Nucleic acid vaccines for varicella-zoster virus (VZV)

Abstract

The present disclosure relates to compositions of nucleic acid vaccines comprising polynucleotide sequences encoding one or more structural proteins of varicella-zoster virus (VZV) and methods of making, making and therapeutic uses for treating, alleviating, ameliorating and / or preventing herpes zoster.

Description

SEQUENCE LISTING

[0001] The instant application is being filed along with an electronic format sequence listing in a computer readable form. The sequence listing file, entitled 22156-89-SequenceListing-2024-01-05.xml, was created on January 5, 2024, and is 176,570 bytes in size. The information contained in the electronic format of the sequence listing is incorporated by reference herein in its entirety. TECHNICAL FIELD

[0002] The present disclosure relates generally to nucleic acid vaccines, in particular compositions, formulations, methods and / or uses of nucleic acid vaccines (e.g., RNA, mRNA, DNA vaccines) encoding one or more proteins, polypeptides, antigenic peptides, fragments or variants thereof of varicella-zoster virus for the prevention, mitigation and / or treatment and / or prophylaxis of shingles and other diseases caused by varicella-zoster virus infection or reactivation, including reducing the physiological effects of infection, reactivation and / or symptoms. BACKGROUND

[0003] Shingles (shingles or herpes zoster (HZ)) is the result of reactivation of latent varicella-zoster virus (VZV), which is the same virus that causes varicella (varicella or chickenpox). Once varicella, caused by primary infection with VZV, resolves, the virus remains latent in the dorsal root or cranial ganglia. When VZV is reactivated, it causes painful maculopapular rash, known as shingles. The seroprevalence of VZV IgG exceeds 90% in most adult populations, meaning that approximately 1 in 3 people will develop shingles in their lifetime. Complications of shingles can persist for weeks, months, or years after the initial symptoms appear.

[0004] Live attenuated vaccines have been approved (e.g., ZOSTAVAX® from Merck, which is no longer approved in the United States) for individuals over the age of 50 to reduce the incidence of shingles in these individuals. However, the protective efficacy of live attenuated vaccines against shingles is modest and decreases with age. There are also approved recombinant subunit vaccines (e.g., SHINGRIX® from GSK Biologicals, which combines glycoprotein E and adjuvant AS01B® B ), which have higher efficacy and do not show a decrease in efficacy with age. However, recombinant subunit vaccines require 2 vaccine doses, 2-6 months apart, and due to the inclusion of a strong adjuvant, there is a greater risk of local and systemic reactogenicity than with live attenuated vaccines.

[0005] Accordingly, there is a need for a safe and effective shingles vaccine. Moreover, there is an unmet medical need in improving shingles vaccine delivery and developing a safe and effective shingles vaccine that is more economical and faster to produce than the currently available vaccines.

[0006] It is an object of the present disclosure to provide polynucleotides (e.g., mRNA) encoding polypeptides or proteins of varicella-zoster virus as vaccines for the prevention and / or treatment of shingles and other diseases caused by varicella-zoster virus infection and / or reactivation. SUMMARY

[0007] The present disclosure provides nucleic acid vaccines, compositions and formulations comprising nucleic acid vaccines, and methods of using them for the prevention of shingles and other diseases caused by varicella-zoster virus (VZV) infection and / or reactivation. The nucleic acid vaccines can include polynucleotides encoding at least one structural protein, polypeptide, antigenic peptide, fragment, or variant thereof of VZV. The viral protein can be, but is not limited to, a structural protein of VZV. Non-limiting examples of the amino acid sequences of these structural proteins are shown in Table 1 (SEQ ID NOs: 1-5). In some embodiments, the structural protein is glycoprotein E (gE) of VZV as shown in Table 1 (SEQ ID NOs: 1-5). In some embodiments, the structural protein is glycoprotein B (gB) of VZV as shown in Table 1 (SEQ ID NO: 58). In some embodiments, the structural protein is glycoprotein C (gC) of VZV as shown in Table 1 (SEQ ID NO: 59). In some embodiments, the structural protein is glycoprotein H (gH) of VZV as shown in Table 1 (SEQ ID NOs: 60-61). In some embodiments, the structural protein is glycoprotein I (gl) of VZV as shown in Table 1 (SEQ ID NO: 62). In some embodiments, the structural protein is glycoprotein K (gK) of VZV as shown in Table 1 (SEQ ID NO: 63). In some embodiments, the structural protein is glycoprotein L (gL) of VZV as shown in Table 1 (SEQ ID NO: 64). In some embodiments, the structural protein is glycoprotein M (gM) of VZV as shown in Table 1 (SEQ ID NO: 65). In some embodiments, the structural protein is glycoprotein N (gN) of VZV as shown in Table 1 (SEQ ID NO: 66).

[0008] Provided herein are nucleic acid vaccines for shingles for use in methods of vaccinating a subject to prevent and / or treat shingles, wherein the nucleic acid vaccine can include at least one polynucleotide encoding at least one structural protein (e.g., glycoprotein E (gE)) of VZV or a fragment thereof.

[0009] The nucleic acid vaccines described herein can be formulated in one or more lipid nanoparticles (LNP).

[0010] Provided herein are pharmaceutical compositions and formulations of nucleic acid vaccines for the treatment and prevention of herpes zoster in humans and animals.

[0011] Provided herein are nucleic acid vaccines for herpes zoster comprising at least one mRNA, wherein the mRNA comprises a coding region having a nucleic acid sequence that is at least 85% identical, or at least 90% identical, or at least 95% identical to a sequence such as, but not limited to, SEQ ID NO: 6, 7, 8, 9, 10, 67, 68, 69, 70, and 71. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 6. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 7. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 8. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 9. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 10. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 67. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 68. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 69. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 70. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 71.

[0012] Provided herein are nucleic acid vaccines for herpes zoster comprising at least one mRNA, wherein the mRNA comprises a coding region having a nucleic acid sequence that is at least 85% identical, or at least 90% identical, or at least 95% identical to a sequence such as, but not limited to, SEQ ID NOs: 11, 12, 13, 14, 15, 72, 73, 74, 75, and 76. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 11. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 12. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 13. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 14. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 15. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 72. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 73. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 74. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 75. In some embodiments, the mRNA of the nucleic acid vaccines disclosed herein comprises a coding region having a nucleic acid sequence as set forth in SEQ ID NO: 76.

[0013] Provided herein are nucleic acid vaccines for herpes zoster comprising at least one mRNA, the mRNA comprising a nucleic acid sequence such as, but not limited to, SEQ ID NOs: 16, 17, 18, 19, and 20.

[0014] Provided herein are methods of preventing and / or treating herpes zoster in a subject by administering a nucleic acid vaccine described herein. In some embodiments, the nucleic acid vaccine can be used as a pre-exposure prophylaxis (PrEP). In some embodiments, the nucleic acid vaccine can be used as a post-exposure prophylaxis (PEP) or as part of a post-exposure prophylaxis (PEP).

[0015] Provided herein are methods of inducing an immune response in a subject by administering an effective amount of a nucleic acid vaccine described herein to generate an immune response. The immune response can be generated by a single administration of a nucleic acid vaccine described herein. In some embodiments, the immune response can be enhanced by at least one booster administration. In some embodiments, the immune response can be enhanced by two, three, or more booster administrations. As another non-limiting example, the immune response can be generated by booster administrations of a nucleic acid vaccine described herein.

[0016] In some embodiments, administering a nucleic acid vaccine to a subject comprises administering to the subject about 1 pg to about 1 mg, about 10 pg to about 1 mg, about 1 pg to about 500 pg, about 1 pg to about 100 pg, or about 10 pg to about 100 pg of the nucleic acid vaccine mRNA.

[0017] In some embodiments, administering a nucleic acid vaccine to a subject comprises administering about 1-5 pg, 1-10 pg, 1-15 pg, 1-20 pg, 1-25 pg, 1-50 pg, 5-10 pg, 5-15 pg, 5-20 pg, 5-25 pg, 5-50 pg, 10-15 pg, 10-20 pg, 10-25 pg, 10-50 pg, 15-20 pg, 20-25 pg, 20-50 pg, 20-60 pg, 20-70 pg, 20-80 pg, 20-90 pg, 20-100 pg, 20-200 pg, 20-300 pg, 20-400 pg, 20-500 pg, 20-600 pg, 20-700 pg, 20-800 pg, 20-900 pg, 20-1000 pg, 25-50 pg, 25-60 pg, 25-70 pg, 25-80 pg, 25-90 pg, 25-100 pg, 25-200 pg, 25-300 pg, 25-400 pg, 25-500 pg, 25-600 pg, 25-700 pg, 25-800 pg, 25-900 pg, 25-1000 pg, 30-50 pg, 30-60 pg, 30-70 pg, 30-80 pg, 30-90 pg, 30-100 pg, 30-200 pg, 30-300 pg, 30-400 pg, 30-500 pg, 30-600 pg, 30-700 pg, 30-800 pg, 30-900 pg, 30-1000 pg, 40-50 pg, 40-60 pg, 40-70 pg, 40-80 pg, 40-90 pg, 40-100 pg, 40-200 pg, 40-300 pg, 40-400 pg, 40-500 pg, 40-600 pg, 40-700 pg, 40-800 pg, 40-900 pg, 40-1000 pg, 50-60 pg, 50-70 pg, 50-80 pg, 50-90 pg, 50-100 pg, 50-200 pg, 50-300 pg, 50-400 pg, 50-500 pg, 50-600 pg, 50-700 pg, 50-800 pg, 50-900 pg, 50-1000 pg, 60-70 pg, 60-80 pg, 60-90 pg, 60-100 pg, 60-200 pg, 60-300 pg, 60-400 pg, 60-500 pg, 60-600 pg, 60-700 pg, 60-800 pg, 60-900 pg, 60-1000 pg, 70-80 pg, 70-90 pg, 70-100 pg, 70-200 pg, 70-300 pg, 70-400 pg, 70-500 pg, 70-600 pg, 70-700 pg, 70-800 pg, 70-900 pg, 70-1000 pg, 80-90 pg, 80-100 pg, 80-200 pg, 80-300 pg, 80-400 pg, 80-500 pg, 80-600 pg, 80-700 pg, 80-800 pg, 80-900 pg, 80-1000 pg, 90-100 pg, 90-200 pg, 90-300 pg, 90-400 pg, 90-500 pg, 90-600 pg, 90-700 pg, 90-800 pg, 90-900 pg, 90-1000 pg, 100-200 pg, 100-300 pg, 100-400 pg, 100-500 pg, 100-600 pg, 100-700 pg, 100-800 pg, 100-900 pg, 100-1000 pg, 200-300 pg, 200-400 pg, 200-500 pg, 200-600 pg, 200-700 pg, 200-800 pg, 200-900 pg, 200-1000 pg, 300-400 pg, 300-500 pg, 300-600 pg, 300-700 pg, 300-800 pg, 300-900 pg, 300-1000 pg, 400-500 pg, 400-600 pg, 400-700 pg, 400-800 pg, 400-900 pg, 400-1000 pg, 500-600 pg, 500-700 pg, 500-800 pg, 500-900 pg, 500-1000 pg, 600-700 pg, 600-800 pg, 600-900 pg, 600-1000 pg, 700-800 pg, 700-900 pg, 700-1000 pg, 800-900 pg, or 800-1000 pg of a nucleic acid vaccine to the subject.μg, 60-1000 μg, 70-80 μg, 70-90 μg, 70-100 μg, 70-200 μg, 70-300 μg, 70-400 μg, 70-500 μg, 70-600 μg, 70-700 μg, 70-800 μg, 70-900 μg, 70-1000 μg, 80-90 μg, 80-100 μg, 80-200 μg, 80-300 μg, 80-400 μg, 80-500 μg, 80-600 μg, 80-700 μg, 80-800 μg, 80-900 μg, 80-1000 μg, 90-100 μg, 90-200 μg, 90-300 μg, 90-400 μg, 90-500 μg, 90-600 μg, 90-700 μg, 90-800 μg, 90-900 μg, 90-1000 μg, 100-200 μg, 100-300 μg, 100-400 μg, 100-500 μg, 100-600 μg, 100-700 μg, 100-800 μg, 100-900 μg, 100-1000 μg, 150-200 μg, 150-300 μg, 150-400 μg, 150-500 μg, 150-600 μg, 150-700 μg, 150-800 μg, 150-900 μg, 150-1000 μg, 200-300 μg, 200-400 μg, 200-500 μg, 200-600 μg, 200-700 μg, 200-800 μg, 200-900 μg, 200-1000 μg, 250-300 μg, 250-400 μg, 250-500 μg, 250-600 μg, 250-700 μg, 250-800 μg, 250-900 μg, 250-1000 μg, 300-400 μg, 300-500 μg, 300-600 μg, 300-700 μg, 300-800 μg, 300-900 μg, 300-1000 μg, 350-400 μg, 350-500 μg, 350-600 μg, 350-700 μg, 350-800 μg, 350-900 μg, 350-1000 μg, 400-500 μg, 400-600 μg, 400-700 μg, 400-800 μg, 400-900 μg, 400-1000 μg, 450-500 μg, 450-600 μg, 450-700 μg, 450-800 μg, 450-900 μg, 450-1000μg, 500-600 μg, 500-700 μg, 500-800 μg, 500-900 μg, 500-1000 μg, 550-600 μg, 550-700 μg, 550-800 μg, 550-900 μg, 550-1000 μg, 600-700 μg, 600-800 μg, 600-900 μg, 600-1000 μg, 650-800 μg, 650-900 μg, 650-1000 μg, 700-800 μg, 700-900 μg, 700-1000 μg, 750-800 μg, 750-900 μg, 750-1000μg, 800-900 μg, 800-1000 μg, 850-900 μg, 850-1000 μg, 900-1000 μg or 950-1000 μg.

[0018] In some implementations of the provided method, administration includes intramuscular (IM) injection of a nucleic acid vaccine into the subject.

[0019] Nucleic acid vaccines can be administered to subjects with a first dose of the nucleic acid vaccine, followed by a second dose of the nucleic acid vaccine approximately 1 to 6 months later.

[0020] This article provides a method for preparing the nucleic acid vaccine described herein.

[0021] Details of several embodiments are set forth in the description below. Other features, objects, and advantages will become apparent from the specification and claims. Attached Figure Description

[0022] Figure 1A and Figure 1B A graph showing the expression of S-001, S-002, S-003, S-004 and S-006 in Huh-7 cells is provided.

[0023] Figure 2A and Figure 2B A graph showing the dose-dependent expression of S-001, S-002, S-003, S-004, and S-006 is provided. Figure 2C The average MFI level of the four experiments is shown.

[0024] Figure 3 A figure is provided showing Western blot analysis of HEK293T cells transfected with mRNA using an anti-gE specific antibody.

[0025] Figure 4 A diagram showing the cellular localization of gE expressed in mRNA-transfected HEK293T cells is provided.

[0026] Figure 5A and Figure 5B A graph showing total gE-specific IgG for S-001 compared to SHINGRIX® and VARIVAX® is provided. Figure 5C and Figure 5D A graph showing cellular responses for S-001 compared to SHINGRIX® and VARIVAX® is provided. DETAILED DESCRIPTION

[0027] The following description sets forth exemplary compositions, methods, parameters, and the like. It is understood, however, that such description is made only by way of example and not as a limitation to the scope of the disclosure.

[0028] I. Compositions of the Disclosure

[0029] Described herein are nucleic acid vaccines, in particular polynucleotides (e.g., mRNA), compositions, formulations, methods, and / or uses thereof for preventing, ameliorating, and / or treating shingles and diseases caused by VZV, of nucleic acid vaccines comprising a polynucleotide encoding one or more proteins, polypeptides, antigenic peptides, fragments, or variants of varicella-zoster virus (VZV). The proteins can be structural proteins of VZV. As non-limiting examples, the structural proteins can be glycoproteins (G) of VZV, such as, but not limited to, glycoprotein E (gE).

[0030] In some embodiments, at least one component of the nucleic acid vaccine is a polynucleotide encoding at least one of a VZV structural protein or polypeptide, or a fragment or variant of a structural protein. The polynucleotide can be an RNA polynucleotide, such as an mRNA polynucleotide.

[0031] In some embodiments, the nucleic acid vaccine comprises at least one mRNA polynucleotide encoding at least one of a VZV structural protein or a fragment or variant of a structural protein.

[0032] In some embodiments, the polynucleotide can be designed to encode one or more polypeptides of interest from VZV or fragments or antigenic peptides or variants thereof. Such polypeptides of interest of VZV can include, but are not limited to, intact polypeptides, multiple polypeptides, or polypeptide fragments or polypeptide variants, which can be independently encoded by one or more regions or portions or the entirety of a polynucleotide from VZV. As used herein, the term “polypeptide of interest” refers to any polypeptide selected to be encoded within a polynucleotide described herein or whose function is affected by a polynucleotide described herein. Any peptide or polypeptide described herein can be antigenic (also referred to as immunogenic).

[0033] As used herein, "polypeptide" refers to a polymer of amino acid residues (natural or non-natural) that are linked together, typically by peptide bonds. As used herein, this term refers to proteins, polypeptides, and peptides of any size, structure, or function or origin. In some embodiments, the polypeptide of interest is an antigen encoded by a polynucleotide as described herein.

[0034] In some embodiments, the encoded polypeptide is less than about 50 amino acids, then the polypeptide is referred to as a peptide. If the polypeptide is a peptide, it is at least about 2, 3, 4, or at least 5 amino acid residues in length. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments of the foregoing, and other equivalents, variants, and analogs. Polypeptides can be single molecules or can be multimeric complexes, such as dimers, trimers, or tetramers. They can also include single- or multi-chain polypeptides, such as antibodies or insulin, and can be associated or linked. Most commonly, disulfide bonds are present in multi-chain polypeptides. The term polypeptide can also apply to amino acid polymers in which one or more of the amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids.

[0035] The term "polypeptide variant" refers to a molecule whose amino acid sequence differs from that of a naturally occurring or reference sequence. Amino acid sequence variants can have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence compared to the naturally occurring or reference sequence. Generally, variants will have at least about 50% identity (homology) to the naturally occurring or reference sequence, and preferably, they will be at least about 80%, or at least about 85%, more preferably at least about 90%, and even more preferably at least about 95% identical (homologous) to the naturally occurring or reference sequence.

[0036] In some embodiments, "variant mimics" are provided. As used herein, the term "variant mimic" is a "variant mimic" that contains one or more amino acids that will mimic an activated sequence. For example, glutamic acid can be used as a mimic for phosphothreonine and / or phosphoserine. Alternatively, a variant mimic can result in inactivation or inactivation products containing the mimic, for example, phenylalanine can act as an inactivating substitution for tyrosine; or alanine can act as an inactivating substitution for serine.

[0037] "Homology" when applied to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for alignment are well known in the art. It will be appreciated that homology depends on the calculation of percent identity, but the value can differ due to the introduction of gaps and penalties in the calculation.

[0038] "homolog" when applied to polypeptide sequences means a corresponding sequence of another species having substantial identity to a second sequence of a second species.

[0039] As used herein, "analog" is meant to include polypeptide variants that differ in one or more amino acid changes, e.g., substitutions, additions or deletions of amino acid residues, but which still retain one or more properties of the parent or starting polypeptide.

[0040] In some embodiments, the present disclosure contemplates several types of polypeptide-based compositions, including variants and derivatives. These include substitution, insertion, deletion, and covalent variants and derivatives. The term "derivative" is used synonymously with the term "variant," but generally refers to a molecule that has been modified and / or altered in any way relative to a reference or starting molecule.

[0041] For example, a sequence tag or amino acid, such as one or more lysines, can be added to the peptide sequences described herein (e.g., at the N- or C-terminus). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or allow biotinylation. Alternatively, amino acid residues located in the carboxy and amino terminal regions of the amino acid sequence of the peptide or protein can be optionally deleted, thereby providing a truncated sequence. Alternatively, certain amino acids can be deleted (e.g., C-terminal or N-terminal residues) depending on the use of the sequence, e.g., to express the sequence as part of a larger sequence that is soluble or attached to a solid support.

[0042] "substitution variant" when referring to a polypeptide is one in which at least one amino acid residue is removed from and inserted into the native or starting sequence at the same location. The substitution can be a single substitution in which only one amino acid in the molecule is substituted, or the substitution can be multiple substitutions in which two or more amino acids in the same molecule are substituted.

[0043] As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid normally found in a sequence with a different amino acid having similar size, charge, or polarity. Examples of conservative substitutions include the replacement of one non-polar (hydrophobic) residue such as isoleucine, valine, and leucine with another non-polar residue. Likewise, a conservative substitution is one in which one polar (hydrophilic) residue is replaced with another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, a basic residue such as lysine, arginine or histidine is replaced with another, or an acidic residue such as aspartic acid or glutamic acid is replaced with another is an additional example of a conservative substitution. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine and / or the substitution of a polar residue for a non-polar residue.

[0044] An "insertion variant," when referring to a polypeptide, refers to those variants in which one or more amino acids are inserted next to an amino acid in the native or starting sequence. "Next to" an amino acid refers to attachment to the alpha-carboxyl or alpha-amino functional group of the amino acid.

[0045] A "deletion variant," when referring to a polypeptide, is one in which one or more amino acids are removed from the native or starting amino acid sequence. Typically, a deletion variant will have one or more amino acids deleted in a particular region of the molecule.

[0046] A "covalent derivative," when referring to a polypeptide, includes modifications of the native or starting protein with organic proteinaceous or non-proteinaceous derivatizing agents and / or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues, or by exploiting post-translational modification mechanisms functionally present in the selected recombinant host cell. The resulting covalent derivatives can be used in procedures for identifying residues important for biological activity, for immunoassays, or for the production of anti-protein antibodies useful in immunoaffinity purification of the recombinant glycoprotein. Such modifications are within the skill of the art and can be made without undue experimentation.

[0047] A "feature," when referring to a polypeptide, is defined as a distinct polypeptide-based component of a molecule. Features of a polypeptide encoded by a polynucleotide described herein include surface manifestations, local conformational shapes, folds, loops, half-loops, domains, half-domains, sites, termini, or any combination thereof.

[0048] As used herein, the term "surface manifestation," when referring to a polypeptide, refers to a polypeptide-based component of a protein that appears on the outermost surface.

[0049] As used herein, the term "local conformational shape" when referring to a polypeptide means the polypeptide-based structure of a protein that is located within a definable space of the protein.

[0050] As used herein, the term "fold" when referring to a polypeptide refers to the conformation of an amino acid sequence that results upon energy minimization. Folding can occur at the secondary or tertiary level of the folding process. Examples of secondary level folding include beta sheets and alpha helices. Examples of tertiary folding include domains and regions that form due to the aggregation or segregation of energy forces. Regions that form in this way include hydrophobic and hydrophilic pockets, among others.

[0051] As used herein, the term "turn" when referring to a polypeptide conformation means a bend that changes the direction of the peptide or polypeptide backbone and can involve one, two, three, or more amino acid residues.

[0052] As used herein, the term "loop" when referring to a polypeptide means a structural feature of a polypeptide that serves to reverse the direction of the peptide or polypeptide backbone. When a loop is present in a polypeptide and merely changes the direction of the backbone, it can comprise four or more amino acid residues. Oliva et al. have identified at least 5 classes of protein loops (J. Mol Bio., 266 (4):814-830; 1997). Loops can be open or closed. Closed loops or "looped" loops can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids between bridging moieties. Such bridging moieties can comprise a cysteine-cysteine bridge (Cys-Cys) typical of polypeptides having disulfide bridges, or alternatively, the bridging moieties can be non-protein based, such as the dibromozylyl reagent used herein.

[0053] As used herein, the term "half-loop" when referring to a polypeptide means a portion of an identified loop that has at least half the number of amino acid residues of the loop from which it is derived. It will be appreciated that loops can not always contain an even number of amino acid residues. Thus, in those instances where a loop contains or is identified as containing an odd number of amino acids, a half-loop of an odd loop will comprise either the integer portion or the next integer portion of the loop (number of amino acids of the loop / 2 + / - 0.5 amino acids).

[0054] As used herein, the term "domain" when referring to a polypeptide means a motif of a polypeptide that has one or more identifiable structural or functional features or properties (e.g., binding ability, use as a site for protein-protein interaction).

[0055] As used herein, when referring to a polypeptide, the term "half-domain" means a portion of an identified domain that has at least half the number of amino acids of the domain from which it is derived. It will be appreciated that domains can not always contain an even number of amino acid residues. Thus, in those instances where a domain contains or is identified as containing an odd number of amino acids, a half-domain of an odd number domain will contain either the whole number portion or the next whole number portion of the domain (the number of amino acids of the domain / 2 + / - 0.5 amino acids). For example, a domain identified as a 7 amino acid domain can yield a 3 amino acid or a 4 amino acid half-domain (7 / 2 = 3.5 + / - 0.5 is 4 or 3). It will also be appreciated that subdomains can be identified within a domain or half-domain that have fewer than all of the structural or functional properties identified in the domain or half-domain from which they are derived. It will also be appreciated that the amino acids comprising any domain type herein need not be contiguous along the backbone of the polypeptide (i.e., amino acids that are not adjacent can be structurally folded to create a domain, half-domain, or subdomain).

[0056] As used herein, when referring to a polypeptide, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain" when it relates to amino acid-based embodiments. A site represents a position within a peptide or polypeptide that can be modified, manipulated, altered, derivatized, or changed within a polypeptide-based molecule described herein.

[0057] As used herein, when referring to a polypeptide, the term "termini" or "terminus" refers to the end of a peptide or polypeptide. Such ends are not limited to only the first or last site of a peptide or polypeptide, but can also include additional amino acids in the terminal region. Polypeptide-based molecules described herein can be characterized as having both an N-terminus (ending in an amino acid with a free amino group (NH2)) and a C-terminus (ending in an amino acid with a free carboxyl group (COOH)). In some cases, proteins described herein are formed from multiple polypeptide chains that are brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These classes of proteins will have multiple N- and C-termini. Alternatively, the termini of a polypeptide can be modified, as appropriate, such that they begin or end with a non-polypeptide-based moiety, such as an organic conjugate.

[0058] Once any feature has been identified or defined as a desired component of a polypeptide encoded by a polynucleotide described herein, a variety of manipulations and / or modifications can be made to the feature by moving, exchanging, inverting, deleting, randomizing, or duplicating. Furthermore, it will be appreciated that manipulations of a feature can result in the same outcome as modifying a molecule described herein. For example, a manipulation involving deletion of a domain will result in a change in the length of the molecule, just as would modifying a nucleic acid to encode a less than full length molecule.

[0059] In polypeptides, the term "modification" refers to a modification as compared to the standard set of 20 amino acids. The modification can be a variety of different modifications. In some embodiments, a region can contain one, two, or more (optionally different) modifications.

[0060] Modifications and manipulations can be accomplished by methods known in the art, such as but not limited to site-directed mutagenesis or a priori incorporation during chemical synthesis. The resulting modified molecules can then be tested for activity using in vitro or in vivo assays, such as those described herein, or any other suitable screening assays known in the art.

[0061] In some embodiments, a polypeptide can comprise a consensus sequence found through multiple rounds of experimentation. As used herein, a "consensus" sequence is a single sequence that represents a collective population of sequences that allow for variation at one or more positions.

[0062] As will be recognized by one of skill in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of a polypeptide of interest. For example, provided herein are any protein fragments (meaning a polypeptide sequence that is at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical to the reference protein). Protein fragments can be of a length containing 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or greater than 100 amino acids. In another example, any protein comprising a segment of about 20, about 30, about 40, about 50, or about 100 amino acids or more that is about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 100% identical to any sequence described herein can be used according to the nucleic acid vaccines described herein. In certain embodiments, a polypeptide used according to the nucleic acid vaccines described herein comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any sequence provided or referenced herein.

[0063] Accordingly, the polynucleotides of the present disclosure encode peptides or polypeptides that contain substitutions, insertions and / or additions, deletions, and covalent modifications relative to a reference sequence, in particular a peptide or polypeptide sequence disclosed herein. The polynucleotides can also contain substitutions, insertions and / or additions, deletions, and covalent modifications relative to a polynucleotide reference sequence.

[0064] A reference molecule (polypeptide or polynucleotide) can share certain identities with a designed molecule (polypeptide or polynucleotide). The term "identity" known in the art refers to the relatedness between sequences of two or more peptides, polypeptides, or polynucleotides as determined by comparing the sequences of the same. In the art, identity also means the degree of sequence relatedness between them, as determined by the number of matches between two or more amino acid residue chains or nucleotide chains. Identity measures the percent of identical matches between shorter sequences of two or more sequences, where gap alignments (if any) are resolved by a particular mathematical model or computer program (e.g., "algorithm"). Identity of related peptides can be readily calculated by known methods. These methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, N.Y., 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, N.Y., 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M. and Griffin, H. G., eds., Humana Press, N.J., 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J. eds., M. Stockton Press, N.Y, 1991; and Carillo et al., SIAM J. Applied Math. 48:1073; 1988.

[0065] In some embodiments, the encoded polypeptide variant can have the same or similar activity as the reference polypeptide. Alternatively, the variant can have an altered activity (e.g., increased or decreased) relative to the reference polypeptide. Generally, a variant of a particular polynucleotide or polypeptide described herein will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity to that particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art. Such tools for alignment include those in the BLAST suite (Stephen F. Altschul et al., Gapped BLAST and PSLBLAST: a new generation of protein database search programs, Nucleic Acids Res. 1997, 25:3389-3402). Other tools are described herein, particularly in the definition of “identity”.

[0066] Varicella-zoster virus (VZV)

[0067] Varicella-zoster virus (VZV) is an alphaherpesvirus that exists as a multilayered structure approximately 200 nm in diameter. The genome (dsDNA) is surrounded by a protein capsid structure that is covered by an amorphous layer of tegument proteins, and both of these structures are surrounded by a lipid envelope containing viral glycoproteins. The VZV genome is the smallest among human herpesviruses, encoding at least 71 unique proteins (ORF0-ORF68), with three additional open reading frames (ORF69-ORF71) of early replication open reading frames (ORF64-ORF62), respectively. Only a subset of the encoded proteins form the structure of the viral particle, including but not limited to nine glycoproteins: ORF5 (glycoprotein K (gK)), ORF9A (glycoprotein N (gN)), ORF14 (glycoprotein C (gC)), ORF31 (glycoprotein B (gB)), ORF37 (glycoprotein H (gH)), ORF50 (glycoprotein M (gM)), ORF60 (glycoprotein L (gL)), ORF67 (glycoprotein I (gl)), and ORF68 (glycoprotein E (gE)).

[0068] VZV is a human pathogen that spreads in the form of varicella or chicken pox to children and then reappears in the form of herpes zoster, also known as shingles (HZ). VZV is one of nine human herpes viruses and is adapted to its human host, so it can infect most people in a given community. Varicella infection is considered an endemic disease worldwide, but it has been largely controlled through vaccination in some countries.

[0069] In its natural human host, varicella infection is spread from the nasopharynx via a limited number of T cells located in the epidermis of the skin. Once there, the infection is transmitted to the basal keratinocytes that make up the innermost layer of the epidermis. The virus gradually infects other cells in its vicinity until it reaches the surface of the skin in the form of VZV vesicles, which are found to contain polykaryocytes or multi-nucleated cells due to the cell-to-cell fusion induced by VZV. As the number of viral particles within the vesicles increases, some of the particles travel retrograde along sensory neurons in the skin to sensory ganglia, where the virus becomes latent or dormant until later in the host's life (years or decades). Without wishing to be bound by theory, under conditions of immunosuppression or aging, VZV can reactivate within the ganglia and spread anterograde back to the skin to cause herpes zoster.

[0070] A subject developing shingles or herpes zoster most commonly has a skin rash in one or two adjacent dermatomes, which can be painful, itchy, and / or tingling (called localized herpes zoster). The rash most often appears on the torso, along the chest dermatome, and usually does not cross the body's midline. The rash develops into clusters of vesicles, with new vesicles continuing to form for three to five days and gradually drying and scabbing over. The vesicles usually heal within two to four weeks, but there can be permanent changes in skin pigmentation and scarring on the skin. When the rash affects three or more dermatomes, it is called disseminated herpes zoster, and it usually occurs only in people with an impaired or suppressed immune system. Disseminated herpes zoster is difficult to distinguish from varicella or chicken pox. Some people can also have a headache, photophobia (sensitivity to bright light), and malaise during the prodromal phase.

[0071] The most common complication of shingles is postherpetic neuralgia (PHN), where pain in the area where the rash once appeared persists for more than 90 days after the onset of the rash. PHN can last for weeks or months, or occasionally for years. The risk of PHN increases with the age of the subject, with older adults more likely to have longer-lasting, more severe pain. Other complications of shingles include, but are not limited to, ophthalmologic involvement with acute or chronic ocular sequelae (ophthalmic shingles), including vision loss; bacterial superinfection of the lesions, usually caused by Staphylococcus aureus, less commonly by group A beta-hemolytic streptococci; cranial and peripheral nerve palsies; and visceral involvement, such as meningoencephalitis, pneumonia, hepatitis, and acute retinal necrosis.

[0072] Subjects with active shingles vesicles or lesions can transmit VZV infection and cause chickenpox in populations who have never had chickenpox or received a chickenpox vaccine.

[0073] According to one aspect of the disclosure, the nucleic acid vaccines described herein encode at least one protein, polypeptide, antigenic peptide, fragment, or variant derived from a glycoprotein (G), a nucleoprotein (N), a phosphoprotein (P), a matrix protein (M), and / or a RNA polymerase (L) of VZV.

[0074] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode more than one fragment, antigenic peptide, or variant of a structural protein of VZV, such as glycoprotein E (gE). gE is the most abundant glycoprotein expressed during VZV infection and / or reactivation, is important for intercellular spread, and has a structural organization containing two consensus N-glycosylation sites and a mucin-like domain (MLD), which is a target for mucin-type O-linked glycosylation. gE-specific antibodies have been detected in adults who have had chickenpox previously and are part of the humoral immune response to VZV vaccine.

[0075] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode a variant of one of the glycoproteins of VZV or a fragment of the variant of the glycoprotein.

[0076] In some embodiments, the nucleic acid vaccines described herein can encode one or more proteins, polypeptides, peptides, fragments, or variants of glycoprotein E (gE), glycoprotein B (gB), glycoprotein C (gC), glycoprotein H (gH), glycoprotein I (gl), glycoprotein K (gK), glycoprotein L (gL), glycoprotein M (gM), and / or glycoprotein N (gN) of VZV. Non-limiting examples of VZV glycoproteins are provided in Table 1.

[0077] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode a full-length polypeptide of glycoprotein E (gE) of VZV, or a fragment or variant of glycoprotein E (gE).

[0078] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein encodes a full-length polypeptide of glycoprotein B (gB) of VZV, or a fragment or variant of glycoprotein B.

[0079] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein encodes a full-length polypeptide of glycoprotein C (gC) of VZV, or a fragment or variant of glycoprotein C.

[0080] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein encodes a full-length polypeptide of glycoprotein H (gH) of VZV, or a fragment or variant of glycoprotein H.

