Nigel McMillan

CD62L as a Therapeutic Target in Chronic Lymphocytic Leukemia

Publisher: American Association for Cancer Research (AACR)

Date: 14-10-2013

DOI: 10.1158/1078-0432.CCR-13-1037

Abstract: Purpose: Despite advances in the treatment of chronic lymphocytic leukemia (CLL), the disease remains incurable with standard therapies and relapse is inevitable. A growing body of evidence indicates that alterations in the adhesion properties of neoplastic cells play a pivotal role in the development and progression of CLL. Experimental Design: The expression of 71 cell surface molecules was examined on CLL peripheral blood mononuclear cells (PBMCs) over 3 weeks in culture. The most highly upregulated marker, CD62L, was examined further for expression on CD5+/CD19+ CLL cells in vitro and in lymph node and bone marrow biopsies. The prosurvival role of CD62L was examined using a functional blocking antibody and therapeutic potential evaluated by comparison with current chemotherapy agents. Results: Blocking CD62L resulted in apoptosis of CLL cells but not PBMCs from healthy donors suggesting a novel role for CD62L in CLL cell survival. The beneficial effect of coculturing CLL cells with bone marrow stromal cells or endothelial cells does not protect CLL cells from anti-CD62L–related toxicity. Moreover, combining fludarabine or mafosfamide with the anti-CD62L in vitro produced an additive effect both with and without stromal cells. Conclusion: This is the first reported data showing that blocking the activation and homing marker, CD62L, regulates CLL cell survival in vitro. These data also suggest that therapeutic antibodies against CD62L may provide additional clinical benefit to patients with CLL receiving current standard chemotherapy protocols. Clin Cancer Res 19(20) 5675–85. ©2013 AACR.

High serum levels of CD178 (soluble FasL) predict for inferior progression free survival in chronic lymphocytic leukemia treated with fludarabine-based chemotherapy

Publisher: Informa UK Limited

Date: 25-06-2019

DOI: 10.1080/10428194.2019.1581936

Self-Adjuvanting Therapeutic Peptide-Based Vaccine Induce CD8⁺ Cytotoxic T Lymphocyte Responses in a Murine Human Papillomavirus Tumor Model

Publisher: Bentham Science Publishers Ltd.

Date: 10-03-2015

DOI: 10.2174/1567201811666141001155729

Abstract: Vaccine candidates for the treatment of human papillomavirus (HPV)-associated cancers are aimed to activate T-cells and induce development of cytotoxic anti-tumor specific responses. Peptide epitopes derived from HPV-16 E7 oncogenic protein have been identified as promising antigens for vaccine development. However, peptide-based antigens alone elicit poor cytotoxic T lymphocyte (CTL) responses and need to be formulated with an adjuvant (immunostimulant) to achieve the desired immune responses. We have reported the ability of polyacrylate 4-arm star-polymer (S4) conjugated with HPV-16 E744-57 (8Qmin) epitope to reduce and eradicate TC-1 tumor in the mouse model. Herein, we have studied the mechanism of induction of immune responses by this polymer-peptide conjugate and found prompt uptake of conjugate by antigen presenting cells, stimulating stronger CD8(+) rather than CD4(+) or NK cell responses.

Outcome of acute hypoxaemic respiratory failure: insights from the LUNG SAFE Study

Publisher: European Respiratory Society (ERS)

Date: 17-12-2020

DOI: 10.1183/13993003.03317-2020

Abstract: Current incidence and outcome of patients with acute hypoxaemic respiratory failure requiring mechanical ventilation in the intensive care unit (ICU) are unknown, especially for patients not meeting criteria for acute respiratory distress syndrome (ARDS). An international, multicentre, prospective cohort study of patients presenting with hypoxaemia early in the course of mechanical ventilation, conducted during four consecutive weeks in the winter of 2014 in 459 ICUs from 50 countries (LUNG SAFE). Patients were enrolled with arterial oxygen tension/inspiratory oxygen fraction ratio ≤300 mmHg, new pulmonary infiltrates and need for mechanical ventilation with a positive end-expiratory pressure of ≥5 cmH 2 O. ICU prevalence, causes of hypoxaemia, hospital survival and factors associated with hospital mortality were measured. Patients with unilateral versus bilateral opacities were compared. 12 906 critically ill patients received mechanical ventilation and 34.9% with hypoxaemia and new infiltrates were enrolled, separated into ARDS (69.0%), unilateral infiltrate (22.7%) and congestive heart failure (CHF 8.2%). The global hospital mortality was 38.6%. CHF patients had a mortality comparable to ARDS (44.1% versus 40.4%). Patients with unilateral-infiltrate had lower unadjusted mortality, but similar adjusted mortality compared to those with ARDS. The number of quadrants on chest imaging was associated with an increased risk of death. There was no difference in mortality comparing patients with unilateral-infiltrate and ARDS with only two quadrants involved. More than one-third of patients receiving mechanical ventilation have hypoxaemia and new infiltrates with a hospital mortality of 38.6%. Survival is dependent on the degree of pulmonary involvement whether or not ARDS criteria are reached.

Effective Delivery of PEGylated siRNA-Containing Lipoplexes to Extraperitoneal Tumours following Intraperitoneal Administration

Publisher: Hindawi Limited

Date: 07-06-2011

DOI: 10.1155/2011/192562

Abstract: Intraperitoneal (i.p.) administration of small interfering RNA (siRNA) has, to date, shown promise in treating tumours located within the peritoneal cavity. The ability of these siRNA molecules to reach extraperitoneal tumours following i.p. administration is, however, yet to be investigated. Here, we examined the impact of PEGylation on the biodistribution of i.p. administered nucleic acids-containing lipoplexes. We showed that in contrast to non-PEGylated liposomes, PEGylated liposomes can deliver siRNA efficiently to extraperitoneal tumours following i.p. administration, resulting in a 45% reduction in tumour size when the oncogene-targeted siRNA was used. This difference was likely contributed by the decreased uptake of PEGylated lipoplexes in the first-pass organs, and, in particular, we observed a 10-fold decrease in the macrophage uptake of these particles compared to non-PEGylated counterparts. Overall, our results indicated the potential of using PEGylated liposomes to deliver siRNA for the treatment of i.p. localized cancer with coexisting extraperitoneal metastasis.

Complete protection by a single-dose skin patch–delivered SARS-CoV-2 spike vaccine

Publisher: American Association for the Advancement of Science (AAAS)

Date: 29-10-2021

DOI: 10.1126/SCIADV.ABJ8065

Abstract: Delivery of a SARS-CoV-2 vaccine to the skin of mice via microarray patches provides complete protection from challenge.

Intranasal delivery of lipid nanoparticle encapsulated SARS-CoV-2 and RSV-targeting siRNAs reduces lung infection

Publisher: Cold Spring Harbor Laboratory

Date: 26-07-2022

DOI: 10.1101/2022.07.25.501479

Abstract: RNA interference (RNAi) is an emerging and promising therapy for a wide range of respiratory viral infections. This highly specific suppression can be achieved by the introduction of short-interfering RNA (siRNA) into mammalian systems, resulting in the effective reduction of viral load. Unfortunately, this has been hindered by the lack of a good delivery system, especially via the intranasal (IN) route. Here, we have developed an IN siRNA encapsulated lipid nanoparticle (LNP) in vivo delivery system that is highly efficient at targeting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and respiratory syncytial virus (RSV) in infected mouse lungs. Importantly, IN siRNA delivery without the aid of LNPs abolishes anti-SARS-CoV-2 activity in vivo . Our approach using LNPs as the delivery vehicle overcomes the significant barriers seen with IN delivery of siRNA therapeutics and is a significant advancement in our ability to delivery siRNAs. The studies presented here demonstrates an attractive alternate therapeutic delivery strategy for the treatment of both future and emerging respiratory viral diseases.

Development of an intravaginal ring for the topical delivery of Aurora kinase A inhibitor, MLN8237

Publisher: Public Library of Science (PLoS)

Date: 27-11-2019

DOI: 10.1371/JOURNAL.PONE.0225774

Self-Catalyzed Degradation of Linear Cationic Poly(2-dimethylaminoethyl acrylate) in Water

Publisher: American Chemical Society (ACS)

Date: 16-04-2011

DOI: 10.1021/BM200219E

Abstract: We report the synthesis of a low cytotoxic polycation that maintains its cationic strength for well over a few hours then degrades into a benign polymer with nontoxic byproducts. Well-defined poly(2-dimethylaminoethyl acrylate) (PDMAEA) of five different molecular weights prepared using reversible addition-fragmentation chain transfer (RAFT) "living" radical polymerization degrades slowly over 200 h (∼8 days). As this degradation is independent of both the polymer molecular weight and solution pH, it is consistent with a self-catalyzed hydrolysis process without the need for an internal or external degradation trigger. In addition, the polymer shows little or no cytotoxicity to HeLa cells for the molecular weights of 5600 and below, even at very high polymer concentrations (equivalent to a nitrogen hosphorus ratio of 200). Therefore, at sufficiently low molecular weights this polymer has the essential attributes (i.e., ability to autodegradable and low toxicity) for a delivery carrier suitable for DNA or siRNA.

Complete protection by a single dose skin patch delivered SARS-CoV-2 spike vaccine

Publisher: Cold Spring Harbor Laboratory

Date: 31-05-2021

DOI: 10.1101/2021.05.30.446357

Abstract: SARS-CoV-2 has infected over 160 million people and resulted in more than 3.3 million deaths, and we still face many challenges in the rollout of vaccines. Here, we use the high-density microarray patch to deliver a SARS-CoV-2 spike subunit vaccine directly to the skin. We show the vaccine, dry-coated on the patch is thermostable, and delivery of spike via HD-MAP induced greater cellular and antibody immune responses, with serum able to potently neutralize clinically relevant isolates including those from the B.1.1.7 and B.1.351 lineages. Finally, a single dose of HD-MAP-delivered spike provided complete protection from a lethal virus challenge, demonstrating that HD-MAP delivery of a SARS-CoV-2 vaccine is superior to traditional needle-and-syringe vaccination and has the potential to greatly impact the ongoing COVID-19 pandemic.

High prevalence of human papillomaviruses in fresh frozen breast cancer samples

Publisher: Wiley

Date: 19-10-2011

DOI: 10.1002/JMV.22223

Abstract: While the etiology of breast cancer remains enigmatic, some recent reports have examined the role of human papillomavirus (HPV) in breast carcinogenesis. The purpose of this study was to determine the prevalence of HPV in breast cancer tissue using PCR analysis and sequencing. Fifty-four (54) fresh frozen breast cancers s les that were removed from a cohort of breast cancer patients were analyzed. S les were tested for HPV using comprehensive PCR primers, and in situ hybridization was performed on paraffin embedded tissue sections. Findings were correlated with clinical and pathological characteristics. The HPV DNA prevalence in the breast cancer s les was 50% (27/54) with sequence analysis indicating all cases to be positive for HPV-18 type. While HPV patients were slightly younger, no correlation was noted for menopausal status or family history. HPV positive tumors were smaller with earlier T staging and demonstrated lesser nodal involvement compared to HPV negative cancers. In situ hybridization analyses proved negative. The high proportion of HPV positive breast cancers detected in this series using fresh frozen tissues cannot be dismissed, however the role of HPV in breast carcinogenesis remains unclear and may ultimately be ascertained by monitoring future breast cancer incidence amongst women vaccinated against high risk HPV types.

Cervical cancer cell lines are sensitive to sub-erythemal UV exposure

Publisher: Elsevier BV

Date: 03-2019

DOI: 10.1016/J.GENE.2018.11.079

Abstract: High risk human papillomavirus (HPV) infections are the causative agent in virtually every cervical cancer as well as a host of other anogenital and oropharyngeal malignancies. These viruses must activate DNA repair pathways to facilitate their replication, while avoiding the cell cycle arrest and apoptosis that can accompany DNA damage. HPV oncoproteins facilitate each of these goals, but also reduce genome stability. Our data dissect the cytotoxic and cytoprotective characteristics of HPV oncogenes in cervical cancer cells. These data show that while the transformation of keratinocytes by HPV oncogene leaves these cells more sensitive to UV, the oncogenes also protect against UV-induced apoptosis. Cisplatin and UV resistant cervical cancer cell lines were generated and probed for their sensitivity to genotoxic agents. Cervical cancer cells can acquire resistance to one DNA crosslinking agent (UV or cisplatin) without gaining broad tolerance of crosslinked DNA. Further, cisplatin resistance may or may not result in sensitivity to PARP1 inhibition.

Potential strategies utilised by papillomavirus to evade host immunity

Publisher: Wiley

Date: 04-1999

DOI: 10.1111/J.1600-065X.1999.TB01288.X

Abstract: The co-evolution of papillomaviruses (PV) and their mammalian hosts has produced mechanisms by which PV might avoid specific and non-specific host immune responses. Low level expression of PV proteins in infected basal epithelial cells, together with an absence of inflammation and of virus-induced cell lysis, restricts the opportunity for effective PV protein presentation to immunocytes by dendritic cells. Additionally, PV early proteins, by a range of mechanisms, may restrict the efficacy of antigen presentation by these cells. Should an immune response be induced to PV antigens, resting keratinocytes (KC) appear resistant to interferon-gamma-enhanced mechanisms of cytotoxic T-lymphocyte (CTL)-mediated lysis, and expression of PV antigens by resting KC can tolerise PV-specific CTL. Thus, KC, in the absence of inflammation, may represent an immunologically privileged site for PV infection. Together, these mechanisms play a part in allowing persistence of PV-induced proliferative skin lesions for months to years, even in immunocompetent hosts.

Shared and Persistent Asymptomatic Cutaneous Human Papillomavirus Infections in Healthy Skin

Publisher: Wiley

Date: 08-2009

DOI: 10.1002/JMV.21529

Abstract: Cutaneous human papillomavirus (HPV) types are commonly found in normal skin, and some of them have been suspected to play a role in the development of non-melanoma skin cancer. This present study is ided into three sections, the aims of this study were to examine if certain HPV-types persist over time and if HPV-types are shared within families. From the first part of the study, swab s les from foreheads were collected for three longitudinal studies from one family with a newborn baby. Five specific HPV-types were isolated from the family with a newborn, with HPV-5 and FA67 being found at various time points and prevalence rates in all four members of the family. Part 2 consisted of a followed up study from two families with a 6 years interval. Six of the family members were found to have at least one of the HPV-types identified in the family 6 years earlier. Many of the HPV-types identified were shared within the families studied. Part 3 of this study involved weekly s les from four healthy females for 4 months. Among the four healthy in iduals, 11%, 65%, and 56% of the weekly s les were HPV-DNA positive with one in idual HPV-negative. All specimens were tested for HPV-DNA by PCR using the broad range HPV-type primer pair FAP59/64. The positive s les were HPV-type determined by cloning and sequencing. Specific cutaneous HPV-types persist over long periods of time in healthy skin in most in iduals investigated and certain HPVs are shared between family members.

Multiple signal pathways in the leukemogenesis and therapeutic implications - Commentary

Publisher: Elsevier BV

Date: 12-2007

DOI: 10.1016/J.LEUKRES.2007.04.006

Silencing of E6/E7 Expression in Cervical Cancer Stem-Like Cells

Publisher: Springer New York

Date: 12-10-2015

DOI: 10.1007/978-1-4939-2013-6_12

Abstract: Accumulating evidence supports the concept that cancer stem cells (CSCs) are responsible for the tumor recurrence and metastasis, the two major causes of cancer-related death. Therefore, CSC-targeted cancer therapy is important for the future development of more effective and advanced cancer therapy. One of the approaches is to specifically silence oncogene expression in CSCs and inhibit their growth. The significance of this approach is its specificity and ability to avoid multi-drug resistance of CSCs. In this chapter, we will describe a method of silencing HPV oncogenes E6/E7 in human cervical CSCs using HeLa cells as a model system.

SiRNA and shRNA as anticancer agents in a cervical cancer model

Publisher: Humana Press

Date: 2008

DOI: 10.1007/978-1-59745-191-8_12

Abstract: We describe the protocols of using siRNAs, or shRNAs delivered by a lentiviral vector, as a means to silence cancer-causing genes. We use cervical cancer as a model to demonstrate the inhibition of the human papillomavirus (HPV) oncogenes E6 and E7 in cervical cancer cells by RNAi and inhibition of the cell growth in vitro and tumor growth in mouse models. The protocols include methods on siRNA and shRNA design, production of lentiviral-vectored shRNA, transfection or transduction of cervical cancer cells with siRNA or shRNA, and detection of the inhibitory effects of siRNA or shRNA both in vitro and in vitro.

Statement in Support of: Virology under the Microscope-a Call for Rational Discourse

Publisher: American Society for Microbiology

Date: 31-05-2023

DOI: 10.1128/JVI.00451-23

Self-Catalyzed Degradable Cationic Polymer for Release of DNA

Publisher: American Chemical Society (ACS)

Date: 31-08-2011

DOI: 10.1021/BM2007423

Abstract: The controlled release of siRNA or DNA complexes from cationic polymers is an important parameter design in polymer-based delivery carriers. In this work, we use the self-catalyzed degradable poly(2-dimethylaminoethyl acrylate) (PDMAEA) to strongly bind, protect, and then release oligo DNA (a mimic for siRNA) without the need for a cellular or external trigger. This self-catalyzed hydrolysis process of PDMAEA forms poly(acrylic acid) and N,N'-dimethylamino ethyl ethanol, both of which have little or no toxicity to cells, and offers the advantage of little or no toxicity to off-target cells and tissues. We found that PDMAEA makes an ideal component of a delivery carrier by protecting the oligo DNA for a sufficiently long period of time to transfect most cells (80% transfection after 4 h) and then has the capacity to release the DNA inside the cells after ~10 h. The PDMAEA formed large nanoparticle complexes with oligo DNA of ~400 nm that protected the oligo DNA from DNase in serum. The nanoparticle complexes showed no toxicity for all molecular weights at a nitrogen hosphorus (N/P) ratio of 10. Only the higher molecular weight polymers at very high N/P ratios of 200 showed significant levels of cytotoxicity. These attributes make PDMAEA a promising candidate as a component in the design of a gene delivery carrier without the concern about accumulated toxicity of nanoparticles in the human body after multiadministration, an issue that has become increasingly more important.

