EA043705B1 - LIPID NANOPARTICLES FOR DELIVERY OF MODIFIED RNA ENCODING VEGF-A POLYPEPTIDE - Google Patents

Description

1. Cross reference to related applications

This application claims priority to U.S. Provisional Patent Application No.

62/579671, filed October 31, 2017, the entire contents of which are incorporated herein by reference.

2. List of sequences

The present invention contains a sequence listing that has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy in question, created on October 31, 2018, is named 09963_0092-00304_SL.txt and is 11396 bytes in size.

3. Technical field

This invention relates to nanoparticles containing a lipid component and a modified RNA encoding a VEGF-A polypeptide. Aspects of the present invention further relate to the use of nanoparticles comprising a lipid moiety and modified RNA encoding a VEGF-A polypeptide to improve wound healing in a subject.

4. Prerequisites for creating the invention

Vascular endothelial growth factor A (VEGF-A) signaling plays a central role in the wound healing process, including revascularization of damaged tissue, improvement of vascular permeability, and formation of new blood vessels (angiogenesis). It remains a challenge to deliver agents that enhance VEGF-A signaling pathways for promising therapeutic effects, such as improving wound healing in a subject.

Various methods have been tried to develop clinically adaptable approaches to increase the amount of VEGF-A proteins in target tissues. However, each of the approaches has significant drawbacks. For example, systemic delivery of VEGF-A protein can result in significant hypotension, and VEGF-A is rapidly degraded. Control of protein expression of virus-encapsulated and naked VEGF-A DNA plasmids is time-limited, and in vivo expression efficiency can be highly variable and independent of dose. The result of these shortcomings is the limited utility of increasing VEGF-A levels as a therapeutic agent.

Another recent development is the delivery of therapeutic RNAs encoding VEGFA proteins. However, delivery of natural RNAs into cells can be challenging due to the relative instability and low cellular permeability of such RNA molecules. In addition, natural RNAs can provoke activation of the immune system (see, for example, Kaczmarek et al., Advances in the delivery of RNA therapeutics: from concept to clinical reality, Genome Med., 2017, 9: 60), which limits their use to deliver VEGF-A proteins to target tissues.

Therefore, there continues to be a need for formulations that provide effective and safe delivery of RNAs encoding VEGF-A proteins. Additionally, there continues to be a need for alternative methods to enhance VEGF-A signaling pathways for promising therapeutic effects, such as improving wound healing in a subject.

5. Brief description of the invention

This invention relates to nanoparticles containing a lipid component and a modified RNA encoding a VEGF-A polypeptide. Aspects of the present invention further relate to the uses of nanoparticles comprising a lipid moiety and modified RNA encoding a VEGF-A polypeptide to improve wound healing in a subject.

Specific embodiments of the present invention are briefly described below. This list is illustrative only and does not exhaust all embodiments presented in the present invention.

Embodiment 1. Nanoparticle containing (i) a lipid component including a compound with the structure

and (ii) a modified RNA containing any of the sequences of SEQ ID NO: 1 and 3-5 encoding a VEGF-A polypeptide having the sequence of SEQ ID NO: 2.

Embodiment 2. The nanoparticle of embodiment 1, wherein the lipid component further comprises a phospholipid, a structural lipid, and/or a PEG lipid.

Embodiment 3. Nanoparticle of Embodiment 2, wherein the phospholipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE), 1 ,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn- 1 043705 glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2 -di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2diphytanoyl- sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3phosphoethanolamine , 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, sodium salt of 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) (DOPG), sphingomyelin and mixtures thereof; the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof; and/or the PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

Embodiment 4. The nanoparticle of Embodiment 1, wherein the lipid component further comprises a phospholipid, which is DSPC, a structural lipid, which is cholesterol, and/or a PEG lipid, which is PEG-DMG.

Embodiment 5. The nanoparticle according to any one of embodiments 1-4, wherein the N:P ratio is from about 2:1 to about 30:1.

Embodiment 6. Nanoparticle of Embodiment 5, wherein the N:P ratio is approximately 5.67:1.

Embodiment 7. Nanoparticle of Embodiment 5, wherein the N:P ratio is approximately 3:1.

Embodiment 8. The nanoparticle according to any one of embodiments 1-4, wherein the weight ratio of the lipid component to the modified RNA is from about 10:1 to about 100:1.

Embodiment 9. The nanoparticle of embodiment 8, wherein the weight ratio of the lipid component to the modified RNA is approximately 20:1.

Embodiment 10. The nanoparticle of embodiment 8, wherein the weight ratio of the lipid component to the modified RNA is approximately 10:1.

Embodiment 11. The nanoparticle according to any one of embodiments 1-4, wherein the nanoparticle has an average diameter of from about 50 to 100 nm.

Embodiment 12. A pharmaceutical composition comprising (a) at least one nanoparticle containing (i) a lipid component comprising a compound with the structure (Compound A) and (ii) a modified RNA containing any of SEQ ID NOs: 1 and 3- 5, encoding a VEGF-A polypeptide having the sequence SEQ ID NO: 2; and (b) a pharmaceutically acceptable excipient.

Embodiment 13. The pharmaceutical composition of embodiment 12, wherein the lipid component further comprises a phospholipid, a structural lipid and/or a PEG lipid.

Embodiment 14. The pharmaceutical composition of Embodiment 13, wherein the phospholipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2dipalmitoyl -sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1 -πpalmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2- di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine ( C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3phosphocholine, 1,2-diphytanoyl-sn -glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt ( DOPG), sphingomyelin and mixtures thereof; the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine,

- 2 043705 ursolic acid, alpha-tocopherol and mixtures thereof; and/or the PEG lipid is selected from the group consisting of

PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid,

PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof.

Embodiment 15. The pharmaceutical composition of Embodiment 12, wherein the lipid component further comprises a phospholipid, which is DSPC, a structural lipid, which is cholesterol, and/or a PEG lipid, which is PEG-DMG.

Embodiment 16. The pharmaceutical composition according to any one of embodiments 12-15, wherein the N:P ratio is from about 2:1 to about 30:1.

Embodiment 17: The pharmaceutical composition of Embodiment 16, wherein the N:P ratio is about 5.67:1.

Embodiment 18. The pharmaceutical composition of embodiment 16, wherein the N:P ratio is approximately 3:1.

Embodiment 19. The pharmaceutical composition according to any one of embodiments 12-18, wherein the weight ratio of the lipid component to the modified RNA is from about 10:1 to about 100:1.

Embodiment 20. The pharmaceutical composition of embodiment 19, wherein the weight ratio of the lipid component to the modified RNA is approximately 20:1.

Embodiment 21. The pharmaceutical composition of embodiment 19, wherein the weight ratio of the lipid component to the modified RNA is approximately 10:1.

Embodiment 22. The pharmaceutical composition according to any one of embodiments 12-21, wherein the nanoparticle has an average diameter of from about 50 to 100 nm.

Embodiment 23. The pharmaceutical composition according to any one of embodiments 12-22, wherein when administered to tissue of a mammal or subject, the pharmaceutical composition results in a maximum observed concentration C _max in plasma and/or tissue of a VEGF-A polypeptide having the sequence SEQ ID NO: 2 , to a level of approximately 450 pg/ml plasma or pg/mg tissue.

Embodiment 24. The pharmaceutical composition according to any one of embodiments 12-22, wherein when administered to tissue of a mammal or subject, the _{pharmaceutical} composition gives the total area under the plasma and/or tissue concentration curve AUC _{0_t} of the VEGF-A polypeptide having the sequence SEQ ID NO: 2, up to approximately 5500 pg*h/ml plasma or pg*h/mg tissue.

Embodiment 25. The pharmaceutical composition according to any one of embodiments 12-22, wherein when administered to tissue of a mammal or subject, the pharmaceutical composition results in a level of VEGF-A polypeptide having the sequence SEQ ID NO: 2 greater than about 400 pg/mg tissue within 8 hours.

Embodiment 26. The pharmaceutical composition according to any one of embodiments 12-22, wherein when administered to tissue of a mammal or subject, the pharmaceutical composition results in a level of VEGF-A polypeptide having the sequence SEQ ID NO: 2 greater than about 1 pg/mg tissue within a period of up to 6 days.

Embodiment 27. The pharmaceutical composition of embodiment 12, wherein the pharmaceutically acceptable excipient is selected from a solvent, dispersion medium, diluent, dispersant, suspension, surfactant, isotonicity agent, thickener or emulsifier, preservative, polymer, peptide, protein, cells, hyaluronidase and mixtures thereof.

Embodiment 28. A method of promoting and/or improving wound healing in a subject, comprising administering to the subject an effective amount of a nanoparticle according to any of embodiments 1-11 or a pharmaceutical composition according to any of embodiments 12-27.

