Publications by authors named "Anastasia Khvorova"

87 Publications

Gene therapy with AR isoform 2 rescues spinal and bulbar muscular atrophy phenotype by modulating AR transcriptional activity.

Sci Adv 2021 Aug 20;7(34). Epub 2021 Aug 20.

Department of Paediatrics, University of Oxford, Oxford, UK.

Spinal and bulbar muscular atrophy (SBMA) is an X-linked, adult-onset neuromuscular condition caused by an abnormal polyglutamine (polyQ) tract expansion in androgen receptor (AR) protein. SBMA is a disease with high unmet clinical need. Recent studies have shown that mutant AR-altered transcriptional activity is key to disease pathogenesis. Restoring the transcriptional dysregulation without affecting other AR critical functions holds great promise for the treatment of SBMA and other AR-related conditions; however, how this targeted approach can be achieved and translated into a clinical application remains to be understood. Here, we characterized the role of AR isoform 2, a naturally occurring variant encoding a truncated AR lacking the polyQ-harboring domain, as a regulatory switch of AR genomic functions in androgen-responsive tissues. Delivery of this isoform using a recombinant adeno-associated virus vector type 9 resulted in amelioration of the disease phenotype in SBMA mice by restoring polyQ AR-dysregulated transcriptional activity.
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http://dx.doi.org/10.1126/sciadv.abi6896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8378820PMC
August 2021

AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma.

J Exp Med 2021 Sep 29;218(9). Epub 2021 Jul 29.

Department of Dermatology, University of Massachusetts Medical School, Worcester, MA.

The STING and absent in melanoma 2 (AIM2) pathways are activated by the presence of cytosolic DNA, and STING agonists enhance immunotherapeutic responses. Here, we show that dendritic cell (DC) expression of AIM2 within human melanoma correlates with poor prognosis and, in contrast to STING, AIM2 exerts an immunosuppressive effect within the melanoma microenvironment. Vaccination with AIM2-deficient DCs improves the efficacy of both adoptive T cell therapy and anti-PD-1 immunotherapy for "cold tumors," which exhibit poor therapeutic responses. This effect did not depend on prolonged survival of vaccinated DCs, but on tumor-derived DNA that activates STING-dependent type I IFN secretion and subsequent production of CXCL10 to recruit CD8+ T cells. Additionally, loss of AIM2-dependent IL-1β and IL-18 processing enhanced the treatment response further by limiting the recruitment of regulatory T cells. Finally, AIM2 siRNA-treated mouse DCs in vivo and human DCs in vitro enhanced similar anti-tumor immune responses. Thus, targeting AIM2 in tumor-infiltrating DCs is a promising new treatment strategy for melanoma.
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http://dx.doi.org/10.1084/jem.20200962DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8329870PMC
September 2021

The NIH Somatic Cell Genome Editing program.

Nature 2021 04 7;592(7853):195-204. Epub 2021 Apr 7.

McGovern Institute for Brain Research, Massachusetts Institute of Technology, Cambridge, MA, USA.

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.
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http://dx.doi.org/10.1038/s41586-021-03191-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8026397PMC
April 2021

Docosanoic acid conjugation to siRNA enables functional and safe delivery to skeletal and cardiac muscles.

Mol Ther 2021 04 19;29(4):1382-1394. Epub 2020 Dec 19.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01604, USA; Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01604, USA. Electronic address:

Oligonucleotide therapeutics hold promise for the treatment of muscle- and heart-related diseases. However, oligonucleotide delivery across the continuous endothelium of muscle tissue is challenging. Here, we demonstrate that docosanoic acid (DCA) conjugation of small interfering RNAs (siRNAs) enables efficient (~5% of injected dose), sustainable (>1 month), and non-toxic (no cytokine induction at 100 mg/kg) gene silencing in both skeletal and cardiac muscles after systemic injection. When designed to target myostatin (muscle growth regulation gene), siRNAs induced ~55% silencing in various muscle tissues and 80% silencing in heart, translating into a ~50% increase in muscle volume within 1 week. Our study identifies compounds for RNAi-based modulation of gene expression in skeletal and cardiac muscles, paving the way for both functional genomics studies and therapeutic gene modulation in muscle and heart.
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http://dx.doi.org/10.1016/j.ymthe.2020.12.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8058398PMC
April 2021

Single-Stranded Phosphorothioated Regions Enhance Cellular Uptake of Cholesterol-Conjugated siRNA but Not Silencing Efficacy.

