Formulation and composition for treatment of obstructive lung diseases

Abstract

The present disclosure provides therapeutic compositions and methods for concurrently downregulating two or more biological targets. In certain embodiments, at least one target is an upstream mediator of an inflammatory cascade, and at least one additional target is involved in tissue remodeling or cellular signal transduction. The disclosed compositions are designed to modulate multiple disease-associated pathways to improve therapeutic outcomes.

Description

RNHA.002WO PATENTFORMULATION AND COMPOSITION FOR TREATMENT OF OBSTRUCTIVELUNG DISEASESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to German Provisional Patent Application No. 102024 135 731.2, filed December 2, 2024, the entire content of which is incorporated by reference herein.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled RNHA002WOSequenceListing.xml, which was created and last modified on December 1, 2025, which is 27,259 bytes in size. The information in the electronic Sequence Listing is hereby incorporated by reference in its entirety.BACKGROUNDFIELD

[0003] The present disclosure relates to the field of multispecific gene-targeting therapeutics for the treatment and prevention of respiratory diseases. More specifically, the disclosure provides dry-powder lipid nanoparticle (LNP) compositions engineered to encapsulate one or more therapeutic nucleic acids, such as siRNA, antisense oligonucleotides, or miRNA constructs, that downregulate two or more molecular targets implicated in respiratory pathology. In certain embodiments, the compositions are optimized for inhalation or pulmonary delivery to enable localized, durable, and coordinated modulation of multiple gene pathways within the respiratory tract. The disclosed formulations and delivery approaches are designed to enhance therapeutic potency, broaden biological activity, and address complex or multifactorial respiratory conditions.DESCRIPTION OF RELATED ART

[0004] Multispecific gene-targeting strategies have been investigated for the treatment of respiratory diseases using a variety of therapeutic modalities, including monoclonal antibodies, bispecific antibodies, and bispecific nanobody constructs. While these protein-based therapies can engage more than one molecular target, they often suffer from significant limitations. Systemic administration can lead to off-target effects and reduced drug concentrations at the site of pulmonary disease. In addition, the complexity of manufacturing multispecific antibodies and nanobodies, along with challenges in achieving synchronized modulation of multiple gene products, can restrict their therapeutic impact. These limitations highlight the need for therapies that provide precise, localized, and coordinated regulation of multiple pathogenic pathways directly within the respiratory tract. Multispecific genesilencing approaches using nucleic-acid-loaded delivery systems offer a promising advancement by enabling targeted pulmonary delivery, reduced systemic exposure, and efficient downregulation of multiple genes involved in complex or multifactorial respiratory conditions.SUMMARY

[0005] Without integrated targeting strategies, current treatments for obstructive and restrictive lung diseases, including, for example, asthma, COPD, and related inflammatory conditions, provide only partial clinical benefit, failing to adequately address both airway inflammation and the underlying tissue remodeling that drives persistent exacerbations. Existing therapies, including mono-specific and bispecific antibody drugs directed at TSLP and downstream cytokines, show limited efficacy in disease and do not reverse structural changes in the lung. In addition, reliance on single-pathway inhibition restricts patient coverage across heterogeneous phenotypes and leaves key pathogenic mechanisms, such as VEGFA-mediated remodeling and ADAMI 7-driven epithelial dysfunction, insufficiently treated. To overcome these limitations, multispecific therapeutic approaches integrating TSLP inhibition with additional targets, including VEGFA and ADAMI 7, are provided herein in several embodiments.

[0006] Several embodiments provide lipid nanoparticle compositions. In some embodiments, the compositions include one or more lipid nanoparticles, a first siRNA thattargets an mRNA encoding thymic stromal lymphopoietin (TSLP), a second siRNA that targets an mRNA encoding disintegrin and metalloprotease 17 (ADAMI 7), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), phosphodiesterase 3 (PDE3), or phosphodiesterase 4 (PDE4). In some embodiments, the first siRNA and the second siRNA are encapsulated within the one or more lipid nanoparticles. In some embodiments, the composition is formulated as a powder. In some embodiments, the first siRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 7-26, or a sequence at least 85% identical thereto. In some embodiments, the second siRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 1-6, or a sequence at least 85% identical thereto. In some embodiments, the compositions further include a third siRNA that targets an mRNA different from the second siRNA target, and which encodes ADAMI 7, VEGF, CTGF, PDE3, or PDE4. In some embodiments, the third siRNA is encapsulated within the one or more lipid nanoparticles. In some embodiments, the third siRNA includes a nucleic acid sequence of any one of SEQ ID NOs: 1-6, or a sequence at least 85% identical thereto. In some embodiments, the second siRNA targets an mRNA encoding ADAMI 7, and the third siRNA targets an mRNA encoding VEGF. In some embodiments, the second siRNA targets an mRNA encoding ADAMI 7, and the third siRNA targets an mRNA encoding CTGF. In some embodiments, the second siRNA targets an mRNA encoding ADAMI 7, and the third siRNA targets an mRNA encoding PDE3. In some embodiments, the second siRNA targets an mRNA encoding ADAMI 7, and the third siRNA targets an mRNA encoding PDE4, wherein the PDE4 is PDE4A, PDE4B, PDE4C, or PDE4D. In some embodiments, the second siRNA targets an mRNA encoding VEGF, and the third siRNA targets an mRNA encoding CTGF. In some embodiments, the second siRNA targets an mRNA encoding VEGF, and the third siRNA targets an mRNA encoding PDE3. In some embodiments, the second siRNA targets an mRNA encoding VEGF, and the third siRNA targets an mRNA encoding PDE4. In some embodiments, the second siRNA targets an mRNA encoding CTGF, and the third siRNA targets an mRNA encoding PDE3. In some embodiments, the second siRNA targets an mRNA encoding CTGF, and the third siRNA targets an mRNA encoding PDE4. In some embodiments, the second siRNA targets an mRNA encoding PDE3, and the third siRNA targets an mRNA encoding PDE4.

[0007] Several embodiments provide lipid nanoparticle compositions that include one or more lipid nanoparticles, a first siRNA that targets expression of an mRNA involved inan upstream inflammatory cascade, and a second siRNA that targets an mRNA encoding for a tissue remodeling factor or an mRNA encoding for a signal transduction regulator. In some embodiments, the first siRNA and the second siRNA are encapsulated within the one or more lipid nanoparticles. In some embodiments, the first siRNA targets an mRNA that encodes TSLP. In some embodiments, the second siRNA targets an mRNA that encoding ADAM 17, VEGF, CTGF, PDE3, or PDE4.

[0008] Several embodiments provide lipid nanoparticle compositions that include one or more lipid nanoparticles, a first siRNA that targets an mRNA encoding TSLP, a second siRNA that targets an mRNA encoding ADAMI 7, VEGF, CTGF, PDE3, or PDE4, and a third siRNA that targets an mRNA different from the second siRNA target, and that encodes ADAMI 7, VEGF, CTGF, PDE3, or PDE4. In some embodiments, the first siRNA, the second siRNA, and the third siRNA are encapsulated within the one lipid nanoparticles. In some embodiments, the first siRNA includes a nucleic acid sequence of any one of SEQ ID NOs: 7- 26, or a sequence at least 85% identical thereto. In some embodiments, the second siRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 1-6, or a sequence at least 85% identical thereto. In some embodiments, the third siRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 1-6, or a sequence at least 85% identical thereto.

[0009] In some embodiments, any of the one or more lipid nanoparticles comprise at least one ionizable lipid. In some embodiments, the at least one ionizable lipid is Cl 2-200, DOTAP (l,2-dioleyl-3-trimethytammonium propane), DODAP (l,2-dioleyl-3- dimethylammonium propane), DOTMA (l,2-di-O-octadecenyl-3 -trimethylammonium propane), DLinDMA, DLin-KC2-DMA, HGT4003, cKK-E12, ICE, DLin-MC3, 7C1, ALC- 0315, SM-102, CL-1, 3060110, OF-02, 7C1, L319, A9, 93O17S, Lipid C24, 1014, Lipidl5, Lipid AX4, Lipid A6, BAMEA-O16B, 98N12-5, 4A3-SC8, 5A2-SC8, or a combination thereof. In some embodiments, the one or more lipid nanoparticles comprise a helper lipid or a stealth lipid. In some embodiments, the helper lipid comprises a sterol, and a phospholipid, ester lipid, lysophospholipid, or a glycerol lipid. In some embodiments, the compositions further include an excipient. In some embodiments, the excipient comprises a mono-, di-, tri-, oligo-, or polysaccharide, sugar alcohol, mono-, di, tri, oligo-, or polypeptide, protein, ester, ether, amide, amine, sulphate, thios, urethane, phosphoester, phosphazene, amino acid, surfactant, lipid, stearate, polymer, or a combination thereof. In some embodiments, thecomposition is formulated as a dry powder having a residual moisture of 10% or less. In some embodiments, the composition is formulated for inhalable administration, enteral administration, transdermal administration, or parenteral administration. In some embodiments, the composition is formulated for respiratory tract administration. In some embodiments, the composition is formulated for administration with an inhaler device.

[0010] Several embodiments provide for methods of delivering the lipid nanoparticle compositions described herein to a respiratory tract of a subject. In some embodiments, the methods include administering any of the compositions described herein to the subject. In some embodiments, the administering is performed using an inhaler device.

[0011] Several embodiments provide for methods of treating or preventing a respiratory tract disease in a subject. In some embodiments, the methods include administering to a lung of the subject any of the lipid nanoparticle compositions described herein. In some embodiments, the administering is performed using an inhaler device. In some embodiments, the respiratory tract disease is asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, bronchiectasis, cystic fibrosis, or a viral infection. In some embodiments, the asthma is allergic asthma, eosinophilic asthma, or non-eosinophilic asthma. In some embodiments, the COPD is emphysema and / or chronic bronchitis. In some embodiments, expression of TSLP is downregulated by the first siRNA through the inhibition of translation of the mRNA encoding for TSLP. In some embodiments, expression of ADAMI 7, VEGF, CTGF, PDE3, or PDE4 is downregulated by the second siRNA through the inhibition of translation of the mRNA encoding for ADAMI 7, VEGF, CTGF, PDE3, or PDE4. In some embodiments, the methods further include administering one or more therapeutic compositions to the subject. In some embodiments, the one or more therapeutic compositions are cystic fibrosis transmembrane conductance regulator (CFTR) modulators and / or potentiators. In some embodiments, the methods decreases and / or alleviates symptoms of the respiratory tract disease and / or restores natural lung physiology.

[0012] Several embodiments provide for uses of any of the compositions described herein for treatment of a respiratory tract disease. In some embodiments, the respiratory tract disease is asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, bronchiectasis, cystic fibrosis, or a viral infection. In some embodiments, the asthmais allergic asthma, eosinophilic asthma, or non-eosinophilic asthma. In some embodiments, the COPD is emphysema and / or chronic bronchitis.

[0013] Several embodiments provide for kits that include any of the lipid nanoparticle compositions described herein, and packaging. In some embodiments, the packaging comprises a capsule, a blister, or a reservoir. In some embodiments, the packaging is multidose or single dose packaging. In some embodiments, the kits further include a nebulizer or an inhaler device.

