RESUMEN
Oral delivery is the most widely used and convenient route of administration of medicine. However, oral administration of hydrophilic macromolecules is commonly limited by low intestinal permeability and pre-systemic degradation in the gastrointestinal (GI) tract. Overcoming some of these challenges allowed emergence of oral dosage forms of peptide-based drugs in clinical settings. Antisense oligonucleotides (ASOs) have also been investigated for oral administration but despite the recent progress, the bioavailability remains low. Given the advancement with highly potent and durable trivalent N-acetylgalactosamine (GalNAc)-conjugated small interfering RNAs (siRNAs) via subcutaneous (s.c.) injection, we explored their activities after oral administration. We report robust RNA interference (RNAi) activity of orally administrated GalNAc-siRNAs co-formulated with permeation enhancers (PEs) in rodents and non-human primates (NHPs). The relative bioavailability calculated from NHP liver exposure was <2.0% despite minimal enzymatic degradation in the GI. To investigate the impact of oligonucleotide size on oral delivery, highly specific GalNAc-conjugated single-stranded oligonucleotides known as REVERSIRs with different lengths were employed and their activities for reversal of RNAi effect were monitored. Our data suggests that intestinal permeability is highly influenced by the size of oligonucleotides. Further improvements in the potency of siRNA and PE could make oral delivery of GalNAc-siRNAs as a practical solution.
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Acetilgalactosamina , ARN Interferente Pequeño , Animales , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/farmacocinética , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Administración Oral , Ratones , Ratas , Interferencia de ARN , Masculino , Disponibilidad Biológica , Humanos , Ratas Sprague-Dawley , Macaca fascicularis , Hígado/metabolismo , Macaca mulattaRESUMEN
Two decades of research on RNA interference (RNAi) have transformed a breakthrough discovery in biology into a robust platform for a new class of medicines that modulate mRNA expression. Here we provide an overview of the trajectory of small-interfering RNA (siRNA) drug development, including the first approval in 2018 of a liver-targeted siRNA interference (RNAi) therapeutic in lipid nanoparticles and subsequent approvals of five more RNAi drugs, which used metabolically stable siRNAs combined with N-acetylgalactosamine ligands for conjugate-based liver delivery. We also consider the remaining challenges in the field, such as delivery to muscle, brain and other extrahepatic organs. Today's RNAi therapeutics exhibit high specificity, potency and durability, and are transitioning from applications in rare diseases to widespread, chronic conditions.
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Acetilgalactosamina , Hígado , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/uso terapéuticoRESUMEN
Homology Directed Repair (HDR)-based genome editing is an approach that could permanently correct a broad range of genetic diseases. However, its utility is limited by inefficient and imprecise DNA repair mechanisms in terminally differentiated tissues. Here, we tested "Repair Drive", a novel method for improving targeted gene insertion in the liver by selectively expanding correctly repaired hepatocytes in vivo. Our system consists of transient conditioning of the liver by knocking down an essential gene, and delivery of an untargetable version of the essential gene in cis with a therapeutic transgene. We show that Repair Drive dramatically increases the percentage of correctly targeted hepatocytes, up to 25%. This resulted in a five-fold increased expression of a therapeutic transgene. Repair Drive was well-tolerated and did not induce toxicity or tumorigenesis in long term follow up. This approach will broaden the range of liver diseases that can be treated with somatic genome editing.
