WO2025012461A1

WO2025012461A1 - Methods and compositions for analyzing messenger rna

Info

Publication number: WO2025012461A1
Application number: PCT/EP2024/069914
Authority: WO
Inventors: Jianping CUI; Michael J. KARMILOWICZ
Original assignee: Sanofi
Priority date: 2023-07-13
Filing date: 2024-07-12
Publication date: 2025-01-16

Abstract

The present disclosure is directed to a method of determining the presence of at least one messenger RNA (mRNA) molecule in a composition, which method 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, to thereby form a test cDNA fragment pattern; and (f) comparing the test cDNA fragment pattern with a control cDNA fragment pattern, wherein the at least one mRNA molecule is present in the composition when the test cDNA fragment pattern comprises the control cDNA fragment pattern. Methods of quantitating and/or assessing the integrity of at least one mRNA molecule in a composition, a process of manufacturing a composition comprising at least one mRNA molecule and primer pairs for use in the present methods are also disclosed.

Description

METHODS AND COMPOSITIONS FOR ANALYZING MESSENGER RNA

CROSS-REFERENCE TO RELATED APPLICATIONS

[1] This application claims priority to European Application No. 23306201.7 filed 13 July 2023, the contents of which is incorporated herein by reference in its entireties.

SEQUENCE LISTING

[2] The present specification makes reference to a Sequence Listing submitted electronically as an .xml file named “PR95288 EP S ANOFI”. The Sequence Listing was generated on 12 July 2023 and is 8.33 KB in size. The entire contents of the sequence listing is incorporated herein by reference.

FIELD

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

BACKGROUND

[4] Messenger RNA (mRNA) is rapidly emerging as a new class of therapeutics, as reflected, for example, in the remarkable success of mRNA-based vaccines for COVID-19. In addition to the high-profile COVID-19 vaccines, mRNA therapeutics are beneficial, e.g., as vaccines for other infectious agents, replacement therapies and regenerative medicine. Scaling up the production of mRNA therapeutics to meet the growing demand requires improved methods for analyzing mRNA therapeutics.

[5] During the manufacturing process of mRNA therapeutics, incomplete mRNA products may be generated. Furthermore, during manufacturing and storage, mRNA can be degraded, for example, by exposure to heat, hydrolysis, oxidation, light and ribonucleases. Accordingly, analytical methods to assess mRNA quality and to ensure the correct identity of the molecules are valuable tools for manufacturing mRNA therapeutics. Analytical methods are also helpful to assess batch-to batch manufacturing and process repeatability as well as the quality of mRNA produced.

[6] However, current analytical methods available to characterize mRNA therapeutics, including mRNA vaccines, and the development of methods for the analysis of large RNA have proven challenging. For example, while Sanger sequencing may be used, e.g., to validate sequences in a cDNA molecule corresponding to an mRNA of interest, this method may prove problematic for distinguishing between multiple sequences, particularly when the mRNAs share a high percentage of sequence identity. The quality of a Sanger sequence is often not reliable for the first 15 to 40 bases, where primer binding occurs. Moreover, sequence quality typically degrades after 700 to 900 bases.

[7] Mass spectrometry-based methods have also been used to analyze RNA. For example, RNase mapping methods have been developed and used in RNA sequence mapping. Enzymatic digestion using ribonucleases such as RNAse T1 generate small oligoribonucleotides that are amenable to chromatographic separation and intact mass measurements. Additional sequence information of the oligoribonucleotides can be obtained using tandem mass spectrometry (MS/MS). However, the use of high-frequency RNAse enzymes for RNA sequence mapping of long mRNA therapeutics results in the production of many small oligoribonucleotides, which map to many different locations throughout the RNA sequence and therefore do not generate unique sequences for sequence mapping. Furthermore, the analysis of RNase sequence mapping MS data is challenging and currently there are limited dedicated software tools available.

[8] Accordingly, there remains an urgent 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

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

[10] In one aspect, the disclosure is directed to a method of determining the presence of at least one messenger RNA (mRNA) molecule in a composition, which method 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, to thereby form a test cDNA fragment pattern; and (f) comparing the test cDNA fragment pattern with a control cDNA fragment pattern, wherein the at least one mRNA molecule is present in the composition when the test cDNA fragment pattern comprises the control cDNA fragment pattern. In some embodiments, the method further comprises determining the integrity of the at least one mRNA, the method further comprising (g) quantifying an amount of at least one test cDNA fragment in the test cDNA fragment pattern; and (h) comparing the amount of the at least one test cDNA fragment to an amount of a control cDNA fragment in the control cDNA fragment pattern, wherein a reduced amount of the at least one test cDNA fragment in comparison to the amount of the control cDNA fragment indicates degradation and thereby reduced integrity of the at least one mRNA molecule in the composition.

