RNAi-mediated silencing of SOD1 profoundly extends survival and functional outcomes in ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative condition, with 20% of familial and 2-3% of sporadic cases linked to mutations in the cytosolic superoxide dismutase (SOD1) gene. Mutant SOD1 protein is toxic to motor neurons, making SOD1 gene lowering a promising approach, supported by preclinical data and the 2023 FDA approval of the GapmeR ASO targeting SOD1, tofersen. Despite the approval of an ASO and the optimism it brings to the field, the pharmacodynamics and pharmacokinetics of therapeutic SOD1 modulation can be improved. Here, we developed a chemically stabilized divalent siRNA scaffold (di-siRNA) that effectively suppresses SOD1 expression in vitro and in vivo. With optimized chemical modification, it achieves remarkable CNS tissue permeation and SOD1 silencing in vivo. Administered intraventricularly, di-siRNASOD1 extended survival in SOD1-G93A ALS mice, surpassing survival previously seen in these mice by ASO modalities, slowed disease progression, and prevented ALS neuropathology. These properties offer an improved therapeutic strategy for SOD1-mediated ALS and may extend to other dominantly inherited neurological disorders.

A combinatorial approach for achieving CNS-selective RNAi.

RNA interference (RNAi) is an endogenous process that can be harnessed using chemically modified small interfering RNAs (siRNAs) to potently modulate gene expression in many tissues. The route of administration and chemical architecture are the primary drivers of oligonucleotide tissue distribution, including siRNAs. Independently of the nature and type, oligonucleotides are eliminated from the body through clearance tissues, where their unintended accumulation may result in undesired gene modulation. Divalent siRNAs (di-siRNAs) administered into the CSF induce robust gene silencing throughout the central nervous system (CNS). Upon clearance from the CSF, they are mainly filtered by the kidneys and liver, with the most functionally significant accumulation occurring in the liver. siRNA- and miRNA-induced silencing can be blocked through substrate inhibition using single-stranded, stabilized oligonucleotides called antagomirs or anti-siRNAs. Using APOE as a model target, we show that undesired di-siRNA-induced silencing in the liver can be mitigated through administration of liver targeting GalNAc-conjugated anti-siRNAs, without impacting CNS activity. Blocking unwanted hepatic APOE silencing achieves fully CNS-selective silencing, essential for potential clinical translation. While we focus on CNS/liver selectivity, coadministration of differentially targeting siRNA and anti-siRNAs can be adapted as a strategy to achieve tissue selectivity in different organ combinations.

Silencing Apoe with divalent-siRNAs improves amyloid burden and activates immune response pathways in Alzheimer's disease.

INTRODUCTION: The most significant genetic risk factor for late-onset Alzheimer's disease (AD) is APOE4, with evidence for gain- and loss-of-function mechanisms. A clinical need remains for therapeutically relevant tools that potently modulate APOE expression. METHODS: We optimized small interfering RNAs (di-siRNA, GalNAc) to potently silence brain or liver Apoe and evaluated the impact of each pool of Apoe on pathology. RESULTS: In adult 5xFAD mice, siRNAs targeting CNS Apoe efficiently silenced Apoe expression and reduced amyloid burden without affecting systemic cholesterol, confirming that potent silencing of brain Apoe is sufficient to slow disease progression. Mechanistically, silencing Apoe reduced APOE-rich amyloid cores and activated immune system responses. DISCUSSION: These results establish siRNA-based modulation of Apoe as a viable therapeutic approach, highlight immune activation as a key pathway affected by Apoe modulation, and provide the technology to further evaluate the impact of APOE silencing on neurodegeneration.

