HK40099133A - Rna constructs and uses thereof - Google Patents

Description

RNA constructs and their uses

This application is a divisional application of Chinese Patent Application No. 202180041726.8, filed on April 22, 2021, entitled “RNA construct and its use”.

Priority requirements

This application claims priority to each of the following applications under 35 U.S.C. § 119, the disclosure of which is incorporated herein by reference in its entirety: International Application No. PCT/EP20/61239, filed April 22, 2020; and International Application No. PCT/EP20/66968, filed June 18, 2020.

Background Technology

The use of RNA polynucleotides as therapeutic agents is an emerging field.

Summary of the Invention

This disclosure identifies certain challenges that may be associated with RNA therapy.

For example, in some embodiments, this disclosure identifies sources of certain problems that may be encountered when expressing polypeptides encoded by RNA therapeutic agents. Among these, this disclosure provides techniques for improving the translation efficiency of RNA encoding a payload and/or the expression of RNA-encoded polypeptide payloads. In some embodiments, translation efficiency and/or the expression of RNA-encoded payloads can be improved using RNA polynucleotides comprising: a Cap1 structure (e.g., _m27,3' ^-O Gppp( _m12' ^{-O )} ApG cap); a 5' UTR comprising the cap proximal sequence disclosed herein; and a sequence encoding the payload. Without being bound by theory, this disclosure proposes that improved translation efficiency and/or polypeptide payload expression can be achieved by preferentially binding eukaryotic translation initiation factor 4E (eIF4E), rather than IFN-induced proteins with a triangular tetrapeptide repeat sequence-1 (IFIT1), to RNA containing a Cap1 structure, such as the _m27,3' ^-O Gppp ( _m12' ^-O ) ApG cap, and/or a 5'UTR containing the cap proximal sequence disclosed herein. For example, in some embodiments, it is proposed that eIF4E can competitively bind to 5' cap-based RNA polynucleotides, at least relative to IFIT1 binding. Furthermore, this disclosure provides certain techniques that may favor eIF4E binding, at least relative to IFIT1 binding, and/or may otherwise enhance translation.

In some embodiments, this disclosure states that the characteristics of specific sequences near the 5' cap (e.g., 5'Cap1 structures) can affect the translation efficiency of the associated payload. Without wishing to be bound by any particular theory, this disclosure proposes that eIF4E competitively binds to RNA polynucleotides against IFIT1, based on the characteristics of one or more nucleotides downstream of the 5' cap, such as the proximal cap sequence as disclosed herein. In some embodiments, this disclosure demonstrates that AGAAU or AGCAC sequences downstream of the 5' cap (e.g., 5'Cap1 structures) can enhance translation. This disclosure proposes that the presence of such sequences (e.g., AGAAU or AGCAC) can increase eIF4E binding, at least relative to IFIT1.

Optionally or additionally, this disclosure records certain advantages of avoiding (e.g., ensuring the absence of) self-hybridization sequences (which in some cases may be referred to as self-complementary sequences) in the RNA polynucleotide encoding the payload. For example, this disclosure demonstrates that such absence can improve the translation (e.g., translation efficiency) of the associated (e.g., RNA-encoded) payload, and/or is necessary for the translation (e.g., translation efficiency) of the associated (e.g., RNA-encoded) payload, and/or for the expression of the polypeptide thereby encoded. Without wishing to be bound by theory, it is believed that self-hybridization sequences (particularly sequences that hybridize with Kozak sequences, 5'UTR elements, and/or 3'UTR elements, or sequences containing one or more Kozak sequences, 5'UTR elements, and/or 3'UTR elements) can interfere with one or more aspects of translation. For example, in some embodiments, it has been proposed that such self-hybridization can inhibit the binding of transcription and/or translation factors to the RNA polynucleotide by self-hybridizing to a complementary sequence in the RNA polynucleotide.

Further, optionally or additionally, in some embodiments, this disclosure defines specific lipid components and/or proportions thereof that may be particularly useful or effective in delivering nucleic acids, especially RNA (e.g., therapeutic RNA or other RNA encoding polypeptides), when administered to a subject (e.g., by injection, such as intramuscular or intravenous injection). For example, in some embodiments, this disclosure demonstrates that lipid ALC-0315 is exceptionally useful for delivery abnormalities as described herein.

This document specifically discloses a composition or pharmaceutical article comprising an RNA polynucleotide comprising: (i) a 5’ cap, which is or contains a cap1 structure, for example, as disclosed herein; (ii) a 5’ UTR sequence containing a proximal cap sequence, for example, as disclosed herein; and (iii) a sequence encoding a payload. Methods for preparing and using them are also disclosed herein, for example, inducing an immune response in a subject.

This article provides a composition or pharmaceutical product comprising an RNA polynucleotide, said RNA polynucleotide comprising:

The sequence includes a 5' cap containing the Cap1 structure; proximal cap sequences containing RNA polynucleotides at positions +1, +2, +3, +4, and +5; and a sequence encoding the payload, wherein:

(i) The Cap1 structure comprises m7G(5')ppp(5')(2'OMeN ₁ )pN ₂ , where N ₁ is the +1 position of the RNA polynucleotide, N ₂ is the +2 position of the RNA polynucleotide, and N ₁ and N ₂ are each independently selected from: A, C, G or U; and

(ii) The cap proximal sequence comprises _N1 and _N2 of the Cap1 structure, and:

(a) Selected from the following sequences: _A3A4X5 (SEQ ID NO: _{1); C3A4X5 (SEQ ID NO:2); A3C4A5 ( SEQ ID NO : 3 ) and A3U4G5} (SEQ ID NO: ₄ ); or

(b) A sequence containing X ₃ Y ₄ X ₅ (SEQ ID NO:7);

Wherein _X3 (nucleotide X at position +3 in SEQ ID NO:7) or _X5 (nucleotide X at position +5 in SEQ ID NO:1 or SEQ ID NO:2) is independently selected from A, G, C or U; and

Where _Y4 (nucleotide Y at position +4 in SEQ ID NO:7) is not C.

This disclosure also provides a composition or pharmaceutical article comprising an RNA polynucleotide, said RNA polynucleotide comprising: a 5' cap; a proximal cap sequence comprising +1, +2, +3, +4, and +5 positions of the RNA polynucleotide; and a sequence encoding a payload, wherein:

(i) The 5' cap includes a Cap1 structure, which includes G*ppp(m ₁ ² ' ^-O )N ₁ pN ₂ , wherein:

N1 is the +1 position of the RNA polynucleotide, _N2 is the +2 position of the RNA polynucleotide, and _N1 and _N2 are each independently selected from A, C, G, or U; and

G* contains the following structures:

The bond representing the first phosphorus atom of the G* group bonded to the ppp group is ^R1 , which is _CH3 , ^R2, which is OH or O- _CH3 , and ^R3 is O- _CH3 ; and

(ii) The cap proximal sequence comprises _N1 and _N2 of the Cap1 structure, and:

(a) Selected from the following sequences: _A3A4X5 (SEQ ID NO: _{1); C3A4X5 (SEQ ID NO:2); A3C4A5 ( SEQ ID NO : 3 ) and A3U4G5} (SEQ ID NO: ₄ ); or

(b) A sequence containing X ₃ Y ₄ X ₅ (SEQ ID NO:7);

Wherein _X3 (nucleotide X at position +3 in SEQ ID NO:7) or _X5 (nucleotide X at position +5 in SEQ ID NO:1 or SEQ ID NO:2) is independently selected from A, G, C or U; and

Where _Y4 (nucleotide Y at position +4 in SEQ ID NO:7) is not C.

This article also provides a composition or pharmaceutical product comprising an RNA polynucleotide, said RNA polynucleotide comprising:

The sequence includes a 5' cap containing the Cap1 structure; proximal cap sequences containing RNA polynucleotides at positions +1, +2, +3, +4, and +5; and a sequence encoding the payload, wherein:

(i) The Cap1 structure comprises m7(3'OMeG)(5')ppp(5')( _2'OMeA1 ) _pG2 , where _A1 is the +1 position of the RNA polynucleotide and _G2 is the +2 position of the RNA polynucleotide; and

(ii) The proximal cap sequence comprises _A1 and _G2 of the Cap1 structure, and includes the following sequences: _A3A4U5 at the +3, +4 and ₊₅ positions of the RNA polynucleotide _, respectively.

This disclosure provides a composition or pharmaceutical product comprising a capped RNA polynucleotide encoding a gene product, said RNA polynucleotide comprising the formula:

Where ^R1 is _CH3 , ^R2 is OH or O- _CH3 , and ^R3 is O- _CH3 .

Wherein _B1 is any nucleobase, preferably A; _B2 is any nucleobase, preferably G; _B3 is any nucleobase, preferably A or C; _B4 is any nucleobase; and _B5 is any nucleobase, and

Among them, when RNA polynucleotides were administered to subjects, the expression levels of the encoded gene products differed by no more than 5-fold between approximately 6 hours and approximately 48 hours after administration.

This document provides a pharmaceutical composition comprising the RNA polynucleotide disclosed herein. In some embodiments, the pharmaceutical composition comprises the composition or pharmaceutical product disclosed herein.

This document also provides a method for manufacturing a pharmaceutical composition, for example, comprising the RNA polynucleotide disclosed herein, the method being by combining the RNA polynucleotide with lipids to form lipid nanoparticles encapsulating the RNA.

This disclosure provides a nucleic acid template suitable for generating cap1-capped RNA, wherein the first five nucleotides transcribed from the template strand of the nucleic acid template comprise the sequence _{N1 pN2 pN3 pN4 pN5} , wherein _N1 is any nucleotide, preferably T; _N2 is any nucleotide, preferably C; _N3 is any nucleotide, preferably T or G; _N4 is any nucleotide; and _N5 is any nucleotide. In some embodiments, the DNA template comprises: a 5' UTR, a sequence encoding the payload, a 3' UTR, and a polyA sequence.

This article provides an in vitro transcription reaction system, including:

(i) Template DNA containing a polynucleotide sequence complementary to the RNA polynucleotide sequence disclosed herein;

(ii) polymerase; and

(iii) RNA polynucleotides.

This article also provides an RNA polynucleotide isolated from the provided in vitro transcription reaction system.

This disclosure provides a method for preparing capped RNA, the method comprising transcribing a nucleic acid template in the presence of a cap structure, wherein the cap structure comprises G*ppp(m ₁ ² ' ^-O )N ₁ pN ₂ ,

_N1 is complementary to the +1 position of the nucleic acid template, and _N2 is complementary to the +2 position of the nucleic acid template. Furthermore, _N1 and _N2 are independently selected from A, C, G, or U.

The +3 position of the nucleic acid template can be any nucleotide, preferably T or G; the +4 position of the nucleic acid template can be any nucleotide; and the +5 position of the nucleic acid template can be any nucleotide.

G* contains the following structure:

The bond representing the first phosphorus atom of the G* group bonded to the ppp group is ^R1 , which is _CH3 , ^R2 is OH or O- _CH3 , and ^R3 is O- _CH3 .

This document also provides a composition comprising a DNA polynucleotide, said DNA polynucleotide containing a sequence complementary to the RNA polynucleotide sequence provided herein. In some embodiments, the DNA polynucleotide disclosed herein can be used to transcribe the RNA polynucleotide disclosed herein.

This disclosure provides a method comprising administering a pharmaceutical composition to a subject, said pharmaceutical composition comprising the RNA polynucleotide disclosed herein, formulated in lipid nanoparticles (LNPs) or lipid complexes (LPXs) particles, for example, as disclosed herein.

This document also provides a method for inducing an immune response in a subject, the method comprising administering to the subject a pharmaceutical composition comprising the RNA polynucleotide disclosed herein, for example, as disclosed herein, formulated in lipid nanoparticles (LNPs) or lipid complexes (LPXs).

This document provides a method for administering a vaccine to a subject by administering a pharmaceutical composition comprising, for example, an RNA polynucleotide disclosed herein formulated in lipid nanoparticles (LNPs) or lipid complexes (LPXs).

This disclosure provides a method for reducing the interaction of IFIT1 with an RNA polynucleotide, the RNA polynucleotide comprising a 5' cap and a proximal cap sequence comprising +1, +2, +3, +4, and +5 positions of the RNA polynucleotide, the method comprising the following steps:

Provide variants of the RNA polynucleotide that differ from the parental RNA polynucleotide in that one or more residues are substituted within the proximal cap sequence, and

It was determined that the variant interacted less with IFIT1 relative to the parental RNA polynucleotide.

This article also provides a method for preparing peptides, the method comprising the following steps:

Provides an RNA polynucleotide containing a 5' cap, proximal cap sequences at +1, +2, +3, +4, and +5 positions of the RNA polynucleotide, and a sequence encoding the payload;

The RNA polynucleotide is characterized in that, when evaluated in an organism to which the RNA polynucleotide or a composition containing the RNA polynucleotide is administered, an increase in the expression of the payload and/or an increase in the duration of expression is observed relative to an appropriate reference.

This article provides a method for increasing the translatability of RNA polynucleotides, wherein the RNA polynucleotide includes a 5' cap, proximal cap sequences comprising +1, +2, +3, +4, and +5 positions of the RNA polynucleotide, and a sequence encoding a payload. The method includes the following steps:

Provide variants of the RNA polynucleotide that differ from the parent RNA polynucleotide in that one or more residues are substituted within the proximal cap sequence; and

It was determined that the expression of the variant was increased relative to the expression of the parental RNA polynucleotide.

This article provides a therapeutic RNA comprising a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding the payload. Improvements include:

The proximal cap sequence includes one or more of the following residues: X at position +1, X at position +2, X at position +3, X at position +4, and X at position +5 of the RNA polynucleotide, which, when administered to a subject as an LNP formulation, demonstrates increased RNA expression. In some embodiments, X is selected from A, C, G, or U.

This disclosure provides a therapeutic RNA comprising a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding a payload, wherein the modification includes one or more of the following residues in the proximal cap sequence: X at position +1 of the RNA polynucleotide, X at position +2 of the RNA polynucleotide, C at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and X at position +5 of the RNA polynucleotide, wherein increased RNA expression is demonstrated when administered to a subject in an LNP formulation. In some embodiments, X is selected from A, C, G, or U.

This article also provides a therapeutic RNA comprising a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding a payload, wherein the improvements include one or more of the following residues in the proximal cap sequence: A at position +1 of the RNA polynucleotide, G at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and U at position +5 of the RNA polynucleotide, which, when administered to subjects in an LNP formulation, demonstrates increased RNA expression.

This disclosure provides a method for increasing the translation of RNA polynucleotides, said RNA polynucleotide comprising a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding a payload, the improvement comprising including one or more of the following residues in the proximal cap sequence: X at position +1 of the RNA polynucleotide, X at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and X at position +5 of the RNA polynucleotide. In some embodiments, X is selected from A, C, G, or U.

This document provides a method for enhancing the translation of RNA polynucleotides, wherein the RNA polynucleotide comprises a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding a payload, the enhancement comprising including one or more of the following residues in the proximal cap sequence: X at position +1 of the RNA polynucleotide, X at position +2 of the RNA polynucleotide, C at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and X at position +5 of the RNA polynucleotide. In some embodiments, X is selected from A, C, G, or U.

This article also provides a method for increasing the translation of RNA polynucleotides, said RNA polynucleotides comprising a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding a payload, the improvement comprising including one or more of the following residues in the proximal cap sequence: A at position +1 of the RNA polynucleotide, G at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and U at position +5 of the RNA polynucleotide.

This article also provides a method for providing a framework for RNA polynucleotides comprising a 5' cap, a cap proximal sequence, and a payload sequence, the method comprising the following steps:

Evaluate at least two variants of the RNA polynucleotide, wherein:

Each variant includes the same 5' cap and payload sequence; and

The variants differ from each other at one or more specific residues in the proximal cap sequence;

The evaluation includes determining the expression level and/or duration of the payload sequence; and

Select at least one combination of the 5' cap and the proximal cap sequence, which shows elevated expression relative to at least one other combination.

Attached Figure Description

Figure 1 illustrates plasma EPO levels in mice at 6, 24, 48, and 72 hours after intravenous administration of a mouse EPO (mEPO) mRNA construct with or without Lig3 in the 3'UTR sequence. Blood samples were collected at 6, 24, 48, and 72 hours post-administration, and mEPO levels were analyzed by ELISA.

Figures 2A-2C are schematic diagrams of self-hybridization of the Lig3 sequence with the 5’UTR (Figures 2A-2B) or 3’UTR (Figure 2C).

Figure 3A-3I shows the structure of the 5' cap that can be incorporated into mRNA.

Figure 4 demonstrates plasma levels at 6, 24, 48, and 72 hours after intravenous administration of the mEPO mRNA construct to mice, which has different nucleotides at +3, +4, or +5 positions. Blood samples were collected at 6, 24, 48, and 72 hours post-administration, and mEPO levels in the samples were analyzed by ELISA.

Figure 5 demonstrates the anti-S protein IgG response at days 7, 14, 21, and 28 following immunization with BNT162a1. BALB/c mice were immunized once with 1, 5, or 10 μg of RBL063.3 IM prepared with LNP. At days 7, 14, 21, and 28 post-immunization, animals were exsanguinated, and serum samples were analyzed to determine the total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) as measured by ELISA. For days 7, 14, 21, and 28, serum dilutions of 1:100 are included in the figure. One dot in the figure represents one mouse, and samples from each mouse were measured in duplicate (group size n = 8; each group includes mean + SEM).

Figure 6 illustrates the anti-S protein IgG response at days 7, 14, 21, and 28 following immunization with BNT162b1. BALB/c mice were immunized once with 0.2, 1, or 5 μg of RBP020.3 IM prepared with LNP. At days 7, 14, 21, and 28 post-immunization, animals were exsanguinated, and serum samples were analyzed to determine the total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) as measured by ELISA. Different serum dilutions are included in the figure for days 7 (1:100), 14 (1:300), 21 (1:900), and 28 (1:2700). One dot in the figure represents one mouse, and samples from each mouse were measured in duplicate (group size n = 8; each group includes mean + SEM).

Figure 7 demonstrates the neutralization of SARS-CoV-2 pseudoviruses at 14, 21, and 28 days post-immunization with BNT162b1. BALB/c mice were immunized once with RBP020.3 IM prepared with 0.2, 1, or 5 μg of LNP. Animals were exsanguinated at 14, 21, and 28 days post-immunization, and serum SARS-CoV-2 pseudovirus neutralization was tested. The figure shows the pVN50 serum dilution (50% reduction in infection events compared to a positive control without serum). One dot in the figure represents one mouse. Samples from each mouse were measured in duplicate. Group size n = 8. Mean + SEM is shown for each group using a horizontal bar with a whisker. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation.

Figure 8 demonstrates the anti-S protein IgG response at days 7, 14, and 21 following immunization with BNT162c1. BALB/c mice were immunized once with 0.2, 1, or 5 μg of RBS004.3 IM prepared with LNP. At days 7, 14, and 21 post-immunization, animals were exsanguinated, and serum samples were analyzed to determine the total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) as measured by ELISA. Different serum dilutions are included in the figure for days 7 (1:100), 14 (1:300), and 21 (1:900). One dot in the figure represents one mouse, and samples from each mouse were measured in duplicate (group size n = 8; each group includes mean + SEM).

Figure 9 demonstrates the neutralization of SARS-CoV-2 pseudoviruses 14 and 21 days post-immunization with BNT162c1. BALB/c mice were immunized once with RBS004.3 IM prepared with LNP at doses of 0.2, 1, or 5 μg. Animals were exsanguinated at 14 and 21 days post-immunization, and serum SARS-CoV-2 pseudovirus neutralization was tested. The figure shows the pVN50 serum dilution (50% reduction in infection events compared to a positive control without serum). One dot in the figure represents one mouse. Samples from each mouse were measured in duplicate. Group size n = 8. Mean + SEM is shown for each group using horizontal bars with thin lines. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation.

Figure 10 demonstrates the anti-S protein IgG response at days 7, 14, 21, and 28 following immunization with RBL063.1 formulated with LNP. BALB/c mice were immunized once with 1, 5, or 10 μg of RBL063.1 IM formulated with LNP. At days 7, 14, 21, and 28 post-immunization, animals were exsanguinated, and serum samples were analyzed to determine the total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) as measured by ELISA. Different serum dilutions are included in the figure for days 7 (1:100), 14 (1:100), 21 (1:300), and 28 (1:900). One dot in the figure represents one mouse, and samples from each mouse were measured in duplicate (group size n = 8; each group includes mean + SEM).

Figure 11 shows the neutralization of SARS-CoV-2 pseudoviruses at 14, 21, and 28 days post-immunization with RBL063.1 formulated with LNP. BALB/c mice were immunized once with 1, 5, or 10 μg of RBL063.1 IM formulated with LNP. At 14, 21, and 28 days post-immunization, animals were exsanguinated, and serum SARS-CoV-2 pseudovirus neutralization was tested. The figure shows the pVN50 serum dilution (50% reduction in infection events compared to a positive control without serum). One dot in the figure represents one mouse. Samples from each mouse were measured in duplicate. Group size n = 8. Mean + SEM is shown for each group using horizontal bars with thin lines. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation.

Figure 12 shows the anti-S protein IgG response at 7, 14, and 21 days post-immunization with BNT162b2 (RBP020.1 formulated with LNP). BALB/c mice were immunized once with 0.2, 1, or 5 μg of RBP020.1 IM formulated with LNP. At days 7, 14, and 21 post-immunization, animals were exsanguinated and serum samples were analyzed to determine the total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) as measured by ELISA. Different serum dilutions are included in the figure for day 7 (1:100), day 14 (1:300), and day 21 (1:1100). One dot in the figure represents one mouse, and samples from each mouse were measured in duplicate (group size n = 8; each group includes mean + SEM).

Figure 13 demonstrates the neutralization of SARS-CoV-2 pseudoviruses at 14 and 21 days post-immunization with BNT162b2 (RBP020.1 prepared by LNP). BALB/c mice were immunized once with 0.2, 1, or 5 μg of RBP020.1 prepared by LNP. Animals were exsanguinated on days 14 and 21 post-immunization, and serum SARS-CoV-2 pseudovirus neutralization was tested. The figure shows the pVN50 serum dilution (50% reduction in infection events compared to a positive control without serum). One dot in the figure represents one mouse. Samples from each mouse were measured in duplicate. Group size n = 8. Mean + SEM is shown for each group using horizontal bars with thin lines. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation.

Figure 14 shows the anti-S protein IgG response at 7, 14, and 21 days post-immunization with RBS004.2 formulated with LNP. BALB/c mice were immunized once with 0.2, 1, or 5 μg of RBS004.2 IM formulated with LNP. At days 7, 14, and 21 post-immunization, animals were exsanguinated, and serum samples were analyzed to determine the total amount of anti-S1 (left) and anti-RBD (right) antigen-specific immunoglobulin G (IgG) as measured by ELISA. Different serum dilutions are included in the figure for days 7 (1:100), 14 (1:300), and 21 (1:900). One dot in the figure represents one mouse, and samples from each mouse were measured in duplicate (group size n = 8; each group includes mean + SEM).

Figure 15 demonstrates the neutralization of SARS-CoV-2 pseudoviruses at 14 and 21 days post-immunization with RBS004.2 formulated with LNP. BALB/c mice were immunized once with 0.2, 1, or 5 μg of RBS004.2 IM formulated with LNP. Animals were exsanguinated at 14 and 21 days post-immunization, and serum SARS-CoV-2 pseudovirus neutralization was tested. The figure shows the pVN50 serum dilution (50% reduction in infection events compared to a positive control without serum). One dot in the figure represents one mouse. Samples from each mouse were measured in duplicate. Group size n = 8. Mean + SEM is shown for each group using horizontal bars with thin lines. LLOQ, lower limit of quantitation. ULOQ, upper limit of quantitation.

Figure 16 shows the activity of ALC-0315 during the screening process.

Figure 17 demonstrates the monitoring of luciferase expression in wild-type (WT) or ApoE knockout C57Bl/6 mice following intramuscular administration, in the presence or absence of ApoE3, at the right side (injection site), dorsal side (injection site), and ventral side (drainage to the liver). Luciferase expression was detected using a Xenolight D-luciferin Rediject at 4, 24, 72, and 96 hours post-administration.

Figure 18 shows luciferase activity following intravenous (IV) and intramuscular (IM) administration in wild-type (WT) or ApoE knockout C57Bl/6 mice with (KO+) or (KO) ApoE3 presence. Luciferase expression was detected using a Xenolight D-luciferin Rediject at 4 hours post-administration.

Detailed Implementation

definition

While this disclosure is described in detail below, it should be understood that it is not limited to the specific methods, schemes, and reagents described herein, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are as defined in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

Unless otherwise stated, the implementation of this disclosure will employ conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA technology, as explained in the literature in the said fields (see, for example, Molecular Cloning: A Laboratory Manual, ^2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

The elements of this disclosure are described below. These elements are listed with specific embodiments; however, it should be understood that they can be combined in any way and in any number to produce additional embodiments. The examples and embodiments described in various ways should not be construed as limiting this disclosure to the explicitly described embodiments. This specification should be understood as disclosing and covering embodiments that combine the explicitly described embodiments with any number of disclosed elements. Furthermore, unless the context otherwise indicates, any permutation and combination of all described elements should be considered as disclosed in this specification. The term “about” means approximately or close to, and in some embodiments, in the context of the numerical values or ranges shown herein, means ±20%, ±10%, ±5%, or ±3% of the listed or claimed numerical values or ranges.

The terms “a” and “an” and “this” as used in the context of describing this disclosure (particularly in the context of the claims), and similar designations, should be understood to cover both singular and plural forms, unless otherwise specified herein or clearly contradicted by the context. The enumeration of ranges of values herein is solely for shorthand purposes to individually refer to each distinct value falling within the range. Each individual value is incorporated into this specification as it is individually enumerated herein, unless otherwise stated herein or otherwise clearly contrary to the context. All methods described herein may be performed in any suitable order. The use of any and all instances or exemplary language (e.g., “as”) provided herein is solely for the purpose of better illustrating this disclosure and does not constitute a limitation on the scope of the claims. No language in this specification should be construed as indicating any unclaimed element necessary for the implementation of this disclosure.

Unless otherwise expressly stated, in the context of this document, the term "comprising" is used to mean that, in addition to the list members introduced by "comprising," other members may optionally exist. However, as a particular embodiment of this disclosure, the term "comprising" is considered to cover the possibility that no other members exist; that is, for the purposes of this embodiment, "comprising" is understood to mean "consisting of" or "substantially consisting of."

Several documents are cited throughout the main body of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturers' specifications, instructions, etc.), whether mentioned above or below, is incorporated herein in its entirety. Nothing herein should be construed as an admission that this disclosure could not have been earlier than such disclosure.

Definitions applicable to all aspects of this disclosure are provided below. Unless otherwise stated, the following terms have the following meanings. Any undefined term has its generally accepted meaning.

Agent: As used herein, the term "agent" can refer to a physical entity or phenomenon. In some embodiments, an agent can be characterized by specific characteristics and/or effects. In some embodiments, an agent can be a compound, molecule, or entity of any chemical class, including, for example, small molecules, peptides, nucleic acids, sugars, lipids, metals, or combinations or complexes thereof. In some embodiments, the term "agent" can refer to a compound, molecule, or entity comprising a polymer. In some embodiments, the term can refer to a compound or entity comprising one or more polymeric moieties. In some embodiments, the term "agent" can refer to a compound, molecule, or entity substantially free of a particular polymer or polymeric moieties. In some embodiments, the term can refer to a compound, molecule, or entity lacking or substantially free of any polymer or polymeric moieties.

Amino acid: In its broadest sense, as used herein, the term "amino acid" refers to a compound and/or substance that can, is, or has been incorporated into a polypeptide chain, for example, by forming one or more peptide bonds. In some embodiments, an amino acid has the general structure _H₂N –C(H)(R)–COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any one of the 20 standard L-amino acids commonly found in naturally occurring peptides. "Non-standard amino acid" refers to any amino acid other than a standard amino acid, whether it is synthetically prepared or obtained from a natural source. In some embodiments, amino acids, including carboxyl- and/or amino-terminal amino acids in polypeptides, may contain structural modifications compared to the general structure described above. For example, in some embodiments, amino acids may be modified by methylation, amidation, acetylation, PEGylation, glycosylation, phosphorylation, and/or substitution (e.g., amino, carboxylic acid, one or more protons and/or hydroxyl groups) compared to the general structure. In some embodiments, this modification can, for example, alter the cycling half-life of a peptide containing the modified amino acid compared to a peptide containing the same unmodified amino acid. In some embodiments, this modification does not significantly alter the relevant activity of the peptide containing the modified amino acid compared to a peptide containing the same unmodified amino acid. As will be clear from the context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments, it may be used to refer to the amino acid residues of a peptide.

