Search

CRISPR-based editing has revolutionized genome engineering despite the observation that many DNA sequences remain challenging to target. Unproductive interactions formed between the single guide RNA's (sgRNA) Cas9-binding scaffold domain and DNA-binding antisense domain are often responsible for such limited editing resolution. To bypass this limitation, we develop a functional SELEX (systematic evolution of ligands by exponential enrichment) approach, termed BLADE (binding and ligand activated directed evolution), to identify numerous, diverse sgRNA variants that bind Streptococcus pyogenes Cas9 and support DNA cleavage. These variants demonstrate surprising malleability in sgRNA sequence. We also observe that particular variants partner more effectively with specific DNA-binding antisense domains, yielding combinations with enhanced editing efficiencies at various target sites. Using molecular evolution, CRISPR-based systems could be created to efficiently edit even challenging DNA sequences making the genome more tractable to engineering. This selection approach will be valuable for generating sgRNAs with a range of useful activities. [Display omitted] • In vitro evolution generates multiple functional gRNA scaffolds • sgRNAs are highly malleable • sgRNA scaffold variants display varied efficacy with different sgRNA spacer domains • Functional sgRNA selection can generate novel sgRNAs with useful properties Bush et al. present a high-throughput selection and RNA evolution scheme that can identify functional sgRNA scaffold domains with distinct properties. These variants provide additional insight into the structure-activity-relationships required for effective Cas9 guided DNA editing. BLADE SELEX promises to facilitate the development of sgRNA variants with new activities. [ABSTRACT FROM AUTHOR]

Coordinated enzymatic reactions regulate blood clot generation. To explore the contributions of various coagulation enzymes in this process, we utilized a panel of aptamers against factors VIIa, IXa, Xa, and prothrombin. Each aptamer dose-dependently inhibited clot formation, yet none was able to completely impede this process in highly procoagulant settings. However, several combinations of two aptamers synergistically impaired clot formation. One extremely potent aptamer combination was able to maintain human blood fluidity even during extracorporeal circulation, a highly procoagulant setting encountered during cardiopulmonary bypass surgery. Moreover, this aptamer cocktail could be rapidly reversed with antidotes to restore normal hemostasis, indicating that even highly potent aptamer combinations can be rapidly controlled. These studies highlight the potential utility of using sets of aptamers to probe the functions of proteins in molecular pathways for research and therapeutic ends. [ABSTRACT FROM AUTHOR]

Coagulation cofactors profoundly regulate hemostasis and are appealing targets for anticoagulants. However, targeting such proteins has been challenging because they lack an active site. To address this, we isolate an RNA aptamer termed T18.3 that binds to both factor V (FV) and FVa with nanomolar affinity and demonstrates clinically relevant anticoagulant activity in both plasma and whole blood. The aptamer also shows synergy with low molecular weight heparin and delivers potent anticoagulation in plasma collected from patients with coronavirus disease 2019 (COVID-19). Moreover, the aptamer's anticoagulant activity can be rapidly and efficiently reversed using protamine sulfate, which potentially allows fine-tuning of aptamer's activity post-administration. We further show that the aptamer achieves its anticoagulant activity by abrogating FV/FVa interactions with phospholipid membranes. Our success in generating an anticoagulant aptamer targeting FV/Va demonstrates the feasibility of using cofactor-binding aptamers as therapeutic protein inhibitors and reveals an unconventional working mechanism of an aptamer by interrupting protein-membrane interactions. [Display omitted] • Aptamers bind with low nanomolar affinity to coagulation cofactor FV/FVa • Aptamers are potent anticoagulants in normal and COVID-19 plasma • Protamine sulfate can rapidly reverse aptamer's anticoagulant activity • Aptamers synergize with low molecular weight heparin (LMWH) Coagulation cofactors are appealing yet challenging targets for anticoagulant development. Soule et al. describe a 2′F-modified RNA aptamer that potently inhibits coagulation cofactor FV/FVa docking to the cellular membrane and demonstrates clinically relevant anticoagulant activity in plasma and whole blood. [ABSTRACT FROM AUTHOR]

Summary: We show that a molecular scaffold can be utilized to convert a receptor binding aptamer into a receptor agonist. Many receptors (including tumor necrosis receptor family members) are activated when they are multimerized on the cell surface. Molecular scaffolds have been utilized to assemble multiple receptor binding peptide ligands to generate activators of such receptors. We demonstrate that an RNA aptamer that recognizes OX40, a member of the tumor necrosis factor receptor superfamily, can be converted into a receptor-activating aptamer by assembling two copies on an olignucleotide-based scaffold. The OX40 receptor-activating aptamer is able to induce nuclear localization of nuclear factor-κB, cytokine production, and cell proliferation, as well as enhance the potency of dendritic cell-based tumor vaccines when systemically delivered to mice. [Copyright &y& Elsevier]

