Extrahepatic Tissues Research Articles

Arsenic Trioxide (ATOIII) Induces NAD(P)H Quinone Oxidoreductase 1 (NQO1) Expression in Hepatic and Extrahepatic Tissues of C57BL/6 Mice.

Arsenic trioxide (ATOIII) has emerged as a potent therapeutic agent for acute promyelocytic leukemia (APL), yet its clinical application is often limited by significant adverse effects. This study investigates the molecular mechanisms underlying ATOIII's impact on cellular detoxification pathways, focusing on the regulation of NAD(P)H/quinone oxidoreductase (NQO1), a crucial enzyme in maintaining cellular homeostasis and cancer prevention. We explored ATOIII's effects on NQO1 expression in C57BL/6 mice and Hepa-1c1c7 cells, both independently and in combination with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a known NQO1 inducer. Our findings revealed that ATOIII significantly increased NQO1 expression in hepatic and extrahepatic tissues, as well as in Hepa-1c1c7 cells, at mRNA, protein, and activity levels. This upregulation occurred both in the presence and absence of TCDD. Mechanistically, we demonstrated that ATOIII promotes the nuclear translocation of both nuclear factor erythroid 2-related factor-2 (NRF2) and aryl hydrocarbon receptor (AHR) transcription factors. Furthermore, ATOIII exposure increased antioxidant response element (ARE)-driven reporter gene activity, indicating a transcriptional mechanism of NQO1 induction. Notably, gene silencing experiments confirmed the critical roles of both NRF2 and AHR in mediating ATOIII-induced NQO1 expression. In conclusion, ATOIII exposure is found to upregulate the NQO1 enzyme through a transcriptional mechanism via AHR- and NRF2- dependent mechanisms, offering valuable insights into its therapeutic mechanisms.

Simplified Lipid Nanoparticles for Tissue- And Cell-Targeted mRNA Delivery Facilitate Precision Tumor Therapy in a Lung Metastasis Mouse Model.

mRNA-based applications have achieved remarkable success in the development of next-generation vaccines and the treatment of diverse liver diseases. Overcoming the challenge of delivering mRNA to extrahepatic tissues, especially specific cells within tissues, is crucial for precision therapy. In this study, a platform is developed for selective mRNA delivery to desired cells within tissues by combining lipid nanoparticle (LNP)-based targeted delivery with mRNA sequence-controlled expression. Through systematic optimization, a three-component LNP platform is developed, enabling targeted mRNA delivery to the lung, liver, and spleen. The incorporation of unique microRNA target sites into the mRNA scaffold further enhances control over protein translation in specific cells within the target tissue. This combined strategy, named SELECT (Simplified LNP with Engineered mRNA for Cell-type Targeting), demonstrates its efficacy in distinguishing mRNA expression between tumor and normal cells based on intracellular microRNA abundance. SELECT encapsulating mRNA encoding a tumor-specific cytotoxic protein, human ELANE, exhibits selective mRNA delivery to tumor lesions and significant inhibition of tumor growth in a mouse model of melanoma lung metastasis. Overall, SELECT has great potential as a new precision tumor treatment approach and also offers promising prospects for other mRNA therapies targeting specific cell types.

Impact of Lipid Tail Length on the Organ Selectivity of mRNA-Lipid Nanoparticles.

The delivery of mRNA molecules to organs beyond the liver is valuable for therapeutic applications. Functionalized lipid nanoparticles (LNPs) using exogenous mechanisms can regulate in vivo mRNA expression profiles from hepatocytes to extrahepatic tissues but lead to process complexity and cost escalation. Here, we report that mRNA expression gradually shifts from the liver to the spleen in an ionizable lipid tail length-dependent manner. Remarkably, this simple chemical strategy held true even when different ionizable lipid head structures were employed. As a potential mechanism underlying this discovery, our data suggest that 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) is enriched on the surface of mRNA/LNPs with short-tail lipids. This feature limits their interaction with biological components, avoiding their rapid hepatic clearance. We also show that spleen-targeting LNPs loaded with SARS-CoV-2 receptor-binding domain (RBD) mRNA can efficiently induce immune responses and neutralize activity following intramuscular vaccination priming and boosting.

