Nucleotide Delivery for Cancer Therapy

Abstract

The present invention relates to mRNAs useful for cancer therapy, and for use in methods of preventing or treating cancer in a subject.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for preventing or treating cancer (hereinafter referred to as cancer) in a subject, comprising administering to the subject an mRNA encoding at least one immunogenic peptide fragment of a polypeptide component of an immune system checkpoint, or a vaccine composition comprising said mRNA, wherein expression of the immunogenic peptide fragment results in an immune response against said checkpoint component, reducing its inhibitory effect. [Background technology]

[0002] 2. Background of the Invention The human immune system can mount a response against cancerous tumors. Harnessing this response is increasingly seen as one of the most promising ways to treat or prevent cancer. The key effector cells of long-term antitumor immune responses are activated tumor-specific effector T cells. However, although cancer patients usually have T cells specific to tumor antigens, the activity of these T cells is frequently suppressed by inhibitory factors and pathways, and cancer remains the leading cause of premature death in developed countries.

[0003] In the past decade, therapies have emerged that specifically target immune system checkpoints. One example is ipilimumab, a fully human IgG1 antibody specific for CTLA-4. Treatment of metastatic melanoma with ipilimumab was associated with an overall response rate of 10.9% and a clinical benefit rate of nearly 30% in a large Phase III trial, with subsequent analyses showing that responses can be durable and long-lasting. However, these figures still indicate that the majority of patients do not benefit from the treatment, leaving room for improvement.

[0004] Thus, there is a need for methods of preventing or treating cancer that enhance T cell anti-tumor responses in a greater percentage of patients without causing undesirable effects such as autoimmune diseases. Summary of the Invention

[0005] The inventors have confirmed in the present invention that the use of mRNA to induce expression by a patient's cells of at least one immunogenic fragment of a polypeptide component of an immune system checkpoint can result in an effective treatment or prevention of cancer.

[0006] Advantageously, multiple copies of the nucleic acid sequence encoding a given immunogenic peptide fragment can be contained in the same mRNA, allowing for greater expression of the peptide in cellular environment, which may be difficult to achieve by direct administration of the peptide itself.Similarly, the nucleic acid sequence encoding different immunogenic peptide fragments can be contained in the same mRNA, allowing for simultaneous expression and targeting of different parts of the same polypeptide component of immune system checkpoint, or different polypeptide components of the same or different checkpoints.In contrast, it can sometimes be difficult to co-formulate multiple different peptides for direct administration.

[0007] The present invention relates to - an open reading frame (ORF) encoding at least one immunogenic peptide fragment of a polypeptide component of an immune system checkpoint; - a 5' end cap at the 5' end, - the 5' untranslated region (UTR) contained 5' to the ORF, - the 3'UTR contained on the 3' side of the ORF, and - 3' tailing sequence at the 3' end The present invention provides an mRNA comprising the

[0008] The immune system checkpoints may be selected from any one or more of the following: - the interaction between IDO1 and its substrates, - interactions between PD1 and PDL1 and / or between PD1 and PDL2, - the interaction between arginase 1 or arginase 2 and its substrate, - the interaction between TDO and its substrates, - the interaction between TGFb1 and its receptors, - the interaction between CTLA4 and CD86 and / or between CTLA4 and CD80, - the interaction between B7-H3 and / or B7-H4 and their respective ligands; - Interaction between HVEM and BTLA, - the interaction between GAL9 and TIM3, - the interaction between MHC class I or II and LAG3, and - Interaction between MHC class I or II and KIR.

[0009] The ORF preferably comprises - at least one copy of a nucleic acid encoding SEQ ID NO: 2 (IO 102) and at least one copy of a nucleic acid encoding SEQ ID NO: 15 (IO 103) and / or SEQ ID NO: 16 (IO 104.1), - at least one copy of a nucleic acid encoding SEQ ID NO: 2 (IO 102) and at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112), (IO 104.1), and at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112), - at least one copy of a nucleic acid encoding SEQ ID NO: 2 (IO 102), and at least one copy of a nucleic acid encoding SEQ ID NO: 15 (IO 103) and / or SEQ ID NO: 16 (IO 104.1), and at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112). Includes.

[0010] The ORF preferably comprises - at least one copy of SEQ ID NO: 272 (IO 102) and at least one copy of SEQ ID NO: 273 (IO 103), - at least one copy of SEQ ID NO: 272 (IO 102) and at least one copy of SEQ ID NO: 277 (IO 112), - at least one copy of SEQ ID NO: 273 (IO 103) and at least one copy of SEQ ID NO: 277 (IO 112), - at least one copy of SEQ ID NO: 272 (IO 102) and at least one copy of SEQ ID NO: 273 (IO 103) and at least one copy of SEQ ID NO: 277 (IO 112) Includes.

[0011] Also provided is a vaccine composition comprising the mRNA of the present invention, wherein the mRNA may optionally be formulated in a lipid nanoparticle composition.

[0012] Also provided are methods for treating or preventing disease, comprising administering to a patient in need thereof a therapeutically or prophylactically effective amount of the mRNA or vaccine composition of the invention. Also provided are methods for producing the mRNA or vaccine composition and kits for their preparation.

[0013] Brief Description of the Sequence Listing Sequences relevant to the present invention are contained in Tables X, 2 and 3.

[0014] Detailed Description of the Invention It is to be understood that various applications of the disclosed products and methods can be modified to suit particular needs in the art, and it is to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[0015] Also, as used in this specification and the appended claims, the singular forms include plural referents unless the content clearly indicates a singular referent. Thus, for example, reference to an "inhibitor" includes two or more such inhibitors, reference to an "oligonucleotide" includes two or more such oligonucleotides, and so forth.

[0016] As used herein, a "subject" includes any mammal, preferably a human.

[0017] "Polynucleotide" is used interchangeably with "nucleic acid" herein to refer to a polymer of nucleosides. Typically, the polynucleotides of the present invention are composed of nucleosides found naturally in DNA or RNA (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine and deoxycytidine) linked by phosphodiester bonds. However, the term also encompasses molecules that contain nucleosides or nucleoside analogs that contain chemically or biologically modified bases, modified backbones, and the like, regardless of whether they are found in natural nucleic acids, and such molecules may be preferred for certain applications. When the present application refers to polynucleotides, it is understood that both DNA and RNA, and in each case both single-stranded and double-stranded forms (and the complement of each single-stranded molecule) are provided, unless otherwise indicated. As used herein, "polynucleotide sequence" can refer to the polynucleotide material itself and / or the sequence information (i.e., the sequence of letters used as abbreviations for bases) that biochemically characterize a particular nucleic acid. Polynucleotide sequences presented herein are presented in the 5' to 3' orientation unless otherwise indicated.

[0018] "Polypeptide" is used herein in the broadest sense to mean a compound of two or more subunit amino acids, amino acid analogs or other peptidomimetics. The term "polypeptide" can include short peptide sequences and also longer polypeptides and proteins. However, smaller portions of longer polypeptides and proteins are typically described as "peptides" or "peptide fragments" of such longer polypeptides or proteins.

[0019] As used herein, the terms "polypeptide sequence" or "amino acid sequence" can refer to the polypeptide substance itself and / or to the sequence information that biochemically characterizes the polypeptide (i.e., the sequence of letters or three-letter codes used as an abbreviation for the names of amino acids). Polypeptide sequences presented herein are presented in the N-terminal to C-terminal direction unless otherwise indicated.

[0020] As used herein, the term "amino acid" refers to natural amino acids, including both the D or L optical isomers, and / or unnatural or synthetic amino acids, as well as amino acid analogs and peptidomimetics.

[0021] All publications, patents, and patent applications cited above or below are hereby incorporated by reference in their entirety.

[0022] Immune system checkpoints and their polypeptide components The activation of effector T cells is usually triggered by T cell receptors that recognize antigen peptides presented by MHC complexes. Thus, the type and level of activation achieved is determined by the balance between signals that stimulate effector T cell responses and signals that inhibit said responses. In this specification, the term "immune system checkpoint" is used to mean any molecular interaction that changes the balance in favor of the inhibition of effector T cell responses. That is, the molecular interaction, when it occurs, negatively regulates the activation of effector T cells. Such an interaction can be direct, such as, for example, the interaction between a cell surface receptor and a ligand that transmits an inhibitory signal into effector T cells. Alternatively, it can be indirect, such as, for example, blocking or inhibiting the interaction between a cell surface receptor and a ligand that transmits an activation signal into effector T cells, or an interaction that promotes the upregulation of inhibitory molecules or cells, or enzymatic depletion of metabolic products required for effector T cells, or a combination thereof.

[0023] Examples of immune system checkpoints include: a. The interaction between IDO1 and its substrates; b. interactions between PD1 and PDL1 and / or between PD1 and PDL2; c. The interaction between Arginase 1 or Arginase 2 and its substrate; d. The interaction between TDO and its substrates; e. The interaction between TGFb1 and its receptor; f. interactions between CTLA4 and CD86 and / or between CTLA4 and CD80; g. Interactions between B7-H3 and / or B7-H4 and their respective ligands; h. the interaction between HVEM and BTLA; i. The interaction between GAL9 and TIM3, j. Interaction between MHC class I or II and LAG3, and k. Interaction between MHC class I or II and KIR.

[0024] Checkpoint (a), i.e., the interaction between IDO1 and its substrate, is a preferred checkpoint for the purposes of the present invention. This checkpoint is a metabolic pathway in cells of the immune system that requires the essential amino acid tryptophan. Lack of tryptophan causes a general suppression of effector T cell function and promotes the conversion of naive T cells to regulatory (i.e., immunosuppressive) T cells (Treg). The protein IDO1 is upregulated in cells of many tumors and is involved in the degradation of tryptophan levels. IDO1 is an enzyme that catalyzes the conversion of L-tryptophan to N-formylkynurenine and is therefore the first and rate-limiting enzyme of tryptophan catabolism by the kynurenine pathway. Thus, IDO1 (which may be referred to as IDO) is a polypeptide component of the immune system checkpoint that may be preferably targeted in the methods of the present invention. The full-length sequence of IDO is shown in Table X. Preferred mRNAs typically contain an ORF that codes for at least one immunogenic peptide fragment of IDO. The fragment can typically comprise or consist of at least 8 or 9 consecutive amino acids of the polypeptide. The fragment can typically consist of up to 50 consecutive amino acids of IDO1, up to 40 consecutive amino acids of IDO1, up to 30 consecutive amino acids of IDO1, or up to 25 consecutive amino acids of IDO1. The consecutive amino acid sequence of IDO1 may be modified by one or more conservative substitutions, so long as the immunogenicity of the starting sequence is retained. Preferred fragments of IDO are disclosed in WO2009 / 143843, WO2017 / 149150, WO2019 / 101954 and PCT / EP2021 / 074064, each of which is incorporated herein by reference. Preferred immunogenic peptide fragments of IDO are also shown in Table X. Preferred immunogenic peptide fragments comprise or consist of any one of the sequences of SEQ ID NOs: 2-13. The most preferred immunogenic peptide fragment comprises or consists of the sequence of SEQ ID NO:2 (a peptide consisting of this sequence may be designated IO 102).

[0025] Checkpoint (b), i.e., the interaction between PD1 and either of its ligands PD-L1 and PD-L2, is another preferred checkpoint for the purposes of the present invention. PD1 is expressed on effector T cells. Engagement with either PD-L1 or PD-L2 generates a signal that downregulates activation. The ligands are expressed by some tumors. In particular, PD-L1 is expressed by many solid tumors, including melanoma. Thus, these tumors may downregulate immune-mediated antitumor effects through activation of the inhibitory PD-1 receptor on T cells. Blocking the interaction between PD1 and one or both of its ligands may eliminate the checkpoint of the immune response and result in enhanced antitumor T cell responses. Thus, each of PD1, PD-L1, and PD-L2 is a polypeptide component of an immune system checkpoint that may be preferably targeted in the methods of the present invention. PD-L1 and PD-L2 are preferred, with PD-L1 being most preferred.

[0026] The full-length sequence of PD-L1 is shown in Table X. A preferred mRNA typically contains an ORF encoding at least one immunogenic peptide fragment of PD-L1. The fragment may typically comprise or consist of at least 8 or 9 consecutive amino acids of the polypeptide. The fragment may typically consist of up to 50 consecutive amino acids of PD-L1, up to 40 consecutive amino acids of PD-L1, up to 30 consecutive amino acids of PD-L1, or up to 25 consecutive amino acids of PD-L1. The consecutive amino acid sequence of PD-L1 may be modified by one or more conservative substitutions, so long as the immunogenicity of the starting sequence is retained. Preferred fragments of PD-L1 are disclosed in WO2013 / 056716, WO2017 / 220602, WO2017 / 149150, WO2019 / 101954 and PCT / EP2021 / 074064, each of which is incorporated herein by reference. Preferred immunogenic peptide fragments of PD-L1 are also shown in Table X. Preferred immunogenic peptide fragments comprise or consist of any one of SEQ ID NOs: 15-100. Particularly preferred are immunogenic peptide fragments comprising or consisting of any one of SEQ ID NOs: 15-25, most preferred are SEQ ID NOs: 15, 16 or 17. The most preferred immunogenic peptide fragment of PD-L1 comprises or consists of the sequence of SEQ ID NO: 15 (a peptide consisting of this sequence may be referred to as IO 103).

[0027] The full length sequence of PD-L2 is shown in Table X. A preferred mRNA typically comprises an ORF encoding at least one immunogenic peptide fragment of PD-L2. The fragment may typically comprise or consist of at least 8 or 9 consecutive amino acids of the polypeptide. The fragment may typically consist of up to 50 consecutive amino acids of PD-L2, up to 40 consecutive amino acids of PD-L2, up to 30 consecutive amino acids of PD-L2, or up to 25 consecutive amino acids of PD-L2. The consecutive amino acid sequence of PD-L2 may be modified by one or more conservative substitutions, so long as the immunogenicity of the starting sequence is retained. Preferred fragments of PD-L2 are disclosed in WO2018 / 077629, which is incorporated herein by reference. Preferred immunogenic peptide fragments of PD-L2 are also shown in Table X. Preferred immunogenic peptide fragments comprise or consist of the sequence of any one of SEQ ID NOs: 102, 103 or 104. The most preferred immunogenic peptide fragment comprises or consists of the sequence of SEQ ID NO:102.

[0028] Checkpoint (c), i.e., the interaction between arginase 1 or arginase 2 and its substrate, is another preferred checkpoint for the purposes of the present invention. Arginase 1 and 2 are enzymes that catalyze the reaction that converts the amino acid L-arginine into L-ornithine and urea. This depletes the microenvironment of arginine, resulting in the suppression of tumor-specific cytotoxic T cell responses. Enhanced arginase activity has been detected in cancer cells of patients with breast, lung, colon or prostate cancer. It has been shown that mouse macrophages transfected with rat arginase gene promote the growth of co-cultured tumor cells both in vitro and in vivo. In clinical settings, the induction of immune responses specific to arginase 1 and / or 2 can support a general anti-cancer immune response in addition to killing cancer cells, by suppressing the immunosuppressive function of arginase-expressing cells, particularly MDSCs and tumor-associated macrophages (TAMs). Thus, each of Arginase 1 and Arginase 2 is a polypeptide component of an immune system checkpoint that may be preferably targeted in the methods of the present invention. Arginase 1 is most preferred.

[0029] The full-length sequence of Arginase 1 is shown in Table X. A preferred mRNA typically comprises an ORF encoding at least one immunogenic peptide fragment of Arginase 1. The fragment typically comprises or can consist of at least 8 or 9 consecutive amino acids of the polypeptide. The fragment typically can consist of up to 50 consecutive amino acids of Arginase 1, up to 40 consecutive amino acids of Arginase 1, up to 30 consecutive amino acids of Arginase 1, or up to 25 consecutive amino acids of Arginase 1. The consecutive amino acid sequence of Arginase 1 may be modified by one or more conservative substitutions, so long as the immunogenicity of the starting sequence is retained. Preferred fragments of Arginase 1 are disclosed in WO2018 / 065563 and WO2020 / 064744, each of which is incorporated herein by reference. Preferred immunogenic peptide fragments of Arginase 1 are also shown in Table X. A preferred immunogenic peptide fragment comprises or consists of any one of SEQ ID NOs: 106 to 158. Particularly preferred are immunogenic peptide fragments comprising or consisting of any one of SEQ ID NOs: 106 to 111, most preferably SEQ ID NOs: 106, 107 or 108. The most preferred immunogenic peptide fragment of Arginase 1 comprises or consists of the sequence of SEQ ID NO: 106 (a peptide consisting of this sequence may be referred to as IO 112).

[0030] The full-length sequence of Arginase 2 is shown in Table X. A preferred mRNA typically contains an ORF encoding at least one immunogenic peptide fragment of Arginase 2. The fragment can typically comprise or consist of at least 8 or 9 consecutive amino acids of the polypeptide. The fragment can typically consist of up to 50 consecutive amino acids of Arginase 2, up to 40 consecutive amino acids of Arginase 2, up to 30 consecutive amino acids of Arginase 2, or up to 25 consecutive amino acids of Arginase 2. The fragment can comprise or consist of 9 to 19 consecutive amino acids of Arginase 2. The consecutive amino acid sequence of Arginase 2 may be modified by one or more conservative substitutions, so long as the immunogenicity of the starting sequence is retained. Preferred fragments of Arginase 2 are disclosed in WO2018 / 065563, WO2020 / 099582, and GB2202547.2, each of which is incorporated herein by reference. Preferred immunogenic peptide fragments of Arginase 2 are also shown in Table X. Preferred immunogenic peptide fragments comprise or consist of any one of the sequences of SEQ ID NOs: 160-220. Particularly preferred are immunogenic peptide fragments comprising or consisting of any one of the sequences of SEQ ID NOs: 160-182, most preferred are SEQ ID NOs: 160, 161, 162, 163 or 164. The most preferred immunogenic peptide fragments of Arginase 2 are peptides of 9 to 19 consecutive amino acids of Arginase 2 comprising or consisting of the sequence of SEQ ID NO: 160 (a peptide consisting of this sequence may be referred to as A2L2), or comprising the amino acids of SEQ ID NO: 163 or 164.

[0031] Checkpoint (d), i.e., the interaction between TDO and its substrate, is also a preferred checkpoint for the purposes of the present invention. Both TDO and IDO catalyze the initial and rate-limiting step of tryptophan oxidation to generate kynurenine, albeit by different mechanisms and without sharing sequence homology. Thus, checkpoint (d) is similar to checkpoint (a), and TDO is a polypeptide component of an immune system checkpoint that may be preferably targeted in the methods of the present invention. The full-length sequence of TDO is shown in Table X. Preferred mRNAs typically contain an ORF encoding at least one immunogenic peptide fragment of TDO. The fragment can typically comprise or consist of at least 8 or 9 consecutive amino acids of the polypeptide. The fragment can typically consist of up to 50 consecutive amino acids of TDO, up to 40 consecutive amino acids of TDO, up to 30 consecutive amino acids of TDO, or up to 25 consecutive amino acids of TDO. The consecutive amino acid sequence of TDO may be modified by one or more conservative substitutions, so long as the immunogenicity of the starting sequence is retained. Preferred fragments of TDO are disclosed in WO2016 / 041560, which is incorporated herein by reference. Preferred immunogenic peptide fragments of TDO are also shown in Table X. Preferred immunogenic peptide fragments comprise or consist of any one of SEQ ID NOs: 222-238. Most preferred immunogenic peptide fragments comprise or consist of up to 25 consecutive amino acids of TDO, including at least any one of SEQ ID NOs: 222-238.

[0032] Checkpoint (e), i.e., the interaction between TGFb and its receptor, is also a preferred checkpoint for the purposes of the present invention. TGFb is a multifunctional cytokine that plays an important role in the control of the immune system. There are four isoforms, of which isoform 1 (TGFb1) is particularly important in T cell immunity. In the context of cancer, TGFb1 neutralizes various immune cells such as cytotoxic T cells (CTLs), tumor-associated neutrophils and natural killer (NK) cells. It is also involved in tumor angiogenesis and metastasis. Thus, TGFb1 is a central inhibitory molecule in the tumor microenvironment (TME), involved in the downregulation of antitumor mechanisms of the immune system, allowing immune evasion by cancer cells. Thus, TGFb1 is a polypeptide component of the immune system checkpoint that can be preferably targeted in the methods of the present invention. TGFb1 is a dimeric cytokine that shares a cysteine ​​knot structure linked to each other by intramolecular disulfide bonds. TGFb1 is synthesized as a monomeric 390 amino acid precursor protein, which is interchangeably referred to as TGFb1 preprotein, TGFb1 precursor, full-length TGFb1, and preproTGFb1. The TGFb1 preprotein monomer has a molecular weight of about 25 kDa. The TGFb1 protein monomer has three distinct domains: a signal peptide (SP: amino acids 1-29), a latency associated peptide (LAP: amino acids 30-278) and a mature peptide (mature TGFb1: amino acids 279-390). The sequences of full-length TGFb1, TGFb1 SP, TGFb1 LAP and mature TGFb1 are shown in Table X. A preferred mRNA typically comprises an ORF encoding at least one immunogenic peptide fragment of full-length TGFb1, TGFb1 SP, TGFb1 LAP or mature TGFb1. The fragment typically can comprise or consist of at least 8 or 9 consecutive amino acids of the polypeptide. The fragment may typically consist of up to 50 consecutive amino acids of the polypeptide, up to 40 consecutive amino acids of the polypeptide, up to 30 consecutive amino acids of the polypeptide, or up to 25 consecutive amino acids of the polypeptide.The contiguous amino acid sequence of the polypeptide may be modified by one or more conservative substitutions, so long as the immunogenicity of the starting sequence is retained. Preferred fragments of the polypeptide are disclosed in WO2020 / 245264, which is incorporated herein by reference. Preferred immunogenic peptide fragments of TGFb1 are also shown in Table X. Preferred immunogenic peptide fragments comprise or consist of the sequence of any one of SEQ ID NOs: 243-271, preferably SEQ ID NOs: 243-247, most preferably SEQ ID NOs: 243 or 244. The most preferred immunogenic peptide fragment comprises or consists of the sequence of SEQ ID NO: 243. (A peptide consisting of this sequence may be referred to as TGFb15).

[0033] Another checkpoint that is preferred for the purposes of the present invention is checkpoint (f), i.e., the interaction between the T cell receptor CTLA-4 and its ligand B7 protein (B7-1 and B7-2). CTLA-4 is normally upregulated on the T cell surface after initial activation, and ligand binding generates a signal that inhibits further / continuous activation. CTLA-4 competes for binding to the B7 protein with the receptor CD28, which is also expressed on the T cell surface but upregulates activation. Thus, by blocking the interaction of B7 protein with CTLA-4, but not with CD28, one of the normal checkpoints of the immune response is eliminated and anti-tumor T cell responses can be enhanced. Thus, CTLA4 and its ligand are examples of polypeptide components of immune system checkpoints that can be preferably targeted in the methods of the present invention. Preferred mRNAs typically contain an ORF that encodes at least one immunogenic peptide fragment of CTLA4 or one of its ligands.

