Nano-in-micro encapsulated therapeutic nucleic acid dry powder and pharmaceutical product containing said dry powder

Abstract

The invention relates to Nano-in-micro (NIM) encapsulated bioactive therapeutic nucleic acid dry powder comprising lipid nanoparticles, the lipid nanoparticles comprising at least an ionizable cationic lipid, a helper lipid, a stealth lipid, and therapeutic nucleic acid, wherein the lipid nanoparticles are encapsulated in a pharmaceutically acceptable excipient, comprising at least one substance selected from the group consisting of monosaccharides, polysaccharides, sugar alcohols, (poly-)peptides, proteins, esters, urethanes, phosphoesters, phosphazenes, amino acids, surfactants, polymers. The invention also relates to the mass ratio of the therapeutic nucleic acid to the excipient is more than 0.10%, preferably between 0.90% and 1.30%.

Description

[0001] Nano-in-Micro encapsulated therapeutic nucleic acid dry powder and pharmaceutical product containing said dry powder

[0002] Description

[0003] The current invention relates a Nano-in-Micro (NIM) encapsulated therapeutic nucleic acid dry powder according to claim 1. The invention further relates to pharmaceutical product, in particular for the use with dry powder inhaler devices, comprising said dry powder in at least one sealed volume of a packaging according to claim 11 .

[0004] Application of drugs directly to their site of action is the optimal way to reduce doses and side effects. For lung diseases such as asthma, pulmonary delivery is therefore favoured. With a relatively low enzyme activity and a slow surface clearance, enzymatically prone substances are perfect candidates for this administration route. In addition, dry powder inhalers enable the delivery of drugs with a high shelf life and provide an fast and easy to use tool for patients along with high compliance. Despite several available treatments for lung diseases, nucleic acid therapy is a promising new tool to address uncontrollable disease variants such as severe, uncontrolled asthma, but also particularly viruses for which no antiviral compounds are available. Knowing the genome of the virus is sufficient to develop nucleic acid-based therapeutics that can inhibit viral replication. Small interfering RNA (siRNA) can silence the translation of messenger RNA into pathologically upregulated proteins and diminish disease symptoms. Other therapeutic nucleic acids aside from siRNA, such as miRNA, shRNA, dsRNA, mRNA, circRNA, saRNA, tracrRNA, IncRNA are equally promising substances on which nucleic acid-based therapeutics could be based on. However, siRNA based and other therapeutic nucleic acid based therapeutics face several challenges associated with the delivery into cells and enzymatic stability. To address these issues, nanoparticles are preferred to protect and encapsulate therapeutic nucleic acids such as siRNA.

[0005] State of the Art

[0006] In WO 2022 / 079105 A1 a method is disclosed how nano-in-micro encapsulated siRNA dry powder can be produced by spray drying from an aqueous suspension, leaving the siRNA intact, i.e. bioactive to a very large extent in the produced dry powder. This is a major advancement in the respective effort to provide for clinical doses of such gene therapy and the respective pharmaceuticals. However, in the state of the art the amount (mass ratio) of siRNA to sugar and in the produced dry powder is still relatively low. The prior art gives the mass ratio of siRNA to sugar and / or sugar alcohol to be preferably between 0.001 % and 0.02 in order to exhibit the high yield or high percentage of intact / bioactive siRNA in the dry powder. With this amount or concentration of siRNA, a treatment of a patient via inhaling the dry powder would require large amounts of repeated doses inhalations. Even if the inhalation of such a large amount of dry powder was possible for a patient, such a treatment or therapy would significantly reduce the patient’s compliance.

[0007] However, increasing the amount or mass ratio of the therapeutic nucleic acid, especially siRNA, in a respective dry powder provides several problems, which need to be overcome. First, that the shielding effect of the excipients or lipid nanoparticles shielding the therapeutic nucleic acid from environmental influences, especially heat, is likely to decrease if the mass ratio of the therapeutic nucleic acid was increased. This makes the therapeutic nucleic acid more prone to degrading influences in the production of the dry powder. As a result, the amount of bioactive therapeutic nucleic acid could be reduced which in turn might not lead to a sufficient net increase of bioactive therapeutic nucleic acid in the dry powder, Second, the increase of therapeutic nucleic acid also requires a respective increase in the mass percentage of lipid nanoparticles. Both changes could lead to even stronger hygroscopic behavior of the dry powder. This would adversely affect the shelf life of the dry powder and the delivery to the site of action, especially if this required passing through a humid and warm environment, such as an oropharynx of a patient, before reaching the site of action, i.e. the lung. Additionally, strong hygroscopic properties would make it increasingly difficult to dry the powder to a level that is acceptable. This might result in the need of higher temperatures during spry drying, which again might cause harm to or destroy the functionality of the therapeutic nucleic acid. In addition, a higher mass ratio of lipid nanoparticles and therapeutic nucleic acid provided in an aqueous suspension for spray drying would make the chemical / physical combination of the lipid nanoparticles with water-soluble excipients, such as sugar and / or sugar alcohol more difficult causing problems with the micro encapsulation of the therapeutic nucleic acid loaded lipid nanoparticles.

