Lysophosphatidic acid triggers inflammation in the liver and white adipose tissue in rat models of 1-acyl-sn-glycerol-3-phosphate acyltransferase 2 deficiency and overnutrition.

AGPAT2 (1-acyl-sn-glycerol-3-phosphate-acyltransferase-2) converts lysophosphatidic acid (LPA) into phosphatidic acid (PA), and mutations of the AGPAT2 gene cause the most common form of congenital generalized lipodystrophy which leads to steatohepatitis. The underlying mechanism by which AGPAT2 deficiency leads to lipodystrophy and steatohepatitis has not been elucidated. We addressed this question using an antisense oligonucleotide (ASO) to knockdown expression of Agpat2 in the liver and white adipose tissue (WAT) of adult male Sprague-Dawley rats. Agpat2 ASO treatment induced lipodystrophy and inflammation in WAT and the liver, which was associated with increased LPA content in both tissues, whereas PA content was unchanged. We found that a controlled-release mitochondrial protonophore (CRMP) prevented LPA accumulation and inflammation in WAT whereas an ASO against glycerol-3-phosphate acyltransferase, mitochondrial (Gpam) prevented LPA content and inflammation in the liver in Agpat2 ASO-treated rats. In addition, we show that overnutrition, due to high sucrose feeding, resulted in increased hepatic LPA content and increased activated macrophage content which were both abrogated with Gpam ASO treatment. Taken together, these data identify LPA as a key mediator of liver and WAT inflammation and lipodystrophy due to AGPAT2 deficiency as well as liver inflammation due to overnutrition and identify LPA as a potential therapeutic target to ameliorate these conditions.

LipidSIM: Inferring mechanistic lipid biosynthesis perturbations from lipidomics with a flexible, low-parameter, Markov modeling framework.

Lipid metabolism is a complex and dynamic system involving numerous enzymes at the junction of multiple metabolic pathways. Disruption of these pathways leads to systematic dyslipidemia, a hallmark of many pathological developments, such as nonalcoholic steatohepatitis and diabetes. Recent advances in computational tools can provide insights into the dysregulation of lipid biosynthesis, but limitations remain due to the complexity of lipidomic data, limited knowledge of interactions among involved enzymes, and technical challenges in standardizing across different lipid types. Here, we present a low-parameter, biologically interpretable framework named Lipid Synthesis Investigative Markov model (LipidSIM), which models and predicts the source of perturbations in lipid biosynthesis from lipidomic data. LipidSIM achieves this by accounting for the interdependency between the lipid species via the lipid biosynthesis network and generates testable hypotheses regarding changes in lipid biosynthetic reactions. This feature allows the integration of lipidomics with other omics types, such as transcriptomics, to elucidate the direct driving mechanisms of altered lipidomes due to treatments or disease progression. To demonstrate the value of LipidSIM, we first applied it to hepatic lipidomics following Keap1 knockdown and found that changes in mRNA expression of the lipid pathways were consistent with the LipidSIM-predicted fluxes. Second, we used it to study lipidomic changes following intraperitoneal injection of CCl4 to induce fast NAFLD/NASH development and the progression of fibrosis and hepatic cancer. Finally, to show the power of LipidSIM for classifying samples with dyslipidemia, we used a Dgat2-knockdown study dataset. Thus, we show that as it demands no a priori knowledge of enzyme kinetics, LipidSIM is a valuable and intuitive framework for extracting biological insights from complex lipidomic data.

Fatty acid conjugation enhances potency of antisense oligonucleotides in muscle.

Enhancing the functional uptake of antisense oligonucleotide (ASO) in the muscle will be beneficial for developing ASO therapeutics targeting genes expressed in the muscle. We hypothesized that improving albumin binding will facilitate traversal of ASO from the blood compartment to the interstitium of the muscle tissues to enhance ASO functional uptake. We synthesized structurally diverse saturated and unsaturated fatty acid conjugated ASOs with a range of hydrophobicity. The binding affinity of ASO fatty acid conjugates to plasma proteins improved with fatty acid chain length and highest binding affinity was observed with ASO conjugates containing fatty acid chain length from 16 to 22 carbons. The degree of unsaturation or conformation of double bond appears to have no influence on protein binding or activity of ASO fatty acid conjugates. Activity of fatty acid ASO conjugates correlated with the affinity to albumin and the tightest albumin binder exhibited the highest activity improvement in muscle. Palmitic acid conjugation increases ASO plasma Cmax and improved delivery of ASO to interstitial space of mouse muscle. Conjugation of palmitic acid improved potency of DMPK, Cav3, CD36 and Malat-1 ASOs (3- to 7-fold) in mouse muscle. Our approach provides a foundation for developing more effective therapeutic ASOs for muscle disorders.