[0034] The same applies to any of the polypeptide components of checkpoints (g)-(k). A preferred mRNA typically contains an ORF encoding at least one immunogenic peptide fragment of any of the polypeptide components of checkpoints (g)-(k).

[0035] In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (a) and (b). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (a) and (c). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (a) and (d). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (a) and (e). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (a) and (f). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (a) and (g). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (a) and (h). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (a) and (i). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (a) and (j). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (a) and (k). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (b) and (c). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (b) and (d). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (b) and (e). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (b) and (f). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (b) and (g).In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (b) and (h). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (b) and (i). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (b) and (j). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (b) and (k). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (c) and (d). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (c) and (e). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (c) and (f). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (c) and (g). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (c) and (h). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (c) and (i). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (c) and (j). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (c) and (k). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (d) and (e). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (d) and (f). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (d) and (g).In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (d) and (h). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (d) and (i). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (d) and (j). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (d) and (k). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (e) and (f). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (e) and (g). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (e) and (h). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (e) and (i). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (e) and (j). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (e) and (k). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (f) and (g). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (f) and (h). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (f) and (i). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (f) and (j). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (f) and (k).In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (g) and (h). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (g) and (i). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (g) and (j). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (g) and (k). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (h) and (i). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (h) and (j). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (h) and (k). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (i) and (j). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (i) and (k). In some embodiments, the ORF encodes at least one immunogenic polypeptide fragment of checkpoints (j) and (k).

[0036] mRNA molecule The present invention relates to - an open reading frame (ORF) encoding at least one immunogenic peptide fragment of a polypeptide component of an immune system checkpoint; - a 5' end cap at the 5' end, - the 5' untranslated region (UTR) contained 5' to the ORF, - the 3'UTR contained on the 3' side of the ORF, and - 3' tailing sequence at the 3' end For each of the mRNA sequences encoding the immunogenic fragments, there may be interspersed cleavage-sensitive sites. The ORF may contain multiple copies of each sequence encoding the different immunogenic peptide fragments, and may optionally contain at least 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more copies of each of said sequences, and preferably the ORF encodes at least 2, 3, 4, 5, 10 or more different immunogenic peptide fragments. In some embodiments, the different immunogenic peptide fragments are different portions of the same immune system checkpoint component polypeptide. In some embodiments, the different immunogenic peptide fragments are fragments of different immune system checkpoint component polypeptides. In some embodiments, the different immunogenic peptide fragments are both different portions of the same immune system checkpoint component polypeptide and fragments of different immune system checkpoint component polypeptides.

[0037] Preferred mRNAs encoding fragments of immune system checkpoint component polypeptides are shown in Table 2. Preferred mRNAs comprise an ORF consisting of or including the sequence of any one of SEQ ID NOs: 272-281.

[0038] The mRNA may be described as an mRNA vaccine against cancer or an mRNA cancer vaccine. The mRNA vaccine is described in International Patent Application No. WO2015 / 164674, which is incorporated herein by reference in its entirety. The mRNA cancer vaccine of the present invention may be a composition, including a pharmaceutical composition. The present invention also includes methods for preparing, producing, formulating and / or using the mRNA cancer vaccine.

[0039] It can be a challenge to generate and deliver immunogenic peptide fragments so that they are effectively presented on MHC molecules to elicit the desired immune response in an individual. In some embodiments, the mRNA of the present invention solves this problem by resulting in the expression of a polypeptide that contains multiple immunogenic peptide fragments, preferably with cleavage sites between them that are recognized by proteases that are abundant in antigen-presenting cells (APCs). These methods mimic antigen processing and can result in more effective antigen presentation than can be achieved with peptide antigens. Expression of immunogenic peptide fragments from RNA as intracellular peptides can provide advantages compared to delivery as exogenous peptides. RNA is delivered intracellularly and expresses the epitope in close proximity to the appropriate cellular machinery for processing of the epitope, which is recognized by the appropriate immune cells. Targeting sequences can also allow for greater specificity in the delivery of peptide epitopes. For example, C-terminal ubiquitin ligase targeting proteins (FBox proteins) can be used to target processing of the polypeptide to the proteasome, more closely mimicking MHC processing. The construct of the present invention may also include a linker, such as a proteolytic cleavage site optimized for APC. These proteolytic sites are advantageous because they facilitate the processing of peptides in APC. When mRNA cancer vaccine is delivered to cells, mRNA is processed into polypeptides by intracellular machinery, which can then process the polypeptides into immunogenic peptide fragments that can stimulate desired immune responses.

[0040] In some embodiments, the mRNA cancer vaccine encodes multiple immunogenic peptide fragments. This may be described as a poly-epitopic mRNA vaccine, since each encoded immunogenic peptide fragment contains at least one epitope. The RNA sequence encoding the immunogenic peptide fragments may be interspersed with sequences encoding amino acid sequences recognized by proteolytic enzymes. Thus, in some embodiments, the mRNA cancer vaccine is an mRNA having an open reading frame encoding a propeptide, since the encoded polypeptide sequence comprises multiple immunogenic peptide fragments linked together, either directly or via a linker such as a cleavage-sensitive site. An exemplary propeptide has the following peptide sequence: T m -Y o -(X1-Y o -X2-Y o -.....X n )- Y o -T m where T is a targeting sequence and m = 0 to 1. The targeting sequence may be included at either the N-terminus, C-terminus or both ends of the central peptide region. When a polypeptide has multiple targeting sequences, the sequences may be the same or different.

[0041] X1, 2, etc. are each independently an immunogenic peptide fragment sequence, n = 0-1000. Each immunogenic peptide fragment sequence represented by X can represent a unique immunogenic peptide fragment sequence in the propeptide, or it can represent a copy of an immunogenic peptide fragment sequence. Thus, the propeptide encoded by the mRNA can be composed of multiple immunogenic peptide fragment sequences, each of which is unique, and / or it can contain multiple copies of each unique immunogenic peptide fragment sequence. In some embodiments, the propeptide can have at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50 or more copies of each unique immunogenic peptide fragment sequence. Preferably, the propeptide has at least 2, 3, 4, 5, 10 or more different immunogenic peptide fragment sequences.

[0042] Y is a linker sequence, preferably a cleavage sensitive sequence, and o = 0-5. Each immunogenic peptide fragment sequence may optionally have one or more linkers and may optionally have cleavage sensitive sites adjacent to the N-terminus and / or C-terminus. In a multi-epitope design, two or more of the immunogenic peptide fragment sequences may have cleavage sensitive sites between them. Alternatively, two or more of the immunogenic peptide fragment sequences may be linked to each other directly or via a linker that is not a cleavage sensitive site. The targeting sequence may also be linked to the immunogenic peptide fragment sequence via a cleavage sensitive site, or it may be linked directly to the immunogenic peptide fragment sequence via a linker that is not a cleavage sensitive site.

[0043] The present invention is primarily directed to mRNAs encoding at least one immunogenic peptide fragment of a polypeptide component of an immune system checkpoint. Preferred RNA sequences are shown in Table 2. However, the mRNA sequences of the present invention may be replaced by corresponding "counterpart" DNA sequences. The DNA sequences may be in either single-stranded or double-stranded form (and the complement of each single-stranded molecule). Suitable "counterpart" DNA sequences are shown in Table 3.

[0044] In some embodiments, the vaccine compositions provided herein comprise two or more mRNA polynucleotides, each encoding a different immunogenic peptide fragment, hi some embodiments, the vaccine compositions comprise two, three, or four mRNA polynucleotides.

[0045] In some embodiments, the vaccine composition comprises a first mRNA polynucleotide encoding a first immunogenic polypeptide fragment and a second mRNA polynucleotide encoding a second immunogenic polypeptide fragment, in some embodiments, the first and second immunogenic polypeptide fragments are protein fragments of the following:

[0046] [Table 1]

[0047] In some embodiments, the first and second immunogenic polypeptide fragments are up to 50 contiguous amino acids of a protein according to the tables above.

[0048] In some embodiments, the first and second immunogenic polypeptide fragments comprise or consist of an amino acid sequence according to the table below.

[0049] [Table 2] TIFF2025508467000003.tif95155

[0050] In some embodiments, the vaccine composition comprises a first mRNA polynucleotide encoding a first immunogenic polypeptide fragment, a second mRNA polynucleotide encoding a second immunogenic polypeptide fragment, and a third mRNA polynucleotide encoding a third immunogenic peptide fragment of a polypeptide component of an immune system checkpoint.

[0051] In some embodiments, the first immunogenic polypeptide fragment is IDO, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of IDO, and optionally comprises or consists of the sequence of any one of SEQ ID NOs: 2-13, preferably SEQ ID NO: 2. The second immunogenic polypeptide fragment is PDL1 or PDL2, preferably PDL1, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of PDL1, and optionally comprises or consists of the sequence of any one of SEQ ID NOs: 15-100, preferably SEQ ID NO: 15 or 16; or PDL2, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of PDL2, and optionally comprises or consists of the sequence of any one of SEQ ID NOs: 102-104. The third immunogenic polypeptide fragment is Arginase 1 or Arginase 2, preferably Arginase 1, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of Arginase 1, and can optionally comprise or consist of any one of SEQ ID NOs: 106-158, preferably SEQ ID NO: 106; or Arginase 2, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of Arginase 2, and can optionally comprise or consist of any one of SEQ ID NOs: 160-220, preferably SEQ ID NO: 150.

[0052] In some embodiments, the vaccine composition comprises a first mRNA polynucleotide encoding a first immunogenic polypeptide fragment and a second mRNA polynucleotide encoding a second immunogenic polypeptide fragment. In some embodiments, the first polynucleotide comprises at least one copy of a nucleic acid encoding SEQ ID NO:2 (IO 102) and the second polynucleotide comprises at least one copy of a nucleic acid encoding SEQ ID NO:15 (IO 103) and / or SEQ ID NO:16 (IO 104.1). In some embodiments, the first polynucleotide comprises at least one copy of a nucleic acid encoding SEQ ID NO:2 (IO 102) and the second polynucleotide comprises at least one copy of a nucleic acid encoding SEQ ID NO:106 (IO 112). In some embodiments, the first polynucleotide comprises at least one copy of a nucleic acid encoding SEQ ID NO: 15 (IO 103) and / or SEQ ID NO: 16 (IO 104.1), and the second polynucleotide comprises at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112).

[0053] In some embodiments, the vaccine composition comprises a first mRNA polynucleotide encoding a first immunogenic polypeptide fragment, a second mRNA polynucleotide encoding a second immunogenic polypeptide fragment, and a third mRNA polynucleotide encoding a third immunogenic peptide fragment of a polypeptide component of an immune system checkpoint. In some embodiments, the first polynucleotide comprises at least one copy of a nucleic acid encoding SEQ ID NO:2 (IO 102), the second polynucleotide comprises at least one copy of a nucleic acid encoding SEQ ID NO:15 (IO 103) and / or SEQ ID NO:16 (IO 104.1), and the third polynucleotide comprises at least one copy of a nucleic acid encoding SEQ ID NO:106 (IO 112).

[0054] In some embodiments, each of the first, second and / or third polynucleotides comprises one or more of SEQ ID NO:272, 273, 274, 275, 276, 277, 278, 279, 280 and 281. In some embodiments, the first polynucleotide comprises at least one copy of SEQ ID NO:272 (IO 102) and the second polynucleotide comprises at least one copy of SEQ ID NO:273 (IO 103). In some embodiments, the first polynucleotide comprises at least one copy of SEQ ID NO:272 (IO 102) and the second polynucleotide comprises at least one copy of SEQ ID NO:277 (IO 112). In some embodiments, the first polynucleotide comprises one copy of SEQ ID NO:273 (IO 103) and the second polynucleotide comprises at least one copy of SEQ ID NO:277 (IO 112). In some embodiments, the first polynucleotide comprises one copy of SEQ ID NO:272 (IO 102), the second polynucleotide comprises at least one copy of SEQ ID NO:273 (IO 103), and the third polynucleotide comprises at least one copy of SEQ ID NO:277 (IO 112).

[0055] Targeting Sequence The mRNA may code for one or more targeting sequences. This may be an endosomal targeting sequence, for example, a portion of the transmembrane domain of lysosomal associated membrane protein (LAMP-1) or a portion of the transmembrane domain of the invariant chain (Ii). The targeting sequence may be a ubiquitination signal attached to one or both ends of the encoded peptide. In other embodiments, the targeting sequence is a ubiquitination signal attached to an internal site and / or either end of the encoded peptide. Thus, the RNA may comprise a nucleic acid sequence encoding a ubiquitination signal at one or both ends of the nucleotides encoding the immunogenic peptide fragment.

[0056] Ubiquitination, a post-translational modification, is the process of attaching ubiquitin to substrate target proteins. Ubiquitination signals are peptide sequences that allow targeting and processing of peptides to one or more proteasomes. By targeting and processing peptides using ubiquitination signals, intracellular processing of peptides can more closely mimic antigen processing in antigen-presenting cells (APCs). The number of ubiquitins added to an antigen can increase the efficiency of the processing step. For example, in polyubiquitination, additional ubiquitin molecules are added after the first ubiquitin molecule is attached to the peptide. The resulting ubiquitin chain is generated by linking the glycine residue of the ubiquitin molecule to the lysine of the ubiquitin attached to the peptide. Each ubiquitin contains an N-terminus and seven lysine residues that can serve as the site of ubiquitination. When four or more ubiquitin molecules are attached to lysine residues on a peptide antigen, the 26S proteasome recognizes the complex, internalizes it, and degrades the protein into small peptides.

[0057] Cleavage sensitive sequence In some embodiments, the immunogenic peptide fragment sequences may be linked by cleavage sensitive sites. Cleavage sensitive sites are peptides that are susceptible to cleavage by enzymes or proteases. These sites are also referred to as protease cleavage sites. Preferably, the protease is an intracellular enzyme. The protease may be a serine protease, a threonine protease, a cysteine ​​protease, an aspartic acid protease, a glutamic acid protease, or a metalloprotease. In some preferred embodiments, the protease is a protease found in antigen presenting cells (APCs). Thus, the protease cleavage sites correspond to highly abundant (highly expressed) proteases in APCs. Cleavage sensitive sites that are sensitive to APC enzymes are referred to as APC cleavage sensitive sites. Proteases expressed in APCs include, but are not limited to, cysteine ​​proteases, such as cathepsin B, cathepsin H, cathepsin L, cathepsin S, cathepsin F, cathepsin Z, cathepsin V, cathepsin O, cathepsin C and cathepsin K, and aspartic proteases, such as cathepsin D, cathepsin E and aspartic endopeptidase.

[0058] The cleavage sensitive site may preferably be a cathepsin B or S sensitive site. Exemplary cathepsin B sensitive sites include, but are not limited to, those described in WO2017 / 020026, which is incorporated herein by reference (see SEQ ID NOs: 12-407 of WO2017 / 020026). Exemplary cathepsin S sensitive sites include, but are not limited to, those described in WO2017 / 020026 (see SEQ ID NOs: 3-5, 408-1122 of WO2017 / 020026). Other cathepsin sensitive sites are known in the art or may be readily determined empirically using digestion assays with no more than routine experimentation.

[0059] Nucleotide Modifications The mRNA cancer vaccine of the present invention comprises one or more polynucleotides, which code for one or more immunogenic peptide fragment sequences. Exemplary polynucleotides may contain at least one chemical modification. Polynucleotides may contain various substitutions and / or insertions. The term "chemical modification" or "chemically modified" as used herein in reference to polynucleotides, where appropriate, refers to modifications in one or more of the position, pattern, proportion or population of adenosine (A), guanosine (G), uridine (U), thymidine (T) or cytidine (C) ribo- or deoxyribonucleosides.

[0060] Modified polynucleotides, when introduced into a cell or organism, can exhibit reduced degradation in the cell or organism compared to unmodified polynucleotides. Modified polynucleotides, when introduced into a cell or organism, can exhibit reduced immunogenicity (e.g., reduced natural response) in the cell or organism. Modifications of polynucleotides are well known in the art and include, for example, those listed in WO2017 / 020026, which is incorporated herein by reference. In general, the modifications discussed in this section are not intended to refer to ribonucleotide modifications in naturally occurring 5'-end mRNA cap moieties.

[0061] A polynucleotide can include naturally occurring or non-naturally occurring modifications, or a polynucleotide can include both naturally occurring and non-naturally occurring modifications. A polynucleotide of the mRNA cancer vaccine of the present invention can include any useful modification, for example, to the sugar, nucleobase, or internucleoside linkage (e.g., the phosphate / phosphodiester linkage / phosphodiester backbone that constitutes the linkage). One or more atoms of a pyrimidine nucleobase can be substituted or replaced by an optionally substituted amino, an optionally substituted thiol, an optionally substituted alkyl (e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro). In certain embodiments, there is a modification (e.g., one or more modifications) in each of the sugar and internucleoside linkage. The modification according to the present invention can be a modification from ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof. Additional modifications are described herein. Non-natural modified nucleotides can be introduced into nucleic acids or polynucleotides, such as polynucleotides of mRNA cancer vaccines, during or after the synthesis of the chain to achieve desired functions or properties. Modifications can be present in internucleotide bonds, purine or pyrimidine bases, or sugars. Modifications can be introduced at the ends of the chain or other sites within the chain using chemical synthesis or polymerase enzymes. Any region of the polynucleotide can be chemically modified.

[0062] The present disclosure provides modified nucleosides and nucleotides. A "nucleoside" as described herein is defined as a compound containing a combination of a sugar molecule (e.g., pentose or ribose) or a derivative thereof and an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). A "nucleotide" as described herein is defined as a nucleoside containing a phosphoric acid group. Modified nucleotides can be synthesized by any useful method described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides). A polynucleotide can include one or more regions of linked nucleosides. Such regions can have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the polynucleotide will include a region of nucleotides.

[0063] Modified nucleotide base pairs include not only standard adenosine-thymine, adenosine-uracil or guanosine-cytosine base pairs, but also base pairs formed between nucleotides containing non-standard or modified bases and / or modified nucleotides, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the non-standard and standard bases or between two complementary non-standard base structures. One example of such a non-standard base pair is the base pair between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of bases / sugars or linkers can be incorporated into the polynucleotides of the invention.

[0064] The mRNA of the present invention may have at least one chemical modification, which is preferably selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine and 2'-O-methyluridine.

[0065] 5' Cap and 3' Tail The present invention relates to messenger RNA (mRNA). As used herein, "messenger RNA" (mRNA) refers to any polynucleotide that encodes at least one peptide or polypeptide of interest and can be translated to produce the encoded peptide polypeptide of interest in vitro, in vivo, in situ or ex vivo. The basic components of an mRNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5'-end cap and a 3'-tailing sequence. The mRNA of the present invention typically includes all of these features.

[0066] A "5' untranslated region (UTR)" is a region of an mRNA that is immediately upstream (i.e., 5') of the start codon (i.e., the first codon of an mRNA transcript that is translated by ribosomes) and does not code for a protein or peptide.

[0067] A "3' untranslated region (UTR)" is a region of an mRNA that is immediately downstream (i.e., 3') of a stop codon (i.e., a codon in an mRNA transcript that indicates the end of translation) and that does not code for a protein or peptide.

[0068] An "open reading frame" is a contiguous stretch of DNA beginning with a start codon [eg, methionine (ATG)] and ending with a stop codon (eg, TAA, TAG, or TGA) that encodes a protein or peptide.

[0069] A 5' end cap is a specially modified nucleotide at the 5' end of some primary transcripts, such as messenger RNA, that promotes stability and translation. It usually consists of a guanine nucleotide linked to the mRNA via an unusual 5' to 5' triphosphate bond. This guanosine is methylated at position 7 by a methyltransferase immediately after cap formation in vivo. It can therefore be referred to as a 7-methylguanylate cap (abbreviated as m7G). A preferred 5' end cap is m7G(5')ppp(5')NlmpNp.

[0070] The 3' tailing sequence is a polyA tail, a polyAG quartet and / or a stem-loop sequence. The 3' tailing sequence is typically 40 to 200 nucleotides in length. In some embodiments, the 3' tailing sequence is a polyA tail. A "polyA tail" is a region of an mRNA that contains multiple consecutive adenosine monophosphates and is downstream of, for example, immediately downstream (i.e., on the 3' side) of the 3' UTR. The polyA tail may contain 10 to 300 adenosine monophosphates. For example, the polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 adenosine monophosphates. In some embodiments, the polyA tail contains 50-250 adenosine monophosphates. In relevant biological contexts (e.g., within a cell, in vivo, etc.), the poly(A) tail functions to protect the mRNA from enzymatic degradation, for example, in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus, and translation.

[0071] In some embodiments, the polynucleotide comprises about 200 to about 3,000 nucleotides (e.g., 200 to 500, 200 to 1,000, 200 to 1,500, 200 to 3,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,500 to 3,000, and 2,000 to 3,000).

[0072] The polynucleotides of the present invention function as mRNA but are distinct from wild-type mRNA in functional and / or structural characteristics. The mRNA cancer vaccines of the present invention can be encoded by in vitro translation (IVT) polynucleotides. As used herein, "in vitro transcription template (IVT)" refers to a deoxyribonucleic acid (DNA) suitable for use in an IVT reaction for the production of messenger RNA (mRNA). In some embodiments, the IVT template encodes a 5' untranslated region, contains an open reading frame, encodes a 3' untranslated region and a polyA tail. The specific nucleotide sequence components and length of the IVT template will depend on the mRNA of interest encoded by the template.

[0073] How mRNA is produced Also disclosed is a method for producing an mRNA cancer vaccine. The mRNA can be produced by any suitable technique known in the art, by any suitable synthesis route. The IVT method is preferred. The in vitro transcription (IVT) method allows template-directed synthesis of RNA molecules of almost any sequence. The size of the RNA molecules that can be synthesized using the IVT method ranges from short oligonucleotides to long nucleic acid polymers of several thousand bases. The IVT method allows the synthesis of large amounts of RNA transcripts (e.g., microgram to milligram amounts) (Beckert et al., Synthesis of RNA by in vitro transcription, Methods Mol Biol. 703:29-41(2011); Rio et al., RNA: A Laboratory Manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press, 2011, 205-220.; Cooper, Geoffery M. The Cell: A Molecular Approach. 4th ed. Washington DC: ASM Press, 2007. 262-299). Generally, IVT uses a DNA template that features a promoter sequence upstream of the sequence of interest. The promoter sequence is most commonly derived from a bacteriophage (e.g., T7, T3 or SP6 promoter sequence), but many other promoter sequences, including those designed de novo, are acceptable. Transcription of the DNA template is typically best achieved by using an RNA polymerase that corresponds to a specific bacteriophage promoter sequence. Exemplary RNA polymerases include, but are not limited to, T7 RNA polymerase, T3 RNA polymerase or SP6 RNA polymerase, among others. IVT generally initiates on double-stranded (ds) DNA, but can also proceed on single strands. Suitable methods include, for example, those described in WO2017 / 020026, which is incorporated herein by reference.

