Bruno M.D.C. Godinho

University of Massachusetts Chan Medical School

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Anastasia Khvorova

University of Massachusetts Chan Medical School

Dimas Echeverria

University of Massachusetts Chan Medical School

Matthew Hassler

Chemnitz University of Technology

Andrew H. Coles

AbbVie (United States)

Julia F. Alterman

University of Massachusetts Chan Medical School

Neil Aronin

University of Massachusetts Chan Medical School

Reka A. Haraszti

University of Tübingen

Caitríona M. O’Driscoll

University College Cork

John F. Cryan

University College Cork

Marian DiFiglia

Massachusetts General Hospital

Cooperative Institutions

University of Massachusetts Chan Medical School

116

UMass Memorial Medical Center

Harvard University

Massachusetts General Hospital

Massachusetts Academy of Math and Science

University College Cork

Biogen (United States)

Howard Hughes Medical Institute

University of Sydney

National Institutes of Health

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Conjugate-mediated Delivery of RNAi-based Therapeutics: Enhancing Pharmacokinetics–Pharmacodynamics Relationships of Medicinal Oligonucleotides

The Royal Society of Chemistry eBooks (2019)

Bruno M.D.C. Godinho Andrew H. Coles Anastasia Khvorova

Therapeutic gene silencing using synthetic small interfering RNA (siRNA) holds great promise for the treatment of genetically-defined disorders by targeting disease-associated gene products for degradation. To date, one of the most clinically advanced configurations in the field consists of fully chemically modified siRNAs conjugated to N-acetylgalactosamine (GalNAc), which enables targeted delivery with potent and long-lasting gene silencing effects in hepatocytes. The revolutionary success of the GalNAc platform has rapidly expanded to various clinical programs to treat liver disorders. This success has spurred much interest in the field to explore other conjugate modalities, which are now being tested for their in vivo utility to achieve meaningful delivery to extrahepatic tissues. Establishing meaningful oligonucleotide delivery and durable gene-silencing effects requires careful consideration of the key aspects that govern the pharmacokinetics–pharmacodynamics (PK–PD) of conjugated oligonucleotides. In this chapter, we provide an overview of the chemical evolution of unformulated RNA interference (RNAi)-based technologies focusing on the major corner stones that determine productive PK–PD relationships: chemical stabilization, conjugation chemistries for modulation of biodistribution, clearance and intracellular localization and the effects of the route of administration.

Biodistribution

Pharmacodynamics

10.1039/9781788015714-00206

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Citations (1)

A High-Throughput Method for Direct Detection of Therapeutic Oligonucleotide-Induced Gene SilencingIn Vivo

Nucleic Acid Therapeutics (2015)

Andrew H. Coles Maire F. Osborn Julia F. Alterman Anton A. Turanov Bruno M.D.C. Godinho

Preclinical development of RNA interference (RNAi)-based therapeutics requires a rapid, accurate, and robust method of simultaneously quantifying mRNA knockdown in hundreds of samples. The most well-established method to achieve this is quantitative real-time polymerase chain reaction (qRT-PCR), a labor-intensive methodology that requires sample purification, which increases the potential to introduce additional bias. Here, we describe that the QuantiGene(®) branched DNA (bDNA) assay linked to a 96-well Qiagen TissueLyser II is a quick and reproducible alternative to qRT-PCR for quantitative analysis of mRNA expression in vivo directly from tissue biopsies. The bDNA assay is a high-throughput, plate-based, luminescence technique, capable of directly measuring mRNA levels from tissue lysates derived from various biological samples. We have performed a systematic evaluation of this technique for in vivo detection of RNAi-based silencing. We show that similar quality data is obtained from purified RNA and tissue lysates. In general, we observe low intra- and inter-animal variability (around 10% for control samples), and high intermediate precision. This allows minimization of sample size for evaluation of oligonucleotide efficacy in vivo.

10.1089/nat.2015.0578

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Self-assembling Modified β-Cyclodextrin Nanoparticles as Neuronal siRNA Delivery Vectors: Focus on Huntington’s Disease

Molecular Pharmaceutics (2012)

Bruno M.D.C. Godinho Julien Ogier Raphael Darcy Caitríona M. O’Driscoll John F. Cryan

