Methods and compositions for analyzing messenger RNA

Abstract

The present disclosure relates to a method of determining the presence of at least one messenger RNA (mRNA) molecule in a composition, the method comprising: (a) reverse transcription of the at least one mRNA molecule to obtain a template comprising at least one first strand of complementary DNA (cDNA); (b) generating at least one double-stranded cDNA molecule from the template; (c) amplifying the at least one double-stranded cDNA molecule; (d) digesting the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain a test cDNA fragment; (e) separating the test cDNA fragments so as to form a test cDNA fragment map; and (f) comparing the test cDNA fragment map to a control cDNA fragment map wherein the at least one mRNA molecule is present in the composition when the test cDNA fragment map comprises the control cDNA fragment map. Also disclosed are methods of quantifying and / or assessing the integrity of at least one mRNA molecule in a composition, processes of making a composition comprising at least one mRNA molecule and a primer pair for use in the methods of the invention.

Description

Cross Reference to Related Applications

[0001] This application claims priority to European application No. 23306201.7, filed July 13, 2023, the contents of which are incorporated by reference in their entirety. SEQUENCE LISTING

[0002] This specification refers to a sequence listing submitted electronically in a.txt file named“PR95288_EP_SANOFI”. The sequence listing was generated on July 12, 2023, and is 8.33 KB in size. The entire contents of the sequence listing are incorporated herein by reference. TECHNICAL FIELD

[0003] The present disclosure relates to methods of analyzing messenger RNA (mRNA). BACKGROUND

[0004] Messenger RNA (mRNA) is rapidly emerging as a new class of therapeutic agents, as reflected in the remarkable success of mRNA-based vaccines for COVID-19, for example. In addition to the well-publicized COVID-19 vaccines, mRNA therapeutics are also beneficial, for example, as vaccines for other infectious agents, replacement therapies, and regenerative medicine. Expanding production of mRNA therapeutics to meet the growing demand requires improved methods for analyzing mRNA therapeutics.

[0005] During the manufacturing process of mRNA therapeutics, incomplete mRNA products can be generated. In addition, mRNA can degrade during manufacturing and storage, for example, by exposure to heat, hydrolysis, oxidation, light, and ribonucleases. Thus, analytical methods that assess mRNA quality and ensure the correct identity of the molecule are valuable tools for manufacturing mRNA therapeutics. Analytical methods also help to assess manufacturing and process reproducibility between batches and the quality of the mRNA produced.

[0006] However, the development of analytical methods currently available to profile mRNA therapeutics, including mRNA vaccines, as well as methods for analyzing large RNAs, has proven challenging. For example, while Sanger sequencing can be used, for example, to verify the sequence in a cDNA molecule corresponding to a mRNA of interest, this method can be problematic in distinguishing multiple sequences, particularly when the mRNAs share a high percentage of sequence identity. The quality of Sanger sequences is typically unreliable for the first 15 to 40 bases where primer binding occurs. In addition, sequence quality typically declines after 700 to 900 bases.

[0007] Mass spectrometry-based methods have also been used to analyze RNA. For example, an RNase mapping method has been developed and used for RNA sequence mapping. Enzymatic digestion using ribonucleases, such as RNase T1, produces small oligoribonucleotides that are amenable to chromatographic separation and intact mass measurement. Additional sequence information for the oligoribonucleotides can be obtained using tandem mass spectrometry (MS / MS). However, RNA sequence mapping of long mRNA therapeutics using high frequency RNases results in the production of many small oligoribonucleotides that map to many different locations in the entire RNA sequence and, thus, do not produce unique sequences for sequence mapping. Furthermore, analysis of RNase sequence mapping MS data is challenging and there are limited specialized software tools currently available.

[0008] Accordingly, there remains a pressing need to develop improved methods and compositions for analyzing mRNA, including, for example, analyzing the identity, integrity, and / or quantity of mRNA manufactured for therapeutic use. SUMMARY

[0009] The present disclosure relates to methods and compositions for analyzing mRNA. The methods and compositions described herein can be used to assess the integrity, presence, and quantity of one or more mRNA species in a composition that can be used as an mRNA therapeutic, for example. The methods of the present invention can also readily distinguish between two or more mRNA species in a composition, including those that share a high percentage of sequence identity.

[0010] In one aspect, the disclosure relates to a method of determining the presence of at least one messenger RNA (mRNA) molecule in a composition, the method comprising: (a) reverse transcribing the at least one mRNA molecule to obtain a template comprising at least one first strand of complementary DNA (cDNA); (b) generating at least one double-stranded cDNA molecule from the template; (c) amplifying the at least one double-stranded cDNA molecule; (d) digesting the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain test cDNA fragments; (e) separating the test cDNA fragments, thereby forming a test cDNA fragment profile; and (f) comparing the test cDNA fragment profile to a control cDNA fragment profile, wherein when the test cDNA fragment profile comprises the control cDNA fragment profile, the at least one mRNA molecule is present in the composition. In some embodiments, the method further comprises determining the integrity of the at least one mRNA, the method further comprising (g) quantifying the amount of at least one test cDNA fragment in the test cDNA fragment profile; and (h) comparing the amount of the at least one test cDNA fragment to the amount of a control cDNA fragment in the control cDNA fragment profile, wherein a decrease in the amount of the at least one test cDNA fragment compared to the amount of the control cDNA fragment is indicative of degradation, and thereby, a decrease in the integrity of the at least one mRNA molecule in the composition.

[0011] In another aspect, the disclosure relates to a method of quantifying at least one messenger RNA (mRNA) molecule in a composition, the method comprising: (a) reverse transcribing the at least one mRNA molecule to obtain a template comprising at least one first strand of complementary DNA (cDNA); (b) generating at least one double-stranded cDNA molecule from the template; (c) amplifying the at least one double-stranded cDNA molecule; (d) digesting the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain test cDNA fragments; (e) separating the test cDNA fragments, thereby forming a test cDNA fragment profile; (f) determining the amount of at least one test cDNA fragment in the test cDNA fragment profile; (g) comparing the amount of the at least one test cDNA fragment to the amount of a control cDNA fragment; and (h) quantifying the amount of the at least one test mRNA molecule in the composition based on the amount of the at least one test cDNA fragment relative to the control cDNA fragment.

[0012] In yet another aspect, the disclosure relates to a process of manufacturing a composition comprising at least one messenger RNA (mRNA) molecule, wherein the process comprises: (a) reverse transcribing the at least one mRNA molecule to obtain a template comprising at least one first strand of complementary DNA (cDNA); (b) generating at least one double-stranded cDNA molecule from the template; (c) amplifying the at least one double-stranded cDNA molecule; (d) digesting the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain test cDNA fragments; (e) isolating test cDNA fragments, thereby forming a test cDNA fragment profile; (f) determining an amount of at least one test cDNA fragment in the test cDNA fragment profile; (g) comparing the amount of the at least one test cDNA fragment to an amount of a control cDNA fragment; and (h) quantifying an amount of the at least one mRNA molecule in the composition based on the amount of the at least one test cDNA fragment relative to the amount of the control cDNA fragment.

[0013] In another aspect, the disclosure relates to a primer pair for use in the methods described herein. In some embodiments, the primer pair comprises a 5'-UTR primer and a 3'-UTR primer, wherein the 5'-UTR primer is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and the 3'-UTR primer is selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO: 3. In some embodiments, the primer pair comprises a primer pair selected from the group consisting of: a) SEQ ID NO: 6 and SEQ ID NO: 2; b) SEQ ID NO: 7 and SEQ ID NO: 2; c) SEQ ID NO: 8 and SEQ ID NO: 2; and d) SEQ ID NO: 9 and SEQ ID NO: 2. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Embodiments of a method for assessing messenger RNA (mRNA) integrity as described in the detailed description are depicted.

[0015] Figure 2A depicts a sequence alignment of the hemagglutinin (HA) gene from A / Wisconsin / 588 / 2019 (Influenza A-H1N1; “Wis”), A / Tasmania / 503 / 2020 (Influenza A-H3N2; “Tan”), B / Washington / 02 / 2019 (Influenza B-Victoria; “Was”), and B / PHUKET / 3073 / 2013 (Influenza B-Yamagata; “Phu”).

[0016] Figure 2 B shows the percent sequence identity of the HA gene from A / Wisconsin / 588 / 2019 (Influenza A-H1N1; “Wis”), A / Tasmania / 503 / 2020 (Influenza A-H3N2; “Tan”), B / Washington / 02 / 2019 (Influenza B-Victoria; “Was”), and B / PHUKET / 3073 / 2013 (Influenza B-Yamagata; “Phu”).

[0017] Figure 3 Depicted is a digital gel image showing four mRNA samples produced by in vitro transcription (IVT) and separated by capillary electrophoresis (CE) as described in Example 1.

[0018] Figure 4 Depicted are four PCR reaction products each prepared using the same template but with different primer pairs (lanes 1-4), a ladder marker (lane L), and a control (left lane) separated by electrophoresis on a 1.2% agarose gel as described in Example 2Bi.

[0019] Figure 5 Depicted are five PCR reaction products each prepared from the same template but with different primer pairs (lanes 1-5) and a ladder marker (lane L) separated by electrophoresis on a 1.2% agarose gel as described in Example 2Bii.

[0020] Figure 6 Depicted are PCR reaction products each prepared using different templates (lanes 1-4) or a mixture of templates from lanes 1-4 (lane 5) separated by electrophoresis on a 1.2% agarose gel as described in Example 2Biii.

[0021] Figure 7Electropherograms depicting four PCR reaction products each prepared from a mixture of four templates digested with Age I (lane 1), Hinc II (lane 2), Afe I (lane 3), or Sac I (lane 4), or uncut (lane Q), and separated by electrophoresis on a 1.2% agarose gel, as described in Example 3i.

[0022] Figure 8 Digital gel images depicting five serially diluted PCR reaction products each prepared from a mixture of four templates, separated by capillary electrophoresis, either uncut (lanes A1-A4) or digested with Age I (lanes A5-A8), Hinc II (lanes A9-A12), Afe I (lanes B1-B4), or Sac I (lanes B1-B8), as described in Example 3i.

[0023] Figure 9 A depicts an electropherogram of a PCR reaction product separated by capillary electrophoresis as described in Example 3i, digested with Afe I, and showing two large peaks corresponding to the 5'-test cDNA fragment (left large peak), the 3'-test cDNA fragment (middle large peak), and uncut cDNA (right large peak), each prepared from a mixture of 4 different templates as described in Example 3i. Two smaller peaks depict a smaller marker (left) and a larger marker (right). Figure 8

[0024] Figure 9 B depicts a PCR reaction product digested with Afe I and separated by electrophoresis on a 1.2% agarose gel, as described in Example 3i.

[0025] Figure 10 Electropherograms depicting four PCR reaction products each prepared from a mixture of four different templates, digested with Ava I (lane 1), Acc I (lane 2), Pfl MI (lane 3), or Stu I (lane 4), or uncut (lane U), and separated by electrophoresis on a 1.2% agarose gel, as described in Example 3ii.

[0026] Figure 11 Digital gel images depicting four serially diluted PCR reaction products each prepared from a mixture of four different templates and separated by capillary electrophoresis, either uncut (lanes A1-A4) or digested with Ava I (lanes B9-B12), Acc I (lanes C1-C4), and Stu I (lanes C5-C8), as described in Example 3ii.

[0027] Figure 12A Digital gel images depicting four serially diluted PCR reaction products each prepared from a mixture of four different templates and separated by capillary electrophoresis, either uncut (lanes A1-A4) or digested with Ava I (lanes B9-B12), Acc I (lanes C1-C4), and Stu I (lanes C5-C8), as described in Example 3ii. Figure 11 ​The electropherogram of the PCR reaction products digested with Ace I and separated by capillary electrophoresis is depicted and shows five large peaks from left to right corresponding to the first 5'-test cDNA fragment (543 bp, from sample I.D. No. 4), the first 3'-test cDNA fragment (654 bp, from sample I.D. No. 3), the second 5'-test cDNA fragment (1335 bp, from sample I.D. No. 3), the second 3'-test cDNA fragment (1452 bp, from sample I.D. No. 4) and uncut DNA (from samples I.D. Nos. 1-4), as described in Example 3 ii.

[0028] Figure 12B Depicted is the mixture of digested PCR reaction products of Figure 11 and ladder (left); the length of DNA fragments that would be obtained if digested with BstUI, and visualized on a virtual 1.2% agarose gel, as described in Example 3 iii.

[0029] Figure 13 Depicted is the mixture of digested PCR reaction products of and ladder (left); the length of DNA fragments that would be obtained if digested with BstUI, and visualized on a virtual 1.2% agarose gel, as described in Example 3 iii.

[0030] Figure 14 Depicted is the mixture of digested PCR reaction products of Figure 13 and ladder (left); the length of DNA fragments that would be obtained if digested with BstUI, and visualized on a virtual 1.2% agarose gel, as described in Example 3 iii. DETAILED DESCRIPTION

[0031] Unless otherwise indicated, the following terms shall have the following definitions. Definitions

[0032] Unless the context clearly dictates otherwise, as used in the present specification and the appended claims, the singular form “a,” “an” and “the” include plural referents. Thus, for example, reference to “a method” includes one or more methods, and / or steps of the type described herein and / or that would be apparent to one of skill in the art upon reading the present disclosure and the like.

[0033] The term“about” is used herein to mean within the typical tolerances of the art. For example,“about” can be understood to be within about 2 standard deviations from the mean. According to certain embodiments,“about,” when used in reference to a measurable value such as an amount of an ingredient, is inclusive of variations that can exist in the values that the minimum and maximum values will carry, to the extent that such values are applicable given the function of the method and / or the composition being used, and / or the nature of the components being used. When“about” precedes a series of numerical values or a range, it is understood that“about” can modify each of the numerical values in that series or range.

[0034] As used herein, the terms“at least,”“less than,”“more than,” or“up to,” preceding a series of numbers or a range of numbers (e.g.,“at least two”) are understood to include the number or numbers that the term“at least,”“less than,” or“more than” precedes, as well as all subsequent numbers or integers that can logically be included, as shown from the context. When the term“at least,”“less than,”“more than,” or“up to” precedes a series of numbers or a range, it is understood that“at least,”“less than,”“more than,” or“up to” can modify each of the numbers in that series or range.

[0035] As used herein in the specification and claims, the term“and / or” should be understood to mean“one or both” of the entities joined by“and / or,” i.e., either the entity named before the term“and / or” or the entity named after the term“and / or,” or both entities. Other entities can optionally be present in addition to the entities explicitly identified by the“and / or” clause, whether related to those explicitly identified or not, unless expressly indicated to the contrary. Thus, as a non-limiting example, a reference to“A and / or B” when used in conjunction with open-ended language such as“comprising” can refer, in one embodiment, to A without B (optionally including entities other than B); in another embodiment, to B without A (optionally including entities other than A); in yet another embodiment, to both A and B (optionally including other entities); and so on.

[0036] As used in the specification and the appended claims, the singular forms“a,”“an” and“the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“a method” includes one or more methods, and / or the steps thereof, and / or equivalents thereof, as will be apparent from the disclosure for those skilled in the art.

[0037] As used herein, the terms “in some embodiments,” “in other embodiments,” “in some other embodiments,” and the like, refer to embodiments of all aspects of the disclosure, unless otherwise expressly specified herein.

[0038] As used herein, the term “integrity” with respect to messenger RNA (mRNA) relates to the amount of degradation of the mRNA molecule. Partial or complete degradation of the mRNA molecule can be assessed, with increased mRNA degradation indicating decreased mRNA integrity. The “integrity” of mRNA can be affected by a variety of factors, including heat, hydrolysis, oxidation, light, and ribonucleases (RNases). RNases are of three main classes: i) endonucleases that cleave RNA internally, ii) 5'-exonucleases that hydrolyze RNA from the 5' end, and iii) 3'-exonucleases that degrade RNA from the 3' end. In some embodiments, degradation begins at an internal portion of the mRNA molecule. In some embodiments, degradation begins at the 5' end of the mRNA molecule. In some embodiments, degradation begins at the 3' end of the mRNA molecule. In some embodiments, the poly(A) tail of the mRNA molecule of the compositions of the disclosure is shortened by de-adenylating enzymes, exposing the mRNA to exonuclease activity. This results in the gradual degradation of the mRNA from the 3' end toward the coding region, resulting in the eventual loss of its functional integrity. mRNA integrity can be evaluated by the methods described herein, including the methods described in the examples.

[0039] As used herein, the term “partial degradation” or “partially degraded” with respect to an individual mRNA molecule means that the mRNA molecule comprises fewer nucleotides than a corresponding full-length mRNA molecule. In some embodiments, a partially degraded mRNA molecule is a fragment of a full-length mRNA molecule that contains 99% or fewer nucleotides of the full-length mRNA molecule, such as 98% or fewer, such as 97% or fewer, such as 96% or fewer, such as 95% or fewer, such as 94% or fewer, such as 93% or fewer, such as 92% or fewer, such as 91% or fewer, such as 90% or fewer, such as 85% or fewer, such as 80% or fewer, such as 70% or fewer, such as 60% or fewer, such as 50% or fewer, such as 40% or fewer, such as 30% or fewer, such as 20% or fewer, such as 10% or fewer, or such as 1% or fewer nucleotides of the corresponding full-length mRNA molecule.

[0040] As used herein, “partial degradation” or “partially degraded” with respect to a population of mRNA molecules of a single species refers to a population comprising full-length mRNA molecules of a single species and fragments thereof. In some embodiments, the fragments in the partially degraded population of mRNA molecules of a single species are 99% or less of the total length of the corresponding full-length species, such as 98% or less, such as 97% or less, such as 96% or less, such as 95% or less, such as 94% or less, such as 93% or less, such as 92% or less, such as 91% or less, such as 90% or less, such as 85% or less, such as 80% or less, such as 70% or less, such as 60% or less, such as 50% or less, such as 40% or less, such as 30% or less, such as 20% or less, such as 10% or less, such as 1% or less, of the total length of the corresponding full-length species. In some embodiments, the fragments in the partially degraded population of mRNA molecules of a single species comprise a mixture of full-length mRNA molecules and fragments of various sizes thereof.

[0041] As used herein, the term “messenger RNA (mRNA)” refers to any polyribonucleotide that encodes a polypeptide or fragment thereof of interest. In some embodiments, the mRNA molecules in the compositions of the disclosure comprise, in addition to other structural features such as a 5'-cap structure and a 3'-poly(A) tail, stabilizing elements such as untranslated regions (UTRs) at the 5' end (5'-UTR) and / or the 3' end (3'-UTR). In cells, 5'-UTRs and 3'-UTRs can be transcribed from genomic DNA and are typical elements of immature mRNA. Structural features unique to mature mRNA, including the 5'-cap and 3'-poly(A) tail, are typically added to the transcribed (immature) mRNA during mRNA processing.

