidos/metabolismoRESUMEN
DNA methylation is a fundamental epigenetic modification regulating gene expression. Aberrant DNA methylation is the most common molecular lesion in cancer cells. However, medical intervention has been limited to the use of broadly acting, small molecule-based demethylating drugs with significant side-effects and toxicities. To allow for targeted DNA demethylation, we integrated two nucleic acid-based approaches: DNMT1 interacting RNA (DiR) and RNA aptamer strategy. By combining the RNA inherent capabilities of inhibiting DNMT1 with an aptamer platform, we generated a first-in-class DNMT1-targeted approach - aptaDiR. Molecular modelling of RNA-DNMT1 complexes coupled with biochemical and cellular assays enabled the identification and characterization of aptaDiR. This RNA bio-drug is able to block DNA methylation, impair cancer cell viability and inhibit tumour growth in vivo. Collectively, we present an innovative RNA-based approach to modulate DNMT1 activity in cancer or diseases characterized by aberrant DNA methylation and suggest the first alternative strategy to overcome the limitations of currently approved non-specific hypomethylating protocols, which will greatly improve clinical intervention on DNA methylation.
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Metilación de ADN , ARN , ARN/genética , ARN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Regulación Neoplásica de la Expresión Génica , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Epigénesis GenéticaRESUMEN
Hypoxia plays a crucial role in tumorigenesis and drug resistance, and it is recognised as a major factor affecting patient clinical outcome. Therefore, the detection of hypoxic areas within the tumour micro-environment represents a useful way to monitor tumour growth and patients' responses to treatments, properly guiding the choice of the most suitable therapy. To date, non-invasive hypoxia imaging probes have been identified, but their applicability in vivo is strongly limited due to an inadequate resistance to the low oxygen concentration and the acidic pH of the tumour micro-environment. In this regard, nucleic acid aptamers represent very powerful tools thanks to their peculiar features, including high stability to harsh conditions and a small size, resulting in easy and efficient tumour penetration. Here, we describe a modified cell-SELEX (Systematic Evolution of Ligands by EXponential enrichment) approach that allows the isolation of specific RNA aptamers for the detection of the hypoxic phenotype in breast cancer (BC) cells. We demonstrated the effectiveness of the proposed method in isolating highly stable aptamers with an improved and specific binding to hypoxic cells. To our knowledge, this is the first example of a cell-SELEX approach properly designed and modified to select RNA aptamers against hypoxia-related epitopes expressed on tumour cell surfaces. The selected aptamers may provide new effective tools for targeting hypoxic areas within the tumour with great clinical potential.
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The transmembrane glycoprotein cluster of differentiation 19 (CD19) is a B cell-specific surface marker, expressed on the majority of neoplastic B cells, and has recently emerged as a very attractive biomarker and therapeutic target for B-cell malignancies. The development of safe and effective ligands for CD19 has become an important need for the development of targeted conventional and immunotherapies. In this regard, aptamers represent a very interesting class of molecules. Additionally referred to as 'chemical antibodies', they show many advantages as therapeutics, including low toxicity and immunogenicity. Here, we isolated a nuclease-resistant RNA aptamer binding to the human CD19 glycoprotein. In order to develop an aptamer also useful as a carrier for secondary reagents, we adopted a cell-based SELEX (Systematic Evolution of Ligands by EXponential Enrichment) protocol adapted to isolate aptamers able to internalise upon binding to their cell surface target. We describe a 2'-fluoro pyrimidine modified aptamer, named B85.T2, which specifically binds to CD19 and shows an exquisite stability in human serum. The aptamer showed an estimated dissociation constant (KD) of 49.9 ± 13 nM on purified human recombinant CD19 (rhCD19) glycoprotein, a good binding activity on human B-cell chronic lymphocytic leukaemia cells expressing CD19, and also an effective and rapid cell internalisation, thus representing a promising molecule for CD19 targeting, as well as for the development of new B-cell malignancy-targeted therapies.
