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EP-4456882-B1 - LIPID-BASED FORMULATIONS FOR ADMINISTRATION OF RNA

EP4456882B1EP 4456882 B1EP4456882 B1EP 4456882B1EP-4456882-B1

Inventors

  • MENINA, Sara
  • MEHRAVAR, Ehsan
  • HEFESHA, Hossam
  • THANKI, Kaushik
  • HAAS, HEINRICH

Dates

Publication Date
20260513
Application Date
20221227

Claims (15)

  1. A composition comprising: (i) RNA; (ii) a cationically ionizable lipid comprising the structure of the following formula (I): wherein each of R 1 and R 2 is independently R 5 or -G 1 -L 1 -R 6 , wherein at least one of R 1 and R 2 is -G 1 -L 1 -R 6 ; each of R 3 and R 4 is independently selected from the group consisting of C 1-6 alkyl, C 2-6 alkenyl, aryl, and C 3-10 cycloalkyl; each of R 5 and R 6 is independently a non-cyclic hydrocarbyl group having at least 10 carbon atoms; each of G 1 and G 2 is independently unsubstituted C 1-12 alkylene or C 2-12 alkenylene; each of L 1 and L 2 is independently selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) x -, -S-S-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, -C(=O)NR a -, -NR a C(=O)NR a -, -OC(=O)NR a - and -NR a C(=O)O-; R a is H or C 1-12 alkyl; m is 0, 1, 2, 3, or 4; and x is 0, 1 or 2; and (iii) at least one polysarcosine-conjugated lipid.
  2. The composition of claim 1, wherein (i) each L 1 is independently selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, and -C(=O)NR a -, preferably each L 1 is independently -O(C=O)- or -(C=O)O-; (ii) L 2 is selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -NR a C(=O)-, and -C(=O)NR a -, preferably L 2 is -O(C=O)- or -(C=O)O-; (iii) R 5 is a straight alkyl or alkenyl group having at least 10 carbon atoms, preferably at least 14 carbon atoms, more preferably at least 16 carbon atoms; (iv) R 5 is a straight alkyl group or a straight alkenyl group having at least 2 carbon-carbon double bonds; (v) R 5 has the following structure: wherein represents the bond by which R 5 is bound to the remainder of the compound; (vi) each G 1 is independently unsubstituted straight C 1-12 alkylene or C 2-12 alkenylene, preferably unsubstituted, straight C 6-12 alkylene or C 6-12 alkenylene, more preferably unsubstituted, straight C 8-12 alkylene or C 8-12 alkenylene, or unsubstituted straight C 6-10 alkylene or C 6-10 alkenylene, such as unsubstituted straight C 8 alkylene; (vii) each R 6 is independently a straight hydrocarbyl group having at least 10 carbon atoms; (viii) each R 6 is attached to L 1 via an internal carbon atom of R 6 ; (ix) each R 6 is independently selected from the group consisting of: and wherein represents the bond by which R 6 is bound to L 1 ; (x) one of R 1 and R 2 is R 5 and the other is -G 1 -L 1 -R 6 , or each of R 1 and R 2 is independently -G 1 -L 1 -R 6 ; (xi) each of R 3 and R 4 is independently C 1-6 alkyl or C 2-6 alkenyl, preferably C 1-4 alkyl or C 2-4 alkenyl, more preferably C 1-3 alkyl, such as methyl or ethyl; (xii) G 2 is unsubstituted C 2-10 alkylene or C 2-10 alkenylene, preferably unsubstituted C 2-6 alkylene or C 2-6 alkenylene, more preferably unsubstituted C 2-4 alkylene or C 2-4 alkenylene, such as ethylene or trimethylene; and/or (xiii) m is 0, 1, 2 or 3, preferably 0 or 2.
  3. The composition of claim 1 or 2, wherein (i) the cationically ionizable lipid comprises the structure of one of the following formulas (IIIa) or (IIIb): wherein each of R 3 and R 4 is independently C 1-4 alkyl or C 2-4 alkenyl, more preferably C 1-3 alkyl, such as methyl or ethyl; R 5 is a straight alkyl or alkenyl group having at least 16 carbon atoms, wherein the alkenyl group preferably has at least 2 carbon-carbon double bonds; each R 6 is independently a straight hydrocarbyl group having at least 10 carbon atoms, wherein R 6 is attached to L 1 via an internal carbon atom of R 6 ; each G 1 is independently unsubstituted, straight C 6-12 alkylene or C 6-12 alkenylene, e.g., unsubstituted, straight C 8-10 alkylene or C 8-10 alkenylene, such as unsubstituted, straight C 8 alkylene; G 2 is unsubstituted C 2-6 alkylene or C 2-6 alkenylene, preferably unsubstituted C 2-4 alkylene or C 2-4 alkenylene, such as ethylene or trimethylene; each of L 1 and L 2 is independently -O(C=O)- or -(C=O)O-; and m is 0, 1, 2 or 3, preferably 0 or 2; (ii) the cationically ionizable lipid comprises one of the following structures (IV-1), (IV-2), and (IV-3): and/or (iii) the cationically ionizable lipid comprises from 20 mol % to 80 mol %, preferably from 25 mol % to 65 mol %, more preferably from 30 mol % to 50 mol %, such as from 40 mol % to 50 mol %, or from 55 mol % to 65 mol % of the total lipid present in the composition.
