Compositions for administering different doses of RNA

Abstract

The present disclosure relates to techniques for preventing or treating coronavirus infections. In particular, the present disclosure relates to methods and agents for vaccination against coronavirus infections and for inducing effective coronavirus antigen-specific immune responses, such as antibody and / or T cell responses.

Description

[Technical Field]

[0001] The present invention provides technology related to pharmaceutical RNA preparations. In particular, the present invention provides technology for providing pharmaceutical RNA preparations suitable for administering different doses of RNA, pharmaceutical RNA preparations and systems suitable for administering different doses of RNA, and methods for using pharmaceutical RNA preparations to administer different doses of RNA. The present invention also provides specific pharmaceutical RNA preparations, including certain dosage forms (e.g., unit dosage forms, ready-to-use forms, shipping forms, etc.). [Background technology]

[0002] Aside from their well-known ability to encode biologically active proteins, nucleic acids such as DNA and RNA have other remarkable properties that make them attractive therapeutic agents: nucleic acid-based therapeutics are easy to manufacture and relatively inexpensive.

[0003] In general, DNA is more stable than RNA, but poses several potential safety risks, such as the induction of anti-DNA antibodies and integration of the transgene into the host genome.

[0004] The use of RNA to deliver foreign genetic information to target cells offers an attractive alternative to DNA. The advantages of RNA include transient expression and non-transforming properties. RNA does not require nuclear penetration for expression, and furthermore, it cannot be integrated into the host genome, thereby eliminating the risk of carcinogenesis.

[0005] Antigen-encoding mRNA can be used as a vaccine to induce protective immunity against infectious diseases. The COVID-19 pandemic has demonstrated the utility and advantages of RNA technology for vaccination, as the first two COVID-19 vaccines in development to receive emergency use authorization from the FDA were RNA-based. Biotechnology's response to the COVID-19 pandemic has highlighted the speed and flexibility of mRNA vaccines, demonstrating that mRNA therapeutics are a powerful tool for addressing epidemic outbreaks caused by newly emerging viruses. The relative simplicity of the development process and the flexibility of the manufacturing platform could significantly accelerate clinical development. Therefore, mRNA-based vaccine technology has attracted much attention during the COVID-19 pandemic.

[0006] The first approved vaccine was developed by BioNTech in collaboration with Pfizer. The RNA in this vaccine, BNT162b2, encodes a full-length spike protein modified by two proline mutations to stabilize the prefusion conformation. The RNA incorporates 1-methyl-pseudouridine, which suppresses innate immune sensing and increases mRNA translation in vivo, and is formulated in lipid nanoparticles (LNPs). BNT162b2 is administered intramuscularly (IM) to adults in two 30 μg doses, 21 days apart.

[0007] Results from a Phase 2 / 3 clinical trial demonstrated a favorable safety profile and robust neutralizing antibody responses in children aged 5 to 11 years who received two 10-μg doses, administered 21 days apart, a smaller dose than the 30-μg dose used in people 12 years of age and older. The antibody responses in participants given the 10-μg dose were comparable to those recorded in a previous Pfizer-BioNTech study in people aged 16 to 25 years who were immunized with the 30-μg dose. The 10-μg dose was carefully selected as the preferred dose due to its safety, tolerability, and immunogenicity in children aged 5 to 11 years.

[0008] Thus, situations may arise in which an RNA therapeutic, such as an RNA vaccine, will be administered at different doses to different patient groups, e.g., patients of different ages, where a smaller dose may be sufficient for administration to younger patients, such as children, compared to adults.

[0009] In principle, it is possible to generate different RNA formulations, each adapted to administer different doses of RNA, however this complicates the manufacturing process, especially since different mixtures need to be prepared.

[0010] Furthermore, providing an RNA formulation with a high concentration of RNA may require excessive dilution for low doses, while providing an RNA formulation with a low concentration of RNA may require administration of a high volume for high doses and may not provide the necessary stability of the RNA.

[0011] Therefore, it would be desirable to provide pharmaceutical RNA preparations that are suitable for administering RNA at different dosages without the need to prepare different mixtures. Furthermore, such pharmaceutical RNA preparations, their manufacture, and their use by clinicians should meet existing pharmaceutical requirements regarding production, packaging, stability, handling, and administration. Summary of the Invention

[0012] According to the present invention, it has been observed that it is possible to provide a single pharmaceutical RNA preparation having an RNA concentration suitable for administering different doses (doses) of RNA and meeting existing pharmaceutical requirements for production, packaging, stability, handling, and administration (e.g., within a certain RNA concentration range as described herein). In particular, it has been observed that it is possible to provide such a single pharmaceutical RNA preparation that allows for the administration of different doses of RNA while at the same time having a dosage volume in a range suitable for administration of an RNA drug and providing the necessary storage stability of the RNA. In some embodiments, such a single pharmaceutical RNA preparation may have one or more desired stability characteristics, e.g., in some embodiments, exhibiting stability through freeze / thaw cycles. Furthermore, such a single pharmaceutical RNA preparation may meet the requirements for pharmaceutical filling processes and may be provided in a volume that allows for the administration of a suitable number of doses from a single container, such as a vial. The present invention also provides a method for providing a pharmaceutical RNA preparation for administering different doses of RNA, comprising: (i) determining different doses at which the RNA will be administered; (ii) determining a concentration of RNA in the pharmaceutical RNA preparation that allows different doses of RNA to be administered in suitable dose volumes of the pharmaceutical RNA preparation, and optionally in suitable dilutions; (iii) determining a suitable formulation of the pharmaceutical RNA preparation to ensure the desired storage stability of the RNA in the pharmaceutical RNA preparation at the determined concentration.

[0013] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of two, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than a factor of two.

[0014] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of two, with a preferred dose volume of 100-400 μl.

[0015] In some embodiments, the different administrations include at least two administrations.

[0016] In some embodiments, the different administrations include at least three administrations.

[0017] In some embodiments, different doses of RNA are administered to different age groups.

[0018] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than two-fold.

[0019] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than three-fold.

[0020] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 5-fold or more.

[0021] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 10-fold or more.

[0022] In some embodiments, the minimum dose volume for one administration and the maximum dose volume for another administration differ by no more than a factor of two.

[0023] In some embodiments, the minimum dose volume for one administration and the maximum dose volume for another administration differ by no more than 1.5-fold.

[0024] In some embodiments, a suitable dose volume is 100-400 μl.

[0025] In some embodiments, a suitable dose volume is 150-350 μl.

[0026] In some embodiments, a suitable dose volume is 200-300 μl.

[0027] In some embodiments, optionally, a dilution of 1:10 or less of the pharmaceutical RNA preparation is performed for at least one administration.

[0028] In some embodiments, optionally, a 1:8 or less dilution of the pharmaceutical RNA preparation is performed for at least one administration.

[0029] In some embodiments, optionally, a 1:6 or less dilution of the pharmaceutical RNA preparation is performed for at least one administration.

[0030] In some embodiments, at least one administration of RNA is administered without diluting the RNA.

[0031] In some embodiments, at least one administration of RNA in which the RNA is administered without dilution comprises a maximum administration of RNA.

[0032] In some embodiments, at least one administration of RNA is administered by diluting the RNA.

[0033] In some embodiments, at least one dose of RNA in which the RNA is diluted and administered comprises a minimum dose of RNA.

[0034] In some embodiments, pharmaceutical RNA preparations are provided as multi-dose preparations, each multi-dose preparation allowing for multiple administrations of a dose of RNA.

[0035] In some embodiments, the multi-dose preparation has a fill volume that is suitable for pharmaceutical manufacturing.

[0036] In some embodiments, multi-dose preparations allow for the administration of a desired number of doses of RNA.

[0037] In some embodiments, the desired number of administrations of RNA is 5 or more, for example, 5-20, 5-15, or 5-10.

[0038] In some embodiments, the multi-dose preparation allows for arbitrary dilution of the pharmaceutical RNA preparation.

[0039] In some embodiments, the pharmaceutical RNA preparation is provided in a vial.

[0040] In some embodiments, the vials used to administer the different doses of RNA are differently labeled.

[0041] In some embodiments, the different indicia include different colored lids.

[0042] In some embodiments, pharmaceutical RNA preparations for administering different doses of RNA have a uniform RNA concentration.

[0043] In some embodiments, the pharmaceutical RNA preparation is a vaccine.

[0044] In some embodiments, the RNA encodes an amino acid sequence comprising the antigen, an immunogenic variant thereof, or an immunogenic fragment of the antigen or immunogenic variant thereof.

[0045] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0046] In some embodiments, the pharmaceutical RNA preparation is for inducing an immune response against SARS-CoV-2.

[0047] In some embodiments, the pharmaceutical RNA preparation is for vaccination against SARS-CoV-2.

[0048] The present invention also relates to the use of a pharmaceutical RNA preparation for administering different doses of RNA (or a method for administering different doses of RNA from a pharmaceutical RNA preparation), comprising: (i) providing a pharmaceutical RNA preparation; (ii) administering different doses of RNA, wherein the different doses of RNA are administered by administering the same and / or different volumes of an optionally diluted pharmaceutical RNA preparation; The concentration of RNA in the pharmaceutical RNA preparation is selected to allow different doses of RNA to be administered in suitable dose volumes of the pharmaceutical RNA preparation, and optionally in suitable dilutions; Pharmaceutical RNA preparations provide for use in which the pharmaceutical RNA preparations are formulated to ensure the desired stability of the RNA in the pharmaceutical RNA preparation.

[0049] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of two, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than a factor of two.

[0050] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of two, with a preferred dose volume of 100-400 μl.

[0051] In some embodiments, the different administrations include at least two administrations.

[0052] In some embodiments, the different administrations include at least three administrations.

[0053] In some embodiments, different doses of RNA are administered to different age groups.

[0054] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than two-fold.

[0055] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than three-fold.

[0056] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 5-fold or more.

[0057] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 10-fold or more.

[0058] In some embodiments, the minimum dose volume for one administration and the maximum dose volume for another administration differ by no more than a factor of two.

[0059] In some embodiments, the minimum dose volume for one administration and the maximum dose volume for another administration differ by no more than 1.5-fold.

[0060] In some embodiments, a suitable dose volume is 100-400 μl.

[0061] In some embodiments, a suitable dose volume is 150-350 μl.

[0062] In some embodiments, a suitable dose volume is 200-300 μl.

[0063] In some embodiments, optionally, a dilution of 1:10 or less of the pharmaceutical RNA preparation is performed for at least one administration.

[0064] In some embodiments, optionally, a 1:8 or less dilution of the pharmaceutical RNA preparation is performed for at least one administration.

[0065] In some embodiments, optionally, a 1:6 or less dilution of the pharmaceutical RNA preparation is performed for at least one administration.

[0066] In some embodiments, at least one administration of RNA is administered without diluting the RNA.

[0067] In some embodiments, at least one administration of RNA in which the RNA is administered without dilution comprises a maximum administration of RNA.

[0068] In some embodiments, at least one administration of RNA is administered by diluting the RNA.

[0069] In some embodiments, at least one dose of RNA in which the RNA is diluted and administered comprises a minimum dose of RNA.

[0070] In some embodiments, pharmaceutical RNA preparations are provided as multi-dose preparations, each multi-dose preparation allowing for multiple administrations of a dose of RNA.

[0071] In some embodiments, the multi-dose preparation has a fill volume that is suitable for pharmaceutical manufacturing.

[0072] In some embodiments, multi-dose preparations allow for the administration of a desired number of doses of RNA.

[0073] In some embodiments, the desired number of administrations of RNA is 5 or more, for example, 5-20, 5-15, or 5-10.

[0074] In some embodiments, the multi-dose preparation allows for arbitrary dilution of the pharmaceutical RNA preparation.

[0075] In some embodiments, the pharmaceutical RNA preparation is provided in a vial.

[0076] In some embodiments, the vials used to administer the different doses of RNA are differently labeled.

[0077] In some embodiments, the different indicia include different colored lids.

[0078] In some embodiments, pharmaceutical RNA preparations for administering different doses of RNA have a uniform RNA concentration.

[0079] In some embodiments, the pharmaceutical RNA preparation is a vaccine.

[0080] In some embodiments, the RNA encodes an amino acid sequence comprising the antigen, an immunogenic variant thereof, or an immunogenic fragment of the antigen or immunogenic variant thereof.

[0081] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0082] In some embodiments, the pharmaceutical RNA preparation is for inducing an immune response against SARS-CoV-2.

[0083] In some embodiments, the pharmaceutical RNA preparation is for vaccination against SARS-CoV-2.

[0084] In some embodiments, the different doses include doses of about 10 μg and about 30 μg.

[0085] In some embodiments, the different dosages include about 3 μg, about 10 μg, and about 30 μg, hi some embodiments, the different dosages include about 3 μg, about 10 μg, about 15 μg, about 25 μg, about 30 μg, about 50 μg, and about 60 μg.

[0086] In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 30 μg / ml to about 100 μg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 50 μg / ml to about 100 μg / ml.

[0087] In some embodiments, the dose volume is from about 200 μl to about 600 μl.

[0088] In some embodiments, the dose volume is about 200 μl to about 300 μl.

[0089] In some embodiments, the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose.

[0090] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) A second administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0091] In some embodiments, a third dose of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0092] The present invention further provides a pharmaceutical RNA preparation for administering different doses of RNA, wherein the concentration of RNA in the pharmaceutical RNA preparation is selected to allow the different doses of RNA to be administered in suitable dose volumes of the pharmaceutical RNA preparation, and optionally in suitable dilutions, and wherein the pharmaceutical RNA preparation is formulated to ensure a desired stability of the RNA in the pharmaceutical RNA preparation.

[0093] Embodiments of the pharmaceutical RNA preparation are as described above.

[0094] The present invention further provides a pharmaceutical RNA preparation for administering different doses of RNA, wherein the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant, wherein the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and wherein the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose.

[0095] Embodiments of the pharmaceutical RNA preparation are as described above.

[0096] The present invention further provides a system for administering different doses of RNA, the system comprising a plurality of vials for administering the different doses of RNA, each of the plurality of vials optionally containing a different volume of a pharmaceutical RNA preparation, wherein the concentration of RNA in the pharmaceutical RNA preparation and the volume of the pharmaceutical RNA preparation in the vial are selected to allow the different doses of RNA to be administered with a suitable dose volume of the pharmaceutical RNA preparation, and optionally a suitable dilution, and wherein the pharmaceutical RNA preparation is formulated to ensure a desired stability of the RNA in the pharmaceutical RNA preparation.

[0097] The system embodiment is described above.

[0098] The present invention provides a system for administering different doses of RNA, the system comprising a plurality of vials for administering the plurality of different doses of RNA, each vial containing a pharmaceutical RNA preparation; the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose; a first vial of the plurality of vials for administering a first administration of about 30 μg of RNA by administering about 300 μl of the undiluted pharmaceutical RNA preparation; The system further provides a second vial of the plurality of vials for administering a second dose of about 10 μg of RNA by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0099] In some embodiments: the volume of the pharmaceutical RNA preparation in a first vial of the plurality of vials is about 2.25 ml for administering a total of at least about 6 doses; The volume of the pharmaceutical RNA preparation in the second vial of the plurality of vials is about 1.3 ml for administering a total of at least about 10 doses.

[0100] In some embodiments, a third vial of the plurality of vials is for administering a third administration of about 3 μg of RNA by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0101] In some embodiments, the volume of the pharmaceutical RNA preparation in the third vial of the plurality of vials is about 0.4 ml for administering a total of at least about 10 doses.

[0102] In some embodiments, the vials for administering different doses of RNA are labeled differently.

[0103] In some embodiments, the different indicia include different colored lids.

[0104] Additional embodiments of the system are described above.

[0105] In some embodiments, the invention provides certain dosage forms (e.g., forms for administration) that may contain, for example, a particular buffer and / or a particular dose of RNA, and in some embodiments, the invention provides a set of such dosage forms having buffer concentrations and RNA concentrations related to each other by a factor that can be achieved, for example, by dilution.

[0106] In some embodiments, provided pharmaceutical RNA preparations comprise a Tris buffer (e.g., free of sodium chloride and / or potassium chloride ions). In some embodiments, provided pharmaceutical RNA preparations comprise sucrose. In some embodiments, provided pharmaceutical RNA preparations comprise no preservatives.

[0107] In some embodiments, a suitable buffer (e.g., a Tris buffer as described herein) is introduced to a provided pharmaceutical RNA preparation during a filtration step, e.g., a tangential flow filtration (TFF) step, which in some embodiments precedes one or more steps of sterile filtration, sterile filling, capping / crimping, labeling, freezing, etc.

[0108] In some embodiments, a provided pharmaceutical RNA preparation or set thereof is disposed in a container (e.g., a vial), and in some such embodiments, such a container is amenable to entry by a syringe (e.g., by a needle attached to a syringe), e.g., through a membrane. In some embodiments, the container is a glass vial (e.g., Type I borosiligate glass or aluminosilicate glass, e.g., sealed with a rubber stopper, such as a bromobutyl rubber stopper (e.g., a 13 mm bromobutyl stopper), and / or an aluminum seal with a flip-off plastic cap). In some embodiments, the container may be present in a pack containing, e.g., about 5 to about 500 vials, e.g., about 5 to about 200 vials, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 10, 175, 180, 185, 190, 195, 200, or more vials, e.g., about 10 vials, or e.g., about 195 vials. In some embodiments, the container is a syringe (e.g., having a chamber and / or needle attached thereto).

[0109] In some embodiments, provided pharmaceutical RNA preparations are stored in the container in which they are placed, e.g., for a specified period of time and / or under specified conditions, in some embodiments, while maintaining certain stability parameters (e.g., one or more of colloidal stability, polydispersity, zeta potential, RNA integrity, etc.). In some embodiments, such storage conditions may include a temperature within a range of about -20°C to about 2-8°C. In some embodiments, provided pharmaceutical RNA preparations exhibit greater stability after storage for a given time (e.g., 1, 2, 3, or 4 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, or 1 year or more) and / or under a given set of conditions than observed for other comparable (e.g., same concentration and / or temperature, and / or in the same state, e.g., frozen or liquid) preparations of the same RNA / LNP composition in a different buffer (e.g., PBS buffer).

[0110] In some embodiments, provided pharmaceutical RNA preparations are manufactured in commercial batch sizes ranging from about 700 to about 1600 L of final product solution, which in some embodiments may correspond to about 300,000 to 700,000 vials (e.g., at 2.25 mL fill volume).

[0111] In some embodiments, vials (e.g., single-dose or multi-dose vials) containing provided pharmaceutical RNA preparations can be shipped frozen, e.g., in some embodiments, at ultra-low temperature conditions in a thermal container with dry ice, or in some embodiments, at -25°C to -15°C (-13°F to 5°F). In some embodiments, vials containing provided pharmaceutical RNA preparations (e.g., single-dose or multi-dose vials) can be shipped at -90°C to -60°C (-130°F to -76°F) or at refrigerated temperatures, e.g., 2°C to 8°C (35°F to 46°F).

[0112] In some embodiments, the frozen pharmaceutical RNA preparations described herein may be stored at refrigerated temperatures (e.g., 2°C to 8°C), e.g., to thaw, and may be stored for extended periods, e.g., at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, or more. In some embodiments, the thawed pharmaceutical RNA preparations described herein can be stored for up to 10 weeks. Alternatively, in some embodiments, the frozen pharmaceutical RNA preparations described herein can be stored in an ultra-low temperature freezer at -90°C to -60°C (-130°F to -76°F). In some embodiments, it may be desirable to refrain from storing the frozen pharmaceutical RNA preparations described herein at -25°C to -15°C (-13°F to 5°F). Once thawed, the frozen pharmaceutical RNA preparations described herein should not be refrozen. In some embodiments, the frozen pharmaceutical RNA preparations described herein can be thawed at room temperature (e.g., up to 25°C (77°F)), e.g., for 30 minutes in some embodiments.

[0113] In some embodiments, vials containing a provided pharmaceutical RNA preparation (e.g., a single-dose vial or a multi-dose vial) may be shipped at refrigerated temperatures (e.g., 2° C. to 8° C.). In some such embodiments, it may be desirable to store such vials at refrigerated temperatures (e.g., 2° C. to 8° C.) upon their arrival.

[0114] In some embodiments, provided pharmaceutical RNA preparations described herein can be stored at room temperature (e.g., 8°C to 25°C (46°F to 77°F)) for a total of 12 hours before dilution or use. In some embodiments, after dilution or first use, such pharmaceutical RNA preparations described herein can be maintained between refrigerated temperatures and room temperature (e.g., 2°C to 25°C (35°F to 77°F)). In some embodiments, provided pharmaceutical RNA preparations after dilution or first use (e.g., in the case of multi-dose vials) can be maintained at such temperatures for about 12 hours before they are discarded. In some embodiments, diluted pharmaceutical RNA preparations as described herein can be maintained at such temperatures for about 6 hours after first use before they are discarded. In some embodiments, one or more doses of the RNA compositions described herein are administered after storage and / or transportation under conditions as described herein.

[0115] In some embodiments, the RNA described herein is a single-stranded RNA that can be translated into a respective protein upon entry into a cell, e.g., a recipient cell. In addition to a wild-type or codon-optimized sequence encoding an amino acid sequence containing the amino acid sequence of a biologically active peptide or polypeptide, e.g., a pharmaceutically active peptide or polypeptide, such as an antigen sequence (epitope-containing peptide or polypeptide), the RNA may contain one or more structural elements optimized for maximum effectiveness of the RNA with respect to stability and translation efficiency (5' cap, 5' UTR, 3' UTR, poly(A)-tail). In some embodiments, the RNA contains all of these elements. In some embodiments, the RNA is encoded by the beta-S-ARCA(D1)(m2 7,2’ -O GppSpG) or m2 7,3’ -O Gppp(m1 2’-O) ApG can be utilized as a specific capping structure at the 5' end of an RNA drug substance. In some embodiments, the 5'-UTR sequence of human alpha-globin mRNA, optionally with an optimized "Kozak sequence" for increased translation efficiency, may be used as the 5'-UTR sequence. The 3'-UTR sequence may be a combination of two sequence elements (FI elements) derived from the "amino-terminal enhancer of split" (AES) mRNA (termed F) and the mitochondrially encoded 12S ribosomal RNA (termed I), located between the coding sequence and the poly(A) tail, to ensure higher maximum protein levels and long-term mRNA persistence. These were identified through an ex vivo selection process for sequences that confer RNA stability and enhance total protein expression (see WO2017 / 060314, incorporated herein by reference). Alternatively, the 3'-UTR may be two repeated 3'-UTRs of human beta-globin mRNA. Additionally or alternatively, in some embodiments, the poly(A) tail may comprise a length of at least 100 adenosine residues (e.g., at least 110 adenosine residues, at least 120 adenosine residues, 130 adenosine residues, or more). In some embodiments, the poly(A) tail may comprise a length of about 100 to about 150 adenosine residues. In some embodiments, the poly(A) tail may comprise an interrupted poly(A) tail. For example, some such embodiments may use a poly(A) tail measuring 110 nucleotides in length, consisting of 30 adenosine residues, followed by a 10-nucleotide linker sequence (of random nucleotides), and another stretch of 70 adenosine residues. This poly(A) tail sequence is designed to improve RNA stability and translation efficiency.