[11] In another aspect, the present disclosure is directed to a method of quantifying at least one messenger RNA (mRNA) molecule in a composition, which method 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, to thereby form a test cDNA fragment pattern; (f) determining an amount of at least one test cDNA fragment in the test cDNA fragment pattern; (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 amount of a control cDNA fragment.

[12] In yet another aspect, the present disclosure is directed 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 doublestranded cDNA molecule; (d) digesting the at least one amplified double-stranded cDNA molecule with at least one restriction enzyme to obtain test cDNA fragment(e) separating the test cDNA fragments, to thereby form a test cDNA fragment pattern; (f) determining an amount of at least one test cDNA fragment in the test cDNA fragment pattern; (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 a control cDNA fragment.

[13] In another aspect, the present disclosure is directed to a primer pair for use in the methods described herein. In some embodiments, the primer pair comprises a 5'-UTR primer and 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 THE DRAWINGS

[14] FIG. 1 depicts an embodiment of a method for assessing messenger RNA (mRNA) integrity as described in the detailed description.

[15] FIG. 2A depicts a sequence alignment of hemagglutinin (HA) genes 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”).

[16] FIG. 2B shows the sequence percent identities of the HA genes 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”).

[17] FIG. 3 depicts 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.

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

[19] FIG. 5 depicts five PCR reaction products, which were each prepared from the same template, but with different primer pairs (lanes 1-5) and a ladder (lane L), separated by electrophoresis on a 1.2% agarose gel as described in Example 2Bii. [20] FIG. 6 depicts PCR reaction products, which were each prepared using a different template (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.

[21] FIG. 7 depicts four PCR reaction products digested with Agel (lane 1), Hindi (lane 2), Afel (lane 3) or SacI (lane 4), which were each prepared from a mixture of four templates or uncut (lane Q) and separated by electrophoresis on a 1.2% agarose gel as described in Example 3i.

[22] FIG. 8 depicts a digital gel image of five serially diluted PCR reaction products separated by capillary electrophoresis, each prepared from a mixture of four templates, which were not digested with a restriction enzyme (lanes A1-A4), or which were digested with Agel (lanes ASAS), Hindi (lanes A9-A12), Afel (lanes B1-B4) or SacI (lanes B1-B8) as described in Example 31.

[23] FIG. 9A depicts an electropherogram of a PCR reaction product digested with Afel, as described for FIG. 8, separated by capillary electrophoresis and showing two large peaks corresponding to a 5'-test cDNA fragment (left large peak), a 3'-test cDNA fragment (middle large peak) and uncut cDNA (right large peak), which were each prepared from a mixture of 4 different templates as described in Example 3i. The two smaller peaks depict a lower marker (left) and an upper marker (right).

[24] FIG. 9B depicts a PCR reaction product digested with Afel and separated by electrophoresis on a 1.2% agarose gel as described in Example 3i.

[25] FIG. 10 depicts four PCR reaction products, which were each prepared from a mixture of four different templates and digested with Aval (lane 1), AccI (lane 2), PflMI (lane 3) or Stul (lane 4), a ladder (lane L), or uncut (lane U) and separated by electrophoresis on a 1.2% agarose gel as described in Example 3 ii.

[26] FIG. 11 depicts a digital gel image of four serially diluted PCR reaction products, prepared from a mixture of four different templates and separated by capillary electrophoresis, which were uncut (lanes A1-A4) or digested with Aval (lanes B9-B12), AccI (lanes C1-C4) and Stul (lanes C5-C8) as described in Example 3 ii.

[27] FIG. 12A depicts an electropherogram of a PCR reaction product digested with AccI as described for FIG. 11, separated by capillary electrophoresis and showing five large peaks from left to right corresponding to a first 5 '-test cDNA fragment (543 bp, from Sample I.D. No. 4), a first 3’-test cDNA fragment (654 bp, from Sample I.D. No. 3), a second 5'-test cDNA fragment (1335 bp, from Sample I.D. No. 3), a second 3'-test cDNA fragment (1452 bp, from Sample I.D. No. 4), and uncut DNA (from Sample I.D. Nos. 1-4), as described in Example 3 ii.

[28] FIG. 12B depicts a PCR reaction product digested with AccI as shown in FIG. 11 or uncut (lane U), separated by electrophoresis on a 1.2% agarose gel as described in Example 3ii.

[29] FIG. 13 depicts predicted PCR reaction products prepared with a different template (lanes A-D) and a ladder (left); the lengths of the DNA fragments, which would be obtained if digested with BstUI, were predicted and visualized on a virtual 1.2% agarose gel, as described in Example 3iii.

[30] FIG. 14 depicts a mixture of the digested PCR reaction products of FIG. 13 in the same lane (right) and a ladder (left); the lengths of the DNA fragments, which would be obtained if digested with BstUI, were predicted and visualized on a virtual 1.2% agarose gel, as described in Example 3 iii.

