RU2792644C2 - Rna particles including polysarcozine - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: group of inventions relates to compositions for delivering RNA to target tissues after administration. A composition for delivering RNA to target cells or tissues is proposed, comprising a plurality of RNA particles, each particle comprising RNA and one or more components that combine with RNA to form RNA particles, where polysarcosine is conjugated with one or several components, and one or more components that are combined with RNA to form particles contain one or more lipids or lipid-like materials. Another option of the said composition is also provided, wherein each particle comprises RNA, a cationic or cationically ionizable lipid or lipid-like material, and a polysarcosine-lipid conjugate or a polysarcosine-lipid-like material conjugate. Also the methods for delivering RNA to cells of a subject and delivering a therapeutic peptide or protein to a subject are disclosed, wherein the RNA encodes a therapeutic peptide or protein, comprising administering of the said composition to the subject. Other options of a method for treating or preventing a disease or disorder in a subject are provided, comprising administering of the said composition to the subject, delivery of the RNA to the subject's cells is beneficial in treating or preventing the disease or disorder, or the RNA encodes a therapeutic peptide or protein.

EFFECT: invention provides increased efficiency of targeting and safety of RNA delivery to target cells.

34 cl, 17 dwg, 4 tbl, 7 ex

Description

Technical field

The present invention relates to RNA particles for delivering RNA to target tissues after administration, in particular after parenteral administration such as intravenous, intramuscular, subcutaneous or intratumoral administration, and compositions comprising such RNA particles. The RNA particles in one embodiment comprise a single stranded RNA, such as mRNA, that encodes a peptide or protein of interest, such as a pharmaceutically active peptide or protein. The RNA is taken up by cells in the target tissue and the RNA is translated into an encoded peptide or protein that can exert its biological activity.

State of the art

The use of RNA to deliver foreign genetic information to target cells is an attractive alternative to DNA. Advantages of using RNA include transient expression and non-transforming nature. RNA does not have to enter the nucleus for expression and, moreover, cannot be integrated into the host genome, thereby eliminating the risk of oncogenesis.

RNA can be delivered to a subject using a variety of delivery systems based primarily on cationic polymers or lipids, which together with RNA form nanoparticles. The nanoparticles are supposed to protect RNA from degradation, enable delivery of RNA to the target site, and facilitate cellular uptake and processing by target cells. For delivery efficiency, in addition to molecular composition, parameters like particle size, charge, or grafting with molecular particles such as polyethylene glycol (PEG) or ligands play a role. PEG grafting is believed to reduce serum interactions, increase serum stability, and increase circulation time, which may be useful in some targeting approaches. Ligands that bind to receptors as a target site can improve targeting efficiency. In addition, pegylation can be used for particle engineering. For example, if lipid nanoparticles (LNPs) are produced by mixing an aqueous RNA phase with an organic lipid phase, a certain fraction of PEG-conjugated lipid is required in the lipid mixture, otherwise the particles stick together during the mixing step. It has been shown that by changing the molar fraction of PEG lipids, including PEG of different molecular weight, it is possible to control the particle size. The particle size can also be controlled by changing the molecular weight of the PEGylated lipid particles. Typical convenient sizes are between 30 and 200 nm (Belliveau et al., 2012, Molecular Therapy–Nucleic Acids 1, e37). The particles thus formed have the additional advantage that, due to the PEG fraction, they interact less with serum components and have a longer circulating half-life, which is desirable in many drug delivery approaches. Without PEG lipids, particles of a distinct size cannot be obtained; particles form large aggregates and sediment.

Thus, in the case of methods in which LNPs are formed from an ethanol and aqueous phase, one of the main roles of PEG lipids is to facilitate particle self-assembly by providing a steric barrier on the surface of the resulting particles formed when nucleic acids are rapidly mixed with ethanolic solutions containing lipids. for RNA binding. The steric hindrance of PEG prevents particle fusion and promotes the formation of a homogeneous population of LNP populations with diameters <100 nm.

PEG is the most widely used and is the gold standard for an "invisible" polymer in drug delivery. PEG lipids are usually included in systems to obtain a homogeneous and stable population of colloidal nanoparticles due to the hydrophilicity of their steric hindrance (the PEG shell prevents electrostatic or van der Waals attraction that leads to aggregation). Pegylation makes it possible to form an aqueous shell around the polymer that protects the RNA complex from opsonization by serum proteins, increase the serum half-life, and also reduce the rapid renal clearance, which leads to an improvement in pharmacokinetic properties. Changing the length of the acyl chains (C18, C16 or C14) of lipids modifies the incorporation stability of the PEG lipid into the particles, leading to modulation of pharmacokinetics. The use of a PEG lipid containing short acyl chains (C14), which dissociates from LNP in vivo with a half-life of <30 min, results in optimal strength of hepatocyte gene silencing (Chen et al., 2014, J. Control. Release, 196: 106 -12; Ambegia et al., 2005, Biochimica et Biophysica Acta, 1669: 155–163). In addition, tight control of particle size can be obtained by changing the parameter of the PEG lipid: a higher MW of PEG or a higher molar fraction of PEG lipids in the particles leads to smaller particles.

Despite these advantages, pegylation of nanoparticles may also lead to some effects that are detrimental to their intended use in drug delivery. PEGylation of liposomes and LNP is known to reduce cellular uptake and endosomal release, thus reducing overall transfection efficiency. Indeed, the PEG shell provides a steric barrier for effective particle binding to the cell and also prevents endosomal release by preventing membrane fusion between the liposome and the endosomal membrane. This is why the type of PEG lipid and the amount of PEG lipid used must always be carefully adjusted. It should be ensured that the in vivo and stabilization aspects are sufficiently obscure on the one hand and that transfection is not obstructed on the other hand. This phenomenon is known as the "PEG dilemma".

In addition to reducing transfection efficiency, pegylation has been associated with accelerated blood clearance (ABC) phenomena induced by anti-PEG antibodies and/or complement activation, as well as storage diseases (Bendele A. et al., 1998, Toxicolocal Sciences, 42, 152-157; Young MA et al., 2007, Translational Research, 149(6), 333-342; S. M. Moghimi, J. Szebeni, 2003, Progress in Lipid Research, 42: 463-478). Ishida et al. and Laverman et al. report that intravenous injection of PEG-attached liposomes into rats can significantly alter the pharmacokinetic properties of a second dose when this second dose is administered after an interval of several days (Laverman P. et al., 2001, J. Pharmacol. Exp. Ther., 298(2 ), 607-12; Ishida et al., 2006, J. Control. Release, 115(3), 251-8). The phenomenon of "accelerated blood purification" (ABC) is inversely related to the content of PEG in liposomes. The presence of anti-PEG antibodies in plasma causes a higher clearance of the mono(nuclear) phagocyte system, which ultimately reduces the effectiveness of the drug.

It is also believed that PEG induces complement activation, which can lead to a hypersensitivity reaction, also known as complement-activated pseudo-allergy (CARPA). It is not yet clear from the literature whether complement activation is caused by the nanoparticle in general or by the presence of PEG in particular.

The presence of PEG in lipid nanoparticles can also induce a specific immune response. Semple et al. reported that liposomes containing PEG-lipid derivatives and encapsulated antisense oligodeoxynucleotide or plasmid DNA elicited a strong immune response that in mice resulted in rapid blood clearance from subsequent doses. The magnitude of this response was sufficient to cause significant morbidity and in some cases mortality. The rapid removal of the liposomal encapsulated ODN from the blood depends on the presence of PEG lipid in the membrane, since the use of non-PEGylated liposomes or liposomes containing a rapidly exchanged PEG lipid abolishes the reaction. Generation of anti-PEG antibodies and putative complement activation was a likely explanation for the rapid removal of vesicles from the blood (Semple et al., 2005, J. Pharmacol. Exp. Ther., 312(3), 1020-6).

Since PEG can induce immune responses, there is a need to avoid it in some applications where multiple injections are required. Examples are therapies using mRNA, such as in the case of protein replacement therapy. In this case, the danger may be particularly high due to the possible inherent immunogenicity of the RNA.

Thus, there remains a need in the art for effective methods and compositions for introducing RNA into cells that avoid the disadvantages associated with the use of PEG. The present disclosure addresses these and other needs.

The inventors surprisingly found that the preparations of RNA particles described in the present description, meet the requirements mentioned above. In particular, polysarcosine-lipid conjugates have been shown to be suitable components for the assembly of RNA particles. Polysarcosine is composed of repeating units of the naturally occurring amino acid sarcosine (N-methylglycine) and is biodegradable. Polysarcosine-lipid conjugates allow the production of RNA particles by a variety of methods resulting in defined properties and controlled size ranges. Production can be carried out with reliable processes that meet the requirements of pharmaceutical production. The particles may have different terminal functional groups for charge modulation or for the introduction of specific molecular species like ligands.

The essence of the invention

In one aspect, the invention relates to a composition comprising a plurality of RNA particles, each particle comprising

(i) RNA

And

(ii) one or more components that combine with RNA to form RNA particles,

and at least one of one or I of several components polysarcosine is conjugated.

In one embodiment, the RNA particles are non-viral RNA particles. In one embodiment, the one or more components that bind to the RNA to form particles include one or more polymers. In one embodiment, one or more polymers include a cationic polymer. In one embodiment, the cationic polymer is an amine-containing polymer. In one embodiment, the one or more polymers include one or more polymers selected from the group consisting of poly-L-lysine, polyamidoamine, polyethyleneimine, chitosan, and poly(β-amino esters).

In one embodiment, the one or more components that bind to the RNA to form particles include one or more lipids or lipid-like materials. In one embodiment, the one or more lipids or lipid-like materials comprise a cationic or cationically ionizable lipid or lipid-like material. In one embodiment, the cationically ionizable lipid or lipid-like material is only positively charged at acidic pH and does not remain cationic at physiological pH. In one embodiment, one or more lipids or lipid-like materials include one or more additional lipids or lipid-like materials. In one embodiment, polysarcosine is conjugated to one or more additional lipids or lipid-like materials.

In another aspect, the invention relates to a composition comprising a plurality of RNA lipid particles, each particle comprising

(a) RNA;

(b) a cationic or cationically ionizable lipid or lipid-like material; And

(c) a polysarcosine-lipid conjugate or a polysarcosine-lipid-like material conjugate.

In one embodiment, each particle also includes

(d) a non-cationic lipid or lipid-like material.

In one embodiment, the cationic or cationically ionizable lipid or lipid-like material comprises from about 20 mole % to about 80 mole % of the total lipid or lipid-like material present in the particles.

In one embodiment, the non-cationic lipid or lipid-like material comprises from about 0 mole % to about 80 mole % of the total lipid or lipid-like material present in the particles.

In one embodiment, the polysarcosine-lipid conjugate or polysarcosine-lipid-like material conjugate comprises from about 0.25 mole % to about 50 mole % of the total lipid or lipid-like material present in the particles.

In one embodiment, the non-cationic lipid or lipid-like material comprises a phospholipid. In one embodiment, the non-cationic lipid or lipid-like material comprises cholesterol or a cholesterol derivative. In one embodiment, the non-cationic lipid or lipid-like material comprises a mixture of a phospholipid and cholesterol or a cholesterol derivative. In one embodiment, the phospholipid is selected from the group consisting of distearoylphosphatidylcholine (DSCP), dipalmitoylphosphatidylcholine (DPPC), or a mixture thereof. In one embodiment, the non-cationic lipid or lipid-like material comprises a mixture of DSCP and cholesterol.

In one embodiment, the polysarcosine-lipid conjugate or polysarcosine-lipid-like material conjugate is of the following general formula (I):

.

.

In one embodiment, the polysarcosine-lipid conjugate or polysarcosine-lipid-like material conjugate is of the following general formula (II):

,

,

where one of R ₁ and R ₂ includes a hydrophobic group, and the other represents H, a hydrophilic group or a functional group, optionally including a targeting particle.

In one embodiment, the polysarcosine-lipid conjugate or polysarcosine-lipid-like material conjugate is of the following general formula (III):

,

,

where R represents H, a hydrophilic group or a functional group, optionally including a targeting particle.

In one embodiment of all aspects of the invention, the particles do not include a polyethylene glycol-lipid conjugate or a polyethylene glycol-lipid-like material conjugate, and preferably do not include polyethylene glycol.

In one embodiment of all aspects of the invention, the RNA is mRNA.

In one embodiment of all aspects of the invention, the cationic or cationically ionizable lipid or lipid-like material comprises N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-( 2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K- DMA) or a mixture thereof.

In one embodiment of all aspects of the invention, the polysarcosine comprises from 2 to 200 sarcosine units.

In one embodiment of all aspects of the invention, the polysarcosine-lipid conjugate or the polysarcosine-lipid-like material conjugate is an element selected from the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-dialkyloxypropyl conjugate, a polysarcosine-phospholipid conjugate, a polysarcosine-ceramide conjugate, and a mixture thereof.

In one embodiment of all aspects of the invention, the particles are nanoparticles.

In one embodiment of all aspects of the invention, the particles include a nanostructured core.

In one embodiment of all aspects of the invention, the particles are from about 30 nm to about 500 nm in size.

In one embodiment of all aspects of the invention, the polysarcosine conjugate inhibits particle aggregation.

In another aspect, the invention relates to a method for delivering RNA to cells of a subject, the method comprising administering to the subject a composition described herein.

In another aspect, the invention relates to a method for delivering a therapeutic peptide or protein to a subject, the method comprising administering to the subject a composition described herein, the RNA encoding the therapeutic peptide or protein.

In another aspect, the invention provides a method for treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a composition as described herein, wherein delivery of the RNA to the subject's cells is beneficial in treating or preventing the disease or disorder.

In another aspect, the invention relates to a method for treating or preventing a disease or disorder in a subject, the method comprising administering to the subject a composition described herein, wherein the RNA encodes a therapeutic peptide or protein, and wherein delivery of the therapeutic RNA peptide or protein to the subject is beneficial in treating or preventing diseases or disorders.

In one embodiment, the subject is a mammal. In one embodiment, the mammal is a human.

Brief description of the drawings

Fig. 1. Relationship between particle size and molar fraction of polysarcosine-conjugated LNPs. Lipid nanoparticles were prepared using mixtures of lipids containing increasing molar fractions of C14PSarc20. Under suitable conditions, stable colloidal particles can be obtained. Although very low PSarc fractions (0.5 and 1%) do not form measurable particles, at 2.5 mole % and above, particles of a conventional size and a low polydispersity index are obtained. The particle size can be fine-tuned by changing the PSarc fraction. The particle size decreases monotonically from about 200-250 nm with 2.5 mole % PSarc to about 50 nm with 20 mole % PSarc.

Fig. 2. Relationship between polysarcosine lengths (polymer units) of PSarc lipids used to form LNPs and in vitro expression of the luciferase-encoding mRNA protein in LNPs in various cell lines. LNPs formulated with mRNA encoding luciferase are tested in lung tumor cells (TC-1), muscle cells (C2C12), hepatocytes (Hep-G2) and macrophages (RAW 264.7). 24 hours after transfection, the bioluminescence signal is measured. Regardless of the cell line, an increase in the number of polymer units in polysarcosine does not lead to a decrease in protein expression levels, as is commonly observed in PEG lipids.

Fig. 3. In vivo efficacy of LNPs containing a constant fraction of PSarc lipid (5%), where the polysarcosine length varies from 11 to 65 units. LNPs derived from mRNA encoding luciferase are injected intravenously into mice (10 μg RNA, n=3). Bioluminescence is measured in vivo and ex vivo. In all cases, the strongest signals are found in the liver. The figure shows data from ex vivo measurements in the liver, which was removed 6 hours after injection. Could not determine a significant effect of polysarcosine length on the level of protein expression in the liver. This allows PSarc particles to be engineered with a wide range of sizes without compromising transfection efficiency.

Fig. 4. Effect of different terminal groups of polysarcosine on particle size and zeta potential. PSarc, consisting of 20 repeating units, with an amino end group, carboxylated or acetylated end group is tested by direct comparison. All other composition parameters are kept constant. The formation of LNPs with all tested end groups is successful, where the correlation between PSarc fraction and particle properties (size and zeta potential) is similar.