Dose-dependent reduction of somatic expansions but not Htt aggregates by di-valent siRNA-mediated silencing of MSH3 in HdhQ111 mice.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by CAG trinucleotide repeat expansions in exon 1 of the HTT gene. In addition to germline CAG expansions, somatic repeat expansions in neurons also contribute to HD pathogenesis. The DNA mismatch repair gene, MSH3, identified as a genetic modifier of HD onset and progression, promotes somatic CAG expansions, and thus presents a potential therapeutic target. However, what extent of MSH3 protein reduction is needed to attenuate somatic CAG expansions and elicit therapeutic benefits in HD disease models is less clear. In our study, we employed potent di-siRNAs to silence mouse Msh3 mRNA expression in a dose-dependent manner in HdhQ111/+ mice and correlated somatic Htt CAG instability with MSH3 protein levels from simultaneously isolated DNA and protein after siRNA treatment. Our results reveal a linear correlation with a proportionality constant of ~ 1 between the prevention of somatic Htt CAG expansions and MSH3 protein expression in vivo, supporting MSH3 as a rate-limiting step in somatic expansions. Intriguingly, despite a 75% reduction in MSH3 protein levels, striatal nuclear HTT aggregates remained unchanged. We also note that evidence for nuclear Msh3 mRNA that is inaccessible to RNA interference was found, and that MSH6 protein in the striatum was upregulated following MSH3 knockdown in HdhQ111/+ mice. These results provide important clues to address critical questions for the development of therapeutic molecules targeting MSH3 as a potential therapeutic target for HD.

Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo.

Metabolic stabilization of therapeutic oligonucleotides requires both sugar and backbone modifications, where phosphorothioate (PS) is the only backbone chemistry used in the clinic. Here, we describe the discovery, synthesis, and characterization of a novel biologically compatible backbone, extended nucleic acid (exNA). Upon exNA precursor scale up, exNA incorporation is fully compatible with common nucleic acid synthetic protocols. The novel backbone is orthogonal to PS and shows profound stabilization against 3'- and 5'-exonucleases. Using small interfering RNAs (siRNAs) as an example, we show exNA is tolerated at most nucleotide positions and profoundly improves in vivo efficacy. A combined exNA-PS backbone enhances siRNA resistance to serum 3'-exonuclease by ~ 32-fold over PS backbone and > 1000-fold over the natural phosphodiester backbone, thereby enhancing tissue exposure (~ 6-fold), tissues accumulation (4- to 20-fold), and potency both systemically and in brain. The improved potency and durability imparted by exNA opens more tissues and indications to oligonucleotide-driven therapeutic interventions.

Extended Nucleic Acid (exNA): A Novel, Biologically Compatible Backbone that Significantly Enhances Oligonucleotide Efficacy in vivo.

Metabolic stabilization of therapeutic oligonucleotides requires both sugar and backbone modifications, where phosphorothioate (PS) is the only backbone chemistry used in the clinic. Here, we describe the discovery, synthesis, and characterization of a novel biologically compatible backbone, extended nucleic acid (exNA). Upon exNA precursor scale up, exNA incorporation is fully compatible with common nucleic acid synthetic protocols. The novel backbone is orthogonal to PS and shows profound stabilization against 3'- and 5'-exonucleases. Using small interfering RNAs (siRNAs) as an example, we show exNA is tolerated at most nucleotide positions and profoundly improves in vivo efficacy. A combined exNA-PS backbone enhances siRNA resistance to serum 3'-exonuclease by ~32-fold over PS backbone and >1000-fold over the natural phosphodiester backbone, thereby enhancing tissue exposure (~6-fold), tissues accumulation (4- to 20-fold), and potency both systemically and in brain. The improved potency and durability imparted by exNA opens more tissues and indications to oligonucleotide-driven therapeutic interventions.

Synthesis of Prepolymers of Poly(glycerol-co-diacids) Based on Sebacic and Succinic Acid Mixtures.