[0014] Several embodiments provide for therapeutic formulations that downregulates two or more targets, for example, target genes, with at least one of which is upstream of an inflammatory cascade, and at least one of which is encoding for a tissue remodeling factor or a signal transduction regulator. In some embodiments, the target genes are downregulated through the inhibition of translation of the corresponding messenger RNAs. In some embodiments, the downregulation is achieved by the mechanism of RNA interference. In some embodiments, one of the upstream genes of the inflammatory cascade is encoding for an alarmin. In some embodiments, the alarmin is thymic stromal lymphopoietin (TSLP). In some embodiments, one of the genes encoding a tissue remodeling factor encode for a disintegrin and metalloprotease 17 (ADAMI 7), also called tumor necrosis factor-a-converting enzyme (TACE), or vascular endothelial growth factor (VEGF), in particular vascular endothelial growth factor A (VEGF A) or one of the signal transduction regulator encodes for a Phosphodiesterase (PDE) in particular, PDE3 or PDE4. In some embodiments, the therapeutic use is for pulmonary diseases. In some embodiments, the therapeutic use is for allergic asthma. In some embodiments, the therapeutic use is for COPD. In some embodiments, the therapeutic use is for idiopathic pulmonary fibrosis. In some embodiments, the therapy is supposed to decrease symptoms and restore the natural lung physiology. In some embodiments, the therapeutic formulation can be administered to the lung. In some embodiments, the active pharmaceutical ingredient (API) is or comprises an RNA capable of initiating the RNAi mechanism, preferably an antisense oligonucleotide, dsRNA, siRNA, or an RNA Conjugate.

[0015] Several embodiments relate to the compositions of the therapeutic formulations described herein having an active pharmaceutical ingredient encapsulate in a carrier particle. In some embodiments, the carrier particle is a nanoparticle. In someembodiments, the encapsulation structure is a lipid structure and / or polymer structure. In some embodiments, the lipids are selected from at least one of a cationic (ionizable) lipid, one or more helper lipids, and / or a stealth lipid. In some embodiments, at least two helper lipids are used and one of the helper lipids is a phospholipid, ester lipid, lysophospholipid, or a glycerol lipid and the second is a sterol. In some embodiments, the cationic (ionizable) lipid is selected from the group consisting of C12-200, DOTAP (1,2- dioleyl-3 -trimethytammonium propane), DODAP (l,2-dioleyl-3 -dimethylammonium propane), DOTMA (l,2-di-O-octadecenyl-3- trimethylammonium propane), DLinDMA, DLin-KC2-DMA, HGT4003, cKK-E12, ICE, DLin-MC3, 7C1, ALC-0315, SM-102, CL-1, 3060110, OF-02, 7C1, L319, A9, 93O17S, Lipid C24, 1014, Lipidl5, Lipid AX4, Lipid A6, BAMEA-016B, 98N12-5, 4A3-SC8, 5A2-SC8 and combinations thereof. In some embodiments, the encapsulation structure is co-processed together with at least one excipient into the form of a dry powder. In some embodiments, the at least one pharmaceutically acceptable excipient is selected from the group consisting of mono-, di- tri- oligo- or polysaccharides, sugar alcohols, mono-, di, tri, oligo- or polypeptides, proteins, esters, ethers, amides, amines, sulphates, thiols urethanes, phosphoesters, phosphazenes, amino acids, surfactants, lipids, stearates, polymers and combination thereof. In some embodiments, the dry powder has a residual moisture of less than 10%, preferably less than 8%, most preferably less than 5%. In some embodiments, the dry powder can at least partially be redispersed. In some embodiments, the composition is formulated for use as a pharmaceutical dosage form, especially for pulmonary delivery. In some embodiments, the composition is formulated for use to adapt the penetration of the particulate system through the natural barriers of the lung and / or airways of the respiratory tract. In some embodiments, the composition is formulated such that the pharmaceutical dosage form can be administered using dry powder inhaler devices.BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The following drawings are for illustrative purposes only and show nonlimiting embodiments. Features from different figures may be combined in several embodiments.

[0017] FIGs. 1A-1C show embodiments of target genes in asthma pathogenesis. FIG. 1A shows a schematic depicting the role of target gene TSLP in asthma pathogenesis.FIG. IB shows a schematic depicting the role of target gene ADAMI 7 in asthma pathogenesis. FIG. 1C shows a schematic depicting the role of target gene VEGF in asthma pathogenesis.

[0018] FIG. 2 shows embodiments of the multimodal treatment opportunities for the therapeutic formulations, including the target genes for three lung disease indications (severe asthma, chronic obstructive pulmonary disease, and idiopathic pulmonary fibrosis).

[0019] FIGs. 3A-3B show embodiments of lung diseases, asthma (FIG. 3A) and idiopathic pulmonary fibrosis (FIG. 3B), including the complex biology related to external stimuli, injuries, and intracellular signaling.

[0020] FIG. 4 shows embodiments of target genes phosphodiesterase 3 (PDE3) and phosphodiesterase 4 (PDE4). FIG. 4 schematic depicting the effects of inhibiting target genes PDE3 and PDE4 on respiratory function at the cellular and tissue level.

[0021] FIG. 5 shows embodiments of target cells for the therapeutic formulations simultaneously targeting TSLP, PDE3, and / or PDE4.

[0022] FIGs. 6A-6C shows embodiments of the compositions with therapeutic formulations delivered to human precision cut lung slices (PCLS). FIG. 6A shows a schematic depicting the pharmacodynamics of anti-TLSP nano-embedded in-microparticles (NEMs) compositions delivered to lung tissue. FIG. 6B shows the fold-change of TSLP gene expression in PCLS samples treated with embodiments of the compositions with therapeutic formulations, as measured by quantitative PCR. FIG. 6C shows TLSP protein expression in PCLS samples treated with embodiments of the compositions with therapeutic formulations, as measured by ELISA.

[0023] FIGs. 7A-7C show embodiments of dual-targeting siRNA compositions evaluated in A549 cells. FIG. 7A shows fold-change in TSLP and PDE4B gene expression in A549 cells pretreated with two siRNAs for 24 hours and triggered with poly(I:C) for 6 hours, as measured by quantitative PCR. FIG. 7B shows fold-change in TSLP and VEGF-A gene expression under the same conditions. FIG. 7C shows fold-change in TSLP and ADAMI 7 gene expression. Single-target controls were transfected with a control siRNA as the second siRNA.

[0024] FIGs. 8A-8D show physicochemical characterization of lipid nanoparticle (LNP) formulations encapsulating dual siRNAs. FIG. 8A shows hydrodynamic diameter for LNPs containing siRNAs targeting TSLP and PDE4B, VEGF-A, or ADAMI 7. FIG. 8B showspolydispersity index of LNPs containing siRNAs targeting TSLP and PDE4B, VEGF-A, or ADAMI 7. FIG. 8C shows zeta potential measurements for single and combined LNPs. FIG. 8D shows encapsulation efficiency for each formulation.

[0025] FIGs. 9A and 9B show gene expression modulation in air-liquid interface (ALI) cultures treated with dual-siRNA LNPs. FIG. 9A shows fold-change in TSLP and PDE4B gene expression after pretreatment with LNPs for 24 hours and poly(I:C) stimulation for 6 hours, as measured by qPCR. FIG. 9B shows fold-change in TSLP and VEGF-A gene expression under the same conditions.

[0026] FIG. 10 shows epithelial barrier integrity in ALI cultures after 48 hours pretreated for 24 hours with siRNAs targeting TSLP and PDE4B.

[0027] FIG. 11 shows particle size distribution of dry powder dual-siRNA LNA formulations for inhalation delivery.

[0028] FIG. 12 shows fine particle fraction (percent of particles in the size range between 1 and 5 pm) and mass median diameter (xso) of dry powder containing combinatorial siRNA LNP for inhalation delivery.

[0029] FIG. 13 shows residual water content of dry powder containing combinatorial siRNA LNP for inhalation delivery.

[0030] FIG. 14 shows x-ray powder diffraction pattern of dry powder containing combinatorial siRNA LNP for inhalation delivery.DETAILED DESCRIPTION

[0031] The foregoing and other aspects of the present disclosure are described in more detail with respect to the description and methodologies provided herein. This description is not intended to be a detailed catalogue of all the ways in which the embodiments of the present disclosure may be implemented, or of all the features that may be added to the present disclosure. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be absent from that embodiment. In addition, variations and additions to the various embodiments described herein, which do not depart from the instant disclosure, are encompassed herein. Hence, the following specification is intended to illustrate some particularembodiments, and not to exhaustively specify all permutations, combinations and variations thereof.

[0032] Lung conditions can be classified as obstructive lung disease or restrictive lung disease. Obstructive lung diseases include conditions that make it hard to exhale all of the air in your lungs. Individuals with restrictive lung disease have trouble fully expanding their lungs with air. Obstructive and restrictive lung disease share the same main symptom: shortness of breath during exertion. Individuals with obstructive lung disease have shortness of breath because it is difficult for them to exhale all the air from their lungs. Due to the damage to the lungs or narrowing of the airways inside the lungs, the exhaled air comes out more slowly than it should. At the end of a full exhalation, too much air may linger in the lungs. As the breathing rate increases, there is even less time to breathe all the air out before inhalation. The most common causes of obstructive lung disease are chronic obstructive pulmonary disease (COPD), emphysema, chronic bronchitis, asthma, bronchiectasis, and / or cystic fibrosis.

[0033] The rationale for multispecific targeting in respiratory diseases includes, for example, improving efficacy by simultaneously and synergistically targeting different diseasedriving pathways, addressing unmet medical needs and broadening patient populations (e.g., Th2, non-Th2, mixed phenotype), addressing targets from different compartments (e.g. cytokines or extracellular matrix targets or intracellular), simplifying the therapeutic development and regulatory pathway compared to developing monotherapies independently for later combination, and / or addressing the pharmaceutical lifecycle management with relation to existing mono-specific drugs.

[0034] Treatment approaches that address obstructive lung diseases on the market so far are antibody therapies, bi-specific antibody therapies, and bi-specific nanobody therapies. The current available bi-specific antibodies target an upstream alarmin, thymic stromal lymphopoietin (TSLP), and downstream cytokines (IL4, IL5, IL13, or IL33) (Figure 6A), primarily addressing eosinophilic asthma, in order to reduce exacerbation rates. However, these treatments do not reverse lung tissue remodeling, a factor contributing to persistently high exacerbation rates. Eosinophilic asthma is a severe form of non-allergic asthma. Eosinophilic granulocytes play an important role in immune defense. These defense cells play a significant role in overreactions of the immune system. If the body produces an excessivenumber of eosinophilic granulocytes, it can cause inflammation in the lungs. These inflammations can then in turn lead to asthma attacks in eosinophilic asthma patients.

[0035] There remains an unmet medical need for treatment or prevention of respiratory diseases that have limited options and / or limited efficacy with current treatments, including, for example, asthma patients suffering noneosinophilic asthma. Tezepelumab is the only effective option for these patients, as other cytokine-dependent drugs have failed as treatment options in non-eosinophilic asthma patients. Additionally, tissue remodeling in asthma leads to increased hospitalization and morbidity, which is a major issue not adequately addressed by current treatments and pipeline drugs. Further, recent in-silico analyses have highlighted top-tier targets, including ADAMI 7, a disintegrin and metalloprotease 17, also called TACE (tumor necrosis factor-a- converting enzyme), which is a 70-kDa enzyme that belongs to the ADAM protein family of disintegrins and metalloproteases, activated by substrate presentation.

[0036] The present disclosure relates to the integration of targets, such as ADAMI 7, with clinically validated targets, such as TSLP, as a therapeutic approach to reduce exacerbation rates and open new pathways in treatment and prevention of respiratory diseases, such as asthma, by promoting lung health restoration. This disruptive approach addresses the root cause of airway inflammation by inhibiting TSLP, while simultaneously reducing tissue remodeling through siRNA targeting VEGF, for example, VEGFA (Vascular endothelial growth factor A), and / or siRNA targeting ADAMI 7. Inhibiting VEGF, in particular VEGFA, as well as ADAMI 7 has shown to be effective in reducing tissue remodeling in pulmonary fibrosis studies. One advantage of this approach relates to the therapeutic pipeline expansion for multiple indications, as TSLP, VEGF, in particular VEGFA, and ADAMI 7 play critical roles across fibrotic and obstructive lung diseases, including idiopathic pulmonary fibrosis (IPF) and COPD. In some embodiments, the present disclosure relates to compositions with therapeutic formulations that address the previously unmet clinical need for treating noneosinophilic asthma patients.

