Varicella-zoster virus mRNA vaccine

Abstract

Provided herein are optimized messenger ribonucleic acid (mRNA) vaccines for varicella zoster virus (VZV). The mRNA vaccines encode one or more VZV antigens, such as gE and variants of gE. Methods of use of the mRNA vaccines are also provided.

Description

[0001] VARICELLA-ZOSTER VIRUS MRNA VACCINE

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of the filing date under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application Serial No. 63 / 517,868, entitled “VARICELLA-ZOSTER VIRUS MRNA VACCINE,” filed on August 4, 2023, and to U.S. Provisional Patent Application Serial No. 63 / 518,096, entitled “VARICELLA-ZOSTER VIRUS MRNA VACCINE,” filed on August 7, 2023; the contents of each of which are incorporated herein by reference in their entireties.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The contents of the electronic sequence listing (M137870287WO00-SEQ-VLJ.xml; Size: 2,685,397 bytes; and Date of Creation: August 2, 2024) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Varicella-zoster virus (VZV) is a human alpha herpesvirus that causes varicella (chickenpox) and zoster (shingles) as the primary infection and establishes latency in sensory ganglia. The primary VZV infection begins by replicating in epithelial cells of the upper respiratory mucosa, followed by widely distributed vesicular rash. The primary VZV infection may result in complications, including viral or secondary bacterial pneumonia. During primary VZV infection, virions may gain access to the nervous system, and latent infection is established and may remain dormant. When viral replication is reactivated (about 10-20% of cases), VZV causes the symptoms of zoster and potentially other serious complications. Varicella epidemics occurred annually in the United States until a varicella vaccine (which is a live attenuated form of the VZV Oka strain) was introduced in 1995, but epidemics continue among children in countries that do not have immunization programs. An estimated 1 million episodes of herpes zoster (shingles) occur annually in just the United States. About every 1 in 3 people in the U.S. will experience shingles in their lifetime.

[0008] There are only a handful of VZV vaccines available in the U.S. For varicella, Merck is the only U.S. supplier of VZV-containing vaccine, including varicella vaccine (VARIVAX®) and a combined measles, mumps, rubella, and varicella (MMR-V) vaccine (PROQUAD®). For zoster there are two vaccines licensed and recommended to prevent shingles ZOSTAVAX® (Merck) and SHINGRIX® (GlaxoSmithKline). Even with vaccination efforts, varicella outbreaks continue to occur even in settings such as schools where most children are vaccinated. Prompt identification, investigation, and control of varicella outbreaks are important as even mild cases can be contagious. While most infections occur amongst unvaccinated individuals, immunity induced by the vaccine may not be lifelong, possibly leaving adults vulnerable to more severe disease. Additionally, some of the currently available vaccines are live attenuated virus, which may not be suitable for all subjects, including pregnant women and persons with moderate or severe acute illnesses. Also, varicella is not suitable or approved for immunocompromised patients, including persons with immunosuppression due to leukemia, lymphoma, generalized malignancy, immune deficiency disease or immunosuppressive therapy. Likewise, persons with moderate or severe cellular immunodeficiency resulting from infection with human immunodeficiency virus (HIV) including those diagnosed with acquired immunodeficiency syndrome (AIDS) should not receive the varicella vaccine. Thus, despite the high risk of morbidity and mortality associated with herpes zoster in immunocompromised individuals, this population is not eligible for vaccination with a live attenuated vaccine, such as ZOSTAVAX®.

[0009] SUMMARY

[0010] In some aspects a varicella-zoster virus (VZV) vaccine comprising a varicella- zoster virus (VZV) vaccine comprising: a messenger ribonucleic acid (mRNA) comprising a 5’UTR, an open reading frame (ORF) encoding a VZV glycoprotein E (gE) protein variant comprising an amino acid sequence of SEQ ID NO: 2, wherein the ORF comprises a nucleic acid sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO: 1, a 3’ UTR, and a poly A tail, wherein all uridines in the mRNA are substituted with 1 -methylpseudouridine; and a lipid nanoparticle (LNP) is provided.

[0011] In some aspects a varicella-zoster virus (VZV) vaccine comprising a messenger ribonucleic acid comprising an open reading frame encoding a VZV glycoprotein E (gE) protein variant, wherein the open reading frame comprises a nucleic acid sequence having at least 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 1 and 20-1018; and a lipid nanoparticle is provided.

[0012] In some aspects, a method comprising: administering to a subject at least one dose of a VZV vaccine comprising: (i) a messenger ribonucleic acid (mRNA) comprising a 5’UTR, an open reading frame (ORF) encoding a VZV glycoprotein E (gE) protein variant comprising an amino acid sequence of SEQ ID NO: 2, wherein the ORF comprises a nucleic acid sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO: 1, a 3’ UTR, and a poly A tail, wherein all uridines in the mRNA are substituted with 1 -methylpseudouridine; and (ii) a lipid nanoparticle (LNP), in an effective amount to induce an immune response in the subject is provided. In some embodiments the subject has, or is at risk of, VZV infection.

[0013] In some embodiments the VZV vaccine is administered intramuscularly. In some embodiments a first dose and a second dose of the VZV vaccine are administered. In some embodiments the at least one dose comprises 10-100 pg of the mRNA, 10-50 pg of the mRNA, 10-40 pg of the mRNA, 10-30 pg of the mRNA, 30-40 pg of the mRNA, or 50- 100 pg of the mRNA.

[0014] In some embodiments the at least one dose is effective to produce a neutralizing antibody response to VZV gE. In some embodiments the at least one dose is effective to produce a CD4+and / or CD8+T cell response to VZV gE. In some embodiments the at least one dose is effective to produce antibodies with antibody-dependent cellular cytotoxicity (ADCC).

[0015] In some embodiments the mRNA vaccine comprises a second mRNA encoding a different VZV antigen. In some embodiments the ORF encodes at least two different VZV antigens.

[0016] In some aspects a method comprising administering to a subject at least one dose of a VZV vaccine comprising: a messenger ribonucleic acid comprising an open reading frame encoding a VZV glycoprotein E (gE) protein variant, wherein the open reading frame comprises a nucleic acid sequence having at least 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 1 and 20-1018; and a lipid nanoparticle is provided.

[0017] In some embodiments, the open reading frame comprises a nucleic acid sequence having at least 96%, at least 97%, at least 99%, or at least 99% identity to the nucleic acid sequence of SEQ ID NO: 1 or any one of SEQ ID NOs: 20-1018. In some embodiments, the open reading frame comprises the nucleic acid sequence of SEQ ID NO: 1 or any one of SEQ ID NOs: 20- 1018.

[0018] In some embodiments, the VZV gE protein variant has a length of no longer than 575, 574, or 573 amino acids.

[0019] In some embodiments, the VZV gE protein variant is truncated at its C-terminal, relative to a full length VZV gE protein. The full length protein is 623 amino acids in length (SEQ ID NO: 10).

[0020] In some embodiments, the VZV gE protein variant comprises a Y569A mutation, relative to a VZV gE protein comprising the amino acid sequence of SEQ ID NO: 10 and / or truncations thereof.

[0021] In some embodiments, the lipid nanoparticle comprises an ionizable amino lipid. In some embodiments, the lipid nanoparticle comprises a neutral lipid, optionally DSPC. In some embodiments, the lipid nanoparticle comprises a sterol lipid, optionally cholesterol. In some embodiments, the lipid nanoparticle comprises a polyethylene glycol (PEG)-modified lipid, optionally PEG2000-DMG. In some embodiments, the lipid nanoparticle comprises 30-60 mol% ionizable amino lipid, 5-20 mol% neutral lipid, 25-55 mol% sterol, and 1-5 mol% PEG-modified lipid. In some embodiments, the open reading frame comprises chemically-modified uracil. In some embodiments, the chemically-modified uracil comprises 1 -methylpseudouridine.

[0022] In some embodiments, the lipid nanoparticle comprises 40-55 mol% ionizable amino lipid, 30-45 mol% sterol, 5-15 mol% neutral lipid, and 1-5 mol% PEG-modified lipid.

[0023] In some embodiments, the ionizable amino lipid comprises a compound of Formula (I): or a salt or isomer thereof, wherein:

[0024] Ri is R”M’R’ or C5-20 alkenyl;

[0025] R2 and R3 are each independently selected from C1-14 alkyl and C2-14 alkenyl;

[0026] R4 is -(CH2)nQ, wherein Q is OH and n is selected from 3, 4, and 5;

[0027] M and M’ are each independently -OC(O)- or -C(O)O-;

[0028] Rs, Re, and R7 are each H;

[0029] R’ is a linear C1-12 alkyl, or C1-12 alkyl substituted with Ce-9 alkyl;

[0030] R” is C3-14 alkyl; m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0031] In some embodiments, the ionizable amino lipid comprises Compound 1: (Compound 1).

[0032] In some embodiments, the ionizable amino lipid comprises a compound of the structure

[0033] A7: (A7).

[0034] In some embodiments, the neutral lipid is 1,2 distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0035] In some embodiments, the sterol is cholesterol.

[0036] In some embodiments, the PEG-modified lipid is PEG2000-DMG.

[0037] In some embodiments, the open reading frame comprises one or more chemically modified nucleotides. In some embodiments, the open reading frame comprises N1 -methylpseudouridine.

[0038] In some embodiments, at least 80% of uracil nucleotides in the open reading frame comprise N1 -methylpseudouridine.

[0039] In some embodiments, 100% of uracil nucleotides in the open reading frame comprise Nl-methylpseudouridine.

[0040] In some embodiments, the open reading frame comprises 5-methylcytidine.

[0041] In some embodiments, at least 80% of cytosine nucleotides in the open reading frame comprise 5-methylcytidine.

[0042] In some embodiments, 100% of cytosine nucleotides in the open reading frame comprise 5 -methylcytidine .

[0043] In some embodiments, the open reading frame comprises 5-methyluridine.

[0044] In some embodiments, at least 80% of uracil nucleotides in the open reading frame comprise 5-methyluridine.

[0045] In some embodiments, 100% of uracil nucleotides in the open reading frame comprise 5- methyluridine.

[0046] In some aspects, the disclosure relates to a pharmaceutical composition comprising a lipid nanoparticle composition as described herein, and a pharmaceutically acceptable excipient.

[0047] In some aspects, the disclosure relates to a method comprising administering to a subject a composition as described herein.

[0048] In some embodiments, the composition is administered intramuscularly.

[0049] BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG. 1 is a schematic illustrating the structure of a naturally occurring (wild-type, WT) VZV gE and several exemplary modified antigens (gE variants) used in Example 1 to design mRNA vaccines.

[0051] FIG. 2 is a graph showing the gE titers following immunization with mRNA vaccines encoding gE variants (shown in (FIG. 1)) and control vaccines (SHINGRIX®, and VARIVAX®), and control PBS on Day 28 (d28), Day 42 (d42), Day 56 (d56) and Day 71 (d71). C57BL / 6J mice were immunized on a two-dose schedule with 2 pg of gE variants 28 days after receiving 1 dose of the VARIVAX® vaccine. gE-specific IgG binding antibody titers were determined by ELISA using recombinant gE coated at 1 pg / ml. Titers were determined using a four-parameter logistic curve fit in GraphPad Prism (GraphPad Software, Inc.) and defined as the reciprocal dilution at approximately OD450nm = 1.5 (normalized to a mouse standard on each plate). The y-axis depicts loglO gE-specific IgG titers. The line is the geometric mean value with 95% Confidence Interval. The horizontal dotted line indicates the lower limit of detection of the assay. FIGs. 3A-3B are graphs showing the gE specific T CD4+ profile following immunization with mRNA vaccines encoding gE variants (shown in FIG. 1) and control vaccines (SHINGRIX®, and VARIVAX®), and control PBS on day 42 (FIG. 3A) and day 71 (FIG. 3B). C57BL / 6J mice were immunized on a two-dose schedule with 2 pg of gE variants 28 days after receiving 1 dose of the VARIVAX® vaccine. gE specific T CD4+ response profile was determined by Intracellular Cytokine staining 6 hours after the incubation of splenocytes stimulated with 1 pg / mL of VZV glycoprotein E peptide pools (JPT). The following cytokines were analyzed: Interferon Gamma (IFNg), Interlukine-2 (IL-2), TNF-Alfa (TNFa), Interlukine-4 (IL-4), Interlukine-5 (IL-5), Interlukine-13 (IL- 13). The splenocytes were analyzed on the Cytek Aurora flow cytometer. The y-axis depicts % CD4+ T cells displaying a specific marker.

[0052] FIGs. 4A-4B are graphs showing the gE specific T CD8+ profile following immunization with mRNA vaccines encoding gE variants (shown in FIG. 1) and control vaccines (SHINGRIX®, and VARIVAX®), and control PBS on day 42 (FIG. 4A) and day 71 (FIG. 4B). C57BL / 6J mice were immunized on a two-dose schedule with 2 pg of mRNA coding for the gE variants, or the SHINGRIX® vaccine, 28 days after receiving 1 dose of the VARIVAX® vaccine. gE specific T CD8+ response profile was determined by Intracellular Cytokine staining 6 hours after the incubation of splenocytes stimulated with 1 pg / mL of VZV glycoprotein E peptide pools (JPT). The following cytokines were analyzed: Interferon Gamma (IFNg), Interlukine-2 (IL-2), TNF-Alfa (TNFa), and CD107a marker was used as a surrogate for CD8+ T cell activation. The splenocytes were analyzed on the Cytek Aurora flow cytometer. The y-axis depicts % CD8+ T cells displaying a specific marker.

[0053] FIG. 5 is a graph illustrating the gE specific Antibody-dependent cellular cytotoxicity (ADCC) activity of serum following immunization with mRNA vaccines encoding gE variants (shown in (FIG. 1)) and control vaccines (SHINGRIX®, and VARIVAX®), and control PBS on day 71. C57BL / 6J mice were immunized on a two-dose schedule with 2 pg of mRNA coding for the gE variants, or the SHINGRIX® vaccine, 28 days after receiving 1 dose of the VARIVAX® vaccine. gE specific ADCC activity of serum was determined by incubating the sera from each animal with CHO cells that express WT gE. After one hour of this incubation the target reporter cell line (Jurkat cells) (Promega) was added to each well and incubated for several hours. The readout of this interaction was accessed by the addition of Bio-Gio Luciferase Assay Reagent. After the addition the Bio-Gio Luciferase Assay Reagent, the Luciferase activity was accessed using the Firestar reader (Biotek). Responses to stimuli (x-axis) were measured using Area Under Curve (AUC) (y-axis). FIG. 6 is a graph showing the results of an ELISA assay. The y-axis measures loglO gE- specific IgG titers following immunization with gEs74-Y569A, using two different UTRs, at different dosages and control vaccine (SHINGRIX®) and control PBS on Day 21 and Day 36.

[0054] FIG. 7 is a graph showing the gE specific T CD4+ profile following immunization with gEs74-Y569A, using two different UTRs, at different dosages and control vaccine (SHINGRIX®) and control PBS. The following cytokines were analyzed: Interferon Gamma (IFNg), Interlukine-2 (IL-2), TNF-Alfa (TNFa), Interlukine-4 (IL-4), Interlukine-5 (IL-5), Interlukine-13 (IL- 13). The y-axis depicts % CD4+ T cells displaying a specific marker. 2-way ANOVA with Dunnett’s multiple comparisons test, GP-style P values are as follows: **** p<0.0001, *** p<0.0005, ** p< 0.005, * p <0.05.

[0055] FIG. 8 is a graph showing the gE specific T CD8+ profile following immunization with gEs74-Y569A, using two different UTRs, at different dosages and control vaccine (SHINGRIX®) and control PBS. The following cytokines were analyzed: Interferon Gamma (IFNg), Interlukine-2 (IL-2), TNF-Alfa (TNFa), and CD107a. The y-axis depicts % CD8+ T cells displaying a specific marker. 2way ANOVA with Dunnett’s multiple comparisons test, GP-style P values are as follows: **** p<0.0001, *** p<0.0005, ** p< 0.005, * p <0.05.

[0056] FIG. 9 is a graph showing the frequency of gE-specific class-switched B cells from spleens . Spleens from mice immunized with gEs74-Y569A, using two different UTRs, at different dosages and control vaccine (SHINGRIX®) and control PBS. * p<0.05, Mann- Whitney test

[0057] DETAILED DESCRIPTION

[0058] Effective optimized mRNA vaccines encoding VZV gE protein variants capable of producing strong immune responses on par with commercially available VZV vaccines are disclosed herein. It has been demonstrated that improved and optimized mRNA vaccines encoding specific gE protein variants are able to generate a significant immune response in vivo. The immune response is significant and to similar levels as, and under some conditions enhanced, relative to the commercially available SHINGRIX® vaccine. As shown in the data presented herein animals receiving the SHINGRIX® vaccine or the mRNA vaccine encoding the gE variant both induced substantial immune responses. Surprisingly, not only did the mRNA vaccine induce similar IgG levels to that of the SHINGRIX® vaccine, but also had significantly elevated IgG levels relative to the animals that received the VARIVAX® vaccine. Therefore, quite surprisingly, the mRNA vaccines disclosed herein encoding gE variants induce a potent antibody response comparable with the current SHINGRIX® vaccine and an enhanced antibody response relative to the VARIVAX® vaccine. mRNA has relatively low toxicity and provides a significant advantage in the field relative to vaccines such as SHINGRIX® vaccines, which are associated with side effects. In some embodiments the improved and optimized mRNA vaccines encode variant gE protein antigens. For instance, in some embodiments the antigen is one of the following: gEl- 540 mRNA (SEQ ID NO: 1094; a gE variant containing amino acids 1-540 of SEQ ID NO: 10, the wild-type, naturally occurring sequence of gE), gE574-Y569A (a gE variant containing amino acids 1-573 and a Y569A mutation; mRNA ORF SEQ ID NO: 1; protein SEQ ID NO: 2), gE dCT (SEQ ID NO: 1095; a gE variant containing amino acids 1-558 of SEQ ID NO: 10).

[0059] Varicella-zoster Virus Proteins

[0060] Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccines that include at least one RNA (e.g., mRNA) polynucleotide encoding a varicella zoster virus (VZV) antigen. There are at least five clades of varicella zoster virus (VZV). Clades 1 and 3 include European / North American strains; clade 2 includes Asian strains, especially from Japan; and clade 5 appears to be based in India. Clade 4 includes some strains from Europe, but its geographic origins need further clarification. Phylogenetic analysis of VZV genomic sequences resolves wild-type strains into 9 genotypes (El, E2, J, Ml, M2, M3, M4, VIII and IX). Sequence analysis of 342 clinical varicella and zoster specimens from 18 European countries identified the following distribution of VZV genotypes: El, 221 (65%); E2, 87 (25%); Ml, 20 (6%); M2, 3 (1%); M4, and 11 (3%). No M3 or J strains were observed. Of 165 clinical varicella and zoster isolates from Australia and New Zealand, 67 of 127 eastern Australian isolates were El, 30 were E2, 16 were J, 10 were Ml, and 4 were M2; 25 of 38 New Zealand isolates were El, 8 were E2, and 5 were Ml.

[0061] VZV is an alpha herpesvirus that exists as a spherical multilayered structure approximately 200 nm in diameter. The viral genome is surrounded by a protein capsid structure that is covered by an amorphous layer of tegument proteins. These two structures are surrounded by a lipid envelope that is studded with viral glycoproteins, each about 8 nm in length, that are displayed on the exterior of the virion, and encloses the 100 nm nucleocapsid which is comprised of 162 hexameric and pentameric capsomeres arranged in an icosahedral form. The tegument, which is comprised of virally -encoded proteins and enzymes, is located in the space between the nucleocapsid and the viral envelope. The viral envelope is acquired from host cell membranes and contains viral-encoded glycoproteins.

[0062] The VZV genome is a single, linear, duplex DNA molecule of 124,884 base pairs having at least 70 open reading frames. The genome has 2 predominant isomers, depending on the orientation of the S segment, P (prototype) and IS (inverted S), which are present with equal frequency for a total frequency of 90-95%. The L segment can also be inverted resulting in a total of four linear isomers (IL and ILS). The VZV genome is the smallest of the human herpesviruses and encodes at least 71 unique proteins (ORFO-ORF68) with three more opening reading frames (ORF69-ORF71) that duplicate earlier open reading frames (ORF64-62, respectively). Only a fraction of the encoded proteins form the structure of the virus particle. Among those proteins are nine glycoproteins: ORF5 (gK), 0RF9A (gN), ORF14 (gC), ORF31 (gB), ORF37 (gH), ORF50 (gM), ORF60 (gL), ORF67 (gl), and ORF68 (gE). The glycoproteins gE, gl, gB, gH, gK, gL, gC, gN, and gM function in different steps of the viral replication cycle. The most abundant glycoprotein found in infected cells, as well as in the mature virion, is glycoprotein E (gE, ORF 68), which is a major component of the virion envelope and is essential for viral replication. Glycoprotein I (gl, ORG 67) forms a complex with gE in infected cells, which facilitates the endocytosis of both glycoproteins and directs them to the trans-Golgi network (TGN) where the final viral envelope is acquired. Glycoprotein I (gl) is required within the TGN for VZV envelopment and for efficient membrane fusion during VZV replication. VZV gE and gl are found complexed together on the infected host cell surface. Glycoprotein B (ORF 31), which is the second most prevalent glycoprotein and thought to play a role in virus entry, binds to neutralizing antibodies. Glycoprotein H is thought to have a fusion function facilitating cell to cell spread of the virus. Antibodies to gE, gB, and gH are prevalent after natural infection and following vaccination and have been shown to neutralize viral activity in vitro.

[0063] Glycoprotein E (gE)

[0064] The Varicella- zoster virus Glycoprotein E (gE) is a viral glycoprotein that harbors two consensus N-glycosylation sites (Asn-X-Ser / Thr), and also contains a mucin-like domain (MLD), which is a peptide stretch that is rich in Pro, Ser, and Thr residues, and constitutes a target for mucin-type O-linked glycosylations. An example of a wild-type Varicella-zoster virus gE protein sequence is provided as SEQ ID NO: 10 (e.g., NCBI GenBank Accession No. Gene ID: 1487709).

[0065] In some embodiments, the VZV protein encoded by an open reading frame of the present disclosure is a VZV gE protein. In some embodiments, the VZV gE protein is a VZV gE protein variant, relative to a wild-type VZV gE protein. In some embodiments, the wild-type VZV gE protein comparator comprises an amino acid sequence of SEQ ID NO: 10 (full length wild type gE). The terms “naturally occurring” and “wild type” are used interchangeably herein to refer to a protein sequence that is comprised of the same type of amino acids found in a protein that is present in nature. A naturally occurring protein is not genetically (or otherwise) modified to substitute, remove, or add any amino acids relative to the protein found in nature. However, a naturally occurring or wild type protein may be produced by recombinant technology or other synthetic techniques. In some embodiments, the VZV gE protein variant is encoded by a mRNA comprising, consisting essentially of or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to or comprises the nucleotide sequence of SEQ ID NOs: 3 or 8. In some embodiments, the VZV gE protein variant is encoded by a mRNA comprising, consisting essentially of or consisting of a nucleotide sequence of about 99% identity to SEQ ID NOs: 3 or 8. In some embodiments, the VZV gE protein variant is encoded by a mRNA comprising , consisting essentially of or consisting of a nucleotide sequence of SEQ ID NOs: 3 or 8.

[0066] In some embodiments, the VZV gE protein variant is encoded by an mRNA having an open reading frame (ORF) comprising, consisting essentially of or consisting of a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity to or comprises the nucleotide sequence of any one of SEQ ID NOs: 1 and 20-1018. In some embodiments, the VZV gE protein variant is encoded by an mRNA having an ORF comprising, consisting essentially of or consisting of a nucleotide sequence of about 99% identity to any one of SEQ ID NOs: 1 and 20-1018. In some embodiments, the VZV gE protein variant is encoded by an mRNA having an ORF comprising, consisting essentially of or consisting of a nucleotide sequence of any one of SEQ ID NOs: 1 and 20-1018.

[0067] In some embodiments, the VZV gE protein variant comprises a C-terminal truncation (e.g., deletion of one or more amino acids) relative to the wild-type VZV gE protein. In some embodiments, the deletion is about 30-80 amino acids. In some embodiments, the deletion is about 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30- 60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50- 60, 60-100, 60-90, 60-80, 60-70, 80-100, 80-90, 90-100 amino acids. For example, the deletion may be a deletion of about 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 amino acids. In some embodiments, the VZV gE protein variant comprises a deletion of amino acids 574-623 with reference to amino acid position numbering of the VZV gE protein of SEQ ID NO: 10. In some embodiments, the VZV gE protein variant has a length of no longer than 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, or 580 amino acids. In some embodiments, the VZV gE protein variant has a length of no longer than 574 amino acids. In some embodiments, the VZV gE protein variant has a length of no longer than 573 amino acids.

[0068] In some embodiments, the VZV gE protein variant comprises a mutation. The mutation may be, for example, an amino acid substitution (e.g., point mutation), insertion (e.g., of one or more amino acids), deletion (e.g., of one or more amino acids), or indel. In some embodiments, the VZV gE protein variant comprises an amino acid substitution at a position corresponding to Y569 with reference to amino acid position numbering of the wild-type VZV gE protein of SEQ ID NO: 10. In some embodiments, the amino acid substitution at a position corresponding to Y569 is Y569A. In some embodiments, the VZV gE protein variant comprises a substitution at position Y569 (e.g., Y569A), and is lacking amino acids 574 or 574-623 and comprises or consists of a sequence of at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100% identity to SEQ ID NO: 2, and optionally having a length of 573 amino acids. In some embodiments, the VZV gE protein variant comprises or consists of a sequence of SEQ ID NO: 2 and having the legnth of SEQ ID NO. 2. In some embodiments the mRNA ORF encoding such a protein variant has a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 1 and 20-1018. In some embodiments, the VZV gE protein variant comprises or consists of the amino acid sequence encoded by the nucleic acid ORFs of any one of SEQ ID NOs: 1 and 20-1018. See, e.g., FIG. 1 “gE574-Y569A”.

[0069] The RNA (e.g., mRNA) of the present disclosure encode a VZV protein of interest, intended to raise an immune response to VZV infection. Thus, the VZV proteins of the present disclosure are antigenic, i.e., they are antigens. Antigenicity or immunogenicity is the ability to be specifically recognized by immune cells, e.g., those that produce antibodies generated as a result of an immune response to a given substance, such as a VZV protein produced by an mRNA molecule of the present disclosure. The mRNA vaccines of the invention encode a VZV protein capable of inducing an immune response (immunogenic compositions, e.g., causing an immune system to produce cellular responses and / or antibodies against the antigen). The compositions of the present disclosure do not comprise antigens per se, but rather comprise RNA (e.g., mRNA) that have an open reading frame encoding a protein antigen (referred to herein simply as a “VZV protein”) that once delivered to subject is expressed by cells in the subject. Delivery of the RNA (e.g., mRNA) is achieved by formulating the RNA in appropriate carriers or delivery vehicles (e.g., lipid nanoparticles) such that upon administration to cells, tissues or subjects, the RNA is taken up by cells which, in turn, express the protein(s) encoded by the RNA.

[0070] It should be understood that the term “protein” encompasses peptides and the term “antigen” encompasses antigenic fragments.

[0071] The vaccines of the present disclosure provide a unique advantage over traditional protein-based vaccination approaches in which protein antigens are purified or produced in vitro, e.g., recombinant protein production technologies. The vaccines of the present disclosure comprise RNA (e.g., mRNA) encoding the desired VZV protein antigen(s), which when introduced into the body, i.e., administered to a mammalian subject (for example a human) in vivo, cause the cells of the body to express the desired antigen(s). In order to facilitate delivery of the RNA (e.g., mRNA) to the cells of the body, the RNA is encapsulated in a lipid nanoparticle (LNP). Upon delivery and uptake by cells of the body, the RNA is translated in the cytosol and protein antigens are generated by the host cell machinery. The proteins are presented and elicit an adaptive humoral and cellular immune response. Neutralizing antibodies are directed against the expressed proteins, and hence the proteins are considered relevant target antigens for vaccine development.

[0072] Many proteins have a quaternary or three-dimensional structure, which includes more than one polypeptide or several polypeptide chains that associate into an oligomeric molecule. As used herein the term “subunit” refers to a single protein molecule, for example, a polypeptide or polypeptide chain resulting from processing of a nascent protein molecule, which subunit assembles (or “coassembles”) with other protein molecules (e.g., subunits or chains) to form a protein complex. Proteins can have a relatively small number of subunits and therefore be described as “oligomeric” or can consist of a large number of subunits and therefore be described as “multimeric”. The subunits of an oligomeric or multimeric protein may be identical, homologous or totally dissimilar and dedicated to disparate tasks.

[0073] Proteins or protein subunits can further comprise domains. As used herein, the term “domain” refers to a distinct functional and / or structural unit within a protein. Typically, a “domain” is responsible for a particular function or interaction, contributing to the overall role of a protein. Domains can exist in a variety of biological contexts. Similar domains (i.e., domains sharing structural, functional and / or sequence homology) can exist within a single protein or can exist within distinct proteins having similar or different functions. A protein domain is often a conserved part of a given protein tertiary structure or sequence that can function and exist independently of the rest of the protein or subunit thereof.

[0074] As used herein, the term “antigen” refers to a full-length protein or a portion, variant and / or truncation thereof, which in whole or in part is immunogenic (i.e., in context of non-self immune system). The antigen may or may not include one or more additional amino acids on the N or C terminus or as a branched chain. An epitope is a part of an antigen to which a receptor on an immune cell (e.g., a T cell receptor or an antibody or an antibody in secreted form) attaches. An epitope may be a peptide, for example, a 7-10 amino acid peptide, or a carbohydrate structure. The art describes protein antigens that are delivered to subjects or immune cells in isolated form, e.g., isolated proteins, however, the design, testing, validation, and production of protein antigens can be costly and time-consuming, especially when producing proteins at large scale. By contrast, mRNA technology is amenable to rapid design and testing of mRNA encoding a variety of antigens. Moreover, rapid production of mRNA coupled with formulation in appropriate delivery vehicles (e.g., lipid nanoparticles), can proceed quickly and can rapidly produce mRNA vaccines at large scale. Potential benefit also arises from the fact that antigens encoded by the mRNAs of the present disclosure are expressed by the cells of the subject, e.g., are expressed by the human body, and thus the subject, e.g., the human body, serves as the “factory” to produce the antigens which, in turn, elicits the desired immune response.