Development of mRNA Lipid Nanoparticles: Targeting and Therapeutic Aspects.

Lipid nanoparticles (LNPs) have emerged as leading non-viral carriers for messenger RNA (mRNA) delivery in clinical applications. Overcoming challenges in safe and effective mRNA delivery to target tissues and cells, along with controlling release from the delivery vehicle, remains pivotal in mRNA-based therapies. This review elucidates the structure of LNPs, the mechanism for mRNA delivery, and the targeted delivery of LNPs to various cells and tissues, including leukocytes, T-cells, dendritic cells, Kupffer cells, hepatic endothelial cells, and hepatic and extrahepatic tissues. Here, we discuss the applications of mRNA-LNP vaccines for the prevention of infectious diseases and for the treatment of cancer and various genetic diseases. Although challenges remain in terms of delivery efficiency, specific tissue targeting, toxicity, and storage stability, mRNA-LNP technology holds extensive potential for the treatment of diseases.

Modulation of AAV9 Galactose Binding Yields Novel Gene Therapy Vectors and Predicts Cross-Species Differences in Glycan Avidity.

Effective use of adeno-associated viruses (AAVs) for clinical gene therapy is limited by their propensity to accumulate in and transduce the liver. This natural liver tropism is associated with severe adverse events at the high doses that can be necessary for achieving therapeutic transgene expression in extrahepatic tissues. To improve the safety and cost of AAV gene therapy, capsid engineering efforts are underway to redirect invivo AAV biodistribution away from the liver toward disease-relevant peripheral organs such as the heart. Building on previous work, we generated a series of AAV libraries containing variations at three residues (Y446, N470, and W503) of the galactose-binding pocket of the AAV9 VP1 protein. Screening of this library in mice identified the XRH family of variants (Y446X, N470R, and W503H), the strongest of which, HRH, exhibited a 6-fold reduction in liver RNA expression and a 10-fold increase in cardiac RNA expression compared with wild-type AAV9 in the mouse. Screening of our library in a nonhuman primate (NHP) revealed reduced performance of AAV9 and two closely related vectors in the NHP liver compared with the mouse liver. Measurement of the galactose-binding capacity of our library further identified those same three vectors as the only strong galactose binders, suggesting an altered galactose presentation between the mouse and NHP liver. N-glycan profiling of these tissues revealed a 9% decrease in exposed galactose in the NHP liver compared with the mouse liver. In this work, we identified a novel family of AAV variants with desirable biodistribution properties that may be suitable for targeting extrahepatic tissues such as the heart. These data also provide important insights regarding species- and tissue-specific differences in glycan presentation that may have implications for the development and translation of AAV gene therapies.

Protein is expressed in all major organs after intravenous infusion of mRNA-lipid nanoparticles in swine

Invivo delivery of mRNA is promising for the study of gene expression and the treatment of diseases. Lipid nanoparticles (LNPs) enable efficient delivery of mRNA constructs, but protein expression has been assumed to be limited to the liver. With specialized LNPs, delivery to extrahepatic tissue occurs in small animal models; however, it is unclear if global delivery of mRNA to all major organs is possible in humans because delivery may be affected by differences in innate immune response and relative organ size. Furthermore, limited studies with LNPs have been performed in large animal models, such as swine, due to their sensitivity to complement activation-related pseudoallergy (CARPA). In this study, we found that exogenous protein expression occurred in all major organs when swine were injected intravenously with a relatively low dose of mRNA encapsulated in a clinically relevant LNP formulation. Exogenous protein was detected in the liver, spleen, lung, heart, uterus, colon, stomach, kidney, small intestine, and brain of the swine without inducing CARPA. Furthermore, protein expression was detected in the bone marrow, including megakaryocytes, hematopoietic stem cells, and granulocytes, and in circulating white blood cells and platelets. These results show that nearly all major organs contain exogenous protein expression and are viable targets for mRNA therapies.