[0042] As used herein, the term “at least one mRNA molecule in the composition” refers to one or more species of mRNA in the compositions of the disclosure that encodes a polypeptide of interest, for example, an HA antigen from the Wis strain. In some embodiments, “at least one mRNA molecule in the composition” refers to more than one species of mRNA in the compositions of the disclosure, wherein each species of mRNA encodes a different polypeptide of interest. In some embodiments, there are four different mRNA molecules in the compositions of the disclosure, wherein each mRNA molecule encodes, for example, a different HA antigen, such as an HA antigen from the Wis strain, an HA antigen from the Tan strain, an HA antigen from the Was strain, and an HA antigen from the Phu strain.

[0043] As used herein, the term “5’-untranslated region” (UTR) refers to a region of an mRNA molecule immediately upstream (i.e., 5’) of a start codon (i.e., the first codon of an mRNA molecule that is translated by a ribosome) that does not encode a polypeptide.

[0044] As used herein, the term “3’-untranslated region” (UTR) refers to a region of an mRNA molecule immediately downstream (i.e., 3’) of a stop codon (i.e., the codon in an mRNA transcript that signals termination of translation) that does not encode a polypeptide.

[0045] As used herein, the term “poly-A tail” is a region of an mRNA molecule that contains multiple consecutive adenosine monophosphates, downstream (e.g., immediately downstream (i.e., 3’)) of a 3’-UTR. A poly-A tail can contain 10 to 300 or more adenosine monophosphates. For example, a poly-A tail can contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, or more adenosine monophosphates. In some embodiments, a poly-A tail contains 50 to 250 adenosine monophosphates. In some embodiments, a poly-A tail functions to protect an mRNA molecule from enzymatic degradation (e.g., in the cytoplasm) and aids in transcription termination and / or export of an mRNA molecule from the nucleus and translation.

[0046] As used herein, the term “5’-cap” refers to an altered nucleotide at the 5’ end of an mRNA molecule, such as a modified guanine (G) nucleotide, which is involved in mRNA stability and translational capacity. In some embodiments, at least one mRNA molecule in a composition of the disclosure does not include a 5’-cap.

[0047] As used herein, the term “RNA transcript” refers to a polyribonucleotide produced by an in vitro transcription reaction using a DNA template and an RNA polymerase. In some embodiments, an RNA transcript includes a coding sequence for a polypeptide of interest, a 5’-UTR, a 3’-UTR, and a poly-A tail. The term “RNA transcript” includes and is interchangeable with the term mRNA or mRNA molecule.

[0048] As used herein, the term “polypeptide” includes a polymeric molecule composed of a linear sequence of amino acids. In some embodiments, a polypeptide can correspond to a molecule encoded by a naturally occurring polynucleotide sequence. A polypeptide can include conservative substitutions, where a naturally occurring amino acid is replaced by an amino acid with similar properties, where such conservative substitutions do not alter the function of the polypeptide.

[0049] As used herein, the term "digest" or "digesting" refers to breaking down, cleaving, or cutting into smaller pieces or components. When referring to cDNA, digestion results in the production of nucleic acid fragments.

[0050] As used herein, the term "cDNA" refers to DNA that is complementary to or identical to an mRNA molecule, except that cDNA includes thymine (T) instead of uracil (U). The term "cDNA" encompasses molecules that can be in single-stranded or double-stranded form.

[0051] As used herein, the term "test cDNA fragment" or "test cDNA fragments" refers to a cDNA, such as an amplified double-stranded cDNA molecule, that has been subjected to a restriction enzyme reaction as described herein. Such cDNA is obtained from a reverse transcription reaction using an mRNA molecule in a composition of the disclosure as a template, as described herein. In some embodiments, the cDNA subjected to the restriction enzyme reaction is cleaved, resulting in test cDNA fragments. In some embodiments, the test cDNA fragments are isolated as described herein to form a test cDNA fragment profile, which can be visualized as described herein. In some embodiments, the cDNA subjected to the restriction enzyme reaction is not cleaved, and results in a test cDNA fragment profile having only one test cDNA fragment, which can correspond to a full-length mRNA of a composition of the disclosure. The test cDNA fragment profile can be visualized as described herein.

[0052] In some embodiments, the cDNA subjected to a restriction enzyme reaction is cleaved to produce only two test cDNA fragments: a 5'-test cDNA fragment corresponding to the region immediately upstream of the start codon in the mRNA template and a 3'-test cDNA fragment corresponding to the region immediately downstream of the stop codon in the mRNA template. In other embodiments, the cDNA subjected to a restriction enzyme reaction is cleaved to produce more than two test cDNA fragments, including a 5'-test cDNA fragment corresponding to the region immediately upstream of the start codon in the mRNA template, an inner fragment, and a 3'-test cDNA fragment corresponding to the region immediately downstream of the stop codon in the mRNA template. Uncut test cDNA fragments, test cDNA fragments produced by restriction enzyme cleavage, and / or test cDNA fragment maps that can be visualized as described herein can be used for analysis, such as determining the presence, quantity, and / or integrity of mRNA molecules in the compositions disclosed herein. As used herein, the term "control cDNA fragment" (or "control cDNA fragments") refers to cDNA, such as an amplified double-stranded cDNA molecule, that has undergone restriction enzyme reactions as described herein to produce control cDNA fragments. In some embodiments, the restriction enzyme-reacted cDNA is obtained, for example, from a reverse transcription reaction as described herein using an undegraded mRNA molecule as a template. In some embodiments, the control cDNA fragment is isolated as described herein to form a control cDNA fragment map, which can be visualized as described herein and compared with a test cDNA fragment map as described herein. In some embodiments, the restriction enzyme-reacted control cDNA is not cleaved, and a control cDNA fragment map containing only one control cDNA fragment is produced, which may correspond to a full-length template mRNA.

[0053] In some embodiments, the restriction enzyme-treated cDNA is cleaved to produce only two control cDNA fragments: a 5'-control cDNA fragment corresponding to the region immediately upstream of the start codon in the mRNA template and a 3'-control cDNA fragment corresponding to the region immediately downstream of the stop codon in the mRNA template. In other embodiments, the restriction enzyme-treated control cDNA is cleaved to produce more than two control cDNA fragments, including a 5'-control cDNA fragment corresponding to the region immediately upstream of the start codon in the mRNA template, an inner fragment, and a 3'-control cDNA fragment corresponding to the region immediately downstream of the stop codon in the mRNA template.

[0054] As used in this article, the term "amplifier" refers to the amplification product of a nucleic acid amplification reaction (e.g., RT-PCR).

[0055] As used herein, the terms “purify,” “purified,” or “purification” mean the removal of unwanted components, material contamination, impurities, or imperfections, such that the resulting product is substantially pure or clean.

[0056] As used herein, the term “substantially” refers to a qualitative condition of being, to nearly total extent or nearly complete. One of ordinary skill in the art of biology will understand that biological and chemical phenomena can not achieve or avoid absolute results. Accordingly, the term “substantially” is used herein to capture the potential lack of perfection inherent in some biological and chemical phenomena.

[0057] As used herein, the term “percent nucleic acid sequence identity” with respect to mRNA molecules in the compositions of the disclosure is defined as the percentage of nucleotides in a first mRNA sequence that are identical with nucleotides in a second mRNA sequence, after aligning the sequences, and introducing gaps if necessary, to achieve the maximum percent sequence identity.

[0058] To determine the percent sequence identity of two nucleotide or amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first nucleotide sequence for optimal alignment). The nucleotides or amino acids at corresponding nucleotide or amino acid positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid as the corresponding position in the second sequence, then the molecules are identical at that position. The percent sequence identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions x 100).

[0059] The determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm of Karlin et al., Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), which is incorporated into the NBLAST procedure and can be used to identify sequences with the desired identity to the nucleotide sequences disclosed herein. For obtaining vacancy alignments for comparison purposes, vacancy BLAST, as described in Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997), can be used. When using BLAST and vacancy BLAST procedures, the default parameters of the respective procedures (e.g., NBLAST) can be used. See procedures provided by the National Center for Biotechnology Information, the National Library of Medicine, and the National Institutes of Health.

[0060] As used in this article, "H1" refers to influenza virus subtype 1 hemagglutinin (HA). Influenza A viruses are divided into Group 1 and Group 2. Groups 1 and 2 are further divided into subtypes, which are classified according to the sequences of two proteins on the viral surface: HA and neuraminidase (NA). Currently, 18 recognized HA subtypes exist (H1-H18). Therefore, H1 differs from other HA subtypes (including H2-H18).

[0061] As used in this article, "H3" refers to influenza virus subtype 3 HA. Therefore, H3 is different from other HA subtypes (including H1, H2, and H4-H18).

[0062] As used in this article, "N1" refers to influenza virus subtype 1 neuraminidase (NA). Influenza A viruses are divided into Group 1 and Group 2. Groups 1 and 2 are further divided into subtypes, which are classified according to the sequences of two proteins on the viral surface: HA and neuraminidase (NA). Currently, 11 recognized NA subtypes exist (N1-N11). Therefore, N1 differs from other NA subtypes (including N2-N11).

[0063] As used in this article, “N2” refers to influenza virus subtype 2 neuraminidase (NA). Therefore, N2 is different from other NA subtypes (including N1 and N3-N11).

[0064] Each year, based on intensive monitoring, the World Health Organization (WHO) selects influenza strains to be included in seasonal vaccine formulations. As used herein, the term "standard of care strain" or "SOC strain" refers to an influenza strain selected by the WHO to be included in a seasonal vaccine formulation. A standard of care strain can include a historical standard of care strain, a current standard of care strain, or a future standard of care strain. Methods of analyzing and manufacturing mRNA molecules

[0065] In one aspect, the disclosure relates to a method of determining the presence of at least one messenger RNA (mRNA) molecule in a composition, the method comprising: (a) reverse transcribing the at least one mRNA molecule to obtain a template comprising at least one first strand of complementary DNA (cDNA); (b) generating at least one double-stranded cDNA molecule from the template; (c) amplifying the at least one double-stranded cDNA molecule; (d) digesting the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain test cDNA fragments; (e) separating the test cDNA fragments, thereby forming a test cDNA fragment profile; and (f) comparing the test cDNA fragment profile to a control cDNA fragment profile, wherein the at least one mRNA molecule is present in the composition when the test cDNA fragment profile comprises the control cDNA fragment profile.

[0066] In some embodiments, the method further comprises determining the integrity of the at least one mRNA molecule in the composition, the method further comprising (g) quantifying the amount of at least one test cDNA fragment in the cDNA fragment profile; and (h) comparing the amount of the at least one cDNA fragment to the amount of the control cDNA fragment, wherein a decrease in the amount of the at least one cDNA fragment compared to the amount of the control cDNA fragment is indicative of degradation, and thereby of a decrease in the integrity of the at least one mRNA molecule in the composition.

[0067] In this aspect, steps (a)-(f) can be performed as described herein. The quantifying (g) and comparing (h) steps can also be performed as described herein.

[0068] In another aspect, the disclosure relates to a method of quantifying at least one mRNA molecule in a composition, the method comprising: (a) reverse transcribing the at least one mRNA molecule to obtain a template comprising at least one first strand of complementary DNA (cDNA); (b) generating at least one double-stranded cDNA molecule from the template; (c) amplifying the at least one double-stranded cDNA molecule; (d) digesting the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain test cDNA fragments; (e) separating the test cDNA fragments, thereby forming a test cDNA fragment profile; (f) determining an amount of at least one test cDNA fragment in the test cDNA fragment profile; (g) comparing the amount of the at least one test cDNA fragment to an amount of a control cDNA fragment; and (h) quantifying an amount of the at least one mRNA molecule in the composition based on the amount of the at least one test cDNA fragment relative to the control cDNA fragment. The foregoing steps (a)-(h) can be performed as described herein.

[0069] In some embodiments, a decrease in the amount of the at least one test cDNA fragment compared to the amount of the control cDNA fragment indicates a decrease in the amount of the at least one mRNA molecule in the composition compared to an amount of at least one control mRNA molecule. In some embodiments, an increase in the amount of the at least one cDNA fragment compared to the amount of the control cDNA fragment indicates an increase in the amount of the at least one mRNA molecule in the composition compared to an amount of at least one control mRNA molecule.

[0070] In another aspect, the disclosure relates to a process of manufacturing a composition comprising at least one mRNA molecule, wherein the process comprises: (a) reverse transcribing the at least one mRNA molecule to obtain a template comprising at least one first strand of complementary DNA (cDNA); (b) generating at least one double-stranded cDNA molecule from the template; (c) amplifying the at least one double-stranded cDNA molecule; (d) digesting the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain test cDNA fragments; (e) separating the test cDNA fragments, thereby forming a test cDNA fragment profile; (f) determining an amount of at least one test cDNA fragment in the test cDNA fragment profile; (g) comparing the amount of the at least one test cDNA fragment to an amount of a control cDNA fragment; and (h) quantifying an amount of the at least one test mRNA molecule in the composition based on the amount of the at least one test cDNA fragment relative to the control cDNA fragment.

[0071] In this aspect, steps (a)-(g) can be performed as described herein. The quantification step described in step (h) can also be performed as described herein.

[0072] In some embodiments of all aspects of the disclosure, at least one mRNA molecule in the composition of the disclosure is not degraded. In some embodiments of all aspects of the disclosure, the amount of the enumerated test cDNA fragments and the amount of the control cDNA fragments are the same.

[0073] As used herein, the phrase "at least one mRNA molecule is not degraded" refers to the integrity of the mRNA molecules in the composition of the disclosure as described herein. In this regard, degradation is assessed as described herein, and the difference between the amount of the test and the amount of the control cDNA fragments and / or the test to control ratio as described herein is less than 10%, such as less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (including all values and subranges therebetween). As used herein, the term "same" with respect to the comparison between the amount of the enumerated one or more test cDNA fragments and the amount of one or more control cDNA fragments indicates that the difference between the amounts is less than 10%, such as less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% (including all values and subranges therebetween). mRNA molecules

[0074] In some embodiments, the at least one mRNA molecule of the composition of the disclosure comprises only one mRNA molecule. In some embodiments, the at least one mRNA molecule comprises a plurality of different mRNA molecules, such as at least two different mRNA molecules, at least three different mRNA molecules, at least four different mRNA molecules, at least five different mRNA molecules, at least six different mRNA molecules, at least seven different mRNA molecules, at least eight different mRNA molecules, at least nine different mRNA molecules, or at least ten different mRNA molecules.

[0075] Thus, in some embodiments, the compositions of the present disclosure are monovalent compositions (e.g., monovalent vaccines) or multivalent compositions (e.g., multivalent vaccines) comprising a plurality of different mRNA molecules. In some embodiments, the compositions are bivalent compositions (e.g., bivalent vaccines) comprising two different kinds of mRNA molecules. In some embodiments, the compositions are trivalent compositions (e.g., trivalent vaccines) comprising three different kinds of mRNA molecules. In some embodiments, the compositions are tetravalent compositions (e.g., tetravalent vaccines) comprising four different kinds of mRNA molecules. In some embodiments, the compositions are pentavalent compositions (e.g., pentavalent vaccines) comprising five different kinds of mRNA molecules. In some embodiments, the compositions are hexavalent compositions (e.g., hexavalent vaccines) comprising six different kinds of mRNA molecules. In some embodiments, the compositions are heptavalent compositions (e.g., heptavalent vaccines) comprising seven different kinds of mRNA molecules. In some embodiments, the compositions are octavalent compositions (e.g., octavalent vaccines) comprising eight different kinds of mRNA molecules. In some embodiments, the compositions are nonavalent compositions (e.g., nonavalent vaccines) comprising nine different kinds of mRNA molecules. In some embodiments, the compositions are decavalent compositions (e.g., decavalent vaccines) comprising ten different kinds of mRNA molecules.

[0076] In some embodiments, each of the different at least one mRNA molecule in the compositions of the present disclosure shares at least 50%, such as 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%, or at least 99% sequence identity with at least one other mRNA molecule in the composition (including all values and subranges therebetween).

[0077] In some embodiments, each of the different mRNA molecules in the composition shares at least 50% sequence identity with at least one other mRNA molecule in the composition.

[0078] In some embodiments, each of the different mRNA molecules in the composition shares at least 75% sequence identity with at least one other mRNA molecule in the composition.

[0079] In some embodiments, each of the different mRNA molecules differs in length from one another by 10 bases or less, 9 bases or less, 8 bases or less, 7 bases or less, 6 bases or less, 5 bases or less, 4 bases or less, 3 bases or less, or 2 bases or less (including all subranges therebetween).

[0080] In some embodiments, the at least one mRNA molecule comprises a plurality of different mRNA molecules, and each of the different mRNA molecules in the composition shares at least 50% sequence identity with at least one other mRNA molecule, and each of the different mRNA molecules differs in length from one another by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less (including all subranges therebetween).

[0081] In some embodiments, the at least one mRNA molecule comprises a plurality of different mRNA molecules, and each of the different mRNA molecules in the composition shares at least 55% sequence identity with at least one other mRNA molecule, and each of the different mRNA molecules differs in length from one another by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less (including all subranges therebetween).

[0082] In some embodiments, the at least one mRNA molecule comprises a plurality of different mRNA molecules, and each of the different mRNA molecules in the composition shares at least 60% sequence identity with at least one other mRNA molecule, and each of the different mRNA molecules differs in length from one another by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less (including all subranges therebetween).

[0083] In some embodiments, the at least one mRNA molecule comprises a plurality of different mRNA molecules, and each of the different mRNA molecules in the composition shares at least 65% sequence identity with at least one other mRNA molecule, and each of the different mRNA molecules differs in length from one another by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less (including all subranges therebetween).

[0084] In some embodiments, the at least one mRNA molecule comprises a plurality of different mRNA molecules, and each of the different mRNA molecules in the composition shares at least 70% sequence identity with at least one other mRNA molecule, and each of the different mRNA molecules differs in length from one another by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less (including all subranges therebetween).

[0085] In some embodiments, the at least one mRNA molecule comprises a plurality of different mRNA molecules, and each of the different mRNA molecules in the composition shares at least 75% sequence identity with at least one other mRNA molecule, and each of the different mRNA molecules differs in length from one another by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less (including all subranges therebetween).

[0086] In some embodiments, the at least one mRNA molecule comprises a plurality of different mRNA molecules, and each of the different mRNA molecules in the composition shares at least 80% sequence identity with at least one other mRNA molecule, and each of the different mRNA molecules differs in length from one another by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less (including all subranges therebetween).