[0037] In some embodiments, the present disclosure relates to compositions with therapeutic formulations that downregulate two or more targets, using the mechanism of RNA interference (RNAi). In some embodiments, the therapeutic formulation includes small interfering RNA (siRNA).

[0038] There are multiple advantages related to using the siRNA technology according to embodiments of the present disclosure. An siRNA technology platform is suitable for combination of target specificities, and has potential for improved efficacy in respiratory diseases due to a broader effect on immune cells, airway remodeling, and mucociliary clearance (MCC). Phosphodiesterases (PDEs) are intracellular targets and not tractable by other biologies. Local treatment will reduce off-target and systemic side effects reported for oral small molecule PDE4 inhibitors (Figure 4). Dry powder inhalators (DPI) utilizing embodiments of the siRNA technology of the present disclosure provide less frequent and faster dosing for better patient compliance and adherence compared other therapeutics on the market, for example, ensifentrine, a phosphodiesterase 3 and 4 inhibitor administered by jet nebulizer. Further, embodiments of the siRNA technology balance out the inhibitory activity against PDE3 versus PDE4, observed in other therapeutics, for example, ensifentrine is 3000 times more potent on PDE3. There are also novel therapeutic options using the siRNA technology according to embodiments of the present disclosure in combination with existing treatment options. For example, treating cystic fibrosis patients with embodiments of the compositions with therapeutic formulations in combination with existing Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) potentiators and / or modulators. Examples of target genes in respiratory pathogenesis are shown in Table 1 (see also Figures 3A and 5).Table 1: Target genes in respiratory pathogenesisCompositions

[0039] Several embodiments provided herein relate to lipid nanoparticle (LNP) compositions suitable for delivering nucleic acids or other therapeutics. In some embodiments, the compositions include one or more lipid nanoparticles formulated to encapsulate or associate with a therapeutic payload. The lipid nanoparticles may comprise a membrane, such as a lipid membrane, that partially or fully surrounds and protects one or more nucleic acids or other therapeutic agents during storage, handling, administration, and / or delivery. The membrane may be a lipid membrane, may include lipid portions, or may be a non-lipid membrane, and may be present in single-layer or multi-layer configurations.

[0040] In some embodiments, the lipid nanoparticles are further embedded within or coated by a matrix comprising one or more excipients, or combinations thereof, that are configured to preserve the structural and functional integrity of the nanoparticles during drying. Such matrices may stabilize the lipid nanoparticles during processes including lyophilization, spray-drying, or other drying techniques, thereby facilitating the manufacture of solid or semisolid dosage forms. The use of a protective matrix helps maintain encapsulation efficiency, membrane integrity, and bioactivity of the therapeutic payload following reconstitution or administration.

[0041] In some embodiments, the compositions are configured to deliver nucleic acid cargo to a target cell, enabling the therapeutic agent to exert its intended biological effect. In various embodiments, the composition is formulated as a dry powder, permitting improved stability, ease of transport, and suitability for non-parenteral delivery platforms. Dry powder LNP formulations may also offer advantages in logistical settings where cold-chain storage is impractical or where long-term shelf stability is desired.

[0042] Several embodiments relate particularly to dry powder lipid nanoparticle compositions comprising a lipid membrane with one or more of the following components: an ionizable amino lipid (e.g., a cationic ionizable amino lipid, CP-LC-0729, or another ionizableamino lipid), cholesterol, DSPC, and a PEG-containing lipid (e.g., PEG-DMG 2000). The compositions may further include one or more siRNA molecules encapsulated or associated with the lipid nanoparticles. In certain embodiments, the lipid nanoparticles are embedded in a matrix comprising lactose, which functions as a stabilizing excipient that preserves nanoparticle structure during the drying process. In these embodiments, the overall composition is formulated as a dry powder suitable for storage and subsequent delivery.

[0043] Embodiments of the compositions provide versatile LNP-based formulations capable of stabilizing and delivering nucleic acids or other therapeutic molecules in a dry powder format. The structural features of the membrane, the choice of lipid components, and the inclusion of excipient matrices allow for tailored stability, delivery, and performance characteristics across a range of therapeutic applications.

[0044] In some embodiments, the compositions include a first small interfering RNA (siRNA) and a second siRNA, each of which may be formulated within the same lipid nanoparticle system. The first siRNA and second siRNA may be co-encapsulated within one or more lipid nanoparticles. In some embodiments, the first and second siRNA are delivered simultaneously to a target cell or tissue. Co-encapsulation may facilitate synergistic genesilencing effects, coordinated modulation of multiple biological pathways, and / or enhanced therapeutic outcomes relative to the use of a single siRNA.

[0045] In some embodiments, each siRNA molecule may have a length ranging from about 17 to about 30 nucleotides, such as from 19 to 23 nucleotides, or approximately 21 nucleotides in length. siRNA molecules may include sense and antisense strands that hybridize to form a duplex, and in some embodiments, the antisense strand is the guide strand that directs RNA-induced silencing complex (RISC) activity. The siRNA may include one or more chemical modifications — such as 2'-O-methyl modifications, 2'-fluoro modifications, phosphorothioate internucleotide linkages, locked nucleic acids (LNA), terminal conjugates, or overhang modifications — to improve nuclease resistance, enhance stability, modulate RISC loading, or reduce off-target effects. Example siRNA sequences are shown in Table 2.Table 2: siRNA Sequences

[0046] The siRNA sequences disclosed herein may include a sense strand and an antisense strand. In some embodiments, the sense and antisense strand form a duplex. In some embodiments, the sequence of the sense and / or antisense strand includes 17 to 23 nucleotides, such as 17, 18, 19, 20, 21, 22, or 23 nucleotides. In some embodiments, the sequence includes a 3' overhang, such as a 2-nucleotide overhang. In some embodiments, the 2-nucleotideoverhang is non-complementary to the target mRNA. In some embodiments, the sequence includes one or more chemical modifications, including, for example, a 2'-O-methyl, 2 '-fluoro, vinyl-phosphonate, locked nucleic acids (LN A), and phosphorothioate linkages.

[0047] In some embodiments, the sense strand and / or the antisense strand comprises 17 to 23 nucleotides selected from the group consisting of unmodified nucleotides and modified nucleosides. In some embodiments, each linkage between the nucleosides is a phosphorothioate, phosphodiester, phosphoramidate, thiophosphoramidate, methylphosphate, methylphosphonate, boranophosphate, or any combination thereof. In some embodiments, the siRNA is at least 85% complementary to a fragment of a target mRNA, such as TSLP, ADAMI 7, VEGF, and / or a PDE. In some embodiments, the siRNA comprises zero, one, or two mismatches to the fragment of a target mRNA. In some embodiments, the mismatches occur at any one or more of positions 1 or 9 through m, wherein m is the total number of nucleotides in the antisense strand. In some embodiments, the mismatches do not occur at a seed region of the siRNA. In some embodiments, the seed region is at positions 2-8. In some embodiments, the modified sugar is selected from the group consisting of 2’-0Me, 2’-F, 2’- MOE, 2’-araF, 2’-OEt, 2’-O-alkyl, LNA, scpBNA, AmNA, cEt, ENA, and GNA. In some embodiments, the antisense strand comprises a 5 ’-phosphate group or a 5 ’-phosphate mimic. In some embodiments, the 5’-phosphate mimic is a 5’-vinylphosphonate.

[0048] In certain embodiments, one or both of the siRNA molecules is a therapeutic siRNA that targets an mRNA of interest. As used herein, an “mRNA of interest” refers to an mRNA whose reduction, degradation, inhibition, or suppression by an siRNA produces a therapeutic, prophylactic, or otherwise beneficial biological effect. The mRNA of interest may encode a protein associated with or related to a disease or disorder, including, for example, a respiratory disease or disorder such as asthma, chronic obstructive pulmonary disease (COPD), bronchitis, allergic airway inflammation, pulmonary fibrosis, or other inflammatory or fibrotic respiratory conditions, although the compositions are not limited to such applications. Targeting disease-associated mRNA transcripts enables selective downregulation of pathogenic protein expression and provides a mechanistically precise therapeutic approach.

[0049] In some embodiments, the siRNA targets an mRNA that encodes thymic stromal lymphopoietin (TSLP), a cytokine implicated in airway inflammation and allergic disease; disintegrin and metalloprotease 17 (ADAMI 7), a sheddase involved in cytokineactivation and epithelial signaling; vascular endothelial growth factor (VEGF), a regulator of angiogenesis; connective tissue growth factor (CTGF), which is implicated in fibrosis and tissue remodeling; phosphodiesterase 3 (PDE3); or phosphodiesterase 4 (PDE4), both of which modulate cyclic nucleotide signaling pathways. Targeting any of these transcripts may confer therapeutic benefits in conditions where aberrant gene expression contributes to disease pathogenesis. In some embodiments, the compositions target an mRNA that encodes a PDE4 isoform, such as PDE4A, PDE4B, PDE4C, and / or PDE4D. In some embodiments, targeting of PDE4B isoform is associated with anti-inflammatory effects in COPD, and minimizes, reduces, and / or ameliorates nausea and / or vomiting associated with PDE4D inhibition.

[0050] In some embodiments, the compositions include multiple siRNA molecules targeting multiple distinct mRNA transcripts, including any combination of the aforementioned targets. For example, the first siRNA may target an mRNA that encodes TSLP, ADAMI 7, VEGF, CTGF, PDE3, or PDE4, and the second siRNA may target a different mRNA that encodes TSLP, ADAMI 7, VEGF, CTGF, PDE3, or PDE4. In some embodiments, the first siRNA targets an mRNA that encodes TSLP, and the second siRNA targets an mRNA that encodes ADAMI 7. In some embodiments, the first siRNA targets an mRNA that encodes TSLP, and the second siRNA targets an mRNA that encodes VEGF. In some embodiments, the first siRNA targets an mRNA that encodes TSLP, and the second siRNA targets an mRNA that encodes CTGF. In some embodiments, the first siRNA targets an mRNA that encodes TSLP, and the second siRNA targets an mRNA that encodes PDE3. In some embodiments, the first siRNA targets an mRNA that encodes TSLP, and the second siRNA targets an mRNA that encodes PDE4. In some embodiments, the first siRNA targets an mRNA that encodes ADAMI 7, and the second siRNA targets an mRNA that encodes VEGF. In some embodiments, the first siRNA targets an mRNA that encodes ADAMI 7, and the second siRNA targets an mRNA that encodes CTGF. In some embodiments, the first siRNA targets an mRNA that encodes ADAMI 7, and the second siRNA targets an mRNA that encodes PDE3. In some embodiments, the first siRNA targets an mRNA that encodes ADAMI 7, and the second siRNA targets an mRNA that encodes PDE4. In some embodiments, the first siRNA targets an mRNA that encodes VEGF, and the second siRNA targets an mRNA that encodes CTGF. In some embodiments, the first siRNA targets an mRNA that encodes VEGF, and the second siRNA targets an mRNA that encodes PDE3. In some embodiments, the first siRNA targets an mRNAthat encodes VEGF, and the second siRNA targets an mRNA that encodes PDE4. In some embodiments, the first siRNA targets an mRNA that encodes CTGF, and the second siRNA targets an mRNA that encodes PDE3. In some embodiments, the first siRNA targets an mRNA that encodes CTGF, and the second siRNA targets an mRNA that encodes PDE4. In some embodiments, the first siRNA targets an mRNA that encodes PDE3, and the second siRNA targets an mRNA that encodes PDE4. In some embodiments, the compositions include a third siRNA, a fourth siRNA, or more, each targeting a different mRNA target, including any of the aforementioned targets.