[0081] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein encodes a full-length polypeptide of glycoprotein I (gl) of VZV, or a fragment or variant of glycoprotein I.

[0082] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein encodes a full-length polypeptide of glycoprotein K (gK) of VZV, or a fragment or variant of glycoprotein K.

[0083] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein encodes a full-length polypeptide of glycoprotein L (gL) of VZV, or a fragment or variant of glycoprotein L.

[0084] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein encodes a full-length polypeptide of glycoprotein M (gM) of VZV, or a fragment or variant of glycoprotein M.

[0085] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein encodes a full-length polypeptide of glycoprotein N (gN) of VZV, or a fragment or variant of glycoprotein N.

[0086] In some embodiments, the polynucleotide of the nucleic acid vaccine described herein encodes more than one glycoprotein of VZV. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein E and glycoprotein B. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein E and glycoprotein C. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein E and glycoprotein H. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein E and glycoprotein I. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein E and glycoprotein K. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein E and glycoprotein L. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein E and glycoprotein M. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein E and glycoprotein N. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein B and glycoprotein C. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein B and glycoprotein H. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein B and glycoprotein I. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein B and glycoprotein K. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein B and glycoprotein L. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein B and glycoprotein M. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein B and glycoprotein N. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein C and glycoprotein H. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein C and glycoprotein I. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein C and glycoprotein K. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein C and glycoprotein L. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein C and glycoprotein M. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein C and glycoprotein N. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein H and glycoprotein I. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein H and glycoprotein K. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein H and glycoprotein L. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein H and glycoprotein M. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein H and glycoprotein N. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein I and glycoprotein K. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein I and glycoprotein L. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein I and glycoprotein M. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein I and glycoprotein N. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein K and glycoprotein L. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein K and glycoprotein M. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein K and glycoprotein N. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein L and glycoprotein M. In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein L and glycoprotein N.In some aspects, the polynucleotide of the nucleic acid vaccine encodes glycoprotein M and glycoprotein N.

[0087] Table 1. Glycoprotein sequences of VZV

[0088]

[0089]

[0090] In some embodiments, the nucleic acid vaccines described herein can encode at least one structural protein having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of the sequences in Table 1 or a fragment of any one of the sequences in Table 1 or a variant of any one of the sequences in Table 1. In one embodiment, the coding sequence of the mRNA vaccines described herein can be based on the coding sequence of the glycoprotein (G) of VZV. In some embodiments, the nucleic acid vaccines described herein can encode a glycoprotein having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence of the glycoprotein (G) of VZV in Table 1 or a variant thereof.

[0091] In some embodiments, the nucleic acid vaccines described herein can encode at least one glycoprotein E (gE) having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of the sequences in Table 1 or a fragment of any one of the sequences in Table 1 or a variant of any one of the sequences in Table 1. In one embodiment, the coding sequence of the mRNA vaccines described herein can be based on the coding sequence of gE of VZV. In some embodiments, the nucleic acid vaccines described herein can encode a gE having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence of gE of VZV in Table 1.

[0092] In some embodiments, the nucleic acid vaccines described herein can encode at least one glycoprotein B (gB) having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of the sequences in Table 1 or a fragment of any one of the sequences in Table 1 or a variant of any one of the sequences in Table 1. In one embodiment, the coding sequence of the mRNA vaccines described herein can be based on the coding sequence of gB of VZV. In some embodiments, the nucleic acid vaccines described herein can encode gB having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence of gB of VZV in Table 1.

[0093] In some embodiments, the nucleic acid vaccines described herein can encode at least one glycoprotein C (gC) having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of the sequences in Table 1 or a fragment of any one of the sequences in Table 1 or a variant of any one of the sequences in Table 1. In one embodiment, the coding sequence of the mRNA vaccines described herein can be based on the coding sequence of gC of VZV. In some embodiments, the nucleic acid vaccines described herein can encode gC having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence of gC of VZV in Table 1.

[0094] In some embodiments, the nucleic acid vaccines described herein can encode at least one glycoprotein H (gH) having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of the sequences in Table 1 or a fragment of any one of the sequences in Table 1 or a variant of any one of the sequences in Table 1. In one embodiment, the coding sequence of the mRNA vaccines described herein can be based on the coding sequence of gH of VZV. In some embodiments, the nucleic acid vaccines described herein can encode gH having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence of gH of VZV in Table 1.

[0095] In some embodiments, the nucleic acid vaccines described herein can encode at least one glycoprotein I (gl) having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of the sequences in Table 1, or a fragment of any one of the sequences in Table 1, or a variant of any one of the sequences in Table 1. In one embodiment, the coding sequence of the mRNA vaccines described herein can be based on the coding sequence of gl of VZV. In some embodiments, the nucleic acid vaccines described herein can encode gl having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence of gl of VZV in Table 1.

[0096] In some embodiments, the nucleic acid vaccines described herein can encode at least one glycoprotein K (gK) having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of the sequences in Table 1, or a fragment of any one of the sequences in Table 1, or a variant of any one of the sequences in Table 1. In one embodiment, the coding sequence of the mRNA vaccines described herein can be based on the coding sequence of gK of VZV. In some embodiments, the nucleic acid vaccines described herein can encode gK having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence of gK of VZV in Table 1.

[0097] In some embodiments, the nucleic acid vaccines described herein can encode at least one glycoprotein L (gL) having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of the sequences in Table 1, or a fragment of any one of the sequences in Table 1, or a variant of any one of the sequences in Table 1. In one embodiment, the coding sequence of the mRNA vaccines described herein can be based on the coding sequence of gL of VZV. In some embodiments, the nucleic acid vaccines described herein can encode gL having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence of gL of VZV in Table 1.

[0098] In some embodiments, the nucleic acid vaccines described herein can encode at least one glycoprotein M (gM) having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of the sequences in Table 1, or a fragment of any one of the sequences in Table 1, or a variant of any one of the sequences in Table 1. In one embodiment, the coding sequence of the mRNA vaccines described herein can be based on the coding sequence of gM of VZV. In some embodiments, the nucleic acid vaccines described herein can encode gM having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence of gM of VZV in Table 1.

[0099] In some embodiments, the nucleic acid vaccines described herein can encode at least one glycoprotein N (gN) having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of any one of the sequences in Table 1, or a fragment of any one of the sequences in Table 1, or a variant of any one of the sequences in Table 1. In one embodiment, the coding sequence of the mRNA vaccines described herein can be based on the coding sequence of gN of VZV. In some embodiments, the nucleic acid vaccines described herein can encode gN having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence of gN of VZV in Table 1.

[0100] In some embodiments, the nucleic acid vaccines can be mRNA vaccines that, upon translation, produce one or more proteins, peptides, fragments, or variants of a structural protein of VZV. Thus, the polynucleotide of the mRNA vaccine is an mRNA polynucleotide that encodes one or more proteins, polypeptides, peptides, fragments, or variants of a structural protein of VZV.

[0101] In some embodiments, the nucleic acid vaccines can be mRNA vaccines that, upon translation, produce one or more proteins, peptides, fragments, or variants of a glycoprotein of VZV. As non-limiting examples, the glycoprotein can be E, B, C, H, I, K, L, M, and / or N. Thus, the polynucleotide of the mRNA vaccine is an mRNA polynucleotide that encodes one or more proteins, polypeptides, peptides, fragments, or variants of a glycoprotein of VZV.

[0102] In one embodiment, the coding sequence of the mRNA vaccine described herein can be based on a coding sequence from a non-gE glycoprotein (G) from VZV. In some embodiments, the nucleic acid vaccine described herein can encode a glycoprotein having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the sequence of a non-gE glycoprotein (G) from VZV.

[0103] In some embodiments, the nucleic acid vaccine described herein comprises an mRNA polynucleotide encoding a protein, polypeptide, antigenic peptide, fragment, or variant of a VZV structural protein, for example, but not limited to, those in Table 1.

[0104] Non-limiting examples of RNA sequences encoding a protein, antigenic peptide, fragment, or variant of a glycoprotein E of VZV are provided in Table 2.

[0105] Table 2. Sequence of gE of VZV

[0106]

[0107]

[0108] In some embodiments, the mRNA polynucleotide encoding a gE of VZV comprises a coding sequence of SEQ ID NO: 6-20 or 67-76, or a variant thereof.

[0109] In some embodiments, the nucleic acid vaccine can comprise a region encoding any one of the sequences listed in Table 1, or a fragment or antigenic peptide or variant thereof. The nucleic acid vaccine can comprise a hybrid or chimeric region, or a mimic or variant. In some embodiments, the nucleic acid vaccine can comprise any one of the polynucleotide sequences listed in Tables 2-3.

[0110] Table 3. Exemplary sequences for use in a nucleic acid vaccine for treating or preventing herpes zoster

[0111]

[0112]

[0113] Any of the sequences mentioned in Tables 1-3, or variants thereof, can also be used in the memory boosting vaccines described herein. In some embodiments, any of the sequences mentioned in Tables 1-3, or variants thereof, can also be used in a boosting vaccine shortly after infection with VZV or VZV re-infection / reactivation.

[0114] In some embodiments, the nucleic acid vaccines described herein encode a protein or fragment or variant thereof that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the protein provided by the amino acid sequence in Table 1. In some embodiments, the nucleic acid vaccines described herein encode a protein or fragment or variant thereof that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the glycoprotein provided by the amino acid sequence in Table 2. In the context of two or more polypeptide sequences, the term “identical” or percent “identity” means two or more sequences that are the same. The percent identity between polypeptide sequences can be performed using algorithms known in the art, such as BLAST and CLUSTAL.

[0115] The sequence of the structural protein or fragment or antigenic peptide or variant of VZV can be obtained from any source. In some embodiments, the sequence of the structural protein or fragment or antigenic peptide or variant of VZV is from a strain that is capable of infecting and / or at risk of infecting a human subject and / or an animal subject.

[0116] In some embodiments, the polynucleotide sequence of the structural protein or fragment or antigenic peptide or variant of VZV can be modified or optimized (e.g., codon optimized) for expression in a particular cell or host organism.

[0117] In some embodiments, the nucleic acid vaccines described herein can be a multivalent vaccine. The multivalent vaccine can include a polynucleotide encoding at least two different proteins, polypeptides, peptides, fragments or variants thereof of VZV. As a non-limiting example, the polynucleotide can encode the same or different structural proteins. As a non-limiting example, the polynucleotide can encode the same class of structural proteins but different types of structural proteins.

[0118] In some embodiments, the nucleic acid vaccine encodes a full-length glycoprotein E of VZV. In some embodiments, the nucleic acid vaccine encodes a fragment of glycoprotein E of VZV. In some embodiments, the nucleic acid vaccine encodes an antigenic peptide of glycoprotein E of VZV. In some embodiments, the nucleic acid vaccine encodes a variant of glycoprotein E of VZV, wherein the N-terminal signal peptide is removed and / or replaced.

[0119] In some embodiments, the nucleic acid vaccine encodes a full-length glycoprotein E of VZV. As non-limiting examples, the nucleic acid vaccine can comprise SEQ ID NO: 6, 11, 16, 67, and / or 72, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 6, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 11, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 16, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 67, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 72, or a fragment or variant thereof.

[0120] In some embodiments, the nucleic acid vaccine encodes a gE of VZV, wherein the gE sequence is truncated as compared to the gE sequence set forth in Table 1.

[0121] In some embodiments, the nucleic acid vaccine encodes a truncated gE of VZV, wherein the truncation begins at amino acid 563 of SEQ ID NO: 2 (e.g., amino acids 563-623 are not encoded).

[0122] In some embodiments, the nucleic acid vaccine encodes a truncated gE of VZV, wherein the truncation begins at amino acid 574 of SEQ ID NO: 2 (e.g., amino acids 574-623 are not encoded).

[0123] In some embodiments, the nucleic acid vaccine encodes a truncated gE of VZV, wherein the truncation begins at amino acid 578 of SEQ ID NO: 2 (e.g., amino acids 578-623 are not encoded).

[0124] In some embodiments, the nucleic acid vaccine encodes a truncated gE of VZV, wherein the truncation begins at amino acid 563 of SEQ ID NO: 2 (e.g., amino acids 563-623 are not encoded), and wherein the wild-type signal peptide of gE is replaced with a human IgG kappa light chain signal peptide.

[0125] In some embodiments, the nucleic acid vaccine encodes a truncated gE of VZV, wherein the truncation begins at amino acid 574 of SEQ ID NO: 2 (e.g., amino acids 574-623 are not encoded), and wherein the wild-type signal peptide of gE is replaced with a human IgG kappa light chain signal peptide.

[0126] In some embodiments, the nucleic acid vaccine encodes a truncated gE of VZV, wherein the truncation begins at amino acid 563 of SEQ ID NO: 2 (e.g., amino acids 563-623 are not encoded), and wherein the wild-type signal peptide of gE is replaced with a human IgG kappa light chain signal peptide. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 9, 14, 19, 70, and / or 75, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 9, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 14, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 19, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 70, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 75, or a fragment or variant thereof.

[0127] In some embodiments, the nucleic acid vaccine encodes a truncated gE of VZV, wherein the truncation begins at amino acid 563 of SEQ ID NO: 2 (e.g., amino acids 563-623 are not encoded), and wherein the wild-type signal peptide of gE is replaced with a human IgG kappa light chain signal peptide. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 9, 14, 19, 70, and / or 75, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 9, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 14, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 19, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 70, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 75, or a fragment or variant thereof.

[0128] In some embodiments, the nucleic acid vaccine encodes a truncated gE of VZV, wherein the gE sequence is truncated compared to the gE sequence set forth in Table 1, and contains at least one mutation or deletion.

[0129] In some embodiments, the nucleic acid vaccine encodes a truncated gE of VZV, wherein the truncation begins at amino acid 563 of SEQ ID NO: 2 (e.g., amino acids 563-623 are not encoded), and wherein the wild-type signal peptide of gE is replaced with a human IgG kappa light chain signal peptide. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 9, 14, 19, 70, and / or 75, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 9, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 14, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 19, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 70, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can comprise SEQ ID NO: 75, or a fragment or variant thereof.

[0130] In some embodiments, the nucleic acid vaccine encodes a truncated gE of VZV, wherein the wild-type signal peptide of gE is replaced with a human IgG kappa light chain signal peptide, wherein the truncation begins at amino acid 578 of SEQ ID NO: 2 (i.e., amino acids 578-623 are not encoded), and wherein the gE of VZV includes a deletion of amino acids 563-565 of SEQ ID NO: 2 (i.e., RMR) and amino acids 568-571 of SEQ ID NO: 2 (i.e., AYRV (SEQ ID NO: 36)). As a non-limiting example, the nucleic acid vaccine can include SEQ ID NO: 8, 13, 18, 69, and / or 74, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can include SEQ ID NO: 8, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can include SEQ ID NO: 13, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can include SEQ ID NO: 18, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can include SEQ ID NO: 69, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can include SEQ ID NO: 74, or a fragment or variant thereof.

[0131] In some embodiments, the nucleic acid vaccine encodes a gE of VZV, wherein the wild-type signal peptide of gE is replaced with a human IgG kappa light chain signal peptide, and wherein the gE of VZV includes a deletion of amino acids 563-565 of SEQ ID NO: 2 (i.e., RMR), amino acids 568-571 of SEQ ID NO: 2 (i.e., AYRV (SEQ ID NO: 36)), amino acids 581-585 of SEQ ID NO: 2 (i.e., YYAGL (SEQ ID NO: 37)), and amino acids 593-598 of SEQ ID NO: 2 (i.e., SESTDT (SEQ ID NO: 38)). As a non-limiting example, the nucleic acid vaccine can include SEQ ID NO: 10, 15, 20, 71, and / or 76, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can include SEQ ID NO: 10, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can include SEQ ID NO: 15, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can include SEQ ID NO: 20, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can include SEQ ID NO: 71, or a fragment or variant thereof. As a non-limiting example, the nucleic acid vaccine can include SEQ ID NO: 76, or a fragment or variant thereof.

[0132] In some embodiments, the nucleic acid vaccine encodes a full-length glycoprotein E of VZV, wherein the nucleic acid vaccine can comprise SEQ ID NO: 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 or a fragment or variant thereof and at least one stop codon. The stop codon(s) can be any stop codon known in the art, such as, but not limited to, the stop codons described herein. As a non-limiting example, the nucleic acid vaccine comprises one stop codon. As a non-limiting example, the nucleic acid vaccine comprises two stop codons. As a non-limiting example, the nucleic acid vaccine comprises two stop codons selected from UGA, UAA, UGA, or UAG. As a non-limiting example, the nucleic acid vaccine comprises two stop codons, and each stop codon is UGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 67 and at least one stop codon. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 67 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 67 and two stop codons, wherein the two stop codons are both UGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 68 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 68 and two stop codons, wherein the two stop codons are both UGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 69 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 69 and two stop codons, wherein the two stop codons are both UGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 70 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 70 and two stop codons, wherein the two stop codons are both UGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 71 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 71 and two stop codons, wherein the two stop codons are both UGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 72 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 72 and two stop codons, wherein the two stop codons are both UGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 73 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 73 and two stop codons, wherein the two stop codons are both UGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 74 and two stop codons.As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 74 and two stop codons, wherein both stop codons are UGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 75 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 75 and two stop codons, wherein both stop codons are UGA. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 76 and two stop codons. As a non-limiting example, the nucleic acid vaccine comprises SEQ ID NO: 76 and two stop codons, wherein both stop codons are UGA.

[0133] Components of nucleic acid vaccines

[0134] In some embodiments, the polynucleotides described herein encode at least one polypeptide of interest, such as one or more proteins, peptides, fragments, or variants thereof of VZV. The proteins, polypeptides, peptides, fragments, or variants thereof of VZV of the present disclosure can be wild-type, in which they are derived from an infectious agent, or modified (e.g., the structural proteins or fragments and variants thereof are engineered, designed, or artificial). They can have any combination of the features described herein.

[0135] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode one or more peptides or polypeptides of interest. Such peptides or polypeptides are structural proteins or fragments or variants thereof of VZV that are used to prevent, ameliorate, and / or treat shingles. As non-limiting examples, these peptides or polypeptides can be used as antigens or antigenic molecules (also preferably as immunogenic molecules). The term “nucleic acid” includes, in its broadest sense, any compound and / or substance comprising a polymer of nucleotides. These polymers are often referred to as polynucleotides.

[0136] Exemplary nucleic acids or polynucleotides include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNAs with a beta-D-ribo configuration, alpha-LNAs (diastereomers of LNAs) with an alpha-L-ribo configuration, 2'-amino-LNAs with 2'-amino functionalization, and 2'-amino-alpha-LNAs with 2'-amino functionalization), ethylene nucleic acids (ENAs), cyclohexene-based nucleic acids (CeNAs), or hybrids or combinations thereof.

[0137] In some embodiments, linear polynucleotides encoding one or more proteins, peptides, fragments, or variants thereof of VZV of the present disclosure (referred to as “IVT polynucleotides”) can be prepared using in vitro transcription (IVT) enzymatic synthesis methods.

[0138] In some embodiments, the nucleic acid vaccine can include a“chimeric polynucleotide” having portions or regions of different sizes and / or encoding different proteins (e.g., structural proteins of VZV). A“chimera” is an entity having two or more discordant or heterogeneous portions or regions. As used herein, a“portion” or“region” of a polynucleotide is defined as any portion of a polynucleotide that is less than the full length of the polynucleotide. As non-limiting examples, the chimeric polynucleotides of the present disclosure can include a region encoding a heterogeneous signal peptide, such as the signal peptide of an immunoglobulin light chain.

[0139] In some embodiments, the nucleic acid vaccine comprises a polynucleotide of about 30 to about 100,000 nucleotides in length (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 30 to 25,000, 30 to 50,000, 30 to 70,000, 100 to 250, 100 to 500, 100 to 1,000, 100 to 1,500, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 500 to 25,000, 500 to 50,000, 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1,000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000, 1,500 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000, and 2,000 to 100,000 nucleotides).

[0140] In some embodiments, the nucleic acid vaccine comprises at least one polynucleotide encoding at least one peptide or polypeptide of interest. In another embodiment, the polynucleotide can be non-coding.

[0141] In some embodiments, the region of the polynucleotide of the nucleic acid vaccine that encodes at least one peptide or polypeptide of interest is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or up to and including 100,000 nucleotides). Such a region can be referred to as a “coding region” or a “region encoding” as used herein.

[0142] In some embodiments, the polynucleotide of the nucleic acid vaccine is or functions as messenger RNA (mRNA). As used herein, the term “messenger RNA (mRNA)” refers to any polynucleotide that encodes at least one peptide or polypeptide of interest and is capable of being translated in vitro, in vivo, in situ, or ex vivo to produce the encoded peptide or polypeptide of interest.

[0143] The minimum length of the region of the polynucleotide of the nucleic acid vaccine can be a length of nucleic acid sequence sufficient to encode a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In another embodiment, the length can be sufficient to encode a peptide of 2-30 amino acids, such as 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length can be sufficient to encode a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or a peptide no more than 40 amino acids long, such as no more than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids long. Examples of dipeptides that can be encoded by a polynucleotide sequence include, but are not limited to, myriocin and anserine.

[0144] The length of the polynucleotide region of a nucleic acid vaccine encoding one or more proteins, peptides, fragments or variants thereof of VZV for preventing, ameliorating and / or treating herpes zoster can be greater than about 30 nucleotides. The length can be, but is not limited to, at least or greater than about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000, or up to and including 100,000 nucleotides.In some embodiments, the region comprises about 30 to about 100,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 1,000, 30 to 1,500, 30 to 3,000, 30 to 5,000, 30 to 7,000, 30 to 10,000, 30 to 25,000, 30 to 50,000, 30 to 70,000, 100 to 250, 100 to 500, 100 to 1,000, 100 to 1,500, 100 to 3,000, 100 to 5,000, 100 to 7,000, 100 to 10,000, 100 to 25,000, 100 to 50,000, 100 to 70,000, 100 to 100,000, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 3,000, 500 to 5,000, 500 to 7,000, 500 to 10,000, 500 to 25,000, 500 to 50,000, 500 to 70,000, 500 to 100,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 3,000, 1,000 to 5,000, 1,000 to 7,000, 1,000 to 10,000, 1,000 to 25,000, 1,000 to 50,000, 1,000 to 70,000, 1,000 to 100,000, 1,500 to 3,000, 1,500 to 5,000, 1,500 to 7,000, 1,500 to 10,000, 1,500 to 25,000, 1,500 to 50,000, 1,500 to 70,000, 1,500 to 100,000, 2,000 to 3,000, 2,000 to 5,000, 2,000 to 7,000, 2,000 to 10,000, 2,000 to 25,000, 2,000 to 50,000, 2,000 to 70,000, and 2,000 to 100,000 nucleotides).

[0145] mRNA component

[0146] The nucleic acid vaccines described herein can be mRNA vaccines. An mRNA vaccine includes at least one mRNA molecule that, when translated, produces at least one peptide or polypeptide of interest for preventing, ameliorating, and / or treating herpes zoster. Generally, an mRNA molecule typically includes at least a coding region, a 5’ untranslated region (UTR), a 3’ UTR, a 5’ cap, and a poly-A tail.

[0147] mRNA components: start and stop codons

[0148] In some embodiments, the mRNA comprises a region that initiates translation. The region can comprise any translation initiation sequence or signal comprising an initiation codon. As non-limiting examples, the region comprises an initiation codon. In some embodiments, the initiation codon can be “ATG,” “ACG,” “AGG,” “ATA,” “ATT,” “CTG,” “GTG,” “TTG,” “AUG,” “AUA,” “AUU,” “CUG,” “GUG,” or “UUG.”

[0149] In some embodiments, the mRNA comprises a region that terminates translation. The region can comprise any translation termination sequence or signal comprising a termination codon. As non-limiting examples, the region comprises a termination codon. In some embodiments, the termination codon can be “TGA,” “TAA,” “TGA,” “TAG,” “UGA,” “UAA,” “UGA,” or “UAG.”

[0150] In some embodiments, the region that initiates or terminates translation can be independently 3 to 40, for example 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length. In addition, these regions can comprise one or more signals and / or restriction sequences in addition to the initiation and / or termination codon.

[0151] In some embodiments, a masking agent can be used to mask the first initiation codon or the alternative initiation codon to increase the chance that translation will initiate on an initiation codon or alternative initiation codon downstream of the masked initiation codon or alternative initiation codon.

[0152] In some embodiments, an initiation codon can be removed from a polynucleotide sequence to cause the polynucleotide to begin translation on a codon that is not an initiation codon. Translation of the polynucleotide can begin on a codon after the removed initiation codon, or on a downstream initiation codon or alternative initiation codon. The polynucleotide sequence with the removed initiation codon can further comprise at least one masking agent for the downstream initiation codon and / or alternative initiation codon to control or attempt to control the initiation of translation, the length of the polynucleotide, and / or the structure of the polynucleotide.

[0153] mRNA components: coding region

[0154] In some embodiments, the coding region of the polynucleotide of the nucleic acid vaccine can encode at least one peptide or polypeptide of interest. Non-limiting examples of the peptide or polypeptide of interest include one or more proteins, polypeptides, peptides, fragments, or variants thereof of VZV for preventing, ameliorating, and / or treating shingles and diseases caused by VZV infection.

[0155] mRNA components: untranslated regions

[0156] The polynucleotides of the nucleic acid vaccines described herein can comprise one or more regions or portions that act as or function as untranslated regions (UTRs). Wild-type UTRs of a gene are transcribed but not translated. In mRNA, the 5’ UTR begins at the transcription start site and continues to the start codon, but does not include the start codon; while the 3’ UTR begins immediately after the stop codon and continues to the transcription termination signal. Without wishing to be bound by theory, UTRs can play a role in the stability of the nucleic acid molecule and translation. Variants of UTRs can be used in which one or more nucleotides (e.g., A, T / U, C, or G) are added to the end of the UTR or removed.

[0157] In some embodiments, the UTRs of the polynucleotides of the nucleic acid vaccines can independently be 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).

[0158] Wild-type 5’ UTRs include features that play a role in translation initiation, as these 5’ UTRs include sequences known to be involved in how ribosomes initiate translation of many genes, such as the Kozak sequence. 5’ UTRs are also known to form secondary structures involved in elongation factor binding. Other non-UTR sequences (e.g., introns or portions of intronic sequences) can also be used as regions or sub-regions that can increase protein production as well as polynucleotide levels.

[0159] Natural or wild-type 3’ UTRs are known to have adenosine and uridine stretches embedded within them. These AU-rich tags are particularly prevalent in genes with high turnover rates. The introduction, removal, or modification of 3’ UTR AU-rich elements (AREs) can be used to modulate the stability of the polynucleotides of the nucleic acid vaccines.

[0160] UTRs from any gene can be incorporated into the polynucleotide region of a nucleic acid vaccine. Alternatively, artificial UTRs that are not variants of wild-type regions can also be used in the polynucleotide of a nucleic acid vaccine. These UTRs or portions thereof can be placed in the same orientation as in the transcript from which they are selected, or the orientation or position can be changed. As used herein, the term "altered" when it refers to a UTR sequence means that the UTR has been altered in some way relative to a reference sequence. As non-limiting examples, a 5' or 3' UTR can be inverted, shortened, lengthened, made from one or more other 5' UTRs or 3' UTRs from different parent sequences.

[0161] In some embodiments, the flanking regions are selected from a family of transcripts whose proteins share common functional, structural, property characteristics. For example, the polypeptides of interest can belong to a family of proteins that are expressed in a particular cell, tissue, or at a certain time during development. UTRs from any of these genes can be exchanged with any other UTR from the same or different protein family to create a new polynucleotide. As used herein, "protein family" is used in the broadest sense to refer to a group of two or more polypeptides of interest that share at least one functional, structural, characteristic, localization, origin, or expression pattern.

[0162] The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5’ UTR having the sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). In some embodiments, the 5’ UTR of the polynucleotide of the nucleic acid vaccine disclosed herein consists of the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). In some embodiments, the 5’ UTR is directly 5’ of the start codon of the sequence encoding the VZV polypeptide of the nucleic acid vaccine. In some embodiments, the 5’ UTR is directly 5’ of the start codon of the sequence encoding the signal peptide of the nucleic acid vaccine. In some embodiments, the 5’ UTR is 1, 2, 3, 4, 5, 6, or more nucleotides from the start codon of the sequence encoding the VZV polypeptide of the nucleic acid vaccine; for example, a spacer of 1, 2, 3, 4, 5, 6, or more nucleotides separates the 5’ UTR from the start codon of the sequence encoding the VZV polypeptide of the nucleic acid vaccine. The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5’ UTR having a sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5’ UTR having a sequence with at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5’ UTR having a sequence with at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5’ UTR having a sequence with at least 91% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5’ UTR having a sequence with at least 92% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5’ UTR having a sequence with at least 93% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5’ UTR having a sequence with at least 94% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA).The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5' UTR having a sequence with at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5' UTR having a sequence with at least 96% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5' UTR having a sequence with at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5' UTR having a sequence with at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5' UTR having a sequence with at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5' UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 39 (DNA) or SEQ ID NO: 40 (RNA).

[0163] The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having the sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). In some embodiments, the 3’ UTR of the polynucleotide of the nucleic acid vaccine disclosed herein consists of the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). In some embodiments, the 3’ UTR is directly 3’ of the last codon of the sequence encoding the VZV polypeptide of the nucleic acid vaccine. In some embodiments, the 3’ UTR is 1, 2, 3, 4, 5, 6, or more nucleotides 3’ from the last codon of the sequence encoding the VZV polypeptide of the nucleic acid vaccine; for example, a spacer of 1, 2, 3, 4, 5, 6, or more nucleotides separates the 3’ UTR from the last codon of the sequence encoding the VZV polypeptide of the nucleic acid vaccine. The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 91% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 92% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 93% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 94% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA).The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 96% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 41 (DNA) or SEQ ID NO: 42 (RNA).

[0164] The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having the sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). In some embodiments, the 3’ UTR of the polynucleotide of the nucleic acid vaccine disclosed herein consists of the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). In some embodiments, the 3’ UTR is directly 3’ of the last codon of the sequence encoding the VZV polypeptide of the nucleic acid vaccine. In some embodiments, the 3’ UTR is 1, 2, 3, 4, 5, 6, or more nucleotides 3’ from the last codon of the sequence encoding the VZV polypeptide of the nucleic acid vaccine; for example, a spacer of 1, 2, 3, 4, 5, 6, or more nucleotides separates the 3’ UTR from the last codon of the sequence encoding the VZV polypeptide of the nucleic acid vaccine. The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 90% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 91% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 92% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 93% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 94% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 95% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA).The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 96% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 97% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 98% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA). The polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 3’ UTR having a sequence with at least 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 43 (DNA) or SEQ ID NO: 44 (RNA).

[0165] mRNA components: Cap and IRES sequences

[0166] In some embodiments, the polynucleotide of the nucleic acid vaccine disclosed herein can comprise a 5’ cap structure. The 5’ cap structure of natural mRNA is involved in nuclear export, increases the stability of the mRNA and binds to the mRNA cap-binding protein (CBP), which is responsible for the stability and translational capacity of the mRNA in the cell by the association of CBP with the poly(A)-binding protein to form the mature, circular mRNA species. The cap further aids in the removal of 5’ proximal introns during mRNA splicing.

[0167] In some embodiments, the 5’ end capping region of the polynucleotide of the nucleic acid vaccine can comprise a single cap or a series of nucleotides forming a cap. The length of the capping region can be 1 to 10 nucleotides, for example 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides. In some examples, the capping region can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides. In some embodiments, the cap is not present.

[0168] In some embodiments, cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, can be used in the nucleic acid vaccine. Cap analogs that can be synthesized chemically (e.g., non-enzymatically) or enzymatically differ in their chemical structure from natural (e.g., endogenous, wild-type, or physiological) 5’ caps, but they retain cap function.

[0169] In some embodiments, the 5’ end cap of the polynucleotide of the nucleic acid vaccine can include an endogenous cap or a cap analog. As non-limiting examples, the 5’ end cap can comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1 -methyl-guanosine (m1G), 2’-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0170] One skilled in the art will appreciate that 5’ capping can be produced enzymatically or by other synthetic methods. Endogenous mRNA molecules are 5’ end capped, producing a 5’-ppp-5’ triphosphate linkage between the terminal guanosine cap residue and the 5’-end transcribed sense nucleotide of the mRNA molecule. This 5’-guanylate cap can then be methylated to produce an N7-methyl-guanosine acid residue. The ribose sugar of the terminal and / or pre-terminal transcribed nucleotide of the 5’ end of the mRNA can also optionally be 2’-O-methylated. Targeting of nucleic acid molecules, such as mRNA molecules, for degradation can be targeted by hydrolysis and cleavage of the 5’-de-capping of the guanylate cap structure.

[0171] The polynucleotides of the nucleic acid vaccines described herein, e.g., mRNA, can be modified to include non-hydrolysable cap structures that prevent de-capping and thus increase mRNA half-life. Because cap structure hydrolysis requires cleavage of the 5’-ppp-5’ phosphodiester linkage, modified nucleotides can be used during the capping reaction. For example, vaccinia virus capping enzymes, available from, e.g., New England Biolabs (Ipswich, MA), can be used with alpha-thio-guanosine nucleotides to produce a thio-phosphate linkage in the 5’-ppp-5’ cap, according to the manufacturer’s instructions. Additional modified guanosine nucleotides can be used, e.g., alpha-methyl-phosphonate and seleno-phosphate nucleotides.

[0172] Additional modifications include, but are not limited to, 2’-O-methylation of the ribose sugar on the 2’-hydroxyl of the sugar ring of the 5’-terminal and / or 5’-pre-terminal nucleotide of the mRNA (as described above). A variety of different 5’-cap structures can be used to produce the 5’-cap of a nucleic acid molecule, such as an mRNA molecule.

[0173] Cap analogs (also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs) differ in their chemical structure from natural (i.e., endogenous, wild-type, or physiological) 5’-caps, yet retain cap function. Cap analogs can be chemically (e.g., non-enzymatically) or enzymatically synthesized and attached to a nucleic acid molecule, such as an mRNA molecule.

[0174] For example, the anti-reverse cap analog (ARCA) cap contains two guanosines linked by a 5'-5'-triphosphate group, one of which contains an N7 methylation and a 3'-O-methylation (i.e., N7, 3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m7G-3'mppp-G; which can be equivalently designated as 3'0-Me-m7G(5')ppp(5')G). The 3'-O atom of the other unmodified guanosine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., mRNA). The N7- and 3'-O-methylated guanosine provides a terminal moiety of the capped nucleic acid molecule (e.g., mRNA).

[0175] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl on the guanosine (i.e., N7, 2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[0176] While cap analogs allow for nucleic acid molecules to be accompanied by capping in in vitro transcription reactions, up to 20% of transcripts can remain uncapped. This, along with structural differences of cap analogs from endogenous 5'-cap structures, can result in reduced translational capacity and reduced cellular stability.