Fig. 5. Determination of in vivo properties of LNPs comprising polysarcosine lipids with different end groups as described in FIG. 4. Use PSarc lipids in a molar fraction of 5% and a length of 20 links. LNPs derived from mRNA encoding luciferase are tested with hepatocytes (Hep-G2), macrophages (RAW 264.7), muscle cells (C2C12) and primary kidney cells (HEK 293 T). 24 hours after transfection, the bioluminescence signal is measured. A bioluminescence signal is obtained for all LNPs and cell lines. The dependence of the signal strength as a function of the cell line is similar for all end groups.

Fig. 6. In vivo performance of LNPs prepared with different end groups described in FIG. 4 and 5. PSarc lipids are used in a 5% molar fraction and 20 units long. LNPs derived from mRNA encoding luciferase are injected intravenously (10 μg RNA, n=3). Bioluminescence is measured in vivo and ex vivo. In all cases, the strongest signals are found in the liver. The figure shows data from ex vivo measurements in a liver extracted 6 hours after injection. Signals of similar strength were determined with all end groups, which indicates that all end groups are suitable for obtaining similar high transfection in vivo.

Fig. 7. Effect of pegylation and polysarcosine conjugation on liposome size. Liposomes are prepared either with DOTMA and DOPE alone (2:1 mol/mol), or lipid mixtures are used that include PEG or pSarc lipid in a 2% molar fraction. Both PEG and pSarc lead to a significant decrease in the measured size, although, however, the polydispersity index is higher (multimodal).

Fig. 8. Formation of lipoplexes using liposomes that include PEG and pSarc as described in FIG. 7. Of all three types of liposomes (one DOTMA and DOPE (2:1 mol/mol), or including PEG or pSarc lipid in a molar fraction of 2%), lipoplexes of limited size and low polydispersity index are formed. Lipoplexes from PEGylated and polysarcosine-conjugated liposomes show an unexpectedly low polydispersity index compared to precursor liposomes where PDI values are high. This shows that pSarc liposomes with a high polydispersity index may also be suitable for the formation of certain RNA lipoplexes of sufficiently small sizes - 50 nm and a PDI of about 0.2.

Fig. 9. Determination of the in vitro properties of lipoplexes prepared from liposomes consisting of either DOTMA and DOPE alone (2:1 mol/mol) or mixtures of the same lipids containing PEG or pSarc lipid in a 2% molar fraction. Lipoplexes with mRNA encoding luciferase are tested with hepatocytes (Hep-G2). 24 hours after transfection, the bioluminescence signal is measured. Although pegylation significantly attenuates the signal, this attenuation is much less pronounced in the presence of pSarc. It turns out that pSarc reduces the efficiency of transfection to a much lesser extent than PEG.

Fig. 10. Determination of in vitro properties of lipoplexes prepared from liposomes consisting either of DOTMA and DOPE alone (2:1 mol/mol) or mixtures of the same lipids, including PEG or pSarc lipid in a molar fraction of 2%. Lipoplexes with mRNA encoding luciferase are tested with muscle cells (C2C12). 24 hours after transfection, the bioluminescence signal is measured. Although pegylation significantly attenuates the signal, this attenuation is much less pronounced in the presence of pSarc. It turns out that pSarc reduces the efficiency of transfection to a much lesser extent than PEG.

Fig. 11. Relationship between particle size and chain length and molar ratio of polysarcosine in the composition.

Fig. 12. Scattering curves (SAXS) of polysarcosine-conjugated lipid particles.

Fig. 13. RNA availability assessed by Quant-It Ribogreen assay.

Fig. 14. Intravenous administration of various doses of mRNA encoding EPO (erythropoietin) loaded into LNP in composition with PSarc or PEG-conjugated lipids.

Fig. 15. Release of liver enzymes as an early marker of liver toxicity after LNP injection in combination with increasing chain length PSarc.

Fig. 16. Complement activation via the C3a complex of pegylated and polysarcosium-conjugated LNPs at theoretical human plasma concentration.

Fig. 17. Image of Cryo-TEM LNPs prepared with DODMA:cholesterol:DSPC:PSarc 23 in the respective mol % 40:45:15:10:5. Scale = 200 nm.

Detailed description

Although the present disclosure is described in detail below, it should be understood that the present disclosure is not limited to the particular methodologies, protocols, and reagents described herein, as they may change. It should also be understood that the terminology used in this specification is used for the purpose of describing only certain embodiments and is not intended to limit the scope of the present disclosure, which will be limited only by the appended claims. Unless otherwise indicated, all technical and scientific terms used in the present description have the same meanings as they are usually given to experts in this field of technology.

Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).Preferably, the terms used herein are defined in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel, and H.

Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

In practice, the present disclosure will use, unless otherwise indicated, the conventional techniques of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as explained in the literature in the art (see, for example, Molecular Cloning: A Laboratory Manual, ^2nd Edition). , J. Sambrook et al.eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Next, the elements of the present disclosure will be described. These elements are listed with specific embodiments, however, it should be borne in mind that they can be combined in any number to create other embodiments. The separately described examples and embodiments should not be construed as limiting the present disclosure to the openly described embodiments. The present description should be understood as disclosing and covering embodiments that combine the openly described embodiments with any number of disclosed elements. In addition, any permutations and combinations of all described elements should be considered as disclosed by this description, unless the context indicates otherwise.

The term "about" means about or about, and in the context of a numerical value or range as defined herein in one embodiment, means ±20%, ±10%, ±5%, or ±3% of the numerical value or range cited or claimed.

Terms in the singular used in the context describing the disclosure (especially in the context of the claims) should be construed as covering both the singular and the plural, unless otherwise indicated in the present description or there is no clear conflict with the context. The listing of ranges of values in this specification is only intended to serve as a way of shortening the reference to each individual value that falls within a range individually. Unless otherwise indicated in the present description, each individual value is included in the description as if it were given separately in the present description. All of the methods described herein may be performed in any suitable order, unless otherwise indicated herein or the context clearly dictates otherwise. The use of any and all examples or exemplary language (eg, "such as") provided herein is intended only to better explain the disclosure and is not intended to limit the scope of the claims. Nothing in the description should be construed as indicating any unclaimed element essential to the practice of the disclosure.

Unless expressly stated otherwise, the term "including" is used in the context of this document to indicate that other members may be present other than the members of the list introduced by "inclusion". However, as a specific embodiment of the present disclosure, the term "comprising" is intended to cover the possibility that no other members are present, i.e. for the purpose of this embodiment, "comprising" should be understood to mean "consisting of".

In the text of this description, some documents are cited. Each of the documents cited in this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) above or below are incorporated herein by reference in their entirety. Nothing in the present description should be construed as an admission that the present disclosure does not give the right to predate.

Definitions

The following are definitions that apply to all aspects of the present disclosure. The following terms have the following meanings, unless otherwise indicated. Any undefined terms have their known meanings.

Terms such as "reduce" or "inhibit" as used herein refer to the ability to cause an overall reduction from a level of, for example, about 5% or more, about 10% or more, about 20% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar expressions includes complete or substantially complete inhibition, i.e. reduction to zero or substantially to zero.

Terms such as "enhance" or "enhance" in one embodiment refer to an increase or increase of at least about 10%, at least about 20%, at least about 30%, at least about 40% , at least about 50%, at least about 80%, or at least about 100%.

The term "physiological pH" as used herein refers to a pH of about 7.4.

When used in this disclosure, "% w/v" refers to percent mass per volume, which is a unit of concentration that measures the amount of a solute in grams (g) expressed as a percentage of the total volume of a solution in milliliters (mL).

As used in this disclosure, "mole %" is defined as the ratio of the number of moles of one component to the total number of moles of all components multiplied by 100.

The term "ionic strength" refers to the mathematical relationship between the number of different kinds of ions in a particular solution and their respective charges. So, the ionic strength I is represented mathematically by the formula

,

,

in which c is the molar concentration of a certain kind of ion, and z is the absolute value of its charge. The sum Σ is taken as the sum of all the different kinds of ions (i) in solution.

According to the disclosure, the term "ionic strength" in one embodiment refers to the presence of monovalent ions. With regard to the presence of divalent ions, in particular divalent cations, their concentration or effective concentration (presence of free ions) due to the presence of chelating agents in one embodiment is low enough to prevent degradation of the RNA. In one embodiment, the concentration or effective concentration of divalent ions is below the catalytic level for the hydrolysis of phosphodiester bonds between RNA nucleotides. In one embodiment, the concentration of free divalent ions is 20 μM or less. In one embodiment, free divalent ions are absent or essentially absent.

The term "osmolality" refers to the concentration of solutes, expressed as the number of osmoles of a solute per kilogram of solvent.

The term "freezing" refers to a phase transition from liquid to solid. It usually occurs when the temperature of the system drops below the critical temperature and is accompanied by a characteristic change in the entropy of the system.

The term "lyophilization" refers to freeze-drying a substance by freezing it and then lowering the ambient pressure to allow the frozen medium in the substance to sublimate from the solid phase directly to the gas phase.

The term "spray drying" refers to the spray drying of a substance by mixing a (heated) gas with a liquid, which is pulverized (sprayed) inside a vessel (spray dryer) where the solvent is evaporated from the droplets formed, leading to a dry powder.

The term "cryoprotectant" refers to a substance that is added to the composition in order to protect the active ingredients during the freezing steps.

The term "lyoprotectant" refers to a substance that is added to the composition in order to protect the active ingredients during the drying steps.

The term "recovery" refers to the addition of a solvent, such as water, to a dry product in order to return it to a liquid state, such as the original liquid state.

The term "recombinant" in the context of the present disclosure means "obtained by genetic engineering". In one embodiment, the "recombinant subject" in the context of the present disclosure does not occur in nature.

The term "naturally occurring" as used herein refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in the body (including viruses) and can be isolated from a source in nature, and that has not been intentionally modified by humans in a laboratory, is naturally occurring. The term "found in nature" means "present in nature" and includes known objects, as well as objects that have not yet been discovered and / or isolated from nature, but which may be discovered and / or isolated in the future from a natural source.

In the context of the present disclosure, the term "particle" refers to a structural entity formed by molecules, or molecular complexes. In one embodiment, the term "particle" refers to a micro- or nano-sized structure, such as a compact micro- or nano-sized structure dispersed in a medium.

In the context of the present disclosure, the term "RNA particle" refers to a particle that contains RNA. Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged RNA are involved in particle formation. This leads to complex formation and spontaneous formation of RNA particles. In one embodiment, the RNA particle is a nanoparticle.

As used in the present disclosure, the term "nanoparticle" refers to a particle having an average diameter suitable for intravenous administration.

The term "average diameter" refers to the average hydrodynamic particle diameter measured by dynamic laser light scattering (DLS), with data analysis using a so-called cumulative algorithm that provides both the results of the so-called Z _mean with the dimension of length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321). In the present description, "average diameter", "diameter" or "size" for particles are used as synonyms with this value Z _average .

The "polydispersity index" is preferably calculated from measurements of dynamic light scattering using the so-called cumulative analysis, as mentioned in the definition of "average diameter". Under certain preconditions, it can be taken as a measure of the particle size distribution in a population of nanoparticles.

In general, the RNA-lipid particles described herein can be obtained by mixing an RNA-containing phase with a lipid-containing phase. This may be mixing an ethanol phase or other water-miscible solvent comprising lipids such as cationic lipids like DODMA and other lipids with an aqueous phase comprising RNA. Another choice is to mix an aqueous phase comprising lipids, for example comprising lipids in the form of liposomes or other types of liquid dispersions, with another aqueous phase comprising RNA.

RNA-containing particles

Various types of RNA particles suitable for delivering RNA particles have previously been described (e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60). In the case of vesicles for non-viral RNA delivery, encapsulation of the RNA nanoparticles physically protects the RNA from degradation and, depending on the specific chemistry, can promote cellular uptake and endosomal release.

The present disclosure describes particles comprising RNA and one or more components that associate with RNA to form RNA particles, and compositions comprising such particles. RNA particles may include RNA that forms a complex in various forms through non-covalent interactions with the particle. The particles described herein are not viral particles, in particular infectious viral particles, i.e. they are not capable of infecting cells like viruses. The RNA-containing particles may be, for example, in the form of protein particles, in the form of polymer-containing particles, or in the form of lipid-containing particles. Suitable proteins, polymers or lipids are included under the term "particle-forming components" or "particle-forming agents". The term "particle-forming components" or "particle-forming agents" refers to any components that associate with RNA to form RNA particles. Such components include any component that may be part of the RNA particles.

Proteins, polymers, lipids, and other hydrophilic, hydrophobic, or amphiphilic compounds are typical constituents of RNA particle compositions.

Due to their high degree of chemical flexibility, polymers are widely used nanoparticle delivery materials. Typically, cationic polymers are used to electrostatically condense negatively charged RNA into nanoparticles. These positively charged groups often consist of amines, which change their protonation state between pH 5.5 and 7.5, which is believed to lead to an ion imbalance resulting in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan, are used for RNA delivery. In addition, some researchers have synthesized polymers specifically for nucleic acid delivery. In particular, poly(β-amino esters) have become widely used in nucleic acid delivery due to their ease of synthesis and biodegradability.

As used herein, "polymer" has its usual meaning, ie. denotes a molecular structure that includes one or more repeating units (monomers) connected by covalent bonds. The repeat units may all be identical, or in some cases they may represent more than one type of repeat unit present in the polymer. In some cases, the polymer is of biological origin, ie. is a biopolymer like protein. In some cases, other groups may also be present in the polymer, for example targeting groups such as those described herein.

If more than one type of repeating unit is present in a polymer, then the polymer is said to be a "copolymer". It should be understood that in any embodiment of the polymer used, in some cases the polymer used may be a copolymer. The repeating units forming the copolymer may be in any order. For example, the repeating units may be random, in alternating order, or as in a "block" copolymer. those. include one or more sections, where each includes a second repeating link (i.e., the second block), etc. Block copolymers may have two (diblock copolymer), three (triblock copolymer) or more different blocks constituting them.

In some embodiments, the polymer is biocompatible. Biocompatible polymers are polymers that generally do not result in significant cell death at moderate concentrations. In some embodiments, the biocompatible polymer is biodegradable, i. the polymer is capable of degrading, chemically and/or biologically, in a physiological environment, such as in the body.

In some embodiments, the particulate-forming polymer may be a protamine or a polyalkyleneimine such as polyethyleneimine.

The term "protamine" refers to any of the various relatively low molecular weight highly basic proteins that are rich in arginine and are found particularly in DNA in place of somatic histones in the spermatozoa of various animals (eg fish). In particular, the term "protamine" refers to proteins found in fish semen that are highly basic, water-soluble and do not coagulate by heating, and yield mainly arginine after hydrolysis. In their purified form, they are used in the preparation of long-acting insulin and to neutralize the anticoagulant effect of heparin.

As used herein, the term "protamine" is intended to include any protamine amino acid sequence derived from or extracted from natural or biological sources, including fragments thereof, and polymeric forms of the amino acid sequence or fragments thereof, as well as (synthesized) polypeptides, which are artificial and created specifically for specific purposes, and cannot be isolated from native or biological sources.

In one embodiment, the polyalkyleneimine comprises polyethyleneimine and/or polypropyleneimine, preferably polyethyleneimine. The preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75*10 ² -10 ⁷ Da, preferably 1000-10 ⁵ Da, more preferably 10000-40000 Da, more preferably 15000-30000 Da, even more preferably 20000-25000 Da.

Preferred according to the disclosure is a linear polyalkyleneimine such as linear polyethyleneimine (PEI).

Lipid-containing particles

In one embodiment, the RNA particles described herein include at least one lipid or lipid-like material as the particle-forming agent. Lipid carriers contemplated for use in the present invention include any substance with which RNA can be associated, for example by complexing with RNA or by forming vesicles in which RNA is enclosed or encapsulated.