Abstract We129: Endothelium-Targeted MiR-122 Inhibition Improves Vascular Endothelial Function In a High-Fat Diet Fed mice

Background: MicroRNA-122-5p (miR-122), a liver-specific miR, levels are increased in the blood during obesity and diabetes and is taken-up by the extra-hepatic tissues including vascular endothelium. Despite miR-122’s role in maintaining hepatic homeostasis, its uptake (from circulation) by other tissues disrupts their function and increases the risk of cardiovascular disorders. Hence, inhibiting extra-hepatic miR-122 is a viable approach to mitigate vascular disorders associated with diabetes. Hypothesis: Endothelium-targeted miR-122 inhibitor selectively targets miR-122 in endothelial cells and improves vascular endothelial function in high-fat-diet (HFD) fed pre-diabetic mice. Methods: To selectively inhibit miR-122 in the endothelial cells, we synthesized gamma-peptide nucleic acid miR-122 inhibitor (γP-122 I) tagged with vascular cell adhesion molecule 1 (VCAM1) targeted peptide (e-γP-122 I). The purity was assessed using HPLC. Pre-diabetic condition was induced by feeding mice with a HFD (for 8 weeks) and e-γP-122-I was injected to the HFD-fed mice (2 weeks after dietary intervention; 5 mg/kg/day, i.p., 6 weeks). MiR-122 expression was measured by real-time PCR. The vascular endothelial function was assessed by determining endothelium-dependent and independent relaxation of phenylephrine-induced precontracted aortic rings. Results were expressed as mean ± s.e.m. Results: The e-γP-122-I was >95% pure. The HFD-fed mice show higher levels of serum and aortic miR-122 expression and impaired vascular endothelial function. e-γP-122-I treatment selectively inhibited aortic miR-122 expression while it has no effect on other organs (heart, kidney etc.) and improved vascular endothelial function and inflammation. Conclusions: e-γP-122-I selectively inhibits miR-122 in the endothelium and improves endothelial function in pre-diabetic mice.

Enhancing siRNA efficacy in vivo with extended nucleic acid backbones.

Therapeutic small interfering RNA (siRNA) requires sugar and backbone modifications to inhibit nuclease degradation. However, metabolic stabilization by phosphorothioate (PS), the only backbone chemistry used clinically, may be insufficient for targeting extrahepatic tissues. To improve oligonucleotide stabilization, we report the discovery, synthesis and characterization of extended nucleic acid (exNA) consisting of a methylene insertion between the 5'-C and 5'-OH of a nucleoside. exNA incorporation is compatible with common oligonucleotide synthetic protocols and the PS backbone, provides stabilization against 3' and 5' exonucleases and is tolerated at multiple oligonucleotide positions. A combined exNA-PS backbone enhances resistance to 3' exonuclease by ~32-fold over the conventional PS backbone and by >1,000-fold over the natural phosphodiester backbone, improving tissue exposure, tissue accumulation and efficacy in mice, both systemically and in the brain. The improved efficacy and durability imparted by exNA may enable therapeutic interventions in extrahepatic tissues, both with siRNA and with other oligonucleotides such as CRISPR guide RNA, antisense oligonucleotides, mRNA and tRNA.