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. • Aptamers can be used to reversibly label and purify cells via FACS or MACS • EGFR aptamer-magnetic bead MACS isolates EGFR+ cells out of complex cell mixtures • An oligonucleotide "antidote" strips off aptamers to recover label-free cells • EGFR aptamer FACS sorting and antidote removal restores native receptor function Cell purification methods rely upon poorly reversible antibody labeling of desired cells or upon depletion of unwanted cells. Powell Gray et al. demonstrate that aptamers can be used to positively purify cells and, unlike antibodies, yield cells in their native state using antidote-mediated reversal of aptamer cell-surface binding. [ABSTRACT FROM AUTHOR]

Biopharmaceuticals have become increasingly attractive therapeutic agents and are often PEGylated to enhance their pharmacokinetics and reduce their immunogenicity. However, recent human clinical trials have demonstrated that administration of PEGylated compounds can evoke anti-PEG antibodies. Considering the ubiquity of PEG in commercial products and the presence of pre-existing anti-PEG antibodies in patients in large clinical trials evaluating a PEG-modified aptamer, we investigated how anti-PEG antibodies effect the therapeutic activities of PEGylated RNA aptamers. We demonstrate that anti-PEG antibodies can directly bind to and inhibit anticoagulant aptamer function in vitro and in vivo. Moreover, in parallel studies we detected the presence of anti-PEG antibodies in nonhuman primates after a single administration of a PEGylated aptamer. Our results suggest that anti-PEG antibodies can limit the activity of PEGylated drugs and potentially compromise the activity of otherwise effective therapeutic agents. • Anti-PEG antibodies bind to PEGylated RNA aptamer RB006 • Anti-PEG antibodies inhibit anticoagulant aptamer activity in vitro and in vivo • After one administration of RB006 anti-PEG antibodies were found in rhesus monkeys The most common approach for pharmacokinetic enhancement of biologically inspired therapies is PEGylation; however, possible limitations of this formulation strategy have arisen. Here, we describe how anti-PEG antibodies can inhibit the therapeutic efficacy of a PEGylated RNA aptamer. These findings further highlight emerging issues between the immune system and PEGylated therapeutics. [ABSTRACT FROM AUTHOR]

Cell isolation from complex mixtures is a key step in many clinical and research applications, but standard isolation methods may affect the cell's biology and are difficult to reverse. Here, we present a method to isolate and restore cells to their native state using an aptamer that binds epidermal growth factor receptor (EGFR+)cells and a complementary antisense oligonucleotide to reverse binding. For complete details on the use and execution of this protocol, please refer to Gray et al. 1 ., Competing Interests: Declaration of interests Duke University has applied for a patent on this technology., (Copyright © 2023. Published by Elsevier Inc.)

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., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Pathological blood clotting, or thrombosis, limits vital blood flow to organs; such deprivation can lead to catastrophic events including myocardial infarction, pulmonary embolism, and ischemic stroke. Prompt restoration of blood flow greatly improves outcomes. We explored whether aptamers could serve as molecular imaging probes to rapidly detect thrombi. An aptamer targeting thrombin, Tog25t, was found to rapidly localize to and visualize pre-existing clots in the femoral and jugular veins of mice using fluorescence imaging and, when circulating, was able to image clots as they form. Since free aptamer is quickly cleared from circulation, contrast is rapidly developed, allowing clot visualization within minutes. Moreover, administration of an antidote oligonucleotide further enhanced contrast development, causing the unbound aptamer to clear within 5min while impacting the clot-bound aptamer more slowly. These findings suggest that aptamers can serve as imaging agents for rapid detection of thrombi in acute care and perioperative settings., Competing Interests: Duke University has submitted patent applications on the use of aptamer-based clot imaging., (© 2023 The Authors.)

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., Competing Interests: Duke University has applied for patents on the strategy to reduce inflammation via nucleic acid scavengers. Lyra Olson, Nicole Hunter, Rachel Rempel, and Bruce Sullenger are listed as inventors on such patents., (© 2022 The Author(s).)

