RU2815001C2 - Lipid-based formulations for rna delivery - Google Patents

Abstract

FIELD: pharmaceutical industry.

SUBSTANCE: group of inventions relates to a composition for treatment or prevention of a disease using mRNA-based therapy; to the method of obtaining it; to its use in the treatment or prevention of disease using mRNA-based therapy. The proposed composition contains (i) particles in a liquid phase, where the particles include mRNA and a lipid composition, and (ii) 1,2-propanediol, wherein the lipid composition includes: (i-a) a cholesterol derivative of formula (I) or pharmaceutically or physiologically acceptable salt thereof

where n is 0, R¹ means a -(CH₂)_q-NH₂ group or a -(CH₂)_r-NH-(CH₂)_s-NH₂ group, R² means a -(CH₂)_t-NH group₂ or a group -(CH₂)_u-NH-(CH₂)_w-NH₂, in which q, r, s, t, u, w are independently an integer from 2 to 6; (i-b) a phosphoglyceride of formula (II) or a pharmaceutically or physiologically acceptable salt thereof

where R⁴ and R⁵ each means a linear alkenyl group containing 10–24 carbon atoms and from 1 to 3 double bonds; (i-c) a pegylated phosphoglyceride of formula (III) or a pharmaceutically or physiologically acceptable salt thereof

where p is an integer from 5 to 200, R⁶ and R⁷ each represent a linear alkyl group containing 10–20 carbon atoms. The proposed method is characterized by the following steps: a) the components of the lipid composition in an organic solvent are dissolved and mixed, then its lyophilization is performed; b) the lyophilized lipid composition is rehydrated by adding water; c) the rehydrated lipid composition is combined with an aqueous solution of RNA; d) 1,2-propanediol is added.

EFFECT: group of inventions improves the efficiency of transfection of compositions based on mRNA and lipids.

14 cl, 3 dwg, 2 tbl, 3 ex

Description

The present invention relates to lipid-based compositions that can be used to effectively administer RNA to a subject.

Various lipid-based formulations are known in the art that can act as vectors for nucleic acids to ensure their delivery to the patient, see, for example, the article: A.S. Silva et al., Current Drug Metabolism, 16, 3-16, (2015) and literature cited therein. A formulation that has been successfully tested in clinical studies of pDNA delivery by inhalation is GL67A (E. Alton et al., Efficacy and Mechanism Evaluation, 3, issue 5, (2016, July)). GL67A contains, along with pDNA as a therapeutically active nucleic acid, GL67 (also known as Genzyme Lipid 67, a cationic cholesterol derivative), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2 -dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG5000 (DMPE-PEG5000).

However, this formulation has limited stability (t½: 6 h) and must therefore be prepared immediately before use. Moreover, it was found that this lipid composition is not effective for RNA transfection in vivo (O. Andries et al., Molecular Pharmaceutics 9, 2136-2145, (2012)). In view of these problems, there is still a need for formulations that can act as effective vectors for RNA delivery in vivo, providing a favorable toxicological profile.

In the context of the present invention, it has been found that the presence of 1,2-propanediol (also referred to as propylene glycol) leads to a significant increase in the transfection efficiency of lipid-based formulations of the type used in the GL67A formulation, which contain RNA as the nucleic acid.

Thus, in the first aspect of the present invention there is provided a composition comprising

(i) particles contained in a liquid phase, wherein the particles include RNA and a lipid composition, wherein the lipid composition includes:

(i-a) a cholesterol derivative of formula (I) or a salt thereof:

Where

n is 0 or 1, preferably 0,

R ¹ means a -(CH ₂ ) _q -NH ₂ group or a -(CH ₂ ) _r -NH-(CH ₂ ) _s -NH ₂ group, where q, r and s are independently equal to an integer from 2 to 6,

R ² means a -(CH ₂ ) _t -NH ₂ group or a -(CH ₂ ) _u -NH-(CH ₂ ) _w -NH ₂ group, where t, u and w are independently equal to an integer from 2 to 6,

R ³ means a linear alkanediyl group containing from 1 to 4 carbon atoms,

(i-b) phosphoglyceride of formula (II) or a salt thereof:

Where

R ⁴ means a linear alkyl group containing from 10 to 20 carbon atoms, or a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 20 carbon atoms,

R ⁵ means a linear alkyl group containing from 10 to 20 carbon atoms, or a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 20 carbon atoms,

And

(i-c) pegylated phosphoglyceride of formula (III) or a salt thereof:

Where

p is equal to an integer from 5 to 200, preferably from 10 to 170 and most preferably from 10 to 140,

R ⁶ means a linear alkyl group containing from 10 to 20 carbon atoms, or a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 20 carbon atoms,

R ⁷ means a linear alkyl group containing from 10 to 20 carbon atoms, or a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 20 carbon atoms,

And

(ii) 1,2-propanediol.

The second aspect of the present invention relates to a method for preparing the composition according to the first aspect of the invention described above, said method comprising the following steps

a) dissolving and mixing the components of the lipid composition in an organic solvent, followed by lyophilization of the lipid composition,

b) rehydrating the lyophilized lipid composition by adding water,

c) combining the rehydrated lipid composition with an aqueous solution of RNA, which provides the formation of particles including RNA and lipid composition, which are contained in the liquid phase,

d) adding 1,2-propanediol.

The addition of 1,2-propanediol can be done in a simple manner, for example, by adding it along with water in step 6, by adding it to the rehydrated lipid composition after step b), by adding it along with an aqueous solution of RNA in step c), or by adding it to the liquid phase after step c), which contains particles including RNA and a lipid composition. It should be understood that the addition can be carried out in one or more stages, for example, at different stages of the process.

Moreover, it has been found that 1,2-propanediol is capable of increasing the stability of particulate RNA formulations when frozen, for example, when such particulate formulation is frozen for storage or processing purposes. In other words, 1,2-propanediol can also act as a cryoprotectant for particles including RNA and lipid composition.

Thus, the third aspect of the invention provides a solid composition comprising:

(i) particles including RNA and lipid composition, and

(ii) 1,2-propanediol,

wherein said solid composition can be prepared by freezing the composition of the first aspect of the present invention as defined above.

In the following, a detailed description of the invention and its objects discussed above will be presented. It should be understood that these objects are closely interrelated. Accordingly, it is apparent that detailed information provided with respect to features of one subject matter of the invention will also apply to other subjects that are based on that feature, unless otherwise indicated.

RNA

The composition and solid composition of the present invention include ribonucleic acid (RNA), more preferably single-stranded RNA, and most preferably mRNA.

Regarding RNA, in principle any type of RNA can be used in the context of the present invention. Preferably, the RNA is single-stranded. The term "single-stranded RNA" refers to a single sequential strand of ribonucleotides, in contrast to RNA molecules in which two or more individual strands form a double-stranded molecule by hybridization of the individual strands. The term "single-stranded RNA" does not exclude the possibility that single-stranded molecules themselves form double-stranded structures, such as secondary (eg, loops and stem-loops) or tertiary structures.

The term "RNA" covers both RNAs that encode an amino acid sequence and RNAs that do not encode an amino acid sequence. It is estimated that more than 80% of the genome contains functional DNA elements that do not code for proteins. These non-coding sequences include DNA regulatory elements (binding sites for transcription factors, regulators and coregulators, etc.) and sequences that encode transcripts that are never translated into proteins. These transcripts, which are encoded by the genome and transcribed into RNA but not translated into proteins, are called non-coding RNAs (ncRNAs). Thus, in one embodiment of the present invention, the RNA is non-coding RNA. Preferably, the non-coding RNA is a single-stranded molecule. Research suggests that ncRNAs play a critical role in gene regulation, maintenance of genome integrity, differentiation and development of cells, and their deregulation is observed in various human diseases. There are different types of ncRNA: short (20-50 nucleotides), medium (50-200 nucleotides) and long (>200 nucleotides) ncRNAs. Short ncRNAs include microRNAs (miRNAs), small interfering RNAs (siRNAs), piwi protein-interacting RNAs (piRNAs), and transcription initiation RNAs (tiRNAs). Examples of medium ncRNAs are small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), transfer RNAs (tRNAs), transcription start site associated RNAs (TSSaRNAs), promoter associated small RNAs (PASRs), and transcript activating sequences promoter (PROMPT). The group of long non-coding RNAs (IncRNAs) includes long intergenic non-coding RNAs (lincRNAs), antisense IncRNAs, intronic IncRNAs, transcribed ultraconserved RNAs (T-UCR) and others (article: Bhan A, Mandal SS, ChemMedChem. (2014, March 26), doi: 10.1002/cmdc.201300534). Of the non-coding RNAs mentioned above, only siRNA is double-stranded. Thus, given that in a preferred embodiment of the present invention the non-coding RNA is single-stranded, preferably the non-coding RNA is not an siRNA. In another embodiment, the RNA is coding RNA, i.e. RNA, which encodes an amino acid sequence. Such RNA molecules are also called mRNA (messenger RNA) and are single-stranded RNA molecules. Nucleic acids can be obtained by chemical and enzymatic methods known to one skilled in the art, or using recombinant DNA methods, or they can be isolated from natural sources, or a combination of the above methods.

Messenger RNAs (mRNAs) are copolymers that consist of nucleoside phosphate structural units, mainly containing adenosine, cytidine, uridine and guanosine as nucleosides, which, as intermediate carriers, transfer genetic information from DNA from the cell nucleus to the cytoplasm, where this information is translated into proteins. Thus, they are suitable as an alternative for gene expression.

In the context of the present invention, the term mRNA means any polyribonucleotide molecule that, when entering a cell, is suitable for the expression of a protein or fragment thereof, or is translated into a protein or fragment thereof. The term "protein" in this context covers any type of amino acid sequence, i.e. chains of two or more amino acids that are linked to each other through peptide bonds and the term also includes peptides and fusion proteins.

mRNA contains a ribonucleotide sequence encoding a protein or fragment thereof whose function in a cell or the cell's environment is necessary or beneficial, for example, a protein whose deficiency or defective form is the primary cause of a disorder or disease, and the presence of which can mitigate or prevent the disorder or a disease or protein that can promote a beneficial process in the body, in the cell or in its environment. The mRNA may contain the sequence for a full-length protein or a functional variant thereof. In addition, the ribonucleotide sequence may encode a protein that acts as a factor, inducer, regulator, stimulant or enzyme, or as a functional fragment thereof, in cases where the function of this protein is necessary for the treatment of a disorder, especially a metabolic disorder, or for the purpose of initiating processes in vivo, such as the formation of new blood vessels, tissues, etc. In this context, a functional variant means a fragment that, within a cell, is capable of exhibiting the function of a protein whose action is required in the cell, or the deficiency or damaged form of which in the cell causes a pathogenic process. In addition, the mRNA may also include additional functional regions and/or 3' or 5' non-coding regions. The 3' and/or 5' non-coding regions may be regions that naturally flank the protein coding sequence or artificial sequences that help stabilize the RNA. Those skilled in the art can determine suitable sequences for these purposes in each case by routine experimentation.

In a preferred embodiment of the present invention, the mRNA contains a 5'-cap structure (five-bar cap, cap-0), consisting of an m7GpppG cap connected to the mRNA through a 5'-5' phosphodiester bond, an additional methyl group in the pre-terminal nucleotide on the 5' side -terminal region of the mRNA (cap-1, directly capped structure analog (ARCA)) and/or internal ribosome entry site (IRES) and/or poly(A) tail in the 3'-terminal region, primarily to improve translation . The mRNA may include additional translation promoting regions, such as, for example, cap-2 structures or histone stem-loop structures.

As noted above, unless otherwise indicated in a specific context, the term “mRNA” as used herein includes modified mRNAs, i.e. The mRNA may be modified mRNA.

In another embodiment of the present invention, the mRNA comprises labeled nucleic acids (preferably nucleotides and/or ribonucleotides), such as, for example, isotopically and/or fluorescently labeled nucleotides. Labeled mRNA molecules play an important role, for example, in studying the intracellular conformation of RNA and DNA molecules.