[0087] In some embodiments, the at least one mRNA molecule comprises a plurality of different mRNA molecules, and each of the different mRNA molecules in the composition shares at least 85% sequence identity with at least one other mRNA molecule, and each of the different mRNA molecules differs in length from one another by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less (including all subranges therebetween).

[0088] In some embodiments, the at least one mRNA molecule comprises a plurality of different mRNA molecules, and each of the different mRNA molecules in the composition shares at least 90% sequence identity with at least one other mRNA molecule, and each of the different mRNA molecules differs in length from one another by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less (including all subranges therebetween).

[0089] In some embodiments, the at least one mRNA molecule comprises a plurality of different mRNA molecules, and each of the different mRNA molecules in the composition shares at least 95% sequence identity with at least one other mRNA molecule, and each of the different mRNA molecules differs in length from one another by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less (including all subranges therebetween).

[0090] In some embodiments, the at least one mRNA molecule comprises a plurality of different mRNA molecules, and each of the different mRNA molecules in the composition shares at least 99% sequence identity with at least one other mRNA molecule, and each of the different mRNA molecules differs in length from one another by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less (including all subranges therebetween).

[0091] In some embodiments, the composition of the disclosure is a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises at least one mRNA molecule in the composition of the disclosure and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition is formulated for parenteral administration, such as intravenous, subcutaneous, intraperitoneal, intradermal, intranasal, inhalation, or intramuscular. In some embodiments, the pharmaceutical composition is formulated for oral or topical administration.

[0092] In some embodiments, the composition comprises naked mRNA molecules. In other embodiments, the composition comprises complexed or encapsulated mRNA molecules. For example, the compositions of the present disclosure can comprise mRNA molecules complexed or encapsulated in a nanoparticle in the form of a liposome. In some embodiments, the mRNA molecules are complexed or encapsulated after determining the integrity or quantity of the mRNA molecules, e.g., as described herein.

[0093] In some embodiments, the pharmaceutical composition is a vaccine against a virus including, but not limited to, an influenza virus, a coronavirus, a respiratory syncytial virus (RSV), a parainfluenza virus, a human immunodeficiency virus (HIV), a herpes virus, a human papillomavirus, a rotavirus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkeypox virus, a parvovirus, an Ebola virus, a dengue virus, a hantavirus, a Zika virus, a West Nile virus, a poliovirus, or a rabies virus. In some embodiments, the vaccine is a monovalent vaccine. In some embodiments, the vaccine is a multivalent vaccine comprising a plurality of different species of mRNA molecules.

[0094] In some embodiments, the vaccine is a bivalent vaccine comprising two different species of mRNA molecules. In some embodiments, the two different species of mRNA molecules in the bivalent vaccine share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity (including all values and subranges therebetween). In some embodiments, the two different species of mRNA molecules in the bivalent vaccine share at least 50% (such as at least 75%) sequence identity. In some embodiments, each of the two different species of mRNA molecules in the bivalent vaccine is from a different strain of the same virus, including but not limited to two different strains of the following: an influenza virus, a coronavirus, a respiratory syncytial virus (RSV), a parainfluenza virus, a human immunodeficiency virus (HIV), a herpes virus, a human papillomavirus, a rotavirus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkeypox virus, a parvovirus, an Ebola virus, a dengue virus, a hantavirus, a Zika virus, a West Nile virus, a poliovirus, or a rabies virus. For example, the bivalent vaccine can comprise a first species of mRNA molecule from a first strain of an influenza virus and a second species of mRNA molecule from a second strain of an influenza virus. In some embodiments, the bivalent vaccine can comprise a first species of mRNA molecule from a first strain of a coronavirus and a second species of mRNA molecule from a second strain of a coronavirus.

[0095] In some embodiments, the bivalent vaccine is a combination vaccine comprising two different kinds of mRNA molecules from different viruses, including but not limited to two different kinds of mRNA molecules from an influenza virus, a coronavirus, RSV, parainfluenza virus, HIV, a herpes virus, a human papillomavirus, a rotavirus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkeypox virus, a parvovirus, an Ebola virus, a dengue virus, a hantavirus, a zika virus, a west nile virus, a poliovirus, or a rabies virus. For example, the bivalent vaccine can comprise a first kind of mRNA molecule from an influenza virus and a second kind of mRNA molecule from a coronavirus. In some embodiments, the bivalent vaccine comprises a first kind of mRNA from RSV and a second kind of mRNA molecule from an influenza virus. In some embodiments, the bivalent vaccine comprises a first kind of mRNA from RSV and a second kind of mRNA molecule from a coronavirus.

[0096] In some embodiments, the vaccine is a trivalent vaccine comprising three different kinds of mRNA molecules. In some embodiments, the three different kinds of mRNA molecules in the trivalent vaccine share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity (including all values and subranges therebetween). In some embodiments, the three different kinds of mRNA molecules in the trivalent vaccine share at least 50% (such as at least 75%) sequence identity. In some embodiments, each of the three different kinds of mRNA molecules in the trivalent vaccine is from a different strain of the same virus, including but not limited to three different kinds of mRNA molecules from an influenza virus, a coronavirus, RSV, parainfluenza virus, HIV, a herpes virus, a human papillomavirus, a rotavirus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkeypox virus, a parvovirus, an Ebola virus, a dengue virus, a hantavirus, a zika virus, a west nile virus, a poliovirus, or a rabies virus.

[0097] In some embodiments, the trivalent vaccine is a combination vaccine, where one or more of the four different species of mRNA molecules are from different viruses, including but not limited to: influenza virus, coronavirus, RSV, parainfluenza virus, HIV, herpes virus, human papillomavirus, rotavirus, norovirus, varicella zoster virus, hepatitis virus, paramyxovirus, monkeypox virus, parvovirus, Ebola virus, dengue virus, hantavirus, zika virus, West Nile virus, poliovirus, or rabies virus. For example, the trivalent vaccine can comprise a first species of mRNA molecule from an influenza virus, a second species of mRNA molecule from a coronavirus, and a third species of mRNA molecule from an RSV. In some embodiments, the trivalent vaccine comprises one species of mRNA molecule from a pneumovirus, another species of mRNA molecule from an influenza virus, and yet another species of mRNA molecule from a coronavirus.

[0098] In some embodiments, the vaccine is a tetravalent vaccine comprising four different species of mRNA molecules. In some embodiments, the four different species of mRNA molecules in the tetravalent vaccine share at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity (including all values and subranges therebetween). In some embodiments, the four different species of mRNA molecules in the tetravalent vaccine share at least 50% (such as at least 75%) sequence identity. In some embodiments, each of the four different species of mRNA molecules in the tetravalent vaccine is from a different strain of the same virus, including but not limited to: influenza virus, coronavirus, RSV, parainfluenza virus, HIV, herpes virus, human papillomavirus, rotavirus, norovirus, varicella zoster virus, hepatitis virus, paramyxovirus, monkeypox virus, parvovirus, Ebola virus, dengue virus, hantavirus, zika virus, West Nile virus, poliovirus, or rabies virus.

[0099] In some embodiments, the tetravalent vaccine is a combination vaccine, where one or more of the four different species of mRNA molecules are from different viruses, including but not limited to: influenza virus, coronavirus, RSV, parainfluenza virus, HIV, herpes virus, human papillomavirus, rotavirus, norovirus, varicella zoster virus, hepatitis virus, paramyxovirus, monkeypox virus, parvovirus, Ebola virus, dengue virus, hantavirus, zika virus, West Nile virus, poliovirus, or rabies virus. For example, the tetravalent vaccine can comprise a first species of mRNA from an RSV virus, a second species of mRNA molecule from a Group I influenza A strain, a third species of mRNA molecule from a Group II influenza A strain, and a fourth species of mRNA molecule from an influenza B strain.

[0100] In some embodiments, the length difference of the mRNA molecule species in the monovalent, bivalent, trivalent, and / or tetravalent vaccine is 10 bases or less, 9 bases or less, 8 bases or less, 7 bases or less, 6 bases or less, 5 bases or less, 4 bases or less, 3 bases or less, or 2 bases or less, including all subranges therebetween.

[0101] In some embodiments, the monovalent, bivalent, trivalent, or tetravalent vaccine is an influenza vaccine, a coronavirus vaccine, an RSV vaccine, a parainfluenza virus vaccine, an HIV vaccine, a herpes virus vaccine, a human papillomavirus vaccine, a rotavirus vaccine, a norovirus vaccine, a varicella zoster virus vaccine, a hepatitis virus vaccine, a paramyxovirus vaccine, a monkeypox virus vaccine, a parvovirus vaccine, an Ebola virus vaccine, a dengue virus vaccine, a hantavirus vaccine, a Zika virus vaccine, a West Nile virus vaccine, a poliovirus vaccine, or a rabies virus vaccine.

[0102] In some embodiments, the multivalent vaccine is a combination vaccine comprising different species of mRNA molecules from different viruses, including, for example, influenza virus, coronavirus, RSV, parainfluenza virus vaccine, HIV vaccine, herpes virus vaccine, human papillomavirus vaccine, rotavirus vaccine, norovirus vaccine, varicella zoster virus vaccine, hepatitis virus vaccine, paramyxovirus vaccine, monkeypox virus vaccine, parvovirus vaccine, Ebola virus vaccine, dengue virus vaccine, hantavirus vaccine, Zika virus vaccine, West Nile virus vaccine, poliovirus vaccine, or rabies virus vaccine.

[0103] In some embodiments, at least one mRNA molecule in the compositions of the disclosure is obtained from a natural source, such as a virus, a cell, a tissue, an organ, or an organism. Viruses that can be used as a source of at least one mRNA molecule in the compositions of the disclosure can include, but are not limited to, influenza virus, coronavirus, RSV, parainfluenza virus vaccine, HIV vaccine, herpes virus vaccine, human papillomavirus vaccine, rotavirus vaccine, norovirus vaccine, varicella zoster virus vaccine, hepatitis virus vaccine, paramyxovirus vaccine, monkeypox virus vaccine, parvovirus vaccine, Ebola virus vaccine, dengue virus vaccine, hantavirus vaccine, Zika virus vaccine, West Nile virus vaccine, poliovirus vaccine, or rabies virus vaccine.

[0104] In some embodiments, at least one mRNA molecule in a composition of the present disclosure encodes an influenza virus protein selected from the group consisting of H1, H3, HA from the B / Victoria lineage, and / or HA from the B / Yamagata lineage. In some embodiments, at least one mRNA molecule in a composition of the present disclosure comprises four mRNA molecules, each mRNA molecule encoding a different influenza virus protein (e.g., tetravalent), such as H1 from a first standard-of-care influenza virus strain, H3 from a second standard-of-care influenza virus strain, HA from a third standard-of-care influenza virus strain from the B / Victoria lineage, and HA from a fourth standard-of-care influenza virus strain from the B / Yamagata lineage.

[0105] In some embodiments, at least one mRNA molecule in a composition of the present disclosure encodes an influenza virus protein selected from the group consisting of N1, N2, NA from the B / Victoria lineage, and / or NA from the B / Yamagata lineage. In some embodiments, at least one mRNA molecule in a composition of the present disclosure comprises four mRNA molecules, each mRNA molecule encoding a different influenza virus protein (e.g., tetravalent), such as N1 from a first standard-of-care influenza virus strain, N2 from a second standard-of-care influenza virus strain, NA from a third standard-of-care influenza virus strain from the B / Victoria lineage, and NA from a fourth standard-of-care influenza virus strain from the B / Yamagata lineage.

[0106] In some embodiments, the source of at least one mRNA molecule in a composition of the present disclosure is an influenza virus, such as a strain of influenza A, a strain of influenza B, or a combination thereof. For example, in some embodiments, the source of at least one mRNA molecule in a composition of the present disclosure is a strain of influenza A, such as A / California / 07 / 2009, A / Japan / 305 / 1957, A / Vietnam / 1194 / 2004, A / Vietnam / 1203 / 2004, A / Netherlands / 219 / 2003, A / HongKong / 1073 / 1999, A / Perth / 16 / 2009, A / Wisconsin / 588 / 2019, and / or A / Tasmania / 503 / 2020. In some embodiments, the source of at least one mRNA molecule in a composition of the present disclosure is from a strain of influenza A, such as A / Wisconsin / 588 / 2019 and / or A / Tasmania / 503 / 2020.

[0107] In some embodiments, the source of at least one mRNA molecule in the compositions of the disclosure is a strain of influenza B, such as B / Brisbane / 2008, B / Malaysia / 2004, B / Victoria / 1987, and / or B / Washington / 02 / 2019 (Victoria lineage) and / or B / PHUKET / 3073 / 2013, B / Florida / 2006, B / Mass / 2012, and / or B / Wisconsin / 2010 (Yamagata lineage). In some embodiments, the source of at least one mRNA molecule in the compositions of the disclosure is a strain of influenza B, such as B / Washington / 02 / 2019 and / or B / PHUKET / 3073 / 2013.

[0108] In some embodiments, the source of at least one mRNA molecule in the compositions of the disclosure comprises a strain of influenza A, such as A / Wisconsin / 588 / 2019 and / or A / Tasmania / 503 / 2020; and a strain of influenza B, such as B / Washington / 02 / 2019 and / or B / PHUKET / 3073 / 2013.

[0109] Cells that can be used as a source of mRNA molecules in the compositions of the disclosure can be prokaryotic (bacterial cells, including species of Escherichia, Bacillus, Staphylococcus, Streptococcus, Pseudomonas), or eukaryotic (including fungi, plants, protozoa and other parasites, and animals (including insect (if Drosophila spp. cells), nematode (including Caenorhabditis elegans cells), mammalian cells (including blood cells (reticulocytes and leukocytes), endothelial cells, epithelial cells, neuronal cells (from central or peripheral nervous system), muscle cells (including myocytes and myoblasts from skeletal, smooth, or cardiac muscle), connective tissue cells (including fibroblasts, adipocytes, chondrocytes, chondroblasts, osteocytes, and osteoblasts), and other stromal cells (e.g., macrophages, dendritic cells, Schwann cells). Mammalian reproductive cells (spermatocytes and oocytes) can also be used as a source of mRNA molecules for use in the methods of the invention.

[0110] Also suitable for use as a source of mRNA are mammalian tissues or organs, such as those derived from brain, kidney, liver, pancreas, blood, bone marrow, muscle, nerve, skin, urogenital, circulatory, lymphoid, gastrointestinal, and connective tissue sources.

[0111] Any of the above prokaryotic or eukaryotic cells, tissues, and organs can be normal, diseased, embryonic, or fetal. Diseased cells can for example include those involved in infectious diseases (caused by bacteria, fungi or yeast, viruses (including AIDS, HIV, HTLV, herpes, hepatitis, etc.), or parasites), genetic or biochemical pathologies (e.g., cystic fibrosis, hemophilia, Alzheimer's disease, muscular dystrophy, or multiple sclerosis), or cancerous. Other cells, cell lines, tissues, organs, and organisms suitable as sources of mRNA for use in the present disclosure will be apparent to one of ordinary skill in the art.

[0112] In some embodiments, a private and / or public database (e.g., GenBank) is used to identify the mRNA of interest used as at least one mRNA molecule in the compositions of the present disclosure.

[0113] The polypeptide encoded by at least one mRNA molecule in the compositions of the present disclosure can be any polypeptide of interest. In some embodiments, the polypeptide of interest encoded by at least one mRNA molecule in the compositions of the present disclosure is a therapeutic protein, such as an antibody, an antigenic protein for use in a vaccine or other biologic, or a protein encoded by the human genome or another genome that has not yet been identified for a therapeutic indication but still has utility in the fields of research and discovery.

[0114] As used herein, a "therapeutic protein" is any polypeptide-based molecule, peptide fragment, or variant thereof, that can be used to treat, cure, alleviate, prevent, or diagnose a disease or medical condition. Therapeutic proteins and peptides can be used to treat or diagnose a disorder or disease in any therapeutic area, such as the areas of hematology, cardiovascular, central nervous system, toxicology (including anti-venom), dermatology, endocrinology, genetics, urogenital, gastrointestinal, musculoskeletal, oncology and immunology, respiratory, sensory, and anti-infective therapeutic areas.

[0115] For example, therapeutic proteins encoded by at least one mRNA molecule in the compositions of the disclosure can be used to treat or diagnose diseases, including cancer, and include proteins such as the following: carcinoembryonic antigen (CEA), New York esophageal squamous cell carcinoma protein 1 (NY-ESO), tyrosinase-related protein 2 (TRP2), tyrosinase, prostate-specific antigen (PSA), six-transmembrane epithelial antigen of the prostate (STEAP), Melan-A, tyrosinase, glycoprotein 100 (gp100), melanoma-associated antigen 1 (MAGEA1), melanoma-associated antigen 1 (MAGEA3), and mucin 1 (MUC1), human epidermal growth factor receptor 2 (HER2), telomerase, and survivin; allergen tolerance, e.g., peanut Ara h 2.02, ovalbumin, grass pollen Phl p 5, dust mite Der p 2; protein replacement, e.g., vasopressin, alpha-1 antitrypsin (AAT), erythropoietin (EPO), surfactant protein B (SPB), forkhead box P3 (FOXP3), herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) (HSV1-TK), vascular endothelial growth factor A (VEGFA), Bcl-2-associated X protein (BAX), as a vaccine component to prevent or treat infectious diseases (including influenza-related antigens, TB-related Hsp65, and RSV antigens).

[0116] In some embodiments, the therapeutic protein encoded by at least one mRNA molecule in the compositions of the disclosure includes one or more antibodies or fragments thereof. The term “antibody” includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments. The term “immunoglobulin” (Ig) is used interchangeably with “antibody” herein.

[0117] “Antibody fragments” comprise a portion of an intact antibody, typically the antigen binding and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; nanobodies; single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0118] In some embodiments, at least one mRNA molecule in the compositions of the present disclosure encodes one or more antigenic proteins for use in a vaccine composition. As used herein, the term “vaccine” refers to a composition that generates a protective immune response or protective immunity in a subject. “Protective immune response” or “protective immunity” refers to an immune response that protects a subject from infection (prevents infection or prevents the occurrence of a disease associated with infection) or reduces the symptoms of an infection (e.g., an influenza virus infection). Vaccines can elicit both prophylactic (preventive) and therapeutic responses. Methods of administration vary depending on the vaccine, but can include inoculation, ingestion, inhalation, or other forms of administration. Inoculation can be delivered by any of a variety of routes, including parenterally, such as intravenously, subcutaneously, intraperitoneally, intradermally, intranasally, by inhalation, or intramuscularly.

