mRNA composition and uses thereof

Abstract

The invention described herein provides designed / engineered 5'UTR, 3'UTR, and poly A signal sequences as mRNA structural elements that, alone or in combination, enhances protein synthesis from mRNA comprising such structural elements.

Description

[0001] mRNA COMPOSITION AND USES THEREOF

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the priority to and the benefit of the filing date of U.S. Provisional Patent Application No. 63 / 652,414, filed on May 28, 2024, the entire content of which is incorporated herein by reference.

[0004] BACKGROUND OF THE INVENTION

[0005] Messenger RNA (mRNA)-based vaccines have recently gained traction in the public eye based on their success combatting the COVID-19 pandemic (Gote et al., Int J Mol Sci. 24(3): 2700, 2023). This technology harnesses the power of mRNA, which instructs cells to produce an antigen with the goal of initiating a protective immune response within the patient.

[0006] There are a multitude of advantages that mRNA-based vaccines and therapies hold against conventional methods - most notably their specificity and manufacturability (Rosa et al., Vaccine. 39(16):2190-2200, 2021). In order to produce material for vaccines or therapeutics, researchers utilize in vitro transcription (IVT) to generate synthetic mRNAs which mimic naturally occurring mRNAs (Sahin et al., Nat Rev Drug Discov. 10:759-80, 2014). These synthetic mRNAs require five key structural components for functionality: 5’ cap, 5’ untranslated region (UTR), coding region of gene of interest (CDS), 3 ’ UTR, and the polyA tail (Kang et al., Adv Drug Deliv Rev. 199: 114961, 2023) [Fig. 1], Each structural component has its own key roles, many of which work together to determine the overall effectiveness of the mRNA-based intervention. Engineering these mRNA structural components to improve therapeutic efficacy is of great interest and is an essential step prior to successful translation of mRNA vaccines and therapeutics.

[0007] SUMMARY OF THE INVENTION

[0008] The invention described herein provides three aspects for improving mRNA development useful for, e.g., vaccine production: translation / expression of mRNA, polyA tail stability, and IVT yield.

[0009] In one aspect, the invention described herein provides designs for the 5 ’UTR and 3 ’UTR that improve translational activity of synthetic mRNAs, and / or transcriptional activity of IVT template DNA.

[0010] In another aspect, the invention described herein provides a multitude of polyA tail sequences for enhancing translational activity of mRNA containing these polyA tails, and the stability of polyA tail.

[0011] In a related aspect, the invention described herein provides mRNA comprising the 5’UTR, 3 ’UTR, and / or polyA tails of the invention (referred to herein as I- mRNA). The I- mRNA of the invention was demonstrated to have improved translation activity, polyA tail stability, and IVT yield compared to benchmark mRNAs without one or more of the 5’UTR, 3 ’UTR, and / or polyA tails of the invention.

[0012] Therefore, the I-mRNA of the invention circumvents multiple obstacles in mRNA design and can be an important tool for the development of mRNA-based vaccines and therapeutics for various diseases, such as infectious diseases and cancers.

[0013] Thus, one aspect of the invention provides a polynucleotide sequence comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 1.

[0014] In a related aspect, the invention provides a polynucleotide sequence comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 2.

[0015] In another related aspect, the invention provides a polynucleotide sequence comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 3.

[0016] In yet another related aspect, the invention provides a variant polynucleotide sequence of SEQ ID NO: 1, comprising, consisting essentially of, or consisting of a sequence that: (1) is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1;

[0017] (2) contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide changes compared to SEQ ID NO: 1;

[0018] (3) has the same RNA secondary structure (e.g., as predicted by MFold) as SEQ ID NO: 1; (4) has a Mean ribosome load (MRL) score of at least about 6, 7, or 8; and / or, (5) is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as SEQ ID NO: 1 as a 5’UTR sequence operably linked to a protein coding sequence, for expression of the protein.

[0019] In yet another related aspect, the invention provides a variant polynucleotide sequence of SEQ ID NO: 2, comprising, consisting essentially of, or consisting of a sequence that: (1) is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 2;

[0020] (2) contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide changes compared to SEQ ID NO: 2;

[0021] (3) has the same RNA secondary structure (e.g., as predicted by MFold) as SEQ ID NO: 2; and / or, (4) has a Mean ribosome load (MRL) score of at least about 6, 7, or 8; and / or, (5) is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as SEQ ID NO: 2 as a 5’UTR sequence operably linked to a protein coding sequence, for expression of the protein.

[0022] In yet another related aspect, the invention provides a variant polynucleotide sequence of SEQ ID NO: 3, comprising, consisting essentially of, or consisting of a sequence that: (1) is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 3;

[0023] (2) contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide changes compared to SEQ ID NO: 3;

[0024] (3) has the same RNA secondary structure (e.g., as predicted by MFold) as SEQ ID NO: 3; and / or, (4) has a Mean ribosome load (MRL) score of at least about 6, 7, or 8; and / or, (5) is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as SEQ ID NO: 3 as a 5’UTR sequence operably linked to a protein coding sequence, for expression of the protein.

[0025] In certain embodiments, the polynucleotide or variant polynucleotide is an RNA.

[0026] In certain embodiments, the RNA is / functions as the 5’UTR (5’ untranslated region) of an mRNA.

[0027] In certain embodiments, the protein coding sequence encodes a protein, such as a fluorescent protein or an oxidative enzyme capable of producing bioluminescence (e.g., a luciferase).

[0028] Another aspect of the invention provides a polynucleotide comprising SEQ ID NO: 4 having an inserted hypoxia response element sequence (HRE), such as an HRE comprising one or more repeats of RCGTG (R = A or G).

[0029] In certain embodiments, the HRE is from the hypoxia-induced egl nine homologue 3 (egln3 / phd3) gene.

[0030] In certain embodiments, the HRE is inserted anywhere within SEQ ID NO: 4, such as inserted immediately before or after nucleotide 87 of SEQ ID NO: 4, or at a nucleotide at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from nucleotide 87 of SEQ ID NO: 4.

[0031] In certain embodiments, the HRE is inserted 5’ to nucleotide 87 of SEQ ID NO: 4. In certain embodiments, the HRE is inserted 3’ to nucleotide 87 of SEQ ID NO: 4.

[0032] In certain embodiments, the polynucleotide of the invention comprises, consists essentially of, or consists of SEQ ID NO: 5.

[0033] In certain embodiments, the polynucleotide is an RNA.

[0034] In certain embodiments, the RNA is / functions as the 3 ’UTR (3 ’ untranslated region) of an mRNA.

[0035] In certain embodiments, the polynucleotide is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as SEQ ID NO: 5 as a 3 ’UTR sequence operably linked to a protein coding sequence, for expression of the protein in a hypoxia tissue.

[0036] In certain embodiments, the hypoxia tissue is a tumor or cancer tissue (such as a melanoma tissue).

[0037] In certain embodiments, the protein coding sequence encodes a protein, such as a fluorescent protein or an oxidative enzyme capable of producing bioluminescence (such as a luciferase).

[0038] Another aspect of the invention provides a polynucleotide having a sequence represented by: Ani - [Nn3 - An2]n4, wherein each N is independently a non-A nucleotide, nl = 10-80, n2 = 10-80, n3 = 2-15, and n4 = 2-20.

[0039] In certain embodiments, nl is about 21-40, about 25-35, or about 30.

[0040] In certain embodiments, n2 is about 21-40, about 25-35, or about 30.

[0041] In certain embodiments, n3 is 2, 3, or 4.

[0042] In certain embodiments, n3 is 2, and Nn3 is UU / TT, CU / CT, GG, or CC.

[0043] In certain embodiments, n4 is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0044] In certain embodiments, nl is 30, n2 is 30, Nn3 is TT, and n4 is 3.

[0045] In certain embodiments, the polynucleotide comprises, consists essentially of, or consists of the polynucleotide sequence of SEQ ID NO: 26.

[0046] In certain embodiments, the polynucleotide of the invention is an RNA.

