| 1. |
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| 2. |
- Coenen-Stass, Anna M. L., et al.
(författare)
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Comprehensive RNA-Sequencing Analysis in Serum and Muscle Reveals Novel Small RNA Signatures with Biomarker Potential for DMD
- 2018
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Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 13, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- Extracellular small RNAs (sRNAs), including microRNAs (miRNAs), are promising biomarkers for diseases such as Duchenne muscular dystrophy (DMD), although their biological relevance is largely unknown. To investigate the relationship between intracellular and extracellular sRNA levels on a global scale, we performed sRNA sequencing in four muscle types and serum from wild-type, dystrophic mdx, and mdx mice in which dystrophin protein expression was restored by exon skipping. Differentially abundant sRNAs were identified in serum (mapping to miRNA, small nuclear RNA [snRNA], and PIWI-interacting RNA [piRNA] loci). One novel candidate biomarker, miR-483, was increased in both mdx serum and muscle, and also elevated in DMD patient sera. Dystrophin restoration induced global shifts in miRNA (including miR-483) and snRNA-fragment abundance toward wild-type levels. Specific serum piRNA-like sRNAs also responded to exon skipping therapy. Absolute miRNA expression in muscle was positively correlated with abundance in the circulation, although multiple highly expressed miRNAs in muscle were not elevated in mdx serum, suggesting that both passive and selective release mechanisms contribute to serum miRNA levels. In conclusion, this study has revealed new insights into the sRNA biology of dystrophin deficiency and identified novel DMD biomarkers.
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| 3. |
- Dash-Wagh, Suvarna, et al.
(författare)
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Intracellular Delivery of Short Interfering RNA in Rat Organ of Corti Using a Cell-penetrating Peptide PepFect6
- 2012
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Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 1:e61
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Tidskriftsartikel (refereegranskat)abstract
- RNA interference (RNAi) using short interfering RNA (siRNA) is an attractive therapeutic approach for treatment of dominant-negative mutations. Some rare missense dominant-negative mutations lead to congenital-hearing impairments. A variety of viral vectors have been tested with variable efficacy for modulating gene expression in inner ear. However, there is concern regarding their safety for clinical use. Here, we report a novel cell-penetrating peptide (CPP)-based nonviral approach for delivering siRNA into inner ear tissue using organotypic cultures as model system. PepFect6 (PF6), a variant of stearyl-TP10, was specially designed for improved delivery of siRNA by facilitating endosomal release. We show that PF6 was internalized by all cells without inducing cytotoxicity in cochlear cultures. PF6/siRNA nanoparticles lead to knockdown of target genes, a housekeeping gene and supporting cell-specific connexin 26. Interestingly, application of PF6/connexin 26 siRNA exhibited knockdown of both connexin 26 and 30 mRNA and their absence led to impaired intercellular communication as demonstrated by reduced transfer of calcein among the PF6/connexin 26-siRNA-treated cells. Thus, we conclude that PF6 is an efficient nonviral vector for delivery of siRNA, which can be applied as a tool for the development of siRNA-based therapeutic applications for hearing impairments.
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| 4. |
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| 7. |
- Freimann, Krista, et al.
(författare)
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Formulation of Stable and Homogeneous Cell-Penetrating Peptide NF55 Nanoparticles for Efficient Gene Delivery In Vivo
- 2018
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Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 10, s. 28-35
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Tidskriftsartikel (refereegranskat)abstract
- Although advances in genomics and experimental gene therapy have opened new possibilities for treating otherwise incurable diseases, the transduction of nucleic acids into the cells and delivery in vivo remain challenging. The high molecular weight and anionic nature of nucleic acids require their packing into nanoparticles for the delivery. The efficacy of nanoparticle drugs necessitates the high bioactivity of constituents, but their distribution in organisms is mostly governed by the physical properties of nanoparticles, and therefore, generation of stable particles with strictly defined characteristics is highly essential. Using previously designed efficient cell-penetrating peptide NF55, we searched for strategies enabling control over the nanoparticle formation and properties to further improve transfection efficacy. The size of the NF55/pDNA nanoparticles correlates with the concentration of its constituents at the beginning of assembly, but characteristics of nanoparticles measured by DLS do not reliably predict the applicability of particles in in vivo studies. We introduce a new formulation approach called cryo-concentration, where we acquired stable and homogeneous nanoparticles for administration in vivo. The cryo-concentrated NF55/pDNA nanoparticles exhibit several advantages over standard formulation: They have long shelf-life and do not aggregate after reconstitution, have excellent stability against enzymatic degradation, and show significantly higher bioactivity in vivo.
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| 8. |
- Gyorgy, Bence, et al.
