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1.
Proc Natl Acad Sci U S A ; 120(11): e2219523120, 2023 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-36893269

RESUMO

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.


Assuntos
COVID-19 , Humanos , Animais , Camundongos , RNA Interferente Pequeno/genética , COVID-19/terapia , SARS-CoV-2/genética , Antivirais/farmacologia , Antivirais/uso terapêutico , Oligonucleotídeos , Pulmão
2.
Nucleic Acids Res ; 50(15): 8418-8430, 2022 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-35920332

RESUMO

The lung is a complex organ with various cell types having distinct roles. Antisense oligonucleotides (ASOs) have been studied in the lung, but it has been challenging to determine their effectiveness in each cell type due to the lack of appropriate analytical methods. We employed three distinct approaches to study silencing efficacy within different cell types. First, we used lineage markers to identify cell types in flow cytometry, and simultaneously measured ASO-induced silencing of cell-surface proteins CD47 or CD98. Second, we applied single-cell RNA sequencing (scRNA-seq) to measure silencing efficacy in distinct cell types; to the best of our knowledge, this is the first time scRNA-seq has been applied to measure the efficacy of oligonucleotide therapeutics. In both approaches, fibroblasts were the most susceptible to locally delivered ASOs, with significant silencing also in endothelial cells. Third, we confirmed that the robust silencing in fibroblasts is broadly applicable by silencing two targets expressed mainly in fibroblasts, Mfap4 and Adam33. Across independent approaches, we demonstrate that intratracheally administered LNA gapmer ASOs robustly induce gene silencing in lung fibroblasts. ASO-induced gene silencing in fibroblasts was durable, lasting 4-8 weeks after a single dose. Thus, lung fibroblasts are well aligned with ASOs as therapeutics.


Assuntos
Células Endoteliais , Fibroblastos/efeitos dos fármacos , Pulmão/citologia , Oligonucleotídeos Antissenso/administração & dosagem , Animais , Fibroblastos/metabolismo , Inativação Gênica , Pulmão/efeitos dos fármacos , Camundongos , Oligonucleotídeos/administração & dosagem , Traqueia/metabolismo
3.
Mol Ther Nucleic Acids ; 35(2): 102230, 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38938759

RESUMO

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.

4.
Mol Ther Nucleic Acids ; 34: 102080, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-38089931

RESUMO

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.

5.
CRISPR J ; 6(6): 570-582, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-38108517

RESUMO

CRISPR-based genome-editing technologies, including nuclease editing, base editing, and prime editing, have recently revolutionized the development of therapeutics targeting disease-causing mutations. To advance the assessment and development of genome editing tools, a robust mouse model is valuable, particularly for evaluating in vivo activity and delivery strategies. In this study, we successfully generated a knock-in mouse line carrying the Traffic Light Reporter design known as TLR-multi-Cas variant 1 (TLR-MCV1). We comprehensively validated the functionality of this mouse model for both in vitro and in vivo nuclease and prime editing. The TLR-MCV1 reporter mouse represents a versatile and powerful tool for expediting the development of editing technologies and their therapeutic applications.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Animais , Camundongos , Sistemas CRISPR-Cas/genética , Modelos Animais de Doenças , Endonucleases/genética , Tecnologia
6.
Nucleic Acid Ther ; 32(1): 66-73, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34928745

RESUMO

Reliable detection and quantification of antisense oligonucleotides (ASOs) in experimental and clinical specimens are essential to understand the biological function of novel oligonucleotide-based therapeutics. In this study, we describe a method to detect and quantify ASOs in biological samples, whereby the ASO acts as a splint to direct the ligation of complementary probes and quantitative real-time PCR was used to monitor ligation products. Low levels of 2'-O-methoxyethyl (2'-O-MOE) gapmer ASO in serum, liver, kidney, lung, heart, muscle, and brain tissues can be detected over a 6-log linear range for detection using this method. This method allows quantification of various types of chemically modified ASOs, including phosphorothioate linkage, 2'-O-methyl, 2'-O-MOE, and locked nucleic acid, as well as siRNAs. This method does not require probe modifications, and can be performed using standard laboratory equipment; making it a fast, sensitive, and reliable technique that can be widely applied. This detection method may find potential applications in detection of therapeutic oligonucleotides in biological samples.