In a preferred embodiment of the present invention, RNA, preferably mRNA, is a molecule that contains modified nucleotides/ribonucleotides. In addition to the four classical ribonucleotides, namely adenosine, guanosine, cytidine and uridine, there are many analogues of each of these nitrogenous bases. Sometimes in this text and in the literature cited, these analogs or RNA/mRNA molecules that include one or more of these analogs are referred to as modified (eg, modified nucleotides or modified ribonucleotides) in the context of the present invention. Some analogues differ from the canonical nitrogenous bases presented above, but they may still exist in nature. Other analogues are of artificial origin. The use of both types of analogues is provided.

Preferred modifications are presented in the following table (Table 1).

In the case of RNA, preferably the mRNA of the present invention, either each of all uridine nucleotides or cytidine nucleotides can be modified in the same form, or in other cases a mixture of each of the modified nucleotides can be used. Modified nucleotides may contain naturally occurring or non-naturally occurring modifications. A mixture of different modified nucleotides can be used. Thus, for example, one portion of the modified nucleotides may contain naturally occurring modifications while another portion contains non-naturally occurring modifications, or a mixture of modified nucleotides containing natural and/or non-naturally occurring modifications may be used. In addition, some of the modified nucleotides may contain a base modification, and another part may contain a sugar modification. Likewise, all base modifications, or all sugar modifications, or any suitable combination of modifications can be used. The stability and/or duration of action of the RNA, preferably the mRNA of the present invention, can be selectively controlled by varying modifications.

In one embodiment of the present invention, at least two different modifications are used for one type of nucleotide, where one type of modified nucleotide contains a functional group through which additional groups can be attached. Nucleotides with different functional groups can also be used to provide binding sites for the attachment of different groups. Thus, a portion of the modified nucleotides may carry, for example, an azide group, an amino group, a hydroxyl group, a thiol group, or several other reactive groups suitable for reaction under the given conditions. It is also possible to use a functional group which, under certain conditions, can activate a bindable natural group so that molecules with functional groups can be attached. Nucleotides modified to provide binding sites can also be introduced as modified adenosine or guanosine. The selection of primarily preferred modifications and the choice of available binding sites depends on the type of groups that need to be introduced and the frequency with which these groups must be present. Thus, the nucleotide content provided by the functional and/or activating groups depends on how high the content of the groups that are to participate in the binding is, which can be determined by a simple method by those skilled in the art. Typically, the content of modified nucleotides with functional and/or activating groups, if present, ranges from 1 to 25% of the modified nucleotides. Those skilled in the art can, if necessary, determine the most suitable groups for each case and their optimal content in the course of routine experiments.

In a preferred embodiment of the present invention, the RNA, preferably the mRNA of the present invention, is characterized in that the modified uridines are selected from 2-thiouridine, 5-methyluridine, pseudouridine, 5-methyluridine-5'-triphosphate (m5U), 5-ioduridine-5'- triphosphate (I5U), 4-thiouridine-5'-triphosphate (S4U), 5-bromouridine-5'-triphosphate (Br5U), 2'-methyl-2'-deoxyuridine-5'-triphosphate (U2'm), 2 '-amino-2'-deoxyuridine-5'-triphosphate (U2'NH2), 2'-azido-2'-deoxyuridine-5'-triphosphate (U2'N3) and 2'-fluoro-2'-deoxyuridine-5 '-triphosphate (U2'F).

In another preferred embodiment of the present invention, the RNA, preferably the mRNA of the present invention, is characterized in that the modified cytidines are selected from 5-methylcytidine, 3-methylcytidine, 2-thiocytidine, 2'-methyl-2'-deoxycytidine-5'-triphosphate (C2 'm), 2'-amino-2'-deoxycytidine-5'-triphosphate (C2'NH2), 2'-fluoro-2'-deoxycytidine-5'-triphosphate (C2'F), 5-iodocytidine-5' -triphosphate (I5U), 5-bromocytidine-5'-triphosphate (Br5U) and 2'-azido-2'-deoxycytidine-5'-triphosphate (C2'N3).

In a preferred embodiment of the present invention, mRNA means an mRNA containing a combination of modified and unmodified nucleotides, preferably an mRNA containing a combination of modified and unmodified nucleotides, as described in international application WO 2011/012316. The mRNA described herein has been reported to exhibit increased stability and decreased immunogenicity. In a preferred embodiment of the present invention, in such a modified mRNA, from 5 to 50% of the cytidyl nucleotides and from 5 to 50% of the uridyl nucleotides are modified. Adenosine- and guanosine-containing nucleotides may be unmodified. Adenosine and guanosine nucleotides may be unmodified or partially modified, and are preferably present in unmodified form. Preferably, 10 to 35% of the cytidyl and uridyl nucleotides are modified, and particularly preferably, the content of the modified cytidyl nucleotides is in the range of 7.5 to 25%, and the content of the modified uridyl nucleotides is in the range of 7.5 to 25%. It has been found that, in fact, relatively low levels, for example only 10% of each of the modified cytidyl and uridyl nucleotides, can achieve the desired properties. More preferably, the modified cytidyl nucleotides are 5-methylcytidyl residues, and the modified uridyl nucleotides are 2-thiouridyl residues. Most preferably, the content of modified cytidyl nucleotides and modified uridyl nucleotides is 25%, respectively.

In some other embodiments of the present invention, in such a modified RNA, preferably, from 5 to 50% of the cytidines are C analogs and from 5 to 50% of the uridines are in U analogs. In some embodiments of the present invention, in such a modified RNA, preferably from 5 to 40% of the mRNA cytidines are C analogs and 5 to 40% of the uridines are U analogs. In some embodiments of the present invention, in such a modified RNA, preferably the mRNA, 5 to 30% of the cytidines are C analogs and 5 to 30% of the uridines are U analogs. In some embodiments of the present invention in such a modified RNA, preferably the mRNA has 10 to 30% of the cytidines are C analogs and 10 to 30% of the uridines are U analogs. In some embodiments of the present invention in such a modified RNA, preferably the mRNA has 5 to 20% of the cytidines are C analogs and 5 to 20% of the uridines are U analogs. In some embodiments of the present invention, such a modified RNA, preferably an mRNA, has 5 to 10% cytidine nucleotides and 5 to 10% uridine nucleotides modified. In some embodiments of the present invention, such a modified RNA, preferably an mRNA, has 25% of the cytidine nucleotides and 25% of the uridine nucleotides modified. In some embodiments of the present invention, adenosine- and guanosine-containing nucleotides may be unmodified. In some embodiments of the present invention, adenosine- and guanosine-containing nucleotides may be unmodified or partially modified, and they are preferably present in unmodified form.

As noted above, in some embodiments of the present invention, the term “U analogs” refers to a single type of U analog. In some embodiments of the present invention, the term “U analogs” refers to two or more types of U analogs. In some embodiments of the present invention, the term “C analogs” refers to a single type of C analogs. In some embodiments of the present invention, the term “C analogs” refers to two or more types of C analogs.

In some embodiments of the present invention, the percentage of cytidines that are cytidine analogs in the RNA, preferably mRNA, is not equal to the percentage of uridines that are uridine analogs in the RNA, preferably mRNA. In some embodiments of the present invention, the percentage of cytidine analogues is lower than the percentage of uridine analogues. As noted above, this may be true in the presence or absence of adenosine and guanosine analogs, however, in some embodiments of the present invention, this is true in the absence of adenosine analogs and guanosine analogs. In some embodiments of the present invention, the RNA, preferably mRNA, as disclosed herein, comprises less than 15%, less than 10%, less than 5%, or less than 2% adenosine analogs, guanosine analogs, or both nucleoside analogs.

In some embodiments, the RNA molecules, preferably mRNA, of the present invention include cytidine analogs and uridine analogs, with 5 to 20% of the cytidines being cytidine analogs and 25 to 45% of the uridines being uridine analogs. In other words, RNA, preferably mRNA, includes modified and unmodified cytidines, as well as modified and unmodified uridines, with 5 to 20% of cytidines including cytidine analogues, while 25 to 45% of uridines including uridine analogues. In other embodiments of the present invention, the RNA, preferably mRNA, includes from 5 to 10% cytidine analogues and from 30 to 40% uridine analogues, for example, from 7 to 9% cytidine analogues, especially about 7, 7.5 or 8% and from 32 to 38% uridine analogues, primarily about 33, 34, 35 or 36%.

In some embodiments, any of the uridine analogs and cytidine analogs described herein may be used, optionally excluding pseudouridine. In some embodiments, the cytidine analogue includes or consists of 5-iodocytidine (e.g., "consists of" if a single type of analogue is used), and the uridine analogue includes or consists of 5-iodouridine (e.g., "consists of" if a single type of analogue is used). the only type of analog).

In some of any of the above embodiments, the term “percentage” of analogues of a given nucleotide refers to the initial percentage (eg, the percentage of analogues in the initial reaction mixture, such as the initial in vitro transcription reaction mixture). In some of any of the above embodiments, the term "percentage" of analogues of a given nucleotide refers to the percentage of the product (eg, the percentage of the compound synthesized or transcribed). Both possible options are considered equally.

The RNA molecules, preferably mRNA, of the present invention can be produced recombinantly in in vivo systems known to those skilled in the art.

In another embodiment, modified RNA molecules, preferably mRNA, of the present invention can be produced in an in vitro system, for example, using an in vitro transcription system known to one skilled in the art. To carry out an in vitro transcription reaction capable of producing RNA, preferably mRNA, a starting mixture of modified and unmodified nucleoside triphosphates is required to produce modified RNA molecules, preferably mRNA, with the desired properties of the present invention. In some embodiments, from 5 to 50% of the cytidines are cytidine analogs in such a starting mixture, and from 5 to 50% of the uridines are uridine analogs in such a starting mixture. In other embodiments, from 5 to 40% of the cytidines are cytidine analogs in such a starting mixture, and from 5 to 40% of the uridines are uridine analogs in such a starting mixture. In other embodiments, from 5 to 30% of the cytidines are cytidine analogs in such a mixture, and from 5 to 30% of the uridines are uridine analogs in such a starting mixture. In yet other embodiments, from 5 to 30% of the cytidines are cytidine analogs in such a mixture, and from 10 to 30% of the uridines are uridine analogs in such a mixture. In yet other embodiments, from 5 to 20% of the cytidines are cytidine analogs in such a starting mixture, and from 5 to 20% of the uridines are uridine analogs in such a starting mixture. In other embodiments, 5 to 10% of the cytidines are cytidine analogs in such a starting mixture, and 5 to 10% of the uridines are uridine analogs in such a starting mixture. In other embodiments, 25% of the cytidines are cytidine analogs in such a starting mixture, and 25% of the uridines are uridine analogs in such a starting mixture. In yet other embodiments, the starting mixture does not include adenosine and/or guanosine analogues. In other embodiments, the starting mixture does not necessarily include one or more adenosine and/or guanosine analogues (or none of them).

In some embodiments, the percentage of cytidine analogue cytidines in the starting mixture is not equal to the percentage of uridine analogue uridines in the starting mixture. In other embodiments, the percentage of cytidine analogs in the starting mixture is lower than the percentage of uridine analogs in the starting mixture. As noted above, this may occur in the presence or absence of adenosine and guanosine analogs in the starting mixture, however, in other embodiments of the invention, this occurs in the absence of adenosine analogs and guanosine analogs in the starting mixture.

In some embodiments, the starting nucleotide mixture for the in vitro transcription system that produces RNA, preferably mRNA, of the present invention includes cydine analogs and uridine analogs, wherein from 5 to 20% of the cytidines in the starting mixture are cytidine analogs and from 25 to 45% of uridines in the initial mixture are uridine analogues. That is, the starting mixture includes modified and unmodified cytidines, as well as modified and unmodified uridines, with 5 to 20% of the cytidines in the starting mixture including cytidine analogs, while 25 to 45% of the uridines in the starting mixture including uridine analogs . In other embodiments of the invention, the starting mixture includes from 5 to 10% cytidine analogs and from 30 to 40% uridine analogs, for example, from 7 to 9% cytidine analogs, especially 7, 7.5 or 8% and, for example, from 32 up to 38% uridine analogues, primarily 33, 34, 35 or 36%.