  4. The composition of any one of claims 1 to 3, wherein (i) the polysarcosine comprises between 2 and 200 sarcosine units, preferably between 5 and 100 sarcosine units, more preferably between 10 and 50 sarcosine units, more preferably between 15 and 40 sarcosine units, more preferably about 23 sarcosine units; (ii) the polysarcosine-conjugated lipid comprises the structure of the following general formula (V): wherein x is the number of sarcosine units; (iii) the polysarcosine-conjugated lipid has the structure of the following general formula (VI): wherein one of R 1 and R 2 comprises a hydrophobic group and the other is H, a hydrophilic group or a functional group optionally comprising a targeting moiety; and x is the number of sarcosine units, wherein, optionally, R 1 is H, a hydrophilic group or a functional group optionally comprising a targeting moiety; and R 2 comprises one or two straight alkyl or alkenyl groups each having at least 12 carbon atoms, preferably at least 14 carbon atoms; (iv) the polysarcosine-conjugated lipid has the structure of the following general formula (VII): wherein R is H, a hydrophilic group or a functional group optionally comprising a targeting moiety; and x is the number of sarcosine units; (v) the polysarcosine-conjugated lipid has the structure of the following formula: wherein n is 23; and/or (vi) the polysarcosine-conjugated lipid comprises from 0.1 mol % to 5 mol %, preferably from 0.5 mol % to 4.5 mol %, more preferably from 1 mol % to 4 mol %, such as 3 mol % to 4 mol %, of the total lipid present in the composition.
  5. The composition of any one of claims 1 to 4, wherein the composition further comprises one or more additional lipids, preferably selected from the group consisting of phospholipids, steroids, and combinations thereof, more preferably the composition comprises the cationically ionizable lipid; a polymer-conjugated lipid, preferably selected from a polysarcosine-conjugated lipid and a pegylated lipid; a phospholipid; and a steroid.
  6. The composition of claim 5, wherein (i) the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins, more preferably selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (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), dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), and diphytanoyl-phosphatidylethanolamine (DPyPE); (ii) the phospholipid comprises from 5 mol % to 40 mol %, preferably from 5 mol % to 20 mol %, more preferably from 5 mol % to 15 mol % of the total lipid present in the composition; (iii) the steroid comprises a sterol such as cholesterol; (iv) the steroid comprises from 10 mol % to 65 mol %, preferably from 20 mol % to 60 mol %, more preferably from 30 mol % to 50 mol % or from 25 mol % to 35 mol % of the total lipid present in the composition; and/or (v) wherein the composition comprises the cationically ionizable lipid, a polysarcosine-conjugated lipid, a phospholipid, and a steroid, wherein the cationically ionizable lipid comprises from 30 mol % to 50 mol %, such as from 40 mol % to 50 mol %, of the total lipid present in the composition; the polysarcosine-conjugated lipid comprises from 1 mol % to 4.5 mol % of the total lipid present in the composition; the phospholipid comprises from 5 mol % to 15 mol % of the total lipid present in the composition; and the steroid comprises from 30 mol % to 50 mol % of the total lipid present in the composition; or the composition comprises the cationically ionizable lipid, a polysarcosine-conjugated lipid, a phospholipid, and a steroid, wherein the cationically ionizable lipid comprises from 55 mol % to 65 mol % of the total lipid present in the composition; the polysarcosine-conjugated lipid comprises from 1 mol % to 4.5 mol % of the total lipid present in the composition; the phospholipid comprises from 5 mol % to 15 mol % of the total lipid present in the composition; and the steroid comprises from 25 mol % to 35 mol % of the total lipid present in the composition.
