Antiviral modified siRNA swarms for treatment of herpes simplex virus infection

Herpes simplex virus type 1 (HSV-1) is a common virus of humans carried by half of the global population. After the primary infection, HSV has the ability to establish life-long latency, wherefrom it can reactivate. The latent state cannot be eliminated with modern pharmaceuticals, nor is there a vaccine available, despite massive efforts. Instead, the treatment focuses on diminishing viral replication. The current treatment, however, is insufficient, as it relies almost solely on acyclovir (ACV), and its derivatives, which share their mechanism of action, making ACV-resistant infections almost untreatable. Unfortunately, such infections are rather common, as severe HSV infections require long-term prophylactic treatment to prevent recurrences, which selects for ACV-resistant variants. The lack of treatment diversity against HSV-1 infections encourages for research on novel therapies. Previously, enzymatically synthetized swarms of small interfering (si)RNA have been established as feasible means to treat HSV infection in vitro and in vivo. They differ from regular siRNA by their enzymatic synthesis and by their substantially longer target sequence. Thus, the emergence of resistance, even during long-term prophylactic treatment, is unlikely. However, as all RNA therapy, siRNA swarms face challenges with RNA stability. Therefore, in this study, the goal was to improve the siRNA swarms by synthesizing novel anti-HSV siRNA swarms with chemical 2′-fluoro-modifications to increase RNA efficacy and stability. The modified siRNA swarms, representing modifications of each nucleotide, were first validated in vitro in cells of the nervous system. The research was continued in a highly translational cell line representing the human cornea, which we first validated for use in antiviral RNAi studies. In both cell types, the modified siRNA swarm(s) proved well tolerated and potent beyond the unmodified counterparts, with only modest effects on the host innate responses, even in the presence of viral challenge. Furthermore, all studied HSV-1 strains, including various clinical isolates, were highly sensitive to both modified and unmodified siRNA swarms, whereas their ACV sensitivity varied, proving the potential of siRNA swarms for future therapeutic use. This study shows that incorporation of modified nucleotides to the anti-HSV siRNA swarms is advantageous, and should therefore be preferred in future studies

Treatment of herpes simplex virus infection using antiviral siRNA swarms with 2’-fluoro-modifications

