In vivo activity of nuclease-resistant siRNAs

Chemical modifications have been incorporated into short interfering RNAs (siRNAs) without reducing their ability to inhibit gene expression in mammalian cells grown in vitro. In this study, we begin to assess the potential utility of 2'-modified siRNAs in mammals. We demonstrate that siRNA modified with 2'-fluoro (2'-F) pyrimidines are functional in cell culture and have a greatly increased stability and a prolonged half-life in human plasma as compared to 2'-OH containing siRNAs. Moreover, we show that the 2'-F containing siRNAs are functional in mice and can inhibit the expression of a target gene in vivo. However, even though the modified siRNAs have greatly increased resistance to nuclease degradation in plasma, this increase in stability did not translate into enhanced or prolonged inhibitory activity of target gene reduction in mice following tail vein injection. Thus, this study shows that 2'-F modified siRNAs are functional in vivo, but that they are not necessarily more potent than unmodified siRNAs in animals.     

Immunostimulatory properties and antiviral activity of modified HBV-specific siRNAs

Sequence-specific gene silencing by small interfering RNA (siRNA) is an intense area of focus in the development of novel therapeutic agents. Currently, there are two major hurdles to achieving clinically effective siRNA-based therapeutics: establishment of an efficient delivery system that transfers the siRNA to the correct tissue(s); and the reduction of unintended immunotoxicity associated with unmodified siRNA. We have developed a novel liver-specific delivery system of apolipoprotein A-I-decorated cationic lipids (DTC-Apo). Here, we show that intravenous injection of an unmodified hepatitis B virus (HBV)-specific siRNA encapsulated in DTC-Apo activates the innate immune response in mice. However, 2′-O-methyl (2′-OMe) modification of siRNA sense-strand uridine or uridine/adenosine residues efficiently abrogated the immunostimulatory properties of the siRNA and also silenced viral replication. In contrast, pyrimidine modification by 2′-OMe or 2′-fluoro (2’-F) substitution failed to circumvent liposome-induced immune recognition. Our findings provide useful information for the design of chemically-modified siRNAs for in vivo applications.     

Synthesis and biological properties of chemically modified siRNAs bearing 1-deoxy-D-ribofuranose in their 3'-overhang region

To elucidate the role of the sugar moiety in the two natural nucleotides of the 3'-overhang region of small interfering RNA (siRNA), we synthesized siRNAs that incorporated two abasic nucleosides, 1-deoxy-D-ribofuranose (R(H)). We improved the method for preparing an O-protected abasic nucleoside, 1-deoxy-2,3,5-tri-O-benzoyl-β-D-ribofuranose, via the reductive cleavage of the anomeric position of 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose. To incorporate R(H) into oligonucleotides by the standard phosphoramidite solid phase method, R(H) was converted into its phosphoramidite derivative and the solid support linked to a controlled pore glass resin. Chemically modified RNAs possessing R(H) at the 3'-overhang region were easily prepared in good yields. siRNAs containing R(H) showed moderate nuclease-resistance and a desirable knockdown effect.     

Toxicological and pharmacokinetic properties of chemically modified siRNAs targeting p53 RNA following intravenous administration

We report the toxicological and pharmacokinetic properties of the synthetic, small interfering RNA I5NP following intravenous administration in rodents and nonhuman primates. I5NP is designed to act via the RNA interference (RNAi) pathway to temporarily inhibit expression of the pro-apoptotic protein p53 and is being developed to protect cells from acute ischemia/reperfusion injuries such as acute kidney injury that can occur during major cardiac surgery and delayed graft function that can occur following renal transplantation. Following intravenous administration, I5NP was very rapidly cleared from plasma was distributed predominantly to the kidney, with very low levels in liver and other tissues. Doses of 800 mg/kg I5NP in rodents, and 1,000 mg/kg I5NP in nonhuman primates, were required to elicit adverse effects, which in the monkey were isolated to direct effects on the blood that included a sub-clinical activation of complement and slightly increased clotting times. In the rat, no additional adverse effects were observed with a rat analogue of I5NP, indicating that the effects likely represent class effects of synthetic RNA duplexes rather than toxicity related to the intended pharmacologic activity of I5NP. Taken together, these data support clinical testing of intravenous administration of I5NP for the preservation of renal function following acute ischemia/reperfusion injury.     

