Aptamer-programmable adeno-associated viral vectors as a novel platform for cell-specific gene transfer.

Adeno-associated viruses (AAVs) are commonly used for in vivo gene therapy. Nevertheless, the wide tropism that characterizes these vectors limits specific targeting to a particular cell type or tissue. Here, we developed new chemically modified AAV vectors (Nε-AAVs) displaying a single site substitution on the capsid surface for post-production vector engineering through biorthogonal copper-free click chemistry. We were able to identify AAV vectors that would tolerate the unnatural amino acid substitution on the capsid without disrupting their packaging efficiency. We functionalized the Nε-AAVs through conjugation with DNA (AS1411) or RNA (E3) aptamers or with a folic acid moiety (FA). E3-, AS1411-, and FA-AAVs showed on average a 3- to 9-fold increase in transduction compared with their non-conjugated counterparts in different cancer cell lines. Using specific competitors, we established ligand-specific transduction. In vivo studies confirmed the selective uptake of FA-AAV and AS1411-AAV without off-target transduction in peripheral organs. Overall, the high versatility of these novel Nε-AAVs might pave the way to tailoring gene therapy vectors toward specific types of cells both for ex vivo and in vivo applications.

Mixed-surface polyamidoamine polymer variants retain nucleic acid-scavenger ability with reduced toxicity.

Nucleic acid-binding polymers can have anti-inflammatory properties and beneficial effects in animal models of infection, trauma, cancer, and autoimmunity. PAMAM G3, a polyamidoamine dendrimer, is fully cationic bearing 32 protonable surface amines. However, while PAMAM G3 treatment leads to improved outcomes for mice infected with influenza, at risk of cancer metastasis, or genetically prone to lupus, its administration can lead to serosal inflammation and elevation of biomarkers of liver and kidney damage. Variants with reduced density of cationic charge through the interspersal of hydroxyl groups were evaluated as potentially better-tolerated alternatives. Notably, the variant PAMAM G3 50:50, similar in size as PAMAM G3 but with half the charge, was not toxic in cell culture, less associated with weight loss or serosal inflammation after parenteral administration, and remained effective in reducing glomerulonephritis in lupus-prone mice. Identification of such modified scavengers should facilitate their development as safe and effective anti-inflammatory agents.

Aptamer-based factor IXa inhibition preserves hemostasis and prevents thrombosis in a piglet model of ECMO.

Extracorporeal membrane oxygenation (ECMO) requires anticoagulation to prevent clotting when the patient's blood contacts the circuit. Unfractionated heparin (UFH) usually prevents clotting but can cause life-threatening bleeding. An anticoagulant that selectively inhibits the contact activation (intrinsic) pathway while sparing the tissue factor (extrinsic) pathway of coagulation might prevent clotting triggered by the circuit while permitting physiologic coagulation at surgical sites. DTRI-178 is an RNA anticoagulant aptamer conjugated to polyethylene glycol that increases its half-life in circulation. This aptamer is based on a previously described molecule (9.3t) that inhibits intrinsic tenase activity by binding to factor IXa on an exosite. Using a piglet model of pediatric venoarterial (VA) ECMO, we compared thromboprevention and blood loss using a single dose of DTRI-178 versus UFH. In each of five experiments, we subjected two litter-matched piglets, one anticoagulated with DTRI-178 and the other with UFH, to simultaneous 12-h periods of VA ECMO. Both anticoagulants achieved satisfactory and comparable thromboprotection. However, UFH piglets had increased surgical site bleeding and required significantly greater blood transfusion volumes than piglets anticoagulated with DTRI-178. Our results indicate that DTRI-178, an aptamer against factor IXa, may be feasible, safer, and result in fewer transfusions and clinical bleeding events in ECMO.

Breast cancer-derived DAMPs enhance cell invasion and metastasis, while nucleic acid scavengers mitigate these effects.