The terms "lipid" and "lipid-like material" are broadly defined herein as molecules that include one or more hydrophobic particles or groups, and optionally also one or more hydrophilic particles or groups. Molecules including hydrophobic particles and hydrophilic particles are also commonly referred to as amphiphilic. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and distinct phases. One such phase consists of lipid bilayers when present in vesicles, multilamellar/monolamellar liposomes or membranes in an aqueous medium. Hydrophobicity can be imparted by the inclusion of apolar groups, which include, but are not limited to, long chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, cycloaliphatic, or heterocyclic groups. Hydrophilic groups may include polar and/or charged groups and include carbohydrates, phosphate, carboxyl, sulfate, amino, sulfhydryl, nitro, hydroxyl, and the like.

As used herein, the term "amphiphilic" refers to a molecule having both a polar moiety and a non-polar moiety. Typically, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar component is soluble in water, while the non-polar component is not soluble in water. In addition, the polar component may have either a formal positive charge or a formal negative charge. On the other hand, the polar component can have both a formal positive and negative charge and be a zwitterion or internal salt. For purposes of the disclosure, an amphiphilic compound may be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

The terms "lipid-like material", "lipid-like compound" or "lipid-like molecule" refer to substances that are structurally and/or functionally related to lipids, but cannot be considered lipids in the strict sense. For example, the term includes compounds that are capable of forming amphiphilic layers when present in vesicles, multilamellar/monolamellar liposomes, or membranes in an aqueous environment, and includes surfactants or synthesized compounds with both hydrophilic and lipophilic groups. Generally speaking, the term refers to molecules that include hydrophilic and hydrophobic groups with different structural organization, which may or may not resemble those in lipids. When used in the present description, the term "lipid" should be considered as covering both lipids and lipid-like materials, unless otherwise specified in the present description or is not dictated clearly by the context.

Specific examples of amphiphilic compounds that may be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

In some embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds which are characterized by being insoluble in water but soluble in many organic solvents. In general, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (formed by the condensation of ketoacyl subunits), steroidal lipids, and prenol lipids (formed by the condensation of isoprene subunits). Although the term "lipid" is sometimes used interchangeably with fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di- and monoglycerides and phospholipids) as well as sterol-containing metabolites such as cholesterol.

Fatty acids or fatty acid residues are a heterogeneous group of molecules formed by a hydrocarbon chain that ends with a carboxyl group; this structure gives the molecule a polar hydrophilic end and a non-polar hydrophobic end, which is insoluble in water. The carbon chain, typically four to 24 carbon atoms in length, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen and sulfur. If the fatty acid contains a double bond, geometric cis- or trans-isomerism is likely, which significantly affects the configuration of the molecule. The cis double bonds force the fatty acid chain to bend, an effect that is complicated by the few double bonds in the chain. The other major classes of lipids in the fatty acid category are fatty acid esters and fatty acid amides.

Glycerolipids include mono-, di-, and tri-substituted glycerols, with the best known triesters of fatty acids and glycerol, called triglycerides. Sometimes the word "triacylglycerol" is used as a synonym for "triglyceride". In these compounds, the three hydroxyl groups of glycerol are typically esterified each with a different fatty acid. Other subclasses of glycerolipids are glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic bond.

Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived tails by ester bonds and one head group by an ester phosphate bond. Examples of glycerophospholipids commonly referred to as phospholipids (although sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).

Sphingolipids are a complex family of compounds that share a common structural feature of the sphingoid base backbone. The most common sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acylsphingoid bases) constitute the main class of derivatives of sphingoid bases with a fatty acid attached by an amide bond. Fatty acids are usually saturated or unsaturated with a chain length of 16 to 26 carbon atoms. The main mammalian phosphosphingolipids are sphingomyelins (ceramidephosphocholines), while insects contain mainly ceramidephosphoethanolamines, and fungi have phytoceramidephosphinositols and mannose-containing head groups. Glycosphingolipids are a heterogeneous family of molecules consisting of one or more sugar residues linked by a glycosidic bond to a sphingoid base. Examples are simple and complex glycosphingolipids such as cerebrosides and gangliosides.

Sterol lipids such as cholesterol and its derivatives or tocopherol and its derivatives are an important component of membrane lipids along with glycerophospholipids and sphingomyelins.

Sugar lipids describe compounds in which fatty acids are attached directly to the sugar backbone, forming structures that are compatible with membrane bilayers. In saccharolipids, a monosaccharide replaces the glycerol backbone present in glycerolipids and glycerophospholipids. The most common saccharolipids are acylated glucosamines, precursors of the lipid A component of lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are glucosamine disaccharides, which are derivatized with as many as seven fatty acyl chains. The minimum lipopolysaccharide required for growth in E. coli is Kdo2-lipid A, a hexaacylated glucosamine disaccharide that is glycosylated with two 3-deoxy-D-mannooctulosonic acid (Kdo) residues.

Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classical enzymes, as well as iterative and multimodular enzymes that share the features of mechanisms with fatty acid synthases. They include a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose chains are usually further modified by glycosylation, methylation, hydroxylation, oxidation, and other processes.

According to the disclosure, lipids and lipid-like materials may be cationic, anionic, or neutral. Neutral lipids or lipid-like materials exist in the form of uncharged or neutral zwitterions at a chosen pH.

Preferably, the RNA particles described herein comprise a cationic or cationically ionizable lipid or lipid-like material. Cationic or ionizable cationic lipids or lipid-like materials can be used with electrostatically linked RNA. Cationically ionizable lipids or lipid-like materials are materials that are preferably only positively charged at acidic pH. This ionization behavior is believed to promote liposome release and reduce toxicity, enhancing efficacy over particles that remain cationic at physiological pH. The particles may also include non-cationic lipids or lipid-like materials. Together, anionic and neutral lipids or lipid-like materials are also referred to herein as non-cationic lipids or lipid-like materials. Optimizing the composition of RNA particles by adding hydrophobic particles other than ionizable/cationic lipid or lipid-like material, such as cholesterol and lipids, enhances particle stability and can significantly improve RNA delivery efficiency.

In one embodiment, the cationic or cationically ionizable lipid or lipid-like material includes a head group that includes at least one nitrogen (N) atom that is positively charged or protonable.

Polysarcosine conjugate

One or more of the particle-forming components described herein, such as polymers, lipids, or lipid-like materials used in the particles described herein, include polysarcosine (poly(N-methylglycine)). Polysarcosine may include acetylated (neutral end groups) or other functionalized end groups. In the case of RNA lipid particles, the polysarcosine is in one embodiment conjugated, preferably covalently linked, to a non-cationic lipid or lipid-like material contained within the particles.

In some embodiments, the terminal groups of the polysarcosine can be functionalized with one or more molecular species that impart certain properties, such as a positive or negative charge, or with a targeting agent that will direct the particle to a specific cell type, collection of cells, or tissue.

A number of targeting agents are known in the art. Non-limiting examples of targeting agents include a peptide, protein, enzyme, nucleic acid, fatty acid, hormone, antibody, carbohydrate, mono-, oligo-, or polysaccharides, peptidoglycan, glycopeptide, or the like. For example, you can use any number of different materials that bind to antigens on the surfaces of target cells. Antibodies to antigens on the surface of target cells will generally exhibit the required specificity for the target. In addition to antibodies, suitable immunoreactive fragments such as Fab, Fab', F(ab')2 or scFv fragments or single domain antibodies (eg V _H H camelid fragments) can also be used. Several antibody fragments are already available in the art for use in generating a targeting mechanism. Similarly, ligands for any of the receptors on the surface of the target cells can suitably be used as the targeting agent. They include any small molecule or biomolecule, natural or synthetic, that specifically binds to a cell surface receptor, protein or glycoprotein found on the surface of the desired target cell.

In some embodiments, the polysarcosine comprises 2 to 200, 2 to 190, 2 to 180, 2 to 170, 2 to 160, 2 to 150, 2 to 140, 2 to 130, 2 to 120, 2 to 110 2 to 100 2 to 90 2 to 80 2 to 70 5 to 200 5 to 190 5 to 180 5 to 170 5 to 160 5 to 150 5 to 140 5 to 130 5 to 120 5 to 110 5 to 100 5 to 90 5 to 80 5 to 70 10 to 200 10 to 190 10 to 180 10 to 170 10 to 160 10 to 150 10 to 140 10 to 130 10 to 120 10 to 110 10 to 100 10 to 90, 10 to 80, or 10 to 70 sarcosine units.

In some embodiments, the polysarcosine corresponds to the general formula (I)

,

,

in which x refers to the number of sarcosine units. The polysarcosine can be linked via one of the bonds to the particle-forming component or the hydrophobic component. The polysarcosine can be linked via another bond to H, a hydrophilic group, an ionizable group, or a linker to a functional group such as a targeting group.

Cationic lipid

In one embodiment, the RNA lipid particles described herein include at least one cationic lipid. As used herein, "cationic lipid" refers to a lipid having a net positive charge. Cationic lipids bind the negatively charged RNA to the lipid matrix by electrostatic interaction. Typically, cationic lipids have a lipophilic group such as a sterol, acyl chain, diacyl or longer acyl chain, and the head group of the lipid usually carries a positive charge. In some embodiments, the cationic lipid only has a net positive charge at a certain pH, in particular an acidic pH, although it preferably has no net positive charge, preferably no charge, i. it is neutral at various, preferably higher pH, such as physiological pH. For the purposes of this disclosure, such "cationic-type ionizable" lipids are included under the term "cationic lipid" unless otherwise required. Examples of cationic lipids include, but are not limited to, N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-(N′, N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propanes; 1,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl(2-hydroxyethyl)dimethylazanium (DMRIE), 1,2-dimyristoyl- sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2( sperminecarboxamide)ethyl]-N,N-dimethyl-l-propanamium (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycylspermine ( DOGS), 3-dimethylamino-2-(cholest-5-en-3-beta-hydroxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5′ -(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl -3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N '-dilinoleylcarbamyl-3-dimethylaminopropane (DL incarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl -[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6 ,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy) bromide -1-propanaminium (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), bromide (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium (GAP-DLRIE), (±)-N-(3-aminopropyl)-N bromide ,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane-1-aminium (DOBAQ), 2-({8-[(3β)-cholest- 5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine ( Octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino ]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), bromide 2 ,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropane-1-ammonium (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis( tetradecyloxy)propane-1-aminium (DMORIE), di((Z)-non-2-en-1-yl)-8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecandioate (L319), N-dodecyl-3-((2-dodecylcarbamoylethyl)-{2-[ (2-dodecylcarbamoylethyl)-2-{(2-dodecylcarbamoylethyl)-[2-(2-dodecylcarbamoylethylamino)ethyl]amino}ethylamino)propionamide (lipidoid 98N _12-5 ), 1-[2-[bis(2-hydroxydodecyl) amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl ]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200). Preferred are DODMA, DOTMA, DOTAP, DODAC and DOSPA. In specific embodiments, at least one cationic lipid is DODMA.

In some embodiments, the cationic lipid may be from about 10 mol% to about 80 mol%, from about 20 mol% to about 60 mol%, from about 25 mol% to about 55 mol%, from about 30 mol% .% to about 50 mol.%, from about 35 mol.% to about 45 mol.%, or about 40 mol.% of all lipids present in the particle.

Additional lipids

In addition to the cationic lipid, the RNA particles described herein may include one or more additional lipids. An additional lipid may be included, which may or may not affect the overall charge of the RNA particles. In some embodiments, the additional lipid is a non-cationic lipid. The non-cationic lipid may include, for example, one or more anionic lipids and/or neutral lipids. As used herein, "neutral lipid" refers to any species in the lipid range that exists in either uncharged or neutral zwitterionic form at the selected pH. In preferred embodiments, the additional lipid comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof, or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol. cholestanone, cholestenon, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

Specific phospholipids that may be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines, or sphingomyelin. Such phospholipids include, in particular, diacylphosphatidylcholines such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dtmyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibegenoylphosphatidylcholine (DBPC), dirachidoylphosphatidylcholine (DAPC), dibegenoylphosphatidylcholine (DBPC), dilauroylphosphatidylcholine (DBPC), dilignoceroylphtadicholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diester PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines such as dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE) , dilauroylphosphatidylethanolamine (DLPE), diphytanoylphosphatidylethanolamine (DPyPE) and other lipid phosphatidylethanolamines with various hydro ophobic chains.

In some preferred embodiments, the additional lipid is DSPC or DSPC and cholesterol.

In some embodiments, the RNA particle includes both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DODMA and the additional lipid is DSPC or DSPC and cholesterol.

Without wishing to be bound by any theory, the amount of at least one cationic lipid compared to the amount of at least one additional lipid can significantly affect important properties of RNA particles such as charge, particle size, stability, tissue selectivity, and bioactivity of the RNA. Accordingly, in some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1: 1.

In some embodiments, a non-cationic lipid, in particular a neutral lipid (for example, one or more phospholipids and/or cholesterol), can be from about 0 mol.% to about 90 mol.%, from about 20 mol.% to about 80 mol. %, from about 25 mol.% to about 75 mol.%, from about 30 mol.% to about 70 mol.%, from about 35 mol.% to about 65 mol.%, or from about 40 mol.% to about 60 they say % of total lipid present in the particle.

In some preferred embodiments, the non-cationic lipid, particularly a neutral lipid, comprises a phospholipid such as DSCP from about 0 mole % to about 90 mole %, from about 20 mole % to about 80 mole %, from about 25 mole % .% to about 75 mol.%, from about 30 mol.% to about 70 mol.%, from about 35 mol.% to about 65 mol.%, or from about 40 mol.% to about 60 mol.% of the total lipid, present in the particle.

In some preferred embodiments, the non-cationic lipid, in particular a neutral lipid, comprises cholesterol or a derivative thereof, from about 10 mol.% to about 80 mol.%, from about 10 mol.% to about 70 mol.%, from about 15 mol. % to about 65 mole %, from about 20 mole % to about 60 mole %, from about 235 mole % to about 55 mole %, or from about 30 mole % to about 50 mole % of the total lipid present in a particle.

In some preferred embodiments, the non-cationic lipid, in particular a neutral lipid, comprises a mixture of (i) a phospholipid such as DSPC in an amount of from about 5 mole % to about 50 mole %, from about 5 mole % to about 45 mole %. %, from about 5 mol.% to about 40 mol.%, from about 5 mol.% to about 35 mol.%, from about 5 mol.% to about 30 mol.%, from about 5 mol.% to about 25 mol.%, or from about 5 mol.% to about 20 mol.% of the total lipid present in the particle; and (ii) cholesterol or a derivative thereof, for example, cholesterol, in an amount of from about 10 mol.% to about 80 mol.%, from about 10 mol.% to about 70 mol.%, from about 15 mol.% to about 65 mol.%, from about 20 mol.% to about 60 mol.%, from about 25 mol.% to about 55 mol.%, or from about 30 mol.% to about 50 mol.% of the total lipid present in the particle. As a non-limiting example, a lipid particle comprising a mixture of phospholipid and cholesterol may include DSPC in an amount of from about 5 mol% to about 50 mol%, from about 5 mol% to about 45 mol%, from about 5 mol% to about 40 mole%, from about 5 mole% to about 35 mole%, from about 5 mole% to about 30 mole%, from about 5 mole% to about 25 mole%, or from about 5 mole% .% to about 20 mol.% of the total lipid present in the particle, and cholesterol in an amount of from about 10 mol.% to about 80 mol.%, from about 10 mol.% to about 70 mol.%, from about 15 mol. % to about 65 mole %, from about 20 mole % to about 60 mole %, from about 25 mole % to about 55 mole %, or from about 30 mole % to about 50 mole % of the total lipid present in a particle.

Polysarcosine lipid conjugate

The RNA particles described herein, such as the RNA particles described above, comprising a cationic lipid and an additional lipid, also include a polysarcosine conjugate, such as a polysarcosine-lipid conjugate. The polysarcosine can form a conjugate, in particular by being covalently linked or linked to any particle-forming component such as a lipid or lipid-like material. A polysarcosine-lipid conjugate is a molecule in which polysarcosine is conjugated to a lipid as described herein, such as a cationic lipid or a cationically ionizable lipid, or an additional lipid. On the other hand, polysarcosine is conjugated to a lipid or lipid-like material that is different from a cationic or cationically ionizable lipid or additional lipid.

