Novel formulations

Abstract

The present invention provides, in particular, an aqueous liquid pharmaceutical formulation comprising a surfactant component and a polynucleotide molecule. The formulation of the present invention is intended for use, for example, as a pharmaceutical for the treatment or prevention of viral infections and diseases associated with viral infections.

Description

[Technical Field]

[0001] (Related applications) This application claims the benefits of U.S. Provisional Application No. 63 / 510,985, filed on 29 June 2023, and International Application No. PCT / US24 / 30546, filed on 22 May 2024. The entire teachings of the above applications are incorporated herein by reference.

[0002] (Sequence Listing) In accordance with 37 CFR §1.52(e)(5), sequence listings submitted via EFS are incorporated herein by reference. The XML file of the sequence listing submitted via EFS includes a file named "4329.3002 WO SEQ Listing.xml", created on June 27, 2024, with a size of 13,174 bytes.

[0003] (Field) The present invention relates to aqueous liquid pharmaceutical formulations comprising a surfactant component and a polynucleotide molecule. In particular, the present invention relates to aqueous liquid pharmaceutical formulations for use as pharmaceuticals for use in the treatment or prevention of viral infections and diseases associated with viral infections, as well as related therapeutic methods. [Background technology]

[0004] (Background of the invention) It is essential that therapeutic agents are delivered to the target tissue and / or cells in an amount that allows them to exert their therapeutic effect. In fact, insufficient delivery of therapeutic agents often hinders the ability of otherwise effective agents to achieve the desired therapeutic outcome. This problem is particularly pronounced with immunoglobulins and polynucleotide molecules, especially biological agents such as genomic DNA (gDNA), complementary DNA (cDNA), mRNA, siRNA, and shRNA, whose high molecular weight and net ionic charge negate further complexity in targeted delivery. Unlike small molecule therapeutics, biological agents do not undergo passive diffusion across the cell membrane.

[0005] Furthermore, when introduced into a target, polynucleotide molecules are susceptible to degradation by endonucleases and exonucleases, which can hydrolyze the phosphodiester bonds of the polynucleotide backbone.

[0006] Therefore, the clinical value of polynucleotide therapeutics depends on delivery technologies that improve the stability of polynucleotides, promote efficient internalization, and increase target affinity (Kulkarni et al., 2021).

[0007] Considering the challenges associated with the delivery of polynucleotide therapeutics, many platform delivery technologies have been developed, particularly in gene therapy products. These include chemically modified antisense oligonucleotides (ASOs), N-acetylgalactosamine (GalNAc) conjugates, adeno-associated virus (AAV) vectors, and lipid delivery systems including lipid nanoparticles (LNPs) and liposomes (Kulkarni et al., 2021).

[0008] ASO refers to polynucleotide molecules that contain many chemical modifications to the backbone, sugar moiety, or nitrogenous base to enhance affinity for target RNA, improve nuclease resistance, and regulate the immunological profile of the polynucleotide (Khvorova and Watts, 2017). GalNAc conjugation promotes the accumulation of related polynucleotide therapeutics, especially in the liver. Specifically, GalNAc constructs target the asialoglycoprotein receptor, which is mainly expressed in hepatocytes of the liver. Subsequent internalization by clathrin-mediated endocytosis and endosomal escape by the polynucleotide molecule result in targeted delivery (Springer and Dowdy, 2018). These chemical modification techniques have significant utility but may complicate the manufacturing process and increase costs. Conversely, the AAV vector system is relatively simple and may result in efficient delivery of polynucleotide molecules to the nucleus. Furthermore, different AAVs exhibit different cell tropisms, and thus this platform technology can be adapted to various target tissues or cells.

[0009] However, lipid delivery systems, including lipid nanoparticles and liposomes, are increasingly being recognized as the most promising delivery systems for polynucleotide molecules. In particular, the biocompatibility of lipid-based formulations and especially the ease of their large-scale manufacture make such formulations an attractive means for research and development. Furthermore, lipid delivery systems are extremely efficient at delivering polynucleotide molecules to target cells.

[0010] However, lipid delivery systems are associated with several problems. For example, lipid delivery systems such as lipid nanoparticles and liposomes often contain aggregation of lipid particles and polyethylene glycol (PEG)-based compounds that hinder subsequent immune recognition and removal (Jokerst et al., 2011). In fact, PEGylated lipid particles appear to have an increased half-life in circulation (Huang and Liu, 2011). However, PEGylated lipid nanoparticles have been reported to strongly inhibit endosomal release of polynucleotide molecules (Song et al., 2002) and to stimulate unwanted immune responses, such as enhanced antibody responses (Garay and Labaune, 2011).

[0011] Alternative polymers to PEG, including naturally derived polymers such as serum albumin and zwitterionic polymers such as poly(carboxybetaine), have been studied (Hoang Thi et al., 2020). However, these polymers do not usually work very favorably and are found in many other common products or pharmaceutical compositions, raising concerns about their immunogenicity. Furthermore, alternative technologies such as the above polymers and XTEN peptides, although they can extend the half-life of lipid particles in vivo, are often inhibitory to the uptake of polynucleotide molecules because of their large size.

[0012] A lipid delivery system containing a polynucleotide molecule must be administered to a subject by a route that is tolerable to the subject, supports patient compliance, and ensures delivery to a target tissue or cell at a concentration suitable for the polynucleotide molecule to exert a therapeutic effect. Usually, a lipid delivery system containing a polynucleotide molecule is administered intravenously, for example, to facilitate systemic administration, or by local injection to a target tissue or organ via, for example, intradermal, subcutaneous, intraocular, intramuscular, intramyocardial, or intratumoral routes.

[0013] In particular, lipid delivery systems containing polynucleotide molecules are not commonly administered topically to the lungs or nose, i.e., via inhalation or intranasal administration (Li et al. 2023). This is despite the fact that these routes of administration are clearly suitable for delivering polynucleotide therapeutics to the respiratory system, especially when polynucleotide therapeutics are useful in treating respiratory system diseases, such as respiratory viral infections or cystic fibrosis.

[0014] However, the administration of lipid delivery systems containing polynucleotide molecules to the respiratory system, particularly via local administration to the lungs or nose, is accompanied by several well-documented challenges.

[0015] Firstly, the lipid delivery system containing polynucleotide molecules must be administered intranasally or by inhalation in a manner that ensures a therapeutically effective dose is delivered to the desired area of ​​the respiratory system, such as the upper or lower respiratory tract. Furthermore, administration should be carried out within a timeframe that supports high patient compliance.

[0016] Furthermore, regulatory requirements stipulate that lipid nanoparticles and liposome drugs are subject to further consideration of vesicle or particle size, size distribution, and morphology (FDA Guidance for Industry, 2018, entitled “Liposome Drug Products Chemistry, Manufacturing, and Controls; Human Pharmacokinetics and Bioavailability; and Labelling Documentation”). In addition, lipid nanoparticles and liposomes are prone to fusion (i.e., smaller lipid particles combining to form larger lipid particles), aggregation, and leakage of contained polynucleotide molecules, each of which can adversely affect the stability of the contained polynucleotide molecules.

[0017] In conclusion, there remains a need to develop liquid pharmaceutical formulations that enable efficient delivery of polynucleotide molecules to target tissues or cells, and that avoid some of the challenges associated with the use of lipid delivery systems, particularly those used for delivering polynucleotide molecules via inhalation or intranasal cavity. [Overview of the Initiative]

[0018] (Summary of the invention) Commonly used excipients are generally well understood and considered pharmacologically inactive. However, the inventors have made the surprising discovery that certain surfactants conventionally used as excipients in pharmaceutical formulations possess specific biophysical and biological activity.

[0019] Therefore, the present invention provides an aqueous liquid pharmaceutical formulation comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) a polynucleotide molecule.

[0020] The present invention further provides aqueous liquid pharmaceutical formulations comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) a polynucleotide molecule, for use as a pharmaceutical for use in the treatment or prevention of viral infections and diseases associated with viral infections.

[0021] In this embodiment, such formulations are suitable for local administration to, for example, the lungs or nose, and other routes.

[0022] In one embodiment, the formulation of the present invention preferably forms a stable colloidal emulsion. [Brief explanation of the drawing]

[0023] (Brief explanation of the drawing) [Figure 1]Figure 1 shows the effects of apical treatment with vehicle (water only), polynucleotide molecules (shRNA 1) in vehicle (water only), or polynucleotide molecules (shRNA 1) in vehicle and three different concentrations of surfactant components (surfactant component 1: 0.005% (w / w) oleic acid and 0.0045% (w / w) polysorbate 80; surfactant component 2: 0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80; and surfactant component 3: 0.2% (w / w) oleic acid and 0.15% (w / w) polysorbate 80) on CXCL10 release into the basal chamber from gas-liquid interface (ALI) cultured nasal epithelium on days 1, 2, and 3 after treatment. [Figure 2] Figure 2 shows the effects of apical treatment with vehicle (water only), polynucleotide molecules (shRNA 1) in vehicle (water only), or polynucleotide molecules (shRNA 1) in vehicle and two different concentrations of surfactant components (0.005% (w / w) oleic acid and 0.0045% (w / w) polysorbate 80; and 0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) on CXCL10 release into apical lavage fluid derived from ALI cultured nasal epithelium on day 1 after treatment (i.e., before viral infection). The effects of these treatments are compared with basal treatment with oseltamivir. [Figure 3] Figure 3 shows the effects of apical treatment with vehicle (water only), polynucleotide molecules (shRNA 1) in vehicle (water only), or polynucleotide molecules (shRNA 1) in vehicle and two different concentrations of surfactant components (0.005% (w / w) oleic acid and 0.0045% (w / w) polysorbate 80; and 0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) on the amount of virus in apical lavage fluid derived from influenza virus-infected ALI cultured nasal epithelium on day 2 post-infection (i.e., day 3 post-treatment). The effects of these treatments are compared with basal treatment with oseltamivir. [Figure 4]Figure 4 shows the effects of apical treatment with vehicle (water only), vehicle (water) with surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) only, vehicle (water) with polynucleotide molecules (shRNA 1), or vehicle (water) with polynucleotide molecules (shRNA 1) and the surfactant components on CXCL10 release into apical lavage fluid derived from ALI cultured nasal epithelium on day 1 after treatment (i.e., before viral infection). The effects of these treatments are compared with basal treatment with oseltamivir. [Figure 5] Figure 5 shows the effects of apical treatment with vehicle (water only), surfactant components in vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) only, and polynucleotide molecules (shRNA 1) in vehicle or polynucleotide molecules (shRNA 1) in vehicle and the surfactant components on the amount of virus in apical lavage fluid derived from influenza virus-infected ALI cultured nasal epithelium on day 2 post-infection (i.e., day 3 post-treatment). The effects of these treatments are compared with basal treatment with oseltamivir. [Figure 6] Figure 6 shows the effect of dsRNA 1 or dsRNA 2 in the vehicle (bufferant: 0.28% (w / w) sodium citrate dihydrate and 0.20% (w / w) citric acid monohydrate), or dsRNA 1 or dsRNA 2 in the vehicle and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) on CXCL10 release into apical lavage solution derived from ALI cultured nasal epithelium on day 1 after treatment (i.e., before viral infection). [Figure 7]Figure 7 shows the effect of dsRNA 1 or dsRNA 2 in the vehicle (bufferant: 0.28% (w / w) sodium citrate dihydrate and 0.20% (w / w) citric acid monohydrate), or dsRNA 1 or dsRNA 2 in the vehicle and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) on the amount of virus in apical lavage fluid derived from influenza virus-infected ALI cultured nasal epithelium on day 1 post-infection (i.e., day 2 post-treatment). [Figure 8] Figure 8 shows the effects of intranasal treatment with a vehicle (water only), a surfactant component in the vehicle combined with further pharmaceutically acceptable excipients (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) (Formulation Example 1A without shRNA 1, hereafter referred to as "Surfactant Formulation"), and polynucleotide molecules (shRNA 1) in the vehicle, or polynucleotide molecules (shRNA 1) in the above-mentioned surfactant formulation (Formulation Example 1A, hereafter referred to as "shRNA 1 containing surfactant formulation") on viral load in nasal tissue from influenza (PR8) infected mice on day 1 and day 5 post-infection. The effects of these treatments are compared with oral treatment with oseltamivir phosphate. [Figure 9] Figure 9 shows the effects of intranasal treatment with a vehicle (water only), a formulation containing surfactant components in the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) and further pharmaceutically acceptable excipients (Formulation Example 1A without shRNA 1, hereafter referred to as "Surfactant Formulation"), and polynucleotide molecules (shRNA 1) in the vehicle, or polynucleotide molecules (shRNA 1) in the above-mentioned surfactant formulation (Formulation Example 1A, hereafter referred to as "Surfactant Formulation with shRNA 1") on neutrophil accumulation in nasal lavage fluid of influenza (PR8) infected mice on day 1 and day 5 post-infection. The effects of these treatments are compared with oral treatment with oseltamivir phosphate. [Figure 10]Figure 10 shows the effects of intranasal treatment with a vehicle (water only), a formulation containing a surfactant component (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) and further pharmaceutically acceptable excipients (Formulation Example 1A without shRNA 1, hereafter referred to as "Surfactant Formulation"), and polynucleotide molecules (shRNA 1) in the vehicle, or polynucleotide molecules (shRNA 1) in the above-mentioned surfactant formulation (Formulation Example 1A, hereafter referred to as "Surfactant Formulation containing shRNA 1") on weight loss observed in influenza (PR8) infected mice for 5 days post-infection. The effects of these treatments are compared with oral treatment with oseltamivir phosphate. [Figure 11] Figure 11 shows the effects of GFP-coding mRNA in a buffer (0.28% (w / w) sodium citrate dihydrate and 0.20% (w / w) citrate monohydrate), or the effects of apical treatment with the GFP-coding mRNA in the buffer and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) on mRNA exposure and the resulting GFP signaling in ALI cultured nasal epithelial cells 24 hours after treatment. [Figure 12] Figure 12 shows the effect of a (β-gal) plasmid encoding β-galactosidase in a buffer (0.28% (w / w) sodium citrate dihydrate and 0.20% (w / w) citrate monohydrate), or the effect of apical treatment with the β-gal plasmid in the buffer and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) on plasmid exposure and resulting β-galactosidase enzyme activity in ALI cultured nasal epithelial cells 24 hours after treatment. [Figure 13]Figure 13(A-D) shows the effect of the presence of three different concentrations of surfactant components (surfactant component 1: 0.005% (w / w) oleic acid and 0.0045% (w / w) polysorbate 80; surfactant component 2: 0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80; and surfactant component 3: 0.2% (w / w) oleic acid and 0.15% (w / w) polysorbate 80) on the polydispersity and particle size of a formulation containing a polynucleotide molecule (shRNA 1) in a vehicle (water). [Figure 14] Figure 14 shows the effects of apical treatment with a vehicle (buffer only), polynucleotide molecules (shRNA 1) in the buffer, surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or shRNA 1 in the vehicle and surfactant components on CXCL10 release into apical lavage solution derived from ALI cultured nasal epithelium on day 1 after treatment (i.e., before viral infection). [Figure 15] Figure 15 shows the effects of vehicle (buffer only), polynucleotide molecules (shRNA 1) in the buffer, surfactant components in the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) or shRNA 1 and surfactant components on the amount of virus in apical lavage fluid derived from human rhinovirus (HRV16) infected ALI cultured nasal epithelium on day 2 post-infection (i.e., day 3 after initial treatment) on day 1 and day 0 pre-infection. [Figure 16] Figure 16 shows the effects of vehicle (buffer only), polynucleotide molecules (shRNA 1) in the buffer, surfactant components in the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) or shRNA 1 and surfactant components on the amount of virus in apical lavage fluid derived from nasal epithelium cultured with respiratory syncytial virus (RSV) A2 culture on day 3 post-infection (i.e., day 4 after initial treatment). [Figure 17]Figure 17 shows the effects of vehicle (buffer only), polynucleotide molecules in the buffer (shRNA 1), surfactant components in the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or apical treatment on day 1 and day 0 before infection with shRNA 1 and surfactant components on cellular integrity, as expressed by transepithelial electrical resistance (TEER), in a cultured nasal epithelial epithelium model infected with respiratory syncytial virus (RSV) A2, on days 0, 1, 2, and 3 post-infection. [Figure 18] Figure 18 shows the effects of intranasal treatment with a vehicle (saline only), surfactant components in the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or polynucleotide molecules (shRNA 1) in the vehicle and surfactant components on the viral load in lung tissue from RSV A2-infected mice four days post-infection. The effects of these treatments are compared with intranasal treatment with ribavirin. [Figure 19] Figure 19 shows the effects of intranasal treatment with a vehicle (saline only), a surfactant component in the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or a polynucleotide molecule (shRNA 1) in the vehicle and a surfactant component on neutrophil accumulation in nasal lavage fluid of mice infected with respiratory syncytial virus (RSV) A2 four days post-infection. The effects of these treatments are compared with intranasal treatment with ribavirin. [Figure 20] Figure 20 shows the effects of intranasal treatment with a vehicle (saline only), surfactant components in the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or polynucleotide molecules (shRNA 1) and surfactant components in the vehicle on weight loss observed in respiratory syncytial virus (RSV) infected mice 4 days post-infection. The effects of these treatments are compared with intranasal treatment with ribavirin. [Figure 21]Figure 21 shows the effects of subcutaneous treatment with a vehicle (physiological saline only), surfactant components in a phosphate buffer (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or polynucleotide molecules (shRNA 1) and surfactant components (2 or 20 mg / mL) in a phosphate buffer on CXCL10 release in mouse serum 24 hours after treatment. [Figure 22] Figure 22 shows the effects of subcutaneous treatment with vehicle (saline only; i.e., untreated), surfactant components in vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), polynucleotide molecules (shRNA 1) in vehicle and surfactant components, surfactant components in vehicle combined with oral oseltamivir treatment, or shRNA 1 in vehicle and surfactant components combined with oral oseltamivir treatment on the viral load in the lung tissue of influenza (PR8) virus-infected mice 5 days post-infection. [Figure 23] Figure 23 shows the effects of subcutaneous treatment with the following on the viral load in the nasal tissue of influenza (PR8) virus-infected mice 5 days post-infection: vehicle (saline only; i.e., untreated), surfactant components in the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), polynucleotide molecules (shRNA 1) in the vehicle and surfactant components, surfactant components in the vehicle combined with oral oseltamivir treatment, or shRNA 1 in the vehicle and surfactant components combined with oral oseltamivir treatment. [Figure 24] Figure 24 shows the effects of subcutaneous treatment with the following on neutrophil accumulation in the lung tissue of influenza (PR8) infected mice 5 days post-infection: vehicle (saline only; i.e., untreated), surfactant components in the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), polynucleotide molecules (shRNA 1) in the vehicle and surfactant components, surfactant components in the vehicle combined with oral oseltamivir treatment, or shRNA 1 in the vehicle and surfactant components combined with oral oseltamivir treatment. [Figure 25] Figure 25 shows the effects of subcutaneous treatment with a vehicle (saline only; i.e., untreated), surfactant components in the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), polynucleotide molecules (shRNA 1) in the vehicle and surfactant components, surfactant components in the vehicle combined with oral oseltamivir treatment, or shRNA 1 in the vehicle and surfactant components combined with oral oseltamivir treatment. [Figure 26] Figure 26 shows the effects of subcutaneous treatment with vehicle (saline only), surfactant components in vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), polynucleotide molecules (shRNA 1) in vehicle and surfactant components, surfactant components in vehicle combined with oral oseltamivir treatment, or shRNA 1 in vehicle and surfactant components combined with oral oseltamivir treatment. [Figure 27] Figure 27 shows the effects of intranasal vaccination with vehicle (i.e., PBS only), recombinant H1N1 (rH1N1) hemagglutinin (HA) in vehicle, rH1N1 HA in vehicle pre-treated with polynucleotide molecules (shRNA 1) and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or rH1N1 HA in vehicle pre-treated with the control adjuvant CPG-ODN on the viral load in the lung tissue of influenza (PR8) infected mice 5 days post-infection. [Figure 28]Figure 28 shows the effects of intranasal vaccination with vehicle (i.e., PBS only), vehicle with recombinant H1N1 (rH1N1) hemagglutinin (HA), vehicle with rH1N1 HA pre-treated with polynucleotide molecules (shRNA 1) and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or vehicle with rH1N1 HA pre-treated with the control adjuvant CPG-ODN on the viral load in the nasal tissue of influenza (PR8) infected mice 5 days post-infection. [Figure 29] Figure 29 shows the effects of intranasal vaccination with vehicle (i.e., PBS only), recombinant H1N1 (rH1N1) hemagglutinin (HA) in vehicle, rH1N1 HA in vehicle after pretreatment with polynucleotide molecules (shRNA 1) and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or rH1N1 HA in vehicle after pretreatment with the control adjuvant CPG-ODN on neutrophil accumulation in lung tissue of influenza (PR8) infected mice 5 days post-infection. [Figure 30] Figure 30 shows the effects of intranasal vaccination with vehicle (i.e., PBS only), vehicle with recombinant H1N1 (rH1N1) hemagglutinin (HA), vehicle with rH1N1 HA pre-treated with polynucleotide molecules (shRNA 1) and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or vehicle with rH1N1 HA pre-treated with the control adjuvant CPG-ODN in influenza (PR8) infected mice 5 days post-infection. [Figure 31]Figure 31 shows the effects of intranasal vaccination with vehicle (i.e., PBS only), recombinant H1N1 (rH1N1) hemagglutinin (HA) in vehicle, rH1N1 HA in vehicle pre-treated with polynucleotide molecules (shRNA 1) and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or rH1N1 HA in vehicle pre-treated with the control adjuvant CPG-ODN on weight loss observed in influenza (PR8) infected mice 5 days post-infection. [Figure 32] Figure 32 shows the effects of apical treatment with GFP-coding mRNA in a buffer (0.28% (w / w) sodium citrate dihydrate and 0.20% (w / w) citrate monohydrate), or with the GFP-coding mRNA in the buffer and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), on mRNA exposure and the resulting GFP signal in ALI cultured nasal epithelial cells 24 hours after treatment. [Figure 33] Figure 33 shows the effect of apical treatment of ALI cultured nasal epithelium with H1N1 hemagglutinin (HA) mRNA in a vehicle (i.e., PBS) or with the H1N1 HA mRNA in a vehicle and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) (i.e., SF) on H1N1 HA protein expression in ALI cultured nasal epithelial cells 48 hours after treatment. [Figure 34] Figure 34 shows the effect of apical treatment of ALI cultured nasal epithelium with H1N1 hemagglutinin (HA) mRNA in a vehicle (i.e., PBS) or with the H1N1 HA mRNA in a vehicle and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) (i.e., SF) on the expression of H1N1 HA protein in ALI cultured nasal epithelial cells 48 hours after treatment. [Figure 35]Figure 35 shows the effect of apical treatment of ALI cultured bronchial epithelium with cystic fibrosis transmembrane conductance regulator (CFTR) encoding mRNA in a vehicle (i.e., PBS only) or with a surfactant component (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) on CFTR protein expression in ALI cultured bronchial epithelial cells 72 hours after treatment. [Figure 36] Figure 36 shows the effects of apical treatment of ALI cultured nasal epithelium with a vehicle (i.e., citrate buffer or phosphate buffer only), surfactant components in the citrate buffer or phosphate buffer (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or both low-shear mixture (i.e., magnetically agitated) and high-shear mixture formulations containing polynucleotide molecules (shRNA 1) and surfactant components in the citrate buffer or phosphate buffer on the amount of virus in the apical lavage solution derived from influenza virus (PR8) infected ALI cultured nasal epithelium on day 2 post-infection (i.e., day 3 post-treatment). [Figure 37] Figure 37 shows the effect of apical treatment of ALI cultured corneal epithelium with a phosphate buffer (0.06% (w / w) sodium dihydrogen phosphate and 0.08% (w / w) disodium hydrogen phosphate) or with the phosphate buffer and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) on siRNA exposure and the resulting fluorescence signal in ALI cultured corneal epithelium cells 4 hours after treatment. [Figure 38] Figure 38 shows the effect of GFP-encoding mRNA in a phosphate buffer (0.06% (w / w) sodium dihydrogen phosphate and 0.08% (w / w) disodium hydrogen phosphate) or the effect of apical treatment of ALI-cultured corneal epithelium with a phosphate buffer and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) on mRNA exposure and the resulting GFP signaling in ALI-cultured corneal epithelium cells 72 hours after treatment. [Figure 39] Figure 39 shows the effects of apical treatment with surfactant components in the buffer (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), shRNA 1 in the buffer, RNA conjugate 1 in the buffer, RNA conjugate 1 and surfactant components in the buffer, RNA conjugate 2 in the buffer, and RNA conjugate 2 and surfactant components in the buffer on CXCL10 release into apical lavage solution derived from ALI cultured nasal epithelium on day 1 after treatment. [Figure 40] Figure 40 shows the effect of the presence of a surfactant component (where the surfactant component is polysorbate 80 (0.045% (w / w)) + caprylic acid (0.05% (w / w)), polysorbate 80 (0.045% (w / w)) + oleic acid (0.05% (w / w)), Brij 35 (0.045% (w / w)) + oleic acid (0.05% (w / w)), Brij 35 (0.45% (w / w)) + caprylic acid (0.5% (w / w)), or polysorbate 80 (4.5% (w / w)) + caprylic acid (5.0% (w / w))) on the polydispersity and particle size of a formulation containing a polynucleotide molecule (shRNA 1) in a vehicle (water). [Figure 41] Figure 41 shows the effect of the presence of a surfactant component (where the surfactant component is polysorbate 80 (0.045% (w / w)) + caprylic acid (0.05% (w / w)), polysorbate 80 (0.045% (w / w)) + oleic acid (0.05% (w / w)), Brij 35 (0.045% (w / w)) + oleic acid (0.05% (w / w)), Brij 35 (0.45% (w / w)) + caprylic acid (0.5% (w / w)), or polysorbate 80 (4.5% (w / w)) + caprylic acid (5.0% (w / w))) on the polydispersity and particle size of a formulation containing a polynucleotide molecule (shRNA 1) in a vehicle (water). [Figure 42]Figure 42 shows the effect of the presence of a surfactant component (where the surfactant component is polysorbate 80 (0.045% (w / w)) + caprylic acid (0.05% (w / w)), polysorbate 80 (0.045% (w / w)) + oleic acid (0.05% (w / w)), Brij 35 (0.045% (w / w)) + oleic acid (0.05% (w / w)), Brij 35 (0.45% (w / w)) + caprylic acid (0.5% (w / w)), or polysorbate 80 (4.5% (w / w)) + caprylic acid (5.0% (w / w))) on the polydispersity and particle size of a formulation containing a polynucleotide molecule (shRNA 1) in a vehicle (water). [Figure 43] Figure 43 shows the effect of the presence of a surfactant component (where the surfactant component is polysorbate 80 (0.045% (w / w)) + caprylic acid (0.05% (w / w)), polysorbate 80 (0.045% (w / w)) + oleic acid (0.05% (w / w)), Brij 35 (0.045% (w / w)) + oleic acid (0.05% (w / w)), Brij 35 (0.45% (w / w)) + caprylic acid (0.5% (w / w)), or polysorbate 80 (4.5% (w / w)) + caprylic acid (5.0% (w / w))) on the polydispersity and particle size of a formulation containing a polynucleotide molecule (shRNA 1) in a vehicle (water). [Figure 44] Figure 44 shows the effect of the presence of a surfactant component (where the surfactant component is polysorbate 80 (0.045% (w / w)) + caprylic acid (0.05% (w / w)), polysorbate 80 (0.045% (w / w)) + oleic acid (0.05% (w / w)), Brij 35 (0.045% (w / w)) + oleic acid (0.05% (w / w)), Brij 35 (0.45% (w / w)) + caprylic acid (0.5% (w / w)), or polysorbate 80 (4.5% (w / w)) + caprylic acid (5.0% (w / w))) on the polydispersity and particle size of a formulation containing a polynucleotide molecule (shRNA 1) in a vehicle (water). [Figure 45]Figures 45(A-F) show the particle structure and diameter formed in a formulation containing the surfactant component of the present invention, as observed under a transmission electron microscope, where the surfactant component includes (i)(A-D) oleic acid (as a fatty acid) + polysorbate 80 (as a nonionic surfactant), (ii)(E) caprylic acid (as a fatty acid) + polysorbate 80 (as a nonionic surfactant), and (iii)(F) caprylic acid (as a fatty acid) + Brij 35 (as a nonionic surfactant). [Figure 46] Figure 46 shows differential scanning calorimetry thermographs of formulations of the present invention containing a surfactant component, particularly polysorbate 80 (0.045% (w / w)) + oleic acid (0.05% (w / w)), including formulations containing only the surfactant component (top panel), formulations containing 2 mg / mL of polynucleotide molecules (shRNA 1) and the surfactant component (middle panel), and formulations containing 20 mg / mL of polynucleotide molecules (shRNA 1) and the surfactant component (bottom panel). [Figure 47] Figure 47 shows the effects of apical treatment with culture medium, surfactant components in phosphate buffer (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), shRNA 1 and surfactant components in phosphate buffer, or preconalil, a known human rhinovirus (HRV) inhibitor in culture medium, on the release of CXCL10, CXCL8, and CCL5 (Figures 47-49, respectively) into apical lavage fluid derived from HRV-infected ALI cultured bronchial epithelium from asthma donors 5 days after viral inoculation. [Figure 48] Figure 48 shows the effects of apical treatment with culture medium, surfactant components in phosphate buffer (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), shRNA 1 and surfactant components in phosphate buffer, or preconalil, a known human rhinovirus (HRV) inhibitor in culture medium, on the release of CXCL10, CXCL8, and CCL5 (Figures 47-49, respectively) into apical lavage fluid derived from HRV-infected ALI cultured bronchial epithelium from asthma donors 5 days after viral inoculation. [Figure 49]Figure 49 shows the effects of apical treatment with culture medium, surfactant components in phosphate buffer (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), shRNA 1 and surfactant components in phosphate buffer, or preconalil, a known human rhinovirus (HRV) inhibitor in culture medium, on the release of CXCL10, CXCL8, and CCL5 (Figures 47-49, respectively) into apical lavage fluid derived from HRV-infected ALI cultured bronchial epithelium from asthma donors 5 days after viral inoculation. [Figure 50] Figure 50 shows the effects of culture medium, surfactant components in phosphate buffer (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), shRNA 1 and surfactant components in phosphate buffer, or apical treatment with preconalil, a known human rhinovirus (HRV) inhibitor, in the culture medium on the viral load in HRV-infected ALI cultured bronchial epithelium from asthma donors two days after viral inoculation. [Figure 51] Figure 51 shows a schematic representation of different types of particles formed by formulations containing lipids and surfactants. [Modes for carrying out the invention]

[0024] (Detailed description of the invention) The present invention is based on findings made by testing the exposure of polynucleotide molecules, such as RNA or DNA molecules, to cells when formulated with specific surfactant components. The present invention is further based on findings made by testing the exposure of various antiviral polynucleotide molecules, such as RNA or DNA molecules, in combination with specific surfactant components, and therefore their antiviral activity.