[0047] In certain embodiments, the RNA is / functions as the polyA sequence of an mRNA. In certain embodiments, the polynucleotide is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as a control polyA sequence of the same length when operably linked to a protein coding sequence, for expression of the protein.

[0048] In certain embodiments, the protein coding sequence encodes a protein, such as a fluorescent protein or an oxidative enzyme capable of producing bioluminescence (such as a luciferase).

[0049] Another aspect of the invention provides a polynucleotide comprising or encoding the polynucleotide of the invention as the 5’UTR, a coding sequence for a gene of interest (GOI), the polynucleotide of the invention as the 3’UTR, and / or the polynucleotide of the invention as the polyA sequence.

[0050] In certain embodiments, the polynucleotide is an mRNA.

[0051] In certain embodiments, the polynucleotide is a closed circular molecule (such as a plasmid, a viral (e.g., adeno viral, AAV, HSV, baculoviral, or lentiviral) vector, or a circular RNA) or a linear molecule.

[0052] In certain embodiments, the GOI encodes an antigen (e.g., an antigen for vaccination against an infectious disease or a cancer), an enzyme or growth factor (e.g., enzyme or growth factor useful for enzyme and hormone replacement therapy), cytokines / chemokines (e.g., cytokines / chemokines for the treatment of cancers, autoimmune diseases, and inflammatory diseases), immune receptors (e.g., chimeric antigen receptors, T cell receptors, B cell receptors, costimulatory receptors / ligands for cancer therapy), antibodies (e.g., immune checkpoint inhibitors for cancer immunotherapy), or tumor inhibitory molecules (e.g., tumor suppressor and angiogenesis regulators for cancer therapy), or transcription factors (e.g., cell reprogramming factors for cell replacement therapy).

[0053] It should be understood that any one embodiment of the invention described herein, including embodiments described only in the examples or claims, can be combined with any one or more other embodiments of the invention, unless such combination is not applicable or proper, or unless the combination is expressly disclaimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIG. 1 is a schematic (not to scale) representation of a synthetic mRNA having the structural components of the invention described herein, with representative approaches to develop each component to produce novel mRNA structures with improved functionality (e.g., transcription, translation, and / or stability).

[0055] FIGS. 2A-2C shows the predicted secondary structures of 5’UTRs JL1, JL2, and JL3 (FIG. 2A), relative mRNA translation activity (FIG. 2B), and transcription activity compared to Reference l ’s 5’UTR sequence (FIG. 2C).

[0056] FIG. 3 A shows images of luciferase expression in normal skin tissues and melanoma skin tissues in mice. FIG. 3B shows total amount of luciferase expression in mice explant tissues after local administration of luciferase mRNA containing the 3’UTR HBA or the 3’UTR HBA / HRE sequence.

[0057] FIG. 4 A shows poly A tail instability during propagation of IVT template DNA plasmid in cells. FIG. 4B shows translation activity of IVT mRNA containing a natural polyA tail A(120) or various designed polyA tails.

[0058] FIGS. 5A-5B show comparisons of in vitro (FIG. 5A) and in vivo (FIG. 5B) translation activities of an I-mRNA of the invention encoding luciferase, composing the 5’UTR JL2, the 3’UTR HBA / HRE, and the polyA tail A(30)-[UU-A(30)]3, and a control luciferase mRNA composed of a control 5’UTRs, a control 3’UTRs, and a control polyA tails contained in an mRNA vaccine (Reference 1 mRNA and Reference 2 mRNA, respectively).

[0059] FIGS. 6A-6C show vector optimization. FIG. 6A shows improved stability of plasmids containing hybrid poly(A)-T2 sequence: E. coli Stbl3™ cells were transformed with pLuc-A o or pLuc-A3o(T2A3o)3 plasmids containing homopolymeric poly(A) sequence (A120) or hybrid poly(A)-T2 sequence (A3o(T2A3o)3). Poly(A) tail length was analyzed for individual bacterial clones. FIG. 6B shows in vitro activity ofLuc-A o and Luc-A3o(T2A3o)3mRNA in transfected BHK-21 cells. Luciferase activity was determined as RLU (relative luminescence units). FIG. 6C shows in vivo imaging of Luc-A o and Luc-A3o(T2A3o)3mRNA expression following i.m. delivery in mice. DET AILED DESCRIPTION OF THE INVENTION

[0060] Improving synthetic mRNA translation, polyA stability, and IVT yield are equally important to enhance the efficacy and manufacturing of mRNA vaccine and therapeutic agents. However, it has been challenging to simultaneously improve upon all these key factors of synthetic mRNA design.

[0061] The invention described herein provides improvements in translation activity, polyA stability, and / or transcription activity of synthetic mRNA products, by utilizing structural components of the synthetic mRNA of the invention, including 5’UTRs, 3 ’UTRs, and polyA tail [see Table 1], These novel 5’UTR, 3’UTR, and polyA tail sequences cooperate to improve translation and transcription activities, and stability of synthetic mRNAs. Specifically, synthetic mRNAs composing the subject 5’UTR, 3’UTR, and / or polyA tail are found to improve the expression of gene of interest (GOI) encoded by the mRNA, compared to the corresponding control mRNAs without the subject 5’UTR, 3’UTR, and / or polyA tail sequences, but are composed of some of the most potent known benchmark 5’UTR, 3’UTR, and polyA tail, such as those disclosed in WO / 2021 / 213945 Al and WO 2021 / 159040 A2 [both incorporated herein by reference].

[0062] More specifically, the I-mRNAs of the invention achieve high IVT yields, and allow for high levels of GOI expression and stability in both in vitro (e.g., mammalian cell lines) and in vivo (e.g., normal and tumor-bearing mice), which are both important parameters for manufacturability and therapeutic efficacy of mRNA vaccine and therapeutic agents.

[0063] Altogether, the I-mRNAs of the invention have broad utility in, for example, generation of efficient and reliable mRNA products for therapeutic and vaccine development.

[0064] Untranslated Regions (UTRs)

[0065] One aspect of the invention provides a 5’ UTR sequence that can be used in the mRNA of the invention as described in further detail below. In a related aspect, the invention also provides a 3’ UTR sequence that can be used in the mRNA of the invention.

[0066] As is generally understood, where mRNAs are designed to encode at least one protein of interest, the polynucleotide may also comprise one or more untranslated regions (UTRs).

[0067] Wild-type untranslated regions of a nucleic acid 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; whereas, the 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal.

[0068] There is 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.

[0069] A 5' UTR is region of an mRNA that is directly upstream (5') from the start codon (the first codon of an mRNA transcript translated by a ribosome). 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, 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.

[0070] One aspect of the invention provides a polynucleotide sequence comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 1. In a related aspect, the invention provides a polynucleotide sequence comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 2. In another related aspect, the invention provides a polynucleotide sequence comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 3. SEQ ID NOs: 1-3 can all function as 5’ UTR of an mRNA of the invention.

[0071] In yet another related aspect, the invention provides a variant polynucleotide sequence of SEQ ID NO: 1, comprising, consisting essentially of, or consisting of a sequence that: (1) is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1;

[0072] (2) contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide changes compared to SEQ ID NO: 1;

[0073] (3) has the same RNA secondary structure (e.g., as predicted by MFold) as SEQ ID NO: 1; (4) has a Mean ribosome load (MRL) score of at least about 6, 7, or 8; and / or, (5) is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as SEQ ID NO: 1 as a 5’UTR sequence operably linked to a protein coding sequence, for expression of the protein.

[0074] In yet another related aspect, the invention provides a variant polynucleotide sequence of SEQ ID NO: 2, comprising, consisting essentially of, or consisting of a sequence that: (1) is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 2;

[0075] (2) contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide changes compared to SEQ ID NO: 2;

[0076] (3) has the same RNA secondary structure (e.g., as predicted by MFold) as SEQ ID NO: 2; and / or, (4) has a Mean ribosome load (MRL) score of at least about 6, 7, or 8; and / or, (5) is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as SEQ ID NO: 2 as a 5’UTR sequence operably linked to a protein coding sequence, for expression of the protein.