Breast cancer (BC) is the most common malignancy in women. Particular subtypes with aggressive behavior are major contributors to poor outcomes. Triple-negative breast cancer (TNBC) is difficult to treat, pro-inflammatory, and highly metastatic. We demonstrate that TNBC cells express TLR9 and are responsive to TLR9 ligands, and treatment of TNBC cells with chemotherapy increases the release of nucleic-acid-containing damage-associated molecular patterns (NA DAMPs) in cell culture. Such culture-derived and breast cancer patient-derived NA DAMPs increase TLR9 activation and TNBC cell invasion in vitro. Notably, treatment with the polyamidoamine dendrimer generation 3.0 (PAMAM-G3) behaved as a nucleic acid scavenger (NAS) and significantly mitigates such effects. In mice that develop spontaneous BC induced by polyoma middle T oncoprotein (MMTV-PyMT), treatment with PAMAM-G3 significantly reduces lung metastasis. Thus, NAS treatment mitigates cancer-induced inflammation and metastasis and represents a novel therapeutic approach for combating breast cancer.

Key Pathogenic Factors in Coronavirus Disease 2019-Associated Coagulopathy and Acute Lung Injury Highlighted in a Patient With Copresentation of Acute Myelocytic Leukemia: A Case Report.

The role of concurrent illness in coronavirus disease 2019 (COVID-19) is unknown. Patients with leukemia may display altered thromboinflammatory responses. We report a 53-year-old man presenting with acute leukemia and COVID-19 who developed thrombotic complications and acute respiratory distress syndrome. Multiple analyses, including rotational thromboelastometry and flow cytometry on blood and bronchoalveolar lavage, are reported to characterize coagulation and immune profiles. The patient developed chemotherapy-induced neutropenia that may have protected his lungs from granulocyte-driven hyperinflammatory acute lung injury. However, neutropenia also alters viral clearing, potentially enabling ongoing viral propagation. This case depicts a precarious equilibrium between leukemia and COVID-19.

Ischemic stroke in COVID-19-positive patients: an overview of SARS-CoV-2 and thrombotic mechanisms for the neurointerventionalist.

Coronavirus disease 2019 (COVID-19) results from infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It was first reported in Wuhan, China in patients suffering from severe pneumonia and acute respiratory distress syndrome and has now grown into the first pandemic in over 100 years. Patients infected with SARS-CoV-2 develop arterial thrombosis including stroke, myocardial infarction and peripheral arterial thrombosis, all of which result in poor outcomes despite maximal medical, endovascular, and microsurgical treatment compared with non-COVID-19-infected patients. In this review we provide a brief overview of SARS-CoV-2, the infectious agent responsible for the COVID-19 pandemic, and describe the mechanisms responsible for COVID-19-associated coagulopathy. Finally, we discuss the impact of COVID-19 on ischemic stroke, focusing on large vessel occlusion.

An Aptamer for Broad Cancer Targeting and Therapy.

Recent advances in chemotherapy treatments are increasingly targeted therapies, with the drug conjugated to an antibody able to deliver it directly to the tumor. As high-affinity chemical ligands that are much smaller in size, aptamers are ideal for this type of drug targeting. Aptamer-highly toxic drug conjugates (ApTDCs) based on the E3 aptamer, selected on prostate cancer cells, target and inhibit prostate tumor growth in vivo. Here, we observe that E3 also broadly targets numerous other cancer types, apparently representing a universal aptamer for cancer targeting. Accordingly, ApTDCs formed by conjugation of E3 to the drugs monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF) efficiently target and kill a range of different cancer cells. Notably, this targeting extends to both patient-derived explant (PDX) cancer cell lines and tumors, with the E3 MMAE and MMAF conjugates inhibiting PDX cell growth in vitro and with the E3 aptamer targeting PDX colorectal tumors in vivo.

RGEN Editing of RNA and DNA: The Long and Winding Road from Catalytic RNAs to CRISPR to the Clinic.

The first clinical studies utilizing RNA-guided endonucleases (RGENs) to therapeutically edit RNA and DNA in cancer patients were recently published. These groundbreaking technological advances promise to revolutionize genetic therapy and, as I discuss, represent the culmination of decades of innovative work to engineer RGENs for such editing applications.

Aptamers as Reversible Sorting Ligands for Preparation of Cells in Their Native State.