[0008] The scientific article “Effect of thermal and shear stress in the spray drying process on the stability of siRNA dry powders” by Jingya Wu et al. (International Journal of Pharmaceutics; 566 (2019) 32-39) discloses that high doses or high mass ratios of siRNA to mannitol (1 / 100) can be spray- dried without significantly effecting the bioactivity of the spray dried siRNA. However, the article only addresses a system in which siRNA and mannitol were dissolved with a weight ratio of 1 / 100 in RNase-free water to a concentration of 30 mg / mL. This spray-dried powder or its redispersion into a solvent would not be able to transfect into cells and could therefore not be internalized into cells as a suitable carrier system is missing.

[0009] In other words, this approach does not teach or suggest the effects of an increased mass ratio of lipid nanoparticles on the system while or after spray-drying and does in general not lead to a powder that could potentially be used in a pharmaceutical use, since the siRNA would have no chance of transfecting into a cell. Equally, the above-cited WO 2022 / 079105 A1 , which teaches a system that is able to transect into cells due to the lipid nanoparticle carriers, only teaches the use of very small quantities of lipid nanoparticles due to the very small amount of siRNA used.

[0010] Accordingly, it is the aim of the present invention to propose a nano-in- micro encapsulated therapeutic nucleic acid dry powder that enables a clinical use or use as a pharmaceutical dosage.

[0011] Invention

[0012] This problem is solved by the Nano-in-micro (NIM) encapsulated bioactive therapeutic nucleic acid dry powder according to claim 1 .

[0013] Advantageous embodiments of the invention are presented in the following description, in the drawings as well as in the dependent claims. Features hereinafter described or claims as device or product features shall also be considered disclosed and claimable as method features, and vice versa.

[0014] The Nano-in-micro (NIM) encapsulated bioactive therapeutic nucleic acid dry powder according to the invention comprise lipid nanoparticles, the lipid nanoparticles comprising from at least an ionizable cationic lipid, a helper lipid, a stealth lipid, and therapeutic nucleic acid, wherein the lipid nanoparticles are encapsulated in a pharmaceutically acceptable excipient, comprising at least one substance selected from the group consisting of monosaccharides, polysaccharides, sugar alcohols, (polypeptides, proteins, esters, urethanes, phosphoesters, phosphazenes, amino acids, surfactants, polymers, wherein the mass ratio of the therapeutic nucleic acid to the excipient is more than 0.10%, especially between 0.90% and 1.30%. It was surprisingly found that the nano-in-micro encapsulated therapeutic nucleic acid, preferably siRNA, could be produced, especially by a spray drying method as disclosed in WO 2022 / 079105 A1 with a mass ratio of siRNA or therapeutic nucleic acid to excipient that is high enough to enable a clinical or pharmaceutical application in humans.

[0015] The disclosure of WO 2022 / 079105 A1 is incorporated into this disclosure by reference with respect to the process / method of spry drying and the analysis of bioactivity of the therapeutic nucleic acid in the dry powder.

[0016] It was proven possible to achieve this result without losing the shielding effect on the therapeutic nucleic acid, thereby again producing a high yield of bioactive / intact spray dried therapeutic nucleic acid. In addition, it was established that the dry powder could be produced as a dry powder without too much or too strong hygroscopic properties. Also very surprisingly, the greatly increased amount of lipid nanoparticles did not affect the spray-dried powder and / or the encapsulated therapeutic nucleic acid, preferably siRNA, in such a fashion that the dry powder would inhibit the use in a pharmaceutical and commercial use, especially for inhalation. The amount of lipid nanoparticles may need to reach levels of approximately 10% mass of the dry powder (see below). It could not have been anticipated that this massive change in the overall formulation would not have significant impact on the spray drying process, the spray-dried powder and / or the state of the therapeutic nucleic acid, preferably siRNA, in the dried state. Especially, in the presence of proportionally less stabilizing excipient, the thermal energy input per therapeutic nucleic acid is much higher during spray drying, and it is therefore surprising that the therapeutic nucleic acid is not damaged in the course of the spray drying.

