Improved parasite vaccines

Abstract

Disclosed herein, in some aspects, are polynucleotides encoding parasite polypeptides. Polynucleotides and polypeptides of the disclosure may be used as vaccines against parasites, such as hookworms. Aspects of the disclosure, also pertain to methods for treating or preventing an infection, such as a hookworm infection, by administering a composition or polynucleotide of the disclosure.

Description

BAYM. P0444WO / BLG 25-024IMPROVED PARASITE VACCINESCROSS REFERENCE TO RELATED PATENTS

[0001] This application claims the benefit of priority to U. S. Provisional Patent Application Serial No. 63 / 733,607, filed 13 December 2024, the contents of which are hereby incorporated by reference in their entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted in ST26 format and is hereby incorporated by reference in its entirety. Said ST26 copy, created on December 12, 2024, is named BAYM_P0444WO_Sequence_Listing.xml and is 72,332 bytes in size.TECHNICAL FIELD AND BACKGROUNDI. Technical Field

[0003] Aspects of this disclosure relate to at least the fields of immunology, vaccine development, public health and medicine.II. Background

[0004] Hookworm infections are a leading cause of non-dietary iron-deficiency anemia worldwide that can hinder physical and cognitive development. These parasitic infections are primarily acquired through skin contact with contaminated soil and occur regularly in tropical and subtropical regions of the globe with limited sanitation infrastructure. More than 100 million people are infected with hookworms (https: / / www.healthdata.org / ), and the associated disease is becoming dominant in parts of Africa and Southeast Asia affected by climate change. Despite the effectiveness of benzimidazole anthelmintics, reinfections can promptly occur post-treatment, with a concern for drug-resistant parasites emerging. Recognizing the limitations of current treatments, vaccines can be a comprehensive solution by providing long-lasting protection, either as isolated interventions or in combination with other measures. However, developing a vaccine has been challenging due to the nature of hookworms as large extracellular parasites with intricate life cycles, and no commercially available helminth vaccine after which to model. Additionally, helminths have evolved sophisticated mechanisms to manipulate the human immune system to prevent acquired immunity, allowing them to survive for years within the host, often leading to reinfection with a similar or even greater disease burden. Apart from a few studies using live attenuated worms or larvae, specific300484454.1 - 1 -BAYM. P0444WO / BLG 25-024antigens crucial for parasite survival and host interaction have been targeted in vaccine programs since the 1980s.

[0005] The initial lead hookworm vaccine antigen, the larvae entry antigen N. americanus Ancylostoma-secreted protein 2 (Na- ASP-2), was discontinued in clinical trials due to an urticarial reaction in some adults from a hookworm endemic region of Brazil, who were later found to have pre-formed anti-AG-ASP-2 IgE. Second-generation vaccine candidates focused on antigens involved in hookworm blood feeding, resulting in the selection of N. americanus aspartic-protease-1 ( / -APR.- I) and N americanus glutathione-S-transferase-1 ( / -GST- I). These candidates were chosen after showing protection efficacy in preclinical studies and after serum screening within hookworm-endemic populations to confirm the absence of antigenspecific IgE. AG- A PR. - I and Aa-GST-l are enzymes that play key roles in parasite blood feeding and hemoglobin digestion and heme binding or detoxification, respectively. Vaccine-induced antibodies that bind to these enzymes can inhibit their function, interfering with the worm’s blood feeding. Vaccination with recombinant Na- APR- 1 and Aa-GST-l protein has been demonstrated to effectively reduce worm burdens in different animal models. Both recombinant protein antigens are being accelerated in clinical trials conducted in adults and children and have been shown to induce robust antibody responses and T-cell responses [26,29,30], Aa-GST-l offers the benefit of its high-yield and low-cost expression a Pichia pastoris yeast system, making it an attractive lead vaccine candidate for global health and resource-poor health systems.

[0006] Although the current generation hookworm protein subunit vaccine formulations provided significant immunogenicity in humans and protective immunity in laboratory animals (with human challenge studies pending), there is an interest in exploring alternative antigen delivery systems. Current limitations associated with subunit vaccines, include challenges in solubility, purification, and formulation, seen during the production of recombinant proteins in heterologous systems. Accordingly, new and improved vaccines are needed to provide accessible and high-quality preventative measures against helminth infections across the globe and particularly to low income countries.SUMMARY

[0007] By leveraging the flexible design of RNA vaccines and protein intracellular trafficking signal sequences, the inventors developed three variants of AG-GST-I as native (cytosolic), secretory, and plasma membrane-anchored (PM) antigens. Compared to recombinant protein immunization, RNA vaccines induced an earlier onset of antigen-specific300484454.1 - 2 -BAYM. P0444WO / BLG 25-024antibodies, superior levels of antigen-specific antibodies, a diverse set of memory T-cell populations, and reduced gut worm burden in immunized animals.

[0008] Aspects of the present disclosure address certain needs by providing at least polynucleotides, polypeptides, and pharmaceutical compositions for the generation of vaccines against helminths. Also disclosed are methods and uses of said polynucleotides, polypeptides, and pharmaceutical compositions to immunize and / or treat individuals at risk of helminth infections or known to have a helminth infection.

[0009] In some aspects, provided herein are compositions comprising one or more polynucleotides, wherein at least one of the one or more polynucleotides comprises a sequence that is at least, at most, exactly, between any two of, or about 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to any one of SEQ ID NOs: 1-7 and 17-25. In some aspects, at least one uridine position of at least one of the one or more polynucleotides is pseudouridine. In some aspects, all uridine positions of at least one of the one or more polynucleotides are pseudouridine. In some aspects, the pseudouridine comprises or excludes N1-methylpseudouridine. In some aspects, the one or more polynucleotides further comprises or excludes a sequence encoding a cell membrane anchor, one or more tags, a signal peptide, one or more linkers, or a combination thereof. In some aspects, the cell membrane anchor comprises a glycosylphosphatidylinositol (GPI) anchor. In some aspects, a GPI anchor is from CD55. In some aspects, a GPI anchor comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 49. In some aspects, a tag comprises or excludes a FLAG tag. In some aspects, a tag comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 51. In some aspects, one or more polynucleotides do not comprise a tag encoding sequence. In some aspects, a signal peptide is from IgG or albumin. In some aspects, a signal peptide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 45, 47, or 53. In some aspects, a linker comprises a polynucleotide sequence that encodes a Lysine (K) Leucine (L) linker. In some aspects, a linker joins a tag, such as a flag tag, and / or a signal peptide sequence to an antigenic polypeptide, such as an APR-1 and / or GST-1 polypeptide. In some aspects, a polynucleotide comprises one or more linker sequences. In some aspects, one or more polynucleotides further comprises or excludes a 5' untranslated region (UTR), Kozak sequence, 3' UTR, a poly(A) tail, or a combination thereof. In some aspects, a 5' UTR comprises a polynucleotide sequence that is at 300484454.1 - 3 -BAYM. P0444WO / BLG 25-024least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 42. In some aspects, a Kozak sequence comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 41. In some aspects, a 3' UTR sequence comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 43. In some aspects, a poly(A) tail comprises 40-150 adenosines. In some aspects, a poly(A) tail comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 44.

[0010] In some aspects, a polynucleotide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 1 or 4. In some aspects, a polynucleotide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 2, 3, or 6. In some aspects, a polynucleotide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100%, or any range derivable therein, identical to SEQ ID NOs: 4 or 7. In some aspects, a polynucleotide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 19, 20, 24, or 25. In some aspects, a polynucleotide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 16, 17, 21, or 22. In some aspects, a polynucleotide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 18 or 23.

[0011] Also provided herein, in some aspects, are compositions comprising a multicistronic polynucleotide comprising at least two sequences that are at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 1-7 and 16-25, wherein the at least two sequences are separated by an internal ribosome entry site (IREs) and / or 2A peptide encoding sequence. In some aspects, provided herein are compositions comprising one, two, three, four, five, or six polynucleotides comprising a sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 1-7 and 16-25.

[0012] In some aspects, one or more polynucleotides further comprise or exclude a 5' cap. In some aspects, a 5' cap comprises or excludes a Cap-1 cap. In some aspects, one or more 300484454.1 - 4 -BAYM. P0444WO / BLG 25-024polynucleotides is an in vitro transcribed polynucleotide. In some aspects, one or more polynucleotides are encoded by a vector. In some aspects, a vector comprises or excludes a T7 promoter. In some aspects, a vector comprises or excludes a pUC57 vector.

[0013] In some aspects, provided herein are compositions comprising a first polynucleotide comprising a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 4 or 7 and a second polynucleotide comprising a nucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 18 or 23.

[0014] In some aspects, compositions provided herein comprise or exclude one or more lipids. In some aspects, one or more lipids comprise or exclude a ionizable lipid, a helper lipid, a PEG-lipid, a cholesterol, or a combination thereof. In some aspects, an ionizable lipid comprises or excludes D-Lin-MC3-DMA, OF-02, SM-102, ALC-0315, LPO1, or a combination thereof. In some aspects, a helper lipid comprises or excludes DSPC, DOPE, or a combination thereof. In some aspects, a PEG-lipid comprises or excludes PEG2000-DMG, ALC-0159, or a combination thereof.

[0015] In some aspects, one or more polynucleotides are encapsulated in one or more lipid nanoparticles (LNPs). In some aspects, provided herein are LNPs comprising or excluding a nitrogen-to-phosphate ratio that is at least, at most, exactly, between any two of, or about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or any value therebetween. In some aspects, LNPs comprise or exclude a nitrogen-to-phosphate ratio of or of about 4:1. In some aspects, LNPs comprise or exclude an average diameter of or of about 40-130 nm. In some aspects, LNPs comprise or exclude an average diameter of or of about 90 nm. In some aspects, LNPs comprise or exclude a poly dispersity index that is at least, at most, exactly, between any two of, or about 15%, 20%, 25%, or 30%. In some aspects, a poly dispersity index of the LNPs is less than or is less than about 15%. In some aspects, at least, at most, exactly, between any two of, or about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of LNPs encapsulate at least one polynucleotide. In some aspects, at least or at least about 85% of the LNPs encapsulate at least one polynucleotide. In some aspects, compositions provided herein further comprises or excludes sterile phosphate buffered saline (PBS) and / or sucrose. In some aspects, compositions comprise or exclude sucrose at a concentration of or of about 8% by weight. In some aspects, one or more polynucleotides are comprised at a concentration of or of about 50-1000 pg / mL. In some aspects, one or more polynucleotides are comprised at a concentration of or of about 200 pg / mL. In some aspects, compositions comprise or exclude a pharmaceutically acceptable300484454.1 - 5 -BAYM. P0444WO / BLG 25-024carrier. In some aspects, a composition is thermostable at room temperature. In some aspects, a composition is frozen at about -80 °C.

[0016] Also provided herein, in some aspects, are compositions comprising one or more polypeptides comprising a sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to any one or more of SEQ ID NOs: 9-15 and 26-40.

[0017] Also provided herein are cells comprising, transduced with, transfected with, and / or transformed with one or more compositions and / or polynucleotides of the disclosure. In some aspects, a cell is a mammalian cell. In some aspects, a cell is a human cell. In some aspects, a cell is a muscle cell, epithelial cell, and / or antigen presenting cell.

[0018] Also provided herein is the use of any one or more compositions, polynucleotides, and / or polypeptides of the disclosure in the manufacture of a medicament for immunizing an individual against a parasite infection. In some aspects, a medicament is formulated to comprise between about 1-200 pg of one or more polynucleotides of the disclosure. In some aspects, a medicament is formulated to comprise a concentration of one or more polynucleotides of the disclosure that is at least, at most, exactly, between any two of, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 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, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200 pg. In some aspects, a medicament is formulated to comprise about 10 pg of one or more polynucleotides of the disclosure. In some aspects, a medicament is formulated for intramuscular administration to an individual. In some aspects, a medicament is formulated for a number of administrations to an individual that is at least, at most, exactly, between any two of, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In some aspects, a medicament is formulated for multiple administrations, with a time between administration that is at least, at most, exactly, between any two of, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more hours, days, or weeks. In some aspects, a medicament is formulated to be substantively non-toxic to an individual.300484454.1 - 6 -BAYM. P0444WO / BLG 25-024

[0019] In some aspects, a medicament is formulated to increase immunoglobulin (Ig) titers in the individual compared to non-immunized levels. In some aspects, Ig titers increase by a value that is at least, at most, exactly, between, or about 10-fold to 10,000,000-fold. In some aspects, Ig titers increase by a value that is at least, at most, exactly, between any two of, or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 200000, 300000, 400000, 500000, 600000, 700000, 800000, 900000, 1000000, 2000000, 3000000, 4000000, 5000000, 6000000, 7000000, 8000000, 9000000, 10000000-fold. In some aspects, an increase in Ig titers is detected as early as week 6 after immunization of an individual. In some aspects, an increase in Ig titers is detected as late as 17 week after immunization of an individual. In some aspects, Ig is measured from blood, plasma, or fecal matter. In some aspects, Ig comprises or excludes IgG2a, IgGl, IgG2c, IgA, or a combination thereof. In some aspects, an Ig inhibits GST and / or APR activity. In some aspects, GST and / or APR activity is inhibited between about 5% to about 50%. In some aspects, a medicament is formulated to increase CD25 expression in immune cells of the individual compared to non-immunized levels. In some aspects, a medicament is formulated to increase expression of a cytokine in immune cells of the individual compared to nonimmunized levels. In some aspects, cytokines comprise or exclude IFN-y, IL-2, TNF-a, IL-4, IL-13, granzyme B, or a combination thereof. In some aspects, immune cells comprise or exclude CD4-positive, CD8-positive, CD3 -positive, CD 19-positive, T central memory, T effector memory, Thl, Th2, B cells, or a combination thereof. In some aspects, upon challenge with a parasite, an immunized individual has a reduced parasite burden that is at least, at most, exactly, between any two of, or about 20% to 100%, or 20% to 60%, or 30% to 50% compared to non-immunized levels. In some aspects, a parasite comprises a helminth. In some aspects, a parasite comprises a nematode. In some aspects, a parasite comprises a hookworm. In some aspects, a parasite comprises a nematode from the Ancylostomatidae family. In some aspects, a parasite comprises or excludes Necator americanus, Ancylostoma duodenale, or a combination thereof. In some aspects, the titer of Ig in non-immunized level samples comprise levels detected in the same individual before immunization or the average level in a sample or population of non-immunized individuals.

[0020] Also provided herein, in some aspects, are methods of immunizing an individual against a parasite infection, the method comprising the steps of administering a composition, polynucleotide, polypeptide, and / or cell of the disclosure to the individual. In some aspects, a composition comprises between about 1-200 pg of the one or more polynucleotides of the 300484454.1 - 7 -BAYM. P0444WO / BLG 25-024disclosure. In some aspects, a composition comprises about 10 pg of the one or more polynucleotides of the disclosure. In some aspects, a composition is administered intramuscularly. In some aspects, a composition is administered to the individual a number of times that is at least, at most, exactly, between any two of, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. In some aspects, a time between administrations is at least, at most, exactly, between any two of, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more hours, days, or weeks. In some aspects, administration of the composition is substantively non-toxic to the individual. In some aspects, administration increases Ig titers in the individual compared to non-immunized levels. In some aspects, Ig titers increase between about 10-fold to 10,000,000-fold. In some aspects, an increase in Ig titers is detected as early as week 6 after immunization. In some aspects, an increase in Ig titers is detected as late as 17 week after immunization. In some aspects, Ig is measured from blood, plasma, or fecal matter. In some aspects, the Ig comprises or excludes IgG2a, IgGl, IgG2c, IgA, or a combination thereof. In some aspects, the Ig inhibits GST and / or APR activity. In some aspects, GST and / or APR activity is inhibited between about 5% to about 50%. In some aspects, administration increases CD25 expression in immune cells of the individual compared to non-immunized levels. In some aspects, administration increases expression of a cytokine in immune cells of the individual compared to non-immunized levels. In some aspects, the cytokines comprise or exclude IFN-y, IL-2, TNF-a, IL-4, IL-13, granzyme B, or a combination thereof. In some aspects, immune cells comprise or exclude CD4-positive, CD8-positive, CD3-positive, CD 19-positive, T central memory, T effector memory, Thl, Th2, B cells, or a combination thereof. In some aspects, upon challenge with a parasite, the individual has a reduced parasite burden that is at least, at most, exactly, between any two of, or about 20% to 100%, or 20% to 60%, or 30% to 50% compared to non-immunized levels. In some aspects, a parasite comprises a helminth. In some aspects, a parasite comprises a nematode. In some aspects, a parasite comprises a hookworm. In some aspects, a parasite comprises a nematode from the Ancylostomatidae family. In some aspects, a parasite comprises or excludes Necator americanus, Ancylostoma duodenale, A. caninum, A. duodenale and A. ceylanicum or a combination thereof. In some aspects, non-immunized levels comprise levels detected in the same individual before immunization or the average level in a sample or population of nonimmunized individuals.

[0021] In some aspects, provided herein are compositions comprising a first polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 and a second polynucleotide comprising a sequence that is identical to SEQ ID NOs: 18 or 23. In some aspects, provided 300484454.1 - 8 -BAYM. P0444WO / BLG 25-024herein is the use of a composition comprising a first polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 and a second polynucleotide comprising a sequence that is identical to SEQ ID NOs: 18 or 23 in the manufacture of a medicament for immunizing an individual against a Necator americanus infection. In some aspects, provided herein is the use of a composition comprising a polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 in the manufacture of a medicament for immunizing an individual against a Necator americanus infection. In some aspects, provided herein is the use of a composition comprising a polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 in the manufacture of a medicament for increasing production of IgG2a antibodies against GST- 1 in an individual. In some aspects, provided herein are methods of immunizing an individual against a Necator americanus infection, the method comprising the steps of: administering to the individual a polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7. In some aspects, provided herein are methods of immunizing an individual against a Necator americanus infection, the method comprising the steps of: administering to the individual a first polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 and a second polynucleotide comprising a sequence that is identical to SEQ ID NOs: 18 or 23. In some aspects, provided herein are methods of increasing production of IgG2a antibodies against GST-1 in an individual, the method comprising the steps of: administering to the individual a polynucleotide comprising a sequence that is identical to SEQ ID NO: 4 or 7.

[0022] Also provided herein are kits comprising the composition, polynucleotides, and / or polypeptides of the disclosure, and / or means for generating the compositions, polynucleotides, and / or polypeptides. Also provided herein are kits suitable for conducting any of the methods disclosed herein, and / or using any of the composition, polynucleotides, and / or polypeptides of the disclosure in the preparation of a medicament.

[0023] The terms “a” and “an” are defined as one or more unless this disclosure explicitly requires otherwise. For example, the use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”

[0024] Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the measurement or quantitation method.

[0025] The terms "comprise" (and any form of comprise, such as "comprises" and "comprising"), "have" (and any form of have, such as "has" and "having"), and "include" (and any form of include, such as "includes" and "including") are open-ended linking verbs. As a result, a composition that "comprises," "has," or "includes" one or more elements possesses 300484454.1 - 9 -BAYM. P0444WO / BLG 25-024those one or more elements, but is not limited to possessing only those elements. Likewise, a method that "comprises," "has," or "includes" one or more steps possesses those one or more steps, but is not limited to possessing only those one or more steps.

[0026] Any configuration of any of compositions or methods can consist of or consist essentially of - rather than comprise / include / have - any of the described steps, elements, and / or features. Thus, in any of the claims, the term "consisting of or "consisting essentially of can be substituted for any of the open-ended linking verbs recited above, in order to change the scope of a given claim from what it would otherwise be using the open-ended linking verb. The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed disclosure.

[0027] As used herein, “or” is inclusive and not exclusive, unless expressly indicated otherwise or indicated otherwise by context, and can have the same meaning as “and / or.” To illustrate, A, B, and / or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.

[0028] As used herein, an ordinal term (e.g., “first,” “second,” “third,” etc.) used to modify an element, such as a structure, a component, an operation, etc., does not by itself indicate any priority or order of the element with respect to another element, but rather merely distinguishes the element from another element having a same name (but for use of the ordinal term).

[0029] Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of “about 0.1 % to about 5%” or “about 0.1 % to 5%” should be interpreted to include not just about 0.1 % to about 5%, but also the individual values (e.g., 1 %, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1 % to 0.5%, 1.1 % to 2.2%, 3.3% to 4.4%) within the indicated range.

[0030] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.300484454.1 - 10 -BAYM. P0444WO / BLG 25-024

[0031] The terms “subject,” “host,” “individual,” and “patient” can be used interchangeably herein to refer to human or veterinary subjects, for example, humans, animals, nonhuman primates, dogs, cats, sheep, mice, horses, and cows. In some aspects, the subject is a human.

[0032] As used herein “treatment” or “treating,” includes any beneficial or desirable effect on the symptoms or pathology of a disease or pathological condition, and may include even minimal reductions in one or more measurable markers of the disease or condition being treated, e.g., parasitic infection, anemia, etc. Treatment may involve either the reduction or amelioration of symptoms of the disease or condition, or the delaying of the progression of the disease or condition. “Treatment” does not necessarily, but can, indicate complete eradication or cure of the disease or condition, or associated symptoms thereof.

[0033] The recitations “sequence identity,” “percent identity,” or for example, “comprising a sequence 90% identical to” or “having at least 90% sequence identity to,” as used herein, refer to the extent that sequences are identical on an amino acid-by-amino acid basis, or a nucleotide-by-nucleotide basis, or over a window of comparison. Thus, a “percentage of sequence identity” can be calculated by comparing two optimally aligned sequences (e.g., nucleic acid) over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U) or the identical amino acid residue (e.g., alanine (Ala), proline (Pro), serine (Ser), threonine (Thr), glycine (Gly), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp), lysine (Lys), arginine (Arg), histidine (His), aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), glutamine (Gin), cysteine (Cys), and methionine (Met)) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[0034] Calculations of sequence similarity or sequence identity between sequences (the terms are used interchangeably herein) can be performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences can be aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In some aspects, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch, J. Mol. Biol., 1970; 48: 444–453) 300484454.1 - 11 -BAYM. P0444WO / BLG 25-024algorithm, which has been incorporated into the GAP program in the GCG software package, using either a BLOSUM 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. 0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0035] The terms “increased” or “increase” or “upregulated” or “upregulate” as used herein generally mean an increase by a statically significant or practical (e.g., for the purposes of characterization) amount relative to a reference. For avoidance of doubt, “increased” may mean an increase, statistical significant or otherwise, of at least 10% as compared to a reference level, including an increase of at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% or more, including, for example at least 1.1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold increase or greater as compared to a reference level, or any value therebetween.

[0036] The term “reduced” or “reduce” or “decrease” or “decreased” or “downregulate” or “downregulated” or “inhibit” as used herein generally mean a decrease by a statistically significant or practical (e.g., for the purposes of characterization) amount relative to a reference. For avoidance of doubt, “reduced” may mean a decrease, statistically significant or otherwise, of at least 10% as compared to a reference level, for example a decrease by at least 20%, at least 30%, at least 40%, at least 50%, or at least 60%, or at least 70%, or at least 80%, at least 90% or more, up to and including a 100% decrease (i.e., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level, or any value therebetween.

[0037] “Therapeutically effective” as used herein can refer to an amount of an agent that is therapeutically effective, or in a broader sense also prophylactically effective, against the progression of a disease, condition, or disorder. A therapeutically effective amount may be an amount that treats a condition, disease, or disorder.

[0038] Certain aspects of the present disclosure are characterized through the following enumerated aspects.

[0039] Aspect l is a composition comprising one or more polynucleotides, wherein at least one of the one or more polynucleotides comprises a sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-7 and 17-25.

[0040] Aspect 2 is the composition of aspect 1, wherein at least one uridine position of at least one of the one or more polynucleotides is pseudouridine.300484454.1 - 12 -BAYM. P0444WO / BLG 25-024

[0041] Aspect 3 is the composition of aspect 1 or 2, wherein all uridine positions of at least one of the one or more polynucleotides are pseudouridine.

[0042] Aspect 4 is the composition of aspect 2 or 3, wherein the pseudouridine comprises N1-methylpseudouridine.

[0043] Aspect 5 is the composition of any one of aspects 1-4, wherein the one or more polynucleotides further comprise a sequence encoding a cell membrane anchor, one or more tags, a signal peptide, one or more linkers, or a combination thereof.

[0044] Aspect 6 is the composition of aspect 5, wherein the cell membrane anchor comprises a glycosylphosphatidylinositol (GPI) anchor.

[0045] Aspect 7 is the composition of aspect 6, wherein the GPI anchor is from CD55.

[0046] Aspect 8 is the composition of aspect 6 or 7, wherein the GPI anchor comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 49.

[0047] Aspect 9 is the composition of any one of aspects 5-8, wherein the tag comprises a FLAG tag.

[0048] Aspect 10 is the composition of any one of aspects 5-9, wherein the tag comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 51.

[0049] Aspect 11 is the composition of any one of aspects 1-8, wherein the one or more polynucleotides do not comprise a tag encoding sequence.

[0050] Aspect 12 is the composition of any one of aspects 5-11, wherein the signal peptide is from IgG or albumin.

[0051] Aspect 13 is the composition of any one of aspects 5-12, wherein the signal peptide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 45, 47, or 53.

[0052] Aspect 14 is the composition of any one of aspects 5-13, wherein the linker comprises a polynucleotide sequence that encodes a Lysine (K) Leucine (L) linker.

[0053] Aspect 15 is the composition of any one of aspects 1-13, wherein the one or more polynucleotides further comprise a 5' untranslated region (UTR), Kozak sequence, 3' UTR, a poly(A) tail, or a combination thereof.

[0054] Aspect 16 is the composition of aspect 15, wherein the 5' UTR comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 42.300484454.1 - 13 -BAYM. P0444WO / BLG 25-024

[0055] Aspect 17 is the composition of aspect 15 or 16, wherein the Kozak sequence comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 41.

[0056] Aspect 18 is the composition of any one of aspects 15-17, wherein the 3' UTR sequence comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 43.

[0057] Aspect 19 is the composition of any one of aspects 15-18, wherein the poly(A) tail comprises 40-150 adenosines.

[0058] Aspect 20 is the composition of any one of aspects 15-19, wherein the poly(A) tail comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 44.

[0059] Aspect 21 is the composition of any one of aspects 1-20, wherein the polynucleotide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 1 or 4.

[0060] Aspect 22 is the composition of any one of aspects 1-20, wherein the polynucleotide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 2, 3, or 6.

[0061] Aspect 23 is the composition of any one of aspects 1-20, wherein the polynucleotide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 4 or 7.

[0062] Aspect 24 is the composition of any one of aspects 1-20, wherein the polynucleotide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 19, 20, 24, or 25.

[0063] Aspect 25 is the composition of any one of aspects 1-20, wherein the polynucleotide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 16, 17, 21, or 22.

[0064] Aspect 26 is the composition of any one of aspects 1-20, wherein the polynucleotide comprises a polynucleotide sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 18 or 23.

[0065] Aspect 27 is the composition of any one of aspects 1-26, comprising a multicistronic polynucleotide comprising at least two sequences that are at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 1-7 and 16-25, wherein the at least two sequences are separated by an internal ribosome entry site (IREs) and / or 2A peptide encoding sequence.300484454.1 - 14 -BAYM. P0444WO / BLG 25-024

[0066] Aspect 28 is the composition of any one of aspects 1-27, wherein the one or more polynucleotides further comprise a 5' cap.

[0067] Aspect 29 is the composition of aspect 28, wherein the 5' cap comprises a Cap-1 cap.

[0068] Aspect 30 is the composition of any one of aspects 1-29, wherein the one or more polynucleotides is in vitro transcribed.

[0069] Aspect 31 is the composition of any one of aspects 1-30, wherein the one or more polynucleotides are encoded by a vector.

[0070] Aspect 32 is the composition of aspect 31, wherein the vector comprises a T7 promoter.

[0071] Aspect 33 is the composition of aspect 31 or 32, wherein the vector comprises a pUC57 vector.

[0072] Aspect 34 is a composition comprising one, two, three, four, five, or six polynucleotides comprising a sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 1-7 and 16-25.

[0073] Aspect 35 is the composition of aspect 34, comprising a first polynucleotide that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 4 or 7 and a second polynucleotide that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 18 or 23.

[0074] Aspect 36 is the composition of any one of aspects 1-35, further comprising one or more lipids.

[0075] Aspect 37 is the composition of aspect 36, wherein the one or more lipids comprise an ionizable lipid, a helper lipid, a PEG-lipid, a cholesterol, or a combination thereof.

[0076] Aspect 38 is the composition of aspect 37, wherein the ionizable lipid comprises D-Lin-MC3-DMA, OF-02, SM-102, ALC-0315, LPO1, or a combination thereof.

[0077] Aspect 39 is the composition of aspect 37 or 38, wherein the helper lipid comprises DSPC, DOPE, or a combination thereof.

[0078] Aspect 40 is the composition of any one of aspects 37-39, wherein the PEG-lipid comprises PEG2000-DMG, ALC-0159, or a combination thereof.

[0079] Aspect 41 is the composition of any one of aspects 36-40, wherein the one or more polynucleotides are encapsulated in one or more lipid nanoparticles (LNPs).300484454.1 - 15 -BAYM. P0444WO / BLG 25-024

[0080] Aspect 42 is the composition of aspect 41, wherein the LNPs comprise a nitrogen-to-phosphate ratio of about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or any value therebetween.

[0081] Aspect 43 is the composition of aspect 42, wherein the LNPs comprise a nitrogen-to-phosphate ratio of or of about 4:1.

[0082] Aspect 44 is the composition of any one of aspects 41-43, wherein the LNPs comprise an average diameter of or of about 40-130 nm.

[0083] Aspect 45 is the composition of any one of aspects 41-44, wherein the LNPs comprise an average diameter of or of about 90 nm.

[0084] Aspect 46 is the composition of any one of aspects 41-45, wherein the LNPs comprise a poly dispersity index that is less than or is less than about 15%, 20%, 25%, or 30%.

[0085] Aspect 47 is the composition of any one of aspects 41-46, wherein the poly dispersity index of the LNPs is less than or is less than about 15%.

[0086] Aspect 48 is the composition of any one of aspects 41-47, wherein at least, or at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the LNPs encapsulate at least one polynucleotide.

[0087] Aspect 49 is the composition of any one of aspects 41-48, wherein at least or at least about 85% of the LNPs encapsulate at least one polynucleotide.

[0088] Aspect 50 is the composition of any one of aspects 1-49, further comprising sterile phosphate buffered saline (PBS) and / or sucrose.

[0089] Aspect 51 is the composition of aspect 50, wherein the sucrose is or is about 8% by weight.

[0090] Aspect 52 is the composition of any one of aspects 1-51, wherein the one or more polynucleotides are comprised at a concentration of or of about 50-1000 pg / mL.

[0091] Aspect 53 is the composition of any one of aspects 1-52, wherein the one or more polynucleotides are comprised at a concentration of or of about 200 pg / mL.

[0092] Aspect 54 is the composition of any one of aspects 1-53, further comprising a pharmaceutically acceptable carrier.

[0093] Aspect 55 is the composition of any one of aspects 1-54, wherein the composition is thermostable at room temperature.

[0094] Aspect 56 is the composition of any one of aspects 1-55, wherein the composition is frozen at -80 °C.300484454.1 - 16 -BAYM. P0444WO / BLG 25-024

[0095] Aspect 57 is a composition comprising one or more polypeptides that include a sequence that is at least, at most, exactly, between any two of, or about 80%, 85%, 90%, 95%, 99%, or 100% identical to any one or more of SEQ ID NOs: 9-15 and 26-40.

[0096] Aspect 58 is a cell comprising, or that has been transduced with, transfected with, and / or transformed with the composition of any one of aspects 1-57.

[0097] Aspect 59 is the cell of aspect 58, wherein the cell is a human cell.

[0098] Aspect 60 is the cell of aspect 58 or 59, wherein the cell is a muscle cell, epithelial cell, and / or antigen presenting cell.

[0099] Aspect 61 is the use of the composition or cell of any one of aspects 1-60 in the manufacture of a medicament for immunizing an individual against a parasite infection.

[0100] Aspect 62 is the use of aspect 61, wherein the medicament is formulated to comprise between about 1-200 pg of polynucleotide.

[0101] Aspect 63 is the use of aspect 61 or 62, wherein the medicament is formulated to comprise about 10 pg of polynucleotide.

[0102] Aspect 64 is the use of any one of aspects 61-63, wherein the medicament is formulated for intramuscular administration to an individual.

[0103] Aspect 65 is the use of any one of aspects 61-64, wherein the medicament is formulated for 1, 2, 3, 4, 5 or more administrations to an individual.

[0104] Aspect 66 is the use of aspect 65, wherein the time between administrations comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more hours, days, or weeks.

[0105] Aspect 67 is the use of any one of aspects 61-66, wherein the medicament is formulated to be substantively non-toxic to an individual.

[0106] Aspect 68 is the use of any one of aspects 61-67, wherein the medicament increases immunoglobulin (Ig) titers in the individual compared to non-immunized levels.

[0107] Aspect 69 is the use of aspect 68, wherein titers increase between about 10-fold to 10,000,000-fold.

[0108] Aspect 70 is the use of aspect 68 or 69, wherein the increase is detected as early as week 6 after immunization of an individual.

[0109] Aspect 71 is the use of any one of aspects 68-70, wherein the increase is detected as late as 17 weeks after immunization of an individual.

[0110] Aspect 72 is the use of any one of aspects 68-71, wherein the Ig is measured from blood, plasma, or fecal matter.

[0111] Aspect 73 is the use of any one of aspects 68-72, wherein the Ig comprises IgG2a, IgGl, IgG2c, IgA, or a combination thereof.300484454.1 - 17 -BAYM. P0444WO / BLG 25-024

[0112] Aspect 74 is the use of any one of aspects 68-73, wherein the Ig inhibits GST and / or APR activity.

[0113] Aspect 75 is the use of aspect 74, wherein GST and / or APR activity is inhibited between about 5% to about 50%.

[0114] Aspect 76 is the use of any one of aspects 61-73, wherein the medicament is formulated to increase CD25 expression in immune cells of the individual compared to nonimmunized levels.