[0025] In particular, the present invention is (i) An apical administration formulation comprising a surfactant component containing a mixture of fatty acids, particularly oleic acid, and a nonionic surfactant, particularly polysorbate 80, combined with one of several diverse antiviral polynucleotide molecules, such as shRNA molecule, dsRNA 1, dsRNA 2, RNA conjugate 1, or RNA conjugate 2, has a potent effect in stimulating the innate immune response as a result of improved polynucleotide molecule delivery, as determined by the production of CXCL10, a surrogate marker of antiviral interferon (IFN) signaling, in gas-liquid interface (ALI) cultured nasal epithelium (see Biological Examples 1-4 and 15, and Figures 1, 2, 4, 6, and 39); (ii) An apical administration formulation containing a surfactant component comprising a mixture of fatty acids, particularly oleic acid, and a nonionic surfactant, particularly polysorbate 80, combined with one of several diverse antiviral polynucleotide molecules, such as shRNA molecules, dsRNA 1, or dsRNA 2, is potent in reducing viral load in infection models including influenza virus-infected ALI cultured nasal epithelium (see Biological Examples 2-4, Figures 3, 5, and 7); (iii) An apical administration formulation comprising a surfactant component, particularly a mixture of fatty acids, especially oleic acid, and a nonionic surfactant, especially polysorbate 80, combined with an antiviral polynucleotide molecule (shRNA 1), has a potent effect in stimulating the innate immune response, as determined by the production of CXCL10, and in reducing viral load in infection models, including human rhinovirus type 16 (HRV16) infected ALI cultured nasal epithelium (see Biological Example 7 and Figures 14 and 15); (iv) An apical administration formulation containing a surfactant component, particularly a mixture of fatty acids, especially oleic acid, and a nonionic surfactant, especially polysorbate 80, combined with an antiviral polynucleotide molecule (shRNA 1), has a potent effect in reducing viral load and improving epithelial cell / barrier integrity, as expressed by transepithelial electrical resistance (TEER), in infection models including human respiratory syncytial virus (RSV) A2-infected ALI cultured nasal epithelium (see Biological Example 8 and Figures 16 and 17); (v) Intranasal administration of a formulation containing a surfactant component, particularly a mixture of fatty acids, especially oleic acid, and a nonionic surfactant, especially polysorbate 80, combined with an antiviral polynucleotide molecule (shRNA 1), has a potent effect in reducing viral load, mitigating virus-induced inflammation, and protecting against virus-induced weight loss in in vivo models, including mice infected with influenza (PR8) virus (see Biological Example 5 and Figures 8-10); and (vi) Intranasal administration of a formulation containing a surfactant component, particularly a mixture of fatty acids, especially oleic acid, and a nonionic surfactant, especially polysorbate 80, combined with an antiviral polynucleotide molecule (shRNA 1), has a potent effect in reducing viral load, mitigating virus-induced inflammation, and protecting against virus-induced weight loss in in vivo models, including human respiratory syncytial virus (RSV) A2 infected mice (see Biological Example 9 and Figures 18-20); (vii) Subcutaneous administration of a formulation containing a surfactant component, particularly a mixture of fatty acids, especially oleic acid, and a nonionic surfactant, especially polysorbate 80, combined with an antiviral polynucleotide molecule (shRNA 1), significantly stimulates the innate immune response, as determined by the production of CXCL10 in uninfected mice, and is potent in reducing viral load, mitigating virus-induced inflammation, and protecting against virus-induced weight loss in in vivo models, including influenza (PR8) virus-infected mice (see Biological Example 10 and Figures 21-26); (viii) Subcutaneous administration of a formulation containing a surfactant component, particularly a mixture of fatty acids, especially oleic acid, and a nonionic surfactant, especially polysorbate 80, combined with an antiviral polynucleotide molecule (shRNA 1), has a potent adjuvant effect on subcutaneous administration of recombinant H1N1 (rH1N1) hemagglutinin (HA), as demonstrated by the enhanced ability of rH1N1 HA vaccination to reduce viral load, mitigate virus-induced inflammation, and protect against virus-induced weight loss in in vivo models, including influenza (PR8) virus-infected mice (see Biological Example 11 and Figures 27-31); (ix) A surfactant component comprising a mixture of fatty acids, particularly oleic acid, and a nonionic surfactant, particularly polysorbate 80, combined with a polynucleotide molecule, particularly siRNA, mRNA, or DNA molecule, that modulates or expresses a marker protein, effectively increases cellular exposure to the polynucleotide molecule, as determined by the modulation or increase of marker protein expression in models including ALI cultured nasal epithelium or ALI cultured corneal epithelium (see Biological Examples 6, 12, and 14, and Figures 11, 12, 32-35, 37, and 38); (x) The in vitro activity of formulations containing a surfactant component, particularly a mixture of fatty acids, especially oleic acid, and a nonionic surfactant, especially polysorbate 80, combined with an antiviral polynucleotide molecule (shRNA 1), is not significantly affected by the type of buffer or shear mixture, as demonstrated by the reduction in viral load in infection models including influenza virus-infected ALI cultured nasal epithelium (see Biological Example 13 and Figure 36); (xi) A apical administration formulation containing a surfactant component comprising a mixture of fatty acids, particularly oleic acid, and a nonionic surfactant, particularly polysorbate 80, combined with an antiviral polynucleotide molecule (shRNA 1), has a potent effect in reducing the human rhinovirus (HRV)-inducible release of pro-inflammatory cytokines CXCL10, CXCL8, and CCL5, as well as reducing viral load, in infection models including ALI cultured bronchial epithelium infected with human rhinovirus type 16 (HRV16) from asthma donors (see Biological Example 16 and Figures 47-50). (xii) A formulation of a polynucleotide molecule, particularly an shRNA molecule, containing a surfactant component comprising a mixture of a specific concentration of fatty acid, particularly oleic acid, and a nonionic surfactant, particularly polysorbate 80, results in a formulation in which the colloidal particle size of the colloidal particles containing the polynucleotide molecule is stable, such that it is represented by low polydispersity and consistent particle size (see Biophysical Example 1 and Figure 13(A-D)); (xiii) Formulations of polynucleotide molecules, particularly shRNA molecules, comprising a surfactant component including caprylic acid or oleic acid as a fatty acid and polysorbate 80 or Brij 35 as a nonionic surfactant, are each produced in which colloidal particles are formed such that they are represented by low polydispersity and consistent particle size, and the particle size of the colloidal particles containing the polynucleotide molecule is stable (see Biophysical Examples 2 and 3 and Figures 40-45(A-F)); and (xiv) Formulations comprising a polynucleotide molecule, particularly shRNA 1, and a surfactant component comprising a mixture of a fatty acid, particularly oleic acid, and a nonionic surfactant, particularly polysorbate 80, produce a stable colloidal emulsion in which the colloidal particles are extremely stable and the stability of the polynucleotide molecule is increased, as indicated by an increase in melting temperature (see Biophysical Example 4 and Figure 46). This is based on the astonishing discovery that...

[0026] (Surfactant component) The aqueous liquid pharmaceutical formulation of the present invention contains a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant.

[0027] (fatty acid) By definition, as used herein, “fatty acid” typically refers to a carboxylic acid molecule containing a carboxylic acid group bonded to an aliphatic hydrocarbon “tail” that is 4 to 24 carbon atoms long. For example, the aliphatic hydrocarbon “tail” may have a carbon atom length of 4 to 22, e.g., 4 to 20, e.g., 4 to 18, e.g., 4 to 16, e.g., 4 to 14, e.g., 4 to 12, e.g., 4 to 10, e.g., 4 to 8, e.g., 4 to 6 carbon atoms. Alternatively, the aliphatic hydrocarbon “tail” may have a carbon atom length of 6 to 24, e.g., 8 to 24, e.g., 10 to 24, e.g., 12 to 24, e.g., 14 to 24 carbon atoms. For example, the aliphatic hydrocarbon “tail” may have a carbon atom length of 6 to 22, e.g., 6 to 20, e.g., 8 to 20, e.g., 8 to 18, e.g., 10 to 18 carbon atoms. In one embodiment, the aliphatic hydrocarbon "tail" is 4 to 6 carbon atoms long, i.e., the fatty acid is a short-chain fatty acid such as butyric acid (4 carbon atoms). Alternatively, the aliphatic hydrocarbon "tail" is 6 to 12 carbon atoms long, i.e., the fatty acid is a medium-chain fatty acid such as caprylic acid (8 carbon atoms) and capric acid (10 carbon atoms). Alternatively, the aliphatic hydrocarbon "tail" is 14 to 24 carbon atoms long, i.e., the fatty acid is a long-chain fatty acid such as oleic acid (18 carbon atoms), stearic acid (18 carbon atoms), and arachidic acid (20 carbon atoms). The aliphatic hydrocarbon "tail" can be saturated or unsaturated. If unsaturated, the aliphatic hydrocarbon "tail" may contain, for example, 1, 2, 3, 4, 5, or 6 C=C double bonds, particularly 1 or 2, especially 1 C=C double bond. Fatty acids can be further classified based on the length and saturation of the aliphatic hydrocarbon "tail".

[0028] Preferably, the fatty acids have a molar mass of about 100 g / mol to about 400 g / mol, for example, about 100 g / mol to about 350 g / mol, for example, about 120 g / mol to about 350 g / mol, for example, about 140 g / mol to about 350 g / mol, for example, about 140 g / mol to about 300 g / mol. Exemplary fatty acids may typically have a molar mass of about 150 g / mol to about 400 g / mol, for example, about 200 g / mol to about 350 g / mol, for example, about 200 g / mol to about 300 g / mol. Further exemplary fatty acids may typically have a molar mass of about 140 g / mol to about 200 g / mol. These include, but are not limited to, arachidic acid, arachidonic acid, lauric acid, linoleic acid, linolenic acid, myristic acid, myristoleic acid, oleic acid, palmitic acid, palmitoleic acid, sapienic acid, stearic acid, and vaccenic acid. A further example is caprylic acid.

[0029] In particular, the fatty acid is oleic acid. Or, the fatty acid is caprylic acid.

[0030] In one embodiment, the fatty acid is not linoleic acid.

[0031] Preferably, the fatty acid has a hydrophilic-lipophilic balance (HLB) in the range of 1 to 4, for example, 1 to 3, for example, 1 to 2.5, for example, 1 to 2, for example, 1 to 1.5.

[0032] As used herein, HLB is defined as 20*(MHL / MHL+MOH) (where MHL is the molecular weight of the hydrophilic portion of the molecule and MOH is the molecular weight of the lipophilic portion of the molecule).

[0033] In the case of fatty acids, MHL is considered to be represented by O, which has a molecular weight of 16. MOH is the remaining molecular weight of the molecule.

[0034] For example, oleic acid has an HLB value of 1, while caprylic acid has an HLB value of 2.2.

[0035] Preferably, fatty acids such as caprylic acid, oleic acid, lauric acid, and palmitic acid have Log P values ​​in the range of 2 to 8. For example, caprylic acid has a Log P value of 3.05, and oleic acid has a Log P value of 7.7. For example, lauric acid has a Log P value of 4.6, and palmitic acid has a Log P value of 7.15.

[0036] As used herein, Log P typically refers to the logarithm of the partition coefficient P of a compound between two immiscible phases, octanol and water. This value is a measure of a compound's lipophilicity, indicating how much the compound prefers a lipid (lipophilic) environment to a water (aqueous) environment. A higher Log P value suggests that the compound is more lipophilic, meaning it dissolves better in lipids or nonpolar solvents than in water.

[0037] Preferably, the fatty acid has a critical micelle concentration (CMC) in the range of 0.001 to 0.01 mM. For example, oleic acid has a CMC value of 0.006 mM.

[0038] As used herein, CMC refers to the concentration of surfactant in the bulk phase beyond which micelles spontaneously begin to form. Below the CMC, surfactants exist primarily as individual molecules dispersed in the solution. When the concentration reaches the CMC, these molecules begin to aggregate to form micelles, which are spherical structures in which the hydrophobic (water-repellent) tails of the surfactant molecules are shielded from water by their hydrophilic (water-absorbing) heads.

[0039] Those skilled in the art will understand that the HLB, LogP, and CMC values ​​of fatty acids and nonionic surfactants (discussed further below) can be readily determined or found by referring to references, for example, the Handbook of Pharmaceutical Excipients, 5th edition (Rowe, Sheskey, and Owen, 2006).

[0040] Preferably, the aqueous liquid pharmaceutical formulation contains a single fatty acid as part of the surfactant component. Alternatively, it contains, for example, a mixture of two (or more) fatty acids as part of the surfactant component.

[0041] (Nonionic surfactant) Exemplary nonionic surfactants typically have a molar mass of about 100 g / mol to about 10,000 g / mol, and in particular, about 100 g / mol to about 2,000 g / mol. Exemplary nonionic surfactants typically contain one or more polyoxyalkylene moieties, such as polyoxyethylene and / or polyoxypropylene moieties.

[0042] Examples of nonionic surfactants include polyoxyalkylenes, particularly poloxamers such as poloxamer 188, poloxamer 407, poloxamer 171, and poloxamer 185.

[0043] Further exemplary nonionic surfactants include alkyl ethers of polyethylene glycol, such as those known by the trade names Brij 35 (polyoxyethylene(23) lauryl ether), Brij 52 (polyoxyethylene(20) cetyl ether), Brij 93 (polyoxyethylene(2) oleyl ether), Brij 97 (polyoxyethylene(10) oleyl ether), Brij L4 (polyoxyethylene(4) lauryl ether), Brij 30 (polyoxyethylene(4) lauryl ether), and Brij 78 (polyoxyethylene(20) stearyl ether).

[0044] Further exemplary nonionic surfactants include alkylphenyl ethers of polyethylene glycol, such as those known by the trade name Triton X-100.

[0045] Specific exemplary nonionic surfactants include fatty acid esters, such as fatty acid esters of polyols. Such fatty acid esters may contain one or more fatty acid chains, for example, one, two, or three fatty acid chains, for example, one fatty acid chain. A specific example is polyoxyethylene sorbitan fatty acid ester. In particular, the nonionic surfactant is polyoxyethylene sorbitan fatty acid ester. Preferred polyoxyethylene sorbitan fatty acid esters include polysorbate 80 (e.g., Tween 80), polysorbate 120, polysorbate 85, polysorbate 65, polysorbate 60, polysorbate 40, and polysorbate 20, and in particular polysorbate 80.

[0046] In one embodiment, the nonionic surfactant is not polysorbate 60.

[0047] In one embodiment, the nonionic surfactant is not polysorbate 85.

[0048] Preferably, nonionic surfactants, such as polysorbate and other highly ethoxylated nonionic surfactants, have a hydrophilic-lipophilic balance (HLB) value of 10 or more, for example, in the range of 10 to 20. For example, polysorbate 20 has an HLB value of 16.7. For example, polysorbate 40 has an HLB value of 15.6. For example, polysorbate 65 has an HLB value of 10.5. For example, polysorbate 120 has an HLB value of 14.9. For example, polysorbate 80 has an HLB value of 15.0. For example, Brij 35 has an HLB value of 16.9. For example, Brij 97 has an HLB value of 12.4.

[0049] Preferably, a nonionic surfactant, such as polysorbate 20, has a Log P value in the range of 1 to 5. For example, polysorbate 80 has a Log P value of 4.7. Preferably, the Log P value of the nonionic surfactant is lower than the Log P value of the fatty acid in the surfactant component.

[0050] Preferably, nonionic surfactants, such as polysorbate 20 and Brij 35, have a critical micelle concentration (CMC) in the range of 0.01 to 0.5 mM. For example, polysorbate 20 has a CMC value of 0.06 mM. For example, polysorbate 80 has a CMC value of 0.012 mM. For example, Brij 35 has a CMC value of 0.09 mM. Preferably, the CMC of the nonionic surfactant is greater than the CMC of the fatty acid in the surfactant component.

[0051] Preferably, the aqueous liquid pharmaceutical formulation contains a single nonionic surfactant as part of the surfactant component. Alternatively, it may contain, for example, a mixture of two (or more) nonionic surfactants as part of the surfactant component.

[0052] (Other aspects) Preferably, the surfactant component is selected from the group consisting of (a) a mixture of oleic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (b) a mixture of lauric acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (c) a mixture of linoleic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (d) a mixture of linolenic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (e) a mixture of palmitic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (f) a mixture of stearic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (g) a mixture of oleic acid or a pharmaceutically acceptable salt thereof and polyoxyalkylene such as poloxamer, (h) a mixture of oleic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol, and (i) a mixture of oleic acid or a pharmaceutically acceptable salt thereof and an alkylphenyl ether of polyethylene glycol.

[0053] Most preferably, the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, particularly a polyoxyethylene sorbitan fatty acid ester selected from polysorbate 80 and polysorbate 20. In particular, the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof, especially oleic acid and polysorbate 80.

[0054] Alternatively, the surfactant component is preferably a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, particularly a polyoxyethylene sorbitan fatty acid ester selected from polysorbate 80 and polysorbate 20. In particular, the surfactant component is a mixture of caprylic acid or a pharmaceutically acceptable salt thereof, especially caprylic acid and polysorbate 80.

[0055] Alternatively, the surfactant component is preferably a mixture of oleic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol, for example, those known by the trade names Brij 35, Brij 52, Brij 93, Brij 97, Brij L4, Brij 30, and Brij 78. In particular, the surfactant component is oleic acid or a pharmaceutically acceptable salt thereof, especially a mixture of oleic acid and Brij 35.

[0056] Alternatively, the surfactant component is preferably a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol, for example, those known by the trade names Brij 35, Brij 52, Brij 93, Brij 97, Brij L4, Brij 30, and Brij 78. In particular, the surfactant component is a mixture of caprylic acid or a pharmaceutically acceptable salt thereof, especially caprylic acid and Brij 35.

[0057] In one embodiment, the surfactant component is not a mixture of linoleic acid and polysorbate 60.

[0058] Pharmaceutically acceptable salt forms of fatty acids that can be used include sodium, potassium, and ammonium salts, particularly sodium salts. Preferably, fatty acids are used as free acids, i.e., in the form of free acids.

[0059] The aqueous liquid pharmaceutical formulation of the present invention should preferably form a stable colloidal emulsion (i.e., an oil-in-water emulsion), such as a stable colloidal nanoemulsion. Typically, a stable colloidal emulsion contains stable colloidal particles (i.e., particles containing an oil phase in an oil-in-water emulsion) having an average particle size of about 10 to about 1000 nm, for example, about 50 to about 1000 nm, for example, about 50 to about 750 nm, for example, about 50 to about 500 nm, for example, about 50 to about 400 nm, for example, about 50 to about 300 nm, for example, about 50 to about 100 nm, or about 100 to about 300 nm, or about 100 to about 250 nm, or about 250 to about 500 nm. Therefore, for example, in a preferred embodiment, the average particle size is about 100 to about 300 nm, for example, about 100 to 200 nm (see Biophysical Example 1). Therefore, preferably, the particles of the colloidal emulsion are droplets formed from a surfactant component and containing polynucleotide molecules. The aforementioned particle size refers to the hydrodynamic diameter (Z-average size) which can be measured as described in Biophysical Example 1.

[0060] Such formulations are preferably obtained by using a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant. This mixture forms an oil phase of a stable colloidal emulsion. Such formulations can be obtained more preferably when the fatty acid is present at a concentration equal to (but ideally close to) the critical micelle concentration (CMC) of the fatty acid, as defined below in this invention. Such formulations can be obtained more preferably when the nonionic surfactant is water miscible and / or has an HLB value of 10 or more, for example, in the range of 10 to 20, and / or is present at the concentrations defined below in this invention. Physical measurements are preferably performed at a temperature of 23°C and a pressure of 1 standard atmosphere.

[0061] Typically, surfactant components can be present in the formulation at concentrations of 0.2 to 30,000 μg / mL, for example, 1 to 30,000 μg / mL, for example, 1 to 20,000 μg / mL, for example, 5 to 20,000 μg / mL, for example, 5 to 15,000 μg / mL, for example, 5 to 10,000 μg / mL, for example, 5 to 5,000 μg / mL (i.e., the total concentration of surfactant components). Preferably, surfactant components are present in the formulation at concentrations of 1 to 3,000 μg / mL, for example, 1 to 2,000 μg / mL, for example, 5 to 2,000 μg / mL, for example, 5 to 1,500 μg / mL, for example, 5 to 1,000 μg / mL, for example, 5 to 500 μg / mL. In one embodiment, the surfactant component is present in the formulation at a concentration of 50-200 μg / mL, for example, 75-150 μg / mL, for example, 90-120 μg / mL, or about 100 μg / mL. In an alternative embodiment, the surfactant component is present in the formulation at a concentration of 500-2000 μg / mL, for example, 750-1500 μg / mL, for example, 900-1200 μg / mL, or about 1000 μg / mL.

[0062] Preferably, fatty acids may be present in the formulation at concentrations of 0.2 to 30,000 μg / mL, for example, 1 to 30,000 μg / mL, for example, 1 to 20,000 μg / mL, for example, 5 to 10,000 μg / mL, and nonionic surfactants may be present in the formulation at concentrations of 0.2 to 20,000 μg / mL, for example, 1 to 20,000 μg / mL, for example, 1 to 15,000 μg / mL, for example, 5 to 5,000 μg / mL. More preferably, fatty acids may be present in the formulation at concentrations of 10-100 ug / mL, for example, 20-80 μg / mL, for example, 25-75 μg / mL, for example, 40-60 μg / mL, or about 50 μg / mL, and nonionic surfactants may be present in the formulation at concentrations of 10-100 ug / mL, for example, 20-80 μg / mL, for example, 25-75 μg / mL, for example, 30-60 μg / mL, for example, 40-50 μg / mL. Alternatively, in another preferred embodiment, fatty acids may be present in the formulation at concentrations of 100-1000 ug / mL, for example, 200-800 μg / mL, for example, 250-750 μg / mL, for example, 400-600 μg / mL, or about 500 μg / mL, and nonionic surfactants may be present in the formulation at concentrations of 100-1000 ug / mL, for example, 200-800 μg / mL, for example, 250-750 μg / mL, for example, 300-600 μg / mL, for example, 400-500 μg / mL.

[0063] Typically, surfactant components can be present in a formulation at concentrations of 0.00002% (w / w) to 3% (w / w), for example, 0.0001% (w / w) to 3% (w / w), for example, 0.0001% (w / w) to 2% (w / w), for example, 0.0005% (w / w) to 2% (w / w), for example, 0.0005% (w / w) to 1.5% (w / w), for example, 0.0005% (w / w) to 1% (w / w), for example, 0.0005% (w / w) to 0.5% (w / w) (where the weight percentage is relative to the total weight of the formulation). Preferably, the surfactant component is present in the formulation at a concentration of 0.0001% (w / w) to 0.3% (w / w), for example, 0.0001% (w / w) to 0.2% (w / w), for example, 0.0005% (w / w) to 0.2% (w / w), for example, 0.0005% (w / w) to 0.15% (w / w), for example, 0.0005% (w / w) to 0.1% (w / w), for example, 0.0005% (w / w) to 0.05% (w / w) (where the weight % is relative to the total weight of the formulation). In one embodiment, the surfactant component is present at a concentration of 0.005% (w / w) to 0.02% (w / w), for example, 0.0075% (w / w) to 0.015% (w / w), for example, 0.009% (w / w) to 0.012% (w / w), or about 0.01% (w / w) (where weight % is relative to the total weight of the formulation). In another embodiment, the surfactant component is present at a concentration of 0.05% (w / w) to 0.2% (w / w), for example, 0.075% (w / w) to 0.15% (w / w), for example, 0.09% (w / w) to 0.12% (w / w), or about 0.1% (w / w) (where weight % is relative to the total weight of the formulation).

[0064] Preferably, fatty acids may be present in the formulation at a concentration of 0.00002% (w / w) to 3% (w / w), for example, 0.0001% (w / w) to 3% (w / w), for example, 0.0001% (w / w) to 2% (w / w), for example, 0.0005% (w / w) to 1% (w / w), and nonionic surfactants may be present in the formulation at a concentration of 0.00002% (w / w) to 2% (w / w), for example, 0.0001% (w / w) to 2% (w / w), for example, 0.0001% (w / w) to 1.5% (w / w), for example, 0.0005% (w / w) to 0.5% (w / w) (where wt%) is relative to the total weight of the formulation). More preferably, fatty acids may be present in the formulation at concentrations of 0.001% (w / w) to 0.01% (w / w), for example, 0.002% (w / w) to 0.008% (w / w), for example, 0.0025% (w / w) to 0.0075% (w / w), for example, 0.004% (w / w) to 0.006% (w / w), or about 0.005% (w / w), and nonionic surfactants may be present at 0.001% (w / w). It may be present in the formulation at concentrations of approximately 0.01% (w / w), for example, 0.002% (w / w) to 0.008% (w / w), for example, 0.0025% (w / w) to 0.0075% (w / w), for example, 0.003% (w / w) to 0.006% (w / w), for example, 0.004% (w / w) to 0.005% (w / w) (where wt%) is relative to the total weight of the formulation. Alternatively, in another preferred embodiment, the fatty acid may be present in the formulation at a concentration of 0.01% (w / w) to 0.1% (w / w), for example, 0.02% (w / w) to 0.08% (w / w), for example, 0.025% (w / w) to 0.075% (w / w), for example, 0.04% (w / w) to 0.06% (w / w), or about 0.05% (w / w), and the nonionic surfactant may be 0. It may be present in the formulation at concentrations of 0.01% (w / w) to 0.1% (w / w), for example, 0.02% (w / w) to 0.08% (w / w), for example, 0.025% (w / w) to 0.075% (w / w), for example, 0.03% (w / w) to 0.06% (w / w), for example, 0.04% (w / w) to 0.05% (w / w) (where the weight % is relative to the total weight of the formulation).

[0065] Preferably, for example, the ratio of the amount of fatty acid or a pharmaceutically acceptable salt thereof, each measured in μg / mL, to the amount of nonionic surfactant is about 5:1 to about 1:5, for example, about 5:1 to about 1:2, for example, about 4:1 to about 1:2, for example, about 2:1 to about 1:2. More preferably, for example, the ratio of the amount of fatty acid or a pharmaceutically acceptable salt thereof, each measured in μg / mL, to the amount of nonionic surfactant is about 3:2 to about 2:3, for example, about 6:5 to about 1:1, for example, about 10:9, or about 11:10.

[0066] As stated above, we do not wish to be constrained by theory, but preferably, when the surfactant component is dispersed in an aqueous formulation, it forms the oil phase of an oil-in-water emulsion. For example, preferably, the surfactant component consists of a fatty acid (e.g., oleic acid) which forms the core (or internal phase) of the particle, and a nonionic surfactant (e.g., polysorbate 80) which forms the oil phase of the emulsion that stabilizes the interface between the oil phase and the aqueous phase. Preferably, the polynucleotide molecule is located inside the stable colloidal particle (i.e., is a component thereof), for example, around the core of the particle, for example, around the core and at the interface between the core of the particle and the nonionic surfactant (which stabilizes the interface between the oil phase and the aqueous phase).

[0067] Figure 51 shows schematic representations of different types of particles formed by formulations containing a surfactant component, which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant. As explained in the paragraph above, and again without wishing to be bound by theory, the inventors believe that the formulations of the present invention suitably form stable colloidal emulsions (e.g., stable colloids or lipid nanoemulsions), as shown in the leftmost of the three diagrams in Figure 51. For images of the particles according to the present invention as observed under a transmission electron microscope, please refer to Figures 45(A) to (F), which will be discussed further below.

[0068] Emulsion particles (i.e., stable colloidal particles containing polynucleotides) can spontaneously self-assemble when present in an aqueous formulation. Preferably, emulsions having particles of a suitable size can be formed by mixing the components. Mixing can be carried out with higher or lower shear depending on the target particle size. For example, emulsions having particle sizes in the range of 100 to 300 nM can be produced by using mixing speeds of 100 to 7000 rpm, or preferably 500 to 3000 rpm (see Biophysical Examples 1 to 3).

[0069] (Polynucleotide molecule) The aqueous liquid pharmaceutical formulation of the present invention contains a polynucleotide molecule. Preferably, the aqueous liquid pharmaceutical formulation contains a single polynucleotide molecule. However, it will be understood that the aqueous liquid formulation of the present invention may contain multiple different polynucleotide molecules, for example, two, three, four, five, six, seven, eight, nine, or ten.

[0070] As used herein, the term "polynucleotide molecule" refers to a molecule containing two or more nucleotides.

[0071] Therefore, the polynucleotide molecule may preferably contain 2 to about 100 nucleotides, for example, 2 to about 90 nucleotides, for example, 2 to about 80 nucleotides, for example, 2 to about 70 nucleotides, for example, 2 to about 60 nucleotides, or for example, 2 to about 50 nucleotides. In one embodiment, the polynucleotide molecule preferably contains about 5 to about 50 nucleotides, for example, about 5 to about 40 nucleotides, for example, 5 to about 30 nucleotides or about 20 to about 40 nucleotides, for example, 10 to about 30 nucleotides or about 20 to about 35 nucleotides, for example, 10 to about 20 nucleotides or about 20 to about 30 nucleotides.

[0072] Alternatively, the polynucleotide molecule may preferably contain more than about 100 nucleotides. Therefore, in one embodiment, the polynucleotide molecule preferably contains more than about 100 nucleotides, for example more than about 200 nucleotides, for example more than about 400 nucleotides, for example more than about 500 nucleotides, for example more than about 750 nucleotides, for example more than about 1000 nucleotides, for example more than about 1250 nucleotides, for example more than about 1500 nucleotides, for example more than about 2000 nucleotides, for example more than about 2500 nucleotides, for example more than about 5000 nucleotides. In one embodiment, the polynucleotide molecule preferably contains about 100 to about 20,000 molecules, for example, about 100 to about 10,000 nucleotides, for example, about 200 to about 8,000 nucleotides, or about 500 to about 10,000 nucleotides, for example, about 2,500 to about 1,000 nucleotides, or about 500 to about 7,500 nucleotides, for example, about 1,000 to about 5,000 nucleotides, for example, about 2,000 to about 5,000 nucleotides, or for example, about 2,000 to about 4,000 nucleotides.

[0073] As used herein, the term “nucleotide” refers to nitrogenous bases, in particular major or standard nitrogenous bases, e.g., adenine, cytosine, guanine, thymine, or uracil; sugar molecules, in particular pentose sugars, e.g., ribose or deoxyribose; and monomeric organic molecules containing a phosphate group or its analogues, e.g., a triphot group. Suitable alternative nitrogenous bases include modified purine nitrogenous bases, e.g., 7-methylguanine, hypoxanthine, and xanthine; modified pyrimidine nitrogenous bases, e.g., 5,6-dihydrouracil, 5-methylcytosine, and 5'-hydroxymethylcytosine; and artificial or synthetic nitrogenous bases. Suitable nucleotides therefore include ribonucleotides and deoxyribonucleotides. Preferably, polynucleotide molecules include ribonucleic acid (RNA) molecules and deoxyribonucleic acid (DNA) molecules.

[0074] As used herein, the term “nucleotide” may further refer to peptide nucleotides, threose nucleotides, glycol nucleotides, serinol nucleotides, or locked nucleotides. Preferably, polynucleotide molecules include peptide nucleic acid (PNA) molecules, threose nucleic acid (TNA) molecules, glycol nucleic acid (GNA) molecules, serinol nucleic acid (SNA) molecules, and locked nucleic acid (LNA) molecules.

[0075] Preferably, the polynucleotide molecule further includes hybrid polynucleotide molecules containing one or more different types of nucleotides from the above-mentioned nucleotides. Particularly preferred hybrid polynucleotide molecules are DNA:RNA hybrid polynucleotide molecules, i.e., polynucleotide molecules containing ribonucleotides and deoxyribonucleotides.

[0076] In particular, polynucleotide molecules are RNA molecules or DNA molecules.

[0077] Polynucleotide molecules may be of any origin, such as viruses, bacteria, archaea, fungi, ribosomes, eukaryotes, or prokaryotes, and may originate from any organism. Polynucleotide molecules may arise from any biological sample and from any organ, tissue, cell, or intracellular compartment. Polynucleotide molecules may be pre-treated before use, for example, by isolation, purification, and / or modification. Polynucleotide molecules, or any number of individual nucleotides within a polynucleotide molecule, may be artificial or synthesized.

[0078] A polynucleotide molecule may consist of a single strand, i.e., be monoheld, for example, RNA, or alternatively, a polynucleotide molecule may consist of two strands, i.e., be double-stranded, for example, dsRNA. In some embodiments, a polynucleotide molecule may consist of three strands, i.e., be triply strung, for example, triply strung DNA. Preferably, a monoheld polynucleotide molecule consists of a sense strand, or a monoheld polynucleotide molecule consists of an antisense strand.

[0079] In one embodiment, a polynucleotide molecule includes a double-stranded region. For example, a double-stranded polynucleotide molecule may form a double helix. Alternatively, two single-stranded polynucleotide molecules may hybridize, i.e., non-covalently, to form a double-stranded polynucleotide molecule. Hybridization may occur between two complementary or partially complementary sequences and between polynucleotide molecules of the same DNA:DNA type or different DNA:RNA type. Alternatively, a single-stranded polynucleotide molecule may hybridize with a second region of the single-stranded polynucleotide molecule to include an intramolecular double-stranded region, for example, a first region that forms a double helix.

[0080] In one embodiment, the polynucleotide molecule includes an intramolecular structure. For example, the polynucleotide molecule may include a helix, a bulge (separation of a double helix region on one strand), an internal loop (separation of a double helix region on both strands), a stem loop or hairpin, a tetraloop (a four-base pair hairpin), a pseudoknot, or a junction.

[0081] In one embodiment, the polynucleotide molecule comprises at least one, for example, one or two 5' or 3' monophosphates, and / or at least one, for example, one or two 5' or 3' diphosphates, and / or at least one, for example, one or two 5' or 3' triphosphates, and / or at least one, for example, one or two 5' or 3' hydroxyl (OH) groups. In particular, the 5' or 3' monophosphate and / or 5' or 3' diphosphate and / or 5' or 3' triphosphate and / or 5' or 3' hydroxyl (OH) groups are located at the 5' and / or 3' ends (one or more) of the single-stranded or double-stranded polynucleotide molecule.

[0082] For example, in one embodiment, the polynucleotide molecule is a polyinosinic acid-polycytidylic acid (poly-I:C), which is typically a synthetic dsRNA with a length of 100 to 10,000, e.g., 200 to 8,000, e.g., 300 to 6,000 base pairs (see, for example, dsRNA 2 of US9682096B2, incorporated herein by reference in the following examples).

[0083] In one embodiment, the polynucleotide molecule is an RNA molecule. Preferably, the polynucleotide molecule that is an RNA molecule is an mRNA molecule, a miRNA molecule, an shRNA molecule, or an siRNA molecule.

[0084] In one embodiment, the polynucleotide molecule is an mRNA molecule. In particular, the polynucleotide molecule is a single-stranded RNA molecule capable of encoding one or more proteins. Preferably, the mRNA molecule can be translated.

[0085] In one embodiment, the mRNA molecule contains approximately 100 to approximately 10,000 nucleotides, for example, approximately 200 to approximately 8,000 nucleotides, for example, approximately 500 to approximately 7,500 nucleotides, for example, approximately 1,000 to approximately 5,000 nucleotides.

[0086] In one embodiment, the mRNA molecule comprises one or more coding regions that can be optionally stabilized by internal base pairs. The coding regions may further comprise regulatory sequences, exon splicing enhancers, or exon splicing silencers. Preferably, the mRNA molecule comprises a 5' untranslated region (5'UTR) and / or a 3' untranslated region (3'UTR). Preferably, the mRNA molecule comprises a 3' tail of adenine nucleotides known as a polyA tail. Preferably, the polyA tail comprises 50 or more, for example, 100 or more adenine nucleotides. Preferably, the polynucleotide molecule, for example, the mRNA molecule, comprises a 5' cap containing a terminal 7-methylguanosine residue linked to the first 5' nucleotide via a 5'-5' triphosphate bond.

[0087] In one embodiment, the mRNA molecule is a cyclic mRNA molecule, for example, due to protein-mediated intramolecular interactions between the 5' cap and the polyA tail of the mRNA molecule.

[0088] In one embodiment, the polynucleotide molecule is a miRNA molecule. In particular, the polynucleotide molecule is a single-stranded RNA molecule that does not encode a protein and functions in RNA silencing and post-transcriptional regulation of gene expression.

[0089] In one embodiment, the miRNA molecule contains approximately 20 to 25 nucleotides. In particular, the miRNA molecule contains 20, 21, 22, 23, 24, or 25 nucleotides.