DETAILED DESCRIPTION

[31] The following terms shall have the definitions set out below unless otherwise indicated.

Definitions

[32] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[33] The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. According to certain embodiments, when referring to a measurable value such as an amount and the like, “about” is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, ±0.9%, ±0.8%, ±0.7%, ±0.6%, ±0.5%, ±0.4%, ±0.3%, ±0.2% or ±0.1% from the specified value as such variations are appropriate to perform the disclosed methods and/or to make and use the disclosed compositions. When “about” is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

[34] As used herein, the term “at least,” “less than,” “more than,” or “up to” prior to a number or series of numbers (e.g., “at least two”) is understood to include the number adjacent to the term “at least,” “less than” or “more than,” and all subsequent numbers or integers that could logically be included, as clear from context. When the term “at least,” “less than,” “more than,” or “up to” is present before 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 the series or range.

[35] The term “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified unless clearly 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 elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[36] As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[37] As used herein, the terms “in some embodiments,” “in other embodiments,” “in some other embodiments,” or the like, refers to embodiments of all aspects of the disclosure, unless the context clearly indicates otherwise.

[38] As used herein, the term “integrity” in reference to messenger RNA (mRNA) relates to the amount of degradation of an mRNA molecule. The mRNA molecule may be assessed for partial or complete degradation, wherein increased mRNA degradation indicates reduced mRNA integrity. The “integrity” of mRNA can be affected by a variety of factors including heat, hydrolysis, oxidation, light and ribonucleases (RNAses). There are three major classes of RNAses: i) endonucleases that cut 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 commences at an internal portion of an mRNA molecule. In some embodiments, degradation commences at the 5'-end of an mRNA molecule. In some embodiments, degradation commences at the 3 '-end of an mRNA molecule. In some embodiments, the poly(A) tail of an mRNA molecule of a composition of the disclosure is shortened by deadenylases, which expose the mRNA to exonuclease activity. This results in the gradual degradation of the mRNA from the 3'-end towards the coding region, leading to the eventual loss of its functional integrity. mRNA integrity may be evaluated by the methods described herein, including the methods described in the examples.

[39] As used herein, the term “partial degradation” or “partially degraded” in reference to a single mRNA molecule refers to an mRNA molecule, which includes fewer nucleotides than that of the corresponding full-length mRNA molecule. In some embodiments, a partially degraded mRNA molecule is a fragment of the full-length mRNA molecule, which contains 99% or fewer nucleotides than that 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 than that of the corresponding full-length mRNA molecule.

[40] As used herein, “partial degradation” or “partially degraded” in reference to a population of mRNA molecules of a single species refers to a population comprising full-length mRNA molecules of the single species and fragments thereof. In some embodiments, the length of the fragments in a partially degraded population of a single species of mRNA molecules is 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 of the corresponding full-length species. In some embodiments, the size of the fragments in a partially degraded population of mRNA molecules of a single species includes a mixture of full- length mRNA molecules and fragments thereof of various sizes.

[41] As used herein, the term “messenger RNA (mRNA)” refers to any polyribonucleotide, which encodes a polypeptide of interest or fragment thereof. In some embodiments, the mRNA molecule in a composition of the disclosure includes stabilizing elements, such as untranslated regions (UTR) at the 5'-end (5'-UTR) and/or at the 3'-end (3'-UTR), in addition to other structural features, such as a 5'-cap structure and a 3'-poly(A) tail. In a cell, the 5'-UTR and the 3'-UTR may be transcribed from a genomic DNA and are typical elements of a premature mRNA. Characteristic structural features of a mature mRNA, including the 5'-cap and the 3'-poly(A) tail, are usually added to the transcribed (premature) mRNA during mRNA processing.

[42] As used herein, the term “at least one mRNA molecule in a composition” refers to one or more species of mRNA in a composition of the disclosure, which encodes a polypeptide of interest, e.g., the HA antigen from the Wis strain. In some embodiments, the “at least one mRNA molecule in a composition” refers to more than one mRNA species in a composition of the disclosure, wherein each mRNA species encodes a different polypeptide of interest. In some embodiments, there are four different mRNA molecules in a composition of the disclosure, wherein each mRNA molecule encodes, e.g., 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.

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

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

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

[46] 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, involved in mRNA stability and translation competency. In some embodiments, the at least one mRNA molecule in a composition of the present disclosure does not include a 5'-cap.

[47] 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 the 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 terms mRNA or mRNA molecule.

[48] As used herein, the term “polypeptide” includes a polymer molecule comprised of multiple amino acids joined in a linear manner. A polypeptide can, in some embodiments, correspond to molecules encoded by a polynucleotide sequence which is naturally occurring. The polypeptide may include conservative substitutions where the naturally occurring amino acid is replaced by one having similar properties, where such conservative substitutions do not alter the function of the polypeptide.

