RESUMO
Chemical modifications are necessary to ensure the metabolic stability and efficacy of oligonucleotide-based therapeutics. Here, we describe analyses of the α-(l)-threofuranosyl nucleic acid (TNA) modification, which has a shorter 3'-2' internucleotide linkage than the natural DNA and RNA, in the context of small interfering RNAs (siRNAs). The TNA modification enhanced nuclease resistance more than 2'-O-methyl or 2'-fluoro ribose modifications. TNA-containing siRNAs were prepared as triantennary N-acetylgalactosamine conjugates and were tested in cultured cells and mice. With the exceptions of position 2 of the antisense strand and position 11 of the sense strand, the TNA modification did not inhibit the activity of the RNA interference machinery. In a rat toxicology study, TNA placed at position 7 of the antisense strand of the siRNA mitigated off-target effects, likely due to the decrease in the thermodynamic binding affinity relative to the 2'-O-methyl residue. Analysis of the crystal structure of an RNA octamer with a single TNA on each strand showed that the tetrose sugar adopts a C4'-exo pucker. Computational models of siRNA antisense strands containing TNA bound to Argonaute 2 suggest that TNA is well accommodated in the region kinked by the enzyme. The combined data indicate that the TNA nucleotides are promising modifications expected to increase the potency, duration of action, and safety of siRNAs.
Assuntos
Ácidos Nucleicos , Animais , Camundongos , Ratos , RNA Interferente Pequeno , Nucleotídeos , Interferência de RNA , AcetilgalactosaminaRESUMO
Lipid nanoparticles have demonstrated utility in hepatic delivery of a range of therapeutic modalities and typically deliver their cargo via low-density lipoprotein receptor-mediated endocytosis. For patients lacking sufficient low-density lipoprotein receptor activity, such as those with homozygous familial hypercholesterolemia, an alternate strategy is needed. Here we show the use of structure-guided rational design in a series of mouse and non-human primate studies to optimize a GalNAc-Lipid nanoparticle that allows for low-density lipoprotein receptor independent delivery. In low-density lipoprotein receptor-deficient non-human primates administered a CRISPR base editing therapy targeting the ANGPTL3 gene, the introduction of an optimized GalNAc-based asialoglycoprotein receptor ligand to the nanoparticle surface increased liver editing from 5% to 61% with minimal editing in nontargeted tissues. Similar editing was noted in wild-type monkeys, with durable blood ANGPTL3 protein reduction up to 89% six months post dosing. These results suggest that GalNAc-Lipid nanoparticles may effectively deliver to both patients with intact low-density lipoprotein receptor activity as well as those afflicted by homozygous familial hypercholesterolemia.
Assuntos
Hipercolesterolemia Familiar Homozigota , Nanopartículas , Animais , Edição de Genes/métodos , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Fígado/metabolismo , Receptores de LDL/genética , Receptores de LDL/metabolismo , Lipoproteínas LDL/metabolismoRESUMO
Conjugation of synthetic triantennary N-acetyl-d-galactosamine (GalNAc) to small interfering RNA (siRNA) mediates binding to the asialoglycoprotein receptor (ASGPR) on the surface of hepatocytes, facilitating liver-specific uptake and siRNA-mediated gene silencing. The natural ß-glycosidic bond of the GalNAc ligand is rapidly cleaved by glycosidases in vivo. Novel GalNAc ligands with S-, and C-glycosides with both α- and ß-anomeric linkages, N-glycosides with ß-anomeric linkage, and the O-glycoside with α-anomeric linkage were synthesized and conjugated to siRNA either on-column during siRNA synthesis or through a high-throughput, post-synthetic method. Unlike natural GalNAc, modified ligands were resistant to glycosidase activity. The siRNAs conjugated to newly designed ligands had similar affinities for ASGPR and similar silencing activity in mice as the parent GalNAc-siRNA conjugate. These data suggest that other factors, such as protein-nucleic acid interactions and loading of the antisense strand into the RNA-induced silencing complex (RISC), are more critical to the duration of action than the stereochemistry and stability of the anomeric linkage between the GalNAc moiety of the ligand conjugated to the sense strand of the siRNA.