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BACKGROUND & AIMS: Current therapy for chronic hepatitis B virus (cHBV) infection involves lifelong treatment. New treatments that enable HBV functional cure would represent a clinically meaningful advance. ALN-HBV and VIR-2218 are investigational RNA interference therapeutics that target all major HBV transcripts. METHODS: We report on: i) the safety of single doses of VIR-2218 (modified from ALN-HBV by enhanced stabilization chemistry plus technology to reduce off-target, seed-mediated binding while maintaining on-target antiviral activity) and ALN-HBV in humanized mice; ii) a cross-study comparison of the safety of single doses of VIR-2218 and ALN-HBV in healthy human volunteers (n = 24 and n = 49, respectively); and iii) the antiviral activity of two doses of 20, 50, 100, 200 mg of VIR-2218 (total n = 24) vs. placebo (n = 8), given 4 weeks apart, in participants with cHBV infection. RESULTS: In humanized mice, alanine aminotransferase (ALT) levels were markedly lower following administration of VIR-2218 compared with ALN-HBV. In healthy volunteers, post-treatment ALT elevations occurred in 28% of participants receiving ALN-HBV compared with none in those receiving VIR-2218. In participants with cHBV infection, VIR-2218 was associated with dose-dependent reductions in hepatitis B surface antigen (HBsAg). The greatest mean reduction of HBsAg at Week 20 in participants receiving 200 mg was 1.65 log IU/ml. The HBsAg reduction was maintained at 0.87 log IU/ml at Week 48. No participants had serum HBsAg loss or hepatitis B surface antibody seroconversion. CONCLUSIONS: VIR-2218 demonstrated an encouraging hepatic safety profile in preclinical and clinical studies as well as dose-dependent HBsAg reductions in patients with cHBV infection. These data support future studies with VIR-2218 as part of combination regimens with a goal of HBV functional cure. TRIAL REGISTRATION: ClinicalTrials.gov identifiers: NCT02826018 and NCT03672188. IMPACT AND IMPLICATIONS: A significant unmet need exists for therapies for chronic HBV (cHBV) infection that achieve functional cure. We report clinical and non-clinical data on two investigational small-interfering RNAs that target HBx, ALN-HBV and VIR-2218, demonstrating that incorporation of enhanced stabilization chemistry plus technology in VIR-2218 reduces its propensity to cause ALT elevations relative to its parent compound, ALN-HBV. We also show that VIR-2218 reduces hepatitis B surface antigen levels in a dose-dependent manner in participants with cHBV infection. These studies support the continued development of VIR-2218 as part of therapeutic regimens for cHBV infection, with the goal of a functional cure, and are important for HBV researchers and physicians.
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Hepatitis B Crónica , Hepatitis B , Humanos , Animales , Ratones , Hepatitis B Crónica/tratamiento farmacológico , Virus de la Hepatitis B , Antígenos de Superficie de la Hepatitis B , Tratamiento con ARN de Interferencia , Ensayos Clínicos Controlados Aleatorios como Asunto , Antivirales , ADN Viral , Antígenos e de la Hepatitis B , Hepatitis B/tratamiento farmacológicoRESUMEN
Adeno-associated virus (AAV)-based gene therapy could be facilitated by the development of molecular switches to control the magnitude and timing of expression of therapeutic transgenes. RNA interference (RNAi)-based approaches hold unique potential as a clinically proven modality to pharmacologically regulate AAV gene dosage in a sequence-specific manner. We present a generalizable RNAi-based rheostat wherein hepatocyte-directed AAV transgene expression is silenced using the clinically validated modality of chemically modified small interfering RNA (siRNA) conjugates or vectorized co-expression of short hairpin RNA (shRNA). For transgene induction, we employ REVERSIR technology, a synthetic high-affinity oligonucleotide complementary to the siRNA or shRNA guide strand to reverse RNAi activity and rapidly recover transgene expression. For potential clinical development, we report potent and specific siRNA sequences that may allow selective regulation of transgenes while minimizing unintended off-target effects. Our results establish a conceptual framework for RNAi-based regulatory switches with potential for infrequent dosing in clinical settings to dynamically modulate expression of virally-delivered gene therapies.
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Dependovirus , Terapia Genética , Interferencia de ARN , Dependovirus/genética , Dependovirus/metabolismo , ARN Interferente Pequeño/metabolismo , Transgenes , ARN Bicatenario , Vectores Genéticos/genéticaRESUMEN
Preclinical mechanistic studies have pointed towards RNA interference-mediated off-target effects as a major driver of hepatotoxicity for GalNAc-siRNA conjugates. Here, we demonstrate that a single glycol nucleic acid or 2'-5'-RNA modification can substantially reduce small interfering RNA (siRNA) seed-mediated binding to off-target transcripts while maintaining on-target activity. In siRNAs with established hepatotoxicity driven by off-target effects, these novel designs with seed-pairing destabilization, termed enhanced stabilization chemistry plus (ESC+), demonstrated a substantially improved therapeutic window in rats. In contrast, siRNAs thermally destabilized to a similar extent by the incorporation of multiple DNA nucleotides in the seed region showed little to no improvement in rat safety suggesting that factors in addition to global thermodynamics play a role in off-target mitigation. We utilized the ESC+ strategy to improve the safety of ALN-HBV, which exhibited dose-dependent, transient and asymptomatic alanine aminotransferase elevations in healthy volunteers. The redesigned ALN-HBV02 (VIR-2218) showed improved specificity with comparable on-target activity and the program was reintroduced into clinical development.