Embodiment 29. The method of embodiment 28, wherein the lipid component of the nanoparticle further comprises a phospholipid, a structural lipid, and a PEG lipid.

Embodiment 30. The method of embodiment 29, wherein the phospholipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE), 1 ,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2- dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1πpalmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O -octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC ), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero- 3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2 -diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3 phospho-rac-(1-glycerol) sodium salt (DOPG) , sphingomyelin and mixtures thereof; the structural lipid is selected from the group

- 3 043705 consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol and mixtures thereof; and/or the PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine,

PEG-modified diacylglycerol, PEG-modified dialkylglycerol and mixtures thereof.

Embodiment 31. The method of embodiment 28, wherein the lipid component further comprises a phospholipid, which is DSPC, a structural lipid, which is cholesterol, and/or a PEG lipid, which is PEG-DMG.

Embodiment 32. The method of any one of embodiments 28-31, wherein the N:P ratio of the nanoparticle is from about 2:1 to about 30:1.

Embodiment 33. The method of embodiment 32, wherein the N:P ratio of the nanoparticle is approximately 5.67:1.

Embodiment 34. The method of embodiment 32, wherein the N:P ratio of the nanoparticle is approximately 3:1.

Embodiment 35. The method of any one of embodiments 28-34, wherein the weight ratio of the lipid component to the modified RNA is from about 10:1 to about 100:1.

Embodiment 36. The method of embodiment 35, wherein the weight ratio of the lipid component to the modified RNA is approximately 20:1.

Embodiment 37. The method of embodiment 35, wherein the weight ratio of the lipid component to the modified RNA is approximately 10:1.

Embodiment 38. The method of any one of embodiments 28-37, wherein the nanoparticle has an average diameter of from about 70 nm to about 80 nm.

Embodiment 39. The method of embodiment 38, wherein the nanoparticle has an average diameter of approximately 72 nm.

Embodiment 40. The method according to any one of embodiments 28-39, wherein administration results in a maximum observed plasma and/or tissue concentration C _max of the VEGF-A polypeptide having the sequence SEQ ID NO: 2, up to about 450 pg/ml plasma or pg/mg tissue.

Embodiment 41. The method of any one of embodiments 28-39, wherein administration results in a total area under the plasma and/or tissue concentration curve AUC _{0_t} of the VEGF-A polypeptide having the sequence SEQ ID NO: 2 of up to about 5500 pg*h/ml plasma or pg*h/mg tissue.

Embodiment 42. The method of any one of embodiments 28-39, wherein administration results in a level of VEGF-A polypeptide having the sequence SEQ ID NO: 2 greater than about 400 pg/mg tissue over 8 hours.

Embodiment 43. The method of any one of embodiments 28-39, wherein administration results in a level of VEGF-A polypeptide having the sequence SEQ ID NO: 2 greater than about 1 pg/mg tissue for 6 days.

Embodiment 44. The method according to any one of embodiments 28-39, wherein the nanoparticle or pharmaceutical composition is administered intradermally.

Embodiment 45. The method of any one of embodiments 28-39, wherein the concentration of the modified RNA is from about 0.01 mg/kg to about 10 mg/kg.

Embodiment 46. The method of any one of embodiments 28-39, wherein administration results in a 5- to 100-fold increase in the production of VEGF-A polypeptide having the sequence of SEQ ID NO: 2 compared to administration of the modified RNA in citrate buffered saline.

Embodiment 47. The method according to any one of embodiments 28-39, wherein the subject suffers from diabetes.

Embodiment 48. The method according to any one of embodiments 28-39, wherein the wound is a surgical wound, a burn, an abrasive wound, a skin biopsy site, a chronic wound, an injury (eg, a traumatic wound), a graft wound, a diabetic wound, a diabetic ulcer ( for example, diabetic foot ulcer), pressure ulcer, bedsore, and combinations thereof.

Embodiment 49. The method according to any one of embodiments 28-39, wherein the pharmaceutical composition contains a pharmaceutically acceptable excipient, preferably a solvent, dispersion medium, diluent, dispersion agent, suspension, surfactant, isotonicity agent, thickener or emulsifier , preservative, polymer, peptide, protein, cell, hyaluronidase and mixtures thereof.

Embodiment 50. A method of inducing the formation of new blood vessels in tissues of a mammal or subject, comprising administering to the tissues of the mammal or subject an effective amount of a nanoparticle according to any of Embodiments 1-11 or a pharmaceutical composition according to any of Embodiments 12-27.

Embodiment 51. A method of inducing angiogenesis in tissues of a mammal or subject, comprising administering to the tissues of the mammal or subject an effective amount of nanoparticles according to any of embodiments 1-11 or a pharmaceutical composition according to any of embodiments 12-27.

Embodiment 52. A method of increasing capillary and/or arteriole density in tissues of a mammal or subject, comprising administering to the tissues of the mammal or subject an effective amount of a nanoparticle according to any of embodiments 1-11 or a pharmaceutical composition according to any of embodiments 12-27.

6. Description of graphic materials

Those skilled in the art will appreciate that the graphics described below are provided for illustrative purposes only.

The drawings are not intended to limit the scope of the present invention in any way.

Fig. 1. In FIG. 1 shows the lipid compound (Compound A) used in the Examples.

Fig. 2A and 2B. Structure diagram (Fig. 2A) of a modified VEGF-A RNA construct and sequence (SEQ ID NO: 1, Fig. 2B) of a representative modified VEGF-A RNA.

Fig. 3. Content of human VEGF-A protein in skin biopsies as a function of time up to 144 hours after intradermal injection of 100 μg of modified VEGF-A RNA prepared in citrated saline (triangles, dotted line) and 3 μg of modified VEGF-A RNA prepared in lipid nanoparticles (LNPs) (circles, solid line), respectively. The lines represent the median at each time point.

Fig. 4. Content of human VEGF-A protein in skin biopsies as a function of time up to 48 hours after intradermal injection of 100 μg of modified VEGF-A RNA prepared in citrated saline (triangles, dotted line) and 3 μg of modified VEGF-A RNA prepared in LLF (circles, solid line), respectively. The lines represent the median at each time point.

Fig. 5. Wound healing percentage after intradermal injections of the following formulations: (1) lipid nanoparticle formulation containing 1 μg of modified VEGF-A RNA (n=6), (2) lipid nanoparticle formulation containing 3 μg of modified RNA VEGF-A (n=6), (3) lipid nanoparticle formulation containing 3 μg of untranslated VEGF-A RNA (n=6), and (4) citrate saline formulation containing 100 μg of modified VEGF-A RNA A (n=7).

Fig. 6. Wound healing percentage after intradermal injections of the following formulations: (1) lipid nanoparticle formulation containing 3 μg of modified VEGF-A RNA (n = 5), (2) lipid nanoparticle formulation containing 3 μg of non-translated modified VEGF-A VEGF-A RNA (n=5), and (3) a citrate saline formulation that does not contain any modified RNA (n=5).

Fig. 7. Wound healing percentage after intradermal injections of the following compositions: (1) a lipid nanoparticle formulation containing 3 μg of modified VEGFA RNA (n=6), (2) a lipid nanoparticle formulation that does not contain any modified RNA (n=5), (3) a lipid nanoparticle formulation containing 3 μg of GFP-modified RNA (n=6), and (4) a citrate saline formulation that does not contain any modified RNA (n=6 ).

Fig. 8. Wound healing percentage after intradermal injections of the following formulations: (1) lipid nanoparticle formulation containing 3 μg of untranslated modified VEGF-A RNA (n=7), (2) citrate saline formulation containing 100 μg modified VEGF-A RNA (n=7), (3) a citrate saline formulation containing 100 μg of untranslated modified VEGF-A RNA (n=7), and (4) a citrate saline formulation that does not contain any modified RNA (n=7).

7. Detailed description

All references mentioned in this disclosure are incorporated in their entirety herein by reference.

Many modifications and other embodiments of the present invention set forth herein will come to mind to one skilled in the art to which the present invention relates, having benefited from the principles presented in the above descriptions and associated drawings. Therefore, it is to be understood that the present invention is not intended to be limited to the specific embodiments disclosed, and modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are used herein, they are used in a general and informative sense only and not for limitation purposes.

Units of measurement, prefixes and symbols can be indicated in the form used in SI. Unless otherwise noted, nucleic acids are written from left to right in a 5'-3' orientation; amino acid sequences are written from left to right in orientation from amino group to carboxy group, respectively. Numeric intervals include numbers that define a range. It is intended that the presentation of ranges of values in this document serve solely as a method of abbreviating the individual indication of each individual value included in a given range. Unless otherwise specified herein, each individual value is included herein as if it were individually mentioned herein. Amino acids in this document may be designated by their commonly used three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Likewise, nucleotides can be designated by their conventional single-letter codes.

