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INTRODUCTION: The apolipoprotein E gene (APOE) is an established central player in the pathogenesis of Alzheimers disease (AD), with distinct apoE isoforms exerting diverse effects. apoE influences not only amyloid-beta and tau pathologies but also lipid and energy metabolism, neuroinflammation, cerebral vascular health, and sex-dependent disease manifestations. Furthermore, ancestral background may significantly impact the link between APOE and AD, underscoring the need for more inclusive research. METHODS: In 2023, the Alzheimers Association convened multidisciplinary researchers at the AAIC Advancements: APOE conference to discuss various topics, including apoE isoforms and their roles in AD pathogenesis, progress in apoE-targeted therapeutic strategies, updates on disease models and interventions that modulate apoE expression and function. RESULTS: This manuscript presents highlights from the conference and provides an overview of opportunities for further research in the field. DISCUSSION: Understanding apoEs multifaceted roles in AD pathogenesis will help develop targeted interventions for AD and advance the field of AD precision medicine. HIGHLIGHTS: APOE is a central player in the pathogenesis of Alzheimers disease. APOE exerts a numerous effects throughout the brain on amyloid-beta, tau, and other pathways. The AAIC Advancements: APOE conference encouraged discussions and collaborations on understanding the role of APOE.

A hexanucleotide (G4C2) repeat expansion (HRE) within intron one of C9ORF72 is the leading genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). C9ORF72 haploinsufficiency, formation of RNA foci, and production of dipeptide repeat (DPR) proteins have been proposed as mechanisms of disease. Here, we report the first example of disease-modifying siRNAs for C9ORF72 driven ALS/FTD. Using a combination of reporter assay and primary cortical neurons derived from a C9-ALS/FTD mouse model, we screened a panel of more than 150 fully chemically stabilized siRNAs targeting different C9ORF72 transcriptional variants. We demonstrate the lack of correlation between siRNA efficacy in reporter assay versus native environment; repeat-containing C9ORF72 mRNA variants are found to preferentially localize to the nucleus, and thus C9ORF72 mRNA accessibility and intracellular localization have a dominant impact on functional RNAi. Using a C9-ALS/FTD mouse model, we demonstrate that divalent siRNAs targeting C9ORF72 mRNA variants specifically or non-selectively reduce the expression of C9ORF72 mRNA and significantly reduce DPR proteins. Interestingly, siRNA silencing all C9ORF72 mRNA transcripts was more effective in removing intranuclear mRNA aggregates than targeting only HRE-containing C9ORF72 mRNA transcripts. Combined, these data support RNAi-based degradation of C9ORF72 as a potential therapeutic paradigm.

Oligonucleotide therapeutics (ASOs and siRNAs) have been explored for modulation of gene expression in the central nervous system (CNS), with several drugs approved and many in clinical evaluation. Administration of highly concentrated oligonucleotides to the CNS can induce acute neurotoxicity. We demonstrate that delivery of concentrated oligonucleotides to the CSF in awake mice induces acute toxicity, observable within seconds of injection. Electroencephalography and electromyography in awake mice demonstrated seizures. Using ion chromatography, we show that siRNAs can tightly bind Ca2+ and Mg2+ up to molar equivalents of the phosphodiester/phosphorothioate bonds independently of the structure or phosphorothioate content. Optimization of the formulation by adding high concentrations (above biological levels) of divalent cations (Ca2+ alone, Mg2+ alone, or Ca2+ and Mg2+) prevents seizures with no impact on the distribution or efficacy of the oligonucleotide. The data here establish the importance of adding Ca2+ and Mg2+ to the formulation for the safety of CNS administration of therapeutic oligonucleotides.

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 that functionalizing DCA with a head group (e.g., phosphatidylcholine [PC]) may enhance delivery to certain tissues. Here, we report the first systematic evaluation of the effect of PC head group chemistry on the extrahepatic distribution of DCA-conjugated siRNAs. We show that functionalizing DCA with a PC head group 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 head group altered the extrahepatic accumulation of siRNA, with the optimal linker length being different for different tissues. Increasing PC head group 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 PC variants for the 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 the extrahepatic distribution of siRNAs.