Mol Ther Nucleic Acids 2020 Sep 25;21:991-1005. Epub 2020 Jul 25.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

Small interfering RNAs (siRNAs) have potential to silence virtually any disease-causing gene but require chemical modifications for delivery to the tissue and cell of interest. Previously, we demonstrated that asymmetric, phosphorothioate (PS)-modified, chemically stabilized, cholesterol-conjugated siRNAs, called hsiRNAs, support rapid cellular uptake and efficient mRNA silencing both in cultured cells and in vivo. Here, we systematically evaluated the impact of number, structure, and sequence context of PS-modified backbones on cellular uptake and RNAi-mediated silencing efficacy. We find that PS enhances cellular internalization in a sequence-dependent manner but only when present in a single-stranded but not double-stranded region. Furthermore, the observed increase in cellular internalization did not correlate with functional silencing improvement, indicating that PS-mediated uptake may drive compounds to non-productive sinks. Thus, the primary contributing factor of PS modifications to functional efficacy is likely stabilization rather than enhanced cellular uptake. A better understanding of the relative impact of different chemistries on productive versus non-productive uptake will assist in improved design of therapeutic RNAs.
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http://dx.doi.org/10.1016/j.omtn.2020.07.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7452107PMC
September 2020

The chemical structure and phosphorothioate content of hydrophobically modified siRNAs impact extrahepatic distribution and efficacy.

Nucleic Acids Res 2020 08;48(14):7665-7680

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01604, USA.

Small interfering RNAs (siRNAs) have revolutionized the treatment of liver diseases. However, robust siRNA delivery to other tissues represents a major technological need. Conjugating lipids (e.g. docosanoic acid, DCA) to siRNA supports extrahepatic delivery, but tissue accumulation and gene silencing efficacy are lower than that achieved in liver by clinical-stage compounds. The chemical structure of conjugated siRNA may significantly impact invivo efficacy, particularly in tissues with lower compound accumulation. Here, we report the first systematic evaluation of the impact of siRNA scaffold-i.e. structure, phosphorothioate (PS) content, linker composition-on DCA-conjugated siRNA delivery and efficacy in vivo. We found that structural asymmetry (e.g. 5- or 2-nt overhang) has no impact on accumulation, but is a principal factor for enhancing activity in extrahepatic tissues. Similarly, linker chemistry (cleavable versus stable) altered activity, but not accumulation. In contrast, increasing PS content enhanced accumulation of asymmetric compounds, but negatively impacted efficacy. Our findings suggest that siRNA tissue accumulation does not fully define efficacy, and that the impact of siRNA chemical structure on activity is driven by intracellular re-distribution and endosomal escape. Fine-tuning siRNA chemical structure for optimal extrahepatic efficacy is a critical next step for the progression of therapeutic RNAi applications beyond liver.
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http://dx.doi.org/10.1093/nar/gkaa595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7430635PMC
August 2020

Cell Type Impacts Accessibility of mRNA to Silencing by RNA Interference.

Mol Ther Nucleic Acids 2020 Sep 12;21:384-393. Epub 2020 Jun 12.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

RNA interference (RNAi) is a potent mechanism that silences mRNA and protein expression in all cells and tissue types. RNAi is known to exert many of its functional effects in the cytoplasm, and thus, the cellular localization of target mRNA may impact observed potency. Here, we demonstrate that cell identity has a profound impact on accessibility of apolipoprotein E (ApoE) mRNA to RNAi. We show that, whereas both neuronal and glial cell lines express detectable ApoE mRNA, in neuronal cells, ApoE mRNA is not targetable by RNAi. Screening of a panel of thirty-five chemically modified small interfering RNAs (siRNAs) did not produce a single hit in a neuronal cell line, whereas up to fifteen compounds showed strong efficacy in glial cells. Further investigation of the cellular localization of ApoE mRNA demonstrates that ApoE mRNA is partially spliced and preferentially localized to the nucleus (∼80%) in neuronal cells, whereas more than 90% of ApoE mRNA is cytoplasmic in glial cells. Such an inconsistency in intracellular localization and splicing might provide an explanation for functional differences in RNAi compounds. Thus, cellular origin might have an impact on accessibility of mRNA to RNAi and should be taken into account during the screening process.
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http://dx.doi.org/10.1016/j.omtn.2020.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340969PMC
September 2020

2'-O-Methyl at 20-mer Guide Strand 3' Termini May Negatively Affect Target Silencing Activity of Fully Chemically Modified siRNA.