Studies on aminoisonucleoside modified siRNAs: stability and silencing activity

A novel class of aminoisonucleoside was synthesized and incorporated into a luciferase gene-targeting siRNA. Structural and functional analyses of such a kind of siRNAs indicated that sense strand modifications with aminoisonucleoside at the 3' or 5' terminal, such as ssIso-1 and ssIso-2, have less effect on RNA duplex thermal and serum stabilities, and their functional activities are also comparable to their native siRNAs. In contrast, antisense strand modifications with aminoisonucleoside at the corresponding positions, such as asIso-2 or asIso-1, bring a striking negative effect on RNA duplex stability but still maintain around 40-50% of gene knockdown.     

RNAi activity of siRNAs modified with 2'-aminoalkyl-substituted fluorinated nucleobases

We recently reported that a 1'-deoxy-1'-(4,6-difluoro-1H-benzimidazol-1-yl)-2'-(beta-aminoethyl)-beta-d-ribofuranose nucleoside appears to be a universal nucleoside which does not differentiate between the four natural nucleosides A, C, G, and U in duplexes. Moreover, ribozymes modified with this nucleoside analog showed a better or at least equal catalytic activity relative to Watson-Crick mismatches.[1] Due to these data, we investigated the ability of this compound to tolerate Watson-Crick mismatches in order to avoid HIV escape mutations in RNA interference. The influence of this nucleoside analog on siRNA efficiency was analyzed with a proven siRNA targeting GFP.     

Catalytic activity and conformation of chemically modified subtilisin Carlsberg in organic media

Subtilisin Carlsberg, an alkaline protease from Bacillus licheniformis, was modified with polyoxyethylene (PEG) or aerosol-OT (AOT), and the solubility, conformation, and catalytic activity of the modified subtilisins in some organic media were compared under the same conditions. The solubility of modified subtilisins depended on the solubility of the modifier. On the other hand, the conformational changes depended on the solubility, rather than the property, of the modifier. When the modified subtilisin was dissolved in water-miscible polar solvents such as dimethylsulfoxide, acetonitrile, and tetrahydrofuran, significant conformational changes occurred. When modified subtilisin was dissolved in water-immiscible organic solvents, such as isooctane and benzene, the solvent did not induce significant conformational changes. The catalytic activity in the transesterification reaction of the N-acetyl-L-phenylalanine ethylester of the modified subtilisin in organic solvents was higher than that of native subtilisin. The high activity of modified subtilisin was thought to be due to a homogeneous reaction by the dissolved enzymes.     

Molecular imaging study on in vivo distribution and pharmacokinetics of modified small interfering RNAs (siRNAs)

Molecular imaging was used to study the biodistribution, pharmacokinetics, and activity of naked small interfering RNAs (siRNAs). siRNAs with riboses chemically modified in the 2' position were compared with unmodified siRNA. In vitro, replacement of the 2'-hydroxyl (2'OH) group of certain nucleotides in an siRNA sequence by a fluorine atom (2'F) on both antisense (AS) and sense (S) strands [2'F(AS/S)], or by a methoxy group (2'OMe) on the S strand [2'OH(AS)/2'OMe(S)], was compatible with RNA interference. Different siRNAs [2'F(AS/S), 2'OH(AS)/2'OMe(S), and 2'OH(AS/S)] were labeled with fluorine-18 (conjugation with [(18)F]FPyBrA), and comparative dynamic and quantitative imaging was performed with positron emission tomography. After intravenous injections of [(18)F]siRNAs in rodents, total radioactivity was rapidly eliminated by the kidneys and the liver. Tissue distribution of the different siRNAs were similar, and their bioavailability (as judged from blood persistence and stability) increased in the order 2'OH(AS/S) = 2'OH(AS)/2'OMe(S) < 2'F(AS/S). However, in our in vivo model, the 2'F(AS/S) siRNA, despite its higher bioavailability, was not able to induce a higher interference effect with respect to the 2'OH(AS/S) siRNA. Molecular imaging approaches, applied in the present work to both natural and chemically modified siRNAs, can contribute to the development of these macromolecules as therapeutic agents.     

Tolerated wobble mutations in siRNAs decrease specificity, but can enhance activity in vivo

RNA interference (RNAi) has become an invaluable tool for functional genomics. A critical use of this tool depends on an understanding of the factors that determine the specificity and activity of the active agent, small interfering RNA (siRNA). Several studies have concluded that tolerance of mutations can be considerable and hence lead to off-target effects. In this study, we have investigated in vivo the toleration of wobble (G:U) mutations in high activity siRNAs against Flap Endonuclease 1 (Fen1) and Aquaporin-4 (Aqp4). Mutations in the central part of the antisense strand caused a pronounced decrease in activity, while mutations in the 5′ and 3′ends were tolerated very well. Furthermore, based on analysis of nine different mutated siRNAs with widely differing intrinsic activities, we conclude that siRNA activity can be significantly enhanced by wobble mutations (relative to mRNA), in the 5′ terminal of the antisense strand. These findings should facilitate design of active siRNAs where the target mRNA offers limited choice of siRNA positions.     