[0075] The vaccines, as provided herein, may include an RNA (e.g., mRNA) or multiple RNAs encoding two or more antigens of the same or different VZV strains. Also provided herein are combination vaccines that include RNA (e.g., mRNA) encoding one or more VZV antigens and one or more antigen(s) of a different organism. Thus, the vaccines of the present disclosure may be combination vaccines that target one or more antigens of the same strain / species, or one or more antigens of different strains / species, e.g., antigens that induce immunity to organisms that are found in the same geographic areas where the risk of VZV infection is high or organisms to which an individual is likely to be exposed to when exposed to VZV.

[0076] Variants of Wild-Type Varicella-Zoster Proteins

[0077] Some embodiments relate to mRNA encoding antigens having one or more mutations (e.g., substitutions) relative to a reference amino acid sequence and / or numbered according to a listed amino acid sequence, which may be referred to as variants. Protein variants are proteins (including full length proteins and peptides) that differ in their amino acid sequence relative to a wild-type, native, or reference amino acid sequence. A protein variant may possess one or more substitutions, deletions, and / or insertions at certain positions within its amino acid sequence, as compared to a wild-type, native, or reference amino acid sequence. In some embodiments, a protein variant has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to a wild-type, native, or reference sequence.

[0078] Some embodiments relate to amino acid or nucleotide sequences having a specified percentage sequence identity to a comparator amino acid or nucleotide sequence, respectively. The term “identity” refers to a relationship between the sequences of two or more polypeptides (e.g. antigens) or polynucleotides (nucleic acids), as determined by comparing the sequences. Identity also refers to the degree of sequence relatedness between or among sequences as determined by the number of matches between strings of two or more amino acid residues or nucleic acid residues. “Percent (%) identity” or “percent (%) sequence identity” as it applies to polypeptide or polynucleotide sequences is defined as the percentage of residues (amino acid residues or nucleic acid residues) in the candidate amino acid or nucleic acid sequence that are identical with the residues in the amino acid sequence or nucleic acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity. Thus, the term “identity” refers to a relationship between two or among three or more sequences (e.g., amino acid sequences or nucleotide sequences) as determined by comparing the sequences to each other. In some embodiments, the full length of the longer of the two or more sequences may be factored into the equation in the determination of sequence identity. For instance, when noted, percent identity may be calculated over the full length of the sequences, including gaps and sequences at either end of the nucleotide or amino acid sequences. In such instances factors such as the length of the nucleic acids or polypeptides may be factored in the calculation of percent identity.

[0079] The percent sequence identity that a candidate sequence (e.g., as present in a claimed protein or nucleic acid) has to a comparator sequence (e.g., having a SEQ ID NO: specified herein) is calculated by (i) aligning the candidate sequence to the comparator sequence, (ii) determining the number of matching residues (amino acids or nucleotides) between the aligned candidate and comparator sequences, and (iii) dividing the number of matching residues by the length of the comparator sequence, including any gaps introduced into the comparator sequence when the two sequences are aligned.

[0080] The skilled artisan will appreciate that to determine whether a candidate protein or nucleic acid comprises an amino acid sequence or nucleotide sequence with a given percentage sequence identity to a comparator sequence, the denominator (length of comparator sequence plus internal gaps) in calculating sequence identity need not include gaps shown at the ends of the comparator sequence in an alignment, as such gaps are added where a candidate sequence contains additional amino acids or nucleotides that extend beyond the portions that align to the N-terminal end and / or C-terminal end (amino acid sequences), or 5' end or 3' end (nucleotide sequences) of the comparator sequence.

[0081] Where an alignment between two sequences is contemplated, the first sequence (e.g., candidate sequence) is aligned to the second sequence (e.g., comparator sequence) using the Needleman- Wunsch algorithm for global alignment of the two sequences. Needleman & Wunsch, J Mol Biol. 1970. 48:443-453. Where two protein sequences are aligned, the Needleman- Wunsch algorithm uses a BLOSUM62 substitution scoring matrix, a Gap Open penalty of 10, a Gap Extend penalty of 0.5, and no End Gap penalties. Where two nucleotide sequences are aligned, the alignment uses an DNAFULL substitution scoring matrix, a Gap Open penalty of 10, a Gap Extend penalty of 0.5, and no End Gap penalties. The skilled artisan will appreciate that at the time of filing the instant specification, these parameters are the default parameters of the EMBOSS Needle pairwise comparison tool provided by European Bioinformatics Institute (see ebi.ac.uk). Other suitable alignment programs may be used to obtain a global alignment using these parameters, such as BLAST, or the Needleman- Wunsch algorithm may be implemented in a scripting language (e.g., Python).

[0082] In some embodiments, an amino acid substitution relative to a reference sequence is denoted by the following formula ZXZ’, wherein “Z” is the amino acid of the reference sequence, “X” indicates the amino acid position of the reference sequence (sequential with the first amino acid of the reference sequence being amino acid position 1), and “Z”’ is the amino acid that is substituted. For example, a T216A substitution of a given sequence would be understood as a substitution of the threonine (T) at amino acid position 216 of the given sequence with an alanine (A).

[0083] A protein variant encoded by an mRNA of the disclosure may contain amino acid changes that confer any of a number of desirable properties, for example, that enhance its immunogenicity, enhance its expression, and / or improve its stability or PK / PD properties in a subject. Protein variants can be made using routine mutagenesis techniques and assayed as appropriate to determine whether they possess the desired property. Assays to determine expression levels and immunogenicity of proteins, including protein variants, are well known in the art. Similarly, PK / PD properties of a protein variant can be measured using art recognized techniques, for example, by determining expression of the protein variant in a vaccinated subject over time and / or by looking at the durability of an induced immune response. The stability of a protein variant encoded by an RNA (e.g., mRNA) may be measured by assaying thermal stability or stability upon urea denaturation or may be measured using in silico prediction, for example. Methods for such experiments and in silico determinations are known in the art. Other methods for determining protein variant expression levels, immunogenicity and / or PK / PD properties of a protein variant may be used.

[0084] In some embodiments, an RNA (e.g., mRNA) comprises an open reading frame that comprises or consists of a nucleotide sequence of any one of the sequences provided herein or comprises or consists of a nucleotide sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a nucleotide sequence of any one of the sequences provided herein.

[0085] In some embodiments, an RNA (e.g., mRNA) comprises an open reading frame that encodes a protein comprising , consisting essentially of or consisting of an amino acid sequence of any one of the sequences provided herein or comprises or consists of an amino acid sequence that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the amino acid sequence of any one of the sequences provided herein. See, e.g., SEQ ID NOs: 2 and 10. As such, polynucleotides and polypeptides containing substitutions, insertions and / or deletions (e.g., indels), and covalent modifications with respect to wild-type, native, or reference sequence, for example, the polypeptide (e.g., protein) sequences disclosed herein, are included within the scope of this disclosure. For example, sequence tags or amino acids, such as one or more lysine(s), can be added to polypeptide sequences (e.g., at the N-terminal and / or C-terminal end). Sequence tags can be used for peptide detection, purification and / or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the N-terminal and / or C-terminal regions of the amino acid sequence of a protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal amino acids) may be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence that is soluble or linked to a solid support. In some embodiments, sequences for (or encoding) signal sequences, termination sequences, transmembrane domains, linkers, multimerization domains (e.g., foldon regions) and the like are substituted with alternative sequences that achieve the same or a similar function. In some embodiments, cavities in the core of proteins can be filled to improve stability, e.g., by introducing larger amino acids. In other embodiments, buried hydrogen bond networks are replaced with hydrophobic resides to improve stability. In yet other embodiments, glycosylation sites are removed and replaced with appropriate residues. Such sequences are readily identifiable to one of skill in the art. It should also be understood that some of the sequences provided herein contain sequence tags or terminal peptide sequences (e.g., at the N-terminal or C-terminal ends) that may be deleted, for example, prior to use in the preparation of an RNA (e.g., mRNA) vaccine.

[0086] As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of VZV proteins provided herein. For example, provided herein is any protein fragment of (meaning a polypeptide sequence at least one amino acid residue shorter than but otherwise identical to) a wild-type, native, or reference sequence, provided that the fragment is immunogenic and confers a protective immune response to VZV. In addition to protein variants that are identical to the wildtype, native, or reference protein but are truncated, in some embodiments, a protein includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations (e.g., substitutions, insertions and / or deletions), as shown in any of the sequences provided or referenced herein. Protein variants can range in length from about 4, 6, or 8 amino acids to full length proteins.

[0087] Non-limiting examples of VZV protein variants are provided in Table 5 and / or Appendix I of US Provisional application no. 63 / 518,096, which is incorporated by reference in its entirety. Signal Peptides

[0088] In some embodiments, an RNA (e.g., mRNA) has an ORF that encodes a signal peptide fused to the VZV protein. Signal peptides, comprising the N-terminal 15-60 amino acids of proteins, are typically needed for the translocation across the membrane on the secretory pathway and, thus, universally control the entry of most proteins both in eukaryotes and prokaryotes to the secretory pathway. In eukaryotes, the signal peptide of a nascent precursor protein (pre-protein) directs the ribosome to the rough endoplasmic reticulum (ER) membrane and initiates the transport of the growing peptide chain across it for processing. ER processing produces mature proteins, wherein the signal peptide is cleaved from precursor proteins, typically by an ER-resident signal peptidase of the host cell, or they remain uncleaved and function as a membrane anchor. A signal peptide may also facilitate the targeting of the protein to the cell membrane.

[0089] A signal peptide may have a length of 15-60 amino acids. For example, a signal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, a signal peptide has a length of 20-60, 25-60, 30-60, 35- 60, 40-60, 45- 60, 50-60, 55-60, 15-55, 20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50, 30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45, 15-40, 20- 40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30, 20-30, 25-30, 15-25, 20-25, or 15-20 amino acids.

[0090] Signal peptides from heterologous genes (which regulate expression of genes other than VZV proteins in nature) are known in the art and can be tested for desired properties and then incorporated into a nucleic acid of the disclosure. Exemplary signal peptides are shown in the sequence tables below.

[0091] Fusion Proteins

[0092] In some embodiments, an RNA (e.g., mRNA) encodes a fusion protein. Thus, an encoded protein may include two or more proteins (e.g., protein and / or protein fragment) joined together with or without a linker. Fusion proteins, in some embodiments, retain the functional property of each independent (nonfusion) protein.

[0093] In some embodiments, a fusion protein comprises 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the VZV proteins encoded by the nucleic acid ORFs: SEQ ID NOs: 1 or 10 or 20-1018. For example, in some embodiments, the RNA (e.g., mRNA) vaccines of the disclosure comprise an ORF that has at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% identity to the ORF of a sequence of any one of SEQ ID NOs: 1 and 20-1018. Linkers and Cleavable Peptides

[0094] In some embodiments, an RNA (e.g., mRNA) that encodes a fusion protein further encodes a linker located between at least one or each domain of the fusion protein. The linker may be, for example, a cleavable linker or protease-sensitive linker. In some embodiments, the linker is selected from the group consisting of F2A linker, P2A linker, T2A linker, E2A linker, and combinations thereof (see, e.g., WO 2017 / 127750). This family of self-cleaving peptide linkers, referred to as 2A peptides, has been described in the art (see, e.g., Kim, J.H. el al. PLoS ONE 2011;6:el8556). In some embodiments, the linker is an F2A linker.

[0095] In some embodiments, the linker is a GS linker. GS linkers are polypeptide linkers that include glycine and serine amino acids repeats. They comprise flexible and hydrophilic residues and can be used to perform fusion of protein subunits without interfering in the folding and function of the protein domains, and without formation of secondary structures. In some embodiments, an RNA (e.g., mRNA) encodes a fusion protein that comprises a GS linker that is 3 to 20 amino acids long. For example, the GS linker may have a length of (or have a length of at least) 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids. In some embodiments, a GS linker is (or is at least) 15 amino acids long (e.g., GGSGGSGGSGGSGGG (SEQ ID NO: 11)). In some embodiments, a GS linker is (or is at least) 8 amino acids long (e.g., GGGSGGGS (SEQ ID NO: 12)). In some embodiments, a GS linker is (or is at least) 7 amino acids long (e.g., GGGSGGG (SEQ ID NO: 13)). In some embodiments, a GS linker is (or is at least) 4 amino acid long (e.g., GGGS (SEQ ID NO: 14)). In some embodiments, the GS linker comprises (GGGS)n (SEQ ID NO: 15), where n is any integer from 1-5. In some embodiments, a GS linker is (or is at least) 4 amino acid long (e.g., GSGG (SEQ ID NO: 16)). In some embodiments, the GS linker comprises (GSGG)n (SEQ ID NO: 17), where n is any integer from 1-5. In some embodiments, a linker is a glycine linker, for example having a length of (or a length of at least) 3 amino acids (e.g., GGG). In some embodiments, a protein encoded by an RNA (e.g., mRNA) includes two or more linkers, which may be the same or different from each other.

[0096] The skilled artisan will appreciate that other art-recognized linkers may be suitable for use in the constructs of the disclosure (e.g., encoded by the nucleic acids of the disclosure). The skilled artisan will likewise appreciate that other polycistronic constructs (RNA (e.g., mRNA) encoding more than one protein separately within the same molecule) may be suitable for use as provided herein. Nucleic Acids Encoding Varicella-zoster Proteins

[0097] Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides. Nucleic acids may be or may include, for example, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA having a P-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'- amino functionalization), ethylene nucleic acid (ENA), cyclohexenyl nucleic acid (CeNA) and / or chimeras and / or combinations thereof.

[0098] RNA (e.g., mRNA) of the present disclosure comprises an open reading frame (ORF) encoding a VZV protein. In some embodiments, the RNA (e.g., mRNA) further comprises a 5' untranslated region (UTR), 3' UTR, a poly(A) tail and / or a 5' cap. In some embodiments, a 5’ cap is a natural cap or 5’ cap analog.

[0099] Messenger RNA

[0100] Messenger RNA (mRNA) is RNA that encodes a (at least one) protein (a naturally- occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. It is understood that, as used herein, the term mRNA does not include not self-amplifying RNA (saRNA) (see, e.g., Bloom K et al. Gene Therapy 2021; 28: 117-129 for a comparison of mRNA and saRNA), which is not mRNA with respect to the gene of interest. saRNAs include alphavirus replicase sequences that encode an RNA-dependent RNA polymerase. The mRNA compositions of the invention do not include alphavirus replicase sequences.

[0101] The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application may recite “T”s in a representative DNA sequence but where the sequence represents mRNA, the “T”s would be substituted for “U”s. Thus, any of the DNAs disclosed and identified by a particular sequence identification number herein also disclose the corresponding mRNA sequence complementary to the DNA, where each “T” of the DNA sequence is substituted with “U.”

[0102] Naturally-occurring eukaryotic mRNA molecules can contain stabilizing elements, including, but not limited to, UTRs at their 5 '-end (5' UTR) and / or at their 3 '-end (3' UTR), in addition to other structural features, such as a 5 '-cap structure or a 3'-poly(A) tail. Both the 5' UTR and the 3' UTR are typically transcribed from the genomic DNA and are elements of the premature mRNA. Characteristic structural features of mature mRNA, such as the 5 '-cap and the 3 '-poly (A) tail are usually added to the transcribed (premature) mRNA during mRNA processing. Exemplary sequences of mRNA that encode proteins as described herein are provided in Table 5 and / or Appendix I of US Provisional application no. 63 / 518,096. In some embodiments, the mRNA comprises an ORF that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or identical to a sequence provided in Table 5 and / or Appendix I of US Provisional application no. 63 / 518,096. In some embodiments, the mRNA comprises a nucleotide sequence that is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or identical to a sequence provided in Table 5 and / or Appendix I of US Provisional application no. 63 / 518,096.

[0103] Untranslated Regions (UTRs)

[0104] The mRNAs of the present disclosure may comprise one or more regions or parts which act or function as an untranslated region. A “5' untranslated region” (UTR) refers to a region of an mRNA that is directly upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a polypeptide. A “3' untranslated region” (UTR) refers to a region of an mRNA that is directly downstream (i.e., 3') from the open reading frame (e.g., downstream from the last amino acid-encoding codon of an open reading frame, where the stop codon is considered part of the 3' UTR, or downstream from the first stop codon signaling translation termination, where that stop codon is considered part of the open reading frame), and which does not encode a polypeptide. When RNA transcripts are being generated, the 5’ UTR may comprise a promoter sequence. Such promoter sequences are known in the art. It should be understood that such promoter sequences will not be present in a vaccine of the disclosure.

[0105] Where mRNAs encode a (at least one) protein, the mRNA may comprise a 5’ UTR and / or 3’ UTR. UTRs of an mRNA are transcribed but not translated. In mRNA, the 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon; the 3' UTR starts immediately following the open reading frame and continues until the transcriptional termination signal. Where an open reading frame ends with a codon encoding an amino acid, the 3' UTR begins with a stop codon, such that no amino acids are added to a polypeptide beyond the last amino acid encoded by the open reading frame. A 3' UTR may further comprise one or more stop codons. There is a growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the polynucleotides of the present disclosure to, among other things, enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites. A variety of 5’ UTR and 3’ UTR sequences are known.

[0106] In some embodiments, the 5' UTR comprises or consists of a sequence provided in Table

[0107] 6 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or comprises a 5' UTR sequence provided in Table 6, or a variant or a fragment thereof.

[0108] In some embodiments, the 3' UTR comprises or consists of a sequence provided in Table

[0109] 7 or a sequence with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity to or comprises a 3' UTR sequence provided in Table 7, or a variant or a fragment thereof.

[0110] It should also be understood that the mRNA of the present disclosure may include any 5’ UTR and / or any 3’ UTR. Exemplary UTR sequences are included herein; however, other UTR sequences may be used or exchanged for any of the UTR sequences described herein.

[0111] In some embodiments, a 5' UTR comprises or consists of a sequence selected from:

[0112] AGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUCG CAACUAGCAAGCUUUUUGUUCUCGCC (SEQ ID NO: 7), GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 1027), GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGACCCCGGCGCCGCC ACC (SEQ ID NO: 1028), GAGGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUCGCAA CUAGCAAGCUUUUUGUUCUCGCC (SEQ ID NO: 4), and

[0113] GGAAAUCGCAAAAUUUGCUCUUCGCGUUAGAUUUCUUUUAGUUUUCUCGCAACU AGCAAGCUUUUUGUUCUCGCC (SEQ ID NO: 1031). In some embodiments, a 3' UTR comprises or consists of, in 5 '-to-3 ' order: (a) the nucleic acid sequence

[0114] UAAAGCUCCCCGGGGGCCUCGGUGGCCUAGCUUCUUGCCCCUUGGGCCUCCCCCC AGCCCCUCCUCCCCUUCCUGCAG (SEQ ID NO: 1032), (b) an identification and ratio determination (IDR) sequence, and (c) the nucleic acid sequence UGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 1033). In some embodiments, each mRNA encoding a distinct protein (z.e., having a different amino acid sequence from proteins encoded by other mRNAs in a composition) comprises or consists of a 3' UTR comprising, in 5'-to-3 ' order: (a) the nucleotide sequence of SEQ ID NO: 1032; (b) a distinct IDR sequence; and (c) the nucleotide sequence of SEQ ID NO: 1033. IDR sequences are described herein in the section entitled “Identification and Ratio Determination (IDR)

[0115] Sequences.”

[0116] In some embodiments, a 5' UTR comprises or consists of a sequence derived from a 5' UTR of a gene selected from HSD17B4, RPL32, ASAHI, ATP5A1, MP68, NDUFA4, NOSIP, RPL31, SLC7A3, TUBB4B and UBQLN2. In some embodiments, the 5' UTR comprises or consists of a sequence derived from the 5' UTR of human hydroxy steroid 17-beta dehydrogenase 4 (HSD17B4). In some embodiments, a 5' UTR comprises or consists of the sequence GGGAGAGUCCCGCAGUCGGCGUCCAGCGGCUCUGCUUGUUCGUGUGUGUGUCGUU GCAGGCCUUAUUCAAGCUUACC (SEQ ID NO: 1034). In some embodiments, a 5' UTR comprises or consists of the sequence

[0117] GUCCCGCAGUCGGCGUCCAGCGGCUCUGCUUGUUCGUGUGUGUGUCGUUGCAGGC CUUAUUC (SEQ ID NO: 1035). In some embodiments, a 5' UTR comprises or consists of the sequence GGGAGAAAGCUUACC (SEQ ID NO: 1036). In some embodiments, a 3' UTR comprises or consists of a sequence derived from a 3' UTR of a gene selected from PSMB3, ALB7, alpha-globin, CASP1, COX6B1, GNAS, NDUFA1 and RPS9. In some embodiments, a 3' UTR comprises or consists of a sequence derived from a 3' UTR of PSMB3 (proteasome 20S subunit beta 3). In some embodiments, a 3' UTR comprises or consists of a sequence derived from a 3' UTR of alpha-globin (MUAG). In some embodiments, a 3' UTR comprises or consists of the sequence

[0118] AGGACUAGUCCCUGUUCCCAGAGCCCACUUUUUUUUCUUUUUUUGAAAUAAAAU AGCCUGUCUUUCAGAUCU (SEQ ID NO: 1037). In some embodiments, a 3' UTR comprises or consists of the sequence GGACUAGUUAUAAGACUGACUAGCCCGAUGGGCCUCCCAACGGGCCCUCCUCCCC UCCUUGCACCGAGAUUAAU (SEQ ID NO: 1038). In some embodiments, the mRNA comprises or consists of a 5' UTR comprising the nucleotide sequence of any one of SEQ ID NOs: 70-72, an open reading frame, one or more stop codons, and a 3' UTR comprising , consisting essentially of or consisting of the nucleotide sequence of SEQ ID NO: 1037 or SEQ ID NO: 1038. In some embodiments, the mRNA further comprises a polyA sequence comprising , consisting essentially of or consisting of at least 64 consecutive adenosine nucleotides. In some embodiments, the mRNA further comprises or consists of a polyC sequence comprising at least 30 consecutive cytidine nucleotides.

[0119] In some embodiments, a 5' UTR comprises or consists of the sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 1051). In some embodiments, a 5' UTR comprises or consists of the sequence GAGAAUAAACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 1039). In some embodiments, a 3' UTR comprises or consists of the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGA GUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACC ACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCU UAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACG AAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACA CC (SEQ ID NO: 1040). In some embodiments, a 3' UTR comprises or consists of the sequence CUCGAGCUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUA CCCCGAGUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCA CUCACCACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAA AACGCUUAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAA UAAACGAAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCC AGCCACACCCUGGAGCUAGC (SEQ ID NO: 1041). In some embodiments, a 3' UTR comprises or consists of the sequence CUGGUACUGCAUGCACGCAAUGCUAGCUGCCCCUUUCCCGUCCUGGGUACCCCGA GUCUCCCCCGACCUCGGGUCCCAGGUAUGCUCCCACCUCCACCUGCCCCACUCACC ACCUCUGCUAGUUCCAGACACCUCCCAAGCACGCAGCAAUGCAGCUCAAAACGCU UAGCCUAGCCACACCCCCACGGGAAACAGCAGUGAUUAACCUUUAGCAAUAAACG AAAGUUUAACUAAGCUAUACUAACCCCAGGGUUGGUCAAUUUCGUGCCAGCCACA CCCUGGAGCUAGC (SEQ ID NO: 1042). In some embodiments, an mRNA comprises or consists of a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 1051 or SEQ ID NO: 1039, an open reading frame, one or more stop codons, and a 3' UTR comprising the nucleotide sequence of any one of SEQ ID NOs: 1039-1041. In some embodiments, an mRNA comprises or consists of a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 1039, an open reading frame, one or more stop codons, and a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 1041. In some embodiments, an mRNA comprises or consists of a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 1039, an open reading frame, the nucleotide sequence UGAUGA, and a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 1041. In some embodiments, the mRNA further comprises or consists of two poly(A) sequences separated by an intervening nucleotide sequence. In some embodiments, the mRNA further comprises or consists of the nucleotide sequence of SEQ ID NO: 1042.

[0120] In some embodiments, a 5' UTR comprises or consists of the sequence GAGGAGACCCAAGCUACAUUUGCUUCUGACACAACUGUGUUCACUAGCAACCUCA AACAGACACCGCCACC (SEQ ID NO: 1043). In some embodiments, a 3' UTR comprises or consists of the sequence GCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCA ACUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUAA UAAAAAACAUUUAUUUUCAUUGC (SEQ ID NO: 1044). In some embodiments, an mRNA comprises or consists of a 5' UTR comprising the nucleotide sequence of SEQ ID NO: 1043, an open reading frame, one or more stop codons, and a 3' UTR comprising the nucleotide sequence of SEQ ID NO: 1044. In some embodiments, the mRNA further comprises or consists of a polyA tail comprising 109 consecutive adenosine nucleotides.

[0121] UTRs may also be omitted from the mRNA described herein. In some embodiments, other UTRs known in the art may be used in the mRNA described herein.

[0122] A 5' UTR does not encode a protein (is non-coding). Natural 5' UTRs have features that play roles in translation initiation. They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes. Kozak sequences have the consensus CCR(A / G)CCAUGG (SEQ ID NO: 1045), where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'.5'UTR also have been known to form secondary structures which are involved in elongation factor binding.

[0123] In some embodiments of the disclosure, a 5’ UTR is a heterologous UTR, i.e., is a UTR found in nature associated with a different ORE In other embodiments, a 5’ UTR is a synthetic UTR, i.e., does not occur in nature. Synthetic UTRs include UTRs that have been mutated to improve their properties, e.g., which increase gene expression as well as those which are completely synthetic. Exemplary 5’ UTRs include Xenopus or human derived a- globin or b- globin (8278063; 9012219), human cytochrome b-245 a polypeptide, and hydroxysteroid (17b) dehydrogenase, and Tobacco etch virus (US8278063, US9012219). CMV immediate-early 1 (IE1) gene (US2014 / 0206753, WO2013 / 185069), the sequence GGGAUCCUACC (SEQ ID NO: 1092) (WO 2014 / 144196) may also be used. In other embodiments, a 5' UTR is a 5' UTR of a TOP gene lacking the 5' TOP motif (the oligopyrimidine tract) (e.g., W02015 / 101414, W02015 / 101415, WO2015 / 062738, WO2015 / 024667, WO2015 / 024667); 5' UTR element derived from ribosomal protein Large 32 (L32) gene (WO / 2015101414, WO2015101415, WO / 2015 / 062738), 5' UTR element derived from the 5' UTR of an hydroxy steroid ( 17-|3) dehydrogenase 4 gene (HSD17B4) (WO201 / 5024667), or a 5' UTR element derived from the 5' UTR of ATP5A1 (WO2015 / 024667) can be used. In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5' UTR.

[0124] A 3' UTR does not encode a protein (is non-coding). Natural or wild type 3' UTRs are known to have stretches of adenosines and uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR binding and thus, stabilization of the message in vivo.

[0125] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of mRNA of the disclosure. When engineering specific nucleic acids, one or more copies of an ARE can be introduced to make nucleic acids of the disclosure less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using nucleic acids of the disclosure and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE- engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hours, 12 hours, 1 day, 2 days, and 7 days post-transfection.

[0126] Those of ordinary skill in the art will understand that 5’ UTRs that are heterologous or synthetic may be used with any desired 3’ UTR sequence. For example, a heterologous or synthetic 5’ UTR may be used with a synthetic 3’ UTR or with a heterologous 3’ UTR.

[0127] Non-UTR sequences may also be used as regions or subregions within a nucleic acid. For example, introns or portions of introns sequences may be incorporated into regions of nucleic acid of the disclosure. Incorporation of intronic sequences may increase protein production as well as nucleic acid levels.

[0128] Combinations of features may be included in flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and / or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly- A tail. A 5' UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes such as the 5' UTRs described in US2010 / 0293625 and WO2015 / 085318A2, each of which is herein incorporated by reference.

[0129] It should be understood that any UTR from any gene may be incorporated into the untranslated regions of a nucleic acid. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type or naturally occurring regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5' or 3' UTR may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3' UTR or 5' UTR may be altered relative to a wild-type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3' or 5') comprise a variant UTR.

[0130] In some embodiments, a double, triple or quadruple UTR such as a 5' UTR or 3' UTR may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3' UTR may be used as described in US2010 / 0129877, which is incorporated herein by reference.

[0131] It is also within the scope of the present disclosure to have patterned UTRs. As used herein “patterned UTRs” are those UTRs which reflect a repeating or alternating pattern, such as AB AB AB or AABB AABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C represent a different UTR at the nucleotide level.

[0132] In some embodiments, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature, or property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new polynucleotide. As used herein, a “family of proteins” is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.

[0133] The untranslated region may also include translation enhancer elements (TEE). As a nonlimiting example, the TEE may include those described in US 2009 / 0226470, herein incorporated by reference, and those known in the art.

[0134] Open Reading Frames

[0135] An open reading frame (ORF) is a continuous stretch of DNA or RNA that (1) begins with a start codon (e.g., ATG or AUG, encoding methionine), and (2) ends with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA) or is immediately followed by a stop codon. A stop codon does not encode an amino acid, such that translation of an ORF terminates when a ribosome reaches the stop codon immediately following the last amino acid-encoding codon in the ORF. A stop codon that results in translation termination may be considered part of the ORF, in which case the ORF ends with the stop codon. Alternatively, the first stop codon immediately following the last amino acid-encoding codon of an ORF may considered part of the 3' untranslated region (3' UTR) of a DNA or RNA, rather than part of the ORF. Those skilled in the art will understand that an ORF sequence that ends in a codon encoding amino acid will be followed by one or more stop codons in a DNA or RNA. An ORF may be followed by multiple stop codons. Inclusion of multiple consecutive stop codons reduces the extent of continued translation that may occur if a stop codon is mutated to a codon encoding an amino acid (readthrough), as a second stop codon may terminate translation even if a first stop codon is mutated and encodes an amino acid, such that only one amino acid is added to the C-terminus of the translated protein. Where multiple stop codons are present at the end, or immediately following, an ORF, the multiple stop codons may comprise the same stop codon (e.g., UGAUGA). Multiple stop codons may comprise different stop codons in series (e.g., UGAUAAUAG). In addition to reducing the extent of readthrough if a first stop codon is mutated, the presence of multiple different stop codons reduces the extent of readthrough if the first stop codon fails to allow translation termination (e.g., if a suppressor tRNA with an anticodon complementary to the first stop codon is present in the cell).