Mol Ther Nucleic Acids 2020 Sep 15;21:266-277. Epub 2020 May 15.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA; Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

Small interfering RNAs (siRNAs) have the potential to treat a broad range of diseases. siRNAs need to be extensively chemically modified to improve their bioavailability, safety, and stability in vivo. However, chemical modifications variably impact target silencing for different siRNA sequences, making the activity of chemically modified siRNA difficult to predict. Here, we systematically evaluated the impact of 3' terminal modifications (2'-O-methyl versus 2'-fluoro) on guide strands of different length and showed that 3' terminal 2'-O-methyl modification negatively impacts activity for >60% of siRNA sequences tested but only in the context of 20- and not 19- or 21-nt-long guide strands. These results indicate that sequence, modification pattern, and structure may cooperatively affect target silencing. Interestingly, the introduction of an extra 2'-fluoro modification in the seed region at guide strand position 5, but not 7, may partially compensate for the negative impact of 3' terminal 2'-O-methyl modification. Molecular modeling analysis suggests that 2'-O-methyl modification may impair guide strand interactions within the PAZ domain of argonaute-2, which may affect target recognition and cleavage, specifically when guide strands are 20-nt long. Our findings emphasize the complex nature of modified RNA-protein interactions and contribute to design principles for chemically modified siRNAs.
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http://dx.doi.org/10.1016/j.omtn.2020.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327867PMC
September 2020

Data on enrichment of chitosan nanoparticles for intranasal delivery of oligonucleotides to the brain.

Data Brief 2020 Feb 3;28:105093. Epub 2020 Jan 3.

Department of Neurology, University of South Florida, Tampa, FL, USA.

Data on preparation and characterization of chitosan-based nanoparticles (NP) carrying small interfering RNA (siRNA) for non-invasive gene therapy is presented. Polyelectrolyte complexation method was carried out in diluted concentrations to obtain relatively small (less than 200 nm) NP. To provide substantial dose of siRNA within tolerable volume of intranasal administration the NP were subjected to enrichment process. Offered here NP fabrication does two steps process comprise provisional and enriched preparations? The differences between these preparations were analyzed with hydrodynamic size distribution and zeta potential measurements. The effect of siRNA lipophilicity on NP physical instability was also tested. Biological evaluation of nanoparticles is described in our published article [1].
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http://dx.doi.org/10.1016/j.dib.2019.105093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6965707PMC
February 2020

Hydrophobically Modified let-7b miRNA Enhances Biodistribution to NSCLC and Downregulates HMGA2 In Vivo.

Mol Ther Nucleic Acids 2020 Mar 18;19:267-277. Epub 2019 Nov 18.

HMS Initiative for RNA Medicine, Department of Pathology, Beth Israel Deaconess Medical Center/Harvard Medical School, Boston, MA, USA. Electronic address:

MicroRNAs (miRNAs) have increasingly been shown to be involved in human cancer, and interest has grown about the potential use of miRNAs for cancer therapy. miRNA levels are known to be altered in cancer cells, including in non-small cell lung cancer (NSCLC), a subtype of lung cancer that is the most prevalent form of cancer worldwide and that lacks effective therapies. The let-7 miRNA is involved in the regulation of oncogene expression in cells and directly represses cancer growth in the lung. let-7 is therefore a potential molecular target for tumor therapy. However, applications of RNA interference for cancer research have been limited by a lack of simple and efficient methods to deliver oligonucleotides (ONs) to cancer cells. In this study, we have used in vitro and in vivo approaches to show that HCC827 cells internalize hydrophobically modified let-7b miRNAs (hmiRNAs) added directly to the culture medium without the need for lipid formulation. We identified functional let-7b hmiRNAs targeting the HMGA2 mRNA, one of the let-7 target genes upregulated in NSCLC, and show that direct uptake in HCC827 cells induced potent and specific gene silencing in vitro and in vivo. Thus, hmiRNAs constitute a novel class of ONs that enable functional studies of genes involved in cancer biology and are potentially therapeutic molecules.
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http://dx.doi.org/10.1016/j.omtn.2019.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6926262PMC
March 2020

Enriched chitosan nanoparticles loaded with siRNA are effective in lowering Huntington's disease gene expression following intranasal administration.