Conjugation of hydrophobic moieties enhances potency of antisense oligonucleotides in the muscle of rodents and non-human primates.

We determined the effect of attaching palmitate, tocopherol or cholesterol to PS ASOs and their effects on plasma protein binding and on enhancing ASO potency in the muscle of rodents and monkeys. We found that cholesterol ASO conjugates showed 5-fold potency enhancement in the muscle of rodents relative to unconjugated ASOs. However, they were toxic in mice and as a result were not evaluated in the monkey. In contrast, palmitate and tocopherol-conjugated ASOs showed enhanced potency in the skeletal muscle of rodents and modest enhancements in potency in the monkey. Analysis of the plasma-protein binding profiles of the ASO-conjugates by size-exclusion chromatography revealed distinct and species-specific differences in their association with plasma proteins which likely rationalizes their behavior in animals. Overall, our data suggest that modulating binding to plasma proteins can influence ASO activity and distribution to extra-hepatic tissues in a species-dependent manner and sets the stage to identify other strategies to enhance ASO potency in muscle tissues.

Double-Stranded RNA Is a Novel Molecular Target in Osteomyelitis Pathogenesis: A Translational Avian Model for Human Bacterial Chondronecrosis with Osteomyelitis.

Osteomyelitis remains a serious inflammatory bone disease that affects millions of individuals worldwide and for which there is no effective treatment. Despite scientific evidence that Staphylococcus bacteria are the most common causative species for human bacterial chondronecrosis with osteomyelitis (BCO), much remains to be understood about the underlying virulence mechanisms. Herein, we show increased levels of double-stranded RNA (dsRNA) in infected bone in a Staphylococcus-induced chicken BCO model and in human osteomyelitis samples. Administration of synthetic [poly(I:C)] or genetic (Alu) dsRNA induces human osteoblast cell death. Similarly, infection with Staphylococcus isolated from chicken BCO induces dsRNA accumulation and cell death in human osteoblast cell cultures. Both dsRNA administration and Staphylococcus infection activate NACHT, LRR and PYD domains-containing protein (NLRP)3 inflammasome and increase IL18 and IL1B gene expression in human osteoblasts. Pharmacologic inhibition with Ac-YVAD-cmk of caspase 1, a critical component of the NLRP3 inflammasome, prevents DICER1 dysregulation- and dsRNA-induced osteoblast cell death. NLRP3 inflammasome and its components are also activated in bone from BCO chickens and humans with osteomyelitis, compared with their healthy counterparts. These findings provide a rationale for the use of chicken BCO as a human-relevant spontaneous animal model for osteomyelitis and identify dsRNA as a new treatment target for this debilitating bone pathogenesis.

TricycloDNA-modified oligo-2'-deoxyribonucleotides reduce scavenger receptor B1 mRNA in hepatic and extra-hepatic tissues--a comparative study of oligonucleotide length, design and chemistry.