[0136] An ORF typically encodes a protein. It will be understood that the sequences disclosed herein may further comprise additional elements, e.g., 5’ and / or 3’ UTRs, but that those elements, unlike the ORF, need not necessarily be present in an RNA (e.g., mRNA) as described herein.

[0137] 5' End Capping

[0138] In some embodiments, an RNA (e.g., mRNA) comprises or consists of a 5' terminal cap. 5 '-capping of polynucleotides may be completed concomitantly during an in vitro transcription reaction using, for example, the following RNA caps to generate the 5 '-guanosine cap structure according to manufacturer protocols: 3'-O-Me-m7G(5')ppp(5') G [the ARCA cap];G(5')ppp(5')A; G(5')ppp(5')G; m7G(5')ppp(5')A; m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). 5'-capping of modified RNA (e.g., mRNA) may be completed post- transcriptionally using, for example, a Vaccinia Virus Capping Enzyme to generate the “Cap 0” structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2'-0 methyl-transferase to generate: m7G(5')ppp(5')G-2'-O-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-O-methylation of the 5 '-antepenultimate nucleotide using a 2'-0 methyltransferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-O- methylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase. Enzymes may be derived from a recombinant source. Other caps may be used. In some embodiments, the cap is a m7GpppGmAG cap. In some preferred embodiments, the cap is a m7GpppGm cap.

[0139] A cap may be, for example, a dinucleotide cap, a trinucleotide cap, or a tetranucleotide cap. In some embodiments, a cap is a dinucleotide cap. In some embodiments, a cap is a trinucleotide cap. In some embodiments, a cap is a tetranucleotide cap.

[0140] A nucleotide cap (e.g., a trinucleotide cap or tetranucleotide cap), in some embodiments, comprises a compound of formula (I) ring Bi is a modified or unmodified Guanine; ring B2 and ring B3 each independently is a nucleobase or a modified nucleobase;

[0141] X2 is O, S(O)P, NR24 or CR25R26 in which p is 0, 1, or 2;

[0142] Yo is O or CReR?;

[0143] Y1 is O, S(O)n, CR6R7, or NRs, in which n is 0, 1 , or 2; each — is a single bond or absent, wherein when each — is a single bond, Yi is O, S(O)n, CReR?, or NRs; and when each — is absent, Y 1 is void;

[0144] Y2 is (OP(O)R4)min which m is 0, 1, or 2, or -0-(CR4oR4i)u-Qo-(CR42R43)v-, in which Qo is a bond, O, S(O)r, NR44, or CR45R46, r is 0, 1 , or 2, and each of u and v independently is 1, 2, 3 or 4; each R2 and R2' independently is halo, LNA, or OR3; each R3 independently is H, C1-C6alkyl, C2-C6 alkenyl, or C2-C6 alkynyl and R3, when being C1-C6alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, is optionally substituted with one or more of halo, OH and C1-C6alkoxyl that is optionally substituted with one or more OH or OC(O)-Ci-C6 alkyl; each R4 and R4' independently is H, halo, C1-C6alkyl, OH, SH, SeH, or BH3'; each of R6, R7, and R8, independently, is -Q1-T1, in which Qi is a bond or C1-C3 alkyl linker optionally substituted with one or more of halo, cyano, OH and C1-C6alkoxy, and Ti is H, halo, OH, COOH, cyano, or Rsi, in which Rsiis C1-C3 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, Ci- Ce alkoxyl, C(O)O-C1-C6alkyl, C3-C8cycloalkyl, C6-C10aryl, NR31R32, (NRaiR32R33)+, 4 to 12- membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and Rsiis optionally substituted with one or more substituents selected from the group consisting of halo, OH, oxo, C1-C6alkyl, COOH, C(O)O-Ci-C6 alkyl, cyano, C1-C6alkoxyl, NR31R32, (NRaiR32R33)+, C3-C8cycloalkyl, C6-C10aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl; each of Rio, R11, R12, R13 R14, and R15, independently, is -Q2-T2, in which Q2 is a bond or C1-C3 alkyl linker optionally substituted with one or more of halo, cyano, OH and C1-C6alkoxy, and T2 is H, halo, OH, NH2, cyano, NO2, N3, Rs2, or ORS2, in which RS2 is C1-C6alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C3-C8cycloalkyl, C6-C10aryl, NHC(0)-CI-C6 alkyl, NR31R32, (NR3iRa2Raa)+, 4 to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and RS2 is optionally substituted with one or more substituents selected from the group consisting of halo, OH, oxo, C1-C6alkyl, COOH, C(O)O-C1-C6alkyl, cyano, Ci - Ce alkoxyl, NR31R32, (NR3iRa2Raa)+, C3-C8cycloalkyl, C6-C10aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6- membered heteroaryl; or alternatively R12 together with R14 is oxo, or R13 together with R15 is oxo, each of R20, R21, R22, and R23 independently is -Q3-T3, in which Q3 is a bond or C1-C3 alkyl linker optionally substituted with one or more of halo, cyano, OH and C1-C6alkoxy, and T3 is H, halo, OH, NH2, cyano, NO2, N3, Rsa, or ORs3, in which Rsa is C1-C6alkyl, C2- C6alkenyl, C2-C6alkynyl, C3-C8cycloalkyl, C6-C10aryl, NHC(O)-CI-C6 alkyl, mono-Ci- C6alkylamino, di-C1-C6alkylamino, 4 to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl, and RS3 is optionally substituted with one or more substituents selected from the group consisting of halo, OH, oxo, C1-C6alkyl, COOH, C(O)O-C1-C6alkyl, cyano, C1-C6alkoxyl, amino, mono-C1-C6alkylamino, di-C1-C6alkylamino, C3-C8cycloalkyl, C6-C10aryl, 4 to 12-membered heterocycloalkyl, and 5- or 6-membered heteroaryl; each of R24, R25, and R26 independently is H or C1-C6alkyl; each of R27 and R28independently is H or OR29; or R27 and R28together form O-R30-O; each R29 independently is H, C1-C6alkyl, C2-C6 alkenyl, or C2-C6 alkynyl and R29, when being C1-C6alkyl, C2-C6 alkenyl, or C2-C6 alkynyl, is optionally substituted with one or more of halo, OH and C1-C6alkoxyl that is optionally substituted with one or more OH or OC(O)-C1-C6alkyl;

[0145] R30 is C1-C6alkylene optionally substituted with one or more of halo, OH and C1-C6alkoxyl; each of R31, R32, and R33, independently is H, C1-C61lkyl, C3-C8cycloalkyl, C6-C10aryl, 4 to 12-membered heterocycloalkyl, or 5- or 6-membered heteroaryl; each of R40, R41, R42, and R43 independently is H, halo, OH, cyano, N3, OP(O)R47R48, or C1-C6alkyl optionally substituted with one or more OP(O)R47R48, or one R41 and one R43, together with the carbon atoms to which they are attached and Qo, form C4-C10cycloalkyl, 4- to 14-membered heterocycloalkyl, C6-C10aryl, or 5- to 14-membered heteroaryl, and each of the cycloalkyl, heterocycloalkyl, phenyl, or 5- to 6-membered heteroaryl is optionally substituted with one or more of OH, halo, cyano, N3, oxo, OP(O)R47R48, C1-C6alkyl, C1-C6haloalkyl, COOH, C(O)O-C1-C6alkyl, C1-C6alkoxyl, C1-C6haloalkoxyl, amino, mono-C1-C6alkylamino, and di-C1-C6alkylamino;

[0146] R44 is H, C1-C6alkyl, or an amine protecting group; each of R45 and R46 independently is H, OP(O)R47R48, or C1-C6alkyl optionally substituted with one or more OP(O)R47R48, and each of R47 and R48, independently is H, halo, C1-C6alkyl, OH, SH, SeH, or BH3 .

[0147] It should be understood that a cap, as provided herein, may include any of the caps described in international publication WO 2017 / 066797, published on 20 April 2017, incorporated by reference herein in its entirety.

[0148] In some embodiments, the B2 middle position can be a non-ribose molecule, such as arabinose.

[0149] In some embodiments R2 is ethyl-based.

[0150] Thus, in some embodiments, a tetranucleotide cap comprises the following structure:

[0151] In other embodiments, a tetranucleotide cap comprises the following structure:

[0152]

[0153] In yet other embodiments, a tetranucleotide cap comprises the following structure:

[0154] (VIII).

[0155] 5 In yet other embodiments, a tetranucleotide cap comprises the following structure:

[0156]

[0157] In some embodiments, R is an alkyl (e.g., C1-C6alkyl). In some embodiments, R is a methyl group (e.g., Ci alkyl). In some embodiments, R is an ethyl group (e.g., C2 alkyl). In some embodiments, R is a hydrogen. In some embodiments, a tetranucleotide cap comprises GGAG.

[0158] In some embodiments, a tetranucleotide cap comprises any one of the following structures:

[0159]

[0160] Poly adenylation Tailing

[0161] A “poly(A) tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR that contains multiple, consecutive adenosine monophosphates. A poly(A) tail may contain 10 to 300 adenosine monophosphates. It can, in some instances, comprise up to about 400 adenine nucleotides. For example, a poly(A) tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some embodiments, a poly(A) tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo) the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, and / or export of the mRNA from the nucleus and translation. In some embodiments, the length of the 3'-poly(A) tail may be an essential element with respect to the stability of the individual mRNA. In some embodiments, a poly(A) tail has a length of about 50, about 100, about 150, about 200, about 250, about 300, about 350, or about 400 nucleotides.

[0162] In some embodiments, a poly(A) tail has a length of 100 nucleotides. In some embodiments, an mRNA comprises a poly(A) sequence that has a length of 50- 75 nucleotides. In some embodiments, an mRNA comprises a poly (A) sequence that comprises 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70 consecutive adenosine nucleotides. In some embodiments, an mRNA comprises a poly(A) sequence comprising 64 consecutive adenosine nucleotides. In some embodiments, the consecutive adenosine nucleotides of a poly(A) sequence are flanked at the 5' and 3' end by nucleotides that are not adenosine nucleotides. In some embodiments, an mRNA comprises a poly(C) sequence, which may comprise 10 to 300 cytidine nucleotides. In some embodiments, the poly(C) sequence comprises 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive cytidine nucleotides. In some embodiments, the poly(C) sequence comprises 30 cytidine nucleotides. In some embodiments, the consecutive cytidine nucleotides of a poly(C) sequence are flanked at the 5' and 3' end by nucleotides that are not cytidine nucleotides.

[0163] In some embodiments, an mRNA comprises two poly(A) sequences separated by an intervening nucleotide sequence. In some embodiments, the intervening nucleotide sequence comprises no more than 3, no more than two, no more than 1, or no adenosine nucleotides. In some embodiments, the intervening sequence comprises 3 adenosine nucleotides. In some embodiments, the intervening sequence does not comprise an adenosine nucleotide. In some embodiments, the intervening sequence is no more than 30, no more than 25, no more than 20, no more than 15, or no more than 10 nucleotides long. In some embodiments, the intervening sequence consists of 10 nucleotides. In some embodiments, the intervening sequence comprises the sequence of GCAUAUGACU (SEQ ID NO: 1019). In some embodiments, the intervening sequence does not begin with an adenosine nucleotide, and does not end with an adenosine nucleotide. In some embodiments, the first poly(A) sequences comprises at least 15, at least 20, at least 25, or at least 30 consecutive adenosine nucleotides. In some embodiments, the second poly (A) sequences comprises at least 55, at least 60, at least 65, or at least 70 consecutive adenosine nucleotides. In some embodiments, the first poly(A) sequence comprises 30 consecutive adenosine nucleotides. In some embodiments, the second poly(A) sequence comprises 70 adenosine nucleotides. In some embodiments, an mRNA comprises the nucleotide sequence AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCAUAUGACUAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAA (SEQ ID NO: 1020).

[0164] In some embodiments, an mRNA comprises a poly(A) sequence that has a length of 90- 120 nucleotides. In some embodiments, an mRNA comprises a poly (A) sequence that comprises 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 190, or 120 consecutive adenosine nucleotides. In some embodiments, an mRNA comprises a poly(A) sequence that comprises at least 109 consecutive adenosine nucleotides. In some embodiments, an mRNA comprises a poly(A) sequence that comprises 109 consecutive adenosine nucleotides. In some embodiments, an mRNA comprises a poly(A) sequence that consists of 109 consecutive adenosine nucleotides.

[0165] Additional Stabilizing Elements

[0166] RNA (e.g., mRNA) provided herein, in some embodiments, includes an additional stabilizing element. Stabilizing elements may include, for example, a histone stem- loop. A stemloop binding protein (SLBP), a 32 kDa protein has been identified. It is associated with the histone stem-loop at the 3'-end of the histone messages in both the nucleus and the cytoplasm. Its expression level is regulated by the cell cycle; it peaks during the S-phase, when histone mRNA levels are also elevated. The protein has been shown to be essential for efficient 3'-end processing of histone pre-mRNA by the U7 snRNR SLBP continues to be associated with the stem-loop after processing, and then stimulates the translation of mature histone mRNAs into histone proteins in the cytoplasm. The RNA binding domain of SLBP is conserved through metazoa and protozoa; its binding to the histone stem-loop depends on the structure of the loop. The minimum binding site includes at least three nucleotides 5’ and two nucleotides 3D relative to the stem- loop.

[0167] In some embodiments, an RNA (e.g., mRNA) includes an open reading frame (coding region), a histone stem-loop, and optionally, a poly(A) sequence or polyadenylation signal. The poly(A) sequence or polyadenylation signal generally should enhance the expression level of the encoded protein. The encoded protein, in some embodiments, is not a histone protein, a reporter protein (e.g., Luciferase, GFP, EGFP, P-Galactosidase, EGFP), or a marker or selection protein (e.g., alpha-Globin, Galactokinase and Xanthine:guanine phosphoribosyl transferase (GPT)).

[0168] In some embodiments, an RNA (e.g., mRNA) includes the combination of a poly(A) sequence or polyadenylation signal and at least one histone stem-loop, even though both represent alternative mechanisms in nature, they act synergistically to increase the protein expression beyond the level observed with either of the individual elements. The synergistic effect of the combination of poly(A) and a histone stem- loop does not depend on the order of the elements or the length of the poly(A) sequence.

[0169] In some embodiments, an RNA (e.g., mRNA) does not include a histone downstream element (HDE). “Histone downstream element” (HDE) includes a purine-rich polynucleotide stretch of approximately 15 to 20 nucleotides 3' of naturally-occurring stem- loops, representing the binding site for the U7 snRNA, which is involved in processing of histone pre-mRNA into mature histone mRNA. In some embodiments, the nucleic acid does not include an intron. An RNA (e.g., mRNA) may or may not contain an enhancer and / or promoter sequence, which may be modified or unmodified or which may be activated or inactivated. In some embodiments, the histone stem-loop is generally derived from histone genes and includes an intramolecular base pairing of two neighbored partially or entirely reverse complementary sequences separated by a spacer, consisting of a short sequence, which forms the loop of the structure. The unpaired loop region is typically unable to base pair with either of the stem loop elements. It occurs more often in RNA, as is a key component of many RNA secondary structures but may be present in single- stranded DNA as well. Stability of the stem-loop structure generally depends on the length, number of mismatches or bulges, and base composition of the paired region. In some embodiments, wobble base pairing (non- Watson-Crick base pairing) may result. In some embodiments, the at least one histone stem-loop sequence comprises a length of 15 to 45 nucleotides.

[0170] In some embodiments, an RNA (e.g., mRNA) has one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES are destabilizing sequences found in the 3 ’UTR. The AURES may be removed from the mRNA. Alternatively, the AURES may remain in the mRNA.

[0171] Sequence Modification

[0172] In some embodiments, an open reading frame encoding a protein of the disclosure is codon optimized. Codon optimization methods are known in the art. An open reading frame of any one or more of the sequences provided herein may be codon optimized. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase RNA (e.g., mRNA) stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g., glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and RNA (e.g., mRNA) degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art - non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. In some embodiments, the open reading frame sequence is optimized using optimization algorithms.

[0173] In some embodiments, a codon optimized sequence shares less than 95% sequence identity to a naturally-occurring or wild-type sequence open reading frame (e.g., a naturally- occurring or wild-type RNA (e.g., mRNA) sequence encoding a VZV protein antigen). In some embodiments, a codon optimized sequence shares less than 90% sequence identity to a naturally- occurring or wild-type sequence (e.g., a naturally-occurring or wild-type RNA (e.g., mRNA) sequence encoding a VZV protein). In some embodiments, a codon optimized sequence shares less than 85% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally- occurring or wild-type RNA (e.g., mRNA) sequence encoding a VZV protein). In some embodiments, a codon optimized sequence shares less than 80% sequence identity to a naturally- occurring or wild-type sequence (e.g., a naturally-occurring or wild-type RNA (e.g., mRNA) sequence encoding a VZV protein). In some embodiments, a codon optimized sequence shares less than 75% sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally- occurring or wild-type RNA (e.g., mRNA) sequence encoding a VZV protein).

[0174] In some embodiments, a codon optimized sequence shares between 65% and 85% (e.g., between about 67% and about 85% or between about 67% and about 80%) sequence identity to a naturally-occurring or wild-type sequence (e.g., a naturally-occurring or wild-type RNA (e.g., mRNA) sequence encoding a VZV protein). In some embodiments, a codon optimized sequence shares between 65% and 75% or about 80% sequence identity to a naturally-occurring or wildtype sequence (e.g., a naturally-occurring or wild-type RNA (e.g., mRNA) sequence encoding a VZV protein).

[0175] In some embodiments, a codon-optimized sequence encodes an antigen that is as immunogenic as, or more immunogenic than (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 100%, or at least 200% more), than a VZV protein encoded by a non-codon-optimized sequence.

[0176] When transfected into mammalian host cells, the modified mRNAs have a stability of between 12-18 hours, or greater than 18 hours, e.g., 24, 36, 48, 60, 72, or greater than 72 hours and are capable of being expressed by the mammalian host cells.

[0177] In some embodiments, a codon optimized RNA (e.g., mRNA) may be one in which the levels of G / C are enhanced. The G / C-content of nucleic acid molecules (e.g., mRNA) may influence the stability of the RNA. RNA (e.g., mRNA) having an increased amount of guanine (G) and / or cytosine (C) residues may be functionally more stable than RNA containing a large amount of adenine (A) and thymine (T) or uracil (U) nucleotides. As an example, WO02 / 098443 discloses a pharmaceutical composition containing an RNA (e.g., mRNA) stabilized by sequence modifications in the translated region. Due to the degeneracy of the genetic code, the modifications work by substituting existing codons for those that promote greater RNA stability without changing the resulting amino acid. The approach is limited to coding regions of the RNA (e.g., mRNA). Some embodiments of mRNAs described herein, comprise a sequence with a %G / C content of 30% - 80%, 40% - 70%, 50% - 60%, 35% - 50%, 50% - 65%, 65% - 70%, 40% - 45%, 45% - 50%, 50% - 55%, 55% - 70%, 70% - 75%, or 75% - 80%. In some embodiments, the nucleic acid sequence of the full-length mRNA comprises a %G / C content of 30% to 80%, 40% - 70%, 50% - 60%, 35% - 50%, 50% - 65%, 65% - 70%, 40% - 45%, 45% - 50%, 50% - 55%, 55% - 70%, 70% - 75%, or 75% - 80%. In some embodiments, the mRNA comprises an ORF with a %G / C content from about 30% to about 80%, about 35% to about 70%, about 40% to about 60%, about 45% to about 55%, about 40% to about 70%, about 50% to about 60%, about 35% to about 50%, about 50% to about 50% to about 65%, about 65% to about 70%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 70%, about 70% to about 75%, or about 75% to about 80%. In some embodiments, the mRNA comprises 5' UTR with a %G / C content from about 30% to about 80%, about 35% to about 70%, about 40% to about 60%, about 45% to about 55%, about 40% to about 70%, about 50% to about 60%, about 35% to about 50%, about 50% to about 50% to about 65%, about 65% to about 70%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 70%, about 70% to about 75%, or about 75% to about 80%. In some embodiments, the mRNA comprises 3' UTR with a %G / C content from about 30% to about 80%, about 35% to about 70%, about 40% to about 60%, about 45% to about 55%, about 40% to about 70%, about 50% to about 60%, about 35% to about 50%, about 50% to about 50% to about 65%, about 65% to about 70%, about 40% to about 45%, about 45% to about 50%, about 50% to about 55%, about 55% to about 70%, about 70% to about 75%, or about 75% to about 80%. In some embodiments, a modified mRNA made by a method described herein comprises a higher %G / C content than a wild-type mRNA sequence. In some embodiments, the %G / C content of the modified mRNA sequence is 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 12% or more, 15% or more, or 20% or more than the %G / C content of the wild-type RNA sequence. In some embodiments, the %G / C content of the modified ORF sequence is 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 12% or more, 15% or more, or 20% or more than the %G / C content of the wild-type ORF sequence. In some embodiments, the %G / C content of the modified 5' UTR sequence is 2% or more, 3% or more, 4% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 12% or more, 15% or more, or 20% or more than the %G / C content of the wild-type 3' UTR sequence.

[0178] Chemically Modified Nucleotides

[0179] The compositions of the present disclosure comprise, in some embodiments, an RNA having an open reading frame encoding a protein, wherein the nucleic acid comprises nucleotides and / or nucleosides that can be standard (unmodified) or modified as is known in the art. In some embodiments, nucleotides and nucleosides of the present disclosure comprise modified nucleotides or nucleosides. Such modified nucleotides and nucleosides can be naturally- occurring modified nucleotides and nucleosides or non-naturally-occurring modified nucleotides and nucleosides. Such modifications can include those at the sugar, backbone, or nucleobase portion of the nucleotide and / or nucleoside as are recognized in the art.

[0180] In some embodiments, a naturally-occurring modified nucleotide or nucleotide of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such naturally-occurring modified nucleotides and nucleotides can be found, inter alia, in the widely recognized MODOMICS database.

[0181] In some embodiments, a non-naturally-occurring modified nucleotide or nucleoside of the disclosure is one as is generally known or recognized in the art. Non-limiting examples of such non-naturally-occurring modified nucleotides and nucleosides can be found, inter alia, in international publication numbers WO2013052523A1; WO2014093924 Al; WO2015051173A2; WO2015051169A2; WO2015089511A2; or WO2017153936A1, each of which is herein incorporated by reference.

[0182] Hence, nucleic acids of the disclosure (e.g., DNA and RNA, such as mRNA) can comprise standard nucleotides and nucleosides, naturally-occurring nucleotides and nucleosides, non-naturally-occurring nucleotides and nucleosides, or any combination thereof.

[0183] Nucleic acids of the disclosure (e.g., DNA and RNA, such as mRNA), in some embodiments, comprise various (more than one) different types of standard and / or modified nucleotides and nucleosides. In some embodiments, a particular region of a nucleic acid contains one, two or more (optionally different) types of standard and / or modified nucleotides and nucleosides.

[0184] In some embodiments, a modified RNA (e.g., mRNA) introduced to a cell or organism, exhibits reduced degradation in the cell or organism, respectively, relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

[0185] In some embodiments, a modified RNA (e.g., mRNA) introduced into a cell or organism, may exhibit reduced immunogenicity in the cell or organism, respectively (e.g., a reduced innate response) relative to an unmodified nucleic acid comprising standard nucleotides and nucleosides.

[0186] Nucleic acids (e.g., RNA, such as mRNA), in some embodiments, comprise non-natural modified nucleotides that are introduced during synthesis or post-synthesis of the nucleic acids to achieve desired functions or properties. The modifications may be present on intemucleotide linkages, purine or pyrimidine bases, or sugars. The modification may be introduced with chemical synthesis or with a polymerase enzyme at the terminal of a chain or anywhere else in the chain. Any of the regions of a nucleic acid may be chemically modified.

[0187] The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids). A “nucleoside” refers to a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as “nucleobase”). A “nucleotide” refers to a nucleoside, including a phosphate group. Modified nucleotides may by synthesized by any useful method, such as, for example, chemically, enzymatically, or recombinantly, to include one or more modified or non-natural nucleosides. Nucleic acids can comprise a region or regions of linked nucleosides. Such regions may have variable backbone linkages. The linkages can be standard phosphodiester linkages, in which case the nucleic acids would comprise regions of nucleotides.

[0188] Modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures, such as, for example, in those nucleic acids having at least one chemical modification. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. Any combination of base / sugar or linker may be incorporated into nucleic acids of the present disclosure.

[0189] In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 1-methyl-pseudouridine (mly), 1-ethyl-pseudouridine (c I \| / ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), and / or pseudouridine (y). In some embodiments, modified nucleobases in nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids) comprise 5-methoxymethyl uridine, 5-methylthio uridine, 1-methoxymethyl pseudouridine, 5-methyl cytidine, and / or 5-methoxy cytidine. In some embodiments, the polyribonucleotide includes a combination of at least two (e.g., 2, 3, 4 or more) of any of the aforementioned modified nucleobases, including but not limited to chemical modifications.

[0190] In some embodiments, a mRNA of the disclosure comprises 1-methyl-pseudouridine (m I \| / ) substitutions at one or more or all uridine positions of the nucleic acid.

[0191] In some embodiments, a mRNA of the disclosure comprises 1-methyl-pseudouridine (m I \| / ) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid. In some embodiments, a mRNA of the disclosure comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid.

[0192] In some embodiments, a mRNA of the disclosure comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid and 5-methyl cytidine substitutions at one or more or all cytidine positions of the nucleic acid.

[0193] In some embodiments, a mRNA of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.

[0194] In some embodiments, mRNAs are uniformly modified (e.g., fully modified, modified throughout the entire sequence) for a particular modification. For example, a nucleic acid can be uniformly modified with 1-methyl-pseudouridine, meaning that all uridine residues in the mRNA sequence are replaced with 1-methyl-pseudouridine. Similarly, a nucleic acid can be uniformly modified for any type of nucleoside residue present in the sequence by replacement with a modified residue such as those set forth above.

[0195] The nucleic acids of the present disclosure may be partially or fully modified along the entire length of the molecule. For example, one or more or all or a given type of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may be uniformly modified in a nucleic acid of the disclosure, or in a predetermined sequence region thereof (e.g., in the mRNA including or excluding the poly (A) tail). In some embodiments, all nucleotides X in a nucleic acid of the present disclosure (or in a sequence region thereof) are modified nucleotides, wherein X may be any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.

[0196] The nucleic acid may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e., any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%). It will be understood that any remaining percentage is accounted for by the presence of unmodified A, G, U, or C. The mRNAs may contain at a minimum 1% and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50% modified nucleotides, at least 80% modified nucleotides, or at least 90% modified nucleotides. For example, the nucleic acids may contain a modified pyrimidine such as a modified uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil (e.g., a 5-substituted uracil). The modified uracil can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the nucleic acid is replaced with a modified cytosine (e.g., a 5-substituted cytosine). The modified cytosine can be replaced by a compound having a single unique structure or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).

[0197] In some embodiments, an mRNA includes N1 -methylpseudouridine. In some embodiments, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of uracil nucleotides in an mRNA comprise N1 -methylpseudouridine. In some embodiments, each uracil nucleotide of an mRNA transcript comprises Nl- methylpseudouridine. In some embodiments, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of uracil nucleotides in an mRNA are substituted with N1 -methylpseudouridine. In some embodiments, each uracil nucleotide of an mRNA transcript is substituted with N1 -methylpseudouridine. . In some embodiments, each uracil nucleotide of an mRNA transcript comprises N1 -methylpseudouridine.

[0198] In some embodiments, an mRNA includes 5 -methylcytidine. In some embodiments, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of cytosine nucleotides in an mRNA comprise 5-methylcytidine. In some embodiments, each cytosine nucleotide of an mRNA transcript comprises 5-methylcytidine. In some embodiments, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of cytosine nucleotides in an mRNA are substituted with 5- methylcytidine. In some embodiments, each cytosine nucleotide of an mRNA transcript is substituted with 5-methylcytidine.

[0199] In some embodiments, an mRNA includes 5-methyluridine. In some embodiments, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of uracil nucleotides in an mRNA comprise 5-methyluridine. In some embodiments, each uracil nucleotide of an mRNA transcript comprises 5-methyluridine. In some embodiments, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of uracil nucleotides in an mRNA are substituted with 5- methyluridine. In some embodiments, each uracil nucleotide of an mRNA transcript is substituted with 5 -methyluridine.

[0200] In some embodiments, an mRNA includes 5-methylcytidine and 5 -methyluridine. In some embodiments, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of uracil nucleotides in an mRNA comprise 5 -methyluridine and at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of cytosine nucleotides in an mRNA comprise 5-methylcytidine. In some embodiments, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of uracil nucleotides in an mRNA are substituted with 5-methyluridine and at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% of cytosine nucleotides in an mRNA are substituted with 5-methylcytidine.

[0201] In some embodiments, each cytosine nucleotide of an mRNA transcript comprises 5- methylcytidine and each uracil nucleotide of an mRNA transcript comprises 5-methyluridine. In some embodiments, each cytosine nucleotide of an mRNA transcript is substituted with 5- methylcytidine and each uracil nucleotide of an mRNA transcript is substituted with 5- methyluridine.

[0202] Unmodified Nucleotides

[0203] In some embodiments, an RNA (e.g., mRNA) is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine. In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard nucleoside residues such as those present in transcribed RNA (e.g., A, G, C, or U). In some embodiments, nucleotides and nucleosides of the present disclosure comprise standard deoxyribonucleosides such as those present in DNA (e.g., dA, dG, dC, or dT).

[0204] In Vitro Transcription (IVT) cDNA encoding the polynucleotides described herein may be transcribed using an in vitro transcription (IVT) system. In vitro transcription of RNA is known in the art and is described in International Publication WO 2014 / 152027, which is incorporated by reference herein in its entirety. In some embodiments, the RNA of the present disclosure is prepared in accordance with any one or more of the methods described in WO 2018 / 053209 and WO 2019 / 036682, each of which is incorporated by reference herein.

[0205] In some embodiments, the RNA transcript is generated using a non- amplified, linearized DNA template in an in vitro transcription reaction to generate the RNA transcript. In some embodiments, the template DNA is isolated DNA. In some embodiments, the template DNA is cDNA. In some embodiments, the cDNA is formed by reverse transcription of a RNA polynucleotide, for example, but not limited to influenza virus mRNA. In some embodiments, cells, e.g., bacterial cells, e.g., E. coli, e.g., DH-1 cells are transfected with the plasmid DNA template. In some embodiments, the transfected cells are cultured to replicate the plasmid DNA which is then isolated and purified. In some embodiments, the DNA template includes a RNA polymerase promoter, e.g., a T7 promoter located 5 ' to and operably linked to the gene of interest.