[0116] The RNA described herein can encode an amino acid sequence that includes the amino acid sequence of a biologically active peptide or polypeptide, e.g., a pharmaceutically active peptide or polypeptide, such as an antigen sequence. The encoded amino acid sequence may include amino acid sequences other than the amino acid sequence of the biologically active peptide or polypeptide. Such other amino acid sequences may support the function or activity of the biologically active peptide or polypeptide. In some embodiments, such other amino acid sequences include amino acid sequences that enhance antigen processing and / or antigen presentation. Alternatively, or additionally, such other amino acid sequences include amino acid sequences that disrupt immune tolerance. [Brief explanation of the drawings]

[0117] [Figure 1A] 1 is a table showing vials containing exemplary compositions as described herein, for use in administering, for example, different doses of RNA to different target populations. [Figure 1B] 1 is a table showing the composition of exemplary stock buffers before and after dilution. In some embodiments, such compositions may be useful for administering different doses of RNA. [Figure 2] 1 is a table showing exemplary stock compositions that can be filled into vials at different volumes (e.g., as described herein) for administering RNA at different doses (e.g., as described herein). [Figure 3] FIG. 1 is a schematic diagram showing various exemplary vaccination regimens. [Figure 4-1]50% neutralization titers of sera collected 7 days after the fourth dose of BNT162b2, Omicron-specific booster (specifically, BA.1-specific booster), or bivalent vaccine are shown. Subjects who previously received two doses of BNT162b2 (30ug) and a third (booster) dose of BNT162b2 (30ug) may receive a second or third (booster) vaccine containing: (i) a 30ug dose of BNT162b2 (encoding the SARS-CoV-2 S protein from the Wuhan strain); (ii) a 60ug dose of BNT162b2; (iii) a 30ug dose of RNA encoding a SARS-CoV-2 S protein with mutations characteristic of the omicron variant (e.g., as described herein (referred to herein as an "omicron-specific RNA vaccine")); (iii) a 60ug dose of RNA encoding a SARS-CoV-2 S protein with mutations characteristic of the omicron variant; (iv) 15ug of BNT162b2 and 15ug of SARS-CoV-2 S protein containing mutations characteristic of the omicron variant. Subjects received either (v) a 30 μg dose of a bivalent vaccine containing 30 μg of BNT162b2 and RNA encoding the SARS-CoV-2 S protein, or (v) a 60 μg dose of a bivalent vaccine containing 30 μg of BNT162b2 and 30 μg of RNA encoding the SARS-CoV-2 S protein containing a mutation characteristic of the Omicron variant. Geometric mean ratios (GMRs) of titers in serum from subjects were collected 7 days after administration of the fourth dose. "b2" refers to serum from subjects who received the Wuhan-specific RNA vaccine as the fourth dose of BNT162b2. "OMI" refers to serum from subjects who received the Omicron-specific fourth dose. "Bivalent" refers to serum from subjects who received a composition containing BNT162b2 and RNA encoding the SARS-CoV-2 S protein containing a mutation characteristic of the Omicron variant (specifically, the BA.1-specific variant) as the fourth dose. Also shown is the fold increase in titer from before administration of the fourth dose to 7 days after administration of the fourth dose (*fold increase). "FFRNT" refers to fluorescent focus reduction neutralization testing. Neutralization data were obtained using the FFRNT assay with viral particles containing the SARS-CoV-2 S protein with mutations characteristic of the variants shown in the figure.LLOQ refers to the lower limit of quantitation, and ULOQ refers to the upper limit of quantitation. (A) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses containing SARS-CoV-2 S proteins with mutations characteristic of the omicron variant (specifically, the BA.1 variant). Sera from subjects previously or currently infected with SARS-CoV-2 are excluded. (B) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses containing SARS-CoV-2 S proteins with mutations characteristic of the omicron variant (specifically, the BA.1 variant) in sera from populations including subjects previously or currently infected with SARS-CoV-2 (as determined, e.g., by antibody testing or PCR assay, respectively). (C) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses containing the Wuhan strain SARS-CoV-2 S protein. Sera from subjects previously or currently infected with SARS-CoV-2 are excluded. (D) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses containing the Wuhan strain of SARS-CoV-2 S protein in sera from populations including individuals previously infected or currently infected with SARS-CoV-2. (E) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses containing the SARS-CoV-2 S protein with mutations characteristic of the delta variant. Sera from subjects previously infected or currently infected with SARS-CoV-2 are excluded. (F) Comparison of neutralizing antibody titers against SARS-CoV-2 pseudoviruses containing the SARS-CoV-2 S protein with mutations characteristic of the delta variant in sera from populations including subjects previously infected or currently infected with SARS-CoV-2.(G) The overall survival of subjects receiving 60ug of BNT162b2, 30ug of RNA encoding a SARS-CoV-2 S protein with mutations characteristic of the Omicron variant (specifically, BA.1 variant, 30ug OMI), 60ug of RNA encoding a SARS-CoV-2 S protein with mutations characteristic of the Omicron variant (specifically, BA.1 variant, 60ug OMI), 30ug of a bivalent vaccine comprising 15ug of BNT162b2 and 15ug of RNA encoding a SARS-CoV-2 S protein with mutations characteristic of the Omicron variant (specifically, BA.1 variant, 30ug Bivalent), or 30ug of BNT162b2 and 30ug of SARS-CoV-2 with mutations characteristic of the Omicron variant, compared to subjects receiving 30ug of BNT162b2 as the fourth dose. Geometric mean rise (GMR) in neutralizing antibodies observed in subjects administered 60ug of a bivalent vaccine (specifically, BA.1 variant, 60ug Bivalent) containing RNA encoding the S protein. Results are shown for both population pools that exclude and include subjects previously or currently infected with SARS-CoV-2. [Figure 4-2] Same as above. [Figure 4-3] Same as above. [Figure 4-4] Same as above. [Figure 4-5] Same as above. [Figure 4-6] Same as above. [Figure 4-7] Same as above. [Figure 5-1]Demonstrating the reactogenicity of certain exemplary RNAs (formulated in LNPs) at a given dose, subjects receiving a 60ug dose of RNA encoding the SARS-CoV-2 S protein are more likely to experience injection site pain and a similar systemic reaction than subjects receiving a 30ug dose of RNA. Subjects were administered 30ug or 60ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain (BNT162b2, corresponding to groups G1 and G2, respectively), 30ug or 60ug of RNA encoding a SARS-CoV-2 S protein with mutations characteristic of the omicron variant (specifically, the BA.1 variant, BNT162b2 OMI, corresponding to groups G3 and G4, respectively), 30ug of a bivalent vaccine comprising 15ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 15ug of RNA encoding a SARS-CoV-2 S protein with mutations characteristic of the omicron variant (specifically, the BA.1 variant, BNT162B2 (15ug) + BNT162b2 OMI (15ug), corresponding to group G5), or 30ug of RNA encoding the SARS-CoV-2 S protein from the Wuhan strain and 30ug of SARS-CoV-2 S protein with mutations characteristic of the omicron variant. Subjects were administered 60 μg of a bivalent vaccine containing the BA.1 variant (specifically, BNT162b2 (30 μg) + BNT162b2 OMI (30 μg), corresponding to group G6) and RNA encoding the SARS-CoV-2 S protein. (A) Local reactions observed within 7 days of injection, including redness, swelling, and pain at the injection site. Increased pain at the injection site was observed in subjects receiving 60 μg of the omicron variant (specifically, the BA.1 variant) or RNA encoding the SARS-CoV-2 S protein containing mutations characteristic of the bivalent vaccine, compared to the other doses tested. (B) Systemic reactions observed within 7 days of injection, including fever, fatigue, headache, chills, vomiting, diarrhea, muscle pain, joint pain, and medication use. Systemic reactions by 7 days were observed to be broadly similar across the different groups. Fatigue tended to be higher after the 60 μg dose compared to the 30 μg dose. [Figure 5-2]Same as above. DETAILED DESCRIPTION OF THE INVENTION

[0118] The table below provides a list of the specific sequences referenced herein. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9] [Table 1-10] [Table 1-11] [Table 1-12] [Table 1-13] [Table 1-14] [Table 1-15]

[0119] Variant-specific vaccines In some embodiments, the RNA disclosed herein encodes an S protein that includes one or more mutations that are characteristic of a SARS-CoV-2 variant. In some embodiments, the RNA disclosed herein encodes an S protein that includes one or more mutations that are characteristic of a SARS-CoV-2 variant of concern (e.g., a SARS-CoV-2 that is prevalent and / or predicted to be prevalent in a relevant jurisdiction). In some embodiments, the present disclosure refers to a SARS-CoV-2 variant that is prevalent and / or spreading rapidly in a relevant jurisdiction. In some embodiments, such variants may be identified based on publicly available data (e.g., data provided in the GISAID initiative database: https: / / www.gisaid.org and / or data provided by the World Health Organization (WHO) (e.g., provided at https: / / www.who.int / activities / tracking-SARS-CoV-2-variants). In some embodiments, such variants refer to variants disclosed herein.

[0120] In some embodiments, the RNA encodes a SARS-CoV-2 S protein that contains one or more mutations characteristic of an alpha variant. The alpha variant was one of the earliest SARS-CoV-2 variants to emerge and quickly become dominant worldwide. The alpha variant (also known as B.1.1.7, VOC202012 / 01, 501Y.V1, or GRY) was initially detected in the United Kingdom. The alpha variant has numerous mutations, including several mutations in the S gene. It has been shown to be inherently more transmissible, with growth rates estimated to be 40-70% higher than other SARS-CoV-2 strains in multiple countries (Volz et al., 2021, Nature, https: / / doi.org / 10.1038 / s41586-021-03470-x, Washington et al., 2021, Cell https: / / doi.org / 10.1016 / j.cell.2021.03.052).

[0121] In some embodiments, the RNA encodes the SARS-CoV-2 S protein of the beta variant (also known as B.1.351 or GH / 501Y.V2). The beta variant was first detected in South Africa. The beta variant has several mutations in the S gene. Three of these mutations are at sites within the RBD associated with immune evasion: N501Y (shared with alpha), E484K, and K417N.

[0122] In some embodiments, the RNA encodes a SARS-CoV-2 S protein containing one or more mutations characteristic of the delta variant. The delta variant (also known as B.1.617.2 or G / 478K.V1) was first documented in India. The delta variant has several point mutations affecting the spike protein, including P681R (a mutation position shared with alpha and adjacent to the furin cleavage site) and L452R, which is within the RBD and has been linked to increased binding to ACE2 and resistance to neutralizing antibodies. There is also a deletion in the spike protein at positions 156 / 157.

[0123] In some embodiments, the RNA disclosed herein comprises a nucleotide sequence encoding the amino acid sequence of a SARS-CoV-2 S protein containing one or more mutations of the omicron variant (also known as B.1.529). B.1.529 was first detected in South Africa in November 2021. Omicron grows approximately 70 times faster than the delta variant and quickly became the dominant strain of SARS-CoV-2 worldwide. Since its initial detection, several omicron sublineages have emerged. Below are listed the current omicron variants of concern, along with certain characteristic mutations associated with each S protein. The BA.4 and BA.5 S proteins share the same set of characteristic mutations, which is why the table below has a single column for "BA.4 or BA.5" and why, in some embodiments, the present disclosure refers to the "BA.4 / 5" S protein. [Table 2-1] [Table 2-2]

[0124] In some embodiments, the RNA described herein comprises a nucleotide sequence encoding a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of an Omicron variant. In some embodiments, the RNA comprises a nucleotide sequence encoding a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations listed in Table 2. In some such embodiments, the one or more mutations may be derived from two or more variants as listed in Table 2. In some embodiments, the RNA comprises a nucleotide sequence encoding a SARS-CoV-2 S protein that includes each of the mutations identified in Table 2 as being characteristic of a particular Omicron variant (e.g., in some embodiments, the RNA comprises a nucleotide sequence encoding a SARS-CoV-2 S protein that includes each of the mutations listed in Table 2 as being characteristic of an Omicron BA.1, BA.2, BA.2.12.1, BA.4 / 5, BA.2.75, BA.2.75.1, BA.4.6, or XBB variant).

[0125] In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes a subset of mutations listed in Table 2. In some embodiments, the RNA encodes a SARS-CoV-2 S protein that includes the mutations listed in Table 2 that are most common for a particular variant (e.g., mutations detected in at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequences collected to date for a given variant sequenced). Mutation prevalence can be determined, for example, based on published sequences (e.g., sequences collected and made publicly available by GISAID).

[0126] In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.4 / 5 variant. In some embodiments, the one or more mutations characteristic of the BA.4 / 5 variant include T19I, Δ24-26, A27S, Δ69 / 70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.4 / 5 variant, excluding R408S. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.4 / 5 variant, excluding R408S.

[0127] In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75 variant. In some embodiments, one or more mutations characteristic of the BA.2.75 variant include T19I, Δ24-26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y, Y505H D614G, H655Y, N679K, P681H, N764K, Q954H, and N969K. In some embodiments, the RNAs described herein encode a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75 variant, excluding N354D. In some embodiments, the RNAs described herein encode a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75 variant, excluding D796Y. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that contains one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75 variant, excluding D796Y and N354D.

[0128] In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.2.75.2 variant. In some embodiments, the one or more mutations characteristic of the BA.2.75.2 variant are T19I, Δ24-26, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, R346T, N354D, S371F, S373P, S375F, T376 A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, F486S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, and D1199N. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.2.75.2 variant, excluding R346T.

[0129] In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of the BA.4.6 variant. In some embodiments, the one or more mutations characteristic of the BA.4.6 variant include T19I, Δ24-26, A27S, Δ69 / 70, G142D, V213G, G339D, R346T, S371F, S373P, S375F, T376A, D405N, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.4.6 variant, excluding R408S. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.4.6 variant, excluding N658S. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that contains one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the BA.4.6 variant, excluding N658S and R408S.

[0130] In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more mutations characteristic of an Omicron-XBB variant. In some embodiments, the one or more mutations characteristic of an Omicron-XBB variant are T19I, Δ24-26, A27S, V83A, G142D, Δ144, H146Q, Q183E, V213E, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405 N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. In some embodiments, one or more mutations characteristic of an Omicron-XBB variant are T19I, Δ24-26, A27S, V83A, G142D, Δ144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D In some embodiments, the RNAs described herein encode a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of Omicron XBB variants, excluding G252V. In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that contains one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the Omicron XBB variant and excluding Q493R.In some embodiments, the RNA described herein encodes a SARS-CoV-2 S protein that includes one or more (e.g., including 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) mutations characteristic of the Omicron XBB variant, excluding Q493R and G252V. In one embodiment, the vaccine antigen described herein comprises, consists essentially of, or consists of the spike protein (S) of SARS-CoV-2, a variant thereof, or a fragment thereof.

[0131] Nucleotide sequence of RBP020.11 (beta-specific vaccine) The nucleotide sequence is shown with individual sequence elements shown in bold. In addition, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon). Red text indicates point mutations in the nucleotide and amino acid sequences. [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0132] The sequence of RBP020.11 is also shown in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5]

[0133] Nucleotide sequence of RBP020.14 (alpha-specific vaccine) The nucleotide sequence is shown with individual sequence elements shown in bold. In addition, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon). Red text indicates point mutations in both the nucleotide and amino acid sequences. [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0134] The sequence of RBP020.14 is also shown in Table 4. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0135] Nucleotide sequence of RBP020.16 (Delta-specific vaccine) The nucleotide sequence is shown with individual sequence elements shown in bold. In addition, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon). Point mutations in the amino acid and nucleotide sequences are shown in red text. [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0136] The sequence of RBP020.16 is also shown in Table 6. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0137] Nucleotide sequence of RBP020.17 (Omicron BA.1-specific vaccine) The nucleotide sequence is shown with individual sequence elements shown in bold. In addition, the sequence of the translated protein is shown in italics below the coding nucleotide sequence (* = stop codon). Red text indicates point mutations in both the nucleotide and amino acid sequences. [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0138] The sequence of RBP020.17 is also shown in Table 5. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5]

[0139] Detailed Description of Specific Embodiments Although the present disclosure is described in further detail below, it should be understood that the disclosure is not limited to the specific methodology, protocols, and reagents described herein, as these may 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 the present 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.

[0140] The elements of the present disclosure are described in more detail below. While these elements are listed with specific embodiments, it should be understood that they can be combined in any manner and in any number to create additional embodiments. The various described examples and preferred embodiments should not be construed as limiting the disclosure to only the explicitly described embodiments. The description should be understood to support and encompass embodiments combining the explicitly described embodiments with any number of disclosed and / or preferred elements. Furthermore, any permutation and combination of elements in all described applications should be considered to be disclosed by the description of this application unless the context dictates otherwise.

[0141] The practice of the present disclosure will employ, unless otherwise indicated, conventional chemical, biochemical, cell biology, immunological, and recombinant DNA techniques described in the art.

[0142] Throughout this specification and the claims that follow, unless the context otherwise requires, the word "comprises," and variations such as "comprises" and "comprising," are understood to imply the inclusion of a stated feature, element, member, whole, or step or group of features, elements, members, wholes, or steps, but not the exclusion of any other feature, element, member, whole, or step or group of features, elements, members, wholes, or steps. The term "consisting essentially of" limits the scope of a claim or disclosure to the specified features, elements, members, wholes, or steps and to those that do not materially affect the basic and novel feature(s) of the claim or disclosure. The term "consisting of" limits the scope of a claim or disclosure to the specified features, elements, members, wholes, or steps. The term "comprising" encompasses the term "consisting essentially of," which in turn encompasses the term "consisting of." Thus, in each occurrence in this application, the term "comprising" can be replaced with the term "consisting essentially of" or "consisting of." Similarly, in each occurrence in this application, the term "consisting essentially of" may be replaced with the term "consisting of."

[0143] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "an," and "the," and similar referents, unless otherwise indicated herein or clearly contradicted by context, should be construed to cover both the singular and the plural.

[0144] All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context.

[0145] Any and all examples provided herein, or the use of exemplary language (e.g., "etc.") are intended merely to better illustrate the disclosure and do not limit the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0146] As used herein, the term "optional" or "optionally" means that the subsequently described event, circumstance, or condition may or may not occur, and that the description includes cases where the event, circumstance, or condition occurs and cases where it does not occur.

[0147] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components, with or without the other. For example, "X and / or Y" should be interpreted as a specific disclosure of each of (i) X, (ii) Y, and (iii) X and Y, as if each were individually set forth herein.

[0148] In the context of the present disclosure, the term "about" denotes an interval of precision that a person skilled in the art would understand to still ensure the technical effect of the feature in question. This term typically denotes a deviation from the indicated numerical value of ±10%, ±5%, ±4%, ±3%, ±2%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2%, ±0.1%, ±0.05%, and for example, ±0.01%. In some embodiments, "about" denotes a deviation of ±10% from the indicated numerical value. In some embodiments, "about" denotes a deviation of ±5% from the indicated numerical value. In some embodiments, "about" denotes a deviation of ±4% from the indicated numerical value. In some embodiments, "about" denotes a deviation of ±3% from the indicated numerical value. In some embodiments, "about" denotes a deviation of ±2% from the indicated numerical value. In some embodiments, "about" denotes a deviation of ±1% from the indicated numerical value. In some embodiments, "about" indicates a ±0.9% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.8% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.7% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.6% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.5% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.4% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.3% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.2% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.1% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.05% deviation from the indicated numerical value. In some embodiments, "about" indicates a ±0.01% deviation from the indicated numerical value. As will be understood by one of ordinary skill in the art, the particular such deviation for the numerical value of a given technical effect will depend on the nature of the technical effect. For example, natural or biological technical effects may generally have larger such deviations than man-made or engineered technical effects.

[0149] The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and unless otherwise indicated herein, each separate value is incorporated herein as if it were individually recited herein.

[0150] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention.

[0151] Methods for Providing Pharmaceutical RNA Preparations In some embodiments, the present invention provides a method for providing a pharmaceutical RNA preparation for administering different doses of RNA, comprising: (i) determining different doses at which the RNA will be administered; (ii) determining a concentration of RNA in the pharmaceutical RNA preparation that allows different doses of RNA to be administered in suitable dose volumes of the pharmaceutical RNA preparation, and optionally in suitable dilutions; (iii) determining a suitable formulation for the pharmaceutical RNA preparation to ensure the desired storage stability of the RNA in the pharmaceutical RNA preparation at the determined concentration.

[0152] The methods of the present invention aim to provide a single pharmaceutical preparation comprising RNA, optionally formulated in particles such as LNPs, which can be used to administer different doses of RNA.

[0153] In some embodiments, at least some of the different doses at which RNA is administered should be significantly different, e.g., 2-fold or more, 2.25-fold or more, 2.5-fold or more, 2.75-fold or more, 3-fold or more, 3.25-fold or more, 3.5-fold or more, 3.75-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 5.5-fold or more, 6-fold or more, 6.5-fold or more, 7-fold or more, 7.5-fold or more, 8-fold or more, 8.5-fold or more, 9-fold or more, 9.5-fold or more, or 10-fold or more.

[0154] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 2-fold or more, 2.25-fold or more, 2.5-fold or more, 2.75-fold or more, 3-fold or more, 3.25-fold or more, 3.5-fold or more, 3.75-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 5.5-fold or more, 6-fold or more, 6.5-fold or more, 7-fold or more, 7.5-fold or more, 8-fold or more, 8.5-fold or more, 9-fold or more, 9.5-fold or more, or 10-fold or more.

[0155] In some embodiments, the dosage volumes of different doses in which the RNA is administered should not differ significantly, for example, by no more than 2-fold, no more than 1.9-fold, no more than 1.8-fold, no more than 1.7-fold, no more than 1.6-fold, no more than 1.5-fold, no more than 1.4-fold, no more than 1.3-fold, no more than 1.2-fold, or no more than 1.1-fold. In some embodiments, the dosage volumes of different doses in which the RNA is administered do not differ, i.e., the same dosage volume is used for different doses.

[0156] In some embodiments, the minimum dose volume for one administration and the maximum dose volume for another administration differ by no more than 2-fold, no more than 1.9-fold, no more than 1.8-fold, no more than 1.7-fold, no more than 1.6-fold, no more than 1.5-fold, no more than 1.4-fold, no more than 1.3-fold, no more than 1.2-fold, or no more than 1.1-fold.

[0157] In some embodiments, the preferred dose volume for each administration is 100-400 μl. In some embodiments, the preferred dose volume for each administration is 150-350 μl. In some embodiments, the preferred dose volume for each administration is 200-300 μl. In some embodiments, the preferred dose volume for each administration is about 200 μl, about 250 μl, or about 300 μl.

[0158] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by a factor of 2 or more (e.g., 2-20, 2-15, 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, or 2-3), and the minimum dose volume for one dose and the maximum dose volume for another dose differ by a factor of no more than 2. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by a factor of 5 or more (e.g., 5-20, 5-15, 5-10, 5-9, 5-8, 5-7, or 5-6), and the minimum dose volume for one dose and the maximum dose volume for another dose differ by a factor of no more than 2. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 10-fold or more (e.g., 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, or 10-11 fold), and the minimum dose volume for one dose and the maximum dose volume for another dose differ by 2-fold or less (e.g., 1-2 fold).

[0159] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 2-fold or more (e.g., 2-10-fold), and the minimum dose volume for one dose and the maximum dose volume for another dose differ by 1.5-fold or less (e.g., 1.5-1-fold). In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 5-fold or more (e.g., 5-10-fold), and the minimum dose volume for one dose and the maximum dose volume for another dose differ by 1.5-fold or less (e.g., 1-1.5-fold). In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 10-fold or more (e.g., 10-20-fold), and the minimum dose volume for one dose and the maximum dose volume for another dose differ by 1.5-fold or less (e.g., 1-1.5-fold).

[0160] In some embodiments, the minimum RNA dose and the maximum RNA dose differ by 2-fold or more (e.g., 2- to 10-fold), and the preferred administration volume is 100-400 μl. In some embodiments, the minimum RNA dose and the maximum RNA dose differ by 2-fold or more (e.g., 2- to 10-fold), and the preferred administration volume is 200-300 μl. In some embodiments, the minimum RNA dose and the maximum RNA dose differ by 5-fold or more (e.g., 5- to 10-fold), and the preferred administration volume is 100-400 μl. In some embodiments, the minimum RNA dose and the maximum RNA dose differ by 5-fold or more (e.g., 5- to 10-fold), and the preferred administration volume is 200-300 μl. In some embodiments, the minimum RNA dose and the maximum RNA dose differ by 10-fold or more (e.g., 10- to 20-fold), and the preferred administration volume is 100-400 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 10-fold or more (eg, 10-20 fold), and the preferred dose volume is 200-300 μl.

[0161] In some embodiments, the different administrations include at least two administrations, in some embodiments, the different administrations include at least three administrations, in some embodiments, the different administrations include at least four administrations, in some embodiments, the different administrations include at least five administrations.

[0162] In some embodiments, different doses of RNA are administered to different patient populations in need thereof, depending, for example, on their physical condition, medical condition, etc. In some embodiments, different doses of RNA are administered to different age groups. In some embodiments, different doses of RNA are administered to different patient groups with different health conditions. In some embodiments, different doses of RNA are administered to different patient groups with different immune conditions, e.g., immune responses, and immunodeficiencies due to cancer or infectious diseases. In some embodiments, a higher dose is administered to adults, e.g., 12 years and older, compared to children, e.g., 5-11 years old, 2-5 years old, or 6 months to 2 years old. In some embodiments, a higher dose is administered to adults, e.g., 12 years old and older, compared to younger children or infants, e.g., 2-5 years old, 6 months to 2 years old, or under 6 months old. In some embodiments, a higher dose is administered to older children, e.g., 5-11 years old, compared to younger children, e.g., 2-5 years old, or 6 months to 5 years old. In some embodiments, higher doses are administered to children aged 2 to under 5 years compared to, for example, 6 months to under 2 years, or to infants and / or toddlers under 6 months of age. In some embodiments, higher doses are administered to immunocompromised patients compared to immunocompetent patients.

[0163] In some embodiments, low doses of RNA are administered to patient populations that may benefit from or desire such low doses. For example, in some embodiments, low doses of RNA are administered to underweight people. In some embodiments, low doses of RNA are administered to poor or malnourished people. In some embodiments, low doses of RNA are administered to individuals who may not tolerate high doses of RNA well (e.g., individuals who have previously experienced adverse reactions after administration of high or full doses of RNA). In some embodiments, low doses of RNA are administered to immunocompromised patients. In some embodiments, low doses of RNA are administered to populations where the cost and / or distribution of higher doses or full doses may be an issue.

[0164] In some embodiments, the pharmaceutical RNA preparation is diluted for at least one administration. In some embodiments, the at least one administration comprises a minimum administration of RNA. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:10 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:9 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:8 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:7 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:6 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:5 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:4 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:3 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:2 for at least one administration.

[0165] In some embodiments, at least one administration of RNA is administered without dilution of the RNA. In some embodiments, the at least one administration comprises a maximum administration of RNA.