Nanomedicine 2020 02 27;24:102119. Epub 2019 Oct 27.

Department of Neurology, University of South Florida, Tampa, FL. Electronic address:

Therapies to lower gene expression in brain disease currently require chronic administration into the cerebrospinal fluid (CSF) by intrathecal infusions or direct intracerebral injections. Though well-tolerated in the short-term, this approach is not tenable for a life-time of administration. Nose-to-brain delivery of enriched chitosan-based nanoparticles loaded with anti-HTT siRNA was studied in a transgenic YAC128 mouse model of Huntington's Disease (HD). A series of chitosan-based nanoparticle (NP) formulations encapsulating anti-HTT small interfering RNA (siRNA) was designed to protect the payload from degradation "en route" to the target. Factors to improve production of effective nanocarriers of anti-HTT siRNA were identified and tested in a YAC128 mouse model of Huntington's disease. Four formulations of nanocarriers were identified to be effective in lowering HTT mRNA expression by at least 50%. Intranasal administration of nanoparticles carrying siRNA is a promising therapeutic alternative for safe and effective lowering of mutant HTT expression.
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http://dx.doi.org/10.1016/j.nano.2019.102119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7098382PMC
February 2020

Loss of huntingtin function slows synaptic vesicle endocytosis in striatal neurons from the htt mouse model of Huntington's disease.

Neurobiol Dis 2020 02 12;134:104637. Epub 2019 Oct 12.

Centre for Discovery Brain Sciences, Hugh Robson Building, University of Edinburgh, Edinburgh EH8 9XD, Scotland, UK. Electronic address:

Huntington's disease (HD) is caused by CAG repeat expansion within the HTT gene, with the dysfunction and eventual loss of striatal medium spiny neurons a notable feature. Since medium spiny neurons receive high amounts of synaptic input, we hypothesised that this vulnerability originates from an inability to sustain presynaptic performance during intense neuronal activity. To test this hypothesis, primary cultures of either hippocampal or striatal neurons were prepared from either wild-type mice or a knock-in HD mouse model which contains 140 poly-glutamine repeats in the huntingtin protein (htt). We identified a striatum-specific defect in synaptic vesicle (SV) endocytosis in htt neurons that was only revealed during high frequency stimulation. This dysfunction was also present in neurons that were heterozygous for the mutant HTT allele. Depletion of endogenous huntingtin using hydrophobically-modified siRNA recapitulated this activity-dependent defect in wild-type neurons, whereas depletion of mutant huntingtin did not rescue the effect in htt neurons. Importantly, this SV endocytosis defect was corrected by overexpression of wild-type huntingtin in homozygous htt neurons. Therefore, we have identified an activity-dependent and striatum-specific signature of presynaptic dysfunction in neurons derived from pre-symptomatic HD mice, which is due to loss of wild-type huntingtin function. This presynaptic defect may render this specific neuronal subtype unable to operate efficiently during high frequency activity patterns, potentially resulting in dysfunctional neurotransmission, synapse failure and ultimately degeneration.
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http://dx.doi.org/10.1016/j.nbd.2019.104637DOI Listing
February 2020

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

Nat Biotechnol 2019 08 2;37(8):884-894. Epub 2019 Aug 2.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA.

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.
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http://dx.doi.org/10.1038/s41587-019-0205-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6879195PMC
August 2019

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

iScience 2019 Jun 27;16:230-241. Epub 2019 May 27.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA; Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.isci.2019.05.029DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562145PMC
June 2019

Nucleic Acid Therapeutics for Neurological Diseases.

Neurotherapeutics 2019 04;16(2):245-247

RNA Therapeutics Institute, UMass Medical School, Worcester, Massachusetts, 01605, USA.

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http://dx.doi.org/10.1007/s13311-019-00736-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6554381PMC
April 2019

The valency of fatty acid conjugates impacts siRNA pharmacokinetics, distribution, and efficacy in vivo.