[0090] In one embodiment, the polynucleotide molecule is an shRNA molecule. In particular, the polynucleotide molecule is a single-stranded RNA molecule in which the first region hybridizes with the second region of a single-stranded RNA molecule to form an intramolecular double-stranded region, for example, a double helix. This hybridization creates a hairpin structure containing a hairpin loop within the RNA molecule.

[0091] In one embodiment, the shRNA contains about 10 to about 70 nucleotides, for example, about 20 to about 70 nucleotides, for example, about 35 to about 70 nucleotides, or about 25 to about 35 nucleotides. In particular, the shRNA molecule contains 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides.

[0092] In one embodiment, the double-stranded region or double helix contains 30 or fewer base pairs, for example, 25 or fewer base pairs, for example, 20 or fewer base pairs, for example, 18 or fewer base pairs, for example, 16 or fewer base pairs, for example, 14 or fewer base pairs, for example, 12 or fewer base pairs, for example, 10 or fewer base pairs, for example, 8 or fewer base pairs, for example, 6 or fewer base pairs, for example, 4 or fewer base pairs. In one embodiment, the double-stranded region or double helix contains about 2 to about 30 base pairs, for example, about 2 to about 25 base pairs, for example, about 4 to about 20 base pairs, for example, about 4 to about 18 base pairs, for example, about 6 to about 18 base pairs, for example, about 8 to about 18 base pairs, for example, about 10 to about 18 base pairs.

[0093] In one embodiment, the double-stranded region contains one or more mispairing bases according to Watson-Crick base pairing. For example, the double-stranded region may contain 1 to 10 mispairing bases, e.g., 1 to 8 mispairing bases, e.g., 1 to 6 mispairing bases, and in particular, 1, 2, 3, 4, 5, or 6 mispairing bases.

[0094] Preferably, the hybridized intramolecular double-stranded regions, for example, the first and second regions that form a double helix, are each 20 nucleotides or less in length, for example, 19 nucleotides or less, or for example, 18 nucleotides or less. Preferably, the first and second regions are each about 5 to about 20 nucleotides in length, for example, about 5 to about 18 nucleotides, for example, about 8 to about 18 nucleotides, or for example, about 10 to about 18 nucleotides.

[0095] Preferably, the hybridized intramolecular double-stranded regions, for example, the first and second regions forming a double helix, are substantially complementary to each other, for example, at least about 80% complementary, particularly at least about 90% complementary, or most preferably 100% complementary.

[0096] The hairpin structure may include a hairpin loop. Preferably, the length of the hairpin loop is 0 or 1 or more nucleotides. For example, the hairpin loop may have a length of 2 or more, 4 or more, 5 or more, 8 or more, 10 or more, 15 or more, 20 or more, 40 or more, 50 or more, or 100 or more nucleotides. In particular, the hairpin loop may have a length of about 1 to about 20, for example, about 1 to about 15, for example, about 2 to about 10, for example, about 4 to about 10 nucleotides.

[0097] In one preferred embodiment, the shRNA molecule includes a blunt end. A blunt end refers to a polynucleotide molecule in which at least one of the double helixes does not protrude, for example, a 3' dinucleotide does not protrude, such that the 5' and 3' strands terminate together. Alternatively, the shRNA molecule may include a 3' protrusion or a 5' protrusion.

[0098] In one embodiment, the shRNA molecule comprises at least one 5' triphot or at least one 5' diphosphate. In particular, the 5' triphot or diphosphate is located at the 5' end of the shRNA molecule. Preferably, the shRNA molecule comprises one 5' triphot or one 5' diphosphate, in particular, wherein the 5' triphot or diphosphate is located at the 5' end.

[0099] In one embodiment, the shRNA molecule, which is a single-stranded RNA molecule forming a hairpin structure including an intramolecular double-stranded region and a hairpin loop, comprises a blunt end and a 5' triphosphate or 5' diphosphate located at the 5' end, where the double-stranded region is about 10 to about 18 nucleotides in length. The shRNA molecule of this embodiment may contain one or more glycosylated nucleotides, each containing a 2'OH modification, and / or one or more skeletal modified nucleotides and / or one or more base-modified nucleotides. In one embodiment, such an shRNA molecule can induce an interferon response in vertebrate cells.

[0100] In one embodiment, an shRNA molecule, which is a single-stranded RNA molecule forming a hairpin structure including an intramolecular double-stranded region and a hairpin loop, includes, consists of, or is essentially composed of, a sequence disclosed in WO2019 / 246450A1, which is incorporated herein by reference. Specifically, an shRNA molecule may include, consist of, or be essentially composed of SEQ ID NO: 1. Alternatively, an shRNA molecule may include, consist of, or be essentially composed of a variant of SEQ ID NO: 1, wherein, as used herein, “variant” means a sequence having, for example, at least about 75% identity with a reference sequence of the same length, for example, at least about 80% identity, for example, at least about 85% identity, and in particular at least about 90% identity, for example, at least about 95%, 98%, or 99% identity with the reference sequence of the same length.

[0101] Alternatively, in one embodiment, the shRNA molecule, which is a single-stranded RNA molecule forming a hairpin structure including an intramolecular double-stranded region and a hairpin loop, contains, consists of, or is essentially composed of SEQ ID NO: 2. Alternatively, the shRNA molecule contains, consists of, or is essentially composed of a variant of SEQ ID NO: 2.

[0102] In one embodiment, an shRNA molecule, which is a single-stranded RNA molecule forming a hairpin structure including an intramolecular double-stranded region and a hairpin loop, includes, consists of, or is essentially composed of, a sequence disclosed in US2023 / 0159923A1, which is incorporated herein by reference. In particular, the shRNA molecule may include, consist of, or be essentially composed of, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. Alternatively, the shRNA molecule may include, consist of, or be essentially composed of, a variant of SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0103] The molecule shRNA 1 used in the following examples is the shRNA molecule of Sequence ID No. 1, which includes a blunt end and a 5' diphosphate at the 5' end. Therefore, in one embodiment, the shRNA molecule is an shRNA molecule having the sequence of Sequence ID No. 1 and including a blunt end and a 5' diphosphate at the 5' end. It will be understood that the shRNA molecule of Sequence ID No. 1 may also include a 5' triphosphate at the 5' end.

[0104] The molecule shRNA 2 is an shRNA molecule of Sequence ID No. 2 that includes a blunt end and a 5' diphosphate at the 5' end. Therefore, in one embodiment, the shRNA molecule is an shRNA molecule that has the sequence of Sequence ID No. 2 and includes a blunt end and a 5' diphosphate at the 5' end. It will be understood that the shRNA molecule of Sequence ID No. 2 may also include a 5' triphosphate at the 5' end.

[0105] In one embodiment, the polynucleotide molecule is an siRNA molecule. In particular, the polynucleotide molecule is a double-stranded RNA molecule that does not encode a protein and functions in RNA silencing and post-transcriptional regulation of gene expression.

[0106] In one embodiment, the siRNA molecule contains about 20 to about 60 nucleotides, for example, about 30 to about 60 nucleotides, for example, about 40 to about 50 nucleotides. In particular, the siRNA molecule contains 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides.

[0107] In one embodiment, the siRNA molecule includes a double-stranded region or double helix containing 30 or fewer base pairs, for example, 25 or fewer base pairs, for example, 20 or fewer base pairs. In one embodiment, the double-stranded region or double helix contains about 10 to about 30 base pairs, for example, about 15 to about 30 base pairs, for example, about 20 to about 30 base pairs, for example, about 20 to about 25 base pairs, or about 25 to about 30 base pairs.

[0108] In one embodiment, the double-stranded region contains one or more mispairing bases according to Watson-Crick base pairing. For example, the double-stranded region may contain 1 to 10 mispairing bases, e.g., 1 to 8 mispairing bases, e.g., 1 to 6 mispairing bases, and in particular, 1, 2, 3, 4, 5, or 6 mispairing bases.

[0109] Preferably, the siRNA molecule contains one or more, for example, two 3' protrusions. Alternatively, the siRNA molecule may contain one or more, for example, two 5' protrusions. Alternatively, the siRNA molecule may contain one or more, for example, one 3' protrusion and / or one or more, for example, one 5' protrusion. Alternatively, the siRNA molecule may contain one or more blunt ends.

[0110] In one embodiment, the siRNA molecule comprises at least one, for example, two 5' monophosphates, or at least one, for example, two 5' diphosphates, or at least one, for example, two 5' triphosphates. In particular, the 5' monophosphate and / or the 5' diphosphate and / or the 5' triphosphate are located at the 5' end of the siRNA molecule.

[0111] Preferably, the siRNA molecule contains at least one, for example, two 3'OH (hydroxyl) groups. In particular, the 3'OH group is located at the 3' end of the siRNA molecule.

[0112] In one embodiment, the siRNA molecule, which is a double-stranded RNA molecule, contains the sequence of SEQ ID NO: 11 or a variant thereof. In particular, one strand of the double-stranded siRNA molecule contains, consists of, or is essentially composed of the sequence of SEQ ID NO: 11 or a variant thereof (see Ren et al., 2019, incorporated herein by reference).

[0113] In one embodiment, the polynucleotide molecule is a guide RNA (gRNA) molecule which includes a CRISPR RNA (crRNA), a nucleotide sequence typically 15-20 nucleotides long that is complementary to the host target DNA, and a tracr RNA (trRNA) that can bind to a Cas nuclease. Preferably, the gRNA molecule is a component of the CRISPR-Cas9 gene editing technology.

[0114] In one embodiment, the polynucleotide molecule is a DNA molecule. Preferably, the DNA molecule is a genomic DNA (gDNA) molecule, for example, a chromosomal DNA molecule or a mitochondrial DNA molecule, a complementary DNA (cDNA) molecule, or an extrachromosomal DNA molecule, for example, a plasmid DNA molecule. In one embodiment, the DNA molecule is a gDNA molecule. In one embodiment, the DNA molecule is a cDNA molecule. In one embodiment, the DNA molecule is an extrachromosomal DNA molecule, in particular a plasmid DNA molecule.

[0115] In one embodiment, the DNA molecule is a coding DNA molecule. For example, the DNA molecule contains one or more coding regions that can be optionally stabilized by internal base pairs. Thus, the DNA molecule can code for one or more proteins. Preferably, the DNA molecule that is a coding DNA molecule can be transcribed. Preferably, the DNA molecule that is a coding DNA molecule further contains one or more promoter sequences, in particular, wherein the one or more promoter sequences are adjacent to the corresponding coding region at the 5' end. Preferably, the DNA molecule that is a coding DNA molecule further contains a termination sequence, in particular, wherein the termination sequence is adjacent to one or more coding regions at the 3' end. Preferably, the coding DNA molecule may further contain any number of non-coding DNA elements, such as those listed below.

[0116] In one embodiment, the DNA molecule is a non-coding DNA molecule, that is, the DNA molecule does not contain a coding region and, as a result, does not code for a protein. Preferably, the non-coding DNA molecule can be transcribed to produce, for example, tRNA, miRNA, siRNA, or ribosomal RNA. Alternatively, the non-coding DNA molecule may contain regulatory sequences that control gene expression, scaffold-binding regions, centromeres, or telomeres. Alternatively, the non-coding DNA molecule may contain non-functional elements, such as introns, pseudogenes, intergenetic DNA, or transposons. Preferably, the non-coding DNA molecule may contain combinations of the above elements.

[0117] In one embodiment, the DNA molecule contains approximately 100 to approximately 20,000 nucleotides, for example, approximately 100 to approximately 15,000 nucleotides, for example, approximately 500 to approximately 15,000 nucleotides, for example, approximately 500 to approximately 10,000 nucleotides, for example, approximately 2,500 to approximately 10,000 nucleotides, for example, approximately 2,500 to approximately 8,000 nucleotides.

[0118] In one embodiment, the polynucleotide molecule has the structure of formula (I): [ka] (In the formula, 5'-P z -(N) b N-3' represents the first nucleic acid sequence; 5'-N(N) b' -3' represents the second nucleic acid sequence; In each case, P is independently a phosphate or an analogue thereof; z is either 2 or 3; In each case, N is any nucleotide or modified nucleotide or its analogue or derivative; b and b' are independently 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; 5'-(E) y (E)-L-(E)(E) y' -3' represents a connector element, where, E is, independently, any nucleotide, modified nucleotide, or debase at each instance; y and y' are independently between 0 and 9, where y + y' is equal to between 0 and 8; L is structure [ka] (Here, X and X' are independently O or S; Y and Y' are independently OR'', SR'', or NRR'; V and V' are independently O, S, or NRR'; q is between 1 and 20; k is between 1 and 20; t is between 1 and 20; M is selected from aliphatic, substituted aliphatic, aryl, substituted aryl, heteroalkyl, heterocyclyl, or substituted heterocyclyl; W is any reactive group; and (d is either 0 or 1) (It is a non-nucleotide segment that has [a certain characteristic].)

[0119] Preferably, the polynucleotide molecule has the structure of formula (II): [ka] (In the formula, 5'-P z -Nu-3' represents the first nucleic acid sequence; 5'-Nu'-3' represents the second nucleic acid sequence; In each case, P is independently a phosphate or an analogue thereof. z is 0, 1, 2, or 3; 5'-(E) y (E)-L-(E)(E) y' -3' represents a connector element, where, E is, independently, any nucleotide, modified nucleotide, or debase at each instance; y and y' are independently between 0 and 9, where y + y' is equal to between 0 and 8; L is structure [ka] (Here, X and X' are independently O or S; Y and Y' are independently OR'', SR'', or NRR'; V and V' are independently O, S, or NRR'; q is between 1 and 20; k is between 1 and 20; t is between 1 and 20; M is selected from aliphatic, substituted aliphatic, aryl, substituted aryl, heteroalkyl, heterocyclyl, or substituted heterocyclyl; W is any reactive or conjugation group; and (d is either 0 or 1) (It is a non-nucleotide segment that has [a certain characteristic].)

[0120] In one embodiment, z is 2. Furthermore, preferably, when z is 2, at least one P is a phosphate analog. Particularly preferably, when z is 2, both P are phosphate analogs. Alternatively, in one embodiment, z is 3. Furthermore, preferably, when z is 3, at least one P is a phosphate analog. Particularly preferably, when z is 3, at least two P are phosphate analogs. For example, preferably, when z is 3, all P are phosphate analogs. In one embodiment, the phosphate analog has the structure: [ka] (Here, Y is O or S, or CH-R (where R = alkyl, aralkyl, heteroaryl, or cycloalkylamine (e.g., piperazine)), X is either O or S, and Z is OH, SH, NHR' (where R' is H, alkyl, aralkyl, or heteroaryl). Includes.

[0121] In one embodiment, b and b' are independently 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; preferably 11, 12, 13, 14, 15, 16, 17, or 18; preferably 13, 14, 15, 16, 17, or 18. In one embodiment, b is 9 and b' is less than, equal to, or greater than 9. In one embodiment, b is 10 and b' is less than, equal to, or greater than 10. In one embodiment, b is 11 and b' is less than, equal to, or greater than 11. In one embodiment, b is 12 and b' is less than, equal to, or greater than 12. In one embodiment, b is 13 and b' is less than, equal to, or greater than 13. In one embodiment, b is 14 and b' is less than, equal to, or greater than 14. In one embodiment, b is 15 and b' is less than, equal to, or greater than 15. In one embodiment, b is 16 and b' is less than, equal to, or greater than 16. In one embodiment, b is 17 and b' is less than, equal to, or greater than 17. In one embodiment, b is 18 and b' is less than, equal to, or greater than 18. In one embodiment, b is equal to b'. In an alternative embodiment, b is not equal to b'. In one embodiment, when b is less than b', the polynucleotide molecule has a 3'-projection. In an alternative embodiment, when b is greater than b', the nucleic acid molecule has a 5'-projection.

[0122] In one embodiment, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. For example, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, and in particular, q is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, in one embodiment, q is 1, 2, 3, 4, or 5, and in particular, q is 1, or q is 2, or q is 3, or q is 4, or q is 5.

[0123] In one embodiment, k and t are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, in particular k and t are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. For example, in one embodiment, k and t are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, in particular k and t are independently 1, 2, 3, 4, or 5. In one embodiment, k is 1, or k is 2, or k is 3, or k is 4, or k is 5. In one embodiment, t is 1, or t is 2, or t is 3, or t is 4, or t is 5. In one embodiment, k and t are the same, and therefore L is symmetric. In an alternative embodiment, k and t are different, and therefore L is asymmetric.

[0124] In one embodiment, R, R', and R'' are independently selected from the group consisting of alkyl, aminoalkyl, carboxamide, polyethylene glycol (PEG), aralkyl, heteroaralkyl, heteroalkyl, substituted or unsubstituted cycloalkyl. In this embodiment, the R and R'' groups may contain functional groups such as amino, hydroxy, azide, or thiol, which can be optionally used in the linkage to a targeting molecule (Tm), such as a vitamin, peptide, antibody, or protein.

[0125] In one embodiment, the R and R'' groups can be peptide groups. Peptide groups include various enzymatically cleavable or non-cleavable peptides. The individual amino acid groups of the peptide can be natural or synthetic amino acids.

[0126] In one embodiment, R and R'' are -CH2-O-CO-R 1 (where R 1 = methyl, isopropyl, t-butyl, -(CH2)n-R 2 (where R 2 is selected from aryl, aralkyl, heteroaryl, heteroaralkyl, alkyl, aminoalkyl, carboxamide, polyethylene glycol (PEG), heteroalkyl, substituted or unsubstituted cycloalkyl)) can be.

[0127] In one embodiment, the reactive group W may be further connected to alkyl, aminoalkyl, carboxamide, polyethylene glycol (PEG), aralkyl, heteroaralkyl, heteroalkyl, substituted or unsubstituted cycloalkyl. In certain embodiments, the R and R'' groups can contain functional groups such as amino, hydroxy, azide, or thiol that can be optionally used for binding to a targeting molecule (Tm) such as a vitamin, peptide, antibody, or protein.

[0128] In one embodiment, M is selected from aliphatic, substituted aliphatic, aryl, substituted aryl, heteroalkyl, heterocyclyl, or substituted heterocyclyl. The terms “aliphatic group” or “aliphatic” refer to a non-aromatic moiety that may contain substituted (e.g., single bonds) or one or more unsaturated units, e.g., double and / or triple bonds. Aliphatic groups may be linear, branched, or cyclic, and may contain carbon, hydrogen, or optionally one or more heteroatoms, and may be substituted or unsubstituted. In addition to aliphatic hydrocarbon groups, aliphatic groups include, for example, polyalkoxyalkyls, e.g., polyalkylene glycols, polyamines, and polyimines. Such aliphatic groups may be further substituted. It is understood that aliphatic groups may include alkyls, substituted alkyls, alkenyls, substituted alkenyls, alkynyls, substituted alkynyls, and substituted or unsubstituted cycloalkyl groups as described herein.

[0129] The term "alkyl" is intended to include both branched and linear substituted or unsubstituted saturated aliphatic hydrocarbon radicals / groups having a specified number of carbon atoms. Preferred alkyl groups have about 1 to about 24 carbon atoms ("C1- 24 This includes ''). Other preferred alkyl groups include about 1 to about 8 carbon atoms ("C1-C8"), for example, about 1 to about 6 carbon atoms ("C1-C6"), or for example, about 1 to about 3 carbon atoms ("C1-C3"). Examples of C1-C6 alkyl radicals include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl, neopentyl, and n-hexyl radicals.

[0130] The term "alkenyl" refers to a linear or branched radical having at least one carbon-carbon double bond. Such radicals preferably have about 2 to about 24 carbon atoms ("C2-C"). 24 It contains ''). Other preferred alkenyl radicals have 2 to about 10 carbon atoms (''C2-C 10"Lower alkenyl" radicals having the 'C2', for example, ethenyl, allyl, propenyl, butenyl, and 4-methylbutenyl. Preferred lower alkenyl radicals contain 2 to about 6 carbon atoms ("C2-C6"). The terms "alkenyl" and "lower alkenyl" encompass radicals having "cis" and "trans" orientations, or alternatively, "E" and "Z" orientations.

[0131] The term "alkynyl" refers to a linear or branched radical having at least one carbon-carbon triple bond. Such radicals preferably have about 2 to about 24 carbon atoms ("C2-C"). 24 It contains ''). Other preferred alkynyl radicals are “lower alkynyl” radicals having 2 to about 10 carbon atoms, such as propargyl, 1-propynyl, 2-propynyl, 1-butyne, 2-butynyl, and 1-pentynyl. Preferred lower alkynyl radicals contain 2 to about 6 carbon atoms ("C2-C6").

[0132] The term "cycloalkyl" refers to a group of 3 to approximately 12 carbon atoms ("C3- 12 The term "cycloalkyl" refers to a saturated carbocyclic radical having 3 to approximately 12 carbon atoms. Examples of such radicals include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0133] The term "alkoxy" refers to a linear or branched oxy-containing radical having an alkyl moiety of 1 to about 24 carbon atoms, or preferably 1 to about 12 carbon atoms. More preferred alkoxy radicals are "lower alkoxy" radicals having 1 to about 10 carbon atoms, and more preferably 1 to about 8 carbon atoms. Examples of such radicals include methoxy, ethoxy, propoxy, butoxy, and tert-butoxy.

[0134] The term “aryl,” alone or in combination, refers to an aromatic system containing one, two, or three rings (where such rings may be bonded together in a drooping manner or condensed). The term “aryl” encompasses aromatic radicals such as phenyl, naphthyl, tetrahydronaphthyl, indanfuranyl, quinazolinyl, pyridyl, and biphenyl.

[0135] The terms "heterocyclyl," "heterocycle," "heterocyclic," or "heterocyclo" refer to saturated, partially unsaturated, and unsaturated heteroatom-containing cyclic radicals, which may also be called "heterocyclyl," "heterocycloalkenyl," and "heteroaryl," respectively, where the heteroatom can be selected from nitrogen, sulfur, and oxygen. Examples of saturated heterocyclyl radicals include saturated 3-6 membered heteromonocyclic groups containing 1-4 nitrogen atoms (e.g., pyrrolidinyl, imidazolidinyl, piperidino, piperazinyl); saturated 3-6 membered heteromonocyclic groups containing 1-2 oxygen atoms and 1-3 nitrogen atoms (e.g., morpholinyl); and saturated 3-6 membered heteromonocyclic groups containing 1-2 sulfur atoms and 1-3 nitrogen atoms (e.g., thiazolidinyl). Examples of partially unsaturated heterocyclyl radicals include dihydrothiophene, dihydropyran, dihydrofuran, and dihydrothiazole. Heterocyclyl radicals, for example, may contain pentavalent nitrogen in tetrazolium and pyridinium radicals. The term "heterocyclic" also includes radicals in which the heterocyclyl radical is condensed with an aryl or cycloalkyl radical. Examples of such condensed bicyclic radicals include benzofuran and benzothiophene.

[0136] The term "heteroaryl" refers to unsaturated aromatic heterocyclyl radicals. Examples of heteroaryl radicals include unsaturated 3-6 membered heteromonocyclic groups containing 1-4 nitrogen atoms, such as pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, pyrimidyl, pyrazinyl, pyridadinyl, triazolyl (e.g., 4H-1,2,4-triazolyl, 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, etc.), and tetrazolyl (e.g., 1H-tetrazolyl, 2H-tetrazolyl, etc.); Unsaturated condensed heterocyclyl groups containing 1 to 5 nitrogen atoms, e.g., indolyl, isoindolyl, indolidinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl (e.g., tetrazolo[1,5-b]pyridazinyl); unsaturated 3 to 6-membered heteromonocyclic groups containing an oxygen atom, e.g., pyranyl, furyl; unsaturated 3 to 6-membered heteromonocyclic groups containing a sulfur atom, e.g., thienyl; unsaturated 3 to 6-membered heteromonocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, e.g., oxazolyl, isoxazolyl, oxadiazolyl (e.g., 1,2,4-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl); Examples include unsaturated condensed heterocyclyl groups containing 1-2 oxygen atoms and 1-3 nitrogen atoms (e.g., benzoxazolyl, benzoxadiazolyl, etc.); unsaturated 3-6 membered heteromonocyclic groups containing 1-2 sulfur atoms and 1-3 nitrogen atoms, e.g., thiazolyl, thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.); and unsaturated condensed heterocyclyl groups containing 1-2 sulfur atoms and 1-3 nitrogen atoms (e.g., benzothiazolyl, benzothiadiazolyl, etc.).

[0137] The terms "aralkyl" or "arylalkyl" refer to aryl-substituted alkyl radicals such as benzyl, diphenylmethyl, triphenylmethyl, phenylethyl, and diphenylethyl. The term "aryloxy" refers to aryl radicals bonded to other radicals via an oxygen atom.

[0138] The term "alkylamino" refers to an amino group substituted with one or two alkyl radicals. Preferred alkylamino radicals have about 1 to about 20 carbon atoms, or preferably 1 to about 12 carbon atoms. More preferred alkylamino radicals are "lower alkylaminos" having an alkyl radical with 1 to about 10 carbon atoms. Most preferred are alkylamino radicals having a lower alkyl radical with 1 to about 8 carbon atoms. Preferred lower alkylaminos may be monosubstituted N-alkylaminos or disubstituted N,N-alkylaminos, such as N-methylamino, N-ethylamino, N,N-dimethylamino, and N,N-diethylamino.

[0139] As used herein, the terms "halogen" or "halo" refer to an atom selected from fluorine, chlorine, bromine, and iodine.

[0140] The term "substituted" refers to the substitution of one or more hydrogen radicals in a given structure with a radical of a specified substituent, including, but not limited to, halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic substituents. It is understood that substituents may be further substituted.

[0141] In one embodiment, the polynucleotide molecule may include, consist of, or essentially consist of SEQ ID NO: 13 and SEQ ID NO: 14, where the 3' end of SEQ ID NO: 13 is connected to the 5' end of SEQ ID NO: 14 via a non-nucleotide moiety L as defined above. Alternatively, the polynucleotide molecule may include, consist of, or essentially consist of a variant of SEQ ID NO: 13 and / or a variant of SEQ ID NO: 14, where the 3' end of SEQ ID NO: 13 is connected to the 5' end of SEQ ID NO: 14 via a non-nucleotide moiety L, where "variant" as used herein refers to a sequence having, for example, at least about 75% identity with a reference sequence of the same name over its entire length, for example, at least about 80% identity, for example, at least about 85% identity, in particular at least about 90% identity, for example, at least about 95%, 98%, or 99% identity.

[0142] Preferably, L is selected from the group consisting of the following: [Table 1] TIFF2026525217000008.tif208170TIFF2026525217000009.tif74170

[0143] For example, in one embodiment, L is L1. In an alternative embodiment, L is L2.

[0144] Therefore, in one embodiment, the polynucleotide molecule may include, consist of, or essentially consist of SEQ ID NOs: 13 and SEQ ID NOs: 14, where the 3' end of SEQ ID NOs: 13 is connected to the 5' end of SEQ ID NOs: 14 via a non-nucleotide moiety L1. Alternatively, the polynucleotide molecule may include, consist of, or essentially consist of variants of SEQ ID NOs: 13 and / or variants of SEQ ID NOs: 14, where the 3' end of SEQ ID NOs: 13 is connected to the 5' end of SEQ ID NOs: 14 via a non-nucleotide moiety L1. In a further embodiment, the polynucleotide molecule may include, consist of, or essentially consist of SEQ ID NOs: 13 and SEQ ID NOs: 14, where the 3' end of SEQ ID NOs: 13 is connected to the 5' end of SEQ ID NOs: 14 via a non-nucleotide moiety L2. Alternatively, the polynucleotide molecule may include, consist of, or essentially consist of, the variant of SEQ ID NO: 13 and / or the variant of SEQ ID NO: 14, where the 3' end of SEQ ID NO: 13 is connected to the 5' end of SEQ ID NO: 14 via a non-nucleotide moiety L2.

[0145] Preferably, polynucleotide molecules may be present in the formulation at concentrations of 0.01 μg / mL to 100,000 μg / mL, for example, 0.1 to 100,000 μg / mL, for example, 1 to 100,000 μg / mL, for example, 1 to 50,000 μg / mL. In one embodiment, polynucleotide molecules may be present in the formulation at concentrations of 1 to 10,000 μg / mL, for example, 1 to 1,000 μg / mL, for example, 1 to 100 μg / mL, for example, 1 to 50 μg / mL, for example, 1 to 20 μg / mL, for example, about 10 μg / mL. In an alternative embodiment, polynucleotide molecules may be present in the formulation at concentrations of 1 to 10,000 μg / mL, for example, 1 to 1,000 μg / mL, for example, 10 to 1,000 μg / mL, for example, 10 to 500 μg / mL, for example, 10 to 250 μg / mL, for example, 50 to 150 μg / mL, for example, about 100 μg / mL. In an alternative embodiment, polynucleotide molecules may be present in the formulation at concentrations of 1 to 10,000 μg / mL, for example, 10 to 10,000 μg / mL, for example, 100 to 10,000 μg / mL, for example, 500 to 5,000 μg / mL, for example, 500 to 2,500 μg / mL, for example, about 1,000 μg / mL. Alternatively, polynucleotide molecules may be present in the formulation at concentrations of 1 to 100,000 μg / mL, for example, 10 to 100,000 μg / mL, for example, 100 to 100,000 μg / mL, for example, 1,000 to 100,000 μg / mL, for example, 5,000 to 75,000 μg / mL, for example, 5,000 to 50,000 μg / mL, for example, 5,000 to 15,000 μg / mL, 10,000 to 20,000 μg / mL, 15,000 to 25,000 μg / mL, or 25,000 to 50,000 μg / mL. Importantly, as will be understood by those skilled in the art, the concentration of polynucleotide molecules in a formulation varies considerably and depends on the type, sequence, structure, and size of the polynucleotide molecule.

[0146] Preferably, polynucleotide molecules may be present in the formulation at a concentration of 0.000001% (w / w) to 10% (w / w), for example, 0.00001% (w / w) to 10% (w / w), for example, 0.0001% (w / w) to 5% (w / w) (where the weight percentage is relative to the total weight of the formulation). In one embodiment, polynucleotide molecules may be present in the formulation at concentrations of 0.0001% (w / w) to 1% (w / w), for example, 0.0001% (w / w) to 0.1% (w / w), for example, 0.0001% (w / w) to 0.01% (w / w), for example, 0.0001% (w / w) to 0.005% (w / w), for example, 0.0001% (w / w) to 0.002% (w / w), for example, about 0.001% (w / w) (where the weight % is relative to the total weight of the formulation). In an alternative embodiment, polynucleotide molecules may be present in the formulation at concentrations of 0.0001% (w / w) to 1% (w / w), for example, 0.0001% (w / w) to 0.1% (w / w), for example, 0.001% (w / w) to 0.1% (w / w), for example, 0.001% (w / w) to 0.05% (w / w), for example, 0.001% (w / w) to 0.025% (w / w), for example, 0.005% (w / w) to 0.015% (w / w), for example, about 0.01% (w / w) (where weight % is relative to the total weight of the formulation). In an alternative embodiment, polynucleotide molecules may be present in the formulation at concentrations of 0.0001% (w / w) to 1% (w / w), for example, 0.001% (w / w) to 1% (w / w), for example, 0.01% (w / w) to 1% (w / w), for example, 0.05% (w / w) to 0.5% (w / w), for example, 0.05% (w / w) to 0.25% (w / w), for example, about 0.1% (w / w) (where weight % is relative to the total weight of the formulation).Alternatively, polynucleotide molecules may be present in the formulation at concentrations of 0.0001% (w / w) to 10% (w / w), for example, 0.001% (w / w) to 10% (w / w), for example, 0.01% (w / w) to 10% (w / w), for example, 0.1% (w / w) to 10% (w / w), for example, 0.5% (w / w) to 7.5% (w / w), for example, 0.5% (w / w) to 5% (w / w), for example, 0.5% (w / w) to 1.5% (w / w), 1% (w / w) to 2% (w / w), 1.5% (w / w) to 2.5% (w / w), or 2.5% (w / w) to 5% (w / w) (where wt%) is relative to the total weight of the formulation).

[0147] Preferably, for example, the ratio of the amount of surfactant component (i.e., the total concentration of surfactant components) to the amount of polynucleotide molecules, each measured in μg / mL, is about 100:1 to about 1:1000, for example, about 100:1 to about 1:750, for example, about 75:1 to about 1:750, for example, about 50:1 to about 1:750, for example, about 50:1 to about 1:500, for example, about 50:1 to about 1:250, for example, about 50:1 to about 1:200, for example, about 40:1 to about 1:200, for example, about 40:1 to about 1:150, for example, about 25:1 to about 1:150, for example, about 25:1 to about 1:100.

[0148] (Nucleic acid modification) In one embodiment, the polynucleotide molecule contains one or more modified nucleotides. For example, the polynucleotide molecule contains two or more, three or more, four or more, five or more, eight or more, or ten or more modified nucleotides.

[0149] Modification of nucleotides can preferably enhance stability, functionality, and / or specificity, and minimize the immunostimulatory properties of the polynucleotide molecule. Preferably, the polynucleotide molecule contains at least one modified nucleotide that confers reduced immunostimulatory activity and at least one modified nucleotide that confers an increased serum half-life. Preferably, the same modified nucleotide produces both effects. Preferably, the polynucleotide molecule contains one or more modified nucleotides that confer enhanced resistance to nuclear enzymes.

[0150] Modified nucleotides include, but are not limited to, those with sugar modification, skeletal modification, and base modification. Suitable modified nucleotides may include any combination of sugar modification, skeletal modification, and base modification.

[0151] Examples of glycosylated nucleotides include, but are not limited to, nucleotides in which a 2'OH- group (ribonucleotide) or a 2'-H group (deoxyribonucleotide) is replaced with a group selected from the group consisting of H, OR, R, halo, SH, SR, NH2, NHR, NR2, or ON (where R is a C1-C6 alkyl, alkenyl, or alkynyl).