In other embodiments, any of the uridine analogs and cytidine analogs described herein may be used, optionally excluding pseudouridine. In some embodiments, the cytidine analogue includes or consists of (eg, "consists of" when a single type of C analogue is used) of 5-iodocytidine, and the uridine analogue includes or consists of (eg, "consists of" when a single type of U analogue is used) of 5-ioduridine.

Typical analogs are described in the tables above. It is apparent that for modified polyribonucleotides encoding the desired polypeptide (module (a)), the terms "analogues" and "degree of modification", unless otherwise specified, are considered to refer to the entire length of the polyribonucleotide encoding the desired polypeptide (module (a)), including 5' and 3' untranslated regions (eg, the degree of modification is based on the initial proportions of analogues in the in vitro transcription reaction mixture, for example, to insert analogues into positions intended for transcription).

Moreover, modified RNA molecules, preferably mRNA, can be chemically synthesized by known chemical synthesis methods on an automated nucleotide sequence synthesizer using a solid phase carrier and standard techniques, or by chemical synthesis of the corresponding DNA sequences and their subsequent transcription in vitro or in vivo.

In another preferred embodiment of the present invention, the mRNA can be combined with target binding sites, target sequences and/or miRNA binding sites to ensure that the desired mRNA is active only in relevant cells. In yet another preferred embodiment of the present invention, the RNA can be combined with a microRNA or short hairpin RNA (shRNA) downstream of the 3' poly(A) tail.

Typically, therapeutic effects can be achieved through the interaction of nucleic acid with cellular compounds and organelles. Such interaction itself may, for example, activate the innate immune system, as is the case with specific CpG oligonucleotides and sequences designed to interact exclusively with toll-like and other extra- or intracellular receptors. Moreover, cellular uptake or introduction of nucleic acids (preferably mRNA) into cells may be intended to express nucleotide sequences such as genes within the nucleic acid (preferably mRNA), may be intended to down-regulate, silence or knockdown expression of an endogenous gene such as consequence of the presence inside the cell of an introduced exogenous nucleic acid, or may be intended to modify endogenous nucleic acid sequences, such as repair, excision, insertion or substitution of selected bases or entire sections of endogenous nucleic acid sequences, or may be intended to suppress virtually any cellular processes due to the presence within cells and interactions with introduced exogenous nucleic acid (preferably mRNA). Overexpression of introduced exogenous nucleic acids (preferably mRNA) may be intended to compensate or complement the expression of endogenous genes, especially in cases where the endogenous gene is defective or "silent", resulting in the absence of a product or an unusable, defective or non-functional expression product genes, as in the case of many metabolic or inherited diseases, such as cystic fibrosis, hemophilia or muscular dystrophy, to name just a few examples. Overexpression of introduced exogenous nucleic acids (preferably mRNA) may also be designed to cause the expression product to interfere with or inhibit any endogenous cellular process, such as regulation of gene expression, signal transduction and other cellular processes. Overexpression of introduced exogenous nucleic acids (preferably mRNA) may also be intended to induce an immune response under the conditions of the particular organism in which the transfected or transduced cell is located, or in which it is intended to be located. Examples include genetic modification of antigen-presenting cells, such as dendritic cells, to present antigen for the purpose of vaccination. Other examples include the overexpression of cytokines in tumors to induce a tumor-specific immune response. Moreover, overexpression of introduced exogenous nucleic acids (preferably mRNA) can also be designed to generate in vivo or ex vivo transiently genetically modified cells for cell therapy, such as engineered T cells, progenitor cells, stem or other cells for regenerative medicine.

Down-regulation, silencing or knockdown of endogenous genes for therapeutic purposes can be achieved, for example, using RNA interference (RNAi) with ribozymes, antisense oligonucleotides, tRNAs, long double-stranded RNAs, and such down-regulation can be sequence-dependent. specific or nonspecific, and can also lead to cell death, as in the case of the introduction of long double-stranded RNA into cells. Down-regulation, silencing or knockdown of endogenous or pre-existing genes may have beneficial effects in the treatment of acquired, inherited or spontaneously occurring diseases, including viral infections and cancer. It may also be possible to introduce nucleic acids into cells as a preventive measure to prevent, for example, viral infection or neoplasms. Downregulation, silencing, or knockdown expression of endogenous genes can occur at the transcriptional and translational levels. Numerous mechanisms known to those skilled in the art include, for example, epigenetic modifications, changes in chromatin structure, selective binding of transcription factors by the introduced nucleic acid, hybridization of the introduced nucleic acid with complementary sequences in genomic DNA, mRNA or other types of RNA due to base pairing , including atypical base pairing mechanisms such as triple helix formation. Similarly, gene repair, base or sequence changes can be performed at the genome level and at the mRNA level, including exon skipping. Base or sequence changes can be accomplished, for example, by RNA-guided site-specific DNA cleavage, cut-and-paste mechanisms by trans-splicing, trans-ribozyme trans-splicing, chimeraplasts, spliceosome-mediated trans-RNA splicing, or using introns group II or redirected introns, or using virus-mediated insertional mutagenesis, or using targeted genomic insertion using prokaryotic, eukaryotic or viral integrase systems. Since nucleic acids are the carriers of the blueprints for the creation of living systems, and since they directly and indirectly participate in many cellular processes, it is theoretically possible to influence any cellular process by introducing nucleic acids into cells from the outside. It is noteworthy that this administration can be carried out directly in vivo and ex vivo in cell or organ cultures, followed by transplantation of the organs or cells thus modified into the recipient. Particles for use in the present invention, when mixed with nucleic acids as therapeutically active agents, can provide beneficial effects for all of the purposes described above.

As mentioned above, RNA, preferably mRNA, may contain a ribonucleotide sequence that encodes a protein or fragment thereof whose function is essential or beneficial in a cell or its environment, for example, a protein whose deficiency or damaged form causes a disorder or disease, and the provision of which is capable of mitigating or preventing a disorder or disease, or a protein that can contribute to a beneficial effect on an organism in a cell or in its environment.

Of course, in recent years, RNA (primarily mRNA) has become increasingly important as a new drug. Unlike DNA-based gene therapies, mRNA does not need to be delivered into the nucleus, but is translated directly into protein in the cytoplasm (J Control Release, 150, 238-247, (2011) and Eur J Pharm Biopharm , 71, 484-489, (2009)).

In addition, there are many known genetic disorders caused by single gene mutation that are candidates for therapeutic use of RNA, preferably mRNA. Disorders caused by mutations in a single gene, such as cystic fibrosis, hemophilia and many others, can be dominant or recessive depending on the likelihood and occurrence of the trait in the offspring. While a dominant allele is characterized by a phenotype in individuals for whom only one copy of that allele is identified, in the case of a recessive allele, an individual must have two copies of it, one from each parent, for the trait to be expressed. In contrast, polygenic disorders are caused by two or more genes, and the underlying disorder is often mild and associated with environmental factors. Examples of polygenic disorders include hypertension, high cholesterol, cancer, neurodegenerative disorders, mental illness, and others. Moreover, in these cases, a therapeutically active RNA, preferably an mRNA that replaces one or more such genes, may have a beneficial effect on such patients. Moreover, a genetic disorder does not have to be inherited from parental genes, but can also be caused by new mutations. In such cases, a therapeutically active RNA, preferably an mRNA that provides the correct gene sequence, may also have a beneficial effect on patients.

An online catalog containing to date 22,993 records of human genes and genetic disorders along with their corresponding genes and descriptions of their phenotypes is available on the ONIM (Online Mendelian Inheritance in Man) page (http://onim.org), gene sequences of each of these are available in the Uniprot database (http://www.uniprot.org). By way of example, but not limited to those listed, the following Table 2 lists some congenital diseases and the corresponding gene(s). Due to the high degree of interaction of cell signaling pathways, a mutation of a particular gene causes many pathogenic symptoms, of which only characteristic ones are listed in Table 2.

In some embodiments of the present invention, the therapeutic protein that is encoded by the RNA, preferably the mRNA of the present invention, is selected from the cellular proteins listed in Table 2. Thus, the RNA molecule, preferably the mRNA of the present invention, can encode a therapeutically active cellular protein, which is encoded the therapeutically active cellular protein is one of the proteins listed in Table 2 or its homolog.

In another embodiment of the present invention, the therapeutically active protein that is encoded by the RNA, preferably the mRNA of the present invention, is selected from the secreted proteins listed in Table 2. Thus, the RNA, preferably the mRNA of the present invention, may encode a therapeutically active fusion protein, wherein the encoded the therapeutically active protein or its homolog is one of those listed in Table 2, and the second protein is a signal peptide that provides secretion of the therapeutically active protein. The signal peptide is a short amino acid sequence of 5 to 30 amino acids in length, located in the N-terminal region of the above therapeutically active protein, and which conducts the hybrid protein along the cellular secretory pathway through certain organelles (i.e., through the endoplasmic reticulum, Golgi apparatus or endosomes). Thus, such a fusion protein is secreted from a cell or from a cell organelle, or incorporated into the cell membrane (eg, polytopic transmembrane proteins) within or on the surface of the cell.

Thus, in preferred embodiments of the present invention, the RNA, preferably the mRNA of the present invention, may encode the following, but not limited to, protein genes that cause, promote, or protect against diseases. Non-limiting examples of disorders that can be treated (or prevented) include the above polypeptide, protein or peptide selected from the group consisting of compounds presented in the following Table 2.

In some embodiments of the present invention, an RNA coding sequence, preferably an mRNA of the present invention, can be transcribed and translated into a portion of a protein or a full-length protein with cellular activity at a level equal to or greater than that of the native protein. In other embodiments, the RNA, preferably the mRNA of the present invention, encodes a therapeutically or pharmaceutically active polypeptide, protein or peptide having a therapeutic or prophylactic effect, wherein said polypeptide, protein or peptide is selected from the group consisting of compounds presented in the following Table 2 RNA, preferably mRNA, more specifically its coding sequence, can be used to express a portion of a protein or a full-length protein with cellular activity at a level equal to or greater than that of the native protein. This may provide treatment for diseases that may benefit from administration of RNA molecules.

In the table presented above. 2 shows examples of genes in which defects lead to diseases that can be treated using RNA, preferably an mRNA of the present invention, wherein the RNA, preferably an mRNA of the present invention includes a ribonucleotide sequence that encodes a full-length protein variant or a functional fragment thereof of the defective gene described above . In particularly preferred embodiments, mention may be made of hereditary diseases which, for example, affect the lungs, such as surfactant-associated protein SPB (surfactant-associated protein B) deficiency, ABCA3 deficiency, cystic fibrosis and α1-antitrypsin deficiency, or which affect for plasma proteins (eg, congenital hemochromatosis (hepcidin deficiency), thrombocytopenic purpura (TPP, ADAMTS 13 deficiency) and which cause coagulation disorders (eg, hemophilia A and B) and complement component deficiencies (eg, protein C deficiency), conditions immunodeficiency, such as, for example, SCID (caused by mutations of various genes, such as: RAG1, RAG2, JAK3, IL7R, CD45, CD3δ, CD3ε), or deficiency due to the absence of adenosine deaminase, for example, (ADA-SCID), septic granulomatosis (eg, caused by mutations of the gp-91-phox gene, p47-phox gene, p67-phox gene or p33-phox gene) and storage diseases such as Gaucher disease, Fabry disease, Krabbe disease, MPS I, MPS II (Hunter syndrome ), MPS VI, glycogen storage disease type II or mucopolysaccharidosis.