[0119] In some embodiments, the polypeptide encoded by at least one mRNA molecule in the compositions of the present disclosure is a variant of a naturally occurring protein. Variants of naturally occurring proteins include substitution variants, conservative amino acid substitutions, insertion variants, and / or deletion variants, and / or covalent derivatives. Exemplary and preferred conservative amino acid substitutions include any of the following: glutamine (Q) for glutamic acid (E), and vice versa; leucine (L) for valine (V), and vice versa; serine (S) for threonine (T), and vice versa; isoleucine (I) for valine (V), and vice versa; lysine (K) for glutamine (Q), and vice versa; isoleucine (I) for methionine (M), and vice versa; serine (S) for asparagine (N), and vice versa; leucine (L) for methionine (M), and vice versa; lysine (L) for glutamic acid (E), and vice versa; alanine (A) for serine (S), and vice versa; tyrosine (Y) for phenylalanine (F), and vice versa; glutamic acid (E) for aspartic acid (D), and vice versa; leucine (L) for isoleucine (I), and vice versa; lysine (K) for arginine (R), and vice versa.

[0120] In some embodiments, the polypeptide encoded by at least one mRNA molecule in the compositions of the present disclosure is an antigenic protein for use in a vaccine, such as an antigenic protein from an influenza virus, a coronavirus, an RSV, a parainfluenza virus, a human immunodeficiency virus (HIV), a herpes virus, a human papillomavirus, a rotavirus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkeypox virus, a parvovirus, an Ebola virus, a dengue virus, a hantavirus, a zika virus, a west nile virus, a poliovirus, or a rabies virus.

[0121] In some embodiments, the influenza vaccine comprises a therapeutic protein, wherein the therapeutic protein is one or more of a hemagglutinin (HA), a nucleoprotein (NP), a neuraminidase (NA) protein, matrix-1 (M1), matrix-2 (M2), non-structural protein-1 (NS1), non-structural protein-2 (NS2) from influenza A and / or influenza B. In some embodiments, the influenza protein is a human-derived influenza protein. In some embodiments, the influenza protein is a swine or bird-derived influenza protein.

[0122] In some embodiments, the therapeutic protein is from an influenza A strain, such as one or more of HI, H2, H5, H6, H8, H9, H11, H13, and HI6 (phylogenetic group I) and / or one or more of H3, H4, H7, HI0, HI5, and H14 (phylogenetic group 2). In some embodiments, the therapeutic protein comprises one or more HA proteins from an influenza B strain, such as a Victoria or Yamagata strain. In some embodiments, the therapeutic protein comprises one or more HA proteins from influenza A and influenza B. In some embodiments, the therapeutic protein comprises HA proteins from group I and group II influenza A and / or HA proteins from Victoria and Yamagata.

[0123] In some embodiments, the composition of the disclosure is a vaccine, and the at least one mRNA molecule of the vaccine composition encodes an influenza virus protein selected from the group consisting of H1 from B / Victoria lineage, H3, HA, and / or HA from B / Yamagata lineage. In some embodiments, the composition of the disclosure is a vaccine, and the at least one mRNA molecule of the vaccine composition comprises four mRNA molecules, each mRNA molecule encoding a different influenza virus protein (e.g., tetravalent), such as H1 from a first standard-of-care influenza virus strain, H3 from a second standard-of-care influenza virus strain, HA from a third standard-of-care influenza virus strain from B / Victoria lineage, and HA from a fourth standard-of-care influenza virus strain from B / Yamagata lineage.

[0124] In some embodiments, the composition of the disclosure is a vaccine, and the at least one mRNA molecule of the vaccine composition encodes an influenza virus protein selected from the group consisting of N1, N2, NA from the B / Victoria lineage, and / or NA from the B / Yamagata lineage. In some embodiments, the composition of the disclosure is a vaccine, and the at least one mRNA molecule of the vaccine composition comprises four mRNA molecules, each mRNA molecule encoding a different influenza virus protein (e.g., tetravalent), such as N1 from a first standard-of-care influenza strain, N2 from a second standard-of-care influenza strain, NA from a third standard-of-care influenza strain (from the B / Victoria lineage), and NA from a fourth standard-of-care influenza strain (from the B / Yamagata lineage).

[0125] In some embodiments, the composition of the disclosure is a vaccine, and the at least one mRNA molecule of the vaccine composition encodes at least one (such as at least two, such as at least three, such as at least four) protein from the following influenza strains: A / Wisconsin / 588 / 2019, A / Tasmania / 503 / 2020, B / Washington / 02 / 2019, and B / PHUKET / 3073 / 2013.

[0126] In some embodiments, the composition of the disclosure is a tetravalent vaccine comprising four mRNA molecules, wherein each of the four mRNA molecules encodes a different HA protein from each of the following influenza strains: A / Wisconsin / 588 / 2019, A / Tasmania / 503 / 2020, B / Washington / 02 / 2019, and B / PHUKET / 3073 / 2013.

[0127] In some embodiments, at least one mRNA molecule in the composition of the disclosure can be structurally or chemically modified to achieve a desired function or property. For example, the sequence of a naturally occurring mRNA can be optimized to match codon frequencies in certain tissue targets and / or host organisms to ensure proper folding; biased G / C content to increase mRNA stability; minimize tandem repeated codons or base runs that can impair expression; customize transcription and translation control regions; insert or remove protein transit sequences; and remove / add post-translational modification sites (e.g., glycosylation sites) in the encoded protein. Sequence optimization tools, algorithms, and sequence optimization services are known in the art; non-limiting examples include services from GeneArt (Life Technologies) and DNA2.0 (Menlo Park Calif.).

[0128] In some embodiments, at least one mRNA molecule in the compositions of the present disclosure can comprise at least one chemically modified nucleotide, including, for example, pseudouridine, methylpseudouridine (e.g., IN-methylpseudouridine), 2-thiouridine, 4'-thiouridine, 5-methylcytosine, 2-thio-l-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thiopseudouridine, 4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, 2'-fluoro ribonucleotides, and 2'-methoxy ribonucleotides. In some embodiments, each uridine in the ribonucleic acid molecule is replaced with pseudouridine, for example, methylpseudouridine, such as IN-methylpseudouridine. Other modifications include incorporation of synthetic UTR sequences from a- or b-globin to increase protein expression or one or more phosphorothioate linkages. Other mRNA modifications are known in the art and described in, for example, Liu A and Wang X (2022) “The Pivotal Role of Chemical Modifications in mRNA Therapeutics.” Front. Cell Dev. Biol. 10:901510. doi: 10.3389 / fcell.2022.901510 and Kim et al. “Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency.” Mol Cell Toxicol. 2022; 18(1): 1-8. doi: 10.1007 / s13273-021-00171-4 and WO 2017 / 070620. Methods of obtaining and preparing mRNA molecules

[0129] At least one mRNA molecule can be obtained by any method known in the art. In some embodiments, at least one mRNA molecule in a composition of the disclosure is obtained by initially isolating total mRNA from a source (e.g., a cell, tissue, organ, or other sample) as described herein, and reverse transcribing the total mRNA into cDNA by methods well known in the art to make a cDNA library (see, e.g., Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th ed., Vol. II, Cold Spring Harbor Laboratory Press, New York.). Briefly, in some embodiments, isolated mRNA can be subjected to a first-strand cDNA synthesis reaction as described herein. Double-stranded cDNA can then be made using any known method, including, e.g., commercially available kits such as the First-Strand cDNA Synthesis Kit (Agilent Technologies, Inc.) and the Second-Strand cDNA Synthesis Kit (ThermoFisher Scientific, Inc.). For example, a first-strand cDNA can be used as a template and a reaction catalyzed using known methods to produce a second-strand cDNA, including, e.g., a combination of E. coli DNA polymerase I with E. coli RNase H and E. coli DNA ligase. E. coli RNase H inserts a nick into the RNA that is complementary to the first-strand cDNA, thereby providing a 3' OH-primer for DNA polymerase I. The 5'-3' exonuclease activity of E. coli DNA polymerase I removes the RNA strand in the synthesis direction, while its polymerase activity replaces the RNA with deoxyribonucleotides. E. coli DNA ligase ligates the nicks to complete the double-stranded cDNA strand. Double-stranded cDNA can be inserted into a plasmid or other vector, transformed into a host cell, and the cDNA library screened for cDNAs of interest. A plasmid or other vector containing a cDNA of interest can be used as a template for, e.g., in vitro transcription to obtain at least one mRNA molecule in a composition of the disclosure as described below.

[0130] In other embodiments, at least one mRNA molecule in a composition of the present disclosure is synthesized by, for example, chemical synthesis known in the art (see, e.g., Gait, M. J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, D.C.: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, Vol. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005. For example, at least one mRNA molecule in a composition of the present disclosure can be made in whole or in part using solid-phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated process in which the molecule is immobilized on a solid support and synthesized stepwise in solution of reactants.

[0131] In other embodiments, at least one mRNA molecule in a composition of the present disclosure can be made in whole or in part using liquid-phase chemical synthesis, i.e., synthesis of nucleic acids of the present disclosure can be made by sequential addition of monomeric building blocks, which can be performed in liquid phase. In some embodiments, use of a combination of solid-phase or liquid-phase chemical synthesis with enzymatic ligation provides an efficient way of generating long-chain nucleic acids that cannot be obtained by chemical synthesis alone.

[0132] In some embodiments, at least one mRNA molecule in a composition of the present disclosure is obtained using in vitro transcription. Templates for transcribing at least one mRNA molecule in a composition of the present disclosure using in vitro transcription can be obtained, for example, from cDNA templates generated by first- and second-strand synthesis as described above, by annealing chemically synthesized oligonucleotides, from linear templates generated by cloning engineered plasmid constructs, and / or from linear templates generated by polymerase chain reaction (PCR).

[0133] For example, double-stranded cDNA corresponding to at least one mRNA molecule in a composition of the disclosure can be cloned into a plasmid, transfected into cells (e.g., bacterial cells, such as E. coli), and cultured to replicate the plasmid DNA. The plasmid DNA is then isolated from the cells and used to produce mRNA by in vitro transcription (IVT).

[0134] In some embodiments, during in vitro transcription, the plasmid DNA template includes an RNA polymerase promoter (e.g., a T7 promoter) located 5' of and operably linked to the cDNA region encoding the polypeptide of interest. In some embodiments, a sequence encoding a poly-A tail is located 3' of the coding region. Examples of suitable plasmids for use as DNA templates for in vitro transcription include pDP from Invitrogen, pGEM from Promega, pBluescript from Stratagene, and pCRII vectors from Invitrogen.

[0135] In some embodiments, immediately downstream of the poly-A tail encoding sequence on the plasmid DNA template is a recognition site for a restriction endonuclease used to linearize the plasmid. Linearization of the plasmid can mitigate transcriptional read-through.

[0136] Linearized DNA templates can be used in an in vitro transcription (IVT) system. In some embodiments, a single linearized DNA template is used in an IVT system to obtain one or more mRNA molecules of the disclosure. In some embodiments, more than one linearized template is used in an IVT system, each corresponding to at least one mRNA molecule in a composition of the disclosure. For example, as described in the examples, a linearized template corresponding to each of one or more mRNA molecules in a tetravalent influenza vaccine composition of the disclosure can be used in an IVT system to simultaneously prepare all four mRNA molecules of an exemplary tetravalent influenza vaccine composition.

[0137] In some embodiments, an IVT system comprises a transcription buffer (e.g., HEPES or Tris, at a pH of, e.g., 7-8.5), magnesium, nucleotide triphosphates (NTPs), an RNase inhibitor, and an RNA polymerase. In some embodiments, the NTPs can be selected from natural and non-natural (modified) NTPs. In some embodiments, the RNA polymerase can be selected from, e.g., bacteriophage RNA polymerases, such as T7 RNA polymerase, T3 RNA polymerase, and SP6 RNA polymerase.

[0138] In some embodiments, dithiothreitol (DTT) and / or 1 mM spermidine are included in the in vitro transcription (IVT) system. In some embodiments, a pyrophosphatase is included in the in vitro transcription reaction to cleave any inorganic pyrophosphate that can be produced after two inorganic phosphate units are incorporated per nucleotide. In this embodiment, magnesium remains in solution and does not precipitate as magnesium pyrophosphate.

[0139] In some embodiments, the in vitro transcription reaction is allowed to proceed, for example, for 4 hours at 37 °C with constant mixing. In some embodiments, the yield ranges, for example, from 1-5 mg RNA / mL transcription reaction. Following the reaction, the mRNA can be purified by any method known in the art following the manufacturer's instructions, including, for example, commercially available silica-based column systems, such as Qiagen RNeasy® kits (QIAGEN) or mRNA Ambion's MEGACLEAR™ kits (Thermo Fisher Scientific). In some embodiments, purification includes removal of linearized plasmid DNA template, for example, separation of the DNA template from the RNA transcript. In one embodiment, the DNA template is removed using poly A capture in a chromatographic process (e.g., an oligo(dT)-based affinity purification step). In this embodiment, the RNA transcript binds to the affinity substrate while the DNA template flows through and is removed. In other embodiments, DNase I is used to enzymatically digest the DNA template immediately after in vitro transcription.

[0140] In some embodiments, the RNA transcript is enzymatically capped at the 5' end after in vitro transcription. Capping can be performed before or after purification of the RNA transcript. Capping can be performed by any method known in the art. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferases can be used to create a classic 5'-5'-triphosphate linkage between the 5' end nucleotide of the mRNA and a guanine cap nucleotide, wherein the cap guanine contains N7 methylation and the 5' end nucleotide of the mRNA contains 2'-O-methylation.

[0141] The RNA transcripts produced by in vitro transcription can be analyzed and characterized as described herein. Analysis can be performed before or after capping and / or before or after purification. RT-PCR

[0142] In some embodiments, at least one mRNA molecule in a composition of the disclosure, obtained for example by in vitro transcription as described above, is used as a template for reverse transcription to make single-stranded cDNA. A reverse transcription (RT) reaction refers to the process of reverse transcribing single-stranded RNA into complementary DNA (cDNA) using any known method. In some embodiments, the RT reaction includes a reverse transcriptase, one or more primers, dNTPs (referring to an equimolar mixture of dATP, dTTP, dCTP, and dGTP), and optionally an RNase inhibitor. General methods and kits including reaction components for reverse transcription are known in the art and can be applied with the methods of the present invention.

[0143] Reverse transcriptases useful in the methods of the disclosure include any polymerase that exhibits reverse transcriptase activity. Suitable reverse transcriptases are known in the art and are commercially available, including, for example, OmniScript (QIAGEN®), avian myeloblastosis virus reverse transcriptase (AMV-RT), Moloney murine leukemia virus reverse transcriptase (MMLV-RT), human immunodeficiency virus reverse transcriptase (HIV-RT), EIAV-RT, RAV2-RT, Tth DNA polymerase, SuperScript I, SuperScript II, SuperScript III (Thermo Fisher Scientific), and mutants, variants, and derivatives thereof.

[0144] In some embodiments, the primers used for the first-strand cDNA synthesis reaction generally rely on the principle of oligo(dT) priming. Oligo(dT) refers to a short single-stranded sequence of deoxythymidine (dT). In some embodiments, the primers comprise a stretch of at least 12 thymidines. In the reverse transcription reaction, the primer binds to the poly(A) tail of the mRNA molecule, and the oligo(dT) initiates reverse transcription at the 3’ end of the transcript. In some embodiments, reverse transcription of the at least one mRNA molecule comprises annealing at least one oligo d(T)n primer to the at least one mRNA molecule of the disclosure.

[0145] Various types of oligo(dT) primers are known in the art and are commercially available. For example, oligo(dT) 20 is a homogenous mixture of 20-mer thymidines, while oligo(dT) 12-18 is a mixture of 12-mer to 18-mer thymidines. The stretch of poly(T) can be any length suitable for hybridizing to the mRNA poly(A) tail and is extended by the reverse transcriptase during the RT reaction. The primer can also be a mixture of oligo(dT) primers with different lengths of poly(T) stretches.

[0146] In some embodiments, the oligo(dT) primer used in the first strand cDNA synthesis reaction is an anchored oligo(dT). Anchored oligo(dT) primers are designed to avoid poly A slippage by ensuring that they anneal at the 3'-UTR / poly A junction. In some embodiments, the anchored oligo(dT) comprises a stretch of poly(T) followed by a nucleotide that is not thymidine (denoted as "V", which can be adenine, cytosine, or guanine). In some embodiments, the primer is used as a mix so as to represent all species of "V" (adenine, cytosine, and guanine). In some embodiments, the non-thymidine nucleotide is the 3' end nucleotide. In other embodiments, the "V" nucleotide is followed by one or more additional nucleotides, which can be thymidine, adenine, cytosine, or guanine (denoted as "N"). In some embodiments, the primer comprises a mix so as to represent all species of "N". The oligo(dT) primer can be denoted as oligo(dT) n where "n" is the number of thymidines in the poly(T) stretch. In some embodiments, "n" is any integer between 5 and 30. The anchored oligo(dT) primer can be denoted as oligo(dT) n V, where "n" is the number of thymidines in the poly(T) stretch, and "V" is adenine, cytosine, and guanine, or denoted as oligo(dT) n VN, where "n" is the number of thymidines in the poly(T) stretch, and "V" is adenine, cytosine, and guanine, and "N" is any nucleotide.

[0147] In some embodiments, the mRNA (such as one mRNA molecule, such as two different mRNA molecules, such as three different mRNA molecules, such as four different mRNA molecules, such as a plurality of more than four different mRNA molecules) is first incubated with the above primer under conditions that denature the mRNA secondary structure (e.g., about 65 °C). The primer / mRNA mixture is then quickly chilled on ice to allow the primer to anneal to the mRNA. Next, the other components of the RT reaction are added to the mixture, including dNTPs, RNase inhibitor, reverse transcriptase, and RT buffer (including, e.g., Tris-HCl, KCl, MgCl2, DTT).

[0148] In some embodiments, the extension reaction is performed under conditions that allow the primers to be extended by a reverse transcriptase, for example at a temperature between about 37°C and 55°C for 15, 30, 45, 60 minutes or longer. For some thermostable reverse transcriptases, the reaction can be performed at higher temperatures. In one embodiment, Superscript Reverse Transcriptase III (SSRTIII) is used to generate full-length cDNA. In some embodiments, the reverse transcriptase can be inactivated with a short incubation at high temperature after annealing and extension (e.g., 5-10 min at > 85°C). In some embodiments, the RNA template is removed. In some embodiments, the template RNA is destroyed by treating the RT reaction with RNase H.