[0152] Further examples of 2'OH- group (ribonucleotide) or 2'-H group (deoxyribonucleotide) modifications include alkoxy or aryloxy modifications, e.g., OR (where R is H, alkyl, e.g., C1-C6 alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); polyethylene glycol (PEG) modifications; "locked" nucleotide modifications (where the 2'OH- group or 2'H- group is connected to the 4' carbon of the same sugar, e.g., via a methylene crosslink); amines, O-amines, and aminoalkoxys, e.g., O(CH2) nExamples of amine modifications include NH2, alkylamino, dialkylamino, heterocyclylamino, arylamino, diarylamino, heteroarylamino, diheteroarylamino, ethylenediamine, or polyamino.

[0153] Further examples of 2'OH- group (ribonucleotide) or 2'-H group (deoxyribonucleotide) modifications include H (in the case of ribonucleotides); halo; amino, e.g., NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH (CH2CH2NH) n Examples include CH2CH2-amine modifications (wherein the amine is NH2, alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino); NHC(O)R modifications (wherein the R is alkyl, e.g., C1-C6 alkyl, cycloalkyl, aryl, aralkyl, heteroaryl, or sugar); cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and optionally substituted alkyls, e.g., C1-C6 alkyl, cycloalkyl, aryl, alkenyl, and alkynyl).

[0154] In one embodiment, the polynucleotide molecule, in particular the RNA molecule, contains one or more sugar-modified nucleotides, each of which includes a 2'OH (or 2'H) modification.

[0155] In one embodiment, the polynucleotide molecule, in particular the RNA molecule, contains one or more sugar-modified nucleotides, each having a 2'OH (or 2'H) modification, selected from the group consisting of 2'-deoxy, 2'-fluoro, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA).

[0156] In one embodiment, the polynucleotide molecule, particularly the RNA molecule, contains one or more sugar-modified nucleotides, each containing a 2'OH modification, selected from the group consisting of 2'-H and 2'-O-methyl. Preferably, the polynucleotide molecule, particularly the RNA molecule, contains one or more nucleotides containing a 2'-O-methyl modification. In one embodiment, all nucleotides in the polynucleotide molecule, particularly the RNA molecule, contain a 2'-O-methyl modification.

[0157] In one embodiment, some or all of the pyrimidine nucleotides of a polynucleotide molecule, particularly an RNA molecule, include 2'OH modifications. In particular, some or all of the pyrimidine nucleotides may include modifications selected from the group consisting of 2'-H and 2'-O-methyl. In one embodiment, some or all of the purine nucleotides of a polynucleotide molecule, particularly an RNA molecule, include 2'OH modifications. In particular, some or all of the purine nucleotides may include modifications selected from the group consisting of 2'-H and 2'-O-methyl.

[0158] In one embodiment, the polynucleotide molecule, particularly the RNA molecule, contains one or more dinucleotides that are readily cleaved by endonucleases, where the 5' nucleotide of the dinucleotide contains a 2'OH (or 2'H) modification. Preferably, the dinucleotides are 5'-UA-3', 5'-UG-3', 5'-CA-3', 5'-UU-3', or 5'-CC-3'.

[0159] Examples of skeletal-modified nucleotides include, but are not limited to, nucleotides that include modifications to the phosphate sugar skeleton of a polynucleotide molecule, and in particular, nucleotides that include modifications to the phosphodiester bond linking the phosphate group of an adjacent nucleotide to the sugar molecule. For example, the phosphodiester bond may be modified to include at least one heteroatom, such as a nitrogen or sulfur heteroatom. In particular, the phosphate ester group of a nucleotide linked to the sugar molecule of an adjacent nucleotide may be replaced with a phosphothioate group.

[0160] In one embodiment, the polynucleotide molecule, in particular the RNA molecule, includes one or more skeletal modified nucleotides in which the skeletal modification is the substitution of a phosphate ester group with a phosphorothioate group of the nucleotide.

[0161] In one embodiment, when the skeletal modification is the substitution of a phosphate ester group of a nucleotide with a phosphorothioate group, the phosphorothioate group is located at the 1st, 2nd, 3rd, 4th, or 5th nucleotide linkage at the 5' and / or 3' end of a polynucleotide molecule, particularly an RNA molecule.

[0162] In one embodiment, a polynucleotide molecule, particularly an RNA molecule, contains one or more skeletal modified nucleotides in which the skeletal modification is the substitution of a ribose (ribonucleotide) or deoxyribose (deoxyribonucleotide) sugar moiety with a pyranose or furanose sugar moiety.

[0163] Examples of base-modified nucleotides include, but are not limited to, nucleotides containing bases that do not exist in nature, rather than naturally occurring bases (adenine, cytosine, guanine, thymine, or uracil). Suitable examples of naturally occurring bases include, but are not limited to, uridine and / or cytidine modified at the 5-position, e.g., 5-(2-amino)propyluridine, 5-bromouridine; adenosine and / or guanosine modified at the 8-position, e.g., 8-bromoguanosine; deazanucleotides, e.g., 7-deaza-adenosine; and O- and N-alkylated nucleotides, e.g., N6-methyladenosine. In particular, these modifications may be combined.

[0164] In one embodiment, the polynucleotide molecule, particularly the RNA molecule, contains one or more base-modified nucleotides.

[0165] In one embodiment, the modified nucleotide, for example, a sugar-modified, skeletal-modified, or base-modified nucleotide, is located proximal to the 5' and / or 3' end of the polynucleotide molecule, for example, within 3, 5, or 10 nucleotides of the terminal.

[0166] In one embodiment, the polynucleotide is not conjugated to any small molecule; for example, the polynucleotide is not conjugated to any organic molecule having a molecular weight in the range of 250 to 1500, for example, 300 to 1000 g / mol.

[0167] (Pharmaceutical preparations) The aqueous liquid pharmaceutical formulation of the present invention contains water as a solvent. Examples of water include, but are not limited to, sterile water or purified water, sterile water for injection, RNase-free water, or bacteriostatic water for injection.

[0168] Preferably, aqueous liquid pharmaceutical formulations are substantially free of any solvent or co-solvent other than water. In particular, aqueous liquid pharmaceutical formulations are free of organic solvents or co-solvents such as ethanol, acetone, dimethyl sulfoxide (DMSO), dichloromethane (DCM), N-methylpyrrolidinone (NMP), N,N'-dimethylformamide (DMF), N,N'-dimethylacetamide (DMAC), 1,3-dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, and benzyl benzoate. As used herein, the term “substantially free” means that the formulation contains less than 2% (w / w), for example, less than 1% (w / w), for example, less than 0.5% (w / w) (where wt% is relative to the total weight of the formulation). Preferably, the formulation does not contain any solvent or co-solvent other than water.

[0169] The aqueous liquid pharmaceutical formulation according to the present invention may further include, but is not limited to, pharmaceutically acceptable excipients, including antioxidants, buffers, diluents, emulsifiers, lubricants, preservatives, solvents, stabilizers, suspending agents, thickeners, tonicity modifiers (osmotic pressure modifiers), vehicles, and wetting agents.

[0170] Suitable antioxidants include, but are not limited to, ascorbic acid (vitamin C), glutathione (reduced form), lipoic acid, uric acid, carotenes including β-carotene, and retinol (vitamin A), cc-tocopherol (vitamin E), ubiquinol (coenzyme Q), butylated hydroxyanisole, butylated hydroxytoluene, propyl gallate, tert-butylhydroquinone, monothioglycerol, lutein, selenium, manganese, zeaxanthin, or combinations thereof.

[0171] The aqueous liquid pharmaceutical formulation of the present invention may contain one or more buffering agents. Suitable buffering agents include, but are not limited to, citric acid, boric acid, formic acid, glycine, alanine, acetic acid, aspartic acid, malic acid, glyoxylic acid, gluconic acid, lactic acid, glycolic acid, oxalic acid, histidine, tartarate, and succinate buffering agents. As used herein, a reference to a "citric acid" buffering agent will be understood to refer to a mixture of citrate and the corresponding acid as a buffering agent system in a ratio corresponding to the target pH, i.e., the pH to which the aqueous liquid pharmaceutical formulation is intended to be buffered. For example, the buffering agent may include sodium citrate dihydrate and citric acid monohydrate. In particular, the buffering agent may be based on a weak organic acid, for example, the buffering agent may be citrate, acetate, lactate, or formate, especially citrate. Further preferred buffering agents are phosphates. For example, the buffering agent may include sodium phosphate and disodium phosphate.

[0172] Suitable pharmaceutically acceptable diluents include, but are not limited to, isotonic saline (0.9% w / v), isotonic dextrose (5% w / v), isotonic mixtures of physiological saline and dextrose (e.g., physiological saline (0.45% w / v) and dextrose (2.5% w / v)), sterile water or purified water, sterile water for injection, or bacteriostatic water for injection. In particular, the diluent may be sterile water or purified water, sterile water for injection, RNase-free water, or bacteriostatic water for injection. For example, in one embodiment, the diluent is sterile water or purified water. In an alternative embodiment, the diluent isotonic saline (0.9% w / v).

[0173] Suitable preservatives include, but are not limited to, edetate and its alkali salts, such as disodium edetate (also known as "EDTA disodium") or calcium edetate (also known as calcium EDTA), phenol, m-cresol, chlorocresol, benzyl alcohol, propylparaben, methylparaben, butylparaben, chlorobutanol, phenylethyl alcohol, benzalkonium chloride, thimerosal, propylene glycol, sorbic acid, benzoic acid derivatives, and combinations thereof.

[0174] Suitable suspending agents include, but are not limited to, gum arabic (rubber), sodium alginate, starch and starch derivatives, xanthan gum, pectin, methylcellulose, hydroxyethylcellulose, sodium carboxymethylcellulose (Avicel RC591), microcrystalline cellulose, hypromellose, hyaluronic acid, and combinations thereof. Particularly preferred suspending agents include microcrystalline cellulose, sodium carboxymethylcellulose (Avicel RC591), hyaluronic acid, and combinations thereof.

[0175] The properties of certain suspending agents can further enhance their suitability as thickeners and / or wetting agents. Therefore, suitable thickeners and / or wetting agents include, but are not limited to, the suspending agents listed above. In particular, suitable thickeners and / or wetting agents include microcrystalline cellulose, sodium carboxymethylcellulose (Avicel RC591), hyaluronic acid, and combinations thereof.

[0176] Suitable tonicity-adjusting (osmotic pressure) agents include, but are not limited to, polyols such as sugars and sugar alcohols, for example, erythritol, glycerol, lactose, maltitol, mannitol, sorbitol, trehalose, and xylitol, as well as salts, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, and calcium chloride. Glycerol is a particularly suitable tonicity-adjusting (osmotic pressure) agent.

[0177] Preferably, the aqueous liquid pharmaceutical formulation according to the present invention has an osmolality of about 50 mOsm to about 750 mOsm, for example, about 100 mOsm to about 600 mOsm, for example, about 100 mOsm to about 500 mOsm. In particular, the aqueous liquid pharmaceutical formulation according to the present invention preferably has an osmolality of about 100 mOsm to about 400 mOsm, for example, about 150 mOsm to about 350 mOsm, for example, about 200 mOsm to about 300 mOsm.

[0178] The pH of the aqueous liquid pharmaceutical formulation according to the present invention is preferably about 4.0 to about 9.0, for example, about 4.0 to about 8.0, for example, about 4.0 to about 7.0, or about 5.0 to about 8.0. In particular, the pH is preferably about 4.0 to about 6.0, for example, about 4.0 to about 5.5. For example, the pH of the aqueous liquid pharmaceutical formulation is about 4.0, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.6, about 4.7, about 4.8, about 4.9, or about 5.0. Alternatively, the pH is preferably about 5.5 to about 8.0, for example, about 6.0 to about 8.0, for example, about 6.5 to about 7.5, or about 7.0 to about 80. For example, the pH of aqueous liquid pharmaceutical formulations is approximately 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. The pH of such pharmaceutical compositions may be adjusted by pH adjusters, which include acidifying agents such as hydrochloric acid, tartaric acid, citric acid, succinic acid, phosphoric acid, ascorbic acid, acetic acid, lactic acid, sulfuric acid, formic acid, and mixtures thereof; or alkaline buffering agents such as ammonium hydroxide, ethylamine, dipropylamine, triethylamine, alkanediamine, ethanolamine, polyalkylene polyamine, heterocyclic amine, alkali metal hydroxides such as sodium hydroxide and potassium hydroxide, alkaline earth metal hydroxides such as magnesium hydroxide and calcium hydroxide, and basic amino acids such as L-arginine, lysine, alanine, leucine, isoleucine, oxylysine, and histidine, and mixtures thereof.

[0179] The aqueous liquid pharmaceutical formulation according to the present invention may preferably have a pH of about 4.0 to about 9.0, for example, about 4.0 to about 8.0. It will be understood by those skilled in the art that an aqueous liquid pharmaceutical formulation suitable for topical administration to the nose may preferably have a pH of about 4.0 to about 9.0, for example, about 4.0 to about 8.0, for example, about 4.0 to about 7.0, for example, about 4.0 to about 6.0, for example, about 4.0 to about 5.0. A particularly suitable buffer for compositions with this desired pH is citrate. Those skilled in the art will further understand that an aqueous liquid pharmaceutical formulation suitable for topical administration to the lungs may preferably have a pH of about 5.5 to about 8.0, for example, about 6.0 to about 8.0, for example, about 6.0 to about 7.0, or about 7.0 to about 8.0. A particularly suitable buffer for compositions with this desired pH is phosphate.

[0180] Preferably, the aqueous liquid pharmaceutical formulation according to the present invention is protein-free. Preferably, the aqueous liquid pharmaceutical formulation according to the present invention is cationic lipopeptide-free, for example, polymyxin, for example, polymyxin B. Furthermore, the aqueous liquid pharmaceutical formulation according to the present invention is preferably inorganic nanoparticle-free, and preferred or typical examples thereof may include inorganic nanoparticles of metal salts such as zinc oxide, which are known in the art, or may include gold, silver, or silica nanoparticles. Furthermore, the aqueous liquid pharmaceutical formulation according to the present invention is preferably lipid nanoparticles (LNPs) or liposomes. The aqueous liquid pharmaceutical formulation according to the present invention is preferably solid or partially solid (e.g., partially solid and partially liquid) nanoparticles. Lipid nanoparticles (LNPs) described in the prior art as nucleic acid delivery systems (e.g., Kulkarni et al., 2021) are particles formed from a core containing cationic lipids, neutral lipids (e.g., cholesterol), and helper lipids that contribute to the particle structure, and an outer shell formed from stabilizer molecules, which can bind to nucleic acids. Helper lipids, which may include phospholipids such as phosphatidylcholine or phosphatidylethanolamine, play a role in enhancing the stability of LNPs by providing structural integrity and promoting the formation of lipid bilayers. Stabilizers, often polyethylene glycol (PEG)-lipids, provide a hydrophilic shield that reduces aggregation and opsonization, thereby extending circulation time in the bloodstream and improving delivery efficiency to target cells. All components in LNPs are necessary for encapsulation efficiency, promote cellular uptake, and ensure their stability in the biological environment. Liposomes are typically spherical vesicles containing lipid bilayers. Preferably, the particles of the stable colloidal emulsion of the present invention actually contain some kind of lipid bilayer.

[0181] Furthermore, preferably, the aqueous liquid pharmaceutical formulation according to the present invention is substantially free of lipid nanoparticles (LNPs) and liposome components. Lipid nanoparticles (LNPs) are typically described as nanoparticles comprising a cationic or ionized lipid core designed to encapsulate nucleic acids such as mRNA or siRNA, forming a complex that can be efficiently delivered to target cells. The core structure is stabilized by neutral lipids such as cholesterol, which help maintain the integrity and fluidity of the lipid bilayer. In addition, helper lipids such as phosphatidylcholine or phosphatidylethanolamine are incorporated to enhance nucleic acid encapsulation efficiency, promote fusion of LNPs with the cell membrane, and thereby improve uptake by target cells. The outer surface of the LNPs is often modified with stabilizer molecules such as polyethylene glycol (PEG)-lipids, which provide a hydrophilic coating that reduces aggregation and opsonization by the immune system. This coating extends the circulation time of LNPs in the bloodstream, increasing their chances of reaching target cells. In summary, these components work synergistically to create a nucleic acid delivery system that can protect genetic material and ensure its effective delivery and expression within target cells. Therefore, for example, preferably, the aqueous liquid pharmaceutical formulation according to the present invention does not contain neutral lipids. Such neutral lipids typically do not have ionizing groups and therefore do not generate an electric charge (whether positive or negative) around a neutral pH (i.e., pH 7.0 to 7.4). In particular, the pharmaceutical formulation does not contain cholesterol or its analogues. Preferably, the aqueous liquid pharmaceutical formulation according to the present invention does not contain cationic lipids such as quaternary ammonium lipids. In particular, the pharmaceutical formulation does not contain cationic lipids such as N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). Such cationic lipids typically have at least one ionizing group that generates a net positive charge around a neutral pH (i.e., pH 7.0 or 7.4). Preferably, the aqueous liquid pharmaceutical formulation according to the present invention does not contain helper lipids such as dioleoylphosphatidylethanolamine (DOPE) or phosphatidylcholine.Furthermore, for example, preferably, the aqueous liquid pharmaceutical formulation according to the present invention does not contain lipids other than fatty acids present therein. In one embodiment where the fatty acid is oleic acid, preferably, the aqueous liquid pharmaceutical formulation does not contain lipids other than oleic acid.

[0182] The aqueous liquid pharmaceutical formulation (or immunostimulant, immunogenic, or vaccine composition) of the present invention may be suitable for parenteral administration, including oral, inhalation, sublingual, oral cavity, intravenous, subcutaneous, topical, transdermal, pulmonary, rectal, vaginal, intraocular, intranasal, intradermal, transmucosal, intraperitoneal, intramuscular, intra-articular, intraorbital, intracardiac, transtracheal, subepidermal, intra-articular, intraspinal, and intrasternal administration.

[0183] In one embodiment, the aqueous liquid pharmaceutical formulation (or immunostimulant, immunogenic, or vaccine composition) of the present invention is suitable for local administration to the lungs or nose. Therefore, the aqueous liquid pharmaceutical formulation of the present invention is suitable for administration by inhalation, for example, for local administration to the lungs by oral inhalation or for intranasal administration. In one embodiment, the aqueous liquid pharmaceutical formulation (or immunostimulant, immunogenic, or vaccine composition) of the present invention is administered locally to the lungs or nose. Therefore, in one embodiment, the aqueous liquid pharmaceutical formulation (or immunostimulant, immunogenic, or vaccine composition) of the present invention is administered by inhalation or intranasal administration.

[0184] It should be noted that the aqueous liquid pharmaceutical formulation of the present invention, which is suitable for local administration to the lungs or nose, may, when administered locally to the lungs via oral inhalation or locally to the nose, result in administration to the pharynx.

[0185] It will be understood that formulations suitable for topical administration to the lungs may contain different pharmaceutically acceptable excipients than those suitable for topical administration to the nose. For example, formulations suitable for topical administration to the nose may contain suspending agents and / or wetting agents and / or thickening agents, such as microcrystalline cellulose, sodium carboxymethylcellulose (Avicel RC591), hyaluronic acid, or mixtures thereof, while formulations suitable for topical administration to the lungs may not contain these.

[0186] In an alternative embodiment, the aqueous liquid pharmaceutical formulation (or immunostimulant, immunogenic, or vaccine composition) of the present invention is suitable for subcutaneous administration and, in particular, by subcutaneous injection. In one embodiment, the aqueous liquid pharmaceutical formulation (or immunostimulant, immunogenic, or vaccine composition) of the present invention is administered subcutaneously, for example, by subcutaneous injection. It will be understood that formulations suitable for subcutaneous administration contain pharmaceutically acceptable excipients suitable for that route, and that these pharmaceutically acceptable excipients are different from those present in formulations suitable for topical administration to the lungs or nose.

[0187] In an alternative embodiment, the aqueous liquid pharmaceutical formulation (or immunostimulant, immunogenic, or vaccine composition) of the present invention is suitable for ocular administration. For example, the aqueous liquid pharmaceutical formulation of the present invention may be suitable for intraocular administration. Alternatively, the aqueous liquid pharmaceutical formulation of the present invention may be suitable for topical administration to the eye. Therefore, in one embodiment, the aqueous liquid pharmaceutical formulation (or immunostimulant, immunogenic, or vaccine composition) of the present invention is administered via an ocular administration route (e.g., via an intraocular route or via topical administration to the eye). It will be understood that formulations suitable for ocular administration contain pharmaceutically acceptable excipients suitable for that route, and that these pharmaceutically acceptable excipients are different from those present in formulations suitable for topical administration to the lung or nose or formulations suitable for subcutaneous administration.

[0188] Preferably, the aqueous liquid pharmaceutical formulation of the present invention is suitable for administration to mammals. More preferably, the aqueous liquid pharmaceutical formulation of the present invention is suitable for administration to humans. In one embodiment, the aqueous liquid pharmaceutical formulation of the present invention is administered to mammals. In particular, the aqueous liquid pharmaceutical formulation of the present invention is administered to humans.

[0189] Preferably, the aqueous liquid pharmaceutical formulations disclosed herein can be administered to a patient or subject once or more times a day, for example, twice, three times, four times, or five times a day. Such treatment can be extended for several weeks or months.

[0190] (medical use) While not wishing to be constrained by theory, the inventors intend that the aqueous liquid pharmaceutical formulations of the present invention (in at least some embodiments) enhance, improve, or make more efficient the delivery of a given polynucleotide molecule to a target cell or tissue, thereby increasing its exposure. Accordingly, in one embodiment, a method is provided for enhancing or improving the delivery of a polynucleotide molecule to a target cell or tissue, comprising formulating a polynucleotide molecule in the aqueous liquid pharmaceutical formulation of the present invention and administering the formulation to a target cell or tissue. As used herein, “target cell or tissue” means a cell or tissue targeted for the administration of a polynucleotide molecule, which may or may not be a cell or tissue targeted for the purpose of eliciting a therapeutic effect. Preferably, the target cell or tissue is a cell or tissue of the lung or nose, in particular a cell or tissue of the nasal or respiratory epithelium.

[0191] Polynucleotide molecules can exert therapeutic effects when they interact with molecules, organelles, cells, or tissues.

[0192] Therefore, in one embodiment, an aqueous liquid pharmaceutical formulation according to the present invention for use as a pharmaceutical is provided. Preferably, the aqueous liquid pharmaceutical formulation according to the present invention is for use as a pharmaceutical for topical administration to the lungs (e.g., by oral inhalation) or topical administration to the nose. Preferably, the aqueous liquid pharmaceutical formulation according to the present invention for use as a pharmaceutical is administered topically to the lungs (e.g., by oral inhalation) or into the nasal cavity. Alternatively, preferably, the aqueous liquid pharmaceutical formulation according to the present invention is for use as a pharmaceutical for subcutaneous administration (e.g., by subcutaneous injection). Alternatively, the aqueous liquid pharmaceutical formulation according to the present invention may be for use as a pharmaceutical for intraocular administration.

[0193] In one embodiment, the aqueous liquid pharmaceutical formulation for use according to the present invention is for therapeutic use, that is, for use in the treatment of a disease; or for preventive use, that is, for use in the prevention of a disease.

[0194] The preferred dose of the aqueous liquid pharmaceutical formulation for use described in the present invention is a therapeutic or prophylactic effective dose that can be determined by those skilled in the art. For example, the aqueous liquid pharmaceutical formulation for use can be administered to a patient or subject in an amount such that the dose of polynucleotide molecules is 0.01 μg / mL to 100,000 μg / mL, for example, 0.1 to 100,000 μg / mL, for example, 1 to 100,000 μg / mL, for example, 1 to 50,000 μg / mL. In one embodiment, the aqueous liquid pharmaceutical formulation for use can be administered to a patient or subject in an amount such that the dose of polynucleotide molecules is 1 to 10,000 μg / mL, for example, 1 to 1,000 μg / mL, for example, 1 to 100 μg / mL, for example, 1 to 50 μg / mL, for example, 1 to 20 μg / mL, for example, about 10 μg / mL. In an alternative embodiment, the aqueous liquid pharmaceutical formulation for use may be administered to a patient or subject in an amount such that the dose of polynucleotide molecules is 1 to 10,000 μg / mL, for example, 1 to 1,000 μg / mL, for example, 10 to 1,000 μg / mL, for example, 10 to 500 μg / mL, for example, 10 to 250 μg / mL, for example, 50 to 150 μg / mL, for example, about 100 μg / mL. Alternatively, aqueous liquid pharmaceutical formulations for use may be administered to a patient or subject in amounts such that the dose of the polynucleotide molecule is 1 to 100,000 μg / mL, for example, 10 to 100,000 μg / mL, for example, 100 to 100,000 μg / mL, for example, 1,000 to 100,000 μg / mL, for example, 5,000 to 75,000 μg / mL, for example, 5,000 to 50,000 μg / mL, for example, 5,000 to 15,000 μg / mL, 10,000 to 20,000 μg / mL, 15,000 to 25,000 μg / mL, or 25,000 to 50,000 μg / mL. Importantly, as will be understood by those skilled in the art, the dose is determined by the route of administration, sequence, structure, and size of the polynucleotide molecule, as well as the indication to be treated.

[0195] Preferably, the aqueous liquid pharmaceutical formulations for use described in the present invention can be administered to a patient or subject once or more times a day, for example, twice, three times, four times, or five times a day. Such treatment can be extended for several weeks or months.

[0196] Preferably, the aqueous liquid pharmaceutical formulation for use according to the present invention is administered to mammals. In particular, the aqueous liquid pharmaceutical formulation of the present invention is administered to humans.

[0197] (immune stimulation) For example, polynucleotide molecules, particularly shRNA molecules, can interact directly or indirectly (e.g., via encoded proteins or other gene products) with molecules or cells of the immune system, leading to the activation of the innate and / or adaptive immune system. For instance, such immune stimulation or activation may have utility in the treatment of infectious diseases or cancer. Other gene products, by definition, include, among others, RNA or cDNA molecules.

[0198] Therefore, in one embodiment, an aqueous liquid pharmaceutical formulation for use according to the present invention is provided, wherein the aqueous liquid pharmaceutical formulation stimulates or activates the innate and / or adaptive immune system and / or elicits a innate and / or adaptive immune response.

[0199] Accordingly, in one embodiment, the polynucleotide molecule is a polynucleotide molecule that stimulates or activates the innate and / or adaptive immune system and / or elicits a innate and / or adaptive immune response, for use in the treatment of diseases or illnesses treated by stimulation or activation of the innate and / or adaptive immune system and / or induction of a innate and / or adaptive immune response, according to the present invention, an aqueous liquid pharmaceutical formulation for use.

[0200] In an alternative embodiment, the present invention provides a method for treating a disease or illness treated by stimulation or activation of the innate and / or adaptive immune system and / or induction of a innate and / or adaptive immune response, comprising administering a therapeutically or prophylactically effective amount of an aqueous liquid pharmaceutical formulation described herein to a subject in need thereof, wherein the polynucleotide molecule is a polynucleotide molecule that stimulates or activates the innate and / or adaptive immune system and / or induces a innate and / or adaptive immune response.

[0201] Furthermore, the present invention provides for the use of aqueous liquid pharmaceutical formulations described herein, in the manufacture of pharmaceuticals for use in the treatment of diseases or illnesses treated by stimulation or activation of the innate and / or adaptive immune system and / or induction of a innate and / or adaptive immune response, wherein the polynucleotide molecule is a polynucleotide molecule that stimulates or activates the innate and / or adaptive immune system and / or induces a innate and / or adaptive immune response.

[0202] (Immune stimulating composition) As discussed above, polynucleotide molecules can interact with molecules or cells of the immune system, leading to the stimulation and / or activation of the innate and / or adaptive immune system.

[0203] Therefore, in one embodiment, an aqueous liquid pharmaceutical formulation for use according to the present invention is provided, for use in the treatment of a disease or illness that is treated by stimulation or activation of the natural and / or adaptive immune system, wherein the polynucleotide molecule is a polynucleotide molecule that stimulates or activates the natural and / or adaptive immune system.

[0204] Furthermore, the present invention provides aqueous liquid pharmaceutical formulations or immunostimulatory compositions for use in stimulating or activating a target immune system. Accordingly, the present invention provides a method for stimulating a target immune system, comprising administering to a target an aqueous liquid formulation or immunostimulatory composition described herein. Furthermore, the use of aqueous liquid pharmaceutical formulations or immunostimulatory compositions in the manufacture of pharmaceuticals for stimulating a target immune system is provided.

[0205] Preferably, the aqueous liquid formulations or immunostimulatory compositions of the present invention are intended for use in stimulating or activating the target antiviral innate and / or adaptive immune response.

[0206] Preferably, the polynucleotide molecule present in the aqueous liquid pharmaceutical formulation for use in stimulating the target immune system is, for example, a directly immunostimulating polynucleotide molecule. In particular, here the polynucleotide molecule is an shRNA molecule. Alternatively, the immunostimulating polynucleotide may be poly(I:C) (see, for example, dsRNA 2 of US9682096B2, which is incorporated herein by reference in the following examples).

[0207] Therefore, in one embodiment, the present invention provides an immunostimulatory composition which is an aqueous liquid formulation comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) an immunostimulatory polynucleotide molecule which can stimulate or is intended for use in stimulating the immune system of a target. Preferably, the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, particularly polysorbate 80. Preferably, the immunostimulatory polynucleotide molecule is an shRNA molecule.

[0208] By definition, the immune system of a subject is considered stimulated when an immune response, including a innate immune response, an antibody response, or a cell-mediated immune response, is initiated, enhanced, or augmented in response to an antigen or immunogen, particularly an exogenous antigen or immunogen. By definition, an immunogen is an immunogenic antigen.

[0209] Preferably, the immune response is stimulated by inducing a pro-inflammatory cytokine response. For example, the immune response may be stimulated by inducing one or more of a pro-inflammatory interleukin response, such as an IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-17, and IL-18 response, or a pro-inflammatory interferon response, such as an IFN-α, IFN-β, and IFN-γ response, or a pro-inflammatory chemokine response, or a pro-inflammatory tumor necrosis factor response, such as a TNF-α and / or TNF-β response, or a combination thereof. In particular, the immune response is stimulated by inducing one or more of an interferon response, such as an IFN-α, IFN-β, and IFN-γ response.

[0210] Preferably, the immune response is stimulated by activating a pattern recognition receptor (PRR). The PRR can induce a pro-inflammatory cytokine response, such as a pro-inflammatory interferon response. For example, the PRR can be any member of a family of PRRs selected from toll-like receptors (TLRs), such as TLR2, TLR3, TLR4, TLR7, TLR8, or TLR9, C-type lectin receptors (CLRs), NOD-like receptors (NLRs), and RIG-I-like receptors (RLRs), such as RIG-I, MDA5, or LGP2. Preferably, the PRR can be any member of the RLRs, such as RIG-I, MDA5, or LGP2, particularly RIG-I.

[0211] (Immunogenic composition) As discussed above, polynucleotide molecules can interact with molecules or cells of the immune system, for example, indirectly (e.g., via a coded protein or other gene product), to effect the induction of a natural and / or adaptive immune response.

[0212] Thus, in one embodiment, there is provided an aqueous liquid pharmaceutical formulation for use in the treatment of a disease or disorder treated by inducing a natural and / or adaptive immune response, the formulation comprising a polynucleotide molecule that induces a natural and / or adaptive immune response of a subject.

[0213] Furthermore, there is provided an aqueous liquid pharmaceutical formulation, immunogenic composition, or vaccine composition for use in inducing an immune response of a subject, wherein the immune response is induced against an immunogen or vaccine immunogen encoded by a polynucleotide molecule. Thus, the invention provides a method of inducing an immune response of a subject, the method comprising administering to the subject an aqueous liquid formulation described herein. Further provided is the use of an aqueous liquid pharmaceutical formulation in the manufacture of a medicament for inducing an immune response of a subject.

[0214] Preferably, the polynucleotide molecule present in the aqueous liquid pharmaceutical formulation or vaccine composition for use in inducing an immune response of a subject encodes an immunogen. Endogenous expression of the immunogen or vaccine immunogen results in the induction of an immune response of the subject against the immunogen. In particular, the polynucleotide molecule is an mRNA molecule or a DNA molecule.

[0215] In one embodiment, the aqueous liquid pharmaceutical formulation or vaccine composition for use in eliciting a target immune response is for therapeutic use, i.e., the immune response is elicited to obtain a therapeutic effect against a disease or illness treated by the elicitation of a natural and / or adaptive immune response, such as cancer. Alternatively, the composition is for prophylactic use, i.e., the immune response is elicited to obtain a protective or preventive effect against a disease or illness treated by the elicitation of a natural and / or adaptive immune response, such as infectious disease.

[0216] Therefore, in one embodiment, the present invention provides an immunogenic composition, which is an aqueous liquid formulation, comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) a polynucleotide molecule encoding an immunogen. The polynucleotide molecule encoding the immunogen is capable of inducing or being used to induce an immune response to the immunogen in a subject. Preferably, the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, particularly polysorbate 80. Preferably, the polynucleotide molecule encoding the immunogen is an mRNA molecule or a DNA molecule.

[0217] Furthermore, a vaccine composition is provided, which is an aqueous liquid formulation, comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) a polynucleotide molecule encoding a vaccine immunogen. The polynucleotide molecule encoding the vaccine immunogen is capable of inducing or for use in inducing an immune response to the vaccine immunogen in a subject. Preferably, the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, particularly polysorbate 80. Preferably, the polynucleotide molecule encoding the vaccine immunogen is an mRNA molecule or a DNA molecule.

[0218] By definition, a vaccine immunogen is an immunogen, i.e., an immunogenic antigen, that can induce a therapeutic, protective, or immune response in a target.