Nucleic acid (NA)-containing damage- and pathogen-associated molecular patterns (DAMPs and PAMPs, respectively) are implicated in numerous pathological conditions from infectious diseases to autoimmune disorders. Nucleic acid-binding polymers, including polyamidoamine (PAMAM) dendrimers, have demonstrated anti-inflammatory properties when administered to neutralize DAMPs/PAMPs. The PAMAM G3 variant has been shown to have beneficial effects in a cutaneous lupus erythematosus (CLE) murine model and improve survival of mice challenged with influenza. Unfortunately, the narrow therapeutic window of cationic PAMAM dendrimers makes their clinical development challenging. An alternative nucleic acid-binding polymer that has been evaluated in humans is a linear β-cyclodextrin-containing polymer (CDP). CDP's characteristics prompted us to evaluate its anti-inflammatory potential in CLE autoimmune and influenza infectious disease mouse models. We report that CDP effectively inhibits NA-containing DAMP-mediated activation of Toll-like receptors (TLRs) in cell culture, improves healing in lupus mice, and does not immunocompromise treated animals upon influenza infection but improves survival even when administered 3 days after infection. Finally, as anticipated, we observe limited toxicity in animals treated with CDP compared with PAMAM G3. Thus, CDP is a new anti-inflammatory agent that may be readily translated to the clinic to combat diseases associated with pathological NA-containing DAMPs/PAMPs., Competing Interests: Declaration of interests Duke University has applied for patents on the strategy to reduce inflammation via nucleic acid scavengers., (Copyright © 2021 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Duke University has issued patents on the anti-FIXa aptamer used in the manuscript, DTRI-178. Dr. Sullenger is listed as an inventor on these patents. Duke University has applied for a use patent for DTRI-178 in extracorporeal circulation. Drs. Reed, Tracy, and Sullenger are listed as inventors on this application. None of the authors have otherwise commercialized or monetized this experimental oligonucleotide., (© 2021 The Authors.)

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., Competing Interests: The authors declare no competing interests. However, Duke University has submitted patents on the applications of nucleic acid scavengers to combat inflammation and cancer., (© 2021 The Authors.)

Reprogramming scar fibroblasts into new heart muscle cells has the potential to restore function to the injured heart. However, the effectiveness of reprogramming is notably low. We have recently demonstrated that the effectiveness of reprogramming fibroblasts into heart muscle cells (cardiomyocytes) is increased by the addition of RNA-sensing receptor ligands. Clinical use of these ligands is problematic due to their ability to induce adverse inflammatory events. To overcome this issue, we sought to determine whether synthetic analogs of natural RNA-sensing receptor ligands, which avoid generating inflammatory insults and are nuclease resistant, would similarly enhance fibroblast reprogramming into cardiomyocytes. Indeed, one such stabilized RNA, ICR2, increased the expression of cardiomyocyte-specific mRNAs in reprogrammed fibroblasts. Moreover, ICR2 enhanced the ability of reprogramming factors to produce cardiomyocytes with mature sarcomeres. Knockdown assays indicated that the effects of ICR2 were mediated by the RNA-sensing receptors Rig-I and TLR3. In addition, ICR2 reduced the effective dose and number of reprogramming factors needed for efficient reprogramming. In summary, the synthetic RNA oligonucleotide ICR2 is a potential therapeutic agent to enhance cardiac reprogramming efficiency., Competing Interests: V.J.D., JL., B.A.S., and C.P.H. are co-founders of Recardia Therapeutics., (© 2020 The Author(s).)

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., Competing Interests: Declaration of Interests Dr. Sullenger is on the scientific advisory board of Rznomics., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Endothelial surface and circulating glycoprotein von Willebrand factor (vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage. To overcome these limitations, we developed a novel anti-vWF aptamer, called DTRI-031, that selectively binds and inhibits vWF-mediated platelet adhesion and arterial thrombosis while enabling rapid reversal of this antiplatelet activity by an antidote oligonucleotide (AO). Aptamer DTRI-031 exerts dose-dependent inhibition of platelet aggregation and thrombosis in whole blood and mice, respectively. Moreover, DTRI-031 can achieve potent vascular recanalization of platelet-rich thrombotic occlusions in murine and canine carotid arteries. Finally, DTRI-031 activity is rapidly (<5 min) and completely reversed by AO administration in a murine saphenous vein hemorrhage model, and murine toxicology studies indicate the aptamer is well tolerated. These findings suggest that targeting vWF with an antidote-controllable aptamer potentially represents an effective and safer treatment for thrombosis patients having platelet-rich arterial occlusions in the brain, heart, or periphery., (Copyright © 2019. Published by Elsevier Inc.)

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 3 rd 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., (Copyright © 2018 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

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.