Analog: As used herein, the term "analog" refers to a substance that shares one or more specific structural features, elements, components, or portions with a reference substance. Typically, an "analog" exhibits significant structural similarity to a reference substance, such as sharing a core or common structure, but also differs in certain discrete ways. In some embodiments, an analog can be a substance that can be generated from a reference substance, for example, through chemical manipulation of the reference substance. In some embodiments, an analog can be generated by performing a synthetic process substantially similar to that used to generate the reference substance (e.g., sharing multiple steps therewith). In some embodiments, an analog is, or can be generated by performing a synthetic process different from that used to generate the reference substance.

Antibody agent: As used herein, the term "antibody agent" refers to a substance that specifically binds to a particular antigen. In some embodiments, the term encompasses polypeptides or polypeptide complexes sufficient to confer a structure to specifically bind an immunoglobulin. For example, in some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements that are considered by those skilled in the art to be complementarity-determining regions (CDRs); in some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to that found in a reference antibody. In some embodiments, the included CDR is substantially identical to a reference CDR, i.e., its sequence is identical to or contains 1-5 amino acid substitutions compared to the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., it exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., it exhibits at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., at least one amino acid in the included CDR is deleted, added, or substituted compared to the reference CDR, but the included CDR has the same amino acid sequence as the reference CDR except for this. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., 1-5 amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR has the same amino acid sequence as the reference CDR except for these defects. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., at least one amino acid in the included CDR is substituted compared to the reference CDR, but the included CDR has the same amino acid sequence as the reference CDR except for these defects. In some embodiments, the included CDR is substantially identical to the reference CDR, i.e., 1-5 amino acids in the included CDR are deleted, added, or substituted compared to the reference CDR, but the included CDR has the same amino acid sequence as the reference CDR except for these defects. In some embodiments, the antibody material is or comprises a polypeptide whose amino acid sequence includes structural elements that are considered by those skilled in the art to be variable domains of immunoglobulins. In some embodiments, the antibody substance is or comprises a polypeptide whose amino acid sequence includes structural elements that, in the opinion of those skilled in the art, correspond to CDR1, 2, and 3 of the variable domain of the antibody; in some such embodiments, the antibody substance is or comprises a polypeptide or polypeptide group whose amino acid sequence together includes structural elements that, in the opinion of those skilled in the art, correspond to the CDRs of the heavy chain and light chain variable regions, for example, heavy chain CDR1, 2, and/or 3 and light chain CDR1, 2, and/or 3. In some embodiments, the antibody substance is a polypeptide protein having a binding domain that is homologous to or substantially homologous to an immunoglobulin-binding domain. In some embodiments, the antibody substance may be or comprises a polyclonal antibody product. In some embodiments, the antibody substance may be or comprises a monoclonal antibody product. In some embodiments, the antibody substance may include one or more constant region sequences having specific biological characteristics, such as those of camels, humans, mice, primates, rabbits, and rats; in many embodiments, the antibody substance may include one or more constant region sequences having human characteristics. In some embodiments, the antibody substance may include one or more sequence elements that, in the opinion of those skilled in the art, are humanized sequences, primate-like sequences, chimeric sequences, etc. In some embodiments, the antibody material may be a typical antibody (e.g., it may contain two heavy chains and two light chains). In some embodiments, the antibody material may be selected from, but is not limited to, intact IgA, IgG, IgE, or IgM antibodies; bispecific or multispecific antibodies (e.g., etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated CDRs or groups thereof; single-chain Fv; peptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masking antibodies (e.g., [missing information]); small modular immunopharmaceuticals (“SMIPs ^™” ); single-chain or tandem dimers; VHH; minibodies; ankyrin repeats or DART; TCR-like antibodies; microproteins; and in some embodiments, the antibody may lack covalent modifications (e.g., glycan attachment) that it would have naturally. In some implementations, antibodies may contain covalent modifications (e.g., linked glycans, payloads [e.g., detectable portions, therapeutic portions, catalytic portions, etc.]) or other side groups [e.g., polyethylene glycol, etc.]. Related: Two events or entities are “related” to each other, as the term is used herein, if the presence, level, extent, type, and/or form of one event or entity is associated with the presence, level, extent, type, and/or form of another event or entity. For example, the presence, level, and/or form of a particular entity (e.g., peptides, genetic traits, metabolites, microorganisms, etc.) is associated with the incidence, susceptibility, or prevalence of a disease, symptom, or disease condition. If an entity is associated with a specific disease, symptom, or condition, it is considered to be related to the severity, stage, or other similar factors (e.g., within a relevant population). In some embodiments, two or more entities are considered physically "related" to each other if they interact directly or indirectly, causing them to be and/or remain physically close to each other. In some embodiments, two or more physically related entities are covalently linked; in other embodiments, two or more physically related entities are not covalently linked but are non-covalently associated, for example, through hydrogen bonds, van der Waals interactions, hydrophobic interactions, magnetism, or combinations thereof.

Binding: As used herein, the term "binding" generally refers to a non-covalent association between two or more entities. "Direct" binding involves physical contact between entities or parts; indirect binding involves physical interaction through physical contact with one or more intermediate entities. Binding between two or more entities can generally be evaluated in a variety of situations – including in isolated cases or in more complex systems studying interacting entities or parts (e.g., when covalently or otherwise associated with a carrier entity and/or in biological systems or cells). Binding between two entities can be considered "specific" if, under the conditions of evaluation, the relevant entities are more likely to associate with each other than with other available binding partners.

Biological Samples: As used herein, the term "biological sample" generally refers to a sample obtained or derived from a biological source of interest (e.g., tissue or organism or cell culture), as described herein. In some embodiments, the source of interest includes organisms such as animals or humans. In some embodiments, biological samples are or include biological tissues or fluids. In some embodiments, biological samples may be or include bone marrow; blood; blood cells; ascites; tissue or fine-needle biopsy samples; cellular body fluids; free-floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; lavage or irrigation solutions, such as catheter lavage or bronchoalveolar lavage fluid; aspirate; scraping fluid; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions and/or excretions; and/or cells therein, etc. In some embodiments, biological samples are or include cells obtained from an individual. In some embodiments, the obtained cells are or include cells from an individual from whom the sample is obtained. In some embodiments, the sample is a “primary sample” obtained directly from the source of interest by any suitable means. For example, in some embodiments, a primary biological sample is obtained by a method selected from biopsy (e.g., fine-needle aspiration or tissue biopsy), surgery, collection of bodily fluids (e.g., blood, lymph, feces, etc.). In some embodiments, as will be apparent from the context, the term “sample” refers to an article obtained by processing (e.g., by removing one or more components and/or by adding one or more substances) a primary sample. For example, filtration using a semi-permeable membrane. Such a “processed sample” may include, for example, nucleic acids or proteins obtained by extraction from a sample or by technical processing of the primary sample (e.g., amplification or reverse transcription of mRNA, isolation and/or purification of certain components, etc.).

Combination therapy: As used herein, the term "combination therapy" refers to a situation where a subject is simultaneously exposed to two or more treatment regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "dose" of the first regimen are administered before any dose of the second regimen is administered); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, "administration" of combination therapy may involve administering one or more agents or methods to a subject receiving other agents or methods in the combination. For clarity, combination therapy does not require the administration of individual drugs together in a single composition (or even necessarily simultaneously), although in some embodiments, two or more agents or their active portions may be administered together in the composition, or even together in a combination compound (e.g., as part of a single chemical complex or covalent entity).

Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to each other, but are similar enough to allow comparisons between them, so that those skilled in the art will understand that reasonable conclusions can be drawn based on observed differences or similarities. In some embodiments, comparable sets of conditions, environments, individuals, or groups are characterized by having many substantially identical features and one or a few different features. Those skilled in the art will understand, in any given context, what degree of similarity is required for two or more such agents, entities, situations, sets of conditions, etc., to be considered comparable. For example, those skilled in the art will understand that an environment set, individual, or group is comparable when it is characterized by a sufficient number and type of substantially identical features to guarantee a reasonable conclusion that differences in results or observed phenomena under different environments, individuals, or groups are caused by or indicate changes in those features.

Corresponding to: As used herein, the term “corresponding to” refers to a relationship between two or more entities. For example, the term “corresponding to” can be used to specify the position/property of a structural element in one compound or composition relative to another compound or composition (e.g., relative to a suitable reference compound or composition). For example, in some embodiments, monomeric residues in a polymer (e.g., amino acid residues in a polypeptide or nucleic acid residues in a polynucleotide) can be identified as “corresponding to” residues in a suitable reference polymer. For example, those skilled in the art will understand that, for simplicity, residues in polypeptides are typically designated using a classical coding system based on a reference-related polypeptide, so the amino acid “corresponding to” the residue at position 190 does not actually need to be the 190th amino acid in a particular amino acid chain, but rather corresponds to the residue found at position 190 in the reference polypeptide; those skilled in the art will readily understand how to identify “corresponding” amino acids. For example, those skilled in the art will recognize that various sequence alignment strategies, including software programs such as BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE, can be used, for example, according to this disclosure, to identify “corresponding” residues in peptides and/or nucleic acids. Those skilled in the art will also understand that, in certain circumstances, the term “corresponding” can be used to describe an event or entity that shares a related similarity with another event or entity (e.g., a suitable reference event or entity). To give just one example, a gene or protein in one organism can be described as “corresponding” to a gene or protein from another organism, in order to indicate in some embodiments that it plays a similar role or performs a similar function and/or indicates that it exhibits a particular degree of sequence similarity or homology, or shares specific characteristic sequence elements.

Designed: As used herein, the term “designed” means a substance (i) whose structure is or is artificially selected; (ii) produced by artificial processes; and/or (iii) different from natural substances or other known substances.

Dosing regimen: Those skilled in the art will understand that the term "dosing regimen" can be used to refer to a set of unit doses (usually more than one) administered individually to a subject, typically at intervals of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen includes multiple doses, each time-separated from the other doses. In some embodiments, the individual doses are separated by time intervals of equal length; in some embodiments, a dosing regimen includes multiple doses and at least two distinct time intervals to separate the individual doses. In some embodiments, all doses in a dosing regimen are the same unit dose amount. In some embodiments, the different doses in a dosing regimen are different amounts. In some embodiments, a dosing regimen includes a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen includes a first dose in a first dose amount, followed by one or more additional doses in a second dose amount identical to the first dose amount. In some embodiments, when administered in a relevant population, the dosing regimen is associated with a desired or beneficial outcome (i.e., it is a therapeutic dosing regimen).

Engineered: Generally, the term “engineered” refers to aspects that have been artificially manipulated. For example, a polynucleotide is considered “engineered” when two or more sequences that are not connected together in nature are directly linked together in an engineered polynucleotide through artificial manipulation, and/or when a particular residue in a polynucleotide is not naturally occurring and/or is linked to an entity or part of it that is not connected in nature through artificial means.

Epitope: As used herein, the term "epitaph" refers to a portion specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. In some embodiments, an epitope comprises a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface-exposed when the antigen adopts an associated three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts such a conformation. In some embodiments, when the antigen adopts an optional conformation (e.g., linearization), at least some of such chemical atoms are physically separated groups.

Expression: As used herein, the term “expression” for a nucleic acid sequence means the production of any gene product from said nucleic acid sequence. In some embodiments, the gene product may be a transcript. In some embodiments, the gene product may be a polypeptide. In some embodiments, the expression of a nucleic acid sequence involves one or more of the following: (1) generating an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, etc.); (3) translating RNA into a polypeptide or protein; and/or (4) post-translational modification of the polypeptide or protein.

Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that demonstrates a statistically significant likelihood of achieving a predetermined therapeutic effect when administered to a relevant population. In some embodiments, the pharmaceutical composition may be specifically formulated for parenteral administration, for example, by subcutaneous, intramuscular, intravenous, or epidural injection, for example, as a sterile solution or suspension, or as a sustained-release formulation.

Polypeptide: As used herein, refers to a polymeric chain of amino acids. In some embodiments, the polypeptide has an amino acid sequence that is naturally occurring. In some embodiments, the polypeptide has an amino acid sequence that is not naturally occurring. In some embodiments, the polypeptide has an engineered amino acid sequence because it is designed and/or generated artificially. In some embodiments, the polypeptide may contain or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, the polypeptide may contain only or consist of natural or non-natural amino acids. In some embodiments, the polypeptide may contain D-amino acids, L-amino acids, or both. In some embodiments, the polypeptide may contain only D-amino acids. In some embodiments, the polypeptide may contain only L-amino acids. In some embodiments, the polypeptide may include one or more side groups or other modifications, for example, modifications or attachments to one or more amino acid side chains at the N-terminus, the C-terminus, or any combination thereof. In some embodiments, such side groups or modifications may be selected from acetylation, amidation, esterification, methylation, polyethylene glycolation, etc., including combinations thereof. In some embodiments, the polypeptide may be cyclic, and/or may contain a cyclic moiety. In some embodiments, the polypeptide is not cyclic and/or does not contain any cyclic portion. In some embodiments, the polypeptide is linear. In some embodiments, the polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to the name of a reference polypeptide, activity, or structure; in such cases, it is used herein to refer to polypeptides that share a related activity or structure and can therefore be considered members of the same class or family of polypeptides. For each such class, exemplary polypeptides within that class are provided in this specification and/or will be recognized by those skilled in the art, whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides of a polypeptide class or family. In some embodiments, members of a polypeptide class or family exhibit significant sequence homology or identity with the reference polypeptide of that class, share common sequence motifs (e.g., characteristic sequence elements), and/or share common activities (in some embodiments at comparable levels or within a specified range); in some embodiments, they are similar to all polypeptides within that class. For example, in some embodiments, the member polypeptide exhibits an overall degree of sequence homology or identity with the reference polypeptide of at least about 30-40%, and typically greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, and/or includes at least one region (e.g., in some embodiments, a conserved region that may be or contains a characteristic sequence element) exhibiting a very high sequence identity, typically greater than 90% or even 95%, 96%, 97%, 98% or 99%. Such conserved regions typically cover at least 3-4, and typically up to 20 or more amino acids; in some embodiments, the conserved region covers at least one segment of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more consecutive amino acids. In some embodiments, the related polypeptide may comprise or consist of fragments of the parent polypeptide.

Prevention: As used herein, when applied in connection with a disease, symptom, and/or condition, means reducing the risk of developing a disease, symptom, and/or condition and/or delaying the onset of one or more features or symptoms of a disease, symptom, or condition. Prevention is considered complete when the onset of a disease, symptom, or condition has been delayed for a predetermined period of time.

Reference: As used herein, a standard or control is described relative to which comparisons are made. For example, in some embodiments, the agent, animal, individual, population, sample, sequence, or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and/or measured substantially simultaneously with the test or assay of interest. In some embodiments, the reference or control is a historical reference or control, optionally contained in a tangible medium. Generally, as understood by those skilled in the art, the reference or control is identified or characterized under conditions or conditions comparable to those conditions or environments being evaluated. Those skilled in the art will understand when sufficient similarity exists to justify dependence and/or comparison with a particular possible reference or control.

Risk: As will be understood from the context, “risk” for a disease, condition, and/or disease status refers to the likelihood that a particular individual will develop a disease, condition, and/or disease status. In some embodiments, risk is expressed as a percentage. In some embodiments, risk ranges from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 up to 100%. In some embodiments, risk is expressed as risk relative to the risk associated with a reference sample or reference sample group. In some embodiments, the reference sample or reference sample group has a known risk of a disease, condition, disease status, and/or event. In some embodiments, the reference sample or reference sample group is drawn from individuals comparable to the particular individual. In some embodiments, the relative risk is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more. In some embodiments, risk may reflect one or more genetic attributes, such as those that may predispose an individual to develop (or not develop) a particular disease, condition, and/or disease status. In some implementations, risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes.

Susceptibility: An individual “susceptible” to a disease, symptom, and/or condition is an individual who has a higher risk of developing a disease, symptom, and/or condition than members of the general public. In some embodiments, an individual susceptible to a disease, symptom, and/or condition may not have been diagnosed with said disease, symptom, and/or condition. In some embodiments, an individual susceptible to a disease, symptom, and/or condition may exhibit symptoms of said disease, symptom, and/or condition. In some embodiments, an individual susceptible to a disease, symptom, and/or condition may not exhibit symptoms of said disease, symptom, and/or condition. In some embodiments, an individual susceptible to a disease, symptom, and/or condition will develop said disease, symptom, and/or condition. In some embodiments, an individual susceptible to a disease, symptom, and/or condition will not develop said disease, symptom, and/or condition.

Vaccination: As used herein, the term "vaccination" refers to the administration of a composition intended to elicit an immune response, such as a disease-related (e.g., pathogenic) substance. In some embodiments, vaccination may be administered before, during, and/or after exposure to a disease-related substance, and in some embodiments, shortly before, during, and/or after exposure to said substance. In some embodiments, vaccination comprises multiple administrations of a vaccine composition at appropriate intervals. In some embodiments, vaccination elicits an immune response to an infectious agent. In some embodiments, vaccination elicits an immune response against a tumor; in some such embodiments, vaccination is "personalized" because it targets, in part or in whole, an epitope (e.g., which may be or include one or more novel epitopes) identified in a particular individual's tumor.

Variant: As used herein in the context of molecules, such as nucleic acids, proteins, or small molecules, the term "variant" refers to a molecule that exhibits significant structural similarity to a reference molecule but differs structurally from the reference molecule, for example, in the presence or absence of the reference molecule or at the level of one or more chemical motifs compared to the reference entity. In some embodiments, the variant also differs functionally from its reference molecule. Generally, whether a particular molecule is properly considered a "variant" of a reference molecule is based on the degree to which it shares structural similarity with the reference molecule. As those skilled in the art will understand, any biological or chemical reference molecule has certain characteristic structural elements. By definition, a variant is a unique molecule that enjoys one or more of these characteristic structural elements but differs from the reference molecule in at least one respect. In some embodiments, a variant polypeptide or nucleic acid may differ from the reference polypeptide or nucleic acid due to one or more differences in the amino acid or nucleic acid sequence and/or one or more differences in the chemical motifs (e.g., carbohydrate, lipid, phosphate ester groups) that are covalent components of the polypeptide or nucleic acid (e.g., linked to the polypeptide or nucleic acid backbone). In some embodiments, the variant peptide or nucleic acid exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 99% overall sequence identity with the reference peptide or nucleic acid. In some embodiments, the variant peptide or nucleic acid does not share at least one characteristic sequence element with the reference peptide or nucleic acid. In some embodiments, the reference peptide or nucleic acid has one or more biological activities. In some embodiments, the variant peptide or nucleic acid enjoys one or more biological activities of the reference peptide or nucleic acid. In some embodiments, the variant peptide or nucleic acid lacks one or more biological activities of the reference peptide or nucleic acid. In some embodiments, the variant peptide or nucleic acid exhibits a reduced level of one or more biological activities compared to the reference peptide or nucleic acid. In some embodiments, a peptide or nucleic acid of interest is considered a “variant” of the reference peptide or nucleic acid if it has the same amino acid or nucleotide sequence as the reference but with minor sequence changes at specific positions. Typically, compared to the reference, the variant contains fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of residues that are substituted, inserted, or deleted. In some embodiments, the variant polypeptide or nucleic acid contains about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residue compared to the reference. Typically, relative to the reference, the variant polypeptide or nucleic acid contains a very small number (e.g., fewer than about 5, about 4, about 3, about 2, or about 1) of substituted, inserted, or deleted functional residues (i.e., residues involved in a specific biological activity). In some embodiments, the variant polypeptide or nucleic acid contains no more than about 5, about 4, about 3, about 2, or about 1 added or deleted residues compared to the reference, and in some embodiments, no added or deleted residues are included. In some embodiments, the variant polypeptide or nucleic acid contains fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, or about 6, and typically fewer than about 5, about 4, about 3, or about 2 additions or deletions, compared to a reference. In some embodiments, the reference polypeptide or nucleic acid is a polypeptide or nucleic acid found in nature.

This disclosure provides an RNA polynucleotide comprising: (i) a 5' cap, which is or includes a cap1 structure, for example, as disclosed herein; (ii) a 5' UTR sequence including a cap proximal sequence, for example, as disclosed herein; and (iii) a sequence encoding a payload. This disclosure also provides compositions and pharmaceutical articles comprising said RNA polynucleotide, and methods for preparing and using said RNA polynucleotide. In some embodiments, the RNA polynucleotide can be used to improve the translation efficiency of RNA encoding a payload and/or the expression of the RNA-encoded payload, said RNA polynucleotide comprising a 5' cap including a Cap1 structure disclosed herein, for example, _m27,3' ^- OGppp( _m12' ^-O )ApG cap; a 5' UTR including the cap proximal sequence disclosed herein; and a sequence encoding a payload. In some embodiments, the absence of a self-hybridization sequence in the RNA polynucleotide encoding the payload can further improve the translation efficiency of the RNA encoding the payload and/or the expression of the payload encoded by the RNA payload.

RNA polynucleotides

As used herein, the terms "polynucleotide" or "nucleic acid" refer to DNA and RNA, such as genomic DNA, cDNA, and mRNA, as well as molecules produced through recombination and chemical synthesis. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the invention, polynucleotides are preferably isolated.

In some embodiments, nucleic acids may be contained in a vector. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, granular vectors, bacteriophage vectors such as λ phage, viral vectors such as retroviruses, adenoviruses, or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). In some embodiments, the vector may be an expression vector; alternatively or additionally, in some embodiments, the vector may be a cloning vector. Those skilled in the art will understand that in some embodiments, the expression vector may be, for example, a plasmid; alternatively or additionally, in some embodiments, the expression vector may be a viral vector. Typically, an expression vector contains the desired coding sequence as well as other appropriate sequences necessary for expressing an operatively linked coding sequence in a particular host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in vitro expression system. Cloning vectors are generally used to engineer and amplify a desired fragment (typically a DNA fragment) and may lack the functional sequences required for expressing the desired fragment.

In some implementations, the nucleic acids described and/or used herein may be or contain recombinant and/or isolated molecules.

Those skilled in the art will understand upon reading this disclosure that the term "RNA" generally refers to a nucleic acid molecule comprising ribonucleotide residues. In some embodiments, the RNA comprises all or most of ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-furanose group. In some embodiments, the RNA may be partially or completely double-stranded RNA; in some embodiments, the RNA may comprise two or more distinct nucleic acid strands (e.g., separate molecules) that partially or completely hybridize with each other. In many embodiments, the RNA is single-stranded, and in some embodiments, it may self-hybridize or otherwise fold into secondary and/or tertiary structures. In some embodiments, the RNA as described and/or used herein does not self-hybridize, at least for certain sequences as described herein. In some embodiments, the RNA can be isolated RNA, such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinant RNA, and/or modified RNA (wherein the term "modification" is understood to mean that one or more residues or other structural elements of the RNA differ from naturally occurring RNA; for example, in some embodiments, modified RNA is distinguished by the addition, deletion, substitution, and/or alteration of one or more nucleotides and/or by one or more portions or features of the nucleotides (e.g., nucleoside or backbone structure or linkages). In some embodiments, modification can be or includes the addition of a non-nucleotide substance to an internal RNA nucleotide or the terminus of the RNA. It is also contemplated herein that the nucleotides in the RNA (e.g., modified RNA) can be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the purposes of this disclosure, these altered RNAs are considered analogs of naturally occurring RNA.

In some embodiments of this disclosure, the RNA is or includes messenger RNA (mRNA) associated with RNA transcripts encoding polypeptides.

In some embodiments, the RNA disclosed herein comprises: a 5' cap containing the 5' cap disclosed herein; a 5' untranslated region (5'-UTR) containing a sequence proximal to the cap, encoding a payload (e.g., a polypeptide); a 3' untranslated region (3'-UTR); and/or a polyadenylate (PolyA) sequence.

In some embodiments, the RNA disclosed herein includes the following components in the 5' to 3' orientation: a 5' cap containing the 5' cap disclosed herein; a 5' untranslated region (5'-UTR) containing a sequence at the proximal end of the cap, a sequence encoding a payload (e.g., a polypeptide); a 3' untranslated region (3'-UTR); and a PolyA sequence.

In some implementations, RNA is produced through in vitro transcription or chemical synthesis. In some implementations, mRNA is produced through in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides.

In some embodiments, the RNA disclosed herein is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

In some implementations, the RNA is "replicon RNA" or simply "replicon," particularly "self-replicating RNA" or "self-amplifying RNA." In some implementations, the replicon or self-replicating RNA originates from or contains elements derived from ssRNA viruses, particularly positive-sense ssRNA viruses such as alphaviruses. Alphaviruses are typical representatives of positive-sense RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphavirus life cycle, see José et al., Future Microbiol., 2009, vol. 4, pp. 837–856). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA usually has a 5' cap and a 3' poly(A) tail. The alphavirus genome encodes non-structural proteins (involved in the transcription, modification, and replication of viral RNA, as well as protein modification) and structural proteins (forming viral particles). There are typically two open reading frames (ORFs) in the genome. Four non-structural proteins (nsP1–nsP4) are typically encoded by a first ORF starting near the 5′ end of the genome, while alphavirus structural proteins are encoded by a second ORF located downstream of the first ORF and extending near the 3′ end of the genome. Typically, the first ORF is larger than the second ORF, in a ratio of approximately 2:1. In alphavirus-infected cells, only the nucleic acid sequences encoding non-structural proteins are translated from the genomic RNA, while the genetic information encoding structural proteins is translated from subgenomic transcripts, which are RNA polynucleotides similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111–124). Post-infection, early in the viral life cycle, the (+) strand of genomic RNA directly acts as messenger RNA for translating the open reading frame encoding the non-structural polyprotein (nsP1234). Alphavirus-derived vectors have been proposed for delivering foreign genetic information to target cells or organisms. In a simplified approach, the open reading frame (OPF) encoding an alphavirus structural protein is replaced by an OPF encoding the protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one molecule encodes a viral replicase, and the other is capable of being trans-replicated by that replicase (hence the name trans-replication system). Trans-replication requires the presence of both nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being trans-replicated by the replicase must contain certain alphavirus sequence elements to allow the alphavirus replicase to recognize and synthesize RNA.

In some embodiments, the RNA described herein may have modified nucleosides. In some embodiments, the RNA contains modified nucleosides in place of at least one (e.g., each) uridine.

As used herein, the term "uracil" describes one of the nucleobases that can appear in nucleic acids that contain RNA. The structure of uracil is:

As used in this article, the term "uridine" describes one of the nucleosides that can appear in RNA. The structure of uridine is:

UTP (uridine 5'-triphosphate) has the following structure:

Pseudo-UTP (pseudouridine-5'-triphosphate) has the following structure:

"Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is linked to a pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

Another exemplary modified nucleoside is N1-methylpseuuridine (m1Ψ), which has the following structure:

N1-Methylpseudouridine-5'-triphosphate (m1ΨTP) has the following structure:

Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the following structure:

.

In some embodiments, one or more uridines in the RNA described herein are replaced by modified nucleosides. In some embodiments, the modified nucleosides are modified uridines.

In some embodiments, the RNA comprises a modified nucleoside replacing at least one uridine. In some embodiments, the RNA comprises a modified nucleoside replacing each uridine.

In some embodiments, the modified nucleoside is independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ). In some embodiments, the modified nucleoside comprises N1-methylpseudouridine (m1Ψ). In some embodiments, the modified nucleoside comprises 5-methyluridine (m5U). In some embodiments, the RNA may comprise more than one type of modified nucleoside, and the modified nucleoside is independently selected from pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ) and N1-methylpseudouridine (m1Ψ). In some embodiments, the modified nucleoside comprises pseudouridine (Ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises N1-methylpseudouridine (m1Ψ) and 5-methyluridine (m5U). In some embodiments, the modified nucleosides include pseudouridine (Ψ), N1-methylpseudouridine (m1Ψ), and 5-methyluridine (m5U).