Synthesis and ex vivo profiling of chemically modified cytomegalovirus CMVpp65 epitopes.

The effect of substituting unnatural hydrophobic amino acids into the critical MHC binding residues of an HLA-A*0201-restricted cytomegalovirus CMVpp65 epitope, NLVPMVATV, has been investigated. A new set of peptides containing the amino acids tert-butyl glycine (Tgl), cyclohexyl glycine (Chg), neo-pentyl glycine (Npg), cyclohexyl alanine (Cha) and cyclo leucine (Cyl), at either position 2, to mimic Leu, or position 9, to mimic Val, have been synthesised. Immunological profiling using class I MHC stabilisation assays to assess MHC binding affinity, and enzyme-linked immunospot (ELISPOT) assays to assess the ability of the modified peptides to re-stimulate a specific cytotoxic T-lymphocyte (CTL) response, compared to the native epitope, have been performed. It was found that the majority of the unnatural substitutions resulted in a decrease in either HLA-A*0201 binding affinity or cytotoxic T-cell activity. However, the HLA-A*0201 binding affinity was unrelated to the ability to re-stimulate a T-cell response. Minimisation and molecular dynamics studies proved helpful in dissecting the ELISPOT responses. Two principal peptide binding modes were found by minimisation, designated kinked and straight. Peptides that bound in a kinked conformation were poor at re-stimulating a T-cell response. Of the peptides that bound in a straight conformation, molecular dynamics (MD) simulations revealed that those capable of re-stimulating the strongest responses had the greatest degree of flexibility (as determined by RMSD values across the MD simulation) around the P6 residue, one of the residues important for T-cell receptor recognition.     

Improved stability and catalytic activity of chemically modified papain in aqueous organic solvents

Papain was modified with the anhydrides of various monocarboxylic (acetic or propionic) and dicarboxylic (citraconic, maleic or succinic) acids. 7–10 of the 11 primary amino groups of the enzyme were modified. The organic solvent tolerances of the modified enzyme forms were increased (especially in the concentration range of 10–60%) in comparison with the unmodified enzyme. Acylation enhanced the catalytic activity and stability of papain both in buffer and in aqueous organic solvents (ethanol and acetonitrile). Decrease of the positive charges on the surface of papain resulted in a higher enzyme stability than when they were replaced by negative charges. The kinetic parameters revealed that in aqueous ethanol the maximum rates (V_max) and Michaelis constants (K_M) of the modified papain forms were increased, and higher catalytic efficiencies (k_cat/K_M) were detected as compared with the native enzyme. The results of near-UV circular dichroism and tryptophan fluorescence spectroscopic studies suggested that the modifications caused only local changes around the aromatic residues. The modified enzyme forms led to higher N-acetyl-l-tyrosine ethyl ester synthesis conversions in aqueous ethanol; acetyl and propionyl papain furnishing the highest productivity.     

Correlations between biological activity and the number of functional groups chemically modified

In interpreting the data obtained by chemical modification of biologically active macromolecules, e.g. enzymes, it has been a common practice to plot the remaining activity against the number of residues modified. An extrapolation of the initial, nearly linear portion of the curve generated to the axis giving numbers of residues often has been presumed to specify the actual number of critical groups modified. That this is usually not the case can be shown if one considers treatment of a typical macromolecule containing a number of a given kind of modifiable residues among which some, including those that are essential for activity, react at one rate, and the remainder react at a different rate. The behavior and character of the plots obtained have been analyzed and are presented with numerical and experimental examples to illustrate those features that should be considered.     

