The design, selection, and evaluation of highly specific and functional siRNA incorporating unlocked nucleobase analogs.

The efficient and specific silencing of genes via RNA interference (RNAi) for functional genomics and therapeutics depends on careful consideration of the factors that affect the functionality of small interfering RNA (siRNA). These factors include (1) the length of sequence available for siRNA targeting of an mRNA, (2) the structural and thermodynamic properties of target and siRNA sequences, (3) the mechanisms of siRNA off-target effects, and (4) the susceptibility of siRNA degradation when exposed to nucleases in serum and inside cells. Incorporation of Unlocked Nucleobase analogs (UNAs) in the siRNA design offers an attractive approach to design highly efficacious siRNAs with dramatically reduced off-target activity. Here, we describe methods and principles pertaining to the design, selection and screening of optimal siRNAs containing UNA.

Pilot trial of onabotulinumtoxina (Botox) in moderate to severe restless legs syndrome.

OBJECTIVE: To evaluate the potential of onabotulinum toxin A (Botox®, Allergan, Inc., Irvine, California) in the treatment of restless legs syndrome (RLS). METHODS: A single-arm, open-label pilot trial of onabotulinum toxin A was conducted to determine its usefulness as an injectable treatment for RLS. RESULTS: The primary outcome measure, change in score on the International Restless Legs Syndrome Scale, showed a statistically significant improvement in RLS during the first 4 weeks after treatment. Two of the secondary outcome measures, pain (measured using a visual analog scale) and patients' global impression of severity of illness also showed improvement. CONCLUSIONS: Onabotulinum toxin A warrants further study in RLS. Increasing the drug dosage, changing the dosing schedule, and increasing the number of injection sites may result in greater activity and longer duration of action.

An amino acid-based amphoteric liposomal delivery system for systemic administration of siRNA.

We demonstrate a systematic and rational approach to create a library of natural and modified, dialkylated amino acids based upon arginine for development of an efficient small interfering RNA (siRNA) delivery system. These amino acids, designated DiLA2 compounds, in conjunction with other components, demonstrate unique properties for assembly into monodisperse, 100-nm small liposomal particles containing siRNA. We show that DiLA2-based liposomes undergo a pH-dependent phase transition to an inverted hexagonal phase facilitating efficient siRNA release from endosomes to the cytosol. Using an arginine-based DiLA2, cationic liposomes were prepared that provide high in vivo siRNA delivery efficiency and are well-tolerated in both cell and animal models. DiLA2-based liposomes demonstrate a linear dose-response with an ED50 of 0.1 mg/kg against liver-specific target genes in BALB/c mice.

RNAi-based therapeutics targeting survivin and PLK1 for treatment of bladder cancer.

Harnessing RNA interference (RNAi) to silence aberrant gene expression is an emerging approach in cancer therapy. Selective inhibition of an overexpressed gene via RNAi requires a highly efficacious, target-specific short interfering RNA (siRNA) and a safe and efficient delivery system. We have developed siRNA constructs (UsiRNA) that contain unlocked nucleobase analogs (UNA) targeting survivin and polo-like kinase-1 (PLK1) genes. UsiRNAs were encapsulated into dialkylated amino acid-based liposomes (DiLA(2)) containing a nor-arginine head group, cholesteryl hemisuccinate (CHEMS), cholesterol and 1, 2-dimyristoyl-phosphatidylethanolamine-polyethyleneglycol 2000 (DMPE-PEG2000). In an orthotopic bladder cancer mouse model, intravesical treatment with survivin or PLK1 UsiRNA in DiLA(2) liposomes at 1.0 and 0.5 mg/kg resulted in 90% and 70% inhibition of survivin or PLK1 mRNA, respectively. This correlated with a dose-dependent decrease in tumor volumes which was sustained over a 3-week period. Silencing of survivin and PLK1 mRNA was confirmed to be RNA-induced silencing complex mediated as specific cleavage products were detected in bladder tumors over the duration of the study. This report suggests that intravesical instillation of survivin or PLK1 UsiRNA can serve as a potential therapeutic modality for treatment of bladder cancer.