[0077] In yet another related aspect, the invention provides a variant polynucleotide sequence of SEQ ID NO: 3, comprising, consisting essentially of, or consisting of a sequence that: (1) is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 3;

[0078] (2) contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide changes compared to SEQ ID NO: 3;

[0079] (3) has the same RNA secondary structure (e.g., as predicted by MFold) as SEQ ID NO: 3; and / or, (4) has a Mean ribosome load (MRL) score of at least about 6, 7, or 8; and / or, (5) is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as SEQ ID NO: 3 as a 5’UTR sequence operably linked to a protein coding sequence, for expression of the protein.

[0080] The MFold web server for nucleic acid folding and hybridization prediction can be access at unafold dot org slash mfold slash applications slash ma-folding-form.php. Also see Zucker, Nucleic Acids Res. 31 (13): 3406-3015, 2003; and Markham et al. , Methods Mol Biol. 453:3-31, 2008 (both incorporated herein by reference). In certain embodiments, the folding temperature is fixed at 37°C. In certain embodiments, all default recommendations of the MFold server are adopted.

[0081] The MRL score of a 5’ UTR sequence can be obtained through accessing optimus5 dot cs dot Washington dot edu slash MRL, which predicts the MRL score due to every possible SNV of the input 5' UTR sequence with length from 25 nt to 100 nt.

[0082] The effectiveness of a potential 5’ UTR sequence can be compared to that of a reference sequence, such as any one of SEQ ID NOs: 1-3, to direct the expression of a marker or reporter gene, such as one with conveniently measured enzymatic activity output such as fluorescent signal. The relative effectiveness of a candidate sequence can be measured against the reference as percentage value of the reporter activity.

[0083] In certain embodiments, the polynucleotide or variant polynucleotide is an RNA.

[0084] In certain embodiments, the RNA is / functions as the 5’UTR (5’ untranslated region) of an mRNA.

[0085] In certain embodiments, the protein coding sequence encodes a protein, such as a fluorescent protein or an oxidative enzyme capable of producing bioluminescence (e.g., a luciferase).

[0086] In some embodiments, the 5' UTR is a synthetic UTR, i.e., does not occur in nature.

[0087] A 3' UTR is region of an mRNA that is directly downstream (3') from the stop codon (the codon of an mRNA transcript that signals a termination of translation). A 3' UTR does not encode a protein (is non-coding).

[0088] One aspect of the invention a polynucleotide (which can function as 3’ UTR) comprising SEQ ID NO: 4 having an inserted hypoxia response element sequence (HRE), such as an HRE comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) repeats of RCGTG (R = A or G) sequence.

[0089] In certain embodiments, the HRE is from the hypoxia-induced egl nine homologue 3 (egln3 / phd3) gene.

[0090] In certain embodiments, the HRE is inserted anywhere within SEQ ID NO: 4. In certain embodiments, the HRE is inserted immediately before or after nucleotide 87 of SEQ ID NO: 4, or at a nucleotide at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from nucleotide 87 of SEQ ID NO: 4.

[0091] In certain embodiments, the HRE is inserted 5’ to nucleotide 87 of SEQ ID NO: 4.

[0092] In certain embodiments, the HRE is inserted 3’ to nucleotide 87 of SEQ ID NO: 4.

[0093] In certain embodiments, the polynucleotide of the invention comprises, consists essentially of, or consists of SEQ ID NO: 5.

[0094] In certain embodiments, the polynucleotide is an RNA. In certain embodiments, the RNA is / functions as the 3 ’UTR (3 ’ untranslated region) of an mRNA.

[0095] In certain embodiments, the polynucleotide is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as SEQ ID NO: 5 as a 3 ’UTR sequence operably linked to a protein coding sequence, for expression of the protein in a hypoxia tissue. In that regard, SEQ ID NO: 5 can be used as a reference sequence (for 100% relative activity / expression of an operably linked reporter gene) to assess the relative percentage of effectiveness associated with a variant 3 ’ UTR.

[0096] In certain embodiments, the hypoxia tissue is a tumor or cancer tissue (such as a melanoma tissue).

[0097] In certain embodiments, the protein coding sequence encodes a protein, such as a fluorescent protein or an oxidative enzyme capable of producing bioluminescence (such as a luciferase).

[0098] In certain embodiments, any 5’ UTR sequence of the invention may be used with any 3’ UTR sequence of the invention.

[0099] In certain embodiments, the 5’ UTR and / or 3’ UTR of the invention is placed in the same orientation as described above. In other embodiment, the 5’ UTR of the invention is altered in orientation or location, such as inverted, shortened, lengthened, made with one or more other 5' UTRs or 3 ' UTRs of the invention.

[0100] 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 reference UTR of the invention 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.

[0101] 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.

[0102] 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 AABBAABBAABB 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 5’ or 3’ UTR at the nucleotide level.

[0103] PolyA Sequence (poly A)

[0104] The 3' poly (A) sequence is important for nuclear export, RNA stability and translational efficiency of eukaryotic messenger RNA (mRNA). The 3' poly (A) sequence is shortened over time and if short enough, the RNA is degraded enzymatically.

[0105] Another aspect of the invention provides a polynucleotide (that may function as a polyA sequence for an mRNA) having a sequence represented by: Ani- [Nn3 - An2]n4, wherein each N is independently a non-A nucleotide, nl = 10-80, n2 = 10-80, n3 = 2-15, and n4 = 2-20.

[0106] In certain embodiments, nl is about 21-40, about 25-35, or about 30.

[0107] In certain embodiments, n2 is about 21-40, about 25-35, or about 30.

[0108] In certain embodiments, n3 is 2, 3, or 4.

[0109] In certain embodiments, n3 is 2, and Nn3 is UU / TT, CU / CT, GG, or CC.

[0110] In certain embodiments, n4 is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0111] In certain embodiments, nl is 30, n2 is 30, Nn3 is TT, and n4 is 3.

[0112] In certain embodiments, the polynucleotide comprises, consists essentially of, or consists of the polynucleotide sequence of SEQ ID NO: 26.

[0113] In some embodiments, the 3’-poly(A) tail of the invention, or “polyA” for short, is typically a stretch of nucleotides added to the 3 '-end of the transcribed mRNA that may, in some instances, comprise up to about 400 adenine nucleotides. 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.

[0114] In certain embodiments, the polynucleotide of the invention is an RNA.

[0115] In certain embodiments, the RNA is / functions as the polyA sequence of an mRNA.

[0116] In certain embodiments, the polynucleotide is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as a control polyA sequence of the same length when operably linked to a protein coding sequence, for expression of the protein.

[0117] In certain embodiments, the protein coding sequence encodes a protein, such as a fluorescent protein or an oxidative enzyme capable of producing bioluminescence (such as a luciferase).

[0118] Nucleic Acids / mRNA (I-mRNA)

[0119] The invention described herein features polynucleotides or nucleic acids, particularly messenger RNA (mRNA) designed to encode a protein of interest, e.g., a protein antigen useful for vaccination against cancer or an infectious pathogen (virus or bacteria), as well as any protein of interest, or a functional subunit, domain or fragments thereof.

[0120] Thus, another aspect of the invention provides a polynucleotide comprising or encoding the polynucleotide of the invention as the 5’UTR, a coding sequence for a gene of interest (GOI), the polynucleotide of the invention as the 3’UTR, and / or the polynucleotide of the invention as the polyA sequence.

[0121] In certain embodiments, the polynucleotide is a closed circular molecule (such as a plasmid, a viral (e.g., adeno viral, AAV, HSV, baculoviral, or lentiviral) vector, or a circular RNA) or a linear molecule.

[0122] In certain embodiments, the polynucleotide is an mRNA.

[0123] In certain embodiments, the GOI encodes an antigen (e.g., an antigen for vaccination against an infectious disease or a cancer), an enzyme or growth factor (e.g., enzyme or growth factor useful for enzyme and hormone replacement therapy), cytokines / chemokines (e.g., cytokines / chemokines for the treatment of cancers, autoimmune diseases, and inflammatory diseases), immune receptors (e.g., chimeric antigen receptors, T cell receptors, B cell receptors, costimulatory receptors / ligands for cancer therapy), antibodies (e.g., immune checkpoint inhibitors for cancer immunotherapy), or tumor inhibitory molecules (e.g., tumor suppressor and angiogenesis regulators for cancer therapy), or transcription factors (e.g., cell reprogramming factors for cell replacement therapy).

