Respirable polynucleotide powder formulations for inhalation - Patents.com

Abstract

The present invention provides respirable dry powder particle formulations comprising a polynucleotide, preferably prepared by spray drying, for delivery to the pulmonary system by inhalation. Preferably, the polynucleotide is RNA.

Description

[Background technology]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 760,232, filed November 13, 2018, the entire contents of which are incorporated herein by reference.

[0002] The development of respirable polynucleotide-containing powder particle formulations, e.g., RNA dry powder particle formulations containing therapeutically relevant levels of RNA for delivery to the pulmonary system by inhalation, would provide a non-invasive and targeted approach in the treatment of disease and an improvement to nebulizers and pressurized metered-dose inhalers. Summary of the Invention

[0003] The present invention provides a respirable dry powder particle formulation comprising a polynucleotide prepared by spray drying for delivery to the pulmonary system by inhalation. Preferably, the polynucleotide is RNA. Preferably, the present invention provides a respirable dry powder particle formulation of a polynucleotide prepared by spray drying for delivery to the pulmonary system by inhalation. The present invention includes formulations and parameters used in spray-dried polynucleotides, such as RNA-containing powders, including mRNA-containing powders, that have chemical, physical and aerosol properties suitable for pulmonary delivery, preferably delivery using a patient-activated dry powder inhaler. For inhaled drugs listed at www.acorda.com, see, for example, the ARCUS® platform.

[0004] Preferably, the respirable polynucleotide dry powder particle formulation for pulmonary delivery comprises: i) at least about 1% by weight of the particle of polynucleotide (e.g., RNA); ii) at least about 10% DPPC by weight of the particle; iii) optionally, one or more of arginine, leucine, isoleucine, or valine, each at least about 1% by weight of the particle, where if more than one amino acid is selected, each amino acid has either the same or a different weight percentage as the other selected amino acids; iv) optionally, at least about 1% NaCl by weight of the particles; v) optionally, at least about 10% Tris by weight of the particles; vi) optionally, at least about 2% EDTA by weight of the particles; vii) optionally, at least about 40% trehalose by weight of the particle; viii) optionally, at least about 2% PEI by weight of the particles; and ix) optionally comprising at least about 40% lactose by weight of the particles; All components of the RNA dry powder add up to 100 weight percent.

[0005] Preferably, the formulation comprises about 1% to about 80% by weight, preferably about 1% to 70% by weight, for example about 1%, 10%, 25%, 50% or about 60% by weight of polynucleotide. Preferably, the polynucleotide is RNA. The term "RNA" is intended to encompass all RNA and modified RNA in all forms and from all sources. Preferably, the RNA is mRNA. The mRNA can be capped or uncapped mRNA. Preferably, the mRNA is capped RNA. In the example using yeast RNA, the yeast RNA is a placeholder for the mRNA.

[0006] Preferably, the polynucleotide can be at least about 10, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 200 or more nucleic acids in length. Preferably, the RNA can be about 10 to about 12,000 or more nucleic acids in length. Preferably, the RNA can be about 20 to about 12,000 or more nucleic acids in length. Preferably, the RNA can be about 50 to about 12,000 or more nucleic acids in length. Preferably, the RNA can be about 100 to about 12,000 or more nucleic acids in length. Preferably, the RNA may have a length of about 20 to about 12,000 monomers, or about 20 to about 10,000 monomers, or about 20 to 8000 monomers, or about 20 to 6000 monomers, or about 20 to about 5000 monomers, or about 20 to about 4000 monomers, or about 20 to about 3600 monomers, or about 20 to about 3200 monomers, or about 20 to about 3000 monomers, or about 20 to about 2800 monomers, or about 20 to about 2600 monomers, or about 20 to 2400 monomers, or about 20 to 2200 monomers, or about 50 to 3200 monomers, or about 50 to 3000 monomers, or about 50 to 2600 monomers.

[0007] Preferably, the formulation contains about 10% to about 30% by weight, preferably about 15% to 25% by weight, for example about 15%, 18%, 20%, 22% or about 25% by weight, of DPPC.

[0008] The present invention provides respirable polynucleotide (e.g., RNA) dry powder formulations that can optionally include about 1%, preferably about 1% to about 20%, preferably about 1% to about 10%, preferably about 1% to about 5% of a cationic excipient, which can be at least one positively charged amino acid or polyethyleneimine (PEI).

[0009] Preferably, the formulation optionally comprises one or more of arginine, leucine, isoleucine, or valine, optionally in an amount of about 1% to about 90% by weight, preferably about 5% to 85% by weight, e.g., about 5%, 20%, 30%, 40%, 50%, 55%, 70%, 74%, 79%, 80%, or about 85% by weight, and where more than one amino acid is selected, each amino acid has the same or a different weight percentage as the other selected amino acids.

[0010] Preferably, the formulation optionally comprises about 1% to about 5% by weight NaCl, preferably about 1% to 3% by weight, for example about 1%, 2% or about 3% by weight.

[0011] Preferably, the formulation optionally comprises about 1% to about 25% by weight, preferably about 5% to 20% by weight, for example about 5%, 10%, 15% or about 20% by weight, of Tris.

[0012] Preferably, the formulation optionally contains about 1% to about 10% by weight, preferably about 2% to 8% by weight, for example about 2%, 3%, 4%, 5%, 6% or about 7% by weight, of EDTA.

[0013] Preferably, the formulation may further comprise about 40% to about 90% by weight, preferably about 45% to 85% by weight, for example about 45% by weight, 50% by weight, 52% by weight, 53% by weight, 54% by weight, 55% by weight, 60% by weight, 65% by weight, 70% by weight, 72% by weight, 74% by weight, 75% by weight, 77% by weight, 79% by weight, 80% by weight or about 82% by weight of trehalose.

[0014] Preferably, the formulation optionally further comprises about 40% to about 90% by weight, preferably about 45% to 85% by weight, for example about 45%, 50%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 72%, 74%, 75%, 77%, 79%, 80% or about 82% by weight of lactose.

[0015] Preferably, the formulation optionally comprises about 1% to about 10% by weight, preferably about 1% to 8% by weight, e.g., about 1%, 2%, 3%, 4%, 5%, 6% or about 7% by weight, of PEI.

[0016] Preferably, the formulation is selected from one of the following formulations listed in Table 1.

[0017] [Table 1]

[0018] The weight percentage is intended to reflect the total amount of solids, lipids, and / or excipients in the dry particle, regardless of residual water, solvents, or impurities. Preferably, all components of the dry particle add up to 100% by weight. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 shows the spray dryer settings used in the development of RNA powder. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] As used herein, the terms "a," "an," and "the" are defined to mean "one or more" and include the plural, unless the context is inappropriate.

[0021] As used herein, the term "comprising" is synonymous with "including," "containing," or "characterized by," is inclusive or open-ended, and does not exclude additional, unrecited elements of a composition or method step. The term "consisting of" excludes elements, steps, or ingredients not otherwise specified. The term "consisting essentially of" limits the scope of the composition or method to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the specified composition or method.

[0022] It should be noted that ratios, concentrations, amounts, and other numerical data may be expressed in a range format herein. Such range formats are used for convenience and brevity, and should therefore be understood to be flexibly interpreted to include not only the numerical values ​​explicitly recited as limits of the range, but also all individual numerical values ​​or subranges subsumed within the range, as if each numerical value and subrange were explicitly recited. By way of example, a concentration range of "about 0.1% to about 5%" should be interpreted to include not only the explicitly recited concentration of about 0.1% to about 5% by weight, but also the individual concentrations (e.g., 1%, 2%, 3%, and 4%) and subranges (e.g., 0.5%, 1.1%, 2.2%, 3.3%, and 4.4%) within the indicated range. The term "about" can include ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%, or more, of the numerical value being modified. Furthermore, "about 'x' to 'y'" includes "about 'x' to about 'y'".

[0023] The term "dry powder" as used herein refers to a composition containing finely dispersed, respirable dry particles that can be dispersed in an inhalation device and then inhaled by a subject. Such dry powders or dry particles may contain up to about 15% water or other solvent, preferably up to about 10% water or other solvent, or preferably be substantially free of water or other solvent, or preferably be anhydrous.

[0024] As used herein, the terms "dry particles" and "particles" refer to respirable particles that may contain up to about 15% water or other solvent, preferably up to 10% water or other solvent, or preferably be substantially free of water or other solvent, or preferably be anhydrous.

[0025] The term "respirable" as used herein refers to dry particles or powders suitable for delivery to the respiratory tract of a subject by inhalation (e.g., pulmonary delivery). Respirable dry powders or dry particles have a mass median aerodynamic diameter (MMAD) of less than about 10 microns, preferably about 5 microns, and more preferably about 3 microns or less. "Mass median aerodynamic diameter" (MMAD) is also referred to herein as "aerodynamic diameter". Experimentally, aerodynamic diameter can be determined by utilizing gravitational settling techniques, which use the time it takes a collection of powder particles to settle a specific distance to directly infer the aerodynamic diameter of the particle. An indirect method for measuring mass median aerodynamic diameter (MMAD) is the multi-stage liquid impinger (MSLI). Aerodynamic diameter d aer is the formula, d aer =d g √ρ tap where d g is the geometric diameter, e.g., MMGD, and ρ is the powder density.

[0026] As used herein, the terms "administration" or "administering" respirable dry particles refer to introducing the respirable dry particles into the airways of a subject.

[0027] As used herein, the term "airways" includes the upper airways (e.g., nasal cavity, nasal cavity, throat, pharynx), respiratory airways (e.g., larynx, trachea, bronchi, bronchioles) and lungs (e.g., respiratory bronchioles, alveolar ducts, alveolar sacs, alveoli). The deep lung or alveoli are typically the desired target of inhaled therapeutic formulations for systemic drug delivery. In one embodiment of the invention, the majority of the particle mass is deposited in the deep lung or alveoli. In another embodiment of the invention, delivery is primarily to the central airways. In other embodiments, delivery is to the upper airways.

[0028] "Pulmonary delivery," as that term is used herein, refers to delivery to the respiratory tract. Pulmonary delivery includes inhalation by a patient capable of inhaling independently, or via a ventilation system, such as a mechanical ventilation (MV) system, or a non-invasive mechanical ventilation system (NIMV), such as a continuous positive airway pressure (CPAP) system.

[0029] As used herein, "gPSD 50 " refers to the average geometric diameter where 50% of the particles have a diameter greater than the recited value and 50% of the particles have a diameter less than the recited value.

[0030] As used herein, the terms "FPF(<5.6)", "FPF(<5.6 microns)", "FPF<5.6 μm" and "fine particle fraction less than 5.6 microns" refer to the fraction of a sample of dry particles having an aerodynamic diameter less than 5.6 microns. For example, a two-stage or three-stage collapse ACI can be used to measure FPF<5.6 microns. The two-stage collapse ACI consists of only the top stage (S0) and the filter stage of the eight-stage ACI, allowing the collection of two separate powder fractions. Specifically, the two-stage collapse ACI is calibrated such that the fraction of powder collected on S0 is composed of non-respirable dry particles having an aerodynamic diameter greater than 5.6 microns. Thus, the fraction of powder that passes through S0 and is deposited on the filter stage is composed of respirable dry particles having an aerodynamic diameter less than 5.6 microns. The airflow rate during such calibration is about 60 L / min. This parameter may also be identified as "FPF TD(<5.6)", where TD means total dose. Similar measurements can be made using an 8-stage ACI. The 8-stage ACI cutoff differs at the standard 60 L / min flow rate, but the FPF TD (<5.6) can be estimated from the 8-stage complete data set. The 8-stage ACI results can also be calculated by the USP method, where the dose collected in the ACI is used instead of what was in the capsule to determine the FPF.