Other disorders for which the RNA, preferably the mRNA of the present invention, can be used include disorders such as SMN1-associated spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), GALT-associated galactosemia, cystic fibrosis (CF), SLC3A1-associated disorders including cinstinuria, COL4A5-associated disorders including familial glomerulonephritis with deafness, galactocerebrosidase deficiency, X-linked adrenoleukodystrophy and adrenomyeloneuropathy, Friedreich's hereditary ataxia, Pelizaeus-Merzbacher disease, TSC1- and TSC2-associated tuberous sclerosis, disease Sanfilippo ( MPS IIIB), CTNS-associated cystinosis, FMR1-associated diseases that include fragile X syndrome, fragile X-associated tremor/ataxia syndrome and fragile X-associated premature ovarian failure syndrome, Prader-Willi syndrome, hereditary hemorrhagic telangiectasia (AT), Niemann-Pick disease type C1, diseases associated with neural waxy lipofuscinosis including juvenile neural waxy lipofuscinosis (JNCL), juvenile Batten disease, Santavuori-Haltia disease, Jansky-Bielschowsky disease, and PTT-1 deficiency and TPP1 associated with TPP1, EIF2B1, EIF2B2, EIF2B3, EIF2B4 and EIF2B5 childhood ataxia with central nervous system hypomyelination/white matter loss, CACNA1A- and CACNB4-associated episodic ataxia type 2, MECP2-associated disorders including classic Rett syndrome, MECP2-associated severe encephalopathy of newborns and RPM-X syndrome, CDKL5-associated atypical Rett syndrome, spinobulbar muscular atrophy (SBMA), Notch-3-associated cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL), SCN1A- and SCN1B- associated seizure disorders, polymerase G-associated disorders, which include Alpers-Huttenlocher syndrome, POLG-associated congenital sensory neuropathy with anhidrosis, dysarthria and ophthalmoparesis, and autosomal dominant and recessive progressive external ophthalmoplegia with deletions in mitochondrial DNA, X-associated hypoplasia adrenal glands, X-associated agammaglobulinemia, Fabry disease, and Wilson disease.

All of these diseases in which the protein, eg enzyme, is defective can be treated using RNA, preferably mRNA, encoding any of the above proteins of the present invention, thereby making available the protein or functional fragment thereof encoded by the defective gene. Transcript replacement therapy/enzyme replacement therapy does not improve the underlying genetic defect but increases the concentration of the enzyme the patient is deficient in. For example, in Pompe disease, transcript replacement therapy/enzyme replacement therapy restores the deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA).

Thus, non-limiting examples of proteins that may be encoded by the mRNA of the present invention include erythropoietin (EPO), growth hormone (hGH), cystic fibrosis transmembrane conductance regulator (CFTR), growth factors such as GM-SCF, G -CSF, MPS, protein C, hepcidin, ABCAZ and surfactant-associated protein B. Other examples of diseases that can be treated using the RNA of the present invention include hemophilia A/B, Fabry disease, CGD, ADAMTS13, Hurler's disease X-mediated -chromosome A-γ-globulinemia, adenosine deaminase-associated immunodeficiency state and neonatal respiratory distress syndrome, which is associated with SP-B. First of all, preferably, the RNA, preferably the mRNA of the present invention, contains a sequence encoding surfactant-associated protein B (SP-B) or erythropoietin. Other examples of proteins that may be encoded by RNA, preferably mRNA, of the present invention include growth factors such as human growth hormone hGH, BMP-2 and angiogenesis factors.

In yet another embodiment, the RNA, preferably mRNA, may contain a ribonucleotide sequence that encodes a full-length antibody or fragment thereof (eg, both heavy and light chains), which can be used in therapeutic regimens, for example, to enhance immunity in a subject. Suitable antibodies and their use in therapy are known in the art.

In another embodiment, the RNA, preferably mRNA, may encode a functional monoclonal or polyclonal antibody that may be useful for targeting and/or inactivating a biological target (eg, a stimulatory cytokine such as tumor necrosis factor). Likewise, the RNA, preferably mRNA, may encode, for example, functional antibodies against nephrotic factor, which is used in the treatment of membranous proliferative glomerulonephritis type II or acute hemolytic uremic syndrome, or alternatively, may encode antibodies against vascular endothelial growth factor (VEGF). ), which are used in the treatment of VEGF-mediated diseases such as cancer.

In one embodiment, the RNA, preferably mRNA, may contain a ribonucteotide sequence encoding an antigen that can preferably be used in therapeutic regimens.

In another embodiment of the present invention, the RNA, preferably mRNA, may contain a ribonucleotide sequence encoding a polypeptide or protein that can be used in genome editing technologies. Genome editing is a type of genetic engineering in which DNA with deletions or substitutions is inserted into an organism's genome using nucleases. Such nucleases create site-specific breaks at desired positions in the genome. Induced breaks undergo repair using non-homologous end joining or homologous recombination, which leads to targeted mutations in the genome, and thereby to genome “editing”. The breaks can be either single-strand breaks or double-strand breaks (DSB), although double-strand breaks (DSB) are preferred. There are many genome editing systems known in the art using various polypeptides or proteins, ie, for example, the CRISPR-Cas system, meganucleases, zinc finger nucleases (ZFNs) and transcription activator-like effector nuclease (TALEN). Genomic engineering methods are described in the review article Trends in Biotechnology 31 (7), 397-405 (2013).

Thus, in a preferred embodiment of the present invention, the RNA, preferably mRNA, may comprise a ribonucleotide sequence encoding a polypeptide or protein of the Cas (CRISPR-associated protein) family of proteins, preferably Cas9 (CRISPR-associated protein 9). Proteins of the Cas family, preferably Cas9, can be used in CRISPR/Cas9-based methods and/or in CRISPR/Cas9-based genome editing technologies. CRISPR/Cas systems for genome editing, genome regulation, and genome targeting are described in a review article by Nat. Biotechnol. 32(4):347–355 (2014).

In another preferred embodiment of the present invention, the RNA, preferably mRNA, may contain a ribonucleotide sequence encoding a meganuclease. Meganucleases are endodeoxyribonucleases that, unlike “conventional endodeoxyribonucleases,” recognize a large recognition site (eg, a double-stranded DNA sequence of 12 to 40 base pairs). As a result, the corresponding site in any given genome exists only a few times, preferably once. Consequently, meganucleases are considered to be the most specific restriction enzymes existing in nature, and accordingly are suitable tools in genome editing technologies.

In yet another preferred embodiment, the RNA, preferably mRNA, contains a ribonucleotide sequence encoding a zinc finger nuclease (ZFN). ZFNs are unnatural restriction enzymes produced by hybridization of a DNA binding zinc finger domain and a DNA cleavage domain. Zinc finger domains can be designed to target specific DNA sequences of interest, allowing zinc finger nucleases to target unique sequences in complex genomes. By taking advantage of the endogenous DNA repair mechanism, ZFNs can be used to precisely modify the genome of higher organisms and hence these nucleases are useful tools in genome editing technologies.

In another preferred embodiment, the RNA, preferably mRNA, may comprise a ribonucleotide sequence encoding a transcription activator-like effector nuclease (TALEN). TALEN nucleases are restriction enzymes that can be engineered to cleave specific DNA sequences. TALEN nucleases are fusion proteins in which the DNA-binding domain of a TAL effector is coupled to a DNA-cleaving domain of a nuclease. Transcription activator-like effectors (TALEs) can be designed to bind virtually any desired DNA sequence. Thus, when coupled with a nuclease, DNA can be cleaved at the specific positions required.

In yet another of the above embodiments, the RNA contains a ribonucleotide sequence that is not intended to be expressed as a protein or polypeptide. Thus, it should be understood that the term RNA does not only mean any polynucleotide sequence that, when incorporated into a cell, is transcribed into a polypeptide/protein or fragment thereof. In contrast, it is also assumed that RNA contains a ribonucleotide sequence that is transcribed only into (functional) RNA, with said RNA being the final product (and therefore no translation required). In this context, the RNA is contemplated to contain a ribonucleotide sequence that preferably provides the genetic information for the siRNA sequence or other desired ribonucleotide sequence.

It should be understood that particles for use in the context of the present invention may include a single type of RNA, but may alternatively include a combination of two or more types of RNA, for example, in the form of particles comprising two or more types of RNA, in a single particle, or in the form mixtures of particles that differ in the type of RNA they contain.

Lipid composition

The composition according to the first aspect of the present invention and the solid composition according to the third aspect of the present invention include particles that include RNA and a lipid composition.

The lipid composition includes (i-a) a cholesterol derivative of formula (I) or a salt thereof, (i-b) a phosphoglycerol of formula (II) or a salt thereof, and (i-c) a PEGylated phosphoglycerol of formula (III) or a salt thereof. Additional lipid components may be present, however, the lipid components in the lipid composition contained in the particles are preferably only components (i-a), (i-b) and (i-c).

Component (i-a) of the lipid composition is a cholesterol derivative of formula (I) or a salt thereof:

Where

n is 0 or 1,

R ¹ means a -(CH ₂ ) _q -NH ₂ group or a -(CH ₂ ) _r -NH-(CH ₂ ) _s -NH ₂ group,

where q, r and s are independently equal to an integer from 2 to 6,

R ² means the group -(CH ₂ ) _t -NH ₂ or -(CH ₂ ) _u -NH-(CH ₂ ) _w -NH ₂ ,

where t, u and w are independently equal to an integer from 2 to 6,

R ³ means a linear alkanediyl group containing from 1 to 4 carbon atoms.

Compounds of this formula and their preparation are described, for example, in US patent 5783565.

The integer n in Formula I is preferably 0.

The integers q, r, s, t, u and w are preferably selected from 3 and 4.

Preferably also R ¹ is a -(CH ₂ ) _q -NH ₂ group and R ² is a -(CH ₂ ) _t -NH ₂ group or a -(CH ₂ ) _u -NH-(CH ₂ ) _w -NH _{2 group} , or R ² means a -(CH ₂ ) _t -NH ₂ group, and R ¹ means a -(CH ₂ ) _q -NH ₂ group or a -(CH ₂ ) _r -NH-(CH ₂ ) _s -NH _{2 group} . In both cases, n is also preferably 0.

Salt forms of the cholesterol derivative of formula (I) can be obtained by protonating with an acid one or more amino groups contained in the compound so that the compound carries a cationic charge. It should be understood that a protonated amino group means a group where an additional hydrogen atom is attached to a nitrogen atom in the amino group, resulting in the formation of a tetravalent nitrogen atom bearing a cationic charge. Suitable nitrogen atoms in the compound of formula (I) are the nitrogen atoms contained in the -N(R ¹ )(R ² ) group and the nitrogen atoms contained in the R ¹ and R ² groups, respectively. It should be understood that the anion acting as a counterion for each protonated amino group is generally a physiologically acceptable anion. In the compositions of the present invention, the compound of formula (I) typically forms a salt with acid groups in the RNA. However, other counterions are not excluded, and examples thereof include chloride, bromide, iodide, sulfate, nitrate, phosphate, hydrogen phosphate, dihydrogen phosphate, carbonate and hydrogen carbonate anions.

It will be apparent to one skilled in the art that the compound of formula (I) as a cholesterol derivative can be represented by the preferred formula (Ia):

or even more preferably formula (Ib):

In formulas (Ia) and (Ib), the definitions and preferred definitions of the groups R ¹ , R ² , R ³ and n are presented above with respect to formula (I).

The most preferred component (ia) of the lipid composition is GL67 or a salt thereof, wherein with respect to formula (I), (Ia) and (Ib), n is 0, R ¹ is the group -(CH ₂ ) ₃ -NH ₂ and R ² means the group -(CH ₂ ) ₄ -NH-(CH ₂ ) ₃ -NH ₂ .

Component (i-b) of the lipid composition is a phosphoglyceride of formula (II) or a salt thereof:

Where

R ⁴ means a linear alkyl group containing from 10 to 24 carbon atoms, or a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 24 carbon atoms,

R ⁵ means a linear alkyl group containing from 10 to 24 carbon atoms, or a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 24 carbon atoms.

Preferably, R ⁴ and R ⁵ each represent a linear alkyl group containing from 14 to 20 atoms, or each represent a linear alkenyl group containing one or two double bonds and from 14 to 20 carbon atoms. More preferably, R ⁴ and R ⁵ each represent a linear alkenyl group containing one double bond and from 14 to 20 carbon atoms.

