Horizontal transfer of RNAi between honeybees and Varroa destructor

Dissertação de mestrado em Biotecnologia Farmacêutica, apresentada à Faculdade de Farmácia da Universidade de CoimbraEste trabalho abrange uma revisão do estado de arte do processo de liofilização e da sua aplicação na área da Nanotecnologia. O foco principal desta revisão é a caracterização dos produtos liofilizados inovadores existentes no mercado e em fase de desenvolvimento clínico, com aplicação em Oncologia. Na área farmacêutica, a exploração das propriedades únicas das nanopartículas tornou-se diferenciadora pela demonstração de efeitos na biodisponibilidade de moléculas ativas e também na proteção contra a degradação enzimática. Mais recentemente, as nanopartículas têm sido usadas para proporcionar um aumento da especificidade e efetividade no direcionamento dos fármacos aos locais alvo, permitindo a diminuição da dose eficaz e da toxicidade geral. Com tudo isto, o desenvolvimento de nanoestruturas para transporte de fármacos tem sido objeto de um interesse crescente, tendo em vista colmatar alguns pontos negativos existentes nos sistemas de dosagem tradicionais. No entanto, a estabilidade das nanopartículas pode constituir um problema, pelo que a sua concepção não é propriamente linear. No caso específico das nanodispersões coloidais, fatores físicos e químicos contribuem para uma baixa estabilidade a longo termo, provocando a destabilização do sistema que limita a sua aplicação clínica. A liofilização surge neste cenário como um processo tecnológico que pode minimizar os problemas de estabilidade das suspensões aquosas de nanopartículas. Trata-se de uma técnica de desidratação muito eficiente quando comparada com as demais, que tem vindo a apresentar melhorias na estabilidade destas suspensões coloidais, e por esta razão merece destaque. Cada produto possui um ciclo de liofilização único em função das suas particularidades, o que faz com que este processo seja uma operação unitária. Para ser percetível a forma como o processo de liofilização é executado cumprindo as boas práticas laboratoriais (BPL/GLP) e de fabrico (BPF/GMP) são explícitos, neste estudo, os requisitos normativos a ter em conta. A liofilização, além de ser útil ao nível da estabilidade, é ainda utilizada para outras finalidades que estão descritas no estudo. De forma a elucidar toda a operação envolvida neste processo é feita uma breve abordagem das etapas envolvidas, dos métodos utilizados, das ix aplicações farmacêuticas e dos principais pros e contras, com o intuito de entender a importância da formulação de nanopartículas liofilizadas. Será ainda dedicado um capítulo ao estudo dos componentes e das condições do processo para conseguir obter resistência às tensões a que as suspensões de nanopartículas são submetidas durante a fase de congelamento na liofilização. Nesta fase, o efeito crioprotetor ou lioprotetor é determinante, dado que na reidratação do produto é mandatório que as propriedades originais das nanopartículas sejam mantidas. A respeito dos avanços recentes com aplicação deste processo, são ainda apresentadas as formulações liofilizadas que estão inseridas no mercado e os ensaios clínicos e pré-clínicos existentes para o tratamento de doenças do foro oncológico, com referência aos produtos protegidos por patente. A relevância deste trabalho traduz-se no facto de o resultado da pesquisa ser orientado em ambiente empresarial e académico, com vista à inserção no processo de valorização da propriedade intelectual de uma spin-off da Universidade de CoimbraThis work aims at conducting a review of the state of art of the lyophilization process and its application in the field of Nanotechnology. The main focus is the characterization of existing innovative freeze-dried products on the market and in clinical evaluation, with applications in Oncology. In the pharmaceutical area, the unique properties of nanoparticles began to demonstrate positive effects on the bioavailability of active molecules and also in protecting against enzymatic degradation. More recently, nanoparticles have been used to provide increased specificity and effectiveness in the targeting of drugs to specific locations, thereby reducing the effective dose and toxicity. With all this, the development of nanostructures for drug delivery has provided the solution for existing weaknesses in traditional dosing systems. However, the stability of the nanoparticles is not exactly a linear topic. In the case of colloidal nanodispersions, physical and chemical factors contribute to a low long term stability, leading to destabilization of the system and thus creating an obstacle to its clinical application. Lyophilization in this scenario appears as a technological process that can minimize stability problems of aqueous suspensions of nanoparticles. It is a very efficient dewatering technique, which has been presenting improvements in the stability of these colloidal suspensions, and therefore noteworthy. Each product has a single lyophilization cycle on the basis of their features, which makes this process a unitary operation. To be noticeable how the freeze-drying process is performed in compliance with the good laboratory practice (BPL/GLP) and manufacturing practice (BPF/GMP), the regulatory requirements to be considered are explicit in this study. This technique, besides being useful for stability issues, it is still used for other purposes which are described in the study. In order to elucidate the entire operation involved in this process, it is made a brief approach of the steps involved, the methods used, the pharmaceutical applications and the main pros and cons of the formulation of lyophilized nanoparticles. The study also comprises a chapter on the components and process conditions to achieve resistance to the stresses to which the nanoparticulate suspensions are subjected during the xi freezing stage of the lyophilization. At this stage, the cryoprotectant or lioprotetor effect is decisive for the rehydration, in order to preserve that the original properties of the nanoparticles. Regarding the recent advances in the application of this process, this study presents the most recent lyophilized formulations on the market and in clinical and preclinical trials for the treatment of oncological diseases, with reference to the proprietary products. The main conclusions of this work will be included in the valuing process of the intellectual property of a spin-off from the University of Coimbra, combining an academic and business perspective on a pharmaceutical process of industrial application

CRISPR/Cas9 Ablation of Integrated HIV-1 Accumulates Proviral DNA Circles with Reformed Long Terminal Repeats

