Diagnostic and Therapeutic Aptamers: A Promising Pathway to Improved Cardiovascular Disease Management.

Despite advances in care, cardiovascular diseases remain the leading cause of death worldwide. As a result, identifying suitable biomarkers for early diagnosis and improving therapeutic and diagnostic strategies is crucial. Because of their significant advantages over other therapeutic approaches, nucleic-based therapies, particularly aptamers, are gaining increased attention. Aptamers are innovative synthetic polymers or oligomers of single-stranded DNA (ssDNA) or RNA molecules that can form 3-dimensional structures and thus interact with their targets with high specificity and affinity. Furthermore, they outperform classical protein-based antibodies in terms of in vitro selection, production, ease of modification and conjugation, high stability, low immunogenicity, and suitability for nanoparticle functionalization for targeted drug delivery. This work aims to review the advances made in the aptamers' field in biomarker detection, diagnosis, imaging, and targeted therapy, which highlight their huge potential in the management of cardiovascular diseases.

Overcoming adaptive resistance to anti-VEGF therapy by targeting CD5L.

Antiangiogenic treatment targeting the vascular endothelial growth factor (VEGF) pathway is a powerful tool to combat tumor growth and progression; however, drug resistance frequently emerges. We identify CD5L (CD5 antigen-like precursor) as an important gene upregulated in response to antiangiogenic therapy leading to the emergence of adaptive resistance. By using both an RNA-aptamer and a monoclonal antibody targeting CD5L, we are able to abate the pro-angiogenic effects of CD5L overexpression in both in vitro and in vivo settings. In addition, we find that increased expression of vascular CD5L in cancer patients is associated with bevacizumab resistance and worse overall survival. These findings implicate CD5L as an important factor in adaptive resistance to antiangiogenic therapy and suggest that modalities to target CD5L have potentially important clinical utility.

Targeted systematic evolution of an RNA platform neutralizing DNMT1 function and controlling DNA methylation.

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.

Minding the gap: Unlockingthe therapeutic potentialof aptamers and making up for lost time.

Aptamers are RNAs that can bind proteins directly and modulate protein-protein interactions. Given their therapeutic potential, aptamers would be expected to capture the interest of both scientists and investors. However, concerns regarding safety, efficacy, and delivery have delayed aptamer development and dampened investor support. Herein, we discuss the major hurdles stalling the translational application of aptamers over recent years and focus on approaches to overcome current barriers and attract the scientific community and investors to the aptamer field.

Structure- and Interaction-Based Design of Anti-SARS-CoV-2 Aptamers.

Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. This study aims to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and interaction-based drug design (SIBDD), a highly specific aptamer to the receptor-binding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variants.

Selection of a Nuclease-Resistant RNA Aptamer Targeting CD19.

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.

Aptamers: Cutting edge of cancer therapies.

The development of an aptamer-based therapeutic has rapidly progressed following the first two reports in the 1990s, underscoring the advantages of aptamer drugs associated with their unique binding properties. In 2004, the US Food and Drug Administration (FDA) approved the first therapeutic aptamer for the treatment of neovascular age-related macular degeneration, Macugen developed by NeXstar. Since then, eleven aptamers have successfully entered clinical trials for various therapeutic indications. Despite some of the pre-clinical and clinical successes of aptamers as therapeutics, no aptamer has been approved by the FDA for the treatment of cancer. This review highlights the most recent and cutting-edge approaches in the development of aptamers for the treatment of cancer types most refractory to conventional therapies. Herein, we will review (1) the development of aptamers to enhance anti-cancer immunity and as delivery tools for inducing the expression of immunogenic neoantigens; (2) the development of the most promising therapeutic aptamers designed to target the hard-to-treat cancers such as brain tumors; and (3) the development of "carrier" aptamers able to target and penetrate tumors and metastasis, delivering RNA therapeutics to the cytosol and nucleus.

Identification of a novel RNA aptamer that selectively targets breast cancer exosomes.

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.

Combined Targeting of Glioblastoma Stem-Like Cells by Neutralizing RNA-Bio-Drugs for STAT3.

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.

Axl-148b chimeric aptamers inhibit breast cancer and melanoma progression.

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.

An Anti-BCMA RNA Aptamer for miRNA Intracellular Delivery.

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.

The Discovery of RNA Aptamers that Selectively Bind Glioblastoma Stem Cells.

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.

Aptamer Chimeras for Therapeutic Delivery: The Challenging Perspectives.

Nucleic acid-based aptamers have emerged as efficient delivery carriers of therapeutics. Thanks to their unique features, they can be, to date, considered one of the best targeting moieties, allowing the specific recognition of diseased cells and avoiding unwanted off-target effects on healthy tissues. In this review, we revise the most recent contributes on bispecific and multifunctional aptamer therapeutic chimeras. We will discuss key examples of aptamer-mediated delivery of nucleic acid and peptide-based therapeutics underlying their great potentiality and versatility. Achieved objectives and challenges will be highlighted as well.