In some embodiments, the polysarcosine-lipid conjugate or polysarcosine-lipid-like material conjugate has the following general formula (II)

,

,

in which one of R ₁ and R ₂ includes a hydrophobic group, and the other represents H, a hydrophilic group, an ionizable group, or a functional group including a targeting group. In one embodiment, the hydrophobic group includes a linear or branched alkyl group or an aryl group, preferably having 10 to 50, 10-40 or 12-20 carbon atoms. In one embodiment, R ₁ or R ₂ , which includes a hydrophobic group, includes a moiety such as a heteroatom, in particular N, connected to one or more linear or branched alkyl groups.

In some embodiments, a polysarcosine-lipid conjugate, or a polysarcosine-lipid-like material conjugate, is of the following general formula (III)

,

,

in which R represents H, a hydrophilic group, an ionizable group or a functional group, optionally including a targeting group.

In the present description, the symbol "x" in the general formulas, for example, the general formulas (II) and (III), refers to the number of sarcosine units, and may be the number defined in the present description.

In some embodiments, the polysarcosine-lipid conjugate or the polysarcosine-lipid-like material conjugate is a member selected from the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-dialkyloxypropyl conjugate, a polysarcosine-phospholipid conjugate, a polysarcosine-ceramide conjugate, and a mixture thereof.

In some cases, the polysarcosine-lipid conjugate may include from about 0.2 mol.% to about 50 mol.%, from about 0.25 mol.% to about 30 mol.%, from about 0.5 mol.% to about 25 mol.%, from about 0.75 mol.% to about 25 mol.%, from about 1 mol.% to about 25 mol.%, from about 1 mol.% to about 20 mol.%, from about 1 mol. % to about 15 mol.%, from about 1 mol.% to about 10 mol.%, from about 1 mol.% to about 5 mol.%, from about 1.5 mol.% to about 25 mol.%, from about 1.5 mole% to about 20 mole%, from about 1.5 mole% to about 15 mole%, from about 1.5 mole% to about 10 mole%, from about 1.5 mole% .% to about 5 mol.%, from about 2 mol.% to about 25 mol.%, from about 2 mol.% to about 20 mol.%, from about 2 mol.% to about 15 mol.%, from about 2 mol.% to about 10 mol.%, or from about 2 mol.% to about 5 mol.% of the total lipid present in the particle.

Typically, the polysarcosine group has 2 to 200, 5 to 200, 5 to 190, 5 to 180, 5 to 170, 5 to 160, 5 to 150, 5 to 140, 5 to 130, 5 to 120, 5 to 110, 5 to 100, 5 to 90, 5 to 80, 10 to 200, 10 to 190, 10 to 180, 10 to 170, 10 to 160, from 10 to 150, 10 to 140, 10 to 130, 10 to 120, 10 to 110, 10 to 100, 10 to 90, or 10 to 80 sarcosine units.

RNA lipid particles

"RNA lipid particle" includes a lipid composition that can be used to deliver RNA to a target site of interest (eg, cell, tissue, organ, etc.). An RNA lipid particle is typically formed from a cationic lipid such as DODMA, one or more non-cationic lipids such as phospholipids (eg DSPC), cholesterol or its analogs, and a polysarcosine-lipid conjugate.

Without wishing to be bound by any theory, it is believed that the cationic lipid and additional lipids combine together with RNA to form aggregates, wherein the nucleic acid binds to the lipid matrix, and this spontaneous aggregation results in colloidally stable particles.

In some embodiments, the RNA lipid particles include more than one type of RNA molecules, where the molecular parameters of the RNA molecules can be similar or different from each other, for example, in terms of molecular weight or fundamental structural elements such as molecular architecture, capping, coding regions, or others. peculiarities.

In some embodiments, the RNA lipid particles include, in addition to RNA, (i) a cationic lipid, which may include from about 10 mol% to about 80 mol%, from about 20 mol% to about 60 mol%, from about 25 mol% to about 55 mol%, from about 30 mol% to about 50 mol%, from about 35 mol% to about 45 mol%, or about 40 mol% of the total lipid present in the particle, (ii ) a non-cationic lipid, in particular a neutral lipid (for example, one or more phospholipids and/or cholesterol), which may include from about 0 mol.% to about 90 mol.%, from about 20 mol.% to about 80 mol.% , from about 25 mol.% to about 75 mol.%, from about 30 mol.% to about 70 mol.%, from about 35 mol.% to about 65 mol.%, or from about 40 mol.% to about 60 mol. .% of the total lipid present in the particle, and (iii) a polysarcosine-lipid conjugate, which may include from about 0.2 mol.% to about 50 mol.%, from about 0.25 mol.% to about 30 mol.% , from about 0.5 mol.% to about 25 mole%, from about 0.75 mole% to about 25 mole%, from about 1 mole% to about 25 mole%, from about 1 mole% to about 20 mole%, from about 1 mol% to about 15 mol%, from about 1 mol% to about 10 mol%, from about 1 mol% to about 5 mol%, from about 1.5 mol% to about 25 mol% , from about 1.5 mol.% to about 20 mol.%, from about 1.5 mol.% to about 15 mol.%, from about 1.5 mol.% to about 10 mol. %, from about 1.5 mol. % to about 5 mol. %, from about 2 mol. % to about 25 mol.%, from about 2 mol.% to about 20 mol.%, from about 2 mol.% to about 15 mol.%, from about 2 mol.% to about 10 mol.%, or from about 2 mol.% up to about 5 mol.% of the total lipid present in the particle.

In some preferred embodiments, the non-cationic lipid, in particular a neutral lipid, comprises a phospholipid in an amount of from about 5 mol% to about 50 mol%, from about 5 mol% to about 45 mol%, from about 5 mol% to about 40 mole%, from about 5 mole% to about 35 mole%, from about 5 mole% to about 30 mole%, from about 5 mole% to about 25 mole%, or from about 5 mole% up to about 20 mol.% of the total lipid present in the particle.

In some preferred embodiments, a non-cationic lipid, in particular a neutral lipid, comprises cholesterol or a derivative thereof in an amount of from about 10 mol.% to about 80 mol.%, from about 10 mol.% to about 70 mol.%, from about 15 mol. % to about 65 mole %, from about 20 mole % to about 60 mole %, from about 25 mole % to about 55 mole %, or from about 30 mole % to about 50 mole % of the total lipid present in a particle.

In some preferred embodiments, the non-cationic lipid, in particular a neutral lipid, comprises a mixture of (i) a phospholipid such as DSPC from about 5 mol% to about 50 mol%, from about 5 mol% to about 45 mol%, from about 5 mole% to about 40 mole%, from about 5 mole% to about 35 mole%, from about 5 mole% to about 30 mole%, from about 5 mole% to about 25 mole% or from about 5 mol.% to about 20 mol.% of the total lipid present in the particle; and (ii) cholesterol or its derivative, for example, cholesterol, from about 10 mol.% to about 80 mol.%, from about 10 mol.% to about 70 mol.%, from about 15 mol.% to about 65 mol.%. %, from about 20 mol.% to about 60 mol.%, from about 25 mol.% to about 55 mol.%, or from about 30 mol.% to about 50 mol.% of the total lipid present in the particle. As a non-limiting example, a lipid particle comprising a mixture of phospholipid and cholesterol may include DSPC from about 5 mol% to about 50 mol%, from about 5 mol% to about 45 mol%, from about 5 mol% to about 40 mole%, from about 5 mole% to about 35 mole%, from about 5 mole% to about 30 mole%, from about 5 mole% to about 25 mole%, or from about 5 mole% up to about 20 mole% of the total lipid present in the particle, and cholesterol from about 10 mole% to about 80 mole%, from about 10 mole% to about 70 mole%, from about 15 mole% to about 65 mol.%, from about 20 mol.% to about 60 mol.%, from about 25 mol.% to about 55 mol.%, or from about 30 mol.% to about 50 mol.% of the total lipid present in the particle.

Typically, the polysarcosine group has 2 to 200, 5 to 200, 5 to 190, 5 to 180, 5 to 170, 5 to 160, 5 to 150, 5 to 140, 5 to 130, 5 to 120, 5 to 110, 5 to 100, 5 to 90, 5 to 80, 10 to 200, 10 to 190, 10 to 180, 10 to 170, 10 to 160, from 10 to 150, 10 to 140, 10 to 130, 10 to 120, 10 to 110, 10 to 100, 10 to 90, 10 to 80 sarcosine units.

In some embodiments, the RNA lipid particles other than RNA include (i) DODMA, which may include from about 10 mol% to about 80 mol%, from about 20 mol% to about 60 mol%, from about 25 mol% up to about 55 mol%, from about 30 mol% to about 50 mol%, from about 35 mol% to about 45 mol%, or about 40 mol% of the total lipid present in the particle, (ii) DSPC, which may include from about 5 mole% to about 50 mole%, from about 5 mole% to about 45 mole%, from about 5 mole% to about 40 mole%, from about 5 mole% to about 35 mol%, from about 5 mol% to about 30 mol%, from about 5 mol% to about 25 mol%, or from about 5 mol% to about 20 mol% of the total lipid present in the particle, (iii) cholesterol, which may include from about 10 mol.% to about 80 mol.%, from about 10 mol.% to about 70 mol.%, from about 15 mol.% to about 65 mol.%, from about 20 mol.% to about 60 mol.%, from about 25 mol.% to about 55 mol.% or from about 30 mol% to about 50 mol% of the total lipid present in the particle, and (iv) a polysarcosine-lipid conjugate, which may include from about 0.2 mol% to about 50 mol%, from about 0 25 mole% to about 30 mole%, about 0.5 mole% to about 25 mole%, about 0.75 mole% to about 25 mole%, about 1 mole% to about 25 mole%, from about 1 mole% to about 20 mole%, from about 1 mole% to about 15 mole%, from about 1 mole% to about 10 mole%, from about 1 mole% to about 5 mol.%, from about 1.5 mol.% to about 25 mol.%, from about 1.5 mol.% to about 20 mol.%, from about 1.5 mol.% to about 15 mol. %, from about 1.5 mol.% to about 10 mol.%, from about 1.5 mol.% to about 5 mol.%, from about 2 mol.% to about 25 mol.%, from about 2 mol. % to about 20 mol.%, from about 2 mol. % to about 15 mol. %, from about 2 mol. % to about 10 mol. % or from about 2 mol.% to about 5 mol.% of the total lipid present in the particle.

In some embodiments, the RNA particles comprise a polysarcosine-lipid conjugate of general formula (II) or (III), DODMA, DSPC, and cholesterol.

RNA particle diameter

The RNA particles described herein have an average diameter that, in one embodiment, ranges from about 30 nm to about 1000 nm, from about 30 nm to about 800 nm, from about 30 nm to about 700 nm, from about 30 nm to about 600 nm, from about 30 nm to about 500 nm, from about 30 nm to about 450 nm, from about 30 nm to about 400 nm, from about 30 nm to about 350 nm, from about 30 nm to about 300 nm, from about 30 nm to about 250 nm, from about 30 nm to about 200 nm, from about 30 nm to about 190 nm, from about 30 nm to about 180 nm, from about 30 nm to about 170 nm, from about 30 nm to about 160 nm, from about 30 nm to about 150 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 50 nm for about 190 nm, about 50 nm to about 180 nm, about 50 nm to about 170 nm, about 50 nm to about 160 nm, or about 50 nm to about 150 nm.

In some embodiments, the RNA particles described herein have an average diameter that ranges from about 40 nm to about 800 nm, from about 50 nm to about 700 nm, from about 60 nm to about 600 nm, from about 70 nm to about 500 nm, from about 80 nm to about 400 nm, from about 150 nm to about 800 nm, from about 150 nm to about 700 nm, from about 150 nm to about 600 nm, from about 200 nm to about 600 nm, from about 200 nm to about 500 nm, or from about 200 nm to about 400 nm.

RNA particles described herein, for example, obtained by the methods described herein, show a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, or less than about 0, 1 or less. As an example, RNA particles can show a polydispersity index ranging from about 0.1 to about 0.3.

RNA

In the present disclosure, the term "RNA" refers to nucleic acid molecules that include ribonucleotide residues. In preferred embodiments, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide with a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. RNA includes, without limitation, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or a change in one or more nucleotides. Such changes may relate to the addition of non-nucleotide material to the internal nucleotides of the RNA or to the end(s) of the RNA. The present invention also contemplates that the nucleotides in RNA may be non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. For the purposes of this disclosure, such altered RNAs are considered analogues of naturally occurring RNA. In certain embodiments, the RNA of the invention comprises a population of different RNA molecules, for example a mixture of different RNA molecules, optionally encoding different peptides and/or proteins. Thus, according to the invention, the term "RNA" may include a mixture of RNA molecules.

In some embodiments of the present disclosure, RNA is messenger RNA (mRNA), which refers to an RNA transcript that codes for a peptide or protein. As established in the art, mRNA typically contains a 5'-untranslated region (5'-UTR), a peptide-coding region, and a 3'-untranslated region (3'-UTR). In some embodiments, RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid that contains deoxyribonucleoids.

In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter for regulating transcription can be any promoter for any RNA polymerase. A DNA template for in vitro transcription can be obtained by cloning a nucleic acid, in particular a cDNA, and inserting it into an appropriate in vitro transcription vector. cDNA can be obtained by reverse transcription of RNA.

et al., Future Microbiol., 2009, vol. 4, pp. 837–856). Общая длина генома многих альфавирусов обычно колеблется от 11000 до 12000 нуклеотидов, и геномная РНК типично имеет 5’-кэп и 3’-поли(A) хвост. Геном альфавирусов кодирует неструктурные белки (вовлеченные в транскрипцию, модификацию и репликацию вирусной РНК и в модификацию белков) и структурные белки (образующие вирусную частицу). В геноме обычно имеются две открытые рамки считывания (ORF). Четыре неструктурных белка (nsP1–nsP4) типично кодированы вместе первой ORF, начинающейся около 5′-конца генома, в то время как структурные белки альфавирусов кодируются вместе второй ORF, которая находится ниже первой ORF и продолжается до почти 3’-конца генома. Типично первая ORF больше второй ORF, причем соотношение составляет приблизительно 2:1. В клетках, инфицированных альфавирусом, только нуклеотидная последовательность, кодирующая неструктурные белки, транслируется из геномной РНК, в то время как генетическая информация, кодирующая структурные белки, может транслироваться из субгеномного транскрипта, который представляет собой молекулу РНК, которая имеет сходство с эукариотической мессенджерной РНК (мРНК; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111–124). После заражения, т.е. на ранних этапах жизненного цикла вируса, (+)-цепочечная геномная РНК действует непосредственно, подобно мессенджерной РНК, на трансляцию открытой рамки считывания, кодирующей неструктурный полипротеин (nsP1234). Происходящие из альфавируса векторы предложены для доставки чужеродной генетической информации в клетки-мишени или организмы-мишени. В простых подходах открытую рамку считывания, кодирующую структурные белки альфавируса, заменяют открытой рамкой считывания, кодирующей белок, представляющий интерес. Системы трансрепликации на основе альфавирусов зависят от элементов нуклеотидной последовательности альфавируса в двух отдельных молекулах нуклеиновой кислоты: одна молекула нуклеиновой кислоты кодирует вирусную репликазу, и другая молекула нуклеиновой кислоты способна реплицироваться указанной репликазой в транс (отсюда обозначение «система трансрепликации»). Трансрепликация требует присутствия обеих этих молекул нуклеиновой кислоты в данной клетке-хозяине. Молекула нуклеиновой кислоты, способная реплицироваться репликазой в транс, должна включать некоторые элементы последовательности альфавируса для возможности узнавания и синтеза РНК репликазой альфавируса.In some embodiments of the present disclosure, the RNA is an RNA replicon or simply a "replicon", in particular a self-replicating RNA. In one particularly preferred embodiment, the replicon or self-replicating RNA is derived from or includes elements from a virus ssRNA, in particular a positive strand ssRNA virus such as an alphavirus. Alphaviruses are typical positive-strand RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for an overview of the life cycle of alphaviruses, see

et al., Future Microbiol., 2009, vol. 4, pp. 837–856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and genomic RNA typically has a 5' cap and a 3' poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and protein modification) and structural proteins (forming the viral particle). There are usually two open reading frames (ORFs) in the genome. The four non-structural proteins (nsP1–nsP4) are typically encoded together with the first ORF starting near the 5' end of the genome, while the structural proteins of alphaviruses are encoded together with the second ORF, which is below the first ORF and extends almost to the 3' end of the genome. Typically, the first ORF is greater than the second ORF, with a ratio of approximately 2:1. In alphavirus-infected cells, only the nucleotide sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins can be translated from the sub-genomic transcript, which is an RNA molecule that shares similarities with eukaryotic messenger RNA ( mRNA, Gould et al., 2010, Antiviral Res., vol. 87 pp. 111–124). After infection, i.e. early in the life cycle of the virus, (+)-strand genomic RNA acts directly, like messenger RNA, to translate the open reading frame encoding the non-structural polyprotein (nsP1234). Alphavirus-derived vectors have been proposed for delivering foreign genetic information to target cells or organisms. In simple approaches, an open reading frame encoding alphavirus structural proteins is replaced with an open reading frame encoding a protein of interest. Alphavirus-based transreplication systems rely on alphavirus nucleotide sequence elements in two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of replicating said replicase in trans (hence the designation "transreplication system"). Transreplication requires the presence of both of these nucleic acid molecules in a given host cell. A nucleic acid molecule capable of being replicated in trans by the replicase must include some elements of the alphavirus sequence in order for the alphavirus replicase to recognize and synthesize RNA.