[0166] In some embodiments, the pharmaceutical RNA preparations are provided as multi-dose preparations, each multi-dose preparation allowing for multiple administrations of a dose of RNA. In some embodiments, the multi-dose preparations include at least two different preparations for administering at least two different doses. In some embodiments, the multi-dose preparations include at least three different preparations for administering at least three different doses. In some embodiments, the multi-dose preparations include at least four different preparations for administering at least four different doses. In some embodiments, the multi-dose preparations include at least five different preparations for administering at least five different doses.

[0167] In some embodiments, a multi-dose preparation comprises several units, such as containers, e.g., vials, for the multi-dose preparation. In some embodiments, each of several multi-dose preparations comprises several units, such as containers, e.g., vials, for the multi-dose preparation. In some embodiments, each multi-dose preparation comprises several units, such as containers, e.g., vials, for the multi-dose preparation. In some embodiments, the several units, such as containers, e.g., vials, comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of the units. In some embodiments, the several units, such as containers, e.g., vials, are provided as a kit. In some embodiments, different multi-dose preparations are provided as a kit. In some embodiments, different multi-dose preparations are provided as a kit, and several units, such as containers, e.g., vials, for each multi-dose preparation are provided in the kit. In some embodiments, the several units, such as containers, e.g., vials, each contain the same fill volume of the pharmaceutical RNA preparation.

[0168] In some embodiments, the multi-dose preparation has a fill volume suitable for pharmaceutical RNA manufacturing. In some embodiments, the multi-dose preparation has a fill volume of 0.2 to 2.5 ml. In some embodiments, the lower fill volume limit is 0.3 ml. In some embodiments, the lower fill volume limit is 0.4 ml. In some embodiments, the lower fill volume limit is 0.5 ml. In some embodiments, the upper fill volume limit is 2.4 ml. In some embodiments, the upper fill volume limit is 2.3 ml.

[0169] In some embodiments, multi-dose preparations allow for the administration of a desired number of doses of RNA, e.g., 5 or more doses of RNA, such as 5-20, 5-15, or 5-10 doses (optionally after suitable dilution of the pharmaceutical RNA preparation).

[0170] In some embodiments, multi-dose preparations allow for any dilution of the pharmaceutical RNA preparation, for example, a dilution factor as described herein.

[0171] In some embodiments, for example, pharmaceutical RNA preparations provided as multi-dose preparations are provided in vials.

[0172] In some embodiments, the vials used to administer the different doses of RNA are differently labeled.

[0173] In some embodiments, the different indicia include different colored lids.

[0174] In some embodiments, the vial stopper does not contain natural rubber latex.

[0175] In some embodiments, pharmaceutical RNA preparations for administering different doses of RNA have a uniform RNA concentration. In some embodiments, the RNA concentration is 0.03 mg / ml to 0.5 mg / ml. In some embodiments, the RNA concentration is 0.05 mg / ml to 0.5 mg / ml. In some embodiments, the RNA concentration is 0.06 mg / ml to 0.4 mg / ml. In some embodiments, the RNA concentration is 0.07 mg / ml to 0.3 mg / ml. In some embodiments, the RNA concentration is 0.08 mg / ml to 0.2 mg / ml. In some embodiments, the RNA concentration is 0.09 mg / ml to 0.15 mg / ml. In some embodiments, the RNA concentration is about 0.03 mg / ml to about 0.1 mg / ml. In some embodiments, the RNA concentration is about 0.05 mg / ml to about 0.1 mg / ml. In some embodiments, the RNA concentration is about 0.1 mg / ml.

[0176] In some embodiments, the pharmaceutical RNA preparation is a vaccine.

[0177] In some embodiments, the RNA encodes an amino acid sequence comprising the antigen, an immunogenic variant thereof, or an immunogenic fragment of the antigen or immunogenic variant thereof.

[0178] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof (e.g., a SARS-CoV-2 S protein comprising one or more mutations of a variant of concern disclosed herein), or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant.

[0179] In some embodiments, the pharmaceutical RNA preparation comprises two or more (e.g., three or more, four or more, or five or more) RNA molecules, each molecule comprising a nucleotide sequence encoding a different variant of the SARS-CoV-2 S protein. In some embodiments, the pharmaceutical RNA preparation comprises a first RNA and a second RNA, wherein the first RNA comprises a nucleotide sequence encoding the Wuhan strain SARS-CoV-2 S protein and the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2 S protein of a SARS-CoV-2 variant of concern (e.g., a variant of concern in the relevant jurisdiction at the time of administration). In some embodiments, the pharmaceutical RNA preparation comprises a first RNA and a second RNA, wherein the first RNA comprises a nucleotide sequence encoding the Wuhan strain SARS-CoV-2 S protein and the second RNA comprises a nucleotide sequence encoding a SARS-CoV-2 S protein of an Omicron variant (e.g., an Omicron variant disclosed herein, e.g., the BA.1 and / or BA.4 / 5 variants). In some embodiments, two or more RNAs are formulated in a particle (e.g., an LNP). In some embodiments, two or more RNAs are co-formulated in the same particle (e.g., the same LNP, e.g., by mixing two or more RNAs prior to formulation). In some embodiments, two or more RNAs are formulated in separate particles (e.g., separate LNPs, e.g., by formulating each RNA separately and then mixing). In some embodiments, the pharmaceutical RNA preparation is for inducing an immune response against SARS-CoV-2.

[0180] In some embodiments, the pharmaceutical RNA preparation is for vaccination against SARS-CoV-2.

[0181] Methods of Using Pharmaceutical RNA Preparations In some embodiments, the present invention provides a method for administering different doses of RNA from a pharmaceutical RNA preparation, comprising: (i) providing a pharmaceutical RNA preparation; (ii) administering different doses of RNA, wherein the different doses of RNA are administered by administering the same and / or different volumes of an optionally diluted pharmaceutical RNA preparation; The concentration of RNA in the pharmaceutical RNA preparation is selected to allow different doses of RNA to be administered in suitable dose volumes of the pharmaceutical RNA preparation, and optionally in suitable dilutions; Methods are provided in which pharmaceutical RNA preparations are formulated to ensure the desired stability of the RNA in the pharmaceutical RNA preparation.

[0182] The methods of the invention are directed to administering different doses of RNA from a single pharmaceutical preparation, optionally including RNA formulated in particles such as, for example, LNPs.

[0183] In some embodiments, at least some of the different doses at which RNA is administered should be significantly different, e.g., 2-fold or more, 2.25-fold or more, 2.5-fold or more, 2.75-fold or more, 3-fold or more, 3.25-fold or more, 3.5-fold or more, 3.75-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 5.5-fold or more, 6-fold or more, 6.5-fold or more, 7-fold or more, 7.5-fold or more, 8-fold or more, 8.5-fold or more, 9-fold or more, 9.5-fold or more, or 10-fold or more. In some embodiments, at least some of the different doses at which RNA is administered should differ by about 2 to about 50-fold, about 2 to about 40-fold, about 2 to about 35-fold, about 2 to about 30-fold, about 2 to about 25-fold, about 2 to about 20-fold, about 2 to about 19-fold, about 2 to about 18-fold, about 2 to about 17-fold, about 2 to about 16-fold, about 2 to about 15-fold, about 2 to about 14-fold, about 2 to about 13-fold, about 2 to about 12-fold, about 2 to about 11-fold, about 2 to about 10-fold, about 2 to about 9-fold, about 2 to about 8-fold, about 2 to about 7-fold, about 2 to about 6-fold, about 2 to about 5-fold, about 2 to about 4-fold, or about 2 to about 3-fold. In some embodiments, at least some of the different doses at which RNA is administered should differ by about 2-fold. In some embodiments, at least some of the different doses at which RNA is administered should differ by about 6.75-fold.

[0184] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 2-fold or more, 2.25-fold or more, 2.5-fold or more, 2.75-fold or more, 3-fold or more, 3.25-fold or more, 3.5-fold or more, 3.75-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 5.5-fold or more, 6-fold or more, 6.5-fold or more, 7-fold or more, 7.5-fold or more, 8-fold or more, 8.5-fold or more, 9-fold or more, 9.5-fold or more, or 10-fold or more.

[0185] In some embodiments, the dosage volumes of different doses of RNA administered should not be significantly different, for example, by no more than 2-fold, no more than 1.9-fold, no more than 1.8-fold, no more than 1.7-fold, no more than 1.6-fold, no more than 1.5-fold, no more than 1.4-fold, no more than 1.3-fold, no more than 1.2-fold, or no more than 1.1-fold. In some embodiments, the dosage volumes of different doses of RNA administered should not differ, i.e., the same dosage volume should be used for different doses.

[0186] In some embodiments, the minimum dose volume for one administration and the maximum dose volume for another administration differ by no more than a factor of 2, no more than a factor of 1.9, no more than a factor of 1.8, no more than a factor of 1.7, no more than a factor of 1.6, no more than a factor of 1.5, no more than a factor of 1.4, no more than a factor of 1.3, no more than a factor of 1.2, or no more than a factor of 1.1. In some embodiments, the minimum dose volume for one administration and the maximum dose volume for another administration differ by no more than a factor of 1 to 2, no more than a factor of 1 to 1.9, no more than a factor of 1 to 1.8, no more than a factor of 1 to 1.7, no more than a factor of 1 to 1.6, no more than a factor of 1 to 1.5, no more than a factor of 1 to 1.4, no more than a factor of 1 to 1.3, no more than a factor of 1 to 1.2, or no more than a factor of 1 to 1.1.

[0187] In some embodiments, the preferred dose volume for each administration is 100-400 μl. In some embodiments, the preferred dose volume for each administration is 150-350 μl. In some embodiments, the preferred dose volume for each administration is 200-300 μl. In some embodiments, the preferred dose volume for each administration is about 200 μl, about 250 μl, or about 300 μl.

[0188] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of 2, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than a factor of 2. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of 5, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than a factor of 2. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of 10, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than a factor of 2.

[0189] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than 2-fold, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than 1.5-fold. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than 5-fold, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than 1.5-fold. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than 10-fold, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than 1.5-fold.

[0190] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than two-fold, and the preferred dose volume is 100 to 400 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than two-fold, and the preferred dose volume is 200 to 300 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than five-fold, and the preferred dose volume is 100 to 400 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than five-fold, and the preferred dose volume is 200 to 300 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than ten-fold, and the preferred dose volume is 100 to 400 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than ten-fold, and the preferred dose volume is 200 to 300 μl.

[0191] In some embodiments, the different administrations include at least two administrations, in some embodiments, the different administrations include at least three administrations, in some embodiments, the different administrations include at least four administrations, in some embodiments, the different administrations include at least five administrations.

[0192] In some embodiments, different doses of RNA are administered to different age groups. In some embodiments, different doses of RNA are administered to different patient groups with different health conditions. In some embodiments, different doses of RNA are administered to different patient groups with different immune states, e.g., immune responsiveness, and immune deficiencies due to cancer or infectious diseases. In some embodiments, a higher dose is administered to adults, e.g., 12 years and older, compared to children, e.g., 5-11 years old, 6 months to under 2 years old, or 2-5 years old. In some embodiments, a higher dose is administered to adults, e.g., 12 years and older, compared to younger children or infants, e.g., 2-5 years old, 6 months to under 2 years old, or under 6 months old. In some embodiments, a higher dose is administered to older children, e.g., 5-11 years old, compared to younger children, e.g., 2-5 years old. In some embodiments, a higher dose is administered to children, e.g., 2-5 years old, compared to toddlers and / or infants, e.g., 6 months to under 2 years old, or under 6 months old. In some embodiments, higher doses are administered to immunocompromised patients compared to immunocompetent patients.

[0193] In some embodiments, the pharmaceutical RNA preparation is diluted for at least one administration. In some embodiments, the at least one administration comprises a minimum administration of RNA. In some embodiments, the pharmaceutical RNA preparation is diluted 1:10-fold or less for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted 1:9-fold or less for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted 1:8-fold or less for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted 1:7-fold or less for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted 1:6-fold or less for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted 1:5-fold or less for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted in the range of 1:2 to 1:20-fold for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted in the range of 1:2 to 1:10-fold for at least one administration. In some embodiments, a 1:2 to 1:5 dilution of the pharmaceutical RNA preparation is performed for at least one administration, and in some embodiments, a 1:5 to 1:7 dilution of the pharmaceutical RNA preparation is performed for at least one administration.

[0194] In some embodiments, at least one administration of RNA is administered without dilution of the RNA. In some embodiments, the at least one administration comprises a maximum administration of RNA.

[0195] In some embodiments, the pharmaceutical RNA preparations are provided as multi-dose preparations, each multi-dose preparation allowing for multiple administrations of a dose of RNA. In some embodiments, the multi-dose preparations include at least two different preparations for administering at least two different doses. In some embodiments, the multi-dose preparations include at least three different preparations for administering at least three different doses. In some embodiments, the multi-dose preparations include at least four different preparations for administering at least four different doses. In some embodiments, the multi-dose preparations include at least five different preparations for administering at least five different doses.

[0196] In some embodiments, a multi-dose preparation comprises several units, such as containers, e.g., vials, for the multi-dose preparation. In some embodiments, each of several multi-dose preparations comprises several units, such as containers, e.g., vials, for the multi-dose preparation. In some embodiments, each multi-dose preparation comprises several units, such as containers, e.g., vials, for the multi-dose preparation. In some embodiments, the several units, such as containers, e.g., vials, comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of the units. In some embodiments, the several units, such as containers, e.g., vials, are provided as a kit. In some embodiments, different multi-dose preparations are provided as a kit. In some embodiments, different multi-dose preparations are provided as a kit, and several units, such as containers, e.g., vials, for each multi-dose preparation are provided in the kit. In some embodiments, the several units, such as containers, e.g., vials, each contain the same fill volume of the pharmaceutical RNA preparation.

[0197] In some embodiments, the multi-dose preparation has a fill volume suitable for pharmaceutical RNA manufacturing. In some embodiments, the multi-dose preparation has a fill volume of 0.2 to 2.5 ml. In some embodiments, the lower fill volume limit is 0.3 ml. In some embodiments, the lower fill volume limit is 0.4 ml. In some embodiments, the lower fill volume limit is 0.5 ml. In some embodiments, the upper fill volume limit is 2.4 ml. In some embodiments, the upper fill volume limit is 2.3 ml.

[0198] In some embodiments, multi-dose preparations allow for the administration of a desired number of doses of RNA, e.g., 5 or more doses of RNA, such as 5-20, 5-15, or 5-10 doses (optionally after suitable dilution of the pharmaceutical RNA preparation).

[0199] In some embodiments, multi-dose preparations allow for any dilution of the pharmaceutical RNA preparation, for example, a dilution factor as described herein.

[0200] In some embodiments, for example, pharmaceutical RNA preparations provided as multi-dose preparations are provided in vials.

[0201] In some embodiments, the vials used to administer the different doses of RNA are differently labeled.

[0202] In some embodiments, the different indicia include different colored lids.

[0203] In some embodiments, pharmaceutical RNA preparations for administering different doses of RNA have a uniform RNA concentration. In some embodiments, the RNA concentration is 0.03 mg / ml to 0.5 mg / ml. In some embodiments, the RNA concentration is 0.05 mg / ml to 0.5 mg / ml. In some embodiments, the RNA concentration is 0.06 mg / ml to 0.4 mg / ml. In some embodiments, the RNA concentration is 0.07 mg / ml to 0.3 mg / ml. In some embodiments, the RNA concentration is 0.08 mg / ml to 0.2 mg / ml. In some embodiments, the RNA concentration is 0.09 mg / ml to 0.15 mg / ml. In some embodiments, the RNA concentration is about 0.03 mg / ml to about 0.1 mg / ml. In some embodiments, the RNA concentration is about 0.05 mg / ml to about 0.1 mg / ml. In some embodiments, the RNA concentration is about 0.1 mg / ml.

[0204] In some embodiments, the pharmaceutical RNA preparation is a vaccine.

[0205] In some embodiments, the RNA encodes an amino acid sequence comprising the antigen, an immunogenic variant thereof, or an immunogenic fragment of the antigen or immunogenic variant thereof.

[0206] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0207] In some embodiments, the pharmaceutical RNA preparation is for inducing an immune response against SARS-CoV-2.

[0208] In some embodiments, the pharmaceutical RNA preparation is for vaccination against SARS-CoV-2.

[0209] In some embodiments, the different dosages include about 10 μg and about 30 μg. In some embodiments, the different dosages include about 3 μg and about 10 μg. In some embodiments, the different dosages include about 3 μg and about 30 μg. In some embodiments, the different dosages include about 3 μg, about 10 μg, and about 30 μg.

[0210] In some embodiments, the different doses include about 10 μg and about 30 μg doses, and the pharmaceutical RNA preparation comprises RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof (e.g., a SARS-CoV-2 S protein comprising one or more mutations characteristic of a variant of concern described herein), or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the different doses include about 3 μg and about 10 μg doses, and the pharmaceutical RNA preparation comprises RNA encoding an amino acid sequence comprising a SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the different dosages include about 3 μg and about 30 μg, and the pharmaceutical RNA preparation comprises RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the different dosages include about 3 μg, about 10 μg, and about 30 μg, and the pharmaceutical RNA preparation comprises RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant is RNA as described herein.

[0211] In some embodiments, the SARS-CoV-2 S protein, an immunogenic variant thereof, or RNA encoding an amino acid sequence comprising an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof is formulated as a nanoparticle. In some embodiments, exemplary nanoparticles include lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles. In some embodiments,

[0212] In some embodiments, RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof is formulated as a LNP. In some embodiments, the LNP comprises one or more cationically ionizable lipids, one or more neutral lipids (e.g., in some embodiments, a sterol, such as cholesterol, and / or a phospholipid), and one or more polymer-conjugated lipids. In some embodiments, the formulation comprises ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride, and water.

[0213] In some embodiments, the different dosages include about 10 μg and about 30 μg dosages, and the pharmaceutical RNA preparation comprises BNT162b2. In some embodiments, the different dosages include about 3 μg and about 10 μg dosages, and the pharmaceutical RNA preparation comprises BNT162b2. In some embodiments, the different dosages include about 3 μg and about 30 μg dosages, and the pharmaceutical RNA preparation comprises BNT162b2. In some embodiments, the different dosages include about 3 μg, about 10 μg, and about 30 μg dosages, and the pharmaceutical RNA preparation comprises BNT162b2.

[0214] In some embodiments, BNT162b2 comprises RNA comprising the sequence of SEQ ID NO: 17. In some embodiments, BNT162b2 is formulated as a nanoparticle. In some embodiments, exemplary nanoparticles include lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles. In some embodiments, BNT162b2 is formulated as an LNP. In some embodiments, the lipid nanoparticles comprise one or more cationically ionizable lipids, one or more neutral lipids (e.g., in some embodiments, a sterol, such as cholesterol, and / or a phospholipid), and one or more polymer-conjugated lipids. In some embodiments, the formulation comprises ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride, and water.

[0215] In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 30 μg / ml to about 100 μg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 50 μg / ml to about 100 μg / ml.

[0216] In some embodiments, the dose volume is about 200 μl to about 300 μl.

[0217] In some embodiments, the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer and about 10% sucrose.

[0218] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) A second administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0219] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation; (ii) A second administration of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0220] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) A second administration of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0221] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) a second administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation; (iii) A third administration of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0222] Pharmaceutical RNA preparations In some embodiments, the invention provides pharmaceutical RNA preparations for administering different doses of RNA, wherein the concentration of RNA in the pharmaceutical RNA preparation is selected to allow the different doses of RNA to be administered in suitable dose volumes of the pharmaceutical RNA preparation, and optionally in suitable dilutions, and wherein the pharmaceutical RNA preparation is formulated to ensure a desired stability of the RNA in the pharmaceutical RNA preparation.

[0223] The pharmaceutical RNA preparations described herein are optionally suitable for administering different doses of RNA from a single pharmaceutical preparation, including, for example, RNA formulated in particles such as LNPs.

[0224] In some embodiments, at least some of the different doses at which RNA is administered should be significantly different, e.g., 2-fold or more, 2.25-fold or more, 2.5-fold or more, 2.75-fold or more, 3-fold or more, 3.25-fold or more, 3.5-fold or more, 3.75-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 5.5-fold or more, 6-fold or more, 6.5-fold or more, 7-fold or more, 7.5-fold or more, 8-fold or more, 8.5-fold or more, 9-fold or more, 9.5-fold or more, or 10-fold or more.

[0225] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 2-fold or more, 2.25-fold or more, 2.5-fold or more, 2.75-fold or more, 3-fold or more, 3.25-fold or more, 3.5-fold or more, 3.75-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 5.5-fold or more, 6-fold or more, 6.5-fold or more, 7-fold or more, 7.5-fold or more, 8-fold or more, 8.5-fold or more, 9-fold or more, 9.5-fold or more, or 10-fold or more.

[0226] In some embodiments, the dosage volumes of different doses of RNA administered should not be significantly different, for example, by no more than 2-fold, no more than 1.9-fold, no more than 1.8-fold, no more than 1.7-fold, no more than 1.6-fold, no more than 1.5-fold, no more than 1.4-fold, no more than 1.3-fold, no more than 1.2-fold, or no more than 1.1-fold. In some embodiments, the dosage volumes of different doses of RNA administered should not differ, i.e., the same dosage volume should be used for different doses.

[0227] In some embodiments, the minimum dose volume for one administration and the maximum dose volume for another administration differ by no more than 2-fold, no more than 1.9-fold, no more than 1.8-fold, no more than 1.7-fold, no more than 1.6-fold, no more than 1.5-fold, no more than 1.4-fold, no more than 1.3-fold, no more than 1.2-fold, or no more than 1.1-fold.

[0228] In some embodiments, the preferred dose volume for each administration is 100-400 μl. In some embodiments, the preferred dose volume for each administration is 150-350 μl. In some embodiments, the preferred dose volume for each administration is 200-300 μl. In some embodiments, the preferred dose volume for each administration is about 200 μl, about 250 μl, or about 300 μl.

[0229] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of 2, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than a factor of 2. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of 5, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than a factor of 2. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of 10, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than a factor of 2.

[0230] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than 2-fold, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than 1.5-fold. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than 5-fold, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than 1.5-fold. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than 10-fold, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than 1.5-fold.

[0231] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than two-fold, and the preferred dose volume is 100 to 400 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than two-fold, and the preferred dose volume is 200 to 300 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than five-fold, and the preferred dose volume is 100 to 400 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than five-fold, and the preferred dose volume is 200 to 300 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than ten-fold, and the preferred dose volume is 100 to 400 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than ten-fold, and the preferred dose volume is 200 to 300 μl.

[0232] In some embodiments, the different administrations include at least two administrations, in some embodiments, the different administrations include at least three administrations, in some embodiments, the different administrations include at least four administrations, in some embodiments, the different administrations include at least five administrations.

[0233] In some embodiments, different dosages of RNA are administered to different age groups. In some embodiments, different dosages of RNA are administered to different patient groups with different health conditions. In some embodiments, different dosages of RNA are administered to different patient groups with different immune conditions, e.g., immune responses, and immunodeficiencies due to cancer or infectious diseases. In some embodiments, a higher dosage is administered to adults, e.g., 12 years and older, compared to children, e.g., 5-11 years old, or 2-5 years old. In some embodiments, a higher dosage is administered to adults, e.g., 12 years old and older, compared to younger children or infants, e.g., 2-5 years old, 6 months to 2 years old, or under 6 months old. In some embodiments, a higher dosage is administered to older children, e.g., 5-11 years old, compared to younger children, e.g., 2-5 years old. In some embodiments, a higher dosage is administered to children, e.g., 2-5 years old, compared to toddlers and / or infants, 6 months to 2 years old, or under 6 months old. In some embodiments, a higher dosage is administered to immunocompromised patients compared to immunocompetent patients.

[0234] In some embodiments, the pharmaceutical RNA preparation is diluted for at least one administration. In some embodiments, the at least one administration comprises a minimum administration of RNA. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:10 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:9 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:8 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:7 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:6 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:5 for at least one administration.

[0235] In some embodiments, at least one administration of RNA is administered without dilution of the RNA. In some embodiments, the at least one administration comprises a maximum administration of RNA.

[0236] In some embodiments, the pharmaceutical RNA preparations are provided as multi-dose preparations, each multi-dose preparation allowing for multiple administrations of a dose of RNA. In some embodiments, the multi-dose preparations include at least two different preparations for administering at least two different doses. In some embodiments, the multi-dose preparations include at least three different preparations for administering at least three different doses. In some embodiments, the multi-dose preparations include at least four different preparations for administering at least four different doses. In some embodiments, the multi-dose preparations include at least five different preparations for administering at least five different doses.

[0237] In some embodiments, a multi-dose preparation comprises several units, such as containers, e.g., vials, for the multi-dose preparation. In some embodiments, each of several multi-dose preparations comprises several units, such as containers, e.g., vials, for the multi-dose preparation. In some embodiments, a multi-dose preparation comprises several units, such as containers, e.g., vials, for the multi-dose preparation. In some embodiments, the several units, such as containers, e.g., vials, comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of the units. In some embodiments, the several units, such as containers, e.g., vials, are provided as a kit. In some embodiments, different multi-dose preparations are provided as a kit. In some embodiments, different multi-dose preparations are provided as a kit, and several units, such as containers, e.g., vials, for each multi-dose preparation are provided in the kit. In some embodiments, the several units, such as containers, e.g., vials, each contain the same fill volume of the pharmaceutical RNA preparation.