[0219] Importantly, an aqueous liquid pharmaceutical formulation for use in eliciting a target immune response may comprise (i) a polynucleotide molecule encoding an immunogen against which an immune response can be elicited, and (ii) a polynucleotide molecule that stimulates the target immune system. That is, the immunogenic composition or vaccine composition according to the present invention may further comprise an immunostimulating polynucleotide molecule. Preferably, the immunostimulating polynucleotide molecule is an shRNA molecule.

[0220] Therefore, in one embodiment, the present invention provides an immunogenic composition, which is an aqueous liquid formulation, comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, (ii) a polynucleotide molecule encoding an immunogen, and (iii) an immunostimulatory polynucleotide molecule. Preferably, the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, particularly polysorbate 80. The polynucleotide molecule encoding the immunogen is capable of eliciting an immune response to the immunogen in a subject, or is intended for use in eliciting such an immune response. Preferably, the polynucleotide molecule encoding the immunogen is an mRNA or DNA molecule. The immunostimulatory polynucleotide molecule is capable of stimulating the immune system in a subject, or is intended for use in stimulating such a system. Preferably, the immunostimulatory polynucleotide molecule is an shRNA molecule.

[0221] Furthermore, a vaccine composition is provided, which is an aqueous liquid formulation, comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, (ii) a polynucleotide molecule encoding a vaccine immunogen, and (iii) an immunostimulatory polynucleotide molecule. Preferably, the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, particularly polysorbate 80. The polynucleotide molecule encoding the vaccine immunogen is capable of eliciting an immune response to the vaccine immunogen in a subject, or is intended for use in eliciting such a response. Preferably, the polynucleotide molecule encoding the vaccine immunogen is an mRNA or DNA molecule. The immunostimulatory polynucleotide molecule is capable of stimulating the immune system in a subject, or is intended for use in stimulating such a system. Preferably, the immunostimulatory polynucleotide molecule is an shRNA molecule.

[0222] Preferably, the immunostimulant or immunogenic composition described above does not contain proteins. Preferably, the composition does not contain cationic lipopeptides such as polymyxin B. Preferably, the composition does not contain neutral lipids. In particular, the composition does not contain cholesterol or its analogues. Preferably, the composition does not contain cationic lipids. Preferably, the composition does not contain helper lipids such as dioleoylphosphatidylethanolamine (DOPE) or phosphatidylcholine. Furthermore, the immunostimulant or immunogenic composition described above does not contain inorganic nanoparticles, lipid nanoparticles (LNPs), or liposomes, and / or substantially does not contain LNPs and liposome components.

[0223] Preferably, the immunostimulant or immunogenic composition described above may further contain pharmaceutically acceptable excipients in relevant amounts, as described above. In fact, the composition may be limited as described above with respect to the aqueous liquid pharmaceutical formulation of the present invention.

[0224] (Adaptation of immune stimulation) Preferably, diseases or illnesses treated by stimulation or activation of the innate and / or adaptive immune system and / or by eliciting a innate and / or adaptive immune response are infectious diseases. Infectious diseases are preferably of bacterial, fungal, parasitic, or viral origin.

[0225] In one embodiment, the aqueous liquid pharmaceutical formulation of the present invention is intended for use in the treatment or prevention of infections caused by bacteria, fungi, or parasites, or diseases associated with such bacterial, fungal, or parasitic infections. That is, the diseases or illnesses treated by stimulation or activation of the natural and / or adaptive immune system and / or induction of a natural and / or adaptive immune response are infections caused by bacteria, fungi, or parasites, or diseases associated with such bacterial, fungal, or parasitic infections.

[0226] Preferably, bacteria, fungi, or parasites infect the brain, circulatory system, endocrine system, eyes, gastrointestinal tract, reproductive tract, kidneys, liver, respiratory system, or skin. Therefore, the diseases associated with the infection are diseases of the brain, circulatory system, endocrine system, eyes, gastrointestinal tract, reproductive tract, kidneys, liver, respiratory system, or skin. In particular, bacteria, fungi, or parasites infect the respiratory system, and the diseases associated with the infection are diseases of the respiratory system.

[0227] For example, bacteria belong to the genera Bordetella, Chlamydophila, Corynebacterium, Coxiella, Escherichia, Haemophilus, Klebsiella, Legionella, Moraxella, Mycobacterium, Mycoplasma, Proteus, Pseudomonas, Serratia, Staphylococcus, and Streptococcus.

[0228] In one embodiment, the aqueous liquid pharmaceutical formulation of the present invention is intended for use in the treatment or prevention of a disease caused by a viral infection or a disease associated with such a viral infection. That is, the disease or illness treated by stimulation or activation of the innate and / or adaptive immune system and / or induction of a innate and / or adaptive immune response is a disease caused by a viral infection or a disease associated with such a viral infection.

[0229] Preferably, the virus is a DNA virus or an RNA virus. Preferably, the virus has a single-stranded genome, a double-stranded genome, or a partially double-stranded genome. Preferably, the single-stranded genome is a sense(+) genome, or the single-stranded genome is an antisense(-) genome. In particular, the virus may have a single-stranded RNA genome, a double-stranded DNA genome, or a double-stranded RNA genome. For example, the virus may have a sense(+) single-stranded RNA genome or an antisense(-) single-stranded RNA genome.

[0230] Preferably, the virus is naked, that is, not enveloped. Or, the virus is enveloped.

[0231] Preferably, the virus belongs to the family Adenoviridae, Arenaviridae, Astroviridae, Bornaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Filoviridae, Flaviviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Orthomyxoviridae, Papillomaviridae, Polyomaviridae, Paramyxoviridae, Parvoviridae, Picornaviridae, Poxviridae, Reoviridae, Retroviridae, Rhabdoviridae, or Togaviridae. In particular, the virus belongs to the family Adenoviridae, Coronaviridae, Herpesviridae, Orthomyxoviridae, Paramyxoviridae, or Picornaviridae.

[0232] Preferably, the virus infects the brain, circulatory system, endocrine system, eyes, digestive tract, reproductive tract, kidneys, liver, respiratory system, or skin. Thus, diseases associated with viral infection are diseases of the brain, circulatory system, endocrine system, eyes, digestive tract, reproductive tract, kidneys, liver, respiratory system, or skin. In particular, the virus infects the respiratory system, and diseases associated with viral infection are diseases of the respiratory system.

[0233] For example, the virus is SARS-CoV-2, and the disease associated with the viral infection is COVID-19. For example, the virus is a seasonal coronavirus, e.g., 229E, NL63, OC43, or HKU1, and the disease associated with the viral infection is a disease associated with seasonal coronavirus infection, e.g., 229E, NL63, OC43, or HKU1. For example, the virus is the influenza virus, and the disease associated with the viral infection is influenza. For example, the virus is the respiratory syncytial virus (RSV), and the disease associated with the viral infection is a disease associated with RSV infection. For example, the virus is human rhinovirus (HRV), and the disease associated with the viral infection is a disease associated with HRV infection. For example, the virus is Middle East Respiratory Syndrome (MERS)-CoV, and the disease associated with the viral infection is MERS. For example, here, the virus is the avian influenza virus, and the disease associated with the viral infection is avian influenza. For example, the virus is the Nipah virus, and the disease associated with the viral infection is a disease associated with Nipah virus infection. For example, if the virus is human parainfluenza virus (HPIV), the disease associated with the viral infection is a disease associated with HPIV infection. For example, if the virus is human metapneumovirus (hMPV), the disease associated with the viral infection is a disease associated with hMPV infection.

[0234] In an alternative embodiment, the virus infects the respiratory system, but the disease associated with the viral infection is a systemic disease or a disease of the brain, circulatory system, endocrine system, eye, gastrointestinal tract, reproductive tract, kidney, liver, skin, or other organ or organ system.

[0235] For example, the virus is the Ebola virus, and the disease associated with the viral infection is Ebola or Ebola virus disease (EVD). For example, the virus is the Lassa virus, and the disease associated with the viral infection is Lassa fever.

[0236] As used herein, "influenza virus" refers to influenza A virus, influenza B virus, influenza C virus, and influenza D virus, for example, influenza A virus or influenza B virus.

[0237] Alternatively, cancer is a disease or illness that is treated by stimulation or activation of the innate and / or adaptive immune system and / or by the induction of an innate and / or adaptive immune response. For example, cancer may be breast cancer, bladder cancer, kidney cancer, lung cancer, prostate cancer, bone cancer, brain tumor, cervical cancer, anal cancer, colon cancer, colorectal cancer, stomach cancer, hematological cancer, such as leukemia, lymphoma, or myeloma, liver cancer, skin cancer, ovarian cancer, pancreatic cancer, testicular cancer, thyroid cancer, vaginal cancer, heart cancer, or sarcoma.

[0238] (Increased gene expression and gene therapy) Alternatively, a polynucleotide molecule, particularly an mRNA or DNA molecule, may be intended to establish the expression of one or more proteins (or other gene products) encoded by the polynucleotide molecule, in particular, where the endogenous expression of the protein (or other gene product) is defective or very low, for example, silent, resulting in insufficient expression of the protein (or other gene product), or the expression of defective or dysfunctional variants of the protein (or other gene product), where the insufficient expression of the protein (or other gene product), or the expression of defective or dysfunctional variants of the protein (or other gene product), contributes to cellular dysfunction and, consequently, to disease.

[0239] In particular, polynucleotide molecules, especially mRNA or DNA molecules, may be intended to establish the expression of one or more proteins (or other gene products) encoded by the polynucleotide molecule in order to complement endogenous gene expression, so as to facilitate the overexpression of proteins (or other gene products) in order to facilitate interference with endogenous cellular processes such as the regulation of gene expression or signaling. Furthermore, the overexpression of proteins (or other gene products) may be intended to activate an immune response or stimulate the immune system, for example, against tumors that overexpress an antigen, or in response to immune cells that present an exogenous antigen.

[0240] Therefore, in one embodiment, an aqueous liquid pharmaceutical formulation for use according to the present invention is provided, wherein the aqueous liquid pharmaceutical formulation increases the endogenous expression of a protein (or other gene product).

[0241] In an alternative embodiment, an aqueous liquid pharmaceutical formulation for use according to the present invention is provided, wherein the aqueous liquid pharmaceutical formulation increases the endogenous expression of a functional protein (or other gene product). For example, the aqueous liquid pharmaceutical formulation may establish the expression of a functional protein (or other gene product), but the endogenous expression of the protein (or other gene product) produces a non-functional or dysfunctional protein (or other gene product), such as a cleaved or misfolded protein (or other gene product).

[0242] As used herein, the term “increase” includes restoring endogenous gene expression, i.e., increasing it from zero or a low value to a “normal” value, and enhancing endogenous gene expression, i.e., increasing it from a “normal” value to a high value.

[0243] Accordingly, in one embodiment, the present invention provides an aqueous liquid pharmaceutical formulation for use according to the present invention, which is a polynucleotide molecule that increases the endogenous expression of a protein (or other gene product) or the endogenous expression of a functional protein (or other gene product), and is intended for use in the treatment of diseases or illnesses that are treated by increasing the endogenous expression of a protein (or other gene product) or by increasing the expression of a functional protein (or other gene product).

[0244] Therefore, in one embodiment, an aqueous liquid pharmaceutical formulation is provided for use in a gene therapy method, wherein a polynucleotide molecule, in particular an mRNA or DNA molecule, encodes a therapeutic gene, protein (or other gene product). Preferably, the use in a gene therapy method is for the treatment of a disease or illness that is treated by increasing the endogenous expression of the therapeutic gene, protein (or other gene product) encoded by the polynucleotide molecule.

[0245] In an alternative embodiment, the present invention provides a method for treating a disease or illness treated by increasing the endogenous expression of a protein (or other gene product) or by increasing the expression of a functional protein (or other gene product), comprising administering a therapeutically or prophylactically effective amount of the aqueous liquid pharmaceutical formulation described herein to a subject in need thereof, wherein the polynucleotide molecule is a polynucleotide molecule that increases the endogenous expression of a protein (or other gene product) or a functional protein (or other gene product).

[0246] Furthermore, a method of gene therapy is provided, comprising administering a therapeutically or prophylactically effective amount of an aqueous liquid pharmaceutical formulation described herein to a subject in need thereof, wherein a polynucleotide molecule, in particular mRNA or DNA molecule, encodes a therapeutic gene, protein (or other gene product). Preferably, the method of gene therapy is for the treatment of a disease or illness that is treated by increasing the endogenous expression of the therapeutic gene, protein (or other gene product) encoded by the polynucleotide.

[0247] The present invention also provides the use of aqueous liquid pharmaceutical formulations described herein, in the manufacture of pharmaceuticals for use in the treatment of diseases or illnesses treated by increasing the endogenous expression of a protein (or other gene product) or by increasing the expression of a functional protein (or other gene product), wherein the polynucleotide molecule is a polynucleotide molecule that increases the endogenous expression of a protein (or other gene product) or increases the endogenous expression of a functional protein (or other gene product).

[0248] Furthermore, the use of aqueous liquid pharmaceutical formulations described herein, in the manufacture of pharmaceuticals for use in gene therapy methods, in which polynucleotide molecules, particularly mRNA or DNA molecules, encode a therapeutic gene, protein (or other gene product), is provided. Preferably, the use in gene therapy methods is for the treatment of a disease or illness that is treated by increasing the endogenous expression of the therapeutic gene, protein (or other gene product) encoded by the polynucleotide.

[0249] Preferably, diseases or illnesses treated by increasing the endogenous expression of a protein (or other gene product) or by increasing the endogenous expression of a functional protein (or other gene product) are monogenic or polygenic diseases or illnesses. For example, diseases or illnesses include blood disorders or illnesses, e.g., anemia, in particular sickle cell anemia; hemophilia, in particular hemophilia A or B; severe combined immunodeficiency (SCID); thalassemia; or von Willebrand disease; hearing disorders or illnesses, e.g., hearing loss; heart disorders or illnesses, e.g., atherosclerosis; coronary heart disease; long QT syndrome; or von Hippel-Lindau syndrome; metabolic disorders or illnesses, e.g., lysosomal storage disorders and diseases, e.g., type 1 diabetes, Gaucher disease; glycogen storage disorders; or obesity; musculoskeletal disorders or illnesses, e.g., This includes muscular dystrophy of the Juchenne type, or achondroplasia; diseases or illnesses of the nervous system or brain, such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Rett syndrome, fragile X syndrome, Huntington's disease, or Parkinson's disease; diseases or illnesses of the gastrointestinal tract or kidneys, such as polycystic kidney disease; diseases or illnesses of the respiratory system, such as asthma, alpha-1 antitrypsin deficiency, chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), pulmonary fibrosis, sarcoidosis, or cystic fibrosis; and diseases or illnesses of the skin, such as albinism, male pattern baldness, or alopecia.

[0250] In particular, diseases or illnesses include respiratory diseases or illnesses such as asthma, alpha-1 antitrypsin deficiency, chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), pulmonary fibrosis, sarcoidosis, or cystic fibrosis.

[0251] For example, the disease or illness is cystic fibrosis, and the polynucleotide molecules present in the formulation according to the present invention increase the endogenous expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein or increase the endogenous expression of functional cystic fibrosis transmembrane conductance regulator (CFTR) protein (see WO2022 / 204270A1 incorporated herein by reference).

[0252] Alternatively, for example, the disease or illness is a PCD, and the polynucleotide molecules present in the formulation according to the present invention increase the endogenous expression of PCD-related proteins, such as dynein axonemal intermediate chain 1, or increase the endogenous expression of functional PCD-related proteins, such as dynein axonemal intermediate chain 1 (see WO2022 / 198099A1 and WO2022 / 204215A1 incorporated herein by reference).

[0253] (Decreased or silencing of gene expression) Alternatively, polynucleotide molecules, particularly miRNA or siRNA molecules, may be intended to downregulate, reduce, silence, or knock down the expression of endogenous genes and, as a result, the encoded proteins (or other gene products) when such overexpression contributes to cellular dysfunction, or when endogenous genes and, as a result, the encoded proteins (or other gene products) are defective or dysfunctional and therefore contribute to cellular dysfunction.

[0254] In particular, polynucleotide molecules, especially miRNA or siRNA molecules, may be intended to downregulate, reduce, silence, or knock down endogenous gene expression in order to decrease endogenous gene expression so that the underexpression of proteins (or other gene products) interferes with other endogenous cellular processes such as the regulation of gene expression or signaling. Furthermore, underexpression of proteins (or other gene products) may be intended to activate an immune response or stimulate the immune system, for example, by the removal of "autoantigens."

[0255] Therefore, in one embodiment, an aqueous liquid pharmaceutical formulation for use according to the present invention is provided, wherein the aqueous liquid pharmaceutical formulation reduces the endogenous expression of a protein (or other gene product). As used herein, the term “reduce” includes restoring endogenous gene expression, i.e., reducing it from a high value to a “normal” value, and impairing endogenous gene expression, i.e., reducing it from a “normal” value to a low value or zero, for example, silencing.

[0256] Therefore, in one embodiment, the aqueous liquid pharmaceutical formulation for use according to the present invention, in which the polynucleotide molecule is a polynucleotide molecule that reduces the endogenous expression of a protein (or other gene product), is for use in the treatment of a disease or illness that is treated by reducing the endogenous expression of a protein (or other gene product).

[0257] In an alternative embodiment, the present invention provides a method for treating a disease or illness that is treated by reducing the endogenous expression of a protein (or other gene product), comprising administering a therapeutically or prophylactically effective amount of the aqueous liquid pharmaceutical formulation described herein to a subject in need thereof, wherein the polynucleotide molecule is a polynucleotide molecule that reduces the endogenous expression of a protein (or other gene product).

[0258] Furthermore, the present invention provides for the use of an aqueous liquid pharmaceutical formulation described herein, in the manufacture of a pharmaceutical for use in the treatment of a disease or illness that is treated by reducing the endogenous expression of a protein (or other gene product), wherein the polynucleotide molecule is a polynucleotide molecule that reduces the endogenous expression of a protein (or other gene product).

[0259] In one embodiment, the disease or illness treated by reducing the endogenous expression of a protein (or other gene product) is an infectious disease. The infectious disease is preferably of bacterial, fungal, parasitic, or viral origin. In particular, the infectious disease is a viral infection or a disease associated with such viral infection. Preferably, the virus infects the respiratory system, and the disease associated with the infection is a disease of the respiratory system.

[0260] (Modification of endogenous nucleic acid sequences) Alternatively, polynucleotide molecules, particularly DNA molecules, may be intended to modify endogenous nucleic acids or polynucleotide sequences, such as RNA or DNA sequences, for example, to repair, excise, insert, or replace bases or stretches in such sequences. For example, a polynucleotide molecule intended to modify endogenous nucleic acids or polynucleotide sequences may be a guide RNA (gRNA) that can function as a component of CRISPR-Cas9 gene editing technology.

[0261] Therefore, in one embodiment, an aqueous liquid pharmaceutical formulation for use according to the present invention is provided, wherein the aqueous liquid pharmaceutical formulation modifies an endogenous nucleic acid sequence, such as an mRNA molecule or a genome. For example, the aqueous liquid pharmaceutical formulation can repair an endogenous nucleic acid sequence, such as a genome, excise a base or stretch therefrom, insert a base or stretch therein, or exchange a base or stretch therewith.

[0262] Therefore, in one embodiment, the aqueous liquid pharmaceutical formulation for use according to the present invention, in which the polynucleotide molecule is a polynucleotide molecule that modifies an endogenous nucleic acid sequence, is for use in the treatment of a disease or illness that is treated by modifying an endogenous nucleic acid sequence.

[0263] Alternatively, in one embodiment, a method is provided for the treatment of a disease or illness treated by modifying an endogenous nucleic acid sequence, comprising administering a therapeutically or prophylactically effective amount of the aqueous liquid pharmaceutical formulation described herein to a subject in need thereof, wherein the polynucleotide molecule is a polynucleotide molecule that modifies an endogenous nucleic acid sequence.

[0264] Furthermore, the present invention provides the use of an aqueous liquid pharmaceutical formulation described herein, in the manufacture of a pharmaceutical for use in the treatment of a disease or illness that is treated by modifying an endogenous nucleic acid sequence, wherein the polynucleotide molecule is a polynucleotide molecule that modifies an endogenous nucleic acid sequence.

[0265] Through these different mechanisms, polynucleotide molecules can interact with, interfere with, modify, or otherwise influence virtually any cellular process.

[0266] (Ocular indications) Preferably, the aqueous liquid pharmaceutical formulation according to the present invention may be used as a pharmaceutical for administration to the eye, for example, by topical administration to the eye.

[0267] When the aqueous liquid pharmaceutical formulation according to the present invention is intended for use as a pharmaceutical for administration to the eye, it is preferably intended for use in the prevention or treatment of eye diseases or illnesses selected from, for example, dry eye-Sjögren's syndrome, Meemann epithelial corneal dystrophy, herpes simplex keratitis (HSK), mucopolysaccharidosis (MPS), dysphagia-ectodermal dysplasia-cleft lip and palate (EEC) syndrome, ocular hypertension and open-angle glaucoma, retinal split, choroidal atrophy, achromatopsia, and recurrent retinoblastoma.

[0268] Also provided is a method for the prevention or treatment of an eye disease or illness selected from, for example, dry eye-Sjögren's syndrome, Meemann's epithelial corneal dystrophy, herpes simplex keratitis (HSK), mucopolysaccharidosis (MPS), dysphagia-ectodermal dysplasia-cleft lip and palate (EEC) syndrome, ocular hypertension and open-angle glaucoma, retinal split, choroidal atrophy, achromatopsia, and recurrent retinoblastoma, comprising administering a therapeutically or prophylactically effective amount of the aqueous liquid pharmaceutical formulation according to the present invention to a subject in need thereof. Similarly, the use of aqueous liquid pharmaceutical formulations according to the present invention is provided in the manufacture of pharmaceuticals for use in the manufacture of pharmaceuticals for use in the treatment of eye diseases or conditions selected from, for example, dry eye-Sjögren's syndrome, Meemann epithelial corneal dystrophy, herpes simplex keratitis (HSK), mucopolysaccharidosis (MPS), dysphagia-ectodermal dysplasia-cleft lip and palate (EEC) syndrome, ocular hypertension and open-angle glaucoma, retinal split, choroidal atrophy, achromatopsia, and recurrent retinoblastoma.

[0269] (Production method) The formulation of the present invention can be produced by mixing the components to create a colloidal emulsion.

[0270] Preferably, the aqueous liquid pharmaceutical formulation of the present invention is produced by the following stepwise process. Those skilled in the art will understand that minor modifications to the process can still result in a process suitable for producing the aqueous liquid pharmaceutical formulation described herein. Furthermore, those skilled in the art will be able to determine optimized parameters for the process below, such as temperature, mixing time, and pH, based on surfactant components, polynucleotide molecules, and their concentrations.

[0271] Therefore, in one embodiment, a process is provided for the production or preparation of an aqueous liquid pharmaceutical formulation, or an immunostimulatory composition, immunogenic composition, or vaccine composition according to the present invention. Preferably, the process may include, consist of, or essentially consist of one or more of the following steps.

[0272] Preferably, first, a surfactant component, which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, for example, a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, is added to water, for example, RNase-free water, at the required concentration, and mixed using an overhead stirrer at an output of 25% to 75%, for example, 40% to 50%, or about 50%. The mixing is continued for any period of time until a homogeneous surfactant component mixture is obtained, for example, about 1 to 60 minutes, for example, about 1 to 30 minutes, for example, about 5 to 20 minutes, for example, about 10 minutes.

[0273] An example of an overhead stirrer is the IKA EURO-STAR Overhead Lab Mixer.

[0274] Next, to obtain the desired final concentration of polynucleotide molecules, a specific volume of polynucleotide molecule stock solution, typically containing polynucleotide molecules at a concentration of about 0.2–20% (w / w) (where wt% is relative to the total weight of the formulation), is dispersed in the homogeneous surfactant component mixture described above. This formulation is then mixed using an overhead stirrer at an output of 25%–75%, for example, 40%–50%, or about 50%, for any period until a homogeneous active surfactant component mixture is produced. Preferably, the mixing may be carried out for about 1–60 minutes, for example, about 1–30 minutes, for example, about 5–20 minutes, for example, about 10 minutes.

[0275] If, in a different process, the formulation is intended to contain further pharmaceutically acceptable excipients, these may be added to water, e.g., RNase-free water, at the required concentration, either simultaneously, stepwise, or in combination thereof, and the mixture may be homogenized until the desired rheology is achieved. For example, homogenization may be carried out at 2000–20000 rpm (revolutions per minute), e.g., 2000–15000, 4000–12000, or 5000–10000, for about 1–20 minutes, e.g., about 1–10 minutes, e.g., about 1–5 minutes. Next, the further pharmaceutically acceptable excipient composition is combined with the above active surfactant component mixture and mixed using an overhead stirrer at an output of 25%–75%, e.g., 40%–50% output, or about 50% output. Mixing is continued for any period until a homogeneous and uniform aqueous liquid pharmaceutical formulation is produced. Preferably, the mixing may be carried out for about 1 to 60 minutes, for example, about 1 to 30 minutes, for example, about 5 to 20 minutes, for example, about 10 minutes.

[0276] An exemplary homogenizer is the Silverson L5M homogenizer.

[0277] Those skilled in the art will understand that the further pharmaceutically acceptable excipients to be included in the aqueous liquid pharmaceutical formulation of the present invention are determined by the surfactant components, polynucleotide molecules, and route of administration. For example, an aqueous liquid pharmaceutical formulation suitable for topical administration to the nose may contain, for example, about 2% (w / w) of carboxymethylcellulose, for example, about 1% (w / w) of hyaluronic acid, and for example, about 2.1% (w / w) of glycerol (where wt%) is relative to the total weight of the formulation. Therefore, these excipients may be present in the above-mentioned further pharmaceutically acceptable excipient composition.

[0278] Finally, the aqueous liquid pharmaceutical formulation is buffered to the desired final pH using, for example, a buffering agent, such as a citrate buffer (for example, containing 0.2% (w / w) citric acid and 0.28% (w / w) sodium citrate, which is suitable for achieving a pH of about 4.0 to 6.0, for example, about 5.0 (where wt% is relative to the total weight of the formulation)). The buffered aqueous liquid pharmaceutical formulation is mixed, for example, using an overhead stirrer as described above, until a uniform pH adjustment is achieved. For example, mixing may be carried out for about 1 to 60 minutes, for example, about 1 to 30 minutes, for example, about 5 to 20 minutes, for example, about 10 minutes.

[0279] Preferably, each of the above steps is carried out at room temperature (i.e., about 20-30°C).