Improved specificity of gene silencing by siRNAs containing unlocked nucleobase analogs.

siRNAs confer sequence specific and robust silencing of mRNA. By virtue of these properties, siRNAs have become therapeutic candidates for disease intervention. However, their use as therapeutic agents can be hampered by unintended off-target effects by either or both strands of the siRNA duplex. We report here that unlocked nucleobase analogs (UNAs) confer desirable properties to siRNAs. Addition of a single UNA at the 5'-terminus of the passenger strand blocks participation of the passenger strand in RISC-mediated target down-regulation with a concomitant increase in guide strand activity. Placement of a UNA in the seed region of the guide strand prevents miRNA-like off-target silencing without compromising siRNA activity. Most significantly, combined substitution of UNA at the 3'-termini of both strands, the addition of a UNA at the 5'-terminus of the passenger strand, and a single UNA in the seed region of the guide strand, reduced the global off-target events by more than 10-fold compared to unmodified siRNA. The reduction in off-target events was specific to UNA placement in the siRNA, with no apparent new off-target events. Taken together, these results indicate that when strategically placed, UNA substitutions have important implications for the design of safe and effective siRNA-based therapeutics.

Gabapentin enacarbil - clinical efficacy in restless legs syndrome.

Restless legs syndrome (RLS) is a sleep-related movement disorder commonly involving an unpleasant urge to move the limbs, typically the legs. Dopaminergic agents represent the first-line therapy for RLS; however, long-term use of such drugs results in worsening symptoms due to "augmentation" or other adverse events. Gabapentin, an analog of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), is an anticonvulsant/analgesic agent. Gabapentin is only mildly effective in relieving RLS symptoms, perhaps a result of its poor absorption from the gastrointestinal (GI) tract. Gabapentin enacarbil is a prodrug of gabapentin specifically designed to enhance absorption via the GI tract, and hence provide improved circulating levels of gabapentin on metabolism. Clinical trials to date have demonstrated favorable safety and (compared to traditional gabapentin) improved pharmacokinetics and efficacy in treating RLS symptoms. Thus, gabapentin enacarbil may prove to be a useful drug in treating RLS. An application of gabapentin enacarbil for treatment of RLS is currently pending with FDA for approval.

Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs.

The efficacy of lipid-encapsulated, chemically modified short interfering RNA (siRNA) targeted to hepatitis B virus (HBV) was examined in an in vivo mouse model of HBV replication. Stabilized siRNA targeted to the HBV RNA was incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP) and administered by intravenous injection into mice carrying replicating HBV. The improved efficacy of siRNA-SNALP compared to unformulated siRNA correlates with a longer half-life in plasma and liver. Three daily intravenous injections of 3 mg/kg/day reduced serum HBV DNA >1.0 log(10). The reduction in HBV DNA was specific, dose-dependent and lasted for up to 7 d after dosing. Furthermore, reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing. The advances demonstrated here, including persistence of in vivo activity, use of lower doses and reduced dosing frequency are important steps in making siRNA a clinically viable therapeutic approach.

High-throughput ribozyme-based assays for detection of viral nucleic acids.

Many reports have suggested that target-activated ribozymes hold potential value as detection reagents. We show that a "half"-ribozyme ligase is activated similarly by three unstructured oligoribonucleotides representing the major sequence variants of a hepatitis C virus 5'-untranslated region (5'-UTR) target and by a structured RNA corresponding to the entire 5'-UTR. Half-ribozyme ligation product was detected both in an ELISA-like assay and in an optical immunoassay through the use of hapten-carrying substrate RNAs. Both assay formats afford a limit of detection of approximately 1 x 10(6) HCV molecules (1.6 attomol, 330 fM), a sensitivity which compares favorably to that provided by standard immunoassays. These data suggest that target-activated ribozyme systems are a viable approach for the sensitive detection of viral nucleic acids using high-throughput platforms.