Human plasmin enzymatic activity is inhibited by chemically modified dextrans

Some synthetic dextran derivatives that mimic the action of heparin/heparan sulfate were shown to promote in vivo tissue repair when added alone to wounds. These biofunctional mimetics were therefore designated as "regenerating agents" in regard to their in vivo properties. In vitro, these biopolymers were able to protect various heparin-binding growth factors against proteolytic degradation as well as to inhibit the enzymatic activity of neutrophil elastase. In the present work, different dextran derivatives were tested for their capacity to inhibit the enzymatic activity of human plasmin. We show that dextran containing carboxymethyl, sulfate as well as benzylamide groups (RG1192 compound), was the most efficient inhibitor of plasmin amidolytic activity. The inhibition of plasmin by RG1192 can be classified as tight binding hyperbolic noncompetitive. One molecule of RG1192 bound 20 molecules of plasmin with a K(i) of 2.8 x 10(-8) m. Analysis with an optical biosensor confirmed the high affinity of RG1192 for plasmin and revealed that this polymer equally binds plasminogen with a similar affinity (K(d) = 3 x 10(-8) m). Competitive experiments carried out with 6-aminohexanoic acid and kringle proteolytic fragments identified the lysine-binding site domains of plasmin as the RG1192 binding sites. In addition, RG1192 blocked the generation of plasmin from Glu-plasminogen and inhibited the plasmin-mediated proteolysis of fibronectin and laminin. Data from the present in vitro investigation thus indicated that specific dextran derivatives can contribute to the regulation of plasmin activity by impeding the plasmin generation, as a result of their binding to plasminogen and also by directly affecting the catalytic activity of the enzyme.     

Mechanisms and optimization of in vivo delivery of lipophilic siRNAs

Cholesterol-conjugated siRNAs can silence gene expression in vivo. Here we synthesize a variety of lipophilic siRNAs and use them to elucidate the requirements for siRNA delivery in vivo. We show that conjugation to bile acids and long-chain fatty acids, in addition to cholesterol, mediates siRNA uptake into cells and gene silencing in vivo. Efficient and selective uptake of these siRNA conjugates depends on interactions with lipoprotein particles, lipoprotein receptors and transmembrane proteins. High-density lipoprotein (HDL) directs siRNA delivery into liver, gut, kidney and steroidogenic organs, whereas low-density lipoprotein (LDL) targets siRNA primarily to the liver. LDL-receptor expression is essential for siRNA delivery by LDL particles, and SR-BI receptor expression is required for uptake of HDL-bound siRNAs. Cellular uptake also requires the mammalian homolog of the Caenorhabditis elegans transmembrane protein Sid1. Our results demonstrate that conjugation to lipophilic molecules enables effective siRNA uptake through a common mechanism that can be exploited to optimize therapeutic siRNA delivery.     

Studies of the ferroxidase activity of native and chemically modified xanthine oxidoreductase.

The O2-utilizing (type O, oxidase) form of xanthine oxidoreductase is primarily responsible for its ferroxidase activity. This form of xanthine oxidoreductase has 1000 times the ferroxidase activity of the serum ferroxidase caeruloplasmin. It has the ability to catalyse the oxidative incorporation of iron into transferrin at very low Fe2+ and O2 concentrations. Furthermore, the pH optimum of the ferroxidase activity of the enzyme is compatible with the conditions of pH that normally exist in the intestinal mucosa, where it has been proposed that xanthine oxidoreductase may facilitate the absorption of ionic iron. Modification of the molybdenum (Mb) centres of the enzyme in vitro by treatment with cyanide, methanol or allopurinol completely abolishes its ferroxidase activity. The feeding of dietary tungsten to rats, which prevents the incorporation of molybdenum into newly synthesized intestinal xanthine oxidoreductase, results in the progressive loss of the ferroxidase activity of intestinal-mucosa homogenates. Removal of the flavin centres from the enzyme also results in the complete loss of ferroxidase activity; however, the ferroxidase activity of the flavin-free form of the enzyme can be restored with artificial electron acceptors that interact with the molybdenum or non-haem iron centres. The presence of superoxide dismutase or catalase in the assay system results in little inhibition of the ferroxidase activity of xanthine oxidoreductase.     

Potent in vivo antimalarial activity of 3,15-di-O-acetylbruceolide against Plasmodium berghei infection in mice

The antimalarial activity of the O-acylated bruceolide derivative, 3,15-di-O-acetylbruceolide, was evaluated against Plasmodium berghei in vivo. The concentration of 3,15-di-O-acetylbruceolide required for 50% suppression (ED50) of P. berghei in mice was 0.46 +/- 0.06 mg/kg/day, whereas bruceolide was only half as effective as 3,15-di-O-acetylbruceolide. Two antimalarial drugs used clinically, chloroquine and artemisinin, demonstrated only low activity corresponding to 1/4 and 1/12 of the ED50 value of 3,15-di-O-acetylbruceolide, respectively. These results may be helpful in the design of better chemotherapeutic bruceolides against falciparum malaria.