[0124] The term "mRNA" means "messenger-RNA," and relates to an RNA transcript which encodes a peptide or protein. The mRNA of the invention comprises a protein coding region for a protein of interest, and one or more of (e.g., all of) a 5’-UTR of the invention, a 3'-UTR of the invention, and / or a polyA sequence of the invention.

[0125] Messenger RNA (mRNA), a subtype of RNA, is a single-stranded molecule of RNA that corresponds to the genetic sequence of a gene. mRNA is created during the process of transcription wherein a single strand of DNA is decoded by RNA polymerase, and mRNA is synthesized, i.e., transcribed. mRNA is read by a ribosome in the process of synthesizing a protein, i.e., translation. Accordingly, messenger RNA (mRNA) is an RNA that encodes a (at least one) protein (a naturally-occurring, non-naturally -occurring, or modified polymer of amino acids, such as any of the ones described above) and can be translated to produce the encoded protein in vitro, in vivo, in situ, or ex vivo. Featured in the instant invention are messenger RNAs (mRNAs), particularly mRNAs designed to encode a protein (such as an antigen) of interest, or a functional subunit, domain or fragments thereof. mRNA may be generated by in vitro transcription from a DNA template. The in vitro transcription methodology is known to the skilled person. For example, there is a variety of in vitro transcription kits commercially available. mRNA can also be directly synthesized chemically without a template, which enables incorporation of one or more modified nucleotides (bases, linkages between nucleotides, etc.) into the mRNA. See Abe et al. , ACS Chem. Biol. 17(6): 1308-1314, 2022.

[0126] According to the invention, mRNA may be further modified by stabilizing modifications and capping.

[0127] The nucleic acids, for example mRNAs, of the invention can, in some embodiments, be formulated in appropriate carriers or delivery vehicles (e.g., lipid nanoparticles), such that the nucleic acids, e.g., mRNAs are suitable for use in vivo. When appropriately formulated, nucleic acids of the invention, e.g., mRNAs, are capable of being delivered to cells and / or tissues within a subject, e.g., a human subject, to effectuate translation of protein encoded by these nucleic acids.

[0128] Nucleic acid molecules are macromolecules comprised of linked nucleotides that carry genetic information and by directing the process of protein synthesis, direct most if not all cellular functions. Nucleic acids comprise a polymer of nucleotides (nucleotide monomers). Thus, nucleic acids are also referred to as polynucleotides (also referred to as polynucleotide chains). The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).

[0129] Nucleosides are the structural subunit of nucleic acids such as DNA and RNA. A nucleoside is composed of a nitrogenous base (a nucleobase), usually either a pyrimidine (cytosine, thymine or uracil) or a purine (adenine or guanine), covalently attached to a five- carbon carbohydrate ribose or “sugar” which is either ribose or deoxyribose. Nucleotides consist of a nitrogenous base, a sugar (ribose or deoxyribose) and one to three phosphate groups. In essence, a nucleotide is simply a nucleoside with an additional phosphate group or groups.

[0130] The nucleic acid molecules, DNA and RNA, are composed of nucleotides that are linked to one another in a chain by chemical bonds known as ester bonds, between the sugar base of one nucleotide and the phosphate group of the adjacent nucleotide. The sugar is the 3' end, and the phosphate is the 5' end of each nucleotide. The phosphate group attached to the 5' carbon of the sugar on one nucleotide forms an ester bond with the free hydroxyl on the 3' carbon of the next nucleotide. These bonds are called phosphodiester bonds, and the sugar-phosphate backbone is described as extending, or growing, in the 5' to 3' direction when the molecule is synthesized.

[0131] The nucleobase portion of nucleic acids features purine bases, adenine (A) and guanine (G), and pyrimidine bases, cytosine (C), thymine (T) in DNA, and uracil (U) in RNA. The sugar portion of nucleic acids features deoxyribose in DNA, ribose in RNA. The five nucleosides are commonly abbreviated to their one-letter codes A, G, C, T and U, respectively. However, thymidine is more commonly written as “dT” (“d” represents “deoxy”) as it contains a 2'- deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) and not ribonucleic acid (RNA). Conversely, uridine is found in RNA and not DNA. The remaining three nucleosides may be found in both RNA and DNA. In RNA, they would be represented as A, C and G whereas in DNA they would be represented as dA, dC and dG.

[0132] 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.” Also consistent with the WIPO ST.26 standard, DNA and RNA having the same sequence but differ only with respect to T’s (in DNA) and U’s (in RNA) are assigned the same SEQ ID NO.

[0133] In certain embodiments, nucleic acids of the invention may be or may include, for example, deoxyribonucleic acids (DNAs), ribonucleic acids (RNAs), e.g. mRNAs, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, 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 acids (ENA), cyclohexenyl nucleic acids (CeNA) and / or chimeras and / or combinations thereof.

[0134] In certain other embodiments, nucleic acids of the invention (e.g., coding sequence for mRNA) may only include DNA.

[0135] In certain other embodiments, nucleic acids of the invention (e.g., mRNA) may only include RNA.

[0136] The compositions of the present disclosure comprise a (at least one) mRNA having an open reading frame (ORF) encoding a gene of interest (GOI), such as a vaccine antigen. In some embodiments, the mRNA further comprises a 5' UTR of the invention, a 3' UTR of the invention, a poly(A) tail of the invention, and / or a 5' cap or cap analog.

[0137] An open reading frame (ORF) is a continuous stretch of DNA or RNA beginning with a start codon (e.g., methionine (ATG or AUG)) and ending with a stop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA). An ORF typically encodes a protein. It will be understood that the sequences disclosed herein may further comprise additional elements, e.g., 5' and 3' UTRs and polyA of the invention, but that those elements, unlike the ORF, need not necessarily be all present in a particular mRNA of the present disclosure.

[0138] It should also be understood that the mRNAs of the invention may include any 5' untranslated region (UTR), any 3' UTR, and / or any polyA sequence of the invention. Exemplary UTR and polyA sequences are provided in the Sequence Listing and Table 1 below. However, other UTR / polyA sequences may be used or exchanged for any of the UTR / polyA sequences of the invention described herein. UTRs may also be omitted from the mRNAs provided herein. Naturally -occurring eukaryotic mRNA molecules can contain stabilizing elements, including, but not limited to untranslated regions (UTR) 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.

[0139] In some embodiments, the mRNA of the invention comprising an open reading frame encoding at least one protein of interest has at least one 5' terminal cap.

[0140] The term "5'-cap" refers to a cap structure found on the 5'-end of an mRNA molecule and generally consists of a guanosine nucleotide connected to the mRNA via an unusual 5' to 5' triphosphate linkage. In one embodiment, this guanosine is methylated at the 7-position.

[0141] The term "conventional 5'-cap" refers to a naturally occurring RNA 5'-cap, preferably to the 7-methylguanosine cap (m7G). In the context of the present invention, the term "5'-cap" includes a 5 '-cap analog that resembles the RNA cap structure and is modified to possess the ability to stabilize RNA if attached thereto, preferably in vivo and / or in a cell.

[0142] Providing an RNA with a 5'-cap or 5'-cap analog may be achieved by in vitro transcription of a DNA template in the presence of said 5'-cap or 5'-cap analog, wherein said 5'- cap is co-transcriptionally incorporated into the generated RNA strand, or the RNA may be generated, for example, by in vitro transcription, and the 5'-cap may be generated post- transcriptionally using capping enzymes, for example, capping enzymes of vaccinia virus.

[0143] 5'-capping of polynucleotides / mRNA may be completed concomitantly during the in vitro transcription reaction using the following chemical RNA cap analogs 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).

[0144] In certain embodiments, the 5 ’cap is Cap 0. 5'-capping of the subject 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). In certain embodiments, the 5 ’cap is Cap 1. The 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.

