WO2024064886A1 - Utilisation de dégrons n-terminaux pour améliorer l'immunogénicité d'un vaccin à lymphocytes t à arn - Google Patents

Utilisation de dégrons n-terminaux pour améliorer l'immunogénicité d'un vaccin à lymphocytes t à arn Download PDF

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Publication number
WO2024064886A1
WO2024064886A1 PCT/US2023/074891 US2023074891W WO2024064886A1 WO 2024064886 A1 WO2024064886 A1 WO 2024064886A1 US 2023074891 W US2023074891 W US 2023074891W WO 2024064886 A1 WO2024064886 A1 WO 2024064886A1
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nucleic acid
acid molecule
rna
ubiquitin
polypeptide
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PCT/US2023/074891
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English (en)
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Daniel Abram Rothenberg
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BioNTech SE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A61P33/06—Antimalarials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/002—Protozoa antigens
    • A61K39/015—Hemosporidia antigens, e.g. Plasmodium antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/95—Fusion polypeptide containing a motif/fusion for degradation (ubiquitin fusions, PEST sequence)

Definitions

  • the present invention relates to a nucleic acid molecule for eliciting an antigen-specific CD8 + T-cell response in a subject comprising a coding sequence encoding a polypeptide comprising an N-terminal degron and an antigenic peptide.
  • the invention further provides a polypeptide comprising an N-terminal degron and an antigenic peptide.
  • the present invention further concerns a host cell comprising the nucleic acid molecule of the invention or the polypeptide of the invention.
  • the invention provides a composition comprising the host cell, and a pharmaceutical composition comprising the nucleic acid molecule or the polypeptide.
  • the invention provides a vaccine composition comprising the nucleic acid molecule, the polypeptide, or the pharmaceutical composition.
  • a vaccine composition comprising the nucleic acid molecule, the polypeptide, or the pharmaceutical composition.
  • different medical use applications for the nucleic acid molecule, the polypeptide, the pharmaceutical composition, and the vaccine composition are provided.
  • Vaccines prevent many millions of illnesses and save numerous lives every year, and recently nucleic acid vaccines such as mRNA vaccines have emerged as promising alternatives to conventional vaccine approaches.
  • mRNA vaccines represent a promising alternative to conventional vaccine approaches because of their potency, capacity for rapid development, and potential for low-cost manufacture and safe administration.
  • the mechanism of action of an mRNA vaccine is similar to the mechanism of viral infection.
  • the mRNA is translated into proteins. These proteins may undergo post-translational modification and eventually degrade, thus generating antigenic peptides that are transported to the endoplasmic reticulum (ER) and loaded onto major histocompatibility complex (MHC) class I molecules for antigen presentation and recognition by specific T cells.
  • MHC major histocompatibility complex
  • secreted proteins are taken up by professional antigen-presenting cells, either residing in the tissue or draining lymph nodes, to then be processed and presented on MHC class II molecules.
  • Recent improvements in mRNA vaccines aim to increase mRNA stability, protein translation, modulate innate and adaptive immunogenicity, and improve mRNA production and delivery into the cells.
  • a further object of the present invention is to provide pharmacological means to improve the CD8 + -specific T cell response. It is also an object of the present invention to provide pharmacological means for improved intracellular processing of peptides encodes by mRNA vaccines.
  • nucleic acid molecules of the invention are achieved by the nucleic acid molecules of the invention. These objects are further achieved by the peptide of the invention, the host cell of the invention, the composition comprising the host cell of the invention, the pharmaceutical composition of the invention, the vaccine composition of the invention, and the medical uses of the invention.
  • the invention provides a nucleic acid molecule for eliciting an antigen-specific CD8 + T-cell response in a subject comprising a coding sequence encoding a polypeptide comprising an N-terminal degron and an antigenic peptide.
  • the invention further provides a nucleic acid molecule for eliciting an antigen-specific CD8 + T-cell response in a subject comprising a coding sequence encoding a polypeptide comprising an antigenic peptide and an N-terminal degron comprising at least one non-deavable ubiquitin.
  • the invention further provides a nucleic acid molecule for eliciting an antigen-specific CD8 + T-cell response in a subject comprising a coding sequence encoding a polypeptide comprising an antigenic peptide and an N-terminal degron comprising at least one cleavable ubiquitin.
  • the research underlying this invention has surprisingly found that the polypeptide encoded by the nucleic acid molecule, and comprising an antigenic peptide and an N-terminal degron significantly improves degradation of the polypeptide.
  • the N-terminal degron advantageously destabilizes the polypeptide, leading to rapid processing by the proteasome system.
  • the N-terminal degron can specifically elicit an antigenspecific CD8 + T-cell response, thereby providing an efficient targeting by cytotoxic CD8 + T cells.
  • the research underlying this invention has surprisingly found that a potent CD8 + T-cell response against selected target proteins can be elicited by the nucleic acid molecules of the invention.
  • the invention further provides an isolated host cell comprising the nucleic acid molecule of the invention or the polypeptide of the invention, and a composition comprising the isolated host cell.
  • the invention further concerns a pharmaceutical composition
  • a pharmaceutical composition comprising the nucleic acid molecule of the invention, or the polypeptide of the invention.
  • the invention further concerns a vaccine composition comprising a nucleic acid molecule of the invention, a polypeptide of the invention, or the pharmaceutical composition of the invention.
  • the invention provides the nucleic acid molecule, the polypeptide, the pharmaceutical composition, or the vaccine composition for use in a method of eliciting an antigen-specific CD8 + T-cell response in a subject in need hereof.
  • the invention also provides the nucleic acid molecule, the polypeptide, the pharmaceutical composition, or the vaccine composition for use in a method for inducing the formation of MHC-I/ peptide complexes in a cell.
  • the nucleic acid molecule, the polypeptide, the pharmaceutical composition, or the vaccine composition for use in a method for stimulating or activating CD8 + T-cells. These uses are particularly relevant in therapeutic and prophylactic medical applications.
  • a nucleic acid molecule for eliciting an antigen-specific CD8 + T-cell response in a subject comprising a coding sequence encoding a polypeptide comprising an N-terminal degron and an antigenic peptide.
  • nucleic acid molecule of any one of the preceding embodiments wherein the nucleic acid molecule is an unmodified mRNA or a modified mRNA, preferably a modified mRNA.
  • nucleic acid molecule of any one of the preceding embodiments wherein the nucleic acid molecule comprises a 5' cap, 5'UTR, a coding region, a 3'UTR, a poly(A) tail, or any combination thereof.
  • nucleic acid molecule of any one of the preceding embodiments comprising a 5'-Cap, a free 5'- triphosphate group, a free 5'-disphosphate group, a free 5'-d I phosphate group, a free 5'- monophosphate group, or a free 5'-OH group, or comprising chemically modified analogues of said 5'-Cap, said 5'- triphosphate group, said free 5'-disphosphate group or said free 5'-monophosphate group.
  • nucleic acid molecule of any one of the preceding embodiments comprising a 5'cap selected from G[5']ppp[5']G, m7G[5']ppp[5']G, m 3 2 ' 2 ' 7 G[5']ppp[5']G, m 2 7 ' 3 '-°G[5']ppp[5']G (3'-ARCA), m 2 7 ' 2 '-°GpppG (2'- ARCA), m 2 7 ' 2 ' °GppSpG (p-S- ARCA), and m 2 7 ' 2 ' °GppSpG (p-S-ARCA) and m 2 7 ' 3 '-°Gppp(mi 2 '-°)ApG.
  • a 5'cap selected from G[5']ppp[5']G, m7G[5']ppp[5']G, m 3 2 ' 2 ' 7 G[5
  • nucleic acid molecule of any one of the preceding embodiments wherein the 3'UTR, if present, comprises an FI element.
  • nucleic acid molecule of any one of the preceding embodiments comprising an interrupted poly(A) sequence.
  • nucleic acid molecule of the preceding embodiment 13 wherein the antigenic peptides are derived from different proteins or different portions of the same protein and fused together, preferably such that a first antigenic peptide is adjacent to a second antigenic peptide or such that a first and second antigenic peptide are separated via a linker.
  • nucleic acid molecule of the preceding embodiment 14, wherein the linker comprises at most 8 amino acids, preferably at most 6 amino acids, more preferably at most 5 amino acids, most preferably 1 to 4 amino acids.
  • a degrading amino acid such as an arginine
  • nucleic acid molecule of the preceding embodiment 20 wherein the non-deavable ubiquitin comprises a glycine substitution at the amino acid position 76 with reference to SEQ ID NO.: 1, preferably wherein the glycine is substituted by an alanine (G76A) (e.g., as shown in SEQ ID NO.: 4).
  • G76A alanine
  • nucleic acid molecule of the preceding embodiment 20 or 21, wherein the non-deavable ubiquitin is immediately followed by a degrading amino acid such as an arginine, e.g., as shown in SEQ ID NO.: 5).
  • nucleic acid molecule of the preceding embodiment 20 or 21, wherein the non-deavable ubiquitin is immediately followed by a proline e.g., as shown in SEQ ID NO.: 3.
  • nucleic acid molecule of the preceding embodiment 25 wherein the destabilizing amino acid is selected from isoleucine, glutamic acid, threonine, glutamine, phenylalanine, leucine, aspartic acid, arginine, lysine, and histidine, preferably selected from arginine, lysine, and histidine (e.g., as shown in SEQ ID NO.: 2).
  • nucleic acid molecule of any one of the preceding embodiments wherein the polypeptide comprises one or more internal lysines, wherein the internal lysines are located outside the ubiquitin sequence, preferably in the antigenic peptide or a linker present in the polypeptide.
  • nucleic acid molecule of any one of the preceding embodiments for use in eliciting an antigenspecific CD8 + T-cell response in a subject.
  • nucleic acid molecule of preceding embodiment 28 wherein the subject suffers from a disease, such as a genetic, metabolic or infectious disease.
  • a nucleic acid molecule for eliciting an antigen-specific CD8 + T-cell response in a subject comprising a coding sequence encoding a polypeptide comprising an antigenic peptide and an N-terminal degron comprising a non-cleavable ubiquitin.
  • nucleic acid molecule of the preceding embodiment 32, wherein the non-cleavable ubiquitin comprises a glycine substitution at the amino acid position 76 with reference to SEQ ID NO.: 1, preferably wherein the glycine is substituted by an alanine (G76A) (e.g., as shown in SEQ ID NO.: 4).
  • G76A alanine
  • nucleic acid molecule of the preceding embodiment 32 or 33, wherein the non-cleavable ubiquitin is immediately followed by a degrading amino acid such as an arginine, e.g., as shown in SEQ ID NO.: 5.
  • nucleic acid molecule of the preceding embodiment 32 or 33, wherein the non-cleavable ubiquitin is immediately followed by a proline e.g., as shown in SEQ ID NO.: 3.
  • nucleic acid molecule of any one of the preceding embodiments 32-36 for use in eliciting an antigenspecific CD8 + T-cell response.
  • nucleic acid molecule of any one of the preceding embodiments 32-37 wherein the subject suffers from a disease, such as a genetic, metabolic or infectious disease.
  • nucleic acid molecule of any one of the preceding embodiments 32-38 wherein the subject is a mammal, preferably a human.
  • nucleic acid molecule of any one of the preceding embodiments 32-39 wherein the nucleic acid molecule elicits a two-fold, preferably a three-fold, more preferably a five-fold increase in the antigenspecific CD8 + T-cell response compared to an antigen-specific CD4+ T-cell immune response.
  • nucleic acid molecule of any one of the preceding embodiments 32-45 wherein the nucleic acid molecule comprises a 5' cap, 5'UTR, a coding region, a 3'UTR, a poly(A) tail, or any combination thereof.
  • nucleic acid molecule of any one of the preceding embodiments 32-46, wherein the 5'UTR, if present, comprises a Kozak sequence.
  • nucleic acid molecule of any one of the preceding embodiments 32-47 comprising a 5'-Cap, a free 5'-triphosphate group, a free 5'-disphosphate group, a free 5'-diphosphate group, a free 5'- monophosphate group, or a free 5'-OH group, or comprising chemically modified analogues of said 5 - Cap, said 5'-tri phosphate group, said free 5'-disphosphate group or said free 5'-monophosphate group.
  • nucleic acid molecule of any one of the preceding embodiments 32-48 comprising a 5'cap selected from G[5']ppp[5']G, m7G[5']ppp[5']G, m 3 2 ' 2 ' 7 G[5']ppp[5']G, m 2 7 ' 3 '-°G[5']ppp[5']G (3'-ARCA), m 2 7 ' 2 '-°GpppG (2'-ARCA), m 2 7 ' 2 ' -0 GppSpG (p-S- ARCA), and m 2 7 ' 2 ' °GppSpG (p-S-ARCA) and m 2 7 ' 3 -°Gppp(mi 2 ' °)ApG.
  • a 5'cap selected from G[5']ppp[5']G, m7G[5']ppp[5']G, m 3 2 ' 2 ' 7
  • nucleic acid molecule of any one of the preceding embodiments 32-50 comprising an interrupted poly(A) sequence.
  • nucleic acid molecule of the preceding embodiment 55 wherein the antigenic peptides are derived from different proteins or different portions of the same protein and fused together, preferably such that a first antigenic peptide is adjacent to a second antigenic peptide or such that a first and second antigenic peptide are separated via a linker.
  • a nucleic acid molecule for eliciting an antigen-specific CD8 + T-cell response in a subject comprising a coding sequence encoding a polypeptide comprising an antigenic peptide and an N-terminal degron comprising a cleavable ubiquitin.
  • nucleic acid molecule of the preceding embodiment 58 wherein the ubiquitin is immediately followed by a destabilizing amino acid.
  • nucleic acid molecule of the preceding embodiment 59 wherein the destabilizing amino acid is selected from isoleucine, glutamic acid, threonine, glutamine, phenylalanine, leucine, aspartic acid, arginine, lysine, and histidine, preferably selected from arginine, lysine, and histidine (e.g., as shown in SEQ ID NO.: 2).
  • nucleic acid molecule of any one of the preceding embodiments 58-61 for use in eliciting an antigenspecific CD8 + T-cell response.
  • nucleic acid molecule of any one of the preceding embodiments 58-63 wherein the subject is a mammal, preferably a human.
  • nucleic acid molecule of any one of the preceding embodiments 58-64 wherein the nucleic acid molecule elicits a two-fold, preferably a three-fold, more preferably a five-fold increase in the antigenspecific CD8 + T-cell response compared to an antigen-specific CD4 + T-cell immune response.
  • nucleic acid molecule of any one of the preceding embodiments 58-65 wherein the presence of an N-terminal degron reduces the half-life of the polypeptide to 1 hour or less, preferably to 30 minutes or less, more preferably to 10 minutes or less compared to the polypeptide without the N-terminal degron.
  • nucleic acid molecule of any one of the preceding embodiments 58-70, wherein the 5'UTR, if present, comprises a Kozak sequence.
  • nucleic acid molecule of any one of the preceding embodiments 58-71 comprising a 5'-cap, a free 5'-triphosphate group, a free 5'-disphosphate group, a free 5'-diphosphate group, a free 5'- monophosphate group, or a free 5'-OH group, or comprising chemically modified analogues of said 5 - cap, said 5'-tri phosphate group, said free 5'-disphosphate group or said free 5'-monophosphate group.
  • nucleic acid molecule of any one of the preceding embodiments 58-72 comprising a 5'cap selected from G[5']ppp[5']G, m7G[5']ppp[5']G, m 3 2 ' 2 ' 7 G[5']ppp[5']G, m 2 7 ' 3 '-°G[5']ppp[5']G (3'-ARCA), m 2 7 ' 2 '-°GpppG (2'-ARCA), m 2 7 ' 2 ' °GppSpG (
  • nucleic acid molecule of any one of the preceding embodiments 58-74 comprising an interrupted poly(A) sequence.
  • antigenic polypeptide is a pathogen-related, tumor-related, or disease-related antigenic peptide.
  • nucleic acid molecule of the preceding embodiment 79 wherein the antigenic peptides are derived from different proteins or different portions of the same protein and fused together, preferably such that a first antigenic peptide is adjacent to a second antigenic peptide or such that a first and second antigenic peptide are separated via a linker.
  • nucleic acid molecule of the preceding embodiment 80 wherein the linker comprises at most 8 amino acids, preferably at most 6 amino acids, more preferably at most 5 amino acids, most preferably 1 to 4 amino acids.
  • a degrading amino acid such as an arginine
  • nucleic acid molecule of any one of the preceding embodiments 58-82 wherein the ubiquitin has an amino acid sequence as shown in SEQ ID NO.: 1, or an amino acid sequence that is at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.: 1, and/or wherein the ubiquitin comprises a lysine substitution, preferably a lysine substitution in one or more of the amino acid positions 6, 11, 27, 29, 33, and 63 or any combination thereof with reference to SEQ ID NO.: 1, more preferably wherein the lysine is substituted at the one or more positions by a degrading amino acid (such as an arginine).
  • a degrading amino acid such as an arginine
  • nucleic acid molecule of any one of the preceding embodiments 1-85 wherein the N-terminal degron comprises two or more ubiquitins, in particular two or more ubiquitins adjacent to each other, wherein the two or more ubiquitins are optionally directly adjacent to each other or separated by a linker.
  • the nucleic acid molecule according to preceding embodiment 86 wherein the two or more ubiquitins can be the same or different ubiquitin sequences.
  • An isolated host cell which comprises the nucleic acid molecule of any one of the preceding embodiments 1-93 and/or the polypeptide of embodiment 94.
  • a composition which comprises the isolated host cell of embodiment 84.
  • a pharmaceutical composition comprising a nucleic acid molecule of any one of the preceding embodiments 1-93 or the polypeptide of embodiment 94 in a pharmaceutically acceptable carrier.
  • a vaccine composition for eliciting an antigen-specific CD8 + T-cell response comprising an effective dose of the nucleic acid molecule of any one of the preceding embodiments 1 to 93, the polypeptide of embodiment 94, or the pharmaceutical composition of embodiment 97. 99.
  • the vaccine composition of the preceding embodiment 98 wherein the nucleic acid molecule is associated with cationic lipids or is encapsulated into a nanoparticle or liposome.
  • nucleic acid molecule of any one of embodiments 1 to 93, the polypeptide of embodiment 94, the pharmaceutical composition of embodiment 97, or the vaccine composition of any one of embodiments 98 to 99 for use in a method of eliciting an antigen-specific CD8 + T-cell response in a subject in need thereof, comprising: administering to the subject an effective amount of the nucleic acid molecule, the pharmaceutical composition, or the vaccine composition, thereby stimulating an antigen-specific CD8 + T- cell response in the subject.
  • nucleic acid molecule of any one of embodiments 1 to 93, the polypeptide of embodiment 94, the pharmaceutical composition of embodiment 97, or the vaccine composition of any one of embodiments 98 to 99 for use in a method for inducing the formation of MHC-I/peptide complexes in a cell, the method comprising administering to a subject an effective amount of the nucleic acid molecule, the pharmaceutical composition, or the vaccine composition.
  • nucleic acid molecule of any one of embodiments 1 to 93, the polypeptide of embodiment 94, the pharmaceutical composition of embodiment 97, or the vaccine composition of any one of embodiments 98 to 99 for use in a method for stimulating or activating CD8 + T-cells, wherein the method comprises administering to a subject an effective amount of the nucleic acid molecule, the pharmaceutical composition, or the vaccine composition.
  • RNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme).
  • RNA molecule can encode a polypeptide (e.g., by a translation process).
  • a gene, a cDNA, or a single-stranded RNA encodes a polypeptide if transcription and translation of mRNA corresponding to that gene produces the polypeptide in a cell or other biological system.
  • a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such a target polypeptide agent.
  • a coding region of a single-stranded RNA encoding a target polypeptide agent refers to a non-coding strand of such a target polypeptide agent, which may be used as a template for transcription of a gene or cDNA.
  • the phrase "nucleic acid molecule encoding a peptide or protein” means that the nucleic acid molecule, if present in the appropriate environment, for example within a cell and/or in a cell-free translation system, can direct the assembly of amino acids to produce the peptide or protein via a process of translation.
  • epitope refers to the part of an antigen that, as used herein, refers to an agent that elicits an immune response; and/or an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody.
  • a "major histocompatibility complex” is either an MHC class I (MHC-I) or MHC class II (MHC-II) molecules and is a protein complex present in all vertebrates.
  • MHC proteins are important for signalling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells.
  • epitopes are the discrete, three-dimensional sites on an antigen, which are presented on MHC class I or II and recognized by T cells via T cell receptors.
  • Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three- dimensional structural characteristics, as well as specific charge characteristics. Conformational and non- conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • the term relates to an immunogenic portion of an antigen comprising the epitope.
  • An epitope of a protein preferably comprises a continuous or discontinuous portion of said protein.
  • epitope of a protein preferably comprises a continuous or discontinuous portion of said protein.
  • epitope of a protein preferably comprises a continuous or discontinuous portion of said protein.
  • epitope of a protein preferably comprises a continuous or discontinuous portion of said protein.
  • epitope of a protein preferably comprises a continuous or discontinuous
  • the epitope in the context of the present invention is a T cell epitope.
  • the peptides (epitopes) are typically about 8 to about 15 amino acids long although longer or shorter peptides can also be effective.
  • the binding peptides (epitopes) are typically about 10 to about 25 amino acids long and are in particular about 13 to about 18 amino acids long, although longer and shorter peptides may also be effective.
  • a "gene” is a DNA sequence in a chromosome that codes for a protein.
  • a gene includes coding sequence (i.e., sequence that encodes a particular protein); in some embodiments, a gene includes non-coding sequence.
  • a gene may include both coding (e.g., exonic) and non-coding (e.g., intronic) sequences.
  • a gene may include one or more regulatory elements that, for example, may control or impact one or more aspects of gene expression (e.g., cell-type- specific expression, inducible expression, etc.).
  • an “immune cell” is any cell of hematopoietic lineage involved in regulating an immune response against an antigen (e.g., a bacterial or viral infection or an auto-antigen).
  • an immune cell is a leukocyte, such as a white blood cell.
  • Immune cells include neutrophils, eosinophils, basophils, lymphocytes, and/or monocytes. Lymphocytes include T lymphocytes and B lymphocytes. Immune cells can also be dendritic cells, natural killer (NK) cells, and/or a mast cell.
  • MG-132 or "MG132” is a commercially available 26S proteasome inhibitor.
  • MHC is a major histocompatibility complex comprising a polymorphic set of proteins.
  • MHC-I is the MHC class I molecule. MHC-I is expressed on the surface of all nucleated cells that present antigenic peptides (epitopes) to CD8 + (including cytotoxic) T cells in the form of proteolytically processed peptides, wherein the epitopes are typically 8-15 amino acids in length.
  • MHC-I/ peptide complex is the epitope-loaded MHC-I, meaning the MHC-I presenting an epitope to specific T cells.
  • operatively linked means that a promoter, or similar regulatory element, is positioned next to an expressible nucleotide sequence or coding region such that the transcription of that coding region is controlled and regulated by that promoter.
  • a plurality of antigenic peptides therefore refers to more than one antigenic peptide.