Herpes simplex virus 1 (HSV-1) is a very common pathogen. Besides mostly harmless oral lesions, HSV-1 causes severe diseases such as neonatal herpes, herpes encephalitis and herpes keratitis, the primary cause of infectious blindness worldwide. The available anti-herpes chemotherapy is efficient but depends on a functional viral thymidine kinase. Long-term treatment, required especially in severe diseases, promotes emergence of thymidine kinase mutant strains. These strains are multi-drug resistant, and may lead to dangerous untreatable exacerbations, demonstrating an evident unmet medical need. Small interfering RNA (siRNA) swarms are a novel antiviral approach with extensive tolerance for pathogen mutations. In contrast to regular siRNAs targeting around twenty nucleotides, swarms can target thousands, and thus overcome major challenges of regular antiviral-siRNAs, such as emergence of resistant mutant strains. The most extensively studied siRNA swarm target is the essential UL29 gene of HSV-1. The UL29 targeting siRNA swarm has proven antiviral efficacy against multiple patient-derived strains in vitro and significant inhibition of virus replication in vivo. Here, the swarm is improved by 2’-fluoro-modifications to achieve advanced stability and potency. In this Master’s thesis, effects of incorporated 2’-fluoro-nucleotides on cellular tolerability, host responses and antiviral efficacy are studied in vitro. According to the results, the modified siRNA swarms are well tolerated and demonstrate high antiviral efficacy in prophylactic and therapeutic settings in vitro. The modified siRNA swarms were better than or equal to the nonmodified siRNA swarms in every studied aspect. Overall, the results encourage for subsequent in vivo experiments utilizing the modified siRNA swarms.Herpes simplex virus tyyppi 1 (HSV-1) on yleinen taudinaiheuttaja, joka tunnetaan parhaiten aiheuttamistaan epämiellyttävistä ja toistuvista yskänrokoista. HSV-1 voi kuitenkin aiheuttaa myös vakavampia tautitiloja, kuten sarveiskalvontulehdusta. HSV-1:n aiheuttama sarveiskalvontulehdus on maailman yleisin sokeuteen johtava infektioperäinen sairaus, johon nykyinen lääkehoito on riittämätön. Herpesinfektioiden nykyinen lääkehoito on tehokasta, mutta edellyttää viruksen oman tymidiinikinaasigeenin toimintaa. Erityisesti vakavammissa sairauksissa vaadittava pitkäaikainen ja ennaltaehkäisevä lääkehoito voi johtaa tymidiinikinaasimutatoituneiden viruskantojen ilmaantumiseen. Nämä lääkeresistentit viruskannat ovat selkeä puute nykyisessä lääkehoidossa, ja siten tärkeä lääkekehityskohde. Pienet häiritsevät RNA:t eli siRNA:t (engl. small interfering RNA) johtavat geenien hiljentymiseen. Niitä voidaan käyttää estämään virusten lisääntymistä kohdistamalla siRNA:t kohdeviruksen välttämättömään geeniin. Tavallisesti siRNA:t on kohdistettu verrattain lyhyeen sekvenssijaksoon. Pieni kohdealue altistaa tehon menetykseen joko lähisukuisten virusten perimän monimuotoisuuden vuoksi tai mutaation kautta. SiRNA-parvet tuotetaan entsymaattisesti pitkästä kohdealueesta, jolloin niiden teho kattaa viruskantojen monimuotoisuuden ja mahdolliset mutaatiot kohdesekvenssissä. SiRNA-parvia on tutkittu lääkkeenä herpesinfektioon sekä in vitro että in vivo erittäin lupaavin tuloksin. SiRNA:t kuitenkin hajoavat nopeasti elimistössä, mikä vähentää hoitomuodon potentiaalia, annostelutavasta riippuen. SiRNA:n kestävyyttä pystytään kuitenkin parantamaan kemiallisilla modifikaatioilla. Tässä Pro Gradu -tutkielmassa selvitettiin siRNA-parviin sisällytettyjen 2’-fluoro-nukleotidien vaikutusta parvien tehoon herpestä vastaan in vitro. Lisäksi selvitettiin solujen luonnollisen immuniteetin vaste muunnelluille siRNA-parville. Tutkimuksissa käytetyt solulinjat edustivat hermostoa ja sarveiskalvoa, jotka ovat olennaisia herpesinfektion kohdekudoksia. Muunnellut siRNA-parvet ovat tulosten perusteella vähintään yhtä turvallisia ja jopa tehokkaampia kuin perinteiset siRNA-parvet. Molempien parvityyppien havaittiin lisäksi estävän virusinfektiota ainakin viiden vuorokauden ajan kerta-annostelun jälkeen. Tulokset valottavat perinteisten ja erityisesti muunneltujen siRNA-parvien potentiaalia terapeuttisena ja ennaltaehkäisevänä lääkehoitomuotona ja kannustavat niiden jatkotutkimuksiin in vivo

Sex Ed to Go- An Analysis of Comprehensive Sexual Education Mobile Phone Applications

The United States has one of the highest teen pregnancy rates among developed countries and the numbers of STIs have increased during the last few years. Mobile phone applications constitute a promising platform to disseminate sexual health information and to reduce adverse health outcomes, particularly among teens. In order to be effective, apps have to follow a comprehensive approach to sexuality education, include concepts of behavior change theories, and adhere to health literacy principles. This study used a mixed-methods approach consisting of a quantitative content analysis and qualitative thematic analysis to assess the quality of sexual education apps available to users on the iOS and Android app market. The results show that there is a dearth of health literate, evidence- and theory-based sexual education apps available to teenagers in the United States. The focus remained on the negative consequences of sex and their prevention, while other topics such as identity or personal safety were hardly addressed. Interactive features were used in only 40% of apps. Furthermore, content was female-oriented and reinforced negative stereotypes and perceived norms that may have a negative impact on the sexual health of teens. These findings suggest that the potential of apps has not yet been fully realized in the context of sexual health promotion. This thesis provides suggestions and guidelines for individuals interested in developing theory-and evidence-based sexual education apps

Klimaethik an den Grenzen realpolitischer Machbarkeit: Rezension zu "From Big Oil to Big Green: Holding the Oil Industry to Account for the Climate Crisis" von Marco Grasso

Marco Grasso: From Big Oil to Big Green: Holding the Oil Industry to Account for the Climate Crisis. Cambridge, MA: MIT Press 2022. 978-0-262-36977-

Image processing methods for limited angle tomography and sparse angle tomography

