| 1. |
- Becquart, Cécile, et al.
(författare)
-
Intracellular Absolute Quantification of Oligonucleotide Therapeutics by NanoSIMS
- 2022
-
Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:29, s. 10549-10556
-
Tidskriftsartikel (refereegranskat)abstract
- Antisense oligonucleotide (ASO)-based therapeutics hold great potential for the treatment of a variety of diseases. Therefore, a better understanding of cellular delivery, uptake, and trafficking mechanisms of ASOs is highly important for early-stage drug discovery. In particular, understanding the biodistribution and quantifying the abundance of ASOs at the subcellular level are needed to fully characterize their activity. Here, we used a combination of electron microscopy and NanoSIMS to assess the subcellular concentrations of a 34S-labeled GalNAc-ASO and a naked ASO in the organelles of primary human hepatocytes. We first cross-validated the method by including a 127I-labeled ASO, finding that the absolute concentration of the lysosomal ASO using two independent labeling strategies gave matching results, demonstrating the strength of our approach. This work also describes the preparation of external standards for absolute quantification by NanoSIMS. For both the 34S and 127I approaches used for our quantification methodology, we established the limit of detection (5 and 2 μM, respectively) and the lower limit of quantification (14 and 5 μM, respectively).
|
|
| 2. |
- Becquart, Cécile
(författare)
-
Quantitative NanoSIMS provides subcellular concentration and distribution of oligonucleotide therapeutics
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Antisense oligonucleotides (ASOs) represent a powerful therapeutic modality that can selectively modulate gene expression. However, ASOs face two major hurdles that restrict their use in the clinic. The first issue is delivery of the ASO to a tissue of therapeutic interest while reducing exposure to unrelated tissues. Additionally, inefficient escape of ASOs from endolysosomal compartments affects their activity since ASOs are unable to reach their intracellular RNA target in the nucleus and/or cytosol. Despites the variety of chemical modifications developed to tackle these delivery issues, it remains challenging to reach particular tissues and/or cell types outside of the liver, and there are still no non-toxic solutions to the endosomal escape problem. To fully realize the therapeutic potential of this class of molecules, it is crucial to understand the mechanisms underlying how ASOs enter cells and exit the endosomal space. Therefore, this thesis focuses on the use of nanoscale secondary ion mass spectrometry (NanoSIMS), in combination with electron microscopy, to investigate the subcellular distribution and accumulation of ASOs. It was necessary to develop a NanoSIMS method capable of absolute quantification of the intracellular exposure of ASO. Thus, external standards were developed to quantify several halogenated compounds (iodine, bromine, and fluorine) as well as a sulfur isotope (34S). Results showed that the uptake of different ASOs was saturable, but conjugation to a N-acetylgalactosamine targeting domain enhanced cellular uptake and improved target knockdown. NanoSIMS data also showed that upon colchicine treatment, the uptake and localization of ASOs were affected. It was also possible to quantifying both the targeting domain and ASO components of an engineered glucagon-like peptide 1 ASO conjugate. That highlighted that fine tuning of ASO chemistry can be used to affect the productive uptake of ASOs. Overall, these findings contribute to a better understanding of the cellular delivery, uptake and trafficking mechanisms of ASOs, which is valuable for the future development of more effective oligonucleotide-based therapeutics.
|
|
| 3. |
- Kay, E., et al.