Monitoring protein modification with allosteric ribozymes.

An allosteric ribozyme is an RNA-based enzyme (ribozyme) whose catalytic activity is modulated by molecular recognition of a protein. The direct coupling of a detectable catalytic event to molecular recognition by an allosteric ribozyme enables simple assays for quantitative protein detection. Most significantly, the mode of development and molecular recognition characteristics of allosteric ribozymes are fundamentally different from antibodies, providing them with functional characteristics that complement those of antibodies. Allosteric ribozymes can be developed using native proteins and, therefore, are often sensitive to protein conformation. In contrast, antibodies tend to recognize a series of adjacent amino acids as a consequence of antigen presentation and typically are not sensitive to protein conformation. Unlike antibody development, the development of allosteric ribozymes is a completely in vitro process that allows the specificity of an allosteric ribozyme to be tightly controlled. These significant differences from antibodies allow the pre-programmed development of conformation-state-specific protein detection reagents that can be used to investigate the activation-state of signal transduction components.

Zeptomole detection of a viral nucleic acid using a target-activated ribozyme.

We describe a strategy for the ultra-sensitive detection of nucleic acids using "half" ribozymes that are devoid of catalytic activity unless completed by a trans-acting target nucleic acid. The half-ribozyme concept was initially demonstrated using a construct derived from a multiple turnover Class I ligase. Iterative RNA selection was carried out to evolve this half-ribozyme into one activated by a conserved sequence present in the hepatitis C virus (HCV) genome. Following sequence optimization of substrate RNAs, this HCV-activated half-ribozyme displayed a maximal turnover rate of 69 min(-1) (pH 8.3) and was induced in rate by approximately 2.6 x 10(9)-fold by the HCV target. It detected the HCV target oligonucleotide in the zeptomole range (6700 molecules), a sensitivity of detection roughly 2.6 x 10(6)-fold greater than that previously demonstrated by oligonucleotide-activated ribozymes, and one that is sufficient for molecular diagnostic applications.

A novel, modification-dependent ATP-binding aptamer selected from an RNA library incorporating a cationic functionality.

An analogue of uridine triphosphate containing a cationic functional group was incorporated into a degenerate RNA library by enzymatic polymerization. In vitro selection experiments using this library yielded a novel receptor that binds ATP under physiological pH and salt conditions in a manner completely dependent on the presence of the cationic functionality. The consensus sequence and a secondary structure model for the ATP binding site were obtained by the analysis of functional sequences selected from a partially randomized pool based on the minimal parental sequence. Mutational studies of this receptor indicated that several of the modified uridines are critical for ATP binding. Analysis of the binding of ATP analogues revealed that the modified RNA receptor makes numerous contacts with ATP, including interactions with the triphosphate group. In contrast, the aptamer repeatedly isolated from natural RNA libraries does not interact with the triphosphate group of ATP. The incorporation of a cationic amine into nucleic acids clearly allows novel interactions to occur during the molecular recognition of ligands, which carries interesting implications for the RNA world hypothesis. In addition, new materials generated from such functionalized nucleic acids could be useful tools in research and diagnostics.

Monitoring post-translational modification of proteins with allosteric ribozymes.

An allosteric hammerhead ribozyme activated specifically by the unphosphorylated form of the protein kinase ERK2 was created through a rational design strategy that relies on molecular recognition of ERK2 to decrease the formation of an alternate, inactive ribozyme conformer. Neither closely related mitogen-activated protein kinases (MAPKs) nor the phosphorylated form of ERK2 induced ribozyme activity. The ribozyme quantitatively detected ERK2 added to mammalian cell lysates and also functioned quantitatively in a multiplexed solution-phase assay. This same strategy was used to construct a second ribozyme selectively activated by the phosphorylated (active) form of ERK2. This approach is generally applicable to the development of ribozymes capable of monitoring post-translational modification of specific proteins.