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ARN Interferente Pequeño , Animales , Ratas , ARN Interferente Pequeño/genéticaRESUMEN
Conjugation of oligonucleotide therapeutics, including small interfering RNAs (siRNAs) or antisense oligonucleotides, to N-acetylgalactosamine (GalNAc) ligands has become the primary strategy for hepatocyte-targeted delivery, and with the recent approvals of GIVLAARI (givosiran) for the treatment of acute hepatic porphyria, OXLUMO (lumasiran) for the treatment of primary hyperoxaluria, and Leqvio (inclisiran) for the treatment of hypercholesterolemia, the technology has been well validated clinically. Although much knowledge has been gained over decades of development, there is a paucity of published literature on the drug metabolism and pharmacokinetic properties of GalNAc-siRNA. With this in mind, the goals of this minireview are to provide an aggregate analysis of these nonclinical absorption, distribution, metabolism, and excretion (ADME) data to build confidence on the translation of these properties to human. Upon subcutaneous administration, GalNAc-conjugated siRNAs are quickly distributed to the liver, resulting in plasma pharmacokinetic (PK) properties that reflect rapid elimination through asialoglycoprotein receptor-mediated uptake from circulation into hepatocytes. These studies confirm that liver PK, including half-life and, most importantly, siRNA levels in RNA-induced silencing complex in hepatocytes, are better predictors of pharmacodynamics (PD) than plasma PK. Several in vitro and in vivo nonclinical studies were conducted to characterize the ADME properties of GalNAc-conjugated siRNAs. These studies demonstrate that the PK/PD and ADME properties of GalNAc-conjugated siRNAs are highly conserved across species, are largely predictable, and can be accurately scaled to human, allowing us to identify efficacious and safe clinical dosing regimens in the absence of human liver PK profiles. SIGNIFICANCE STATEMENT: Several nonclinical ADME studies have been conducted in order to provide a comprehensive overview of the disposition and elimination of GalNAc-conjugated siRNAs and the pharmacokinetic/pharmacodynamic translation between species. These studies demonstrate that the ADME properties of GalNAc-conjugated siRNAs are well correlated and predictable across species, building confidence in the ability to extrapolate to human.
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Acetilgalactosamina , Porfirias Hepáticas , Acetilgalactosamina/farmacocinética , Receptor de Asialoglicoproteína/metabolismo , Hepatocitos/metabolismo , Humanos , Porfirias Hepáticas/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
In order to achieve efficient therapeutic post-transcriptional gene-silencing mediated by the RNA interference (RNAi) pathway, small interfering RNAs (siRNAs) must be chemically modified. Several supra-RNA structures, with the potential to stabilize siRNAs metabolically have been evaluated for their ability to induce gene silencing, but all have limitations or have not been explored in therapeutically relevant contexts. Covalently closed circular RNA transcripts are prevalent in eukaryotes and have potential as biomarkers and disease targets, and circular RNA mimics are being explored for use as therapies. Here we report the synthesis and evaluation of small circular interfering RNAs (sciRNAs). To synthesize sciRNAs, a sense strand functionalized with the trivalent N-acetylgalactosamine (GalNAc) ligand and cyclized using 'click' chemistry was annealed to an antisense strand. This strategy was used for synthesis of small circles, but could also be used for synthesis of larger circular RNA mimics. We evaluated various sciRNA designs in vitro and in vivo. We observed improved metabolic stability of the sense strand upon circularization and off-target effects were eliminated. The 5'-(E)-vinylphosphonate modification of the antisense strand resulted in GalNAc-sciRNAs that are potent in vivo at therapeutically relevant doses. Physicochemical studies and NMR-based structural analysis, together with molecular modeling studies, shed light on the interactions of this novel class of siRNAs, which have a partial duplex character, with the RNAi machinery.