We report the evaluation of 20-, 18-, 16- and 14-mer phosphorothioate (PS)-modified tricycloDNA (tcDNA) gapmer antisense oligonucleotides (ASOs) in T(m), cell culture and animal experiments and compare them to their gap-matched 20-mer 2'-O-methoxyethyl (MOE) and 14-mer 2',4'-constrained ethyl (cEt) counterparts. The sequence-matched 20-mer tcDNA and MOE ASOs showed similar T(m) and activity in cell culture under free-uptake and cationic lipid-mediated transfection conditions, while the 18-, 16- and 14-mer tcDNA ASOs were moderate to significantly less active. These observations were recapitulated in the animal experiments where the 20-mer tcDNA ASO formulated in saline showed excellent activity (ED(50) 3.9 mg/kg) for reducing SR-B1 mRNA in liver. The tcDNA 20-mer ASO also showed better activity than the MOE 20-mer in several extra-hepatic tissues such as kidney, heart, diaphragm, lung, fat, gastrocnemius and quadriceps. Interestingly, the 14-mer cEt ASO showed the best activity in the animal experiments despite significantly lower T(m) and 5-fold reduced activity in cell culture relative to the 20-mer tcDNA and MOE-modified ASOs. Our experiments establish tcDNA as a useful modification for antisense therapeutics and highlight the role of chemical modifications in influencing ASO pharmacology and pharmacokinetic properties in animals.

Innate and adaptive immune cell interaction drives inflammasome activation and hepatocyte apoptosis in murine liver injury from immune checkpoint inhibitors.

Immune checkpoints (CTLA4 & PD-1) are inhibitory pathways that block aberrant immune activity and maintain self-tolerance. Tumors co-opt these checkpoints to avoid immune destruction. Immune checkpoint inhibitors (ICIs) activate immune cells and restore their tumoricidal potential, making them highly efficacious cancer therapies. However, immunotolerant organs such as the liver depend on these tolerogenic mechanisms, and their disruption with ICI use can trigger the unintended side effect of hepatotoxicity termed immune-mediated liver injury from ICIs (ILICI). Learning how to uncouple ILICI from ICI anti-tumor activity is of paramount clinical importance. We developed a murine model to recapitulate human ILICI using CTLA4+/- mice treated with either combined anti-CTLA4 + anti-PDL1 or IgG1 + IgG2. We tested two forms of antisense oligonucleotides to knockdown caspase-3 in a total liver (parenchymal and non-parenchymal cells) or in a hepatocyte-specific manner. We also employed imaging mass cytometry (IMC), a powerful multiplex modality for immunophenotyping and cell interaction analysis in our model. ICI-treated mice had significant evidence of liver injury. We detected cleaved caspase-3 (cC3), indicating apoptosis was occurring, as well as Nod-like receptor protein 3 (NLRP3) inflammasome activation, but no necroptosis. Total liver knockdown of caspase-3 worsened liver injury, and induced further inflammasome activation, and Gasdermin-D-mediated pyroptosis. Hepatocyte-specific knockdown of caspase-3 reduced liver injury and NLRP3 inflammasome activation. IMC-generated single-cell data for 77,692 cells was used to identify 22 unique phenotypic clusters. Spatial analysis revealed that cC3+ hepatocytes had significantly closer interactions with macrophages, Kupffer cells, and NLRP3hi myeloid cells than other cell types. We also observed zones of three-way interaction between cC3+ hepatocytes, CD8 + T-cells, and macrophages. Our work is the first to identify hepatocyte apoptosis and NLRP3 inflammasome activation as drivers of ILICI. Furthermore, we report that the interplay between adaptive and innate immune cells is critical to hepatocyte apoptosis and ILICI.

Chop/Ddit3 depletion in ß cells alleviates ER stress and corrects hepatic steatosis in mice.

Type 2 diabetes (T2D) is a metabolic disorder characterized by hyperglycemia, hyperinsulinemia, and insulin resistance (IR). During the early phase of T2D, insulin synthesis and secretion by pancreatic ß cells is enhanced, which can lead to proinsulin misfolding that aggravates endoplasmic reticulum (ER) protein homeostasis in ß cells. Moreover, increased circulating insulin may contribute to fatty liver disease. Medical interventions aimed at alleviating ER stress in ß cells while maintaining optimal insulin secretion are therefore an attractive therapeutic strategy for T2D. Previously, we demonstrated that germline Chop gene deletion preserved ß cells in high-fat diet (HFD)-fed mice and in leptin receptor-deficient db/db mice. In the current study, we further investigated whether targeting Chop/Ddit3 specifically in murine ß cells conferred therapeutic benefits. First, we showed that Chop deletion in ß cells alleviated ß cell ER stress and delayed glucose-stimulated insulin secretion (GSIS) in HFD-fed mice. Second, ß cell-specific Chop deletion prevented liver steatosis and hepatomegaly in aged HFD-fed mice without affecting basal glucose homeostasis. Third, we provide mechanistic evidence that Chop depletion reduces ER Ca2+ buffering capacity and modulates glucose-induced islet Ca2+ oscillations, leading to transcriptional changes of ER chaperone profile ("ER remodeling"). Last, we demonstrated that a GLP1-conjugated Chop antisense oligonucleotide strategy recapitulated the reduction in liver triglycerides and pancreatic insulin content. In summary, our results demonstrate that Chop depletion in ß cells provides a therapeutic strategy to alleviate dysregulated insulin secretion and consequent fatty liver disease in T2D.