Proprotein convertase subtilisin/kexin type 9 deficiency in extrahepatic tissues: emerging considerations.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is primarily secreted by hepatocytes. PCSK9 is critical in liver low-density lipoprotein receptors (LDLRs) metabolism. In addition to its hepatocellular presence, PCSK9 has also been detected in cardiac, cerebral, islet, renal, adipose, and other tissues. Once perceived primarily as a "harmful factor," PCSK9 has been a focal point for the targeted inhibition of both systemic circulation and localized tissues to treat diseases. However, PCSK9 also contributes to the maintenance of normal physiological functions in numerous extrahepatic tissues, encompassing both LDLR-dependent and -independent pathways. Consequently, PCSK9 deficiency may harm extrahepatic tissues in close association with several pathophysiological processes, such as lipid accumulation, mitochondrial impairment, insulin resistance, and abnormal neural differentiation. This review encapsulates the beneficial effects of PCSK9 on the physiological processes and potential disorders arising from PCSK9 deficiency in extrahepatic tissues. This review also provides a comprehensive analysis of the disparities between experimental and clinical research findings regarding the potential harm associated with PCSK9 deficiency. The aim is to improve the current understanding of the diverse effects of PCSK9 inhibition.

Selenoprotein P increases upon selenium and coenzyme Q10 supplementation and is associated with telomere length, quality of life and reduced inflammation and mortality

BackgroundSelenoprotein P (SELENOP) transports selenium to extrahepatic tissues and is a biomarker of selenium status. Low soil selenium leads to low dietary selenium intake. A consequence is an increased risk of cardiovascular disease. ObjectiveTo investigate clinical aspects associated with SELENOP deficiency, including biomarkers of inflammation, quality of life, and mortality within 12 years, and the effect of dietary selenium and coenzyme Q10 supplementation on SELENOP. MethodsSELENOP was determined at inclusion and after four years of supplementation in 403 elderly community-living participants low in selenium receiving selenium yeast (200 μg/day) and coenzyme Q10 (200 mg/day), or placebo. Pre-intervention, the average serum selenium level was 67 μg/L. T-tests, repeated measures of variance, Cox proportional regressions analyses, Kaplan-Meier graphs and ANCOVA analyses were applied. Associations with biomarkers of inflammation, telomere length, quality of life and mortality were investigated. Benchmark modelling was used to determine the serum selenium concentration at which the saturation levels of SELENOP and GPx3 was achieved. Comparison with GPx3 and serum selenium to identify increased mortality risk was performed, and the effect of supplementation on SELENOP levels were evaluated. ResultsInverse associations were observed between the level of SELENOP at inclusion and biomarkers for inflammation. At follow-up, shorter telomere lengths were seen in those with low levels of SELENOP at inclusion, whereas high levels of SELENOP were associated with better quality of life and decreased mortality. SELENOP had increased prognostic power compared to GPx3 and selenium. Saturation of SELENOP was achieved at a serum selenium level of 146 μg/L, and for GPx3 at 99 μg/L. Supplementation induced higher levels of SELENOP. ConclusionSignificant associations between SELENOP and inflammation, length of telomeres, quality of life, and mortality were observed. Thus, selenium supplementation improved SELENOP expression, thereby facilitating systemic selenium bioavailability and resulting in the observed positive health effects.

An Early Gestation Plasma Inflammasome in Rural Bangladeshi Women.

Circulating α1-acid glycoprotein (AGP) and C-reactive protein (CRP) are commonly measured to assess inflammation, but these biomarkers fail to reveal the complex molecular biology of inflammation. We mined the maternal plasma proteome to detect proteins that covary with AGP and CRP. In 435 gravida predominantly in <12-week gestation, we correlated the relative quantification of plasma proteins assessed via a multiplexed aptamer assay (SOMAScan®) with AGP and CRP, quantified by immunoassay. We defined a plasma inflammasome as protein correlates meeting a false discovery rate <0.05. We examined potential pathways using principal component analysis. A total of 147 and 879 of 6431 detected plasma proteins correlated with AGP and CRP, respectively, of which 61 overlapped with both biomarkers. Positive correlates included serum amyloid, complement, interferon-induced, and immunoregulatory proteins. Negative correlates were micronutrient and lipid transporters and pregnancy-related anabolic proteins. The principal components (PCs) of AGP were dominated by negatively correlated anabolic proteins associated with gestational homeostasis, angiogenesis, and neurogenesis. The PCs of CRP were more diverse in function, reflecting cell surface and adhesion, embryogenic, and intracellular and extra-hepatic tissue leakage proteins. The plasma proteome of AGP or CRP reveals wide proteomic variation associated with early gestational inflammation, suggesting mechanisms and pathways that merit future research.