[0115] Aspect 77 is the use of any one of aspects 61-76, wherein the medicament is formulated to increase expression of a cytokine in immune cells of the individual compared to non-immunized levels.

[0116] Aspect 78 is the use of aspect 77, wherein the cytokines comprise IFN-y, IL-2, TNF-a, IL-4, IL- 13, granzyme B, or a combination thereof.

[0117] Aspect 79 i s the use of any one of aspects 76-79, wherein the immune cell s compri se CD4-positive, CD8-positive, CD3 -positive, CD 19-positive, T central memory, T effector memory, Thl, Th2, B cells, or a combination thereof.

[0118] Aspect 80 is the use of any one of aspects 61-79, wherein upon challenge with a parasite, an immunized individual has a reduced parasite burden of between about 20% to 100%, or 20% to 60%, or 30% to 50% compared to non-immunized levels.

[0119] Aspect 81 is the use of any one of aspects 61-80, wherein the parasite comprises a helminth.

[0120] Aspect 82 is the use of any one of aspects 61-81, wherein the parasite comprises a nematode.

[0121] Aspect 83 is the use of any one of aspects 61-82, wherein the parasite comprises a hookworm.

[0122] Aspect 84 is the use of any one of aspects 61-83, wherein the parasite comprises a nematode from the Ancylostomatidae family.

[0123] Aspect 85 is the use of any one of aspects 61-84, wherein the parasite comprises Necator americanus, Ancylostoma duodenale, or a combination thereof.

[0124] Aspect 86 is the use of any one of aspects 68-85, wherein the non-immunized levels comprise levels detected in the same individual before immunization or the average level in a sample or population of non-immunized individuals.

[0125] Aspect 87 is a method of immunizing an individual against a parasite infection, the method comprising the steps of administering the composition or cell of any one of aspects 1-60 to the individual.300484454.1 - 18 -BAYM. P0444WO / BLG 25-024

[0126] Aspect 88 is the method of aspect 87, wherein the composition comprises between about 1-200 pg of the one or more polynucleotides.

[0127] Aspect 89 is the method of aspect 87 or 88, wherein the composition comprises about 10 pg of the one or more polynucleotides.

[0128] Aspect 90 is the method of any one of aspects 87-89, wherein the composition is administered intramuscularly.

[0129] Aspect 91 is the method of any one of aspects 87-90, wherein the composition is administered 1, 2, 3, 4, 5 or more times to the individual.

[0130] Aspect 92 is the method of aspect 91, wherein the time between administrations comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more hours, days, or weeks.

[0131] Aspect 93 is the method of any one of aspects 87-92, wherein administration of the composition is substantively non-toxic to the individual.

[0132] Aspect 94 is the method of any one of aspects 87-93, wherein administration increases Ig titers in the individual compared to non-immunized levels.

[0133] Aspect 95 is the method of aspect 94, wherein titers increase between about 10-fold to 10,000,000-fold.

[0134] Aspect 96 is the method of aspect 94 or 95, wherein the increase is detected as early as week 6 after immunization.

[0135] Aspect 97 is the method of any one of aspects 87-96, wherein the increase is detected as late as 17 weeks after immunization.

[0136] Aspect 98 is the method of any one of aspects 87-97, wherein the Ig is measured from blood, plasma, or fecal matter.

[0137] Aspect 99 is the method of any one of aspects 87-98, wherein the Ig comprises IgG2a, IgGl, IgG2c, IgA, or a combination thereof.

[0138] Aspect 100 is the method of any one of aspects 87-99, wherein the Ig inhibits GST and / or APR activity.

[0139] Aspect 101 is the method of aspect 100, wherein GST and / or APR activity is inhibited between about 5% to about 50%.

[0140] Aspect 102 is the method of any one of aspects 87-101, wherein the administration increases CD25 expression in immune cells of the individual compared to non-immunized levels.

[0141] Aspect 103 is the method of any one of aspects 87-102, wherein the administration increases expression of a cytokine in immune cells of the individual compared to nonimmunized levels.300484454.1 - 19 -BAYM. P0444WO / BLG 25-024

[0142] Aspect 104 is the method of aspect 103, wherein the cytokines comprise IFN-y, IL- 2, TNF-a, IL-4, IL- 13, granzyme B, or a combination thereof.

[0143] Aspect 105 is the method of any one of aspects 102-104, wherein the immune cells comprise CD4-positive, CD8-positive, CD3-positive, CD 19-positive, T central memory, T effector memory, Thl, Th2, B cells, or a combination thereof.

[0144] Aspect 106 is the method of any one of aspects 87-105, wherein upon challenge with a parasite, the individual has a reduced parasite burden of between about 20% to 100%, or 20% to 60%, or 30% to 50% compared to non-immunized levels.

[0145] Aspect 107 is the method of any one of aspects 87-106, wherein the parasite comprises a helminth.

[0146] Aspect 108 is the method of any one of aspects 87-107, wherein the parasite comprises a nematode.

[0147] Aspect 109 is the method of any one of aspects 87-108, wherein the parasite comprises a hookworm.

[0148] Aspect 110 is the method of any one of aspects 87-109, wherein the parasite comprises a nematode from the Ancylostomatidae family.

[0149] Aspect 111 is the method of any one of aspects 87-110, wherein the parasite comprises Necator americanus, Ancylostoma duodenale, A. caninum, A. duodenale and A. ceylanicum or a combination thereof.

[0150] Aspect 112 is the method of any one of aspects 87-111, wherein the non-immunized levels comprise levels detected in the same individual before immunization or the average level in a sample or population of non-immunized individuals.

[0151] Aspect 113 is a composition, comprising a first polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 and a second polynucleotide comprising a sequence that is identical to SEQ ID NOs: 18 or 23.

[0152] Aspect 114 is the use of a composition comprising a first polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 and a second polynucleotide comprising a sequence that is identical to SEQ ID NOs: 18 or 23 in the manufacture of a medicament for immunizing an individual against a Necator americanus infection.

[0153] Aspect 115 is the use of a composition comprising a polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 in the manufacture of a medicament for immunizing an individual against a Necator americanus infection.300484454.1 - 20 -BAYM. P0444WO / BLG 25-024

[0154] Aspect 116 is the use of a composition comprising a polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 in the manufacture of a medicament for increasing production of IgG2a antibodies against GST-1 in an individual.

[0155] Aspect 117 is a method of immunizing an individual against a Necator americanus infection, the method comprising the steps of: administering to the individual a polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7.

[0156] Aspect 118 is a method of immunizing an individual against a Necator americanus infection, the method comprising the steps of: administering to the individual a first polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 and a second polynucleotide comprising a sequence that is identical to SEQ ID NOs: 18 or 23.

[0157] Aspect 119 is a method of increasing production of IgG2a antibodies against GST-1 in an individual, the method comprising the steps of: administering to the individual a polynucleotide comprising a sequence that is identical to SEQ ID NO: 4 or 7.

[0158] It is specifically contemplated that any limitation discussed with respect to one aspect of the disclosure may apply to any other aspect of the disclosure. Furthermore, any composition of the disclosure may be used in any method of the disclosure, and any method of the disclosure may be used to produce or to utilize any composition of the disclosure. Any aspect discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. For example, any step in a method described herein can apply to any other method. Moreover, any method described herein may have an exclusion of any step or combination of steps. Aspects of an aspect set forth in the Examples are also aspects that may be implemented in the context of aspects discussed elsewhere in a different Example or elsewhere in the application, such as in the Summary, Detailed Description, Claims, and Brief Description of the Drawings.

[0159] Any method in the context of a therapeutic, diagnostic, or physiologic purpose or effect may also be described in “use” claim language such as “Use of’ any compound, composition, or agent discussed herein for achieving or implementing a described therapeutic, diagnostic, or physiologic purpose or effect.

[0160] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific aspects of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.300484454.1 - 21 -BAYM. P0444WO / BLG 25-024BRIEF DESCRIPTION OF THE DRAWINGS

[0161] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific aspects presented herein.

[0162] FIGs. 1A-1E depict in vitro validation of mRNAs encoding different forms of Na-GST-1. FIG. 1A is a schematic representation of Na-GST-1 mRNA vaccine candidates: native (n), secretory (s), or plasma membrane-anchored (pm), and their migration in a 1.5% agarose gel. In the diagram, “SS” represents the signal peptide sequence, and “GPI” denotes the glycosylphosphatidylinositol attachment sequence. Unlabeled regions indicate the 5' and 3' UTRs, while “PPP” marks the triphosphate linkage connecting the 7-methylguanosine cap to the first base of the mRNA. FIG. 1B are immunocytochemistry images of transfected DC2.4 cells. Images were captured 20 hours post-transfection. Alexa Fluor 488-conjugated mouse anti-FLAG was used for immunostaining. Cells were both fixed and permeabilized for immunostaining within membrane-bound organelles or only fixed for immunostaining in the cytosol and PM. Scale bars represent 25 pm. Schematics depict expected protein localization in each case. FIG. 1C depicts Western blots against FLAG from lysates of cells and supernatant from transfected DC2.4 cells. FIG. ID shows results of flow cytometry analysis of transfected DC2.4 cells immunostained with Cy3 conjugated mouse anti-FLAG antibody. MFI: Median fluorescence intensity. FIG. IE is a bar chart showing cell viability of transfected DC2.4 cells. The dotted line indicates the average cell viability level of the untreated cells (Neg.)

[0163] FIGs. 2A-2G show antigen-specific IgG titers induced by Na-GST-1 mRNA vaccine candidates. FIG. 2A is a schematic illustrating the immunization schedule used for BALB / C mice (N=8 per group) and sample collection schedule (blood, feces, and spleens); blood and stool samples were collected at each of the prime (day 0), boost (day 21), and sacrifice (day 42) time points. FIGs. 2B-2E show Na-GST-1-specific total IgG and IgG subclass titers in mouse sera measured by indirect ELISA. IgG subclasses were assessed only for sera collected after the second immunization (day 42). FIG. 2F shows IgGl: IgG2 ratio for groups showing induction of both IgG subclasses. FIG. 2G shows Na-GST-1-specific total IgG measured in mouse feces by indirect ELISA. In FIGs. 2A-2E, the dashed line indicates the titer cutoff. Each data point represents the average of technical duplicates. Statistical analysis was performed using the Kruskal -Wallis test, followed by Dunn’s test for pairwise comparison of groups. *p < 0.05, ** p < 0.01, ***p < 0.001, ****p < 0.0001.300484454.1 - 22 -BAYM. P0444WO / BLG 25-024

[0164] FIGs. 3A-3C show the percentage of CD25-expressing lymphocytes and cytokineproducing T cells after splenocyte restimulation with rNa-GST-1 protein. FIG. 3A shows the percentage of CD4-positive, CD8-positive, or CD19-positive cells that were also CD25-positive. FIG.3B shows the percentage of CD4-positive cells identified as also IFNγ-positive, IL2-positive, TNFα-positive, IL4-positive, or IL13-positive. FIG.3C shows the percentage of CD8-positive cells that were also identifies as IFNγ-positive, IL2-positive, TNFα-positive, or granzyme B-positive. Dashed lines indicate the adjusted baseline (zero), with non-stimulated cell values subtracted from / -GST-I -stimulated cells. Statistical analysis was performed using the Kruskal -Wallis test, followed by Dunn’s test for pairwise comparison of groups. *p < 0.05, ** p < 0.01, ***p < 0.001.

[0165] FIGs. 4A-4C show neutralization of Na-GST-1 enzymatic activity after incubation with purified IgG from immunized mice. FIG. 4A depicts SDS-PAGE of purified IgG from pooled sera. One microgram of IgG was loaded per sample. Total yield after purification is indicated below the gel. FIG. 4B shows results of a GST-1 neutralization assay using multiple amounts of purified IgG from each sample. FIG. 4C shows a WB of pooled sera from the pmNa-GST-1 mRNA group against rNA-GST-1 or total proteins from L3 larvae, adult A. caninum, and adult N. americanus.

[0166] FIG. 5 shows the percentage of memory effector and memory central cytokineproducing CD4-positive T cells after splenocyte restimulation with rNa-GST-1 protein. Statistical significance was assessed using Kruskal-Wallis test for multiple comparisons. *p < 0.05, ** p < 0.01, ***p < 0.001. ****p < 0.0001.

[0167] FIG. 6 shows the percentage of memory effector and memory central cytokineproducing CD8-positive T cells after splenocyte restimulation with rNa-GST-1 protein. Statistical significance was assessed using Kruskal-Wallis test for multiple comparisons. *p < 0.05, ** p < 0.01, ***p < 0.001. ****p < 0.0001.

[0168] FIGs. 7A-7D show schematics illustrating mRNA constructs, mRNA vaccine manufacturing, lipids for lipid nano particles (LNPs), and methods of making the same. FIG.7A depicts a schematic of an mRNA construct, comprising a 5' Cap, 5' untranslated region (UTR), signal peptide, gene of interest, 3' UTR, and Poly A tail. FIG. 7B depicts a schematic for preparing a purified capped mRNA vaccine. FIG. 7C depicts illustrative LNP lipids. FIG.7D depicts a schematic representation of an illustrative LNP.

[0169] FIGs. 8A-8D show how mice inoculated with GST-1 mRNA vaccines obtained immunogenicity towards and protection against challenge with murine parasite Nippostrongylus brasiliensis. FIG. 8A shows a schematic immunization schedule of BALB / C 300484454.1 - 23 -BAYM. P0444WO / BLG 25-024mice and sample collection for experiments performed (El and E2, respectively). Each experiment included 5 groups as follows: 1) LNP control; 2) mRNA encoding cytoplasmic GST-1 [10 pg] / LNP; 3) mRNA encoding plasma membrane bound GST-1 [10 pg] / LNP; 4) mRNA encoding secreted GST-1 [10 pg] / LNP; and 5) recombinant Na-GST-1 [25 pg] + Alhydrogel control (N=5 mice per group). Immunization was administered at week 1 and week 3, with 50 pL / dose injected intramuscularly in the gluteal muscle. mRNA / LNP vaccine formulations were prepared at a concentration of 200 pg / ml mRNA in sterile PBS with 8% sucrose and stored at -80 °C until use. FIG. 8B shows IgGl titers at week 6 of El and E2, and week 16 of E2. FIG. 8C shows IgG2c titers at week 6 of El and E2, and week 16 of E2. FIG.8D shows how vaccination provided significant gut worm (“Nippo”) burden control at week 7 of El, and how pmGST-1 mRNA vaccine provided significant gut worm burden control at week 17.

[0170] FIGs. 9A-9C depict in vitro validation of mRNAs encoding different forms of Na-APR-1. FIG.9A is a schematic representation of Na-APR-1 mRNA vaccine candidates: native (top), secretory (middle), and plasma membrane-anchored (bottom). FIG. 9B and FIG. 9C depict western blot (WB) analysis of protein production from constructs tsfullNa-APR-1 (tissue plasminogen secretory full-length protein), tsNa-APR-1 (tissue plasminogen secretory mature protein), asfullNa-APR-1 (albumin secretory full-length protein), pmNa-APR-1 (GPI plasma membrane anchored mature protein), and asNa-APR-1 (albumin secretory mature protein) when analyzed with anti-flag antibody (FIG. 9B), or when serum samples were analyzed using anti-APR antibody (FIG. 9C).

[0171] FIGs. 10A-10B describe bivalent Na-GST-1 and Na-APR-1 mRNA vaccines and associated in vivo trials. FIG. 10A is a schematic representation of pmNa-GST-1 and pmNa-APR-1 mRNA vaccine candidates. FIG. 10B is a schematic representation of preclinical trials testing the efficacy of bivalent vaccines in providing immunogenicity towards and protection against challenge with parasites, e.g., Nippostrongylus brasiliensis.

[0172] FIGs. 11A-11B depict a GST-1 consensus protein sequence alignment with N americanus, A. duodenale, A. ceylanicum, and A. canium GST-1 (FIG. 11 A), and percentage identity and similarity for GST-1 between these four species (FIG. 11B).DETAILED DESCRIPTION

[0173] It is to be understood that the present disclosure is not limited to particular aspects described, as such may, of course, vary. It is also to be understood that the terminology used300484454.1 - 24 -BAYM. P0444WO / BLG 25-024herein is for the purpose of describing particular aspects only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0174] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this technology belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present technology, the preferred methods, devices and materials are now described. All technical and patent publications cited herein are incorporated herein by reference in their entirety.

[0175] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, e.g., Green and Sambrook eds. (2012) Molecular Cloning: A Laboratory Manual, 4th edition; the series Ausubel et al. eds. (2015) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N. Y.); MacPherson et al. (2015) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; McPherson et al. (2006) PCR: The Basics (Garland Science); Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Greenfield ed. (2014) Antibodies, A Laboratory Manual; Freshney (2010) Culture of Animal Cells: A Manual of Basic Technique, 6th edition; Gait ed. (1984) Oligonucleotide Synthesis; U. S. Pat. No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Herdewijn ed. (2005) Oligonucleotide Synthesis: Methods and Applications; Hames and Higgins eds. (1984) Transcription and Translation; Buzdin and Lukyanov ed. (2007) Nucleic Acids Hybridization: Modem Applications; Immobilized Cells and Enzymes (IRL Press (1986)); Grandi ed. (2007) In Vitro Transcription and Translation Protocols, 2nd edition; Guisan ed. (2006) Immobilization of Enzymes and Cells; Perbal (1988) A Practical Guide to Molecular Cloning, 2nd edition; Miller and Calos eds, (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); Lundblad and Macdonald eds. (2010) Handbook of Biochemistry and Molecular Biology, 4th edition; and Herzenberg et al. eds (1996) Weir's Handbook of Experimental Immunology, 5th edition.

[0176] It also is to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art. It is to be inferred without explicit recitation and unless otherwise intended, that when the present 300484454.1 - 25 -BAYM. P0444WO / BLG 25-024technology relates to a polypeptide, protein, polynucleotide, cell, or antibody, an equivalent or a biologically equivalent of such is intended within the scope of the present technology.

[0177] Accordingly, disclosed herein are polynucleotides, polypeptides, compositions, and / or cells comprising and / or encoding engineered parasite antigens, as well as methods for use of the same in treatment or prophylaxis of disease (e.g., infectious diseases, immunization). Particular aspects relate to mRNA vaccines for providing immunity against parasites, such as for example, a nematode or hookworm. Aspects of the disclosure provide improved vaccines against parasites at least by inducing a faster antibody response and unique immune profiles. In some aspects, plasma membrane anchoring of a parasite antigen, and / or expressing a solubilized form improves immunization.I. Hookworms

[0178] Parasites are organisms that live on or inside a host organism, deriving nutrients at the host’s expense. Among these, hookworms are a significant type of parasitic nematode that primarily infect the intestines of their hosts. The term hookworm encompasses multiple nematode species within the family Ancylostomatidae, such as for example, Ancylostoma duodenale and Necator americanus. Hookworms are known for their hook-like mouthparts which they use to attach to the intestinal wall and feed on the host's blood. The adult hookworms then ingest the blood relying on host hemoglobin as a nutrient source

[0013] , This feeding behavior can lead to various health issues in the host, including anemia, malnutrition, and general weakness.

[0179] Hookworm infections are typically transmitted through contact with contaminated soil, where the larvae penetrate the skin, usually through the feet. Once inside the host, the larvae travel through the bloodstream to the lungs, ascend the respiratory tract, and are swallowed to reach the intestines where they mature into adult worms. Effective control and treatment of hookworm infections are crucial, especially in regions with poor sanitation, to prevent the associated health complications and improve overall public health.II. Polynucleotides

[0180] The present disclosure relates, at least in part, to polynucleotides encoding one or more wild type or engineered antigens that can stimulate an immune response in an individual. Illustrative polynucleotides of the disclosure include, but are not limited to, antigen-encoding RNA polynucleotides. In some aspects, polynucleotides of the disclosure comprise an mRNA vaccine.300484454.1 - 26 -BAYM. P0444WO / BLG 25-024

[0181] Illustrative nucleic acids or polynucleotides include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-α-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA), or hybrids or combinations thereof.

[0182] In some aspects, linear polynucleotides encoding one or more antigens of the Polynucleotides of the disclosure of the present disclosure are made using in vitro transcription (IVT) enzymatic synthesis methods and are referred to as “in vitro transcribed polynucleotides” herein.

[0183] In some aspects, a polynucleotide of the disclosure may include from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000).

[0184] In some aspects, polynucleotides of the present disclosure may encode at least one peptide or polypeptide of interest. In some aspects, the length of a region encoding at least one peptide polypeptide of interest of the polynucleotides is greater than 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including300484454.1 - 27 -BAYM. P0444WO / BLG 25-024100,000 nucleotides). As used herein, such a region may be referred to as a “coding region” or “region encoding.”

[0185] In some aspects, polynucleotides of the present disclosure is or functions as a messenger RNA (mRNA). As used herein, the term “messenger RNA” (mRNA) refers to any polynucleotide which encodes at least one peptide or polypeptide of interest and which is capable of being translated to produce the encoded peptide polypeptide of interest in vitro, in vivo, in situ, or ex vivo.

[0186] In some aspects, polynucleotides of the present disclosure may be structurally modified or chemically modified. As used herein, a “structural” modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted, resulting in a structural modification to the polynucleotide.

[0187] In some aspects, the polynucleotides of the present disclosure, such as in vitro transcribed polynucleotides, may have a uniform chemical modification of all or any of the same nucleoside type or a population of modifications produced by mere downward titration of the same starting modification in all or any of the same nucleoside type, or a measured percent of a chemical modification of all any of the same nucleoside type but with random incorporation, such as where all uridines are replaced by a uridine analog, e.g., pseudouridine. In some aspects, the polynucleotides may have a uniform chemical modification of two, three, or four of the same nucleoside type throughout the entire polynucleotide (e.g., all uridines and cytosines are modified in the same way).A. RNA, mRNA

[0188] RNA(s) is the usual abbreviation for ribonucleic acid(s). It is a nucleic acid molecule, i.e., a polymer consisting of nucleotide monomers. These nucleotides are usually adenosine-monophosphate (AMP), uridine-monophosphate (UMP), guanosinemonophosphate (GMP) and cytidine-monophosphate (CMP) monomers or analogs thereof, which are connected to each other along a so-called “backbone.” The backbone is formed by300484454.1 - 28 -BAYM. P0444WO / BLG 25-024phosphodiester bonds between the sugar, i.e., ribose, of a first and a phosphate moiety of a second, adjacent monomer. The specific order of the monomers, i.e., the order of the bases linked to the sugar / phosphate-backbone, is called the RNA sequence. Usually RNA may be obtainable by transcription of a DNA sequence, for example, but not limited to, inside a cell. In eukaryotic cells, transcription is typically performed inside the nucleus or the mitochondria. In vivo transcription of DNA usually results in the so-called premature RNA which has to be processed into so-called messenger-RNA, usually abbreviated as mRNA. Processing of the premature RNA, for example, but not limited to, in eukaryotic organisms, comprises a variety of different posttranscriptional modifications such as splicing, 5 '-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA. The mature messenger RNA usually provides the nucleotide sequence that may be translated into an amino acid sequence of a particular peptide or protein. Typically, a mature mRNA comprises a 5'-cap, a 5' UTR, an open reading frame (ORF), a 3' UTR, a poly(A) sequence, and / or any combination thereof.

[0189] In addition to messenger RNA, several non-coding types of RNA exist which may be involved in regulation of transcription and / or translation, and immunostimulation. The term “RNA” further encompasses RNA molecules, such as viral RNA, retroviral RNA and replicon RNA, small interfering RNA (siRNA), antisense RNA, CRISPR / Cas9 guide RNA, ribozymes, aptamers, riboswitches, immunostimulating RNA, transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), microRNA (miRNA), and Piwi-interacting RNA (piRNA).B. Polynucleotide Architecture

[0190] Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5' cap and a poly-A tail. Polynucleotides of the present disclosure may function as mRNA but are distinguished from wild-type mRNA in their functional and / or structural design features which serve to overcome existing problems of effective polypeptide production using nucleic-acid based therapeutics.

[0191] FIG. 7A shows an illustrative mRNA construct of general structure representing one or more polynucleotides of the present disclosure. Such mRNA constructs are useful in compositions of the disclosure, such as mRNA vaccines. According to FIG. 7A, a polynucleotide may contain a 5' cap, a 5' untranslated region (UTR), a signal peptide (SP), a gene of interest (GOI) coding region, a 3' UTR, a 3' tailing sequences, such as a polyA tail, or a combination thereof.300484454.1 - 29 -BAYM. P0444WO / BLG 25-0241. Transgene

[0192] In some aspects, disclosed herein are compositions and methods that relate to the use of vectors to deliver transgenes. In some aspects, a vector comprises a transgene. As used herein, a “transgene” or “gene of interest” refers to a heterologous polynucleotide that acts as a template for translation of a polypeptide. In some aspects a transgene may comprise a native (wild-type), truncated, and / or mutated sequence. In some aspects, a transgene has been modified to be secreted or anchored to the plasma membrane.

[0193] In some aspects, a transgene of the disclosure comprises Na-GST-1. Na-GST-1 is an enzyme involved in the detoxification process of helminth parasites and serves as a protective antigen for vaccine development against hookworm infections. In some aspects, an Na-GST-1 sequence may comprise a native (cytosolic) Na-GST-1 (nNa-GST-1), a soluble Na-GST-1 (sNa-GST-1), which may contain an IgG signal peptide, for example, for endoplasmic reticulum (ER) import and secretion, and / or a plasma membrane bound Na-GST-1 (pmNa-GST-1), which may comprise an albumin signal peptide, for example, for ER import and GPI attachment. In some aspects, a transgene described herein may include a tag sequence, such as a flag tag sequence. In some aspects, a tag is optional and can be removed from any sequence described herein that comprises a tag sequence.

[0194] In some aspects, a transgene comprises, consists essentially of, or consists of a polynucleotide sequence that is, or is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to one or more of SEQ ID NOs: 1-7.SEQ ID NO: 1 - nNa-GST-1 construct (NA) - AUGGUUCAUUAUAAAUUGACGUAUUUUGCCAUCAGGGGUGCCGGGGAAUGCGCGAGACAAAU AUUUGCGCUCGCAGAUCAGGAGUUUGAAGAUGUACGCCUUGAUAAAGAACAAUUCGCCAAGG UAAAACCUGAUUUGCCCUUUGGGCAAGUCCCUGUACUGGAAGUCGAUGGGAAGCAGCUCGCA CAGAGUCUCGCUAUUUGUCGGUAUCUUGCAAGGCAAUUCGGGUUUGCAGGGAAAUCAACUUU UGACGAGGCAGUAGUUGACUCCCUGGCUGACCAGUAUUCAGAUUAUCGAGUGGAGAUCAAGU CAUUUUUUUACACCGUUAUCGGAAUGCGAGAGGGAGAUGUGGAACAACUUAAAAAGGAAGUC CUUCUCCCUGCGAGAGAUAAAUUCUUUGGGUUUAUUACAAAAUUUUUGAAAAAAAGUCCGAG UGGCUUUCUUGUAGGCGACAGUCUGACCUGGGUGGACCUGCUUGUGUCCGAGCAUAAUGCCA CCAUGCUGACUUUUGUCCCUGAGUUCCUCGAAGGUUAUCCUGAAGUAAAAGAACACAUGGAG AAGAUUAGAGCAAUUCCUAAGCUCAAGAAGUGGAUAGAAACGCGCCCUGAAACCUUGUUCGA CUACAAAGAUGAUGACGAUAAGUAAUAA (SEQ ID NO: 1 )SEQ ID NO: 2 - sNa-GST-1 construct (NA) - AUGGGCUGGUCCUGCAUCAUCUUGUUCUUGGUGGCUACUGCCACUGGAGUACACAGCAAGCU UGUUCAUUAUAAAUUGACGUAUUUUGCCAUCAGGGGUGCCGGGGAAUGCGCGAGACAAAUAU300484454.1 - 30 -BAYM. P0444WO / BLG 25-024UUGCGCUCGCAGAUCAGGAGUUUGAAGAUGUACGCCUUGAUAAAGAACAAUUCGCCAAGGUA AAACCUGAUUUGCCCUUUGGGCAAGUCCCUGUACUGGAAGUCGAUGGGAAGCAGCUCGCACA GAGUCUCGCUAUUUGUCGGUAUCUUGCAAGGCAAUUCGGGUUUGCAGGGAAAUCAACUUUUG ACGAGGCAGUAGUUGACUCCCUGGCUGACCAGUAUUCAGAUUAUCGAGUGGAGAUCAAGUCA UUUUUUUACACCGUUAUCGGAAUGCGAGAGGGAGAUGUGGAACAACUUAAAAAGGAAGUCCU UCUCCCUGCGAGAGAUAAAUUCUUUGGGUUUAUUACAAAAUUUUUGAAAAAAAGUCCGAGUG GCUUUCUUGUAGGCGACAGUCUGACCUGGGUGGACCUGCUUGUGUCCGAGCAUAAUGCCACC AUGCUGACUUUUGUCCCUGAGUUCCUCGAAGGUUAUCCUGAAGUAAAAGAACACAUGGAGAA GAUUAGAGCAAUUCCUAAGCUCAAGAAGUGGAUAGAAACGCGCCCUGAAACCUUGUUCGACU ACAAAGAUGAUGACGAUAAGUAAUAA (SEQ ID NO: 2 )SEQ ID NO: 3 - sNa-GST-1 construct without FLAG tag (NA) - AUGGGCUGGUCCUGCAUCAUCUUGUUCUUGGUGGCUACUGCCACUGGAGUACACAGCAAGCU UGUUCAUUAUAAAUUGACGUAUUUUGCCAUCAGGGGUGCCGGGGAAUGCGCGAGACAAAUAU UUGCGCUCGCAGAUCAGGAGUUUGAAGAUGUACGCCUUGAUAAAGAACAAUUCGCCAAGGUA AAACCUGAUUUGCCCUUUGGGCAAGUCCCUGUACUGGAAGUCGAUGGGAAGCAGCUCGCACA GAGUCUCGCUAUUUGUCGGUAUCUUGCAAGGCAAUUCGGGUUUGCAGGGAAAUCAACUUUUG ACGAGGCAGUAGUUGACUCCCUGGCUGACCAGUAUUCAGAUUAUCGAGUGGAGAUCAAGUCA UUUUUUUACACCGUUAUCGGAAUGCGAGAGGGAGAUGUGGAACAACUUAAAAAGGAAGUCCU UCUCCCUGCGAGAGAUAAAUUCUUUGGGUUUAUUACAAAAUUUUUGAAAAAAAGUCCGAGUG GCUUUCUUGUAGGCGACAGUCUGACCUGGGUGGACCUGCUUGUGUCCGAGCAUAAUGCCACC AUGCUGACUUUUGUCCCUGAGUUCCUCGAAGGUUAUCCUGAAGUAAAAGAACACAUGGAGAA GAUUAGAGCAAUUCCUAAGCUCAAGAAGUGGAUAGAAACGCGCCCUGAAACCUUGUUC (SEQ ID NO: 3 )SEQ ID NO: 4 - pmAa-GST-1 construct (NA) - AUGAAAUGGGUCACCUUUAUCAGCCUGCUGUUCCUGUUCAGCAGCGCCUACAGCGACUACAA AGACGAUGACGACAAGAAGCUUGUUCAUUAUAAAUUGACGUAUUUUGCCAUCAGGGGUGCCG GGGAAUGCGCGAGACAAAUAUUUGCGCUCGCAGAUCAGGAGUUUGAAGAUGUACGCCUUGAU AAAGAACAAUUCGCCAAGGUAAAACCUGAUUUGCCCUUUGGGCAAGUCCCUGUACUGGAAGU CGAUGGGAAGCAGCUCGCACAGAGUCUCGCUAUUUGUCGGUAUCUUGCAAGGCAAUUCGGGU UUGCAGGGAAAUCAACUUUUGACGAGGCAGUAGUUGACUCCCUGGCUGACCAGUAUUCAGAU UAUCGAGUGGAGAUCAAGUCAUUUUUUUACACCGUUAUCGGAAUGCGAGAGGGAGAUGUGGA ACAACUUAAAAAGGAAGUCCUUCUCCCUGCGAGAGAUAAAUUCUUUGGGUUUAUUACAAAAU UUUUGAAAAAAAGUCCGAGUGGCUUUCUUGUAGGCGACAGUCUGACCUGGGUGGACCUGCUU GUGUCCGAGCAUAAUGCCACCAUGCUGACUUUUGUCCCUGAGUUCCUCGAAGGUUAUCCUGA AGUAAAAGAACACAUGGAGAAGAUUAGAGCAAUUCCUAAGCUCAAGAAGUGGAUAGAAACGC GCCCUGAAACCUUGUUCAAGCUUCCUAACAAAGGCAGCGGCACCACCUCUGGCACCACAAGA CUGCUGUCUGGCCACACCUGUUUCACACUGACCGGCCUGCUGGGCACACUGGUUACAAUGGG ACUGCUGACCUAAUAA (SEQ ID NO: 4 )SEQ ID NO: 5 - nNa-GST-1 protein (NA) - AUGGUUCAUUAUAAAUUGACGUAUUUUGCCAUCAGGGGUGCCGGGGAAUGCGCGAGACAAAU AUUUGCGCUCGCAGAUCAGGAGUUUGAAGAUGUACGCCUUGAUAAAGAACAAUUCGCCAAGG UAAAACCUGAUUUGCCCUUUGGGCAAGUCCCUGUACUGGAAGUCGAUGGGAAGCAGCUCGCA CAGAGUCUCGCUAUUUGUCGGUAUCUUGCAAGGCAAUUCGGGUUUGCAGGGAAAUCAACUUU UGACGAGGCAGUAGUUGACUCCCUGGCUGACCAGUAUUCAGAUUAUCGAGUGGAGAUCAAGU CAUUUUUUUACACCGUUAUCGGAAUGCGAGAGGGAGAUGUGGAACAACUUAAAAAGGAAGUC CUUCUCCCUGCGAGAGAUAAAUUCUUUGGGUUUAUUACAAAAUUUUUGAAAAAAAGUCCGAG UGGCUUUCUUGUAGGCGACAGUCUGACCUGGGUGGACCUGCUUGUGUCCGAGCAUAAUGCCA CCAUGCUGACUUUUGUCCCUGAGUUCCUCGAAGGUUAUCCUGAAGUAAAAGAACACAUGGAG300484454.1 - 31 -BAYM. P0444WO / BLG 25-024AAGAUUAGAGCAAUUCCUAAGCUCAAGAAGUGGAUAGAAACGCGCCCUGAAACCUUGUUC(SEQ ID NO: 5 )SEQ ID NO: 6 - sV / - ST-l protein (NA) - GUUCAUUAUAAAUUGACGUAUUUUGCCAUCAGGGGUGCCGGGGAAUGCGCGAGACAAAUAUU UGCGCUCGCAGAUCAGGAGUUUGAAGAUGUACGCCUUGAUAAAGAACAAUUCGCCAAGGUAA AACCUGAUUUGCCCUUUGGGCAAGUCCCUGUACUGGAAGUCGAUGGGAAGCAGCUCGCACAG AGUCUCGCUAUUUGUCGGUAUCUUGCAAGGCAAUUCGGGUUUGCAGGGAAAUCAACUUUUGA CGAGGCAGUAGUUGACUCCCUGGCUGACCAGUAUUCAGAUUAUCGAGUGGAGAUCAAGUCAU UUUUUUACACCGUUAUCGGAAUGCGAGAGGGAGAUGUGGAACAACUUAAAAAGGAAGUCCUU CUCCCUGCGAGAGAUAAAUUCUUUGGGUUUAUUACAAAAUUUUUGAAAAAAAGUCCGAGUGG CUUUCUUGUAGGCGACAGUCUGACCUGGGUGGACCUGCUUGUGUCCGAGCAUAAUGCCACCA UGCUGACUUUUGUCCCUGAGUUCCUCGAAGGUUAUCCUGAAGUAAAAGAACACAUGGAGAAG AUUAGAGCAAUUCCUAAGCUCAAGAAGUGGAUAGAAACGCGCCCUGAAACCUUGUUC (SEQ ID NO: 6 )SEQ ID NO: 7 - pmAa-GST-1 protein (NA) - GUUCAUUAUAAAUUGACGUAUUUUGCCAUCAGGGGUGCCGGGGAAUGCGCGAGACAAAUAUU UGCGCUCGCAGAUCAGGAGUUUGAAGAUGUACGCCUUGAUAAAGAACAAUUCGCCAAGGUAA AACCUGAUUUGCCCUUUGGGCAAGUCCCUGUACUGGAAGUCGAUGGGAAGCAGCUCGCACAG AGUCUCGCUAUUUGUCGGUAUCUUGCAAGGCAAUUCGGGUUUGCAGGGAAAUCAACUUUUGA CGAGGCAGUAGUUGACUCCCUGGCUGACCAGUAUUCAGAUUAUCGAGUGGAGAUCAAGUCAU UUUUUUACACCGUUAUCGGAAUGCGAGAGGGAGAUGUGGAACAACUUAAAAAGGAAGUCCUU CUCCCUGCGAGAGAUAAAUUCUUUGGGUUUAUUACAAAAUUUUUGAAAAAAAGUCCGAGUGG CUUUCUUGUAGGCGACAGUCUGACCUGGGUGGACCUGCUUGUGUCCGAGCAUAAUGCCACCA UGCUGACUUUUGUCCCUGAGUUCCUCGAAGGUUAUCCUGAAGUAAAAGAACACAUGGAGAAG AUUAGAGCAAUUCCUAAGCUCAAGAAGUGGAUAGAAACGCGCCCUGAAACCUUGUUC (SEQ ID NO: 7 )