  7. The composition of any one of claims 1 to 6, wherein (i) at least a portion of (i) the RNA, (ii) the cationically ionizable lipid, and, if present, (iii) the one or more additional lipids is present in nanoparticles, such as lipid nanoparticles (LNPs), wherein, optionally, the nanoparticles have a size of from 30 nm to 500 nm; (ii) the RNA is mRNA; (iii) the RNA (a) comprises a modified nucleoside in place of uridine, wherein the modified nucleoside is preferably selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U); (b) has a coding sequence which is codon-optimized; and/or (c) has a coding sequence whose G/C content is increased compared to the wild-type coding sequence; and/or (iv) the RNA comprises at least one of the following, preferably all of the following: a 5' cap; a 5' UTR; a 3' UTR; and a poly-A sequence, wherein, optionally, (a) the poly-A sequence comprises at least 100 A nucleotides, wherein the poly-A sequence preferably is an interrupted sequence of A nucleotides; and/or (b) the 5' cap is a cap1 or cap2 structure.
  8. The composition of any one of claims 1 to 7, wherein the RNA encodes one or more peptides or proteins, wherein preferably the one or more peptides or proteins are pharmaceutically active peptides or proteins and/or comprise an epitope for inducing an immune response against an antigen in a subject, wherein, optionally, (i) the pharmaceutically active peptide or protein and/or the antigen or epitope is derived from or is a SARS-CoV-2 spike (S) protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof; and/or (ii) the RNA comprises an open reading frame (ORF) encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or the immunogenic variant thereof, wherein, optionally, (a) the SARS-CoV-2 S protein variant has proline residue substitutions at positions 986 and 987 of SEQ ID NO: 11; (b) the SARS-CoV-2 S protein variant has at least 80% identity to the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 11 or the amino acid sequence of amino acids 17 to 1273 of SEQ ID NO: 12; (c) the fragment comprises the receptor binding domain (RBD) of the SARS-CoV-2 S protein; or (d) the fragment of (i) the SARS-CoV-2 S protein or (ii) the immunogenic variant of the SARS-CoV-2 S protein has at least 80% identity to the amino acid sequence of amino acids 327 to 528 of SEQ ID NO: 11.
  9. The composition of any one of claims 1 to 8 for use in therapy.
  10. The composition of any one of claims 1 to 8 for use in a method for delivering RNA to cells of a subject, the method comprising administering the composition to a subject.
  11. The composition of any one of claims 1 to 8 for use in a method for delivering a pharmaceutically active peptide or protein to a subject, the method comprising administering the composition to a subject, wherein the RNA encodes the pharmaceutically active peptide or protein.
  12. The composition of any one of claims 1 to 8 for use in a method for treating or preventing a disease or disorder in a subject, the method comprising administering the composition to a subject, wherein delivering the RNA to cells of the subject is beneficial in treating or preventing the disease or disorder.
  13. The composition of any one of claims 1 to 8 for use in a method for treating or preventing a disease or disorder in a subject, the method comprising administering the composition to a subject, wherein the RNA encodes a pharmaceutically active peptide or protein and wherein delivering the pharmaceutically active peptide or protein to the subject is beneficial in treating or preventing the disease or disorder.
  14. The composition of any one of claims 1 to 8 for use in inducing an immune response in a subject.
  15. The composition for use of any one of claims 10 to 13, wherein the subject is a mammal, and wherein, optionally, the mammal is a human.