[0031] As used herein, the terms "FPF(<3.4)", "FPF(<3.4 microns)", "FPF<3.4 μm" and "fine particle fraction less than 3.4 microns" refer to the fraction of the mass of respirable dry particles having an aerodynamic diameter less than 3.4 microns. For example, a three-stage disintegration ACI can be used to measure both FPF less than 5.6 microns and FPF less than 3.4 microns. A three-stage disintegration ACI consists of collection stages S0, S2 and a filter stage, providing the fraction of powder with aerodynamic diameters greater than 5.6 microns, less than 5.6 microns and less than 3.4 microns. This parameter may also be identified as "FPF TD(<3.4)", where TD means total dose. Similar measurements can be performed using an eight-stage ACI. The eight-stage ACI results can also be calculated by the USP method, where the dose collected in the ACI is used instead of that which was in the capsule to determine the FPF. Other cutoff values ​​for FPF (i.e., <5.0 microns, etc.) can be similarly utilized by utilizing different stage configurations of the ACI or by extrapolating from the results obtained for a particular set of stages and cutoff diameters.

[0032] A preferred "excipient", as that term is used herein, is an excipient that can be taken into the lungs. Such an excipient can be generally regarded as safe (GRAS) by the U.S. Food and Drug Administration. Such an excipient includes water.

[0033] The terms "patient" or "subject", as used interchangeably herein, are individuals to whom the compositions of the invention may be administered. Examples of such individuals include elderly humans, adult humans, and pediatric humans. Pediatric humans include individuals from birth to 18 years of age. Pediatric children include, but are not limited to, the following subpopulations: neonates, including newborn individuals up to about 28 days or 1 month of age; infants, including individuals from the neonatal stage to 12 months of age; toddlers, including individuals 1-3 years of age; preschoolers, including individuals 3-5 years of age, school-age children, including individuals 6-10 years of age, and adolescents, including individuals 11-14 years of age. Pediatric children may be referred to as having an age range of about 6 to about 11 years of age, about 12 to about 17 years of age, or about 6 to about 17 years of age. Immature human children (premature infants) include individuals with a gestational age of less than about 37 weeks. Elderly humans may be at least 50 years of age, such as at least 65 years of age, such as 70 years of age or older.

[0034] The invention further provides for packaging the respirable dry powder in the presence of a desiccant, such as silica gel desiccant; zeolite; alumina; bauxite; anhydrous calcium sulfate; water-absorbent clay; molecular sieve; and any mixture thereof. The desiccant may be packaged in a sachet, pouch or pack and placed in the package with the capsule. The desiccant may also be incorporated into the package itself, such as by coating, absorption or adsorption. For example, the film sealing the blister pack containing the capsule may contain the desiccant, such as by incorporation, absorption or adsorption. The package may be further sealed with a moisture barrier, such as a bottle or blister with a foil seal.

[0035] The desiccant may be added in an amount effective to reduce moisture in the package during storage, depending on the size of the container and the internal exposure conditions to humidity. For example, 1 g of silica gel is sufficient to protect a package containing four No. 00 capsules (HPMC capsules). It is preferred that the humidity or volatile content in the package is reduced to less than 5% by weight, preferably less than 3% by weight, for example less than 1% by weight. Preferably, the desiccant is added in an amount that maintains or increases the FPF value after storage at 40° C. and 75% relative humidity for 2 weeks, for example 4 weeks.

[0036] The term "size 00 FPF<5.6 μm(%)" is intended to mean the percentage of particles in the tested capsule that have an aerodynamic diameter of less than 5.6 microns. Preferably, "size 00 FPF<5.6 μm(%)" is at least about 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or 100%. Preferably, "size 00 FPF<5.6 μm(%)" after storage is at least about 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or 100%.

[0037] The term "size 00 FPF<3.4 μm(%)" is intended to mean the percentage of particles in the tested capsule that have an aerodynamic diameter of less than 3.4 microns. Preferably, "size 00 FPF<3.4 μm(%)" is at least about 35%, preferably at least 50%, more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or 100%. Preferably, "size 00 FPF<3.4 μm(%)" after storage is at least about 50%, preferably more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, or 100%.

[0038] The term "nucleic acid" in its broadest sense includes any compound and / or substance that comprises a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides described herein include, but are not limited to, ribonucleic acid (RNA), small interfering RNA (siRNA), microRNA, deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, e.g., LNA with a β-D-ribo configuration, α-LNA (a diastereomer of LNA) with an α-L-ribo configuration, 2'-amino LNA with a 2'-amino functionalization, and 2'-amino-α-LNA with a 2'-amino functionalization) or hybrids thereof.

[0039] As used herein, the term "polynucleotide" is generally used to refer to a nucleic acid (e.g., DNA or RNA). When RNA, such as mRNA, is specifically referred to, the term polyribonucleotide may be used. Terms such as polynucleotide, polyribonucleotide, nucleic acid, ribonucleic acid, DNA, RNA, mRNA, and the like include molecules that may be composed of standard or unmodified residues; non-standard or modified residues (e.g., analogs); and mixtures of standard and non-standard (e.g., analogs) residues. In certain embodiments, a polynucleotide or polyribonucleotide is a modified polynucleotide or polyribonucleotide. In the context of this disclosure, for each RNA (polyribonucleotide) sequence listed herein, the corresponding DNA (polydeoxyribonucleotide or polynucleotide) sequence is contemplated, and vice versa. "Polynucleotide" may be used interchangeably with "oligomer."

[0040] As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide that encodes a protein or polypeptide of interest and can be translated to produce the encoded protein or polypeptide of interest in vitro, in vivo, in situ, or ex vivo. "Protein" and "polypeptide" are used interchangeably herein and are intended to include proteins and fragments thereof.

[0041] As used herein, the term "translation" is the process by which ribosomes make polypeptides. In translation, messenger RNA (mRNA) is decoded by transfer RNA (tRNA) in the ribosome complex to generate a specific amino acid chain or polypeptide. The coding region of a polynucleotide sequence (DNA or RNA), also known as a coding sequence or CDS, can be converted into a protein or a fragment thereof by the process of translation.

[0042] As used herein, the term "codon-optimized" refers to a native coding sequence (or a purposefully designed variant of a native coding sequence) that has been redesigned by selecting different codons without changing the encoded protein amino acid sequence. Codon-optimized sequences can increase the protein expression level of the encoded protein, among other benefits (Gustafsson et al., Codon bias and heterologous protein expression. 2004, Trends Biotechnol 22:346-53). Variables such as high codon adaptation index (CAI), LowU method, mRNA secondary structure, cis-regulatory sequences, GC content, and many other similar variables have been shown to correlate somewhat with protein expression levels (Villalobos et al., Gene Designer: a synthetic biology tool for constructing artificial DNA segments. 2006, BMC Bioinformatics 7:285). High CAI (codon adaptation index) method selects the most frequently used synonymous codons for the entire protein coding sequence. The most frequently used codon for each amino acid is inferred from 74,218 protein-coding genes from the human genome. The Low U method targets only U-containing codons that can be replaced with synonymous codons that have fewer U moieties. When there are several options for replacement, the more frequently used codon is selected. The remaining codons in the sequence are not changed by the Low U method. This method can be used in conjunction with the disclosed mRNA to find, for example, 5-methoxyuridine or N 1 A coding sequence can be designed that is synthesized with -methylpseudouridine.

[0043] As used herein, "modified" refers to a change in the state or structure of a molecule disclosed herein. A molecule can be altered in many ways, for example, chemically, structurally, or functionally. Preferably, a polynucleotide or polypeptide of the present disclosure is modified compared to a naturally occurring polynucleotide or polypeptide, or compared to a reference polypeptide or polynucleotide sequence. For example, the mRNA disclosed herein can be modified by codon optimization or by the insertion of non-natural nucleosides or nucleotides. A polypeptide can be modified, for example, by site-specific amino acid deletion or substitution to change the properties of the polypeptide.

[0044] As used herein, the term "homology" refers to the overall relatedness between polymer molecules, such as between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. In some embodiments, polymer molecules are considered to be "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical or similar. The term "homologous" necessarily refers to a comparison between at least two sequences (polynucleotide or polypeptide sequences). According to the present disclosure, two polynucleotide sequences are considered to be homologous if the polypeptides they encode are at least about 50%, 60%, 70%, 80%, 90%, 95%, or even 99% identical over at least a stretch of at least about 20 amino acids. In some embodiments, homologous polynucleotide sequences are characterized by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. For polynucleotide sequences less than 60 nucleotides in length, homology is determined by the ability to encode a stretch of at least 4-5 uniquely specified amino acids. According to the present disclosure, two protein sequences are considered to be homologous if the proteins are at least about 50%, 60%, 70%, 80%, or 90% identical over at least a stretch of at least about 20 amino acids.

[0045] As used herein, the term "identity" refers to the overall relatedness between polymer molecules, e.g., between oligonucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or between polypeptide molecules. Calculation of the percent identity of two polynucleotide sequences can be performed, for example, by aligning the two sequences for optimal comparison purposes. In certain embodiments, the length of the sequences aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. If a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps that need to be introduced for optimal alignment of the two sequences and the length of each gap. Comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm. Commonly used methods for determining percent identity between sequences include, but are not limited to, those disclosed in Carillo, H. and Lipman, D., SIAM J Applied Math., 48:1073 (1988), which is incorporated herein by reference. Techniques for determining identity are codified in publicly available computer programs. Exemplary computer software for determining homology between two sequences include, but are not limited to, the GCG program package, Devereux, J., et al., Nucleic Acids Research, 12(1), 387 (1984), BLASTP, BLASTN, and FASTA Altschul, SF et al., J. Molec. Biol., 215, 403 (1990).

[0046] An "effective amount" of an mRNA sequence encoding an open reading frame (ORF) protein or its corresponding composition can be the amount of mRNA that results in ORF protein production in a cell. Preferably, protein production using the mRNA composition described herein is more efficient than a composition containing the corresponding wild-type mRNA encoding the ORF protein. The increased efficiency can be demonstrated by an increase in cell transfection (i.e., the percentage of cells transfected with the nucleic acid), an increase in protein translation from the nucleic acid, a decrease in nucleic acid degradation (e.g., as demonstrated by an increase in the duration of protein translation from the modified nucleic acid), or a decrease in the innate immune response of the host cell. When referring to the ORF protein described herein, an effective amount is the amount of ORF protein that overcomes ORF protein deficiency in a cell. When the mRNA is an mRNA vaccine, an effective amount includes an amount that induces immunity against infections such as influenza virus or respiratory syncytial virus, or cancer, etc.

[0047] As used herein, the term "in vitro" refers to events that take place not in a living organism (e.g., an animal, plant, or microorganism), but in an artificial environment, such as in a test tube or reaction vessel, in cell culture, in a Petri dish, etc.

[0048] As used herein, the term "in vivo" refers to events that take place within an organism (eg, an animal, its cells or tissues).

[0049] As used herein, the term "isolated" refers to a substance or entity that is separated from at least some of the components with which it is associated (whether in nature or in an experimental environment). Isolated substances can have various levels of purity relative to the substances with which they are associated. Isolated substances and / or entities can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of other components with which they were originally associated. In some embodiments, isolated agents are greater than about 80%, greater than about 85%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, or greater than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. Substantially isolated: "Substantially isolated" means that the compound is substantially separated from the environment in which it was formed or detected. Partial isolation can include, for example, compositions enriched for the compounds described herein. Substantial isolation can include compositions that contain at least about 50% by weight, at least about 60% by weight, at least about 70% by weight, at least about 80% by weight, at least about 90% by weight, at least about 95% by weight, at least about 97% by weight, or at least about 99% by weight of the compounds described herein or salts thereof. Methods for isolating compounds and salts thereof are routine in the art.

[0050] As used herein, the term "prevent" refers to partially or completely delaying the onset of an infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or clinical manifestations of a particular infection, disease, disorder, and / or condition; partially or completely delaying the onset of one or more symptoms, characteristics, or symptoms of a particular infection, disease, disorder, and / or condition; partially or completely delaying the progression from an infection, a particular disease, disorder, and / or condition; and / or reducing the risk of developing a pathology associated with an infection, disease, disorder, and / or condition.

[0051] As used herein, the term "substantially" refers to a qualitative condition indicating the entire or nearly entire extent or degree of a characteristic or property of interest. Those skilled in the biological arts will understand that biological and chemical phenomena rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0052] As used herein, the term "therapeutically effective amount" means an amount of an agent (e.g., a nucleic acid, protein or peptide, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) delivered that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and / or condition, to treat, ameliorate the symptoms of, diagnose, prevent, and / or delay the onset of the infection, disease, disorder, and / or condition.

