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Inhalation February 2025 25 base modifications of mRNA vaccines) was awarded a Nobel Prize in 2023 [12]. Once the siRNA enters the cytosol (a process that involves several hurdles as discussed in the drug delivery system section of this article), it is loaded into the RISC. e RISC separates the guide strand from the passenger strand, with the passenger strand exiting the complex while the guide strand remains. Once loaded, the RISC-guide strand complex can bind to a mRNA molecule through Watson and Crick base pairing (recognized by a Nobel Prize in 1962 [13]). is binding leads to mRNA degrada- tion, while the guide siRNA strand is preserved and can degrade additional mRNA molecules, giving it an enzymatic character [14, 15]. Since RNAi utilizes cellular mechanisms available in almost all eukaryotic cells, it opens a wide range of potential applications. When conventional drugs such as small molecules or therapeutic proteins can- not achieve sufficient treatment, siRNA offers a key solution by silencing the underlying mRNA, thereby reducing the production of disease-causing proteins. Importantly, this approach addresses the disease at a different stage, compared to classical drugs. RNAi is also being explored for treating viral infec- tions. Since viruses cannot replicate independently, they must enter the cytosolic compartments of host cells, where they interact with RNAi machinery. e recurrent outbreaks of coronaviruses, such as SARS- CoV-2, highlight the need for deep understanding and effective treatments. Betacoronaviruses have complex RNA structures and sophisticated repli- cation mechanisms, making them challenging yet critical targets for therapeutic development [9]. Pre- vious studies from our research group have demon- strated the effective treatment of SARS-CoV-2 using polyplex formulations, forming the basis of our cur- transcripts from CoV replication can theoretically be targeted with RNAi. Our research indicates that targeting open reading frame 1 (ORF1) significantly impacts SARS-CoV-2 replication. Identifying con- served sequences within ORF1a/b allows for poten- tial treatment against various betacoronaviruses [6]. Due to poor absorption and degradation issues, we are exploring polymer-based nucleic acid (NA) car- riers, such as biocompatible polyspermines as an promising alternative to the "gold standard" polyeth- yleneimine (PEI), which exhibits increased toxicity as discussed below [7, 8]. Inhalable nanoparticles offer a patient-friendly ther- apy targeting betacoronaviruses, which first infect the respiratory tract [9]. siRNA can specifically target viral RNAs without interfering with cellular func- tions, offering both therapeutic and prophylactic benefits. is approach could be crucial for stopping virus propagation early in an outbreak [6]. The API: siRNA inducing RNA interference (RNAi) siRNA is a clinically approved active pharmaceutical ingredient (API) that utilizes the cell's internal RNAi machinery, a discovery that was recognized with the Nobel Prize in 2006 [10]. siRNA typically consists of 21-23 nucleotide strands, excluding overhangs, and is a rigid, double-stranded RNA molecule [11]. Among these two strands, only one is responsible for the effect and is referred to as the "guide" or "antisense" strand, which is complementary to a specific mRNA sequence. e other strand, known as the "passenger" or "sense" strand, resembles partly the target mRNA sequence (as most approved RNA therapies involve chemical modifications to the sugar), phosphate and/ or base components to enhance stability and reduce the inflammatory response. is discovery (regarding Figure 1 Schematic representation of an experimentational approach to investigate the aerosolizability of siRNA therapies against betacoronaviruses in a sophisticated, three-dimensional air-liquid interface (ALI). Vibrating Mesh Nebulizer e.g., Aerogen ® Pro or PARI eFlow ® Rapid PBAE Polyplexes bearing an ACE-2 antibody while encapsulating siRNA Betacoronaviruses e.g., SARS-CoV, SARS- Cov-2 or HCoV-OC43 Calu-3 Cells cultured at ALI Submerged Cell Culture Air-Liquid Interface Transfection Infection Polymerase Chain Reaction