[0145] In certain embodiments, the 5 ’cap is Cap 2. The 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 methyl-transferase.

[0146] In certain embodiments, the 5 ’cap is Cap 3. The 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.

[0147] All enzymes used for capping may be derived from a recombinant source.

[0148] In some embodiments, the mRNA of the invention further comprising a histone stemloop. A stem-loop 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 snRNP. 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 3' relative to the stem-loop.

[0149] In some embodiments, the mRNA of the invention comprising a coding region, at least one histone stem-loop, and a poly(A) sequence or poly adenylation signal of the invention described herein. 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, GPP, EGFP, b-Galactosidase, EGFP), or a marker or selection protein (e.g. alpha-Globin, Galactokinase and Xanthine: guanine phosphoribosyl transferase (GPT)).

[0150] In some embodiments, the mRNA of the invention comprising the combination of a poly(A) sequence or poly adenylation signal of the invention, and at least one histone stem-loop, even though both represent alternative mechanisms in nature, acts 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 at least one histone stem-loop does not depend on the order of the elements or the length of the poly(A) sequence.

[0151] In some embodiments, the mRNA of the invention 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 stemloops, 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.

[0152] An mRNA of the invention 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. 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.

[0153] In some embodiments, the mRNA of the invention has one or more AU-rich sequences removed. These sequences, sometimes referred to as AURES are destabilizing sequences found in the 3'UTR.

[0154] In some embodiments, the mRNA of the invention comprises non-UTR sequences. 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.

[0155] In some embodiments, the untranslated region may also include translation enhancer elements (TEE). As a non-limiting example, the TEE may include those described in US Application No.20090226470, herein incorporated by reference in its entirety, and those known in the art.

[0156] Signal Peptides

[0157] In some embodiments, the mRNA of the invention comprises an ORF that encodes a signal peptide fused to the protein of interest encoded by the mRNA.

[0158] 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 a 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.

[0159] 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.

[0160] Signal peptides from heterologous genes (which regulate expression of genes other than the gene of interest) are known in the art, and can be tested for desired properties and then incorporated into a nucleic acid of the disclosure.

[0161] It should be understood that any one of the proteins encoded by the mRNA described herein may or may not comprise a signal sequence, and may or may not retain the first Met.

[0162] Sequence Optimization

[0163] In some embodiments, an ORF encoding a protein (e.g., an antigen for a vaccine) of the disclosure is codon optimized.

[0164] Codon optimization methods are known in the art. For example, an ORF 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 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 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.

[0165] 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 (ORF) sequence is optimized using optimization algorithms.

[0166] In some embodiments, a codon optimized sequence shares less than 95% sequence identity to a naturally -occurring or wild-type sequence ORF. In some embodiments, a codon optimized sequence shares less than 90% sequence identity to a naturally-occurring or wild-type sequence. In some embodiments, a codon optimized sequence shares less than 85% sequence identity to a naturally -occurring or wild-type sequence. In some embodiments, a codon optimized sequence shares less than 80% sequence identity to a naturally-occurring or wild-type sequence. In some embodiments, a codon optimized sequence shares less than 75% sequence identity to a naturally -occurring or wild-type sequence.

[0167] 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. In some embodiments, a codon optimized sequence shares between 65% and 75% or about 80% sequence identity to a naturally-occurring or wildtype sequence.

[0168] When transfected into mammalian host cells, the mRNA of the invention typically has 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.

[0169] In some embodiments, a codon optimized RNA 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 having an increased amount of guanine (G) and / or cytosine (C) residues may be functionally more stable than mRNA 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 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.

[0170] Chemically Unmodified Nucleotides

[0171] In some embodiments, the mRNA of the invention is not chemically modified and comprises the standard ribonucleotides consisting of adenosine, guanosine, cytosine and uridine.

[0172] 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).

[0173] 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).

[0174] Chemical Modifications

[0175] The mRNA of the invention may comprise 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.

[0176] 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.

[0177] 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 aha, in the widely recognized MODOMICS database.

[0178] 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 published US application Nos. PCT7US2012 / 058519; PCT / US2013 / 075177;

[0179] PCT / US2014 / 058897; PCT / US2014 / 058891; PCT / US2014 / 070413; PCT7US2015 / 36773; PCT / US2015 / 36759; PCT7US2015 / 36771; or PCT / IB2017 / 051367 all of which are incorporated by reference herein.

[0180] Hence, nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids) can comprise standard nucleotides and nucleosides, naturally- occurring nucleotides and nucleosides, non-naturally -occurring nucleotides and nucleosides, or any combination thereof.

[0181] Nucleic acids of the disclosure (e.g., DNA nucleic acids and RNA nucleic acids, such as mRNA nucleic acids), 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.

[0182] In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), 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.

[0183] In some embodiments, a modified RNA nucleic acid (e.g., a modified mRNA nucleic acid), 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.

[0184] Nucleic acids (e.g., RNA nucleic acids, such as mRNA nucleic acids), 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.

[0185] The present disclosure provides for modified nucleosides and nucleotides of a nucleic acid (e.g., RNA nucleic acids, such as mRNA nucleic acids).

[0186] 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”).

[0187] 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 (Ml\| / ), 1-ethyl-pseudouridine (ely), 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, an 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, an 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.

[0192] In some embodiments, an mRNA of the disclosure comprises pseudouridine (y) substitutions at one or more or all uridine positions of the nucleic acid.

[0193] In some embodiments, an 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.

[0194] In some embodiments, an mRNA of the disclosure comprises uridine at one or more or all uridine positions of the nucleic acid.

[0195] 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.

[0196] 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.

[0197] 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.

[0198] 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).

[0199] In vitro Transcription of RNA 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.

[0200] 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 an 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 an RNA polymerase promoter, e.g., a T7 promoter located 5 ' to and operably linked to the gene of interest.

[0201] In some embodiments, an in vitro transcription template encodes a 5' untranslated (UTR) region of the invention, contains an open reading frame, and encodes a 3' UTR of the invention, and / or a poly(A) tail of the invention. The particular nucleic acid sequence composition and length of an in vitro transcription template will depend on the mRNA encoded by the template.

[0202] 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. 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.

[0203] A “3' untranslated region” (UTR) refers to a region of an mRNA that is directly downstream (i.e., 3') from the stop codon (i.e., the codon of an mRNA transcript that signals a termination of translation) that does not encode a polypeptide.

[0204] An “open reading frame” is a continuous stretch of DNA beginning with a start codon (e.g., methionine (ATG)), and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a polypeptide.

[0205] A “poly(A) tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR. A poly(A) tail may contain 10 to 300 monophosphates. 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 monophosphates. In some embodiments, a poly(A) tail contains 50 to 250 mostly 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.

[0206] In some embodiments, a nucleic acid / mRNA of the invention includes 200 to 3,000 nucleotides. For example, a nucleic acid / mRNA of the invention may include 200 to 500, 200 to 1000, 200 to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to 3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to 3000 nucleotides).

[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 for in vitro transcription. 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] Chemical Synthesis

[0212] Solid-phase chemical synthesis. Nucleic acids / mRNAs of 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. Liquid Phase Chemical Synthesis. The synthesis of nucleic acids of the present disclosure by the sequential addition of monomer building blocks may also be carried out in a liquid phase.

[0213] Combination of Synthetic Methods. 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.

[0214] Ligation of Nucleic Acid Regions or Subregions

[0215] Assembling nucleic acids by a ligase may also be used to produce the mRNA of the invention. DNA or RNA ligases promote intermol ecul ar 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.

[0216] Purification

[0217] 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).

[0218] 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.

[0219] 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.

[0220] In some embodiments, the nucleic acids may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.

[0221] Quantification

[0222] 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, broncheo alveolar 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.

[0223] Assays may be performed using construct specific probes, cytometry, qRT-PCR, realtime 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.

[0224] 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.

[0225] 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).