In this study, poly(glycerol-co-diacids) prepolymers were produced using different ratios of glycerol (G), sebacic acid (S), and succinic acid (Su) (molar ratios: GS 1:1, GSSu 1:0.9:0.1, GSSu 1:0.8:0.2, GSSu 1:0.5:0.5, GSSu 1:0.2:0.8, GSSu 1:0.1:0.9, GSu 1:1). All polycondensation reactions were performed at 150 °C until reaching a degree of polymerization of ≈55%, inferred by the water volume collected from a reactor. We concluded that the reaction time is correlated with the ratio of diacids used, that is, the increase in succinic acid is proportional to a decrease in the duration of the reaction. In fact, the reaction of poly(glycerol sebacate) (PGS 1:1) is twice as slow as the reaction of poly(glycerol succinate) (PGSu 1:1). The obtained prepolymers were analyzed by electrospray ionization mass spectrometry (ESI-MS) and 1H and 13C nuclear magnetic resonance (NMR). Besides its catalytic influence in poly(glycerol)/ether bond formation, the presence of succinic acid also contributes to a mass growth of ester oligomers, the formation of cyclic structures, a greater number of oligomers detected, and a difference in mass distribution. When compared with PGS (1:1), and even at lower ratios, the prepolymers produced with succinic acid presented mass peak characteristics of oligomer species with a glycerol unit as its end group in higher abundance. Generally, the most abundant oligomers have molecular weights between 400 and 800 g/mol.

Divalent siRNAs are bioavailable in the lung and efficiently block SARS-CoV-2 infection.

The continuous evolution of SARS-CoV-2 variants complicates efforts to combat the ongoing pandemic, underscoring the need for a dynamic platform for the rapid development of pan-viral variant therapeutics. Oligonucleotide therapeutics are enhancing the treatment of numerous diseases with unprecedented potency, duration of effect, and safety. Through the systematic screening of hundreds of oligonucleotide sequences, we identified fully chemically stabilized siRNAs and ASOs that target regions of the SARS-CoV-2 genome conserved in all variants of concern, including delta and omicron. We successively evaluated candidates in cellular reporter assays, followed by viral inhibition in cell culture, with eventual testing of leads for in vivo antiviral activity in the lung. Previous attempts to deliver therapeutic oligonucleotides to the lung have met with only modest success. Here, we report the development of a platform for identifying and generating potent, chemically modified multimeric siRNAs bioavailable in the lung after local intranasal and intratracheal delivery. The optimized divalent siRNAs showed robust antiviral activity in human cells and mouse models of SARS-CoV-2 infection and represent a new paradigm for antiviral therapeutic development for current and future pandemics.

Different methods of synthesizing poly(glycerol sebacate) (PGS): A review.

Poly(glycerol sebacate) (PGS) is a biodegradable elastomer that has attracted increasing attention as a potential material for applications in biological tissue engineering. The conventional method of synthesis, first described in 2002, is based on the polycondensation of glycerol and sebacic acid, but it is a time-consuming and energy-intensive process. In recent years, new approaches for producing PGS, PGS blends, and PGS copolymers have been reported to not only reduce the time and energy required to obtain the final material but also to adjust the properties and processability of the PGS-based materials based on the desired applications. This review compiles more than 20 years of PGS synthesis reports, reported inconsistencies, and proposed alternatives to more rapidly produce PGS polymer structures or PGS derivatives with tailor-made properties. Synthesis conditions such as temperature, reaction time, reagent ratio, atmosphere, catalysts, microwave-assisted synthesis, and PGS modifications (urethane and acrylate groups, blends, and copolymers) were revisited to present and discuss the diverse alternatives to produce and adapt PGS.

Chemical engineering of therapeutic siRNAs for allele-specific gene silencing in Huntington's disease models.