[0177] In exemplary aspects of the present disclosure, polynucleotides (e.g., mRNA) can be capped post-transcriptionally using enzymes. For example, a recombinant vaccinia virus capping enzyme and a recombinant 2'-O-methyltransferase can produce a canonical 5'-5'-triphosphate linkage between the 5'-terminal nucleotide and the guanine cap nucleotide of an mRNA, wherein the cap guanine contains an N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2'-O-methyl. This structure is referred to as a Cap 1 structure. In some embodiments, Cap 1 structures provide higher translational capacity and cellular stability and reduced cellular activation of proinflammatory cytokines compared to other 5' cap analog structures known in the art, for example. Cap structures include 7mG(5')ppp(5')N, pN2p (Cap 0), 7mG(5')ppp(5')N1mpNp (Cap 1), and 7mG(5')-ppp(5')N1mpN2mp (Cap 2).

[0178] In one embodiment, the polynucleotides of the nucleic acid vaccines described herein comprise a Cap 1 structure.

[0179] Because polynucleotides (e.g., mRNA) can be capped post-transcriptionally, and because this process is more efficient, up to 100% of polynucleotides (e.g., mRNA) can be capped. In contrast to this, about 80% when cap analogs are linked to mRNA during in vitro transcription reactions.

[0180] In some embodiments, the polynucleotide of the nucleic acid vaccine can contain a sequence that enhances capping of the polynucleotide sequence. As non-limiting examples, the sequence can be at least 70%, 75%, 80%, 85%, 90%, 99%, or more identical to the sequence provided in SEQ ID NO: 56 (DNA) or SEQ ID NO: 57 (RNA). As non-limiting examples, the sequence comprises SEQ ID NO: 56 (DNA) or SEQ ID NO: 57 (RNA).

[0181] In some embodiments, the polynucleotide of the nucleic acid vaccine can contain an internal ribosome entry site (IRES) sequence. Without wishing to be bound by theory, IRES plays an important role in initiating protein synthesis in the absence of a 5' cap structure. The IRES can act as the sole ribosome binding site or can be used as one of multiple ribosome binding sites for an mRNA.

[0182] mRNA components: tailing region

[0183] In some embodiments, the polynucleotide of the nucleic acid vaccine, e.g., mRNA, comprises a tailing region. Non-limiting examples of tailing regions include a poly-A sequence, a poly-C sequence, and / or a polyA-G quartet.

[0184] In some embodiments, the mRNA comprises a chain terminating nucleoside. Non-limiting examples of chain terminating nucleosides include 2'-O methyl, F, and locked nucleic acid (LNA).

[0185] In some embodiments, the tailing region sequence of the polynucleotide of the nucleic acid vaccine can range in length from none to 500 nucleotides (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). If the tailing region is a poly-A tail, the length can be described in units of or as a function of poly-A binding protein binding.

[0186] In some embodiments, the poly-A tail can also be added after the construct is exported from the nucleus.

[0187] In some embodiments, long chains of adenine nucleotides (poly-A tails) can be added to polynucleotides such as mRNA molecules during RNA processing to increase stability. Immediately following transcription, the 3’ end of the transcript can be cleaved to release a 3’ hydroxyl. Poly-A polymerase then adds a chain of adenine nucleotides to the RNA. This process is known as polyadenylation, adding a poly-A tail of length that can be between, for example, about 80 to about 250 residues, including poly-A tails of length about 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 residues.

[0188] In some embodiments, when present, the length of the poly-A tail is greater than 30 nucleotides in length (e.g., at least or greater than about 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the poly-A tail region thereof comprises about 30 to about 3,000 nucleotides (e.g., 30 to 50, 30 to 100, 30 to 250, 30 to 500, 30 to 750, 30 to 1,000, 30 to 1,500, 30 to 2,000, 30 to 2,500, 50 to 100, 50 to 250, 50 to 500, 50 to 750, 50 to 1,000, 50 to 1,500, 50 to 2,000, 50 to 2,500, 50 to 3,000, 100 to 500, 100 to 750, 100 to 1,000, 100 to 1,500, 100 to 2,000, 100 to 2,500, 100 to 3,000, 500 to 750, 500 to 1,000, 500 to 1,500, 500 to 2,000, 500 to 2,500, 500 to 3,000, 1,000 to 1,500, 1,000 to 2,000, 1,000 to 2,500, 1,000 to 3,000, 1,500 to 2,000, 1,500 to 2,500, 1,500 to 3,000, 2,000 to 3,000, 2,000 to 2,500, and 2,500 to 3,000 nucleotides).

[0189] In some embodiments, the length of the poly-A tail is about 100 nucleotides (SEQ ID NO: 45).

[0190] In some embodiments, the poly-A tail is designed relative to the length of the entire polynucleotide or the length of a particular region of the polynucleotide. The design can be based on the length of the coding region, the length of a particular feature or region, or based on the length of the final product expressed from the polynucleotide.

[0191] In this case, the poly-A tail can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer than the polynucleotide or feature thereof. The poly-A tail can also be designed to be part of the polynucleotide to which it belongs. In this case, the poly-A tail can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the total length of the construct, the region of the construct, or the total length of the construct minus the poly-A tail. Furthermore, conjugation of the engineered binding site and the polynucleotide to a Poly-A binding protein can enhance expression.

[0192] In some embodiments, there can be a spacer region in the polynucleotide, such as but not limited to a polyadenylation sequence. There can be one or more such spacer regions.

[0193] In some embodiments, the spacer region can be between 3-25 nucleotides, i.e., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. The spacer region can be derived from another sequence, such as but not limited to a restriction site. As a non-limiting example, the restriction site can be Spel and can comprise the sequence ACTAGT.

[0194] The polynucleotide, including regions thereof, can have a “patterned polyadenylation sequence”. Patterned polyadenylation sequences are those polynucleotides that have a pattern of residues or regions of residues within, across, or between regions of the polynucleotide.

[0195] Patterns of polyadenylation sequences that begin and end within a defined region. Patterns of polyadenylation sequences that span portions or regions are those that begin in one portion or region and end in another adjacent portion or region. Patterns of polyadenylation sequences between portions or regions are those that begin and end in one portion or region and repeat in a different portion or region, which is not necessarily adjacent to the first region or portion.

[0196] Regions or sub-regions of a pattern can have a simple alternating pattern, such as ABAB[AB]n, where each "A" and each "B" represents a different sequence (e.g., adenosine residues, restriction sites, spacer sequences). The pattern can be repeated n times, where n = 3-300. Further, each A or B can represent 1-2500 units (e.g., nucleosides) in the pattern. The pattern can also be an alternating multiple, such as AABBAABB[AABB]n (alternating doublet) or AAABBBAAABBB[AAABBB]n (alternating triplet) pattern. The pattern can be repeated n times, where n = 3-300.

[0197] Different patterns can also be mixed together to form a second order pattern. For example, a single alternating pattern can be combined with a triple alternating pattern to form a second order alternating pattern A'B'. One example is [ABABAB][AAABBBAAABBB][ABABAB][AAABBBAAABBB][ABABAB][AAABBBAAABBB], where [ABABAB] is A' and [AAABBBAAABBB] is B'. In a similar manner, these patterns can be repeated n times, where n = 3-300.

[0198] Patterns can include three or more different sequences to form an ABCABC[ABC]n pattern. These three component patterns can also be multiples, such as AABBCCAABBCC[AABBCC]n, and can be designed to be combined with other patterns, such as ABCABCAABBCCABCABCAABBCC, and can be higher order patterns.

[0199] The regions or sub-regions of positions, percentages, and population modifications need not reflect equal contributions from each class of sequence (e.g., adenosine residues, restriction sites, spacer sequences). They can form series such as "1-2-3-4", "1-2-4-8", where each integer represents the number of units of a particular sequence type. Alternatively, they can be odd numbers only, such as "1-3-3-1-3-1-5" or even numbers only "2-4-2-4-6-4-8" or a mixture of odd and even units, such as "1-3-4-2-5-7-3-3-4".

[0200] In some embodiments, the tailing region comprises a spacer sequence as part of the tailing sequence.

[0201] Signal sequence

[0202] In some embodiments, the polynucleotides of the nucleic acid vaccines can also encode additional features that can facilitate transport of the polypeptides to the therapeutically relevant site. One such feature that aids in protein transport is a signal sequence. As used herein, a “signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, of about 9 to 200 nucleotides (3-60 amino acids) in length that is incorporated into the 5’ end of a coding region or the N-terminus of a coded polypeptide, respectively. In some embodiments, the addition of these sequences results in the transport of the coded polypeptide to the endoplasmic reticulum via one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidases after the protein is transported.

[0203] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein include a signal sequence comprising SEQ ID NO: 46 (DNA) or SEQ ID NO: 47 (RNA). In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode a signal sequence comprising SEQ ID NO: 48.

[0204] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein include a signal sequence comprising SEQ ID NO: 49 (DNA) or SEQ ID NO: 50 (RNA). In some embodiments, the polynucleotides of the nucleic acid vaccines described herein encode a signal sequence comprising SEQ ID NO: 51.

[0205] Codon optimization

[0206] The polynucleotides of the nucleic acid vaccines, regions or portions or subregions thereof, can be codon optimized. Methods of codon optimization are known in the art and can be used in the work to achieve one or more of several goals. These goals include, but are not limited to, matching codon frequencies in the target and host organism to ensure proper folding, altering GC content to increase mRNA stability or reduce secondary structure, minimizing tandem repeated codons or runs of bases that can impair gene construction or expression, customizing transcription and translation control regions, inserting or removing protein transport sequences, removing / adding post-translational modification sites (e.g., glycosylation sites) in the encoded protein, adding, removing, or shuffling protein domains, inserting or deleting restriction sites, modifying ribosome binding sites and mRNA degradation sites to modulate translation rates to allow proper folding of various domains of the protein, or to reduce or eliminate problematic secondary structures within the polynucleotide. Codon optimization tools, algorithms, and services are known in the art, non-limiting examples of which include, but are not limited to, services and / or proprietary methods from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.). In some embodiments, the ORF sequences are optimized using optimization algorithms. Codon choices for each amino acid are given in Table 4.

[0207] Table 4. Codon Substitution

[0208]

[0209] In some embodiments, the nucleic acid vaccine is vectorized after codon optimization. Non-limiting examples of vectors include, but are not limited to, plasmids, viruses, cosmids, and artificial chromosomes.

[0210] Modifications

[0211] The nucleic acid vaccines of the present disclosure, including mRNA vaccines, can include one or more modifications. The term “modification” or, where appropriate, “modified” refers to a modification to an A, G, U, or C ribonucleotide. Generally, in this context, these terms are not intended to refer to ribonucleotide modifications in the naturally occurring 5’ end mRNA cap moiety. In polypeptides, the term “modification” refers to a modification compared to the standard set of 20 amino acids.

[0212] As described herein, a “nucleoside” is defined as a compound containing a combination of a sugar molecule (e.g., a pentose or ribose) or derivative thereof and an organic base (e.g., a purine or pyrimidine) or derivative thereof (also referred to herein as a “nucleobase”). As described herein, a “nucleotide” is defined as a nucleoside comprising a phosphate group or other backbone linkage (internucleoside linkage).

[0213] The modification can be various different modifications. In some embodiments, the coding region, untranslated region, flanking region, and / or terminal or tailing region can contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the nucleic acid vaccines of the present disclosure comprise one or more modifications that make the nucleic acid molecule more resistant to degradation in a cell and / or more stable in a cell when introduced into the cell compared to an unmodified polynucleotide.

[0214] The polynucleotides of the nucleic acid vaccines described herein can include any available modification, such as a modification to the sugar, nucleobase, or internucleoside linkage (e.g., to a linking phosphate / diester bond / phosphodiester backbone). One or more atoms of a pyrimidine nucleobase can be replaced or substituted, for example, with an optionally substituted amino group, an optionally substituted thiol, an optionally substituted alkyl group (e.g., methyl or ethyl), an optionally substituted or halogen (e.g., chlorine or fluorine) atom or group. 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 disclosure can be a modification of 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.

[0215] In some embodiments, the modifications include 2’-O-methyl modified or 2’-O- methoxyethyl modified nucleotides (2’-OMe and 2’-MOE modifications, respectively).

[0216] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein can comprise at least one modification described herein.

[0217] The polynucleotides of the nucleic acid vaccines described herein can include a combination of modifications to sugars, nucleobases, and / or internucleoside linkages.

[0218] Modifications to polynucleotides (e.g., RNA polynucleotides, such as mRNA polynucleotides) that can be used in the vaccines of the present disclosure include, but are not limited to, any modifications as described in PCT Publication WO2017070626, the contents of which are incorporated by reference herein in their entirety, including, for example, modifications or deletions of nucleotides (or codons) that encode one or more N-linked glycosylation sites in the translated polypeptide. Modifications that can be used in the vaccines of the present disclosure can also include any modifications as described in PCT Publication WO2018200892, the contents of which are incorporated by reference herein in their entirety. The vaccines of the present disclosure can further comprise features or modifications described in PCT Patent Application Publications WO2020255063, WO2020182869, WO2016011222, WO2016011226, WO2016005004, WO2016000792, WO2015176737, WO2015085318, WO2015048744, and WO2015034925, and U.S. Patent Application Publications US20200254086, US20200206362, US20180311336, and US20180303929; the contents of each of which are incorporated by reference herein in their entirety.

[0219] For example, a polynucleotide, including an mRNA molecule of the nucleic acid vaccines described herein, can include the following modifications. The internucleoside linkages of the polynucleotide can be partially or fully modified. The polynucleotide can comprise modifications to one or more nucleobases. The polynucleotide can comprise 5-methylcytosine in place of all cytosine nucleobases / cytidine nucleotides. Further, the polynucleotide can have one or more modifications to one or more sugar subunits of the nucleosides. The sugar modifications can be one or more locked nucleic acids (LNAs) or 2’-O-methoxyethyl modifications (“2’-MOE”). The polynucleotide can be designed with a patterned array of sugar, nucleobase, or linkage modifications. In some embodiments, the polynucleotide can comprise modifications to maximize stability. In some embodiments, the polynucleotide can be fully 2’-MOE-sugar modified.

[0220] Modified nucleobases

[0221] Modified nucleosides and nucleotides can include modified nucleobases. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleobases found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine.

[0222] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 5 s 2 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 5 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 1 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 5 s 2 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 1 s 4 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 3 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 5 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 1 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 3 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 3 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 5 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 5 s 2 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 5 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 2 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 5 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 5 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 5 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 3 U), 1 -thio-methyl-pseudouridine, 5-thio-2-thio-uridine (s 51-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2- methoxycarbonylvinyl)uridine, and 5-[3-(l-E-propenylamino)uridine.

[0223] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having a modified cytosine include 5-aza-cytidine, 6-aza- cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl-cytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5-halogeno-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio- cytidine (s 2 C), 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l-methyl-l-deaza-pseudoisocytidine, l-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2- methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4- methoxy-l-methyl-pseudoisocytidine, k2C, a-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m 5 Cm), N4-acetyl-2'-O-methyl-cytidine (ac 4 Cm), N4,2'-O-dimethyl-cytidine (m 4 Cm), 5-formyl-2'-O-methyl-cytidine (f 5 Cm), N4,N4,2'-O-trimethyl-cytidine (m 4 2Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0224] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m 1 A), 2-methyl-adenosine (m 2 A), N6-methyl-adenosine (m 6 A), 2-methylthio-N6-methyl-adenosine (ms 2 m 6 A), N6-isopentenyl-adenosine (i 6 A), 2-methylthio-N6-isopentenyl-adenosine (ms 2 i 6 A), N6-(cis-hydroxyisopentenyl)adenosine (io 6 A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2 io 6 A), N6-glycinamido-carbonyl-adenosine (g 6 A), N6-threoninamido-carbonyl-adenosine (t 6 A), N6-methyl-N6-threoninamido-carbonyl-adenosine (m 6 t 6 A), 2-methylthio-N6-threoninamido-carbonyl-adenosine (ms 2 g 6 A), N6,N6-dimethyl-adenosine (m 6 2A), N6-hydroxynorvalinamido-carbonyl-adenosine (hn 6 A), 2-methylthio-N6-hydroxynorvalinamido-carbonyl-adenosine (ms 2 hn 6 A), N6-acetyl-adenosine (ac 6 A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a- thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N6,N6,2'-O-trimethyl-adenosine (m 6 2Am), 1,2'-O-dimethyl-adenosine (m 1Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2’-F-ara-adenosine, 2’-F-adenosine, 2’-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0225] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), iso-wyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanine, queuosine (Q), epoxy-queuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanine (preQ0), 7-aminomethyl-7-deaza-guanine (preQ1), gualosine (G + ), 7-deaza-8-aza-guanine, 6-thio-guanine, 6-thio-7-deaza-guanine, 6-thio-7-deaza-8-aza-guanine, 7-methyl-guanine (m 7 G), 6-thio-7-methyl-guanine, 7-methyl-inosine, 6-methoxy-guanine, 1-methyl-guanine (m 1 G), N2-methyl-guanine (m 2 G), N2,N2-dimethyl-guanine (m 2 2G), N2,7-dimethyl-guanine (m 2,7 G), N2,N2,7-trimethyl-guanine (m 2,2,7 G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thio-guanine, N2-methyl-6-thio-guanine, N2,N2-dimethyl-6-thio-guanine, a-thio-guanine, 2'-O-methyl-guanine (Gm), N2-methyl-2'-O-methyl-guanine (m 2 Gm), N2,N2-dimethyl-2'-O-methyl-guanine (m 2 2Gm), 1-methyl-2'-O-methyl-guanine (m 1 Gm), N2,7-dimethyl-2'-O-methyl-guanine (m 2,7 Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m 1 Im), and 2'-O-ribosylguanosine (phosphate) (Gr(p)).

[0226] The nucleobases of the nucleotides can be independently selected from purines, pyrimidines, purine or pyrimidine analogs. For example, the nucleobases can each be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleobases can also include, for example, naturally occurring and synthetic derivatives of the bases, including pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2- thiocytosine, 5-propynyluracil and cytosine; 6-azo uracil, cytosine and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8- halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8- substituted adenines and guanines; 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines; 7-methylguanine and 7-methyladenine, 8-azaguanine and 8- azadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazadenine, 7-deazadenine, 3- deazadenine, pyrazolo[3,4-d]pyrimidine, imidazo[l,5-a]l,3,5-triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine, and 1,3,5 triazine.

[0227] Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) can be introduced at various positions in the polynucleotides described herein. One of ordinary skill in the art will understand that nucleotide analogs or other modifications can be at any position of a polynucleotide such that the function of the polynucleotide is not substantially reduced. The polynucleotides of the present disclosure can contain from about 1% to about 100% modified nucleotides (relative to the total nucleotide content, or relative to one or more types of nucleotides, i.e., any one or more of A, G, T / U, or C), or any intermediate percentage (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%).

[0228] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein can be modified to be circular nucleic acids. The ends of the polynucleotides can be joined by a chemical reagent or enzyme, resulting in a circular polynucleotide that has no free ends. Circular polynucleotides are expected to be more stable than linear counterparts and resistant to exonuclease digestion. The circular polynucleotides can further comprise other structural and / or chemical modifications with respect to A, G, T / U, or C ribo / deoxyribonucleotides.

[0229] In some embodiments, the polynucleotides are at least 50% modified, e.g., at least 50% of the nucleotides are modified. In some embodiments, the polynucleotides are at least 75% modified, e.g., at least 75% of the nucleotides are modified. It will be understood that any modification to any portion of a nucleotide or nucleoside will constitute a modification, as the nucleotides (sugar, base, and phosphate moieties, e.g., linkages) can each be modified.

[0230] In some embodiments, the polynucleotide is modified by at least 10% in only one component of the nucleotides, the component being the nucleobase, sugar, or bond between nucleosides. For example, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the nucleobases, sugars, or bonds of a polynucleotide described herein can be modified.

[0231] As non-limiting examples, all of the uracil nucleosides of the polynucleotide of the nucleic acid vaccine are modified. The modifications can be the same or different. In some embodiments, all of the guanine nucleosides of the polynucleotide of the nucleic acid vaccine are modified. The modifications can be the same or different. In some embodiments, all of the guanine nucleosides of the polynucleotide of the nucleic acid vaccine are modified. The modifications can be the same or different. In some embodiments, all of the cytosine nucleosides of the polynucleotide of the nucleic acid vaccine are modified. The modifications can be the same or different. In some embodiments, all of the adenine nucleosides of the polynucleotide of the nucleic acid vaccine are modified. The modifications can be the same or different.

[0232] In one embodiment of the disclosure, the polynucleotide of the nucleic acid vaccine is modified to comprise N1-methyl-pseudo-uridine nucleotides.

[0233] Sugar modifications

[0234] Modified nucleosides and nucleotides that can be incorporated into a polynucleotide (e.g., an RNA or mRNA as described herein) can be modifications on the sugar of the ribonucleic acid. For example, the 2’ hydroxyl (OH) group can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2’-position include, but are not limited to, H, halogen, optionally substituted C 1-6 alkyl; optionally substituted C 1-6 alkoxy; optionally substituted C 6-10 aryloxy; optionally substituted C 3-8 cycloalkyl; optionally substituted C 3-8 cycloalkoxy; optionally substituted C 6-10 aryloxy; optionally substituted C 6-10 aryl-C 1-6 alkoxy, optionally substituted C 1-12 (heterocyclyl)oxy; sugar (e.g., ribose, pentose, or any sugar described herein); polyethylene glycol (PEG), -O(CH2CH2O) n CH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20); a “locked” nucleic acid (LNA), where the 2’-hydroxyl is tethered to the 3’-position of the sugar via a C 1-6alkylene or C 1-6 heteroalkylene bridge to the 4'-carbon of the same ribose, where exemplary bridges include methylene, propylene, ether, or amino bridges; aminoalkyl; aminoalkoxy; amino and amino acids.

[0235] In some embodiments, the mRNA of a polynucleotide, such as a nucleic acid vaccine described herein, comprises at least one sugar modification. Typically, RNA comprises a glycosyl ribose, which is a 5-membered ring with an oxygen. Exemplary, non-limiting modified nucleotides include replacement of the oxygen in the ribose (e.g., with S, Se, or an alkylene such as methylene or ethylene); addition of a double bond (e.g., replacement of the ribose with a cyclopentenyl or cyclohexenyl); ring contraction of the ribose (e.g., forming a 4-membered ring of cyclobutane or oxetane); ring expansion of the ribose (e.g., forming a 6- or 7-membered ring with an additional carbon or heteroatom, such as for an anhydrohexitol, altitol, mannitol, cyclohexyl, cyclohexenyl, and morpholino also with phosphoramidate backbones); polycyclic forms (e.g., tricyclic; and “unlocked” forms such as glycol nucleic acids (GNAs) (e.g., R-GNA or S-GNA, where the ribose is replaced by glycol units linked to the phosphodiester bond), threose nucleic acids (TNAs, where the ribose is replaced by a-L-threofuranosyl-(3’→2’)), and peptide nucleic acids (PNAs, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone). The glycosyl can also contain one or more carbons with opposite stereochemical configuration to the corresponding carbon in ribose. Thus, polynucleotide molecules (including mRNA) as described herein can include nucleotides containing, for example, arabinose as the sugar.

[0236] Non-limiting examples of sugar modifications can include the modifications provided in Table 5. The polynucleotides of the present disclosure can have one or more nucleotides bearing a modification as provided in Table 5. In some embodiments, each nucleotide of a polynucleotide described herein bears any one of the modifications as provided in Table 5, or does not bear any of the modifications as provided in Table 5.

[0237] Table 5. Nucleotide sugar modifications

[0238]

[0239]

[0240]

[0241]

[0242]

[0243] In some embodiments, at least one of the 2' position of the sugar of a nucleotide of the polynucleotide (OH in RNA or H in DNA) is substituted with -OMe, referred to as 2'-OMe. In some embodiments, at least one of the 2' position of the sugar of a nucleotide of the polynucleotide (OH in RNA or H in DNA) is substituted with -F, referred to as 2'-F.

[0244] Internucleoside Linkage

[0245] The polynucleotides of the present disclosure can include any modification to the internucleoside linkage (e.g., to the linking phosphate / phosphodiester bond / phosphodiester backbone). In the context of a polynucleotide backbone, the phrases “phosphate” and “phosphodiester” are used interchangeably. The backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with different substituents. Further, modified nucleosides and nucleotides can include an entire replacement of the unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, methylphosphonate, phosphoroselenoate, boranophosphate, boranophosphate ester, phosphonooxidate, phosphoramidate, diphosphoramidate, alkyl or aryl phosphonates, and phosphotriesters. Both non-linking oxygens of a phosphorodithioate are replaced with sulfur. Phosphate linkers can also be modified by replacing the linking oxygen with nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene-phosphonates).

[0246] Alpha-thio-substituted phosphate moieties are provided to confer stability to RNA and DNA polynucleotides through non-natural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and thus have longer half-lives in cellular environments. Phosphorothioate-linked polynucleotide molecules are also expected to reduce innate immune responses through weaker binding / activation of cellular innate immune molecules.

[0247] In particular embodiments, the modified nucleosides include alpha-thio nucleosides (e.g., 5'-0-(1 -thio phosphate)-adenosine, 5'-0-(1 -thio phosphate)-cytidine (alpha-thio-cytidine), 5'-0-(1 -thio phosphate)-guanosine, 5'-0-(1 -thio phosphate)-uridine, or 5'-0-(1 -thio phosphate)-pseudouridine).

[0248] In some embodiments, the polynucleotide comprises at least one phosphorothioate bond or methylphosphonate bond between nucleotides.

[0249] In some embodiments, the polynucleotide comprises at least one 5’-(E)-vinylphosphonate (5’-E-VP) (a phosphate mimic) as a modification.

[0250] In one embodiment of the disclosure, the polynucleotide (e.g., mRNA) of the herpes zoster nucleic acid vaccine can be modified.

[0251] Valence

[0252] The valency of the nucleic acid vaccines of the disclosure can vary. “Valency” refers to the number of antigenic components in the nucleic acid vaccine or polynucleotide of the nucleic acid vaccine. The antigenic components of the nucleic acid vaccine can be on the same polynucleotide, or they can be on different polynucleotides. In some embodiments, the nucleic acid vaccine can be monovalent. In some embodiments, the nucleic acid vaccine can be bivalent. In some embodiments, the nucleic acid vaccine can be trivalent. In some embodiments, the nucleic acid vaccine can be multivalent, which can comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more than 25 antigens or antigenic portions, such as but not limited to antigenic peptides. As a non-limiting example, the antigenic peptides can be one or more fragments or variants of VZV structural proteins.

[0253] Synthesis

[0254] Enzymatic methods

[0255] In vitro transcription-enzymatic synthesis

[0256] A cDNA encoding a polynucleotide of a nucleic acid vaccine described herein can be transcribed using an in vitro transcription (IVT) system. The system generally comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase. The NTPs can be made in-house, can be selected from a supplier, or can be synthesized as described herein. The NTPs can be selected from, but are not limited to, those described herein, including natural and non-natural (modified) NTPs. The polymerase can be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase, and polymerase variants.

[0257] In some embodiments, DNA template is removed from the IVT reaction using DNase I enzyme. The digested DNA and nucleotides are then removed during oligo dT purification of the mRNA. This purification method is based on the affinity of the poly-A tail of the mRNA to the poly-dT column bed. Centrifugation can be used, but can not be required to remove the digested DNA and nucleotides. Following purification by reverse phase column (e.g., SDVB) to remove double stranded RNA from the mRNA, ultrafiltration can be used followed by one or more filtration steps. Following purification, residual DNA can be measured by PCR to the regions outside the region of the plasmid transcribed into mRNA to confirm that the DNA has been removed. In some embodiments, where a certain concentration of product is required, diafiltration methods can be used followed by one or more filtration steps to remove any bioburden (e.g., biological molecules or other biological material).

[0258] Any number of RNA polymerases or variants can be used in the synthesis of the polynucleotides of the nucleic acid vaccines described herein. RNA polymerases can be modified by insertion or deletion of amino acids of the RNA polymerase sequence.

[0259] Polynucleotide or nucleic acid synthesis reactions can be performed by enzymatic methods that utilize a polymerase enzyme. DNA polymerases that are currently known can be divided into different families based on amino acid sequence comparisons and crystal structure analysis. The DNA polymerase I (pol I) or A polymerase family, which includes the Klenow fragment of E. coli, Bacillus DNA polymerase I, Thermus aquaticus (Taq) DNA polymerase, and T7 RNA and DNA polymerases, are the best studied of these families. Another large family is the DNA polymerase A (pol a) or B polymerase family, which includes all eukaryotic replicative DNA polymerases and polymerases from bacteriophages T4 and RB69. Although they employ similar catalytic mechanisms, these polymerase families differ in substrate specificity, efficiency of substrate analog incorporation, extent and rate of primer extension, mode of DNA synthesis, exonuclease activity, and sensitivity to inhibitors.

[0260] Solid phase chemical synthesis

[0261] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein can be manufactured, in whole or in part, using solid phase techniques. Solid phase chemical synthesis of polynucleotides or nucleic acids is an automated method in which molecules are immobilized on a solid support and synthesized stepwise in a solution of reactants. Impurities and excess reagents are washed away, and no purification is required after each step. Automation of the process is served by a computer-controlled solid phase synthesizer. Solid phase synthesis allows for rapid production of polynucleotides or nucleic acids on a relatively large scale, leading to the commercial availability of some polynucleotides or nucleic acids.

[0262] In some embodiments, automated solid phase synthesis is used, in which the chain is synthesized in the 3' to 5' direction. The hydroxyl group at the 3' end of the nucleoside is attached to a solid support via a chemically or photo-cleavable linker. Activated nucleoside monomers, such as 2'-deoxynucleosides (dA, dC, dG, and dT), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, are added sequentially to the nucleoside bound to the support. At the end of synthesis, a cleaving agent such as ammonia or ammonium hydroxide is added to remove all protecting groups and release the polynucleotide chain from the solid support. Light can also be used to cleave the polynucleotide chain. The product can then be further purified with high pressure liquid chromatography (HPLC) or electrophoresis.

[0263] Liquid phase chemical synthesis

[0264] The synthesis of the polynucleotides of the nucleic acid vaccines described herein by sequential addition of monomers to building blocks can be performed in liquid phase. Covalent bonds are formed between monomers or between the terminal functional group of the growing chain and the incoming monomer. Functional groups not involved in the reaction must be temporarily protected. After the addition of each monomer building block, the reaction mixture must be purified before the next monomer building block is added. The functional group at one end of the chain must be deprotected to enable reaction with the next monomer building block. Liquid phase synthesis is laborious and time consuming and cannot be automated. Despite these limitations, liquid phase synthesis can be used for large scale preparation of short polynucleotides. Since the system is homogeneous, large excesses of reagents are not required and it is cost effective in this regard.

[0265] Quantification and purification

[0266] In some embodiments, the polynucleotides of the nucleic acid vaccines described herein can be quantified in exosomes or when derived from one or more bodily fluids. As used herein, "bodily fluid" includes peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, bronchial alveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural fluid and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretion, mucosal secretion, watery stool, pancreatic juice, lavage fluid from sinus cavities, bronchopulmonary aspirate, blastocyl cavity fluid, lavage fluid from sinus cavities, bronchopulmonary aspirate, blastocyl cavity fluid, and umbilical cord blood. Alternatively, the exosomes can be recovered from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testicle, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

[0267] In the exosome quantification method, no more than 2 mL of sample is obtained from the subject and exosomes are isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or a combination thereof. In the assay, the level or concentration of the polynucleotide can be the level of expression, presence, absence, truncation, or alteration of the administered construct. It is advantageous to correlate the level to one or more clinical phenotypes or to the determination of a human disease biomarker. The determination can be performed using construct-specific probes, cell counts, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or a combination thereof, while exosomes can be isolated using immuno-histochemical methods such as enzyme-linked immunoabsorbent assay (ELISA) methods. Exosomes can also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or a combination thereof.

[0268] These methods enable researchers to monitor the level of polynucleotide remaining or delivered in real time. This is possible because the polynucleotides described herein are different from the endogenous form due to structural modifications.

[0269] In some embodiments, the polynucleotide can be quantified using methods such as, but not limited to, ultraviolet-visible spectroscopy (UV / Vis). A non-limiting example of a UV / Vis spectrometer is a NANODROP® spectrometer (ThermoFisher, Waltham, Mass.). The quantified polynucleotide can be analyzed to determine if the polynucleotide can have the appropriate size, check that the polynucleotide has not degraded. Degradation of the polynucleotide can be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC-based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE), and capillary gel electrophoresis (CGE).

[0270] Purification of the polynucleotides of the nucleic acid vaccines described herein can include, but is not limited to, polynucleotide clean-up, quality assurance, and quality control. The clean-up can be performed by methods known in the art, such as, but not limited to, AGEN-COURT® beads (Beckman Coulter Genomics, Danvers, Mass.), poly-T beads, LNA™ oligo-T capture probes (EX- IQON® Inc, Vedbaek, Denmark), or HPLC-based purification methods, such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term “purified” when used in reference to a polynucleotide such as “purified polynucleotide” means a polynucleotide that is separated from at least one contaminant. As used herein, a “contaminant” is any substance that makes another substance unsuitable, impure, or inferior. Thus, a purified polynucleotide (e.g., DNA and RNA) exists in a form or environment different from that in which it is found in nature, or in a form or environment different from that in which it existed prior to being subjected to a process or purification method.

[0271] Quality assurance and / or quality control checks can be performed using methods such as, but not limited to, gel electrophoresis, UV absorption, or analytical HPLC.

[0272] II. Pharmaceutical Compositions and Delivery

[0273] The nucleic acid vaccines described herein can be used as a therapeutic or prophylactic, e.g., pre-exposure prophylaxis and post-exposure prophylaxis (PEP). In some embodiments, the present disclosure provides a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a nucleic acid vaccine (i.e., a nucleic acid vaccine for herpes zoster). Accordingly, the pharmaceutical compositions comprising the nucleic acid vaccines described herein can be used to prevent, alleviate, and / or treat herpes zoster.

[0274] Provided herein are nucleic acid vaccines and pharmaceutical compositions thereof, which can be used in combination with one or more pharmaceutically acceptable excipients. The pharmaceutical compositions can optionally comprise one or more additional active substances, e.g., therapeutically and / or prophylactically active substances. The pharmaceutical compositions of the nucleic acid vaccines described herein can be sterile and / or non-pyrogenic.

[0275] In some embodiments, the compositions are administered to a human, human patient, or subject. For purposes of the present disclosure, the phrase “active ingredient” generally refers to a nucleic acid vaccine or a polynucleotide contained therein, e.g., a polynucleotide encoding one or more proteins, peptides, fragments, or variants of VZV for preventing, alleviating, and / or treating herpes zoster, to be delivered as described herein.