Huntington's disease (HD) is a rare autosomal dominant neurodegenerative disease caused by the expression of a toxic Huntingtin (HTT) protein. The use of short interfering RNAs (siRNAs) to silence the mutant protein is one of the most promising therapeutic strategies under investigation. The biggest caveat to siRNA-based approaches is the lack of efficient and nontoxic delivery vectors for siRNA delivery to the central nervous system. In this study, we investigated the potential of modified amphiphilic β-cyclodextrins (CDs), oligosaccharide-based molecules, as novel siRNA neuronal carriers. We show that CDs formed nanosize particles which were stable in artificial cerebrospinal fluid. Moreover, these complexes were able to reduce the expression of the HTT gene in rat striatal cells (ST14A-HTT120Q) and in human HD primary fibroblasts. Only limited toxicity was observed with CD·siRNA nanoparticles in any of the in vitro models used. Sustained knockdown effects were observed in the striatum of the R6/2 mouse model of HD after single direct injections of CD·siRNA nanoparticles. Repeated brain injections of CD·siRNA complexes resulted in selective alleviation of motor deficits in this mouse model. Together these data support the utility of modified β-CDs as efficient and safe siRNA delivery vectors for RNAi-based therapies for neuropsychiatric and neurodegenerative disorders.

10.1021/mp3003946

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Desenvolvimento de novas metodologias para determinação de antibióticos em águas de consumo (estudo exploratório)

Saúde & tecnologia/Saúde & tecnologia (2008)

Bruno M.D.C. Godinho João Mário Pedro Anabela Graça

It is nowadays well established that antibiotics are constantly released to the environment as a consequence of their broad use in human and animal therapy. The presence of these substances in superficial, underground and residual waters, have being detected by several authors. Resistant bacterial strains and toxicity risks are the major concerning impact for the environment which demands a quality control of the waters. Thus, the development of new methodologies for environmental antibiotic determination is an important issue. The suitability of a Solid Phase extraction technique using a C-18 reversed phase packed material and several elution solvents were tested for detection of Tetracycline (TC), Chloramphenicol (CPA) and Streptomycin (STP) in standard water samples. The chromatographic separation was held in High Pressure Liquid Chromatography equipment with a diode array detector, using a C-18 reversed phase column (30cm x 3.9mm d.i., 5μm particle size). The specific absorption wavelenght for each antibiotic was montitorized: 357, 277, and 257nm for TC, CPA and STP. The elution solvent was determinant for the extraction step, as we achieve 77,1% of recovery for TC and 15,3% for CPA; no recovery was achieved for STP. Therefore, a method of analysis for the TC and CPA was established, however a separated method of analysis is suggested for the analysis of STP, due to its high polar behavior.

Gradient elution

Solid phase extraction

Chromatography detector

10.25758/set.134

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A combinatorial approach for achieving CNS-selective RNAi

Nucleic Acids Research (2024)

Chantal Ferguson Bruno M.D.C. Godinho Dimas Echeverria Matthew Hassler Lorenc Vangjeli

Abstract 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.

Trans-acting siRNA

RNA Silencing

10.1093/nar/gkae100

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

Alzheimer s & Dementia (2024)

Chantal Ferguson Samuel Hildebrand Bruno M.D.C. Godinho Julianna Buchwald Dimas Echeverria

Abstract 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.

Apolipoprotein E

Amyloid (mycology)

10.1002/alz.13703

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Citations (9)

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

bioRxiv (Cold Spring Harbor Laboratory) (2023)

Ken Yamada Vignesh Hariharan Jillian Caiazzi Rachael Miller Chantal Furguson

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.

Phosphodiester bond

Exonuclease III

10.1101/2023.05.26.542506

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Citations (6)

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

Research Square (Research Square) (2023)

Vignesh Hariharan Jillian Caiazzi Rachael Miller Chantal Ferguson Ellen Sapp

Abstract 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.

Phosphodiester bond

Exonuclease III

10.21203/rs.3.rs-2987323/v1

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Citations (3)

RNAi therapeutics for brain cancer: current advancements in RNAi delivery strategies

Molecular BioSystems (2015)

Meenakshi Malhotra André Toulouse Bruno M.D.C. Godinho David John Mc Carthy John F. Cryan

Malignant primary brain tumors are aggressive cancerous cells that invade the surrounding tissues of the central nervous system. The current treatment options for malignant brain tumors are limited due to the inability to cross the blood-brain barrier. The advancements in current research has identified and characterized certain molecular markers that are essential for tumor survival, progression, metastasis and angiogenesis. These molecular markers have served as therapeutic targets for the RNAi based therapies, which enable site-specific silencing of the gene responsible for tumor proliferation. However, to bring about therapeutic success, an efficient delivery carrier that can cross the blood-brain barrier and reach the targeted site is essential. The current review focuses on the potential of targeted, non-viral and viral particles containing RNAi therapeutic molecules as delivery strategies specifically for brain tumors.

Brain tumor

10.1039/c5mb00278h

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

Nature Biotechnology (2019)

Julia F. Alterman Bruno M.D.C. Godinho Matthew Hassler Chantal Ferguson Dimas Echeverria

Trans-acting siRNA

RNA Silencing

10.1038/s41587-019-0205-0

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Citations (173)