Ligand conjugated antisense oligonucleotide for the treatment of transthyretin amyloidosis: preclinical and phase 1 data.

AIMS: Amyloidogenic transthyretin (ATTR) amyloidosis is a fatal disease characterized by progressive cardiomyopathy and/or polyneuropathy. AKCEA-TTR-LRx (ION-682884) is a ligand-conjugated antisense drug designed for receptor-mediated uptake by hepatocytes, the primary source of circulating transthyretin (TTR). Enhanced delivery of the antisense pharmacophore is expected to increase drug potency and support lower, less frequent dosing in treatment. METHODS AND RESULTS: AKCEA-TTR-LRx demonstrated an approximate 50-fold and 30-fold increase in potency compared with the unconjugated antisense drug, inotersen, in human hepatocyte cell culture and mice expressing a mutated human genomic TTR sequence, respectively. This increase in potency was supported by a preferential distribution of AKCEA-TTR-LRx to liver hepatocytes in the transgenic hTTR mouse model. A randomized, placebo-controlled, phase 1 study was conducted to evaluate AKCEA-TTR-LRx in healthy volunteers (ClinicalTrials.gov: NCT03728634). Eligible participants were assigned to one of three multiple-dose cohorts (45, 60, and 90 mg) or a single-dose cohort (120 mg), and then randomized 10:2 (active : placebo) to receive a total of 4 SC doses (Day 1, 29, 57, and 85) in the multiple-dose cohorts or 1 SC dose in the single-dose cohort. The primary endpoint was safety and tolerability; pharmacokinetics and pharmacodynamics were secondary endpoints. All randomized participants completed treatment. No serious adverse events were reported. In the multiple-dose cohorts, AKCEA-TTR-LRx reduced TTR levels from baseline to 2 weeks after the last dose of 45, 60, or 90 mg by a mean (SD) of -85.7% (8.0), -90.5% (7.4), and -93.8% (3.4), compared with -5.9% (14.0) for pooled placebo (P < 0.001). A maximum mean (SD) reduction in TTR levels of -86.3% (6.5) from baseline was achieved after a single dose of 120 mg AKCEA-TTR-LRx . CONCLUSIONS: These findings suggest an improved safety and tolerability profile with the increase in potency achieved by productive receptor-mediated uptake of AKCEA-TTR-LRx by hepatocytes and supports further development of AKCEA-TTR-LRx for the treatment of ATTR polyneuropathy and cardiomyopathy.

Lineage 2 west nile virus as cause of fatal neurologic disease in horses, South Africa.

Serologic evidence suggests that West Nile virus (WNV) is widely distributed in horses in southern Africa. However, because few neurologic cases have been reported, endemic lineage 2 strains were postulated to be nonpathogenic in horses. Recent evidence suggests that highly neuroinvasive lineage 2 strains exist in humans and mice. To determine whether neurologic cases are being missed in South Africa, we tested 80 serum or brain specimens from horses with unexplained fever (n = 48) and/or neurologic signs (n = 32) for WNV. From March 2007 through June 2008, using reverse transcription-PCR (RT-PCR) and immunoglobulin (Ig) M ELISA, we found WNV RNA or IgM in 7/32 horses with acute neurologic disease; 5 horses died or were euthanized. In 5/7 horses, no other pathogen was detected. DNA sequencing for all 5 RT-PCR-positive cases showed the virus belonged to lineage 2. WNV lineage 2 may cause neurologic disease in horses in South Africa.