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Silenciador del Gen , Interferencia de ARN , ARN Circular , ARN Interferente Pequeño , Animales , Femenino , Ratones , Ratones Endogámicos C57BLRESUMEN
RNA interference (RNAi) offers the potential to treat disease at the earliest onset by selectively turning off the expression of target genes, such as intracellular oncogenes that drive cancer growth. However, the development of RNAi therapeutics as anti-cancer drugs has been limited by both a lack of efficient and target cell-specific delivery systems and the necessity to overcome numerous intracellular barriers, including serum/lysosomal instability, cell membrane impermeability, and limited endosomal escape. Here, we combine two technologies to achieve posttranscriptional gene silencing in tumor cells: Centyrins, alternative scaffold proteins binding plasma membrane receptors for targeted delivery, and small interfering RNAs (siRNAs), chemically modified for high metabolic stability and potency. An EGFR Centyrin known to internalize in EGFR-positive tumor cells was site-specifically conjugated to a beta-catenin (CTNNb1) siRNA and found to drive potent and specific target knockdown by free uptake in cell culture and in mice inoculated with A431 tumor xenografts (EGFR amplified). The generalizability of this approach was further demonstrated with Centyrins targeting multiple receptors (e.g., BCMA, PSMA, and EpCAM) and siRNAs targeting multiple genes (e.g., CD68, KLKb1, and SSB1). Moreover, by installing multiple conjugation handles, two different siRNAs were fused to a single Centyrin, and the conjugate was shown to simultaneously silence two different targets. Finally, by specifically pairing EpCAM-binding Centyrins that exhibited optimized internalization profiles, we present data showing that an EpCAM Centyrin CTNNb1 siRNA conjugate suppressed tumor cell growth of a colorectal cancer cell line containing an APC mutation but not cells with normal CTNNb1 signaling. Overall, these data demonstrate the potential of Centyrin-siRNA conjugates to target cancer cells and silence oncogenes, paving the way to a new class of anticancer drugs.
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Técnicas de Transferencia de Gen , Interferencia de ARN , ARN Interferente Pequeño/genética , Animales , Línea Celular Tumoral , Técnicas de Silenciamiento del Gen , Silenciador del Gen , Genes erbB-1 , Terapia Genética , Humanos , Ligandos , Ratones , ARN Mensajero , ARN Interferente Pequeño/administración & dosificación , Tenascina/genética , Ensayos Antitumor por Modelo de Xenoinjerto , beta Catenina/genéticaRESUMEN
One hallmark of trivalent N-acetylgalactosamine (GalNAc)-conjugated siRNAs is the remarkable durability of silencing that can persist for months in preclinical species and humans. Here, we investigated the underlying biology supporting this extended duration of pharmacological activity. We found that siRNA accumulation and stability in acidic intracellular compartments is critical for long-term activity. We show that functional siRNA can be liberated from these compartments and loaded into newly generated Argonaute 2 protein complexes weeks after dosing, enabling continuous RNAi activity over time. Identical siRNAs delivered in lipid nanoparticles or as GalNAc conjugates were dose-adjusted to achieve similar knockdown, but only GalNAc-siRNAs supported an extended duration of activity, illustrating the importance of receptor-mediated siRNA trafficking in the process. Taken together, we provide several lines of evidence that acidic intracellular compartments serve as a long-term depot for GalNAc-siRNA conjugates and are the major contributor to the extended duration of activity observed in vivo.
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Acetilgalactosamina/metabolismo , Receptor de Asialoglicoproteína/metabolismo , Portadores de Fármacos , Silenciador del Gen , Prealbúmina/genética , ARN Interferente Pequeño/metabolismo , Acetilgalactosamina/química , Animales , Proteínas Argonautas/genética , Receptor de Asialoglicoproteína/genética , Transporte Biológico , Estabilidad de Medicamentos , Femenino , Glicoconjugados/química , Glicoconjugados/metabolismo , Hepatocitos/citología , Hepatocitos/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Hígado/citología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Nanopartículas/química , Nanopartículas/metabolismo , Prealbúmina/antagonistas & inhibidores , Prealbúmina/metabolismo , ARN Interferente Pequeño/genética , Factores de TiempoRESUMEN
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.