In some embodiments, the nucleoside that replaces one or more (e.g., all) uridines in the RNA modification can be any one or more of the following: 3-methyluridine ( ^m3 U), 5-methoxyuridine ( ^mo5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine ( ^s2 U), 4-thio-uridine ( ^s4 U), 4-thio-pseuuridine, 2-thio-pseuuridine, 5-hydroxyuridine ( ^ho5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5-oxyacetic acid ( ^cmo5 U), uridine 5-oxyacetic acid methyl ester ( ^mcmo5 U), 5-carboxymethyluridine ( ^cm5 U), 1-carboxymethyl-pseuuridine, 5-carboxyhydroxymethyluridine ( ^chm5 5-Carboxyhydroxymethyl-uridine methyl ester (mchm ⁵ U), 5-methoxycarbonylmethyl-uridine (mcm ⁵ U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm ⁵ s ² U), 5-aminomethyl-2-thio-uridine (nm ⁵ s ² U), 5-methylaminomethyl-uridine (mnm ⁵ U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm ⁵ s ² U), 5-methylaminomethyl-2-seleno-uridine (mnm ⁵ se ² U), 5-carbamoylmethyl-uridine (ncm ⁵ U), 5-carboxymethylaminomethyl-uridine (cmnm ⁵ U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm ⁵ s ² U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauronic acid methyl-uridine (τm ⁵ U), 1-tauronic acid methyl-pseudouridine, 5-tauronic acid methyl-2-thio-uridine (τm5s2U), 1-tauronic acid methyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m ⁵ s ² U), 1-methyl-4-thio-pseudouridine (m ¹ s ⁴ ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m ³ ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-denitro-pseudouridine, 2-thio-1-methyl-1-denitro-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m ⁵ D), 2-Thio-dihydrouridine, 2-Thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp ³ U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ³ ψ), 5-(isopentenylaminomethyl)uridine (inm ⁵ U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm ⁵ s ² U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m ⁵ Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s ² Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm ⁵ Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm ⁵ Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm ⁵ Um), 3,2′-O-dimethyl-uridine (m ³ Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm ⁵ Um), 1-thio-uridine, deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine, 5-(2-methoxycarbonylvinyl)uridine, 5-[3-(1-E-propenylamino)uridine or any other modified uridine known in the art.

In some embodiments, the RNA comprises other modified nucleosides, or further modified nucleosides, such as modified cytidine. For example, in some embodiments, 5-methylcytidine partially or completely, preferably completely, replaces cytidine in the RNA. In some embodiments, the RNA comprises 5-methylcytidine and one or more selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), and 5-methyl-uridine (m5U). In some embodiments, the RNA comprises 5-methylcytidine and N1-methyl-pseudouridine (m1ψ). In some embodiments, the RNA comprises 5-methylcytidine replacing each cytidine, and N1-methyl-pseudouridine (m1ψ) replacing each uridine.

In some embodiments, RNA encoding a payload such as a vaccine antigen is expressed in the cells of the treated subject to provide the payload, e.g., the vaccine antigen. In some embodiments, the RNA is transiently expressed in the subject's cells. In some embodiments, the RNA is in vitro transcribed RNA. In some embodiments, the payload such as the vaccine antigen is expressed on the cell surface. In some embodiments, the payload such as the vaccine antigen is expressed and presented against an MHC background. In some embodiments, the expression of the payload such as the vaccine antigen enters the extracellular space, i.e., secreted vaccine antigen.

In the context of this disclosure, the term "transcription" refers to a process in which the genetic code in a DNA sequence is transcribed into RNA. The RNA can then be translated into peptides or proteins.

According to the invention, the term "transcription" includes "in vitro transcription," wherein the term "in vitro transcription" refers to a process in which RNA, particularly mRNA, is synthesized in vitro in a cell-free system, preferably using a suitable cell extract. Preferably, a cloning vector is used to produce the transcript. These cloning vectors are generally designated as transcription vectors and are covered by the term "vector" according to the invention. According to the invention, the RNA used is preferably in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, the in vitro transcription according to the invention is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning nucleic acids, particularly cDNA, and introducing them into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

Regarding RNA, the terms "expression" or "translation" refer to a process within the cell's ribosomes through which the mRNA chain directs the assembly of amino acid sequences to produce peptides or proteins.

In some embodiments, after administration of the RNA described herein, for example, formulated as RNA lipid particles, at least a portion of the RNA is delivered to target cells. In some embodiments, at least a portion of the RNA is delivered to the cytosol of the target cells. In some embodiments, the RNA is translated by the target cells to produce a peptide or protein encoded therein. In some embodiments, the target cells are spleen cells. In some embodiments, the target cells are antigen-presenting cells, such as specialized antigen-presenting cells in the spleen. In some embodiments, the target cells are dendritic cells or macrophages. The RNA particles described herein, such as RNA lipid particles, can be used to deliver RNA to such target cells. Therefore, this disclosure also relates to a method of delivering RNA to target cells in a subject, the method comprising administering the RNA particles described herein to the subject. In some embodiments, the RNA is delivered to the cytosol of the target cells. In some embodiments, the RNA is translated by the target cells to produce a peptide or protein encoded therein.

"Encoding" refers to the inherent characteristics of a specific nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, so that it can be used as a template for the synthesis of other polymers or macromolecules in biological processes. These polymers or macromolecules have defined nucleotide sequences (i.e., rRNA, tRNA, and mRNA) or defined amino acid sequences and the resulting biological characteristics. Therefore, if the transcription and translation of the mRNA corresponding to a gene produces a protein in a cell or other biological system, then that gene encodes a protein. The coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing, as well as the non-coding strand used as a template for gene or cDNA transcription, can both be referred to as encoding the protein or other product of that gene or cDNA.

In some embodiments, the nucleic acid compositions described herein, such as compositions comprising mRNA encapsulated in lipid nanoparticles, are characterized by (e.g., when administered to a subject) sustained expression of the encoded polypeptide. For example, in some embodiments, such compositions are characterized in that, when administered to a human, they achieve detectable polypeptide expression in a biological sample (e.g., serum) from that human, and in some embodiments, this expression persists for at least 36 hours or longer, including, for example, at least 48 hours, at least 60 hours, at least 72 hours, at least 96 hours, at least 120 hours, at least 148 hours, or longer.

In some embodiments, the RNA encoding the vaccine antigen administered according to the present invention is non-immunogenic. RNA encoding an immunostimulant can be administered according to the present invention to provide an adjunctive effect. The RNA encoding the immunostimulant can be standard RNA or non-immunogenic RNA.

As used herein, the term "non-immunogenic RNA" refers to RNA that, when administered to, for example, a mammal, does not induce an immune system response, or induces a response that is weaker than that induced by the same RNA that has not undergone modifications and treatments to render the non-immunogenic RNA non-immunogenic—that is, weaker than that induced by standard RNA (stdRNA). In a preferred embodiment, non-immunogenic RNA, also referred to herein as modified RNA (modRNA), is rendered non-immunogenic by incorporating a modified nucleoside that inhibits RNA-mediated activation of innate immune receptors into the RNA and removing the double-stranded RNA (dsRNA).

To impart non-immunogenicity to non-immunogenic RNA by incorporating a modified nucleoside, any modified nucleoside can be used, as long as it reduces or inhibits the immunogenicity of the RNA. Modified nucleosides that inhibit RNA-mediated activation of innate immune receptors are particularly preferred. In some embodiments, the modified nucleoside comprises replacing one or more uridines with a nucleoside containing a modified nucleobase. In some embodiments, the modified nucleobase is a modified uracil. In some embodiments, the nucleoside containing the modified nucleobase is selected from 3-methyluridine ( ^m3 U), 5-methoxyuridine ( ^mo5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine ( ^s2 U), 4-thio-uridine ( ^s4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine ( ^ho5 U), 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid ( ^cmo5 U), uridine 5-oxyacetic acid methyl ester ( ^mcmo5 U), 5-carboxymethyluridine ( ^cm5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyluridine ( ^chm5 U), 5-carboxyhydroxymethyluridine methyl ester ( ^mchm5 U), 5-methoxycarbonylmethyl-uridine (mcm ⁵ U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm ⁵ s ² U), 5-aminomethyl-2-thio-uridine (nm ⁵ s ² U), 5-methylaminomethyl-uridine (mnm ⁵ U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm ⁵ s ² U), 5-methylaminomethyl-2-seleno-uridine (mnm ⁵ se ² U), 5-carbamoylmethyl-uridine (ncm ⁵ U), 5-carboxymethylaminomethyl-uridine (cmnm ⁵ U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm ⁵ s ² U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-tauronic acid methyl-uridine (τm ⁵ U), 1-Taurine methyl-pseudouridine, 5-Taurine methyl-2-thio-uridine (τm5s2U), 1-Taurine methyl-4-thio-pseudouridine, 5-Methyl- ² -thio-uridine ( ^m5s2U ), 1-Methyl- ^{4-thio-pseudouridine (m1s4ψ )} , 4-Thio-1-methyl-pseudouridine, 3-Methyl-pseudouridine ( ^m3ψ ), 2-Thio-1-methyl-pseudouridine, 1-Methyl-1-denitro-pseudouridine, 2-Thio-1-methyl-1-denitro-pseudouridine, dihydrouridine (D), dihydrouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine ( ^m5s2U), D), 2-Thio-dihydrouridine, 2-Thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp ³ U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp ³ ψ), 5-(isopentenylaminomethyl)uridine (inm ⁵ U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm ⁵ s ² U), α-thio-uridine, 2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m ⁵ Um), 2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s ² Um), 5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm ⁵ Um), 5-carbamoylmethyl-2′-O-methyl-uridine (ncm ⁵ Um), 5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm ⁵ Um), 3,2′-O-dimethyl-uridine (m ³ Um), 5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm ⁵ Um), 1-thio-uridine, deoxythymidine, 2′-F-arasu-uridine, 2′-F-uridine, 2′-OH-arasu-uridine, 5-(2-methoxycarbonylvinyl)uridine and 5-[3-(1-E-propenylamino)uridine. In a particularly preferred embodiment, the nucleoside containing the modified nucleobase is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U), especially N1-methyl-pseudouridine.

In some embodiments, replacing one or more uridines with a nucleoside containing a modified nucleobase includes replacing at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the uridine.

During mRNA synthesis via in vitro transcription (IVT) using T7 RNA polymerase, a large number of aberrant products, including double-stranded RNA (dsRNA), are generated due to the enzyme's unconventional activity. dsRNA induces inflammatory cytokines and activates effector enzymes, leading to inhibition of protein synthesis. dsRNA can be removed from RNA such as IVT RNA using, for example, ion-pair reversed-phase HPLC, using a non-porous or porous C-18 polystyrene-divinylbenzene (PS-DVB) matrix. Alternatively, dsRNA contamination can be eliminated from IVT RNA products using an enzyme-based method employing *E. coli* RNase III, which specifically hydrolyzes dsRNA but not ssRNA. Furthermore, dsRNA can be separated from ssRNA by using a cellulose material. In some embodiments, the RNA product is contacted with a cellulose material, and ssRNA is separated from the cellulose material under conditions that allow dsRNA to bind to the cellulose material but prevent ssRNA from binding to it.

When used herein, "removal" or "elimination" refers to the characteristic of separating a population of a first substance, such as non-immunogenic RNA, from a population of adjacent second substances, such as dsRNA, wherein the population of the first substance need not be completely devoid of the second substance, and the population of the second substance need not be completely devoid of the first substance. However, it is characterized by the population of the first substance from which the second substance has been removed having a measurably lower content of the second substance compared to a mixture of the unseparated first and second substances.

In some embodiments, removal of dsRNA from non-immunogenic RNA includes removing dsRNA such that less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, less than 0.3%, or less than 0.1% of the RNA in the non-immunogenic RNA composition is dsRNA. In some embodiments, the non-immunogenic RNA contains no or substantially no dsRNA. In some embodiments, the non-immunogenic RNA composition comprises a purified product of nucleoside-modified single-stranded RNA. For example, in some embodiments, the purified product of nucleoside-modified single-stranded RNA is substantially free of double-stranded RNA (dsRNA). In some embodiments, the purified product contains at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% nucleoside-modified single-stranded RNA relative to all other nucleic acid molecules (DNA, dsRNA, etc.).

In some embodiments, non-immunogenic RNA exhibits higher translation efficiency in cells compared to standard RNA having the same sequence. In some embodiments, the fold increase in translation is 2-fold relative to its unmodified counterpart. In some embodiments, the fold increase in translation is 3-fold. In some embodiments, the fold increase in translation is 4-fold. In some embodiments, the fold increase in translation is 5-fold. In some embodiments, the fold increase in translation is 6-fold. In some embodiments, the fold increase in translation is 7-fold. In some embodiments, the fold increase in translation is 8-fold. In some embodiments, the fold increase in translation is 9-fold. In some embodiments, the fold increase in translation is 10-fold. In some embodiments, the fold increase in translation is 15-fold. In some embodiments, the fold increase in translation is 20-fold. In some embodiments, the fold increase in translation is 50-fold. In some embodiments, the fold increase in translation is 100-fold. In some embodiments, the fold increase in translation is 200-fold. In some embodiments, the fold increase in translation is 500-fold. In some embodiments, the translation enhancement factor is 1000x. In some embodiments, the translation enhancement factor is 2000x. In some embodiments, the factor is 10-1000x. In some embodiments, the factor is 10-100x. In some embodiments, the factor is 10-200x. In some embodiments, the factor is 10-300x. In some embodiments, the factor is 10-500x. In some embodiments, the factor is 20-1000x. In some embodiments, the factor is 30-1000x. In some embodiments, the factor is 50-1000x. In some embodiments, the factor is 100-1000x. In some embodiments, the factor is 200-1000x. In some embodiments, the translation enhancement includes any other significant amount or range of amounts.

In some embodiments, non-immunogenic RNA exhibits significantly lower innate immunogenicity compared to standard RNA having the same sequence. In some embodiments, non-immunogenic RNA exhibits a 2-fold less innate immune response than its unmodified counterpart. In some embodiments, the fold reduction in innate immunogenicity is 3-fold. In some embodiments, the fold reduction in innate immunogenicity is 4-fold. In some embodiments, the fold reduction in innate immunogenicity is 5-fold. In some embodiments, the fold reduction in innate immunogenicity is 6-fold. In some embodiments, the fold reduction in innate immunogenicity is 7-fold. In some embodiments, the fold reduction in innate immunogenicity is 8-fold. In some embodiments, the fold reduction in innate immunogenicity is 9-fold. In some embodiments, the fold reduction in innate immunogenicity is 10-fold. In some embodiments, the fold reduction in innate immunogenicity is 15-fold. In some embodiments, the fold reduction in innate immunogenicity is 20-fold. In some embodiments, the fold reduction in innate immunogenicity is 50-fold. In some embodiments, the fold reduction in innate immunogenicity is 100-fold. In some embodiments, the fold reduction in innate immunogenicity is 200-fold. In some embodiments, the fold reduction in innate immunogenicity is 500-fold. In some embodiments, the fold reduction in innate immunogenicity is 1000-fold. In some embodiments, the fold reduction in innate immunogenicity is 2000-fold.

The term "exhibiting significantly less innate immunogenicity" refers to a detectable reduction in innate immunogenicity. In some embodiments, the term refers to a reduction such that an effective amount of non-immunogenic RNA can be administered without triggering a detectable innate immune response. In some embodiments, the term refers to a reduction such that repeated administration of non-immunogenic RNA can be performed without inducing an innate immune response sufficient to detectably reduce the production of the protein encoded by the non-immunogenic RNA. In some embodiments, the reduction allows for repeated administration of non-immunogenic RNA without inducing an innate immune response sufficient to eliminate the detectable production of the protein encoded by the non-immunogenic RNA.

"Immunogenicity" is the ability of a foreign substance, such as RNA, to elicit an immune response in a human or other animal. The innate immune system is a component of the immune system, and it is relatively nonspecific and immediate. Together with the adaptive immune system, it is one of the two main components of the vertebrate immune system. As used herein, "endogenous" refers to any substance that originates from or is produced within an organism, cell, tissue, or system.

As used herein, the term “exogenous” means any substance introduced from or produced outside an organism, cell, tissue, or system.

As used in this article, the term “expression” is defined as the transcription and/or translation of a specific nucleotide sequence.

As used herein, the terms “connected,” “fused,” or “fusion” are used interchangeably. These terms refer to two or more elements, components, or domains that are connected together.

Codon optimization

In some embodiments, the payload (e.g., a peptide) described herein is encoded by a coding sequence that has undergone codon optimization and/or has an increased G/C content compared to a wild-type coding sequence. In some embodiments, one or more regions of the coding sequence are codon-optimized and/or have increased G/C content compared to the corresponding sequence region of the wild-type coding sequence. In some embodiments, the increase in codon optimization and/or G/C content preferably does not alter the sequence of the encoded amino acid sequence.

The term "codon optimization" is understood by those skilled in the art to refer to altering codons in the coding region of a nucleic acid molecule to reflect typical codon usage in the host organism, but preferably without altering the amino acid sequence encoded by the nucleic acid molecule. In the context of this disclosure, it is preferred to use the RNA polynucleotides described herein to codon-optimize the coding region for optimal expression in the subject to be treated. Codon optimization is based on the finding that translation efficiency is also determined by the varying frequencies of tRNA occurrence in the cell. Therefore, the RNA sequence can be modified to insert codons that yield frequently occurring tRNAs instead of "rare codons."

In some embodiments, the guanosine/cytosine (G/C) content of the coding region of the RNA (e.g., the payload sequence) is increased compared to the G/C content of the corresponding coding sequence encoding the wild-type RNA, wherein the amino acid sequence encoded by the RNA is preferably unmodified compared to the wild-type RNA. This modification of the RNA sequence is based on the fact that the sequence of any RNA region to be translated is important for efficient translation of that mRNA. Sequences with increased G (guanosine)/C (cytosine) content are more stable than sequences with increased A (adenosine)/U (uracil) content. Given that several codons encode one and the same amino acid (so-called degeneracy of the genetic code), the codons most favorable for stability (so-called alternative codon usage) can be determined. Depending on the amino acids encoded by the RNA, there are various possibilities for modifications to the RNA sequence compared to its wild-type sequence. In particular, codons containing A and/or U nucleosides can be modified by replacing these codons with other codons that encode the same amino acid but do not contain A and/or U or contain a lower amount of A and/or U nucleosides.

In some implementations, the G/C content of the coding region of the RNA described herein is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, or even more compared to the G/C content of the coding region of wild-type RNA.

5' cap

RNA capping has been extensively studied, for example, in Decroly E et al. (2012) Nature Reviews 10:51-65; and Ramanathan A. et al. (2016) Nucleic Acids Res; 44(16):7511–7526, the full contents of each of which are incorporated herein by reference. The 5’ cap includes Cap-0 (also referred to as “Cap0” in this paper), Cap-1 (also referred to as “Cap1” in this paper), or Cap-2 (also referred to as “Cap2” in this paper). See, for example, Figure 1 of Ramanathan A et al. and Figure 1 of Decroly E et al.

As used herein, the term "5'-cap" refers to a structure found at the 5'-end of RNA, such as mRNA, and generally comprises a guanosine nucleotide (also referred to as Gppp or G(5')ppp(5')) linked to RNA, such as mRNA, via a 5'-to-5'-triphosphate bond. In some embodiments, the guanosine nucleotide included in the 5'-cap may be modified, for example, by methylation at one or more positions (e.g., at the 7-position) on the base (guanine) and/or by methylation at one or more positions on the ribose. In some embodiments, the guanosine nucleotide included in the 5'-cap comprises 3'O methylation of the ribose (3'OMeG). In some embodiments, the guanosine nucleotide included in the 5'-cap comprises methylation at the 7-position of guanine (m7G). In some embodiments, the guanosine nucleotide included in the 5'-cap comprises methylation at the 7-position of guanine and 3'O methylation of the ribose (m7(3'OMeG)).

In some embodiments, providing RNA having a 5'-cap or a 5'-cap analogue disclosed herein can be achieved via in vitro transcription, wherein the 5'-cap is co-transcribed into the RNA strand, or it can be ligated to RNA post-transcriptionally using a capping enzyme. In some embodiments, using a cap disclosed herein, for example, using cap1 or a cap1 analogue, improves the capping efficiency of RNA compared to co-transcriptional capping with a suitable reference comparison. In some embodiments, improved capping efficiency can increase RNA translation efficiency and/or translation rate, and/or increase the expression of encoded polypeptides.

In some embodiments, the RNA described herein comprises a 5'-cap or a 5'-cap analogue, such as Cap0, Cap1, or Cap2. In some embodiments, the provided RNA does not contain an uncapped 5'-triphosphate. In some embodiments, the RNA may be capped using a 5'-cap analogue. In some embodiments, the RNA described herein comprises Cap0. In some embodiments, the RNA described herein comprises Cap1, for example, as described herein. In some embodiments, the RNA described herein comprises Cap2.

In some embodiments, the Cap0 structure contains a 7-methylated guanosine nucleotide (m7G) of guanine. In some embodiments, the Cap0 structure is linked to RNA via a 5'-to-5'-triphosphate bond and is also referred to herein as m7Gppp or m7G(5')ppp(5').

In some embodiments, the Cap1 structure comprises a 7-methylated guanosine nucleotide (m7G) of guanine and a 2'O (2'OMeN ₁ ) methylated first nucleotide in RNA. In some embodiments, the Cap1 structure is linked to RNA via a 5'-to-5'-triphosphate bond and is also referred to herein as m7Gppp(2'OMeN ₁ ) or m7G(5')ppp(5')(2'OMeN ₁ ). In some embodiments, N ₁ is selected from A, C, G, or U. In some embodiments, N ₁ is A. In some embodiments, N ₁ is C. In some embodiments, N ₁ is G. In some embodiments, N ₁ is U.

In some embodiments, the m7G(5')ppp(5')(2'OMeN ₁ )Cap1 structure contains a second nucleotide N ₂ , which is a cap-proximal nucleotide at position 2 and is selected from A, G, C, or U (m7G(5')ppp(5')(2'OMeN ₁ )N ₂ ). In some embodiments, N ₂ is A. In some embodiments _, N 2 is C. In some embodiments, N ₂ is G. In some embodiments, N ₂ is U.

In some implementations, the cap1 structure is or contains m7G(5')ppp(5')(2'OMeA ₁ )pG ₂ , where A is the cap proximal nucleotide at the +1 position and G is the cap proximal nucleotide at the +2 position, and has the following structure:

In some embodiments, the cap1 structure is or contains m7G(5')ppp(5')(2'OMeA ₁ )pU ₂ , where A is the cap proximal nucleotide at position 1 and U is the cap proximal nucleotide at position 2, and has the following structure:

In some embodiments, the cap1 structure is or contains m7G(5')ppp(5')(2'OMeG ₁ )pG ₂ , where G is the cap proximal nucleotide at position 1 and G is the cap proximal nucleotide at position 2, and has the following structure:

In some embodiments, the Cap1 structure comprises a 7-methylated guanosine nucleotide (m7G) and one or more additional modifications, such as methylation on the ribose and a 2'O methylated first nucleotide in the RNA. In some embodiments, the Cap1 structure comprises a 7-methylated guanosine nucleotide and a 3'O methylation on the ribose (m7(3'OMeG)); and a 2'O methylated first nucleotide in the RNA (2'OMeN ₁ ). In some embodiments, the Cap1 structure is linked to the RNA via a 5'-to-5'-triphosphate bond and is also referred to herein as m7(3'OMeG)ppp(2'OMeN ₁ ) or m7(3'OMeG)(5')ppp(5')(2'OMeN ₁ ). In some embodiments, N ₁ is selected from A, C, G, or U. In some embodiments, N ₁ is A. In some embodiments, N ₁ is C. In some embodiments, N ₁ is G. In some embodiments, N ₁ is U.

In some embodiments, the m7(3'oMeG)(5')ppp(5')(2'OMeN ₁ )Cap1 structure contains a second nucleotide N ₂ , which is a cap-proximal nucleotide at position 2 and is selected from A, G, C, or U (m7(3'OMeG)(5')ppp(5')(2'OMeN ₁ )N ₂ ). In some embodiments, N ₂ is A. In some embodiments, N ₂ is C. In some embodiments, N ₂ is G. In some embodiments, N ₂ is U.

In some embodiments, the cap1 structure is or contains m7(3'OMeG)(5')ppp(5')(2'OMeA ₁ )pG ₂ , where A is the cap proximal nucleotide at position 1 and G is the cap proximal nucleotide at position 2, and has the following structure:

In some embodiments, the cap1 structure is or contains m7(3'OMeG)(5')ppp(5')(2'OMeG ₁ )pG ₂ , where G is the cap proximal nucleotide at position 1 and G is the cap proximal nucleotide at position 2, and has the following structure:

In some embodiments, the second nucleotide in the Cap1 structure may include one or more modifications, such as methylation. In some embodiments, the Cap1 structure containing a second nucleotide with 2'O methylation is a Cap2 structure.

In some embodiments, RNA polynucleotides containing a Cap1 structure exhibit increased translation efficiency, increased translation rate, and/or increased expression of the encoded payload relative to a suitable reference comparison. In some embodiments, RNA polynucleotides containing a Cap1 structure having m7(3'OMeG)(5')ppp(5')(2'OMeA ₁ )pG ₂ (where A is the cap proximal nucleotide at position 1 and G is the cap proximal nucleotide at position 2) exhibit increased translation efficiency relative to RNA polynucleotides containing a Cap1 structure having m7(3'OMeG)(5')ppp(5')(2'OMeG ₁ )pG ₂ (where G ₁ is the cap proximal nucleotide at position 1 and G ₂ is the cap proximal nucleotide at position 2). In some embodiments, the increased translation efficiency is evaluated when the RNA polynucleotide is administered to cells or an organism.

In some implementations, the cap analog used in the RNA polynucleotide is _m27,3' ^-O Gppp( _m12' ^-O )ApG (sometimes also called _m27,3'O G(5')ppp(5') ^{m2' -O} ApG or m7(3'OMeG)(5')ppp(5')(2'OMeA)pG), which has the following structure:

The following is an example Cap1 RNA, which contains RNA and _m27,3`OG ( ⁵ ')ppp(5') ^m2'- OApG:

Here is another example of Cap1 RNA:

In some embodiments, the RNA polynucleotide disclosed herein includes the Cap shown in any of Figures 3A-3I. In some embodiments, the RNA polynucleotide disclosed herein includes the Cap shown in Figure 3A. In some embodiments, the RNA polynucleotide disclosed herein includes the Cap shown in Figure 3B. In some embodiments, the RNA polynucleotide disclosed herein includes the Cap shown in Figure 3C. In some embodiments, the RNA polynucleotide disclosed herein includes the Cap shown in Figure 3D. In some embodiments, the RNA polynucleotide disclosed herein includes the Cap shown in Figure 3E. In some embodiments, the RNA polynucleotide disclosed herein includes the Cap shown in Figure 3F. In some embodiments, the RNA polynucleotide disclosed herein includes the Cap shown in Figure 3G. In some embodiments, the RNA polynucleotide disclosed herein includes the Cap shown in Figure 3H. In some embodiments, the RNA polynucleotide disclosed herein includes the Cap shown in Figure 3I.

5’UTR and cap proximal sequence

In some embodiments, the RNA disclosed herein contains a 5'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA polynucleotide such as mRNA. An untranslated region (UTR) can be present at the 5' (upstream) end of an open reading frame (5'-UTR) and/or the 3' (downstream) end of an open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end, upstream of the start codon of the protein-coding region. The 5'-UTR is located downstream of the 5'-cap (if present), for example, directly adjacent to the 5'-cap.

In some embodiments, the 5’ UTR disclosed herein includes a cap proximal sequence, for example, as disclosed herein. In some embodiments, the cap proximal sequence includes a sequence adjacent to the 5’ cap. In some embodiments, the cap proximal sequence includes nucleotides at positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide.

In some embodiments, the Cap structure comprises one or more polynucleotides in the proximal cap sequence. In some embodiments, the Cap structure comprises an m7 guanosine cap of an RNA polynucleotide and nucleotide +1 ( _N1 ). In some embodiments, the Cap structure comprises an m7 guanosine cap of an RNA polynucleotide and nucleotide +2 ( _N2 ). In some embodiments, the Cap structure comprises an m7 guanosine cap of an RNA polynucleotide and nucleotides +1 and +2 ( _N1 and _N2 ).

Those skilled in the art will understand upon reading this disclosure that, in some embodiments, one or more residues in the proximal cap sequence (e.g., one or more of residues +1, +2, +3, +4, and/or +5) may be included in the RNA because they are already included in the cap body (e.g., the Cap1 structure, etc.); or, in some embodiments, at least some residues in the proximal cap sequence may be enzymatically added (e.g., by a polymerase such as T7 polymerase). For example, in some exemplary embodiments using the _m27,3' ^-O Gppp( _m12' ^-O )ApG cap, +1 and +2 are the ( _m12' ^-O )A and G residues of the cap, while +3, +4, and +5 are added by a polymerase (e.g., T7 polymerase).