Dietary Cholest-4-en-3-one, a Cholesterol Metabolite of Gut Microbiota, Alleviates Hyperlipidemia, Hepatic Cholesterol Accumulation, and Hyperinsulinemia in Obese, Diabetic db/db Mice.

Previous studies have shown that dietary cholest-4-en-3-one (4-cholestenone, 4-STN) exerts anti-obesity and lipid-lowering effects in mice. However, its underlying mechanisms are not fully understood. In the present study, we evaluated whether 4-STN supplementation would protect obese diabetic db/db mice from obesity-related metabolic disorders. After four weeks of feeding of a 0.25% 4-STN-containing diet, dietary 4-STN was found to have significantly alleviated hyperlipidemia, hepatic cholesterol accumulation, and hyperinsulinemia; however, the effect was not sufficient to improve hepatic triglyceride accumulation or obesity. Further analysis demonstrated that dietary 4-STN significantly increased the content of free fatty acids and neutral steroids in the feces of db/db mice, indicating that the alleviation of hyperlipidemia by 4-STN was due to an increase in lipid excretion. In addition, dietary 4-STN significantly reduced the levels of desmosterol, a cholesterol precursor, in the plasma but not in the liver, suggesting that normalization of cholesterol metabolism by 4-STN is partly attributable to the suppression of cholesterol synthesis in extrahepatic tissues. In addition, dietary 4-STN increased the plasma and hepatic levels of 4-STN metabolites cholestanol (5α-cholestan-3β-ol) and coprostanol (5β-cholestan-3β-ol). Our results show that dietary 4-STN alleviates obesity-related metabolic disorders, such as hyperlipidemia, hepatic cholesterol accumulation, and hyperinsulinemia, in db/db mice.

Liver-specific glucocorticoid action in alcoholic liver disease: Study of glucocorticoid receptor knockout and knockin mice

Background and aimGlucocorticoids are the only first-line drugs for severe alcoholic hepatitis (AH), with limited efficacy and various side effects on extrahepatic tissues. Liver-targeting glucocorticoid therapy may have multiple advantages over systemic glucocorticoid for AH. The aim of this study was to determine the role of hepatocellular glucocorticoid receptor (GR) in alcohol-associated steatosis (AS) and AH. Materials and methodsAS was induced by a high-fat diet plus binge alcohol in adult male and female mice with liver-specific knockout (LKO) and heterozygote of GR. AH was induced by chronic-plus-binge in middle-aged male mice with liver-specific knockin of GR. Changes in hepatic mRNA and protein expression were determined by quantitative real-time polymerase chain reaction and Western blot. ResultsGR-LKO aggravated steatosis and decreased hepatic expression and circulating levels of albumin in both genders of AS mice but only increased markers of liver injury in male AS mice. Marked steatosis in GR-LKO mice was associated with induction of lipogenic genes and down-regulation of bile acid synthetic genes. Hepatic protein levels of GR, hepatocyte nuclear factor 4 alpha, and phosphorylated signal transducer and activator of transcription 3 were gene-dosage-dependently decreased, whereas that of lipogenic ATP citrate lyase was increased in male GR heterozygote and LKO mice. Interestingly, hepatic expression of estrogen receptor alpha (ERα) was induced, and the essential estrogen-inactivating enzyme sulfotransferase 1e1 was gene-dosage-dependently down-regulated in GR heterozygote and knockout AS mice, which was associated with induction of ERα-target genes. Liver-specific knockin of GR protected against liver injury and steatohepatitis in middle-aged AH mice. ConclusionsHepatic GR deficiency plays a crucial role in the pathogenesis of AS induced by high-fat diet plus binge, and liver-specific overexpression/activation of GR protects against chronic-plus-binge-induced AH in middle-aged mice. Hepatocellular GR is important for protection against AS and AH.