[0149] In some embodiments, one or more first-strand cDNAs prepared corresponding to each species of mRNA molecule in the compositions of the disclosure as described above are used directly as templates for a polymerase chain reaction (PCR) to generate double-stranded cDNA, which is then amplified. PCR is a well-known technique in the art. PCR is used to amplify nucleic acids by subjecting a reaction mixture to cycles of: 1) nucleic acid denaturation, 2) oligonucleotide primer annealing, and 3) nucleic acid polymerization. In some embodiments, the reaction conditions for amplification include thermal cycling, i.e., alternating the temperature of the reaction mixture to facilitate each step of the PCR cycle. In some embodiments, PCR is extended by multiple cycles of denaturation, annealing, and replication, and optionally augmented with an initial extended denaturation step and a final extended extension (polymerization) step. In some embodiments, thermal cycling occurs between about 23°C to about 100°C, such as a temperature range of about 37°C to about 95°C. In some embodiments, nucleic acid denaturation occurs between about 90°C to about 100°C, such as about 94°C. In some embodiments, annealing occurs between about 37°C to about 75°C, such as about 55°C. In some embodiments, polymerization occurs between about 55°C to about 80°C, such as about 72°C. The number of thermal cycles can vary, depending on, for example, the amount of DNA product desired. In some embodiments, the number of PCR cycles ranges from about 5 to about 99. In some embodiments, the number of PCR cycles is greater than about 15, 20, 25, 30, 35, or 40. In some embodiments, the number of PCR cycles is about 20, about 25, about 30, about 35, or about 40 cycles (including all values and subranges therebetween).

[0150] In some embodiments, components of the PCR reaction mixture include a DNA template (e.g., cDNA as described herein), a thermostable DNA polymerase, primers, and dNTPs. Thermostable polymerases are isolated from a variety of thermophilic bacteria, such as Thermus aquaticus (Taq), Thermus brockianus (Tbr), Thermus flavus (Tfl), Thermus rubber (Tru), Thermus thermophilus (Tth), Thermococcus litoralis (Tli) and other species of Thermococcus, Thermoplasma acidophilum (Tac), Thermotoga neapolitana (Tne), Thermotoga maritime (Tma) and other species of Thermotoga, Pyrococcus furiosus (Pfu), Pyrococcus woesei (Pwo) and other species of Pyrococcus (e.g., Phusion® (New England BioLabs®)), Bacullus sterothermophilus (Bst), Sulfolobus acidocaldarius (Sac), Sulfolobus solfataricus (Sso), Pyrodictium occultum (Poc), Pyrodictium abyssi (Pab), and Methanobacterium thermoautotrophicum (Mth) and mutants, variants, or derivatives thereof. Many DNA polymerases are known in the art and are commercially available.

[0151] In some embodiments, the amplified double-stranded cDNA is purified. Commercially available purification kits can be used, such as QIAquick® PCR Purification Kit (Qiagen). Oligonucleotide primers

[0152] In some embodiments, oligonucleotide primers useful for PCR have a length of about 15 to about 30 bases, are not palindromic (self-complementary), and are not complementary to other primers that can be used in the reaction mixture. In some embodiments, primers used to facilitate reverse transcription of a first nucleic acid molecule complementary to a portion of an mRNA template (e.g., a cDNA molecule) as described herein can also be used to facilitate replication of nucleic acids (e.g., PCR amplification of DNA). Any primers can be synthesized by one of ordinary skill in the art or can be purchased from any of a number of commercial suppliers.

[0153] In some embodiments, primers used for PCR are different from reverse transcription primers. In some embodiments, PCR primers are sequence-specific primers. Desired sequence-specific primers for use in the present methods can be designed by one of ordinary skill using methods known in the art, including, for example, using primer design software, such as the web-based tool Primer3 WEB.

[0154] In some embodiments, a pair of sequence-specific PCR primers can be used to amplify only one of the cDNAs corresponding to mRNA molecules in a composition of the disclosure. In other embodiments, a pair of sequence-specific primers can be used to amplify at least two of the cDNAs corresponding to at least two mRNA molecules of the disclosure. In other embodiments, a pair of sequence-specific primers can be used to amplify all mRNA molecules of interest in a composition of the disclosure.

[0155] In some embodiments, primers used to amplify double-stranded cDNA molecules anneal to regions of cDNA corresponding to 3' UTRs and 5' UTRs of one or more mRNA molecules in a composition of the disclosure. In some embodiments, a single pair of such primers is capable of annealing to more than one cDNA corresponding to mRNA in a composition of the disclosure.

[0156] For example, in some embodiments, where the composition of the disclosure comprises more than one mRNA molecule, e.g., four different mRNA molecules, each encoding four different HA antigen proteins from four different influenza A strains, as described herein, primers annealing to the cDNA nucleotides corresponding to each of the 3’-UTR and 5’-UTR of the mRNA molecules can be used to obtain and amplify double stranded cDNA from the single stranded cDNA molecules. In some embodiments, the 3’-UTR primer is set forth in SEQ ID NO: 2 or SEQ ID NO: 3. In some embodiments, the 3’-UTR primer is set forth in SEQ ID NO: 2. In some embodiments, the 3’-UTR primer of SEQ ID NO: 2 or SEQ ID NO: 3 can be paired with a 5’-UTR primer selected from SEQ ID NO: 1, 6, 7, 8, or 9. In some embodiments, the 3’-UTR primer of SEQ ID NO: 2 can be paired with a 5’-UTR primer selected from SEQ ID NO: 1, 6, 7, 8, or 9. In some embodiments, the 3’-UTR primer of SEQ ID NO: 2 can be paired with a 5’-UTR primer selected from SEQ ID NO: 6, 7, 8, or 9. In some embodiments, the primer comprises a 3’-UTR primer as set forth in SEQ ID NO: 2 paired with a 5’-UTR primer as set forth in SEQ ID NO: 6. Such sequence specific primers can be beneficially used in PCR to avoid, for example, false background products. Restriction fragment analysis

[0157] The methods disclosed herein can be used to determine the presence of at least one mRNA molecule in a composition. In some embodiments, the method comprises digesting at least one amplified double stranded cDNA molecule as described herein with at least one restriction enzyme to obtain test cDNA fragments. The test cDNA fragments are then separated to form a test cDNA fragment profile. The test profile is then compared to a control cDNA fragment profile, wherein the at least one mRNA molecule is present in the composition of the disclosure when the test cDNA fragment profile comprises the control cDNA fragment profile.

[0158] Restriction enzymes can be obtained from bacteria or produced by recombinant techniques, and are readily available through many commercial sources. In some embodiments, the restriction enzymes that can be used to obtain the test and control cDNA fragments of the disclosure are bacterial enzymes that bind and cleave DNA at specific target sequences. Restriction enzymes can bind DNA at specific recognition sites consisting of short palindromic sequences and cleave the DNA within the site, e.g., AGCT (for Alul), GAATTC (for EcoRI).

[0159] In some embodiments, the predicted nucleic acid sequence of at least one amplified double-stranded cDNA molecule of the disclosure is analyzed to identify at least one restriction site, e.g., GAATTC, such as at least two restriction sites, such as at least three restriction sites, such as at least four restriction sites, based on the nucleic acid sequence of the template from which at least one mRNA was transcribed, to obtain test and / or control cDNA fragments. In some embodiments, it can be desirable to identify the absence of a restriction site, e.g., the absence of an AluI or EcoRI site.

[0160] Double-stranded cDNA molecules can be digested with appropriate restriction enzymes, and the resulting fragments subjected to separation, as described herein. Those skilled in the art will understand how to analyze a nucleic acid sequence of interest, such as an amplified double-stranded cDNA molecule of the disclosure, to identify suitable restriction enzymes or combinations of restriction enzymes (e.g., two different restriction enzymes, three different restriction enzymes, four different restriction enzymes, or more) for use in the methods described herein.

[0161] For example, in some embodiments, software, such as Geneious Prime software (Biomatters, Inc.), or other methods known to those skilled in the art, can be used to identify one or more restriction enzyme sites in a cDNA of the disclosure. In some embodiments, software or other methods known to those skilled in the art can be used to select a desired set of restriction enzymes, e.g., all known commercially available restriction enzymes or a desired subset thereof. Restriction enzymes that cleave a cDNA of the disclosure, e.g., at 0-5 sites (such as 0-4 sites, such as 0-3 sites, such as 0-2 sites, such as 0-1 site, such as at 1 or more restriction sites) can be identified. In some embodiments, regions of cleavage are identified in order to obtain a particular number of fragments of distinguishable lengths. For example, in some embodiments, only restriction enzymes that cleave a cDNA within a specified region (e.g., a specified internal region of the cDNA or any other region of the cDNA) are identified. In other embodiments, restriction enzymes that do not cleave a cDNA of the disclosure within a specified region, but do cleave the cDNA outside of the specified region are identified. In other embodiments, enzymes that cleave anywhere within a cDNA of the disclosure can be identified. In some embodiments, the software displays a list of fragments produced by the selected restriction enzymes.

[0162] For a multivalent composition, each of the different amplified double-stranded cDNA molecules can be analyzed to identify at least one restriction site for each of the different amplified double-stranded cDNA molecules. In some embodiments, the predicted nucleic acid sequence of the different amplified double-stranded cDNA molecules is analyzed to identify at least one restriction site for each of the different amplified double-stranded cDNA molecules based on the nucleic acid sequence of the template that transcribed the different mRNA. In some embodiments, the at least one restriction site identified for each of the different amplified double-stranded cDNA molecules is present in only one of the different amplified double-stranded cDNA molecules. In some embodiments, the at least one restriction site identified for each of the different amplified double-stranded cDNA molecules is present in more than one (e.g., two) of the different amplified double-stranded cDNA molecules.

[0163] In some embodiments, a restriction enzyme is used that generates distinctly distinguishable cDNA fragments for each of the different amplified double-stranded cDNA molecules. For example, the difference in length of the DNA fragments generated for each of the different amplified double-stranded cDNA molecules can be at least 50 bases, such as at least 100 bases, at least 150 bases, at least 200 bases, at least 250 bases, at least 300 bases, or at least 350 bases or more (including all values and subranges therebetween). In some embodiments, the difference in length of the DNA fragments generated for each of the different amplified double-stranded cDNA molecules is at least 400 bases, such as at least 450 bases, at least 500 bases, at least 550 bases, at least 600 bases, at least 650 bases, at least 700 bases, at least 750 bases, at least 800 bases, at least 850 bases, at least 900 bases, at least 950 bases, or at least 1000 bases (including all values and subranges therebetween). In some embodiments, a restriction enzyme that can digest only one (or two) cDNA molecule(s) (corresponding to only one (or two) mRNA molecule(s) in a composition containing multiple mRNA molecules) is more likely to more clearly distinguish and / or quantify the generated cDNA fragments.

[0164] In some embodiments, a restriction enzyme that generates a limited number of fragments is more likely, such as a restriction enzyme that generates only 6 fragments (such as only 5 fragments, such as only 4 fragments, such as only 3 fragments, such as only 2 fragments, such as only 1 fragment) for each of the different amplified double-stranded cDNA molecules.

[0165] Suitable restriction enzymes include, for example, Aarl, Aatll, Absl, AccI, AceII, AceIII, Acil, Acll, Acyl, Affel, Aflll, Aflll, Age I, Agsl, AhaIII, Alul, AlwNI, AmaCSI, ApaBI, Apal, ApaLI, Apol, ApyPI, AquII, AquIII, AquIV, Ascl, Asi256I, Asp718I, Asul, Asul, Aval, AvaII, AvrII, Bahl, BamHI, Bbel, BbvCI, Bbvl, Bbvll, BccI, Bce83I, BceAI, BceAI, BcefI, BciVI, BcII, Betl, Bfll, BglI, BglII, Binl, Bpu10I, BsaAI, BsaBI, BsbI, BscAI, BscXI, BscXI, BscXI, BscXI, BseMII, BseMII, BsePI, BsePI, BsePI, BseRI, BseSI, BseYI, BseYI, BsgI, BsiI, BsiYI, BsmAI, BsmFI, BsmI, BsmI, BsmI, BsmI, BsmI, BsmI, Bsp120I, Bsp1407I, BspCNI, BspD6I, BspHI, BspKT6I, BspLU11I, BspMI, BspMII, BsrBI, BsrDI, BsrI, BstAPI, BstEII, BstF5I, BstNI, BstUI, BstXI, BtgZI, BthCI, BtrI, BtsI, BtsIMutI, Cac8I, CauII, CchII, CdpI, Cfr10I, CfrI, Chal, CjeNIII, Clal, CstMI, CviAII, CviJI, CviQI, CviRI, Ddel, Dral, Dralll, DralRl, DralRl, Drdl, DrdIV, DrdV, Dsal, Eam1105I, EciI, Eco31I, Eco47III, Eco56I, Eco57I, Eco57MI, Eco78I, EcoHI, EcoICRI, EcoNI, EcoRI, EcoRII, EcoRV, EcoT22I, EsaBC3I, Esp3I, EspI, Fail, FatI, FauI, Fmul, Fnu4HI, FnuDII, FokI, Fsel, Fsel, FspAI, GdiII, Gsal, Gsul, Haell, Haelll, Haelll, HauII, Hgal, HgiAI,HgiCI, Hhal, HindII, HindIII, HinfI, HinP1I, HpaI, HpaII, HphI, Hpy178III, Hpy188I, Hpy8I, Hpy99I, KasI, Kpnl, Ksp632I, Lmnl, LpnI, MaeI, MaeII, MaeIII, MaqI, MauBI, MboI, MboII, McaTI, McrI, MfeI, Mlul, MlyI, MmeI, MnlI, MseI, MslI, MspGI, MstI, MwoI, NaeI, NarI, Ncol, Ndel, NgoAVII, NheI, NlaCI, NlaIII, NlaIV, Nli3877I, NmeA6CIII, NmeAIII, NotI, NruI, NspBII, NspI, OliI, PabI, PacI, PasI, PasI, PflMI, PfoI, PlaDI, PleI, PmaCI, PmeI, Ppu10I, PpuMI, PshAI, PsiI, Psp03I, PspOMII, PspPRI, PspXI, PssI, PstI, PvuI, PvuII, RceI, RdeGBII, RlaII, RleAI, RpaB5I, RpaBI, Rpal, RsaI, RsrII, SacI, SacII, Sail, SanDI, SapI, SauI, ScaI, ScII, ScrFI, SdeAI, SduI, SecI, SelI, SetI, SexAI, SfaNI, SfeI, Sfil, SgfI, SgrAI, SgrDI, SgrAI, SgrDI, SimI, SmaI, SmlI, SnaBI, Spel, SphI, SplI, SrfI, Sse232I, Sse8387I, Sse8647I, SsoII, SspD5I, SspI, SstE37I, Sth132I, Sth302II, StsI, Stul, StyI, Swal, TaiI, TaqI, TaqII, TaqIII, TatI, TauI, Tfil, TkoI, TkoII, TseI, TsoI, Tsp45I, Tsp4CI, TspDTI, TspEI, TspGWI, TspRI, Tth111I, Tth111II, UnbI, VpaK11AI, VspI, WviI, Xbal, Xcai, Xcml, Xhol, XhoII, Xmal, XmaIII, Xmnl, and / or Zral.

[0166] In some embodiments, the at least one restriction enzyme comprises BstUI. In some embodiments, the at least one restriction enzyme comprises Age I, HinII, Affel, and Sac I. In some embodiments, the at least one restriction enzyme comprises Ava I, Acc I, PfIMI, and Stu I.

[0167] In some embodiments, at least one amplified double-stranded cDNA molecule of the disclosure is digested by combining the amplified double-stranded cDNA molecule with one or more selected restriction enzymes, water, and buffer in a 10 µl to 50 µl reaction. In some embodiments, one unit of a restriction endonuclease is used to completely digest 1 µg of substrate DNA in 1 hour. In some embodiments, a 10-fold excess of enzyme is added to the reaction to ensure complete cleavage. In some embodiments, the digestion reaction is performed at 37°C for 1-4 hours. However, some restriction enzymes require higher (e.g., 50-65°C) temperatures, while other restriction enzymes require lower (e.g., 25°C) incubation temperatures.

[0168] In some embodiments, at least one restriction enzyme of the disclosure is used to digest each of the species of amplified double-stranded cDNA molecules corresponding to the different species of mRNA molecules of the disclosure. For example, in some embodiments, the at least one restriction enzyme digests each of a plurality of amplified double-stranded cDNA molecules. In some embodiments, one restriction enzyme digests two of a plurality of amplified double-stranded cDNA molecules. In some embodiments, one restriction enzyme digests only one of a plurality of amplified double-stranded cDNA molecules.

[0169] In some embodiments, a composition of the disclosure is a tetravalent vaccine comprising a plurality of amplified double-stranded cDNA molecules, wherein the plurality of amplified double-stranded cDNA molecules comprises a first amplified double-stranded cDNA molecule corresponding to a first species of mRNA molecule in the tetravalent vaccine, a second amplified double-stranded DNA molecule corresponding to a second species of mRNA molecule in the tetravalent vaccine, a third amplified double-stranded DNA molecule corresponding to a third species of mRNA molecule in the tetravalent vaccine, and a fourth amplified double-stranded DNA molecule corresponding to a fourth species of mRNA molecule in the tetravalent vaccine, wherein the at least one restriction enzyme digests the first amplified double-stranded DNA molecule, the second amplified double-stranded DNA molecule, the third amplified double-stranded DNA molecule, and the fourth amplified double-stranded DNA molecule. In some embodiments, the at least one restriction enzyme is BstUI.

[0170] In some cases, it can not be possible or can be challenging to identify a single restriction enzyme that digests each of a plurality of amplified double-stranded cDNA molecules. Alternatively, a restriction enzyme can generate fragments of similar size and thus difficult to distinguish upon isolation for two or more of a plurality of amplified double-stranded cDNA molecules. In yet further alternatives, a restriction enzyme can generate small fragments that are difficult to quantify for one or more of a plurality of amplified double-stranded cDNA molecules of the disclosure. In such embodiments, two or more restriction enzymes can be used, wherein each restriction enzyme digests only one or only two of a plurality of amplified double-stranded cDNA molecules. In these embodiments, test cDNA fragments of distinguishable size and easy to quantify can be obtained for each of a plurality of amplified double-stranded cDNA molecules.

[0171] For example, in some embodiments, the composition of the disclosure is a tetravalent vaccine, and the plurality of amplified double-stranded cDNA molecules comprises a first amplified double-stranded cDNA molecule corresponding to a first species of mRNA molecule in the tetravalent vaccine, a second amplified double-stranded cDNA molecule corresponding to a second species of mRNA molecule in the tetravalent vaccine, a third amplified double-stranded cDNA molecule corresponding to a third species of mRNA molecule in the tetravalent vaccine, and a fourth amplified double-stranded cDNA molecule corresponding to a fourth species of mRNA molecule in the tetravalent vaccine, wherein the at least one restriction enzyme comprises a first, a second, a third, and a fourth restriction enzyme, wherein the first restriction enzyme digests the first amplified double-stranded cDNA molecule, the second restriction enzyme digests the second amplified double-stranded cDNA molecule, the third restriction enzyme digests the third amplified double-stranded cDNA molecule, and the fourth restriction enzyme digests the fourth amplified double-stranded cDNA molecule. In some embodiments, the four restriction enzymes comprise Age I, Hinc II, Affe I, and Sac I.

