Engineering cell communication from nanoscale to tissue levels towards therapeutic applications

Cell communication which is vital for the proper function, development, and survival of multicellular organisms is often disrupted in diseases. This thesis aims to enhance our understanding of cell communication by employing advanced tools that facilitate the manipulation and analysis of biological systems, spanning from the nanoscale to tissue levels, towards therapeutic applications.

In Paper I, we utilized a microfluidic device to artificially create neuromuscular junctions and gain insights into nerve-muscle communication. We simulated endocrine signaling and the formation of neuromuscular junctions by using myotubes derived from primary mouse myoblasts and motor neurons derived from embryonic stem cells. Transducing skeletal muscle with PGC-1α1, increased the neuromuscular junction formation and size. Neurturin emerged as a mediator in the PGC-1α1-depentent retrograde signaling from muscle to motor neurons. This discovery may pave the way for potential therapies in diseases where neuromuscular junctions are affected early on.

In Paper II, we employed DNA origami nanotechnology to harness the spatial organization of insulin receptors and control multivalent receptor activation. This innovative approach involved assembling insulin into nanoclusters on the surface of DNA origami nanostructures. Beyond in vitro assessments, we extended our investigation to in vivo studies, utilizing a zebrafish model of diabetes. Our findings not only demonstrate the effectiveness of insulin nanoclusters but also highlight the applicability of DNA origami nanostructures in the field of nanomedicine.

In Paper III, we monitored the biodistribution and clearance dynamics of fluorescently labelled DNA origami nanostructures in live zebrafish embryos. We coupled advanced imaging techniques with single-cell RNA sequencing to gain insight into the interactions of DNA nanostructures with biological systems in vivo. This work serves as a guide for evaluating DNA-origami based nanomedicines in animal models.

In Paper IV, we introduce a new method for monitoring the stability of DNA origami nanostructures by using the proximity ligation assay. We were able to detect the preservation of proximity between selected regions when nanostructures were bound to cultured cells as a validation of nanostructure integrity. This approach holds great potential for applications both in vitro and in vivo.

List of scientific papers

I. Richard Mills*, Hermes Taylor-Weiner*, Jorge C. Correia, Leandro Z. Agudelo, Ilary Allodi, Christina Kolonelou, Vicente Martinez-Redondo, Duarte M. S Ferreira, Susanne Nichterwitz, Laura H. Comley, Vanessa Lundin, Eva Hedlund, Jorge L. Ruas, Ana I. Teixeira. Neurturin is a PGC-1𝜶1-controlled myokine that promotes motor neuron recruitment and neuromuscular junction formation. Mol Metab. 7, 12–22 (2018). *These authors contributed equally to this work.
https://doi.org/10.1016/j.molmet.2017.11.001

II. Joel Spratt*, José M. Dias*, Christina Kolonelou, Georges Kiriako, Enya Engström, Ekaterina Petrova, Christos Karampelias, Igor Cervenka, Natali Papanicolaou, Antonio Lentini, Björn Reinius, Olov Andersson, Elena Ambrosetti, Jorge L. Ruas, Ana I. Teixeira. Multivalent insulin receptor activation using insulin–DNA origami nanostructures. Nat Nanotechnol. (2023). *These authors contributed equally to this work.
https://doi.org/10.1038/s41565-023-01507-y

III. Christina Kolonelou, Lars Bräutigam, Steven Edwards, Enya Engström, José M. Dias, Joel Spratt, Christos Karampelias, Stefan Wennmalm, Hjalmar Brismar, Olov Andersson, Ana I. Teixeira. Biodistribution of DNA-origami nanostructures in live zebrafish embryos with single-cell resolution. [Manuscript]

IV. Christina Kolonelou, Ana I. Teixeira. A proximity ligation assay-based method to analyse the integrity of DNA origami nanostructures. [Manuscript]