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

[50] As used herein, the term “cDNA” refers to a DNA that is complementary or identical to an mRNA molecule, except that cDNA includes thymine (T) rather than uracil (U). The term “cDNA” encompasses molecules that may be in either single-stranded or double-stranded form.

[51] As used herein, the term “test cDNA fragment” or “test cDNA fragments” refer to a cDNA, such as an amplified double-stranded cDNA molecule, which has been subjected to a restriction enzyme reaction as described herein. Such cDNA is obtained from a reverse transcription reaction as described herein using an mRNA molecule in a composition of the disclosure as a template. In some embodiments, the cDNA subjected to a restriction enzyme reaction is cleaved resulting in test cDNA fragments. In some embodiments, the test cDNA fragments are separated as described herein to form a test cDNA fragment pattern, which may be visualized, as described herein. In some embodiments, the cDNA subjected to a restriction enzyme reaction is not cleaved and results in a test cDNA fragment pattern having only one test cDNA fragment, which may correspond to a full-length mRNA of the composition of the disclosure. The test cDNA fragment pattern may be visualized as described herein. [52] In some embodiments, the cDNA, which is subjected to a restriction enzyme reaction, is cleaved to yield only two test cDNA fragments, i.e., a 5'-test cDNA fragment corresponding to a region of the mRNA template that is directly upstream from the start codon and a 3 '-test cDNA fragment corresponding to a region of the mRNA template that is directly downstream from the stop codon. In other embodiments, the cDNA, which is subjected to a restriction enzyme reaction, is cleaved to yield more than two test cDNA fragments, including a 5'-test cDNA fragment corresponding to a region of the mRNA template that is directly upstream from the start codon, internal fragments, and a 3 '-test cDNA fragment corresponding to a region of the mRNA template that is directly downstream from the stop codon. An uncleaved test cDNA fragment, test cDNA fragments resulting from restriction enzyme cleavage, and/or a test cDNA fragment pattern, which may be visualized as described herein, may be used to analyze, e.g., determine the presence, quantity and/or integrity of an mRNA molecule in a composition of the disclosure, as described herein. As used herein, the term “control cDNA fragment” or “control cDNA fragments” refer to a cDNA, such as an amplified double-stranded cDNA molecule, which has been subjected to a restriction enzyme reaction as described herein to yield the control cDNA fragments. In some embodiments, the cDNA subjected to the restriction enzyme reaction is obtained, e.g., from a reverse transcription reaction as described herein using an mRNA molecule as a template, which is not degraded. In some embodiments, the control cDNA fragments are separated as described herein to form a control cDNA fragment pattern, which may be visualized, as described herein and compared to a test cDNA fragment pattern as described herein. In some embodiments, the control cDNA subjected to a restriction enzyme reaction is not cleaved and results in a control cDNA fragment pattern having only one control cDNA fragment, which may correspond to a full-length template mRNA.

[53] In some embodiments, the cDNA, which is subjected to a restriction enzyme reaction, is cleaved to yield only two control cDNA fragments, i.e., a 5'-control cDNA fragment corresponding to a region of the mRNA template that is directly upstream from the start codon and a 3 '-control cDNA fragment corresponding to a region of the mRNA template that is directly downstream from the stop codon. In other embodiments, the control cDNA, which is subjected to a restriction enzyme reaction, is cleaved to yield more than two control cDNA fragments, including a 5 '-control cDNA fragment corresponding to a region of the mRNA template that is directly upstream from the start codon, internal fragments, and a 3 '-control cDNA fragment corresponding to a region of the mRNA template that is directly downstream from the stop codon.

[54] As used herein, the term “amplicon” refers to the amplified product of a nucleic acid amplification reaction, e.g., RT-PCR.

[55] As used herein, the term “purify,” “purified,’ or “purification” means to make substantially pure or clear from unwanted components, material defilement, admixture or imperfection.

[56] As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena may not achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in some biological and chemical phenomena.

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

[58] To determine the sequence percent identity of two nucleotide or amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of a first nucleotide sequence). 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 sequence percent 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).

[59] The determination of percent identity between two sequences may be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin et al., Proc. Natl. Acad. Set. USA, 90:5873- 5877 (1993), which is incorporated into the NBLAST program, and which may be used to identify sequences having the desired identity to nucleotide sequences of the disclosure. To obtain gapped alignments for comparison purposes, Gapped BLAST may be utilized as described in Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., NBLAST) may be used. See the programs provided by National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health.