7.1. Definitions

Unless specifically stated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Unless otherwise noted, the techniques used or contemplated herein are standard techniques well known to one of ordinary skill in the art. In the practice of the present invention, unless otherwise indicated, conventional techniques of microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA technology will be used, which are within the competence of one skilled in the art. The materials, methods, and examples are illustrative and not limiting only. The following is presented for illustrative purposes and is not intended to limit the scope of the present invention.

In some embodiments, the numerical parameters set forth in the present specification (which incorporates the claims in their entirety) are approximate values that may vary depending on the desired properties to be obtained in a particular embodiment. In some embodiments, numerical parameters must be interpreted based on the number of significant digits reported and by applying normal rounding techniques. Although the numerical ranges and parameters defining the broad scope of some embodiments of the present invention are approximate values, the numerical values indicated in the specific examples are reported to the greatest possible accuracy. The numerical values presented in some embodiments of the present invention may contain certain errors necessarily resulting from the standard deviation found in their respective test measurements. It is intended that the presentation of ranges of values in this document serve solely as a method of abbreviating the individual indication of each individual value included in a given range. Unless otherwise specified herein, each individual value is included herein as if it were individually mentioned herein.

For convenience, certain terms used throughout the present invention (including the description, examples, and appended claims) are collected herein. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

In some embodiments, numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions and results, etc., which are used to describe and claim certain embodiments of the present invention, should be understood as modified in some cases by the term about. One skilled in the art will understand the meaning of a term roughly in the context of the meaning it defines. In some embodiments, the term is roughly used to indicate that the value includes a standard deviation from the average value for the device or method used to determine the value. In some embodiments, the numerical parameters set forth in the present specification (which incorporates the claims in their entirety) are approximate values that may vary depending on the desired properties to be obtained in a particular embodiment. In some embodiments, numerical parameters must be interpreted based on the number of significant digits reported and by applying normal rounding techniques. Although the numerical ranges and parameters defining the broad scope of some embodiments of the present invention are approximate values, the numerical values indicated in the specific examples are reported to the greatest possible accuracy. The numerical values presented in some embodiments of the present invention may contain certain errors necessarily resulting from the standard deviation found in their respective test measurements.

As used herein, the term administration refers to placing a nanoparticle and/or a pharmaceutical composition containing at least one nanoparticle into the tissue of a mammal or subject in a manner or manner that results in at least partial localization of the nanoparticle and/or composition at a desired site or site in the tissue. In some embodiments, nanoparticles containing a lipid moiety and modified RNA can be administered intradermally. In some embodiments, at least a portion of the protein expressed by the modified RNA is localized to a desired location in the target tissue or cell through intradermal administration.

The term pharmaceutical composition refers to a mixture that contains therapeutically active ingredient(s) and a carrier or excipient, such as a pharmaceutically acceptable carrier or excipient that is conventional in the art. For example, the pharmaceutical composition used herein typically contains at least a lipid component, a modified RNA according to the invention, and a suitable excipient.

The term compound includes all isotopes and isomers of the structure depicted. Isotopes refer to atoms that have the same atomic number but different mass numbers due to different numbers of neutrons in the nuclei. For example, isotopes of hydrogen include tritium and deuterium. In addition, the compound, salt or complex of the present invention can be prepared in combination with solvent or water molecules to form solvates and hydrates using standard methods. Isomer means any geometric isomer, tautomer, zwitterion, stereoisomer, enantiomer or diastereomer of a compound. Compounds may include one or more chiral centers and/or double bonds and thus may exist as stereoisomers such as double bond isomers or diastereomers. The present disclosure covers any and all isomers of the compounds described herein, including stereomerically pure forms and enantiomeric and stereoisomeric mixtures, such as racemates. Mixtures of enantiomers and stereoisomers of compounds and methods for separating them into their constituent enantiomers or stereoisomers are well known in the art.

The terms provide, have and include are non-restrictive linking verbs. Any forms or tenses of one or more of these words, such as provides, provides, has, having, includes and including, are also non-limiting. For example, any method that provides, has or includes one or more steps is not limited to having only such one or more steps and may also include other steps not listed. Likewise, any composition that provides, has or includes one or more features is not limited to having only those one or more features and may include other features not listed. The use of any and all types of examples or illustrative phrases (eg, such as) provided in connection with certain embodiments herein is intended solely to better illuminate the present invention and not to state a limitation on the scope of the present invention otherwise claimed. Nothing in this specification should be construed as indicating that any unstated element is essential to the practice of the present invention.

The term consisting essentially of admits the presence of additional materials or steps that do not significantly affect the main and novel feature(s) of the claimed invention.

The term consisting of refers to the compositions, methods and their respective ingredients/components described herein, which exclude any element not mentioned in such embodiment description.

The term delivery means ensuring the presence of an object of interest at the required location. For example, delivery of a therapeutic agent to a subject may involve administering a pharmaceutical composition containing at least one nanoparticle that includes modified RNA to the subject (eg, by the intradermal route). Administration of a pharmaceutical composition containing at least one nanoparticle into tissue of a mammal or subject may involve contacting one or more cells with the pharmaceutical composition.

The terms disease or disorder are used interchangeably herein and refer to any change in the condition of the body or certain organs that suspends or impairs the performance of functions and/or causes symptoms such as discomfort, dysfunction, distress or even death of the affected individual or those in contact with the individual. Disease or disorder may also include depression, ailment, disease, disorder, faintness, disease, suffering, infirmity or defeat.

As used herein, the term effective amount refers to an amount of a therapeutic agent (eg, modified RNA) or pharmaceutical composition sufficient to reduce at least one or more symptoms of a disease or disorder or to provide a desired effect. For example, it may be an amount that produces a therapeutically significant reduction in a symptom or clinical indicator associated with wound healing.

As used herein, expression of a nucleic acid sequence refers to one or more of the following events: (1) formation of an RNA template from a DNA sequence (eg, through transcription); (2) processing of the RNA transcript (eg, through splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of RNA into a polypeptide or protein; and (4) post-translational modification of the polypeptide or protein.

- 7 043705

As used herein, the term lipid component means that component of a nanoparticle that includes one or more lipids. For example, the lipid component may include one or more cationic/ionizable, PEGylated, structural or other lipids, such as phospholipids. In one embodiment, the lipid component includes Compound A (FIG. 1).

In this context, the term modified RNA refers to RNA molecules containing one, two, or more than two nucleoside modifications relative to adenosine (A) ((2R,3R,4S,5R)-2(6-amino-9H-purine-9- yl)-5-(hydroxymethyl)oxolan-3,4-diol), guanosine (G) (2-amino-9-[3,4dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-3H-purine-6 -one), cytidine (C) (4-amino-1-[3,4dihydroxy-5-(hydroxymethyl)tetrahydrofuran-2-yl]pyrimidin-2-one) and uridine (U) (1-[(3R,4S ,5R)3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]pyrimidin-2,4-dione) or compared to AMP, GMP, CMP and UMP in RNA molecules or parts thereof. Non-limiting examples of nucleoside modifications are presented elsewhere in this specification. When the nucleotide sequence of a particular claimed RNA is otherwise identical to the sequence of a naturally occurring RNA molecule, the modified RNA is intended to be an RNA molecule with at least one modification different from that of its natural counterpart. The difference may be either a chemical change in the nucleoside/nucleotide or the position of the change within the sequence. In one embodiment, the modified RNA is a modified messenger RNA (or modified mRNA). In some embodiments, the modified RNA includes at least one UMP that is modified to form N1-methyl-pseudo-UMP. In some embodiments, all UMPs in the modified RNA have been replaced with N1-methyl-pseudo-UMP.

As used herein, the term nanoparticle is a particle containing one or more lipids and one or more therapeutic agents. Nanoparticles are typically measured in micrometers or smaller in size and may be bilayered lipid particles. In some embodiments, the average diameter of the nanoparticle (e.g., hydrodynamic diameter) is from about 50 to about 100 nm, such as from about 60 to about 90 nm, from 70 to 80 nm in diameter, as measured by dynamic light scattering (see NIST Special Publication 1200-6, Measuring the Size of Nanoparticles in Aqueous Media Using Batch Mode Dynamic Light Scattering). In some embodiments, the average hydrodynamic diameter of the nanoparticle is about 71 nm, 72 nm, 73 nm, 74 nm, 75 nm, 76 nm, 77 nm, 78 nm, 79 nm, 80 nm, 81 nm, 82 nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm or 90 nm. In some embodiments, the therapeutic agent is a modified RNA. In some embodiments, the nanoparticles comprise Compound A, as shown in FIG. 1, and modified RNA.