Aptamer-miR-34c Conjugate Affects Cell Proliferation of Non-Small-Cell Lung Cancer Cells.

MicroRNAs (miRNAs) are key regulators of different human processes that represent a new promising class of cancer therapeutics or therapeutic targets. Indeed, in several tumor types, including non-small-cell lung carcinoma (NSCLC), the deregulated expression of specific miRNAs has been implicated in cell malignancy. As expression levels of the oncosuppressor miR-34c-3p are decreased in NSCLC compared to normal lung, we show that reintroduction of miR-34c-3p reduces NSCLC cell survival in vitro. Further, in order to deliver the miR-34c-based therapeutic selectively to tumor cells, we took advantage of a reported nucleic acid aptamer (GL21.T) that binds and inhibits the AXL transmembrane receptor and is rapidly internalized in the target cells. By applying methods successfully used in our laboratory, we conjugated miR-34c to the GL21.T aptamer as targeting moiety for the selective delivery to AXL-expressing NSCLC cells. We demonstrate that miR-34c-3p and the GL21.T/miR-34c chimera affect NSCLC cell proliferation and are able to overcome acquired RTK-inhibitor resistance by targeting AXL receptor. Thus, the GL21.T/miR-34c chimera exerts dual inhibition of AXL at functional and transcriptional levels and represents a novel therapeutic tool for the treatment of NSCLC.

Nucleic Acid Aptamers Targeting Epigenetic Regulators: An Innovative Therapeutic Option.

Epigenetic mechanisms include DNA methylation, posttranslational modifications of histones, chromatin remodeling factors, and post transcriptional gene regulation by noncoding RNAs. All together, these processes regulate gene expression by changing chromatin organization and DNA accessibility. Targeting enzymatic regulators responsible for DNA and chromatin modifications hold promise for modulating the transcriptional regulation of genes that are involved in cancer, as well as in chronic noncommunicable metabolic diseases like obesity, diabetes, and cardiovascular diseases. Increasingly studies are emerging, leading to the identification of specific and effective molecules targeting epigenetic pathways involved in disease onset. In this regard, RNA interference, which uses small RNAs to reduce gene expression and nucleic acid aptamers are arising as very promising candidates in therapeutic approach. Common to all these strategies is the imperative challenge of specificity. In this regard, nucleic acid aptamers have emerged as an attractive class of carrier molecules due to their ability to bind with high affinity to specific ligands, their high chemical flexibility as well as tissue penetration capability. In this review, we will focus on the recent progress in the field of aptamers used as targeting moieties able to recognize and revert epigenetics marks involved in diseases onset.

Nucleic acids delivering nucleic acids.

Nucleic acid therapeutics, including siRNAs, miRNAs/antimiRs, gRNAs and ASO, represent innovative and highly promising molecules for the safe treatment of a wide range of pathologies. The efficiency of systemic treatments is impeded by 1) the need to overcome physical and functional barriers in the organism, and 2) to accumulate in the intracellular active site at therapeutic concentrations. Although oligonucleotides either as modified naked molecules or complexed with delivery carriers have revealed to be effectively delivered to the affected target cells, this is restricted to topic treatments or to a few highly vascularized tissues. Therefore, the development of effective strategies for therapeutic nucleic acid selective delivery to target tissues is of primary importance in order to reduce the occurrence of undesired effects on non-target healthy tissues and to permit their translation to clinic. Due to their high affinity for specific ligands, high tissue penetration and chemical flexibility, short single-stranded nucleic acid aptamers are emerging as very attractive carriers for various therapeutic oligonucleotides. Yet, different aptamer-based bioconjugates, able to provide accumulation into target tissues, as well as efficient processing of therapeutic oligonucleotides, have been developed. In this respect, nucleic acid aptamer-mediated delivery strategies represent a powerful approach able to increase the therapeutic efficacy also highly reducing the overall toxicity. In this review, we will summarize recent progress in the field and discuss achieved objectives and optimization of aptamers as delivery carriers of short oligonucleotides.

STAT3 Gene Silencing by Aptamer-siRNA Chimera as Selective Therapeutic for Glioblastoma.

Glioblastoma (GBM) is the most frequent and aggressive primary brain tumor in adults, and despite advances in neuro-oncology, the prognosis for patients remains dismal. The signal transducer and activator of transcription-3 (STAT3) has been reported as a key regulator of the highly aggressive mesenchymal GBM subtype, and its direct silencing (by RNAi oligonucleotides) has revealed a great potential as an anti-cancer therapy. However, clinical use of oligonucleotide-based therapies is dependent on safer ways for tissue-specific targeting and increased membrane penetration. The objective of this study is to explore the use of nucleic acid aptamers as carriers to specifically drive a STAT3 siRNA to GBM cells in a receptor-dependent manner. Using an aptamer that binds to and antagonizes the oncogenic receptor tyrosine kinase PDGFRß (Gint4.T), here we describe the design of a novel aptamer-siRNA chimera (Gint4.T-STAT3) to target STAT3. We demonstrate the efficient delivery and silencing of STAT3 in PDGFRß+ GBM cells. Importantly, the conjugate reduces cell viability and migration in vitro and inhibits tumor growth and angiogenesis in vivo in a subcutaneous xenograft mouse model. Our data reveals Gint4.T-STAT3 conjugate as a novel molecule with great translational potential for GBM therapy.