[0074] Codon Optimization The mRNA disclosed herein can be fully or partially codon-optimized for human expression and / or to reduce immune recognition.Codon optimization methods are known in the art and can be useful in efforts to achieve various results, such as: matching the codon frequency in target and host organisms to ensure proper folding; biasing GC content to increase mRNA stability or reduce secondary structure; minimizing tandem repeat codons or base sequences that may impair gene assembly or expression; customizing (individualizing) transcriptional and translational control regions; inserting or removing protein transport sequences; removing / adding post-translational modification sites (e.g., glycosylation sites) in encoded proteins; adding, removing or shuffling protein domains; inserting or removing restriction sites; modifying ribosome binding sites and mRNA degradation sites; adjusting the translation rate to allow various domains of protein to fold properly; or reducing or eliminating problematic secondary structures in polynucleotides. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services and / or proprietary methods from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA). Preferably, the ORF sequence is optimized using an optimization algorithm.

[0075] A codon-optimized sequence may share less than 95%, less than 90%, less than 85%, less than 80% or less than 75% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest). A codon-optimized sequence may share between 65% and 85% sequence identity to a naturally occurring or wild-type sequence (e.g., a naturally occurring or wild-type mRNA sequence encoding a polypeptide or protein of interest).

[0076] An exemplary codon-optimized RNA may have an increased G / C level. The G / C content of a nucleic acid molecule may affect the stability of the RNA. RNA with an increased amount of guanine (G) and / or cytosine (C) residues may be more functionally stable than nucleic acids containing large amounts of adenine (A) and thymine (T) or uracil (U) nucleotides. WO02 / 098443, which is incorporated herein by reference, discloses pharmaceutical compositions containing mRNAs stabilized by sequence modifications in the translation region. The modifications are made by replacing existing codons with those that enhance RNA stability, without changing the resulting amino acid due to the degeneracy of the genetic code. This approach is limited to the coding region of the RNA.

[0077] Methods for preventing or treating cancer Also disclosed are methods for preventing or treating cancer, comprising administering the mRNA described herein.

[0078] The cancer may be prostate cancer, brain cancer, breast cancer, colorectal cancer, pancreatic cancer, ovarian cancer, lung cancer, cervical cancer, liver cancer, head / neck / throat cancer, skin cancer, bladder cancer or blood cancer. The cancer may take the form of a tumor or a blood-borne cancer. The tumor may be solid. The tumor is typically malignant and may be metastatic. The tumor may be an adenoma, adenocarcinoma, blastoma, carcinoma, desmoid tumor, desmoplastic small round cell tumor, endocrine tumor, germ cell tumor, lymphoma, leukemia, sarcoma, Wilms' tumor, lung tumor, colon tumor, lymphoid tumor, breast tumor or melanoma.

[0079] Blastoma types include hepatoblastoma, glioblastoma, neuroblastoma or retinoblastoma. Carcinoma types include colorectal or hepatocellular carcinoma, pancreatic cancer, prostate cancer, gastric cancer, esophageal cancer, cervical cancer, and head and neck cancer, and adenocarcinoma. Sarcoma types include Ewing's sarcoma, osteosarcoma, rhabdomyosarcoma, or any other soft tissue sarcoma. Melanoma types include lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentigo melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, amelanotic melanoma, soft tissue malignant melanoma, melanoma with small nevus-like cells, melanoma with features of Spitz nevus, and uveal melanoma. Lymphoma and leukemia types include precursor T-cell leukemia / lymphoma, acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, follicular lymphoma, diffuse large B-cell lymphoma, mantle cell lymphoma, chronic lymphocytic leukemia / lymphoma, MALT lymphoma, Burkitt lymphoma, mycosis fungoides, peripheral T-cell lymphoma, nodular sclerosing Hodgkin lymphoma, mixed-cellularity subtype of Hodgkin lymphoma. Lung tumor types include non-small cell lung cancer (adenocarcinoma, squamous cell carcinoma, and large cell carcinoma) and small cell lung cancer tumors.

[0080] The method of the present invention functions by activating or enhancing anti-cancer T cell responses in a subject. This is achieved by enhancing cancer or tumor-specific effector T cell activation by blocking or inhibiting one or more immune checkpoints. The method of the present invention blocks or inhibits one or more immune checkpoints by administering an mRNA, which results in the expression of an immunogenic peptide fragment of a component of the checkpoint, thereby inducing an immune response in the subject against said component, thereby blocking or inhibiting the activity of the checkpoint. It can therefore alternatively be described as a vaccine against said component of said checkpoint. The component of the checkpoint targeted by said immune response is preferably expressed by tumor cells and can also be expressed by normal cells with an immune inhibitory effect. The immune response therefore has a dual effect, that it blocks and inhibits the activity of the checkpoint, and also directly attacks the tumor.

[0081] The mRNA can advantageously code for immunogenic peptide fragments of multiple checkpoint components from the same or different checkpoints. By blocking or inhibiting multiple immune system checkpoints, the method of the present invention reduces side effects or complications and produces a greater antitumor response compared to other methods. The antitumor response is typically greater than expected when only a single checkpoint is targeted. Also, there is less chance of efficacy being reduced by antidrug responses. Because the first approach (the vaccine) actively benefits from such responses, which can also produce long-lasting effects. These advantages are also true when both approaches target the same immune system checkpoint.

[0082] The mRNA or mRNA cancer vaccine can be administered by any route, for example, intranasally, intravenously (IV), intradermally, intramuscularly (IM) or intraperitoneally. In some embodiments, the administration is a single dose. IV and IM are preferred. In some embodiments, the vaccine is administered to the subject multiple times.

[0083] The mRNA cancer vaccine can be used as a therapeutic or preventive agent. The effective amount of the polynucleotide of the mRNA cancer vaccine of the present invention that is administered to a cell, tissue or subject can be sufficient for immune activation, particularly antigen-specific immune activation. A prophylactically effective amount can be a therapeutically effective amount that prevents the progression of cancer at a clinically acceptable level. Administration can be a single dose, or the vaccine can be administered to a subject multiple times.

[0084] Compositions and methods are provided for the treatment or prevention of a disease or condition in humans and other mammals. The active therapeutic agent includes an mRNA cancer vaccine, a cell containing the mRNA cancer vaccine, or a polypeptide translated from a polynucleotide contained in the mRNA cancer vaccine.

[0085] The mRNA cancer vaccine can induce translation of a polypeptide (e.g., antigen or immunogen) in a cell, tissue, or organism. Such translation can occur in vivo, ex vivo, in culture, or in vitro. The cell, tissue, or organism is contacted with an effective amount of a composition containing an mRNA cancer vaccine that contains a polynucleotide having at least one translatable region that encodes an immunogenic peptide fragment as described herein. An "effective amount" of an mRNA cancer vaccine is provided based, at least in part, on the target tissue, the target cell type, the means of administration, the physical characteristics of the polynucleotide (e.g., the size and extent of modified nucleosides) and other components of the mRNA cancer vaccine as well as other determinants. In general, an effective amount of an mRNA cancer vaccine composition will result in an induced or enhanced immune response depending on the production of the encoded immunogenic peptide fragment in the cell, and is preferably more efficient than a composition containing a corresponding unmodified polynucleotide that encodes the same antigen or peptide antigen. Increased production may be demonstrated by an increase in cell transfection (i.e., the percentage of cells transfected with the mRNA cancer vaccine), an increase in protein translation from the polynucleotide, a decrease in nucleic acid degradation (e.g., as demonstrated by an increase in the duration of protein translation from a modified polynucleotide), or a change in the antigen-specific immune response of the host cell.

[0086] The method may also include the administration of one or more other therapeutic substances. The other substances may preferably be "immunomodulators", which means any substance that, when administered to a subject, blocks or inhibits the action of immune system checkpoints, thereby leading to the upregulation of immune effector responses in the subject, typically T cell effector responses, which preferably include anti-tumor T cell effector responses. The immunomodulators used in the method of the present invention may block or inhibit any of the immune system checkpoints. The substance may be an antibody or any other suitable substance that leads to the blocking or inhibition. Thus, the substance may generally be referred to as an inhibitor of the checkpoints.

[0087] As used herein, "antibody" includes whole antibodies and any antigen-binding fragment (i.e., "antigen-binding portion") or single chains thereof. Antibodies can be polyclonal or monoclonal and can be produced by any suitable method. Examples of binding fragments included in the term "antigen-binding portion" of an antibody include Fab fragments, F(ab')2 fragments, Fab' fragments, Fd fragments, Fv fragments, dAb fragments and isolated complementarity determining regions (CDRs). Single chain antibodies, such as scFv, and heavy chain antibodies, such as VHH and camelid antibodies, are also intended to be included in the term "antigen-binding portion" of an antibody.

[0088] Preferred antibodies that block or inhibit the interaction of CTLA-4 with B7 protein include ipilumumab, tremelimumab, or any of the antibodies disclosed in WO2014 / 207063. Other molecules include polypeptides or soluble mutant CD86 polypeptides.

[0089] Preferred antibodies that block or inhibit the interaction between PD1 and PD-L1 include nivolumab, pembrolizumab, lambrolizumab, pidiluzumab and AMP-224. Anti-PD-L1 antibodies include MEDI-4736 and MPDL3280A. Pembrolizumab and nivolumab are particularly preferred.

[0090] Other suitable inhibitors include small molecule inhibitors (SMIs), which are typically small organic molecules.Preferred inhibitors of IDO1 include epacadostat (INCB24360), indoximod, GDC-0919 (NLG919) and F001287.Other inhibitors of IDO1 include 1-methyltryptophan (1MT).

[0091] Immunomodulatory agents such as antibodies or SMIs can be formulated with pharma- ceutically acceptable excipients or other auxiliary substances for administration to a subject. Suitable excipients and auxiliary substances are known in the art. Suitable forms for manufacturing, packaging and selling immunotherapeutic compositions are also known in the art.

[0092] mRNA cancer vaccine and other therapeutic substances can be administered simultaneously or sequentially.When the other therapeutic substances are administered simultaneously, they can be administered in the same or different formulations, but are administered simultaneously.When the administration of the other therapeutic substances and the mRNA cancer vaccine is separated in time, the other therapeutic substances are administered sequentially with each other and with the mRNA cancer vaccine.The time interval between the administration of these compounds can be several minutes, or it can be longer, for example, several hours, several days, several weeks, several months.

[0093] The present invention provides a method comprising administering an mRNA cancer vaccine according to the present invention to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The compositions of the present invention are typically formulated in dosage unit form for ease of administration and uniformity of dosage. However, it is understood that the total daily usage of the compositions of the present invention can be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level, prophylactically effective dose level or suitable imaging dose level for any particular patient will depend on a variety of factors, including the severity of the disorder and disease being treated; the activity of the specific compound used; the specific composition used; the age, weight, general health, sex and diet of the patient; the administration time, route of administration and excretion rate of the specific compound used; the duration of treatment; drugs used in combination or simultaneously with the specific compound used; and similar factors well known in the art.

[0094] The composition according to the present invention may be administered at a daily dose of about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 0.05 mg / kg, about 0.005 mg / kg to about 0.05 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg in order to obtain the desired therapeutic, diagnostic, prophylactic or imaging effect. The compound may be administered one or more times daily at a dosage level sufficient to deliver 10 mg / kg (where kg is the unit of body weight of the subject) (see, e.g., the unit dosage ranges set forth in International Publication No. WO2013078199, which is incorporated herein by reference in its entirety).

[0095] The desired dosage can be delivered three times a day, twice a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage can be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations). When multiple administrations are used, a split-dosing regimen can be used, for example, as described herein. The mRNA cancer vaccine pharmaceutical composition described herein can be formulated into dosage forms described herein, for example, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous) dosage forms.

[0096] The RNA (e.g., mRNA) vaccine composition may be administered at a dose of about 0.0001 mg / kg to about 100 mg / kg, about 0.001 mg / kg to about 0.05 mg / kg, about 0.005 mg / kg to about 0.05 mg / kg, about 0.001 mg / kg to about 0.005 mg / kg, about 0.05 mg / kg to about 0.5 mg / kg, about 0.01 mg / kg to about 50 mg / kg, about 0.1 mg / kg to about 40 mg / kg, about 0.5 mg / kg to about 30 mg / kg, about 0.01 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 10 mg / kg, or about 1 mg / kg to about 25 mg / kg per day to achieve the desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be administered once or more per day, weekly, monthly, etc., at dosage levels sufficient to deliver 10 mg / kg (where kg is a unit of subject body weight) (see, e.g., the unit dosage ranges set forth in International Publication No. WO2013078199, which is incorporated herein by reference in its entirety). The desired dosage may be delivered three times per day, twice per day, once per day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every two months, every three months, every six months, etc. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more administrations).

[0097] When multiple doses are used, split dosing regimens, e.g., as described herein, may be used. In some embodiments, the RNA vaccine composition may be administered at a dosage level sufficient to deliver 0.0005 mg / kg to 0.01 mg / kg, e.g., about 0.0005 mg / kg to about 0.0075 mg / kg, e.g., about 0.0005 mg / kg, about 0.001 mg / kg, about 0.002 mg / kg, about 0.003 mg / kg, about 0.004 mg / kg, or about 0.005 mg / kg. In some embodiments, the RNA vaccine composition may be administered once or twice (or more) at a dosage level sufficient to deliver 0.025 mg / kg to 0.250 mg / kg, 0.025 mg / kg to 0.500 mg / kg, 0.025 mg / kg to 0.750 mg / kg, or 0.025 mg / kg to 1.0 mg / kg.

[0098] The RNA vaccine composition may be used in the following dosage forms: 0.0100 mg, 0.025 mg, 0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg, 0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg, 0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg, 0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg, 0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, The compound may be administered twice (e.g., on days 0 and 7, 0 and 14, 0 and 21, 0 and 28, 0 and 60, 0 and 90, 0 and 120, 0 and 150, 0 and 180, 0 and 3 months, 0 and 6 months, 0 and 9 months, 0 and 12 months, 0 and 18 months, 0 and 2 years, 0 and 5 years, or 0 and 10 years) at a dosage level or total dose sufficient to deliver a total dose of 0.850 mg, 0.875 mg, 0.900 mg, 0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher or lower dosages and administration frequencies are also encompassed by the present disclosure. For example, the RNA vaccine composition can be administered three or four times.

[0099] The RNA vaccine composition may be administered twice (e.g., on days 0 and 7, 0 and 14, 0 and 21, 0 and 28, 0 and 60, 0 and 90, 0 and 120, 0 and 150, 0 and 180, 0 and 3 months later, 0 and 6 months later, 0 and 9 months later, 0 and 12 months later, 0 and 18 months later, 0 and 2 years later, 0 and 5 years later, or 0 and 10 years later) at a dosage level or total dose sufficient to deliver a total dose of 0.010 mg, 0.025 mg, 0.100 mg, or 0.400 mg.

[0100] An mRNA vaccine for use in a method of vaccinating a subject may be administered to a subject as a single dose of 10 μg / kg to 400 μg / kg of an effective amount of the nucleic acid vaccine to vaccinate the subject.An RNA vaccine for use in a method of vaccinating a subject may be administered to a subject as a single dose of 10 μg to 400 μg of an effective amount of the nucleic acid vaccine to vaccinate the subject.

[0101] Composition, Formulation, Encapsulation Also disclosed are pharmaceutical compositions comprising the mRNA cancer vaccines and the mRNA cancer vaccine compositions and / or complexes, optionally in combination with one or more pharma- ceutically acceptable excipients. Vaccines can be formulated or administered alone or in combination with one or more other components. For example, vaccine compositions of the present invention can include other components, including, but not limited to, adjuvants. Optionally, vaccines do not contain adjuvants.

[0102] The pharmaceutical composition may optionally contain one or more additional active substances, such as therapeutically and / or prophylactically effective substances. The pharmaceutical composition of the present invention may be sterile and / or pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical products are described, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005, which is incorporated herein by reference in its entirety. The composition may typically be administered to a human, human patient, or subject. For the purposes of this disclosure, the term "active ingredient" generally refers to the mRNA cancer vaccine or the polynucleotide contained therein, such as a polynucleotide encoding an immunogenic peptide fragment, such as an mRNA, delivered as described herein.

[0103] The formulations of the pharmaceutical compositions described herein may be prepared by any method known in the art or hereafter developed. In general, such preparation methods include combining the active ingredient with an excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, dividing, shaping and / or packaging the product into the desired single-dose or multi-dose units. The relative amounts of the active ingredient, pharma- ceutically acceptable excipient and / or any additional ingredients in a pharmaceutical composition according to the invention will vary depending on the identity, size and / or condition of the subject being treated, as well as the route by which the composition is administered. For example, the composition may contain 0.1% to 100%, e.g., 0.5% to 50%, 1 to 30%, 5 to 80%, at least 80% (w / w) of the active ingredient.

[0104] The mRNA cancer vaccine may be formulated using one or more excipients to (1) enhance stability, (2) increase cell transfection, (3) enable sustained or delayed release (e.g., from a depot formulation), (4) modify biodistribution (e.g., to target specific tissues or cell types), (5) enhance translation of the encoded protein in vivo, and / or (6) modify the release profile of the encoded protein (antigen) in vivo. In addition to conventional excipients, such as any solvent, dispersion medium, diluent or other liquid vehicle, dispersion or suspension aid, surfactant, isotonicity agent, thickener or emulsifier, preservative, the excipients of the present invention include, but are not limited to, lipid-like substances, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with mRNA cancer vaccines (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof.

[0105] The mRNA and / or composition disclosed herein may include stabilizing elements. Naturally occurring eukaryotic mRNA molecules have been found to contain stabilizing elements, including but not limited to 5' and 3' UTRs, 5' caps and 3' tails, as described elsewhere herein. Other stabilizing elements that may be included in the mRNA disclosed herein may include, for example, histone stem loops. In some embodiments, the histone stem loops are generally derived from histone genes. The histone stem loops include intramolecular base pairing of two adjacent partially or completely reverse-complementary sequences separated by a spacer of short sequences, forming a loop of the structure. One or more AU-rich sequences may be removed from the mRNA. Such sequences may cause destabilization. The RNA vaccine may or may not contain enhancer and / or promoter sequences, which may be modified or unmodified, or activated or inactivated.

[0106] In a preferred embodiment, the mRNA cancer vaccine can be formulated in a lipid polycation complex. The formation of the lipid polycation complex can be accomplished by methods known in the art and / or as described in US Patent Application Publication No. 20120178702, which is incorporated herein by reference in its entirety. As a non-limiting example, the polycation can include cationic peptides or polypeptides, such as, but not limited to, polylysine, polyornithine and / or polyarginine, and cationic peptides described in International Publication No. WO2012013326 or US Patent Application Publication No. US20130142818, each of which is incorporated herein by reference in its entirety. In another embodiment, the mRNA cancer vaccine can be formulated in a lipid polycation complex, which can further include a non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE). Liposome formulation can be influenced by cationic lipid component selection, cationic lipid saturation, PEGylation nature, total component ratio and biophysical parameters such as, but not limited to, size.In one example by Semple et al. (Semple et al., Nature Biotech. 2010 28: 172-176; incorporated herein by reference in its entirety), liposome formulation was composed of 57.1% cationic lipid, 7.1% dipalmitoyl phosphatidylcholine, 34.3% cholesterol and 1.4% PEG-c-DMA.In another example, modifying the composition of cationic lipid can more effectively deliver siRNA to various antigen-presenting cells (Basha et al., Mol Ther. 2011 19:2186-2200; incorporated herein by reference in its entirety).

[0107] The liposome formulation may comprise about 35 to about 45% cationic lipid, about 40 to about 50% cationic lipid, about 50 to about 60% cationic lipid, and / or about 55 to about 65% cationic lipid. In some embodiments, the lipid to mRNA ratio in the liposome may be about 5:1 to about 20:1, about 10:1 to about 25:1, about 15:1 to about 30:1, and / or at least 30:1. The ratio of PEG in a lipid nanoparticle (LNP) formulation may be increased or decreased, and / or the carbon chain length of the PEG lipid may be modified from C14 to C18 to modify the pharmacokinetics and / or in vivo distribution of the LNP formulation. As a non-limiting example, the LNP formulation may contain a lipid molar ratio of about 0.5% to about 3.0%, about 1.0% to about 3.5%, about 1.5% to about 4.0%, about 2.0% to about 4.5%, about 2.5% to about 5.0%, and / or about 3.0% to about 6.0% PEG-DOMG (R-3-[(co-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) relative to the cationic lipid, DSPC, and cholesterol. In another embodiment, PEG-c-DOMG may be replaced with a PEG lipid, such as, but not limited to, PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-dimyristoyl-sn-glycerol) and / or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

[0108] The mRNA cancer vaccine formulation comprising the polynucleotide may be a nanoparticle that may include at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipid, and aminoalcohol lipid. In another embodiment, the lipid may be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol lipid. The aminoalcohol cationic lipid may be a lipid described in U.S. Patent Application Publication No. US20130150625 (incorporated herein by reference in its entirety) and / or a lipid produced by the method described therein. As non-limiting examples, cationic lipids include 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 in US20130150625); 2-amino-3-[ (9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 4 in US20130150625); or any pharma- ceutically acceptable salt or stereoisomer thereof.

[0109] Lipid nanoparticle formulations typically contain lipids, in particular ionizable cationic lipids such as 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) or di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioic acid (L319), as well as neutral lipids, sterols, and molecules that may reduce particle aggregation, such as PEG or PEG-modified lipids. The lipid nanoparticle formulation can consist essentially of (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a sterol, e.g., cholesterol, and (iv) a PEG lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid. The formulation may comprise a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319) in an amount of about 25% to about 75% on a molar basis, for example, about 35% to about 65%, about 45% to about 65%, about 60%, about 57.5%, about 50%, or about 40% on a molar basis.

[0110] The formulation may contain about 0.5% to about 15% on a molar basis, for example, about 3 to about 12%, about 5 to about 10% or about 15%, about 10% or about 7.5% neutral lipid on a molar basis. Exemplary neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE and SM. The formulation may contain about 5% to about 50% on a molar basis (e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about 38.5%, about 35% or about 31%) of sterol. An exemplary sterol is cholesterol. The formulation may contain about 0.5% to about 20% on a molar basis (e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about 0.5%, about 1.5%, about 3.5% or about 5%) of PEG or PEG-modified lipid. The PEG or PEG-modified lipid may comprise a PEG molecule with an average molecular weight of 2,000 Da, or less than 2,000, such as about 1,500 Da, about 1,000 Da, or about 500 Da. Exemplary PEG-modified lipids include, but are not limited to, PEG-distearoylglycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG), PEG-cDMA (discussed in more detail in Reyes et al., J. Controlled Release, 107, 276-287 (2005), the contents of which are incorporated herein by reference in their entirety).