Polyacrylate-Based Delivery System for Self-adjuvanting Anticancer Peptide Vaccine

Publisher: American Chemical Society (ACS)

Date: 22-12-2014

DOI: 10.1021/JM501514H

Abstract: Vaccination can provide a safe alternative to chemotherapy by using the body's natural defense mechanisms to create a potent immune response against tumor cells. Peptide-based therapeutic vaccines against human papillomavirus (HPV)-related cancers are usually designed to elicit cytotoxic T cell responses by targeting the HPV-16 E7 oncoprotein. However, peptides alone lack immunogenicity, and an additional adjuvant or external delivery system is required. In this study, we developed new polymer-peptide conjugates to create an efficient self-adjuvanting system for peptide-based therapeutic vaccines. These conjugates reduced tumor growth and eradicated E7-positive TC-1 tumors in mice after a "single shot" immunization, without the help from an external adjuvant. The new conjugates had a significantly higher anticancer efficacy than the antigen formulated with a commercial adjuvant. Furthermore, the polymer-peptide conjugates were promptly taken up by antigen presenting cells, including dendritic cells and macrophages, and efficiently activated CD4(+) T-helper cells and CD8(+) cytotoxic T lymphocyte cells.

A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19.

Publisher: Elsevier BV

Date: 07-2021

DOI: 10.1016/J.YMTHE.2021.05.004

Infectious clones produce sars-cov-2 that causes severe pulmonary disease in infected k18-human ace2 mice

Publisher: American Society for Microbiology

Date: 27-04-2021

DOI: 10.1128/MBIO.00819-21

Abstract: To develop COVID-19 countermeasures, powerful research tools are essential. We produced a SARS-COV-2 reverse genetic (RG) infectious clone toolkit that will benefit a variety of investigations.

Short interfering RNA induced generation and translation of stable 5' mRNA cleavage intermediates.

Publisher: Elsevier BV

Date: 08-2016

DOI: 10.1016/J.BBAGRM.2016.06.005

Abstract: Sequence-specific degradation of homologous mRNA is the main mechanism by which short-interfering RNAs (siRNAs) suppress gene expression. Generally, it is assumed that the mRNA fragments resulting from Ago2 cleavage are rapidly degraded, thus making the transcript translation-incompetent. However, the molecular mechanisms involved in the post-cleavage mRNA decay are not completely understood and the fate of cleavage intermediates has been poorly studied. Using specific siRNAs and short-hairpin RNAs (shRNAs) we show that the 5' and 3' mRNA cleavage fragments of human papilloma virus type 16 (HPV-16) E6/7 mRNA, over-expressed in cervical malignancies, are unevenly degraded. Intriguingly, the 5' mRNA fragment was more abundant and displayed a greater stability than the corresponding 3' mRNA fragment in RNAi-treated cells. Further analysis revealed that the 5' mRNA fragment was polysome-associated, indicating its active translation, and this was further confirmed by using tagged E7 protein to show that C-terminally truncated proteins were produced in treated cells. Overall, our findings provide new insight into the degradation of siRNA-targeted transcripts and show that RNAi can alter protein expression in cells as a result of preferential stabilization and translation of the 5' cleavage fragment. These results challenge the current model of siRNA-mediated RNAi and provide a significant step forward towards understanding non-canonical pathways of siRNA gene silencing.

Human PKR transfected into murine cells stimulates expression of genes under control of the HIV1 or HTLV-1 LTR (vol 214, pg 653, 1995)

Publisher: Elsevier BV

Date: 07-1998

DOI: 10.1006/VIRO.1998.9277

Co-administration of Oral Cholera Vaccine With Oral Polio Vaccine Among Bangladeshi Young Children: A Randomized Controlled Open Label Trial to Assess Interference

Publisher: Oxford University Press (OUP)

Date: 22-09-2022

DOI: 10.1093/CID/CIAC782

Abstract: Cholera remains a public health threat for low- and middle-income countries, particularly in Asia and Africa. Shanchol™, an inactivated oral cholera vaccine (OCV) is currently in use globally. OCV and oral poliovirus vaccines (OPV) could be administered concomitantly, but the immunogenicity and safety of coadministration among children aged 1–3 years is unknown. We undertook an open-label, randomized, controlled, inequality trial in Dhaka city, Bangladesh. Healthy children aged 1–3 years were randomly assigned to 1 of 3 groups: bivalent OPV (bOPV)-alone, OCV-alone, or combined bOPV + OCV and received vaccines on the day of enrollment and 28 days later. Blood s les were collected on the day of enrollment, day 28, and day 56. Serum poliovirus neutralizing antibodies and vibriocidal antibodies against Vibrio cholerae O1 were assessed using microneutralization assays. A total of 579 children aged 1‒3 years were recruited, 193 children per group. More than 90% of the children completed visits at day 56. Few adverse events following immunization were recorded and were equivalent among study arms. On day 28, 60% (90% confidence interval: 53%–67%) and 54% (46%–61%) of participants with co-administration of bOPV + OCV responded to polioviruses type 1 and 3, respectively, compared to 55% (47%–62%) and 46% (38%–53%) in the bOPV-only group. Additionally, & % of participants showed a ≥4-fold increase in vibriocidal antibody titer responses on day 28, comparable to the responses observed in OCV-only arm. Co-administration of bOPV and OCV is safe and effective in children aged 1–3 years and can be cost-beneficial. ClinicalTrials.gov (NCT03581734).

Toward Typhoid Fever Elimination

Publisher: Elsevier BV

Date: 06-2022

DOI: 10.1016/J.IJID.2022.03.036

Abstract: Salmonella enterica serotype Typhi (S Typhi) causes typhoid fever and is responsible for an estimated 9 million cases and 110,000 deaths globally per annum. Typhoid fever is endemic in areas where water, sanitation, and hygiene (WaSH) infrastructure is poor. Serious complications develop in approximately 10%-15% of patients if left untreated, and this is driven by inadequate diagnostic methods and the high burden of antibiotic-resistant strains, complicating clinical management and ultimately prognosis. Asymptomatic chronic carriers, in addition to acutely infected patients, contribute to continued transmission through the shedding of the organism in the feces. The high morbidity and mortality of typhoid fever in low- and middle-income countries reinforce the need for an integrated control approach, which may ultimately lead to elimination of the disease in the 21

Statement in Support of: “Virology under the Microscope—a Call for Rational Discourse”

Publisher: American Society for Microbiology

Date: 22-06-2023

DOI: 10.1128/MSPHERE.00165-23

Weaning from mechanical ventilation in intensive care units across 50 countries (WEAN SAFE): a multicentre, prospective, observational cohort study

Publisher: Elsevier BV

Date: 05-2023

DOI: 10.1016/S2213-2600(22)00449-0

Evoking potent RNAi response using novel 2 '-OMe-phosphorodithioated modified siRNAs

Publisher: American Association for Cancer Research (AACR)

Date: 10-2014

DOI: 10.1158/1538-7445.AM2014-1406

Abstract: Improving small interfering RNA (siRNA) efficacy in target cell populations remains a critical challenge to bringing siRNA therapy into the clinic. There is currently an unmet need to develop a reliable strategy to globally enhance siRNA stability and potency. Here, we report a novel chemical modification, consisting of phosphorodithioate (PS2) and 2′-O-Methyl (2′-OMe) MePS2 on a single nucleotide, that significantly enhances potency and resistance to nuclease degradation for a variety of siRNA sequences. We show a 3.5-fold improvement in gene silencing in tumors following systemic delivery of MePS2-modified siRNAs using DOPC nanoliposomes compared to unmodified counterparts. We found that this enhanced potency stems from an unforeseen increase in loading of siRNAs to the RNA-induced silencing complex (RISC), likely due to the unique interaction between 2′-OMe and PS2 moieties. We subsequently demonstrate the therapeutic utility of MePS2 siRNAs in orthotopic mouse models of chemoresistant ovarian cancer, focusing on targeting GRAM domain containing 1B (GRAMD1B), a protein whose role in taxane resistance is first reported here. Efficient silencing of GRAMD1B (& %) was achieved in tumors following systemic delivery of these MePS2-modified siRNAs and a synergistic anti-tumor effect was observed when combined with paclitaxel treatment. Given that limited success has been achieved thus far toward broadly enhancing siRNA potency with chemically modified siRNAs, our finding represents an important step forward in bringing siRNA therapeutics into the clinic. Citation Format: Sherry Wu, Xianbin Yang, Martin Egli, Kshipra Ghaupure, Hiroto Hatakeyama, Michael McGuire, Rajesha Rupaimoole, Takahito Miyake, Morgan Taylor, Sunila Pradeep, Archana Nagaraja, Malgorzata Sierant, Richa Singhania, Cristian Rodriguez-Aguayo, Nigel McMillan, Gabriel Lopez-Berestein, Prahlad Ram, Barbara Nawrot, Anil K. Sood. Evoking potent RNAi response using novel 2′-OMe-phosphorodithioated modified siRNAs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research 2014 Apr 5-9 San Diego, CA. Philadelphia (PA): AACR Cancer Res 2014 (19 Suppl):Abstract nr 1406. doi:10.1158/1538-7445.AM2014-1406

Prevalence and types of high-risk human papillomaviruses in head and neck cancers from Bangladesh

Publisher: Springer Science and Business Media LLC

Date: 25-11-2017

DOI: 10.1186/S12885-017-3789-0

Systemic Delivery of CRISPR/Cas9 Targeting HPV Oncogenes Is Effective at Eliminating Established Tumors

Publisher: Elsevier BV

Date: 12-2019

DOI: 10.1016/J.YMTHE.2019.08.012

Human Papillomavirus DNA Detected in Peripheral Blood Samples From Healthy Australian Male Blood Donors

Publisher: Wiley

Date: 20-08-2009

DOI: 10.1002/JMV.21592

Abstract: Recent studies have shown that human papillomavirus (HPV) DNA can be found in circulating blood, including peripheral blood mononuclear cells (PBMCs), sera, plasma, and arterial cord blood. In light of these findings, DNA extracted from PBMCs from healthy blood donors were examined in order to determine how common HPV DNA is in blood of healthy in iduals. Blood s les were collected from 180 healthy male blood donors (18-76 years old) through the Australian Red Cross Blood Services. Genomic DNA was extracted and specimens were tested for HPV DNA by PCR using a broad range primer pair. Positive s les were HPV-type determined by cloning and sequencing. HPV DNA was found in 8.3% (15/180) of the blood donors. A wide variety of different HPV types were isolated from the PBMCs belonging to the cutaneous beta and gamma papillomavirus genera and mucosal alpha papillomaviruses. High-risk HPV types that are linked to cancer development were detected in 1.7% (3/180) of the PBMCs. Blood was also collected from a healthy HPV-positive 44-year-old male on four different occasions in order to determine which blood cell fractions harbor HPV. PBMCs treated with trypsin were negative for HPV, while non-trypsinized PBMCs were HPV-positive. This suggests that the HPV in blood is attached to the outside of blood cells via a protein-containing moiety. HPV was also isolated in the B cells, dendritic cells, NK cells, and neutrophils. To conclude, HPV present in PBMCs could represent a reservoir of virus and a potential new route of transmission.

Dendrosome-based delivery of siRNA against E6 and E7 oncogenes in cervical cancer

Publisher: Elsevier BV

Date: 06-2010

DOI: 10.1016/J.NANO.2009.12.001

Abstract: Although small interfering RNA (siRNA) treatment holds great promise for the treatment of cancers, the field has been held back by the availability of suitable delivery vehicles. For cervical cancer the E6 and E7 oncogenes are ideal siRNA targets for treatment. The purpose of the present study was to explore the potential of dendrosomes for the delivery of siRNA targeting E6 and E7 proteins of cervical cancer cells in vitro. Optimization of dendrimer generation and nitrogen-to-phosphate (N/P) ratio was carried out using dendrimer-fluorescein isothiocyanate oligo complexes. The optimized N/P ratios were used in formulating complexes between dendrimers and siRNA targeting green fluorescence protein (siGFP). Although formulation 4D100 (dendrimer-siRNA complex) displayed the highest GFP knockdown, it was also found to be highly toxic to cells. In the final formulation 4D100 was encapsulated into dendrosomes so as to mask these toxic effects. The optimized dendrosomal formulation (DF), DF3 was found to possess a siGFP-entrapment efficiency of 49.76% +/- 1.62%, vesicle size of 154 +/- 1.73 nm, and zeta potential of +3.21 +/- 0.07 mV. The GFP knockdown efficiency of DF3 (dendrosome) was found to be almost identical to that of 4D100, but the former was completely nontoxic to the cells. DF3 containing siRNA against E6 and E7 was found to knock down the target genes considerably, as compared with the other formulations tested. Our results imply that dendrosomes hold potential for the delivery of siRNA and that a suitable targeting strategy could be useful for applications in vivo. siRNA treatment holds great promise for the treatment of cancers, but overall, the availability of suitable delivery vehicles remains a major issue. The purpose of this study was to explore the potential of dendrosomes for the delivery of siRNA targeting specific proteins in cervical cancer cells in vitro. The results suggest that dendrosomes hold potential for the delivery of siRNA and a suitable targeting strategy could be useful for applications in vivo.

Both treated and untreated tumors are eliminated by short hairpin RNA-based induction of target-specific immune responses

Publisher: Proceedings of the National Academy of Sciences

Date: 19-05-2009

DOI: 10.1073/PNAS.0812085106

Abstract: RNA interference (RNAi) for cancer treatment relies on the ability to directly kill cancer cells via down-regulation of target genes, but issues of delivery and efficacy have limited clinical adoption. Furthermore, current studies using immune-deficient animal models disregard potential interactions with the adaptive immune system. It has previously been observed that certain viral antigens appear to be more rapidly presented to the immune system than normal proteins due to the production of defective ribosomal products by the virus. Given that RNAi could potentially result in the generation of truncated mRNAs, we wondered whether a similar mechanism of immune presentation of a target gene was possible. Here we show that RNAi-cleaved mRNAs can be translated into incomplete protein, and if cleavage was downstream of cytotoxic T cell epitopes, resulted in increased presentation of target protein and the generation of a tumor-protective immune response. We show that mice inoculated with tumor cells treated with such short hairpin RNAs (shRNAs) were protected from subsequent challenge with untreated tumors. However, protection was only found if shRNAs were targeted downstream of the dominant cytotoxic T cell (CTL) epitope. Our work suggests that RNAi can alter immunity to targets and shows that not all tumor cells require direct RNAi exposure for treatment to be effective in vivo, pointing the way to a new class of RNAi-based therapy.

The human papillomavirus E7 protein is able to inhibit the antiviral and anti-growth functions of interferon-alpha

Publisher: Elsevier BV

Date: 11-2000

DOI: 10.1006/VIRO.2000.0584

Inhibition of Aurora A and Aurora B Is Required for the Sensitivity of HPV-Driven Cervical Cancers to Aurora Kinase Inhibitors.

Publisher: American Association for Cancer Research (AACR)

Date: 09-2017

DOI: 10.1158/1535-7163.MCT-17-0159

Abstract: The activity and efficacy of Aurora inhibitors have been reported in a wide range of cancer types. The most prominent Aurora inhibitor is alisertib, an investigational Aurora inhibitor that has been the subject of more than 30 clinical trials. Alisertib has inhibitory activity against both Aurora A and B, although it is considered to be primarily an Aurora A inhibitor in vivo. Here, we show that alisertib inhibits both Aurora A and B in vivo in preclinical models of HPV-driven cervical cancer, and that it is the inhibition of Aurora A and B that provides the selectivity and efficacy of this drug in vivo in this disease setting. We also present formal evidence that alisertib requires progression through mitosis for its efficacy, and that it is unlikely to combine with drugs that promote a G2 DNA damage checkpoint response. This work demonstrates that inhibition of Aurora A and B is required for effective control of HPV-driven cancers by Aurora kinase inhibitors. Mol Cancer Ther 16(9) 1934–41. ©2017 AACR.

The human papillomavirus type 16 E7 protein binds human interferon regulatory factor-9 via a novel PEST domain required for transformation

Publisher: Mary Ann Liebert Inc

Date: 07-2006

DOI: 10.1089/JIR.2006.26.455

Abstract: It is critical that viruses are able to avoid the antiviral activities of interferon (IFN). We have shown previously that the human papillomavirus (HPV) is able to avoid IFN-alpha via interaction of the HPV-16 E7 protein with IFN regulatory factor-9 (IRF-9). Here, we investigated the details of the interaction using HPV-16 E7 peptide mapping to show that IRF-9 binds HPV-16 E7 in a domain encompassing amino acids 25-36. A closer examination of this region indicates this is a novel proline, glutamate, serine, and threonine-rich (PEST) domain, with a PEST score of 8.74. We have also mapped the region of interaction within IRF-9 and found that amino acids 354-393 play an important role in binding to HPV-16 E7. This region of IRF-9 encompasses the IRF association domain (IAD), a region important for protein-protein interaction central to IRF function. Finally, we used alanine-scanning mutagenesis to determine if E7-IRF-9 interaction was important for E7-mediated cellular transformation and found that the HPV-16 E7 mutants Y25A, E26A, S31A, S32A, and E35A, but not L28A and N29A, caused loss of transformation ability. Preliminary data suggest loss of IRF-9 interaction with E7 mutants correlated with transformation. Our work suggests E7-IRF-9 interaction is important for the transforming ability of HPV-16 E7 and that HPV-16 E7 may interact with other IRF proteins that have IAD domains.