[0051] In some embodiments, the compositions include multiple siRNA molecules targeting multiple distinct mRNA transcripts, including any of the targets listed above. In certain aspects, each siRNA may be present in an amount sufficient to provide a therapeutically effective level of gene silencing, such as reduction of the target mRNA by at least 20%, 30%, 50%, 70%, 80%, 90%, or greater, as measured using any suitable analytical method. The ratio of the first siRNA to the second siRNA (or to a third siRNA or more) may vary and may include, for example, weight ratios ranging from 1 :1 to 1 :10, 1 :5 to 5:1, or other ratios appropriate for the intended biological effect.

[0052] In some embodiments, the siRNA molecules are encapsulated within one or more lipid nanoparticles, and encapsulation efficiency may range from 50% to greater than 95%, depending on formulation parameters. Encapsulation may protect the siRNA from degradation, promote cellular uptake, and facilitate delivery into the cytoplasm where RISC- mediated gene silencing occurs. The siRNA molecules may be present in a concentration suitable for inhalation, parenteral administration, or other delivery routes, depending on the formulation.

[0053] Examples of suitable siRNA include, without limitation, siRNA sequences specifically engineered to target human TSLP transcripts (e.g., targeting exon regions, 3' untranslated regions (UTRs), or splice junctions), siRNA directed to human ADAMI 7 catalytic domain mRNA, siRNA targeting VEGF-A isoforms, or siRNA sequences validated for PDE3A / B or PDE4D transcripts. Additional examples may include siRNA variants optimized for thermodynamic asymmetry, sequence-specific RISC loading, or reduced innate immune activation.

[0054] These embodiments collectively provide flexible compositions capable of incorporating one or more siRNA species for single-target or multi-target therapeutic strategies. The ability to tailor siRNA length, chemical modifications, target selection, ratios, and encapsulation parameters allows for broad applicability across a range of diseases and tissue types, including complex disorders that benefit from simultaneous modulation of multiple gene expression pathways.

[0055] In some embodiments, the lipid nanoparticle composition includes one or more lipid nanoparticles. In some embodiments, the one or more lipid nanoparticles include a helper lipid or a stealth lipid. In some embodiments, the helper lipid includes a sterol, and a phospholipid, ester lipid, lysophospholipid, or a glycerol lipid.

[0056] In some embodiments, the composition further includes an excipient. In some embodiments, the excipient includes a mono-, di-, tri-, oligo-, or polysaccharide, sugar alcohol, mono-, di, tri, oligo-, or polypeptide, protein, ester, ether, amide, amine, sulphate, thios, urethane, phosphoester, phosphazene, amino acid, surfactant, lipid, stearate, polymer, or a combination thereof.

[0057] In some embodiments, the composition is formulated as a powder. In some embodiments, the composition is formulated as a dry powder having a residual moisture of 10% or less.

[0058] In some embodiments, the composition is formulated for inhalable administration, enteral administration, transdermal administration, or parenteral administration. In some embodiments, the composition is formulated for respiratory tract administration. In some embodiments, the composition is formulated for administration with an inhaler device.Methods of delivering compositions

[0059] Several embodiments provided herein relate to methods of delivering a lipid nanoparticle composition to a respiratory tract of a subject. In some embodiments, the method of delivering a lipid nanoparticle composition to a respiratory tract of a subject further includes administering any of the compositions described herein to the subject. In some embodiments, the administering is performed using an inhaler device.Methods of treating respiratory diseases

[0060] Several embodiments provided herein relate to methods of treating, preventing, inhibiting, reducing the severity of, delaying the onset of, halting the progression of, or reversing a respiratory disease or disorder in a subject in need thereof. In some embodiments, the methods include administering to the lung, airway, bronchi, bronchioles, alveoli, or any region of the respiratory tract of the subject any one of the lipid nanoparticle (LNP) compositions described herein. In some embodiments, the administering is performed by pulmonary delivery. In some embodiments, the administering is performed using an inhalation device, such as a dry-powder inhaler (DPI), metered-dose inhaler (MDI), nebulizer, vibrating mesh nebulizer, soft-mist inhaler, jet nebulizer, ultrasonic nebulizer, intratracheal instillation device, or intrabronchial catheter.

[0061] In some embodiments, the methods include administering a therapeutically effective amount of the LNP composition to the subject. The term “therapeutically effective amount” refers to an amount sufficient to reduce, ameliorate, or prevent one or more symptoms of the respiratory disease, or to alter the biological activity or expression of one or more target mRNA molecules within the respiratory epithelium. In some embodiments, the therapeutically effective amount of the siRNA within the LNP composition is between 0.01 mg / kg and 10 mg / kg, between 0.05 mg / kg and 5 mg / kg, between 0.1 mg / kg and 1 mg / kg, or between 1 mg and 100 mg total dose delivered to the lung. In some embodiments, the therapeutically effective amount of the siRNA within the LNP composition is between 1 pg and 10 mg, 1 pg and 5 mg, between 1 pg and 50 pg, between 10 pg and 50 pg, between 10 pg and 500 pg, between 10 pg and 1 mg, between 50 pg and 5 mg, between 100 pg and 500 pg, between 100 pg and 2 mg, or between 500 pg and 5 mg total dose delivered to the lung. In some embodiments, the dose of the siRNA per administration (per inhalation or nebulization session) is between 1 pg and 10 mg, 1 pg and 5 mg, between 1 pg to 50 pg, 10 pg and 10 mg, between 10 pg and 50 pg, between 10 pg and 500 pg, between 10 pg and 1 mg, between 50 pg and 5 mg, between 100 pg and 500 pg, between 100 pg and 2 mg, or between 500 pg and 5 mg.

[0062] In some embodiments, the effective amount of an siRNA for a human subject is 1, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 pg, or 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg or any amount within the range defined by any two aforementionedamounts. In some embodiments, the effective amount of an siRNA for a human subject is 1, 10, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 ng / kg, or 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 pg / kg or any amount within the range defined by any two aforementioned amounts. In some embodiments, the effective amount of an siRNA is dosed more than one time. In some embodiments, the siRNA dose is administered every 1, 2, 3, 4, 5, 6, 7 days, or 1, 2, 3, 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1, 2, 3, 4, 5 years, or any period or combination thereof within the range defined by any two aforementioned times. In some embodiments, at least one loading dose and at least one maintenance dose is administered to the subject, where the at least one loading dose is a higher dose of the siRNA than the at least one maintenance dose.

[0063] In some embodiments, the dosing schedule comprises administration once daily, twice daily, three times weekly, once weekly, once every two weeks, or once every four weeks, or as needed. In some embodiments, the dosing regimen comprises an induction phase followed by a maintenance phase. For example, in some embodiments the induction phase comprises administering the LNP composition once daily for 3-14 days, and the maintenance phase comprises administering the composition once weekly or once every other week. In some embodiments, the dosing frequency is adjusted based on disease severity, biomarkers such as protein expression levels, lung function measurements (e.g., FEV1), symptom scores, or exacerbation frequency.

[0064] In some embodiments, the respiratory disease or disorder is selected from asthma (including, for example, sever asthma), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF), bronchiectasis, cystic fibrosis, or a viral infection of the respiratory tract, including influenza, SARS-CoV-2, respiratory syncytial virus (RSV), parainfluenza, adenovirus, rhinovirus, metapneumovirus, or any combination thereof. In some embodiments, the asthma is allergic asthma, eosinophilic asthma, non-eosinophilic asthma, steroid-resistant asthma, or late-onset asthma (Figure 2). In some embodiments, the COPD is emphysema, chronic bronchitis, or mixed emphysema-bronchitis phenotype.

[0065] In some embodiments, the subject is a human subject. In some embodiments, the subject is a non-human mammalian subject, such as a rodent, rabbit, dog, cat, sheep, pig, or non-human primate. In some embodiments, the subject is a pediatric subject,an adult subject, or an elderly subject. In some embodiments, the subject has a history of exacerbations, hospitalization for respiratory disease, or corticosteroid dependence.

[0066] In some embodiments, expression of TSLP is downregulated by the first siRNA through RNA interference (RNAi), resulting in decreased translation of the mRNA encoding TSLP. In some embodiments, expression of ADAMI 7, VEGF, CTGF, PDE3, or PDE4 is downregulated by the second siRNA through the inhibition of translation of the corresponding mRNA. In some embodiments, the first and second siRNA are co-encapsulated within the same LNP population. In some embodiments, the first and second siRNA are separately encapsulated in distinct LNPs and administered together or sequentially.

[0067] In some embodiments, the siRNA sequences are between 18 and 30 nucleotides in length, between 19 and 25 nucleotides, or between 20 and 23 nucleotides. In some embodiments, the siRNA includes one or more chemical modifications, including 2'-O- methyl, 2 '-fluoro, locked nucleic acid (LN A), phosphor othioate linkages, or conjugated ligands to improve stability or uptake. In some embodiments, the antisense strand comprises a seed region specifically complementary to the target mRNA. In some embodiments, the siRNA reduces expression of the target mRNA by at least 20%, 30%, 50%, 70%, 80%, 90%, 95%, or 99% relative to baseline.

[0068] In some embodiments, the method further includes administering one or more additional therapeutic agents or co-therapies to the subject. In some embodiments, the one or more therapeutic compositions include cystic fibrosis transmembrane conductance regulator (CFTR) modulators, CFTR potentiators, CFTR correctors, anti-inflammatory agents, bronchodilators, beta-agonists, muscarinic antagonists, corticosteroids, leukotriene receptor antagonists, immune modulators, or antiviral agents. In some embodiments, the CFTR modulators are lumacaftor, ivacaftor, tezacaftor, elexacaftor, or combinations thereof. In some embodiments, the LNP-siRNA compositions are administered concurrently, sequentially, or in alternation with such agents.

[0069] In some embodiments, the method results in a therapeutic benefit, including but not limited to decreasing, alleviating, reducing, preventing, or reversing one or more symptoms of a respiratory disease or disorder. In some embodiments, the method restores normal lung physiology, reduces mucus plugging, decreases airway hyperresponsiveness, improves mucociliary clearance, reduces inflammatory cytokines, reduces airway remodeling,or improves lung function parameters including FEV1, FVC, or peak expiratory flow. In some embodiments, the treatment reduces the frequency or severity of exacerbations or hospitalizations.Kits

[0070] In some embodiments, the present disclosure provides kits comprising any one of the lipid nanoparticle (LNP) compositions described herein, together with packaging, labeling, and / or instructions for use. In some embodiments, the kit comprises a pharmaceutical composition including one or more LNPs encapsulating one or more siRNA molecules as described herein. In some embodiments, the composition is formulated in a liquid, dry powder, lyophilized, or reconstitutable form. In some embodiments, the composition is provided in a container configured for pulmonary delivery.

[0071] In some embodiments, the packaging comprises a capsule, blister, cartridge, vial, ampoule, reservoir, sachet, pouch, syringe, or unit-dose container suitable for storing the LNP composition. In some embodiments, the packaging is single-dose packaging, such as a single-use capsule, blister, vial, or ampoule configured to deliver one therapeutic dose of the LNP formulation. In other embodiments, the packaging is multidose packaging, including multi-reservoir cartridges, multi-dose vials, pressurized canisters, or multi-use nebulizer reservoirs. In some embodiments, the packaging is moisture-resistant, oxygen-impermeable, or light-protective to maintain stability and prevent degradation of the siRNA or LNP components.