[0238] In some embodiments, the multi-dose preparation has a fill volume suitable for pharmaceutical RNA manufacturing. In some embodiments, the multi-dose preparation has a fill volume of 0.2 to 2.5 ml. In some embodiments, the lower limit of the fill volume is 0.3 ml. In some embodiments, the lower limit of the fill volume is 0.4 ml. In some embodiments, the lower limit of the fill volume is 0.5 ml. In some embodiments, the upper limit of the fill volume is 2.4 ml. In some embodiments, the upper limit of the fill volume is 2.3 ml.

[0239] In some embodiments, multi-dose preparations allow for the administration of a desired number of doses of RNA, e.g., 5 or more doses of RNA, such as 5-20, 5-15, or 5-10 doses (optionally after suitable dilution of the pharmaceutical RNA preparation).

[0240] In some embodiments, multi-dose preparations allow for any dilution of the pharmaceutical RNA preparation, for example, a dilution factor as described herein.

[0241] In some embodiments, for example, pharmaceutical RNA preparations provided as multi-dose preparations are provided in vials.

[0242] In some embodiments, the vials used to administer the different doses of RNA are differently labeled.

[0243] In some embodiments, the different indicia include different colored lids.

[0244] In some embodiments, pharmaceutical RNA preparations for administering different doses of RNA have a uniform RNA concentration. In some embodiments, the RNA concentration is 0.03 mg / ml to 0.5 mg / ml. In some embodiments, the RNA concentration is 0.05 mg / ml to 0.5 mg / ml. In some embodiments, the RNA concentration is 0.06 mg / ml to 0.4 mg / ml. In some embodiments, the RNA concentration is 0.07 mg / ml to 0.3 mg / ml. In some embodiments, the RNA concentration is 0.08 mg / ml to 0.2 mg / ml. In some embodiments, the RNA concentration is 0.09 mg / ml to 0.15 mg / ml. In some embodiments, the RNA concentration is about 0.03 mg / ml to about 0.1 mg / ml. In some embodiments, the RNA concentration is about 0.05 mg / ml to about 0.1 mg / ml. In some embodiments, the RNA concentration is about 0.1 mg / ml.

[0245] In some embodiments, the pharmaceutical RNA preparation is a vaccine.

[0246] In some embodiments, the RNA encodes an amino acid sequence comprising the antigen, an immunogenic variant thereof, or an immunogenic fragment of the antigen or immunogenic variant thereof.

[0247] In some embodiments, the RNA present in the pharmaceutical RNA preparation is formulated in a nanoparticle. In some embodiments, exemplary nanoparticles include lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles. In some embodiments, the RNA present in the pharmaceutical RNA preparation is formulated in a lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises one or more cationically ionizable lipids, one or more neutral lipids (e.g., in some embodiments, a sterol, such as cholesterol, and / or a phospholipid), and one or more polymer-conjugated lipids.

[0248] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0249] In some embodiments, the pharmaceutical RNA preparation is for inducing an immune response against SARS-CoV-2.

[0250] In some embodiments, the pharmaceutical RNA preparation is for vaccination against SARS-CoV-2.

[0251] In some embodiments, the different dosages include about 10 μg and about 30 μg. In some embodiments, the different dosages include about 3 μg and about 10 μg. In some embodiments, the different dosages include about 3 μg and about 30 μg. In some embodiments, the different dosages include about 3 μg, about 10 μg, and about 30 μg.

[0252] In some embodiments, the different doses include about 10 μg and about 30 μg, and the pharmaceutical RNA preparation comprises RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the different doses include about 3 μg and about 10 μg, and the pharmaceutical RNA preparation comprises RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the different doses include about 3 μg and about 30 μg, and the pharmaceutical RNA preparation comprises RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the different dosages include about 3 μg, about 10 μg, and about 30 μg, and the pharmaceutical RNA preparation comprises RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant is RNA as described herein. In some embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant is formulated as a nanoparticle. In some embodiments, exemplary nanoparticles include lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles.In some embodiments, RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof is formulated as a LNP. In some embodiments, the LNP comprises one or more cationically ionizable lipids, one or more neutral lipids (e.g., in some embodiments, a sterol, such as cholesterol, and / or a phospholipid), and one or more polymer-conjugated lipids. In some embodiments, the formulation comprises ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride, and water.

[0253] In some embodiments, the different doses include about 10 μg and about 30 μg doses, and the pharmaceutical RNA preparation comprises BNT162b2 or a variant thereof, such as, in some embodiments, a variant encoding a SARS-CoV-2 S protein from a variant of an ancestral strain. In some embodiments, the different doses include about 3 μg and about 10 μg doses, and the pharmaceutical RNA preparation comprises BNT162b2 or a variant thereof, such as, in some embodiments, a variant encoding a SARS-CoV-2 S protein from a variant of an ancestral strain. In some embodiments, the different doses include about 3 μg and about 30 μg doses, and the pharmaceutical RNA preparation comprises BNT162b2 or a variant thereof, such as, in some embodiments, a variant encoding a SARS-CoV-2 S protein from a variant of an ancestral strain. In some embodiments, the different dosages include about 3 μg, about 10 μg, and about 30 μg dosages, and the pharmaceutical RNA preparation comprises BNT162b2 or a variant thereof, such as, in some embodiments, a variant encoding a SARS-CoV-2 S protein from an ancestral strain variant. In some embodiments, the BNT162b2 comprises RNA comprising the sequence of SEQ ID NO: 17. In some embodiments, the BNT162b2 encoding a SARS-CoV-2 S protein from an ancestral strain variant comprises RNA comprising a sequence that is at least 85% identical (e.g., at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or more) to SEQ ID NO: 17. In some embodiments, the BNT162b2 is formulated as nanoparticles. In some embodiments, exemplary nanoparticles include lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles. In some embodiments, BNT162b2 is formulated as LNPs.In some embodiments, the LNPs comprise one or more cationically ionizable lipids, one or more neutral lipids (e.g., in some embodiments, a sterol, such as cholesterol, and / or a phospholipid), and one or more polymer-conjugated lipids. In some embodiments, the formulation comprises ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride, and water.

[0254] In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml.

[0255] In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 30 μg / ml to about 100 μg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 50 μg / ml to about 100 μg / ml. In some embodiments, the administration volume is about 200 μl to about 300 μl.

[0256] In some embodiments, the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer and about 10% sucrose.

[0257] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) A second administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0258] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation; (ii) A second administration of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0259] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) A second administration of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0260] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) a second administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation; (iii) A third administration of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0261] In some embodiments, the invention provides a pharmaceutical RNA preparation for administering different doses of RNA, wherein the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant, wherein the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and wherein the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose.

[0262] In some embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises BNT162b2. In some embodiments, BNT162b2 comprises RNA comprising the sequence of SEQ ID NO: 17.

[0263] In some embodiments, RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant is formulated as a nanoparticle. In some embodiments, exemplary nanoparticles include lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles. In some embodiments, RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant is formulated as a LNP. In some embodiments, the LNP comprises one or more cationically ionizable lipids, one or more neutral lipids (e.g., in some embodiments, a sterol, e.g., cholesterol, and / or a phospholipid), and one or more polymer-conjugated lipids. In some embodiments, the formulation comprises ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride, and water.

[0264] In some embodiments, the different dosages include about 10 μg and about 30 μg. In some embodiments, the different dosages include about 3 μg and about 10 μg. In some embodiments, the different dosages include about 3 μg and about 30 μg. In some embodiments, the different dosages include about 3 μg, about 10 μg, and about 30 μg.

[0265] In some embodiments, the dose volume is about 200 μl to about 300 μl.

[0266] In some embodiments, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) A second administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0267] In some embodiments, (i) a first administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation; (ii) A second administration of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0268] In some embodiments, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) A second administration of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0269] In some embodiments, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) a second administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation; (iii) A third administration of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0270] In some embodiments, the present invention provides (a) about 0.1 mg / mL of RNA comprising an open reading frame encoding a polypeptide comprising a SARS-CoV-2 protein or an immunogenic fragment or variant thereof, wherein the RNA is formulated in a lipid nanoparticle comprising a cationic ionizable lipid, a neutral lipid, a steroid, and a polyethylene glycol (PEG)-lipid; (b) about 10 mM Tris buffer; (c) about 300 mM sucrose.

[0271] In some embodiments, the present invention provides (a) about 0.1 mg / mL of RNA comprising an open reading frame encoding a polypeptide comprising a SARS-CoV-2 protein or an immunogenic fragment or variant thereof; (b) ALC-0315 at about 1.43 mg / ml; (c) about 0.18 mg / ml ALC-0159; (d) about 0.31 mg / ml DSPC; (e) cholesterol of about 0.62 mg / ml; (f) sucrose at about 103 mg / ml; (g) tromethamine (Tris base) at about 0.20 mg / ml; (h) about 1.32 mg / ml of Tris(hydroxymethyl)aminomethane hydrochloride (Tris HCl), and A composition is provided that includes: (i) a suitable amount of water.

[0272] In some embodiments, the pH of the composition is pH 7.4.

[0273] In some embodiments, the present invention provides (a) about 0.1 mg / mL of RNA comprising an open reading frame encoding a polypeptide comprising a SARS-CoV-2 protein or an immunogenic fragment or variant thereof, wherein the RNA is formulated in a lipid nanoparticle comprising a cationic ionizable lipid, a neutral lipid, a steroid, and a polyethylene glycol (PEG)-lipid; (b) about 10 mM Tris buffer; (c) about 300 mM sucrose; and providing a sealed multi-dose vial containing a stock composition comprising:

[0274] In some embodiments, the pH of the stock composition is pH 7.4.

[0275] In some embodiments, the RNA and lipid nanoparticles remain stable at refrigerated temperatures for at least 10 weeks.

[0276] In some embodiments, the vial contains about 2.25 mL of the stock composition.

[0277] In some embodiments, a vial containing about 2.25 mL of stock composition contains 6 doses.

[0278] In some embodiments, the present invention provides a tray comprising 10 sealed multi-dose vials, the vials containing approximately 2.25 mL of stock composition.

[0279] In some embodiments, the present invention provides a package comprising 20 such trays.

[0280] In some embodiments, the vial contains about 1.3 mL of the stock composition.

[0281] In some embodiments, a vial containing about 1.3 mL of stock composition contains 10 doses.

[0282] In some embodiments, the present invention provides a tray comprising 10 sealed multi-dose vials, the vials containing approximately 1.3 mL of stock composition.

[0283] In some embodiments, the present invention provides a package comprising 20 such trays.

[0284] In some embodiments, the vial contains about 0.4 mL of the stock composition.

[0285] In some embodiments, a vial containing about 0.4 mL of stock composition contains 10 doses.

[0286] In some embodiments, the present invention provides a tray comprising 10 sealed multi-dose vials, the vials containing approximately 0.4 mL of stock composition.

[0287] In some embodiments, the present invention provides a package comprising 20 such trays.

[0288] In some embodiments, the present invention provides a syringe containing a pharmaceutical agent having an injection volume of about 300 μL, comprising: Pharmaceuticals, (a) about 0.1 mg / mL of RNA comprising an open reading frame encoding a polypeptide comprising a SARS-CoV-2 protein or an immunogenic fragment or variant thereof, wherein the RNA is formulated in a lipid nanoparticle comprising a cationic ionizable lipid, a neutral lipid, a steroid, and a polyethylene glycol (PEG)-lipid; (b) about 10 mM Tris buffer; (c) about 300 mM sucrose.

[0289] In some embodiments, the syringe is a low dead volume syringe.

[0290] In some embodiments, the present invention provides a multi-dose vial containing about 2.6 mL of diluted pharmaceutical agent, comprising: Diluted medicines, (a) about 50 μg / mL of RNA comprising an open reading frame encoding a polypeptide comprising a SARS-CoV-2 protein or an immunogenic fragment or variant thereof, wherein the RNA is formulated in a lipid nanoparticle comprising a cationic ionizable lipid, a neutral lipid, a steroid, and a polyethylene glycol (PEG)-lipid; (b) about 5 mM Tris buffer; (c) about 150 mM sucrose; (d) about 0.45% sodium chloride.

[0291] In some embodiments, the present invention provides a syringe containing a diluted pharmaceutical agent in an injection volume of about 200 uL, comprising: Diluted medicines, (a) about 50 μg / mL of RNA comprising an open reading frame encoding a polypeptide comprising a SARS-CoV-2 protein or an immunogenic fragment or variant thereof, wherein the RNA is formulated in a lipid nanoparticle comprising a cationic ionizable lipid, a neutral lipid, a steroid, and a polyethylene glycol (PEG)-lipid; (b) about 5 mM Tris buffer; (c) about 150 mM sucrose; (d) 0.45% sodium chloride.

[0292] In some embodiments, the syringe is a low dead volume syringe.

[0293] In some embodiments, the present invention provides a multi-dose vial containing about 2.7 mL of diluted pharmaceutical agent, comprising: Diluted medicines, (a) about 14.8 μg / mL of RNA comprising an open reading frame encoding a polypeptide comprising a SARS-CoV-2 protein or an immunogenic fragment or variant thereof, wherein the RNA is formulated in a lipid nanoparticle comprising a cationic ionizable lipid, a neutral lipid, a steroid, and a polyethylene glycol (PEG)-lipid; (b) about 1.5 mM Tris buffer; (c) about 44.4 mM sucrose; (d) 0.77% sodium chloride; and providing a multi-dose vial comprising:

[0294] In some embodiments, the present invention provides a syringe containing a diluted pharmaceutical agent in an injection volume of about 200 uL, comprising: Diluted medicines, (a) approximately 14.8 μg / mL of RNA comprising an open reading frame encoding a polypeptide comprising a SARS-CoV-2 protein or an immunogenic fragment or variant thereof, wherein the RNA is formulated in a lipid nanoparticle comprising a cationic ionizable lipid, a neutral lipid, a steroid, and a polyethylene glycol (PEG)-lipid; (b) about 1.5 mM Tris buffer; (c) about 44.4 mM sucrose; (d) about 0.77% sodium chloride.

[0295] In some embodiments, the syringe is a low dead volume syringe.

[0296] system In some embodiments, the invention provides a system for administering different doses of RNA, the system comprising a plurality of vials for administering the different doses of RNA, each of the plurality of vials optionally containing a different volume of a pharmaceutical RNA preparation, the concentration of RNA in the pharmaceutical RNA preparation and the volume of the pharmaceutical RNA preparation in the vial being selected to allow the different doses of RNA to be administered with a suitable dose volume of the pharmaceutical RNA preparation, and optionally a suitable dilution, and the pharmaceutical RNA preparation is formulated to ensure a desired stability of the RNA in the pharmaceutical RNA preparation.

[0297] The systems described herein are suitable for administering different doses of RNA from a single pharmaceutical preparation, and the systems include multiple vials (including any type of suitable container), each of which contains a pharmaceutical RNA preparation, the RNA optionally formulated in particles such as LNPs. In some embodiments, one or more of the multiple vials contain different volumes of the pharmaceutical RNA preparation, and the volume of the pharmaceutical RNA preparation in the vial can be selected to allow for administration of different doses of RNA with suitable dose volumes, and optionally suitable dilution of the pharmaceutical RNA preparation, and administration of a desired number of doses.

[0298] In some embodiments, at least some of the different doses at which RNA is administered should be significantly different, e.g., 2-fold or more, 2.25-fold or more, 2.5-fold or more, 2.75-fold or more, 3-fold or more, 3.25-fold or more, 3.5-fold or more, 3.75-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 5.5-fold or more, 6-fold or more, 6.5-fold or more, 7-fold or more, 7.5-fold or more, 8-fold or more, 8.5-fold or more, 9-fold or more, 9.5-fold or more, or 10-fold or more.

[0299] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by 2-fold or more, 2.25-fold or more, 2.5-fold or more, 2.75-fold or more, 3-fold or more, 3.25-fold or more, 3.5-fold or more, 3.75-fold or more, 4-fold or more, 4.5-fold or more, 5-fold or more, 5.5-fold or more, 6-fold or more, 6.5-fold or more, 7-fold or more, 7.5-fold or more, 8-fold or more, 8.5-fold or more, 9-fold or more, 9.5-fold or more, or 10-fold or more.

[0300] In some embodiments, the dosage volumes of different doses of RNA administered should not be significantly different, for example, by no more than 2-fold, no more than 1.9-fold, no more than 1.8-fold, no more than 1.7-fold, no more than 1.6-fold, no more than 1.5-fold, no more than 1.4-fold, no more than 1.3-fold, no more than 1.2-fold, or no more than 1.1-fold. In some embodiments, the dosage volumes of different doses of RNA administered should not differ, i.e., the same dosage volume should be used for different doses.

[0301] In some embodiments, the minimum dose volume for one administration and the maximum dose volume for another administration differ by no more than 2-fold, no more than 1.9-fold, no more than 1.8-fold, no more than 1.7-fold, no more than 1.6-fold, no more than 1.5-fold, no more than 1.4-fold, no more than 1.3-fold, no more than 1.2-fold, or no more than 1.1-fold.

[0302] In some embodiments, the preferred dose volume for each administration is 100-400 μl. In some embodiments, the preferred dose volume for each administration is 150-350 μl. In some embodiments, the preferred dose volume for each administration is 200-300 μl. In some embodiments, the preferred dose volume for each administration is about 200 μl, about 250 μl, or about 300 μl.

[0303] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of 2, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than a factor of 2. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of 5, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than a factor of 2. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than a factor of 10, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than a factor of 2.

[0304] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than 2-fold, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than 1.5-fold. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than 5-fold, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than 1.5-fold. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than 10-fold, and the minimum dose volume for one dose and the maximum dose volume for another dose differ by no more than 1.5-fold.

[0305] In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than two-fold, and the preferred dose volume is 100 to 400 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than two-fold, and the preferred dose volume is 200 to 300 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than five-fold, and the preferred dose volume is 100 to 400 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than five-fold, and the preferred dose volume is 200 to 300 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than ten-fold, and the preferred dose volume is 100 to 400 μl. In some embodiments, the minimum dose of RNA and the maximum dose of RNA differ by more than ten-fold, and the preferred dose volume is 200 to 300 μl.

[0306] In some embodiments, the different administrations include at least two administrations, in some embodiments, the different administrations include at least three administrations, in some embodiments, the different administrations include at least four administrations, in some embodiments, the different administrations include at least five administrations.

[0307] In some embodiments, different doses of RNA are administered to different age groups. In some embodiments, different doses of RNA are administered to different patient groups with different health conditions. In some embodiments, different doses of RNA are administered to different patient groups with different immune conditions, e.g., immune responses, and immunodeficiencies due to cancer or infectious diseases. In some embodiments, a higher dose is administered to adults, e.g., 12 years or older, compared to children, e.g., 5-11 years old, or 2-5 years old. In some embodiments, a higher dose is administered to adults, e.g., 12 years old or older, compared to younger children or infants, e.g., 2-5 years old, 6 months to 2 years old, or under 6 months old. In some embodiments, a higher dose is administered to older children, e.g., 5-11 years old, compared to younger children, e.g., 2-5 years old. In some embodiments, a higher dose is administered to children, e.g., 2-5 years old, compared to toddlers and / or infants, 6 months to 2 years old, or under 6 months old. In some embodiments, a higher dose is administered to immunocompromised patients compared to immunocompetent patients.

[0308] In some embodiments, the pharmaceutical RNA preparation is diluted for at least one administration. In some embodiments, the at least one administration comprises a minimum administration of RNA. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:10 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:9 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:8 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:7 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:6 for at least one administration. In some embodiments, the pharmaceutical RNA preparation is diluted no more than 1:5 for at least one administration.

[0309] In some embodiments, at least one administration of RNA is administered without dilution of the RNA. In some embodiments, the at least one administration comprises a maximum administration of RNA.

[0310] In some embodiments, the pharmaceutical RNA preparations are provided as multi-dose preparations, each multi-dose preparation allowing for multiple administrations of a dose of RNA. In some embodiments, the multi-dose preparations include at least two different preparations for administering at least two different doses. In some embodiments, the multi-dose preparations include at least three different preparations for administering at least three different doses. In some embodiments, the multi-dose preparations include at least four different preparations for administering at least four different doses. In some embodiments, the multi-dose preparations include at least five different preparations for administering at least five different doses.

[0311] In some embodiments, a multi-dose preparation comprises several units, such as containers, e.g., vials, for the multi-dose preparation. In some embodiments, each of several multi-dose preparations comprises several units, such as containers, e.g., vials, for the multi-dose preparation. In some embodiments, a multi-dose preparation comprises several units, such as containers, e.g., vials, for the multi-dose preparation. In some embodiments, the several units, such as containers, e.g., vials, comprise two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more of the units. In some embodiments, the several units, such as containers, e.g., vials, are provided as a kit. In some embodiments, different multi-dose preparations are provided as a kit. In some embodiments, different multi-dose preparations are provided as a kit, and several units, such as containers, e.g., vials, for each multi-dose preparation are provided in the kit. In some embodiments, the several units, such as containers, e.g., vials, each contain the same fill volume of the pharmaceutical RNA preparation.

[0312] In some embodiments, the multiple vials or multi-dose preparations have a fill volume suitable for pharmaceutical RNA manufacturing. In some embodiments, the multiple vials or multi-dose preparations have a fill volume of 0.2 to 2.5 ml. In some embodiments, the lower fill volume limit is 0.3 ml. In some embodiments, the lower fill volume limit is 0.4 ml. In some embodiments, the lower fill volume limit is 0.5 ml. In some embodiments, the upper fill volume limit is 2.4 ml. In some embodiments, the upper fill volume limit is 2.3 ml.

[0313] In some embodiments, the vial or multi-dose preparation allows for the administration of a desired number of doses of RNA, e.g., 5 or more doses of RNA, such as 5-20, 5-15, or 5-10 doses (optionally after suitable dilution of the pharmaceutical RNA preparation).

[0314] In some embodiments, the vial or multi-dose preparation allows for any dilution of the pharmaceutical RNA preparation, for example, a dilution factor as described herein.

[0315] In some embodiments, the vials used to administer the different doses of RNA are differently labeled.

[0316] In some embodiments, the different indicia include different colored lids.

[0317] In some embodiments, pharmaceutical RNA preparations for administering different doses of RNA have a uniform RNA concentration. In some embodiments, the RNA concentration is between 0.05 mg / ml and 0.5 mg / ml. In some embodiments, the RNA concentration is between 0.06 mg / ml and 0.4 mg / ml. In some embodiments, the RNA concentration is between 0.07 mg / ml and 0.3 mg / ml. In some embodiments, the RNA concentration is between 0.08 mg / ml and 0.2 mg / ml. In some embodiments, the RNA concentration is between 0.09 mg / ml and 0.15 mg / ml. In some embodiments, the RNA concentration is about 0.1 mg / ml.

[0318] In some embodiments, the pharmaceutical RNA preparation is a vaccine.

[0319] In some embodiments, the RNA encodes an amino acid sequence comprising the antigen, an immunogenic variant thereof, or an immunogenic fragment of the antigen or immunogenic variant thereof.

[0320] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

[0321] In some embodiments, the pharmaceutical RNA preparation is for inducing an immune response against SARS-CoV-2.

[0322] In some embodiments, the pharmaceutical RNA preparation is for vaccination against SARS-CoV-2.

[0323] In some embodiments, the different dosages include about 10 μg and about 30 μg. In some embodiments, the different dosages include about 3 μg and about 10 μg. In some embodiments, the different dosages include about 3 μg and about 30 μg. In some embodiments, the different dosages include about 3 μg, about 10 μg, and about 30 μg.

[0324] In some embodiments, the different doses include about 10 μg and about 30 μg, and the pharmaceutical RNA preparation comprises RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the different doses include about 3 μg and about 10 μg, and the pharmaceutical RNA preparation comprises RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the different doses include about 3 μg and about 30 μg, and the pharmaceutical RNA preparation comprises RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the different dosages include about 3 μg, about 10 μg, and about 30 μg, and the pharmaceutical RNA preparation comprises RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant. In some embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant is RNA as described herein. In some embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant is formulated as a nanoparticle. In some embodiments, exemplary nanoparticles include lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles.In some embodiments, RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof is formulated as a LNP. In some embodiments, the LNP comprises one or more cationically ionizable lipids, one or more neutral lipids (e.g., in some embodiments, a sterol, such as cholesterol, and / or a phospholipid), and one or more polymer-conjugated lipids. In some embodiments, the formulation comprises ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride, and water.

[0325] In some embodiments, the different dosages include about 10 μg and about 30 μg, and the pharmaceutical RNA preparation comprises BNT162b2. In some embodiments, the different dosages include about 3 μg and about 10 μg, and the pharmaceutical RNA preparation comprises BNT162b2. In some embodiments, the different dosages include about 3 μg and about 30 μg, and the pharmaceutical RNA preparation comprises BNT162b2. In some embodiments, the different dosages include about 3 μg, about 10 μg, and about 30 μg, and the pharmaceutical RNA preparation comprises BNT162b2. In some embodiments, the BNT162b2 comprises RNA comprising the sequence of SEQ ID NO: 17. In some embodiments, the BNT162b2 is formulated as nanoparticles. In some embodiments, exemplary nanoparticles include lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles. In some embodiments, BNT162b2 is formulated as LNPs. In some embodiments, the LNPs comprise one or more cationically ionizable lipids, one or more neutral lipids (e.g., in some embodiments, a sterol, such as cholesterol, and / or a phospholipid), and one or more polymer-conjugated lipids. In some embodiments, the formulation comprises ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride, and water.