Salt forms of the compound of formula (II) include internal salt forms wherein the acid group proton -OH attached to the phosphorus atom P as shown in formula (II) protonates the amino group shown in that formula. Such salt forms also include salts formed by an acidic -OH group with a base, or salts formed by an amino group with an acid. Again, it should be understood that salts are generally salts that are pharmaceutically acceptable. As base salts, mention should be made of alkali metal salts such as sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, or ammonium salts. As typical salts formed with an acid, mention should be made of the salt formed with the acid groups of RNA, but other salts are not excluded, and as typical salts, salts of inorganic acids such as chloride, bromide or iodide, sulfates, nitrates, phosphates, hydrophosphates or dihydrogen phosphates, carbonates and bicarbonates.

The most preferred component (i-b) of the lipid composition is 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE) or a salt thereof.

Component (i-c) of the lipid composition is a pegylated phosphoglyceride of formula (III) or a salt thereof:

Where

p is equal to an integer from 5 to 200, preferably from 10 to 170 and most preferably from 10 to 140,

R ⁶ means a linear alkyl group containing from 10 to 20 carbon atoms, or a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 20 carbon atoms,

R ⁷ means a linear alkyl group containing from 10 to 20 carbon atoms, or a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 20 carbon atoms.

Preferably, R ⁶ and R ⁷ each represent a linear alkyl group containing from 10 to 20 atoms, more preferably, preferably R ⁶ and R ⁷ each represent a linear alkyl group containing from 10 to 16 atoms.

Thus, also preferably in formula (III), R ⁶ and R ⁷ each represent a linear alkyl group containing from 10 to 16 atoms, and p is an integer from 10 to 140.

Salt forms of the compound of formula (III) are generally pharmaceutically acceptable salts. Typical salts of the compound of formula (III) are salts formed with a base, and mention should be made, for example, of alkali metal salts such as sodium or potassium salts, alkaline earth metal salts such as calcium or magnesium salts, and ammonium salts.

Strictly preferred component (ic) in the lipid composition is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG (DMPE-PEG) or a salt thereof, wherein the PEG (polyethylene glycol) moiety contains from 10 to 140 repeating units, i.e. in this strictly preferred embodiment of the present invention, according to formula (III), R ⁶ and R ⁷ each represent a linear alkyl group containing 13 atoms, and p is equal to an integer from 10 to 140. The most preferred component (ic) is 1,2 -dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG5000 (DMPE-PEG5000) or a salt thereof, where 5000 represents the number average molecular weight of the PEG moiety, corresponding to an average p-value of approximately 113.

Thus, it should be understood that a primarily preferred lipid composition for use in the context of the present invention is one in which:

component (i-a) is GL67 or its salt,

component (i-b) is 1,2-dioleyl-sn-glycero-3-phosphoethanolamine-PEG (DOPE) or a salt thereof, and

component (i-c) is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG (DMPE-PEG) or a salt thereof, wherein the PEG (polyethylene glycol) moiety contains from 10 to 140 repeating units, and more preferably 1,2 -dimyristoyl-5-glycero-3-phosphoethanolamine-PEO5000 (DMPE-PEG5000) or its salt.

In the lipid composition contained in the particles of the present invention, the molar ratio of components (i-a), (i-b) and (i-c), that is, (i-a)/(i-b)/(i-c), is preferably 1:(0.5 to 5):(0.01 to 1), more preferably 1:(1 to 5):(0.01 to 0.5) and most preferably 1:(1 to 3):(0.02 up to 0.2).

The lipid composition preferably contains components (i-a), (i-b) and (i-c), which are lipid components only.

As stated above, a strictly preferred composition according to the first aspect of the present invention is a composition which includes:

(i) particles contained in a liquid phase, where the particles include mRNA and a lipid composition, and where the lipid composition includes:

(i-a) GL67 or its salt,

(i-b) 1,2-dioleyl-sn-glycero-3-phosphoethanolamine-PEG (DOPE) or its salt,

(i-c) 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG (DMPE-PEG) or a salt thereof, wherein the PEG (polyethylene glycol) moiety contains from 10 to 140 repeating units, and more preferably 1,2-dimyristoyl- sn-glycero-3-phosphoethanolamine-PEG5000 (DMPE-PEG5000) or its salt,

in the molar ratio of components (i-a)/(i-b)/(i-c), 1:(1 to 3):(0.02 to 0.2) and

(ii) 2-propanediol.

Likewise, a strongly preferred embodiment of the third aspect of the present invention relates to a solid composition comprising (i) particles comprising RNA and the above lipid composition, and (ii) 1,2-propanediol, wherein the solid composition is prepared by freezing the above strongly preferred composition.

Particles

The composition according to the first aspect of the present invention and the solid composition according to the third aspect of the invention include particles comprising RNA and a lipid composition as described above.

The particles typically include nanoparticles comprising RNA and a lipid composition, or microparticles comprising RNA and a lipid composition. As will be apparent to one skilled in the art, the conjunction “or” is used in this context in a non-exclusive manner unless specifically stated otherwise. Thus, reference to nano- or microparticles includes compositions containing nanoparticles, compositions containing microparticles, and compositions containing both types of particles, nanoparticles and microparticles. The term nanoparticles as used in this context generally refers to particles with a diameter in the nanometer range, that is, a diameter ranging from 1 nm or more to less than 1000 nm. The term microparticles as used herein generally refers to particles with a diameter in the micrometer range, that is, a diameter of 1000 nm or more to 100 μm or less. Preferably, the particles consist of nanoparticles comprising RNA and a lipid composition, or microparticles comprising RNA and a lipid composition.

The particles contained in the compositions of the present invention preferably have an average particle diameter in the range of 1 to 5000 nm, more preferably 10 to 4000 nm, and most preferably 50 to 3000 nm.

The upper limit of the diameter of individual particles in the compositions of the present invention is preferably 20 μm, more preferably 10 μm, and most preferably 5 μm. Thus, as is apparent from the above, a strictly preferred particulate composition is characterized by an average particle diameter in the range of 50 to 3000 nm, and a maximum particle diameter of 5 μm.

Particle diameters and the average diameter of nano- or microparticles in the composition described in this context can be determined by a simple method using dynamic light scattering (DLS). Typically, the particle diameters and average diameters described in this context are referred to as the suspended hydrodynamic particle diameters, which are determined by dynamic light scattering. Since the effect of temperature is taken into account when measuring on a device (eg Malvern ZetaSizer) and when presenting the results, the measured diameters are largely independent of temperature. However, the measurement is usually carried out at room temperature (25°C). As a suspension medium for measuring DLS, if necessary, you can use, for example, water or water containing 1,2-propanediol. In the case of a frozen solid composition, particle diameters are usually determined after thawing the composition. In cases where average particle size or average particle diameter is specified, the average usually means z-average unless otherwise noted.

In the particles contained in the compositions of the present invention, the acidic groups of the RNA typically protonate the amino groups contained in the lipid composition so that the anionic RNA molecules and the cationic lipid molecules can interact, preferentially resulting in the formation of complexes of RNA molecules and lipid molecules.

The N/P ratio, that is, the ratio of the number of nitrogen atoms of the cholesterol derivative of formula (I) in the lipid composition to the number of P atoms of the phosphate groups in the RNA, is preferably in the range of 1 to 100, more preferably 1 to 30, and most preferably 2 until 20.

Preferably, the particles are in the form of liposomes or lipid nanoparticles.

In view of the above, a strictly preferred composition according to the first aspect of the present invention is a composition which includes:

(i) nano- or microparticles contained in a liquid phase, where the nano- or microparticles include mRNA and a lipid composition, and where the lipid composition includes:

(i-a) GL67 or its salt,

(i-b) 1,2-dioleyl-sn-glycero-3-phosphoethanolamine-PEG (DOPE) or its salt,

(i-c) 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG (DMPE-PEG) or a salt thereof, wherein the PEG (polyethylene glycol) moiety contains from 10 to 140 repeating units, and more preferably 1,2-dimyristoyl- sn-glycero-3-phosphoethanolamine-PEG5000 (DMPE-PEG5000) or its salt,

in the molar ratio of components (i-a)/(i-b)/(i-c), 1:(from 1 to 3):(from 0.02 to 0.2), and where

the N/P ratio, that is, the ratio of the number of nitrogen atoms N in the cholesterol derivative of formula (I) in the lipid composition to the number of P atoms of phosphate groups in the mRNA, is in the range from 2 to 20, and

(ii) 1,2-propanediol.

Likewise, a strictly preferred embodiment of the present invention according to the third aspect relates to a solid composition comprising (i) nano- or microparticles comprising RNA and the lipid composition specified in the previous paragraph, and (ii) 1,2-propanediol, wherein said the solid composition is obtained by freezing the above strictly preferred composition.

In addition to the RNA and lipid composition, the particles in the compositions of the present invention may include one or more additional components, for example, excipients or additives, which are typically pharmaceutically acceptable components. For example, such additional components may facilitate transport to specific sites or accelerate the subsequent uptake of particles into specific sites after they are administered to a patient, or they may help stabilize the particles or the therapeutically active agent contained therein.

In addition to the RNA and lipid composition, the particles contained in the compositions of the first and third aspects of the present invention may include, as an optional additive or additives, one or more components that exhibit an effector function during the delivery of the therapeutic agent, and preferably during delivery RNA as a therapeutic agent into the cell. Such components may include, but are not limited to, polyanions, other lipids, shielding oligomers or polymers, poloxamers (also known as pluronics), poloxamines, targeting ligands, endosomolytic agents, cell penetrating agents and signal peptides, magnetic and non-magnetic nanoparticles , RNase inhibitors, fluorescent dyes, radioactive isotopes or contrast agents for diagnostic imaging. The term "effector function" means any function that contributes to the desired biological effect of the therapeutically active agent in the composition exerted near or within the biological target or on the environment of the biological target. For example, nucleic acid delivery compositions have been developed to include non-coding nucleic acids or non-nucleic acid polyanions as “excipient materials” (Kichler et al., J Gene Med, 7, 1459-1467 (2005). Such excipient materials are useful for reducing the dose of nucleic acid required to produce a desired biological effect while maintaining the degree or level of that effect obtained with a higher dose of nucleic acid in the absence of such excipient material.Non-nucleic acid polyanions are also used to achieving prolonged gene expression in vivo with reduced toxicity (see Uchida et al., J Control Release, 155, 296-302 (2011)).

Other typical shielding polymers described in the literature that can be used as components of a particulate formulation containing a complex of ribonucleic acid with a cationic excipient include hydroxyethyl starch (HES: see Noga et al., Journal of Controlled Release, 159(1), 92-103 (2012)), PAS/PA polypeptide (Pro, Ala, Ser (or Ala, Ser) polypeptide), see Schlapschy et al., Protein Eng Des Sel. 26(8), 489-501 (Aug, 2013)) or polysarcosine (Psar: see Heller et al., Macromol Biosci, 14, 1380-1395 (2014)).

Targeting ligands can be used, for example, in particulate formulations for the delivery of ribonucleic acid for the purpose of selective and enhanced transfection of target cells (see the chapter by Philipp and Wagner in the book "Gene and Cell Therapy Therapeutic Mechanisms and Strategy", 3rd ed. , Chapter 15. CRC Press, Taylor & Francis Group LLC, Boca Raton (2009)). A targeting ligand can be any compound that provides the compositions of the present invention with the function of recognizing a target and/or binding to a target directly or indirectly. Typical targeting ligands are prostacyclin analogs disclosed in international patent WO 2011/076391, such as iloprost or treprostinil. The antibody may also act as a targeting ligand. As ligands for the particles, mention may be made of folic acid and N-acetylgalactosamine. In a broader sense, a target is a discrete biological entity to which a targeting ligand can bind specifically through molecular interaction, and where such binding will ultimately result in the selective accumulation of a therapeutically active agent, such as a formulated nucleic acid, in tissue. target and/or in the target cell or its environment. Targeting ligands can be associated, for example, with the terminal residues of the shielding polymer.