[0276] While the description of pharmaceutical compositions provided herein is primarily concerned with pharmaceutical compositions suitable for administration to humans, it is to be understood that such compositions are generally suitable for administration to any other animal for which the compounds, derivatives, prodrugs, salts, solvates, hydrates, isomers, clathrates, polymorphs, metabolites and / or impurities are intended. Modifications can be made to the pharmaceutical compositions described and / or otherwise used for administration to humans to make them suitable for administration to various animals, and such modifications are within the scope of the present disclosure. Subjects to which the pharmaceutical compositions are intended to be administered include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals, such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially relevant birds, such as domesticated fowl, chickens, ducks, geese, and / or turkeys.

[0277] Formulation

[0278] Pharmaceutical formulations can additionally include a pharmaceutically acceptable excipient, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and / or other additives, as such materials are known in the art to be appropriate in a given pharmaceutical formulation. Various excipients and techniques for formulating pharmaceutical compositions are known in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 21stEdition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of conventional excipient media is contemplated within the scope of the present disclosure, unless any conventional excipient medium can not be compatible with the substance or derivative thereof, for example, by generating any undesirable biological effects or interacting in a deleterious manner with any other component of the pharmaceutical composition. st Edition, A. R. Gennaro, Lippincott, Williams &Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety). The use of conventional excipient media is contemplated within the scope of the present disclosure, unless any conventional excipient medium can not be compatible with the substance or derivative thereof, for example, by generating any undesirable biological effects or interacting in a deleterious manner with any other component of the pharmaceutical composition.

[0279] The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association 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 desired single- or multi-dose unit.

[0280] Pharmaceutical compositions according to the present disclosure can be manufactured, packaged, and / or sold in bulk, as single unit dosages, and / or as a plurality of single unit dosages. As used herein, a “unit dosage” is a discrete amount of the pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of the active ingredient generally will be equal to that which would be used to effect the intended purpose in association with appropriate dosing instructions. The relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any 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 further depending on the route of administration of the composition. For example, the composition can comprise between 0.1% and 100%, e.g., between 0.5% and 50%, between 1% and 30%, between 5% and 80%, at least 80% (w / w) of the active ingredient.

[0281] The relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any 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 further depending on the route of administration of the composition. For example, the composition can comprise between 0.1% and 100%, e.g., between 0.5% and 50%, between 1% and 30%, between 5% and 80%, at least 80% (w / w) of the active ingredient.

[0282] In some embodiments, the formulations described herein can comprise at least one nucleic acid vaccine composition, e.g., a varicella nucleic acid vaccine, e.g., a varicella mRNA vaccine. As non-limiting examples, the formulation can contain 1, 2, 3, 4, or 5 nucleic acid vaccine compositions having different sequences, e.g., 1, 2, 3, 4, or 5 mRNA vaccine compositions having different sequences. In some embodiments, the formulation contains at least two nucleic acid vaccine (e.g., mRNA vaccine) compositions having different sequences. In some embodiments, the formulation contains at least three nucleic acid vaccine (e.g., mRNA vaccine) compositions having different sequences. In some embodiments, the formulation contains at least four nucleic acid vaccine (e.g., mRNA vaccine) compositions having different sequences. In some embodiments, the formulation contains at least five nucleic acid vaccine (e.g., mRNA vaccine) compositions having different sequences.

[0283] One or more excipients can be used to formulate the nucleic acid vaccine compositions of the present disclosure to: (1) increase stability; (2) increase cell transfection; (3) allow sustained or delayed release (e.g., from a depot formulation of the nucleic acid vaccine composition); (4) alter biodistribution (e.g., target the nucleic acid vaccine composition to a particular tissue or cell type); (5) increase in vivo translation of the encoded protein; and / or (6) alter the in vivo release profile of the encoded protein.

[0284] In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicle, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, other than the excipients of the present disclosure can include, but are not limited to, lipids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the nucleic acid vaccine composition (e.g., for implantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof. Thus, the formulations of the present disclosure can include one or more excipients, each excipient in an amount that together increases the stability of the nucleic acid vaccine composition and / or increases the cell transfection of the nucleic acid vaccine composition. Further, the nucleic acid vaccine compositions of the present disclosure can be formulated using self-assembling nucleic acid nanoparticles. Pharmaceutically acceptable carriers, excipients, and delivery agents for nucleic acids that can be used to formulate with the nucleic acid vaccine compositions of the present disclosure are disclosed in PCT Patent Application Publication WO 2013 / 090648, the contents of which are incorporated by reference in their entirety.

[0285] Lipids

[0286] The nucleic acid vaccine compositions of the present disclosure can be formulated using one or more lipids.

[0287] The synthesis of lipids has been extensively described, and formulations containing these compounds are particularly suitable for the delivery of oligonucleotides or nucleic acids (see Mahon et al., Bioconjug Chem. 2010, 21 : 1448-1454; Schroeder et al., J Intern Med. 2010, 267:9-21; Akinc et al., Nat Biotechnol. 2008, 26:561-569; Love et al., Proc Natl Acad Sci U S A. 2010, 107: 1864-1869; Siegwart et al., Proc Natl Acad Sci U S A. 2011, 108: 12996-3001; the contents of all of these references are incorporated by reference in their entirety).

[0288] While these lipids have been used to effectively deliver double-stranded small interfering RNA molecules in rodents and non-human primates (see Akinc et al., Nat Biotechnol. 2008, 26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci U S A. 2008, 105:11915-11920; Akinc et al., Mol Ther. 2009, 17:872-879; Love et al., Proc Natl Acad Sci U S A. 2010, 107:1864-1869; Leuschner et al., Nat Biotechnol. 2011, 29:1005-1010; the contents of all of which are incorporated by reference herein in their entirety), the present disclosure contemplates their formulation and use in delivering at least one pharmaceutically acceptable carrier, including nucleic acid vaccines. Complexes, micelles, liposomes, or particles containing these lipids can be prepared and thus can result in effective delivery of nucleic acid vaccine compositions upon injection of the lipoid formulation via local and / or systemic routes of administration. Lipoid complexes containing nucleic acid vaccine compositions can be administered by various means, including but not limited to intravenous (IV), intramuscular (IM), subcutaneous (SC), intraparenchymal (IPa), intrathecal (IT), or intracerebroventricular (ICV) administration.

[0289] In vivo delivery of nucleic acids can be influenced by a number of parameters, including but not limited to formulation composition, nature of particle polyethyleneglycolation, degree of loading, ratio of polynucleotide to lipid, and biophysical parameters such as, but not limited to, particle size (Akinc et al., Mol Ther. 2009, 17:872-879; the contents of which are incorporated by reference herein in their entirety). As one example, small changes in the anchor length of poly(ethyleneglycol) (PEG) lipids can have a dramatic impact on in vivo efficacy. Formulations with different lipids, including but not limited to penta[3-(1-laurylamidopropanoyl)]- triethylenetetraamine hydrochloride (TETA-5LAP; also known as 98N12-5, see Murugaiah et al., Analytical Biochemistry, 2010, 401:61; the contents of which are incorporated by reference herein in their entirety), C12-200 (including derivatives and variants), and MD1 can be tested for in vivo activity.

[0290] A lipidoid referred to herein as "98N12-5" is disclosed by Akinc et al., Mol Ther. 2009, 17:872-879, and the contents of which are incorporated herein by reference in their entirety.

[0291] A lipidoid referred to herein as "C12-200" is disclosed by Love et al., Proc Natl Acad Sci U S A. 2010, 107:1864-1869, and Liu and Huang, Molecular Therapy. 2010, 669-670; the contents of both of which are incorporated herein by reference in their entirety. In addition to nucleic acid vaccine compositions, lipidoid formulations can include particles comprising 3 or 4 or more components. As one example, a formulation with certain lipidoids includes, but is not limited to, 98N12-5, and can contain 42% lipidoid, 48% cholesterol, and 10% PEG (C14 alkyl chain length). As another example, a formulation with certain lipidoids includes, but is not limited to, C12-200, and can contain 50% lipidoid, 10% distearoylphosphatidylcholine, 38.5% cholesterol, and 1.5% PEG-DMG.

[0292] In some embodiments, nucleic acid vaccine compositions formulated with lipidoids are used for systemic intravenous administration. For example, a final optimized intravenous formulation uses a nucleic acid vaccine composition and comprises a lipid molar composition of 42% 98N12-5, 48% cholesterol, and 10% PEG-lipid, a final weight ratio of total lipid to nucleic acid vaccine composition of about 7.5: 1, and a C14 alkyl chain length on the PEG lipid, with an average particle size of about 50-60 nm, such an intravenous formulation can result in greater than 90% distribution of the formulation in the liver. (See Akinc et al., Mol Ther. 2009, 17:872-879; the contents of which are incorporated herein by reference in their entirety). In another example, an intravenous formulation using C12-200 lipidoids (see PCT Patent Application Publication WO2010129709, the contents of which are incorporated herein by reference in their entirety) can have a molar ratio of 50 / 10 / 38.5 / 1.5 of C12-200 / distearoylphosphatidylcholine / cholesterol / PEG-DMG, a weight ratio of total lipid to nucleic acid of 7: 1, and an average particle size of 80 nm, can be effective to deliver a nucleic acid vaccine composition (see Love et al., Proc Natl Acad Sci U S A. 2010, 107:1864-1869; the contents of which are incorporated herein by reference in their entirety).

[0293] In some embodiments, formulations containing MD1 lipids can be used to effectively deliver nucleic acid vaccine compositions to hepatocytes in vivo. The properties of optimized lipoid formulations for intramuscular or subcutaneous routes can vary significantly depending on the target cell type and the ability of the formulation to diffuse through the extracellular matrix into the bloodstream. While particle sizes less than 150 nm can be required for effective hepatocyte delivery due to the size of endothelial fenestrae (see, Akinc et al., Mol Ther. 2009, 17:872-879, the contents of which are incorporated by reference in their entirety), the use of lipoid-formulated nucleic acid vaccine compositions to deliver formulations to other cell types, including but not limited to endothelial cells, bone marrow cells, and muscle cells, can not be subject to similar size limitations.

[0294] Delivery of siRNA to other non-hepatocyte cells such as bone marrow cells and endothelial cells in vivo using lipoid formulations has been reported (see Akinc et al., Nat Biotechnol. 2008, 26:561-569; Leuschner et al., Nat Biotechnol. 2011, 29:1005-1010; Cho et al. Adv. Funct. Mater. 2009, 19:3112-3118; Eighth International Judah Folkman Conference, Cambridge, MA, October 8-9, 2010; the contents of each of which are incorporated by reference in their entirety). To effectively deliver to bone marrow cells, such as monocytes, lipoid formulations can have similar component molar ratios. Different ratios of lipoids and other components, including but not limited to distearoylphosphatidylcholine, cholesterol, and PEG-DMG, can be used to optimize formulation of nucleic acid vaccine compositions for delivery to different cell types, including but not limited to hepatocytes, bone marrow cells, muscle cells, and the like. For example, component molar ratios can include, but are not limited to, 50% C12-200, 10% distearoylphosphatidylcholine, 38.5% cholesterol, and 1.5% PEG-DMG (see Leuschner et al., Nat Biotechnol 2011, 29:1005-1010; the contents of which are incorporated by reference in their entirety). The use of lipoid formulations for local delivery of nucleic acids to cells by subcutaneous or intramuscular delivery can not require all formulation components required for systemic delivery, and thus can include only lipoids and nucleic acid vaccine compositions.

[0295] Liposomes

[0296] Nucleic acid vaccine compositions of the present disclosure can be formulated using one or more liposomes.

[0297] In some embodiments, the pharmaceutical composition of the nucleic acid vaccine composition comprises a liposome. Liposomes are artificially prepared vesicles that can consist primarily of a lipid bilayer and can be used as delivery vehicles to administer nutrients and pharmaceutical agents. Liposomes can have different sizes, such as but not limited to multilamellar vesicles (MLVs) (which can be several hundred nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments), small unilamellar vesicles (SUVs) which can be less than 50 nm in diameter, and large unilamellar vesicles (LUVs) which can be between 50 and 500 nm in diameter. Liposome designs can include, but are not limited to, opsonins or ligands in order to improve liposome attachment to unhealthy tissue or activation events such as, but not limited to, endocytosis. Liposomes can contain low or high pH in order to improve delivery of pharmaceutical agents.

[0298] Formation of liposomes can depend on physicochemical properties such as, but not limited to, the embedded pharmaceutical agent and the liposome components, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the embedded material and its potential toxicity, any other processes involved during application and / or delivery of the vesicles, the optimized particle size, polydispersity, and shelf life of the vesicles for the intended application, and the reproducibility between batches and the possibility of safely and effectively producing liposome products on a large scale.

[0299] In some embodiments, the pharmaceutical composition comprising the nucleic acid vaccine described herein can include, but is not limited to, liposomes such as those formed from 1,2-dioleoyl-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), SMARTICLES® / NOV340 (Marina Biotech, Bothell), 1,2-dilinoleyl-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (U.S. Patent Application Publication US20100324120; the contents of which are incorporated by reference herein in their entirety), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (e.g., for siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006, 5(12): 1708-1713); the contents of which are incorporated by reference herein in their entirety), hyaluronan-coated liposomes (Quiet Therapeutics, Israel), and liposomes that can deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, PA).

[0300] In some embodiments, a pharmaceutical composition comprising a nucleic acid vaccine described herein can include, but is not limited to, liposomes such as those formed from the synthesis of stable plasmid-lipid particles (SPLPs) or stable nucleic acid-lipid particles (SNALPs) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see 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; de Fougerolles Hum Gene Ther. 2008, 19:125-132; the contents of each of which are incorporated herein in their entirety). Wheeler et al.’s initial manufacturing method was a detergent dialysis method which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method. In addition to the nucleic acid vaccine composition, the liposome formulation can be composed of 3 to 4 lipid components. As a non-limiting example, the liposome can include, but is not limited to, 55% cholesterol, 20% distearoylphosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleoyloxy-N,N-dimethylammonium propane (DODMA) as described by Jeffs et al. In another example, certain liposome formulations can contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be 1,2-distearyloxy-N,N-dimethylammonium propane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA) as described by Heyes et al.In another example, the nucleic acid-lipid particle can comprise a cationic lipid at about 50 mol% to about 85 mol% of the total lipid present in the particle; a non-cationic lipid at about 13 mol% to about 49.5 mol% of the total lipid present in the particle; and a conjugated lipid that inhibits particle aggregation at about 0.5 mol% to about 2 mol% of the total lipid present in the particle, as described in WO2009127060 by Maclachlan et al.; the contents of which are incorporated herein by reference in their entirety. In another example, the nucleic acid-lipid particle can be any of the nucleic acid-lipid particles disclosed in US2006008910 by Maclachlan et al.; the contents of which are incorporated herein by reference in their entirety. As a non-limiting example, the nucleic acid-lipid particle can comprise a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits particle aggregation as described herein.

[0301] In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated in a lipid vesicle, which can have a cross-linking between the functionalized lipid bilayers.

[0302] In some embodiments, the liposome can comprise a sugar-modified lipid disclosed in US Patent No. US5595756 by Bally et al., the contents of which are incorporated herein by reference in their entirety. The lipid can be a ganglioside and a cerebrosides in an amount of about 10 mol%.

[0303] In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated in a liposome comprising a cationic lipid. The molar ratio of nitrogen atoms in the cationic lipid of the liposome to the phosphate in the nucleic acid vaccine composition (N:P ratio) can be between 1:1 to 20:1, as described in PCT Patent Application Publication No. WO2013006825, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the liposome can have a N:P ratio greater than 20:1 or less than 1:1.

[0304] In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated in a lipid-poly cation complex. Formation of the lipid-poly cation complex can be accomplished by methods known in the art and / or described in U.S. Publication No. 20120178702, the contents of which are incorporated by reference herein in their entirety. As non-limiting examples, the poly cation can include a cationic peptide or polypeptide, such as, but not limited to, polylysine, polyornithine, and / or polyarginine, and the cationic peptides described in PCT Patent Application Publication No. WO2012013326; the contents of which are incorporated by reference herein in their entirety. In some embodiments, the nucleic acid vaccine compositions can be formulated in a lipid-poly cation complex, which complex can further comprise a neutral lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0305] Liposome formulations can be influenced by, but are not limited to, the selection of the cationic lipid component, the degree of saturation of the cationic lipid, the nature of the PEGylation, the ratio of all components, and biophysical parameters such as size. In one example of Semple et al. (Semple et al. Nature Biotech. 2010, 28: 172-176; the contents of which are incorporated by reference herein in their entirety), the liposome formulation consisted of 57.1% cationic lipid, 7.1% dipalmitoyl phosphatidyl choline, 34.3% cholesterol, and 1.4% PEG-c-DMA.

[0306] In some embodiments, the pharmaceutical compositions can be formulated with any of the amphipathic liposomes disclosed in Panzner’s PCT Patent Application Publication No. WO2008043575 and Essler et al. (Marina Biotech) U.S. Patent No. US 8,580,297, the contents of which are incorporated by reference herein in their entirety. The amphipathic liposomes can comprise a mixture of lipids, which mixture includes a cationic amphiphile, an anionic amphiphile, and optionally one or more neutral amphiphiles. The amphipathic liposomes can comprise an amphiphilic compound based on an amphiphilic molecule, the head group of which is substituted by one or more amphoteric groups. In some embodiments, the pharmaceutical compositions can be formulated with an amphipathic lipid comprising one or more amphoteric groups having an isoelectric point between 4 and 9, as disclosed in Essler et al. (Marina Biotech) U.S. Patent Application Publication No. US20140227345, the contents of which are incorporated by reference herein in their entirety.

[0307] In some embodiments, the pharmaceutical composition can be formulated with liposomes comprising a sterol derivative as disclosed in U.S. Patent No. US7312206 to Panzner et al. (Novosom), the contents of which are incorporated by reference in their entirety herein. In some embodiments, the pharmaceutical composition can be formulated with amphipathic liposomes comprising at least one amphipathic cationic lipid, at least one amphipathic anionic lipid, and at least one neutral lipid, or the liposomes comprise at least one amphipathic lipid having both positive and negative charges, and at least one neutral lipid, wherein the liposomes are stable at pH 4.2 and pH 7.5, as disclosed in U.S. Patent No. 7780983 to Panzner et al. (Novosom), the contents of which are incorporated by reference in their entirety herein. In some embodiments, the pharmaceutical composition can be formulated with liposomes comprising a serum-stable lipid mixture taught in U.S. Patent Application Publication No: US 20110076322 to Panzner et al. (the contents of which are incorporated by reference in their entirety herein) capable of encapsulating the nucleic acid vaccine compositions of the disclosure. The lipid mixture comprises phosphatidylcholine and phosphatidylethanolamine in a ratio ranging from about 0.5 to about 8. The lipid mixture can also include pH-sensitive anionic and cationic amphiphiles such that the mixture is amphoteric, negatively charged or neutral at pH 7.4, and positively charged at pH 4. The drug / lipid ratio can be adjusted to target the liposomes to specific organs or other sites in the body. In some embodiments, the liposomes loaded with the nucleic acid vaccine compositions of the disclosure as cargo are prepared by the method disclosed in U.S. Patent Application Publication No: US 20120021042 to Panzner et al., the contents of which are incorporated by reference in their entirety herein. The method includes the steps of mixing an aqueous solution of a polyanionic active agent and an alcoholic solution of one or more amphiphiles and buffering the mixture to an acidic pH, wherein the one or more amphiphiles are prone to form amphoteric liposomes at the acidic pH, thereby forming amphoteric liposomes in the form of a suspension encapsulating the active agent.

[0308] Lipoplex

[0309] The nucleic acid vaccine compositions of the disclosure can be formulated using one or more lipoplexes.

[0310] In some embodiments, the nucleic acid vaccine compositions can be formulated into lipoplexes such as, but not limited to, ATUPLEX® from Silence Therapeutics (London, UK) TM Systems, DACC systems, DBTC systems, and other siRNA-lipoplex technologies from STEMFECT® (Cambridge, MA) STEMFECT TMand non-targeted delivery of nucleic acids based on polyethyleneimine (PEI) or protamine (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., J. Immunother., 2011, 34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl Acad Sci U S A. 2007, 6;104:4095-4100; de Fougerolles Hum Gene Ther. 2008, 19:125-132; the contents of each of which are incorporated by reference in their entirety).

[0311] Lipid Nanoparticle (LNP)

[0312] In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated in a lipid nanoparticle (LNP). Generally, an LNP can be characterized as a small solid or semi-solid particle with an outer lipid layer having a hydrophilic outer surface exposed to the non-LNP environment, an internal space that can be aqueous (vesicle-like) or non-aqueous (micelle-like), and at least one hydrophobic intermembrane space. The LNP membrane can be lamellar or non-lamellar and can comprise 1, 2, 3, 4, 5, or more layers. In some embodiments, an LNP can include cargo or a payload into its internal space, into the intermembrane space, on its outer surface, or any combination thereof.

[0313] LNP useful herein are known in the art and generally comprise cholesterol (helps stability and promotes membrane fusion), a phospholipid (which provides structure to the LNP bilayer and can also help endosomal escape), a polyethylene glycol (PEG) derivative (which reduces LNP aggregation and "protects" the LNP from non-specific endocytosis by immune cells), and an ionizable lipid (which complex the negatively charged RNA and enhances endosomal escape), which form an LNP forming composition.

[0314] The components of the LNP can be selected based on the desired target, tropism, cargo, size, or other desired feature or property.

[0315] The LNP can be a lipid nanoparticle described in PCT Patent Application Publication No. WO2012170930, the contents of which are incorporated by reference herein in their entirety.

[0316] In some embodiments, the nucleic acid vaccine compositions of the present disclosure can be formulated in LNP comprising at least one cationic lipid.

[0317] In some embodiments, the cationic lipid useful in the formulations of the present disclosure can be selected from, but not limited to, the cationic lipids described in PCT Patent Application Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865, and WO2008103276, U.S. Patent Nos. 7,893,302, 7,404,969, and 8,283,333, and U.S. Patent Publication Nos. US20100036115 and US20120202871, the contents of each of which are incorporated by reference in their entirety. The cationic lipid can also be selected from, but not limited to, Formula A described in PCT Patent Application Publication Nos. WO2012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, and WO2012044638, the contents of each of which are incorporated by reference in their entirety. Alternatively, the cationic lipid can be selected from, but not limited to, Formulas CLI-CLXXIX of PCT Patent Application No. WO2008103276, Formulas CLI-CLXXIX of U.S. Patent No. 7,893,302, Formulas CLI-CLXXXXII of U.S. Patent No. 7,404,969, and Formulas I-VI of U.S. Patent Publication No. US20100036115; the contents of each of which are incorporated by reference in their entirety. The cationic lipid can be a multivalent cationic lipid, such as the cationic lipids disclosed in U.S. Patent No. 7,223,887 to Gaucheron et al., the contents of which are incorporated by reference in their entirety. As described in U.S. Patent No. 7,223,887 to Gaucheron et al., the cationic lipid can have a positively charged head group comprising two quaternary amino groups, and a hydrophobic portion comprising four hydrocarbon chains. The cationic lipid can be biodegradable, for example, the biodegradable lipids disclosed in U.S. Patent Application Publication No. US20130195920 to Maier et al., the contents of which are incorporated by reference in their entirety. The cationic lipid can have one or more biodegradable groups located in the lipid portion of the cationic lipid, as described in Formulas I-IV in US20130195920 to Maier et al.

[0318] In some embodiments, the cationic lipid can also be a cationic lipid disclosed in US20130156845 to Manoharan et al. and US20130129785 to Manoharan et al., WO2012047656 to Wasan et al., WO2010144740 to Chen et al., WO2013086322 to Ansell et al., or WO2012016184 to Manoharan et al., the contents of each of which are incorporated by reference herein in their entirety.

[0319] As non-limiting examples, the cationic lipid can be selected from (20Z,23Z)-N,N- dimethylnonacos-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacos-17,20-dien-9-amine, (1 Z,19Z)-N,N-dimethylpentacos-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyl-docos-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylheneicos-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-8-amine, (17Z,20Z)-N,N-dimethylhexacos-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacos-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriacont-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriacont-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacos-18-ene-10-amine, (17Z)-N,N-dimethylhexacos-17-ene-9-amine, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacos-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylundec-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacos-20-ene-10-amine, (15Z)-N,N-dimethylheptadec-15-ene-10-amine, (14Z)-N,N-dimethylnonadec-14-ene-10-amine, (17Z)-N,N-dimethylnonadec-17-ene-10-amine, (24Z)-N,N-dimethyltritriacont-24-ene-10-amine, (20Z)-N,N-dimethylnonacos-20-ene-10-amine, (22Z)-N,N-dimethylhentriacont-22-ene-10-amine, (16Z)-N,N-dimethylpentadec-16-ene-8-amine, (12Z,15Z)-N,N-dimethylheneicos-12,15-dien-4-amine, (13Z,16Z)-N,N-dimethyl-docos-13,16-dien-5-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-8-amine, (17Z,20Z)-N,N-dimethylhexacos-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacos-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriacont-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriacont-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacos-18-ene-10-amine, (17Z)-N,N-dimethylhexacos-17-ene-9-amine, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacos-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylundec-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacos-20-ene-10-amine, (15Z)-N,N-dimethylheptadec-15-ene-10-amine, (14Z)-N,N-dimethylnonadec-14-ene-10-amine, (17Z)-N,N-dimethylnonadec-17-ene-10-amine, (24Z)-N,N-dimethyltritriacont-24-ene-10-amine, (20Z)-N,N-dimethylnonacos-20-ene-10-amine, (22Z)-N,N-dimethylhentriacont-22-ene-10-amine, (16Z)-N,N-dimethylpentadec-16-ene-8-amine, (12Z,15Z)-N,N-dimethylheneicos-12,15-dien-4-amine, (13Z,16Z)-N,N-dimethyl-docos-13,16-dien-5-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracos-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-8-amine, (17Z,20Z)-N,N-dimethylhexacos-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacos-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriacont-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriacont-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacos-18-ene-10-amine, (17Z)-N,N-dimethylhexacos-17-ene-9-amine, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacos-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylundec-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacos-20-ene-10-amine, (15Z)-N,N-dimethylheptadec-15-ene-10-amine, (14Z)-N,N-dimethylnonadec-14-ene-10-amine, (17Z)-N,N-dimethylnonadec-17-ene-10-amine, (24Z)-N,N-dimethyltritriacont-24-ene-10-amine, (20Z)-N,N-dimethylnonacos-20-ene-10-amine, (22Z)-N,N-dimethylhentriacont-22-ene-10-amine, (16Z)-N,N-dimethylpentadec-16-ene-8-amine, (12Z,15Z)-NN,N-dimethyl-2-nonyl heneicos-12,15-dien-1 -amine, (13Z,16Z)-N,N-dimethyl-3- nonyl docosa-13,16-dien-1 -amine, N,N-dimethyl-1 -[(1 S,2R)-2-octylcyclopropyl]heptadecan-8- amine, 1 -[(1 S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1 -[(1 S,2R)-2- octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21 -[(1 S,2R)-2-octylcyclopropyl]heneicosan-10-amine, N,N-dimethyl-1 -[(1 S,2S)-2-{[(1 R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1 -[(1 S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1 R,2S)-2-undecylcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(1 S,2R)-2-octylcyclopropyl]heptyl}dodecan-1 -amine, 1 -[(1 R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine, 1 -[(1 S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-1 -[(1 S,2R)-2-octylcyclopropyl]pentadecan-8-amine, R-N,N-dimethyl-1 -[(9Z,12Z)-octadeca-9,12-dien-1 -yloxy]-3-(octyloxy)propan-2-amine, S-N,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]propan-2-amine, 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)-octadeca-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)-eicosa-11,14-dien-1-yloxy]-N,N- dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-eicosa-11,14- dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-eicosa-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)-docosa-13-en-1-yloxy]- 3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docosa-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-(1-methyloctyl)oxy]-3-[(9Z,12Z)- octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N- dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1- (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-dimethylnonacos-11,20,23-trien-10- amine, or a pharmaceutically acceptable salt or stereoisomer thereof.

[0320] Lipid nanoparticle (LNP) compositions

[0321] In some embodiments, the lipid nanoparticle can comprise at least one cationic lipid, at least one non-cationic lipid, at least one sterol, at least one additional LNP functional component, or any combination thereof. In some embodiments, the lipid nanoparticle can comprise at least one cationic lipid, at least one non-cationic lipid, at least one sterol, and at least one additional LNP functional component. In some embodiments, the LNP can comprise at least one cationic lipid, at least one non-cationic lipid, and at least one sterol. In some embodiments, the LNP can comprise at least one cationic lipid, at least one non-cationic lipid, and at least one additional LNP functional component. In some embodiments, the LNP can comprise at least one non-cationic lipid, at least one sterol, and at least one additional LNP functional component. In some embodiments, the LNP can comprise at least one cationic lipid and at least one non-cationic lipid. In some embodiments, the LNP can comprise at least one cationic lipid and at least one sterol. In some embodiments, the LNP can comprise at least one cationic lipid and at least one additional LNP functional component. In some embodiments, the LNP can comprise at least one non-cationic lipid and at least one sterol. In some embodiments, the LNP can comprise at least one non-cationic lipid and at least one additional LNP functional component. In some embodiments, the LNP can comprise at least one sterol and at least one additional LNP functional component. In some embodiments, the LNP can comprise at least one cationic lipid. In some embodiments, the LNP can comprise at least one non-cationic lipid. In some embodiments, the LNP can comprise a sterol. In some embodiments, the LNP can comprise an additional LNP functional component.

[0322] In some embodiments, the at least one cationic lipid can comprise any of at least one ionizable cationic lipid, at least one amino lipid, at least one saturated cationic lipid, at least one unsaturated cationic lipid, at least one zwitterionic lipid, at least one multivalent cationic lipid, or any combination thereof. In some embodiments, the LNP can be substantially free of the at least one cationic lipid. In some embodiments, the LNP can not contain the at least one cationic lipid in any amount.