In some embodiments of the present disclosure, the RNA in the RNA particles described herein is at a concentration of from about 0.002 mg/mL to about 5 mg/mL, from about 0.002 mg/mL to about 2 mg/mL, from about 0.005 mg/mL. ml to about 2 mg/ml, from about 0.01 mg/ml to about 1 mg/ml, from about 0.05 mg/ml to about 0.5 mg/ml, or from about 0.1 mg/ml to about 0.5 mg/ml. In specific embodiments, the RNA is at a concentration of from about 0.005 mg/ml to about 0.1 mg/ml, from about 0.005 mg/ml to about 0.09 mg/ml, from about 0.005 mg/ml to about 0.08 mg/ml ml, from about 0.005 mg/ml to about 0.07 mg/ml, from about 0.005 mg/ml to about 0.06 mg/ml, or from about 0.005 mg/ml to about 0.05 mg/ml.

In one embodiment, the RNA may have modified ribonucleotides. Examples of modified ribonucleotides include, without limitation, 5-methylcystidine, pseudouridine (ψ), N1-methylpseudouridine (m ¹ ψ) or 5-methiuridine (m ⁵ U).

In some embodiments, the RNA according to the present disclosure includes a 5' cap. In one embodiment, the RNA of the present disclosure does not have uncapped 5'-triphosphates. In one embodiment, the RNA may be modified with a 5' cap analog. The term "5' cap" refers to a structure found at the 5' end of an RNA molecule, and usually consisting of a guanosine nucleotide linked to the mRNA via a 5'-5' triphosphate bond. In one embodiment, this guanosine is methylated at position 7. The provision of a 5'-capped RNA or 5'-cap analog can be achieved by in vitro transcription in which the 5'-cap is co-expressed by transcription into the RNA strand, or can be attached to the RNA after transcription. using capping enzymes.

In some embodiments, the RNA of the present disclosure includes a 5'UTR and/or a 3'UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) may be present 5' (above) the open reading frame (5'-UTR) and/or 3' (below) the open reading frame (3'-UTR). The 5'-UTR, if present, is located 5' upstream of the start codon of the protein-coding region. The 5'-UTR is located below the 5'-cap (if present), for example, in close proximity to the 5'-cap. The 3'UTR, if present, is located 3' downstream of the termination codon of the protein-coding region, but the term "3'UTR" preferably does not include the poly-(A) tail. Thus, the 3'-UTR is located upstream of the poly-(A) sequence (if present), for example, in close proximity to the poly-(A) sequence.

In some embodiments, the RNA according to the present disclosure includes the sequence 3'-poly-(A). The term "poly-(A) sequence" refers to a sequence of adenyl (A) residues that is typically located at the 3' end of an RNA molecule. According to the disclosure, in one embodiment, the poly-(A) sequence comprises at least about 20, at least about 40, at least about 80, at least about 100, and up to about 500, up to about 400, up to about 300, up to about 200 or up to about 150 A nucleotides, and in particular about 120 A nucleotides.

In the context of the present disclosure, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into a peptide or protein.

In relation to RNA, the term "expression" or "translation" refers to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of an amino acid sequence to form a peptide or protein.

The RNA may be coding RNA, ie. RNA encoding a peptide or protein. Said RNA may express the encoded peptide or protein. Alternatively, said RNA may be a non-coding RNA such as antisense RNA, micro RNA (miRNA) or siRNA.

The RNA used in the present invention may be a pharmaceutically active RNA. A "pharmaceutically active RNA" is an RNA that encodes a pharmaceutically active peptide or protein or is pharmaceutically active in itself, for example, it has one or more pharmaceutical activities, such as those described for pharmaceutically active proteins, for example, immunostimulatory activity. For example, the RNA may be one or more strands of RNA interference (RNAi). Such agents include short interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) or precursor siRNAs or miRNA-like RNAs targeted to a target transcript, eg, the subject's endogenous disease-associated transcript.

Some aspects of the disclosure include targeted delivery of the RNA disclosed herein to specific cells or tissues. In one embodiment, the disclosure includes targeting the lymphatic system, in particular secondary lymphoid organs, more specifically the spleen. Targeting the lymphatic system, in particular secondary lymphoid organs, more specifically the spleen, is particularly preferred if the RNA administered is RNA encoding an antigen or epitope to induce an immune response. In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen presenting cell, such as a professional antigen presenting cell of the spleen. In one embodiment, the target cell is a dendritic cell in the spleen. The "lymphatic system" is part of the circulatory system and an important part of the immune system, comprising a network of lymph vessels that carry lymph. The lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature precursor cells. The thymus and bone marrow make up the primary lymphoid organs. Secondary or peripheral lymphoid organs, which include the lymph nodes and spleen, retain mature naive lymphocytes and initiate an adaptive immune response.

Lipid-based RNA delivery systems are preferred for the liver. Accumulation in the liver is caused by the intermittent nature of the hepatic vasculature or by lipid (liposome-lipid conjugates or cholesterol) metabolism. In one embodiment, the target organ is the liver and the target tissue is liver tissue. Delivery to such a target tissue is preferred, in particular if the presence of the RNA or encoded peptide or protein is desired in that organ and/or if it is desired to express large amounts of the encoded peptide or protein and/or if the systemic presence of the encoded peptide or protein is desired or required. especially in significant quantities.

In one embodiment, following administration of the RNA particles described herein, at least a portion of the RNA is delivered to the target cell or organ. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is an RNA encoding a peptide or protein, and the RNA is translated by the target cell to produce the peptide or protein. In one embodiment, the target cell is a liver cell. In one embodiment, the target cell is a muscle cell. In one embodiment, the target cell is an endothelial cell. In one embodiment, the target cell is a tumor cell or a cell in the tumor microenvironment. In one embodiment, the target cell is a blood cell. In one embodiment, the target cell is a cell in the lymph nodes. In one embodiment, the target cell is a cell in the lung. In one embodiment, the target cell is a blood cell. In one embodiment, the target cell is a skin cell. In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell in the spleen. In one embodiment, the target cell is a T cell. In one embodiment, the target cell is a B cell. In one embodiment, the target cell is an NK cell. In one embodiment, the target cell is a monocyte. Thus, the RNA particles described herein can be used to deliver RNA to such target cells. Accordingly, the present disclosure also relates to a method for delivering RNA to a target cell of a subject, comprising administering to the subject the RNA particles described herein. In one embodiment, the RNA is an RNA encoding a peptide or protein, and the RNA is translated by the target cell to produce the peptide or protein.

In one embodiment, the RNA encodes a pharmaceutically active peptide or protein.

According to the disclosure, the term "RNA encodes" means that RNA, if present in an appropriate environment, such as in cells of a target tissue, can direct the assembly of amino acids to produce the peptide or protein (which) it encodes during the translation process. In one embodiment, the RNA is capable of interacting with the cellular translation machinery to allow translation of the peptide or protein. The cell may produce the encoded peptide or protein intracellularly (eg, in the cytoplasm), may secrete the encoded peptide or protein, or may produce it on the surface.

According to the disclosure, the term "peptide" includes oligo- and polypeptides and refers to substances that include about two or three, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids connected to each other by peptide bonds. The term "protein" refers to large peptides, in particular peptides having at least about 151 amino acids, but the terms "peptide" and "protein" are used interchangeably herein.

A "pharmaceutically active peptide or protein" or "therapeutic peptide or protein" has a positive or beneficial effect on a condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In one embodiment, the pharmaceutically active peptide or protein has curative or palliative properties and may be administered to ameliorate, alleviate, alleviate, reverse, delay the onset or lessen the severity of one or more symptoms of a disease or disorder. The pharmaceutically active peptide or protein may have prophylactic properties and may be used to delay the onset of a disease or reduce the severity of such a disease or condition. The term "pharmaceutically active peptide or protein" includes complete proteins or polypeptides and may also refer to their pharmaceutically active fragments. It may also include pharmaceutically active analogs of the peptide or protein.

Examples of pharmaceutically active proteins include, but are not limited to, cytokines and their derivatives, such as fusion cytokines (like albumin-fused cytokines), and immune system proteins, such as immunologically active compounds (e.g., interleukins, colony stimulating factor (CSF), granulocytic colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, seletins, homing receptors, T-cell receptors, chimeric antigen receptors (CAR ), immunoglobulins, including antibodies or bispecific antibodies, e.g. for immune stimulation or production of neutralizing antibodies in case of viral/bacterial infection, soluble major histocompatibility complex antigens, immunologically active antigens such as bacterial, parasite or viral antigens, allergens, self-antigens, antibodies ), hormones (insulin, thyroid hormone, catecholamines, gonadotropins, tropic hormones, prolactin, oxytocin, dopamine, bovine somatotropin, leptins, etc.), growth hormones (eg, human growth hormone), growth factors (eg, epidermal growth factor, nerve growth factor, insulin-like growth factor, etc.) .p.), growth factor receptors, enzymes (tissue plasminogen activator, streptokinase, biosynthesized or degradative cholesterol), steroidogenic enzymes, kinases, phosphodiesterases, methylases, demethylases, dehydrogenases, cellulases, proteases, lipases, phospholipases, aromatases, cytochromes, adenylate or guanylate cyclases, neuroamidases, lysosomal enzymes, etc.), receptors (steroid hormone receptors, peptide receptors), binding proteins (proteins that bind growth hormone or growth factors, etc.), transcription and translation factors, proteins, tumor growth inhibitors (such as proteins that inhibit angiogenesis), structural proteins (such as collagen, fibrin, fibrinogen, elastin, tubulin, actin, and myosin), blood proteins and (thrombin, serum albumin, factor VII, factor VIII, insulin, factor IX, factor X, tissue plasminogen activator, protein C, von Willebrand factor, antithrombin III, glucocerebrosidase, erythropoietin, granulocyte colony stimulating factor (GCSF) or modified factor VIII, anticoagulants, etc.

The term "immunologically active compound" refers to any compound that alters the immune response, for example, by inducing and/or inhibiting the maturation of immune cells, inducing and/or inhibiting cytokine biosynthesis, and/or altering humoral immunity by stimulating the production of antibodies by B cells. The immunologically active compounds have potent immunostimulatory activity, including, but not limited to, antiviral and antitumor activity, and can also downregulate other aspects of the immune response, such as shifting the immune response away from the TH2 immune response, which is useful in the treatment of a wide range of TH2 mediated diseases. Immunologically active compounds may be useful as vaccine adjuvants.

In one embodiment, the pharmaceutically active peptide or protein comprises a cytokine. The term "cytokine" refers to a class of small proteins (~5-20 kDa) that are important in cell signaling. Their release has an effect on the behavior of the surrounding cells. Cytokines are involved in autocrine signaling, paracrine signaling, and endocrine signaling as immunomodulators. Cytokines include chemokines, interferons, interleukins, lympholeukins, and tumor necrosis factors, but generally not hormones or growth factors (despite some overlap in terminology). Cytokines are produced by a wide variety of cells, including immune cells like macrophages, B-lymphocytes, T-lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by more than one cell type. Cytokines act through receptors and are especially important in the immune system; cytokines modulate the balance between humoral and cellular immune responses, and they regulate the maturation, growth, and reactivity of individual cell populations. Some cytokines enhance or inhibit the action of other cytokines in complex ways.

In one embodiment, the pharmaceutically active protein of the invention is a cytokine that is involved in the regulation of lymphoid homeostasis, preferably a cytokine that is involved in and preferably induces or enhances the development, priming, expansion, differentiation and/or survival of T cells. In one embodiment, the cytokine is an intelukin. In one embodiment, the pharmaceutically active protein of the invention is an interleukin selected from the group consisting of IL-2, IL-7, IL-12, IL-15 and IL-21.

In one embodiment, the pharmaceutically active peptide or protein comprises a replacement protein. In this embodiment, the present invention relates to a method of treating a subject with a protein replacement disorder (eg, from protein deficiency disorders) comprising administering to the subject the RNA described herein encoding the replacement protein. The term "protein replacement" refers to the administration of a protein (including functional variants thereof) to a subject deficient in such protein. The term also refers to the administration of a protein to a subject otherwise requiring or benefiting from the provision of a protein, such as those suffering from a protein deficiency. The term "disorder characterized by protein deficiency" refers to any disorder that occurs with a pathology caused by the absence or insufficient amounts of protein. This term covers protein folding disorders, i.e. conformational disturbances that result in a biologically inactive protein product. Protein deficiencies can be involved in infectious disease, immunosuppression, organ failure, iron problems, radiation sickness, nutritional deficiencies, poisoning, or other environmental or external injury.

In one embodiment, the pharmaceutically active peptide or protein comprises one or more antigens or one or more epitopes, i.e. administering a peptide or protein to a subject produces an immune response in the subject against one or more antigens or one or more epitopes that may be therapeutically wholly or partially protective.

The term "antigen" refers to an agent that includes an epitope against which it can generate an immune response. The term "antigen" includes, in particular, proteins and peptides. In one embodiment, the antigen is presented by cells of the immune system, such as antigen presenting cells like dendrites or macrophages. The antigen or its processing product, such as a T cell epitope, is in one embodiment bound to a T or B cell receptor or an immunoglobulin molecule, such as an antibody. Accordingly, the antigen or its processing product can specifically interact with antibodies or T-lymphocytes (T-cells). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, and the epitope is derived from such an antigen.

The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule that contains epitopes that will stimulate the host's immune system to elicit a cellular antigen-specific immune response and/or a humoral antibody response against the disease. Therefore, an antigen associated with a disease, or an epitope thereof, can be used for therapeutic purposes. Disease-associated antigens may be associated with infection via microbes, typically microbial antigens, or associated with cancer, typically tumors.

The term "tumor antigen" refers to an element of cancer cells that can be derived from the cytoplasm, cell surface, and cell nucleus. In particular, it refers to those antigens that are produced within cells or as surface antigens on tumor cells.

The term "viral antigen" refers to any component of a virus that has antigenic properties, i.e. capable of inducing an immune response in an individual. The viral antigen may be a viral ribonucleoprotein or an envelope protein.

The term "antigen of a bacterium" refers to any component of a bacterium that has antigenic properties, i. capable of inducing an immune response in an individual. The bacterial antigen may be derived from the cell wall or cytoplasmic membrane of the bacterium.

The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope may be recognized by T cells, B cells, or antibodies. An antigen epitope may include a continuous or discontinuous portion of the antigen and may be from about 5 to about 100, for example, from about 5 to about 50, more preferably from about 8 to about 30, most preferably from about 10 to about 25 amino acids in length, for example, the epitope may preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids in length. In one embodiment, the epitope is from about 10 to about 25 amino acids in length. The term "epitope" includes T-cell epitopes.