  • a plurality of antigenic peptides refers to at least two antigenic peptides, preferably at least three, at least four, or at least five antigenic peptides.
  • polypeptide polypeptide
  • nucleic acid molecule when used in the context of a nucleic acid molecule means a nucleic acid molecule having nucleotide sequences that are not naturally joined together and can be made by artificially combining two otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.
  • Recombinant nucleic acid molecules include vectors comprising an amplified or assembled polynucleotide, which can be used to transform or transfect a suitable host cell.
  • a host cell that comprises the recombinant nucleic acid molecule is referred to as a "recombinant host cell.”
  • the nucleic acid molecule is then expressed in the recombinant host cell to produce a "recombinant polypeptide.”
  • a recombinant nucleic acid molecule can also comprise a non-coding function.
  • a “string” can be a nucleic acid molecule or polypeptide of the invention.
  • a string may comprise a plurality of antigenic peptides strung like beads on a string.
  • a "string” can comprise a plurality of antigenic peptides, wherein at least some of these antigenic peptides may be separated by linker sequences.
  • a "subject" is a mammal, preferably a human, of either gender (a male or a female).
  • the subject may be of any age.
  • the subject is female.
  • the subject is male.
  • the subject is a patient having a disease, in particular a female patient having disease and/or a male patient having a disease.
  • treating when used in the context of a disease or disease condition means ameliorating, improving or remedying a disease, disorder, or symptom of a disease or condition associated with the disease, or can mean completely or partially stopping, on a molecular level, the biochemical basis of the disease, such as halting replication of a virus, etc. It describes an act that leads to the elimination, reduction, alleviation, reversal, or prevention or delay of onset or recurrence of any symptom of a disease.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors comprise plasmids, cosmid vectors, phagemids such as lambda phage, virus genomes including retroviral, adenoviral or baculoviral vectors, artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or PI artificial chromosomes (PAC) and functional portions thereof.
  • BAC bacterial artificial chromosomes
  • YAC yeast artificial chromosomes
  • PAC PI artificial chromosomes
  • plasmid refers to a circular double stranded DNA into which additional DNA segments may be ligated.
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • Expression vectors comprise plasmids as well as viral vectors and generally contain a desired coding sequence and appropriate non-coding sequences necessary for the expression of the operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammal) or in in vitro expression systems.
  • Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragments.
  • nucleic acid comprises deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof.
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • the term comprises genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules.
  • a nucleic acid is DNA.
  • a nucleic acid is RNA.
  • a nucleic acid is a mixture of DNA and RNA.
  • a nucleic acid may be present as a single-stranded or double-stranded and linear or covalently circularly closed molecule.
  • a nucleic acid can be isolated.
  • isolated nucleic acid means, according to the present disclosure, that the nucleic acid (i) was amplified in vitro, for example via polymerase chain reaction (PCR) for DNA or in vitro transcription (using, e.g., an RNA polymerase) for RNA, (ii) was produced recombinantly by cloning, (iii) was purified, for example, by cleavage and separation by gel electrophoresis, or (iv) was synthesized, for example, by chemical synthesis.
  • PCR polymerase chain reaction
  • RNA polymerase RNA polymerase
  • nucleoside (abbreviated herein as "N") relates to compounds which can be thought of as nucleotides without a phosphate group. While a nucleoside is a nudeobase linked to a sugar (e.g., ribose or deoxyribose), a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.
  • the five standard nucleosides which usually make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine and guanosine.
  • the five nucleosides are commonly abbreviated to their one letter codes U, A, T, C and G, respectively.
  • thymidine is more commonly written as “dT” ("d” represents “deoxy") as it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA).
  • uridine is found in RNA and not DNA.
  • the remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G, whereas in DNA they would be represented as dA, dC and dG.
  • a modified purine (A or G) or pyrimidine (C, T, or U) base moiety is, in some embodiments, modified by one or more alkyl groups, e.g., one or more Cl-4 alkyl groups, e.g., one or more methyl groups.
  • modified purine or pyrimidine base moieties include N7-alkyl-guanine, N6-alkyl-adenine, 5-alkyl-cytosine, 5- alkyl-uracil, and N(l)-alkyl-uracil, such as N7-C 1 -4 alkyl-guanine, N6-C 1 -4 alkyl-adenine, 5-C 1 -4 alkyl-cytosine, 5-C 1 -4 alkyl-uracil, and N(l)-Cl-4 alkyl-uracil, preferably N7-methyl-guanine, N6-methyl-adenine, 5-methyl- cytosine, 5-methyl-uracil, and N(l)-methyl-uracil.
  • DNA relates to a nucleic acid molecule which is entirely or at least substantially composed of deoxyribonucleotide residues.
  • the DNA contains all or a majority of deoxyribonucleotide residues.
  • deoxyribonucleotide refers to a nucleotide which lacks a hydroxyl group at the 2'-position of a p-D-ribofuranosyl group.
  • DNA encompasses without limitation, double stranded DNA, single stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, as well as modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nudeotide material to internal DNA nucleotides or to the end(s) of DNA. It is also contemplated herein that nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. For the present disclosure, these altered DNAs are considered analogs of naturally-occurring DNA.
  • a molecule contains "a majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule.
  • the total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).
  • DNA may be recombinant DNA and may be obtained by cloning of a nucleic acid, in particular cDNA.
  • the cDNA may be obtained by reverse transcription of RNA.
  • RNA relates to a nucleic acid molecule which includes ribonucleotide residues.
  • the nucleic acid molecule as disclosed herein is an RNA as disclosed herein.
  • the RNA contains all or a majority of ribonucleotide residues.
  • ribonucleotide refers to a nucleotide with a hydroxyl group at the 2'-position of a p-D-ribofuranosyl group.
  • RNA encompasses without limitation, double stranded RNA, single stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, as well as modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides. Such alterations may refer to addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of RNA. It is also contemplated herein that nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides.
  • altered/modified nucleotides can be referred to as analogs of naturally occurring nucleotides, and the corresponding RNAs containing such altered/modified nucleotides (i.e., altered/modified RNAs) can be referred to as analogs of naturally occurring RNAs.
  • a molecule contains "a majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is more than 50% (such as at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), based on the total number of nucleotide residues in the molecule.
  • the total number of nucleotide residues in a molecule is the sum of all nucleotide residues (irrespective of whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).
  • RNA includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), trans-amplifying RNA (taRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA) and immunostimulatory RNA (isRNA).
  • RNA refers to mRNA.
  • in vitro transcription means that the transcription (i.e., the generation of
  • RNA is conducted in a cell-free manner. I.e., IVT does not use living/cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)).
  • IVT does not use living/cultured cells but rather the transcription machinery extracted from cells (e.g., cell lysates or the isolated components thereof, including an RNA polymerase (preferably T7, T3 or SP6 polymerase)).
  • the term '"RNA includes “mRNA”.
  • mRNA means “messenger-RNA” and includes a “transcript” which may be generated by using a DNA template.
  • mRNA encodes a peptide or polypeptide.
  • mRNA is single-stranded but may contain self-complementary sequences that allow parts of the mRNA to fold and pair with itself to form double helices.
  • dsRNA means double-stranded RNA and is RNA with two partially or completely complementary strands.
  • the mRNA relates to an RNA transcript which encodes a peptide or polypeptide.
  • the mRNA which preferably encodes a peptide or polypeptide has a length of at least 15 nucleotides (such as at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 600, at least 700, at least 800, at least 900 nucleotides), preferably up to 15,000, such as up to 12,000, up to 8,000, up to 6,000 nucleotides, or up to 4,000 nucleotides.
  • nucleotides such as at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 120, at least 140, at least 160, at least 180, at least 200, at least 250, at least 300, at least 350, at least 400,
  • mRNA generally contains a 5' untranslated region (5'-UTR), a peptide/ polypeptide coding region, and a 3' untranslated region (3'-UTR).
  • the mRNA is produced by in vitro transcription or chemical synthesis.
  • the mRNA is produced by in vitro transcription using a DNA template.
  • the in vitro transcription methodology is known to the skilled person; cf., e.g., Molecular Cloning: A Laboratory Manual, 4th Edition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012.
  • in vitro transcription kits are commercially available, e.g., from Thermo Fisher Scientific (such as TranscriptAidTM T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc. (such as HiScribeTM T7 kit, HiScribeTM T7 ARCA mRNA kit), Promega (such as RiboMAXTM, HeLaScribe®, Riboprobe® systems), Jena Bioscience (such as SP6 or T7 transcription kits), and Epicentre (such as AmpliScribeTM).
  • Thermo Fisher Scientific such as TranscriptAidTM T7 kit, MEGAscript® T7 kit, MAXIscript®), New England BioLabs Inc.
  • HiScribeTM T7 kit such as HiScribeTM T7 kit, HiScribeTM T7 ARCA mRNA kit
  • Promega such as RiboMAXTM, HeLaScribe®, Riboprobe® systems
  • Jena Bioscience such as SP6 or T
  • correspondingly modified nucleotides such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and/or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be affected in and/or added to the mRNA after transcription.
  • RNA is in vitro transcribed RNA (IVT-RNA) and may be obtained by in vitro transcription of an appropriate DNA template.
  • the promoter for controlling transcription can be any promoter for any RNA polymerase.
  • RNA polymerases are the T7, T3, and SP6 RNA polymerases.
  • the in vitro transcription is controlled by a T7 or SP6 promoter.
  • a DNA template for in vitro transcription may be obtained by cloning of a nucleic acid, in particular cDNA, and introducing it into an appropriate vector for in vitro transcription.
  • the cDNA may be obtained by reverse transcription of RNA.
  • the RNA is "replicon RNA” or simply a “replicon”, in particular "self-replicating RNA” or “self-amplifying RNA”.
  • the replicon or self-replicating RNA is derived from or comprises elements derived from an ssRNA virus, in particular a positive-stranded ssRNA virus such as an alphavirus.
  • Alphaviruses are typical representatives of positive-stranded RNA viruses.
  • Alphaviruses replicate in the cytoplasm of infected cells (for review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856).
  • the total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5'-cap, and a 3' poly(A) tail.
  • the genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome.
  • the four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3' terminus of the genome.
  • the first ORF is larger than the second ORF, the ratio being roughly 2:1.
  • the genomic RNA In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124). Following infection, i.e. at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234).
  • mRNA eukaryotic messenger RNA
  • Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms.
  • the open reading frame encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest.
  • Alphavirus-based trans-replication (transamplification) systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system).
  • Trans-replication requires the presence of both these nucleic acid molecules in a given host cell.
  • the nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow recognition and RNA synthesis by the alphaviral replicase.
  • the RNA (in particular, mRNA) described herein contains one or more modifications, e.g., in order to increase its stability and/or increase translation efficiency and/or decrease immunogenicity and/or decrease cytotoxicity.
  • the RNA (in particular, mRNA) may be modified within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without altering the sequence of the expressed peptide or polypeptide.
  • Such modifications are described, for example, in WO 2007/036366 and PCT/EP2019/056502, and include the following: a 5'-cap structure; an extension or truncation of the naturally occurring poly(A) tail; an alteration of the 5'- and/or 3'-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA; the replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., to alter, preferably increase, the GC content of the RNA).
  • UTR 5'-cap structure
  • an extension or truncation of the naturally occurring poly(A) tail an alteration of the 5'- and/or 3'-untranslated regions (UTR) such as introduction of a UTR which is not related to the coding region of said RNA
  • UTR 5'- and/or 3'-untranslated regions
  • codon optimization e.g., to alter, preferably increase,
  • a combination of the above described modifications i.e., incorporation of a 5'-cap structure, incorporation of a poly-A sequence, unmasking of a poly-A sequence, alteration of the 5'- and/or 3'- UTR (such as incorporation of one or more 3'-UTRs), replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and/or pseudouridine (4 1 ) or N(l)- methylpseudouridine (mlQJ) or 5-methyluridine (m5U) for uridine), and codon optimization, has a synergistic influence on the stability of RNA (preferably mRNA) and increase in translation efficiency.
  • synthetic nucleotides e.g., 5-methylcytidine for cytidine and/or pseudouridine (4 1 ) or N(l)- methylpseudouridine (mlQJ) or 5-methyluridine (m5
  • the RNA (in particular, mRNA) described in the present disclosure contains a combination of at least two, at least three, at least four or all five of the above-mentioned modifications, i.e., (I) incorporation of a 5'-cap structure, (ii) incorporation of a poly-A sequence, unmasking of a poly-A sequence; (ill) alteration of the 5'- and/or 3'-UTR (such as incorporation of one or more 3'-UTRs); (iv) replacing one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and/or pseudouridine (4 1 ) or N(l)-methylpseudouridine (mlQJ) or 5-methyluridine (m5U) for uridine), and (v) codon optimization.
  • synthetic nucleotides e.g., 5-methylcytidine for cytidine and/or pseudouridine (4 1 ) or
  • the RNA (in particular, mRNA) described herein comprises a 5'-cap structure.
  • the "5'cap” is a cap structure on the 5'-end of mRNAs, which is present in eukaryotic organisms.
  • Naturally occurring cap structures comprise a ribo-guanosine residue that is methylated at position N7 of the guanine base, abbreviated 7m Gppp.
  • 7m Gppp ribo-guanosine residue that is methylated at position N7 of the guanine base
  • the presence of the 7m Gppp fragment on the 5'-end is essential for mRNA maturation, it protects the mRNAs from degradation by exonucleases, facilitates transport of mRNAs from the nucleus to the cytoplasm and plays a key role in assembly of the translation initiation complex.
  • a 5' cap may be or comprise a dinucleotide cap analog such as G[5']ppp[5']G, m7G[5']ppp[5']G, m 3 2 ' 2 ' 7 G[5']ppp[5']G, m 2 7 - 3 '-°G[5']ppp[5']G (3'-ARCA), m 2 7 ' 2 '-°GpppG (2'- ARCA), m 2 7 ' 2 ' °GppSpG (p-S- ARCA), and m 2 7 ' 2 ' °GppSpG (p-S-ARCA) and m 2 7 ' 3 ' °Gppp(mi 2 ' °)ApG (CleanCap413).
  • a dinucleotide cap analog such as G[5']ppp[5']G, m7G[5']ppp[5']G, m 3 2
  • the RNA does not have uncapped 5'-triphosphates.
  • the RNA in particular, mRNA may comprise a conventional 5'-cap and/or a 5'-cap analog.
  • inventional 5'-cap refers to a cap structure found on the 5'-end of an RNA molecule and generally comprises a guanosine 5'- triphosphate (Gppp) which is connected via its triphosphate moiety to the 5'-end of the next nucleotide of the RNA (i.e., the guanosine is connected via a 5' to 5' triphosphate linkage to the rest of the RNA).
  • Gppp guanosine 5'- triphosphate
  • the guanosine may be methylated at position N7 (resulting in the cap structure m7Gppp).
  • 5'-cap analog includes a 5'-cap which is based on a conventional 5'-cap but which has been modified at either the 2 1 - or 3'-position of the m7guanosine structure in order to avoid an integration of the 5'-cap analog in the reverse orientation (such 5'-cap analogs are also called anti-reverse cap analogs (ARCAs)).
  • ARCAs anti-reverse cap analogs
  • Particularly preferred 5'-cap analogs are those having one or more substitutions at the bridging and non-bridging oxygen in the phosphate bridge, such as phosphoroth ioate modified 5'-cap analogs at the p-phosphate (such as m 2 7 ' 2 O G(5')ppSp(5')G (referred to as beta-S-ARCA or p-S-ARCA)), as described in PCT/EP2019/056502.
  • phosphoroth ioate modified 5'-cap analogs at the p-phosphate such as m 2 7 ' 2 O G(5')ppSp(5')G (referred to as beta-S-ARCA or p-S-ARCA)
  • RNA in particular, mRNA
  • a 5'-cap structure as described herein may be achieved by in vitro transcription of a DNA template in presence of a corresponding 5' -cap compound, wherein said 5'-cap structure is co-transcriptionally incorporated into the generated RNA (in particular, mRNA) strand, or the RNA (in particular, mRNA) may be generated, for example, by in vitro transcription, and the 5'-cap structure may be attached to the RNA post- transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.
  • capping enzymes for example, capping enzymes of vaccinia virus.
  • the RNA comprises a 5'-cap structure selected from the group consisting of m 2 7 ' 2 O G(5')ppSp(5')G (in particular its DI diastereomer), m 2 7 ' 3 '°G(5')ppp(5')G, and m 2 7 ' 3 ' °Gppp(mi 2 ' °)ApG.
  • RNA comprises m 2 7 ' 2 '°G(5')ppSp(5')G (in particular its DI diastereomer) as 5' -cap structure.
  • RNA comprises m 2 7 ' 3 ' °Gppp(mi 2 ' °)ApG as 5'-cap structure.
  • the RNA comprises a capO, capl, or cap2, preferably capl or cap2.
  • capO means the structure "m 7 GpppN", wherein N is any nucleoside bearing an OH moiety at position 2 1 .
  • capl means the structure "m 7 GpppNm”, wherein Nm is any nucleoside bearing an OCH3 moiety at position 2'.
  • cap2 means the structure "m 7 GpppNmNm", wherein each Nm is independently any nucleoside bearing an OCH 3 moiety at position 2 1 .
  • the 5'-cap analog beta-S-ARCA (0-S-ARCA) has the following structure:
  • the "DI diastereomer of beta-S-ARCA" or "beta-S-ARCA(Dl)” is the diastereomer of beta-S-ARCA which elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)) and thus exhibits a shorter retention time.
  • the HPLC preferably is an analytical HPLC.
  • a Supelcosil LC-18-T RP column preferably of the format: 5 pm, 4.6 x 250 mm is used for separation, whereby a flow rate of 1.3 ml/min can be applied.
  • VWD UV-detection
  • FLD fluorescence detection
  • the 5'-cap analog m 2 7 ' 3 ' °Gppp(mi 2 ' °)ApG (also referred to as m 2 7 ' 3 O G(5')ppp(5')m 2 ' °ApG) which is a building block of a capl has the following structure:
  • An exemplary capO mRNA comprising 0-S-ARCA and mRNA has the following structure:
  • An exemplary capO mRNA comprising m2 7 ' 3 '°G(5')ppp(5')G and mRNA has the following structure:
  • An exemplary capl mRNA comprising m2 7 ' 3 ' °Gppp(mi 2 ' °)ApG and mRNA has the following structure:
  • poly-A tail or "poly-A sequence” or “poly(A)-ta II” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA (in particular, mRNA) molecule.
  • poly(A)-tail refers to a chain of adenine nucleotides that is added to a mRNA molecule during RNA processing to increase the stability of the molecule. This process, called polyadenylation, adds a poly(A)-tail that is usually between 100 and 250 residues long. Poly(A) tails play an important role in the translation and stability of the mRNA.
  • RNA having an unmasked poly-A sequence is translated more efficiently than RNA having a masked poly-A sequence.
  • poly(A) tail relates to a sequence of adenyl (A) residues which typically is located on the 3'-end of an RNA molecule and "unmasked poly-A sequence” means that the poly-A sequence at the 3' end of an RNA molecule ends with an A of the poly-A sequence and is not followed by nucleotides other than A located at the 3' end, i.e. downstream, of the poly-A sequence.
  • a long poly-A sequence of about 120 base pairs results in an optimal transcript stability and translation efficiency of RNA.
  • Poly-A tails or poly-A sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs (in particular, mRNAs) described herein.
  • An "interrupted poly(A)-tail” is a poly(A)-tail comprising non-adenine nucleotides at regular or irregularly spaced intervals.
  • the interrupting sequence is a trinucleotide, dinucleotide or mononucleotide interrupting sequence.
  • the poly(A) tail comprises or contains one non-adenine nucleotide or one consecutive stretch of 2 to 10 non-adenine nucleotides every 8 to 50 consecutive adenine nucleotides.
  • the poly(A) tail comprises or contains 1, 2, 3, 4, or 5 consecutive non-adenine nucleotides every 8-50 consecutive adenine nucleotides. In some embodiments, wherein the poly(A) tail comprises or contains more than one non-adenine nucleotide or more than one consecutive stretch of 2-10 non-adenine nucleotides. In some embodiments, a poly(A) sequence measuring 110 nucleotides in length, consisting of a stretch of 30 adenosine residues, followed by a 10-nucleotides linking sequence and another 70 adenosine residues is used. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. In nature, an uninterrupted poly-A tail is typical.
  • RNAs in particular, mRNAs
  • RNAs can have a poly-A tail attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase. It has been demonstrated that a poly-A tail of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (5') of the poly-A tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009- 4017).
  • the poly-A tail may be of any length.
  • a poly-A tail comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides.
  • nucleotides in the poly-A tail typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly-A tail are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate).
  • consists of means that all nucleotides in the poly-A tail, i.e., 100% by number of nucleotides in the poly-A tail, are A nucleotides.
  • a nucleotide or “A” refers to adenylate.
  • a poly-A tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.
  • the DNA sequence encoding a poly-A tail (coding strand) is referred to as poly(A) cassette.
  • the poly(A) cassette present in the coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al may be used in the present disclosure.
  • a poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on DNA level, constant propagation of plasmid DNA in E. coli and is still associated, on RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed.
  • the poly-A tail contained in an RNA (in particular, mRNA) molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • the poly(A) tail comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence of 10 nucleotides.
  • no nucleotides other than A nucleotides flank a poly-A tail at its 3'-end, i.e., the poly-A tail is not masked or followed at its 3'-end by a nucleotide other than A.
  • a poly-A tail may comprise at least 20, at least 30, at least 40, at least 80, or at least
  • the poly- A tail may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly-A tail comprises at least 100 nucleotides. In some embodiments, the poly-A tail comprises about 150 nucleotides. In some embodiments, the poly-A tail comprises about 120 nucleotides.
  • the term "untranslated region" or “UTR” relates to a region in a DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA molecule, such as an mRNA molecule.
  • An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5‘- UTR) and/or 3' (downstream) of an open reading frame (3'-UTR).
  • a 5'-UTR if present, is located at the 5'-end, upstream of the start codon of a protein-encoding region.
  • a 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap.
  • a 3'-UTR if present, is located at the 3'-end, downstream of the termination codon of a protein-encoding region, but the term "3'-UTR" does generally not include the poly-A sequence.
  • the 3'-UTR is upstream of the poly-A sequence (if present), e.g., directly adjacent to the poly- A sequence.
  • the "3'UTR sequence" or "3'UTR” or “3'-UTR” is a 3' untranslated region known to regulate mRNA-based processes, such as mRNA localization, mRNA stability, and translation.
  • 3' UTRs can establish 3' UTR-mediated protein-protein interactions (PPIs), and thus can transmit genetic information encoded in 3' UTRs to proteins.
  • PPIs protein-protein interactions
  • This function has been shown to regulate diverse protein features, including protein complex formation or posttranslational modifications, but is also expected to alter protein conformations.
  • Incorporation of a 3'-UTR into the 3'-non translated region of an RNA (preferably mRNA) molecule can result in an enhancement in translation efficiency.