[0111] The formulations herein may comprise 25-75% cationic lipid selected from 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and Di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% neutral lipid, 5-50% sterol and 0.5-20% PEG or PEG-modified lipid (where % is on a molar basis). In one embodiment, the formulation of the invention comprises 35-65% cationic lipid selected from 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and Di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% neutral lipid, 15-45% sterol and 0.5-10% PEG or PEG-modified lipid (where % is on a molar basis). The formulations herein may comprise 45-65% cationic lipid selected from 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and Di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% neutral lipid, 25-40% sterol and 0.5-10% PEG or PEG-modified lipid (where % is on a molar basis).The formulations herein may comprise about 60% cationic lipid selected from 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and Di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5% neutral lipid, about 31% sterol and about 1.5% PEG or PEG-modified lipid (where % is on a molar basis).

[0112] The formulations herein may comprise about 50% cationic lipid selected from 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and Di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% neutral lipid, about 38.5% sterol and about 1.5% PEG or PEG-modified lipid (where % is on a molar basis).

[0113] The formulations herein may comprise about 50% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% neutral lipid, about 35% sterol, about 4.5% or about 5% PEG or PEG-modified lipid and about 0.5% targeting lipid (where % is on a molar basis).

[0114] The formulations herein may comprise about 40% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% neutral lipid, about 40% sterol and about 5% PEG or PEG-modified lipid (where % is on a molar basis). In one embodiment, the formulation comprises about 57.2% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.1% neutral lipid, about 34.3% sterol and about 1.4% PEG or PEG-modified lipid.

[0115] The formulations herein may comprise about 57.5% cationic lipid selected from a PEG lipid, which is PEG-cDMA (PEG-cDMA is discussed in further detail in Reyes et al., J. Controlled Release, 107, 276-287 (2005), the contents of which are incorporated herein by reference in their entirety), about 7.5% neutral lipid, about 31.5% sterol, and about 3.5% PEG or PEG-modified lipid (wherein the % are on a molar basis).

[0116] In a preferred embodiment, the lipid nanoparticle formulation consists essentially of a lipid mixture comprising, in molar ratios, about 20-70% cationic lipid, 5-45% neutral lipid, 20-55% cholesterol, and 0.5-15% PEG-modified lipid, more preferably, in molar ratios, about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% cholesterol, and 0.5-15% PEG-modified lipid. In certain embodiments, the molar ratio of lipids is about 50 / 10 / 38.5 / 1.5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG, PEG-DSG, or PEG-DPG), 57.2 / 7.1134.3 / 1.4 (cationic lipid / neutral lipid, e.g., DPPC / Chol / PEG-modified lipid, e.g., PEG-cDMA), 40 / 15 / 40 / 5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG), 50 / 10 / 35 / 4.5 / 0.5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., ...0 / 10 / 35 / 4.5 / 0.5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DPG), 50 / 10 / 35 / 4.5 / 0.5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g. % of SG), 50 / 10 / 35 / 5 (molar % of cationic lipid / neutral lipid, such as DSPC / Chol / PEG-modified lipid, such as PEG-DMG), 40 / 10 / 40 / 10 (molar % of cationic lipid / neutral lipid, such as DSPC / Chol / PEG-modified lipid, such as PEG-DMG or PEG-cDMA), 35 / 15 / 40 / 10 (molar % of cationic lipid / neutral lipid, such as DSPC / Chol / PEG-modified lipid, such as PEG-DMG or PEG-cDMA) or 52 / 13 / 30 / 5 (molar % of cationic lipid / neutral lipid, such as DSPC / Chol / PEG-modified lipid, such as PEG-DMG or PEG-cDMA).

[0117] Exemplary lipid nanoparticle compositions and methods for their production are described, for example, in Semple et al., (2010) Nat. Biotechnol. 28: 172-176; Jayarama et al., (2012), Angew. Chem. Int. Ed., 51: 8529-8533; and Maier et al., (2013) Molecular Therapy 21, 1570-1578, the contents of each of which are incorporated by reference herein in their entirety.

[0118] The lipid nanoparticle formulations described herein can include cationic lipids, PEG lipids, and structural lipids, and can optionally include non-cationic lipids. As a non-limiting example, the lipid nanoparticles can include about 40-60% cationic lipids, about 5-15% non-cationic lipids, about 1-2% PEG lipids, and about 30-50% structural lipids. As another non-limiting example, the lipid nanoparticles can include about 50% cationic lipids, about 10% non-cationic lipids, about 1.5% PEG lipids, and about 38.5% structural lipids. As yet another non-limiting example, the lipid nanoparticles can include about 55% cationic lipids, about 10% non-cationic lipids, about 2.5% PEG lipids, and about 32.5% structural lipids. In one embodiment, the cationic lipid can be any cationic lipid described herein, including, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA, and L319.

[0119] The lipid nanoparticle formulations described herein can be four-component lipid nanoparticles. The lipid nanoparticles can include cationic lipids, non-cationic lipids, PEG lipids, and structured lipids. As a non-limiting example, the lipid nanoparticles can include about 40-60% cationic lipids, about 5-15% non-cationic lipids, about 1-2% PEG lipids, and about 30-50% structured lipids. As another non-limiting example, the lipid nanoparticles can include about 50% cationic lipids, about 10% non-cationic lipids, about 1.5% PEG lipids, and about 38.5% structured lipids. As yet another non-limiting example, the lipid nanoparticles can include about 55% cationic lipids, about 10% non-cationic lipids, about 2.5% PEG lipids, and about 32.5% structured lipids. In one embodiment, the cationic lipid can be any cationic lipid described herein, such as, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA, and L319.

[0120] The lipid nanoparticle formulations described herein may include cationic lipids, non-cationic lipids, PEG lipids, and structured lipids. As a non-limiting example, the lipid nanoparticles include about 50% cationic lipid DLin-KC2-DMA, about 10% non-cationic lipid DSPC, about 1.5% PEG lipid PEG-DOMG, and about 38.5% structured lipid cholesterol. As a non-limiting example, the lipid nanoparticles include about 50% cationic lipid DLin-MC3-DMA, about 10% non-cationic lipid DSPC, about 1.5% PEG lipid PEG-DOMG, and about 38.5% structured lipid cholesterol. As a non-limiting example, the lipid nanoparticles include about 50% cationic lipid DLin-MC3-DMA, about 10% non-cationic lipid DSPC, about 1.5% PEG lipid PEG-DMG, and about 38.5% structured lipid cholesterol. As yet another non-limiting example, the lipid nanoparticles comprise about 55% cationic lipid L319, about 10% non-cationic lipid DSPC, about 2.5% PEG lipid PEG-DMG, and about 32.5% structural lipid cholesterol.

[0121] The relative amounts of active ingredient, pharma- ceutically acceptable excipients and / or additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size and / or condition of the subject being treated, and also depending on the route by which the composition is administered. For example, the composition may contain 0.1%-99% (w / w) active ingredient. For example, the composition may contain 0.1%-100%, e.g., 0.5%-50%, 1%-30%, 5%-80%, at least 80% (w / w) active ingredient.

[0122] The mRNA cancer vaccine composition may comprise the polynucleotide described herein, formulated in lipid nanoparticles comprising MC3, cholesterol, DSPC and PEG2000-DMG, trisodium citrate buffer, sucrose and water for injection.As a non-limiting example, the composition comprises 2.0 mg / mL drug (e.g., polynucleotide encoding H10N8 influenza virus), 21.8 mg / mL MC3, 10.1 mg / mL cholesterol, 5.4 mg / mL DSPC, 2.7 mg / mL PEG2000-DMG, 5.16 mg / mL trisodium citrate, 71 mg / mL sucrose and about 1.0 mL water for injection.

[0123] RNA vaccines may be formulated using one or more liposomes, lipoplexes or lipid nanoparticles. Pharmaceutical compositions of RNA vaccines may include liposomes. Liposomes are artificially prepared vesicles, which are composed primarily of lipid bilayers and may be used as delivery vehicles for the administration of nutrients and pharmaceutical formulations. Liposomes may be of various sizes, including but not limited to, multilamellar vesicles (MLVs), which may have diameters of hundreds of nanometers and may contain a series of concentric bilayers separated by narrow aqueous compartments, small single-cell vesicles (SUVs), which may have diameters of less than 50 nm, and large single-cell vesicles (LUVs), which may have diameters of 50-500 nm. Liposome designs may include, but are not limited to, opsonins or ligands to improve attachment of liposomes to non-healthy tissues or to activate events such as, but not limited to, endocytosis. Liposomes may contain low or high pH to improve delivery of pharmaceutical formulations. The formation of liposomes may depend on the physicochemical properties, including but not limited to, the encapsulated pharmaceutical agent and liposomal components, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the encapsulated substance and its potential toxicity, any additional processes involved during application and / or delivery of the vesicles, optimizing size, polydispersity and shelf life of the vesicles for the intended use, and the feasibility of large-scale production and batch-to-batch reproducibility of a safe and efficient liposomal product.

[0124] As non-limiting examples, liposomes such as synthetic membrane vesicles can be produced by the methods, devices, and apparatus described in U.S. Patent Application Publication Nos. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373, and US20130183372, the contents of each of which are incorporated by reference in their entirety. The pharmaceutical compositions described herein may include, but are not limited to, liposomes formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) and MC3 (US20100324120; incorporated herein by reference in its entirety), and liposomes capable of delivering small molecule drugs, such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA).

[0125] The pharmaceutical compositions described herein may include, but are not limited to, liposomes formed by synthesis of stabilized plasmid-lipid particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), which have been shown and described to be suitable for oligonucleotide delivery in vitro and in vivo (Wheeler et al., Gene Therapy. 1999 6:271-281; ​​Zhang et al., Gene Therapy. 1999 6: 1438-1447; Jeffs et al., Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2: 1002-1007; Zimmermann et al., Nature. 2006 441: 111-114; Heyes et al., J Contr Rel. 2005 107:276-287; Semple et al., Nature Biotech. 2010 28: 172-176; Judge et al., J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19: 125-132; US Patent Application Publication No. US20130122104; all of which are incorporated by reference in their entirety). The original manufacturing method by Wheeler et al. was the detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method. Liposomal formulations are composed of three to four lipid components in addition to the polynucleotide.

[0126] As an example, the liposomes may contain, but are not limited to, 55% cholesterol, 20% disteroylphosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) as described in Jeffs et al. As another example, a particular liposome formulation may contain, but is not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid may be, but is not limited to, 1,2-distearoyloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA) as described in Heyes et al.

[0127] The liposome formulation may contain about 25.0% to about 40.0% cholesterol, about 30.0% to about 45.0% cholesterol, about 35.0% to about 50.0% cholesterol, and / or about 48.5% to about 60% cholesterol. In a preferred embodiment, the formulation may contain a percentage of cholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0% and 43.5%. In some embodiments, the formulation may contain about 5.0% to about 10.0% DSPC and / or about 7.0% to about 15.0% DSPC.

[0128] The pharmaceutical composition may comprise a liposome that may be formed to deliver a polynucleotide that may code for at least one immunogen (antigen) or any other polypeptide of interest. The RNA vaccine may be encapsulated by the liposome, and / or it may be contained within an aqueous core, which may then be encapsulated by the liposome (see International Publication Nos. WO2012031046, WO2012031043, WO2012030901 and WO2012006378, and U.S. Patent Application Publication Nos. US20130189351, US20130195969 and US20130202684; the entire contents of each of which are incorporated herein by reference).

[0129] Liposomes can be formulated for targeted delivery. As a non-limiting example, liposomes can be formulated for targeted delivery to the liver. Liposomes used for targeted delivery can include, but are not limited to, the liposomes and liposome manufacturing methods described in US Patent Application Publication No. US20130195967, the contents of which are incorporated herein by reference in their entirety. In another embodiment, polynucleotides that can code for immunogens (antigens) can be formulated in cationic oil-in-water emulsions, where the emulsion particles comprise cationic lipids and oil cores that can interact with polynucleotides to immobilize the molecules in the emulsion particles (see International Publication No. WO2012006380, the entire contents of which are incorporated herein by reference).

[0130] RNA vaccines can also be formulated in water-in-oil emulsions that contain a continuous hydrophobic phase in which a hydrophilic phase is dispersed. As a non-limiting example, the emulsion can be produced by the method described in International Publication No. WO201087791, the contents of which are incorporated herein by reference in their entirety. The lipid formulation can include at least a cationic lipid, a lipid that can enhance transfection, and at least one lipid that contains a hydrophilic head group linked to the lipid moiety (International Publication No. WO2011076807 and US Patent Application Publication No. 20110200582; the contents of each of which are incorporated herein by reference in their entirety). In another embodiment, the polynucleotide encoding the immunogen can be formulated in lipid vesicles that can have crosslinks between functionalized lipid bilayers (see US Patent Application Publication No. 20120177724; the contents of which are incorporated herein by reference in their entirety).

[0131] Polynucleotides can be formulated in liposomes as described in International Patent Publication No. WO2013086526, the contents of which are incorporated herein by reference in their entirety. RNA vaccines can be encapsulated in liposomes using a reverse pH gradient and / or optimized internal buffer composition as described in International Patent Publication No. WO2013086526, the contents of which are incorporated herein by reference in their entirety.

[0132] RNA vaccine pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (e.g., for siRNA delivery to ovarian cancer (Landen et al., Cancer Biology & Therapy 2006 5(12)1708-1713, the contents of which are incorporated herein by reference in their entirety)), and hyaluronic acid-coated liposomes (Quiet Therapeutics, Israel).

[0133] The cationic lipid can be a low molecular weight cationic lipid, such as those described in U.S. Patent Application No. 20130090372, the contents of which are incorporated herein by reference in their entirety.

[0134] RNA vaccines can be formulated in lipid vesicles that can have crosslinks between functionalized lipid bilayers.

[0135] RNA vaccines can be formulated in liposomes that contain cationic lipids. As described in International Publication No. WO2013006825 (which is incorporated herein by reference in its entirety), liposomes can have a molar ratio (N:P ratio) of nitrogen atoms in cationic lipids to phosphates in RNA of 1:1 to 20:1. In another embodiment, liposomes can have an N:P ratio of greater than 20:1 or less than 1:1.

[0136] The RNA vaccine can be formulated in lipid-polycation complexes. The formation of lipid-polycation complexes can be achieved by methods known in the art and / or as described in US Patent Application Publication No. 20120178702, which is incorporated herein by reference in its entirety. By way of non-limiting example, polycations include, but are not limited to, cationic peptides or polypeptides, such as polylysine, polyornithine and / or polyarginine, and cationic peptides as described in International Publication No. WO2012013326 or US Patent Application Publication No. US20130142818, each of which is incorporated herein by reference in its entirety. In another embodiment, the RNA vaccine can be formulated in lipid-polycation complexes, which can further include non-cationic lipids, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0137] RNA vaccines can be formulated in aminoalcohol lipidoids. Aminoalcohol lipidoids that can be used in the present invention can be prepared by the methods described in U.S. Pat. No. 8,450,298, which is incorporated herein by reference in its entirety. The liposome formulation can be influenced by the choice of cationic lipid component, the degree of cationic lipid saturation, the nature of PEGylation, the ratio of all components, and biophysical parameters, including but not limited to size. In one example by Semple et al. (Semple et al., Nature Biotech. 2010 28: 172-176; incorporated herein by reference in its entirety), the liposome formulation consisted of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA. In another example, modification of the cationic lipid composition could more effectively deliver siRNA to various antigen-presenting cells (Basha et al., Mol Ther. 2011 19:2186-2200; incorporated herein by reference in its entirety). In some embodiments, the liposome formulation may contain about 35 to about 45% cationic lipid, about 40 to about 50% cationic lipid, about 50 to about 60% cationic lipid, and / or about 55 to about 65% cationic lipid. In some embodiments, the lipid to mRNA ratio in the liposome may be about 5:1 to about 20:1, about 10:1 to about 25:1, about 15:1 to about 30:1, and / or at least 30:1. In some embodiments, the ratio of PEG in a lipid nanoparticle (LNP) formulation can be increased or decreased and / or the carbon chain length of the PEG lipid can be modified from C14 to C18 to modify the pharmacokinetics and / or in vivo distribution of the LNP formulation.As a non-limiting example, the LNP formulation may contain a lipid molar ratio of about 0.5% to about 3.0%, about 1.0% to about 3.5%, about 1.5% to about 4.0%, about 2.0% to about 4.5%, about 2.5% to about 5.0%, and / or about 3.0% to about 6.0% PEG-DOMG (R-3-[(co-methoxy-poly(ethylene glycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) relative to the cationic lipid, DSPC, and cholesterol. In another embodiment, PEG-c-DOMG may be replaced with PEG lipids such as, but not limited to, PEG-DSG (1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-dimyristoyl-sn-glycerol) and / or PEG-DPG (1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art, such as, but not limited to, DLin-MC3-DMA, D Lin-DMA, C 12-200 and DLin-KC2-DMA.

[0138] RNA vaccines can be formulated in lipid nanoparticles, such as those described in International Publication No. WO2012170930, which is incorporated by reference in its entirety.

[0139] The mRNA vaccine formulation comprising the polynucleotide may be a nanoparticle that may include at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipid, and aminoalcohol lipid. In another embodiment, the lipid may be a cationic lipid, such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and aminoalcohol lipid. The aminoalcohol cationic lipid may be a lipid described in U.S. Patent Application Publication No. US20130150625 (incorporated herein by reference in its entirety) and / or a lipid produced by the method described therein. As non-limiting examples, cationic lipids include 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 in US20130150625); 2-amino-3-[ (9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 4 in US20130150625); or any pharma- ceutically acceptable salt or stereoisomer thereof.

[0140] Lipid nanoparticle formulations typically contain lipids, in particular ionizable cationic lipids such as 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) or di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioic acid (L319), as well as neutral lipids, sterols, and molecules that may reduce particle aggregation, such as PEG or PEG-modified lipids.

[0141] The lipid nanoparticle formulation can consist essentially of (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a sterol, e.g., cholesterol, and (iv) a PEG lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% sterol, and 0.5-15% PEG lipid. In one embodiment, the formulation may comprise a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319) at about 25% to about 75% on a molar basis, for example, about 35% to about 65%, about 45% to about 65%, about 60%, about 57.5%, about 50% or about 40% on a molar basis.

[0142] In one embodiment, the formulation comprises about 0.5% to about 15% on a molar basis, for example, about 3 to about 12%, about 5 to about 10% or about 15%, about 10% or about 7.5% neutral lipid on a molar basis. Exemplary neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE and SM. In one embodiment, the formulation comprises about 5% to about 50% on a molar basis (e.g., about 15 to about 45%, about 20 to about 40%, about 40%, about 38.5%, about 35% or about 31%) of sterol. An exemplary sterol is cholesterol. In one embodiment, the formulation comprises about 0.5% to about 20% on a molar basis (e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about 0.5%, about 1.5%, about 3.5% or about 5% on a molar basis) of PEG or PEG-modified lipid. In one embodiment, the PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of 2,000 Da. In other embodiments, the PEG or PEG-modified lipid comprises PEG molecules with an average molecular weight of less than 2,000, e.g., about 1,500 Da, about 1,000 Da or about 500 Da. Exemplary PEG-modified lipids include, but are not limited to, PEG-distearoylglycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG), PEG-cDMA (discussed in further detail in Reyes et al., J. Controlled Release, 107, 276-287 (2005), the contents of which are incorporated herein by reference in their entirety).

[0143] In one embodiment, the formulation comprises 25-75% cationic lipid selected from 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and Di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 0.5-15% neutral lipid, 5-50% sterol and 0.5-20% PEG or PEG-modified lipid (where % is on a molar basis). In one embodiment, the formulation comprises 35-65% cationic lipid selected from 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and Di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 3-12% neutral lipid, 15-45% sterol and 0.5-10% PEG or PEG-modified lipid (where % is on a molar basis).

[0144] In one embodiment, the formulation may comprise 45-65% cationic lipid selected from 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), Dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and Di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), 5-10% neutral lipid, 25-40% sterol and 0.5-10% PEG or PEG-modified lipid (where % is on a molar basis).

[0145] In one embodiment, the formulation comprises about 60% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.5% neutral lipid, about 31% sterol and about 1.5% PEG or PEG-modified lipid (where % is on a molar basis).

[0146] In one embodiment, the formulation comprises about 50% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% neutral lipid, about 38.5% sterol and about 1.5% PEG or PEG-modified lipid (where % is on a molar basis).

[0147] In one embodiment, the formulation comprises about 50% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 10% neutral lipid, about 35% sterol, about 4.5% or about 5% PEG or PEG-modified lipid and about 0.5% targeting lipid (where % is on a molar basis).

[0148] In one embodiment, the formulation comprises about 40% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 15% neutral lipid, about 40% sterol and about 5% PEG or PEG-modified lipid (where % is on a molar basis).

[0149] In one embodiment, the formulation comprises about 57.2% cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA) and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), about 7.1% neutral lipid, about 34.3% sterol and about 1.4% PEG or PEG-modified lipid.

[0150] In one embodiment, the formulation comprises about 57.5% cationic lipid selected from PEG lipids, which are PEG-cDMA (PEG-cDMA is discussed in more detail in Reyes et al., J. Controlled Release, 107, 276-287 (2005), the entire contents of which are incorporated herein by reference), about 7.5% neutral lipid, about 31.5% sterol, and about 3.5% PEG or PEG-modified lipid (wherein % is on a molar basis). In a preferred embodiment, the lipid nanoparticle formulation consists essentially of a lipid mixture of about 20-70% cationic lipid, 5-45% neutral lipid, 20-55% cholesterol, and 0.5-15% PEG-modified lipid in molar ratio, more preferably about 20-60% cationic lipid, 5-25% neutral lipid, 25-55% cholesterol, and 0.5-15% PEG-modified lipid in molar ratio. In certain embodiments, the molar ratio of lipids is about 50 / 10 / 38.5 / 1.5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG, PEG-DSG, or PEG-DPG), 57.2 / 7.1134.3 / 1.4 (cationic lipid / neutral lipid, e.g., DPPC / Chol / PEG-modified lipid, e.g., PEG-cDMA), 40 / 15 / 40 / 5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DMG), 50 / 10 / 35 / 4.5 / 0.5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., ...0 / 10 / 35 / 4.5 / 0.5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g., PEG-DPG), 50 / 10 / 35 / 4.5 / 0.5 (cationic lipid / neutral lipid, e.g., DSPC / Chol / PEG-modified lipid, e.g. % of SG), 50 / 10 / 35 / 5 (molar % of cationic lipid / neutral lipid, such as DSPC / Chol / PEG-modified lipid, such as PEG-DMG), 40 / 10 / 40 / 10 (molar % of cationic lipid / neutral lipid, such as DSPC / Chol / PEG-modified lipid, such as PEG-DMG or PEG-cDMA), 35 / 15 / 40 / 10 (molar % of cationic lipid / neutral lipid, such as DSPC / Chol / PEG-modified lipid, such as PEG-DMG or PEG-cDMA) or 52 / 13 / 30 / 5 (molar % of cationic lipid / neutral lipid, such as DSPC / Chol / PEG-modified lipid, such as PEG-DMG or PEG-cDMA).