Gene editing may be used to excise the human immunodeficiency virus type 1 (HIV-1) provirus from the host cell genome, possibly eradicating the infection. Here, using cells acutely or latently infected by HIV-1 and treated with long terminal repeat (LTR)-targeting CRISPR/Cas9, we show that the excised HIV-1 provirus persists for a few weeks and may rearrange in circular molecules. Although circular proviral DNA is naturally formed during HIV-1 replication, we observed that gene editing might increase proviral DNA circles with restored LTRs. These extrachromosomal elements were recovered and probed for residual activity through their transfection in uninfected cells. We discovered that they can be transcriptionally active in the presence of Tat and Rev. Although confirming that gene editing is a powerful tool to eradicate HIV-1 infection, this work highlights that, to achieve this goal, the LTRs must be cleaved in several pieces to avoid residual activity and minimize the risk of reintegration in the context of genomic instability, possibly caused by the off-target activity of Cas9. IMPORTANCE The excision of HIV-1 provirus from the host cell genome has proven feasible in vitro and, to some extent, in vivo. Among the different approaches, CRISPR/Cas9 is the most promising tool for gene editing. The present study underlines the remarkable effectiveness of CRISPR/Cas9 in removing the HIV-1 provirus from infected cells and investigates the fate of the excised HIV-1 genome. This study demonstrates that the free provirus may persist in the cell after editing and in appropriate circumstances may reactivate. As an episome, it might be transcriptionally active, especially in the presence of Tat and Rev. The persistence of the HIV-1 episome was strongly decreased by gene editing with multiple targets. Although gene editing has the potential to eradicate HIV-1 infection, this work highlights a potential issue that warrants further investigation

Large-scale field application of RNAi technology reducing Israeli acute paralysis virus disease in honey bees (Apis mellifera, Hymenoptera: Apidae).

The importance of honey bees to the world economy far surpasses their contribution in terms of honey production; they are responsible for up to 30% of the world's food production through pollination of crops. Since fall 2006, honey bees in the U.S. have faced a serious population decline, due in part to a phenomenon called Colony Collapse Disorder (CCD), which is a disease syndrome that is likely caused by several factors. Data from an initial study in which investigators compared pathogens in honey bees affected by CCD suggested a putative role for Israeli Acute Paralysis Virus, IAPV. This is a single stranded RNA virus with no DNA stage placed taxonomically within the family Dicistroviridae. Although subsequent studies have failed to find IAPV in all CCD diagnosed colonies, IAPV has been shown to cause honey bee mortality. RNA interference technology (RNAi) has been used successfully to silence endogenous insect (including honey bee) genes both by injection and feeding. Moreover, RNAi was shown to prevent bees from succumbing to infection from IAPV under laboratory conditions. In the current study IAPV specific homologous dsRNA was used in the field, under natural beekeeping conditions in order to prevent mortality and improve the overall health of bees infected with IAPV. This controlled study included a total of 160 honey bee hives in two discrete climates, seasons and geographical locations (Florida and Pennsylvania). To our knowledge, this is the first successful large-scale real world use of RNAi for disease control

Bidirectional Transfer of RNAi between Honey Bee and <em>Varroa destructor</em>: <em>Varroa</em> Gene Silencing Reduces <em>Varroa</em> Population

<div><p>The mite <em>Varroa destructor</em> is an obligatory ectoparasite of the honey bee (<em>Apis mellifera</em>) and is one of the major threats to apiculture worldwide. We previously reported that honey bees fed on double-stranded RNA (dsRNA) with a sequence homologous to that of the Israeli acute paralysis virus are protected from the viral disease. Here we show that dsRNA ingested by bees is transferred to the <em>Varroa</em> mite and from mite on to a parasitized bee. This cross-species, reciprocal exchange of dsRNA between bee and <em>Varroa</em> engendered targeted gene silencing in the latter, and resulted in an over 60% decrease in the mite population. Thus, transfer of gene-silencing-triggering molecules between this invertebrate host and its ectoparasite could lead to a conceptually novel approach to <em>Varroa</em> control.</p> </div

Expression of an Entire Bacterial Operon in Plants1[W][OA]

Multigene expression is required for metabolic engineering, i.e. coregulated expression of all genes in a metabolic pathway for the production of a desired secondary metabolite. To that end, several transgenic approaches have been attempted with limited success. Better success has been achieved by transforming plastids with operons. IL-60 is a platform of constructs driven from the geminivirus Tomato yellow leaf curl virus. We demonstrate that IL-60 enables nontransgenic expression of an entire bacterial operon in tomato (Solanum lycopersicum) plants without the need for plastid (or any other) transformation. Delivery to the plant is simple, and the rate of expressing plants is close to 100%, eliminating the need for selectable markers. Using this platform, we show the expression of an entire metabolic pathway in plants and delivery of the end product secondary metabolite (pyrrolnitrin). Expression of this unique secondary metabolite resulted in the appearance of a unique plant phenotype disease resistance. Pyrrolnitrin production was already evident 2 d after application of the operon to plants and persisted throughout the plant's life span. Expression of entire metabolic pathways in plants is potentially beneficial for plant improvement, disease resistance, and biotechnological advances, such as commercial production of desired metabolites

Mean (+ SE) number of <i>Varroa</i> mites per bee in four treatments.

<p>Different letters above columns indicate significant differences between treatments (<i>P</i><0.05).</p

List of <i>Varroa</i> dsRNA sequences used in <i>Varroa</i> gene silencing.

<p>This table lists the <i>Varroa</i> dsRNA sequences numbers, genes function, and the mixtures they are contained in. The full sequences are located in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003035#ppat.1003035.s003" target="_blank">Table S1</a>.</p