Assuntos
Receptor de Asialoglicoproteína , Galactosamina , RNA Interferente Pequeno , Complexo de Inativação Induzido por RNA , Animais , Camundongos , Acetilgalactosamina/química , Receptor de Asialoglicoproteína/metabolismo , Glicosídeo Hidrolases/metabolismo , Glicosídeos/metabolismo , Hepatócitos/metabolismo , Ligantes , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/metabolismoRESUMO
A critical challenge for the successful development of RNA interference-based therapeutics therapeutics has been the enhancement of their in vivo metabolic stability. In therapeutically relevant, fully chemically modified small interfering RNAs (siRNAs), modification of the two terminal phosphodiester linkages in each strand of the siRNA duplex with phosphorothioate (PS) is generally sufficient to protect against exonuclease degradation in vivo. Since PS linkages are chiral, we systematically studied the properties of siRNAs containing single chiral PS linkages at each strand terminus. We report an efficient and simple method to introduce chiral PS linkages and demonstrate that Rp diastereomers at the 5' end and Sp diastereomers at the 3' end of the antisense siRNA strand improved pharmacokinetic and pharmacodynamic properties in a mouse model. In silico modeling studies provide mechanistic insights into how the Rp isomer at the 5' end and Sp isomer at the 3' end of the antisense siRNA enhance Argonaute 2 (Ago2) loading and metabolic stability of siRNAs in a concerted manner.
Assuntos
Organofosfatos , RNA Interferente Pequeno , Animais , Isomerismo , Camundongos , Interferência de RNA , Estabilidade de RNA , RNA de Cadeia Dupla , RNA Interferente Pequeno/metabolismoRESUMO
Gene-editing technologies, which include the CRISPR-Cas nucleases1-3 and CRISPR base editors4,5, have the potential to permanently modify disease-causing genes in patients6. The demonstration of durable editing in target organs of nonhuman primates is a key step before in vivo administration of gene editors to patients in clinical trials. Here we demonstrate that CRISPR base editors that are delivered in vivo using lipid nanoparticles can efficiently and precisely modify disease-related genes in living cynomolgus monkeys (Macaca fascicularis). We observed a near-complete knockdown of PCSK9 in the liver after a single infusion of lipid nanoparticles, with concomitant reductions in blood levels of PCSK9 and low-density lipoprotein cholesterol of approximately 90% and about 60%, respectively; all of these changes remained stable for at least 8 months after a single-dose treatment. In addition to supporting a 'once-and-done' approach to the reduction of low-density lipoprotein cholesterol and the treatment of atherosclerotic cardiovascular disease (the leading cause of death worldwide7), our results provide a proof-of-concept for how CRISPR base editors can be productively applied to make precise single-nucleotide changes in therapeutic target genes in the liver, and potentially in other organs.
Assuntos
Sistemas CRISPR-Cas , LDL-Colesterol/sangue , Edição de Genes , Modelos Animais , Pró-Proteína Convertase 9/genética , Adenina/metabolismo , Animais , Células Cultivadas , Feminino , Hepatócitos/metabolismo , Humanos , Fígado/enzimologia , Mutação com Perda de Função , Macaca fascicularis/sangue , Macaca fascicularis/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Pró-Proteína Convertase 9/sangue , Pró-Proteína Convertase 9/metabolismo , Fatores de TempoRESUMO
The 5'-monophosphate group plays an important role in strand selection during gene silencing mediated by small-interfering RNA. We show that blocking of 5' phosphorylation of the sense strand by introducing a 5'-morpholino modification improves antisense strand selection and RNAi activity. The 5'-morpholino modification of the antisense strand triggers complete loss of activity.