(författare)
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CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer's Disease
- 2018
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Ingår i: Molecular Therapy Nucleic Acids. - : CELL PRESS. - 2162-2531. ; 11, s. 429-440
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Tidskriftsartikel (refereegranskat)abstract
- The APPswe (Swedish) mutation in the amyloid precursor protein (APP) gene causes dominantly inherited Alzheimer's disease (AD) as a result of increased beta-secretase cleavage of the amyloid-beta (A beta) precursor protein. This leads to abnormally high A beta levels, not only in brain but also in peripheral tissues of mutation carriers. Here, we selectively disrupted the human mutant APP(sw) allele using CRISPR. By applying CRISPR/Cas9 from Streptococcus pyogenes, we generated allele-specific deletions of either APP(sw) or APP(WT). As measured by ELISA, conditioned media of targeted patient-derived fibroblasts displayed an approximate 60% reduction in secreted A beta. Next, coding sequences for the APP(sw)-specific guide RNA (gRNA) and Cas9 were packaged into separate adeno-associated viral (AAV) vectors. Site-specific indel formation was achieved both in primary neurons isolated from APP(sw) transgenic mouse embryos (Tg2576) and after co-injection of these vectors into hippocampus of adult mice. Taken together, we here present proof-of-concept data that CRISPR/Cas9 can selectively disrupt the APP(sw) allele both ex vivo and in vivo-and thereby decrease pathogenic A beta. Hence, this system may have the potential to be developed as a tool for gene therapy against AD caused by APPswe and other point mutations associated with increased A beta.
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| 9. |
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| 11. |
- Hanson, Britt, et al.
(författare)
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Non-uniform dystrophin re-expression after CRISPR-mediated exon excision in the dystrophin/utrophin double-knockout mouse model of DMD
- 2022
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Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier. - 2162-2531. ; 30, s. 379-397
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Tidskriftsartikel (refereegranskat)abstract
- Duchenne muscular dystrophy (DMD) is the most prevalent inherited myopathy affecting children, caused by genetic loss of the gene encoding the dystrophin protein. Here we have investigated the use of the Staphylococcus aureus CRISPR-Cas9 system and a double-cut strategy, delivered using a pair of adeno-associated virus serotype 9 (AAV9) vectors, for dystrophin restoration in the severely affected dystrophin/utrophin double-knockout (dKO) mouse. Single guide RNAs were designed to excise Dmd exon 23, with flanking intronic regions repaired by non-homologous end joining. Exon 23 deletion was confirmed at the DNA level by PCR and Sanger sequencing, and at the RNA level by RT-qPCR. Restoration of dystrophin protein expression was demonstrated by western blot and immunofluorescence staining in mice treated via either intraperitoneal or intravenous routes of delivery. Dystrophin restoration was most effective in the diaphragm, where a maximum of 5.7% of wild-type dystrophin expression was observed. CRISPR treatment was insufficient to extend lifespan in the dKO mouse, and dystrophin was expressed in a within-fiber patchy manner in skeletal muscle tissues. Further analysis revealed a plethora of non-productive DNA repair events, including AAV genome integration at the CRISPR cut sites. This study highlights potential challenges for the successful development of CRISPR therapies in the context of DMD.
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| 12. |
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| 13. |
- Kadekar, Sandeep, et al.
(författare)
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Synthetic design of asymmetric miRNA with engineered 3′-overhang to improve strand selection
- 2019
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Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier. - 2162-2531. ; 16, s. 597-604
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Tidskriftsartikel (refereegranskat)abstract
- We have developed a novel miRNA design that significantly improves strand selection within the RISC complex by engineering the 3′-end by adding extra nucleotides. Addition of seven nucleotides at the 3′-ends of the miR or miR* strand resulted in a thermodynamic asymmetry at either of the two-ends, which resulted in selective RISC recruitment as demonstrated by the stem-loop quantitative PCR experiment. Such selective recruitment was also corroborated at the protein level by Western blot analysis. In order to investigate the functional effect due to selective recruitment, we performed apoptosis and metastasis studies using human colon carcinoma cells (HCT116) and human osteosarcoma cells (MG63). These experiments indicated that the recruitment of miR strand is responsible for inducing apoptosis as well as to inhibit invasiveness of cancer cells. Recruitment of miR* strand, on the other hand, showed opposite effect. To the best of our knowledge, our strand engineering strategy is the first report of improved strand selection of desired miRNA strand by RISC without using any chemical modifications or mismatches. We believe such structural modifications of miR34a could mitigate some of the off-target effects of miRNA therapy and would also allow a better understanding of sequence-specific gene regulation. Such a design could also be adapted to other miRNA to enhance their therapeutic potential.
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| 14. |
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| 15. |
- Konstantinidis, Evangelos, et al.