COMPARISON OF THE GENOMES OF 2 SYMPATRIC IRIDESCENT VIRUSES (TYPE-9 AND TYPE-16)

Publisher: Springer Science and Business Media LLC

Date: 09-1990

DOI: 10.1007/BF01310758

LOCALIZATION OF THE HUMAN INTERFERON-INDUCED, DS-RNA ACTIVATED P68 KINASE GENE (PRKR) TO CHROMOSOME-2P21-P22

Publisher: Elsevier BV

Date: 06-1993

DOI: 10.1006/GENO.1993.1263

Abstract: The interferon-induced dsRNA-activated protein kinase (PRKR) belongs to a subclass of serine/threonine kinases, involved in the regulation of protein synthesis by phosphorylation of the alpha subunit of initiation factor eIF2. Somatic cell hybrids segregating human chromosomes were used to assign this kinase to human chromosome 2. Fluorescence in situ hybridization confirmed this assignment and further localized the gene (PRKR) to the boundary region of bands p21 and 22.

Phosphorylation of HIV Tat by PKR increases interaction with TAR RNA and enhances transcription

Publisher: Springer Science and Business Media LLC

Date: 2005

DOI: 10.1186/1743-422X-2-17

A phase I/II study to evaluate safety, tolerability and immunogenicity of Hillchol®, an inactivated single Hikojima strain based oral cholera vaccine, in a sequentially age descending population in Ba

Publisher: Elsevier BV

Date: 07-2021

DOI: 10.1016/J.VACCINE.2021.06.069

Analysis of a hit‐and‐run tumor model by HPV in oropharyngeal cancers

Publisher: Wiley

Date: 04-11-2023

DOI: 10.1002/JMV.28260

Abstract: Several viruses are known to be associated with the development of certain cancers, including human papilloma virus (HPV), an established causative agent for a range of anogenital and head and neck cancers. However, the causality has been based on the presence of the virus, or its genetic material, in the s led tumors. We have long wondered if viruses cause cancer via a “hit and run” mechanism such that they are no longer present in the resulting tumors. Here, we hypothesize that the absence of viral genes from the tumor does not necessarily exclude the viral aetiology. To test this, we used an HPV‐driven oropharyngeal cancer (OPC) tumor model and CRISPR to delete the viral oncogene, E7. Indeed, the genetic removal of HPV E7 oncogene eliminates tumors in vivo. Remarkably, E7 deleted tumors recurred over time and develop new mutations not previously seen in HPV + OPC tumors. Importantly, a number of these new mutations are found to be already present in HPV − OPC tumors.

Development of novel lipidic particles for siRNA delivery that are highly effective after 12 months storage

Publisher: Public Library of Science (PLoS)

Date: 08-02-2019

DOI: 10.1371/JOURNAL.PONE.0211954

Passive and pH-gradient loading of doxycycline into nanoliposomes using modified freeze-drying of a monophase solution method for enhanced antibacterial activity

Publisher: Springer Science and Business Media LLC

Date: 30-01-2022

DOI: 10.1007/S11696-021-02036-5

BPV1 E2 Protein Enhances Packaging of Full-Length Plasmid DNA in BPV1 Pseudovirions

Publisher: Elsevier BV

Date: 07-2000

DOI: 10.1006/VIRO.2000.0348

A COMPARISON OF THE STRUCTURAL POLYPEPTIDES OF 3 IRIDESCENT VIRUSES (TYPE-6, TYPE-9, AND TYPE-16) AND THE MAPPING OF THE DNA REGION CODING FOR THEIR MAJOR CAPSID POLYPEPTIDES

Publisher: Springer Science and Business Media LLC

Date: 03-1992

DOI: 10.1007/BF01317153

Abstract: The aim of the present study was to establish whether in healthy human subjects the actions of group I muscle afferents arising from the same spinal segments as the soleus innervation (e.g., common peroneal nerve CPN) or from more proximal spinal segments (femoral nerve FN) on the soleus H-reflex are modified by changes in hip position. Control and conditioned soleus H-reflexes were elicited and recorded via conventional methods. In seated subjects, CPN and FN stimulation resulted in similar effects to the soleus H-reflex to that previously reported in healthy subjects. However, during hip angle changes, CPN stimulation at the C-T interval of 2 ms resulted in soleus H-reflex depression only when the hip was flexed at 30 degrees , whereas with the hip flexed or extended at 10 degrees the H-reflex was facilitated. CPN stimulation delivered at 100 ms also induced soleus H-reflex facilitation regardless of the hip angle tested. The heteronymous reflex facilitation (conditioned H-reflex with FN stimulation) did not vary systematically with hip angle changes. These findings indicate that hip proprioceptors interact with spinal inhibitory interneurons to enhance spinal reflex excitability under static conditions. This neural switch might constitute an important feature of movement regulation in humans.

Lowering the siRNA delivery barrier: Alginate scaffolds and immune stimulation

Publisher: Jenny Stanford Publishing

Date: 27-03-2013

DOI: 10.1201/B14773

Retraction to “Dendrimer nanocarriers as versatile vectors in gene delivery” [Nanomedicine: NBM 2010;6:25–34]

Publisher: Elsevier BV

Date: 12-2010

DOI: 10.1016/J.NANO.2010.11.001

Molecular basis of pathogenesis, prognosis and therapy in chronic lymphocytic leukaemia

Publisher: Informa UK Limited

Date: 04-02-2008

DOI: 10.4161/CBT.7.2.5262

Abstract: B-cell chronic lymphocytic leukemia (CLL) is caused by the abnormal accumulation of non-functional B-cells in peripheral blood and bone marrow. However, the precise aetiology and mechanism of the disease are unclear. Recently, progress has been made in the identification of both the genetic deficiencies and environmental factors that may underlie CLL. This has provided some clues to the nature of the disease, but no definitive cures. Although treatment has increased remission time, at present the disease is not curable by conventional therapy. Further studies of the pathogenesis of CLL are needed, as are the development of suitable cell lines and animal models in which to study it. This review summarises the most recent progress in CLL with emphasis on molecular events and possible implications in therapy.

Will diverse Tat interactions lead to novel antiretroviral drug targets?

Publisher: Bentham Science Publishers Ltd.

Date: 12-2006

DOI: 10.2174/138945006779025338

Abstract: More than fifteen years following the description of Tat as a critical HIV gene expression regulatory protein, additional roles for Tat in HIV replication have been described, including reverse transcription. Tat achieves function through direct interaction with viral proteins, including reverse transcriptase, and numerous cellular proteins including cyclin T1, RNA polymerase II, protein kinase R (PKR), p300/CBP, and P/CAF. Despite our advanced knowledge of how Tat operates, this has not yet resulted in the discovery of effective agents capable of targeting various Tat functions. Nevertheless, Tat remains an attractive, virus-specific molecule and detailed understanding of specific protein interaction holds promise for future drug discovery.

Lipidic Systems for In Vivo siRNA Delivery

Publisher: Springer Science and Business Media LLC

Date: 09-09-2009

DOI: 10.1208/S12248-009-9140-1

Targeted Drug Delivery to the Virus-Infected Airway; Complications and Remedies

Publisher: Bentham Science Publishers Ltd.

Date: 10-03-2015

DOI: 10.2174/1567201811666140918114528

Abstract: Drug delivery to the airway and lower respiratory tract by aerosol inhalation has become a successful, non-invasive method of preventing and treating local disease of the lung. Consequently, it has been a promising route for clinical trials using highly specific and novel therapies to overcome viral pulmonary infection such as RNA interference, neutralising monoclonal antibodies and microparticle treatments. Yet despite this great potential, this form of delivery has proven somewhat ineffective due to airway remodeling, inflammation and mucus hypersecretion that results from viral symptoms in the respiratory tract. Here we review the research into the delivery technologies available as well as the types of therapeutics used for respiratory virus disease and examine how virus infection-induced airway inflammation modulates its success. We discuss the future of aerosol administration and present potential alternative methods for efficient drug delivery so as to improve postinfection virus control therapies.

Piperacillin Encapsulation in Nanoliposomes Using Modified Freeze-Drying of a Monophase Solution Method: Preparation, Characterization and In Vitro Antibacterial Activity

Publisher: Springer Science and Business Media LLC

Date: 06-05-2020

DOI: 10.1007/S00284-020-02008-0

Patient derived xenografts as models for head and neck cancer

Publisher: Elsevier BV

Date: 10-2018

DOI: 10.1016/J.CANLET.2018.07.023

Abstract: Translational cancer research has benefitted significantly from the generation of preclinical models that recapitulate the native tumour environment. While conventional cell models have contributed substantially to the current understanding of cancer biology and therapeutic development, a missing link between cell culture and their clinical applications is evident. Patient derived xenograft (PDX) models represent this missing link as they enable the examination of patient tumour tissue in a native environment without significantly affecting the cellular complexity, genomics, and stromal architecture of the neoplasms. The use of PDXs to model head and neck cancer (HNC) begets the development of novel therapeutics, increased understanding of tumorigenesis and the advent of personalised treatments cancer patients. There has been an increase in attempts to generate viable PDXs for HNCs in recent years. This concise review summarizes the current developments in the field of PDXs for HNCs.

RNA interference for the treatment of cancer

Publisher: Portico

Date: 2006

DOI: 10.1358/DNP.2006.19.6.985937

Abstract: RNA interference (RNAi) is the latest new technology in the field of genetic medicine in which specific genes can be turned off, or silenced, so as to affect a therapeutic outcome. It can be highly specific, works in the nanomolar range and is far more effective than the antisense approaches popular 10-15 years ago. Here we review the field and explore the potential role of RNAi in cancer therapy, highlighting recent progress and examining the hurdles that must be overcome before this promising technology is ready for clinical use.

Hyperactivating p53 in Human Papillomavirus-Driven Cancers: A Potential Therapeutic Intervention.

Publisher: Springer Science and Business Media LLC

Date: 05-04-2022

DOI: 10.1007/S40291-022-00583-5

Papillomavirus virus-like particles activate the PI3-kinase pathway via alpha-6 beta-4 integrin upon binding

Publisher: Elsevier BV

Date: 09-2006

DOI: 10.1016/J.VIROL.2006.05.002

Abstract: We have previously shown that human papillomavirus virus-like particles (VLPs) are able to activate the Ras/MAP kinase pathway. Ras can also elicit an anti-apoptotic signal via PI3-kinase so we investigated this further. Here we show that binding of VLPs from HPV types 6b, 18, 31, 35 and BPV1 results in activation of PI3-kinase. Activation was achieved by either L1 or L1/L2 VLPs and was dependent on both VLP-cell interaction and correct conformation of the virus particle. VLP-induced PI3-kinase activity resulted in efficient downstream signaling to Akt and consequent phosphorylation of FKHR and GSK3beta. We also present evidence that PV signaling is activated via the alpha6beta4 integrin. These data suggest that papillomaviruses use a common receptor that is able to signal through to Ras. Combined activation of the Ras/MAP kinase and PI3-kinase pathways may be beneficial for the virus by increasing cell numbers and producing an environment more conducive to infection.

The interplay between WASH practices and vaccination with oral cholera vaccines in protecting against cholera in urban Bangladesh: Reanalysis of a cluster-randomized trial

Publisher: Elsevier BV

Date: 03-2023

DOI: 10.1016/J.VACCINE.2023.02.054

Development of a novel method for formulating stable sirna-loaded lipid particles for in vivo use

Publisher: Springer Science and Business Media LLC

Date: 21-11-2009

DOI: 10.1007/S11095-008-9766-1

Abstract: A simple yet novel method was developed to prepare stable PEGylated siRNA-loaded lipid particles which are suitable for in vivo use. PEGylated siRNA-loaded lipid particles were formulated by hydration of a freeze-dried matrix. The effect of various formulation parameters on the size and homogeneity of resulting particles was studied. Particles prepared using this method were compared to those prepared using an established post-insertion procedure for the entrapment efficiency, stability, in vitro biological activity as well as in vivo biodistribution. Using this hydration method, a particle size of less than 200 nm can be obtained with high siRNA entrapment efficiency (>90%) and high gene-silencing efficiency. Following intravenous administration into mice, these particles achieved a similar degree of accumulation in subcutaneous tumours but displayed less liver uptake compared to the post-insertion formulations. Importantly, in contrast to post-insertion preparations, particles made by hydration method retained 100% of their gene-silencing efficiency after storage at room temperature for 1 month. This paper describes a simple method of formulating PEGylated siRNA-loaded lipid particles. Given the ease of preparation, long term stability and favourable characteristics for in vivo delivery, our work represents an advance in lipid formulation of siRNA for in vivo use.

Efficiency of delivery of DNA to cells by bovine papillomavirus type-1 L1/L2 pseudovirions

Publisher: Springer Science and Business Media LLC

Date: 07-2001

DOI: 10.1007/S002530100655

Abstract: To investigate the efficiency of encapsidation of plasmid by papillomavirus virus-like particles (PV VLPs), and the infectivity of the resultant PV pseudovirions, Cos-1 cells were transfected with an 8-kb plasmid incorporating a green fluorescent protein (GFP) reporter gene (pGSV), and infected with bovine PV (BPV-1) L1/L2 recombinant vaccinia virus to produce BPV1 pseudovirions. Approximately 1 in 1.5 x 10(4) of dense (1.35 g/ml) PV pseudovirions and 0.3 in 10(4) of less-dense (1.29 g/ml) pseudovirions packaged an intact pGSV plasmid. The majority (>75%) of packaged plasmids contained deletions, and the deletions affected all tested genes. After exposure of Cos-1 cells to BPV-1 pseudovirions at an MOI of 40,000:1, 6% of cells expressed GFP, giving a calculated efficiency of delivery of the pGSV plasmid, by pseudovirions which had packaged an intact plasmid, of approximately 5%. Plasmid delivery was not effected by purified pGSV plasmid, was blocked by antiserum against BPV-1, and was not blocked by DNase treatment of pseudovirions, confirming that delivery was mediated by DNA within the pseudovirion. We conclude that a major limitation to the use of PV pseudovirions as a gene delivery system is that intact plasmid DNA is not efficiently selected for packaging by VLPs in cell-based pseudovirions production systems.

Hyperbranched polymers as delivery vectors for oligonucleotides

Publisher: Wiley

Date: 05-04-2012

DOI: 10.1002/POLA.26055

Prophylactic intranasal administration of lipid nanoparticle formulated siRNAs reduce SARS-CoV-2 and RSV lung infection

Publisher: Elsevier BV

Date: 06-2023

DOI: 10.1016/J.JMII.2023.02.010

In vivo comparison of local versus systemic delivery of immunostimulating siRNA in HPV-driven tumours

Publisher: Wiley

Date: 12-11-2014

DOI: 10.1038/ICB.2013.75

Abstract: Small interfering RNAs (siRNAs) to inhibit oncogene expression and also to activate innate immune responses via Toll-like receptor (TLR) recognition have been shown to be beneficial as anti-cancer therapy in certain cancer models. In this study, we investigated the effects of local versus systemic delivery of such immune-stimulating Dicer-substrate siRNAs (IS-DsiRNAs) on a human papillomavirus (HPV)-driven tumour model. Localized siRNA delivery using intratumour injection of siRNA was able to increase siRNA delivery to the tumour compared with intravenous (IV) delivery and potently activated innate immune responses. However, IV injection remained the more effective delivery route for reducing tumour growth. Although IS-DsiRNAs activated innate immune cells and required interferon-α (IFNα) for full effect on tumour growth, we found that potent silencing siRNA acting independently of IFNα were overall more effective at inhibiting TC-1 tumour growth. Other published work utilising IS-siRNAs have been carried out on tumour models with low levels of major histocompatibility complex (MHC)-class 1, a target of natural killer cells that are potently activated by IS-siRNA. As TC-1 cells used in our study express high levels of MHC-class I, the addition of the immunostimulatory motifs may not be as beneficial in this particular tumour model. Our data suggest that selection of siRNA profile and delivery method based on tumour environment is crucial to developing siRNA-based therapies.