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Morfolinos/química , ARN Interferente Pequeño/química , Animales , Apolipoproteína B-100 , Apolipoproteínas B/genética , Proteínas Argonautas/genética , Silenciador del Gen , Humanos , Ratones , Modelos Moleculares , Morfolinos/síntesis química , Morfolinos/genética , Interferencia de ARN , ARN Interferente Pequeño/síntesis química , ARN Interferente Pequeño/genéticaRESUMEN
Clinical application of siRNA-based therapeutics outside of the liver has been hindered by the inefficient delivery of siRNA effector molecules into extra-hepatic organs and cells of interest. To understand the parameters that enable RNAi activity in vivo, it is necessary to develop a systematic approach to identify which cells within a tissue are permissive to oligonucleotide internalization and activity. In the present study, we evaluate the distribution and activity within the lung of chemically stabilized siRNA to characterize cell-type tropism and structure-activity relationship. We demonstrate intratracheal delivery of fully modified siRNA for RNAi-mediated target knockdown in lung CD11c+ cells (dendritic cells, alveolar macrophages) and alveolar epithelial cells. Finally, we use an allergen-induced model of lung inflammation to demonstrate the capacity of inhaled siRNA to induce target knockdown in dendritic cells and ameliorate lung pathology.
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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.
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Acetilgalactosamina/efectos adversos , Acetilgalactosamina/química , Desoxirribonucleótidos/efectos adversos , Desoxirribonucleótidos/química , Flúor/química , ARN Interferente Pequeño/efectos adversos , ARN Interferente Pequeño/química , Animales , Femenino , Flúor/efectos adversos , Humanos , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
An efficient multicomponent orthogonal protocol was developed for post-synthetic oligonucleotide modification using commercially available 2'- O-methyl ester and 2'- O-propargyl nucleoside scaffolds. Amidation of methyl esters with primary amines was achieved in the presence of 2'-propargyl groups which were utilized for subsequent copper catalyzed cycloaddition with GalNAc-azide. The methodology was applied to generate siRNA composed of multiple amide and triazole conjugates. Computational methods were used to illustrate the impact of substitution at the 2'-position. This a powerful post-oligomerization technique for rapidly introducing diversity to oligonucleotide design.
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Acetilgalactosamina/química , Amidas/química , Azidas/química , Cobre/química , Reacción de Cicloadición/métodos , Oligonucleótidos/química , ARN Interferente Pequeño/química , Acetilgalactosamina/síntesis química , Amidas/síntesis química , Azidas/síntesis química , Catálisis , Química Clic/métodos , Esterificación , Células HeLa , Humanos , Modelos Moleculares , Oligonucleótidos/síntesis química , ARN Interferente Pequeño/síntesis química , Triazoles/síntesis química , Triazoles/químicaRESUMEN
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.
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Silenciador del Gen , ARN Interferente Pequeño/genética , Acetilgalactosamina/genética , Animales , Secuencia de Bases , Biotecnología , Células Cultivadas , Femenino , Hepatocitos/metabolismo , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Oligonucleótidos/química , Oligonucleótidos/genética , Interferencia de ARN , ARN Interferente Pequeño/químicaRESUMEN
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.
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Acetilgalactosamina/química , Hígado/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/toxicidad , Acetilgalactosamina/toxicidad , Animales , Hígado/metabolismo , Masculino , Interferencia de ARN , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
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.
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Acetilgalactosamina , Interferencia de ARN , ARN Interferente Pequeño , Acetilgalactosamina/química , Animales , Proteínas Argonautas/genética , Regulación de la Expresión Génica , Silenciador del Gen , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Ratones , Ratones Transgénicos , ARN Mensajero/genética , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , ARN Interferente Pequeño/genéticaRESUMEN
(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.
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Organofosfonatos/química , Organofosfonatos/síntesis química , ARN Interferente Pequeño/química , Animales , Proteínas Argonautas/química , Proteínas Argonautas/deficiencia , Proteínas Argonautas/genética , Secuencia de Bases , Técnicas de Química Sintética , Silenciador del Gen , Ratones , Modelos Moleculares , Dominios Proteicos , ARN Interferente Pequeño/genética , EstereoisomerismoRESUMEN
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.
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Acetilgalactosamina , Receptor de Asialoglicoproteína/genética , Regulación de la Expresión Génica , Interferencia de ARN , ARN Interferente Pequeño/genética , Acetilgalactosamina/química , Animales , Receptor de Asialoglicoproteína/química , Receptor de Asialoglicoproteína/metabolismo , Modelos Animales de Enfermedad , Portadores de Fármacos , Sistemas de Liberación de Medicamentos , Evaluación Preclínica de Medicamentos , Femenino , Silenciador del Gen , Hepatocitos/metabolismo , Humanos , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Ratones , Ratones Noqueados , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Interferente Pequeño/químicaRESUMEN
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.