[0323] In some embodiments, the at least one cationic lipid can be selected from, but not limited to, at least one of 1,3-bis-(l,2-bis-tetradecyloxy-propyl-3-dimethylethoxy bromide)-propane-2-ol ((R)-PLC-2), 2-(diphenylamino)ethane-l-ol (17-10), 2-(didodecylamino)ethane-l-ol (17-11), 3-(didodecylamino)propane-l-ol (17-12), 4-(didodecylamino)butane-l-ol (17-13), 2-(hexyl((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethane-l-ol (17-2), 2-(nonyl((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethane-l-ol (17-3), 2-(dodecyl((9Z,12Z)-octadeca-9,12-dien-l-yl)amino)ethane-l-ol (17-4), 2-(((9Z,12Z)-octadeca-9,12-dien-l-yl)(tetradecyl)amino)ethane-l-ol (17-5), 2-(((9Z,12Z)-octadeca-9,12-dien-l-yl)(octadecyl)amino)ethane-l-ol (17-6), 2-(ditetradecylamino)ethane-l-ol (17-7), 2-(di((Z)-octadeca-9-en-l-yl)amino)ethane-l-ol (17-8), (9Z,12Z)-N-(2-methoxyethyl)-N-((9Z,12Z)-octadeca-9,12-dien-l-yl)octadeca-9,12-dien-l-amine (17-9), N-nonyl-N-(2-(piperazin-l-yl)ethyl)nonane-l-amine (19-1), N-dodecyl-N-(2-(piperazin-l-yl)ethyl)dodecane-l-amine (19-2), (9Z,12Z)-N-((9Z,12Z)-octadeca-9,12-dien-l-yl)-N-(2-(piperazin-l-yl)ethyl)octadeca-9,12-dien-l-amine (19-3), N-dodecyl-N-(2-(4-methylpiperazin-l-yl)ethyl)dodecane-l-amine-1,2-(didodecylamino)ethane-l-ol (19-4), N-dodecyl-N-(2-(4-(4-methoxybenzyl)piperazin-l-yl)ethyl)dodecane-l-amine (19-5), (9Z,12Z)-N-(2-(4-dodecylpiperazin-l-yl)ethyl)-N-((9Z,12Z)-octadeca-9,12-dien-l-yl)octadeca-9,12-dien-l-amine (19-6), (3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraeny-19-yloxy)-N,N-dimethylpropane-l-amine) (1-1),N-(2-(dihexadecylamino)ethyl)-N-hexadecanecarboxamide (20-7), N-(2- (dihexadecylamino)ethyl)-N-hexadecanecarboxamide (20-8), 8,8'-((2- (dihexadecylamino)ethyl)azanediyl)diheptadecanoic acid dipentyl ester (20-9), N-(2- (didodecylamino)ethyl)-N-dodecylglycine (20-1), dinonyl 8,8'-((2- (dodecyl(2-hydroxyethyl)amino)ethyl)azanediyl)dioctanoate (20-10), 3-((2- (ditetradecylamino)ethyl)(dodecyl)amino)propane-1-ol (20-11), 2-((2- (ditetradecylamino)ethyl)(tetradecyl)amino)ethane-1-ol (20-12), 2-((2-(di((9Z,12Z)- octadeca-9,12-dien-1-yl)amino)ethyl)(dodecyl)amino)ethane-1-ol (20-13), 2-((2-(di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl)((9Z,12Z)-octadeca-9,12-dien-1- yl)amino)ethane-1-ol (20-14), 2-((2-(dihexadecylamino)ethyl)(hexyl)amino)ethane-1- ol (20-15), 2-((2-(dinonylamino)ethyl)(nonyl)amino)ethane-1-ol (20-16), 2-((2- (didecylamino)ethyl)(nonyl)amino)ethane-1-ol (20-17), 2-((2-(dinonylamino)ethyl)(dodecyl)amino)ethane-1-ol (20-18), 2-((2-(didecylamino)ethyl)amino)ethane-1- ol (20-19), pentyl 6-(dodecyl(2-(dodecyl(2-hydroxyethyl)amino)ethyl)amino)hexanoate (20-2), 2-((2-(didecylamino)ethyl)(dodecyl)amino)ethane-1-ol (20-20), 3-((2- (didecylamino)ethyl)(dodecyl)amino)propane-1-ol (20-21), 4-((2-(didecylamino)ethyl)(dodecyl)amino)butane-1-ol (20-22), (Z)-2-((2-(didecylamino)ethyl)(dodec-6-en-1-yl)amino)ethane-1-ol (20-23), 2-((2-(didecylamino)ethyl)(tetradecyl)amino)ethane-1-ol (20-24), 2-((2- (didecylamino)ethyl)((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethane-1-ol (20-25), pentyl 6-((2-(didecylamino)ethyl)(2-hydroxyethyl)amino)hexanoate (20-3), dipentyl 6,6'- ((2-(dodecyl(2-hydroxyethyl)amino)ethyl)azanediyl)di hexanoate (20-4), diheptyl 6,6'- ((2-((6-(heptyloxy)-6-oxohexyl)(2-hydroxyethyl)amino)ethyl)azanediyl)di hexanoate (20-5), pentyl 6-((2-(dinonylamino)ethyl)(2-hydroxyethyl)amino)hexanoate (20-6),6-(dodecyl(2-(dodecyl(2-hydroxyethyl)amino)ethyl)amino)heptanoic acid (20-7), 8-((2-(dododecylamino)ethyl)(2-hydroxyethyl)amino)octanoic acid (20-8), heptadecan-9-yl 8-((2-(dododecylamino)ethyl)(2-hydroxyethyl)amino)octanoate (20-9), 1-(2,2-di((9Z,12Z)-octadeca-9,12-dien-1-yl)cyclopropyl)-N,N-dimethylmethanamine (21-1), 3,3-di((9Z,12Z)-octadeca-9,12-dien-1-yl)cyclobutyl 4-(dimethylamino)butanoate (21-2), 3,3-di((9Z,12Z)-octadeca-9,12-dien-1-yl)cyclopentyl 3-(dimethylamino)propanoate (21-3), 3,3-di((9Z,12Z)-octadeca-9,12-dien-1-yl)cyclopentyl 4-(dimethylamino)butanoate (21-4), 1-(2,3-di((8Z,11Z)-heptadeca-8,11-dien-1-yl)cyclopropyl)-N,N-dimethylmethanamine (21-6), poly{4-((2-(dimethylamino)ethyl)thio)tetrahydro-2H-pyran-2-one}-r-poly{4-(octylthio)tetrahydro-2H-pyran-2-one} (A7), (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[1,3]dioxol-5-amine (ALN100), (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN1001), ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine)) (ALNY-100), dimyristyltrimethylammonium propane (amphipol 6), BADACA, N,N-dihydroxyethylmethyl-N-2-(cholestryloxy carbonylamino)ethylammonium bromide (BHEM-Chol), N,N-bis-(2-hydroxyethyl)-N-methyl-N-(2-cholestryloxy carbonylamino-ethyl)ammonium bromide (BHEM-Chol1), 2-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (butyl-CLinDMA),(2R)-2-{4-[(3β)-cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)- octadeca-9, 12-dien-1-yloxypropan-1-amine (Butyl-CLinDMA (2R)), (2R)-2-{4-[(3β)- cholest-5-en-3-yloxy]butoxy}-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1- yloxy]propan-1-amine (Butyl-CLinDMA (2S)), 1,1'-(2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl) bis(dodecan-2-ol) (C12-200), 1,1'-((2-(4-(2-((2-(bis(2- hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl) bis(dodecan-2-ol) (C12-200), Cholesteryl-succinylsilane (C2), (9Z,9'Z,12Z,12'Z)-2- ((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3- diyl bis(octadeca-9,12-dienoate) (Cationic Lipid A2), (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyloctadeca-9,12- dienoate (Cationic Lipid A3), 1-(3-cholesteryl)-oxycarbonyl-aminomethylimidazole (CHIM), [(2-morpholin-4-yl-ethylcarbamoyl)methyl]-cholesteryl carbamate (Chol-C3N-Mo2), [(2-morpholin-4-yl-ethylcarbamoyl)-ethyl]-cholesteryl carbamate (Chol-DMC3N-Mo2), [1-methyl-2-(2-morpholin-4-yl-ethylcarbamoyl)-propyl]-cholesteryl carbamate (Chol-C4N-Mo2), 1,17-bis(2-octylcyclopropyl)heptadecan-9-yl 4-(dimethylamino)butanoate (CL), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)-butanoate (CLOl), cholesteryl 3-(dimethylamino)propanoate (CL06), cholesteryl 2- (dimethylamino)acetate (CL08), N,N-dimethyl-2,3-bis(((9Z,12Z)-octadeca-9,12-dien-1- yl)oxy)propan-1-amine (CL-1),N-methyl-2-(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)-N-(2-((((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)ethyl)ethan-1-amine (CL-11), (3R,4R)-3,4-bis(((Z)-hexadec-9-en-1-yl)oxy)-1-methylpyrrolidine (Compound CL-12) (CL-12), 2-(dimethylamino)-N-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl)acetamide (CL-13), 3-(dimethylamino)propane-1,2-diyl (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate) (CL-14), (9Z,12Z)-bis((9Z,12Z)-octadeca-9,12-dien-1-yl)amine (CL-15), 7-hydroxy 7-(4-((1-methylpiperidine-4-carbonyl)oxy)butyl)tridecane-1,13-diyl didodecanoate (CL15B6), 7-hydroxy 7-(4-((1-methylpiperidine-4-carbonyl)oxy)butyl)tridecane-1,13-diyl ditetradecanoate (CL15C6), 7-hydroxy 7-(4-((1-methylpiperidine-4-carbonyl)oxy)butyl)tridecane-1,13-diyl dipalmitate (CL15D6), 7-hydroxy 7-(4-((1-methylpiperidine-4-carbonyl)oxy)butyl)tridecane-1,13-diyl dioleate (CL15H6), bis(2-(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)ethyl)amine (CL-16), (9Z,12Z)-N-methyl-N-(2-(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)ethyl)octadeca-9,12-dien-1-amine (CL-17), (9Z,12Z)-N-(3-(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)propyl)octadeca-9,12-dien-1-amine (CL-18), (1-methylpiperidin-3-yl)methyl bis((11Z,14Z)-eicosa-11,14-dien-1-yl)carbamate (CL-19), N-methyl-N,N-bis(2-((Z)-hexadec-9-enyloxy)ethyl)amine (CL-2), (13Z,16Z)-N,N-dimethyl-4-((9Z,12Z)-octadeca-9,12-dien-1-yl)docosa-3,13,16-trien-1-amine (CL-20), (S)-2-amino-3-hydroxy-N,N-bis(2-(((Z)-octadec-9-en-1-yl)oxy)ethyl)propanamide (CL-21), N,N-dihexadecyl-N'-(3-triethoxysilylpropyl)succinamide (CL3),trans- 1 -Methyl-3,4-bis((((Z)-octadeca-9-en- 1 -yl)oxy)methyl)pyrrolidine (CL-3), trans- 1 -Methylpyrrolidine-3,4-diyl)bis(methylene)(9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate) (CL-4), 7-(4-(Diisopropylamino)butyl)-7-hydroxytridecane- 1, 13 -diyl dimyristate (CL4C6), 7-(4-(Diisopropylamino)butyl)-7-hydroxytridecane- 1, 13 -diyl dipalmitate (CL4D6), 11 -(4-(Diisopropylamino)butyl)- 11 -hydroxyheneicosane- 1,21 -diyl dioleate (CL4H10), 7-(4-(Diisopropylamino)butyl)-7-hydroxytridecane- 1, 13 -diyl dioleate (CL4H6), 9-(4-(Diisopropylamino)butyl)-7-hydroxyheptadecane- 1, 17 -diyl dioleate (CL4H8), (6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31 -tetraen- 19 -yl 4-(dimethylamino)butanoate (CL-5), 2-(Dimethylamino)-N-(2-(((Z)-octadeca-9-en- 1 - yl)oxy)ethyl)-N-((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)acetamide (CL-53), 3-((2-(((Z)-octadeca-9-en- 1 - yl)oxy)ethyl)((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)amino)propan- 1 -ol (CL-54), 1 -Methyl-3,3- bis((((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)oxy)methyl)azetidine (CL-55), 1 -Methyl-3,3-bis(2-(((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)oxy)ethyl)azetidine (CL-56), 1 -Methyl-3,3-bis(2-(((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)oxy)propyl)azetidine (CL-57), 2-(3,3-Di((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)azetidin- 1 -yl)ethan- 1 -ol (CL-58), 2-(3,3-Di((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)azetidin- 1 -yl)propan- 1 -ol (CL-59), 3-(Di((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)amino)propan- 1 -ol (CL-6), 3-(Dimethylamino)propyl 3,3-di((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)azetidine- 1 -carboxylate (CL-60), 2-(Di((Z)-octadeca-9-en- 1 -yl)amino)ethan- 1 -ol (CL-61),3-(di((Z)-octadeca-9-en-1-yl)amino)propan-1-ol (CL-62), (11Z,14Z)-2-((dimethylamino)methyl)-2-((9Z,12Z)-octadeca-9,12-dien-1-yl)icosa-11,14-dien-1-ol (CL-63), (11Z,14Z)-2-(dimethylamino)-2-((9Z,12Z)-octadeca-9,12-dien-1-yl)icosa-11,14-dien-1-ol (CL-64), 3-(dimethylamino)-2,2-bis((((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)methyl)propan-1-ol (CL-65), (9Z,12Z)-N-(2-(((Z)-octadeca-9-en-1-yl)oxy)ethyl)octadeca-9,12-dien-1-amine (CL-7), 1-methyl-3,3-di((9Z,12Z)-octadeca-9,12-dien-1-yl)azetidine (CL-8), N,2-dimethyl-1,3-bis(((9Z,12Z)-octadeca-9,12-dien-1-yl)oxy)propan-2-amine (CL-9), 3-dimethylamino-2-(cholest-5-en-3beta-oxobutan-4-oxo)-1-(cis,cis-9,12-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-oxo-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), cetyltrimethylammonium bromide (CTAB), 1,2-diarachidonoyloxy-(N,N-dimethyl)-propyl-3-amine (DAraDMA), O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl)diethanolamine chloride (DC-6-14), 3beta-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-Chol), dimethyl dioctadecyl ammonium (DDA), dimethyl dioctadecyl ammonium bromide (DDAB), DDDAB, l,2-didocosa-hexaenoyloxy-(N,N-dimethyl)-propyl-3-amine (DDocDMA), N-(2-(dimethylamino)ethyl)-4,5-bis(dodecylthio)pentanamide (DEDPA), 3-dimethylamino-2-(cholest-5-en-3beta-oxopento-3-oxa-ene-5-oxo)-1-(cis,cis-9,12-octadecadienoxy)propane (DEG-CLinDMA), 1,6-dioleoyl triethylenetetramine (dio-TETA),N1,N19-bis((S,23E,25E,27E,29E)-16-((2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6- trimethylcyclohex-1 -en-1 -yl)nona-2,4,6,8-tetraenoyl amino)-24,28-dimethyl-15,22-dioxo- 30-(2,6,6-trimethylcyclohex-1 -en-1 -yl)-4,7,10-trioxa-14,21 -diazatriaconta-23,25,27,29- tetraen-1 -yl)-4,7,10,13,16-pentaoxanona-1,19-diamide (diVA-PEG-diVA), DiLin-N- methylpiperazine (DL-033), DiLin-N,N-dimethylglycine (DL-036), Dioleyl-N,N- dimethylglycine (DL-048), 3-((1,3-bis(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)propan-2- yl)amino)propanoic acid (DLAPA), 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), 1 -linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 3-(N,N- dilinoleylamino)-1,2-propanediol (DLinAP), 1,2-N,N'-dilinoleylcarbamoyl-3- dimethylaminopropane (DLincarbDAP), 1,2-dilinoleylcarbamoyloxy-3- dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyl-oxy-3-(dimethylamino)acetyloxypropane (DLin-DAC), 1,2-dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2- dilinoleyl-oxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyl-oxy-3- dimethylaminopropane (DLinDMA 1 ), 1,2-dilinoleyl-oxy-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), Dilinoleyl-4-aminobutanoic acid (DLinFAB), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA), 2,2-dilinoleyl-4- dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-dilinoleyl-oxy-3- morpholino propane (DLin-MA), (6Z,9Z,28Z,31 Z)-heptatriaconta-6,9,28,31 -tetraen-19-yl 4-1,2-dilinoleyls-3-(N-methylpiperazinyl)propane (DLin-MPZ), dilinoleyl-3- piperidinylpropanamine (DLinPip), 1,2-dilinoleyls-3-(3'-hydroxypiperidinyl)- propanamine (DLinPip-3OH), 1,2-dilinoleyls-3-(4'-hydroxypiperidinyl)- propanamine (DLinPip-4OH), 1,2-dilinoleyls-3-hydroxypropane (DLinPO), 1,2- dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1,2-dilinoleyl-3- trimethylaminopropane (DLinTAP), 1,2-dilinoleyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyls-3-trimethylaminopropane (DLinTMA), 1,2-dilinoleyls-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 3-((1,3- bis(((9Z,12Z,15Z)-octadeca-9,12,15-trienoyl)oxy)propan-2-yl)amino)propanoic acid (DLLAPA), 1,2-dilinoleyls-3-(N,N-dimethyl)-propanamine (DLmDEA), 1,2- dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dilauroyl-sn-glycero-3- glycerol (DLPG), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), dimyristyl phosphatidylserine (DMPS), N-[1-(2,3-dimyristyloxy)propyl]-N,N- dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE), 1,2-dimyristyl-3- dimethyl-hydroxyethylammonium bromide (DMRIE1), 1,2-dimyristyl-3- trimethylammonium propane (DMTAP), 3-(N,N-dioleylamino)-1,2-propanediol (DOAP), 3-((1,3-bis(oleoyloxy)propan-2-yl)amino)propanoic acid (DOAPA), 1,2-N,N'- dioleoylcarbamoyl-3-dimethylaminopropane (DOcarbDAP), 1,2-dioleoylcarbamoyl- 3-dimethylammonium-propane (DOCDAP), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), N,N- dihydroxyethyl Ν,Ν-dioctadecylammonium chloride (DODEAC), N,N-dimethyl-2,3- dioleyloxypropylamine (DODMA), dioleoyl-4-aminobutyric acid (DOFAB), dioleo-1,2-dioleoyl-3-N-pyridonium-propane bromide salt (DOP6P), 1,2-dioleoyl-3- dimethyl-hydroxyethyl ammonium bromide (DORI), 1,2-dioleyloxypropyl-3- dimethyl-hydroxyethyl ammonium bromide (DORIE), 1,2-dioleyloxypropyl-3- dimethyl-hydroxybutyl ammonium bromide (DORIE-HB), 1,2-dioleyloxypropyl-3- dimethyl-hydroxypropyl ammonium bromide (DORIE-HP), 1,2-dioleyloxypropyl-3- dimethyl-hydroxy-pentyl ammonium bromide (DORIE-Hpe), 2,3-dioleyloxy-N-[2 (spermylcarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 1,3-dioleoyloxy-2-(6-carboxy-spermyl)-propylamide (DOSPER), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP1), N-[5'-(2',3'-dioleoyl)uridine]-N',N',N'-trimethylammonium tosylate (DOTAU), 1-[2-(9(Z)-octadecenoyloxy)ethyl]-2-(8(Z)-heptadecenyl-3-(2- hydroxyethyl)imidazolinium chloride (DOTIM), N-(1-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), dioleylphosphatidyluridine phosphatidylcholine (DOUPC), 1,2-diphytanoyloxy-(N,N-dimethyl)-butan-4-amine (DPan-C2-DMA), 1,2-diphytanoyloxy-3-(N,N-dimethyl)-propan-1-amine (DPanDMA), 2,3-bis(dodecylthio)propyl (2-(dimethylamino)ethyl)carbamate (DPDEC), dipalmitoyl-4-aminobutyric acid (DPFAB), 1,2-dipalmitoyloxypropyl-3-dimethyl- hydroxyethyl ammonium bromide (DPRIE), 1,2-dipalmitoyl-3-trimethylammonium propane (DPTAP), 1-[2-(hexadecanoyloxy)ethyl]-2-pentadecyl-3-(2- hydroxyethyl)imidazolinium chloride (DPTIM), 3-((1,3-bis(stearyloxy)propan-2- yl)amino)propanoic acid (DSAPA), distearyldimethylammonium (DSDMA), 1,2- distearyloxy-N,N-dimethylaminopropane (DSDMA1), 1,2-distearyloxypropyl-3- dimethyl-hydroxyethyl ammonium bromide (DSRIE), 1,2-distearyloxy-3- trimethylammonium propane (DSTAP), ditetradecyltrimethylammonium (DTDTMA), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (EDOPC),N2-[N2,N5-bis(3-aminopropyl)-L-ornithyl]-N,N-dioctadecyl-L-glutamine tetrahydrotrifluoroacetate (GC33), Cholesta-5-en-3-ol (3P)-, 3-[(3-aminopropyl)[4-[(3-aminopropyl)amino]butyl]carbamate] (GL67), Glycerol monooleate (GMO), Guanidino-dialkyl-carboxylic acid (GUADACA), 2-(bis(2-(tetradecanoyloxy)ethyl)amino)-N-(2-hydroxyethyl)-N,N-dimethyl-2-oxoethanaminium bromide (HEDC), 2,2'-(tert-butoxycarbonylazanediyl)bis(ethane-2,1-diyl) ditetradecanoate (HEDC-BOC-TN), 1-(2-(((3S,10R,13R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3- yl)disulfanyl)ethyl)guanidine (HGT4002), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl) tetracosa-15,18-dien-1-amine (HGT5000), (15Z,18Z)-N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-1-yl) tetracosa-4,15,18-trien-1-amine (HGT5001), Histaminyl-cholesterol hemisuccinate (HisChol), Histidinyl cholesteryl hemisuccinate (Hist-Chol), Hydrogenated soy PC (HSPC), Imidazole cholesteryl ester (ICE), 3-(didodecylamino)-N1,N1,4-tridodecyl-1-piperazinethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,N4,N4-tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-bis-tridecyl-15,18,21,24-tetraaza-triacontane (KL25), N,N-di-n-tetradecyl, N-methyl-N-(2-guanidino)ethyl ammonium chloride (Lipid 1), N,N-di-n-octadecyl, N-methyl-N-(2-guanidino)ethyl ammonium chloride (Lipid 2), 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate (Lipid A), (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate (Lipid A1),2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (Lipid A2), ((5-((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(octane-8,l-diyl) bis(decanoate) (Lipid B), 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-l,3-diyl (9Z,9'Z,12Z,12'Z)-bis( octadeca-9, 12-dienoate) (Lipid C), 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)- 13-(octanoyloxy)tridecyl-3-octylundecanoate (Lipid D), (6Z, 16Z)-12-((Z)-dec-4-en- 1-yl)dohca-6, 16-diene-11-yl 5-(dimethylamino)pentanoate (Lipid I), Bisstearyl-(2-hydroxy-3-propylamino)aminopolyllysine (Lipid T), (3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan- 1 -amine (MC3 ether), (MC3 thioether) described in U.S. Provisional Application No. 61 / 384,050, (4-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N- dimethylbutane-l -amine (MC4 ether), 3-((2-(((9Z, 12Z)-octadeca-9, 12-dienoyl)oxy)ethyl)amino)propanoic acid (MLAPA), 3-((2-(((9Z, 12Z, 15Z)-octadeca-9, 12, 15-trienoyl)oxy)ethylamino)propanoic acid (MLLAPA), monymcolylglycerol (MMG), 3-((2-(oleoyloxy)ethyl)amino)propanoic acid (MOAPA), 4-(2-aminoethyl)-morpholino-cholesterol hemi succinate (MoChol), 1,2-dioleoyl-3-N-morpholine-propane (MoDO), methylpyridyl-dialkyl-carboxylic acid (MPDACA), monomyristoylphosphatidylcholine (MPPC), 3-((2-(stearoyloxy)ethyl)amino)propanoic acid (MSAPA), N1-[2-((lS)-l-[(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylformamido)ethyl]-3,4-di[oleoyloxy]-benzamide (MVL5), 2-({8-[(3beta)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien- 1-yloxy]propan- 1 -amine (Octyl-CLinDMA),(2R)-2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)- octadeca-9, 12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA (2R)), phosphatidylcholine (PC), 1,3-bis-(1,2-bis-tetradecyloxy-propyl-3-dimethylethoxy bromide)-propane-2-ol (PCL-2), palmitoyl-oleoyl-nor-arginine (PONA), stearylamine (STA), 2-(((tert-butyldimethylsilyl)oxy)methyl)-2-(hydroxymethyl)propane-1,3-diol (Synthesis Example 1 (A)), 3-((tert- butyl(dimethyl)silyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl (9Z)- tetradec-9-enoate (Synthesis Example 1 (B)), 3-hydroxy-2,2-bis(((9Z)-tetradec-9- enoyloxy)methyl)propyl (9Z)-tetradec-9-enoate (Synthesis Example 1 (C)), 3-((4- (dimethylamino)butanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl (9Z)- tetradec-9-enoate (Synthesis Example 1 (D)), 3-(5-(bis(2-hydroxydodecyl)amino) pentan-2-yl)-6-(5-((2-hydroxydodecyl)(2-hydroxyundecyl)amino)pentan-2-yl)-1,4- dioxane-2,5-dione) (Target 24), trehalose 6'6'-dibehenate (TDB), 1,1'-(2-(4-(2-((2- (bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl) ethylazanediyl)dodecan-2-ol (Tech G1), 3-((1,3-bis(((9Z,12Z)-octadeca-9,12-dienoyl)oxy)- 2-((((9Z,12Z)-octadeca-9,12-dienoyl)oxy)methyl)propan-2-yl)amino)propanoic acid (TLAPA), (1-(2,3-linoleyloxypropoxy)-2-(linoleyloxy)-(N,N-dimethyl)-propyl-3- amine) (TLinDMA), 3-((1,3-bis(((9Z,12Z,15Z)-octadeca-9,12,15-trienoyl)oxy)-2- ((((9Z,12Z,15E)-octadeca-9,12,15-trienoyl)oxy)methyl)propan-2-yl)amino)propanoic acid (TLLAPA), N-(a-trimethylammonioacetyl)-dibehenoyl-D-glutamate chloride (TMAG),3-((1,3-bis(((Z)-octadec-9-enoyl)oxy)-2-((((Z)-octadec-9-enoyl)oxy)methyl)propan-2- yl)amino)propanoic acid (TOAPA), 3-((1,3-bis(stearoyloxy)-2- ((stearoyloxy)methyl)propan-2-yl)amino)propanoic acid (TSAPA), 1, N19-bis((16E,18E,20E,22E)- 17,21-dimethyl-15-oxo-23-(2,6,6-trimethylcyclohex-1-en-1-yl)-4,7,10-trioxa-14-azatriaconta-16,18,20,22- tetraen-1-yl)-4,7,10,13,16-pentaoxanonadecane-1,19-diamide (VA-PEG-VA), 2,2-dilinoleyl-4- dimethylaminoethyl-[1,3]-dioxolane (XTC), 1,2-di-γ-linolenyloxy-N,N-dimethylaminopropane (γ- DLenDMA), a-D-tocopherol hemisuccinoyl, (9Z,9'Z,12Z,12'Z)-2-((2-(((3- (dimethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diyl bis(octadeca-9,12- dienoate), 2-(((13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)dodeca-13,16-dienoyl)oxy)propane- 1,3-diyl dioctanoate, 2-(((13Z,16Z)-4-(((3-(dimethylamino)propoxy)carbonyl)oxy)dodeca-13,16- dienoyl)oxy)propane-1,3-diyl dioctanoate, 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl dioctanoate, 2-((4-(((3-((2-(((1 -isopropylpiperidine-4-carbonyl)oxy)methyl)-1,4-phenylene)bis(oxy))bis(octane-8,1 - diyl) bis(decanoate), 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane- 1,3-diyl dodecanoate, 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3- diyl dodecanoate, 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3- diyl dodecanoate, 2-((4-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane- 1,3-diyl dimyristate, 2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane- 1,3-diyl dimyristate, 2-((4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3- diyl dimyristate, (Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3- diyl dioleate, (9Z,9'Z,12Z,12'Z,15Z,15'Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl bis(octadeca-9,12,15-trienoate), (9Z,9'Z,12Z,12'Z)-2-((4-(((3- (diethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1,3-diyl bis(octadeca-9,12-dienoate), (9Z,9'Z,12Z,12'Z)-2-((4-(((3-(dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane- 1,3-diyl bis(octadeca-9,12-dienoate), N,N,N-trimethyl-5-oxo-5-(3-((3-pentyloctanoyl)oxy)-2,2-bis(((3- pentyloctanoyl)oxy)methyl)propoxy)pentan-1 -ium iodide, 3-((5-(dimethylamino)pentanoyl)oxy)-2,2- bis(((3-pentyloctanoyl)oxy)methyl)propyl 3-pentyloctanoate, 3-dimethylaminopropyl carbonate (9Z,12Z)- octadeca-19,22-dien-11 -yl, 2-(((N,N-dimethyl-beta-alaninyl)oxy]methyl}-2-[(octanoyloxy)methyl)propane- 1,3-diyl (9Z,9'Z)bis-tetracosa-9-enate, O’,O1-(2-(7-dodecyl-14-methyl-3,9-dioxo-2,4,8,10-tetraoxa-14- azapentadecyl)propane-1,3-diyl)8-dimethyldioctanedioate,8-dimethyl O', O1-(2-(((1-methylpyrrolidine-3-carbonyl)oxy)methyl)propane-1,3- diyl) dioctanedioate, 1-(3-((6,6-bis((2-propylpentyl)oxy)hexanoyl)oxy)-2-(((1,4- dimethylpiperidine-4-carbonyl)oxy)methyl)propyl) 8-methyloctanedioate, (9Z,12Z)-5- (((3-(dimethylamino)propoxy)carbonyl)oxy)-7-octylpentadecylocta-9,12-dienoate, 5- (((3-(dimethylamino)propoxy)carbonyl)oxy)-7-octylpentadecyloctanoate, 1-(3-((6,6-bis((2- propylpentyl)oxy)hexanoyl)oxy)-2-(((1,4-dimethylpiperidine-4-carbonyl)oxy)methyl)propyl) 10- octyldecanedioate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-5-octyltridecyldecanoate, 1- (16-(((4,4-bis(octyloxy)butanoyl)oxy)methyl)-9-dodecyl-2-methyl-7,13-dioxo-6,8,12,14- tetraoxa-2-azanonadec-17-yl) 8-methyloctanedioate, 3-((5-(dimethylamino)pentanoyl)oxy)-2,2- bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate, 3-((5- (dimethylamino)pentanoyl)oxy)-2,2-bis(((3-pentyloctanoyl)oxy)methyl)propyl 3-pentyloctanoate, (9Z,9'Z,12Z,12'Z)-2-(((3-(diethylamino)propanoyl)oxy)methyl)propane-1,3-diyl bis(octadeca-9,12-dienoate), ((2-(((4-(dimethylamino)butanoyl)oxy)methyl)-1,4- phenylene)bis(oxy))bis(octane-8,1-diyl) bis(decanoate), 1-(3-((4,4-bis(octyloxy)butanoyl)oxy)- 2-(((1-methylpyrrolidine-3-carbonyl)oxy)methyl)propyl) 8-methyloctanedioate, 3-((4,4-bis(octyloxy) butanoyl)oxy)-2-((palmitoyloxy)methyl)propyl 1-methylpyrrolidine-3-carboxylate, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((tetradecanoyloxy)methyl)propyl 1- methylpyrrolidine-3-carboxylate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13- (octanoyloxy)tridecyl 9-pentyldodecanoate, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2- ((dodecanoyloxy)methyl)propyl 1-methylpyrrolidine-3-carboxylate, 3-(((3-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl 7- hexyltridecanoate, 2-(5-(3-((1-methylpyrrolidine-3-carbonyl)oxy)-2- ((tetradecanoyloxy)methyl)propoxy)-5-oxopentyl)propane-1,3-diyl dioctanoate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl 5- heptyldodecanoate, 2-(5-(3-((1-methylpyrrolidine-3-carbonyl)oxy)-2- ((palmitoyloxy)methyl)propoxy)-5-oxopentyl)propane-1,3-diyl dioctanoate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13-hydroxytridecyl 5- heptyldodecanoate, 2-(((1-methylpyrrolidine-3-carbonyl)oxy)methyl)propane-1,3- diyl bis(6,6-bis(octyloxy)hexanoate), (9Z,12Z)-3-(((3-dimethylamino)propoxy)carbonyl)oxy)-13-(octanoyloxy)tridecyl octadeca-9,12-dienoate, 3-((5- (dimethylamino)pentanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl (9Z)-octadec-9-enoate, 2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3- aza nonadec-19-yl)propane-1,3-diyl dioctanoate, ((2-(((1-methylpiperidine-4- carbonyl)oxy)methyl)-1,4-phenylene)bis(oxy))bis(octane-8,1-diyl)bis(decanoate), 2-(((3-(dimethylamino)propanoyl)oxy)methyl)propane-1,3-diyl bis(4,4-bis(octyloxy) butanoate), (9Z,12Z)-2-(((11Z,14Z)-2-((3-(dimethylamino)propanoyl)oxy)undeca-11,14- dien-1-yl)oxy)ethyloctadeca-9,12-dienoate, 2-(((1,3-dimethylpyrrolidine-3- carbonyl)oxy)methyl)propane-1,3-diyl bis(4,4-bis(octyloxy)butanoate), (13Z,16Z)- 4-(((3-(dimethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-1-yl heptadecan- 9-yl succinate, 2,2-bis(heptyloxy)ethyl 3-((3-ethyl-10-((9Z,12Z)-octadeca-9,12-dien-1-yl)- 8,15-dioxo-7,9,14-trioxa-3-azahexadecan-17-yl)disulfonyl)propanoate, 2- (((1-methylpyrrolidine-3-carbonyl)oxy)methyl)propane-1,3-diyl bis(4,4-bis(octyloxy) butanoate,1 -(3-((1,3-dimethylpyrrolidine-3-carbonyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyl- oxy)methyl)propyl) 10-octyldecanedioate, (13Z,16Z)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)docosa-13,16-dien-1 -yl 2,2-bis(heptyloxy)acetate, (13Z,16Z)-4-(((2-(dimethylamino)ethoxy)carbonyl)oxy)docosa-13,16-dien-1 -yl 2,2-bis(heptyloxy)acetate, (20,23R)-2-methyl-9-[(9Z,12Z)-octadeca-9,12-dien-1 -yl]-7-oxo-6,8,11 -trioxa-2- azanonacosan-20-en-23-yl 3-(dimethylamino)propanecarbonyl acetate, (11 Z,14Z)-1 -{[(9Z,12R)-12-hydroxyoctadeca-9-en-1 -yl] 2,2-bis(heptyloxy)acetate, (12Z,15Z)-1 -((((9Z,12Z)-octadeca-9,12-dien-1 -yloxy)carbonyl)oxy)heneicosa-12,15-dien-3-yl 3-(dimethylamino)propanoate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(dimethylamino)propyl)carbamoyl)oxy)methyl)propyl octadeca-9,12-dienoate, (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)heneicosa-12,15-dien-1 -yl 9-pentyl- tetradecanoate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1,2,2,6,6-pentamethylpiperidin-4- yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)heneicosa-12,15-dien-1 -yl 7-hexyl- tridecanoate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1 -methylpiperidin-4- yl)methoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)heneicosa-12,15-dien-1 -yl 5-heptyl- dodecanoate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1 -ethylpiperidin-4- yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, (12Z,15Z)-3-((4-(dimethylamino)butanoyl)oxy)heneicosa-12,15-dien-1 -yl 3-octyl- undecanoate formate,3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl (9Z)-hexadec-9-enoate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-methylazetidin-3-yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, (9Z,12Z)-(12Z,15Z)-3-((3-(dimethylamino)propanoyl)oxy)heneicos-12,15-dien-1-yl octadeca-9,12-dienoate, 2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecyl 4,4-bis((2-ethylhexyl)oxy)butyrate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-methylpyrrolidin-3-yl)oxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, (9Z,12Z)-3-(((2-(dimethylamino)ethoxy)carbonyl)oxy)pentadecyl octadeca-9,12-dienoate, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, 3-(dimethylamino)propyl triacontan-11-yl carbonate triacontan-11-ol, (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(pyrrolidin-1-yl)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, (9Z,12Z)-3-(((3-(ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl octadeca-9,12-dienoate, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 4-((diethylamino)methyl)benzoate, (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl octadeca-9,12-dienoate, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 3-((dimethylamino)methyl)benzoate, (9Z,12Z)-3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl octadeca-9,12-dienoate,3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1-methylpiperidine-3-carboxylate, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1-methylpiperidine-4-carboxylate, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1,4-dimethylpiperidine-4-carboxylate, 3-((4-(dimethylamino)butanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl (9Z)-hexadec-9-enoate, 2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azahexadec-16-yl)propane-1,3-diyl dioctanoate, (9Z,9'Z,12Z,12'Z)-2-(((4-(piperidin-1-yl)butanoyl)oxy)methyl)propane-1,3-diyl bis(octadeca-9,12-dienoate), 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 4-methylmorpholine-2-carboxylate, (2R)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1-methylpyrrolidine-2-carboxylate, (2S)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1-methylpyrrolidine-2-carboxylate, (9Z,9'Z,12Z,12'Z)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propane-1,3-diyl bis(octadeca-9,12-dienoate), (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((((1-ethylpiperidin-3-yl)methoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1-(cyclopropylmethyl)piperidine-4-carboxylate, 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)propyl 1-isopropylpiperidine-4-carboxylate,(9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3-(dimethylamino)propyl)oxy)methyl)propyl octadeca-9,12-dienoate, 4-(dimethylamino)butyl carbonate (6Z,9Z,26Z,29Z)-tricosa-6,9,26,29-tetraen-18-yl, 3-((6-(dimethylamino)hexanoyl)oxy)-2,2-bis(((9Z)-tetradec-9-enoyloxy)methyl)propyl (9Z)-tetradec-9-enoate, 2,5-bis((9Z,12Z)-octadeca-9,12-dienyloxy)benzyl 3-(dimethylamino)propyl carbonate, (9Z,9'Z,12Z,12'Z)-2-(((4-(pyrrolidin-1-yl)butanoyl)oxy)methyl)propane-1,3-diyl bis(octadeca-9,12-dienoate), 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 5-heptyldodecanoate, acetic acid (7R,9Z)-18-({[3-(dimethylamino)propyloxy]carbonyl}oxy)octacosan-9-en-7-yl, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 9-pentyltetradecanoate, (9Z,12Z)-3-((6,6-bis(octyloxy)hexanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 7-hexyltridec-6-enoate, (9Z,12Z)-3-(2,2-bis(heptyloxy)acetyloxy)-2-((((2-(dimethylamino)ethoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 3-octylundec-2-enoate, (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((5-heptyldodeanoyl)oxy)methyl)propyl octadeca-9,12-dienoate, 3-(((3-dimethylamino)propoxy)carbonyl)oxy)pentadecyl 3-octylundecanoate, (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((9-pentyltetradecanoyl)oxy)methyl)propyl octadeca-9,12-dienoate, diacetic acid (7R,9Z,26Z,29R)-18-({[3-(dimethylamino)propoxy]carbonyl}oxy)tricosa-9,26-dien-7,29-diyl, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 8,8-bis((2-propylpentyl)oxy)octanoate,(9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((7- hexyltridecanoyl)oxy)methyl)propyl octadeca-9,12-dienoate, 3-(((3- (ethyl(methyl)amino)propoxy)carbonyl)oxy)pentadecyl 8,8-bis((2- propylpentyl)oxy)octanoate, (9Z,12Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((3- octylundecanoyl)oxy)methyl)propyl octadeca-9,12-dienoate, 3-(((3- (diethylamino)propoxy)carbonyl)oxy)pentadecyl 8,8-bis((2- propylpentyl)oxy)octanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 8,8- dibutoxyoctanoate, 3-((5-(dimethylamino)pentanoyl)oxy)-2,2-bis(((9Z)- tetradec-9-enoyl)oxy)methyl)propyl (9Z)-tetradec-9-enoate, 3- (dimethylamino)propyl carbonate (6Z,9Z,26Z,29Z)-tricosen-6,9,26,29-tetraen-18-yl, 2,5- bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl 3-(dimethylamino)propanoate, (9Z,9'Z,12Z,12'Z)-2-(((3-(4-methylpiperazin-1-yl)propanoyl)oxy)methyl)propane-1,3- diyl bis(octadeca-9,12-dienoate), 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 8,8- bis(octyloxy)octanoate, 3-(dimethylamino)propyl octacosan-11-yl carbonate, 2,4- bis((9Z,12Z)-octadeca-9,12-dienyloxy)benzyl 4-(dimethylamino)butanoate, (9Z,12Z)-3- (((3-(diethylamino)propoxy)carbonyl)oxy)-2-(((2-heptylundecanoyl)oxy)methyl)propyl octadeca-9,12-dienoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 6,6- bis((2-ethylhexyl)oxy)hexanoate, 2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)propane-1,3- diyl bis(2-heptylundecanoate), 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 6,6- bis(hexyloxy)hexanoate, 4-methyl-2,5-bis((9Z,12Z)-octadeca-9,12-dien-1-yloxy)benzyl 4- (dimethylamino)butanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 6,6- bis(octyloxy)hexanoate, 4-(dimethylamino)butyl 4-methyl-2,5-bis((9Z,12Z)-octadeca-9,12- dienyloxy)benzyl carbonate,3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 4,4-bis((2- propylpentyl)oxy)butyrate, 2-(12-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3- azaoctadecan-18-yl)propane-1,3-diyl dioctanoate, 2-(5-oxo-5-((3-(((3- ((3-(piperidin-1-yl)propoxy)carbonyl)oxy)pentadecyl)oxy)pentyl)oxy)pentyl) propane-1,3-diyl dioctanoate, 3-(dimethylamino)propyl 4-methyl-2,5-bis((9Z,12Z)- octadeca-9,12-dien-1-yloxy)benzyl carbonate, 3-(((3-(ethyl(methyl)amino)propoxy) carbonyl)oxy)pentadecyl 4,4-bis((2-propylpentyl)oxy)butyrate, 2-(11-dodecyl-3- ethyl-9,15-dioxo-8,10,14-trioxa-3-azanonadecan-19-yl)propane-1,3-diyl dioctanoate, 2-(10-dodecyl-3-ethyl-8,15-dioxo-7,9,14-trioxa-3-azanonadecan-19-yl)propane- 1,3-diyl dioctanoate, 2-(5-((4-((((1-methylpiperidin-4-yl)oxy)carbonyl)oxy) hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl dioctanoate, 2-(5-((4-((((1- ethylpiperidin-3-yl)methoxy)carbonyl)oxy)hexadecyl)oxy)-5-oxopentyl)propane-1,3- diyl dioctanoate, 2-(5-((4-(((((R)-1-methylpyrrolidin-3-yl)oxy)carbonyl)oxy) hexadecyl)oxy)-5-oxopentyl)propane-1,3-diyl dioctanoate, 2-(5-((4-(((((S)-1-4,4-bis(octyloxy)butyl 5-(((3-(diethylamino)propoxy)carbonyl)oxy)heptadecanoate, 6-((6,6-bis(octyloxy)hexanoyl)oxy)-4-(((3-(diethylamino)propoxy)carbonyl)oxy)hexyloctanoate, (12Z,15Z)-3-(((3-(diethylamino)propoxy)carbonyl)oxy)heneicosa-12,15-dien-1-yl 6,6-bis(octyloxy)hexanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)tridecyl 6,6-bis(octyloxy)hexanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)undecyl 6,6-bis(octyloxy)hexanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 5-(4,6-diheptyl-1,3-dioxan-2-yl)pentanoate, 3-((5-(diethylamino)pentanoyl)oxy)pentadecyl 6,6-bis(octyloxy)hexanoate, 1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yl 1,4-dimethylpiperidine-4-carboxylate, 3-((3-(1-methylpiperidin-4-yl)propanoyl)oxy)pentadecyl 6,6-bis(octyloxy)hexanoate, 1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yl 1,3-dimethylpyrrolidine-3-carboxylate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 4,4-bis((2-ethylhexyl)oxy)butyrate, 2-(((1,3-dimethylpyrrolidine-3-carbonyl)oxy)methyl)propane-1,3-diyl bis(8-(octanoyloxy)octanoate), ((2-((((3-(dimethylamino)propoxy)carbonyl)oxy)methyl)-1,4-phenylene)bis(oxy))bis(octane-8,1-diyl) bis(decanoate), (2R)-1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yl pyrrolidine-2-carboxylate, (2S)-1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yl 1-methylpyrrolidine-2-carboxylate, (2R)-1-((6,6-bis(octyloxy)hexanoyl)oxy)pentadecan-3-yl 1-methylpyrrolidine-2-carboxylate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 6,6-bis((3-ethylpentyl)oxy)hexanoate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 6,6-bis((2-propylpentyl)oxy)hexanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 6,6-bis((2-propylpentyl)oxy)hexanoate, 3-(((2-(diethylamino)ethoxy)carbonyl)oxy)pentadecyl 6,6-bis(octyloxy)hexanoate,3-(((3-morpholinopropoxy)carbonyl)oxy)pentadecyl 6,6-bis(octyloxy)hexanoate, 3-((((1-methylpiperidin-4-yl)methoxy)carbonyl)oxy)pentadecyl 6,6-bis(octyloxy)hexanoate, 3-(((3-(4-methylpiperazin-1-yl)propoxy)carbonyl)oxy)pentadecyl 6,6-bis(octyloxy)hexanoate, 3-(((3-(diethylamino)propoxy)carbonyl)oxy)pentadecyl 4,4-bis(octyloxy)butyrate, 2-(((4-(dimethylamino)butyryl)oxy)methyl)-2-((dodecanoyloxy)methyl)propane-1,3-diyl (9Z,9'Z)bis-tetradec-9-enoate, (9Z,9'Z,12Z,12'Z)-2-(((4-(dimethylamino)butyryl)oxy)methyl)propane-1,3-diyl bis(octadeca-9,12-dienoate), 3-(((4-(diethylamino)butoxy)carbonyl)oxy)pentadecyl 6,6-bis(octyloxy)hexanoate, 3-(((3-(piperazin-1-yl)propoxy)carbonyl)oxy)pentadecyl 6,6-bis(octyloxy)hexanoate, 3-(((3-piperidin-1-yl)propoxy)carbonyl)oxy)pentadecyl 6,6-bis(octyloxy)hexanoate, 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)pentadecyl 4,4-bis(octyloxy)butyrate, (9Z,9'Z,12Z,12'Z)-2-(9-dodecyl-2-methyl-7,12-dioxo-6,8,13-trioxa-2-azatetradec-14-yl)propane-1,3-diyl bis(octadeca-9,12-dienoate), (9Z,12Z)-10-dodecyl-3-ethyl-14-(2-((9Z,12Z)-octadeca-9,12-dienoyloxy)ethyl)-8,13-dioxo-7,9-dioxa-3,14-diazahexadec-16-yl octadeca-9,12-dienoate, 2-((2-(((3-(diethylamino)propoxy)carbonyl)oxy)tetradecanoyl)oxy)propane-1,3-diyl dioctanoate, 2-(9-dodecyl-2-methyl-7,13-dioxo-6,8,12-trioxa-2-azanonadec-19-yl)propane-1,3-diyl dioctanoate, 2-((decanoyloxy)methyl)-2-(((4-(dimethylamino)butyryl)oxy)methyl)propane-1,3-diyl (9Z,9'Z)bis-tetradec-9-enoate, (9Z,9'Z,12Z,12'Z)-2-(((3-morpholinopropanoyl)oxy)methyl)propane-1,3-diyl bis(octadeca-9,12-dienoate), 3-(dimethylamino)propyl carbonate (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl,2,5-bis((9z,12z)-octadeca-9,12-dien-1-yloxy)benzyl 4-(dimethylamino)butanoate, 2-(10-dodecyl-3-ethyl-8,14-dioxo-7,9,13-trioxa-3-azaoctadecan-18-yl)propane-1,3-diyl dioctanoate, (9z,9'z,12z,12'z)-2-(((1,3-dimethylpyrrolidine-3-carbonyl)oxy)methyl)propane-1,3-diyl bis(octadeca-9,12-dienoate), ((5-((dimethylamino)methyl)benzene-1,2,3-triyl)tris(oxy))tris(decan-10,1-diyl) trioctanoate, o',o-(((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(propane-3,1-diyl)) 9-dioctyldinonanedioate, (9z,12z)-3-(3-((dimethylamino)methyl)-5-(3-((3-octylundecanoyl)oxy)propoxy)phenoxy)propyloctadeca-9,12-dienoate, ((((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(propane-3,1-diyl))bis(oxy))bis(4- oxobutane-4,1-diyl) bis(decanoate), (r)-4-(3-((r)-3,4-bis(octanoyloxy)butoxy)-5- ((dimethylamino)methyl)phenoxy)butane-1,2-diyl dioctanoate, (s)-4-(3-((s)-3,4-bis(octanoyloxy)butoxy)-5-((dimethylamino)methyl)phenoxy)butane-1,2-diyl dioctanoate, (r)-4-(3-((s)-3,4-bis(octanoyloxy)butoxy)-5-((dimethylamino)methyl)phenoxy)butane-1,2-diyl dioctanoate, 4,4'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-1,2-diyl) tetraoctanoate, 6,6'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))dihexadecyl dibutyrate, di((9z,12z)-octadeca-9,12-dien-1-yl) 5,5'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy)) dipentanoate, (((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(6- oxohexane-6,1-diyl) bis(decanoate), (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene) bis(8-(octanoyloxy)octanoate), (5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene) bis(10-(octanoyloxy)decanoate), (((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(6- oxohexane-6,1-diyl) dioctanoate,((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(8-oxooctane-8,1-diyl) bis(decanoate), (9Z,9'Z,12Z,12'Z)-(((5-((dimethylamino)methyl)-1,3-phenylene)bis(methylene))bis(oxy))bis(4- oxobutane-4,1-diyl) bis(octadeca-9,12-dienoate), 0',0-((5-((dimethylamino)methyl)-1,3- phenylene)bis(methylene)) 8-dinonyldioctanedioate, 0,0'-((5-((dimethylamino)methyl)-1,3- phenylene)bis(methylene)) bis(10-(octanoyloxy)decyl) disuccinate, 0,0'-((5-((dimethylamino)methyl)- 1,3-phenylene)bis(methylene)) bis((9Z,12Z)-octadeca-9,12-dien-1-yl) disuccinate, (9Z,9'Z,12Z,12'Z)- (5-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)-1,3-phenylene)bis(methylene) bis(octadeca-9,12-dienoate), (9Z,12Z)-4-(3-((dimethylamino)methyl)-5-(4-(oleoyloxy) butoxy)phenoxy)butyloctadeca-9,12-dienoate, (9Z,9'Z,12Z,12'Z,15Z,15'Z)-((5-((dimethylamino)methyl)- 1,3-phenylene)bis(oxy))bis(butane-4,1-diyl) bis(octadeca-9,12,15-trienoate), ((5-((dimethylamino)methyl)- 1,3-phenylene)bis(oxy))bis(butane-4,1-diyl) ditetradecanoate, (Z)-((5-((dimethylamino)methyl)-1,3- phenylene)bis(oxy))bis(butane-4,1-diyl) dioleate, ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hexane-6,1-diyl) didodecanoate, (9Z,9'Z,12Z,12'Z)-((((5-((diethylamino)methyl)-1,3-phenylene)bis(oxy))bis(ethane-2,1-diyl))bis(oxy))bis(ethane-2,1-diyl) bis(octadeca-9,12-dienoate), 8,8'-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy)) didecanoic acid didecyl ester, ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(propane-3,1-diyl) bis(3-octylundecanoate), (9Z,9'Z,12Z,12'Z)-((5-((diethylamino)methyl-2-methyl-1,3-phenylene)bis(oxy))bis(butane-4,1-diyl) bis(octadeca-9,12-dienoate),((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl) didodecanoate, ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl) dipelargonate, (9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl-2-methyl-1,3-phenylene)bis(oxy))bis(butane-4,1-diyl) dioctadeca-9,12-dienoate, (8Z,8'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hexane-bis(dodec-8-enoate), (9Z,9'Z,12Z,12'Z)-((5-((3-hydroxyazetidin-1-yl)methyl)-1,3-phenylene)bis(oxy))bis(butane-4,1-diyl) dioctadeca-9,12-dienoate, ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hexane-6,1-diyl) dioctanoate, ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hexane-6,1-diyl) dipelargonate, (9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl) dioctadeca-9,12-dienoate, (9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(hexane-6,1-diyl) dioctadeca-9,12-dienoate, ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(decane-10,1-diyl) dihexanoate, ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(decane-10,1-diyl) dioctanoate, ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl) dioctanoate, ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(octane-8,1-diyl) dihexanoate, (9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(ethane-2,1-diyl) dioctadeca-9,12-dienoate, (9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(propane-3,1-diyl) dioctadeca-9,12-dienoate, (9Z,9'Z,12Z,12'Z)-((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butane-4,1-diyl) dioctadeca-9,12-dienoate,(5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene) ditridecanoate, (9Z,9'Z,12Z,12'Z)-(5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene) dioctadeca-9,12-dienoate, (2,6-bis((9Z,12Z)-octadeca-9,12-dien-l-yloxy)pyridin-4-yl)methyl 3- (dimethylamino)propanoate, (9Z,9'Z,12Z,12'Z)-5-(((3- (dimethylamino)propanoyl)oxy)methyl)-l,3-phenylene bis(octadeca-9,12-dienoate), l-(3,5-bis((9Z,12Z)-octadeca-9,12-dien-l-yloxy)phenyl)-N,N-dimethylmethanamine, 3,5-bis((9Z,12Z)-octadeca-9,12-dien-l-yloxy)benzyl 3-(dimethylamino)propanoate, l-(3,5-bis(4,4-bis(octyloxy)butyloxy)phenyl)-N,N-dimethylmethanamine, ( (( (5-((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(butan-4, 1-diyl))bis(oxy))bis(propane-3,2, 1-triyl) tetraoctanoate, ((5-(((4- (dimethylamino)butanoyl)oxy)methyl)-l,3-phenylene)bis(oxy))bis(octan-8, 1-diyl) bis(decanoate), ((5-(((3-(dimethylamino)propanoyl)oxy)methyl)-l,3- phenylene)bis(oxy))bis(octan-8, 1-diyl) bis(decanoate), (9Z,9'Z,12Z,12'Z)-((5- (3-morpholinopropyl)-l,3-phenylene)bis(oxy))bis(butan-4, 1-diyl) bis(octadeca-9,12- dienoate), (9Z,9'Z,12Z,12'Z)-((5-(3-(dimethylamino)propyl)-l,3- phenylene)bis(oxy))bis(butan-4, 1-diyl) bis(octadeca-9,12-dienoate), (9Z,9'Z,12Z,12'Z)- ((5-(3-(piperidin-l-yl)propyl)-l,3-phenylene)bis(oxy))bis(butan-4, 1-diyl) bis(octadeca-9,12-dienoate), (5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene) bis(9-pentyltetradecanoate), (5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene) bis(7-hexyltridecanoate), (5-((dimethylamino)methyl)-l,3-phenylene)bis(methylene) bis(5-heptyldodecanoate), ((5-((dimethylamino)methyl)-l,3-phenylene)bis(oxy))bis(butan-4, 1-diyl) bis(3-octylundecanoate),((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butan-4,1-diyl) bis(5- heptyldodecanoate), ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butan-4,1- diyl) bis(9-pentyltetradecanoate), ((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butan-4,1-diyl) bis(7-hexyltridecanoate), (9Z,9'Z,12Z,12'Z)-((5-(pyrrolidin-1-ylmethyl)-1,3-phenylene)bis(oxy))bis(butan-4,1-diyl) bis( octadeca-9,12-dienoate), (((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(methylene))bis(propane-3,2,1-triyl) tetraoctanoate, (((5-((dimethylamino)methyl)-1,3-phenylene)bis(oxy))bis(butan-4,1-diyl))bis(propane-3,2,1-triyl) tetraoctanoate, (9Z,12Z)-4-(3-((dimethylamino)methyl-5-(4-((3-octylundecanoyl)oxy)butoxy)phenoxy)butyl