In addition, endosomolytic agents such as endosomolytic peptides (Plank et al., Adv Drug Deliv Rev, 34, 21-35 (1998)) or any other compound intended to enhance endosomal release are useful components of the compositions of the present invention. endocytosed nucleic acid. Similarly, useful components of the composition of the present invention as mediators of intracellular delivery of nucleic acid into the cell are cell penetrating peptides (in another context also known as protein transduction domains) (see Lindgren et al., Trends Pharmacol Sci, 21, 99 -103 (2000)). The so-called TAT peptide falls into this classification and also functions to promote intranuclear localization (Rudolph et al., J Biol Chem, 278, 11411-11418 9999 (2003).

The particles are preferably characterized by an active substance content expressed as the weight of RNA as active substance relative to the total weight of particles in the particle composition, ranging from 0.1 to 95 wt. %, more preferably from 0.5 to 80 wt. %, most preferably from 1 to 50 wt. %.

Compositions Comprising Particles and 1,2-Propanediol The composition of the first aspect of the present invention and the solid composition of the third aspect of the present invention include particles comprising RNA and a lipid composition mixed with 1,2-propanediol. It should be understood that the information presented above with respect to suitable and preferred RNA variants, lipid compositions and particles containing them also applies in the context of the following discussion.

Since the composition according to the first aspect of the present invention contains RNA as a therapeutically active agent and is intended for administering a therapeutically active agent to a patient, it can be called a therapeutic composition or a pharmaceutical composition.

In the composition of the first aspect of the present invention, which includes particles comprising RNA and a lipid composition in a liquid phase, the particles comprising RNA and a lipid composition are preferably dispersed in a liquid phase. When the term "dispersed" is used, it is meant that the particles form a dispersed phase in a continuous liquid phase. It is generally preferred that the dispersion is a biphasic dispersion with one continuous liquid phase and particles containing the RNA and the lipid composition dispersed therein as a dispersed phase.

The composition according to the first aspect of the present invention, in which particles comprising RNA and a lipid composition are contained in a liquid phase, preferably includes particles in an amount that provides a concentration of RNA in the particles from 0.01 to 50 mg/ml, more preferably from 0.02 up to 30 mg/ml and most preferably from 0.05 to 10 mg/ml based on the total volume of the composition.

Typically, 1,2-propanediol is contained as an additional component in the liquid phase of the composition according to the first aspect of the present invention, which contains particles comprising RNA and a lipid composition, preferably in a suspended state. The 1,2-propanediol is preferably dissolved in the liquid phase. However, it may be in associated form with particles contained in the liquid phase.

In the composition of the first aspect of the present invention, which includes particles contained in a liquid phase, 1,2-propanediol is preferably present in a concentration of from 0.1 to 50 w/v. %, more preferably from 0.2 to 30 wt./vol. %, most preferably from 0.3 to 20 wt./vol. %, where the percentage value means the mass of 1,2-propanediol in grams per 100 ml of the total volume of the composition.

The liquid phase in which the particles are contained, preferably in a dispersed state, usually contains water as a solvent. Water provides preferably 50% or more, most preferably 70% or more of the volume (based on the total volume of the liquid phase at 20°C). More preferably, water and 1,2-propanediol are the only solvents contained in the liquid phase.

As typical, additional, optional additives in the liquid phase composition, one or more additives selected from salts, sugars, organic solvents and buffers may be mentioned. As buffer substances, substances that are commonly used in pharmaceutical or biological applications can be selected.

The solid composition of the third aspect of the present invention contains the same components as the composition that can be frozen to form a solid composition. Thus, the information presented above with respect to suitable and preferred embodiments of RNA, lipid composition, particles containing RNA and lipid composition, 1,2-propanediol and liquid phase and components thereof also applies equally to a composition comprising a liquid phase, and to a solid composition. However, it will be apparent to one skilled in the art that a liquid phase may be present in a solid composition. Accordingly, the solid composition preferably contains a dispersion of the particulate therapeutic agent composition within a solid continuous phase of the frozen composition. In connection with the above, 1,2-propanediol is preferably contained in a continuous phase in which the particulate composition is dispersed.

In accordance with the above, a strictly preferred composition according to the first aspect of the invention is a composition which includes:

(i) nano- or microparticles dispersed in a liquid phase comprising water as a solvent, wherein the nano- or microparticles comprise mRNA and a lipid composition, wherein the lipid composition includes:

(i-a) GL67 or its salt,

(i-b) 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE),

(i-c) is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG (DMPE-PEG) or a salt thereof, wherein the PEG (polyethylene glycol) moiety contains from 10 to 140 repeating units, and more preferably 1,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG5000 (DMPE-PEG5000) or its salt,

in the molar ratio of components (i-a)/(i-b)/(i-c) 1: (from 1 to 3): (from 0.02 to 0.2),

and where the N/P ratio, that is, the ratio of the number of nitrogen atoms N in the cholesterol derivative of formula (I) in the lipid composition to the number of phosphate groups P in the mRNA, is 2:1, and

(ii) 1,2-propanediol as an additional component in the liquid phase in which nano- or microparticles are suspended,

and where the composition contains 1,2-propanediol in an amount of from 0.3 to 20 wt./vol. % 1,2-propanediol based on the total volume of the composition.

Likewise, a strictly preferred embodiment of the third aspect of the present invention relates to a solid composition comprising (i) nano- or microparticles containing RNA and the lipid composition described in the preceding paragraph, and (ii) 1,2-propanediol, which solid composition obtained by freezing the above strictly preferred composition.

Application in pharmaceutical field

As stated above, the RNA molecules, preferably mRNA, that are present in the compositions of the present invention, as defined above, are primarily useful in medical settings and in the treatment of certain diseases, and especially in RNA-based therapies. Thus, the compositions of the present invention comprising RNA molecules are useful as pharmaceutical compositions.

First of all, the compositions of the first aspect of the present invention are suitable for administration to a subject. Thus, RNA, especially mRNA, contained in such compositions can also be delivered into the body of a subject. The preferred route of administration of the composition is into or through the respiratory tract, especially pulmonary or nasal administration.

However, it will be apparent to one skilled in the art that the composition of the first aspect of the present invention can also be administered by other routes of administration known in the art for particulate formulations of a therapeutically active agent, such as intravenous administration in the form of a dispersion.

Devices for forming an aerosol form from a composition comprising particles contained in a liquid, or for spraying such a composition, are known in the art, and such devices are commercially available. They can be used to administer the composition of the first aspect of the invention into or through the respiratory tract, especially the lungs. For nasal administration, for example, a nasal spray or nasal infusion device can be used.

Thus, another aspect of the invention relates to a device for forming an aerosol from a particulate composition contained in a liquid or for spraying such a composition, wherein said device includes a composition according to the first aspect of the invention. The device is preferably an inhaler selected from a metered dose inhaler, a nebulizer and a nasal spray device.

By such administration to a subject, the RNA contained in the particles can be delivered to target cells through or through the respiratory tract. The term "delivered to target cells" preferably means the transfer of RNA, preferably single-stranded RNA, such as mRNA, into a cell.

The composition can be administered to a subject at a suitable dose. The dosage regimen is determined by the attending physician and clinical factors. As is well known in the medical field, dosages for any subject depend on many factors, including the subject's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and concurrent administration of other drugs. A typical dose of therapeutically active substances may, for example, range from 1 ng to several grams. In the preferred case of mRNA therapy, dosages of mRNA to produce expression or inhibit expression should fall within this range, but dosages below or above this typical range are contemplated, primarily taking into account the above-mentioned factors. Typically, the regimen for regular administration of a pharmaceutical composition should be in the range of 0.01 μg to 10 mg per kilogram of body weight per day. Accordingly, if the dosage regimen involves a continuous infusion, the dose should also be in the range of 1 mcg to 10 mg per kilogram of body weight. The progress of treatment can be monitored with periodic evaluation. Dosages may vary, but the preferred dosage for administering the mRNA as components of the composition of the present invention is approximately 10 ⁶ to 10 ¹⁹ copies of the mRNA molecule.

The present invention also relates to a method of treatment comprising administering a composition according to the first aspect of the invention to a patient, preferably by administration into or through the respiratory tract, more preferably using respiratory or nasal administration. Thus, the RNA contained in said composition may have a prophylactic or therapeutic effect. It should be noted that the term "patient" includes animals and humans.

By administering the composition of the present invention, especially according to the first aspect of the present invention, it is possible to treat or prevent diseases. The term “disease” refers to any putative pathological condition that can be treated, prevented or vaccinated against using the composition of the present invention. These diseases may, for example, be hereditary, acquired, infectious or non-infectious, age-related, cardiovascular, metabolic, intestinal, tumor (especially cancer) or genetic diseases. The basis of the disease may be, for example, violations of physiological processes, molecular processes, biochemical reactions in the body, which, in turn, may be based, for example, on the genetic apparatus of the body, behavioral, social or environmental factors, such as exposure to chemicals or radiation .

The terms "treatment" or "cure" as used in this context generally mean achieving the desired pharmacological and/or physiological effect. Accordingly, the treatment of the present invention may relate to the treatment of (acute) conditions of a particular disease, but may also relate to prophylactic treatment in relation to the complete or partial prevention of a disease or symptom thereof. Preferably, the term "treatment" should be understood as a therapeutic effect with respect to partial or complete cure of the disease and/or side effects and/or symptoms associated with the disease. In this regard, the term "acute" means that the subject is experiencing symptoms of the disease. In other words, the subject who needs treatment actually requires treatment, and the term "acute treatment" in the context of the present invention refers to measures taken to actually treat the disease after the onset of the disease or symptoms of the disease. Treatment may also be prophylactic or preventative measures, that is, measures taken to prevent disease, for example, to prevent infection and/or the onset of disease. The progress of treatment can be monitored with periodic evaluation.

Generally, RNA, preferably mRNA, is included in an effective amount in the composition of the present invention. The term "effective amount" refers to an amount sufficient to induce a detectable therapeutic response in a subject to whom administration of the pharmaceutical composition is prescribed. In accordance with the above, the content of RNA, preferably mRNA, in the pharmaceutical composition according to the above-mentioned first aspect of the present invention is not limited as long as it is suitable for treatment as described above. As stated above, the composition of the first aspect of the invention, in which particles comprising RNA and a lipid composition are contained in a liquid phase, preferably includes particles in an amount sufficient to provide a concentration of RNA contained in the particles at a level of from 0.01 to 50 mg/ml , more preferably from 0.02 to 30 mg/ml and most preferably from 0.05 to 10 mg/ml based on the total volume of the composition.

According to the present invention, the term "pharmaceutical composition" refers to the composition of the present invention, especially the composition of the first aspect of the present invention, intended for administration to a subject. Typical subjects include a mammal such as a dog, cat, pig, cattle, sheep, horse, rodent such as a rat, mouse and guinea pig, or a primate such as a gorilla, chimpanzee and human. In the most preferred embodiment of the present invention, the subject is a human.

The pharmaceutical composition of the present invention can be used in RNA-based therapy. As stated above, the RNA molecule, preferably an mRNA, includes a protein coding sequence and accordingly can be used in RNA-based therapy, wherein the RNA, preferably the mRNA, encodes a therapeutically or pharmaceutically active polypeptide or protein having a therapeutic or prophylactic effect. Thus, in preferred embodiments, the pharmaceutical composition of the invention can be used in RNA-based therapy in the treatment or prevention of the disease listed above in table. 2. Accordingly, the RNA-based therapy of the present invention can be used in treatment or prevention as indicated in Table 1 above. 2.

Thus, the pharmaceutical composition of the present invention can be used in RNA-based therapy in cases where the defective genes described in the table above. 2, lead to a disease that can be treated or prevented using transcript replacement/enzyme replacement therapy using an RNA molecule, preferably an mRNA, of the present invention, where the RNA molecule encodes a full-length protein variant or a functional fragment thereof that compensates for the described defective gene.