CCL2 and CXCL2 enhance survival of primary chronic lymphocytic leukemia cellsin vitro

Publisher: Informa UK Limited

Date: 19-04-2012

DOI: 10.3109/10428194.2012.672735

Abstract: Chronic lymphocytic leukemia (CLL) is predominantly a disease of accumulation rather than rapid proliferation. To date, no cell lines exist, as CLL cells undergo rapid apoptosis when cultured in vitro, suggesting that a favorable in vivo microenvironment is required. To identify survival signals we cultured primary CLL peripheral blood mononuclear cells (PBMCs) at high density, which has previously been shown to dramatically improve survival. Using antibody arrays we measured the level of 42 cytokines in culture supernatants and showed that inerleukin-6 (IL-6), IL-8, CXCL2 and CCL2 were highly up-regulated in culture. This is the first report to describe a role for CCL2 and CXCL2 in CLL cell survival. Importantly, CXCL2, IL-6 and IL-8 were significantly up-regulated in primary patient plasma. The addition of either CXCL2 or CCL2 enhanced CLL cell survival, while antibodies blocking these chemokines reduced survival. Co-culture of CLL cells and PBMC accessory cells separated by transwells provided a similar degree of survival protection compared to normal culture, whereas CLL cells cultured alone died rapidly. Interestingly, CCL2 and CXCL2 appeared to be produced by CLL cells but only when co-cultured with accessory cells. Thus, we speculate that accessory cells release soluble factors that promote the production of these pro-survival chemokines from CLL cells and physical interactions are not required. Our data support the concept that the CLL microenvironment is critical, and suggests that soluble factors are more important than physical interactions.

Synthetic lethal screen identifies Aurora A as a selective target in HPV driven cervical cancer

Publisher: Elsevier BV

Date: 11-2014

DOI: 10.1016/S0959-8049(14)70420-4

Intra-tumor injection of lentiviral-vector delivered shRNA targeting human papillomavirus E6 and E7 oncogenes reduces tumor growth in a xenograft cervical cancer model in mice

Publisher: Sciedu Press

Date: 02-08-2012

DOI: 10.5430/JST.V2N4P4

Combined synthetic and recombinant techniques for the development of lipoprotein-based, self-adjuvanting vaccines targeting human papillomavirus type-16 associated tumors

Publisher: Elsevier BV

Date: 12-2015

DOI: 10.1016/J.BMCL.2015.10.049

Abstract: Human papillomaviruses (HPVs) are associated with various cancers, with HPV16 linked to more than half of cervical cancer cases. Vaccines to prevent HPV infection and cancer development have proven effective, but are not useful in in iduals with prior HPV exposure. Treatment vaccines to eradicate or control HPV-associated lesions are therefore desirable for these patients. Herein we describe the development of a process to enable the production of semisynthetic vaccines based on the site-specific attachment of synthetic bacterial lipid analogs (e.g., Pam2Cys) to a non-oncogenic mutant HPV16 E7 protein to generate molecularly defined vaccines. Many cytotoxic lymphocyte (CTL) epitopes from E7 are delivered by this approach potentially ensuring that large numbers of immunized in iduals can generate CTLs to clear HPV infected cells. Delivery of this construct reduced the growth of HPV16-associated tumors in a TC1 mouse model, the effects of which were better than the potent CTL epitope HPV16 E7(44-57) administered with Montanide ISA51 adjuvant.

The Therapeutic Potential of CRISPR/Cas9 Systems in Oncogene-Addicted Cancer Types: Virally Driven Cancers as a Model System

Publisher: Elsevier BV

Date: 09-2017

DOI: 10.1016/J.OMTN.2017.06.006

Diagnosis, Management, and Future Control of Cholera

Publisher: American Society for Microbiology

Date: 21-09-2022

DOI: 10.1128/CMR.00211-21

Abstract: Cholera, caused by Vibrio cholerae , persists in developing countries due to inadequate access to safe water, sanitation, and hygiene. There are approximately 4 million cases and 143,000 deaths each year due to cholera.

Cell specific therapy on target

Publisher: Springer Science and Business Media LLC

Date: 08-2014

DOI: 10.1038/NNANO.2014.155

Inhibition of cervical cancer cell growth in vitro and in vivo with lentiviral-vector delivered short hairpin RNA targeting human papillomavirus E6 and E7 oncogenes

Publisher: Springer Science and Business Media LLC

Date: 30-06-2006

DOI: 10.1038/SJ.CGT.7700971

Abstract: In this study, we investigated the suppressive effect of a short hairpin RNA delivered by a lentiviral vector (LV-shRNA) against human papillomavirus (HPV) type 18 E6 on the expression of the oncogenes E6 and E7 in cervical cancer HeLa cells both in vitro and in vivo. The LV-shRNA effectively delivered the shRNA to HeLa cells and lead to a dose-dependent reduction of E7 protein and the stabilization of E6 target proteins, p53 and p21. Low-dose infection of HeLa cells with LV-shRNA caused reduced cell growth and the induction of senescence, whereas a high-dose infection resulted in specific cell death via apoptosis. Transplant of HeLa cells infected with a low dose of LV-shRNA into Rag-/- mice significantly reduced the tumor weight, whereas transplant of cells infected with a high dose resulted in a complete loss of tumor growth. Systemic delivery of LV-shRNA into mice with established HeLa cell lung metastases led to a significant reduction in the number of tumor nodules. Our data collectively suggest that lentiviral delivery is an effective way to achieve stable suppression of E6/E7 oncogene expression and induce inhibition of tumor growth both in vitro and in vivo. These results encourage further investigation of this form of RNA interference as a promising treatment for cervical cancer.

Nucleocapsid Specific Diagnostics for the Detection of Divergent SARS-CoV-2 Variants

Publisher: Frontiers Media SA

Date: 10-06-2022

DOI: 10.3389/FIMMU.2022.926262

Abstract: Since the start of the COVID-19 pandemic, multiple waves of SARS-CoV-2 variants have emerged. Of particular concern is the omicron variant, which harbors 28 mutations in the spike glycoprotein receptor binding and N-terminal domains relative to the ancestral strain. The high mutability of SARS-CoV-2 therefore poses significant hurdles for development of universal assays that rely on spike-specific immune detection. To address this, more conserved viral antigens need to be targeted. In this work, we comprehensively demonstrate the use of nucleocapsid (N)-specific detection across several assays using previously described nanobodies C2 and E2. We show that these nanobodies are highly sensitive and can detect ergent SARS-CoV-2 ancestral, delta and omicron variants across several assays. By comparison, spike-specific antibodies S309 and CR3022 only disparately detect SARS-CoV-2 variant targets. As such, we conclude that N-specific detection could provide a standardized universal target for detection of current and emerging SARS-CoV-2 variants of concern.

A blueprint for eliminating cholera by 2030

Publisher: Springer Science and Business Media LLC

Date: 22-07-2022

DOI: 10.1038/S41591-022-01898-W

Can gene editing and silencing technologies play a role in the treatment of head and neck cancer?

Publisher: Elsevier BV

Date: 05-2017

DOI: 10.1016/J.ORALONCOLOGY.2017.02.016

Abstract: Conventional treatment strategies have done little to improve the prognosis or disease-free survival in head and neck cancer (HNC) patients. Recent progress in our understanding of molecular aspects of head and neck squamous cell carcinoma (HNSCC) has provided insights into the potential use of molecular targeted therapies in combination with current treatment strategies. Here we review the current understanding of treatment modalities for both HPV-positive and HPV-negative HNSCCs with the potential to use gene editing and silencing technologies therapeutically. The development of sequence-specific RNA interference (RNAi) with its strong gene-specific silencing ability, high target specificity, greater potency and reduced side effects, has shown it to be a promising therapeutic candidate for treating cancers. CRISPR/Cas gene editing is the newest technology with the ability to delete, mutate or replace genes of interest and has great potential for treating HNSCCs. We also discuss the major challenge in using these approaches in HNSCC that being the choice of target and the ability to deliver the payload. Finally, we highlight the potential combination of RNAi or CRIPSR/Cas with current treatment strategies and outline the possible path to the clinic.

Human papillomavirus type spectrum in normal skin of individuals with or without a history of frequent sun exposure

Publisher: Microbiology Society

Date: 11-2008

DOI: 10.1099/VIR.0.2008/003665-0

Abstract: Cutaneous human papillomavirus (HPV) has been widely detected in healthy skin. Previous studies have found that UV radiation can activate several HPV types, and a possible role for cutaneous HPV in the development of non-melanoma skin cancer has been suggested. This study investigated the prevalence and type-spectrum of cutaneous HPV in relation to UV radiation by studying forehead skin swab s les from 50 healthy males frequently exposed to the sun and 50 healthy males who were not frequently exposed to the sun. A questionnaire including ethnic background of the participants, history of cancers and a self-assessment of sun-exposure was also conducted and analysed. PCR with the FAP primer pair was carried out to detect HPV DNA in s les. HPV prevalence was higher in in iduals who spent more time outdoors and in in iduals with a history of skin cancers ( P =0.044 and P =0.04, respectively). Furthermore, in iduals wearing sunglasses as a means of sun protection had a lower prevalence of HPV ( P =0.018). Interestingly, HPV-76 was only detected in the group without frequent sun-exposure ( P =0.001). These results suggest that increased UV radiation exposure may be a factor leading to a difference in prevalence of cutaneous HPV types.

Genetic deletion of HPV E7 oncogene effectively eliminates HPV driven oropharyngeal cancer tumours

Publisher: Research Square Platform LLC

Date: 22-06-2022

DOI: 10.21203/RS.3.RS-1743660/V1

Abstract: The major HPV oncogenes E6 and E7 are known for its notoriety in driving the carcinogenic process in human papilloma virus (HPV) driven cancers. It is well-established that the removal of E7 d ens HPV cancer cell growth and proliferation. This has made E7 one of the most attractive targets for HPV cancers. Seminal work from our laboratory employed in vivo CRISPR editing treatment to delete E7, resulting in the effective elimination of HPV positive (HPV+) cervical cancer tumours in vivo . We have also successfully delayed HPV+ tumour growth in vivo with aurora kinase (AURK) inhibitors, an effect which is strongly sensitized by the presence of E7 expression. Unlike our observations in cervical cancer cells, in vitro targeting of E6/E7 have only resulted in partial killing of HPV+ oropharyngeal cancer (OPC) cells. The effect of E7 removal on HPV+ OPC tumours in vivo have not been explored. In this study we investigated a staggered combination of aurora kinase inhibition, using alisertib, followed by CRISPR editing of E7, to determine if this would lead to better HPV+ OPC cell killing in vitro and in vivo . Remarkably, genetic deletion of E7 alone was sufficient to effectively eliminate established HPV+ OPC tumours in vivo suggesting that E7 is essential in the maintenance of HPV+ OPC cancers.

The human papillomavirus E7 oncoprotein abrogates signaling mediated by interferon-α

Publisher: Elsevier BV

Date: 07-1999

DOI: 10.1006/VIRO.1999.9771

Identification of the alpha(6) integrin as a candidate receptor for papillomaviruses

Publisher: American Society for Microbiology

Date: 03-1997

DOI: 10.1128/JVI.71.3.2449-2456.1997

Abstract: Papillomaviruses (PVs) bind in a specific and saturable fashion to a range of epithelial and other cell lines. Treatment of cells with trypsin markedly reduces their ability to bind virus particles, suggesting that binding is mediated via a cell membrane protein. We have investigated the interaction of human PV type 6b L1 virus-like particles (VLPs) with two epithelial cell lines, CV-1 and HaCaT, which bind VLPs, and a B-cell line (DG75) previously shown not to bind VLPs. Immunoprecipitation of a mixture of PV VLPs with [35S]methionine-labeled cell extracts and with biotin-labeled cell surface proteins identified four proteins from CV-1 and HaCaT cells of 220, 120, 87, and 35 kDa that reacted with VLPs and were not present in DG75 cells. The alpha6beta4 integrin complex has subunits corresponding to the VLP precipitated proteins, and the tissue distribution of this complex suggested that it was a candidate human PV receptor. Monoclonal antibodies (MAbs) to the alpha6 or beta4 integrin subunits precipitated VLPs from a mixture of CV-1 cell proteins and VLPs, whereas MAbs to other integrin subunits did not. An alpha6 integrin-specific MAb (GoH3) inhibited VLP binding to CV-1 and HaCaT cells, whereas an anti-beta4 integrin MAb and a range of integrin-specific and other MAbs did not. Furthermore, human laminin, the natural ligand for the alpha6beta4 integrin, was able to block VLP binding. By use of sections of monkey esophagus, the distribution of alpha6 integrin expression in the basal epithelium was shown to coincide with the distribution of bound VLPs. Taken together, these data suggest that VLPs bind specifically to the alpha6 integrin subunit and that integrin complexes containing alpha6 integrin complexed with either beta1 or beta4 integrins may act as a receptor for PV binding and entry into epithelial cells.

HIV-1 TAR RNA HAS AN INTRINSIC ABILITY TO ACTIVATE INTERFERON-INDUCIBLE ENZYMES

Publisher: Elsevier BV

Date: 11-1994

DOI: 10.1006/VIRO.1994.1601

Abstract: The TAR sequence at the 5'-termini of all HIV-1 mRNA species forms a stable structure that is responsible for both transcriptional and translational regulation of HIV-1. Previously we and others reported that purified TAR RNA synthesized by in vitro transcription could activate two interferon-induced enzymes, the protein kinase (PKR) and 2-5A-synthetase. Because the PKR- and 2-5A-systems block protein synthesis initiation and induce RNA decay, respectively, these findings suggested mechanisms for the control of HIV-1 replication by the interferon system. To determine if contaminating dsRNA from in vitro transcription reactions was responsible for this effect, as suggested by Gunnery et al. 1990, (Proc., Natl. Acad. Sci. USA 87, 8687), we have reexamined these findings using chemically synthesized TAR (nucleotides +1 to +57). TAR RNA is shown here to have an intrinsic ability to activate PKR and 2-5A-synthetase. In contrast, a mutant form of TAR designed to have a disrupted secondary structure did not stimulate either enzyme. Chemically synthesized TAR mimicked other dsRNA species in its ability to activate and inhibit PKR at low and high RNA concentrations, respectively. HIV-1 TAT protein inhibited activation of PKR by HIV-1 TAR RNA suggesting an escape mechanism for the virus.

Silencing oncogene expression in cervical cancer stem-like cells inhibits their cell growth and self-renewal ability

Publisher: Springer Science and Business Media LLC

Date: 09-09-2011

DOI: 10.1038/CGT.2011.58

Abstract: Accumulating evidence supports the concept that cancer stem cells (CSCs) are responsible for tumor initiation and maintenance. They are also considered as an attractive target for advanced cancer therapy. Using a sphere culture method that favors the growth of self-renewal cells, we have isolated sphere-forming cells (SFCs) from cervical cancer cell lines HeLa and SiHa. HeLa-SFCs were resistant to multiple chemotherapeutic drugs and were more tumorigenic, as evidenced by the growth of tumors following injection of immunodeficient mice with 1 × 10(4) cells, compared with 1 × 10(6) parental HeLa cells required to grow tumors of similar size in the same time frame. These cells showed an expression pattern of CD44(high)/CD24(low) that resembles the CSC surface biomarker of breast cancer. We further demonstrated that HeLa-SFCs expressed a higher level (6.9-fold) of the human papillomavirus oncogene E6, compared with that of parental HeLa cells. Gene silencing of E6 with a lentiviral-short-hairpin RNA (shRNA) profoundly inhibited HeLa-SFC sphere formation and cell growth. The inhibition of cell growth was even greater than that for sphere formation after E6 silence, suggesting that the loss of self-renewing ability may be more important. We then measured the expression of self-renewal genes, transformation growth factor-beta (TGF-β) and leukemia-inhibitory factor (LIF), in shRNA-transduced HeLa-SFCs and found that expression of all three TGF-β isoforms was significantly downregulated while LIF remained unchanged. Expression of the Ras gene (a downstream component of TGF-β) was also markedly decreased, suggesting that the growth-inhibitory effect could be via the TGF-β pathway. The above data indicate RNA interference-based therapy may offer a new approach for CSC-targeted cancer therapy.

RNA Interference for the Treatment of Papillomavirus Disease

Publisher: Bentham Science Publishers Ltd.

Date: 28-12-2012

DOI: 10.2174/1874357901206010204

cGAS-STING responses are dampened in high-risk HPV type 16 positive head and neck squamous cell carcinoma cells

Publisher: Elsevier BV

Date: 07-2019

DOI: 10.1016/J.MICPATH.2019.05.004

Abstract: Head and neck cancers (HNCs) are a major health problem and a leading cause of morbidity and mortality worldwide. More than 90% of these tumours are head and neck squamous cell carcinomas (HNSCCs). Amongst the common risk factors for HNCs (tobacco and alcohol use), there is a strong association of human papillomavirus (HPV) with HNSCCs. HPV type 16 (HPV 16), the major high-risk HPV type, is most commonly associated with HPV-driven HNSCCs. The promiscuous nature of the major HPV oncogene, E7, allows its interaction with a myriad of host proteins including STING, a component of the viral DNA-sensing cyclic GMP-AMP synthase (cGAS) - stimulator of interferon genes (STING) machinery. Sensing of viral DNA by the cGAS-STING machinery results in a type I interferon (IFN)-mediated anti-viral response. Amelioration of IFN responses resulting from the direct blockade of STING by E7 was first demonstrated in high-risk HPV type 18 (HPV 18) positive (+) cervical squamous cell carcinoma (CESC) cells. However, the role of E7 from HPV 16 (HPV 16E7) in antagonising cGAS-STING responses have not been investigated, let alone in the context of HNSCCs. Here, we show that HPV 16E7+, but not HPV 16E7 negative (-), HNSCC cells respond poorly to cGAS-STING activation stimulus. We further confirm that this inhibition occurred via the highly conserved LXCXE motif in 16E7. This finding contributes to the better understanding of role of high-risk HPV E7 in blocking cGAS-STING pathway, especially in the context of HNSCCs.