[0226] Pharmaceutical Formulations

[0227] Provided herein are compositions (e.g., pharmaceutical compositions), kits, reagents, and uses thereof for producing proteins from mRNA in vitro and / or in vivo. The compositions provided herein can be used as therapeutic or prophylactic agents. They may also be used in medicine to prevent and / or treat diseases, such as infectious diseases or cancer.

[0228] In some embodiments, the mRNA as described herein can be administered to a subject (e.g., a mammalian subject, such as a human subject), and the mRNAs are translated in vivo to produce a protein of interest.

[0229] 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 composition, and other determinants, such as age, body weight, height, sex and general health of the subject. Typically, for example, in the context of vaccination, an effective amount of a composition for vaccination provides an induced or boosted immune response as a function of antigen production in the cells of the subject.

[0230] In some embodiments, an effective amount of the composition containing the mRNA of the invention having at least one chemical modifications are more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same protein. For example, 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.

[0231] 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.

[0232] 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.

[0233] Additional suitable pharmaceutical carriers and diluents, as well as pharmaceutical necessities for their use, are described in Remington's Pharmaceutical Sciences.

[0234] In some embodiments, the compositions (comprising polynucleotides and their encoded polypeptides) in accordance with the present disclosure 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.

[0235] A composition may be administered with other prophylactic or therapeutic compounds.

[0236] 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 prophylactic (vaccine) composition. 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, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25 years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60 years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95 years or more than 99 years. 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, 6 months or 1 year.

[0237] In some embodiments, a composition may be administered intramuscularly, intranasally or intradermally.

[0238] Provided herein are pharmaceutical compositions including RNA and / or complexes optionally in combination with one or more pharmaceutically acceptable excipients.

[0239] The RNA may be formulated or administered alone or in conjunction with one or more other components. For example, a composition may comprise other components including, but not limited to, adjuvants.

[0240] In some embodiments, a composition does not include an adjuvant (they are adjuvant free).

[0241] An RNA may be formulated or administered in combination with one or more pharmaceutically-acceptable excipients. In some embodiments, the composition is a vaccine compositions comprising at least one additional active substance, 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).

[0242] In some embodiments, a composition is administered to humans, human patients or subjects.

[0243] Formulations of the 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) 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.

[0244] 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.

[0245] In some embodiments, an mRNA 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 profile 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.

[0246] EXAMPLES

[0247] The experiments below demonstrate that several designed 5’UTR, 3’UTR, and polyA tail sequences of the invention enhance efficacy of mRNA vaccines and therapeutics, through enhanced expression / translation of the synthetic mRNA vaccines and therapeutics, polyA tail- conferred mRNA stability, as well as IVT yields.

[0248] Example 1 5’ UTR Sequence Designs

[0249] Utilizing algorithm-supported sequence and structure design, three unique 5 ’UTR sequences referred to as JL1, JL2, and JL3 were designed [SEQ 1, 2, 3], These sequences all have enhanced / high score (e.g., MRL score of over 5, 6, 7, or 8) based on the Mean ribosome load (MRL) score prediction software [optimus5.cs. washington.edu / MRL]. JL1-JL3 all have MRL score of over 8. Enhanced MRL scores are bioinformatically predicted to have more ribosomes associated with the mRNA, which in turn can predict enhanced translation efficacy (Sample et al., Nat Biotechnol. 37:803-9, 2019).

[0250] An additional consideration for a well-functioning 5 ’UTR is the secondary structure of 5 ’UTR, which regulates the initiation of mRNA translation (Hedaya et al., Nature Communications. 14:6166, 2023). The M-Fold software [unafold.org / mfold / applications / ma- folding-form.php] was used to predict secondary structure of the designed 5’ UTR region sequences (see Markham et al., Methods Mol Biol. 453:3-31, 2008) [Fig. 2A],

[0251] In FIG. 2B, a reference / reporter luciferase mRNA containing JL1, JL2, or JL3 showed a comparable translation activity compared to the same luciferase mRNA containing 5 ’UTR of Reference 1 ’s mRNA. Interestingly, DNA templates for IVT of luciferase mRNA containing JL2, JL3, or Reference l’s 5 ’UTR produced significantly greater amounts of luciferase mRNA by IVT than DNA template for IVT of luciferase mRNA containing JL1 [Fig. 2C].

[0252] Example 2 3 ’ UTR Sequence Designs

[0253] The naturally occurring 3 ’UTR of human hemoglobin alpha (hHBA) mRNA (referred to as HBA) [SEQ 4], is canonically known for enhancing mRNA translation, and is broadly used in synthetic mRNAs (see Holcik et al., PNAS 94:2410-2414, 1997). However, such synthetic mRNAs containing hHBA 3 ’UTR sequence are not evenly expressed in vivo in all types of tissues, depending on the presence of certain normal or abnormal conditions. For example, various inflammatory conditions, such as cancers and trauma, lead to the development of severe tissue hypoxia (Eltzschig et al. , NEJM. 364(7): 656-665, 2011). Cells exposed to hypoxia down- regulate the expression of multiple genes at transcription and translation levels, and simultaneously up-regulate the expression of hypoxia-induced genes (Cavadas et al., Scientific Reports. 6:31355, 2016), leading to improved survival and proliferation of cells after low oxygen insult (Brugarolas et al., Genes & Development 18:2893-2904, 2004). The expression of hypoxia-induced genes is known to dependent on the presence of hypoxia response element sequence (HRE) in non-coding regions of these hypoxia-induced genes (Semenza, Trends Pharmacol Sci. 33(4): 207-214, 2012).

[0254] To overcome reduced expression of synthetic mRNAs with hHBA 3’UTR in hypoxic tissues, a 3’UTR was designed to contain an hHBA sequence, into which was inserted the HRE of hypoxia-induced egl nine homologue 3 (egln3 / phd3) gene (see Pescador et al., Biochemical Journal. 390, 189-197, 2005), referred to herein as HBA / HRE [SEQ 5, with the non-HBA sequence double underlined].

[0255] In FIG. 3, luciferase mRNA containing 3 ’UTR HBA was significantly less expressed in hypoxic melanoma skin tissues than in normal skin tissues. However, luciferase mRNA containing 3’UTR HBA / HRE was equally well expressed in melanoma and normal skin tissues. Furthermore, normal skin tissues expressed comparable levels of luciferase mRNAs containing either 3’UTR HBA / HRE or 3’UTR HBA, whereas hypoxic melanoma skin tissues expressed significantly higher level of luciferase mRNA containing 3’UTR HBA / HRE than that containing 3 ’UTR HBA [Fig. 3],

[0256] Example 3 PolyA Sequence Designs

[0257] A growing body of evidence shows that the expression of synthetic mRNAs is positively correlated to the length of polyA tail (Holtkamp et al., Blood. 108(13):4009-4017, 2006; Grier, et al., Molecular Therapy -Nucleic Acids 5:e306, 2016). However, the control of polyA tail length turns out to be extremely difficult for the expansion and IVT synthesis of synthetic mRNAs with long polyA tails.

[0258] This example demonstrates that certain engineered polyA tail signal sequences confer improved stability of polyA tail and translation activity of synthetic mRNAs containing such engineered polyA tails.

[0259] To this end, a large panel of IVT template DNA constructs were designed and generated to contain various lengths of polyA tails, each containing two consecutive non-A nucleotides [Table 2] All these IVT DNA constructs maintained the original polyA tail sequences during propagation in cells independent of their length [FIG. 4A], Interestingly, luciferase mRNA containing polyA tail with two consecutive non-A nucleotides improved the level of luciferase expression in proportion to the length of the polyA tail [Fig. 4B],

[0260] Example 3 I -mRNA Designs

[0261] Synthetic mRNAs comprising the new 5’UTR, 3’UTR, and polyA tail of the invention have comparable or significantly better translation activities in vitro and in vivo than corresponding control mRNAs composed of the benchmark 5’UTR, 3’UTR, and polyA tail contained in Reference 1 & 2 mRNAs useful as COVID- 19 vaccines [FIG. 5],

[0262] Altogether, the mRNA structural components rationally designed herein provide improved stability and translation activity of synthetic mRNA comprising such structural elements. Thus, these structural components have a great potential to improve the therapeutic efficacy of the various mRNA-based therapeutic agents, such as mRNA vaccines and therapeutics against infections diseases and cancers.