Small interfering RNAs are a new class of drugs, exhibiting sequence-driven, potent, and sustained silencing of gene expression in vivo. We recently demonstrated that siRNA chemical architectures can be optimized to provide efficient delivery to the CNS, enabling development of CNS-targeted therapeutics. Many genetically-defined neurodegenerative disorders are dominant, favoring selective silencing of the mutant allele. In some cases, successfully targeting the mutant allele requires targeting single nucleotide polymorphism (SNP) heterozygosities. Here, we use Huntington's disease (HD) as a model. The optimized compound exhibits selective silencing of mutant huntingtin protein in patient-derived cells and throughout the HD mouse brain, demonstrating SNP-based allele-specific RNAi silencing of gene expression in vivo in the CNS. Targeting a disease-causing allele using RNAi-based therapies could be helpful in a range of dominant CNS disorders where maintaining wild-type expression is essential.

PK-modifying anchors significantly alter clearance kinetics, tissue distribution, and efficacy of therapeutics siRNAs.

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.

Intrathecal Delivery of Therapeutic Oligonucleotides for Potent Modulation of Gene Expression in the Central Nervous System.

Therapeutic oligonucleotides hold tremendous potential for treating central nervous system (CNS) disorders. The route of administration of oligonucleotides significantly impacts both distribution and silencing efficiency. Here, we describe a technically simple, clinically relevant method to administer oligonucleotide compounds into the CNS via direct intrathecal injections. This method achieves distribution throughout the CNS rapidly and permits high-throughput testing of oligonucleotide efficacy and potency in mammals.

Imaging Net Retrograde Axonal Transport In Vivo: A Physiological Biomarker.

OBJECTIVE: The objective of this study is to develop a novel method for monitoring the integrity of motor neurons in vivo by quantifying net retrograde axonal transport. METHODS: The method uses single photon emission computed tomography to quantify retrograde transport to spinal cord of tetanus toxin fragment C (125 I-TTC) following intramuscular injection. We characterized the transport profiles in 3 transgenic mouse models carrying amyotrophic lateral sclerosis (ALS)-associated genes, aging mice, and SOD1G93A transgenic mice following CRISPR/Cas9 gene editing. Lastly, we studied the effect of prior immunization of tetanus toxoid on the transport profile of TTC. RESULTS: This technique defines a quantitative profile of net retrograde axonal transport of TTC in living mice. The profile is distinctly abnormal in transgenic SOD1G93A mice as young as 65 days (presymptomatic) and worsens with disease progression. Moreover, this method detects a distinct therapeutic benefit of gene editing in transgenic SOD1G93A mice well before other clinical parameters (eg, grip strength) show improvement. Symptomatic transgenic PFN1C71G/C71G ALS mice display gross reductions in net retrograde axonal transport, which is also disturbed in asymptomatic mice harboring a human C9ORF72 transgene with an expanded GGGGCC repeat motif. In wild-type mice, net retrograde axonal transport declines with aging. Lastly, prior immunization with tetanus toxoid does not preclude use of this assay. INTERPRETATION: This assay of net retrograde axonal transport has broad potential clinical applications and should be particularly valuable as a physiological biomarker that permits early detection of benefit from potential therapies for motor neuron diseases. ANN NEUROL 2022;91:716-729.

Comparative route of administration studies using therapeutic siRNAs show widespread gene modulation in Dorset sheep.

siRNAs comprise a class of drugs that can be programmed to silence any target gene. Chemical engineering efforts resulted in development of divalent siRNAs (di-siRNAs), which support robust and long-term efficacy in rodent and nonhuman primate brains upon direct cerebrospinal fluid (CSF) administration. Oligonucleotide distribution in the CNS is nonuniform, limiting clinical applications. The contribution of CSF infusion placement and dosing regimen on relative accumulation, specifically in the context of large animals, is not well characterized. To our knowledge, we report the first systemic, comparative study investigating the effects of 3 routes of administration - intrastriatal (i.s.), i.c.v., and intrathecal catheter to the cisterna magna (ITC) - and 2 dosing regimens - single and repetitive via an implanted reservoir device - on di-siRNA distribution and accumulation in the CNS of Dorset sheep. CSF injections (i.c.v. and ITC) resulted in similar distribution and accumulation across brain regions. Repeated dosing increased homogeneity, with greater relative deep brain accumulation. Conversely, i.s. administration supported region-specific delivery. These results suggest that dosing regimen, not CSF infusion placement, may equalize siRNA accumulation and efficacy throughout the brain. These findings inform the planning and execution of preclinical and clinical studies using siRNA therapeutics in the CNS.