In this context, the term polydispersity index (pDI) is a measure of the size distribution of nanoparticles in a sample of nanoparticles (see NIST Special Publication 1200-6, Measuring the Size of Nanoparticles in Aqueous Media Using Batch Mode Dynamic Light Scattering). In some embodiments, the polydispersity index value is from about 0.10 to about 0.20, such as about 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19 or 0.20.

In this context, the term N:P ratio represents the molar ratio of ionizable (in the physiological pH range) nitrogen atoms in lipids and phosphate groups in RNA, for example, in a nanoparticle comprising a lipid component and a modified RNA.

As used herein, the term nucleic acid in its broadest sense includes any compound and/or substance that is part of a polymer of nucleotides linked by a phosphodiester bond. Such polymers are often called oligonucleotides or polynucleotides depending on their size. The terms polynucleotide sequence and nucleotide sequence are used interchangeably herein.

As used herein, the term PEG lipid or pegylated lipid refers to a lipid containing a polyethylene glycol component.

The phrase pharmaceutically acceptable is used herein to designate those compounds, materials, formulations and/or dosage forms that, based on careful medical evaluation, are suitable for use in contact with tissues of humans and animals without causing excessive toxicity, irritation, or an allergic reaction. or other problems or complications commensurate with a reasonable benefit/risk ratio. Drug regulatory authorities (eg, EMA, US-FDA) provide guidance and approve pharmaceutically acceptable compounds, materials, formulations and/or dosage forms. Examples are listed in Pharmacopoeias.

The phrase pharmaceutically acceptable excipient is used herein to mean a pharmaceutically acceptable material selected from a solvent, dispersion medium, diluent, dispersant, suspension, surfactant, isotonicity agent, thickener or emulsifier, preservative, polymer, peptide, protein, cells, hyaluronidase and mixtures thereof. In some embodiments, the solvent is an aqueous solvent.

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As used herein, the term phospholipid is a lipid that includes a phosphate moiety and one or more carbon chains, such as unsaturated fatty acid chains. A phospholipid may include one or more multiple (eg, double or triple) bonds (eg, one or more unsaturated bonds). Certain phospholipids may facilitate membrane fusion. For example, a cationic phospholipid may interact with one or more negatively charged membrane phospholipids (eg, a cellular or intracellular membrane). Fusion of a phospholipid with a membrane may allow one or more constituent parts of the lipid-containing composition to pass through the membrane, allowing, for example, delivery of one or more constituent parts into a cell.

As used herein, a polypeptide means a polymer of amino acid residues (natural or non-natural) linked together, most commonly by peptide bonds. The term as used herein refers to proteins, polypeptides and peptides of any size, structure or function. The polypeptide may be a single molecule or may be a multimolecular complex such as a dimer, trimer, or tetramer. They may also include single chain or multi-chain polypeptides, such as antibodies or insulin, and may be associated or linked. Disulfide bonds are most often found in multi-chain polypeptides. The term polypeptide can also be applied to polymers of amino acids in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acids.

The protein used herein is a polymer consisting of virtually any of 20 amino acids. Although the term polypeptide is often used to refer to relatively large polypeptides, and the term peptide is often used to refer to small polypeptides, the use of these terms overlaps and varies in the prior art. The terms peptide(s), protein(s), and polypeptide(s) are sometimes used interchangeably herein.

The term subject refers to an animal, such as a human, to which treatment, including prophylactic treatment, is provided using the methods and compositions described herein. In the case of treating conditions or disease states specific to a particular animal, such as a human subject, the term subject refers to that particular animal.

The term tissue refers to a group or layer of similar specialized cells that work together to perform certain specialized functions.

As used herein, the terms treat, treat, or treat refer to the improvement or elimination of a disease or disorder or at least one overt symptom thereof. In some embodiments, treating or treating refers to improving or eliminating at least one measurable physical parameter, not necessarily discernible by the patient.

It should be understood that the present invention is not limited to the specific methods, protocols, reagents, etc. described herein and may vary accordingly. The terminology used herein is provided solely for the purpose of describing specific embodiments, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

7.2. Lipid components

The nanoparticles contain a lipid component, including compound A (Fig. 1). Additional compounds are disclosed in WO 2017/049245 A2 (see, for example, compounds 1-147 in WO 2017/049245 A2), which is incorporated herein by reference in its entirety. The lipid components may also include a variety of other lipids, such as a phospholipid, a structural lipid, and/or a PEGlipid. Phospholipids

The lipid component of the nanoparticle may include one or more phospholipids, such as one or more (poly)unsaturated lipids. Phospholipids can assemble into one or more lipid bilayers. Typically, phospholipids may contain a phospholipid moiety and one or more fatty acid moieties.

The phospholipid useful for use in the formulations and methods may be selected from the non-limiting group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2 -dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di- O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diαrahidonoyl-sn-glycero3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn -glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2-disteαroyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diαrachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, sodium salt 1,2- 9 043705 dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol ) (DOPG) and sphingomyelin. In some embodiments, the lipid component includes DSPC. In some embodiments, the lipid component includes DOPE. In some embodiments, the lipid component includes both DSPC and

DOPE.

Structural lipids

The lipid component of a nanoparticle may include one or more structural lipids. Structural lipids can be selected from, but are not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, and mixtures thereof. In some embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipid includes cholesterol and a corticosteroid (such as prednisolone, dexamethasone, prednisone, and hydrocortisone) or a combination thereof. In some embodiments, the lipid component includes cholesterol.

PEG lipids.

The lipid component of the nanoparticle may include one or more PEG or PEG-modified lipids. Such lipids may otherwise be referred to as pegylated lipids. PEGlipid is a lipid modified with polyethylene glycol. The PEG lipid can be selected from the non-limiting group consisting of PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, the PEG lipid may be PEG-c-DOMG-, PEG-DMG- (1,2dimyristoyl-OT-glycerol methoxypolyethylene glycol, obtained from Avanti Polar Lipids, Alabaster, Alabama), PEG-DLPE-, PEG-DMPE-, PEG-DPPC- or PEG-DSPE-lipid. In some embodiments, the lipid component includes PEG-DMG.

7.3. Modified RNAs encoding VEGF-A polypeptides

In the field of therapeutics, diagnostics, reagents and biological assays, there is great interest in the ability to deliver a nucleic acid, such as ribonucleic acid (RNA), into a cell, whether in vitro, in vivo, in situ or ex vivo, in such a way as to provide intracellular translation of the nucleic acid and production of the encoded polypeptide of interest.

Naturally occurring RNAs are synthesized from four basic ribonucleotides: ATP, CTP, UTP, and GTP, but may contain nucleotides that have undergone posttranscriptional modification. In addition, approximately one hundred different nucleoside modifications have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J., The RNA Modification Database: 1999 update, Nucl Acids Res, (1999) 27: 196-197).

According to the present invention, such RNAs are preferably modified to avoid the disadvantages of other prior art RNA molecules (eg, activation of the innate immune system response and rapid degradation upon administration). Therefore, such polynucleotides are called modified RNA. In some embodiments, the modified RNA, once administered to a subject, does not elicit a response from the innate immune system. In some embodiments, the half-life of the modified RNA is extended compared to unmodified RNA.

In preferred embodiments, the RNA molecule is messenger RNA (mRNA). As used herein, the term messenger RNA (mRNA) refers to any polynucleotide that encodes a polypeptide of interest and which can be translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.

As shown in FIG. 2A, the major components of an mRNA molecule typically include at least a coding region, a 5' untranslated region (UTR), a 3' untranslated region (UTR), a 5' cap, and a poly(A) tail. Based on this modular wild-type structure, the present invention expands the scope of functionality of traditional mRNA molecules by providing polynucleotides or primary RNA constructs that retain modular organization, but which contain one or more structural and/or chemical modifications or changes that impart beneficial properties to the polynucleotide. properties, including, in some embodiments, not inducing a significant response from the innate immune system in the cell into which the polynucleotide is introduced.

Modified RNAs may include any suitable modification relative to the standard RNA nucleotide chain, such as modification of a sugar, nucleotide base (e.g., one or more nucleotide base modifications, such as substitution or substitution of an atom in a pyrimidine nucleotide base with an optionally substituted amino group, optionally substituted thiol, optionally substituted alkyl (eg, methyl or ethyl) or halogen (eg, chlorine or fluorine)), or internucleoside linkage (eg, one or more modifications in the phosphodiester backbone).

As non-limiting examples, in some embodiments the implementation is modified

- 10 043705 RNA may include, for example, at least one uridine monophosphate (UMP), which is modified to form N1-methyl-pseudo-UMP. In some embodiments, N1-methylpseudo-UMP is present in place of UMP at a percentage of UMP in the sequence of 0.1%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9%, and 100%. In some embodiments, all UMPs have been replaced with N1-methyl-pseudo-UMP.

