| 1. |
- Bost, Jeremy
(author)
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Characterizing and exploiting the endocytic pathway for macromulecular delivery
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Macromolecular drugs with cytosolic or nuclear targets often exhibit low therapeutic activity due, at least in part, to their inability to escape endosomal compartments following cellular internalization. There exist a range of strategies to address this inefficient delivery by attempting to bolster the endosomal escape of the therapeutic cargo. Many of these delivery strategies apply to a broad range of molecular species, including proteins and nucleic acids. Two strategies of particular interest involve the use of extracellular vesicles (EVs) or endosomolytic small molecule compounds (SMCs). EVs are nanoscale, membrane-bound particles produced by all cell types and present in all bodily fluids. As a biological nanoparticulate species, EVs are inherently capable of delivering the material they contain to cells. Further, EVs can be modified through recombinant protein-based engineering strategies which can bestow a range of functional utilities such as fusogenicity, preferential cargo loading, and molecular targeting. However, the use of EVs as a scalable therapeutic modality is hampered by an inability to reliably mass-produce a homogenous population of these nanoparticles in vitro. SMCs, on the other hand, are easily synthesized at scale and can function in a stochastic manner dependent on an appropriate co-dosing strategy with their complementary therapeutic cargo. However, the mechanisms underlying SMC-mediated macromolecular delivery can be difficult to elucidate due to a lack of high-resolution characterization techniques. In this thesis, two issues - one underpinning each strategy - are investigated. First, the effects of culture media composition on the production of proteinloaded EVs in vitro are explored, with the ultimate aim of increasing EV output while characterizing the cellular biology driving the EV production. Certain serum components can differentially affect EV biogenesis by influencing ceramide-dependent EV biogenesis. In the second project, a functional screen of a novel family of SMCs is conducted to identify several chemical analogs in this family that demonstrate endosomolytic activity. Thereafter, superresolution and real-time microscopic assays are employed to determine the mechanism and consequence of the novel compounds during their co-treatment with a splice-switching oligonucleotide (SSO). SSOs are clinically relevant small-RNA therapeutics that alter the production of splice variants for a given genetic transcript. The novel SMCs bolster SSO activity by disrupting the structure of endosomes in a manner dependent on the acidification of the endosomal compartments, suggesting the SMCs display a buffering capacity at certain concentrations. The findings herein strengthen the potential of each delivery strategy as a therapeutically relevant approach to functionally delivering macromolecular cargo to cells.
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| 2. |
- Bost, Jeremy P., et al.
(author)
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Novel endosomolytic compounds enable highly potent delivery of antisense oligonucleotides
- 2022
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In: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 5:1
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Journal article (peer-reviewed)abstract
- The therapeutic and research potentials of oligonucleotides (ONs) have been hampered in part by their inability to effectively escape endosomal compartments to reach their cytosolic and nuclear targets. Splice-switching ONs (SSOs) can be used with endosomolytic small molecule compounds to increase functional delivery. So far, development of these compounds has been hindered by a lack of high-resolution methods that can correlate SSO trafficking with SSO activity. Here we present in-depth characterization of two novel endosomolytic compounds by using a combination of microscopic and functional assays with high spatiotemporal resolution. This system allows the visualization of SSO trafficking, evaluation of endosomal membrane rupture, and quantitates SSO functional activity on a protein level in the presence of endosomolytic compounds. We confirm that the leakage of SSO into the cytosol occurs in parallel with the physical engorgement of LAMP1-positive late endosomes and lysosomes. We conclude that the new compounds interfere with SSO trafficking to the LAMP1-positive endosomal compartments while inducing endosomal membrane rupture and concurrent ON escape into the cytosol. The efficacy of these compounds advocates their use as novel, potent, and quick-acting transfection reagents for antisense ONs.
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| 3. |
- Klionsky, Daniel J., et al.
(author)
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Guidelines for the use and interpretation of assays for monitoring autophagy
- 2012
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In: Autophagy. - : Informa UK Limited. - 1554-8635 .- 1554-8627. ; 8:4, s. 445-544
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Research review (peer-reviewed)abstract
- In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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| 4. |
- Sork, Helena, et al.
(author)
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Lipid-based Transfection Reagents Exhibit Cryo-induced Increase in Transfection Efficiency
- 2016
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In: Molecular Therapy Nucleic Acids. - : Elsevier BV. - 2162-2531. ; 5
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Journal article (peer-reviewed)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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