(författare)
-
NanoSIMS Imaging Reveals the Impact of Ligand-ASO Conjugate Stability on ASO Subcellular Distribution
- 2022
-
Ingår i: Pharmaceutics. - : MDPI AG. - 1999-4923. ; 14:2
-
Tidskriftsartikel (refereegranskat)abstract
- The delivery of antisense oligonucleotides (ASOs) to specific cell types via targeted en-docytosis is challenging due to the low cell surface expression of target receptors and inefficient escape of ASOs from the endosomal pathway. Conjugating ASOs to glucagon-like peptide 1 (GLP1) leads to efficient target knockdown, specifically in pancreatic β-cells. It is presumed that ASOs dissociate from GLP1 intracellularly to enable an ASO interaction with its target RNA. It is unknown where or when this happens following GLP1-ASO binding to GLP1R and endocytosis. Here, we use correlative nanoscale secondary ion mass spectroscopy (NanoSIMS) and transmission electron microscopy to explore GLP1-ASO subcellular trafficking in GLP1R overexpressing HEK293 cells. We isotopically label both eGLP1 and ASO, which do not affect the eGLP1-ASO conjugate function. We found that the eGLP1 peptide and ASO are not detected at the same level in the same endosomes, within 30 min of GLP1R-HEK293 cell exposure to eGLP1-ASO. When we utilized different linker chemistry to stabilize the GLP1-ASO conjugate, we observed more ASO located with GLP1 compared to cell incubation with the less stable conjugate. Overall, our work suggests that the ASO separates from GLP1 relatively early in the endocytic pathway, and that linker chemistry might impact the GLP1-ASO function. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.
|
|
| 4. |
- Palmgren, Henrik, et al.
(författare)
-
Elevated Adipocyte Membrane Phospholipid Saturation Does Not Compromise Insulin Signaling
- 2023
-
Ingår i: DIABETES. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 72:10, s. 1350-1363
-
Tidskriftsartikel (refereegranskat)abstract
- Increased saturated fatty acid (SFA) levels in membrane phospholipids have been implicated in the development of metabolic disease. Here, we tested the hypothesis that increased SFA content in cell membranes negatively impacts adipocyte insulin signaling. Preadipocyte cell models with elevated SFA levels in phospholipids were generated by disrupting the ADIPOR2 locus, which resulted in a striking twofold increase in SFA-containing phosphatidylcholines and phosphatidylethanolamines, which persisted in differentiated adipocytes. Similar changes in phospholipid composition were observed in white adipose tissues isolated from the ADIPOR2-knockout mice. The SFA levels in phospholipids could be further increased by treating ADIPOR2-deficient cells with palmitic acid and resulted in reduced membrane fluidity and endoplasmic reticulum stress in mouse and human preadipocytes. Strikingly, increased SFA levels in differentiated adipocyte phospholipids had no effect on adipocyte gene expression or insulin signaling in vitro. Similarly, increased adipocyte phospholipid saturation did not impair white adipose tissue function in vivo, even in mice fed a high-saturated fat diet at thermoneutrality. We conclude that increasing SFA levels in adipocyte phospholipids is well tolerated and does not affect adipocyte insulin signaling in vitro and in vivo.
|
|
| 5. |
- Vo, Duc Duy, et al.
(författare)
-
Building of neomycin-nucleobase-amino acid conjugates for the inhibition of oncogenic miRNAs biogenesis
- 2018
-
Ingår i: Organic and biomolecular chemistry. - : Royal Society of Chemistry (RSC). - 1477-0520 .- 1477-0539. ; 16:34, s. 6262-6274
-
Tidskriftsartikel (refereegranskat)abstract
- MicroRNAs (miRNAs) are a recently discovered category of small RNA molecules that regulate gene expression at the post-transcriptional level. Accumulating evidence indicates that miRNAs are aberrantly expressed in a variety of human cancers, thus being oncogenic. The inhibition of oncogenic miRNAs (defined as the blocking of miRNAs' production or function) would find application in the therapy of different types of cancer in which these miRNAs are implicated. In this work, we describe the design and synthesis of new small-molecule RNA ligands with the aim of inhibiting Dicer-mediated processing of oncogenic miRNAs. One of the synthesized compound (4b) composed of the aminoglycoside neomycin conjugated to an artificial nucleobase and to amino acid histidine is able to selectively decrease miR-372 levels in gastric adenocarcinoma (AGS) cells and to restore the expression of the target LATS2 protein. This activity led to the inhibition of proliferation of these cells. The study of the interactions of 4b with pre-miR-372 allowed for the elucidation of the molecular mechanism of the conjugate, thus leading to new perspectives for the design of future inhibitors.
|
|