[0151] Exemplary lipid nanoparticle compositions and methods for their production are described, for example, in Semple et al., (2010) Nat. Biotechnol. 28: 172-176; Jayarama et al., (2012), Angew. Chem. Int. Ed., 51: 8529-8533; and Maier et al., (2013) Molecular Therapy 21, 1570-1578, the contents of each of which are incorporated by reference herein in their entirety.

[0152] In one embodiment, the lipid nanoparticle formulations described herein can include cationic lipids, PEG lipids, and structural lipids, and can optionally include non-cationic lipids. As a non-limiting example, the lipid nanoparticles can include about 40-60% cationic lipids, about 5-15% non-cationic lipids, about 1-2% PEG lipids, and about 30-50% structural lipids. As another non-limiting example, the lipid nanoparticles can include about 50% cationic lipids, about 10% non-cationic lipids, about 1.5% PEG lipids, and about 38.5% structural lipids. As yet another non-limiting example, the lipid nanoparticles can include about 55% cationic lipids, about 10% non-cationic lipids, about 2.5% PEG lipids, and about 32.5% structural lipids. In one embodiment, the cationic lipid can be any cationic lipid described herein, including, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA, and L319.

[0153] In one embodiment, the lipid nanoparticle formulations described herein can be four-component lipid nanoparticles. The lipid nanoparticles can include cationic lipids, non-cationic lipids, PEG lipids, and structured lipids. As a non-limiting example, the lipid nanoparticles can include about 40-60% cationic lipids, about 5-15% non-cationic lipids, about 1-2% PEG lipids, and about 30-50% structured lipids. As another non-limiting example, the lipid nanoparticles can include about 50% cationic lipids, about 10% non-cationic lipids, about 1.5% PEG lipids, and about 38.5% structured lipids. As yet another non-limiting example, the lipid nanoparticles can include about 55% cationic lipids, about 10% non-cationic lipids, about 2.5% PEG lipids, and about 32.5% structured lipids. In one embodiment, the cationic lipid can be any cationic lipid described herein, including, but not limited to, DLin-KC2-DMA, DLin-MC3-DMA, and L319.

[0154] In one embodiment, the lipid nanoparticle formulations described herein may include cationic lipids, non-cationic lipids, PEG lipids, and structured lipids. As a non-limiting example, the lipid nanoparticles include about 50% cationic lipid DLin-KC2-DMA, about 10% non-cationic lipid DSPC, about 1.5% PEG lipid PEG-DOMG, and about 38.5% structured lipid cholesterol. As a non-limiting example, the lipid nanoparticles include about 50% cationic lipid DLin-MC3-DMA, about 10% non-cationic lipid DSPC, about 1.5% PEG lipid PEG-DOMG, and about 38.5% structured lipid cholesterol. As a non-limiting example, the lipid nanoparticles include about 50% cationic lipid DLin-MC3-DMA, about 10% non-cationic lipid DSPC, about 1.5% PEG lipid PEG-DMG, and about 38.5% structured lipid cholesterol. As yet another non-limiting example, the lipid nanoparticles comprise about 55% cationic lipid L319, about 10% non-cationic lipid DSPC, about 2.5% PEG lipid PEG-DMG, and about 32.5% structural lipid cholesterol.

[0155] In one embodiment, the cationic lipids are those described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2008103276, WO2013086373 and WO2013086354; U.S. Pat. No. 7,893,302; The cationic lipids may be selected from, but are not limited to, those cationic lipids described in U.S. Patent Application Publication Nos. 7,404,969, 8,283,333 and 8,466,122, and U.S. Patent Application Publication Nos. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541 and US20130225836, the contents of each of which are incorporated by reference in their entirety. In another embodiment, the cationic lipid may be selected from, but is not limited to, Formula A as described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, and WO2013116126, or U.S. Patent Application Publication Nos. US20130178541 and US20130225836, the contents of each of which are incorporated by reference in their entirety. In yet another embodiment, the cationic lipid is of formula CLI-CLXXIX of International Publication No. WO2008103276, formula CLI-CLXXIX of U.S. Pat. No. 7,893,302, formula CLI-CLXXIX of U.S. Pat. No. 7,404,The compound may be selected from, but is not limited to, formulas CLI-CLXXXXII of U.S. Patent Application Publication No. 969, formulas I-VI of U.S. Patent Application Publication No. US20100036115, and formula I of U.S. Patent Application Publication No. US20130123338 (the entire contents of each of which publications are incorporated herein by reference). As non-limiting examples, the cationic lipid may be selected from the following: (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhenicosa- 12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-8-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z, 24Z)-N,N-Dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-Dimethylheptacosa-18-en-10-amine, (17Z)-N,N-Dimethylhexacosa-17-en-9-amine, (19Z,22Z)-N,N-Dimethyloctacosa-19,22-dien-7-amine, N,N-Dimethylheptacosa-10-amine, (20Z,23Z)-N-Ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(HZ,14Z)-1-nonylcosa-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-10-amine, (15Z)-N,N-dimethylheptacos-15-en-10-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethyltritriacont-22-en-10-amine, (16Z )-N,N-Dimethylpentacosa-16-en-8-amine, (12Z,15Z)-N,N-Dimethyl-2-nonylnicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-Dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-Dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecan-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-Dimethylnonadecan-10-amine, N,N-Dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-Dimethyl-21-[(1S ,2R)-2-octylcyclopropyl]henicosan-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecane-5-amine, N,N-dimethyl-3-{7 -[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy] 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)- 3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine )propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(l-methoyloctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2 R)-l-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-l-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine or a pharma-ceutically acceptable salt or stereoisomer thereof.

[0156] In one embodiment, the lipid may be a cleavable lipid, such as those described in International Publication No. WO2012170889, the entire contents of which are incorporated herein by reference. In another embodiment, the lipid may be a cationic lipid, such as, but not limited to, Formula (I) of U.S. Patent Application No. US20130064894, the entire contents of which are incorporated herein by reference.

[0157] Cationic lipids may be synthesized by methods known in the art and / or as described in International Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, WO2013086373 and WO2013086354, the contents of each of which are incorporated herein by reference in their entirety.

[0158] The cationic lipid can be a trialkyl cationic lipid. Non-limiting examples of trialkyl cationic lipids and methods of making and using trialkyl cationic lipids are described in International Patent Publication No. WO2013126803, the contents of which are incorporated herein by reference in their entirety.

[0159] The LNP formulation of the RNA vaccine may contain PEG-c-DOMG at a lipid molar ratio of 3%. In another embodiment, the LNP formulation of the RNA vaccine may contain PEG-c-DOMG at a lipid molar ratio of 1.5%. The pharmaceutical composition of the RNA vaccine may include at least one of the PEGylated lipids described in International Publication No. WO2012099755, the contents of which are incorporated herein by reference in their entirety.

[0160] The LNP formulation may contain PEG-DMG 2000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000). In one embodiment, the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art, and at least one other component. In another embodiment, the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art, DSPC, and cholesterol. As a non-limiting example, the LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC, and cholesterol. As another non-limiting example, the LNP formulation can contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40:10:48 (see, e.g., Geall et al., Nonviral delivery of self-amplifying RNA vaccines, PNAS 2012; PMID:22908294; incorporated herein by reference in its entirety).

[0161] The LNP formulation may be formulated by the methods described in International Publication Nos. WO2011127255 or WO2008103276, the contents of each of which are incorporated herein by reference in their entirety. As a non-limiting example, the RNA vaccines described herein may be encapsulated in an LNP formulation as described in WO2011127255 and / or WO2008103276, the contents of each of which are incorporated herein by reference in their entirety.

[0162] The RNA vaccines described herein may be formulated in nanoparticles delivered by parenteral routes as described in US Patent Application Publication No. US20120207845, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the RNA vaccines may be formulated in lipid nanoparticles produced by the methods described in US Patent Application Publication No. US20130156845 or International Publication No. WO2013093648 or WO2012024526, the contents of each of which are incorporated herein by reference in their entirety. The lipid nanoparticles described herein may be produced in a sterile environment by the systems and / or methods described in US Patent Application Publication No. US20130164400, the contents of which are incorporated herein by reference in their entirety.

[0163] The LNP formulations may be formulated in nanoparticles, such as the nucleic acid-lipid particles described in U.S. Patent No. 8,492,359, the entire contents of which are incorporated herein by reference. As a non-limiting example, the lipid particles may include one or more active or therapeutic agents, one or more cationic lipids that constitute about 50 mol % to about 85 mol % of the total lipids present in the particle, one or more non-cationic lipids that constitute about 13 mol % to about 49.5 mol % of the total lipids present in the particle, and one or more conjugated lipids that inhibit particle aggregation, that constitute about 0.5 mol % to about 2 mol % of the total lipids present in the particle. The nucleic acid in the nanoparticle may be a polynucleotide as described herein and / or known in the art.

[0164] The LNP formulation may be formulated by the methods described in International Publication No. WO2011127255 or WO2008103276, the contents of each of which are incorporated herein by reference in their entirety. As a non-limiting example, the modified RNA described herein may be encapsulated in an LNP formulation as described in WO2011127255 and / or WO2008103276, the contents of each of which are incorporated herein by reference in their entirety. The LNP formulation described herein may include a polycationic composition. As a non-limiting example, the polycationic composition may be selected from formula 1-60 of U.S. Patent Application Publication No. US20050222064, the contents of which are incorporated herein by reference in their entirety.

[0165] The LNP formulations comprising the polycationic composition can be used to deliver the modified RNA described herein in vivo and / or in vitro. In one embodiment, the LNP formulations described herein can further comprise a permeability enhancing molecule. Non-limiting permeability enhancing molecules are described in U.S. Patent Application Publication No. US20050222064, the entire contents of which are incorporated herein by reference.

[0166] The RNA vaccine pharmaceutical composition may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLES® (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al., Cancer Biology & Therapy 2006 5(12)1708-1713, the contents of which are incorporated herein by reference in their entirety)), and hyaluronic acid-coated liposomes (Quiet Therapeutics, Israel). In one embodiment, the RNA vaccine may be formulated in a lyophilized gel-phase liposome composition as described in U.S. Patent Application Publication No. US2012060293, the contents of which are incorporated herein by reference in their entirety.

[0167] The RNA vaccine may be conjugated to the following: cationic or polycationic compounds, such as protamine, nucleolin, spermine or spermidine, or other cationic peptides or proteins, such as poly-L-lysine (PLL), polyarginine, basic polypeptides, cell penetrating peptides (CPPs), such as HIV-binding peptides, HIV-1 Tat (HIV), Tat derived peptides, penetratin, VP22 derived or similar peptides, pestivirus Erns, HSV, VP22 (herpes simplex), MAP, KALA or protein transduction domains (PTD), PpT620, proline rich peptides, arginine rich peptides, lysine rich peptides, MPG-peptides, Pep-1, L-oligomers, calcitonin peptides, antennapedia derived peptides [especially from Drosophila antennapedia], pAntp, plsl, FGF, lactoferrin, transportan, buforin-2, Bac715-24, SynB, SynB(l), pVEC, hCT derived peptides, SAP, histones, cationic polysaccharides such as chitosan, polybrene, cationic polymers such as polyethyleneimine (PEI), cationic lipids such as DOTMA: [l-(2,3-siloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Choi, BGTC, CTAP, DOPC, DODAP, DOPE: dioleylphosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: dioctadecylamidoglycylspermine, DIMRI: dimyristoxypropyldimethylhydroxyethylammonium bromide, DOTAP: dioleoyloxy-3-(trimethylammonio)propane, DC-6-14: 0,0-ditetradecanoyl-N-alpha-trimethylammonioacetyl)diethanolamine chloride, CLIP 1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac-[2(2,3-Dihexadecyloxypropyloxymethyloxy)ethyl]-trimethylammonium, CLIP9: rac-[2(2,3-Dihexadecyloxypropyloxysuccinyloxy)ethyl]-trimethylammonium, Oligofectamine, or cationic or polycationic polymers such as modified polyamino acids, such as β-amino acid polymers or inverse polyamides, modified polyethylenes, such as PVP (poly(N-ethyl-4-vinylpyridinium bromide)), modified acrylates, such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), modified amidoamines, such as pAMAM (poly(amidoamine)), modified polybeta aminoesters (PBAE), such as diamine end-modified 1,4 butanediol diacrylate-co-5 -amino-1-pentanol polymers, dendrimers such as polypropylamine dendrimers or dendrimers based on pAMAM, polyimines such as PEL poly(ethyleneimine), poly(propyleneimine), polyallylamine, polymers based on sugar backbones such as cyclodextrin-based polymers, dextran-based polymers, chitosan, polymers based on silane backbones such as PMOXA-PDMS copolymers, block polymers consisting of a combination of one or more cationic blocks (e.g. selected from the cationic polymers mentioned above) and one or more hydrophilic or hydrophobic blocks (e.g. polyethylene glycol).

[0168] Alternatively, the RNA vaccine is not conjugated to a cationic or polycationic compound.

[0169] The nanoparticle formulation may comprise a phosphate conjugate. The phosphate conjugate can increase the in vivo circulation time and / or increase the targeted delivery of nanoparticles. The phosphate conjugate used in the present invention can be prepared by the method described in International Application No. WO2013033438 or US Patent Application Publication No. US20130196948 (each of which is incorporated herein by reference in its entirety). As a non-limiting example, the phosphate conjugate can comprise any one of the compounds of formula described in International Application No. WO2013033438 (each of which is incorporated herein by reference in its entirety).

[0170] The nanoparticle formulation may include a polymer conjugate. The polymer conjugate may be a water-soluble conjugate. The polymer conjugate may have a structure as described in US Patent Application Publication No. 20130059360, the entire contents of which are incorporated herein by reference. In one embodiment, the polymer conjugate with the polynucleotide of the present invention may be prepared using the method and / or segmented polymerization reagent as described in US Patent Application Publication No. 20130072709, the entire contents of which are incorporated herein by reference. In another embodiment, the polymer conjugate may have a pendant side group that includes a ring moiety, such as, but not limited to, the polymer conjugate described in US Patent Application Publication No. US20130196948, the entire contents of which are incorporated herein by reference.

[0171] Nanoparticle formulations can include conjugates that facilitate delivery of nanoparticles of the present invention in a subject. In addition, the conjugates can inhibit phagocytic clearance of nanoparticles in a subject. In one embodiment, the conjugates can be "self-peptides" designed from human membrane protein CD47 [e.g., "self-particles" as described in Rodriguez et al. (Science 2013 339, 971-975), which is incorporated herein by reference in its entirety]. As shown by Rodriguez et al., self-peptides delay macrophage-mediated clearance of nanoparticles, which facilitates delivery of nanoparticles. In another embodiment, the conjugate can be the membrane protein CD47 (see, e.g., Rodriguez et al., Science 2013 339, 971-975, which is incorporated herein by reference in its entirety). Similar to "self-peptides," Rodriguez et al. showed that CD47 can increase circulating particle rates in subjects compared to scrambled peptides and PEG-coated nanoparticles.

[0172] The RNA vaccine can be formulated in nanoparticles that contain a conjugate that facilitates delivery of the nanoparticles of the present invention in a subject. The conjugate can be CD47 membrane, or the conjugate can be derived from CD47 membrane protein, such as the "self-peptide" described above. In another embodiment, the nanoparticle can contain a conjugate of PEG and CD47 or a derivative thereof. In yet another embodiment, the nanoparticle can contain both the "self-peptide" described above and the membrane protein CD47. The "self-peptide" and / or CD47 protein can be conjugated to a virus-like particle or pseudovirus particle, as described herein for delivery of the RNA vaccine.

[0173] The formulation herein may comprise an RNA vaccine pharmaceutical composition comprising a polynucleotide of the present invention and a conjugate that may have degradable bonds.Non-limiting examples of conjugates include aromatic moieties that contain ionizable hydrogen atoms, spacer moieties and water-soluble polymers.Non-limiting examples include pharmaceutical compositions that comprise conjugates that have degradable bonds and delivery methods of such pharmaceutical compositions are described in US Patent Application Publication No. US20130184443 (the entire contents of which are incorporated herein by reference).

[0174] The nanoparticle formulation can be a carbohydrate nanoparticle comprising a carbohydrate carrier and an RNA vaccine.As a non-limiting example, the carbohydrate carrier can include, but is not limited to, anhydrous modified phytoglycogen or glycogen-type substances, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydrous modified phytoglycogen beta-dextrin (see, for example, International Publication No. WO2012109121; the entire contents of which are incorporated herein by reference).

[0175] Nanoparticle formulations can be coated with surfactants or polymers to improve particle delivery. In one embodiment, nanoparticles can be coated with hydrophilic coatings, including but not limited to PEG coatings, and / or coatings with neutral surface charges. Hydrophilic coatings can aid in the delivery of nanoparticles with larger payloads, such as RNA vaccines in the central nervous system. By way of non-limiting example, nanoparticles comprising hydrophilic coatings and methods for making such nanoparticles are described in U.S. Patent Application Publication No. US20130183244, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the lipid nanoparticles of the present invention can be hydrophilic polymer particles. Non-limiting examples of hydrophilic polymer particles and methods for making hydrophilic polymer particles are described in U.S. Patent Application Publication No. US20130210991, the contents of which are incorporated herein by reference in their entirety. In another embodiment, the lipid nanoparticles of the present invention can be hydrophobic polymer particles.

[0176] Lipid nanoparticle formulations can be improved by replacing cationic lipids with biodegradable cationic lipids known as rapidly eliminated lipid nanoparticles (reLNPs). Ionizable cationic lipids, such as but not limited to DLinDMA, DLin-KC2-DMA and DLin-MC3-DMA, have been shown to accumulate over time in plasma and tissues and can be a potential source of toxicity. Rapid metabolism of rapidly eliminated lipids can improve the tolerability and therapeutic index of lipid nanoparticles by an order of magnitude, from a 1 mg / kg dose to a 10 mg / kg dose in rats. The inclusion of an enzyme-degradable ester bond can improve the degradation and metabolic profile of the cationic component while still maintaining the activity of the reLNP formulation. The ester bond can be located within the lipid chain or it can be located at the end of the lipid chain. Any carbon in the lipid chain can be replaced by an internal ester bond. In one embodiment, the internal ester bond can be located on either side of a saturated carbon.

[0177] In one embodiment, an immune response can be elicited by delivering lipid nanoparticles that can include a nano species, a polymer, and an immunogen (US Patent Application Publication No. 20120189700 and International Publication No. WO2012099805; each of which is incorporated herein by reference in its entirety). The polymer can encapsulate or partially encapsulate the nano species. The immunogen can be a recombinant protein, modified RNA, and / or polynucleotide as described herein. In one embodiment, the lipid nanoparticles can be formulated for use in a vaccine, including but not limited to a vaccine against a pathogen. The lipid nanoparticles can be designed to modify the surface properties of the particle so that it can penetrate mucosal barriers. Mucus is present in mucosal tissues, including but not limited to the oral cavity (e.g., buccal and esophageal mucosa and tonsillar tissue), eye, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., membranes of nose, pharynx, trachea and bronchus), and reproductive (e.g., membranes of vagina, cervix and urethra). Nanoparticles larger than 10-200 nm, which are preferred due to their higher drug encapsulation efficiency and the fact that they can result in sustained delivery of a wide variety of drugs, are believed to be too large to rapidly diffuse into the mucosal barrier. Most of the trapped particles are removed from the mucosal tissue within seconds or hours, as mucus is continuously secreted, released, disposed of, or digested and recycled. Large polymeric nanoparticles (200 nm-500 nm in diameter) densely coated with low molecular weight polyethylene glycol (PEG) diffuse 4-6 times slower in mucosa than the same particles in water (Lai et al., PNAS 2007 104(5): 1482-487; Lai et al., Adv Drug Deliv Rev. 2009 61(2): 158-171; each of which is incorporated by reference in its entirety). Nanoparticle transport can be measured using permeation kinetics and / or fluorescence microscopy techniques, including but not limited to fluorescence recovery after photobleaching (FRAP) and high-resolution multiple particle tracking (MPT).As non-limiting examples, compositions capable of penetrating mucosal barriers can be prepared as described in U.S. Pat. No. 8,241,670 or International Patent Publication No. WO2013110028, the contents of each of which are incorporated herein by reference in their entirety.

[0178] Lipid nanoparticles designed to penetrate mucus may include polymeric materials (i.e., polymer cores) and / or polymer-vitamin conjugates and / or triblock copolymers. Polymeric materials include, but are not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyleneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric materials may be biodegradable and / or biocompatible. Non-limiting examples of biocompatible polymers are described in International Patent Publication No. WO2013116804, the contents of which are incorporated herein by reference in their entirety. The polymeric materials may further be irradiated. As a non-limiting example, the polymeric material may be gamma irradiated (see, e.g., International Application No. WO201282165, which is incorporated herein by reference in its entirety). Non-limiting examples of specific polymers include the following: poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), poly(L-lactic-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-P poly(EO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacrylates, polyurethanes, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethylene glycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG),Polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohol (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitrocellulose, hydroxypropyl cellulose, carboxymethyl cellulose, polymers of acrylic acid such as poly(methyl (meth)acrylate) (PMMA), poly(ethyl (meth)acrylate), poly(butyl (meth)acrylate), acrylate), poly(isobutyl (meth)acrylate), poly(hexyl (meth)acrylate), poly(isodecyl (meth)acrylate), poly(lauryl (meth)acrylate), poly(phenyl (meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamer, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate, polyvinylpyrrolidone. The lipid nanoparticles can be coated with copolymers, including but not limited to block copolymers (e.g., branched polyether-polyamide block copolymers described in International Publication No. WO2013012476, which is incorporated by reference in its entirety) and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymers (see, e.g., U.S. Patent Application Publication Nos. 20120121718 and 20100003337, and U.S. Patent No. 8,263,665; each of which is incorporated by reference in its entirety);or attached thereto. The copolymer can be a polymer that is generally regarded as safe (GRAS), and the formation of the lipid nanoparticles can be by a method such that no new chemicals are produced. For example, the lipid nanoparticles can include poloxamer-coated PLGA nanoparticles that can still rapidly penetrate human mucus without the production of new chemicals (Yang et al., Angew. Chem. Int. Ed. 2011 50:2597-2600; the entire contents of which are incorporated herein by reference).

[0179] A non-limiting scalable method for producing nanoparticles capable of penetrating human mucus is described in Xu et al. (see, e.g., J Control Release 2013, 170(2):279-86, the entire contents of which are incorporated herein by reference). The vitamin of the polymer-vitamin conjugate can be vitamin E. The vitamin portion of the conjugate can be replaced with other suitable moieties, such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, hydrophobic moieties or hydrophobic moieties of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains).

[0180] Lipid nanoparticles designed to penetrate mucus can include surface altering agents, such as, but not limited to, polynucleotides, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants, e.g., dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrins), nucleic acids, polymers (e.g., heparin, polyethylene glycol, and poloxamers), mucolytic agents (e.g., N-acetylcysteine, Artemisia anguicida, bromelain, papain, Clerodendrum, acetylcysteine, bromhexine, carbocysteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin beta 4 dornase alpha, neltenexin, erdosteine), and various DNases, e.g., rhDNase. The surface-altering agent may be embedded or entangled with the particle surface or disposed on the surface of the lipid nanoparticle (e.g., by coating, adsorption, covalent bonding or other process) (see, e.g., U.S. Patent Application Publication No. 20100215580 and U.S. Patent Application Publication Nos. 20080166414 and US20130164343; the contents of each of which are incorporated by reference in their entirety).