Genetic deletion of HPV E7 oncogene effectively regresses HPV driven oral squamous carcinoma tumour growth

Publisher: Elsevier BV

Date: 11-2022

DOI: 10.1016/J.BIOPHA.2022.113782

Abstract: The major HPV oncogenes, E6 and E7, are known for its notoriety in driving the carcinogenic process in human papilloma virus (HPV) driven cancers. It is well-established that the removal of E7 d ens HPV cancer cell growth and proliferation. This has made E7 an attractive target for HPV cancers. Seminal work from our laboratory employed a CRISPR editing approach to delete E7 which resulted in the effective elimination of HPV

HPV-associated head and neck cancers in the Asia Pacific: A critical literature review & meta-analysis

Publisher: Elsevier BV

Date: 12-2015

DOI: 10.1016/J.CANEP.2015.09.013

Abstract: Malignancies of the upper aero-digestive tract are a major public health problem, especially in the Asia Pacific. Certain Human papillomaviruses (HPVs) are well-established risk factors for carcinoma of the uterine cervix and for a subset of head and neck carcinomata: however their true importance in different populations and anatomical subsites remains unclear. The major risk factors in Asia Pacific remain smoked/smokeless tobacco, areca nut, alcohol abuse and poor diet, with limited evidence for HPVs. We review published studies of association of HPV with anatomical site-specific Head & Neck Squamous Cell Carcinoma (HNSCC) in these populations and attempt a meta-analysis. From MEDLINE/PubMed/WEB-of SCIENCE/EMBASE/Scopus databases we found 67 relevant studies with a total of 7280 cases: 15 case-control studies met our inclusion criteria for meta-analysis, totaling 1106 cases & 638 controls. HPV detection rates, s le site and size, and methods of tissue preservation and HPV detection were tabulated for each study. Studies were heterogeneous in terms of s le selection and method of detection of HPVs. Most were of limited quality. Averaging data from 67 studies of HNSCC, the prevalence of HPV of any subtype is approximately 36%. PCR (polymerase chain reaction) was the most used detection method and HPV16 the most common genotype reported. Meta-analyses of case-control studies from this region reveal significant heterogeneity but suggest higher HPV prevalence in oropharyngeal cancer (OR: 14.66 95%CI: 6.09-35.26) compared to oral cavity cancer and laryngeal cancer (OR: 4.06 95%CI: 3.05-5.39 & OR: 3.23 95%CI: 1.37-7.61) respectively. In view of the significant association of HPV with HNSCC, studies with accurate subsite classification and more sensitive detection methods are necessary. Accurate data from this geographical region are essential to inform public health policies and treatment decisions, especially as studies from Europe and North America reveal HPV-driven cancers to be less aggressive, permitting treatment de-intensification.

CXCL1 gene silencing in skin using liposome-encapsulated siRNA delivered by microprojection array.

Publisher: Elsevier BV

Date: 11-2014

DOI: 10.1016/J.JCONREL.2014.08.021

Abstract: The barrier morphology of skin provides major obstacles for the application of siRNA for gene silencing, which current delivery technologies do not effectively overcome. Emerging technologies utilise microprojection array devices to penetrate into the skin epidermis and dermis for delivery of drug payloads. Delivery of siRNA by such devices has been proven in principle, yet requires optimisation for clinical applications. Herein, we demonstrate the use of Nanopatch™ microprojection arrays to deliver liposome-encapsulated siRNA to overcome skin barrier, and in vivo siRNA delivery hurdles. This application provided effective silencing of CXCL1 expression induced by the co-delivery of Fluvax 2012® by microprojection array. Liposomes encapsulating siRNA were dry-coated onto microprojection arrays, and remained intact after elution from arrays in vitro. Microprojection arrays facilitated the delivery of fluorescently-labelled nucleic acids through murine ear stratum corneum to the epidermis and dermis, with diffusion from microprojections into adjacent skin evident within 30s. CXCL1 mRNA, induced by delivery of Fluvax by microprojection array, was reduced by 75% up to 20 h post-treatment by co-delivery of liposome-encapsulated CXCL1-specific siRNA, but not by arrays co-delivering liposome-encapsulated control siRNA. CXCL1 protein expression in explant cultures from skin treated with arrays bearing CXCL1 specific or control siRNA was similarly reduced. These results as a test case have many implications for gene silencing in skin and inflammation, with the benefit of targeted delivery using microprojection arrays to deliver liposome-encapsulated siRNA.

Papillomavirus in healthy skin of Australian animals

Publisher: Microbiology Society

Date: 11-2006

DOI: 10.1099/VIR.0.82195-0

Abstract: Papillomaviruses are a group of ubiquitous viruses that are often found in normal skin of humans, as well as a range of different vertebrates. In this study, swab s les collected from the healthy skin of 225 Australian animals from 54 species were analysed for the presence of papillomavirus DNA with the general skin papillomavirus primer pair FAP59/FAP64. A total of five putative and potential new animal papillomavirus types were identified from three different animal species. The papillomaviruses were detected in one monotreme and two marsupial species: three from koalas, and one each from an Eastern grey kangaroo and an echidna. The papillomavirus prevalence in the three species was 14 % (10/72) in koalas, 20 % (1/5) in echidnas and 4 % (1/23) in Eastern grey kangaroos. Phylogenetic analysis was performed on the putative koala papillomavirus type that could be cloned and it appears in the phylogenetic tree as a novel putative papillomavirus genus. The data extend the range of species infected by papillomaviruses to the most primitive mammals: the monotremes and the marsupials.

RNAi to treat SARS‐CoV‐2—variant proofing the next generation of therapies

Publisher: EMBO

Date: 14-03-2022

DOI: 10.15252/EMMM.202215811

Liposomal Delivery of miR-34b-5p Induced Cancer Cell Death in Thyroid Carcinoma

Publisher: MDPI AG

Date: 11-12-2018

DOI: 10.3390/CELLS7120265

Abstract: This study aims to determine the functional roles of microRNA-34b-5p (miR-34b) in the suppression of anaplastic thyroid carcinoma. We used hydration-of-freeze-dried-matrix (HFDM) formulated liposomes (liposome-loaded miR-34b) for effective delivery of miR-34b to anaplastic thyroid carcinoma in vitro and in vivo. Real time polymerase chain was used to determine the level of miR-34b. Immunocytochemistry, Western blot and ELISA were carried out to determine the effect of this manipulation on VEGF-A expression. In addition, an in vivo xenotransplantation mouse model was used to investigate the functional roles of overexpression of miR-34b in the carcinoma. In anaplastic thyroid carcinoma cells, miR-34b expression was low and significant overexpression (p 0.05) was noted following transfection with liposome-loaded miR-34b. The miR-34b overexpressed thyroid carcinoma cell lines showed reduction in VEGF-A protein expression, decreased cell proliferation, decreased wound healing, reduced cell cycle progression and increased apoptosis (p 0.05). In in vivo experiments, when compared to control groups, smaller tumours formed upon intravenous administration of liposome-loaded miR-34b. To conclude, the current study confirmed the tumour suppressor properties of miR-34b via VEGF-A regulation in anaplastic thyroid carcinoma. In addition, delivery of miR-34b using cationic liposome could be a useful therapeutic strategy for targeting therapy in the carcinoma.

A novel method for the development of plasmid DNA-loaded nanoliposomes for cancer gene therapy

Publisher: Springer Science and Business Media LLC

Date: 28-07-2021

DOI: 10.1007/S13346-021-01034-0

Abstract: We aimed to develop a simple yet novel method to prepare plasmid DNA-loaded nanoliposomes for cancer gene therapy. Murine interleukin-12 (mIL-12) pDNA-loaded nanoliposomes were prepared via novel freeze-drying of a monophase solution method. The physicochemical characteristics, cytotoxicity, and transfection efficiency of the prepared nanoliposomes in murine CT-26 colon carcinoma cells were evaluated. Furthermore, tumor progression and survival rate in CT-26 colon carcinoma-bearing BALB/c mice subsequent to direct intratumoral injections were investigated over a period of 40 days. Using this preparation method, nanoliposomes with particle size of around 300 nm and zeta potential of 96.5 mV were obtained. The transmission electron microscope results showed that the liposomes were nano-sized and almost spherical. The agarose gel retardation assay revealed the pDNA encapsulation in the nanoliposomes. The nanoliposomes with 72.4% encapsulation efficiency and low cell toxicity could significantly improve mIL-12 expression by approximately 25-fold relative to the naked mIL-12 pDNA. There was a significant tumor growth inhibition after repeated injections of mIL-12 pDNA-loaded nanoliposomes. This is the first study on the freeze-drying of a monophase solution method as a simple yet novel technique for the preparation of pDNA-loaded nanoliposomes. Given the ease of preparation method and promising in vitro and in vivo characteristics, this investigation demonstrates advances in pDNA lipid formulation for cancer gene therapy.

Loss of PKR activity in chronic lymphocytic leukemia

Publisher: Wiley

Date: 2004

DOI: 10.1002/IJC.11714

Abstract: There are a number of observations that suggest the dsRNA-activated protein kinase, PKR, may play an active role in formation and maintenance of leukemia, including nonrandom chromosomal deletions in acute leukemia as well as truncations and deletions of the PKR gene in some leukemia cell lines. However, there is little direct evidence from patient material that this is so. Here we show that full-length PKR is present but not active in 21 of 28 patient s les from B-cell chronic lymphocytic leukemia (B-CLL). PKR from these patients was unable to auto-activate or phosphorylate substrates but was able to bind dsRNA. Furthermore, the lack of PKR activation was not due to differing levels of the PKR activator, PACT nor of the PKR inhibitor, p58(IPK). We compared PKR status with clinical parameters and disease staging. No differences were found between the 2 groups in terms of staging (modified Rai or Binet), age, CD38 status, p53 status, 11q23 deletion status or CEP12 deletion status. However, there was a significant correlation between deletion in 13q14.3 and lack of PKR activity. We show that B-CLL cells appear to contain a soluble inhibitor of PKR, as lysates from cells lacking PKR activity were able to inhibit exogenous PKR in mixing experiments. Finally, we show suppression of PKR activity was still present following ultrafilitration through a 10,000 Da cutoff filter but was lost upon extraction with phenol/chloroform or by high salt washing. This data suggests loss of PKR activity may contribute to the formation and/or maintenance of CLL.

Pristine large pore benzene-bridged mesoporous organosilica nanoparticles as an adjuvant and co-delivery platform for eliciting potent antitumor immunity

Publisher: Elsevier BV

Date: 06-2020

DOI: 10.1016/J.MTADV.2020.100069

Self-Adjuvanting Polymer–Peptide Conjugates As Therapeutic Vaccine Candidates against Cervical Cancer

Publisher: American Chemical Society (ACS)

Date: 22-07-2013

DOI: 10.1021/BM400626W

Abstract: Dendrimers are structurally well-defined, synthetic polymers with sizes and physicochemical properties often resembling those of biomacromolecules (e.g., proteins). As a result, they are promising candidates for peptide-based vaccine delivery platforms. Herein, we established a synthetic pathway to conjugate a human papillomavirus (HPV) E7 protein-derived peptide antigen to a star-polymer to create a macromolecular vaccine candidate to treat HPV-related cancers. These conjugates were able to reduce tumor growth and eradicate E7-expressing TC-1 tumors in mice after a single immunization, without the help of any external adjuvant.

CRISPR/Cas9-loaded stealth liposomes effectively cleared established HPV16-driven tumours in syngeneic mice

Publisher: Public Library of Science (PLoS)

Date: 07-01-2021

DOI: 10.1371/JOURNAL.PONE.0223288

Abstract: Gene-editing has raised the possibility of being able to treat or cure cancers, but key challenges remain, including efficient delivery, in vivo efficacy, and its safety profile. Ideal targets for cancer therapy are oncogenes, that when edited, cause cell death. Here, we show, using the human papillomavirus (HPV) type 16 cancer cell line TC1, that CRISPR/Cas9 targeting the E7 oncogene and packaged in PEGylated liposomes cleared established tumours in immunocompetent mice. Treatment caused no significant toxicity in the spleen or liver. An ideal therapeutic outcome would be the induction of an immunogenic cell death (ICD), such that recurrent tumours would be eliminated by the host immune system. We show here for the first time that CRISPR/Cas9-mediated cell death via targeting E7 did not result in ICD. Overall, our data show that in vivo CRISPR/Cas targeting of oncogenes is an effective treatment approach for cancer.

Statement in Support of: “Virology under the Microscope—a Call for Rational Discourse”

Publisher: American Society for Microbiology

Date: 27-06-2023

DOI: 10.1128/MBIO.00815-23

Human Papillomavirus in Benign Prostatic Hyperplasia and Prostatic Adenocarcinoma Patients

Publisher: Springer Science and Business Media LLC

Date: 16-01-2011

DOI: 10.1007/S12253-010-9357-4

Abstract: The aim of this study was to determine the prevalence of human papillomavirus (HPV) types in tissue and HPV antibodies in prostatic disease. Prostate tissue s les were collected from 51 patients diagnosed with adenocarcinoma and 11 with benign prostatic hyperplasia (BPH). All tissue s les were confirmed by histology. Plasma s les were available for 52 prostate patients. We investigated HPV DNA prevalence by PCR, and PCR positive s les were HPV type determined by sequencing. Prevalence of antibodies against twenty-seven HPV proteins from fourteen different HPV types was assessed in the plasma s les. The HPV DNA prevalence in the tissue s les was 14% (7/51) for prostate cancer s les and 27% (3/11) for BPHs. HPV-18 was the only type detected in tissue s les (10/62). No significant difference in HPV prevalence between the prostate cancer and BPH s les was found. HPV-positive cells were identified in eight of our thirteen prostate tissue slides (3/3 BPH and 5/10 adenocarcinoma) by in situ hybridisation, and the positive cells were found in epithelial cells and peripheral blood cells. Serology data showed no significant increase in levels of antibodies against any of the HPV-18 proteins tested for in prostatic disease patients. Antibodies against HPV-1, HPV-4, HPV-6 and HPV-11 were significantly higher in the group of males with prostatic disease. Our study did not show an association between prostatic disease and either presence of HPV DNA in s les or previous exposure of high-risk HPV.

Valproic acid combined with cytosine arabinoside in elderly patients with acute myeloid leukemia has in vitro but limited clinical activity

Publisher: Informa UK Limited

Date: 11-01-2012

DOI: 10.3109/10428194.2011.642302

Abstract: Elderly patients with acute myeloid leukemia (AML) have a poor prognosis. The authors examined the in vitro and clinical activity of the histone deacetylase inhibitor valproic acid (VA) combined with cytosine arabinoside (AraC) in elderly patients with AML unsuited to intensive therapy. For the in vitro studies, primary AML cells from 11 patients were treated with AraC and VA and analyzed for apoptosis, cytostatic effects, differentiation and acetyl histone H3 induction. VA (alone and with AraC) enhanced apoptosis and induced acetyl histone H3. VA inhibited cell proliferation. For the clinical trial, 15 patients were treated with VA and subcutaneous AraC and assessed for toxicity and response. No complete or partial remissions were achieved. In conclusion, VA has in vitro activity against AML and has additional activity with AraC. However, in this study, this combination demonstrated limited clinical activity in elderly patients with AML.

Long-term sialidase-specific immune responses after natural infection with cholera: Findings from a longitudinal cohort study in Bangladesh

Publisher: Frontiers Media SA

Date: 22-12-2022

DOI: 10.3389/FIMMU.2022.1067737

Abstract: Immune responses that target sialidase occur following natural cholera and have been associated with protection against cholera. Sialidase is a neuraminidase that facilitates the binding of cholera toxin (CT) to intestinal epithelial cells. Despite this, little is known about age-related sialidase-specific immune responses and the impact of nutritional status and co-infection on sialidase-specific immunity. We enrolled 50 culture-confirmed Vibrio cholerae O1 cholera cases presenting to the icddr,b Dhaka hospital with moderate to severe dehydration. We evaluated antibody responses out to 18 months (day 540) following cholera. We assessed immune responses targeting sialidase, lipopolysaccharide (LPS), cholera toxin B subunit (CtxB), and vibriocidal responses. We also explored the association of sialidase-specific immune responses to nutritional parameters and parasitic co-infection of cases. This longitudinal cohort study showed age-dependent differences in anti-sialidase immune response after natural cholera infection. Adult patients developed plasma anti-sialidase IgA and IgG responses after acute infection (P& .05), which gradually decreased from day 30 on. In children, no significant anti-sialidase IgA, IgM, and IgG response was seen with the exception of a late IgG response at study day 540 (p=0.05 compared to adults). There was a correlation between anti-sialidase IgA with vibriocidal titers, as well as anti-sialidase IgA and IgG with anti-LPS and anti-CtxB antibody responses in adult patients, whereas in children, a significant positive correlation was seen only between anti-sialidase IgA and CtxB IgA responses. Stunted children showed significantly lower anti-sialidase IgA, IgG, and IgM antibody responses and higher LPS IgG and IgM antibody responses than healthy children. The anti-sialidase IgA and IgG responses were significantly higher in cases with concomitant parasitic infection. Our data suggest that cholera patients develop age-distinct systemic and mucosal immune responses against sialidase. The stunted children have a lower anti-sialidase antibody response which may be associated with gut enteropathy and the neuraminidase plays an important role in augmented immune response in cholera patients infected with parasites.

Rational Design of Immunostimulatory siRNAs

Publisher: Elsevier BV

Date: 04-2010

DOI: 10.1038/MT.2010.4

Targeting of high mobility group A2 by small interfering RNA-loaded nanoliposome-induced apoptosis and migration inhibition in gastrointestinal cancer cells.