[0017] A difference compared to the spray drying process according the disclosure of WO 2022 / 079105 A1 was that prior to adding the lipid nanoparticles with the encapsulated siRNA or therapeutic nucleic acid to the water excipients, preferably a 10% lactose solution in purified water, the concentration of lipid nanoparticles was raised. The concentration was determined by standard procedure and was necessary since the lipid nanoparticles can be produced only with a limited concentration. In a preferred variation of the known method, the concentration was raised to 60mM to 70mM from the concentration level of about 16mM of the direct preparation.

[0018] Surprisingly, the concentrated lipid nanoparticles could be combined with the water-soluble excipient without significant dispersion problems and could be spray dried without significant negative impact on the state or activity of the spray dried siRNA or therapeutic nucleic acid.

[0019] The used Ionizable Cationic Lipids can comprise or consist of lipids that can carry a positive charge under certain pH conditions. Their role includes facilitating nucleic acid encapsulation in lipid nanoparticles (LNPs), mediating endosomal membrane disruption for nucleic acid release, and possibly supporting endosomal uptake. Examples include lipids like 1 ,2-dioleoyl-3-dimethylammonium-propane (DODAP), DLin- MC3-DMA, being a key component in the formulation of LNPs used in the Pfizer-BioNTech and Moderna COVID-19 vaccines, and other similar ionizable lipids.

[0020] The used stealth lipids, often PEGylated lipids or their alternatives are added to LNPs to improve their stability, prolong circulation time in the body, and help evade the immune system. Examples of stealth lipids include PEG-lipids (e.g., DSPE-PEG2000), Polyvinylpyrrolidone (PVP) lipids, Polyvinyl alcohol (PVA) lipids, Poloxamer lipids, and Polysarcosine lipids. These examples all serve the purpose of providing a stealth characteristic to the LNPs, which enhances their stability and improves biodistribution. The helper lipids support the stability of LNPs during storage and circulation. They can include a range of lipids such as sterols, phospholipids, and glycerolipids. Examples include cholesterol (a common sterol), DOPE (Dioleoylphosphatidylethanolamine, a type of phospholipid), and DOPC (Dioleoylphosphatidylcholine, another type of phospholipid). These lipids are typically non-cationic and help to improve the fluidity and flexibility of LNPs, as well as overall stability. In a preferred embodiment of the invention, two helper lipids are used. When using two helper lipids it is especially advantageous that one of the helper lipids is cholesterol.

[0021] The choice of the respective set of lipids is largely dependent on the specific formulation and desired properties of the LNPs. Different applications may require adjustments in the types and ratios of these components.

[0022] As suggested in WO 2022 / 079105 A1 the first spray dry processes were undertaken with sugar and / or sugar alcohol as the pharmaceutically acceptable excipient. However, the excipient could also be chosen or combined from at least one of following substances: monosaccharides, polysaccharides, sugar alcohols, (poly-)peptides, proteins, esters, urethanes, phosphoesters, phosphazenes, amino acids, surfactants, polymers. The excipients generally have a smaller impact on the spraydrying process and can therefore be adapted to the special needs of the respective dry powder.

[0023] The excipient composition, on the other hand, has a significant influence on the LNP stabilizing properties of the dry powder formulation. Depending on the overall composition of the LNPs (lipids and cargo) and the overall LNP concentration in the dry powder, the excipient composition is adjusted to best preserve the LNPs and ensure long-term storage stability.

[0024] Additionally, the excipient composition influences the physicochemical properties of the dry powder such as particle size and particle size distribution, moisture content, surface characteristics and morphology, density, flow properties, disintegration time and solubility, crystallinity and polymorphism, drug distribution and homogeneity, electrostatic properties, and thermal properties.

[0025] The combination of excipients significantly determines solubility of the dry powder and subsequently ensures the uptake into the cells.

[0026] Additionally, they determine the inhalable properties of the dry powder like dose delivered, mass median aerodynamic diameter and fine particle fraction.

