Methods for assembling B-cell lymphoma specific and internalizing aptamer-siRNA nanoparticles via the sticky bridge.

Structured functional RNA entities, including aptamers and siRNAs, have amazing versatility in structure and function. These molecules can serve as powerful, attractive building blocks for the bottom-up assembly of complex nanostructures. Here, we describe novel cell-type specific and internalizing B-cell activating factor receptor (BAFF-R) aptamer-siRNA delivery systems for B-cell lymphoma therapy, in which both the aptamer and the Dicer substrate siRNA (DsiRNA) portions are conjugated through a "sticky bridge." The BAFF-R is overexpressed on the surface of B-cell malignancies, allowing binding and internalization of the aptamer-stick-siRNA nanoparticles. STAT3 siRNAs are encapsulated within the nanoparticles delivered by the BAFF-R aptamers and are localized to the cytoplasm, resulting in robust gene silencing of STAT3 mRNAs in a variety of B-cell lines. Moreover, these nanoparticles do not induce cell proliferation and apoptosis. Collectively, aptamer-mediated delivery strategies provide a toolset to become a more widely used therapeutic modality for the treatment of diseases.

Noncoding oligonucleotides: the belle of the ball in gene therapy.

Gene therapy carries the promise of cures for many diseases based on manipulating the expression of a person's genes toward the therapeutic goal. The relevance of noncoding oligonucleotides to human disease is attracting widespread attention. Noncoding oligonucleotides are not only involved in gene regulation, but can also be modified into therapeutic tools. There are many strategies that leverage noncoding oligonucleotides for gene therapy, including small interfering RNAs, antisense oligonucleotides, aptamers, ribozymes, decoys, and bacteriophage phi 29 RNAs. In this chapter, we will provide a broad, comprehensive overview of gene therapies that use noncoding oligonucleotides for disease treatment. The mechanism and development of each therapeutic will be described, with a particular focus on its clinical development. Finally, we will discuss the challenges associated with developing nucleic acid therapeutics and the prospects for future success.

Gene therapy: a promising approach to treating spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a severe autosomal recessive disease caused by a genetic defect in the survival motor neuron 1 (SMN1) gene, which encodes SMN, a protein widely expressed in all eukaryotic cells. Depletion of the SMN protein causes muscle weakness and progressive loss of movement in SMA patients. The field of gene therapy has made major advances over the past decade, and gene delivery to the central nervous system (CNS) by in vivo or ex vivo techniques is a rapidly emerging field in neuroscience. Despite Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis being among the most common neurodegenerative diseases in humans and attractive targets for treatment development, their multifactorial origin and complicated genetics make them less amenable to gene therapy. Monogenic disorders resulting from modifications in a single gene, such as SMA, prove more favorable and have been at the fore of this evolution of potential gene therapies, and results to date have been promising at least. With the estimated number of monogenic diseases standing in the thousands, elucidating a therapeutic target for one could have major implications for many more. Recent progress has brought about the commercialization of the first gene therapies for diseases, such as pancreatitis in the form of Glybera, with the potential for other monogenic disease therapies to follow suit. While much research has been carried out, there are many limiting factors that can halt or impede translation of therapies from the bench to the clinic. This review will look at both recent advances and encountered impediments in terms of SMA and endeavor to highlight the promising results that may be applicable to various associated diseases and also discuss the potential to overcome present limitations.

Aptamer-based therapeutics: new approaches to combat human viral diseases.

Viruses replicate inside the cells of an organism and continuously evolve to contend with an ever-changing environment. Many life-threatening diseases, such as AIDS, SARS, hepatitis and some cancers, are caused by viruses. Because viruses have small genome sizes and high mutability, there is currently a lack of and an urgent need for effective treatment for many viral pathogens. One approach that has recently received much attention is aptamer-based therapeutics. Aptamer technology has high target specificity and versatility, i.e., any viral proteins could potentially be targeted. Consequently, new aptamer-based therapeutics have the potential to lead a revolution in the development of anti-infective drugs. Additionally, aptamers can potentially bind any targets and any pathogen that is theoretically amenable to rapid targeting, making aptamers invaluable tools for treating a wide range of diseases. This review will provide a broad, comprehensive overview of viral therapies that use aptamers. The aptamer selection process will be described, followed by an explanation of the potential for treating virus infection by aptamers. Recent progress and prospective use of aptamers against a large variety of human viruses, such as HIV-1, HCV, HBV, SCoV, Rabies virus, HPV, HSV and influenza virus, with particular focus on clinical development of aptamers will also be described. Finally, we will discuss the challenges of advancing antiviral aptamer therapeutics and prospects for future success.

Nanoparticle-Based Delivery of RNAi Therapeutics: Progress and Challenges.

RNA interference (RNAi) is an evolutionarily conserved, endogenous process for post-transcriptional regulation of gene expression. Although RNAi therapeutics have recently progressed through the pipeline toward clinical trials, the application of these as ideal, clinical therapeutics requires the development of safe and effective delivery systems. Inspired by the immense progress with nanotechnology in drug delivery, efforts have been dedicated to the development of nanoparticle-based RNAi delivery systems. For example, a precisely engineered, multifunctional nanocarrier with combined passive and active targeting capabilities may address the delivery challenges for the widespread use of RNAi as a therapy. Therefore, in this review, we introduce the major hurdles in achieving efficient RNAi delivery and discuss the current advances in applying nanotechnology-based delivery systems to overcome the delivery hurdles of RNAi therapeutics. In particular, some representative examples of nanoparticle-based delivery formulations for targeted RNAi therapeutics are highlighted.