  • a synergistic effect may be achieved by incorporating two or more of such 3'-UTRs (which are preferably arranged in a head-to-tail orientation; cf., e.g., Holtkamp et al., Blood 108, 4009-4017 (2006)).
  • the 3'-UTRs may be autologous or heterologous to the RNA (e.g., mRNA) into which they are introduced.
  • the 3'-UTR is derived from a globin gene or mRNA, such as a gene or mRNA of alpha2-globin, alphal-globin, or beta-globin, e.g., beta-globin, e.g., human beta-globin.
  • the RNA may be modified by the replacement of the existing 3'-UTR with or the insertion of one or more, e.g., two copies of a 3'-UTR derived from a globin gene, such as alpha2-globin, alphal-globin, beta-globin, e.g., beta-globin, e.g., human beta-globin.
  • a globin gene such as alpha2-globin, alphal-globin, beta-globin, e.g., beta-globin, e.g., human beta-globin.
  • the "5' UTR sequence" or “5'UTR” s to a 5'-untranslated region which lies within the noncoding genome upstream of a coding sequence and plays an important role in regulating gene expression.
  • 5'-UTR sequences may be numerous cis-regulatory elements present that can interact with the transcriptional machinery to regulate mRNA abundance.
  • the 5'-untranslated region may contain various RNA-based regulatory elements including the secondary structures, RNA-binding protein motifs, upstream open-reading frames (uORFs), internal ribosome entry sites, terminal oligo pyrimidine (TOP) tracts, and G-quadruplexes. These elements can alter the efficiency of mRNA translation; some can also affect mRNA transcript levels via changes in stability or degradation.
  • a 5'-UTR is or comprises a modified human alphaglobin 5'-UTR.
  • a 3'-UTR comprises a first sequence from the amino terminal enhancer of split (AES) messenger RNA and a second sequence from the mitochondrial encoded 12S ribosomal RNA.
  • the RNA (in particular, mRNA) described herein may have modified ribonucleotides in order to increase its stability and/or decrease immunogenicity and/or decrease cytotoxicity.
  • uridine in the RNA (in particular, mRNA) described herein is replaced (partially or completely, preferably completely) by a modified nucleoside.
  • the modified nucleoside is a modified uridine.
  • the modified uridine replacing uridine is selected from the group consisting of pseudouridine (qj), Nl-methyl-pseudouridine (mlqj), 5-methyl-uridine (m5U), and combinations thereof.
  • the modified nucleoside replacing (partially or completely, preferably completely) uridine in the RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza- uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-th io-u rid ine (s4U), 4-thio-pseudouridine, 2- thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridineor 5- bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5- carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine
  • RNA preferably mRNA which is modified by pseudouridine (replacing partially or completely, preferably completely, uridine)
  • QJ-modified RNA which is modified by pseudouridine (replacing partially or completely, preferably completely, uridine)
  • mlUJ-modified means that the RNA (preferably mRNA) contains N(l)-methylpseudouridine (replacing partially or completely, preferably completely, uridine).
  • m5U-modified means that the RNA (preferably mRNA) contains 5-methyluridine (replacing partially or completely, preferably completely, uridine).
  • RNA preferably mRNA
  • N(l)-methylpseudouridine replacing completely uridine
  • the codons of the RNA (in particular, mRNA) described in the present disclosure may further be optimized, e.g., to increase the GC content of the RNA and/or to replace codons which are rare in the cell (or subject) in which the peptide or polypeptide of interest is to be expressed by codons which are synonymous frequent codons in said cell (or subject).
  • the amino acid sequence encoded by the RNA (in particular, mRNA) described in the present disclosure is encoded by a coding sequence which is codon- optimized and/or the G/C content of which is increased compared to wild type coding sequence.
  • This also includes embodiments, wherein one or more sequence regions of the coding sequence are codon-optimized and/or increased in the G/C content compared to the corresponding sequence regions of the wild type coding sequence.
  • the codon-optimization and/or the increase in the G/C content preferably does not change the sequence of the encoded amino acid sequence.
  • coding regions may be codon-optimized for optimal expression in a subject to be treated using the RNA (in particular, mRNA) described herein. Codon-optimization is based on the finding that the translation efficiency is also determined by a different frequency in the occurrence of tRNAs in cells. Thus, the sequence of RNA (in particular, mRNA) may be modified such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons".
  • the guanosine/cytosine (G/C) content of the coding region of the RNA (in particular, mRNA) described herein is increased compared to the G/C content of the corresponding coding sequence of the wild type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence encoded by the wild type RNA.
  • This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that RNA. Sequences having an increased G (guanosine)/C (cytosine) content are more stable than sequences having an increased A (adenosine)/U (uracil) content.
  • codons which contain A and/or U nucleotides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleotides.
  • the G/C content of the coding region of the RNA (in particular, mRNA) described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G/C content of the coding region of the wild type RNA.
  • non-immunogenic RNA refers to RNA that does not induce a response by the immune system upon administration, e.g., to a mammal, or induces a weaker response than would have been induced by the same RNA that differs only in that it has not been subjected to the modifications and treatments that render the non-immunogenic RNA non-immunogenic, i.e., than would have been induced by standard RNA (stdRNA).
  • stdRNA standard RNA
  • non-immunogenic RNA is rendered non-immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and/or limiting the amount of double-stranded RNA (dsRNA), e.g., by limiting the formation of double-stranded RNA (dsRNA), e.g., during in vitro transcription, and/or by removing double-stranded RNA (dsRNA), e.g., following in vitro transcription.
  • dsRNA double-stranded RNA
  • non-immunogenic RNA is rendered non-immunogenic by incorporating modified nucleosides suppressing RNA-mediated activation of innate immune receptors into the RNA and/or by removing double-stranded RNA (dsRNA), e.g., following in vitro transcription.
  • dsRNA double-stranded RNA
  • any modified nucleoside may be used as long as it lowers or suppresses immunogenicity of the RNA.
  • modified nucleosides that suppress RNA-mediated activation of innate immune receptors.
  • the modified nucleosides comprise a replacement of one or more uridines with a nucleoside comprising a modified nucleobase.
  • the modified nucleobase is a modified uracil.
  • the nucleoside comprising a modified nucleobase is selected from the group consisting of 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2- thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine ⁇ e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1- carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-
  • the nucleoside comprising a modified nudeobase is pseudouridine (qj), Nl-methyl- pseudouridine (mlqj) or 5-methyl-uridine (m5U), in particular Nl-methyl-pseudouridine.
  • the replacement of one or more uridines with a nucleoside comprising a modified nucleobase comprises a replacement of at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% of the uridines.
  • dsRNA double-stranded RNA
  • IVT in vitro transcription
  • dsRNA double-stranded RNA
  • formation of dsRNA can be limited during synthesis of mRNA by in vitro transcription (IVT), for example, by limiting the amount of uridine triphosphate (UTP) during synthesis.
  • UTP may be added once or several times during synthesis of mRNA.
  • dsRNA can be removed from RNA such as IVT RNA, for example, by ion-pair reversed phase HPLC using a non-porous or porous C-18 polystyrene-divinylbenzene (PS- DVB) matrix.
  • PS- DVB polystyrene-divinylbenzene
  • an enzymatic based method using E. coli RNaselll that specifically hydrolyzes dsRNA but not ssRNA, thereby eliminating dsRNA contaminants from IVT RNA preparations can be used.
  • dsRNA can be separated from ssRNA by using a cellulose material.
  • an RNA preparation is contacted with a cellulose material and the ssRNA is separated from the cellulose material under conditions which allow binding of dsRNA to the cellulose material and do not allow binding of ssRNA to the cellulose material.
  • Suitable methods for providing ssRNA are disclosed, for example, in WO 2017/182524.
  • remove or “removal” refers to the characteristic of a population of first substances, such as non-immunogenic RNA, being separated from the proximity of a population of second substances, such as dsRNA, wherein the population of first substances is not necessarily devoid of the second substance, and the population of second substances is not necessarily devoid of the first substance.
  • a population of first substances characterized by the removal of a population of second substances has a measurably lower content of second substances as compared to the non-separated mixture of first and second substances.
  • the amount of double-stranded RNA is limited, e.g., dsRNA (especially dsmRNA) is removed from non-immunogenic RNA , such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, less than 0.1%, less than 0.05%, less than 0.03%, less than 0.01%, less than 0.005%, less than 0.004%, less than 0.003%, less than 0.002%, less than 0.001%, or less than 0.0005% of the RNA in the non-immunogenic RNA composition is dsRNA.
  • dsRNA double-stranded RNA
  • the non-immunogenic RNA is free or essentially free of dsRNA.
  • the non-immunogenic RNA (especially mRNA) composition comprises a purified preparation of single-stranded nucleoside modified RNA.
  • the non-immunogenic RNA (especially mRNA) composition comprises single-stranded nucleoside modified RNA (especially mRNA) and is substantially free of double stranded RNA (dsRNA).
  • the non-immunogenic RNA (especially mRNA) composition comprises at least 90%, at least 91%, at least 92%, at least 93 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, at least 99.99%, at least 99.991%, at least 99.992%, , at least 99.993%,, at least 99.994%, , at least 99.995%, at least 99.996%, at least 99.997%, or at least 99.998% single stranded nucleoside modified RNA, relative to all other nucleic acid molecules (DNA, dsRNA, etc.).
  • RNA may be spotted onto a membrane, e.g., nylon blotting membrane. The membrane may be blocked, e.g., in TBS-T buffer (20 mM TRIS pH 7.4, 137 mM NaCI, 0.1% (v/v) TWEEN-20) containing 5% (w/v) skim milk powder.
  • the membrane may be incubated with dsRNA-specific antibody, e.g., dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). After washing, e.g., with TBS-T, the membrane may be incubated with a secondary antibody, e.g., HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody may be detected.
  • dsRNA-specific antibody e.g., dsRNA-specific mouse mAb (English & Scientific Consulting, Szirak, Hungary). After washing, e.g., with TBS-T, the membrane may be incubated with a secondary antibody, e.g., HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch, Cat #715-035-150), and the signal provided by the secondary antibody may be detected.
  • a secondary antibody e.g.
  • the non-immunogenic RNA (especially mRNA) is translated in a cell more efficiently than standard RNA with the same sequence.
  • translation is enhanced by a factor of 2- fold relative to its unmodified counterpart.
  • translation is enhanced by a 3-fold factor.
  • translation is enhanced by a 4-fold factor.
  • translation is enhanced by a 5-fold factor.
  • translation is enhanced by a 6-fold factor.
  • translation is enhanced by a 7-fold factor.
  • translation is enhanced by an 8-fold factor.
  • translation is enhanced by a 9-fold factor.
  • translation is enhanced by a 10-fold factor.
  • translation is enhanced by a 15-fold factor. In some embodiments, translation is enhanced by a 20-fold factor. In some embodiments, translation is enhanced by a 50-fold factor. In some embodiments, translation is enhanced by a 100-fold factor. In some embodiments, translation is enhanced by a 200-fold factor. In some embodiments, translation is enhanced by a 500-fold factor. In some embodiments, translation is enhanced by a 1000-fold factor. In some embodiments, translation is enhanced by a 2000-fold factor. In some embodiments, the factor is 10-1000-fold. In some embodiments, the factor is 10-100-fold. In some embodiments, the factor is 10-200-fold. In some embodiments, the factor is 10-300-fold.
  • the factor is 10-500-fold. In some embodiments, the factor is 20-1000- fold. In some embodiments, the factor is 30-1000-fold. In some embodiments, the factor is 50-1000-fold. In some embodiments, the factor is 100-1000-fold. In some embodiments, the factor is 200-1000-fold. In some embodiments, translation is enhanced by any other significant amount or range of amounts.
  • the non-immunogenic RNA exhibits significantly less innate immunogenicity than standard RNA with the same sequence. In some embodiments, the non-immunogenic RNA (especially mRNA) exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In some embodiments, innate immunogenicity is reduced by a 3-fold factor. In some embodiments, innate immunogenicity is reduced by a 4-fold factor. In some embodiments, innate immunogenicity is reduced by a 5- fold factor. In some embodiments, innate immunogenicity is reduced by a 6-fold factor. In some embodiments, innate immunogenicity is reduced by a 7-fold factor. In some embodiments, innate immunogenicity is reduced by an 8-fold factor.
  • innate immunogenicity is reduced by a 9-fold factor. In some embodiments, innate immunogenicity is reduced by a 10-fold factor. In some embodiments, innate immunogenicity is reduced by a 15-fold factor. In some embodiments, innate immunogenicity is reduced by a 20-fold factor. In some embodiments, innate immunogenicity is reduced by a 50-fold factor. In some embodiments, innate immunogenicity is reduced by a 100-fold factor. In some embodiments, innate immunogenicity is reduced by a 200-fold factor. In some embodiments, innate immunogenicity is reduced by a 500-fold factor. In some embodiments, innate immunogenicity is reduced by a 1000-fold factor. In some embodiments, innate immunogenicity is reduced by a 2000-fold factor.
  • the term "exhibits significantly less innate immunogenicity" refers to a detectable decrease in innate immunogenicity.
  • the term refers to a decrease such that an effective amount of the non-immunogenic RNA (especially mRNA) can be administered without triggering a detectable innate immune response.
  • the term refers to a decrease such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the non-immunogenic RNA.
  • the decrease is such that the non-immunogenic RNA (especially mRNA) can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the non-immunogenic RNA.
  • Immunogenicity is the ability of a foreign substance, such as RNA, to provoke an immune response in the body of a human or other animal.
  • the innate immune system is the component of the immune system that is relatively unspecific and immediate. It is one of two main components of the vertebrate immune system, along with the adaptive immune system.
  • RNA described herein may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked RNA, or delivery mediated by delivery vehicles. Some aspects of the disclosure involve the targeted delivery of the RNA disclosed herein to certain cells or tissues.
  • RNA in particular, mRNA
  • at least a portion of the RNA is delivered to a target cell or target organ.
  • at least a portion of the RNA is delivered to the cytosol of the target cell.
  • the RNA in particular, mRNA
  • the target cell is a muscle cell.
  • the target cell is a cell in the liver. In some embodiments, the target cell is a cell in the lung. In some embodiments, the disclosure involves targeting the lymphatic system, in particular secondary lymphoid organs, more specifically spleen. In some embodiments, the target cell is a cell in the lymph nodes. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. Thus, RNA (in particular, mRNA) compositions/formulations described herein may be used for delivering RNA to such target cell.
  • RNA in particular, mRNA compositions/formulations described herein may be used for delivering RNA to such target cell.
  • the "lymphatic system” is part of the circulatory system and an important part of the immune system, comprising a network of lymphatic vessels that carry lymph.
  • the lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and the circulating lymph.
  • the primary or central lymphoid organs generate lymphocytes from immature progenitor cells.
  • the thymus and the bone marrow constitute the primary lymphoid organs.
  • Secondary or peripheral lymphoid organs which include lymph nodes and the spleen, maintain mature naive lymphocytes and initiate an adaptive immune response.
  • RNA-based RNA delivery systems have an inherent preference to the liver, where, depending on the composition of the RNA delivery systems used, RNA expression in the liver can be obtained. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or the lipid metabolism (liposomes and lipid or cholesterol conjugates).
  • the target organ for RNA expression is liver and the target tissue is liver tissue.
  • the delivery to such target tissue is preferred, in particular, if presence of RNA or of the encoded peptide or polypeptide in this organ or tissue is desired and/or if it is desired to express large amounts of the encoded peptide or polypeptide and/or if systemic presence of the encoded peptide or polypeptide, in particular in significant amounts, is desired or required.
  • RNA may be administered with one or more delivery vehicles that protect the RNA from degradation, maximize delivery to on-target cells and minimize exposure to off-target cells.
  • RNA delivery vehicles may complex or encapsulate RNA and include a range of materials, including polymers and lipids.
  • such RNA delivery vehicles may form particles with RNA.
  • RNA in particular mRNA, described herein may be present in particles comprising (i) the RNA, and (ii) at least one cationic or cationically ionizable compound such as a polymer or lipid complexing the RNA. Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged RNA are involved in particle formation. This results in complexation and spontaneous formation of RNA particles.
  • RNA containing particles have been described previously to be suitable for delivery of RNA in particulate form (cf ., e.g., Kaczmarek, J. C. et al., 2017, Genome Medicine 9, 60).
  • nanoparticle encapsulation of RNA physically protects RNA from degradation and, depending on the specific chemistry, can aid in cellular uptake and endosomal escape.
  • the term "particle” relates to a structured entity formed by molecules or molecule complexes, in particular particle forming compounds.
  • the particle contains an envelope (e.g., one or more layers or lamellas) made of one or more types of amphiphilic substances (e.g., amphiphilic lipids).
  • amphiphilic substance means that the substance possesses both hydrophilic and lipophilic properties.
  • the envelope may also comprise additional substances (e.g., additional lipids) which do not have to be amphiphilic.
  • the particle may be a monolamellar or multilamellar structure, wherein the substances constituting the one or more layers or lamellas comprise one or more types of amphiphilic substances (in particular selected from the group consisting of amphiphilic lipids) optionally in combination with additional substances (e.g., additional lipids) which do not have to be amphiphilic.
  • the term “particle” relates to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure. According to the present disclosure, the term “particle” includes nanoparticles.
  • An "RNA particle” can be used to deliver RNA to a target site of interest (e.g., cell, tissue, organ, and the like).
  • An RNA particle may be formed from lipids comprising at least one cationic or cationically ionizable lipid.
  • the cationic or cationically ionizable lipid combines together with the RNA to form aggregates, and this aggregation results in colloidally stable particles.
  • RNA particles described herein include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.
  • a lipoplex (LPX) described herein is obtainable from mixing two aqueous phases, namely a phase comprising RNA and a phase comprising a dispersion of lipids.
  • the lipid phase comprises liposomes.
  • liposomes are self -closed unilamellar or multilamellar vesicular particles wherein the lamellae comprise lipid bilayers and the encapsulated lumen comprises an aqueous phase.
  • a prerequisite for using liposomes for nanoparticle formation is that the lipids in the mixture as required are able to form lamellar (bilayer) phases in the applied aqueous environment.
  • liposomes comprise unilamellar or multilamellar phospholipid bilayers enclosing an aqueous core (also referred to herein as an aqueous lumen). They may be prepared from materials possessing polar head (hydrophilic) groups and nonpolar tail (hydrophobic) groups.
  • cationic lipids employed in formulating liposomes designed for the delivery of RNA are amphiphilic in nature and consist of a positively charged (cationic) amine head group linked to a hydrocarbon chain or cholesterol derivative via glycerol.
  • lipoplexes are multilamellar liposome-based formulations that form upon electrostatic interaction of cationic liposomes with RNAs.
  • formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact RNA- lipoplexes.
  • an LPX particle comprises an amphiphilic lipid, in particular cationic or cationically ionizable amphiphilic lipid, and RNA (especially mRNA) as described herein.
  • electrostatic interactions between positively charged liposomes made from one or more amphiphilic lipids, in particular cationic or cationically ionizable amphiphilic lipids
  • negatively charged RNA especially mRNA results in complexation and spontaneous formation of RNA lipoplex particles.
  • Positively charged liposomes may be generally synthesized using a cationic or cationically ionizable amphiphilic lipid, such as DOTMA and/or DODMA, and optionally additional lipids, such as DOPE or DSPC.
  • a cationic or cationically ionizable amphiphilic lipid such as DOTMA and/or DODMA
  • additional lipids such as DOPE or DSPC.
  • an RNA (especially mRNA) lipoplex particle is a nanoparticle.
  • a lipid nanoparticle is obtainable from direct mixing of RNA in an aqueous phase with lipids in a phase comprising an organic solvent, such as ethanol.
  • lipids or lipid mixtures can be used for particle formation, which do not form lamellar (bilayer) phases in water.
  • LNPs comprise or consist of a cation ic/cation ical ly ionizable lipid and helper lipids such as phospholipids, cholesterol, and/or polymer-conjugated lipids (e.g., polyethylene glycol (PEG) lipids).
  • PEG polyethylene glycol
  • the RNA in the RNA LNPs described herein the RNA (in particular, mRNA) is bound by cationically ionizable lipid that occupies the central core of the LNP.
  • polymer-conjugated lipid forms the surface of the LNP, along with phospholipids.
  • the surface comprises a bilayer.
  • cholesterol and cationically ionizable lipid in charged and uncharged forms can be distributed throughout the LNP.
  • RNA e.g., mRNA
  • RNA e.g., mRNA
  • the RNA may be adhered to the outer surface of the particle (surface RNA (especially surface mRNA)) and/or may be contained in the particle (encapsulated RNA (especially encapsulated mRNA)).
  • the particles (e.g., LNPs and LPXs) described herein have a size (such as a diameter) in the range of about 10 to about 2000 nm, such as at least about 15 nm (e.g., at least about 20 nm, at least about 25 nm, at least about 30 nm, at least about 35 nm, at least about 40 nm, at least about 45 nm, at least about 50 nm, at least about 55 nm, at least about 60 nm, at least about 65 nm, at least about 70 nm, at least about 75 nm, at least about 80 nm, at least about 85 nm, at least about 90 nm, at least about 95 nm, or at least about 100 nm) and/or at most about 1900 nm (e.g., at most about 1800 nm, at most about 1700 nm, at most about 1600 nm, at most about 1500 nm, at most about 1400
  • the particles (e.g., LNPs and LPXs) described herein have a size (such as a diameter) in the range of from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm.
  • the particles (e.g., LNPs and LPXs) described herein have an average diameter that in some embodiments ranges from about 50 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 50 nm to about 700 nm, from about 50 nm to about 600 nm, from about 50 nm to about 500 nm, from about 50 nm to about 450 nm, from about 50 nm to about 400 nm, from about 50 nm to about 350 nm, from about 50 nm to about 300 nm, from about 50 nm to about 250 nm, from about 50 nm to about 200 nm, from about 100 nm to about 1000 nm, from about 100 nm to about 800 nm, from about 100 nm to about 700 nm, from about 100 nm to about 600 nm, from about 100 nm to about 500 nm, from about 100 nm to about 450
  • the particles e.g., LNPs and LPXs
  • the particles have an average diameter that in some embodiments ranges from about 40 nm to about 200 nm, such as from about 50 nm to about 180 nm, from about 60 nm to about 160 nm, from about 80 nm to about 150 nm or from about 80 nm to about 120 nm.
  • the particles described herein are nanoparticles.
  • nanoparticle relates to a nano-sized particle comprising nucleic acid (especially mRNA) as described herein and at least one cationic or cationically ionizable lipid, wherein all three external dimensions of the particle are in the nanoscale, i.e., at least about 1 nm and below about 1000 nm.
  • the size of a particle is its diameter.
  • RNA particles (especially mRNA particles) described herein may exhibit a polydispersity index (PDI) less than about 0.5, less than about 0.4, less than about 0.3, less than about 0.2, less than about 0.1, or less than about 0.05.
  • PDI polydispersity index
  • the RNA particles can exhibit a polydispersity index in a range of about 0.01 to about 0.4 or about 0.1 to about 0.3.
