Self-delivering RNAi compounds as therapeutic agents in the central nervous system to enhance axonal regeneration after injury.

The therapeutic use of RNAi has grown but often faces several hurdles related to delivery systems, compound stability, immune activation, and on-target/off-tissue effects. Self-delivering RNAi (sdRNA) molecules do not require delivery agents or excipients. Here we demonstrate the ability of sdRNA to reduce the expression of PTEN (phosphatase and tensin homolog) to stimulate regenerative axon regrowth in the injured adult CNS. PTEN-targeting sdRNA compounds were tested for efficacy in vivo by intravitreal injection after adult rat optic nerve injury. We describe critical steps in lead compound generation through the optimization of nucleotide modifications, enhancements for stability in biological matrices, and screening for off-target immunostimulatory activity. The data show that PTEN expression in vivo can be reduced using sdRNA and this enhances regeneration in adult CNS neurons after injury, raising the possibility that this method could be utilized for other clinically relevant nervous system indications.

Visualization of self-delivering hydrophobically modified siRNA cellular internalization.

siRNAs are a new class of therapeutic modalities with promising clinical efficacy that requires modification or formulation for delivery to the tissue and cell of interest. Conjugation of siRNAs to lipophilic groups supports efficient cellular uptake by a mechanism that is not well characterized. Here we study the mechanism of internalization of asymmetric, chemically stabilized, cholesterol-modified siRNAs (sd-rxRNAs®) that efficiently enter cells and tissues without the need for formulation. We demonstrate that uptake is rapid with significant membrane association within minutes of exposure followed by the formation of vesicular structures and internalization. Furthermore, sd-rxRNAs are internalized by a specific class of early endosomes and show preferential association with epidermal growth factor (EGF) but not transferrin (Tf) trafficking pathways as shown by live cell TIRF and structured illumination microscopy (SIM). In fixed cells, we observe ∼25% of sd-rxRNA co-localizing with EGF and <5% with Tf, which is indicative of selective endosomal sorting. Likewise, preferential sd-rxRNA co-localization was demonstrated with EEA1 but not RBSN-containing endosomes, consistent with preferential EGF-like trafficking through EEA1-containing endosomes. sd-rxRNA cellular uptake is a two-step process, with rapid membrane association followed by internalization through a selective, saturable subset of the endocytic process. However, the mechanistic role of EEA1 is not yet known. This method of visualization can be used to better understand the kinetics and mechanisms of hydrophobic siRNA cellular uptake and will assist in further optimization of these types of compounds for therapeutic intervention.

Diphencyprone Treatment of Alopecia Areata: Postulated Mechanism of Action and Prospects for Therapeutic Synergy with RNA Interference.

Diphencyprone (DPCP) is a potent topical sensitizing agent that has been used since the late 1970s by physicians for the treatment of alopecia areata (AA), viral warts (human papillomavirus) and cutaneous metastases of melanoma. Although to date the compound is not approved as a drug by the FDA or EMA, physicians have continued to use DPCP because of its proven effects in these dermatological conditions. The use of the drug has been highly variable because of differences in compounding, and as a result, the literature reports vary widely in the concentrations used for sensitization and challenge treatment with DPCP. The efficacy of DPCP has generally been ascribed to immunological reactions by the host. Inducing inflammation with a contact sensitizer is counterintuitive to treating AA, an autoimmune disorder. We have hypothesized that the body's attempt to downregulate the inflammation caused by the contact sensitizer may also ameliorate AA. Studies using microarray and miRNA profiling may provide information about how DPCP induces inflammation in human skin at different times. Gene targets and microRNAs identified through these data may be modulated by an RNA interference approach to enhance DPCP efficacy and response rates. In addition, this approach may result in the discovery and development of drugs that are more potent and selective for the treatment of AA.

Novel hydrophobically modified asymmetric RNAi compounds (sd-rxRNA) demonstrate robust efficacy in the eye.