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Messenger RNAs (mRNAs) present a great potential as therapeutics for the treatment and prevention of a wide range of human pathologies, allowing for protein replacement, vaccination, cancer immunotherapy, and genomic engineering. Despite advances in the design of mRNA-based therapeutics, a key aspect for their widespread translation to clinic is the development of safe and effective delivery strategies. To this end, non-viral delivery systems including peptide-based complexes, lipidic or polymeric nanoparticles, and hybrid formulations are attracting growing interest. Despite displaying somewhat reduced efficacy compared to viral-based systems, non-viral carriers offer important advantages in terms of biosafety and versatility. In this review, we provide an overview of current mRNA therapeutic applications and discuss key biological barriers to delivery and recent advances in the development of non-viral systems. Challenges and future applications of this novel therapeutic modality are also discussed.
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ARN Mensajero/administración & dosificación , Animales , Sistemas de Liberación de Medicamentos , Técnicas de Transferencia de Gen , HumanosRESUMEN
4-Nonylphenol (4-NP) is an emerging environmental pollutant widely diffused in waters and sediments. It mainly derives from the degradation of alkyl phenol ethoxylates, compounds commonly employed as industrial surfactants. 4-NP strongly contaminates foods and waters for human use; thus, it displays a wide range of toxic effects not only for aquatic organisms but also for mammals and humans. After ingestion through the diet, it tends to accumulate in body fluids and tissues. One of the main organs where 4-NP and its metabolites are concentrated is the liver, where it causes, even at low doses, oxidative stress and apoptosis. In the present study, we analyzed the effects of 4-NP on a human hepatic cell line (HepG2) to deepen the knowledge of its cytotoxic mechanism. We found that 4-NP, in a range of concentration from 50 to 100 µM, significantly reduced cell viability; it caused a partial block of proliferation and induced apoptosis with activation of caspase-3 and overexpression of p53. Moreover, 4-NP induced-apoptosis seemed to involve both an ER-stress response, with the appearance of high level of GRP78, CHOP and the spliced XBP1, and a dysregulation of mitochondrial physiology, characterized by an overexpression of main markers of mitochondrial dynamics. Our data support the idea that a daily consumption of 4-NP-contaminated foods may lead to local damages at the level of gastrointestinal system, including liver, with negative consequences for the organ physiology.
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Apoptosis/efectos de los fármacos , Citotoxinas/toxicidad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Hígado/metabolismo , Mitocondrias Hepáticas/metabolismo , Fenoles/toxicidad , Chaperón BiP del Retículo Endoplásmico , Células Hep G2 , Humanos , Hígado/patología , Mitocondrias Hepáticas/patologíaRESUMEN
Despite the therapeutic utility of small interfering RNA (siRNA) molecules, the development of a safe and reliable method to selectively target diseased organs and tissues is still a critical need for their translation to the clinic. Here we describe how nucleic acid-based aptamers against cell surface epitopes may be used to address this issue. We discuss the most recent examples and advances in the field of aptamer siRNA delivery and provide a fast and simple protocol for the design and generation of aptamer-siRNA chimeras. The described approach is based on the annealing of the targeting aptamer, and the antisense strand through "stick" complementary sequences elongated at their 3' end, and the subsequent paring with the sense strand. Such a protocol allows a modular non-covalent generation of the constructs and permits an efficient delivery of the siRNA moiety into aptamer target cells.
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Aptámeros de Nucleótidos/genética , Técnicas de Transferencia de Gen , Interferencia de ARN , ARN Interferente Pequeño/genética , Factor de Transcripción STAT3/genética , Aptámeros de Nucleótidos/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , ARN Interferente Pequeño/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proyectos de Investigación , Factor de Transcripción STAT3/metabolismo , Flujo de TrabajoRESUMEN
Breast cancer is a leading cause of cancer mortality in women. Despite advances in its management, the identification of new options for early-stage diagnosis and therapy of this tumor still represents a crucial challenge. Increasing evidence indicates that extracellular vesicles called exosomes may have great potential as early diagnostic biomarkers and regulators of many cancers, including breast cancer. Therefore, exploiting molecules able to selectively recognize them is of great interest. Here, we developed a novel differential SELEX strategy, called Exo-SELEX, to isolate nucleic acid aptamers against intact exosomes derived from primary breast cancer cells. Among the obtained sequences, we optimized a high-affinity aptamer (ex-50.T) able to specifically recognize exosomes from breast cancer cells or patient serum samples. Furthermore, we demonstrated that the ex.50.T is a functional inhibitor of exosome cellular uptake and antagonizes cancer exosome-induced cell migration in vitro. This molecule provides an innovative tool for the specific exosome detection and the development of new therapeutic approaches for breast cancer.