[0280] The present invention is further defined by the following clauses: Clause 1. An aqueous liquid pharmaceutical preparation comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) a polynucleotide molecule. Clause 2. The aqueous liquid pharmaceutical preparation according to Clause 1, wherein the fatty acid is selected from the group consisting of arachidic acid, arachidonic acid, caprylic acid, lauric acid, linoleic acid, linolenic acid, myristic acid, myristoleic acid, oleic acid, palmitic acid, palmitoleic acid, sapienic acid, stearic acid, and vaccenic acid. Clause 3. The aqueous liquid pharmaceutical preparation according to Clause 2, wherein the fatty acid is selected from caprylic acid and oleic acid. Clause 4. The aqueous liquid pharmaceutical preparation according to Clause 3, wherein the fatty acid is caprylic acid. Clause 5. The aqueous liquid pharmaceutical preparation according to Clause 3, wherein the fatty acid is oleic acid. Clause 6. An aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 5, wherein the nonionic surfactant is selected from the group consisting of polyoxyalkylenes such as poloxamers, alkyl ethers of polyethylene glycol, alkylphenyl ethers of polyethylene glycol, and fatty acid esters such as polyoxyethylene sorbitan fatty acid esters. Clause 7. The aqueous liquid pharmaceutical preparation according to Clause 6, wherein the nonionic surfactant is selected from the alkyl ether of polyethylene glycol and the polyoxyethylene sorbitan fatty acid ester. Clause 8. The aqueous liquid pharmaceutical preparation according to Clause 7, wherein the nonionic surfactant is an alkyl ether of polyethylene glycol. Clause 9. The aqueous liquid pharmaceutical formulation according to Clause 8, wherein the nonionic surfactant is Brij 35 (polyoxyethylene (23) lauryl ether), Brij 52 (polyoxyethylene (20) cetyl ether), Brij 93 (polyoxyethylene (2) oleyl ether), Brij 97 (polyoxyethylene (10) oleyl ether), Brij L4 (polyoxyethylene (4) lauryl ether), Brij 30 (polyoxyethylene (4) lauryl ether), or Brij 78 (polyoxyethylene (20) stearyl ether). Clause 10. The aqueous liquid pharmaceutical preparation according to Clause 9, wherein the nonionic surfactant is Brij 35 (polyoxyethylene (23) lauryl ether). Clause 11. The aqueous liquid pharmaceutical preparation according to Clause 7, wherein the nonionic surfactant is a polyoxyethylene sorbitan fatty acid ester. Clause 12. The aqueous liquid pharmaceutical preparation according to Clause 11, wherein the nonionic surfactant is polysorbate 80, polysorbate 120, polysorbate 85, polysorbate 65, polysorbate 60, polysorbate 40, or polysorbate 20. Clause 13. The aqueous liquid pharmaceutical preparation according to Clause 12, wherein the nonionic surfactant is polysorbate 80. Clause 14. The surfactant component is (a) a mixture of oleic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (b) a mixture of lauric acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (c) a mixture of linoleic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (d) a mixture of linolenic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (e) a mixture of palmitic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (f) a mixture of stearic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (g) oleic acid or a pharmaceutically acceptable salt thereof and poloxamer An aqueous liquid pharmaceutical preparation according to Clause 1, selected from the group consisting of (h) a mixture of polyoxyalkylenes such as (i) oleic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol, (j) a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and an alkylphenyl ether of polyethylene glycol, (k) a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, (l) a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and a polyoxyalkylene such as poloxamer, and (m) a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol. Clause 15. The aqueous liquid pharmaceutical preparation according to Clause 14, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester. Clause 16. The aqueous liquid pharmaceutical preparation according to Clause 15, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester selected from polysorbate 80 and polysorbate 20. Clause 17. The aqueous liquid pharmaceutical preparation according to Clause 16, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and polysorbate 80. Clause 18. The aqueous liquid pharmaceutical preparation according to Clause 14, wherein the surfactant component is a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester. Clause 19. The aqueous liquid pharmaceutical preparation according to Clause 18, wherein the surfactant component is a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester selected from polysorbate 80 and polysorbate 20. Clause 20. The aqueous liquid pharmaceutical preparation according to Clause 19, wherein the surfactant component is a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and polysorbate 80. Clause 21. The aqueous liquid pharmaceutical preparation according to Clause 14, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol. Clause 22. The aqueous liquid pharmaceutical formulation according to Clause 21, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol selected from Brij 35 (polyoxyethylene (23) lauryl ether), Brij 52 (polyoxyethylene (20) cetyl ether), Brij 93 (polyoxyethylene (2) oleyl ether), Brij 97 (polyoxyethylene (10) oleyl ether), Brij L4 (polyoxyethylene (4) lauryl ether), Brij 30 (polyoxyethylene (4) lauryl ether), or Brij 78 (polyoxyethylene (20) stearyl ether). Clause 23. The aqueous liquid pharmaceutical preparation according to Clause 22, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and Brij 35 (polyoxyethylene (23) lauryl ether). Clause 24. The aqueous liquid pharmaceutical preparation according to Clause 14, wherein the surfactant component is a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol. Clause 25. The aqueous liquid pharmaceutical formulation according to Clause 24, wherein the surfactant component is a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol selected from Brij35 (polyoxyethylene (23) lauryl ether), Brij52 (polyoxyethylene (20) cetyl ether), Brij93 (polyoxyethylene (2) oleyl ether), Brij97 (polyoxyethylene (10) oleyl ether), BrijL4 (polyoxyethylene (4) lauryl ether), Brij30 (polyoxyethylene (4) lauryl ether), or Brij78 (polyoxyethylene (20) stearyl ether). Clause 26. The aqueous liquid pharmaceutical preparation according to Clause 25, wherein the surfactant component is a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and Brij 35 (polyoxyethylene (23) lauryl ether). Clause 27. An aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 26, wherein the fatty acid is in the form of a free acid. Clause 28. An aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 27, wherein the concentration of the surfactant component in the preparation is 0.2 to 30,000 μg / mL, for example, 1 to 30,000 μg / mL, for example, 1 to 20,000 μg / mL, 5 to 20,000 μg / mL, 5 to 15,000 μg / mL, 5 to 10,000 μg / mL, or 5 to 5,000 μg / mL. Clause 29. The aqueous liquid pharmaceutical preparation according to Clause 28, wherein the concentration of the surfactant component in the preparation is 1 to 3000 μg / mL, for example, 1 to 2000 μg / mL, 5 to 2000 μg / mL, 5 to 1500 μg / mL, 5 to 1000 μg / mL, or 5 to 500 μg / mL. Clause 30. The aqueous liquid pharmaceutical preparation according to Clause 29, wherein the concentration of the surfactant component in the preparation is 50 to 200 μg / mL, for example, 75 to 150 μg / mL, for example, 90 to 120 μg / mL, or about 100 μg / mL. Clause 31. The aqueous liquid pharmaceutical preparation according to Clause 28, wherein the concentration of the surfactant component in the preparation is 500 to 2000 μg / mL, for example, 750 to 1500 μg / mL, for example, 900 to 1200 μg / mL, or about 1000 μg / mL. Clause 32. The fatty acid is present in the preparation at a concentration of 10-100 ug / mL, for example, 20-80 μg / mL, for example, 25-75 μg / mL, for example, 40-60 μg / mL, or about 50 μg / mL; and The aqueous liquid pharmaceutical preparation according to Clause 28, wherein the nonionic surfactant is present in the preparation at a concentration of 10 to 100 ug / mL, for example, 20 to 80 μg / mL, for example, 25 to 75 μg / mL, for example, 30 to 60 μg / mL, for example, 40 to 50 μg / mL. Clause 33. The fatty acid is present in the formulation at a concentration of 100-1000 ug / mL, for example, 200-800 μg / mL, for example, 250-750 μg / mL, for example, 400-600 μg / mL, or about 500 μg / mL; and The aqueous liquid pharmaceutical preparation according to Clause 28, wherein the nonionic surfactant is present in the preparation at a concentration of 100 to 1000 ug / mL, for example, 200 to 800 μg / mL, for example, 250 to 750 μg / mL, for example, 300 to 600 μg / mL, for example, 400 to 500 μg / mL. Clause 34. An aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 33, wherein the ratio of the amount of fatty acid or a pharmaceutically acceptable salt thereof, each measured in μg / mL, to the amount of nonionic surfactant is about 5:1 to about 1:5, about 5:1 to about 1:2, about 4:1 to about 1:2, or about 2:1 to about 1:2. Clause 35. The aqueous liquid pharmaceutical formulation according to Clause 34, wherein the ratio of the amount of fatty acid or a pharmaceutically acceptable salt thereof, each measured in μg / mL, to the amount of nonionic surfactant is about 3:2 to about 2:3, for example, about 6:5 to about 1:1, for example, about 10:9, or about 11:10. Clause 36. An aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 35, wherein the ratio of the amount of surfactant component to polynucleotide molecules, each measured in μg / mL, is approximately 100:1 to approximately 1:1000, approximately 100:1 to approximately 1:750, approximately 75:1 to approximately 1:750, approximately 50:1 to approximately 1:750, approximately 50:1 to approximately 1:500, approximately 50:1 to approximately 1:250, approximately 50:1 to approximately 1:200, approximately 40:1 to approximately 1:200, approximately 40:1 to approximately 1:150, approximately 25:1 to approximately 1:150, for example, approximately 25:1 to approximately 1:100. Clause 37. An aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 36, wherein the polynucleotide molecule is a single-stranded polynucleotide molecule. Clause 38. An aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 36, wherein the polynucleotide molecule is a double-stranded polynucleotide molecule. Clause 39. An aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 38, wherein the polynucleotide molecule includes an intramolecular structure. Clause 40. An aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 39, wherein the polynucleotide molecule is a ribonucleic acid (RNA) molecule. Clause 41. The aqueous liquid pharmaceutical preparation according to Clause 40, wherein the RNA molecule is an mRNA, miRNA, shRNA, or siRNA molecule. Clause 42. The aqueous liquid pharmaceutical preparation according to Clause 41, wherein the RNA molecule is an mRNA molecule. Clause 43. The aqueous liquid pharmaceutical formulation according to Clause 42, wherein the mRNA molecule comprises about 100 to about 10,000 nucleotides, for example, about 200 to about 8,000 nucleotides, for example, about 500 to about 7,500 nucleotides, for example, about 1,000 to about 5,000 nucleotides. Clause 44. The aqueous liquid pharmaceutical preparation according to Clause 41, wherein the RNA molecule is a miRNA molecule. Clause 45. The aqueous liquid pharmaceutical preparation according to Clause 44, wherein the miRNA molecule contains approximately 20 to approximately 25 nucleotides. Clause 46. The aqueous liquid pharmaceutical preparation according to Clause 45, wherein the miRNA molecule comprises 20, 21, 22, 23, 24, or 25 nucleotides. Clause 47. The aqueous liquid pharmaceutical preparation according to Clause 41, wherein the RNA molecule is an shRNA molecule. Clause 48. The aqueous liquid pharmaceutical preparation according to Clause 47, wherein the shRNA molecule comprises about 10 to about 70 nucleotides, for example, about 20 to about 70 nucleotides, for example, about 35 to about 70 nucleotides, or about 25 to about 35 nucleotides. Clause 49. The aqueous liquid pharmaceutical preparation according to Clause 48, wherein the shRNA molecule comprises 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides. Clause 50. An aqueous liquid pharmaceutical preparation according to any one of Clauses 47 to 49, wherein the shRNA molecule includes a double-stranded region containing 30 or fewer base pairs, for example, 25 or fewer base pairs, for example, 20 or fewer base pairs, for example, 18 or fewer base pairs, for example, 16 or fewer base pairs, for example, 14 or fewer base pairs, for example, 12 or fewer base pairs, for example, 10 or fewer base pairs, for example, 8 or fewer base pairs, for example, 6 or fewer base pairs, for example, 4 or fewer base pairs. Clause 51. An aqueous liquid pharmaceutical preparation according to any one of Clauses 47 to 50, wherein the shRNA molecule comprises a double-stranded region containing one or more mispaired bases. Clause 52. An aqueous liquid pharmaceutical preparation according to any one of Clauses 47 to 51, wherein the shRNA molecule contains a blunt end. Clause 53. The aqueous liquid pharmaceutical preparation according to any one of Clauses 47 to 52, wherein the shRNA molecule comprises at least one 5' triphosphate or at least one 5' diphosphate, in particular, wherein the 5' triphosphate or 5' diphosphate is located at the 5' terminus. Clause 54. An aqueous liquid pharmaceutical preparation according to any one of Clauses 47 to 53, wherein the shRNA molecule comprises (i) a blunt end, (ii) a 5' triphosphate or 5' diphosphate moiety located at the 5' end, and (iii) a double-stranded region approximately 10 to approximately 18 nucleotides in length. Clause 55. An aqueous liquid pharmaceutical preparation according to any one of Clauses 47 to 54, wherein the shRNA molecule comprises or consists of SEQ ID NO: 1 or a variant of SEQ ID NO: 1. Clause 56. An aqueous liquid pharmaceutical preparation according to any one of Clauses 47 to 54, wherein the shRNA molecule includes, or comprises, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or a variant of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10. Clause 57. An aqueous liquid formulation according to any one of Clauses 54 to 56, wherein the shRNA molecule can induce an interferon response in vertebrate cells. Clause 58. The aqueous liquid pharmaceutical preparation according to Clause 41, wherein the RNA molecule is an siRNA molecule. Clause 59. The aqueous liquid pharmaceutical formulation according to Clause 58, wherein the siRNA molecule comprises about 20 to about 60 nucleotides, for example, about 30 to about 60 nucleotides, for example, about 40 to about 50 nucleotides. Clause 60. The aqueous liquid pharmaceutical formulation according to Clause 59, wherein the siRNA molecule comprises 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 nucleotides. Clause 61. An aqueous liquid pharmaceutical preparation according to any one of Clauses 58 to 60, wherein the siRNA molecule includes a double-stranded region having 30 or fewer base pairs, for example, 25 or fewer base pairs, for example, 20 or fewer base pairs. Clause 62. An aqueous liquid pharmaceutical preparation according to any one of Clauses 58 to 61, wherein the siRNA molecule comprises a double-stranded region containing one or more mispairing bases. Clause 63. An aqueous liquid pharmaceutical formulation according to any one of Clauses 58 to 62, wherein the siRNA molecule comprises one or more, for example, two 3' projections. Clause 64. An aqueous liquid pharmaceutical formulation according to any one of Clauses 58 to 63, wherein the siRNA molecule comprises at least one, for example, two 5' monophosphates, or at least one, for example, two 5' diphosphates, or at least one, for example, two 5' triphosphates. Clause 65. An aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 39, wherein the polynucleotide molecule is a deoxyribonucleic acid (DNA) molecule. Clause 66. The aqueous liquid pharmaceutical formulation according to Clause 65, wherein the DNA molecule is a genomic DNA (gDNA) molecule, a complementary DNA (cDNA) molecule, or an extrachromosomal DNA molecule, such as a plasmid DNA molecule. Clause 67. The aqueous liquid pharmaceutical preparation according to Clause 66, wherein the DNA molecule is a gDNA molecule. Clause 68. The aqueous liquid pharmaceutical preparation according to Clause 66, wherein the DNA molecule is a cDNA molecule. Clause 69. The aqueous liquid pharmaceutical formulation according to Clause 66, wherein the DNA molecule is an extrachromosomal DNA molecule, for example, a plasmid DNA molecule. Clause 70. An aqueous liquid pharmaceutical preparation according to any one of Clauses 65 to 69, wherein the DNA molecule comprises about 100 to about 20,000 nucleotides, for example, about 100 to about 15,000 nucleotides, for example, about 500 to about 15,000 nucleotides, for example, about 500 to about 10,000 nucleotides, for example, about 2,500 to about 10,000 nucleotides, for example, about 2,500 to about 8,000 nucleotides. Clause 71. The aqueous liquid pharmaceutical preparation according to Clause 1, wherein the polynucleotide molecule has the structure of formula (I): [ka] (In the formula, 5'-P z -(N) b N-3' represents the first nucleic acid sequence; 5'-N(N) b' -3' represents the second nucleic acid sequence; In each case, P is independently a phosphate or an analogue thereof; z is either 2 or 3; In each case, N is any nucleotide or modified nucleotide or its analogue or derivative; b and b' are independently 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; [ka] This represents a connector element, where, E is, independently, any nucleotide, modified nucleotide, or debase at each instance; y and y' are independently between 0 and 9, where y + y' is equal to between 0 and 8; L is structure [ka] (Here, X and X' are independently O or S; Y and Y' are independently OR'', SR'', or NRR'; V and V' are independently O, S, or NRR'; q is between 1 and 20; k is between 1 and 20; t is between 1 and 20; M is selected from aliphatic, substituted aliphatic, aryl, substituted aryl, heteroalkyl, heterocyclyl, or substituted heterocyclyl; W is any reactive group; and (d is either 0 or 1) (It is a non-nucleotide segment that has [a certain characteristic].) Clause 72. The aqueous liquid pharmaceutical preparation according to Clause 71, wherein the polynucleotide molecule has the structure of formula (II): [ka] (In the formula, 5'-P z -Nu-3' represents the first nucleic acid sequence; 5'-Nu'-3' represents the second nucleic acid sequence; In each case, P is independently a phosphate or an analogue thereof. z is 0, 1, 2, or 3; [ka] This represents a connector element, where, E is, independently, any nucleotide, modified nucleotide, or debase at each instance; y and y' are independently between 0 and 9, where y + y' is equal to between 0 and 8; L is [ka] (Here, X and X' are independently O or S; Y and Y' are independently OR'', SR'', or NRR'; V and V' are independently O, S, or NRR'; q is between 1 and 20; k is between 1 and 20; t is between 1 and 20; M is selected from aliphatic, substituted aliphatic, aryl, substituted aryl, heteroalkyl, heterocyclyl, or substituted heterocyclyl; W is any reactive or conjugation group; and (d is either 0 or 1) (It is a non-nucleotide segment that has [a certain characteristic].) Clause 73. The aqueous liquid pharmaceutical preparation according to Clause 1, wherein the polynucleotide molecule comprises, consists of, or is essentially composed of SEQ ID NO: 13 and SEQ ID NO: 14, wherein the 3' end of SEQ ID NO: 13 is connected to the 5' end of SEQ ID NO: 14 via a non-nucleotide portion L1. Clause 74. The aqueous liquid pharmaceutical preparation according to Clause 1, wherein the polynucleotide molecule comprises, consists of, or is essentially composed of SEQ ID NO: 13 and SEQ ID NO: 14, wherein the 3' end of SEQ ID NO: 13 is connected to the 5' end of SEQ ID NO: 14 via a non-nucleotide portion L2. Clause 75. An aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 74, wherein the polynucleotide molecule comprises one or more sugar-modified nucleotides, each having a 2'OH (or 2'H) modification. Clause 76. The aqueous liquid pharmaceutical formulation according to Clause 75, wherein the 2'OH (or 2'H) modification is selected from the group consisting of 2'-deoxy, 2'-fluoro, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-amino-propyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and 2'-ON-methylacetamide (2'-O-NMA). Clause 77. The aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 76, wherein the polynucleotide molecule comprises one or more skeletal modified nucleotides, wherein the skeletal modification is the substitution of a phosphate ester group with a phosphorothioate group of the nucleotide. Clause 78. An aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 77, wherein the polynucleotide molecule comprises one or more base-modified nucleotides. Clause 79. The aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 78, wherein the aqueous liquid pharmaceutical preparation is in the form of a stable colloidal emulsion. Clause 80. An aqueous liquid pharmaceutical preparation in the form of a stable colloidal emulsion comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) a polynucleotide molecule. Clause 81. The aqueous liquid pharmaceutical formulation according to Clause 79 or Clause 80, wherein the average particle size of the stable colloidal particles is about 10 to about 1000 nm, for example, about 50 to about 1000 nm, for example, about 50 to about 750 nm, for example, about 50 to about 500 nm, for example, about 50 to about 400 nm, for example, about 50 to about 300 nm. Clause 82. The aqueous liquid pharmaceutical preparation according to Clause 81, wherein the average particle size of the stable colloidal particles is approximately 100 to approximately 300 nm. Clause 83. The aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 82, wherein the aqueous liquid pharmaceutical preparation is suitable for parenteral administration, including oral, inhalation, sublingual, oral cavity, intravenous, subcutaneous, topical, transdermal, pulmonary, rectal, vaginal, ocular, nasal cavity, intradermal, transmucosal, intraperitoneal, intramuscular, intra-articular, intraorbital, intracardiac, transtracheal, subepidermal, intra-articular, intraspinal, or intrasternal. Clause 84. The aqueous liquid pharmaceutical preparation according to Clause 83, wherein the aqueous liquid pharmaceutical preparation is suitable for local administration to the lungs or nose. Clause 85. The aqueous liquid pharmaceutical preparation according to Clause 83, wherein the aqueous liquid pharmaceutical preparation is suitable for subcutaneous administration, for example, subcutaneous injection. Clause 86. The aqueous liquid pharmaceutical preparation according to Clause 83, wherein the aqueous liquid pharmaceutical preparation is suitable for ocular administration, for example, intraocular administration or topical administration to the eye. Clause 87. An aqueous liquid pharmaceutical preparation as described in any one of Clauses 1 to 86, for use as a pharmaceutical product. Clause 88. Aqueous liquid pharmaceutical formulations for use as described in Clause 87, for topical administration to the lungs (e.g., by oral inhalation) or topical administration to the nose. Clause 89. Aqueous liquid pharmaceutical formulation for use as described in Clause 87, for subcutaneous administration. Clause 90. Aqueous liquid pharmaceutical formulations for use as described in Clause 87, for ocular administration, e.g., intraocular administration or topical administration to the eye. Clause 91. An aqueous liquid pharmaceutical formulation for use according to any one of Clauses 87 to 90, for use in the treatment of a disease or illness treated by stimulation or activation of the innate and / or adaptive immune system and / or induction of a innate and / or adaptive immune response, wherein the polynucleotide molecule is a polynucleotide molecule that stimulates or activates the innate and / or adaptive immune system and / or induces a innate and / or adaptive immune response. Clause 92. A method for the treatment of a disease or illness treated by stimulation or activation of the innate and / or adaptive immune system and / or induction of a innate and / or adaptive immune response, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an aqueous liquid pharmaceutical preparation described in any one of Clauses 1 to 86, wherein the polynucleotide molecule is a polynucleotide molecule that stimulates or activates the innate and / or adaptive immune system and / or induces a innate and / or adaptive immune response. Clause 93. Use of an aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 86, in the manufacture of a pharmaceutical preparation for use in the treatment of a disease or illness treated by stimulation or activation of the natural and / or adaptive immune system and / or induction of a natural and / or adaptive immune response, wherein the polynucleotide molecule is a polynucleotide molecule that stimulates or activates the natural and / or adaptive immune system and / or induces a natural and / or adaptive immune response. Clause 94. An aqueous liquid pharmaceutical preparation, method, or use described in any one of Clauses 91 to 93, wherein the disease or illness is caused by or related to a viral infection. Clause 95. An aqueous liquid pharmaceutical formulation, method, or use for use as described in Clause 94, wherein the virus infects the respiratory system and the disease associated with the infection is a disease of the respiratory system. Article 96. Whether the virus is SARS-CoV-2 and the disease associated with the viral infection is COVID-19; or The virus is a seasonal coronavirus, e.g., 229E, NL63, OC43, or HKU1, and the disease associated with the viral infection is a disease associated with seasonal coronavirus infection, e.g., 229E, NL63, OC43, or HKU1; or The virus is an influenza virus, and the disease associated with the viral infection is influenza; or The virus is respiratory syncytial virus (RSV), and the disease associated with the viral infection is a disease associated with RSV infection; or The virus is human rhinovirus (HRV), and the disease associated with the viral infection is a disease associated with HRV infection; or The virus is Middle East Respiratory Syndrome (MERS)-CoV, and the disease associated with the viral infection is MERS; or The virus is an avian influenza virus, and the disease associated with the viral infection is avian influenza; or The virus is Nipah virus, and the disease associated with the viral infection is a disease associated with Nipah virus infection; or The virus is human parainfluenza virus (HPIV), and the disease associated with the viral infection is a disease associated with HPIV infection; or The aqueous liquid pharmaceutical formulation, method, or use for use described in Clause 95, wherein the virus is human metapneumovirus (hMPV) and the disease associated with the viral infection is a disease associated with hMPV infection. Clause 97. An aqueous liquid pharmaceutical preparation for use according to any one of Clauses 87 to 90, wherein the polynucleotide molecule is a polynucleotide molecule that increases the endogenous expression of a protein or other gene product or increases the expression of a functional protein or other gene product, for use in the treatment of a disease or illness that is treated by increasing the endogenous expression of a protein or other gene product or by increasing the expression of a functional protein or other gene product. Clause 98. A method for treating a disease or illness treated by increasing the endogenous expression of a protein or other gene product or by increasing the expression of a functional protein or other gene product, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the aqueous liquid pharmaceutical preparation described in any one of Clauses 1 to 86, wherein the polynucleotide molecule is a polynucleotide molecule that increases the endogenous expression of a protein or other gene product or a functional protein or other gene product. Clause 99. Use of an aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 86, in the manufacture of a pharmaceutical preparation for use in the treatment of a disease or illness treated by increasing the endogenous expression of a protein or other gene product or by increasing the expression of a functional protein or other gene product, wherein the polynucleotide molecule is a polynucleotide molecule that increases the endogenous expression of a protein or other gene product or a functional protein or other gene product. Clause 100. An aqueous liquid pharmaceutical formulation, method, or use for use according to any one of Clauses 97 to 99, wherein the disease or illness is a respiratory disease or illness, such as asthma, alpha-1 antitrypsin deficiency, chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), pulmonary fibrosis, sarcoidosis, or cystic fibrosis. Clause 101. An aqueous liquid pharmaceutical formulation for use in any one of Clauses 87 to 90, for use in a method of gene therapy in which the polynucleotide molecule, in particular the mRNA or DNA molecule, encodes a therapeutic gene, protein, or other gene product. Clause 102. An aqueous liquid pharmaceutical formulation for use as described in Clause 101, for use in the treatment of a disease or illness that is treated by increasing the endogenous expression of a therapeutic gene, protein, or other gene product encoded by the polynucleotide molecule. Clause 103. An aqueous liquid pharmaceutical preparation for use according to any one of Clauses 87 to 90, wherein the polynucleotide molecule is a polynucleotide molecule that reduces the endogenous expression of a protein or other gene product, for use in the treatment of a disease or illness that is treated by reducing the endogenous expression of a protein or other gene product. Clause 104. A method for treating a disease or illness that is treated by reducing the endogenous expression of the protein or other gene product, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the aqueous liquid pharmaceutical preparation described in any one of Clauses 1 to 86, wherein the polynucleotide molecule is a polynucleotide molecule that reduces the endogenous expression of the protein or other gene product. Clause 105. Use of an aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 86, in the manufacture of a pharmaceutical preparation for use in the treatment of a disease or illness that is treated by reducing the endogenous expression of the protein or other gene product, wherein the polynucleotide molecule is a polynucleotide molecule that reduces the endogenous expression of the protein or other gene product. Clause 106. An aqueous liquid pharmaceutical preparation for use according to any one of Clauses 87 to 90, wherein the polynucleotide molecule is a polynucleotide molecule that modifies an endogenous nucleic acid sequence, for use in the treatment of a disease or illness treated by modification of an endogenous nucleic acid sequence. Clause 107. A method for treating a disease or illness treated by modifying an endogenous nucleic acid sequence, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of an aqueous liquid pharmaceutical preparation described in any one of Clauses 1 to 86, wherein the polynucleotide molecule is a polynucleotide molecule that modifies an endogenous nucleic acid sequence. Clause 108. Use of an aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 86, in the manufacture of a pharmaceutical preparation for use in the treatment of a disease or illness treated by modification of an endogenous nucleic acid sequence, wherein the polynucleotide molecule is a polynucleotide molecule that modifies an endogenous nucleic acid sequence. Clause 109. An immunostimulatory composition, which is an aqueous liquid formulation, comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) an immunostimulatory polynucleotide molecule. Clause 110. The immunostimulatory composition according to Clause 109, wherein the immunostimulatory polynucleotide molecule is an shRNA molecule. Clause 111. An immunostimulatory composition according to Clause 109 or Clause 110, for use in stimulating or activating the immune system of a target. Clause 112. An immunostimulatory composition according to Clause 111, for use in stimulating or activating a target antiviral innate and / or adaptive immune response. Clause 113. An immunogenic composition, which is an aqueous liquid formulation, comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) a polynucleotide molecule encoding an immunogen. Clause 114. A vaccine composition that is an aqueous liquid formulation comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) a polynucleotide molecule which encodes a vaccine immunogen. Clause 115. The immunogenic composition or vaccine composition according to Clause 113 or Clause 114, wherein the polynucleotide molecule encoding the immunogen or vaccine immunogen is an mRNA molecule or a DNA molecule. Clause 116. An immunogenic composition or vaccine composition according to any one of Clauses 113 to 115, further comprising an immunostimulatory polynucleotide molecule. Clause 117. The immunogenic composition or vaccine composition according to Clause 116, wherein the immunostimulatory polynucleotide molecule is an shRNA molecule. Clause 118. An immunogenic composition or vaccine composition according to any one of Clauses 113 to 117, wherein the composition does not contain lipid nanoparticles (LNPs) or liposomes, and / or substantially does not contain LNPs and liposome components. Clause 119. An immunogenic composition or vaccine composition according to any one of Clauses 113 to 118, for use in eliciting a target immune response, wherein the immune response is elicited against an immunogen or vaccine immunogen encoded by the polynucleotide molecule. Clause 120. An immunogenic composition or vaccine composition for use as described in Clause 119, for therapeutic use, i.e., for use in inducing an immune response to obtain a therapeutic effect against a disease or illness that is treated by the induction of a natural and / or adaptive immune response. Clause 121. An immunogenic composition or vaccine composition for use as described in Clause 120, wherein the disease or illness is cancer. Clause 122. An immunogenic composition or vaccine composition for use as described in Clause 119, for prophylactic use, i.e., for use in inducing an immune response to obtain a protective effect against a disease or illness treated by the induction of a natural and / or adaptive immune response. Clause 123. An immunogenic composition or vaccine composition for use as described in Clause 122, wherein the disease or illness is an infectious disease. Clause 124. The aqueous liquid pharmaceutical preparation described in any one of Clauses 1 to 86, wherein the aqueous liquid pharmaceutical preparation does not contain protein. Clause 125. The aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 86, wherein the aqueous liquid pharmaceutical preparation does not contain a cationic lipopeptide, for example, the aqueous liquid pharmaceutical preparation does not contain polymyxin B. Clause 126. The aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 86, wherein the aqueous liquid pharmaceutical preparation does not contain inorganic nanoparticles. Clause 127. The aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 86, wherein the aqueous liquid pharmaceutical preparation does not contain lipid nanoparticles (LNPs) or liposomes. Clause 128. The aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 86, wherein the aqueous liquid pharmaceutical preparation does not contain neutral lipids, for example, the aqueous liquid pharmaceutical preparation does not contain cholesterol or its analogues. Clause 129. The aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 86, wherein the aqueous liquid pharmaceutical preparation does not contain cationic lipids. Clause 130. The aqueous liquid pharmaceutical preparation according to any one of Clauses 1 to 86, wherein the aqueous liquid pharmaceutical preparation does not contain helper lipids, for example, dioleoylphosphatidylethanolamine (DOPE) or phosphatidylcholine. Clause 131. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) one or more buffers; and (iv) a diluent. Clause 132. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) one or more buffers selected from citrates, acetates, lactates, or formates and phosphates; and (iv) a diluent selected from isotonic saline (0.9% w / v), isotonic dextrose (5% w / v), an isotonic mixture of physiological saline and dextrose (e.g., physiological saline (0.45% w / v) and dextrose (2.5% w / v)), sterile water or purified water, sterile water for injection, or bacteriostatic water for injection. Clause 133. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) one or more buffers selected from citrates and phosphates; and (iv) a diluent selected from isotonic saline (0.9% w / v) and sterile water or purified water. Clause 134. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) a citrate; and (iv) an isotonic saline solution (0.9% w / v). Clause 135. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) a citrate; and (iv) sterile water or purified water. Clause 136. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) a phosphate; and (iv) an isotonic saline solution (0.9% w / v). Clause 137. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) a phosphate; and (iv) sterile water or purified water. Clause 138. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) one or more buffering agents; (iv) a diluent; (v) optionally one or more suspending agents; (vi) optionally one or more wetting agents or thickeners; and (vii) optionally one or more osmotic pressure or tonicity modifiers. Clause 139. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) one or more buffers selected from citrates and phosphates; (iv) a diluent selected from isotonic saline (0.9% w / v) and sterile water or purified water; (v) optionally one or more suspending agents; (vi) optionally one or more wetting agents or thickeners; and (vii) optionally one or more osmotic pressure or tonicity modifiers. Clause 140: An aqueous liquid pharmaceutical preparation according to any one of Clauses 131 to 139, in the form of a stable colloidal emulsion. [Examples]

[0281] (Examples) The abbreviations used herein are defined below (see Table 1). Any abbreviations not defined are intended to convey their generally accepted meaning. Table 1: Abbreviations [Table 2]

[0282] The molecular structures used in this specification are shown below (see Table 2). Table 2: Structure of polynucleotide molecules [Table 3]

[0283] (material) All starting materials and solvents were obtained from commercially available sources.

[0284] (Examples of formulations) (Formulation Example 1A: Example of an aqueous liquid pharmaceutical formulation containing shRNA 1)

[0285] Aqueous liquid pharmaceutical formulations with a pH of approximately 4.7 or lower can be prepared, for example, by following the protocol generally described in Biophysical Example 1: [Table 4] This formulation can be administered intranasally via a nasal spray device having a spray volume of 100 μL, suitable for one or two sprays per nostril. This formulation may be suitable for the treatment or prevention of viral infections or diseases associated with such viral infections.

[0286] (Formulation Example 1B: Example of an aqueous liquid pharmaceutical formulation containing dsRNA 1) Aqueous liquid pharmaceutical formulations with a pH of approximately 4.7 or lower can be prepared, for example, by following the protocol generally described in Biophysical Example 1: [Table 5]

[0287] This formulation can be administered intranasally via a nasal spray device having a spray volume of 100 μL, suitable for one or two sprays per nostril. This formulation may be suitable for the treatment or prevention of viral infections or diseases associated with such viral infections.

[0288] (Formulation Example 1C: Example of an aqueous liquid pharmaceutical formulation containing dsRNA-2) Aqueous liquid pharmaceutical formulations with a pH of approximately 4.7 or lower can be prepared, for example, by following the protocol generally described in Biophysical Example 1: [Table 6]

[0289] This formulation can be administered intranasally via a nasal spray device having a spray volume of 100 μL, suitable for one or two sprays per nostril. This formulation may be suitable for the treatment or prevention of viral infections or diseases associated with such viral infections.

[0290] (Formulation Example 1D: Example of an aqueous liquid pharmaceutical formulation containing shRNA 1) Aqueous liquid pharmaceutical formulations with a pH of approximately 7.2 or lower can be prepared, for example, by following the protocol generally described in Biophysical Example 1: [Table 7]

[0291] This formulation can be administered by inhalation, or by mouth to the lungs, for example, via an air jet nebulizer. This formulation may be suitable for the treatment or prevention of viral infections or diseases associated with such viral infections.

[0292] (Formulation Example 1E: Example of an aqueous liquid pharmaceutical formulation containing shRNA 1) Aqueous liquid pharmaceutical formulations with a pH of approximately 4.7 or lower can be prepared, for example, by generally following the protocols described in Biophysical Examples 1 and 2: [Table 8]

[0293] This formulation can be administered by inhalation, orally, or to the lungs, for example, via an air jet nebulizer. This formulation may be suitable for the treatment or prevention of viral infections or diseases associated with such viral infections.

[0294] (Formulation Example 2: Example of an aqueous liquid pharmaceutical formulation containing shRNA 1) Aqueous liquid pharmaceutical formulations with a pH of approximately 7.2 or lower can be prepared, for example, by generally following the protocols described in Biophysical Examples 1 and 2: [Table 9]

[0295] This formulation can be administered subcutaneously, for example, by subcutaneous injection. The formulation may be suitable for the treatment or prevention of viral infections or diseases associated with such viral infections.

[0296] (Formulation Example 3A: Example of an aqueous liquid pharmaceutical formulation containing GFP-encoding mRNA) Aqueous liquid pharmaceutical formulations with a pH of approximately 4.7 or lower can be prepared, for example, by generally following the protocols described in Biophysical Examples 1 and 2: [Table 10]

[0297] This formulation can be administered intranasally, for example, via a nasal spray device having a spray capacity of 100 μL suitable for one or two sprays per nostril. Alternatively, this formulation can be administered into the eyes.

[0298] (Formulation Example 3B: Example of an aqueous liquid pharmaceutical formulation containing a pSV-β-galactosidase control vector (DNA)) Aqueous liquid pharmaceutical formulations with a pH of approximately 4.7 or lower can be prepared, for example, by generally following the protocols described in Biophysical Examples 1 and 2: [Table 11]

[0299] This formulation can be administered into the nasal cavity via a nasal spray device having a spray capacity of 100 μL, suitable for one or two sprays per nostril.

[0300] (Formulation Example 3C: Example of an aqueous liquid pharmaceutical formulation containing H1N1 hemagglutinin (HA) encoding mRNA) Aqueous liquid pharmaceutical formulations with a pH of approximately 7.2 or lower can be prepared, for example, by generally following the protocols described in Biophysical Examples 1 and 2: [Table 12]

[0301] This formulation can be administered into the nasal cavity via a nasal spray device having a spray capacity of 100 μL, suitable for one or two sprays per nostril.

[0302] (Formulation Example 3D: Example of an aqueous liquid pharmaceutical formulation containing cystic fibrosis transmembrane conductance regulator (CFTR) encoding mRNA) Aqueous liquid pharmaceutical formulations with a pH of approximately 7.2 or lower can be prepared, for example, by generally following the protocols described in Biophysical Examples 1 and 2: [Table 13]

[0303] This formulation can be administered intranasally via a nasal spray device having a spray volume of 100 μL, suitable for, for example, one or two sprays per nostril. This formulation may be suitable for the treatment or prevention of cystic fibrosis.

[0304] (Formulation Example 3E: Example of an aqueous liquid pharmaceutical formulation containing Silencer(trademark) Cy(trademark)3-labeled negative control No. 1 siRNA (Cy3-labeled siRNA)) Aqueous liquid pharmaceutical formulations with a pH of approximately 7.2 or lower can be prepared, for example, by generally following the protocols described in Biophysical Examples 1 and 2: [Table 14]

[0305] This preparation can be administered to the eyes.

[0306] (Formulation Example 3F: Example of an aqueous liquid pharmaceutical formulation containing GFP-encoding mRNA) Aqueous liquid pharmaceutical formulations with a pH of approximately 7.2 or lower can be prepared, for example, by generally following the protocols described in Biophysical Examples 1 and 2: [Table 15]

[0307] This formulation can be administered intranasally, for example, via a nasal spray device having a spray capacity of 100 μL suitable for one or two sprays per nostril. Alternatively, this formulation can be administered into the eyes.

[0308] (Formulation Example 4A: Example of an aqueous liquid pharmaceutical formulation containing RNA conjugate 1) Aqueous liquid pharmaceutical formulations with a pH of approximately 4.7 or lower can be prepared, for example, by generally following the protocols described in Biophysical Examples 1 and 2: [Table 16]

[0309] This formulation can be administered intranasally via a nasal spray device having a spray volume of 100 μL, suitable for one or two sprays per nostril. This formulation may be suitable for the treatment or prevention of viral infections or diseases associated with such viral infections.

[0310] (Formulation Example 4B: Example of an aqueous liquid pharmaceutical formulation containing RNA conjugate 2) Aqueous liquid pharmaceutical formulations with a pH of approximately 4.7 or lower can be prepared, for example, by generally following the protocols described in Biophysical Examples 1 and 2: [Table 17]

[0311] This formulation can be administered intranasally via a nasal spray device having a spray volume of 100 μL, suitable for one or two sprays per nostril. This formulation may be suitable for the treatment or prevention of viral infections or diseases associated with such viral infections.

[0312] (Biological examples) (Biological Example 1: Evaluation of shRNA 1-mediated CXCL10 production in ALI-cultured human nasal epithelium) (Experimental method) Primary human nasal, tracheal, bronchial, or small airway epithelial cells can be cultured and differentiated at the gas-liquid interface (ALI) to form pseudostratified mucovicillary airway epithelium, composed of ciliated cells, goblet cells, club cells, and / or basal cells with an arrangement that closely reflects the in vivo cellular composition. This in vitro model of human airway epithelium (HAE) cultured at ALI (HAE-ALI) closely replicates key features of infected and / or immunostimulated upper and lower airways in vivo. Therefore, HAE-ALI cultures are used, for example, to study respiratory virus-host cell interactions and immune responses in the context of influenza virus infection.

[0313] ALI cultured pooled donor human nasal epithelium (provided by Epithelix Sarl (Geneva, Switzerland)) was maintained at the gas-liquid interface using MucilAir® culture medium in a Costar Transwell insert (Corning, NY, USA), according to the manufacturer's instructions.