[0053] As used herein, a "total daily dose" is the amount given or prescribed in a 24 hour period. It may be administered as a single unit dose.

[0054] As used herein, the term "treat" refers to partial or complete relief, remission, improvement, mitigation, delay in onset, inhibition of progression, reduction in severity, and / or reduction in incidence of one or more symptoms or characteristics of a protein deficiency. Treatment may be administered to subjects who do not exhibit symptoms of said protein deficiency and / or who exhibit only early signs of protein deficiency, for the purpose of reducing the risk of developing pathologies associated with a disease, disorder, and / or condition.

[0055] As used herein, the term "transfect" or "transfection" refers to the intracellular introduction of a nucleic acid into a cell, or preferably a target cell. The introduced nucleic acid may be stably or transiently maintained in the target cell. The term "transfection efficiency" refers to the relative amount of nucleic acid taken up by the target cell subject to transfection. In practice, transfection efficiency is estimated by the amount of reporter nucleic acid product expressed by the target cell after transfection. Compositions with high transfection efficiency are preferred, especially those that minimize adverse effects caused by transfection of non-target cells and tissues.

[0056] As used herein, the term "target cell" refers to a cell or tissue to which the disclosed compositions are directed or targeted. In some embodiments, the target cell lacks a protein or enzyme of interest. For example, if it is desired to deliver a nucleic acid to an epithelial cell or a lung cell, the epithelial cell or the lung cell refers to the target cell. In some embodiments, the disclosed nucleic acids and compositions transfect target cells on a discriminatory basis (i.e., do not transfect non-target cells).

[0057] Following transfection of one or more target cells with the compositions and nucleic acids described herein, expression of proteins encoded by such nucleic acids is preferably stimulated to enhance the ability of such target cells to express the protein of interest. For example, transfection of a target cell with mRNA will allow expression of a modified protein product following translation of the nucleic acid. The nucleic acids of the compositions and / or methods provided herein preferably encode products (e.g., proteins, enzymes, polypeptides, peptides, functional RNA, and / or antisense molecules), preferably encoding products whose in vivo production is desired.

[0058] Respirable polynucleotide dry powder particle formulations The present invention provides a respirable dry powder particle formulation comprising a polynucleotide prepared by spray drying for delivery to the pulmonary system by inhalation. Preferably, the polynucleotide is RNA. Preferably, the present invention provides a respirable RNA dry powder particle formulation prepared by spray drying for delivery to the pulmonary system by inhalation. The present invention provides a respirable RNA dry powder particle formulation prepared by spray drying for delivery to the pulmonary system by inhalation. The present invention includes formulations and parameters for spray drying RNA-containing powders, such as mRNA-containing powders, with suitable chemical, physical and aerosol properties for delivery using a dry powder inhaler system, preferably a patient-actuated dry powder inhaler system, such as the ARCUS® platform described at www.civitis.com. In the examples, yeast RNA was used as a placeholder for mRNA.

[0059] Preferably, the respirable RNA dry powder particle formulation for pulmonary delivery comprises: i) at least about 1% RNA by weight of the particle; ii) at least about 10% DPPC by weight of the particle; iii) optionally, one or more of arginine, leucine, isoleucine, or valine, each at least about 1% by weight of the particle, where if more than one amino acid is selected, each amino acid has either the same or a different weight percentage as the other selected amino acids; iv) optionally, at least about 1% NaCl by weight of the particles; v) optionally, at least about 10% Tris by weight of the particles; vi) optionally, at least about 2% EDTA by weight of the particles; vii) optionally, at least about 40% trehalose by weight of the particle; viii) optionally, at least about 2% PEI by weight of the particles; and ix) optionally comprising at least about 40% lactose by weight of the particles; All components of the RNA dry powder add up to 100 weight percent.

[0060] Preferably, the formulation comprises about 1% to about 80% by weight, preferably about 1% to 70% by weight, for example about 1%, 10%, 25%, 50% or about 60% by weight, of RNA.

[0061] Preferably, the RNA can be at least about 10, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 125, 150, 200 or more nucleic acids in length. Preferably, the RNA can be about 10 to about 12,000 or more nucleic acids in length. Preferably, the RNA can be about 20 to about 12,000 or more nucleic acids in length. Preferably, the RNA can be about 50 to about 12,000 or more nucleic acids in length. Preferably, the RNA can be about 100 to about 12,000 or more nucleic acids in length. Preferably, the RNA may have a length of about 20 to about 12,000 monomers, or about 20 to about 10,000 monomers, or about 20 to 8000 monomers, or about 20 to 6000 monomers, or about 20 to about 5000 monomers, or about 20 to about 4000 monomers, or about 20 to about 3600 monomers, or about 20 to about 3200 monomers, or about 20 to about 3000 monomers, or about 20 to about 2800 monomers, or about 20 to about 2600 monomers, or about 20 to 2400 monomers, or about 20 to 2200 monomers, or about 50 to 3200 monomers, or about 50 to 3000 monomers, or about 50 to 2600 monomers.

[0062] Preferably, the formulation contains about 10% to about 30% by weight, preferably about 15% to 25% by weight, for example about 15%, 18%, 20%, 22% or about 25% by weight, of DPPC.

[0063] Preferably, the formulation optionally comprises one or more of arginine, leucine, isoleucine, or valine, optionally in an amount of about 1% to about 90% by weight, preferably about 5% to 85% by weight, e.g., about 5%, 20%, 30%, 40%, 50%, 55%, 70%, 74%, 79%, 80%, or about 85% by weight, and where more than one amino acid is selected, each amino acid has the same or a different weight percentage as the other selected amino acids.

[0064] Preferably, the formulation optionally comprises about 1% to about 5% by weight NaCl, preferably about 1% to 3% by weight, for example about 1%, 2% or about 3% by weight.

[0065] Preferably, the formulation optionally comprises about 1% to about 25% by weight, preferably about 5% to 20% by weight, for example about 5%, 10%, 15% or about 20% by weight, of Tris.

[0066] Preferably, the formulation optionally contains about 1% to about 10% by weight, preferably about 2% to 8% by weight, for example about 2%, 3%, 4%, 5%, 6% or about 7% by weight, of EDTA.

[0067] Preferably, the formulation may further comprise about 40% to about 90% by weight, preferably about 45% to 85% by weight, for example about 45% by weight, 50% by weight, 52% by weight, 53% by weight, 54% by weight, 55% by weight, 60% by weight, 65% by weight, 70% by weight, 72% by weight, 74% by weight, 75% by weight, 77% by weight, 79% by weight, 80% by weight or about 82% by weight of trehalose.

[0068] Preferably, the formulation optionally further comprises about 40% to about 90% by weight, preferably about 45% to 85% by weight, for example about 45%, 50%, 52%, 53%, 54%, 55%, 60%, 65%, 70%, 72%, 74%, 75%, 77%, 79%, 80% or about 82% by weight of lactose.

[0069] Preferably, the formulation optionally comprises about 1% to about 10% by weight, preferably about 1% to 8% by weight, e.g., about 1%, 2%, 3%, 4%, 5%, 6% or about 7% by weight, of PEI.

[0070] Preferably, the formulation is selected from the formulations listed in Table 1.

[0071] The weight percentage is intended to reflect the total amount of solids, lipids, and / or excipients in the dry particle, regardless of residual water, solvents, or impurities. Preferably, all components of the dry particle add up to 100% by weight.

[0072] Respirable dry particles of the invention preferably have an MMAD of about 10 microns or less, for example, an MMAD of about 0.5 microns to about 10 microns. Preferably, dry particles of the invention have an MMAD of about 7 microns or less (e.g., about 0.5 microns to about 7 microns), preferably about 1 micron to about 7 microns, or about 2 microns to about 7 microns, or about 3 microns to about 7 microns, or about 4 microns to about 7 microns, or about 5 microns to about 7 microns, or about 1 micron to about 6 microns, or about 1 micron to about 5 microns, or about 2 microns to about 5 microns, or about 2 microns to about 4 microns, or about 3 microns.

[0073] Respirable dry particles of the present invention preferably have a gPSD of about 10 microns or less. 50 , for example, a gPSD of about 1 micron to about 7 microns 50 Preferably, the dry particles of the present invention have a gPSD of about 6 microns or less, for example, from about 1 micron to about 6 microns, preferably from about 1 micron to about 5 microns, or from about 1 micron to about 4 microns, or from about 1 micron to about 3 microns, or from about 1 micron to about 2 microns, or from about 2 microns to about 5 microns, or from about 2 microns to about 4 microns, or about 3 microns. 50 has.

[0074] The fine particle fraction of a powder less than 5.6 microns, or the FPF of a powder<5.6 μm, corresponds to the percentage of particles in the powder that have an aerodynamic diameter of less than 5.6 μm. The FPF<5.6 μm of the powder of the present invention is preferably about 40% or more. In certain embodiments, the FPF<5.6 μm of the powder is at least about 45%, 50%, 55%, 60%, 65%, 70%, 75%, or more. In one embodiment, the FPF<5.6 μm is about 40% to about 80%. In one embodiment, the FPF<5.6 μm is 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%.

[0075] The fine particle fraction of a powder less than 3.4 microns, or the FPF of a powder<3.4 μm, corresponds to the percentage of particles in the powder that have an aerodynamic diameter of less than 3.4 μm. In one embodiment, the FPF of a powder of the present invention<3.4 μm is about 30% or more. In one embodiment, the FPF of a powder<3.4 μm is at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or more. In one embodiment, the FPF<3.4 μm is about 40% to about 80%. In one embodiment, the FPF<3.4 μm is 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, or 80%.

[0076] Preferably, the powder of the present invention has a density of about 0.4 g / cm 3 For example, the powder has a tap density of 0.02 to 0.20 g / cm 3 , 0.02~0.15g / cm 3 Between 0.03 and 0.12 g / cm3 Between 0.05 and 0.15 g / cm 3 , or about 0.15 g / cm 3 A tap density of less than about 0.10 g / cm 3 Tap density of less than about 0.15 g / cm 3 In one embodiment, the powder of the present invention has a tap density of less than about 0.2 g / cm 3 Preferably, the tap density is about 0.02 to 0.175 g / cm. 3 Preferably, the tap density is about 0.06 to 0.175 g / cm 3 It is.

[0077] Tap density can be measured using equipment known to those skilled in the art, such as a dual platform microprocessor controlled tap density tester (Vankel, NC) or a GEOPYC™ instrument (Micrometrics Instrument Corp., Norcross, GA, 30093). Tap density is a standard measure of envelope mass density. Tap density can be determined using the USP bulk and tap density method, United States Pharmacopeia Regulations, Rockville, Md., 10th Supplement, 4950-4951, 1999. Features that can cause low tap density include irregular surface texture and porous structure. The envelope mass density of an isotropic particle is defined as the mass of the particle divided by the minimum spherical envelope volume within which the particle can be enclosed. In one embodiment of the present invention, the particle has a mass density of about 0.4 g / cm 3 has an envelope mass density of less than

[0078] Preferably, the respirable dry powder and dry particle formulations of the invention contain less than about 15% by weight, less than about 13% by weight, less than about 11.5% by weight, less than about 10% by weight, less than about 9% by weight, less than about 8% by weight, less than about 7% by weight, less than about 6% by weight, less than about 5% by weight, less than about 4% by weight, less than about 3% by weight, less than about 2% by weight, less than about 1% by weight, or are anhydrous.

[0079] Preferably, the dry particle formulations of the present invention can contain less than about 6% and more than about 1%, less than about 5.5% and more than about 1.5%, less than about 5% and more than about 2%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5% water or solvent.

[0080] Treatment indications Respirable polynucleotide formulations (e.g., mRNA formulations) described herein can be used for treatment. For example, respirable polynucleotide formulations described herein can be administered to animals or human patients via pulmonary administration, and the polynucleotides are translated in vivo to produce therapeutic peptides in the animals or subjects. Thus, respirable mRNA formulations of the present invention can be used for treatment or prevention of diseases or conditions in humans and other mammals.