Assuntos
Morfolinos/química , RNA Interferente Pequeno/química , Animais , Apolipoproteína B-100 , Apolipoproteínas B/genética , Proteínas Argonautas/genética , Inativação Gênica , Humanos , Camundongos , Modelos Moleculares , Morfolinos/síntese química , Morfolinos/genética , Interferência de RNA , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/genéticaRESUMO
For oligonucleotide therapeutics, chemical modifications of the sugar-phosphate backbone are frequently used to confer drug-like properties. Because 2'-deoxy-2'-fluoro (2'-F) nucleotides are not known to occur naturally, their safety profile was assessed when used in revusiran and ALN-TTRSC02, two short interfering RNAs (siRNAs), of the same sequence but different chemical modification pattern and metabolic stability, conjugated to an N-acetylgalactosamine (GalNAc) ligand for targeted delivery to hepatocytes. Exposure to 2'-F-monomer metabolites was low and transient in rats and humans. In vitro, 2'-F-nucleoside 5'-triphosphates were neither inhibitors nor preferred substrates for human polymerases, and no obligate or non-obligate chain termination was observed. Modest effects on cell viability and mitochondrial DNA were observed in vitro in a subset of cell types at high concentrations of 2'-F-nucleosides, typically not attained in vivo. No apparent functional impact on mitochondria and no significant accumulation of 2'-F-monomers were observed after weekly administration of two GalNAc-siRNA conjugates in rats for â¼2 years. Taken together, the results support the conclusion that 2'-F nucleotides can be safely applied for the design of metabolically stabilized therapeutic GalNAc-siRNAs with favorable potency and prolonged duration of activity allowing for low dose and infrequent dosing.
Assuntos
Acetilgalactosamina/efeitos adversos , Acetilgalactosamina/química , Desoxirribonucleotídeos/efeitos adversos , Desoxirribonucleotídeos/química , Flúor/química , RNA Interferente Pequeno/efeitos adversos , RNA Interferente Pequeno/química , Animais , Feminino , Flúor/efeitos adversos , Humanos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2'/O2' atoms are the most common sites for modification. Compared to 2'-O-substituents, ribose 4'-C-substituents lie in proximity of both the 3'- and 5'-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2'-F and 2'-OMe with 4'-Cα- and 4'-Cß-OMe, and 2'-F with 4'-Cα-methyl modification. The α- and ß-epimers of 4'-C-OMe-uridine and the α-epimer of 4'-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4'α-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2'-substituent (H, F, OMe). The 4'ß-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2'-F,4'-Cα-OMe-U, 2'-F,4'-Cß-OMe-U, 2'-OMe,4'-Cα-OMe-U, 2'-OMe,4'-Cß-OMe-U or 2'-F,4'-Cα-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4'ß-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2'-F,4'-Cα-OMe-U or 2'-F,4'-Cß-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2'-/4'-C-modifications.
Assuntos
Oligonucleotídeos/química , Estabilidade de RNA/genética , RNA Interferente Pequeno/química , Termodinâmica , Humanos , Modelos Moleculares , Conformação de Ácido Nucleico , Oligonucleotídeos/genética , Fosfatos/química , Interferência de RNA , Ribonucleases/química , Ribose/química , Uridina/química , Uridina/genéticaRESUMO
We report rapid, potent reversal of GalNAc-siRNA-mediated RNA interference (RNAi) activity in vivo with short, synthetic, high-affinity oligonucleotides complementary to the siRNA guide strand. We found that 9-mers with five locked nucleic acids (LNAs) have the highest potency across several targets. Our modular, sequence-specific approach, named REVERSIR, may enhance the therapeutic profile of any long-acting GalNAc-siRNA (short interfering RNA) conjugate by enabling control of RNAi pharmacology.
Assuntos
Inativação Gênica , RNA Interferente Pequeno/genética , Acetilgalactosamina/genética , Animais , Sequência de Bases , Biotecnologia , Células Cultivadas , Feminino , Hepatócitos/metabolismo , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Oligonucleotídeos/química , Oligonucleotídeos/genética , Interferência de RNA , RNA Interferente Pequeno/químicaRESUMO
Small interfering RNAs (siRNAs) conjugated to a trivalent N-acetylgalactosamine (GalNAc) ligand are being evaluated in investigational clinical studies for a variety of indications. The typical development candidate selection process includes evaluation of the most active compounds for toxicity in rats at pharmacologically exaggerated doses. The subset of GalNAc-siRNAs that show rat hepatotoxicity is not advanced to clinical development. Potential mechanisms of hepatotoxicity can be associated with the intracellular accumulation of oligonucleotides and their metabolites, RNA interference (RNAi)-mediated hybridization-based off-target effects, and/or perturbation of endogenous RNAi pathways. Here we show that rodent hepatotoxicity observed at supratherapeutic exposures can be largely attributed to RNAi-mediated off-target effects, but not chemical modifications or the perturbation of RNAi pathways. Furthermore, these off-target effects can be mitigated by modulating seed-pairing using a thermally destabilizing chemical modification, which significantly improves the safety profile of a GalNAc-siRNA in rat and may minimize the occurrence of hepatotoxic siRNAs across species.