  • the N/P ratio gives the ratio of the nitrogen groups in the lipid to the number of phosphate groups in the RNA. It is correlated to the charge ratio, as the nitrogen atoms (depending on the pH) are usually positively charged and the phosphate groups are negatively charged.
  • the N/P ratio where a charge equilibrium exists, depends on the pH. Lipid formulations are frequently formed at N/P ratios larger than four up to twelve, because positively charged nanoparticles are considered favorable for transfection. In that case, RNA is considered to be completely bound to nanoparticles.
  • RNA particles (especially mRNA particles) described herein can be prepared using a wide range of methods that may involve obtaining a colloid from at least one cationic or cationically ionizable lipid and mixing the colloid with RNA to obtain RNA particles.
  • the term "colloid” as used herein relates to a type of homogeneous mixture in which dispersed particles do not settle out.
  • the insoluble particles in the mixture are microscopic, with particle sizes between 1 and 1000 nanometers.
  • the mixture may be termed a colloid or a colloidal suspension. Sometimes the term “colloid” only refers to the particles in the mixture and not the entire suspension.
  • colloids comprising at least one cationic or cationically ionizable lipid methods are applicable herein that are conventionally used for preparing liposomal vesicles and are appropriately adapted.
  • the most commonly used methods for preparing liposomal vesicles share the following fundamental stages: (I) lipids dissolution in organic solvents, (II) drying of the resultant solution, and (ill) hydration of dried lipid (using various aqueous media).
  • film hydration method lipids are firstly dissolved in a suitable organic solvent, and dried down to yield a thin film at the bottom of the flask. The obtained lipid film is hydrated using an appropriate aqueous medium to produce a liposomal dispersion.
  • an additional downsizing step may be included.
  • Reverse phase evaporation is an alternative method to the film hydration for preparing liposomal vesicles that involves formation of a water-in-oil emulsion between an aqueous phase and an organic phase containing lipids. A brief sonication of this mixture is required for system homogenization. The removal of the organic phase under reduced pressure yields a milky gel that turns subsequently into a liposomal suspension.
  • RNA (especially mRNA) lipoplex particles described herein are obtainable by adding RNA (especially mRNA) to a colloidal liposome dispersion.
  • colloidal liposome dispersion is, in some embodiments, formed as follows: an ethanol solution comprising lipids, such as cationic or cationically ionizable lipids (like DOTMA and/or DODMA) and additional lipids, is injected into an aqueous solution under stirring.
  • lipids such as cationic or cationically ionizable lipids (like DOTMA and/or DODMA) and additional lipids
  • lipids such as cationic or cationically ionizable lipids (like DOTMA and/or DODMA) and additional lipids
  • lipids such as cationic or cationically ionizable lipids (like DOTMA and/or DODMA) and additional lipids
  • RNA lipoplex particles described herein are obtainable without a step of extrusion.
  • extruding refers to the creation of particles having a fixed, cross-sectional profile. In particular, it refers to the downsizing of a particle, whereby the particle is forced through filters with defined pores.
  • LNPs comprise four components: cationically ionizable lipids, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer-conjugated lipid.
  • LNPs may be prepared by mixing lipids dissolved in ethanol rapidly with RNA in an aqueous buffer. While RNA particles described herein may comprise polymer-conjugated lipids such as PEG lipids, provided herein are also RNA particles which do not comprise PEG lipids, or do not comprise any polymer-conjugated lipids.
  • the LNPs comprising RNA and at least one cationic or cationically ionizable lipid described herein are prepared by (a) preparing an RNA solution containing water and a buffering system; (b) preparing an ethanolic solution comprising the cationic or cationically ionizable lipid and, if present, one or more additional lipids; and (c) mixing the RNA solution prepared under (a) with the ethanolic solution prepared under (b), thereby preparing the formulation comprising LNPs. After step (c) one or more steps selected from diluting and filtrating, such as tangential flow filtrating, can follow.
  • the LNPs comprising RNA and at least one cationic or cationically ionizable lipid described herein are prepared by (a') preparing liposomes or a colloidal preparation of the cationic or cationically ionizable lipid and, if present, one or more additional lipids in an aqueous phase; and (b') preparing an RNA solution containing water and a buffering system; and (c') mixing the liposomes or colloidal preparation prepared under (a') with the RNA solution prepared under (b'). After step (c') one or more steps selected from diluting and filtrating, such as tangential flow filtrating, can follow.
  • compositions comprising RNA (especially mRNA) and at least one cationic or cationically ionizable lipid which associates with the RNA to form RNA particles and formulations comprising such particles.
  • RNA particles may comprise RNA which is complexed in different forms by non-covalent interactions to the particle.
  • the particles described herein are not viral particles, in particular infectious viral particles, i.e., they are not able to virally infect cells.
  • Suitable cationic or cationically ionizable lipids are those that form RNA particles and are included by the term “particle forming components” or “particle forming agents”.
  • the term “particle forming components” or “particle forming agents” relates to any components which associate with RNA to form RNA particles. Such components include any component which can be part of RNA particles.
  • RNA particles (especially mRNA particles) comprise more than one type of RNA molecules, where the molecular parameters of the RNA molecules may be similar or different from each other, like with respect to molar mass or fundamental structural elements such as molecular architecture, capping, coding regions or other features.
  • each RNA species is separately formulated as an individual particulate formulation.
  • each individual particulate formulation will comprise one RNA species.
  • the individual particulate formulations may be present as separate entities, e.g., in separate containers.
  • Such formulations are obtainable by providing each RNA species separately (typically each in the form of an RNA- containing solution) together with a particle-forming agent, thereby allowing the formation of particles.
  • Respective particles will contain exclusively the specific RNA species that is being provided when the particles are formed (individual particulate formulations).
  • a composition such as a pharmaceutical composition comprises more than one individual particle formulation.
  • Respective pharmaceutical compositions are referred to as mixed particulate formulations.
  • Mixed particulate formulations according to the present disclosure are obtainable by forming, separately, individual particulate formulations, followed by a step of mixing of the individual particulate formulations.
  • a formulation comprising a mixed population of RNA-containing particles is obtainable.
  • Individual particulate populations may be together in one container, comprising a mixed population of individual particulate formulations.
  • RNA species of the pharmaceutical composition are formulated together as a combined particulate formulation.
  • Such formulations are obtainable by providing a combined formulation (typically combined solution) of all RNA species together with a particle-forming agent, thereby allowing the formation of particles.
  • a combined particulate formulation will typically comprise particles which comprise more than one RNA species.
  • different RNA species are typically present together in a single particle.
  • polymers are commonly used materials for nanopartide-based delivery.
  • cationic polymers are used to electrostatically condense the negatively charged RNA into nanoparticles.
  • These positively charged groups often consist of amines that change their state of protonation in the pH range between 5.5 and 7.5, thought to lead to an ion imbalance that results in endosomal rupture.
  • Polymers such as poly-L-lysine, polyamidoamine, protamine and polyethyleneimine, as well as naturally occurring polymers such as chitosan have all been applied to nucleic acid delivery and are suitable as cationic polymers herein.
  • some investigators have synthesized polymers specifically for nucleic acid delivery. Poly(P-amino esters), in particular, have gained widespread use in nucleic acid delivery owing to their ease of synthesis and biodegradability.
  • Such synthetic polymers are also suitable as cationic polymers herein.
  • a "polymer”, as used herein, is given its ordinary meaning, i.e., a molecular structure comprising one or more repeat units (monomers), connected by covalent bonds.
  • the repeat units can all be identical, or in some cases, there can be more than one type of repeat unit present within the polymer.
  • the polymer is biologically derived, i.e., a biopolymer such as a protein.
  • additional moieties can also be present in the polymer, for example targeting moieties.
  • the polymer is said to be a "copolymer". It is to be understood that the polymer being employed herein can be a copolymer.
  • the repeat units forming the copolymer can be arranged in any fashion. For example, the repeat units can be arranged in a random order, in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions each comprising a first repeat unit (e.g., a first block), and one or more regions each comprising a second repeat unit (e.g., a second block), etc.
  • Block copolymers can have two (a diblock copolymer), three (a triblock copolymer), or more numbers of distinct blocks.
  • the polymer is biocompatible.
  • Biocompatible polymers are polymers that typically do not result in significant cell death at moderate concentrations.
  • the biocompatible polymer is biodegradable, i.e., the polymer is able to degrade, chemically and/or biologically, within a physiological environment, such as within the body.
  • polymer may be protamine or polyalkyleneimine.
  • protamine refers to any of various strongly basic proteins of relatively low molecular weight that are rich in arginine and are found associated especially with DNA in place of somatic histones in the sperm cells of various animals (as fish).
  • protamine refers to proteins found in fish sperm that are strongly basic, are soluble in water, are not coagulated by heat, and yield chiefly arginine upon hydrolysis. In purified form, they are used in a long-acting formulation of insulin and to neutralize the anticoagulant effects of heparin.
  • the term "protamine” as used herein is meant to comprise any protamine amino acid sequence obtained or derived from natural or biological sources including fragments thereof and multimeric forms of said amino acid sequence or fragment thereof as well as (synthesized) polypeptides which are artificial and specifically designed for specific purposes and cannot be isolated from native or biological sources.
  • the polyalkyleneimine comprises polyethylenimine and/or polypropylenimine, preferably polyethyleneimine.
  • a preferred polyalkyleneimine is polyethyleneimine (PEI).
  • the average molecular weight of PEI is preferably 0.75- 10 2 to 10 7 Da, preferably 1000 to 10 5 Da, more preferably 10000 to 40000 Da, more preferably 15000 to 30000 Da, even more preferably 20000 to 25000 Da.
  • linear polyalkyleneimine such as linear polyethyleneimine (PEI).
  • Cationic polymers contemplated for use herein include any cationic polymers which are able to electrostatically bind nucleic acid.
  • cationic polymers contemplated for use herein include any cationic polymers with which nucleic acid can be associated, e.g., by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.
  • Particles described herein may also comprise polymers other than cationic polymers, i.e., non-cationic polymers and/or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as noncationic polymers.
  • lipid and "lipid-like material” are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually insoluble or poorly soluble in water, but soluble in many organic solvents. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment.
  • Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s).
  • the hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.
  • hydrophobic refers to any a molecule, moiety or group which is substantially immiscible or insoluble in aqueous solution.
  • hydrophobic group includes hydrocarbons having at least 6 carbon atoms.
  • the monovalent radical of a hydrocarbon is referred to as hydrocarbyl herein.
  • the hydrophobic group can have functional groups (e.g., ether, ester, halide, etc.) and atoms other than carbon and hydrogen as long as the group satisfies the condition of being substantially immiscible or insoluble in aqueous solution.
  • hydrocarbon includes non-cydic, e.g., linear (straight) or branched, hydrocarbyl groups, such as alkyl, alkenyl, or alkynyl as defined herein. It should be appreciated that one or more of the hydrogen atoms in alkyl, alkenyl, or alkynyl may be substituted with other atoms, e.g., halogen, oxygen or sulfur. Unless stated otherwise, hydrocarbon groups can also include a cyclic (alkyl, alkenyl or alkynyl) group or an aryl group, provided that the overall polarity of the hydrocarbon remains relatively nonpolar.
  • alkyl refers to a saturated linear or branched monovalent hydrocarbon moiety which may have one to thirty, typically one to twenty, often six to eighteen carbon atoms.
  • exemplary nonpolar alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, hexyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, and the like.
  • alkenyl refers to a linear or branched monovalent hydrocarbon moiety having at least one carboncarbon double bond in which the total carbon atoms may be six to thirty, typically six to twenty often six to eighteen.
  • the maximal number of carbon-carbon double bonds in the alkenyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenyl group by 2 and, if the number of carbon atoms in the alkenyl group is uneven, rounding the result of the division down to the next integer.
  • the maximum number of carbon-carbon double bonds is 4.
  • the alkenyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carboncarbon double bonds.
  • alkynyl refers to a linear or branched monovalent hydrocarbon moiety having at least one carboncarbon triple bond in which the total carbon atoms may be six to thirty, typically six to twenty, often six to eighteen.
  • Alkynyl groups can optionally have one or more carbon-carbon double bonds.
  • the maximal number of carbon-carbon triple bonds in the alkynyl group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkynyl group by 2 and, if the number of carbon atoms in the alkynyl group is uneven, rounding the result of the division down to the next integer.
  • the maximum number of carbon-carbon triple bonds is 4.
  • the alkynyl group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, more preferably 1 or 2 carbon-carbon triple bonds.
  • alkylene refers to a saturated linear or branched divalent hydrocarbon moiety which may have one to thirty, typically two to twenty, often four to twelve carbon atoms.
  • exemplary nonpolar alkylene groups include, but are not limited to, methylene, ethylene, trimethylene, hexamethylene, decamethylene, dodeca methylene, tetradeca methylene, hexadeca methylene, octadecmethylene, and the like.
  • alkenylene refers to a linear or branched divalent hydrocarbon moiety having at least one carboncarbon double bond in which the total carbon atoms may be two to thirty, typically two to twenty, often four to twelve.
  • the maximal number of carbon-carbon double bonds in the alkenylene group can be equal to the integer which is calculated by dividing the number of carbon atoms in the alkenylene group by 2 and, if the number of carbon atoms in the alkenylene group is uneven, rounding the result of the division down to the next integer.
  • the maximum number of carbon-carbon double bonds is 4.
  • the alkenylene group has 1 to 6 (such as 1 to 4), i.e., 1, 2, 3, 4, 5, or 6, carbon-carbon double bonds.
  • cycloalkyl represents cyclic non-aromatic versions of “alkyl” and "alkenyl” with preferably 3 to 14 carbon atoms, such as 3 to 12 or 3 to 10 carbon atoms, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 carbon atoms (such as 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms), more preferably 3 to 7 carbon atoms.
  • Exemplary cycloalkyl groups include cyclopropyl, cyclopropenyl, cyclobutyl, cydobutenyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, cycloheptenyl, cyclooctyl, cydooctenyl, cydononyl, cyclononenyl, cylcodecyl, cylcodecenyl, and adamantyl.
  • the cycloalkyl group may consist of one ring (monocyclic), two rings (bicyclic), or more than two rings (polycyclic).
  • aryl refers to a monoradical of an aromatic cyclic hydrocarbon.
  • the aryl group contains 3 to 14 (e.g., 5, 6, 7, 8, 9, or 10, such as 5, 6, or 10) carbon atoms which can be arranged in one ring (e.g., phenyl) or two or more condensed rings (e.g., naphthyl).
  • exemplary aryl groups include cyclopropenylium, cyclopentadienyl, phenyl, indenyl, naphthyl, azulenyl, fluorenyl, anthryl, and phenanthryl.
  • aryl refers to a monocyclic ring containing 6 carbon atoms or an aromatic bicyclic ring system containing 10 carbon atoms. Preferred examples are phenyl and naphthyl. Aryl does not encompass fullerenes.
  • aromatic as used in the context of hydrocarbons means that the whole molecule has to be aromatic.
  • a monocyclic aryl is hydrogenated (either partially or completely) the resulting hydrogenated cyclic structure is classified as cycloalkyl for the purposes of the present disclosure.
  • a bi- or polycyclic aryl such as naphthyl
  • the resulting hydrogenated bi- or polycyclic structure is classified as cycloalkyl for the purposes of the present disclosure (even if one ring, such as in 1,2-dihydronaphthyl, is still aromatic).
  • amphiphilic refers to a molecule having both a polar portion and a non-polar portion. Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt.
  • the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.
  • lipid-like material lipid-like compound or “lipid-like molecule” relates to substances, in particular amphiphilic substances, that structurally and/or functionally relate to lipids but may not be considered as lipids in a strict sense.
  • the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties.
  • the term includes molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids.
  • lipid-like compounds capable of spontaneous integration into cell membranes include functional lipid constructs such as synthetic function-spacer-lipid constructs (FSL), synthetic function-spacer-sterol constructs (FSS) as well as artificial amphipathic molecules.
  • FSL synthetic function-spacer-lipid constructs
  • FSS synthetic function-spacer-sterol constructs
  • Lipids comprising two long alkyl chains and a polar head group are generally cylindrical. The area occupied by the two alkyl chains is similar to the area occupied by the polar head group.
  • Such lipids have low solubility as monomers and tend to aggregate into planar bilayers that are water insoluble.
  • Traditional surfactant monomers comprising only one linear alkyl chain and a hydrophilic head group are generally cone shaped. The hydrophilic head group tends to occupy more molecular space than the linear alkyl chain.
  • surfactants tend to aggregate into spherical or elliptoid micelles that are water soluble. While lipids also have the same general structure as surfactants - a polar hydrophilic head group and a nonpolar hydrophobic tail - lipids differ from surfactants in the shape of the monomers, in the type of aggregates formed in solution, and in the concentration range required for aggregation. As used herein, the term "lipid” is to be construed to cover both lipids and lipid-like materials unless otherwise indicated herein or clearly contradicted by context.
  • lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterol lipids and prenol lipids (derived from condensation of isoprene subunits).
  • lipid is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides.
  • Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as steroids, i.e., sterol-containing metabolites such as cholesterol or a derivative thereof.
  • cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2 1 - hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.
  • Fatty acids, or fatty acid residues are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water.
  • the carbon chain typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more cis double bonds in the chain.
  • Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.
  • Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides.
  • triacylglycerol is sometimes used synonymously with "triglyceride”.
  • the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids.
  • Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.
  • the glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head” group by a phosphate ester linkage.
  • Examples of glycerophospholipids usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).
  • Sphingolipids are a complex family of compounds that share a common structural feature, a sphingoid base backbone.
  • the major sphingoid base in mammals is commonly referred to as sphingosine.
  • Ceramides N-acyl- sphingoid bases
  • the fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms.
  • the major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannosecontaining headgroups.
  • the glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.
  • Sterol lipids such as cholesterol and its derivatives, or tocopherol and its derivatives, are an important component of membrane lipids, along with the glycerophospholipids and sphingomyelins.
  • Saccharolipids describe compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers.
  • a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids.
  • the most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria.
  • Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E.
  • Kdo2-Lipid A a hexaacylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.
  • Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.
  • lipids and lipid-like materials may be cationic, anionic or neutral.
  • Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.
  • the RNA compositions and formulations and RNA particles described herein comprise at least one cationic or cationically ionizable lipid as particle forming agent.
  • Cationic or cationically ionizable lipids contemplated for use herein include any cationic or cationically ionizable lipids (including lipid-like materials) which are able to electrostatically bind nucleic acid.
  • cationic or cationically ionizable lipids contemplated for use herein can be associated with nucleic acid, e.g., by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.
  • a "cationic lipid” refers to a lipid or lipid-like material having a net positive charge. Cationic lipids bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.
  • a cationic lipid has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH.
  • This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.
  • a "cationically ionizable lipid” refers to a lipid or lipid-like material which has a net positive charge or is neutral, i.e., which is not permanently cationic.
  • the cationically ionizable lipid is either positively charged or neutral.
  • cationically ionizable lipids are covered by the term "cationic lipid" unless contradicted by the circumstances.
  • the cationic or cationically ionizable lipid comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated, e.g., under physiological conditions.
  • cationic or cationically ionizable lipids include, but are not limited to N,N-dimethyl-2,3- dioleyloxypropylamine (DODMA), l,2-dioleoyl-3-trimethylammonium propane (DOTAP); 1,2-di-O-octadecenyl- 3-trimethylammonium propane (DOTMA), 3-(N— (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC- Chol), dimethyldioctadecylammonium (DDAB); l,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2- diacyloxy-3-dimethylammonium propanes; l,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), l,2-distearyloxy-N
  • the cationic or cationically ionizable lipid is DOTMA. In some embodiments, the cationic or cationically ionizable lipid is DODMA.
  • DOTMA is a cationic lipid with a quaternary amine headgroup.
  • the structure of DOTMA may be represented as follows:
  • DODMA is an ionizable cationic lipid with a tertiary amine headgroup.
  • the structure of DODMA may be represented as follows:
  • the cationic or cationically ionizable lipid may comprise from about 10 mol % to about 95 mol %, from about 20 mol % to about 95 mol %, from about 20 mol % to about 90 mol %, from about 30 mol % to about 90 mol %, from about 40 mol % to about 90 mol %, or from about 40 mol % to about 80 mol % of the total lipid present in the particle.
  • RNA compositions and formulations and RNA particles described herein may also comprise lipids (including lipid-like materials) other than cationic or cationically ionizable lipids (also collectively referred to herein as cationic lipids), i.e., non-cationic lipids (including non-cationic or non-cationically ionizable lipids or lipid-like materials).
  • cationic lipids also collectively referred to herein as cationic lipids
  • non-cationic lipids including non-cationic or non-cationically ionizable lipids or lipid-like materials.
  • anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids.
  • Optimizing the formulation of RNA particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to a cationic or cationically ionizable lipid may enhance particle stability and efficacy of RNA delivery.
  • the one or more additional lipids may or may not affect the overall charge of the RNA particles.
  • the one or more additional lipids are a non-cationic lipid or lipid-like material.
  • the non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids.
  • an "anionic lipid” refers to any lipid that is negatively charged at a selected pH.
  • a "neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH.
  • the RNA compositions and formulations and RNA particles described herein comprise a cationic or cationically ionizable lipid and one or more additional lipids.
  • the amount of the cationic or cationically ionizable lipid compared to the amount of the one or more additional lipids may affect important RNA particle characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the RNA. Accordingly, in some embodiments, the molar ratio of the cationic or cationically ionizable lipid to the one or more additional lipids is from about 10:0 to about 1:9, about 4:1 to about 1:2, about 4:1 to about 1:1, about 3: 1 to about 1:1, or about 3:1 to about 2: 1.
  • the one or more additional lipids comprised in the RNA compositions and formulations and RNA particles described herein comprise one or more of the following: neutral lipids, steroids, and combinations thereof.
  • the one or more additional lipids comprise a neutral lipid which is a phospholipid.
  • the phospholipid is selected from the group consisting of phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines and sphingomyelins. Specific phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin.
  • Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoylphosphatidylcholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleo
  • the neutral lipid is selected from the group consisting of DSPC, DOPC, DMPC, DPPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC. In some embodiments, the neutral lipid is DOPE.
  • the additional lipid comprises one of the following: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.
  • cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.
  • the RNA compositions and formulations and RNA particles described herein comprise (1) a cationic or cationically ionizable lipid, and a phospholipid such as DSPC or DOPE or (2) a cationic or cationically ionizable lipid and a phospholipid such as DSPC or DOPE and cholesterol.
  • the RNA particles (especially the particles comprising mRNA) described herein comprise (1) DOTMA and DOPE, (2) DOTMA, DOPE and cholesterol, (3) DODMA and DOPE or (4) DODMA, DOPE and cholesterol.
  • DSPC is a neutral phospholipid.
  • the structure of DSPC may be represented as follows:
  • DOPE is a neutral phospholipid.
  • the structure of DOPE may be represented as follows:
  • the structure of cholesterol may be represented as follows:
  • RNA compositions and formulations and RNA particles described herein do not include a polymer conjugated lipid such as a pegylated lipid.
  • a polymer conjugated lipid such as a pegylated lipid.
  • pegylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.
  • the additional lipid (e.g., one or more phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 2 mol % to about 80 mol %, from about 5 mol % to about 80 mol %, from about 5 mol % to about 60 mol %, from about 5 mol % to about 50 mol %, from about 7.5 mol % to about 50 mol %, or from about 10 mol % to about 40 mol % of the total lipid present in the particle.
  • the additional lipid (e.g., one or more phospholipids and/or cholesterol) comprises about 10 mol %, about 15 mol %, or about 20 mol % of the total lipid present in the particle.
  • the additional lipid comprises a mixture of: (i) a phospholipid such as DOPE; and (ii) cholesterol or a derivative thereof.
  • the molar ratio of the phospholipid such as DOPE to the cholesterol or a derivative thereof is from about 9:0 to about 1:10, about 2: 1 to about 1:4, about 1: 1 to about 1:4, or about 1:1 to about 1:3.
  • RNA compositions and formulations and RNA particles described herein may comprise at least one polymer-conjugated lipid.
  • a polymer-conjugated lipid is typically a molecule comprising a lipid portion and a polymer portion conjugated thereto.
  • a polymer-conjugated lipid is a PEG- conjugated lipid, also referred to herein as pegylated lipid or PEG-lipid.
  • pegylated lipid refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.
  • a polymer-conjugated lipid is a polysa rcosine-conjugated lipid, also referred to herein as sarcosinylated lipid or pSar-lipid.
  • sarcosinylated lipid refers to a molecule comprising both a lipid portion and a polysarcosine portion.
  • a polymer-conjugated lipid is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer.
  • a polymer- conjugated lipid can reduce its association with serum proteins and/or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.
  • RNA compositions/formulations and RNA particles described herein comprise a PEG- conjugated lipid.
  • the PEG-conjugated lipid is a lipid having the structure of the following general formula: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein: each of R 12 and R 13 is each independently a straight or branched, alkyl or alkenyl chain containing from 10 to 30 carbon atoms, wherein the alkyl/alkenyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.
  • each of R 12 and R 13 is independently a straight alkyl chain containing from 10 to 18 carbon atoms, preferably from 12 to 16 carbon atoms.
  • R 12 and R 13 are identical. In some embodiments, each of R 12 and R 13 is a straight alkyl chain containing 12 carbon atoms. In some embodiments, each of R 12 and R 13 is a straight alkyl chain containing 14 carbon atoms. In some embodiments, each of R 12 and R 13 is a straight alkyl chain containing 16 carbon atoms.
  • R 12 and R 13 are different. In some embodiments, one of R 12 and R 13 is a straight alkyl chain containing 12 carbon atoms and the other of R 12 and R 13 is a straight alkyl chain containing 14 carbon atoms.
  • w has a mean value ranging from 40 to 50, such as a mean value of 45.
  • w is within a range such that the PEG portion of the pegylated lipid has an average molecular weight of from about 400 to about 6000 g/mol, such as from about 1000 to about 5000 g/mol, from about 1500 to about 4000 g/mol, or from about 2000 to about 3000 g/mol.
  • each of R 12 and R 13 is a straight alkyl chain containing 14 carbon atoms and w has a mean value of 45.
  • PEG-conjugated lipids include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2' ,3 di(tetradecanoyloxy)propyl-l-O-(co-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)prop
  • PEG-DSG
  • the PEG-conjugated lipid is or comprises 2-[(polyethylene glycol)- 2000]-N,N-ditetradecylacetamide.
  • the pegylated lipid has the following structure:
  • the PEG-conjugated lipid is DMG-PEG 2000, e.g., having the following structure: wherein n has a mean value ranging from 30 to 60, such as about 50.
  • the PEG- conjugated lipid is PEG2000-C-DMA which preferably refers to 3-N-[(w-methoxy polyethylene glycol)2000)carbamoyl]-l,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA) or methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate (2000).
  • RNA compositions/formulations described herein may comprise one or more PEG- conjugated lipids or pegylated lipids as described in WO 2017/075531 and WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • the pegylated lipid comprises from about 1 mol % to about 10 mol %, preferably from about 1 mol % to about 5 mol %, more preferably from about 1 mol % to about 2.5 mol % of the total lipid present in the RNA compositions/formulations and RNA particles described herein.
  • the RNA described herein may be present in RNA lipoplex particles.
  • Lipoplexes are electrostatic complexes which are generally formed by mixing preformed cationic lipid liposomes with anionic RNA. Formed lipoplexes possess distinct internal arrangements of molecules that arise due to the transformation from liposomal structure into compact RNA-lipoplexes.
  • the RNA lipoplex particles include both a cationic lipid and an additional lipid.
  • the cationic lipid is DOTMA and the additional lipid is DOPE.
  • the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3: 1 to about 1: 1.
  • the molar ratio may be about 3:1, about 2.75: 1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25: 1, or about 1:1.
  • the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.
  • RNA lipoplex particles described herein have an average diameter that in some embodiments ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm.
  • the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 675 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm.
  • the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In some embodiments, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.
  • RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration.
  • Spleen targeting RNA lipoplex particles are described in WO 2013/143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen-presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in the spleen occurs.
  • RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen.
  • RNA lipoplex particles of the disclosure may be used for targeting RNA, e.g., RNA encoding an antigen or at least one epitope, to the lymphatic system, in particular secondary lymphoid organs, more specifically spleen.
  • the target cell is a spleen cell.
  • the target cell is an antigen presenting cell such as a professional antigen presenting cell in the spleen.
  • the target cell is a dendritic cell in the spleen.
  • the electric charge of the RNA lipoplex particles of the present disclosure is the sum of the electric charges present in the at least one cationic lipid and the electric charges present in the RNA.
  • the charge ratio is the ratio of the positive charges present in the at least one cationic lipid to the negative charges present in the RNA.
  • concentration of RNA and the at least one cationic lipid amount can be determined using routine methods by one skilled in the art.
  • the charge ratio of positive charges to negative charges in the RNA lipoplex particles is from about 1.6:2 to about 1:2, or about 1.6:2 to about 1.1:2. In specific embodiments, the charge ratio of positive charges to negative charges in the RNA lipoplex particles at physiological pH is about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.
  • RNA described herein is present in the form of lipid nanoparticles (LNPs).
  • LNP lipid nanoparticles
  • the LNP may comprise any lipid capable of forming a particle to which the one or more RNA molecules are attached, or in which the one or more RNA molecules are encapsulated.
  • LNPs typically comprise four components: cationically ionizable lipid, neutral lipids such as phospholipids, a steroid such as cholesterol, and a polymer-conjugated lipid such as PEG-lipid.
  • LNPs may be prepared by mixing lipids dissolved in ethanol with RNA in an aqueous buffer.
  • the RNA in the RNA LNPs described herein the RNA is bound by cationically ionizable lipid that occupies the central core of the LNP.
  • Polymer-conjugated lipid forms the surface of the LNP, along with phospholipids.
  • the surface comprises a bilayer.
  • cholesterol and cationically ionizable lipid in charged and uncharged forms can be distributed throughout the LNP.
  • the LNP comprises one or more cationically ionizable lipids, and one or more stabilizing lipids.
  • Stabilizing lipids include neutral lipids and polymer-conjugated lipids.
  • the LNP comprises a cationically ionizable lipid, a neutral lipid, a steroid, a polymer- conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.
  • the LNP comprises from 40 to 60 mol percent, 40 to 55 mol percent, from 45 to 55 mol percent, or from 45 to 50 mol percent of the cationically ionizable lipid.
  • the neutral lipid is present in a concentration ranging from 5 to 15 mol percent, from 7 to 13 mol percent, or from 9 to 11 mol percent.
  • the steroid is present in a concentration ranging from 30 to 50 mol percent, from 30 to 45 mol percent, from 35 to 45 mol percent or from 35 to 43 mol percent.
  • the LNP comprises from 1 to 10 mol percent, from 1 to 5 mol percent, or from 1 to 2.5 mol percent of the polymer-conjugated lipid.
  • the LNP comprises from 45 to 55 mol percent of a cationically ionizable lipid; from 5 to 15 mol percent of a neutral lipid; from 30 to 45 mol percent of a steroid; from 1 to 5 mol percent of a polymer-conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.
  • the mol percent is determined based on total mol of lipid present in the lipid nanoparticle. In some embodiments, the mol percent is determined based on total mol of cationically ionizable lipid, neutral lipid, steroid and polymer-conjugated lipid present in the lipid nanoparticle.
  • the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.
  • the steroid is cholesterol
  • the polymer conjugated lipid is a pegylated lipid, e.g., a pegylated lipid as described above.
  • the cationically ionizable lipid component of the LNPs has the structure of Formula (HI):
  • G 1 and G 2 are each independently unsubstituted C 1 -C 12 alkylene or C 1 -C 12 alkenylene;
  • G 3 is C 1 -C 24 alkylene, C 1 -C 24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
  • R a is H or C 1 -C 12 alkyl
  • R 1 and R 2 are each independently C6-C 24 alkyl or C6-C 24 alkenyl
  • R 4 is C 1 -C 12 alkyl
  • R 5 is H or Ci-Ce alkyl; and x is 0, 1 or 2.
  • the lipid has one of the following structures (IIIA) or (IIIB):
  • A is a 3 to 8-membered cycloalkyl or cycloalkylene ring
  • R 6 is, at each occurrence, independently H, OH or C 1 -C 24 alkyl; n is an integer ranging from 1 to 15.
  • the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).
  • the lipid has one of the following structures (IIIC) or (HID):
  • the lipid has one of the following structures (HIE) or (IIIF):
  • the lipid has one of the following structures (IIIG),
  • n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4.
  • n is 3, 4, 5 or 6.
  • n is 3.
  • n is 4. In some embodiments, n is 5. In some embodiments, n is 6.
  • y and z are each independently an integer ranging from 2 to 10.
  • y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.
  • R 6 is H.
  • R 6 is Ci -C 24 alkyl.
  • R 6 is OH.
  • G 3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G 3 is linear C 1 -C 24 alkylene or linear C 1 -C 24 alkenylene.
  • R 1 or R 2 is C6-C 24 alkenyl.
  • R 1 and R 2 each, independently have the following structure: wherein:
  • R 7a and R 7b are, at each occurrence, independently H or C 1 -C 12 alkyl; and a is an integer from 2 to 12, wherein R 7a , R 7b and a are each selected such that R 1 and R 2 each independently comprise from 6 to 20 carbon atoms.
  • a is an integer ranging from 5 to 9 or from 8 to 12.
  • At least one occurrence of R 7a is H.
  • R 7a is H at each occurrence.
  • at least one occurrence of R 7b is Ci-Cs alkyl.
  • Ci-Cs alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R 1 or R 2 has one of the following structures:
  • R 4 is methyl or ethyl.
  • the cationic lipid of Formula (III) has one of the structures set forth in the table below.
  • Table 1 Representative Compounds of Formula (III)
  • RNA described herein is formulated in an LNP composition
  • an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, a neutral lipid, a steroid, and a polymer conjugated lipid.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, a neutral lipid, a steroid, and a polymer conjugated lipid.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, a neutral lipid, a steroid, and a polymer conjugated lipid.
  • RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, a neutral lipid, a steroid, and a polymer conjugated lipid.
  • RNA described herein is formulated in an LNP composition comprising ALC-0366, a neutral lipid, a steroid, and a polymer conjugated lipid.
  • RNA described herein is formulated in an LNP composition comprising ALC-0315, a neutral lipid, a steroid, and a polymer conjugated lipid.
  • the neutral lipid is DSPC.
  • the steroid is cholesterol.
  • the polymer conjugated lipid is a pegylated lipid, e.g., DMG-PEG 2000, PEG2000-C-DMA, or ALC- 0159.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, a neutral lipid, a steroid, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, a neutral lipid, a steroid, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, a neutral lipid, a steroid, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, a neutral lipid, a steroid, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising ALC-0366, a neutral lipid, a steroid, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising ALC-0315, a neutral lipid, a steroid, and a pegylated lipid.
  • the neutral lipid is DSPC.
  • the steroid is cholesterol.
  • the pegylated lipid is DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and a pegylated lipid.
  • a cationically ionizable lipid e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and a pegylated lipid.
  • RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and a pegylated lipid.
  • the pegylated lipid is DMG-PEG 2000, PEG2000-C-DMA, or ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and DMG-PEG 2000. In some embodiments, RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and DMG-PEG 2000.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and DMG-PEG 2000.
  • RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and DMG-PEG 2000.
  • RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and DMG-PEG 2000.
  • RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and DMG-PEG 2000.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and PEG2000-C-DMA.
  • a cationically ionizable lipid e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and PEG2000-C-DMA.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and PEG2000-C-DMA.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and PEG2000-C-DMA.
  • RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and PEG2000-C-DMA.
  • RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and PEG2000-C-DMA.
  • RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and PEG2000-C-DMA.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid, e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and ALC-0159.
  • a cationically ionizable lipid e.g., a cationically ionizable lipid as shown above, DSPC, cholesterol, and ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid of Formula III, DSPC, cholesterol, and ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising a cationically ionizable lipid shown in the above tables, DSPC, cholesterol, and ALC-0159. In some embodiments, RNA described herein is formulated in an LNP composition comprising 3D-P-DMA, DSPC, cholesterol, and ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising ALC-0366, DSPC, cholesterol, and ALC-0159.
  • RNA described herein is formulated in an LNP composition comprising ALC-0315, DSPC, cholesterol, and ALC-0159.
  • 3D-P-DMA (6Z,16Z)-12-((Z)-dec-4-en-l-yl)docosa-6,16-dien-ll-yl 5-(dimethylamino)pentanoate
  • ALC-0366 ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butylocta noate)
  • ALC-0315 ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl)bis(2-hexyldecanoate) / 6-[N-6-(2- hexyldecanoyloxy)hexyl-N-(4-hydroxybutyl)amino]hexyl 2-hexyldecanoate
  • PEG2000-C-DMA 3-N-[(w-Methoxy polyethylene glycol)2000) carbamoyl]-l,2-dimyristyloxy-propylamine (MPEG-(2 kDa)-C-DMA or Methoxy-polyethylene glycol-2,3-bis(tetradecyloxy)propylcarbamate (2000)) wherein n has a mean value ranging from 30 to 60, such as about 50.
  • ALC-0159 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide / 2-[2-(w-methoxy (polyethyleneglycol2000) ethoxy]-N,N-ditetradecylacetamide
  • the N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N/P value is about 6.
  • compositions comprising one or more RNAs described herein, e.g., in the form of RNA particles, may comprise salts, buffers, or other components as further described below.
  • a salt for use in the compositions described herein comprises sodium chloride.
  • sodium chloride functions as an ionic osmolality agent for preconditioning RNA prior to mixing with lipids.
  • the compositions described herein may comprise alternative organic or inorganic salts.
  • Alternative salts include, without limitation, potassium chloride, dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, disodium phosphate, monosodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and sodium salts of ethylenediaminetetraacetic acid (EDTA).
  • potassium chloride dipotassium phosphate, monopotassium phosphate, potassium acetate, potassium bicarbonate, potassium sulfate, disodium phosphate, monosodium phosphate, sodium acetate, sodium bicarbonate, sodium sulfate, lithium chloride, magnesium chloride, magnesium phosphate, calcium chloride, and sodium salts of ethylenediaminetetraacetic acid (EDTA).
  • EDTA ethylenediaminetetraacetic acid
  • compositions for storing RNA particles such as for freezing RNA particles comprise low sodium chloride concentrations, or comprises a low ionic strength.
  • the sodium chloride is at a concentration from 0 mM to about 50 mM, from 0 mM to about 40 mM, or from about 10 mM to about 50 mM.
  • the RNA particle compositions described herein have a pH suitable for the stability of the RNA particles and, in particular, for the stability of the RNA.
  • the use of a buffer system maintains the pH of the particle compositions described herein during manufacturing, storage and use of the compositions.
  • the buffer system may comprise a solvent (in particular, water, such as deionized water, in particular water for injection) and a buffering substance.
  • the buffering substance may be selected from 2-[4-(2- hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES), 2-amino-2-(hydroxymethyl)propane-l,3-diol (Tris), acetate, and histidine.
  • HEPES 2-amino-2-(hydroxymethyl)propane-l,3-diol
  • the buffering substance is HEPES.
  • the buffering substance is Tris.
  • compositions in particular, RNA compositions/formulations described herein may also comprise a cryoprotectant and/or a surfactant as stabilizer to avoid substantial loss of the product quality and, in particular, substantial loss of RNA activity during storage, freezing, and/or lyophilization, for example to reduce or prevent aggregation, particle collapse, RNA degradation and/or other types of damage.
  • a cryoprotectant and/or a surfactant as stabilizer to avoid substantial loss of the product quality and, in particular, substantial loss of RNA activity during storage, freezing, and/or lyophilization, for example to reduce or prevent aggregation, particle collapse, RNA degradation and/or other types of damage.
  • the cryoprotectant is a carbohydrate.
  • carbohydrate refers to and encompasses monosaccharides, disaccharides, trisaccharides, oligosaccharides and polysaccharides.
  • the cryoprotectant is a monosaccharide.
  • monosaccharide refers to a single carbohydrate unit (e.g., a simple sugar) that cannot be hydrolyzed to simpler carbohydrate units.
  • monosaccharide cryoprotectants include glucose, fructose, galactose, xylose, ribose and the like.
  • the cryoprotectant is a disaccharide.
  • disaccharide refers to a compound or a chemical moiety formed by 2 monosaccharide units that are bonded together through a glycosidic linkage, for example through 1-4 linkages or 1-6 linkages. A disaccharide may be hydrolyzed into two monosaccharides.
  • Exemplary disaccharide cryoprotectants include sucrose, trehalose, lactose, maltose and the like. In some embodiments, the cryoprotectant is sucrose.
  • trisaccharide means three sugars linked together to form one molecule. Examples of a trisaccharides include raffinose and melezitose.
  • the cryoprotectant is an oligosaccharide.
  • oligosaccharide refers to a compound or a chemical moiety formed by 3 to about 15, such as 3 to about 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure.
  • Exemplary oligosaccharide cryoprotectants include cyclodextrins, raffinose, melezitose, maltotriose, stachyose, acarbose, and the like. An oligosaccharide can be oxidized or reduced.
  • the cryoprotectant is a cyclic oligosaccharide.
  • cyclic oligosaccharide refers to a compound or a chemical moiety formed by 3 to about 15, such as 6, 7, 8, 9, or 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a cyclic structure.
  • Exemplary cyclic oligosaccharide cryoprotectants include cyclic oligosaccharides that are discrete compounds, such as a cyclodextrin, 0 cyclodextrin, or y cyclodextrin.
  • exemplary cyclic oligosaccharide cryoprotectants include compounds which include a cyclodextrin moiety in a larger molecular structure, such as a polymer that contains a cyclic oligosaccharide moiety.
  • a cyclic oligosaccharide can be oxidized or reduced, for example, oxidized to dicarbonyl forms.
  • the term "cyclodextrin moiety", as used herein refers to cyclodextrin (e.g., an a, P, or y cyclodextrin) radical that is incorporated into, or a part of, a larger molecular structure, such as a polymer.
  • a cyclodextrin moiety can be bonded to one or more other moieties directly, or through an optional linker.
  • a cyclodextrin moiety can be oxidized or reduced, for example, oxidized to dicarbonyl forms.
  • Carbohydrate cryoprotectants e.g., cyclic oligosaccharide cryoprotectants
  • the cryoprotectant is a derivatized cyclic oligosaccharide, e.g., a derivatized cyclodextrin, e.g., 2-hydroxypropyl-p-cydodextrin, e.g., partially etherified cyclodextrins (e.g., partially etherified 3 cyclodextrins).
  • An exemplary cryoprotectant is a polysaccharide.
  • polysaccharide refers to a compound or a chemical moiety formed by at least 16 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure, and includes polymers that comprise polysaccharides as part of their backbone structure. In backbones, the polysaccharide can be linear or cyclic.
  • Exemplary polysaccharide cryoprotectants include glycogen, amylase, cellulose, dextran, maltodextrin and the like.
  • RNA particle compositions may include sucrose.
  • sucrose functions to promote cryoprotection of the compositions, thereby preventing RNA (especially mRNA) particle aggregation and maintaining chemical and physical stability of the composition.
  • RNA particle compositions may include alternative cryoprotectants to sucrose.
  • Alternative stabilizers include, without limitation, trehalose and glucose.
  • an alternative stabilizer to sucrose is trehalose or a mixture of sucrose and trehalose.
  • a preferred cryoprotectant is selected from the group consisting of sucrose, trehalose, glucose, and a combination thereof, such as a combination of sucrose and trehalose.
  • the cryoprotectant is sucrose.
  • chelating agents refer to chemical compounds that are capable of forming at least two coordinate covalent bonds with a metal ion, thereby generating a stable, water-soluble complex. Without wishing to be bound by theory, chelating agents reduce the concentration of free divalent ions, which may otherwise induce accelerated RNA degradation in the present disclosure.
  • chelating agents include, without limitation, ethylenediaminetetraacetic acid (EDTA), a salt of EDTA, desferrioxamine B, deferoxamine, dithiocarb sodium, penicillamine, pentetate calcium, a sodium salt of pentetic acid, succimer, trientine, nitrilotriacetic acid, trans-diaminocyclohexanetetraacetic acid (DCTA), diethylenetriaminepentaacetic acid (DTPA), and bis(aminoethyl)glycolether-N,N,N',N'-tetraacetic acid.
  • the chelating agent is EDTA or a salt of EDTA.
  • the chelating agent is EDTA disodium dihydrate. In some embodiments, the EDTA is at a concentration from about 0.05 mM to about 5 mM, from about 0.1 mM to about 2.5 mM or from about 0.25 mM to about 1 mM.
  • RNA particle compositions described herein do not comprise a chelating agent.
  • the present invention provides a nucleic acid molecule for eliciting an antigen-specific CD8 + T-cell response in a subject comprising a coding sequence encoding a polypeptide comprising an N-terminal degron and one or more antigenic peptides.
  • N-terminal is meant in this context in relation to the one or more antigenic peptides, i.e. in the polypeptide of the invention, the N-terminal degron lies more N-terminal as compared to the antigenic peptides (as shown, e.g., in Fig. 2b).
  • the N-terminal degron forms the N- terminus of the polypeptide (as shown, e.g., in Fig. 2b).
  • the term “following” is used with respect to elements of the polypeptide, this is meant to refer to the N- to C-terminus direction, i.e. if it is stated herein that the N-terminal degron (e.g., ubiquitin) is "immediately followed” by the antigenic peptide, this means that the antigenic peptide is attached to the C-terminus of the N-degron.
  • the polypeptide of the invention can also be referred to as a fusion protein comprising an N-terminal degron and one or more antigenic peptides.
  • the N-terminal degron significantly destabilizes the polypeptide and improves its degradation via the proteasome, thereby increasing the amount of degradation products that are available for antigen presentation as shown by the examples described below and corresponding data depicted in Fig. 2 to 6.
  • the N-terminal degron can specifically elicit an antigen-specific CD8 + T-cell response, thereby providing an efficient targeting by cytotoxic CD8 + T-cells.