In some embodiments, the modified RNAs contain a modification in the 5' cap, such as a 5'-diguanosine cap. In some embodiments, the modified RNAs contain a modification in the coding region. In some embodiments, the modified RNAs contain a modification in the 5'UTR. In some embodiments, the modified RNAs contain a modification in the 3'UTR. In some embodiments, the modified RNAs contain a modification in the poly(A) tail. In some embodiments, the modified RNAs contain any combination of modifications in the coding region, 5' cap, 5' UTR, 3' UTR, or poly(A) tail. In some embodiments, the modified RNA may optionally be treated with alkaline phosphatase.

In some embodiments, the modified RNA encodes a vascular endothelial growth factor (VEGF) polypeptide, any of the large family of VEGF proteins that typically play a central role in regulating wound healing. VEGF functions also include roles in activation of nitric oxide (NO) signaling, angiogenesis during embryonic development and postnatal angiogenesis, tumor angiogenesis, arteriogenesis, endothelial replication, and as a switch of cell differentiation in multipotent cardiovascular progenitor cells.

Those skilled in the art will appreciate that for any given VEGF gene, one or more variants or isoforms may exist. Non-limiting examples of VEGF-A polypeptides according to the present invention are listed in table. 1. Those skilled in the art will appreciate that the sequences disclosed in Table. 1 contain potential flanking regions. They are encoded in each nucleotide sequence either in the 5' direction (upstream of transcription) or in the 3' direction (downstream of transcription) from the open reading frame. The open reading frame is specifically and specifically disclosed by stating a nucleotide reference sequence. The 5' and 3' flanking regions can also be further characterized using one or more available databases or algorithms. The databases annotate features characteristic of the flanking regions of the sequences in NCBI and are available from the state of the art.

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Table 1

Homo sapiens VEGF-A mRNA isoforms

Description No. NM No. no NP Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 1 NM_001171623.1 NP_001165094.1 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 1 ΝΜ_0010253 66.2 NP_001020537.2 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 2 NM_001171624.1 NP_001165095.1 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 2 NM_003376.5 NP_003 367.4 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 3 NM_001171625.1 NP_001165096.1 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 3 NM_001025367.2 NP_00102053 8.2 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 4 NM_001171626.1 NP_001165097.1 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 4 NM_001025368.2 NP_001020539.2 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 4 NM_001317010.1 NP_001303939,l Vascular endothelial growth factor A mRNA (VEGF-A) Homo sapiens, variant NM_001171627.1 NP_001165098.1

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transcript 5 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 5 NM_001025369.2 NP_001020540.2 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 6 NM_001171628.1 NP_001165099.1 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 6 NM_001025370.2 NP_001020541.2 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA, transcript variant 7 NM_001171629.1 NP_001165100.1 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA, transcript variant 7 NM_001033756.2 NP_001028928.1 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 8 NM_001171630.1 NP_001165101.1 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 8 NM_001171622.1 NP_001165093.1 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 9 NM_001204385,l NP_001191314.1 Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA transcript variant 9 NM_0012043 84.1 NP_001191313,l Homo sapiens vascular endothelial growth factor A (VEGF-A) mRNA, transcript variant 10 NM_001287044.1 NP_001273 973.1

Those skilled in the art will appreciate that RNA molecules encoding VEGF-A polypeptides, such as human VEGF-A polypeptide, can be designed according to the VEGF-A mRNA isoforms listed in Table 1. Numerous isoforms of the others are generally known to one of ordinary skill in the art. members of the VEGF family.

In one embodiment, the present invention provides a modified RNA encoding a VEGF-A polypeptide (eg, SEQ ID NO: 2). In some embodiments, the modified RNA encodes a VEGF-A polypeptide, wherein the modified PFQC comprises any of the sequences of SEQ ID NOs: 1 and 3-5. In some embodiments, the modified PFQC further comprises a 5' cap, 5' UTR, 3' UTR, poly(A) tail, or any combination thereof. In some embodiments, the 5' cap, 5' UTR, 3' UTR, poly(A) tail, or any combination thereof may include one or more modified nucleotides.

In some embodiments, the modified PFQC encoding a VEGFA polypeptide may have the structure depicted in FIG. 2B, which is SEQ ID NO: 1. In some embodiments, the modified PFQC encoding a VEGF-A polypeptide may comprise any of the sequences of SEQ ID NO: 3-5.

7.4. Compositions containing a lipid component and modified RNA

Some embodiments provide nanoparticles that contain a lipid component and a modified PFQC.

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In some embodiments, the lipid component of the nanoparticle may include Compound A (FIG. 1). In some embodiments, the lipid component of the nanoparticle may further include a phospholipid, a structural lipid, and/or a PEG lipid, as disclosed herein. For example, in some embodiments, the lipid component of the nanoparticle may include DSPC, cholesterol, PEG-DMG, and mixtures thereof.

The constituent parts of the lipid component can be presented in certain proportions. In some embodiments, the lipid component of the nanoparticle includes Compound A, a phospholipid, a structural lipid, and a PEG lipid. In some embodiments, the lipid component of the nanoparticle includes from about 30 mol% to about 60 mol% Compound A, from about 0 mol% to about 30 mol% phospholipid, from about 18.5 mol% to about 48 .5 mol% structural lipid and from about 0 mol% to about 10 mol% PEG lipid, provided that the total mole% does not exceed 100%. In some embodiments, the lipid component of the nanoparticle includes from about 35 mol.% to about 55 mol.% compound A, from about 5 mol.% to about 25 mol.% phospholipid, from about 30 mol.% to about 40 mol.% % structural lipid and from about 0 mol.% to about 10 mol.% PEG lipid. In some embodiments, the lipid component includes about 50 mol% Compound A, about 10 mol% phospholipid, about 38.5 mol% structural lipid, and about 1.5 mol% PEG lipid. In some embodiments, the phospholipid may be DOPE. In some embodiments, the structural lipid may be cholesterol. In some embodiments, the PEG lipid may be PEG-DMG.

In some embodiments, the γ-modified RNA nanoparticle component may include a modified RNA encoding a VEGF-A polypeptide as described herein (eg, SEQ ID NO: 2). In some embodiments, the modified RNA nanoparticle component may include a modified RNA comprising any of the sequences of SEQ ID NOs: 1 and 3-5. In some embodiments, the modified RNA further comprises a 5' cap, 5' UTR, 3' UTR, poly(A) tail, or any combination thereof. In some embodiments, the 5' cap, 5' UTR, 3' UTR, poly(A) tail, or any combination thereof may include one or more modified nucleotides.

In some embodiments, the relative amounts of the lipid component and the modified RNA in the nanoparticle may vary. In some embodiments, the weight ratio of the lipid component to the modified RNA in the nanoparticle can be from about 5:1 to about 100:1, such as 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 , 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1, 25:1, 30:1, 35 :1, 40:1, 45:1, 50:1, 60:1, 70:1, 80:1, 90:1, and 100:1. For example, the weight ratio of the lipid component to the modified RNA can be from about 10:1 to about 40:1. In some embodiments, the weight ratio is about 20:1. In some embodiments, the weight ratio is about 10:1.

In some embodiments, the relative amounts of the lipid component and modified RNA in the nanoparticle may be determined by a particular N:P ratio. The compositional N:P ratio refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. Generally, a lower N:P ratio is preferred. In some embodiments, the N:P ratio can be from about 2:1 to about 30:1, such as 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1 , 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In some embodiments, the N:P ratio can be from about 2:1 to about 8:1. For example, the N:P ratio may be about 3.0:1, about 3.5:1, about 4.0:1, about 4.5:1, about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. In some embodiments, the N:P ratio may be approximately 3:1. In some embodiments, the N:P ratio may be about 5.67:1.

Lipid nanoparticles can be prepared using methods well known in the art (see, for example, Belliveau et al., Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA, Mol. Ther. Nucleic Acids, 2012, 1 (8):e37; Zhigaltsev et al., Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing, Langmuir, 2012, 28(7):3633-3640).

In some embodiments, the nanoparticles may further comprise a pharmaceutically acceptable excipient, which, as used herein, includes, without limitation, any and all types of solvents, dispersion media, diluents or other liquid carriers, dispersion additives, or suspensions, surfactants, isotonicity agents, thickeners or emulsifiers, preservatives, etc., suitable for the particular dosage form required. Excipients may also include, but are not limited to, polymers, core/shell nanoparticles, peptides, proteins, cells, hyaluronidase, nanoparticle mimics, and combinations thereof. Various excipients for the formulation of pharmaceutical compositions and methods for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, ^22nd Edition, Edited by Allen, Loyd V., Jr.