Although antibodies are routinely used to label and isolate a desired cell type from a more complex mixture of cells, via either fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS), such antibody labeling is not easily reversible. We describe an FACS and MACS compatible method to reversibly label and purify cells using aptamers. Magnetic beads loaded with the epidermal growth factor receptor (EGFR)-binding antagonistic aptamer E07 specifically isolated EGFR-expressing cells, and pure, label-free cells were recovered via treatment with an "antidote" oligonucleotide complementary to the aptamer. Additionally, while FACS sorting cells with E07 or EGFR antibody yielded EGFR(+) cells with impeded EGFR signaling, stripping off the aptamer via antidote treatment restored receptor function, returning cells to their native state, which was not possible with the antibody. The ability to reversibly label or isolate cells without compromising their function is a valuable, versatile tool with important implications for both the laboratory and clinic.

Toll-like receptor activation as a biomarker in traumatically injured patients.

BACKGROUND: Surgical insult and trauma have been shown to cause dysregulation of the immune and inflammatory responses. Interaction of damage-associated molecular patterns (DAMPs) with toll-like receptors (TLRs) initiates innate immune response and systemic inflammatory responses. Given that surgical patients produce high levels of circulating damage-associated molecular patterns, we hypothesized that plasma-activated TLR activity would be correlated to injury status and could be used to predict pathological conditions involving tissue injury. METHODS: An observational study was performed using samples from a single-institution prospective tissue and data repository from a Level-1 trauma center. In vitro TLR 2, 3, 4, and 9 activation was determined in a TLR reporter assay after isolation of plasma from peripheral blood. We determined correlations between plasma-activated TLR activity and clinical course measures of severity. RESULTS: Eighteen patients were enrolled (median Injury Severity Score 15 [interquartile range 10, 23.5]). Trauma resulted in significant elevation in circulation high mobility group box 1 as well as increase of plasma-activated TLR activation (2.8-5.4-fold) compared to healthy controls. There was no correlation between circulating high mobility group box 1 and trauma morbidity; however, the plasma-activated TLR activity was correlated with acute physiology and chronic health evaluation II scores (R square = 0.24-0.38, P < 0.05). Patients who received blood products demonstrated significant increases in the levels of plasma-activated TLRs 2, 3, 4, and 9 and had a trend toward developing systemic inflammatory response syndrome. CONCLUSIONS: Further studies examining TLR modulation and signaling in surgical patients may assist in predictive risk modeling and reduction in morbidity and mortality.

Combination of aptamer and drug for reversible anticoagulation in cardiopulmonary bypass.

Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with heparin-induced thrombocytopenia-associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, we solved X-ray crystal structures of the aptamer bound to factor Xa (FXa). The finding that 11F7t did not bind the catalytic site suggested that it could complement small-molecule FXa inhibitors. We demonstrate that combinations of 11F7t and catalytic-site FXa inhibitors enhance anticoagulation in purified reaction mixtures and plasma. Aptamer-drug combinations prevented clot formation as effectively as UFH in human blood circulated in an extracorporeal oxygenator circuit that mimicked CPB, while avoiding side effects of UFH. An antidote could promptly neutralize the anticoagulant effects of both FXa inhibitors. Our results suggest that drugs and aptamers with shared targets can be combined to exert more specific and potent effects than either agent alone.

Tunable cytotoxic aptamer-drug conjugates for the treatment of prostate cancer.

Therapies that can eliminate both local and metastatic prostate tumor lesions while sparing normal organ tissue are desperately needed. With the goal of developing an improved drug-targeting strategy, we turned to a new class of targeted anticancer therapeutics: aptamers conjugated to highly toxic chemotherapeutics. Cell selection for aptamers with prostate cancer specificity yielded the E3 aptamer, which internalizes into prostate cancer cells without targeting normal prostate cells. Chemical conjugation of E3 to the drugs monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF) yields a potent cytotoxic agent that efficiently kills prostate cancer cells in vitro but does not affect normal prostate epithelial cells. Importantly, the E3 aptamer targets tumors in vivo and treatment with the MMAF-E3 conjugate significantly inhibits prostate cancer growth in mice, demonstrating the in vivo utility of aptamer-drug conjugates. Additionally, we report the use of antidotes to block E3 aptamer-drug conjugate cytotoxicity, providing a safety switch in the unexpected event of normal cell killing in vivo.

Polymer-Mediated Inhibition of Pro-invasive Nucleic Acid DAMPs and Microvesicles Limits Pancreatic Cancer Metastasis.