Effects of intestine-specific deletion of FGF15 on the development of fatty liver disease with vertical sleeve gastrectomy.

Vertical sleeve gastrectomy (SGx) is a type of bariatric surgery to treat morbid obesity and metabolic dysfunction-associated steatotic liver disease (MASLD). The molecular mechanisms of SGx to improve MASLD are unclear, but increased bile acids (BAs) and FGF19 (mouse FGF15) were observed. FGF15/19 is expressed in the ileum in response to BAs and is critical in not only suppressing BA synthesis in the liver but also promoting energy expenditure. We hypothesized the reduction of obesity and resolution of MASLD by SGx may be mediated by FGF15/19. First, we conducted hepatic gene expression analysis in obese patients undergoing SGx, with the results showing increased expression of FGF19 in obese patients' livers. Next, we used wild-type and intestine-specific Fgf15 knockout mice (Fgf15ile-/-) to determine the effects of FGF15 deficiency on improving the metabolic effects. SGx improved metabolic endpoints in both genotypes, evidenced by decreased obesity, improved glucose tolerance, and reduced MASLD progression. However, Fgf15ile-/- mice showed better improvement compared to wild-type mice after SGx, suggesting that other mediators than FGF15 are also responsible for the beneficial effects of FGF15 deficiency. Further gene expression analysis in brown adipose tissue suggests increased thermogenesis. FGF15 deficiency, the larger BA pool and higher levels of secondary BAs may increase energy expenditure in extrahepatic tissues, which may be responsible for improved metabolic functions following SGx.

Phosphatidylcholine head group chemistry alters the extrahepatic accumulation of lipid-conjugated siRNA

Small interfering RNAs (siRNAs) are revolutionizing the treatment of liver-associated indications. Yet, robust delivery to extrahepatic tissues remains a challenge. Conjugating lipids (e.g., docosanoic acid, DCA) to siRNA supports extrahepatic delivery, but tissue accumulation remains lower than that achieved in liver by approved siRNA therapeutics. Early evidence suggests functionalizing DCA with a headgroup (e.g., phosphatidylcholine, PC) may enhance delivery to certain tissues. Here, we report the first systematic evaluation of the effect of PC headgroup chemistry on the extrahepatic distribution of DCA-conjugated siRNAs. We show that functionalizing DCA with a PC headgroup enhances siRNA accumulation in heart, muscle, lung, pancreas, duodenum, urinary bladder, and fat. Varying the size of the linker between the phosphate and choline moiety of the PC headgroup altered the extrahepatic accumulation of siRNA, with optimal linker length being different for different tissues. Increasing PC headgroup valency also improved extrahepatic accumulation in a tissue-specific manner. This study demonstrates the structural impact of the PC moiety on the biodistribution of lipid-conjugated siRNA and introduces multiple novel phosphocholine linker variants for chemical optimization of DCA-conjugated siRNA. These chemical variants can be used in the context of other lipids to increase the repertoire of conjugates for extrahepatic distribution of siRNAs.

Recent Progress of Small Interfering RNA Delivery on the Market and Clinical Stage.

Small interfering RNAs (siRNAs) are promising therapeutic strategies, and five siRNA drugs have been approved by the Food and Drug Administration (FDA) and the European Commission (EC). This marks a significant milestone in the development of siRNA for clinical applications. The approved siRNA agents can effectively deliver siRNAs to the liver and treat liver-related diseases. Currently, researchers have developed diverse delivery platforms for transporting siRNAs to different tissues such as the brain, lung, muscle, and others, and a large number of siRNA drugs are undergoing clinical trials. Here, these delivery technologies and the latest advancements in clinical applications are summarized, and this Review provides a concise overview of the strategies employed for siRNA delivery to both hepatic and extrahepatic tissues.