[0072] In some embodiments, the kit further comprises a delivery device suitable for administering the LNP composition to the respiratory tract of a subject. In some embodiments, the delivery device is an inhalation device, such as a nebulizer, vibrating mesh nebulizer, jet nebulizer, ultrasonic nebulizer, metered-dose inhaler (MDI), dry powder inhaler (DPI), breath-actuated inhaler, soft-mist inhaler, or intratracheal delivery apparatus. In some embodiments, the delivery device is preconfigured or pre-filled with the LNP composition. In other embodiments, the delivery device is separate from the composition but packaged together within the kit for use in combination.

[0073] In some embodiments, the kit further includes instructions for use, including printed materials, electronic documents, QR-code-linked digital instructions, or labeling thatdescribes administration techniques, dosing schedules, storage requirements, or indications for treatment or prevention of a respiratory disease. In some embodiments, the instructions specify that the LNP composition is to be administered once daily, twice daily, weekly, or according to an induction-and-maintenance dosing regimen.

[0074] In some embodiments, the kit comprises additional components useful for preparing, handling, or administering the LNP composition. These components may include reconstitution solutions, diluents, sterile water, saline, measuring devices, droppers, adapters, or connectors designed for coupling the packaging container to the inhalation device. In some embodiments, the kit includes a sterile filter, a spacer device for use with an MDI, or a reservoir cup for use with a nebulizer.

[0075] In some embodiments, the kit is configured as a patient-ready system, wherein all components necessary for administering the LNP composition are provided. In other embodiments, the kit is configured as a clinician-ready system, such as for use in hospitals, urgent care settings, or clinical research studies.

[0076] In some embodiments, the kit is provided in a sealed outer package or secondary packaging configured to ensure regulatory compliance, transport stability, track- and-trace identification, or tamper evidence.Terms

[0077] The term “composition” as used herein has its plain and ordinary meaning and shall include any combination (e.g., mixture or other combination) of two or more products, substances, or compounds. Composition may be, for example, a solution, a suspension, liquid, a gel, powder, a paste, a lyocake, aqueous, non-aqueous, or any combination thereof. In some embodiments, the compositions are formulated for inhalation, for example, as a dry powder formulation, spray dried particles, lipid nanoparticle dry powders, engineered nanoparticles, aqueous solutions aqueous suspensions, buffer formulations, lipid nanoparticle suspensions, polymeric nanoparticle or liposome suspensions, nebulized liquid formulations, pressurized metered-dose inhalation, soft-mist inhalation, and the like.

[0078] The term “excipient” as used herein has its plain and ordinary meaning and shall include a substance or component other than the active ingredient of a pharmaceutical composition or medicine, such as inert substances, compounds, or materials added to apharmaceutical composition to provide, without limitation, bulk, consistency, stability, binding ability, lubrication, disintegrating ability etc., to the composition. Excipients with desirable properties include but are not limited to preservatives, adjuvants, stabilizers, solvents, buffers, diluents, solubilizing agents, detergents, surfactants, chelating agents, antioxidants, alcohols, ketones, aldehydes, ethylenediaminetetraacetic acid (EDTA), citric acid, salts, sodium chloride, sodium bicarbonate, sodium phosphate, sodium borate, sodium citrate, potassium chloride, potassium phosphate, magnesium sulfate sugars, dextrose, fructose, mannose, lactose, galactose, sucrose, sorbitol, cellulose, serum, amino acids, polysorbate 20, polysorbate 80, sodium deoxycholate, sodium taurodeoxycholate, magnesium stearate, octylphenol ethoxylate, benzethonium chloride, thimerosal, gelatin, esters, ethers, 2-phenoxyethanol, urea, or vitamins, or any combination thereof. The amount of the excipient may be found in a pharmaceutical composition at a percentage of 0%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% w / w or any percentage by weight in a range defined by any two of the aforementioned numbers.

[0079] The term “nanoparticle” or “nanocarrier” as used herein has its ordinary meaning and shall include a matter particle that is 1 to 1000 nanometers in diameter. Nanoparticles can be composed of multiple substances and can contain compositions, molecules, and / or nucleic acids within its core.

[0080] The term “lipid nanoparticle (LNP)” as used herein has its ordinary meaning and shall include a spherical particle composed of lipids that is 1 to 1000 nanometers in diameter. Lipid nanoparticles can be composed of phospholipids, ionizable lipids, polyethylene glycol-derived lipids (PEGylated lipids), sterols, cholesterols, or a combination thereof. Lipid nanoparticles can contain compositions, molecules, and / or nucleic acids within its core. A lipid nanoparticle may be referred to herein as an encapsulation structure.

[0081] The terms “encapsulate”, “encapsulates”, “encapsulated”, “encapsulating”, and “encapsulation” as used herein have their ordinary meaning and shall include to enclose, encase, envelop, wrap or surround another object partially or completely as in a capsule. In certain embodiments, the terms refer to the nucleic acids including siRNAs surrounded partially or completely by the one or more LNPs.

[0082] The term “pharmaceutically active ingredient” or “active pharmaceutical ingredient (API)” as used herein has its ordinary meaning and shall include a component that provides an active (e.g., biologically active) or other direct effect on the treatment, mitigation, cure, prevention or diagnosis of a disease of a subject or an effect on a physiological function of a subject.

[0083] The term “lipids” as used herein has its ordinary meaning and shall include a group of hydrophobic or amphiphilic organic molecules. Lipids can include but are not limited to monoglycerides, diglycerides, fats (triglycerides), fatty acids, phospholipids, and sterols.

[0084] The term “cationic lipid” as used herein has its ordinary meaning and shall include a lipid molecule that is positively charged in an acidic pH condition and neutral at physiological pH.

[0085] The term “helper lipids” as used herein has its ordinary meaning and shall include a class of lipid molecules that contribute to the stability and delivery efficacy of a lipid nanoparticle.

[0086] The term “stealth lipid” as used herein has its ordinary meaning and shall include a class of lipid molecules (PEGylated lipids) that are modified with polyethylene glycol (PEG), or PEG alternatives, including, for example, polysarcosine, poly(2-oxazoline), poly(glycerol), poly(N-vinylpyrrolidone), zwitterionic polymers such as poly(carboxybetaine) or poly(sulfobetaine), polysaccharides such as dextran or hyaluronic acid, or peptide-based hydrophilic coatings.

[0087] The term “polymer” as used herein has its ordinary meaning and shall include a molecule composed of multiple repeating subunits (monomers), or block copolymer.

[0088] The term “dried state”, “dried”, or “dry” (e.g., dry powder) as used herein has its ordinary meaning and shall include the condition of particulates and / or nanoparticles that have been altered / processed to remove water / liquid through, for example, spray drying and / or lyophilization. A dried state may include a partially dried state, including for example a composition that includes 20% or less of moisture content, including for example, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or 0.5% or less moisture content, and thus a dried state does not necessarily refer to a state that is entirely devoid of moisture.

[0089] The term “hydrated state” as used herein has its ordinary meaning and shall include the condition of particulates and / or nanoparticles in a liquid solution, in suspension, or dispersion.

[0090] The term “powder” as used herein has its ordinary meaning and shall include a solid substance composed of particulates and / or nanoparticles.

[0091] The term “dry powder” as used herein has its ordinary meaning and shall include particulates and / or nanoparticles in a dry state (e.g., fine granular solid state).

[0092] The term “colloidal suspension” or “colloid” as used herein has its ordinary meaning and shall include a mixture in which microscopically dispersed insoluble particles of one substance are suspended through a different substance. Colloidal suspensions can include aerosols, liquids, and gels.

[0093] The term “inhaler” or “inhaler device” as used herein has its ordinary meaning and shall include a delivery device used to administer a substance such as a pharmaceutical composition or medicine to the lungs or other tissue of a subject through the subject’s breathing or through forced ventilation. Types of inhalers include but are not limited to meter-dosed inhalers (MDI), dry powder inhalers (DPI), soft mist inhalers, smart inhalers, and nebulizers. The term “nebulizer” as used herein has its ordinary meaning and shall include a delivery device used to administer a pharmaceutical composition or medicine in the form of a inhalable mist to respiratory tract (for example, the nasal cavities, airways, and / or lungs) of a subject. A nebulizer may use ultrasonic power, vibrating meshes, oxygen, or compressed air to break solutions and suspensions into aerosol droplets that include a mixture of gas and solid and / or liquid particulate. Nebulizers can include a mouthpiece or face mask for the subject to breathe the inhalable mist over a period of time. The term “soft mist inhaler” as used herein has its ordinary meaning and shall include a device that provides a metered dose to the user and that imposes pressure on a composition, causing composition to spray out of a nozzle, thus forming a soft mist to be inhaled.

[0094] The term “dry powder inhaler” as used herein has its ordinary meaning and shall include a delivery device used to administer a pharmaceutical composition or medicine in the form of a dry powder to the respiratory tract (for example, nasal cavities, airways, and / or lungs) of a subject through the subject’s breathing. The dry powder can be delivered to lungs of the subject without the use of an aerosol and / or a propellant as the subject takes an inhalationthrough a mouthpiece of the dry powder inhaler. Tissue other than the lung can be targeted using a dry powder inhaler.

[0095] A “nucleic acid” sequence has its ordinary meaning and shall include a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sequence. The term captures sequences that include any of the known base analogues of DNA and RNA such as, but not limited to 4-acetylcytosine, 8-hydroxy-N6- methyladenosine, aziridinylcytosine, pseudoisocytosine, 5 -(carboxyhydroxyl- methyl) uracil, 5 -fluorouracil, 5 -bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5 -carboxymethylaminomethyluracil, dihydrouracil, inosine, N6-isopentenyladenine, 1 -methyladenine, 1 -methylpseudouracil, 1 -methylguanine, 1- methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5- methylcytosine, N6-methyladenine, 7-methylguanine, 5 -methylaminomethyluracil, 5- methoxy- aminomethyl-2 -thiouracil, beta-D-mannosylqueosine, 5'- methoxy carbonylmethyluracil, 5 -methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil- 5-oxyacetic acid methylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5- methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5 -methyluracil, N- uracil-5- oxyacetic acid methylester, uracil-5 -oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

[0096] The term “oligonucleotide” as used herein has its ordinary meaning and shall include a nucleic acid sequence comprising from about 2 to about to about 100 nucleotides (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100 nucleotides, or a range defined by any of the foregoing values). In the context of siRNA, an oligonucleotide refers to a strand of the siRNA, such as the sense strand (S strand) or the antisense strand (AS strand).

[0097] The term “polynucleotide” has its ordinary meaning and shall include a polymeric form of nucleotides of any length, including DNA, RNA, or analogs thereof. A nucleotide may include mono-, di-, tri-, oligo-, and poly- forms. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is apolynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[0098] The term “messenger RNA (mRNA)” as used herein has its ordinary meaning and shall include a single-stranded RNA sequence that corresponds to a DNA sequence of a gene. mRNA is synthesized during transcription from the DNA sequence of the gene by an RNA polymerase and utilized by ribosomes during translation to synthesize a protein encoded by the gene.

[0099] The terms “small interfering RNA (siRNA)”, “short interfering RNA”, or “silencing RNA”, as used herein interchangeably, have their ordinary meaning and shall include a non-coding double-stranded RNA sequence comprising from about 10 to about 30 nucleotides (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or a range defined by any of the foregoing values). siRNA regulates gene expression via RNA interference (RNAi) by degrading messenger RNA (mRNA) thereby preventing translation of the target gene into protein.