[0326] In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 30 μg / ml to about 100 μg / ml. In some embodiments, the concentration of RNA in the pharmaceutical RNA preparation is about 50 μg / ml to about 100 μg / ml.

[0327] In some embodiments, the dose volume is about 200 μl to about 300 μl.

[0328] In some embodiments, the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose.

[0329] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) A second administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0330] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation; (ii) A second administration of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0331] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) A second administration of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0332] In some embodiments, the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose, (i) a first administration of about 30 μg of RNA is administered by administering about 300 μl of undiluted pharmaceutical RNA preparation; (ii) a second administration of about 10 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation; (iii) A third administration of about 3 μg of RNA is administered by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0333] In some embodiments, the invention provides a system for administering different doses of RNA, the system comprising a plurality of vials for administering the plurality of different doses of RNA, each vial comprising a pharmaceutical RNA preparation; The system provides that the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant, the concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer, about 10% sucrose.

[0334] In some embodiments, the RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof comprises BNT162b2. In some embodiments, BNT162b2 comprises RNA comprising the sequence of SEQ ID NO: 17.

[0335] In some embodiments, RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant is formulated as a nanoparticle. In some embodiments, exemplary nanoparticles include lipid nanoparticles, polyplexes (PLX), lipidated polyplexes (LPLX), liposomes, or polysaccharide nanoparticles. In some embodiments, RNA encoding an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or its immunogenic variant is formulated as a LNP. In some embodiments, the LNP comprises one or more cationically ionizable lipids, one or more neutral lipids (e.g., in some embodiments, a sterol, e.g., cholesterol, and / or a phospholipid), and one or more polymer-conjugated lipids. In some embodiments, the formulation comprises ALC-0315 (4-hydroxybutyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate), ALC-0159 (2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), cholesterol, sucrose, trometamol (Tris), trometamol hydrochloride, and water.

[0336] In some embodiments, the different dosages include about 10 μg and about 30 μg. In some embodiments, the different dosages include about 3 μg and about 10 μg. In some embodiments, the different dosages include about 3 μg and about 30 μg. In some embodiments, the different dosages include about 3 μg, about 10 μg, and about 30 μg.

[0337] In some embodiments, the dose volume is about 200 μl to about 300 μl.

[0338] In some embodiments, a first vial of the plurality of vials is for administering a first administration of about 30 μg of RNA by administering about 300 μl of undiluted pharmaceutical RNA preparation; A second vial of the plurality of vials is for administering a second dose of about 10 μg of RNA by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0339] In some embodiments, a first vial of the plurality of vials is for administering a first administration of about 10 μg of RNA by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation; A second vial of the plurality of vials is for administering a second dose of about 3 μg of RNA by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0340] In some embodiments, a first vial of the plurality of vials is for administering a first administration of about 30 μg of RNA by administering about 300 μl of undiluted pharmaceutical RNA preparation; A second vial of the plurality of vials is for administering a second dose of about 3 μg of RNA by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0341] In some embodiments, a first vial of the plurality of vials is for administering a first administration of about 30 μg of RNA by administering about 300 μl of undiluted pharmaceutical RNA preparation; a second vial of the plurality of vials for administering a second administration of about 10 μg of RNA by diluting the pharmaceutical RNA preparation about 1:1 and administering about 200 μl of the diluted pharmaceutical RNA preparation; A third vial of the plurality of vials is for administering a third dose of about 3 μg of RNA by diluting the pharmaceutical RNA preparation about 1:5.75 and administering about 200 μl of the diluted pharmaceutical RNA preparation.

[0342] In some embodiments, the volume of the pharmaceutical RNA preparation for administering about 30 μg of RNA doses in multiple vials is about 2.25 ml for administering at least 6 total doses.

[0343] In some embodiments, the volume of the pharmaceutical RNA preparation for administering about 10 μg of RNA doses in multiple vials is about 1.3 ml for administering at least 10 total doses.

[0344] In some embodiments, the volume of the pharmaceutical RNA preparation for administering about 3 μg of RNA doses in multiple vials is about 0.4 ml for administering at least 10 total doses.

[0345] In some embodiments, the vials for administering different doses of RNA are labeled differently.

[0346] In some embodiments, the different indicia include different colored lids.

[0347] In some embodiments, the vials described herein are glass vials (e.g., Type 1 borosilicate glass or aluminosilicate glass) sealed with, for example, an aluminum seal with a bromobutyl rubber stopper and a flip-off plastic cap, optionally in pack sizes of 195 vials or 10 vials.

[0348] definition The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated. Any undefined terms have their art-recognized meanings.

[0349] As used herein, terms such as "reduce" or "inhibit" refer to the ability to cause an overall decrease in levels, for example, by about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, or about 75% or more. The term "inhibit" or similar phrases includes complete or essentially complete inhibition, i.e., a reduction to zero or a reduction to essentially zero.

[0350] As used herein, terms such as "enhance" refer to the ability to cause an overall increase or enhancement in levels, e.g., by at least about 5% or more, about 10% or more, about 15% or more, about 20% or more, about 25% or more, about 30% or more, about 40% or more, about 50% or more, about 75% or more, or about 100% or more.

[0351] "Physiological pH," as used herein, refers to a pH of about 7.4. In some embodiments, the physiological pH is 7.3 to 7.5. In some embodiments, the physiological pH is 7.35 to 7.45. In some embodiments, the physiological pH is 7.3, 7.35, 7.4, 7.45, or 7.5.

[0352] As used in this disclosure, "% w / v" refers to weight-volume percent, which is a unit of concentration that measures the amount of solute in grams (g) expressed as a percentage of the total volume of the solution in milliliters (mL).

[0353] As used in this disclosure, "wt. %" refers to weight percent, which is a unit of concentration that measures the amount of a substance in grams (g) expressed as a percentage of the total weight of the total composition in grams (g).

[0354] As used in this disclosure, "mol %" is defined as the ratio of moles of one component to the total moles of all components multiplied by 100.

[0355] As used in this disclosure, "mol % total lipid" is defined as the ratio of moles of one lipid component to the total moles of all lipids multiplied by 100. In this context, in some embodiments, the term "total lipid" includes lipids and lipid-like materials.

[0356] The term "ionic strength" refers to the mathematical relationship between the number of different ionic species in a particular solution and their respective charges. Thus, ionic strength, I, is mathematically represented by the formula:

number

[0357] According to the present disclosure, the term "ionic strength" in some embodiments refers to the presence of monovalent ions. With respect to the presence of divalent ions, particularly divalent cations, due to the presence of chelating agents, their concentration or effective concentration (presence of free ions) is, in some embodiments, sufficiently low to prevent degradation of nucleic acids. In some embodiments, the concentration or effective concentration of divalent ions is below the catalytic level for hydrolysis of phosphodiester bonds between nucleotides, such as RNA nucleotides. In some embodiments, the concentration of free divalent ions is 20 μM or less. In some embodiments, free divalent ions are absent or essentially absent.

[0358] "Osmolality" refers to the concentration of a particular solvent expressed as the number of osmoles of solvent per kilogram of solvent.

[0359] The term "lyophilizing" or "lyophilization" refers to the freeze-drying of a substance by freezing the substance and then reducing the surrounding pressure (e.g., to less than 15 Pa, e.g., less than 10 Pa, less than 5 Pa, or 1 Pa or less) to allow the freezing medium in the substance to sublime directly from the solid phase to the gas phase. Thus, the terms "lyophilizing" and "freeze-drying" are used interchangeably herein.

[0360] The term "spray drying" refers to spray drying a substance by mixing a (heated) gas with an atomized (atomized) fluid in a vessel (spray dryer) where the solvent from the formed droplets evaporates, resulting in a dry powder.

[0361] The term "reconstitute" relates to the addition of a solvent, such as water, to a dried product to return it to a liquid state, such as its original liquid state.

[0362] The term "recombinant" in the context of the present disclosure means "made by genetic engineering." In some embodiments, a "recombinant entity" in the context of the present disclosure is not naturally occurring.

[0363] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that exists in an organism (including a virus), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring. The term "found in nature" means "existing in nature" and includes known objects as well as objects that have not yet been discovered and / or isolated from nature, but may be discovered and / or isolated from natural sources in the future.

[0364] As used herein, the terms "room temperature" and "ambient temperature" are used interchangeably herein and refer to a temperature of at least about 15°C, e.g., from about 15°C to about 35°C, from about 15°C to about 30°C, from about 15°C to about 25°C, or from about 17°C to about 22°C. Such temperatures include 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, and 22°C.

[0365] The term "EDTA" refers to ethylenediaminetetraacetic acid disodium salt. All concentrations are given in terms of EDTA disodium salt.

[0366] The term "cryoprotectant" relates to a substance added to a formulation to protect the active ingredient during the freezing step.

[0367] The term "lyoprotectant" relates to a substance added to a formulation to protect the active ingredient during the drying step.

[0368] According to the present disclosure, the term "peptide" refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids linked together via peptide bonds. The term "polypeptide" refers to large peptides, particularly peptides having at least about 151 amino acids. Although both "peptide" and "polypeptide" are protein molecules, the terms "protein" and "polypeptide" are generally used synonymously herein.

[0369] The term "biological activity" refers to a response of a biological system to a molecule. Such a biological system can be, for example, a cell or an organism. In some embodiments, such a response is therapeutically or pharmaceutically useful.

[0370] The term "portion" refers to a fraction. With respect to a particular structure such as an amino acid sequence or a protein, the term "portion" may refer to a contiguous or discontinuous fraction of that structure.

[0371] The terms "portion" and "fragment" are used interchangeably herein and refer to a continuous element. For example, a portion of a structure such as an amino acid sequence or protein refers to a continuous element of that structure. When used in the context of a composition, the term "portion" refers to a portion of the composition. For example, a portion of a composition can be any portion between 0.1% and 99.9% of the composition (e.g., 0.1%, 0.5%, 1%, 5%, 10%, 50%, 90%, or 99%).

[0372] "Fragment" refers to a portion of an amino acid sequence (peptide or polypeptide), i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. C-terminally truncated fragments (N-terminal fragments) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. N-terminally truncated fragments (C-terminal fragments) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains the initiation codon responsible for initiating translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence comprises, for example, at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence. Fragments of an amino acid sequence include, for example, amino acid sequences of up to 8, in particular up to 10, up to 12, up to 15, up to 20, up to 30 or up to 55 consecutive amino acids of the amino acid sequence.

[0373] "Variant," as used herein with reference to an amino acid sequence (peptide or polypeptide), means an amino acid sequence that differs from a parent amino acid sequence by at least one amino acid (e.g., a different amino acid, or a modification of the same amino acid). The parent amino acid sequence can be a naturally occurring or wild-type (WT) amino acid sequence, or can be a modified version of the wild-type amino acid sequence. In some embodiments, the variant amino acid sequence has at least one amino acid difference compared to the parent amino acid sequence, e.g., 1 to about 20 amino acid differences compared to the parent, e.g., 1 to about 10 or 1 to about 5 amino acid differences.

[0374] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variants. A wild-type amino acid sequence, peptide, or polypeptide has an amino acid sequence that has not been intentionally modified.

[0375] For purposes of this disclosure, a "variant" of an amino acid sequence (peptide, or polypeptide) can include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all mutants, splice variants, post-translationally modified variants, conformations, isoforms, allelic variants, species variants, and species homologs, particularly those that occur naturally. The term "variant" particularly includes fragments of an amino acid sequence.

[0376] Amino acid insertion variants include the insertion of a single or two or more amino acids into a specific amino acid sequence. In amino acid sequence variants with insertions, one or more amino acid residues are inserted at a specific site in the amino acid sequence, although random insertion is also possible with appropriate screening of the resulting product. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants, including deletions at the N- and / or C-termini of a protein, are also referred to as N- and / or C-terminal truncation variants. Amino acid substitution variants are characterized by at least one residue in the sequence being removed and another residue being inserted in its place. Modifications at positions within the amino acid sequence that are not conserved between homologous peptides or peptides and / or replacement of amino acids with other amino acids with similar properties are preferred. In some embodiments, amino acid changes in peptide and polypeptide variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve substitutions of members of a family of amino acids related by their side chains. Naturally occurring amino acids are generally classified into four families: acidic (aspartate, glutamate), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes jointly classified as aromatic amino acids. In some embodiments, conservative amino acid substitutions include substitutions within the following groups: -Glycine, alanine; -valine, isoleucine, leucine; -Aspartic acid, glutamic acid; -Asparagine, glutamine; -Serine, threonine; -lysine, arginine; and -Phenylalanine, tyrosine.

[0377] In some embodiments, the degree of similarity, e.g., identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence will be at least about 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the degree of similarity or identity is given over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is, in some embodiments, given over, e.g., at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 consecutive amino acids. In some embodiments, the degree of similarity or identity is given over the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, e.g., sequence identity, can be performed using tools known in the art, e.g., using best sequence alignment, e.g., Align using standard settings, preferably EMBOSS::Needle, Matrix:Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0378] "Sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences refers to the percentage of amino acids that are identical between the sequences. "Sequence identity" between two nucleic acid sequences refers to the percentage of nucleotides that are identical between the sequences.

[0379] "% identical" and "% identity" or similar terms are intended to refer specifically to the percentage of nucleotides or amino acids that are identical in optimal alignment between the compared sequences. The percentage is purely statistical; differences between the two sequences may, but are not necessarily, randomly distributed over the entire length of the compared sequences. Comparison of two sequences is usually performed by comparing the sequences after optimal alignment over a segment or "comparison window" to identify local regions of corresponding sequences. Optimal alignment for comparison can be performed manually or with the aid of the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, with the aid of the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, with the aid of the similarity search algorithm of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 88, 2444, or with the aid of computer programs that use such algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics software package (Genetics Computer Group, 575 Science Drive, Madison, Wis.)). In some embodiments, the percent identity of two sequences is determined using the BLASTN or BLASTP algorithm, available at the National Center for Biotechnology Information (NCBI) website (e.g., blast.ncbi.nlm.nih.gov / Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq).In some embodiments, the algorithm parameters used for the BLASTN algorithm on the NCBI website include: (i) expectation threshold set to 10, (ii) word size set to 28, (iii) maximum match within query range set to 0, (iv) match / mismatch score set to 1, -2, (v) gap cost set to linear, and (vi) a filter for low complexity regions is used. In some embodiments, the algorithm parameters used for the BLASTP algorithm on the NCBI website include: (i) expectation threshold set to 10, (ii) word size set to 3, (iii) maximum match within query range set to 0, (iv) matrix set to BLOSUM62, (v) gap costs set to Existence:11, Extension:1, and (vi) conditional composition score matrix adjustment.

[0380] Percentage identity is obtained by determining the number of identical positions where the compared sequences correspond, dividing this number by the number of positions being compared (e.g., the number of positions in the reference sequence), and multiplying this result by 100.

[0381] In some embodiments, the degree of similarity or identity is given for a region that is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference sequence. For example, if the reference nucleic acid sequence consists of 200 nucleotides, the degree of identity is given in some embodiments for at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 consecutive nucleotides. In some embodiments, the degree of similarity or identity is given for the entire length of the reference sequence.

[0382] Homologous amino acid sequences according to the present disclosure exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and for example at least 95%, at least 98, or at least 99% of the amino acid residues.

[0383] The amino acid sequence variants described herein may be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or polypeptides with substitutions, additions, insertions, or deletions is described, for example, in Molecular Cloning: A Laboratory Manual, 4 th Edition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Furthermore, the peptides, polypeptides, and amino acid variants described herein can be readily prepared with the aid of known peptide synthesis techniques, such as, for example, solid phase synthesis and similar methods.

[0384] In some embodiments, a fragment or variant of an amino acid sequence (peptide or polypeptide) is a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. With respect to an antigen or antigen sequence, one particular function is one or more immunogenic activities exhibited by the amino acid sequence from which the fragment or variant is derived. As used herein, the term "functional fragment" or "functional variant" particularly refers to a variant molecule or sequence that includes an amino acid sequence that is modified by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence, yet is still able to perform one or more functions of the parent molecule or sequence, such as eliciting an immune response. In some embodiments, modifications in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter the characteristics of the molecule or sequence. In different embodiments, the function of the functional fragment or functional variant may be reduced but still significantly present, for example, the function of the functional fragment or functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however, in other embodiments, the function of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0385] An amino acid sequence (peptide, or polypeptide) "derived from" a specified amino acid sequence (peptide, or polypeptide) refers to the origin of the first amino acid sequence. In some embodiments, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that antigens suitable for use herein can be modified such that the sequence varies from the naturally occurring sequence or the native sequence from which they are derived while retaining the desired activity of the native sequence.

[0386] In some embodiments, "isolated" means removed (e.g., purified) from a natural state or from an artificial composition, such as a composition from a production process. For example, a nucleic acid, peptide, or polypeptide that is naturally present in a living animal is not "isolated," but the same nucleic acid, peptide, or polypeptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid, peptide, or polypeptide can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0387] The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of this disclosure, the term "transfection" also includes the introduction of nucleic acids into or the uptake of nucleic acids by such cells, which may be present in a subject, e.g., a patient, or in vitro, e.g., outside the patient. Thus, according to the present disclosure, cells for transfection of nucleic acids described herein can be present in vitro or in vivo, e.g., the cells can form part of an organ, tissue, and / or body of a patient. According to the present disclosure, transfection can be transient or stable. In some applications of transfection, transient expression of the transfected genetic material is sufficient. RNA can be transfected into cells to transiently express its encoded protein. Because nucleic acids introduced during the transfection process are typically not integrated into the nuclear genome, the foreign nucleic acid is diluted or degraded through mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must be performed.Such stable transfection can be achieved, for example, by using a virus-based system or a transposon-based system for transfection.Generally, the nucleic acid encoding the antigen is transiently transfected into the cell.RNA can be transfected into the cell to transiently express its encoded protein.

[0388] The present disclosure includes analogs of peptides or polypeptides. According to the present disclosure, a peptide or polypeptide analog is a modified form of the peptide or polypeptide from which it is derived, retaining at least one functional property of the peptide or polypeptide. For example, a pharmacologically active analog of a peptide or polypeptide retains at least one pharmacological activity of the peptide or polypeptide from which it is derived. Such modifications include any chemical modification, including single or multiple substitutions, deletions, and / or additions of carbohydrates, lipids, and / or any molecules associated with the peptide or polypeptide, such as the peptide or polypeptide. In some embodiments, a "peptide or polypeptide analog" includes those modified forms resulting from glycosylation, acetylation, phosphorylation, amidation, palmitoylation, myristoylation, isoprenylation, lipidation, alkylation, derivatization, introduction of protecting / blocking groups, proteolytic cleavage, or conjugation to an antibody or another cellular ligand. The term "analog" also covers all functional chemical equivalents of the peptides and polypeptides.

[0389] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining of two or more elements or components or domains.

[0390] As used herein, "endogenous" refers to any material that originates from or is produced within an organism, cell, tissue, or system.

[0391] As used herein, the term "exogenous" refers to any material introduced or produced from outside an organism, cell, tissue, or system.

[0392] According to various embodiments of the present disclosure, a nucleic acid, such as an RNA, encoding a peptide or polypeptide is taken up or introduced, i.e., transfected or transduced, into a cell, which may be present in vitro or in a subject, resulting in expression of the peptide or polypeptide. The cell may, for example, express the encoded peptide or polypeptide intracellularly (e.g., in the cytoplasm and / or in the nucleus), may secrete the encoded peptide or polypeptide, and / or may express it on its surface.

[0393] In accordance with the present disclosure, "nucleic acid expression" and "nucleic acid code" or similar terms are used interchangeably herein and, with respect to a particular peptide or polypeptide, mean that a nucleic acid, when present in an appropriate environment, e.g., a cell, can be expressed to produce that peptide or polypeptide.

[0394] As used herein, the term "expression" includes the transcription and / or translation of a particular nucleotide sequence.

[0395] In the context of the present disclosure, the term "transcription" relates to the process by which the genetic code in a DNA sequence is transcribed into RNA (particularly mRNA). RNA can then be translated into peptides or polypeptides.

[0396] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or polypeptide.

[0397] The medical preparations described herein, particularly kits, may include educational materials or instructions. As used herein, "educational materials" or "instructions" includes publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present invention. The educational materials of the kits of the present invention can be, for example, affixed to a container containing the composition of the present invention or shipped together with a container containing the composition. Alternatively, the educational materials may be shipped separately from the container, with the intention that the educational materials and the composition will be used cooperatively by the recipient.

[0398] Prodrugs of certain compounds described herein are those compounds that, upon administration to an individual, undergo chemical conversion under physiological conditions to provide the specified compound. Furthermore, prodrugs can be converted to the specified compound by chemical or biochemical methods in an ex vivo environment. For example, prodrugs can be slowly converted to the specified compound when placed in a transdermal patch reservoir, for example, using suitable enzymes or chemical reagents. Exemplary prodrugs are in vivo hydrolyzable esters (using alcohol or carboxy groups contained in certain compounds) or amides (using amino or carboxy groups contained in certain compounds). Specifically, any amino group contained in certain compounds that has at least one hydrogen atom can be converted to a prodrug form. Typical N-prodrug forms include carbamates, Mannich bases, enamines, and enaminones.

[0399] In this specification, the structural formula of a compound may represent a particular isomer of the compound. However, it should be understood that the present invention includes all isomers and mixtures of isomers, such as structurally occurring geometric isomers, optical isomers based on asymmetric carbons, stereoisomers, tautomers, etc., and is not limited to the description of the formula.

[0400] "Isomers" are compounds that have the same molecular formula but differ in structure ("structural isomers") or in the geometric (spatial) arrangement of functional groups and / or atoms ("stereoisomers"). "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A "racemic mixture" or "racemate" contains equal amounts of a pair of enantiomers and is designated by the prefix (±). "Diastereomers" are stereoisomers that are non-superimposable and are not mirror images of each other. "Tautomers" are structural isomers of the same chemical substance that, even when pure, spontaneously and reversibly interconvert into each other due to the migration of individual atoms or atomic groups; i.e., tautomers are in dynamic chemical equilibrium with each other. An example of a tautomer is a keto-enol tautomer. "Conformers" are stereoisomers that can formally be interconverted only by rotation about a single bond, and in particular include those resulting in different three-dimensional configurations of (hetero)cyclic rings, such as the chair, half-chair, boat, and twist-boat forms of cyclohexane.

[0401] The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic light scattering (DLS) with data analysis using a so-called cumulative algorithm, which results in a so-called Z dimension with a length dimension. 平均 , and the dimensionless polydispersity index (PDI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the "average diameter," "diameter," or "size" of a particle is Z 平均 Used synonymously with this value.

[0402] In some embodiments, the "polydispersity index" can be calculated based on dynamic light scattering measurements by so-called cumulative analysis, as mentioned in the definition of "average diameter." Under certain prerequisites, it can be taken as a measure of the overall size distribution of the nanoparticles.

[0403] The "radius of gyration" of the particle around the axis of rotation (R g) is the radial distance from the axis of rotation of a point at which, assuming the total mass of the particle is concentrated, its moment of inertia about a given axis is the same as its actual mass distribution. Mathematically, R g is the root mean square distance of a particle's components from either its center of mass or a given axis. For example, if a particle is at a fixed distance s from its center of mass, i Mass m located at i For a macromolecule consisting of n mass elements (i=1, 2, 3, ..., n), R g is the s over all mass elements i 2 is the mass-averaged square root of and can be calculated as follows:

number

number

number

[0404] The "hydrodynamic radius" (sometimes called the "Stokes radius" or "Stokes-Einstein radius") of a particle is the radius of a hypothetical hard sphere diffusing at the same rate as the particle. The hydrodynamic radius is related to the particle's mobility, taking into account not only size but also solvent effects. For example, a smaller charged particle with stronger hydration may have a larger hydrodynamic radius than a larger charged particle with weaker hydration. This is because the smaller particle draws in a greater number of water molecules as it passes through the solution. Because the actual dimensions of a particle in a solvent cannot be measured directly, the hydrodynamic radius may be defined by the Stokes-Einstein equation:

number

[0405] As used herein, the expression "light scattering" refers to the physical process by which light is forced to deviate from a straight line trajectory by one or more paths due to localized inhomogeneities in the medium through which it passes.

[0406] The term "UV" means ultraviolet light and refers to wavelengths between 10 nm and 400 nm, i.e., the band of the electromagnetic spectrum shorter than visible light but longer than X-rays.