(författare)
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CRISPR-Cas9 treatment partially restores amyloid-β 42/40 in human fibroblasts with the Alzheimer's disease PSEN1 M146L mutation
- 2022
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Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 28, s. 450-461
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Tidskriftsartikel (refereegranskat)abstract
- Presenilin 1 (PS1) is a central component of γ-secretase, an enzymatic complex involved in the generation of the amyloid-β (Aβ) peptide that deposits as plaques in the Alzheimer’s disease (AD) brain. The M146L mutation in the PS1 gene (PSEN1) leads to an autosomal dominant form of early-onset AD by promoting a relative increase in the generation of the more aggregation-prone Aβ42. This change is evident not only in the brain but also in peripheral cells of mutation carriers. In this study we used the CRISPR-Cas9 system from Streptococcus pyogenes to selectively disrupt the PSEN1M146L allele in human fibroblasts. A disruption of more than 50% of mutant alleles was observed in all CRISPR-Cas9-treated samples, resulting in reduced extracellular Aβ42/40 ratios. Fluorescence resonance energy transfer-based conformation and western blot analyses indicated that CRISPR-Cas9 treatment also affects the overall PS1 conformation and reduces PS1 levels. Moreover, our guide RNA did not lead to any detectable editing at the highest-ranking candidate off-target sites identified by ONE-seq and CIRCLE-seq. Overall, our data support the effectiveness of CRISPR-Cas9 in selectively targeting the PSEN1M146L allele and counteracting the AD-associated phenotype. We believe that this system could be developed into a therapeutic strategy for patients with this and other dominant mutations leading to early-onset AD.
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| 16. |
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| 17. |
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| 18. |
- Paernaste, Ly, et al.
(författare)
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The Formation of Nanoparticles between Small Interfering RNA and Amphipathic Cell-Penetrating Peptides
- 2017
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Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 7, s. 1-10
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Tidskriftsartikel (refereegranskat)abstract
- Cell-penetrating peptides (CPPs) are delivery vectors widely used to aid the transport of biologically active cargoes to intracellular targets. These cargoes include small interfering RNAs (siRNA) that are not naturally internalized by cells. Elucidating the complexities behind the formation of CPP and cargo complexes is crucial for understanding the processes related to their delivery. In this study, we used modified analogs of the CPP transportan10 and investigated the binding properties of these CPPs to siRNA, the formation parameters of the CPP/siRNA complexes, and their stabiliy to enzymatic degradation. We conclude that the pH dependent change of the net charge of the CPP may very well be the key factor leading to the high delivery efficiency and the optimal binding strength between CPPs to siRNAs, while the hydrophobicity, secondary structure of the CPP, and the positions of the positive charges are responsible for the stability of the CPP/siRNA particles. Also, CPPs with distinct hydrophobic and hydrophilic regions may assemble into nanoparticles that could be described as coreshell formulations.
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| 19. |
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| 20. |
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| 21. |
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| 22. |
- Sork, Helena, et al.
(författare)
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Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency
- 2016
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Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 5
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Tidskriftsartikel (refereegranskat)abstract
- The advantages of lipid-based transfection reagents have permitted their widespread use in molecular biology and gene therapy. This study outlines the effect of cryo-manipulation of a cationic lipid-based formulation, Lipofectamine 2000, which, after being frozen and thawed, showed orders of magnitude higher plasmid delivery efficiency throughout eight different cell lines, without compromising cell viability. Increased transfection efficiency with the freeze-thawed reagent was also seen with 2'-O-methyl phosphorothioate oligonucleotide delivery and in a splice-correction assay. Most importantly, a log-scale improvement in gene delivery using the freeze-thawed reagent was seen in vivo. Using three different methods, we detected considerable differences in the polydispersity of the different nucleic acid complexes as well as observed a clear difference in their surface spreading and sedimentation, with the freeze-thawed ones displaying substantially higher rate of dispersion and deposition on the glass surface. This hitherto overlooked elevated potency of the freeze-thawed reagent facilitates the targeting of hard-to-transfect cells, accomplishes higher transfection rates, and decreases the overall amount of reagent needed for delivery. Additionally, as we also saw a slight increase in plasmid delivery using other freeze-thawed transfection reagents, we postulate that freeze-thawing might prove to be useful for an even wider variety of transfection reagents.
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| 23. |
- Stenler, S., et al.
(författare)
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Micro-minicircle gene therapy : Implications of size on fermentation, complexation, shearing resistance, and expression
- 2014
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Ingår i: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 3, s. e140-
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Tidskriftsartikel (refereegranskat)abstract
- The minicircle (MC), composed of eukaryotic sequences only, is an interesting approach to increase the safety and efficiency of plasmid-based vectors for gene therapy. In this paper, we investigate micro-MC (miMC) vectors encoding small regulatory RNA. We use a construct encoding a splice-correcting U7 small nuclear RNA, which results in a vector of 650 base pairs (bp), as compared to a conventional 3600 bp plasmid carrying the same expression cassette. Furthermore, we construct miMCs of varying sizes carrying different number of these cassettes. This allows us to evaluate how size influences production, supercoiling, stability and efficiency of the vector. We characterize coiling morphology by atomic force microscopy and measure the resistance to shearing forces caused by an injector device, the Biojector. We compare the behavior of miMCs and plasmids in vitro using lipofection and electroporation, as well as in vivo in mice. We here show that when the size of the miMC is reduced, the formation of dimers and trimers increases. There seems to be a lower size limit for efficient expression. We demonstrate that miMCs are more robust than plasmids when exposed to shearing forces, and that they show extended expression in vivo.
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