[0206] In some embodiments, an in vitro transcription template encodes a 5' untranslated (UTR) region, contains an open reading frame, and encodes a 3' UTR and a poly(A) tail. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.

[0207] An in vitro transcription system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase.

[0208] The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs.

[0209] Any number of RNA polymerases or variants may be used in the method of the present disclosure. The polymerase may be selected from, but is not limited to, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNA polymerase, and / or mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids and / or modified nucleotides, including chemically modified nucleic acids and / or nucleotides. Some embodiments exclude the use of DNase.

[0210] In some embodiments, the RNA transcript is capped via enzymatic capping. In some embodiments, the RNA comprises 5' terminal cap, for example, 7mG(5’)ppp(5’)NlmpNp.

[0211] In some embodiments, the RNA polymerase is an RNA polymerase variant, such as those described in WO 2020 / 172239, incorporated herein by reference in its entirety. RNA polymerase variants of the present disclosure include at least one amino acid substitution, relative to the wild type (WT) RNA polymerase. A WT T7 RNA polymerase is represented by SEQ ID NO: 1021 :

[0212] MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFERQLKAGEVADNAAAK PLI TTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPEAVAYI TIKTTLACLTSADNTTVQAVASAIGRAIEDE ARFGRI RDLEAKHFKKNVEEQLNKRVGHVYKKAFMQWEADMLSKGLLGGEAWS S WHKED S I HVGVRC I EMLI E S TG MVSLHRQNAGWGQDSETIELAPEYAEAIATRAGALAGI SPMFQPCWPPKPWTGI TGGGYWANGRRPLALVRTHSK KALMRYEDVYMPEVYKAINIAQNTAWKINKKVLAVANVI TKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKR AAAAVYRKDKARKSRRI SLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTKGLLTLAKGKP IGKEG YYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPLENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLP LAFDGSCSGIQHFSAMLRDEVGGRAVNLLP SETVQDIYGIVAKKVNEILQADAINGTDNEWTVTDENTGEI SEKVK LGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGKGLMFTQPNQAAGYMAKLIWESVSV TWAAVEAMNWLKSAAKLLAAEVKDKKTGEILRKRCAVHWVTPDGFPVWQEYKKP IQTRLNLMFLGQFRLQPTINTN KDSEIDAHKQESGIAPNFVHSQDGSHLRKTWWAHEKYGIESFALIHDSFGTIPADAANLFKAVRETMVDTYESCDV LADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA (SEQ ID NO: 1021) .

[0213] For example, with reference to WT T7 RNA polymerase having an amino acid sequence of SEQ ID NO: 1021, the glycine at position 47 is considered a “wild-type amino acid,” whereas a substitution of the glycine for alanine at position 47 is considered an “amino acid substitution” that has a high-helix propensity. In some embodiments, the RNA polymerase variant is a T7 RNA polymerase variant comprising at least one (one or more) amino acid substitution relative to WT RNA polymerase (e.g., WT T7 RNA polymerase having an amino acid sequence of SEQ ID NO: 1021.

[0214] In some embodiments, a RNA polymerase variant comprises a RNA polymerase that includes an (at least one) amino acid modification causes a loop structure of the RNA polymerase variant to undergo a conformational change to a helix structure as the RNA polymerase variant transitions from an initiation complex to an elongation complex. In some embodiments, the amino acid modification is an amino acid substitution at one or more of positions 42, 43, 44, 45, 46, and 47, relative to the wild-type RNA polymerase, wherein the wildtype RNA polymerase comprises the amino acid sequence of SEQ ID NO: 1021. The amino acid substitution, in some embodiments, is a high propensity amino acid substitution. Examples of high-helix propensity amino acids include alanine, isoleucine, leucine, arginine, methionine, lysine, glutamine, and / or glutamate. In some embodiments, the amino acid substitution at position 47 is G47A.

[0215] In some embodiments, a RNA polymerase variant comprise a RNA polymerase that includes an additional C-terminal amino acid, relative to the wild-type RNA polymerase. The additional C-terminal amino acid, in some embodiments, is selected from glycine, alanine, threonine, proline, glutamine, and serine. In some embodiments, the additional C-terminal amino acid (e.g., at position 884 relative to wild-type RNA polymerase comprising the amino acid sequence of SEQ ID NO: 1021) is glycine.

[0216] Also provided herein are co-transcriptional capping methods for ribonucleic acid (RNA) synthesis, using any of the RNA polymerase variants described herein. That is, RNA is produced in a “one-pot” reaction, without the need for a separate capping reaction. Thus, the methods, in some embodiments, comprise reacting a polynucleotide template with a RNA polymerase variant, nucleoside triphosphates, and a cap under in vitro transcription reaction conditions to produce RNA transcript. Multivalent Vaccines

[0217] The compositions, as provided herein, may include RNA (e.g., mRNA) or multiple RNAs (e.g., mRNAs) encoding two or more antigens of the same or different species. In some embodiments, composition includes an RNA (e.g., mRNA) or multiple RNAs (e.g., mRNAs) encoding two or more VZV proteins. In some embodiments, the RNA (e.g., mRNA) may encode 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or more VZV proteins.

[0218] In some embodiments, two or more different RNA (e.g., mRNA) encoding antigens may be formulated in the same lipid nanoparticle. In other embodiments, two or more different RNA (e.g., mRNA) encoding antigens may be formulated in separate lipid nanoparticles (each RNA (e.g., mRNA) formulated in a single lipid nanoparticle). Lipid nanoparticles may then be combined and administered as a single vaccine composition (e.g., comprising multiple RNAs (e.g., mRNAs) encoding multiple antigens) or may be administered separately.

[0219] Identification and Ratio Determination (IDR) Sequences

[0220] An Identification and Ratio Determination (IDR) sequence is a sequence of a biological molecule (e.g., nucleic acid or protein) that, when combined with the sequence of a target biological molecule, serves to identify the target biological molecule. Typically, an IDR sequence is a heterologous sequence that is incorporated within or appended to a sequence of a target biological molecule and can be used as a reference to identify the target molecule. Thus, in some embodiments, a nucleic acid (e.g., mRNA) comprises (i) a target sequence of interest (e.g., a coding sequence encoding a therapeutic and / or antigenic peptide or protein); and optionally (ii) a unique IDR sequence. In all embodiments the IDR sequence is optional and not required unless otherwise specified.

[0221] An RNA species (e.g., RNA having a given coding sequence) may comprise an IDR sequence that differs from the IDR sequence of other RNA species (e.g., RNA(s) having different coding sequence(s)). Each IDR sequence thus identifies a particular RNA species, and so the abundance of IDR sequences may be measured to determine the abundance of each RNA species in a composition. Use of distinct IDR sequences to identify RNA species allows for analysis of multivalent RNA compositions (e.g., containing multiple RNA species) containing RNA species with similar coding sequences and / or lengths, which could otherwise be difficult to distinguish using PCR- or chromatography-based analysis of full-length RNAs.

[0222] Each RNA species in a multivalent RNA composition may comprise an IDR sequence that is not a sequence isomer of an IDR sequence of another RNA species in a multivalent RNA composition (e.g., the IDR sequence does not have the same number of adenosine nucleotides, the same number of cytosine nucleotides, the same number of guanine nucleotides, and the same number of uracil nucleotides, as another IDR sequence in the composition, even if those sequences have different sequences). Having identical nucleotide compositions causes sequence isomers to have the same mass, presenting a challenge to distinguishing sequence isomers using mass-based identification methods (e.g., mass spectrometry).

[0223] Each RNA species in a multivalent RNA composition may comprise an IDR sequence having a mass that differs from the mass of IDR sequences of each other RNA species in a multivalent RNA composition. For example, the mass of each IDR sequence may differ from the mass of other IDR sequences by at least 9 Da, at least 25 Da, at least 25 Da, or at least 50 Da. Use of IDR sequences with distinct masses allows RNA fragments comprising different IDR sequences to be distinguished using mass-based analysis methods (e.g., mass spectrometry), which do not require reverse transcription, amplification, or sequencing of RNAs.

[0224] Each RNA species in an RNA composition may comprises an IDR sequence with a different length. For example, each IDR sequence may have a length independently selected from 0 to 25 nucleotides. The length of a nucleic acid influences the rate at which the nucleic acid traverses a chromatography column, and so the use of IDR sequences of different lengths on different RNA species allows RNA fragments having different IDR sequences to be distinguished using chromatography-based methods (e.g., LC-UV).

[0225] IDR sequences may be chosen such that no IDR sequence comprises a start codon, ‘AUG’ . Lack of a start codon in an IDR sequence prevents undesired translation of nucleotide sequences within and / or downstream from the IDR sequence.

[0226] IDR sequences may be chosen such that no IDR sequence comprises a recognition site for a restriction enzyme. In one example, no IDR sequence comprises a recognition site for Xbal, ‘UCUAG’. Lack of a recognition site for a restriction enzyme (e.g., Xbal recognition site ‘UCUAG’) allows the restriction enzyme to be used in generating and modifying a DNA template for in vitro transcription, without affecting the IDR sequence or sequence of the transcribed RNA.

[0227] Non-limiting examples of distinct IDR sequences include: GAGAUUGAGUGUAGUGACUAG (SEQ ID NO: 1022), GAGAUUGAGUGUAGUGAC (SEQ ID NO: 1023), GAGAUUGAGUGUAGUG (SEQ ID NO: 1024), GAUUGAGACUACGGG (SEQ ID NO: 1025), and CAUAGACACUACG (SEQ ID NO: 1026). In some embodiments of the compositions described herein, each mRNA encoding a distinct protein comprises a 3' UTR comprising a distinct IDR sequence selected from SEQ ID NOs: 1022-1026. Nucleic Acid Production

[0228] Chemical Synthesis

[0229] Nucleic acids the present disclosure may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Solid-phase synthesis is useful in site-specific introduction of chemical modifications in the nucleic acid sequences.

[0230] The synthesis of nucleic acids of the present disclosure by the sequential addition of monomer building blocks may be carried out in a liquid phase.

[0231] The synthetic methods discussed above each has its own advantages and limitations. Attempts have been conducted to combine these methods to overcome the limitations. Such combinations of methods are within the scope of the present disclosure. The use of solid-phase or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient way to generate long chain nucleic acids that cannot be obtained by chemical synthesis alone. Ligation

[0232] Assembling nucleic acids by a ligase may also be used. DNA or RNA ligases promote intermolecular ligation of the 5’ and 3’ ends of polynucleotide chains through the formation of a phosphodiester bond. Nucleic acids such as chimeric polynucleotides and / or circular nucleic acids may be prepared by ligation of one or more regions or subregions. DNA fragments can be joined by a ligase catalyzed reaction to create recombinant DNA with different functions. Two oligodeoxynucleotides, one with a 5’ phosphoryl group and another with a free 3’ hydroxyl group, serve as substrates for a DNA ligase.

[0233] Purification

[0234] Purification of the nucleic acids described herein may include, but is not limited to, nucleic acid clean-up, quality assurance and quality control. Clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQON® 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 relation to a nucleic acid such as a “purified nucleic acid” refers to one that is separated from at least one contaminant. A “contaminant” is any substance that makes another unfit, impure or inferior. Thus, a purified nucleic acid (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method. A quality assurance and / or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.

[0235] In some embodiments, the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.

[0236] Quantification

[0237] In some embodiments, the nucleic acids of the present disclosure may be quantified in exosomes or when derived from one or more bodily fluid. Bodily fluids include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. Alternatively, exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

[0238] Assays may be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof.

[0239] These methods afford the investigator the ability to monitor, in real time, the level of nucleic acids remaining or delivered. This is possible because the nucleic acids of the present disclosure, in some embodiments, differ from the endogenous forms due to the structural or chemical modifications.

[0240] In some embodiments, the nucleic acid may 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, MA). The quantified nucleic acid may be analyzed in order to determine if the nucleic acid may be of proper size, check that no degradation of the nucleic acid has occurred. Degradation of the nucleic acid may 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).

[0241] Lipid Compositions

[0242] In some embodiments, the nucleic acids are formulated as a lipid composition, such as a composition comprising a lipid nanoparticle, a liposome, and / or a lipoplex. In some embodiments, nucleic acids are formulated as lipid nanoparticle (LNP) compositions. Lipid nanoparticles typically comprise amino lipid, non-cationic lipid, structural lipid, and PEG lipid components along with the nucleic acid cargo of interest. The lipid nanoparticles can be generated using components, compositions, and methods as are generally known in the art, see for example PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551;

[0243] PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016 / 000129; PCT / US2016 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 052117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575; PCT / US2016 / 069491; PCT / US2016 / 069493; and PCT / US2014 / 066242, all of which are incorporated by reference herein in their entirety.

[0244] In some embodiments, the lipid nanoparticle comprises at least one ionizable amino lipid, at least one non-cationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG)- modified lipid.

[0245] In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable amino lipid, 5-25% non-cationic lipid, 25-55% structural lipid, and 0.5-15% PEG-modified lipid.

[0246] In some embodiments, the lipid nanoparticle comprises a molar ratio of 20-60% ionizable amino lipid, 5-30% non-cationic lipid, 10-55% structural lipid, and 0.5-15% PEG-modified lipid.

[0247] In some embodiments, the lipid nanoparticle comprises 40-50 mol% ionizable lipid, optionally 45-50 mol%, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%.

[0248] In some embodiments, the lipid nanoparticle comprises 20-60 mol% ionizable amino lipid. For example, the lipid nanoparticle may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable amino lipid. In some embodiments, the lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable amino lipid. In some embodiments, the lipid nanoparticle comprises 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, or 55 mol% ionizable amino lipid. In some embodiments, the lipid nanoparticle comprises 45 - 55 mole percent (mol%) ionizable amino lipid. For example, lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable amino lipid.

[0249] Ionizable amino lipids

[0250] Formula (Al)

[0251] In some embodiments, the ionizable amino lipid of a lipid nanoparticle is a compound of

[0252] Formula (Al): t; wherein Raa, Raβ, Ra', and Raδare each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;

[0253] R2and R3are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl;

[0254] R4is selected from the group consisting of -(CH2)nOH, wherein n is selected from the — 5 — group consisting wherein ? denotes a point of attachment; wherein R10is N(R)2; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R5is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0255] M and M’ are each independently selected from the group consisting of -C(O)O- and -OC(O)-;

[0256] R’ is a Ci-12 alkyl or C2-12 alkenyl;

[0257] 1 is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13. In some embodiments of the compounds of Formula (Al), R’ais R’branched; R’branched -sdenotes a point of attachment; Raa, Raβ, Ray, and Raδare each H; R2and R3are each C1-14 alkyl; R4is -(CH2)nOH; n is 2; each R5is H; each R6is H; M and M’ are each - C(O)O-; R’ is a C1-12 alkyl; 1 is 5; and m is 7.

[0258] In some embodiments of the compounds of Formula (Al), R’ais R’branched; R’branched -s denotes a point of attachment; Raa, Raβ, Ray, and Raδare each H; R2and R3are each C1-14 alkyl; R4is -(CH2)nOH; n is 2; each R5is H; each R6is H; M and M’ are each -

[0259] C(O)O-; R’ is a C1-12 alkyl; 1 is 3; and m is 7.

[0260] In some embodiments of the compounds of Formula (Al), R’ais R’branched; R’branched -sdenotes a point of attachment; Raais C2-12 alkyl; Raβ, Ray, and Raδare each H; R2and R3are each Ci-14 alkyl; R4i R10NH(CI-6 alkyl); n2 is 2; R5 is H; each R6is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; 1 is 5; and m is 7.

[0261] In some embodiments of the compounds of Formula (Al), R’ais R’branched; R’branched -sdenotes a point of attachment; Raa, Raβ, and Raδare each H; Rayis C2-12 alkyl; R2and R3are each C1-14 alkyl; R4is -(CH2)nOH; n is 2; each R5is H; each R6is H; M and

[0262] M’ are each -C(O)O-; R’ is a C1-12 alkyl; 1 is 5; and m is 7.

[0263] In some embodiments, the compound of Formula (Al) is selected from:

[0264]

[0265] In some embodiments, the ionizable amino lipid of Formula (Al) is a compound of Formula (Ala): (Ala), or its N-oxide, or a salt or isomer thereof, wherein R’ais R,branched; wherein

[0266] R’ branched |s;denotes a point of attachment; wherein RaP, Ray, and Raδare each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;

[0267] R2and R3are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl;

[0268] R4is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and denotes a point of attachment; wherein R10is N(R)2; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R5is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are each independently selected from the group consisting of -C(O)O- and

[0269] -OC(O)-;

[0270] R’ is a Ci-12 alkyl or C2-12 alkenyl;

[0271] 1 is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0272] In some embodiments, the ionizable amino lipid of Formula (Al) is a compound of Formula (Alb): (Alb), or its N-oxide, or a salt or isomer thereof, wherein R’ais R,branched; wherein

[0273] R’ branched ^s; whereindenotes a point of attachment; wherein Raa, Raβ, Ray, and Raδare each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;

[0274] R2and R3are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl;

[0275] R4is -(CH2)nOH, wherein n is selected from the group consisting of 1, 2, 3, 4, and 5; each R5is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0276] M and M’ are each independently selected from the group consisting of -C(O)O- and -OC(O)-;

[0277] R’ is a C1-12 alkyl or C2-12 alkenyl;

[0278] 1 is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0279] In some embodiments of Formula (Al) or (Alb), R’ais R’brancbed;R’branched -sdenotes a point of attachment; Raβ, Ray, and Raδare each H; R2and R3 are each C1-14 alkyl; R4is -(CH2)nOH; n is 2; each R5is H; each R6is H; M and M’ are each - C(O)O-; R’ is a C1-12 alkyl; 1 is 5; and m is 7. In some embodiments of Formula (Al) or (Alb), R’ais R’branched; R’branched -s denotes a point of attachment; Raβ, Ray, and Raδare each H; R2and R3are each C1-14 alkyl; R4is -(CH2)nOH; n is 2; each R5is H; each R6is H; M and M’ are each -

[0280] C(O)O-; R’ is a C1-12 alkyl; 1 is 3; and m is 7.

[0281] In some embodiments of Formula (Al) or (Alb), R’ais R’branched; R’branched -s denotes a point of attachment; Raβand Raδare each H; Rayis C2-12 alkyl;

[0282] R2and R3are each C1-14 alkyl; R4is -(CH2)nOH; n is 2; each R5is H; each R6is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; 1 is 5; and m is 7.

[0283] In some embodiments, the ionizable amino lipid of Formula (Al) is a compound of

[0284] Formula (Ale): (Ale), or its N-oxide, or a salt or isomer thereof, wherein R’ais R’branched; wherein

[0285] R’branched ^s wjiereindenotes a point of attachment; wherein Raa, Raβ, Rayand Raδare each independently selected from the group consisting of H, C2-12 alkyl, and C2-12 alkenyl;

[0286] R2and R3are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl;

[0287] .

[0288] R4is wherein denotes a point of attachment; wherein R10is

[0289] N(R)2; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R5is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; M and M’ are each independently selected from the group consisting of -C(O)O- and -OC(O)-;

[0290] R’ is a Ci-12 alkyl or C2-12 alkenyl;

[0291] 1 is selected from the group consisting of 1, 2, 3, 4, and 5; and m is selected from the group consisting of 5, 6, 7, 8, 9, 10, 11, 12, and 13.

[0292] In some embodiments, R’ais R’branched; R’branchedis denotes a point of attachment; Raβ, Ray, and Raδare each H; Raais C2-12 alkyl; R2and R3are each C1-14 alkyl; R4is denotes a point of attachment; R10is NH(CI-6 alkyl); n2 is 2; each R5is H; each R6is H; M and M’ are each -C(O)O-; R’ is a C1-12 alkyl; 1 is 5; and m is 7.

[0293] In some embodiments, the compound of Formula (Ale) is:

[0294] Formula (All)

[0295] In some embodiments, the ionizable amino lipid is a compound of Formula (All): (All), or its N-oxide, or a salt or isomer thereof,

[0296] ’branched ; wherein denotes a point of attachment; Rayand Raδare each independently selected from the group consisting of H, C1-12 alkyl, and C2-12 alkenyl, wherein at least one of Rayand Ra° is selected from the group consisting of Ci- 12 alkyl and C2-12 alkenyl;

[0297] Rbyand Rbδare each independently selected from the group consisting of H, C1-12 alkyl, and C2-12 alkenyl, wherein at least one of Rbyand Rb5is selected from the group consisting of Ci- 12 alkyl and C2-12 alkenyl;

[0298] R2and R3are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl;

[0299] R4is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and wherein denotes a point of attachment; wherein R10is N(R)2; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a C1-12 alkyl or C2-12 alkenyl;

[0300] Yais a C3-6 carbocycle;

[0301] R*”ais selected from the group consisting of C1-15 alkyl and C2-15 alkenyl; and s is 2 or 3; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;

[0302] 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0303] In some embodiments, the ionizable amino lipid of Formula (All) is a compound of Formula (All- a): (All-a), or its N-oxide, or a salt or isomer thereof, wherein R’ais R’branchedOr R’cyclic; wherein denotes a point of attachment; Rayand Raδare each independently selected from the group consisting of H, C1-12 alkyl, and C2-12 alkenyl, wherein at least one of Rayand Ra° is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;

[0304] Rb■ and Rb5are each independently selected from the group consisting of H, C1-12 alkyl, and C2-12 alkenyl, wherein at least one of Rbyand Rb5is selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;

[0305] R2and R3are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl;

[0306] R4is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and , wherein denotes a point of attachment; wherein R10is N(R)2; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a C1-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;

[0307] 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0308] In some embodiments, the ionizable amino lipid of Formula (All) is a compound of Formula (All-b): (All-b), or its N-oxide, or a salt or isomer thereof, wherein R’ais R’branchedOr R’cyclic; wherein

[0309] ’branched wherein denotes a point of attachment;

[0310] Rayand Rbyare each independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;

[0311] R2and R3are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl; R4is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and denotes a point of attachment; wherein R10is N(R)2; each R is independently selected from the group consisting of

[0312] Ci-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a C1-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;

[0313] 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0314] In some embodiments, the ionizable amino lipid of Formula (All) is a compound of

[0315] Formula (AII-c): wherein R’ais R’branchedOr R’cyclic; wherein

[0316] R’ branched |s ; andR’b is;denotes a point of attachment; wherein Rayis selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;

[0317] R2and R3are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl;

[0318] R4is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and , enotes a point of attachment; wherein R10is N(R)2; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10;

[0319] R’ is a C1-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;

[0320] 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9. In some embodiments, the ionizable amino lipid of Formula (All) is a compound of

[0321] Formula (All-d): a (All-d), or its N-oxide, or a salt or isomer thereof, wherein R’ais R’branchedOr R’cyclic; wherein

[0322] R’ branched |g ;andR’ b js;wherein denotes a point of attachment; wherein Rayand Rbyare each independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;

[0323] R4is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5, and wherein denotes a point of attachment; wherein R10is N(R)2; each R is independently selected from the group consisting of C1-6 alkyl, C2-3 alkenyl, and H; and n2 is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10; each R’ independently is a C1-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;

[0324] 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0325] In some embodiments, the ionizable amino lipid of Formula (All) is a compound of Formula (All-e): wherein R’ais R’branchedor R’ cyclic.wherein

[0326] R’ branched |gand R’bis:wherein denotes a point of attachment; wherein Rayis selected from the group consisting of C1-12 alkyl and C2-12 alkenyl;

[0327] R2and R3are each independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl;

[0328] R4is -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5;

[0329] R’ is a C1-12 alkyl or C2-12 alkenyl; m is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9;

[0330] 1 is selected from 1, 2, 3, 4, 5, 6, 7, 8, and 9.

[0331] In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), m and 1 are each independently selected from 4, 5, and 6. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e), m and 1 are each 5.

[0332] In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), each R’ independently is a C1-12 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e), each R’ independently is a C2-5 alkyl.

[0333] In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), R’bis: and R2and R3are each independently a C1-14 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e),

[0334] R’bis: R2and R3are each independently a Ce-io alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e), R’bis: R2and R3are each a C8alkyl.

[0335] In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- and R3are each independently a Ce-io alkyl. In some embodiments of the compound of Formula embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e), alkyl, and R2and R3are each a

[0336] C8alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), R’braIlchedis: and RayandRbYare a Ci-12 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c),

[0337] (AII-d), or (AII-e), R’branchedis:and Rayand Rby each a C2-6 alkyl.

[0338] In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), m and 1 are each independently selected from 4, 5, and 6 and each R’ independently is a C1-12 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII-d), or (All-e), m and 1 are each 5 and each R’ independently is a C2-5 alkyl.

[0339] In some embodiments of the compound of (All), (All-a), (All-b), (AII-c), (AII-d), or

[0340] (AII-e), R’branchedis;m and 1 arc each independently selected from 4, 5, and 6, each R’ independently is a C1-12 alkyl, and Rayand Rby' are each a C1-12 alkyl. In some embodiments of the compound of Formula (All), (All-a), (All-b),

[0341] (AII-c), (AII-d), or (AII-e), R’branchedis:m and 1 are each 5, each R’ independently is a C2-5 alkyl, and Rayand Rbyare each a C2-6 alkyl.

[0342] In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (AII-e), R’branchedisand R’b ^s: mand 1 are each independently selected from 4, 5, and 6, R’ is a C1-12 alkyl, Rayis a C1-12 alkyl and R2and R3are each independently a C6-10alkyl.

[0343] In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (AII-e), R’branchedis; andR,bis: mand 1 are each 5, R’ is a C2-

[0344] 5 alkyl, Rayis a C2-6 alkyl, and R2and R3are each a C8alkyl.

[0345] In some embodiments of the compound of (All), (All-a), (All-b), (AII-c), (AII-d), or

[0346] .

[0347] (AII-e), R4is wherein R10is NH(CI-6 alkyl) and n2 is 2. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e), R4is wherein R10is NH(CH3) and n2 is 2.

[0348] In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), R’branchedis; , R is:,m and1 each independently selected from 4, 5, and 6, each R’ independently is a C1-12 alkyl, Rayand Rbyare

[0349] . each a C1-12 alkyl, and R4is wherein R10is NH(CI-6 alkyl), and n2 is 2. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All- e), R’branchedis; m and 1 are each 5, each R’ independently is a C2-5alkyl, Rayand Rbyare each a C2-6 alkyl, and R4is wherein R10is NH(CH3) and n2 is 2.

[0350] In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), R’branchedis; andR’b ^s; ,m and1areeach independently selected from 4, 5, and 6, R’ is a C1-12 alkyl, R2and R3are each independently a

[0351] .

[0352] C6-10alkyl, Rayis a C1-12 alkyl, and R4is wherein R10is NH(CI-6 alkyl) and n2 is 2. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), R’branchedis;andR’b is; ,m and1areeach 5, R’ is a C2-

[0353] 5 alkyl, Rayis a C2-6 alkyl, R2and R3are each a C8alkyl, and R4is wherein

[0354] R10is NH(CH3) and n2 is 2. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), R4is -(CH2)nOH and n is 2, 3, or 4. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e), R4is -(CH2)nOH and n is 2.

[0355] In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (AII- d), or (All-e), R’branchediS;m and 1 each independently selected from 4, 5, and 6, each R’ independently is a C1-12 alkyl, Rayand Rby' are each a C1-12 alkyl, R4is -(CH2)nOH, and n is 2, 3, or 4. In some embodiments of the compound of Formula (All), (All-a), (All-b), (AII-c), (All-d), or (All-e), R’branchedis:

[0356] . .

[0357] R is: m and 1 are each 5, each R’ independently is a C2-5 alkyl, Rayand R7are each a C2-6 alkyl, R4is -(CH2)nOH, and n is 2.

[0358] In some embodiments, the ionizable amino lipid of Formula (All) is a compound of

[0359] Formula (All-f):

[0360] (All-f), or its N-oxide, or a salt or isomer thereof, wherein R’ais R’branchedOr R’cyclic; wherein wherein denotes a point of attachment;

[0361] RαYis a C1-12 alkyl;

[0362] R2and R3are each independently a C1-14 alkyl;

[0363] R4is -(CH2)nOH wherein n is selected from the group consisting of 1, 2, 3, 4, and 5;

[0364] R’ is a C1-12 alkyl; m is selected from 4, 5, and 6; and

[0365] 1 is selected from 4, 5, and 6.

[0366] In some embodiments of the compound of Formula (All-f), m and 1 are each 5, and n is 2, 3, or 4.

[0367] In some embodiments of the compound of Formula (All-f) R’ is a C2-5 alkyl, Rayis a C2-6 alkyl, and R2and R3are each a Ce-io alkyl. In some embodiments of the compound of Formula (All-f), m and 1 are each 5, n is 2, 3, or 4, R’ is a C2-5 alkyl, Rayis a C2-6 alkyl, and R2and R3are each a Ce-io alkyl.

[0368] In some embodiments, the ionizable amino lipid of Formula (All) is a compound of Formula (All-g): isomer thereof; wherein

[0369] Rayis a C2-6 alkyl;

[0370] R’ is a C2-5 alkyl; and

[0371] R4is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 3, 4, and 5, and wherein denotes a point of attachment, R10is NH(CI-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3.

[0372] In some embodiments, the ionizable amino lipid of Formula (All) is a compound of

[0373] Formula (All-h): isomer thereof; wherein

[0374] Rayand Rbyare each independently a C2-6alkyl; each R’ independently is a C2-5alkyl; and

[0375] R4is selected from the group consisting of -(CH2)nOH wherein n is selected from the group consisting of 3, 4, and 5, and , denotes a point of attachment, R10is NH(CI-6 alkyl), and n2 is selected from the group consisting of 1, 2, and 3. In some embodiments of the compound of Formula (All-g) or (All-h), R4is wherein R10is NH(CH3) and n2 is 2.

[0376] In some embodiments of the compound of Formula (All-g) or (All-h), R4is -(CH2)nOH.