Circulating lactate is a fuel source for liver metabolism but may exacerbate metabolic diseases such as nonalcoholic steatohepatitis (NASH). Indeed, haploinsufficiency of lactate transporter monocarboxylate transporter 1 (MCT1) in mice reportedly promotes resistance to hepatic steatosis and inflammation. Here, we used adeno-associated virus (AAV) vectors to deliver thyroxin binding globulin (TBG)-Cre or lecithin-retinol acyltransferase (Lrat)-Cre to MCT1fl/fl mice on a choline-deficient, high-fat NASH diet to deplete hepatocyte or stellate cell MCT1, respectively. Stellate cell MCT1KO (AAV-Lrat-Cre) attenuated liver type 1 collagen protein expression and caused a downward trend in trichrome staining. MCT1 depletion in cultured human LX2 stellate cells also diminished collagen 1 protein expression. Tetra-ethylenglycol-cholesterol (Chol)-conjugated siRNAs, which enter all hepatic cell types, and hepatocyte-selective tri-N-acetyl galactosamine (GN)-conjugated siRNAs were then used to evaluate MCT1 function in a genetically obese NASH mouse model. MCT1 silencing by Chol-siRNA decreased liver collagen 1 levels, while hepatocyte-selective MCT1 depletion by AAV-TBG-Cre or by GN-siRNA unexpectedly increased collagen 1 and total fibrosis without effect on triglyceride accumulation. These findings demonstrate that stellate cell lactate transporter MCT1 significantly contributes to liver fibrosis through increased collagen 1 protein expression in vitro and in vivo, while hepatocyte MCT1 appears not to be an attractive therapeutic target for NASH.

Identifying therapeutic oligonucleotides that are cross-reactive to experimental animal species can dramatically accelerate the process of preclinical development and clinical translation. Here, we identify fully chemically-modified small interfering RNAs (siRNAs) that are cross-reactive to Janus kinase 1 (JAK1) in humans and a large variety of other species. We validated the identified siRNAs in silencing JAK1 in cell lines and skin tissues of multiple species. JAK1 is one of the four members of the JAK family of tyrosine kinases that mediate the signaling transduction of many inflammatory cytokine pathways. Dysregulation of these pathways is often involved in the pathogenesis of various immune disorders, and modulation of JAK family enzymes is an effective strategy in the clinic. Thus, this work may open up unprecedented opportunities for evaluating the modulation of JAK1 in many animal models of human inflammatory skin diseases. Further chemical engineering of the optimized JAK1 siRNAs may expand the utility of these compounds for treating immune disorders in additional tissues.

Inherited retinal dystrophies caused by dominant mutations in photoreceptor (PR) cell expressed genes are a major cause of irreversible vision loss. Oligonucleotide therapy has been of interest in diseases that conventional medicine cannot target. In the early days, small interfering RNAs (siRNAs) were explored in clinical trials for retinal disorders with limited success due to a lack of stability and efficient cellular delivery. Thus, an unmet need exists to identify siRNA chemistry that targets PR cell expressed genes. Here, we evaluated 12 different fully chemically modified siRNA configurations, where the valency and conjugate structure were systematically altered. The impact on retinal distribution following intravitreal delivery was examined. We found that the increase in valency (tetravalent siRNA) supports the best PR accumulation. A single intravitreal administration induces multimonths efficacy in rodent and porcine retinas while demonstrating a good safety profile. The data suggest that this configuration can treat retinal diseases caused by PR cell expressed genes with 1–2 intravitreal injections per year.

Abstract Inhibition of Janus kinase (JAK) family enzymes is a popular strategy for treating inflammatory and autoimmune skin diseases. In the clinic, small molecule JAK inhibitors show distinct efficacy and safety profiles, likely reflecting variable selectivity for JAK subtypes. Absolute JAK subtype selectivity has not yet been achieved. Here, we rationally design small interfering RNAs (siRNAs) that offer sequence-specific gene silencing of JAK1, narrowing the spectrum of action on JAK-dependent cytokine signaling to maintain efficacy and improve safety. Our fully chemically modified siRNA supports efficient silencing of JAK1 expression in human skin explant and modulation of JAK1-dependent inflammatory signaling. A single injection into mouse skin enables five weeks of duration of effect. In a mouse model of vitiligo, local administration of the JAK1 siRNA significantly reduces skin infiltration of autoreactive CD8+ T cells and prevents epidermal depigmentation. This work establishes a path toward siRNA treatments as a new class of therapeutic modality for inflammatory and autoimmune skin diseases.