octadeca-9,12-dienoate, bis(1,3-bis(octanoyloxy)propan-2-yl) O,O'-((5-((dimethylamino)methyl)-1,3-phenylene)methylene) disuccinate, (5-((dimethylamino)methyl)-1,3-phenylene)methylene bis(6-(((nonyloxy)carbonyl)oxy)hexanoate), 2-(3-(4-(5-((dimethylamino)methyl)-2-methyl-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)phenoxy)butoxy)-3- oxopropyl)propane-1,3-diyl dihexanoate, 3-((dimethylamino)methyl)-5-(((8- (octanoyloxy)octanoyl)oxy)methyl)benzyl 3-octylundecanoate, ((5-((diethylamino)methyl)benzene-1,2,3-triyl)tris(oxy))tris(decane-10,1-diyl) tris(octanoate), 1-(3,5-bis((Z)-octadec-9-en-1-yloxy)phenyl)-N,N-dimethylmethanamine, N'-methyl-N',N",N"-tris((2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-propane-1,3-diamine, 1,17-bis(2-((2-pentylcyclopropyl)methyl)cyclopropyl)heptadecan-9-yl 4-(dimethylamino)butanoate, (7Z)-17-{[4-(dimethylamino)butanoyl]oxy}hexacos-7-enoate, (Z)-6-(2-(dimethylamino)-3-(octadec-9-en-1-yloxy)propoxy)hexanoate,2-(dilaurylamino)-1-(4-(N-(2-(dilaurylamino)ethyl)-N-laurylglycyl)piperazin-1-yl)ethan-1-one, 3-((3-(1-(3-((2-(dilaurylamino)ethyl)(dodecyl)amino)propanoyl)piperidin-4-yl)propyl)(dodecyl)amino)propanoic acid, 3-((3-(4-(3-((2-(dilaurylamino)ethyl)(dodecyl)amino)propanoyl)piperazin-1-yl)-3-oxopropyl)(dodecyl)amino)propanoic acid, 3-((2-(dilaurylamino)ethyl)(dodecyl)amino)-1-(4-(3-(dilaurylamino)propyl)piperidin-1-yl)propan-1-one, 4-((3-(1-(3-((2-(dilaurylamino)ethyl)(dodecyl)amino)propanoyl)piperidin-4-yl)propyl)(dodecyl)amino)butyric acid, 4-((2-(1-(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)piperidin-4-yl)ethyl)(dodecyl)amino)butyric acid, 4-(((1-(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)piperidin-3-yl)methyl)(dodecyl)amino)butyric acid, 4-((2-(1-(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)piperidin-3-yl)ethyl)(dodecyl)amino)butyric acid, 4-((2-(1-(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)piperidin-4-yl)ethyl)(dodecyl)amino)butyric acid, 2-(dilaurylamino)-1-(4-(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)piperazin-1-yl)ethan-1-one, 2-((2-(dilaurylamino)ethyl)(dodecyl)amino)-1-(3-(2-(dilaurylamino)ethyl)piperidin-1-yl)ethan-1-one, 4,4'-((2-(4-(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)piperazin-1-yl)-2-oxoethyl)azanediyl)dibutyric acid, 4-(dodecyl(2-(4-(N-dodecyl-N-(2-(dodecyl(4-oxo-4-(pentyloxy)butyl(buryl))amino)ethyl)glycyl)piperazin-1-yl)-2-oxoethyl)amino)butyric acid, 2-((2-(dilaurylamino)ethyl)(dodecyl)amino)-1-(3-((dilaurylamino)methyl)pyrrolidin-1-yl)ethan-1-one, 2-((2-(dilaurylamino)ethyl)(dodecyl)amino)-1-(4-(dilaurylglycyl)piperazin-1-yl)ethan-1-one, 2-((2-(dilaurylamino)ethyl)(dodecyl)amino)-1-(3-(2-(dilaurylamino)ethyl)pyrrolidin-1-yl)ethan-1-one,4-((3-(4-(3-((2-(diphenylamino)ethyl)(phenyl)amino)propanoyl)piperazin-1-yl)-3- oxopropyl)(phenyl)amino)butanoic acid, 3-((2-(1-(N-(2-(diphenylamino)ethyl)-N- phenylglycyl)piperidin-4-yl)ethyl)(phenyl)amino)propyl hexanoate, 5-((2-(1-(N-(2- (diphenylamino)ethyl)-N-phenylglycyl)piperidin-4-yl)ethyl)(phenyl)amino)pentanoic acid butyl ester, 2-((2-(diphenylamino)ethyl)(phenyl)amino)-1-(4- (diphenylglycyl)piperazin-1-yl)ethan-1-one, 6-((2-(1-(N-(2- (diphenylamino)ethyl)-N-phenylglycyl)piperidin-4-yl)ethyl)(phenyl)amino)hexanoic acid propyl ester, 7-((2-(1-(N-(2-(diphenylamino)ethyl)-N- phenylglycyl)piperidin-4-yl)ethyl)(phenyl)amino)heptanoic acid ethyl ester, 8-((2-(1-(N-(2- (diphenylamino)ethyl)-N-phenylglycyl)piperidin-4-yl)ethyl)(phenyl)amino)octanoic acid methyl ester, 3-((2-(4-(N-(2-(diphenylamino)ethyl)-N- phenylglycyl)piperazin-1-yl)-2-oxoethyl)(phenyl)amino)propyl hexanoate, 5-((2-(4-(N-(2- (diphenylamino)ethyl)-N-phenylglycyl)piperazin-1-yl)-2-oxoethyl)(phenyl)amino)pentanoic acid butyl ester, 6-((2-(4-(N-(2-(diphenylamino)ethyl)-N- phenylglycyl)piperazin-2-oxoethyl)(phenyl)amino)hexanoic acid propyl ester, 7-((2-(4-(N-(2- (diphenylamino)ethyl)-N-phenylglycyl)piperazin-1-yl)-2-oxoethyl)(phenyl)amino)heptanoic acid ethyl ester, 3-(diphenylamino)-1-(4-(3-((2- (diphenylamino)ethyl)(phenyl)amino)propanoyl)piperazin-1-yl)propan-1-one, 2-((2- (diphenylamino)ethyl)(phenyl)amino)-1-(4-(diphenylglycyl)piperazin-1-yl)ethan-1-one, 2- (diphenylamino)-1-(4-(2-((2-(diphenylamino)ethyl)(phenyl)amino)ethyl)piperidin-1-yl)ethan-1- one, 2-(diphenylamino)-1-(4-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperazin-1-yl)ethan-1- one, 2-((2-(diphenylamino)ethyl)(phenyl)amino)-1-(4-(2- (diphenylamino)ethyl)piperidin-1-yl)ethan-1-one, 8-((2-(4-(diphenylglycyl)piperazin-1-yl)-2- oxoethyl)(2-((8-methoxy-8-oxooctyl)(phenyl)amino)ethyl)amino)octanoic acid methyl ester,8-((2-(diphenylamino)ethyl)(2-(4-(diphenylglycyl)piperazin-1-yl)-2-oxoethyl)amino)octanoic acid methyl ester, 8-((2-((2-(4-(diphenylglycyl)piperazin-1-yl)-2-oxoethyl)(phenyl)amino)ethyl)(phenyl)amino)octanoic acid methyl ester, 4-((2-(4-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperazin-2-oxoethyl)(phenyl)amino)butanoic acid methyl ester, 8-((2-(4-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperazin-1-yl)-2-oxoethyl)(phenyl)amino)octanoic acid methyl ester, 2-((2-(diphenylamino)ethyl)(phenyl)amino)-1-(5-(diphenylglycyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)ethan-1-one, 1,2-(diphenylamino)-1-(5-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)ethan-1-one, N1,N1,N2-tris(phenyl)-N2-(2-(piperazin-1-yl)ethyl)ethane-1,2-diamine, N1,N1,N2-tris((Z)-octadec-9-en-1-yl)-N2-(2-(piperazin-1-yl)ethyl)ethane-1,2-diamine, 2-(diphenylamino)-1-(4-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperazin-1-yl)ethan-1-one, N1,N1,N2-tris(phenyl)-N2-(2-(piperazin-1-yl)ethyl)ethane-1,2-diamine, N1,N1,N2-tris(phenyl)-N2-(2-(piperazin-1-yl)ethyl)ethane-1,2-diamine, N1,N1,N2-tris(phenyl)-N2-(2-(piperazin-1-yl)ethyl)ethane-1,2-diamine, N1-(2-(4-(2-(diphenylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tris(phenyl)ethane-1,2-diamine, N1-(2-(4-(2-(diphenylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tris(phenyl)ethane-1,2-diamine, N1-(2-(4-(2-(diphenylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tris(phenyl)ethane-1,2-diamine,N1-(2-(4-(2-(dilaurylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecyl ethane-1,2-diamine, 2-(didodecylamino)-1-(4-(2-((2-(didodecylamino)ethyl)(dodecyl)amino)ethyl)piperazin-1-yl)ethan-1-one, N1-(2-(4-(2-(di((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecyl ethane-1,2-diamine, N1-(2-(4-(2-(di((Z)-octadeca-9-en-1-yl)amino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecyl ethane-1,2-diamine, N1,N1,N2-tridodecyl-N2-(2-(4-(2-(dodecyl((9Z,12Z)-octadeca-9,12-dien-1-yl)amino)ethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine, N1-(2-(4-(2-(didodecylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecyl ethane-1,2-diamine, N1-(2-(4-(2-(di(dodecyl)amino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecyl ethane-1,2-diamine, N1-(2-(4-(2-(di((Z)-dodec-6-en-1-yl)amino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecyl ethane-1,2-diamine, (Z)-N1-(2-(4-(2-(dodec-6-en-1-yl(dodecyl)amino)ethyl)piperazin-1-yl)ethyl)-N,N2,N2-tridodecyl ethane-1,2-diamine, N1-(2-(4-(2-(dilaurylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecyl ethane-1,2-diamine, N1-(2-(4-(2-(dioctylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecyl ethane-1,2-diamine, N1-(2-(4-(2-(dihexylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecyl ethane-1,2-diamine, N1-(2-(4-(2-(didodecylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecyl ethane-1,2-diamine, 2-((2-(didodecylamino)ethyl)(dodecyl)amino)-1-(4-(2-(didodecylamino)ethyl)piperazin-1-yl)ethan-1-one, N1-(2-(4-(2-(didodecylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecyl ethane-1,2-diamine, N1-(2-(4-(2-(dilaurylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecyl ethane-1,2-diamine,N1-(2-(4-(2-(bisdo-decylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-trihexylethane-1,2-diamine, 12,12'-((2-(4-(2-((2-(bisdo-decylamino)ethyl)(dodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl) dodecanedioic acid dimethyl ester, 12-((2-(4-(2-((2-(bisdo-decylamino)ethyl)(dodecyl)amino)ethyl)piperazin-1-yl)ethyl)(dodecyl)amino)dodecanoic acid methyl ester, 6,6'-((2-(4-(2-((2-(bisdo-decylamino)ethyl)(dodecyl)amino)ethyl)piperazin-1-yl)ethyl)azanediyl) dipentanoic acid, 6-((2-(4-(2-((2-(bisdo-decylamino)ethyl)(dodecyl)amino)ethyl)piperazin-1-yl)ethyl)(dodecyl)amino)hexanoic acid pentyl ester, 6-((2-(4-(2-((2-(bisdo-decylamino)ethyl)(dodecyl)amino)ethyl)piperazin-1-yl)ethyl)(dodecyl)amino)hexanoic acid pentyl ester, 2-(bisdo-decylamino)-1-(4-(N-(2-(bisdo-decylamino)ethyl)-N-dodecylglycyl)piperazin-1-yl)ethan-1-one, 2-(bisdo-decylamino)-1-(4-(N-(2-(bisdo-decylamino)ethyl)-N-nonylglycyl)piperazin-1-yl)ethan-1-one, 2-(bisdo-decylamino)-N-(2-(4-(2-(bisdo-decylamino)ethyl)piperazin-1-yl)ethyl)-N-dodecylacetamide, ((2-((3'S,4R)-3,4-dihydroxypyrrolidin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl)(9Z,9'Z,12Z,12'Z)-bis( octadeca-9,12-dienoic acid), 2-amino-N,N-dihexadecyl-3-(1H-imidazol-5-yl)propanamide, (2-amino-N,N-dihexadecyl-3-(1H-imidazol-5-yl)propanamide, (9Z)-19-[2-(dimethylamino)ethyl]heptacos-9-enoic acid methyl ester, 8-(2-{9-[2-(dimethylamino)ethyl]octadecyl}cyclopropyl)octanoic acid methyl ester, (9Z)-19-[2-(dimethylamino)ethyl]octacos-9-enoic acid methyl ester, 8-(2-{11-[(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoic acid ethyl ester, 8-(2-{11-[(dimethylamino)methyl]octadecyl}cyclopropyl)octanoic acid ethyl ester, bis((9Z,12Z)-octadeca-9,12-dien-1-yl) 3-(((2-(dimethylamino)ethoxy)carbonyl)amino)pentanedioic acid ester, 6-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(tetradecyl)amino)hexanoic acid heptyl ester,8-(2-{11-[(dimethylamino)methyl]nonadecyl}cyclopropyl)octanoic acid ethyl ester, 8-((2-(1-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperidin-4-yl)ethyl)(phenyl)amino)pentanoic acid, 8-(2-{11-[(dimethylamino)methyl]eicosyl}cyclopropyl)octanoic acid ethyl ester, 8-(2-{9-[(dimethylamino)methyl]pentadecyl}cyclopropyl)octanoic acid ethyl ester, 3-((2-(1-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperidin-4-yl)ethyl)(phenyl)amino)propyl decanoate, 6-((2-(4-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperazin-1-yl)-2-oxoethyl)(phenyl)amino)hexanoic acid heptyl ester, 8-(2-{9-[(dimethylamino)methyl]hexadecyl}cyclopropyl)octanoic acid ethyl ester, 8-((2-(4-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperazin-2-oxoethyl)(phenyl)amino)pentanoic acid, 8-(2-{9-[(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoic acid ethyl ester, 6-(2-(8-(2-(dimethylamino)-3-(phenyloxy)propoxy)octyl)cyclopropyl)hexanoic acid methyl ester, (9Z)-21-(dimethylamino)heptacos-9-enoic acid methyl ester, (9Z)-21-{[4-(dimethylamino)butyryl]oxy}heptacos-9-enoic acid methyl ester, (2R)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]dodecan-2-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, 8-(2-{9-[(dimethylamino)methyl]octadecyl}cyclopropyl)octanoic acid ethyl ester, 3-((2-(4-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperazin-1-yl)-2-oxoethyl)(phenyl)amino)propyl decanoate, 4-(2-{11-[(dimethylamino)methyl]eicosyl}cyclopropyl)butyric acid ethyl ester, 8-(2-{7-[(dimethylamino)methyl]hexadecyl}cyclopropyl)octanoic acid ethyl ester, 3-((3-(1-(3-((2-(diphenylamino)ethyl)(phenyl)amino)propanoyl)piperidin-4-yl)propyl)(phenyl)amino)propyl hexanoate, 6-(2-{9-[(dimethylamino)methyl]pentadecyl}cyclopropyl)hexanoic acid ethyl ester, 3-((3-(4-(3-((2-(diphenylamino)ethyl)(phenyl)amino)propanoyl)piperazin-1-yl)-3-oxopropyl)(phenyl)amino)propyl hexanoate, 6-(2-{9-[(dimethylamino)methyl]hexadecyl}cyclopropyl)hexanoic acid ethyl ester,3-((2-(diphenylamino)ethyl)(phenyl)amino)-1-(4-(3-(diphenylamino)propyl)piperidin-1-yl)propan-1-one, 4-((3-(1-(3-((2-(diphenylamino)ethyl)(phenyl)amino)propanoyl)piperidin-4-yl)propyl)(phenyl)amino)butanoic acid, 6-(2-{9-[(dimethylamino)methyl]heptadecyl}cyclopropyl)hexanoic acid, 4-((2-(1-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperidin-4-yl)ethyl)(phenyl)amino)butanoic acid, 6-(2-{9-[(dimethylamino)methyl]octadecyl}cyclopropyl)hexanoic acid, 4-(((1-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)pyrrolidin-3-yl)methyl)(phenyl)amino)butanoic acid, (9Z)-21-[(dimethylamino)methyl]heptacos-9-enoic acid, 4-((2-(1-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)pyrrolidin-3-yl)ethyl)(phenyl)amino)butanoic acid, (9Z)-21-[(dimethylamino)methyl]octacos-9-enoic acid, ((2-((3'S,4R)-3,4-dihydroxypyrrolidin-1-yl)acetyl)azanediyl)bis(ethane-2,1-diyl) (9Z,9'Z,12Z,12'Z)-bis(octadeca-9,12-dienoate), 4-((2-(1-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperidin-3-yl)ethyl)(phenyl)amino)butanoic acid, (9Z)-21-[(dimethylamino)methyl]nonacos-9-enoic acid, 6-(2-(8-(2-(dimethylamino)-3-(heptyloxy)propoxy)octyl)cyclopropyl)hexanoic acid, (9Z)-21-{[4-(dimethylamino)butanoyl]oxy}octacos-9-enoic acid, (9Z)-21-(dimethylamino)octacos-9-enoic acid, 2-(didodecylamino)-1-(4-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperazin-1-yl)ethanol, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]nonan-2-amine, (18Z,21Z)-N,N-dimethylheptacos-18,21-dien-10-amine, (9Z)-21-[(dimethylamino)methyl]triacont-9-enoic acid, (9Z)-19-[(dimethylamino)methyl]pentacos-9-enoic acid, (9Z)-19-[(dimethylamino)methyl]hexacos-9-enoic acid, (9Z)-19-[(dimethylamino)methyl]heptacos-9-enoic acid, (9Z)-19-[(dimethylamino)methyl]octacos-9-enoic acid,(5Z)-17-[(dimethylamino)methyl]hexacos-5-enoic acid ethyl ester, (9Z)-17-[(dimethylamino)methyl]hexacos-9-enoic acid ethyl ester, 2-((2-(diphenylamino)ethyl)(phenyl)amino)-1-(3-(2-(diphenylamino)ethyl)piperidin-1-yl)ethan-1-one, (7Z)-17-[(dimethylamino)methyl]tricos-7-enoic acid ethyl ester, 4,4'-((2-(4-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperazin-1-yl)-2-oxoethyl)azanediyl) dibutyric acid diphenyl ester, 4-(phenyl(2-(4-(N-phenyl-N-(2-(phenyl(4-oxo-4-(pentyloxy)butyl)amino)ethyl)glycyl)piperazin-1-yl)-2-oxoethyl)amino)butyric acid phenyl ester, (7Z)-17-[(dimethylamino)methyl]tetracos-7-enoic acid ethyl ester, (7Z)-17-[(dimethylamino)methyl]pentacos-7-enoic acid ethyl ester, 2-((2-(diphenylamino)ethyl)(phenyl)amino)-1-(3-((diphenylamino)methyl)pyrrolidin-1-yl)ethan-1-one, trans-3-[(3,7-dimethyloctyl)oxy]-1-methyl-4-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]pyrrolidine, 6-(2-(8-(2-(dimethylamino)-3-(hexyloxy)propoxy)octyl)cyclopropyl)hexanoic acid methyl ester, (9Z)-21-{[4-(dimethylamino)butanoyl]oxy}nonacos-9-enoic acid methyl ester, (9Z)-21-(dimethylamino)nonacos-9-enoic acid methyl ester, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]tridecan-2-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (7Z)-17-[(dimethylamino)methyl]hexacos-7-enoic acid ethyl ester, 2-((2-(diphenylamino)ethyl)(phenyl)amino)-1-(3-(2-(diphenylamino)ethyl)pyrrolidin-1-yl)ethan-1-one, 6-(2-{11-[(dimethylamino)methyl]heneicosyl}cyclopropyl)hexanoic acid methyl ester, 10-(2-{7-[(dimethylamino)methyl]hexadecyl}cyclopropyl)decanoic acid methyl ester, 8-(2-{11-[(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoic acid methyl ester, 8-(2-{11-[(dimethylamino)methyl]octadecyl}cyclopropyl)octanoic acid methyl ester, 8-(2-{11-[(dimethylamino)methyl]nonadecyl}cyclopropyl)octanoic acid methyl ester, 8-(2-{11-[(dimethylamino)methyl]eicosyl}cyclopropyl)octanoic acid methyl ester,4-((3-(4-(3-((2-(diphenylamino)ethyl)(phenyl)amino)propionyl)piperazin-1-yl)-3- oxopropyl)(phenyl)amino)butanoic acid, 8-(2-{9-[(dimethylamino)methyl]pentadecyl}cyclopropyl)octanoic acid methyl ester, 8-(2-{9-[(dimethylamino)methyl]hexadecyl}cyclopropyl)octanoic acid methyl ester, 3-((2-(1-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperidin-4-yl)ethyl)(phenyl)amino)propyl hexanoate, 8-(2-{9-[(dimethylamino)methyl]heptadecyl}cyclopropyl)octanoic acid methyl ester, 8-(2-(dimethylamino)-3-((6-((2-phenylcyclopropyl)methoxy)-6-oxohexyl)oxy)propoxy)octanoic acid methyl ester, 5-((2-(1-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperidin-4-yl)ethyl)(phenyl)amino)pentanoic acid butyl ester, trans-1-methyl-3-[(12Z)-octadeca-12-en-1-yloxy]-4-(phenyloxy)pyrrolidine, (9Z)-21-{[4-(dimethylamino)butanoyl]oxy}triaconta-9-enoic acid methyl ester, (9Z)-21-(dimethylamino)triaconta-9-enoic acid methyl ester, 2-((2-(diphenylamino)ethyl)(phenyl)amino)-1-(4-(diphenylglycyl)piperazin-1-yl)ethan-1-one, N-(2-(diphenylamino)ethyl)-N-phenylglycine methyl ester, 1-((2R,3S,5R)-3-(bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)tetrahydro-methanesulfonate, (Z)-16-(3-(decyloxy)-2-(dimethylamino)propoxy)hexadec-7-enoic acid methyl ester, (2S)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]nonan-2-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, 6-((2-(1-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperidin-4-yl)ethyl)(phenyl)amino)hexanoic acid propyl ester, 7-(2-(dimethylamino)-3-((6-((2-phenylcyclopropyl)methoxy)-6-oxohexyl)oxy)propoxy)heptanoic acid methyl ester, (7Z)-19-[(dimethylamino)methyl]octacos-7-enoic acid methyl ester, (8Z)-19-[(dimethylamino)methyl]octacos-11-enoic acid methyl ester, 7-((2-(1-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperidin-4-yl)ethyl)(phenyl)amino)heptanoic acid ethyl ester, (2-phenylcyclopropyl)methyl 6-(2-(dimethylamino)-3-((5-methoxy-5-oxopentyl)oxy)propoxy)hexanoate,8-((2-(1-(N-(2-(dionylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)octanoic acid methyl ester, (9Z)-21-[(dimethylamino)methyl]heptacos-9-enoic acid methyl ester, (2-octylcyclopropyl)methyl 6-(2-(dimethylamino)-3-(4-methoxy-4-oxobutoxy)propoxy)hexanoate, (9Z)-21-[(dimethylamino)methyl]octacos-9-enoic acid methyl ester, 3-((2-(4-(N-(2-(dionylamino)ethyl)-N-nonylglycyl)piperazin-1-yl)-2-oxoethyl)(nonyl)amino)propyl hexanoate, (Z)-8-(2-(dimethylamino)-3-((6-oxo-6-(undec-2-en-1-yloxy)hexyl)oxy)propoxy)octanoic acid methyl ester, (9Z)-21-[(dimethylamino)methyl]nonacos-9-enoic acid methyl ester, 5-((2-(4-(N-(2-(dionylamino)ethyl)-N-nonylglycyl)piperazin-1-yl)-2-oxoethyl)(nonyl)amino)pentanoic acid butyl ester, (Z)-7-(2-(dimethylamino)-3-((6-oxo-6-(undec-2-en-1-yloxy)hexyl)oxy)propoxy)heptanoic acid methyl ester, 6-((2-(4-(N-(2-(dionylamino)ethyl)-N-nonylglycyl)piperazin-1-yl)-2-oxoethyl)(nonyl)amino)hexanoic acid propyl ester, (9Z)-21-[(dimethylamino)methyl]triacont-9-enoic acid methyl ester, (Z)-undec-2-en-1-yl 6-(2-(dimethylamino)-3-((5-methoxy-5-oxopentyl)oxy)propoxy)hexanoate, (9Z)-19-[(dimethylamino)methyl]pentacos-9-enoic acid methyl ester, 7-((2-(4-(N-(2-(dionylamino)ethyl)-N-nonylglycyl)piperazin-1-yl)-2-oxoethyl)(nonyl)amino)heptanoic acid ethyl ester, (Z)-undec-2-en-1-yl 6-(2-(dimethylamino)-3-(4-methoxy-4-oxobutoxy)propoxy)hexanoate, 6-(2-(dimethylamino)-3-((6-((2-octylcyclopropyl)methoxy)-6-oxohexyl)oxy)propoxy)hexanoic acid methyl ester, (9Z)-19-[(dimethylamino)methyl]hexacos-9-enoic acid methyl ester, 3-(dionylamino)-1-(4-(3-((2-(dionylamino)ethyl)(nonyl)amino)propanoyl)piperazin-1-yl)propan-1-one, (9Z)-19-[(dimethylamino)methyl]heptacos-9-enoic acid methyl ester, 2-((2-(dionylamino)ethyl)(nonyl)amino)-1-(4-(bistetradecylglycyl)piperazin-1-yl)ethan-1-one,(Z)-6-(2-(dimethylamino)-3-((6-oxo-6-(undec-2-en-1-yloxy)hexyl)oxy)propoxy)hexanoic acid methyl ester, 8-(2-(dimethylamino)-3-((8-(2-(6-methoxy-6-oxohexyl)cyclopropyl)octyl)oxy)propoxy)octanoic acid methyl ester, 8-(2-{9-[(dimethylamino)methyl]octadecyl}cyclopropyl)octanoic acid methyl ester, 2-(dinonylamino)-1-(4-(2-((2-(dinonylamino)ethyl)(nonyl)amino)ethyl)piperidin-1-yl)ethan-1-one, trans-1-methyl-3-[(9Z)-octadeca-9-en-1-yloxy]-4-(octyloxy)pyrrolidine, (9Z)-19-{[4-(dimethylamino)butanoyl]oxy}pentacos-9-enoic acid methyl ester, (9Z)-19-(dimethylamino)pentacos-9-enoic acid methyl ester, (Z)-16-(2-(dimethylamino)-3-(nonyloxy)propoxy)hexadec-7-enoic acid methyl ester, (2S)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]decan-2-amine, (12Z,15Z)-N,N-dimethylheneicosa-12,15-dien-4-amine, 7-(2-(dimethylamino)-3-((8-(2-(6-methoxy-6-oxohexyl)cyclopropyl)octyl)oxy)propoxy)heptanoic acid methyl ester, (9Z)-19-[(dimethylamino)methyl]octacos-9-enoic acid methyl ester, 2-((2-(dinonylamino)ethyl)(nonyl)amino)-1-(4-(2-(dinonylamino)ethyl)piperidin-1-yl)ethan-1-one, 8-((2-(4-(dinonylglycyl)piperazin-1-yl)-2-oxoethyl)(2-((8-methoxy-8-oxooctyl)(nonyl)amino)ethyl)amino)octanoic acid methyl ester, 6-(2-(8-(2-(dimethylamino)-3-((5-methoxy-5-oxopentyl)oxy)propoxy)octyl)cyclopropyl)hexanoic acid methyl ester, 8-{2-[11-(dimethylamino)heptadecyl]cyclopropyl}octanoic acid ethyl ester, 8-((2-(dinonylamino)ethyl)(2-(4-(dinonylglycyl)piperazin-1-yl)-2-oxoethyl)amino)octanoic acid methyl ester, 6-(2-(8-(2-(dimethylamino)-3-(4-methoxy-4-oxobutoxy)propoxy)octyl)cyclopropyl)hexanoic acid methyl ester, 8-{2-[11-(dimethylamino)octadecyl]cyclopropyl}octanoic acid ethyl ester, 8-((2-((2-(4-(dinonylglycyl)piperazin-1-yl)-2-oxoethyl)(nonyl)amino)ethyl)(nonyl)amino)octanoic acid methyl ester, 8-{2-[11-(dimethylamino)nonadecyl]cyclopropyl}octanoic acid ethyl ester,(Z)-16-(2-(dimethylamino)-3-((8-methoxy-8-oxooctyl)oxy)propoxy)hexadec-7-enoic acid methyl ester, 4-((2-(4-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperazin-1-yl)-2-oxoethyl)(phenyl)amino)butyric acid methyl ester, 8-{2-[11-(dimethylamino)undecyl]cyclopropyl}octanoic acid ethyl ester, (Z)-16-(2-(dimethylamino)-3-((7-methoxy-7-oxoheptyl)oxy)propoxy)hexadec-7-enoic acid methyl ester, 8-((2-(4-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperazin-1-yl)-2-oxoethyl)(phenyl)amino)octanoic acid methyl ester, 8-{2-[9-(dimethylamino)pentadecyl]cyclopropyl}octanoic acid ethyl ester, (Z)-16-(2-(dimethylamino)-3-((5-methoxy-5-oxopentyl)oxy)propoxy)hexadec-7-enoic acid methyl ester, (11E,20Z,23Z)-N,N-dimethylnonacos-11,20,23-trien-10-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, 8-{2-[9-(dimethylamino)hexadecyl]cyclopropyl}octanoic acid ethyl ester, 2-((2-(diphenylamino)ethyl)(phenyl)amino)-1-(5-(N-(2-(diphenylamino)ethyl)-N- phenylglycyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)ethan-1-one, (Z)-16-(2-(dimethylamino)-3-(4-methoxy-4-oxobutoxy)propoxy)hexadec-7-enoic acid methyl ester, 6-(2-(8-(2-(dimethylamino)-3-((6-methoxy-6-oxohexyl)oxy)propoxy)octyl)cyclopropyl)hexanoic acid methyl ester, 8-{2-[9-(dimethylamino)heptadecyl]cyclopropyl}octanoic acid ethyl ester, 2-(diphenylamino)-1-(5-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)-2,5-diazabicyclo[2.2.1]heptan-2-yl)ethan-1-one, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N1,N1,N2-tris((9Z,12Z)-octadeca-9,12-dien-1-yl)-N2-(2-(piperazin-1-yl)ethyl)ethane-1,2-diamine, 8-{2-[9-(dimethylamino)octadecyl]cyclopropyl}octanoic acid ethyl ester, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine, (Z)-16-(2-(dimethylamino)-3-((6-methoxy-6-oxohexyl)oxy)propoxy)hexadec-7-enoic acid methyl ester, N1,N1,N2-tris((Z)-octadeca-9-en-1-yl)-N2-(2-(piperazin-1-yl)ethyl)ethane-1,2-diamine,N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine, methyl 8-(2-(dimethylamino)-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)octanoate, ethyl 4-{2-[11-(dimethylamino)heneicosyl]cyclopropyl}butyrate, trans-1-methyl-3-[((9Z,12Z)-octadeca-9,12-dienyl)oxy]-4-octyloxy-pyrrolidine, methyl (9Z)-19-(dimethylamino)hexacos-9-enoate, methyl (9Z)-19-{[4-(dimethylamino)butanoyl]oxy}hexacos-9-enoate, methyl (Z)-16-(2-(dimethylamino)-3-(heptyloxy)propoxy)hexadec-7-enoate, (2R)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]dodecan-2-amine, (13Z,16Z)-N,N-dimethyl-docosa-13,16-dien-5-amine, N,N-dimethyl-1-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine, methyl 7-(2-(dimethylamino)-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)heptanoate, ethyl 8-{2-[7-(dimethylamino)hexadecyl]cyclopropyl}octanoate, 2-(didodecylamino)-N-dodecyl-N-(2-(piperazin-1-yl)ethyl)acetamide, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N1-(2-(piperazin-1-yl)ethyl)-N1,N2,N2-tritetradecyl-ethane-1,2-diamine, methyl 6-(2-(dimethylamino)-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)hexanoate, ethyl 6-{2-[9-(dimethylamino)pentadecyl]cyclopropyl}hexanoate, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N1,N2-tridodecyl-N2-(2-(piperazin-1-yl)ethyl)ethane-1,2-diamine, methyl 5-(2-(dimethylamino)-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)pentanoate, ethyl 6-{2-[9-(dimethylamino)hexadecyl]cyclopropyl}hexanoate, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]heneicosan-10-amine, N,N,N2-trinonyl-N2-(2-(piperazin-1-yl)ethyl)ethane-1,2-diamine, methyl 4-(2-(dimethylamino)-3-((8-(2-((2-pentylcyclopropyl)methyl)cyclopropyl)octyl)oxy)propoxy)butyrate,6-{2-[9-(dimethylamino)heptadecyl]cyclopropyl}hexanoic acid ethyl ester, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N1,N1,N2-trihexyl-N2-(2-(piperazin-1-yl)ethyl)ethane-1,2-diamine, 8-(2-(dimethylamino)-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propoxy)octanoic acid methyl ester, 6-{2-[9-(dimethylamino)octadecyl]cyclopropyl}hexanoic acid ethyl ester, N1-(2-(4-(2-(didodecylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tris((9Z,12Z)-octadeca-9,12-dien-1-yl)ethane-1,2-diamine, 7-(2-(dimethylamino)-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propoxy)heptanoic acid methyl ester, (9Z)-21-(dimethylamino)heptacos-9-enoic acid ethyl ester, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, 1-methyl 18-[(2Z)-non-2-en-1-yl] 9-{[4-(dimethylamino)butanoyl]oxy}octadecanedioate, N1-(2-(4-(2-(didodecylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tris((Z)-octadeca-9-en-1-yl)ethane-1,2-diamine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]nonadecan-8-amine, 6-(2-(dimethylamino)-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propoxy)hexanoic acid methyl ester, (9Z)-21-(dimethylamino)octacos-9-enoic acid ethyl ester, (9Z)-19-{[4-(dimethylamino)butanoyl]oxy}heptacos-9-ene dioate dimethyl ester, N1-(2-(4-(2-(dimyristylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tris(myristyl)ethane-1,2-diamine, 5-(2-(dimethylamino)-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propoxy)pentanoic acid methyl ester, 8-{[4-(dimethylamino)butanoyl]oxy}-15-(2-octylcyclopropyl)pentadecanoic acid ethyl ester, (9Z)-21-(dimethylamino)nonacos-9-enoic acid ethyl ester, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N1-(2-(4-(2-(didodecylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tris(myristyl)ethane-1,2-diamine, 9-{[4-(dimethylamino)butanoyl]oxy}-16-(2-octylcyclopropyl)hexadecanoic acid methyl ester,4-(2-(dimethylamino)-3-((9Z,12Z)-octadeca-9,12-dien-1-yloxy)propoxy)butanoic acid methyl ester, (9Z)-21-(dimethylamino)triacont-9-enoic acid ethyl ester, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, 8-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy)octanoic acid methyl ester, (9Z)-19-(dimethylamino)pentacos-9-enoic acid ethyl ester, (18Z,21Z)-8-{[4-(dimethylamino)butanoyl]oxy}heptacos-18,21-dienoic acid ethyl ester, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (9Z)-19-{[4-(dimethylamino)butanoyl]oxy}heptacos-9-enoic acid methyl ester, (9Z)-19-(dimethylamino)heptacos-9-enoic acid methyl ester, 2-(didodecylamino)-1-(4-(2-((2-(didodecylamino)ethyl)(dodecyl)amino)ethyl)piperazin-1-yl)ethan-1-one, (Z)-16-(2-(dimethylamino)-3-(hexyloxy)propoxy)hexadec-7-enoic acid methyl ester, (2S)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]dodecan-2-amine, (16Z,19Z)-N,N-dimethylpentacos-16,19-dien-8-amine, N1-(2-(4-(2-(dinonylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tritetradecylethan-1,2-diamine, 7-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy)heptanoic acid methyl ester, (19Z,22Z)-9-{[4-(dimethylamino)butanoyl]oxy}octacos-19,22-dienoic acid methyl ester, (9Z)-19-(dimethylamino)hexacos-9-enoic acid ethyl ester, (22Z)-N,N-dimethylhentriacont-22-en-10-amine, N1-(2-(4-(2-(di((Z)-octadec-9-en-1-yl)amino)ethyl)piperazin-1-yl)ethyl)-N1-tridodecylethan-1,2-diamine, 5-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy)pentanoic acid methyl ester, (9Z)-19-(dimethylamino)heptacos-9-enoic acid ethyl ester, (2-butylcyclopropyl)methyl 12-{[4-(dimethylamino)butanoyl]oxy}henicosaonate, (20Z)-N,N-dimethylnonatricosa-20-en-10-amine, N1,N1,N2-tridodecyl-N2-(2-(4-(2-(dodecyl((9Z,12Z)-octadeca-9,12-dien-yl)amino)ethyl)piperazin-1-yl)ethyl)ethane-1,2-diamine,4-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy)butyric acid methyl ester, (9Z)-19-(dimethylamino)octadeca-9-enoic acid ethyl ester, (2-octylcyclopropyl)methyl 8-{[4-(dimethylamino)butanoyl]oxy}heptadecanoate, (24Z)-N,N-dimethyltricosa-24-ene-10-amine, N1-(2-(4-(2-(bistridecylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecylpropane-1,2-diamine, (5Z)-17-(dimethylamino)hexacos-5-enoic acid ethyl ester, (Z)-8-(2-(dimethylamino)-3-(octadeca-9-en-1-yloxy)propoxy)octanoic acid methyl ester, (2Z)-hept-2-en-1-yl 12-{[4-(dimethylamino)butanoyl]oxy}heneicosanoate, (17Z)-N,N-dimethylnonaconta-17-ene-10-amine, N1-(2-(4-(2-(di((Z)-dodec-6-en-1-yl)amino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecylpropane-1,2,-diamine, (9Z)-17-(dimethylamino)hexa-9-enoic acid ethyl ester, (Z)-7-(2-(dimethylamino)-3-(octadeca-9-en-1-yloxy)propoxy)heptanoic acid methyl ester, (2Z)-undec-2-en-1-yl 8-{[4-(dimethylamino)butanoyl]oxy}heptadecanoate, (14Z)-N,N-dimethylnonaconta-14-ene-10-amine, (7Z)-17-(dimethylamino)tricosa-7-enoic acid ethyl ester, (Z)-N1-(2-(4-(2-(dodec-6-en-1-yl(dodecyl)amino)ethyl)piperazin-N,N-tridodecylpropane-1,2-diamine, (Z)-5-(2-(dimethylamino)-3-(octadeca-9-en-1-yloxy)propoxy)pentanoic acid methyl ester, (2-hexylcyclopropyl)methyl 10-{[4-(dimethylamino)butanoyl]oxy}nonadecanoate, (15Z)-N,N-dimethylheptaconta-15-ene-10-amine, (7Z)-17-(dimethylamino)tetra-7-enoic acid ethyl ester, (Z)-4-(2-(dimethylamino)-3-(octadeca-9-en-1-yloxy)propoxy)butyric acid methyl ester, (2Z)-non-2-en-1-yl 10-{[4-(dimethylamino)butanoyl]oxy}nonadecanoate, (20Z)-N,N-dimethylheptaconta-20-ene-10-amine, N1-(2-(4-(2-(dioctylamino)ethyl)piperazin-1-yl)ethyl)-N1,N2,N2-tridodecylpropane-1,2-diamine, 6-(2-(dimethylamino)-3-((8-(2-octylcyclopropyl)octyl)oxy)propoxy)hexanoic acid methyl ester,6-[2-(9-{[4-(dimethylamino)butanoyl]oxy}octadecyl)cyclopropyl]hexanoic acid ethyl ester, (7Z)-17-(dimethylamino)pentacos-7-ene acid ethyl ester, 1-[(11Z,14Z)-1- nonylicos-11,14-dien-1-yl]pyrrolidine, (7Z)-17-(dimethylamino)hexacos-7-ene acid ethyl ester, (20Z,23Z)-N-ethyl-N-methylnonacos-20,23-dien-10-amine, N,N- dimethylheptacos-10-amine, 6-{2-[11-(dimethylamino)heneicosyl]cyclopropyl}hexanoic acid methyl ester, 6-[2-(11-{[4-(dimethylamino)butanoyl]oxy}heneicosyl)cyclopropyl]hexanoic acid methyl ester, (2-octylcyclopropyl)methyl 6-(3-(decyloxy)-2- (dimethylamino)propoxy)hexanoate, 8-{2-[9-(dimethylamino)octadecyl]cyclopropyl}octanoic acid methyl ester, 8-[2-(9-{[4-(dimethylamino)butanoyl]oxy}octadecyl)cyclopropyl]octanoic acid methyl ester, 7-(2-(8-(2-(dimethylamino)-3-(octyloxy)propoxy)octyl)cyclopropyl)heptanoic acid methyl ester, heptadecan-9-yl 8-((2-hydroxyethyl)(tetradecyl)amino)octanoate, 2-((2-(dododecylamino)ethyl)(dodecyl)amino)-1-(4-(2-(dododecylamino)ethyl)piperazin-1-yl)ethan-1-one, (2S)-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]undecan-2-amine, (17Z,20Z)-N,N-dimethylhexacos-17,20-dien-9-amine, (18Z)-heptacos-18-ene-10-yl 4- (dimethylamino)butanoate, (2S)-1-({6-[3-cholest-5-en-3-yloxy]hexyl}oxy)-N,N-dimethyl-3-[(9Z)-octadeca-9-en-1-yloxy]propan-2-amine, 10-{2-[7- (dimethylamino)hexadecyl]cyclopropyl}decanoic acid methyl ester, 10-[2-(7-{[4- (dimethylamino)butanoyl]oxy}hexadecyl)cyclopropyl]decanoic acid methyl ester, (2S)-N,N-dimethyl-1-({8-[(1R,2R)-2-{[(1S,2S)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)tridecan-2-amine, (2-octylcyclopropyl)methyl 6-(2-(dimethylamino)-3-(nonyloxy)propoxy)hexanoate, (19Z,22Z)-N,N-dimethyloctacos-19,22-dien-7-amine, 4-((N-(2-(dinonylamino)ethyl)-N-nonylglycyl)oxy)pentan-2-yl dinonylglycinate, 3-hydroxybutan-2-yl-(2-(dimethylamino)ethyl)-N-nonyl,di(hexadecan-9-yl) 8,8'-(26,28-dimethyl-11,24,30,43-tetraoxo-10,25,29,44- tetraoxa-19,35-diazapentacontane-19,35-diyl) dioctanoate, di(hexadecan-9-yl) 8,8'- (26,27-dimethyl-11,24,29,42-tetraoxo-10,25,28,43-tetraoxa-19,34-diazapentacontane- 19,34-diyl) dioctanoate, di(hexadecan-9-yl) 8,8'-(11,24,29,42-tetraoxo-10,25,28,43- tetraoxa-19,34-diazapentacontane-19,34-diyl) dioctanoate, di(hexadecan-9-yl) 8,8'- ((piperazin-1,4-diylbis(5-oxopentane-5,1-diyl))bis((8-(nonyloxy)-8-oxooctyl)azanediyl)) dioctanoate, di(hexadecan-9-yl) 15,18-dimethyl-9,24-bis(8-(nonyloxy)-8-oxooctyl)- 14,19-dioxo-9,15,18,24-tetraazadotriacontanedioate, di(hexadecan-9-yl) 15,19-dimethyl- 9,25-bis(8-(nonyloxy)-8-oxooctyl)-14,20-dioxo-9,15,19,25-tetraazatridecane- dioate, di(hexadecan-9-yl) 15,18-diethyl-9,24-bis(8-(nonyloxy)-8-oxooctyl)-14,19-dioxo- 9,15,18,24-tetraazadotriacontanedioate, N,N-dimethyl-3-{[(9Z,12Z)-octadeca-9,12- dien-1-yloxy]methyl}dodecan-1-amine, 8-[2-(11-{[4-(dimethylamino)butanoyl]oxy} octadecyl)cyclopropyl]octanoic acid methyl ester, 8-{2-[11-(dimethylamino)heptadecyl] cyclopropyl}octanoic acid methyl ester (Compound 18), hexadecan-9-yl 8-((2- hydroxyethyl)(8-(nonyloxy)-8-oxooctyl)amino)octanoate, (2-octylcyclopropyl)methyl 6-(2-(dimethylamino)-3-(heptyloxy)propoxy)hexanoate, (17Z)-N,N-dimethyl- hexacos-17-ene-9-amine, N1-(2-(4-(2-(bodiphenylamino)ethyl)piperazin-1-yl)ethyl)- N1,N2,N2-trihexylethane-1,2-diamine, N,N-dimethyl-2-{[(9Z,12Z)-octadeca-9,12-dien-1- yloxy]methyl}undecan-1-amine, 8-{2-[11-(dimethylamino)octadecyl]cyclopropyl} octanoic acid methyl ester, (2-octylcyclopropyl)methyl 6-(2-(dimethylamino)-3-2-((2-(diphenylamino)ethyl)(phenyl)amino)ethyl tetradecanoate, 2-((2-(diphenylamino)ethyl)(phenyl)amino)ethyl nonanoate, N-(2-(diphenylamino)ethyl)-N-phenylglycine tetradecyl ester, N-(2-(diphenylamino)ethyl)-N-phenylglycine nonyl ester, 4-(2-((2-(diphenylamino)ethyl)(phenyl)amino)acetamido)butyl pentanoate, 1,1'-(piperazine-1,4-diyl)bis(5-(didecylamino)pentan-1-one, 2-((2-(diphenylamino)ethyl)(phenyl)amino)-N-tetradecylacetamide, N-decyl-2-((2-(diphenylamino)ethyl)(phenyl)amino), N1-(3-(3-(diphenylamino)propoxy)propyl)-N1,N2,N2-triphenyl ethane-1,2-diamine, N1-(2-(diphenylamino)ethyl)-N,N8,N8-triphenyl octane-1,8-diamine, 8-[2-(11-{[4-(dimethylamino)butanoyl]oxy}nonadecyl)cyclopropyl]octanoic acid methyl ester, 8-{2-[11-(dimethylamino)nonadecyl]cyclopropyl}octanoic acid methyl ester, (Z)-undec-2- en-1-yl 6-(3-(decyloxy)-2-(dimethylamino)propoxy)hexanoate, (2R,12Z,15Z)-N,N-dimethyl-1-(undecyloxy)heneicos-12,15-dien-2-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, 2-(diphenylamino)-N-(4-(2-((2-(diphenylamino)ethyl)(phenyl)amino)-N-methylacetamido)butyl)-N-methylacetamide, 7,10-dimethyl-13,16-diphenyl-6,11-dioxo-4-tetradecyl-4,7,10,13,16-pentazapentacosa- yl decanoate, 2-(diphenylamino)-N-(2-(2-((2-(diphenylamino)ethyl)(phenyl)amino)-N- ethylacetamido)ethyl)-N-ethylacetamide, 2-(diphenylamino)-N-(3-(2-((2-(diphenylamino)ethyl)(phenyl)amino)-N-methylacetamido)propyl)-N-methylacetamide, 2-((2-(di((Z)-non-3-en-1-yl)amino)ethyl)((Z)-non-3-en-1-yl)amino)-N-(2-(2-(diphenylamino)-N-methylacetamido)ethyl)-N-methylacetamide, 2-(diphenylamino)-N-(2-(2-((2-(diphenylamino)ethyl)(phenyl)amino)acetamido)ethyl)acetamide, 8,11-dimethyl-5,14,17-triphenyl-7,12-dioxo-5,8,11,14,17-pentazahexacosan-5-yl pentanoate,2-((2-(dionylamino)ethyl)(nonyl)amino)-N-methyl-N-(2-(methylamino)ethyl)acetamide, 2-(dionylamino)-N-(2-(2-((2-(dionylamino)ethyl)(nonyl)amino)-N-methylacetamido)ethyl)-N- methylacetamide, 2-(dionylamino)-N-methyl-N-(2-(methylamino)ethyl)acetamide, 2- ((N-(2-(dionylamino)ethyl)-N-nonylglycyl)oxy)ethyldionylglycinate, 2-hydroxyethyldionylglycinate, methyl 8-[2-(11-{[4-(dimethylamino)butanoyl]oxy}eicosyl)cyclopropyl]octanoate, methyl 8-{2-[11-(dimethylamino)eicosyl]cyclopropyl}octanoate, (Z)-undec-2-en-1-yl 6-(2-(dimethylamino)-3-(nonyloxy)propoxy)hexanoate, (2R,12Z,15Z)-1-(hexadecyloxy)-N,N-dimethylheneicos-12,15-dien-2-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, tert-butyl 4-(didecylamino)butanoate, 1,1-(piperazin-1,4-diyl)bis(4-(didecylamino)butane-1- one), heptyl 5-(4-(N-(2-(dionylamino)ethyl)-N-nonylglycyl)piperazin-1-yl)-5-oxopentanoate, 5-(heptyloxy)-5-oxopentanoic acid, heptyl 5-(4-(N-(2-(dionylamino)ethyl)-N-nonylglycyl)piperazin-1-yl)-5-oxopentanoate, 5-(heptyloxy)-5-oxopentanoic acid, (Z)-4-((2-(4-(N-(2-(dionylamino)ethyl)-N-nonylglycyl)piperazin-1-yl)-2-oxoethyl)(tetradecyl)amino)but-2-en-1-yl nonanoate, (Z)-4-hydroxybut-2-en-1-yl nonanoate, (Z)-3-((2-(4-(N-(2-(dionylamino)ethyl)-N-nonylglycyl)piperazin-1-yl)-2-oxoethyl)(tetradecan-9-en-1-yl)amino)propyl decanoate, (Z)-tetradecan-9-en-1-yl methanesulfonate, methyl 8-[2-(9-{[4-(dimethylamino)butanoyl]oxy}pentadecyl)cyclopropyl]octanoate, methyl 8-{2-[9-(dimethylamino)pentadecyl]cyclopropyl}octanoate, (Z)-undec-2-en-1-yl 6-(2-(dimethylamino)-3-(heptyloxy)propoxy)hexanoate, (2R,12Z,15Z)-1-(hexyloxy)-N,N-dimethylheneicos-12,15-dien-2-amine, (16Z,19Z)-N,N-dimethylpentacos-16,19-dien-6-amine,8-((2-(4-(N-(2-(di((Z)-non-3-en-1-yl)amino)ethyl)-N-((Z)-non-3-en-1-yl)glycyl)piperazin-1-yl)-2-oxoethyl)(nonyl)amino)octanoic acid methyl ester, 4-(nonylglycyl)piperazine-1-carboxylic acid tert-butyl ester, 3-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazin-1-yl)-2-oxoethyl)(tetradecyl)amino)propyl (Z)-deca-3-enoic acid ester (Z)-deca-3-en-1-ol, 2-((2-(di((Z)-non-3-en-1-yl)amino)ethyl)((Z)-non-3-en-1-yl)amino)-1-(4-(dinonylglycyl)piperazin-1-yl)ethan-1-one (Z)-1-bromonon-4-ene, 3-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-oxoethyl)(dodecyl)amino)propyl octanoic acid ester tert-butyl dodecyl glycine ester, S-4-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazin-1-yl)-2-oxoethyl)(nonyl)amino)thiobutanoic acid pentyl ester, 3-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-1-yl)ethyl)(nonyl)amino)propyl 3-methylhexanoic acid ester, tert-butyl 4-(2-((3-((3-methylhexanoyl)oxy)propyl)(nonyl)amino)ethyl)piperidine, 1,3-((2-(1-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperidin-4-yl)ethyl)(nonyl)amino)-2-methylpropyl hexanoic acid ester, 3-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-oxoethyl)(nonyl)amino)propyl 3-methylhexanoic acid ester, 3-((2-(4-(N-(2-(dinonylamino)ethyl)-N-nonylglycyl)piperazine-oxoethyl)(nonyl)amino)-2-methylpropyl hexanoic acid ester, 8-[2-(9-{[4-(dimethylamino)butanoyl]oxy}hexadecyl)cyclopropyl]octanoic acid methyl ester, 8-{2-[9-(dimethylamino)hexadecyl]cyclopropyl}octanoic acid methyl ester, (Z)-undec-2-en-1-yl 6-(2-(dimethylamino)-3-(hexyloxy)propoxy)hexanoic acid ester, (2R,12Z,15Z)-1-(decyloxy)-N,N-dimethylheneicos-12,15-dien-2-amine, (17Z,20Z)-N,N-dimethylhexacos-17,20-dien-7-amine, 2-((2-(dinonylamino)ethyl)(nonyl)amino)ethyl 1-(dinonylglycyl)piperidine-4-carboxylic acid ester,1 -(2-(dilaurylamino)ethyl)-4-(2-((2-(dilaurylamino)ethyl)(dodecyl)amino)ethyl)cyclohexane-1,4-dicarboxylate 2-(dilaurylamino)ethane-1 -ol, 12-((2-(1 -(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)pyrrolidin-3-yl)ethyl)(tetradecyl)amino)dodecanoic acid methyl ester, 3-(2-((12-methoxy-12-oxododecyl)(tetradecyl)amino)ethyl)pyrrolidine-1 -carboxylic acid tert-butyl ester, 3-((2-(1 -(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)pyrrolidin-3-yl)ethyl)(tetradecyl)amino)propyl decanoate, 3-(2-((3-(decanoyloxy)propyl)(tetradecyl)amino)ethyl)pyrrolidine-1 -carboxylic acid tert-butyl ester, 6-((2-(1 -(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)pyrrolidin-3-yl)ethyl)(tetradecyl)amino)heptanoic acid, 3-(2-((6-(heptyloxy)-6-oxohexyl)(tetradecyl)amino)ethyl)pyrrolidine-1 -carboxylic acid tert-butyl ester, 8-((2-(1 -(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)pyrrolidin-3-yl)ethyl)(tetradecyl)amino)octanoic acid, 3-(2-(tetradecylamino)ethyl)pyrrolidine-1 -carboxylic acid tert-butyl ester, 12-((2-(1 -(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)piperidin-3-yl)ethyl)(tetradecyl)amino)dodecanoic acid methyl ester, 3-(2-((12-methoxy-12-oxododecyl)(tetradecyl)amino)ethyl)piperidine-1 -carboxylic acid butyl ester, 3-((2-(1 -(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)piperidin-3-yl)ethyl)(tetradecyl)amino)propyl decanoate, 6-((2-(1 -(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)piperidin-3-yl)ethyl)(tetradecyl)amino)heptanoic acid, 8-((2-(1 -(N-(2-(dilaurylamino)ethyl)-N-dodecylglycyl)piperidin-3-yl)ethyl)(tetradecyl)amino)octanoic acid, 6-((2-(4-(2-((2-(dododecylamino)ethyl)(dodecyl)amino)ethyl)piperazin-1 -yl)ethyl)(dodecyl)amino)hexanoic acid, 6-bromohexanoic acid, 8-[2-(9-{[4-(dimethylamino)butanoyl]oxy}heptadecyl)cyclopropyl]octanoic acid methyl ester, 8-{2-[9-(dimethylamino)heptadecyl]cyclopropyl}octanoic acid methyl ester, (2S,12Z,15Z)-N,N-dimethyl-1 -(octyloxy)heneicosane-12,15-diene-2-amine, (2-octylcyclopropyl)methyl 6-(2-(dimethylamino)-3-(octyloxy)propoxy)hexanoate,(18Z,21Z)-N,N-dimethylheptacos-18,21-dien-8-amine, trans-1-methyl-3,4-bis(((Z)-hexadec-9-enoyloxy)methyl)pyrrolidine, (Z)-nonyl 4-((2-(4-(N-(2-(dilaurylamino)ethyl)-N-laurylglycyl)piperazin-1-yl)-2-oxoethyl)(tetradecyl)amino)butanoate, trans-1-methyl-3,4-bis(((9Z,12Z)-octadeca-9,12-dienoyloxy)methyl)pyrrolidine, methyl 12-((2-(4-(N-(2-(dilaurylamino)ethyl)-N-laurylglycyl)piperazin-1-yl)-2-oxoethyl)(tetradecyl)amino)dodecanoate, (7Z)-ethyl 17-[2-(dimethylamino)ethyl]hexacos-7-enoate, trans-1-methyl-3,4-bis(((Z)-octadeca-9-enoyloxy)methyl)pyrrolidine, methyl 6-(2-(1-(N-(2-(dimethylamino)ethyl)eicosyl)cyclopropyl)hexanoate, methyl 12-((2-(1-(N-(2-(dilaurylamino)ethyl)-N-laurylglycyl)piperidin-4-yl)ethyl)(tetradecyl)amino)dodecanoate, methyl 10-(2-N-2-(dimethylamino)ethyl]hexadecyl}cyclopropyl)decanoate, methyl 8-(2-{N1-2-(dimethylamino)ethyl]heptadecyl}cyclopropyl)octanoate, di-nonylglycyl 2-(1-(N-(2-(dilaurylamino)ethyl)-N-laurylglycyl)piperidin-4-yl)ethyl ester, tert-butyl 4-(2-((dilaurylglycyl)oxy)ethyl)piperidine-1-carboxylate, methyl 8-(2-{N1-2-(dimethylamino)ethyl]octadecyl}cyclopropyl)octanoate, methyl 8-(2-(N1-2-(dimethylamino)ethyl]nonadecyl}cyclopropyl)octanoate, 1-(piperazine-1,4-diyl)bis(2-(dilaurylamino)ethan-1-one), methyl 8-[2-{N1-2-(dimethylamino)ethyl]eicosyl}cyclopropyl)octanoate, methyl 8-(2-{9-[2-(dimethylamino)ethyl]pentadecyl}cyclopropyl)octanoate, (7Z)-methyl 19-{[4-(dimethylamino)butanoyl]oxy}octacos-7-enoate, (7Z)-methyl 19-(dimethylamino)octacos-7-enoate, cis-1-methyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-4-(octyloxy)pyrrolidine, 2-(didodecylamino)-1-(4-(N-(2-(didodecylamino)ethyl)-N-dodecylglycyl)piperazin-1-yl)ethan-1-one, (Z)-undec-2-en-1-yl 6-(2-(dimethylamino)-3-(octyloxy)propoxy)hexanoate,(N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]decane-2-amine (compound 11), (19Z,22Z)-N,N-dimethyloctacosane-19,22-diene-9-amine, methyl 8-(2-{9-[2-(dimethylamino)ethyl]hexadecyl}cyclopropyl)octanoate, 5-((2-(4-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperazin- yloxy)ethyl)(phenyl)amino)pentyl methyl carbonate, methyl 8-(2-{9-[2-(dimethylamino)ethyl]heptadecyl}cyclopropyl)octanoate, methyl (7Z)-19-[2-(dimethylamino)ethyl]octadeca-7-enoate, (Z)-pent-2-en-1-yl 4-((2-(4-(N-(2-(diphenylamino)ethyl)-N- phenylglycyl)piperazin-1-yl)-2-oxoethyl)(phenyl)amino)butanoate, methyl (11Z)-19-[2-(dimethylamino)ethyl]octadeca-11-enoate, methyl (9Z)-21-[2-(dimethylamino)ethyl]heptacos-9-enoate, methyl (9Z)-21-[2-(dimethylamino)ethyl]octacos-9-enoate, methyl (9Z)-21-[2-(dimethylamino)ethyl]nonacos-9-enoate, 2-(1-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperidin-3-yl)ethyldiphenylglycinate, methyl (9Z)-21-[2-(dimethylamino)ethyl]triacont-9-enoate, (1-(N-(2-(diphenylamino)ethyl)-N-phenylglycyl)piperidin-3-yl)methyldiphenylglycinate, methyl (9Z)-19-[2-(dimethylamino)ethyl]pentacos-9-enoate, methyl (9Z)-19-[2-(dimethylamino)ethyl]hexacos-9-enoate, methyl 6-(2-(8-(3-(decyloxy)-2-(dimethylamino)propoxy)octyl)cyclopropyl)hexanoate, methyl (11Z)-19-{[4-(dimethylamino)butanoyl]oxy}octadeca-11-enoate, methyl (11Z)-19-(dimethylamino)octadeca-11-enoate, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]dodecan-2-amine, (14Z,17Z)-N,N-dimethyltricos-14,17-diene-4-amine, methyl di((9Z,12Z)-octadeca-9,12-dienyl)amine, methyl (9Z)-19-{[4-(dimethylamino)butanoyl]oxy}octadeca-9-enoate, methyl (9Z)-19-(dimethylamino)octadeca-9-enoate, (Z)-methyl 17-(2-(dimethylamino)-3-(octyloxy)propoxy)heptadeca-8-enoate,(3R,4R)-3,4-bis((Z)-hexadec-9-enyloxy)-1-methylpyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]undecan-2-amine, (20Z,23Z)-octacos-20,23-dien-10-yl 4-(dimethylamino)butanoate, (20Z,23Z)-N,N-dimethyloctacos-20,23-dien-10-amine, 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1- amine, 3-((6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yloxy)-N,N-dimethylpropan-1- amine, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate), (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 5-(dimethylamino)pentanoate, (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 5-(dimethylamino)pentanoate, (6Z,16Z)-12-((Z)-dec-4-enyl)docosa-6,16-dien-11-yl 5-(dimethylamino)pentanoate, L-arginine-a-(2,3-dilauryloxy)propionamide, L-lysine-a-(2,3-dilauryloxy)propionamide, 2,3-dioleyloxypropylamine, 2,3-distearyloxypropylamine, 2,3-dilauryloxypropylamine, dilinoleylmethyl 4-(dimethylamino)propyl ether), dilinoleylmethyl 4-(dimethylamino)butyl ether), and 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane.

