A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene Silencing<i>In Vivo</i>

Coles, Andrew H.; Osborn, Maire F.; Alterman, Julia F.; Turanov, Anton A.; Kennington, Lori; Chase, Kathryn; Aronin, Neil; Khvorova, Anastasia

doi:10.1089/nat.2015.0578

Cited by 40 publications

(52 citation statements)

References 28 publications

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“…Wild-type mice (FVB/NJ, n = 8 per group) were injected into the right striatum with aCSF, a non-targeting control hsiRNA (PC-DHA-hsiRNA NTC , 4 nmol, 50 µg), DHA-hsiRNA Htt (2 nmol, 25 µg), and PC-DHA-hsiRNA Htt (2 nmol, 25 µg). After one week, the levels of Htt mRNA expression in the ipsilateral striatum and cortex were measured by the QuantiGene® assay, normalized to a housekeeping gene ( Hprt1 ), and presented as percentage of aCSF-treated control 48 . Huntingtin mRNA levels are reduced by 72% and 77% in the striatum after administration of 2 nmol of DHA-hsiRNA or PC-DHA-hsiRNA Htt , respectively (Figure 3a).…”

Section: Resultsmentioning

confidence: 99%

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Synthesis and Evaluation of Parenchymal Retention and Efficacy of a Metabolically Stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA Conjugate in Mouse Brain

Nikan¹,

Osborn²,

Coles³

et al. 2017

Bioconjugate Chem.

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Ligand-conjugated siRNAs have potential to achieve targeted delivery and efficient silencing in neurons following local administration in the central nervous system (CNS). We recently described the activity and safety profile of a docosahexaenoic acid (DHA)-conjugated, hydrophobic siRNA (DHA-hsiRNA) targeting Huntingtin (Htt) mRNA in mouse brain. Here, we report the synthesis of an amide-modified, phosphocholine-containing DHA-hsiRNA conjugate (PC-DHA-hsiRNA), which closely resembles the endogenously esterified biological structure of DHA. We hypothesized that this modification may enhance neuronal delivery in vivo. We demonstrate that PC-DHA-hsiRNA silences Htt in mouse primary cortical neurons and astrocytes. After intrastriatal delivery, Htt-targeting PC-DHA-hsiRNA induces ~80% mRNA silencing and 71% protein silencing after one week. However, PC-DHA-hsiRNA did not substantially outperform DHA-hsiRNA under the conditions tested. Moreover, at the highest locally administered dose (4 nmol, 50 µg), we observe evidence of PC-DHA-hsiRNA-mediated reactive astrogliosis. Lipophilic ligand conjugation enables siRNA delivery to neural tissues, but rational design of functional, non-toxic siRNA conjugates for CNS delivery remains challenging.

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Section: Resultsmentioning

confidence: 99%

“…mRNA was quantified from both cells and tissue punches using the QuantiGene® 2.0 DNA Assay (Affymetrix) exactly as described previously 1, 48 . Probe sets (Mouse Htt or Hprt ) were diluted and used according to the manufacturer’s recommended protocol.…”

Section: Methodsmentioning

confidence: 99%

Synthesis and Evaluation of Parenchymal Retention and Efficacy of a Metabolically Stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA Conjugate in Mouse Brain

Nikan¹,

Osborn²,

Coles³

et al. 2017

Bioconjugate Chem.

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“…Thus, for an accurate mRNA quantification and evaluation of in vivo silencing efficiency, the brains are processed immediately after harvest (Alterman et al ., 2015; Coles et al ., 2016; Didiot et al ., 2016). Brains are cut in 300 μM coronal sections in PBS at −1 to 0.5 °C using a vibratome according to the manufacturer’s instructions.…”

Section: Materials and Reagentsmentioning

confidence: 99%

“…Brain samples are processed for mRNA quantification following the method from Alterman et al ., 2015; Coles et al ., 2016; Didiot et al ., 2016.…”

Section: Materials and Reagentsmentioning

confidence: 99%

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Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System

Haraszti¹,

Coles²,

Aronin³

et al. 2017

BIO-PROTOCOL

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Efficient delivery of oligonucleotide therapeutics, i.e., siRNAs, to the central nervous system represents a significant barrier to their clinical advancement for the treatment of neurological disorders. Small, endogenous extracellular vesicles were shown to be able to transport lipids, proteins and RNA between cells, including neurons. This natural trafficking ability gives extracellular vesicles the potential to be used as delivery vehicles for oligonucleotides, i.e., siRNAs. However, robust and scalable methods for loading of extracellular vesicles with oligonucleotide cargo are lacking. We describe a detailed protocol for the loading of hydrophobically modified siRNAs into extracellular vesicles upon simple co-incubation. We detail methods of the workflow from purification of extracellular vesicles to data analysis. This method may advance extracellular vesicles-based therapies for the treatment of a broad range of neurological disorders.

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The valency of fatty acid conjugates impacts siRNA pharmacokinetics, distribution, and efficacy in vivo

Biscans

Coles

Echeverria

et al. 2019

Journal of Controlled Release

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Lipid-conjugated small-interfering RNAs (siRNAs) exhibit accumulation and gene silencing in extrahepatic tissues, providing an opportunity to expand therapeutic siRNA utility beyond the liver. Chemically engineering lipids may further improve siRNA delivery and efficacy, but the relationship between lipid structure/configuration and siRNA pharmacodynamics is unclear. Here, we synthesized a panel of mono-, di-, and tri-meric fatty acid-conjugated siRNAs to systematically evaluate the impact of fatty acid structure and valency on siRNA clearance, distribution, and efficacy. Fatty acid valency significantly altered the physicochemical properties of conjugated siRNAs, including hydrophobicity and micelle formation, which affected distribution. Trivalent lipid-conjugated siRNAs were predominantly retained at the site of injection with minimal systemic exposure, whereas monovalent lipid-conjugated siRNAs were quickly released into the circulation and accumulated primarily in kidney. Divalent lipid-conjugated siRNAs showed intermediate behavior, and preferentially accumulated in liver with functional distribution to lung, heart, and fat. The chemical structure of the conjugate, rather than overall physicochemical properties (i.e. hydrophobicity), predicted the degree of extrahepatic tissue accumulation necessary for productive gene silencing. Our findings will inform chemical engineering strategies for enhancing the extrahepatic delivery of lipophilic siRNAs.

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A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene SilencingIn Vivo

Cited by 40 publications

References 28 publications

Synthesis and Evaluation of Parenchymal Retention and Efficacy of a Metabolically Stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA Conjugate in Mouse Brain

Synthesis and Evaluation of Parenchymal Retention and Efficacy of a Metabolically Stable O-Phosphocholine-N-docosahexaenoyl-l-serine siRNA Conjugate in Mouse Brain

Loading of Extracellular Vesicles with Chemically Stabilized Hydrophobic siRNAs for the Treatment of Disease in the Central Nervous System

The valency of fatty acid conjugates impacts siRNA pharmacokinetics, distribution, and efficacy in vivo

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