Chemically modified small interfering RNAs (siRNAs) are promising therapeutics guiding sequence-specific silencing of disease genes. Identifying chemically modified siRNA sequences that effectively silence target genes remains challenging. Such determinations necessitate computational algorithms. Machine learning is a powerful predictive approach for tackling biological problems but typically requires datasets significantly larger than most available siRNA datasets. Here, we describe a framework applying machine learning to a small dataset (356 modified sequences) for siRNA efficacy prediction. To overcome noise and biological limitations in siRNA datasets, we apply a trichotomous, two-threshold, partitioning approach, producing several combinations of classification threshold pairs. We then test the effects of different thresholds on random forest machine learning model performance using a novel evaluation metric accounting for class imbalances. We identify thresholds yielding a model with high predictive power, outperforming a linear model generated from the same data, that was predictive upon experimental evaluation. Using a novel model feature extraction method, we observe target site base importances and base preferences consistent with our current understanding of the siRNA-mediated silencing mechanism, with the random forest providing higher resolution than the linear model. This framework applies to any classification challenge involving small biological datasets, providing an opportunity to develop high-performing design algorithms for oligonucleotide therapies.

Atom-economical, eco-efficient, metal- and chemical oxidant-free formation of C–C and C–N bond from C(sp2)−H and C(sp3)−H of arenes toward the direct synthesis of biaryls and anilides or N-benzylamides under mild electro-oxidative conditions is described. The products of C–C and C–N coupling are obtained in up to 88% yields. Aromatic substrates that are oxidized at potentials less positive than +2 V or have bulky bromine or iodine substituents undergo homo-coupling reactions by anodic oxidation to form biaryls or dimers. Aromatic substrates that are difficult to oxidize (Eox > +2 V) preferentially form anilides and N-benzylamides upon anodic oxidation. The presence of a chlorine substituent on the aromatic ring leads to the formation of both biaryls and anilides during electro-oxidation.

Although an increasing number of small interfering RNA (siRNA) therapies are reaching the market, the challenge of efficient extra-hepatic delivery continues to limit their full therapeutic potential. Drug delivery vehicles and hydrophobic conjugates are being used to overcome the delivery bottleneck. Previously, we reported a novel dendritic conjugate that can be appended efficiently to oligonucleotides, allowing them to bind albumin with nanomolar affinity. Here, we explore the ability of this novel albumin-binding conjugate to improve the delivery of siRNA in vivo. We demonstrate that the conjugate binds albumin exclusively in circulation and extravasates to various organs, enabling effective gene silencing. Notably, we show that the conjugate achieves a balance between hydrophobicity and safety, as it significantly reduces the side effects associated with siRNA interactions with blood components, which are commonly observed in some hydrophobically conjugated siRNAs. In addition, it reduces siRNA monocyte uptake, which may lead to cytokine/inflammatory responses. This work showcases the potential of using this dendritic conjugate as a selective albumin binding handle for the effective and safe delivery of nucleic acid therapeutics. We envision that these properties may pave the way for new opportunities to overcome delivery hurdles of oligonucleotides in future applications.

Oligonucleotide therapeutics offer great promise in the treatment of previously untreatable neurodegenerative disorders; however, there are some challenges to overcome in pre-clinical studies. (1) They carry a well-established dose-related acute neurotoxicity at the time of administration. (2) Repeated administration into the cerebrospinal fluid may be required for long-term therapeutic effect. Modifying oligonucleotide formulation has been postulated to prevent acute toxicity, but a sensitive and quantitative way to track seizure activity in pre-clinical studies is lacking. The use of intracerebroventricular (i.c.v.) catheters offers a solution for repeated dosing; however, fixation techniques in large animal models are not standardized and are not reliable. Here we describe a novel surgical technique in a sheep model for i.c.v. delivery of neurotherapeutics based on the fixation of the i.c.v. catheter with a 3D-printed anchorage system composed of plastic and ceramic parts, compatible with magnetic resonance imaging, computed tomography, and electroencephalography (EEG). Our technique allowed tracking electrical brain activity in awake animals via EEG and video recording during and for the 24-h period after administration of a novel oligonucleotide in sheep. Its anchoring efficiency was demonstrated for at least 2 months and will be tested for up to a year in ongoing studies.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Chemically modified siRNAs distinguish between mutant and normal huntingtin based on a single nucleotide difference and lower mutant huntingtin specifically in patient derived cells and in a mouse model of Huntington’s disease.

Effective systemic delivery of small interfering RNAs (siRNAs) to tissues other than liver remains a challenge. siRNAs are small (∼15 kDa) and therefore rapidly cleared by the kidneys, resulting in limited blood residence times and tissue exposure. Current strategies to improve the unfavorable pharmacokinetic (PK) properties of siRNAs rely on enhancing binding to serum proteins through extensive phosphorothioate modifications or by conjugation of targeting ligands. Here, we describe an alternative strategy for enhancing blood and tissue PK based on dynamic modulation of the overall size of the siRNA. We engineered a high-affinity universal oligonucleotide anchor conjugated to a high-molecular-weight moiety, which binds to the 3′ end of the guide strand of an asymmetric siRNA. Data showed a strong correlation between the size of the PK-modifying anchor and clearance kinetics. Large 40-kDa PK-modifying anchors reduced renal clearance by ∼23-fold and improved tissue exposure area under the curve (AUC) by ∼26-fold, resulting in increased extrahepatic tissue retention (∼3- to 5-fold). Furthermore, PK-modifying oligonucleotide anchors allowed for straightforward and versatile modulation of blood residence times and biodistribution of a panel of chemically distinct ligands. The effects were more pronounced for conjugates with low lipophilicity (e.g., N-Acetylgalactosamine [GalNAc]), where significant improvement in uptake by hepatocytes and dose-dependent silencing in the liver was observed.