[0195] In some aspects, a polynucleotide of the disclosure encodes for a / -GST-I polypeptide. In some aspects, a 7Va-GST-l polypeptide comprises, consists essentially of, or consists of a sequence that is, or is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to one or more of SEQ IDNOs: 8-15.SEQ ID NO: 8 - consensus-GST-1 protein only (AA) - MVHYKLTYFDIRGAGECARQIFALAGQEFEDXRLTKEXFAPXKPKXPFGQVPVLEVDGKQLA QSLAICRYLARQFGXAGKSPFDEAVVDSLADQXSDXRVEIKPYFYTAIGMREGDLEQLKKEV LLPAREKFFGFJTKFLKXNPSGFLVGDSVTWVDXLISEXIATXLXFXPEXLDGYPEVKAHME KVRAIPNLKKWIEXXPXRPF (SEQ ID NO: 8 )SEQ ID NO: 9 - nNa-GST-1 protein only (AA) - MVHYKLTYFAIRGAGECARQIFALADQEFEDVRLDKEQFAKVKPDLPFGQVPVLEVDGKQLA QSLAICRYLARQFGFAGKSTFDEAVVDSLADQYSDYRVEIKSFFYTVIGMREGDVEQLKKEV LLPARDKFFGFITKFLKKSPSGFLVGDSLTWVDLLVSEHNATMLTFVPEFLEGYPEVKEHME KIRAIPKLKKWIETRPETLF (SEQ ID NO: 9 )300484454.1 - 32 -BAYM. P0444WO / BLG 25-024SEQ ID NO: 10 - i a-GST-1 with FLAG tag (AA) - MVHYKLTYFAIRGAGECARQIFALADQEFEDVRLDKEQFAKVKPDLPFGQVPVLEVDGKQLA QSLAICRYLARQFGFAGKSTFDEAVVDSLADQYSDYRVEIKSFFYTVIGMREGDVEQLKKEV LLPARDKFFGFITKFLKKSPSGFLVGDSLTWVDLLVSEHNATMLTFVPEFLEGYPEVKEHME KIRAIPKLKKWIETRPETLFDYKDDDDK (SEQ ID NO: 10)SEQ ID NO: 11 - s\tf-GST-l protein only (AA) - VHYKLTYFAIRGAGECARQIFALADQEFEDVRLDKEQFAKVKPDLPFGQVPVLEVDGKQLAQ SLAICRYLARQFGFAGKSTFDEAVVDSLADQYSDYRVEIKSFFYTVIGMREGDVEQLKKEVL LPARDKFFGFITKFLKKSPSGFLVGDSLTWVDLLVSEHNATMLTFVPEFLEGYPEVKEHMEK IRAIPKLKKWIETRPETLF (SEQ ID NO: 11)SEQ ID NO: 12 - sAa-GST-1 with signal peptide (AA) - MGWSCI ILFLVATATGVHSKLVHYKLTYFAIRGAGECARQIFALADQEFEDVRLDKEQFAKV KPDLPFGQVPVLEVDGKQLAQSLAICRYLARQFGFAGKSTFDEAVVDSLADQYSDYRVEIKS FFYTVIGMREGDVEQLKKEVLLPARDKFFGFITKFLKKSPSGFLVGDSLTWVDLLVSEHNAT MLTFVPEFLEGYPEVKEHMEKIRAIPKLKKWIETRPETLF (SEQ ID NO: 12)SEQ ID NO: 13 - sAa-GST-1 with signal peptide and FLAG tag (AA) - MGWSCI ILFLVATATGVHSKLVHYKLTYFAIRGAGECARQIFALADQEFEDVRLDKEQFAKV KPDLPFGQVPVLEVDGKQLAQSLAICRYLARQFGFAGKSTFDEAVVDSLADQYSDYRVEIKS FFYTVIGMREGDVEQLKKEVLLPARDKFFGFITKFLKKSPSGFLVGDSLTWVDLLVSEHNAT MLTFVPEFLEGYPEVKEHMEKIRAIPKLKKWIETRPETLFDYKDDDDK (SEQ ID NO: 13)SEQ ID NO: 14 - pm\tf-GST-l protein only (AA) - VHYKLTYFAIRGAGECARQIFALADQEFEDVRLDKEQFAKVKPDLPFGQVPVLEVDGKQLAQ SLAICRYLARQFGFAGKSTFDEAVVDSLADQYSDYRVEIKSFFYTVIGMREGDVEQLKKEVL LPARDKFFGFITKFLKKSPSGFLVGDSLTWVDLLVSEHNATMLTFVPEFLEGYPEVKEHMEK IRAIPKLKKWIETRPETLF (SEQ ID NO: 14)SEQ ID NO: 15 - pmAa-GST-1 with signal peptide, FLAG tag, and anchor (AA) - MKWVTFISLLFLFSSAYSDYKDDDDKKLVHYKLTYFAIRGAGECARQIFALADQEFEDVRLD KEQFAKVKPDLPFGQVPVLEVDGKQLAQSLAICRYLARQFGFAGKSTFDEAVVDSLADQYSD YRVEIKSFFYTVIGMREGDVEQLKKEVLLPARDKFFGFITKFLKKSPSGFLVGDSLTWVDLL VSEHNATMLTFVPEFLEGYPEVKEHMEKIRAIPKLKKWIETRPETLFKLPNKGSGTTSGTTR LLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 15)

[0196] In some aspects, a transgene of the disclosure comprises Na-APR-1. Na-APR-1 is a key enzyme involved in the digestion of host hemoglobin by the hookworm Necator americanus and is a candidate antigen for hookworm vaccine development. In some aspects, / -APR.-I sequence may comprise a secretory version or a plasma membrane bound version of Na- APR-1. In some aspects, an / -APR.-I comprises one or more point mutations coding for DI 13 A and / or D300A. In some aspects, an M / -APR.-I lacks the coding sequence for the first 73 amino acids of the wild-type Na-APR-1 (referred to as Na-APR-1 M74). In some aspects, an300484454.1 - 33 -BAYM. P0444WO / BLG 25-024 / -APR- I lacks the coding sequence for the first 16 amino acids of the wild-type Na- APR- 1 (referred to as Na- APR- 1 FL).

[0197] In some aspects, a transgene comprises, consists essentially of, or consists of a polynucleotide sequence that is, or is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to one or more of SEQ ID NO: 16-25.SEQ ID NO: 16 - sNa-APR-l(M74) construct (NA) - AUGGGCUGGUCCUGCAUCAUCUUGUUCUUGGUGGCUACUGCCACUGGAGUACACAGCAAGCU UGCUAGUAAACUUCAGUCCGCUAACGAGAUCGACGAAUUGCUCAGAAAUUACAUGGAUGCUC AGUAUUACGGCGUGAUUCAGAUAGGAACGCCUGCACAGAAUUUUACUGUCAUAUUCGCCACC GGAAGUUCCAAUUUGUGGGUCCCCUCUAGGAAAUGUCCCUUCUAUGAUAUAGCAUGUAUGUU GCAUCACCGCUAUGACAGUGGGGCUUCUUCCACGUAUAAGGAGGACGGUAGGAAAAUGGCAA UUCAAUAUGGUACCGGAUCCAUGAAAGGAUUCAUCAGUAAGGACAUUGUUUGCAUCGCAGGG AUUUGUGCCGAGGAGCAACCUUUCGCGGAAGCCACGUCUGAACCCGGUCUUACUUUCAUUGC UGCAAAGUUCGAUGGGAUAUUGGGGAUGGCCUUCCCUGAGAUCGCGGUCCUGGGUGUUACGC CGGUAUUUCACACAUUCAUUGAGCAAAAAAAGGUGCCCUCUCCGGUAUUUGCGUUCUGGCCA AAUCGCAACCCAGAGUCUGAAAUCGGAGGUGAAAUUACUUUCGGGGGCGUAGACACCAGGAG GUACGUUGAACCUAUCACUUGGACCCCUGUGACACGGAGAGGCUACUGGCAAUUCAAAAUGG ACAUGGUACAAGGUGGAAGUUCCUCAAUCGCAUGCCCAAACGGUUGCCAAGCUAUCGCGGCU ACCGGCACUUCCUUGAUUGCUGGGCCAAAGGCCCAGGUUGAGGCCAUACAAAAGUAUAUUGG UGCUGAGCCACUGAUGAAAGGCGAAUAUAUGAUACCGUGUGACAAGGUGCCAUCCCUUCCAG AUGUGAGUUUCAUAAUAGACGGGAAAACUUUUACCCUGAAGGGUGAAGACUACGUGCUUACC GUUAAAGCGGCUGGAAAAUCUAUAUGCCUCAGCGGCUUCAUGGGCAUGGACUUCCCUGAAAA AAUUGGAGAGCUUUGGAUACUGGGAGACGUCUUCAUCGGAAAGUAUUACACCGUUUUCGAUG UUGGCCAGGCAAGAGUGGGUUUUGCUCAAGCGAAAUCCGAAGACGGCUUCCCAGUUGGCACC CCAGUCCGAACCUUCCGCCAGCUCCAGGAAGAUUCCGAUUCAGACGAAGAUGAUGUUUUUAC AUUUGACUAUAAGGAUGAUGAUGACAAAUAAUAA (SEQ ID NO: 16 )SEQ ID NO: 17 - sNa-APR-l(FL) construct (NA) - AUGGGCUGGUCCUGCAUCAUCUUGUUCUUGGUGGCUACUGCCACUGGAGUACACAGCAAGCU UAGCGUUCAUAGGAGACUGUUCCACCAGGCCCGACGCCAUGUCACCAGUGUCUCCCUGUCCC GGCAACCGACCUUGAGAGAACGCCUUAUAGCGUCUGGGUCCUGGGAAGACUACCAGAAACAG CGCUACCAUUACCAGAAGAAGAUCCUUGCCAAGUAUGCUGCAAACAAAGCUAGUAAACUUCA GUCCGCUAACGAGAUCGACGAAUUGCUCAGAAAUUACAUGGAUGCUCAGUAUUACGGCGUGA UUCAGAUAGGAACGCCUGCACAGAAUUUUACUGUCAUAUUCGCCACCGGAAGUUCCAAUUUG UGGGUCCCCUCUAGGAAAUGUCCCUUCUAUGAUAUAGCAUGUAUGUUGCAUCACCGCUAUGA CAGUGGGGCUUCUUCCACGUAUAAGGAGGACGGUAGGAAAAUGGCAAUUCAAUAUGGUACCG GAUCCAUGAAAGGAUUCAUCAGUAAGGACAUUGUUUGCAUCGCAGGGAUUUGUGCCGAGGAG CAACCUUUCGCGGAAGCCACGUCUGAACCCGGUCUUACUUUCAUUGCUGCAAAGUUCGAUGG GAUAUUGGGGAUGGCCUUCCCUGAGAUCGCGGUCCUGGGUGUUACGCCGGUAUUUCACACAU UCAUUGAGCAAAAAAAGGUGCCCUCUCCGGUAUUUGCGUUCUGGCCAAAUCGCAACCCAGAG UCUGAAAUCGGAGGUGAAAUUACUUUCGGGGGCGUAGACACCAGGAGGUACGUUGAACCUAU CACUUGGACCCCUGUGACACGGAGAGGCUACUGGCAAUUCAAAAUGGACAUGGUACAAGGUG GAAGUUCCUCAAUCGCAUGCCCAAACGGUUGCCAAGCUAUCGCGGCUACCGGCACUUCCUUG AUUGCUGGGCCAAAGGCCCAGGUUGAGGCCAUACAAAAGUAUAUUGGUGCUGAGCCACUGAU300484454.1 - 34 -BAYM. P0444WO / BLG 25-024GAAAGGCGAAUAUAUGAUACCGUGUGACAAGGUGCCAUCCCUUCCAGAUGUGAGUUUCAUAA UAGACGGGAAAACUUUUACCCUGAAGGGUGAAGACUACGUGCUUACCGUUAAAGCGGCUGGA AAAUCUAUAUGCCUCAGCGGCUUCAUGGGCAUGGACUUCCCUGAAAAAAUUGGAGAGCUUUG GAUACUGGGAGACGUCUUCAUCGGAAAGUAUUACACCGUUUUCGAUGUUGGCCAGGCAAGAG UGGGUUUUGCUCAAGCGAAAUCCGAAGACGGCUUCCCAGUUGGCACCCCAGUCCGAACCUUC CGCCAGCUCCAGGAAGAUUCCGAUUCAGACGAAGAUGAUGUUUUUACAUUUGACUAUAAGGA UGAUGAUGACAAAUAAUAA (SEQ ID NO: 17 )SEQ ID NO: 18 - pmNa-APR-l(M74) construct (NA) - AUGAAAUGGGUCACCUUUAUCAGCCUGCUGUUCCUGUUCAGCAGCGCCUACAGCGACUACAA AGACGAUGACGACAAGAAGCUUGCUAGUAAACUUCAGUCCGCUAACGAGAUCGACGAAUUGC UCAGAAAUUACAUGGAUGCUCAGUAUUACGGCGUGAUUCAGAUAGGAACGCCUGCACAGAAU UUUACUGUCAUAUUCGCCACCGGAAGUUCCAAUUUGUGGGUCCCCUCUAGGAAAUGUCCCUU CUAUGAUAUAGCAUGUAUGUUGCAUCACCGCUAUGACAGUGGGGCUUCUUCCACGUAUAAGG AGGACGGUAGGAAAAUGGCAAUUCAAUAUGGUACCGGAUCCAUGAAAGGAUUCAUCAGUAAG GACAUUGUUUGCAUCGCAGGGAUUUGUGCCGAGGAGCAACCUUUCGCGGAAGCCACGUCUGA ACCCGGUCUUACUUUCAUUGCUGCAAAGUUCGAUGGGAUAUUGGGGAUGGCCUUCCCUGAGA UCGCGGUCCUGGGUGUUACGCCGGUAUUUCACACAUUCAUUGAGCAAAAAAAGGUGCCCUCU CCGGUAUUUGCGUUCUGGCCAAAUCGCAACCCAGAGUCUGAAAUCGGAGGUGAAAUUACUUU CGGGGGCGUAGACACCAGGAGGUACGUUGAACCUAUCACUUGGACCCCUGUGACACGGAGAG GCUACUGGCAAUUCAAAAUGGACAUGGUACAAGGUGGAAGUUCCUCAAUCGCAUGCCCAAAC GGUUGCCAAGCUAUCGCGGCUACCGGCACUUCCUUGAUUGCUGGGCCAAAGGCCCAGGUUGA GGCCAUACAAAAGUAUAUUGGUGCUGAGCCACUGAUGAAAGGCGAAUAUAUGAUACCGUGUG ACAAGGUGCCAUCCCUUCCAGAUGUGAGUUUCAUAAUAGACGGGAAAACUUUUACCCUGAAG GGUGAAGACUACGUGCUUACCGUUAAAGCGGCUGGAAAAUCUAUAUGCCUCAGCGGCUUCAU GGGCAUGGACUUCCCUGAAAAAAUUGGAGAGCUUUGGAUACUGGGAGACGUCUUCAUCGGAA AGUAUUACACCGUUUUCGAUGUUGGCCAGGCAAGAGUGGGUUUUGCUCAAGCGAAAUCCGAA GACGGCUUCCCAGUUGGCACCCCAGUCCGAACCUUCCGCCAGCUCCAGGAAGAUUCCGAUUC AGACGAAGAUGAUGUUUUUACAUUUAAGCUUCCUAACAAAGGCAGCGGCACCACCUCUGGCA CCACAAGACUGCUGUCUGGCCACACCUGUUUCACACUGACCGGCCUGCUGGGCACACUGGUU ACAAUGGGACUGCUGACCUAAUAA (SEQ ID NO: 18 )SEQ ID NO: 19 - tsNa-APR-l(M74) construct (NA) - AUGGAUGCAAUGAAGCGGGGGCUCUGCUGUGUGCUGCUGCUGUGUGGAGCAGUCUUCGUUUC GGCCAAGCUUGCUAGUAAACUUCAGUCCGCUAACGAGAUCGACGAAUUGCUCAGAAAUUACA UGGAUGCUCAGUAUUACGGCGUGAUUCAGAUAGGAACGCCUGCACAGAAUUUUACUGUCAUA UUCGCCACCGGAAGUUCCAAUUUGUGGGUCCCCUCUAGGAAAUGUCCCUUCUAUGAUAUAGC AUGUAUGUUGCAUCACCGCUAUGACAGUGGGGCUUCUUCCACGUAUAAGGAGGACGGUAGGA AAAUGGCAAUUCAAUAUGGUACCGGAUCCAUGAAAGGAUUCAUCAGUAAGGACAUUGUUUGC AUCGCAGGGAUUUGUGCCGAGGAGCAACCUUUCGCGGAAGCCACGUCUGAACCCGGUCUUAC UUUCAUUGCUGCAAAGUUCGAUGGGAUAUUGGGGAUGGCCUUCCCUGAGAUCGCGGUCCUGG GUGUUACGCCGGUAUUUCACACAUUCAUUGAGCAAAAAAAGGUGCCCUCUCCGGUAUUUGCG UUCUGGCCAAAUCGCAACCCAGAGUCUGAAAUCGGAGGUGAAAUUACUUUCGGGGGCGUAGA CACCAGGAGGUACGUUGAACCUAUCACUUGGACCCCUGUGACACGGAGAGGCUACUGGCAAU UCAAAAUGGACAUGGUACAAGGUGGAAGUUCCUCAAUCGCAUGCCCAAACGGUUGCCAAGCU AUCGCGGCUACCGGCACUUCCUUGAUUGCUGGGCCAAAGGCCCAGGUUGAGGCCAUACAAAA GUAUAUUGGUGCUGAGCCACUGAUGAAAGGCGAAUAUAUGAUACCGUGUGACAAGGUGCCAU CCCUUCCAGAUGUGAGUUUCAUAAUAGACGGGAAAACUUUUACCCUGAAGGGUGAAGACUAC GUGCUUACCGUUAAAGCGGCUGGAAAAUCUAUAUGCCUCAGCGGCUUCAUGGGCAUGGACUU CCCUGAAAAAAUUGGAGAGCUUUGGAUACUGGGAGACGUCUUCAUCGGAAAGUAUUACACCG300484454.1 - 35 -BAYM. P0444WO / BLG 25-024UUUUCGAUGUUGGCCAGGCAAGAGUGGGUUUUGCUCAAGCGAAAUCCGAAGACGGCUUCCCA GUUGGCACCCCAGUCCGAACCUUCCGCCAGCUCCAGGAAGAUUCCGAUUCAGACGAAGAUGA UGUUUUUACAUUUGACUAUAAGGAUGAUGAUGACAAAUAAUAA SEQ ID NO: 19 ) SEQ ID NO: 20 - tsNa-APR-l(FL) construct (NA) - AUGGAUGCAAUGAAGCGGGGGCUCUGCUGUGUGCUGCUGCUGUGUGGAGCAGUCUUCGUUUC GGCCAAGCUUAGCGUUCAUAGGAGACUGUUCCACCAGGCCCGACGCCAUGUCACCAGUGUCU CCCUGUCCCGGCAACCGACCUUGAGAGAACGCCUUAUAGCGUCUGGGUCCUGGGAAGACUAC CAGAAACAGCGCUACCAUUACCAGAAGAAGAUCCUUGCCAAGUAUGCUGCAAACAAAGCUAG UAAACUUCAGUCCGCUAACGAGAUCGACGAAUUGCUCAGAAAUUACAUGGAUGCUCAGUAUU ACGGCGUGAUUCAGAUAGGAACGCCUGCACAGAAUUUUACUGUCAUAUUCGCCACCGGAAGU UCCAAUUUGUGGGUCCCCUCUAGGAAAUGUCCCUUCUAUGAUAUAGCAUGUAUGUUGCAUCA CCGCUAUGACAGUGGGGCUUCUUCCACGUAUAAGGAGGACGGUAGGAAAAUGGCAAUUCAAU AUGGUACCGGAUCCAUGAAAGGAUUCAUCAGUAAGGACAUUGUUUGCAUCGCAGGGAUUUGU GCCGAGGAGCAACCUUUCGCGGAAGCCACGUCUGAACCCGGUCUUACUUUCAUUGCUGCAAA GUUCGAUGGGAUAUUGGGGAUGGCCUUCCCUGAGAUCGCGGUCCUGGGUGUUACGCCGGUAU UUCACACAUUCAUUGAGCAAAAAAAGGUGCCCUCUCCGGUAUUUGCGUUCUGGCCAAAUCGC AACCCAGAGUCUGAAAUCGGAGGUGAAAUUACUUUCGGGGGCGUAGACACCAGGAGGUACGU UGAACCUAUCACUUGGACCCCUGUGACACGGAGAGGCUACUGGCAAUUCAAAAUGGACAUGG UACAAGGUGGAAGUUCCUCAAUCGCAUGCCCAAACGGUUGCCAAGCUAUCGCGGCUACCGGC ACUUCCUUGAUUGCUGGGCCAAAGGCCCAGGUUGAGGCCAUACAAAAGUAUAUUGGUGCUGA GCCACUGAUGAAAGGCGAAUAUAUGAUACCGUGUGACAAGGUGCCAUCCCUUCCAGAUGUGA GUUUCAUAAUAGACGGGAAAACUUUUACCCUGAAGGGUGAAGACUACGUGCUUACCGUUAAA GCGGCUGGAAAAUCUAUAUGCCUCAGCGGCUUCAUGGGCAUGGACUUCCCUGAAAAAAUUGG AGAGCUUUGGAUACUGGGAGACGUCUUCAUCGGAAAGUAUUACACCGUUUUCGAUGUUGGCC AGGCAAGAGUGGGUUUUGCUCAAGCGAAAUCCGAAGACGGCUUCCCAGUUGGCACCCCAGUC CGAACCUUCCGCCAGCUCCAGGAAGAUUCCGAUUCAGACGAAGAUGAUGUUUUUACAUUUGA CUAUAAGGAUGAUGAUGACAAAUAAUAA (SEQ ID NO: 20 )SEQ ID NO: 21 - sNa-APR-l(M74) protein (NA) - GCUAGUAAACUUCAGUCCGCUAACGAGAUCGACGAAUUGCUCAGAAAUUACAUGGAUGCUCA GUAUUACGGCGUGAUUCAGAUAGGAACGCCUGCACAGAAUUUUACUGUCAUAUUCGCCACCG GAAGUUCCAAUUUGUGGGUCCCCUCUAGGAAAUGUCCCUUCUAUGAUAUAGCAUGUAUGUUG CAUCACCGCUAUGACAGUGGGGCUUCUUCCACGUAUAAGGAGGACGGUAGGAAAAUGGCAAU UCAAUAUGGUACCGGAUCCAUGAAAGGAUUCAUCAGUAAGGACAUUGUUUGCAUCGCAGGGA UUUGUGCCGAGGAGCAACCUUUCGCGGAAGCCACGUCUGAACCCGGUCUUACUUUCAUUGCU GCAAAGUUCGAUGGGAUAUUGGGGAUGGCCUUCCCUGAGAUCGCGGUCCUGGGUGUUACGCC GGUAUUUCACACAUUCAUUGAGCAAAAAAAGGUGCCCUCUCCGGUAUUUGCGUUCUGGCCAA AUCGCAACCCAGAGUCUGAAAUCGGAGGUGAAAUUACUUUCGGGGGCGUAGACACCAGGAGG UACGUUGAACCUAUCACUUGGACCCCUGUGACACGGAGAGGCUACUGGCAAUUCAAAAUGGA CAUGGUACAAGGUGGAAGUUCCUCAAUCGCAUGCCCAAACGGUUGCCAAGCUAUCGCGGCUA CCGGCACUUCCUUGAUUGCUGGGCCAAAGGCCCAGGUUGAGGCCAUACAAAAGUAUAUUGGU GCUGAGCCACUGAUGAAAGGCGAAUAUAUGAUACCGUGUGACAAGGUGCCAUCCCUUCCAGA UGUGAGUUUCAUAAUAGACGGGAAAACUUUUACCCUGAAGGGUGAAGACUACGUGCUUACCG UUAAAGCGGCUGGAAAAUCUAUAUGCCUCAGCGGCUUCAUGGGCAUGGACUUCCCUGAAAAA AUUGGAGAGCUUUGGAUACUGGGAGACGUCUUCAUCGGAAAGUAUUACACCGUUUUCGAUGU UGGCCAGGCAAGAGUGGGUUUUGCUCAAGCGAAAUCCGAAGACGGCUUCCCAGUUGGCACCC CAGUCCGAACCUUCCGCCAGCUCCAGGAAGAUUCCGAUUCAGACGAAGAUGAUGUUUUUACA UUU (SEQ ID NO: 21 )300484454.1 - 36 -BAYM. P0444WO / BLG 25-024 SEQ ID NO: 22 - sNa-APR-l(FL) protein (NA) - AGCGUUCAUAGGAGACUGUUCCACCAGGCCCGACGCCAUGUCACCAGUGUCUCCCUGUCCCG GCAACCGACCUUGAGAGAACGCCUUAUAGCGUCUGGGUCCUGGGAAGACUACCAGAAACAGC GCUACCAUUACCAGAAGAAGAUCCUUGCCAAGUAUGCUGCAAACAAAGCUAGUAAACUUCAG UCCGCUAACGAGAUCGACGAAUUGCUCAGAAAUUACAUGGAUGCUCAGUAUUACGGCGUGAU UCAGAUAGGAACGCCUGCACAGAAUUUUACUGUCAUAUUCGCCACCGGAAGUUCCAAUUUGU GGGUCCCCUCUAGGAAAUGUCCCUUCUAUGAUAUAGCAUGUAUGUUGCAUCACCGCUAUGAC AGUGGGGCUUCUUCCACGUAUAAGGAGGACGGUAGGAAAAUGGCAAUUCAAUAUGGUACCGG AUCCAUGAAAGGAUUCAUCAGUAAGGACAUUGUUUGCAUCGCAGGGAUUUGUGCCGAGGAGC AACCUUUCGCGGAAGCCACGUCUGAACCCGGUCUUACUUUCAUUGCUGCAAAGUUCGAUGGG AUAUUGGGGAUGGCCUUCCCUGAGAUCGCGGUCCUGGGUGUUACGCCGGUAUUUCACACAUU CAUUGAGCAAAAAAAGGUGCCCUCUCCGGUAUUUGCGUUCUGGCCAAAUCGCAACCCAGAGU CUGAAAUCGGAGGUGAAAUUACUUUCGGGGGCGUAGACACCAGGAGGUACGUUGAACCUAUC ACUUGGACCCCUGUGACACGGAGAGGCUACUGGCAAUUCAAAAUGGACAUGGUACAAGGUGG AAGUUCCUCAAUCGCAUGCCCAAACGGUUGCCAAGCUAUCGCGGCUACCGGCACUUCCUUGA UUGCUGGGCCAAAGGCCCAGGUUGAGGCCAUACAAAAGUAUAUUGGUGCUGAGCCACUGAUG AAAGGCGAAUAUAUGAUACCGUGUGACAAGGUGCCAUCCCUUCCAGAUGUGAGUUUCAUAAU AGACGGGAAAACUUUUACCCUGAAGGGUGAAGACUACGUGCUUACCGUUAAAGCGGCUGGAA AAUCUAUAUGCCUCAGCGGCUUCAUGGGCAUGGACUUCCCUGAAAAAAUUGGAGAGCUUUGG AUACUGGGAGACGUCUUCAUCGGAAAGUAUUACACCGUUUUCGAUGUUGGCCAGGCAAGAGU GGGUUUUGCUCAAGCGAAAUCCGAAGACGGCUUCCCAGUUGGCACCCCAGUCCGAACCUUCC GCCAGCUCCAGGAAGAUUCCGAUUCAGACGAAGAUGAUGUUUUUACAUUU (SEQ ID NO: 22 )SEQ ID NO: 23 - pmNa-APR-l(M74) protein (NA) - GCUAGUAAACUUCAGUCCGCUAACGAGAUCGACGAAUUGCUCAGAAAUUACAUGGAUGCUCA GUAUUACGGCGUGAUUCAGAUAGGAACGCCUGCACAGAAUUUUACUGUCAUAUUCGCCACCG GAAGUUCCAAUUUGUGGGUCCCCUCUAGGAAAUGUCCCUUCUAUGAUAUAGCAUGUAUGUUG CAUCACCGCUAUGACAGUGGGGCUUCUUCCACGUAUAAGGAGGACGGUAGGAAAAUGGCAAU UCAAUAUGGUACCGGAUCCAUGAAAGGAUUCAUCAGUAAGGACAUUGUUUGCAUCGCAGGGA UUUGUGCCGAGGAGCAACCUUUCGCGGAAGCCACGUCUGAACCCGGUCUUACUUUCAUUGCU GCAAAGUUCGAUGGGAUAUUGGGGAUGGCCUUCCCUGAGAUCGCGGUCCUGGGUGUUACGCC GGUAUUUCACACAUUCAUUGAGCAAAAAAAGGUGCCCUCUCCGGUAUUUGCGUUCUGGCCAA AUCGCAACCCAGAGUCUGAAAUCGGAGGUGAAAUUACUUUCGGGGGCGUAGACACCAGGAGG UACGUUGAACCUAUCACUUGGACCCCUGUGACACGGAGAGGCUACUGGCAAUUCAAAAUGGA CAUGGUACAAGGUGGAAGUUCCUCAAUCGCAUGCCCAAACGGUUGCCAAGCUAUCGCGGCUA CCGGCACUUCCUUGAUUGCUGGGCCAAAGGCCCAGGUUGAGGCCAUACAAAAGUAUAUUGGU GCUGAGCCACUGAUGAAAGGCGAAUAUAUGAUACCGUGUGACAAGGUGCCAUCCCUUCCAGA UGUGAGUUUCAUAAUAGACGGGAAAACUUUUACCCUGAAGGGUGAAGACUACGUGCUUACCG UUAAAGCGGCUGGAAAAUCUAUAUGCCUCAGCGGCUUCAUGGGCAUGGACUUCCCUGAAAAA AUUGGAGAGCUUUGGAUACUGGGAGACGUCUUCAUCGGAAAGUAUUACACCGUUUUCGAUGU UGGCCAGGCAAGAGUGGGUUUUGCUCAAGCGAAAUCCGAAGACGGCUUCCCAGUUGGCACCC CAGUCCGAACCUUCCGCCAGCUCCAGGAAGAUUCCGAUUCAGACGAAGAUGAUGUUUUUACA UUU (SEQ ID NO: 23 )SEQ ID NO: 24 - tsNa-APR-l(M74) protein (NA) - GCUAGUAAACUUCAGUCCGCUAACGAGAUCGACGAAUUGCUCAGAAAUUACAUGGAUGCUCA GUAUUACGGCGUGAUUCAGAUAGGAACGCCUGCACAGAAUUUUACUGUCAUAUUCGCCACCG GAAGUUCCAAUUUGUGGGUCCCCUCUAGGAAAUGUCCCUUCUAUGAUAUAGCAUGUAUGUUG CAUCACCGCUAUGACAGUGGGGCUUCUUCCACGUAUAAGGAGGACGGUAGGAAAAUGGCAAU300484454.1 - 37 -BAYM. P0444WO / BLG 25-024UCAAUAUGGUACCGGAUCCAUGAAAGGAUUCAUCAGUAAGGACAUUGUUUGCAUCGCAGGGA UUUGUGCCGAGGAGCAACCUUUCGCGGAAGCCACGUCUGAACCCGGUCUUACUUUCAUUGCU GCAAAGUUCGAUGGGAUAUUGGGGAUGGCCUUCCCUGAGAUCGCGGUCCUGGGUGUUACGCC GGUAUUUCACACAUUCAUUGAGCAAAAAAAGGUGCCCUCUCCGGUAUUUGCGUUCUGGCCAA AUCGCAACCCAGAGUCUGAAAUCGGAGGUGAAAUUACUUUCGGGGGCGUAGACACCAGGAGG UACGUUGAACCUAUCACUUGGACCCCUGUGACACGGAGAGGCUACUGGCAAUUCAAAAUGGA CAUGGUACAAGGUGGAAGUUCCUCAAUCGCAUGCCCAAACGGUUGCCAAGCUAUCGCGGCUA CCGGCACUUCCUUGAUUGCUGGGCCAAAGGCCCAGGUUGAGGCCAUACAAAAGUAUAUUGGU GCUGAGCCACUGAUGAAAGGCGAAUAUAUGAUACCGUGUGACAAGGUGCCAUCCCUUCCAGA UGUGAGUUUCAUAAUAGACGGGAAAACUUUUACCCUGAAGGGUGAAGACUACGUGCUUACCG UUAAAGCGGCUGGAAAAUCUAUAUGCCUCAGCGGCUUCAUGGGCAUGGACUUCCCUGAAAAA AUUGGAGAGCUUUGGAUACUGGGAGACGUCUUCAUCGGAAAGUAUUACACCGUUUUCGAUGU UGGCCAGGCAAGAGUGGGUUUUGCUCAAGCGAAAUCCGAAGACGGCUUCCCAGUUGGCACCC CAGUCCGAACCUUCCGCCAGCUCCAGGAAGAUUCCGAUUCAGACGAAGAUGAUGUUUUUACA UUU (SEQ ID NO: 24 )SEQ ID NO: 25 - tsNa-APR-l(FL) protein (NA) - AGCGUUCAUAGGAGACUGUUCCACCAGGCCCGACGCCAUGUCACCAGUGUCUCCCUGUCCCG GCAACCGACCUUGAGAGAACGCCUUAUAGCGUCUGGGUCCUGGGAAGACUACCAGAAACAGC GCUACCAUUACCAGAAGAAGAUCCUUGCCAAGUAUGCUGCAAACAAAGCUAGUAAACUUCAG UCCGCUAACGAGAUCGACGAAUUGCUCAGAAAUUACAUGGAUGCUCAGUAUUACGGCGUGAU UCAGAUAGGAACGCCUGCACAGAAUUUUACUGUCAUAUUCGCCACCGGAAGUUCCAAUUUGU GGGUCCCCUCUAGGAAAUGUCCCUUCUAUGAUAUAGCAUGUAUGUUGCAUCACCGCUAUGAC AGUGGGGCUUCUUCCACGUAUAAGGAGGACGGUAGGAAAAUGGCAAUUCAAUAUGGUACCGG AUCCAUGAAAGGAUUCAUCAGUAAGGACAUUGUUUGCAUCGCAGGGAUUUGUGCCGAGGAGC AACCUUUCGCGGAAGCCACGUCUGAACCCGGUCUUACUUUCAUUGCUGCAAAGUUCGAUGGG AUAUUGGGGAUGGCCUUCCCUGAGAUCGCGGUCCUGGGUGUUACGCCGGUAUUUCACACAUU CAUUGAGCAAAAAAAGGUGCCCUCUCCGGUAUUUGCGUUCUGGCCAAAUCGCAACCCAGAGU CUGAAAUCGGAGGUGAAAUUACUUUCGGGGGCGUAGACACCAGGAGGUACGUUGAACCUAUC ACUUGGACCCCUGUGACACGGAGAGGCUACUGGCAAUUCAAAAUGGACAUGGUACAAGGUGG AAGUUCCUCAAUCGCAUGCCCAAACGGUUGCCAAGCUAUCGCGGCUACCGGCACUUCCUUGA UUGCUGGGCCAAAGGCCCAGGUUGAGGCCAUACAAAAGUAUAUUGGUGCUGAGCCACUGAUG AAAGGCGAAUAUAUGAUACCGUGUGACAAGGUGCCAUCCCUUCCAGAUGUGAGUUUCAUAAU AGACGGGAAAACUUUUACCCUGAAGGGUGAAGACUACGUGCUUACCGUUAAAGCGGCUGGAA AAUCUAUAUGCCUCAGCGGCUUCAUGGGCAUGGACUUCCCUGAAAAAAUUGGAGAGCUUUGG AUACUGGGAGACGUCUUCAUCGGAAAGUAUUACACCGUUUUCGAUGUUGGCCAGGCAAGAGU GGGUUUUGCUCAAGCGAAAUCCGAAGACGGCUUCCCAGUUGGCACCCCAGUCCGAACCUUCC GCCAGCUCCAGGAAGAUUCCGAUUCAGACGAAGAUGAUGUUUUUACAUUU (SEQ ID NO: 25 )