PURPOSE: The major challenges of developing an RNAi therapeutic include efficient delivery to and entry into the cell type of interest. Conventional ("naked" and chemically stabilized) small interfering RNAs (siRNAs) have been used in the eye in the past but they demonstrated limited clinical efficacy. Here we investigated a recently developed class of small, hydrophobic, asymmetric RNAi compounds. These compounds, termed "self-delivering rxRNAs" (sd-rxRNA(®)), are extensively modified, have a small duplex region of <15 base pairs, contain a fully phosphorothioated single-stranded tail, and readily enter cells and tissues without the requirement for a delivery vehicle. METHODS: We compared sd-rxRNA compounds with stabilized siRNAs in vitro (in ARPE-19 cells) and in vivo (intravitreal injection in mouse and rabbit eyes). Specifically, we investigated the retinal uptake, distribution, efficacy, and preliminary safety of sd-rxRNAs. RESULTS: Treatment with sd-rxRNAs resulted in uniform cellular uptake and full retina penetration in both animal models while no detectable cellular uptake was observed with stabilized siRNAs either in vitro or in vivo. Further, both in vitro and in vivo delivery (without any transfection reagent or formulation) resulted in a significant reduction of the targeted mRNA levels, which lasted 14-21 days in vivo. Retinal morphology and function were unaltered following a single administration of sd-rxRNAs. CONCLUSION: These data support the potential of developing sd-rxRNAs as a therapeutic for ocular disease.

Rabenosyn-5 defines the fate of the transferrin receptor following clathrin-mediated endocytosis.

Cell surface receptors and other proteins internalize through diverse mechanisms at the plasma membrane and are sorted to different destinations. Different subpopulations of early endosomes have been described, raising the question of whether different internalization mechanisms deliver cargo into different subsets of early endosomes. To address this fundamental question, we developed a microscopy platform to detect the precise position of endosomes relative to the plasma membrane during the uptake of ligands. Axial resolution is maximized by concurrently applied total internal reflection fluorescence and epifluorescence-structured light. We found that transferrin receptors are delivered selectively from clathrin-coated pits on the plasma membrane into a specific subpopulation of endosomes enriched in the multivalent Rab GTPase and phosphoinositide-binding protein Rabenosyn-5. Depletion of Rabenosyn-5, but not of other early endosomal proteins such as early endosome antigen 1, resulted in impaired transferrin uptake and lysosomal degradation of transferrin receptors. These studies reveal a critical role for Rabenosyn-5 in determining the fate of transferrin receptors internalized by clathrin-mediated endocytosis and, more broadly, a mechanism whereby the delivery of cargo from the plasma membrane into specific early endosome subpopulations is required for its appropriate intracellular traffic.

Potent and systematic RNAi mediated silencing with single oligonucleotide compounds.

RNA interference (RNAi) has been established as an important tool for functional genomics studies and has great promise as a therapeutic intervention for human diseases. In mammalian cells, RNAi is conventionally induced by 19-27-bp RNA duplexes generated by hybridization of two complementary oligonucleotide strands (oligos). Here we describe a novel class of RNAi molecules composed of a single 25-28-nucleotide (nt) oligo. The oligo has a 16-nt mRNA targeting region, followed by an additional 8-10 nt to enable self-dimerization into a partially complementary duplex. Analysis of numerous diverse structures demonstrates that molecules composed of two short helices separated by a loop can efficiently enter and activate the RNA-induced silencing complex (RISC). This finding enables the design of highly effective single-oligo compounds for any mRNA target.

Monoalkylglycerol ether lipids promote adipogenesis.

The molecular mechanisms that lead to the generation of adipose tissue (adipogenesis) are of basic and biomedical interest. Cellular models of adipogenesis have proven extremely valuable in defining biomolecules-primarily genes and proteins-that regulate adipogenesis. Here, the analysis of differentiating adipocytes using an untargeted metabolomics approach led to the discovery of the monoalkylglycerol ethers as a natural class of adipocyte differentiation factors.