The purpose of this thesis was two-fold: Firstly, to evaluate if limited angle tomography is suitable for clinical implant planning. Secondly, to improve clinical image quality and workflow of the limited angle tomography by developing new imaging processing algorithms. Conventional computed tomography (CT) design is not optimal in the sense of cost, workflow or dose for two reasons. Firstly, CT devices are typically expensive and bulky devices because they require a stable X-ray production, rigid gantry with accurate and repeatable movements, high scanning speed, solid patient support and a low-noise X-ray detector. Secondly, current non-regularized reconstruction techniques require high dose per projection image as well as a huge number of projection image. This also limits the usage of the CT imaging to serious trauma cases and other lethal diseases. To overcome the limitations mentioned above, new approaches have been introduced to replace conventional CT imaging. For example, year 2007 a dental imaging technology company Palodex Group released an upgrade kit for standard panoramic X-ray device, called Volumetric Tomography (VT), which is based on limited angle tomography. In the first article, we demonstrated that limited angle tomography is able to give similar clinical information as CT devices in dental implant planning. Therefore, the implant planning could be executed more cost and dose effectively when suitable algorithms are applied throughout the reconstruction process. Since limited angle tomography system applies small number of X-ray images taken from a limited aperture, new image processing methods are required for clinically suitable image quality. For that reason, two novel imaging processing methods and one analyzing method were created and documented in this work. In the second article, a new image processing method based on modification of the constrained least-square filter for extremely sparse situations was introduced. In this method, called Wiener-filter based iterative reconstruction technique (WIRT), we considered the uncertainty of the interpolation as noise and utilized the regularization only in the regions where the uncertainty is the dominating factor. In the third article, a new sinogram estimation algorithm called sinogram interpolation technique (SINT) was created, where the missing sinogram columns were estimated based on the known columns. In the fourth article, a method named mutual information based technology (MINT) was developed to estimate the imaging geometry directly from the projection data. In this method, the imaging angles can be estimated based on the projection images without any external markers or additional constructions to the device. Therefore, this method simplifies workflow and the improves imaging angle accuracy significantly.Tämän väitöstyön tarkoituksena oli arvioida alhaisen sädeannoksen tuottavan tietokonetomografialaitteen käyttöä implanttisuunnittelussa sekä luoda uusia kuvanlaatua ja käytettävyyttä parantavia menetelmiä tähän konseptiin sekä yleisesti alhaisen säteilyn omaaviin tietokonetomografialaitteisiin. Tietokonetomografian tarkoitus on luoda kolmeulotteinen malli kohteesta perustuen useisiin röntgenkuviin, jotka ovat otettu eri suunnista kohdetta. Verrattuna tavalliseen röntgenkuvaukseen tietokonetomografia lisää röntgenkuvauksen kliinistä hyötyä, sillä se mahdollistaa kappaleiden muotojen ja niiden välisten etäisyyksien mittaamisen. Perinteinen tietokonetomografia ei kuitenkaan aina ole optimaalinen kustannuksen, käytettävyyden tai annoksen suhteen. Tämä johtuu siitä, että se perustuu mekaanisesti tarkkaan laitteistoon, joka suunnataan oikeassa ja ennalta määrätyssä kulmassa potilaaseen nähden ja kykenee ottamaan useita, jopa satoja, röntgenkuvia mahdollisimman lyhyessä ajassa. Tästä syystä tietokonetomografialaitteet ovat perinteisesti olleet hyvin raskaita ja kalliita laitteita, jotka tuottavat potilaalle korkean sädeannoksen. Edellä mainituista syistä on viimevuosina kehitelty uusia kolmeulotteisia kuvausmenetelmiä, joissa laitteisto on pyritty kehittämään kevyemmäksi ja sädeannokseltaan pienemmäksi kuin perinteiset tietokonetomografialaitteet. Pieni röntgenkuvamäärä asettaa kuitenkin suuria haasteita kolmeulotteisen tiedon muodostamiseen. Koska nykyiset tietokonetomografiassa käytetyt kuvankäsittelyoperaatiot on suunniteltu suurille säde-annoksille, ne eivät sellaisenaan sovellu pienannoksiseen tomografialaskentaan. Tässä työssä pyrittiin toteuttamaan, arvioimaan ja dokumentoimaan uusia kuvankäsittelyoperaatioita jotka soveltuvat pienemmän sädeannoksen kuvaukseen eli ns. harva- ja rajoitetun kulman tomografiaan. Väitöskirja sisältää kolme täysin uutta kuvankäsittelymenetelmää joista kaksi parantaa kliinistä kuvanlaatua ja kolmas laitteiston käytettävyyttä. Tässä väitöskirjassa osoitetaan myös että vaikka pienannoksinen tietokonetomografialaite ei tuota niin tarkkaa kuvaa kuin perinteinen tietokonetomografialaite, sillä saadaan riittävän tarkka tieto hammasimplanttisuunniteluun