In other embodiments, the pharmaceutical composition of the present invention can be used in the RNA-based therapy of the present invention, wherein the RNA, preferably mRNA, encodes a therapeutically or pharmaceutically active polypeptide, protein or peptide having a therapeutic or prophylactic effect, wherein said polypeptide, protein or peptide is selected from group encoded by genes, as indicated in table. 2.

The pharmaceutical composition of the present invention is primarily suitable for use in RNA-based therapy in the treatment or prevention of lung diseases. Typical diseases include alpha-1 antitrypsin deficiency, asthma, cystic fibrosis, dysfunction of surfactant metabolism, or primary ciliary dyskinesia, as listed in Table 1 above. 2.

In other exemplary embodiments, the pharmaceutical composition of the present invention can be used in RNA-based therapy in the treatment or prevention of lysosomal diseases such as Gaucher disease, Fabry disease, mucopolysaccharidosis (MPS) I, mucopolysaccharidosis II (Gunther syndrome), mucopolysaccharidosis VI and diseases glycogen storage disease, such as glycogen storage disease type I (von Gierke disease), type II (Pompe disease), type III (Measles disease), type IV (Andersen disease), type V (McArdle disease), type VI (Hers disease), type VII (Taury disease), type VIII, type IX, type X, type XI (Fanconi-Bickel syndrome), type XI or type 0. Transcript replacement therapy/enzyme replacement therapy does not have a beneficial effect on genetic defect that causes the disease, but increases the concentration of the enzyme, the deficiency of which is observed in the patient. For example, in Pompe disease, transcript replacement therapy/enzyme replacement therapy restores the concentration of a deficient lysosomal enzyme, alpha-glucosidase acid (GAA).

According to additional examples, the RNA-based therapeutic regimens of the present invention can be used to treat cancer, cardiovascular disease, viral infection, immune dysfunction, autoimmune disease, neurological disorder, inherited metabolic disorder or genetic disorder, or any disease where in which a protein or protein fragment produced in a cell can have a beneficial effect on a patient. Examples of cancer include head and neck cancer, breast cancer, kidney cancer, bladder cancer, lung cancer, prostate cancer, bone cancer, brain cancer, cervical cancer, anal cancer, colon cancer, colorectal cancer, appendix cancer , eye cancer, stomach cancer, leukemia, lymphoma, liver cancer, skin cancer, ovarian cancer, penile cancer, pancreatic cancer, testicular cancer, thyroid cancer, vaginal cancer, vulvar cancer, endometrial cancer, heart cancer and sarcoma. Examples of cardiovascular diseases include atherosclerosis, coronary heart disease, pulmonary heart disease, and cardiomyopathy. Examples of immune dysfunctions and autoimmune diseases include, but are not limited to, rheumatic diseases, multiple sclerosis and asthma. Examples of viral infections include, but are not limited to, infections caused by human immunodeficiency virus, herpes simplex virus, human papillomavirus, and hepatitis B and C viruses. Examples of neurological disorders include, but are not limited to, Parkinson's disease, multiple sclerosis and dementia. Examples of inherited metabolic disorders include, but are not limited to, Gaucher disease and phenylketonuria.

Methods of obtaining

Suitable methods for preparing particles comprising a nucleic acid and a lipid composition in a liquid phase are known to those skilled in the art, and these methods can be optimized to produce compositions according to the first aspect of the present invention, where the particles include RNA in admixture with the lipid composition described above.

For example, particulate liposomes comprising RNA and a lipid composition can be prepared in a simple manner by rehydrating the lipid composition, that is, rehydrating the lipid films as necessary, followed by homogenization techniques such as sonication or extrusion. An alternative approach is to infuse the lipid composition dissolved in an organic solvent into water or an aqueous solution. A typical method that can be used, for example, to form lipid nanoparticles is the solvent displacement method.

As an alternative method for preparing the compositions of the invention where the particles are contained in a liquid phase, mention may be made of precipitation methods such as nanoparticle precipitation.

1,2-Propanediol can be added in a simple manner to the liquid phase containing the particles comprising the RNA and lipid composition, or which is intended to produce the particles. The addition of 1,2-propanediol to the liquid phase can be accomplished at one or more different stages, including steps before, during or after the preparation of particles containing the RNA and lipid composition in the liquid phase.

Thus, as stated above, another aspect of the invention relates to a method for preparing a composition according to the first aspect of the invention, said method comprising the following steps:

a) dissolving and mixing the components of the lipid composition in an organic solvent, followed by lyophilization of the lipid composition,

b) rehydrating the lyophilized lipid composition by adding water,

c) combining the rehydrated lipid composition with an aqueous solution of RNA, thereby forming particles comprising the RNA and the lipid composition, which are contained in a liquid phase, and

d) adding 1,2-propanediol.

The addition of 1,2-propanediol can be done in a simple manner, for example, by adding it simultaneously with water in step b), by adding it to the rehydrated lipid composition after step b), by adding it simultaneously with an aqueous solution of RNA in step c), or when it is added to the liquid phase after step c), which contains particles including RNA and a lipid composition. It should be understood that the addition can be carried out at one or more stages, for example, at different stages of the process.

The solid composition of the third aspect of the invention can be prepared by a method comprising:

the first step of producing a composition according to the first aspect of the invention described above by a method including

a) dissolving and mixing the components of the lipid composition in an organic solvent, followed by lyophilization of the lipid composition,

b) rehydrating the lyophilized lipid composition by adding water,

c) combining the rehydrated lipid composition with an aqueous solution of RNA, thereby forming particles comprising the RNA and the lipid composition, which are contained in a liquid phase, and

d) adding 1,2-propanediol,

and a second step of freezing the composition obtained in the first step.

In this method, the addition of 1,2-propanediol can also be carried out in a simple way, for example, by adding simultaneously with water in step b), by adding 1,2-propanediol simultaneously with an aqueous solution of RNA in step c), or by adding it in step c) into the liquid phase, which contains particles containing RNA and a lipid composition. It should be understood that the addition can be carried out at one or more stages, for example, at different stages of the process.

The freezing step as a second step is typically carried out by exposing the suspension composition to sufficiently low temperatures (eg -10°C or less, preferably -20°C or less) in a suitable container. The cooling medium can be, for example, cooled air or cooled liquids.

In this regard, as discussed above, the solid composition of the third aspect of the present invention is a composition in which particles including RNA can be stored. In this case, it should be understood that the composition of the first aspect of the invention can also be recovered from the solid composition of the third aspect of the invention when the solid composition is thawed.

Thus, in another aspect of the present invention there is also provided a method for preparing a composition according to the above first aspect, said method comprising:

the first step of obtaining a composition according to the above first object by a method including:

a) dissolving and mixing the components of the lipid composition in an organic solvent, followed by lyophilization of the lipid composition,

b) rehydration of the lipid composition by adding water,

c) combining the rehydrated lipid composition with an aqueous solution of RNA to form particles comprising the RNA and the lipid composition that are contained in a liquid phase, and

d) adding 1,2-propanediol,

a second step of freezing the composition obtained in the first step; and a third step of thawing the frozen composition obtained in the second step.

A summary of the important aspects of the invention is presented in the following paragraphs. It should be understood that the elements presented in these paragraphs form part of the general disclosure of the present invention, so that information presented in the previous part of the description, for example, regarding additional preferred embodiments or additional features, also applies to the elements described in the following paragraphs.

1. Composition including

(i) particles contained in a liquid phase, where the particles include RNA and a lipid composition, and where the lipid composition includes:

(i-a) a cholesterol derivative of formula (I) or a salt thereof:

Where

n is 0 or 1, preferably 0,

R ¹ means a -(CH ₂ ) _q -NH ₂ group or a -(CH ₂ ) _r -NH-(CH ₂ ) _s -NH ₂ group,

where q, r and s are independently equal to an integer from 2 to 6,

R ² means the group -(CH ₂ ) _t -NH ₂ or -(CH ₂ ) _u -NH-(CH ₂ ) _w -NH ₂ ,

where t, u and w are independently equal to an integer from 2 to 6,

R ³ means a linear alkanediyl group containing from 1 to 4 carbon atoms,

(i-b) phosphoglyceride of formula (II) or a salt thereof:

Where

R ⁴ means a linear alkyl group containing from 10 to 24 carbon atoms, or a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 24 carbon atoms,

R ⁵ means a linear alkyl group containing from 10 to 24 carbon atoms, or a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 24 carbon atoms.

(i-c) pegylated phosphoglyceride of formula (III) or a salt thereof:

Where

p is equal to an integer from 5 to 200, preferably from 10 to 170 and most preferably from 10 to 140,

R ⁶ means a linear alkyl group containing from 10 to 20 carbon atoms, or a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 20 carbon atoms,

R ⁷ means a linear alkyl group containing from 10 to 20 carbon atoms, or a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 20 carbon atoms,

And

(ii) 1,2-propanediol.

2. The composition according to claim 1, where the RNA is mRNA.

3. The composition according to claim 1 or claim 2, where in formula (I), n is 0, and R ¹ means a -(CH ₂ ) ₃ -NH ₂ group, and R ² means a -(CH ₂ ) ₄ - group NH-(CH ₂ ) ₃ -NH ₂ .

4. Composition according to claim 1 or claim 2, wherein the cholesterol derivative of formula (I) is GL67.

5. The composition according to any one of claims 1 to 4, wherein in formula (II), R ⁴ and R ⁵ each means a linear alkenyl group containing one double bond and from 14 to 20 carbon atoms.

6. The composition according to any one of claims 1 to 4, wherein the phosphoglyceride of formula (II) is 1,2-dioleyl-sn-glycero-3-phosphoethanolamine (DOPE).

7. The composition according to any of paragraphs 1 to 6, where in formula (III), R ⁶ and R ⁷ each means a linear alkyl group containing from 10 to 16 carbon atoms and p is equal to an integer from 10 to 140.

8. The composition according to any one of claims 1 to 6, wherein the PEGylated phosphoglyceride of formula (III) is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG (DMPE-PEG), wherein the PEG (polyethylene glycol) fragment contains from 10 to 140 repeating units and more preferably 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-PEG5000 (DMPE-PEG5000).

9. The composition according to any of paragraphs 1-8, where the molar ratio of components (i-a)/(i-b)/(i-c) in the lipid composition is

1:(0.5 to 5):(0.01 to 1),

more preferably 1:(1 to 5):(0.01 to 0.5)

most preferably 1:(1 to 3):(0.02 to 0.2).

10. The composition according to any of paragraphs 1-9, where the particles are nano- or microparticles.

11. The composition according to any one of claims 1 to 10, wherein the particles have an average particle diameter ranging from 1 to 5000 nm, more preferably from 10 to 4000 nm, and most preferably from 50 to 3000 nm.

12. The composition according to any one of claims 1 to 11, wherein the particles have a maximum particle diameter of 20 µm, more preferably 10 µm and most preferably 5 µm.

13. The composition according to any one of paragraphs 1 to 12, wherein the N/P ratio, that is, the ratio of the number of nitrogen atoms in the cholesterol derivative of formula (I) to the number of P atoms of phosphate groups in RNA, is preferably in the range from 1 to 100, more preferably from 1 to 30 and most preferably 2 to 20.

14. The composition according to any one of claims 1 to 13, wherein the particles are characterized by an active substance content expressed as the weight of RNA relative to the total weight of the particles in the range of 0.1 to 95%, more preferably 0.5 to 80%, most preferably from 1 to 50%.

15. The composition according to any one of paragraphs 1-14, which includes particles comprising RNA and a lipid composition in an amount that provides an RNA concentration of from 0.01 to 50 mg/ml, more preferably from 0.02 to 30 mg/ml and most preferably from 0.05 to 10 mg/ml based on the total volume of the composition.

16. The composition according to any one of claims 1-15, wherein the particles comprising RNA and a lipid composition are dispersed in a liquid phase.

17. The composition according to any one of claims 1 to 16, wherein the 1,2-propanediol is contained in a liquid phase in which the particles are contained.

18. The composition according to any of paragraphs 1-17, where 1,2-propanediol is contained in a concentration of from 0.1 to 50 wt./vol. %, more preferably from 0.2 to 30 wt./vol. %, most preferably from 0.3 to 20 wt./vol. %, based on the total volume of the composition.