[0314] As shown in Figure 1, ALI cultures were administered via apical injection over 30 minutes using either 50 μL of vehicle control (water only), shRNA 1 in the vehicle (2 mg / mL), or shRNA 1 in the vehicle (2 mg / mL) along with surfactant component 1 (0.005% (w / w) oleic acid and 0.0045% (w / w) polysorbate 80), surfactant component 2 (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or surfactant component 3 (0.2% (w / w) oleic acid and 0.15% (w / w) polysorbate 80). (The mixture was then removed using a pipette.) Samples were collected from the base chamber on days 1, 2, and 3 after treatment (stored at -80°C). CXCL10 concentration was quantified by an enzyme-linked immunosorbent assay using the MSD multiplex platform (U-PLEX Human IP-10 Assay, Catalog #K151UFK-2; n=3, mean ± SEM).

[0315] The processed formulations used in this example were produced largely as described in Biophysical Example 1.

[0316] (result) As shown in Figure 1, the level of CXCL10, a marker of antiviral IFN production, was determined in basal chamber samples. shRNA 1 alone (in water) strongly induced CXCL10 expression over 3 days post-treatment compared to a water control that did not induce CXCL10 expression. Surfactant component 1, containing shRNA 1 and the lowest surfactant concentration, did not enhance the induction of CXCL10 expression compared to shRNA 1 alone. Conversely, shRNA 1, surfactant component 2, and surfactant component 3 induced significantly higher levels of CXCL10 expression compared to shRNA 1 in water. In particular, surfactant component 3 (containing shRNA 1), containing the highest surfactant concentration, only slightly increased CXCL10 induction compared to surfactant component 2 (containing shRNA 1).

[0317] (Biological Example 2: Evaluation of CXCL10 production and viral load in influenza virus-infected ALI cultured human epithelial cells treated with shRNA 1) (Experimental method) ALI cultured pooled donor human nasal epithelium (provided by Epithelix Sarl (Geneva, Switzerland)) was maintained at the gas-liquid interface using MucilAir® culture medium in a Costar Transwell insert (Corning, NY, USA) according to the manufacturer's instructions. On day 0, influenza virus inoculum (PR8 strain; 100 μL; diluted in MucilAir® culture medium to obtain a final MOI of 0.1) was added to the apical surface of the epithelium for 1 hour (34°C / 5% CO2). Subsequently, the virus inoculum was removed and the insert was placed in sterile PBS (Ca 2+ / Mg 2+ It was washed (including)

[0318] The following processed formulations were produced in general as described in Biophysical Example 1.

[0319] One day before viral inoculation, 50 μL of vehicle control (water only), shRNA 1 in vehicle (2 mg / mL), or shRNA 1 in vehicle (2 mg / mL) and surfactant component 1 (0.005% (w / w) oleic acid and 0.0045% (w / w) polysorbate 80) or surfactant component 2 (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) were administered apex-to-apex for 30 minutes (then removed using a pipette). Then, 30 minutes before viral inoculation, these treatments were reapplied (then removed using a pipette), and then incubated with the viral inoculation material for 60 minutes. Simultaneously, oseltamivir carboxylate (10 μM) was added to the bottom chamber of the sample wells the day before viral inoculation and on day 0. Sampling was performed on days 0 and 2 by adding 300 μL of culture medium to the apical surface of each well for 10 minutes (stored at -80°C). On day 0 (day 1 after treatment), the CXCL10 concentration before viral infection was quantified by the method described in Biological Example 1 (n=3, mean ± SEM).

[0320] Two days after virus inoculation (third day after treatment), the viral load was measured to 50% of the tissue culture infectious dose (TCID). 50 Quantitative analysis was performed using TCID. Briefly, the apical wash was thawed, and serial dilutions of the apical wash sample in a medium containing 0.1 μg / ml TPCK trypsin were applied to plates covered with MDCK cells (80% confluence). These were then incubated at 35°C and 5% CO2 for 2-3 days, particularly until CPE induced by the vehicle control became visible. 50 This was calculated using the Reid-Münch equation (Bullen et al., 2022) (n=3, mean ± range).

[0321] Specifically, the first dilution with CPE-positive % ≥ 50 ("high dilution") and the first dilution with CPE-positive % < 50 ("low dilution") were identified, and the proportional distance (PD) was calculated using the following formula: PD = (Percentage of CPE-positive wells at high dilution - 50%) / (Percentage of CPE-positive wells at high dilution - Percentage of CPE-positive wells at low dilution) Log TCID 50 =Log dilution large+PD

[0322] (result) As shown in Figure 2, shRNA 1 alone did not induce the expression of CXCL10, a marker of antiviral IFN production. However, surfactant component 1, containing shRNA 1 and the lowest surfactant concentration, enhanced CXCL10 induction compared to the vehicle control (water) or shRNA 1 alone. Surfactant component 2, containing shRNA 1 and a relatively high surfactant concentration, induced a further significant increase in CXCL10 expression compared to shRNA 1 alone and both shRNA 1 and surfactant component 1. In particular, oseltamivir control (10 μM) did not induce CXCL10 expression because it is a direct antiviral agent and does not stimulate the innate immune system.

[0323] As shown in Figure 3, high levels of influenza replication were detected in apical lavage after treatment with vehicle controls 48 hours after inoculation (mean: 3.5 Log, TCID). 50 ( / mL). When comparing the detected viral load after each treatment with that after treatment with the vehicle control (water), only shRNA 1 did not show a significant antiviral effect, and similarly, neither shRNA 1 nor surfactant component 1, which contained the lowest surfactant concentration, significantly reduced the viral load. However, as indicated by a 0.7 Log reduction in viral load, shRNA and surfactant component 2 had a significant antiviral effect. The assay control oseltamivir (10 μM) showed a similar antiviral effect (0.7 Log reduction), as predicted (Boda et al., 2018).

[0324] (Biological Example 3: Evaluation of CXCL10 production and viral load in influenza virus-infected ALI cultured human epithelial cells treated with shRNA 1 or surfactant alone) (Experimental method) ALI-cultured human nasal epithelium was maintained as described in Biological Example 2 and inoculated with influenza virus (PR8 strain).

[0325] One day before viral inoculation, 50 μL of vehicle control (water only), surfactant components from the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), shRNA 1 from the vehicle, or shRNA 1 from the vehicle and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) were administered apex-to-apex for 30 minutes (then removed using a pipette). Then, 30 minutes before viral inoculation, these treatments were reapplied (then removed using a pipette), and then incubated with the viral inoculation material for 60 minutes. Simultaneously, oseltamivir carboxylate (10 μM) was added to the bottom chamber of the sample wells the day before viral inoculation and on day 0. On the second day (48 hours after inoculation), sampling was performed by adding 300 μL of culture medium to the apical surface of each well for 10 minutes (stored at -80°C).

[0326] The viral load on day 2 post-infection (day 3 post-treatment) (n=3, mean ± range) and the CXCL10 level on day 0 pre-infection (day 1 post-treatment) (n=3, mean ± SEM) were calculated as described in Biological Example 2 above.

[0327] The processed formulations used in this example were produced largely as described in Biophysical Example 1.

[0328] (result) As shown in Figure 4, the vehicle control (water), shRNA 1 alone, and especially the surfactant component alone did not induce CXCL10 expression on day 1 after treatment. Conversely, shRNA 1 and the surfactant component significantly induced CXCL10 expression. As previously discussed, the oseltamivir control (10 μM), being a direct antiviral agent, did not induce CXCL10 expression.

[0329] As shown in Figure 5, high levels of influenza replication were detected in apical lavage after treatment with vehicle controls 48 hours after inoculation (mean: approximately 3.4 Log, TCID). 50 ( / mL). Neither shRNA 1 alone nor, in particular, the surfactant component alone reduced the viral load compared to the vehicle control. However, both shRNA 1 and the surfactant component had very potent antiviral effects, as represented by a 3.2 Log reduction in viral load compared to water alone (vehicle control). Importantly, both shRNA 1 and the surfactant component induced a more significant reduction in viral load than the assay control oseltamivir (10 μM), which resulted in a 2.2 Log reduction in viral load compared to the vehicle control.

[0330] (Biological Example 4: Evaluation of CXCL10 production and viral load in influenza virus-infected ALI cultured human epithelial cells after treatment with dsRNA 1 or dsRNA 2) (Experimental method) ALI-cultured human nasal epithelium was maintained as described in Biological Example 2 and inoculated with influenza virus (PR8 strain).

[0331] One day before virus inoculation, the ALI culture was inoculated with 50 μL of vehicle control (buffer: 0.20% (w / w) citrate monohydrate and 0.28% (w / w) sodium citrate dihydrate in water), surfactant components in the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), dsRNA 1 (10 μg / mL) or dsRNA 2 (100 μg / mL) in the vehicle, or dsRNA 1 (10 μg / mL) or dsRNA in the vehicle. 2 (100 μg / mL) and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) were administered apex-to-apex over 30 minutes (then removed using a pipette). Then, 30 minutes before virus inoculation, these treatments were reapplied (then removed using a pipette), and the mixture was incubated with the virus inoculation material for 60 minutes. Sampling was performed on day 0 (24 hours after treatment) and day 1 (24 hours after virus inoculation and 48 hours after treatment) by adding 300 μL of culture medium to the apex surface of each well over 10 minutes (stored at -80°C).

[0332] The viral load (n=3, mean ± range) and CXCL10 level (n=3, mean ± SEM) were calculated as described in Biological Example 2 above. The viral loads detected after each treatment were compared with the viral loads detected after treatment with the vehicle control.

[0333] The processed formulations used in this example were produced largely as described in Biophysical Example 1.

[0334] (result) As shown in Figure 6, the surfactant component alone did not induce CXCL10 expression in the apical wash compared to the vehicle control (buffer). Furthermore, dsRNA 1 did not induce CXCL10 expression compared to the vehicle control. However, both dsRNA 1 and the surfactant component induced strong CXCL10 expression. Only dsRNA 2 induced significant CXCL10 expression, but formulations containing both dsRNA 2 and the surfactant component induced a further increase in the observed induction of CXCL10 expression.

[0335] As shown in Figure 7, high levels of influenza replication were detected in the apical lavage solution after treatment with the vehicle control (buffer). Only dsRNA 1 did not show a significant antiviral effect. In particular, only the surfactant component appeared to produce a mild antiviral effect, as indicated by a 0.7 Log reduction in viral load compared to the vehicle control. Both dsRNA 1 and the surfactant component exhibited potent antiviral activity, as indicated by a significant reduction in viral load (a 1.8 Log reduction compared to dsRNA 1). Only dsRNA 2 showed significant antiviral activity compared to the vehicle control. However, formulations of dsRNA 2 and the surfactant component enhanced the antiviral activity of dsRNA 2, as indicated by a 2.8 Log reduction in viral load compared to the surfactant component alone and a 1.5 Log reduction in viral load compared to dsRNA 2 alone.

[0336] (Biological Example 5: Evaluation of viral load, virus-induced inflammation, and weight loss in a mouse model of influenza virus infection treated with shRNA 1) (Experimental method) Non-fasting mice (male BALB / C, 20-30g) were anesthetized with isoflurane (5% in O2) and intranasal infection with influenza virus (PR8 strain) or a virus dilution (DMEM, 2% FBS, 12.5% ​​sucrose). Influenza virus (PR8 strain; 10 μL per nostril, 2 × 10⁶ 2PFU was administered by drop (10 μl into each nostril) to each mouse. After infection, each mouse was weighed daily (n=5, mean ± SEM).

[0337] Vehicle control (water), surfactant components in the vehicle (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) and further pharmaceutically acceptable excipients (formulation example 1A without shRNA 1, hereafter referred to as "surfactant formulation"), or treatment formulations of shRNA 1 in the vehicle (2 mg / mL) or shRNA 1 in the vehicle (2 mg / mL) with surfactant components and further pharmaceutically acceptable excipients (formulation example 1A, hereafter referred to as "shRNA 1 and surfactant formulation") were produced in general as described in Biophysical Example 1 below.

[0338] On day 0, 3 days, 1 day, and 1 hour prior to viral infection, the vehicle control or test formulation was administered intranasally (10 μL into each nasal cavity) using a pipette. As a drug control, oseltamivir phosphate was formulated in PBS and orally administered to control mice once daily (10 mg / kg) 1 hour prior to viral infection and 1 day after inoculation.

[0339] Mice were euthanized on day 1 and day 5 post-infection (intraperitoneal injection of pentobarbiton overdose). The airways were then lavaged by inserting a cannula into the trachea and washing the lungs with 0.5 mL of PBS. Immediately after bronchoalveolar lavage, a catheter was inserted through the tracheal opening used for bronchoalveolar lavage into the posterior nostril(s) to collect nasal lavage fluid (NLF). PBS (1 mL) was gently perfused into the nasal cavity, and NLF was collected from the anterior nostril(s). The isolated NLF was centrifuged at 1500 rpm for 10 minutes at 4°C, and the supernatant was aliquoted at -80°C (350 μL) for subsequent cytokine analysis. The cell pellet was resuspended in 1.6 mL of PBS, and the total and differential cell counts of NLF cells were analyzed. The total and differential cell counts of the NLF fluid samples were measured using an XT-2000iV analyzer (Sysmex). The results are expressed as cells / mL, e.g., neutrophils / mL (total and differential count) (n=5, mean ± SEM)

[0340] Immediately after NLF recovery, the thoracic cavity of the euthanized mice was opened, and the right lung lobe of each mouse was extracted and homogenized twice for 20 seconds in DMEM (containing 1% BSA and 25% sucrose; 10 mL per gram of lung). The homogenate was transferred to a sterile tube and spun at 2000 rpm for 5 minutes at 4°C. The clarified homogenate was then transferred to a cooled cryovial, rapidly frozen in liquid nitrogen, and stored at -80°C. After the extraction of the right lung lobe, nasal tissue was removed from each animal. Dissection involved first removing the mandible and head skin, followed by the removal of the head. Next, the palette and the bone protecting the brain and olfactory bulb were removed to expose the nasal tissue. The extracted nasal tissue was homogenized twice for 20 seconds in DMEM (containing 1% BSA and 25% sucrose; 10 mL per gram of tissue). Similar to the lung homogenate, the nasal tissue homogenate was transferred to a sterile tube, spun at 2000 rpm for 5 minutes at 4°C, then transferred to a cryovial, rapidly frozen in liquid nitrogen, and stored at -80°C.

[0341] Serial dilutions of supernatants collected from lung and nasal tissue homogenates using a medium containing 0.1 μg / ml TPCK trypsin were applied to plates covered with MDCK cells (80% confluence), and the seeded cells were incubated at 37°C for 1 hour. Next, the seeding material was removed from the wells, the cells were washed twice with PBS, and an overlay of 1% methylcellulose agar (containing growth medium and 0.1 μg / ml TPCK trypsin) was applied to each well. Once the agar had solidified, the plates were incubated with 5% CO2 at 37°C for 3 days, and the resulting plaques were counted. After removing the agar overlay and staining the cells with crystal violet, a second count was performed (n=5, mean ± SEM, PFU / group).

[0342] (result) High levels of influenza virus replication were detected in the nasal tissue of mice treated with a vehicle control (water only) on day 1 post-inoculation, but only a slight decrease in viral load was observed on day 5 post-inoculation. As shown in Figure 8, the surfactant formulation alone did not exhibit antiviral effects. However, only shRNA 1 showed a moderate antiviral effect, as represented by a 41% decrease in viral load (compared to water only) on both day 1 and day 5 post-inoculation, while the formulations of shRNA 1 and the surfactant formulation produced a more significant antiviral effect, as represented by a 60% and 51% decrease in viral load (compared to the surfactant formulation alone) on day 1 and day 5 post-inoculation, respectively. Therefore, shRNA 1 and the surfactant formulation had significantly stronger antiviral effects than the oseltamivir control, inducing a 38% and 33% decrease in viral load (compared to the vehicle) on day 1 and day 5 post-inoculation, respectively.

[0343] Furthermore, as shown in Figure 9, significant neutrophil accumulation was observed in the noses of influenza virus-infected mice after vehicle treatment on day 1 post-inoculation, but the neutrophil count decreased moderately by day 5 post-inoculation. shRNA 1 in water induced a moderate decrease in neutrophil accumulation, as represented by a 41% and 67% decrease in neutrophil counts (compared to water alone) on day 1 and day 5 post-inoculation, respectively. However, formulations of shRNA 1 and surfactant resulted in a more significant decrease in neutrophil accumulation, as represented by an 85% and 82% decrease in neutrophil counts (compared to surfactant formulations alone, which did not induce a decrease in neutrophil accumulation compared to vehicle) on day 1 and day 5 post-inoculation, respectively. Oseltamivir control also resulted in a decrease in neutrophil accumulation, albeit to a lesser degree than the shRNA 1 and surfactant formulations, as represented by a 57% and 69% decrease in neutrophil counts on day 1 and day 5 post-inoculation, respectively.

[0344] Finally, influenza virus infection resulted in significant weight loss in influenza virus-infected mice treated with the vehicle. As shown in Figure 10, surfactant formulations alone did not prevent this influenza virus-induced weight loss. However, while shRNA 1 in the vehicle had a moderate protective effect against weight loss, shRNA 1 and surfactant formulations further prevented influenza virus-induced weight loss. In fact, shRNA 1 and surfactant formulations had a protective effect similar to that of oseltamivir controls.

[0345] (Biological Example 6: Evaluation of mRNA and plasmid DNA exposure to ALI-cultured human nasal epithelium) (Experimental method) ALI cultured pooled donor human nasal epithelium (provided by Epithelix Sarl (Geneva, Switzerland)) was maintained at the gas-liquid interface using MucilAir® culture medium in a Costar Transwell insert (Corning, NY, USA), according to the manufacturer's instructions.

[0346] ALI cultures were apical-administered with either 50 μL of vehicle control in a citrate buffer (0.20% (w / w) citrate monohydrate and 0.28% (w / w) sodium citrate dihydrate), i.e., 0.5 mg / mL of GFP-coding mRNA (Vetnal), or the test formulation in the aforementioned buffer, i.e., 0.5 mg / mL of GFP-coding mRNA (Vetnal) and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), for 4 hours. After the treatment was removed, the apical surface of the ALI cultures was washed with 100 μL of culture medium. After overnight incubation (24 hours after treatment), ALI cultured nasal epithelial cells were harvested by gentle agitation in sterile PBS and then fixed with 4% paraformaldehyde in PBS. Samples from two independent inserts were combined and analyzed by flow cytometry using a BD Accuri™ instrument (Becton Dickinson) with a 530 / 30 nm bandpass (BP) filter. MFI was calculated, and the MFI (autofluorescence) of the untreated control was subtracted.

[0347] Alternatively, ALI cultures were apical-administered with 50 μL of a vehicle control in a citrate buffer (0.20% (w / w) citrate monohydrate and 0.28% (w / w) sodium citrate dihydrate), i.e., 0.033 μg / mL pSV-β-galactosidase control vector (Promega, E1081) (β-gal), or the test formulation in the aforementioned buffer, i.e., 0.033 μg / mL pSV-β-galactosidase control vector (Promega, E1081) (β-gal) and a surfactant component (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) for 4 hours. After that, the treatment was removed and the apical surface of the ALI cultures was washed with 100 μL of culture medium. ALI-cultured nasal epithelial cells from the three inserts were lysed together in reporter lysis buffer according to the manufacturer's instructions (Promega, #E2000, β-galactosidase enzyme assay system using reporter lysis buffer). After incubation overnight with the substrate provided in the kit, β-galactosidase enzyme activity (24 hours after treatment) was determined by reading the absorbance at 420 nm.

[0348] The processed formulations used in this example were produced largely as described in Biophysical Example 1.

[0349] (result) As shown in Figure 11, treatment of ALI-cultured nasal epithelial cells with a formulation of GFP-coding mRNA and a surfactant component resulted in significant MFI (particularly compared to background autofluorescence). Furthermore, the MFI observed when treated with GFP-coding mRNA and the surfactant component was significantly higher than the MFI observed when treated with GFP-coding mRNA alone. These data suggest that formulations of GFP-coding mRNA and the surfactant component promote greater exposure of epithelial cells to the mRNA, leading to greater GFP expression and consequently higher levels of fluorescence.

[0350] Furthermore, as shown in Figure 12, formulations of plasmids encoding β-galactosidase and surfactant components appear to similarly increase the exposure of ALI-cultured nasal epithelial cells to the plasmid.

[0351] These data support the idea that the formulations of the present invention may be suitable for improving the delivery of a wide variety of polynucleotide molecules, including DNA, and thereby increasing their exposure.

[0352] (Biological Example 7: Evaluation of viral load and virus-induced inflammation in human epithelial cells cultured with human rhinovirus-infected ALI after treatment with shRNA 1) (Experimental method) ALI cultured pooled donor human nasal epithelium (provided by Epithelix Sarl (Geneva, Switzerland)) was maintained at the gas-liquid interface using MucilAir® culture medium in a Costar Transwell insert (Corning, NY, USA) according to the manufacturer's instructions. On day 0 of the experiment, human rhinovirus type 16 (HRV16) inoculum (400,000 PFU / mL in 100 μL of MucilAir® culture medium to obtain a final MOI of approximately 0.2) was added to the apical surface of the epithelium for 1 hour (34°C / 5% CO2). Subsequently, the virus inoculum was removed, and the insert was immersed in 100 μL of sterile PBS (Ca 2+ / Mg 2+ It was washed (including)

[0353] One day before viral inoculation, 50 μL of vehicle control (citric acid buffer: 0.20% (w / w) citrate monohydrate and 0.28% (w / w) sodium citrate dihydrate), shRNA 1 (2 mg / mL) from the vehicle, surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or shRNA 1 (2 mg / mL) and surfactant components were administered apical over 30 minutes (then almost completely removed using a pipette without additional washing). Then, 30 minutes before viral inoculation, these treatments were reapplied (then removed using a pipette), and then incubated with the viral inoculation material for 60 minutes. On day 0 (before viral inoculation) and day 2, sampling was performed by adding 300 μL of culture medium to the apical surface of each well over 10 minutes (stored at -80°C). On day 0 (day 1 after initial treatment), the CXCL10 concentration before viral infection was quantified by the method described in Biological Example 1 (n=3, mean ± SEM).

[0354] On the second day after virus inoculation (third day after initial treatment), the viral load should be measured to 50% of the tissue culture infectious dose (TCID). 50 Quantitative analysis was performed using TCID. Briefly, the apical wash was thawed, and serial dilutions of the apical wash sample in 1% FBS DMEM medium containing 15 mM MgCl2 were applied to plates covered with HeLa cells (ATCC®, Manassas, #CCL-2, 80% confluence). These were then incubated at 34°C and 5% CO2 for 5 days, particularly until CPE induced by the vehicle control was visible. 50 This was calculated using the Reid-Münch equation (Bullen et al., 2022) (n=3, mean ± range).

[0355] Specifically, the first dilution with CPE-positive % ≥ 50 ("high dilution") and the first dilution with CPE-positive % < 50% ("low dilution") were identified, and the proportional distance (PD) was calculated using the following formula: PD = (Percentage of CPE-positive wells at high dilution - 50%) / (Percentage of CPE-positive wells at high dilution - Percentage of CPE-positive wells at low dilution) Log TCID 50 =Log dilution large+PD

[0356] The processed formulations used in this example were produced in general as described in Biophysical Examples 1 and 2.

[0357] (result) As shown in Figure 14, shRNA 1 alone (i.e., in the vehicle) slightly induced CXCL10 expression, a marker of antiviral IFN production. However, shRNA 1 and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80) induced a considerably more significant increase in CXCL10 expression compared to the vehicle control.

[0358] Furthermore, as shown in Figure 15, high levels of HRV16 replication were detected in the apical lavage fluid 48 hours after inoculation, following treatment with the vehicle control (mean: 3.1 Log, TCID). 50 ( / mL). shRNA 1 alone (i.e., in the vehicle) did not show a significant antiviral effect. However, while treatment with the surfactant component alone had a moderate antiviral effect, shRNA 1 and the surfactant component had a specific and potent antiviral effect, as indicated by a 2.8 Log reduction in viral load compared to the vehicle control.

[0359] (Biological Example 8: Evaluation of viral load and cell integrity in human epithelial cells cultured with ALI and infected with human respiratory syncytial virus (RSV) after treatment with shRNA 1) (Experimental method) ALI cultured pooled donor human nasal epithelium (provided by Epithelix Sarl (Geneva, Switzerland)) was maintained at the gas-liquid interface using MucilAir® culture medium in a Costar Transwell insert (Corning, NY, USA) according to the manufacturer's instructions. On day 0 of the experiment, a viral inoculum of human respiratory syncytial virus A2 (RSV A2) (40,000 PFU / mL in 100 μL of MucilAir® culture medium to obtain a final MOI of approximately 0.02) was added to the apical surface of the epithelium for 1 hour (37°C / 5% CO2). Subsequently, the viral inoculum was removed, and the insert was sterilized in 100 μL of sterile PBS (Ca 2+ / Mg 2+ It was washed (including)

[0360] One day before viral inoculation, 50 μL of vehicle control (citric acid buffer: 0.20% (w / w) citrate monohydrate and 0.28% (w / w) sodium citrate dihydrate), shRNA 1 (2 mg / mL) from the vehicle, surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or shRNA 1 (2 mg / mL) and surfactant components were administered apical over 30 minutes (then almost completely removed using a pipette without additional washing). Then, 30 minutes before viral inoculation, these treatments were reapplied (then removed using a pipette), and then incubated with the viral inoculation material for 60 minutes. On day 0 (before viral inoculation) and day 3, sampling was performed by adding 300 μL of culture medium to the apical surface of each well over 10 minutes. All apical lavage solutions were collected in sterile Eppendorf tubes containing 100 μL of 50% sucrose solution (stored at -80°C).

[0361] The processed formulations used in this example were produced in general as described in Biophysical Examples 1 and 2.

[0362] Three days after viral inoculation (four days after initial treatment), the viral load was quantified by plaque assay. In short, HEp-2 cells were measured at 5-10 × 10⁶. 4 Cells were seeded at a cell / well density in 24-well plates (Corning, NY, USA) and grown in 10% FBS DMEM for 48 hours prior to infection until 100% confluence was achieved.

[0363] Next, the virus-infected samples were thawed at room temperature, and 10-fold serial dilutions were prepared in serum-free DMEM. HEp-2 cell-derived growth medium was aspirated and replaced with 300 μL of serially diluted virus recovery, and the mixture was infected at 37°C / 5% CO2 for 4 hours. After infection, the virus medium was aspirated and replaced with 1 mL of plaque assay overlay (0.3% Avicel RC-591 [FMC Biopolymer UK, Girvan, Scotland] in MEM with FBS added to a final concentration of 2%), and the mixture was left at 37°C / 5% CO2 for 7 days.

[0364] The cells were then fixed with ice-cold methanol for 10 minutes, the methanol was removed, and the fixed cells were washed with sterile PBS (×2). The cells were then stained with 200 μL of 0.1% crystal violet solution (in distilled water) for 1 hour. After removing the crystal violet solution and rinsing the cells with water, the plaques were counted and the viral load was determined (n=3, mean ± range).

[0365] In addition to the plaque assay described above, transepithelial electrical resistance (TEER) was measured to examine the integrity of tight junction dynamics in ALI-cultured pseudostratified epithelium before RSV A2 infection and 3 days post-infection. This TEER measurement is a surrogate measure of epithelial damage. Specifically, chopstick electrodes were placed in the apical and basal chambers, and TEER was measured using a dedicated voltage / ohmmeter (EVOM2, an epithelial voltage / ohmmeter for TEER). TEER was measured in Ohm / cm². 2 It is represented as follows.

[0366] (result) As shown in Figure 16, high levels of human RSV A2 replication were detected in the apical wash after treatment with the vehicle control 72 hours after inoculation (mean: 6.4 Log, PFU / mL). shRNA 1 alone (i.e., in the vehicle) did not show a significant antiviral effect. However, when comparing the detected viral load with the viral load after treatment with the vehicle (buffer), only the surfactant component showed a moderate antiviral effect, while shRNA 1 and the surfactant component showed a significant antiviral effect, as indicated by a 1.6 Log reduction in viral load.

[0367] Furthermore, as shown in Figure 17, TEER decreased after human RSV A2 infection following treatment with a vehicle control (buffer). As mentioned above, this indicates virus-induced epithelial damage. Treatment of cells with shRNA 1 alone (i.e., in the vehicle) after infection mitigated the decrease in TEER and / or restored the decrease in TEER. However, treatment with the surfactant component alone significantly increased TEER (measured 72 hours after infection) compared to TEER measured before infection, suggesting a protective effect and increased epithelial cell integrity in the presence of the surfactant component. This protective effect is even more pronounced after treatment with shRNA 1 in the surfactant component, as evidenced by the further increase in TEER.

[0368] (Biological Example 9: Evaluation of viral load, virus-induced inflammation, and weight loss in a mouse model infected with respiratory syncytial virus (RSV) treated with shRNA 1) (Experimental method) Non-fasting mice (male BALB / C, 20-30g) were anesthetized with isoflurane (5% in O2) and RSV (5 x 10) was administered. 6 PFU (mouse) was inoculated intranasally. RSV virus (RSV A2 strain; 25 μL per nostril, 2.5 × 10⁴ 6 PFU was administered by drop (25 μl into each nostril) to each mouse. After infection, each mouse was weighed daily (n=5, mean ± SEM).

[0369] Treatment formulations were produced in general the same manner as described in Biophysical Examples 1 and 2, using physiological saline, surfactant components in phosphate buffer (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), and shRNA 1 (2 mg / mL) in the vehicle, along with surfactant components and further pharmaceutically acceptable excipients (see Formulation Example 1A).

[0370] On both day 0, four days and one day prior to viral infection, the vehicle control or test formulation was administered intranasally (25 μL into each nostril) using a pipette. As a drug control, ribavirin was formulated in PBS and administered intranasally to control mice twice daily, three hours before viral infection and from day 0 to 4 after inoculation (12.5 mg / kg).

[0371] Four days after infection, mice were euthanized (intraperitoneal injection of an overdose of pentobarbiton). Subsequently, the airways were lavaged by inserting a cannula into the trachea and washing the lungs with 0.5 mL of PBS. Immediately after bronchoalveolar lavage, a catheter was inserted into the posterior nostril(s) through the tracheal opening used for bronchoalveolar lavage to collect nasal lavage fluid (NLF). PBS (1 mL) was gently perfused into the nasal cavity, and NLF was collected from the anterior nostril(s). The isolated NLF was centrifuged at 1500 rpm for 10 minutes at 4°C, and the supernatant was aliquoted at -80°C (350 μL) for subsequent cytokine analysis. The cell pellet was resuspended in 1.6 mL of PBS, and the total and differential cell counts of NLF cells were analyzed. The total and differential cell counts of the NLF fluid samples were measured using an XT-2000iV analyzer (Sysmex). The results are expressed as cells / mL, e.g., neutrophils / mL (total and differential count) (n=5, mean ± SEM)

[0372] Immediately after NLF recovery, the thoracic cavity of the euthanized mice was opened, and the right lung lobe of each mouse was extracted and homogenized twice for 20 seconds in DMEM (containing 1% BSA and 25% sucrose; 10 mL per gram of lung). The homogenate was transferred to a sterile tube and spun at 2000 rpm for 5 minutes at 4°C. The clarified homogenate was then transferred to a cooled cryovial, rapidly frozen in liquid nitrogen, and stored at -80°C. After the extraction of the right lung lobe, nasal tissue was removed from each animal. Dissection involved first removing the mandible and head skin, followed by the removal of the head. Next, the palette and the bone protecting the brain and olfactory bulb were removed to expose the nasal tissue. The extracted nasal tissue was homogenized twice for 20 seconds in DMEM (containing 1% BSA and 25% sucrose; 10 mL per gram of tissue). Similar to the lung homogenate, the nasal tissue homogenate was transferred to a sterile tube, spun at 2000 rpm for 5 minutes at 4°C, then transferred to a cryovial, rapidly frozen in liquid nitrogen, and stored at -80°C.

[0373] Plaque assays were performed to determine viral load. HEp2 cells were grown in 24-well plates and then infected in DMEM containing 10% (v / v) FBS until 100% confluence was achieved. Supernatants collected from lung and nasal tissue homogenates were thawed at room temperature, and serial dilutions were prepared in serum-free DMEM. Growth medium derived from HEp2 cells was aspirated and replaced with 300 μL of serial dilution lung homogenate (along with a positive control of stock RSV only), and the cells were infected at 37°C / 5% CO2 for 4 hours. Subsequently, the infection medium was aspirated and replaced with 500 μL of plaque assay overlay ((1% (w / v) methylcellulose, 2% (v / v) FBS, 1% (w / v) pen / strep, 0.5 μg / ml amphotericin B in MEM)), and the cells were left at 37°C / 5% CO2 for 7 days.

[0374] Next, the cells were fixed with ice-cold methanol for 10 minutes, and then washed twice with sterile PBS. Anti-RSV F-protein antibody [2F7] was diluted to a concentration of 1:150 in blocking buffer (5% (w / v) milk powder (Marvel) in 0.05% (v / v) PBS-Tween 20), and 150 μL was added to the cells at room temperature for 2 hours with shaking. After washing the cells twice with PBS, 150 μL of secondary antibody (goat anti-mouse / HRP conjugate), diluted 1:400 in blocking buffer, was added to the cells at room temperature for 1 hour with shaking. The secondary antibody solution was removed, and the cells were washed twice with PBS. 150 μL of metal-enhanced chromogenic substrate DAB, prepared in ultrapure water, was applied to the cells until plaques were visualized. Plaque was counted visually and confirmed using an optical microscope, enabling the calculation of plaque formation units per 1 mL (n=5, mean ± SEM, PFU / group).

