Targeted nanoparticle mediated delivery of nucleic acids into muscle stem cells for prevention of critical illness myopathy (B06#)

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The genetic manipulation of cells in vivo is still challenging although numerous approaches are well established and investigated in vitro. One reason is the fact that targeted nanoparticles have to overcome different barriers subsequent to intravenous administration. Immunogenic recognition must be avoided, crossing of the endothelial barrier and internalization into the targeted cells are hurdles to be taken before the nanoparticles can transfer their payload to the site of action. In the case of nucleic acids as payload, further difficulties have to be taken into account: They are easily degraded and unable to cross membranes because of their hydrophilicity. Nanoparticles protect the genetic material and promote its endosomal uptake and release to the cytoplasm of the targeted cell in an active form. To import new information into cells, messenger RNA (mRNA) can be more sufficient as mRNA does not have to enter the cell nucleus. Thereby, delivery across the nuclear membrane is not necessary. Polymers with cationic charges can be used to bind, protect and transfer the negatively charged genetic material. This principle has been known since decades. However, a lot of gene carrier failed in vivo although being efficient in cell culture experiments. For an efficient gene delivery to targeted cells, multi-functional nanoparticles for the delivery of mRNA encoding GFP will be developed. The used polymers not only bind the genetic material by electrostatic interactions, due to their hydrophobic nature they also form particles. For in vivo applications, a second functional layer will be introduced to prevent side reactions and clearance. The cationic particles will be shielded by these block copolymers where one block contains pH value dependent anionic charges to bind to the cationic particle and the second block introduces stealth moieties. Functional end groups allow the introduction of targeting moieties to enhance the targeted gene delivery. The particles will be tested in an advanced cell culture setup using a fluidic approach combined with co-cultivation of immune as well as endothelial cells. Different aspects, such as cellular targeting and uptake of nanoparticles, endosomal release and intercellular delivery of mRNA as well as efficiency of nanoparticle mediated gene delivery will be addressed. Design parameters like size, surface charge, particle density, and the balance between active and passive targeting will be investigated within the project. “