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, where _N1 and _N2 are any nucleotides, such as A, C, G, or U. In some embodiments, _N1 is A. In some embodiments, _N1 is C. In some embodiments, _N1 is G. In some embodiments, _N1 is U. In some embodiments, _N2 is A. In some embodiments, _N2 is C. In some embodiments, _N2 is G. In some embodiments, _N2 is U.

In some implementations, _N1 is A and _N2 is A. In some implementations, _N1 is A and N2 is C. In some implementations, _N1 is _A and _N2 is G. In some implementations, _N1 is A and _N2 is U.

In some implementations, _N1 is C and _N2 is A. In some implementations, _N1 is C and _N2 is C. In some implementations, _N1 is _C and N2 is G. In some implementations, _N1 is C and _N2 is U.

In some implementations, _N1 is G and _N2 is A. In some implementations, _N1 is G and N2 is C. In some implementations, N1 _{is G} and _N2 is G. In some implementations, _N1 is G and _N2 is U.

In some embodiments, _N1 is U and _N2 is A. In some embodiments, _N1 is U and N2 is C. In some embodiments, _N1 is _U and _N2 is G. In some embodiments, _N1 is U and _N2 is U.

In some implementations, the proximal cap sequence includes _N1 and _N2 , as well as _N3 , _N4 , and _N5 of the Cap structure, where _N1 to _N5 correspond to the +1, +2, +3, +4, and/or +5 positions of the RNA polynucleotide.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is A. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is C. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is G. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is U. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is A. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is G. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is C. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is U. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is A. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is C. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is G. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is U. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is A. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is C. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is G. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is A. In some embodiments, _N4 is U. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is A. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is C. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is G. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is U. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is A. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is G. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is C. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is U. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is A. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is C. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, _C , G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is _G. In _some embodiments, _N5 is C. _{( This text} is _{repeated three times} in the original _. )

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is A. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is C. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is G. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is C. In some embodiments, _N4 is U. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is A. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is C. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is G. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is U. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is A. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is G. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is C. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is U. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is A. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is C. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is G. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is U. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is A. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is C. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is G. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A and _N2 is G. In some embodiments, _N3 is G. In some embodiments, _N4 is U. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is A. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is C. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is G. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is U. In some embodiments, _N5 is A.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is A. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is G. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is C. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is U. In some embodiments, _N5 is G.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is A. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is C. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is G. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is U. In some embodiments, _N5 is C.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is A. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is C. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is G. In some embodiments, _N5 is U.

In some embodiments, _N1 , _N2 , _N3 , _N4 , or _N5 is any nucleotide, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _N3 is U. In some embodiments, _N4 is U. In some embodiments, _N5 is U.

In some embodiments, the 5’ UTR disclosed herein includes a cap proximal sequence, for example, as disclosed herein. In some embodiments, the cap proximal sequence includes a sequence adjacent to the 5’ cap. In some embodiments, the cap proximal sequence includes nucleotides at positions +1, +2, +3, +4, and/or +5 of the RNA polynucleotide.

In some embodiments, the Cap structure comprises one or more polynucleotides in the proximal cap sequence. In some embodiments, the Cap structure comprises an m7 guanosine cap of an RNA polynucleotide and nucleotide +1 ( _N1 ). In some embodiments, the Cap structure comprises an m7 guanosine cap of an RNA polynucleotide and nucleotide +2 ( _N2 ). In some embodiments, the Cap structure comprises an m7 guanosine cap of an RNA polynucleotide and nucleotides +1 and +2 ( _N1 and _N2 ).

In some embodiments, _N1 and _N2 are each independently selected from A, C, G, or U. In some embodiments, _N1 is A. In some embodiments, _N1 is C. In some embodiments, _N1 is G. In some embodiments, _N1 is U. In some embodiments, _N2 is A. In some embodiments, _N2 is C. In some embodiments, _N2 is G. In some embodiments, _N2 is U.

In some embodiments, _N1 and _N2 are each independently selected from A, C, G, or U. In some embodiments, _N1 is A. In some embodiments, _N1 is C. In some embodiments, _N1 is G. In some embodiments, _N1 is U. In some embodiments, _N2 is A. In some embodiments, _N2 is C. In some embodiments, _N2 is G. In some embodiments, _N2 is U.

In some implementations, _N1 is A and _N2 is A. In some implementations, _N1 is A and N2 is C. In some implementations, _N1 is _A and _N2 is G. In some implementations, _N1 is A and _N2 is U.

In some implementations, _N1 is C and _N2 is A. In some implementations, _N1 is C and _N2 is C. In some implementations, _N1 is _C and N2 is G. In some implementations, _N1 is C and _N2 is U.

In some implementations, _N1 is G and _N2 is A. In some implementations, _N1 is G and N2 is C. In some implementations, N1 _{is G} and _N2 is G. In some implementations, _N1 is G and _N2 is U.

In some implementations, _N1 is U and _N2 is A. In some implementations, _N1 is U and N2 is C. In some implementations, _N1 is _U and _N2 is G. In some implementations, _N1 is U and _N2 is U.

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, and a sequence comprising: _{A3 A4 X5} (SEQ ID NO: 1). In some embodiments, _N1 and _N2 are each independently selected from: A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _X5 is selected from A, C, G, or U. In some embodiments, _X5 is A. In some embodiments, _X5 is C. In some embodiments, _X5 is G. In some embodiments, _X5 is U.

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, and a sequence comprising: _C3A4X5 (SEQ ID NO: ₂ ). In some embodiments, _N1 and _N2 are each independently selected from A, C, _G , or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _X5 is selected from A, C, G, or U. In some embodiments, _X5 is A. In some embodiments, _X5 is C. In some embodiments, _X5 is G. In some embodiments, _X5 is U.

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, and a sequence comprising: _{X3 Y4 X5} (SEQ ID NO: 7). In some embodiments, _N1 and _N2 are each independently selected from: A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _X3 and _X5 are each independently selected from: A, C, G, or U. In some embodiments, _X3 and/or _X5 is A. In some embodiments, _X3 and/or _X5 is C. In some embodiments, _X3 and/or _X5 is G. In some embodiments, _X3 and/or _X5 is U. In some embodiments, _Y4 is not C. In some embodiments, _Y4 is A. In some embodiments, _Y4 is G. In some embodiments, _Y4 is U.

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, and a sequence comprising: _{X3 Y4 X5} (SEQ ID NO: 7). In some embodiments, _N1 and _N2 are each independently selected from: A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _X3 and _X5 are each independently selected from: A, C, G, or U. In some embodiments, _X3 and/or _X5 is A. In some embodiments, _X3 and/or _X5 is C. In some embodiments, _X3 and/or _X5 is G. In some embodiments, _X3 and/or _X5 is U. In some embodiments, _Y4 is not G. In some embodiments, _Y4 is A. In some embodiments, _Y4 is C. In some embodiments, _Y4 is U.

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, and a sequence comprising: _A3C4A5 (SEQ ID NO: ₃ ). In some embodiments, _N1 and _N2 are each independently selected from: A, C, _G , or U. In some embodiments, _N1 is A, and _N2 is G.

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, and a sequence comprising: _{A3 U4 G5} (SEQ ID NO:4). In some embodiments, _N1 and _N2 are each independently selected from: A, C, G, or U. In some embodiments, _N1 is A and _N2 is G.

In some embodiments, the Cap structure comprises one or more polynucleotides in the proximal cap sequence. In some embodiments, the Cap structure comprises an m7 guanosine cap of an RNA polynucleotide and nucleotide +1 ( _N1 ). In some embodiments, the Cap structure comprises an m7 guanosine cap of an RNA polynucleotide and nucleotide +2 ( _N2 ). In some embodiments, the Cap structure comprises an m7 guanosine cap of an RNA polynucleotide and nucleotides +1 and +2 ( _N1 and _N2 ).

In some embodiments, _N1 and _N2 are any nucleotides, such as A, C, G, or U. In some embodiments, _N1 is A. In some embodiments, _N1 is C. In some embodiments, _N1 is G. In some embodiments, _N1 is U. In some embodiments, _N2 is A. In some embodiments, _N2 is C. In some embodiments, _N2 is G. In some embodiments, _N2 is U.

In some embodiments, _N1 and _N2 are any nucleotides, such as A, C, G, or U. In some embodiments, _N1 is A. In some embodiments, _N1 is C. In some embodiments, _N1 is G. In some embodiments, _N1 is U. In some embodiments, _N2 is A. In some embodiments, _N2 is C. In some embodiments, _N2 is G. In some embodiments, _N2 is U.

In some implementations, _N1 is A and _N2 is A. In some implementations, _N1 is A and _N2 is C. In some implementations, _N1 is A and _N2 is G. In some implementations, _N1 is A and _N2 is U.

In some implementations, _N1 is C and _N2 is A. In some implementations, _N1 is C and _N2 is C. In some implementations, _N1 is _C and N2 is G. In some implementations, _N1 is C and _N2 is U.

In some implementations, _N1 is G and _N2 is A. In some implementations, _N1 is G and N2 is C. In some implementations, N1 _{is G} and _N2 is G. In some implementations, _N1 is G and _N2 is U.

In some implementations, _N1 is U and _N2 is A. In some implementations, _N1 is U and N2 is C. In some implementations, _N1 is _U and _N2 is G. In some implementations, _N1 is U and _N2 is U.

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, and a sequence comprising: _{A3 A4 X5} (SEQ ID NO: 1). In some embodiments, _N1 and _N2 are any nucleotides, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _X5 is selected from A, C, G, or U. In some embodiments, _X5 is A. In some embodiments, _X5 is C. In some embodiments, _X5 is G. In some embodiments, _X5 is U.

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, and a sequence comprising: _C3A4X5 (SEQ ID NO:2). In some embodiments, _N1 and _N2 are any nucleotides, such as A, _C , _G , or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _X5 is any nucleotide, such as A, C, G, or U. In some embodiments, _X5 is A. In some embodiments, _X5 is C. In some embodiments, _X5 is G. In some embodiments, _X5 is U.

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, and a sequence comprising: _{X3 Y4 X5} (SEQ ID NO: 7). In some embodiments, _N1 and _N2 are any nucleotides, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _X3 and _X5 are any nucleotides, such as A, C, G, or U. In some embodiments, _X3 and/or _X5 is A. In some embodiments, _X3 and/or _X5 is C. In some embodiments, _X3 and/or _X5 is G. In some embodiments, _X3 and/or _X5 is U. In some embodiments, _Y4 is not C. In some embodiments, _Y4 is A. In some embodiments, _Y4 is G. In some embodiments, _Y4 is U.

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, and a sequence comprising: _{X3 Y4 X5} (SEQ ID NO: 7). In some embodiments, _N1 and _N2 are any nucleotides, such as A, C, G, or U. In some embodiments, _N1 is A, and _N2 is G. In some embodiments, _X3 and _X5 are any nucleotides, such as A, C, G, or U. In some embodiments, _X3 and/or _X5 is A. In some embodiments, _X3 and/or _X5 is C. In some embodiments, _X3 and/or _X5 is G. In some embodiments, _X3 and/or _X5 is U. In some embodiments, _Y4 is not G. In some embodiments, _Y4 is A. In some embodiments, _Y4 is C. In some embodiments, _Y4 is U.

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, and _a sequence comprising: _A3C4A5 (SEQ ID NO:3). In some embodiments, _N1 and _N2 are any nucleotides, such as A, C, _G , or U. In some embodiments, _N1 is A, and _N2 is G.

In some embodiments, the proximal cap sequence comprises _N1 and _N2 of the Cap structure, and a sequence comprising: _A3U4G5 (SEQ ID NO: ₄ ). In some embodiments, _N1 and _N2 are any nucleotides, such as A, _C , G, or U. In some embodiments, _N1 is A, and _N2 is G.

An exemplary 5’UTR may contain a human alpha globin (hAg) 5’UTR or a fragment thereof, a TEV 5’UTR or a fragment thereof, an HSP70 5’UTR or a fragment thereof, or a c-Jun 5’UTR or a fragment thereof.

In some embodiments, the RNA disclosed herein comprises an hAg 5’UTR or a fragment thereof. In some embodiments, the RNA disclosed herein comprises an hAg 5’UTR having 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the human alpha globin 5’UTR provided in SEQ ID NO:11. In some embodiments, the RNA disclosed herein comprises an hAg 5’UTR having 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the human alpha globin 5’UTR provided in SEQ ID NO:12. In some embodiments, the RNA disclosed herein comprises an hAg 5’UTR provided in SEQ ID NO:12.

3’UTR

In some embodiments, the RNA disclosed herein contains a 3'-UTR. If present, the 3'-UTR is located at the 3' end, downstream of the stop codon of the protein-coding region, but the term "3'-UTR" preferably does not include the poly(A) sequence. Thus, the 3'-UTR is located upstream of the poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

In some embodiments, the RNA disclosed herein comprises a 3’UTR, which contains an F element and/or an I element. In some embodiments, the 3’UTR or its proximal sequence contains a restriction site. In some embodiments, the restriction site is a BamHI site. In some embodiments, the restriction site is an XhoI site.

In some embodiments, the RNA disclosed herein comprises a 3’UTR having 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the 3’UTR provided in SEQ ID NO:13. In some embodiments, the RNA disclosed herein comprises the 3’UTR provided in SEQ ID NO:13.

PolyA

In some embodiments, the RNA disclosed herein comprises a polyadenylated nucleotide (PolyA) sequence, for example, as described herein. In some embodiments, the PolyA sequence is located downstream of the 3'-UTR, for example, adjacent to the 3'-UTR.

As used herein, the term "poly(A) sequence" or "poly-A tail" refers to a continuous or discontinuous sequence of adenosine residues, typically located at the 3' end of an RNA polynucleotide. Poly(A) sequences are known to those skilled in the art and can follow the 3'-UTR in the RNA described herein. A continuous poly(A) sequence is characterized by a continuous sequence of adenosine residues. Continuous poly(A) sequences are typical in nature. The RNAs disclosed herein may have a poly(A) sequence that is ligated to a free 3' end of the RNA after transcription by a template-independent RNA polymerase, or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.

It has been shown that the poly(A) sequence of approximately 120 A nucleotides has a beneficial effect on RNA levels in transfected eukaryotic cells as well as on protein levels translated from an open reading frame located upstream (5’) of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

The poly(A) sequence can have any length. In some embodiments, the poly(A) sequence comprises, is substantially composed of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, "substantially composed of" means that the majority of the nucleotides in the poly(A) sequence are A nucleotides, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the poly(A) sequence are A nucleotides, but the remaining nucleotides are allowed to be nucleotides other than A nucleotides, such as U nucleotides (uridine monophosphate), G nucleotides (guanosine monophosphate), or C nucleotides (cytidine monophosphate). In this context, "composed of" means that all the nucleotides in the poly(A) sequence are A nucleotides, that is, 100% of the nucleotides in the poly(A) sequence are A nucleotides. The term "A nucleotide" or "A" refers to adenosine monophosphate.

In some implementations, a poly(A) sequence is ligated during RNA transcription, for example, during the preparation of in vitro transcribed RNA, based on a DNA template containing repeating dT nucleotides (deoxythymidines) in a strand complementary to the coding strand. The DNA sequence (coding strand) encoding the poly(A) sequence is called a poly(A) box.

In some embodiments, the poly(A) box present in the DNA coding strand is essentially composed of dA nucleotides, but interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). The length of such a random sequence can be 5-50, 10-30, or 10-20 nucleotides. Such a box is disclosed in WO 2016/005324 A1, which is incorporated herein by reference. Any poly(A) box disclosed in WO 2016/005324A1 can be used in this invention. A poly(A) box encompassing such a box, which is essentially composed of dA nucleotides but interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT) having an equal distribution and a length of, for example, 5-50 nucleotides, exhibits constant proliferation of plasmid DNA at the DNA level in *E. coli* and remains associated with beneficial properties supporting RNA stability and translation efficiency at the RNA level. In some implementations, the poly(A) sequence contained in the RNA polynucleotide described herein consists primarily of A nucleotides, but is interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

In some implementations, there are no nucleotides other than A on the 3'-flanking side of the poly(A) sequence; that is, the poly(A) sequence is not masked or followed by nucleotides other than A at its 3'-end.

In some embodiments, the poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist substantially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, the poly(A) sequence comprises about 120 nucleotides.

In some embodiments, the RNA disclosed herein comprises a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO:14, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the nucleotide sequence of SEQ ID NO:14.

In some implementations, the RNA disclosed herein contains the poly(A) sequence of SEQ ID NO:14.

Payload

In some embodiments, the RNA polynucleotides disclosed herein comprise a sequence encoding a payload, for example, as described herein. In some embodiments, the sequence encoding the payload comprises a promoter sequence. In some embodiments, the sequence encoding the payload comprises a sequence encoding a secretion signal peptide.

In some implementations, the payload is selected from: protein substitute peptides; antibody substances; cytokines; antigenic peptides; gene editing components; regenerative medicine components or combinations thereof.

In some embodiments, the payload is or comprises a protein substitute peptide. In some embodiments, the protein substitute peptide includes peptides aberrantly expressed in a disease or condition. In some embodiments, the protein substitute peptide includes intracellular proteins, extracellular proteins, or transmembrane proteins. In some embodiments, the protein substitute peptide includes an enzyme.

In some implementations, the diseases or conditions in which the peptide expression is abnormal include, but are not limited to: rare diseases, metabolic disorders, muscular dystrophy, cardiovascular diseases, or monogenic diseases.

In some embodiments, the payload is or contains an antibody substance. In some embodiments, the antibody substance binds to a polypeptide expressed on a cell. In some embodiments, the antibody substance comprises a CD3 antibody, a Claudin 6 antibody, or a combination thereof.

In some embodiments, the payload is or contains cytokines or fragments or variants thereof. In some embodiments, the cytokines include: IL-12 or fragments or variants or fusions thereof, IL-15 or fragments or variants or fusions thereof, GMCSF or fragments or variants thereof; or IFN-α or fragments or variants thereof.

In some embodiments, the payload is or contains an antigenic peptide or its immunogenic variant or immunogenic fragment. In some embodiments, the antigenic peptide contains one epitope from an antigen. In some embodiments, the antigenic peptide contains multiple different epitopes from an antigen. In some embodiments, the antigenic peptide containing multiple different epitopes from an antigen is multiepitope-containing.

In some implementations, the antigenic peptide includes: antigenic peptides derived from allergens, viral antigenic peptides, bacterial antigenic peptides, fungal antigenic peptides, parasitic antigenic peptides, antigenic peptides derived from infectious agents, antigenic peptides derived from pathogens, tumor antigenic peptides, or self-antigenic peptides.

In some implementations, the viral antigenic peptide includes HIV antigenic peptide, influenza antigenic peptide, coronavirus antigenic peptide, rabies antigenic peptide, or Zika virus antigenic peptide.

In some embodiments, the viral antigenic polypeptide is or comprises a coronavirus antigenic polypeptide. In some embodiments, the coronavirus antigen is or comprises the SARS-CoV-2 protein. In some embodiments, the SARS-CoV-2 protein comprises the SARS-CoV-2 spike (S) protein, or its immunogenic variant or immunogenic fragment. In some embodiments, the SARS-CoV-2 protein, or its immunogenic variant or immunogenic fragment, comprises proline residues at positions 986 and 987.

In some embodiments, the SARS-CoV-2S peptide has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the SARS-CoV-2S peptide disclosed herein. In some embodiments, the SARS-CoV-2S peptide has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with SEQ ID NO:9.

In some embodiments, the SARS-CoV-2S polypeptide is encoded by RNA having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with the SARS-CoV-2S polynucleotide disclosed herein. In some embodiments, the SARS-CoV-2S polypeptide is encoded by RNA having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with SEQ ID NO:10.

In some embodiments, the payload is or comprises a tumor antigen peptide or its immunogenic variants or immunogenic fragments. In some embodiments, the tumor antigen peptide comprises tumor-specific antigens, tumor-associated antigens, tumor neoantigens, or combinations thereof. In some embodiments, the tumor antigen peptides include p53, ART-4, BAGE, ss-ligin/m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MA GE-A9, MAGE-A10, MAGE-A11 or MAGE-A12, MAGE-B, MAGE-C, MART-1/Melan-A, MC1R, myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minimal BCR-abL, Plac-1, Pm1/RARa, PRAME, protease 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE, WT, WT-1, or combinations thereof.

In some implementations, the tumor antigen peptide includes tumor antigens derived from cancer, sarcoma, melanoma, lymphoma, leukemia, or combinations thereof.

In some implementations, the tumor antigen peptide contains melanoma tumor antigen.

In some implementations, the tumor antigen peptide includes a prostate cancer antigen.

In some implementations, the tumor antigen peptide contains HPV16-positive head and neck cancer antigen.

In some implementations, the tumor antigen peptide contains a breast cancer antigen.

In some implementations, the tumor antigen peptide contains ovarian cancer antigen.

In some implementations, the tumor antigen peptide contains lung cancer antigen.

In some implementations, the tumor antigen peptide contains NSCLC antigen.

In some embodiments, the payload is or contains a self-antigen polypeptide or its immunogenic variants or immunogenic fragments. In some embodiments, the self-antigen polypeptide contains an antigen that is normally expressed on cells and recognized as a self-antigen by the immune system. In some embodiments, the self-antigen polypeptide includes: a multiple sclerosis antigen polypeptide, a rheumatoid arthritis antigen polypeptide, a lupus antigen polypeptide, a celiac disease antigen polypeptide, a Sjogren's syndrome antigen polypeptide, or ankylosing spondylitis antigen polypeptide, or a combination thereof.

Exemplary polynucleotides

In some embodiments, the RNA polynucleotide described herein, or a composition or pharmaceutical article comprising such RNA polynucleotide, comprises the nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide comprises a sequence having at least 80% identity with the nucleotide sequence disclosed herein. In some embodiments, the RNA polynucleotide comprises a sequence encoding a polypeptide having at least 80% identity with the polypeptide sequence disclosed herein. Exemplary nucleotide and polypeptide sequences are provided, for example, in Table 1 or in this section titled “Exemplary Polynucleotides” or Example 2.

In some embodiments, the RNA polynucleotide described herein, or a composition or pharmaceutical article containing the RNA polynucleotide, is transcribed from a DNA template. In some embodiments, the DNA template used to transcribe the RNA polynucleotide described herein contains a sequence complementary to the RNA polynucleotide.

In some embodiments, the payload described herein is encoded by an RNA polynucleotide comprising the nucleotide sequences disclosed herein, such as those in Table 1 or in this section titled “Exemplary Polynucleotides” or Example 2. In some embodiments, the RNA polynucleotide comprises a polypeptide payload encoding a polypeptide payload sequence having at least 80% identity with the polypeptide payload sequence disclosed herein. In some embodiments, the payload described herein is encoded by an RNA polynucleotide transcribed from a DNA template comprising a sequence complementary to the RNA polynucleotide.

Table 1: Exemplary sequences of RNA constructs disclosed in this paper

RBL063.1 (SEQ ID NO: 28 Nucleotide; SEQ ID NO: 9 Amino Acid)

Structure β-S-ARCA(D1)-hAg-Kozak-S1S2-PP-FI-A30L70

Encoding the viral spike protein (S1S2 protein) of the SARS-CoV-2 antigen (full-length S1S2 protein, sequence variant) SEQ ID NO:28

RBL063.2 (SEQ ID NO: 29 Nucleotide; SEQ ID NO: 9 Amino Acid)

Structure β-S-ARCA(D1)-hAg-Kozak-S1S2-PP-FI-A30L70

The viral spike protein (S1S2 protein) encoding the antigen SARS-CoV-2 (full-length S1S2 protein, sequence variant)

SEQ ID NO:29

BNT162a1; RBL063.3 (SEQ ID NO: 30 Nucleotide; SEQ ID NO: 21 Amino Acid)

The structure β-S-ARCA(D1)-hAg-Kozak-RBD-GS-fibritin-FI-A30L70 encodes the viral spike protein (S protein) of the SARS-CoV-2 antigen (partial sequence, receptor-binding domain (RBD) of the S1S2 protein)

SEQ ID NO:30

BNT162b2; RBP020.1 (SEQ ID NO: 31 Nucleotide; SEQ ID NO: 9 Amino Acid)

Structure m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG)-hAg-Kozak-S1S2-PP-FI-A30L70

The viral spike protein (S1S2 protein) encoding the antigen SARS-CoV-2 (full-length S1S2 protein, sequence variant)

SEQ ID NO:31

RBP020.2 (SEQ ID NO: 10 Nucleotide; SEQ ID NO: 9 Amino Acid) (see Table 1)

Structure m ₂ ^7,3'-O Gppp(m ₁ ^2'-O )ApG)-hAg-Kozak-S1S2-PP-FI-A30L70

The viral spike protein encoding the antigen SARS-CoV-2 (S1S2 protein) (full-length S1S2 protein, sequence variant) BNT162b1; RBP020.3 (SEQ ID NO:32; SEQ ID NO:21 amino acids)

Structure _m2 ^7,3'-O Gppp( _m1 ^2'-O )ApG)-hAg-Kozak-RBD-GS-minor fibrin-FI-A30L70

The viral spike protein (S1S2 protein) encoding the antigen SARS-CoV-2 (partial sequence, fused to the receptor-binding domain (RBD) of the S1S2 protein in the minor fibrin)

SEQ ID NO:32

RBS004.1 (SEQ ID NO:33; SEQ ID NO:9 amino acid)

Structure β-S-ARCA(D1)-replicaase-S1S2-PP-FI-A30L70

The viral spike protein (S protein) encoding the antigen SARS-CoV-2 (full-length S1S2 protein, sequence variant) SEQ ID NO:33

RBS004.2 (SEQ ID NO:34; SEQ ID NO:9 amino acid)

Structure β-S-ARCA(D1)-replicaase-S1S2-PP-FI-A30L70

Encoding the viral spike protein (S protein) of the SARS-CoV-2 antigen (full-length S1S2 protein, sequence variant) SEQ ID NO:34

BNT162c1; RBS004.3 (SEQ ID NO:35; SEQ ID NO:21 amino acid)

Structure β-S-ARCA(D1)-replicaase-RBD-GS-minor fibrin-FI-A30L70

The viral spike protein (S protein) encoding the antigen SARS-CoV-2 (partial sequence, receptor-binding domain (RBD) of the S1S2 protein)

SEQ ID NO:35

RBS004.4(SEQ ID NO:36; SEQ ID NO:37)

The structure β-S-ARCA(D1)-replicaase-RBD-GS-minor fibrin-TM-FI-A30L70 encodes the viral spike protein (S protein) of the SARS-CoV-2 antigen (partial sequence, receptor-binding domain (RBD) of the S1S2 protein)

SEQ ID NO:36

SEQ ID NO:37

BNT162b3c(SEQ ID NO:38; SEQ ID NO:39)

Structure _m2 ^7,3'-O Gppp( _m1 ^2'-O )ApG-hAg-Kozak-RBD-GS-minor fibrin-GS-TM-FI-A30L70

The viral spike protein (S1S2 protein) encoding the antigen SARS-CoV-2 (partial sequence, fused to the receptor-binding domain (RBD) of the S1S2 protein in the minor fibrin, fused to the transmembrane domain (TM) of the S1S2 protein); the intrinsic S1S2 protein secretion signal peptide (aa 1-19) at the N-terminus of the antigen sequence.

SEQ ID NO:38

SEQ ID NO:39

BNT162b3d(SEQ ID NO:40; SEQ ID NO:41)

Structure _m2 ^7,3'-O Gppp( _m1 ^2'-O )ApG-hAg-Kozak-RBD-GS-minor fibrin-GS-TM-FI-A30L70

The viral spike protein (S1S2 protein) encoding the antigen SARS-CoV-2 (partial sequence, fused to the receptor-binding domain (RBD) of the S1S2 protein into the minor fibrin, fused to the transmembrane domain (TM) of S1S2); the immunoglobulin secretion signal peptide (aa 1-22) at the N-terminus of the antigen sequence.

SEQ ID NO:40

SEQ ID NO:41

Particles containing nucleic acids

The nucleic acids described in this article, such as RNA encoding a payload, can be formulated into particles for drug delivery.

In the context of this disclosure, the term "particle" refers to a structured entity formed of molecules or molecular complexes. In some embodiments, the term "particle" refers to a micrometer or nanometer-sized structure, such as a dense micrometer or nanometer-sized structure dispersed in a medium. In some embodiments, a particle is a particle containing nucleic acids, such as a particle containing DNA, RNA, or a mixture thereof.

Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acids are involved in particle formation. This leads to the recombination and spontaneous formation of nucleic acid particles. In some embodiments, the nucleic acid particles are nanoparticles.

As used in this disclosure, "nanoparticle" means a particle having an average diameter suitable for parenteral administration.

Nucleic acid particles can be used to deliver nucleic acids to target sites of interest (e.g., cells, tissues, organs, etc.). Nucleic acid particles can be formed from at least one cation or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine or a mixture thereof, and nucleic acid. Nucleic acid particles include formulations based on lipid nanoparticles (LNPs) and lipid complexes (LPXs).

Unbound by any theory, it is believed that cationic or cationically ionizable lipids or lipid-like materials and/or cationic polymers bind together with nucleic acids to form aggregates, and this aggregation results in colloidally stable particles.

In some embodiments, the particles described herein further comprise at least one lipid or lipid-like material other than a cationic or cationically ionizable lipid or lipid-like material, at least one polymer other than a cationic polymer, or a mixture thereof.

In some embodiments, nucleic acid particles comprise more than one type of nucleic acid molecule, wherein the molecular parameters of the nucleic acid molecules may be similar or different from each other, for example, regarding molar mass or basic structural elements such as molecular structure, capping, coding regions, or other features. The average diameter of the nucleic acid particles described herein may be from about 30 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 70 nm to about 600 nm, from about 90 nm to about 400 nm, or from about 100 nm to about 300 nm in some embodiments.

The nucleic acid particles described herein may exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. For example, the nucleic acid particles may exhibit a polydispersity index in the range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

Regarding RNA lipid particles, the N/P ratio gives the proportion of nitrogen groups in the lipid to the number of phosphate groups in the RNA. It is related to the charge ratio, as nitrogen atoms (depending on pH) are generally positively charged, while phosphate groups are negatively charged. When charge balance is present, the N/P ratio depends on pH. Lipid formulations are typically formed with an N/P ratio greater than 4 and at most 12, because positively charged nanoparticles are considered favorable for transfection. In this case, complete RNA binding to the nanoparticles is assumed.

The nucleic acid particles described herein can be prepared using a wide range of methods, which may include obtaining a colloid from at least one cation or cationic ionizable lipid or lipid-like material and/or at least one cationic polymer, and mixing the colloid with nucleic acids to obtain nucleic acid particles.

As used herein, the term "colloid" refers to a type of homogeneous mixture in which dispersed particles do not precipitate. The insoluble particles in the mixture are microscopic, with a particle size between 1 and 1000 nanometers. This mixture may be referred to as a colloid or a colloidal suspension. Sometimes the term "colloid" refers only to the particles in the mixture rather than the entire suspension.

For the preparation of colloids comprising at least one cation or cation-ionizable lipid or lipid-like material and/or at least one cationic polymer, conventional methods for preparing liposome vesicles, with appropriate modifications, are applicable here. The most common methods for preparing liposome vesicles involve the following basic steps: (i) dissolving the lipid in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipid (using various aqueous media).

In membrane hydration methods, lipids are first dissolved in a suitable organic solvent and then dried to form a thin film at the bottom of a flask. The resulting lipid membrane is then hydrated using a suitable aqueous medium to produce a liposome dispersion. Additional shrinkage steps may also be included.

Reverse-phase evaporation is an alternative method for membrane hydration in the preparation of liposome vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. This mixture is briefly sonicated to homogenize the system. Removal of the organic phase under reduced pressure produces an emulsion gel, which subsequently becomes a liposome suspension.

The term "ethanol injection technique" refers to a process in which a lipid-containing ethanol solution is rapidly injected into an aqueous solution via a needle. This action disperses the lipids throughout the solution and promotes the formation of lipid structures, such as lipid vesicles or liposomes. Generally, the RNA-lipid complex particles described herein can be obtained by adding RNA to a colloidal liposome dispersion. In some embodiments, such colloidal liposome dispersions are formed using the ethanol injection technique as follows: an ethanol solution containing lipids, such as cationic lipids, and additional lipids is injected into an aqueous solution under stirring. In some embodiments, the RNA-lipid complex particles described herein are obtained without an extrusion step.

The term "extruding" or "extrusion" refers to the production of particles with a fixed cross-sectional profile. In particular, it refers to the reduction in particle size, thereby forcing the particles through a filter with defined pores.

Other methods that do not contain organic solvents can also be used to prepare colloids according to this disclosure.

LNPs typically consist of four components: ionizable cationic lipids, neutral lipids such as phospholipids, steroids such as cholesterol, and polymer-conjugated lipids such as polyethylene glycol (PEG)-lipids. Each component is responsible for payload protection and enables efficient intracellular delivery. LNPs can be prepared by rapidly mixing lipids dissolved in ethanol with nucleic acids in an aqueous buffer.

The term "average diameter" refers to the average hydrodynamic diameter of a particle, measured by dynamic laser scattering (DLS) and data analysis using a so-called cumulant algorithm. The result provides a so-called Zaverage with a given length dimension, and a dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the terms "average diameter," "diameter," or "size" of the particle are used synonymously with this value of Zaverage.

The "polydispersity index" is preferably based on dynamic light scattering measurements and calculated through the so-called cumulative analysis mentioned in the definition of "average diameter". Under certain prerequisites, it can be used as a measure of the overall size distribution of nanoparticles.

Different types of nucleic acid-containing particles have been previously described as suitable for delivering nucleic acids in particulate form (e.g., Kaczmarek, J.C. et al., 2017, Genome Medicine 9, 60). For non-viral nucleic acid delivery mediators, the encapsulation of nucleic acids in nanoparticles physically protects the nucleic acids from degradation and, depending on specific chemistry, can facilitate cellular uptake and endosome escape.

This disclosure describes particles comprising nucleic acids, at least one cation or cationically ionizable lipid or lipid-like material, and/or at least one cationic polymer associated with nucleic acids to form nucleic acid particles, and compositions comprising such particles. The nucleic acid particles may comprise nucleic acids complexed with the particles in various forms through non-covalent interactions. The particles described herein are not viral particles, particularly infectious viral particles, i.e., they cannot virally infect cells.

Suitable cationic or cationically ionizable lipids or lipid-like materials, as well as cationic polymers, are those that form nucleic acid particles and are included in the term "particle-forming component" or "particle-forming substance." The term "particle-forming component" or "particle-forming substance" refers to any component that associates with nucleic acids to form nucleic acid particles. This type of component includes any component that can be part of a nucleic acid particle.

Some embodiments described herein relate to compositions, methods, and uses comprising one or more, such as 2, 3, 4, 5, 6, or even more, nucleic acid substances such as RNA tumors, for example, a) a nucleic acid comprising a first nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein, wherein the amino acid positions in at least one fragment of the parent viral protein are modified to include amino acids found at corresponding amino acid positions in one or more viral protein variants; and b) a nucleic acid comprising a second nucleotide sequence encoding an amino acid sequence comprising at least one fragment of a parent viral protein, wherein the amino acid positions in at least one fragment of the parent viral protein are modified to include amino acids found at corresponding amino acid positions in one or more viral protein variants.

In particulate formulations, it is possible to formulate each nucleic acid substance as a separate particulate formulation. In this case, each individual particulate formulation will contain one nucleic acid substance. A single particulate formulation can exist as a separate entity, for example, in a separate container. Such formulations can be obtained by providing each nucleic acid substance (usually in the form of a solution containing nucleic acid) along with a particle-forming substance, thereby allowing particle formation. Each particle will contain only the specific nucleic acid substance (the individual particulate formulation) provided during particle formation.

In some embodiments, the composition, such as a pharmaceutical composition, comprises more than one individual particulate formulation. Each pharmaceutical composition is referred to as a mixed particulate formulation. The mixed particulate formulation according to the invention can be obtained by separately forming individual particulate formulations as described above, and then mixing the individual particulate formulations. Through the mixing step, a formulation comprising a mixed population containing nucleic acid particles is obtained. The individual particulate populations can be contained together in a single container, comprising a mixed population of individual particulate formulations.

Alternatively, it is possible to formulate different nucleic acid substances together as combined microparticle formulations. Such formulations are obtained by providing a combination of different RNA substances (typically a combination solution) along with particle-forming substances, thereby allowing particle formation. In contrast to mixed microparticle formulations, combined microparticle formulations typically contain particles containing more than one type of RNA substance. In combined microparticle compositions, different RNA substances are usually present together within a single particle.

Cationic polymeric materials (e.g., polymers)

Given their high chemical flexibility, polymeric materials are commonly used for nanoparticle-based delivery. Typically, cationic materials are used to electrostatically aggregate negatively charged nucleic acids into nanoparticles. These positively charged groups are often composed of amines, which alter their protonation state in a pH range of 5.5–7.5, presumably causing ionic imbalance and leading to endosome rupture. Polymers such as poly-L-lysine, polyamide amines, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been used in nucleic acid delivery and are suitable as cationic materials useful in some embodiments described herein. Furthermore, some researchers have synthesized polymeric materials specifically for nucleic acid delivery. In particular, poly(β-amino esters) have gained widespread use in nucleic acid delivery due to their ease of synthesis and biodegradability. In some embodiments, such synthetic materials may be suitable for use as cationic materials as described herein.

As used herein, "polymeric material" has its general meaning, namely, a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. In some embodiments, these repeating units may be all identical; or, in some cases, more than one type of repeating unit may be present within the polymeric material. In some cases, polymeric materials are biologically derived, such as biopolymers like proteins. In some cases, additional portions may also be present in the polymeric material, such as targeting portions, as described herein.

Those skilled in the art will recognize that when a polymer (or polymeric portion) contains more than one type of repeating unit, the polymer (or polymeric portion) is referred to as a "polymer". In some embodiments, the polymer (or polymeric portion) used according to this disclosure may be a copolymer. The repeating units forming the copolymer may be arranged in any manner. For example, in some embodiments, the repeating units may be arranged in a random order; alternatively or additionally, in some embodiments, the repeating units may be arranged in an alternating order, or as a "block" copolymer, i.e., comprising one or more regions, each region containing a first repeating unit (e.g., a first block), and one or more regions, each region containing a second repeating unit (e.g., a second block), and so on. Block copolymers may have two (diblock copolymers), three (triblock copolymers), or more different blocks.

In some embodiments, the polymeric materials used according to this disclosure are biocompatible. Biocompatible materials are those that do not typically cause significant cell death at moderate concentrations. In some embodiments, the biocompatible materials are biodegradable, i.e., capable of chemical and/or biodegradation in physiological environments such as in vivo.

In some embodiments, the polymeric material may be or contains protamine or polyalkylene imine, particularly protamine.

As those skilled in the art will know, the term "protamine" is commonly used to refer to a variety of relatively low molecular weight, strongly basic proteins rich in arginine, which are found in the sperm cells of various animals (such as fish) to be specifically associated with DNA, replacing somatic cell histones. In particular, the term "protamine" is often used to refer to proteins found in fish sperm that are strongly basic, soluble in water, do not coagulate upon heating, and primarily produce arginine upon hydrolysis. In purified forms, they are used in long-acting formulations of insulin and to neutralize the anticoagulant effect of heparin.

In some embodiments, as used herein, the term "protamine" refers to any protamine amino acid sequence obtained from or derived from natural or biological sources, including fragments thereof and/or multimeric forms of said amino acid sequence or fragments thereof, as well as (synthetic) polypeptides that are artificial, specifically designed for a particular purpose, and cannot be isolated from natural or biological sources.

In some embodiments, the polyalkylene imide comprises polyethyleneimine and/or polypropyleneimine, preferably polyethyleneimine. In some embodiments, the preferred polyalkylene imide is polyethyleneimine (PEI). In some embodiments, the average molecular weight of PEI is preferably 0.75 × 10²–10⁷ Da, preferably 1000–10⁵ Da, more preferably 10000–40000 Da, more preferably 15000–30000 Da, and even more preferably 20000–25000 Da.

According to certain embodiments of this disclosure, linear polyalkylene imides, such as linear polyethyleneimine (PEI), are preferred.

The cationic materials (e.g., polymeric materials, including polycationic polymers) considered herein include those capable of electrostatically binding nucleic acids. In some embodiments, the cationic polymers considered herein include any cationic polymeric material with which nucleic acids can associate, for example, by forming a complex with the nucleic acid or forming vesicles therein that surround or encapsulate the nucleic acid.

In some embodiments, the particles described herein may comprise polymers other than cationic polymers, i.e., non-cationic polymers and/or anionic polymers. Anionic and neutral polymers are collectively referred to herein as non-cationic polymers.

Lipids and lipid-like materials

The terms "lipid" and "lipid-like material" are used herein to refer to molecules comprising one or more hydrophobic moieties or groups and optionally one or more hydrophilic moieties or groups. Molecules comprising both hydrophobic and hydrophilic moieties are also commonly referred to as amphiphilic molecules. Lipids are generally poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of these phases consists of lipid bilayers, as they are present in vesicles, multilayer/monolayer liposomes, or membranes in an aqueous environment. Hydrophobicity can be imparted by including nonpolar groups, including but not limited to long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. In some embodiments, the hydrophilic groups may comprise polar and/or charged groups and include carbohydrate, phosphate, carboxyl, sulfate, amino, thiol, nitro, hydroxyl, and other similar groups.

As used herein, the term "amphiphilic" refers to a molecule having both a polar and a nonpolar moiety. Typically, amphiphilic compounds have a polar head attached to a long hydrophobic tail. In some embodiments, the polar moiety is soluble in water, while the nonpolar moiety is insoluble in water. Furthermore, the polar moiety may have a formal positive or negative charge. Alternatively, the polar moiety may have both a formal positive and negative charge and may be a zwitterion or an internal salt. For the purposes of this disclosure, the amphiphilic compound may be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

The terms “lipid-like material,” “lipid-like compound,” or “lipid-like molecule” refer to substances that are structurally and/or functionally related to lipids but cannot be strictly considered lipids. For example, the term includes compounds capable of forming an amphiphilic layer when present in an aqueous environment in vesicles, multilayer/monolayer liposomes, or membranes, and includes surfactants or synthetic compounds having both hydrophilic and hydrophobic portions. Generally, the term refers to molecules containing hydrophilic and hydrophobic portions with different structural organization, which may be similar to or different from the structural organization of lipids. As used herein, the term “lipid” is interpreted to encompass both lipids and lipid-like materials unless otherwise stated herein or clearly contradicted by the context.

Specific examples of amphiphilic compounds that may be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

In some embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be classified into eight classes: fatty acids, glycerides, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and isopentenol lipids (derived from the condensation of isopentenyl subunits). Although the term "lipid" is sometimes used synonymously with fat, fat is a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as metabolites containing sterols such as cholesterol.

Fatty acids, or fatty acid residues, are a group of distinct molecules formed by hydrocarbon chains terminated by carboxylic acid groups; this arrangement imparts polar, hydrophilic ends to the molecule, as well as nonpolar, hydrophobic ends that are insoluble in water. The carbon chains, typically 4–24 carbons in length, can be saturated or unsaturated and can be linked to functional groups containing oxygen, halogens, nitrogen, and sulfur. If fatty acids contain double bonds, they may exhibit cis or trans geometric isomerism, which significantly affects the molecular configuration. Cis double bonds cause the fatty acid chain to bend, an effect that is compounded by the presence of more double bonds in the chain. Other major lipid classes within the fatty acid category are fatty esters and fatty amides.

Glycerides include mono-, di-, and tri-substituted glycerols, the most well-known being fatty acid triesters of glycerol, called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, the three hydroxyl groups of glycerol are typically esterified by different fatty acids. Other subclasses of glycerides are represented by glycosylglycerols, characterized by the presence of one or more sugar residues linked to glycerol via glycosidic bonds.

Glycerophospholipids are amphiphilic molecules (containing both hydrophobic and hydrophilic regions) comprising a glycerol core linked to two fatty acid-derived "tails" via ester bonds and to a "head" group via phosphate ester bonds. Examples of glycerophospholipids commonly referred to as phospholipids (although sphingomyelin is also classified as a phospholipid) include phosphatidylcholine (also known as PC, GPCho, or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

Sphingolipids are a complex family of compounds sharing a common structural feature: the sphingosine base skeleton. The main sphingosine base in mammals is generally called sphingosine. Ceramides (N-acyl-sphingosine bases) are the major subclass of sphingosine base derivatives, consisting of fatty acids linked by an amide. These fatty acids are typically saturated or monounsaturated, with chain lengths of 16–26 carbon atoms. The main phosphosphingolipid in mammals is sphingomyelin (ceramide phosphocholine), while insects primarily contain ceramide phosphoethanolamine, and fungi possess phytoceramide phosphoinositol and a head group containing mannose. Glycosphingolipids are a diverse family of molecules comprising one or more sugar residues linked to a sphingosine base via a glycosidic bond. Examples of these include simple and complex glycosphingolipids such as cerebrosides and gangliosides.

Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, together with glycerophospholipids and sphingomyelins, are important components of membrane lipids.

Glycolipids are compounds in which fatty acids are directly linked to a glycoskeletal backbone, forming a structure compatible with the membrane bilayer. In glycolipids, monosaccharides replace the glycerol backbone present in glycerides and glycerophospholipids. The most familiar glycolipid is the acylated glucosamine precursor of the lipid A component of lipopolysaccharides in Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine, derived from up to seven fatty-acyl chains. The smallest lipopolysaccharide required for E. coli growth is Kdo2-lipid A, a hexaacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

Polyketides are synthesized by polymerizing acetyl and propionyl subunits using classical enzymes and iterative, modular enzymes that share the mechanistic feature with fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources and exhibit great structural diversity. Many polyketides are cyclic molecules, and their backbones are often further modified through glycosylation, methylation, hydroxylation, oxidation, or other processes.

According to this disclosure, lipids and lipid-like materials can be cationic, anionic, or neutral. Neutral lipids or lipid-like materials exist as uncharged or neutral zwitterions at a selected pH.

Cations or cation-ionizable lipids or lipid-like materials

In some embodiments, the nucleic acid particles described and/or used according to this disclosure may comprise at least one cationic or cationically ionizable lipid or lipid-like material as a particle-forming substance. The cationic or cationically ionizable lipid or lipid-like material intended for use herein includes any cationic or cationically ionizable lipid or lipid-like material capable of electrostatically binding nucleic acids. In some embodiments, the cationic or cationically ionizable lipid or lipid-like material intended for use herein may associate with nucleic acids, for example, by forming a complex with nucleic acids or forming vesicles therein surrounding or encapsulating nucleic acids.

As used herein, “cationic lipid” or “cationic lipid-like material” refers to lipids or lipid-like materials with a net positive charge. Cationic lipids or lipid-like materials bind negatively charged nucleic acids through electrostatic interactions. Generally, cationic lipids have lipophilic moieties, such as sterols, acyl chains, diacyl groups, or multiple acyl chains, and the head group of the lipid is usually positively charged.

In some embodiments, cationic lipids or lipid-like materials have a net positive charge only at certain pH levels, particularly at acidic pH levels, while at different, preferably higher, pH levels such as physiological pH levels, they preferably have no net positive charge, or more preferably no charge, i.e., they are neutral. Compared to particles that remain cationic at physiological pH levels, this ionizable behavior is believed to enhance potency by facilitating endosome escape and reducing toxicity.

For the purposes of this disclosure, unless it contradicts the context, the term "cationic lipid or lipid-like material" includes such "cationically ionizable" lipids or lipid-like materials.

In some embodiments, the cation or cation-ionizable lipid or lipid-like material includes a head group comprising at least one positively charged or protonable nitrogen atom (N).

Examples of cationic lipids include, but are not limited to: ((4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), and 1,2-di-O-octadecenyl-3-trimethylammonium propane. ne, DOTMA), 3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB), 1,2-dioleoyl-3-dimethylammonium-propane (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, 1,2-dialkyloxy-3-dimethylammonium propane Nium propane), dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (D... MEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2-dilinyl... DLinDMA (1,2-dilinoleyloxy-N,N-dimethylaminopropane), DOGS (dioctadecylamidoglycyl spermine), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12-octadecadienyloxy)propane -9,12-octadecadienoxy)propane, CpLinDMA), 2-[5′-(cholest-5-en-3-β-oxy)-3′-oxapentoxy)-3-dimethyl-1-(cis,cis-9′,12′-octadecadienoxy)propane, CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine, DMOBA), 1,2-N,N′-dioleylcarbamoyl-3-dimethylaminopropane, CpLinDMA ylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N′-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane ( 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptadecyl-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate (heptatriaconta-6,9 ,28,31-tetraen-19-yl-4-(dimethylamino)butanoate,DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide,DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide,(±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide bromide, GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide, GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide, GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(decadecyloxy)-1-propanaminium bromide, GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide, GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(decadecyloxy)-1-propanaminium bromide, GAP-DMRIE, N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(decadecyloxy)-1-propanaminium bromide, GAP-DMRIE, GAP-DMRIE, GAP-DMRIE, GAP-DMRIE, GAP-DMRIE, GAP-DMRIE, GAP-DMRIE, N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(decadecyloxy)-1-propanaminium bromide ... Tetraalkyloxy)-1-propanamine bromide (N-(2-Aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide, βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium, DOBAQ), 2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecyl-9,12-dien-1-yloxy]propan-1-amine (2-({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadecyl-9,12-dien-1-yloxy]propan-1-amine) Octyl-ClinDMA, 1,2-dimyristoyl-3-dimethylammonium-propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamidoyl)ethyl]-3,4-di[oleyloxy]-benzamide (N1-[2-((1S)-1- [(3-aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-amine (N,N-dimethyl-2,3-bis(dodecyloxy)propan-1-aminium bromide) an-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamine (N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}-ethylamino)propionamid e,lipidoid 98N12-5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazine-1-yl]ethyl]amino]dodecane-2-ol (1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazine-1-yl]ethyl]amino]dodecane-2-ol)

hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol,lipidoid C12-200), or heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (heptadecan-9-yl8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, SM-102).

In some embodiments, the cationic lipid is or comprises heptadecano-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102). In some embodiments, the cationic lipid is or comprises the cationic lipid shown in the following structure.

In some embodiments, the cationic lipid is or contains ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester), also referred to herein as ALC-0315.

In some embodiments, the cationic lipids may comprise about 10 mol% to about 100 mol%, about 20 mol% to about 100 mol%, about 30 mol% to about 100 mol%, about 40 mol% to about 100 mol%, or about 50 mol% to about 100 mol% of the total lipids present in the particles.

In some specific embodiments, the particles used according to this disclosure include ALC-0315, for example, in the range of about 40-55 molar percentages of total lipids by weight.

Additional lipids or lipid-like materials

In some embodiments, the particles described herein comprise (e.g., in addition to cationic lipids such as ALC315), one or more lipids or lipid-like materials other than cationic or cationic ionizable lipids or lipid-like materials, such as non-cationic lipids or lipid-like materials (including non-cationic ionizable lipids or lipid-like materials). Anionic and neutral lipids or lipid-like materials are collectively referred to herein as non-cationic lipids or lipid-like materials. Optimizing the formulation of nucleic acid particles by adding other hydrophobic portions such as cholesterol and lipids in addition to ionizable/cationic lipids or lipid-like materials can enhance particle stability and nucleic acid delivery efficiency.

Additional lipids or lipid-like materials may be incorporated, which may or may not affect the total charge of the nucleic acid particles. In some embodiments, the additional lipids or lipid-like materials are non-cationic lipids or lipid-like materials. Non-cationic lipids may comprise, for example, one or more anionic lipids and/or neutral lipids. As used herein, “anionic lipid” means any lipid that carries a negative charge at a selected pH. As used herein, “neutral lipid” means any of a variety of lipid species that are present at a selected pH in an uncharged or neutral zwitterionic form. In a preferred embodiment, the additional lipid comprises one of the following neutral lipid components: (1) phospholipids, (2) cholesterol or a derivative thereof; or (3) a mixture of phospholipids and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholesterol, cholesterolone, coprosterol, cholesterol-2'-hydroxyethyl ether, cholesterol-4'-hydroxybutyl ether, tocopherol and its derivatives, and mixtures thereof.

Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin. These phospholipids particularly include diacylphosphatidylcholine, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), and dicarboxylic acid phosphatidylcholine. Diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), diilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), and 1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline (1,2-di-O-octadecenyl-sn-glycerol-3-phosphate choline) 1-Oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16LysoPC), and phosphatidylethanolamines, especially diacylphosphatidylethanolamines such as dioleoylphosphatidylethanolamine (DOPE), distearate, etc. Acyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and other phosphatidylethanolamine lipids with different hydrophobic chains.

In some preferred embodiments, the additional lipid is DSPC or DSPC and cholesterol.

In some implementations, nucleic acid particles include cationic lipids and additional lipids.

In some embodiments, the particles described herein comprise polymer-conjugated lipids such as polyethylene glycol-modified lipids. The term "polyethylene glycol-modified lipid" refers to a molecule comprising a lipid moiety and a polyethylene glycol moiety. Polyethylene glycol-modified lipids are known in the art. In some embodiments, the polyethylene glycol-modified lipid is ALC-0159, also referred to herein as (2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide).

Not wanting to be bound by theory, the amount of at least one cationic lipid, compared to the amount of at least one additional lipid, can affect important nucleic acid particle characteristics such as charge, particle size, stability, tissue selectivity, and nucleic acid bioactivity. Therefore, in some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

In some embodiments, non-cationic lipids, particularly neutral lipids (e.g., one or more phospholipids and/or cholesterol), may comprise about 0 mol% to about 90 mol%, about 0 mol% to about 80 mol%, about 0 mol% to about 70 mol%, about 0 mol% to about 60 mol%, or about 0 mol% to about 50 mol% of the total lipids present in the particles.

In some embodiments, the particles used according to this disclosure may include, for example, ALC-0315, DSPC, CHOL, and ALC-0159, wherein ALC-0315 comprises about 40-55 mol%; DSPC comprises about 5-15 mol%; CHOL comprises about 30-50 mol%; and ALC-0159 comprises about 1-10 mol%.

Lipid complex particles

In some embodiments of this disclosure, RNA may be present in RNA-lipid complex particles.

In the context of this disclosure, the term "RNA-lipid complex particle" refers to a particle comprising lipids, particularly cationic lipids, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA lead to the complexation and spontaneous formation of RNA-lipid complex particles. Positively charged liposomes can generally be synthesized using cationic lipids such as DOTMA and additional lipids such as DOPE. In some embodiments, the RNA-lipid complex particle is a nanoparticle.

In some embodiments, the RNA-lipid complex particles comprise cationic lipids and additional lipids. In an exemplary embodiment, the cationic lipid is DOTMA, and the additional lipid is DOPE.

In some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In specific embodiments, the molar ratio may be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is about 2:1.

The average diameter of the RNA-lipid complex particles described herein is, in some embodiments, about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In specific embodiments, the average diameter of the RNA-lipid complex particles is approximately 200 nm, approximately 225 nm, approximately 250 nm, approximately 275 nm, approximately 300 nm, approximately 325 nm, approximately 350 nm, approximately 375 nm, approximately 400 nm, approximately 425 nm, approximately 450 nm, approximately 475 nm, approximately 500 nm, approximately 525 nm, approximately 550 nm, approximately 575 nm, approximately 600 nm, approximately 625 nm, approximately 650 nm, approximately 700 nm, approximately 725 nm, approximately 750 nm, approximately 775 nm, approximately 800 nm, approximately 825 nm, approximately 850 nm, approximately 875 nm, approximately 900 nm, approximately 925 nm, approximately 950 nm, approximately 975 nm, or approximately 1000 nm. In one embodiment, the average diameter of the RNA-lipid complex particles is approximately 250 nm to approximately 700 nm. In another embodiment, the average diameter of the RNA-lipid complex particles is about 300 nm to about 500 nm. In an exemplary embodiment, the average diameter of the RNA-lipid complex particles is about 400 nm.

In some embodiments, the RNA-lipid complex particles described herein and/or compositions comprising RNA-lipid complex particles can be used to deliver RNA to target tissues after parenteral administration, particularly after intravenous administration. In some embodiments, RNA-lipid complex particles can be prepared using liposomes, which can be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. In some embodiments, the aqueous phase has an acidic pH. In some embodiments, the aqueous phase contains acetic acid, for example, in an amount of about 5 mM. Liposomes can be used to prepare RNA-lipid complex particles by mixing them with RNA. In some embodiments, the liposomes and RNA-lipid complex particles comprise at least one cationic lipid and at least one additional lipid. In some embodiments, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP). In some embodiments, the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE), cholesterol (Chol), and/or 1,2-dioleoyl-sn-glycerol-3-phosphate choline (DOPC). In some embodiments, the at least one cationic lipid comprises 1,2-di-O-octadecenoyl-3-trimethylammonium propane (DOTMA), and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE). In some embodiments, the liposome and RNA-lipid complex particles comprise 1,2-di-O-octadecenoyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycerol-3-phosphate ethanolamine (DOPE).