[0027] According to a preferred embodiment of the dry powder therapeutic nucleic acid comprising at least one substance selected from the group consisting of siRNA, miRNA, shRNA, dsRNA, mRNA, circRNA, saRNA, tracrRNA, IncRNA. The therapeutic nucleic acid enable a very broad range of applications, especially pharmaceutical uses of the respective dry powders. The therapeutic nucleic acids are structurally similar enough to siRNA so that the established principles of a nano-in-micro encapsulation by spray drying can be translated to different types of therapeutic nucleic acids with the inventive high mass ratio of the therapeutic nucleic acid to the excipient.

[0028] For example, antisense oligonucleotides (ASOs) as a sub-group of the therapeutic nucleic acids are single-stranded DNA or RNA molecules designed to be complementary to a specific target mRNA sequence. Their characteristics, such as a smaller number of nucleotides, require optimization for nanoparticle formulation. While small interfering RNA (siRNA) does not necessarily require chemical modification of the nucleotides, modifications of ASOs are often required to ensure stability within the cell.

[0029] In general, siRNA and ASOs can be used for similar purposes, such as gene silencing, but they have different mechanisms of action. ASOs bind directly to complementary mRNA sequences, preventing translation or promoting degradation through RNase H-mediated mechanisms. In contrast, siRNA is incorporated into the RNA-induced silencing complex (RISC), which then binds to and degrades the target mRNA. Consequently, the kinetics of degradation differ between these two approaches.

[0030] According to a preferred embodiment of the dry powder, at least 50%, preferably at least 75%, more preferably at least 85% of the therapeutic nucleic acid is bioactive in the dry powder state and / or after re-dispersion. The bioactivity of the spray-dried therapeutic nucleic acid can be assessed as disclosed in WO 2022 / 079105 A1. In a preferred embodiment, the therapeutic nucleic acid in the dry powder is structurally fully intact. In other words, the therapeutic nucleic acid within the dry powder is free of degradation products. The high percentage of bioactive therapeutic nucleic acid is especially important for the high mass ratio of therapeutic nucleic acid of the dry powder. Because even if it was technologically possible to spray dry a powder with a high initial amount or ratio of therapeutic nucleic acid the bioactivity after the spray drying determines whether the powder can be practically or conveniently used, for example as a pharmaceutical dosage for a dry powder inhaler (DPI).

[0031] According to another advantageous embodiment of the invention, the residual moisture of the dry powder is less than 7.5%, preferably less than 5%, in particular less than 3%. This leads to a longer shelf life of the resulting dry powder. Preferably, an additional drying process can reach the residual moisture after the initial spray drying.

[0032] In accordance with an advantageous variation of the invention, the lipid mass ratio of the dry powder is above 8%, preferably above 10%. This is advantageous because high lipid mass ratio supports a high mass ratio of therapeutic nucleic acid. Surprisingly even with such a high amount or mass ratio of lipids in the dry powder the hygroscopic properties of the dry powder were still within the acceptable range to be used as a dry powder for a dry inhaler.

[0033] According to another preferred embodiment, the Mass Median Diameter (MMD) of the dry powder is between 1 pm and 6pm. With this preferred MMD, the use of the dry powder is optimized for pulmonary delivery by inhalation.

[0034] In another preferred embodiment, the dry powder, in particular in a redispersed state within an aqueous medium, exhibits cell transfectability into living cells. As expressed above, for a therapeutic effect of the Nano- in-micro (NIM) encapsulated bioactive therapeutic nucleic acid, it is not only important to administer a substance near target cells that has a sufficiently high rate or amount of therapeutically active ingredient, such as the therapeutic nucleic acid. Further, an uptake or internalisation of the therapeutic nucleic acid is the key factor in enabling the therapeutic effect of the therapeutic nucleic acid. Thus, providing the cell transfectability into living cells the dry powder or it re-dispersed form in an aqueous medium is a very important aspect for using the dry powder in a therapeutic application.

[0035] According to a further embodiment of the invention, the excipient comprises or consist of sugar and / or sugar alcohol, preferably lactose, mannitol trehalose and / or leucin. It is believed that the above-identified excipients have an especially advantageous effect on the resulting dry powder. The respective mechanisms are, however, not yet fully understood or proven. The remaining excipients of claim 1 might are in general equally suitable for producing the dry powder.