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Non-small-cell lung cancer (NSCLC) is the most common type of lung cancer worldwide, with the highest incidence in developed countries. NSCLC patients often face resistance to currently available therapies, accounting for frequent relapses and poor prognosis. Indeed, despite great recent advancements in the field of NSCLC diagnosis and multimodal therapy, most patients are diagnosed at advanced metastatic stage, with a very low overall survival. Thus, the identification of new effective diagnostic and therapeutic options for NSCLC patients is a crucial challenge in oncology. A promising class of targeting molecules is represented by nucleic-acid aptamers, short single-stranded oligonucleotides that upon folding in particular three dimensional (3D) structures, serve as high affinity ligands towards disease-associated proteins. They are produced in vitro by SELEX (systematic evolution of ligands by exponential enrichment), a combinatorial chemistry procedure, representing an important tool for novel targetable biomarker discovery of both diagnostic and therapeutic interest. Aptamer-based approaches are promising options for NSCLC early diagnosis and targeted therapy and may overcome the key obstacles of currently used therapeutic modalities, such as the high toxicity and patients' resistance. In this review, we highlight the most important applications of SELEX technology and aptamers for NSCLC handling.
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Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Técnica SELEX de Producción de Aptámeros/métodos , Antineoplásicos/química , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/diagnóstico , Carcinoma de Pulmón de Células no Pequeñas/genética , Sistemas de Liberación de Medicamentos/métodos , Humanos , Neoplasias Pulmonares/diagnóstico , Neoplasias Pulmonares/genética , Nanoestructuras/química , ARN/químicaRESUMEN
An important drawback in the management of glioblastoma (GBM) patients is the frequent relapse upon surgery and therapy. A likely explanation is that conventional therapies poorly affect a small population of stem-like cancer cells (glioblastoma stem cells, GSCs) that remain capable of repopulating the tumour mass. Indeed, the development of therapeutic strategies able to hit GSCs while reducing the tumour burden has become an important challenge to increase a patient's survival. The signal transducer and activator of transcription-3 (STAT3) has been reported to play a pivotal role in maintaining the tumour initiating capacity of the GSC population. Therefore, in order to impair the renewal and propagation of the PDGFRß-expressing GSC population, here we took advantage of the aptamer-siRNA chimera (AsiC), named Gint4.T-STAT3, that we previously have shown to efficiently antagonize STAT3 in subcutaneous PDGFRß-positive GBM xenografts. We demonstrate that the aptamer conjugate is able to effectively and specifically prevent patient-derived GSC function and expansion. Moreover, because of the therapeutic potential of using miR-10b inhibitors and of the broad expression of the Axl receptor in GBM, we used the GL21.T anti-Axl aptamer as the targeting moiety for anti-miR-10b, showing that, in combination with the STAT3 AsiC, the aptamer-miR-10b antagonist treatment further enhances the inhibition of GSC sphere formation. Our results highlight the potential to use a combined approach with targeted RNA therapeutics to inhibit GBM tumour dissemination and relapse.
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Celiac disease (CD) is a common intestinal inflammatory disease involving both a genetic background and environmental triggers. The ingestion of gluten, a proteic component of several cereals, represents the main hexogen factor implied in CD onset that involves concomitant innate and adaptive immune responses to gluten. Immunogenicity of some gluten sequences are strongly enhanced as the consequence of the deamidation of specific glutamine residues by type 2 transglutaminase (TG2), a ubiquitous enzyme whose expression is up-regulated in the intestine of CD patients. A short gluten sequence resistant to intestinal proteases, the α-gliadin peptide 31-43, seems to modulate TG2 function in the gut; on the other hand, the enzyme can affect the biological activity of this peptide. In addition, an intense auto-immune response towards TG2 is a hallmark of CD. Auto-antibodies exert a range of biological effects on several cells, effects that in part overlap with those induced by peptide 31-43. In this review, we delineate a scenario in which TG2, anti-TG2 antibodies and peptide 31-43 closely relate to each other, thus synergistically participating in CD starting and progression.