[0324] In some embodiments, the at least one non-cationic lipid comprises at least one phospholipid, at least one fusogenic lipid, at least one anionic lipid, at least one helper lipid, at least one neutral lipid, or any combination thereof. In some embodiments, the LNP can be substantially free of the at least one non-cationic lipid. In some embodiments, the LNP can not contain the at least one non-cationic lipid in any amount.

[0325] In some embodiments, the at least one non-cationic lipid can be selected from, but not limited to, at least one of: 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), DSPC but with 3 unsaturated double bonds per tail (18:3 PC), acylcarnitine (AC), 1-hexadecyl-sn-glycero-3-phosphocholine (CI 6 Lyso PC), N-oleoyl-SPM (CI 8: l), N-tetracosanoyl SPM (C24:0), N-nervacyl C (N-nervacyl C) (C24: l), Cetylphosphate (Cet-P), Cholesterol Hemisuccinate (CHEMS), Cholesterol (Chol), Cholesterol Hemisuccinic acid (Chol-C12), 12-Cholestyloxy carbonylaminododecanoic acid (Chol-C13N), Cholesterol Hemioxalate (Chol-C2), Cholesterol Hemomalonate (Chol-C3), N-(Cholestyloxy carbonyl)glycine (Chol-C3N), Cholesterol Hemoglutarate (Chol-C5), Cholesterol Hemoadipate (Chol-C6), Cholesterol Hemepimelate (Chol-C7), Cholesterol Hemosuberate (Chol-C8), Cardiolipin (CL), 1,2-Bis(tricos-10,12-dienoyl)-sn-glycero-3-phosphocholine (DC8-9PC), Dicetylphosphate (DCP), Dihexadecylphosphate (DCP1), 1,2-Dipalmitoyl glycerol-3-hemisuccinate (DGSucc), Short-chain bis-n-heptadecanoyl phosphatidylcholine (DHPC), Dipalmitoyl phosphatidyl ethanolamine (DHPE), 1,2-Dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), l,2-Dilauroyl-sn-glycero-3-PE (DLPE), Dimyristoyl glycerol hemisuccinate (DMGS), Dimyristoyl phosphatidylcholine (DMPC), Dimyristoyl phosphatidyl ethanolamine (DMPE), Dimyristoyl phosphatidyl glycerol (DMPG), Dioleyloxybenzyl alcohol (DOBA), 1,2-Dioleoyl glycerol-3-hemisuccinate (DOGHEMS), N-[2-(2-{2-[2-(2,3-bis-octadec-9-enyloxy-propoxy)-ethoxy]-ethoxy}-ethyl]-3-(3,4,5-trihydroxy-6-hydroxymethyl-tetrahydro-pyran-2-yl thiol)-propionamide (DOGP4aMan), Dioleoyl phosphatidylcholine (DOPC), Dioleoyl phosphatidyl ethanolamine (DOPE), Dioleoyl-phosphatidyl ethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), Dioleoyl phosphatidyl glycerol (DOPG), 1,2-dioleoyl-sn-glycero-3-(phospho-L-serine) (DOPS), cell fusogenic phospholipid (DPhPE), dipalmitoyl phosphatidylcholine (DPPC), dipalmitoyl phosphatidyl ethanolamine (DPPE), dipalmitoyl phosphatidylglycerol (DPPG), dipalmitoyl phosphatidylserine (DPPS), distearoyl phosphatidylcholine (DSPC), distearoyl-phosphatidyl-ethanolamine (DSPE), distearoyl phosphoethanolamine imidazole (DSPEI), 1,2-dierucoyl-sn-glycero-phosphocholine (DUPC), egg phosphatidylcholine (EPC), N-histidyl cholesteryl carbamate (HCChol), histidyl distearoyl glycerol (HDSG), N-histidyl cholesteryl hemisuccinate (HistChol), 1,2-dipalmitoyl glycerol-hemisuccinate-Na-histidyl-hemisuccinate (HistSuccDG), N-(5'-hydroxy-3'-oxypentyl)-10,12-pentacosadiynoic amide (h-Pegl-PCDA), 2-[l-hexyloxyethyl]-2-devinyl pyro-phyllotoxin-a (HPPH), hydrogenated soy phosphatidylcholine (HSPC), 1,2-dipalmitoyl glycerol-Oa-histidyl-Na-hemisuccinate (IsohistsuccDG), mannosialized dipalmitoyl phosphatidyl ethanolamine (ManDOG), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidomethyl)cyclohexane-carboxamide] (MCC-PE), l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), l-myristoyl-2-hydroxy-sn-glycero-phosphocholine (MHPC), thiol-reactive maleimide headgroup lipid, l,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide (MPB-PE), nervonic acid (NA), sodium cholate (NaChol), 1,2-dioleoyl-sn-glycero-3-[phosphoethanolamine-N-dodecanoyl (NC12-DOPE), ND98, N- glutaryl phosphatidyl ethanolamine (NG-PE), N-hydroxysuccinimidyl (NHS- 'x'), dicarboxylic acid derivatized phosphatidyl ethanolamine (NwPE- 'x'), oleic acid (OA), l-oleoyl-2-cholesteryl hemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), phosphatidic acid (PA), phosphatidyl ethanolamine lipid (PE), PE lipid conjugated to polyethylene glycol (PEG), polyethylene glycol-distearoyl phosphatidyl ethanolamine lipid (PEG-PE), phosphatidyl glycerol (PG), partially hydrogenated soy phosphatidyl choline (PHSPC), phosphatidyl inositol lipid (PI), phosphatidylinositol-4-phosphate (PIP), palmitoyloleoyl phosphatidyl choline (POPC), phosphatidyl ethanolamine (POPE), palmitoyloleoyl phosphatidyl glycerol (POPG), phosphatidyl serine (PS), lissaminerhodamine B-phosphatidyl ethanolamine lipid (Rh-PE), purified soy derived phospholipid mixture (S100), phosphatidyl choline (SM), 18-1-trans PE, l-stearoyl-2-oleoyl-phosphatidyl ethanolamine (SOPE), soy phosphatidyl choline (SPC), sphingomyelin (SPM), a-a'-trehalose 6,6'-dibehenate (TDB), l,2-ditricosanoyl-sn-glycero-3-phosphoethanolamine (transDOPE), ((23S,5R)-3-(bis(hexadecyloxy)methoxy)-5-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-l(2H)-yl)tetrahydrofuran-2-yl)methyl methyl phosphate, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-dibehenoyl-sn-glycero-3-phosphocholine, 1,2-dibehenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-sn-glycero-3-phosphocholine, 1,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dioleyl-sn-glycero-3-phosphoethanolamine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 16-O-monomethyl PE, 16-O-dimethyl PE, and dioleoyl phosphatidyl ethanolamine.