Nucleic acid binding polymers (NABPs) have been extensively used as vehicles for DNA and RNA delivery. More recently, we discovered that a subset of these NABPs can also serve as anti-inflammatory agents by capturing pro-inflammatory extracellular nucleic acids and associated protein complexes that promote activation of toll-like receptors (TLRs) in diseases such as lupus erythematosus. Nucleic-acid-mediated TLR signaling also facilitates tumor progression and metastasis in several cancers, including pancreatic cancer (PC). In addition, extracellular DNA and RNA circulate on or within lipid microvesicles, such as microparticles or exosomes, which also promote metastasis by inducing pro-tumorigenic signaling in cancer cells and pre-conditioning secondary sites for metastatic establishment. Here, we explore the use of an NABP, the 3rd generation polyamidoamine dendrimer (PAMAM-G3), as an anti-metastatic agent. We show that PAMAM-G3 not only inhibits nucleic-acid-mediated activation of TLRs and invasion of PC tumor cells in vitro, but can also directly bind extracellular microvesicles to neutralize their pro-invasive effects as well. Moreover, we demonstrate that PAMAM-G3 dramatically reduces liver metastases in a syngeneic murine model of PC. Our findings identify a promising therapeutic application of NABPs for combating metastatic disease in PC and potentially other malignancies.

Differential Induction of Immunogenic Cell Death and Interferon Expression in Cancer Cells by Structured ssRNAs.

Activation of the RNA-sensing pattern recognition receptor (PRR) in cancer cells leads to cell death and cytokine expression. This cancer cell death releases tumor antigens and damage-associated molecular patterns (DAMPs) that induce anti-tumor immunity. However, these cytokines and DAMPs also cause adverse inflammatory and thrombotic complications that can limit the overall therapeutic benefits of PRR-targeting anti-cancer therapies. To overcome this problem, we generated and evaluated two novel and distinct ssRNA molecules (immunogenic cell-killing RNA [ICR]2 and ICR4). ICR2 and ICR4 differentially stimulated cell death and PRR signaling pathways and induced different patterns of cytokine expression in cancer and innate immune cells. Interestingly, DAMPs released from ICR2- and ICR4-treated cancer cells had distinct patterns of stimulation of innate immune receptors and coagulation. Finally, ICR2 and ICR4 inhibited in vivo tumor growth as effectively as poly(I:C). ICR2 and ICR4 are potential therapeutic agents that differentially induce cell death, immune stimulation, and coagulation when introduced into tumors.

Aptamers as Therapeutics.

Aptamers are single-stranded nucleic acid molecules that bind to and inhibit proteins and are commonly produced by systematic evolution of ligands by exponential enrichment (SELEX). Aptamers undergo extensive pharmacological revision, which alters affinity, specificity, and therapeutic half-life, tailoring each drug for a specific clinical need. The first therapeutic aptamer was described 25 years ago. Thus far, one aptamer has been approved for clinical use, and numerous others are in preclinical or clinical development. This review presents a short history of aptamers and SELEX, describes their pharmacological development and optimization, and reviews potential treatment of diseases including visual disorders, thrombosis, and cancer.

Polycationic Nanofibers for Nucleic Acid Scavenging.

Dying cells release nucleic acids (NA) and NA-containing complexes that activate inflammatory pathways of immune cells. Sustained activation of these pathways contributes to chronic inflammation frequently encountered in autoimmune and inflammatory diseases. In this study, grafting of cationic polymers onto a nanofibrous mesh enabled local scavenging of negatively charged pro-inflammatory molecules in the extracellular space. Nucleic acid scavenging nanofibers (NASFs) formed from poly(styrene-alt-maleic anhydride) conjugated with 1.8 kDa bPEI resulted in nanofibers of diameters 486 ± 9 nm. NASFs inhibited the NF-κB response stimulated by the negatively charged agonists, CpG and poly(I:C), in Ramos-blue cells but not Pam3CSK4, a nonanionic agonist. Moreover, NASFs significantly impeded NF-κB activation in cells stimulated with damage-associated molecular pattern molecules (DAMPs) released from doxorubicin killed cancer cells. In vivo application of NASFs to open wounds demonstrated nucleic acid scavenging in wounds of diabetic mice infected with Pseudomonas aeruginosa, suggesting the in vivo efficacy of NASFs. This simple technique of generating NASF results in effective localized anti-inflammation in vitro and local nucleic acid scavenging in vivo.

