| 1. |
- 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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| 2. |
- Morrison, Jamie, et al.
(författare)
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Standardized Preclinical In Vitro Blood-Brain Barrier Mouse Assay Validates Endocytosis-Dependent Antibody Transcytosis Using Transferrin-Receptor-Mediated Pathways
- 2023
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Ingår i: Molecular Pharmaceutics. - : American Chemical Society (ACS). - 1543-8384 .- 1543-8392. ; 20:3, s. 1564-1576
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Tidskriftsartikel (refereegranskat)abstract
- The presence of the blood-brain barrier (BBB) creates a nigh-on impenetrable obstacle for large macromolecular therapeutics that need to be delivered to the brain milieu to treat neurological disorders. To overcome this, one of the strategies used is to bypass the barrier with what is referred to as a "Trojan Horse" strategy, where therapeutics are designed to use endogenous receptor-mediated pathways to piggyback their way through the BBB. Even though in vivo methodologies are commonly used to test the efficacy of BBB-penetrating biologics, comparable in vitro BBB models are in high demand, as they benefit from being an isolated cellular system devoid of physiological factors that can on occasion mask the processes behind BBB transport via transcytosis. We have developed an in vitro BBB model (In-Cell BBB-Trans assay) based on the murine cEND cells that help delineate the ability of modified large bivalent IgG antibodies conjugated to the transferrin receptor binder scFv8D3 to cross an endothelial monolayer grown on porous cell culture inserts (PCIs). Following the administration of bivalent antibodies into the endothelial monolayer, a highly sensitive enzyme-linked immunosorbent assay (ELISA) is used to determine the concentration in the apical (blood) and basolateral (brain) chambers of the PCI system, allowing for the evaluation of apical recycling and basolateral transcytosis, respectively. Our results show that antibodies conjugated to scFv8D3 transcytose at considerably higher levels compared to unconjugated antibodies in the In-Cell BBB-Trans assay. Interestingly, we are able to show that these results mimic in vivo brain uptake studies using identical antibodies. In addition, we are able to transversely section PCI cultured cells, allowing for the identification of receptors and proteins that are likely involved in the transcytosis of the antibodies. Furthermore, studies using the In-Cell BBB-Trans assay revealed that transcytosis of the transferrin-receptor-targeting antibodies is dependent on endocytosis. In conclusion, we have designed a simple, reproducible In-Cell BBB-Trans assay based on murine cells that can be used to rapidly determine the BBB-penetrating capabilities of transferrin-receptor-targeting antibodies. We believe that the In-Cell BBB-Trans assay can be used as a powerful, preclinical screening platform for therapeutic neurological pathologies.
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| 3. |
- Stenler, Sofia
(författare)
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Characterization and applications in muscle of a Minicircle vector for Nonviral gene therapy
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In gene therapy, the aim is to change the behaviour of a cell by introduction of genetic material, often DNA encoding a protein or a therapeutic RNA. The purpose can be to replace a malfunctioning copy of a gene, as in clinical trials for treatment of X-linked severe combined immunodeficiency, or introduce a new gene into the body to help fight a disease, as has been done in clinical trials for e.g. leukaemia and lymphoma where immune cells has been modified to recognize and destroy cancer. In order to alter the behaviour, the genetic material must be transported into the cell and reach the nucleus. The two main ways to achieve this is either using viral vectors, where engineered viruses carry the therapeutic DNA, or nonviral vectors, which are commonly based on plasmids produced in bacteria. This thesis focuses on nonviral vectors. Nonviral vectors are generally considered safer and more easily produced than viral vectors, but are less efficient in delivery and long term expression. This is thought to be partly due to the plasmid backbone, i.e. sequences needed only for propagation in the bacteria such as origin of replication and selection markers, commonly antibiotics resistance genes. Bacterially produced DNA sequences have a different methylation pattern than eukaryotic DNA. It has been shown that this can induce an immune response, especially in combination with the use of lipids for transfection. Also for naked delivery of plasmids, the expression is transient, which could be due to epigenetic phenomenon. A way to optimize the plasmid vector is to remove the bacterial backbone by recombination in the production bacteria. The resulting vector is called the minicircle (MC). In one of studies included in this thesis, we investigate how the size of the MC vector affects coiling and relate these findings to analysis of other aspects such as robustness, expression efficiency and transfection. We find that reducing the size of the MC affects the configuration of the vector, causing an increased frequency of dimer and trimer formation during production. We also find that there seems to be a lower size limit for efficient expression. However, the smaller sizes also result in a vector which is more robust than conventional plasmids when exposed to shearing forces, and shows extended expression in vivo. In the two other studies, we evaluate the vector for use in muscle. A comparison of the MC to a conventional plasmid for expression of a growth factor in heart and skeletal muscle in the mouse shows that the smaller size allows for a higher effective dose, and thus, higher gene expression. The third study demonstrates that it is possible to use the MC to express small regulatory RNAs for splice-switching, targeting Duchenne muscular dystrophy, and that treatment with these MCs in mouse muscle results in increased dystrophin levels. However, development of suitable delivery methods is required to realize the full potential of MCs in vivo. Thus, the smaller size enabling a higher dose, prolonged expression and increased robustness, and the fact that the MC construct is devoid of bacterial sequences and antibiotics resistance gene make the MC vector an attractive alternative for nonviral gene. However, for use where systemic treatment is needed, delivery must be enhanced. Consequently, the vector might be more suitable for treatments where only local expression is required, such as single organ treatment, DNA vaccination or ex vivo treatments.
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| 4. |
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| 5. |
- Stenler, Sofia, 1980-, et al.
(författare)
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Over the BBB and into the cell : Pursuing intracellular targets for immunotherapy of Parkinson’s disease
- 2019
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- The aim of our research is to modify therapeutic antibodies so that they can reach their dementia target inside cells located on the other side of the blood brain barrier. While the aggregates associated with Alzheimer’s are located extracellularly and thus readily available for antibodies that have crossed the BBB barrier, this is not the case for Parkinson’s disease. In this study, we focus on developing a peptide shuttle that can deliver antibodies not only over the BBB but also into neuronal cells where the Tau and a-synuclein aggregates can be found.For this purpose, we have investigated the use of a peptide which binds to a receptor that co- localizes with the aggregates. Our in-house experience suggests that the peptide is not an efficient BBB transporter despite the fact that some groups have used it as such, but that it might be more suitable as a transporter for intracellular delivery.We have successfully expressed recombinant antibodies with the peptide on the N-terminal of an antibody targeting the aggregates associated with Parkinson’s disease. Our initial studies indicate that the tyrosine on the N-terminal of the peptide needs to be free and unmodified to be able to enhance uptake into neuronal cells. This hinders the use of the normal labelling method which attaches radiolabelled iodine to tyrosines where the affinity for peptide target would be destroyed. We have been pursuing alternative methods, such as using click chemistry to attach the peptide which will leave the antibody free to be radiolabelled, as well as methods to detect unlabelled antibodies in vivo and in vitro.We have assessed the peptide-assisted increase in uptake in appropriate neuronal cell line models. Furthermore, we have studied uptake and retention in brain in mouse models for Parkinson’s disease.
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