[60] As used herein, “Hl” refers to an influenza virus subtype 1 hemagglutinin (HA). Type A influenza viruses are divided into Groups 1 and 2. Groups 1 and 2 are further divided into subtypes, which refers to classification of a virus based on the sequences of two proteins on the surface of the virus HA and neuraminidase (NA). Currently, there are 18 recognized HA subtypes (Hl -Hl 8). Hl is thus distinct from the other HA subtypes, including H2-H18.

[61] As used herein, “H3” refers to an influenza virus subtype 3 HA. H3 is thus distinct from the other HA subtypes, including Hl, H2 and H4-H18.

[62] As used herein, “Nl” refers to an influenza virus subtype 1 neuraminidase (NA). Type A influenza viruses are divided into Groups 1 and 2. Groups 1 and 2 are further divided into subtypes, which refers to classification of a virus based on the sequences of two proteins on the surface of the virus HA and neuraminidase (NA). Currently, there are 11 recognized NA subtypes (Nl-Nl 1). Nl is thus distinct from the other NA subtypes, including N2-N11.

[63] As used herein, “N2” refers to an influenza virus subtype 2 neuraminidase (NA). N2 is thus distinct from the other NA subtypes, including Nl and N3-N11.

[64] Each year, based on intensive surveillance efforts, the World Health Organization (WHO) selects influenza strains to be included in the seasonal vaccine preparations. As used herein, the term “standard of care strain” or “SOC strain” refers to an influenza strain that is selected by the WHO to be included in the seasonal vaccine preparations. 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

[65] In one aspect, the present disclosure is directed 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, to thereby form a test cDNA fragment pattern; and (f) comparing the test cDNA fragment pattern with a control cDNA fragment pattern, wherein the at least one mRNA molecule is present in the composition when the test cDNA fragment pattern comprises the control cDNA fragment pattern.

[66] 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 an amount of the at least one test cDNA fragment in the cDNA fragment pattern; and (h) comparing the amount of the at least one cDNA fragment to an amount of the control cDNA fragment, wherein a reduced amount of the at least one cDNA fragment in comparison to the amount of the control cDNA fragment indicates degradation and thereby reduced integrity of the at least one mRNA molecule in the composition.

[67] In this aspect, steps (a)-(f) may be conducted as described herein. The quantitation (g) and comparison (h) steps may also be conducted as described herein.

[68] In another aspect, the present disclosure is directed to a method of quantifying at least one mRNA molecule in a composition, which method 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, to thereby form a test cDNA fragment pattern; (f) determining an amount of at least one test cDNA fragment in the test cDNA fragment pattern; (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 a control cDNA fragment. Foregoing steps (a)-(h) may be conducted as described herein.

[69] In some embodiments, a reduced amount of the at least one test cDNA fragment in comparison to the amount of the control cDNA fragment indicates a reduction in an amount of the at least one mRNA molecule in the composition in comparison to an amount of at least one control mRNA molecule. In some embodiments, an increased amount of the of the at least one cDNA fragment in comparison to the amount of the control cDNA fragment indicates an increase in an amount of the at least one mRNA molecule in the composition in comparison to an amount of at least one control mRNA molecule. [70] In another aspect, the present disclosure is directed 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, to thereby form a test cDNA fragment pattern; (f) determining an amount of at least one test cDNA fragment in the test cDNA fragment pattern; (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 amount of a control cDNA fragment.

[71] In this aspect, steps (a)-(g) may be conducted as described herein. The quantitation step described in step (h) may also be conducted as described herein.

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

[73] As used herein, the phrase “the at least one mRNA molecule is not degraded” refers to the integrity of the mRNA molecule in a composition of the disclosure as described herein. In this aspect, degradation is assessed as described herein and a difference between an amount of the test and an amount of the control cDNA fragments and/or test and control ratios as described herein are 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 “the same” in reference to a comparison between the amounts of the recited test cDNA fragment(s) test and control cDNA fragment(s) 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. rnRNAs molecules

[74] In some embodiments, the at least one mRNA molecule of a composition of the present 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 different five 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.

[75] Accordingly, in some embodiments, a composition of the present disclosure is a monovalent composition (e.g., a monovalent vaccine) or a multivalent composition (e.g., a multivalent vaccine) comprising a plurality of different mRNA molecules. In some embodiments, the composition is a bivalent composition (e.g., a bivalent vaccine) comprising two different species of mRNA molecules. In some embodiments, the composition is a trivalent composition (e.g., a trivalent vaccine) comprising three different species of mRNA molecules. In some embodiments, the composition is a quadrivalent composition (e.g., a quadrivalent vaccine) comprising four different species of mRNA molecules. In some embodiments, the composition is a pentavalent composition (e.g., a pentavalent vaccine) comprising five different species of mRNA molecules. In some embodiments, the composition is a hexavalent composition (e.g., a hexavalent vaccine) comprising six different species of mRNA molecules. In some embodiments, the composition is a heptavalent composition (e.g., a heptavalent vaccine) comprising seven different species of mRNA molecules. In some embodiments, the composition is an octavalent composition (e.g., an octavalent vaccine) comprising eight different species of mRNA molecules. In some embodiments, the composition is a nonavalent composition (e.g., a nonavalent vaccine) comprising nine different species of mRNA molecules. In some embodiments, the composition is a decavalent composition (e.g., a decavalent vaccine) comprising ten different species of mRNA molecules.