[0172] In other embodiments, the composition of the disclosure can be a tetravalent vaccine, and the plurality of amplified double-stranded DNA molecules comprises a first amplified double-stranded cDNA molecule corresponding to a first species of mRNA molecule in the tetravalent vaccine, a second amplified double-stranded cDNA molecule corresponding to a second species of mRNA molecule in the tetravalent vaccine, a third amplified double-stranded cDNA molecule corresponding to a third species of mRNA molecule in the tetravalent vaccine, and a fourth amplified double-stranded cDNA molecule corresponding to a fourth species of mRNA molecule in the tetravalent vaccine, wherein the at least one restriction enzyme comprises a first restriction enzyme and a second restriction enzyme, wherein the first restriction enzyme digests the first amplified double-stranded cDNA molecule and the second amplified double-stranded cDNA molecule, and the second restriction enzyme digests the third amplified double-stranded cDNA molecule and the fourth amplified double-stranded cDNA molecule.

[0173] In other embodiments, the composition of the present disclosure can be a tetravalent vaccine, and the plurality of amplified double stranded DNA molecules comprises a first amplified double stranded cDNA molecule corresponding to a first species of mRNA molecule in the tetravalent vaccine, a second amplified double stranded cDNA molecule corresponding to a second species of mRNA molecule in the tetravalent vaccine, a third amplified double stranded cDNA molecule corresponding to a third species of mRNA molecule in the tetravalent vaccine, and a fourth amplified double stranded cDNA molecule corresponding to a fourth species of mRNA molecule in the tetravalent vaccine, wherein the at least one restriction enzyme comprises a first restriction enzyme and a second restriction enzyme, wherein the first restriction enzyme digests the first amplified double stranded cDNA molecule, and the second restriction enzyme digests the second amplified double stranded cDNA molecule, the third amplified double stranded cDNA molecule, and the fourth amplified double stranded cDNA molecule.

[0174] In other embodiments, the composition of the present disclosure can be a tetravalent vaccine, and the plurality of amplified double stranded DNA molecules comprises a first amplified double stranded cDNA molecule corresponding to a first species of mRNA molecule in the tetravalent vaccine, a second amplified double stranded cDNA molecule corresponding to a second species of mRNA molecule in the tetravalent vaccine, a third amplified double stranded cDNA molecule corresponding to a third species of mRNA molecule in the tetravalent vaccine, and a fourth amplified double stranded cDNA molecule corresponding to a fourth species of mRNA molecule in the tetravalent vaccine, wherein the at least one restriction enzyme comprises a first restriction enzyme, a second restriction enzyme, and a third restriction enzyme, wherein the first restriction enzyme digests the first amplified double stranded cDNA molecule, the second restriction enzyme digests the second amplified double stranded cDNA molecule, and the third restriction enzyme digests the third amplified double stranded cDNA molecule and the fourth amplified double stranded cDNA molecule. isolating

[0175] In some embodiments, the test cDNA fragments produced by the restriction enzyme digestion described above are isolated. Isolation can be accomplished by various methods known to those of skill in the art. For example, methods of liquid chromatography, such as HPLC or UPLC, can be used to isolate the test cDNA fragments of the present disclosure. HPLC and UPLC rely on a pump to pass a pressurized liquid solvent containing the sample mixture through a column packed with a solid adsorbent material. Each component in the sample interacts differently with the adsorbent material, resulting in different flow rates for the different components, resulting in separation of the components as they flow out of the column.

[0176] In some embodiments, electrophoresis can be used to separate the test cDNA fragments disclosed herein. In electrophoresis, biomolecules (e.g., nucleic acids, proteins, or amino acids) are separated based on charge, size, and shape. In gel electrophoresis, a physical gel is used as the separation medium. A gel electrophoresis apparatus includes a gel casting tray for preparing the gel, a casting comb for preparing wells, a buffer tank, positive and negative electrodes, and a voltage supply unit. Negatively charged molecules (e.g., nucleic acids) move from the cathode to the anode. If a high degree of molecular separation or isolation is desired, a higher concentration gel with smaller pore sizes can be prepared. Nucleic acids separated on the gel matrix can be further separated using an intercalation dye (e.g., ethidium bromide, SYBR). ® After staining with green or acridine orange, the molecules are observed using a UV transilluminator. The separated molecules appear as bands on the gel matrix. In some embodiments, the electrophoretic separation method disclosed herein includes agarose gel electrophoresis.

[0177] In some embodiments, capillary electrophoresis (CE) is used to separate the test cDNA fragments disclosed herein. CE is a modification of gel electrophoresis. In CE, similar to gel electrophoresis, the separation of molecules (e.g., the cDNA test fragments disclosed herein) is based on the molecule's charge, size, and shape. However, CE is performed in a capillary containing a gel material (such as polyacrylamide) or a liquid polymer (such as hydroxymethyl cellulose). In some embodiments, the capillary may be made of molten silica with an inner diameter ranging from 50 to 100 μm and a length ranging from 25 to 100 cm. The sample is injected into the capillary containing the polymer material, and separation is faster than with conventional gel electrophoresis. CE can provide greater resolution than gel electrophoresis, and the separation can be more accurate. In some embodiments, molecules separated using CE are detected via an automated spectrophotometric detector. In other embodiments, molecules separated using CE are labeled as described herein and detected using any known method (e.g., via a fluorescence detector). In some embodiments, commercially available automated capillary electrophoresis platforms (e.g., Agilent 5200, 5300, and 5400 fragment analyzer systems (Agilent Technologies)) can be used to separate the test and / or control cDNA fragments disclosed herein. In some embodiments, cDNA fragments can be visualized as electrophoretic maps using, for example, Agilent 5200, 5300, and / or 5400 fragment analyzer system software. In other embodiments, cDNA fragments can be visualized as digital or “virtual” electrophoretic agarose gels using, for example, the 5200, 5300, and 5400 fragment analyzer systems (Agilent Technologies).

[0178] In some embodiments, the isolated test cDNA fragments comprise 5'-test cDNA fragments and 3 '-test cDNA fragments. In some embodiments, the isolated cDNA test fragments further comprise one or more fragments internal to the 5'-test cDNA fragments and 3 '-test cDNA fragments located in the intact double-stranded cDNA molecule prior to restriction enzyme digestion and isolation.

[0179] In some embodiments, the test cDNA fragments and the control cDNA fragments in the control cDNA fragment profile are labeled prior to or after isolation. In some embodiments, the label comprises a DNA intercalating label as described above. In some embodiments, the label is a fluorescent dye, including, for example, 6-FAM™ (blue), VIC (green), NED™ (yellow / black), PET (red), and LIZ (orange). Degradation analysis

[0180] In one aspect, the disclosure provides a method of assessing the integrity or degradation of at least one test mRNA molecule disclosed herein. In some embodiments, at least one test cDNA fragment in a test cDNA fragment profile is quantified as described herein, for example, to assess the degree of degradation, if any, of at least one mRNA molecule disclosed herein. The amount of the quantified test cDNA fragment in the test cDNA fragment profile is then compared to the amount of the corresponding control cDNA fragment in the control cDNA fragment profile. A decrease in the amount of the at least one test cDNA fragment compared to the amount of the corresponding control cDNA fragment is indicative of degradation, and thus, a decrease in the integrity of the at least one test mRNA molecule in the composition. In some embodiments, the test cDNA fragments and the control cDNA fragments are labeled as described herein. In some embodiments, the quantification is determined by determining the intensity of the labeled control cDNA fragment and the intensity of the labeled test cDNA fragment as described herein. A decrease in the intensity of the labeled test cDNA fragment compared to the intensity of the labeled control cDNA fragment is indicative of degradation of the corresponding at least one mRNA molecule in the composition of the disclosure. In some embodiments, a change in intensity of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% (including all values and subranges therebetween) is indicative of degradation. In some embodiments, if a decrease in intensity of at least 10% between the test cDNA and the control cDNA fragment is detected, the corresponding mRNA molecule is discarded. In some embodiments, the control cDNA fragment corresponds to an mRNA molecule obtained at time point 0 (TP0) and the test cDNA fragment corresponds to an mRNA molecule obtained at a subsequent time point X (TPx), where TP0 < TPx. In some embodiments, the test cDNA fragment corresponds to an mRNA molecule obtained at a time point X (TPx) and the control cDNA fragment corresponds to an mRNA molecule obtained at a subsequent time point Y (TPy), where TPx < TPy. In some embodiments, the test cDNA fragment corresponds to an mRNA molecule obtained at a time point X (TPx) and the control cDNA fragment corresponds to an mRNA molecule obtained at a time point Y (TPy), where TPx < TPy. Xobtained mRNA molecule. In some embodiments, the mRNA molecule is prepared at time point zero, e.g., by in vitro transcription, and the double-stranded cDNA is also prepared at this initial time. In contrast, in some embodiments, the double-stranded test cDNA is prepared from the same batch of mRNA but at a later time, e.g., 2 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, or more (including all values and subranges therebetween) after the initial batch of mRNA is prepared. This embodiment can be used to assess, e.g., mRNA storage conditions to determine whether, e.g., degradation of the mRNA molecule occurs over time.

[0181] In some embodiments, the quantified test cDNA fragments are 3 '-quantified test cDNA fragments, and the quantified control cDNA fragments are 3 '-quantified control cDNA fragments. In some embodiments, the quantified test cDNA fragments are 5 '-quantified test cDNA fragments, and the quantified control cDNA fragments are 5 '-quantified control cDNA fragments. In some embodiments, the quantified test cDNA fragments are fragments internal to the 3 '-test cDNA fragments and the 5 '-test cDNA fragments, and the quantified control cDNA fragments are corresponding fragments internal to the 3 '-control cDNA fragments and the 5 '-control cDNA fragments.

[0182] In some embodiments, degradation, if any, is indicated at the 3 'end of the mRNA molecule, but not internal or at the 5 'end. In other embodiments, degradation, if any, of the mRNA molecule is indicated at the 5 'end, but not at the 3 'end or internal. In some embodiments, degradation, if any, of the mRNA molecule is indicated internal, but not at the 3 'end or the 5 'end.

[0183] In other embodiments, the amount of quantified 3 '-test cDNA fragments is compared to the amount of quantified 5 '-test cDNA fragments, e.g., where each fragment is included in the cDNA test fragment profile, e.g., in the same lane of an agarose gel. In this embodiment, the quantified 5 '-test cDNA fragments are used as a control, and a decrease in the amount of 3 '-test cDNA fragments compared to the amount of "control" 5 '-test DNA fragments indicates degradation of the corresponding mRNA molecule at the 3'end. In other embodiments, the quantified 5 '-test cDNA fragments are used as a control, and compared to, e.g., internal fragments to assess possible internal degradation, where a decrease in the amount of internal fragments compared to the amount of "control" 5 '-test DNA fragments indicates degradation of the corresponding mRNA molecule at the internal region. In other embodiments, the 3 '-test cDNA fragments are used as a control, and compared to 5 '-test cDNA fragments or internal cDNA fragments to assess possible 5'end or internal degradation, where a decrease in the amount of 5 '-test cDNA fragments or internal cDNA fragments compared to the amount of "control" 3 '-test DNA fragments indicates degradation of the corresponding mRNA molecule at the 5'end or internal region. In other embodiments, the internal cDNA fragments are used as a control fragment, and compared to 3 '-test cDNA fragments or 5 '-test cDNA fragments to assess possible 5'end and / or 3'end degradation, where a decrease in the amount of 3 '-test cDNA fragments or 5 '-test cDNA fragments compared to the amount of "control" internal fragments indicates degradation of the corresponding mRNA molecule at the 3'end or 5'end.

[0184] In some embodiments, a decrease of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% (including all values and subranges therebetween) between the test cDNA and control cDNA fragments indicates degradation of the corresponding mRNA molecule of the compositions of the disclosure. In some embodiments, if a decrease of at least 10% between the test cDNA and control cDNA fragments is detected, the corresponding mRNA molecule is discarded. In certain embodiments, the test cDNA and control cDNA fragments are labeled as described herein.

[0185] In other embodiments, the amount of the quantified 3 '-test cDNA fragment is determined relative to the amount of the quantified 5 '-test cDNA fragment ("test ratio"), wherein each test cDNA fragment is included in the cDNA test fragment profile. In this embodiment, the test ratio is compared to the ratio of the amount of the quantified 3 '-control cDNA fragment to the amount of the quantified 5 '-control cDNA fragment ("control ratio"). In this embodiment, a change in the ratio between the test ratio and the control ratio of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% (including all values and subranges therebetween) is indicative of degradation of at least one mRNA molecule in the composition of the disclosure.

[0186] In Figure 1 the foregoing embodiments are illustrated. As Figure 1 shown in , two lanes containing cDNA labeled with ethidium bromide and illuminated on an agarose gel are depicted. The lane designated TP0 depicts a control fragment profile containing two fragments (5 '-control cDNA fragment and 3 '-control cDNA fragment) obtained from a restriction digest of double-stranded cDNA corresponding to a template control mRNA molecule prepared using in vitro transcription (IVT) at time point 0 (TP0). The lane designated TP X depicts a cDNA fragment profile containing two fragments (5 '-test cDNA fragment and 3 '-test cDNA fragment) obtained from a restriction digest of double-stranded cDNA corresponding to the same template mRNA molecule used to prepare the control cDNA fragments, but which has been stored for a period of time (TP X ).

[0187] The relative intensity of each illuminated fragment is determined. The ratio of the relative fluorescence intensity of the 5 '-control fragment to the 3 '-control fragment (RF TP0 ) is 1.5. The ratio of the relative fluorescence intensity of the 5 '-test cDNA fragment to the 3 '-test cDNA fragment (RF TPX ) is 2.5. Thus, the difference between RF TP0 and RF TPX (ARF) is 1, and the percent change (ARF / RF TP0 ) is 66.7%. Since this change between the test ratio and the control ratio is 10% or greater, it is indicative of degradation of the mRNA molecule at least at the 3'end.

[0188] In some embodiments, the test ratio comprises a 5'-cDNA fragment and an internal test cDNA fragment, and the control ratio comprises a 5'-control cDNA fragment and an internal control cDNA fragment. In some embodiments, the test ratio comprises a 3'-test cDNA fragment and an internal test cDNA fragment, and the control ratio comprises a 3'-control cDNA fragment and an internal control cDNA fragment. quantification

[0189] The test cDNA and control cDNA fragments of the present disclosure can be quantified by any method known in the art. Quantification can be relative and / or absolute. For example, in some embodiments, the relative quantification of a cDNA fragment can be determined by labeling the cDNA fragments with an intercalating dye (e.g., staining the gel) or a fluorescent dye, visualizing the cDNA fragments with a transilluminator, and assessing the relative fluorescence intensity of the fragments using, for example, ImageJ software (National Institutes of Health) (NIH, imagej.nih.gov / ij / download.html) on an agarose gel. In other embodiments, the relative fluorescence intensity of a test cDNA and / or control cDNA fragment can be assessed by comparing the intensity of the fragment to a DNA ladder (e.g., Invitrogen DNA Ladder (Thermo Fisher Scientific)) comprising known amounts of fragments. Relative quantification of the test cDNA and control cDNA fragments of the present disclosure can also be determined during fragment separation using known techniques. For example, in some embodiments, the amount of test cDNA and control cDNA fragments can be quantified using capillary electrophoresis, for example, using the commercially available 5300 P / ACE® MDQ Analytical System (Agilent Technologies).

[0190] In some embodiments, absolute quantification of the test cDNA and control cDNA fragments of the present disclosure can be determined during fragment separation. For example, in some embodiments, the amount of test cDNA and control cDNA fragments can be quantified using liquid chromatography, such as high performance liquid chromatography (HPLC). In alternative embodiments, fragments can be quantified by purifying the fragments of interest from, for example, an agarose gel, for example, using a commercially available kit (such as the QIAquick® Gel Extraction Kit (QIAGEN)), and determining the absolute quantification of the purified cDNA fragments using, for example, a spectrophotometer (such as the NanoDrop™ Spectrophotometer (Thermo Fisher Scientific)). compositions

[0191] In some embodiments, the disclosure relates to a primer pair comprising a 5'-UTR primer and a 3'-UTR primer. In some embodiments, the 5'-UTR primer is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, and the 3'-UTR primer is selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3. In some embodiments, any primer pair comprising a 5'-UTR primer and a 3'-UTR primer that can be suitable for use in the methods described in the disclosure can be used.

[0192] In some embodiments, the disclosure relates to a primer pair selected from the group consisting of: a) SEQ ID NO: 6 and SEQ ID NO: 2; b) SEQ ID NO: 7 and SEQ ID NO: 2; c) SEQ ID NO: 8 and SEQ ID NO: 2; and d) SEQ ID NO: 9 and SEQ ID NO: 2.

[0193] In some embodiments, the primers are detectably labeled. Any detectable label used in conjunction with nucleic acid technology can be used, including, for example, labels that generate a signal from a chemical reaction, such as a change in light absorbance, a change in fluorescence, generation of chemiluminescence or electrochemiluminescence, a change in reflectance, refractive index, or light scattering, accumulation or release of a detectable label from a surface, oxidation or reduction or redox species, a change in current or potential, magnetic field, etc. In some embodiments, the detectable label includes a DNA intercalating label, including, for example, ethidium bromide, SYBR® green, or acridine orange. In some embodiments, the label is a fluorescent dye, such as, for example, 6-FAM™ (blue), VIC (green), NED™ (yellow / black), PET (red), and LIZ (orange).

[0194] Labeled primers are not naturally occurring molecules; that is, the combination of a nucleic acid primer coupled with a label does not exist in nature.