Use of L-asparagine and N-phosphonacetyl-L-asparagine to investigate the linkage of catalysis and homotropic cooperativity in E. coli aspartate transcarbomoylase.

The mechanism of domain closure and the allosteric transition of Escherichia coli aspartate transcarbamoylase (ATCase) are investigated using L-Asn, in the presence of carbamoyl phosphate (CP), and N-phosphonacetyl-L-asparagine (PASN). ATCase was found to catalyze the carbamoylation of L-Asn with a K(m) of 122 mM and a maximal velocity 10-fold lower than observed with the natural substrate, L-Asp. As opposed to L-Asp, no cooperativity was observed with respect to L-Asn. Time-resolved small-angle X-ray scattering (SAXS) and fluorescence experiments revealed that the combination of CP and L-Asn did not convert the enzyme from the T to the R state. PASN was found to be a potent inhibitor of ATCase exhibiting a K(D) of 8.8 microM. SAXS experiments showed that PASN was able to convert the entire population of molecules to the R state. Analysis of the crystal structure of the enzyme in the presence of PASN revealed that the binding of PASN was similar to that of the R-state complex of ATCase with N-phosphonaceyl-L-aspartate, another potent inhibitor of the enzyme. The linking of CP and L-Asn into one molecule, PASN, correctly orients the asparagine moiety in the active site to induce domain closure and the allosteric transition. This entropic effect allows for the high affinity binding of PASN. However, the binding of L-Asn, in the presence of a saturating concentration of CP, does not induce the closure of the two domains of the catalytic chain, nor does the enzyme undergo the transition to the high-activity high- affinity R structure. These results imply that Arg229, which interacts with the beta-carboxylate of L-Asp, plays a critical role in the orientation of L-Asp in the active site and demonstrates the requirement of the beta-carboxylate of L-Asp in the mechanism of domain closure and the allosteric transition in E. coli ATCase.

N-phosphonacetyl-L-isoasparagine a potent and specific inhibitor of Escherichia coli aspartate transcarbamoylase.

The synthesis of a new inhibitor, N-phosphonacetyl-L-isoasparagine (PALI), of Escherichia coli aspartate transcarbamoylase (ATCase) is reported, as well as structural studies of the enzyme.PALI complex. PALI was synthesized in 7 steps from beta-benzyl L-aspartate. The KD of PALI was 2 microM. Kinetics and small-angle X-ray scattering experiments showed that PALI can induce the cooperative transition of ATCase from the T to the R state. The X-ray structure of the enzyme.PALI complex showed 22 hydrogen-bonding interactions between the enzyme and PALI. The kinetic characterization and crystal structure of the ATCase.PALI complex also provides detailed information regarding the importance of the alpha-carboxylate for the binding of the substrate aspartate.

T-state inhibitors of E. coli aspartate transcarbamoylase that prevent the allosteric transition.

Escherichia coli aspartate transcarbamoylase (ATCase) catalyzes the committed step in pyrimidine nucleotide biosynthesis, the reaction between carbamoyl phosphate (CP) and l-aspartate to form N-carbamoyl-l-aspartate and inorganic phosphate. The enzyme exhibits homotropic cooperativity and is allosterically regulated. Upon binding l-aspartate in the presence of a saturating concentration of CP, the enzyme is converted from the low-activity low-affinity T state to the high-activity high-affinity R state. The potent inhibitor N-phosphonacetyl-l-aspartate (PALA), which combines the binding features of Asp and CP into one molecule, has been shown to induce the allosteric transition to the R state. In the presence of only CP, the enzyme is the T structure with the active site primed for the binding of aspartate. In a structure of the enzyme-CP complex (T(CP)), two CP molecules were observed in the active site approximately 7A apart, one with high occupancy and one with low occupancy. The high occupancy site corresponds to the position for CP observed in the structure of the enzyme with CP and the aspartate analogue succinate bound. The position of the second CP is in a unique site and does not overlap with the aspartate binding site. As a means to generate a new class of inhibitors for ATCase, the domain-open T state of the enzyme was targeted. We designed, synthesized, and characterized three inhibitors that were composed of two phosphonacetamide groups linked together. These two phosphonacetamide groups mimic the positions of the two CP molecules in the T(CP) structure. X-ray crystal structures of ATCase-inhibitor complexes revealed that each of these inhibitors bind to the T state of the enzyme and occupy the active site area. As opposed to the binding of Asp in the presence of CP or PALA, these inhibitors are unable to initiate the global T to R conformational change. Although the best of these T-state inhibitors only has a K(i) value in the micromolar range, the structural information with respect to their mode of binding provides important information for the design of second generation inhibitors that will have even higher affinity for the active site of the T state of the enzyme.