Native RNA purification method for small RNA molecules based on asymmetrical flow field-flow fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules.Peer reviewe

Native RNA Purification Method for Small RNA Molecules Based on Asymmetrical Flow Field-Flow Fractionation

RNA molecules provide promising new possibilities for the prevention and treatment of viral infections and diseases. The rapid development of RNA biology and medicine requires advanced methods for the purification of RNA molecules, which allow fast and efficient RNA processing, preferably under non-denaturing conditions. Asymmetrical flow field-flow fractionation (AF4) enables gentle separation and purification of macromolecules based on their diffusion coefficients. The aim of the study was to develop an AF4 method for efficient purification of enzymatically produced antiviral small interfering (si)RNA molecules and to evaluate the overall potential of AF4 in the separation of short single-stranded (ss) and double-stranded (ds) RNA molecules. We show that AF4 separates monomeric ssRNA from dsRNA molecules of the same size and monomeric ssRNA from multimeric forms of the same ssRNA. The developed AF4 method enabled the separation of enzymatically produced 27-nt siRNAs from partially digested substrate dsRNA, which is potentially toxic for mammalian cells. The recovery of AF4-purified enzymatically produced siRNA molecules was about 70%, which is about 20% higher than obtained using anion-exchange chromatography. The AF4-purified siRNAs were not toxic for mammalian cells and fully retained their biological activity as confirmed by efficient inhibition of herpes simplex virus 1 replication in cell culture. Our work is the first to develop AF4 methods for the separation of short RNA molecules

Use of Evidence-Based Best Practices and Behavior Change Techniques in Breast Cancer Apps: Systematic Analysis

Background: Theoretically designed mobile health (mHealth) breast cancer interventions are essential for achieving positive behavior change. In the case of breast cancer, they can improve the health outcomes of millions of women by increasing prevention and care efforts. However, little is known about the theoretical underpinnings of breast cancer apps available to the general public. Objective: Given that theories may strengthen mHealth interventions, this study aimed to identify breast cancer apps designed to support behavior change, to assess the extent to which they address content along the cancer care continuum and contain behavior change techniques, and to assess the degree to which star rating is related to theory-based design. Methods: Using a criteria-based screening process, we searched 2 major app stores for breast cancer apps designed to promote behavior change. Apps were coded for content along the cancer care continuum and analyzed for behavior change techniques. The Mann-Whitney U test was used to examine the relationship between star ratings and the use of behavior change techniques in apps with star ratings compared to those without ratings. Results: The search resulted in a total of 302 apps, of which 133 were identified as containing breast cancer content. Only 9.9% (30/302) of apps supported behavior change and were further analyzed. These apps were disproportionally focused on behaviors to enhance early detection, whereas only a few apps supported care management, treatment, and posttreatment behaviors. Regarding theories, 63% (19/30) of apps customized content to users, 70% (21/30) established a health-behavior link, and 80% (24/30) provided behavior change instructions. Of the 30 apps, 15 (50%) prompted intention formation whereas less than half of the apps included goal setting (9/30, 30%) and goal reviewing (7/30, 23%). Most apps did not provide information on peer behavior (7/30, 23%) or allow for social comparison (6/30, 20%). None of the apps mobilized social norms. Only half of the apps (15/30, 50%) were user rated. The results of the Mann-Whitney U test showed that apps with star ratings contained significantly more behavior change techniques (median 6.00) than apps without ratings. The analysis of behavior change techniques used in apps revealed their shortcomings in the use of goal setting and social influence features. Conclusions: Our findings indicate that commercially available breast cancer apps have not yet fully realized their potential to promote behavior change, with only a minority of apps focusing on behavior change, and even fewer including theoretical design to support behavior change along the cancer care continuum. These shortcomings are likely limiting the effectiveness of apps and their ability to improve public health. More attention needs to be paid to the involvement of professionals in app development and adherence to theories and best practices in app design to support individuals along the cancer care continuum