Challenging cancer targets for aptamer delivery.

The extraordinary boost in the understanding of the genetic and epigenetic mechanisms underlying the development and progression of different types of cancer, is offering an unprecedented hope for the development of precise therapeutics able to interfere or replace the expression of target genes. In the last decade, the design of stable, safe and effective RNA-based therapeutics has been significantly improved increasing the number of molecules now in preclinical or in clinical trials for cancer gene therapy. However, with few exclusions as liver and hematological malignancies which are easy accessible to drugs, the development of effective systemic approaches for the delivery of RNA therapeutics to target cells is still unmet. To be effective, targeting carriers must be able to overcome both functional and physical barriers to safely carry and accumulate the therapeutic through the organism selectively to the tumor site, penetrate the target cancer mass, promote the uptake and localization in the appropriate intracellular compartment ultimately leading to the effective modulation of gene expression. Nucleic acid aptamers are folded single stranded oligonucleotides that bind at high affinity and high specificity their targets (proteins, lipids, small molecules etc), coupling the advantages of binding specificity proper of antibodies to the chemical nature of nucleic acids, sometimes also termed "nucleic acid antibodies". In several cases, aptamers targeting cell surface receptors are recycled into the cell together with the bound receptor enabling to drive conjugated therapeutics to cancer cells in a receptor-dependent manner. Therefore, besides other in vivo delivery strategies, the use of aptamers as precise and effective targeting moieties for anticancer RNA-based therapeutics has rapidly emerged and has been successfully addressed by several laboratories. In this Review, we will focus on the most recent and challenging progresses in the field that highlights the precision and flexibility of aptamer-based chimeras paving the way to the development of safe and effective carriers for cancer gene therapeutics.

Therapeutic Targeting of AXL Receptor Tyrosine Kinase Inhibits Tumor Growth and Intraperitoneal Metastasis in Ovarian Cancer Models.

Despite substantial improvements in the treatment strategies, ovarian cancer is still the most lethal gynecological malignancy. Identification of drug treatable therapeutic targets and their safe and effective targeting is critical to improve patient survival in ovarian cancer. AXL receptor tyrosine kinase (RTK) has been proposed to be an important therapeutic target for metastatic and advanced-stage human ovarian cancer. We found that AXL-RTK expression is associated with significantly shorter patient survival based on the The Cancer Genome Atlas patient database. To target AXL-RTK, we developed a chemically modified serum nuclease-stable AXL aptamer (AXL-APTAMER), and we evaluated its in vitro and in vivo antitumor activity using in vitro assays as well as two intraperitoneal animal models. AXL-aptamer treatment inhibited the phosphorylation and the activity of AXL, impaired the migration and invasion ability of ovarian cancer cells, and led to the inhibition of tumor growth and number of intraperitoneal metastatic nodules, which was associated with the inhibition of AXL activity and angiogenesis in tumors. When combined with paclitaxel, in vivo systemic (intravenous [i.v.]) administration of AXL-aptamer treatment markedly enhanced the antitumor efficacy of paclitaxel in mice. Taken together, our data indicate that AXL-aptamers successfully target in vivo AXL-RTK and inhibit its AXL activity and tumor growth and progression, representing a promising strategy for the treatment of ovarian cancer.

SERS-active metal-dielectric nanostructures integrated in microfluidic devices for label-free quantitative detection of miRNA.

In this work, SERS-based microfluidic PDMS chips integrating silver-coated porous silicon membranes were used for the detection and quantitation of microRNAs (miRNAs), which consist of short regulatory non-coding RNA sequences typically over- or under-expressed in connection with several diseases such as oncogenesis. In detail, metal-dielectric nanostructures which provide noticeable Raman enhancements were functionalized according to a biological protocol, adapted and optimized from an enzyme-linked immunosorbent assay (ELISA), for the detection of miR-222. Two sets of experiments based on different approaches were designed and performed, yielding a critical comparison. In the first one, the labelled target miRNA is revealed through hybridization to a complementary thiolated DNA probe, immobilized on the silver nanoparticles. In the second one, the probe is halved into shorter strands (half1 and half2) that interact with the complementary miRNA in two steps of hybridization. Such an approach, taking advantage of the Raman labelling of half2, provides a label-free analysis of the target. After suitable optimisation of the procedures, two calibration curves allowing quantitative measurements were obtained and compared on the basis of the SERS maps acquired on the samples loaded with several miRNA concentrations. The selectivity of the two-step assay was confirmed by the detection of target miR-222 mixed with different synthetic oligos, simulating the hybridization interference coming from similar sequences in real biological samples. Finally, that protocol was applied to the analysis of miR-222 in cellular extracts using an optofluidic multichamber biosensor, confirming the potentialities of SERS-based microfluidics for early-cancer diagnosis.