[0100] The terms “microRNA (miRNA)” or “microRNA”, as used herein interchangeably, have their ordinary meaning and shall include a non-coding single stranded RNA sequence comprising from about 10 to about 30 nucleotides (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or a range defined by any of the foregoing values). Typically, miRNA include a non-coding single stranded RNA sequence comprising from about 19 to about 25 nucleotides (e.g., about 19, 20, 21, 22, 23, 24, or 25 nucleotides, or a range defined by any of the foregoing values). miRNA regulates gene expression via RNA silencing and post-transcriptional regulation of gene expression by binding complementary sequences of mRNA, and thereby silencing the mRNA by cleaving the mRNA strand into two pieces, destabilizing the mRNA by shortening the poly(A) tail, and / or reducing translation of the mRNA into protein.

[0101] The terms “polypeptide” and “protein” have their ordinary meaning and shall include a polymer of amino acid residues and are not limited to a minimum length. A peptide may include mono-, di-, tri-, oligo-, and poly- forms. Polypeptides, including therapeutic proteins and other peptides, e.g., linkers, tags, capsid proteins, may include amino acid residues including natural and / or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation,acetylation, phosphorylation, and the like. In some respects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0102] Amino acids generally can be grouped according to the following common side- chain properties: (1) hydrophobic: Norleucine, Met, Ala, Vai, Leu, He; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Amino acids may include proteinogetic or nonproteinogetic forms.

[0103] Amino acid substitutions may also refer to one or more changes in a polypeptide sequence. The changes may include replacement of one amino acid in a polypeptide with another amino acid, insertion of one or amino acids, and / or deletion of one or more amino acids, or any combination thereof. Non-conservative amino acid substitutions will involve exchanging a member of one of these classes for another class.

[0104] The term “recombinant,” has its ordinary meaning and shall include a polynucleotide that is a product of various steps of alteration including but not limited to restriction enzyme digestion followed by ligation steps, alterations introduced by procedures such as polymerase chain reaction (PCR) (for example, introduction of restriction enzyme sites and nucleotide substitutions), and / or a combination of polynucleotides and proteins that is not found in nature.

[0105] The term “gene” has its ordinary meaning and shall include a polynucleotide containing at least one open reading frame that is capable of encoding a particular gene product. Any of the polynucleotide sequences described herein may be used to identify larger fragments or full-length coding sequences of the genes with which they are associated.

[0106] The terms “encode”, “encodes”, “encoded”, and “encoding” as used herein interchangeably, have their ordinary meaning and shall include containing the DNA sequence, RNA sequence, and / or peptide sequence information associated with a gene or protein. Encoded DNA sequence information can be utilized to synthesize an mRNA sequence of the gene during transcription Encoded DNA and / or RNA sequence information can be utilized to synthesize a protein of the gene during transcription and / or translation.

[0107] The term “expression” as used herein has its ordinary meaning as and shall include one or more processes by which a polynucleotide is transcribed from a DNA template (such as into an mRNA or other RNA transcript) and / or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides can be collectively referred to as “gene product.”

[0108] The terms “regulate”, “regulating”, “regulated” and “regulation” as used herein interchangeably, have their ordinary meaning and shall include modulation of expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence relative to its expression level in a wild-type state.

[0109] The terms “upregulate”, “upregulating”, “upregulated”, and “upregulation” as used herein interchangeably, have their ordinary meaning and shall include an increased expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence (e.g., protein) relative to its expression level in a wild-type state.

[0110] The terms “downregulate”, downregulating”, “downregulated”, and “downregulation” as used herein interchangeably, have their ordinary meaning and shall include an decreased expression level of a polynucleotide (e.g., RNA such as mRNA) and / or polypeptide sequence (e.g., protein) relative to its expression level in a wild-type state.

[0111] The terms “target” or “target gene” as used herein interchangeably, has their ordinary meaning and shall include a DNA sequence, RNA sequence, and / or protein sequence of interest for regulation.

[0112] The term “upstream” as used herein, has its ordinary meaning and shall include a DNA sequence proximal to the 5’ end of a DNA sequence and / or before a DNA sequence encoding a gene.

[0113] The term “downstream” as used herein, has its ordinary meaning and shall include a DNA sequence proximal to the 3’ end of a DNA sequence and / or after a DNA sequence encoding a gene.

[0114] The term “inflammatory cascade” as used herein has its ordinary meaning and shall include a multi-step immune response to a stimulus, including but not limited to an injury and / or an infection, such as, for example, an epithelial injury (see for example, Figure 3B). In some embodiments, the term refers to the multi-step immune response to a stimulusaffecting the lungs and / or respiratory tract of a subject. In some embodiments, a gene associated with an inflammatory cascade is TSLP.

[0115] The term “tissue remodeling factor” as used herein has its ordinary meaning and shall include genes encoding proteins, for example growth factors, associated with the reorganization or renovation of existing tissues. In some embodiments, tissue remodeling factors include ADAMI 7, VEGF, and CTGF.

[0116] The term “signal transduction regulator” as used herein has its ordinary meaning and shall include genes encoding proteins associated with modulating processes by which chemical and / or physical signals are transmitted through a cell as a series of molecular events. In some embodiments, signal transduction regulators include PDE3 and PDE4.

[0117] The term “thymic stromal lymphopoietin (TSLP)” as used herein has its ordinary meaning and refers to a gene encoding an alarmin cytokine. In some embodiments, TSLP is associated with an inflammatory cascade, for example as shown in Figure 1A.

[0118] The term “a disintegrin and metalloprotease 17 (ADAMI 7)”, a disintegrin and metalloprotease 17, as used herein has its ordinary meaning and refers to a gene encoding an 70-kDa enzyme that belongs to the ADAM protein family of disintegrins and metalloproteases, for example, as shown in Figure IB. The term ADAMI 7 can be used herein interchangeably with tumor necrosis factor-a- converting enzyme (TACE). In some embodiments, ADAMI 7 is a tissue remodeling factor.

[0119] The term “vascular endothelial growth factor (VEGF)”, as used herein has its ordinary meaning and refers to a gene or genes encoding a sub-family of growth factors that stimulate the formation of blood vessels, for example as shown in Figure 1C. The VEGF family includes vascular endothelial growth factor A (VEGF A), vascular endothelial growth factor B (VEGFB), vascular endothelial growth factor C (VEGFC), vascular endothelial growth factor D (VEGFD), and placenta growth factor (PGF). In some embodiments, VEGF is a tissue remodeling factor. In some embodiments, VEGF is VEGF A.

[0120] The term “connective tissue growth factor (CTGF)”, as used herein has its ordinary meaning and refers to a gene encoding a matricellular protein of the CCN family of extracellular matrix-associated heparin-binding proteins. The term CTGF can be used herein interchangeably with CCN2. In some embodiments, CTGF is a tissue remodeling factor.

[0121] The term “phosphodiesterase 3 (PDE3)”, as used herein has its ordinary meaning and refers to a gene encoding a phosphodiesterase involved in regulation of cardiac and vascular smooth muscle contractility. In some embodiments, PDE3 is a signal transduction regulator. In some embodiments, PDE3 includes PDE3A and PDE3B isoforms.

[0122] The term “phosphodiesterase 4 (PDE4)”, as used herein has its ordinary meaning and refers to a gene encoding a phosphodiesterase involved in hydrolyzing and degrading cyclic adenosine monophosphate (cAMP). In some embodiments, PDE4 is a signal transduction regulator. In some embodiments, PDE4 includes PDE4A, PDE4B, PDE4C, and PDE4D isoforms.

[0123] The term “administering” as used herein has its plain and ordinary meaning and shall include one or more steps that may be taken to introduce a composition to a cell, cell population, tissue, respiratory tract, lungs, airway, or other organ or body part of a subject. Administering includes “instructing administration.”

[0124] As used herein, “therapeutically effective” has its ordinary meaning and shall include an amount of a pharmaceutically active compound(s) that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with diseases or conditions. For example, an effective amount in reference to diseases is that amount which is sufficient to block or prevent onset; or if disease pathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. In any case, an effective amount may be given in single or divided doses.

[0125] The terms “treating,” “treatment,” “therapeutic,” or “therapy”, as used herein interchangeably, have their plain and ordinary meanings and do not necessarily mean total cure or abolition of the disease or condition. Any alleviation of any undesired signs or symptoms of a disease or condition, to any extent can be considered treatment and / or therapy. To “treat” a disease as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

[0126] As used herein, and unless otherwise specified, the terms “prevent,” “preventing” and “prevention” have their ordinary meaning and shall include the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s),prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein. The terms encompass the inhibition or reduction of a symptom of the particular disease. In certain embodiments, subjects with familial history of a disease are potential candidates for preventive regimens. In certain embodiments, subjects who have a history of recurring symptoms are also potential candidates for prevention. In this regard, the term “prevention” may be interchangeably used with the term “prophylactic treatment.”

[0127] The term “subject”, “individual”, or “patient”, as used herein interchangeably, have their plain and ordinary meanings and shall include a vertebrate, optionally a mammal, such as a human. Mammals include, but are not limited to, murine, simian, human, farm animal, sport animal, and pets. Tissues, cells, and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.EXAMPLES

[0128] Some aspects of the embodiments discussed above are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the present disclosure. Those in the art will appreciate that many other embodiments also fall within the scope of the invention, as it is described herein above and in the claims.Example 1 : Expression of TSLP in Human Precision Cut Ling Slices

[0129] This example demonstrates examples of compositions of therapeutic formulations delivered to human precision cut lung slices (PCLS). Figure 6A shows a schematic depicting the pharmacodynamics of anti-TLSP nano-embedded in-microparticles (NEMs) compositions delivered to lung tissue. Figure 6B shows the fold-change of TSLP gene expression in PCLS samples treated with embodiments of the compositions with therapeutic formulations (dry powder formulated composition having anti-TSLP siRNA), as measured by quantitative PCR. Figure 6C shows TLSP protein expression in PCLS samples treated with embodiments of the compositions with therapeutic formulations (dry powder formulated composition having anti-TSLP siRNA), as measured by ELISA.Example 2: Simultaneous gene expression knockdown of two target genes by dual target LNPs encapsulating two siRNAs

[0130] In this non-limiting example, target gene expression was measured using quantitative PCR (qPCR) before and after A549 cells were treated with dual target LNPs encapsulating two siRNAs. The encapsulated two siRNAs each separately target mRNA encoding one of the target genes (TSLP, PDE4B, VEGF-A. ADAMI 7, and VEGF). The A549 cells pretreated with two siRNAs for 24 hours and triggered with poly(I:C) for 6 hours. FIG. 7A shows fold-change in TSLP and PDE4B gene expression in A549 cells pretreated with two siRNAs for 24 hours and triggered with poly(I:C) for 6 hours, as measured by quantitative PCR. FIG. 7B shows fold-change in TSLP and VEGF-A gene expression under the same conditions. FIG. 7C shows fold-change in TSLP and ADAMI 7 gene expression. Single-target controls were transfected with a control siRNA as the second siRNA.

[0131] The data demonstrates that there is simultaneous gene expression knockdown of the two target genes in cells / tissue treated with LNPs encapsulating two siRNAs in all the three dual target LNP formulations tested.Table 3: Dual target LNP formulationsExample 3: Target gene protein expression in cells / tissue treated with dual target LNPs encapsulating two siRNAs

[0132] In this non-limiting example, target gene protein expression levels were observed using enzyme-linked immunosorbent assays (ELISA) in A549 cells treated with dual target LNPs encapsulating two siRNAs. Target gene protein expression levels were measured by ELISA 24 hours after poly(I:C) stimulation in cells pretreated with two siRNAs for 24 hours. Dual target LNP formulations utilized in this non-limiting example include LNP formulations including siRNA targeting: 1) TSLP and PDE4B mRNA; 2) TSLP and VEGF-A mRNA; and 3) TSLP and ADAMI 7.Example 4: Formulation of dual target LNPs and triple target LNPs

[0133] In this non-limiting example, methods of formulating dual target LNPs and target LNPs were performed. The quality of the dual target LNPs was also assessed by measuring the size, zeta potential, and encapsulation efficiency of the LNPs.