[76] In some embodiments, each of the different at least one mRNA molecule in a composition of the 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.

[77] 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.

[78] 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. [79] In some embodiments, each of the different mRNA molecules differ in length from each other 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.

[80] 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 differ in length from each other by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less including all subranges therebetween.

[81] 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 differ in length from each other by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less including all subranges therebetween.

[82] 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 differ in length from each other by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less including all subranges therebetween.

[83] 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 differ in length from each other by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less including all subranges therebetween.

[84] 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 differ in length from each other by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less including all subranges therebetween.

[85] 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 differ in length from each other by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less including all subranges therebetween.

[86] 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 differ in length from each other by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less including all subranges therebetween.

[87] 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 differ in length from each other by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less including all subranges therebetween.

[88] 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 differ in length from each other by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less including all subranges therebetween.

[89] 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 differ in length from each other by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less including all subranges therebetween.

[90] 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 differ in length from each other by 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases or less including all subranges therebetween.

[91] 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 a 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, by inhalation, or intramuscular. In some embodiments, the pharmaceutical composition is formulated for oral or topical administration.

[92] In some embodiments, the compositions comprise naked mRNA molecules. In other embodiments, the compositions comprise complexed or encapsulated mRNA molecules. For example, the compositions of the disclosure may comprise mRNA molecules that are complexed in liposomal form or encapsulated in a nanoparticle. In some embodiments, the mRNA molecules are complexed or encapsulated after determining, e.g., the integrity or quantity of the mRNA molecule as described herein.

[93] 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 herpesvirus, a human papilloma virus, a rotavirus virus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkey pox 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 multiple different species of mRNA molecules.

[94] 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 an influenza virus, a coronavirus, a respiratory syncytial virus (RSV), a parainfluenza virus, a human immunodeficiency virus (HIV), a herpesvirus, a human papilloma virus, a rotavirus virus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkey pox 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 may include a first species of an mRNA molecule from a first strain of an influenza virus and a second species of an mRNA molecule from a second strain of an influenza virus. In some embodiments, the bivalent vaccine may include a first species of an mRNA molecule from a first strain of a coronavirus and a second species of an mRNA molecule from a second strain of a coronavirus.

[95] In some embodiments, the bivalent vaccine is a combination vaccine comprising two different species of mRNA molecules from different viruses, including but not limited to, two different species of mRNA molecules from an influenza virus, a coronavirus, a RSV, a parainfluenza virus, a HIV, a herpesvirus, a human papilloma virus, a rotavirus virus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkey pox 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 may include a first species of an mRNA molecule from an influenza virus and a second species of an mRNA molecule from a coronavirus. In some embodiments, the bivalent vaccine comprises a first species of an mRNA from a RSV and a second species of an mRNA molecule from an influenza virus. In some embodiments, the bivalent vaccine comprises a first species of an mRNA from a RSV and a second species of an mRNA molecule from a coronavirus.

[96] In some embodiments, the vaccine is a trivalent vaccine comprising three different species of mRNA molecules. In some embodiments, the three different species 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 species 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 species of mRNA molecules in the trivalent vaccine is from a different strain of the same virus, including but not limited to, three different species of mRNA molecules from an influenza virus, a coronavirus, a RSV, a parainfluenza virus, a HIV, a herpesvirus, a human papilloma virus, a rotavirus virus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkey pox virus, a parvovirus, an Ebola virus, a dengue virus, a hantavirus, a Zika virus, a west Nile virus, a poliovirus, or a rabies virus.

[97] In some embodiments, the trivalent vaccine is a combination vaccine, wherein one or more of the three different species of mRNA molecules is from a different virus, including but not limited to, an influenza virus, a coronavirus, a RSV, a parainfluenza virus, a HIV, a herpesvirus, a human papilloma virus, a rotavirus virus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkey pox 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 trivalent vaccine may include a first species of an mRNA molecule from an influenza virus, a second species of an mRNA molecule from a coronavirus and a third species of an mRNA molecule from a RSV. In some embodiments, the trivalent vaccine comprises one species of an mRNA molecule from a pneumonia virus, another species of an mRNA molecule from an influenza virus and yet another species of an mRNA molecule from a coronavirus.