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Autoanticuerpos/inmunología , Enfermedad Celíaca/inmunología , Proteínas de Unión al GTP/inmunología , Transglutaminasas/inmunología , Animales , Gliadina/inmunología , Humanos , Mucosa Intestinal/inmunología , Fragmentos de Péptidos/inmunología , Proteína Glutamina Gamma Glutamiltransferasa 2RESUMEN
Tumor mass consists of a complex ensemble of malignant cancer cells and a wide variety of resident and infiltrating cells, secreted factors, and extracellular matrix proteins that are referred as tumor microenvironment (TME). Cancer associated fibroblasts (CAFs) are key TME components that support tumor growth, generating a physical barrier against drugs and immune infiltration, and contributing to regulate malignant progression. Thus, it is largely accepted that therapeutic approaches aimed at hampering the interactions between tumor cells and CAFs can enhance the effectiveness of anti-cancer treatments. In this view, nucleic acid therapeutics have emerged as promising molecules. Here, we summarize recent knowledge about their role in the regulation of CAF transformation and tumor-promoting functions, highlighting their therapeutic utility and challenges.
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microRNAs (miRNAs) are small non-coding RNAs acting as negative regulators of gene expression and involved in tumor progression. We recently showed that inhibition of the pro-metastatic miR-214 and simultaneous overexpression of its downstream player, the anti-metastatic miR-148b, strongly reduced metastasis formation. To explore the therapeutic potential of miR-148b, we generated a conjugated molecule aimed to target miR-148b expression selectively to tumor cells. Precisely, we linked miR-148b to GL21.T, an aptamer able to specifically bind to AXL, an oncogenic tyrosine kinase receptor highly expressed on cancer cells. Axl-148b conjugate was able to inhibit migration and invasion of AXL-positive, but not AXL-negative, cancer cells, demonstrating high efficacy and selectivity in vitro. In parallel, expression of ALCAM and ITGA5, two miR-148b direct targets, was reduced. More importantly, axl-148b chimeric aptamers were able to inhibit formation and growth of 3D-mammospheres, to induce necrosis and apoptosis of treated xenotransplants, as well as to block breast cancer and melanoma dissemination and metastatization in mice. Relevantly, axl aptamer acted as specific delivery tool for miR-148b, but it also actively contributed to inhibit metastasis formation, together with miR-148b. In conclusion, our data show that axl-148b conjugate is able to inhibit tumor progression in an axl- and miR-148b-dependent manner, suggesting its potential development as therapeutic molecule.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/fisiopatología , Línea Celular Tumoral , Proliferación Celular/fisiología , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/metabolismo , Melanoma/fisiopatología , MicroARNs/metabolismo , Células Neoplásicas Circulantes , Células Tumorales Cultivadas/metabolismo , Células Tumorales Cultivadas/fisiologíaRESUMEN
Despite the benefits associated with radiotherapy and chemotherapy for glioblastoma (GBM) treatment, most patients experience a relapse following initial therapy. Recurrent or progressive GBM usually does not respond anymore to standard therapy, and this is associated with poor patient outcome. GBM stem cells (GSCs) are a subset of cells resistant to radiotherapy and chemotherapy and play a role in tumor recurrence. The targeting of GSCs and the identification of novel markers are crucial issues in the development of innovative strategies for GBM eradication. By differential cell SELEX (systematic evolution of ligands by exponential enrichment), we have recently described two RNA aptamers, that is, the 40L sequence and its truncated form A40s, able to bind the cell surface of human GSCs. Both aptamers were selective for stem-like growing GBM cells and are rapidly internalized into target cells. In this study, we demonstrate that their binding to cells is mediated by direct recognition of the ephrin type-A receptor 2 (EphA2). Functionally, the two aptamers were able to inhibit cell growth, stemness, and migration of GSCs. Furthermore, A40s was able to cross the blood-brain barrier (BBB) and was stable in serum in in vitro experiments. These results suggest that 40L and A40s represent innovative potential therapeutic tools for GBM.