[0195] Primers can be synthesized according to any method known in the art, such as, for example, chemical synthesis as described herein, or purchased from any of a number of commercial suppliers. Examples Example 1. Selection of mRNA

[0196] A quadrivalent (QIV) mRNA vaccine was generated and characterized that contains four different species of mRNA molecules, each encoding a hemagglutinin (HA) antigen from a different influenza strain, as summarized in Table 1. Table 1. QIV mRNA vaccine targets

[0197] Figure 2 A depicts a sequence alignment of the HA gene of each of the four influenza strains described in Table 1. As shown in Figure 2 B, the HA genes share a range of percent identity from 47.2% up to 76.4%. As described in the present disclosure, the inventive method can be used to readily distinguish cDNAs representing mRNA molecules with high sequence identity (e.g., greater than 75%) as exemplified below. The inventive method exemplified herein can also be used to determine the presence, integrity, and quantity of different mRNA molecules in an mRNA therapeutic composition (such as the QIV mRNA vaccine used in these examples). Example 2. In vitro transcription

[0198] The mRNA of the QIV mRNA vaccine was produced by in vitro transcription (IVT). Briefly, plasmid DNA encoding four mRNAs (including a poly-A tail) were each linearized and added to an IVT premix containing transcription buffer (Tris-HCl pH 8.0, MgCl2, dithiothreitol (DTT), Teknova, Inc.), T7 RNA polymerase, nucleotide triphosphates (NTPs, Roche Holding AG), RNase inhibitor, and nuclease-free water. Each reaction mixture was incubated at 37 °C for 90 minutes to obtain IVT mRNA. Following incubation, the plasmid DNA template was removed by adding a DNase pre-mix (containing DNase, DNase buffer, and nuclease-free water), followed by a 15-minute incubation at 37 °C. The IVT mRNA was then purified using a Qiagen® RNeasy® kit according to the manufacturer’s protocol.

[0199] The concentration of each of the four IVT mRNAs was determined using a NanoDrop™ spectrophotometer (Thermo Fisher Scientific). As shown in Table 2 below, the concentrations ranged from 1222 ng / µl to 2587 ng / µl. Table 2. Quantification of IVT mRNA

[0200] Quality of IVT mRNA was assessed by capillary electrophoresis (CE) using an Agilent 5300 Fragment Analyzer (Agilent Technologies) with Agilent-DNF-471-1000 RNA Kit according to the manufacturer’s instructions. Briefly, the Fragment Analyzer system was prepared to incorporate a capillary array containing RNA separation gels (DNF-265-0240) mixed with intercalating dye (DNF-600-U030). Samples were heat denatured and added to wells in a sample plate containing diluent marker (DNF-369-0004). RNA ladder marker (DNF-382-U020) was used as a control. The plate was loaded into the Fragment Analyzer, then fragments were separated and visualized on a digital gel image as shown in FIG. 1. Figure 3

[0201] Figure 3 As expected, the range of IVT mRNA was from about 1950 to 2000 nucleotides. In addition, no additional smaller fragments (indicator of problematic transcription) or larger fragments (indicator of DNA template contamination) were detected. Thus, the IVT mRNA was considered to be of high quality. Example 2A. First-Strand cDNA Synthesis

[0202] First-strand complementary DNA (cDNA) was synthesized from each of the four IVT mRNA samples using reverse transcription (RT). RT was performed using the Invitrogen SuperScript™ III First-Strand Synthesis Reagent according to the manufacturer’s protocol (Thermo Fisher Scientific). Briefly, IVT mRNA (1000 ng) from each sample was used as a template for different oligo(dT) primers (100 µM). Messenger RNA primer annealing was performed by incubating the mRNA / oligo(dT) mixture at 65°C for five minutes, followed by a quick chill on ice. A cDNA pre-mix containing RNase inhibitor, SuperScript™ III Reverse Transcriptase, dNTPs, DTT, and first-strand buffer was added to the IVT mRNA / oligo(dT) mixture and incubated at 50°C for 50 minutes. The reaction was terminated by heating to 85°C for 5 minutes. Each mRNA template was removed by adding RNase H to the terminated reaction, followed by incubation at 37°C for 20 minutes. Example 2B. PCR Evaluation of First-Strand cDNA i. PCR

[0203] ​The first strand cDNAs obtained as described above were used as templates for polymerase chain reaction (PCR). For each first strand cDNA template, four PCR reaction mixtures were prepared, each containing a different reverse primer, i.e., SEQ ID NOs: 2-5 (Table 3), paired with SEQ ID NO: 1 (Table 3) to assess primer specificity. A fifth PCR reaction mixture was also prepared as a positive control, containing the plasmid encoding sample I.D. number 1 as the DNA template, with primers SEQ ID NOs: 1 and 2.

[0204] The SEQ ID NOs: 1 and 2 primers were designed by evaluating each of the 4 individual cDNA sequences corresponding to mRNA molecule sample I.D. numbers 1-4 using the web-based tool Primer3 WEB, which allows selection of multiple input parameters such as primer length, melting temperature (Tm), and GC content. The specificity of the resulting PCR primers was evaluated by cDNA alignment with the other 3 cDNA sequences in the QIV sample.

[0205] PCR was performed using Phusion® Hot Start Flex 2X Master Mix (New England BioLabs®) according to the manufacturer's protocol. After 30 PCR cycles, the reaction products were separated by electrophoresis on a 1.2% agarose gel and photographed.

[0206] Figure 4 Four of the PCR reaction products are shown, all of which were generated from the first strand cDNA template representing sample I.D. number 1 and the four different primer pairs described above. As shown in Figure 4 all of the PCR reactions produced full-length amplicons. As further described below, the PCR primers were then optimized to mitigate background products. Table 3. Primers used for 1st strand cDNA evaluation ii. PCR optimization

[0207] A series of truncated 5'-UTR PCR primers were designed to improve the specificity of the PCR amplification reaction. The truncated primers from SEQ ID NO: 1 are shown in Table 4 below. Four PCR reaction mixtures were prepared, each containing one of the truncated 5'-UTR primers (SEQ ID NO: 6, 7, 8, or 9) paired with the 3'-UTR primer (3'-UTR-12) of SEQ ID NO: 2 depicted in Table 3. To directly compare, a PCR reaction mixture containing the untruncated 5'-UTR of SEQ ID NO: 1 (5'-UTR + 15, Table 4) paired with the 3'-UTR(-12) (SEQ ID NO: 2) primer was also prepared. First strand cDNA synthesized from IVT mRNA of sample I.D. number 1 was used as template in all five PCR reactions. DNA Polymerase, Hot Start Taq 2X Master Mix (Intact® Genomics, Inc.) was included in each PCR reaction mixture.

[0208] After 30 PCR cycles, the resulting products were separated by electrophoresis on a 1.2% agarose gel and photographed. As shown in FIG. 2, the desired full-length amplicon was observed in all products. However, less background product was observed from the PCR reactions produced with the truncated 5'-UTR primers (lanes 2-5) compared to the PCR reaction produced with the 5'-UTR untruncated primer (lane 1). Figure 5 Table 4. 5' PCR primers used for PCR optimization iii. PCR evaluation of individual first strand cDNA and mixtures of first strand cDNA

[0209] The truncated 5'-UTR of SEQ ID NO: 6 was paired with the 3'-UTR of SEQ ID NO: 2 and used to prepare additional PCR reactions to evaluate each of the individual first strand cDNAs produced from the 4 IVT mRNA templates. A PCR reaction containing a mixture of all four first strand cDNA templates ("QIV cDNA template mixture") was also prepared. DNA Polymerase, Hot Start Taq 2X Master Mix (Intact® Genomics, Inc.) was included in each of the five PCR reaction mixtures.

[0210] After 30 PCR cycles, the resulting products were separated by electrophoresis on a 1.2% agarose gel and photographed. As shown in FIG. 2, the desired full-length amplicon was observed in all products. However, less background product was observed from the PCR reactions produced with the truncated 5'-UTR primers (lanes 2-5) compared to the PCR reaction produced with the 5'-UTR untruncated primer (lane 1). Figure 6 ​As shown in Figure 2, each of the PCR reaction products obtained using either individual first strand cDNA as template (lanes 1-4) or QIV cDNA template mixture (lane 5) produced the desired amplicon with little apparent non-specific amplification. Example 3. Restriction fragment length polymorphism (RFLP) i. Identification of restriction enzymes (RE) for one of the four cDNA species in the mixture

[0211] As described above in Example 2Bii, PCR reaction products obtained using individual first strand cDNA templates representing IVT mRNA samples I.D. Nos. 1-4 were sequenced to identify restriction enzyme (RE) sites for restriction fragment length polymorphism (RFLP) analysis. RE sites for four REs (i.e., Age I, Hinc II, Afe I, and Sac I, respectively) were identified, each of which only recognized one of the four template cDNAs.

[0212] Subsequently, PCR reaction products obtained using the QIV cDNA template mixture were digested with Age I, Hinc II, Afe I, or Sac I (New England BioLabs®) according to the manufacturer’s protocol. Equal amounts of PCR reaction product (1500 ng / 50 µl final volume) were used for each RE digestion. Following RE digestion, PCR reaction products were purified using QIAGEN® PCR purification reagent according to the manufacturer’s protocol and then eluted with 100 µl of water. Restriction enzyme digestion products (60 ng) were separated by electrophoresis on a 1.2% agarose gel and photographed. For comparison, undigested PCR reaction product prepared from the mixture of the four cDNAs was also loaded on the gel.

[0213] As shown in Figure 2, each of the PCR reaction products obtained using either individual first strand cDNA as template (lanes 1-4) or QIV cDNA template mixture (lane 5) produced the desired amplicon with little apparent non-specific amplification. Figure 7 and Table 5, Age I cleavage represented only the cDNA for sample I.D. No. 1, producing two fragments (1137 bp and 804 bp). Similarly, Hinc II, Afe I, and Sac I cleavage represented only the cDNAs for samples I.D. Nos. 2, 3, and 4, respectively, each producing two fragments. Table 5. Restriction enzymes targeting one of the four PCR reaction products

[0214] As an alternative to separating the cDNA fragments by agarose gel electrophoresis, capillary electrophoresis (CE) was used to separate and quantify the cDNA fragments generated by RE digestion. First, serial 2-fold dilutions of the PCR reaction products digested with Age I, Hinc II, Afe I, and Sac I were prepared in Tris-EDTA (TE) buffer (6, 3, 1.5, and 0.75 ng / µl) and then separated by CE using an Agilent 5300 Fragment Analyzer.

[0215] Figure 8 A digital gel image of the digested PCR products obtained from the QIV cDNA template mixture and separated by CE is depicted. Similar to agarose gel electrophoresis, the digital gel image of the digested PCR reaction products depicts two fragments cut with Age I, Hinc II, Afe I, and Sac I, respectively.

[0216] Figure 9 A depicts an electropherogram obtained with an Agilent 5300 Fragment Analyzer. Two peaks corresponding to the 1165 bp and 824 bp fragments obtained from Afe I digestion and a peak corresponding to the uncut cDNA are observed, Afe I recognizing only the cDNA in the PCR reaction product corresponding to mRNA sample I.D. number 3. Figure 9 B depicts a photograph of an agarose gel showing the same digestion products for comparison.

[0217] The Agilent Fragment Analyzer also reported that 16.3% of the total sample was attributed to the 5'-test cDNA fragment of 824 bp, 20.9% of the total sample was attributed to the 3'-test cDNA fragment of 1165 bp, and 58.2% of the total sample was attributed to the remaining three uncut cDNAs. As described above in the detailed description, the relative concentrations of the 5'-test and 3'-test cDNA fragments can be used to assess the integrity of the mRNA sample by comparing the relative concentrations of each fragment to, for example, the relative concentration of the photograph fragment. For example, the relative amount of the 3'-test cDNA fragment can be used as a 3'-control cDNA fragment and compared to the 5'-test cDNA fragment. In this example, the difference between the relative amounts of the 3'-test cDNA fragment (as a control) and the 5'-test cDNA fragment was 4.6% (20.9%-16.3%). Since this difference did not reach at least 10%, degradation of mRNA sample number 3 was not indicated. ii. RE identifying two cDNA species in a mixture of cDNA species

[0218] Additional RE sites recognized by Ava I, Acc I, Stu I and Pfl M I were identified in the cDNAs corresponding to mRNA sample I.D. numbers 1-4. Ava I recognized only RE sites in the cDNAs corresponding to sample I.D. numbers 1 and 2. Ava I cleaved the cDNA corresponding to sample I.D. number 2 at two sites located in the sequence coding part, i.e. at position 198 from the 5' end and at position 783 from the 3' end. Thus, after digestion with Ava I, three fragments of 198 bp, 960 bp and 783 bp in length, respectively, were generated (starting from the 5' end). Thus, the 960 bp fragment is located internally between the 5'-test cDNA fragment of 198 bp and the 3'-test cDNA fragment of 783 bp. Ava I cleaved the cDNA corresponding to sample I.D. number 1 at one site, resulting in two fragments (5' test cDNA fragment and 3'-test cDNA fragment). Acc I and Stu I recognized only one RE site in each of the cDNAs corresponding to sample I.D. numbers 3 and 4, respectively, while Pfl M I recognized only one RE site in each of the cDNAs corresponding to sample I.D. numbers 2 and 4, respectively.

[0219] The four PCR reaction products obtained using the QIV cDNA template mix as described in Example 2Bii above were each digested with Ava I, Acc I, Stu I or Pfl M I. The RE reactions were performed as described above in Example 3ii. The restriction enzyme digestion products (60 ng) were separated by electrophoresis on a 1.2% agarose gel and photographed. For comparison, also a mixture of the four cDNAs without digestion was loaded on the gel.

[0220] From Figure 10 It can be seen that digestion with Ava I, Acc I, Stu I or Pfl M I each resulted in a different fragment pattern. Ava I resulted in five fragments, while Acc I, Stu I and Pfl M I each resulted in a pattern of fragments with four different sizes, as shown in the table below. Table 6. Restriction enzymes targeting two of the four PCR reaction products

[0221] For the restriction enzymes Age I, Hinc II, Afe I and Sac I, CE was used to separate and quantify the DNA fragments generated by digestion with Ava I, Acc I and Stu I, as described in Example 3i above. Figure 11 A digital gel image of the digested diluted PCR products obtained from the QIV cDNA template mix is depicted. From Figure 11 It can be seen that the digital gel image resulted in the same fragment pattern as shown in the agarose gel of Figure 10 .

[0222] Figure 12A An electropherogram of the PCR reaction products separated by CE digested with Ace I is depicted as Figure 11 The electropherogram shows five large peaks from left to right corresponding to the first 5'-test cDNA fragment (543 bp, corresponding to Sample I.D. No. 4), the first 3'-test fragment (654 bp, corresponding to Sample I.D. No. 3), the second 5'-test fragment (1335 bp, corresponding to Sample I.D. No. 3), the second 3'-test fragment (1452 bp, corresponding to Sample I.D. No. 4), and the uncut cDNA (corresponding to Sample I.D. Nos. 1-4).

[0223] The Agilent Fragment Analyzer also reported that 17.1% of the total sample was attributed to the 5'-test cDNA fragment of 1335 bp (Sample I.D. No. 3), 10.6% of the total sample was attributed to the 3'-test cDNA fragment of 654 bp (Sample I.D. No. 3), 7.9% of the total sample was attributed to the 5'-test cDNA fragment of 543 bp (Sample I.D. No. 4), 17.4% of the total sample was attributed to the 3'-test cDNA fragment of 1452 bp (Sample I.D. No. 4), and 44.0% of the total sample was attributed to the remaining 2 uncut cDNAs. Thus, the fragment profile generated by Ace I, Hinc II, Afe I, and Sac I can be used to confirm the presence of each mRNA in the QIV influenza vaccine.

[0224] In addition, the relative amounts of the 5'-test cDNA fragment and the 3'-test cDNA fragment can be compared to assess the integrity of the mRNA of Sample I.D. Nos. 3 and 4. For example, the relative amount of the 5'-test cDNA fragment of Sample I.D. No. 3 can be used as a control. In this example, the difference between the relative amounts of the 5'-test cDNA fragment (as control) and the 3'-test cDNA fragment of Sample I.D. No. 3 is 6.5% (17.1%-10.6%). Since this difference does not reach at least 10%, degradation of the mRNA Sample No. 3 is not indicated. Similarly, the relative amount of the 3'-test fragment of Sample I.D. No. 4 can be used as a control. Since the difference between the relative amounts of the 3'-test cDNA fragment (as control) and the 5'-test fragment is 9.5% (17.4%-7.9%), degradation of the mRNA of Sample I.D. No. 4 is not indicated. iii. Restriction enzymes (REs) that identify the 4 cDNAs in the PCR reaction products

[0225] PCR reaction products obtained using four separate first strand synthesis cDNA templates representing IVT mRNA from four additional samples (Samples A-D) were prepared as described above for Samples I.D. Nos. 1-4. The PCR products were sequenced to identify restriction enzyme (RE) sites for restriction fragment length polymorphism (RFLP) analysis. RE sites were identified in all four cDNAs recognized by BstUI.

[0226] The length of the DNA fragments that would be generated from these products if the PCR reaction products were digested with BstUI was predicted using Geneious Prime software (Biomatters Corporation). The Geneious Prime software uses DNA sequence files to enable the generation of virtual gels of restriction enzymes. The predicted fragments for each PCR product after theoretical digestion with BstUI were visualized on a virtual agarose gel. Figure 13 The predicted fragment pattern for each PCR product after theoretical digestion is depicted. As shown in Table 6 below, the predicted fragment lengths for the PCR product representing Sample A after theoretical digestion with BstUI predicted the generation of 3 fragments of lengths 318 bp, 670 bp, and 713 bp. The PCR product representing Sample B after theoretical BstUI digestion was predicted to generate 3 fragments of lengths 1262 bp, 153 bp, and 286 bp. The PCR products representing Sample C and Sample D after theoretical BstUI digestion were also predicted to generate three fragments of lengths 830 bp, 704 bp, 215 bp (Sample C) and 380, 702, and 673 bp (Sample D). Table 7. Fragment lengths for PCR products representing IVT mRNA (Samples A-D) cleaved with BstUI. * 5’ fragment; ** 3’ fragment

[0227] Figure 14 The fragment pattern for all PCR products after theoretical digestion with BstUI as described above loaded onto a single lane is depicted. From Table 6 above and Figure 14 As can be seen, some of the predicted fragment lengths are nearly identical, i.e., the PCR product representing Sample A after theoretical digestion has a predicted fragment length of 670 bp and the PCR product representing Sample D after theoretical digestion has a similar predicted size fragment of 673 bp. In addition, the PCR products representing Sample D, Sample C, and Sample A after theoretical digestion each contain similar predicted size fragments of 702, 704, and 713 bp, respectively. Further, the PCR reaction products representing Sample B and Sample A after theoretical BstUI digestion are predicted to generate smaller fragments, i.e., 286 and 318 bp, respectively.

[0228] As discussed in the present disclosure, fragments of similar size can be more difficult to distinguish, making it difficult to make a profile determination of a given mRNA. Further, as noted herein, shorter fragments, such as those less than 400 bp, can be more difficult to quantify. Thus, while in the present example a single restriction enzyme was identified that theoretically cleaves each of the PCR products representing four different mRNAs, in some embodiments, it can be more desirable to be able to use a restriction enzyme that digests only one or two cDNAs, e.g., corresponding to only one or two mRNA molecules in a composition containing multiple mRNA molecules (as described in detail), to more clearly distinguish and / or quantify the generated cDNA fragments.