[0181] The mucus-penetrating lipid nanoparticles may comprise at least one polynucleotide as described herein. The polynucleotide may be encapsulated within the lipid nanoparticle and / or disposed on the surface of the particle. The polynucleotide may be covalently bound to the lipid nanoparticle. The mucus-penetrating lipid nanoparticle formulation may comprise a plurality of nanoparticles. Additionally, the formulation may contain particles that may interact with mucus and alter the structure and / or adhesive properties of the surrounding mucus, thereby reducing mucoadhesion and facilitating delivery of the mucus-penetrating lipid nanoparticles to mucosal tissues.

[0182] Mucus-penetrating lipid nanoparticles can be hypotonic formulations that include mucosal permeability-enhancing coatings. The formulation can be hypotonic to the epithelium to which it is delivered. Non-limiting examples of hypotonic formulations can be found in International Patent Publication No. WO2013110028, the contents of which are incorporated herein by reference in their entirety. To facilitate delivery through mucosal barriers, RNA vaccine formulations can include or be hypotonic solutions. It has been found that hypotonic solutions increase the rate at which mucus-inert particles, such as but not limited to mucus-penetrating particles, can reach the vaginal epithelium surface (see, for example, Ensign et al., Biomaterials 2013 34(28):6922-9, the contents of which are incorporated herein by reference in their entirety).In one embodiment, the RNA vaccine is delivered using lipoplexes, including but not limited to the ATUPLEX™ system, DACC system, DBTC system, and other siRNA-lipoplex technologies from Silence Therapeutics (London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, Mass.), and polyethylenimine (PEI) or protamine based targeted and non-targeted delivery of nucleic acids (Aleku et al., Cancer Res. 2008 68:9788-9798; Strumberg et al., Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 2006 13: 1222-1234; Santel et al., Gene Ther 2006 13: 1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al., Microvasc Res 2010 80:286-293 Weide et al., J Immunother. 2009 32:498-507; Weide et al., J Immunother. 2008 31: 180-188; Pascolo Expert Opin. Biol. Ther. 4: 1285-1294; Fotin-Mleczek et al., 2011 J. Immunother. 34: 1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl Acad Sci US A. 2007 6; 104:4095-4100; deFougerolles Hum Gene Ther. 2008 19: 125-132; the contents of each of which are incorporated herein by reference in their entireties.

[0183] Additionally, such formulations can be constructed or engineered to be passively or actively targeted in vivo to a variety of cell types, including, but not limited to, hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes (Akinc et al., Mol Ther. 2010 18: 1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge et al., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res 2010 80:286-293; Santel et al., Gene Ther 2006 13: 1222-1234; Santel et al., Gene Ther 2006 13: 1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske and Cullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18: 1127-1133; all of which are incorporated herein by reference in their entirety). One example of passive targeting of formulations to hepatocytes includes lipid nanoparticle formulations based on DLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA, which have been shown to bind to apolipoprotein E and promote the in vivo binding and uptake of these formulations into hepatocytes (Akinc et al., Mol Ther. 2010 18: 1357-1364; all of which are incorporated herein by reference in their entirety).The formulations can also be selectively targeted by expression of various ligands on their surface, exemplified by, but not limited to, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeting approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011 8: 197-206; Musacchio and Torchilin, Front Biosci. 2011 16: 1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25: 1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 27: 1-61). 5:309-319; Akinc et al., Mol Ther. 2010 18: 1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820: 105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68; Peer et al., Proc Natl Acad Sci US A. 2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 2011 18: 1127-1133; all of which are incorporated by reference in their entireties.

[0184] The RNA vaccine may be formulated as a solid lipid nanoparticle. The solid lipid nanoparticle (SLN) may be spherical with an average diameter of 10-1000 nm. The SLN has a solid lipid core matrix, which is capable of solubilizing lipophilic molecules and may be stabilized with surfactants and / or emulsifiers. In another embodiment, the lipid nanoparticle may be a self-assembled lipid-polymer nanoparticle (Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; the entire contents of which are incorporated herein by reference). As a non-limiting example, the SLN may be the SLN described in International Patent Publication No. WO2013105101, the entire contents of which are incorporated herein by reference). As another non-limiting example, the SLN is manufactured by the method or process described in International Patent Publication No. WO2013105101, the entire contents of which are incorporated herein by reference.

[0185] Liposomes, lipoplexes or lipid nanoparticles can be used to improve the efficacy of polynucleotide-based protein production, because these formulations can enhance cell transfection by RNA vaccines and / or enhance the translation of encoded proteins. One such example includes the use of lipid encapsulation to enable effective systemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720; incorporated herein by reference in its entirety). Liposomes, lipoplexes or lipid nanoparticles can also be used to enhance the stability of polynucleotides.

[0186] The RNA vaccine of the present invention can also be formulated for controlled release and / or targeted delivery. As used herein, "controlled release" refers to a release profile of a pharmaceutical composition or compound that is adapted to a specific release pattern that produces a therapeutic effect. In one embodiment, the RRNA vaccine can be encapsulated in a delivery agent described herein and / or known in the art for controlled release and / or targeted delivery. As used herein, the term "encapsulation" refers to enclosing, surrounding, or enveloping. With respect to the formulation of the compound of the present invention, the encapsulation can be substantial, complete, or partial. The term "substantially encapsulated" means that at least 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9, or more than 99.999% of the pharmaceutical composition or compound of the present invention can be enclosed, surrounded, or encapsulated in the delivery agent. By "partially encapsulated" is meant that less than 10, 10, 20, 30, 40, 50 percent of a pharmaceutical composition or compound of the present invention may be enclosed, surrounded or enveloped within the delivery agent.

[0187] Advantageously, encapsulation can be determined by measuring the leakage or activity of the pharmaceutical composition or compound of the present invention using fluorescence and / or electron microscopy.For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or more than 99.99% of the pharmaceutical composition or compound of the present invention is encapsulated in the delivery agent.The controlled release formulation includes, but is not limited to, triblock copolymers.As a non-limiting example, the formulation can include two different types of triblock copolymers (International Publication Nos. WO2012131104 and WO2012131106; the entire contents of each of which are incorporated herein by reference).

[0188] The RNA vaccine can be encapsulated in lipid nanoparticles or rapidly eliminated lipid nanoparticles, which can then be encapsulated in polymers, hydrogels, and / or surgical sealants described herein and / or known in the art. As non-limiting examples, the polymers, hydrogels, or surgical sealants can be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, FL), HYLENEX® (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELL® (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEAL® (Baxter International, Inc Deerfield, IL).

[0189] The lipid nanoparticles can be encapsulated within any polymer known in the art that can form a gel when injected into a subject. As another non-limiting example, the lipid nanoparticles can be encapsulated within a polymer matrix that can be biodegradable.

[0190] The RNA vaccine formulation for controlled release and / or targeted delivery may also include at least one controlled release coating. Controlled release coatings include, but are not limited to, OPADRY®, polyvinylpyrrolidone / vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS®, and cellulose derivatives such as ethyl cellulose aqueous dispersions (AQUACOAT® and SURELEASE®). In one embodiment, the RNA vaccine controlled release and / or targeted delivery formulation may include at least one degradable polyester that may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In another embodiment, the degradable polyester may include PEG conjugation to form a PEGylated polymer. RNA vaccine controlled release and / or targeted delivery formulations comprising at least one polynucleotide may contain at least one PEG and / or PEG-related polymer derivatives as described in U.S. Pat. No. 8,404,222, the entirety of which is incorporated herein by reference.

[0191] The RNA vaccine controlled release delivery formulation comprising at least one polynucleotide may be a controlled release polymer system as described in US20130130348, which is incorporated herein by reference in its entirety. The RNA vaccine of the present invention may be encapsulated in a therapeutic nanoparticle, referred to herein as a "therapeutic nanoparticle RRNA vaccine". The therapeutic nanoparticle may be formulated by methods described herein and known in the art, for example, but not limited to, the following publications: International Publication Nos. WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, U.S. Patent Application Publication Nos. US20110262491, US20100104645, US20100262491 ... Nos. 0100087337, US20100068285, US20110274759, US20100068286, US20120288541, US20130123351 and US20130230567, and U.S. Patent Nos. 8,206,747, 8,293,276, 8,318,208 and 8,318,211, the contents of each of which are incorporated herein by reference in their entirety. In another embodiment, the therapeutic polymeric nanoparticles may be identified by the methods described in U.S. Patent Application Publication No. 20120140790, the contents of which are incorporated herein by reference in their entirety.

[0192] Therapeutic nanoparticle RNA vaccines can be formulated for sustained release. "Sustained release" as used herein refers to a pharmaceutical composition or compound that is adapted for a release rate over a specific period of time. Periods include, but are not limited to, hours, days, weeks, months, and years. As a non-limiting example, sustained release nanoparticles can include a polymer and a therapeutic agent, such as, but not limited to, a polynucleotide of the present invention (see International Publication No. 2010075072 and U.S. Patent Application Publication Nos. US20100216804, US20110217377, and US20120201859; the contents of each of which are incorporated herein by reference in their entirety). In another non-limiting example, the sustained release formulation may include materials that allow sustained bioavailability, such as, but not limited to, crystals, polymeric gels, and / or particle suspensions (see U.S. Patent Application Publication No. US20130150295, the contents of which are incorporated herein by reference in their entirety). In one embodiment, the therapeutic nanoparticle RNA vaccine may be formulated to be target specific. As a non-limiting example, the therapeutic nanoparticle may include a corticosteroid (see International Publication No. WO2011084518, the contents of which are incorporated herein by reference in their entirety). As non-limiting examples, therapeutic nanoparticles can be formulated into nanoparticles as described in International Publication Nos. WO2008121949, WO2010005726, WO2010005725, WO2011084521 and U.S. Patent Application Publication Nos. US20100069426, US20120004293 and US20100104655, the contents of each of which are incorporated by reference in their entirety.

[0193] The nanoparticles of the present invention may comprise a polymer matrix. As a non-limiting example, the nanoparticles may comprise two or more polymers, such as, but not limited to, polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumerate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), or combinations thereof. In one embodiment, the therapeutic nanoparticles comprise a diblock copolymer. In one embodiment, the diblock copolymer may include PEG in combination with, for example, but not limited to, the following polymers: polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumerate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polylysine, poly(ethyleneimine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), or combinations thereof. In another embodiment, the diblock copolymer may include those diblock copolymers described in European Patent Publication No. 2003-203634, the contents of which are incorporated herein by reference in their entirety. In yet another embodiment, the diblock copolymer can be a high X diblock copolymer, such as those described in International Patent Publication No. WO2013120052, the contents of which are incorporated herein by reference in their entirety.

[0194] As a non-limiting example, the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see US Patent Application Publication No. US20120004293 and US Patent No. 8,236,330; each of which is incorporated herein by reference in its entirety). In another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA, or PEG and PLGA (see US Patent Application Publication No. WO2012166923; each of which is incorporated herein by reference in its entirety). In yet another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle or a target-specific stealth nanoparticle as described in US Patent Application Publication No. US20130172406 (each of which is incorporated herein by reference in its entirety).

[0195] Therapeutic nanoparticles can include multi-block copolymers (see, e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910 and U.S. Patent Application Publication No. US20130195987; the contents of each of which are incorporated by reference in their entirety herein). In yet another non-limiting example, the lipid nanoparticle comprises the block copolymer PEG-PLGA-PEG (e.g., thermosensitive hydrogel (PEG-PLGA-PEG) was used as a TGF-β gene delivery vehicle in Lee et al., Thermosensitive Hydrogel as a Tgf-β Gene Delivery Vehicle Enhances Diabetic Wound Healing. Pharmaceutical Research, 2003 20(12): 1995-2000; Li et al., Controlled Gene Delivery System Based on Thermosensitive Biodegradable Hydrogel. Pharmaceutical Research 2003 20(6):884-888; and Chang et al., Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymer enhances gene delivery efficiency in rat skeletal muscle. J Controlled Release. 2007 118:245-253, each of which is incorporated herein by reference in its entirety).

[0196] The RNA vaccine of the present invention can be formulated in lipid nanoparticles that contain PEG-PLGA-PEG block copolymers.Therapeutic nanoparticles can contain multiblock copolymers (see, for example, U.S. Patent Nos. 8,263,665 and 8,287,910 and U.S. Patent Application Publication No. US20130195987; the entire contents of each of which are incorporated herein by reference).The block copolymers described herein can be included in polyion complexes that contain non-polymeric micelles and block copolymers (see, for example, U.S. Patent Application Publication No. 20120076836; the entire contents of which are incorporated herein by reference).

[0197] The therapeutic nanoparticles may comprise at least one acrylic polymer, including, but not limited to, acrylic acid, methacrylic acid, copolymers of acrylic acid and methacrylic acid, methyl methacrylate copolymers, ethoxyethyl methacrylate, cyanoethyl methacrylate, aminoalkyl methacrylate copolymers, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates, and combinations thereof.

[0198] The therapeutic nanoparticles may include at least one poly(vinyl ester) polymer. The poly(vinyl ester) polymer may be a copolymer, such as a random copolymer. As a non-limiting example, the random copolymer may have a structure as described, for example, in International Application No. WO2013032829 or U.S. Patent Application Publication No. US20130121954, the contents of which are incorporated herein by reference in their entirety. In one embodiment, the poly(vinyl ester) polymer may be conjugated to a polynucleotide as described herein. In another embodiment, the poly(vinyl ester) polymer that may be used in the present invention may be one described in a publication that is incorporated herein by reference in its entirety.

[0199] The therapeutic nanoparticles may include at least one diblock copolymer. The diblock copolymer may be, but is not limited to, a polylactic acid-polyethylene glycol copolymer (see, e.g., International Patent Publication No. WO2013044219; incorporated herein by reference in its entirety). As a non-limiting example, the therapeutic nanoparticles may be used to treat cancer (see, e.g., International Patent Publication No. WO2013044219; incorporated herein by reference in its entirety). In one embodiment, the therapeutic nanoparticles may include at least one cationic polymer as described herein and / or known in the art.

[0200] The therapeutic nanoparticles may comprise at least one amine-containing polymer, including but not limited to polylysine, polyethyleneimine, poly(amidoamine) dendrimers, poly(beta amino esters) (see, e.g., U.S. Pat. No. 8,287,849, the entire contents of which are incorporated herein by reference), and combinations thereof. In another embodiment, the nanoparticles described herein may comprise amine cationic lipids, such as those described in International Patent Application No. WO2013059496, the entire contents of which are incorporated herein by reference). In one aspect, the cationic lipids may have amino-amine or amino-amide moieties.

[0201] The therapeutic nanoparticles may include at least one degradable polyester that may contain polycationic side chains. Degradable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In another embodiment, the degradable polyester may include PEG conjugation to form a PEGylated polymer.

[0202] The therapeutic nanoparticles may include the conjugation of at least one targeting ligand, which may be any ligand known in the art, including but not limited to, a monoclonal antibody (Kirpotin et al., Cancer Res. 2006 66:6732-6740; incorporated herein by reference in its entirety).

[0203] Therapeutic nanoparticles can be formulated in aqueous solutions that can be used to target cancer (see International Publication No. WO2011084513 and U.S. Patent Application Publication No. US20110294717; each of which is incorporated herein by reference in its entirety). Therapeutic nanoparticle RNA vaccines (e.g., therapeutic nanoparticles comprising at least one RNA vaccine) can be formulated using methods described in U.S. Patent No. 8,404,799 to Podobinski et al., the contents of which are incorporated herein by reference in their entirety.

[0204] The RNA vaccines can be encapsulated in and / or linked to and / or bound to synthetic nanocarriers, including those described in International Publication Nos. WO2010005740, WO2010030763, WO201213501, WO2012149252, WO2012149255, WO2012149259, WO2012149265, WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405, WO2012149501, WO2012149601, WO2012149701, WO2012149801, WO2012149902, WO2012149102, WO2012149253, WO2012149259, WO2012149265, WO2012149268, WO2012149282, WO2012149301, WO2012149393, WO2012149405, WO2012149501, WO2012149601, WO2012149702, WO2012149703, WO2012149704, WO2012149705, WO2012149706, WO2012149707, WO2012149708, WO2012149709, WO2012149 Synthetic nanocarriers include, but are not limited to, those described in WO2012149411, WO2012149454 and WO2013019669, and U.S. Patent Application Publication Nos. US20110262491, US20100104645, US20100087337 and US20120244222, the contents of each of which are incorporated by reference in their entirety. Synthetic nanocarriers may be formulated using methods known in the art and / or described herein. As non-limiting examples, synthetic nanocarriers can be formulated according to the methods described in International Publication Nos. WO2010005740, WO2010030763, and WO201213501, and U.S. Patent Application Publication Nos. US20110262491, US20100104645, US20100087337, and US2012024422, each of which is incorporated by reference in its entirety. In another embodiment, synthetic nanocarrier formulations can be lyophilized according to the methods described in International Publication No. WO2011072218 and U.S. Patent No. 8,211,473, each of which is incorporated by reference in its entirety. In yet another embodiment, the formulations of the present invention, including but not limited to synthetic nanocarriers, may be lyophilized or reconstituted (reconstituted) by the methods described in U.S. Patent Application Publication No. US20130230568, the contents of which are incorporated herein by reference in their entirety.In one embodiment, the synthetic nanocarriers may contain reactive groups for releasing (liberating) the polynucleotides described herein (see International Publication No. WO20120952552 and U.S. Patent Application Publication No. US20120171229; each of which is incorporated by reference in its entirety).

[0205] The synthetic nanocarriers may contain immune stimulants to enhance immune responses by delivery of the synthetic nanocarriers. As a non-limiting example, the synthetic nanocarriers may contain Th1 immune stimulants that may enhance Th1-based immune system responses (see International Publication No. WO2010123569 and U.S. Patent Application Publication No. US20110223201; each of which is incorporated herein by reference in its entirety). The synthetic nanocarriers may be formulated for targeted release. In one embodiment, the synthetic nanocarriers are formulated to release polynucleotides at a specified pH and / or after a desired time interval. As a non-limiting example, synthetic nanoparticles can be formulated to release RNA vaccines after 24 hours and / or at pH 4.5 (see International Publication Nos. WO2010138193 and WO2010138194 and U.S. Patent Application Publication Nos. US20110020388 and US20110027217; each of which is incorporated by reference in its entirety herein).

[0206] Synthetic nanocarriers can be formulated for controlled and / or sustained release of the polynucleotides described herein. As a non-limiting example, synthetic nanocarriers for sustained release can be formulated by methods known in the art and / or described herein and / or described in International Publication No. WO2010138192 and US Patent Application Publication No. 20100303850, each of which is incorporated herein by reference in its entirety. In one embodiment, the RNA vaccine can be formulated for controlled and / or sustained release, where the formulation comprises at least one polymer that is a crystallizable side chain (CYSC) polymer. CYSC polymers are described in US Patent No. 8,399,007, which is incorporated herein by reference in its entirety.

[0207] The synthetic nanocarriers may be formulated for use as a vaccine. In one embodiment, the synthetic nanocarriers may encapsulate at least one polynucleotide encoding at least one antigen. As a non-limiting example, the synthetic nanocarriers may include at least one antigen and an excipient (for a vaccine formulation) (see International Publication No. WO2011150264 and US Patent Application Publication No. US20110293723; each of which is incorporated herein by reference in its entirety). As another non-limiting example, the vaccine formulation may include at least two synthetic nanocarriers with the same or different antigens and excipients (see International Publication No. WO2011150249 and US Patent Application Publication No. US20110293701; each of which is incorporated herein by reference in its entirety). Vaccine formulations may be selected by methods described herein and / or known in the art and / or described in International Publication No. WO2011150258 and U.S. Patent Application Publication No. US20120027806, each of which is incorporated by reference in its entirety.

[0208] The synthetic nanocarrier may comprise at least one polynucleotide encoding at least one adjuvant. As a non-limiting example, the adjuvant may comprise dimethyldioctadecylammonium-bromide, dimethyldioctadecylammonium-chloride, dimethyldioctadecylammonium-phosphate or dimethyldioctadecylammonium-acetate (DDA), and the apolar fraction of the total lipid extract of mycobacteria or a portion of the apolar fraction (see, for example, U.S. Patent No. 8,241,610; each of which is incorporated herein by reference in its entirety). In another embodiment, the synthetic nanocarrier may comprise at least one polynucleotide and an adjuvant. As a non-limiting example, the synthetic nanocarrier comprising an adjuvant may be formulated by the methods described in International Publication No. WO2011150240 and U.S. Patent Application Publication No. US20110293700 (each of which is incorporated herein by reference in its entirety). The synthetic nanocarrier may encapsulate at least one polynucleotide that codes for a peptide, fragment or region from a virus. As non-limiting examples, synthetic nanocarriers include, but are not limited to, the nanocarriers described in International Publication Nos. WO2012024621, WO201202629, WO2012024632 and US Patent Application Publication Nos. US20120064110, US20120058153 and US20120058154 (each of which is incorporated herein by reference in its entirety). The synthetic nanocarrier may be coupled to a polynucleotide that may be capable of inducing humoral and / or cytotoxic T lymphocyte (CTL) response (see, for example, International Publication No. WO2013019669; which is incorporated herein by reference in its entirety). In one embodiment, the RNA vaccine may be encapsulated within and / or linked to and / or bound to a zwitterionic lipid. Non-limiting examples of zwitterionic lipids and methods of using zwitterionic lipids are described in U.S. Patent Application Publication No. US20130216607, the contents of which are incorporated herein by reference in their entirety.In one aspect, zwitterionic lipids can be used in the liposomes and lipid nanoparticles described herein. In one embodiment, RNA vaccines can be formulated in colloidal nanocarriers as described in U.S. Patent Application Publication No. US20130197100, the contents of which are incorporated herein by reference in their entirety.

[0209] The nanoparticles may be optimized for oral administration. The nanoparticles may include at least one cationic biopolymer, such as, but not limited to, chitosan or its derivatives. As a non-limiting example, the nanoparticles may be formulated by the methods described in U.S. Patent Application Publication No. 20120282343, which is hereby incorporated by reference in its entirety. In some embodiments, the LNPs include the lipid KL52 [an amino-lipid disclosed in U.S. Patent Application Publication No. 2012 / 0295832, which is hereby expressly incorporated by reference in its entirety]. By incorporating such lipids, the activity and / or safety of LNP administration (measured by examining one or more of ALT / AST, white blood cell count, and cytokine induction) may be improved. KL52-containing LNPs may be administered intravenously and / or in one or more doses. In some embodiments, administration of KL52-containing LNPs results in comparable or improved mRNA and / or protein expression compared to LNPs containing MC3.