From the RNA world to the clinic.

The study of RNA has continually emphasized the structural and functional versatility of RNA molecules. This versatility has inspired translational and clinical researchers to explore the utility of RNA-based therapeutic agents for a wide variety of medical applications. Several RNA therapeutics, with diverse modes of action, are being evaluated in large late-stage clinical trials, and many more are in early clinical development. Hundreds of patients are enrolled in large trials testing messenger RNAs to combat cancer, small interfering RNAs to treat renal and hepatic disorders, and aptamers to combat ocular and cardiovascular disease. Results from these studies are generating considerable interest among the biomedical community and the public and will be important for the future development of this emerging class of therapeutic agents.

In Vivo Selection Against Human Colorectal Cancer Xenografts Identifies an Aptamer That Targets RNA Helicase Protein DHX9.

The ability to selectively target disease-related tissues with molecules is critical to the design of effective therapeutic and diagnostic reagents. Recognizing the differences between the in vivo environment and in vitro conditions, we employed an in vivo selection strategy to identify RNA aptamers (targeting motifs) that could localize to tumor in situ. One of the selected molecules is an aptamer that binds to the protein DHX9, an RNA helicase that is known to be upregulated in colorectal cancer. Upon systemic administration, the aptamer preferentially localized to the nucleus of cancer cells in vivo and thus has the potential to be used for targeted delivery.

2'Fluoro Modification Differentially Modulates the Ability of RNAs to Activate Pattern Recognition Receptors.

Although the use of RNAs has enormous therapeutic potential, these RNA-based therapies can trigger unwanted inflammatory responses by the activation of pattern recognition receptors (PRRs) and cause harmful side effects. In contrast, the immune activation by therapeutic RNAs can be advantageous for treating cancers. Thus, the immunogenicity of therapeutic RNAs should be deliberately controlled depending on the therapeutic applications of RNAs. In this study, we demonstrated that RNAs containing 2'fluoro (2'F) pyrimidines differentially controlled the activation of PRRs. The activity of RNAs that stimulate toll-like receptors 3 and 7 was abrogated by the incorporation of 2'F pyrimidine. By contrast, incorporation of 2'F pyrimidines enhanced the activity of retinoic acid-inducible gene 1-stimulating RNAs. Furthermore, we found that transfection with RNAs containing 2'F pyrimidine and 5' triphosphate (5'ppp) increased cell death and interferon-ß expression in human cancer cells compared with transfection with 2'hydroxyl 5'ppp RNAs, whereas RNAs containing 2'O-methyl pyrimidine and 5'ppp completely abolished the induction of cell death and cytokine expression in the cells. Our findings suggest that incorporation of 2'F and 2'O-methyl nucleosides is a facile approach to differentially control the ability of therapeutic RNAs to activate or limit immune and inflammatory responses depending on therapeutic applications.

Targeting Two Coagulation Cascade Proteases with a Bivalent Aptamer Yields a Potent and Antidote-Controllable Anticoagulant.

Potent and rapid-onset anticoagulation is required for several clinical settings, including cardiopulmonary bypass surgery. In addition, because anticoagulation is associated with increased bleeding following surgery, the ability to rapidly reverse such robust anticoagulation is also important. Previously, we observed that no single aptamer was as potent as heparin for anticoagulating blood. However, we discovered that combinations of two aptamers were as potent as heparin. Herein, we sought to combine two individual anticoagulant aptamers into a single bivalent RNA molecule in an effort to generate a single molecule that retained the potent anticoagulant activity of the combination of individual aptamers. We created four bivalent aptamers that can inhibit Factor X/Xa and prothrombin/thrombin and anticoagulate plasma, as well as the combination of individual aptamers. Detailed characterization of the shortest bivalent aptamer indicates that each aptamer retains full binding and functional activity when presented in the bivalent context. Finally, reversal of this bivalent aptamer with a single antidote was explored, and anticoagulant activity could be rapidly turned off in a dose-dependent manner. These studies demonstrate that bivalent anticoagulant aptamers represent a novel and potent approach to actively and reversibly control coagulation.