[0229] While the foregoing disclosure has been described in some detail and explained with the intent and effect of illustrative and example embodiments, it will be apparent to those skilled in the art, by reading this disclosure, that various changes in form and detail can be made without departing from the true scope of the disclosure and that it is intended to cover all such changes and modifications within the scope of the appended claims. For example, all constructs, methods, and / or features, steps, elements or other aspects of their components can be used in various combinations.

[0230] All patents, patent applications, websites, other publications or files, accession numbers, and the like cited herein are hereby incorporated by reference for all purposes to the same extent as if each individual item were specifically and individually indicated to be incorporated by reference.

Claims

1. A method for determining the presence of at least one messenger RNA (mRNA) molecule in a composition, the method comprising: (a) Reverse transcription of at least one mRNA molecule to obtain a template containing at least one first strand of complementary DNA (cDNA); (b) Generate at least one double-stranded cDNA molecule from the template; (c) Amplify at least one double-stranded cDNA molecule; (d) Digest the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain a test cDNA fragment; (e) Isolate these test cDNA fragments to form a test cDNA fragment map; and (f) Compare the cDNA fragment map of the test with the control cDNA fragment map. When the test cDNA fragment map includes the control cDNA fragment map, the at least one mRNA molecule is present in the composition.

2. The method of claim 1, further comprising determining the integrity of the at least one mRNA molecule in the composition, the method further comprising: (g) Quantify the amount of at least one test cDNA fragment in the test cDNA fragment map; as well as (h) Compare the amount of the at least one test cDNA fragment with the amount of the control cDNA fragment in the control cDNA fragment map. The reduction in the amount of the at least one test cDNA fragment compared to the amount of the control cDNA fragment indicates degradation, and thus indicates a decrease in the integrity of the at least one mRNA molecule in the composition.

3. A method for quantifying at least one messenger RNA (mRNA) molecule in a composition, the method comprising: (a) Reverse transcription of at least one mRNA molecule to obtain a template containing at least one first strand of complementary DNA (cDNA); (b) Generate at least one double-stranded cDNA molecule from the template; (c) Amplify at least one double-stranded cDNA molecule; (d) Digest the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain a test cDNA fragment; (e) Isolate these test cDNA fragments to form a test cDNA fragment map; (f) Determine the amount of at least one test cDNA fragment in the test cDNA fragment map; (g) Compare the amount of the at least one test cDNA fragment with the amount of a control cDNA fragment; and (h) Quantify the amount of the at least one mRNA molecule in the composition based on the amount of the at least one test cDNA fragment relative to the amount of the control cDNA fragment.

4. A process for manufacturing a composition comprising at least one messenger RNA (mRNA) molecule, wherein the process includes: (a) Reverse transcription of at least one mRNA molecule to obtain a template containing at least one first strand of complementary DNA (cDNA); (b) Generate at least one double-stranded cDNA molecule from the template; (c) Amplify at least one double-stranded cDNA molecule; (d) Digest the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain a test cDNA fragment; (e) Isolate these test cDNA fragments to form a test cDNA fragment map; (f) Determine the amount of at least one test cDNA fragment in the test cDNA fragment map; (g) Compare the amount of the at least one test cDNA fragment with the amount of a control cDNA fragment; and (h) Quantify the amount of the at least one mRNA molecule in the composition based on the amount of the at least one test cDNA fragment relative to the amount of the control cDNA fragment.

5. The method of claim 3 or 4, wherein a reduction in the amount of the at least one test cDNA fragment compared to the amount of the control cDNA fragment indicates a reduction in the amount of the at least one mRNA molecule in the composition compared to the amount of the at least one control mRNA molecule.

6. The method of any of the preceding claims, wherein the at least one mRNA molecule is the product of in vitro transcription of an unamplified DNA template.

7. The method of any of the preceding claims, wherein the test cDNA fragment is separated by liquid chromatography or electrophoresis.

8. The method of claim 7, wherein the liquid chromatography method includes high performance liquid chromatography (HPLC) or ultra-high performance liquid chromatography (UPLC).

9. The method of claim 7, wherein the electrophoresis comprises capillary electrophoresis.

10. The method of claim 7, wherein the electrophoresis comprises gel electrophoresis.

11. The method of claim 10, wherein the gel electrophoresis is agarose gel electrophoresis.

12. The method of claim 10, wherein the gel electrophoresis is capillary gel electrophoresis.

13. The method of any one of claims 2-12, wherein the at least one test cDNA fragment comprises a 5'-test cDNA fragment and a 3'-test cDNA fragment.

14. The method of claim 13, wherein the 5'-test cDNA fragment and the 3'-test cDNA fragment contain a detectable marker.

15. The method of claim 14, wherein the detectable marker is a fluorescent dye.

16. The method of claim 15, wherein the detectable marker is a DNA embedding marker.

17. The method of any one of claims 14-16, wherein the control cDNA fragment is a labeled 5'-test cDNA fragment or a labeled 3'-test cDNA fragment.

18. The method of claim 17, wherein the quantification comprises comparing the intensity of the labeled 3'-test cDNA fragment with that of the labeled 5'-test cDNA fragment.

19. The method of claim 18, wherein a decrease in the intensity of the labeled 3'-test cDNA fragment compared to the intensity of the labeled 5'-test cDNA fragment, or a decrease in the intensity of the labeled 5'-test cDNA fragment compared to the intensity of the labeled 3'-test cDNA fragment, indicates degradation of the at least one mRNA molecule in the composition.

20. The method of any one of claims 13-16, wherein a change in the ratio of the intensity of the labeled 5'-test cDNA fragment to the intensity of the labeled 3'-test cDNA fragment, compared to a control ratio, indicates degradation of the at least one mRNA molecule in the composition.

21. The method of claim 20, wherein the cDNA fragment of the control ratio corresponds to the mRNA molecule obtained at time point 0 (TP0), and the cDNA fragment of the test ratio corresponds to the mRNA molecule obtained at a subsequent time point X (TP0). X mRNA molecules obtained.

22. The method of claim 20 or 21, wherein the ratio of the intensity of the labeled 5'-test cDNA fragment to the intensity of the labeled 3'-test cDNA fragment changes by at least 10% compared to the control ratio.

23. The method of any one of claims 2-12, wherein the control cDNA fragment and the at least one test cDNA fragment contain a detectable marker. The quantification includes comparing the intensity of the at least one labeled test cDNA fragment with the intensity of the labeled control cDNA fragment. The decrease in the intensity of the at least one labeled test cDNA fragment compared to the intensity of the control cDNA fragment indicates the degradation of the at least one mRNA molecule in the composition.

24. The method of claim 23, wherein the at least one test cDNA fragment comprises a 5'-test cDNA fragment and a 3'-test cDNA fragment.

25. The method of claim 23 or 24, wherein the detectable marker is a fluorescent dye.

26. The method of claim 25, wherein the detectable marker is a DNA embedding marker.

27. The method of any one of claims 2-12 and 23-26, wherein the control cDNA fragment corresponds to the mRNA molecule obtained at time point 0 (TP0), and the test cDNA fragment corresponds to the mRNA molecule obtained at a subsequent time point X (TP0). X mRNA molecules obtained.

28. The method as described in any of the preceding claims, wherein the composition is a pharmaceutical composition.

29. The method as claimed in any of the preceding claims, wherein the composition is a vaccine.

30. The method of claim 29, wherein the vaccine is selected from the group consisting of: a monovalent vaccine, a bivalent vaccine comprising two different types of mRNA molecules, a trivalent vaccine comprising three different types of mRNA molecules, and a quadrivalent vaccine comprising four different types of mRNA molecules.

31. The method of claim 29 or 30, wherein the vaccine is an influenza vaccine.

32. The method of claim 30, wherein in the bivalent vaccine, the two different types of mRNA molecules share at least 50% sequence identity.

33. The method of claim 30, wherein in the bivalent vaccine, the two different types of mRNA molecules share at least 75% sequence identity.

34. The method of claim 30, wherein in the trivalent vaccine, at least two of the three different types of mRNA molecules share at least 50% sequence identity.

35. The method of claim 30, wherein in the trivalent vaccine, at least two of the three different types of mRNA molecules share at least 75% sequence identity.

36. The method of claim 30, wherein in the quadrivalent vaccine, at least two of the four different types of mRNA molecules share at least 50% sequence identity.

37. The method of claim 30, wherein in the quadrivalent vaccine, at least two of the four different types of mRNA molecules share at least 75% sequence identity.

38. The method of any one of claims 30-37, wherein the length difference of the different types of mRNA molecules is 10 bases or less, 9 bases or less, 8 bases or less, 7 bases or less, 6 bases or less, 5 bases or less, 4 bases or less, 3 bases or less, or 2 bases or less.

39. The method of any of the preceding claims, wherein reverse transcription of the at least one mRNA molecule comprises annealing at least one oligod(T)n primer to the at least one mRNA molecule.

40. The method of any of the preceding claims, wherein amplification of the double-stranded cDNA comprises annealing the 5'-UTR primer and the 3'-UTR primer to the double-stranded cDNA.

41. The method of claim 40, wherein the 5'-UTR primer is SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO:

9.

42. The method of claim 41, wherein the 3'-UTR primer is SEQ ID NO: 2 or SEQ ID NO:

3.

43. The method of any of the preceding claims, wherein the at least one mRNA molecule comprises a plurality of different mRNA molecules, wherein each of the different mRNA molecules shares at least 50% sequence identity with at least one other mRNA molecule, and wherein the length difference between each of the different mRNA molecules is 10 bases or less, 9 bases or less, 8 bases or less, 7 bases or less, 6 bases or less, 5 bases or less, 4 bases or less, 3 bases or less, or 2 bases or less.

44. The method of any of the preceding claims, wherein the at least one mRNA molecule comprises a plurality of different mRNA molecules, wherein each of the different mRNA molecules shares at least 75% sequence identity with at least one other mRNA molecule, and wherein the length difference between each of the different mRNA molecules is 10 bases or less, 9 bases or less, 8 bases or less, 7 bases or less, 6 bases or less, 5 bases or less, 4 bases or less, 3 bases or less, or 2 bases or less.

45. The method of any one of claims 43 or 44, wherein the at least one restriction enzyme digests each of the plurality of amplified double-stranded cDNA molecules.

46. ​​The method of any one of claims 43 or 44, wherein the at least one restriction enzyme digests at least two of the plurality of amplified double-stranded cDNA molecules.

47. The method of any one of claims 43 or 44, wherein the at least one restriction enzyme digests only one of the plurality of amplified double-stranded cDNA molecules.

48. The method of any one of claims 43 or 44, wherein the vaccine is a quadrivalent vaccine, and the plurality of amplified double-stranded cDNA molecules comprises a first amplified double-stranded cDNA molecule corresponding to a first type of mRNA molecule in the quadrivalent vaccine, a second amplified double-stranded cDNA molecule corresponding to a second type of mRNA molecule in the quadrivalent vaccine, a third amplified double-stranded cDNA molecule corresponding to a third type of mRNA molecule in the quadrivalent vaccine, and a fourth amplified double-stranded cDNA molecule corresponding to a fourth type of mRNA molecule in the quadrivalent vaccine, and wherein the at least one restriction enzyme digests the first amplified double-stranded cDNA molecule, the second amplified double-stranded cDNA molecule, the third amplified double-stranded cDNA molecule, and the fourth amplified double-stranded cDNA molecule.

49. The method of any one of claims 43 or 44, wherein the vaccine is a quadrivalent vaccine, and the plurality of amplified double-stranded cDNA molecules comprises a first amplified double-stranded cDNA molecule corresponding to a first type of mRNA molecule in the quadrivalent vaccine, a second amplified double-stranded cDNA molecule corresponding to a second type of mRNA molecule in the quadrivalent vaccine, a third amplified double-stranded cDNA molecule corresponding to a third type of mRNA molecule in the quadrivalent vaccine, and a fourth amplified double-stranded cDNA molecule corresponding to a fourth type of mRNA molecule in the quadrivalent vaccine, and wherein the at least one restriction enzyme comprises a first restriction enzyme and a second restriction enzyme, wherein the first restriction enzyme digests the first amplified double-stranded cDNA molecule and the second amplified double-stranded cDNA molecule, and the second restriction enzyme digests the third amplified double-stranded cDNA molecule and the fourth amplified double-stranded cDNA molecule.

50. The method of any one of claims 43 or 44, wherein the vaccine is a quadrivalent vaccine, and the plurality of amplified double-stranded cDNA molecules comprises a first amplified double-stranded cDNA molecule corresponding to a first type of mRNA molecule in the quadrivalent vaccine, a second amplified double-stranded cDNA molecule corresponding to a second type of mRNA molecule in the quadrivalent vaccine, a third amplified double-stranded cDNA molecule corresponding to a third type of mRNA molecule in the quadrivalent vaccine, and a fourth amplified double-stranded cDNA molecule corresponding to a fourth type of mRNA molecule in the quadrivalent vaccine, and wherein the at least one restriction enzyme comprises a first, a second, a third, and a fourth restriction enzyme, wherein the first restriction enzyme digests the first amplified double-stranded cDNA molecule, the second restriction enzyme digests the second amplified double-stranded cDNA molecule, the third restriction enzyme digests the third amplified double-stranded cDNA molecule, and the fourth restriction enzyme digests the fourth amplified double-stranded cDNA molecule.

51. The method of any one of the preceding claims, wherein the at least one restriction enzyme comprises at least one of the following: AarI, AatII, AbsI, AccI, AceII, AceIII, AciI, AclI, AcyI, AffeI, AflII, AflIII, AgeI, AgsI, AhaIII, AluI, AlwNI, AmaCSI, ApaBI, ApaI, ApaLI, ApoI, ApyPI, AquII, AquIII, AquIV, AscI, Asi256I, Asp718I, AsuI, AsuII, AvaI, AvaII, AvrII, BalI, BamH I, BbeI, BbvCI, BbvI, BbvII, BccI, Bce83I, BceAI, BceAI, BcefI, BciVI, BclI, BetI, BfiI, BglI, BglII, BinI, Bpu10I, BsaAI, BsaBI, BsbI, BscAI, BscX Bsc smI, BsmI, Bsp120I, Bsp1407I, BspCNI, BspD6I, BspHI, BspKT6I, BspLU11I, BspMI, BspMII, BsrBI, BsrDI, BsrI, BstAPI, BstEII, BstF5I, BstNI, BstU I, BstXI, BtgZI, BthCI, BtrI, BtsI, BtsIMutI, Cac8I, CauII, CchII, CdpI, Cfr10I, CfrI, ChaI, CjeNIII, ClaI, CstMI, CviAII, CviJI, CviQI, CviRI, Dd eI, DraII, DraIII, DraRI, DraRI, DrdI, DrdIV, DrdV, DsaI, Eam1105I, EciI, Eco31I, Eco47III, Eco56I, Eco57I, Eco57MI, Eco78I, EcoHI, EcoICRI, Eco NI, EcoRI, EcoRII, EcoRV, EcoT22I, EsaBC3I, Esp3I, EspI, FaiI, FatI, FauI, FmuI, Fnu4HI, FnuDII, FokI, FseI, FseI, FspAI, GdiII, GsaI, GsuI, HaeI,HaeII, HaeIII, HauII, HgaI, HgiAI, HgiCI, HhaI, HincII, HindII, HindIII, HinfI, HinP1I, HpaI, HpaII, HphI, Hpy178III, Hpy188I, Hpy8I, Hpy99I, KasI, KpnI, Ksp632I, LmnI, LpnI, MaeI, MaeII, MaeIII, MaqI, MauBI, MboI, MboII, McaTI, McrI, MfeI, MluI, MlyI, MmeI, MnlI, MseI, MslI, MspGI, MstI, MwoI, NaeI, NarI, NcoI, NdeI, NgoAVII, NheI, NlaCI, NlaIII, NlaIV, Nli3877I, NmeA6CIII, NmeAIII, NotI, NruI, NspBII, NspI, OliI, PabI, PacI, PasI, PasI, PflMI, PfoI, PlaDI, PleI, PmaCI, PmeI, Ppu10I, PpuMI, PshAI, PsiI, Psp03I, PspOMII, PspPRI, PspXI, PssI, PstI, PvuI, PvuII, RceI, RdeGBII, RlaII, RleAI, RpaB5I, RpaBI, RpaI, RsaI, RsrII, SacI, SacII, SalI, SanDI, SapI, SauI, ScaI, SciI, ScrFI, SdeAI, SduI, SecI, SelI, SetI, SexAI, SfaNI, SfeI, SfiI, SgfI, SgrAI, SgrDI, SgrAI, SgrDI, SimI, SmaI, SmlI, SnaBI, SpeI, SphI, SplI, SrfI, Sse232I, Sse8387I, Sse8647I, SsoII, SspD5I, SspI, SstE37I, Sth132I, Sth302II, StsI, StuI, StyI, SwaI, TaiI, TaqI, TaqII, TaqIII, TatI, TauI, TfiI, TkoI, TkoII, TseI, TsoI, Tsp45I, Tsp4CI, TspDTI, TspEI, TspGWI, TspRI, Tth111I, Tth111II, UnbI, VpaK11AI, VspI, WviI, XbaI, XcaI, XcmI, XhoI, XhoII, XmaI, XmaIII, XmnI and / or ZraI.

52. The method as described in any of the preceding claims, wherein the at least one restriction enzyme comprises BstUI.

53. The method as described in any of the preceding claims, wherein the at least one restriction enzyme comprises AgeI, HincII, AffeI, and SacI.

54. The method of any of the preceding claims, wherein the at least one restriction enzyme comprises AvaI, AccI, PfIMI, and StuI.

55. The method as described in any one of the preceding claims, wherein the method further comprises: After step (a), purify at least one first strand of the cDNA; And / or purify the at least one amplified double-stranded cDNA after step (c); and / or purify the digested amplified double-stranded cDNA after step (d).

56. A primer pair comprising a 5'-UTR primer and a 3'-UTR primer, wherein the 5'-UTR primer is selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9, and the 3'-UTR primer is selected from the group consisting of: SEQ ID NO: 2 and SEQ ID NO:

3.

57. A primer pair selected from the group consisting of: a) SEQ ID NO: 6 and SEQ ID NO: 2; b) SEQ ID NO: 7 and SEQ ID NO: 2; c) SEQ ID NO: 8 and SEQ ID NO: 2; and d) SEQ ID NO: 9 and SEQ ID NO:

2.

58. The primer pair of claim 56 or 57, wherein the primers are labeled with a detectable marker.

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