Assuntos
Acetilgalactosamina/química , Fígado/efeitos dos fármacos , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/toxicidade , Acetilgalactosamina/toxicidade , Animais , Fígado/metabolismo , Masculino , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Significant progress has been made in the advancement of RNAi therapeutics by combining a synthetic triantennary N-acetylgalactosamine ligand targeting the asialoglycoprotein receptor with chemically modified small interfering RNA (siRNA) designs, including the recently described Enhanced Stabilization Chemistry. This strategy has demonstrated robust RNAi-mediated gene silencing in liver after subcutaneous administration across species, including human. Here we demonstrate that substantial efficacy improvements can be achieved through further refinement of siRNA chemistry, optimizing the positioning of 2'-deoxy-2'-fluoro and 2'-O-methyl ribosugar modifications across both strands of the double-stranded siRNA duplex to enhance stability without compromising intrinsic RNAi activity. To achieve this, we employed an iterative screening approach across multiple siRNAs to arrive at advanced designs with low 2'-deoxy-2'-fluoro content that yield significantly improved potency and duration in preclinical species, including non-human primate. Liver exposure data indicate that the improvement in potency is predominantly due to increased metabolic stability of the siRNA conjugates.
Assuntos
Acetilgalactosamina , Interferência de RNA , RNA Interferente Pequeno , Acetilgalactosamina/química , Animais , Proteínas Argonautas/genética , Regulação da Expressão Gênica , Inativação Gênica , Hepatócitos/metabolismo , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genéticaRESUMO
(E)-Vinylphosphonate ((E)-VP), a metabolically stable phosphate mimic at the 5'-end of the antisense strand, enhances the in vivo potency of siRNA. Here we describe a straightforward synthetic approach to incorporate a nucleotide carrying a vinylphosphonate (VP) moiety at the 5'-end of oligonucleotides under standard solid-phase synthesis and deprotection conditions by utilizing pivaloyloxymethyl (POM) protected VP-nucleoside phosphoramidites. The POM protection enhances scope and scalability of 5'-VP-modified oligonucleotides and, in a broader sense, the synthesis of oligonucleotides modified with phosphonate moieties. Trivalent N-acetylgalactosamine-conjugated small interfering RNA (GalNAc-siRNA) comprising (E)-geometrical isomer of VP showed improved RISC loading with robust RNAi-mediated gene silencing in mice compared to the corresponding (Z)-isomer despite similar tissue accumulation. We also obtained structural insights into why bulkier 2'-ribosugar substitutions such as 2'-O-[2-(methylamino)-2-oxoethyl] are well tolerated only when combined with 5'-(E)-VP.
Assuntos
Organofosfonatos/química , Organofosfonatos/síntese química , RNA Interferente Pequeno/química , Animais , Proteínas Argonautas/química , Proteínas Argonautas/deficiência , Proteínas Argonautas/genética , Sequência de Bases , Técnicas de Química Sintética , Inativação Gênica , Camundongos , Modelos Moleculares , Domínios Proteicos , RNA Interferente Pequeno/genética , EstereoisomerismoRESUMO
The hepatocyte-specific asialoglycoprotein receptor (ASGPR) is an ideal candidate for targeted drug delivery to the liver due to its high capacity for substrate clearance from circulation together with its well-conserved expression and function across species. The development of GalNAc-siRNA conjugates, in which a synthetic triantennary N-acetylgalactosamine-based ligand is conjugated to chemically modified siRNA, has enabled efficient, ASGPR-mediated delivery to hepatocytes. To investigate the potential impact of variations in receptor expression on the efficiency of GalNAc-siRNA conjugate delivery, we evaluated the pharmacokinetics and pharmacodynamics of GalNAc-siRNA conjugates in multiple pre-clinical models with reduced receptor expression. Despite greater than 50% reduction in ASGPR levels, GalNAc conjugate activity was retained, suggesting that the remaining receptor capacity was sufficient to mediate efficient uptake of potent GalNAc-siRNAs at pharmacologically relevant dose levels. Collectively, our data support a broad application of the GalNAc-siRNA technology for hepatic targeting, including disease states where ASGPR expression may be reduced.