[0036] In a further embodiment of the invention, the lipid nanoparticles are according to an Onpattro®-formulation or patisiran formulation. The use of Onpattro® being an already clinically approved substance and the respective Onpattro®-formulation being readily available, makes the use of Onpattro® and derivatives therefrom good starting points for the LNP as they are expected to receive clinical approval more conveniently. The respective lipid nanoparticles are disclosed in US 8,158,601 B2, which is hereby included by reference into the current disclosure. The respective formulation is documented by the EMA under the product name EU / 3 / 11 / 857 and was given the non-proprietary name “PATISIRAN”.

[0037] In an alternative, preferred embodiment of the invention, the the ionizable cationic lipid is selected from the group consisting of C12-200, DOTAP

[0038] (1 ,2-dioleoyl-3-trimethytammonium propane), DODAP (1 ,2-dioleoyl-3- dimethylammonium propane), DOTMA (1 ,2-di-O-octadecenyl-3- trimethylammonium propane), DLinDMA (N, N-dilinoleyl-N, N- dimethylammonium chloride), DLin-KC2-DMA (6Z,9Z,28Z, 31 - heptatriaconta-6,9,28,31-tetraen-19-yloxy(N,N- dimethylcarbamoylmethyl)amine), HGT4003 (chemical name not specified), CKK-E12 (C2-dimethylamino)ethyl methanethiosulfonate), ICE (4A3-SC8), ALC-0315 ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen- 19-yloxy (N,N-dimethylcarbamoylmethyl)amine), SM-102 (heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-((pentylamino)methyl)hexyl)amino)octanoate), and combinations thereof. Said inonizable cationic lipids enable a very effective transfection into cells.

[0039] In another preferred embodiment, the dry powder is used as a pharmaceutical dosage form, in particular for pulmonary delivery. In this respect, it is especially advantageous that the dry powder, when produced by a spray drying process, such as disclosed in WO 2022 / 079105 A1 , results in a particle size and size distribution, which is suitable for pulmonary delivery. This allows for a direct use of the powder without further processing, i.e. the dry powder can directly be packaged and used. This among other things achieved by the fact that the particles of the dry powder have a mass ratio of therapeutic nucleic acid to excipient of more than 0.10%, especially between 0.90% and 1.30%. It is very surprising that the amount of lipid nanoparticles that can make up for 10% or more of the mass of the dry powder, which are necessary to achieve the respective high mass ration of therapeutic nucleic acid does not affect the spray-drying process, the resulting dry powder or the state and functionality of the therapeutic nucleic acid in the dry and / or re-dispersed state.

[0040] According to another aspect of the invention, the therapeutic nucleic acid of the dry powder is active in silencing the translation of messenger RNA and / or silencing the replication of viral RNA into proteins causing lung diseases. This enables very potent application opportunities, since the dry powder is, in some cases directly after the spray drying, is in a condition that can be used with a DPI. Thus, the treatment of lung diseases and the use of respective therapeutic nucleic acid is a promising application of the inventive dry powder.

[0041] In a further preferred embodiment of the dry powder, the therapeutic nucleic acid is active in promoting a protein expression of a natural, especially autologous, protein. This enables the use of the dry powder in protein replacement therapy applications, where the body is not able to produce a certain protein on its own. By promoting the expression of the natural, especially autologous, protein the lack of natural, especially autologous, protein can be overcome. In these applications, the clinical picture is triggered by the absence of the endogenous or autologous protein. The body produces said protein incorrectly, not in a functional form or not at all. A possible application is protein replacement therapy for cystic fibrosis, in which the therapeutic nucleic acid promotes the expression of the membrane protein Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), which is not correctly produced by the cells. In a further preferred embodiment of the invention, the therapeutic nucleic acid is active in promoting a protein expression of a protein that is characteristic to a specific virus and recognizable by the immune system. In this embodiment, the dry powder can be used in vaccine applications similar to the SARS-CoV-2 vaccinations in which an mRNA was used as a therapeutic nucleic acid that promoted the expression of the spike protein of SARS-CoV-2. The immune system recognizes those characteristic spike proteins as foreign and reacts to them. Among other things, it forms antibodies against the virus specific spike protein.