  • the research underlying this invention has surprisingly found that a particularly potent antigen-specific CD8 + T-cell specific response against selected target proteins can be elicited by the nucleic acid molecule of the invention.
  • N-terminal degron Polypeptides comprising the N-terminal degron are advantageously turned over more quickly and are more immunogenic than corresponding reference constructs lacking the N-terminal degron.
  • N-terminal degrons have the potential to be deployed in all T-cell-targeting vaccines to enhance immunogenicity and polarize away from CD4+ T-cell responses towards CD8 + T-cell responses.
  • Antigen-specific CD8 + T-cell response means that in response to immunization with the nucleic acid molecule of the invention, antigen-specific CD8 + T-cells can be detected.
  • the antigen-specific CD8 + T-cell response is an antigen-specific CD8 + T-cell specific response, which means that the response is CD8 + T-cell specific, i.e. more antigen-specific CD8 + compared to antigen-specific CD4+ T-cells are activated.
  • antigen-specific CD8 + T-cell specific response means that substantially lower (compared to reference nucleic acid molecule not containing the N-degron), substantially no or no antigenspecific CD4 + immune response is elicited by the nucleic acid molecule of the invention.
  • the antigenic peptide comprised in the polypeptide encoded by the nucleic acid molecule of the invention makes the elicited immune response antigen-specific.
  • Eliciting an antigen-specific CD8 + specific T-cell response is particular advantageous for therapeutic or prophylactic vaccinations against certain diseases. For example, it may be in particular desirable in cases where an antigen-specific CD4+ T cell response could worsen the disease. Exemplary diseases that can be worsened by an antigen-specific CD4+ T cell response are human immunodeficiency virus infections or Epstein-Barr virus infections. Eliciting an antigen-specific CD8 + T-cell response may also be particularly desirable in multi-component vaccines, where individual components are directed to producing different immune responses.
  • the nucleic acid molecule of the invention encodes the polypeptide of the invention.
  • the features and embodiments defined herein for the polypeptide encoded by the nucleic acid molecule are meant to be equally disclosed also for the corresponding nucleic acid molecule of the invention and vice versa.
  • the nucleic acid molecule is a recombinant nucleic acid molecule, i.e., wherein the sequence is not naturally joined together, but is artificially combined.
  • the nucleic acid molecule is an unmodified mRNA or a modified mRNA, preferably a modified mRNA.
  • the modified mRNA has an increased stability and/or translation compared to an unmodified mRNA.
  • the nucleic acid molecule comprises a 5' cap, a 5'UTR, a coding region, a 3'UTR, a poly(A) tail, or any combination thereof.
  • the 5' cap, 5'UTR, coding region, and 3'UTR, and poly(A) tail are not specifically limited and can be any one of the 5' cap, the 5'UTR, the coding region, the 3'UTR, and the poly(A) tail disclosed herein and defined above.
  • the 5'UTR if present, comprises a Kozak sequence, preferably an optimized Kozak sequence to increase translational efficiency.
  • Kozak sequences are known to increase the efficiency of translation of some RNA transcripts but are not necessarily required for all RNAs to enable efficient translation.
  • a Kozak sequence typically extends from approximately position -6 to position +6, where +1 is assigned to the adenine of the START codon. The Kozak sequence is known to affect transcription initiation.
  • the nucleic acid molecule comprises a 5'-cap, a free 5'-triphosphate group, a free 5'- disphosphate group, a free 5'-diphosphate group, a free 5'- monophosphate group, or a free 5'-OH group, or comprising chemically modified analogues of said 5'-cap, said 5'-tri phosphate group, said free 5'-disphosphate group or said free 5'-monophosphate group.
  • the 5'cap is selected from G[5']ppp[5']G, m7G[5']ppp[5']G, m 3 2 ' 2 ' 7 G[5']ppp[5']G, m 2 7 - 3 '-°G[5']ppp °GppSpG (p-S- ARCA), and m 2 7 ' 2 ' °GppSpG (p-S-ARCA)
  • the nucleic acid molecule comprises a 3'UTR comprising an FI element, preferably derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I).
  • An "FI element” is a sequence in the 3' -untranslated region known to improve mRNA stability and translation efficiency. The FI element can be positioned in the 3'UTR. Exemplary FI elements suitable for use in the nucleic acid molecule of the invention are descripted in the patent application WO 2017/059902 Al. The FI element thus can be placed between the coding sequence and the poly(A) sequence to assure higher maximum protein levels and prolonged persistence of the mRNA.
  • the nucleic acid molecule comprises an interrupted poly(A) sequence (i.e. interrupted poly(A) tail) disclosed herein and defined above.
  • the poly(A) sequence essentially consists of dA nucleotides but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • the poly-A tail contained in a nucleic acid (in particular, mRNA) molecule described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • Antigenic peptide as used herein is a peptide comprising an antigen.
  • An "antigen” is a substance, preferably a peptide that is a target of an immune response and/or that will elicit an immune response. The same applies to the antigenic peptide.
  • an “antigenic peptide” is any peptide that reacts specifically with, i.e. binds to antibodies or T-lymphocytes (T-cells), in particular T-cell receptors.
  • T-cells T-lymphocytes
  • the term “antigenic peptide” comprises any molecule which comprises at least one epitope such as a B cell or T cell epitope.
  • the term “antigenic peptide” encompasses full-length protein and fragments of the protein, i.e.
  • the antigenic peptide can be an antigenic fragment (or "antigenic peptide fragment").
  • An antigenic peptide fragment as used herein, can be an epitope, but can also comprise more than one epitope.
  • an antigen in the context of the present invention is a molecule which, optionally after processing, induces an immune reaction, which is preferably specific for the antigen or cells expressing the antigen.
  • Antigens may include or may be derived from allergens, viruses, bacteria, fungi, parasites and other infectious agents and pathogens or an antigen may also be a tumor antigen.
  • the polypeptide encoded by the nucleic acid molecule of the invention comprises several, i.e. more than one antigenic peptides.
  • the term "antigenic peptide" as used herein is different from and does not encompass the N-terminal degron.
  • “Fragment” as used herein, with reference to an amino acid sequence (polypeptide or protein), relates to a part of a naturally occurring protein amino acid sequence, e.g., a partial sequence that has been shortened at the N-terminus and/or C-terminus compared to the naturally occurring full length sequence.
  • An amino acid sequence whose sequence represents two or more discontinuous sequences derived from the same parental amino acid sequence fused together is considered to be two or more fragments of that parental sequence.
  • full length with respect to a given polypeptide means the form of the polypeptide naturally translated from the coding sequence, beginning with the ATG start codon, which encodes the first methionine in the amino acid sequence, and ending at the TGA, TAG, or TTA stop codon, or whichever stop codon employed by the organism.
  • the antigenic peptides can be artificial peptides, i.e. not being derived from naturally occurring proteins.
  • the antigenic peptides are derived from naturally occurring proteins, i.e. are full-length naturally occurring proteins and/or are fragments of such proteins.
  • the antigenic peptide is an immunogenic peptide.
  • the antigenic polypeptide is a pathogen- related, tumor-related, or disease-related antigenic peptide.
  • the antigenic peptide is a full-length protein or fragment thereof.
  • an "immunogenic peptide” is a peptide capable of eliciting an immune response in a subject.
  • the antigenic peptide is an immunogenic peptide.
  • the polypeptide comprises a plurality of antigenic peptides, such as concatenated antigenic peptides.
  • the antigenic peptides are each a full-length protein.
  • the antigenic peptides are fragments derived from full-length proteins, wherein the fragments can be antigenic peptide fragments.
  • at least one antigenic peptide is a full- length protein, while at least one antigenic peptide is a fragment derived from a full-length protein.
  • the antigenic peptides do not comprise a full length protein.
  • amino acid sequence whose sequence represents two or more discontinuous sequences derived from the same parental amino acid sequence fused together is considered to be two or more antigenic peptides or antigenic peptide fragments of that parental sequence.
  • the antigenic peptides can also be artificial peptides, i.e. are not derived from naturally occurring proteins.
  • antigenic peptides are concatenated antigenic peptides that are derived from different proteins or different portions of the same protein and fused together, preferably such that a first antigenic peptide is adjacent to a second antigenic peptide or such that a first and second antigenic peptide are separated via a linker.
  • a first antigenic peptides is adjacent to a second antigenic peptide.
  • the antigenic peptides are linked via a linker sequence.
  • all antigenic peptides comprised in the polypeptide are adjacent to another antigenic peptide or separated therefrom by way of a linker.
  • a “linker” is a non-naturally occurring amino acid sequence present between, and thereby separating, two peptides of a protein or two peptides of different proteins. Non-naturally occurring means in this context not naturally-occurring in the protein sequence adjacent to the peptide sequence from which the peptides are derived. In preferred embodiments, some antigenic peptides are separated by a linker, while other antigenic peptides are not separated by a linker.
  • linker as used herein does not encompass the N-terminal degron.
  • the linker sequence is not particularly limited but can consist of a single amino acid or of two or more amino acids. In some embodiments, the linker sequence consists of 1-20 amino acids, 2-19 amino acids, 3-18 amino acids, 4-17 amino acids, 5-16 amino acids, or 6-15 amino acids.
  • the linker can comprise at most 8 amino acids, preferably at most 6 amino acids, more preferably at most 5 amino acids, most preferably 1 to 4 amino acids. In this context, it is understood that the linker sequence preferably is not naturally occurring adjacent to the antigenic peptides linked by the linker.
  • the antigen-specific CD8 + T- cell response triggered by the nucleic acid molecule disclosed herein can measured by an ELISpot assay and/or multimer staining following a MACS separation to separate CD4+ T cells from CD8 + T cells. These methods are well known in the art.
  • An "antigen-specific CD8 + T-cell response" is an antigen -specific activation of CD8 + T cells, i.e., CD8 + T cells directed to specific antigens, through major histocompatibility complex class I (MHC-I) presentation.
  • MHC-I major histocompatibility complex class I
  • the antigen-specific CD8 + T-cell response is an antigen-specific CD8 + T-cell specific response. This means that the proportion of CD8 + - vs.
  • CD4 + -immune response is higher compared to immunization with a corresponding polypeptide not comprising an N-terminal degron of the invention.
  • antigen-specific CD8 + T-cell specific response means that more antigen -specific CD8 + compared to antigen-specific CD4+ T- cells are activated.
  • substantially no or no antigen-specific CD4 + immune response is elicited.
  • a preferred method to evaluate an antigen-specific response is to immunize mice with the nucleic acid molecule of the invention, or the polypeptide of the invention. Afterwards, the immune cells of the immunized mice are analyzed quantitively by separating CD4 + from CD8 + cells via MACS separation, and performing an ELISpot on each set. The successfully induced T cells respond with cytokine (IFNy) production.
  • an MHC-I tetramer staining known in the art can be performed to demonstrate CD8 + T cell responding to specific antigenic peptides.
  • Ubiquitin is a regulatory protein found in most tissues of eukaryotic organisms, which gave it its name as it is found ubiquitously. Ubiquitin is highly conserved across species and typically has about 76 amino acids and is around 8.6 kDa. Ubiquitin serves in the natural environment of an organism as a protein tag marking and therefore serving to rapidly remove unwanted or damaged proteins by acting as a marker for protein degradation. Not only protein can become ubiquitinated, but also ubiquitin attached to other proteins can become further ubiquitinated to form a poly-ubiquitin chain.
  • lysines within the ubiquitin or the N- terminus of ubiquitin are ubiquitinated and result in a poly-ubiquitin chain which targets the poly-ubiquitinated protein for proteasomal degradation.
  • Ubiquitin is typically a 76-amino acid protein, generated from a precursor that is processed by deubiquitinating enzymes (DUBs) to expose the glycine— g lyci ne sequence at the ubiquitin C-terminus, its site of attachment to target molecules.
  • DRBs deubiquitinating enzymes
  • ATP-dependent ubiquitin activation can be catalyzed by the El (ubiquitin-activating) enzyme, which adenylates the ubiquitin C-terminus, allowing the subsequent formation of a high-energy thioester bond between the glycine residue of ubiquitin and the cysteine residue on the El active site.
  • Ubiquitin is then transferred from the El cysteinyl side chain to a cysteinyl group on one of several E2 (ubiquitin-conjugating) enzymes.
  • E3 ubiquitin-ligase
  • This naturally occurring mechanism i.e. the ubiquitination of peptides, has divers biological functions such as cell cycle control, transcriptional regulation, signal transduction, inflammatory response, membrane trafficking, receptor endocytosis and downregulation, apoptosis, and development.
  • nucleic acid molecule encoding for a polypeptide comprising a ubiquitin and an antigenic peptide significantly increases the antigen-specific CD8 + T- cell response. Further and without wishing to be bound by theory, the ubiquitin leads to improved degradation of the polypeptide, leading to an increased amount of antigenic peptides present for antigen processing and presentation.
  • ubiquitin as used herein has a sequence shown in SEQ ID NO.: 1. In some embodiments, ubiquitin has an amino acid sequence that is at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO.: 1. In particular, ubiquitin as used herein may comprise one or more mutations. It is understood, that the mutation will not abrogate the function of ubiquitin to mark a protein for degradation.
  • ubiquitin comprises a mutation substituting the glycine at position 76 with reference to SEQ ID NO.: 1 and/or a mutation substituting a lysine at amino acid position 6, 11, 27, 29, 33, 48 or 63 or any combination thereof with reference to SEQ ID NO.: 1 as long as one lysine remains in the ubiquitin.
  • further additional mutations that result in a ubiquitin sequence with less than 100% identity with reference to SEQ ID NO.: 1 are encompassed, wherein the presence of such a mutated ubiquitin in an N-terminal degron results in destabilization and degradation of the polypeptide.
  • the ubiquitin is a cleavable or non-cleavable ubiquitin as disclosed herein.
  • Specifying amino acid positions "with reference to SEQ ID NO.: 1" means the amino acid position that corresponds to the amino acid position in SEQ ID NO.: 1 that the number refers to. This can easily be determined by aligning a modified ubiquitin sequence with the reference ubiquitin sequence of SEQ ID NO.: 1.
  • the N-terminal degron comprises a ubiquitin as disclosed herein. It is however understood that the N-terminal degron can comprise two or more ubiquitins directly adjacent to each other, e.g., a first ubiquitin immediately followed by a second ubiquitin.
  • N-terminal degron is a sequence that destabilize a polypeptide and targets it for degradation.
  • the N-terminal degron is located towards, in particular at the N-terminus of a polypeptide comprising one or more antigenic peptides.
  • the N-terminal degron degradation may operate through the N-end rule pathway (also referred to as “N-degron” or “degron”) or the ubiquitin fusion degradation (UFD) pathway (also referred to as "UFD-degron”) as exemplarily shown in Fig. 1.
  • the N-terminal degron comprises a ubiquitin having the amino acid sequence shown in SEQ ID NO.: 1, or a ubiquitin comprising a mutation substituting the glycine at position 76 with reference to SEQ ID NO.: 1 and/or a mutation substituting the lysine at position 6, 11, 27, 29, 33, 48 or 63 or any combination thereof with reference to SEQ ID NO.: 1 as long as one lysine remains in the ubiquitin, or a ubiquitin having a sequence that is at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO 1.
  • the presence of an N-terminal degron reduces the half-life of the polypeptide to 1 hour or less, preferably to 30 minutes or less, more preferably to 10 minutes or less compared to the polypeptide without the N-terminal degron.
  • the N-terminal degron degradation operates through the N-end rule pathway ("N- degron").
  • the destabilizing effect of the N-end rule pathway depends on the N-terminal amino acid of the polypeptide as shown in Fig. 1A. If the N-terminal amino acid is a destabilizing amino acid ("d" in Fig. 1A), such as isoleucine, glutamic acid, threonine, glutamine, phenylalanine, leucine, aspartic acid, arginine, lysine, and histidine, preferably selected from arginine, lysine, and histidine, the half-life of the polypeptide can be reduced to several minutes.
  • a destabilizing amino acid such as isoleucine, glutamic acid, threonine, glutamine, phenylalanine, leucine, aspartic acid, arginine, lysine, and histidine, preferably selected from arginine, lysine, and his
  • a cleavable ubiquitin is fused to the N-terminus of a polypeptide, wherein the ubiquitin is immediately followed by a destabilizing amino acid.
  • the cleavable ubiquitin immediately followed by a destabilizing amino acid can be cleaved by a deubiquitinase (DUB) enzyme, and resulting in the release of a polypeptide with the destabilizing N-terminal amino acid at position 1.
  • DAB deubiquitinase
  • the destabilizing N-terminal amino acid may be introduced between the cleavable ubiquitin and the antigenic peptide or may be the first amino acid of the antigenic peptide.
  • the polypeptide released after ubiquitin cleavage additionally contains internal lysines, such as e.g., N-terminal proximal lysines. This means that the released polypeptide is not ubiquitin, but the polypeptide comprising the antigenic peptide.
  • the internal lysins may be introduced after the destabilizing amino acid and before the antigenic peptide starts (e.g., as a linker or as part of a linker), included in the antigenic peptide following the destabilizing amino acid (naturally or introduced by mutation), or in a linker separating antigenic peptides.
  • a "cleavable ubiquitin” is a ubiquitin located at the N-terminus of a polypeptide comprising one or more antigenic peptides, wherein the ubiquitin can be cleaved by a deubiquitinating enzyme (DUB). It is understood that in context of the cleavable ubiquitin, the ubiquitin comprises an intact DUB cleavage site, thus that preferably the glycine at position 76 with reference to SEQ ID NO.: 1, i.e. the last glycine of the ubiquitin sequence, is not mutated.
  • DUB deubiquitinating enzyme
  • the cleavable ubiquitin comprises a destabilizing amino acid at the C-terminus, wherein following cleavage of the ubiquitin the destabilizing amino acid remains at the N- terminus of the polypeptide comprising the antigenic peptide.
  • the destabilizing amino acid is immediately following the last amino acid of ubiquitin. It is further understood in context of the cleavable ubiquitin that following the ubiquitin cleavage by DUB the destabilizing amino acid remains at the N- terminus of the polypeptide comprising one or more antigenic peptides.
  • the ubiquitin has a sequence as shown in SEQ ID NO.: 1 and has 76 amino acids and the destabilizing amino acid immediately follows after amino acid position 76 of ubiquitin. This means that cleavage of the ubiquitin results in removal of the ubiquitin sequence and the first amino acid of the resulting polypeptide starts with the destabilizing amino acid at position 1.
  • an “internal lysine” is a lysine located within the sequence of the polypeptide comprising one or more antigenic peptides, wherein the "internal lysine” is located outside the ubiquitin.
  • the internal lysine can be located within an antigenic peptide, or within an optional linker (e.g., after a destabilizing amino acid, or between antigenic peptides).
  • the internal lysine is located within the antigenic peptide.
  • the internal lysine is a linker or located within a linker.
  • the internal lysin may be an N-terminal proximal lysine.
  • N-terminal proximal lysine is a lysine located close to the N-terminus of the polypeptide comprising one or more antigenic peptides.
  • the N-terminal proximal lysine can be located within an antigenic peptide or within an optional linker.
  • one or more internal lysines are included in the polypeptide encoding one or more antigenic peptides, either because they are naturally present in the antigenic peptide(s), or because the antigenic peptide(s) were modified to include one or more lysines, or because they are introduced as linker, or because they are present in linker between antigenic peptides.
  • Internal lysines are present in polypeptides with a cleavable ubiquitin and may be present in polypeptides with a non-deavable ubiquitin.
  • a “destabilizing amino acid” is an amino acid located at the C-terminus of the cleavable ubiquitin that remains after ubiquitin cleavage in the resulting polypeptide and, when exposed as N-terminus of the polypeptide after cleavage of the ubiquitin, leads to enhanced proteasomal degradation of the polypeptide comprising the destabilizing amino acid.
  • the destabilizing amino acid is located immediately following the last amino acid of the cleavable ubiquitin, and following cleavage of the ubiquitin the destabilizing amino acid remains is at amino acid position 1 at the N-terminus of the polypeptide comprising one or more antigenic peptides.
  • destabilization of the polypeptide by the cleavable ubiquitin depends on the destabilizing amino acid at the N-terminus of a polypeptide released after the cleavage of the ubiquitin by DUBs.
  • Positively charged polar amino acids such as arginine, lysine, and histidine are particularly preferred destabilizing amino acids, reducing the half-life of proteins from over one day down to several minutes. Since protein translation begins with a starting methionine residue, destabilizing amino acids cannot be generated by adding the destabilizing amino acid upstream of the start codon.
  • a ubiquitin is added to the N-terminus of the protein followed immediately by the destabilizing amino acid.
  • the N-terminal ubiquitin can be cleaved by a DUB enzyme, revealing the destabilizing N-terminal amino acid, resulting in an enhanced proteasomal degradation rate as shown in Fig. 4 compared to a polypeptide without the cleavable ubiquitin and without the destabilizing amino acid.
  • the destabilizing amino acid is located at amino acid position 77 with reference to SEQ ID NO.: 2 immediately following a cleavable ubiquitin consisting of 76 amino acids (e.g., the ubiquitin of SEQ ID NO.: 1). It is understood, in context of the disclosure, that following cleavage of the ubiquitin the destabilizing amino acid is located at amino acid position 1 of the resulting polypeptide comprising one or more antigenic peptides.
  • the destabilizing amino acid is selected from isoleucine, glutamic acid, threonine, glutamine, phenylalanine, leucine, aspartic acid, arginine, lysine, and histidine.
  • the destabilizing amino acid is selected from arginine, lysine, and histidine. In some embodiments, the destabilizing amino acid id arginine. Destabilizing amino acids may be introduced immediately following the ubiquitin and before the antigenic peptide sequence starts (e.g., as a linker or as the first amino acid of a linker) or may be the first amino acid sequence of the antigenic peptide.
  • the cleavable ubiquitin comprises a sequence shown in SEQ ID NO.: 1, or a sequence being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in said SEQ ID NO.: 1, with the proviso that the ubiquitin sequence is immediately followed by an amino acid selected from isoleucine, glutamic acid, threonine, glutamine, phenylalanine, leucine, aspartic acid, arginine, lysine, and histidine, preferably selected from arginine, lysine, and histidine.
  • the amino acid at position 77 is a destabilizing amino acid.
  • the cleavable ubiquitin has a sequence shown in SEQ ID NO.: 2, or a sequence having at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in SEQ ID NO.: 2 with the proviso that immediately following the cleavable ubiquitin a destabilizing amino acid, such as arginine, is present.
  • the N-terminal degron degradation may operate through the ubiquitin fusion degradation (UFD) pathway (“UFD-degron”) (see e.g., Fig. IB).
  • UFD-degron ubiquitin fusion degradation
  • a UFD-degron functions through a different mechanism.
  • a non-deavable ubiquitin is fused to the N- terminus of the polypeptide.
  • a "non-cleavable ubiquitin” is a ubiquitin located at the N-terminus of a polypeptide comprising an antigenic peptide, wherein the ubiquitin cannot be cleaved by a deubiquitinating enzyme (DUB).