[0198] In some aspects, a polynucleotide of the disclosure encodes for a / -APR.-I polypeptide. In some aspects, a 7Va-GST-l polypeptide comprises, consists essentially of, or consists of a sequence that is, or is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to one or more of SEQ ID NOs: 26-40.300484454.1 - 38 -BAYM. P0444WO / BLG 25-024 SEQ ID NO: 26 - sNa-APR-l(M74) protein only (AA) - ASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQNFTVIFATGSSNLWVPSRKCPFYDIACML HHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISKDIVCIAGICAEEQPFAEATSEPGLTFIA AKFDGILGMAFPEIAVLGVTPVFHTFIEQKKVPSPVFAFWPNRNPESEIGGEITFGGVDTRR YVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPNGCQAI AATGTS L I AGPKAQVEAI QKY I G AEPLMKGEYMIPCDKVPSLPDVSFIIDGKTFTLKGEDYVLTVKAAGKSICLSGFMGMDFPEK IGELWILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFPVGTPVRTFRQLQEDSDSDEDDVFTF (SEQ ID NO: 26)SEQ ID NO: 27 - sNa-APR-l(M74) with signal peptide (AA) - MGWSCI ILFLVATATGVHSKLASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQNFTVIFAT GSSNLWVPSRKCPFYDIACMLHHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISKDIVCIAG I CAEEQPFAEATS E PGLTF I AAKFDG I LGMAFPE I AVLGVTPVFHTF I EQKKVPS PVFAFWP NRNPES E I GGE I TFGGVDTRRYVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPNGCQAI AA TGTSLIAGPKAQVEAIQKYIGAEPLMKGEYMIPCDKVPSLPDVSFI IDGKTFTLKGEDYVLT VKAAGKSICLSGFMGMDFPEKIGELWILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFPVGT PVRTFRQLQEDSDSDEDDVFTF (SEQ ID NO: 27)SEQ ID NO: 28 - sNa-APR-l(M74) with signal peptide and FLAG tag (AA) - MGWSCI ILFLVATATGVHSKLASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQNFTVIFAT GSSNLWVPSRKCPFYDIACMLHHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISKDIVCIAG I CAEEQPFAEATS E PGLTF I AAKFDG I LGMAFPE I AVLGVTPVFHTF I EQKKVPS PVFAFWP NRNPES E I GGE I TFGGVDTRRYVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPNGCQAI AA TGTSLIAGPKAQVEAIQKYIGAEPLMKGEYMIPCDKVPSLPDVSFI IDGKTFTLKGEDYVLT VKAAGKSICLSGFMGMDFPEKIGELWILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFPVGT PVRTFRQLQEDSDSDEDDVFTFDYKDDDDK (SEQ ID NO: 28)SEQ ID NO: 29 - sNa-APR-l(FL) protein only (AA) - SVHRRLFHQARRHVTSVSLSRQPTLRERLIASGSWEDYQKQRYHYQKKILAKYAANKASKLQ SANEIDELLRNYMDAQYYGVIQIGTPAQNFTVIFATGSSNLWVPSRKCPFYDIACMLHHRYD SGASSTYKEDGRKMAIQYGTGSMKGFISKDIVCIAGICAEEQPFAEATSEPGLTFIAAKFDG I LGMAFPE I AVLGVTPVFHTF I EQKKVPS PVFAFWPNRNPES E I GGE I TFGGVDTRRYVE P I TWTPVTRRGYWQFKMDMVQGGSSSIACPNGCQAIAATGTSLIAGPKAQVEAIQKYIGAEPLM KGEYMIPCDKVPSLPDVSFIIDGKTFTLKGEDYVLTVKAAGKSICLSGFMGMDFPEKIGELW ILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFPVGTPVRTFRQLQEDSDSDEDDVFTF(SEQ ID NO: 29)SEQ ID NO: 30 - sNa-APR-l(FL) with signal peptide (AA) - MGWSCI ILFLVATATGVHSKLSVHRRLFHQARRHVTSVSLSRQPTLRERLIASGSWEDYQKQ RYHYQKKILAKYAANKASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQNFTVIFATGSSNL WVPSRKCPFYDIACMLHHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISKDIVCIAGICAEE QPFAEATS E PGLTF I AAKFDG I LGMAFPE I AVLGVTPVFHTF I EQKKVPS PVFAFWPNRNPE SEI GGE I TFGGVDTRRYVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPNGCQAI AATGTS L IAGPKAQVEAIQKYIGAEPLMKGEYMIPCDKVPSLPDVSFI IDGKTFTLKGEDYVLTVKAAG KSICLSGFMGMDFPEKIGELWILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFPVGTPVRTF RQLQEDSDSDEDDVFTF (SEQ ID NO: 30)300484454.1 - 39 -BAYM. P0444WO / BLG 25-024 SEQ ID NO: 31 - sNa-APR-l(FL) with signal peptide and FLAG tag (AA) - MGWSCI ILFLVATATGVHSKLSVHRRLFHQARRHVTSVSLSRQPTLRERLIASGSWEDYQKQ RYHYQKKILAKYAANKASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQNFTVIFATGSSNL WVPSRKCPFYDIACMLHHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISKDIVCIAGICAEE QPFAEATS E PGLTF I AAKFDG I LGMAFPE I AVLGVTPVFHTF I EQKKVPS PVFAFWPNRNPE SEI GGE I TFGGVDTRRYVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPNGCQAI AATGTS L IAGPKAQVEAIQKYIGAEPLMKGEYMIPCDKVPSLPDVSFI IDGKTFTLKGEDYVLTVKAAG KSICLSGFMGMDFPEKIGELWILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFPVGTPVRTF RQLQEDSDSDEDDVFTFDYKDDDDK (SEQ ID NO: 31)SEQ ID NO: 32 - pmNa-APR-l(M74) with anchor (AA) - ASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQNFTVIFATGSSNLWVPSRKCPFYDIACML HHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISKDIVCIAGICAEEQPFAEATSEPGLTFIA AKFDGILGMAFPEIAVLGVTPVFHTFIEQKKVPSPVFAFWPNRNPESEIGGEITFGGVDTRR YVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPNGCQAI AATGTS L I AGPKAQVEAI QKY I G AEPLMKGEYMIPCDKVPSLPDVSFIIDGKTFTLKGEDYVLTVKAAGKSICLSGFMGMDFPEK IGELWILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFPVGTPVRTFRQLQEDSDSDEDDVFT FKLPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 32) SEQ ID NO: 33 - pmNa-APR-l(M74) with signal peptide and anchor (AA) - MKWVTFISLLFLFSSAYSKLASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQNFTVIFATG SSNLWVPSRKCPFYDIACMLHHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISKDIVCIAGI CAEEQPFAEATS E PGLTF I AAKFDG I LGMAFPE I AVLGVTPVFHTF I EQKKVPS PVFAFWPN RNPES E I GGE I TFGGVDTRRYVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPNGCQAI AAT GTSLIAGPKAQVEAIQKYIGAEPLMKGEYMIPCDKVPSLPDVSFI IDGKTFTLKGEDYVLTV KAAGKSICLSGFMGMDFPEKIGELWILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFPVGTP VRTFRQLQEDSDSDEDDVFTFKLPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (S EQ ID NO: 33)SEQ ID NO: 34 - pmNa-APR-l(M74) with signal peptide, FLAG tag, and anchor (AA) - MKWVTFISLLFLFSSAYSDYKDDDDKKLASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQN FTVIFATGSSNLWVPSRKCPFYDIACMLHHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISK DIVCIAGICAEEQPFAEATSEPGLTFIAAKFDGILGMAFPEIAVLGVTPVFHTFIEQKKVPS PVFAFWPNRNPES E I GGE I TFGGVDTRRYVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPN GCQAIAATGTSLIAGPKAQVEAIQKYIGAEPLMKGEYMIPCDKVPSLPDVSFI IDGKTFTLK GEDYVLTVKAAGKSICLSGFMGMDFPEKIGELWILGDVFIGKYYTVFDVGQARVGFAQAKSE DGFPVGTPVRTFRQLQEDSDSDEDDVFTFKLPNKGSGTTSGTTRLLSGHTCFTLTGLLGTLV TMGLLT (SEQ ID NO: 34)SEQ ID NO: 35 - tsNa-APR-l(M74) protein only (AA) - ASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQNFTVIFATGSSNLWVPSRKCPFYDIACML HHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISKDIVCIAGICAEEQPFAEATSEPGLTFIA AKFDGILGMAFPEIAVLGVTPVFHTFIEQKKVPSPVFAFWPNRNPESEIGGEITFGGVDTRR YVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPNGCQAI AATGTS L I AGPKAQVEAI QKY I G AEPLMKGEYMIPCDKVPSLPDVSFIIDGKTFTLKGEDYVLTVKAAGKSICLSGFMGMDFPEK IGELWILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFPVGTPVRTFRQLQEDSDSDEDDVFTF (SEQ ID NO: 35)300484454.1 - 40 -BAYM. P0444WO / BLG 25-024 SEQ ID NO: 36 - tsNa-APR-l(M74) with signal peptide (AA) - MDAMKRGLCCVLLLCGAVFVSAKLASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQNFTVI FATGSSNLWVPSRKCPFYDIACMLHHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISKDIVC I AG I CAEEQPFAEATS E PGLTF I AAKFDG I LGMAFPE I AVLGVTPVFHTF I EQKKVPS PVFA FWPNRNPES E I GGE I TFGGVDTRRYVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPNGCQA IAATGTSLIAGPKAQVEAIQKYIGAEPLMKGEYMIPCDKVPSLPDVSFI IDGKTFTLKGEDY VLTVKAAGKSICLSGFMGMDFPEKIGELWILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFP VGTPVRTFRQLQEDSDSDEDDVFTF (SEQ ID NO: 36)SEQ ID NO: 37 - tsNa-APR-l(M74) with signal peptide and FLAG tag (AA) - MDAMKRGLCCVLLLCGAVFVSAKLASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQNFTVI FATGSSNLWVPSRKCPFYDIACMLHHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISKDIVC I AG I CAEEQPFAEATS E PGLTF I AAKFDG I LGMAFPE I AVLGVTPVFHTF I EQKKVPS PVFA FWPNRNPES E I GGE I TFGGVDTRRYVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPNGCQA IAATGTSLIAGPKAQVEAIQKYIGAEPLMKGEYMIPCDKVPSLPDVSFI IDGKTFTLKGEDY VLTVKAAGKSICLSGFMGMDFPEKIGELWILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFP VGTPVRTFRQLQEDSDSDEDDVFTFDYKDDDDK (SEQ ID NO: 37)SEQ ID NO: 38 - tsNa-APR-l(FL) protein only (AA) - SVHRRLFHQARRHVTSVSLSRQPTLRERLIASGSWEDYQKQRYHYQKKILAKYAANKASKLQ SANEIDELLRNYMDAQYYGVIQIGTPAQNFTVIFATGSSNLWVPSRKCPFYDIACMLHHRYD SGASSTYKEDGRKMAIQYGTGSMKGFISKDIVCIAGICAEEQPFAEATSEPGLTFIAAKFDG I LGMAFPE I AVLGVTPVFHTF I EQKKVPS PVFAFWPNRNPES E I GGE I TFGGVDTRRYVE P I TWTPVTRRGYWQFKMDMVQGGSSSIACPNGCQAIAATGTSLIAGPKAQVEAIQKYIGAEPLM KGEYMIPCDKVPSLPDVSFIIDGKTFTLKGEDYVLTVKAAGKSICLSGFMGMDFPEKIGELW ILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFPVGTPVRTFRQLQEDSDSDEDDVFTF(SEQ ID NO: 38)SEQ ID NO: 39 - tsNa-APR-l(FL) with signal peptide (AA) - MDAMKRGLCCVLLLCGAVFVSAKLSVHRRLFHQARRHVTSVSLSRQPTLRERLIASGSWEDY QKQRYHYQKKILAKYAANKASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQNFTVIFATGS SNLWVPSRKCPFYDIACMLHHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISKDIVCIAGIC AEEQPFAEATS E PGLTF I AAKFDG I LGMAFPE I AVLGVTPVFHTF I EQKKVPS PVFAFWPNR NPES E I GGE I TFGGVDTRRYVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPNGCQAI AATG TSLIAGPKAQVEAIQKYIGAEPLMKGEYMIPCDKVPSLPDVSFI IDGKTFTLKGEDYVLTVK AAGKSICLSGFMGMDFPEKIGELWILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFPVGTPV RTFRQLQEDSDSDEDDVFTF (SEQ ID NO: 39)SEQ ID NO: 40 - tsNa-APR-l(FL) with signal peptide and FLAG tag (AA) - MDAMKRGLCCVLLLCGAVFVSAKLSVHRRLFHQARRHVTSVSLSRQPTLRERLIASGSWEDY QKQRYHYQKKILAKYAANKASKLQSANEIDELLRNYMDAQYYGVIQIGTPAQNFTVIFATGS SNLWVPSRKCPFYDIACMLHHRYDSGASSTYKEDGRKMAIQYGTGSMKGFISKDIVCIAGIC AEEQPFAEATS E PGLTF I AAKFDG I LGMAFPE I AVLGVTPVFHTF I EQKKVPS PVFAFWPNR NPES E I GGE I TFGGVDTRRYVE P I TWTPVTRRGYWQFKMDMVQGGS S S I ACPNGCQAI AATG TSLIAGPKAQVEAIQKYIGAEPLMKGEYMIPCDKVPSLPDVSFI IDGKTFTLKGEDYVLTVK AAGKSICLSGFMGMDFPEKIGELWILGDVFIGKYYTVFDVGQARVGFAQAKSEDGFPVGTPV RTFRQLQEDSDSDEDDVFTFDYKDDDDK (SEQ ID NO: 40)300484454.1 - 41 -BAYM. P0444WO / BLG 25-0242. 5 '-CAP

[0199] A 5' cap is typically a modified nucleotide, particularly a guanine nucleotide, added to the 5' end of an RNA molecule. A 5' cap structure of a natural mRNA is involved in nuclear export, increasing mRNA stability and binding the mRNA Cap Binding Protein (CBP), which is responsible for mRNA stability in the cell and translation competency through the association of CBP with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns during mRNA splicing.

[0200] Endogenous mRNA molecules may be 5 '-end capped generating a 5 '-ppp-5 '-triphosphate linkage between a terminal guanosine cap residue and the 5 '-terminal transcribed sense nucleotide of the mRNA molecule. This 5 '-guanylate cap may then be methylated to generate anN7-methyl-guanylate residue. The ribose sugars of the terminal and / or anteterminal transcribed nucleotides of the 5' end of the mRNA may optionally also be 2'-O-methylated. 5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA molecule, for degradation.

[0201] In some aspects, polynucleotides (e.g., antigen-encoding polynucleotides featured in the Polynucleotides of the disclosure) may be designed to incorporate a cap moiety. Modifications to the polynucleotides of the present disclosure may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5 '-ppp-5' phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, Mass.) may be used with a-thio-guanosine nucleotides according to the manufacturer’s instructions to create a phosphorothioate linkage in the 5 '-ppp-5' cap. Another example includes, a CleanCap® Reagent M6 (Trilink BioTechnologies, San Diego, CA) that may be used for co-transcri phonal capping with m6AG (3'0Me) by following the manufacturer’s instructions. Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.

[0202] Additional modifications include, but are not limited to, 2'-O-methylation of the ribose sugars of 5 '-terminal and / or 5 '-anteterminal nucleotides of the polynucleotide (as mentioned above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5 '-cap structures can be used to generate the 5 '-cap of a nucleic acid molecule, such as a polynucleotide which functions as an mRNA molecule.

[0203] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5 '-caps in their chemical structure, while retaining cap 300484454.1 - 42 -BAYM. P0444WO / BLG 25-024function. Cap analogs may be chemically (i.e. non-enzymatically) or enzymatically synthesized and / or linked to the polynucleotides of the disclosure. For example, the AntiReverse Cap Analog (ARC A) cap contains two guanines linked by a 5 '-5 '-triphosphate group, wherein one guanine contains an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5 '-triphosphate-5 '-guanosine (m7G-3'mppp-G; which may equivalently be designated 3' O-Me-m7G(5')ppp(5')G). The 3'-0 atom of the other, unmodified, guanine becomes linked to the 5 '-terminal nucleotide of the capped polynucleotide. The N7- and 3'-0 methylated guanine provides the terminal moiety of the capped polynucleotide. Another illustrative cap is mCAP, which is similar to ARCA but has a 2' O-methyl group on guanosine (i.e., N7,2'-O-dimethyl-guanosine-5 '-triphosphate-5 '-guanosine, m7Gm-ppp-G).

[0204] Polynucleotides disclosed herein may also be capped post-manufacture (whether IVT or chemical synthesis), using enzymes. For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0 methyltransferase enzyme can create a canonical 5 '-5'-triphosphate linkage between the 5 '-terminal nucleotide of a polynucleotide and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5 '-terminal nucleotide of the mRNA contains a 2'-O-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5 'cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), and 7mG(5')-ppp(5')NlmpN2mp (cap 2).

[0205] As a non-limiting example, capping chimeric polynucleotides post-manufacture may be more efficient as nearly 100% of the chimeric polynucleotides may be capped. This is in contrast to -80% when a cap analog is linked to a chimeric polynucleotide in the course of an in vitro transcription reaction.

[0206] In some aspects, 5' terminal caps may include endogenous caps or cap analogs, such as, for example, a guanine analog. Useful guanine analogs include, but are not limited to, inosine, Nl-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

[0207] A 5' cap may include a naturally occurring cap, a synthetic cap or an optimized cap. Non-limiting examples of optimized caps include the caps taught by Rhoads in U. S. Pat. No.7,074,596 and International Patent Publication No. WO2008157668, WO2009149253 and WO2013103659, the contents of each of which are herein incorporated by reference in their entirety. In some aspects, a 5' cap is added using a 5 '-5 '-triphosphate linkage. In some aspects, 300484454.1 - 43 -BAYM. P0444WO / BLG 25-024a 5' cap may be methylated, for example, but not limited to, m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5' cap, typically the 5 '-end of an RNA. A naturally occurring 5' cap is m7GpppN.

[0208] Further examples of 5' cap structures include, but are not limited to, glyceryl, inverted deoxy abasic residue (moiety), 4', 5' methylene nucleotide, l-(beta-D-erythrofuranosyl) nucleotide, 4'-thio nucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides, alpha-nucleotide, modified base nucleotide, threo-pentofuranosyl nucleotide, acyclic 3',4'-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3 '-3 '-inverted nucleotide moiety, 3 '-3 '-inverted abasic moiety, 3'-2'-inverted nucleotide moiety, 3'-2'-inverted abasic moiety, 1,4-butanediol phosphate, 3'-phosphoramidate, hexylphosphate, aminohexyl phosphate, 3 '-phosphate, 3'phosphorothioate, phosphorodithioate, or bridging or non-bridging methylphosphonate moiety.

[0209] In certain aspects, 5' cap structures are CAP1 (methylation of the ribose of the adjacent nucleotide of m7G), CAP2 (methylation of the ribose of the 2ndnucleotide downstream of the m7G), CAP3 (methylation of the ribose of the 3rdnucleotide downstream of the m7G) and CAP4 (methylation of the ribose of the 4thnucleotide downstream of the m7G).

[0210] In certain aspects, a 5' cap structure may be formed by a cap analog. A 5' cap analog refers to a non-extendable di-nucleotide that has cap functionality which means that it facilitates translation or localization, and / or prevents degradation of the RNA molecule when incorporated at the 5' end of the RNA molecule. Non-extendable means that the cap analog will be incorporated only at the 5' terminus because it does not have a 5' triphosphate and therefore cannot be extended in the 3' direction by a template dependent RNA polymerase.

[0211] 5' cap analogs include, but are not limited to, m7GpppG, m7GpppA, m7GpppC; unmethylated cap analogs (for example, but not limited to, GpppG); dimethylated cap analog (for example, but not limited to, m2,7GpppG), trimethylated cap analog (for example, but not limited to, m2,2,7GpppG), demethylated symmetrical cap analogs (for example, but not limited to, m7Gpppm7G), or anti-reverse cap analogs (for example, but not limited to, ARCA; m7,2'OmeGpppG, m7,2'dGpppG, m7,3'OmeGpppG, m7,3'dGpppG and their tetraphosphate derivatives) (Stepinski et al., 2001. RNA 7(10): 1486-95).

[0212] Further 5' cap analogs have been described previously in U. S. Pat. No. 7,074,596, WO 2008 / 016473, WO 2008 / 157688, WO 2009 / 149253, WO 2011 / 015347, WO 2013 / 059475, and US 2019 / 0010485 Al (all of which are incorporated herein by reference in their entirety for the purposes described herein). The synthesis of N7-(4-chlorophenoxy ethyl) 300484454.1 - 44 -BAYM. P0444WO / BLG 25-024substituted dinucleotide cap analogs has been described in Kore et al., 2013 (see e.g., Bioorg. Med. Chem. 21(15):4570-4), the contents of which are incorporated herein by reference in their entirety for the purposes described herein.

[0213] In certain aspects, 5' cap analogs comprise G[5']ppp[5']G, m7G[5']ppp[5']G, m32’2’7G[5']ppp[5']G, m27’3'oG[5']ppp[5']G (3'-ARCA), m27’2'-°GpppG (2'-ARCA), m27’2'-°GppspGDl (P-S-ARCA DI), m27’2'°GppspG D2 (P-S-ARCA D2), and / or any combinations thereof.

[0214] In certain aspects, a 5' cap analog is added with an initial concentration in the range of about 1 to 20 mM, 1 to 17.5 mM, 1 to 15 mM, 1 to 12.5 mM, 1 to 10 mM, 1 to 7.5 mM, 1 to 5 mM or 1 to 2.5 mM. In certain aspects, a 5' cap analog is added with an initial concentration of about 5 to 20 mM, 7.5 to 20 mM, 10 to 20 mM or 12.5 to 20 mM.3. Flanking untranslated regions, 5' UTR and 3' UTR

[0215] In certain aspects, any of the constructs described herein can include an untranslated region (UTR), such as a 5' UTR or a 3' UTR. UTRs of a gene are transcribed but not translated. A 5' UTR starts at the transcription start site and continues to the start codon but does not include the start codon. A 3' UTR starts immediately following the stop codon and continues until the transcriptional termination signal. The regulatory and / or control features of a UTR can be incorporated into any of the constructs, particles, polynucleotides, compositions, kits, or methods as described herein to enhance or otherwise modulate the expression of a gene product for gene therapy.

[0216] Natural 5' UTRs include a sequence that plays a role in translation initiation. In certain aspects, a 5' UTR can comprise sequences, 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 sequence CCR(A / G)CCAUGG (SEQ ID NO: 41), where R is a purine (A or G) three bases upstream of the start codon (AUG), and the start codon is followed by another “G” In certain aspects, 5' UTRs also form secondary structures that are involved in elongation factor binding. In certain aspects, a 5' UTR is included in any of the constructs described herein. Non-limiting examples of 5' UTRs, including those from the following genes: P-globin, albumin, serum amyloid A, Apolipoprotein A / BZE, transferrin, alpha fetoprotein, erythropoietin, and Factor VIII, can be used to enhance expression of a nucleic acid molecule, such as an mRNA.

[0217] By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of the300484454.1 - 45 -BAYM. P0444WO / BLG 25-024polynucleotides of the disclosure. For example, introduction of 5' UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein A / B / E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, could be used to enhance expression of a nucleic acid molecule, such as a polynucleotides, in hepatic cell lines or the liver of an indiviual. Likewise, use of 5' UTR from other tissue-specific mRNA to improve expression in that tissue is possible for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C / EBP, AML1, G-CSF, GM-CSF, CDllb, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (SP-AB / C / D).

[0218] In some aspects, a 5' UTR comprises, consists essentially of, or consists of a polynucleotide sequence that is, or is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to one or more of SEQ IDNO: 42.SEQ ID NO: 42 - Modified Xenopus 0-globin 5' UTR - AGGGAAUACAAGCUACUUGUUCUUUUUGCACUCGAGCCACC (SEQ ID NO: 42)

[0219] 3' UTRs are known to have stretches of adenosines and uridines (in the RNA form) or thymidines (in the DNA form) embedded in them. These AU-rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU-rich elements (AREs) can be separated into three classes (see e.g., Chen et al., Mol. Cell. Biol. 15:5777-5788, 1995; Chen etal., Mol. Cell Biol. 15:2010-2018, 1995, each of which is incorporated herein by reference for the purposes described herein): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. For example, c-Myc and MyoD mRNAs contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U / A) (U / A) nonamers. GM-CSF and TNF-alpha mRNAs are examples that contain class II AREs. Class III AREs are less well defined. These U-rich regions do not contain an AUUUA motif, two well-studied examples of this class are c-Jun and myogenin mRNAs.

[0220] Most proteins binding to the AREs are known to destabilize the messenger RNA, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes. Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules may lead to HuR binding and thus, stabilization of the RNA.300484454.1 - 46 -BAYM. P0444WO / BLG 25-024

[0221] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of polynucleotides of the disclosure. When engineering specific polynucleotides, one or more copies of an ARE can be introduced to make polynucleotides of the disclosure less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using polynucleotides of the disclosure and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.

[0222] In some aspects, a 3' UTR comprises, consists essentially of, or consists of a polynucleotide sequence that is, or is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to one or more of SEQ IDNO: 43.SEQ ID NO: 43 - Human -globin 3' UTR - UCUAGAGCUCGCUUUCUUGCUGUCCAAUUUCUAUUAAAGGUUCCUUUGUUCCCUAAGUCCAA CUACUAAACUGGGGGAUAUUAUGAAGGGCCUUGAGCAUCUGGAUUCUGCCUGCGGCCGC(SEQ ID NO: 43 )

[0223] In certain aspects, non-ARE sequences may be incorporated into the 5' or 3' UTRs. In certain aspects, introns or portions of intron sequences may be incorporated into the flanking regions of the polynucleotides in any of the constructs, particles, polynucleotides, compositions, kits, and methods provided herein. Incorporation of intronic sequences may increase protein production as well as mRNA levels.

[0224] It should be understood that any UTR from any gene may be incorporated into the regions of the polynucleotide. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present disclosure to provide artificial UTRs which are not variants of wild type regions. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5' or 3' UTR may be inverted, shortened, lengthened, made with one or more other 5' UTRs or 3' UTRs. As used herein, the term “altered” as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference300484454.1 - 47 -BAYM. P0444WO / BLG 25-024sequence. For example, a 3' or 5' UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an “altered” UTR (whether 3' or 5') comprise a variant UTR.4. Start Codon Region

[0225] In some aspects, polynucleotides of the disclosure may comprise regions that are analogous to or function like a start codon region.

[0226] In some aspects, the translation of a polynucleotide may initiate on a codon which is not the start codon AUG. Translation of the polynucleotide may initiate on an alternative start codon such as, but not limited to, ACG, AGG, AAG, CTG / CUG, GTG / GUG, AT A / AU A, ATT / AUU, TTG / UUG (see Touriol et al. Biology of the Cell 95 (2003) 169-178 and Matsuda and Mauro PLoS ONE, 2010 5:11; the contents of each of which are herein incorporated by reference in their entirety). As a non-limiting example, the translation of a polynucleotide begins on the alternative start codon ACG. As another non-limiting example, polynucleotide translation begins on the alternative start codon CTG or CUG. As yet another non-limiting example, the translation of a polynucleotide begins on the alternative start codon GTG or GUG. Nucleotides flanking a codon that initiates translation such as, but not limited to, a start codon or an alternative start codon, are known to affect the translation efficiency, the length and / or the structure of the polynucleotide. (See e.g., Matsuda and Mauro PLoS ONE, 2010 5:11; the contents of which are herein incorporated by reference in its entirety). Masking any of the nucleotides flanking a codon that initiates translation may be used to alter the position of translation initiation, translation efficiency, length and / or structure of a polynucleotide.5. Stop Codon Region

[0227] In some aspects, polynucleotides of the present disclosure may comprise at least one or more stop codons before the 3' untranslated region (UTR). The stop codon may be selected from TGA, TAA and TAG. In some aspects, a polynucleotide includes the stop codon TGA and one additional stop codon. In some aspects, the additional stop codon may be TAA. In some aspects, a polynucleotide includes 1, 2, 3, 4, 5, or more stop codons.6. Polyadenylation sequences

[0228] In certain aspects, a construct provided herein can include a polyadenylation (poly(A)) signal sequence. Most nascent eukaryotic mRNAs possess a poly(A) tail at their 3' end, which is added during a complex process that includes cleavage of the primary transcript 300484454.1 - 48 -BAYM. P0444WO / BLG 25-024and a coupled polyadenylation reaction driven by the poly(A) signal sequence (see, e.g., Proudfoot et al., Cell 108:501-512, 2002, which is incorporated herein by reference for the purposes described herein). A poly(A) tail confers mRNA stability and transferability (see e.g., Molecular Biology of the Cell, Third Edition by B. Alberts et al., Garland Publishing, 1994, which is incorporated herein by reference for the purposes described herein). In some aspects, a poly(A) signal sequence is positioned 3' to a coding sequence.