[0134] FIGs. 8A-8D show physicochemical characterization of lipid nanoparticle (LNP) formulations encapsulating dual siRNAs. FIG. 8A shows hydrodynamic diameter for LNPs containing siRNAs targeting TSLP and PDE4B, VEGF-A, or ADAMI 7. FIG. 8B shows polydispersity index of LNPs containing siRNAs targeting TSLP and PDE4B, VEGF-A, or ADAMI 7. FIG. 8C shows zeta potential measurements for single and combined LNPs. FIG. 8D shows encapsulation efficiency for each formulation.Example 5: Characterization of cells grown in an air-liquid interface (ALI) cell culture treated with a dual target LNP

[0135] In this non-limiting example, target gene mRNA expression (FIGS. 9A-9B) and protein expression levels were measured in cells grown in an ALI cell culture treated with a dual target LNP.

[0136] FIGs. 9A and 9B show gene expression modulation in air-liquid interface (ALI) cultures treated with dual-siRNA LNPs. FIG. 10A shows fold-change in TSLP and PDE4B gene expression after pretreatment with LNPs for 24 hours and poly(I:C) stimulation for 6 hours, as measured by qPCR. FIG. 10B shows fold-change in TSLP and VEGF-A gene expression under the same conditions.

[0137] The tight junction integrity of the treated epithelial cells was observed using transepithelial electrical resistance (TEER) measurements to assess tissue remodeling effects of LNPs with siRNAs targeting mRNA encoding for tissue remodeling factors.

[0138] FIG. 10 shows epithelial barrier integrity in ALI cultures after 48 hours pretreated for 24 hours with siRNAs targeting TSLP and PDE4B.Example 6: Characterization of dual-siRNA formulations for inhalation delivery

[0139] In this non-limiting example, the dual-siRNA formulations were characterized for inhalation delivery. FIG. 11 shows dry powder characteristics of dual-siRNA formulations for inhalation delivery.Example 7: Formulation and characterization of dry powder dual target and triple target LNPs

[0140] In this non-limiting example, methods of formulating dry powder dual target LNPs and triple target LNPs were performed. Dry powder dual target LNPs and triple target LNPs were characterized via laser diffraction to support inhalation. FIG. 12 shows fine particle fraction (percent of particles in the size range between 1 and 5 pm) and mass median diameter (xso) of dry powder containing combinatorial siRNA LNP for inhalation delivery. FIG. 13 shows residual water content of dry powder containing combinatorial siRNA LNP for inhalation delivery. FIG. 14 shows x-ray powder diffraction pattern of dry powder containing combinatorial siRNA LNP for inhalation delivery.

[0141] Any titles or subheadings used herein are for organizational purposes and should not be used to limit the scope of embodiments disclosed herein. All literature and similar materials cited in this application, including but not limited to, patents, patent applications, articles, books, treatises, and internet web pages are expressly incorporated by reference in their entirety for any purpose, including the disclosures specifically referenced herein. When definitions of terms in incorporated references appear to differ from the definitions provided in the present teachings, the definition provided in the present teachings shall control. It will be appreciated that there is an implied “about” prior to the temperatures, concentrations, times, etc. discussed in the present teachings, such that slight and insubstantial deviations are within the scope of the present teachings herein.

[0142] Although embodiments described herein have been disclosed in the context of certain embodiments and examples, the present disclosure extends beyond the specifically disclosed embodiments to other alternative embodiments and / or uses of the disclosure and obvious modifications and equivalents thereof. It is also contemplated that various combinations or sub-combinations of the specific features and aspects of the embodiments may be made and still fall within the scope of the disclosure. It should be understood that various features and aspects of the disclosed embodiments can be combined with, or substituted for,one another in order to form varying modes or embodiments. Thus, it is intended that the scope of the present disclosure should not be limited by the particular disclosed embodiments described above.

[0143] The terminology used in the description presented herein is not intended to be interpreted in any limited or restrictive manner. Rather, the terminology is simply being utilized in conjunction with a detailed description of embodiments of the systems, methods, and related components. Furthermore, embodiments may include several novel features, no single one of which is solely responsible for its desirable attributes or is believed to be essential to practicing the embodiments herein described.

[0144] Changes and modifications in the embodiments described herein can be carried out without departing from the principles of the present disclosure. Each of the disclosed aspects and examples of the present disclosure may be considered individually or in combination with other aspects, examples, and variations of the disclosure. In addition, unless otherwise specified, none of the steps of the methods of the present disclosure are confined to any particular order of performance.

[0145] While the compositions and methods described herein may be susceptible to various modifications and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. Embodiments are not to be limited to the particular forms or methods disclosed, but rather intended is to cover modifications, equivalents, and alternatives falling within the spirit and scope of the various examples and embodiments described herein and / or in the appended claims. Further, the disclosure herein of any particular feature, aspect, method, property, characteristic, quality, attribute, element, or the like in connection with an example can be used in all other examples set forth herein. Any methods disclosed herein need not be performed in the order recited. The use of sequential, or time-ordered language, such as “then,” “next,” “after,” “subsequently,” and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to facilitate the flow of the text and is not intended to limit the sequence of operations performed. Thus, some examples may be performed using the sequence of operations described herein, while other examples may be performed following a different sequence of operations.

[0146] Conditional language used herein, such as, among others, “can,” “might,” “may,” “e.g.,” and the like, unless specifically stated otherwise, or otherwise understood within the context as used, is generally intended to convey that some examples include, while other examples do not include, certain features, elements, and / or states. Thus, such conditional language is not generally intended to imply that features, elements, blocks, and / or states are in any way required for one or more examples or that one or more examples necessarily include logic for deciding, with or without author input or prompting, whether these features, elements and / or states are included or are to be performed in any particular example.

[0147] Where compositions or methods “comprise” or “include” (the two being interchangeable) certain features or steps, such compositions or methods may also “consist essentially of such features or steps if identified as such in the claims. Where compositions or methods “comprise” or “include” (the two being interchangeable) certain features or steps, such compositions or methods may also “consist” of such features or steps if identified as such in the claims.

[0148] The methods disclosed herein may include certain actions taken by a practitioner; however, the methods can also include any user (which may not be a practitioner) or third-party instruction of those actions, either expressly or by implication. For example, actions such as “administering a composition” include “instructing administration of a composition.”

[0149] The ranges disclosed herein also encompass any and all overlap, sub-ranges, and combinations thereof. Language such as “up to,” “at least,” “greater than,” “less than,” “between,” and the like includes the number recited. Numbers preceded by a term such as “about” or “approximately” include the recited numbers and should be interpreted based on the circumstances (e.g., as accurate as reasonable under the circumstances, for example ±5%, ±10%, ±15%, etc.). For example, “about 4 inches” includes “4 inches.” Phrases preceded by a term such as “substantially” include the recited phrase and should be interpreted based on the circumstances (e.g., as much as reasonably possible under the circumstances). For example, “substantially linear” includes “linear.” Unless stated otherwise, all measurements are at standard conditions including temperature and pressure. The phrase “at least one of’ is intended to require at least one item from the subsequent listing, not one type of each itemfrom each item in the subsequent listing. For example, “at least one of A, B, and C” can include A; B; C; A and B; A and C; B and C; or A, B, and C.

[0150] All numbers expressing quantities of ingredients, reaction conditions, and so forth used in the specification are to be understood as being modified in all instances by the term ‘about.’ Accordingly, unless indicated to the contrary, the numerical parameters set forth herein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0151] Unless otherwise defined, all terms (including technical and scientific terms) are to be given their ordinary and customary meaning, and are not to be limited to a special or customized meaning unless expressly so defined herein. It should be noted that the use of particular terminology when describing certain features or aspects of the disclosure should not be taken to imply that the terminology is being re-defined herein to be restricted to include any specific characteristics of the features or aspects of the disclosure with which that terminology is associated. Terms and phrases used in this application, and variations thereof, especially in the appended claims, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing, the term ‘including’ should be read to mean ‘including, without limitation,’ ‘including but not limited to,’ or the like; the term ‘comprising’ as used herein is synonymous with ‘including,’ ‘containing,’ or ‘characterized by,’ and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps; the term ‘having’ should be interpreted as ‘having at least;’ the term ‘includes’ should be interpreted as ‘includes but is not limited to;’ the term ‘example’ is used to provide example instances of the item in discussion, not an exhaustive or limiting list thereof; adjectives such as ‘known’, ‘normal’, ‘standard’, and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but instead should be read to encompass known, normal, or standard technologies that may be available or known now or at any time in the future; and use of terms like ‘preferably,’ ‘preferred,’ ‘desired,’ or ‘desirable,’ and words of similar meaning should not be understood as implying that certain features are critical, essential, or even important tothe structure or function of the invention, but instead as merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment of the invention. Likewise, a group of items linked with the conjunction ‘and’ should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as ‘and / or’ unless expressly stated otherwise. Similarly, a group of items linked with the conjunction ‘or’ should not be read as requiring mutual exclusivity among that group, but rather should be read as ‘and / or’ unless expressly stated otherwise.

[0152] As used in the claims below and throughout this disclosure, by the phrase “consisting essentially of’ is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase “consisting essentially of’ indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.

[0153] With respect to the use of substantially any plural and / or singular terms herein, these shall be translated from the plural to the singular and / or from the singular to the plural as is appropriate to the context and / or application. The various singular / plural permutations may be expressly set forth herein for sake of clarity. The indefinite article “a” or “an” does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0154] Furthermore, although the foregoing has been described in some detail by way of illustrations and examples for purposes of clarity and understanding, changes and modifications may be practiced. Therefore, the description and examples should not be construed as limiting the scope of the invention to the specific embodiments and examples described herein, but rather to also cover modifications and alternatives coming with the true scope and spirit of the inventions described herein.

Claims

WHAT IS CLAIMED IS:

1. A lipid nanoparticle composition, comprising: one or more lipid nanoparticles; a first siRNA that targets an mRNA encoding thymic stromal lymphopoietin (TSLP); a second siRNA that targets an mRNA encoding disintegrin and metalloprotease 17 (ADAMI 7), vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), phosphodiesterase 3 (PDE3), or phosphodiesterase 4 (PDE4); wherein the first siRNA and the second siRNA are encapsulated within the one or more lipid nanoparticles.

2. The composition of claim 1, wherein the composition is formulated as a powder.

3. The composition of any one of claims 1-2, wherein the first siRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 7-26, or a sequence at least 85% identical thereto.

4. The composition of any one of claims 1-3, wherein the second siRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 1-6, or a sequence at least 85% identical thereto.

5. The composition of any one of claims 1-4, further comprising a third siRNA that targets an mRNA different from the second siRNA target, and which encodes ADAMI 7, VEGF, CTGF, PDE3, or PDE4, wherein the third siRNA is encapsulated within the one or more lipid nanoparticles.

6. The composition of claim 5, wherein the third siRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 1-6, or a sequence at least 85% identical thereto.

7. The composition of claim 5, wherein the second siRNA targets an mRNA encoding ADAMI 7, and wherein the third siRNA targets an mRNA encoding VEGF.

8. The composition of claim 5, wherein the second siRNA targets an mRNA encoding ADAMI 7, and wherein the third siRNA targets an mRNA encoding CTGF.

9. The composition of claim 5, wherein the second siRNA targets an mRNA encoding ADAMI 7, and wherein the third siRNA targets an mRNA encoding PDE3.