The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" includes MHC class I and MHC class II molecules and refers to a complex of genes that are present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune responses, wherein the MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells. MHC-encoded proteins are expressed on the surface of cells and reflect both self antigens (peptide fragments from the cell itself) and non-self antigens (eg fragments of invading microorganisms) to T cells. In the case of MHC class I/peptide complexes, binding peptides are typically about 8 to about 10 amino acids in length, although longer or shorter peptides may be effective. In the case of MHC class II/peptide complexes, binding peptides are typically about 10 to about 25 amino acids in length, and in particular about 13 to about 18 amino acids in length, although longer or shorter peptides may be effective.

The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTL, CD8+ T cells), which include cytolysis T cells. The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes the antigen targeted by the T cell, in particular when presented on the surface of antigen presenting cells or diseased cells such as cancer cells, in the context of MHC molecules, and preferably exhibits T cell effector functions. T cells are considered specific for an antigen if the cells kill target cells expressing the antigen. The specificity of T cells can be assessed using various standard methods such as chromium release assay or proliferation assay. On the other hand, the synthesis of lymphokines (such as interferon-γ) can be measured. In some embodiments of the present disclosure, the RNA encodes at least one epitope.

In some embodiments, the epitope is derived from a tumor antigen. The tumor antigen may be a "standard" antigen that is generally known to be expressed in various cancers. The tumor antigen may also be a "neoantigen" that is specific for a tumor in an individual and has not been previously recognized by the immune system. A neoantigen or neoepitope can lead to one or more cancer-specific mutations in the genome of cancer cells, resulting in an amino acid substitution. Examples of tumor antigens include, without limitation, p53, ART-4, BAGE, beta-catenin/m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, claudin family cell surface proteins , such as CLAUD ΓΝ-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 /neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE- A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A 10, MAGE-A 1 1 or MAGE-A12, MAGE-B, MAGE-C, MART-1/melan-A, MC1R , myosin/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor BCR-abL, Pml/RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, Survivin, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE, WT and WT-1.

Cancer mutations change with each individual. Thus, cancer mutations that encode new epitopes (neoepitopes) are attractive targets in the development of vaccine and immunotherapy compositions. The effectiveness of tumor immunotherapy is based on the selection of cancer-specific antigens and epitopes that can induce a strong immune response in the host. RNA can be used to deliver patient-specific tumor epitopes to a patient. Dendritic cells (DC) found in the spleen are antigen-presenting cells of particular interest for RNA expression of immunogenic epitopes or antigens such as tumor epitopes. The use of multiple epitopes has been shown to promote therapeutic efficacy in anticancer vaccine formulations. Tumor methanome rapid sequencing can provide a variety of epitopes for individualized vaccines, which can be encoded by the RNA described herein, for example, as a single peptide, with the epitopes optionally separated by linkers. In some embodiments of the present disclosure, the RNA encodes at least one epitope, at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes. Exemplary embodiments include RNA that encodes for at least five epitopes (called "pentatope"), RNA that encodes for at least ten epitopes (called "decatope"), RNA that encodes for at least twenty epitopes (called "eicosatope") .

Compositions Including RNA Particles

The term "multiple RNA particles" or "multiple RNA-lipid particles" refers to a population of a certain number of particles. In some embodiments, the term refers to a population greater than 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ , 10 ¹² , 10 ¹³ , 10 ¹⁴ , 10 ¹⁵ , 10 ¹⁶ , 10 ¹⁷ , 10 ¹⁸ , 10 ¹⁹ , 10 ²⁰ , 10 ²¹ , 10 ²² , or 10 ²³ or more particles.

It will be apparent to those skilled in the art that the plurality of particles may include any fraction of the above ranges or any range herein.

In embodiments, the composition of the present disclosure is liquid or solid. Non-limiting examples of a solid composition include a frozen form, a lyophilized form, or a spray-dried form. In a preferred embodiment, the composition is liquid.

According to the present disclosure, the compositions described herein may include salts such as organic or inorganic salts, including, but not limited to, sodium chloride, potassium chloride, dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, disodium phosphate, monosodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and sodium salts of ethylenediaminetetraacetic acid (EDTA) and amino acids.

The compositions described herein may also include a stabilizer in order to avoid significant loss of product quality, and in particular, significant loss of RNA activity during storage, freezing, lyophilization and/or spray drying, for example, to reduce or prevent aggregation, flattening of particles, destruction of RNA and/or other types of damage.

In one embodiment, the stabilizer is a cryoprotectant or lyoprotectant.

In one embodiment, the stabilizer is a carbohydrate. The term "carbohydrate" as used herein refers to and encompasses monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides.

In one embodiment, the stabilizer is an amino acid or a surfactant (eg, poloxamer).

According to the present disclosure, the RNA particle compositions described herein have a pH suitable for the stability of the RNA particles, and in particular for the stability of the RNA. In one embodiment, the RNA particle compositions described herein have a pH of about 4.0 to about 8.0, or about 5.0 to about 7.5. Without wishing to be bound by any theory, the use of a buffer maintains the pH of the composition during manufacture, storage and use of the composition. In some embodiments of the present disclosure, the buffer may be sodium bicarbonate, monosodium phosphate, monopotassium phosphate, dipotassium phosphate, [tris(hydroxymethyl)methylamino]propane sulfonic acid (TAPS), 2-(bis(2-hydroxyethyl)amino)acetic acid (bicine), 2-amino -2-(hydroxymethyl)propane-1,3-diol (tris), N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine (tricine), 3-[[1,3-dihydroxy-2 -(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid (TAPSO), 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES), 2-[ [1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid (TES), 1,4-piperazinediethanesulfonic acid (PIPES), dimethylarsinic acid, 2-morpholin-4-ylethanesulfonic acid (MES) , 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO) or phosphate buffered saline (PBS). Other suitable buffering systems may be acetic acid alone or with a salt, citric acid alone or with a salt, boric acid alone or with a salt and one phosphoric acid or with a salt, or amino acids and amino acid derivatives.

Some embodiments of the present disclosure involve the use of a chelating agent in the composition described in the present description. Chelating agents refer to chemical compounds that are capable of forming at least two coordination covalent bonds with a metal ion, whereby a stable water-soluble complex is generated. Without wishing to be bound by any theory, chelating agents reduce the concentration of free divalent ions, which may otherwise cause accelerated degradation of the RNA of the present disclosure. Examples of suitable chelating agents include, without limitation, ethylenediaminetetraacetic acid (EDTA), EDTA salt, desferrioxamine B, deferoxamine, dithiocarbsodium, penicillamine, calcium pentetate, pentetic acid sodium, succimer, trientine, nitrilotriacetic acid, trans-diaminocyclohexanetetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), N,N,N',N'-tetraacetic acid bis(aminoethyl)glycol ester, iminodiacetic acid, citric acid, tartaric acid, fumaric acid, or salts thereof. In some embodiments, the chelating agent is EDTA or an EDTA salt. In an exemplary embodiment, the chelating agent is EDTA disodium salt dihydrate.

In some embodiments, the EDTA is at a concentration of about 0.05 mM to about 5 mM, about 0.1 mM to about 2.5 mM, or about 0.25 mM to about 1 mM.

Pharmaceutical compositions

The compositions comprising the RNA particles described herein are useful as or for the preparation of pharmaceutical compositions or drugs for treatment or prophylaxis.

In one aspect, the RNA particles described herein are present in a pharmaceutical composition. In another aspect, the composition described in the present description is a pharmaceutical composition.

The particles of the present disclosure may be administered in the form of any suitable pharmaceutical composition.

The term "pharmaceutical composition" refers to a composition comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. Said pharmaceutical composition is used to treat, prevent, or lessen the severity of a disease or disorder by administering said pharmaceutical composition to a subject. A pharmaceutical composition is also known in the art as a pharmaceutical formulation. In the context of the present disclosure, the pharmaceutical composition includes the RNA particles described in the present description.

Pharmaceutical compositions of the present disclosure may include one or more adjuvants, or may be administered with one or more adjuvants. The term "adjuvant" refers to a compound that prolongs, enhances or accelerates an immune response. Adjuvants include a heterogeneous group of mixtures such as emulsions (eg Freund's adjuvants), inorganic compounds (such as alum), bacterial products (such as pertussis toxin), or immunostimulatory complexes. Examples of adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines such as monoxins, lymphokines, interleukins, chemokines. Chemokines can be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, INFa, INF-γ, GM-CSF, LT-a. Other known adjuvants are aluminum hydroxide, Freund's adjuvant or an oil such as Montanide® ISA51. Other adjuvants suitable for use in the present disclosure include lipopeptides such as Pam3Cys as well as lipophilic components such as saponins, trehalose-6,6-dibehenate (TDB), monophosphoryl lipid A (MPL), monomycoloyl glycerol (MMG), or the adjuvant glucopyranosyl lipid. (G.L.A.).

Pharmaceutical compositions according to the present disclosure are generally used in a "pharmaceutically acceptable amount" and in a "pharmaceutically acceptable formulation". The term "pharmaceutically acceptable" refers to the absence of toxicity in a material that does not interact with the active ingredient of the pharmaceutical composition.

The term "pharmaceutically effective amount" refers to the amount at which the desired response or desired effect is achieved, alone or together with other doses. In the case of treating a certain disease, the desired response preferably refers to the inhibition of the course of the disease. This includes slowing down the progress of the disease and in particular interrupting or reversing the progress of the disease. The desired response in the treatment of a disease may also be to delay the onset or prevent the onset of said disease or said condition. The effective amount of the particles or compositions described herein will depend on the condition being treated, the severity of the disease, the individual parameters of the patient, including age, physiological state, size and weight, duration of treatment, type of concomitant therapy (if any), specific method introduction and similar factors. Accordingly, the doses of the administered particles or compositions described herein may depend on many such parameters. In the event that the patient's response is insufficient at the initial dose, higher doses may be used (or effectively higher doses achieved by another more localized route of administration).

Pharmaceutical compositions of the present disclosure may contain salts, buffers, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure include one or more pharmaceutically acceptable carriers, diluents and/or excipients.

Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben, and thimerosal. The term "excipient", as used in the present description, refers to a substance that may be present in the pharmaceutical composition of the present disclosure, but is not an active ingredient. Examples of excipients include, without limitation, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavors, or colorants.

The term "diluent" refers to a diluting and/or thinning agent. In addition, the term "diluent" includes any one or more liquids, liquid or solid suspension and/or mixed media. Examples of suitable diluents include ethanol, glycerin and water.

The term "carrier" refers to a component, which may be natural, synthetic, organic, inorganic, with which the active ingredient is combined in order to facilitate, enhance or enable the administration of a pharmaceutical composition. The carrier when used in the present invention may be one or more compatible liquid or solid excipients, diluents, or encapsulating agents that are suitable for administration to a subject. Suitable carriers include, without limitation, sterile water, Ringer's solution, Ringer's lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and in particular biocompatible polylactides, lactide-glycolide copolymers, or polyoxyethylene/polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises an isotonic saline solution.

Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985).

Pharmaceutical carriers, excipients or diluents may be selected based on the intended route of administration and common pharmaceutical practice.

Routes of Administration of Pharmaceutical Compositions

In one embodiment, the pharmaceutical compositions described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intramuscularly, or intratumorally. In some embodiments, the pharmaceutical composition is formulated for topical administration or systemic administration. Systemic administration may include enteral administration, which includes absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration by any means other than through the gastrointestinal tract, such as intravenous injection. In a preferred embodiment, the pharmaceutical compositions are formulated for systemic administration. In another preferred embodiment, systemic administration is by intravenous injection.

Application of pharmaceutical compositions

The RNA particles described herein can be used in the therapeutic or prophylactic treatment of various diseases, in particular, diseases in which the provision of a peptide or protein to a subject results in a therapeutic or prophylactic effect. For example, providing an antigen or epitope that is derived from a virus may be useful in treating a viral disease caused by said virus. Providing a tumor antigen or epitope may be useful in the treatment of cancer, wherein the cancer cells express said tumor antigen. Providing a functional protein or enzyme may be useful in the treatment of a genetic disorder characterized by a dysfunctional protein, such as lysosomal storage diseases (eg, mucopolysaccharidoses) or factor deficiencies. The provision of a cytokine or fusion cytokine may be useful in modulating the tumor microenvironment.

The term "disease" (also referred to herein as a "disorder") refers to an abnormal condition that affects the body of an individual. The disease is often interpreted as a medical condition associated with specific symptoms and signs. The disease may be caused by factors coming from an external source, such as an infectious disease, or it may be caused by an internal dysfunction, such as an autoimmune disease. In humans, the term "disease" is often used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems, or death of a person suffering from the disease, or similar problems in those who come into contact with that person. In a broader sense, it sometimes includes injuries, disabilities, disorders, disorders, syndromes, infections, isolated symptoms, deviant behavior, and atypical changes in structure and function, although in other contexts and for other purposes these may be considered as distinct categories. Illnesses usually affect people not only physically but emotionally, as acquiring and living with many diseases can change one's outlook on life and one's personality.

In the context of the present invention, the term "treatment" or "therapeutic intervention" refers to a plan of treatment and care for a subject in order to combat a condition such as a disease or disorder. The term is intended to include the full range of treatments for a given condition from which a subject suffers, such as administering a therapeutically effective compound to relieve symptoms or complications, to delay the progression of a disease, disorder or condition, to alleviate or eliminate the disease, disorder or condition, and to prevention of a condition, wherein prevention is to be understood as a plan of treatment and care for a subject to combat a disease, disorder, or condition, and includes the administration of active compounds to prevent the onset of symptoms or complications.

The term "therapeutic treatment" refers to any treatment that improves health and prolongs (increases) life expectancy of an individual. Said treatment may eliminate a disease in an individual, halt or slow the development of a disease in an individual, inhibit or slow the development of a disease in an individual, reduce the frequency or severity of symptoms in an individual, and/or reduce relapses in an individual who is currently ill or who has had the disease in the past.

The terms "prophylactic treatment" or "preventive treatment" refers to any treatment that is intended to prevent the onset of a disease in an individual. The terms "prophylactic treatment" or "preventive treatment" in the present description are used interchangeably.

The terms "individual" and "subject" are used in the present description interchangeably. They refer to a human or other mammal (such as a mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate) or any other non-mammalian animal, including birds (chickens), fish, or any other a species of animal that may be affected or susceptible to a disease or disorder (e.g., cancer, infectious diseases), but may or may not have the disease or disorder, and may need a preventive intervention, such as vaccination, or may need interventions, such as a protein replacement. In many embodiments, the individual is a human. Unless otherwise indicated, the terms "individual" and "subject" do not designate a specific age and thus include adults, the elderly, children and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient".

The term "patient" means an individual or subject for treatment, in particular, a diseased individual or subject.

In one embodiment of the disclosure, the purpose is to provide protection against an infectious disease by vaccination.

In one embodiment of the disclosure, the purpose is to provide the subject, in particular the subject in need thereof, with secreted therapeutic proteins such as antibodies, bispecific antibodies, cytokines, cytokines fused to proteins, enzymes.

In one embodiment of the disclosure, the purpose is to provide the subject, in particular the subject in need thereof, with protein replacement therapy such as the production of erythropoietin, factor VII, von Willebrand factor, β-galactosidase, alpha-N-acetylglucosaminidase.

In one embodiment of the disclosure, the goal is to modulate/reprogram immune cells in the blood.

One skilled in the art will recognize that one of the principles of immunotherapy and vaccination is based on the fact that an immune protective response to a disease is elicited by immunizing a subject with an antigen or epitope that is immunologically relevant for the disease being treated. Accordingly, the pharmaceutical compositions described herein are useful for inducing or enhancing an immune response. The pharmaceutical compositions described herein are therefore useful in the prophylactic and/or therapeutic treatment of a disease involving an antigen or epitope.

The term "immunization" or "vaccination" describes the process of introducing an antigen into an individual for the purpose of inducing an immune response, for example, for therapeutic or prophylactic reasons.

The citation of documents and studies referenced herein does not imply that any of the foregoing is prior art. All statements regarding the content of these documents are based on information available to Applicants and do not constitute any admission of the correctness of the content of these documents.

The following description is provided to enable those skilled in the art to make and apply the various embodiments. Descriptions of specific devices, methods, and applications are provided as examples only. Various modifications of the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Thus, the various embodiments are not intended to be limited to the examples described herein and shown, but are to be presented within the scope of the claims.