[0407] The expression "multi-angle light scattering" or "MALS" as used herein relates to a technique for measuring light scattered at multiple angles by a sample. "Multi-angle" in this context means that the scattered light can be detected at different, distinct angles, measured, for example, by a single detector moving over a range that includes a selected specific angle, or by an array of detectors fixed at specific angular positions. In certain embodiments, the light source used in MALS is a laser light source (MALLS: Multi-Angle Laser Light Scattering). Based on the MALS signal of a composition containing particles and by using an appropriate formalism (e.g., Zimm plot, Berry plot, or Debye plot), the radius of gyration (R g), and thus it is possible to determine the size of the particle. Preferably, the Zimm plot is a graphical representation using the following equation:

number

number

number

[0408] The term "dynamic light scattering" or "DLS," as used herein, refers to a technique for determining particle size and size distribution profiles, particularly with respect to the hydrodynamic radius of the particles. A monochromatic light source, usually a laser, is emitted into a sample through a polarizer. The scattered light then enters through a second polarizer and is detected, and the resulting image is projected onto a screen. Particles in solution collide with the light and diffract it in all directions. The diffracted light from the particles can either interfere constructively (bright areas) or destructively (dark areas). This process is repeated over short time intervals, and the resulting set of speckle patterns is analyzed by an autocorrelator, which compares the light intensity at each spot over time.

[0409] As used herein, the term "static light scattering" or "SLS" refers to a technique for determining particle size and size distribution profiles, particularly with respect to the particle's radius of gyration and / or molar mass. A high-intensity monochromatic light, usually a laser, is projected into a solution containing the particles. One or more detectors are used to measure the scattering intensity at one or more angles. Angular dependence is required to obtain accurate measurements of both the molar mass and size of all macromolecular radii. Therefore, simultaneous measurements at several angles relative to the direction of incident light, known as multi-angle light scattering (MALS) or multi-angle laser light scattering (MALLS), are generally considered the standard practice of static light scattering.

[0410] nucleic acid The term "nucleic acid" includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), combinations thereof, and modified forms thereof. This term includes genomic DNA, cDNA, mRNA, recombinantly produced, and chemically synthesized molecules. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is RNA. In some embodiments, the nucleic acid is a mixture of DNA and RNA. The nucleic acid can exist as a single-stranded or double-stranded molecule and as a linear or covalently closed circular molecule. The nucleic acid can be isolated. The term "isolated nucleic acid," according to the present disclosure, means that the nucleic acid has been (i) amplified in vitro, e.g., via polymerase chain reaction (PCR) of DNA or in vitro transcription of RNA (e.g., using RNA polymerase), (ii) recombinantly produced by cloning, (iii) purified, e.g., by cleavage and separation by gel electrophoresis, or (iv) synthesized, e.g., by chemical synthesis.

[0411] The term "nucleoside" (abbreviated herein as "N") refers to a compound that can be considered a nucleotide without a phosphate group. A nucleoside is a nucleic acid base (e.g., ribose or deoxyribose) linked to a sugar, while a nucleotide is composed of a nucleoside and one or more phosphate groups. Examples of nucleosides include cytidine, uridine, pseudouridine, adenosine, and guanosine.

[0412] The five standard nucleosides that typically make up naturally occurring nucleic acids are uridine, adenosine, thymidine, cytidine, and guanosine. The five nucleosides are commonly abbreviated by the single-letter codes U, A, T, C, and G, respectively. However, thymidine is more commonly written as "dT" (the "d" stands for "deoxy") because it contains a 2'-deoxyribofuranose moiety rather than the ribofuranose ring found in uridine. This is because thymidine is found in deoxyribonucleic acid (DNA) but not ribonucleic acid (RNA). Conversely, uridine is found in RNA but not DNA. The remaining three nucleosides can be found in both RNA and DNA. They are represented as A, C, and G in RNA, but dA, dC, and dG in DNA.

[0413] The modified purine (A or G) or pyrimidine (C, T, or U) base moiety may, in some embodiments, be one or more alkyl groups, e.g., one or more C 1-4 Specific examples of modified purine or pyrimidine base moieties include N 7 -Alkyl-guanine, N 6 -alkyl-adenine, 5-alkyl-cytosine, 5-alkyl-uracil, and N(1)-alkyl-uracil, e.g., N 7 -C 1-4 Alkyl-guanine, N 6 -C 1-4 Alkyl-adenine, 5-C 1-4 Alkyl-cytosine, 5-C 1-4 Alkyl-uracil and N(1)-C 1-4 Alkyl-uracil, preferably N 7 -methyl-guanine, N 6 -methyl-adenine, 5-methyl-cytosine, 5-methyl-uracil, and N(1)-methyl-uracil.

[0414] In this disclosure, the term "DNA" refers to a nucleic acid molecule containing deoxyribonucleotide residues. In a preferred embodiment, DNA contains all or most deoxyribonucleotide residues. As used herein, "deoxyribonucleotide" refers to a nucleotide lacking a hydroxyl group at the 2' position of the β-D-ribofuranosyl group. DNA includes, but is not limited to, double-stranded DNA, single-stranded DNA, isolated DNA such as partially purified DNA, essentially pure DNA, synthetic DNA, recombinantly produced DNA, and modified DNA that differs from naturally occurring DNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal DNA nucleotides or to the end(s) of the DNA. It is also contemplated herein that the nucleotides in DNA may be non-standard nucleotides, such as chemically synthesized nucleotides or ribonucleotides. In this disclosure, these modified DNAs are considered analogs of naturally occurring DNA. A molecule contains a "majority of deoxyribonucleotide residues" if the content of deoxyribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, etc.), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in the molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0415] The DNA may be recombinant DNA and may be obtained by cloning a nucleic acid, in particular cDNA, which may be obtained by reverse transcription of RNA.

[0416] The term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, the RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of the RNA. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In the present disclosure, these modified / modified nucleotides may be referred to as analogs of naturally occurring nucleotides, and the corresponding RNA containing such modified / modified nucleotides (i.e., modified / modified RNA) may be referred to as analogs of naturally occurring RNA. A molecule contains a "majority of ribonucleotide residues" if the content of ribonucleotide residues in the molecule is greater than 50% (e.g., at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, etc.), based on the total number of nucleotide residues in the molecule. The total number of nucleotide residues in the molecule is the sum of all nucleotide residues (whether the nucleotide residues are standard (i.e., naturally occurring) nucleotide residues or analogs thereof).

[0417] "RNA" includes mRNA, tRNA, ribosomal RNA (rRNA), small nuclear RNA (snRNA), self-amplifying RNA (saRNA), single-stranded RNA (ssRNA), dsRNA, inhibitory RNA (such as antisense ssRNA, small interfering RNA (siRNA), or microRNA (miRNA)), activating RNA (such as small activating RNA), and immunostimulatory RNA (isRNA). In some embodiments, "RNA" refers to mRNA.

[0418] As used herein, the term "in vitro transcription" or "IVT" means that transcription (i.e., production of RNA) is performed in a cell-free manner. That is, IVT uses transcription machinery extracted from cells (e.g., cell lysates or isolated components thereof, including RNA polymerase (preferably T7, T3, or SP6 polymerase)) rather than living / cultured cells.

[0419] mRNA According to the present disclosure, the term "mRNA" means "messenger RNA" and includes "transcripts" that can be produced by using a DNA template. Generally, mRNA encodes a peptide or polypeptide.

[0420] Although mRNA is single-stranded, it may contain self-complementary sequences that allow part of the mRNA to fold back on itself and pair with itself to form a double helix.

[0421] According to the present disclosure, "dsRNA" means double-stranded RNA, which is RNA having two partially or completely complementary strands.

[0422] In a preferred embodiment of the present disclosure, mRNA refers to an RNA transcript that encodes a peptide or polypeptide.

[0423] In some embodiments, preferably the mRNA encoding the peptide or polypeptide has a length of at least 45 nucleotides (e.g., at least 60, at least 90, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1,000, at least 1,500, at least 2,000, at least 2,500, at least 3,000, at least 3,500, at least 4,000, at least 4,500, at least 5,000, at least 6,000, at least 7,000, at least 8,000, at least 9,000 nucleotides), preferably up to 15,000, e.g., up to 14,000, up to 13,000, up to 12,000 nucleotides, up to 11,000 nucleotides, or up to 10,000 nucleotides.

[0424] As established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide / polypeptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, mRNA is produced by in vitro transcription or chemical synthesis. In some embodiments, mRNA is produced by in vitro transcription using a DNA template. In vitro transcription methodologies are known to those skilled in the art and are described, for example, in Molecular Cloning: A Laboratory Manual, 4 thEdition, M.R. Green and J. Sambrook eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 2012. Additionally, various in vitro transcription kits are commercially available from, for example, Thermo Fisher Scientific (e.g., TranscriptAid™ T7 kit, MEGAscript™ T7 kit, MAXIscript™), New England BioLabs Inc. (e.g., HiScribe™ T7 kit, HiScribe™ T7 ARCA mRNA kit), Promega (e.g., RiboMAX™, HeLaScribe™, Riboprobe™ systems), Jena Bioscience (e.g., SP6 or T7 transcription kits), and Epicentre (e.g., AmpliScribe™). To provide modified mRNA, the corresponding modified nucleotides, such as modified naturally occurring nucleotides, non-naturally occurring nucleotides and / or modified non-naturally occurring nucleotides, can be incorporated during synthesis (preferably in vitro transcription), or modifications can be imparted to and / or added to the mRNA after transcription.

[0425] In some embodiments, the mRNA is in vitro transcribed mRNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. Specific examples of RNA polymerases are T7, T3, and SP6 RNA polymerases. Preferably, in vitro transcription is controlled by a T7 or SP6 promoter. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0426] In some embodiments of the present disclosure, the mRNA is a "replicon mRNA" or simply a "replicon," particularly a "self-replicating mRNA" or "self-amplifying mRNA." In certain embodiments, the replicon or self-replicating mRNA is derived from or contains elements derived from a ssRNA virus, particularly a positive-strand ssRNA virus such as an alphavirus. Alphaviruses are typical representatives of positive-strand RNA viruses. Alphaviruses replicate within the cytoplasm of infected cells (for a review of the alphavirus life cycle, see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856). The total genome length of many alphaviruses typically ranges from 11,000 to 12,000 nucleotides, and the genomic RNA typically has a 5' cap and a 3' poly(A) tail. The genome of an alphavirus encodes nonstructural proteins (involved in viral RNA transcription, modification, and replication and protein modification) as well as structural proteins (which form the virus particle). The genome typically contains two open reading frames (ORFs). The four nonstructural proteins (nsP1-nsP4) are typically encoded together by the first ORF, which begins near the 5' end of the genome, while the alphavirus structural proteins are encoded together by the second ORF, which is found downstream of the first ORF and extends toward the 3' end of the genome. Typically, the first ORF is larger than the second ORF, with a ratio of approximately 2:1. In cells infected by alphaviruses, only the nucleic acid sequences encoding the nonstructural proteins are translated from the genomic RNA, while the genetic information encoding the structural proteins can be translated from subgenomic transcripts, which are RNA molecules similar to eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87, pp. 111-124). After infection, i.e., early in the viral life cycle, the (+)-strand genomic RNA acts directly as a messenger RNA for the translation of an open reading frame encoding a nonstructural polyprotein (nsP1234).Alphavirus-derived vectors have been proposed for the delivery of foreign genetic information to target cells or organisms. In a simple approach, the open reading frame encoding the alphavirus structural proteins is replaced by an open reading frame encoding a protein of interest. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes the viral replicase and the other nucleic acid molecule is replicable by that replicase in trans (hence the name trans-replication system). Trans-replication requires the presence of both of these nucleic acid molecules in a given host cell. The nucleic acid molecule replicable by the replicase in trans must contain certain alphavirus sequence elements to enable recognition and RNA synthesis by the alphavirus replicase.

[0427] In some embodiments of the present disclosure, the mRNA contains one or more modifications, for example, to increase its stability, and / or increase translation efficiency, and / or reduce immunogenicity, and / or reduce cytotoxicity. For example, to increase mRNA expression, the mRNA may be modified within the coding region, i.e., the sequence encoding the expressed peptide or polypeptide, preferably without modifying the sequence of the expressed peptide or polypeptide. Such modifications are described, for example, in WO2007 / 036366 and PCT / EP2019 / 056502, and include: 5' cap structures; extension or truncation of naturally occurring poly(A) tails; modification of the 5' and / or 3' untranslated regions (UTRs), such as the introduction of UTRs not associated with the coding region of the RNA; replacement of one or more naturally occurring nucleotides with synthetic nucleotides; and codon optimization (e.g., modifying, preferably increasing, the GC content of the RNA).

[0428] In some embodiments, the mRNA comprises a 5' cap structure. In some embodiments, the mRNA does not have an uncapped 5' triphosphate. In some embodiments, the mRNA may comprise a conventional 5' cap and / or a 5' cap analog. The term "conventional 5' cap" refers to the cap structure found on the 5' end of an mRNA molecule, and generally consists of a guanosine 5' triphosphate (Gppp) connected via its triphosphate moiety to the 5' end of the next nucleotide in the mRNA (i.e., the guanosine is connected to the remainder of the mRNA via a 5'-to-5' triphosphate linkage). The guanosine is N 7 can be methylated at the cap structure m 7 The term "5' cap analog" is based on the traditional 5' cap, but to avoid incorporation of the 5' cap analog in the reverse orientation, m 7 These include 5' caps modified at either the 2' or 3' position of the guanosine structure (such 5' cap analogs are also called anti-reverse cap analogs (ARCAs)). Particularly preferred 5' cap analogs are phosphorothioate-modified 5' cap analogs at the β-phosphate (e.g., m2), as described in PCT / EP2019 / 056502. 7,2’ O and those having one or more substitutions at the bridging and non-bridging oxygens in the phosphate bridge, such as G(5')ppSp(5')G (referred to as beta-S-ARCA or β-S-ARCA). Providing mRNA with a 5' cap structure as described herein can be achieved by in vitro transcription of a DNA template in the presence of the corresponding 5' cap compound, with the 5' cap structure being co-transcriptionally incorporated into the generated mRNA strand, or mRNA can be generated, for example, by in vitro transcription, and the 5' cap structure can be attached to the mRNA post-transcriptionally using a capping enzyme, for example, vaccinia virus capping enzyme.

[0429] In some embodiments, the mRNA is m2 7,2’OG(5')ppSp(5')G (specifically its D1 diastereomer), m2 7,3’O G(5')ppp(5')G, and m2 7,3’-O Gppp(m1 2’-O In some embodiments, the RNA encoding the antigen- or epitope-containing peptide or polypeptide comprises a 5' cap structure selected from the group consisting of m2 7,2’O It contains G(5')ppSp(5')G (specifically, its D1 diastereomer) as the 5' cap structure.

[0430] In some embodiments, the mRNA comprises cap0, cap1, or cap2, preferably cap1 or cap2. According to the present disclosure, the term "cap0" refers to the structure "m 7 GpppN" where N is any nucleoside having an OH moiety at the 2' position. According to the present disclosure, the term "cap1" refers to the structure "m 7 GpppNm, where Nm is any nucleoside having an OCH3 moiety at the 2' position. According to the present disclosure, the term "cap2" refers to the structure "m 7 GpppNmNm" where each Nm is independently any nucleoside having an OCH3 moiety at the 2' position.

[0431] The 5' cap analog beta-S-ARCA (β-S-ARCA) has the following structure: [ka] The "D1 diastereomer of beta-S-ARCA" or "beta-S-ARCA(D1)" is the diastereomer of beta-S-ARCA that elutes first on an HPLC column compared to the D2 diastereomer of beta-S-ARCA (beta-S-ARCA(D2)), and therefore exhibits a shorter retention time. The HPLC is preferably analytical HPLC. In some embodiments, a Supelcosil LC-18-T RP column, preferably 5 μm, 4.6 × 250 mm, is used for the separation, thereby enabling a flow rate of 1.3 ml / min. In some embodiments, a gradient of methanol in ammonium acetate is used, e.g., a linear gradient of 0 to 25% methanol in 0.05 M ammonium acetate (pH = 5.9) within 15 min. UV detection (VWD) can be performed at 260 nm, and fluorescence detection (FLD) can be performed with excitation at 280 nm and detection at 337 nm.

[0432] 5' cap analog m2, a building block of cap1 7,3’-O Gppp(m1 2’-O )ApG(m2 7,3’O G(5')ppp(5')m 2’-O ApG (also called ApG) has the following structure: [ka] An exemplary cap0 mRNA containing β-S-ARCA and mRNA has the following structure: [ka] m2 7,3’O An exemplary cap0 mRNA containing G(5')ppp(5')G and mRNA has the following structure: [ka] m2 7,3’-O Gppp(m1 2’-O ) An exemplary cap1 mRNA containing ApG and mRNA has the following structure: [ka]

[0433] In some embodiments, the RNA comprises a poly-A tail. As used herein, the term "poly-A tail" or "poly-A sequence" typically refers to an uninterrupted or interrupted sequence of adenylate residues located at the 3' end of an mRNA molecule. Poly-A tails or poly-A sequences are known to those skilled in the art and may follow the 3'-UTR in the mRNAs described herein. An uninterrupted poly-A tail is characterized by consecutive adenylate residues. Uninterrupted poly-A tails are typical in nature. The RNAs disclosed herein can have a poly-A tail attached to the free 3' end of an mRNA by a template-independent RNA polymerase after transcription, or a poly-A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0434] PolyA tails of approximately 120 nucleotides have been demonstrated to have a beneficial effect on the levels of mRNA in transfected eukaryotic cells, as well as on the levels of protein translated from open reading frames located upstream (5') of the polyA tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0435] The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, consists essentially 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, "essentially consisting" means that the majority of nucleotides in the poly-A tail, 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%, by number of nucleotides in the poly-A tail, are A nucleotides, while allowing the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consisting of" means that all nucleotides in the poly A tail are A nucleotides, i.e., 100% by number of nucleotides in the poly A tail. The term "A nucleotide" or "A" refers to adenylate.

[0436] In some embodiments, the poly(A) tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template containing repetitive dT nucleotides (deoxythymidylate) on the strand complementary to the coding strand. The DNA sequence encoding the poly(A) tail (coding strand) is referred to as a poly(A) cassette.

[0437] In some embodiments, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by a random sequence of four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such cassettes are disclosed in WO 2016 / 005324 A1, which is incorporated herein by reference. Any poly(A) cassette disclosed in WO 2016 / 005324 A1 may be used in the present disclosure. A poly(A) cassette consisting essentially of dA nucleotides but interrupted by a random sequence with an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of, for example, 5 to 50 nucleotides, exhibits consistent growth of plasmid DNA in E. coli at the DNA level and, at the RNA level, remains assembled and contains beneficial properties for supporting RNA stability and translation efficiency. Thus, in some embodiments, the poly-A tails included in the mRNA molecules described herein consist essentially of A nucleotides, but are 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.

[0438] In some embodiments, no nucleotides other than A nucleotides are adjacent to the polyA tail at its 3' end, i.e., the polyA tail is not masked or followed at its 3' end by nucleotides other than A.

[0439] In some embodiments, the polyA tail 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 polyA tail may consist essentially 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 polyA tail 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 polyA tail comprises the polyA tail set forth in SEQ ID NO: 8. In some embodiments, the polyA tail comprises at least 100 nucleotides. In some embodiments, the polyA tail comprises about 150 nucleotides. In some embodiments, the polyA tail comprises about 120 nucleotides.

[0440] In some embodiments, an mRNA according to the present disclosure comprises a 5'-UTR and / or a 3'-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 a corresponding region in an RNA molecule, such as an mRNA molecule. An untranslated region (UTR) can be located 5' (upstream) of an open reading frame (5'-UTR) and / or 3' (downstream) of an open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end of a protein-coding region, upstream of the start codon. The 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. If present, the 3'-UTR is located at the 3' end of a protein-coding region, downstream of the termination codon, although the term "3'-UTR" generally does not include a polyA sequence. Thus, the 3'-UTR is upstream of the polyA sequence (if present), e.g., directly adjacent to the polyA sequence. The incorporation of 3'-UTR into the 3' untranslated region of RNA (preferably mRNA) molecules can improve translation efficiency.Synergistic effects can be achieved by incorporating two or more of these 3'-UTRs (preferably arranged in a head-to-tail orientation; see, for example, Holtkamp et al. Blood 108, 4009-4017 (2006)).3'-UTRs can be homologous or heterologous to the RNA (e.g., mRNA) into which they are introduced.In certain embodiments, 3'-UTRs are derived from globin genes or mRNAs, such as alpha2-globin, alpha1-globin, or beta-globin, for example, beta-globin, for example, human beta-globin genes or mRNAs. For example, an RNA (e.g., an mRNA) can be modified by replacing or inserting an existing 3'-UTR with one or more, e.g., two, copies of a 3'-UTR from a globin gene, such as alpha2-globin, alpha1-globin, beta-globin, e.g., beta-globin, e.g., human beta-globin.

[0441] A particularly preferred 5'-UTR comprises the nucleotide sequence of SEQ ID NO: 6. A particularly preferred 3'-UTR comprises the nucleotide sequence of SEQ ID NO: 7.

[0442] In some embodiments, the RNA comprises a 5'-UTR comprising the nucleotide sequence of SEQ ID NO:6, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6.

[0443] In some embodiments, the RNA comprises a 3'-UTR comprising the nucleotide sequence of SEQ ID NO:7, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7.

[0444] The mRNA may have modified ribonucleotides to increase its stability, and / or reduce immunogenicity, and / or reduce cytotoxicity. For example, in some embodiments, uridines in the mRNA described herein are replaced (partially or completely, preferably completely) by modified nucleosides. In some embodiments, the modified nucleosides are modified uridines.

[0445] In some embodiments, the modified uridine substituting for uridine is selected from the group consisting of pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), 5-methyl-uridine (m5U), and combinations thereof.

[0446] In some embodiments, the modified nucleoside that replaces (partially or completely, preferably completely) uridine in the mRNA is 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5- Bromo-uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-Methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm5U), 1-taurinomethyl-uridine 5-Taurinomethyl-2-thio-uridine (m5s2U), 1-Taurinomethyl-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-deaza-pseudouridine, 2-Thio-1-methyl-1-deaza-pseudouridine, Dihydrouridine (D), Dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 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 (acp3U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3ψ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine The uridine may be any one or more of lysine (ψm), 2-thio-2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0447] RNA (preferably mRNA) modified with pseudouridine (partially or completely, preferably completely replacing uridine) is referred to herein as "Ψ-modified," although the term "mΨ-modified" means that the RNA (preferably mRNA) contains N(1)-methylpseudouridine (partially or completely, preferably completely replacing uridine). Furthermore, the term "m5U-modified" means that the RNA (preferably mRNA) contains 5-methyluridine (partially or completely, preferably completely replacing uridine). Such Ψ- or mΨ- or m5U-modified RNAs typically exhibit reduced immunogenicity compared to their unmodified forms and are therefore preferred in applications where induction of an immune response is avoided or minimized. In some embodiments, the RNA (preferably mRNA) contains N(1)-methylpseudouridine completely replacing uridine.

[0448] The codons of the mRNA used in the present disclosure can be further optimized, for example, to increase the GC content of the RNA and / or to replace codons that are rare in a cell (or subject) in which a peptide or polypeptide of interest is expressed with codons that are synonymous and frequent in that cell (or subject). In some embodiments, the amino acid sequence encoded by the mRNA used in the present disclosure is encoded by a coding sequence that is codon-optimized and / or has an increased G / C content compared to a wild-type coding sequence. This includes embodiments in which one or more sequence regions of the coding sequence are codon-optimized and / or have an increased G / C content compared to the corresponding sequence region of a wild-type coding sequence. In some embodiments, the codon optimization and / or increased G / C content preferably does not alter the sequence of the encoded amino acid sequence.

[0449] The term "codon optimization" refers to the modification of codons in the coding region of a nucleic acid molecule to reflect the typical codon usage of a host organism, preferably without modifying the amino acid sequence encoded by the nucleic acid molecule.In the context of the present disclosure, the coding region can be codon-optimized for optimal expression in a subject treated with the mRNA described herein.Codon optimization is based on the discovery that translation efficiency is also determined by the different frequencies of occurrence of tRNA in cells.Therefore, the sequence of mRNA can be modified so that instead of "rare codons", codons that frequently occur and that are available for tRNA are inserted.

[0450] In some embodiments, the guanosine / cytosine (G / C) content of the coding region of an mRNA described herein is increased compared to the G / C content of the corresponding coding sequence of a wild-type mRNA, and the amino acid sequence encoded by the mRNA is preferably unmodified compared to the amino acid sequence encoded by the wild-type RNA. This modification of the mRNA sequence is based on the fact that the sequence of any RNA region to be translated is important for the efficient translation of that mRNA. Sequences with an increased G (guanosine) / C (cytosine) content are more stable than sequences with an increased A (adenosine) / U (uracil) content. Given the fact that several codons encode one and the same amino acid (the so-called degeneration of the genetic code), it is possible to determine the codons most favorable for stability (the so-called alternative codon usage). Depending on the amino acid encoded by the mRNA, there are various possibilities for modifying the mRNA sequence compared to its wild-type sequence. In particular, codons containing A and / or U nucleotides can be modified by replacing these codons with other codons that encode the same amino acid but do not contain A and / or U nucleotides or contain a lower content of A and / or U nucleotides.

[0451] In various embodiments, the G / C content of the coding region of the mRNA 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 the wild-type RNA.

