Iodine-124 PET quantification of organ-specific delivery and expression of NIS-encoding RNA

Matthias Miederer¹, Stefanie Pektor¹, Isabelle Miederer¹, Nicole Bausbacher¹, Isabell Sofa Keil², Hossam Hefesha³, Heinrich Haas³, Ugur Sahin^2,3, Mustafa Diken^2,3
1Department of Nuclear Medicine, University Medical Center of Johannes Gutenberg University, Mainz, Germany
²TRON - Translational Oncology at the University Medical Center, Johannes Gutenberg University Mainz gGmbH, Mainz, Germany
³Biopharmaceutical New Technologies (BioNTech) SE, Mainz, Germany

https://doi.org/10.1186/s13550-021-00753-2

Summary
RNA-based vaccination strategies tailoring immune response to specific reactions have become an important pillar for a broad range of applications. Recently, the use of lipid-based nanoparticles opened the possibility to deliver RNA to specific sites within the body, overcoming the limitation of rapid degradation in the bloodstream. 
Nanoparticles show promising potency as delivery vehicles for a variety of molecules, leading to fundamentally new applications and therapeutic strategies. Due to their complex chemical composition and relevant interaction with plasma proteins, the pharmacokinetic properties and delivery properties of nanoparticles are variable and remain challenging to adapt for optimal conditions. Accomplishing precise RNA delivery to target tissue using nanoparticles would serve as a versatile platform that enables easy and transient expression of any protein in principal. RNA is currently already in use to selectively activate the immune system against specific target proteins for cancer therapy. 
In the article, the authors have investigated whether small animal PET/MRI can be employed to image the biodistribution of RNA-encoded protein. For this purpose, a reporter RNA coding for the sodium-iodide-symporter (NIS) was assembled with liposomes at different charge ratios, and functional NIS protein translation was imaged and quantified in vivo and ex vivo by Iodine-124 PET upon intravenous administration in mice.

Results from the nanoScan PET/MRI
For the small animal imaging, the authors have used a nanoScan PET/MRI 1T, which provided a perfect option to follow the uptake of Iodine-124 not just in the thyroids, but also in the NIS-expressing tissues. Moreover, it could be accompanied by the help of MRI, to identify internal organs, like spleen.
Groups of n = 3 animals were intravenously injected with RNA-lipoplexes containing 20 µg NIS RNA. Six hours later 6.64 ± 0.66 MBq Iodine-124 was injected intravenously. Three hours after Iodine-124 injection, mice were anesthetized with 2% isoflurane and static imaging was performed over 20 min. For anatomic imaging MRI measurements (Material Map for coregistration of the PET scan; 3D Gradient Echo External Averaging (GRE-EXT), Multi Field of View (FOV); slice thickness: 0.6 mm; TE: 2 ms; TR: 15 ms; flip angle: 25 deg) were performed afterward. Additionally, one animal per group was imaged dynamically for one hour. PET data were reconstructed with Teratomo 3D (4 iterations, 6 subsets, voxel size 0.4 mm), co-registered to the MR and corrected for decay.
Figure 2. shows the PET/MRI of Iodine-124 distribution in vivo. (A) Coronal slices of PET/MRI fusion and volumes of interests (red) for spleen and lung are shown in representative animals. From left to right: targeting of spleen with non-coding RNA, targeting of spleen with NIS RNA, targeting of lung with non-coding RNA, targeting of lung with NIS-RNA. (B) Calculated organ uptake from the volumes of interests. Data are shown as mean + SD of n = 3 mice. (C) Representative in vivo bioluminescence images of Luc-RNA lipoplexes after targeting the spleen and lung. (D) Maximum intensity projections of PET images after application of lung-targeting NIS-RNA lipoplexes (right) in comparison with non-coding control (left). (E) Time activity curve of Iodine-124 uptake in the lung over 60 min immediately after Iodine-124 injection (6 h after administration of NIS-RNA lipoplexes targeting the lung).

• In this study, two RNA-lipoplex systems for systemic NIS-RNA delivery were compared by small animal PET/MRI of Iodine-124 uptake. One system with net anionic charge is known to mediate translation primarily within the spleen, and the other with net positive charge is known to yield translation primarily within the lungs.
• Tha authors have shown highly specific targeting, delivery and expression of RNA to spleen and lung by anionic and cationic RNA-lipoplex nanoparticles, respectively, through the use of the NIS reporter gene system and Iodine-124 uptake as imaged by PET/MRI. Combining NIS reporter gene imaging with in vivo small animal PET/MRI thus represents a powerful tool to monitor the distribution and extent of expression of RNA targeted specifically to any tissue over time.