[0229] As used herein, “polyadenylation” refers to the covalent linkage of a polyadenylyl moiety, or its modified variant, to an RNA molecule. In eukaryotic organisms, most mRNA molecules are polyadenylated at the 3' end. A 3' poly(A) tail is a long sequence of adenine nucleotides (e.g., about 50, 60, 70, 100, 200, 500, 1000, 2000, 3000, 4000, or 5000) added to the pre-mRNA through the action of an enzyme, polyadenylate polymerase. In certain aspects, a poly(A) tail is added onto transcripts that contain a specific sequence, e.g., a poly(A) signal. A poly(A) tail and associated proteins aid in protecting mRNA from degradation by exonucleases. Polyadenylation also plays a role in transcription termination, export of the mRNA from the nucleus, and translation. Polyadenylation typically occurs in the nucleus immediately after transcription of DNA into RNA, but also can occur later in the cytoplasm. After transcription has been terminated, an mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. A cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, adenosine residues are added to the free 3' end at the cleavage site.

[0230] As used herein, a “poly(A) signal sequence” or “polyadenylation signal sequence” is a sequence that triggers the endonuclease cleavage of an mRNA and the addition of a series of adenosines to the 3' end of the cleaved mRNA.

[0231] There are several poly(A) signal sequences that can be used in some aspects, including those from bovine growth hormone (bGH) (Woychik et al., Proc. Natl. Acad Sci. U. S. A. 81(13):3944-3948, 1984; U. S. Patent No. 5,122,458, each of which is incorporated herein by reference for the purposes described herein), mouse-P-globin, mouse-a-globin (Orkin et al., EMBO J 4(2):453-456, 1985; Thein et al., Blood 71 (2):313-319, 1988, each of which is incorporated herein by reference for the purposes described herein), human collagen, polyoma virus (Batt et al., Mol. Cell Biol. 15(9):4783-4790, 1995, which is incorporated herein by reference for the purposes described herein), the Herpes simplex virus thymidine kinase gene (HSV TK), IgG heavy-chain gene polyadenylation signal (US 2006 / 0040354, which is incorporated herein by reference for the purposes described herein), human growth hormone (hGH) (Szymanski et al., Mol Therapy 15(7): 1340- 1347, 2007, which is incorporated herein 300484454.1 - 49 -BAYM. P0444WO / BLG 25-024by reference for the purposes described herein), and / or the group consisting of SV40 poly(A) site, such as the SV40 late and early poly(A) site (see e.g., Schek et al., Mol Cell Biol.12(12):5386-5393, 1992, which is incorporated herein by reference for the purposes described herein).

[0232] In certain aspects, the poly(A) signal sequence can be AATAAA. The AATAAA sequence may be substituted with other hexanucleotide sequences with homology to AATAAA and that are capable of signaling polyadenylation, including ATTAAA, AGTAAA, CATAAA, TATAAA, GATAAA, ACTAAA, AATATA, AAGAAA, AATAAT, AAAAAA, AATGAA, AATCAA, AACAAA, AATCAA, AATAAC, AATAGA, AATTAA, or AATAAG (see, e g., WO 06 / 12414, which is incorporated herein by reference for the purposes described herein). In certain aspects, a poly(A) signal sequence can be a synthetic polyadenylation site (see, e.g., the pCl-neo expression construct of Promega that is based on Levitt et al., Genes Dev.3(7): 1019-1025, 1989, which is incorporated herein by reference for the purposes described herein).

[0233] In some aspects, a polynucleotide of the disclosure may comprise a polyA tail ranging from absent to 500 nucleotides in length (e.g., at least 40, 50, 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Where the tailing region is a polyA tail, the length may be determined in units of or as a function of polyA Binding Protein binding. In this aspect, the polyA tail is long enough to bind at least 4 monomers of PolyA Binding Protein. PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are particularly functional.

[0234] In some aspects, a poly(A) tail comprises, consists essentially of, or consists of a polynucleotide sequence that is, or is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range derivable therein, identical to one or more of SEQ IDNO: 44.SEQ ID NO: 44 - Segmented poly(A) tail -. AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAU GCAUAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 44 )7. Signal Sequences

[0235] In some aspects, polynucleotides of the disclosure may also encode additional features which facilitate trafficking of the polypeptides they encode to therapeutically relevant300484454.1 - 50 -BAYM. P0444WO / BLG 25-024sites. One such feature which aids in protein trafficking is the signal sequence. As used herein, a “signal sequence” or “signal peptide” is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3-60 amino acids) in length which is incorporated at the 5' (or N-terminus) of the coding region or polypeptide encoded, respectively. Addition of these sequences result in trafficking of the encoded polypeptide to the endoplasmic reticulum through one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported.

[0236] Additional signal sequences which may be utilized in the compositions of the present disclosure include those taught in, for example, databases such as those found at http: / / www.signalpeptide.de / or http: / / proline.bic.nus.edu.sg / spdb / . Those described in U. S. Pat. Nos. 8,124,379; 7,413,875 and 7,385,034 are also within the scope of the disclosure and the contents of each are incorporated herein by reference in their entirety.

[0237] In some aspects, a signal sequence comprises a sequence that is or is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 45-48 or 53-54.SEQ ID NO: 45 - Illustrative human IgG signal peptide (NA) AUGGGCUGGUCCUGCAUCAUCUUGUUCUUGGUGGCUACUGCCACUGGAGUACACAGC (SEQ ID NO: 45 )SEQ ID NO: 46 - Illustrative human IgG signal peptide (AA)MGWSCI ILFLVATATGVHS (SEQ ID NO: 46 )SEQ ID NO: 47 - Illustrative albumin signal peptide (NA) AUGAAAUGGGUCACCUUUAUCAGCCUGCUGUUCCUGUUCAGCAGCGCCUACAGC (SEQ ID NO: 47 )SEQ ID NO: 48 - Illustrative albumin signal peptide (AA) MKWVTFISLLFLFSSAYS (SEQ ID NO: 48 )SEQ ID NO: 53 - Illustrative tissue-type plasminogen activator (tPA) signal peptide (NA) AUGGAUGCAAUGAAGCGGGGGCUCUGCUGUGUGCUGCUGCUGUGUGGAGCAGUCUUCGUUUC GGCC (SEQ ID NO: INSERT)SEQ ID NO: 54 - Illustrative tissue-type plasminogen activator (tPA) signal peptide (AA) MDAMKRGLCCVLLLCGAVFVSA (SEQ ID NO: INSERT)8. Transmembrane domain

[0238] In some aspects, a polynucleotide of the disclosure encodes a transmembrane domain. As used herein, the term “transmembrane domain” or “anchor” refers to the domain 300484454.1 - 51 -BAYM. P0444WO / BLG 25-024of a peptide, polypeptide or protein that is capable of spanning the plasma membrane of a cell. These domains can be used to anchor a polypeptide on the cell membrane.

[0239] Transmembrane regions of proteins are highly hydrophobic or lipophilic domains that are the proper size to span the lipid bilayer of the cellular membrane, thereby anchoring proteins or peptides in the cell membrane. They will typically, but not always, comprise 15-30 amino acids. See Chou et al. (1999 Biotechnology and Bioengineering 65(2): 160-169), which describes using several transmembrane domains from different source proteins to express a different protein on the cell membrane. One skilled in the art can adapt the method performed in Chou et al., for example, to optimize or screen different transmembrane domains and / or GPI-anchor domains for use with the present disclosure.

[0240] Transmembrane proteins may contain from one or multiple transmembrane domains. For example, receptor tyrosine kinases, certain cytokine receptors, receptor guanylyl cyclases and receptor serine / threonine protein kinases contain a single transmembrane domain. However, various other proteins including channels and adenylyl cyclases contain numerous transmembrane domains.

[0241] Many cell surface receptors are classified as “seven transmembrane domain” proteins, as they contain membrane spanning regions. Transmembrane protein receptors include, but are not limited to insulin receptor, insulin-like growth factor receptor, human growth hormone receptor, glucose transporters, transferrin receptor, epidermal growth factor receptor, low density lipoprotein receptor, epidermal growth factor receptor, leptin receptor, interleukin receptors, e.g. IL-1 receptor, IL-2 receptor, etc.

[0242] Described herein are examples of transmembrane domains, but the transmembrane domain of the polypeptides disclosed herein can be any amino acid sequence that will span the plasma cell membrane and can anchor other domains to the membrane. Characteristics of transmembrane domains include generally consecutive hydrophobic amino acids that may be followed by charged amino acids. Therefore, upon analysis of the amino acid sequence of a particular protein, the localization and number of transmembrane domains within the protein may be predicted by those skilled in art. A transmembrane domain may comprise hydrophobic regions or amphipathic regions. Hydrophobic regions contain hydrophobic amino acids, which include, but are not limited to, phenylalanine, methionine, isoleucine, leucine, valine, cysteine, tryptophan, alanine, threonine, glycine and serine and include hydrophobic alpha-helices. Amphipathic regions may have both hydrophobic and hydrophilic amino acids and moieties and include amphipathic alpha-helices. Hydrophilic amino acids include, but are not limited to, arginine, aspartate, lysine, glutamate, asparagine, glutamine, histidine, tyrosine and proline.300484454.1 - 52 -BAYM. P0444WO / BLG 25-024Transmembrane domains that form stable alpha helices have been previously described in the art.

[0243] Transmembrane domains include, but are not limited to, those from: a member of the tumor necrosis factor receptor superfamily, CD30, platelet derived growth factor receptor (PDGFR, e.g. amino acids 514-562 of human PDGFR; Chestnut et al. 1996 J Immunological Methods 193:17-27; also see Gronwald et al. 1988 PNAS 85:3435); nerve growth factor receptor, Murine B7-1 (Freeman et al. 1991 J Exp Med 174:625-631), asialoglycoprotein receptor Hl subunit (ASGPR; Speiss et al. 1985 J Biol Chem 260:1979-1982), CD27, CD40, CD120a, CD120b, CD80 (Freeman et al. 1989 J Immunol 143:2714-22) lymphotoxin beta receptor, galactosyltransferase (E. G. GenBank accession number AF 155582), sialyly transferase (E. G. GenBank accession number NM-003032), aspartyl transferase 1 (Aspl; e.g. GenBank accession number AF200342), aspartyl transferase 2 (Asp2; e.g. GenBank accession number NM-012104), syntaxin 6 (e.g. GenBank accession number NM-005819), ubiquitin, dopamine receptor, insulin B chain, acetylglucosaminyl transferase (e.g. GenBank accession number NM-002406), APP (e.g. GenBank accession number A33292), a G-protein coupled receptor, thrombomodulin (Suzuki et al. 1987 EMBO J 6, 1891) and TRAIL receptor. In some aspects, a transmembrane domain is from a human protein. For the purposes of the present disclosure all or part of a transmembrane domain from one or more proteins comprising a transmembrane domain may be utilized. Examples of transmembrane domains are also described in Patent Publication US20040126859, which is incorporated in its entirety herein by reference.

[0244] A wide range of cell-surface proteins, including enzymes, coat proteins, surface antigens, and adhesion molecules, are attached to plasma membranes via GPI anchors (Burikofer et al. 2002 FASEB J 15:545). GPI is a post-translationally added lipid anchor; therefore, unlike conventional polypeptide anchors which have different transmembrane domains and connect to specific cytoplasmic extensions, GPI anchors use a common lipid structure to attach to the membrane, which is irrespective of the proteins linked with it (Englund et al., Annul Rev. Biochem. 62:121 (1993)). GPI anchor signal sequences have been identified for many proteins (for example, see Cares et al., Science 243:1196 (1989)). The GPI anchor signals have been successfully engineered onto the C-terminus of other un-GPI anchored proteins, and these GPI anchored proteins are coated on the cell surface and are functional. (Anderson et al., P. N. A. S. 93:5894 (1996); Brunschwig et al., J. Immunother. 22:390 (1999)). GPI anchors are proposed to function in protein targeting, transmembrane signaling, and in the uptake of small molecules (endocytosis). GPI anchors of plasma membrane proteins are present 300484454.1 - 53 -BAYM. P0444WO / BLG 25-024in eukaryotes from protozoa and fungi to vertebrates. For examples of GPI anchor domains, which may be utilized with the present disclsoure, see Doering, T. L. et al. (1990) J. Biol. Chem. 265:611-614; McConville, M. J. et al. (1993) Biochem. J. 294:305-324; and PCT Publication WO 03 / 017944), all of which are incorporated herein by reference in their entirety.

[0245] Without wishing to be limited by theoretical considerations, immediately following protein synthesis, a protein comprising a GPI modification signal is anchored to the ER lumen by a hydrophobic sequence approximately 15-20 amino acids in length. Alberts et al., Molecular Biology Of The Cell, 3rd Edition, p. 591 (1994). A GPI anchor is pre-assembled in the ER and following GPI attachment, the modified protein is glycosylated and shuttled to the exterior surface of the plasma membrane. The process of covalently attaching a GPI anchor to the C-terminus of a peptide is catalyzed by enzymes in the rough ER. Enzymes of the ER cleave the original membrane-anchor sequence and then the new carboxyl-terminus is attached to the amino group of ethanolamine. The anchor typically comprises a phosphoethanolamine (EthN-P), several sugars, including N-acetylglucosamine (GlcNAc) and mannose, linked to an inositol phospholipid (Ikezawa 2002 Biol Pharm Bull 25: 409-417). Furthermore, the inositol phospholipid typically contains 1 -alkyl, 2-acyl glycerol. The inositol phospholipids in anchors, however, can vary. For example, inositol phospholipids of proteins expressed on erythrocytes have an additional inositol-associated fatty acid that provides an additional point of attachment to the plasma membrane. Such anchors are described as being “two footed.” Accordingly, GPI anchors that may be used with the present disclosure can be “one footed,” “two footed” or “three footed.”

[0246] Essentially any GPI-anchor signal sequence can be used in accordance with the present disclosure. GPI-anchor signal sequences are known in the art and / or can be determined using methods known in the art, e.g., using the Big-P predictor analysis available at http: / / mendel.imp.univie.ac.at / gpi / gpi_server.html and described in Eisenhaber et al. 1999 J Mol Biol 292: 741-758; Eisenhaber et al. 2003 Nucleic Acids Research 31: 3631-3634; Eisenhaber et al. Protein Engineering 14: 17-25; Eisenhaber et al. 2000 TIBS 25: 340-341; and Eisenhaber et al. 1998 Protein Engineering 11: 1155-1161, all of which are incorporated herein by reference in their entirety. In some aspects, a transmembrane domain comprises a GPI anchor. In some aspects, a GPI anchor comprises a cecay-accelerating factor (DAF; also known as CD55) GPI anchor.

[0247] In some aspects, a transmembrane domain comprises a sequence that is or is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NOs: 49-50.300484454.1 - 54 -BAYM. P0444WO / BLG 25-024SEQ ID NO: 49 - Illustrative DAE GPI anchor (NA) CCUAACAAAGGCAGCGGCACCACCUCUGGCACCACAAGACUGCUGUCUGGCCACACCUGUUU CACACUGACCGGCCUGCUGGGCACACUGGUUACAAUGGGACUGCUGACC (SEQ ID NO: 49 )SEQ ID NO: 50 - Illustrative DAF GPI anchor (AA) PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO: 50 )9. Codon Optimization

[0248] Polynucleotides of the disclosure and their regions or parts or subregions may be codon optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include 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, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park Calif.) and / or proprietary methods. In some aspects, an open reading frame sequence may be codon optimized using optimization algorithms. Codon options for each amino acid are given in US patent application US20220193223A1, which is incorporated herein by reference in its entirety.10. IRES Sequences

[0249] In some aspects, polynucleotides of the disclosure may contain an internal ribosome entry site (IRES). First identified as a feature Picoma virus RNA, IRES plays an important role in initiating protein synthesis in absence of the 5' cap structure. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. Polynucleotides containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes (“multicistronic nucleic acid molecules”). When polynucleotides are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the disclosure include without limitation, those from picomaviruses (e.g. FMDV),300484454.1 - 55 -BAYM. P0444WO / BLG 25-024pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SLY) or cricket paralysis viruses (CrPV).11. Additional sequences

[0250] In some aspects, a polynucleotide of the disclosure may comprise one or more 2A elements. In some aspects, a 2A element may be a T2A, P2A, E2A, and / or F2A element.

[0251] In some aspects, a polynucleotide polypeptide of the disclosure may comprise a linker. In some aspects, a linker codes for a polypeptide comprising a KL sequence

[0252] In some aspects, constructs provided herein can optionally include a sequence encoding a reporter polypeptide and / or protein (“a reporter sequence”). Non-limiting examples of reporter sequences include DNA sequences encoding: a beta-lactamase, a betagalactosidase (LacZ), an alkaline phosphatase, a thymidine kinase, a green fluorescent protein (GFP), a red fluorescent protein, an mCherry fluorescent protein, a yellow fluorescent protein, a chloramphenicol acetyltransferase (CAT), and a luciferase. Additional examples of reporter sequences are known in the art. When associated with control elements which drive their expression, the reporter sequence can provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence, or other spectrographic assays, fluorescent activating cell sorting (FACS) assays and / or immunological assays (e.g., enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry).

[0253] In some aspects, a reporter sequence is the Lacz gene, and the presence of a construct carrying the Lacz gene in a cell is detected by assays for beta-galactosidase activity. In some aspects, a reporter sequence is a fluorescent protein (e.g., mCherry, green fluorescent protein (GFP)) or luciferase. In aspects where a reporter sequence is a fluorescent protein or luciferase, the presence of a construct carrying the fluorescent protein or luciferase in a cell may be measured by fluorescent imaging techniques (e.g., fluorescent microscopy or FACS) or light production in a luminometer (e.g., a spectrophotometer or an IVIS imaging instrument). In some aspects, a reporter sequence can be used to verify tissue-specific targeting capabilities and / or tissue-specific promoter regulatory and / or control activity of any of the constructs described herein.

[0254] In some aspects, a reporter sequence is a FLAG tag (e.g., a lx, 2x, 3x FLAG tag), and the presence of a construct carrying the FLAG tag in a cell is detected by protein binding or detection assays (e.g., Western blots, immunohistochemistry, radioimmunoassay (RIA),300484454.1 - 56 -BAYM. P0444WO / BLG 25-024mass spectrometry). Sequences described herein that include one or more tags, such as a FLAG tag, are also expressly contemplated in forms that do not comprise the tag and / or that comprise alternative tag sequences.

[0255] In some aspects, a reporter sequence is an HA tag, and the presence of a construct carrying the HA tag in a cell is detected by protein binding or detection assays (e.g., Western blots, immunohistochemistry, radioimmunoassay (RIA), mass spectrometry). In some aspects, multiple HA tags are used in tandem, such as 1, 2, 3, 4, 5 or more HA tags. Tandem tags may be connected by a flexible linker, such as a glycine-serine linker.

[0256] In some aspects, a tag comprises a sequence that is or is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 51-52.SEQ ID NO: 51 - Illustrative FLAG tag (NA)GACUACAAAGAUGAUGACGAUAAG (SEQ ID NO: 51)SEQ ID NO: 52 - Illustrative FLAG tag (AA)DYKDDDDK (SEQ ID NO: 52)III. In vitro transcription

[0257] In some aspects, polynucleotides of the disclosure, such as mRNA vaccine, may be in vitro transcribed. The term “in vitro transcription” relates to a process wherein RNA is synthesized in a cell-free system (in vitro). RNA is commonly obtained by enzymatic DNA dependent in vitro transcription of an appropriate DNA template, which is often a linearized plasmid DNA template. The promoter for controlling RNA in vitro transcription can be any promoter for any DNA dependent RNA polymerase. Particular examples of DNA dependent RNA polymerases are the bacteriophage enzymes T7, T3, and / or SP6 RNA polymerases.

[0258] Methods for RNA in vitro transcription are known in the art (see for example Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle et al. (2013) Methods Enzymol. 530: 101-14; all of which are incorporated herein by reference in their entirety). Reagents used in said methods may include: a linear DNA template with a promoter sequence that has a high binding affinity for its respective RNA polymerase; ribonucleoside triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); a cap analog (for example, but not limited to, m7G(5')ppp(5')G (m7G)); other modified nucleotides; DNA-dependent RNA polymerase (for example, but not limited to, T7, T3 or SP6 RNA polymerase); ribonuclease (RNase) inhibitor to inactivate any contaminating RNase; pyrophosphatase to degrade pyrophosphate, which inhibits transcription; MgCh, which supplies Mg2+as a cofactor for the300484454.1 - 57 -BAYM. P0444WO / BLG 25-024RNA polymerase; antioxidants (for example, but not limited to, DTT); polyamines such as spermidine; and a buffer to maintain a suitable pH value.

[0259] Common buffer systems used in RNA in vitro transcription include 4-(2-hydroxy-ethyl)-l -piperazineethanesulfonic acid (HEPES) and tris(hydroxymethyl) amino-methane (Tris). The pH value of the buffer is commonly adjusted to a pH value of about 6 to 8.5. Some commonly used transcription buffers comprise 80 mM HEPES / KOH, pH 7.5 and 40 mM Tris / HCl, pH 7.5.

[0260] The transcription buffer can also contain a magnesium salt such as MgCh commonly in a range between 5-50 mM. Magnesium ions (Mg2+) are an essential component in an RNA in vitro transcription buffer system because free Mg2+acts as cofactor in the catalytic center of the RNA polymerase and is critical for the RNA polymerization reaction. In diffuse binding, fully hydrated Mg2+ions also interact with the RNA product via nonspecific long-range electrostatic interactions.

[0261] RNA in vitro transcription reactions are typically performed as batch reactions in which all components are combined and then incubated to allow the synthesis of RNA molecules until the reaction terminates. In addition, fed-batch reactions were developed to increase the efficiency of the RNA in vitro transcription reaction (see e.g., Kern et al. (1997) Biotechnol. Prag. 13: 747-756; and Kern et al. (1999) Biotechnol. Prog. 15: 174-184; both of which are incorporated herein by reference in their entirety). In a fed-batch system, all components are combined, but then additional amounts of some of the reagents are added over time (for example, but not limited to, NTPs, and / or MgCh) to maintain constant reaction conditions.

[0262] Moreover, the use of a bioreactor (transcription reactor) for the synthesis of RNA molecules by in vitro transcription has been reported (see e.g., WO 1995 / 08626; incorporated herein by reference in its entirety). The bioreactor may be configured such that reactants are delivered via a feed line to the reactor core and RNA products are removed by passing through an ultrafiltration membrane (having a nominal molecular weight cut-off, for example, but not limited to, 100,000 Daltons) to the exit stream.

[0263] The concentration of the nucleic acid template comprised in the in vitro transcription mixture may be in a range from about 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or about 1 to 10 nM. In certain aspects, the concentration of the nucleic acid template may be from about 10 to 30 nM. In certain aspects, the concentration of the nucleic acid template may be about 20 nM. In certain aspects, a concentration of the nucleic acid template may be of about 1 to 200 pg / ml, about 10 to 100 pg / ml, or about 20 to 50 pg / ml.300484454.1 - 58 -BAYM. P0444WO / BLG 25-024

[0264] DNA encoding the polynucleotides described herein may be transcribed using an in vitro transcription system. The system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to incorporate polynucleotides (e.g., modified nucleic acids).A. Ribonucleoside triphosphates

[0265] The ribonucleoside triphosphates (NTPs) GTP, ATP, CTP and UTP are the monomers which are polymerized during the RNA polynucleotide synthesis process. They may be provided with a monovalent or divalent cation as counterion. In certain aspects, the monovalent cation is selected from the group consisting of Li+, Na+, K+, NH4+or tris(hydroxymethyl)-aminomethane (Tris). In certain aspects, the divalent cation is selected from the group consisting of Mg2+, Ba2+, and Mn2+. In certain aspects, the monovalent cation is Na+or tri s(hydroxymethyl)-aminom ethane (Tris).

[0266] In certain aspects, a part of, or all of, at least one ribonucleoside triphosphate in the RNA synthesis is replaced with a modified nucleoside triphosphate as defined below.1. Modified nucleoside triphosphate

[0267] The term “modified nucleoside triphosphate” as used herein refers to chemical modifications comprising backbone modifications as well as sugar modifications or base modifications. These modified nucleoside triphosphates are also termed herein as “nucleotide analogs.”

[0268] In this context, the modified nucleoside triphosphates as defined herein are nucleotide analogs / modifications, for example, but not limited to, backbone modifications, sugar modifications or base modifications. A backbone modification in connection with the present disclosure is a modification, in which phosphates of the backbone of the nucleotides are chemically modified. A sugar modification in connection with the present disclosure is a chemical modification of the sugar of the nucleotides. Furthermore, a base modification in connection with the present disclosure is a chemical modification of the base moiety of the nucleotides. In this context nucleotide analogs or modifications are preferably selected from nucleotide analogs which are applicable for RNA synthesis and / or translation.300484454.1 - 59 -BAYM. P0444WO / BLG 25-0241. Backbone modifications

[0269] The phosphate backbone may further be modified in the modified nucleosides and nucleotides. The phosphate groups of the backbone can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the full replacement of an unmodified phosphate moiety with a modified phosphate. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. In certain aspects, the phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylene-phosphonates).2. Sugar modifications

[0270] The modified nucleosides and nucleotides, which may be used in the context of the present disclosure, can be modified in the sugar moiety. For example, but not limited to, the 2' hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. Examples of “oxy”-2' hydroxyl group modifications include, but are not limited to, alkoxy or aryloxy (-OR, for example, but not limited to, R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), — O(CH2CH2O)nCH2CH2OR; “locked” nucleic acids (LNA) in which the 2' hydroxyl is connected, for example, but not limited to, by a methylene bridge, to the 4' carbon of the same ribose sugar; and amino groups ( — 0-amino, wherein the amino group, for example, but not limited to, NRR, can be alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroaryl amino, ethylene diamine, polyamino) or aminoalkoxy.

[0271] “Deoxy” modifications include hydrogen, amino (for example, but not limited to, NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or the amino group can be attached to the sugar through a linker, wherein the linker comprises one or more of the atoms C, N, and / or O.

[0272] The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleotide can include nucleotides containing, for example, but not limited to, arabinose as the sugar.300484454.1 - 60 -BAYM. P0444WO / BLG 25-0243. Base modifications

[0273] The modified nucleosides and nucleotides, which in certain aspects, may be used in aspects of the present disclosure, can further be modified in the nucleobase moiety. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine and uracil. For example, the nucleosides and nucleotides described herein can be chemically modified on the major groove face. In certain aspects, the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.

[0274] In certain aspects of the present disclosure, nucleotide analogs / modifications may be selected from base modifications including, but not limited to, 2-amino-6-chloropurineriboside-5'-triphosphate, 2-Aminopurine-riboside-5'-triphosphate; 2-aminoadenosine-5'-triphosphate, 2'-Amino-2'-deoxycytidinetriphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5 '-triphosphate, 2'-Fluorothymidine-5'-triphosphate, 2'-O-Methyl inosine-5 '-triphosphate, 4-thiouridine-5 '-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoally luridine-5 '-triphosphate, 5-bromocytidine-5'triphosphate, 5-bromouridine-5'-triphosphate, 5-Bromo-2'deoxycytidine-5'-triphosphate, 5-Bromo-2'-deoxyuridine-5 'triphosphate, 5-iodocytidine-5'-triphosphate, 5-Iodo-2'deoxycytidine-5'-triphosphate, 5-iodouridine-5 'triphosphate, 5-Iodo-2'-deoxyuridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'triphosphate, 5-Propyny l-2'-deoxycytidine-5 '-triphosphate, 5-Propyny l-2'-deoxyuridine-5'-triphosphate, 6-azacytidine-5 '-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurineriboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine-5 '-triphosphate, 8-azaadenosine-5 '-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, Nl-methyladenosine-5'-triphosphate, Nl-methylguanosine-5 '-triphosphate, N6-methyladenosine-5 '-triphosphate, 06-methylguanosine-5'-triphosphate, pseudouridine-5 '-triphosphate, puromycin-5 '-triphosphate, xanthosine-5 '-triphosphate, and / or any combinations thereof. In certain aspects, nucleotides for base modifications comprise 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, pseudouridine-5 '-triphosphate, and / or any combinations thereof. In some aspects, a modified nucleoside comprises Nl-Methylpseudouridine-5'-Triphosphate (m1ΨTP).

[0275] In certain aspects, modified nucleosides comprise pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propyny 1-uridine, 1-propyny 1 -pseudouridine, 5 -taurinom ethyluridine, 1-taurinomethy 1-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1 -taurinom ethyl-4-thio-uri dine, 5-methyl- 300484454.1 - 61 -BAYM. P0444WO / BLG 25-024uridine, 1-methyl-pseudouridine, 4-thio-l -methylpseudouridine, 2-thio-l -methylpseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2-thio-l-methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thiodihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxypseudouridine, 4-methoxy-2-thio-pseudouridine, and / or any combinations thereof.

[0276] In certain aspects, modified nucleosides comprise 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine, 2-thio-5-methyl-cytidine, 4-thiopseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-l -methylpseudoisocytidine, and / or any combinations thereof.

[0277] In certain aspects, modified nucleosides comprise 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7 -deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-( cishydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6, N6-dimethyladenosine, 7-methyladenine, 2-methylthioadenine, 2-methoxy-adenine, and / or any combinations thereof.

[0278] In certain aspects, modified nucleosides comprise inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thioguanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-azaguanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2-m ethylguanosine, N2, N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2, N2-dimethyl-6-thio-guanosine, and / or any combinations thereof.

[0279] In certain aspects, a nucleotide can be modified on the major groove face and can include replacing hydrogen on C-5 of uracil with a methyl group or a halo group.

[0280] In certain aspects, a modified nucleoside comprises 5'-O-(l-Thiophosphate)-Adenosine, 5'-O-(l-Thiophosphate)-Cytidine, 5′-O-(1-Thiophosphate)-Guanosine, 5'-O(l-Thiophosphatej-Uridine, 5'-O-(l-Thiophosphate)Pseudouridinem, and / or any combinations thereof.300484454.1 - 62 -BAYM. P0444WO / BLG 25-024

[0281] In certain aspects the modified nucleotides comprise nucleoside modifications, for example, but not limited to, 6-aza-cytidine, 2-thio-cytidine, a-thio-cytidine, Pseudoisocytidine, 5-aminoallyl-uridine, 5-iodo-uridine, Nl-methylpseudouridine, 5,6-dihydrouridine, a-thio-uridine, 4-thiouridine, 6-aza-uridine, 5-hydroxy-uridine, deoxythymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, a-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytidine, 8-oxo-guanosine, 7-deaza-guanosine, Nl-methyl-adenosine, 2-amino-6-Chloro-purine, N6-methyl-2-amino-purine, Pseudo-isocytidine, 6-Chloro-purine, N6-methyl-adenosine, a-thioadenosine, 8-azido-adenosine, 7-deaza-adenosine. Further modified nucleotides have been described previously in WO 2013 / 052523 (the contents of which are incorporated herein by reference in their entirety).B. Pyrophosphatase

[0282] A pyrophosphatase is an acid anhydride hydrolase that hydrolyses diphosphate bonds. In an in vitro transcription reaction it serves to hydrolyze the bonds within the diphosphate released upon incorporation of the ribonucleoside triphosphates into the nascent RNA chain. In certain aspects, the concentration of the pyrophosphatase may be from about 1 to 20 units / mL, 1 to 15 units / mL, 1 to 10 units / mL, 1 to 5 units / mL, or 1 to 2.5 units / mL. In certain aspects, the concentration of the pyrophosphatase may be about 1 unit / mL or about 5 units / mL.C. Ribonuclease inhibitor

[0283] A ribonuclease inhibitor inhibits the action of a ribonuclease which degrades RNA. In certain aspects, the concentration of the ribonuclease inhibitor may be from about 1 to 500 units / mL, 1 to 400 units / mL, 1 to 300 units / mL, 1 to 200 units / mL, or 1 to 100 units / mL. In certain aspects, the concentration of the ribonuclease inhibitor may be about 200 units / mL.D. Antioxidant

[0284] An antioxidant inhibits the oxidation of other molecules. Suitable antioxidants for use with the present disclosure include, but are not limited to, DTT (dithiothreitol), TCEP (tris(2-carboxyethyl)phosphine), NAC (N-acetylcysteine), beta-mercaptoethanol, glutathione, cysteine and cystine. In certain aspects, DTT may be used in an in vitro transcription reaction.

[0285] In certain aspects, the concentration of DTT may be about 1 to 50 mM, 5 to 48 mM, 8 to 47 mM, 10 to 46 mM, 15 to 45 mM, 18 to 44 mM, 20 to 43 mM, 23 to 42 mM, 25 to 41 mM or 28 to 40 mM. In certain aspects, the concentration of DTT may be 40 mM.300484454.1 - 63 -BAYM. P0444WO / BLG 25-024E. RNA Polymerases Useful for Synthesis

[0286] Any number of RNA polymerases or variants may be used in the synthesis of polynucleotides disclosed herein.

[0287] The RNA polymerase is an enzyme which catalyzes the transcription of a DNA template into RNA. In certain aspects, suitable RNA polymerases for use in methods and / or compositions of the present disclosure include, but are not limited to, T7, T3, SP6 and E. coli RNA polymerase. In certain aspects, a T7 RNA polymerase is used. In certain aspects, an RNA polymerase for use in the present disclosure is a recombinant RNA polymerase, meaning that it is added to the RNA in vitro transcription reaction as a single component and not as part of a cell extract which contains other components in addition to the RNA polymerase. The skilled person knows that the choice of the RNA polymerase depends on the promoter present in the DNA template which has to be bound by the suitable RNA polymerase. In certain aspects, the concentration of the RNA polymerase may be from about 1 to 100 nM, 1 to 90 nM, 1 to 80 nM, 1 to 70 nM, 1 to 60 nM, 1 to 50 nM, 1 to 40 nM, 1 to 30 nM, 1 to 20 nM, or about 1 to 10 nM. In certain aspects, the concentration of the RNA polymerase may be from about 10 to 50 nM, 20 to 50 nM, or 30 to 50 nM. In certain aspects, the RNA polymerase concentration may be about 40 nM. In certain aspects, a concentration of 500 to 10000 U / mL of the RNA polymerase may be used. In certain aspects, a concentration of 1000 to 7500 U / mL, or a concentration of 2500 to 5000 Units / mL of the RNA polymerase may be used. The person skilled in the art will understand that the choice of the RNA polymerase concentration is also influenced by the concentration of the DNA template.