[0377] Formula (AIII)

[0378] In some embodiments, the ionizable amino lipids of a lipid nanoparticle may be one or more of compounds of Formula (AIII): (AIII), or their N-oxides, or salts or isomers thereof,

[0379] Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

[0380] R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0381] R4 is selected from the group consisting of hydrogen, a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a carbocycle, heterocycle, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(0)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(0)N(R)2, -N(R)C(S)N(R)2, -N(R)R8, -N(R)S(O)2R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -0C(0)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(R)N(R)2C(0)0R, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R6is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0382] M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group, in which M” is a bond, C1-13 alkyl or C2-13 alkenyl; R? is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0383] Rs is selected from the group consisting of C3-6 carbocycle and heterocycle;

[0384] R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-15 alkyl and C3-15 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13; and wherein when R4 is -(CH2)nQ, -(CH2)nCHQR, -CHQR, or -CQ(R)2, then (i) Q is not -N(R)2 when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl when n is 1 or 2.

[0385] In some embodiments, another subset of compounds of Formula (AIII) includes those in which:

[0386] Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

[0387] R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0388] R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CFDnCHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and a 5- to 14-membered heterocycloalkyl having one or more heteroatoms selected from N, O, and S which is substituted with one or more substituents selected from oxo (=0), OH, amino, mono- or di- alkylamino, and C1-3 alkyl, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each Re is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0389] M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group;

[0390] R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0391] Rs is selected from the group consisting of C3-6 carbocycle and heterocycle;

[0392] R9 is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

[0393] In some embodiments, another subset of compounds of Formula (AIII) includes those in which:

[0394] Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

[0395] R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0396] R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CH2)nCHQR -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heterocycle having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2,-N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -0C(0)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(0R)C(0)N(R)2, -N(0R)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R)2, and each n is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5- to 14-membered heterocycle and (i) R4 is -(CH2)nQ in which n is 1 or 2, or (ii) R4 is -(CH2)nCHQR in which n is 1, or (iii) R4 is -CHQR, and -CQ(R)2, then Q is either a 5- to 14-membered heteroaryl or 8- to 14-membered heterocycloalkyl; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each Re is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0397] M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group;

[0398] R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0399] Rs is selected from the group consisting of C3-6 carbocycle and heterocycle;

[0400] R9is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

[0401] In some embodiments, another subset of compounds of Formula (AIII) includes those in which:

[0402] Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’; R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0403] R4 is selected from the group consisting of a C3-6 carbocycle, -(CH2)nQ, -(CFDnCHQR, -CHQR, -CQ(R)2, and unsubstituted C1-6 alkyl, where Q is selected from a C3-6 carbocycle, a 5- to 14-membered heteroaryl having one or more heteroatoms selected from N, O, and S, -OR, -O(CH2)nN(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -CRN(R)2C(O)OR, -N(R)R8, -O(CH2)nOR, -N(R)C(=NR9)N(R)2, -N(R)C(=CHR9)N(R)2, -0C(0)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(0R)C(0)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR9)N(R)2, -N(OR)C(=CHR9)N(R)2, -C(=NR9)R, -C(O)N(R)OR, and -C(=NR9)N(R)2, and each n is independently selected from 1, 2, 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each Re is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0404] M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group;

[0405] R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0406] Rs is selected from the group consisting of C3-6 carbocycle and heterocycle;

[0407] R9is selected from the group consisting of H, CN, NO2, C1-6 alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C2-6 alkenyl, C3-6 carbocycle and heterocycle; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C2-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof. In some embodiments, another subset of compounds of Formula (AIII) includes those in which

[0408] Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

[0409] R2 and R3 are independently selected from the group consisting of H, C2-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle;

[0410] R4 is -(CH2)nQ or -(CH2)nCHQR, where Q is -N(R)2, and n is selected from 3, 4, and 5; each R5 is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each Re is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0411] M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group;

[0412] R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C1-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

[0413] In some embodiments, another subset of compounds of Formula (AIII) includes those in which

[0414] Ri is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, -R*YR”, -YR”, and -R”M’R’;

[0415] R2 and R3 are independently selected from the group consisting of C1-14 alkyl, C2-14 alkenyl, -R*YR”, -YR”, and -R*OR”, or R2 and R3, together with the atom to which they are attached, form a heterocycle or carbocycle; R4 is selected from the group consisting of -(CH2)nQ, -(CthKCHQR, -CHQR, and -CQ(R)2, where Q is -N(R)2, and n is selected from 1, 2, 3, 4, and 5; each Rs is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each Re is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H;

[0416] M and M’ are independently selected from -C(O)O-, -OC(O)-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -S-S-, an aryl group, and a heteroaryl group;

[0417] R7 is selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R is independently selected from the group consisting of C1-3 alkyl, C2-3 alkenyl, and H; each R’ is independently selected from the group consisting of C1-18 alkyl, C2-18 alkenyl, -R*YR”, -YR”, and H; each R” is independently selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; each R* is independently selected from the group consisting of C1-12 alkyl and C1-12 alkenyl; each Y is independently a C3-6 carbocycle; each X is independently selected from the group consisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.

[0418] In certain embodiments, a subset of compounds of Formula (AIII) includes those of Formula (AIII- A): (AIII- A), or its N-oxide, or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; Mi is a bond or M’; R4 is hydrogen, unsubstituted C1-3 alkyl, or -(CFDnQ, in which Q is -OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2,-NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group,; and R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl. For example, m is 5, 7, or 9. For example, Q is OH, -NHC(S)N(R)2, or -NHC(O)N(R)2. For example, Q is -N(R)C(O)R, or -N(R)S(O)2R.

[0419] In certain embodiments, a subset of compounds of Formula (AIII) includes those of

[0420] Formula (AIII-B): (AIII-B), or its N-oxide, or a salt or isomer thereof in which all variables are as defined herein. For example, m is selected from 5, 6, 7, 8, and 9;

[0421] R4 is hydrogen, unsubstituted C1-3 alkyl, or -(CH2)nQ, in which Q is H, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2,-NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a hetero aryl group; and R2and R3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-i4 alkenyl. For example, m is 5, 7, or 9. For example, Q is OH, -NHC(S)N(R)2, or -NHC(O)N(R)2. For example, Q is -N(R)C(O)R, or -N(R)S(O)2R.

[0422] In certain embodiments, a subset of compounds of Formula (AIII) includes those of Formula (AIII-C): (AIII-C), or its N-oxide, or a salt or isomer thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; Mi is a bond or M’; R4 is hydrogen, unsubstituted C1-3 alkyl, or -(CH2)nQ, in which n is 2, 3, or 4, and Q is OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)R8, -NHC(=NR9)N(R)2,-NHC(=CHR9)N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M’ are independently selected from -C(O)O-, -OC(O)-, -OC(O)-M”-C(O)O-, -C(O)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a hetero aryl group; and R2and R3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-i4 alkenyl.

[0423] In some embodiments, the compounds of Formula (AIII) are of Formula (AIII-D), (AIII-D), or their N-oxides, or salts or isomers thereof, wherein R4 is as described herein.

[0424] In another embodiment, the compounds of Formula (AIII) are of Formula (AIII-E), (AIII-E), or their N-oxides, or salts or isomers thereof, wherein R4 is as described herein.

[0425] In another embodiment, the compounds of Formula (AIII) are of Formula (AIII-F) or (AIII-G): isomers thereof, wherein R4 is as described herein.

[0426] In another embodiment, the compounds of Formula (AIII) are of Formula (AIII-H):

[0427] (AIII-H) or their N-oxides, or salts or isomers thereof, wherein M is -C(O)O- or -OC(O)-, M” is C1-6 alkyl or C2-6 alkenyl, R2 and R3 are independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl, and n is selected from 2, 3, and 4.

[0428] In a further embodiment, the compounds of Formula (AIII) are of Formula (AIII-I): (AIII-I), or their N-oxides, or salts or isomers thereof, wherein n is 2, 3, or 4; and m, R’, R”, and R2 through Re are as described herein. For example, each of R2 and R3 may be independently selected from the group consisting of C5-14 alkyl and C5-

[0429] 14 alkenyl.

[0430] In some embodiments, an ionizable amino lipid of the disclosure comprises a compound having structure: (Compound 1).

[0431] In some embodiments, an ionizable amino lipid of the disclosure comprises a compound having structure: (Compound 2).

[0432] In a further embodiment, the compounds of Formula (AIII) are of Formula (AIII-J), (AIII-J), or their N-oxides, or salts or isomers thereof, wherein 1 is selected from 1, 2, 3, 4, and 5; m is selected from 5, 6, 7, 8, and 9; Mi is a bond or

[0433] M’; M and M’ are independently selected from -C(0)0-, -0C(0)-, -0C(0)-M”-C(0)0-, -C(0)N(R’)-, -P(O)(OR’)O-, -S-S-, an aryl group, and a heteroaryl group; and R2 and R3 are independently selected from the group consisting of H, C1-14 alkyl, and C2-14 alkenyl. For example, M” is C1-6 alkyl (e.g., C1-4 alkyl) or C2-6 alkenyl (e.g. C2-4 alkenyl). For example, R2 and R3 are independently selected from the group consisting of C5-14 alkyl and C5-14 alkenyl.

[0434] In some embodiments, the ionizable amino lipids are one or more of the compounds described in U.S. Application Nos. 62 / 220,091, 62 / 252,316, 62 / 253,433, 62 / 266,460, 62 / 333,557, 62 / 382,740, 62 / 393,940, 62 / 471,937, 62 / 471,949, 62 / 475,140, and 62 / 475,166, and PCT Application No. PCT / US2016 / 052352.

[0435] The central amine moiety of a lipid according to Formula (AIII), (AIII-A), (AIII-B), (AIII-C), (AIII-D), (AIII-E), (AIII-F), (AIII-G), (AIII-H), (AIII-I), or (AIII- J) may be protonated at a physiological pH. Thus, a lipid may have a positive or partial positive charge at physiological pH. Such amino lipids may be referred to as cationic lipids, ionizable lipids, cationic amino lipids, or ionizable amino lipids. Amino lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.

[0436] Formula (AIV)

[0437] In some embodiments, the ionizable amino lipids of a lipid nanoparticle may be one or more of compounds of formula (AIV), salts or isomers thereof, wherein t is 1 or 2;

[0438] Ai and A2 are each independently selected from CH or N;

[0439] Z is CH2 or absent wherein when Z is CH2, the dashed lines (1) and (2) each represent a single bond; and when Z is absent, the dashed lines (1) and (2) are both absent;

[0440] Ri, R2, R3, R4, and R5 are independently selected from the group consisting of C5-20 alkyl, C5-20 alkenyl, -R”MR’, -R*YR”, -YR”, and -R*OR”;

[0441] Rxi and Rx2 are each independently H or C1-3 alkyl; each M is independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)O-, -C(O)N(R’)-, -N(R’)C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR’)O-, -S(O)2-, -C(O)S-, -SC(O)-, an aryl group, and a heteroaryl group;

[0442] M* is C1-C6alkyl, W1and W2are each independently selected from the group consisting of -O- and -N(Re)-; each Re is independently selected from the group consisting of H and C1-5 alkyl;

[0443] X1, X2, and X3are independently selected from the group consisting of a bond, -CH2-, -(CH2)2-, -CHR-, -CHY-, -C(O)-, -C(O)O-, -OC(O)-, -(CH2)n-C(O)-, -C(O)-(CH2)n-, -(CH2)n-C(O)O-, -OC(O)-(CH2)n-, -(CH2)n-OC(O)-, -C(O)O-(CH2)n-, -CH(OH)-, -C(S)-, and -CH(SH)-; each Y is independently a C3-6 carbocycle; each R* is independently selected from the group consisting of C1-12 alkyl and C2-i2 alkenyl; each R is independently selected from the group consisting of C1-3 alkyl and a C3-6 carbocycle; each R’ is independently selected from the group consisting of C1-12 alkyl, C2-i2 alkenyl, and H; each R” is independently selected from the group consisting of C3-12 alkyl, C3-12 alkenyl and -R*MR’ ; and n is an integer from 1-6; wherein when ring then i) at least one of X1, X2, and X3is not -CH2-; and / or ii) at least one of Ri, R2, R3, R4, and R5 is -R”MR’.

[0444] In some embodiments, the compound is of any of formulae (AIVa)-(AIVh):

[0445]

[0446] In some embodiments, the ionizable amino lipid is or a salt thereof.

[0447] The central amine moiety of a lipid according to Formula (AIV), (AlVa), (AlVb), (AIVc), (AlVd), (AlVe), (AlVf), (AlVg), or (AlVh) may be protonated at a physiological pH.

[0448] Thus, a lipid may have a positive or partial positive charge at physiological pH.

[0449] Formula (AV)

[0450] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: (AV), or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0451] R1is optionally substituted C1-C24 alkyl or optionally substituted C2-C24 alkenyl;

[0452] R2and R3are each independently optionally substituted C1-C36 alkyl;

[0453] R4and R5are each independently optionally substituted C1-C6alkyl, or R4and R5join, along with the N to which they are attached, to form a heterocyclyl or heteroaryl;

[0454] L1, L2, and L3are each independently optionally substituted Ci-C is alkylene;

[0455] G1is a direct bond,

[0456] G2and G3are each independently -(C=O)O- or -O(C=O)-; and n is an integer greater than 0.

[0457] Formula (AVI)

[0458] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: (AVI) or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

[0459] G1is -N(R3)R4or -OR5;

[0460] R1is optionally substituted branched, saturated or unsaturated C12-C36 alkyl;

[0461] R2is optionally substituted branched or unbranched, saturated or unsaturated C12- C36 alkyl when L is -C(=O)-; or R2is optionally substituted branched or unbranched, saturated or unsaturated C4-C36 alkyl when L is C6-C12 alkylene, C6-C12 alkenylene, or C2-C6 alkynylene;

[0462] R3and R4are each independently H, optionally substituted branched or unbranched, saturated or unsaturated C1-C6alkyl; or R3and R4are each independently optionally substituted branched or unbranched, saturated or unsaturated C1-C6alkyl when L is C6-C12 alkylene, C6-C12 alkenylene, or C2-C6 alkynylene; or R3and R4, together with the nitrogen to which they are attached, join to form a heterocyclyl;

[0463] R5is H or optionally substituted C1-C6alkyl;

[0464] L is -C(=O)-, C6-C12 alkylene, C6-C12 alkenylene, or C2-C6 alkynylene; and n is an integer from 1 to 12.

[0465] Formula (AVII)

[0466] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: (A VII), or a pharmaceutically acceptable salt thereof, wherein: each Rlais independently hydrogen, Rlc, or Rld; each Rlbis independently Rlcor Rld; each Rlcis independently -[CH2]2C(O)X1R3; each RldIs independently -C(O)R4; each R2is independently -[C(R2a)2]cR2b; each R2ais independently hydrogen or C1-C6alkyl;

[0467] R2bis -N(LI-B)2; -(OCH2CH2)6OH; or -(OCH2CH2)bOCH3; each R3and R4is independently C6-C30 aliphatic; each I.3 is independently C1-C10 alkylene; each B is independently hydrogen or an ionizable nitrogen-containing group; each X1is independently a covalent bond or O; each a is independently an integer of 1-10; each b is independently an integer of 1-10; and each c is independently an integer of 1-10.

[0468] Formula (AVIII)

[0469] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: ( ), or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0470] X is N, and Y is absent; or X is CR, and Y is NR;

[0471] L1is -O(C-O)R1, -(C=O)OR1, -C(=O)R1, -OR1, -S(O)XR1, -S-SR1, -C(=O)SR1, - SC(=O)R1, -NRaC(=O)R1, -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRc, or - NRaC(=O)OR1;

[0472] L2is -O(C=O)R2, -(C=O)OR2, -C(=O)R2, -OR2, -S(O)XR2, -S-SR2, -C(=O)SR2, - SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, -OC(=O)NReRf; -NRdC(=O)OR2or a direct bond to R2;

[0473] L3is -O(C=O)R3or -(C=O)OR3;

[0474] G1and G2are each independently C2-C12 alkylene or C2-C12 alkenylene; G3is C1-C24 alkylene, C2-C24 alkenylene, C1-C24 heteroalkylene or C2-C24 heteroalkenylene when X is CR, and Y is NR; and G3is C1-C24 heteroalkylene or C2-C24 heteroalkenylene when X is N, and Y is absent;

[0475] Ra, Rb, Rdand Reare each independently H or C1-C12 alkyl or C1-C12 alkenyl;

[0476] Rcand Rfare each independently C1-C12 alkyl or C2-C12 alkenyl; each R is independently H or C1-C12 alkyl;

[0477] R1, R2and R3are each independently C1-C24 alkyl or C2-C24 alkenyl; and x is 0, 1 or 2, and wherein each alkyl, alkenyl, alkylene, alkenylene, heteroalkylene and heteroalkenylene is independently substituted or unsubstituted unless otherwise specified.

[0478] Formula (AIX)

[0479] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0480] L1and L2are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, - C(=O)S-, -SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, -NRaC(=O)NRa-, -OC(=O)NRa-, -NRaC(=O)O- or a direct bond;

[0481] G1is C1-C2 alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -NRaC(=O)- or a direct bond;

[0482] G2is -C(O)-, -(CO)O-, -C(=O)S-, -C(=O)NRa- or a direct bond;

[0483] G3is C1-C6alkylene;

[0484] Rais H or C1-C12 alkyl;

[0485] Rlaand Rlbare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) Rlais H or C1-C12 alkyl, and Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond;

[0486] R2aand R2bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R2ais H or C1-C12 alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon-carbon double bond; R3aand R3bare, at each occurrence, independently either (a): H or C1-C12 alkyl; or (b) R3ais H or C1-C12 alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;

[0487] R4aand R4bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0488] R5and R6are each independently H or methyl;

[0489] R7is H or C1-C20 alkyl;

[0490] R8is OH, -N(R9)(C=O)R10, -(C=O)NR9R10, -NR9R10, -(C=O)ORn lor -O(C=O)R", provided that G3is C4-C6 alkylene when R8is -NR9R10,

[0491] R9and R10are each independently H or C1-C12 alkyl;

[0492] R" is aralkyl; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2, wherein each alkyl, alkylene and aralkyl is optionally substituted.

[0493] Formula (AX)

[0494] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: ( ), or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0495] X and X' are each independently N or CR;

[0496] Y and Y' are each independently absent, -O(C=O)-, -(C=O)O- or NR, provided that: a) Y is absent when X is N; b) Y' is absent when X' is N; c) Y is -O(C=O)-, -(C=O)O- or NR when X is CR; and d) Y' is -O(C=O)-, -(C=O)O- or NR when X' is CR

[0497] L1and L1are each independently

[0498] L2and L2are each independently -O(C=O)R2, -(C=O)OR2, -C(=O)R2, -OR2, -S(O)ZR2, -S-SR2, -C(=O)SR2, -SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, - OC(=O)NReRf; -NRdC(=O)OR2or a direct bond to R2;

[0499] G1. G1, G2and G2are each independently C2-C12 alkylene or C2-C12 alkenylene; G is C2-C24 heteroalkylene or C2-C24 heteroalkenylene;

[0500] Ra, Rb, Rdand Reare, at each occurrence, independently H, C1-C12 alkyl or C2-C12 alkenyl;

[0501] Rcand Rfare, at each occurrence, independently C1-C12 alkyl or C2-C12 alkenyl;

[0502] R is, at each occurrence, independently H or C1-C12 alkyl;

[0503] R1and R2are, at each occurrence, independently branched C6-C24 alkyl or branched C6-C24 alkenyl; z is 0, 1 or 2, and wherein each alkyl, alkenyl, alkylene, alkenylene, hetero alkylene and heteroalkenylene is independently substituted or unsubstituted unless otherwise specified.

[0504] Formula (AXI)

[0505] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0506] L1is -O(C=O)R1, -(C=O)OR1, -C(=O)R1, -OR1, -S(O)XR1, -S-SR1, - C(=O)SR1, -SC(=O)R1, -NRaC(=O)R1, -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRcor -NRaC(=O)OR1;

[0507] L2is -O(C=O)R2, -(C=O)OR2, -C(=O)R2, -OR2, -S(O)XR2, -S-SR2, - C(=O)SR2, -SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, -OC(=O)NReRf-, -NRdC(=O)OR2- or a direct bond to R2;

[0508] G1and G2are each independently C2-C12 alkylene or C2-C12 alkenylene;

[0509] G3is C1-C24 alkylene, C2-C24 alkenylene, C3-C8cycloalkylene or C3-C8cycloalkenylene;

[0510] Ra, Rb, Rdand Reare each independently H or C1-C12 alkyl or C1-C12 alkenyl;

[0511] Rcand Rfare each independently C1-C12 alkyl or C2-C12 alkenyl;

[0512] R1and R2are each independently branched C6-C24 alkyl or branched C6-C24 alkenyl;

[0513] R3is -N(R4)R5;

[0514] R4is C1-C12 alkyl;

[0515] R5is substituted C1-C12 alkyl; and x is 0, 1 or 2, and wherein each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl and aralkyl is independently substituted or unsubstituted unless otherwise specified.

[0516] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: (AXIb), or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0517] L1is -O(C=O)R1, -(C=O)OR1, -C(=O)R1, -OR1, -S(O)XR1, -S-SR1, -C(=O)SR1, -SC(=O)R1, -NRaC(=O)R1, -C(=O)NRbRc, -NRaC(=O)NRbRc, -OC(=O)NRbRcor -NRaC(=O)OR1;

[0518] L2is -O(C=O)R2, -(C=O)OR2, -C(=O)R2, -OR2, -S(O)XR2, -S-SR2, -C(=O)SR2, -SC(=O)R2, -NRdC(=O)R2, -C(=O)NReRf, -NRdC(=O)NReRf, -OC(=O)NReRf; -NRdC(=O)OR2or a direct bond to R2;

[0519] Glaand G2bare each independently C2-C12 alkylene or C2-C12 alkenylene;

[0520] Glband G2bare each independently C1-C12 alkylene or C2-C12 alkenylene;

[0521] G3is C1-C24 alkylene, C2-C24 alkenylene, C3-C8cycloalkylene or C3-C8cycloalkenylene;

[0522] Ra, Rb, Rdand Reare each independently H or C1-C12 alkyl or C2-C12 alkenyl;

[0523] Rcand Rfare each independently C1-C12 alkyl or C2-C12 alkenyl;

[0524] R1and R2are each independently branched C6-C24 alkyl or branched C6-C24 alkenyl;

[0525] R3ais -C(=O)N(R4a)R5aor -C(=O)OR6;

[0526] R3bis -NR4bC(=O)R5b;

[0527] R4ais C1-C12 alkyl;

[0528] R4bis H, C1-C12 alkyl or C2-C12 alkenyl;

[0529] R5ais H, Ci-Cs alkyl or C2-C8 alkenyl;

[0530] R5bis C2-C12 alkyl or C2-C12 alkenyl when R4bis H; or R5bis C1-C12 alkyl or C2-C12 alkenyl when R4bis C1-C12 alkyl or C2-C12 alkenyl;

[0531] R6is H, aryl or aralkyl; and x is 0, 1 or 2, and wherein each alkyl, alkenyl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, aryl and aralkyl is independently substituted or unsubstituted.

[0532] Formula (AXII)

[0533] In some embodiments, the lipid nanoparticle comprises a lipid having the structure:

[0534] (AXII), or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein:

[0535] G1is -OH, -R3R4, -(C=O)R5or -R3(C=O)R5; G2is -CH2- or -(C=O)-;

[0536] R is, at each occurrence, independently H or OH;

[0537] R1and R2are each independently optionally substituted branched, saturated or unsaturated C12-C36 alkyl;

[0538] R3and R4are each independently H or optionally substituted straight or branched, saturated or unsaturated C1-C6alkyl;

[0539] R5is optionally substituted straight or branched, saturated or unsaturated C1-C6alkyl; and n is an integer from 2 to 6.

[0540] Formula (AXIII)

[0541] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: (AXIII), or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of G1or G2is, at each occurrence, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O) , -S-S-, -C(=O)S-, SC(=O)-, -N(Ra)C(=O)-, -C(=O)N(Ra)-, -N(Ra)C(=O)N(Ra)-, -OC(=O)N(Ra)- or -N(Ra)C(=O)O-, and the other of G1or G2is, at each occurrence, -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O), -S-S-, -C(=O)S-, -SC(=O)-, -N(Ra)C(=O)-, -C(=O)N(Ra)-, -N(Ra)C(=O)N(Ra)-, -OC(=O)N(Ra)- or -N(Ra)C(=O)O- or a direct bond;

[0542] L is, at each occurrence, ~O(C=O)-, wherein ~ represents a covalent bond to X;

[0543] X is CRa;

[0544] Z is alkyl, cycloalkyl or a monovalent moiety comprising at least one polar functional group when n is 1; or Z is alkylene, cycloalkylene or a polyvalent moiety comprising at least one polar functional group when n is greater than 1 ;

[0545] Rais, at each occurrence, independently H, C1-C12 alkyl, C1-C12 hydroxylalkyl, C1-C12 aminoalkyl, C1-C12 alkylaminylalkyl, C1-C12 alkoxyalkyl, C1-C12 alkoxycarbonyl, C1-C12 alkylcarbonyloxy, C1-C12 alkylcarbonyloxyalkyl or C1-C12 alkylcarbonyl;

[0546] R is, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R together with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;

[0547] R1and R2have, at each occurrence, the following structure, respectively: a1and a2are, at each occurrence, independently an integer from 3 to 12; b1and b2are, at each occurrence, independently 0 or 1 ; c1and c2are, at each occurrence, independently an integer from 5 to 10; d1and d2are, at each occurrence, independently an integer from 5 to 10; y is, at each occurrence, independently an integer from 0 to 2; and n is an integer from 1 to 6, wherein each alkyl, alkylene, hydroxylalkyl, aminoalkyl, alkylaminylalkyl, alkoxyalkyl, alkoxycarbonyl, alkylcarbonyloxy, alkylcarbonyloxyalkyl and alkylcarbonyl is optionally substituted with one or more substituent.

[0548] Formula (AXIV)

[0549] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: or a pharmaceutically acceptable salt, prodrug or stereoisomer thereof, wherein: one of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, -SC(=O)-, - RaC(=O)-, -C(=O)Ra-, RaC(=O)Ra-, -OC(=O)Ra- or - RaC(=O)O-, and the other of L1or L2is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, - RaC(=O)-, -C(=O)Ra-, RaC(=O)Ra-, -OC(=O)Ra- or -NRaC(=O)O- or a direct bond;

[0550] G1and G2are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

[0551] G3is C1-C24 alkylene, C1-C24 alkenylene, C3-C8cycloalkylene, C3-C8cycloalkenylene;

[0552] Rais H or C1-C12 alkyl;

[0553] R1and R2are each independently C6-C24 alkyl or C6-C24 alkenyl;

[0554] R3is H, OR5, CN, -C(=O)OR4, -OC(=O)R4or - R5C(=O)R4;

[0555] R4is C1-C12 alkyl;

[0556] R5is H or C1-C6alkyl; and x is 0, 1 or 2.

[0557] Formula (AXV)

[0558] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: AXV), or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0559] L1and L2are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, -SC(=O)-, -RaC(=O)-, -C(=O)Ra-, -RaC(=O)Ra-, -OC(=O)Ra-, -RaC(=O)O- or a direct bond;

[0560] G1is C1-C2 alkylene, -(C=O)-, -O(C=O)-, -SC(=O)-, -RaC(=O)- or a direct bond:

[0561] G2is -C(=O)-, -(C=O)O-, -C(=O)S-, -C(=O)NRa- or a direct bond;

[0562] G3is C1-C6alkylene;

[0563] Rais H or C1-C12 alkyl;

[0564] Rlaand Rlbare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) Rlais H or C1-C12 alkyl, and Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond;

[0565] R2aand R2bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R2ais H or C1-C12 alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0566] R3aand R3bare, at each occurrence, independently either (a): H or C1-C12 alkyl; or (b) R3ais H or C1-C12 alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R and the carbon atom to which it is bound to form a carbon-carbon double bond;

[0567] R4aand R4bare, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0568] R5and R6are each independently H or methyl;

[0569] R7is C4-C20 alkyl; R8and R9are each independently C1-C12 alkyl; or R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.

[0570] Formula (AXVI)

[0571] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

[0572] L1and L2are each independently -O(C=O)-, -(C=O)O- or a carbon-carbon double bond;

[0573] Rlaand Rlbare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) Rlais H or C1-C12 alkyl, and Rlbtogether with the carbon atom to which it is bound is taken together with an adjacent Rlband the carbon atom to which it is bound to form a carbon-carbon double bond;

[0574] R2aand R2bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R2ais H or C1-C12 alkyl, and R2btogether with the carbon atom to which it is bound is taken together with an adjacent R2band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0575] R3aand R3bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R3ais H or C1-C12 alkyl, and R3btogether with the carbon atom to which it is bound is taken together with an adjacent R3band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0576] R4aand R4bare, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R4ais H or C1-C12 alkyl, and R4btogether with the carbon atom to which it is bound is taken together with an adjacent R4band the carbon atom to which it is bound to form a carbon-carbon double bond;

[0577] R5and R6are each independently methyl or cyclo alkyl;

[0578] R7is, at each occurrence, independently H or C1-C12 alkyl; R8and R9are each independently unsubstituted C1-C12 alkyl; or R8and R9, together with the nitrogen atom to which they are attached, form a 5, 6 or 7- membered heterocyclic ring comprising one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; and e is 1 or 2, provided that: at least one of Rla, R2a, R3aor R4ais C1-C12 alkyl, or at least one of L1or L2is -O(C=O)- or -(C=O)O-; and

[0579] Rlaand Rlbare not isopropyl when a is 6 or n-butyl when a is 8.

[0580] Formula (AXVII)

[0581] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: pharmaceutically acceptable salt thereof, wherein

[0582] Ri and R2 are the same or different, each a linear or branched alkyl with 1-9 carbons, or as alkenyl or alkynyl with 2 to 11 carbon atoms,

[0583] Li and L2 are the same or different, each a linear alkyl having 5 to 18 carbon atoms, or form a heterocycle with N,

[0584] Xi is a bond, or is -CG-G- whereby L2-CO-O-R2 is formed,

[0585] X2 is S or O,

[0586] L3 is a bond or a lower alkyl, or form a heterocycle with N,

[0587] R3 is a lower alkyl, and

[0588] R4 and R5 are the same or different, each a lower alkyl.

[0589] Compounds (Al)-(All)

[0590] In some embodiments, the lipid nanoparticle comprises an ionizable lipid having the structure: pharmaceutically acceptable salt thereof.

[0591] In some embodiments, the lipid nanoparticle comprises a lipid having the structure:

[0592] (A2), or a pharmaceutically acceptable salt thereof.