The ketone body β-Hydroxybutyrate as a fuel source of chondrosarcoma cells

Chondrosarcoma (CS) is a malignant bone tumor arising from cartilage-producing cells. The conventional subtype of CS typically develops within a dense cartilaginous matrix, creating an environment deficient in oxygen and nutrients, necessitating metabolic adaptation to ensure proliferation under stress conditions. Although ketone bodies (KBs) are oxidized by extrahepatic tissue cells such as the heart and brain, specific cancer cells, including CS cells, can undergo ketolysis. In this study, we found that KBs catabolism is activated in CS cells under nutrition-deprivation conditions. Interestingly, cytosolic β-hydroxybutyrate dehydrogenase 2 (BDH2), rather than mitochondrial BDH1, is expressed in these cells, indicating a specific metabolic adaptation for ketolysis in this bone tumor. The addition of the KB, β-Hydroxybutyrate (β-HB) in serum-starved CS cells re-induced the expression of BDH2, along with the key ketolytic enzyme 3-oxoacid CoA-transferase 1 (OXCT1) and monocarboxylate transporter-1 (MCT1). Additionally, internal β-HB production was quantified in supplied and starved cells, suggesting that CS cells are also capable of ketogenesis alongside ketolysis. These findings unveil a novel metabolic adaptation wherein nutrition-deprived CS cells utilize KBs for energy supply and proliferation.

Burden, Outcome, and Comorbidities of Extrahepatic Manifestations in Hepatitis B Virus Infections.

Hepatitis B virus (HBV) infections affect approximately 296 million people around the world, and the prevalence of any past or present HBV infection during the years 2015-2018 was as high as 4.3%. Acute HBV infection often presents with nonspecific symptoms and is usually self-limited, but 5% of patients can have persistent infections leading to chronic HBV infection and the risk of turning into chronic HBV infection is significantly higher in babies with vertical transmission (95%). Patients with chronic HBV infection are usually asymptomatic, but 15 to 40% of chronic HBV carriers develop cirrhosis and/or hepatocellular carcinoma. In addition to liver-related disorders, HBV is also associated with several extrahepatic complications, including glomerulonephritis, cryoglobulinemia, neurologic disorders, psychological manifestations, polyarthritis, and dermatologic disorders. Making the diagnosis of HBV can be challenging since patients with chronic infections can remain symptom-free for decades before developing cirrhosis or hepatocellular carcinoma, and patients with acute HBV infection may have only mild, nonspecific symptoms. Therefore, understanding how this virus causes extrahepatic complications can help clinicians consider this possibility in patients with diverse symptom presentations. The pathophysiology of these extrahepatic disorders likely involves immune-related tissue injury following immune complex formation and inflammatory cascades. In some cases, direct viral infection of extrahepatic tissue may cause a clinical syndrome. Currently, the American Association for the Study of Liver Diseases recommends treatment of chronic HBV infections with interferon therapy and/or nucleos(t)ide analogs, and this treatment has been reported to improve some extrahepatic disorders in some patients with chronic HBV infection. These extrahepatic complications have a significant role in disease outcomes and increase medical costs, morbidity, and mortality. Therefore, understanding the frequency and pathogenesis of these extrahepatic complications provides important information for both specialists and nonspecialists and may help clinicians identify patients at an earlier stage of their infection.