J Control Release 2019 05 30;302:116-125. Epub 2019 Mar 30.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01604, USA; Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01604, USA. Electronic address:

Lipid-conjugated small-interfering RNAs (siRNAs) exhibit accumulation and gene silencing in extrahepatic tissues, providing an opportunity to expand therapeutic siRNA utility beyond the liver. Chemically engineering lipids may further improve siRNA delivery and efficacy, but the relationship between lipid structure/configuration and siRNA pharmacodynamics is unclear. Here, we synthesized a panel of mono-, di-, and tri-meric fatty acid-conjugated siRNAs to systematically evaluate the impact of fatty acid structure and valency on siRNA clearance, distribution, and efficacy. Fatty acid valency significantly altered the physicochemical properties of conjugated siRNAs, including hydrophobicity and micelle formation, which affected distribution. Trivalent lipid-conjugated siRNAs were predominantly retained at the site of injection with minimal systemic exposure, whereas monovalent lipid-conjugated siRNAs were quickly released into the circulation and accumulated primarily in kidney. Divalent lipid-conjugated siRNAs showed intermediate behavior, and preferentially accumulated in liver with functional distribution to lung, heart, and fat. The chemical structure of the conjugate, rather than overall physicochemical properties (i.e. hydrophobicity), predicted the degree of extrahepatic tissue accumulation necessary for productive gene silencing. Our findings will inform chemical engineering strategies for enhancing the extrahepatic delivery of lipophilic siRNAs.
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http://dx.doi.org/10.1016/j.jconrel.2019.03.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546539PMC
May 2019

Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines.

J Extracell Vesicles 2018 23;7(1):1535750. Epub 2018 Nov 23.

Institute of Biomedicine and Molecular Immunology (IBIM), National Research Council (CNR) of Italy, Palermo, Italy.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.
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http://dx.doi.org/10.1080/20013078.2018.1535750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6322352PMC
November 2018

Rac1 Activity Is Modulated by Huntingtin and Dysregulated in Models of Huntington's Disease.

J Huntingtons Dis 2019 ;8(1):53-69

Department of Neurology, Laboratory of Cellular Neurobiology, Massachusetts General Hospital, Charlestown, MA, USA.

Background: Previous studies suggest that Huntingtin, the protein mutated in Huntington's disease (HD), is required for actin based changes in cell morphology, and undergoes stimulus induced targeting to plasma membranes where it interacts with phospholipids involved in cell signaling. The small GTPase Rac1 is a downstream target of growth factor stimulation and PI 3-kinase activity and is critical for actin dependent membrane remodeling.

Objective: To determine if Rac1 activity is impaired in HD or regulated by normal Huntingtin.

Methods: Analyses were performed in differentiated control and HD human stem cells and HD Q140/Q140 knock-in mice. Biochemical methods included SDS-PAGE, western blot, immunoprecipitation, affinity chromatography, and ELISA based Rac activity assays.

Results: Basal Rac1 activity increased following depletion of Huntingtin with Huntingtin specific siRNA in human primary fibroblasts and in human control neuron cultures. Human cells (fibroblasts, neural stem cells, and neurons) with the HD mutation failed to increase Rac1 activity in response to growth factors. Rac1 activity levels were elevated in striatum of 1.5-month-old HD Q140/Q140 mice and in primary embryonic cortical neurons from HD mice. Affinity chromatography analysis of striatal lysates showed that Huntingtin is in a complex with Rac1, p85α subunit of PI 3-kinase, and the actin bundling protein α-actinin and interacts preferentially with the GTP bound form of Rac1. The HD mutation reduced Huntingtin interaction with p85α.

Conclusions: These findings suggest that Huntingtin regulates Rac1 activity as part of a coordinated response to growth factor signaling and this function is impaired early in HD.
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http://dx.doi.org/10.3233/JHD-180311DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398565PMC
March 2020

Gene Silencing With siRNA (RNA Interference): A New Therapeutic Option During Ex Vivo Machine Liver Perfusion Preservation

Liver Transpl 2019 01;25(1):140-151

Department of Surgery, Division of Organ Transplantation, UMass Memorial Medical Center, University of Massachusetts, Worcester, MA.