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Pharmaceutical Press; which is incorporated herein by reference in its entirety). It is within the scope of the present invention that the use of a conventional excipient vehicle may be contemplated, except that any conventional excipient vehicle may be incompatible with the substance or its derivatives, such as producing any undesirable biological effect or otherwise interacting in a harmful manner with any ( i) other component(s) of the pharmaceutical composition.

In some embodiments, the nanoparticles may contain a pharmaceutically effective amount of a lipid moiety and a modified RNA, the formulations further comprising a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient is selected from a solvent, dispersion medium, diluent, dispersant, suspension, surfactant, isotonicity agent, thickener or emulsifier, preservative, core/shell nanoparticle, polymer, peptide, protein, cells, hyaluronidase and mixtures thereof. In some embodiments, the solvent is an aqueous solvent. In some embodiments, the solvent is a non-aqueous solvent.

The present invention also provides that the pharmaceutical composition contains one or more lipid nanoparticles containing a lipid component and a modified RNA as disclosed herein, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical compositions contain a plurality of lipid nanoparticles as described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutically acceptable excipient is selected from a solvent, dispersion medium, diluent, dispersant, suspension, surfactant, isotonicity agent, thickener or emulsifier, preservative, core/shell nanoparticle, polymer, peptide, protein , cells, hyaluronidases and mixtures thereof. In some embodiments, the solvent is an aqueous solvent. In some embodiments, the solvent is a non-aqueous solvent. 7.5. Improved wound healing in subject

VEGF-A signaling plays a central role in wound healing processes, including revascularization of damaged tissue, improvement of vascular permeability, and formation of new blood vessels. The purpose of the present invention is to treat subjects who suffer from diseases resulting from impaired wound healing processes.

In some embodiments, the nanoparticles of this invention are administered to a subject who is suffering from a disease that affects vascular structures. Vascular structures are most often affected by penetrating trauma, burns, or surgery. Diabetes negatively impacts multiple components that support wound healing, and a patient with diabetic wound healing typically exhibits altered blood flow due to vascular dysfunction. Therefore, in a subject with a skin ulcer, including diabetic ulcers, wound healing is typically impaired or delayed. In some embodiments, the nanoparticles disclosed herein are administered to a subject suffering from diabetes. In the context of this invention, a wound may be, for example, a surgical wound, a burn, an abrasion wound, a skin biopsy site, a chronic wound, an injury (e.g., a traumatic wound), a graft wound, a diabetic wound, a diabetic ulcer (e.g., a diabetic foot ulcer), pressure ulcer, bedsore and their combinations.

In some embodiments, nanoparticles comprising a lipid moiety and modified RNA (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5) can be used to improve wound healing in mammalian tissue or subject.

In some embodiments, the nanoparticles disclosed herein can be used to induce the formation of new blood vessels in tissues of a mammal or subject. In some embodiments, the nanoparticles disclosed herein can be used to induce angiogenesis in tissues of a mammal or subject.

In some additional embodiments, the nanoparticles disclosed herein can be used to treat vascular injury resulting from trauma or surgery. In some embodiments, the nanoparticles disclosed herein can be used to treat a disease, including skin grafting and tissue grafting.

Other aspects of the present invention relate to the administration of nanoparticles to subjects in need thereof. In some embodiments, nanoparticles disclosed herein are administered intradermally to enhance wound healing in tissues of a mammal or subject.

In certain embodiments, the nanoparticles disclosed herein can be administered at dose levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 40 mg/kg, about 0.5 mg/kg to about 30 mg/kg, about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about

- 15043705 mg/kg, or from about 1 mg/kg to about 25 mg/kg of modified RNA per day, based on the subject's body weight, one or more times per day, to obtain the desired therapeutic effect.

In some embodiments, the nanoparticles disclosed herein are administered to a subject in a single administration. In some embodiments, the nanoparticles disclosed herein are administered to a subject in a fixed dosage over multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more). In each of the embodiments of this paragraph, multiple administrations may be separated from each other by short (1-5 minutes), medium (6-30 minutes) or long (more than 30 minutes, hours or even days) periods of time.

The nanoparticles can be administered to a subject at any administration dosage effective to treat the disease, disorder and/or condition. The exact dosage required will vary from subject to subject depending on the species, age and general condition of the subject, the severity of the disease, the particular drug, its route of administration, its mode of action, and the like. However, it will be understood that the total daily dosage of the formulations may be determined by the attending physician within the exercise of sound medical judgment. The specific pharmaceutically effective dose size for any particular patient will depend on a variety of factors, including the severity of the disease, the specific formulation used, age, body weight, general health, sex and diet of the patient, time of administration, route of administration, duration of treatment and similar factors, well famous in the field of medicine.

All claims in this document are incorporated by reference into the specification in their entirety as additional embodiments.

8. Examples

8.1. Example 1

Preparation of nanoparticle and citrate saline formulations.

Lipids and Modified RNAs: A lipid stock solution in ethanol was prepared from Compound A, distearoylphosphatidylcholine (DSPC, Avanti Polar Lipids), cholesterol (Sigma) and polyethylene glycol modified lipid (mPEG ₂ ooo-DMG from NOF Corporation). Lipids were mixed in 99.5% ethanol to a total lipid concentration of 12.5 mM. The composition was Compound A, DSPC, cholesterol, DMG-PEG in a ratio of 50:10:38.5:1.5 mol%. Modified VEGF-A RNA (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5) was thawed and diluted to 6.25 mM in sodium acetate buffer and HyClone water at concentrations , corresponding to a charge ratio (N:P) of 5.67 or 3 in the final composition. The resulting compositions after dilution were as follows:

LNP formulations were prepared by rapidly mixing an ethanol solution containing lipids and an aqueous solution of modified VEGF-A RNA in a microfluidic device, followed by dialysis in phosphate-buffered saline (PBS). Briefly, the modified VEGF-A RNA solution and the lipid solution were injected into a microfluidic mixing device at an aqueous to ethanol volumetric ratio of 3:1 and a flow rate of 12-14 ml/min using two syringes driven by syringe pumps. Ethanol was removed by dialysis of LNP formulations in PBS buffer overnight using membranes with an upper permeability limit of 10 kDa. LNP compositions were concentrated using centrifuging filter devices with an upper permeability limit of 30 kDa and the following properties were studied: particle size (72 nm), concentration of modified VEGF-A RNA (0.35 mg/ml), polydispersity coefficient (0. 14) and encapsulation (94%). Compositions

LNPs were diluted in PBS to final concentrations of 0.33 mg/ml and sterilized by filtration. Compositions

LNPs were stored in the refrigerator.

Compositions of citrate saline solution.

Citrate saline formulations were prepared by diluting the melted solution of modified VEGF-A RNA with HyClone water and concentrated buffer to a final formulation of 10 mM sodium citrate and 130 mM sodium chloride at pH 6.5.

8.2. Example 2

Evaluation of human VEGF-A protein production following intradermal injection of modified human VEGF-A RNA in mice.

Nanoparticle citrate saline formulations containing modified VEGF-A RNA and Compound A were prepared as in Example 1. In this example, the modified VEGF-A RNA had the sequence SEQ ID NO: 3.

Male db/db mice (C57BL/6J, BKS.Cg-m+/+Leprdb/BomTac Homozygous, Taconic Denmark) were anesthetized with isoflurane. These mice are an established model of type II diabetes and have impaired wound healing compared to wild-type mice. The backs of the mice were shaved and the remaining hair was removed using a hair removal cream. Modified VEGF-A RNA (100 μg) in citrate saline or modified VEGF-A RNA (3 μg) in LNP (see Example 1) was administered intradermally in 4 separate injections (10 μl each, total volume 40 μl) into within a circular area measuring 0.785 mm ² . At predetermined time points after intradermal injections, mice were anesthetized and injected skin areas were collected and quickly frozen in liquid nitrogen and stored at 80°C. All samples were analyzed for human VEGF-A protein.

Samples from db/db mice injected with 100 μg of modified VEGF-A RNA prepared in citrated saline were collected at 6, 24, 48, 72, and 144 h postinjection. Samples from db/db mice injected with 3 μg of modified VEGF-A RNA formulated in LNP were collected at 3, 6, 24, 48, 72, 144 h after injection.

Quantification of human VEGF-A protein in mouse skin.

To prepare skin samples for human VEGF-A protein analysis, frozen tissue biopsies were supplemented with Tris lysis buffer containing phosphatase I and II inhibitors and a protease inhibitor (Meso Scale Discovery (MSD), Rockville, MD, USA) and frozen at approximately 20 °C until homogenization. Steel balls (3 mm) were then added and the samples were homogenized using a Precellys homogenizer. Homogenates were centrifuged and supernatants were stored at 80°C until analysis.