RNA-lipid complex particles targeting the spleen are described in WO 2013/143683, which are incorporated herein by reference. RNA-lipid complex particles with a net negative charge have been found to preferentially target spleen tissue or spleen cells such as antigen-presenting cells, particularly dendritic cells. Therefore, RNA accumulation and/or RNA expression occur in the spleen after administration of the RNA-lipid complex particles. Therefore, the RNA-lipid complex particles of this disclosure can be used to express RNA in the spleen. In one embodiment, no or substantially no RNA accumulation and/or RNA expression occurs in the lungs and/or liver after administration of the RNA-lipid complex particles. In some embodiments, RNA accumulation and/or RNA expression occur in dedicated antigen-presenting cells, such as those in the spleen, after administration of the RNA-lipid complex particles. Therefore, the RNA-lipid complex particles of this disclosure can be used to express RNA in such antigen-presenting cells. In some embodiments, the antigen-presenting cells are dendritic cells and/or macrophages.

Lipid nanoparticles (LNP)

In some embodiments, nucleic acids such as RNA described herein are administered in the form of lipid nanoparticles (LNPs). LNPs may comprise any lipid capable of forming particles, with one or more nucleic acid molecules attached to the lipid, or one or more nucleic acid molecules encapsulated within the lipid.

In some embodiments, the LNP comprises one or more cationic lipids, and one or more stable lipids. Stable lipids include neutral lipids and polyethylene glycol-modified lipids.

In some embodiments, the LNP comprises cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and RNA encapsulated within or associated with lipid nanoparticles.

In some embodiments, the LNP comprises 40-55 mol%, 40-50 mol%, 41-49 mol%, 41-48 mol%, 42-48 mol%, 43-48 mol%, 44-48 mol%, 45-48 mol%, 46-48 mol%, 47-48 mol%, or 47.2-47.8 mol% cationic lipids. In some embodiments, the LNP comprises about 47.0, 47.1, 47.2, 47.3, 47.4, 47.5, 47.6, 47.7, 47.8, 47.9, or 48.0 mol% cationic lipids.

In some embodiments, neutral lipids are present at concentrations of 5-15 mol%, 7-13 mol%, or 9-11 mol%. In some embodiments, neutral lipids are present at concentrations of about 9.5, 10, or 10.5 mol%.

In some embodiments, the steroid is present at a concentration of 30-50 mol%, 35-45 mol%, or 38-43 mol%. In some embodiments, the steroid is present at a concentration of about 40, 41, 42, 43, 44, 45, or 46 mol%.

In some embodiments, the LNP comprises 1-10 mol%, 1-5 mol%, or 1-2.5 mol% of a polymer-conjugated lipid.

In some embodiments, the LNP comprises 40-50 mol% cationic lipids; 5-15 mol% neutral lipids; 35-45 mol% steroids; 1-10 mol% polymer-conjugated lipids; and RNA, which is encapsulated within or associated with lipid nanoparticles.

In some implementations, the molar percentage is determined based on the total molar amount of lipids present in the lipid nanoparticles.

In some embodiments, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is DSPC.

In some implementations, the steroid is cholesterol.

In some embodiments, the polymer-conjugated lipid is a polyethylene glycol-modified lipid. In some embodiments, the polyethylene glycol-modified lipid has the following structure:

Or its pharmaceutically acceptable salts, tautomers, or stereoisomers, wherein:

R12 and R13 are each independently a straight-chain or branched, saturated or unsaturated alkyl chain containing 10-30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and the average value of w is 30-60. In some embodiments, R12 and R13 are each independently a straight-chain, saturated alkyl chain containing 12-16 carbon atoms. In some embodiments, the average value of w is 40-55. In some embodiments, the average w is about 45. In some embodiments, R12 and R13 are each independently a straight-chain, saturated alkyl chain containing about 14 carbon atoms, and the average value of w is about 45.

In some embodiments, the PEGylated lipid is DMG-PEG 2000, for example, having the following structure:

In some embodiments, the cationic lipid component of LNP has the structure of formula (III):

Or its pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer, wherein:

One of L1 or L2 is –O(C＝O)-, -(C＝O)O-, -C(＝O)-, -O-, -S(O)x-, -S-S-, -C(＝O)S-, SC(＝O)-, -NRaC(＝O)-, -C(＝O)NRa-, NRaC(＝O)NRa-, -OC(＝O)NRa- or -NRaC(＝O)O-, and the other of L1 or L2 is –O(C＝O)-, -(C＝O)O-, -C(＝O)-, -O-, -S(O)x-, -S-S-, -C(＝O)S-, SC(＝O)-, -NRaC(＝O)-, -C(＝O)NRa-, NRaC(＝O)NRa-, -OC(＝O)NRa- or -NRaC(＝O)O- or a direct bond;

G1 and G2 are each independently an unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, or C3-C8 alkenylene.

Ra is H or a C1-C12 alkyl group;

R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;

R3 is H, OR5, CN, -C(=O)OR4, -OC(=O)R4 or –NR5C(=O)R4;

R4 is a C1-C12 alkyl group;

R5 is H or a C1-C6 alkyl group; and

x is 0, 1, or 2.

In some of the foregoing embodiments of formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

in:

A is a 3-8 membered cycloalkyl or cyclohexane ring;

In each occurrence, R6 is independently H, OH, or a C1-C24 alkyl group;

n is an integer from 1 to 15.

In some of the aforementioned embodiments of formula (III), the lipid has structure (IIIA), while in other embodiments, the lipid has structure (IIIB).

In other embodiments of formula (III), the lipid has one of the following structures (IIIC) or (IIID):

Where y and z are each an independent integer from 1 to 12.

In any of the foregoing embodiments of formula (III), one of L1 or L2 is -O(C=O)-. For example, in some embodiments, L1 and L2 are each -O(C=O)-. In some different embodiments of any of the foregoing, L1 and L2 are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments, L1 and L2 are each -(C=O)O-.

In some different embodiments of formula (III), the lipid has one of the following structures (IIIE) or (IIIF):

In some of the aforementioned embodiments of formula (III), the lipid has one of the following structures: (IIIG), (IIIH), (IIII), or (IIIJ):

In some of the foregoing embodiments of equation (III), n is an integer from 2 to 12, such as 2-8 or 2-4. For example, in some embodiments, n is 3, 4, 5, or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

In some other of the foregoing embodiments of equation (III), y and z are each independently an integer from 2 to 10. For example, in some embodiments, y and z are each independently an integer from 4 to 9 or from 4 to 6.

In some of the foregoing embodiments of formula (III), R6 is H. In other foregoing embodiments, R6 is a C1-C24 alkyl group. In other embodiments, R6 is OH.

In some embodiments of formula (III), G3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3 is a linear C1-C24 alkylene or a linear C1-C24 alkenylene.

In some other aforementioned embodiments of formula (III), R1 or R2, or both, are C6-C24 alkenyl groups. For example, in some embodiments, R1 and R2 each independently have the following structures:

in:

In each occurrence, R7a and R7b are independently H or C1-C12 alkyl groups; and

a is an integer between 2 and 12.

R7a, R7b, and a are each selected, so that R1 and R2 each independently contain 6-20 carbon atoms. For example, in some embodiments, a is an integer of 5-9 or 8-12.

In some of the foregoing embodiments of formula (III), R7a is H at least once. For example, in some embodiments, R7a is H in every occurrence. In other different foregoing embodiments, R7b is a C1-C8 alkyl group at least once. For example, in some embodiments, the C1-C8 alkyl group is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, or n-octyl.

In different embodiments of formula (III), R1 or R2 or both have one of the following structures:

In some of the aforementioned embodiments of formula (III), R3 is OH, CN, -C(=O)OR4, -OC(=O)R4, or –NHC(=O)R4. In some embodiments, R4 is methyl or ethyl.

In various embodiments, the cationic lipid of formula (III) has one of the structures shown in the table below.

Representative compounds of formula (III).

In some embodiments, LNP comprises lipids of formula (III), RNA, neutral lipids, steroids, and PEGylated lipids. In some embodiments, the lipid of formula (III) is compound III-3. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is ALC-0159.

In some embodiments, cationic lipids are present in the LNP at an amount of about 40 to about 50 mol percent. In some embodiments, neutral lipids are present in the LNP at an amount of about 5 to about 15 mol percent. In some embodiments, steroids are present in the LNP at an amount of about 35 to about 45 mol percent. In some embodiments, PEGylated lipids are present in the LNP at an amount of about 1 to about 10 mol percent.

In some embodiments, LNP comprises about 40 to about 50 molar percentages of compound III-3, about 5 to about 15 molar percentages of DSPC, about 35 to about 45 molar percentages of cholesterol, and about 1 to about 10 molar percentages of ALC-0159.

In some embodiments, LNP comprises about 47.5 mole percent of compound III-3, about 10 mole percent of DSPC, about 40.7 mole percent of cholesterol, and about 1.8 mole percent of ALC-0159.

In various implementations, the cationic lipid has one of the structures shown in the table below.

In some embodiments, the LNP comprises the cationic lipids shown in the table above, such as cationic lipids of formula (B) or (D), particularly cationic lipids of formula (D), RNA, neutral lipids, steroids, and PEGylated lipids. In some embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In some embodiments, the PEGylated lipid is DMG-PEG 2000.

In some embodiments, the LNP comprises a cationic lipid, which is an ionizable lipid-like material. In some embodiments, the cationic lipid has the following structure:

The N/P value is preferably at least about 4. In some embodiments, the N/P value ranges from 4 to 20, 4 to 12, 4 to 10, 4 to 8, or 5 to 7. In some embodiments, the N/P value is about 6.

The average diameter of the LNP described herein can be about 30 nm to about 200 nm or about 60 nm to about 120 nm in some embodiments.

Pharmaceutical Composition

In some embodiments, the pharmaceutical composition comprises the RNA polynucleotide disclosed herein formulated as particles. In some embodiments, the particles are or comprise lipid nanoparticles (LNPs) or lipid complexes (LPXs) particles.

In some embodiments, the RNA polynucleotides disclosed herein may be administered in a pharmaceutical composition or in a pharmaceutical medicament, and may be administered in the form of any suitable pharmaceutical composition.

In some embodiments, the pharmaceutical compositions described herein are immunogenic compositions for inducing an immune response. For example, in some embodiments, the immunogenic composition is a vaccine.

In some embodiments, the RNA polynucleotides disclosed herein can be administered in a pharmaceutical composition, which may contain a pharmaceutically acceptable carrier and optionally include one or more adjuvants, stabilizers, etc. In some embodiments, the pharmaceutical composition is used for therapeutic or prophylactic treatment.

The term "adjuvant" refers to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants comprise a heterogeneous group of compounds such as oil emulsions (e.g., Freund's adjuvant), minerals (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immunostimulatory complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines such as monokines, lymphokines, interleukins, and chemokines. Cytokines may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, and LT-α. Other known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as ISA51. Other suitable adjuvants for use in this disclosure include lipopeptides such as Pam3Cys.

The pharmaceutical compositions according to this disclosure are generally used in a "pharmaceuticalally effective amount" and in a "pharmaceutically acceptable formulation".

The term "pharmaceutical acceptable" means that a substance is non-toxic and does not interact with the active ingredient in a pharmaceutical composition.

The term "pharmaceuticalally effective amount" or "therapeutically effective amount" refers to the amount that, alone or in conjunction with further doses, achieves a desired response or desired effect. In the case of treating a specific disease, the desired response preferably involves inhibiting the disease process. This includes slowing the progression of the disease, and in particular, interrupting or reversing the progression of the disease. The desired response in treating a disease can also be delaying the onset of the disease or disease condition or preventing the onset of the disease or disease condition. The effective amount of the compositions described herein depends on the disease condition to be treated, the severity of the disease, the individual parameters of the patient, including age, physical condition, size and weight, duration of treatment, type of concomitant therapy (if present), specific route of administration, and similar factors. Therefore, the dosage of the compositions described herein can depend on several such parameters. In cases where the response in a patient is insufficient with the initial dose, a higher dose may be used (or a higher dose that is effectively achieved through a different, more localized route of administration).

In some embodiments, the pharmaceutical compositions disclosed herein may comprise salts, buffers, preservatives, and other optional therapeutic agents. In some embodiments, the pharmaceutical compositions disclosed herein comprise one or more pharmaceutically acceptable carriers, diluents, and/or excipients.

Suitable preservatives for use in the pharmaceutical compositions of this disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, p-hydroxybenzoate and thimerosal.

As used herein, the term "excipient" refers to a substance that may be present in the pharmaceutical compositions of this disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

The term "diluent" refers to a diluting and/or thinning agent. Furthermore, the term "diluent" includes any one or more fluid, liquid, or solid suspensions and/or mixtures. Examples of suitable diluents include ethanol, glycerol, and water.

The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, in which an active component is incorporated to promote, enhance, or achieve the administration of a pharmaceutical composition. As used herein, a carrier may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalene, and particularly biocompatible lactide polymers, lactide/glycolic acid copolymers, or polyoxyethylene/polyoxypropylene copolymers. In some embodiments, the pharmaceutical compositions of this disclosure comprise isotonic saline.

Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical field and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985).

Drug carriers, excipients, or diluents can be selected based on the intended route of administration and standard pharmaceutical practices.

In some embodiments, the pharmaceutical compositions described herein can be administered intravenously, intra-arterially, subcutaneously, intradermally, or intramuscularly. In some embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration, which includes absorption via the gastrointestinal tract, or parenteral administration. As used herein, the term "parenteral administration" means administration by any means other than through the gastrointestinal tract, such as intravenous injection. In a preferred embodiment, the pharmaceutical compositions are formulated for intramuscular administration. In another embodiment, the pharmaceutical compositions are formulated for systemic administration, for example, for intravenous administration.

Characterization

In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in a organism administering a composition or pharmaceutical product containing the RNA polynucleotide, an increased expression of the payload is observed relative to an appropriate reference.

In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in a organism administering a composition or pharmaceutical article containing the RNA polynucleotide, an increased duration of expression of the payload (e.g., prolonged expression) is observed relative to a suitable reference.

In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in a organism administering a composition or pharmaceutical product containing the RNA polynucleotide, a reduced interaction between the RNA polynucleotide and IFIT1 is observed relative to a suitable reference.

In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in a organism administering a composition or pharmaceutical product containing the RNA polynucleotide, an increase in the translation of the RNA polynucleotide is observed relative to a suitable reference.

In some embodiments, the reference comparison includes organisms administered other similar RNA polynucleotides without the m7(3'OMeG)(5')ppp(5')(2'OMeA ₁ )pG ₂ cap. In some embodiments, the reference comparison includes organisms administered other similar RNA polynucleotides without the cap proximal sequence disclosed herein. In some embodiments, the reference comparison includes organisms administered other similar RNA polynucleotides having a self-hybridization sequence.

In some embodiments, the RNA polynucleotides disclosed herein are characterized in that, when evaluated in a organism administering a composition or pharmaceutical article containing the RNA polynucleotide, an increase in the expression of the payload and an increase in the duration of expression (e.g., prolonged expression) are observed relative to a suitable reference.

In some embodiments, elevated expression is determined at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration of the composition or pharmaceutical product containing RNA polynucleotide. In some embodiments, elevated expression is determined at least 24 hours after administration of the composition or pharmaceutical product containing RNA polynucleotide. In some embodiments, elevated expression is determined at least 48 hours after administration of the composition or pharmaceutical product containing RNA polynucleotide. In some embodiments, elevated expression is determined at least 72 hours after administration of the composition or pharmaceutical product containing RNA polynucleotide. In some embodiments, elevated expression is determined at least 96 hours after administration of the composition or pharmaceutical product containing RNA polynucleotide. In some embodiments, elevated expression is determined at least 120 hours after administration of the composition or pharmaceutical product containing RNA polynucleotide.

In some embodiments, elevated expression is determined approximately 24-120 hours after administration of the composition or pharmaceutical product containing RNA polynucleotide. In some embodiments, elevated expression is determined approximately 24-110 hours, approximately 24-100 hours, approximately 24-90 hours, approximately 24-80 hours, approximately 24-70 hours, approximately 24-60 hours, approximately 24-50 hours, approximately 24-40 hours, approximately 24-30 hours, approximately 30-120 hours, approximately 40-120 hours, approximately 50-120 hours, approximately 60-120 hours, approximately 70-120 hours, approximately 80-120 hours, approximately 90-120 hours, approximately 100-120 hours, or approximately 110-120 hours after administration of the composition or pharmaceutical product containing RNA polynucleotide.

In some embodiments, the expression increase of the payload is at least 2 to at least 10 times. In some embodiments, the expression increase of the payload is at least 2 times. In some embodiments, the expression increase of the payload is at least 3 times. In some embodiments, the expression increase of the payload is at least 4 times. In some embodiments, the expression increase of the payload is at least 6 times. In some embodiments, the expression increase of the payload is at least 8 times. In some embodiments, the expression increase of the payload is at least 10 times.

In some embodiments, the expression increase of the payload is about 2 to about 50 times. In some embodiments, the expression increase of the payload is about 2 to about 45 times, about 2 to about 40 times, about 2 to about 30 times, about 2 to about 25 times, about 2 to about 20 times, about 2 to about 15 times, about 2 to about 10 times, about 2 to about 8 times, about 2 to about 5 times, about 5 to about 50 times, about 10 to about 50 times, about 15 to about 50 times, about 20 to about 50 times, about 25 to about 50 times, about 30 to about 50 times, about 40 to about 50 times, or about 45 to about 50 times.

In some embodiments, after administration of the composition or pharmaceutical product containing the RNA polynucleotide, the elevated expression of the effective load (e.g., an increased duration of expression) persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours. In some embodiments, the elevated expression of the effective load persists for at least 24 hours after administration. In some embodiments, the elevated expression of the effective load persists for at least 48 hours after administration. In some embodiments, the elevated expression of the effective load persists for at least 72 hours after administration. In some embodiments, the elevated expression of the effective load persists for at least 96 hours after administration. In some embodiments, the elevated expression of the effective load persists for at least 120 hours after administration of the composition or pharmaceutical product containing the RNA polynucleotide.

In some embodiments, the elevated expression of the effective load persists for about 24-120 hours after administration of the composition or pharmaceutical product containing the RNA polynucleotide. In some embodiments, the elevated expression of the effective load persists for about 24-110 hours, about 24-100 hours, about 24-90 hours, about 24-80 hours, about 24-70 hours, about 24-60 hours, about 24-50 hours, about 24-40 hours, about 24-30 hours, about 30-120 hours, about 40-120 hours, about 50-120 hours, about 60-120 hours, about 70-120 hours, about 80-120 hours, about 90-120 hours, about 100-120 hours, or about 110-120 hours after administration of the composition or pharmaceutical product containing the RNA polynucleotide.

use

This document discloses a method for preparing and using RNA polynucleotides comprising a 5' cap; a 5' UTR comprising a cap proximal structure; and a sequence encoding a payload.

In some embodiments, an in vitro transcription reaction system is disclosed herein, comprising: (i) template DNA containing a polynucleotide sequence complementary to the RNA polynucleotide sequence disclosed herein; (ii) a polymerase; and (iii) an RNA polynucleotide. In some embodiments, the polymerase is or comprises a T7 polymerase. In some embodiments, the reaction further comprises a 5' cap or a 5' cap analogue. In some embodiments, the 5' cap analogue is or comprises a Cap1 structure. In some embodiments, the RNA polynucleotide comprises a cap containing a Cap1 structure; a proximal cap sequence comprising +1, +2, +3, +4, and +5 positions of the RNA polynucleotide; and a sequence encoding a payload. In some embodiments, the Cap1 structure comprises m7G(5')ppp(5')(2'OMeN1)pN2, wherein N1 is the +1 position of the RNA polynucleotide, N2 is the +2 position of the RNA polynucleotide, and wherein N1 and N2 are each independently selected from: A, C, G, or U.

In some embodiments, this document also discloses a method for preparing capped RNA, the method comprising transcribing a nucleic acid template in the presence of a cap structure, wherein the cap structure comprises G*ppp(m ₁ ² ' ^-O )N ₁ pN ₂ ,

_N1 is complementary to the +1 position of the nucleic acid template, and _N2 is complementary to the +2 position of the nucleic acid template. Furthermore, _N1 and _N2 are independently selected from A, C, G, or U.

The RNA comprises: _N3 , which is complementary to the +3 position of the nucleic acid template and is any nucleotide, preferably A or C; _N4 , which is complementary to the +4 position of the nucleic acid template and is a nucleotide selected from A, G, and U, preferably T; and _N5 , which is complementary to the +5 position of the nucleic acid template and is any nucleotide.

G* contains the following structures:

The bond representing the first phosphorus atom of the G* group bonded to the ppp group is ^R1 , which is _CH3 , ^R2 is OH or O- _CH3 , and ^R3 is O- _CH3 .

In some embodiments, this document discloses a method for preparing a polypeptide, the method comprising the steps of: providing an RNA polynucleotide comprising a 5' cap, proximal cap sequences comprising +1, +2, +3, +4, and +5 positions of the RNA polynucleotide, and a sequence encoding a payload; wherein the RNA polynucleotide is characterized in that, when evaluated in an organism administered the RNA polynucleotide or a composition comprising the RNA polynucleotide, an increased expression of the payload and/or an increased duration of expression is observed relative to a suitable reference.

In some embodiments, a method is disclosed herein that includes administering a pharmaceutical composition to a subject, said pharmaceutical composition comprising RNA polynucleotides formulated in lipid nanoparticles (LNPs) or lipid complexes (LPXs) particles disclosed herein.

In some embodiments, this document discloses a method for inducing an immune response in a subject, the method comprising administering a pharmaceutical composition to the subject, the pharmaceutical composition comprising RNA polynucleotides formulated in lipid nanoparticles (LNPs) or lipid complexes (LPXs) particles disclosed herein.

In some embodiments, this document discloses a method for administering a vaccine to a subject, the method comprising administering a pharmaceutical composition to the subject comprising RNA polynucleotides formulated in lipid nanoparticles (LNPs) or lipid complexes (LPXs) particles disclosed herein.

In some embodiments, this document provides a method for reducing the interaction between an RNA polynucleotide and IFIT1, said RNA polynucleotide comprising a 5' cap and a proximal cap sequence comprising +1, +2, +3, +4, and +5 positions of the RNA polynucleotide, said method comprising the following steps:

Provide variants of the RNA polynucleotide that differ from the parental RNA polynucleotide in that one or more residues are substituted within the proximal cap sequence, and

It was determined that the variant interacts less with IFIT1 relative to the parental RNA polynucleotide. In some embodiments, administering the RNA polynucleotide or a composition containing the RNA polynucleotide to cells or organisms is determined.

In some embodiments, this document discloses a method for increasing the translatability of an RNA polynucleotide, said RNA polynucleotide comprising a 5' cap, a proximal cap sequence comprising +1, +2, +3, +4, and +5 positions of the RNA polynucleotide, and a sequence encoding a payload, said method comprising the steps of: providing a variant of the RNA polynucleotide that differs from a parent RNA polynucleotide in that one or more residues are substituted within the proximal cap sequence; and determining that the expression of said variant is increased relative to the expression of the parent RNA polynucleotide. In some embodiments, determining includes administering the RNA polynucleotide or a composition comprising the RNA polynucleotide to cells or an organism. In some embodiments, increased translatability is evaluated by an increase in payload expression and/or persistence of expression. In some embodiments, an increase in expression is determined at least 6 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration. In some embodiments, the increase in expression is at least 2 to 10 times. In some embodiments, the increase in expression is at least about 2 to 50 times. In some implementations, the elevated expression persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration.

In some embodiments, this document provides a therapeutic RNA comprising a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding a payload, wherein the modification comprises: including one or more of the following residues in the proximal cap sequence: X at position +1 of the RNA polynucleotide, X at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and X at position +5 of the RNA polynucleotide, wherein increased RNA expression is demonstrated when administered to a subject as an LNP formulation. In some embodiments, X is selected from A, C, G, or U.

In some embodiments, this document provides a therapeutic RNA comprising a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding a payload, wherein the modification comprises including one or more of the following residues in the proximal cap sequence: A at position +1 of the RNA polynucleotide, G at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and U at position +5 of the RNA polynucleotide, wherein increased RNA expression is demonstrated when administered to a subject as an LNP formulation.

In some embodiments, this document provides a therapeutic RNA comprising a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding a payload, wherein the modification comprises including one or more of the following residues in the proximal cap sequence: X at position +1 of the RNA polynucleotide, X at position +2 of the RNA polynucleotide, C at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and X at position +5 of the RNA polynucleotide, wherein increased RNA expression is demonstrated when administered to a subject as an LNP formulation. In some embodiments, X is selected from A, C, G, or U.

In some embodiments, this document provides a method for enhancing the translation of RNA polynucleotides, said RNA polynucleotide comprising a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding a payload, the enhancement comprising including one or more of the following residues in the proximal cap sequence: X at position +1 of the RNA polynucleotide, X at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and X at position +5 of the RNA polynucleotide. In some embodiments, X is selected from A, C, G, or U.

In some embodiments, this document provides a method for increasing the translation of RNA polynucleotides, said RNA polynucleotides comprising a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding a payload, the improvement comprising including one or more of the following residues in the proximal cap sequence: A at position +1 of the RNA polynucleotide, G at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and U at position +5 of the RNA polynucleotide.

In some embodiments, this document provides a method for enhancing the translation of RNA polynucleotides, said RNA polynucleotides comprising a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding a payload, the enhancement comprising including one or more of the following residues in the proximal cap sequence: X at position +1 of the RNA polynucleotide, X at position +2 of the RNA polynucleotide, C at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and X at position +5 of the RNA polynucleotide. In some embodiments, X is selected from A, C, G, or U.

In some embodiments of any of the methods disclosed herein, an immune response is induced in a subject. In some embodiments of any of the methods disclosed herein, the immune response is a prophylactic immune response or a therapeutic immune response.

In some implementations of any of the methods disclosed herein, the subjects are mammals.

In some implementations of any of the methods disclosed herein, the subject is a human being.

In some implementations of any of the methods disclosed herein, the subject suffers from the disease or condition disclosed herein.

In some embodiments of any of the methods disclosed herein, vaccination produces an immune response to the agent. In some embodiments, the immune response is a prophylactic immune response.

In some implementations of any of the methods disclosed herein, the subject suffers from the disease or condition disclosed herein.

In some embodiments of any of the methods disclosed herein, a dose of the pharmaceutical composition is administered.

In some embodiments of any of the methods disclosed herein, multiple doses of the pharmaceutical composition are administered.

In some embodiments of any of the methods disclosed herein, the methods further include administering one or more therapeutic agents. In some embodiments, one or more therapeutic agents are administered before, after, or simultaneously with the administration of a pharmaceutical composition comprising an RNA polynucleotide.

In some implementations, this document also provides a method for providing a framework for an RNA polynucleotide comprising a 5' cap, a proximal cap sequence, and a payload sequence, the method comprising the following steps:

Evaluate at least two variants of the RNA polynucleotide, wherein:

Each variant includes the same 5' cap and payload sequence; and

The variants differ from each other at one or more specific residues in the proximal cap sequence;

The evaluation includes determining the expression level and/or duration of the payload; and

Select at least one combination of the 5' cap and the proximal cap sequence, which shows elevated expression relative to at least one other combination.

In some implementations, the evaluation includes administering the RNA construct or a composition containing the RNA construct to cells or organisms.

In some embodiments, an increase in payload expression is detected at time points of at least 6 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration. In some embodiments, the expression increase is at least 2 to 10 times. In some embodiments, the expression increase is about 2 to about 50 times.

In some implementations, the elevated expression of the effective load after administration lasts for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours.

In some embodiments of any of the methods disclosed herein, the RNA polynucleotide includes one or more features of the RNA polynucleotides provided herein.

In some embodiments of any of the methods disclosed herein, the composition comprising RNA polynucleotides comprises the pharmaceutical composition provided herein.