Assuntos
Acetilgalactosamina , Receptor de Asialoglicoproteína/genética , Regulação da Expressão Gênica , Interferência de RNA , RNA Interferente Pequeno/genética , Acetilgalactosamina/química , Animais , Receptor de Asialoglicoproteína/química , Receptor de Asialoglicoproteína/metabolismo , Modelos Animais de Doenças , Portadores de Fármacos , Sistemas de Liberação de Medicamentos , Avaliação Pré-Clínica de Medicamentos , Feminino , Inativação Gênica , Hepatócitos/metabolismo , Humanos , Cirrose Hepática/genética , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Camundongos , Camundongos Knockout , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno/químicaRESUMO
Covalent attachment of a synthetic triantennary N-acetylagalactosamine (GalNAc) ligand to chemically modified siRNA has enabled asialoglycoprotein (ASGPR)-mediated targeted delivery of therapeutically active siRNAs to hepatocytes in vivo. This approach has become transformative for the delivery of RNAi therapeutics as well as other classes of investigational oligonucleotide therapeutics to the liver. For efficient functional delivery of intact drug into the desired subcellular compartment, however, it is critical that the nucleic acids are stabilized against nucleolytic degradation. Here, we compared two siRNAs of the same sequence but with different modification pattern resulting in different degrees of protection against nuclease activity. In vitro stability studies in different biological matrices show that 5'-exonuclease is the most prevalent nuclease activity in endo-lysosomal compartments and that additional stabilization in the 5'-regions of both siRNA strands significantly enhances the overall metabolic stability of GalNAc-siRNA conjugates. In good agreement with in vitro findings, the enhanced stability translated into substantially improved liver exposure, gene silencing efficacy and duration of effect in mice. Follow-up studies with a second set of conjugates targeting a different transcript confirmed the previous results, provided additional insights into kinetics of RISC loading and demonstrated excellent translation to non-human primates.
Assuntos
Acetilgalactosamina/farmacocinética , Rim/metabolismo , Fígado/metabolismo , RNA Interferente Pequeno/farmacocinética , Acetilgalactosamina/administração & dosagem , Acetilgalactosamina/metabolismo , Animais , Área Sob a Curva , Sistemas de Liberação de Medicamentos/métodos , Humanos , Fígado/citologia , Masculino , Taxa de Depuração Metabólica , Camundongos Endogâmicos C57BL , Interferência de RNA , RNA Interferente Pequeno/administração & dosagem , RNA Interferente Pequeno/metabolismoRESUMO
We designed novel 4'-modified 2'-deoxy-2'-fluorouridine (2'-F U) analogues with the aim to improve nuclease resistance and potency of therapeutic siRNAs by introducing 4'-C-methoxy (4'-OMe) as the alpha (C4'α) or beta (C4'ß) epimers. The C4'α epimer was synthesized by a stereoselective route in six steps; however, both α and ß epimers could be obtained by a nonstereoselective approach starting from 2'-F U. 1H NMR analysis and computational investigation of the α-epimer revealed that the 4'-OMe imparts a conformational bias toward the North-East sugar pucker, due to intramolecular hydrogen bonding and hyperconjugation effects. The α-epimer generally conceded similar thermal stability as unmodified nucleotides, whereas the ß-epimer led to significant destabilization. Both 4'-OMe epimers conferred increased nuclease resistance, which can be explained by the close proximity between 4'-OMe substituent and the vicinal 5'- and 3'-phosphate group, as seen in the X-ray crystal structure of modified RNA. siRNAs containing several C4'α-epimer monomers in the sense or antisense strands triggered RNAi-mediated gene silencing with efficiencies comparable to that of 2'-F U.
