260. Long-Term Neurologic Correction in the Pompe Disease Mice By Intrathecal Gene Therapy (original) (raw)
Related papers
Journal of Biological Chemistry, 2011
RNA-based drugs are an emerging class of therapeutics. They have the potential to regulate proteins, chromatin, as well as bind to specific proteins of interest in the form of aptamers. These aptamers are protected from nuclease attack by chemical modifications that enhance their stability for in vivo usage. However, nucleases are ubiquitous, and as we have yet to characterize the entire human microbiome it is likely that many nucleases are yet to be identified. Any novel, unusual enzymes present in vivo might reduce the efficacy of RNA-based therapeutics, even when they are chemically modified. We have previously identified an RNA-based aptamer capable of neutralizing a broad spectrum of clinical HIV-1 isolates and are developing it as a vaginal and rectal microbicide candidate. As a first step we addressed aptamer stability in the milieu of proteins present in these environments. Here we uncover a number of different nucleases that are able to rapidly degrade 2-F-modified RNA. We demonstrate that the aptamer can be protected from the nuclease(s) present in the vaginal setting, without affecting its antiviral activity, by replacement of key positions with 2-O-Me-modified nucleotides. Finally, we show that the aptamer can be protected from all nucleases present in both vaginal and rectal compartments using Zn 2؉ cations. In conclusion we have derived a stable, antiviral RNA-based aptamer that could form the basis of a pre-exposure microbicide or be a valuable addition to the current tenofovir-based microbicide candidate undergoing clinical trials.
Nucleic acid aptamers are single-stranded DNA or RNA oligonucleotide sequences that bind to a specific target molecule with high affinity and specificity through their ability to adopt 3-dimensional structure in solution. Aptamers have huge potential as targeted therapeutics, diagnostics, delivery agents and as biosensors. However, aptamers composed of natural nucleotide monomers are quickly degraded in vivo and show poor pharmacodynamic properties. To overcome this, chemically-modified nucleic acid aptamers are developed by incorporating modified nucleotides after or during the selection process by Systematic Evolution of Ligands by EXponential enrichment (SELEX). This review will discuss the development of chemically-modified aptamers and provide the pros and cons, and new insights on in vitro aptamer selection strategies by using chemically-modified nucleic acid libraries. Abbreviations: SELEX, Systematic Evolution of Ligands by EXponential enrichment; US FDA, United States Food and Drug Administration; AMD, Age-related macular degeneration; VEGF, Vascular endothelial growth factor protein, 2 0-NH 2 , 2 0-Amino; 2 0-OH, 2 0-Hydroxyl; K d , Equilibrium dissociation constant; 2 0-OMe, 2 0-O-Methyl; Bfgf, Basic fibroblast growth factor; 2 0-F, 2 0-Fluoro; PSMA, Prostate specific membrane antigen; IFN-g, Interferon-gamma; KGF, Keratinocyte growth factor; 4 0-S, 4 0-Thio; 2 0-FANA, 2 0-Fluroarabino nucleic acid; HNA, 1,5-Anhydro hexitol nucleic acid; TAR, transactivation responsive element; TNA, Threose nucleic acid; LNA, Locked nucleic acid; SOMAmers, Slow Off-rate Modified Aptamers KEYWORDS Aptamers; chemical antibodies; chemicallymodified aptamers; in vitro selection; modified nucleotides; nucleic acid ligands; SELEX thesis. Generally, a web-based secondary structure prediction algorithm (e.g. mfold, 12 RNAfold 13) is used as a tool to assist with the positioning of chemically-modified nucleotides and to truncate the overall size of the selected aptamers during chemical synthesis. Such chemically-fabricated
DNA and RNA Aptamers: From Tools for Basic Research Towards Therapeutic Applications
Combinatorial Chemistry & High Throughput Screening, 2006
The systematic evolution of ligands by exponential enrichment (SELEX) is a combinatorial oligonucleotide library-based in vitro selection approach in which DNA or RNA molecules are selected by their ability to bind their targets with high affinity and specificity, comparable to those of antibodies. Nucleic acids with high affinity for their targets have been selected against a wide variety of compounds, from small molecules, such as ATP, to membrane proteins and even whole organisms. Recently, the use of the SELEX technique was extended to isolate oligonucleotide ligands, also known as aptamers, for a wide range of proteins of importance for therapy and diagnostics, such as growth factors and cell surface antigens. The number of aptamers generated as inhibitors of various target proteins has increased following automatization of the SELEX process. Their diagnostic and therapeutic efficacy can be enhanced by introducing chemical modifications into the oligonucleotides to provide resistance against enzymatic degradation in body fluids. Several aptamers are currently being tested in preclinical and clinical trials, and aptamers are in the process of becoming a new class of therapeutic agents. Recently, the anti-VEGF aptamer pegaptanib received FDA approval for treatment of human ocular vascular disease.