[0263] Table 1. RNA sequence information

[0264] Example 4 Seasonal influenza mRNA vaccine candidates with a hybrid poly (A) sequence

[0265] This study shows systematic optimization of seasonal influenza mRNA vaccine candidates that encompasses vector development with a novel hybrid poly(A) sequence. Methods

[0266] Plasmids Construction

[0267] Luciferase reporter plasmids: synthetic DNA fragments including luciferase gene, 5’ or 3’ UTR sequences, and ultramers containing A30 and T2A30 blocks (IDT) were cloned into pUC- GW-Kan plasmid (Azenta, Morrisville, NC, USA).

[0268] Plasmid Stability

[0269] In order to compare A120 and AsofUAso^ tail stability, E. coli Stbl3™ cells were transformed with luciferase reporter plasmids (pLuc) containing either homopolymeric poly(A) sequence (A120) or a hybrid poly(A) sequence including T2 spacers (AsofUAso^). After transformation, cells were plated on kanamycin selection plates and incubated at 37°C overnight. Colonies were chosen at random and incubated in LB media at 37°C overnight. Plasmids were purified from overnight culture and analyzed by Sanger sequencing. mRNA Production

[0270] Plasmids were linearized via BspQI and purified by phenol-chloroform extraction followed by sodium acetate precipitation and three 70% ethanol washes. Pellets were resuspended in nuclease free water. mRNA was generated by in vitro transcription (IVT) using linear plasmid DNA template (50 pg / ml) and IVT reaction mixture containing Tris-HCl (40mM), MgCh (24mM) (Invitrogen)), ribonucleoside triphosphate mix (6 mM each NTP, with Nl- methylpsuedouridine 5 ’ -triphosphate used in place of UTP), yeast inorganic pyrophosphatase (2 U / ml), RNase inhibitor (1000 U / ml), and T7 RNA Polymerase (5000 U / ml) (Hongene Biotech, Shanghai, China) at 30°C for 3 hrs. DNA template in the IVT reaction was removed by DNase treatment at 30°C for 30 min (Hongene Biotech). After DNase treatment, mRNA was precipitated with LiCl (Invitrogen). Pellets were washed in 70% ethanol, air-dried and resuspended in nuclease free water. Post-transcriptional capping reaction included the following components: vaccinia capping system (200 U / ml), capping buffer with total 2mM of MgCh, GTP (0.75 mM), S -adenosylmethionine (0.32 mM), RNase inhibitor (500 U / ml), and 2 -O- methyltransferase (1000 U / ml) (all from Hongene Biotech). The capping reaction was carried out by incubating mRNA with vaccinia virus capping enzyme and 2-O-methyltransferase (Hongene Biotech,) at 37°C at for 1.5 hrs, which added a 7-methylguanylate cap structure (Cap 1) to the 5’ end of mRNA. Newly capped mRNA was precipitated with LiCl (Invitrogen), and pellets were washed in 70% ethanol, air-dried and resuspended in nuclease free water at concentration of 1 pg / pl. mRNA concentration was confirmed via nanodrop. Correct size and mRNA purity were determined by RNA gel electrophoresis. mRNA was stored at -80°C.

[0271] Formulation

[0272] Ready To Use (RTU) Lipid Nano Particles (LNP). The RTU LNP mRNA vaccines were formulated in a two-vial system composed of a lyophilized mRNA vial and a separate LNP dispersion vial. For generation of combination vaccines comprising several mRNA species, the individual mRNA preparations were pre-mixed prior to lyophilization. The LNP dispersion was generated using a microfluidic process (Dolomite Microfluidics, Unchained Labs, Pleasanton, CA, USA). Four LNP components: 2-dimyristoyl-rac-glycero-3 -methoxypolyethylene gly col- 2000 (Avanti, Alabaster, AL, USA), cholesterol (Sigma-Aldrich St. Louis, MO, USA)), 1,2- distearoyl-sn-glycero-3 -phosphocholine (Avanti, Alabaster, AL, USA), and cationic lipid (inhouse code Lipid No. 4 (10,000) cells per well in a 96-well cell culture treated plate in 100 pL growth media and incubated overnight at 37°C, 5% CO2. Next day, cells were transfected in triplicate with 100 ng / well of Luc-A o or Luc-A3o(T2-A3o)3 mRNA using Lipofectamine MessengerMax (Invitrogen) according to manufacturer’s instructions. Transfected cells were incubated for 24 hrs at 37°C, 5% CO2 prior to luciferase activity assay. Luciferase activity was measured with Bio-Gio™ reagent (Promega, Madison, WI, USA) added to the transfected cells at 1 : 1 volume ratio. Plates were incubated at room temperature in the dark on an orbital shaker for 15 min prior to reading on SpectraMax ID5 plate reader (Molecular Devices, San Jose, CA).

[0273] In Vivo Immunogenicity Studies

[0274] All in vivo experiments were conducted in the vivarium operated by Mispro, Durham, NC, in accordance with the approved IACUC protocols. Naive 6 to 8-weeks-old female BALB / c mice were sourced from The Jackson Laboratory (Bar Harbor, ME, USA). Mice were given food and water ad libitum throughout the course of all studies

[0275] Luciferase activity in vivo was detected after intramuscular (I.M.) delivery of RTU LNP formulated Luc-A o or Luc-A3o(T2A3o)3 mRNA (5 pg per injection). In vivo imaging was performed 16 hrs after injection, using VivoGlo™ Luciferin (Promega) substrate on Pearl Trilogy Imaging System (LI-COR Biosciences, Lincoln, NE, USA). Statistical analysis. The assays were performed with duplicate technical replicates for all biological replicates. Statistical analysis was performed using GraphPad Prism (10) software. Differences in mean values among more than two groups were determined using ANOVA with Tukey’s test for mean comparisons. P < 0.05(*), P < 0.01(**), P < 0.001(***) and P < 0.0001(****) indicate statistically significant differences; not significant (n.s.).

[0276] Results

[0277] Vector Optimization

[0278] Segmented or heteromeric poly(A) tail sequences have been shown to increase plasmid stability and mRNA expression. Here a novel segmented poly(A) tail was developed by minimizing the length of the linkers placed in between homomeric adenosine stretches. A homomeric tail length of approximately 120 A’s is considered to be optimal for maintaining plasmid stability and maximizing translation efficiency of synthetic mRNAs as longer tails do not significantly increase protein expression. This hybrid poly(A) sequence A3o(T2A3o)3 was tested against a homomeric poly(A) tail sequence of 120 adenosine residues (A120).

[0279] Plasmid stability was evaluated by transforming sequence confirmed pLuc-A o and pLuc-A3o(T2A3o)3 plasmids into E. coli Stbl3™ cells. Only 2 of 7 pLuc-A o colonies contained full-length poly(A) tail, while 11 of 12 pLuc-A3o(T2A3o)3 clones contained the full-length hybrid poly(A) tail. The corresponding mutation rates of pLuc-A o or pLuc-A3o(T2A3o)3 plasmids were 71.4% and 8.3%, respectively (FIG. 6A). In addition, the only mutated pLuc-A3o(T2A3o)3 plasmid isolate had a single A nucleotide deletion whereas the deletion mutations of poly(A) tails seen in pLuc-A o plasmids ranged from 48 to 84 bases.

[0280] In vitro translation activities of mRNAs encoded by pLuc-A o or pLuc-A3o(T2A3o)3 plasmids were measured by luciferase assay in BHK-21 cells. The cells transfected with Luc- A3O(T2A3O)3 mRNA had approximately 2-fold higher luciferase activity than those transfected with Luc-A o mRNA (FIG. 6B), confirming improved translational activity of Luc-A3o(T2A3o)3 mRNA compared with conventional Luc-A o mRNA. Finally, robust in vivo luciferase expression was confirmed in mouse muscle tissue after injection of RTU LNP formulated Luc- A3O(T2A3O)3 as well as Luc-A o mRNA (FIG. 6C).

