Cholesterol-functionalized DNA/RNA heteroduplexes cross the blood-brain barrier and knock down genes in the rodent CNS
Nagata, T;Dwyer, CA;Yoshida-Tanaka, K;Ihara, K;Ohyagi, M;Kaburagi, H;Miyata, H;Ebihara, S;Yoshioka, K;Ishii, T;Miyata, K;Miyata, K;Powers, B;Igari, T;Yamamoto, S;Arimura, N;Hirabayashi, H;Uchihara, T;Hara, RI;Wada, T;Bennett, CF;Seth, PP;Rigo, F;Yokota, T;
PMID: 34385691 | DOI: 10.1038/s41587-021-00972-x
Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5' end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood-brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration.
Didiot MC, Ferguson CM, Ly S, Coles AH, Smith AO, Bicknell AA, Hall LM, Sapp E, Echeverria D, Pai AA, DiFiglia M, Moore MJ, Hayward LJ, Aronin N, Khvorova A.
PMID: 30184490 | DOI: 10.1016/j.celrep.2018.07.106
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.
Sci Adv. 2018 Oct 17;4(10):eaat3386.
Ämmälä C, Drury WJ 3rd, Knerr L, Ahlstedt I, Stillemark-Billton P, Wennberg-Huldt C, Andersson EM, Valeur E, Jansson-Löfmark R, Janzén D, Sundström L, Meuller J, Claesson J, Andersson P, Johansson C, Lee RG, Prakash TP, Seth PP, Monia BP, Andersson S.
PMID: 30345352 | DOI: 10.1126/sciadv.aat3386
Antisense oligonucleotide (ASO) silencing of the expression of disease-associated genes is an attractive novel therapeutic approach, but treatments are limited by the ability to deliver ASOs to cells and tissues. Following systemic administration, ASOs preferentially accumulate in liver and kidney. Among the cell types refractory to ASO uptake is the pancreatic insulin-secreting β-cell. Here, we show that conjugation of ASOs to a ligand of the glucagon-like peptide-1 receptor (GLP1R) can productively deliver ASO cargo to pancreatic β-cells both in vitro and in vivo. Ligand-conjugated ASOs silenced target genes in pancreatic islets at doses that did not affect target gene expression in liver or other tissues, indicating enhanced tissue and cell type specificity. This finding has potential to broaden the use of ASO technology, opening up novel therapeutic opportunities, and presents an innovative approach for targeted delivery of ASOs to additional cell types.
Ryan, KA;Bewley, KR;Watson, RJ;Burton, C;Carnell, O;Cavell, BE;Challis, A;Coombes, NS;Davies, ER;Edun-Huges, J;Emery, K;Fell, R;Fotheringham, SA;Gooch, KE;Gowan, K;Handley, A;Harris, DJ;Hesp, R;Hunter, L;Humphreys, R;Johnson, R;Kennard, C;Knott, D;Lister, S;Morley, D;Ngabo, D;Osman, KL;Paterson, J;Penn, EJ;Pullan, ST;Richards, KS;Summers, S;Thomas, SR;Weldon, T;Wiblin, NR;Rayner, EL;Vipond, RT;Hallis, B;Salguero, FJ;Funnell, SGP;Hall, Y;
PMID: 37014911 | DOI: 10.1371/journal.ppat.1011293
The mutation profile of the SARS-CoV-2 Omicron (lineage BA.1) variant posed a concern for naturally acquired and vaccine-induced immunity. We investigated the ability of prior infection with an early SARS-CoV-2 ancestral isolate (Australia/VIC01/2020, VIC01) to protect against disease caused by BA.1. We established that BA.1 infection in naïve Syrian hamsters resulted in a less severe disease than a comparable dose of the ancestral virus, with fewer clinical signs including less weight loss. We present data to show that these clinical observations were almost absent in convalescent hamsters challenged with the same dose of BA.1 50 days after an initial infection with ancestral virus. These data provide evidence that convalescent immunity against ancestral SARS-CoV-2 is protective against BA.1 in the Syrian hamster model of infection. Comparison with published pre-clinical and clinical data supports consistency of the model and its predictive value for the outcome in humans. Further, the ability to detect protection against the less severe disease caused by BA.1 demonstrates continued value of the Syrian hamster model for evaluation of BA.1-specific countermeasures.
Miao, H;Wu, F;Li, Y;Qin, C;Zhao, Y;Xie, M;Dai, H;Yao, H;Cai, H;Wang, Q;Song, X;Li, L;
PMID: 36563164 | DOI: 10.1126/sciadv.abq7289
Understanding how long noncoding RNAs (lncRNAs) cooperate with splicing factors (SFs) in alternative splicing (AS) control is fundamental to human biology and disease. We show that metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a well-documented AS-implicated lncRNA, regulates AS via two SFs, polypyrimidine tract-binding protein 1 (PTBP1) and PTB-associated SF (PSF). MALAT1 stabilizes the interaction between PTBP1 and PSF, thereby forming a functional module that affects a network of AS events. The MALAT1-stabilized PTBP1/PSF interaction occurs in multiple cellular contexts; however, the functional module, relative to MALAT1 only, has more dominant pathological significance in hepatocellular carcinoma. MALAT1 also stabilizes the PSF interaction with several heterogeneous nuclear ribonucleoparticle proteins other than PTBP1, hinting a broad role in AS control. We present a model in which MALAT1 cooperates with distinct SFs for AS regulation and pose that, relative to analyses exclusively performed for lncRNAs, a comprehensive consideration of lncRNAs and their binding partners may provide more information about their biological functions.