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Type 2 transglutaminase (TG2) is a ubiquitous enzyme able to modify gliadin peptides introduced into the organism through the diet. By means of its catalytic activity, TG2 seems to have an important pathogenetic role in celiac disease (CD), an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals. A strong autoimmune response to TG2 characterizes CD development. Anti-TG2 antibodies specifically derange the uptake of the α-gliadin peptide 31-43 by control, but not by celiac dermal fibroblasts, underlying some different constitutive features regarding TG2 in healthy and celiac subjects. Our aim was to investigate whether these differences depended on a different TG2 subcellular distribution and whether peptide 31-43 differentially regulated TG2 expression and activity in cells of the two groups of subjects. We found that TG2 was more abundantly associated with membranes of celiac fibroblasts than of control cells, in particular with the early endosomal and autophagic compartments. We also found that peptide 31-43 differentially affected TG2 expression and activity in the two groups of cells, activating TG2 more in control than in celiac cells and inducing TG2 expression in celiac cells, but not in control ones. The different TG2 subcellular localization and the different way the peptide 31-43 modulates TG2 activity and availability into control and CD cells suggested that TG2 is involved in the definition of a constitutive CD cellular phenotype, thus having an important and still undefined role in CD pathogenesis.
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Enfermedad Celíaca/enzimología , Enfermedad Celíaca/metabolismo , Proteínas de Unión al GTP/metabolismo , Transglutaminasas/metabolismo , Adolescente , Adulto , Anticuerpos , Autoanticuerpos/inmunología , Enfermedad Celíaca/inmunología , Dieta Sin Gluten , Fibroblastos/metabolismo , Gliadina/inmunología , Voluntarios Sanos , Humanos , Péptidos , Proteína Glutamina Gamma Glutamiltransferasa 2 , Piel/metabolismo , Adulto JovenRESUMEN
B cell maturation antigen is highly expressed on malignant plasma cells in human multiple myeloma and has recently emerged as a very promising target for therapeutic interventions. Nucleic-acid-based aptamers are small oligonucleotides with high selective targeting properties and functional advantages over monoclonal antibodies, as both diagnostic and therapeutic tools. Here, we describe the generation of the first-ever-described nuclease resistant RNA aptamer selectively binding to B cell maturation antigen. We adopted a modified cell-based systematic evolution of ligands by exponential enrichment approach allowing the enrichment for internalizing aptamers. The selected 2'Fluoro-Pyrimidine modified aptamer, named apt69.T, effectively and selectively bound B cell maturation antigen-expressing myeloma cells with rapid and efficient internalization. Interestingly, apt69.T inhibited APRIL-dependent nuclear factor κB (NF-κB) pathway in vitro. Moreover, the aptamer was conjugated to microRNA-137 (miR-137) and anti-miR-222, demonstrating high potential against tumor cells. In conclusion, apt69.T is a novel tool suitable for direct targeting and delivery of therapeutics to B cell maturation antigen-expressing myeloma cells.
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Glioblastoma (GBM) is the most aggressive primary brain tumor in adults. Despite progress in surgical and medical neuro-oncology, prognosis for GBM patients remains dismal, with a median survival of only 14-15 months. The modest benefit of conventional therapies is due to the presence of GBM stem cells (GSCs) that cause tumor relapse and chemoresistance and, therefore, that play a key role in GBM aggressiveness and recurrence. So far, strategies to identify and target GSCs have been unsuccessful. Thus, the development of an approach for GSC detection and targeting would be fundamental for improving the survival of GBM patients. Here, using the cell-systematic evolution of ligand by exponential (SELEX) methodology on human primary GSCs, we generated and characterized RNA aptamers that selectively bind GSCs versus undifferentiated GBM cells. We found that the shortened version of the aptamer 40L, which we have called A40s, costained with CD133-labeled cells in human GBM tissue, suggestive of an ability to specifically recognize GSCs in fixed human tissues. Of note, both 40L and A40s were rapidly internalized by cells, allowing for the delivery of the microRNA miR-34c and the anti-microRNA anti-miR-10b, demonstrating that these aptamers can serve as selective vehicles for therapeutics. In conclusion, the aptamers 40L and A40s can selectively target GSCs. Given the crucial role of GSCs in GBM recurrence and therapy resistance, these aptamers represent innovative drug delivery candidates with a great potential in the treatment of GBM.