Towards a Trait-Based Approach to Potentiate Yield under Drought in Legume-Rich Annual Forage Mixtures.

Mediterranean annual forage mixtures are facing the impact of climate change, especially higher frequencies of winter-time drought. Increased mixture plasticity to climate variability is needed to mitigate this impact. However, little information exists regarding the specificities and complementarities of each forage species component to potentiate mixture resilience under drought. In this study, we identified traits with breeding potential under water scarcity through a detailed characterization of leaf and root-related parameters of 10 legume and grass species components of Mediterranean annual forage mixtures, complemented by their photosynthetic response evaluation under well-watered and water deficit conditions. This integrated approach also allowed us to identify the most resilient species to water deficit. In particular, we found that the highest canopy height and root to shoot ratio of grass components complemented well the highest aerial and root biomass and superior photosynthetic performance of the legume components. Trifolium squarrosum and Triticosecale showed the most adequate combination of traits and the best photosynthetic performance under water deficit within each species family. Although some of these traits are not commonly used in annual forage selection, they may in part explain the potential higher resilience of the grass-legume mixture under water deficit and should be considered in forage breeding.

A divalent siRNA chemical scaffold for potent and sustained modulation of gene expression throughout the central nervous system.

Sustained silencing of gene expression throughout the brain using small interfering RNAs (siRNAs) has not been achieved. Here we describe an siRNA architecture, divalent siRNA (di-siRNA), that supports potent, sustained gene silencing in the central nervous system (CNS) of mice and nonhuman primates following a single injection into the cerebrospinal fluid. Di-siRNAs are composed of two fully chemically modified, phosphorothioate-containing siRNAs connected by a linker. In mice, di-siRNAs induced the potent silencing of huntingtin, the causative gene in Huntington's disease, reducing messenger RNA and protein throughout the brain. Silencing persisted for at least 6 months, with the degree of gene silencing correlating to levels of guide strand tissue accumulation. In cynomolgus macaques, a bolus injection of di-siRNA showed substantial distribution and robust silencing throughout the brain and spinal cord without detectable toxicity and with minimal off-target effects. This siRNA design may enable RNA interference-based gene silencing in the CNS for the treatment of neurological disorders.

Serum Deprivation of Mesenchymal Stem Cells Improves Exosome Activity and Alters Lipid and Protein Composition.

Exosomes can serve as delivery vehicles for advanced therapeutics. The components necessary and sufficient to support exosomal delivery have not been established. Here we connect biochemical composition and activity of exosomes to optimize exosome-mediated delivery of small interfering RNAs (siRNAs). This information is used to create effective artificial exosomes. We show that serum-deprived mesenchymal stem cells produce exosomes up to 22-fold more effective at delivering siRNAs to neurons than exosomes derived from control cells. Proteinase treatment of exosomes stops siRNA transfer, indicating that surface proteins on exosomes are involved in trafficking. Proteomic and lipidomic analyses show that exosomes derived in serum-deprived conditions are enriched in six protein pathways and one lipid class, dilysocardiolipin. Inspired by these findings, we engineer an "artificial exosome," in which the incorporation of one lipid (dilysocardiolipin) and three proteins (Rab7, Desmoplakin, and AHSG) into conventional neutral liposomes produces vesicles that mimic cargo delivering activity of natural exosomes.

Hydrophobicity drives the systemic distribution of lipid-conjugated siRNAs via lipid transport pathways.