[0281] A common problem for mRNA technologies is ensuring optimum length and uniformity of poly(A) tail sequences. Native poly(A) sequences containing homomeric A stretches pose considerable manufacturing risks due to plasmid instability. While mRNA molecules with longer poly(A) tails have increased stability and expression within cells, the longer homomeric poly(A) tail sequences are harder to maintain in plasmid. This study developed a new heteromeric poly(A) sequence, referred to as hybrid poly(A), with the non-A spacer element minimized to two nucleotides: T2 in the plasmid template transcribed into U2 or T2 in mRNA or N1 -methylpseudo uridine modified mRNA. We also minimized the length of the individual homo meric A stretches to 30 As, in line with the model of consecutive poly(A)-binding protein (PABP) molecules bound to the same poly(A) tract as a series of repeating units covering approximately 27. Our hybrid poly(A) sequence is functional both in terms of improved plasmid stability and efficient expression in vitro and in vivo (FIGs. 6A-6C). While we have not observed sequence instability of the plasmids described in this study, the use of the hybrid poly(A) sequence significantly reduced the bacterial colony screening effort during cloning and ensured consistent poly(A) tail length of the mRNAs included in our vaccines.

Claims

CLAIMS1. A polynucleotide sequence comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 1.

2. A polynucleotide sequence comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 2.

3. A polynucleotide sequence comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 3.

4. A variant polynucleotide sequence of SEQ ID NO: 1, comprising, consisting essentially of, or consisting of a sequence that:(1) is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 1;(2) contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide changes compared to SEQ ID NO: 1;(3) has the same RNA secondary structure (e.g. , as predicted by MFold) as SEQ ID NO: 1;(4) has a Mean ribosome load (MRL) score of at least about 6, 7, or 8; and / or,(5) is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as SEQ ID NO: 1 as a 5’UTR sequence operably linked to a protein coding sequence, for expression of the protein.

5. A variant polynucleotide sequence of SEQ ID NO: 2, comprising, consisting essentially of, or consisting of a sequence that:(1) is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 2;(2) contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide changes compared to SEQ ID NO: 2;(3) has the same RNA secondary structure (e.g. , as predicted by MFold) as SEQ ID NO: 2; and / or,(4) has a Mean ribosome load (MRL) score of at least about 6, 7, or 8; and / or,(5) is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as SEQ ID NO: 2 as a 5’UTR sequence operably linked to a protein coding sequence, for expressionof the protein.

6. A variant polynucleotide sequence of SEQ ID NO: 3, comprising, consisting essentially of, or consisting of a sequence that:(1) is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to SEQ ID NO: 3;(2) contains at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide changes compared to SEQ ID NO: 3;(3) has the same RNA secondary structure (e.g. , as predicted by MFold) as SEQ ID NO: 3; and / or,(4) has a Mean ribosome load (MRL) score of at least about 6, 7, or 8; and / or,(5) is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as SEQ ID NO: 3 as a 5’UTR sequence operably linked to a protein coding sequence, for expression of the protein.

7. The polynucleotide or variant polynucleotide of any one of claims 1-6, which is an RNA.

8. The polynucleotide or variant polynucleotide of claim 7, wherein the RNA is / functions as the 5’UTR (5’ untranslated region) of an mRNA.

9. The polynucleotide or variant polynucleotide of any one of claims 4-8, wherein the protein coding sequence encodes a protein, such as a fluorescent protein or an oxidative enzyme capable of producing bioluminescence (e.g., a luciferase).

10. A polynucleotide comprising SEQ ID NO: 4 having an inserted hypoxia response element sequence (HRE), such as an HRE comprising one or more repeats of RCGTG (R = A or G).

11. The polynucleotide of claim 10, wherein the HRE is from the hypoxia-induced egl nine homologue 3 (egln3 / phd3) gene.

12. The polynucleotide of claim 10 or 11, wherein the HRE is inserted anywhere within SEQ ID NO: 4, such as inserted immediately before or after nucleotide 87 of SEQ ID NO: 4, or at a nucleotide at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides from nucleotide 87 of SEQ ID NO: 4.

13. The polynucleotide of claim 10 or 11, wherein the HRE is inserted 5’ to nucleotide 87 ofSEQ ID NO: 4.

14. The polynucleotide of claim 10 or 11, wherein the HRE is inserted 3’ to nucleotide 87 of SEQ ID NO: 4.

15. The polynucleotide of any one of claims 10-14, comprising, consisting essentially of, or consisting of SEQ ID NO: 5.

16. The polynucleotide of claim 15, which is an RNA.

17. The polynucleotide of claim 16, wherein the RNA is / functions as the 3’UTR (3’ untranslated region) of an mRNA.

18. The polynucleotide of any one of claims 10-17, wherein the polynucleotide is at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as SEQ ID NO: 5 as a 3’UTR sequence operably linked to a protein coding sequence, for expression of the protein in a hypoxia tissue.

19. The polynucleotide of claim 18, wherein the hypoxia tissue is a tumor or cancer tissue (such as a melanoma tissue).

20. The polynucleotide of claim 18 or 19, wherein the protein coding sequence encodes a protein, such as a fluorescent protein or an oxidative enzyme capable of producing bioluminescence (such as a luciferase).

21. A polynucleotide comprising: Ani- [Nn3 - An2]n4, wherein each N is independently a non- A nucleotide, nl = 10-80, n2 = 10-80, n3 = 2-15, and n4 = 2-20.

22. The polynucleotide of claim 21, wherein nl is about 21-40, about 25-35, or about 30.

23. The polynucleotide of claim 21 or 22, wherein n2 is about 21-40, about 25-35, or about 30.

24. The polynucleotide of any one of claims 21-23, wherein n3 is 2, 3, or 4.

25. The polynucleotide of claim 24, wherein n3 is 2, and Nn3 is UU / TT, CU / CT, GG, or CC.

26. The polynucleotide of any one of claims 21-25, wherein n4 is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

27. The polynucleotide of any one of claims 21-26, wherein nl is 30, n2 is 30, Nn3 is TT, and n4 is 3.

28. The polynucleotide of claim 27, comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 26.

29. The polynucleotide of any one of claims 21-28, which is an RNA.

30. The polynucleotide of claim 29, wherein the RNA is / functions as the polyA sequence of an mRNA.

31. The polynucleotide of claim 29 or 30, wherein the polynucleotide is at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% as effective as a control polyA sequence of the same length when operably linked to a protein coding sequence, for expression of the protein.

32. The polynucleotide of claim 31 , wherein the protein coding sequence encodes a protein, such as a fluorescent protein or an oxidative enzyme capable of producing bioluminescence (such as a luciferase).

33. A polynucleotide comprising or encoding the polynucleotide of any one of claims 1-9 as the 5’UTR, a coding sequence for a gene of interest (GOI), the polynucleotide of any one of claims 10-20 as the 3’UTR, and the polynucleotide of any one of claims 21-32 as the polyA sequence.

34. The polynucleotide of claim 33, which is an mRNA.

35. The polynucleotide of claim 33 or 34, which is a closed circular molecule (such as a plasmid, a viral (e.g., adeno viral, AAV, HSV, baculoviral, or lentiviral) vector, or a circular RNA) or a linear molecule.

36. The polynucleotide of any one of claims 33-35, wherein the GOI encodes an antigen (e.g., an antigen for vaccination against an infectious disease or a cancer), an enzyme or growth factor (e.g., enzyme or growth factor useful for enzyme and hormone replacement therapy), cytokines / chemokines (e.g., cytokines / chemokines for the treatment of cancers, autoimmune diseases, and inflammatory diseases), immune receptors (e.g., chimeric antigen receptors, T cell receptors, B cell receptors, costimulatory receptors / ligands for cancer therapy), antibodies (e.g., immune checkpoint inhibitors for cancer immunotherapy), or tumor inhibitory molecules (e.g., tumor suppressor and angiogenesis regulators for cancer therapy), or transcription factors (e.g., cell reprogramming factors for cell replacement therapy).

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