[0210] In some embodiments, RNA vaccines can be delivered using smaller LNPs. Such particles may have a diameter of less than 0.1 μm to 100 nm, e.g., less than 0.1 μm, less than 1.0 μm, less than 5 μm, less than 10 μm, less than 15 μm, less than 20 μm, less than 25 μm, less than 30 μm, less than 35 μm, less than 40 μm, less than 50 μm, less than 55 μm, less than 60 μm, less than 65 μm, less than 70 μm, less than 75 μm, less than 80 μm, less than 85 μm, less than 90 μm, less than 95 μm, less than 100 μm, less than 125 μm, less than 150 μm, less than 175 μm, less than 200 μm, less than 225 μm, less than 250 μm, less than 275 μm, less than 300 μm, less than 325 μm, less than 350 μm, less than 375 μm, less than 400 μm, less than 425 μm, less than 450 μm, less than 50 ... The diameter may be, but is not limited to, less than 475 μm, less than 500 μm, less than 525 μm, less than 550 μm, less than 575 μm, less than 600 μm, less than 625 μm, less than 650 μm, less than 675 μm, less than 700 μm, less than 725 μm, less than 750 μm, less than 775 μm, less than 800 μm, less than 825 μm, less than 850 μm, less than 875 μm, less than 900 μm, less than 925 μm, less than 950 μm, less than 975 μm.

[0211] RNA molecules have the following diameters, and are used and delivered in small LNPs: about 1 nm to about 100 nm, about 1 nm to about 10 nm, about 1 nm to about 20 nm, about 1 nm to about 30 nm, about 1 nm to about 40 nm, about 1 nm to about 50 nm, about 1 nm to about 60 nm, about 1 nm to about 70 nm, about 1 nm to about 80 nm, about 1 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 30 nm, about 5 nm to about 40 nm, about 5 nm to about 50 nm, about 5 nm to about 60 nm, about 5 nm to about 70 nm, about 5 nm to about 80 nm, about 1 nm to about 90 nm, about 5 nm to about 100 nm, about 5 nm to about 10 nm, about 5 nm to about 20 nm, about 5 nm to about 30 nm, about 5 nm to about 40 nm, about 5 nm to about 50 nm, about 5 nm to about 60 nm, about 5 nm to about 70 nm, about 5 nm to about 80 nm, about 5 The invention relates to a method for preparing an aqueous solution of at least one nanometer of the present invention and to a method for preparing an aqueous solution of at least one nanometer of the present invention. The aqueous solution may be selected from the group consisting of nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers, nanometers

[0212] Such LNPs can be synthesized using methods involving microfluidic mixers. Exemplary microfluidic mixers can include, but are not limited to, slit interdigital micromixers, such as those manufactured by Microinnova (Allerheiligen bei Wildon, Austria), and / or staggered herringbone micromixers (SHM) (Zhigaltsev, IV et al.). Bottom-up design and synthesis of limit-size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing has been published (Langmuir. 2012. 28:3633-40; Belliveau, NM et al., Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA. Molecular Therapy-Nucleic Acids. 2012. I:e37; Chen, D. et al., Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012. 134(16):6948-51; each of which is incorporated herein by reference in its entirety). In some embodiments, the LNP production method involving SHM further comprises mixing at least two input streams, where the mixing occurs via microstructure-induced chaotic advection (MICA). According to this method, the fluid streams flow through channels that are in a herringbone pattern, causing rotational flow and mixing of the fluids around each other. The method also includes a fluid mixing surface, where the surface changes direction during fluid circulation.Methods of generating LNPs using SHM include those disclosed in U.S. Patent Application Publication Nos. 2004 / 0262223 and 2012 / 0276209, each of which is expressly incorporated by reference in its entirety.

[0213] The RNA vaccines of the present invention may be formulated in lipid nanoparticles made using a micromixer, such as, but not limited to, a Slit Interdigital Micro structured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or a Caterpillar (CPMM) or an Impinging-jet (IJMM) (by Institut fiir Mikrotechnik Mainz GmbH, Mainz Germany). The RNA vaccine of the present invention can be formulated in lipid nanoparticles made using microfluidic technology (see Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006 442: 368-373; and Abraham et al., Chaotic Mixer for Microchannels. Science, 2002 295: 647-651; each of which is incorporated herein by reference in its entirety). As a non-limiting example, controlled microfluidic formulations include passive methods for mixing constant pressure-driven flow in microchannels at low Reynolds numbers (e.g., Abraham et al., Chaotic Mixer for Microchannels. Science, 2002 295: 647-651; each of which is incorporated herein by reference in its entirety). The RNA vaccines of the present invention may be formulated in lipid nanoparticles produced using micromixer chips, such as, but not limited to, those from Harvard Apparatus (Holliston, MA) or Dolomite Microfluidics (Royston, UK).The micromixer chip can be used for rapid mixing of two or more fluid streams using a split and recombine mechanism.

[0214] The RNA vaccine of the present invention can be formulated for delivery using drug-loaded microspheres as described in International Patent Publication No. WO2013063468 or U.S. Patent No. 8,440,614 (each of which is incorporated herein by reference in its entirety). The microspheres can contain compounds of formula (I), (II), (III), (IV), (V) or (VI) as described in International Patent Publication No. WO2013063468 (the contents of which are incorporated herein by reference in their entirety). In another embodiment, amino acids, peptides, polypeptides, lipids (APPLs) are useful for delivering the RNA vaccine of the present invention to cells (see International Patent Publication No. WO2013063468; the contents of which are incorporated herein by reference in their entirety).

[0215] The RNA vaccines of the present invention can be formulated in lipid nanoparticles having a diameter of about 10 to about 200 nm, for example, but not limited to, the following diameters: about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, approximately 40 to approximately 50 nm, approximately 40 to approximately 60 nm, approximately 40 to approximately 70 nm, approximately 40 to approximately 80 nm, approximately 40 to approximately 90 nm, approximately 40 to approximately 100 nm, approximately 50 to approximately 60 nm, approximately 50 to approximately 70 nm, approximately 50 to approximately 80 nm, approximately 50 to approximately 90 nm, approximately 50 to approximately 100 nm, approximately 50 to approximately 150 nm, approx. 50 ~ approx. 200 nm, approx. 60 ~ approx. 70 nm, approx. 60 ~ approx. 80 nm, approx. 60 ~ approx. 90 nm, approx. 60 ~ approx. 100 nm, approx. 60 ~ approx. 150 nm, approx. nm, about 80 to about 90 nm, about 80 to about 100 nm, about 80 to about 150 nm, about 80 to about 200 nm, about 90 to about 100 nm, about 90 to about 150 nm and / or about 90 to about 200 nm.

[0216] The lipid nanoparticles may have a diameter of about 10-500 nm. In one embodiment, the lipid nanoparticles may have a diameter of greater than 100 nm (i.e., greater than 100 nm), greater than 150 nm, greater than 200 nm, greater than 250 nm, greater than 300 nm, greater than 350 nm, greater than 400 nm, greater than 450 nm, greater than 500 nm, greater than 550 nm, greater than 600 nm, greater than 650 nm, greater than 700 nm, greater than 750 nm, greater than 800 nm, greater than 850 nm, greater than 900 nm, greater than 950 nm, or greater than 1000 nm.

[0217] The lipid nanoparticles can be limit size lipid nanoparticles described in International Patent Publication No. WO2013059922 (the entire contents of which are incorporated herein by reference).Limit size lipid nanoparticles can include a lipid bilayer surrounding an aqueous core or a hydrophobic core, where the lipid bilayer can include phospholipids, such as, but not limited to, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, cerebroside, C8-C20 fatty acid diacylphosphatidylcholine and l-palmitoyl-2-oleoylphosphatidylcholine (POPC).In another embodiment, the limit size lipid nanoparticles can include polyethylene glycol lipids, such as, but not limited to, DLPE-PEG, DMPE-PEG, DPPC-PEG and DSPE-PEG.

[0218] The RNA vaccine can be delivered, localized, and / or concentrated at a specific site using the delivery method described in International Patent Publication No. WO2013063530 (the entire contents of which are incorporated herein by reference).As a non-limiting example, before, simultaneously with, or after delivery of the RNA vaccine to the subject, empty polymer particles can be administered to the subject.When the empty polymer particles come into contact with the subject, they change volume and remain, embed, immobilize, or capture at a specific site in the subject.

[0219] The RNA vaccine can be formulated in an active agent release system (see, for example, U.S. Patent Application Publication No. US20130102545; the entire contents of which are incorporated herein by reference). The active agent release system can include 1) at least one nanoparticle bound to an oligonucleotide inhibitor chain hybridized to a catalytically active nucleic acid, and 2) a compound bound to at least one substrate molecule bound to a therapeutically active substance (e.g., a polynucleotide described herein), where the therapeutically active substance is released by cleavage of the substrate molecule by the catalytically active nucleic acid.

[0220] The RNA vaccine can be formulated in nanoparticles that comprise an inner core that comprises non-cellular material and an outer surface that comprises a cell membrane. The cell membrane can be from a cell or can be from a virus. As a non-limiting example, the nanoparticle can be produced by the method described in International Patent Publication No. WO2013052167, which is incorporated herein by reference in its entirety. As another non-limiting example, the nanoparticle described in International Patent Publication No. WO2013052167, which is incorporated herein by reference in its entirety, can be used to deliver the RNA vaccine described herein.

[0221] RNA vaccines can be formulated in porous nanoparticle-supported lipid bilayers (protocells), which are described in International Patent Publication No. WO2013056132, the contents of which are incorporated herein by reference in their entirety.

[0222] The RNA vaccines described herein can be formulated in polymeric nanoparticles described in U.S. Patent Nos. 8,420,123 and 8,518,963 and European Patent No. EP2073848B1 (each of which is incorporated herein by reference in its entirety) or prepared by the methods described therein. As a non-limiting example, the polymeric nanoparticles can have a high glass transition temperature, such as the nanoparticles described in U.S. Patent No. 8,518,963 (each of which is incorporated herein by reference in its entirety) or prepared by the methods described therein. As another non-limiting example, polymeric nanoparticles for oral and parenteral formulations can be prepared by the methods described in European Patent No. EP2073848B1 (each of which is incorporated herein by reference in its entirety).

[0223] The RNA vaccines described herein may be formulated in nanoparticles used for imaging. The nanoparticles may be liposomal nanoparticles, such as those described in U.S. Patent Application Publication No. US20130129636, the entire contents of which are incorporated herein by reference. As a non-limiting example, the liposomes may include gadolinium(III) 2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N'-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl}-acetate and a neutral fully saturated phospholipid component (see, e.g., U.S. Patent Application Publication No. US20130129636, the entire contents of which are incorporated herein by reference). Nanoparticles that may be used in the present invention may be liposomal nanoparticles, such as those described in U.S. Patent Application Publication No. US20130130348, the entire contents of which are incorporated herein by reference). The nanoparticles of the invention can be formed by the methods described in US Pat. No. 6,399,413, the contents of which are incorporated herein by reference. The nanoparticles of the invention can include nutrients, such as nutrients whose deficiency can lead to adverse health effects ranging from anemia to neural tube defects (see, e.g., the nanoparticles described in International Patent Publication No. WO2013072929, the contents of which are incorporated herein by reference in their entirety). By way of non-limiting example, the nutrient can be iron in the form of ferrous, ferric salts, or elemental iron, iodine, folic acid, vitamins, or micronutrients.

[0224] The RNA vaccine of the present invention can be formulated in swellable nanoparticles. The swellable nanoparticles can be, but are not limited to, those described in U.S. Patent No. 8,440,231 (the entire contents of which are incorporated herein by reference).As a non-limiting embodiment, the swellable nanoparticles can be used to deliver the RNA vaccine of the present invention to the pulmonary system (see, for example, U.S. Patent No. 8,440,231; the entire contents of which are incorporated herein by reference).

[0225] The RNA vaccines of the present invention may be formulated in polyanhydride nanoparticles, such as, but not limited to, those described in U.S. Pat. No. 8,449,916, the contents of which are incorporated herein by reference in their entirety.

[0226] The nanoparticles and microparticles of the present invention can be geometrically engineered to modulate macrophage and / or immune responses. In one embodiment, the geometrically engineered particles can have various shapes, sizes and / or surface charges to incorporate the polynucleotides of the present invention for targeted delivery, such as, but not limited to, pulmonary delivery (see, for example, International Publication No. WO2013082111, the contents of which are incorporated herein by reference in their entirety). Other physical features that geometrically designed particles can have include, but are not limited to, fenestration, angled arms, asymmetry and surface roughness, charge (which can alter interactions with cells and tissues). As a non-limiting example, the nanoparticles of the present invention can be produced by the methods described in International Publication No. WO2013082111, the contents of which are incorporated herein by reference in their entirety.

[0227] The nanoparticles of the present invention can be water-soluble nanoparticles, such as those described in International Publication No. WO2013090601, the contents of which are incorporated herein by reference in their entirety. The nanoparticles can be inorganic nanoparticles with small zwitterionic ligands to exhibit good water solubility. The nanoparticles can also have a small hydrodynamic diameter (HD), stability over time, pH and salinity, and low levels of non-specific protein binding. In one embodiment, the nanoparticles of the present invention can be developed by the methods described in U.S. Patent Application Publication No. US20130172406, the contents of which are incorporated herein by reference in their entirety. The nanoparticles of the present invention can be stealth nanoparticles or target-specific stealth nanoparticles, such as, but not limited to, those described in U.S. Patent Application Publication No. US20130172406, the contents of which are incorporated herein by reference in their entirety. The nanoparticles of the present invention can be produced by the methods described in U.S. Patent Application Publication No. US20130172406, the contents of which are incorporated herein by reference in their entirety.

[0228] The stealth or target-specific stealth nanoparticles may comprise a polymer matrix. The polymer matrix may comprise two or more polymers, such as polyethylene, polycarbonate, polyanhydride, polyhydroxy acid, polypropyl fumerate, polycaprolactone, polyamide, polyacetal, polyether, polyester, poly(orthoester), polycyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylate, polycyanoacrylate, polyurea, polystyrene, polyamine, polyester, polyanhydride, polyether, polyurethane, polymethacrylate, polyacrylate, polycyanoacrylate, or combinations thereof. The nanoparticles may be nanoparticle-nucleic acid hybrid structures with a dense nucleic acid layer. As a non-limiting example, the nanoparticle-nucleic acid hybrid structures may be produced by the method described in U.S. Patent Application Publication No. US20130171646, the entire contents of which are incorporated herein by reference. The nanoparticles can include nucleic acids, such as, but not limited to, polynucleotides described herein and / or known in the art.

[0229] The nanoparticles may be embedded in the core of the nanostructure. Alternatively, the nanoparticles may be coated with a low-density porous 3D structure or coating capable of carrying or binding at least one payload inside or on the surface of the nanostructure. Non-limiting examples of nanostructures comprising at least one nanoparticle are described in International Patent Publication No. WO2013123523, the contents of which are incorporated herein by reference in their entirety.

[0230] Other formulations include those described in US20170136121 A1, US9221891 B2, EP2971033 B1 and US20160331828 A1, which are incorporated by reference in their entireties.

[0231] Also provided is a method for producing an mRNA encoding at least one immunogenic peptide fragment, the method comprising: (a) binding a first polynucleotide comprising an open reading frame encoding an immunogenic peptide fragment and a second polynucleotide comprising a 5'-UTR to a polynucleotide conjugated (bound) to a solid support; (b) ligating the 3' end of the second polynucleotide to the 5' end of the first polynucleotide under suitable conditions including a DNA ligase, thereby generating a first ligation product; (c) ligating the 5' end of a third polynucleotide comprising a 3' UTR to the 3' end of the first ligation product under suitable conditions comprising an RNA ligase, thereby generating a second ligation product; (d) releasing the second ligation product from the solid support, thereby generating an mRNA encoding an immunogenic peptide fragment. Includes.

[0232] Also provided is a kit for producing an mRNA cancer vaccine, which may contain one or more containers containing one or more polynucleotides comprising a 5'-ORF, one or more polynucleotides comprising a 3'-ORF, one or more polynucleotides comprising a poly(A) tail, a ligase enzyme, and instructions for ligating one or more polynucleotides comprising an ORF encoding a patient-specific epitope to one or more polynucleotides comprising a 5'-ORF, a 3'-ORF and a poly(A) tail.

[0233] Sequence information [Table X] TIFF2025508467000004.tif237165TIFF2025508467000005.tif247165TIFF2025508467000006.tif245166TIFF2025508467000007.tif247166T IFF2025508467000008.tif246163TIFF2025508467000009.tif247165TIFF2025508467000010.tif245166TIFF2025508467000011.tif59164[Table 2] TIFF2025508467000012.tif127164[Table 3] TIFF2025508467000013.tif127164

[0234] The present invention is illustrated by the following examples, which should not be construed as limiting.

[0235] Working Example Example 1. Production of polynucleotides The polynucleotides may be produced by any suitable method, including, for example, those disclosed in WO2014152027, the entirety of which is incorporated herein by reference along with each of its priority applications.

[0236] Polynucleotides may be purified by any suitable method, including, for example, those methods disclosed in WO2014152031, the entirety of which is incorporated herein by reference, along with each of its priority applications.

[0237] Polynucleotides may be detected and characterized by any suitable method, including, for example, those disclosed in WO2014144039, the entirety of which is incorporated by reference herein, along with each of its priority applications.

[0238] The polynucleotide can be characterized using a method selected from the group consisting of polynucleotide mapping, reverse transcriptase sequencing, charge distribution analysis and detection of RNA impurities, wherein the characterization comprises determining the sequence of the RNA transcript, determining the purity of the RNA transcript or determining the charge heterogeneity of the RNA transcript. Such methods are taught, for example, in WO2014144711 (the entirety of which is incorporated herein by reference together with each of its priority applications).

[0239] Example 2. Chimeric Polynucleotide Synthesis The two regions or portions of the polynucleotide may be joined or linked using triphosphate chemistry as described in Example 2 of WO2017020026, which is incorporated by reference in its entirety along with each of its priority applications.

[0240] Example 3: PCR for cDNA production PCR for the production of cDNA can be performed using 2xKAPA HIFI™ HotStart ReadyMix from Kapa Biosystems (Woburn, MA). The system includes 12.5 μl 2xKAPA ReadyMix, 0.75 μl forward primer (10 μM), 0.75 μl reverse primer (10 μM), template cDNA - 100 ng, and dH20 diluted to 25.0 μl. The reaction conditions are 95°C for 5 minutes, 98°C for 20 seconds, then 58°C for 15 seconds, then 72°C for 45 seconds, then 25 cycles of 72°C for 5 minutes, then 4°C until termination.

[0241] Reactions may be cleaned up (up to 5 μg) using Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, CA) following the manufacturer's instructions. Larger reactions should be purified using larger volumes of product. After purification, the cDNA is quantified using NANODROP™ and analyzed by agarose gel electrophoresis to ensure that the cDNA is of the expected size. The cDNA is then sent for sequencing analysis before proceeding to the in vitro transcription reaction.

[0242] Example 4. In vitro transcription (IVT) The in vitro transcription reaction produces polynucleotides containing uniformly modified polynucleotides. Such uniformly modified polynucleotides may include regions or portions of the polynucleotides of the invention. An input nucleotide triphosphate (NTP) mixture is prepared in the tissue using natural and non-natural NTPs.

[0243] An exemplary in vitro transcription reaction is described in Example 4 of WO2017020026, the entirety of which is incorporated herein by reference along with each of its priority applications. IVT can be used to generate mRNA of the invention from a suitable cDNA template.

[0244] Example 5. mRNA for vaccine compositions The methods of Examples 1-4 are used to construct an mRNA comprising an ORF encoding at least one immunogenic peptide fragment of a polypeptide component of an immune system checkpoint, wherein the polypeptide component of an immune system checkpoint is selected from any one or more of the following: a. IDO; preferably, an immunogenic peptide fragment thereof is up to 50 consecutive amino acids of IDO, which optionally comprises or consists of the sequence of any one of SEQ ID NOs: 2-13, preferably SEQ ID NO: 2; b. PDL1 or PDL2, preferably PDL1; preferably, an immunogenic peptide fragment thereof is up to 50 consecutive amino acids of PDL1, which optionally comprises or consists of the sequence of any one of SEQ ID NOs: 15-100, preferably SEQ ID NO: 15 or 16; or PDL2; preferably, an immunogenic peptide fragment thereof is up to 50 consecutive amino acids of PDL2, which optionally comprises or consists of the sequence of any one of SEQ ID NOs: 102-104; c. Arginase 1 or Arginase 2, preferably Arginase 1; preferably, the immunogenic peptide fragment thereof is up to 50 consecutive amino acids of Arginase 1, which optionally comprises or consists of the sequence of any one of SEQ ID NOs: 106-158, preferably SEQ ID NO: 106; or Arginase 2; preferably, the immunogenic peptide fragment thereof is up to 50 consecutive amino acids of Arginase 2, which optionally comprises or consists of the sequence of any one of SEQ ID NOs: 160-220, preferably SEQ ID NO: 160; d. TDO; preferably, an immunogenic peptide fragment thereof is up to 50 consecutive amino acids of TDO, which optionally comprises or consists of the sequence of any one of SEQ ID NOs: 222-238; or e. TGFb; preferably, the immunogenic peptide fragment is up to 50 contiguous amino acids of TGFb, which optionally comprises or consists of the sequence of any one of SEQ ID NOs: 240-271.

[0245] The methods of Examples 1-4 are used to construct an mRNA containing an ORF including: - at least one copy of a nucleic acid encoding SEQ ID NO: 2 (IO 102) and at least one copy of a nucleic acid encoding SEQ ID NO: 15 (IO 103) and / or SEQ ID NO: 16 (IO 104.1); - at least one copy of a nucleic acid encoding SEQ ID NO: 2 (IO 102) and at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112); - at least one copy of a nucleic acid encoding SEQ ID NO: 15 (IO 103) and / or 16 (IO 104.1) and at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112); - at least one copy of a nucleic acid encoding SEQ ID NO: 2 (IO 102), at least one copy of a nucleic acid encoding SEQ ID NO: 15 (IO 103) and / or SEQ ID NO: 16 (IO 104.1), and at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112).

[0246] The mRNA is formulated into a vaccine composition by any suitable method, in particular using a lipid nanoparticle carrier comprising, in molar ratio, about 20-60% cationic lipid, 5-25% non-cationic lipid, 25-55% sterol, and 0.5-15% PEG-modified lipid.