Abstract 2055: Delivery of oligonucleotide therapeutic drugs: Universal detection reagents specific for nucleic acid modifications

Abstract Nucleic acid therapies, including oligonucleotide therapeutics (ONTs) and vaccines, have increased in development, respectively, since their inception as an emerging drug modality and after the first mRNA COVID-19 vaccine was granted EUA approval in 2020. RNA-based therapies, such as antisense oligonucleotides (ASO), RNAi, aptamers, and mRNA, are designed to modulate protein synthesis in patients and have applications in oncology, gene therapy, and infectious disease prevention. Strategies for ONT drug delivery have advanced over recent years with the goal of overcoming limitations when targeting extrahepatic tissues. Improving the “drug-likeness” of ONTs is facilitated by chemical modifications to the sugar-phosphate backbone and/or nitrogenous bases. Modifications are designed to increase ONT drug stability, uptake, and efficacy, but may increase the likelihood of toxicity. First generation chemical modifications include phosphorothioate (PS) modification of the phosphate portion of the backbone, whereas second generation modifications include adding substituent groups to the 2’ position of the ribose portion of the backbone, specifically 2’-O-methyl (OME), 2’-O-methoxy-ethyl (MOE), and 2’-fluoro modifications of RNA. Third generation modifications include peptide nucleic acids (PNA), locked nucleic acids (LNA), morpholino phosphoroamidate (PMO) modifications, and others. We have developed, optimized, and validated a library of monoclonal antibody reagents that detect these chemical modifications independent of nucleic acid composition, structure, strandedness, configuration, or platform. These “universal detection reagents” can be used in (1) immunohistochemistry (IHC) to determine biodistribution, (2) immunofluorescence (IF) to determine intracellular localization, (3) ELISA to determine anti-drug antibody binding, immunogenicity, and drug ranking, (4) immunoprecipitation to determine protein binding in serum and/or tissue lysates, (5) in vivo cell culture studies to determine potency, and (6) other immunoassays. While in situ hybridization (ISH) is an established method for the detection and localization of ONTs in cells and tissues, this method has limitations that include lack of utility (as unique probes must be designed individually for each ONT), high costs in time and materials to produce probes, the inability of ISH to bind and detect short ONT drug sequences, and difficulties encountered when repeated sequences are present within the ONT sequence. Here we report differential sensitivity and specificity of panels of antibodies specific for PS and MOE modifications. We demonstrate the performance of these antibodies by various in vitro assays that show the utility of these specialty reagents to facilitate the collection of analytical data to support ADMET studies useful to ensure ONT approval by regulatory agencies. Citation Format: Todd Giardiello, Amy Anderson, Renee Salvo, Carl Ascoli. Delivery of oligonucleotide therapeutic drugs: Universal detection reagents specific for nucleic acid modifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2055.

Intrathecal administration of a novel siRNA modality extends survival and improves motor function in the SOD1G93A ALS mouse model

Antisense oligonucleotides (ASOs) were the first modality to pioneer targeted gene knockdown in the treatment of amyotrophic lateral sclerosis (ALS) caused by mutant superoxide dismutase 1 (SOD1). RNA interference (RNAi) is another mechanism of gene silencing in which short interfering RNAs (siRNAs) effectively degrade complementary transcripts. However, delivery to extrahepatic tissues like the CNS has been a bottleneck in the clinical development of RNAi. Herein, we identify potent siRNA duplexes for the knockdown of human SOD1 in which medicinal chemistry and conjugation to anaccessory oligonucleotide (ACO) enable activity in CNS tissues. Local delivery via intracerebroventricular or intrathecal injection into SOD1G93A mice delayed disease progression and extended animal survival with superior efficacy compared with an ASO resembling tofersen in sequence and chemistry. Treatment also prevented disease-related declines in motor function, including improvements in animal mobility, muscle strength, and coordination. The ACO itself does not target any specific complementary nucleic acid sequence; rather, it imparts benefits conducive to bioavailability and delivery through its chemistry. The complete conjugate (i.e., siRNA-ACO) represents a novel modality for delivery of duplex RNA (e.g., siRNA) to the CNS that is currently being tested in the clinic for treatment of ALS.