  • DUBB deubiquitinating enzyme
  • the ubiquitin can be made non-cleavable e.g., by introducing mutations. Cleavage of the ubiquitin by e.g., DUBs can be prevented by a C-terminal glycine mutation, preferably at amino acid position 76 with reference to the amino acid sequence shown in SEQ ID NO.: 1.
  • the N-terminal degron is a ubiquitin fusion degradation (UFD) degron.
  • the ubiquitin is a non-cleavable ubiquitin.
  • the non-cleavable ubiquitin comprises a glycine mutation at the amino acid position 76 with reference to SEQ ID NO.: 1, such that the glycine is deleted or mutated to another amino acid.
  • the non-cleavable ubiquitin has with reference to SEQ ID NO.: 1 at amino acid position 76 an alanine (e.g., as shown in SEQ ID NO.: 4).
  • an additional amino acid may be added immediately after the ubiquitin, termed "degrading amino acid”.
  • a “degrading amino acid” is defined as an amino acid that when added to the C-terminus of ubiquitin (i.e.
  • a degrading amino acid is added immediately after the ubiquitin to further enhance degradation of the polypeptide.
  • the degrading amino acid may be introduced between ubiquitin and the antigenic polypeptide or may be the first amino acid of the antigenic polypeptide.
  • the degrading amino acid is arginine or valine, preferably arginine as shown in SEQ ID NO.: 5.
  • the non-cleavable ubiquitin has a sequence as shown in SEQ ID NO.: 5 or a ubiquitin comprising a mutation substituting the lysine at position 6, 11, 27, 29, 33, or 63 or any combination thereof with reference to SEQ ID NO.: 1 or a sequence having at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at 99% identity to SEQ ID NO.: 5 with the proviso that the non-cleavable ubiquitin sequence has a glycine to alanine mutation at position 76 of SEQ ID NO.: 5 C'G76A") or at a corresponding position and that an arginine is immediately following the alanine.
  • the non-cleavable ubiquitin has an alanine at position 76 and an arginine at position 77 with reference to SEQ ID NO.: 5 (or at corresponding positions).
  • a non-cleavable ubiquitin is also referred to herein as "UbiAR”.
  • cleavage of the ubiquitin by DUBs can be inhibited when the ubiquitin is immediately followed by a proline.
  • the non-cleavable ubiquitin comprises a proline immediately following the ubiquitin, i.e. at the C-terminus of the non-cleavable ubiquitin (e.g., as shown in Fig. IB).
  • the ubiquitin sequence is immediately followed by a proline which results in the ubiquitin being non-cleavable.
  • the proline may be introduced between the ubiquitin and the antigenic polypeptide or may be the first amino acid of the antigenic polypeptide.
  • the ubiquitin has the sequences as shown in SEQ ID NO.: 1 and the proline immediately following the ubiquitin resulting e.g., in an amino acid sequence as shown in SEQ ID NO.: 3.
  • the non-cleavable ubiquitin has the sequence shown in SEQ ID NO.: 3.
  • the non-cleavable ubiquitin comprises a sequence comprising a mutation substituting the lysine at position 6, 11, 27, 29, 33, or 63 or any combination thereof with reference to SEQ ID NO.: 3.
  • Such a non-cleavable ubiquitin is also referred to herein as "Ubi-P".
  • the non-deavable ubiquitin can comprise therefore at least one, at least 2, at least 3, at least 4, at least 5, at least 6 or 7 lysines.
  • one or more of the seven lysines can be mutated, e.g., being substituted by another amino acid, such as arginine.
  • at least the lysine at amino acid position 48 is not mutated (an exemplary embodiment being SEQ ID NO.: 7).
  • the non-deavable ubiquitin sequence can comprise a glycine mutation at amino acid position 76 with reference to SEQ ID NO.: 1 immediately followed by a proline, meaning the amino acid at position 76 with reference to SEQ ID NO.: 1 is not glycine, but e.g., an alanine, and the immediately following amino acid is proline, such as shown e.g., in SEQ ID NO.: 23.
  • the non-cleavable ubiquitin comprises one C-terminal valine as shown in SEQ ID NO.: 24. In some embodiments, the non-cleavable ubiquitin comprises a G76V mutation with reference to SEQ ID NO.: 24. In some embodiments, the C-terminal valine is immediately followed by a degrading amino acid such as valine or arginine, meaning the glycine at amino acid position 76 is replaced by a valine (G76V) and the degrading amino acid following the position 76 with reference to SEQ ID NO.: 24 is valine or arginine as e.g., shown in SEQ ID NO.: 25 or 26.
  • a degrading amino acid such as valine or arginine
  • the N-terminal degron preferably comprises two N-terminal ubiquitins, wherein one ubiquitin comprises a G76V mutation with reference to SEQ ID NO.: 24 immediately followed by a valine or arginine, immediately followed by a second ubiquitin comprising a G76V mutation with reference to SEQ ID NO.: 24 immediately followed by a valine or arginine.
  • the N-terminal degron preferably comprises three N-terminal ubiquitins, wherein one ubiquitin comprises a G76V mutation with reference to SEQ ID NO.: 24 immediately followed by a valine or arginine, immediately followed by a second ubiquitin comprising a G76V mutation with reference to SEQ ID NO.: 24 immediately followed by a valine or arginine, immediately followed by a third ubiquitin comprising a G76V mutation with reference to SEQ ID NO.: 24 immediately followed by a valine or arginine.
  • the valine or arginine can be located between the last ubiquitin and an antigenic peptide or can be the first amino acid of the antigenic peptide.
  • the ubiquitin comprises a lysine substitution. In some embodiments, the ubiquitin comprises a plurality of lysine substitutions. In some embodiments, the ubiquitin sequence comprises one or more lysine substitutions selected from the positions 6, 11, 27, 29, 33, and 63 with reference to SEQ ID NO.: 1. The lysine can be replaced by any other amino acid. Preferably, the one or more lysine substitutions are selected from the group consisting of K6R, KIIR, K27R, K29R, K33R, and K63R, or any combination thereof with reference to SEQ ID NO.: 1.
  • the ubiquitin sequence is selected from the sequences shown in SEQ ID NOs.: 7-13. It is however understood that the ubiquitin can comprise any combination of lysine substitutions at positions 6, 11, 27, 29, 33, and 63 with reference to SEQ ID NO.: 1. In some embodiments, the ubiquitin comprises 2, 3, 4, 5, or 6 lysine substitutions in any of the positions 6, 11, 27, 29, 33, and 63.
  • the ubiquitin comprises lysine substitutions at the positions 6, 11, Z1 , 29, 33, and 63.
  • the lysine is replaced by arginine.
  • the ubiquitin comprises the sequence shown in SEQ ID NO.: 7 or a sequence being at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identical to the sequence shown in said SEQ ID NO, with the proviso that the ubiquitin is able to be ubiquitinated and mark the protein for degradation.
  • the non-cleavable ubiquitin comprises a sequence as shown in SEQ ID NO.: 8 or a sequence having at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in said SEQ ID NO, with the proviso that the ubiquitin is able to be ubiquitinated and mark the protein for degradation.
  • the non-cleavable ubiquitin comprises a sequence as shown in SEQ ID NO.: 9 or a sequence having at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in said SEQ ID NO, with the proviso that the ubiquitin is able to be ubiquitinated and mark the protein for degradation.
  • the non-cleavable ubiquitin comprises a sequence as shown in SEQ ID NO.: 10 or a sequence having at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in said SEQ ID NO.
  • the non-cleavable ubiquitin comprises a sequence as shown in SEQ ID NO.: 11 or a sequence having at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in said SEQ ID NO, with the proviso that the ubiquitin is able to be ubiquitinated and mark the protein for degradation.
  • the non-cleavable ubiquitin comprises a sequence as shown in SEQ ID NO.: 12 or a sequence having at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in said SEQ ID NO, with the proviso that the ubiquitin is able to be ubiquitinated and mark the protein for degradation.
  • the non-cleavable ubiquitin comprises a sequence as shown in SEQ ID NO.: 13 or a sequence having at least 80%, at least 90%, at least 95, at least 96%, at least 97%, at least 98%, or at least 99% identity to the sequence shown in said SEQ ID NO, with the proviso that the ubiquitin is able to be ubiquitinated and mark the protein for degradation.
  • the non-cleavable ubiquitin or the cleavable ubiquitin is followed by one or more internal lysines, such as N-terminal proximal lysines, wherein the one or more internal lysines are preferably positioned within one or more of the antigenic peptides or within optional linkers.
  • a ubiquitin amino acid position refers to ubiquitin as shown in SEQ ID NO.: 1 unless indicated otherwise.
  • SEQ ID NO.: 1 has 76 amino acids.
  • position 76 refers to the last glycine of ubiquitin in reference to amino acid sequence of SEQ ID NO.: 1.
  • positions 6, 11, 27, 29, 33, 48 and 63 refer to the seven lysine positions present in ubiquitin in reference to amino acid sequence of SEQ ID NO.: 1.
  • position 43 refers to a leucine in reference to amino acid sequence of SEQ ID NO.: 1.
  • destabilizing amino acids immediately following ubiquitin e.g., in cleavable ubiquitin
  • degrading amino acids e.g., in non-deavable ubiquitin
  • amino acid position 77 the amino acid at position 77 is not considered to be part of ubiquitin.
  • this position 77 becomes after cleavage of ubiquitin position 1 of the released polypeptide comprising one or more antigenic peptides, wherein the released polypeptide is not ubiquitin.
  • the polypeptide comprises malaria antigens ⁇ Plasmodium antigens) for eliciting a Plasmodium-spedtf'K CD8 + immune response.
  • the polypeptide comprises one or more Plasmodium T-cell antigens, such as at least 2 and at most 10 Plasmodium T-cell antigens.
  • the encoded polypeptide comprises at least 25 amino acids and at most 1100 amino acids. In some embodiments, the encoded polypeptide comprises at least 25 amino acids and at most 500 amino acids. In some embodiments, the polypeptide has a sequence as shown in SEQ ID NO.: 27.
  • the polypeptide disclosed herein comprises next to an N-terminal degron a malarial T-cell peptide string construct that includes the following antigenic peptides in order: (I) an antigenic Plasmodium CSP (circumsporozoite protein) polypeptide fragment; (ii) an antigenic PiasmodiumlPPP (Thrombospondin-related adhesion protein) polypeptide fragment; (iii) an antigenic Plasmodium UIS3 (Upregulated in infective sporozoites gene 3) polypeptide fragment; (iv) an antigenic Plasmodium ETRAMP10.3 polypeptide fragment; and (v) an antigenic Plasmodium LSAP2 polypeptide fragment, wherein the Piasmodium ⁇ s preferably Plasmodium falciparum and more preferably Plasmodium falciparum isolate 3D7.
  • the Piasmodium ⁇ s preferably Plasmodium falciparum and more preferably Plasmodium falciparum isolate 3D7.
  • the polypeptide includes an amino acid sequence with at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to an amino acid sequence according to SEQ ID NO.: 27.
  • the polypeptide is encoded by the DNA sequence as shown in SEQ ID NO.: 28, or DNA sequence with at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO.: 28.
  • the polypeptide is encoded by the RNA sequence as shown in SEQ ID NO.: 29, or RNA sequence with at least 85% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence identity to SEQ ID NO.: 29.
  • the nucleic acid molecule is for use in eliciting an antigen-specific CD8 + T-cell response.
  • the nucleic acid molecule for use in eliciting an antigen-specific CD8 + T-cell response is a nucleic acid molecule encoding for the polypeptide comprising the cleavable ubiquitin disclosed herein.
  • the nucleic acid molecule for use in eliciting an antigen-specific CD8 + T-cell response is a nucleic acid molecule encoding for the polypeptide comprising the non-deavable ubiquitin disclosed herein.
  • the nucleic acid molecule has the advantage of polarizing towards an antigen-specific CD8 + T-cell response, allowing improved modulation of the immune response.
  • the nucleic acid molecule can enhance the antigen-specific CD8 + T-cell response.
  • the nucleic acid molecule elicits a two-fold, preferably a three-fold, more preferably a five-fold, increase in the antigen-specific CD8 + T-cell response compared to an antigen-specific CD4 + T-cell immune response.
  • the subject suffers from a disease, such as a genetic, metabolic or infectious disease.
  • the nucleic acid molecule is for use in the prevention and/or treatment of genetic, metabolic, or infectious diseases.
  • the subject suffers from cancer.
  • the disease may be an infectious disease, such as a viral disease, e.g., a herpes virus infection, a malaria infection, or shingles.
  • the subject can be a mammal, preferably a human.
  • the invention further provides a nucleic acid molecule for eliciting an antigen-specific CD8 + T-cell response in a subject comprising a coding sequence encoding a polypeptide comprising an antigenic peptide and an N- terminal degron comprising at least one non-deavable ubiquitin.
  • nucleic acid molecule defined herein and above are meant to be equally disclosed also for the corresponding nucleic acid molecule encoding the polypeptide comprising the non-cleavable ubiquitin.
  • the invention further provides a nucleic acid molecule for eliciting an antigen-specific CD8 + T-cell response in a subject comprising a coding sequence encoding a polypeptide comprising an antigenic peptide and an N- terminal degron comprising at least one cleavable ubiquitin.
  • the nucleic acid molecule is for use in eliciting an antigen-specific CD8 + T-cell immune response.
  • nucleic acid molecule defined herein and above are meant to be equally disclosed also for the corresponding nucleic acid molecule encoding the polypeptide comprising the non-cleavable ubiquitin.
  • the invention further provides a polypeptide encoded by the nucleic acid molecule disclosed herein.
  • the polypeptide encoded by the nucleic acid molecule comprises a ubiquitin, preferably a cleavable or non-cleavable ubiquitin.
  • the polypeptide is for use in eliciting an antigen-specific CD8 + T- cell immune response.
  • polypeptide of the invention is the polypeptide encoded by the nucleic acid molecule disclosed herein.
  • the invention further provides an isolated host cell which comprises the nucleic acid molecule disclosed herein and/or the polypeptide disclosed herein.
  • the isolated host cell comprises the nucleic acid molecule disclosed herein or the polypeptide disclosed herein.
  • the isolated host cell comprises the nucleic acid molecule disclosed herein and the polypeptide disclosed herein.
  • the isolated host cell comprises the nucleic acid molecule disclosed herein.
  • the isolated host cell comprises the polypeptide disclosed herein.
  • the invention further provides a composition comprising the isolated host cell disclosed herein.
  • the invention further provides a pharmaceutical composition comprising the nucleic acid molecule disclosed herein or the polypeptide disclosed herein in a pharmaceutically acceptable carrier.
  • the pharmaceutical composition can comprise the features and embodiments defined above for pharmaceutical compositions.
  • nucleic acid molecule and/or polypeptide described herein may be administered in pharmaceutical compositions or medicaments and may be administered in the form of any suitable pharmaceutical composition.
  • the pharmaceutical composition is for therapeutic or prophylactic treatments, e.g., for use in treating or preventing a disease involving an antigen.
  • composition relates to a composition comprising a therapeutically effective agent, preferably together with pharmaceutically acceptable carriers, diluents and/or excipients. Said pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease by administration of said pharmaceutical composition to a subject.
  • a “pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” is a solvent, dispersion medium, coating, antibacterial agent and antifungal agent, isotonic agent, and absorption delaying agent, and the like, that is compatible with pharmaceutical administration.
  • the use of such media and agents for pharmaceutically active substances is well known in the art.
  • the pharmaceutically acceptable carrier or excipient is not naturally occurring.
  • compositions of the present disclosure may comprise one or more adjuvants or may be administered with one or more adjuvants.
  • adjuvant relates to a compound which prolongs, enhances or accelerates an immune response.
  • adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvants), mineral compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immune-stimulating complexes.
  • adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines.
  • the chemokines may be IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-10, IL-12, INFa, INF-y, GM-CSF, LT-a.
  • Further known adjuvants are aluminum hydroxide, Freund's adjuvant or oil such as Montanide® ISA51.
  • Suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys, as well as lipophilic components, such as saponins, trehalose-6,6-dibehenate (TDB), monophosphoryl lipid-A (MPL), monomycoloyl glycerol (MMG), or glucopyranosyl lipid adjuvant (GLA).
  • lipopeptides such as Pam3Cys
  • lipophilic components such as saponins, trehalose-6,6-dibehenate (TDB), monophosphoryl lipid-A (MPL), monomycoloyl glycerol (MMG), or glucopyranosyl lipid adjuvant (GLA).
  • compositions of the present disclosure may be in a storable form (e.g., in a frozen or lyophilized/freeze-dried form) or in a "ready-to-use form" (i.e., in a form which can be immediately administered to a subject, e.g., without any processing such as diluting).
  • a storable form e.g., in a frozen or lyophilized/freeze-dried form
  • ready-to-use form i.e., in a form which can be immediately administered to a subject, e.g., without any processing such as diluting.
  • a frozen pharmaceutical composition has to be thawed, or a freeze- dried pharmaceutical composition has to be reconstituted, e.g., by using a suitable solvent (e.g., deionized water, such as water for injection) or liquid (e.g., an aqueous solution).
  • a suitable solvent e.g., deionized water, such as water for injection
  • liquid e.g., an aqueous solution.
  • the pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and in "a pharmaceutically acceptable preparation".
  • An "effective amount” is an amount of a given substance that is sufficient in quantity to produce a desired effect, including an improvement or remediation of the disease, disorder, or symptoms of the disease or condition.
  • an effective amount of the vaccine composition for eliciting an antigen-specific CD8 + T- cell response in a subject is an amount capable to achieve a detectable increase in antigen -specific CD8 + T cells upon administration to the subject.
  • pharmaceutically acceptable refers to the non-toxicity of a material which does not interact with the action of the active component of the pharmaceutical composition.
  • pharmaceutically effective amount refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses.
  • the desired reaction may relate to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in some embodiments, interrupting or reversing the progress of the disease.
  • the desired reaction in a treatment of a disease may also be delay of the onset or a prevention of the onset of said disease or said condition, or symptoms thereof.
  • An effective amount of the pharmaceutical compositions described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the pharmaceutical compositions described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.
  • compositions of the present disclosure may contain buffers, preservatives, and optionally other therapeutic agents.
  • the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents and/or excipients.
  • Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, paraben and thimerosal.
  • excipient refers to a substance which may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient.
  • excipients include without limitation, carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or colorants.
  • the term "diluent” relates a diluting and/or thinning agent.
  • the term “diluent” includes any one or more of fluid, liquid or solid suspension and/or mixing media. Examples of suitable diluents include ethanol, glycerol and water.
  • carrier refers to a component which may be natural, synthetic, organic, inorganic in which the active component is combined in order to facilitate, enhance or enable administration of the pharmaceutical composition.
  • a carrier as used herein may be one or more compatible solid or liquid fillers, diluents or encapsulating substances, which are suitable for administration to subject.
  • Suitable carriers include, without limitation, sterile water, Ringer, Ringer lactate, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and, in particular, biocompatible lactide polymers, lactide/glycolide copolymers or polyoxyethylene/polyoxy-propylene copolymers.
  • the pharmaceutical composition of the present disclosure includes isotonic saline.
  • Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985). Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, dermally, intranodally, or intramuscularly. In some embodiments, the pharmaceutical compositions described herein may be administered intramuscularly. In some embodiments, the pharmaceutical composition is formulated for local administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration” refers to the administration in any manner other than through the gastrointestinal tract, such as by intravenous injection. In some embodiments, the pharmaceutical compositions are formulated for systemic administration.
  • systemic administration is by intravenous administration.
  • pharmaceutical compositions are formulated for intramuscular administration.
  • intramuscular administration comprises administration into the upper arm, in particular into the musculus deltoideus. If more than one dose, e.g., three doses, of a pharmaceutical composition described herein is administered, the different administrations may be into the same arm.
  • the present invention also provides a vaccine composition for eliciting an antigen-specific CD8 + T-cell response comprising an effective dose of the nucleic acid molecule disclosed herein, the polypeptide disclosed herein, or the pharmaceutical composition disclosed herein.
  • the nucleic acid molecule is associated with cationic lipids or is encapsulated into a nanoparticle or liposome, preferably as disclosed above.
  • the vaccine composition is for use in eliciting an antigen-specific CD8 + T-cell response and/or for use in a therapeutic or prophylactic treatment.
  • the invention further provides the nucleic acid molecule disclosed herein, the polypeptide disclosed herein, the pharmaceutical composition disclosed herein, or the vaccine composition disclosed herein for use in a method of eliciting an antigen-specific CD8 + T-cell response in a subject in need thereof, comprising: administering to the subject an effective amount of the nucleic acid molecule, the pharmaceutical composition, or the vaccine composition, thereby stimulating an antigen-specific CD8 + T-cell response in the subject.
  • the invention further provides the nucleic acid molecule disclosed herein, the polypeptide disclosed herein, the pharmaceutical composition disclosed herein, or the vaccine composition disclosed herein for use in a method for inducing the formation of MHC-I/ peptide complexes in a cell, the method comprising administering to a subject an effective amount of the nucleic acid molecule, the pharmaceutical composition, or the vaccine composition.
  • the invention further provides the nucleic acid molecule disclosed herein, the polypeptide disclosed herein, the pharmaceutical composition disclosed herein, or the vaccine composition disclosed herein for use in a method for stimulating or activating CD8 + T-cells, wherein the method comprises administering to a subject an effective amount of the nucleic acid molecule, the pharmaceutical composition, or the vaccine composition.
  • the administration is intravenously. In some embodiments, the administration is intraarterially, subcutaneously, intradermally, dermally, intranodally, or intramuscularly. In some embodiments, the administration is intramuscularly. In some embodiments, the admiration is a local administration or systemic administration.
  • Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. In some embodiments, the administration is a systemic administration. In some embodiments, the systemic administration is by intravenous administration.
  • SEQ ID NO.: 1 is an exemplary amino acid sequence of ubiquitin suitable for use in the N-terminal degron disclosed herein.
  • the ubiquitin sequence comprises 76 amino acids.
  • SEQ ID NO.: 2 is an exemplary amino acid sequence of a cleavable ubiquitin suitable for use in the N-terminal degron disclosed herein.
  • the ubiquitin sequence comprises a destabilizing amino acid at the C-terminus.
  • SEQ ID NO.: 3 is an exemplary amino acid sequence of a non-deavable ubiquitin suitable for use in the N- terminal degron disclosed herein.
  • the ubiquitin sequence comprises a proline at the C-terminus.
  • SEQ ID NO.: 4 is an exemplary amino acid sequence of a non-deavable ubiquitin suitable for use in the N- terminal degron disclosed herein, comprising a G76A mutation.
  • SEQ ID NO.: 5 is an exemplary amino acid sequence of a non-deavable ubiquitin suitable for use in the N- terminal degron disclosed herein comprising a G76A mutation immediately followed by an arginine.
  • SEQ ID NO.: 6 is an exemplary amino acid sequence of GFP.
  • SEQ ID NO.: 7 is an exemplary amino acid sequence of a non-deavable ubiquitin suitable for use in the N- terminal degron disclosed herein based on SEQ ID NO.: 5 and comprising K6R, KIIR, K27R, K29R, K33R, and K63R substitutions.