[0247] Example 6. Clinical formulation of lipoplexes The following describes the clinical formulation of lipoplexes described in Example 12 of US10485884 (incorporated herein by reference in its entirety). The formulation involves two steps: pre-formulating a given RNA using isotonic sodium chloride solution as a diluent, and adding a predetermined amount of liposomes to form lipoplexes. For pre-formulation, first, 4 ml of sodium chloride (0.9% w / w in water) solution is removed from a NaCl vial with a syringe and added to the RNA. Then, 400 μL of liposomes (2.8 mg / mL total lipid in water) are removed from the liposome vial and injected into the solution of RNA and sodium chloride using a cannula (0.9 mm inner diameter). The resulting RNA lipoplex formulation (5.5 ml) can be administered by direct parenteral injection of the desired dose or after preparation for intravenous infusion. For this purpose, 5.0 mL is taken from the RNA lipoplex preparation and diluted in an infusion bag containing 50 ml of isotonic sodium chloride solution. This protocol allows the production of lipoplex preparations with particle sizes of approximately 300-500 nm in a robust and reproducible manner. The materials and components that can be used are as follows:

[0248] component: RNA: 0.5 mg / ml in 10 mM HEPES and 0.1 mM EDTA Diluent: 0.9% NaCl Liposomes: 2.68 mM DOTMA, 1.34 mM DOPE, particle size (Z ave ) 300~500 nm Syringe: 5mL syringes: (e.g., Omnifix, 5mL, Luer Lock, B. Braun Melsungen AG (Melsungen, Germany) 1mL syringe: Injekt-F Tuberculin, 1 mL, Luer Lock, B. Braun Melsungen AG (Melsungen, Germany) needle: · 0.9×44 mm, 20 G 1 / 2", BD Microlance 3, Becton Dickinson SA (Fraga, Spain)

[0249] The size of the RNA lipoplex particles produced according to the above method ranges from 300 nm to 500 nm.

[0250] The mRNA sequences described herein can be formulated in the lipoplexes described above. For example, any one of SEQ ID NOs: 272, 273, 274, 275, 276, 277, 278, 279, 280 and 281 can be formulated in the lipoplexes described above. Furthermore, two or more different mRNA sequences can be formulated in the same lipoplex (e.g., SEQ ID NO: 272 and SEQ ID NO: 273).

[0251] Example 7. Formulation of modified mRNA using lipidoids The following describes the formulation of modified mRNA using lipidoids as described in Example 8 of US10898571 (incorporated herein by reference in its entirety). Modified mRNA (mmRNA) is formulated for in vitro experiments by mixing mmRNA and lipidoid in a fixed ratio prior to addition to cells. In vivo formulations may require the addition of additional components to facilitate systemic circulation. Standard formulation processes used for siRNA-lipidoid formulations are used as a starting point to test the ability of these lipidoids to form particles suitable for in vivo implementation. Initial mmRNA-lipidoid formulations can consist of particles composed of 42% lipidoid, 48% cholesterol and 10% PEG, with further optimization of ratios possible. After particle formation, mmRNA is added and combined with the complex. Encapsulation efficiency is determined using a standard dye exclusion assay.

[0252] A. Lipid synthesis Six lipids to be formulated with modified RNA are synthesized by methods outlined in the art: DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 and DLin-MC3-DMA. DLin-DMA and precursors are synthesized as described in Heyes et al., J. Control Release, 2005, 107, 276-287. DLin-K-DMA and DLin-KC2-DMA and precursors are synthesized as described in Semple et al., Nature Biotechnology, 2010, 28, 172-176. 98N12-5 and precursors are synthesized as described in Akinc et al., Nature Biotechnology, 2008, 26, 561-569.

[0253] C12-200 and precursors are synthesized following the method outlined in Love et al., PNAS, 2010, 107, 1864-1869. 2-Epoxydodecane (5.10 g, 27.7 mmol, 8.2 eq.) is added to a vial containing amine 200 (0.723 g, 3.36 mmol, 1 eq.) and a stir bar. The vial is sealed and warmed to 80° C. The reaction is stirred at 80° C. for 4 days. The mixture is then purified by silica gel chromatography using a gradient from pure dichloromethane (DCM) to DCM:MeOH 98:2. The target compound is further purified by RP-HPLC to give the desired compound.

[0254] DLin-MC3-DMA and precursors are synthesized according to the method described in WO2010054401, which is incorporated herein by reference in its entirety. A mixture of dilinoleylmethanol (1.5 g, 2.8 mmol, 1 eq.), N,N-dimethylaminobutyric acid (1.5 g, 2.8 mmol, 1 eq.), DIPEA (0.73 mL, 4.2 mmol, 1.5 eq.) and TBTU (1.35 g, 4.2 mmol, 1.5 eq.) in 10 mL of DMF is stirred at room temperature for 10 h. The reaction mixture is then diluted with ether and washed with water. The organic layer is dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The crude product is purified by silica gel chromatography using a gradient of DCM to DCM:MeOH 98:2. The target compound is then subjected to an additional RP-HPLC purification using a YMC-Pack C4 column to obtain the target compound.

[0255] B. Formulation of Modified RNA Nanoparticles A solution of synthetic lipids, 1,2-distearoyl-3-phosphatidylcholine (DSPC) (Avanti Polar Lipids, Alabaster, Ala.), cholesterol (Sigma-Aldrich, Taufkirchen, Germany) and α-[3'-(1,2-dimyristoyl-3-propanoxy)-carboxamido-propyl]-ω-methoxy-polyoxyethylene (PEG-c-DOMG) (NOF, Bouwelven, Belgium) is prepared at a concentration of 50 mM in ethanol and stored at -20°C. The lipids are combined to obtain a molar ratio of 50:10:38.5:1.5 (lipid:DSPC:cholesterol:PEG-c-DOMG) and diluted with ethanol to a final lipid concentration of 25 mM. A modified mRNA stock solution is prepared by diluting an aqueous solution of modified mRNA at a concentration of 1-2 mg / mL in 50 mM sodium citrate buffer at pH 3. The lipid and modified mRNA formulations are prepared by mixing synthetic lipid solution and modified mRNA solution to obtain total lipid to modified mRNA weight ratios of 10:1, 15:1, 20:1 and 30:1. The lipid ethanol solution is rapidly injected into the modified mRNA aqueous solution to obtain a suspension containing 33% ethanol. The solution is injected manually (MI) or with the aid of a syringe pump (SP) (Harvard Pump 33 Dual Syringe Pump Harvard Apparatus Holliston, Mass.). To remove ethanol and to perform buffer exchange, the formulation is dialyzed twice against 200 times the volume of the primary product of phosphate buffered saline (PBS) (pH 7.4) using a Slide-A-Lyzer cassette with a molecular weight cut-off (MWCO) of 10 kD (Thermo Fisher Scientific Inc. Rockford, Ill.). The first dialysis is performed at room temperature for 3 hours, and then the formulation is dialyzed overnight at 4°C. The resulting nanoparticle suspension is filtered through a 0.2 μm sterile filter (Sarstedt, Niimbrecht, Germany) into glass vials and sealed with a crimp seal.

[0256] C. Formulation Characterization A Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) is used to measure the particle size, polydispersity index (PDI) and zeta potential of the modified mRNA nanoparticles. Here, 1x PBS is used for particle size measurement and 15 mM PBS is used for zeta potential measurement. The concentration of the modified mRNA nanoparticle formulation is determined using UV-Vis spectroscopy. 100 μL of the diluted formulation in 1x PBS is added to 900 μL of a 4:1 (v / v) mixture of methanol and chloroform. After mixing, the absorbance spectrum of the solution is recorded at 230 nm to 330 nm on a DU 800 spectrophotometer (Beckman Coulter, Beckman Coulter, Inc., Brea, Calif.). The modified RNA concentration in the nanoparticle formulation is calculated based on the extinction coefficient of the modified RNA used in the formulation and the difference between the absorbance at a wavelength of 260 nm and the baseline value at a wavelength of 330 nm.

[0257] To evaluate the encapsulation of modified RNA by nanoparticles, the QUANT-IT™ RIBOGREEN® RNA assay (Invitrogen Corporation Carlsbad, Calif.) is used. Samples are diluted to a concentration of about 5 μg / mL in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). 50 μL of the diluted sample is transferred to a polystyrene 96-well plate, and then either 50 μL of TE buffer or 50 μL of 2% Triton X-100 solution is added. The plate is incubated at a temperature of 37° C. for 15 minutes. RIBOGREEN® reagent is diluted 1:100 in TE buffer, and 100 μL of this solution is added to each well. Fluorescence intensity is measured at an excitation wavelength of about 480 nm and an emission wavelength of about 520 nm using a fluorescence plate reader (Wallac Victor 1420 Multilablel Counter; Perkin Elmer, Waltham, Mass.). Determine the percentage of free modified RNA by subtracting the fluorescence value of the reagent blank from each of the fluorescence values ​​of the samples and dividing the fluorescence intensity of the intact sample (no Triton X-100 added) by the fluorescence value of the disrupted sample (caused by the addition of Triton X-100).

[0258] D. In Vitro Incubation Human embryonic kidney epithelial cells (HEK293) and hepatocellular carcinoma epithelial cells (HepG2) (LGC standards GmbH, Wesel, Germany) are seeded in 96-well plates (Greiner Bio-one GmbH, Frickenhausen, Germany). Plates for HEK293 cells are precoated with collagen type 1. HEK293 are seeded at a density of 30,000 cells per well and HepG2 at a density of 35,000 cells per well in 100 μl cell culture medium. For HEK293, the cell culture medium is DMEM, 10% FCS, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 1× non-essential amino acids (Biochrom AG, Berlin, Germany) and 1.2 mg / ml sodium bicarbonate (Sigma-Aldrich, Munich, Germany). For HepG2, the culture medium is MEM (Gibco Life Technologies, Darmstadt, Germany), 10% FCS, supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate and 1× non-essential amino acids (Biochrom AG, Berlin, Germany). Formulations containing mRNA (mRNA sequence selected from SEQ ID NOs: 272-281, polyA tail of approximately 160 nucleotides not shown in sequence, 5′ cap, Cap1) are added in quadruplicates immediately after cell seeding and incubated.

Claims

1. - an open reading frame (ORF) encoding at least one immunogenic peptide fragment of a polypeptide component of an immune system checkpoint; - a 5' end cap at the 5' end, - the 5' untranslated region (UTR) contained 5' to the ORF; - the 3'UTR contained 3' to the ORF, and - 3' tailing sequence at the 3' end mRNA containing

2. The ORF encodes at least 2, 3, 4, 5, 10 or more immunogenic peptide fragments that are not identical to one another, each of which (i) different portions of the same immune system checkpoint component polypeptide; or (ii) fragments of different immune system checkpoint component polypeptides; 10. The mRNA of claim 1, which may optionally be a component of the same or a different immune system checkpoint. (a) the ORF comprises multiple copies of each sequence encoding an immunogenic peptide fragment, optionally at least 2, 3, 4, 5, 10, 20, 30, 40, 50 or more copies of each said sequence; and / or (b) for each mRNA sequence encoding an immunogenic fragment, there is an intervening sequence encoding a cleavage-sensitive site, preferably a cathepsin B cleavage site; and / or (c) the ORF is codon-optimized for human expression and / or to reduce immune recognition, and / or 3. The mRNA of claim 1, wherein the mRNA comprises at least one chemical modification, optionally selected from pseudouridine, N1-methylpseudouridine, 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2'-O-methyluridine.

4. a. the 5' cap is a 7-methylguanylate cap, preferably m7G(5')ppp(5')NlmpNp; b) The mRNA of claim 1, wherein the tailing sequence is a poly-A tail, a poly-AG quartet and / or a stem-loop sequence, preferably a poly-A tail, typically 40 to 200 nucleotides in length.

5. Immune system checkpoints include: a. Interaction between IDO1 and its substrates; b. Interaction between PD1 and PDL1 and / or between PD1 and PDL2; c. The interaction between arginase 1 or arginase 2 and its substrate; d. Interaction between TDO and its substrate; e. Interaction between TGFb1 and its receptors; f. Interaction between CTLA4 and CD86 and / or between CTLA4 and CD80; g. Interactions between B7-H3 and / or B7-H4 and their respective ligands; h. Interaction between HVEM and BTLA; i. The interaction between GAL9 and TIM3 j. Interaction between MHC class I or II and LAG3, and k. Interaction between MHC class I or II and KIR The mRNA of claim 1, selected from any one or more of the following:

6. The polypeptide component of the immune system checkpoint is: a. IDO, preferably an immunogenic peptide fragment of which is up to 50 consecutive amino acids of IDO, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 2 to 13, preferably SEQ ID NO: 2; b. PDL1 or PDL2, preferably PDL1; preferably PDL1, an immunogenic peptide fragment of which is up to 50 consecutive amino acids of PDL1, optionally comprising or consisting of the sequence of any one of SEQ ID NOS: 15-100, preferably SEQ ID NOS: 15 or 16; or PDL2; preferably PDL2, an immunogenic peptide fragment of which is up to 50 consecutive amino acids of PDL2, optionally comprising or consisting of the sequence of any one of SEQ ID NOS: 102-104; c. Arginase 1 or Arginase 2, preferably Arginase 1; preferably Arginase 1, the immunogenic peptide fragment of which is up to 50 consecutive amino acids of Arginase 1, optionally comprising or consisting of the sequence of any one of SEQ ID NOS: 106-158, preferably SEQ ID NOS: 106; or Arginase 2; preferably Arginase 2, the immunogenic peptide fragment of which is up to 50 consecutive amino acids of Arginase 2, optionally comprising or consisting of the sequence of any one of SEQ ID NOS: 160-220, preferably SEQ ID NOS: 160; d. TDO, preferably an immunogenic peptide fragment thereof of up to 50 consecutive amino acids of TDO, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 222-238; or e. TGFb; preferably, the immunogenic peptide fragment thereof is up to 50 consecutive amino acids of TGFb, which optionally comprises or consists of the sequence of any one of SEQ ID NOs: 240-271. selected from any one or more of In some cases, the ORF is - at least one immunogenic polypeptide fragment of (a) and (b); - at least one immunogenic polypeptide fragment of (a) and (c), - at least one immunogenic polypeptide fragment of (a) and (d); - at least one immunogenic polypeptide fragment of (a) and (e); - at least one immunogenic polypeptide fragment of (b) and (c); - at least one immunogenic polypeptide fragment of (b) and (d); - at least one immunogenic polypeptide fragment of (b) and (e); - at least one immunogenic polypeptide fragment of (c) and (d); - at least one immunogenic polypeptide fragment of (c) and (e); - at least one immunogenic polypeptide fragment of (d) and (e), or - at least one immunogenic polypeptide fragment of (a), (b) and (c); The mRNA of claim 1 .

7. ORF is - at least one copy of a nucleic acid encoding SEQ ID NO: 2 (IO 102) and at least one copy of a nucleic acid encoding SEQ ID NO: 15 (IO 103) and / or SEQ ID NO: 16 (IO 104.1); - at least one copy of a nucleic acid encoding SEQ ID NO: 2 (IO 102) and at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112); - at least one copy of a nucleic acid encoding SEQ ID NO: 15 (IO 103) and / or 16 (IO 104.1) and at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112); or - at least one copy of a nucleic acid encoding SEQ ID NO: 2 (IO 102), and at least one copy of a nucleic acid encoding SEQ ID NO: 15 (IO 103) and / or SEQ ID NO: 16 (IO 104.1), and at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112); The mRNA of claim 1 .

8. the ORF comprises one or more of SEQ ID NOs: 272, 273, 274, 275, 276, 277, 278, 279, 280 and 281; and / or ORF is - at least one copy of SEQ ID NO: 272 (IO 102) and at least one copy of SEQ ID NO: 273 (IO 103), - at least one copy of SEQ ID NO: 272 (IO 102) and at least one copy of SEQ ID NO: 277 (IO 112), - one copy of SEQ ID NO: 273 (IO 103) and at least one copy of SEQ ID NO: 277 (IO 112), or - 1 copy of SEQ ID NO: 272 (IO 102) and at least 1 copy of SEQ ID NO: 273 (IO 103) and at least 1 copy of SEQ ID NO: 277 (IO 112) The mRNA of claim 1 .

9. 14. A vaccine composition comprising the mRNA of any one of claims 1 to 13 formulated in a lipid nanoparticle composition, optionally wherein the lipid nanoparticles have an average diameter of 50 to 200 nm, Further, optionally, the lipid nanoparticle composition comprises a cationic lipid, a PEG-modified lipid, a sterol, and a non-cationic lipid, and optionally, the lipid nanoparticle carrier comprises, in molar ratios, about 20-60% cationic lipid, 5-25% non-cationic lipid, 25-55% cholesterol, and 0.5-15% PEG-modified lipid; 10. The vaccine composition of claim 1, further optionally comprising an adjuvant.

10. A vaccine composition comprising a first mRNA and a second mRNA, wherein the first mRNA comprises a first open reading frame (ORF) encoding a first immunogenic peptide fragment of a polypeptide component of an immune system checkpoint, and the second mRNA comprises a second ORF encoding a second immunogenic peptide fragment of a polypeptide component of an immune system checkpoint; wherein each of the first ORF and the second ORF is - a 5' end cap at the 5' end, - the 5' untranslated region (UTR) contained 5' to the ORF; - the 3'UTR contained 3' to the ORF, and - 3' tailing sequence at the 3' end The vaccine composition comprising:

11. the first immunogenic polypeptide fragment is IDO, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of IDO, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 2-13, preferably SEQ ID NO: 2; and the second immunogenic polypeptide fragment is PDL1 or PDL2, preferably PDL1, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of PDL1, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 15-100, preferably SEQ ID NO: 15 or 16; or PDL2, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of PDL2, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 102-104; the first immunogenic polypeptide fragment is IDO, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of IDO, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 2-13, preferably SEQ ID NO: 2; and the second immunogenic polypeptide fragment is Arginase 1 or Arginase 2, preferably Arginase 1, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of Arginase 1, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 106-158, preferably SEQ ID NO: 106; or Arginase 2, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of Arginase 2, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 160-220, preferably SEQ ID NO: 160; the first immunogenic polypeptide fragment is IDO, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of IDO, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 2-13, preferably SEQ ID NO: 2; and the second immunogenic polypeptide fragment is TDO, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of TDO, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 222-238; the first immunogenic polypeptide fragment is IDO, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of IDO, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 2-13, preferably SEQ ID NO: 2; and the second immunogenic polypeptide fragment is TGFb, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of TGFb, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 240-271; The first immunogenic polypeptide fragment is PDL1 or PDL2, preferably PDL1, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of PDL1, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 15-100, preferably SEQ ID NO: 15 or 16; or PDL2, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of PDL2, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 102-104; and the second immunogenic the polypeptide fragment is Arginase 1 or Arginase 2, preferably Arginase 1, and preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of Arginase 1, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 106-158, preferably SEQ ID NO: 106; or Arginase 2, and preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of Arginase 2, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 160-220, preferably SEQ ID NO: 160; the first immunogenic polypeptide fragment is PDL1 or PDL2, preferably PDL1, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of PDL1, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 15-100, preferably SEQ ID NO: 15 or 16; or PDL2, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of PDL2, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 102-104; and the second immunogenic polypeptide fragment is TDO, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of TDO, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 222-238; the first immunogenic polypeptide fragment is PDL1 or PDL2, preferably PDL1, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of PDL1, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 15-100, preferably SEQ ID NO: 15 or 16; or PDL2, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of PDL2, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 102-104; and the second immunogenic polypeptide fragment is TGFb, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of TGFb, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 240-271; the first immunogenic polypeptide fragment is Arginase 1 or Arginase 2, preferably Arginase 1, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of Arginase 1, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 106-158, preferably SEQ ID NO: 106; or Arginase 2, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of Arginase 2, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 160-220, preferably SEQ ID NO: 160; and the second immunogenic polypeptide fragment is TDO, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of TDO, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 222-238; the first immunogenic polypeptide fragment is Arginase 1 or Arginase 2, preferably Arginase 1, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of Arginase 1, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 106-158, preferably SEQ ID NO: 106; or Arginase 2, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of Arginase 2, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 160-220, preferably SEQ ID NO: 160; and the second immunogenic polypeptide fragment is TGFb, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of TGFb, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 240-271; the first immunogenic polypeptide fragment is TDO, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of TDO, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 222-238; and the second immunogenic polypeptide fragment is TGFb, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of TGFb, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 240-271; The vaccine composition of claim 10.

12. a third mRNA comprising a third open reading frame (ORF) encoding a third immunogenic peptide fragment of a polypeptide component of an immune system checkpoint; Optionally, the first immunogenic polypeptide fragment is IDO, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of IDO, which optionally comprises or consists of the sequence of any one of SEQ ID NOs: 2-13, preferably SEQ ID NO: 2; the second immunogenic polypeptide fragment is PDL1 or PDL2, preferably PDL1, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of PDL1, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 15-100, preferably SEQ ID NO: 15 or 16; or PDL2, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of PDL2, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 102-104; and the third immunogenic polypeptide fragment is Arginase 1 or Arginase 2, preferably Arginase 1, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of Arginase 1, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 106-158, preferably SEQ ID NO: 106; or Arginase 2, preferably wherein the immunogenic peptide fragment is up to 50 consecutive amino acids of Arginase 2, optionally comprising or consisting of the sequence of any one of SEQ ID NOs: 160-220, preferably SEQ ID NO: 150; The vaccine composition of claim 11.

13. - the first ORF comprises at least one copy of a nucleic acid encoding SEQ ID NO: 2 (IO 102) and the second ORF comprises at least one copy of a nucleic acid encoding SEQ ID NO: 15 (IO 103) and / or SEQ ID NO: 16 (IO 104.1); - the first ORF comprises at least one copy of a nucleic acid encoding SEQ ID NO: 2 (IO 102) and the second ORF comprises at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112); - the first ORF comprises at least one copy of a nucleic acid encoding SEQ ID NO: 15 (IO 103) and / or 16 (IO 104.1), and the second ORF comprises at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112); or - the first ORF comprises at least one copy of a nucleic acid encoding SEQ ID NO: 2 (IO 102), the second ORF comprises at least one copy of a nucleic acid encoding SEQ ID NO: 15 (IO 103) and / or SEQ ID NO: 16 (IO 104.1), and the third ORF comprises at least one copy of a nucleic acid encoding SEQ ID NO: 106 (IO 112); A vaccine composition according to claim 11 or 12.

14. each of the first, second and / or third ORFs comprises one or more of SEQ ID NOs: 272, 273, 274, 275, 276, 277, 278, 279, 280 and 281; or - the first ORF comprises at least one copy of SEQ ID NO: 272 (IO 102) and the second ORF comprises at least one copy of SEQ ID NO: 273 (IO 103); - the first ORF comprises at least one copy of SEQ ID NO: 272 (IO 102) and the second ORF comprises at least one copy of SEQ ID NO: 277 (IO 112); - the first ORF comprises one copy of SEQ ID NO: 273 (IO 103) and the second ORF comprises at least one copy of SEQ ID NO: 277 (IO 112); or - the first ORF comprises one copy of SEQ ID NO: 272 (IO 102), the second ORF comprises at least one copy of SEQ ID NO: 273 (IO 103), and the third ORF comprises at least one copy of SEQ ID NO: 277 (IO 112); The vaccine composition of claim 10.

15. The mRNA of claim 1 for use in a method for treating or preventing a disease, comprising administering a therapeutically or prophylactically effective amount of the mRNA of claim 1 to a patient in need of such treatment or prevention, wherein the disease is cancer.