[0375] (result) As shown in Figure 18, high levels of RSV A2 were detected in the lung tissue of mice treated with a vehicle control (i.e., saline) four days after inoculation. In particular, treatment of mice with the surfactant component formulation alone had no antiviral effect in the lungs. However, treatment of mice with the shRNA 1 and surfactant component formulation showed a significant antiviral effect, as evidenced by a 54% reduction in viral load four days after inoculation compared to treatment with the surfactant component alone. Furthermore, the antiviral effect of the shRNA 1 and surfactant component formulation was comparable to that of ribavirin, which, as expected, had a potent antiviral effect, as evidenced by a 66% reduction in viral load four days after inoculation compared to treatment with a vehicle control (i.e., saline).

[0376] Furthermore, as shown in Figure 19, significant neutrophil accumulation was observed in the lungs of RSV A2-infected mice four days post-inoculation after treatment with a vehicle control (i.e., saline). Treatment of mice with the surfactant component alone did not show a significant decrease in neutrophil count. However, treatment of mice with the shRNA 1 and surfactant component resulted in a significant decrease in the degree of neutrophil accumulation, as evidenced by a 57% decrease in neutrophil count four days post-inoculation compared to treatment with the vehicle control (i.e., saline) and a 55% decrease in neutrophil count four days post-inoculation compared to treatment with the surfactant component alone. In particular, treatment of mice with the shRNA 1 and surfactant component resulted in a decrease in neutrophil accumulation to the same extent as the decrease observed after treatment with ribavirin. Specifically, ribavirin treatment resulted in a 61% decrease in neutrophil count four days post-inoculation compared to treatment with the vehicle control (i.e., saline).

[0377] Finally, as shown in Figure 20, RSV A2 infection resulted in significant weight loss in mice treated with a vehicle control (i.e., saline). Treatment of mice with surfactant formulations did not limit or prevent RSV A2 infection-induced weight loss. In contrast, treatment of mice with shRNA 1 and surfactant formulations and treatment of mice with ribavirin controls both provided protection against the RSV A2 infection-induced weight loss. The degree of this protection was similar between the shRNA 1 and surfactant formulations and the ribavirin controls.

[0378] (Biological Example 10: Evaluation of CXCL10 release in uninfected mice and viral load, virus-induced inflammation, and weight loss in influenza virus-infected mouse models after subcutaneous treatment with shRNA 1) (Experimental method) Non-fasted male BALB / C mice (20-30 g) were subcutaneously injected with physiological saline, surfactant components in phosphate buffer (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), shRNA1 (2 mg / mL) and surfactant components, or shRNA1 (20 mg / mL) and surfactant components at a dose of 10 mL / kg. Twenty-four hours after administration (n=4), blood samples were collected from the lateral tail vein of infected mice and placed in serum tubes. Each serum sample was maintained at room temperature for 45 minutes to allow coagulation, and then centrifuged at 2000 g for 15 minutes at 4°C. The resulting supernatant was extracted, aliquoted (2 × 50 μL), and stored at -80°C for transport.

[0379] Immediately after blood collection, each mouse was euthanized by intraperitoneal pentobarbitone overdose. The trachea was then isolated by a midline neck incision and separation of the muscle layer. A small incision was made in the trachea, a plastic cannula was inserted, and secured with sutures. The airway was then lavaged by washing the lung with 0.5 mL of PBS. This procedure was repeated until a total volume of 1.6 mL was recovered. The isolated BALF was then centrifuged at 1500 rpm for 10 minutes at 4°C, and the supernatant was aliquoted at -80°C (400 μL). CXCL10 levels in serum and BALF were determined using a mouse CXCL10 kit with an MSD multiscanner (Meso Scale Diagnostics).

[0380] Separately, non-fasting mice (male BALB / C, 20-30g) were intranasally infected with influenza (PR8 strain) under isoflurane (5% in O2) anesthesia. Influenza virus (PR8 strain; 10 μL per nostril, 2 × 10⁶ 2 PFU was administered by drop (10 μl into each nostril) to each mouse. After infection, each mouse was weighed daily (n=5, mean ± SEM).

[0381] Four days and one day prior to viral infection on day 0, mice were subcutaneously administered either physiological saline (i.e., untreated), surfactant components in a phosphate buffer (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or a treatment formulation of shRNA 1 (2 mg / mL) and surfactant components in a phosphate buffer at a dose of 10 mL / kg. As a drug control, oseltamivir phosphate was formulated in PBS and orally administered to control mice four hours prior to viral infection (10 mg / kg). In this case, the mice were also treated with subcutaneous injection of the surfactant component alone four days and one day prior to viral infection on day 0. Furthermore, one group of mice was treated with oral oseltamivir and with the combination therapy of subcutaneously administered shRNA 1 and surfactant components.

[0382] The treatment formulations used in this example were produced in general as described in Biophysical Examples 1 and 2.

[0383] Five days after infection, mice were euthanized (intraperitoneal injection of pentobarbiton overdose). Subsequently, the airways were lavaged by inserting a cannula into the trachea and washing the lungs with 0.5 mL of PBS. Immediately after bronchoalveolar lavage, a catheter was inserted into the posterior nostril(s) through the tracheal opening used for bronchoalveolar lavage to collect nasal lavage fluid (NLF). PBS (1 mL) was gently perfused into the nasal cavity, and NLF was collected from the anterior nostril(s). The isolated NLF was centrifuged at 1500 rpm for 10 minutes at 4°C, and the supernatant was aliquoted at -80°C (350 μL) for subsequent cytokine analysis. The cell pellet was resuspended in 1.6 mL of PBS, and the total and differential cell counts of NLF cells were analyzed. The total and differential cell counts of the NLF fluid samples were measured using an XT-2000iV analyzer (Sysmex). The results are expressed as cells / mL, e.g., neutrophils / mL (total and differential count) (n=5, mean ± SEM)

[0384] Immediately after NLF recovery, the thoracic cavity of the euthanized mice was opened, and the right lung lobe of each mouse was extracted and homogenized twice for 20 seconds in DMEM (containing 1% BSA and 25% sucrose; 10 mL per gram of lung). The homogenate was transferred to a sterile tube and spun at 2000 rpm for 5 minutes at 4°C. The clarified homogenate was then transferred to a cooled cryovial, rapidly frozen in liquid nitrogen, and stored at -80°C. After the extraction of the right lung lobe, nasal tissue was removed from each animal. Dissection involved first removing the mandible and head skin, followed by the removal of the head. Next, the palette and the bone protecting the brain and olfactory bulb were removed to expose the nasal tissue. The extracted nasal tissue was homogenized twice for 20 seconds in DMEM (containing 1% BSA and 25% sucrose; 10 mL per gram of tissue). Similar to the lung homogenate, the nasal tissue homogenate was transferred to a sterile tube, spun at 2000 rpm for 5 minutes at 4°C, then transferred to a cryovial, rapidly frozen in liquid nitrogen, and stored at -80°C.

[0385] Serial dilutions of supernatants collected from lung and nasal tissue homogenates using a medium containing 0.1 μg / ml TPCK trypsin were applied to plates covered with MDCK cells (80% confluence), and the seeded cells were incubated at 37°C for 1 hour. Next, the seeding material was removed from the wells, the cells were washed twice with PBS, and an overlay of 1% methylcellulose agar (containing growth medium and 0.1 μg / ml TPCK trypsin) was applied to each well. Once the agar had solidified, the plates were incubated with 5% CO2 at 37°C for 3 days, and the resulting plaques were counted. After removing the agar overlay and staining the cells with crystal violet, a second count was performed (n=5, mean ± SEM, PFU / group).

[0386] (result) As shown in Figure 21, subcutaneous injection of saline did not induce significant CXCL10 expression in treated mice. Subcutaneous treatment of mice with surfactant components alone significantly induced CXCL10 expression compared to treatment with saline. However, subcutaneous injection of a formulation containing shRNA 1 and surfactant components resulted in a considerably more significant increase in CXCL10 expression compared to treatment with either saline or surfactant components alone. It should be noted that there was no significant difference in the level of CXCL10 expression induction between a 2 mg / mL shRNA 1 dose and a 20 mg / mL shRNA 1 dose. The data shown are from 24 hours post-injection.

[0387] In a mouse model of influenza virus infection, high levels of influenza virus replication were detected in both the lungs (see Figure 22) and nasal tissue (see Figure 23) of untreated mice (i.e., those injected only with physiological saline). Furthermore, as shown in Figures 22 and 23, treatment of mice with the surfactant component formulation did not show any significant antiviral effect in either the lung or nasal tissue. In contrast, treatment of mice with the shRNA 1 and surfactant component formulation showed a clear and potent antiviral effect, as evidenced by a 29% reduction in viral load in the lungs and a 45% reduction in viral load in the nasal tissue at 5 days post-inoculation compared to treatment with the surfactant component alone. Treatment of mice with the control drug oseltamivir also resulted in a reduction in viral load, as evidenced by a 41% reduction in viral load in the lungs and a 49% reduction in viral load in the nasal tissue at 5 days post-inoculation compared to treatment with the surfactant component alone. It should be noted that the antiviral effect of the control drug oseltamivir was comparable to that of the shRNA 1 and surfactant component formulation. Finally, the combination treatment of oral oseltamivir, subcutaneous shRNA 1, and a surfactant component showed an even more significant antiviral effect compared to treatment with the surfactant component alone, as evidenced by a 73% reduction in viral load in the lungs and a 67% reduction in viral load in nasal tissue five days after vaccination.

[0388] Furthermore, significant neutrophil accumulation was observed in both the lung tissue (see Figure 24) and nasal tissue (see Figure 25) of influenza virus-infected mice treated with saline alone (i.e., untreated). Additionally, as shown in Figures 24 and 25, treatment of mice with surfactant component formulations alone did not reduce neutrophil accumulation in either the lung or nasal tissue. However, treatment of mice with shRNA 1 and surfactant component formulations resulted in a significant reduction in neutrophil accumulation in both the lung and nasal tissue, as evidenced by a 40% reduction in neutrophil counts in the lungs and a 56% reduction in neutrophil counts in the nasal tissue at 5 days post-inoculation compared to treatment with surfactant component alone. Treatment of mice with the control drug oseltamivir also resulted in a reduction in neutrophil accumulation in both the lung and nasal tissue, as evidenced by a 60% reduction in neutrophil counts in the lungs and a 70% reduction in neutrophil counts in the nasal tissue at 5 days post-inoculation compared to treatment with surfactant component alone. More importantly, the combination treatment of oral oseltamivir with subcutaneous shRNA 1 and a surfactant component induced an even more significant reduction in neutrophil accumulation, as evidenced by an 80% reduction in neutrophil counts in the lungs and a 74% reduction in neutrophil counts in nasal tissue at 5 days post-inoculation, compared to treatment with the surfactant component alone.

[0389] Finally, as shown in Figure 26, influenza virus infection resulted in significant weight loss in mice treated with a vehicle control (i.e., saline). Subcutaneous treatment of mice with surfactant formulations limited or prevented influenza virus infection-induced weight loss. However, both subcutaneous treatment of mice with shRNA 1 and surfactant formulations and subcutaneous treatment of mice with oral oseltamivir control provided protective effects against the influenza virus infection-induced weight loss. Furthermore, combination treatment of oral oseltamivir with subcutaneous shRNA 1 and surfactant completely prevented influenza virus infection-induced weight loss.

[0390] (Biological Example 11: Evaluation of the adjuvant effect of shRNA 1 and surfactant components on mice vaccinated with recombinant H1N1 hemagglutinin (HA) and infected with influenza virus) (Experimental method) Non-fasted mice (male BALB / C, 20-30 g) were intranasally treated with PBS, shRNA 1 in phosphate buffer and surfactant components (0.05% (w / w) oleic acid and 0.045% (w / w) polysorbate 80), or CPG-ODN solution (InvivoGen), which is a control adjuvant and TLR9 agonist (10 μL / nostril). The treatment formulations used in this example were produced in general as described in Biophysical Examples 1 and 2.

[0391] On day 0 after adjuvant administration, mice were treated under isoflurane with either a vehicle (i.e., PBS) or recombinant hemagglutinin (HA) (1 mg / mL in PBS) derived from influenza virus PR8 strain (SinoBiologics), with a volume of 10 μL delivered per nostril. This treatment was administered by drip infusion, alternating between the two until the required amount (10 μg HA / 10 μL) was delivered. Qualitative evaluations for each group were recorded by the level of piloerection in each animal after the priming dose. All animals received a booster dose (adjuvant and HA antigen) again on day 28. On day 56, each animal received influenza virus (PR8 strain; 2 × 10⁶). 2 PFU was administered intranasally (10 μL). After infection, each animal was weighed daily and changes in body weight were monitored. On day 61 (i.e., day 5 post-infection), terminal blood samples were collected by venous puncture (via the caudal vein) and placed in LiHep tubes. Each blood sample was gently mixed, centrifuged (4°C, 2000 g for 5 minutes), and the resulting plasma was extracted, aliquoted, and stored at -80°C.

[0392] Immediately after blood collection, the mice were euthanized (intraperitoneal injection of an overdose of pentobarbiton). The airways were then lavaged by inserting a cannula into the trachea and washing the lungs with 0.5 mL of PBS. Immediately after bronchoalveolar lavage, a catheter was inserted through the tracheal opening used for bronchoalveolar lavage into the posterior nostril(s) to collect nasal lavage fluid (NLF). The nasal cavity was gently perfused with PBS (1 mL), and NLF was collected from the anterior nostril(s). The isolated NLF was centrifuged at 1500 rpm for 10 minutes at 4°C, and the supernatant was aliquoted at -80°C (350 μL) for subsequent cytokine analysis. The cell pellet was resuspended in 1.6 mL of PBS, and the total and differential cell counts of NLF cells were analyzed. The total and differential cell counts of the NLF fluid samples were measured using an XT-2000iV analyzer (Sysmex). The results are expressed as cells / mL, e.g., neutrophils / mL (total and differential count) (n=6, mean ± SEM)

[0393] Immediately after NLF recovery, the thoracic cavity of the euthanized mice was opened, and the right lung lobe of each mouse was extracted and homogenized twice for 20 seconds in DMEM (containing 1% BSA and 25% sucrose; 10 mL per gram of lung). The homogenate was transferred to a sterile tube and spun at 2000 rpm for 5 minutes at 4°C. The clarified homogenate was then transferred to a cooled cryovial, rapidly frozen in liquid nitrogen, and stored at -80°C. After the extraction of the right lung lobe, nasal tissue was removed from each animal. Dissection involved first removing the mandible and head skin, followed by the removal of the head. Next, the palette and the bone protecting the brain and olfactory bulb were removed to expose the nasal tissue. The extracted nasal tissue was homogenized twice for 20 seconds in DMEM (containing 1% BSA and 25% sucrose; 10 mL per gram of tissue). Similar to the lung homogenate, the nasal tissue homogenate was transferred to a sterile tube, spun at 2000 rpm for 5 minutes at 4°C, then transferred to a cryovial, rapidly frozen in liquid nitrogen, and stored at -80°C.

[0394] Serial dilutions of supernatants collected from lung and nasal tissue homogenates using a medium containing 0.1 μg / ml TPCK trypsin were applied to plates covered with MDCK cells (80% confluence), and the seeded cells were incubated at 37°C for 1 hour. Next, the seeding material was removed from the wells, the cells were washed twice with PBS, and an overlay of 1% methylcellulose agar (containing growth medium and 0.1 μg / ml TPCK trypsin) was applied to each well. Once the agar had solidified, the plates were incubated with 5% CO2 at 37°C for 3 days, and the resulting plaques were counted. After removing the agar overlay and staining the cells with crystal violet, a second count was performed (n=5, mean ± SEM, PFU / group).

[0395] (result) Mice treated with recombinant H1N1 (rH1N1) hemagglutinin (HA) vaccine alone (i.e., PBS) showed a moderate but not statistically significant reduction in viral load in both the lungs (see Figure 27) and nasal tissue (see Figure 28). Mice treated with rH1N1 HA vaccine and the control adjuvant CPG-ODN similarly experienced a moderate reduction in viral load in both the lungs and nasal tissue. However, most precisely, mice treated with rH1N1 HA vaccine and a formulation of shRNA 1 and a surfactant component showed a considerably larger and statistically very significant reduction in viral load in both the lungs and nasal tissue. Therefore, the antiviral effect of rH1N1 HA vaccine after treatment with the shRNA 1 and surfactant component formulation was considerably greater than the antiviral effect observed after treatment with the same vaccine with the control adjuvant CPG-ODN.

[0396] Furthermore, five days post-vaccination, significant neutrophil accumulation was observed in both the lungs (see Figure 29) and nasal tissue (see Figure 30) of influenza virus-infected mice treated with the vehicle (i.e., PBS). Mice vaccinated with rH1N1 HA alone (i.e., in PBS) showed a significant decrease in neutrophil accumulation in both lungs and nasal tissue, and in particular, mice vaccinated with rH1N1 HA after treatment with the control adjuvant CPG-ODN showed a similar degree of decrease in neutrophil accumulation. That is, the control adjuvant CPG-ODN did not enhance the decrease in neutrophil accumulation induced by rH1N1 HA vaccination. In contrast, mice treated with rH1N1 HA vaccination after treatment with a formulation of shRNA 1 and a surfactant component showed an even more significant reduction in neutrophil accumulation in both lung and nasal tissues, as represented by a 40% reduction in neutrophil accumulation in the lungs and an 83% reduction in neutrophil accumulation in the nasal tissues compared to treatment with the vehicle (i.e., PBS) on day 5 post-vaccination. In other words, the formulation of shRNA 1 and a surfactant component enhanced the reduction in neutrophil accumulation associated with rH1N1 HA vaccination.

[0397] Finally, as shown in Figure 31, influenza virus infection resulted in significant weight loss in mice treated with the vehicle (i.e., PBS). Intranasal vaccination with rH1N1 HA moderately reduced influenza infection-induced weight loss. The control adjuvant CPG-ODN did not enhance the protective effect of rH1N1 HA vaccination and, in fact, resulted in mice suffering greater weight loss than mice treated with rH1N1 HA vaccination alone (i.e., PBS). In contrast, treatment of mice with rH1N1 HA vaccination after treatment with a formulation of shRNA 1 and a surfactant component resulted in a more significant protective effect against influenza virus infection-induced weight loss, and in fact, almost completely reduced such weight loss.

[0398] (Biological Example 12: Evaluation of mRNA exposure to ALI-cultured human nasal epithelium) (Experimental method) Following the favorable results of the above-described biological example 6, the inventors further characterized the presented data regarding exposure to GFP-encoding mRNA, which supports the use of the formulation of the present inve...

Claims

1. (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) a polynucleotide molecule, an aqueous liquid pharmaceutical preparation in the form of a stable colloidal emulsion.

2. The aqueous liquid pharmaceutical formulation according to claim 1, wherein the fatty acid is selected from the group consisting of arachidic acid, arachidonic acid, caprylic acid, lauric acid, linoleic acid, linolenic acid, myristic acid, myristoleic acid, oleic acid, palmitic acid, palmitoleic acid, sapienic acid, stearic acid, and vaccenic acid.

3. The aqueous liquid pharmaceutical formulation according to claim 2, wherein the fatty acid is selected from caprylic acid and oleic acid.

4. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 3, wherein the nonionic surfactant is selected from the group consisting of polyoxyalkylenes such as poloxamers, alkyl ethers of polyethylene glycol, alkylphenyl ethers of polyethylene glycol, and fatty acid esters such as polyoxyethylene sorbitan fatty acid esters.

5. The aqueous liquid pharmaceutical formulation according to claim 4, wherein the nonionic surfactant is selected from an alkyl ether of polyethylene glycol and a polyoxyethylene sorbitan fatty acid ester.

6. The surfactant component is (a) a mixture of oleic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (b) a mixture of lauric acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (c) a mixture of linoleic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (d) a mixture of linolenic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (e) a mixture of palmitic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (f) a mixture of stearic acid or a pharmaceutically acceptable salt thereof and polyoxyethylene sorbitan fatty acid ester, (g) oleic acid or a pharmaceutically acceptable salt thereof and poloxamer, etc. An aqueous liquid pharmaceutical formulation according to claim 1, selected from the group consisting of (h) a mixture of polyoxyalkylenes, (i) a mixture of oleic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol, (j) a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester, (k) a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and a polyoxyalkylene such as poloxamer, (l) a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol, and (m) a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol.

7. The aqueous liquid pharmaceutical preparation according to claim 6, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester.

8. The aqueous liquid pharmaceutical preparation according to claim 7, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and polysorbate 80.

9. The aqueous liquid pharmaceutical preparation according to claim 6, wherein the surfactant component is a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and a polyoxyethylene sorbitan fatty acid ester.

10. The aqueous liquid pharmaceutical preparation according to claim 9, wherein the surfactant component is a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and polysorbate 80.

11. The aqueous liquid pharmaceutical formulation according to claim 6, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol.

12. The aqueous liquid pharmaceutical formulation according to claim 11, wherein the surfactant component is a mixture of oleic acid or a pharmaceutically acceptable salt thereof and Brij 35 (polyoxyethylene (23) lauryl ether).

13. The aqueous liquid pharmaceutical formulation according to claim 6, wherein the surfactant component is a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and an alkyl ether of polyethylene glycol.

14. The aqueous liquid pharmaceutical formulation according to claim 13, wherein the surfactant component is a mixture of caprylic acid or a pharmaceutically acceptable salt thereof and Brij 35 (polyoxyethylene (23) lauryl ether).

15. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 14, wherein the fatty acid is in the form of a free acid.

16. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 15, wherein the concentration of the surfactant component in the preparation is 0.2 to 30,000 μg / mL, for example, 1 to 30,000 μg / mL, for example, 1 to 20,000 μg / mL, 5 to 20,000 μg / mL, 5 to 15,000 μg / mL, 5 to 10,000 μg / mL, or 5 to 5,000 μg / mL.

17. An aqueous liquid pharmaceutical preparation according to any one of claims 1 to 16, wherein the ratio of the amount of fatty acid or a pharmaceutically acceptable salt thereof, measured in μg / mL units, to the amount of nonionic surfactant is about 5:1 to about 1:5, about 5:1 to about 1:2, about 4:1 to about 1:2, or about 2:1 to about 1:

2.

18. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 17, wherein the polynucleotide molecule is a ribonucleic acid (RNA) molecule.

19. The aqueous liquid pharmaceutical formulation according to claim 18, wherein the RNA molecule is an mRNA, miRNA, shRNA, or siRNA molecule.

20. The aqueous liquid pharmaceutical formulation according to claim 19, wherein the RNA molecule is an shRNA molecule.

21. The aqueous liquid pharmaceutical formulation according to claim 20, wherein the shRNA molecule comprises (i) a blunt end, (ii) a 5' triphosphate or 5' diphosphate moiety located at the 5' end, and (iii) a double-stranded region having a length of about 10 to about 18 nucleotides.

22. The aqueous liquid pharmaceutical formulation according to claim 20 or claim 21, wherein the shRNA molecule includes or consists of SEQ ID NO: 1 or a variant of SEQ ID NO:

1.

23. The aqueous liquid pharmaceutical formulation according to claim 20 or claim 21, wherein the shRNA molecule comprises or consists of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10, or a variant of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO:

10.

24. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 17, wherein the polynucleotide molecule is a deoxyribonucleic acid (DNA) molecule.

25. The aqueous liquid pharmaceutical formulation according to claim 24, wherein the DNA molecule is a genomic DNA (gDNA) molecule, a complementary DNA (cDNA) molecule, or an extrachromosomal DNA molecule, such as a plasmid DNA molecule.

26. The aqueous liquid pharmaceutical formulation according to claim 1, wherein the polynucleotide molecule has the structure of formula (I): 【Chemistry 1】 (In the formula, 5'-P z -(N) b N-3' represents the first nucleic acid sequence; 5'-N(N) b' -3' represents the second nucleic acid sequence; In each case, P is independently a phosphate or an analogue thereof; z is either 2 or 3; In each case, N is any nucleotide or modified nucleotide or its analogue or derivative; b and b' are independently 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18; 【Chemistry 2】 This represents a connector element, where, E is, independently, any nucleotide, modified nucleotide, or debase at each instance; y and y' are independently between 0 and 9, where y+y' is equal to 0 and 8; L is structure 【Transformation 3】 (Here, X and X' are independently O or S; Y and Y' are independently OR'', SR'', or NRR'; V and V' are independently O, S, or NRR'; q is between 1 and 20; k is between 1 and 20; t is between 1 and 20; M is selected from aliphatic, substituted aliphatic, aryl, substituted aryl, heteroalkyl, heterocyclyl, or substituted heterocyclyl; W is any reactive group; and (d is either 0 or 1) (It is a non-nucleotide segment that has [a certain characteristic].)

27. The aqueous liquid pharmaceutical formulation according to claim 1, wherein the polynucleotide molecule comprises, consists of, or is essentially composed of SEQ ID NO: 13 and SEQ ID NO: 14, wherein the 3' end of SEQ ID NO: 13 is connected to the 5' end of SEQ ID NO: 14 via the non-nucleotide portion L1.

28. The aqueous liquid pharmaceutical formulation according to claim 1, wherein the polynucleotide molecule comprises, consists of, or is essentially composed of, the 3' end of SEQ ID NO: 13 and SEQ ID NO: 14, wherein the 3' end of SEQ ID NO: 13 is connected to the 5' end of SEQ ID NO: 14 via the non-nucleotide portion L2.

29. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 28, wherein the polynucleotide molecule comprises one or more sugar-modified nucleotides, each having a 2'OH (or 2'H) modification.

30. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 29, wherein the polynucleotide molecule comprises one or more skeletal modified nucleotides, and in particular, the skeletal modification comprises substitution of a phosphate ester group with a phosphorothioate group of a nucleotide.

31. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 30, wherein the polynucleotide molecule comprises one or more base-modified nucleotides.

32. The aqueous liquid pharmaceutical formulation according to any one of claims 1 to 31, wherein the average particle size of the stable colloidal particles is about 10 to about 1000 nm, for example, about 50 to about 1000 nm, for example, about 50 to about 750 nm, for example, about 50 to about 500 nm, for example, about 50 to about 400 nm, for example, about 50 to about 300 nm.

33. The aqueous liquid pharmaceutical formulation according to claim 32, wherein the average particle size of the stable colloidal particles is about 100 to about 300 nm.

34. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 33, wherein the aqueous liquid pharmaceutical preparation is suitable for local administration to the lungs or nose.

35. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 33, wherein the aqueous liquid pharmaceutical preparation is suitable for subcutaneous administration, for example, subcutaneous injection.

36. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 33, wherein the aqueous liquid pharmaceutical preparation is suitable for ocular administration, for example, intraocular administration or topical administration to the eye.

37. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 36, wherein the aqueous liquid pharmaceutical preparation does not contain protein.

38. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 37, wherein the aqueous liquid pharmaceutical preparation does not contain cationic lipopeptides, for example, the aqueous liquid pharmaceutical preparation does not contain polymyxin B.

39. The aqueous liquid pharmaceutical formulation according to any one of claims 1 to 38, wherein the aqueous liquid pharmaceutical formulation does not contain inorganic nanoparticles.

40. The aqueous liquid pharmaceutical formulation according to any one of claims 1 to 39, wherein the aqueous liquid pharmaceutical formulation does not contain lipid nanoparticles (LNPs) or liposomes.

41. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 40, wherein the aqueous liquid pharmaceutical preparation does not contain neutral lipids, for example, the aqueous liquid pharmaceutical preparation does not contain cholesterol or its analogues.

42. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 41, wherein the aqueous liquid pharmaceutical preparation does not contain cationic lipids.

43. The aqueous liquid pharmaceutical preparation according to any one of claims 1 to 42, wherein the aqueous liquid pharmaceutical preparation does not contain helper lipids, for example, the aqueous liquid pharmaceutical preparation does not contain dioleoylphosphatidylethanolamine (DOPE) or phosphatidylcholine.

44. An aqueous liquid pharmaceutical preparation according to any one of claims 1 to 43, for use as a pharmaceutical product.

45. The aqueous liquid pharmaceutical formulation for use according to claim 44, wherein the polynucleotide molecule is a polynucleotide molecule that stimulates or activates the innate and / or adaptive immune system and / or elicits a innate and / or adaptive immune response, for use in the treatment of diseases or illnesses treated by stimulation or activation of the innate and / or adaptive immune system and / or induction of a innate and / or adaptive immune response.

46. A method for treating a disease or illness treated by stimulation or activation of the innate and / or adaptive immune system and / or induction of a innate and / or adaptive immune response, comprising administering to a subject in need thereof a therapeutically or prophylactically effective amount of the aqueous liquid pharmaceutical preparation described in any one of claims 1 to 43, wherein the polynucleotide molecule is a polynucleotide molecule that stimulates or activates the innate and / or adaptive immune system and / or induces a innate and / or adaptive immune response.

47. Use of an aqueous liquid pharmaceutical preparation according to any one of claims 1 to 43, in the manufacture of a pharmaceutical preparation for use in the treatment of a disease or illness treated by stimulation or activation of the innate and / or adaptive immune system and / or induction of a innate and / or adaptive immune response, wherein the polynucleotide molecule is a polynucleotide molecule that stimulates or activates the innate and / or adaptive immune system and / or induces a innate and / or adaptive immune response.

48. An aqueous liquid pharmaceutical formulation, method, or use for use according to any one of claims 45 to 47, wherein the disease or illness is caused by a viral infection or is associated with such a viral infection.

49. The virus is SARS-CoV-2, and the disease associated with the viral infection is COVID-19; or The virus is a seasonal coronavirus, e.g., 229E, NL63, OC43, or HKU1, and the disease associated with the viral infection is a disease associated with seasonal coronavirus infection, e.g., 229E, NL63, OC43, or HKU1; or The virus is an influenza virus, and the disease associated with the viral infection is influenza; or The virus is respiratory syncytial virus (RSV), and the disease associated with the viral infection is a disease associated with RSV infection; or The virus is human rhinovirus (HRV), and the disease associated with the viral infection is a disease associated with HRV infection; or The virus is Middle East Respiratory Syndrome (MERS)-CoV, and the disease associated with the viral infection is MERS; or The virus is an avian influenza virus, and the disease associated with the viral infection is avian influenza; or The virus is Nipah virus, and the disease associated with the viral infection is a disease associated with Nipah virus infection; or The virus is human parainfluenza virus (HPIV), and the disease associated with the viral infection is a disease associated with HPIV infection; or The virus is human metapneumovirus (hMPV), and the disease associated with the infection is a disease associated with hMPV infection. The aqueous liquid pharmaceutical formulation, method, or use for use according to claim 48.

50. The aqueous liquid pharmaceutical formulation for use according to claim 44, wherein the polynucleotide molecule is a polynucleotide molecule that increases the endogenous expression of a protein or other gene product or increases the endogenous expression of a functional protein or other gene product, for use in the treatment of a disease or illness that is treated by increasing the endogenous expression of a protein or other gene product or by increasing the expression of a functional protein or other gene product.

51. A method for treating a disease or illness that is treated by increasing the endogenous expression of a protein or other gene product or by increasing the expression of a functional protein or other gene product, comprising administering a therapeutically or prophylactically effective amount of the aqueous liquid pharmaceutical preparation described in any one of claims 1 to 43 to a subject in need thereof, wherein the polynucleotide molecule is a polynucleotide molecule that increases the endogenous expression of a protein or other gene product or increases the endogenous expression of a functional protein or other gene product.

52. Use of an aqueous liquid pharmaceutical preparation according to any one of claims 1 to 43, in the manufacture of a pharmaceutical preparation for use in the treatment of a disease or illness treated by increasing the endogenous expression of a protein or other gene product or by increasing the expression of a functional protein or other gene product, wherein the polynucleotide molecule is a polynucleotide molecule that increases the endogenous expression of a protein or other gene product or a functional protein or other gene product.

53. An aqueous liquid pharmaceutical formulation, method, or use for use according to any one of claims 50 to 52, wherein the disease or illness is a respiratory disease or illness, for example, asthma, alpha-1 antitrypsin deficiency, chronic obstructive pulmonary disease (COPD), primary ciliary dyskinesia (PCD), pulmonary fibrosis, sarcoidosis, or cystic fibrosis.

54. An immunostimulatory composition which is an aqueous liquid formulation in the form of a stable colloidal emulsion, comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) an immunostimulatory polynucleotide molecule.

55. An immunostimulatory composition according to claim 54, for use in stimulating or activating an antiviral innate and / or adaptive immune response in a subject.

56. An immunogenic composition in aqueous liquid formulation, comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) a polynucleotide molecule encoding an immunogen.

57. A vaccine composition in aqueous liquid formulation, comprising (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant, and (ii) a polynucleotide molecule which encodes a vaccine immunogen.

58. The immunogenic composition or vaccine composition according to claim 56 or claim 57, further comprising an immunostimulating polynucleotide molecule.

59. An immunogenic composition or vaccine composition according to any one of claims 56 to 58, for use in inducing an immune response in a target, wherein the immune response is induced against an immunogen or vaccine immunogen encoded by the polynucleotide molecule.

60. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) one or more buffers; and (iv) a diluent.

61. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) one or more buffers selected from citrates and phosphates; and (iv) a diluent selected from isotonic saline (0.9% w / v) and sterile water or purified water.

62. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) one or more buffering agents; (iv) a diluent; (v) optionally one or more suspending agents; (vi) optionally one or more wetting agents or thickeners; and (vii) optionally one or more osmotic pressure or tonicity modifiers.

63. An aqueous liquid pharmaceutical preparation comprising or essentially consisting of: (i) a surfactant component which is a mixture of a fatty acid or a pharmaceutically acceptable salt thereof and a nonionic surfactant; (ii) a polynucleotide molecule; (iii) one or more buffers selected from citrates and phosphates; (iv) a diluent selected from isotonic saline (0.9% w / v) and sterile water or purified water; (v) optionally one or more suspending agents; (vi) optionally one or more wetting agents or thickeners; and (vii) optionally one or more osmotic pressure or tonicity modifiers.

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