Millions of individuals are prescribed platelet inhibitors, such as aspirin and clopidogrel, to reduce their risk of thrombosis-related clinical events. Unfortunately many platelet inhibitors are contraindicated in surgical settings because of their inherent bleeding risk complicating the treatment of patients who require surgery. We describe the development of a potent antiplatelet agent, an RNA aptamer-termed Ch-9.14-T10 that binds von Willebrand factor (VWF) with high affinity and inhibits thrombosis in a murine carotid artery damage model. As expected, when this potent antiplatelet agent is administered, it greatly increases bleeding from animals that are surgically challenged. To improve this antiplatelet agent's safety profile, we describe the generation of antidotes that can rapidly reverse the activity of Ch-9.14-T10 and limit blood loss from surgically challenged animals. Our work represents the first antidote controllable antiplatelet agent, which could conceivably lead to improved medical management of patients requiring antiplatelet medication who also need surgery.

Osteopontin (OPN) is a secreted phosphoprotein which mediates tumorigenesis, local growth, and metastasis in a variety of cancers. It is a potential therapeutic target for the regulation of cancer metastasis. RNA aptamer technology targeting OPN may represent a clinically viable therapy. In this study, we characterize the critical sequence of an RNA aptamer, termed OPN-R3, directed against human OPN. It has a K(d) of 18 nmol/l and binds specifically to human OPN as determined by RNA electrophoretic mobility assays. In MDA-MB231 human breast cancer cells examined under fluorescence microscopy, OPN-R3 ablates cell surface binding of OPN to its cell surface CD44 and alpha(v)beta(3) integrin receptors. Critical enzymatic components of the OPN signal transduction pathways, PI3K, JNK1/2, Src and Akt, and mediators of extracellular matrix degradation, matrix metalloproteinase 2 (MMP2) and uroplasminogen activator (uPA), are significantly decreased following exposure to OPN-R3. OPN-R3 inhibits MDA-MB231 in vitro adhesion, migration, and invasion characteristics by 60, 50, and 65%, respectively. In an in vivo xenograft model of breast cancer, OPN-R3 significantly decreases local progression and distant metastases. On the basis of this "proof-of-concept" study, we conclude that RNA aptamer targeting of OPN has biologically relevance for modifying tumor growth and metastasis.

We have developed and validated a new tumor-targeting gene therapy strategy based upon the targeting and replacement of human telomerase reverse transcriptase (hTERT) RNA, using a trans-splicing ribozyme. By constructing novel adenoviral vectors harboring the hTERT-targeting trans-splicing ribozymes with the downstream reporter gene (Ad-Ribo-LacZ) or suicide gene (Ad-Ribo-HSVtk) driven by the cytomegalovirus (CMV) promoter, we demonstrated that this viral system selectively marks tumor cells expressing hTERT or sensitizes tumor cells to prodrug treatments. We confirmed that Ad-Ribo-LacZ successfully and selectively delivered a ribozyme that performed a highly specific trans-splicing reaction into hTERT-expressing cancer cells, both in vitro and in a peritoneal carcinomatosis nude mouse model. We also determined that the hTERT-specific expression of the suicide gene in the Ad-Ribo-HSVtk, and treatment with the corresponding prodrug, reduced tumor progression with almost the same efficacy as the strong constitutive CMV promoter-driven adenovirus, both in cancer cell lines and in nude mouse HT-29 xenografts. These observations provide the basis for a novel approach to cancer gene therapy, and demonstrate that trans-splicing ribozymes can be employed as targeting anti-cancer agents which recognize cancer-specific transcripts and reprogram them, thereby combating cancerous cells.

Due to the prevalence of tumor chemoresistance, the clinical response of advanced non-small cell lung cancer (NSCLC) to chemotherapy is poor. We suppressed tumor resistance to doxorubicin (Dox) in A549 cells, a human NSCLC cell line, both in vitro and in vivo in a lung tumor xenograft model, using a novel adenoviral expression system to deliver an RNA aptamer (A-p50) that specifically inhibits nuclear factor-kappaB (NF-kappaB) activation. By achieving selective, targeted, and early inhibition of NF-kappaB activity, we demonstrate that NF-kappaB plays a critical role in Dox-induced chemoresistance by regulating genes involved in proliferation (Ki-67), response to DNA damage (GADD153), antiapoptosis (Bcl-XL), and pH regulation (CA9). This Dox-induced NF-kappaB activation and subsequent chemoresistance is dependent on expression of p53. We also demonstrate that NF-kappaB promotes angiogenesis in the presence of Dox via the hypoxia-inducible factor-1alpha/vascular endothelial growth factor (HIF-1alpha/VEGF) pathway, revealing a previously unknown mechanism of NSCLC resistance to Dox. These studies provide important insights into the mechanisms of Dox-induced chemoresistance, and they demonstrate a novel, effective, and clinically practical strategy for interfering with these processes.