[0288] Polynucleotides of the disclosure may also be synthesized. For example, using Solid-Phase Chemical Synthesis, Liquid Phase Chemical Synthesis, Small Region Synthesis, Ligation of Polynucleotide Regions or Subregions, or a Combination of Synthetic Methods may be employed to produce one or more of the polynucleotides of the disclosure.F. Solid-phase chemical synthesis

[0289] Polynucleotides of the present disclosure may be manufactured in whole or in part using solid phase techniques.

[0290] Solid-phase chemical synthesis of polynucleotides or nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution. Impurities and excess reagents are washed away and no purification is required after each step. The automation of the process is amenable on a computer controlled solid-phase synthesizer. Solid-phase synthesis allows rapid production of polynucleotides or300484454.1 - 64 -BAYM. P0444WO / BLG 25-024nucleic acids in a relatively large scale that leads to the commercial availability of some polynucleotides or nucleic acids. Furthermore, it is useful in site-specific introduction of chemical modifications in the polynucleotide or nucleic acid sequences. It is an indispensable tool in designing modified derivatives of natural nucleic acids.

[0291] In automated solid-phase synthesis, the chain is synthesized in 3' to 5' direction. The hydroxyl group in the 3' end of a nucleoside is tethered to a solid support via a chemically cleavable or light-cleavable linker. Activated nucleoside monomers, such as 2'-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, are added to the support-bound nucleoside sequentially. Currently, most widely utilized monomers are the 3 '-phosphorami dite derivatives of nucleoside building blocks. The 3' phosphorus atom of the activated monomer couples with the 5' oxygen atom of the support-bound nucleoside to form a phosphite triester. To prevent side reactions, all functional groups not involved in the coupling reaction, such as the 5' hydroxyl group, the hydroxyl group on the 3' phosphorus atom, the 2' hydroxyl group in ribonucleosides monomers, and the amino groups on the purine or pyrimidine bases, may all be blocked with protection groups. The next step may involve oxidation of the phosphite triester to form a phosphate triester or phosphotriester, where the phosphorus atom is pentavalent. The protection group on the 5' hydroxyl group at the end of the growing chain may then be removed, ready to couple with an incoming activated monomer building block. At the end of the synthesis, a cleaving agent such as ammonia or ammonium hydroxide may be added to remove all the protecting groups and release the polynucleotide chains from the solid support. Light may also be applied to cleave the polynucleotide chain. The product can then be further purified with high pressure liquid chromatography (HPLC) or electrophoresis.

[0292] Scientific studies and research are going on to further improve the solid-phase synthesis method. For example, instead of the well-established 3'-to-5' synthesis, U. S. Pat. No.8,309,707 and US Pat. Publication No. 2013 / 0072670, the contents of which are incorporated herein by reference in their entirety for the purposes described herein, disclosed a 5'-to-3' synthesis of RNA utilizing a novel phosphoramidite and a novel nucleoside derivative, thereby allowing easy modifications of the synthetic RNA at the 3' end. PCT application PCT / US2013 / 026045 published as WO2013123125A1, the contents of which are incorporated herein by reference in their entirety for the purposes described herein, describes assembly of a target nucleic acid sequence from a plurality of subsequences, wherein resins with the subsequences are placed in an emulsion droplet. The subsequences are cleaved off the resins and assemble within the emulsion droplet. To reduce the cost of solid supports, a reusable CPG 300484454.1 - 65 -BAYM. P0444WO / BLG 25-024solid support has been developed with a hydroquinone-O, O'-diacetic acid linker (Q-linker) (Pon et al., Nucleic Acid Research, vol. 27, 1531-1538 (1999), the contents of which are incorporated herein by reference in their entirety for the purposes described herein).G. Liquid phase chemical synthesis

[0293] In certain aspects, synthesis of recombinant polynucleotides or circular polynucleotides of the present disclosure by the sequential addition of monomer building blocks may be carried out in a liquid phase. In liquid phase chemical synthesis, a covalent bond is formed between the monomers or between a terminal functional group of the growing chain and an incoming monomer. Functional groups not involved in the reaction must be temporarily protected. After the addition of each monomer building block, the reaction mixture may be purified before adding the next monomer building block. The functional group at one terminal of the chain may be deprotected to be able to react with the next monomer building blocks. A liquid phase synthesis is labor- and time-consuming and is not automated. Despite the limitations, liquid phase synthesis is still useful in preparing short polynucleotides in a large scale. Because the system is homogenous, it does not require a large excess of reagents and is cost effective in this respect.H. Nucleotide purification

[0294] In some aspects, the polynucleotides of the disclosure may be purified. The term “purified” when used in relation to a polynucleotide such as a “purified polynucleotide” refers to one that is separated from at least one contaminant. As used herein, a “contaminant” is any substance which makes another unfit, impure, and / or inferior. Thus, a purified polynucleotide (for example, but not limited to, 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.

[0295] Purification of the polynucleotides of the disclosure described herein may include, but is not limited to, polynucleotide 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, Mass.), poly-T beads, LNA™ 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), hydrophobic interaction HPLC (HIC-HPLC), and / or any combinations thereof.300484454.1 - 66 -BAYM. P0444WO / BLG 25-024

[0296] A quality assurance and / or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, sequencing, and / or analytical HPLC. In certain aspects, the polynucleotides may be sequenced by methods including, but not limited, to reverse-transcriptase-PCR.IV. Pharmaceutical Compositions

[0297] The present disclosure provides, at least, pharmaceutical compositions comprising one or more polynucleotides of the disclosure. The polynucleotides may be combined with one or more pharmaceutically acceptable excipients. Pharmaceutical compositions may optionally comprise one or more additional active substances, e.g., therapeutically and / or prophylactically active substances. Pharmaceutical compositions of the present disclosure may be sterile and / or pyrogen-free. General considerations in the formulation and / or manufacture of pharmaceutical agents 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).

[0298] In some aspects, compositions are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase “active ingredient” generally refers to the polynucleotides of the disclosure, e.g., antigen-encoding polynucleotides, for example, RNA polynucleotides, to be delivered as described herein.

[0299] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, rats, and / or birds.

[0300] Formulations of the pharmaceutical 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 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-dose300484454.1 - 67 -BAYM. P0444WO / BLG 25-024unit. Accordingly, the formulations of the present disclosure may include one or more excipients, each in an amount that may increases the stability of the polynucleotides, increases cell transfection by the polynucleotides, increases the expression of polynucleotides encoded protein, and / or alters the release profile of polynucleotide encoded proteins. Further, the polynucleotides of the present disclosure may be formulated using self-assembled nucleic acid nanoparticles.

[0301] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition. Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, surface active agents and / or emulsifiers, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in the pharmaceutical formulations of the disclosure.

[0302] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and / or condition of the subject treated and further depending upon the mode 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 to 50%, between 1 to 30%, between 5 to 80%, or at least 80% (w / w) active ingredient.

[0303] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a “unit dose” refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be 300484454.1 - 68 -BAYM. P0444WO / BLG 25-024administered to a subject and / or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

[0304] In some aspects, the formulations described herein may contain at least one polynucleotide, e.g., antigen-encoding polynucleotide. As a non-limiting example, the formulations may contain 1, 2, 3, 4, 5, or more polynucleotides of the disclosure. In some aspects, formulations of the disclosure described herein may comprise more than one type of polynucleotides, e.g., antigen-encoding polynucleotide. In some aspects, a formulation may comprise a chimeric polynucleotide in linear and circular form. In some aspects, a formulation may comprise one or more in vitro transcribed polynucleotides.

[0305] In some aspects, a formulation may contain polynucleotides encoding proteins selected from categories such as, but not limited to, human proteins, veterinary proteins, bacterial proteins, parasite proteins, helminth proteins, biological proteins, antibodies, immunogenic proteins, therapeutic peptides and proteins, secreted proteins, plasma membrane proteins, cytoplasmic and cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease and / or proteins associated with nonhuman diseases, or a combination thereof. In some aspects, a formulation contains at least one polynucleotide encoding proteins. In some aspects, a formulation contains at least 1, 2, 3, 4, 5, or more polynucleotides encoding proteins.

[0306] The polynucleotides of the disclosure may be 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 of the present disclosure can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with polynucleotides of the disclosure (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof.

[0307] In some aspects, a particle size of the lipid nanoparticle may be increased and / or decreased. The change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of the modified polynucleotides delivered to an individual.300484454.1 - 69 -BAYM. P0444WO / BLG 25-024A. Lipidoids

[0308] The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of polynucleotides (see Mahon et al., Bioconjug Chem. 2010 21:1448-1454; Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol. 2008 26:561-569; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-3001; all of which are incorporated herein by reference in their entireties).

[0309] While lipidoids have been used to effectively deliver double stranded small interfering RNA molecules in rodents and non-human primates (see Akinc et al., Nat Biotechnol. 2008 26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci USA. 2008 105:11915-11920; Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 2011 29:1005-1010; all of which is incorporated herein in their entirety), the present disclosure describes their formulation and use for delivering polynucleotides described herein, e.g., in vitro transcribed polynucleotides.

[0310] Complexes, micelles, liposomes or particles can be prepared containing lipidoids and therefore, can result in an effective delivery of the polynucleotide, as judged by the production of an encoded protein, production of reactive antibodies, and / or induction of an immune response, in some examples, following the injection of a lipidoid formulation via localized and / or systemic routes of administration. Lipidoid complexes comprising polynucleotides can be administered by various means including, but not limited to, intravenous, intramuscular, or subcutaneous means.

[0311] In vivo delivery of nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, polynucleotide to lipid ratio, and biophysical parameters such as, but not limited to, particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety). As an example, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids may result in significant effects on in vivo efficacy. Formulations with the different lipidoids, including, but not limited to penta[3-(l-laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401:61 (2010); herein incorporated by reference in its entirety), C12-200 (including derivatives and variants), and MD1, can be tested for in vivo activity.

[0312] The characteristics of optimized lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. For example, a 300484454.1 - 70 -BAYM. P0444WO / BLG 25-024particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see, Akinc et al., Mol Ther. 2009 17:872-879 herein incorporated by reference in its entirety). Use of a lipidoid-formulated polynucleotide of the disclosure to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may or may not be similarly size-limited.

[0313] Use of lipidoid formulations to deliver siRNA in vivo to other non-hepatocyte cells such as myeloid cells and endothelium has been reported (see Akinc et al., Nat Biotechnol.200826:561-569; Leuschner et al., Nat Biotechnol. 2011 29:1005-1010; Cho et al. Adv. Funct. Mater. 2009 19:3112-3118; 8th International Judah Folkman Conference, Cambridge, Mass. Oct. 8-9, 2010; each of which is herein incorporated by reference in its entirety). Different ratios of lipidoids and other components including, but not limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG, may be used to optimize the formulation of the polynucleotides of the disclosure for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc. For example, the component molar ratio may include, but is not limited to, 50% C12-200, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and %1.5 PEG-DMG (see Leuschner et al., Nat Biotechnol 2011 29:1005-1010; herein incorporated by reference in its entirety). Effective delivery to myeloid cells, such as monocytes, lipidoid formulations may or may not have a similar component molar ratio. The use of lipidoid formulations for the localized delivery of nucleic acids to cells (such as, but not limited to, adipose cells and muscle cells) via either subcutaneous or intramuscular delivery, may not require all of the formulation components desired for systemic delivery, and as such may comprise only the lipidoid and polynucleotides of the disclosure.

[0314] Combinations of different lipidoids may be used to improve the efficacy of polynucleotides directed protein production as the lipidoids may be able to increase cell transfection; and / or increase the translation of encoded protein (see Whitehead et al., Mol. Ther. 2011, 19: 1688-1694, herein incorporated by reference in its entirety).B. Liposomes, Lipoplexes, and Lipid Nanoparticles

[0315] The polynucleotides of the disclosure may be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In some aspects, pharmaceutical compositions comprising polynucleotides of the disclosure include liposomes, lipoplexes, or lipid nanoparticles. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and / or pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a300484454.1 - 71 -BAYM. P0444WO / BLG 25-024multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments; a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter; and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.

[0316] The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and / or delivery of the vesicles, the optimization size, poly dispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.

[0317] As a non-limiting example, liposomes such as synthetic membrane vesicles may be prepared by the methods, apparatus and devices described in US Patent Publication No. US20130177638, US20130177637, US20130177636, US20130177635, US20130177634, US20130177633, US20130183375, US20130183373 and US20130183372, the contents of each of which are herein incorporated by reference in their entirety. For example, a NanoAssemblr Ignite™ instrument (Precision Nanosystems) may be used to generate a lipid nanoparticle of the disclosure.

[0318] In some aspects, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from 1, 2-di oleyloxy -N, N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), l,2-dilinoleyloxy-3 -dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXIL® from Janssen Biotech, Inc. (Horsham, Pa.).

[0319] In some aspects, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo 300484454.1 - 72 -BAYM. P0444WO / BLG 25-024(see Wheeler et al. Gene Therapy. 1999 6:271-281; Mang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006441:111-114; Heyes et al. J ContrRel. 2005 107:276-287; Semple et al. Nature Biotech. 201028:172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; U. S. Patent Publication No US20130122104; each of which is incorporated herein by reference in its entirety). The original manufacture method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method. The liposome formulations are composed of 3 to 4 lipid components in addition to the polynucleotide. As an example a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1, 2-di oleyloxy -N, N-dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be l,2-distearloxy-N, N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or l,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.

[0320] In some aspects, liposome formulations may comprise from about 25.0% cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol to about 45.0% cholesterol, from about 35.0% cholesterol to about 50.0% cholesterol and / or from about 48.5% cholesterol to about 60% cholesterol (w / w). In some aspects, formulations may comprise a percentage of cholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 37.5%, 38.5%, 39.0% and 43.5% (w / w). In some aspects, formulations may comprise from about 5.0% to about 10.0% DSPC and / or from about 7.0% to about 15.0% DSPC (w / w).

[0321] In some aspects, pharmaceutical compositions may include liposomes which may be formed to deliver polynucleotides which may encode at least one polypeptide of the disclosure, e.g., an immunogen (antigen), or any other polypeptide or combination of polypeptides of interest. The polynucleotides of the disclosure may be encapsulated by the liposome and / or it may be contained in an aqueous core which may then be encapsulated by the liposome (see International Pub. Nos. W02012031046, W02012031043, W02012030901 and W02012006378 and US Patent Publication No. US20130189351, US20130195969 and US20130202684; the contents of each of which are herein incorporated by reference in their entirety).

[0322] In some aspects, polynucleotides of the disclosure may be formulated in a water-in-oil emulsion comprising a continuous hydrophobic phase in which the hydrophilic phase is 300484454.1 - 73 -BAYM. P0444WO / BLG 25-024dispersed. As a non-limiting example, the emulsion may be made by the methods described in International Publication No. W0201087791, the contents of which are herein incorporated by reference in its entirety.

[0323] In some aspects, a lipid formulation may include at least a cationic lipid, a lipid which may enhance transfection, and / or a least one lipid which contains a hydrophilic head group linked to a lipid moiety (International Pub. No. WO2011076807 and U. S. Pub. No.20110200582; the contents of each of which is herein incorporated by reference in their entirety). In some aspects, polynucleotides of the disclosure, e.g., encoding an immunogen, may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers (see U. S. Pub. No. 20120177724, the contents of which is herein incorporated by reference in its entirety).

[0324] In some aspects, polynucleotides of the disclosure may be formulated in a lipsome as described in International Patent Publication No. WO2013086526, the contents of which is herein incorporated by reference in its entirety. Polynucleotides of the disclosure may be encapsulated in a liposome using reverse pH gradients and / or optimized internal buffer compositions as described in International Patent Publication No. WO2013086526, the contents of which is herein incorporated by reference in its entirety.

[0325] In some aspects, pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3 -phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

[0326] In some aspects, a cationic lipid may be a low molecular weight cationic lipid such as those described in US Patent Application No. 20130090372, the contents of which are herein incorporated by reference in its entirety.

[0327] In some aspects, polynucleotides of the disclosure may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.

[0328] In some aspects, polynucleotides of the disclosure may be formulated in a liposome comprising a cationic lipid. The liposome may have a molar ratio of nitrogen atoms in the cationic lipid to the phosphates in the RNA (N: P ratio) of between 1: 1 and 20: 1 as described in International Publication No. W02013006825, herein incorporated by reference in its entirety. In some aspects, a liposome may have a N: P ratio of greater than 20: 1 or less than 1: 1. In some aspects, a liposome may have a N: P ratio of 4:1.300484454.1 - 74 -BAYM. P0444WO / BLG 25-024

[0329] In some aspects, polynucleotides of the disclosure may be formulated in a lipidpolycation complex. The formation of a lipid-polycation complex may be accomplished by methods known in the art and / or as described in U. S. Pub. No. 20120178702, herein incorporated by reference in its entirety. As a non-limiting example, a polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and / or polyarginine and the cationic peptides described in International Pub. No. WO2012013326 or US Patent Pub. No. US20130142818; each of which is herein incorporated by reference in its entirety. In some aspects, polynucleotides of the disclosure may be formulated in a lipid-polycation complex which may further include a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

[0330] In some aspects, polynucleotides of the disclosure may be formulated in an aminoalcohol lipidoid. Aminoalcohol lipidoids which may be used may be prepared by the methods described in U. S. Pat. No. 8,450,298, herein incorporated by reference in its entirety.

[0331] A liposome formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. In one example by Semple et al. (Semple et al. Nature Biotech. 2010 28:172-176; herein incorporated by reference in its entirety), the liposome formulation was composed of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol, and 1.4% PEG-c-DMA (w / w). As another example, changing the composition of the cationic lipid could more effectively deliver siRNA to various antigen presenting cells (Basha et al. Mol Ther. 2011 19:2186-2200; herein incorporated by reference in its entirety). In some aspects, liposome formulations may comprise from about 35 to about 45% cationic lipid, from about 40% to about 50% cationic lipid, from about 50% to about 60% cationic lipid and / or from about 55% to about 65% cationic lipid. In some aspects, the ratio of lipid to mRNA in liposomes may be from about 5: 1 to about 20:1, from about 10:1 to about 25:1, from about 15:1 to about 30:1 and / or at least 30:1.

[0332] In some aspects, the ratio of PEG in the lipid nanoparticle (LNP) formulations may be increased or decreased and / or the carbon chain length of the PEG lipid may be modified from C14 to C18 to alter the pharmacokinetics and / or biodistribution of the LNP formulations. As a non-limiting example, LNP formulations may contain from about 0.5% to about 3.0%, from about 1.0% to about 3.5%, from about 1.5% to about 4.0%, from about 2.0% to about 4.5%, from about 2.5% to about 5.0% and / or from about 3.0% to about 6.0% of the lipid molar ratio (w / w) of PEG-c-DOMG (R-3-[(co-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-l,2-dimyristyloxypropyl-3-amine) (also referred to herein as PEG-DOMG) as compared to the 300484454.1 - 75 -BAYM. P0444WO / BLG 25-024cationic lipid, DSPC and cholesterol. In some aspects, PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG-DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG (1,2-Dimyristoyl-sn-glycerol) and / or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). A cationic lipid may be selected from any lipid known in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, Cl 2-200 and DLin-KC2-DMA.

[0333] In some aspects, polynucleotides of the disclosure may be formulated in a lipid nanoparticle such as those described in International Publication No. W02012170930, the contents of which is herein incorporated by reference in its entirety.

[0334] In some aspects, a polynucleotide formulation of the disclosure comprises a nanoparticle which may comprise at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids and amino alcohol lipids. In other aspects, the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationic lipid may be the lipids described in and / or made by the methods described in US Patent Publication No. US20130150625, herein incorporated by reference in its entirety. As a non-limiting example, the cationic lipid may be 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien- 1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-l-yloxy]methyl}propan-l-ol (Compound 1 in U S20130150625); 2-amino-3 -[(9Z)-octadec-9-en- 1 -yloxy]-2- { [(9Z)-octadec-9-en- 1 -yloxy]methyl}propan-l-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2-[(octyloxy)methyl]propan-l-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]methyl}propan-l-ol (Compound 4 in US20130150625); or any pharmaceutically acceptable salt or stereoisomer thereof.

[0335] Lipid nanoparticle formulations typically comprise a lipid, in particular, an ionizable cationic lipid, for example, 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-l-yl) 9-((4 (dimethylamino)butanoyl)oxy)heptadecanedioate (L319), and further comprise a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example a PEG or PEG-modified lipid. In some aspects, formulations may comprise from about 25.0% to about 70.0% of an ionizable lipid (w / w). In some aspects, formulations may comprise about 50.0% of an ionizable lipid. In some aspects, an ionizable lipid is one described in International Publication No. WO202052589A (incorporated herein 300484454.1 - 76 -BAYM. P0444WO / BLG 25-024by reference in its entirety). In some aspects, an ionizable lipid comprises D-Lin-MC3-DMA, OF-02, SM-102, ALC-0315, LPO1, or a combination thereof. In some aspects, an ionizable lipid is one described in Gautam et al., (2023) Nat Commun 14, 6468, which is incorporated herein by reference in its entirety.

[0336] Illustrative lipid nanoparticle compositions and methods of making same are described, for example, in Semple et al. (2010) Nat. Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed., 51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578 (the contents of each of which are incorporated herein by reference in their entirety). In some aspects, the cationic lipid may be selected from, but not limited to, a cationic lipid described in International Publication Nos. W02012040184, WO2011153120, WO2011149733, WO2011090965, WO2011043913, WO2011022460, WO2012061259, WO2012054365, WO2012044638, W02010080724, W0201021865, W02008103276, WO2013086373 and WO2013086354, U. S. Pat. Nos. 7,893,302, 7,404,969, 8,283,333, and 8,466,122 and US Patent Publication No. US20100036115, US20120202871, US20130064894, US20130129785, US20130150625, US20130178541 and US20130225836; the contents of each of which are herein incorporated by reference in their entirety. As a nonlimiting example, the cationic lipid may be selected from (20Z,23Z)-N, N-dimethylnonacosa-20,23 -dien- 10-amine, ( 17Z,20Z)-N, N-dimemylhexacosa- 17,20-dien-9-amine, ( 1Z, 19Z)-N5N-dimethylpentacosa- 16, 19-dien-8-amine, (13Z, 16Z)-N, N-dimethyldocosa- 13,16-dien-5-amine, ( 12Z, 15Z)-N, N-dimethylhenicosa- 12,15-dien-4-amine, ( 14Z, 17Z)-N, N-dimethyltricosa- 14, 17-dien-6-amine, (15Z, 18Z)-N, N-dimethyltetracosa-l 5, 18-dien-7-amine, ( 18Z,21 Z)-N, N-dimethylheptacosa- 18,21 -then- 10-amine, (15Z, 18Z)-N, N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N, N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N, N-dimethyloctacosa- 19,22-dien-9-amine, (18Z,21 Z)-N, N-dimethylheptacosa- 18,21 -dien-8-amine, (17Z,20Z)-N, N-dimethylhexacosa- 17,20-dien-7-amine, ( 16Z, 19Z)-N, N-dimethylpentacosa- 16,19-dien-6-amine, (22Z,25Z)-N, N-dimethylhentriaconta-22,25-dien- 10-amine, (21 Z,24Z)-N, N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N, N-dimetylheptacos-18-en- 10-amine, ( 17Z)-N, N-dimethylhexacos- 17-en-9-amine, ( 19Z,22Z)-N, N-dimethyloctacosa- 19,22-dien-7-amine, N, N-dimethylheptacosan- 10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23 -dien- 10-amine, 1-[(11 Z,14Z)-l-nonylicosa-l 1, 14-dien-l -yl] pyrrolidine, (20Z)-N, N-dimethylheptacos-20-en-l 0-amine, (15Z)-N, N-dimethyl eptacos-15-en- 10-amine, ( 14Z)-N, N-dimethylnonacos- 14-en- 10-amine, ( 17Z)-N, N-dimethylnonacos- 17-en- 10-amine, (24Z)-N, N-dimethyltritriacont-24-en- 10-amine, (20Z)-N, N-dimethylnonacos-20-en-l O-amine, (22Z)-N, N-dimethylhentriacont-22-en-l 0-amine, (16Z)-N, N- 300484454.1 - 77 -BAYM. P0444WO / BLG 25-024dimethylpentacos- 16-en-8-amine, (12Z, 15Z)-N, N-dimethyl-2-nonylhenicosa- 12, 15-dien- 1 -amine, ( 13Z, 16Z)-N, N-dimethyl-3 -nonyldocosa- 13,16-dien- 1 -amine, N, N-dimethyl- 1 -[(1 S,2R)-2-octylcyclopropyl]eptadecan-8-amine, l-[(lS,2R)-2-hexylcyclopropyl]-N, N-dimethylnonadecan-10-amine, N, N-dimethyl-l-[(lS,2R)-2-octylcyclopropyl]nonadecan-10-amine, N, N-dimethyl-21 -[( 1 S,2R)-2-octylcyclopropyl]henicosan- 10-amine, N, N-dimethyl- 1 -[(1 S,2S)-2-{[(lR,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N, N-dimethyl-l-[(lS,2R)-2-octylcyclopropyl]hexadecan-8-amine, N, N-dimethyl-[(lR,2S)-2-undecylcyclopropyl]tetradecan-5-amine, N, N-dimethyl-3-{7-[(l S,2R)-2-octylcyclopropyl]heptyl} dodecan-1 -amine, l-[(lR,2S)-2-heptylcyclopropyl]-N, N-dimethyloctadecan-9-amine, l-[(lS,2R)-2-decylcyclopropyl]-N, N-dimethylpentadecan-6-amine, N, N-dimethyl-l-[(l S,2R)-2-octylcyclopropyl]pentadecan-8-amine, R — N, N-dimethyl-l-[(9Z,12Z)-octadeca-9,12-then-l-yloxy]-3-(octyloxy)propan-2-amine, S — N, N-dimethyl-l-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-(octyloxy)propan-2-amine, l-{2-[(9Z,12Z)-octadeca-9, 12-dien- 1 -yloxy]-l -[(octyloxy)methyl]ethyl (pyrrolidine, (2S) — N, N-dimethyl- 1 - [(9Z,12Z)-octadeca-9,12-dien-l-yloxy]-3-[(5Z)-oct-5-en-l-yloxy]propan-2-amine, l-{2-[(9Z, 12Z)-octadeca-9, 12-dien- 1 -yloxy]- 1 -[(octyloxy )m ethyl] ethyl } azetidine, (2 S)- 1 - (hexyloxy)-N, N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N, N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien- l-yloxy]propan-2-amine, N, N-dimethyl-l-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine. N, N-dimethyl-l-[(9Z)-octadec-9-en-l-yloxy]-3-(octyloxy)propan-2-amine; (2S) — N, N-dimethyl-1 -[(6Z,9Z, 12Z)-octadeca-6,9, 12-trien- 1 -yloxy]-3 -(octyloxy)propan-2-amine, (2 S)- 1 - [(1 lZ,14Z)-icosa-l l,14-dien-l-yloxy]-N, N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)- 1 -(hexyloxy)-3-[(l lZ,14Z)-icosa-l l,14-dien-l-yloxy]-N, N-dimethylpropan-2-amine, 1- [(1 lZ,14Z)-icosa-l l,14-dien-l-yloxy]-N, N-dimethyl-3-(octyloxy)propan-2-amine, 1- [(13Z, 16Z)-docosa- 13,16-dien- 1 -yloxy]-N, N-dimethyl-3 -(octyloxy )propan-2-amine, (2S)- 1 -[(13Z, 16Z)-docosa- 13,16-dien- 1 -yloxy]-3 -(hexyloxy)-N, N-dimethylpropan-2-amine, (2S)- 1 - [(13Z)-docos- 13 -en- 1 -yloxy]-3 -(hexyloxy)-N, N-dimethylpropan-2-amine, 1 -[( 13Z)-docos- 13 -en- 1 -yloxy]-N, N-dimethyl-3 -(octyloxy )propan-2-amine, 1 -[(9Z)-hexadec-9-en- 1 -yloxy]-N, N-dimethyl-3-(octyloxy)propan-2-amine, (2R) — N, N-dimethyl-H(l-metoylo ctyl)oxy]-3-[(9Z,12Z)-octadeca-9,l 2-dien- l-yloxy]propan-2-amine, (2R)-l-[(3,7-dimethyloctyl)oxy]-N, N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, N, N-dimethyl-l-(octyloxy)-3-({8-[(lS,2S)-2-{[(lR,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N, N-dimethyl-l-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11E,2OZ,23Z)-N, N- 300484454.1 - 78 -BAYM. P0444WO / BLG 25-024dimethylnonacosa-ll,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.

[0337] In some aspects, a pharmaceutical composition comprising polynucleotides of the disclosure may include at least one of the PEGylated lipids described in International Publication No. WO2012099755, the contents of which is herein incorporated by reference in its entirety.

[0338] In some aspects, the LNP formulation may contain PEG-DMG 2000 (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000). In some aspects, a LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art and at least one other component. In some aspects, a LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art, DSPC, and / or cholesterol. As a non-limiting example, a LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol. As another non-limiting example a LNP formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40:10:48 (see e.g., Geall et al., Nonviral delivery of self-amplifying RNA vaccines, PNAS 2012; PMID: 22908294; herein incorporated by reference in its entirety).

[0339] In some aspects, LNP formulations may be formulated by the methods described in International Publication Nos. WO2011127255 or W02008103276, the contents of each of which is herein incorporated by reference in their entirety. As a non-limiting example, polynucleotides of the disclosure described herein may be encapsulated in LNP formulations as described in WO2011127255 and / or W02008103276; each of which is herein incorporated by reference in their entirety.

[0340] In some aspects, polynucleotides of the disclosure described herein may be formulated in a nanoparticle to be delivered by a parenteral route as described in U. S. Pub. No. US20120207845; the contents of which are herein incorporated by reference in its entirety. In some aspects, polynucleotides of the disclosure may be formulated in a lipid nanoparticle made by the methods described in US Patent Publication No US20130156845 or International Publication No WO2013093648 or WO2012024526, each of which is herein incorporated by reference in its entirety.

[0341] Lipid nanoparticles described herein may be made in a sterile environment by the system and / or methods described in US Patent Publication No. US20130164400, herein incorporated by reference in its entirety.300484454.1 - 79 -BAYM. P0444WO / BLG 25-024

[0342] As a non-limiting example, polynucleotides described herein may be encapsulated in LNP formulations as described in WO2011127255 and / or W02008103276; the contents of each of which are herein incorporated by reference in their entirety.

[0343] Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier. Mucus is located on mucosal tissue such as, but not limited to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes). Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers. Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosal tissue within seconds or within a few hours. Large polymeric nanoparticles (200 nm to 500 nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4- to 6-fold lower than the same particles diffusing in water (Lai et al. PNAS 2007 104(5): 1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2): 158-171; each of which is herein incorporated by reference in their entirety). The transport of nanoparticles may be determined using rates of permeation and / or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT). As a non-limiting example, compositions which can penetrate a mucosal barrier may be made as described in U. S. Pat. No. 8,241,670 or International Patent Publication No. WO2013110028, the contents of each of which are herein incorporated by reference in its entirety.

[0344] In some aspects, polynucleotides of the disclosure may be delivered using smaller LNPs. In some aspects a LNP or a population of LNPs may may comprise a diameter or average diameter from below 0.1 pm up to 100 nm such as, but not limited to, less than 0.1 pm, less than 1.0 pm, less than 5 pm, less than 10 pm, less than 15 pm, less than 20 pm, less than 25 pm, less than 30 pm, less than 35 pm, less than 40 pm, less than 50 pm, less than 55 pm, less than 60 pm, less than 65 pm, less than 70 pm, less than 75 pm, less than 80 pm, less than 85 pm, less than 90 pm, less than 95 pm, less than 100 pm, less than 125 pm, less than 150 pm, less than 175 pm, less than 200 pm, less than 225 pm, less than 250 pm, less than 275 pm, less than 300 pm, less than 325 pm, less than 350 pm, less than 375 pm, less than 400 pm, less than 425 pm, less than 450 pm, less than 475 pm, less than 500 pm, less than 525 pm, less 300484454.1 - 80 -BAYM. P0444WO / BLG 25-024than 550 pm, less than 575 pm, less than 600 pm, less than 625 pm, less than 650 pm, less than 675 pm, less than 700 pm, less than 725 pm, less than 750 pm, less than 775 pm, less than 800 pm, less than 825 pm, less than 850 pm, less than 875 pm, less than 900 pm, less than 925 pm, less than 950 pm, less than 975 pm, 1000 pm. In some aspects, a population of LNPs comprise an average diameter of about 90 nm. In some aspects, a population of LNPs comprise an average diameter of about 90 nm with a poly dispersity index of less than about 15%.

[0345] In some aspects, polynucleotides of the disclosure may be delivered using LNPs which may comprise a diameter from about 1 nm to about 100 nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, from about 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1 nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm to about 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90 nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10 nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, from about 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5 nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm to about 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nm, from about 20 to about 50 nm, from about 30 to about 50 nm, from about 40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about 60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, from about 30 to about 70 nm, from about 40 to about 70 nm, from about 50 to about 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm, from about 30 to about 80 nm, from about 40 to about 80 nm, from about 50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about 90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, from about 50 to about 90 nm, from about 60 to about 90 nm and / or from about 70 to about 90 nm.

