Biologics-based technologies for highly efficient and targeted RNA delivery

Anastasiya Kostyusheva; Sergey Brezgin; Natalia Ponomareva; Anastasiia Frolova; Alexander Lunin; Ekaterina Bayurova; Andrey Tikhonov; Olga Slatinskaya; Polina Demina; Artyom Kachanov; Gulalek Babayeva; Irina Khan; Dmitry Khochenkov; Yulia Khochenkova; Darina Sokolova; Denis Silachev; Georgy Maksimov; Evgeny Khaydukov; Vadim S Pokrovsky; Andrey A Zamyatnin Jr; Alessandro Parodi; Ilya Gordeychuk; Vladimir Chulanov; Dmitry Kostyushev

doi:10.1016/j.ymthe.2024.11.004

Biologics-based technologies for highly efficient and targeted RNA delivery

Mol Ther. 2024 Nov 6:S1525-0016(24)00731-7. doi: 10.1016/j.ymthe.2024.11.004. Online ahead of print.

Authors

Anastasiya Kostyusheva¹, Sergey Brezgin², Natalia Ponomareva², Anastasiia Frolova², Alexander Lunin³, Ekaterina Bayurova³, Andrey Tikhonov¹, Olga Slatinskaya⁴, Polina Demina⁵, Artyom Kachanov¹, Gulalek Babayeva⁶, Irina Khan⁶, Dmitry Khochenkov⁷, Yulia Khochenkova⁸, Darina Sokolova⁹, Denis Silachev¹⁰, Georgy Maksimov⁴, Evgeny Khaydukov⁵, Vadim S Pokrovsky⁶, Andrey A Zamyatnin Jr¹¹, Alessandro Parodi¹², Ilya Gordeychuk³, Vladimir Chulanov¹³, Dmitry Kostyushev¹⁴

Affiliations

¹ Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119435, Russia.
² Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119435, Russia; Division of Biotechnology, Sirius University of Science and Technology, Sochi 354340, Russia.
³ Chumakov Federal Scientific Center for Research and Development of Immunobiological Products, Russian Academy of Sciences (Polio Institute), Moscow 142782, Russia.
⁴ Faculty of Biology, Lomonosov Moscow State University, Moscow 119234, Russia.
⁵ Moscow Pedagogical State University, Moscow 119435, Russia; Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Moscow 117997, Russia.
⁶ Blokhin National Medical Research Center of Oncology, Moscow 115522, Russia; RUDN University, Moscow 117198, Russia.
⁷ Blokhin National Medical Research Center of Oncology, Moscow 115522, Russia; Togliatti State University, Togliatti 445020, Russia.
⁸ Blokhin National Medical Research Center of Oncology, Moscow 115522, Russia.
⁹ Division of Biotechnology, Sirius University of Science and Technology, Sochi 354340, Russia; Blokhin National Medical Research Center of Oncology, Moscow 115522, Russia; RUDN University, Moscow 117198, Russia.
¹⁰ A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia; V.I. Kulakov National Medical Research Center of Obstetrics, Gynecology and Perinatology, Moscow 117198, Russia.
¹¹ Division of Biotechnology, Sirius University of Science and Technology, Sochi 354340, Russia; A.N. Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow 119234, Russia; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119192, Russia.
¹² Division of Biotechnology, Sirius University of Science and Technology, Sochi 354340, Russia.
¹³ Department of Infectious Diseases, First Moscow State Medical University (Sechenov University), Moscow 119435, Russia.
¹⁴ Laboratory of Genetic Technologies, Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, First Moscow State Medical University (Sechenov University), Moscow 119435, Russia; Division of Biotechnology, Sirius University of Science and Technology, Sochi 354340, Russia; Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, Moscow 119192, Russia. Electronic address: dkostushev@gmail.com.

PMID: 39511888
DOI: 10.1016/j.ymthe.2024.11.004

Abstract

The demand for RNA-based therapeutics is increasing globally. However, their use is hampered by the lack of safe and effective delivery vehicles. Here, we developed technologies for highly efficient delivery of RNA cargo into programmable extracellular vesicle-mimetic nanovesicles (EMNVs) by fabricating hybrid EMNV-liposomes (Hybs). Tissue targeting is endowed by highly efficient genetic platforms based on truncated CD63 (ΔCD63) or PTGFRN proteins. For the first time we reveal their efficiency in functionalizing EMNVs, resulting in >10-fold enhancement of nanoparticle internalization in vitro and >2-fold in vivo. RNA delivery using Hybs demonstrated efficiency of >85% in human and mouse cell lines. Comparative analysis of EMNVs and Hyb lysosome colocalization and stability suggested that Hybs enter the lysosomal compartment and escape over time, whereas EMNVs primarily avoid it. Finally, we used these technologies to generate liver-targeting Hybs loaded with therapeutic small interfering RNA and demonstrated the robust efficiency of this system in vitro and in vivo. These technologies can be adapted for manufacturing a wide range of next-generation vehicles for highly efficient, safe delivery of RNA into desired organs and tissues for therapeutic and prophylactic applications.

Keywords: RNA-interference; drug delivery; exosome-mimetic nanoparticles; exosomes; extrusion; functionalization; in vivo biodistribution; knockdown; nanoparticle-tracking analysis; size-exclusion chromatography.