Nucleic Acid Aptamers as Potential Therapeutic and Diagnostic Agents for Lymphoma.

Lymphomas are cancers that arise from white blood cells and usually present as solid tumors. Treatment of lymphoma often involves chemotherapy, and can also include radiotherapy and/or bone marrow transplantation. There is an un-questioned need for more effective therapies and diagnostic tool for lymphoma. Aptamers are single stranded DNA or RNA oligonucleotides whose three-dimensional structures are dictated by their sequences. The immense diversity in function and structure of nucleic acids enable numerous aptamers to be generated through an iterative in vitro selection technique known as Systematic Evolution of Ligands by EXponential enrichment (SELEX). Aptamers have several biochemical properties that make them attractive tools for use as potential diagnostic and pharmacologic agents. Isolated aptamers may directly inhibit the function of target proteins, or they can also be formulated for use as delivery agents for other therapeutic or imaging cargoes. More complex aptamer identification methods, using whole cancer cells (Cell-SELEX), may identify novel targets and aptamers to affect them. This review focuses on recent advances in the use of nucleic acid aptamers as diagnostic and therapeutic agents and as targeted delivery carriers that are relevant to lymphoma. Some representative examples are also discussed.

Aptamer-mediated inhibition of Mycobacterium tuberculosis polyphosphate kinase 2.

Inorganic polyphosphate (polyP) plays a number of critical roles in bacterial persistence, stress, and virulence. PolyP intracellular metabolism is regulated by the polyphosphate kinase (PPK) protein families, and inhibition of PPK activity is a potential approach to disrupting polyP-dependent processes in pathogenic organisms. Here, we biochemically characterized Mycobacterium tuberculosis (MTB) PPK2 and developed DNA-based aptamers that inhibit the enzyme's catalytic activities. MTB PPK2 catalyzed polyP-dependent phosphorylation of ADP to ATP at a rate 838 times higher than the rate of polyP synthesis. Gel filtration chromatography suggested MTB PPK2 to be an octamer. DNA aptamers were isolated against MTB PPK2. Circular dichroism revealed that aptamers grouped into two distinct classes of secondary structure; G-quadruplex and non-G-quadruplex. A selected G-quadruplex aptamer was highly selective for binding to MTB PPK2 with a dissociation constant of 870 nM as determined by isothermal titration calorimetry. The binding between MTB PPK2 and the aptamer was exothermic yet primarily driven by entropy. This G-quadruplex aptamer inhibited MTB PPK2 with an IC(50) of 40 nM and exhibited noncompetitive inhibition kinetics. Mutational mechanistic analysis revealed an aptamer G-quadruplex motif is critical for enzyme inhibition. The aptamer was also tested against Vibrio cholerae PPK2, where it showed an IC(50) of 105 nM and insignificant inhibition against more distantly related Laribacter hongkongensis PPK2.

Identification of a DNA aptamer that inhibits sclerostin's antagonistic effect on Wnt signalling.

Sclerostin is an extracellular negative regulator of bone formation that is a recognized therapeutic target for osteoporosis therapy. In the present study, we performed DNA aptamer selection against sclerostin, then characterized aptamer-sclerostin binding and the ability to inhibit sclerostin function in cell culture. We show that a selected DNA aptamer was highly selective for binding to sclerostin with affinities in the nanomolar range as determined by solid-phase assays and by isothermal titration calorimetry. Binding between sclerostin and the aptamer was exothermic and enthalpically driven. CD confirmed that the aptamer had temperature-dependent parallel G-quadruplex characteristics. The aptamer was stabilized with 3' inverted thymidine to investigate efficacy at inhibiting sclerostin function in cell culture. The stabilized DNA aptamer showed potent and specific dose-dependent inhibition of sclerostin's antagonistic effect on Wnt activity using a reporter assay. Taken together, the present findings suggest an alternative approach to inhibiting sclerostin function with therapeutic potential.

Differential inhibitory activities and stabilisation of DNA aptamers against the SARS coronavirus helicase.

The helicase from severe acute respiratory syndrome coronavirus (SARS-CoV) possesses NTPase, duplex RNA/DNA-unwinding and RNA-capping activities that are essential for viral replication and proliferation. Here, we have isolated DNA aptamers against the SARS-CoV helicase from a combinatorial DNA library. These aptamers show two distinct classes of secondary structure, G-quadruplex and non-G-quadruplex, as shown by circular dichroism and gel electrophoresis. All of the aptamers that were selected stimulated ATPase activity of the SARS-CoV helicase with low-nanomolar apparent K(m) values. Intriguingly, only the non-G-quadruplex aptamers showed specific inhibition of helicase activities, whereas the G-quadruplex aptamers did not inhibit helicase activities. The non-G-quadruplex aptamer with the strongest inhibitory potency was modified at the 3'-end with biotin or inverted thymidine, and the modification increased its stability in serum, particularly for the inverted thymidine modification. Structural diversity in selection coupled to post-selection stabilisation has provided new insights into the aptamers that were selected for a helicase target. These aptamers are being further developed to inhibit SARS-CoV replication.