Description

Technical Field The present disclosure relates to compositions comprising RNA and a cationically ionizable lipid for delivering RNA to target tissues after administration, in particular after parenteral administration. The present disclosure also relates to methods for delivering RNA to cells of a subject or for treating or preventing a disease or disorder in a subject, wherein the methods comprise administering to a subject a composition of the present disclosure. The RNA compositions in some embodiments comprise single-stranded RNA such as mRNA which encodes a peptide or polypeptide of interest, such as a pharmaceutically active peptide or polypeptide. The RNA is taken up by cells of a target tissue and the RNA is translated into the encoded peptide or polypeptide, which may exhibit its physiological activity. Thus, the present disclosure also relates to methods for delivering a pharmaceutically active peptide or protein to a subject or for treating or preventing a disease or disorder in a subject, wherein the methods comprise administering to a subject an RNA composition of the present disclosure, wherein the RNA encodes the pharmaceutically active peptide or protein. Background The use of RNA to deliver foreign genetic information into target cells offers an attractive alternative to DNA. The advantages of RNA include transient expression and non-transforming character. RNA does not require nucleus infiltration for expression and moreover cannot integrate into the host genome, thereby eliminating the risk of oncogenesis. RNA can be delivered to a target through different vehicles, based mostly upon cationic polymers or lipids, which combine with the RNA to form nanoparticles. The nanoparticles are intended to protect the RNA from degradation, enable delivery of the RNA to the target site and facilitate cellular uptake and processing by the target cells. The efficiency of RNA delivery depends, in part, on the molecular composition of the nanoparticle and can be influenced by numerous parameters, including particle size, formulation, and charge or grafting with molecular moieties, such as polyethylene glycol (PEG) or other ligands. Nogueira et al. (ACS Appl. Nano Mater. 2020, 3, 10634-10645) discloses polysarcosine-functionalized lipid nanoparticles for therapeutic mRNA delivery. There is a need of providing formulations for the delivery of pharmaceutically active RNA to a target tissue where the delivered RNA is efficiently translated into the peptide or polypeptide it codes for. The inventors surprisingly found that the RNA formulations described herein fulfill the above-mentioned requirements. Summary The present invention generally provides a composition comprising RNA, a cationically ionizable lipid comprising the structure of formula (1) shown below, and at least one polysarcosine-conjugated lipid. The inventors found that RNA formulations containing this cationically ionizable lipid of formula (I) can be manufactured in a robust and reproducible manner. They are suitable as RNA delivery systems and display comparable characteristics with respect to known systems. The RNA formulations described herein are useful as RNA delivery vehicles for in vivo application, such as for pharmaceutical application. In a first aspect, the invention provides a composition comprising: (i) RNA; and (ii) a cationically ionizable lipid comprising the structure of the following formula (I): wherein each of R1 and R2 is independently R5 or -G1-L1-R6, wherein at least one of R1 and R2 is -G1-L1-R6;each of R3 and R4 is independently selected from the group consisting of C1-6 alkyl, C2-6 alkenyl, aryl, and C3-10 cycloalkyl;each of R5 and R6 is independently a non-cyclic hydrocarbyl group having at least 10 carbon atoms;each of G1 and G2 is independently unsubstituted C1-12 alkylene or C2-12 alkenylene;each of L1 and L2 is independently selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa- and -NRaC(=O)O-;Ra is H or C1-12 alkyl;m is 0, 1, 2, 3, or 4; andx is 0, 1 or 2. In some embodiments, each L1 is independently selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa-. In some embodiments, each L1 is independently -O(C=O)- or -(C=O)O-. In some embodiments, L2 is selected from the group consisting of -O(C=O)-, -(C=O)O-, -C(=O)-, -C(=O)S-, -SC(=O)-, -NRaC(=O)-, and -C(=O)NRa-. In some embodiments, L2 is -O(C=O)- or -(C=O)O-. In some embodiments, R5 is a straight alkyl or alkenyl group having at least 10 carbon atoms, preferably at least 14 carbon atoms, more preferably at least 16 carbon atoms. In some embodiments, R5 is a straight alkyl group or a straight alkenyl group having at least 2 carbon-carbon double bonds. In some embodiments, R5 has the following structure: wherein represents the bond by which R5 is bound to the remainder of the compound. In some embodiments, ea