Publisher: Wiley

Date: 02-12-2019

DOI: 10.1002/JCB.28196

Abstract: Considering the complex nature of gastrointestinal cancer, different methods including surgery, radiotherapy, and chemotherapy are considered for the treatment. Novel strategies including silencing of oncogenes using safe delivery systems could be considered as a novel approach in colorectal cancer treatment. The aim of this study was to investigate the silencing effect of high mobility group A2 (HMGA2) small interfering RNA (siRNA)-loaded nanoliposomes on gastrointestinal cancers. The siRNA-lipoplexes were prepared using dioleoyl trimethylammonium propane (DOTAP)/cholesterol (Chol)/1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) through the freeze-drying of a monophase solution method. The size, polydispersity index (PDI), and zeta-potential of nanoliposomes were determined using Zetasizer analyzer. The morphology of the nanoliposomes was determined by transmission electron microscopy (TEM). The agarose gel-retardation assay was carried out to confirm the loading of siRNAs into liposome. The silencing of the HMGA2 in cancer cells was evaluated by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The effect of liposomes on cell cytotoxicity was studied by MTT assay. The inhibitory effect of siRNA-loaded liposomes was evaluated by a wound-healing assay. The apoptosis induction was investigated via the annexin V ropidium iodide assay. The size, PDI, and zeta-potential of the prepared liposomes were found to be 350 nm, 0.67, and 86.3 mV, respectively. They were spherical in shape and could efficiently associate with siRNA. The results of gene silencing showed that the optimum condition of HMGA2 silencing was 80 pmol HMGA2 and 24 hours after treatment in each cancer cell lines. MTT assays indicated that silencing of HMGA2 in optimal condition could reduce the viability of the cancer cells more than 60% in the three cell lines. The result of the apoptosis assay showed more than 50% of the cell deaths related to the apoptosis in all three cell lines. The gene expression evaluation confirmed that apoptosis was induced via the intrinsic pathway inducing both caspase-3 and -9 expressions. Also, the reduction in Bcl2 expression confirmed the activation apoptosis pathway in the treated cancer cells. The wound-healing assay showed the suppression of cancer cell migration after treatment with the prepared nanoliposomes. The results of this study showed the HMGA2 siRNA-loaded nanoliposomes could be effective in the treatment of gastrointestinal cancers.

Efficient biodistribution and gene silencing in the lung epithelium via intravenous liposomal delivery of siRNA

Publisher: Elsevier BV

Date: 2013

DOI: 10.1038/MTNA.2013.22

Assessment of the accuracy and reproducibility of adrenal volume measurements using MRI and its relationship with corticosteroid phenotype: a normal volunteer pilot study

Publisher: Wiley

Date: 04-2013

DOI: 10.1111/CEN.12193

Abstract: The significant role of corticosteroids in hypertension and cardiovascular disease highlights the importance of the adrenal gland in these disorders. The ability to correlate corticosteroid production with adrenal volume offers a novel research tool and intermediate phenotype in cardiovascular disease. The aim of this study was to develop and validate the use of magnetic resonance imaging (MRI) in adrenal volume assessment and investigate whether this associates with corticosteroid production. Twenty normotensive men underwent noncontrast 1·5T MRI scanning of adrenals, measurement of blood pressure and plasma corticosteroids. Left adrenal volume was calculated twice using standard segmentation software by four independent observers with differing levels of clinical expertise and segmentation experience. To optimize this process, adrenal 'phantoms' with known fixed volumes underwent MRI scanning and analysis by two observers. Intra-observer coefficients of repeatability (CoRs) in phantoms ranged from 0·23 to 0·43 ml (interobserver CoR 0·48 ml). In the subject group, mean adrenal volumes were 3·99-5·82 ml with intra-observer CoRs 0·27-1·94 ml. Interobserver variability was 2·73 ml. Segmentation expertise was the main factor affecting variability, with experienced observers having the lowest CoRs clinical knowledge was a factor when combined with segmentation experience. Mean adrenal volume correlated with plasma glucocorticoids (r = 0·523, P < 0·05) and aldosterone (r = 0·515, P < 0·05) for the most experienced observer only. Measurement of adrenal volume using MRI is challenging most accurate volumes are achieved using a single observer with both segmentation experience and anatomical knowledge. The data also provide novel preliminary evidence that adrenal gland volume may be associated with plasma corticosteroid concentrations supporting further study of adrenal volume and steroid production across a range of blood pressures.

Synthetic lethal screen identifies Aurora A as a selective target in HPV driven cervical cancer

Publisher: American Association for Cancer Research (AACR)

Date: 08-2015

DOI: 10.1158/1538-7445.AM2015-945

Abstract: HPV has been identified as the definitive agent in cancers of the cervix, penis, vulva, vagina, anus, skin, eye, and head and neck, and is responsible for 5% of the total cancer burden worldwide. HPV oncogenes disable a number of tumour suppressor pathways, including p53 and Rb, contributing to the transformed phenotype. We have performed an siRNA screen using the kinome (779 genes) library to identify genes that when depleted are synthetically lethal with HPV transformation. The primary and validations screens have confirmed Aurora A kinase (AURKA) as a potential synthetic lethal target selective for HPV transformed cells. In vitro research using the investigational selective small molecule AURKA inhibitor alisertib found alisertib to be significantly more potent towards the HPV transformed cells, and selectively promoted apoptosis in the HPV cancers. The drug was shown to target the HPVE7 oncogene, the level of expression of this oncogene possibly influencing sensitivity. Apoptosis was sensitive to Mcl-1 but not Bcl-2 over expression, indicating that the mechanism is associated with the proteolytic destruction of Mcl-1 in the extended mitosis in the alisertib treated HPV cancer lines. Xenograft experiments with cervical cancer cell lines showed alisertib inhibited growth of HPV and non-HPV xenografts during treatment. The non-HPV cancer growth was delayed, but in two separate HPV cancers models, regression and no resumption of growth was detected at even 50 days post-treatment. A second transgenic model of premalignant disease driven solely by HPVE7 similarly demonstrated sensitivity to drug treatment. These findings provide preclinical evidnce that alisertib warrants evaluation as a potential targeted compound with activity in HPV-transformed cervical cancer and premalignant disease that may have application to other HPV driven cancers. Citation Format: Brian G. Gabrielli, Fawzi Bokhari, Max Ranall, Zay Yar Oo, Alex Stevenson, Weili Wang, Sara McKee, Graham Leggatt, Paul Leo, Thomas J. Gonda, Nigel AJ McMillan. Synthetic lethal screen identifies Aurora A as a selective target in HPV driven cervical cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research 2015 Apr 18-22 Philadelphia, PA. Philadelphia (PA): AACR Cancer Res 2015 (15 Suppl):Abstract nr 945. doi:10.1158/1538-7445.AM2015-945

Polymeric siRNA delivery vectors: knocking down cancers with polymeric-based gene delivery systems

Publisher: Wiley

Date: 09-09-2015

DOI: 10.1002/JCTB.4508

A “hit-and-run” affair – A possible link for cancer progression in virally driven cancers

Publisher: Elsevier BV

Date: 2021

DOI: 10.1016/J.BBCAN.2020.188476

Potent Inhibition of Hendra Virus Infection via RNA Interference and Poly I:C Immune Activation

Publisher: Public Library of Science (PLoS)

Date: 14-05-2013

DOI: 10.1371/JOURNAL.PONE.0064360

RNA-based gene targeting therapies for human papillomavirus driven cancers

Publisher: Elsevier BV

Date: 12-2021

DOI: 10.1016/J.CANLET.2021.10.005

HIV-1 Tat Directly Interacts with the Interferon-Induced, Double-Stranded RNA-Dependent Kinase, PKR

Publisher: Elsevier BV

Date: 11-1995

DOI: 10.1006/VIRO.1995.0014

Abstract: We present evidence that the HIV-1 Tat protein and the RNA-dependent cellular protein kinase, PKR, interact with each other both in vitro and in vivo. Using GST fusion chromatography, we demonstrate that PKR, interacts directly with the HIV-1 Tat protein. The region in Tat sufficient for binding PKR maps within amino acids 20 to 72. In in vitro assays, the two-exon form of Tat (Tat 86) was phosphorylated by PKR, while the one exon form of Tat (Tat 72) inhibited PKR autophosphorylation and substrate phosphorylation. The ability of Tat to interact with PKR was demonstrated in both yeast and mammalian cells. Expression of PKR in yeast results in a growth suppressor phenotype which was reversed by coexpression of a one exon form of Tat. Expression of Tat 72 in HeLa cells resulted in direct interaction with PKR as detected by coimmunprecipitation with a Tat antibody. Tat and PKR also form a coimmunoprecipitable complex in cell-free extracts prepared from productively infected T lymphocytes. The interaction of Tat with PKR provides a potential mechanism by which HIV could suppress the interferon system.

Targeting Aurora Kinase to Enhance the Curative Potential of Radiotherapy in HPV Related Cancers

Publisher: Elsevier BV

Date: 09-2019

DOI: 10.1016/J.IJROBP.2019.06.321

Aurora kinases are a novel therapeutic target for HPV-positive head and neck cancers

Publisher: Elsevier BV

Date: 11-2018

DOI: 10.1016/J.ORALONCOLOGY.2018.09.006

Abstract: Human papilloma virus (HPV) is the main culprit in cancers of the cervix, penis, anus, skin, eye and head and neck. Current treatments for HPV cancers have not altered survival outcomes for 30 years and there is a significant lack of targeted therapeutic agents in the management of advanced HPV-related HNSCC. Here we show that survival and maintenance of HPV-positive HNC cells relies on the continuous expression of the major HPV oncogene, E7, and that Aurora kinases are critical for survival of high-risk HPV-positive HNC cells. To assess the role of HPV E7 on HNC cell survival, RNA interference (RNAi) of the E7 gene was initially performed. Using an Aurora kinase inhibitor, Alisertib, the role of Aurora kinases in the carcinogenesis of HPV E7 positive HNC tumour lines was then investigated. An in vivo HNC xenograft model was also utilised to assess loss of tumour volume in response to RNAi E7 gene silencing and Alisertib treatment. RNAi silencing of the HPV E7 gene inhibited the growth of HPV-positive HNC cells and in vivo tumour load. We show that HPV E7 oncogene expression confers sensitivity to Alisertib on HNC cells where Alisertib-mediated loss in in vitro cell viability and in vivo tumour load is dependent on E7 expression. Moreover, Aurora kinase inhibition induced degradation of MCL-1 in HPV E7-expressing HNC cells. Overall, we show that Aurora kinases are a novel therapeutic target for HPV-positive HNCs. It might be feasible to combine Aurora kinase and MCL-1 inhibitors for future HNC therapies.

Future of RNAi-based therapies for human papillomavirus-associated cervical cancer

Publisher: Future Medicine Ltd

Date: 11-2007

DOI: 10.2217/17460794.2.6.587

Abstract: Over 99% of cervical cancers are associated with infection of high-risk type human papillomaviruses (HPV). These viruses infect epithelial cells lining the cervix and express the early viral genes E6 and E7, which are oncogenes and are primarily responsible for the transformation of the epithelial cells. The continuous expression of those genes is essential for maintenance of the cancer cell phenotype and viability. These viral genes can be silenced using oligonucleotide-based techniques, for ex le RNAi, antisense RNA and ribozymes. In spite of promising results in vitro and in vivo, in mice, these methods have thus far proved unsuccessful in humans, owing to the lack of an effective delivery system amongst other limitations. In this review we will discuss potential gene-silencing strategies in cervical cancer that would target both viral genes such as E6 and E7, and cellular genes that become deregulated such as E2F, p53, Akt, mTor, NF-κB or Bcl-2. By investigating these approaches we may generate an effective treatment for HPV-induced cervical cancer using gene silencing.

Application of the predicted heat strain model in development of localized, threshold-based heat stress management guidelines for the construction industry

Publisher: Oxford University Press (OUP)

Date: 26-12-2014

DOI: 10.1093/ANNHYG/MET070

Abstract: Existing heat stress risk management guidelines recommended by international standards are not practical for the construction industry which needs site supervision staff to make instant managerial decisions to mitigate heat risks. The ability of the predicted heat strain (PHS) model [ISO 7933 (2004). Ergonomics of the thermal environment analytical determination and interpretation of heat stress using calculation of the predicted heat strain. Geneva: International Standard Organisation] to predict maximum allowable exposure time (D lim) has now enabled development of localized, action-triggering and threshold-based guidelines for implementation by lay frontline staff on construction sites. This article presents a protocol for development of two heat stress management tools by applying the PHS model to its full potential. One of the tools is developed to facilitate managerial decisions on an optimized work-rest regimen for paced work. The other tool is developed to enable workers' self-regulation during self-paced work.

Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry

Publisher: Elsevier BV

Date: 05-2003

DOI: 10.1016/S0042-6822(03)00114-4

Abstract: Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC(50) (50% inhibition) was only 0.5 microg/ml for heparin, 1 microg/ml for dextran sulfate, and 5-10 microg/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate.

RNA TRANSCRIPT MAPPING OF THE WISEANA IRIDESCENT VIRUS GENOME

Publisher: Elsevier BV

Date: 06-1994

DOI: 10.1016/0168-1702(94)90082-5

Abstract: The replication of Wiseana iridescent virus (WIV) was studied in Lymantria dispar tissue culture cells. Using a combination of [35S]methionine pulse-labeling and Northern blotting with WIV DNA probes, a transcriptional map of the genome was constructed. WIV has a wide dispersal of immediate-early genes with seven different regions identified. WIV has been reported to have extensive repetitive DNA sequences but no early transcription was observed in these regions. Although fine-mapping is required, some early regions (Bam L and Eco O) have been identified which are transcriptionally active at 6- and 12-h but are shut down by 24 h. These regions could provide probes for early genes and the hypothesized switch from nuclear to cytoplasmic replication for iridoviruses.

RNA Interference for Viral Infections

Publisher: Bentham Science Publishers Ltd.

Date: 08-2012

DOI: 10.2174/138945012803530161

Abstract: The treatment of viral infections has relied on pre-emptive vaccination or use of a limited range of anti-viral drugs. However, the majority of viruses have no available drugs and treatment is merely supportive. RNA interference (RNAi) offers the ability to directly and rapidly treat virus infections via the targeting of viral genes. Indeed, clinical trials have already been undertaken with promising results. Here we review the current state of the RNAi field for the treatment of viral infections such as HIV, human papillomavirus and HCV. We also review novel strategies including the concept of targeting self-genes to limit viral infection and activating the immune system for improved outcomes. Finally we examine innovative approaches being pursued at the Australian Infectious Diseases Research Centre including the use of high-throughput siRNA screens to identify new antiviral targets.

Expression of the α6 Integrin Confers Papillomavirus Binding upon Receptor-Negative B-Cells

Publisher: Elsevier BV

Date: 09-1999

DOI: 10.1006/VIRO.1999.9825

Inhibition of Aurora B kinase activity triggers senescence that can be bypassed by blocking p53 and RB function, promoting replication stress

Publisher: American Association for Cancer Research (AACR)

Date: 12-2019

DOI: 10.1158/1535-7163.TARG-19-A060

Abstract: Aurora B kinase has a major role in regulating progression through mitosis and partitioning the replicated genome at exit from mitosis and cytokinesis. We have previously reported that Aurora A and B inhibitors selectively targeted HPV-driven tumours, and that the HPV E7 oncogene is a major determinant of this selectivity. Another group has shown that sensitivity to Aurora B inhibitor AZD2811 is dependent on loss of the retinoblastoma protein (RB). Here we have investigated the outcomes of inhibition of Aurora B using selective and pan Aurora inhibitors. We show that inhibition of Aurora A and B promotes more cell death, although a subset of cells are protected from the cytotoxic effects as a consequence of Aurora A inhibition slowing the cell cycle. Aurora B inhibition promotes senescence, although this requires at least two cycles of failed cytokinesis in all cell types, including primary fibroblasts. Loss of RB function either by HPV E7 or CDK4 R24C mutant over-expression, and loss of p53 function have somewhat different effects on the outcomes of Aurora B inhibition, although both reduced the proportion of cells that entered senescence. Loss of either also promoted replication stress which was not observed in RB and p53 proficient cells, the level of replication stress was not enhanced by Aurora A co-inhibition. Selective Aurora B inhibitor was also less toxic to proliferating PBMC derive CD3+ T cells than the pan Aurora inhibitor. Together these data indicate that short term treatment with Aurora B selective inhibitor is sufficient to promote cell cycle arrest and senescence in RB and p53 proficient cells, although proliferating T cells appear to tolerate this. RB and p53 pathway defective cells are less sensitive to this senescence trigger, but undergo replication stress. Citation Format: Ariel Andrews, Ramyashree Prasanna Kumar, Deborah Nazareth, Anna Ehmann, John Hooper, Nigel McMillan, Brian Gabrielli. Inhibition of Aurora B kinase activity triggers senescence that can be bypassed by blocking p53 and RB function, promoting replication stress [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics 2019 Oct 26-30 Boston, MA. Philadelphia (PA): AACR Mol Cancer Ther 2019 (12 Suppl):Abstract nr A060. doi:10.1158/1535-7163.TARG-19-A060

Aurora a is critical for survival in HPV-transformed cervical cancer

Publisher: American Association for Cancer Research (AACR)

Date: 12-2015

DOI: 10.1158/1535-7163.MCT-15-0506

Abstract: Human papillomavirus (HPV) is the causative agent in cervical cancer. HPV oncogenes are major drivers of the transformed phenotype, and the cancers remain addicted to these oncogenes. A screen of the human kinome has identified inhibition of Aurora kinase A (AURKA) as being synthetically lethal on the background of HPV E7 expression. The investigational AURKA inhibitor MLN8237/Alisertib selectively promoted apoptosis in the HPV cancers. The apoptosis was driven by an extended mitotic delay in the Alisertib-treated HPV E7–expressing cells. This had the effect of reducing Mcl-1 levels, which is destabilized in mitosis, and increasing BIM levels, normally destabilized by Aurora A in mitosis. Overexpression of Mcl-1 reduced sensitivity to the drug. The level of HPV E7 expression influenced the extent of Alisertib-induced mitotic delay and Mcl-1 reduction. Xenograft experiments with three cervical cancer cell lines showed Alisertib inhibited growth of HPV and non-HPV xenografts during treatment. Growth of non-HPV tumors was delayed, but in two separate HPV cancer cell lines, regression with no resumption of growth was detected, even at 50 days after treatment. A transgenic model of premalignant disease driven solely by HPV E7 also demonstrated sensitivity to drug treatment. Here, we show for the first time that targeting of the Aurora A kinase in mice using drugs such as Alisertib results in a curative sterilizing therapy that may be useful in treating HPV-driven cancers. Mol Cancer Ther 14(12) 2753–61. ©2015 AACR.