Assuntos
Inativação Gênica , Interferência de RNA , Estabilidade de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , Ribonucleotídeos/química , Ribonucleotídeos/metabolismo , Desnaturação de Ácido Nucleico , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , RNA Interferente Pequeno/genética , Terapêutica com RNAi , Ribonucleotídeos/genética , Termodinâmica , Uridina/química , Uridina/metabolismoRESUMO
Here we report the investigation of glycol nucleic acid (GNA), an acyclic nucleic acid analogue, as a modification of siRNA duplexes. We evaluated the impact of (S)- or (R)-GNA nucleotide incorporation on RNA duplex structure by determining three individual crystal structures. These structures indicate that the (S)-nucleotide backbone adopts a conformation that has little impact on the overall duplex structure, while the (R)-nucleotide disrupts the phosphate backbone and hydrogen bonding of an adjacent base pair. In addition, the GNA-T nucleobase adopts a rotated conformation in which the 5-methyl group points into the minor groove, rather than the major groove as in a normal Watson-Crick base pair. This observation of reverse Watson-Crick base pairing is further supported by thermal melting analysis of GNA-C and GNA-G containing duplexes where it was demonstrated that a higher thermal stability was associated with isoguanine and isocytosine base pairing, respectively, over the canonical nucleobases. Furthermore, it was also shown that GNA nucleotide or dinucleotide incorporation increases resistance against snake venom phosphodiesterase. Consistent with the structural data, modification of an siRNA with (S)-GNA resulted in greater in vitro potencies over identical sequences containing (R)-GNA. A walk of (S)-GNA along the guide and passenger strands of a GalNAc conjugate duplex targeting mouse transthyretin (TTR) indicated that GNA is well tolerated in the seed region of both strands in vitro, resulting in an approximate 2-fold improvement in potency. Finally, these conjugate duplexes modified with GNA were capable of maintaining in vivo potency when subcutaneously injected into mice.
Assuntos
Glicóis/química , Ácidos Nucleicos/química , RNA Interferente Pequeno/química , Animais , Cristalografia por Raios X , Inativação Gênica/efeitos dos fármacos , Concentração Inibidora 50 , Camundongos , Modelos Biológicos , Ácidos Nucleicos Heteroduplexes/química , RNA Interferente Pequeno/farmacologia , Receptores de Albumina/efeitos dos fármacos , TemperaturaRESUMO
ATTR amyloidosis is a systemic, debilitating and fatal disease caused by transthyretin (TTR) amyloid accumulation. RNA interference (RNAi) is a clinically validated technology that may be a promising approach to the treatment of ATTR amyloidosis. The vast majority of TTR, the soluble precursor of TTR amyloid, is expressed and synthesized in the liver. RNAi technology enables robust hepatic gene silencing, the goal of which would be to reduce systemic levels of TTR and mitigate many of the clinical manifestations of ATTR that arise from hepatic TTR expression. To test this hypothesis, TTR-targeting siRNAs were evaluated in a murine model of hereditary ATTR amyloidosis. RNAi-mediated silencing of hepatic TTR expression inhibited TTR deposition and facilitated regression of existing TTR deposits in pathologically relevant tissues. Further, the extent of deposit regression correlated with the level of RNAi-mediated knockdown. In comparison to the TTR stabilizer, tafamidis, RNAi-mediated TTR knockdown led to greater regression of TTR deposits across a broader range of affected tissues. Together, the data presented herein support the therapeutic hypothesis behind TTR lowering and highlight the potential of RNAi in the treatment of patients afflicted with ATTR amyloidosis.
Assuntos
Neuropatias Amiloides Familiares/terapia , Fígado/metabolismo , Pré-Albumina/antagonistas & inibidores , RNA Mensageiro/antagonistas & inibidores , RNA Interferente Pequeno/administração & dosagem , Neuropatias Amiloides Familiares/genética , Neuropatias Amiloides Familiares/metabolismo , Neuropatias Amiloides Familiares/patologia , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Benzoxazóis/farmacologia , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Feminino , Expressão Gênica , Humanos , Fígado/patologia , Macaca fascicularis , Masculino , Camundongos , Camundongos Transgênicos , Pré-Albumina/genética , Pré-Albumina/metabolismo , Interferência de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacocinéticaRESUMO
Small interfering RNA (siRNA)-mediated silencing requires siRNA loading into the RNA-induced silencing complex (RISC). Presence of 5'-phosphate (5'-P) is reported to be critical for efficient RISC loading of the antisense strand (AS) by anchoring it to the mid-domain of the Argonaute2 (Ago2) protein. Phosphorylation of exogenous duplex siRNAs is thought to be accomplished by cytosolic Clp1 kinase. However, although extensive chemical modifications are essential for siRNA-GalNAc conjugate activity, they can significantly impair Clp1 kinase activity. Here, we further elucidated the effect of 5'-P on the activity of siRNA-GalNAc conjugates. Our results demonstrate that a subset of sequences benefit from the presence of exogenous 5'-P. For those that do, incorporation of 5'-(E)-vinylphosphonate (5'-VP), a metabolically stable phosphate mimic, results in up to 20-fold improved in vitro potency and up to a threefold benefit in in vivo activity by promoting Ago2 loading and enhancing metabolic stability.