Yu Q, Hardin H, Chu YH, Rehrauer W, Lloyd RV.
PMID: 31119524 | DOI: 10.1007/s12022-019-9578-3
Parathyroid adenomas are slow growing benign neoplasms associated with hypercalcemia, while atypical parathyroid adenomas and parathyroid carcinomas are uncommon tumors and their histologic features may overlap with parathyroid adenomas. LncRNAs participate in transcription and in epigenetic or post-transcriptional regulation of gene expression, and probably contribute to carcinogenesis. We analyzed a group of normal, hyperplastic, and neoplastic parathyroid lesions to determine the best immunohistochemical markers to characterize these lesions and to determine the role of selected lncRNAs in tumor progression. A tissue microarray consisting of 111 cases of normal parathyroid (n = 14), primary hyperplasia (n = 15), secondary hyperplasia (n = 10), tertiary hyperplasia (n = 11), adenomas (n = 50), atypical adenomas (n = 7), and carcinomas (n = 4) was used. Immunohistochemical staining with antibodies against chromogranin A, synaptophysin, parathyroid hormone, and insulinoma-associated protein 1(INSM1) was used. Expression of lncRNAs including metastasis-associated lung adenocarcinoma transcript one (MALAT1), HOX transcript antisense intergenic RNA (HOTAIR), and long intergenic non-protein coding regulator of reprograming (Linc-ROR or ROR) was also analyzed by in situ hybridization and RT-PCR. All of the parathyroid tissues were positive for parathyroid hormone, while most cases were positive for chromogranin A (98%). Synaptophysin was expressed in only 12 cases (11%) and INMS1 was negative in all cases. ROR was significantly downregulated during progression from normal, hyperplastic, and adenomatous parathyroid to parathyroid carcinomas. These results show that parathyroid hormone and chromogranin A are useful markers for parathyroid neoplasms, while synaptophysin and INSM1 are not very sensitive broad-spectrum markers for these neoplasms. LincRNA ROR may function as a tumor suppressor during parathyroid tumor progression.
Handley, A;Ryan, K;Davies, E;Bewley, K;Carnell, O;Challis, A;Coombes, N;Fotheringham, S;Gooch, K;Charlton, M;Harris, D;Kennard, C;Ngabo, D;Weldon, T;Salguero, F;Funnell, S;Hall, Y;
| DOI: 10.20944/preprints202302.0171.v1
The Golden Syrian hamster (Mesocricetus auratus) is now commonly used in preclinical research for the study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and the assessment of vaccines, drugs and therapeutics. Here we show that hamsters inoculated via the intranasal route with the same infectious virus dose of prototypical SARS-CoV-2 administered in a different volume present with different clinical signs, weight loss and viral shedding, with a reduced volume resulting in reduced severity of disease similar to that obtained by a 500-fold reduction in challenge dose. The tissue burden of virus and the severity of pulmonary pathology were also significantly affected by different challenge inoculum volumes. These findings suggest that direct comparison between the severity of SARS-CoV-2 variants or studies assessing the efficacy of treatments determined by hamster studies cannot be made unless both the challenge dose and inoculation volume are matched when using the intranasal route. Additionally, analysis of sub-genomic and total genomic RNA PCR data demonstrated no link between sub-genomic and live viral titres and that sub-genomic analyses do not provide any information beyond that provided by more sensitive total genomic PCR.
Barutcu, AR;Wu, M;Braunschweig, U;Dyakov, BJA;Luo, Z;Turner, KM;Durbic, T;Lin, ZY;Weatheritt, RJ;Maass, PG;Gingras, AC;Blencowe, BJ;
PMID: 35182477 | DOI: 10.1016/j.molcel.2021.12.010
The nucleus is highly compartmentalized through the formation of distinct classes of membraneless domains. However, the composition and function of many of these structures are not well understood. Using APEX2-mediated proximity labeling and RNA sequencing, we surveyed human transcripts associated with nuclear speckles, several additional domains, and the lamina. Remarkably, speckles and lamina are associated with distinct classes of retained introns enriched in genes that function in RNA processing, translation, and the cell cycle, among other processes. In contrast to the lamina-proximal introns, retained introns associated with speckles are relatively short, GC-rich, and enriched for functional sites of RNA-binding proteins that are concentrated in these domains. They are also highly differentially regulated across diverse cellular contexts, including the cell cycle. Thus, our study provides a resource of nuclear domain-associated transcripts and further reveals speckles and lamina as hubs of distinct populations of retained introns linked to gene regulation and cell cycle progression.