RNA interference (RNAi) is a natural process of posttranscriptional gene regulation that has raised a lot of attention culminating with the Nobel Prize in Medicine in 2006. RNAi-based therapeutics have been tested in experimental transplantation to reduce ischemia/reperfusion injury (IRI) with success. Modulation of genes of the innate immune system, as well as apoptotic genes, and those involved in the nuclear factor kappa B pathways can reduce liver injury in rodent liver pedicle clamping and transplantation models of IRI. However, in vivo use of RNAi faces limitations regarding the method of administration, uptake, selectivity, and stability. Machine perfusion preservation, a more recent alternative approach for liver preservation showing superior results to static cold preservation, could be used as a platform for gene interference therapeutics. Our group was the first to demonstrate uptake of small interfering RNA (siRNA) during liver machine preservation under both normothermic and hypothermic perfusion. Administering siRNA in the perfusion solution during ex vivo machine preservation has several advantages, including more efficient delivery, lower doses and cost-saving, and none/fewer side effects to other organs. Recently, the first RNAi drug was approved by the US Food and Drug Administration for clinical use, opening a new avenue for new drugs with different clinical applications. RNAi has the potential to have transformational therapeutic applications in several areas of medicine including transplantation. We believe that machine preservation offers great potential to be the ideal delivery method of siRNA to the liver graft, and future studies should be initiated to improve the clinical applicability of RNAi in solid organ transplantation.
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http://dx.doi.org/10.1002/lt.25383DOI Listing
January 2019

Diverse lipid conjugates for functional extra-hepatic siRNA delivery in vivo.

Nucleic Acids Res 2019 02;47(3):1082-1096

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01604, USA.

Small interfering RNA (siRNA)-based therapies are proving to be efficient for treating liver-associated disorders. However, extra-hepatic delivery remains challenging, limiting therapeutic siRNA utility. We synthesized a panel of fifteen lipid-conjugated siRNAs and systematically evaluated the impact of conjugate on siRNA tissue distribution and efficacy. Generally, conjugate hydrophobicity defines the degree of clearance and the liver-to-kidney distribution profile. In addition to primary clearance tissues, several conjugates achieve significant siRNA accumulation in muscle, lung, heart, adrenal glands and fat. Oligonucleotide distribution to extra-hepatic tissues with some conjugates was significantly higher than with cholesterol, a well studied conjugate, suggesting that altering conjugate structure can enhance extra-hepatic delivery. These conjugated siRNAs enable functional gene silencing in lung, muscle, fat, heart and adrenal gland. Required levels for productive silencing vary (5-200 μg/g) per tissue, suggesting that the chemical nature of conjugates impacts tissue-dependent cellular/intracellular trafficking mechanisms. The collection of conjugated siRNA described here enables functional gene modulation in vivo in several extra-hepatic tissues opening these tissues for gene expression modulation. A systemic evaluation of a panel of conjugated siRNA, as reported here, has not previously been investigated and shows that chemical engineering of lipid siRNAs is essential to advance the RNA therapeutic field.
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http://dx.doi.org/10.1093/nar/gky1239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379722PMC
February 2019

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

Nucleic Acids Res 2019 02;47(3):1070-1081

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA.

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.
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http://dx.doi.org/10.1093/nar/gky1232DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379714PMC
February 2019

RNAi modulation of placental sFLT1 for the treatment of preeclampsia.

Nat Biotechnol 2018 Nov 19. Epub 2018 Nov 19.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts, USA.

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.
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http://dx.doi.org/10.1038/nbt.4297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6526074PMC
November 2018

Exosomes Produced from 3D Cultures of MSCs by Tangential Flow Filtration Show Higher Yield and Improved Activity.

Mol Ther 2018 12 22;26(12):2838-2847. Epub 2018 Sep 22.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA; Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA. Electronic address:

Exosomes can deliver therapeutic RNAs to neurons. The composition and the safety profile of exosomes depend on the type of the exosome-producing cell. Mesenchymal stem cells are considered to be an attractive cell type for therapeutic exosome production. However, scalable methods to isolate and manufacture exosomes from mesenchymal stem cells are lacking, a limitation to the clinical translation of exosome technology. We evaluate mesenchymal stem cells from different sources and find that umbilical cord-derived mesenchymal stem cells produce the highest exosome yield. To optimize exosome production, we cultivate umbilical cord-derived mesenchymal stem cells in scalable microcarrier-based three-dimensional (3D) cultures. In combination with the conventional differential ultracentrifugation, 3D culture yields 20-fold more exosomes (3D-UC-exosomes) than two-dimensional cultures (2D-UC-exosomes). Tangential flow filtration (TFF) in combination with 3D mesenchymal stem cell cultures further improves the yield of exosomes (3D-TFF-exosomes) 7-fold over 3D-UC-exosomes. 3D-TFF-exosomes are seven times more potent in small interfering RNA (siRNA) transfer to neurons compared with 2D-UC-exosomes. Microcarrier-based 3D culture and TFF allow scalable production of biologically active exosomes from mesenchymal stem cells. These findings lift a major roadblock for the clinical utility of mesenchymal stem cell exosomes.
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http://dx.doi.org/10.1016/j.ymthe.2018.09.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277553PMC
December 2018

Nuclear Localization of Huntingtin mRNA Is Specific to Cells of Neuronal Origin.