List of implementation plans

1. A composition or pharmaceutical product comprising an RNA polynucleotide, said RNA polynucleotide comprising:

The sequence includes a 5' cap containing the Cap1 structure; proximal cap sequences containing RNA polynucleotides at positions +1, +2, +3, +4, and +5; and a sequence encoding the payload, wherein:

(i) The Cap1 structure comprises m7G(5')ppp(5')(2'OMeN ₁ )pN ₂ , wherein N ₁ is the +1 position of the RNA polynucleotide, N ₂ is the +2 position of the RNA polynucleotide, and wherein N ₁ and N ₂ are each independently selected from: A, C, G or U; and

(ii) The cap proximal sequence comprises _N1 and _N2 of the Cap1 structure, and:

(a) Selected from the following sequences: _A3A4X5 (SEQ ID NO: _{1); C3A4X5 (SEQ ID NO:2); A3C4A5 ( SEQ ID NO : 3 ) and A3U4G5} (SEQ ID NO: ₄ ); or

(b) A sequence containing X ₃ Y ₄ X ₅ (SEQ ID NO:7);

Wherein _X3 (nucleotide X at position +3 in SEQ ID NO:7) or _X5 (nucleotide X at position +5 in SEQ ID NO:1 or SEQ ID NO:2) is independently selected from A, G, C or U; and

Where _Y4 (nucleotide Y at position +4 in SEQ ID NO:7) is not C.

2. The composition or pharmaceutical product of embodiment 1, wherein:

(i) The _N1 position is A, the _N2 position is G, and the Cap1 structure contains m7G(5')ppp(5')(2'OMeA ₁ )pG ₂ ;

(ii) The _N1 position is A, the _N2 position is U, and the Cap1 structure contains m7G(5')ppp(5')(2'OMeA ₁ )pU ₂ ; or

(iii) The _N1 position is G, the _N2 position is G, and the Cap1 structure contains m7G(5')ppp(5')(2'OMeG ₁ )pG ₂ .

3. The composition or pharmaceutical product of embodiment 1 or 2, wherein the methylated guanosine (m7G) in the Cap1 structure further comprises one or more modifications, for example, wherein the m7G in the Cap1 structure comprises methylated ribose.

4. The composition or pharmaceutical product of embodiment 3, wherein the m7G in Cap1 comprises 3’O methylation (m7(3’OMeG)).

5. A composition or pharmaceutical article comprising an RNA polynucleotide, said RNA polynucleotide comprising: a 5' cap; a proximal cap sequence comprising +1, +2, +3, +4, and +5 positions of the RNA polynucleotide; and a sequence encoding a payload, wherein:

(i) The 5' cap includes a Cap1 structure, which includes G*ppp(m ₁ ² ' ^-O )N ₁ pN ₂ , wherein:

N1 is the +1 position of the RNA polynucleotide, _N2 is the +2 position of the RNA polynucleotide, and _N1 and _N2 are each independently selected from A, C, G, or U; and

G* contains the following structures:

The bond representing the first phosphorus atom of the G* group bonded to the ppp group is ^R1 , which is _CH3 , ^R2, which is OH or O- _CH3 , and ^R3 is O- _CH3 ; and

(ii) The proximal sequence of the cap contains _N1 and _N2 of the Cap1 structure, and:

(a) Selected from the following sequences: _A3A4X5 (SEQ ID NO:1) _{; C3A4X5} (SEQ ID NO _{: 2} ); _A3C4A5 (SEQ _{ID NO} :3 _{) or A3U4G5} (SEQ ID NO: ₄ ); or

(b) A sequence containing X ₃ Y ₄ X ₅ (SEQ ID NO:7);

Wherein _X3 (nucleotide X at position +3 in SEQ ID NO:7) or _X5 (nucleotide X at position +5 in SEQ ID NO:1 or SEQ ID NO:2) is independently selected from A, G, C or U; and

Where _Y4 (nucleotide Y at position +4 in SEQ ID NO:7) is not C.

6. The composition or pharmaceutical product of embodiment 5, wherein:

(i) The _N1 position is A, the _N2 position is G, and the Cap1 structure contains m7G(5')ppp(5')(2'OMeA ₁ )pG ₂ ;

(ii) The _N1 position is A, the _N2 position is U, and the Cap1 structure contains m7G(5')ppp(5')(2'OMeA ₁ )pU ₂ ; or

(iii) The _N1 position is G, the _N2 position is G, and the Cap1 structure contains m7G(5')ppp(5')(2'OMeG ₁ )pG ₂ .

7. The composition or pharmaceutical article of any of the foregoing embodiments, wherein the proximal cap sequence comprises _N1 and _N2 of the Cap1 structure, and a sequence comprising _A3A4X5 (SEQ ID NO: ₁ ), wherein optionally _X5 is any _nucleotide , such as A, C, G or U, preferably U.

8. A composition or pharmaceutical article of any one of embodiments 1-6, wherein the proximal cap sequence comprises _N1 and _N2 of the Cap1 structure, and a sequence comprising _C3A4X5 (SEQ ID NO: ₂ ), optionally wherein _{X5 is} any nucleotide, such as A, C, G or U.

9. A composition or pharmaceutical article of any one of embodiments 1-6, wherein the proximal cap sequence comprises _N1 and _N2 of the Cap1 structure, and a sequence comprising _{X3 Y4 X5} (SEQ ID NO:7), wherein _X3 or _X5 is any nucleotide, such as A, C, G or U, and _Y4 is not C.

10. A composition or pharmaceutical article of any one of embodiments 1-6, wherein the proximal cap sequence comprises _N1 and _N2 of the Cap1 structure, and _a sequence comprising _A3C4A5 (SEQ ID NO: ₃ ).

11. A composition or pharmaceutical article of any one of embodiments 1-6, wherein the proximal _cap sequence comprises _N1 and _N2 of the Cap1 structure, and a sequence comprising _A3U4G5 (SEQ ID NO: ₄ ).

12. A composition or pharmaceutical product comprising an RNA polynucleotide, said RNA polynucleotide comprising:

The sequence includes a 5' cap containing the Cap1 structure; proximal cap sequences containing RNA polynucleotides at positions +1, +2, +3, +4, and +5; and a sequence encoding the payload, wherein:

(i) The Cap1 structure comprises m7(3'OMeG)(5')ppp(5')( _2'OMeA1 ) _pG2 , where _A1 is the +1 position of the RNA polynucleotide and _G2 is the +2 position of the RNA polynucleotide; and

(ii) The proximal cap sequence comprises _A1 and _G2 of the Cap1 structure, and includes the following sequences: _A3A4U5 at the +3, +4 and ₊₅ positions of the RNA polynucleotide _, respectively.

13. The composition or pharmaceutical product of any of the foregoing embodiments, wherein the RNA polynucleotide comprises a 5’UTR and the cap proximal sequence is located in the 5’UTR.

14. A composition or pharmaceutical product comprising a capped RNA polynucleotide encoding a gene product, said RNA polynucleotide comprising the formula:

Where ^R1 is _CH3 , ^R2 is OH or O- _CH3 , and ^R3 is O- _CH3 .

Wherein _B1 is any nucleobase, preferably A; _B2 is any nucleobase, preferably G; _B3 is any nucleobase, preferably A or C; _B4 is any nucleobase; and _B5 is any nucleobase, and

Specifically, when the RNA polynucleotide was administered to the subject, the expression level of the encoded gene product differed by no more than 5-fold between approximately 6 hours and approximately 48 hours after administration.

15. The composition or pharmaceutical product of embodiment 14, wherein, when the RNA polynucleotide is administered to a subject, the expression of the gene product is detectable at least 72 hours after administration.

16. The composition or pharmaceutical product according to embodiment 14 or 15, wherein:

The RNA polynucleotide contains a 5'UTR; and/or

The RNA polynucleotide further comprises a 3’UTR sequence; and/or a polyA sequence.

17. The composition or pharmaceutical product of any of the foregoing embodiments, wherein the RNA polynucleotide does not contain a self-hybridization sequence, optionally wherein the self-hybridization sequence is a sequence capable of hybridizing with a sequence in the 5’UTR or 3’UTR of the RNA polynucleotide, for example, wherein the self-hybridization sequence is or contains SEQ ID NO:8.

18. The composition or pharmaceutical product of any of the foregoing embodiments, wherein the 5’UTR comprises human alpha globin (hAg) 5’UTR or a fragment thereof, TEV 5’UTR or a fragment thereof, HSP70 5’UTR or a fragment thereof, or c-Jun 5’UTR or a fragment thereof.

19. The composition or pharmaceutical product of any of the foregoing embodiments, wherein the 5’UTR comprises the human α-globin 5’UTR provided in SEQ ID NO:11, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with it.

20. A composition or pharmaceutical article of any one of embodiments 1-18, wherein the 5’UTR comprises the human α-globin 5’UTR provided in SEQ ID NO:12, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with it.

21. The composition or pharmaceutical product of any of the foregoing embodiments, wherein the 5’UTR further comprises a T7 RNA polymerase promoter sequence.

22. The composition or pharmaceutical product of any of the foregoing embodiments, wherein the 5' cap structure is co-transcribed or not enzymatically added.

23. The composition or pharmaceutical product of any of the foregoing embodiments, wherein the RNA polynucleotide comprises a 3’UTR or a fragment thereof, optionally wherein the 3’UTR sequence comprises SEQ ID NO:13, or a sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with it.

24. A composition or pharmaceutical article of any one of embodiments 16-23, wherein the 3’UTR or its proximal sequence contains a restriction site, optionally wherein the restriction site is a BamHI site or an XhoI site.

25. The composition or pharmaceutical product of any of the foregoing embodiments, wherein the PolyA sequence comprises at least 100 nucleotides, optionally wherein:

(i) The polyA sequence is an interrupted sequence of adenine nucleotides; and/or

(ii) The polyA sequence contains 30 adenine nucleotides followed by 70 adenine nucleotides, with the 30 and 70 adenine nucleotides separated by a linker sequence.

26. The composition or pharmaceutical article of embodiment 25, wherein the polyA sequence comprises the sequence of SEQ ID NO:14.

27. The composition or pharmaceutical article of any one of embodiments 16-26, wherein the 5' cap, 5' UTR, sequence encoding the payload, 3' UTR and polyA sequence are arranged in a 5' to 3' orientation.

28. The composition or pharmaceutical product of any of the foregoing embodiments, wherein the RNA polynucleotide comprises a Kozak sequence upstream of the sequence encoding the payload.

29. The composition or pharmaceutical article of any of the foregoing embodiments, wherein the sequence encoding the payload comprises a promoter sequence and/or wherein the sequence encoding the payload comprises a sequence encoding a secretion signal peptide.

30. The composition or pharmaceutical product of any of the foregoing embodiments, wherein the RNA polynucleotide comprises a sequence encoding a payload selected from: protein substitute peptides; antibody substances; cytokines; antigenic peptides; gene editing components; regenerative medicine components or combinations thereof.

31. The composition or pharmaceutical product of any of the foregoing embodiments, characterized in that, when evaluated in a organism to which the composition or pharmaceutical product comprising RNA polynucleotide is administered, an increase in the expression of the effective load and/or an increase in the duration of expression is observed relative to a suitable reference.

32. The composition or pharmaceutical article of embodiment 30, wherein the effective load is or comprises a protein-substituted polypeptide, optionally wherein:

Protein substitute peptides include peptides that are abnormally expressed in diseases or conditions;

Protein substitutes include intracellular proteins, extracellular proteins, or transmembrane proteins; and/or

Protein substitutes for peptides include enzymes.

33. The composition or pharmaceutical product of embodiment 32, wherein the disease or condition in which the peptide expression is abnormal includes, but is not limited to: rare diseases, metabolic disorders, muscular dystrophy, cardiovascular diseases or monogenic diseases.

34. The composition or pharmaceutical article of embodiment 30, wherein the effective load is or comprises an antibody substance, optionally wherein the antibody substance binds to a polypeptide expressed on a cell.

35. The composition or pharmaceutical product of embodiment 34, wherein the antibody substance comprises a CD3 antibody, a Claudin 6 antibody, or a combination thereof.

36. The composition or pharmaceutical article of embodiment 30, wherein the effective load is or comprises a cytokine or a fragment or variant thereof, optionally wherein the cytokine comprises: IL-12 or a fragment or variant thereof or a fusion thereof, IL-15 or a fragment or variant thereof or a fusion thereof, GMCSF or a fragment or variant thereof; or IFN-α or a fragment or variant thereof.

37. The composition or pharmaceutical article of embodiment 30, wherein the effective load is or comprises an antigenic polypeptide or an immunogenic variant or immunogenic fragment thereof, optionally wherein the antigenic polypeptide comprises one epitope from an antigen or multiple different epitopes from an antigen.

38. The composition or pharmaceutical article of embodiment 37, wherein the antigenic polypeptide comprising multiple different epitopes from the antigen is multi-epitope.

39. The composition or pharmaceutical product of embodiment 37 or 38, wherein the antigenic polypeptide includes: an antigenic polypeptide derived from an allergen, a viral antigenic polypeptide, a bacterial antigenic polypeptide, a fungal antigenic polypeptide, a parasitic antigenic polypeptide, an antigenic polypeptide derived from an infectious source, an antigenic polypeptide derived from a pathogen, a tumor antigenic polypeptide, or a self-antigenic polypeptide.

40. The composition or pharmaceutical article of embodiment 39, wherein the viral antigen polypeptide comprises an HIV antigen polypeptide, an influenza antigen polypeptide, a coronavirus antigen polypeptide, a rabies antigen polypeptide, or a Zika virus antigen polypeptide, optionally wherein the viral antigen polypeptide is or comprises a coronavirus antigen polypeptide.

41. The composition or pharmaceutical article of embodiment 40, wherein the coronavirus antigen is or comprises the SARS-CoV-2 protein, optionally wherein the SARS-CoV-2 protein comprises the SARS-CoV-2 spike (S) protein, or an immunogenic variant or immunogenic fragment thereof.

42. The composition or pharmaceutical article of embodiment 41, wherein the SARS-CoV-2 protein, or its immunogenic variant or immunogenic fragment, contains proline residues at positions 986 and 987.

43. The composition or pharmaceutical product of embodiment 41 or 42, wherein the SARS-CoV-2S polypeptide:

(i) has at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity with SEQ ID NO:9; or

(ii) Encoded by RNA having at least 99%, 98%, 97%, 96%, 95%, 90%, 85% or 80% identity with SEQ ID NO:10.

44. The composition or pharmaceutical article of embodiment 30, wherein the effective load is or comprises a tumor antigen polypeptide or an immunogenic variant or immunogenic fragment thereof, optionally wherein the tumor antigen polypeptide comprises a tumor-specific antigen, a tumor-associated antigen, a tumor neoantigen, or a combination thereof.

45. The composition or pharmaceutical product of embodiment 43 or 44, wherein the tumor antigen peptide comprises p53, ART-4, BAGE, ss-ligin/m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap100, HAGE, HER-2/neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAG E-A8, MAGE-A9, MAGE-A10, MAGE-A11 or MAGE-A12, MAGE-B, MAGE-C, MART-1/Melan-A, MC1R, myosin/m, MUC1, MUM-1, -2, -3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minimal BCR-abL, Plac-1, Pm1/RARa, PRAME, protease 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, SURVIVIN, TEL/AML1, TPI/m, TRP-1, TRP-2, TRP-2/INT2, TPTE, WT, WT-1, or combinations thereof.

46. The composition or pharmaceutical product of any one of embodiments 44-45, wherein the tumor antigen polypeptide comprises a tumor antigen derived from cancer, sarcoma, melanoma, lymphoma, leukemia, or a combination thereof.

47. The composition or pharmaceutical product of embodiment 46, wherein the tumor antigen polypeptide comprises: melanoma tumor antigen; prostate cancer antigen; HPV16 positive head and neck cancer antigen; breast cancer antigen; ovarian cancer antigen; lung cancer antigen, such as NSCLC antigen.

48. The composition or pharmaceutical article of embodiment 39, wherein the active load is or comprises a self-antigen polypeptide or an immunogenic variant or immunogenic fragment thereof, optionally wherein the self-antigen polypeptide comprises an antigen that is normally expressed on cells and recognized as a self-antigen by the immune system.

49. The composition or pharmaceutical product of embodiment 48, wherein the self-antigen polypeptide comprises: multiple sclerosis antigen polypeptide, rheumatoid arthritis antigen polypeptide, lupus antigen polypeptide, celiac disease antigen polypeptide, Sjögren's syndrome antigen polypeptide or ankylosing spondylitis antigen polypeptide, or a combination thereof.

50. The composition or pharmaceutical product of any of the foregoing embodiments, wherein the RNA polynucleotide comprises a modified nucleoside instead of uridine, optionally wherein the modified nucleoside is selected from pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) and 5-methyl-uridine (m5U).

51. The composition or pharmaceutical article of any of the foregoing embodiments, wherein the RNA polynucleotide further comprises one or more additional sequences, such as one or more additional payloads, and/or one or more regulatory elements.

52. The composition or pharmaceutical article of any of the foregoing embodiments, wherein the RNA polynucleotide is characterized in that, when administered to an organism, it increases the translation efficiency of the effective load compared to other similar RNA polynucleotides without the m7(3'OMeG)(5')ppp(5')(2'OMeA ₁ )pG ₂ cap.

53. The composition or pharmaceutical article of any of the foregoing embodiments, wherein the RNA polynucleotide is characterized in that, upon administration to a organism, it increases the expression level and/or duration of expression of the encoded payload compared to other similar RNA polynucleotides without the m7(3'OMeG)(5')ppp(5')(2'OMeA ₁ )pG ₂ cap, without the cap proximal sequence disclosed herein, and/or having a self-hybridization sequence, optionally wherein:

(i) An increase in expression is determined at least 6 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration, optionally wherein the increase in expression is at least 2 to 10 times; or

(ii) The elevated expression persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours.

54. The composition or pharmaceutical product of any of the foregoing embodiments, wherein the RNA is formulated or to be formulated as a liquid, a solid or a combination thereof; wherein the RNA is formulated or to be formulated for injection; or wherein the RNA is formulated or to be formulated for intramuscular injection.

55. A pharmaceutical composition comprising an RNA polynucleotide of any of the foregoing embodiments, formulated as particles, optionally wherein the particles are or comprise lipid nanoparticles (LNPs) or lipid complexes (LPXs) particles.

56. The pharmaceutical composition of embodiment 55, wherein the lipid nanoparticles comprise various of the following: cationic lipids; sterols; neutral lipids; and lipid conjugates.

57. The pharmaceutical composition of embodiment 56, wherein the cationic lipid is or comprises ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(2-decanoic acid hexyl ester), the sterol is cholesterol, the neutral lipid is or comprises phospholipid, and the lipid conjugate is or comprises polyethylene glycol (PEG) lipid.

58. The pharmaceutical composition of embodiment 57, wherein the phospholipid is or comprises distearate phosphatidylcholine (DSPC).

59. The pharmaceutical composition of embodiment 57, wherein the (PEG)-lipid is or comprises 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide.

60. The pharmaceutical composition of any one of embodiments 56-58, wherein the lipid nanoparticles comprise various of the following: a. ((4-hydroxybutyl)azanidinediyl)bis(hexane-6,1-diyl)bis(hexyl 2-decanoate); b. cholesterol; c. distearate phosphatidylcholine (DSPC); and d. 2-[(polyethylene glycol)-2000]-N,N-bistetradecylacetamide.

61. The pharmaceutical composition of any one of embodiments 56-60, wherein:

The concentration of neutral lipids is 5-15 mol% of total lipids;

The concentration of cationic ionizable lipids is 40-55 mol% of total lipids;

The concentration of steroids was 30-50 mol% of total lipids; and/or

The concentration of PEGylated lipids is 1-10 mol% of the total lipids.

62. The pharmaceutical composition of any one of embodiments 56-61, wherein the lipid nanoparticles comprise 40-55 mol% of cationic ionizable lipids; 5-15 mol% of neutral lipids; 30-50 mol% of steroids; and 1-10 mol% of polyethylene glycol-modified lipids.

62. The pharmaceutical composition of embodiment 55, wherein the RNA-lipid complex particles can be obtained by mixing RNA polynucleotides with liposomes.

63. A composition or pharmaceutical product of any one of embodiments 1-54, or a pharmaceutical composition of any one of embodiments 55-62, wherein the RNA is mRNA or saRNA.

64. A pharmaceutical composition according to any one of embodiments 55-63, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and/or excipients.

65. A pharmaceutical composition according to any one of embodiments 55-64, packaged as a kit, optionally wherein the kit further comprises instructions for use of the pharmaceutical composition for inducing an immune response in a subject.

66. A method of manufacturing a pharmaceutical composition according to any one of embodiments 55-64, comprising combining an RNA polynucleotide with a lipid to form lipid nanoparticles encapsulating the RNA.

67. A nucleic acid template suitable for producing cap1-capped RNA, wherein the first five nucleotides transcribed from the template strand of the nucleic acid template comprise the sequence _{N1 pN2 pN3 pN4 pN5} , wherein _N1 is any nucleotide, preferably T; _N2 is any nucleotide, preferably C; _N3 is any nucleotide, preferably T or G; _N4 is any nucleotide; and _N5 is any nucleotide.

68. The nucleic acid template of embodiment 67, wherein the DNA template comprises: a 5’ UTR, a sequence encoding a payload, a 3’ UTR, and a polyA sequence.

69. An in vitro transcription reaction system, comprising:

(i) Template DNA comprising a polynucleotide sequence complementary to the RNA polynucleotide sequence provided in any one of embodiments 1-54;

(ii) Polymerase (e.g., T7 polymerase); and

(iii) RNA polynucleotides.

70. The in vitro transcription reaction system of embodiment 69 further comprises a 5' cap or a 5' cap analogue, optionally wherein the 5' cap or 5' cap analogue is or comprises a Cap1 structure.

71. The in vitro transcription reaction system of embodiment 69, wherein the RNA polynucleotide comprises a cap containing a Cap1 structure; a proximal cap sequence containing +1, +2, +3, +4, and +5 positions of the RNA polynucleotide; and a sequence encoding a payload, optionally wherein the Cap1 structure comprises m7G(5')ppp(5')(2'OMeN ₁ )pN ₂ , wherein N ₁ is the +1 position of the RNA polynucleotide, N ₂ is the +2 position of the RNA polynucleotide, and wherein N ₁ and N ₂ are each independently selected from: A, C, G, or U.

72. RNA polynucleotides isolated from the in vitro transcription reaction system provided in any of embodiments 69-71.

73. A method for preparing capped RNA, the method comprising transcribing a nucleic acid template in the presence of a cap structure, wherein the cap structure comprises G*ppp(m ₁ ² ' ^-O )N ₁ pN ₂ ,

_N1 is complementary to the +1 position of the nucleic acid template, and _N2 is complementary to the +2 position of the nucleic acid template. Furthermore, _N1 and _N2 are independently selected from A, C, G, or U.

The +3 position of the nucleic acid template can be any nucleotide, preferably T or G; the +4 position of the nucleic acid template can be any nucleotide; and the +5 position of the nucleic acid template can be any nucleotide.

G* contains the following structures:

The bond representing the first phosphorus atom of the G* group bonded to the ppp group is ^R1 , which is _CH3 , ^R2 is OH or O- _CH3 , and ^R3 is O- _CH3 .

74. A composition comprising a DNA polynucleotide, said DNA polynucleotide comprising a sequence complementary to an RNA polynucleotide sequence provided in any one of embodiments 1-54.

75. The DNA polynucleotide composition of embodiment 74, which can be used to transcribe RNA polynucleotides and/or which are placed in a vector.

76. A method comprising:

A pharmaceutical composition comprising an RNA polynucleotide formulated in lipid nanoparticles (LNPs) or lipid complexes (LPXs) particles, according to any of the administration embodiments 55-64, for administration to a subject.

77. A method for inducing an immune response in a subject, the method comprising administering to the subject a pharmaceutical composition comprising an RNA polynucleotide formulated in lipid nanoparticles (LNPs) or lipid complexes (LPXs) as described in any of embodiments 55-64.

78. The method of implementation scheme 77, wherein an immune response is induced in a subject, optionally wherein the immune response is a prophylactic immune response or a therapeutic immune response.

79. A method for administering a vaccine to a subject by administering a pharmaceutical composition comprising an RNA polynucleotide formulated in lipid nanoparticles (LNPs) or lipid complexes (LPXs) as described in any of embodiments 55-64.

80. The method of implementation plan 79, wherein vaccination produces an immune response, optionally wherein the immune response is a prophylactic immune response.

81. A method for reducing the interaction between an RNA polynucleotide and IFIT1, said RNA polynucleotide comprising a 5' cap and a proximal cap sequence comprising +1, +2, +3, +4, and +5 positions of the RNA polynucleotide, said method comprising the following steps:

Variants of the RNA polynucleotide are provided, differing from the parental RNA polynucleotide in that one or more residues are substituted within the proximal cap sequence, and

The variant is determined to have reduced interaction with IFIT1 relative to the parental RNA polynucleotide, optionally including administration of the RNA polynucleotide or a composition containing the RNA polynucleotide to cells or organisms.

82. A method for preparing polypeptides, comprising the following steps:

Provides an RNA polynucleotide containing a 5' cap, proximal cap sequences at +1, +2, +3, +4, and +5 positions of the RNA polynucleotide, and a sequence encoding the payload;

The RNA polynucleotide is characterized in that, when evaluated in an organism to which the RNA polynucleotide or a composition containing the RNA polynucleotide is administered, an increase in the expression of the payload and/or an increase in the duration of expression is observed relative to a suitable reference.

83. A method for increasing the translatability of RNA polynucleotides, said RNA polynucleotide comprising a 5' cap, proximal cap sequences comprising +1, +2, +3, +4, and +5 positions of the RNA polynucleotide, and a sequence encoding a payload, said method comprising the following steps:

Provide a variant of the RNA polynucleotide that differs from the parental RNA polynucleotide in that one or more residues are substituted within the proximal cap sequence; and

The expression of the variant is determined to be increased relative to the expression of the parental RNA polynucleotide, optionally including administration of the RNA polynucleotide or a composition containing the RNA polynucleotide to cells or organisms.

84. The method of implementation scheme 83, wherein increased translatability is evaluated by the increase in the expression of the payload and/or the persistence of the expression.

85. The method of implementing Implementation 84, wherein:

(i) An increase in expression is determined at least 6 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration, optionally wherein the increase in expression is at least 2 to 10 times; or

(ii) The elevated expression persists for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours.

86. In therapeutic RNA, which includes a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding the payload, improvements include:

The proximal cap sequence includes one or more of the following residues: A at position +1 of the RNA polynucleotide, G at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and U at position +5 of the RNA polynucleotide.

When LNP formulations were administered to subjects, increased RNA expression was confirmed.

87. A method for increasing the translation of RNA polynucleotides, said RNA polynucleotide comprising a 5' cap containing a Cap1 structure, a proximal cap sequence, and a sequence encoding a payload, the improvement comprising:

The proximal cap sequence includes one or more of the following residues: A at position +1 of the RNA polynucleotide, G at position +2 of the RNA polynucleotide, A at position +3 of the RNA polynucleotide, A at position +4 of the RNA polynucleotide, and U at position +5 of the RNA polynucleotide.

88. The method of any one of embodiments 76-87, wherein one or more doses of the pharmaceutical composition are administered.

89. The method of any one of embodiments 76-88, wherein the method further comprises administering one or more therapeutic agents.

90. The method of embodiment 89, wherein one or more therapeutic agents are administered before, after, or simultaneously with administration of a pharmaceutical composition comprising an RNA polynucleotide.

91. The method of any one of embodiments 76-90, wherein the subject or organism is a mammal.

92. The method of implementation scheme 91, wherein the subject or organism is a human.

93. The method of any one of embodiments 76-93, wherein the subject suffers from the disease or condition disclosed herein.

94. A method for providing a framework for an RNA polynucleotide comprising a 5' cap, a proximal cap sequence, and a payload sequence, the method comprising the steps of:

Evaluate at least two variants of the said RNA polynucleotide, wherein:

Each variant includes the same 5' cap and payload sequence; and

The variants differ from each other at one or more specific residues in the proximal cap sequence;

The evaluation includes determining the expression level and/or duration of the payload sequence; and

Select at least one combination of the 5' cap and the proximal cap sequence, which shows elevated expression relative to at least one other combination.

95. The method of embodiment 94, wherein the evaluation includes administering an RNA construct or a composition containing an RNA construct to cells or organisms.