Nucleic Acids Research, 2009
The envelope glycoprotein of human immunodeficiency virus (HIV) consists of an exterior glycoprotein (gp120) and a trans-membrane domain (gp41) and has an important role in viral entry into cells. HIV-1 entry has been validated as a clinically relevant anti-viral strategy for drug discovery. In the present work, several 2'-F substituted RNA aptamers that bind to the HIV-1 BaL gp120 protein with nanomole affinity were isolated from a RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. From two of these aptamers we created a series of new dual inhibitory function anti-gp120 aptamer-siRNA chimeras. The aptamers and aptamer-siRNA chimeras specifically bind to and are internalized into cells expressing HIV gp160. The Dicer-substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells (PBMCs). Moreover, we have introduced a 'sticky' sequence onto a chemically synthesized aptamer which facilitates attachment of the Dicer substrate siRNAs for potential multiplexing. Our results provide a set of novel inhibitory agents for blocking HIV replication and further validate the use of aptamers for delivery of Dicer substrate siRNAs.
Aptamers are single stranded nucleic acids that specifically recognize and bind tightly to their cognate targets due to their stable three dimensional structures. Nucleic acid aptamers have been developed for various applications, including diagnostics, molecular imaging and biomarker discovery along with target validation, therapeutics, and drug delivery. Since its discovery in the early 1990s, aptamer technology has progressed tremendously. Automated selection procedures now allow rapid identification of DNA and RNA sequences that can target a broad range of extra and intracellular proteins with nanomolar affinities and high specificities. The unique binding properties of nucleic acids, which are amenable to various modifications, make aptamers perfectly suitable for different areas of biotechnology. RNA aptamers represent an emerging class of pharmaceuticals with great potential for targeted cancer diagnostics and therapy. Several RNA aptamers that bind cancer cell-surface antigens with high affinity and specificity have been described. However, their clinical potential has yet to be realized the demand for diagnostic assays to assist in the management of existing and emerging diseases is increasing, and aptamers could potentially fulfill molecular recognition needs in those assays. Compared with the bellwether antibody technology, aptamer research is still in its infancy, but it is progressing at a fast pace. The potential of aptamers may be realized in the near future in the form of aptamer-based diagnostic products in the market. In such products, aptamers may play a key role either in conjunction with, or in place of antibodies. It is also likely that existing diagnostic formats may change according to the need to better harness the unique features. This review summarizes aptamers as valuable tools for diagnostics, target validation, drug discovery, and even therapeutic approaches. Keywords RNA Aptamers, SELEX, RNA Therapy, Drug Delivery
Poly-Target Selection Identifies Broad-Spectrum RNA Aptamers
Molecular Therapy - Nucleic Acids, 2018
Aptamer selections often yield distinct subpopulations, each with unique phenotypes that can be leveraged for specialized applications. Although most selections aim to attain ever higher specificity, we sought to identify aptamers that recognize increasingly divergent primate lentiviral reverse transcriptases (RTs). We hypothesized that aptamer subpopulations in libraries pre-enriched against a single RT may exhibit broadspectrum binding and inhibition, and we devised a multiplexed poly-target selection to elicit those phenotypes against a panel of primate lentiviral RTs. High-throughput sequencing and coenrichment/codepletion analysis of parallel and duplicate selection trajectories rapidly narrowed the list of candidate aptamers by orders of magnitude and identified dozens of priority candidates for further screening. Biochemical characterization validated a novel aptamer motif and several rare and unobserved variants of previously known motifs that inhibited recombinant RTs to varying degrees. These broad-spectrum aptamers also suppressed replication of viral constructs carrying phylogenetically diverse RTs. The poly-target selection and coenrichment/codepletion approach described herein is a generalizable strategy for identifying cross-reactivity among related targets from combinatorial libraries.
Biochemistry, 2009
The transactivating responsive (TAR) element is a RNA hairpin located in the 5 0 untranslated region of HIV-1 mRNA. It is essential for full-length transcription of the retroviral genome and therefore for HIV-1 replication. Hairpin aptamers that generate highly stable and specific complexes with TAR were previously identified, thus decreasing the level of TAR-dependent expression in cultured cells [Kolb, G., et al. (2006) RNA Biol. 3, 150-156]. We performed genomic SELEX against TAR using a human RNA library to identify human transcripts that might interact with the retroviral genome through loop-loop interactions and potentially contribute to the regulation of TAR-mediated processes. We identified a genomic aptamer termed a1 that folds as a hairpin with an apical loop complementary to five nucleotides of the TAR hexanucleotide loop. Surface plasmon resonance experiments performed on a truncated or mutated version of the a1 aptamer, in the presence of the Rop protein of Escherichia coli, indicate the formation of a highly stable a1-TAR kissing complex. The 5 0 ACCCAG loop of a1 constitutes a new motif of interaction with the TAR loop.