[98] In some embodiments, the vaccine is a quadrivalent vaccine comprising four different species of mRNA molecules. In some embodiments, the four different species of mRNA molecules in the quadrivalent 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 quadrivalent 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 quadrivalent vaccine is from a different strain of the same virus, including but not limited to, an influenza virus, a coronavirus, a RSV, a parainfluenza virus, a HIV, a herpesvirus, a human papilloma virus, a rotavirus virus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkey pox virus, a parvovirus, an Ebola virus, a dengue virus, a hantavirus, a Zika virus, a west Nile virus, a poliovirus, or a rabies virus.

[99] In some embodiments, the quadrivalent vaccine is a combination vaccine, wherein one or more of the four different species of mRNA molecules is from a different virus, including but not limited to, an influenza virus, a coronavirus, a RSV, a parainfluenza virus, a HIV, a herpesvirus, a human papilloma virus, a rotavirus virus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkey pox 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 quadrivalent vaccine may include a first species of an mRNA from a RSV virus, a second species of an mRNA molecule from a Group I influenza A strain, a third species of an mRNA molecule from a Group II influenza A strain and a fourth species of an mRNA molecule from an influenza B strain.

[100] In some embodiments, the species of mRNA molecules in the monovalent, bivalent, trivalent and/or quadrivalent vaccine differ in length 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. [101] In some embodiments, the monovalent, bivalent, trivalent or quadrivalent vaccine is an influenza vaccine, a coronavirus vaccine, a RSV vaccine, a parainfluenza virus vaccine, a HIV vaccine, a herpesvirus vaccine, a human papilloma virus vaccine, a rotavirus virus vaccine, a norovirus vaccine, a varicella zoster virus vaccine, a hepatitis virus vaccine, a paramyxovirus vaccine, a monkey pox 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.

[102] In some embodiments, a multivalent vaccine is a combination vaccine comprising different species of mRNA molecules from different viruses, including for example, an influenza virus, a coronavirus, a RSV, a parainfluenza virus vaccine, a HIV vaccine, a herpesvirus vaccine, a human papilloma virus vaccine, a rotavirus virus vaccine, a norovirus vaccine, a varicella zoster virus vaccine, a hepatitis virus vaccine, a paramyxovirus vaccine, a monkey pox 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.

[103] In some embodiments, the at least one mRNA molecule in a composition of the disclosure is obtained from natural sources, such as viruses, cells, tissues, organs or organisms. Viruses that may be used as sources of the at least one mRNA molecule in a composition of the disclosure may include, but are not limited to, an influenza virus, a coronavirus, a RSV, a parainfluenza virus vaccine, a HIV vaccine, a herpesvirus vaccine, a human papilloma virus vaccine, a rotavirus virus vaccine, a norovirus vaccine, a varicella zoster virus vaccine, a hepatitis virus vaccine, a paramyxovirus vaccine, a monkey pox 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.

[104] In some embodiments, the at least one mRNA molecule in a composition of the disclosure encodes an influenza virus protein selected from Hl, H3, HA from a B/ Victori a lineage, and/or HA from a B/Yamagata lineage. In some embodiments, the at least one mRNA molecule in a composition of the disclosure comprises four mRNA molecules, each mRNA molecule encoding a different influenza virus protein (e.g., quadrivalent), such as an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B/Victoria lineage, and an HA from a fourth standard of care influenza virus strain from the B/Yamagata lineage. 1 [105] In some embodiments, the at least one mRNA molecule in a composition of the disclosure encodes an influenza virus protein selected from Nl, N2, NA from a B/ Victori a lineage, and/or NA from a B/Yamagata lineage. In some embodiments, the at least one mRNA molecule in a composition of the disclosure comprises four mRNA molecules, each mRNA molecule encoding a different influenza virus protein (e.g., quadrivalent), such as a Nl from a first standard of care influenza virus strain, a N2 from a second standard of care influenza virus strain, an NA from a third standard of care influenza virus strain from the B/Victoria lineage, and an NA from a fourth standard of care influenza virus strain from the B/Yamagata lineage.

[106] In some embodiments, the source of the at least one mRNA molecule in a composition of the disclosure is an influenza virus, such as a strain of Influenza A, a strain of Influenza B or combinations thereof. For example, in some embodiments, the source of the at least one mRNA molecule in a composition of the 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 the 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.

[107] In some embodiments, the source of the at least one mRNA molecule in a composition of the present 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 the at least one mRNA molecule in a composition of the present disclosure is a strain of Influenza B, such as B/Washington/02/2019 and/or B/PHUKET/3073/2013.

[108] In some embodiments, the source of the at least one mRNA molecule in a composition of the present 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.

[109] Cells that may be used as sources of the mRNA molecules in a composition of the disclosure may be prokaryotic (bacterial cells, including species of Escherichia, Bacillus, Staphylococcus, Streptococcus, Pseudomonas), or eukaryotic (including fungi, plants, protozoans and other parasites, and animals including insects such as Drosophila spp. cells, nematodes including Caenorhabditis elegans cells), mammalian cells including blood cells (reticulocytes and leukocytes), endothelial cells, epithelial cells, neuronal cells (from the central or peripheral nervous systems), 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 germ cells (spermatocytes and oocytes) may also be used as sources of mRNA molecules for use in the instant methods.