[0452] Combinations of the above modifications, i.e., incorporation of a 5' cap structure, incorporation of a polyA sequence, unmasking of a polyA sequence, modification of the 5'-UTR and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine for cytidine and / or pseudouridine (ψ), or N(1)-methylpseudouridine (mΨ) or 5-methyluridine (m5U) for uridine), and codon optimization, have a synergistic effect on RNA (preferably mRNA) stability and increased translation efficiency. Thus, in some embodiments, the mRNA used in this disclosure includes a combination of at least two, at least three, at least four, or all five of the above-mentioned modifications, i.e., (i) incorporation of a 5' cap structure, (ii) incorporation of a polyA sequence, unmasking of a polyA sequence, (iii) alteration of the 5'- and / or 3'-UTR (such as incorporation of one or more 3'-UTRs), (iv) replacement of one or more naturally occurring nucleotides with synthetic nucleotides (e.g., 5-methylcytidine and / or pseudouridine (Ψ) for cytidine, or N(1)-methylpseudouridine (mΨ), or 5-methyluridine (m5U) for uridine), and (v) codon optimization.

[0453] Some aspects of the present disclosure involve targeted delivery of the mRNA disclosed herein to specific cells or tissues. In some embodiments, the present disclosure involves targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen. Targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen, is particularly preferred when the administered mRNA encodes an antigen or epitope to induce an immune response. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. The "lymphatic system" is part of the circulatory system and is an important part of the immune system, including a network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphoid organs, a conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and bone marrow constitute primary lymphoid organs. Secondary or peripheral lymphoid organs, including lymph nodes and the spleen, maintain mature naive lymphoid cells and initiate adaptive immune responses.

[0454] Lipid-based mRNA delivery systems have an inherent preference for the liver. Liver accumulation is caused by discontinuities in the liver vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates). In some embodiments, the target organ is the liver and the target tissue is liver tissue. Delivery to such target tissue is preferred, particularly when the presence of the mRNA or the encoded peptide or polypeptide in this organ or tissue is desired, and / or when abundant expression of the encoded peptide or polypeptide is desired, and / or when systemic presence of the encoded peptide or polypeptide is desired or required, especially in significant amounts.

[0455] In some embodiments, after administration of an mRNA particle described herein, at least a portion of the mRNA is delivered to a target cell or target organ. In some embodiments, at least a portion of the mRNA is delivered to the cytoplasm of the target cell. In some embodiments, the mRNA encodes a peptide or polypeptide, and the mRNA is translated by the target cell to produce the peptide or polypeptide. In some embodiments, the target cell is a cell in the liver. In some embodiments, the target cell is a muscle cell. In some embodiments, the target cell is an endothelial cell. In some embodiments, the target cell is a tumor cell or a cell in the tumor microenvironment. In some embodiments, the target cell is a blood cell. In some embodiments, the target cell is a cell in a lymph node. In some embodiments, the target cell is a cell in the lung. In some embodiments, the target cell is a blood cell. In some embodiments, the target cell is a cell in the skin. In some embodiments, the target cell is a spleen cell. In some embodiments, the target cell is an antigen-presenting cell, such as a professional antigen-presenting cell in the spleen. In some embodiments, the target cell is a dendritic cell in the spleen. In some embodiments, the target cell is a T cell. In some embodiments, the target cell is a B cell. In some embodiments, the target cells are NK cells. In some embodiments, the target cells are monocytes. The RNA particles described herein can be used to deliver mRNA to such target cells.

[0456] Pharmaceutically active peptides or polypeptides "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0457] In some embodiments, RNA as used in this disclosure comprises a nucleic acid sequence that encodes a peptide or polypeptide, for example, a pharmaceutically active peptide or polypeptide.

[0458] In some embodiments, the RNA used in the present disclosure comprises a nucleic acid sequence encoding a peptide or polypeptide, preferably a pharmaceutically active peptide or polypeptide, and is capable of expressing the peptide or polypeptide, particularly when transferred into a cell or subject. Thus, in some embodiments, the nucleic acid used in the present disclosure contains a coding region (open reading frame (ORF)) encoding a peptide or polypeptide, e.g., encoding a pharmaceutically active peptide or polypeptide. In this regard, an "open reading frame" or "ORF" is a continuous stretch of codons beginning with a start codon and ending with a stop codon. Such nucleic acids encoding pharmaceutically active peptides or polypeptides are also referred to herein as "pharmaceutically active nucleic acids." In particular, such mRNAs encoding pharmaceutically active peptides or polypeptides are also referred to herein as "pharmaceutically active mRNAs." In some embodiments, the RNA used in the present disclosure comprises a nucleic acid sequence encoding two or more peptides or polypeptides, e.g., two, three, four, or more peptides or polypeptides.

[0459] According to the present disclosure, the term "pharmaceutically active peptide or polypeptide" refers to a peptide or polypeptide that can be used to treat an individual in whom expression of the peptide or polypeptide would be beneficial, e.g., in ameliorating disease symptoms. Preferably, a pharmaceutically active peptide or polypeptide has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease. In some embodiments, a pharmaceutically active peptide or polypeptide, when administered to an individual in a therapeutically effective amount, has a positive or beneficial effect on the individual's condition or disease state. A pharmaceutically active peptide or polypeptide can have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease. The term "pharmaceutically active peptide or polypeptide" includes the entire peptide or polypeptide and may refer to a pharmaceutically active fragment thereof. It can also include pharmaceutically active variants and / or analogs of the peptide or polypeptide.

[0460] Specific examples of pharmaceutically active peptides and polypeptides include, but are not limited to, immune stimulants such as cytokines, hormones, adhesion molecules, immunoglobulins, immunoactive compounds, growth factors, protease inhibitors, enzymes, receptors, apoptosis regulators, transcription factors, tumor suppressor proteins, structural proteins, reprogramming factors, genome engineering proteins, and blood proteins.

[0461] An "immunostimulant" is any substance that stimulates the immune system by inducing activation or increasing the activity of any of the components of the immune system, particularly immune effector cells. Immune stimulants can be pro-inflammatory (e.g., when treating infections or cancer) or anti-inflammatory (e.g., when treating autoimmune diseases).

[0462] In one embodiment, the immunostimulatory substance is a cytokine or a variant thereof. Examples of cytokines include interferons such as interferon-alpha (IFN-α) or interferon-gamma (IFN-γ), interleukins such as IL2, IL7, IL12, IL15, and IL23, colony-stimulating factors such as M-CSF and GM-CSF, and tumor necrosis factors. In another embodiment, the immunostimulatory substance comprises an adjuvant-type immunostimulatory substance such as an APC Toll-like receptor agonist or a costimulatory / cell adhesion membrane protein. Examples of Toll-like receptor agonists include costimulatory / adhesion proteins such as CD80, CD86, and ICAM-1.

[0463] The term "cytokine" refers to proteins with a molecular weight of approximately 5 to 60 kDa that are involved in cell signaling (e.g., paracrine, endocrine, and / or autocrine signaling). Specifically, upon release, cytokines affect the behavior of cells surrounding their release site. Examples of cytokines include lymphokines, interleukins, chemokines, interferons, and tumor necrosis factors (TNFs). According to the present disclosure, cytokines do not include hormones or growth factors. Cytokines differ from hormones in that (i) they typically act at much more variable concentrations than hormones and (ii) they are generally produced by a wide range of cells (almost all nucleated cells can produce cytokines). Interferons are typically characterized by antiviral, antiproliferative, and immunomodulatory activities. Interferons are proteins that modify and regulate intracellular gene transcription by binding to interferon receptors on the surface of regulated cells, thereby preventing intracellular viral replication. Interferons can be grouped into two types: Specific examples of cytokines include erythropoietin (EPO), colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factors (TNFs), bone morphogenetic proteins (BMPs), interferon alpha (IFNα), interferon beta (IFNβ), interferon gamma (INFγ), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interleukin 11 (IL-11), interleukin 12 (IL-12), interleukin 15 (IL-15), and interleukin 21 (IL-21), and variants and derivatives thereof.

[0464] According to the present disclosure, cytokine can be naturally occurring cytokine or its functional fragment or variant.Cytokine can be human cytokine and can be derived from any vertebrate, particularly any mammal.A particularly preferred cytokine is interferon-α.

[0465] An immunostimulatory agent can be provided to a subject by administering to the subject RNA encoding the immunostimulatory agent in a formulation for preferential delivery of the RNA to the liver or liver tissue. Delivery of RNA to such target organs or tissues is preferred, particularly when it is desired to express large amounts of the immunostimulatory agent and / or when a particularly significant amount of the immunostimulatory agent is desired or required systemically.

[0466] RNA delivery systems have an inherent preference for the liver. This concerns lipid-based particles, cationic and neutral nanoparticles, especially lipid nanoparticles.

[0467] Examples of immune stimulants suitable for targeting the liver are cytokines involved in the proliferation and / or maintenance of T cells. Examples of suitable cytokines include IL2 or IL7, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants, such as extended PK cytokines.

[0468] In another embodiment, the RNA encoding the immunostimulant can be administered in a formulation that preferentially delivers the RNA to the lymphatic system, particularly to secondary lymphatic organs, more particularly to the spleen. Delivery of the immunostimulant to such a target tissue is particularly preferred when the presence of the immunostimulant in this organ or tissue is desired (for example, when the immunostimulant, such as cytokines, is required to induce an immune response, particularly during T cell priming, or for the activation of resident immune cells), but the immunostimulant is not desired to be present systemically, especially in significant amounts (for example, because the immunostimulant has systemic toxicity).

[0469] Examples of suitable immune stimulants are cytokines involved in T cell priming, including IL12, IL15, IFN-α, or IFN-β, fragments and variants thereof, and fusion proteins of these cytokines, fragments, and variants, such as extended PK cytokines.

[0470] Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, parasites, and tumor cells. In a typical scenario, a cell infected with a virus releases interferons to mount an antiviral defense in nearby cells.

[0471] Based on the type of receptor through which they signal, interferons are typically divided into three classes: type I interferons, type II interferons, and type III interferons.

[0472] All type I interferons bind to a specific cell surface receptor complex known as the IFN-α / β receptor (IFNAR), which consists of the IFNAR1 and IFNAR2 chains.

[0473] The type I interferons present in humans are IFNα, IFNβ, IFNε, IFNκ, and IFNω. Generally, type I interferons are produced when the body recognizes an invading virus. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, which leads to the expression of proteins that prevent the virus from producing and replicating its RNA and DNA.

[0474] IFNα proteins are primarily produced by plasmacytoid dendritic cells (pDCs). They are primarily involved in innate immunity against viral infections. The genes responsible for their synthesis are derived from 13 subtypes called IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21. These genes are found together in a cluster on chromosome 9.

[0475] IFN-β proteins are produced in large amounts by fibroblasts. They have antiviral activity, primarily involved in the innate immune response. Two types of IFN-β, IFN-β1 and IFN-β3, have been described. Natural and recombinant forms of IFN-β1 have antiviral, antibacterial, and anticancer properties.

[0476] Type II interferons (IFNγ in humans), also known as immune interferons, are activated by IL12 and are also released by cytotoxic T cells and T helper cells.

[0477] Type III interferons signal through a receptor complex consisting of IL10R2 (also called CRF2-4) and IFNLR1 (also called CRF2-12). Although more recently discovered than type I and type II IFNs, recent information demonstrates the importance of type III IFNs in several types of viral or fungal infections.

[0478] In general, type I and type II interferons are responsible for regulating and activating the immune response.

[0479] According to the present disclosure, the type I interferon is preferably IFNα or IFNβ, more preferably IFNα.

[0480] According to the present disclosure, the interferon can be a naturally occurring interferon, or a functional fragment or variant thereof. The interferon can be a human interferon and can be derived from any vertebrate, particularly any mammal.

[0481] Interleukins (ILs) are a group of cytokines (secreted proteins and signaling molecules) that can be divided into four major groups based on distinct structural features. However, their amino acid sequence similarity is rather weak (typically 15-25% identity). The human genome encodes over 50 interleukins and related proteins.

[0482] According to the present disclosure, the interleukin can be a naturally occurring interleukin, or a functional fragment or variant thereof. The interleukin can be a human interleukin and can be derived from any vertebrate, particularly any mammal.

[0483] The immunostimulatory polypeptides described herein can be prepared as fusion or chimeric polypeptides comprising an immunostimulatory substance portion and a heterologous polypeptide (i.e., a polypeptide that is not an immunostimulatory substance). The immunostimulatory substance may be fused to an extended PK group that increases its circulating half-life. Non-limiting examples of extended PK groups are described below. It should be understood that other PK groups that increase the circulating half-life of immunostimulatory substances, such as cytokines or variants thereof, are also applicable to the present disclosure. In certain embodiments, the extended PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

[0484] As used herein, the term "PK" is an acronym for "pharmacokinetics" and encompasses the properties of a compound, including, by way of example, absorption, distribution, metabolism, and elimination by a subject. As used herein, an "extended PK group" refers to a protein, peptide, or moiety that, when fused to or administered together with a biologically active molecule, increases the circulating half-life of the biologically active molecule. Examples of extended PK groups include serum albumin (e.g., HSA), immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (such as those disclosed in U.S. Publication Nos. 2005 / 0287153 and 2007 / 0003549). Other exemplary extended PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7):903-15, which is incorporated herein by reference in its entirety. As used herein, an "extended PK" immunostimulant refers to an immunostimulant moiety combined with an extended PK group. In some embodiments, the extended PK immunostimulant is a fusion protein in which the immunostimulant moiety is linked or fused to the extended PK group.

[0485] In certain embodiments, the serum half-life of the extended PK immunostimulant is increased compared to the immunostimulant alone (i.e., the immunostimulant not fused to the extended PK group). In certain embodiments, the serum half-life of the extended PK immunostimulant is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or 1000% longer than the serum half-life of the immunostimulant alone. In certain embodiments, the serum half-life of the extended PK immunostimulant is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50-fold longer than the serum half-life of the immunostimulant alone. In certain embodiments, the serum half-life of the extended PK immunostimulant is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

[0486] As used herein, "half-life" refers to the time required for the serum or plasma concentration of a compound, such as a peptide or polypeptide, to be reduced by 50% in vivo, e.g., due to degradation and / or clearance or sequestration by natural mechanisms. Suitable extended PK immunostimulants for use herein are stabilized in vivo, and their half-life is increased, e.g., by fusion to serum albumin (e.g., HSA or MSA), which resists degradation and / or clearance or sequestration. Half-life can be determined by any method known per se, such as pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and may, for example, generally include administering a suitable dose of an amino acid sequence or compound to a subject, periodically collecting blood or other samples from the subject, determining the level or concentration of the amino acid sequence or compound in the blood samples, and calculating, from a plot of the data thus obtained, the time until the level or concentration of the amino acid sequence or compound is reduced by 50% compared to the initial level at the time of administration. Further details are provided in standard handbooks such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). See also Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

[0487] In certain embodiments, the extended PK group comprises serum albumin or a fragment thereof, or a variant of serum albumin or a fragment thereof (all of which are encompassed by the term "albumin" for purposes of this disclosure). The polypeptides described herein may be fused to albumin (or a fragment or variant thereof) to form an albumin fusion protein. Such albumin fusion proteins are described in U.S. Publication No. 20070048282.

[0488] As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) to at least one molecule of a protein, such as a Therapeutic protein, particularly an immunostimulant. Albumin fusion proteins can be produced by translation of a nucleic acid in which a polynucleotide encoding a Therapeutic protein is joined in-frame with a polynucleotide encoding albumin. The Therapeutic protein and albumin are once part of the albumin fusion protein, and each can be referred to as a "portion," "region," or "moiety" of the albumin fusion protein (e.g., a "Therapeutic protein portion" or an "albumin protein portion"). In highly preferred embodiments, the albumin fusion protein comprises at least one molecule of a Therapeutic protein (including, but not limited to, the mature form of a Therapeutic protein) and at least one molecule of albumin (including, but not limited to, the mature form of albumin). In some embodiments, the albumin fusion protein is processed by host cells, such as cells of a target organ for the administered RNA (e.g., liver cells), and secreted into the circulation. Processing of the nascent albumin fusion protein in the secretory pathway of the host cell used to express the RNA may include, but is not limited to, signal peptide cleavage, disulfide bond formation, proper folding, carbohydrate addition and processing (e.g., N- and O-linked glycosylation), specific proteolytic cleavage, and / or assembly into multimeric proteins. The albumin fusion protein is preferably encoded by the RNA in an unprocessed form, particularly with a signal peptide at the N-terminus, and subsequent secretion by the cell is preferably in a processed form, particularly with the signal peptide cleaved. In a most preferred embodiment, the "processed form of the albumin fusion protein" refers to an albumin fusion protein product that has undergone N-terminal signal peptide cleavage, also referred to herein as the "mature albumin fusion protein."

[0489] In preferred embodiments, albumin fusion proteins comprising a Therapeutic protein have greater plasma stability than the plasma stability of the same Therapeutic protein when not fused to albumin. Plasma stability typically refers to the period of time a Therapeutic protein is administered in vivo and transported into the bloodstream, and the period of time it takes for the Therapeutic protein to be degraded and cleared from the bloodstream, ultimately entering organs such as the kidneys or liver that clear the Therapeutic protein from the body. Plasma stability is calculated in terms of the half-life of the Therapeutic protein in the bloodstream. The half-life of a Therapeutic protein in the bloodstream can be readily determined by common assays known in the art.

[0490] As used herein, "albumin" collectively refers to an albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or a fragment or variant thereof, particularly the mature form of human albumin, or albumin or a fragment thereof from another vertebrate, or a variant of these molecules. Albumin may be derived from any vertebrate, particularly any mammal, such as human, bovine, ovine, or porcine. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein may be derived from a different animal than the therapeutic protein portion.

[0491] In certain embodiments, the albumin is human serum albumin (HSA), or a fragment or variant thereof, such as those disclosed in US 5,876,969, WO2011 / 124718, WO2013 / 075066, and WO2011 / 0514789.

[0492] The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are broader and encompass human serum albumin (and fragments and variants thereof) as well as albumins from other species (and fragments and variants thereof).

[0493] As used herein, a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of a Therapeutic protein refers to a fragment of albumin of sufficient length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein, such that the plasma stability of the Therapeutic protein portion of the albumin fusion protein is extended or prolonged compared to the plasma stability of the unfused state.

[0494] The albumin portion of the albumin fusion protein may comprise the full-length albumin sequence, or may comprise one or more fragments thereof that are capable of stabilizing or extending therapeutic activity or plasma stability. Such fragments may be 10 or more amino acids in length, or may comprise about 15, 20, 25, 30, 50, or more consecutive amino acids from the albumin sequence, or may comprise some or all of a particular domain of albumin. For example, one or more fragments of HSA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.

[0495] Generally speaking, an albumin fragment or variant is at least 100 amino acids in length, preferably at least 150 amino acids in length.

[0496] According to the present disclosure, the albumin can be naturally occurring albumin, or a fragment or variant thereof. The albumin can be human albumin and can be derived from any vertebrate, particularly any mammal.

[0497] Preferably, the albumin fusion protein comprises albumin as the N-terminal moiety and a Therapeutic protein as the C-terminal moiety. Alternatively, albumin fusion proteins comprising albumin as the C-terminal moiety and a Therapeutic protein as the N-terminal moiety may be used. In other embodiments, the albumin fusion protein has a Therapeutic protein fused to both the N- and C-termini of albumin. In a preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are the same Therapeutic protein. In another preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are different Therapeutic proteins. In some embodiments, the different Therapeutic proteins are both cytokines.

[0498] In some embodiments, the therapeutic protein(s) are conjugated to albumin via (a) a peptide linker(s). The linker peptide between the fusion moieties can provide greater physical separation between the moieties, thus maximizing the accessibility of the therapeutic protein moiety to bind to its cognate receptor, for example. The linker peptide can be composed of amino acids that make it flexible or more rigid. The linker sequence can be cleavable by proteases or chemically.

[0499] As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the Fc domains (or Fc portions) of each of its two heavy chains. As used herein, the term "Fc domain" refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain, wherein the Fc domain does not include an Fv domain. In certain embodiments, an Fc domain begins at the hinge region immediately upstream of the papain cleavage site of the antibody and ends at the C-terminus. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc domain comprises at least one of a hinge (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, an Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain comprises a CH2 domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH3 domain or portion thereof. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH3 domain (or portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or portion thereof) and a CH2 domain (or portion thereof). In certain embodiments, an Fc domain lacks at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). An Fc domain, as used herein, generally refers to a polypeptide comprising all or a portion of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and / or CH3 domains, as well as fragments of such peptides comprising, for example, only the hinge, CH2, and CH3 domains.The Fc domain can be derived from any species and / or any subtype of immunoglobulin, including, but not limited to, human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. Fc domains encompass naturally occurring Fc and Fc variant molecules. As described herein, it will be understood by those skilled in the art that any Fc domain can be modified to vary in amino acid sequence from the native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., FcγR binding).

[0500] The Fc domains of the polypeptides described herein can be derived from different immunoglobulin molecules. For example, the Fc domain of the polypeptide can comprise a CH2 and / or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the Fc domain can comprise a chimeric hinge region derived, in part, from an IgG1 molecule and, in part, from an IgG3 molecule. In another example, the Fc domain can comprise a chimeric hinge derived, in part, from an IgG1 molecule and, in part, from an IgG4 molecule.

[0501] In certain embodiments, the extended PK group comprises an Fc domain or fragment thereof, or a variant of an Fc domain or fragment thereof (all of which are encompassed by the term "Fc domain" for purposes of this disclosure). An Fc domain does not contain a variable region that binds to an antigen. Fc domains suitable for use in the present disclosure can be obtained from several different sources. In certain embodiments, the Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is derived from a human IgG1 constant region. However, it is understood that the Fc domain can be derived from the immunoglobulin of another mammalian species, including, for example, a rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species.

[0502] Furthermore, the Fc domain (or a fragment or variant thereof) can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

[0503] Various Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of public deposits. Constant region domains, including Fc domain sequences, can be selected that lack specific effector functions and / or have specific modifications to reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and suitable Fc domain sequences (e.g., hinge, CH2, and / or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using art-recognized techniques.

[0504] In certain embodiments, the extended PK group is a serum albumin binding protein, such as those described in US2005 / 0287153, US2007 / 0003549, US2007 / 0178082, US2007 / 0269422, US2010 / 0113339, WO2009 / 083804, and WO2009 / 133208, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is transferrin, as disclosed in US7,176,278 and US8,158,579, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is a serum immunoglobulin-binding protein, such as those disclosed in US2007 / 0178082, US2014 / 0220017, and US2017 / 0145062 (which are incorporated by reference in their entireties). In certain embodiments, the extended PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as those disclosed in US2012 / 0094909 (which is incorporated by reference in its entirety). Methods of making fibronectin-based scaffold domain proteins are also disclosed in US2012 / 0094909. A non-limiting example of an Fn3-based extended PK group is Fn3(HSA), i.e., an Fn3 protein that binds to human serum albumin.

[0505] In certain embodiments, extended PK immunostimulatory agents suitable for use in accordance with the present disclosure may employ one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that connects two or more domains (e.g., an extended PK portion and an immunostimulatory portion) within the linear amino acid sequence of a polypeptide chain. For example, a peptide linker may be used to connect an immunostimulatory agent portion to an HSA domain.

[0506] Linkers suitable for fusing an extended PK group to, for example, an immunostimulatory agent are well known in the art. Exemplary linkers include a glycine-serine-polypeptide linker, a glycine-proline-polypeptide linker, and a proline-alanine polypeptide linker. In certain embodiments, the linker is a glycine-serine-polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

[0507] In some embodiments, the pharmaceutically active peptide or polypeptide comprises a replacement protein. In these embodiments, the present disclosure provides a method of treating a subject having a disorder requiring protein replacement (e.g., a protein deficiency disorder), comprising administering to the subject a nucleic acid as described herein encoding the replacement protein. The term "protein replacement" refers to the introduction of a protein (including a functional variant thereof) into a subject having a deficiency of such a protein. The term also refers to the introduction of a protein into a subject who would otherwise require or benefit from providing the protein, e.g., suffering from a protein deficiency. The term "disorder characterized by protein deficiency" refers to any disorder exhibiting pathology caused by a lack of protein or an insufficient amount of protein. This term encompasses protein folding disorders, i.e., conformational disorders, that result in biologically inactive protein products. Protein deficiency may be involved in infectious disease, immunosuppression, organ failure, glandular disorders, radiation sickness, nutritional deficiency, poisoning, or other environmental or external damage.

[0508] The term "hormone" refers to a class of signaling molecules produced by glands, where signal transduction typically involves the following steps: (i) synthesis of the hormone in a specific tissue; (ii) storage and secretion; (iii) transport of the hormone to its target; (iv) binding of the hormone by a receptor; (v) signal relay and amplification; and (vi) degradation of the hormone. Hormones differ from cytokines in that (1) hormones typically act at less variable concentrations and (2) are generally made by specific types of cells. In some embodiments, a "hormone" is a peptide or polypeptide hormone, such as insulin, vasopressin, prolactin, adrenocorticotropic hormone (ACTH), thyroid hormone, growth hormone (such as human growth hormone or bovine somatotropin), oxytocin, atrial natriuretic peptide (ANP), glucagon, somatostatin, cholecystokinin, gastrin, and leptin.