[0081] The respirable mRNA formulations of the invention can be used to induce translation of synthetic or recombinant polynucleotides to produce polypeptides in a cell population (e.g., lung epithelial cells). Effective amounts of the formulations of the invention will depend, at least in part, on the target tissue, the target cell type, the means of administration, the physical characteristics of the nucleic acid (e.g., size and degree of nucleoside substitution), and other determinants.

[0082] Thus, the present invention provides a respirable formulation comprising a polynucleotide (e.g., mRNA) for use in a method of inducing in vivo translation of a recombinant polypeptide in a mammalian subject in need thereof, wherein an effective amount of RNA in the respirable formulation has at least one translatable region encoding a desired polypeptide and is administered to the subject using the delivery methods described herein. The mRNA is provided in the formulation in an amount and under other conditions such that the nucleic acid is localized to one or more cells of the subject and the recombinant polypeptide is translated intracellularly from the nucleic acid. The cell in which the nucleic acid is localized or the tissue in which the cell resides can be targeted with one or more administrations of the nucleic acid.

[0083] The subject to which the therapeutic agent is administered is either suffering from or at risk of developing a disease, disorder, or adverse condition. Methods are provided for identifying, diagnosing, and classifying subjects based on evidence that may include clinical diagnosis, biomarker levels, genome-wide association studies (GWAS), and other methods known in the art.

[0084] Preferably, the administered respirable polynucleotide formulation (e.g., an mRNA formulation) results in the production of one or more recombinant polypeptides that provide a functional activity that is substantially absent in the cell in which the recombinant polypeptide is translated. For example, the missing functional activity can be enzymatic, structural, or gene regulatory in nature.

[0085] Preferably, the administered replacement nucleic acid directs the production of one or more recombinant polypeptides that replace a polypeptide (or polypeptides) that is substantially absent from the cell in which the recombinant polypeptide is translated, such absence being likely due to genetic mutation of the encoding gene or its regulatory pathway.

[0086] Preferably, the administered formulation directs the production of antibodies, e.g., neutralizing antibodies, useful for modulating, directly or indirectly, the activity of a biological moiety present on the surface of a cell or secreted from the cell.

[0087] The formulations of the present invention also provide improved methods and compositions for treating diseases associated with protein deficiency, for example, using mRNA therapy to express modified proteins. The present invention provides a method of treating protein deficiency, comprising administering to a subject in need of treatment, a formulation of the present invention comprising, for example, an mRNA sequence as described herein encoding a stabilized modified human protein or an active fragment of such a stabilized modified human protein, at an effective dose and dosing interval such that the intensity, severity, or frequency of at least one symptom or characteristic of the protein deficiency is reduced or the onset is delayed. The formulations of the present invention also provide modified proteins encoded by the mRNA sequences that have improved properties compared to wild-type human proteins, such as improved stability and resistance to proteolysis, and increased half-life.

[0088] Preferably, pulmonary administration of the mRNA compositions described herein increases therapeutic protein expression or activity in a subject compared to a control level, preferably the control level is a baseline serum therapeutic protein expression or activity level in the subject prior to treatment and / or the control level represents the average serum protein expression or activity level in untreated patients.

[0089] Preferably, the proteins encoded by the mRNA formulations described herein are generated from heterologous mRNA constructs that include an open reading frame (ORF), also referred to herein as a "coding sequence" (CDS), that encodes a therapeutic protein. Preferably, the coding sequence is codon-optimized. Preferably, the coding sequence is optimized to have a theoretical minimum of uridines that can encode a therapeutic protein. Preferably, the mRNA constructs described herein include one or more of the following features: 5' cap, 5' UTR, 5' UTR enhancer sequence, Kozak sequence or partial Kozak sequence, 3' UTR, an open reading frame encoding a therapeutic protein, and a polyA tail. Preferably, the mRNA constructs described herein can provide highly efficient expression of modified proteins. Expression can be in vitro, ex vivo, or in vivo.

[0090] Preferably, the mRNA described herein comprises a Kozak sequence and / or a 3'UTR. As understood in the art, a Kozak sequence is a short consensus sequence centered on the translation start site of eukaryotic mRNA that allows efficient initiation of translation of the mRNA. The ribosomal translation machinery recognizes the AUG start codon in the context of the Kozak sequence. The Kozak sequence may be inserted upstream of the coding sequence of the therapeutic protein of interest and downstream of the 5'UTR, or may be inserted upstream of the coding sequence of the therapeutic protein of interest and downstream of the 5'UTR. Preferably, the mRNA described herein comprises a 3' tail region that may help protect the mRNA from exonuclease degradation. The tail region may be a 3' poly(A) and / or a 3' poly(C) region. Preferably, the tail region is a 3' poly(A) tail. As used herein, a "3' poly(A) tail" is a polymer of consecutive adenine nucleotides that may vary in size, for example, from 10 or more adenosines.

[0091] The 3' poly(A) tail can be added using a variety of methods known in the art, such as by adding the tail to synthetic or in vitro transcribed RNA using poly(A) polymerase. Other methods include the use of a transcription vector encoding a polyA tail or the use of a ligase (e.g., via splint ligation using T4 RNA ligase and / or T4 DNA ligase), where poly(A) can be ligated to the 3' end of the sense RNA. Preferably, a combination of any of the above methods is utilized.

[0092] Preferably, the mRNA formulations described herein include a 5' cap. 5' ends capped with various groups and their analogs are known in the art. The 5' cap can be selected from m7GpppA, m7GpppC; unmethylated cap analogs (e.g., GpppG); dimethylated cap analogs (e.g., m2,7GpppG), trimethylated cap analogs (e.g., m2,2,7GpppG), dimethylated symmetric cap analogs (e.g., m7Gpppm7G), or anti-reverse cap analogs (e.g., ARCA; m7,2'OmeGpppG, m72'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives) (see, e.g., Jemielity, J. et al., RNA 9:1108-1122 (2003)). The 5' cap can be an ARCA cap (3'-OMe-m7G(5')pppG). The 5' cap can be mCAP (m7G(5')ppp 5')G,N 7 -methyl-guanosine-5'-triphosphate-5'-guanosine). The 5' cap can be resistant to hydrolysis.

[0093] Preferably, the mRNA formulation described herein comprises one or more chemically modified nucleotides.Examples of nucleic acid monomers include non-natural, modified and chemically modified nucleotides, such as any such nucleotides known in the art.It has been shown that mRNA sequences that comprise chemically modified nucleotides improve mRNA expression, expression rate, half-life and / or expressed protein concentration.The mRNA sequences that comprise chemically modified nucleotides are also useful for optimizing protein localization, thereby avoiding adverse biological responses, such as immune responses and / or degradation pathways.

[0094] Examples of modified or chemically modified nucleotides include 5-hydroxycytidine, 5-alkylcytidine, 5-hydroxyalkylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-alkoxycytidine, 5-alkynylcytidine, 5-halocytidine, 2-thiocytidine, N 4 -Alkylcytidine, N 4 -Aminocytidine, N 4 -Acetylcytidine and N 4 ,N 4 -Dialkylcytidines.

[0095] Examples of modified or chemically modified nucleotides include 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 5-bromocytidine, 5-iodocytidine, 2-thiocytidine, N 4 -Methylcytidine, N 4 -Aminocytidine, N 4 -Acetylcytidine, and N 4 ,N 4 -Dimethylcytidine.

[0096] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-alkyluridine, 5-hydroxyalkyluridine, 5-carboxyuridine, 5-carboxyalkylester uridine, 5-formyluridine, 5-alkoxyuridine, 5-alkynyluridine, 5-halouridine, 2-thiouridine and 6-alkyluridine.

[0097] Examples of modified or chemically modified nucleotides include 5-hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5-methoxyuridine (also referred to herein as "5MeOU"), 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine, 2-thiouridine and 6-methyluridine.

[0098] Examples of modified or chemically modified nucleotides include 5-methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-thiouridine, 5-carbamoylmethyluridine, 5-carbamoylmethyl-2'-O-methyluridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethyluridine, 5-methyldihydrouridine, 5-taurinomethyluridine, 5-taurinomethyl-2-thiouridine, 5-(isopentenylaminomethyl)uridine, 2'-O-methylpseudouridine, 2-thio-2'O-methyluridine and 3,2'-O-dimethyluridine.

[0099] Examples of modified or chemically modified nucleotides include N 6 -Methyladenosine, 2-aminoadenosine, 3-methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8-oxoadenosine, 8-bromoadenosine, 2-methylthio-N 6 -Methyladenosine, N 6 -Isopentenyl adenosine, 2-methylthio-N 6 -Isopentenyl adenosine, N6 -(cis-Hydroxyisopentenyl)adenosine, 2-methylthio-N 6 -(cis-hydroxyisopentenyl)adenosine, N 6 -Glycinylcarbamoyl adenosine, N 6 -Threonylcarbamoyl-adenosine, N 6 -Methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N 6 -Threonylcarbamoyl-adenosine, N 6 ,N 6 -Dimethyladenosine, N6-hydroxynorvalylcarbamoyl adenosine, 2-methylthio-N 6 -Hydroxynorvalylcarbamoyl-adenosine, N 6 -Acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenosine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine, N 6 ,2'-O-Dimethyl-adenosine,N 6 ,N 6 ,2'-O-trimethyl-adenosine, 1,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine, 2-amino-N 6 -methyl-purine, 1-thio-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N 6 -(19-amino-pentaoxanonadecyl)-adenosine.

[0100] Examples of modified or chemically modified nucleotides include N 1 -Alkylguanosine, N 2 -Alkylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8-bromoguanosine, O 6 -Alkylguanosine, xanthosine, inosine and N 1 -Alkyl inosines.

[0101] Examples of modified or chemically modified nucleotides include N 1 -Methylguanosine, N 2-Methylguanosine, Thienoguanosine, 7-Deazaguanosine, 8-Oxoguanosine, 8-Bromoguanosine, O 6 -Methylguanosine, xanthosine, inosine and N 1 -methylinosine.

[0102] An example of a modified or chemically modified nucleotide is pseudouridine. 1 -Alkylpseudouridine, N 1 -Cycloalkylpseudouridines, N 1 -Hydroxypseudouridine, N 1 -Hydroxyalkylpseudouridine, N 1 -Phenylpseudouridine, N 1 -Phenylalkylpseudouridine, N 1 -Aminoalkylpseudouridines, N 3 -Alkylpseudouridine, N 6 -Alkylpseudouridine, N 6 -Alkoxypseudouridine, N 6 -Hydroxypseudouridine, N 6 -Hydroxyalkylpseudouridine, N 6 -Morpholinopseudouridine, N 6 -Phenylpseudouridine and N 6 Examples of pseudouridines include N-halopseudouridine. 1 -Alkyl-N 6 -Alkylpseudouridine, N 1 -Alkyl-N 6 -Alkoxypseudouridine, N 1 -Alkyl-N 6 -Hydroxypseudouridine, N 1 -Alkyl-N 6 -Hydroxyalkylpseudouridine, N 1 -Alkyl-N 6 -Morpholinopseudouridine, N 1 -Alkyl-N 6 -Phenylpseudouridine and N 1 -Alkyl-N 6-halopseudouridine. In these examples, the alkyl, cycloalkyl and phenyl substituents may be unsubstituted or further substituted with alkyl, halo, haloalkyl, amino or nitro substituents.

[0103] Examples of pseudouridines include N 1 -methylpseudouridine (also referred to herein as "N1MPU"), N 1 -Ethylpseudouridine, N 1 -Propylpseudouridine, N 1 -Cyclopropylpseudouridine, N 1 -Phenylpseudouridine, N 1 -Aminomethylpseudouridine, N 3 -Methylpseudouridine, N 1 -Hydroxypseudouridine and N 1 -Hydroxymethylpseudouridine is included.

[0104] Examples of nucleic acid monomers include modified and chemically modified nucleotides, including any such nucleotides known in the art.