MUC13 protects colorectal cancer cells from death by activating the NF-Kb pathway and is a potential therapeutic target

Publisher: American Association for Cancer Research (AACR)

Date: 15-07-2016

DOI: 10.1158/1538-7445.AM2016-3564

Abstract: MUC13 is a transmembrane mucin glycoprotein that is overexpressed by many cancers, although its functions are not fully understood. NF-κB is a key transcription factor promoting cancer cell survival, but therapeutically targeting this pathway has proved difficult because NF-κB has pleiotropic functions. Here, we report that MUC13 prevents colorectal cancer cell death by promoting two distinct pathways of NF-kB activation, consequently up-regulating BCL-XL. MUC13 promoted TNF-induced NF-κB activation by interacting with TNFR1 and the E3 ligase, cIAP1, to increase ubiquitination of RIPK1. MUC13 also promoted genotoxin-induced NF-κB activation by increasing phosphorylation of ATM and SUMOylation of NEMO. Moreover, elevated expression of cytoplasmic MUC13 and NF-κB correlated with colorectal cancer progression and metastases. Our demonstration that MUC13 enhances NF-κB signalling in response to both TNF and DNA damaging agents provides a new molecular target for specific inhibition of NF-κB activation. As proof of principle, silencing MUC13 sensitized colorectal cancer cells to death in response to cytotoxic drugs and inflammatory signals and abolished chemotherapy-induced enrichment of CD133+ CD44+ cancer stem cells, slowed xenograft growth in mice, and synergized with 5-fluourouracil to induce tumor regression. Therefore, these data indicate that combining chemotherapy and MUC13 antagonism could improve the treatment of metastatic cancers. Citation Format: Yong H. Sheng, Yaowu He, sumaira Z. hasnain, Ran Wang, Hui Tong, Daniel T. Clarke, Rohan Lourie, Iulia Oancea, kuanyau wong, John W. Lumley, Timothy H. Florin, Philip Sutton, John. D. Hooper, Nigel A. Mcmillan, Michael A. Mcguckin. MUC13 protects colorectal cancer cells from death by activating the NF-κb pathway and is a potential therapeutic target. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research 2016 Apr 16-20 New Orleans, LA. Philadelphia (PA): AACR Cancer Res 2016 (14 Suppl):Abstract nr 3564.

Systemic delivery of E6/7 siRNA using novel lipidic particles and its application with cisplatin in cervical cancer mouse models

Publisher: Springer Science and Business Media LLC

Date: 12-08-2011

DOI: 10.1038/GT.2010.113

Abstract: Small interfering RNA (siRNA) shows great promise in cancer therapy, but its effectiveness in vivo still remains a crucial issue for its transition into the clinics. Although the successful use of polyethylene glycol (PEG)ylated lipidic delivery systems have already been reported, most of the formulation procedures used are labour intensive and also result in unstable end products. We have previously developed a simple yet efficient hydration-of-freeze-dried-matrix (HFDM) method to entrap siRNA within lipid particles, in which the products exhibited superior stability. Here, we show that these HFDM-formulated particles are stable in the presence of serum and can deliver siRNA efficiently to tumours after intravenous administration. Using these particles, around 50% knockdown of the target gene expression was observed in tumours. With the use of siRNA targeting the E6/7 oncogenes expressed in cervical cancer, we showed a 50% reduction in tumour size. This level of tumour growth suppression was comparable to that achieved from cisplatin at the clinically used dose. Overall, our results demonstrate the feasibility of using HFDM-formulated particles to systematically administer E6/7-targeted siRNA for cervical cancer treatment. The simplicity of preparation procedure along with superior product stability obtained from our method offers an innovative approach for the in vivo delivery of siRNA.

Human Papillomavirus Type 6b Virus-Like Particles Are Able To Activate the Ras-MAP Kinase Pathway and Induce Cell Proliferation

Publisher: American Society for Microbiology

Date: 05-2001

DOI: 10.1128/JVI.75.9.4150-4157.2001

Abstract: The initial step in viral infection is the attachment of the virus to the host cell via an interaction with its receptor. We have previously shown that a receptor for human papillomavirus is the α6 integrin. The α6 integrin is involved in the attachment of epithelial cells with the basement membrane, but recent evidence suggests that ligation of many integrins results in intracellular signaling events that influence cell proliferation. Here we present evidence that exposure of A431 human epithelial cells to human papillomavirus type 6b L1 virus-like particles (VLPs) results in a dose-dependent increase in cell proliferation, as measured by bromodeoxyuridine incorporation. This proliferation is lost if VLPs are first denatured or incubated with a monoclonal antibody against L1 protein. The MEK1 inhibitor PB98059 inhibits the VLP-mediated increase in cell proliferation, suggesting involvement of the Ras-MAP kinase pathway. Indeed, VLP binding results in rapid phosphorylation of the β4 integrin upon tyrosine residues and subsequent recruitment of the adapter protein Shc to β4. Within 30 min, the activation of Ras, Raf, and Erk2 was observed. Finally, the upregulation of c- myc mRNA was observed at 60 min. These data indicate that human papillomavirus type 6b is able to signal cells via the Ras-MAP kinase pathway to induce cell proliferation. We hypothesize that such a mechanism would allow papillomaviruses to infect hosts more successfully by increasing the potential pool of cells they are able to infect via the initiation of proliferation in resting keratinocyte stem and suprabasal cells.

A Novel Method for Screening Viral Interferon-Resistance Genes

Publisher: Mary Ann Liebert Inc

Date: 08-2004

DOI: 10.1089/1079990041689610

Abstract: Many viruses have evolved mechanisms to antagonize the interferon (IFN) system, targeting all the major components involved in receptor binding and signaling. Although a number of these vital proteins are homologous to cellular proteins involved in IFN downregulation (e.g., viral IFN regulatory factors [vIRFs]), many share little resemblance to known proteins. To determine the IFN-blocking properties of these proteins, functional assays are required. Here, we present a new and rapid functional screening method, based on the 2fTGH cell line, which is able to determine viral gene products that inhibit the IFN-alpha/Jak-Stat signaling pathway. Expression cloning of viral IFN-blocking genes into 2fTGH and consequent selection with IFN-alpha and 6-thioguanine result in the outgrowth of cells that are no longer responsive to IFN-alpha. We also demonstrate that selection occurs if members of the Jak-Stat signaling pathway are lost. To show the utility of our system, we have used a known suppressor of IFN signaling, the human papillomavirus (HPV) E7 gene. Expression of E7 causes the loss of ability of 2fTGH cells to respond to IFN-alpha treatment because of a functional disruption of the signaling pathway. This approach offers a new strategy for identifying novel viral genes or new functions of already described viral genes that have a role in IFN-alpha signaling inhibition.

Potential use of salivary mRNA-based biomarkers to predict oral squamous cell carcinoma

Publisher: Elsevier BV

Date: 12-2022

DOI: 10.1016/J.ORALONCOLOGY.2022.106215

RNA Interference against Human Papillomavirus Oncogenes in Cervical Cancer Cells Results in Increased Sensitivity to Cisplatin

Publisher: American Society for Pharmacology & Experimental Therapeutics (ASPET)

Date: 24-08-2005

DOI: 10.1124/MOL.105.014191

Abstract: Targeted inhibition of oncogenes in tumor cells is a rational approach toward the development of cancer therapies based on RNA interference (RNAi). Tumors caused by human papillomavirus (HPV) infection are an ideal model system for RNAi-based cancer therapies because the oncogenes that cause cervical cancer, E6 and E7, are expressed only in cancerous cells. We investigated whether targeting HPV E6 and E7 oncogenes yields cancer cells more sensitive to chemotherapy by cisplatin, the chemotherapeutic agent currently used for the treatment of advanced cervical cancer. We have designed siRNAs directed against the HPV E6 oncogene that simultaneously targets both E6 and E7, which results in an 80% reduction in E7 protein and reactivation of the p53 pathway. The loss of E6 and E7 resulted in a reduction in cellular viability concurrent with the induction of cellular senescence. Interference was specific in that no effect on HPV-negative cells was observed. We demonstrate that RNAi against E6 and E7 oncogenes enhances the chemotherapeutic effect of cisplatin in HeLa cells. The IC50 for HeLa cells treated with cisplatin was 9.4 microM, but after the addition of a lentivirus-delivered shRNA against E6, the IC50 was reduced almost 4-fold to 2.4 microM. We also observed a decrease in E7 expression with a concurrent increase in p53 protein levels upon cotreatment with shRNA and cisplatin over that seen with in idual treatment alone. Our results provide strong evidence that loss of E6 and E7 results in increased sensitivity to cisplatin, probably because of increased p53 levels.

Engineered extracellular vesicles directed to the spike protein inhibit SARS-CoV-2

Publisher: Elsevier BV

Date: 03-2022

DOI: 10.1016/J.OMTM.2022.01.015

Serum Levels Of CD178 (Soluble FasL) Predict Treatment Response and Survival In Chronic Lymphocytic Leukaemia (CLL)

Publisher: American Society of Hematology

Date: 15-11-2013

DOI: 10.1182/BLOOD.V122.21.2866.2866

Abstract: CLL cells have prolonged survival in vivo, but rapidly undergo apoptosis when cultured ex vivo indicating the importance of the tumour microenvironment in resistance to apoptosis. Chemokines and their receptors have been identified as playing a critical role in the CLL microenvironment and we have previously identified CCL2, CXCL2, IL-6 and IL-8 as pro-survival factors in our in vitro long-term CLL culture model. We examined the in vivo significance of these and other chemokines by examining serum levels in a cohort of uniformly treated patients with CLL. Serum s les were collected from 32 Australian patients participating in the German CLL Study Group CLL8 trial prior to treatment and were assessed for circulation levels of several cytokines and chemokines. Serum s les from 7 age matched healthy donors were used as controls. Serum concentration of CCL21, CXCL12, CXCL13, CCL19 and CXCL2 were examined using enzyme-linked immunosorbent assays (ELISA) and levels of CCL2, CCL3, CCL4, sCD54, sCD178, IL6 and IL-8 were examined using flex sets of BD Cytometric Bead Arrays System. Levels of these cytokines and chemokines were compared both to the healthy donor control s les and correlated with known prognostic and clinical factors. The levels were also evaluated in respect to response to therapy, progression-free survival (PFS) and overall survival (OS). 32 patients were studied: median age 62yrs, 84% male, Binet stage B 63%, stage C 37%. 15 received FCR and 17 FC with a median PFS of 45 months and median OS not reached. Compared to controls, patients with CLL had significantly higher serum levels of CXCL13 (117pg/ml vs 15pg/ml, p=0.0001), CCL3 (273pg/ml vs 103pg/ml, p=0.0007), CCL4 (13pg/ml vs 0pg/ml, p=0.0057), CD178 (45pg/ml vs 26pg/ml, p=0.01) and IL-6 (5pg/ml vs 0, p=0.02) and significantly lower levels of CXCL12 (803pg/ml vs 1177pg/ml, p=0.003) and sCD154 (345pg/ml vs 2121pg/ml p=0.0004). CCL2 levels did not correlate with any baseline clinical feature, response to therapy, PFS or OS. CXCL2 levels reduced with more advanced Rai stage disease (p=0.0083) but did not predict PFS or OS. CD178 levels did not correlate with any baseline clinical feature but predicted response to therapy with lower levels predicting improved response (p=0.04). Higher CD178 also strongly predicted worse PFS (HR 1.026, p .001) and OS (HR 1.02, p=0.018), a consistent effect whether treated with FC (HR 1.023, p=0.024) or FCR (HR 1.026, p=0.026). No other chemokine levels predicted PFS. Baseline serum levels of CD178 (soluble FasL) may be a useful predictor of response to therapy and outcome in CLL. Further studies to confirm these findings and to define the biological mechanism of FasL overexpression and function are required. We thank the German CLL Study Group and Roche for access to patient s les and data from the CLL8 study, and the Leukaemia Foundation of Australia for grant support. Mollee: Roche Australia: Sponsorship for attending ICML Lugano 2013 Other.

THE INTERFERON-INDUCIBLE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN-KINASE SELF-ASSOCIATES IN-VITRO AND IN-VIVO

Publisher: Proceedings of the National Academy of Sciences

Date: 29-08-1995

DOI: 10.1073/PNAS.92.18.8283

Abstract: The interferon-inducible double-stranded (ds) RNA-activated protein kinase (PKR) exhibits antiviral, anticellular, and antitumor activities. The mechanisms of its enzymatic activation by autophosphorylation and of the observed transdominant inhibitory phenotype of enzymatically inactive mutants have invoked PKR dimerization. Here we present direct evidence in support of PKR-PKR interaction. We show that radiolabeled PKR can specifically interact with matrix-bound unlabeled PKR in the absence of dsRNA. The self-association activity resides, in part, in the N-terminal region of 170 residues, which also constitutes the dsRNA-binding domain (DRBD). DRBD can bind to matrix-bound PKR or to matrix-bound DRBD. Dimerization of DRBD was directly demonstrated by chemical crosslinking. Affinity chromatography and electrophoretic mobility supershift assays demonstrated that mutants that fail to bind dsRNA can still exhibit protein-protein interaction. The PKR-PKR interaction could also be observed in a two-hybrid transcriptional activation assay in mammalian cells and consequently is likely to be an important feature of PKR activity in vivo.

Critical risk-benefit assessment of the novel anti-cancer aurora a kinase inhibitor alisertib (MLN8237): A comprehensive review of the clinical data

Publisher: Elsevier BV

Date: 11-2017

DOI: 10.1016/J.CRITREVONC.2017.09.006

Abstract: Many current anticancer chemotherapeutics suffer from significant side effects, which have led to the exploration of more targeted therapies. This resulted in the exploration of inhibitors of Aurora A kinase as a potential anti-cancer treatment. Alisertib (MLN8237) has proven to be a potent Aurora A kinase inhibitor that had the highest safety profile among its therapeutic family. Phase I/II/III clinical trials with Alisertib have been carried out and reported promising efficacy, yet serious side effects. This article attempts to assess the clinical effect of Alisertib administration in various cancer phenotypes while describing the reported side effects. Alisertib clinical data were systematically retrieved from Medline, CINAHL, PubMed, and Cochrane Central Register of Controlled Trials and analyzed for quality, relevance, and originality in three stages prior to inclusion. Overall, seven studies met inclusion criteria and enrolled a total of 630 patients. The reported "potential" clinical effect of Alisertib in various tumours is promising as it improved time to disease progression, progression-free survival, and the duration of disease stability. The achieved improvement therefore rationalizes its further investigation as a novel anticancer therapy. However, the administration of the drug was associated with serious haematological disturbances in a relatively high percentage of patients. The evidence of the anti-tumour effect of Alisertib administration is compelling in various types of malignancies. The reported side effects were serious but manageable in many cases. Topical or more targeted routes of administration are suggested when possible to overcome off-target events with systematic administration of the drug.

siRNA‐induced immunostimulation through TLR7 promotes antitumoral activity against HPV‐driven tumors in vivo

Publisher: Wiley

Date: 22-03-2011

DOI: 10.1038/ICB.2011.19

Abstract: Oncogene-specific downregulation mediated by RNA interference (RNAi) is a promising avenue for cancer therapy. In addition to specific gene silencing, in vivo RNAi treatment with short interfering RNAs (siRNAs) can initiate immune activation through innate immune receptors including Toll-like receptors, (TLRs) 7 and 8. Two recent studies have shown that activation of innate immunity by addition of tri-phosphate motifs to oncogene-specific siRNAs, or by co-treatment with CpG oligos, can potentiate siRNA antitumor effects. To date, there are no reports on applying such approach against human papillomavirus (HPV)-driven cancers. Here, we characterized the antitumor effects of non-modified siRNAs that can target a specific oncogene and/or recruit the innate immune system against HPV-driven tumors. Following the characterization of silencing efficacy and TLR7 immunostimulatory potential of 15 siRNAs targeting the HPV type 16 E6/E7 oncogenes, we identified a bifunctional siRNA sequence that displayed both potent gene silencing and active immunostimulation effect. In vivo systemic administration of this siRNA resulted in reduced growth of established TC-1 tumors in C57BL/6 mice. Ablation of TLR7 recruitment via 2'O-methyl modification of the oligo backbone reduced these antitumor effects. Further, a highly immunostimulatory, but non-HPV targeting siRNA was also able to exert antitumoral effects although for less prolonged time compared with the bifunctional siRNA. Collectively, our work demonstrates for the first time that siRNA-induced immunostimulation can have antitumoral effects against HPV-driven tumors in vivo, even independent of gene silencing efficacy.