96. Implement the method of 94 or 95, wherein:

Elevated expression was detected at time points of at least 6 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours after administration, optionally wherein the elevation was at least 2 to 10 times; and/or

The elevated expression lasts for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, or at least 120 hours.

97. The method of any one of embodiments 76-96, wherein the RNA polynucleotide comprises one or more features of the RNA polynucleotide provided in any one of embodiments 1-54.

98. The method of any one of embodiments 76-97, wherein the composition comprises the pharmaceutical composition of any one of embodiments 55-64.

Example

Example 1 - Providing an improved RNA cassette for sustained expression of encoded proteins

This embodiment describes the generation of RNA boxes that, in vivo, have improved expression levels of the coding payload and/or increased expression duration of the coding payload. The methods used in this embodiment are described below.

method:

mRNA preparation

For the template, a purified codon-optimized plasmid encoding mouse erythropoietin (mEPO) was used. The plasmid corresponding to the 5' untranslated region sequence of tobacco etch virus 5' leader RNA (TEV) or human α-globin (hAg) mRNA was linearized using BspQI (New England Biolabs, Cat#R0712L) to generate the template. Transcription was performed using a MEGAscript T7 RNA polymerase kit (Thermo Fisher Scientific, Cat#AMB1334-5), replacing the UTP with N1-methylpseudouridine (m1Ψ) 5'-triphosphate (TriLink, Cat#N-1081). The mRNA was co-transcribed and capped using the anti-reverse Cap1 analog CleanCap413 (TriLink, Cat#N-7413) at a final concentration of 3 mM. To obtain the desired transcript produced using the cap analogue, the initial GTP concentration in the transcription reaction was reduced from 7.5 mM to 1.5 mM, and incubated at 37°C for 30 min in a hybridization chamber. The initial concentration of additional nucleotides, including ATP, CTP, and m1ΨTP, corresponded to the final 7.5 mM concentration. After incubation for 30, 60, 90, and 120 min, an additional 1.5 mM GTP was added to the mixture, and the mixture was incubated for another 30 min at 37°C. The mRNA was transcribed to a 3' poly(A) tail containing 100 nt. To remove the template, 1/10 volume of DNA Turbo DNase (Thermo Fisher Scientific, Cat#AM1907) was added to the reaction mixture, and the mixture was incubated at 37°C for 15 min. The synthesized mRNA was separated from the reaction mixture by precipitation with half the reaction volume of 8 M LiCl solution (Sigma-Aldrich, Cat#L7026). After cooling at -20°C for at least 1 hour, RNA particles were collected by centrifugation at 17,000 × g for 5 minutes at 4°C. The RNA particles were washed twice with at least 200 μl of ice-cold 75% ethanol solution and then dissolved in nuclease-free water. The concentration and mass of in vitro transcribed mRNA were measured on a NanoDrop 2000C spectrophotometer (Thermo Fisher Scientific, Cat#ND-2000c). Samples of denatured IVT mRNA were analyzed by electrophoresis on an agarose gel containing 0.005% (v/v) GelRed ^™ nucleic acid gel staining agent (Masek T et al., (2005) AnalBiochem 336:46-50). Aliquots of the mRNA sample were stored in silicated tubes at -20°C. All mRNA was purified by cellulose as described (Baiersdorfer M. et al. (2019) Mol Ther Nucleic Acids 15(15):26-35).

Mouse EPO-specific enzyme-linked immunosorbent assay (ELISA)

To quantify mouse EPO levels, plasma samples were collected at specified time points from mice injected with a complex of IVT mRNA encoding mouse erythropoietin and TransIT mRNA (Mirusbio, Cat#MIR2255) and analyzed using a mouse erythropoietin DuoSet ELISA kit (R&D Systems, Cat#DY959). Flat-bottomed 96-well plates were pre-coated with 2 μg/ml rat anti-mouse EPO capture antibody (100 μl/well) and incubated overnight at room temperature (RT). The plates were washed three times with PBS containing 0.05% Tween-20 and incubated for 2 hours at RT with 1% BSA (bovine serum albumin) (Sigma-Aldrich, Cat#2153) solution to prevent non-specific antibody binding, followed by a second wash. A seven-point standard curve was applied using serially diluted buffer (2-fold) and a high standard at 4000 pg/ml. Add 50 μl of plasma sample and standards diluted in 1% BSA solution to appropriate wells and incubate at RT for 2 hours. After washing the plate, dispense 100 μl of rat biotinylated anti-mouse EPO detection antibody in 1 μg/mL 1% BSA solution into each well and incubate at RT for 2 hours. Wash the plate and then incubate with 100 μl of horseradish peroxidase conjugated streptavidin diluted (1:200) in 1% BSA solution for 20 min at room temperature. After washing, add 100 μl of TMB 2-component microwell peroxidase substrate solution (Medac GmbH, Cat#50-76-11) to each well (100 μl/well). The sample was incubated at room temperature for 5 min, and 2M sulfuric acid (R&D Systems, Cat#DY994) was added to terminate the reaction. The absorbance was measured at 450 nm and 570 nm using an Infinite 200Pro microplate reader (Tecan).

Animal program

All experiments were conducted in accordance with federal animal research policies, using 6–12-week-old female BALB/c mice from Charles River Laboratories (Sandhofer, Germany). To determine mRNA translation in vivo, 1–3 μg of cap1-TEV-mEPO mRNA or cap1-hAg-mEPO mRNA, complexed with TransIT and encoded with the backbone and nucleoside modified with TransIT, was intravenously injected into mice (3 mice/group). Blood was collected at 6, 24, 48, and 72 h post-mRNA injection as described (Kariko Ke et al., (2012) Mol. Ther. 20:948-953; Mahin AJ et al., (2016) Methods Mol Biol 1428:297-306) to avoid the influence of sampling on animal hematological parameters. In summary, blood (18 μL) was collected via tail vein puncture, mixed with 2 μL of 0.2 M EDTA, and centrifuged in a 20 μL Lrummond microcapsule (Sigma-Aldrich). After breaking the capillary, the plasma was recovered, and plasma mEPO levels were measured using the mEPO DuoSet ELISA Development Kit (R&D Systems, Minneapolis, MN, USA) and an Infinite 200 Pro microplate reader (Tecan, Mannedorf, Switzerland). See the EPO-ELISA description for details.

result

This embodiment evaluated the effect of sequence elements in RNA on the expression level and/or duration of the RNA-encoded payload. The first evaluation focused on the effect of self-hybridization sequences in the 3'UTR sequence of the RNA polynucleotide. RNA constructs with or without a self-hybridization sequence termed "Lig3" were generated and intravenously injected into mice. Blood samples were collected from the animals at 6, 24, 48, and 72 h post-mRNA injection, and the expression of the polypeptide EPO encoded by the RNA constructs was evaluated.

As shown in Figure 1, EPO expression from the construct lacking the Lig3 sequence was more robust. At 24 and 48 hours post-drug administration, EPO expression levels in these animals were higher than in the control (RNA with Lig3), and the duration of EPO expression was also longer, with significant amounts detected up to 72 hours post-drug administration. This data confirms that the absence of self-hybridization sequences in the 3'UTR that can hybridize with other RNA elements, such as the 5'UTR or the 3'UTR itself (as shown in Figure 2), is beneficial for enhancing the expression of the RNA-encoded payload.

Next, other sequence elements that may affect the expression of the RNA-encoded payload were evaluated. One of the RNA structural elements required for translation and/or stability is the 5' cap. Different 5' cap structures that can be used for RNA are shown in Figures 3A-3H. It has been previously shown that when the mRNA has a cap0 structure, the IFIT1 (interferon-induced tetratricopeptide 1) protein can bind to the 5' end of the mRNA with very high affinity (PLoSpathogens (2013) 9, e1003663). When the mRNA has a cap1 structure, the affinity of IFIT1 for interacting with the mRNA is reduced, and this allows translation factors such as the elongation factor eIF4E to bind preferentially. Subsequently, it has also been reported that the first four transcribed nucleotides of the 5' end of the mRNA are located in a small grove of IFIT1 (PNAS (2017) 114(11): E2106-E2115). Based on these observations, we hypothesize that the presence of the Cap1 structure on RNA and the nature of the 5' cap proximal RNA sequence (also referred to as the "cap proximal sequence" in this paper) can affect the expression of the RNA-encoded payload.

To test this hypothesis, RNAs with different residues at positions +3, +4, and +5 of the RNA sequence were generated and tested. Positions +1 and +2 of the tested RNA sequences were A and G, respectively. Animals were intravenously injected with the various RNA constructs shown in Figure 4. Blood was collected from the animals at 6, 24, 48, and 72 h post-injection to evaluate the expression of the polypeptide EPO encoded by the RNA constructs.

EPO expression levels at 6 and 24 hours post-RNA administration were comparable in animals administered various constructs. At 48 hours post-administration, some constructs resulted in better in vivo EPO expression compared to others (comparing AGAAU and AGACA at 48 hours). At 72 hours, the differences in in vivo EPO expression among animals administered different construct doses were even more pronounced, with the AGACA construct showing more than a 10-fold reduction in EPO expression compared to several constructs, including AGAAU, AGAAC, AGCAA, and AGCAC.

This data confirms that the properties of the 5' cap proximal RNA sequence have a significant impact on the in vivo expression level and/or duration of the RNA-encoded payload. In some embodiments, this data indicates that RNA constructs with an optimized cap proximal RNA sequence have increased in vivo payload expression and/or duration of expression compared to comparatives, such as RNA sequences without an optimized cap proximal sequence, allowing for administration of lower doses of said RNA or compositions containing said RNA to organisms.

Example 2: Exemplary Coronavirus Vaccine Construct

This embodiment describes certain coronavirus vaccine RNAs. The example constructs include sequences encoding at least one epitope of the coronavirus spike protein, as well as various other structural elements and/or features. Exemplary RNAs, including, for example, the cap1 structure and proximal cap sequence described herein, are recorded as expressing well and exhibiting strong immunogenicity.

Table 2: Exemplary sequences used in this embodiment.

Primary pharmacodynamic studies were conducted in BALB/c mice to test the immunogenicity of the candidate vaccines shown in the table below.

Table 3: Candidate Vaccines

vaccine mRNA type Antigens encoded by vaccines BNT162a1 uRNA The RBD (receptor-binding domain) of the SARS-CoV-2 spike protein (S protein). BNT162b1 modRNA The RBD (receptor-binding domain) of the SARS-CoV-2 spike protein (S protein). BNT162b2 modRNA Modifications of the SARS-CoV-2 spike protein (S protein) BNT162c1 saRNA The RBD (receptor-binding domain) of the SARS-CoV-2 spike protein (S protein).

In this study, eight female BALB/c mice in each of four groups were immunized once with three different doses of animal test material or with buffer (control group; see Table 4). While the clinical trial material was diluted in saline, the animal test material was diluted in PBS containing 300 mM sucrose. This was because it was the material's own storage buffer, ensuring the test items were representative of the vaccine to be used in the planned clinical trial. Immunization was performed using a 20 μL dose volume (IM).

Table 4: Research Design

Group number Animal number vaccine dosage World Immunization Day Dose volume [μL]/route Blood collection day End of life cycle 1 8 buffer solution 0 20/IM 7,14,21 28 2 8 Low 0 20/IM 7,14,21 28 3 8 middle 0 20/IM 7,14,21 28 4 8 high 0 20/IM 7,14,21 28

Blood samples were collected from immunized animals on days 7, 14, 21, and 28, and antibody immune responses were analyzed by ELISA and pseudovirus-based neutralization assay (pVNT).

The SARS-CoV-2-S-specific antibody response against the recombinant S1 subunit or RBD was detected by ELISA. In short, high-protein-binding 96-well plates (MaxiSorp ELISA plate, VWR International GmbH, Cat. No. 7341284) were coated overnight at 4°C with 100 μL of coating buffer (50 mM sodium carbonate-sodium bicarbonate buffer, pH 9.6) per well containing 100 ng of recombinant S1 subunit (Sino Biological Inc., Cat. No. 40591-V08H) or RBD (Sino Biological Inc., Cat. No. 40592-V02H). The plates were washed three times with 300 μL/well of 1x phosphate-buffered saline (PBS, VWR International GmbH, Cat. No. 0780-10L) supplemented with 0.01% Tween 20 (Carl Roth GmbH & Co. KG, Cat. No. 9127.1), and blocked for 1 hour at 37°C on a microplate shaker with 250 μL/well of 1x casein blocking buffer (Sigma-Aldrich GmbH, Cat. No. B6429-500 ml). The plates were then washed three times again with 300 μL/well of 1x PBS supplemented with 0.01% Tween 20, and incubated for 1 hour at 37°C on a microplate shaker with mouse serum samples diluted in 1x casein blocking buffer. The plates were washed three times with 300 μL/well of 1x PBS supplemented with 0.01% Tween 20, followed by incubation at 37°C for 45 min on a microplate shaker with peroxidase-conjugated goat anti-mouse secondary antibody (Jackson Immuno Research Ltd., Cat. No. 115-036-071; diluted 1:7500 in 1x casein blocking buffer). The plates were then washed three times with 300 μL/well of 1x PBS supplemented with 0.01% Tween 20, and 100 μL/well of TMB substrate (Biotrend Chemiekalien GmbH, Cat. No. 4380A) was added. The plates were incubated at room temperature for 8 min, and the reaction was terminated by adding 100 μL of 25% sulfuric acid (VWR International GmbH, Cat. No. 1007161000). The plate was read on an ELISA reader, and the recorded absorbance at 450 nm was corrected by subtracting the reference absorbance at 620 nm.

Functional antibody responses to candidate vaccines were detected using pVNT. pVNT used a replication-deficient vesicular stomatitis virus (VSV) lacking the genetic information for the VSV envelope glycoprotein G, but containing an open reading frame (ORF) of green fluorescent protein (GFP). VSV/SARS-CoV-2 pseudoviruses were generated according to a published protocol (Hoffmann et al., Cell, 2020; PMID32142651). The pseudoviruses contained the SARS-CoV-2S protein, which mediates cell entry. Therefore, the pseudoviruses could be inactivated by neutralizing antibodies binding to SARS-CoV-2S. This inactivation could be analyzed using in vitro methods.

In summary, ⁴ x 10⁴ Vero 76 cells (CRL-1587 ^™ ) were seeded per well in a 96-well plate (Greiner Bio-One GmbH, Cat. No. 655160) supplemented with 150 μL/well of DMEM (Thermo Fisher Scientific, Cat. No. 61965059) containing 10% fetal bovine serum (FBS, Sigma-Aldrich GmbH, Cat. No. F7524). Cells were incubated at 37°C and 7.5% CO₂ for 4–6 hours. _{Simultaneously} , mouse serum samples were diluted 1:6 to 1:768 in DMEM/10% FBS using a 2-fold dilution procedure. The diluted serum samples were combined with an equal volume of titrated and pre-diluted VSV/SARS CoV-2 pseudovirus supernatant, resulting in serum dilutions from 1:12 to 1:1536. The pseudovirus/serum dilution mixture was incubated at 750 rpm for 5 min at RT on a microplate shaker, followed by another 5 min at RT without shaking. 50 μL/well of the pseudovirus/serum dilution mixture was added to each well of inoculated Vero-76 cells, with each well containing a pseudovirus volume equivalent to 200 infectious units (IU). Each serum dilution was tested in replicate wells. Cells were incubated at 37°C and 7.5% _CO2 for 16–24 h. Vero 76 cells incubated with pseudovirus in the absence of mouse serum served as a positive control. Vero 76 cells not incubated with pseudovirus served as a negative control. After incubation, the cell culture plates were removed from the incubator and placed in an IncuCyte live cell analysis system (Essen Bioscience), and incubated for 30 min prior to analysis. Bright-field scanning and GFP fluorescence were performed using a 4× objective. To calculate the neutralizing titer, the number of GFP-positive cells infected per well was compared to the pseudovirus positive control. Multiplying the mean of the pseudovirus positive control by 0.5 represents a pseudovirus neutralization rate of 50% (pVN50). Serum samples with mean values below this cutoff value showed >50% virus neutralization activity.

Immunogenicity study of BNT162a1 (RBL063.3)

To investigate the efficacy of the LNP-formulated uRNA vaccine encoding BNT162a1, BALB/c mice were immunized once with IM as described in Table 3. The immunogenicity of the RNA vaccine was studied by monitoring antibody immune responses.

ELISA data at 7, 14, 21 and 28 days after the first immunization showed early, dose-dependent immune activation against the S1 protein and receptor-binding domain (Figure 5).

Immunogenicity study of BNT162b1 (RBP020.3)

To investigate the efficacy of the LNP-formulated modRNA vaccine encoding BNT162b1, BALB/c mice were immunized once with IM as described in Table 3. The immunogenicity of the RNA vaccine was studied by monitoring antibody immune responses.

ELISA data at 7, 14, 21, and 28 days post-immunization showed early, dose-dependent immune activation against the S1 protein and receptor-binding domain (Figure 6). Serum obtained at 14, 21, and 28 days post-immunization showed high neutralization of SARS-CoV-2 pseudoviruses, particularly from sera from mice immunized with 1 or 5 μg BNT162b1, and were strongly correlated with an increase in IgG antibody titers (Figure 7).

Immunogenicity study of BNT162c1 (RBS004.3)

To investigate the efficacy of the LNP-formulated saRNA vaccine encoding BNT162c1, BALB/c mice were immunized once with IM as described in Table 3. The immunogenicity of the RNA vaccine was studied by monitoring antibody immune responses.

ELISA data at 7, 14, and 21 days post-immunization showed early, dose-dependent immune activation against the S1 protein and receptor-binding domain (Figure 8). Serum samples obtained at 14 and 21 days post-immunization showed dose-dependent neutralizing activity against SARS-CoV-2 pseudoviruses (Figure 9).

Immunogenicity study of LNP-prepared uRNA encoding viral S protein-V8 (SEQ ID NO: 7,8) (RBL063.1) Inquiry

To investigate the efficacy of the LNP-formulated uRNA vaccine encoding viral S protein-V8 (RBL063.1), BALB/c mice were immunized once with IM as described in Table 3. The immunogenicity of the RNA vaccine was studied by monitoring antibody immune responses.

ELISA data were available at 7, 14, 21, and 28 days post-immunization, showing early, dose-dependent immune activation against the S1 protein and receptor-binding domain (Figure 10). Serum samples obtained at 14, 21, and 28 days post-immunization showed dose-dependent neutralizing activity against SARS-CoV-2 pseudoviruses (Figure 11).

Immunogenicity study of BNT162b2 (RBP020.1)

To investigate the efficacy of the vaccine BNT162b2 (RBP020.1), the immunogenicity of the construct was studied. For this purpose, a dose titration study was initiated in BALB/c mice, during which the immune response was analyzed, with a focus on antibody-mediated immune responses.

ELISA data were available at 7, 14, and 21 days post-immunization, showing early, dose-dependent immune activation against the S1 protein and receptor-binding domain (Figure 12). Serum samples obtained at 14 and 21 days post-immunization showed dose-dependent neutralizing activity against SARS-CoV-2 pseudoviruses (Figure 13).

Immunogenicity of LNP-prepared saRNA encoding viral S protein-V9 (SEQ ID NO: 7,9) (RBS004.2) Research

To investigate the efficacy of the LNP-formulated saRNA vaccine encoding V9, BALB/c mice were immunized once with IM as described in Table 3. The immunogenicity of the RNA vaccine was studied by monitoring antibody immune responses.

ELISA data were available at 7, 14, and 21 days post-immunization, showing early, dose-dependent immune activation against the S1 protein and receptor-binding domain (Figure 14). Serum samples obtained at 14 and 21 days post-immunization showed dose-dependent neutralizing activity against SARS-CoV-2 pseudoviruses (Figure 15).

The data above confirm in vivo immune responses against the full-length S protein ("V8"/"V9") with the trimeric domain ("V5") and mutations in all tested platforms (including vaccines BNT162a1, BNT162b1, BNT162b2, and BNT162c1). Antibody immune responses were observed at a very early time point (i.e., 7 days post-immunization) by ELISA. Importantly, the induced antibodies were able to effectively neutralize SARS-CoV-2 pseudovirus infection in vitro. Furthermore, the use of extremely low immunization doses (0.2 μg/mouse) to induce antibody responses on both the modRNA platform (BNT162b1, BNT162b2) and the saRNA platform (BNT162c1) demonstrates the high potency of the candidate vaccines.

In mice, BNT162b2 induced higher antigen-specific titers compared to BNT162b1, which is encoded by the same RNA platform. As expected, immunogenicity to antigens varies across different RNA platforms in mice. In mice, the most immunogenic platform based on antigen-specific antibody induction was modRNA, followed by saRNA. The uRNA platform induced the lowest antigen-specific antibody titers.

Example 3: LNP formulation

An exemplary LNP delivery system has been developed to efficiently and safely deliver therapeutic nucleic acids into the cytosol of various cell types after local administration in vivo. Early formulation work was conducted using several promising LNP formulations and alternative RNAs encoding luciferase. The aim of the experiments was to correlate the effects of different ionizable cationic lipids on the efficacy of LNPs in delivering RNA in vivo. Formulations were compared in terms of RNA encapsulation efficiency, apparent pKa, LNP size, and polydispersity.

Among the screened cationic lipids, ALC-0315 exhibited suitable physical characteristics in terms of particle size, homogeneity, and RNA encapsulation efficiency.

Building on this, the ALC-0315/DSPC/CHOL/ALC-0159 prototype was submitted for in vivo screening. The results shown in Figure 16 summarize the in vivo testing using two independent experimental batches of luciferase (Luc) RNA. The results confirmed improved potency of the ALC-0315 prototype compared to the internal benchmark (ALC-0218). Based on these studies, ALC-0315 was identified as a highly efficient cationic lipid, and its application in further product development studies is proposed.

The formulation screening procedure described above involved intravenous administration, resulting in primary delivery to the liver. The mechanism by which LNPs are taken up into hepatocytes is through binding to endogenous apolipoproteins, followed by receptor-mediated endocytosis, such as via low-density lipoprotein receptors. To investigate whether intramuscular administration involves the same mechanism, LNPs containing Luc RNA and ALC-0315 were administered intravenously (0.3 mg/kg) and intramuscularly (0.2 mg/kg) to ApoE knockout mice in the presence or absence of recombinant human ApoE3. As a control, wild-type C57Bl/6 mice were also treated via different routes of administration. Before administration, RNA-LNPs and recombinant human ApoE3 (1 mg of encapsulated mRNA and 1 mg of ApoE3) were pre-incubated at room temperature (RT) for 1 hour. Luc expression was monitored at 4, 24, 72, and 96 hours post-administration (Figure 17).

When administered intravenously to mice, Luc expression was detected in wild-type C57Bl/6 mice. In ApoE knockout mice, Luc expression was significantly reduced, but when pre-incubated with exogenous ApoE, Luc expression recovered to a level similar to that in wild-type mice (Figure 18).

In vivo Luc expression experiments using a mouse model showed that, in the case of intramuscular administration, the absorption of RNA-LNP involves a similar mechanism to intravenous administration, and this applies not only to hepatocytes but also to local cells at the administration site.

Ultimately, in vivo experiments following intramuscular administration of ALC-0315/DSPC/CHOL/ALC-0159 demonstrated minimal problems (drainage) in terms of biodistribution, immunogenicity (vaccine activity), and tolerability.

Equivalent

Those skilled in the art will recognize or be able to determine multiple equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. It should be understood that the invention encompasses all variations, combinations, and arrangements, wherein one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims are incorporated into another claim referencing the same basic claim (or any other related claim) unless otherwise stated, or unless obvious to those skilled in the art to create a contradiction or inconsistency. Furthermore, it should be understood that any embodiment or aspect of the invention may be expressly excluded from the claims, regardless of whether the specific exclusion is mentioned in the specification. The scope of the invention is not intended to be limited to the foregoing description, but rather as shown in the appended claims.

Claims (16)

1. A composition or pharmaceutical product comprising an RNA polynucleotide, said RNA polynucleotide comprising: (a) A 5' cap containing the Cap1 structure; (b) The proximal cap sequence containing the +1, +2, +3, +4 and +5 positions of the RNA polynucleotide; (c) The 5’UTR sequence containing the nucleotide sequence of SEQ ID NO:27; (d) Encode the sequence of payloads; (e) The 3’UTR sequence containing the nucleotide sequence of SEQ ID NO:13; and (f) A polyA sequence containing the nucleotide sequence of SEQ ID NO:14; in (i) The Cap1 structure comprises m7(3'OMeG)(5')ppp(5')( _2'OMeA1 ) _pG2 , where _A1 is the +1 position of the RNA polynucleotide and _G2 is the +2 position of the RNA polynucleotide; and (ii) The cap proximal sequence comprises _A1 and _G2 of the Cap1 structure, and comprises the following sequence: _A3A4U5 (SEQ ID NO: ₅ ) at the +3, +4 and +5 positions of the RNA polynucleotide _, respectively. 2. The composition or pharmaceutical product of claim 1, wherein the RNA polynucleotide comprises a 5' cap, a cap proximal sequence, a 5' UTR, a sequence encoding the payload, a 3' UTR, and a polyA sequence in the 5' to 3' direction. 3. The composition or pharmaceutical product of claim 1, wherein the RNA polynucleotide comprises a modified nucleoside instead of uridine. 4. The composition or pharmaceutical product of claim 3, wherein the modified nucleoside is or contains N1-methyl-pseudouridine (m1ψ). 5. The composition or pharmaceutical article of claim 1, wherein the sequence encoding the payload is or comprises the following sequences: a sequence encoding a protein substitute polypeptide; a sequence encoding an antibody substance; a sequence encoding a cytokine; a sequence encoding an antigen polypeptide; a sequence encoding a gene editing component; a sequence encoding a regenerative medicine component; or a combination thereof. 6. The composition or pharmaceutical article of claim 5, wherein the sequence encoding the payload is or comprises a sequence encoding an antigenic polypeptide. 7. The composition or pharmaceutical article of claim 6, wherein the antigenic polypeptide comprises a plurality of different epitopes from one or more antigens. 8. The composition or pharmaceutical product of claim 6, wherein the antigenic polypeptide comprises: viral antigenic polypeptide, bacterial antigenic polypeptide, fungal antigenic polypeptide, parasitic antigenic polypeptide, antigenic polypeptide from an infectious source, or antigenic polypeptide from a pathogen. 9. The composition or pharmaceutical product of claim 8, wherein the RNA polynucleotide is or comprises mRNA. 10. A pharmaceutical composition comprising an RNA polynucleotide formulated in lipid nanoparticles (LNPs), said lipid nanoparticles comprising cationicly ionizable lipids, neutral lipids, steroids, and polymerically conjugated lipids, said RNA polynucleotide comprising: (a) A 5' cap containing the Cap1 structure; (b) The proximal cap sequence containing the +1, +2, +3, +4 and +5 positions of the RNA polynucleotide; (c) The 5’UTR sequence containing the nucleotide sequence of SEQ ID NO:27; (d) Encode the sequence of payloads; (e) The 3’UTR sequence containing the nucleotide sequence of SEQ ID NO:13; and (f) A polyA sequence containing the nucleotide sequence of SEQ ID NO:14; in (i) The Cap1 structure comprises m7(3'OMeG)(5')ppp(5')( _2'OMeA1 ) _pG2 , where _A1 is the +1 position of the RNA polynucleotide and _G2 is the +2 position of the RNA polynucleotide; and (ii) The cap proximal sequence comprises _A1 and _G2 of the Cap1 structure, and comprises the following sequence: _A3A4U5 (SEQ ID NO: ₅ ) at the +3, +4 and +5 positions of the RNA polynucleotide _, respectively. 11. The pharmaceutical composition of claim 10, wherein The concentration of cationic ionizable lipids in LNP is in the range of approximately 40–approximately 55 mol%. The concentration of steroids in LNP is in the range of approximately 30 to approximately 50 mol%. The concentration of neutral lipids in LNP is in the range of about 5 to about 15 mol%, and The concentration of polymer-conjugated lipids in LNP is in the range of about 1 to about 10 mol%. 12. The pharmaceutical composition of claim 10, wherein it is formulated as a liquid. 13. The pharmaceutical composition of claim 10, wherein it is formulated as a solid. 14. The composition or pharmaceutical product of claim 6, wherein the antigenic polypeptide comprises an antigenic polypeptide derived from an allergen. 15. The composition or pharmaceutical product of claim 6, wherein the antigenic polypeptide comprises a tumor antigenic polypeptide. 16. The composition or pharmaceutical product of claim 6, wherein the antigenic polypeptide comprises a self-antigen polypeptide.