[0346] In some aspects, LNPs may be synthesized using methods comprising microfluidic mixers. Exemplary microfluidic mixers may include, but are not limited to a slit interdigital micromixer including, but not limited to those manufactured by Microinnova (Allerheiligen bei Wildon, Austria) and / or a staggered herringbone micromixer (SHM) (Zhigaltsev, I. V. et al., Bottom-up design and synthesis of limit size lipid nanoparticle systems with aqueous and triglyceride cores using millisecond microfluidic mixing have been published (Langmuir.2012. 28:3633-40; Belliveau, N. M. et al., Microfluidic synthesis of highly potent limit-size lipid nanoparticles for in vivo delivery of siRNA. Molecular Therapy-Nucleic Acids. 2012.1:e37; Chen, D. et al., Rapid discovery of potent siRNA-containing lipid nanoparticles enabled by controlled microfluidic formulation. J Am Chem Soc. 2012. 134(16):6948-51; each of 300484454.1 - 81 -BAYM. P0444WO / BLG 25-024which is herein incorporated by reference in its entirety). In some aspects, methods of LNP generation comprising SHM, may further comprise the mixing of at least two input streams wherein mixing occurs by microstructure-induced chaotic advection (MICA). According to this method, fluid streams flow through channels present in a herringbone pattern causing rotational flow and folding the fluids around each other. This method may also comprise a surface for fluid mixing wherein the surface changes orientations during fluid cycling. Methods of generating LNPs using SHM include those disclosed in U. S. Application Publication Nos.2004 / 0262223 and 2012 / 0276209, each of which is expressly incorporated herein by reference in their entirety.

[0347] In some aspects, polynucleotides of the disclosure may be formulated in lipid nanoparticles created using a micromixer such as, but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2) or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar (CPMM) or Impinging j et (UMM) from the Institut fur Mikrotechnik Mainz GmbH, Mainz Germany). In some aspects, polynucleotides of the disclosure may be formulated in lipid nanoparticles created using a NanoAssemblr Ignite™ instrument (Precision Nanosystems) and a Genvoy ILM lipid reagent (Precision Nanosystems, Cat. NWW0042).

[0348] In some aspects, polynucleotides of the disclosure may be formulated in lipid nanoparticles created using microfluidic technology (see Whitesides, George M. The Origins and the Future of Microfluidics. Nature, 2006442: 368-373; and Abraham et al. Chaotic Mixer for Microchannels. Science, 2002 295: 647-651; each of which is herein incorporated by reference in its entirety). As a non-limiting example, controlled microfluidic formulation includes a passive method for mixing streams of steady pressure-driven flows in micro channels at a low Reynolds number (See e.g., Abraham et al. Chaotic Mixer for Microchannels. Science, 2002295: 647-651; which is herein incorporated by reference in its entirety).

[0349] In some aspects, polynucleotides of the disclosure may be formulated in lipid nanoparticles created using a micromixer chip such as, but not limited to, those from Harvard Apparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). A micromixer chip can be used for rapid mixing of two or more fluid streams with a split and recombine mechanism.

[0350] In some aspects, polynucleotides of the disclosure may be formulated in lipid nanoparticles having a diameter from about 10 to about 100 nm such as, but not limited to, about 10 to about 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10 to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about 10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm, about 20 to about 40 nm, about 20 to about 50 nm, 300484454.1 - 82 -BAYM. P0444WO / BLG 25-024about 20 to about 60 nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about 90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 to about 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30 to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about 40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm, about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100 nm, about 50 to about 60 nm, about 50 to about 70 nm about 50 to about 80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 to about 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60 to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about 70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nm and / or about 90 to about 100 nm. In some aspects, lipid nanoparticles may have a diameter from about 10 to 500 nm.

[0351] In some aspects, a lipid nanoparticle may be a limit size lipid nanoparticle described in International Patent Publication No. WO2013059922, the contents of which are herein incorporated by reference in its entirety. The limit size lipid nanoparticle may comprise a lipid bilayer surrounding an aqueous core or a hydrophobic core; where the lipid bilayer may comprise a phospholipid such as, but not limited to, diacylphosphatidylcholine, a diacylphosphatidylethanolamine, a ceramide, a sphingomyelin, a dihydrosphingomyelin, a cephalin, a cerebroside, a C8-C20 fatty acid diacylphosphatidylcholine, and l-palmitoyl-2-oleoyl phosphatidylcholine (POPC). In another aspect the limit size lipid nanoparticle may comprise a polyethylene glycol-lipid such as, but not limited to, DLPE-PEG, DMPE-PEG, DPPC-PEG and DSPE-PEG.C. Excipients

[0352] Pharmaceutical formulations of the disclosure may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but are not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, flavoring agents, stabilizers, antioxidants, osmolality adjusting agents, pH adjusting agents, and the like, as suited to the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, Md., 2006; incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by300484454.1 - 83 -BAYM. P0444WO / BLG 25-024producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.

[0353] In some aspects, a pharmaceutically acceptable excipient may be at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some aspects, an excipient is approved for use for humans and for veterinary use. In some aspects, an excipient may be approved by United States Food and Drug Administration. In some aspects, an excipient may be of pharmaceutical grade. In some aspects, an excipient may meet the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0354] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in pharmaceutical compositions. The composition may also include excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents.

[0355] Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and / or combinations thereof.

[0356] Illustrative granulating and / or dispersing agents include, but are not limited to, potato starch, com starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (VEEGUM®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and / or combinations thereof.

[0357] Illustrative surface active agents and / or emulsifiers include, but are not limited to, natural emulsifiers (e.g., acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), 300484454.1 - 84 -BAYM. P0444WO / BLG 25-024colloidal clays (e.g., bentonite [aluminum silicate] and VEEGUM® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g., carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g., polyoxyethylene sorbitan monolaurate [TWEEN®20], polyoxyethylene sorbitan [TWEEN®60], polyoxyethylene sorbitan monooleate [TWEEN®80], sorbitan monopalmitate [SPAN®40], sorbitan monostearate [SPAN®60], sorbitan tristearate [SPAN®65], glyceryl monooleate, sorbitan monooleate [SPAN®80]), polyoxyethylene esters (e.g., polyoxyethylene monostearate [MYRJ®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g., CREMOPHOR®), polyoxyethylene ethers, (e.g., polyoxyethylene lauryl ether [BRIJ®30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC®F 68, POLOXAMER®188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and / or combinations thereof.

[0358] Illustrative binding agents include, but are not limited to, starch (e.g., cornstarch and starch paste); gelatin; sugars (e.g., sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); amino acids (e.g., glycine); natural and synthetic gums (e.g., acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (VEEGUM®), and larch arabolactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water, alcohol; etc.; and combinations thereof.

[0359] Illustrative preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and / or other preservatives. Oxidation is a potential degradation pathway for mRNA, especially for liquid mRNA formulations. In order to prevent oxidation, antioxidants can be added to the formulation. Illustrative antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, ascorbyl palmitate, benzyl alcohol, butylated hydroxyanisole, 300484454.1 - 85 -BAYM. P0444WO / BLG 25-024EDTA, m-cresol, methionine, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, thioglycerol and / or sodium sulfite. Illustrative chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and / or trisodium edetate. Illustrative antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and / or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and / or sorbic acid. Illustrative alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and / or phenylethyl alcohol. Illustrative acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and / or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL®115, GERMABEN®II, NEOLONE™, KATHON™, and / or EUXYL®.

[0360] In some aspects, the pH of a formulation of the disclosure may be maintained between pH 5 and pH 8 to improve stability of the polynucleotides. Illustrative buffers to control pH may include, but are not limited to sodium phosphate, sodium citrate, sodium succinate, histidine (or histidine-HCl), sodium carbonate, and / or sodium malate. In some aspects, the illustrative buffers listed above may be used with additional monovalent counterions (including, but not limited to potassium). Divalent cations may also be used as buffer counterions; however, these are not always preferred due to complex formation and / or mRNA degradation.

[0361] Illustrative buffering agents may also include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium 300484454.1 - 86 -BAYM. P0444WO / BLG 25-024gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and / or combinations thereof.

[0362] Illustrative lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.

[0363] Illustrative oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, chamomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macadamia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughly, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree, thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyl dodecanol, oleyl alcohol, silicone oil, and / or combinations thereof.

[0364] Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents can be present in the composition, according to the judgment of the formulator.

[0365] Illustrative additives include physiologically biocompatible buffers (e.g., trimethylamine hydrochloride), addition of chelants (such as, for example, DTPA or DTPA-bisamide) or calcium chelate complexes (as for example calcium DTPA, CaNaDTPA-bisamide), or, optionally, additions of calcium or sodium salts (for example, calcium chloride, calcium ascorbate, calcium gluconate or calcium lactate). In addition, antioxidants and suspending agents may be used.300484454.1 - 87 -BAYM. P0444WO / BLG 25-024D. Cryoprotectants

[0366] In some aspects, formulations of the disclosure may comprise one or more cryoprotectant. As used herein, there term “cryoprotectant” refers to one or more agent that when combined with a given substance, helps to reduce or eliminate damage to that substance that occurs upon freezing. In some aspects, cryoprotectants are combined with polynucleotides of the disclosure and / or compositions of the disclosure, e.g., lipid nanoparticles comprising polynucleotides of the disclosure, to stabilize them during freezing. Frozen storage of polynucleotides of the disclosure between -20 °C. and -80 °C. may be advantageous for long term (e.g., 36 months) stability of the polynucleotides and / or compositions of the disclosure. In some aspects, cryoprotectants are included in formulation to stabilize the polynucleotides and / or compositions of the disclosure through freeze / thaw cycles and under frozen storage conditions. Cryoprotectants of the present disclosure may include, but are not limited to sucrose, trehalose, lactose, glycerol, dextrose, raffinose and / or mannitol. Trehalose is listed by the Food and Drug Administration as being generally regarded as safe (GRAS) and is commonly used in commercial pharmaceutical formulations.

[0367] In some aspects, a cryoprotectant comprises from between about 1% to about 40% of a composition (w / w). In some aspects, a cryoprotectant comprises about 8% (w / w) of a composition. In some aspects, a cryoprotectant comprises about 8% sucrose in PBS (w / w).E. Adjuvants

[0368] Adjuvants or immune potentiators, may also be administered with or in combination with one or more polynucleotides of the disclosure. Advantages of adjuvants include the enhancement of the immunogenicity of antigens, modification of the nature of the immune response, the reduction of the antigen amount needed for a successful immunization, the reduction of the frequency of booster immunizations needed and an improved immune response in vaccines for the elderly and immunocompromised. Adjuvants may be coadministered by any route, e.g., intramuscular, subcutaneous, intravenous, and / or intradermal routes.

[0369] Adjuvants useful in the present disclosure may include, but are not limited to, natural or synthetic. They may be organic or inorganic. Adjuvants may be selected from any of the following groups: 1) mineral salts, e.g., aluminum hydroxide and aluminum or calcium phosphate gels; 2) emulsions including: oil emulsions and surfactant based formulations, e.g., microfluidised detergent stabilized oil-in-water emulsion, purified saponin, oil-in-water emulsion, stabilized water-in-oil emulsion; 3) particulate adjuvants, e.g., virosomes300484454.1 - 88 -BAYM. P0444WO / BLG 25-024(unilamellar liposomal vehicles incorporating influenza hemagglutinin), structured complex of saponins and lipids, polylactide co-glycolide (PLG); 4) microbial derivatives; 5) endogenous human immunomodulators; 6) inert vehicles, such as gold particles; 7) microorganism derived adjuvants; 8) tensoactive compounds; 9) carbohydrates; or combinations thereof.

[0370] Adjuvants for DNA nucleic acid vaccines have been disclosed in, for example, Kobiyama, et al Vaccines, 2013, 1(3), 278-292, the contents of which are incorporated herein by reference in their entirety. Any of the adjuvants disclosed by Kobiyama may be used in the polynucleotides and / or compositions of the disclosure of the present disclosure.

[0371] Other adjuvants which may be utilized with the polynucleotides and / or compositions of the disclosure include any of those listed on the web-based vaccine adjuvant database, Vaxjo; http: / / www.violinet.org / vaxjo / and described in for example Sayers, et al., J. Biomedicine and Biotechnology, volume 2012 (2012), Article ID 831486, 13 pages, the content of which is incorporated herein by reference in its entirety.

[0372] Selection of appropriate adjuvants will be evident to one of ordinary skill in the art. Specific adjuvants may include, without limitation, cationic liposome-DNA complex JVRS-100, aluminum hydroxide vaccine adjuvant, aluminum phosphate vaccine adjuvant, aluminum potassium sulfate adjuvant, alhydrogel, ISCOM(s)™, Freund's Complete Adjuvant, Freund's Incomplete Adjuvant, CpG DNA Vaccine Adjuvant, Cholera toxin, Cholera toxin B subunit, Liposomes, Saponin Vaccine Adjuvant, DDA Adjuvant, Squalene-based Adjuvants, Etx B subunit Adjuvant, IL-12 Vaccine Adjuvant, LTK63 Vaccine Mutant Adjuvant, TiterMax Gold Adjuvant, Ribi Vaccine Adjuvant, Montanide ISA 720 Adjuvant, Corynebacterium-derived P40 Vaccine Adjuvant, MPL™ Adjuvant, AS04, AS02, Lipopolysaccharide Vaccine Adjuvant, Muramyl Dipeptide Adjuvant, CRL1005, Killed Corynebacterium parvum Vaccine Adjuvant, Montanide ISA 51, Bordetella pertussis component Vaccine Adjuvant, Cationic Liposomal Vaccine Adjuvant, Adamantylamide Dipeptide Vaccine Adjuvant, Arlacel A, VSA-3 Adjuvant, Aluminum vaccine adjuvant, Polygen Vaccine Adjuvant, Adj urn er™, Algal Glucan, Bay R1005, Theramide®, Stearyl Tyrosine, Specol, Algammulin, Avridine®, Calcium Phosphate Gel. CTA1-DD gene fusion protein, DOC / Alum Complex. Gamma Inulin, Gerbu Adjuvant, GM-CSF, GMDP, Recombinant hlFN-gamma / Interferon-g, Interleukin- ip, Interleukin-2, Interleukin-7, Sclavo peptide, Rehydragel LV, Rehydragel HP A, Loxoribine, MF59, MTP-PE Liposomes, Murametide, Murapalmitine, D-Murapalmitine, NAGO, NonIonic Surfactant Vesicles, PMMA, Protein Cochleates, QS-21, SPT (Antigen Formulation), nanoemulsion vaccine adjuvant, AS03, Quil-A vaccine adjuvant, RC529 vaccine adjuvant, LTR1920 Vaccine Adjuvant, E. coli heat-labile toxin, LT, amorphous aluminum 300484454.1 - 89 -BAYM. P0444WO / BLG 25-024hydroxyphosphate sulfate adjuvant, Calcium phosphate vaccine adjuvant, Montanide Incomplete Seppic Adjuvant, Imiquimod, Resiquimod, AF03, Flagellin, Poly(I: C), ISCOMATRIX®, Abisco-100 vaccine adjuvant, Albumin-heparin microparticles vaccine adjuvant. AS-2 vaccine adjuvant, B7-2 vaccine adjuvant, DHEA vaccine adjuvant, Immunoliposomes Containing Antibodies to Costimulatory Molecules, SAF-1, Sendai Proteoliposomes, Sendai-containing Lipid Matrices, Threonyl muramyl dipeptide (TMDP), Ty Particles vaccine adjuvant, Bupivacaine vaccine adjuvant, DL-PGL (Polyester poly (DL-lactide-co-glycolide)) vaccine adjuvant, IL- 15 vaccine adjuvant, LTK72 vaccine adjuvant, MPL-SE vaccine adjuvant, non-toxic mutant E112K of Cholera Toxin mCT-E112K, and / or Matrix- S.

[0373] Other adjuvants which may be co-administered with the polynucleotides and / or compositions of the disclosure include, but are not limited to interferons, TNF-alpha, TNF-beta, chemokines such as CCL21, eotaxin, HMGB1, SA100-8alpha, GCSF, GMCSF, granulysin, lactoferrin, ovalbumin, CD-40L, CD28 agonists, PD-1, soluble PD1-L1 or -L2, or interleukins such as IL-1, IL-2, IL-4, IL-6, IL-7, IL-10. IL-12, IL-13, IL-21. IL-23, IL-15, IL-17, and IL-18.F. Valency

[0374] Polynucleotides of the disclosure may vary in their valency. Valency refers to the number of antigenic components in the compositions of the disclosure, e.g., nucleic acid vaccine (e.g., mRNA vaccine). In some aspects, polynucleotides of the disclosure are monovalent. In some aspects, polynucleotides of the disclosure are divalent. In some aspects, polynucleotides of the disclosure are trivalent. In some aspects, polynucleotides of the disclosure are multi-valent. Multivalent polynucleotides (e.g., mRNA vaccines) may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens or antigenic moieties (e.g., antigenic peptides, etc.). The antigenic components of polynucleotides of the disclosure may be on a single polynucleotide or on separate polynucleotides.G. Dosage

[0375] In some aspects, an amount of polynucleotide (e.g., RNA) administered to an individual can range from 0.1 pg to 300 pg, 0.5 pg to 200 pg, or 1 pg to 100 pg, such as about 1 pg, about 3 pg, about 10 pg, about 30 pg, about 50 pg, or about 100 pg may be administered per dose. In some aspects, a polynucleotide or composition of the disclosure is administered of a single dose. In some aspects, a polynucleotide or composition of the disclosure is300484454.1 - 90 -BAYM. P0444WO / BLG 25-024administered as a priming dose followed by one or more booster doses. A booster dose or the first booster dose may be administered 7 to 28 days or 14 to 24 days following administration of the priming dose.

[0376] In some aspects, an amount of polynucleotide (e.g., RNA) described herein of 60 pg or lower, 50 pg or lower, 40 pg or lower, 30 pg or lower, 20 pg or lower, 10 pg or lower, 5 pg or lower, 2.5 pg or lower, or 1 pg or lower may be administered per dose.

[0377] In some aspects, an amount of polynucleotide (e.g., RNA) described herein of at least 0.25 pg, at least 0.5 pg, at least 1 pg, at least 2 pg, at least 3 pg, at least 4 pg, at least 5 pg, at least 10 pg, at least 20 pg, at least 30 pg, or at least 40 pg may be administered per dose.

[0378] In some aspects, an amount of polynucleotide (e.g., RNA) described herein of 0.25 pg to 60 pg, 0.5 pg to 55 pg, 1 pg to 50 pg, 5 pg to 40 pg, or 10 pg to 30 pg may be administered per dose.

[0379] In some aspects, an amount of polynucleotide (e.g., RNA) described herein of about 10 pg is administered per dose. In some aspects, an amount of polynucleotide (e.g., RNA) described herein of about 20 pg is administered per dose. In some aspects, at least two of such doses are administered. For example, a second dose may be administered about 21 days following administration of the first dose.

[0380] In some aspects, a composition of the disclosure comprises polynucleotides of the disclosure (e.g., RNA comprise in a LNP) at a concentration between about 50-1000 pg / mL. In some aspects, a composition of the disclosure comprises polynucleotides of the disclosure (e.g., RNA comprise in a LNP) at a concentration of about 50 pg / mL, 60 pg / mL, 70 pg / mL, 80 pg / mL, 90 pg / mL, 100 pg / mL, 110 pg / mL, 120 pg / mL, 130 pg / mL, 140 pg / mL, 150 pg / mL, 160 pg / mL, 170 pg / mL, 180 pg / mL, 190 pg / mL, 200 pg / mL, 210 pg / mL, 220 pg / mL, 230 pg / mL, 240 pg / mL, 250 pg / mL, 260 pg / mL, 270 pg / mL, 280 pg / mL, 290 pg / mL, 300 pg / mL, 310 pg / mL, 320 pg / mL, 330 pg / mL, 340 pg / mL, 350 pg / mL, 360 pg / mL, 370 pg / mL, 380 pg / mL, 390 pg / mL, 400 pg / mL, 410 pg / mL, 420 pg / mL, 430 pg / mL, 440 pg / mL, 450 pg / mL, 460 pg / mL, 470 pg / mL, 480 pg / mL, 490 pg / mL, 500 pg / mL, 510 pg / mL, 520 pg / mL, 530 pg / mL, 540 pg / mL, 550 pg / mL, 560 pg / mL, 570 pg / mL, 580 pg / mL, 590 pg / mL, 600 pg / mL, 610 pg / mL, 620 pg / mL, 630 pg / mL, 640 pg / mL, 650 pg / mL, 660 pg / mL, 670 pg / mL, 680 pg / mL, 690 pg / mL, 700 pg / mL, 710 pg / mL, 720 pg / mL, 730 pg / mL, 740 pg / mL, 750 pg / mL, 760 pg / mL, 770 pg / mL, 780 pg / mL, 790 pg / mL, 800 pg / mL, 810 pg / mL, 820 pg / mL, 830 pg / mL, 840 pg / mL, 850 pg / mL, 860 pg / mL, 870 pg / mL, 880 pg / mL, 890 pg / mL, 900 pg / mL, 910 pg / mL, 920 pg / mL, 930 pg / mL, 940 pg / mL, 950 pg / mL, 960 pg / mL, 970 pg / mL, 980 pg / mL, 990 pg / mL, or 1000 pg / mL.300484454.1 - 91 -BAYM. P0444WO / BLG 25-024V. Therapeutics

[0381] Polynucleotides of the disclosure may be used as therapeutic or prophylactic agents. They may be provided for use in medicine and / or for the priming of immune effector cells, e.g., to stimulate / transfect PBMCs ex vivo and re-infuse the activated cells. For example, a polynucleotide of the disclosure may be administered to a subject, wherein the polynucleotides is translated in vivo to produce an antigen. Provided are compositions, methods, kits, and reagents for diagnosis, treatment or prevention of a disease or condition in humans and other mammals. The active therapeutic agents of the disclosure include polynucleotides of the disclosure, cells containing polynucleotides of the disclosure, or polypeptides translated from polynucleotides of the disclosure.

[0382] Provided herein are methods of inducing translation of a polypeptide (e.g., antigen or immunogen) in a cell, tissue, or organism using polynucleotides or compositions of the disclosure. Such translation can be in vivo, ex vivo, in culture, or in vitro. In some aspects, translation is in vivo. In some aspects, a cell, tissue, or organism is contacted with an effective amount of a composition containing a polynucleotide of the disclosure that has one or more translatable regions encoding a polypeptide of interest (e.g., antigen or immunogen).

[0383] An “effective amount” of composition is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the composition or polynucleotide, and other determinants. In general, an effective amount of the polynucelotide composition provides an induced or boosted immune response as a function of antigen production in the cell, preferably more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen. Increased antigen production may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the polynucleotide), increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, e.g., by increased duration of protein translation from a modified polynucleotide), or altered innate immune response of the host cell.

[0384] Some aspects of the disclosure are directed to methods of inducing in vivo translation of a polypeptide antigen in a mammalian subject in need thereof. Therein, an effective amount of a polypeptide composition that has at least one structural or chemical modification and a translatable region encoding the polypeptide (e.g., antigen or immunogen) is administered to the subject using a delivery method described herein. The polynucleotide may be provided in an amount and under other conditions such that the polynucleotide is300484454.1 - 92 -BAYM. P0444WO / BLG 25-024translated in the cell. The cell in which the polynucleotide is localized, or the tissue in which the cell is present, may be targeted with one or more instances of administration.

[0385] In certain aspects, administered polynucleotides of the disclosure direct production of one or more polypeptides that provide a functional immune system-related activity which is substantially absent in the cell, tissue, or organism in which the polypeptide is translated.

[0386] The polypeptides described herein may be engineered for localization within the cell, potentially within a specific compartment such as the cytoplasm or nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell. In some aspects, polynucleotides and their encoded polypeptides in accordance with the present disclosure may be used for treatment of a disease and / or condition, such as parasitic infections. The subject to whom a composition of the disclosure or therapeutic agent may be administered suffers from or may be at risk of developing a disease, disorder, or deleterious condition.

[0387] In some aspects, a therapeutic agent may be administered in addition to a composition of the disclosure. Compositions of the disclosure and / or therapeutic agents can be administered simultaneously, for example in a combined unit dose (e.g., providing simultaneous delivery of two or more agents). The compositions of the disclosure and / or therapeutic agents can also be administered at a specified time interval, such as, but not limited to, an interval of minutes, hours, days or weeks. Generally, the compositions of the disclosure and / or therapeutic agents may be concurrently bioavailable, e.g., detectable, in a subject. In some aspects, the compositions of the disclosure and / or therapeutic agents may be administered essentially simultaneously, for example two unit dosages administered at the same time, or a combined unit dosage of the two agents. In other aspects, the compositions of the disclosure and / or therapeutic agents may be delivered in separate unit dosages. The compositions of the disclosure and / or therapeutic agents may be administered in any order, or as one or more preparations that includes two or more compositions of the disclosure and / or therapeutic agents. In some aspects, at least one administration of one of the compositions of the disclosure and / or therapeutic agents, e.g., a first administration, may be made within minutes, one, two, three, or four hours, or even within one or two days of another administration, e.g., a second administration. In some aspects, combinations can achieve synergistic results, e.g., greater than additive results, e.g., at least, at most, about, or any value between any two of 25, 50, 75, 100, 200, 300, 400, 500% than additive results.300484454.1 - 93 -BAYM. P0444WO / BLG 25-024A. Modulation of the Immune Response

[0388] According to the present disclosure, polynucleotides disclosed herein, e.g., RNA vaccines, may act as a single composition as a vaccine. As used herein, a “vaccine” refers to a composition, for example, a substance or preparation that stimulates, induces, causes or improves immunity in an organism, e.g., an animal organism, for example, a mammalian organism (e.g., a human.) Preferably, a vaccine provides immunity against one or more diseases or disorders in the organism, including prophylactic and / or therapeutic immunity. Illustrative vaccines includes one or more agents that resembles an infectious agent, e.g., a disease-causing microorganism, and can be made, for example, from live, attenuated, modified, weakened or killed forms of disease-causing microorganisms, or antigens derived therefrom, including combinations of antigenic components. In some aspects, a vaccine stimulates, induces causes or improves immunity in an organism or causes or mimics infection in the organism without inducing any disease or disorder. A vaccine may introduce an antigen into the tissues, extracellular space, or cells of a subject and elicit an immune response, thereby protecting the subject from a particular disease or pathogen infection. The polynucleotides disclosed herein may encode an antigen and when the polynucleotides are expressed in cells, a desired immune response is achieved.

[0389] Polynucleotides of the 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.

[0390] Polynucleotides of the disclosure may also be administered as a second line treatment after the standard first line treatments, such as antiparasitic drugs. In this regard, the polynucleotides of the disclosure of the present disclosure are useful in settings where resistance to first line treatments has developed and disease and / or infection persists and induces chronic disease.

[0391] The use of RNA in or as a vaccine overcomes the disadvantages of conventional genetic vaccination involving incorporating DNA into cells in terms of safeness, feasibility, applicability, and effectiveness to generate immune responses. RNA molecules are considered to be significantly safer than DNA vaccines, as RNAs are more easily degraded. They are cleared quickly out of the organism and cannot integrate into the genome and influence the cell’s gene expression in an uncontrollable manner. It is also less likely for RNA vaccines to cause severe side effects like the generation of autoimmune disease or anti-DNA antibodies (Bringmann A. et al., Journal of Biomedicine and Biotechnology (2010), vol. 2010, article ID623687). Transfection with RNA requires only insertion into the cell’s cytoplasm, which is 300484454.1 - 94 -BAYM. P0444WO / BLG 25-024easier to achieve than into the nucleus. However, RNA is susceptible to RNase degradation and other natural decomposition.

[0392] Various attempts to increase the stability and shelf life of RNA vaccines. U. S. publication 2005 / 0032730 to Von Der Mulbe et al. (incorporated herein by reference in its entirety) discloses improving the stability of mRNA vaccine compositions by increasing G(guanosine) / C(cytosine) content of the mRNA molecules; U. S. Pat. No. 5,580,859 to Feigner et al. (incorporated herein by reference in its entirety) teaches incorporating polynucleotide sequences coding for regulatory proteins that bind to and regulate the stability of mRNA. While not wishing to be bound by theory, it is believed that the polynucleotides disclosed herein will result in improved stability and therapeutic efficacy due at least in part to the specificity, purity, and selectivity of the construct designs.

[0393] In some aspects, a polynucleotide may encode at least one polypeptide of interest (e.g., an antigen) and may be provided to an individual to stimulate the immune system to protect the individual against a disease-causing agents (e.g., a parasite). As a non-limiting example, a polynucleotide encoding an immunogen may be delivered to a vertebrate in a dose amount large enough to be immunogenic to the vertebrate (see International Pub. No. WO2012006372 and WO2012006369 and US Publication No. US20130149375 and US20130177640; the contents of each of which are herein incorporated by reference in their entirety).

[0394] In some aspects, polynucleotides may be utilized in various settings depending on the prevalence of the infection or the degree or level of an unmet medical need. As a nonlimiting example, the polynucleotides disclosed herein may be utilized to treat and / or prevent helminth (e.g., hookworm) infection. Symptoms of helminth infection include itchy rash, respiratory and / or gastrointestinal problems, iron deficiency anemia, blood loss, abdominal pain, diarrhea, dysentery, general malaise, weakness, impaired growth and physical development, intestinal obstruction, or a combination thereof.

[0395] In another non-limiting example, the polynucleotides of the present disclosure may be formulated with an excipient for administration as described herein and / or known in the art (see e.g., U. S. Publication No. US20120213812, which is herein incorporated by reference in its entirety).

[0396] In one aspect, polynucleotides of the disclosure may be administrated with other prophylactic or therapeutic compounds. As a non-limiting example, the 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 300484454.1 - 95 -BAYM. P0444WO / BLG 25-024administration of the prophylactic 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, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 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, 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, or any value therebetween.

[0397] In one aspect, polynucleotides of the disclosure may be administered intranasally similar to the administration of live vaccines. In another aspect, polynucleotides may be administered intramuscularly or intradermally similarly to the administration of inactivated vaccines known in the art.B. Anti-Parasitic Agents

[0398] In some aspects, an anti-parasitic agent may be administered before, concurrently, or after administration of a composition of the disclosure (e.g., a polynucleotide). Illustrative anti-parasitic agents include, but are not limited to, antinematodes (e.g., mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine, ivermectin), anticestodes (e.g., niclosamide, praziquantel, albendazole), antitrematodes (e.g., praziquantel), antiamoebics (e.g., rifampin, amphotericin B), and antiprotozoal s (e.g., melarsoprol, eflornithine, metronidazole, tinidazole).VI. Transfection

[0399] As used herein, the term “transfection” is defined as the introduction of an extracellular nucleic acid into a host cell by any means known in the art, including, but not limited to, calcium phosphate co-precipitation, liposome / lipid nanoparticle fusion, microinjection, microparticle bombardment, electroporation. The terms “uptake of nucleic acid by a host cell,” “taking up of nucleic acid by a host cell,” “uptake of particles comprising nucleic acid by a host cell,” and “taking up of particles comprising nucleic acid by a host cell”300484454.1 - 96 -BAYM. P0444WO / BLG 25-024denote any process wherein an extracellular nucleic acid, with or without accompanying material, enters a host cell.

[0400] A variety of methods are known in the art and suitable for transfection of nucleic acid into a cell. The polynucleotides of the present disclosure may be formulated, using the methods described herein. The formulations may comprise polynucleotides which may be modified and / or unmodified. The formulations may further comprise, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, a sustained-release delivery depot, and / or any combinations thereof.

[0401] The formulated polynucleotides may be delivered to the cell using routes of administration known in the art and described herein. Examples of typical methods include, but are not limited to, naked delivery, lipidoid mediate transfer, liposome-, lipoplexes, and / or lipid nanoparticle-mediated transfer, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, microinjection, microprojectile mediated transfer (for example, but not limited to, nanoparticles), cationic polymer mediated transfer (for example, but not limited to, DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion.

