12 research outputs found
Influence of Polyethylene Glycol Lipid Desorption Rates on Pharmacokinetics and Pharmacodynamics of siRNA Lipid Nanoparticles
Lipid nanoparticles (LNPs) encapsulating short interfering RNAs that target hepatic genes are advancing through clinical trials, and early results indicate the excellent gene silencing observed in rodents and nonhuman primates also translates to humans. This success has motivated research to identify ways to further advance this delivery platform. Here, we characterize the polyethylene glycol lipid (PEG-lipid) components, which are required to control the self-assembly process during formation of lipid particles, but can negatively affect delivery to hepatocytes and hepatic gene silencing in vivo. The rate of transfer from LNPs to plasma lipoproteins in vivo is measured for three PEG-lipids with dialkyl chains 14, 16, and 18 carbons long. We show that 1.5 mol % PEG-lipid represents a threshold concentration at which the chain length exerts a minimal effect on hepatic gene silencing but can still modify LNPs pharmacokinetics and biodistribution. Increasing the concentration to 2.5 and 3.5 mol % substantially compromises hepatocyte gene knockdown for PEG-lipids with distearyl (C18) chains but has little impact for shorter dimyristyl (C14) chains. These data are discussed with respect to RNA delivery and the different rates at which the steric barrier disassociates from LNPs in vivo
Branched Multifunctional Polyether Polyketals: Variation of Ketal Group Structure Enables Unprecedented Control over Polymer Degradation in Solution and within Cells
Multifunctional biocompatible and biodegradable nanomaterials
incorporating
specific degradable linkages that respond to various stimuli and with
defined degradation profiles are critical to the advancement of targeted
nanomedicine. Herein we report, for the first time, a new class of
multifunctional dendritic polyether polyketals containing different
ketal linkages in their backbone that exhibit unprecedented control
over degradation in solution and within the cells. High-molecular-weight
and highly compact polyÂ(ketal hydroxyethers) (PKHEs) were synthesized
from newly designed α-epoxy-ω-hydroxyl-functionalized
AB<sub>2</sub>-type ketal monomers carrying structurally different
ketal groups (both cyclic and acyclic) with good control over polymer
properties by anionic ring-opening multibranching polymerization.
Polymer functionalization with multiple azide and amine groups was
achieved without degradation of the ketal group. The polymer degradation
was controlled primarily by the differences in the structure and torsional
strain of the substituted ketal groups in the main chain, while for
polymers with linear (acyclic) ketal groups, the hydrophobicity of
the polymer may play an additional role. This was supported by the
log <i>P</i> values of the monomers and the hydrophobicity
of the polymers determined by fluorescence spectroscopy using pyrene
as the probe. A range of hydrolysis half-lives of the polymers at
mild acidic pH values was achieved, from a few minutes to a few hundred
days, directly correlating with the differences in ketal group structures.
Confocal microscopy analyses demonstrated similar degradation profiles
for PKHEs within live cells, as seen in solution and the delivery
of fluorescent marker to the cytosol. The cell viability measured
by MTS assay and blood compatibility determined by complement activation,
platelet activation, and coagulation assays demonstrate that PKHEs
and their degradation products are highly biocompatible. Taken together,
these data demonstrate the utility this new class of biodegradable
polymer as a highly promising candidate in the development of multifunctional
nanomedicine
Allele-Selective Inhibition of Mutant Atrophin‑1 Expression by Duplex and Single-Stranded RNAs
Dentatorubral-pallidoluysian
atrophy (DRPLA) is a progressive neurodegenerative
disorder that currently has no curative treatments. DRPLA is caused
by an expansion of a CAG trinucleotide repeat region within the protein-encoding
sequence of the atrophin-1 (<i>ATN-1</i>) gene. Inhibition
of mutant ATN-1 protein expression is one strategy for treating DRPLA,
and allele-selective gene silencing agents that block mutant expression
over wild-type expression would be lead compounds for therapeutic
development. Here we develop an assay for distinguishing mutant from
wild-type ATN-1 protein by gel electrophoresis. We use this assay
to evaluate duplex RNAs and single-stranded silencing RNAs (ss-siRNAs)
for allele-selective inhibition of ATN-1 protein expression. We observed
potent and allele-selective inhibition by RNA duplexes that contain
mismatched bases relative to the CAG target and have the potential
to form miRNA-like complexes. ss-siRNAs that contained mismatches
were as selective as mismatch-containing duplexes. We also report
allele-selective inhibition by duplex RNAs containing unlocked nucleic
acids or abasic substitutions, although selectivities are not as high.
