5 research outputs found

    CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression

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    Sjuksköterskeyrket innebär ofta långvarig stress och bristfällig arbetsmiljö. Personalbrist, hög arbetsbelastning och bristande inflytande på arbetsplatsen är orsaker till att sjuksköterskor väljer att sluta inom yrket. För lite forskning gällande den upplevda stressen på arbetsplatsen inom sjuksköterskeyrket råder. Därför finns anledning till ökad kunskap kring ämnet. Syftet med studien är att undersöka i hur hög utsträckning sjuksköterskor upplever stress i sin arbetsmiljö, och studien är baserad på 1044 yrkesverksamma sjuksköterskor över hela landet som är medlemmar i Facebookgruppen ”Sjuksköterskan”. Dessa personer har fått svara på enkäten Work Stress Questionnaire med 21 frågor. Resultatet från denna studie visar att arbetsbelastningen ökat och personalen har inte möjlighet att påverka beslut som tas på arbetsplatsen. Konflikter är också förekommande där chefen, i de flesta fall, inte gör något för att lösa dessa konflikter. Sjuksköterskorna sätter höga krav på sig själva och är mycket engagerade i arbetet, men har ofta svårt att sätta gränser. Resultatet visar också att en hög andel har svårt att hinna med familj, vänner och fritidsintressen. Det finns tidigare studier med samma mätinstrument som visar att arbetsbelastningen och ansvarstagandet har ökat under de senaste åren. Eventuellt kan detta i slutändan innebära en risk för ökad utbrändhet och fler sjukskrivningar

    Gene targeting and delivery of therapeutic oligonucleotides

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    Research in gene therapeutic strategies involving oligonucleotides (ONs) constitutes a growing field with several clinical products already approved and many more under intense investigation. The recent advances have mainly involved the Antisense ON platform, with the aim of modulating gene expression on the RNA level. In contrast, targeting DNA using antigene ONs is a much less explored therapeutic option, but has been shown to be able to provide a potent alternative for the modulation of gene expression. Many genetic diseases originate in mutations in the genome, and by directly targeting these using anti-gene ONs could potentially have advantages over RNA based options, including lower dosage and reduced toxicity. In this thesis, several anti-gene therapeutic approaches involving ONs are presented, optimized and evaluated both in vitro and in cells. In Paper I, a novel approach for the generation of Zorro-LNA using click chemistry is developed, in order to join the two antigene ONs involved in a 3´-5´- 5´-3´orientation. This strategy replaces the use of reverse LNA phosphoroamidites and provides a screening platform suitable for optimizing new Zorro-LNA constructs. In paper II, single stranded ONs targeting the CAG-repeat region in the Huntingtin gene is used to down-regulate the mutant protein responsible for Huntington´s disease. The ONs are active in patient-derived fibroblasts and can efficiently reduce both mRNA and protein levels up to seven days following transfection and naked uptake delivery strategies. We also demonstrate, in different assays, that the mode of action is through ON binding to DNA, and not through RNA interactions. In paper III, we optimize different bisLNA anti-gene ON constructs for efficient DNA strand invasion using novel chemistries and intercalators. We demonstrate the selective binding through both Hoogsteen and Watson-Crick hydrogen bonding to supercoiled DNA in a physiological environment. In addition, the DNA binding is assessed in bacterial cells and detected using rolling circle amplification. Paper IV evaluates the specific delivery to cancer cells using aptamer-mediated uptake of LNA-containing ONs. The effect on the aptamer plasticity is investigated using chemical modifications, both in the cargo ON and in the aptamer itself, as well as the influence of the construct properties on cellular uptake in two different cell lines. Taken together, the results presented in this thesis aims at advancing the anti-gene based ON therapeutic strategies in terms of efficacy and specific delivery

    Engineered Exosomes for Delivery of Therapeutic siRNAs to Neurons

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    Extracellular vesicles (EVs), exosomes and microvesicles, transfer endogenous RNAs between neurons over short and long distances. We have explored EVs for siRNA delivery to brain. (1) We optimized siRNA chemical modifications and siRNA conjugation to lipids for EV-mediated delivery. (2) We developed a GMP-compatible, scalable method to manufacture active EVs in bulk. (3) We characterized lipid and protein content of EVs in detail. (4) We established how protein and lipid composition relates to siRNA delivering activity of EVs, and we reverse engineered natural exosomes (small EVs) into artificial exosomes based on these data. We established that cholesterol-conjugated siRNAs passively associate to EV membrane and can be productively delivered to target neurons. We extensively characterized this loading process and optimized exosome-to-siRNA ratios for loading. We found that chemical stabilization of 5\u27-phosphate with 5\u27-E-vinylphosphonate and chemical stabilization of all nucleotides with 2\u27-O-methyl and 2\u27-fluoro increases the accumulation of siRNA and the level of mRNA silencing in target cells. Therefore, we recommend using fully modified siRNAs for lipid-mediated loading to EVs. Later, we identified that α-tocopherol-succinate (vitamin E) conjugation to siRNA increases productive loading to exosomes compared to originally described cholesterol. Low EV yield has been a rate-limiting factor in preclinical development of the EV technology. We developed a scalable EV manufacturing process based on three-dimensional, xenofree culture of mesenchymal stem cells and concentration of EVs from conditioned media using tangential flow filtration. This process yields exosomes more efficient at siRNA delivery than exosomes isolated via differential ultracentrifugation from two-dimensional cultures of the same cells. In-depth characterization of EV content is required for quality control of EV preparations as well as understanding composition–activity relationship of EVs. We have generated mass-spectrometry data on more than 3000 proteins and more than 2000 lipid species detected in exosomes (small EVs) and microvesicles (large EVs) isolated from five different producer cells: two cell lines (U87 and Huh7) and three mesenchymal stem cell types (derived from bone marrow, adipose tissue and umbilical cord Wharton’s jelly). These data represent an indispensable resource for the community. Furthermore, relating composition change to activity change of EVs isolated from cells upon serum deprivation allowed us to identify essential components of siRNA-delivering exosomes. Based on these data we reverse engineered natural exosomes into artificial exosomes consisting of dioleoyl-phosphatidylcholine, cholesterol, dilysocardiolipin, Rab7, AHSG and Desmoplakin. These artificial exosomes reproduced efficient siRNA delivery of natural exosomes both in vitro and in vivo. Artificial exosomes may facilitate manufacturing, quality control and cargo loading challenge that currently impede the therapeutic EV field

    Detection of cruciform DNA in vivo

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