Examples

Example 1 Materials and Methods

materials

Coding luciferase or secreted luciferase NanoLuc® (secNLuc) mRNA is obtained from an RNA Biochemistry unit (BioNTech RNA Pharmaceuticals, Mainz, Germany) (mRNA concentration 2 to 5 mg/ml in water or 10 mM Hepes; 0.1 mM EDTA; pH 7 ,0).

Cationic ionizable lipid DODMA (1,2-dioleyloxy-N,N-dimethyl-3-aminopropane) and helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) are purchased from Merck. Helper lipid DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine) is obtained from Avanti Polar Lipids. Cholesterol by Sigma Aldrich. Sodium dodecyl sulfate (SDS) was obtained from Sigma Adlrich.

Prior to preparation (lipid nanoparticles), lipids are dissolved in absolute ethanol (Carl Roth) to a concentration of 5 to 100 mM, and lipid ethanol solutions are stored at -20°C.

Before obtaining (lipid nanoparticles) ethanolic solution of lipids, sterile citrate buffer 100 mm, pH 5.4, and RNA balance at room temperature.

Protocol 1 for obtaining lipid nanoparticles

Lipid nanoparticles are prepared by mixing an ethanol phase containing lipids with an aqueous phase containing RNA using a NanoAssemblr™ Benchtop Instrument microfluidic mixer (Precision NanoSystems, Vancouver, BC). One volume of an ethanol-containing lipid mixture with 9 mM total lipids and 3 volumes of RNA, 0.15 mg/ml in 100 mM citrate buffer, pH 5.4, was mixed with a microfluidic cartridge at a combined flow rate of 12 ml/min. The resulting mixture is mixed directly with 2 volumes of 100 mM citrate buffer, pH 5.4. Unless otherwise indicated, the particles are dialyzed against phosphate buffered saline (PBS) for 2.5 hours in a 10K MWCO dialysis cassette (Slide-A-Lyser, ThermoFisher Scientific). The particles are then concentrated again by ultrafiltration using Amicon® ultracentrifuge filters (30 kDa NMWL, Merck Millipore) to a theoretical RNA concentration of about 0.2-0.5 mg/ml. Physico-chemical properties (size, polydispersity, zeta potential, RNA availability and total RNA concentration) are checked on the day of receipt. After full characterization, slides are stored at 4°C for no longer than 2 days. Lipid particles before testing in vitro or injection in vivo dissolved in PBS to the desired concentration of RNA.

Protocol 2 for obtaining lipid nanoparticles

Lipid nanoparticles are prepared by mixing an aqueous phase containing lipids with an aqueous phase containing RNA. The pSarc liposomes are prepared by injecting 600 µl of 75 mM ethanol of all lipids containing cationic lipid, helper lipids with or without pSarc in various molar fractions, into a total volume of 14.4 ml of water with stirring for 30 minutes. The liposomes are then added to RNA in water at N/P 4 followed by rapid shaking and stirring formation of pSarc-LPX with a final RNA concentration of 0.05 mg/mL. Physico-chemical properties (size, polydispersity, RNA availability and total RNA concentration) are checked on the day of receipt. After full characterization, slides are stored at 4°C for no longer than 2 days. Lipid nanoparticles before testing in vitro are dissolved in water to the desired concentration of RNA.

Particle size measurement

Particle size and polydispersity (PDI) of lipid nanoparticles are measured by dynamic light scattering. The preparations are diluted with PBS to a final RNA concentration of 0.005 mg/ml. Measurements are made on 120 µl of the diluted sample in triplicate in a 96-well plate. The size is measured with a plate reader II DynaPro, technology WYATT, GmbH (Dernbach, Germany).

Measurement of zeta potential (electrophoretic mobility)

RNA-lipid nanoparticles are diluted to an RNA concentration of 0.01 mg/ml in 1 ml of PBS 0.1x. Get three samples of 1.05 ml for each drug in plastic cuvettes. The electrophoretic mobility of particles is measured by laser Doppler electrophoresis using a ζ-Wallis device (Corduan technologies, France). For each sample, an average resolution measurement with 1 sequence of 10 cycles is used. Measurements with low signal-to-noise ratios or marginal mobility µ (>3 or <-3 µm*cm/V*S) are excluded from the final analysis.

RiboGreen analysis for RNA availability and total RNA concentration

RNA-lipid nanoparticles are always concentrated to a final concentration of about 0.2-0.5 mg/ml RNA. Quant-iT RiboGreen RNA assay (Thermo Fischer Scientific) was used to quantify RNA availability as well as total RNA concentration in the preparation. Briefly, encapsulation efficiency using the RNA-binding dye RiboGreen was determined by comparing the fluorescence of samples in the presence and absence of 2% Triton X-100. In the absence of detergent, fluorescence can only be measured from available free RNA, while in the presence of detergent, fluorescence is measured from total RNA. Fluorescence of samples in the presence of Triton X-100 detergent is also used to calculate the total RNA concentration from the calibration curve.

Liquid nanoparticle samples or PBS (negative control) were diluted 1x with TE buffer (Thermo Fisher Scientist) to an mRNA concentration of 2 to 5 μg/ml.

Aliquots of each diluted sample are further diluted 1:1 with 1x TE buffer (measurement of available mRNA) or 1:1 with 1x TE buffer containing 2% Triton X-100 (measurement of total mRNA, both available in the particle and free mRNA). Samples are obtained in duplicate. Samples are incubated for 10 minutes at 37°C to ensure sufficient lipid dissociation. Then, 1.1 Quant-iT RiboGreen RNA Reagent (1:100 dilution from stock solution in TE buffer) was added to each sample, and dye fluorescence was measured at 485 nm excitation and 535 nm emission (Tecan Infinite M200 Multimode Plate Reader Spectrophotometer Pro Multimode Plate Reader).

RNA availability is determined by the following formula:

The total RNA concentration is determined using the RNA calibration curve in 1x TE buffer with 2% Triton X-100.

Agarose gel electrophoresis

Agarose gel electrophoresis is performed to evaluate free RNA. The gel was cast using 1 g of agarose dissolved in 100 ml of 1x TAE (Tris-acetate-EDTA) buffer (ThermoFisher), 1 ml of 5% sodium hypochlorite and 10 μl of GelRed Nucleic Acid Gel Stain (Biotium). The gel is allowed to set for at least 25 minutes at room temperature. The gel is then placed in an electrophoresis chamber and TAE 1x running buffer (ThermoFisher) is used. Prior to loading, samples are incubated at 40° C. with or without 2% Triton X-100 for total or free RNA, respectively. The gel runs at 80V for 40 minutes. Electrophoregrams are obtained on the Chemidoc XRS imaging system (Bio-Rad).

In vitro transfection assay and cell viability assay

Cells are seeded in a colorless 96-well plate (flat-bottomed wells) at 5,000 cells/well for C2C12 cells and 20,000 cells/well for HepG2 and TC1 cells. Cells are kept at 37°C and 5% CO2, but C2C12 at 7.5% CO2. After 18-24 hours, the supernatant is discarded and replaced with 90 µl of appropriate media supplemented with 10% non-inactivated FCS. The preparations are diluted with PBS to a final concentration of 1-10 µg/ml. Then, 10 µl of the lipid particle solution was added to the cells, and a final medium volume of 100 µl was obtained. The total amount of RNA in the wells ranges from 33 to 100 ng. The plates are centrifuged for 5 min at 500g at room temperature. After 24 hours of incubation with cells, a Bright-Glo™ luciferase assay (Cat. #E260, Promega GmbH, Mannheim, Germany) was performed according to the instructions. Bioluminescence (RLU) signals were measured using a Tecan Infinite M200 Pro Multimode Plate Reader and luciferase expression was calculated by subtracting the background of non-transfected cells (using PBS as control).

To measure cell viability, the same procedure is followed. After 24 hours of incubation of the preparation with cells, an assay is performed with CellTiter-Glo™ (cat. G9242, Promega GmbH, Mannheim, Germany) according to the instructions. Include control with PBS for 100% viability and DMSO for toxicity. Viability is calculated as follows:

In vivo transfection in mice

Mice are anesthetized with isoflurane and 200 μl of test drugs with 0.05 mg/ml luciferase-encoding mRNA are injected intravenously into the retroorbital sinus using an insulin syringe pre-equipped with a 30G cannula. The mouse is observed for signs of pain, distress, and distress until it regains consciousness.

At the time of measurement (6 hours and 24 hours after the dose), mice are injected intraperitoneally with a solution of D-luciferin at 100 μg/kg body weight. Mice are then anesthetized with isoflurane and placed on a warm mat (37° C.) in an IVIS® Spectrum imaging chamber (Perkin Elmer) with a constant supply of isoflurane/oxygen through individual anesthetic masks. Five minutes after the injection of luciferin, bioluminescence detection is performed using a camera. Mice are then sacrificed by vertebral dislocation, organs like liver, lung, spleen, heart, kidney, brain, lymph nodes are harvested and measured again ex-vivo with the IVIS® Spectrum imaging device. The resulting images are analyzed using the program "LivingImage" (Perkin Elmer). Areas of interest (ROI) around organs are delineated to quantify total flux [p/s]. Serum is obtained from the collected blood by centrifugation of whole blood at 1000×g for 3 min. Determine the levels of liver enzymes alanine aminotransferase (ALT), aspartate aminotransferase (AST) and LDH using a high-performance analyzer for clinical and chemical analyzes Indiko, Thermo.

For EPO experiments, Balb/c mice (n=5) are dosed intravenously with mRNA from 30 to 3 μg. Whole blood is taken at 3, 6, 24 and 48 hours and plasma is obtained by centrifugation at 13,000× rpm for 3 minutes. EPO secretion is determined using the DuoSet Mouse Erythropoietin ELISA kit (R&D systems).

Small angle X-ray scattering

Small-angle X-ray scattering (SAXS) experiments are carried out at the German synchrotron EMBL (DESY) Hamburg [P12]. The distance from the sample to the detector can be adjusted from 1.6 to 6 m to enable measurements from q = 0.6 Å-1 to q = 3 Å-1. Concentrated suspensions of LNP are loaded into place in quartz capillaries using a syringe.

Complement activation

In vitro C3a levels are determined using the Human C3a EIA kit (Quidel). Briefly, LNP and controls (positive (cremophor El) and negative (1x PBS and EDTA (18 mM)) are incubated with complement in healthy human serum (NHS, Quidel) at a ratio of 20:80 (sample: NHS) for 1 hour at 37° C. LNP tested at 5x, 1x and 0.02x relative to the theoretical plasma concentration from a dose of mRNA 1 mg/kg EIA with a set of C3a EIA performed according to the manufacturer's protocol.

Cryo-TEM

Samples were stored on amorphous ice supported by holey carbon films on 200 mesh copper meshes (QuantiFoil® R2/1). The glass transition is carried out in liquid ethane at -180° C. using a Leica EM GP. The grids are stored in liquid nitrogen until transferred to an electron microscope for imaging. Cryogenic TEM imaging is performed with a Zeiss Libra® 120 under liquid N2 cryo conditions on perforated carbon copper grids. The microscope is used at an accelerating voltage of 120 kV and images are taken with a Gatan UltraScan® ccd camera. Each grid is imaged at multiple scales to evaluate the overall distribution of the sample.

Example 2 Preparation of RNA lipid nanoparticles comprising pSarc

Receive mRNA-lipid nanoparticles using polypeptide based on amino acids - lipid : lipids conjugated with polysarcosine.

LNP is prepared by mixing an ethanol phase containing DODMA lipids, cholesterol, DSPC and C14pSarc20 in a molar ratio of 40:50-X:10:X, and 3 volumes of RNA, 0.15 mg/ml, in 100 mM citrate buffer, pH 5, 4.

Table 1. Physicochemical properties of RNA-lipid nanoparticles obtained with different molar fraction (%) С14pSarc20

Composition Ratio N/P Z-Av (nm) PdI RNA Availability (%) DODMA:Chol:DSPC:C14PSARC20 40:49.5:10:0.5 2.7 -- -- 51.2 DODMA:Chol:DSPC:C14PSARC20 40:49:10:1 2.7 -- -- 72.5 DODMA:Chol:DSPC:C14PSARC20 40:48.5:10:2.5 2.7 222.8 0.159 74.2 DODMA:Chol:DSPC:C14PSARC20 40:45:10:5 2.7 117.8 0.177 52.3 DODMA:Chol:DSPC:C14PSARC20 40:42.5:10:7.5 2.7 129.4 0.195 75.9 DODMA:Chol:DSPC:C14PSARC20 40:40:10:10 2.7 81 0.193 68.3 DODMA:Chol:DSPC:C14PSARC20 40:35:10:15 2.7 63.1 0.173 62.0 DODMA:Chol:DSPC:C14PSARC20 40:30:10:20 2.7 57.3 0.238 72.2

Fig. 1 shows the relationship between particle size and molar fraction of polysarcosine-conjugated LNPs. Lipid nanoparticles are prepared using a mixture of lipids including increasing molar fractions of C14PSarc20. Under suitable conditions, stable colloidal particles can be obtained. Although very small fractions of PSarc (0.5 and 1%) do not form measurable particles, at 2.5 mole % or more particles of distinct size and low polydispersity index are obtained. The particle size can be precisely controlled by changing the PSarc fraction. The particle size decreases monotonically from about 200-250 nm with 2.5 mol% PSarc to about 50 nm with 20 mol% PSarc.

Example 3 PSarc Lipid Particles with Polymerized Sarcosine with Different Numbers of Units

LNP is prepared by mixing one volume of an ethanol phase containing DODMA lipids, cholesterol, DSPC and C14pSarcX with a polymer of various lengths (X = 11, 20, 34 or 65) at a molar ratio of 40:45:10:5, and 3 volumes of RNA, 0 15 mg/ml, in 100 mM citrate buffer, pH 5.4.

Table 2. Physicochemical properties of RNA-lipid nanoparticles obtained with a molar fraction of C14pSarcX 5% with polymers of various lengths

Composition Attitude N/P Z-Av (nm) PdI Zeta potential Availability of RNA, % Free RNA (%) DODMA:Chol:DSPC:C14PSARC11 40:45:10:5 2.7 106.8 0.317 2.09 ± 1.43 72.5 1.6 DODMA:Chol:DSPC:C14PSARC20 40:45:10:5 2.7 107.5 0.174 2.6±0.7 75.9 5.1 DODMA:Chol:DSPC:C14PSARC34 40:45:10:5 2.7 83.4 0.202 2.04 ± 1.82 72.2 16.8 DODMA:Chol:DSPC:C14PSARC65 40:45:10:5 2.7 114.7 0.237 1.15 ± 2.43 69.1 16.0

Fig. 2 shows the relationship between the length of the polysarcosine (polymer units) of pSarc lipids used to form LNPs and the in vitro expression of LNP proteins encoding luciferase mRNA in various cell lines. LNP in combination with mRNA encoding luciferase is tested in lung tumor cells (TC-1), muscle cells (C2C12), hepatocytes (Hep-G2) and macrophages (RAW 264.7). 24 hours after transfection, the bioluminescence signal is measured. Regardless of the cell line, an increase in the number of units in polysarcosine does not lead to a decrease in protein expression levels, as is commonly observed for PEG lipids.

Fig. 3 shows the in vivo performance of LNPs comprising a constant fraction of PSarc lipid (5%), where the polysarcosine length varies from 11 to 65 units. LNP in composition with mRNA encoding luciferase is injected intravenously into mice (10 μg RNA, n=3). Bioluminescence is measured in vivo and ex vivo. In all cases, the strongest signals are found in the liver. The figure shows ex vivo measurements in livers that were extracted 6 hours after injection. It was not possible to determine a significant effect of polysarcosine length on the level of protein expression in the liver. This allows particles to be engineered using PSarc with a wide range of particle sizes without compromising transfection efficiency.

Example 4 Effect of Polysarcosine Lipid End Group

LNP is obtained by mixing one volume of an ethanol phase containing DODMA lipids, cholesterol, DSPC and C14pSarc20 with different end groups (NH ₂ , COOH and C ₂ H ₃ O) at a molar ratio of 40:50-x:10:x, and 3 volumes RNA, 0.15 mg/ml, in 100 mM citrate buffer, pH 5.4.