Pnpla3 silencing with antisense oligonucleotides ameliorates nonalcoholic steatohepatitis and fibrosis in Pnpla3 I148M knock-in mice.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD) is becoming a leading cause of advanced chronic liver disease. The progression of NAFLD, including nonalcoholic steatohepatitis (NASH), has a strong genetic component, and the most robust contributor is the patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 encoding the 148M protein sequence variant. We hypothesized that suppressing the expression of the PNPLA3 148M mutant protein would exert a beneficial effect on the entire spectrum of NAFLD. METHODS: We examined the effects of liver-targeted GalNAc3-conjugated antisense oligonucleotide (ASO)-mediated silencing of Pnpla3 in a knock-in mouse model in which we introduced the human PNPLA3 I148M mutation. RESULTS: ASO-mediated silencing of Pnpla3 reduced liver steatosis (p = 0.038) in homozygous Pnpla3 148M/M knock-in mutant mice but not in wild-type littermates fed a steatogenic high-sucrose diet. In mice fed a NASH-inducing diet, ASO-mediated silencing of Pnpla3 reduced liver steatosis score and NAFLD activity score independent of the Pnpla3 genotype, while reductions in liver inflammation score (p = 0.018) and fibrosis stage (p = 0.031) were observed only in the Pnpla3 knock-in 148M/M mutant mice. These responses were accompanied by reduced liver levels of Mcp1 (p = 0.026) and Timp2 (p = 0.007) specifically in the mutant knock-in mice. This may reduce levels of chemokine attracting inflammatory cells and increase the collagenolytic activity during tissue regeneration. CONCLUSION: This study provides the first evidence that a Pnpla3 ASO therapy can improve all features of NAFLD, including liver fibrosis, and suppress the expression of a strong innate genetic risk factor, Pnpla3 148M, which may open up a precision medicine approach in NASH.

Peripheral androgen receptor gene suppression rescues disease in mouse models of spinal and bulbar muscular atrophy.

Spinal and bulbar muscular atrophy (SBMA) is caused by the polyglutamine androgen receptor (polyQ-AR), a protein expressed by both lower motor neurons and skeletal muscle. Although viewed as a motor neuronopathy, data from patients and mouse models suggest that muscle contributes to disease pathogenesis. Here, we tested this hypothesis using AR113Q knockin and human bacterial artificial chromosome/clone (BAC) transgenic mice that express the full-length polyQ-AR and display androgen-dependent weakness, muscle atrophy, and early death. We developed antisense oligonucleotides that suppressed AR gene expression in the periphery but not the CNS after subcutaneous administration. Suppression of polyQ-AR in the periphery rescued deficits in muscle weight, fiber size, and grip strength, reversed changes in muscle gene expression, and extended the lifespan of mutant males. We conclude that polyQ-AR expression in the periphery is an important contributor to pathology in SBMA mice and that peripheral administration of therapeutics should be explored for SBMA patients.

Characterization of target mRNA reduction through in situ RNA hybridization in multiple organ systems following systemic antisense treatment in animals.

Advances in the medicinal chemistry of antisense oligonucleotide drugs have been instrumental in achieving and optimizing antisense activity in cell types other than hepatocytes, the cell type that is most sensitive to antisense effects following systemic treatment. To broadly characterize the effects of antisense drugs on target messenger RNA (mRNA) levels in different organs and cell types in animals, we have developed a sensitive RNA in situ hybridization technique using the noncoding RNA metastasis associated lung adenocarcinoma transcript 1 (MALAT1) as a surrogate target. We have used this technique to evaluate the effects of 2'-O-methoxy ethyl (MOE) and constrained ethyl bicyclic nucleic acid (cEt) gapmer antisense oligonucleotides (ASOs). ASO tissue distribution was also characterized using immunohistochemical techniques, and MALAT1 mRNA reductions were confirmed by quantitative real time-polymerase chain reaction. Our findings demonstrate that systemic antisense drug administration in both mice and non-human primates resulted in marked reductions in MALAT1 RNA in many tissues and cell types other than liver including kidney, muscle, lung, adipose, adrenal gland, and peripheral nerve tissue. As expected, ASOs with cEt chemistry were more efficacious than MOE ASO in all tissues examined.