Search

Summary Human toll-like receptor 8 (TLR8) activation induces a potent T helper-1 (Th1) cell response critical for defense against intracellular pathogens, including protozoa. The receptor harbors two distinct binding sites, uridine and di- and/or trinucleotides, but the RNases upstream of TLR8 remain poorly characterized. We identified two endolysosomal endoribonucleases, RNase T2 and RNase 2, that act synergistically to release uridine from oligoribonucleotides. RNase T2 cleaves preferentially before, and RNase 2 after, uridines. Live bacteria, P. falciparum-infected red blood cells, purified pathogen RNA, and synthetic oligoribonucleotides all required RNase 2 and T2 processing to activate TLR8. Uridine supplementation restored RNA recognition in RNASE2−/− or RNASET2−/− but not RNASE2−/− RNASET2−/− cells. Primary immune cells from RNase T2-hypomorphic patients lacked a response to bacterial RNA but responded robustly to small-molecule TLR8 ligands. Our data identify an essential function of RNase T2 and RNase 2 upstream of TLR8 and provide insight into TLR8 activation.

REP 2139 is a nucleic acid polymer (NAP) currently under clinical development for chronic hepatitis B (HBV) therapy. This preclinical study investigated different REP 2139 analogs that would display reduced accumulation in the serum and tissues, while retaining an antiviral effect against HBV infection. REP 2139 analogs were evaluated in human plasma, CD-1 mice, cynomolgus monkeys, and Pekin ducks. Discrete ribose transformation to 2′OH in selected riboadenosines resulted in a slow degradation in acidified human plasma that plateaued after 48 hr. REP 2165, a REP 2139 analog containing three unmodified riboadenosines equally spaced throughout the polymer, showed similar plasma clearance and tissue distribution as REP 2139 in mice and cynomolgus monkeys after a single dose. Interestingly, after repeated administration, accumulation of REP 2165 in plasma and organs was reduced, indicating a dramatically faster rate of clearance from organs after therapy was ended in both species. Both REP 2139 and REP 2165 were well tolerated at clinically relevant doses, with no alterations in liver, kidney, or hematological function. In chronic duck HBV (DHBV) infection, REP 2165 displayed significantly reduced liver accumulation after repeated dosing but retained antiviral activity similar to REP 2139. These results indicate the therapeutic potential of REP 2165 against chronic HBV infection in patients is similar to REP 2139, but with significantly reduced drug accumulation and improved tissue clearance. Keywords: nucleic acid polymer, HBV, pharmacokinetics

Small interfering RNAs (siRNAs) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. The development of siRNA-based therapeutics requires in-depth knowledge of the manufacturing process as well as adequate analytical methods to characterize this class of molecules. Here the impurity formation during the annealing of siRNA was investigated. Two siRNAs containing common chemical RNA modifications (2'-O-methyl, 2'-deoxy-2'-fluoro, 2'-deoxy-ribose, and phosphorothioate linkages) were used to determine major side reactions-such as 2',3'-isomerization, strand scission, and HF elimination-depending on annealing parameters such as RNA concentration, presence of cations, temperature, and time. Individual impurities were characterized using analytical size exclusion chromatography, denaturing and nondenaturing ion-pair reversed-phase high-performance liquid chromatography, differential scanning calorimetry, and ultraviolet spectrometry. The degradation pathways described in this work can lead to significantly reduced product quality and compromised drug activity. The data reported here provide background to successfully address challenges associated with the manufacture of siRNAs and other nucleic acid therapeutics such as aptamers, spiegelmers, and decoy and antisense oligonucleotides.

A manufacturing and purification process for duplex oligonucleotides was established, which shortens and simplifies currently used procedures, yielding a product of higher purity. The reported procedure is based on nondenaturing anion-exchange (AEX) chromatography, which is performed on the annealed duplex rather than the individual single strands. The duplex is formed early in the process by annealing of the crude single strands directly after solid-phase synthesis. Two 30 μmol manufacturing runs using duplex purification were performed on 2 different AEX resins and compared with a manufacturing run of the same scale using conventional single-strand chromatography. The same pooling strategy was employed for all purifications. Content of optimal duplex (duplex exclusively comprising full-length single strands) was 90.5% and 90.2% for the batches obtained by duplex purification and 86.1% for the batch obtained by single-strand purification. Maximum chromatographic recoveries were 67% for the duplex purification and 68% for the single-strand purification. Hence, the manufacture of small interfering RNA (siRNA) using duplex purification was simpler and faster than conventional single-strand purification and provided better purity and similar yield of final siRNA.

Small interfering RNAs (siRNA) are emerging as a novel therapeutic modality for the specific inhibition of target gene expression. siRNA are typically formed by annealing of two complementary single stranded oligoribonucleotides. Compared to purity determination of non-hybridized single strands by denaturing chromatographic methods, characterization of the hybridized duplex is challenging. Here we are reporting a non-denaturing ion pairing-reversed phase (IP-RP) chromatography method capable of separating optimal duplex (full-length single strands only) from non-optimal duplex variants (containing shortmers, longmers and 2',5'-isomers) using ultraviolet- and mass spectrometric detection. The impact of different annealing conditions on siRNA composition was investigated. Optimized annealing conditions lead to a significant increase in optimal duplex, while total duplex content remained constant. The non-denaturing method reported herein showed high mass spectrometric sensitivity and superior separation efficiencies compared to other IP-RP buffer systems. The method is useful for in-process control and release testing of therapeutic double stranded nucleic acids such as siRNA.

5-Aminoallyl-2'-fluoro-dUTP, 5-aminoallyl-UTP, and N(6)-([6-aminohexyl]carbamoylmethyl)-ATP were systematically tested for their suitability for the systematic evolution of ligands by exponential enrichment (SELEX) process with the aim of introducing additional functionalities to RNA libraries. All three aminomodified nucleoside triphosphates proved to be compatible with the enzymatic steps required for SELEX and maintained strict Watson-Crick basepairing. Complementary RNA molecules modified with the two uridine analogues show a significantly increased melting temperature, whereas the introduction of N(6)-([6-aminohexyl]carbamoylmethyl)-ATP leads to a decreased T(m) and thus less stable basepairing. The chemical synthesis of 5-aminoallyl-2'-fluoro-dUTP is reported in detail.