[0326] In some embodiments, the LNP comprises an ionizable lipid or lipid-like material. As non-limiting examples, the ionizable lipid can be C12-200, CKK-E12, 5A2-SC8, BAMEA-016B, or 7C1. Other ionizable lipids are known in the art and can be used herein.

[0327] In some embodiments, the LNP comprises a phospholipid. As non-limiting examples, the phospholipid (helper) can be DOPE, DSPC, DOTAP, or DOTMA.

[0328] In some embodiments, the LNP comprises a PEG derivative. As non-limiting examples, the PEG derivative can be lipid-anchored, e.g., the PEG is C14-PEG2000, C14-PEG1000, C14-PEG3000, C14-PEG5000, C12-PEG1000, C12-PEG2000, C12-PEG3000, C12-PEG5000, C16-PEG1000, C16-PEG2000, C16-PEG3000, C16-PEG5000, C18-PEG1000, C18-PEG2000, C18-PEG3000, or C18-PEG5000.

[0329] In some embodiments, the at least one sterol comprises at least one cholesterol or cholesterol derivative. In some embodiments, the LNP can be substantially free of the at least one sterol. In some embodiments, the LNP can be free of the at least one sterol in any amount.

[0330] In some embodiments, the at least one additional LNP functional component comprises at least one component that reduces particle aggregation, at least one component that reduces clearance of the LNP from circulation in a subject, at least one component that increases the ability of the LNP to cross a mucus layer, at least one component that reduces an immune response of a subject to the administered LNP, at least one component that alters the membrane fluidity of the LNP, at least one component that aids in the stability of the LNP, or any combination thereof. In some embodiments, the LNP can be substantially free of the at least one additional LNP functional component. In some embodiments, the LNP can be free of the at least one additional LNP functional component in any amount.

[0331] In some embodiments, the additional LNP functional component can comprise a polymer. In some embodiments, the polymer comprising the additional LNP functional component can comprise at least one polyethylene glycol (PEG), at least one polypropylene glycol (PPG), poly(2-oxazoline) (POZ), at least one polyamide (ATTA), at least one cationic polymer, or any combination thereof.

[0332] In some implementations, the average molecular weight of the polymer portion (e.g., PEG) can be between 500 and 20,000 Daltons. In some embodiments, the molecular weight of the polymer can be about 500 to 20,000, 1,000 to 20,000, 1,500 to 20,000, 2,000 to 20,000, 2,500 to 20,000, 3,000 to 20,000, 3,500 to 20,000, 4,000 to 20,000, 4,500 to 20,000, 5,000 to 20,000, 5,500 to 20,000, 6,000 to 20,000, 6,500 to 20,000, 7,000 to 20,000, 7,500 to 20,000, 8,000 to 20,000, 8,500 to 20,000, 8,500 to 20,000. 000, 9,000 to 20,000, 9,500 to 20,000, 10,000 to 20,000, 10,500 to 20,000, 11,000 to 20,000, 11,500 to 20,000, 12,000 to 20,000, 12,500 to 20,000, 13,000 to 20,000, 13,500 to 20,000, 14,000 to 20,000, 14,500 to 20,000, 15,000 to 20,000, 15,500 to 20,000, 16,000 to 20,000, 16,500 to 20,000, 17,000 to 20,000 00, 17,500 to 20,000, 18,000 to 20,000, 18,500 to 20,000, 19,000 to 20,000, 19,500 to 20,000, 500 to 19,500, 1,000 to 19,500, 1,500 to 19,500, 2,000 to 19,500, 2,500 to 19,500, 3,000 to 19,500, 3,500 to 19,500, 4,000 to 19,500, 4,500 to 19,500, 5,000 to 19,500, 5,500 to 19,500, 6,000 to 19,500, 6,500 to 19,500 00, 7,000 to 19,500, 7,500 to 19,500, 8,000 to 19,500, 8,500 to 19,500, 9,000 to 19,500, 9,500 to 19,500, 10,000 to 19,500, 10,500 to 19,500, 11,000 to 19,500, 11,500 to 19,500, 12,000 to 19,500, 12,500 to 19,500, 13,000 to 19,500, 13,500 to 19,500, 14,000 to 19,500, 14,500 to 19,500, 15,000 to 19,500, 15,500 to 19,500, 16,000 to 19,500, 16,500 to 19,500, 17,000 to 19,500, 17,500 to 19,500, 18,000 to 19,500, 18,500 to 19,500, 19,000 to 19,500, 1,500 to 19,000, 2,000 to 19,000, 2,500 to 19,000, 3,000 to 19,000, 3,500 to 19,000, 4,000 to 19,000, 4,500 to 19,000, 5,000 to 19,000, 5,500 to 19,000, 6,000 to 19,000, 6,500 to 19,000, 7,000 to 19,000, 7,500 to 19,000, 8,000 to 19,000, 8,500 to 19,000, 9,000 to 19,000, 9,500 to 19,000, 10,000 to 19,000, 10,500 to 19,000, 11,000 to 19,000, 11,500 to 19,000, 12,000 to 19,000, 12,500 to 19,000, 13,000 to 19,000, 13,500 to 19,000, 14,000 to 19,000, 14,500 to 19,000, 15,000 to 19,000, 15,500 to 19,000, 16,000 to 19,000, 16,500 to 19,000, 17,000 to 19,000, 17,500 to 19,000, 18,000 to 19,000, 18,500 to 19,000, 1,500 to 18,500, 2,000 to 18,500, 2,500 to 18,500, 3,000 to 18,500, 3,500 to 18,500, 4,000 to 18,500, 4,500 to 18,500, 5,000 to 18,500, 5,500 to 18,500, 6,000 to 18,500, 6,500 to 18,500, 7,000 to 18,500, 7,500 to 18,500, 8,000 to 18,500, 8,500 to 18,500, 9,000 to 18,500, 9,500 to 18,500, 10,000 to 18,500, 10,500 to 18,500, 11,000 to 18,500, 11,500 to 18,500, 12,000 to 18,500, 12,500 to 18,500, 13,000 to 18,500, 13,500 to 18,500, 14,000 to 18,500, 14,500 to 18,500, 15,000 to 18,500, 15,500 to 18,500, 16,000 to 18,500, 16,500 to 18,500, 17,000 to 18,500, 17,500 to 18,500, 18,000 to 18,500, 1,500 to 18,000, 2,000 to 18,000, 2,500 to 18,000, 3,000 to 18,000, 3,500 to 18,000, 4,000 to 18,000, 4,500 to 18,000, 5,000 to 18,000, 5,500 to 18,000, 6,000 to 18,000, 6,500 to 18,000, 7,000 to 18,000, 7,500 to 18,000, 8,000 to 18,000, 8,500 to 18,000, 9,000 to 18,000, 9,500 to 18,000, 10,000 to 18,000, 10,500 to 18,000, 11,000 to 18,000, 11,500 to 18,000, 12,000 to 18,000, 12,500 to 18,000, 13,000 to 18,000, 13,500 to 18,000, 14,000 to 18,000, 14,500 to 18,000, 15,000 to 18,000, 15,500 to 18,000, 16,000 to 18,000, 16,500 to 18,000, 17,000 to 18,000, 17,500 to 18,000, 1,500 to 17,500, 2,000 to 17,500, 2,500 to 17,500, 3,000 to 17,500, 3,500 to 17,500, 4,000 to 17,500, 4,500 to 17,500, 5,000 to 17,500, 5,500 to 17,500, 6,000 to 17,500, 6,500 to 17,500, 7,000 to 17,500, 7,500 to 17,500, 8,000 to 17,500, 8,500 to 17,500, 9,000 to 17,500, 9,500 to 17,500, 10,000 to 17,500, 10,500 to 17,500, 11,000 to 17,500, 11,500 to 17,500, 12,000 to 17,500, 12,500 to 17,500, 13,000 to 17,500, 13,500 to 17,500, 14,000 to 17,500, 14,500 to 17,500, 15,000 to 17,500, 15,500 to 17,500, 16,000 to 17,500, 16,500 to 17,500, 17,000 to 17,500, 1,500 to 17,000, 2,000 to 17,000, 2,500 to 17,000, 3,000 to 17,000, 3,500 to 17,000, 4,000 to 17,000, 4,500 to 17,000, 5,000 to 17,000, 5,500 to 17,000, 6,000 to 17,000, 6,500 to 17,000, 7,000 to 17,000, 7,500 to 17,000, 8,000 to 17,000, 8,500 to 17,000, 9,000 to 17,000, 9,500 to 17,000, 10,000 to 17,000, 10,500 to 17,000, 11,000 to 17,000, 11,500 to 17,000, 12,000 to 17,000, 12,500 to 17,000, 13,000 to 17,000, 13,500 to 17,000, 14,000 to 17,000, 14,500 to 17,000, 15,000 to 17,000, 15,500 to 17,000, 16,000 to 17,000, 16,500 to 17,000, 1,500 to 16,500, 2,000 to 16,500, 2,500 to 16,500, 3,000 to 16,500, 3,500 to 16,500, 4,000 to 16,500, 4,500 to 16,500, 5,000 to 16,500, 5,500 to 16,500, 6,000 to 16,500, 6,500 to 16,500, 7,000 to 16,500, 7,500 to 16,500, 8,000 to 16,500, 8,500 to 16,500, 9,000 to 16,500, 9,500 to 16,500, 10,000 to 16,500, 10,500 to 16,500, 11,000 to 16,500, 11,500 to 16,500, 12,000 to 16,500, 12,500 to 16,500, 13,000 to 16,500, 13,500 to 16,500, 14,000 to 16,500, 14,500 to 16,500, 15,000 to 16,500, 15,500 to 16,500, 16,000 to 16,500, 1,500 to 16,000, 2,000 to 16,000, 2,500 to 16,000, 3,000 to 16,000, 3,500 to 16,000, 4,000 to 16,000, 4,500 to 16,000, 5,000 to 16,000, 5,500 to 16,000, 6,000 to 16,000, 6,500 to 16,000, 7,000 to 16,000, 7,500 to 16,000, 8,000 to 16,000, 8,500 to 16,000, 9,000 to 16,000, 9,500 to 16,000, 10,000 to 16,000, 10,500 to 16,000, 11,000 to 16,000, 11,500 to 16,000, 12,000 to 16,000, 12,500 to 16,000, 13,000 to 16,000, 13,500 to 16,000, 14,000 to 16,000, 14,500 to 16,000, 15,000 to 16,000, 15,500 to 16,000, 1,500 to 15,500, 2,000 to 15,500, 2,500 to 15,500, 3,000 to 15,500, 3,500 to 15,500, 4,000 to 15,500, 4,500 to 15,500, 5,000 to 15,500, 5,500 to 15,500, 6,000 to 15,500, 6,500 to 15,500, 7,000 to 15,500, 7,500 to 15,500, 8,000 to 15,500, 8,500 to 15,500, 9,000 to 15,500, 9,500 to 15,500, 10,000 to 15,500, 10,500 to 15,500, 11,000 to 15,500, 11,500 to 15,500, 12,000 to 15,500, 12,500 to 15,500, 13,000 to 15,500, 13,500 to 15,500, 14,000 to 15,500, 14,500 to 15,500, 15,000 to 15,500, 1,500 to 15,000, 2,000 to 15,000, 2,500 to 15,000, 3,000 to 15,000, 3,500 to 15,000, 4,000 to 15,000, 4,500 to 15,000, 5,000 to 15,000, 5,500 to 15,000, 6,000 to 15,000, 6,500 to 15,000, 7,000 to 15,000, 7,500 to 15,000, 8,000 to 15,000, 8,500 to 15,000, 9,000 to 15,000, 9,500 to 15,000, 10,000 to 15,000, 10,500 to 15,000, 11,000 to 15,000, 11,500 to 15,000, 12,000 to 15,000, 12,500 to 15,000, 13,000 to 15,000, 13,500 to 15,000, 14,000 to 15,000, 14,500 to 15,000, 1,500 to 14,500, 2,000 to 14,500, 2,500 to 14,500, 3,000 to 14,500, 3,500 to 14,500, 4,000 to 14,500, 4,500 to 14,500, 5,000 to 14,500, 5,500 to 14,500, 6,000 to 14,500, 6,500 to 14,500, 7,000 to 14,500, 7,500 to 14,500, 8,000 to 14,500, 8,500 to 14,500, 9,000 to 14,500, 9,500 to 14,500, 10,000 to 14,500, 10,500 to 14,500, 11,000 to 14,500, 11,500 to 14,500, 12,000 to 14,500, 12,500 to 14,500, 13,000 to 14,500, 13,500 to 14,500, 14,000 to 14,500, 1,500 to 14,000, 2,000 to 14,000, 2,500 to 14,000, 3,000 to 14,000, 3,500 to 14,000, 4,000 to 14,000, 4,500 to 14,000, 5,000 to 14,000, 5,500 to 14,000, 6,000 to 14,000, 6,500 to 14,000, 7,000 to 14,000, 7,500 to 14,000, 8,000 to 14,000, 8,500 to 14,000, 9,000 to 14,000, 9,500 to 14,000, 10,000 to 14,000, 10,500 to 14,000, 11,000 to 14,000, 11,500 to 14,000, 12,000 to 14,000, 12,500 to 14,000, 13,000 to 14,000, 13,500 to 14,000, 1,500 to 13,500, 2,000 to 13,500, 2,500 to 13,500, 3,000 to 13,500, 3,500 to 13,500, 4,000 to 13...

Claims

1. A polynucleotide encoding at least one structural protein or fragment, antigenic peptide, or variant thereof of varicella-zoster virus (VZV), wherein the at least one structural protein is glycoprotein E (gE), and wherein the polynucleotide comprises a first sequence region having a nucleic acid sequence that is at least 80% identical to SEQ ID NO:

6.

2. The polynucleotide of claim 1, wherein the first sequence region consists of SEQ ID NO:

6.

3. A polynucleotide encoding at least one structural protein or fragment, antigenic peptide, or variant thereof of VZV, wherein the at least one structural protein is glycoprotein (G), and wherein the polynucleotide comprises a first sequence region having a nucleic acid sequence that is at least 80% identical to a member of the group consisting of SEQ ID NOs: 7, 8, 9, and 10.

4. The polynucleotide of claim 3, wherein the first sequence region consists of SEQ ID NO:

7.

5. The polynucleotide of claim 3, wherein the first sequence region consists of SEQ ID NO:

8.

6. The polynucleotide of claim 3, wherein the first sequence region consists of SEQ ID NO:

9.

7. The polynucleotide of claim 3, wherein the first sequence region consists of SEQ ID NO:

10.

8. The polynucleotide of any one of claims 3-7, wherein the polynucleotide further comprises a second sequence region comprising the nucleic acid sequence of SEQ ID NO: 47 or 50.

9. The polynucleotide of any one of claims 1-8, wherein at least 50% of the polynucleotide sequence is codon-optimized.

10. The polynucleotide of claim 9, wherein the polynucleotide is DNA, or RNA, or mRNA.

11. The polynucleotide of claim 10, wherein the polynucleotide is mRNA.

12. The polynucleotide of claim 11, comprising a 5' UTR and a 3' UTR, wherein the 5' UTR comprises SEQ ID NO: 40 and the 3' UTR comprises SEQ ID NO: 42 or 44.

13. The polynucleotide of claim 12, wherein at least one uridine is modified to an Nl-methyl pseudouridine.

14. The polynucleotide of claim 13, wherein all uridines are modified to Nl-methyl pseudouridines.

15. A polynucleotide comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 16, 17, 18, 19, and 20.

16. A polypeptide comprising at least one structural protein or fragment, antigenic peptide, or variant thereof of VZV, wherein the at least one structural protein is glycoprotein E (gE), and wherein the polypeptide comprises a first sequence region comprising SEQ ID NOs: 2, 52, 53, 54, and 55.

17. The polypeptide of claim 16, wherein the first sequence region comprises SEQ ID NO:

2.

18. The polypeptide of claim 16, wherein the first sequence region comprises SEQ ID NO:

52.

19. The polypeptide of claim 16, wherein the first sequence region comprises SEQ ID NO:

53.

20. The polypeptide of claim 16, wherein the first sequence region comprises SEQ ID NO:

54.

21. The polypeptide of claim 16, wherein the first sequence region comprises SEQ ID NO:

55.

22. A nucleic acid vaccine comprising the polynucleotide of any one of claims 1-15 or encoding the polypeptide of any one of claims 16-21.

23. The nucleic acid vaccine of claim 22, formulated in a lipid nanoparticle (LNP).

24. A pharmaceutical composition comprising the nucleic acid vaccine of claims 22-23 and a pharmaceutically acceptable excipient.

25. A method of treating and / or preventing herpes zoster in a subject, the method comprising administering the nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24.

26. The method of claim 25, further comprising administering an immunoglobulin.

27. The method of claim 26, wherein the subject is exposed to varicella virus.

28. The method of claim 27, wherein the exposure is a bite exposure.

29. The method of claim 27, wherein the exposure is a non-bite exposure.

30. The method of any one of claims 25-29, wherein the subject is a human.

31. A method of preventing herpes zoster in a subject, the method comprising administering the nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24.

32. The method of claim 31, comprising administering to the subject a first dose of the nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24 alone.

33. The method of claim 32, further comprising administering a second dose to the subject between 1 and 5 weeks after the first dose.

34. The method of claim 32, further comprising administering a third dose to the subject between 1 and 5 weeks after the first dose.

35. The method of claim 33, further comprising administering a third dose to the subject between 1 and 5 weeks after the second dose.

36. A method of inducing an immune response in a subject, the method comprising administering the nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24.

37. The method of claim 36, wherein the immune response comprises a T cell response and / or a B cell response.

38. The method of claim 36, further comprising administering a booster after the first administration.

39. The method of claim 38, wherein the booster is administered once per year.

40. A method of reducing or ameliorating one or more physiological effects or symptoms of herpes zoster in a subject, the method comprising administering to the subject the nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24.

41. The method of claim 40, wherein the subject is a human patient.

42. The method of claim 40, wherein the subject is a mammal.

43. The nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24 for use in the treatment and / or prevention of herpes zoster in a subject in need thereof.

44. The nucleic acid vaccine or pharmaceutical composition for use of claim 43, for use in combination with an immunoglobulin.

45. The nucleic acid vaccine or pharmaceutical composition for use of claim 43 or 44, wherein the subject is exposed to varicella virus.

46. The nucleic acid vaccine or pharmaceutical composition for use of claim 45, wherein the exposure is a bite exposure.

47. The nucleic acid vaccine or pharmaceutical composition for use of claim 45, wherein the exposure is a non-bite exposure.

48. The nucleic acid vaccine or pharmaceutical composition for use of any one of claims 43-47, wherein the subject is a human.

49. The nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24 for use in the prevention of herpes zoster in a subject in need thereof.

50. The nucleic acid vaccine or pharmaceutical composition for use of claim 49, wherein the nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24 is for administration to the subject as a separate first dose.

51. The nucleic acid vaccine or pharmaceutical composition for use of claim 50, wherein the nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24 is for administration to the subject as a second dose between 1 and 5 weeks after the first dose.

52. The nucleic acid vaccine or pharmaceutical composition for use of claim 50, wherein the nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24 is for administration to the subject as a third dose between 1 and 5 weeks after the first dose.

53. The nucleic acid vaccine or pharmaceutical composition for use of claim 51, wherein the nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24 is for administration to the subject as a third dose between 1 and 5 weeks after the second dose.

54. The nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24 for use in inducing an immune response in a subject in need thereof.

55. The nucleic acid vaccine or pharmaceutical composition for use of claim 54, wherein the immune response comprises a T cell response and / or a B cell response.

56. The nucleic acid vaccine or pharmaceutical composition for use of claim 54 or 55, for use in combination with a booster for use after the first administration.

57. The nucleic acid vaccine or pharmaceutical composition for use of claim 56, wherein the booster is administered once a year.

58. The nucleic acid vaccine of any one of claims 22-23 or the pharmaceutical composition of claim 24, for use in reducing or ameliorating one or more physiological effects or symptoms of shingles in a subject in need thereof.

59. The nucleic acid vaccine or pharmaceutical composition for use of claim 58, wherein the subject is a human patient.

60. The nucleic acid vaccine or pharmaceutical composition for use of claim 58, wherein the subject is a mammal.

Images