Assuntos
Oligonucleotídeos Antissenso , Contenções , Fígado , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/uso terapêutico , Oligonucleotídeos Fosforotioatos , Reação em Cadeia da Polimerase em Tempo Real
7.
BMB Rep ; 55(8): 407-412, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35725014

RESUMO

A well-controlled inflammatory response is crucial for the recovery from injury and maintenance of tissue homeostasis. The anti-inflammatory response of 2-methoxycinnamaldehyde (2-MCA), a natural compound derived from cinnamon, has been studied; however, the underlying mechanism on macrophage has not been fully elucidated. In this study, LPS-stimulated production of TNF-α and NO was reduced by 2-MCA in macrophages. 2-MCA significantly activated the NRF2 pathway, and expression levels of autophagy-associated proteins in macrophages, including LC3 and P62, were enhanced via NRF2 activation regardless of LPS treatment, suggesting the occurrence of 2-MCA-mediated autophagy. Moreover, evaluation of autophagy flux using luciferase-conjugated LC3 revealed that incremental LC3 and P62 levels are coupled to enhanced autophagy flux. Finally, reduced expression levels of TNF-α and NOS2 by 2-MCA were reversed by autophagy inhibitors, such as bafilomycin A1 and NH4Cl, in LPS-stimulated macrophages. In conclusion, 2-MCA enhances autophagy flux in macrophages via NRF2 activation and consequently reduces LPS-induced inflammation. [BMB Reports 2022; 55(8): 407-412].


Assuntos
Lipopolissacarídeos , Fator 2 Relacionado a NF-E2 , Acroleína/análogos & derivados , Autofagia , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/toxicidade , Macrófagos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
8.
Cell Death Differ ; 29(6): 1199-1210, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-34974535

RESUMO

Ninjurin1 (Ninj1), an adhesion molecule, regulates macrophage function in hyaloid regression, multiple sclerosis, and atherosclerosis. However, its biological relevance and the mechanism underlying its function in vascular network integrity have not been studied. In this study, we investigated the role of Ninj1 in physiological (postnatal vessel formation) and pathological (endotoxin-mediated inflammation and diabetes) conditions and developed a strategy to regulate Ninj1 using specific micro (mi)RNAs under pathological conditions. Ninj1-deficient mice exhibited decreased hyaloid regression, tip cell formation, retinal vascularized area, recruitment of macrophages, and endothelial apoptosis during postnatal development, resulting in delayed formation of the vascular network. Five putative miRNAs targeting Ninj1 were selected using the miRanda algorithm and comparison of expression patterns. Among them, miR-125a-5p showed a profound inhibitory effect on Ninj1 expression, and miR-125a-5p mimic suppressed the cell-to-cell and cell-to-matrix adhesion of macrophages and expression of pro-inflammatory factors mediated by Ninj1. Furthermore, miR-125a-5p mimic inhibited the recruitment of macrophages into inflamed retinas in endotoxin-induced inflammation and streptozotocin-induced diabetes in vivo. In particular, miR-125a-5p mimic significantly attenuated vascular leakage in diabetic retinopathy. Taken together, these findings suggest that Ninj1 plays a pivotal role in macrophage-mediated vascular integrity and that miR-125a-5p acts as a novel regulator of Ninj1 in the management of inflammatory diseases and diabetic retinopathy.


Assuntos
Moléculas de Adesão Celular Neuronais , Retinopatia Diabética , MicroRNAs , Fatores de Crescimento Neural , Animais , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Endotoxinas/metabolismo , Inflamação/genética , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo
9.
Nat Med ; 28(1): 117-124, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34949835

RESUMO

Expansions of a G4C2 repeat in the C9ORF72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two devastating adult-onset neurodegenerative disorders. Using C9-ALS/FTD patient-derived cells and C9ORF72 BAC transgenic mice, we generated and optimized antisense oligonucleotides (ASOs) that selectively blunt expression of G4C2 repeat-containing transcripts and effectively suppress tissue levels of poly(GP) dipeptides. ASOs with reduced phosphorothioate content showed improved tolerability without sacrificing efficacy. In a single patient harboring mutant C9ORF72 with the G4C2 repeat expansion, repeated dosing by intrathecal delivery of the optimal ASO was well tolerated, leading to significant reductions in levels of cerebrospinal fluid poly(GP). This report provides insight into the effect of nucleic acid chemistry on toxicity and, to our knowledge, for the first time demonstrates the feasibility of clinical suppression of the C9ORF72 gene. Additional clinical trials will be required to demonstrate safety and efficacy of this therapy in patients with C9ORF72 gene mutations.


Assuntos
Proteína C9orf72/genética , Mutação , Oligonucleotídeos Antissenso/genética , Animais , Proteína C9orf72/metabolismo , Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Transgênicos , Neurônios/metabolismo
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