[0402] The polynucleotides of the present disclosure may be delivered to a cell naked. As used herein in, “naked” refers to delivering polynucleotides free from agents which promote transfection. For example, but not limited to, the polynucleotides delivered to the cell may contain no modifications and / or delivery agents. The naked polynucleotides may be delivered to the cell using routes of administration known in the art and described herein. In certain aspects, the polynucleotides are delivered naked and the cells uptake them by endocytosis, for example.EXAMPLES

[0403] The following examples are included to demonstrate preferred aspects of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific aspects which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.300484454.1 - 97 -BAYM. P0444WO / BLG 25-024Example 1 - Material and methodsA. RNA production

[0404] Human codon-optimized Na-gst-1 gene (GenBank: FJ711440) was synthesized by Twist Biosciences (San Francisco, CA) and subsequently cloned into plasmid backbones containing, from 5' to 3', an AG T7 promoter (CleanCap® compatible), 5' untranslated region (UTR), Kozak sequence, cloning site, 3' UTR, and segmented poly(A) tail

[0042] , To generate secretory 7Va-GST-l, the plasmid also included an IgG signal peptide sequence upstream of the cloning site

[0043] , For anchored z-GST-1, the plasmid contained albumin signal peptide and CD55 GPI attachment sequences upstream and downstream of the cloning site

[0044] , Additionally, a FLAG tag (DYKDDDDK (SEQ ID NO: 52) coding sequence was added to the 5' (following the signal peptide) for anchored z-GST-1, and to the 3' end for the other constructs. Plasmids were then linearized and utilized as template for in-vitro transcription reactions (co-transcription capping) following the protocol of CleanCap® Reagent M6 (Trilink BioTechnologies, San Diego, CA), with rUTP substituted for m1ΨTP

[0045] , After DNAse I treatment, mRNA was purified using Monarch® Spin RNA Cleanup Kit (NEB, Ipswich, MA). To assess size and integrity, mRNA was heated at 70 °C for 10 min before loading into a 1.5% agarose gel.B. In vitro RNA transfection and protein localization1. Cell culture

[0405] DC2.4 murine dendritic cell line was maintained in RPMI 1640+L-glutamine supplemented with 10% fetal bovine serum (FBS), antibiotics, 1 mM non-essential amino acids, 10 mM HEPES, and 55 pM beta-mercaptoethanol under 5% CO2 at 37 °C.2. Immunocytochemistry (IC)

[0406] Around 1.2E5 DC2.4 cells were seeded per well in a 24-well plate. The next day, transfections were conducted by mixing 500 ng mRNA with Lipofectamine MessengerMAX [Thermo Fisher Scientific (TFS), Waltham, MA], following the manufacturer’s instructions. After 20 hours, cells were either fixed or fixed and permeabilized using Cytofix™ fixation buffer or Cytofix / Cytoperm™ fixation / permeabilization buffer (BD, Franklin Lakes, NJ) for 30 min at 4 °C. Cells were washed three times with staining buffer (2% FBS in phosphate buffered saline (PBS) for only-fixed cells) or Perm / Wash™ buffer (BD) and then incubated with anti-FLAG monoclonal antibody conjugated with Alexa Fluor 488 (TFS, Cat. MA1-142-A488) for 60 min at 4 °C. The antibody was diluted 1:250 in either staining buffer or Perm / Wash™ buffer. Following a second wash step, images were captured using an inverted fluorescence microscope.300484454.1 - 98 -BAYM. P0444WO / BLG 25-0243. Western blot (WB) analysis for FLAG tag detection

[0407] About 2.5E5 DC2.4 cells were seeded per well in a 12-well plate and transfected with 1 pg mRNA-Lipofectamine MessengerMAX complexes on the following day. After a 20-hour incubation period, cells were washed once with PBS and detached using cell dissociation reagent Accutase® (MilliporeSigma, Burlington, MA). The harvested cells were resuspended with 50 pL RIPA buffer and incubated on ice for 30 min with agitation. Subsequently, the samples were centrifuged at 13,000 x g for 20 min. After quantification with BCA Assay (TFS), 10 pg total protein per sample were loaded onto a 4-12% Bis-Tris SDS-PAGE gel. Monoclonal anti-FLAG M2 (MilliporeSigma, Cat. F3165, 1:1000) was used as primary antibody, and alkaline phosphatase goat anti-mouse (KPL, Cat. 5220-0357, 1:3000) as secondary antibody. Washing steps were performed with PBST (0.05% Tween-20 in PBS), and detection was carried out using NBT / BCIP substrate.4. FLAG tag immunostaining and flow cytometry

[0408] After transfection in a 12-well plate, cells were detached, washed with PBS, and counted. Around 1.0E5 cells were seeded per well in a laminar wash 96-well plate (Curiox Biosystems, Seoul, South Korea) and incubated for 20 min at 4 °C. Once settled, cells underwent ten wash cycles with staining buffer (2% FBS in PBS) using a Laminar Wash HT2000 (Curiox Biosystems) for all washing steps. The cells were then resuspended in 70 pL Cytofix / Cytoperm™ buffer and incubated for 20 min at 4 °C, followed by 10 wash cycles with Perm / Wash™ buffer. Subsequently, cells were resuspended in 25 pL Cytofix / Cytoperm™ buffer containing 1 pL mouse Fc block (BD) and incubated for 5 min at 4 °C. Immediately after, 45 pL Cytofix / Cytoperm™ buffer containing anti-FLAG M2-Cy3 (MilliporeSigma, Cat. A9594, 1: 100) was added to the cells which were incubated for more 30 min at 4 °C, followed by a final wash step of 15 cycles. Finally, cells were analyzed for FLAG staining using a Guava Muse Flow Cytometer (Cytek Biosciences, Fremont, CA).C. RNA formulation into lipid nanoparticles (LNPs)

[0409] / -GST- I mRNAs were formulated with Genvoy ILM lipid reagent (Precision Nanosystems, Cat. NWW0042) at a nitrogen-to-phosphate ratio of 4:1 using a NanoAssemblr Ignite™ instrument (Precision Nanosystems). The resulting mRNA / LNP complexes were concentrated using 30 kDa spin filter columns and sterilized through 0.2 pm disk filters. The average LNP size was 90 nm with a poly dispersity index of less than 15%, as determined by dynamic light scattering using a Zetasizer Nano ZS90 (Malvern Panalytical, UK). The mRNA concentration was measured using a RiboGreen RNA Assay kit (Thermo Fisher Scientific, USA Cat. R11490). Experiments with and without TritonXIOO detergent indicated that the 300484454.1 - 99 -BAYM. P0444WO / BLG 25-024loading efficiency of the encapsulated mRNA was over 85% for all mRNA LNP formulations. In a final step, mRNA / LNP vaccine formulations were diluted to a concentration of 200 pg / mL mRNA in sterile PBS with 8% sucrose and stored at -80 °C until use.D. Recombinant Aa-GST-1 protein vaccine

[0410] / -GST- I protein was produced in Pichia pastoris and purified as published elsewhere

[0022] , The protein was adsorbed to aluminum hydroxide adjuvant (Alhydrogel, Croda Denmark) in a glucose imidazole buffer (10% glucose, 10 mM imidazole, pH 7.4).E. Immunogenicity study1. Immunization

[0411] Immunization and sampling procedures strictly adhered to the Guide for the Care and Use of Laboratory Animals

[0046] , The protocol under number AN-5765 was approved by the Institutional Animal Care and Use Committee (IACUC) at Baylor College of Medicine.

[0412] Forty female BALB / c mice (cAnNTac, Taconic Biosciences), aged 6-8 weeks, were divided into 5 groups, each consisting of 8 individuals. Two intramuscular immunizations were administered three weeks apart (switching leg for the second injection), followed by euthanasia at day 42. Each group received either placebo (empty LNPs), 10 pg mRNA / LNPs, or 20 pg Aa-GST-1 protein with 160 pg of aluminum hydroxide. For serum collection, blood samples were obtained on immunization days and at the time of euthanasia. Fecal pellets from the large intestine and spleens were collected after euthanasia. Splenocytes were processed for flow cytometry as published elsewhere

[0047] ,2. ELISA

[0413] Ninety-six-well flat bottom plates were coated overnight at 4 °C with 100 pL of 0.25 pg / mL r / -GST-l diluted in KPL coating solution (SeraCare Life Sciences, Milford, MA), followed by blocking with 200 pL dilution buffer (0.1% BSA in PBST) for two hours at room temperature. After a single wash with PBST, wells were incubated with 100 pL of diluted sera in duplicates for two hours at room temperature. Mouse sera were serially diluted threefold, ranging from 1:200 to 1:437,400. Naive mouse sera were also included in all plates, serving as the cutoff. After incubation, plates were washed four times and incubated with 100 pL of either goat anti-mouse IgG HRP, goat anti-mouse IgGl HRP or goat anti-mouse IgG2 HRP (Lifespan Bioscience, Shirley, MA) in dilution buffer for one hour at room temperature. After five washes, wells were incubated with 100 pL TMB substrate for 15 min. Reactions were stopped with 100 pL IM HC1, and absorbance was measured at 450 nm using a BioTek Epoch 2 spectrophotometer (Agilent, Santa Clara, CA). For data analysis, duplicates were averaged, and titers were calculated using a four-parameter logistic regression curve. Titer 300484454.1 - 100 -BAYM. P0444WO / BLG 25-024cutoff values were determined by adding the average of naive mouse control to three times its standard deviation.

[0414] To perform fecal IgA and IgG ELISA, pellets were solubilized in extraction buffer (10% goat serum in PBS) at a ratio of 100 pL buffer per 10 mg pellet, followed by vortexing until complete disruption. Samples were clarified by centrifugation at 13,000 g for 10 min. For ELISA, fecal samples were serially diluted two-fold, ranging from 1:8 to 1:1024. In addition to goat anti -mouse IgG HRP, samples were incubated with goat anti -mouse IgA HRP (Southern Biotech, Birmingham, AL).3. Splenocyte immunostaining and flow cytometry

[0415] Around 1.0E6 splenocytes per mouse were seeded per well into 96-well plates. Cells were subjected to three conditions (in cRPMI medium): restimulated with 10 pg / mL Na-GST-1, stimulated with PMA / I (positive stimulation control), or unstimulated. Plates were incubated for 48 hours at 5% CO2 and 37 °C, with Brefeldin A (BD) added during the final five hours of incubation. Subsequently, the cells were transferred to a laminar wash 96-well plate, and all wash cycles were executed using a Laminar Wash HT2000. Once settled on the plate, cells underwent 10 wash cycles with IX PBS and resuspended in viability dye-containing IX PBS. After 30 min incubation at 4 °C, cells underwent 10 wash cycles with staining buffer. CD16 / CD32 Fc receptors were blocked with 2 pL mouse Fc Block (BD), followed by five-minute incubation before addition of the surface marker antibody cocktail (CD3, CD4, CD8, CD25, CD44, and CD62L) and a further incubation of 30 min at 4 °C. After 10 wash cycles, cells were resuspended in Cytofix / Cytoperm™ buffer and incubated for 20 min at 4 °C, followed by 10 additional wash cycles with Perm / Wash™ buffer. Subsequently, an intracellular marker antibody cocktail (IL-2, IL-4, IL- 13, IFN-y, TNF-a, and granzyme B) was added. Following incubation for 30 min at 4 °C, cells underwent 15 wash cycles before being transferred to 96-well culture plates. Samples were finally analyzed using an Aurora Spectral Flow Cytometer (Cytek). Single-stained cells and bead controls were used to unmix the raw data. Fluorescence minus one (FMO) of the samples and untreated controls were used to gate the cell populations using FlowJo software. The percentages of unstimulated cell populations were subtracted from the values of / -GST- I stimulated cells to obtain the results.F. Statistical analysis

[0416] Statistical analysis was conducted using GraphPad Prism software. The Kruskal-Wallis test was performed initially to assess overall differences among groups, followed by Dunn's test for multiple comparisons to identify specific pairwise differences. For flow cytometry data, p-values were corrected for multiple comparisons to control Type I error. In 300484454.1 - 101 -BAYM. P0444WO / BLG 25-024contrast, ELISA data were analyzed using uncorrected p-values to prioritize the detection of biologically relevant differences and to mitigate the risk of Type II errors (false negatives), which were observed when corrections were applied. Data are presented as mean ± standard deviation, with differences considered statistically significant at p < 0.05.G. Neutralization assay of Aa-GST-1 thiol transferase activity

[0417] Total IgG was purified from pooled sera of each immunization group using Nab™ Protein G spin columns (TFS). The neutralization assay of / -GST- I thiol-transferase activity was performed using the GST Fluorometric Activity Assay Kit (Abeam, Cambridge, UK) following the manufacturer’s protocol, but including modifications as published elsewhere

[0048] , Specifically, three concentrations of purified IgG (10, 5, and 2.5 pg in a 10 pL volume) were incubated in triplicates with 0.225 pg / -GST- I protein (in 90 pL GST Assay Buffer) in a 96-well black flat bottom plate for 1 hour at 37 °C with agitation. Then, 10 pL of glutathione followed by 100 pL of monochlorobimane (MCB) solution was added per well. Fluorescence was measured at Ex / Em = 380 / 460 nm in kinetic mode every 5 min for 1 hour. The time point of 20 min was selected within the linear range to calculate / -GST- I activity.Example 2 - Altering the intracellular trafficking of Necator americanus GST-1 antigen yielded novel hookworm mRNA vaccine candidates

[0418] Leveraging the advantages of RNA technology, three mRNA vaccine candidates were strategically designed to direct the accumulation of / -GST- I in the cytosol, to be secreted, or to be anchored in the plasma membrane (PM) of transfected cells. Following immunization of BALB / c mice, both antigen-specific antibody titers and cellular response of splenocytes were quantified to evaluate the immune response to the three mRNA vaccine candidates, also in comparison with the well-studied recombinant / -GST- I (r / -GST- l) produced in Pichia pastoris

[0022] ,A. Signal sequences altered the intracellular trafficking of Aa-GST-1

[0419] / -GST- I, comprising 206 amino acids with a molecular weight of 23.68 kDa, does not contain a predicted signal peptide according to the SignalP 6.0 server

[0040] , This suggests that / -GST- I predominantly accumulates in the cytosol. Based on this premise, three mRNA vaccine candidates for / -GST- I were developed in-house, encoding native / -GST-1 (nAa-GST-1), secretory / -GST- I (sAa-GST-1), and PM-anchored / -GST- I (pm / -GST-1). s / -GST- l included a signal peptide for endoplasmic reticulum (ER) import and secretion, while prmVa-GST-1 contained both a signal peptide for ER import and a GPI attachment300484454.1 - 102 -BAYM. P0444WO / BLG 25-024sequence for plasma membrane (PM) anchoring. These mRNAs were successfully generated by co-capping in-vitro transcription (FIG. 1A).

[0420] To determine the site of antigen trafficking post-translation, two distinct immunocytochemistry (IC) protocols were applied to transfected DC2.4 mouse dendritic cells. The first protocol involved cell fixation-and-permeabilization, allowing for immunostaining of FLAG-tagged / -GST- I within membrane-bound organelles. In contrast, the second protocol involved only cell fixation, restricting immunostaining to the cytosol and PM due to the inability of antibodies to cross internal organelle membranes of DC2.4 cells. Analysis revealed that nAG-GST-l exhibited a nucleocytoplasmic distribution, easily visible in fixed-and-permeabilized cells (FIG. IB). Differently, sAa-GST-1, which contained an ER import signal peptide, was localized in the endomembrane system, as evidenced by its absence in the cytosol of cells that underwent only fixation. Secretion of sAa-GST-1 into the supernatant was also confirmed by Westen blotting (WB) (FIG. IC). Furthermore, pmAa-GST-1 was successfully anchored to the PM, covering the outer layer of transfected cells (FIG. IB).

[0421] Given the variations in translation efficiency observed in IC images among the three mRNA candidates, cells were immunostained after fixation-and-permeabilization, followed by flow cytometry analysis to measure protein expression. Consistent with IC results, the median fluorescence intensity of cells expressing sAa-GST-1 and prmVa-GST-1 was higher compared to cells expressing nAa-GST-1 (FIG. ID). It is important to note, however, that the spatial distribution of the different antigens could also have influenced the measured fluorescent signal. For example, prmVa-GST-1 is thought to be more exposed to antibody binding during immunostaining, potentially leading to a higher fluorescence intensity. Regardless of translation efficiency, all DC2.4 cells transfected with mRNAs exhibited comparable viability to cells treated only with the transfection reagent, suggesting that the different forms of Na-GST-1 are non-toxic protein products (FIG. IE).B. sAa-GST-1 and pmAa-GST-1 boosted the production of antigen-specific IgG

[0422] After in vitro validation, mRNAs were encapsulated in LNPs for immunization in mice. Forty mice were divided into 5 groups, each comprising 8 individuals, and immunized twice (FIG.2A). Two control groups received either recombinant AG-GST- I protein produced in / < pastoris (rAa-GST-1) or empty LNPs. Twenty-one days after the first immunization dose, sAG-GST- l and prmVa-GST-1 induced higher levels of serum antigen-specific IgG titers compared to the other three groups (FIG. 2B). Following the second dose, IgG titers in nNa-GST-1 group reached comparable levels with riVa-GST-l. The group immunized with pr Va- 300484454.1 - 103 -BAYM. P0444WO / BLG 25-024GST-1 continued to show a higher titer in antigen-specific IgG titers compared to both nNa-GST-1 and r / -GST- l (FIG. 2C) until the end of the study (Day 42). Based on recently published data from the PARIS study, it is reasonable to expect that the observed antibody responses to the mRNA antigens will be long-lasting (Srivastava et al., 2024; Immunity, 57 (3), 587-599).

[0423] The role of immunoglobulin class or subclass in the response to / -GST- I remains unclear. However, in line with previous studies, the inventors measured serum antigen-specific IgGl and IgG2a subclasses, as crude correlates to T helper 2 (Th2) and T helper 1 (Thl) cells [22,49,50], Significant levels of IgGl and IgG2a were observed across all mRNA groups compared to the empty LNP control (FIG.2D). An increase in IgGl was seen for pm / -GST-1 compared to r7Va-GST-l, while IgG2a was only induced in the mRNA groups, with pmM / -GST-1 inducing higher titers than nM / -GST-l (FIG. 2E). IgGl to IgG2a ratio suggested that prmVa-GST-1 induces the most balanced IgG response (FIG.2F).

[0424] Total IgG was also measured in fecal samples collected from the large intestine of mice. Elevated titers of antigen-specific IgG were observed only in the prmVa-GST-1 group (FIG. 2G). No differences in antigen-specific IgA levels were detected among any vaccination groups compared to the control.C. pnEVa-GST-1 mRNA induced the most diverse set of T cell populations

[0425] To investigate the antigen-specific T-cell populations elicited after immunization, splenocytes were cultured and restimulated with r / -GST- l protein, followed by flow cytometry analysis. CD25 was used as an indicator of antigen-specific activation for CD4+ and CD8+ T cells, and as a marker of maturation and efficient antigen presentation for B cells [43,44], While CD25-expressing CD8+ T cells increased in the groups immunized with n / -GST- l and prmVa-GST-1 mRNAs, no significant differences were observed for CD4+ T cells when comparing the vaccine groups and the LNP control. Additionally, an increase in CD25+ B cells was observed only for the prmVa-GST-1 group (FIG. 3A).

[0426] For cytokine-producing CD4+ T cells, higher expression of IFN-y was observed in the n / -GST- l and prmVa-GST-1 groups, indicating a strong Thl response (FIG. 3B). In the prmVa-GST-1 group, expressions of TNF-a, IL-2, and IL-4 were also up-regulated, suggesting a mixed Thl / Th2 response. An increase in IL-13 was only observed in the r / -GST- l group. Similar results were also seen in memory and effector CD4+ T cells (FIG. 5). Overall, pmM / -GST-1 mRNA was particularly more effective in inducing a robust and diverse cellular response, which does not seem to be directly associated with humoral response, as s / -GST-1 mRNA induced high antibody titers.300484454.1 - 104 -BAYM. P0444WO / BLG 25-024

[0427] Increased expression of IFN-y and TNF-a was also observed in CD8+ T cells from both n / -GST- l and pm / -GST- l groups, with IL-2 also elevated in the latter (FIG. 3C).Furthermore, when examining central memory CD8+ cells, upregulation of these three cytokines was observed in the n / -GST-l and pm / -GST-l groups, while IFN-y was specifically increased in pm / -GST- l when analyzing effector memory CD8+ cells (FIG. 6).Importantly, granzyme B expression was also increased, reinforcing that the intracellular accumulation and anchoring of / -GST- I enhances T cytotoxic cellular responses.D. mRNA vaccine candidates induced neutralizing antibodies against Na- GST-1

[0428] Protection by targeting / -GST-I is thought to be mediated by antigen-specific antibodies that neutralize the capacity of this enzyme in detoxifying pro-oxidants generated during digestion of host hemoglobin by hookworms. To evaluate the effectiveness of mRNA vaccine-induced antibodies in this context, the inhibition of glutathione-S-transferase activity of / -GST- I was assessed in vitro using a commercial fluorometric GST assay kit with modifications, as published elsewhere

[0048] , The assay was inconsistent when using whole serum. Thus, purified IgG was used as in previous publications

[0048] , For each group, purified IgG from pooled sera was mixed with a fixed amount of recombinant / -GST- I protein (0.225 pg), followed by the addition of glutathione and a fluorescent thiol substrate.

[0429] After purification with protein G columns, the total IgG yield was similar across all mRNA groups, ranging from 1.8 to 2.1 mg / mL. Antibodies induced by s / -GST- l mRNA and r / -GST- l protein exhibited superior neutralization activity against / -GST- I compared to the other vaccine groups (FIG. 4A). This suggested that the extracellular presence of / -GST-1 as a free antigen enhanced the likelihood of generating highly specific antibodies. Since antibodies induced by pm / -GST- l mRNA exhibited the lowest neutralization activity, it is possible that the GPI anchor impacts the availability of certain structural epitopes essential for neutralization of thiol transferase activity.

[0430] To confirm that mRNA vaccines induced antibodies capable of recognizing wildtype GST-1 proteins, the inventors tested worm extracts from Ancylostoma caninum (L3 larvae and adults) arxdNecator americanus (adults) against pooled sera from the pmNa-GST-1 mRNA group. In line with the ELISA results for rNa-GST-1, antibodies detected wild-type GST-1 in all samples (FIG. 4B). Cross-reactivity with caninum was expected due to the high identity and similarity of / -GST- l with GST-1 proteins from other hookworm species (FIG. 11).300484454.1 - 105 -BAYM. P0444WO / BLG 25-024E. Discussion

[0431] Signal sequences, including signal peptides, transmembrane domains, and anchoring attachment sequences, among others, play a crucial role in guiding post-translational modifications and intracellular trafficking of proteins within living cells. In recombinant expression systems, removing or incorporating these sequences directly impacts these processes, altering at least protein localization. By leveraging these aspects, the inventors designed multiple mRNA vaccine variants of the same antigen, each with distinct intracellular distribution and, subsequently, exposure to the immune system. Through this approach, the inventors successfully expressed / -GST- I in its native (mostly cytoplasmic), secreted, or membrane-anchored forms using recombinant mRNAs.

[0432] In these mRNA molecules, the incorporation of signal peptide sequences likely enhanced the translation efficiency of Aa-GST-1. Given that hookworms are extracellular parasites and have not undergone host-induced codon bias, the introduction of mammal-derived signal peptides may have been advantageous for translating a hookworm gene. Indeed, both prmVa-GST-1 and sAa-GST-1 seemed to be more highly expressed than mVa-GST-1 in-vitro, potentially influencing in-vivo results as well. However, antibody titers do not increase in direct proportion to expression levels, suggesting that spatial distribution — rather than translation efficiency alone — may play a more critical role in driving the humoral response. Additionally, native Aa-GST- 1, which mostly accumulated in the cytoplasm, elicited a stronger CD8+ T cell than extracellular Aa-GST-1 (sAa-GST-1 and rAa-GST-1), compared to the LNP control. Altogether, these results indicated that spatial distribution plays a more crucial role in tailoring the immune response than mRNA translation efficiency. These findings highlighted the importance of understanding antigen localization before RNA vaccine design. Importantly, if a humoral response is the primary protective mechanism against a pathogen, extracellular antigen exposure increases humoral responses, a hallmark of subunit vaccines. Differently, if a cytotoxic T response is necessary, cytoplasmic accumulation of an antigen may enhance proteasome degradation and peptide loading into MHC-1.

[0433] The rationale for using / -GST- I as a vaccine antigen stems from its pivotal role in detoxifying harmful substances, widespread expression in hookworm tissues, and immunogenicity [22,51], Significant attention has been directed to detoxification of heme, a byproduct of hemoglobin, within the gastrointestinal track [22,52,53], Although / -GST- I activity has been detected in excretory / secretory products after in-vitro hookworm culture

[0054] , it remains unproven how / -GST- I is secreted from the hookworm’s cells, given the absence of a classical signal peptide in its sequence. It is hypothesized that cytoplasmic GSTs utilize 300484454.1 - 106 -BAYM. P0444WO / BLG 25-024non-classical secretion pathways or unconventional mechanisms for secretion [55,56], As previously suggested, / -GST- I could be excreted with conjugated molecules attached as a “molecular dispatch mechanism” or in exosome-like vesicles [54,56,57],

[0434] In the context of / -GST- I mRNA vaccines, antigen secretion or extracellular exposure enhanced antibody titers in mice. Previous preclinical studies with hamsters and rNa-GST-1 required a three-dose immunization schedule to boost antibody production

[0022] , Indeed, after a single immunization with r / -GST- l in mice, antigen-specific antibodies were not significantly detected, whereas notable titers were already achieved with pm / -GST- l and s / -GST- l mRNAs. While antibody levels with rM / -GST- l increased after two doses, they remained inferior to those induced by pm / -GST- l and s / -GST- l mRNAs. Despite high antibody titers, pmM / -GST- l mRNA generated the lowest proportion of antibodies that neutralized Aa-GST-l’s thiol transferase activity. GPI-anchored proteins are located to the outer side of the cell membrane, with their orientation being influenced by the lipid environment. This could have impacted the exposure of certain epitopes or active sites, making them less accessible than in soluble forms of Aa-GST-1. Circulating extracellular antigens (sM / -GST- l and rM / -GST- l ), on the other hand, induced the highest proportion of neutralizing antibodies, possibly due to more frequent direct encounters between them and naive B cells. This direct binding, followed by antigen internalization and MHC-II presentation to T-helper cells, could have led to the production of antibodies with higher affinity. Since the inventors only evaluated the thiol transferase activity of M / -GST- I, it is too early to draw definitive conclusions about neutralizing antibodies. Thus, while high levels of antigen-specific anti-GST-1 antibody are suspected to achieve protective immunity, further experiments to establish this aspect as a true correlate of protection are needed. Future challenge experiments will be pivotal to determine if the in vitro observations correlate with in vivo efficacy outcomes.

[0435] The high genetic conservation of GST-1 across hookworm species highlights its potential as a broadly protective antigen. The results disclosed herein showed that antibodies induced by pm / -GST- l mRNA vaccine effectively recognized GST-1 in both L3 larvae and adult A. caninum. This cross-reactivity is important, as N. americanus and A. caninum share significant sequence identity and similarity with other medically important hookworm species, such as A. duodenale and A. ceylanicum. Given this conservation, the / -GST-I -based vaccines has the potential to provide cross-species protection, targeting the primary hookworms affecting diverse geographic regions worldwide.

[0436] In the development of vaccines based on Aa-GST-1, effective immunity relies not only on the generation of plasma cells to produce antigen-specific antibodies, but also on the 300484454.1 - 107 -BAYM. P0444WO / BLG 25-024establishment of memory cells that can quickly reestablish antibody production upon hookworm reinfection. Despite comparable antigen-specific antibody titers between s / -GST-1 and prmVa-GST-1, the former did not induce significant counts of memory T cells relative to the LNP control. This observation suggests that immediate antibody levels induced by both vaccines may not be correlated with the specific subsets of T helper cells evaluated. Beyond these T cells results, s / -GST- l mostly reflected the immunoprofile of r z-GST-1, except for the upregulation of IL-13. While antibody production was similar, prmVa-GST-1 may still outperform s / -GST- l in conferring enduring protection for stimulating higher counts of memory CD4+ subsets, which are pivotal in the expansion and differentiation of B cells. In healthy adults vaccinated with co-administered / -GST-I and z-APR-1, CD4+ cells producing IL-2 and TNF were correlated with / -GST- I IgG levels

[0030] , Here, all vaccine candidates except s / -GST-l induced higher counts of memory effector CD4+ cells expressing IL-2, while TNF-a was up-regulated by prmVa-GST-1 in memory effector CD4+ T cells and by prmVa-GST-1 and rM / -GST- l in memory central CD4+ T cells. Nevertheless, considering both antibody titers and thiol -transferase neutralization, sM / -GST- l outperformed the other mRNA vaccine candidates under our study conditions.

[0437] These mRNA vaccine design strategies for enhancing antigen ...

Claims

BAYM. P0444WO / BLG 25-024CLAIMS1. A composition, comprising a polynucleotide comprising a sequence that is identical to SEQ ID NOs: 2 or 4.

2. A composition comprising one or more polynucleotides, wherein at least one of the one or more polynucleotides comprises a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to any one of SEQ ID NOs: 1-7 and 17-25.

3. The composition of claim 2, wherein at least one uridine position of at least one of the one or more polynucleotides is pseudouridine.

4. The composition of claim 2, wherein the one or more polynucleotides further comprise a sequence encoding a cell membrane anchor, one or more tags, a signal peptide, one or more linkers, or a combination thereof.

5. The composition of claim 4, wherein the cell membrane anchor comprises a glycosylphosphatidylinositol (GPI) anchor.

6. The composition of claim 5, wherein the GPI anchor is from CD55.

7. The composition of claim 5, wherein the GPI anchor comprises a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NO: 49.

8. The composition of claim 4, wherein the signal peptide is from IgG or albumin.

9. The composition of claim 2, comprising a multicistronic polynucleotide comprising at least two sequences that are at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one of SEQ ID NOs: 1-7 and 16-25, wherein the at least two sequences are separated by an internal ribosome entry site (IREs) and / or 2A peptide encoding sequence.

10. The composition of claim 2, wherein the one or more polynucleotides further comprise a 5' cap.

11. The composition of claim 2, wherein the one or more polynucleotides is in vitro transcribed.300484454.1 - 116 -BAYM. P0444WO / BLG 25-02412. The composition of claim 2, wherein the one or more polynucleotides are encoded by a vector.

13. A composition comprising one, two, three, four, five, or six polynucleotides comprising a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 1-7 and 16-25.

14. The composition of claim 13, comprising a first polynucleotide comprising a polynucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 4 or 7 and a second polynucleotide comprising a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to SEQ ID NOs: 18 or 23.

15. The composition of claim 2, further comprising one or more lipids.

16. The composition of claim 15, wherein the one or more lipids comprise a ionizable lipid, a helper lipid, a PEG-lipid, a cholesterol, or a combination thereof.

17. The composition of claim 15, wherein the one or more polynucleotides are encapsulated in one or more lipid nanoparticles (LNPs).

18. The composition of claim 17, wherein the LNPs comprise a nitrogen-to-phosphate ratio of about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, or any value therebetween.

19. The composition of claim 17, wherein the LNPs comprise an average diameter of or of about 40-130 nm.

20. The composition of claim 17, wherein the LNPs comprise a poly dispersity index that less than or is less than about 15%, 20%, 25%, or 30%.

21. The composition of claim 17, wherein at least, or at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the LNPs encapsulate at least one polynucleotide.

22. The composition of claim 2, further comprising sterile phosphate buffered saline (PBS) and / or sucrose.

23. The composition of claim 2, wherein the one or more polynucleotides are comprised at a concentration of or of about 50-1000 pg / mL.

24. The composition of claim 2, further comprising a pharmaceutically acceptable carrier.300484454.1 - 117 -BAYM. P0444WO / BLG 25-02425. The composition of claim 2, wherein the composition is thermostable at room temperature.

26. The composition of claim 2, wherein the composition is frozen at -80 °C.

27. A composition comprising one or more polypeptides comprising a sequence that is at least 80%, 85%, 90%, 95%, 99%, or 100% identical to any one or more of SEQ ID NOs: 9-15 and 26-40.

28. A cell comprising, transduced with, transfected with, and / or transformed with the composition of claim 2.

29. The cell of claim 28, wherein the cell is a human cell.

30. The cell of claim 28, wherein the cell is a muscle cell, epithelial cell, and / or antigen presenting cell.

31. Use of the composition or cell of any one of claims 1-30 in the manufacture of a medicament for immunizing an individual against a parasite infection.

32. A method of immunizing an individual against a parasite infection, the method comprising the steps of administering the composition claim 2 to the individual.

33. The method of claim 32, wherein the composition comprises between about 1-200 pg of the one or more polynucleotides.

34. The method of claim 32, wherein the composition is administered 1, 2, 3, 4, 5 or more times to the individual.

35. The method of claim 34, wherein the time between administrations comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more hours, days, or weeks.

36. The method of claim 32, wherein administration increases Ig titers in the individual compared to non-immunized levels.

37. The method of claim 36, wherein titers increase between about 10-fold to 10,000,000-fold.300484454.1 - 118 -BAYM. P0444WO / BLG 25-02438. The method of claim 36, wherein the increase is detected as early as week 6 after immunization.

39. The method of claim 32, wherein the increase is detected as late as 17 week after immunization.

40. The method of claim 32, wherein the Ig is measured from blood, plasma, or fecal matter.

41. The method of claim 32, wherein the Ig comprises IgG2a, IgGl, IgG2c, IgA, or a combination thereof.

42. The method of claim 32, wherein the Ig inhibits GST and / or APR activity.

43. The method of claim 42, wherein GST and / or APR activity is inhibited between about 5% to about 50%.

44. The method of claim 32, wherein the administration increases CD25 expression in immune cells of the individual compared to non-immunized levels.

45. The method of claim 32, wherein the administration increases expression of a cytokine in immune cells of the individual compared to non-immunized levels.

46. The method of claim 45, wherein the cytokines comprise IFN-y, IL-2, TNF-a, IL-4, IL-13, granzyme B, or a combination thereof.

47. The method of claim 44, wherein the immune cells comprise CD4-positive, CD8-positive, CD3 -positive, CD 19-positive, T central memory, T effector memory, Thl, Th2, B cells, or a combination thereof.

48. The method of claim 32, wherein upon challenge with a parasite, the individual has a reduced parasite burden of between about 20% to 100%, or 20% to 60%, or 30% to 50% compared to non-immunized levels.

49. The method of claim 32, wherein the parasite comprises a helminth.

50. The method of claim 32, wherein the parasite comprises a nematode.

51. The method of claim 32, wherein the parasite comprises a hookworm.300484454.1 - 119 -BAYM. P0444WO / BLG 25-02452. The method of claim 32, wherein the parasite comprises a nematode from the Ancylostomatidae family.

53. The method of claim 32, wherein the parasite comprises Necator americanus, Ancylostoma duodenale, A. caninum, A. duodenale and A. ceylanicum or a combination thereof.

54. The method of claim 32, wherein the non-immunized levels comprise levels detected in the same individual before immunization or the average level in a sample or population of non-immunized individuals.

55. A composition, comprising a first polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 and a second polynucleotide comprising a sequence that is identical to SEQ ID NOs: 18 or 23.

56. Use of a composition comprising a first polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 and a second polynucleotide comprising a sequence that is identical to SEQ ID NOs: 18 or 23 in the manufacture of a medicament for immunizing an individual against a Necator americanus infection.

57. Use of a composition comprising a polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 in the manufacture of a medicament for immunizing an individual against a Necator americanus infection.

58. Use of a composition comprising a polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 in the manufacture of a medicament for increasing production of IgG2a antibodies against GST-1 in an individual.

59. A method of immunizing an individual against a Necator americanus infection, the method comprising the steps of: administering to the individual a polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7.

60. A method of immunizing an individual against a Necator americanus infection, the method comprising the steps of: administering to the individual a first polynucleotide comprising a sequence that is identical to SEQ ID NOs: 4 or 7 and a second polynucleotide comprising a sequence that is identical to SEQ ID NOs: 18 or 23.300484454.1 - 120 -BAYM. P0444WO / BLG 25-02461. A method of increasing production of IgG2a antibodies against GST-1 in an individual, the method comprising the steps of: administering to the individual a polynucleotide comprising a sequence that is identical to SEQ ID NO: 4 or 7.300484454.1 - 121 -

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