[0105] Examples of modified and chemically modified nucleotide monomers include any such nucleotides known in the art, such as 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'-deoxyribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl nucleotides, and inverted deoxy abasic monomer residues.

[0106] Examples of modified and chemically modified nucleotide monomers include 3'-terminal stabilized nucleotides, 3'-glyceryl nucleotides, 3'-inverted abasic nucleotides and 3'-inverted thymidine.

[0107] Examples of modified and chemically modified nucleotide monomers include locked nucleic acid nucleotides (LNA), 2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'-methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides and 2'-O-methyl nucleotides. In an exemplary embodiment, the modified monomer is a locked nucleic acid nucleotide (LNA).

[0108] Examples of modified and chemically modified nucleotide monomers include 2',4'-constrained 2'-O-methoxyethyl (cMOE) and 2'-O-ethyl (cEt) modified DNA.

[0109] Examples of modified and chemically modified nucleotide monomers include 2'-amino nucleotides, 2'-O-amino nucleotides, 2'-C-allyl nucleotides and 2'-O-allyl nucleotides.

[0110] Examples of modified and chemically modified nucleotide monomers include N 6 -methyl adenosine nucleotides.

[0111] Examples of modified and chemically modified nucleotide monomers include nucleotide monomers having the modified bases 5-(3-amino)propyluridine, 5-(2-mercapto)ethyluridine, 5-bromouridine; 8-bromoguanosine, or 7-deazaadenosine.

[0112] Examples of modified and chemically modified nucleotide monomers include 2'-O-aminopropyl substituted nucleotides.

[0113] Examples of modified and chemically modified nucleotide monomers include replacing the 2'-OH group of the nucleotide with 2'-R, 2'-OR, 2'-halogen, 2'-SR or 2'-amino, where R can be H, alkyl, alkenyl, or alkynyl.

[0114] The above examples of base modifications can be combined with further modifications of the nucleoside or nucleotide structure, including sugar modifications and linkage modifications. Certain modified or chemically modified nucleotide monomers can be found in nature.

[0115] Preferred nucleotide modifications include N 1 -methylpseudouridine and 5-methoxyuridine.

[0116] The formulation of the present invention may also include a polynucleotide (e.g., DNA, RNA, cDNA, mRNA) encoding a modified human protein of interest, which may be operably linked to one or more regulatory nucleotide sequences in an expression construct, such as a vector or plasmid. In certain embodiments, such a construct is a DNA construct. The regulatory nucleotide sequence is generally suitable for the host cell used for expression. A large number of types of suitable expression vectors and suitable regulatory sequences for various host cells are known in the art.

[0117] Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, a promoter sequence, a leader or signal sequence, a ribosome binding site, a transcriptional start and end sequence, a translational start and end sequence, and an enhancer or activator sequence. Constitutive or inducible promoters known in the art are contemplated by embodiments of the present disclosure. The promoter may be either a naturally occurring promoter or a hybrid promoter that combines elements of two or more promoters.

[0118] Preferably, the polynucleotides described herein encoding the modified therapeutic proteins described herein can generate an immune response or can be used in gene therapy. Gene therapy is a technique that uses genes to treat or prevent disease and can be used to treat disorders by inserting genes into the cells of a subject. In some embodiments, the polynucleotides encoding the modified therapeutic proteins described herein replace mutated genes that cause disease. In other embodiments, the polynucleotides encoding the modified therapeutic proteins described herein are used to inactivate or "knock out" mutated genes that are functioning improperly. In yet other embodiments, the polynucleotides encoding the modified therapeutic proteins described herein introduce new genes into a subject to help fight disease.

[0119] An effective amount of an mRNA formulation may be an amount sufficient to treat a protein deficiency in a cell and / or a patient. A therapeutically effective dose may be an amount of an agent or formulation sufficient to cause a therapeutic effect. A therapeutically effective dose may be administered in one or more separate administrations and by different routes. Generally, a therapeutically effective amount is sufficient to achieve a significant benefit in a subject (e.g., treating, regulating, curing, preventing and / or ameliorating phenylketonuria). For example, a therapeutically effective amount may be an amount sufficient to achieve a desired therapeutic and / or prophylactic effect. Generally, the amount of a therapeutic agent administered to a subject in need of administration of the therapeutic agent will depend on the characteristics of the subject. Such characteristics include the subject's condition, disease severity, general health, age, sex and weight. Those skilled in the art will be able to easily determine the appropriate dosage depending on these and other relevant factors. Furthermore, both objective and subjective assays may be used, in some cases, to identify optimal dosage ranges.

[0120] The methods provided herein contemplate single and multiple administration of therapeutically effective amounts of respirable mRNA formulations described herein. Respirable formulations comprising mRNA sequences encoding ORF proteins described herein can be administered at regular intervals depending on the nature, severity and extent of the subject's condition. Preferably, therapeutically effective amounts of respirable mRNA formulations can be administered at regular intervals (e.g., yearly, 6 monthly, 4 monthly, 3 monthly, 2 monthly, monthly), biweekly, weekly, daily, twice a day, three times a day, four times a day, five times a day, six times a day, or continuously.

[0121] The respirable polynucleotide formulations of the invention can also be used for intracellular expression of, for example, antibodies, where the RNA (sequence) encodes an antibody or comprises at least one coding region that encodes at least one antibody, respectively. Antibody-encoding RNA according to the invention includes any RNA that encodes an antibody. More generally, the RNA (for intracellular expression) of the invention comprises at least one coding region, where the at least one coding region encodes at least one antibody. When the RNA molecule of the invention comprises more than one coding region, the second, third, etc. coding region may also encode an antibody that may be the same or different from the first antibody coding region. Preferably, the RNA comprising the formulation of the invention comprises at least two coding regions, all of which encode the same or different antibodies. Preferably, the RNA of the formulation may encode more than one antibody within the same coding region.

[0122] The antibody-encoding RNA of the formulation of the invention may be single- or double-stranded, linear or circular, or most preferably in the form of mRNA. The antibody-encoding RNA is preferably in the form of single-stranded RNA, and even more preferably in the form of mRNA.

[0123] The antibody-encoding RNA according to the present invention is preferably 50 to 15,000 nucleotides in length, more preferably 50 to 10,000 nucleotides in length, even more preferably 500 to 10,000 nucleotides in length, and most preferably 500 to 7,000, 500 to 5,000, or 700 to 3,000 nucleotides in length.

[0124] The antibodies encoded by the respirable RNA preparation according to the invention can be selected from all antibodies, e.g. produced by recombinant methods or naturally occurring and known to the skilled artisan from the prior art, in particular from antibodies that are (can be) used for therapeutic or diagnostic or research purposes or found in specific diseases, e.g. cancer diseases, infectious diseases.

[0125] The antibodies encoded by the RNA preparations of the invention typically include all antibodies known to those skilled in the art (mentioned above), such as naturally occurring antibodies or antibodies generated in a host organism by immunization, antibodies isolated and identified from naturally occurring antibodies or antibodies generated in a host organism by (conventional) immunization, or antibodies prepared by recombinant methods, generated with the aid of molecular biology methods, as well as chimeric antibodies, human antibodies, humanized antibodies, bispecific antibodies, intrabodies, i.e. antibodies expressed intracellularly and possibly localized in a specific cellular compartment, and fragments of the above antibodies. In this respect, the term antibody should be understood in its broadest sense. In this context, an antibody generally comprises a light chain and a heavy chain, both of which have a variable domain and a constant domain. The light chain comprises an N-terminal variable domain V L and the C-terminal constant domain C L In contrast, the heavy chain of an IgG antibody contains an N-terminal variable domain V H and three constant domains C H 1. C H 2 and C H It can be divided into three.

[0126] The antibodies encoded by the RNA according to the invention particularly preferably comprise so-called full-length antibodies, i.e. antibodies comprising both the complete heavy chain and the complete light chain as described above. RNAs alternatively encoding one or more antibody fragments of the above-mentioned antibodies instead of the corresponding full-length antibodies can furthermore be provided in the context of the present invention. Examples of such antibody fragments are any antibody fragments known to the skilled artisan, such as the Fab, Fab', F(ab')2, Fc, Facb, pFc', Fd and Fv fragments of the above-mentioned antibodies.

[0127] Respirable formulations that include, but are not limited to, antibody-encoding RNAs encode, inter alia, antibodies that bind to antigens or specific nucleic acids. Antigens are typically molecules that are recognized by the immune system as foreign and traditionally provoke an immune reaction or immune response with the formation of antibodies specifically directed against them. However, antigens can also include endogenous molecules or structures that are incorrectly recognized by the immune system as foreign, thereby triggering an immune response, particularly in the case of autoimmune diseases.

[0128] Antigens typically include these proteins, peptides, or epitopes of these proteins or peptides. In this context, epitopes (also called "antigenic determinants") are small regions (molecular segments) typically having a length of 5-15, rarely 25, preferably 6-9 amino acids, on the surface of such protein or peptide structures. Antigens may further include lipids, carbohydrates, etc. In the context of the present invention, antigens also include, for example, so-called immunogens, i.e. antigens that provide immunity to an organism transfected with them. Examples of antigens include, but are not limited to, cell surface antigens, tumor antigens, etc. For example, according to the present invention, antibodies may be directed to the following antigens (typically present in vertebrates), such as tumor specific surface antigens (TSSAs), e.g., 5T4, α5β1-integrin, 707-AP, AFP, ART-4, B7H4, BAGE, β-catenin / m, Bcr-abl, MN / C IX-antigen, CA125, CAMEL, CAP-1, CASP-8, CD4, CD19, CD20, CD22, CD25, CDC27 / m, CD 30, CD33, CD52, CD56, CD80, CDK4 / m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN, EpCam, E TV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2 / neu, HLA-A*0201-R170I, HPV-E7, HSP70-2M, It can bind to sequences such as HAST-2, hTERT (or hTRT), iCE, IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR / FUT, MAGE, MART-1 / Melan-A, MART-2 / Ski, MC1R, myosin / m, MUC1, MUM-1, -2, -3, NA88-A, NY-ESO1, PAP, proteinase-3, p190 minor bcr-abl, Pml / RARα, PRAME, PSA, PSM, PSMA, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, survivin, TEL / AML1, TGFβ, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, VEGF and WT1, or for example NY-Eso-1 or NY-Eso-B.Other antigens target immune checkpoint inhibitor proteins, including CTLA4 and PD-1 or PD1 ligand.

[0129] Tumor antigens, for example, can typically be responsible for metastasis. In this context, such tumor antigens are of particular interest, which cause modified cell-cell interactions compared to the native state.

[0130] As discussed above with respect to protein replacement therapy, the antibodies encoding the RNA respirable formulations of the present invention may contain modified RNA for any purpose, such as increasing expression, improving stabilization of secondary structure if necessary, stabilizing the RNA against degradation, and reducing the immunogenicity of the respirable RNA.

[0131] Preferably, diseases of the respiratory system that can be treated using the formulations of the present invention include, but are not limited to, asthma, pulmonary arterial hypertension, chronic obstructive pulmonary disease (COPD), respiratory distress syndrome (RDS), chronic bronchitis, acute bronchitis, emphysema, cystic fibrosis, pneumonia, tuberculosis, lung cancer, acute respiratory distress syndrome (ARDS), influenza, respiratory syncytial virus, pneumoconiosis, interstitial lung disease (ILD) (e.g., sarcoidosis, idiopathic pulmonary fibrosis and autoimmune diseases), pulmonary embolism, pleural effusion and mesothelioma.

[0132] example The present invention will be better understood in conjunction with the following examples. However, it should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Various changes and modifications will be apparent to those skilled in the art, and such changes and modifications, including but not limited to those related to the formulations and / or methods of the present invention, can be made without departing from the spirit of the present invention and the scope of the appended claims.

[0133] Early development Studies R1 (batch number 449043), R2 (batch number 483016), and R3 (batch number 483022) were conducted as part of the initial development work to produce an RNA delivery powder. These three studies aimed to investigate whether it was possible to spray dry a powder containing yeast RNA (as a placeholder for mRNA) with suitable physical and aerosol properties for delivery. By varying the formulation and parameters used to spray dry the RNA powder, it was found that using product filter bags conditioned to a pre-dried 15% RH environment could produce a suitable powder.