Five compounds that showed >8-fold allele selectivity for mutant <i>ATN-1</i> were also selective for inhibiting the expression
of two other trinucleotide repeat disease genes, ataxin-3 (<i>ATXN-3</i>) and huntingtin (<i>HTT</i>). These data
demonstrate that the expanded trinucleotide repeat within <i>ATN-1</i> mRNA is a potential target for compounds designed
to achieve allele-selective inhibition of ATN-1 protein, and one agent
may allow the targeting of multiple disease genes
Targeted Delivery of RNAi Therapeutics With Endogenous and Exogenous Ligand-Based Mechanisms
Lipid nanoparticles (LNPs) have proven to be highly efficient carriers of short-interfering RNAs (siRNAs) to hepatocytes in vivo; however, the precise mechanism by which this efficient delivery occurs has yet to be elucidated. We found that apolipoprotein E (apoE), which plays a major role in the clearance and hepatocellular uptake of physiological lipoproteins, also acts as an endogenous targeting ligand for ionizable LNPs (iLNPs), but not cationic LNPs (cLNPs). The role of apoE was investigated using both in vitro studies employing recombinant apoE and in vivo studies in wild-type and apoE−/− mice. Receptor dependence was explored in vitro and in vivo using low-density lipoprotein receptor (LDLR−/−)–deficient mice. As an alternative to endogenous apoE-based targeting, we developed a targeting approach using an exogenous ligand containing a multivalent N-acetylgalactosamine (GalNAc)-cluster, which binds with high affinity to the asialoglycoprotein receptor (ASGPR) expressed on hepatocytes. Both apoE-based endogenous and GalNAc-based exogenous targeting appear to be highly effective strategies for the delivery of iLNPs to liver
A rapid two-step method for isolation of functional primary mouse hepatocytes: cell characterization and asialoglycoprotein receptor based assay development
Therapeutic RNAi targeting PCSK9 acutely lowers plasma cholesterol in rodents and LDL cholesterol in nonhuman primates
Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates low density lipoprotein receptor (LDLR) protein levels and function. Loss of PCSK9 increases LDLR levels in liver and reduces plasma LDL cholesterol (LDLc), whereas excess PCSK9 activity decreases liver LDLR levels and increases plasma LDLc. Here, we have developed active, cross-species, small interfering RNAs (siRNAs) capable of targeting murine, rat, nonhuman primate (NHP), and human PCSK9. For in vivo studies, PCSK9 and control siRNAs were formulated in a lipidoid nanoparticle (LNP). Liver-specific siRNA silencing of PCSK9 in mice and rats reduced PCSK9 mRNA levels by 50-70%. The reduction in PCSK9 transcript was associated with up to a 60% reduction in plasma cholesterol concentrations. These effects were shown to be mediated by an RNAi mechanism, using 5′-RACE. In transgenic mice expressing human PCSK9, siRNAs silenced the human PCSK9 transcript by >70% and significantly reduced PCSK9 plasma protein levels. In NHP, a single dose of siRNA targeting PCSK9 resulted in a rapid, durable, and reversible lowering of plasma PCSK9, apolipoprotein B, and LDLc, without measurable effects on either HDL cholesterol (HDLc) or triglycerides (TGs). The effects of PCSK9 silencing lasted for 3 weeks after a single bolus i.v. administration. These results validate PCSK9 targeting with RNAi therapeutics as an approach to specifically lower LDLc, paving the way for the development of PCSK9-lowering agents as a future strategy for treatment of hypercholesterolemia
Development of Lipidoid–siRNA Formulations for Systemic Delivery to the Liver
RNA interference therapeutics afford the potential to silence target gene expression specifically, thereby blocking production of disease-causing proteins. The development of safe and effective systemic small interfering RNA (siRNA) delivery systems is of central importance to the therapeutic application of siRNA. Lipid and lipid-like materials are currently the most well-studied siRNA delivery systems for liver delivery, having been utilized in several animal models, including nonhuman primates. Here, we describe the development of a multicomponent, systemic siRNA delivery system, based on the novel lipid-like material 98N12-5(1). We show that in vivo delivery efficacy is affected by many parameters, including the formulation composition, nature of particle PEGylation, degree of drug loading, and biophysical parameters such as particle size. In particular, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids can result in significant effects on in vivo efficacy. The lead formulation developed is liver targeted (>90% injected dose distributes to liver) and can induce fully reversible, long-duration gene silencing without loss of activity following repeat administration