[0042] The above problem is also solved by a pharmaceutical product, in particular for the use with dry inhaler devices, comprising a packaging enclosing at least one sealed volume, preferably multiple sealed volumes, wherein the dry powder of any of the previously described embodiments is enclosed in the at least one sealed volume. This allows a very convenient use of the dry powder for a patient. The packaging can be built and shaped in such a form that in can be used with one or more different types of dry inhaler devices. In a preferred embodiment, the packaging might be realized as a blister packaging. Preferably, the packaging comprises multiple sealed volumes. According to a preferred embodiment, the packaging is constructed and shaped in such a way that it can be used in magazine function together with the dry inhaler device, allowing a sealed volume to be “loaded” into an application position and / or mechanically opened upon activating a preparation mechanism of the dry inhaler, such as a loading lever. Preferably, the packaging is built to release the dry powder into a stream of gas, preferably air, upon activating a release mechanism of the dry inhaler device. In an alternative preferred embodiment, the packaging may have the form and function of a capsule, in which the dry powder is enclosed. Fiqures

[0043] Aspects of the invention are described by with respect to the figures, showing examples and advantageous embodiments of the invention.

[0044] Those figures show:

[0045] Fig. 1 : A Scanning Electron Microscopy Image of an embodiment of the inventive dry powder;

[0046] Fig. 2: The results of an siRNA integrity test a the dry powder according to the invention;

[0047] Fig. 3: shows particle size distribution of the dry powder according to the invention after releasing it from a capsule based dry powder inhaler;

[0048] Fig. 4: shows an in vitro gene silencing of the enhanced green fluorescent protein (EGFP) in H1299-EGFP cell line, a human epithelial-like lung cancer cell line carrying a plasmid for EGFP;

[0049] Fig. 5: shows in vivo murine asthma model showing the downregulation of Type 2 asthma related cytokines and %eosinophiles in bronchoalveolar lavage (BAL) fluid in vivo following an asthma NIM treatment with an inventive dry powder;

[0050] Fig. 6: shows the residual moisture levels assessed by Karl Fischer

[0051] (KF) titration of different dry powder formulations according to the invention.

[0052] Fig. 7: shows the fine particle fraction (FPF), percentage of powder consisting of a MMD between 1-5 pm, of the dry powder according to the invention. Fig. 1 shows a Scanning Electron Microscopy (SEM) image of the dry powder (01 ) according to the invention. The particles (02) of the dry powder show a spherical shape. As can be seen from the scale bar (03), which represent a length / distance of 10pm, that the particles (02) have a diameter of less than 10pm.

[0053] Fig. 2 shows the result of a therapeutic nucleic acid integrity test (Bioanalyser). The second and the third column represent the comparison between fresh and spry dried lipid nanoparticles with the respective therapeutic nucleic acid. The test bands / lines at the values 4 and 20 of the ladder are representative of structurally and functionally intact therapeutic nucleic acid. It is shown in Fig. 2 that no internal lines between the ones of 4 and 20 are present for the dry powder. This means that no degradation of the therapeutic nucleic acid has occurred and that the therapeutic nucleic acid is intact in the dry powder.

[0054] Fig. 3 shows the particle size distribution of the dry powder according to the invention after releasing it from a capsule based dry powder inhaler. Assessing the Mass Median Diameter (MMD) via a laser diffraction equipped with an inhalation module (Sympatec). It is shown that the MMD lies between 1-6 pm, which required for inhaled dry powder pulmonary delivery.

[0055] Fig. 4 shows an in vitro gene silencing of the enhanced green fluorescent protein (EGFP) in H1299-EGFP cell line, a human epithelial-like lung cancer cell line carrying a plasmid for EGFP. The gene silencing efficiency of different dry powder formulations according to the invention show no significant differences to the fresh nanoparticle formulation before spray drying, preserving the silencing efficiency.

[0056] Fig. 5 in vivo asthma model shows the downregulation of Type 2 asthma related cytokines and %eosinophiles in bronchoalveolar lavage (BAL) fluid in vivo following an asthma NEM treatment. An OVA-induced acute asthma mouse model was used. Over two weeks, animals of the treatment groups - i.e. one group treated with active, and one group treated with non-coding oligonucleotide - were sensitized with OVA and adjuvant, followed by three intranasal OVA challenges to induce asthmatic symptoms. Prior to these challenges, three doses of 2 mg of anti-GATA3

[0057] NEM dry powder, along with a noncoding control NEM, were administered intratracheally to assess whether the NEMs could reduce allergic reactions. The animals were sacrificed 24 hours after the last treatment. Bronchoalveolar lavage (BAL) fluid was collected from the animals. Fig. 6 shows the residual moisture levels assessed by Karl Fischer (KF) titration of different dry powder formulations according to the invention. All values stay below 3.5% water content in the final formulation.