Sodagar, A;Javed, R;Tahir, H;Razak, SIA;Shakir, M;Naeem, M;Yusof, AHA;Sagadevan, S;Hazafa, A;Uddin, J;Khan, A;Al-Harrasi, A;
PMID: 35883527 | DOI: 10.3390/biom12070971
The number of deaths has been increased due to COVID-19 infections and uncertain neurological complications associated with the central nervous system. Post-infections and neurological manifestations in neuronal tissues caused by COVID-19 are still unknown and there is a need to explore how brainstorming promoted congenital impairment, dementia, and Alzheimer's disease. SARS-CoV-2 neuro-invasion studies in vivo are still rare, despite the fact that other beta-coronaviruses have shown similar properties. Neural (olfactory or vagal) and hematogenous (crossing the blood-brain barrier) pathways have been hypothesized in light of new evidence showing the existence of SARS-CoV-2 host cell entry receptors into the specific components of human nerve and vascular tissue. Spike proteins are the primary key and structural component of the COVID-19 that promotes the infection into brain cells. Neurological manifestations and serious neurodegeneration occur through the binding of spike proteins to ACE2 receptor. The emerging evidence reported that, due to the high rate in the immediate wake of viral infection, the olfactory bulb, thalamus, and brain stem are intensely infected through a trans-synaptic transfer of the virus. It also instructs the release of chemokines, cytokines, and inflammatory signals immensely to the blood-brain barrier and infects the astrocytes, which causes neuroinflammation and neuron death; and this induction of excessive inflammation and immune response developed in more neurodegeneration complications. The present review revealed the pathophysiological effects, molecular, and cellular mechanisms of possible entry routes into the brain, pathogenicity of autoantibodies and emerging immunotherapies against COVID-19.
Primard, C;Monchâtre-Leroy, E;Del Campo, J;Valsesia, S;Nikly, E;Chevandier, M;Boué, F;Servat, A;Wasniewski, M;Picard-Meyer, E;Courant, T;Collin, N;Salguero, F;Le Vert, A;Guyon-Gellin, D;Nicolas, F;
| DOI: 10.3389/fimmu.2023.1188605
Spike-based COVID-19 vaccines induce potent neutralizing antibodies but their efficacy against SARS-CoV-2 variants decreases. OVX033 is a recombinant protein composed of the full-length nucleocapsid (N) protein of SARS-CoV-2 genetically fused to oligoDOM , a self-assembling domain which improves antigen immunogenicity. OVX033 including N as an antigenic target is proposed as new vaccine candidate providing broad-spectrum protection against sarbecoviruses. OVX033 demonstrated its ability to trigger cross-reactive T cell responses and cross-protection against three variants of SARS-CoV-2 (B.1 Europe, Delta B.1.617.2, and Omicron B.1.1.529) in a hamster challenge model, as evidenced by lower weight loss, lower lung viral loads, and reduced lung histopathological lesions.
Case reports in pathology
Butler, JT;Chellappan, R;Litovsky, S;Leal, SM;Benson, PV;
PMID: 37180570 | DOI: 10.1155/2023/9998749
While rare, coronary stent infections present with significant mortality-with most infections and further complications occurring within months of percutaneous coronary intervention (PCI). Here, we discuss a post-COVID-19 patient who presented approximately one year after PCI for declotting of an arteriovenous graft (AVG). Upon admission, the patient was found to be bacteremic with multilobar pneumonia and an infection of the AVG. Empiric antibiotics were started, and blood cultures were subsequently positive for MRSA. Removal of the AVG was unsuccessful, and two days after admission, the patient passed. Autopsy revealed a perivascular abscess in the RCA near the origin of the stent with a ground section of the RCA with stent revealing abundant calcific atherosclerosis and marked necrosis of the artery wall. The cause of death was determined to be sepsis complicating coronary artery disease and chronic renal failure.
Mei, X;Li, J;Wang, Z;Zhu, D;Huang, K;Hu, S;Popowski, KD;Cheng, K;
PMID: 36759564 | DOI: 10.1038/s41563-023-01475-7
The surge of fast-spreading SARS-CoV-2 mutated variants highlights the need for fast, broad-spectrum strategies to counteract viral infections. In this work, we report a physical barrier against SARS-CoV-2 infection based on an inhalable bioadhesive hydrogel, named spherical hydrogel inhalation for enhanced lung defence (SHIELD). Conveniently delivered via a dry powder inhaler, SHIELD particles form a dense hydrogel network that coats the airway, enhancing the diffusional barrier properties and restricting virus penetration. SHIELD's protective effect is first demonstrated in mice against two SARS-CoV-2 pseudo-viruses with different mutated spike proteins. Strikingly, in African green monkeys, a single SHIELD inhalation provides protection for up to 8 hours, efficiently reducing infection by the SARS-CoV-2 WA1 and B.1.617.2 (Delta) variants. Notably, SHIELD is made with food-grade materials and does not affect normal respiratory functions. This approach could offer additional protection to the population against SARS-CoV-2 and other respiratory pathogens.