[0134] In study R2, excipients were changed from R1 to evaluate whether excipient combinations would cause powder particles from R1 to adhere to the product filter bag. Trehalose, a sugar, was used in place of the previous amino acid. Additionally, EDTA and Tris buffer replaced NaCl as it was believed to aid in more rapid dissolution of the RNA into the aqueous phase. RNA loading was kept constant at 10%. During spray drying of these components, vibration of the product filter bag caused partial release of powder. After approximately 100 minutes of spray drying, enough powder had adhered to the bag to cause the system pressure to become positive; therefore, the run was terminated early. Collected powder had 61% FPF<5.6μm and 10% gPSD 50 It was found to have a mean geometric diameter in microns (d50) of 3.3 μm and aerosol and physical properties suitable for use in dry powder inhaler systems, particularly the ARCUS® platform.

[0135] Study R3 was run with a formulation similar to that of R2. The main change from the previous study was to condition the product filter bag in a dry 15% RH environment immediately prior to run. Conditioning the bag allowed the powder particles to be better released from the bag so that the powder could be successfully collected as product. This significantly increased the yield. The resulting powder had 61% FPF<5.6μm and gPSD 50 was 2.0 μm and had similar properties to those of R2.

[0136] Nebulization of RNA containing trehalose and leucine Studies R4 (Batch No. 483028) and R5 (Batch No. 483045) were formulated using either the amino acid leucine or the sugar trehalose, respectively, as the primary excipient. Both of these excipients have been used widely in the past in spray drying operations and tend to produce powders with suitable physical and aerosol properties. The R4 and R5 studies were also conducted to initially confirm that RNA can be spray dried with these excipients to produce powders with desirable properties.

[0137] Studies R4 and R5 yielded powders with desirable properties, thus confirming that RNA was successfully spray dried with these excipients. In R4, 75% FPF < 5.6 μm and gPSD of 2.5 μm 50 R5 produced powders with 71% FPF<5.6 μm and gPSD of 3.8 μm. 50 Further powder properties are given in Examples 4 and 5 below.

[0138] Increased RNA loading Studies R5 (Batch No. 483045) and R9 (Batch No. 483085) were formulated using trehalose as the primary excipient and contained 1% and 25% RNA, respectively. Studies R11 (Batch No. 483109) and R14 (Batch No. 483132) were formulated using isoleucine as the primary excipient and contained 50% and 25% RNA, respectively. In both cases, increasing the RNA loading in the particles increased the residual solvent content. In studies R5 and R9, the TGA-120°C (TGA-120: total weight loss or volatility due to heating the sample to 120°C with a heating ramp of 20°C / min) increased from 2.93% to 3.50%, while in studies R14 and R11, the residual solvent content increased from 1.73% to 3.55%. For formulations with higher RNA loading, the weight continued to decrease after 120°C by TGA. This was also observed when TGA was performed on pure yeast RNA.

[0139] The FPF <5.6um and <3.4um decreased with increasing RNA loading. The change was even more dramatic when isoleucine was the primary excipient, decreasing from 60% to 49% for FPF <5.6um and from 48% to 38% for FPF <3.4um. There was no significant decrease in FPF with trehalose when RNA loading was increased from 1% to 25%.

[0140] Adding charged compounds to the formulation Several studies were conducted with positively charged excipients, including R1 (batch number 449043), R6 (batch number 483071) and R12 (batch number 483119).

[0141] Reduction of maximum solubility of RNA by PEI In preparation for running Study R6 (Batch No. 483071), 1% RNA, 74% trehalose, and 2% NaCl were dissolved in water. After the three compounds were dissolved, 5% PEI was added to the aqueous phase. The PEI dissolved successfully and Study R6 proceeded. Addition of 5% PEI to the aqueous phase of Study R8 (Batch No. 483084), consisting of 10% RNA, 74% trehalose, and 2% NaCl, caused the RNA to precipitate. This indicates that the presence of PEI reduces the maximum solubility of the RNA. This can be used in later studies to generate larger, more porous particles by precipitating the RNA from solution after atomization.

[0142] Various product collection methods The formulations and process parameters for studies R6 (batch number 483071), R7 (batch number 483072) and R10 (batch number 483107) were nearly identical except for the method of product collection. (See Examples 1 and 3 below.) R6 used the filter baghouse setup described below in Example 2 (equipment setup) and Example 3 (process parameters). R7 and R10 used a cyclone to collect the final product. This was done to determine if the time required to start collecting the powder could be reduced.

[0143] For batches that used the cyclone, the time required to prime the system was reduced because it was no longer necessary to cover the filter bag house and product filter before powder began to accumulate in the collection vessel. For R7 and R10, it took only 7 and 12 minutes, respectively, for powder to begin collecting. For batch R6, the system had to be run for approximately 30 minutes before powder began to collect.

[0144] In R7 and R10, the majority of the smaller particles did not deposit in the collection vessel. Instead, the smaller particles exited the top of the cyclone and flowed into the filter bag house. This resulted in a decrease in yield from 23.8% in R6 to 12.8% in R7 and 15.2% in R10, respectively (see Table 6 in Example 5 below). This also resulted in a decrease in gPSD for R7 and R10 compared to R6. 50 This explains the increase in and decrease in FPF.

[0145] In contrast to the filter baghouse, there was no significant change in the solid properties of the powder recovered in the cyclone.

[0146] Example 1 - Materials and Methods The formulations used in studies investigating dry powder formulations for delivery of RNA are listed in Table 19.

[0147] [Table 2]

[0148] Materials used in studies investigating the formulation of dry powders for delivery of RNA are listed in Table 2.

[0149] [Table 3]

[0150] The manufacturers of the reagents used in the study are listed below.

[0151] reagent 1. RNA (Roche Diagnostics GmbH, Mannheim, Germany) 2.DPPC (Lipoid GmbH, Steinhausen, Switzerland) 3. NaCl (BDH, VWR, Radnor, PA, USA) 4. Trehalose (Sigma Aldrich, St. Louis, MO, USA) 5. Arginine (Amresco Life Sciences, VWR, Radnor, PA, USA) 6. Isoleucine (Sigma Aldrich, St. Louis, MO, USA) 7. Leucine (Sigma Aldrich, St. Louis, MO, USA) 8. Valine (Sigma Aldrich, St. Louis, MO, USA) 9. Tris(hydroxymethyl)aminomethane (G Biosciences, St. Louis, MO, USA) 10. Ethylenediaminetetraacetic acid (BDH, VWR, Radnor, PA, USA) 11. Polyethyleneimine (Aldrich Chemistry, Sigma, St. Louis, MO, USA)

[0152] Example 2 - Device Settings The spray dryer used in this evaluation consisted of a size 1 spray dryer (GEA, NIRO PSD1, Dusseldorf, Germany) with a single bag product filter bag house. The product filter bag with the single bag product filter bag house was model number P034582-016-210 (Donaldson Filtration Solutions, Bloomington, MN) made of polyester with a PTFE bag (5.87 inch diameter x 37.50 inch length) with a flat width of 9.23 inch. The air cap used in this evaluation was model number 67147 (Spraying Systems Co., Wheaton, IL) and the fluid cap used in this evaluation was model number 2850 (Spraying Systems Co., Wheaton, IL).

[0153] Example 3 – Spray drying process parameters Powders were made using a size 1 spray dryer (GEA, NIRO PSD1, Dusseldorf, Germany). Table 3 lists typical process parameter values ​​used for the production of RNA-containing powders. Process parameters for each specific run are listed in Table 4.

[0154] [Table 4] [Table 5]

[0155] Example 4 - Stability data for spray-dried powders containing RNA The stability data for the spray-dried powder containing RNA is shown in Table 5.

[0156] [Table 6-1] [Table 6-2] [Table 6-3]

[0157] Example 5 - Results for spray-dried powder containing RNA Table 6 shows the results obtained for the spray-dried powder containing RNA.

[0158] [Table 7]

[0159] Example 6 - Discussion and Observations of the Data and Results of Examples 1-5 The spray drying results above show that a light / dispersible powder can be formed. ·High loadings are achievable with sufficient FPF<5.6μm and low residual solvent. · RNA powders can be formulated with positively charged cationic excipients. R1 and R2 had to be discontinued due to powder build-up on the filter bags. · The powder adhering to the filter bags of R1 and R2 rapidly absorbed moisture once exposed to the atmosphere. For R3, the filter bag was placed in a room with RH below 15% for 2 days before use. · In both R2 and R3, silica gel is required to maintain the FPF observed at t=0. High mRNA and salt content are most likely the reason for the lower FPF without desiccant. The presence of silica gel reduces the amount of water in the product over time. · Due to the decrease in the atomizing gas velocity of R5, a thin layer of moisture formed inside the pipe at the exit of the drying cylinder. This may be the reason for the significant drop in yield between R4 and R5. The moisture layer may also be the reason for the increase in residual moisture detected by TGA. · In both R4 and R5, silica gel is required to maintain the FPF observed at t=0. Reduced mRNA and salt content increases the stability of the desiccant-free product. · In R7, the filter bag was pulsed twice every 20 minutes to reduce system pressure build-up. · In R7, smaller particles were not collected by the cyclone, resulting in an increased gPSD(50) and a smaller FPF. If the size distribution is increased overall, the cyclone yields become more similar. The solid state data for R6 and R7 are very similar. The use of cyclones and filter baghouses primarily affects size distribution and yield rather than solid state properties. · Addition of cationic excipients to the aqueous phase of formulation 8 precipitated the mRNA out of solution at 10% mRNA. This did not occur at 1% mRNA (R6) or in the absence of cationic excipients (R5 and R9). · In R7, the system was pulsed twice every 20 minutes to prevent an increase in system pressure (this was done once every 10 minutes in R10). · In R10, R7 and R10, smaller particles were not collected by the cyclone, resulting in an increased gPSD(50) and a smaller FPF. If the size distribution is increased overall, the cyclone yields become more similar. · R6, R7 and R10 have similar solid state data. The use of cyclones and filter baghouses primarily affects size distribution and yield rather than solid state properties. The density of the powder from R11 was significantly denser than R5 and R9. Under similar process conditions, increasing the yeast RNA loading from 25 to 50% decreased the FPF. In R12, an alternative positively charged excipient was used after the first cationic excipient precipitated the yeast RNA. This may not be an issue for GT mRNA, as yeast RNA is high MW and has a variable distribution. · R12 had a bimodal gPSD that could be attributed to the crystallization of one of the amino acid excipients. In R13, the diameter of the new filter bag was too large, causing the bag to fall off during operation. Contacted core filtration to change the size of the new bag will increase the mouth diameter or add a snap band to the top of the bag. RNA was successfully loaded from 1% to 50%. · Yeast RNA is highly soluble in water and compatible with excipients. A variety of excipients were tested, including a number of amino acids and sugars. To optimize drug uptake, powders were formulated with cationic excipients. · Up to 60-80% fine particle fraction (FPF<5.6μm) was generated. Physical stability testing of the amorphous phase under accelerated storage conditions is ongoing. Chemical stability (assessed at 40°C / 75% RH for 1-6 months). Formulations with and without desiccant have been used in long-term stability studies.

[0160] Example 7 - Investigations with spray-dried powder formulations for delivery of RNA and mRNA Objectives - The objective of this study was to investigate various parameters and formulations in studies referred to herein as Studies R16-R43, using materials and procedures similar to those described in Examples 1-5, to spray dry mRNA-containing powders with chemical, physical and aerosol properties suitable for delivery using a dry powder inhaler system, specifically for use with the ARCUS® platform. In some batches, yeast RNA was used as a placeholder for the mRNA.

[0161] The specific dry powder formulations tested are listed in Table 7.

[0162] [Table 8]

[0163] Materials and Methods Table 8 lists the materials used to manufacture the spray-dried powders of the formulations investigated in this Example 7 for delivery of RNA and mRNA.

[0164] [Table 9]

[0165] Table 9 lists the abbreviations used in this Example 7.