Efficient delivery of therapeutic RNA beyond the liver is the fundamental obstacle preventing its clinical utility. Lipid conjugation increases plasma half-life and enhances tissue accumulation and cellular uptake of small interfering RNAs (siRNAs). However, the mechanism relating lipid hydrophobicity, structure, and siRNA pharmacokinetics is unclear. Here, using a diverse panel of biologically occurring lipids, we show that lipid conjugation directly modulates siRNA hydrophobicity. When administered in vivo, highly hydrophobic lipid-siRNAs preferentially and spontaneously associate with circulating low-density lipoprotein (LDL), while less lipophilic lipid-siRNAs bind to high-density lipoprotein (HDL). Lipid-siRNAs are targeted to lipoprotein receptor-enriched tissues, eliciting significant mRNA silencing in liver (65%), adrenal gland (37%), ovary (35%), and kidney (78%). Interestingly, siRNA internalization may not be completely driven by lipoprotein endocytosis, but the extent of siRNA phosphorothioate modifications may also be a factor. Although biomimetic lipoprotein nanoparticles have been explored for the enhancement of siRNA delivery, our findings suggest that hydrophobic modifications can be leveraged to incorporate therapeutic siRNA into endogenous lipid transport pathways without the requirement for synthetic formulation.

RNAi modulation of placental sFLT1 for the treatment of preeclampsia.

Preeclampsia is a placentally induced hypertensive disorder of pregnancy that is associated with substantial morbidity and mortality to mothers and fetuses. Clinical manifestations of preterm preeclampsia result from excess circulating soluble vascular endothelial growth factor receptor FLT1 (sFLT1 or sVEGFR1) of placental origin. Here we identify short interfering RNAs (siRNAs) that selectively silence the three sFLT1 mRNA isoforms primarily responsible for placental overexpression of sFLT1 without reducing levels of full-length FLT1 mRNA. Full chemical stabilization in the context of hydrophobic modifications enabled productive siRNA accumulation in the placenta (up to 7% of injected dose) and reduced circulating sFLT1 in pregnant mice (up to 50%). In a baboon preeclampsia model, a single dose of siRNAs suppressed sFLT1 overexpression and clinical signs of preeclampsia. Our results demonstrate RNAi-based extrahepatic modulation of gene expression with nonformulated siRNAs in nonhuman primates and establish a path toward a new treatment paradigm for patients with preterm preeclampsia.

Transvascular Delivery of Hydrophobically Modified siRNAs: Gene Silencing in the Rat Brain upon Disruption of the Blood-Brain Barrier.

Effective transvascular delivery of therapeutic oligonucleotides to the brain presents a major hurdle to the development of gene silencing technologies for treatment of genetically defined neurological disorders. Distribution to the brain after systemic administrations is hampered by the low permeability of the blood-brain barrier (BBB) and the rapid clearance kinetics of these drugs from the blood. Here we show that transient osmotic disruption of the BBB enables transvascular delivery of hydrophobically modified small interfering RNA (hsiRNA) to the rat brain. Intracarotid administration of 25% mannitol and hsiRNA conjugated to phosphocholine-docosahexanoic acid (PC-DHA) resulted in broad ipsilateral distribution of PC-DHA-hsiRNAs in the brain. PC-DHA conjugation enables hsiRNA retention in the parenchyma proximal to the brain vasculature and enabled active internalization by neurons and astrocytes. Moreover, transvascular delivery of PC-DHA-hsiRNAs effected Htt mRNA silencing in the striatum (55%), hippocampus (51%), somatosensory cortex (52%), motor cortex (37%), and thalamus (33%) 1 week after administration. Aside from mild gliosis induced by osmotic disruption of the BBB, transvascular delivery of PC-DHA-hsiRNAs was not associated with neurotoxicity. Together, these findings provide proof-of-concept that temporary disruption of the BBB is an effective strategy for the delivery of therapeutic oligonucleotides to the brain.