Cell Rep 2018 09;24(10):2553-2560.e5

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

Huntington's disease (HD) is a monogenic neurodegenerative disorder representing an ideal candidate for gene silencing with oligonucleotide therapeutics (i.e., antisense oligonucleotides [ASOs] and small interfering RNAs [siRNAs]). Using an ultra-sensitive branched fluorescence in situ hybridization (FISH) method, we show that ∼50% of wild-type HTT mRNA localizes to the nucleus and that its nuclear localization is observed only in neuronal cells. In mouse brain sections, we detect Htt mRNA predominantly in neurons, with a wide range of Htt foci observed per cell. We further show that siRNAs and ASOs efficiently eliminate cytoplasmic HTT mRNA and HTT protein, but only ASOs induce a partial but significant reduction of nuclear HTT mRNA. We speculate that, like other mRNAs, HTT mRNA subcellular localization might play a role in important neuronal regulatory mechanisms.
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http://dx.doi.org/10.1016/j.celrep.2018.07.106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6245656PMC
September 2018

Functional features defining the efficacy of cholesterol-conjugated, self-deliverable, chemically modified siRNAs.

Nucleic Acids Res 2018 11;46(20):10905-10916

Advirna, 60 Prescott Street, Worcester, MA 01605, USA.

Progress in oligonucleotide chemistry has produced a shift in the nature of siRNA used, from formulated, minimally modified siRNAs, to unformulated, heavily modified siRNA conjugates. The introduction of extensive chemical modifications is essential for conjugate-mediated delivery. Modifications have a significant impact on siRNA efficacy through interference with recognition and processing by RNAi enzymatic machinery, severely restricting the sequence space available for siRNA design. Many algorithms available publicly can successfully predict the activity of non-modified siRNAs, but the efficiency of the algorithms for designing heavily modified siRNAs has never been systematically evaluated experimentally. Here we screened 356 cholesterol-conjugated siRNAs with extensive modifications and developed a linear regression-based algorithm that effectively predicts siRNA activity using two independent datasets. We further demonstrate that predictive determinants for modified and non-modified siRNAs differ substantially. The algorithm developed from the non-modified siRNAs dataset has no predictive power for modified siRNAs and vice versa. In the context of heavily modified siRNAs, the introduction of chemical asymmetry fully eliminates the requirement for thermodynamic bias, the major determinant for non-modified siRNA efficacy. Finally, we demonstrate that in addition to the sequence of the target site, the accessibility of the neighboring 3' region significantly contributes to siRNA efficacy.
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http://dx.doi.org/10.1093/nar/gky745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237813PMC
November 2018

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

Mol Ther 2018 11 8;26(11):2580-2591. Epub 2018 Aug 8.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA 01605, USA; Department of Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ymthe.2018.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6225091PMC
November 2018

Heavily and fully modified RNAs guide efficient SpyCas9-mediated genome editing.

Nat Commun 2018 07 6;9(1):2641. Epub 2018 Jul 6.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, 01605, USA.

RNA-based drugs depend on chemical modifications to increase potency and to decrease immunogenicity in vivo. Chemical modification will likely improve the guide RNAs involved in CRISPR-Cas9-based therapeutics as well. Cas9 orthologs are RNA-guided microbial effectors that cleave DNA. Here, we explore chemical modifications at all positions of the crRNA guide and tracrRNA cofactor. We identify several heavily modified versions of crRNA and tracrRNA that are more potent than their unmodified counterparts. In addition, we describe fully chemically modified crRNAs and tracrRNAs (containing no 2'-OH groups) that are functional in human cells. These designs will contribute to Cas9-based therapeutics since heavily modified RNAs tend to be more stable in vivo (thus increasing potency). We anticipate that our designs will improve the use of Cas9 via RNP and mRNA delivery for in vivo and ex vivo purposes.
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http://dx.doi.org/10.1038/s41467-018-05073-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6035171PMC
July 2018

Optimized Cholesterol-siRNA Chemistry Improves Productive Loading onto Extracellular Vesicles.

Mol Ther 2018 08 21;26(8):1973-1982. Epub 2018 Jun 21.

RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, MA, USA; Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA, USA. Electronic address:

Extracellular vesicles are promising delivery vesicles for therapeutic RNAs. Small interfering RNA (siRNA) conjugation to cholesterol enables efficient and reproducible loading of extracellular vesicles with the therapeutic cargo. siRNAs are typically chemically modified to fit an application. However, siRNA chemical modification pattern has not been specifically optimized for extracellular vesicle-mediated delivery. Here we used cholesterol-conjugated, hydrophobically modified asymmetric siRNAs (hsiRNAs) to evaluate the effect of backbone, 5'-phosphate, and linker chemical modifications on productive hsiRNA loading onto extracellular vesicles. hsiRNAs with a combination of 5'-(E)-vinylphosphonate and alternating 2'-fluoro and 2'-O-methyl backbone modifications outperformed previously used partially modified siRNAs in extracellular vesicle-mediated Huntingtin silencing in neurons. Between two commercially available linkers (triethyl glycol [TEG] and 2-aminobutyl-1-3-propanediol [C7]) widely used to attach cholesterol to siRNAs, TEG is preferred compared to C7 for productive exosomal loading. Destabilization of the linker completely abolished silencing activity of loaded extracellular vesicles. The loading of cholesterol-conjugated siRNAs was saturated at ∼3,000 siRNA copies per extracellular vesicle. Overloading impaired the silencing activity of extracellular vesicles. The data reported here provide an optimization scheme for the successful use of hydrophobic modification as a strategy for productive loading of RNA cargo onto extracellular vesicles.
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http://dx.doi.org/10.1016/j.ymthe.2018.05.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6094392PMC
August 2018

Novel Cluster and Monomer-Based GalNAc Structures Induce Effective Uptake of siRNAs in Vitro and in Vivo.

Bioconjug Chem 2018 07 2;29(7):2478-2488. Epub 2018 Jul 2.

Center for Translational Biomedicine , Skolkovo Institute of Science and Technology , Skolkovo , 143026 Moscow , Russia.

GalNAc conjugation is emerging as a dominant strategy for delivery of therapeutic oligonucleotides to hepatocytes. The structure and valency of the GalNAc ligand contributes to the potency of the conjugates. Here we present a panel of multivalent GalNAc variants using two different synthetic strategies. Specifically, we present a novel conjugate based on a support-bound trivalent GalNAc cluster, and four others using a GalNAc phosphoramidite monomer that was readily assembled into tri- or tetravalent designs during solid phase oligonucleotide synthesis. We compared these compounds to a clinically used trivalent GalNAc cluster both in vitro and in vivo. In vitro, cluster-based and phosphoramidite-based scaffolds show a similar rate of internalization in primary hepatocytes, with membrane binding observed as early as 5 min. All tested compounds provided potent, dose-dependent silencing, with 2-4% of injected dose recoverable from liver after 1 week. The two preassembled trivalent GalNAc clusters showed higher tissue accumulation and gene silencing relative to di-, tri-, or tetravalent GalNAc conjugates assembled via phosphoramidite chemistry.
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http://dx.doi.org/10.1021/acs.bioconjchem.8b00365DOI Listing
July 2018

Chitosan-Mangafodipir nanoparticles designed for intranasal delivery of siRNA and DNA to brain.

J Drug Deliv Sci Technol 2018 Feb 21;43:453-460. Epub 2017 Nov 21.

Department of Neurology, University of South Florida, Tampa, FL, USA.

The overall objective of the present research was to develop a nanocarrier system for non-invasive delivery to brain of molecules useful for gene therapy. Manganese-containing nanoparticles (mNPs) carrying -eGFP siRNA were tested in cell cultures of eGFP-expressing cell line of mouse fibroblasts (NIH3T3). The optimal mNPs were then tested in mice. Following intranasal instillation, mNPs were visualized by 7T MRI throughout brain at 24 and 48 hrs. mNPs were effective in significantly reducing GFP mRNA expression in Tg GFP+ mice in olfactory bulb, striatum, hippocampus and cortex. Intranasal instillation of mNPS loaded with dsDNA encoding RFP also resulted in expression of the RFP in multiple brain regions. In conclusion, mNPs carrying siRNA, or dsDNA were capable of delivering the payload from nose to brain. This approach for delivery of gene therapies to humans, if successful, will have a significant impact on disease-modifying therapeutics of neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.jddst.2017.11.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5967853PMC
February 2018
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