[0593] In some embodiments, the lipid nanoparticle comprises a lipid having the structure:

[0594] (A3), or a pharmaceutically acceptable salt thereof.

[0595] In some embodiments, the lipid nanoparticle comprises a lipid having the structure:

[0596] (A4), or a pharmaceutically acceptable salt thereof.

[0597] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: (A5), or a pharmaceutically acceptable salt thereof.

[0598] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: (A6), or a pharmaceutically acceptable salt thereof.

[0599] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: (A7), or a pharmaceutically acceptable salt thereof.

[0600] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: (A8), or a pharmaceutically acceptable salt thereof.

[0601] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: (A9), or a pharmaceutically acceptable salt thereof.

[0602] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: (A 10), or a pharmaceutically acceptable salt thereof.

[0603] In some embodiments, the lipid nanoparticle comprises a lipid having the structure: (Al 1), or a pharmaceutically acceptable salt thereof.

[0604] Non-cationic lipids

[0605] In certain embodiments, the lipid nanoparticles described herein comprise one or more non-cationic lipids. Non-cationic lipids may be phospholipids. In some embodiments, the lipid nanoparticle comprises 5-25 mol% non-cationic lipid. For example, the lipid nanoparticle may comprise 5-20 mol%, 5-15 mol%, 5-10 mol%, 10-25 mol%, 10-20 mol%, 10-25 mol%, 15-25 mol%, 15-20 mol%, or 20-25 mol% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises 5 mol%, 10 mol%, 15 mol%, 20 mol%, or 25 mol% non-cationic lipid.

[0606] In some embodiments, a non-cationic lipid of the disclosure comprises 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC), l,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero- phosphocholine (DMPC), l,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1- palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3- phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3- phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2- dilinolenoyl-sn-glycero-3-phosphocholine,l,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2- didocosahexaenoyl-sn-glycero-3-phosphocholine, l,2-diphytanoyl-sn-glycero-3- phosphoethanolamine (ME 16.0 PE), l,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2- dilinoleoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinolenoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1 ,2- didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac- (1-glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof.

[0607] In some embodiments, the lipid nanoparticle comprises 5-15 mol%, 5-10 mol%, or 10-15 mol% DSPC. For example, the lipid nanoparticle may comprise 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 mol% DSPC.

[0608] In certain embodiments, the lipid composition of the lipid nanoparticle composition disclosed herein can comprise one or more phospholipids, for example, one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties.

[0609] A phospholipid moiety can be selected, for example, from the non-limiting group consisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol, phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.

[0610] A fatty acid moiety can be selected, for example, from the non-limiting group consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid can interact with one or more negatively charged phospholipids of a membrane (e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane can allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue.

[0611] Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triple bond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing a lipid bilayer of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye).

[0612] Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidy glycerols, and phosphatidic acids. Phospholipids also include phospho sphingolipid, such as sphingomyelin.

[0613] In some embodiments, a phospholipid comprises l,2-distearoyl-sn-glycero-3- phosphocholine (DSPC), l,2-Distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), l,2-dilinoleoyl-sn-glycero-3- phosphocholine (DLPC), 1,2-dimyristoyl-sn-gly cero-phosphocholine (DMPC), 1,2-dioleoyl-sn- glycero-3-phosphocholine (DOPC), l,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2- diundecanoyl-sn-glycero-phosphocholine (DUPC), l-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC), l,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2 cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl- sn-glycero-3-phosphocholine (C16 Lyso PC), l,2-dilinolenoyl-sn-glycero-3-phosphocholine,l,2- diarachidonoyl-sn-glycero-3-phosphocholine, l,2-didocosahexaenoyl-sn-glycero-3- phosphocholine, l,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1,2- distearoyl-sn-glycero-3-phosphoethanolamine, l,2-dilinoleoyl-sn-glycero-3- phosphoethanolamine, 1 ,2-dilinolenoyl-sn-glycero-3-phosphoethanolamine, 1 ,2-diarachidonoyl- sn-glycero-3-phosphoethanolamine, l,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, l,2-dioleoyl-sn-glycero-3-phospho-rac-(l -glycerol) sodium salt (DOPG), sphingomyelin, or mixtures thereof. Formula (HI)

[0614] In certain embodiments, a phospholipid is an analog or variant of DSPC. In certain embodiments, a phospholipid is a compound of Formula (HI): (HI), or a salt thereof, wherein: each R1is independently optionally substituted alkyl; or optionally two R1are joined together with the intervening atoms to form optionally substituted monocyclic carbocyclyl or optionally substituted monocyclic heterocyclyl; or optionally three R1are joined together with the intervening atoms to form optionally substituted bicyclic carbocyclyl or optionally substitute bicyclic heterocyclyl; n is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0615] A is of the formula: each instance of L2is independently a bond or optionally substituted Ci-6 alkylene, wherein one methylene unit of the optionally substituted Ci-6 alkylene is optionally replaced with O, N(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN); each instance of R2is independently optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl; optionally wherein one or more methylene units of R2are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), - OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O), OS(O), S(O)O, - OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or - N(RN)S(O)2O; each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

[0616] Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2. In certain embodiments, the compound is not of the formula: wherein each instance of R2is independently unsubstituted alkyl, unsubstituted alkenyl, or unsubstituted alkynyl.

[0617] In some embodiments, the phospholipids may be one or more of the phospholipids described in PCT Application No. PCT / US2018 / 037922.

[0618] In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25% noncationic lipid relative to the other lipid components. For example, the lipid nanoparticle may comprise a molar ratio of 5-30%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, 20-25%, or 25-30% non-cationic lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, 25%, or 30% non-cationic lipid.

[0619] In some embodiments, the lipid nanoparticle comprises a molar ratio of 5-25% phospholipid relative to the other lipid components. For example, the lipid nanoparticle may comprise a molar ratio of 5-30%, 5-15%, 5-10%, 10-25%, 10-20%, 10-25%, 15-25%, 15-20%, 20-25%, or 25-30% phospholipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 5%, 10%, 15%, 20%, 25%, or 30% phospholipid lipid. Structural lipids

[0620] The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids. As used herein, the term “structural lipid” includes sterols and also to lipids containing sterol moieties.

[0621] Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol.

[0622] In some embodiments, the structural lipids may be one or more of the structural lipids described in U.S. Application No. 16 / 493,814. In some embodiments, the lipid nanoparticle comprises a molar ratio of 25-55% structural lipid relative to the other lipid components. For example, the lipid nanoparticle may comprise a molar ratio of 10- 55%, 25-50%, 25-45%, 25-40%, 25-35%, 25-30%, 30-55%, 30- 50%, 30-45%, 30-40%, 30-35%, 35-55%, 35-50%, 35-45%, 35-40%, 40-55%, 40-50%, 40-45%, 45-55%, 45-50%, or 50-55% structural lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55% structural lipid.

[0623] In some embodiments, the lipid nanoparticle comprises 30-45 mol% sterol, optionally 35- 40 mol%, for example, 30-31 mol%, 31-32 mol%, 32-33 mol%, 33-34 mol%, 34-35 mol%, 35- 36 mol%, 36-37 mol%, 37-38 mol%, 38-39 mol%, or 39-40 mol%. In some embodiments, the lipid nanoparticle comprises 25-55 mol% sterol. For example, the lipid nanoparticle may comprise 25-50 mol%, 25-45 mol%, 25-40 mol%, 25-35 mol%, 25-30 mol%, 30-55 mol%, 30- 50 mol%, 30-45 mol%, 30-40 mol%, 30-35 mol%, 35-55 mol%, 35-50 mol%, 35-45 mol%, 35- 40 mol%, 40-55 mol%, 40-50 mol%, 40-45 mol%, 45-55 mol%, 45-50 mol%, or 50-55 mol% sterol. In some embodiments, the lipid nanoparticle comprises 25 mol%, 30 mol%, 35 mol%, 40 mol%, 45 mol%, 50 mol%, or 55 mol% sterol.

[0624] In some embodiments, the lipid nanoparticle comprises 35 - 40 mol% cholesterol. For example, the lipid nanoparticle may comprise 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, or 40 mol% cholesterol.

[0625] Polyethylene glycol (PEG)-Lipids

[0626] The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more polyethylene glycol (PEG) lipids.

[0627] As used herein, the term “PEG-lipid” or “PEG-modified lipid” refers to polyethylene glycol (PEG) -modified lipids. Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, and PEG-modified l,2-diacyloxypropan-3- amines. Such lipids are also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0628] In some embodiments, the PEG-lipid includes, but not limited to 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), l,2-distearoyl-sn-glycero-3- phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEGDAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1, 2- dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the PEG-lipid is selected from the group consisting of a PEG- modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the PEG-modified lipid is PEG- DMG, PEG-c-DOMG (also referred to as PEG-DOMG), PEG-DSG, and / or PEG-DPG.

[0629] In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about Ci4 to about C22, preferably from about C14 to about C16. In some embodiments, a PEG moiety, for example an mPEG-Nth, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some embodiments, the PEG-lipid is PEG2k-DMG.

[0630] In some embodiments, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG- DSG and PEG-DSPE.

[0631] PEG-lipids are known in the art, such as those described in U.S. Patent No. 8158601 and International Publ. No. WO 2015 / 130584 A2, which are incorporated herein by reference in their entirety.

[0632] In general, some of the other lipid components (e.g., PEG lipids) of various formulae described herein may be synthesized as described International Patent Application No. PCT / US2016 / 000129, filed December 10, 2016, entitled “Compositions and Methods for Delivery of Therapeutic Agents,” which is incorporated by reference in its entirety.

[0633] The lipid component of a lipid nanoparticle composition may include one or more molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, a PEG lipid may be PEG-c-DOMG, PEG- DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.

[0634] In some embodiments the PEG-modified lipids are a modified form of PEG DMG. PEG- DMG has the following structure:

[0635] In some embodiments, PEG lipids can be PEGylated lipids described in International

[0636] Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety. Any of these exemplary PEG lipids described herein may be modified to comprise a hydroxyl group on the PEG chain. In certain embodiments, the PEG lipid is a PEG-OH lipid. As generally defined herein, a “PEG-OH lipid” (also referred to herein as “hydroxy-PEGylated lipid”) is a PEGylated lipid having one or more hydroxyl (-OH) groups on the lipid. In certain embodiments, the PEG-OH lipid includes one or more hydroxyl groups on the PEG chain. In certain embodiments, a PEG-OH or hydroxy-PEGylated lipid comprises an -OH group at the terminus of the PEG chain. Each possibility represents a separate embodiment.

[0637] Formula (PI)

[0638] In certain embodiments, a PEG lipid is a compound of Formula (PI): (PI), or salts thereof, wherein:

[0639] R3is -OR°;

[0640] R° is hydrogen, optionally substituted alkyl, or an oxygen protecting group; r is an integer between 1 and 100, inclusive;

[0641] L1is optionally substituted C1-10 alkylene, wherein at least one methylene of the optionally substituted C1-10 alkylene is independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, O, N(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN);

[0642] D is a moiety obtained by click chemistry or a moiety cleavable under physiological conditions; m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10;

[0643] A is of the formula: each instance of L2is independently a bond or optionally substituted Ci-6 alkylene, wherein one methylene unit of the optionally substituted Ci-6 alkylene is optionally replaced with O, N(RN), S, C(O), C(O)N(RN), NRNC(O), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, or NRNC(O)N(RN); each instance of R2is independently optionally substituted C1-30 alkyl, optionally substituted C1-30 alkenyl, or optionally substituted C1-30 alkynyl; optionally wherein one or more methylene units of R2are independently replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), - OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), NRNC(S)N(RN), S(O) , OS(O), S(O)O, - OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), N(RN)S(O)2N(RN), OS(O)2N(RN), or - N(RN)S(O)2O; each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group;

[0644] Ring B is optionally substituted carbocyclyl, optionally substituted heterocyclyl, optionally substituted aryl, or optionally substituted heteroaryl; and p is 1 or 2.

[0645] In certain embodiments, the compound of Formula (PI) is a PEG-OH lipid (z.e., R3is - OR°, and R° is hydrogen). In certain embodiments, the compound of Formula (PI) is of Formula (PI-OH): (PI-OH), or a salt thereof.

[0646] Formula (PH)

[0647] In certain embodiments, a PEG lipid is a PEGylated fatty acid. In certain embodiments, a PEG lipid is a compound of Formula (PII). In some embodiments, compounds of Formula (PII) have the following formula:

[0648] (PII), or a salts thereof, wherein:

[0649] R3is-OR°;

[0650] R° is hydrogen, optionally substituted alkyl or an oxygen protecting group; r is an integer between 1 and 100, inclusive;

[0651] R5is optionally substituted C10-40 alkyl, optionally substituted C10-40 alkenyl, or optionally substituted C10-40 alkynyl; and optionally one or more methylene groups of R5are replaced with optionally substituted carbocyclylene, optionally substituted heterocyclylene, optionally substituted arylene, optionally substituted heteroarylene, N(RN), O, S, C(O), C(O)N(RN), - NRNC(O), NRNC(O)N(RN), C(O)O, OC(O), OC(O)O, OC(O)N(RN), NRNC(O)O, C(O)S, SC(O), C(=NRN), C(=NRN)N(RN), NRNC(=NRN), NRNC(=NRN)N(RN), C(S), C(S)N(RN), NRNC(S), - NRNC(S)N(RN), S(O), OS(O), S(O)O, OS(O)O, OS(O)2, S(O)2O, OS(O)2O, N(RN)S(O), - S(O)N(RN), N(RN)S(O)N(RN), OS(O)N(RN), N(RN)S(O)O, S(O)2, N(RN)S(O)2, S(O)2N(RN), - N(RN)S(O)2N(RN), OS(O)2N(RN), or N(RN)S(O)2O; and each instance of RNis independently hydrogen, optionally substituted alkyl, or a nitrogen protecting group. In certain embodiments, the compound of Formula (PII) is of Formula (PII-OH):

[0652] O (PII-OH), or a salt thereof. In some embodiments, r is 40-50.

[0653] In yet other embodiments the compound of Formula (PII) is:

[0654] In some embodiments, the lipid composition of the pharmaceutical compositions disclosed herein does not comprise a PEG-lipid.

[0655] In some embodiments, the PEG-lipids may be one or more of the PEG lipids described in U.S. Application No. US 15 / 674,872.

[0656] In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5-15% PEG lipid relative to the other lipid components. For example, the lipid nanoparticle may comprise a molar ratio of 0.5-10%, 0.5-5%, 1-15%, 1-10%, 1-5%, 2-15%, 2-10%, 2-5%, 5-15%, 5-10%, or 10-15% PEG lipid. In some embodiments, the lipid nanoparticle comprises a molar ratio of 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% PEG- lipid.

[0657] In some embodiments, the lipid nanoparticle comprises 1-5% PEG-modified lipid, optionally 1-3 mol%, for example 1.5 to 2.5 mol%, 1-2 mol%, 2-3 mol%, 3-4 mol%, or 4-5 mol%. In some embodiments, the lipid nanoparticle comprises 0.5-15 mol% PEG-modified lipid. For example, the lipid nanoparticle may comprise 0.5-10 mol%, 0.5-5 mol%, 1-15 mol%, 1-10 mol%, 1-5 mol%, 2-15 mol%, 2-10 mol%, 2-5 mol%, 5-15 mol%, 5-10 mol%, or 10-15 mol%. In some embodiments, the lipid nanoparticle comprises 0.5 mol%, 1 mol%, 2 mol%, 3 mol%, 4 mol%, 5 mol%, 6 mol%, 7 mol%, 8 mol%, 9 mol%, 10 mol%, 11 mol%, 12 mol%, 13 mol%, 14 mol%, or 15 mol% PEG-modified lipid.

[0658] Some embodiments comprise adding PEG to a composition comprising an LNP encapsulating a nucleic acid (e.g., which already includes PEG in the amounts listed above). In embodiments comprise adding about 0.5mo% or more PEG to an LNP composition, such as about lmol%, about 1.5mol%, about 2mol%, about 2.5mol%, about 3mol%, about 3.5mol%, about 4mol%, about 5mol%, or more after formation of an LNP composition (e.g., which already contains PEG in amount listed elsewhere herein).

[0659] In some embodiments, the lipid nanoparticle comprises 20-60 mol% ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% sterol, and 0.5-15 mol% PEG-modified lipid.

[0660] In some embodiments, a LNP of the disclosure comprises an ionizable amino lipid of Compound 1, wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG lipid is DMG-PEG.

[0661] In some embodiments, a LNP of the disclosure comprises an ionizable amino lipid of Compound 2, wherein the non-cationic lipid is DSPC, the structural lipid that is cholesterol, and the PEG lipid is DMG-PEG.

[0662] In some embodiments, a LNP comprises an ionizable amino lipid of any of Formula (AIII), (AIV), or (AV), a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising PEG-DMG.

[0663] In some embodiments, a LNP comprises an ionizable amino lipid of any of Formula (AIII), (AIV), or (AV), a phospholipid comprising DSPC, a structural lipid, and a PEG lipid comprising a compound having Formula (PII).

[0664] In some embodiments, a LNP comprises an ionizable amino lipid of Formula (AIII), (AIV), or (AV), a phospholipid comprising a compound having Formula (HI), a structural lipid, and the PEG lipid comprising a compound having Formula (PI) or (PII).

[0665] In some embodiments, a LNP comprises an ionizable amino lipid of Formula (AIII), (AIV), or (AV), a phospholipid comprising a compound having Formula (HI), a structural lipid, and the PEG lipid comprising a compound having Formula (PI) or (PII).

[0666] In some embodiments, a LNP comprises an ionizable amino lipid of Formula (AIII), (AIV), or (AV), a phospholipid having Formula (HI), a structural lipid, and a PEG lipid comprising a compound having Formula (PII).

[0667] In some embodiments, the lipid nanoparticle comprises 49 mol% ionizable amino lipid,

[0668] 10 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.

[0669] In some embodiments, the lipid nanoparticle comprises 49 mol% ionizable amino lipid,

[0670] 11 mol% DSPC, 38.5 mol% cholesterol, and 1.5 mol% DMG-PEG.

[0671] In some embodiments, the lipid nanoparticle comprises 48 mol% ionizable amino lipid, 11 mol% DSPC, 38.5 mol% cholesterol, and 2.5 mol% DMG-PEG.

[0672] In some embodiments, a LNP comprises an N:P ratio of from about 2:1 to about 30:1.

[0673] In some embodiments, a LNP comprises an N:P ratio of about 6:1.

[0674] In some embodiments, a LNP comprises an N:P ratio of about 3:1, 4:1, or 5:1. In some embodiments, a LNP comprises a wt / wt ratio of the ionizable amino lipid component to the RNA of from about 10:1 to about 100:1.

[0675] In some embodiments, a LNP comprises a wt / wt ratio of the ionizable amino lipid component to the RNA of about 20:1.

[0676] In some embodiments, a LNP comprises a wt / wt ratio of the ionizable amino lipid component to the RNA of about 10:1.

[0677] Some embodiments comprise a composition having one or more LNPs having a diameter of about 150 nm or less, such as about 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less. Some embodiments comprise a composition having a mean LNP diameter of about 150 nm or less, such as about 140 nm, 130 nm, 120 nm, 110 nm, 100 nm, 90 nm, 80 nm, 70 nm, 60 nm, 50 nm, 40 nm, 30 nm, or 20 nm or less. In some embodiments, the composition has a mean LNP diameter from about 30nm to about 150nm, or a mean diameter from about 60nm to about 120nm.

[0678] A LNP may comprise or one or more types of lipids, including but not limited to amino lipids (e.g., ionizable amino lipids), neutral lipids, non-cationic lipids, charged lipids, PEG- modified lipids, phospholipids, structural lipids and sterols. In some embodiments, a LNP may further comprise one or more cargo molecules, including but not limited to nucleic acids (e.g., mRNA, plasmid DNA, DNA or RNA oligonucleotides, siRNA, shRNA, snRNA, snoRNA, IncRNA, etc.), small molecules, proteins and peptides.

[0679] In some embodiments, the composition comprises a liposome. A liposome is a lipid particle comprising lipids arranged into one or more concentric lipid bilayers around a central region. The central region of a liposome may comprises an aqueous solution, suspension, or other aqueous composition.

[0680] In some embodiments, a lipid nanoparticle may comprise two or more components (e.g., amino lipid and nucleic acid, PEG-lipid, phospholipid, structural lipid). For instance, a lipid nanoparticle may comprise an amino lipid and a nucleic acid. Compositions comprising the lipid nanoparticles, such as those described herein, may be used for a wide variety of applications, including the stealth delivery of therapeutic payloads with minimal adverse innate immune response.

[0681] Effective in vivo delivery of nucleic acids represents a continuing medical challenge. Exogenous nucleic acids (z.e., originating from outside of a cell or organism) are readily degraded in the body, e.g., by the immune system. Accordingly, effective delivery of nucleic acids to cells often requires the use of a particulate carrier (e.g., lipid nanoparticles). The particulate carrier should be formulated to have minimal particle aggregation, be relatively stable prior to intracellular delivery, effectively deliver nucleic acids intracellularly, and illicit no or minimal immune response. To achieve minimal particle aggregation and pre-delivery stability, many conventional particulate carriers have relied on the presence and / or concentration of certain components (e.g., PEG-lipid). However, it has been discovered that certain components may decrease the stability of encapsulated nucleic acids (e.g., mRNA molecules). The reduced stability may limit the broad applicability of the particulate carriers. As such, there remains a need for methods by which to improve the stability of nucleic acid (e.g., mRNA) encapsulated within lipid nanoparticles.

[0682] In some embodiments, the lipid nanoparticles comprise one or more of ionizable molecules, polynucleotides, and optional components, such as structural lipids, sterols, neutral lipids, phospholipids and a molecule capable of reducing particle aggregation (e.g., polyethylene glycol (PEG), PEG-modified lipid), such as those described above.

[0683] In some embodiments, a LNP described herein may include one or more ionizable molecules (e.g., amino lipids or ionizable lipids). The ionizable molecule may comprise a charged group and may have a certain pKa. In certain embodiments, the pKa of the ionizable molecule may be greater than or equal to about 6, greater than or equal to about 6.2, greater than or equal to about 6.5, greater than or equal to about 6.8, greater than or equal to about 7, greater than or equal to about 7.2, greater than or equal to about 7.5, greater than or equal to about 7.8, greater than or equal to about 8. In some embodiments, the pKa of the ionizable molecule may be less than or equal to about 10, less than or equal to about 9.8, less than or equal to about 9.5, less than or equal to about 9.2, less than or equal to about 9.0, less than or equal to about 8.8, or less than or equal to about 8.5. Combinations of the above referenced ranges are also possible (e.g., greater than or equal to 6 and less than or equal to about 8.5). Other ranges are also possible. In embodiments in which more than one type of ionizable molecule are present in a particle, each type of ionizable molecule may independently have a pKa in one or more of the ranges described above.

[0684] In general, an ionizable molecule comprises one or more charged groups. In some embodiments, an ionizable molecule may be positively charged or negatively charged. For instance, an ionizable molecule may be positively charged. For example, an ionizable molecule may comprise an amine group. As used herein, the term “ionizable molecule” has its ordinary meaning in the art and may refer to a molecule or matrix comprising one or more charged moiety. As used herein, a “charged moiety” is a chemical moiety that carries a formal electronic charge, e.g., monovalent (+1, or -1), divalent (+2, or -2), trivalent (+3, or -3), etc. The charged moiety may be anionic (z.e., negatively charged) or cationic (z.e., positively charged). Examples of positively-charged moieties include amine groups (e.g., primary, secondary, and / or tertiary amines), ammonium groups, pyridinium group, guanidine groups, and imidizolium groups. In a particular embodiment, the charged moieties comprise amine groups. Examples of negatively- charged groups or precursors thereof, include carboxylate groups, sulfonate groups, sulfate groups, phosphonate groups, phosphate groups, hydroxyl groups, and the like. The charge of the charged moiety may vary, in some cases, with the environmental conditions, for example, changes in pH may alter the charge of the moiety, and / or cause the moiety to become charged or uncharged. In general, the charge density of the molecule and / or matrix may be selected as desired.

[0685] In some cases, an ionizable molecule (e.g., an amino lipid or ionizable lipid) may include one or more precursor moieties that can be converted to charged moieties. For instance, the ionizable molecule may include a neutral moiety that can be hydrolyzed to form a charged moiety, such as those described above. As a non-limiting specific example, the molecule or matrix may include an amide, which can be hydrolyzed to form an amine, respectively. Those of ordinary skill in the art will be able to determine whether a given chemical moiety carries a formal electronic charge (for example, by inspection, pH titration, ionic conductivity measurements, etc.), and / or whether a given chemical moiety can be reacted (e.g., hydrolyzed) to form a chemical moiety that carries a formal electronic charge.

[0686] The ionizable molecule (e.g., amino lipid or ionizable lipid) may have any suitable molecular weight. In certain embodiments, the molecular weight of an ionizable molecule is less than or equal to about 2,500 g / mol, less than or equal to about 2,000 g / mol, less than or equal to about 1,500 g / mol, less than or equal to about 1,250 g / mol, less than or equal to about 1,000 g / mol, less than or equal to about 900 g / mol, less than or equal to about 800 g / mol, less than or equal to about 700 g / mol, less than or equal to about 600 g / mol, less than or equal to about 500 g / mol, less than or equal to about 400 g / mol, less than or equal to about 300 g / mol, less than or equal to about 200 g / mol, or less than or equal to about 100 g / mol. In some instances, the molecular weight of an ionizable molecule is greater than or equal to about 100 g / mol, greater than or equal to about 200 g / mol, greater than or equal to about 300 g / mol, greater than or equal to about 400 g / mol, greater than or equal to about 500 g / mol, greater than or equal to about 600 g / mol, greater than or equal to about 700 g / mol, greater than or equal to about 1000 g / mol, greater than or equal to about 1,250 g / mol, greater than or equal to about 1,500 g / mol, greater than or equal to about 1,750 g / mol, greater than or equal to about 2,000 g / mol, or greater than or equal to about 2,250 g / mol. Combinations of the above ranges (e.g., at least about 200 g / mol and less than or equal to about 2,500 g / mol) are also possible. In embodiments in which more than one type of ionizable molecules are present in a particle, each type of ionizable molecule may independently have a molecular weight in one or more of the ranges described above. In some embodiments, the percentage (e.g., by weight, or by mole) of a single type of ionizable molecule (e.g., amino lipid or ionizable lipid) and / or of all the ionizable molecules within a particle may be greater than or equal to about 15%, greater than or equal to about 16%, greater than or equal to about 17%, greater than or equal to about 18%, greater than or equal to about 19%, greater than or equal to about 20%, greater than or equal to about 21%, greater than or equal to about 22%, greater than or equal to about 23%, greater than or equal to about 24%, greater than or equal to about 25%, greater than or equal to about 30%, greater than or equal to about 35%, greater than or equal to about 40%, greater than or equal to about 42%, greater than or equal to about 45%, greater than or equal to about 48%, greater than or equal to about 50%, greater than or equal to about 52%, greater than or equal to about 55%, greater than or equal to about 58%, greater than or equal to about 60%, greater than or equal to about 62%, greater than or equal to about 65%, or greater than or equal to about 68%. In some instances, the percentage (e.g., by weight, or by mole) may be less than or equal to about 70%, less than or equal to about 68%, less than or equal to about 65%, less than or equal to about 62%, less than or equal to about 60%, less than or equal to about 58%, less than or equal to about 55%, less than or equal to about 52%, less than or equal to about 50%, or less than or equal to about 48%. Combinations of the above referenced ranges are also possible (e.g., greater than or equal to 20% and less than or equal to about 60%, greater than or equal to 40% and less than or equal to about 55%, etc.). In embodiments in which more than one type of ionizable molecule is present in a particle, each type of ionizable molecule may independently have a percentage (e.g., by weight, or by mole) in one or more of the ranges described above. The percentage (e.g., by weight, or by mole) may be determined by extracting the ionizable molecule(s) from the dried particles using, e.g., organic solvents, and measuring the quantity of the agent using high pressure liquid chromatography (i.e., HPLC), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), or mass spectrometry (MS). Those of ordinary skill in the art would be knowledgeable of techniques to determine the quantity of a component using the above-referenced techniques. For example, HPLC may be used to quantify the amount of a component, by, e.g., comparing the area under the curve of a HPLC chromatogram to a standard curve.

[0687] It should be understood that the terms “charged” or “charged moiety” does not refer to a “partial negative charge" or “partial positive charge" on a molecule. The terms “partial negative charge" and “partial positive charge" are given their ordinary meaning in the art. A “partial negative charge" may result when a functional group comprises a bond that becomes polarized such that electron density is pulled toward one atom of the bond, creating a partial negative charge on the atom. Those of ordinary skill in the art will, in general, recognize bonds that can become polarized in this way. According to the disclosures herein, a lipid composition may comprise one or more lipids as described herein. Such lipids may include those useful in the preparation of lipid nanoparticle formulations as described above or as known in the art.

[0688] In some embodiments, the nucleic acids are in (e.g., formulated as) a lipid composition, such as a composition comprising a lipid nanoparticle. In some embodiments, nucleic acids of the present disclosure are in (e.g., formulated as) lipid nanoparticle (LNP) compositions. Lipid nanoparticles typically comprise an ionizable amino lipid, non-cationic lipid (e.g., a phospholipid), structural lipid, and PEG lipid components along with the nucleic acid cargo (i.e., mRNA) of interest. A lipid nanoparticles of the present disclosure can be generated using components, compositions, and methods as are generally known in the art, see for example PCT / US2016 / 052352; PCT / US2016 / 068300; PCT / US2017 / 037551; PCT / US2015 / 027400; PCT / US2016 / 047406; PCT / US2016 / 000129; PCT / US2016 / 014280; PCT / US2017 / 038426; PCT / US2014 / 027077; PCT / US2014 / 055394; PCT / US2016 / 052117; PCT / US2012 / 069610; PCT / US2017 / 027492; PCT / US2016 / 059575; PCT / US2016 / 069491; PCT / US2016 / 069493; and PCT / US2014 / 066242, all of which are incorporated by reference herein in their entirety.

[0689] In some embodiments, a lipid nanoparticle comprises at least one ionizable amino lipid, at least one non-cationic lipid, at least one sterol, and / or at least one polyethylene glycol (PEG)- modified lipid.