Heparin and protamine are the standard anticoagulant-antidote regimen used in almost every cardiopulmonary bypass (CPB) procedure even though both are associated with an array of complications and toxicities. Here we demonstrate that an anticoagulant aptamer-antidote pair targeting factor IXa can replace heparin and protamine in a porcine CPB model and also limit the adverse effects on thrombin generation, inflammation, and cardiac physiology associated with heparin and protamine use. These results demonstrate that targeting clotting factors upstream of thrombin in the coagulation cascade can potentially reduce the perioperative pathologies associated with CPB and suggest that the aptamer-antidote pair to FIXa may improve the outcome of patients undergoing CPB. In particular, this novel anticoagulant-antidote pair may prove to be useful in patients diagnosed with heparin-induced thrombocytopenia or those who have been sensitized to protamine, particularly patients who have insulin-dependent diabetes.

Retroposable elements such as retroviral and lentiviral vectors have been employed for many gene therapy applications. Unfortunately, such gene transfer vectors integrate genes into many different DNA sequences and unintended integration of the vector near a growth-promoting gene can engender pathological consequences. For example, retroviral vector-mediated gene transfer induced leukemia in 2 of 11 children treated for severe combined immunodeficiency, raising significant safety issues for gene transfer strategies that cannot be targeted to specific sequences. Here, we examine the use of a mobile retroposable genetic element that can be targeted to introduce therapeutic sequences site specifically into mutant genes. The data demonstrate that the mobile group II intron from Lactococcus lactis can be targeted to insert into and repair mutant lacZ (approved gene symbol GLB1) and beta-globin (approved gene symbol HBB) genes with high efficiency and fidelity in model systems in bacteria. These results suggest that these mobile genetic elements represent a novel class of agents for performing targeted genetic repair.

A variety of mutations in the p53 tumor suppressor gene have been found in over half of human tumors. Thus, restoration of wild-type p53 activity by repair of mutant RNA has been previously suggested as an approach to cancer treatment. To explore the potential utility of RNA repair for cancer therapy, we developed a group I intron-based ribozyme that can replace mutant p53 RNA with a wild-type RNA sequence attached to the 3' end of the ribozyme by trans-splicing reaction. First, RNA mapping analysis demonstrated that the leader sequences upstream of the AUG start codon in the mutant p53 RNA appeared to be particularly accessible to the ribozymes. Then, the trans-splicing ribozyme specifically recognizing the most accessible sequence induced functional p53 activity, resulting in an 8- and a 2.6-fold induction of transactivation of p53-responsive promoters in two mutant p53-related ovarian cancer cell lines, SKOV3 cells and 2774 cells, respectively, by repairing defective p53 RNA. The repair efficiency of the mutant p53 RNA was almost 10% in 2774 cells. Moreover, the ribozyme activated the expression level of endogenous p21 and Bax genes in the cells. Furthermore, apoptosis was efficiently triggered in the human cancer cells transfected with the specific ribozyme. These results suggest that a trans-splicing ribozyme could be a potent anti-cancer agent that can revert the defective p53-related neoplastic phenotype., (Copyright The American Society of Gene Therapy)

RNA aptamers against a variety of clinically relevant target proteins have been generated. For example, we previously isolated an RNA aptamer that inhibits the function of the E2F family of transcription factors that play a critical role in the control of cell proliferation. However, the development of this and other aptamers for gene therapy applications has been complicated by the fact that expression of RNA aptamers in the context of flanking sequences can inhibit the ability of an aptamer to fold into its functional conformation. Insertion of the E2F aptamer into a tRNA expression cassette resulted in the production of high levels of chimeric tRNA that contains a misfolded and inactive aptamer in transfected mammalian cells. To overcome this problem, we randomized the sequence flanking the aptamer and selected for chimeric tRNAs that retained high affinity binding to E2F1. This expression cassette SELEX strategy yielded RNAs that bind E2F with high affinity (IC50 of 15 nM) and which can be expressed at high levels in mammalian cells. Moreover, these chimeric tRNA-E2F aptamers are functional and can inhibit E2F-mediated transactivation by up to 80% in human 293 cells. Expression cassette SELEX should greatly facilitate the use of aptamers for a variety of gene therapy applications.