[0166] [Table 10]

[0167] Equipment settings and process parameters The spray dryer used in the studies listed in Table 10 consisted of a size 1 spray dryer (GEA, NIRO, Dusseldorf, Germany) equipped with a single bag product filter bag house. The product filter bags used in the studies listed in Table 10, except for R26, were model number P034582-016-210 (Donaldson Filtration Solutions, Bloomington, MN) made of polyester with a PTFE bag (5.87 inches diameter x 37.50 inches length) with a flat width of 9.23 inches. The filter bag used in R26 was model number 18-9088 (Franklin Products, Haw River, NC) made of PTFE (9.313 inches wide x 37.5 inches long). The air cap used in this evaluation was model number 67147 (Spraying Systems Co., Wheaton, Ill.) and the fluid cap used in this evaluation was model number 2850 (Spraying Systems Co., Wheaton, Ill.) Table 10 lists the target process parameter values ​​used to produce the RNA-containing powder.

[0168] [Table 11-1]

[0169] [Table 11-2]

[0170] The spray dryer used for the studies listed in Table 11 is a Buchi Mini Spray Dryer B-290 (Buchi, New Castle, Del.). The target process parameters for formulations R27-R29, R35, R37, R39, and R40 are listed in Table 11.

[0171] [Table 12]

[0172] Spraying of RNA containing isoleucine, valine and leucine - R14, R16, and R17 were formulated with 25% RNA and contained isoleucine, valine, and leucine, respectively, as the primary excipients. The three amino acids had different particle size distributions. When isoleucine was the primary excipient, the particle size distribution was right tailed and slightly bimodal. When valine was the primary excipient, the particle size distribution was bimodal. When leucine was the primary excipient, the distribution was unimodal and right tailed. The FPF<5.6 μm of the three powders was similar and the FPF<3.4 μm was relatively similar. The FPF<5.6 μm was 60% for R14, 57% for R16, and 57% for R17, and the FPF<3.4 μm was 48% for R14, 42% for R16, and 45% for R17. All three powders produced were crystalline, but they had different crystal structures. The residual solvent content of the three powders produced is relatively low, ranging from 0.80%. The residual solvent content is 1.73% for R14, 2.53% for R16, and 2.35% for R17. Overall, the powders made with amino acids as the primary excipient are relatively similar to each other.

[0173] Spray drying of amino acids with and without positively charged excipients R24 and R17 were formulated with 25% RNA and leucine as the primary excipient, with and without arginine, respectively. Studies R12 and R16 were formulated with 25% RNA and valine as the primary excipient, with and without arginine, respectively. When valine was the primary excipient, the addition of arginine reduced the gPSD 50 The gPSD decreased by 0.3 μm from 3.7 μm in R16 to 3.4 μm in R12. When leucine was the main excipient, 50increased by 0.4 μm from 1.6 μm for R17 to 2.0 μm for R24. When valine and leucine were the primary excipients, the addition of arginine reduced the FPF<5.6 μm and FPF<3.4 μm. FPF<5.6 μm was reduced by 3% when valine was the primary excipient and by 12% when leucine was the primary excipient. FPF<3.4 μm was reduced by 3% when valine was the primary excipient and by 9% when leucine was the primary excipient. The addition of arginine to valine and leucine increased the residual solvent content. When valine was the primary excipient, the residual solvent content increased by 0.61%, and when leucine was the primary excipient, the residual solvent content increased by 0.15%. Powders containing leucine and valine as primary excipients remained crystalline when arginine was added, but the crystal structure was altered. It is unlikely that trends can be drawn from the four studies detailed here. Unlike changing process parameters or increasing the amount of an excipient, the addition of a new excipient can change many of the ways that formulations interact during mixing, atomization, and drying.

[0174] Spray drying of RNA with trehalose, lactose and SD-30 R9, R22, and R23 were formulated with 25% RNA and contained trehalose, lactose, and SD-30 as the main excipients, respectively. Both powders containing trehalose and lactose had normal particle size distributions. The particle size distribution of trehalose has a slightly longer tail than that of lactose. The particle size distribution of SD-30 was bimodal. The FPF<5.6 μm and FPF<3.4 μm of the three powders were relatively similar. The FPF<5.6 μm was 68% for R9, 67% for R22, and 64% for R23, and the FPF<3.4 μm was 54% for R9, 49% for R22, and 53% for R23. All three powders produced were amorphous with a single peak at 21 (2Θ). The residual solvent content of the three powders produced was relatively high, with a range of 0.43%. The residual solvent content is 3.50% for R9, 3.93% for R22, and 3.92% for R23. Overall, the powders produced with sugar as the primary excipient are relatively similar to each other.

[0175] Comparison of amino acids and sugars as primary excipients R14, R16 and R17 were formulated with 25% RNA and amino acids as the primary excipient. Studies R9, R22 and R23 were also formulated with 25% RNA but contained sugar as the primary excipient. Studies that incorporated sugar as the primary excipient had a higher FPF<5.6μm and a higher FPF<3.4μm than studies that incorporated amino acids as the primary excipient. The FPF<5.6μm for powders incorporated with sugar ranged from 64%-68% and for powders incorporated with amino acids ranged from 57%-60%. The FPF<3.4μm for powders incorporated with sugar ranged from 49%-54% and for powders incorporated with amino acids ranged from 42%-48%. Powders containing sugar also had a higher residual solvent content than powders containing amino acids. The residual solvent content of the powders containing sugar ranged from 3.50% to 3.93%, and the residual solvent content of the powders containing amino acids ranged from 1.73% to 2.53%. The powders containing sugar were amorphous, and the powders containing amino acids were crystalline.

[0176] Increase in outlet temperature R5, R25 and R30 were formulated with 25% RNA, trehalose as the main excipient, and outlet temperatures of 40°C, 45°C and 60°C, respectively. Studies R17 and R34 were formulated with 25% RNA, leucine as the main excipient, and outlet temperatures of 40°C and 60°C, respectively. Studies R22 and R36 were formulated with 25% RNA, lactose as the main excipient, and outlet temperatures of 40°C and 60°C, respectively. In all three cases, increasing the spray dryer outlet temperature increased the gPSD of the produced powder. 50 The gPSD increased with a 5°C increase in outlet temperature. 50 increased by 0.4 μm from 2.7 μm for R9 to 3.1 μm for R25, while a 20°C increase in outlet temperature 50The trend is not linear as the gPSD increased by 3.3 μm from 2.7 μm for R9 to 6.0 μm for R30. Increasing the outlet temperature also has a greater effect on powders containing sugars as the main excipient compared to powders containing amino acids as the main excipient. When lactose is the main excipient, increasing the outlet temperature by 20°C reduces the gPSD 50 The gPSD increased by 2.3 μm from 3.7 μm for R22 to 6.0 μm for R36. When leucine was the main excipient, increasing the outlet temperature by 20°C reduced the gPSD 50 increased by 0.3 μm from 1.6 μm in R17 to 1.9 μm in R34.

[0177] Powders containing leucine as the primary excipient had a normally distributed particle size distribution at 40°C. Increasing the outlet temperature to 60°C increased the width of the particle size distribution. Powders containing sugar as the primary excipient also had a normally distributed particle size distribution at 40°C. Increasing the outlet temperature to 60°C resulted in a left tailed and slightly bimodal particle size distribution. The trend is not linear as a 5°C increase in outlet temperature only increased the width of the particle size distribution.

[0178] Increasing the outlet temperature also increased the FPF<5.6μm and FPF<3.4μm. When trehalose was the primary excipient, increasing the outlet temperature by 5°C increased the FPF slightly. This increased the FPF<5.6μm from 68% to 69% and the FPF<3.4μm from 54% to 57%. When the outlet temperature was increased from 40°C to 60°C, the change in FPF was more pronounced. The FPF<5.6μm increased from 68% to 78% and the FPF<3.4μm increased from 54% to 68%. When leucine and lactose were the primary excipients, increasing the outlet temperature had less effect on the FPF. When leucine was the primary excipient, increasing the outlet temperature by 20°C increased the FPF<5.6μm from 57% to 63% and the FPF<3.4μm from 45% to 50%. When lactose was the primary excipient, FPF<5.6 μm increased from 67% to 68% and FPF<3.4 μm increased from 49% to 56%.

[0179] Increasing the outlet temperature reduced the residual solvent content of the powder. When trehalose was the primary excipient and the outlet temperature was increased by 20°C, the residual solvent content decreased by 0.51%, from 3.50% in R9 to 2.99% in R30. When lactose was the primary excipient, the residual solvent content decreased by 1.40%, from 3.93% in R22 to 2.53% in R36. When leucine was the primary excipient, the residual solvent content decreased by 0.34%, from 2.35% in R17 to 2.01% in R34. When trehalose was the primary excipient and the outlet temperature was increased by 5°C, the residual solvent content increased by 0.13%, from 3.50% in R9 to 3.63% in R25. This result is opposite to what would be expected, since increasing the temperature of the spray dryer should increase the evaporation rate of the solvent in the spray dryer. It may be the result of experimental error.

[0180] Increased atomized gas velocity R30 and R32 were formulated with 25% RNA and trehalose as the main excipient at nebulization gas rates of 30 g / min and 40 g / min, respectively. Increasing the nebulization gas rate reduced the gPSD 50 The particle size decreased by 1.66 μm from 5.99 μm for R30 to 4.33 μm for R32. At an atomization gas rate of 30 g / min, the particle distribution was left tailed and slightly bimodal. Increasing the atomization gas rate to 40 g / min resulted in a more bimodal particle distribution. Increasing the atomization gas rate also decreased the FPF of the powder. FPF<5.6 μm decreased by 10% from 78% for R30 to 68% for R32. FPF<3.4 μm decreased by 8% from 68% for R30 to 60% for R32. Residual solvent content also decreased slightly with increasing atomization gas rate. Residual solvent content decreased by 0.18% from 2.99% for R30 to 2.81% for R32.

[0181] Unheated Liquid Feed Stream R18 and R24 were formulated with 25% RNA, leucine as the primary excipient, and arginine as the positively charged excipient. In study R18, the liquid skid circulating bath and the atomizing tower circulating bath were not turned on. When the organic and aqueous solutions were mixed in the static mixer, a white powder precipitated out of solution. The white powder was likely leucine or arginine. In study R24, when the liquid skid circulating bath and the atomizing tower circulating bath were set at 60°C, nothing precipitated out of solution. Some formulations require that the liquid feed stream be heated before entering the static mixer.

[0182] Comparison of Polyester and PTFE Product Filter Bags R12 and R26 were formulated with 25% RNA, valine as the main excipient, and arginine as the positively charged excipient. Study R12 used a filter bag made of polyester felt with a PTFE membrane. This is the same filter bag used in studies R1-R25, R27-R28, R30-R34, and R36. Study R26 used a filter bag made entirely of PTFE. The powders produced in these studies had similar solid state data. Residual solvent content increased by only 0.1%, from 3.14% in R12 to 3.24% in R26. Data obtained from DSC were similar. Low T1 was 50.21°C in R12 and 48.58°C in R26. Low T2 for R12 was 66.87°C in R12 and 65.06 in R26. Aerosol data between the two powders is less consistent. gPSD 50The FPF increased by 1.2 μm from 3.4 μm in R12 to 4.6 μm in R26. The particle size distribution is bimodal in both R12 and R26, but the first peak is slightly larger in R12 and the second peak is slightly larger in R26. This may be due to more particles of a particular size adhering to the polyester filter bag. The FPF also increased from R12 in R26. The FPF<5.6 μm increased by 1% from 54% in R12 to 55% in R26, and the FPF<3.4 μm increased by 7% from 39% in R12 to 46% in R26.

[0183] With conventional filter bags, it typically takes about 30 minutes before powder begins to build up in the IBC. With the new filter bags, it only took about 10 minutes before powder began to build up in the IBC. The new filter bags also significantly increased yields. Yields increased by 15.5%, from 22.3% for R12 to 37.8% for R26.

[0184] Comparison of yeast RNA and mRNA R29, R35 and R37 were formulated with 1% mRNA and contained trehalose, leucine and lactose as the main excipients, respectively. R28, R39 and R40 were formulated with 1% RNA and contained trehalose, leucine and lactose as the main excipients, respectively.