Structure of the E.coli aspartate transcarbamoylase trapped in the middle of the catalytic cycle.

Snapshots of the catalytic cycle of the allosteric enzyme aspartate transcarbamoylase have been obtained via X-ray crystallography. The enzyme in the high-activity high-affinity R state contains two catalytic chains in the asymmetric unit that are different. The active site in one chain is empty, while the active site in the other chain contains an analog of the first substrate to bind in the ordered mechanism of the reaction. Small angle X-ray scattering shows that once the enzyme is converted to the R state, by substrate binding, the enzyme remains in the R state until substrates are exhausted. Thus, this structure represents the active form of the enzyme trapped at two different stages in the catalytic cycle, before the substrates bind (or after the products are released), and after the first substrate binds. Opening and closing of the catalytic chain domains explains how the catalytic cycle occurs while the enzyme remains globally in the R-quaternary structure.

Structural basis for ordered substrate binding and cooperativity in aspartate transcarbamoylase.

X-ray structures of aspartate transcarbamoylase in the absence and presence of the first substrate carbamoyl phosphate are reported. These two structures in conjunction with in silico docking experiments provide snapshots of critical events in the function of the enzyme. The ordered substrate binding, observed experimentally, can now be structurally explained by a conformational change induced upon the binding of carbamoyl phosphate. This induced fit dramatically alters the electrostatics of the active site, creating a binding pocket for aspartate. Upon aspartate binding, a further change in electrostatics causes a second induced fit, the domain closure. This domain closure acts as a clamp that both facilitates catalysis by approximation and also initiates the global conformational change that manifests homotropic cooperativity.

Alkaline phosphatase from the hyperthermophilic bacterium T. maritima requires cobalt for activity.

The hyperthermophilic bacterium Thermotoga maritima encodes a gene sharing sequence similarities with several known genes for alkaline phosphatase (AP). The putative gene was isolated and the corresponding protein expressed in Escherichia coli, with and without a predicted signal sequence. The recombinant protein showed phosphatase activity toward the substrate p-nitrophenyl-phosphate with a k(cat) of 16 s(-1) and a K(m) of 175 microM at a pH optimum of 8.0 when assayed at 25 degrees C. T. maritima phosphatase activity increased at high temperatures, reaching a maximum k(cat) of 100 s(-1), with a K(m) of 93 microM at 65 degrees C. Activity was stable at 65 degrees C for >24 h and at 90 degrees C for 5 h. Phosphatase activity was dependent on divalent metal ions, specifically Co(II) and Mg(II). Circular dichroism spectra showed that the enzyme gains secondary structure on addition of these metals. Zinc, the most common divalent metal ion required for activity in known APs, was shown to inhibit the T. maritima phosphatase enzyme at concentrations above 0.3 moles Zn: 1 mole monomer. All activity was abolished in the presence of 0.1 mM EDTA. The T. maritima AP primary sequence is 28% identical when compared with E. coli AP. Based on a structural model, the active sites are superimposable except for two residues near the E. coli AP Mg binding site, D153 and K328 (E. coli numbering) corresponding to histidine and tryptophan in T. maritima AP, respectively. Sucrose-density gradient sedimentation experiments showed that the protein exists in several quaternary forms predominated by an octamer.