10. The composition of claim 5, wherein the second siRNA targets an mRNA encoding ADAMI 7, and wherein the third siRNA targets an mRNA encoding PDE4, wherein the PDE4 is PDE4A, PDE4B, PDE4C, or PDE4D.

11. The composition of claim 5, wherein the second siRNA targets an mRNA encoding VEGF, and wherein the third siRNA targets an mRNA encoding CTGF.

12. The composition of claim 5, wherein the second siRNA targets an mRNA encoding VEGF, and wherein the third siRNA targets an mRNA encoding PDE3.

13. The composition of claim 5, wherein the second siRNA targets an mRNA encoding VEGF, and wherein the third siRNA targets an mRNA encoding PDE4.

14. The composition of claim 5, wherein the second siRNA targets an mRNA encoding CTGF, and wherein the third siRNA targets an mRNA encoding PDE3.

15. The composition of claim 5, wherein the second siRNA targets an mRNA encoding CTGF, and wherein the third siRNA targets an mRNA encoding PDE4.

16. The composition of claim 5, wherein the second siRNA targets an mRNA encoding PDE3, and wherein the third siRNA targets an mRNA encoding PDE4.

17. A lipid nanoparticle composition, comprising: one or more lipid nanoparticles; a first siRNA that targets expression of an mRNA involved in an upstream inflammatory cascade; a second siRNA that targets an mRNA encoding for a tissue remodeling factor or an mRNA encoding for a signal transduction regulator; wherein the first siRNA and the second siRNA are encapsulated within the one or more lipid nanoparticles.

18. The composition of claim 17, wherein the first siRNA targets an mRNA that encodes TSLP.

19. The composition of any one of claims 17-18, wherein the second siRNA targets an mRNA that encoding ADAM 17, VEGF, CTGF, PDE3, or PDE4.

20. A lipid nanoparticle composition, comprising: one or more lipid nanoparticles; a first siRNA that targets an mRNA encoding TSLP;a second siRNA that targets an mRNA encoding ADAMI 7, VEGF, CTGF, PDE3, or PDE4; a third siRNA that targets an mRNA different from the second siRNA target, and that encodes ADAMI 7, VEGF, CTGF, PDE3, or PDE4; wherein the first siRNA, the second siRNA, and the third siRNA are encapsulated within the one lipid nanoparticles.

21. The composition of claim 20, wherein the first siRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 7-26, or a sequence at least 85% identical thereto.

22. The composition of any one of claims 20-21, wherein the second siRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 1-6, or a sequence at least 85% identical thereto.

23. The composition of any one of claims 20-22, wherein the third siRNA comprises a nucleic acid sequence of any one of SEQ ID NOs: 1-6, or a sequence at least 85% identical thereto.

24. The composition of any one of claims 1-23, wherein the one or more lipid nanoparticles comprise at least one ionizable lipid.

25. The composition of claim 24, wherein the at least one ionizable lipid is C12- 200, DOTAP (l,2-dioleyl-3-trimethytammonium propane), DODAP (l,2-dioleyl-3- dimethylammonium propane), DOTMA (l,2-di-O-octadecenyl-3 -trimethylammonium propane), DLinDMA, DLin-KC2-DMA, HGT4003, cKK-E12, ICE, DLin-MC3, 7C1, ALC- 0315, SM-102, CL-1, 3060110, OF-02, 7C1, L319, A9, 93O17S, Lipid C24, 1014, Lipidl5, Lipid AX4, Lipid A6, BAMEA-016B, 98N12-5, 4A3-SC8, 5A2-SC8, or a combination thereof.

26. The composition of any one of claims 1-25, wherein the one or more lipid nanoparticles comprise a helper lipid or a stealth lipid.

27. The composition of claim 26, wherein the helper lipid comprises a sterol, and a phospholipid, ester lipid, lysophospholipid, or a glycerol lipid.

28. The composition of any one of claims 1-27, further comprising an excipient.

29. The composition of claim 28, wherein the excipient comprises a mono-, di-, tri- , oligo-, or polysaccharide, sugar alcohol, mono-, di, tri, oligo-, or polypeptide, protein, ester,ether, amide, amine, sulphate, thios, urethane, phosphoester, phosphazene, amino acid, surfactant, lipid, stearate, polymer, or a combination thereof.

30. The composition of any one of claims 1-29, wherein the composition is formulated as a dry powder having a residual moisture of 10% or less.

31. The composition of any one of claims 1-30, wherein the composition is formulated for inhalable administration, enteral administration, transdermal administration, or parenteral administration.

32. The composition of any one of claims 1-30, wherein the composition is formulated for respiratory tract administration.

33. The composition of any one of claims 1-30, wherein the composition is formulated for administration with an inhaler device.

34. A method of delivering a lipid nanoparticle composition to a respiratory tract of a subject, the method comprising administering the composition of any one of claims 1-33 to the subject.

35. The method of claim 34, wherein the administering is performed using an inhaler device.

36. A method of treating or preventing a respiratory tract disease in a subject, the method comprising administering to a lung of the subject the lipid nanoparticle composition of any one of claims 1-33.

37. The method of claim 36, wherein the administering is performed using an inhaler device.

38. The method of any one of claims 36-37, wherein the respiratory tract disease is asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, bronchiectasis, cystic fibrosis, or a viral infection.

39. The method of claim 38, wherein the asthma is allergic asthma, eosinophilic asthma, or non-eosinophilic asthma.

40. The method of claim 38, wherein the COPD is emphysema and / or chronic bronchitis.

41. The method of any one of claims 36-40, wherein expression of TSLP is downregulated by the first siRNA through the inhibition of translation of the mRNA encoding for TSLP; and wherein expression of ADAMI 7, VEGF, CTGF, PDE3, or PDE4 isdownregulated by the second siRNA through the inhibition of translation of the mRNA encoding for ADAMI 7, VEGF, CTGF, PDE3, or PDE4.

42. The method of any one of claims 36-41, further comprising administering one or more therapeutic compositions to the subject, wherein the one or more therapeutic compositions are cystic fibrosis transmembrane conductance regulator (CFTR) modulators and / or potentiators.

43. The method of any one of claims 36-42, wherein the method decreases and / or alleviates symptoms of the respiratory tract disease and / or restores natural lung physiology.

44. Use of the composition of any one of claims 1-33 for treatment of a respiratory tract disease.

45. The use of claim 44, wherein the respiratory tract disease is asthma, chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis, bronchiectasis, cystic fibrosis, or a viral infection.

46. The use of claim 45, wherein asthma is allergic asthma, eosinophilic asthma, or non-eosinophilic asthma.

47. The use of claim 45, wherein the COPD is emphysema and / or chronic bronchitis.

48. A kit comprising the lipid nanoparticle composition of any one of claims 1-33 and packaging.

49. The kit of claim 48, wherein the packaging comprises a capsule, a blister, or a reservoir.

50. The kit of any one of claims 48-49, wherein the packaging is multidose or single dose packaging.

51. The kit of any one of claims 48-50, further comprising a nebulizer or an inhaler device.

52. A therapeutic formulation that downregulates two or more targets, especially target genes, with at least one of which is upstream of an inflammatory cascade, and at least one of which is encoding for a tissue remodeling factor or a signal transduction regulator.

53. The therapeutic formulation of claim 52, wherein the target genes are downregulated through the inhibition of translation of the corresponding messenger RNAs.

54. The therapeutic formulation of claim 52 or claim 53, wherein the downregulation is achieved by the mechanism of RNA interference.

55. The therapeutic formulation of any one of claims 52-54, wherein one of the upstream genes of the inflammatory cascade is encoding for an alarmin.

56. The therapeutic formulation of claim 55, where the alarmin is thymic stromal lymphopoietin (TSLP).

57. The therapeutic formulation of any one of claims 52-56, wherein one of the genes encoding a tissue remodeling factor encode for a disintegrin and metalloprotease 17 (ADAMI 7), also called tumor necrosis factor-a-converting enzyme (TACE), or vascular endothelial growth factor (VEGF), in particular vascular endothelial growth factor A (VEGFA) or one of the signal transduction regulator encodes for a Phosphodiesterase (PDE) in particular, PDE3 or PDE4.

58. The therapeutic formulation of any one of claims 52-57, where the therapeutic use is for pulmonary diseases.

59. The therapeutic formulation of any one of claims 52-58, where the therapeutic use is for allergic asthma.

60. The therapeutic formulation of any one of claims 52-59, where the therapeutic use is for COPD.

61. The therapeutic formulation of any one of claims 52-60, where the therapeutic use is for idiopathic pulmonary fibrosis.

62. The therapeutic formulation of any one of claims 52-61, where the therapy is supposed to decrease symptoms and restore the natural lung physiology.

63. The therapeutic formulation of any one of claims 52-62, where the therapeutic formulation can be administered to the lung.

64. The therapeutic formulation of any one of claims 52-63, where the active pharmaceutical ingredient (API) is or comprises an RNA capable of initiating the RNAi mechanism, preferably an antisense oligonucleotide, dsRNA, siRNA, or an RNA Conjugate.

65. A composition with a therapeutic formulation according to any one of claims 52-64, wherein the active pharmaceutic ingredient (API) is encapsulated in a carrier particle.

66. The composition according to claim 65, wherein the particulate is a nanoparticle.

67. The composition according to claim 65 or claim 66, wherein the encapsulation structure is a lipid structure and / or polymer structure.

68. The composition according to one of claims 65 to 67, wherein the lipids are selected from at least one of a cationic (ionizable) lipid, one or more helper lipids, and / or a stealth lipid.

69. The composition according to claim 68, wherein at least two helper lipids are used and one of the helper lipids is a phospholipid, ester lipid, lysophospholipid, or a glycerol lipid and the second is a sterol.

70. The composition according to claim 69, wherein the cationic (ionizable) lipid is selected from the group consisting of Cl 2-200, DOTAP (1,2- dioleyl-3-trimethytammonium propane), DODAP (1 ,2-dioleyl-3 -dimethylammonium propane), DOTMA (1,2-di-O- octadecenyl-3 -trimethylammonium propane), DLinDMA, DLin-KC2-DMA, HGT4003, cKK- E12, ICE, DLin-MC3, 7C1, ALC-0315, SM-102, CL-1, 3060110, OF-02, 7C1, L319, A9, 93O17S, Lipid C24, 1014, Lipidl5, Lipid AX4, Lipid A6, BAMEA-016B, 98N12-5, 4A3- SC8, 5A2-SC8 and combinations thereof.

71. The composition according to one of claims 65-70, wherein the encapsulation structure is co-processed together with at least one excipient into the form of a dry powder.

72. The composition according to claim 71, wherein the at least one pharmaceutically acceptable excipient is selected from the group consisting of mono-, di- trioligo- or polysaccharides, sugar alcohols, mono-, di, tri, oligo- or polypeptides, proteins, esters, ethers, amides, amines, sulphates, thiols urethanes, phosphoesters, phosphazenes, amino acids, surfactants, lipids, stearates, polymers and combination thereof.

73. The composition according to claim 71 or claim 72, wherein the dry powder has a residual moisture of less than 10%, preferably less than 8%, most preferably less than 5%.

74. The composition according to one of claims 65 to 73, wherein the dry powder can at least partially be redispersed.

75. The composition according to one of claims 65 to 74, wherein the composition is formulated for use as a pharmaceutical dosage form, especially for pulmonary delivery.

76. The composition according to one of claims 65 to 75, wherein the composition is formulated for use to adapt the penetration of the particulate system through the natural barriers of the lung and / or airways of the respiratory tract.

77. The composition according to one of claims 65 to 77, wherein the composition is formulated such that the pharmaceutical dosage form can be administered using dry powder inhaler devices.

Images