  • SEQ ID NO.: 8 is an exemplary amino acid sequence of a non-deavable ubiquitin suitable for use in the N- terminal degron disclosed herein based on SEQ ID NO.: 5 and comprising a K6R substitution.
  • SEQ ID NO.: 9 is an exemplary amino acid sequence of a non-cleavable ubiquitin suitable for use in the N- terminal degron disclosed herein based on SEQ ID NO.: 5 and comprising a KIIR substitution.
  • SEQ ID NO.: 10 is an exemplary amino acid sequence of a non-cleavable ubiquitin suitable for use in the N- terminal degron disclosed herein based on SEQ ID NO.: 5 and comprising a K27R substitution.
  • SEQ ID NO.: 11 is an exemplary amino acid sequence of a non-cleavable ubiquitin suitable for use in the N- terminal degron disclosed herein based on SEQ ID NO.: 5 and comprising a K29R substitution.
  • SEQ ID NO.: 12 is an exemplary amino acid sequence of a non-cleavable ubiquitin suitable for use in the N- terminal degron disclosed herein based on SEQ ID NO.: 5 and comprising a K33R substitution.
  • SEQ ID NO.: 13 is an exemplary amino acid sequence of a non-cleavable ubiquitin suitable for use in the N- terminal degron disclosed herein based on SEQ ID NO.: 5 and comprising a K63R substitution.
  • SEQ ID NO.: 14 is an exemplary amino acid sequence of a non-cleavable ubiquitin suitable for use in the N- terminal degron disclosed herein based on SEQ ID NO.: 5 and comprising a L43A substitution and K6R, KIIR, K27R, K29R, K33R, K48R and K63R substitutions.
  • SEQ ID NO.: 15 is an exemplary amino acid sequence of a cleavable ubiquitin suitable for use in the N-terminal degron disclosed herein based on SEQ ID NO.: 2 and comprising a L43A substitution and K6R, KIIR, K27R, K29R, K33R, K48R and K63R substitutions.
  • SEQ ID NO.: 16 is an exemplary amino acid sequence comprising the cleavable ubiquitin sequence shown in SEQ ID NO.: 2 fused to the GFP sequence shown in SEQ ID NO.: 6.
  • SEQ ID NO.: 17 is an exemplary amino acid sequence of a non-cleavable ubiquitin sequence shown in SEQ ID NO.: 5 fused to the GFP sequence shown in SEQ ID NO.: 6.
  • SEQ ID NO.: 18 is an exemplary amino acid sequence of a non-cleavable ubiquitin sequence shown in SEQ ID NO.: 13 fused to the GFP sequence shown in SEQ ID NO.: 6.
  • SEQ ID NO.: 19 is an exemplary amino acid sequence of a non-cleavable ubiquitin sequence shown in SEQ ID NO.: 7 fused to the GFP sequence shown in SEQ ID NO.: 6.
  • SEQ ID NO.: 20 is an exemplary amino acid sequence of a non-cleavable ubiquitin sequence shown in SEQ ID NO.: 14 fused to the GFP sequence shown in SEQ ID NO.: 6.
  • SEQ ID NO.: 21 is an exemplary amino acid sequence of a cleavable ubiquitin sequence shown in SEQ ID NO.: 15 fused to the GFP sequence shown in SEQ ID NO.: 6.
  • SEQ ID NO.: 22 is an exemplary amino acid sequence of a signal peptide HSV-lgD SP.
  • SEQ ID NO.: 23 is an exemplary amino acid sequence of a non-cleavable ubiquitin sequence comprising a glycine mutation at amino acid position 76 with reference to SEQ ID NO.: 1 immediately followed by a proline.
  • SEQ ID NO.: 24 is an exemplary amino acid sequence of a non-cleavable ubiquitin comprises a C-terminal valine.
  • SEQ ID NO.: 25 is an exemplary amino acid sequence of a non-cleavable ubiquitin comprises a C-terminal valine immediately followed by an arginine.
  • SEQ ID NO.: 26 is an exemplary amino acid sequence of a non-cleavable ubiquitin comprises a C-terminal valine immediately followed by a valine.
  • SEQ ID NO.: 27 is an exemplary amino acid sequence of a polypeptide according to the invention comprising the N-terminal degron of SEQ ID NO.: 2 followed by the following antigenic peptides: (I) an antigenic Plasmodium CSP (circumsporozoite protein) polypeptide fragment; (ii) an antigenic PiasmodiumTriPP (Thrombospondin-related adhesion protein) polypeptide fragment; (iii) an antigenic Plasmodium UIS3 (Upregulated in infective sporozoites gene 3) polypeptide fragment; (iv) an antigenic Plasmodium ETRAM Pl 0.3 polypeptide fragment; and (v) an antigenic Plasmodium LSAP2 polypeptide fragment.
  • antigenic Plasmodium CSP circumsporozoite protein
  • PiasmodiumTriPP Thrombospondin-related adhesion protein
  • an antigenic Plasmodium UIS3 Upregulated in infective sporozoites gene 3
  • SEQ ID NO.: 28 is an exemplary DNA sequence encoding for the SEQ ID NO.: 27.
  • SEQ ID NO.: 29 is an exemplary RNA sequence encoding for the SEQ ID NO.: 27.
  • HEK293 cells were transfected with 5 ⁇ g mRNA using Lipofectamine MessengerMax using manufacturer's protocol using as shown in Fig.2b:
  • SP+M1TD (2.) reference mRNA construct
  • SP+M1TD reference mRNA construct
  • CSP, TRAP, UIS3, ETRAMP10.3, and LSAP2 signal peptides derived from malaria
  • LSAP2 antigenic peptides derived from malaria
  • MIMD MHC internal transmembrane domain
  • mRNA construct comprising a cleavable ubiquitin at the N-terminus immediately followed by a destabilizing amino acid, and antigenic peptides derived from malaria (CSP, TRAP, UIS3, ETRAMP10.3, and LSAP2) separated by linkers with the first antigenic malaria peptide comprising N-terminal proximal lysines (N- term proximal lysines).
  • the cells were harvested, pelleted, and lysed in an 8M urea lysis buffer. Lysates were cleared of insoluble material by centrifugation and stored at -80C.
  • Protein concentrations of the supernatants were measured using a BCA kit according to manufacturer's protocols, and concentrations were normalized. Cysteine sulfhydryls were reduced with TCEP and alkylated with iodoacetamide. Proteins were digested using trypsin overnight at 37°C at a ratio of 1 ⁇ g trypsin per 50 ⁇ g total protein. Digested peptides were further desalted using C18 solid phase extraction. Following desalting, peptides were analyzed by targeted LC-MS/MS analysis to quantify the relative abundance of the transfected protein in the cell lysate.
  • Fig. 2a This demonstrates that the degron leads increased turnover of the polypeptide (as measured by the increase in abundance in the presence of MG132) compared to the SP+MITD lacking the degron.
  • Example 2 Immunogenicity of the polypeptide comprising the N-terminal degron and antigens
  • HLA A2.1 transgenic mice were immunized with 2.5 ⁇ g RNA as described in Example 1 intramuscularly. Seven days after immunization, spleens were harvested and serum was collected.
  • Single cell suspensions were generated from collected spleens for ELISpot analysis. Spleens were decanted onto 70 pm filters placed on 50 mL conical vials and gently dissociated using the plunger of a 3 mL syringe and rinsing with ⁇ 6 mL of RPMI media to release splenocytes into the tube. Splenocytes were centrifuged for 10 min at 4°C at 1300 revolutions per minute (rpm), the supernatant was decanted and red blood cells were lysed with ammonium-chloride-potassium (ACK) Lysing Buffer for 3 min at RT.
  • ACK ammonium-chloride-potassium
  • the ACK lysis was stopped by adding 9 mL of RPMI media and cells were centrifuged again and resuspended in 5 mL of serum-free assay media (X-VIVO + 1% Pen-Strep + 1% Glutamax) and refiltered into a fresh tube over a fresh 70 pm filter. Cells were then counted using a Nexcelom Cellaca-MX.
  • ELISpot assays with fresh splenocytes were performed according to the manufacturer's protocol (with minor modifications as described below) using R&D systems' mouse IFN-y ELISpot kit. Briefly, 96-well ELISpot plates were blocked with serum-free assay media (X-VIVO + 1% Pen-Strep + 1% Glutamax) for at least 1 h at 37°C. Next, 100 pL of the splenocyte solution (3 x 10 5 cells) were transferred to the respective well of the 96-well ELISpot plate. Another 100 pL of overlapping peptide pools or controls were added in the following concentrations:
  • SP+MITD peptide pool (57 peptides, 15mer/llmer overlap): 0.3 pM final concentration per peptide
  • Concanavalin A (ConA): 0.3 pM final concentration
  • splenocytes were stimulated with ConA.
  • medium with DMSO equivalent to the highest volume of peptide mix was added. All stimulations were performed in triplicate.
  • the degron efficiently elicits an immune response compared to the SP+MITD lacking the degron, providing an improved immunogenicity.
  • HEK293 cells 10 6 HEK293 cells were plated in each well of a 12 well plate. 24 hours after plating, 1 ⁇ g of either GFP (control), UbiR-GFP (cleavable ubiquitin), or UbiAR-GFP (non-cleavable ubiquitin) mRNA was transfected into cells using Lipofectamine MessengerMax reagent according to manufacturer's protocols. At the same time as transfection, either 0 nM or 100 nM proteasome inhibitor MG132 was added to the cells. Fluorescence from the cells was quantitatively measured using the Incucyte platform for 3 days post-transfection. For quantification, GFP integrated intensity was normalized to cell confluence.
  • the GFP fused to cleavable ubiquitin (UbiR) or non-cleavable ubiquitin (UbiAR) is degraded significantly faster compared to GFP.
  • the presence of different concentrations of the proteasome inhibitor MG132 shows that the degradation of both N-terminal degron constructs occurs via the proteasomal system as increasing inhibition of the proteasome inhibitor MG132 results decreased degradation of the UbiR-GFP and UbiAR-GFP.
  • HEK293 cells were transfected with 5 ⁇ g mRNA, using cells using Lipofectamine MessengerMax reagent according to manufacturer's protocols:
  • the cells were harvested, pelleted, and lysed in a urea lysis buffer. Lysates were cleared of insoluble material by centrifugation and stored at -80°C. After thawing, expression of the mRNA-encoded protein was assessed. Protein concentrations of the supernatants were measured using a BCA kit according to manufacturer's protocols, and concentrations were normalized. Cysteine sulfhydryls were reduced with TCEP and alkylated with iodoacetamide. Proteins were digested using trypsin overnight at 37°C at a ratio of 1 ⁇ g trypsin per 50 ⁇ g total protein. Digested peptides were further desalted using C18 solid phase extraction.
  • peptides were analyzed by targeted LC-MS/MS analysis to quantify the relative abundance of the transfected protein in the cell lysate.
  • the results are shown in Fig. 5, the relative expression of GFP peptides in cells expressing UbiR-GFP and UbiAR-GFP is significantly decreased due to the effective degradation. This timepoint was taken at 24 hours after transfection of the GFP constructs. Two tryptic peptides derived from GFP are shown.
  • Example 5 N-terminal dearons reduce overall expression and HLA-II presentation, but increase HLA-I presentation
  • HEK293 or A375 cells were transfected with 250 ⁇ g mRNA, using Lipofectamine MessengerMax reagent according to manufacturer's protocols:
  • HEK293 cells were transfected with 250 ⁇ g mRNA, using Lipofectamine MessengerMax reagent according to manufacturer's protocols:
  • SP+MITD comprising a signal peptide (HSV-lgD SP as shown in SEQ ID NO.: 22), GFP, and an MHC internal transmembrane domain (MITD) (SEC-MITD).
  • Lysates from the A375 transfection were applied to agarose beads functionalized with the pan-HLA-DR antibody L243.
  • Lysates from the HEK293 transfection were applied to agarose beads functionalized with the pan-HLA-I antibody W6/32. HLA pulldown was performed for at least 3 h.
  • the supernatant from the A375 transfection was saved for expression analysis (see below), the beads were washed, and bound HLA-peptide complexes were eluted with 10% acetic acid.
  • the eluate was ultrafiltered through a 10 kDa molecular weight cutoff filter, cysteine sulfhydryls were reduced with TCEP and alkylated with iodoacetamide, and peptides were further desalted by C18 solid phase extraction. Following desalting, peptides were analyzed by targeted LC-MS/MS analysis to quantify HLA peptides from the transfected protein.
  • mRNA-encoded protein was done using the supernatants from the HLA pulldown above. Protein concentrations of the supernatants were measured using a BCA kit according to manufacturer's protocols, and concentrations were normalized. Cysteine sulfhydryls were reduced with TCEP and alkylated with iodoacetamide. Detergents from the lysis buffer were removed by SP3 cleanup with carboxylate magnetic beads as described in Hughes CS, Moggridge S, Muller T, Sorensen PH, Morin GB, Krijgsveld J. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc.
  • the presence of an N-terminal degron such as UbiAR UFD (i.e. UbiAR-GFP) and UbiR N-deg (i.e. UbiR-GFP) lead to significantly enhanced HLA-I presentation and significantly decreased HLA-II presentation compared to GFP alone, demonstrating that the N-terminal degron allows to module the immune response by specifically eliciting an antigen-specific CD8 + T cell response.
  • the timepoint shown in Figure 6 was 24 hours after transfection of the GFP constructs, and the expression + HLA-II data was taken from the same sample (A375 cells), while the HLA-I data was taken from a different sample (HEK293T cells).
  • a ubiquitin comprising a leucine substitution at the 43 th position is used, wherein the leucine at position 43 of the ubiquitin sequence is replaced by alanine (L43A) as shown in SEQ ID NO.: 14 or 15, wherein the SEQ ID NO.: 14 shows a non-cleavable ubiquitin (UbiAR) comprising a L43A mutation, and SEQ ID NO.:15 shows a cleavable ubiquitin (UbiR) comprising a L43A mutation.
  • This mutation leads to degradation of the ubiquitin rather than its recycling.
  • UbiAR(L43A)-GFP is shown in SEQ ID NO.: 20 and UbiR(L43A)-GFP is shown in SEQ ID NO.: 21. These sequences further comprise K6R, KIIR, K27R, K29R, K33R, and K63R mutations.
  • mice are immunized with 2.5 ⁇ g RNA intramuscularly. Seven days after immunization, spleens are harvested and serum is collected.
  • Spleen processing is done as described above in Example 2 for ELISpot analysis as described in Example 2. Briefly, single cell suspensions are generated from collected spleens for ELISpot analysis. Spleens are decanted onto 70 pm filters placed on 50 mL conical vials and gently dissociated using the plunger of a 3 mL syringe and rinsing with ⁇ 6 mL of RPMI media to release splenocytes into the tube. Splenocytes are centrifuged for 10 min at 4°C at 1300 revolutions per minute (rpm), the supernatant is decanted and red blood cells are lysed with ammonium-chloride-potassium (ACK) Lysing Buffer for 3 min at RT.
  • ACK ammonium-chloride-potassium
  • the ACK lysis is stopped by adding 9 mL of RPMI media and cells are centrifuged again and resuspended in 5 mL of serum-free assay media (X- VIVO + 1% Pen-Strep + 1% Glutamax) and refiltered into a fresh tube over a fresh 70 pm filter. Cells are then counted using a Nexcelom Cellaca-MX. Pre-isolated cells are saved or CD4 vs CD8 enrichment is performed using Stem Cell's EasySepTM mouse CD8a positive selection kit II and mouse CD4 positive selection kit II with EasyEightsTM EasySepTM magnet according to the manufacturers protocol.
  • ELISpot assays with fresh splenocytes are performed according to the manufacturer's protocol (with minor modifications as described below) using R&D systems' mouse IFN-y ELISpot kit as described above in Example 2. Briefly, 96-well ELISpot plates are blocked with serum-free assay media (X-VIVO + 1% Pen-Strep + 1% Glutamax) for at least 1 h at 37°C. Next, 100 pL of the splenocyte solution (3 x 105 cells) are transferred to the respective well of the 96-well ELISpot plate. Another 100 pL of overlapping peptide pools or controls are added in the following concentrations: 1. GFP peptide pool (57 peptides, ISmer/llmer overlap): 0.3 pM final concentration per peptide
  • Concanavalin A (ConA): 0.3 pM final concentration
  • the GFP peptide pool contains overlapping peptides that span GFP.
  • the Ubiquitin peptide pool contains overlapping peptides that span UbiR and UbiAR.
  • the splenocytes are stimulated with ConA.
  • medium with DMSO equivalent to the highest volume of peptide mix is added. All stimulations are performed in triplicate.
  • Plates are incubated overnight in a 37°C humidified incubator with 5% CO2 and after approximately 20 h, cells are removed from the plates and the detection protocol was initiated.
  • the detection antibody, Streptavidin- Alkaline Phosphatase (AP), and the ready-to-use substrate are added to the wells according to the manufacturer's protocol with an overnight incubation at 4°C.
  • an ELISpot plate reader ImmunoSpot® S6 Core Analyzer, CTL
  • the counting size threshold is adjusted for this prime/boost experiment to a minimum spot size of 0.0075 sq.mm and a maximum spot size of 0.1080 sq.mm. Spots falling into this size range are counted.
  • Example 7 Analyzing N-terminal dearons comprising lysine mutations
  • the examples described herein demonstrate the efficient destabilization of the polypeptides when using the N- terminal degron disclosed herein.
  • the N-terminal degron leads to rapid degradation of the polypeptides, thereby eliciting an antigen-specific CD8 + T cell response, allowing to module the immune response upon vaccination.
  • Fig. 1 depicts an exemplary schematic representation of the N-end rule pathway ("N-degron”, i.e., cleavable ubiquitin) and the ubiquitin fusion degradation (UFD) pathway (“UFD degron”, i.e. non-cleavable ubiquitin).
  • N-degron i.e., cleavable ubiquitin
  • UFD degron ubiquitin fusion degradation
  • the degradation of the polypeptide depends on the N-terminal amino acid.
  • a ubiquitin is fused to the N-terminus of a polypeptide followed immediately by a destabilizing amino acid.
  • the N-terminal ubiquitin (cleavable ubiquitin) can be cleaved by a deubiquitinase enzyme, revealing the destabilizing N- terminal amino acid at the N-terminus of the polypeptide comprising the antigenic peptide.
  • the destabilizing amino acid and internal lysines (exemplarily here lysines at positions 15 and 17 (K 15 - 17 )) are ubiquitinated resulting in a poly-ubiquitin chain, targeting the poly- ubiquitinated polypeptide for degradation.
  • the degradation of the polypeptide is caused by the presence of a non-deavable ubiquitin (due to the presence of a proline following the ubiquitin sequence or due to the mutation of the glycine at position 76 to another amino acid, with reference to SEQ ID NO.: 1, such a G76A,) fused to the N-terminus of the polypeptide.
  • a non-deavable ubiquitin due to the presence of a proline following the ubiquitin sequence or due to the mutation of the glycine at position 76 to another amino acid, with reference to SEQ ID NO.: 1, such a G76A, fused to the N-terminus of the polypeptide.
  • lysines within the ubiquitin are ubiquitinated resulting in a poly-ubiquitin chain, targeting the poly-ubiquitinated polypeptide for degradation.
  • Fig. 2a depicts a diagram showing the relative protein abundance under the presence or absence of MG132 of (1) a control, (2) a reference polypeptide (termed “SP+MITD”) comprising a signal peptide (SP), antigenic peptide comprising epitopes derived from malaria (CSP, TRAP, UIS3, ETRAMP10.3, and LSAP2), and a MHC internal transmembrane domain (MITD), and (3) a polypeptide (termed “degron”) comprising a cleavable ubiquitin followed by a destabilizing amino acid, and antigenic peptide comprising epitopes derived from malaria (CSP, TRAP, UIS3, ETRAMP10.3, and LSAP2).
  • SP+MITD a reference polypeptide comprising a signal peptide (SP), antigenic peptide comprising epitopes derived from malaria
  • CSP, TRAP, UIS3, ETRAMP10.3, and LSAP2 a MHC internal transmembran
  • the antigenic polypeptide comprises internal lysines (N-terminal proximal lysines "N-term proximal lysines”) in the first encoded antigenic peptide CSP.
  • Fig. 2b depicts a schematic representation of the "SP+MITD” and "degron” polypeptides.
  • Fig. 3a and 3b shows the increased immunogenicity of the "degron" polypeptide compared to the "SP+MITD" polypeptide.
  • the black dots represent stimulation with the peptide pool corresponding to the antigen on the X- axis, and white dots represent stimulation with a DMSO control.
  • Fig. 4 depicts a diagram of a real time incucyte® microscopy live-cell analysis of samples expressing either GFP (GFP), cleavable ubiquitin fused to GFP (UbiR-GFP), or non-cleavable ubiquitin fused to GFP (UbiAR-GFP) upon increasing MG132 treatments leading to increasing inhibition of the proteasome.
  • GFP GFP
  • UbiR-GFP cleavable ubiquitin fused to GFP
  • UbiAR-GFP non-cleavable ubiquitin fused to GFP
  • Fig. 5 depicts a diagram showing the relative expression of GFP peptides in samples expressing GFP (GFP), non-cleavable ubiquitin fused to GFP (UbiAR), and cleavable ubiquitin fused to GFP (UbiR).
  • the timepoint was taken at 24 hours after transfection of the GFP constructs (i.e., of GFP, UbiAR, and UbiR).
  • Fig. 6 depicts a diagram showing the relative abundance of GFP in samples expressing GFP (GFP), non- cleavable ubiquitin (UbiAR UFD) fused to GFP, and cleavable ubiquitin (UbiR N-deg) fused to GFP.
  • GFP GFP
  • UbiAR UFD non- cleavable ubiquitin
  • UbiR N-deg cleavable ubiquitin
  • GFP MHC class II (HLA-II) presentation and MHC class I (HLA-I) presentation has been quantified in samples expressing (1) GFP (GFP), (2) GFP fused to a signal peptide (SP) and a MHC internal Transmembrane domain (MITD) (SEC+MITD), (3) non-cleavable ubiquitin (UbiAR UFD) fused to GFP, and (3) cleavable ubiquitin (UbiR N-deg) fused to GFP.
  • the timepoints were taken 24 hours after transfection of the GFP constructs (i.e., GFP, UbiAR UFD, and UbiR-N-deg).
  • A375 cells were used for the GFP expression and GFP HLA-II presentation data, while HEK293T cells were used for the GFP HLA-I presentation.

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Abstract

La présente invention concerne une molécule d'acide nucléique permettant de déclencher une réponse à lymphocytes T CD8+ spécifique à un antigène chez un sujet comprenant une séquence codante codant pour un polypeptide comprenant un dégron N-terminal et un peptide antigénique et des applications d'utilisation médicale pour la molécule d'acide nucléique selon l'invention.
PCT/US2023/074891 2022-09-23 2023-09-22 Utilisation de dégrons n-terminaux pour améliorer l'immunogénicité d'un vaccin à lymphocytes t à arn WO2024064886A1 (fr)

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