Preeclampsia (PE) is a rising, potentially lethal complication of pregnancy. PE is driven primarily by the overexpression of placental soluble fms-like tyrosine kinase 1 (sFLT1), a validated diagnostic and prognostic marker of the disease when normalized to placental growth factor (PlGF) levels. Injecting cholesterol-conjugated, fully modified, small interfering RNAs (siRNAs) targeting sFLT1 mRNA into pregnant mice or baboons reduces placental sFLT1 and ameliorates clinical signs of PE, providing a strong foundation for the development of a PE therapeutic. siRNA delivery, potency, and safety are dictated by conjugate chemistry, siRNA duplex structure, and chemical modification pattern. Here, we systematically evaluate these parameters and demonstrate that increasing 2′-O-methyl modifications and 5′ chemical stabilization and using sequence-specific duplex asymmetry and a phosphocholine-docosanoic acid conjugate enhance placental accumulation, silencing efficiency and safety of sFLT1-targeting siRNAs. The optimization strategy here provides a framework for the chemical optimization of siRNAs for PE as well as other targets and clinical indications.

The Extracellular RNA Communication Consortium (ERCC) was launched to accelerate progress in the new field of extracellular RNA (exRNA) biology and to establish whether exRNAs and their carriers, including extracellular vesicles (EVs), can mediate intercellular communication and be utilized for clinical applications. Phase 1 of the ERCC focused on exRNA/EV biogenesis and function, discovery of exRNA biomarkers, development of exRNA/EV-based therapeutics, and construction of a robust set of reference exRNA profiles for a variety of biofluids. Here, we present progress by ERCC investigators in these areas, and we discuss collaborative projects directed at development of robust methods for EV/exRNA isolation and analysis and tools for sharing and computational analysis of exRNA profiling data.

Oligonucleotídeos sintéticos, como os small interfering RNAs (siRNAs), providenciam uma forma simples e eficiente de modular a expressão de qualquer gene. siRNAs utilizam um mecanismo endógeno, chamado ARN interferência, para degradar ARN mensageiros que estejam associados a condições patológicas. A capacidade de silenciar genes com elevada especificidade e potência faz dos siRNAs fármacos ideais com um elevado potencial para transformar o ramo da medicina e a forma como se faz desenvolvimento farmacêutico. Contudo, os primeiros ensaios clínicos com esta tecnologia não tiveram sucesso imediato, o que reduziu temporariamente o entusiasmo da comunidade científica. A maioria destes estudos iniciais não atingiram a eficácia clínica desejada. A introdução prematura de fármacos que não se encontravam devidamente estabilizados e a utilização de estratégias de administração inadequadas foram as principais causas dos primeiros fracassos. Avanços recentes na síntese química de oligonucleotídeos, como a melhor compreensão dos processos biológicos que definem a farmacocinética/farmacodinâmica destes fármacos, resultou numa mudança drástica no panorama clínico desta nova modalidade terapêutica. O número de ensaios clínicos tem aumentado significativamente ao longo dos últimos anos, em paralelo com o aumento da eficácia terapêutica destes medicamentos. Em 2018 foi testemunhado um marco importante para o ramo de desenvolvimento destes fármacos, com a aprovação do primeiro produto baseado em liposomas, Patisiran (OnpattroTM), pela Food and Drug Administration e pela European Medicines Agency. Aproximadamente um ano mais tarde foi aprovado o primeiro fármaco que utiliza a estratégia de conjugados que possibilita a internalização específica em hepatócitos, Givosiran (GIVLAARITM). A recente aprovação destes dois fármacos trouxe uma esperança renovada ao ramo de ARN interferência, alimentando o interesse nesta estratégia como poderosa ferramenta terapêutica para doenças do foro genético. Este artigo de revisão pretende providenciar uma perspetiva geral sobre o panorama clínico dos fármacos para silenciamento génico, contextualizando o papel dos avanços tecnológicos que permitiram a definição desta modalidade como uma nova classe farmacêutica.