Assuntos
Acetilgalactosamina/química , Organofosfonatos/química , Interferência de RNA , RNA Interferente Pequeno/química , Compostos de Vinila/química , Animais , Apolipoproteínas B/antagonistas & inibidores , Apolipoproteínas B/genética , Apolipoproteínas B/metabolismo , Proteínas Argonautas/antagonistas & inibidores , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Células Cultivadas , Fator IX/antagonistas & inibidores , Fator IX/genética , Fator IX/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Lipoproteínas LDL/sangue , Camundongos , Camundongos Endogâmicos C57BL , Organofosfonatos/farmacologia , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Complexo de Inativação Induzido por RNA/química , Complexo de Inativação Induzido por RNA/metabolismo , Fatores de Transcrição/metabolismo , Compostos de Vinila/farmacologiaRESUMO
Although judicious use of chemical modifications has contributed to the success of nucleic acid therapeutics, poor systemic stability remains a major hurdle. The introduction of functional groups around the phosphate backbone can enhance the nuclease resistance of oligonucleotides (ONs). Here, we report the synthesis of enantiomerically pure (R)- and (S)-5'-C-methyl (C5'-Me) substituted nucleosides and their incorporation into ONs. These modifications generally resulted in a decrease in thermal stability of oligonucleotide (ON) duplexes in a manner dependent on the stereoconfiguration at C5' with greater destabilization characteristic of (R)-epimers. Enhanced stability against snake venom phosphodiesterase resulted from modification of the 3'-end of an ON with either (R)- or (S)-C5'-Me nucleotides. The (S)-isomers with different 2'-substituents provided greater resistance against 3'-exonucleases than the corresponding (R)-isomers. Crystal structure analyses of RNA octamers with (R)- or (S)-5'-C-methyl-2'-deoxy-2'-fluorouridine [(R)- or (S)-C5'-Me-2'-FU, respectively] revealed that the stereochemical orientation of the C5'-Me and the steric effects that emanate from the alkyl substitution are the dominant determinants of thermal stability and are likely molecular origins of resistance against nucleases. X-ray and NMR structural analyses showed that the (S)-C5'-Me epimers are spatially and structurally more similar to their natural 5' nonmethylated counterparts than the corresponding (R)-epimers.
Assuntos
Exorribonucleases/química , Oligonucleotídeos/química , Pirimidinas/química , Sequência de Bases , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico , Estereoisomerismo , TermodinâmicaRESUMO
5'-Phosphorylation is a critical step in the cascade of events that leads to loading of small interfering RNAs (siRNAs) into the RNA-induced silencing complex (RISC) to elicit gene silencing. 5'-Phosphorylation of exogenous siRNAs is generally accomplished by a cytosolic Clp1 kinase, and in most cases, the presence of a 5'-monophosphate on synthetic siRNAs is not a prerequisite for activity. Chemically introduced, metabolically stable 5'-phosphate mimics can lead to higher metabolic stability, increased RISC loading, and higher gene silencing activities of chemically modified siRNAs. In this study, we report the synthesis of 5'-C-malonyl RNA, a 5'-monophosphate bioisostere. A 5'-C-malonyl-modified nucleotide was incorporated at the 5'-terminus of chemically modified RNA oligonucleotides using solid-phase synthesis. In vitro silencing activity, in vitro metabolic stability, and in vitro RISC loading of 5'-C-malonyl siRNA was compared to corresponding 5'-phosphorylated and 5'-nonphosphorylated siRNAs. The 5'-C-malonyl siRNAs showed sustained or improved in vitro gene silencing and high levels of Ago2 loading and conferred dramatically improved metabolic stability to the antisense strand of the siRNA duplexes. In silico modeling studies indicate a favorable fit of the 5'-C-malonyl group within the 5'-phosphate binding pocket of human Ago2MID domain.