[0509] The term "adhesion molecule" refers to proteins located on the surface of cells and involved in binding of cells to other cells or to the extracellular matrix (ECM). Adhesion molecules are typically transmembrane receptors and can be classified as calcium-independent (e.g., integrins, immunoglobulin superfamily, lymphocyte homing receptors) and calcium-dependent (cadherins and selectins). Specific examples of adhesion molecules include integrins, lymphocyte homing receptors, selectins (e.g., P-selectin), and addressins.

[0510] Integrins are also involved in signal transduction. In particular, upon ligand binding, integrins regulate cell signaling pathways, for example, transmembrane protein kinase pathways such as receptor tyrosine kinases (RTKs). Such regulation can result in cell growth, division, survival, or differentiation, or apoptosis. Specific examples of integrins include α1β1, α2β1, α3β1, α4β1, α5β1, α6β1, α7β1, α L β2, α M β2, α IIb β3, α Vβ1, α V β3, α V β5, α V β6, α V β8, and α6β4.

[0511] The term "immunoglobulin" or "immunoglobulin superfamily" refers to molecules involved in cell recognition, binding, and / or adhesion processes. Molecules belonging to this superfamily share the characteristic of containing regions known as immunoglobulin domains or immunoglobulin folds. Members of the immunoglobulin superfamily include antibodies (e.g., IgG), T cell receptors (TCRs), major histocompatibility complex (MHC) molecules, co-receptors (e.g., CD4, CD8, CD19), antigen receptor accessory molecules (e.g., CD3-γ, CD3-δ, CD3-ε, CD79a, CD79b), costimulatory or inhibitory molecules (e.g., CD28, CD80, CD86), and others.

[0512] The term "immunoactive compound" relates to any compound that modifies the immune response, for example, by inducing and / or suppressing immune cell maturation, inducing and / or suppressing cytokine biosynthesis, and / or modifying humoral immunity by stimulating antibody production by B cells. Immunoactive compounds have potent immunostimulatory activity, including, but not limited to, antiviral and antitumor activity, and can also downregulate other aspects of the immune response, for example, shifting the immune response away from a TH2 immune response, which is useful for treating a wide range of TH2-mediated diseases. Immunoactive compounds may be useful as vaccine adjuvants. Specific examples of immunoactive compounds include interleukins, colony-stimulating factors (CSFs), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, tumor necrosis factor (TNF), interferons, integrins, addressins, selectins, homing receptors, and antigens, particularly tumor-associated antigens, pathogen-associated antigens (such as bacterial, parasitic, or viral antigens), allergens, and autoantigens. The immunoactive compound may be a vaccine antigen, i.e., an antigen whose inoculation into a subject induces an immune response.

[0513] In some embodiments, the RNA used in this disclosure comprises a nucleic acid sequence that encodes a peptide or polypeptide comprising an epitope for inducing an immune response to an antigen in a subject. A "peptide or polypeptide comprising an epitope for inducing an immune response to an antigen in a subject" is also designated herein as a "vaccine antigen," "peptide and protein antigen," or simply "antigen."

[0514] In some embodiments, the RNA encoding the vaccine antigen is a single-stranded 5'-capped mRNA that is translated into the respective protein upon entry into the cells of a subject receiving the RNA, e.g., antigen-presenting cells (APCs). Preferably, the RNA contains structural elements optimized for maximum effectiveness of the RNA with respect to stability and translation efficiency (5'-cap, 5'-UTR, 3'-UTR, poly(A) sequence).

[0515] In some embodiments, beta-S-ARCA(D1) is utilized as a specific capping structure at the 5' end of the RNA. In some embodiments, the 5'-UTR comprises the nucleotide sequence of SEQ ID NO:6 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:6. In some embodiments, the 3'-UTR comprises the nucleotide sequence of SEQ ID NO:7 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:7. In some embodiments, the poly(A) sequence is 110 nucleotides long and consists of a stretch of 30 adenosine residues, followed by a 10-nucleotide linker sequence and another 70 adenosine residues. This poly(A) sequence was designed to improve RNA stability and translation efficiency in dendritic cells. In some embodiments, the poly(A) sequence comprises the nucleotide sequence of SEQ ID NO:8 or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO:8.

[0516] In some embodiments, RNA encoding the vaccine antigen is expressed in cells of a subject to provide the vaccine antigen. In some embodiments, expression of the vaccine antigen is on the cell surface. In some embodiments, the vaccine antigen is presented in the context of an MHC. In some embodiments, RNA encoding the vaccine antigen is transiently expressed in cells of a subject. In some embodiments, RNA encoding the vaccine antigen is administered systemically. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in expression of RNA encoding the vaccine antigen in the spleen. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in expression of RNA encoding the vaccine antigen in antigen-presenting cells, preferably professional antigen-presenting cells. In some embodiments, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in no or essentially no expression of RNA encoding the vaccine antigen in the lung and / or liver. In some embodiments, systemic administration of RNA encoding the vaccine antigen results in expression of RNA encoding the vaccine antigen in the spleen that is at least five times higher than the level of expression in the lung.

[0517] A vaccine antigen comprises an epitope for inducing an immune response against the antigen in a subject. Thus, a vaccine antigen comprises an antigen sequence for inducing an immune response against the antigen in a subject. Such an antigen sequence may correspond to a protein of a target antigen or a disease-related antigen, such as an infectious agent (e.g., a viral or bacterial antigen) or a tumor antigen, or an immunogenic variant thereof, or an immunogenic fragment of the target antigen or disease-related antigen or its immunogenic variant. Thus, the antigen sequence may comprise at least an epitope of the target antigen or disease-related antigen or its immunogenic variant.

[0518] Antigen sequences, e.g., epitopes, suitable for use in accordance with the present disclosure will typically be derived from the target antigen, i.e., the antigen to which an immune response is elicited. For example, the antigen sequence contained within a vaccine antigen may be the target antigen or a fragment or variant of the target antigen.

[0519] The antigen sequence or its processing product, e.g., a fragment thereof, can bind to an antigen receptor, such as a TCR or CAR, carried by an immune effector cell. In some embodiments, the antigen sequence is selected from the group consisting of an antigen expressed by a target cell, or a fragment thereof, to which the immune effector cell is targeted, or a variant of the antigen sequence or fragment.

[0520] Vaccine antigens that can be provided to a subject according to the present disclosure by administering RNA encoding the vaccine antigen preferably result in the induction of an immune response in the subject provided with the vaccine antigen, e.g., stimulation, priming, and / or expansion of immune effector cells. The immune response, e.g., stimulated, primed, and / or expanded immune effector cells, is preferably directed against a target antigen, particularly a target antigen expressed by diseased cells, diseased tissues, and / or diseased organs, i.e., a disease-associated antigen. Thus, the vaccine antigen can include a disease-associated antigen, or a fragment or variant thereof. In some embodiments, such a fragment or variant is immunologically equivalent to the disease-associated antigen.

[0521] In the context of the present disclosure, the term "antigen fragment" or "antigen variant" refers to an agent that results in the induction of an immune response, e.g., stimulation, priming, and / or expansion of immune effector cells, where the immune response, e.g., stimulation, priming, and / or expansion of immune effector cells, targets an antigen, i.e., a disease-associated antigen, particularly when presented by diseased cells, diseased tissues, and / or diseased organs. Thus, a vaccine antigen may correspond to or comprise a disease-associated antigen, or may correspond to or comprise a fragment of a disease-associated antigen, or may correspond to or comprise an antigen that is homologous to a disease-associated antigen or a fragment thereof. When a vaccine antigen comprises a fragment of a disease-associated antigen or an amino acid sequence homologous to a fragment of a disease-associated antigen, the fragment or amino acid sequence may comprise an epitope of the disease-associated antigen or a sequence homologous to an epitope of the disease-associated antigen to which the antigen receptor of an immune effector cell is targeted. Thus, according to the present disclosure, a vaccine antigen can comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-associated antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen that can induce, for example, a stimulating, priming, and / or expanding immune response against immune effector cells bearing antigen receptors that bind to the antigen or cells expressing the antigen. Vaccine antigens (like disease-associated antigens) preferably provide relevant epitopes for binding by antigen receptors present on immune effector cells. In some embodiments, vaccine antigens or fragments thereof (like disease-associated antigens) are expressed on the surface of cells, such as antigen-presenting cells (optionally in the context of an MHC) so as to provide relevant epitopes for binding by immune effector cells. Vaccine antigens can be recombinant antigens.

[0522] In some embodiments of all aspects of the invention, RNA encoding a vaccine antigen is expressed in cells of a subject to provide the antigen or its processing products for binding by antigen receptors expressed by immune effector cells, which binding results in stimulation, priming, and / or expansion of the immune effector cells. An "antigen" according to the present disclosure encompasses any substance that elicits an immune response and / or any substance against which an immune mechanism, such as an immune or cellular and / or humoral response, is directed. This also includes situations in which an antigen is processed into antigenic peptides and an immune response or mechanism is directed against one or more antigenic peptides, particularly when presented in the context of an MHC molecule. In particular, "antigen" relates to any substance, such as a peptide or polypeptide, that specifically reacts with antibodies or T lymphocytes (T cells). The term "antigen" can include molecules that contain at least one epitope, such as a T cell epitope. In some embodiments, an antigen is a molecule that, optionally after processing, induces an immune response that may be specific to the antigen (including cells expressing the antigen). In some embodiments, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0523] In some embodiments, the antigen is presented or present on the surface of a cell of the immune system, such as an antigen-presenting cell, such as a dendritic cell or macrophage. In some embodiments, the antigen or its processing product, such as a T cell epitope, is bound by an antigen receptor. Thus, the antigen or its processing product can specifically react with immune effector cells, such as T lymphocytes (T cells).

[0524] The term "autoantigen" or "self-antigen" refers to an antigen that originates within a subject's body (i.e., an autoantigen may also be called a "self-antigen") and that produces an abnormally vigorous immune response against this normal part of the body. Such a vigorous immune response against an autoantigen can cause an "autoimmune disease."

[0525] According to the present disclosure, any suitable antigen that is a candidate for an immune response may be used, and the immune response may include a humoral immune response, a cellular immune response, or both. In the context of some embodiments of the present disclosure, the antigen is presented by a cell, such as an antigen-presenting cell, in the context of an MHC molecule, resulting in an immune response against the antigen. The antigen may correspond to a naturally occurring antigen or a product derived from a naturally occurring antigen. Such naturally occurring antigens may include or be derived from allergens, viruses, bacteria, fungi, parasites, and other infectious agents, and the pathogen or antigen may also be a tumor antigen. According to the present disclosure, the antigen may correspond to a naturally occurring product, for example, a viral protein, or a portion thereof.

[0526] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Disease-associated antigens include pathogen-associated antigens, i.e., antigens associated with infection by microorganisms, typically microbial antigens (such as bacterial or viral antigens), or antigens associated with cancer, typically tumors, e.g., tumor antigens.

[0527] In some embodiments, the antigen is a tumor antigen, i.e., part of a tumor cell, particularly one that occurs primarily intracellularly or as a surface antigen on tumor cells. In another embodiment, the antigen is a pathogen-associated antigen, i.e., an antigen derived from a pathogen, e.g., a virus, bacterium, unicellular organism, or parasite, e.g., a viral antigen such as a viral ribonucleoprotein or coat protein. In some embodiments, the antigen should be presented by an MHC molecule that results in modulation, particularly activation of cells of the immune system, such as CD4+ and CD8+ lymphocytes, particularly through modulation of T cell receptor activity.

[0528] The term "tumor antigen" or "tumor-associated antigen" refers to a component of a cancer cell that may originate from the cytoplasm, cell surface, or cell nucleus. In particular, it refers to an antigen that is produced intracellularly or as a surface antigen on a tumor cell. For example, tumor antigens include carcinoembryonic antigen, α1-fetoprotein, isoferritin, and fetal sulfoglycoprotein, α2-H-ferroprotein, and γ-fetoprotein, as well as various viral tumor antigens. According to some embodiments of the present disclosure, tumor antigens include any antigen that is characteristic of a tumor or cancer, and tumor or cancer cells, in terms of type and / or expression level.

[0529] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0530] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0531] The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., a portion or fragment of a molecule that is recognized by the immune system, e.g., by antibodies, T cells, or B cells, particularly when presented in the context of an MHC molecule. An epitope of a protein can include a continuous or discontinuous portion of the protein and can be about 5 to about 100, about 5 to about 50, about 8 to about 30, or about 10 to about 25 amino acids in length; for example, an epitope can preferably be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope in the context of the present disclosure is a T cell epitope.

[0532] Terms such as "epitope," "fragment of an antigen," "immunogenic peptide," and "antigenic peptide" are used interchangeably herein and may refer, for example, to an antigen or an incomplete representation of an antigen that is capable of eliciting an immune response against a cell that expresses or contains the antigen and presents the antigen. In some embodiments, the term refers to an immunogenic portion of an antigen. In some embodiments, it is the portion of the antigen that is recognized (i.e., specifically bound) by a T cell receptor, particularly when presented in the context of an MHC molecule. Certain preferred immunogenic portions bind to MHC class I or class II molecules. The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by a T cell, a B cell, or an antibody. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and can be about 5 to about 100, e.g., about 5 to about 50, about 8 to about 30, or about 8 to about 25 amino acids in length; for example, an epitope can be 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0533] The term "T cell epitope," when presented in the context of an MHC molecule, refers to a portion or fragment of a protein that is recognized by T cells. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during the immune response; they bind peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, the bound peptide is typically about 8 to about 10 amino acids in length, although longer or shorter peptides can be effective. For class II MHC / peptide complexes, the binding peptides are typically about 10 to about 25 amino acids in length, particularly about 13 to about 18 amino acids in length, although longer and shorter peptides may be effective.

[0534] Peptide and polypeptide antigens can be 2 to 100 amino acids in length, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

[0535] A peptide or polypeptide antigen can be any peptide or polypeptide that is capable of inducing or increasing the ability of the immune system to develop antibody and T-cell responses against the peptide or polypeptide.

[0536] In some embodiments, vaccine antigens, i.e., antigens whose inoculation into a subject induces an immune response, are recognized by immune effector cells. In some embodiments, when recognized by immune effector cells, the vaccine antigen, in the presence of appropriate costimulatory signals, can induce stimulation, priming, and / or expansion of immune effector cells bearing antigen receptors that recognize the vaccine antigen. In the context of embodiments of the present disclosure, vaccine antigens are presented or present on the surface of cells, such as, for example, antigen-presenting cells.

[0537] In some embodiments, the antigen is expressed in diseased cells (such as cancer cells or infected cells).

[0538] In some embodiments, the antigen is presented by a diseased cell (such as a cancer cell or an infected cell). In some embodiments, the antigen receptor is a TCR that binds to an epitope of the antigen presented in the context of an MHC. In some embodiments, binding of the TCR, when expressed by and / or present on a T cell, to an antigen presented by a cell, such as an antigen-presenting cell, results in stimulation, priming, and / or expansion of the T cell. In some embodiments, binding of the TCR, when expressed by and / or present on a T cell, to an antigen presented on a diseased cell results in cytolysis and / or apoptosis of the diseased cell, and the T cell releases cytotoxic factors, e.g., perforin and granzymes.

[0539] In some embodiments, the antigen is expressed on the surface of a diseased cell (such as a tumor cell or an infected cell). In some embodiments, the antigen receptor is a CAR that binds to an extracellular domain or an epitope within the extracellular domain of the antigen. In some embodiments, the CAR binds to a native epitope of the antigen present on the surface of a living cell. In some embodiments, binding of the CAR, when expressed by and / or present on a T cell, to an antigen presented on a cell, such as an antigen-presenting cell, results in stimulation, priming, and / or expansion of the T cell. In some embodiments, binding of the CAR, when expressed by and / or present on a T cell, to an antigen presented on a diseased cell results in cytolysis and / or apoptosis of the diseased cell, and the T cell preferably releases cytotoxic factors, such as perforin and granzymes.

[0540] According to some embodiments, the amino acid sequence that enhances antigen processing and / or presentation is fused to the antigenic peptide or polypeptide (antigen sequence) either directly or via a linker. Thus, in some embodiments, the RNA described herein comprises at least one coding region that encodes an antigenic peptide or polypeptide and an amino acid sequence that enhances antigen processing and / or presentation.

[0541] In some embodiments, antigens for vaccination that may be administered in the form of RNA encoding therefor include naturally occurring antigens or fragments thereof, such as epitopes thereof.

[0542] Such amino acid sequences that enhance antigen processing and / or antigen presentation are preferably, but not limited to, located at the C-terminus of the antigenic peptide or antigenic polypeptide (and optionally at the C-terminus of the amino acid sequence that breaks immune tolerance). Amino acid sequences that enhance antigen processing and / or antigen presentation as defined herein preferably improve antigen processing and antigen presentation. In some embodiments, amino acid sequences that enhance antigen processing and / or antigen presentation as defined herein include, but are not limited to, sequences derived from the human MHC class I complex (HLA-B51, haplotype A2, B27 / B51, Cw2 / Cw3), particularly a sequence comprising the amino acid sequence of SEQ ID NO: 2, or a functional variant thereof.

[0543] In some embodiments, the amino acid sequence that enhances antigen processing and / or antigen presentation comprises the amino acid sequence of SEQ ID NO:2, an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:2, or a functional fragment of the amino acid sequence of SEQ ID NO:2, or an amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the amino acid sequence of SEQ ID NO:2. In some embodiments, the amino acid sequence that enhances antigen processing and / or antigen presentation comprises the amino acid sequence of SEQ ID NO:2.

[0544] Thus, in some embodiments, the RNA described herein comprises at least one coding region encoding an antigenic peptide or polypeptide and an amino acid sequence that enhances antigen processing and / or antigen presentation, wherein the amino acid sequence that enhances antigen processing and / or antigen presentation is preferably fused to the antigenic peptide or polypeptide, more preferably to the C-terminus of the antigenic peptide or polypeptide as described herein.

[0545] Furthermore, a secretory sequence, for example a sequence comprising the amino acid sequence of SEQ ID NO: 1, may be fused to the N-terminus of the antigenic peptide or antigenic polypeptide.

[0546] Amino acid sequences derived from tetanus toxoid of Clostridium tetani can be used to overcome self-tolerance mechanisms to efficiently initiate immune responses to self-antigens by providing T cell help during priming.

[0547] Tetanus toxoid heavy chains bind promiscuously to MHC class II alleles and bind to CD4 in almost all tetanus-vaccinated individuals. + It is known that these antigens contain epitopes that can induce memory T cells. In addition, the combination of tetanus toxoid (TT) helper epitopes with tumor-associated antigens induces CD4 T cell activation during priming. + It is known that administration of tumor-associated antigens alone improves immune stimulation by providing mediated T cell help, thereby improving immune stimulation compared to application of tumor-associated antigens alone. + To reduce the risk of stimulating T cells, CD8 + The entire fragment C of tetanus toxoid is not used because it is known to contain a T cell epitope. To ensure binding to as many MHC class II alleles as possible, two peptide sequences containing promiscuous binding helper epitopes were instead selected. Based on data from ex vivo studies, the well-known epitope p2 (QYIKANSKFIGITEL; TT 830-844 ) and p16(MTNSVDDALINSTKIYSYFPSVISKVNQGAQG;TT 578-609 ) was selected. The p2 epitope has already been used in peptide vaccination in clinical trials to boost anti-melanoma activity.

[0548] Preclinical data show that an RNA vaccine encoding both a tumor antigen and a promiscuous tetanus toxoid sequence can inhibit CD8 T cell proliferation directed against the tumor antigen. +These results demonstrate that the tetanus sequences induce enhanced T cell responses and improved tolerance breaking. Immune monitoring data from patients vaccinated with vaccines containing sequences fused in-frame wit...

Claims

1. A method for providing a pharmaceutical RNA preparation for administering different doses of RNA, (i) The step of determining different doses in which the RNA in the pharmaceutical RNA preparation is administered, (ii) A step of determining the concentration of the RNA in the pharmaceutically acceptable RNA preparation, which enables the administration of the different doses of the RNA in a suitable dose volume, (iii) The step of determining a suitable formulation of the pharmaceutically acceptable RNA preparation to ensure the desired storage stability of the RNA in the pharmaceutically acceptable RNA preparation at the determined concentration, The method wherein the RNA encodes an amino acid sequence comprising the SARS-CoV-2 S protein, an immunogenic variant thereof, or an immunogenic fragment of the SARS-CoV-2 S protein or an immunogenic variant thereof.

2. The method according to claim 1, wherein the concentration of the RNA in the pharmaceutical RNA preparation allows for suitable dilution of the pharmaceutical RNA preparation.

3. The method according to claim 1 or 2, wherein the minimum dose and the maximum dose of the RNA differ by more than two times, and the minimum dose volume and the maximum dose volume differ by less than two times.

4. The method according to claim 1, wherein the RNA in different doses is administered to different age groups and / or different conditions.

5. The method according to claim 1, wherein the minimum dose and the maximum dose of the RNA differ by more than two times.

6. The method according to claim 1, wherein the minimum dose volume for one dose and the maximum dose volume for another dose differ by 1.5 times or less.

7. The method according to claim 1, wherein the preferred administration volume is 100 to 400 μl.

8. (a) The pharmaceutical RNA preparation is diluted to a ratio of 1:10 or less, and at least one of the different doses is achieved. (b) The method according to claim 1, wherein at least one dose of the RNA is achieved without diluting the pharmaceutically acceptable RNA preparation.

9. (a) The different doses include doses of approximately 3 μg, approximately 10 μg, and approximately 30 μg. (b) The concentration of the RNA in the pharmaceutical RNA preparation is about 0.1 mg / ml, and / or (c) The method according to claim 1, wherein the administration volume is approximately 200 μl to approximately 300 μl.

10. The method according to claim 1, wherein the RNA in the pharmaceutical RNA preparation is formulated in about 10 mM Tris buffer and about 10% sucrose.

11. The concentration of the RNA in the pharmaceutical RNA preparation is approximately 0.1 mg / ml, and the RNA in the pharmaceutical RNA preparation is formulated in approximately 10 mM Tris buffer and approximately 10% sucrose. (i) A first dose of the RNA, approximately 30 μg, is administered by administering approximately 300 μl of undiluted pharmaceutically acceptable RNA preparation. (ii) The method according to claim 1, wherein a second dose of the RNA, approximately 10 μg, is administered by diluting the pharmaceutically acceptable RNA preparation in a ratio of approximately 1:1 and administering approximately 200 μl of the diluted pharmaceutically acceptable RNA preparation.

12. The method according to claim 11, wherein a third dose of the RNA, approximately 3 μg, is administered by diluting the pharmaceutical RNA preparation to approximately 1:5.75 and administering approximately 200 μl of the diluted pharmaceutical RNA preparation.

13. The method according to claim 1, wherein the RNA is formulated in lipid nanoparticles containing a cationically ionizable lipid, a neutral lipid, a steroid, and a polyethylene glycol (PEG)-lipid.

14. The method according to claim 13, wherein the pH of the pharmaceutical RNA preparation is pH 7.

4.

15. The method according to claim 13 or 14, characterized in that the RNA and the lipid nanoparticles remain stable at refrigeration temperatures for at least 10 weeks.

16. The method according to claim 1, wherein the pharmaceutically acceptable RNA preparation is provided in multiple vials, and the vials used to administer different doses of the RNA are labeled with different lid colors.

17. The method according to claim 1, wherein the pharmaceutically acceptable RNA preparation is provided in a multi-dose vial.

18. The aforementioned multi-dose vial, (a) Six doses of 2.25 mL of the pharmaceutically acceptable RNA preparation and 30 μg of RNA; (b) 10 doses of 1.3 mL of the pharmaceutically acceptable RNA preparation and 10 μg of RNA; or (c) The method according to claim 17, comprising 0.4 mL of the pharmaceutically acceptable RNA preparation and 10 doses of 3 μg of RNA.

19. The aforementioned multi-dose vial, (a) 2.6 mL of the pharmaceutically acceptable RNA preparation, (i) 50 μg / mL RNA comprising an open reading frame encoding a polypeptide containing the SARS-CoV-2 protein or an immunogenic fragment or variant thereof, wherein the RNA is formulated in lipid nanoparticles containing cationically ionizable lipids, neutral lipids, steroids, and polyethylene glycol (PEG)-lipids, and the RNA and (ii) 5 mM Tris buffer, (iii) 150 mM sucrose and, (iv) The pharmaceutically acceptable RNA preparation comprising 0.45% sodium chloride, or (b) 2.7 mL of the pharmaceutically acceptable RNA preparation, (i) 14.8 μg / mL of RNA comprising an open reading frame encoding a polypeptide containing the SARS-CoV-2 protein or an immunogenic fragment or variant thereof, wherein the RNA is formulated in lipid nanoparticles containing cationically ionizable lipids, neutral lipids, steroids, and PEG-lipids, and the RNA and (ii) 1.5 mM Tris buffer, (iii) 44.4 mM sucrose, The method according to claim 17, comprising the pharmaceutically acceptable RNA preparation comprising (iv) 0.77% sodium chloride.

20. A method for providing multiple different dosages of pharmaceutically acceptable RNA preparations, (i) A step of providing a stock RNA preparation at a first concentration, (ii) The step of preparing at least one dilution of the stock RNA preparation such that each dilution achieves a predetermined concentration selected to deliver a predetermined dose when administered in a predetermined volume, A method in which each predetermined volume is within the range of approximately 100 to 400 μl.

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