Human VEGF-A concentrations were determined using a sandwich immunoassay with electrochemical luminescence detection. The MSD® 96-well MULTI-ARRAY® Human VEGF-A Assay Kit (Mesoscale, Rockville, MD) was used to measure VEGF-A concentrations in tissue homogenates. This assay detects only human VEGF-A protein and thus only analyzes VEGF-A expressed from modified RNA. The assay was performed according to the instructions in the kit. Standards were serially diluted in MSD diluents. High concentration samples were diluted with MSD diluents prior to analysis to fit the standard curve, and the plates were read on a Sector Imager 6000 from Meso Scale Discovery.

Results.

In fig. Figures 3 and 4 show the time profiles and level of production of human VEGF-A protein after intradermal injection of modified VEGF-A RNA prepared in citrate saline (100 μg) and LNP (3 μg), respectively. Efficient protein production was observed within 6 and 3 hours, respectively (Figure 4). Specifically, the LNP formulation resulted in the production of more than approximately 400 pg/mg VEGF-A protein within 8 hours (Figure 4). In addition, the LNP formulation resulted in the production of more than about 1 pg/mg VEGF-A protein over a period of up to 6 days (Fig. 3). VEGF-A protein production was significantly increased when modified VEGF-A RNA was prepared in LNP compared to citrate saline (Figures 3 and 4).

In table Table 2 shows the pharmacokinetic parameters obtained after intradermal injection of modified VEGF-A RNA prepared in citrate saline solution (100 μg) and LNP (3 μg). _Cmax increased 13.7-fold with modified VEGF-A RNA prepared in LNP, despite the fact that the dose was only 3% of modified VEGF-A RNA prepared in citrate saline. The total area under the AUC _{0_t} concentration curve increased 6.6-fold with 3 μg of modified VEGF-A RNA prepared _in LNP compared with 100 μg of modified VEGF-A RNA prepared in citrate saline.

Table 2. Pharmacokinetic parameters calculated from human VEGF-A concentrations,

- 17 043705 obtained in two time series with modified human VEGF-A RNA in citrate saline and LNP, respectively. The total area under the concentration curve (AUC _0-t ) was calculated based on the profile of the median data measured at points up to 144 hours after dosing.

table 2

Group Dose (mcg) Tschakh (h) Stakh (pg/mg tissue) AUC ₍ ot) (pg*h/mg tissue) tl/2 (h) LLF 3 6 424 5296 50 Citrate saline solution 100 24 31 806 40

8.3. Example 3

Effect of intradermal injection of modified human VEGF-A RNA on wound healing.

Materials and methods.

Lipid nanoparticle and citrate saline formulations containing modified VEGF-A RNA and Compound A were prepared as in Example 1. In this example, the modified VEGF-A RNA had the sequence SEQ ID NO: 4. In addition, the untranslated modified VEGF-A RNA had the sequence SEQ ID NO: 4. A (SEQ ID NO: 6) was used to prepare specific nanoparticle or saline citrate formulations as shown in the figures.

Male 12-week-old db/db mice (B6.BKS(D)-Leprdb/J) from Jackson Lab USA were analyzed for blood glucose after 4 hours of fasting and then randomized to treatment groups. Mice were anesthetized with isoflurane. The dorsal surface of each mouse was shaved using an electric clipper, and then the remaining hair was removed using a hair remover. The skin was washed with descutane and ethanol. A full-thickness wound on the back of each mouse was created using a 10-mm biopsy punch and then excised under sterile conditions and covered with a Tegaderm dressing. On the 3rd day after injury, the Tegaderm dressing was removed. The nanoparticle or citrate saline formulation was administered intradermally in 4 separate injections (10 μL each, total volume 40 μL) at locations close to the wound edge. The wound was then covered with a new Tegaderm dressing. During the observation period, mice were kept separately to avoid interference with wound healing.

Wound healing was determined from a time series (ie, every 3/4th day until day 17 post-wound) of digital photographs taken at a fixed distance and under indirect illumination. Wound area was determined by tracing the wound edge using Image J image analysis software and then calculated as a percentage of area from baseline on day 3 postwounding immediately before dosing.

Statistical evaluation was performed using an unpaired two-tailed Student's t test, and p values <0.05 were considered significant.

Results.

As shown in FIG. 5, on day 7, a lipid nanoparticle formulation containing 1 μg or 3 μg of modified VEGF-A RNA significantly improved wound healing compared with a lipid nanoparticle formulation containing 3 μg of untranslated VEGF-A, as well as a formulation containing based on citrate saline solution containing 100 μg of modified VEGF-A RNA. Additionally, at day 10, a lipid nanoparticle formulation containing 1 μg or 3 μg of modified VEGF-A RNA significantly improved wound healing compared with a lipid nanoparticle formulation containing 3 μg of untranslated modified VEGF-A RNA. At day 10, there was no significant difference between the lipid nanoparticle formulation containing 1 μg or 3 μg of modified VEGF-A RNA and the citrate saline formulation containing 100 μg of modified VEGF-A RNA.

In a separate experiment shown in FIG. 6, on day 7, a lipid nanoparticle formulation containing 3 μg of modified VEGF-A RNA significantly improved wound healing compared to a lipid nanoparticle formulation containing 3 μg of untranslated modified VEGF-A RNA, as well as a formulation based on citrate saline solution, which does not contain any modified RNA. Additionally, at day 10, a lipid nanoparticle formulation containing 3 μg of modified VEGF-A RNA significantly improved wound healing compared to a lipid nanoparticle formulation containing 3 μg of untranslated modified VEGF-A RNA.

In fig. Figure 7 shows another wound healing experiment. On the 7th day, a composition based on lipid

- 18 043705 nanoparticles containing 3 μg of modified VEGF-A RNA significantly improved wound healing compared to a lipid nanoparticle formulation containing 3 μg of GFP-modified RNA, a lipid nanoparticle formulation that did not contain any modified RNA, or a citrate saline formulation that did not contain any modified RNA. Additionally, at day 10, a lipid nanoparticle formulation containing 3 μg of modified VEGF-A RNA significantly improved wound healing compared to a lipid nanoparticle formulation containing 3 μg of GFP-modified RNA.

As shown in FIG. 8, on days 7 or 10, there were no significant differences in healing after intradermal injections of the following four formulations: (1) a lipid nanoparticle formulation containing 3 μg of untranslated modified VEGF-A RNA, (2) a citrate saline formulation, containing 100 μg of modified VEGF-A RNA, (3) a citrate saline formulation containing 100 μg of nontranslated modified VEGFA RNA, and (4) a citrate saline formulation that does not contain any modified RNA.

9. Sequences

9.1. SEQ ID NO: 1: modified RNA encoding VEGF-A.

5'^7MeG_PppG2OM_eGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCC ACCAUGAACUUUCUGCUGUCUUGGGUGCAUUGGAGCCUUGCCUUGCUGCUCUA CCUCCACCAUGCCAAGUGGUCCCAGGCUGCACCCAUGGCAGAAGGAGGAGGGC AGAAUCAUCACGAAGUGGUGAAGUUCAUGGAUGUCUAUCAGCGCAGCUACUGC CAUCCAAUCGAGACCCUGGUGGAC AUCUUCCAGGAGUACCCUGAUGAGAUCGA GUACAUCUUCAAGCCAUCCUGUGUGCCCCUGAUGCGAUGCGGGGGCUGCUGCA AUGACGAGGGCCUGGAGUGUGCCCACUGAGGAGUCCAACAUCACCAUGCAG AUUAUGCGGAUCAAACCUCACCAAGGCCAGCAUAGGAGAGAUGAGCUUCCU ACAGCACAACAAAUGUGAAUGCAGACCAAAGAAAGAUAGA U CCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGG GCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAGoh3' (SEQ GO NO: 1) where

A, C, G and U=AMP, CMP, GMP and N1-methyl-pseudo-UMP, respectively, Me=methyl, p=inorganic phosphate.