A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19

Publisher: Cold Spring Harbor Laboratory

Date: 19-04-2021

DOI: 10.1101/2021.04.19.440531

Abstract: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in humans. Despite several emerging vaccines, there remains no verifiable therapeutic targeted specifically to the virus. Here we present a highly effective siRNA therapeutic against SARS-CoV-2 infection using a novel lipid nanoparticle delivery system. Multiple small-interfering RNAs (siRNAs) targeting highly conserved regions of the SARS-CoV-2 virus were screened and three candidate siRNAs emerged that effectively inhibit virus by greater than 90% either alone or in combination with one another. We simultaneously developed and screened two novel lipid nanoparticle formulations for the delivery of these candidate siRNA therapeutics to the lungs, an organ that incurs immense damage during SARS-CoV-2 infection. Encapsulation of siRNAs in these LNPs followed by in vivo injection demonstrated robust repression of virus in the lungs and a pronounced survival advantage to the treated mice. Our LNP-siRNA approaches are scalable and can be administered upon the first sign of SARS-CoV-2 infection in humans. We suggest that an siRNA-LNP therapeutic approach could prove highly useful in treating COVID-19 disease as an adjunctive therapy to current vaccine strategies.

Immune responses in children after vaccination with a typhoid Vi-tetanus toxoid conjugate vaccine in Bangladesh

Publisher: Elsevier BV

Date: 05-2023

DOI: 10.1016/J.VACCINE.2023.04.014

ASGCT 22nd Annual Meeting Abstracts

Publisher: Elsevier BV

Date: 04-2019

DOI: 10.1016/J.YMTHE.2019.04.004

Multiantigenic peptide–polymer conjugates as therapeutic vaccines against cervical cancer

Publisher: Elsevier BV

Date: 09-2016

DOI: 10.1016/J.BMC.2016.07.036

Abstract: Immunotherapy is one of the most promising strategies for the treatment of cancer. Human papillomavirus (HPV) is responsible for virtually all cases of cervical cancer. The main purpose of a therapeutic HPV vaccine is to stimulate CD8(+) cytotoxic T lymphocytes (CTLs) that can eradicate HPV infected cells. HPV oncoproteins E6 and E7 are continuously expressed and are essential for maintaining the growth of HPV-associated tumor cells. We designed polymer-based multi-antigenic formulations/constructs that were comprised of the E6 and E7 peptide epitopes. We developed an N-terminus-based epitope conjugation to conjugate two unprotected peptides to poly tert-butyl acrylate. This method allowed for the incorporation of the two antigens into a polymeric dendrimer in a strictly equimolar ratio. The most effective formulations eliminated tumors in up to 50% of treated mice. Tumor recurrence was not observed up to 3months post initial challenge.

Plasma Circulating Tumor HPV DNA and HPV-Related Oropharynx Cancer-A Caution

Publisher: American Medical Association (AMA)

Date: 10-2022

DOI: 10.1001/JAMAOTO.2022.2452

Morphological and molecular analysis of a breast cancer cluster at the ABC Studio in Toowong

Publisher: Elsevier BV

Date: 08-2012

DOI: 10.1097/PAT.0B013E3283559E67

RNAi Targeting of Human Metapneumovirus <b><i>P</i></b> and <b><i>N</i></b> Genes Inhibits Viral Growth

Publisher: S. Karger AG

Date: 2018

DOI: 10.1159/000491927

Abstract: b i Background/Aims: /i /b Human metapneumovirus (hMPV) is an important human respiratory pathogen and is implicated in an array of respiratory illnesses, ranging from asymptomatic infection to severe bronchiolitis. Currently, there is no reliable vaccine or specific antiviral therapy for hMPV infection and treatment is supportive. The use of ribonucleic acid interference has the potential to change that with the targeting of essential viral genes via small interfering RNAs (siRNAs) offering the ability to directly and rapidly treat viral infections. b i Method: /i /b The human lung carcinoma epithelial cell line, A549, was transfected with siRNAs targeting the i N /i and i P /i genes before infecting with hMPV A2 CAN97-83. Viral growth inhibition was then measured by the viral plaque assay and nucleoprotein ( i N /i ) and phosphoprotein ( i P /i ) gene knockdown was determined by real-time PCR. b i Results: /i /b In vitro prophylactic use of siRNAs targeting the 3′-abundantly expressed i N /i and i P /i genes of hMPV resulted in potent, sequence-specific viral inhibition. The viral inhibition was specific and not mediated by an anti-viral interferon-β response or cell death. b i Conclusion: /i /b The findings presented here confirmed the highly potent, sequence-specific antiviral effect of siRNAs targeting the N and P gene of hMPV. These results may facilitate the development of a novel therapeutic agent for hMPV control.

National Hospital-Based Sentinel Surveillance for Cholera in Bangladesh: Epidemiological Results from 2014 to 2021

Publisher: American Society of Tropical Medicine and Hygiene

Date: 14-08-2023

DOI: 10.4269/AJTMH.23-0074

Abstract: Despite focusing on cholera burden, epidemiologic studies in Bangladesh tend to be limited in geographic scope. National-level cholera surveillance data can help inform cholera control strategies and assess the effectiveness of preventive measures. Hospital-based sentinel surveillance among patients with suspected diarrhea in different sites across Bangladesh has been conducted since 2014. We selected an age-stratified s le of 20 suspected cholera cases each week from each sentinel site, tested stool for the presence of Vibrio cholerae O1/O139 by culture, and characterized antibiotic susceptibility in a subset of culture-positive isolates. We estimated the odds of being culture positive among suspected cholera cases according to different potential risk factors. From May 4, 2014 through November 30, 2021, we enrolled 51,414 suspected cases from our sentinel surveillance sites. We confirmed V. cholerae O1 in 5.2% of suspected cases through microbiological culture. The highest proportion of confirmed cholera cases was from Chittagong (9.7%) and the lowest was from Rangpur Division (0.9%). Age, number of purges, duration of diarrhea, occupation, and season were the most relevant factors in distinguishing cholera-positive suspected cases from cholera-negative suspected cases. Nationwide surveillance data show that cholera is circulating in Bangladesh and the southern region is more affected than the northern region. Antimicrobial resistance patterns indicate that multidrug resistance (resistance to three or more classes of antibiotics) of V. cholerae O1 could be a major threat in the future. Alignment of these results with Bangladesh’s cholera-control program will be the foundation for future research into the efficacy of cholera-control initiatives.

Human PKR transfected into murine cells stimulates expression of genes under control of the HIV1 or HTLV-1 LTR

Publisher: Elsevier BV

Date: 12-1995

DOI: 10.1006/VIRO.1995.0080

Abstract: We have analyzed the effect of transfection into murine NIH/3T3 cells of the human dsRNA-activated kinase PKR on the expression of the beta-galactosidase reporter gene, placed under control of the HIV1 or the HTLV-I LTR. beta-Galactosidase expression is stimulated when the reporter plasmids are cotransfected with wild-type PKR but inhibited when cotransfected with a catalytically inactive mutant PKR. In the case of HIV1, beta-galactosidase expression was not stimulated when cotransfection was carried out with PKR harboring mutations in the dsRNA binding domains, indicating that stimulation depends on the classical mode of PKR activation through dsRNA binding. In contrast, the dsRNA binding mutants of PKR could still partially stimulate beta-galactosidase expression from the HTLV-I LTR, suggesting that PKR activation in this case may involve different/additional mechanisms. These results show that, in addition to the known down-regulation of protein synthesis through elF2 phosphorylation, PKR can also positively stimulate gene expression in vivo, most probably through phosphorylation of a substrate distinct from elF2.

RETRACTED: Dendrimer nanocarriers as versatile vectors in gene delivery

Publisher: Elsevier BV

Date: 02-2010

DOI: 10.1016/J.NANO.2009.05.005

Abstract: The successful delivery of nucleic acids to particular target sites is the challenge that is being addressed using a variety of viral and nonviral delivery systems, both of which have distinct advantages and disadvantages. Nonviral vectors offer the advantage of safety and flexibility over viral vectors, although they lack efficiency. Dendrimers are novel, three-dimensional polymers that have the ability to interact with various forms of nucleic acids such as plasmid DNA, antisense oligonucleotides, and RNA to form complexes that protect the nucleic acid from degradation. The interaction between the dendrimers and the nucleic acids is purely electrostatic where the cationic dendrimer condenses the anionic nucleic acids. Because cell membranes are negatively charged, the net positive charge of the dendrimer nucleic acid complex determines the transfection efficiency, although highly cationic systems are also cytotoxic. The nature of the dendrimer nucleic acid complex depends on various factors like stoichiometry, concentration of dendrimer-amines and nucleic acid-phosphates, as well as bulk solvent properties like pH, salt concentration, buffer strength, and dynamics of mixing. This article aims to review the role of dendrimers as novel gene delivery vectors both in vitro and in vivo. Dendrimer-based transfection reagents have become routine tools for in vitro transfection, but in vivo delivery of therapeutic nucleic acids remains a challenge. This review discusses the role of dendrimers as novel gene delivery vectors both in vitro and in vivo. Dendrimer based transfection reagents have become routine tools for in vitro transfection but in vivo delivery of therapeutic nucleic acids remains a challenge.

Loss of HPV type 16 E7 restores cGAS-STING responses in human papilloma virus-positive oropharyngeal squamous cell carcinomas cells

Publisher: Elsevier BV

Date: 08-2021

DOI: 10.1016/J.JMII.2020.07.010

Abstract: Human papilloma viruses (HPV) are the main culprit in cervical and oropharyngeal cancers. HPV positive (+) cancers are regarded as 'oncogene addicted', displaying an absolute requirement for the continued expression of the oncogenes for their viability owing their survival, and thus making these genes salient targets for developing specific therapeutic agents. There is a strong association between HPV and oropharyngeal squamous cell carcinomas (OPSCC), a subset of head and neck cancers (HNCs). Alarmingly, HPV-associated OPSCC are on the rise globally, and the number of cases of HPV + OPSCCs surpasses that of cervical cancer in the USA. Here, we show that major HPV oncogenes, E6 and E7, are essential for the survival of HPV positive (+) OPSCCs, making these oncogenes salient targets for HPV-driven OPSCCs. HPV E7 is known to interact with STING, a component of the viral DNA-sensing cGAS-STING machinery which activates a pro-typical anti-viral type I interferon (IFN) response. Our recent work showed that E7 from HPV type 16 is responsible for the blockade of cGAS-STING responses in HPV + OPSCC cells. In this study, we show that CRISPR/Cas9-mediated loss of E7 from HPV + OPSCC cells, SCC2 and SCC104, restored cGAS-STING responses. Future work could involve HPV oncogene targeting leading to HPV + OPSCC tumour regression and that the combined use of STING agonists would induce favourable tumour clearance by activating appropriate anti-tumour responses.

Development of novel lipidic particles for siRNA delivery that are highly effective after 12 months storage

Publisher: Cold Spring Harbor Laboratory

Date: 13-08-2018

DOI: 10.1101/390641

Abstract: Liposomes are versatile and well-proven as a means to deliver nucleic acids into cells. Most of the formulation procedures used are labour intensive and result in unstable end products. We have previously reported on the development of a simple, yet efficient, hydration-of-freeze-dried-matrix (HFDM) method to entrap siRNA within lipid particles. Here we show that the particles are stable up to 12 months after storage room temperature (RT), 4°C or - 20°C. While RT storage results in changes in particle size and polydispersity, gene silencing of all particles was similar to freshly prepared particles following storage for 3, 6, 9 or 12 months at all temperatures. This is the first report of such long-term stability in siRNA-loaded liposomes.

Vaginal delivery of siRNA using a novel PEGylated lipoplex-entrapped alginate scaffold system

Publisher: Elsevier BV

Date: 11-2011

DOI: 10.1016/J.JCONREL.2011.02.002

Abstract: Sustained vaginal delivery of siRNA has been precluded by the mucosal barrier lining the vaginal tract. In contrast to prior reports, we showed that conventional lipoplexes administered intravaginally are unable to reach the vaginal epithelium under normal physiological conditions. Here we have developed a novel alginate scaffold system containing muco-inert PEGylated lipoplexes to provide a sustained vaginal presence of lipoplexes in vivo and to facilitate the delivery of siRNA/oligonucleotides into the vaginal epithelium. These PEGylated lipoplex-entrapped alginate scaffolds (PLAS) were fabricated using a freeze-drying method and the entrapment efficiency, release rate, and efficacy were characterized. We demonstrated that the PLAS system had an entrapment efficiency of ~50%, which released PEGylated lipoplexes gradually both in vitro and in vivo. While the presence of alginate diminished the cell uptake efficiency of PEGylated lipoplexes in vitro, as expected, we showed a six-fold increase their uptake into the vaginal epithelium compared to existing transfection systems following intravaginal administration in mice. A significant knockdown of Lamin A/C level was also observed in vaginal tissues using siLamin A/C-containing PLAS system in vivo. Overall, our results indicated the potential of the biodegradable PLAS system for the sustained delivery of siRNA/oligonucleotides to vaginal epithelium.

Identification of a histone family gene signature for predicting the prognosis of cervical cancer patients

Publisher: Springer Science and Business Media LLC

Date: 28-11-2017

DOI: 10.1038/S41598-017-16472-5

Abstract: Heterogeneity in terms of tumor characteristics, prognosis, and survival among cancer patients is an unsolved issue. Here, we systematically analyzed the aberrant expression patterns of cervical cancer using RNA-Seq data from The Cancer Genome Atlas (TCGA). We incorporated gene profiling, molecular signatures, functional and pathway information with gene set enrichment and protein-protein interaction (PPI) network analysis, to identify sub-networks of genes. Those identified genes relating to DNA replication and DNA repair-mediated signaling pathways associated with systemic lupus erythematosus (SLE). Next, we combined cross-validated prognostic scores to build an integrated prognostic model for survival prediction. The combined approach revealed that the DNA repair-mediated including the functional interaction module of 18 histone genes (Histone cluster 1 H2A, B and H4), were significantly correlated with the survival rate. Furthermore, five of these histone genes were highly expressed in three cervical cancer cohorts from the Oncomine database. Comparison of high and low histone variant-expressing human cervical cancer cell lines revealed different responses to DNA damage, suggesting protective functions of histone genes against DNA damage. Collectively, we provide evidence that two SLE-associated gene sets (HIST1H2BD and HIST1H2BJ and HIST1H2BD, HIST1H2BJ, HIST1H2BH, HIST1H2AM and HIST1H4K) can be used as prognostic factors for survival prediction among cervical cancer patients.

MUTATIONAL ANALYSIS OF THE DOUBLE-STRANDED-RNA (DSRNA) BINDING DOMAIN OF THE DSRNA-ACTIVATED PROTEIN-KINASE, PKR

Publisher: Elsevier BV

Date: 02-1995

DOI: 10.1074/JBC.270.6.2601

Inhibition of cervical cancer cell growth in vitro and in vivo with dual shRNAs

Publisher: Springer Science and Business Media LLC

Date: 19-11-2011

DOI: 10.1038/CGT.2010.72

Abstract: RNA interference (RNAi)-based gene silencing is widely used in laboratories for gene function studies and also holds a great promise for developing treatments for diseases. However, in vivo delivery of RNAi therapy remains a key issue. Lentiviral vectors have been employed for stable gene transfer and gene therapy and therefore are expected to deliver a stable and durable RNAi therapy. But this does not seem to be true in some disease models. Here, we showed that lentivirus delivered short-hairpin RNA (shRNA) against human papillomavirus (HPV) E6/E7 oncogenes were effective for only 2 weeks in a cervical cancer model. However, using this vector to carry two copies of the same shRNA or two shRNAs targeting at two different but closely related genes (HPV E6 and vascular endothelial growth factor) was more effective at silencing the gene targets and inhibiting cell or even tumor growth than their single shRNA counterparts. The cancer cells treated with dual shRNA were also more sensitive to chemotherapeutic drugs than single shRNA-treated cells. These results suggest that a multi-shRNA strategy may be a more attractive approach for developing an RNAi therapy for this cancer.

Hyperactivation of p53 using CRISPRa kills human papillomavirus-driven cervical cancer cells

Publisher: Springer Science and Business Media LLC

Date: 06-12-2022

DOI: 10.1007/S11262-022-01960-2

Svendborg Gymnasium

Location: Denmark

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Griffith University Griffith Health

Location: Australia

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University Of Otago

Location: New Zealand

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Delivery Of Gene-silencing Nucleotides

Start Date: 2007

End Date: 2009

Funder: National Health and Medical Research Council

Comprehensive Cell Imaging Facility

Start Date: 2011

End Date: 2011

Funder: Australian Research Council

Linkage Projects - Grant ID: LP0882320

Start Date: 2008

End Date: 02-2009

Amount: $217,750.00

Funder: Australian Research Council

Discovery Projects - Grant ID: DP0985215

Start Date: 2009

End Date: 12-2012

Amount: $400,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE0989436

Start Date: 2009

End Date: 03-2010

Amount: $400,000.00

Funder: Australian Research Council

Linkage Infrastructure, Equipment And Facilities - Grant ID: LE110100172

Start Date: 2011

End Date: 12-2011

Amount: $330,000.00

Funder: Australian Research Council