10 research outputs found
Improving Drug Discovery by Nucleic Acid Delivery in Engineered Human Microlivers
The liver plays a central role in metabolism; however, xenobiotic metabolism variations between human hepatocytes and those in model organisms create challenges in establishing functional test beds to detect the potential drug toxicity and efficacy of candidate small molecules. In the emerging areas of RNA interference, viral gene therapy, and genome editing, more robust, long-lasting, and predictive human liver models may accelerate progress. Here, we apply a new modality to a previously established, functionally stable, multi-well bioengineered microliverâfabricated from primary human hepatocytes and supportive stromal cellsâin order to advance both small molecule and nucleic acid therapeutic pipelines. Specifically, we achieve robust and durable gene silencing in vitro to tune the human metabolism of small molecules, and demonstrate its capacity to query the potential efficacy and/or toxicity of candidate therapeutics. Additionally, we apply this engineered platform to test siRNAs designed to target hepatocytes and impact human liver genetic and infectious diseases. Mancio-Silva et al. show that nucleic acid-mediated silencing of primary human hepatocytes can be leveraged in an in vitro engineered human liver model to fine-tune metabolism and to assess safety and efficacy of RNAi-based therapeutics.National Cancer Institute (Grant P30-CA14051
Reduction in urinary oxalate excretion in mouse models of Primary Hyperoxaluria by RNA interference inhibition of liver lactate dehydrogenase activity
In vitro and in vivo characterization of VIR-2218, an investigational RNAi therapeutic targeting hepatitis B virus
Preclinical Development of a Subcutaneous ALAS1 RNAi Therapeutic for Treatment of Hepatic Porphyrias Using Circulating RNA Quantification
The acute hepatic porphyrias are caused by inherited enzymatic deficiencies in the heme biosynthesis pathway. Induction of the first enzyme 5-aminolevulinic acid synthase 1 (ALAS1) by triggers such as fasting or drug exposure can lead to accumulation of neurotoxic heme intermediates that cause disease symptoms. We have demonstrated that hepatic ALAS1 silencing using siRNA in a lipid nanoparticle effectively prevents and treats induced attacks in a mouse model of acute intermittent porphyria. Herein, we report the development of ALN-AS1, an investigational GalNAc-conjugated RNAi therapeutic targeting ALAS1. One challenge in advancing ALN-AS1 to patients is the inability to detect liver ALAS1 mRNA in the absence of liver biopsies. We here describe a less invasive circulating extracellular RNA detection assay to monitor RNAi drug activity in serum and urine. A striking correlation in ALAS1 mRNA was observed across liver, serum, and urine in both rodents and nonhuman primates (NHPs) following treatment with ALN-AS1. Moreover, in donor-matched human urine and serum, we demonstrate a notable correspondence in ALAS1 levels, minimal interday assay variability, low interpatient variability from serial sample collections, and the ability to distinguish between healthy volunteers and porphyria patients with induced ALAS1 levels. The collective data highlight the potential utility of this assay in the clinical development of ALN-AS1, and in broadening our understanding of acute hepatic porphyrias disease pathophysiology
siRNA Conjugates Carrying Sequentially Assembled Trivalent <i>N-</i>Acetylgalactosamine Linked Through Nucleosides Elicit Robust Gene Silencing <i>In Vivo</i> in Hepatocytes
Asialoglycoprotein
receptor (ASGPR) mediated delivery of triantennary <i>N</i>-acetylgalactosamine (GalNAc) conjugated short interfering
RNAs (siRNAs) to hepatocytes is a promising paradigm for RNAi therapeutics.
Robust and durable gene silencing upon subcutaneous administration
at therapeutically acceptable dose levels resulted in the advancement
of GalNAc-conjugated oligonucleotide-based drugs into preclinical
and clinical developments. To systematically evaluate the effect of
display and positioning of the GalNAc moiety within the siRNA duplex
on ASGPR binding and RNAi activity, nucleotides carrying monovalent
GalNAc were designed. Evaluation of clustered and dispersed incorporation
of GalNAc units to the sense (S) strand indicated that sugar proximity
is critical for ASGPR recognition, and location of the clustered ligand
impacts the intrinsic potency of the siRNA. An array of nucleosidic
GalNAc monomers resembling a trivalent ligand at or near the 3âČ
end of the S strand retained <i>in vitro</i> and <i>in vivo</i> siRNA activity, similar to the parent conjugate
design. This work demonstrates the utility of simple, nucleotide-based,
cost-effective siRNAâGalNAc conjugation strategies
Metabolism of 13C5-hydroxyproline in mouse models of Primary Hyperoxaluria and its inhibition by RNAi therapeutics targeting liver glycolate oxidase and hydroxyproline dehydrogenase
siRNA Conjugates Carrying Sequentially Assembled Trivalent <i>N-</i>Acetylgalactosamine Linked Through Nucleosides Elicit Robust Gene Silencing <i>In Vivo</i> in Hepatocytes
Asialoglycoprotein
receptor (ASGPR) mediated delivery of triantennary <i>N</i>-acetylgalactosamine (GalNAc) conjugated short interfering
RNAs (siRNAs) to hepatocytes is a promising paradigm for RNAi therapeutics.
Robust and durable gene silencing upon subcutaneous administration
at therapeutically acceptable dose levels resulted in the advancement
of GalNAc-conjugated oligonucleotide-based drugs into preclinical
and clinical developments. To systematically evaluate the effect of
display and positioning of the GalNAc moiety within the siRNA duplex
on ASGPR binding and RNAi activity, nucleotides carrying monovalent
GalNAc were designed. Evaluation of clustered and dispersed incorporation
of GalNAc units to the sense (S) strand indicated that sugar proximity
is critical for ASGPR recognition, and location of the clustered ligand
impacts the intrinsic potency of the siRNA. An array of nucleosidic
GalNAc monomers resembling a trivalent ligand at or near the 3âČ
end of the S strand retained <i>in vitro</i> and <i>in vivo</i> siRNA activity, similar to the parent conjugate
design. This work demonstrates the utility of simple, nucleotide-based,
cost-effective siRNAâGalNAc conjugation strategies