[0690] In some embodiments, a lipid nanoparticle comprises 20-60 mole percent (mol%) ionizable amino lipid, 5-25 mol% non-cationic lipid, 25-55 mol% structural lipid, and 0.5-15 mol% PEG-modified lipid.

[0691] In some embodiments, a lipid nanoparticle comprises 20-60 mol% ionizable amino lipid, 5-30 mol% non-cationic lipid, 10-55 mol% structural lipid, and 0.5-15 mol% PEG-modified lipid.

[0692] In some embodiments, a lipid nanoparticle comprises 40-50 mol% ionizable lipid, optionally 45-50 mol%, for example, 45-46 mol%, 46-47 mol%, 47-48 mol%, 48-49 mol%, or 49-50 mol% for example about 45 mol%, 45.5 mol%, 46 mol%, 46.5 mol%, 47 mol%, 47.5 mol%, 48 mol%, 48.5 mol%, 49 mol%, or 49.5 mol%.

[0693] In some embodiments, a lipid nanoparticle comprises 20-60 mol% ionizable amino lipid. For example, a lipid nanoparticle may comprise 20-50 mol%, 20-40 mol%, 20-30 mol%, 30-60 mol%, 30-50 mol%, 30-40 mol%, 40-60 mol%, 40-50 mol%, or 50-60 mol% ionizable amino lipid. In some embodiments, a lipid nanoparticle comprises 20 mol%, 30 mol%, 40 mol%, 50 mol%, or 60 mol% ionizable amino lipid. In some embodiments, a lipid nanoparticle comprises 35 mol%, 36 mol%, 37 mol%, 38 mol%, 39 mol%, 40 mol%, 41 mol%, 42 mol%, 43 mol%, 44 mol%, 45 mol%, 46 mol%, 47 mol%, 48 mol%, 49 mol%, 50 mol%, 51 mol%, 52 mol%, 53 mol%, 54 mol%, or 55 mol% ionizable amino lipid.

[0694] In some embodiments, a lipid nanoparticle comprises 45-55 mol% ionizable amino lipid. For example, lipid nanoparticle may comprise 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 mol% ionizable amino lipid.

[0695] Ionizable amino lipids

[0696] In some embodiments, the ionizable lipid is a compound of Formula (IL*) or a salt thereof, wherein:

[0697] R1is -OH, -NRN-C4-IO cycloalkenyl optionally substituted with one or more oxo or - N(RNRN);

[0698] RNis H or C1-6 alkyl;

[0699] RN’ is H or C1-6 alkyl;

[0700] RN” is H or C1-6 alkyl; o is 1, 2, 3, or 4; n is 4, 5, 6, 7, or 8; m is 4, 5, 6, 7, or 8;

[0701] M is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R2;

[0702] M’ is -C(=O)-O-* or -O-C(=O)-*, wherein * indicates attachment to R3;

[0703] R2i

[0704] R2ais -H or Ci-io alkyl;

[0705] R2bis -H or Ci-io alkyl;

[0706] R2Cis Ci-8 alkyl or C2-8 alkenyl;

[0707] Y^ Y R3C

[0708] R3isR3aR3b .

[0709] R3ais H or Ci-10 alkyl;

[0710] R3bis H or C1-8 alkyl; and

[0711] R3Cis Ci-10 alkyl or C2-8 alkenyl. In some embodiments, the ionizable lipid is of Formula (IL**-I): or a salt thereof, wherein:

[0712] R1is -OH; o is 2, 3, or 4; n is 4, 5, 6, 7, or 8;

[0713] M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8;

[0714] M’ is -C(=O)-O-*, wherein * indicates attachment to R3;

[0715] R2Cis C4-8 alkyl;

[0716] R3ais C7-10 alkyl; and

[0717] R3Cis C3-5 alkyl.

[0718] In some embodiments, the ionizable lipid is of Formula (IL**-III): or a salt thereof, wherein:

[0719] R1is NRN-C4-IO cycloalkenyl optionally substituted with one or more oxo or -N(RNRN);

[0720] RNis H;

[0721] RN’ is C1-2 alkyl;

[0722] RN” is H; o is 2, 3, or 4; n is 6, 7, or 8;

[0723] M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8;

[0724] M’ is -C(=O)-O-*, wherein * indicates attachment to R3;

[0725] R2ais C7-10 alkyl;

[0726] R2Cis C4-6 alkyl;

[0727] R3ais C1-3 alkyl; and

[0728] R3Cis C4-6 alkyl.

[0729] In some embodiments, the ionizable lipid is of Formula (IL**-IV): or a salt thereof, wherein:

[0730] R1is OH; o is 2, 3, or 4; n is 6, 7, or 8;

[0731] M is -C(=O)-O-*, wherein * indicates attachment to R2; m is 6, 7, or 8;

[0732] M’ is -C(=O)-O-*, wherein * indicates attachment to R3;

[0733] R2bis C3-5 alkyl;

[0734] R2Cis C2-4 alkyl;

[0735] R3ais C7-10 alkyl; and

[0736] R3Cis C4-6 alkyl.

[0737] In some embodiments, the ionizable lipid is of Formula (IL*-I): ( ) or a salt thereof, wherein: R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*; and R3ais Ci-8 alkyl.

[0738] In some embodiments, ionizable lipid is of Formula (IL*-Ia): or a salt thereof, wherein:

[0739] R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais Ci-8 alkyl.

[0740] In some embodiments, the ionizable lipid is of Formula (IL*-Ia’): or a salt thereof, wherein: o, M, M’, R2cand R3care as defined for variable IL*; and R3ais Ci-8 alkyl.

[0741] In some embodiments, the ionizable lipid is of Formula (IL*-IIa):

[0742] (IL*-IIa) or a salt thereof, wherein:

[0743] R1, o, m, n, M, M’, R2c, and R3care as defined for Formula IL*; and R3ais Ci-8 alkyl.

[0744] In some embodiments, the ionizable lipid is of Formula (IL*-II’):

[0745] (IL*-IF) or a salt thereof, wherein: o, M, M’, R2cand R3care as defined for variable IL*; and R3ais Ci-8 alkyl.

[0746] In some embodiments, the ionizable lipid is of Formula (IL*-III):

[0747] (IL*-III) or a salt thereof, wherein:

[0748] R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;

[0749] R2ais a Ci-8 alkyl; and

[0750] R3ais Ci-8 alkyl.

[0751] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa):

[0752] (IL*-IIIa) or a salt thereof, wherein:

[0753] R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;

[0754] R2bis a Ci-8 alkyl; and

[0755] R3ais Ci-8 alkyl.

[0756] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa): or a salt thereof, wherein:

[0757] R1, o, M, M’, R2c, and R3care as defined for variable IL*;

[0758] R2ais a Ci-8 alkyl; and

[0759] R3ais Ci-8 alkyl.

[0760] In some embodiments, the ionizable lipid is of Formula (IL*-IIIa’): or a salt thereof, wherein:

[0761] R1, o, M, M’, R2c, and R3care as defined for variable IL*;

[0762] R2ais a Ci-8 alkyl; and

[0763] R3ais Ci-8 alkyl.

[0764] In some embodiments, the ionizable lipid is of Formula (IL*-IIIb): or a salt thereof, wherein:

[0765] R\ o, M, M’, R2c, and R3care as defined for variable IL*;

[0766] R2ais a Ci-8 alkyl; and

[0767] R3ais Ci-8 alkyl.

[0768] In some embodiments, the ionizable lipid is of Formula (IL*-IIIb’):

[0769] (IL*-IIIb’) or a salt thereof, wherein:

[0770] R\ o, M, M’, R2c, and R3care as defined for variable IL*;

[0771] R2ais a Ci-8 alkyl; and

[0772] R3ais Ci-8 alkyl.

[0773] In some embodiments, the ionizable lipid is of Formula (IL*-IV):

[0774] (IL*-IV) or a salt thereof, wherein:

[0775] R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;

[0776] R2bis a Ci-8 alkyl; and

[0777] R3ais Ci-8 alkyl.

[0778] In some embodiments, the ionizable lipid is of Formula (IL*-IVa):

[0779] (IL*-IVa) or a salt thereof, wherein:

[0780] R1, o, m, n, M, M’, R2c, and R3care as defined for variable IL*;

[0781] R2bis a Ci-8 alkyl; and

[0782] R3ais Ci-8 alkyl.

[0783] In some embodiments, the ionizable lipid is of Formula (IL*-Iva’): or a salt thereof, wherein: o, M, M’, R2C, and R3care as defined for variable IL*;

[0784] R2ais a Ci-8 alkyl; and

[0785] R3ais Ci-8 alkyl.

[0786] Variables o, R1, RN, RN, RNof Ionizable Lipid

[0787] In some embodiments of the ionizable lipid, o is 1.

[0788] In some embodiments of the ionizable lipid, o is 2.

[0789] In some embodiments of the ionizable lipid, o is 3.

[0790] In some embodiments of the ionizable lipid, o is 4.

[0791] In some embodiments of the ionizable lipid, R1is -OH.

[0792] In some embodiments of the ionizable lipid, RNis H.

[0793] In some embodiments of the ionizable lipid, RNis methyl.

[0794] In some embodiments of the ionizable lipid, RNis ethyl.

[0795] In some embodiments of the ionizable lipid, R1is -NRN-cyclobutenyl, wherein the cyclobutenyl is optionally substituted with one or more oxo or -N(RNRN).

[0796] In some embodiments of the ionizable lipid, RNis H.

[0797] In some embodiments of the ionizable lipid, RNis methyl.

[0798] In some embodiments of the ionizable lipid, RNis ethyl.

[0799] In some embodiments of the ionizable lipid, RNis H.

[0800] In some embodiments of the ionizable lipid, RNis methyl.

[0801] In some embodiments of the ionizable lipid, RNis ethyl.

[0802] In some embodiments of the ionizable lipid, RNis H and RNis methyl.

[0803] In some embodiments of the ionizable lipid,

[0804] In some embodiments of the ionizable lipid, Variables m and n of the Ionizable Lipid

[0805] In some embodiments of the ionizable lipid, m is 4.

[0806] In some embodiments of the ionizable lipid, m is 5.

[0807] In some embodiments of the ionizable lipid, m is 6.

[0808] In some embodiments of the ionizable lipid, m is 7.

[0809] In some embodiments of the ionizable lipid, m is 8.

[0810] In some embodiments of the ionizable lipid, m is 4.

[0811] In some embodiments of the ionizable lipid, n is 5.

[0812] In some embodiments of the ionizable lipid, n is 6.

[0813] In some embodiments of the ionizable lipid, n is 7.

[0814] In some embodiments of the ionizable lipid, n is 8.

[0815] In some embodiments of the ionizable lipid, n is 5 and m is 7.

[0816] In some embodiments of the ionizable lipid, n is 7 and m is 7.

[0817] In some embodiments of the ionizable lipid, m is 6 and n is 6.

[0818] Variables M and M’

[0819] In some embodiments of the ionizable lipid, M is -O-C(=O)-*, wherein * indicates attachment to R2.

[0820] In some embodiments of the ionizable lipid, M is -C(=O)-O-* wherein * indicates attachment to R2.

[0821] In some embodiments of the ionizable lipid, M’ is -O-C(=O)-*, wherein * indicates attachment to R3.

[0822] In some embodiments of the ionizable lipid, M’ is -C(=O)-O-* wherein * indicates attachment to R3.

[0823] In some embodiments of the ionizable lipid, M is -O-C(=O)-*, wherein * indicates attachment to R2, and M’ is -C(=O)-O-* wherein * indicates attachment to R3

[0824] Variables R2, R2a, R2b, R2c

[0825] R2aR2b

[0826] In some embodiments of the ionizable lipid, R is

[0827] In some embodiments of the ionizable lipid, R2ais hydrogen.

[0828] In some embodiments of the ionizable lipid, R2ais methyl.

[0829] In some embodiments of the ionizable lipid, R2ais ethyl.

[0830] In some embodiments of the ionizable lipid, R2ais propyl.

[0831] In some embodiments of the ionizable lipid, R2ais butyl.

[0832] In some embodiments of the ionizable lipid, R2ais pentyl. In some embodiments of the ionizable lipid, R2ais hexyl.

[0833] In some embodiments of the ionizable lipid, R2ais heptyl.

[0834] In some embodiments of the ionizable lipid, R2ais octyl.

[0835] In some embodiments of the ionizable lipid, R2bis hydrogen.

[0836] In some embodiments of the ionizable lipid, R2bis methyl.

[0837] In some embodiments of the ionizable lipid, R2bis ethyl.

[0838] In some embodiments of the ionizable lipid, R2bis propyl.

[0839] In some embodiments of the ionizable lipid, R2bis butyl.

[0840] In some embodiments of the ionizable lipid, R2bis pentyl.

[0841] In some embodiments of the ionizable lipid, R2bis hexyl.

[0842] In some embodiments of the ionizable lipid, R2bis heptyl.

[0843] In some embodiments of the ionizable lipid, R2bis octyl.

[0844] In some embodiments of the ionizable lipid, R2ais hydrogen and R2bis hydrogen.

[0845] In some embodiments of the ionizable lipid, R2ais hexyl and R2bis hydrogen.

[0846] In some embodiments of the ionizable lipid, R2ais octyl and R2bis hydrogen.

[0847] In some embodiments of the ionizable lipid, R2ais hydrogen and R2bis butyl.

[0848] In some embodiments of the ionizable lipid, R2cis methyl.

[0849] In some embodiments of the ionizable lipid, R2cis ethyl.

[0850] In some embodiments of the ionizable lipid, R2cis propyl.

[0851] In some embodiments of the ionizable lipid, R2cis butyl.

[0852] In some embodiments of the ionizable lipid, R2cis pentyl.

[0853] In some embodiments of the ionizable lipid, R2cis hexyl.

[0854] In some embodiments of the ionizable lipid, R2cis heptyl.

[0855] In some embodiments of the ionizable lipid, R2cis octyl.

[0856] In some embodiments of the ionizable lipid, R2is -(Ci-6 alkylene)-(C3-s cycloalkyl)-Ci-6 alkyl.

[0857] In some embodiments of the ionizable lipid, R2is -(Ci-6 alkylene)-(cyclohexyl)-Ci-6 alkyl.

[0858] In some embodiments of the ionizable lipid, R2is -(Ci-6 alkylene)-(cyclopentyl)-Ci-6 alkyl.

[0859] Variables R3, R3a, R3b, andR3c

[0860] In some embodiments of the ionizable lipid, R3is R3aR3b

[0861] In some embodiments of the ionizable lipid, R3ais hydrogen. In some embodiments of the ionizable lipid, R3ais methyl.

[0862] In some embodiments of the ionizable lipid, R3ais ethyl.

[0863] In some embodiments of the ionizable lipid, R3ais propyl.

[0864] In some embodiments of the ionizable lipid, R3ais butyl.

[0865] In some embodiments of the ionizable lipid, R3ais pentyl.

[0866] In some embodiments of the ionizable lipid, R3ais hexyl.

[0867] In some embodiments of the ionizable lipid, R3ais heptyl.

[0868] In some embodiments of the ionizable lipid, R3ais octyl.

[0869] In some embodiments of the ionizable lipid, R3bis hydrogen.

[0870] In some embodiments of the ionizable lipid, R3bis methyl.

[0871] In some embodiments of the ionizable lipid, R3bis ethyl.

[0872] In some embodiments of the ionizable lipid, R3bis propyl.

[0873] In some embodiments of the ionizable lipid, R3bis butyl.

[0874] In some embodiments of the ionizable lipid, R3bis pentyl.

[0875] In some embodiments of the ionizable lipid, R3bis hexyl.

[0876] In some embodiments of the ionizable lipid, R3bis heptyl.

[0877] In some embodiments of the ionizable lipid, R3bis octyl.

[0878] In some embodiments of the ionizable lipid, R3ais octyl and R3bis hydrogen.

[0879] In some embodiments of the ionizable lipid, R3ais ethyl and R3bis hydrogen.

[0880] In some embodiments of the ionizable lipid, R3ais hexyl and R3bis hydrogen.

[0881] In some embodiments of the ionizable lipid, R3cis methyl.

[0882] In some embodiments of the ionizable lipid, R3cis ethyl.

[0883] In some embodiments of the ionizable lipid, R3cis propyl.

[0884] In some embodiments of the ionizable lipid, R3cis butyl.

[0885] In some embodiments of the ionizable lipid, R3cis pentyl.

[0886] In some embodiments of the ionizable lipid, R3cis hexyl.

[0887] In some embodiments of the ionizable lipid, R3cis heptyl.

[0888] In some embodiments of the ionizable lipid, R3cis octyl.

[0889] It is understood that, for an ionizable lipid, variables o, R1, RN, RN, RN, m, n, M, M’, R2, R2a, R2b, R2C, R3, R3a, R3b, and R3ccan each be, where applicable, selected from the groups described herein, and any group described herein for any of variables 0,-R1, RN, RN, RN, m, n, M, M’, R2, R2a, R2b, R2C, R3, R3a, R3b, and R3ccan be combined, where applicable, with any group described herein for one or more of the remainder of variables o, R1, RN, RN, RN, m, n, M, M’, R2, R2a, R2b, R2C, R3, R3a, R3b, and R3c. In some embodiments, the ionizable lipid is a compound selected from:

[0890] In some embodiments, the ionizable lipid is

[0891] In some embodiments, the ionizable lipid is

[0892] In some embodiments, the ionizable lipid is

[0893] Without wishing to be bound by theory, it is understood that an ionizable lipid may have a positive or partial positive charge at physiological pH. Such lipids may be referred to as cationic or ionizable (amino)lipids. Lipids may also be zwitterionic, i.e., neutral molecules having both a positive and a negative charge.

[0894] Stabilizing compounds

[0895] Some embodiments of the compositions described herein are stabilized pharmaceutical compositions. Various non-viral delivery systems, including nanoparticle formulations, present attractive opportunities to overcome many challenges associated with mRNA delivery. Lipid nanoparticles (LNPs) have drawn particular attention in recent years as various LNP formulations have shown promise in a variety of pharmaceutical applications. However, lipids have been shown to degrade nucleic acids, including mRNA, and lipid nanoparticle formulations undergo rapid loss of purity when stored as refrigerated liquids. Moreover, the storage stability of mRNA encapsulated within LNPs is lower than that of unencapsulated mRNA.

[0896] A class of compounds has been found to stabilize nucleic acids within a lipid carrier such as an LNP, an unexpected and unprecedented discovery which enables applications including extended refrigerated liquid shelf-life, extended in-use periods at room temperature, and extended in-use stability at physiological temperatures up to higher temperatures such as 40°C. Such stabilizing compounds solve a critical problem, as current manufacturing processes and formulations experience a 5-10% purity loss during LNP formation and processing that is typical with current large-scale LNP production.

[0897] In some embodiments, the stabilized pharmaceutical composition comprises a nucleic acid formulation comprising a nucleic acid and a stabilizing compound (e.g., a compound of Formula (I), of Formula (II), or a tautomer or solvate thereof). In some embodiments, the stabilized pharmaceutical composition comprises a nucleic acid formulation comprising a nucleic acid and a lipid, and a compound of Formula (I): tautomer or solvate thereof, wherein:

[0898] = is a single bond or a double bond;

[0899] R1is H; R2is OCH3, or together with R3is OCH2O; R3is OCH3, or together with R2is

[0900] OCH2O; R4is H; R5is H or OCH3; R6is OCH3; R7is H or OCH3; R8is H; R9is H or CH3; and X is a pharmaceutically acceptable anion, e.g., a halide such as chloride.

[0901] In some embodiments, the compound of Formula (I) has the structure of: or a tautomer or solvate thereof.

[0902] In some embodiments, the stabilized pharmaceutical composition comprises a nucleic acid formulation comprising a nucleic acid and a lipid, and a compound of Formula (II): (II), or a tautomer or solvate thereof, wherein:

[0903] R10is H; R11is H; R12together with R13is OCH2O; R14is H; R15together with R16is OCH2O; R17is H; and X is a pharmaceutically acceptable anion, e.g., a halide such as chloride.

[0904] In some embodiments, the compound of Formula (II) has the structure of:

[0905] Stabilizing compounds of Formulas (I), (la), (lb), (Ic), (II), and (Ila) are described in International Application No. PCT / US2022 / 025967, which is incorporated by reference herein in its entirety.

[0906] In some embodiments, the nucleic acid formulation comprises lipid nanoparticles. In some embodiments, the nucleic acid is mRNA.

[0907] In some embodiments, the stabilizing compound (“the compound”) has a purity of at least 70%, 80%, 90%, 95%, or 99%. In some embodiments, the compound contains fewer than lOOppm of elemental metals. In some embodiments, the stabilized pharmaceutical composition (“the composition”) comprises a pharmaceutically acceptable metal chelator, e.g., EDTA (ethylenediaminetetraacetic acid) or DTPA (diethylenetriaminepentaacetic acid).

[0908] In some embodiments, the composition is an aqueous solution. In some embodiments, the compound is present at a concentration between about O.lmM and about lOmM in the aqueous solution. In some embodiments, the aqueous solution has a pH of or about 5 to 8, including pH of about 5, 5.5, 6, 6.5, 7, 7.5, or 8. In some embodiments, the aqueous solution does not comprise NaCl. In some embodiments, the aqueous solution comprises NaCl in a concentration of or about 150mM. In some embodiments, the aqueous solution comprises a phosphate buffer, a tris buffer, an acetate buffer, a histidine buffer, or a citrate buffer.

[0909] In some embodiments, microbial growth in the composition is inhibited by the compound.

[0910] In some embodiments, the composition is characterized as having a mRNA purity level of greater than 60%, greater than 70%, greater than 80%, or greater than 90% main peak mRNA purity after at least thirty days of storage. In some embodiments, the composition comprises a mRNA purity level of greater than 50% main peak mRNA purity after at least six months of storage. In some embodiments, the storage is at room temperature.

[0911] In some embodiments, the composition comprises a lipid nanoparticle encapsulating a mRNA, and the composition comprises less than 50%, less than 60%, less than 70%, less than 80%, less than 90%, or less than 95% RNA fragments after at least thirty days of storage. In some embodiments, the storage temperature is greater than room temperature. In some embodiments, the storage temperature is about 4°C.

[0912] In some embodiments, the compound interacts with the nucleic acid comprised within a lipid nanostructure (e.g., a lipid nanoparticle, liposome, or lipoplex), e.g., via pi-pi stacking and / or by changing backbone helicity of the nucleic acid. In some embodiments, the compound intercalates with a nucleic acid. In some embodiments, the compound binds with a nucleic acid, e.g., reversible binding, and / or binding to the stranded regions of the nucleic acid. In some embodiments, the compound self-associates, binds to nucleic acid ribose contacts, and / or binds to nucleic acid base contacts. In some embodiments, the compound does not substantially bind to nucleic acid phosphate contacts. In some embodiments, the positive charge of the compound contributes to nucleic acid binding. In some embodiments, the interacts with the nucleic acid with a binding affinity defined by an equilibrium dissociation constant of less than 10'3M (e.g., less than 10'4M, less than 10'5M, less than 10'5M, less than 10'7M, less than 10'8M, or less than 10'9M).

[0913] In some embodiments, the compound interacts with a nucleic acid and provides shielding from solvent, e.g., water. In some embodiments, the compound shields ribose from solvent more than the compound shields the phosphate groups of the nucleic acid. In some embodiments, the solvent exposure is measured by the solvent accessible surface area (SASA). In some embodiments, a stabilizing compound decreases the solvent accessible area of ribose to about 5- 10 nm2. In some embodiments, a stabilizing compound decreases the solvent accessible area of ribose to about 6-8 nm2. In some embodiments, a stabilizing compound decreases the solvent accessible area of phosphate to about 9-12 nm2. In some embodiments, a stabilizing compound decreases the solvent accessible area of phosphate to about 10-11 nm2. In some embodiments, a nucleic acid that is conformationally stabilized by the compound exhibits thermal unfolding temperatures (measured by circular dichroism or DSC, for example) that are higher than in the absence of the compound. In some embodiments, the compound confers increased stability, e.g., thermal stability, to the nucleic acid in a folded structure, e.g., relative to its unfolded or less folded or more linear form. In some embodiments, the compound causes compaction of the nucleic acid upon interaction with the nucleic acid. In some embodiments, the compound causes a decrease in the hydrodynamic radius of the nucleic acid molecule upon interaction with the nucleic acid. In some embodiments, a stabilizing compound causes compaction or a decrease in the hydrodynamic radius of a nucleic acid molecule by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more. In some embodiments, a stabilizing compound causes compaction or a decrease in the hydrodynamic radius of a nucleic acid molecule when the compound is in a concentration of 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 15 pM, 20 pM, 25 pM, 30 pM, 35 pM, 40 pM, 45 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, or 100 pM. mRNA- Lipid Adducts

[0914] It has been determined that certain ionizable lipids are susceptible to the formation of lipid-polynucleotide adducts. In particular, ionizable lipids that comprise a tertiary amine group may decompose into one or both of a secondary amine and a reactive aldehyde species capable of interacting with polynucleotides (such as mRNA) to form an ionizable lipid-polynucleotide adduct impurity that can be detected by reverse phase ion pair chromatography (RP-IP HPLC). For example, oxidation of the tertiary amine may lead to N-oxide formation that can undergo acid / base-catalyzed hydrolysis at the amine to generate aldehydes and secondary amines which may form adducts with mRNA. Thus, in some aspects, the ionizable lipid-polynucleotide adduct impurity is an aldehyde-mRNA adduct impurity.

[0915] It also has been determined that such adducts may disrupt mRNA translation and impact the activity of lipid nanoparticle (LNP) formulated mRNA products. Thus, it can be advantageous to prepare and use LNP compositions with a reduced content of ionizable lipid- polynucleotide adduct impurity, such as wherein less than about 20%, less than about 10%, less than about 5%, or less than about 1%, of the mRNA is in the form of ionizable lipid- polynucleotide adduct impurity, as may be measured by RP-IP HPLC. Thus, in accordance with some aspects, an LNP composition is provided wherein less than about 10%, less than about 5%, or less than about 1%, of the mRNA is in the form of ionizable lipid-polynucleotide adduct impurity, including less than 10%, less than 5%, or less than 1%, as may be measured by RP-IP HPLC. In some aspects, an amount of lipid aldehydes in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of N- oxide compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of transition metals, such as Fe, in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of alkyl halide compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of anhydride compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of ketone compounds in the composition is less than about 50 ppm, including less than 50 ppm. Additionally or alternatively, in some aspects an amount of conjugated diene compounds in the composition is less than about 50 ppm, including less than 50 ppm.

[0916] In some aspects, the composition is stable against the formation of ionizable lipidpolynucleotide adduct impurity. In some aspects, an amount of ionizable lipid-poly nucleotide adduct impurity in the composition increases at an average rate of less than about 2% per day when stored at a temperature of about 25 °C or below, including at an average rate of less than 2% per day. In some aspects, an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when stored at a temperature of about 5 °C or below, including at an average rate of less than 0.5% per day. In some aspects, an amount of ionizable lipid-polynucleotide adduct impurity in the composition increases at an average rate of less than about 0.5% per day when stored at a refrigerated temperature, optionally wherein the refrigerated temperature is about 5 °C.

[0917] Lipid vehicle (e.g., LNP) compositions with a reduced content of ionizable lipid- polynucleotide adduct impurity can be prepared by methods that inhibit formation of one or both of N-oxides and aldehydes. Such methods may comprise treating a composition comprising an ionizable lipid comprising a tertiary amine group to inhibit formation of one or both of N-oxides and aldehydes, such as by treating the composition with a reducing agent; treating the composition with a chelating agent; adjusting the pH of the composition; adjusting the temperature of the composition; and adjusting the buffer in the composition. Such methods may comprise, prior to combining the ionizable lipid with a polynucleotide, one or more of treating the ionizable lipid with a scavenging agent; treating the ionizable lipid with a reductive treatment agent; treating the ionizable lipid with a reducing agent; treating the ionizable lipid with a chelating agent; treating the polynucleotide with a reducing agent; and treating the polynucleotide with a chelating agent. In accordance with any of the foregoing, the scavenging agent, reductive treatment agent, and / or reducing agent may be an agent that reacts with aldehyde, ketone, anhydride and / or diene compounds. A scavenging agent may comprise one or more selected from (O-(2,3,4,5,6- Pentafluorobenzyl)hydroxylamine hydrochloride) (PFBHA), methoxyamine (e.g., methoxyamine hydrochloride), benzyloxyamine (e.g., benzyloxyamine hydrochloride), ethoxyamine (e.g., ethoxyamine hydrochloride), 4-[2-(aminooxy)ethyl]morpholine dihydrochloride, butoxyamine (e.g., tert-butoxyamine hydrochloride), 4-Dimethylaminopyridine (DMAP), 1,4- diazabicyclo[2.2.2]octane (DABCO), Triethylamine (TEA), Piperidine 4-carboxylate (BPPC), and combinations thereof. A reductive treatment agent may comprise a boron compound (e.g., sodium borohydride and / or bis(pinacolato)diboron). A reductive treatment agent may comprise a boron compound, such as one or both of sodium borohydride and bis(pinacolato)diboron). A chelating agent may comprise immobilized iminodiacetic acid. A reducing agent may comprise an immobilized reducing agent, such as immobilized diphenylphosphine on silica (Si-DPP), immobilized thiol on agarose (Ag-Thiol), immobilized cysteine on silica (Si-Cysteine), immobilized thiol on silica (Si-Thiol), or a combination thereof. A reducing agent may comprise a free reducing agent, such as potassium metabisulfite, sodium thioglycolate, tris(2- carboxyethyl)phosphine (TCEP), sodium thiosulfate, N-acetyl cysteine, glutathione, dithiothreitol (DTT), cystamine, dithioerythritol (DTE), dichlorodiphenyltrichloroethane (DDT), homocysteine, lipoic acid, or a combination thereof.

[0918] In accordance with any of the foregoing, the pH may be, or adjusted to be, a pH of from about 7 to about 9.

[0919] In accordance with any of the foregoing, a buffer may be selected from sodium phosphate, sodium citrate, sodium succinate, histidine, histidine-HCl, sodium malate, sodium carbonate, and TRIS (tris(hydroxymethyl)aminomethane). In accordance with any of the foregoing, a buffer may be TRIS and may be, or adjusted to be, from about 20 mM to about 150 mM TRIS.

[0920] In accordance with any of the foregoing, the temperature of the composition may be, or adjusted to be, 25 °C or less.

[0921] The composition may also comprise a free reducing agent or antioxidant.