9.2. SEQ ID NO: 2: Amino acid sequence of human VEGF-A isoform VEGF-165.

MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHP IETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQG QHIGEMSFLQHNKCECRPKKDRARQENPCGPCSERRKHLFVQDPQTCKCSCKNTDSR CKARQLELNERTCRCDKPRR (SE Q ID NO: 2)

- 19 043705

9.3. SEQ ID NO: 3: Modified RNA encoding VEGF-A.

5'^7MeGpppG2OM_eAGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGC CACCAUGAACUUUCUGCUGUCUUGGGUGCAUUGGAGCCUUGCCUUGCUGCUCU ACCUCCACCAUGCCAAGUGGUCCCAGGCUGCACCCAUGGCAGAAGGAGGAGGG CAGAAUCAUCACGAAGUGGUGAAGUUCAUGGAUGUCUAUCAGCGCAGCUACUG CCAUCCAAUCGAGACCCUGGUGGAC AUCUUCCAGGAGUACCCUGAUGAGAUCG AGUACAUCUUCAAGCCAUCCUGUGUGCCCCUGAUGCGAUGCGGGGGCUGCUGC AAUGACGAGGGCCUGGAGUGUGCCCACUGAGGAGUCCAACAUCACCAUGCA GAUUAUGCGGAUCAAACCUCACCAAGGCCAGCACAUAGGAGAGAUGAGCUUCC UACAGCACAACAAAUGUGAAUGCAGACCAAAGAAAGAUAGA GCAAGACAAGAA AAUCCCUGUGGGCCUUGCUCAGAGCGGAGAAAGCAUUUUUGUACAAGAUCC GCAGACGUGUAAAUGUUCCUGCAAAAACACAGACUCGCGUUGCAAGGCGAGGC AGCUUGAGUUAAACGAACGUACUUGCAGAUGUGACAAGCCGAGGCGGUGAUAA UAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGC CC CUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUG GGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

AAUCUAGoh3' (SEQ ID NO: 3) where

A, C, G and U=AMP, CMP, GMP and N1-methyl-pseudo-UMP, respectively, Me=methyl, p=inorganic phosphate.

9.4. SEQ ID NO: 4: modified RNA encoding VEGF-A (VEGF-01-012).

5'^7MeGpp_PGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAUAAUAAGAGCCACC AUGAACUUUCUCCUUUCUUGGGUGCAUUGGAGCCUUGCCUUGUUACUCUACCU CCACCACGCCAAGUGGUCCCAGGCGCACCCAUGGCAGAAGGAGGAGGGCAGA AUCAUCACGAAGUGGUGAAGUUCAUGGACGUCUAUCAGCGCAGCUACUGCCAU CCAAUCGAGACACUGGUGGAC AUCUUCCAGGAGUACCCUGAUGAGAUCGAGUA CAUCUUCAAGCCAUCCUGUGUGCCCCUGAUGCGAUGCGGCGGCUGCUGCAAUG ACGAGGGCCUGGAGUGUGCCUACUGAGGAGUCCAACAUCACCAUGCAGAUU AUGCGGAUCAAACCUCACCAAGGCCAGCACAUAGGAGAGAUGAGCUUCCUACA GCACAACAAAUGUGAAUGCAGACCAAAGAAAGAUAGA C UCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGC GGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

CUAG3' (SEQ ID NO: 4) where

A, C, G and U=AMP, CMP, GMP and N1-methyl-pseudo-UMP respectively, p=inorganic phosphate.

- 20 043705

9.5. SEQ ID NO: 5: Modified RNA encoding VEGF-A.

5' ^7Me Gp _PP AGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC AUGAACUUUCUGCUGUCUUGGGUGCAUUGGAGCCUUGCCUUGCUGCUCUACCU CCACCAUGCCAAGUGGUCCCAGGCUGCACCCAUGGCAGAAGGAGGAGGGCAGA AUCAUCACGAAGUGGUGAAGUUCAUGGAUGUCUAUCAGCGCAGCUACUGCCAU CCAAUCGAGACCCUGGUGGACAUCUUCCAGGAGUACCCUGAUGAGAUCGAGUA CAUCUUCAAGCCAUCCUGUGUGCCCCUGAUGCGAUGCGGGGGCUGCUGCAAUG ACGAGGGCCUGGAGUGUGUGCCCACUGAGGAGUCCAACAUCACCAUGCAGAUU AUGCGGAUCAAACCUCACCAAGGCCAGCACAUAGGAGAGAUGAGCUUCCUACA GCACAACAAAUGUGAAUGCAGACCAAAGAAAGAUAGAGCAAGACAAGAAAAUC CCUGUGGGCCUUGCUCAGAGCGGAGAAAGCAUUUGUUUGUACAAGAUCCGCAG ACGUGUAAAUGUUCCUGCAAAAACACAGACUCGCGUUGCAAGGCGAGGCAGCU UGAGUUAAACGAACGUACUUGCAGAUGUGACAAGCCGAGGCGGUGAUAAUAG GCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUC CUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGC GGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAU CUAG3' (SEQ ID NO: 5) где А, С, G и U=AMP, CMP, GMP и N1-метил-псевдо -UMP respectively, p=inorganic phosphate.

9.6. SEQ ID NO: 6: Untranslated modified VEGF-A RNA.

5' ^7Me Gpp _P GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC ACGAACUUUGUGCUCUCUUGGGUGCAUUGGAGCCUUGCCUUGCUGCUCUACCU CCACCACGCCAAGUGGUCCCAGGCCGCACCCACGGCAGAAGGAGGAGGGCAGA AUCAUCACGAAGUGGUGAAGUUCACGGACGUCUAUCAGCGCAGCUACUGCCAU CCAAUCGAGACCCUCGUGGACAUCUUCCAGGAGUACCCUCACGAGAUCGAGUA CAUCUUCAAGCCAUCCUGUGUGCCCCUGACGCGACGCGGGGGCUGCUGCAACG ACGAGGGCCUCGAGUGUGUGCCCACCGAGGAGUCCAACACCACCACGCAGAUU ACGCGGAUCAAACCUCACCAAGGCCAGCACAUAGGAGAGACGAGCUUCCUACA GCACAACAAACGUGAACGCAGACCAAAGAAAGAUAGAGCAAGACAAGAAAAUC CCUGUGGGCCUUGCUCAGAGCGGAGAAAGCAUUUGUUUGUACAAGAUCCGCAG ACGUGUAAACGUUCCUGCAAAAACACAGACUCGCGUUGCAAGGCGAGGCAGCU UGAGUUAAACGAACGUACUUGCAGACGUGACAAGCCGAGGCGGUGAUAAUAGG UUGGAGCCUCGGUGGCCACGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCC UCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCG GCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA3' (SEQ ID NO: 6) где А, С, G и U=AMP, CMP, GMP и N1-метил-псевдо- UMP respectively, p=inorganic phosphate.

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Claims (15)

CLAIM 1. Nanoparticle containing (i) a lipid component including a compound with the structure and (ii) a modified RNA containing any of the sequences of SEQ ID NO: 1 and 3-5 encoding a VEGF-A polypeptide having the sequence of SEQ ID NO: 2. 2. Nanoparticle according to claim 1, where the lipid component additionally contains a phospholipid, a structural lipid and/or a PEG lipid. 3. Nanoparticle according to claim 2, where the phospholipid is selected from the group consisting of 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dilinoleoyl-sn-glycero-3phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC), 1,2-dipalmitoyl-sn -glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycerophosphocholine (DUPC), 1-πpalmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O- Octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) , 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero -3-phosphoethanolamine (ME 16.0 PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3 phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero3-phosphoethanolamine, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG ), sphingomyelin and mixtures thereof; the structural lipid is selected from the group consisting of cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, and mixtures thereof; and/or the PEG lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. 4. A nanoparticle according to claim 1, wherein the lipid component further comprises a phospholipid, which is DSPC, a structural lipid, which is cholesterol, and/or a PEGlipid, which is PEG-DMG. 5. Nanoparticle according to any one of claims 1-4, where the molar ratio of ionizable nitrogen atoms in the lipid component and phosphate groups in the RNA in the physiological pH range (N:P ratio) is from about 2:1 to about 30:1 . 6. Nanoparticle according to claim 5, wherein the N:P ratio is approximately 5.67:1. 7. Nanoparticle according to claim 5, wherein the N:P ratio is approximately 3:1. 8. Nanoparticle according to any one of claims 1-4, where the weight ratio of the lipid component and the modified RNA is from about 10:1 to about 100:1. 9. Nanoparticle according to claim 8, where the mass ratio of the lipid component and modified RNA is approximately 20:1. 10. Nanoparticle according to claim 8, where the mass ratio of the lipid component and modified RNA is approximately 10:1. 11. Nanoparticle according to any one of claims 1 to 4, wherein the nanoparticle has an average diameter of from about 50 to about 100 nm. 12. A pharmaceutical composition containing (a) an effective amount of nanoparticles according to any one of claims 1-11 and (b) a pharmaceutically acceptable excipient. 13. The pharmaceutical composition according to claim 12, where the pharmaceutically acceptable excipient is selected from a solvent, dispersion medium, diluent, dispersant, suspension, surfactant, isotonicity agent, thickener or emulsifier, preservative, polymer, peptide, protein , cells, hyaluronidases and mixtures thereof. 14. A method of promoting and/or improving wound healing in a subject, comprising administering to the subject an effective amount of a nanoparticle according to any one of claims 1 to 11 or a pharmaceutical composition according to any one of claims 12, 13. 15. The method of claim 14, wherein the nanoparticle has an average diameter of from about 70 nm to about 80 nm, possibly about 72 nm. -