Table 3. Physicochemical properties of RNA-lipid nanoparticles obtained with different molar fractions (%) of C14pSarc20 with 3 different end groups

Composition Molar ratio N/P Size
[d.nm] PdI ζ [mV] σ Access-
ness (%) Σ Free RNA DODMA:Chol:DSPC:C14PSARC20-NH2 40:47,5:10:
2.5 2.7 187 0.325 10 1.04 64.9 3.1 63.7 DODMA:Chol:DSPC:C14PSARC20-NH2 40:45:10:5 2.7 110 0.175 4.5 1 61.7 2.0 0.0 DODMA:Chol:DSPC:C14PSARC20-NH2 40:40:10:10 2.7 95.6 0.201 1.4 1.13 72.8 0.2 14.5 DODMA:Chol:DSPC:C14PSARC20-COOH 40:47,5:10:
2.5 2.7 193 0.131 6.9 1.73 56.6 1.6 0.0 DODMA:Chol:DSPC:C14PSARC20-COOH 40:45:10:5 2.7 97.1 0.162 0.7 1.21 17.4 0.3 0.0 DODMA:Chol:DSPC:C14PSARC20-COOH 40:40:10:10 2.7 69.7 0.199 -2.2 1 12.9 0.8 0.0 DODMA:Chol:DSPC:C14PSARC20-C2H3O 40:47,5:10:
2.5 2.7 240 0.141 2.2 0.76 57.1 0.4 0.0 DODMA:Chol:DSPC:C14PSARC20-C2H3O 40:45:10:5 2.7 139 0.17 5.2 0.73 57.4 1.9 13.5 DODMA:Chol:DSPC:C14PSARC20-C2H3O 40:40:10:10 2.7 69.3 0.226 1.1 1.65 49.3 1.6 0.0

Fig. 4 shows the effect of various polysarcosine end groups on particle size and zeta potential. PSarc, consisting of 20 repeating units with an amino group, a carboxyl group, or an acetyl end group, is tested by direct comparison. All other drug parameters are kept constant. An LNP formulation with test end groups is successful when the correlation between the PSarc fraction and particle properties (size and zeta potential) is similar.

Fig. 5 shows the in vitro determination of the properties of LNPs comprising polysarcosine lipids with various end groups described in FIG. 4. Use pSarc lipids with a molar fraction of 5% and a length of 20 units. LNP in combination with mRNA encoding luciferase is tested in hepatocytes (Hep-G2), macrophages (RAW 264.7), muscle cells (C2C12), embryonic kidney cells (HEK 293 T). The bioluminescence signal is measured 24 hours after transfection. Receive the bioluminescence signal for all LNP and cell lines. The dependence of the signal strength as a function of the cell line is similar for all end groups.

Fig. 6 shows the in vivo performance of LNP in the various end group formulations described in FIG. 5 and 6. pSarc lipids with a molar fraction of 5% and a length of 20 links are used. LNP in composition with mRNA encoding luciferase is injected intravenously (10 μg RNA, n=3). Bioluminescence is measured in vivo and ex vivo. In all cases, the strongest signal is found in the liver. The figure shows ex vivo measurements in livers that were extracted 6 hours after injection. Signals of similar strength are determined with all end groups, indicating that all end groups are suitable for obtaining a similar high transfection in vivo.

Example 5 Preparation of PSarc RNA Lipid Nanoparticles with Various Cationic Lipids

The results of subsequent experiments show the versatility of polysarcosine for the formation of RNA-lipid nanoparticles with various types of cationic particles.

Table 4. Physicochemical properties of pegylated or polysarcosine-conjugated RNA-lipid nanoparticles obtained with various cationic particles, as well as with non-standard DOPE ratios

Composition Molar relation N/P Size [d.nm] PdI ζ [mV] σ Availability (%) σ Free RNA KC2:DOPE:CHOL:PEG 40:20:38:2 4 107.2 0.14 -1.17 1.97 48.4 2 8.4 KC2:DOPE:CHOL:C14 pSarcC 20 40:20:35:5 4 0 0 1.89 2.17 4.1 0.2 0 C12-200:DOPE:Chol:PEG 40:40:18:2 16 81.55 0.128 -0.44 2.05 18.8 0.3 0 C12-200:DOPE:cold: C14 pSarcC 20 40:40:15:5 16 155.75 0.234 6.04 1.36 3.4 0.2 0

Example 6 pSarc liposomes and RNA lipoplexes

The results of the following experiments show that the inclusion of polysarcosine-conjugated lipids is suitable for the formation of liposomes and secretive RNA lipoplexes. Under appropriate conditions, small particles with high transfection efficiency are formed.

pSarc liposomes are prepared by injecting 600 µl of an ethanolic lipid solution at 75 mM of all lipids containing cationic, helper lipids and pSarc or PEG into a total volume of 14.4 ml of water with stirring for 30 minutes. Liposomes are added to RNA in water at N/P 4 followed by rapid formation with shaking and stirring pSarc-LPX.

Fig. 7 shows the effect of pegylation and polysarcosine conjugation on liposome size. Liposomes are prepared either with DOTMA and DOPE alone (2:1, mol/mmol), or lipid mixtures are used that include PEG or pSarc lipid in a 2% molar fraction. Both PEG and pSarc lead to a significant decrease in the measured size, however, at the same time, the polydispersity index is higher (multimodal).

Fig. 8 shows the formation of lipoplexes using liposomes that include the PEG and pSarc described in FIG. 7. Of all three types of liposomes (one DOTMA and DOPE (2:1, mol/mol) or including PEG or pSarc lipid in a molar fraction of 2%), lipoplexes of limited size and low polydispersity index are formed. Lipoplexes from PEGylated and polysarcosine-conjugated liposomes show an unexpectedly low polydispersity index compared to progenitor liposomes where PDI values were high. This shows that pSarc liposomes with a high polydispersity index may also be suitable for the formation of well-defined RNA lipoplexes smaller than 50 nm and a PDI of about 0.2.

Fig. 9 describes the determination of the in vitro properties of lipoplexes prepared from liposomes consisting of either DOTMA and DOPE alone (2:1, mol/mol) or mixtures of the same lipids, including PEG or pSarc lipid in a 2% molar fraction. Lipoplexes in composition with mRNA encoding luciferase are tested in hepatocytes (Hep-G2). 24 hours after transfection, the bioluminescence signal is measured. Although pegylation significantly reduces the signal, this decrease is much less pronounced in the presence of PSarc. It appears that PSarc reduces the efficiency of transfection to a much lesser extent than PEG.

Fig. 10 describes the determination of the in vitro properties of lipoplexes prepared from liposomes consisting of either DOTMA and DOPE alone (2:1, mol/mol) or mixtures of the same lipids, including PEG or pSarc lipid in a 2% molar fraction. Lipoplexes in composition with mRNA encoding luciferase are tested in muscle cells (C2C12). 24 hours after transfection, the bioluminescence signal is measured. Although pegylation significantly reduces the signal, this decrease is much less pronounced in the presence of PSarc. It appears that PSarc reduces the efficiency of transfection to a much lesser extent than PEG.

Example 7 Additional testing of pSarc particles

Fig. 11 shows the ratio between particle size and chain length of polysarcosine and the molar ratio in the preparation. Whereas with short chain polysarcosine, particle formation is only possible at higher molar ratios, with long chain polysarcosine, particle formation is possible at molar ratios of 1%. In general, particle size decreases with increasing chain length or molar ratio of the polysarcosine present in the formulation.

Fig. 12 explains the scatter curves (SAXS) of lipid nanoparticle formulations with polysarcosine. LNP is included in compositions with polysarcosine in variable chain length (11-34 units) and in various molar ratios (2.5-10%). LNP scatter curves show that LNPs have low internal organization which decreases with increasing chain length or pSar molar ratio. The presence of two peaks indicates that significant contributions are made by the shape factor of the individual lipid bilayers.

Fig. 13 shows RNA availability as assessed by the Quant-It Ribogreen assay. PSarc-LNPs show high RNA availability regardless of chain length and polysarcosine molar ratio.

Fig. 14 shows the results of intravenous administration of various doses of mRNA encoding EPO (erythropoietin) loaded into LNP in lipid formulation conjugated to either PSarc or PEG. Plasma was withdrawn at 3, 6, 24 and 48 hours and EPO was quantified by ELISA. The results show that polysarcosine can directly replace other latent particles like PEG-conjugated lipids without compromising efficacy. PSarc can even promote long-term protein secretion, which would be an advantage for protein replacement therapy.

Fig. 15 shows the release of liver enzymes as an early marker of liver toxicity. Liver enzymes such as alanine aminotransferase (ALT), aspartate aminotransferase (AST) and LDH are measured in serum 6 and 24 hours after LNP injection in the progressive chain PSarc formulation. The results show that the increase in PSarc chain length does not trigger any release of liver enzymes (horizontal lines show the range of values obtained from healthy mice), demonstrating that this biobased polymer is safe to use.

Fig. 16 illustrates complement activation by the C3a complex of pegylated and polysarcosine-conjugated LNPs at theoretical human plasma concentrations. Lipid preparations and controls (positive and negative) are incubated with human serum in a ratio of 20:80 (sample:serum) for 1 hour at 37°C.

The data show reduced levels of the C3a complex with PSarc-LNP when compared to PEG-LNP at higher doses, namely five times the suggested human dose. Lower doses do not trigger the formation of the C3a complex when compared to PBS (drug buffer). These results suggest that PSarc may be less immunogenic than PEG-conjugated lipids.

Fig. 17 shows a Crto-TEM image of LNP in combination with DODMA:cholesterol:DSPC:PSarc 23 in the respective mol % 40:45:10:5. The morphology of polysarcosine-conjugated LNPs shows small multilamellar vesicles in which mRNA can reside between closely spaced bilayers. Scale = 200 nm.

In summary, the results once again show that pSarc is a versatile platform for preparing small RNA particle preparations, regardless of the method, for efficient RNA delivery.

Claims (48)

1. A composition for delivering RNA to target cells or target tissues, comprising a plurality of RNA particles, each particle comprising (i) RNA And (ii) one or more components that combine with RNA to form RNA particles, wherein at least one of the one or more components is conjugated with polysarcosine, wherein the one or more components that bind to the RNA to form particles comprise one or more lipids or lipid-like materials. 2. Composition according to claim 1, wherein the RNA particles are non-viral RNA particles. 3. A composition according to claim 1 or 2, wherein the one or more lipids or lipid-like materials comprise a cationic or cationically ionizable lipid or lipid-like material. 4. The composition of claim 3, wherein the cationically ionizable lipid or lipid-like material is only positively charged at acidic pH and does not remain cationic at physiological pH. 5. A composition according to claim 3 or 4, wherein the one or more lipids or lipid-like materials comprise one or more additional lipids or lipid-like materials. 6. The composition of claim 5, wherein at least one of one or more additional lipids or lipid-like materials is conjugated with polysarcosine. 7. A composition for delivering RNA to target cells or target tissues, comprising a plurality of RNA lipid particles, each particle comprising (a) RNA; (b) a cationic or cationically ionizable lipid or lipid-like material; And (c) a polysarcosine-lipid conjugate or a polysarcosine-lipid-like material conjugate. 8. Composition according to claim 7, wherein each particle also includes (d) a non-cationic lipid or lipid-like material. 9. The composition according to any one of paragraphs. 1-8, wherein the particles do not include a polyethylene glycol-lipid conjugate or a polyethylene glycol-lipid-like material conjugate, and preferably do not include polyethylene glycol. 10. The composition according to any one of paragraphs. 7-9, wherein the cationic or cationically ionizable lipid or lipid-like material comprises from about 20 mole % to about 80 mole % of the total lipid or lipid-like material present in the particles. 11. Composition according to any one of paragraphs. 8-10, wherein the non-cationic lipid or lipid-like material comprises from about 0 mole % to about 80 mole % of the total lipid or lipid-like material present in the particles. 12. The composition according to any one of paragraphs. 7-11, wherein the polysarcosine-lipid conjugate or polysarcosine-lipid-like material conjugate comprises from about 0.25 mole % to about 50 mole % of the total lipid or lipid-like material present in the particles. 13. The composition according to any one of paragraphs. 1-12, wherein the RNA is mRNA. 14. The composition according to any one of paragraphs. 3-13, wherein the cationic or cationically ionizable lipid or lipid-like material comprises N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), bromide N,N-distearyl-N,N-dimethylammonium (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(1-(2 ,3-dioleyloxy)propyl)-N,N,N-trimethylammonium (DOTMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLenDMA), 2 ,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2,2-dilinoleyl-4-dimethylaminomethyl[1,3]-dioxolane (DLin-K-DMA ) or a mixture of them. 15. The composition according to any one of paragraphs. 8-14, wherein the non-cationic lipid or lipid-like material comprises a phospholipid. 16. The composition according to any one of paragraphs. 8-15, wherein the non-cationic lipid or lipid-like material comprises cholesterol or a cholesterol derivative. 17. The composition according to any one of paragraphs. 8-16, wherein the non-cationic lipid or lipid-like material comprises a mixture of a phospholipid and cholesterol or a cholesterol derivative. 18. Composition according to any one of paragraphs. 15-17, wherein the phospholipid is selected from the group consisting of distearoylphosphatidylcholine (DSCP), dipalmitoylphosphatidylcholine (DPPC), or a mixture thereof. 19. The composition according to any one of paragraphs. 8-18, wherein the non-cationic lipid or lipid-like material comprises a mixture of DSCP and cholesterol. 20. The composition according to any one of paragraphs. 1-19, wherein the polysarcosine comprises from 2 to 200 sarcosine units. 21. The composition according to any one of paragraphs. 7-20, wherein the polysarcosine-lipid conjugate or polysarcosine-lipid-like material conjugate has the following general formula (I):

, where x is the number of sarcosine units and has values from 2 to 200. 22. The composition according to any one of paragraphs. 7-21, wherein the polysarcosine-lipid conjugate or polysarcosine-lipid-like material conjugate has the following general formula (II):

, where x is the number of sarcosine units and has values from 2 to 200 and one of R ₁ and R ₂ includes a hydrophobic group and the other is H, a hydrophilic group or a functional group, optionally including a targeting particle. 23. The composition according to any one of paragraphs. 7-22, wherein the polysarcosine-lipid conjugate or polysarcosine-lipid-like material conjugate has the following general formula (III):

, where x is the number of sarcosine units and has values from 2 to 200 and R is H, a hydrophilic group or a functional group, optionally including a targeting particle. 24. The composition according to any one of paragraphs. 1-23, wherein the polysarcosine-lipid conjugate or the polysarcosine-lipid-like material conjugate is an element selected from the group consisting of a polysarcosine-diacylglycerol conjugate, a polysarcosine-dialkyloxypropyl conjugate, a polysarcosine-phospholipid conjugate, a polysarcosine-ceramide conjugate, and a mixture thereof. 25. The composition according to any one of paragraphs. 1-24, wherein the particles are nanoparticles. 26. The composition according to any one of paragraphs. 1-25, wherein the particles include a nanostructured core. 27. The composition according to any one of paragraphs. 1-26, with particles having a size of from about 30 nm to about 500 nm. 28. The composition according to any one of paragraphs. 1-27, wherein the polysarcosine conjugate inhibits particle aggregation. 29. A method for delivering RNA to cells of a subject, comprising administering to the subject a composition according to any one of paragraphs. 1-28. 30. A method for delivering a therapeutic peptide or protein to a subject, comprising administering to the subject a composition according to any one of paragraphs. 1-28, wherein the RNA encodes a therapeutic peptide or protein. 31. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition according to any one of paragraphs. 1-28 in a therapeutically effective amount, wherein delivery of the RNA to the subject's cells is beneficial in treating or preventing the disease or disorder. 32. A method of treating or preventing a disease or disorder in a subject, comprising administering to the subject a composition according to any one of paragraphs. 1-28 in a therapeutically effective amount, wherein the RNA encodes a therapeutic peptide or protein, and wherein delivery of the therapeutic RNA peptide or protein to a subject is beneficial in treating or preventing a disease or disorder. 33. The method according to any one of paragraphs. 29-32, wherein the subject is a mammal. 34. The method of claim 33, wherein the mammal is a human.

Images