[0922] Pharmaceutical Formulations

[0923] Provided herein are compositions (e.g., pharmaceutical compositions), methods, kits and reagents for prevention or treatment of VZV in humans and other mammals, for example. The compositions provided herein can be used as therapeutic or prophylactic agents. They may be used in medicine to prevent and / or treat a VZV infection. In some embodiments, the VZV vaccine containing RNA as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the RNA polynucleotides are translated in vivo to produce an antigenic polypeptide (antigen).

[0924] An “effective amount” of a composition (e.g., comprising RNA) is based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the RNA (e.g., length, nucleotide composition, and / or extent of modified nucleosides), other components of the vaccine, and other determinants, such as age, body weight, height, sex and general health of the subject. Typically, an effective amount of a composition provides an induced or boosted immune response as a function of antigen production in the cells of the subject. In some embodiments, an effective amount of the composition containing RNA polynucleotides having at least one chemical modifications are more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen or a peptide antigen. Increased antigen production may be demonstrated by increased cell transfection (the percentage of cells transfected with the RNA vaccine), increased protein translation and / or expression from the polynucleotide, decreased nucleic acid degradation (as demonstrated, for example, by increased duration of protein translation from a modified polynucleotide), or altered antigen specific immune response of the host cell.

[0925] The term "pharmaceutical composition" refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo. A "pharmaceutically acceptable carrier," after administered to or upon a subject, does not cause undesirable physiological effects. The carrier in the pharmaceutical composition must be "acceptable" also in the sense that it is compatible with the active ingredient and can be capable of stabilizing it. One or more solubilizing agents can be utilized as pharmaceutical carriers for delivery of an active agent. Examples of a pharmaceutically acceptable carrier include, but are not limited to, biocompatible vehicles, adjuvants, additives, and diluents to achieve a composition usable as a dosage form. Examples of other carriers include colloidal silicon oxide, magnesium stearate, cellulose, and sodium lauryl sulfate. Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

[0926] In some embodiments, the compositions (comprising polynucleotides and their encoded polypeptides) in accordance with the present disclosure may be used for treatment or prevention of a VZV infection. A composition may be administered prophylactically or therapeutically as part of an active immunization scheme to healthy individuals or early in infection during the incubation phase or during active infection after onset of symptoms. In some embodiments, the amount of RNA provided to a cell, a tissue or a subject may be an amount effective for immune prophylaxis.

[0927] A composition may be administered with other prophylactic or therapeutic compounds. As a non-limiting example, a prophylactic or therapeutic compound may be an adjuvant or a booster. As used herein, when referring to a prophylactic composition, such as a vaccine, the term “booster” refers to an extra administration of the vaccine composition and may include a traditional boost, seasonal boost or a pandemic shift boost. A booster (or booster vaccine) may be given after an earlier administration of the prophylactic composition. The time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, one year, or more. In some embodiments, the time of administration between the initial administration of the prophylactic composition and the booster is at least 6 months. In exemplary embodiments, the time of administration between the initial administration of the prophylactic composition and the booster may be, but is not limited to, 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or 6 months. As is described herein, the booster may comprise the same or different mRNAs as compared to the earlier administration of the prophylactic composition. In some embodiments, the booster may comprise a combination of the same mRNA from the earlier administration of the prophylactic composition and at least one different mRNA. In some embodiments, the ratio of the mRNA from the earlier administration of the prophylactic composition and the at least one different mRNA is 1 : 1 , 1 :2, 1 :4, 4: 1 , or 2: 1. In one embodiment, the ratio is 1 : 1. In some embodiments, the booster may comprise different mRNAs as compared to the earlier administration of the prophylactic compositions. In some embodiments, such a booster may comprise 1, 2, 3, 4 or more mRNAs that were not present in the prophylactic composition. In some embodiments, the ratio of two mRNA polynucleotides (none of which were in the prophylactic composition) in the booster is 1:1, 1:2, 1:4, 4:1, or 2:1. In one embodiment, the ratio is 1:1. A boost or booster dose may be administered more than once, for example 2, 3, 4, 5, 6 or more times after the initial prophylactic (prime) dose. In some embodiments, a subsequent boost is administered within weeks, e.g., within 3-4 weeks of the first (or previous) boost. In some embodiments, a second boost is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks after the first (or previous) boost. The booster, in some embodiments is monovalent (e.g., the mRNA encodes a single antigen). In some embodiments, the booster is multivalent (e.g., the mRNA encodes more than one antigen).

[0928] In some embodiments, the booster dose is 5 pg-30 pg, 5 pg-25 pg, 5 pg-20 pg, 5 pg-15 pg, 5 pg-10 pg, 10 pg-30 pg, 10 pg-25 pg, 10 pg-20 pg, 10 pg-15 pg, 15 pg-30 pg, 15 pg-25 pg, 15 pg-20 pg, 20 pg-30 pg, 25 pg-30 pg, or 25 pg-300 pg. In some embodiments, the booster dose is 10 pg-60 pg, 10 pg-55 pg, 10 pg-50 pg, 10 pg-45 pg, 10 pg-40 pg, 10 pg-35 pg, 10 pg-

[0929] 30 pg, 10 pg-25 pg, 10 pg-20 pg, 15 pg-60 pg, 15 pg-55 pg, 15 pg-50 pg, 15 pg-45 pg, 15 pg-

[0930] 40 pg, 15 pg-35 pg, 15 pg-30 pg, 15 pg-25 pg, 15 pg- 20 pg, 20 pg-60 pg, 20 pg-55 pg, 20 pg- 50 pg, 20 pg-45 pg, 20 pg-40 pg, 20 pg-35 pg, 20 pg-30 pg, 20 pg-25 pg, 25 pg-60 pg, 25 pg-

[0931] 55 pg, 25 pg-50 pg, 25 pg-45 pg, 25 pg-40 pg, 25 pg-35 pg, 25 pg-30 pg, 30 pg-60 pg, 30 pg-

[0932] 55 pg, 30 pg-50 pg, 30 pg-45 pg, 30 pg-40 pg, 30 pg-35 pg, 35 pg-60 pg, 35 pg-55 pg, 35 pg-

[0933] 50 pg, 35 pg-45 pg, 35 pg-40 pg, 40 pg-60 pg, 40 pg-55 pg, 40 pg-50 pg, 40 pg-45 pg, 45 pg-

[0934] 60 pg, 45 pg-55 pg, 45 pg-50 pg, 50 pg-60 pg, 50 pg-55 pg, or 55 pg-60 pg. In some embodiments, the booster dose is at least 10 pg and less than 25 pg of the composition. In some embodiments, the booster dose is at least 5 pg and less than 25 pg of the composition. For example, the booster dose is 5 pg, 10 pg, 15 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 55 pg, 60 pg, 65 pg, 70 pg, 75 pg, 80 pg, 85 pg, 90 pg, 95 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, 150 pg, 160 pg, 170 pg, 180 pg, 190 pg, 200 pg, 250 pg, or 300 pg. In some embodiments, the booster dose is 50 pg.

[0935] In some embodiments, a composition may be administered intramuscularly, intranasally or intradermally, similarly to the administration of inactivated vaccines known in the art.

[0936] A composition may be utilized in various settings depending on the prevalence of the infection or the degree or level of unmet medical need. As a non-limiting example, the RNA vaccines may be utilized to treat and / or prevent a variety of infectious disease. RNA vaccines have superior properties in that they produce much larger antibody titers, better neutralizing immunity, produce more durable immune responses, and / or produce responses earlier than commercially available vaccines.

[0937] Provided herein are pharmaceutical compositions including RNA and / or complexes optionally in combination with one or more pharmaceutically acceptable excipients.

[0938] The RNA may be formulated or administered alone or in conjunction with one or more other components. For example, an immunizing composition may comprise other components including, but not limited to, adjuvants.

[0939] In some embodiments, an immunizing composition does not include an adjuvant (it is adjuvant free). An RNA may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients. In some embodiments, vaccine compositions comprise at least one additional active substances, such as, for example, a therapeutically-active substance, a prophylactically-active substance, or a combination of both. Vaccine compositions may be sterile, pyrogen-free or both sterile and pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents, such as vaccine compositions, may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0940] In some embodiments, an immunizing composition is administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the RNA vaccines or the polynucleotides contained therein, for example, RNA polynucleotides (e.g., mRNA polynucleotides) encoding antigens.

[0941] Formulations of the vaccine compositions described herein may 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 (e.g., mRNA polynucleotide) 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 a desired single- or multi-dose unit.

[0942] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, at least 80% (w / w) active ingredient.

[0943] In some embodiments, an RNA is formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation); (4) alter the biodistribution (e.g., target to specific tissues or cell types); (5) increase the translation of encoded protein in vivo; and / or (6) alter the release profde of encoded protein (antigen) in vivo. In addition to traditional excipients such as 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, excipients can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with the RNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof. Methods of Prophylaxis / Treatment

[0944] Provided herein are a compositions (e.g., vaccines), methods, kits and reagents for prevention and / or treatment of a VZV infection in humans and other mammals. The compositions can be used as therapeutic or prophylactic agents. In some embodiments, the compositions are used to provide prophylactic protection from a VZV infection. In some embodiments, the compositions are used to treat a VZV infection. In some embodiments, embodiments, the compositions are used in the priming of immune effector cells, for example, to activate peripheral blood mononuclear cells (PBMCs) ex vivo, which are then infused (reinfused) into a subject.

[0945] A subject may be any mammal, including non-human primate and human subjects. Typically, a subject is a human subject. In some embodiments, the subject has, or is at risk of, VZV infection. In some embodiments, the subject has a VZV infection. In some embodiments, the subject is at risk of VZV infection. If the subject is at risk of having VZV infection, administering a composition of the present disclosure to the subject refers to reducing the risk of the subject having VZV infection or preventing the subject from developing VZV infection.

[0946] In some embodiments, a composition is administered to a subject (e.g., a mammalian subject, such as a human subject) in an effective amount to induce an antigen- specific immune response. The RNA encoding the VZV protein is expressed and translated in vivo to produce the antigen, which then stimulates an immune response in the subject.

[0947] Prophylactic protection from a VZV can be achieved following administration of a composition of the present disclosure. The compositions can be administered once, twice, three times, four times or more but it is likely sufficient to administer the composition once (optionally followed by a single booster). It is possible, although less desirable, to administer a composition to an infected individual to achieve a therapeutic response. Dosing may need to be adjusted accordingly.

[0948] A method of eliciting an immune response in a subject against a VZV protein (or multiple antigens) is provided in aspects of the present disclosure. In some embodiments, a method involves administering to the subject a vaccine comprising a RNA (e.g., mRNA) having an open reading frame encoding a VZV protein (or multiple antigens), thereby inducing in the subject an immune response specific to the VZV protein (or multiple antigens), wherein antiantigen antibody titer in the subject is increased following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against the antigen. An “anti-antigen antibody” is a serum antibody the binds specifically to the antigen. A prophylactically effective dose is an effective dose that prevents infection with the virus at a clinically acceptable level. In some embodiments, the effective dose is a dose listed in a package insert for the vaccine. A traditional vaccine, as used herein, refers to a vaccine other than the RNA (e.g., mRNA) vaccines of the present disclosure. For instance, a traditional vaccine includes, but is not limited, to live microorganism vaccines, killed microorganism vaccines, subunit vaccines, protein antigen vaccines, DNA vaccines, virus like particle (VLP) vaccines, etc. In exemplary embodiments, a traditional vaccine is a vaccine that has achieved regulatory approval and / or is registered by a national drug regulatory body, for example the Food and Drug Administration (FDA) in the United States or the European Medicines Agency (EMA).

[0949] In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log to 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against VZV or an unvaccinated subject. In some embodiments, the anti-antigen antibody titer in the subject is increased 1 log, 2 log, 3 log, 4 log, 5 log, or 10 log following vaccination relative to anti-antigen antibody titer in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against VZV or an unvaccinated subject.

[0950] A method of eliciting an immune response in a subject against VZV is provided in other aspects of the disclosure. The method involves administering to the subject a composition comprising an RNA (e.g., mRNA) comprising an open reading frame encoding a VZV protein, thereby inducing in the subject an immune response specific to VZV, wherein the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine against VZV at 2 times to 100 times the dosage level relative to the composition.

[0951] In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at twice the dosage level relative to a composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at three times the dosage level relative to a composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 4 times, 5 times, 10 times, 50 times, or 100 times the dosage level relative to a composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 10 times to 1000 times the dosage level relative to a composition of the present disclosure. In some embodiments, the immune response in the subject is equivalent to an immune response in a subject vaccinated with a traditional vaccine at 100 times to 1000 times the dosage level relative to a composition of the present disclosure.

[0952] In other embodiments, the immune response is assessed by determining antibody titer in the subject. In other embodiments, the ability of serum or antibody from an immunized subject is tested for its ability to neutralize viral uptake or reduce VZV transformation of human B lymphocytes. In other embodiments, the ability to promote a robust T cell response(s) is measured using art recognized techniques.

[0953] Other aspects the disclosure provide methods of eliciting an immune response in a subject against VZV by administering to the subject composition comprising an mRNA having an open reading frame encoding a VZV protein, thereby inducing in the subject an immune response specific to the VZV protein, wherein the immune response in the subject is induced 2 days to 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine against VZV. In some embodiments, the immune response in the subject is induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine at 2 times to 100 times the dosage level relative to a composition of the present disclosure.

[0954] In some embodiments, the immune response in the subject is induced 2 days, 3 days, 1 week, 2 weeks, 3 weeks, 5 weeks, or 10 weeks earlier relative to an immune response induced in a subject vaccinated with a prophylactically effective dose of a traditional vaccine.

[0955] Also provided herein are methods of eliciting an immune response in a subject against VZV by administering to the subject an mRNA having an open reading frame encoding at least one VZV protein, wherein the mRNA does not include a stabilization element, and wherein an adjuvant is not co-formulated or co-administered with the vaccine.

[0956] A composition may be administered by any route that results in a therapeutically effective outcome. These include, but are not limited, to intradermal, intramuscular, intranasal, and / or subcutaneous administration. The present disclosure provides methods comprising administering RNA (e.g., mRNA) vaccines to a subject in need thereof. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. The RNA (e.g., mRNA) is typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the RNA (e.g., mRNA) may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.

[0957] The effective amount of the mRNA (e.g., an effective dose), as provided herein, may be as low as 20 pg, administered for example as a single dose or as two 10 pg doses (e.g., a first effective vaccine dose and a second effective vaccine dose). In some embodiments, the first effective vaccine dose and the second effective vaccine dose are the same amount. In some embodiments, the first effective vaccine dose and the second effective vaccine dose are different amounts. In some embodiments, the effective amount is a total dose of 5 pg-30 pg, 5 pg-25 pg, 5 pg-20 pg, 5 pg-15 pg, 5 pg-10 pg, 10 pg-30 pg, 10 pg-25 pg, 10 pg-20 pg, 10 pg-15 pg, 15 pg-30 pg, 15 pg-25 pg, 15 pg-20 pg, 20 pg-30 pg, 25 pg-30 pg, or 25 pg-300 pg. In some embodiments, the effective dose (e.g., effective amount) is at least 10 pg and less than 25 pg of the composition. In some embodiments, the effective dose (e.g., effective amount) is at least 5 pg and less than 25 pg of the composition. For example, the effective amount may be a total dose of 5 pg, 10 pg, 15 pg, 20 pg, 25 pg, 30 pg, 35 pg, 40 pg, 45 pg, 50 pg, 55 pg, 60 pg, 65 pg, 70 pg, 75 pg, 80 pg, 85 pg, 90 pg, 95 pg, 100 pg, 110 pg, 120 pg, 130 pg, 140 pg, 150 pg, 160 pg, 170 pg, 180 pg, 190 pg, 200 pg, 250 pg, or 300 pg. In some embodiments, the effective amount (e.g., effective dose) is a total dose of 10 pg. In some embodiments, the effective amount is a total dose of 20 pg (e.g., two 10 pg doses). In some embodiments, the effective amount is a total dose of 25 pg. In some embodiments, the effective amount is a total dose of 30 pg. In some embodiments, the effective amount is a total dose of 50 pg. In some embodiments, the effective amount is a total dose of 60 pg (e.g., two 30 pg doses). In some embodiments, the effective amount is a total dose of 75 pg. In some embodiments, the effective amount is a total dose of 100 pg. In some embodiments, the effective amount is a total dose of 150 pg. In some embodiments, the effective amount is a total dose of 200 pg. In some embodiments, the effective amount is a total dose of 250 pg. In some embodiments, the effective amount is a total dose of 300 pg.

[0958] In some embodiments, a composition of the present disclosure is administered in at least one (e.g., at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten) dose(s). In some embodiments, a composition of the present disclosure is administered in at least one dose, at least two doses, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, at least eight doses, at least nine doses, or at least ten doses. In some embodiments, a composition of the present disclosure is administered in at least one dose. In some embodiments, a composition of the present disclosure is administered in at least two doses. In some embodiments, a composition of the present disclosure is administered in at least three doses. In some embodiments, a composition of the present disclosure is administered in a first dose. In some embodiments, a composition of the present disclosure is administered in a first dose and a second dose.

[0959] In some embodiments, the at least one dose comprises 10-100 (e.g., 10-100, 10-90, 10- 80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90, 90-100) pg of the mRNA. In some embodiments, the at least one dose comprises 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90, 90-100 pg of the mRNA. In some embodiments, the at least one dose comprises 10-100 pg of the mRNA.

[0960] In some embodiments, the at least one dose is effective to produce a neutralizing antibody response to VZV gE. As described herein, the phrase “neutralizing antibody” refers to an antibody that binds to the viral antigen and prevents the virus from infecting cells.

[0961] In some embodiments, the at least one dose is effective to produce a CD4+and / or CD8+T cell response to VZV gE. In some embodiments, the at least one dose is effective to produce a CD4+T cell response to VZV gE. In some embodiments, the at least one dose is effective to produce a CD8+T cell response to VZV gE. In some embodiments, the at least one dose is effective to produce a CD4+and CD8+T cell response to VZV gE. In some embodiments, the at least one dose is effective to produce a CD4+or CD8+T cell response to VZV gE.

[0962] In some embodiments, a CD4+T cell response is measured by the percentage of CD4+T cells expressing IFNy, IL-2, TNFa, IL-4, IL-5, and / or IL- 13.

[0963] In some embodiments, a CD8+T cell response is measured by the percentage of CD8+T cells expressing CD 107a, IFNy, IL-2, and / or TNF. In some embodiments, a CD4+T cell response is measured by the percentage of CD8+T cells expressing CD 107a, IFNy, IL-2, and / or TNFa.

[0964] In some embodiments, the at least one dose is effective to produce antibodies with antibody-dependent cellular cytotoxicity (ADCC). As described herein, the terms “antibodydependent cellular cytotoxicity” or “ADCC” refers to a type of immune reaction in which a target cell or microbe is coated with antibodies and killed by certain types of white blood cells, which bind to the antibodies and release substances that kill the target cells or microbes. Primary infection by VZV often results in childhood chickenpox. Following the resolution of the chickenpox, the virus may establish latency in the neurons of the cranial nerve, dorsal root and autonomic ganglia along the entire neuraxis. During latency, the symptoms and virus become undetectable. The virus may later activate and become an active infection. The VZV vaccine disclosed herein may be used to vaccinate a naive subject who has not previously been infected with VZV. Alternatively, a VZV vaccine may be administered to a subject having a latent VZV infection capable of reactivation. The VZV vaccine may be useful in that context to prevent development or severity of viral infection if that virus is reactivated and / or prevent new VZV infections. Thus, in some embodiments, the at least one dose of mRNA vaccine is administered to a subject having or at risk of having a VZV infection. In some embodiments, the mRNA vaccine is administered to the subject in one or more doses. For example, the mRNA vaccine is administered to the subject as a primary vaccine and / or as a booster vaccine.

[0965] The RNA (e.g., mRNA) described herein can be formulated into a dosage form described herein, such as an intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intradermal, intracardiac, intraperitoneal, and subcutaneous).

[0966] Vaccine Efficacy

[0967] Some aspects of the present disclosure provide formulations of the compositions (e.g., RNA vaccines), wherein the mRNA is formulated in an effective amount to produce an antigen specific immune response in a subject (e.g., production of antibodies specific to a VZV antigen). “An effective amount” is a dose of the mRNA effective to produce an antigen- specific immune response. Also provided herein are methods of inducing an antigen- specific immune response in a subject.

[0968] As used herein, an immune response to a vaccine of the present disclosure is the development in a subject of a humoral and / or a cellular immune response to a (one or more) VZV protein(s) encoded by the RNA (e.g., mRNA) present in the vaccine. For purposes of the present disclosure, a “humoral” immune response refers to an immune response mediated by antibody molecules, including, e.g., secretory (IgA) or IgG molecules, while a “cellular” immune response is one mediated by T-lymphocytes (e.g., CD4+ helper and / or CD8+ T cells (e.g., CTLs) and / or other white blood cells. One important aspect of cellular immunity involves an antigenspecific response by cytolytic T-cells (CTLs). CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells. CTLs help induce and promote the destruction of intracellular microbes or the lysis of cells infected with such microbes. Another aspect of cellular immunity involves and antigen- specific response by helper T-cells. Helper T-cells act to help stimulate the function and focus the activity nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface. A cellular immune response also leads to the production of cytokines, chemokines, and other such molecules produced by activated T-cells and / or other white blood cells including those derived from CD4+ and CD8+ T-cells. Humoral immune responses may be further divided into Thl and Th2 responses, resulting the production of Thl -type cytokines and Th2-type cytokines, respectively. Thl -type cytokines tend to produce the proinflammatory responses responsible for killing intracellular parasites and for perpetuating autoimmune responses. The main Thl cytokine is interferon gamma. Excessive proinflammatory responses (e.g., Thl-based responses), in some embodiments, can lead to uncontrolled tissue damage, and are counteracted by the Th2-type cytokines. The Th2-type cytokines include interleukins 4, 5, and 13, which are associated with the promotion of IgE and eosinophilic responses in atopy, and also interleukin- 10, which is antiinflammatory. In excess, Th2 responses will counteract the Thl mediated microbicidal action. Accordingly, in some embodiments, the vaccines provided herein elicit a balanced Thl and Th2 response. In some embodiments, administration of the vaccines provided herein may result in a Thl7 response. T helper 17 cells (Thl7) are a subset of pro-inflammatory T helper cells defined by their production of interleukin 17. Thl7 cells maintain mucosal barriers and contribute to pathogen clearance at the mucosal surfaces. The Thl7-type cytokines target innate immune cells and epithelial cells to produce G-CSF and 11-8, leading to neutrophil production and recruitment. In some embodiments, the compositions (e.g., vaccines) of the present disclosure produce a Thl response. In some embodiments, the compositions (e.g., vaccines) of the present disclosure produce a Th2 response. In some embodiments, the compositions (e.g., vaccines) of the present disclosure produce a Thl7 response. In some embodiments, the compositions (e.g., vaccines) of the present disclosure produce Thl and Th2 responses, Thl and Thl7 responses, Th2 and Thl7 responses, or Thl, Th2, and Th 17 responses.

[0969] In some embodiments, the antigen- specific immune response is characterized by measuring an anti-VZV antigen antibody titer produced in a subject administered a composition as provided herein. An antibody titer is a measurement of the amount of antibodies within a subject, for example, antibodies that are specific to a particular antigen or epitope of an antigen. Antibody titer is typically expressed as the inverse of the greatest dilution that provides a positive result. Enzyme-linked immunosorbent assay (ELISA) is a common assay for determining antibody titers, for example.

[0970] A variety of serological tests can be used to measure antibody against encoded antigen of interest, for example, a VZV antigen. These tests include the hemagglutination-inhibition test, complement fixation test, fluorescent antibody test, enzyme-linked immunosorbent assay (ELISA), and plaque reduction neutralization test (PRNT). Each of these tests measures different antibody activities. In exemplary embodiments, A plaque reduction neutralization test, or PRNT (e.g., PRNT50 or PRNT90) is used as a serological correlate of protection. PRNT measures the biological parameter of in vitro virus neutralization and is the most serologically virus-specific test among certain classes of viruses, correlating well to serum levels of protection from virus infection. The basic design of the PRNT allows for virus-antibody interaction to occur in a test tube or microtiter plate, and then measuring antibody effects on viral infectivity by plating the mixture on virus- susceptible cells, preferably cells of mammalian origin. The cells are overlaid with a semi- solid media that restricts spread of progeny virus. Each virus that initiates a productive infection produces a localized area of infection (a plaque), that can be detected in a variety of ways. Plaques are counted and compared back to the starting concentration of virus to determine the percent reduction in total virus infectivity. In PRNT, the serum sample being tested is usually subjected to serial dilutions prior to mixing with a standardized amount of virus. The concentration of virus is held constant such that, when added to susceptible cells and overlaid with semi-solid media, individual plaques can be discerned and counted. In this way, PRNT end-point titers can be calculated for each serum sample at any selected percent reduction of virus activity.

[0971] In functional assays intended to assess vaccinal immunogenicity, the serum sample dilution series for antibody titration should ideally start below the “seroprotective” threshold titer. Regarding VZV neutralizing antibodies, a seropositivity threshold of 1:10 can be considered a seroprotection threshold in certain embodiments.

[0972] PRNT end-point titers are expressed as the reciprocal of the last serum dilution showing the desired percent reduction in plaque counts. The PRNT titer can be calculated based on a 50% or greater reduction in plaque counts (PRNT50). A PRNT50 titer is preferred over titers using higher cut-offs (e.g., PRNT90) for vaccine sera, providing more accurate results from the linear portion of the titration curve.

[0973] There are several ways to calculate PRNT titers. The simplest and most widely used way to calculate titers is to count plaques and report the titer as the reciprocal of the last serum dilution to show >50% reduction of the input plaque count as based on the back-titration of input plaques. Use of curve fitting methods from several serum dilutions may permit calculation of a more precise result. There are a variety of computer analysis programs available for this (e.g., SPSS or GraphPad Prism).

[0974] In some embodiments, an ant...

Claims

What is claimed is:CLAIMS1. A varicella- zoster virus (VZV) vaccine comprising:(i) a messenger ribonucleic acid (mRNA) comprising a 5’UTR, an open reading frame (ORF) encoding a VZV glycoprotein E (gE) protein variant comprising an amino acid sequence of SEQ ID NO: 2, wherein the ORF comprises a nucleic acid sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO: 1, a 3’ UTR, and a poly A tail, wherein all uridines in the mRNA are substituted with 1 -methylpseudouridine; and(ii) a lipid nanoparticle (LNP).

2. The VZV vaccine of claim 1, wherein the ORF comprises a nucleic acid sequence having at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleic acid sequence of SEQ ID NO: 1.

3. The VZV vaccine of claim 2, wherein the ORF comprises the nucleic acid sequence of SEQ ID NO: 1.

4. The VZV vaccine of any one of claims 1-3, wherein the VZV gE protein variant has a length of no longer than 575, 574, or 573 amino acids.

5. The VZV vaccine of any one of claims 1-4, wherein the LNP comprises an ionizable amino lipid.

6. The VZV vaccine of claim 5, wherein the ionizable amino lipid comprises a compound of Formula (I):whereinRi is selected from the group consisting of C5-30 alkyl, C5-20 alkenyl, and -R"M'R';R2 and R3 are independently selected from the group consisting of C1-14 alkyl and C2-14 alkenyl;R4is -(CH2)nQ, wherein Q is -OR, and n is selected from 1, 2, 3, 4, and 5; each R5 is H;each R6 is H;M and M' are independently selected from -C(O)O- and -OC(O)-;R7is HR is H;R' is selected from the group consisting of Ci-is alkyl and C2-18 alkenyl;R" is selected from the group consisting of C3-14 alkyl and C3-14 alkenyl; and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

7. The VZV vaccine of any one of claims 1-5, wherein the LNP comprises a neutral lipid, optionally DSPC.

8. The VZV vaccine of any one of claims 1-5, wherein the LNP comprises a sterol lipid, optionally cholesterol.

9. The VZV vaccine of any one of claims 1-5, wherein the LNP comprises a polyethylene glycol (PEG) -modified lipid, optionally PEG2000-DMG.

10. The VZV vaccine of any one of claims 1-9, wherein the mRNA vaccine comprises a second mRNA encoding a different VZV antigen.

11. The VZV vaccine of any one of claims 1-9, wherein the ORF encodes at least two different VZV antigens.

12. A method comprising: administering to a subject at least one dose of a VZV vaccine comprising:(i) a messenger ribonucleic acid (mRNA) comprising a 5’UTR, an open reading frame (ORF) encoding a VZV glycoprotein E (gE) protein variant comprising an amino acid sequence of SEQ ID NO: 2, wherein the ORF comprises a nucleic acid sequence having at least 95% identity to the nucleic acid sequence of SEQ ID NO: 1, a 3’ UTR, and a poly A tail, wherein all uridines in the mRNA are substituted with 1 -methylpseudouridine; and(ii) a lipid nanoparticle (LNP), in an effective amount to induce an immune response in the subject.

13. The method of claim 12, wherein the subject has, or is at risk of, VZV infection.

14. The method of claim 12 or 13, wherein the VZV vaccine is administered intramuscularly.

15. The method of any one of claims 12-14, comprising administering a first dose and a second dose of the VZV vaccine.

16. The method of any one of claims 12-15, wherein the at least one dose comprises 10-100 pg of the mRNA.

17. The method of any one of claims 12-15, wherein the at least one dose comprises 10-50 pg of the mRNA.

18. The method of any one of claims 12-15, wherein the at least one dose comprises 10-40 pg of the mRNA.

19. The method of any one of claims 12-15, wherein the at least one dose comprises 10-30 pg of the mRNA.

20. The method of any one of claims 12-15, wherein the at least one dose comprises 30-40 pg of the mRNA.

21. The method of any one of claims 12-15, wherein the at least one dose comprises 50-100 pg of the mRNA.

22. The method of any one of claims 12-21, wherein the at least one dose is effective to produce a neutralizing antibody response to VZV gE.

23. The method of any one of claims 12-21, wherein the at least one dose is effective to produce a CD4+and / or CD8+T cell response to VZV gE.

24. The method of any one of claims 12-21, wherein the at least one dose is effective to produce antibodies with antibody-dependent cellular cytotoxicity (ADCC).

25. The method of any one of claims 12-24, wherein the mRNA vaccine comprises a second mRNA encoding a different VZV antigen.

26. The method of any one of claims 12-24, wherein the ORF encodes at least two different VZV antigens.

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