[0185] Replacing RNA with mRNA results in gPSD 50 When trehalose was the main excipient, there was a slight change in gPSD 50 increased by 0.1 μm from 1.3 μm in R28 to 1.4 μm in R29. When leucine was the main excipient, the gPSD 50 The gPSD decreased by 0.4 μm from 1.7 μm for R39 to 1.3 μm for R35. 50 gPSD increased by 1.0 μm from 2.2 μm in R40 to 1.2 μm in R37. 50The changes in gPSD are most likely due to variations in the flow rates of the aqueous and organic feed streams. The flow rates of both the aqueous and organic feed streams were set to 5 mL / min for all studies performed at Buchi. During the runs, the flow rates frequently deviated from the set point by 0.5 mL / min for extended periods of time. This offset was responsible for the gPSD 50 causes changes in

[0186] The changes in FPF<5.6μm and FPF<3.4μm are gPSD 50 The results can be correlated with the change in FPF. When trehalose was the primary excipient and 1% RNA was used, the capsules did not rotate when ACI-3 was performed. When trehalose was the primary excipient and 1% mRNA was used, the capsules rotated when ACI-3 was performed. Therefore, when comparing FPF data for R28 and R29, it is better to use the 2 week stability data because the capsules rotated with RNA and mRNA. The average FPF value is used for R28 and R29 because they are stable with and without desiccant. When trehalose was the primary excipient, the FPF<5.6μm decreased 2% from 58% in R28 to 56% in R29, and the FPF<3.4μm decreased 6% from 49% in R28 to 43% in R29. When leucine was the primary excipient, FPF<5.6 μm increased by 19% (from 60% in R39 to 79% in R35) and FPF<3.4 μm increased by 19% (from 50% in R39 to 69% in R35).When lactose was the primary excipient, FPF<5.6 μm increased by 28% (from 37% in R40 to 65% in R37) and FPF<3.4 μm increased by 33% (from 24% in R40 to 57% in R37).gPSD 50 A linear trend is observed when changes in FPF correlate with changes in FPF<5.6μm. The trend line can be calculated using the equation (ΔFPF<5.6μm)=-26.9*(gPSD 50 ) + 3.4 and R 2 = 0.92. 50 A linear trend is also observed when the change in FPF correlates with the change in FPF<3.4μm. The trend line can be calculated using the equation (ΔFPF<3.4μm)=-35.1*(gPSD 50 ) + 0.1 and R 2= 0.95. Using these trends, the gPSD 50 If there was no change in FPF, a 3.4% increase in FPF<5.6 μm and a 0.1% increase in FPF<3.4 μm would be expected.

[0187] Powders produced with sugar as the primary excipient had increased residual solvent content when RNA was replaced with mRNA. When trehalose was the primary excipient, the residual solvent content increased by 0.39%, from 2.21% in R28 to 2.60% in R29. When lactose was the primary excipient, the residual solvent content increased by 0.13%, from 2.36% in R40 to 2.49% in R37. The increase in residual solvent content was greater when trehalose was the primary excipient. This was due to the gPSD 50 was increased when RNA was replaced by mRNA and decreased when lactose was the primary excipient. 50 An increase in gPSD means that the ratio of total liquid flow rate to atomizing gas velocity is increasing. A higher ratio of total liquid flow rate to atomizing gas velocity increases the residual solvent content. 50 As the concentration of leucine in R39 decreased, the residual solvent content increased, meaning that the powders produced with mRNA had a higher residual solvent content than those produced with RNA. When leucine was the main excipient, the residual solvent content decreased by 0.038%, from 0.500% in R39 to 0.462% in R35. This change is small enough to be considered noise.

[0188] When RNA was replaced with mRNA for all three excipients, the crystal structure of the powders produced did not change. When trehalose and lactose were used as the primary excipients, the DSC data were very similar. When lactose was the primary excipient, the DSC data were very similar before 90°C, but deviated slightly thereafter. On this basis, powders produced with RNA will likely be similar to those produced with mRNA.

[0189] Particle characterization and stability data Table 13 provides particle characterization of the formulations listed above. Table 14 provides stability data and particle characterization from T=0 to T=6 months for the formulations listed and described above.

[0190] [Table 13-1] [Table 13-2]

[0191] [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4]

[0192] Example 8 - Particle size distribution of spray-dried powders containing RNA and mRNA - Table 15 provides the particle size distribution of the spray dried powders of Formulations 1-14 and 17-40 described in Examples 1-7.

[0193] [Table 15]

[0194] Example 9 - Automatic vs. Manual Spray Dryer Operation Table 16 shows the results of the automatic versus manual spray dryer runs.

[0195] [Table 16]

[0196] Example 10 – Additional excipient testing Table 17 shows the results of additional excipient testing of Formulations 12, 16 and 17.

[0197] [Table 17]

[0198] Example 11 – Capped mRNA containing lactose Table 18 shows the results of spray drying capped mRNA with lactose in a Buchi spray dryer and a size 1 spray dryer.

[0199] [Table 18]

[0200] Table 20 shows the particle characterization results of capped mRNA formulations containing lactose from R44-R46.

[0201] [Table 19]

[0202] Table 21 provides the particle size distribution of the spray dried powders of R44-R46.

[0203] [Table 20]

[0204] The patent and scientific literature referred to herein establishes knowledge available to those skilled in the art. All U.S. patents and published or unpublished U.S. patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are incorporated by reference.

[0205] Although the present invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims. It will also be understood that any of the embodiments described herein are not mutually exclusive and may be combined in various manners without departing from the scope of the invention as encompassed by the appended claims. The present invention includes the following preferred embodiments. (1) A respirable dry powder particle formulation for pulmonary delivery, comprising: i) at least about 1% RNA by weight of the particle; ii) at least about 10% DPPC by weight of the particle; iii) optionally, one or more of arginine, leucine, isoleucine, or valine, each at least about 1% by weight of the particle, where if more than one amino acid is selected, each amino acid has either the same or a different weight percentage as the other selected amino acids; iv) optionally, at least about 1% NaCl by weight of the particles; v) optionally, at least about 10% Tris by weight of the particles; vi) optionally, at least about 2% EDTA by weight of the particles; vii) optionally, at least about 40% trehalose by weight of the particle; ix) optionally, at least about 40% lactose by weight of the particles; and viii) optionally, at least about 2% PEI by weight of the particles; The above dry powder particle formulation, wherein all components of the RNA dry powder add up to 100% by weight. (2) The formulation of (1), wherein the powder has an FPF of at least about 40% or more <5.6 μm. (3) The formulation of (1) or (2), wherein the powder has an FPF of at least about 45%, 50%, 55%, 60%, 65%, 70%, 75% or more <5.6 μm. (4) The formulation according to any one of (1) to (3), wherein the powder has an FPF of at least about 30% or more <3.4 μm. (5) The formulation of any one of (1) to (4), wherein the powder has an FPF of at least about 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more <3.4 μm. (6) gPSD 50 The formulation according to any one of (1) to (5), wherein the particle size is about 1 micron to about 10 microns. (7) A formulation according to any one of (1) to (6) selected from Table 1. JPEG0007632959000028.jpg204154 (8) A method for treating a patient suffering from a respiratory disease, comprising administering at least one formulation according to (1) by pulmonary delivery. (9) The method according to (8), wherein the patient is an adult human or a pediatric human. (10) The method according to (8), wherein the disease is selected from asthma, pulmonary arterial hypertension, chronic obstructive pulmonary disease (COPD), respiratory distress syndrome (RDS), chronic bronchitis, acute bronchitis, emphysema, cystic fibrosis, pneumonia, tuberculosis, lung cancer, acute respiratory distress syndrome (ARDS), pneumoconiosis, interstitial lung disease (ILD) (e.g., sarcoidosis, idiopathic pulmonary fibrosis, and autoimmune diseases), pulmonary embolism, pleural effusion, and mesothelioma. (11) A respirable dry powder particle formulation for pulmonary delivery, comprising: i) at least about 1% RNA by weight of the particle; ii) at least about 10% DPPC by weight of the particle; iii) optionally, one or more of arginine, leucine, isoleucine, or valine, each at least about 1% by weight of the particle, where if more than one amino acid is selected, each amino acid has either the same or a different weight percentage as the other selected amino acids; iv) optionally, at least about 1% NaCl by weight of the particles; v) optionally, at least about 10% Tris by weight of the particles; vi) optionally, at least about 2% EDTA by weight of the particles; vii) optionally, at least about 40% trehalose by weight of the particle; viii) optionally, at least about 2% PEI by weight of the particles; and ix) optionally comprising at least about 40% lactose by weight of the particles; The above dry powder particle formulation, wherein all components of the RNA dry powder add up to 100% by weight. (12) A respirable dry powder particle formulation for pulmonary delivery, comprising: i) at least about 1% by weight of the particle of polynucleotide; ii) at least about 10% DPPC by weight of the particle; iii) optionally, one or more of arginine, leucine, isoleucine, or valine, each at least about 1% by weight of the particle, where if more than one amino acid is selected, each amino acid has either the same or a different weight percentage as the other selected amino acids; iv) optionally, at least about 1% NaCl by weight of the particles; v) optionally, at least about 10% Tris by weight of the particles; vi) optionally, at least about 2% EDTA by weight of the particles; vii) optionally, at least about 40% trehalose by weight of the particle; ix) optionally, at least about 40% lactose by weight of the particles; and viii) optionally, at least about 2% PEI by weight of the particles; The above dry powder particle formulation, wherein all components of the RNA dry powder add up to 100% by weight. (13) The method according to (12), wherein the polynucleotide is ribonucleic acid (RNA), small interfering RNA (siRNA), microRNA, deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), or locked nucleic acid (LNA). (14) The method according to (1), wherein the RNA is about 10 to about 12,000 nucleotides in length. (15) The method according to (12), wherein the polynucleotide is about 10 to about 12,000 nucleotides in length.

Claims

1. 1. A respirable dry powder particle formulation for pulmonary delivery comprising: i) at least 1% by weight of the particle is RNA; ii) 10% to 30% DPPC by weight of the particles; iii) 5% to 85% leucine by weight of the particle; and iv) 1% to 3% NaCl by weight of the particles; It consists of: All components of the dry powder particles add up to 100% by weight, At least 40% or more of the powder particles have an FPF of <5.6 μm; At least 30% or more of the powder particles have an FPF of <3.4 μm; and gPSD ５０ is between 1 micron and 10 microns; Dry powder particle formulation.

2. 2. The formulation of claim 1, wherein at least 45%, 50%, 55%, 60%, 65%, 70%, 75% or more of the powder particles have an FPF<5.6 μm.

3. 3. The formulation of claim 1 or 2, wherein at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% or more of the powder particles have an FPF<3.4 μm.

4. RNA:leucine:DPPC:NaCl (1:79:18:2), RNA:leucine:DPPC:NaCl (25:55:18:2), or Uncapped mRNA:Leucine:DPPC:NaCl (1:79:18:2) The formulation according to any one of claims 1 to 3, comprising:

5. The formulation of any one of claims 1 to 4, wherein the RNA is 10 to 12,000 nucleotides in length.

6. 6. The formulation of any one of claims 1 to 5 for use in a method of treating a patient suffering from a respiratory disease, wherein the formulation is administered by pulmonary delivery.

7. 7. The formulation for use according to claim 6, wherein the patient is an adult human or a pediatric human.

8. 8. The formulation for use according to claim 6 or 7, wherein the disease is selected from the group consisting of asthma, pulmonary arterial hypertension, chronic obstructive pulmonary disease (COPD), respiratory distress syndrome (RDS), chronic bronchitis, acute bronchitis, emphysema, cystic fibrosis, pneumonia, tuberculosis, lung cancer, acute respiratory distress syndrome (ARDS), pneumoconiosis, interstitial lung disease (ILD), pulmonary embolism, pleural effusion and mesothelioma.

9. 9. The formulation for use according to claim 8, wherein the interstitial lung disease (ILD) comprises sarcoidosis, idiopathic pulmonary fibrosis or an autoimmune disease.

Images