[0058] Fig. 7 shows the fine particle fraction (FPF), percentage of powder consisting of a MMD between 1-5 pm, of the dry powder according to the invention after releasing it from a capsule based dry powder inhaler.

[0059] Assessing the FPF via a laser diffraction equipped with an inhalation module (Sympatec). It is shown that the high FPF are achieved.

Claims

Claims1 . Nano-in-micro (NIM) encapsulated bioactive therapeutic nucleic acid dry powder comprising lipid nanoparticles, the lipid nanoparticles comprising at least an ionizable cationic lipid, a helper lipid, a stealth lipid, and therapeutic nucleic acid, wherein the lipid nanoparticles are encapsulated in a pharmaceutically acceptable excipient, comprising at least one substance selected from the group consisting of monosaccharides, polysaccharides, sugar alcohols, (poly-)peptides, proteins, esters, urethanes, phosphoesters, phosphazenes, amino acids, surfactants, polymers, characterized in that the mass ratio of the therapeutic nucleic acid to the excipient is more than 0.10%, preferably between 0.90% and 1.30%.

2. Dry powder according to claim 1 characterized in that the therapeutic nucleic acid comprising at least one substance selected from the group consisting of siRNA, miRNA, shRNA, dsRNA, mRNA, circRNA, saRNA, tracrRNA, IncRNA.

3. Dry powder according to claim 1 or 2, characterized in that at least 50%, preferably 75%, most preferably at least 85% of the therapeutic nucleic acid is bioactive in the dry powder state and / or after re-dispersion.

4. Dry powder according to any of the preceding claims, characterized in thatresidual moisture of the dry powder is less than 7.5%, in particular less than 5%, preferably less than 3%.

5. Dry powder according to any of the preceding claims, characterized in that the lipid mass ratio of the dry powder is above 8%, preferably above 10%.

6. Dry powder according to any of the preceding claims, characterized in that the Mass Median Diameter (MMD) of the dry powder is between1 pm and 6pm.

7. Dry powder according to any of the preceding claims, characterized in that the dry powder, in particular in a re-dispersed state within an aqueous medium, exhibits cell transfectability into living cells.

8. Dry powder according to any of the previous claims, characterized in that the excipient comprises or consists of a sugar and / or alcohol sugar, preferably lactose, mannitol trehalose and / or leucin.

9. Dry powder according to any of the preceding claims, characterized in that, the ionizable cationic lipid is selected from the group consisting of C12-200, DOTAP (1 ,2-dioleoyl-3-trimethytammonium propane), DODAP (1 ,2-dioleoyl-3-dimethylammonium propane), DOTMA (1 ,2- di-O-octadecenyl-3-trimethylammonium propane), DLinDMA (N,N- dilinoleyl-N,N-dimethylammonium chloride), DLin-KC2-DMA (6Z,9Z,28Z,31Z-heptatriaconta-6,9,28,31 -tetraen-19-yloxy(N,N- dimethylcarbamoylmethyl)amine), HGT4003 (chemical name notspecified), CKK-E12 (C2-dimethylamino)ethyl methanethiosulfonate), ICE (4A3-SC8), ALC-0315 ((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy (N,N- dimethylcarbamoylmethyl)amine), SM-102 (heptadecan-9-yl 8-((2- hydroxyethyl)(6-oxo-6- ((pentylamino)methyl)hexyl)amino)octanoate), and combinations thereof.

10. Dry powder according to claims 1 to 9, used as a pharmaceutical dosage form, especially for pulmonary delivery.11 . Dry powder according to claim 1 to 10, wherein the therapeutic nucleic acid is active in silencing the translation of messenger RNA and / or silencing the replication of viral RNA into proteins causing lung diseases.

12. Dry powder according to any of the preceding claims, characterized in that the therapeutic nucleic acid is active in promoting a protein expression of a natural, especially autologous, protein.

13. Dry powder according to any of the preceding claims, characterized in that the therapeutic nucleic acid is active in promoting a protein expression of a protein that is characteristic to a specific virus and recognizable by the immune system.

14. Pharmaceutical product, in particular for the use with dry powder inhaler devices, comprising a packaging enclosing at least one sealed volume, preferably multiple sealed volumes, wherein a drypowder is enclosed in the sealed volume, characterized by a dry powder of any of the preceding claims.

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