19. The composition according to any one of claims 1-18, wherein the liquid phase in which the particles are contained includes water.

20. Composition according to claim 19, where water is provided preferably 50 vol. % or more, preferably 70 vol. % or more volume based on the total volume of the liquid phase.

21. The composition according to any one of claims 1-20, wherein water and 1,2-propanediol are the only solvents contained in the liquid phase.

22. The composition according to any one of claims 1-21, wherein the liquid phase in which the particles are contained includes a buffer substance.

23. A method for producing a composition according to any of paragraphs 1-22, wherein said method includes the following steps:

a) dissolving and mixing the components of the lipid composition in an organic solvent, followed by lyophilization of the lipid composition,

b) rehydration of the lipid composition by adding water,

c) combining the rehydrated lipid composition with an aqueous solution of RNA to form particles comprising the RNA and the lipid composition that are contained in a liquid phase, and

d) adding 1,2-propanediol.

24. A solid composition comprising

(i) particles including RNA and lipid composition, and

(ii) 1,2-propanediol,

and said composition is obtained by freezing the composition according to any one of paragraphs 1 to 22.

25. A composition according to any one of claims 1-22 for use in the treatment or prevention of a disease using RNA-based therapy.

26. A composition for use according to claim 25, wherein the disease to be treated or prevented is a lung disease.

27. A composition for use according to claim 25 or claim 26, wherein the treatment or prophylaxis comprises administering the composition into or through the respiratory tract, preferably pulmonary or nasal administration.

28. A method of treatment comprising administering a composition according to any one of claims 1 to 22 to a patient, preferably into or through the respiratory tract, more preferably into the lungs or nasally.

29. The method according to claim 28 for the treatment of lung disease.

30. A device for forming an aerosol from a particulate composition contained in a liquid, or for spraying such a composition, wherein said device includes the composition of claims 1-22.

31. The device of claim 30, wherein the device is an inhaler selected from a metered dose inhaler, a nebulizer, or a nasal spray device.

Example I

Application of complexes to ALI cultures

Methods

Formation of the RNA-lipid complex at the N/P5 (and N/P3) ratio

Stock solutions of GL67, DOPE and DMPE-PEG5000 were prepared at a concentration of 10-20 mg/ml of each drug in a mixture of tert-butanol/water, 9:1. Lipids were mixed in a molar ratio of 1:2:0.05 (GL67/DOPE/DMPE-PEG5000), with a total lipid mass of 5 mg, frozen at -80°C overnight and lyophilized for at least 96 hours. using the dry lipid mixture was rehydrated by adding 400 μl of 5 v/v. % propylene glycol (with a total lipid concentration of 12.5 mg/ml) followed by incubation at RT for 30 min without shaking, then stirred on a Vortex mixer for 10 s and sonicated in a water bath at 37°C within 10 min. The resulting mixture of liposomes was diluted to form a complex with mRNA. For this purpose, 69.65 μL (87.69 μL for N/P3 ratio) of water was mixed with 10.24 μL of propylene glycol and stirred on a Vortex mixer for 3 s. Then 45.10 μl (27.06 μl for N/P3 ratio) of the liposome mixture was added, the mixture was mixed on a Vortex mixer for 10 s, equilibrated at 39°C for 3 min. 125 μL of mRNA solution in water (conc. 0.5 mg/mL) was equilibrated at 39°C for 3 min. To assemble the complex, diluted mRNA was drawn into a syringe with a thin needle (BD Micro-Fine insulin syringe, 0.5 ml U40, 8 mm, 07468060 Becton Dickinson) and quickly injected into the diluted liposomes, immediately mixed on a Vortex mixer for 10 s and incubated at RT for 10 min to assemble the complex. The complexes were then stored on ice.

ALI culture treatment

The MucilAir™-ALI culture was obtained from Epithelix (France). A mucus wash was performed 5 days before transfection to remove accumulated mucus on the upper surface. 200 μL of PBS 1 solution was added to the top side and incubated at 37°C for 20 min. To separate the mucus from the top surface, three back-and-forth movements were performed on the top surface using a pipette and 100 μl of the top liquid. PBS ^-/- from the upper surface of the MucilAir™ culture was removed by gentle suction without damaging the epithelium. On the day of transfection, ALI cultures were washed again with 200 μl of PBS ^−/− and then washed with 200 μl of the appropriate mRNA complex diluent (water or 5% propylene glycol). The diluent was immediately removed from the top surface of the ALI cultures by gentle suction without damaging the epithelium. Each well was then transfected with 3 μg of total mRNA complex in a volume of 50 μl. The formulations were diluted with an appropriate diluent, added to the top surface of the ALI culture and incubated for 6 hours at 37°C and 5% CO ₂ . The complex solution was then removed, the cell layer was washed with PBS ^-/- , and the ALI cultures were incubated for 24 hours at 37°C and 5% CO ₂ .

Detection of tdTomato signal using fluorescence microscopy

Fluorescence imaging was used to detect a positive tdTomato signal 24 h after transfection. The corresponding image in each well was acquired using the following parameters: exposure 500 ms, gain 1.5, intensity 30%.

results

The mRNA encoding tdTomato was used to treat cultures with ALI. Treated wells were analyzed for the presence of tdTomato protein 24 h after treatment. In fig. 1 shows typical images.

As shown in FIG. 1, the addition of propylene glycol significantly increases the level of mRNA that is delivered to cells and translated into protein. This effect can be observed at both an N/P ratio of 3 and an N/P ratio of 5.

Example II

Nasal administration of complexes

Methods

Formation of the RNA-lipid complex

Complexes were formed as described previously in Example I.

Animal treatment

Balb/c mice (Charles River Laboratories) were treated with complexes at a dose of 10 μg/40 μl of mRNA. The solution was administered one drop at a time into the animal's nostrils after anesthesia using isoflurane inhalation (Isothesia, Henry Shine, Germany).

Detection of luciferase levels in nasal tissue

5 hours after administration, 1.5 mg of D-luciferin dissolved in 50 μl of PBS was injected intranasally (nose inhalation method) into the nostrils of each animal. Bioluminescent imaging was performed using IVIS 100 in vivo imaging system (Perkin Elmer, USA). Animals were sacrificed by cervical dislocation immediately after imaging. Images were analyzed using Living Image software (Perkin Elmer, USA) by measuring radiation in a given region of interest (ROI). Values are given as average radiation [p/s/cm ² /sr] (photons/s/cm ² /steradian).

results

To treat Balb/c mice by nasal inhalation, mRNA encoding firefly luciferase was used as part of a complex at N/P 5. The results of analysis of nasal tissue for luciferase activity 5 hours after treatment (Fig. 2) indicate that a solution of the complexes in 5% propylene glycol results in increased reporter protein activity in nasal tissue compared to the same complexes in the absence of propylene glycol (0% PG, water).

Example III

Particle size stability during freeze-thaw cycle

Methods

Particle Formation

Particles were formed as described in example 1.

Freezing a particle solution

Once formed, the particles were frozen at -20°C for at least 16 hours.

Size Measurement

The hydrodynamic diameter of particles was determined by the dynamic light scattering method on a ZetaSizer Nano ZS particle analyzer (Malvern instruments). For correct measurements, instead of the composition of the buffer solution used by the software to calculate the viscosity of solutions, if necessary, the device was set up using a solution containing 1,2-propanediol.

results

The particles were formulated. After their preparation, the size of freshly prepared particles in solution was measured in the presence or absence of 1,2-propanediol. Particles were frozen at -20°C for 16 h and thawed when maintained at RT. Then the particle size in the thawed solutions was measured again. To maintain activity, the deviation in particle size before freezing and after thawing should be minimal. As shown in FIG. 3, for particles that were frozen in a solution not containing 1,2-propanediol, a significant increase in size was observed due to aggregation processes during freezing. In the presence of 1,2-propanediol, the particle size remains stable after a freeze-thaw cycle.

Figures

In fig. Figure 1 shows the tdTomato signal level after transfection using GL67 complexes in various excipient solutions (water (0% propylene glycol) or 5% propylene glycol) at N/P ratios of 3 or 5.

In fig. Figure 2 shows luciferase activity in the nasal cavity of Balb/c mice 5 hours after treatment with GL67 complexes in water or 5% propylene glycol solution.

In fig. Figure 3 shows the particle size before freezing and after thawing in the presence or absence of 1,2-propanediol.

Claims (40)

1. A composition for treating or preventing a disease using mRNA-based therapy, comprising (i) particles contained in a liquid phase, where the particles include mRNA and a lipid composition, and where the lipid composition includes: (i-a) a cholesterol derivative of formula (I) or a pharmaceutically acceptable or physiologically acceptable salt thereof: Where n is 0, R ¹ means a -(CH ₂ ) _q -NH ₂ group or a -(CH ₂ ) _r -NH-(CH ₂ ) _s -NH ₂ group, where q, r and s are independently equal to an integer from 2 to 6, R ² means a -(CH ₂ ) _t -NH ₂ group or a -(CH ₂ ) _u -NH-(CH ₂ ) _w -NH ₂ group, where t, u and w are independently equal to an integer from 2 to 6, (i-b) phosphoglyceride of formula (II) or a pharmaceutically acceptable or physiologically acceptable salt thereof: Where R ⁴ means a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 24 carbon atoms, R ⁵ means a linear alkenyl group containing from 1 to 3 double bonds and from 10 to 24 carbon atoms, (i-c) pegylated phosphoglyceride of formula (III) or a pharmaceutically acceptable or physiologically acceptable salt thereof: Where p is equal to an integer from 5 to 200, R ⁶ means a linear alkyl group containing from 10 to 20 carbon atoms, R ⁷ means a linear alkyl group containing from 10 to 20 carbon atoms, And (ii) 1,2-propanediol. 2. Composition according to claim 1, wherein the cholesterol derivative of formula (I) is GL67. 3. Composition according to claim 1 or 2, wherein the phosphoglyceride of formula (II) is 1,2-dioleyl- sn -glycero-3-phosphoethanolamine. 4. Composition according to any one of paragraphs. 1-3, wherein the PEGylated phosphoglyceride of formula (III) is 1,2-dimyristoyl -sn -glycero-3-phosphoethanolamine-PEG5000. 5. Composition according to any one of paragraphs. 1-4, where the molar ratio of components (i-a):(i-b):(i-c) in the lipid composition is 1:(0.5 to 5):(0.01 to 1). 6. Composition according to any one of paragraphs. 1-5, where the particles are characterized by an average particle diameter in the range from 1 to 5000 nm. 7. Composition according to any one of paragraphs. 1-6, where the N/P ratio, that is, the ratio of the number of nitrogen atoms in the cholesterol derivative of formula (I) to the number of P atoms of phosphate groups in the mRNA, ranges from 1 to 100. 8. Composition according to any one of paragraphs. 1-7, where 1,2-propanediol is contained in a concentration of from 0.1 to 50 wt./vol.% based on the total volume of the composition. 9. Composition according to any one of paragraphs. 1-8, wherein the liquid phase in which the particles are contained includes water. 10. Composition according to any one of paragraphs. 1-9, where in the PEGylated phosphoglyceride of formula (III) p is equal to an integer from 10 to 170 and preferably from 10 to 140. 11. Method for obtaining the composition according to any one of paragraphs. 1-10, and this method includes the following steps: a) dissolving and mixing the components of the lipid composition in an organic solvent, followed by lyophilization of the lipid composition, b) rehydrating the lyophilized lipid composition by adding water, c) combining the rehydrated lipid composition with an aqueous solution of RNA to form particles comprising the RNA and the lipid composition that are contained in a liquid phase, and d) adding 1,2-propanediol. 12. Use of the composition according to any one of paragraphs. 1-10 in the treatment or prevention of disease using mRNA-based therapy. 13. Use according to claim 12, wherein the disease to be treated or prevented is a lung disease. 14. Use according to claim 12 or 13, wherein the treatment or prophylaxis comprises administering the composition into or through the respiratory tract, preferably into the lungs or nasally.