[HO] Also suitable for use as mRNA sources are mammalian tissues or organs such as those derived from brain, kidney, liver, pancreas, blood, bone marrow, muscle, nervous, skin, genitourinary, circulatory, lymphoid, gastrointestinal and connective tissue sources.

[111] Any of the above prokaryotic or eukaryotic cells, tissues and organs may be normal, diseased, embryonic or fetal. Diseased cells may, for example, include those involved in infectious diseases (caused by bacteria, fungi or yeast, viruses (including AIDS, HIV, HTLV, herpes, hepatitis and the like) or parasites), in 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 mRNAs for use in the present disclosure will be apparent to one of ordinary skill in the art.

[112] In some embodiments, an mRNA of interest for use as the at least one mRNA molecule in a composition of the disclosure is identified using private and/or public databases, e.g., GenBank.

[113] The polypeptide encoded by the at least one mRNA molecule in a composition of the disclosure may be any polypeptide of interest. In some embodiments, the polypeptide of interest encoded by the at least one mRNA molecule in a composition of the 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 for which no therapeutic indication has been identified, but which nonetheless has utility in areas of research and discovery.

[114] As used herein, a “therapeutic protein” is any polypeptide-based molecule, peptide fragment or variant thereof, which may be used to treat, cure, mitigate, prevent, or diagnose a disease or medical condition. Therapeutic proteins and peptides may be utilized to treat or diagnose conditions or diseases in any therapeutic area such as, blood, cardiovascular, central nervous system, poisoning (including antivenoms), dermatology, endocrinology, genetic, genitourinary, gastrointestinal, musculoskeletal, oncology, and immunology, respiratory, sensory and anti-infective therapeutic fields.

[115] For example, the therapeutic proteins encoded by the at least one mRNA molecule in a composition of the disclosure may be useful for treating or diagnosing diseases including cancer, and include proteins such as carcinoembryonic antigen (CEA), New York esophageal squamous cell carcinoma 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 (gplOO), Melanoma-associated antigen 1 (MAGEA1), Melanoma- associated antigen 1 (MAGEA3) and Mucin 1 (MUC1), human epidermal growth factor receptor 2 (HER2), telomerase, and surviving; Allergy tolerization, e.g., peanut Ara h 2.02, ovalbumin, grass pollen Phi 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 vaccine components for preventing or treating infectious diseases including Influenza-associated antigen, TB-associated Hsp65 and RSV-antigen.

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

[117] An “antibody fragment” comprises a portion of an intact antibody, typically the antigen binding and/or the 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.

[118] In some embodiments, the at least one mRNA molecule in a composition of the 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 a protective immunity in a subject. A “protective immune response” or “protective immunity” refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection) or reduces the symptoms of infection (for instance, an infection by an influenza virus). Vaccines may elicit both prophylactic (preventative) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Inoculations can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous, intraperitoneal, intradermal, intranasal, by inhalation, or intramuscular.

[119] In some embodiments, the polypeptide encoded by the at least one mRNA molecule in a composition of the disclosure is a variant of a naturally occurring protein. Variants of naturally occurring proteins include substitutional variants, conservative amino acid substitutions, insertional variants, and/or deletional variants and/or covalent derivatives. Exemplary and preferred conservative amino acid substitutions include any of: 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.

[120] In some embodiments, the polypeptide encoded by the at least one mRNA molecule in a composition of the disclosure is an antigenic protein for use in a vaccine, such as an antigenic protein from an influenza virus, a coronavirus, a RSV, a parainfluenza virus, a human immunodeficiency virus (HIV), a herpesvirus, a human papilloma virus, a rotavirus virus, a norovirus, a varicella zoster virus, a hepatitis virus, a paramyxovirus, a monkey pox virus, a parvovirus, an Ebola virus, a dengue virus, a hantavirus, a Zika virus, a west Nile virus, a poliovirus, or a rabies virus.

[121] 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, a matrix- 1 (Ml), a matrix-2 (M2), a non-structural protein- 1 (NS1) a non-structural protein-2 (NS2) from influenza A and/or influenza B. In some embodiments, the influenza protein is of human origin. In some embodiments, the influenza protein is of swine or bird origin. [122] In some embodiments, the therapeutic protein is from an influenza A strain, such as one or more of HI, H2, H5, H6, H8, H9, Hl 1, Hl 3 and HI6 (phylogenetic group I) and/or one or more of H3, H4, H7, HIO, 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 protein from influenza A and influenza B. In some embodiments, the therapeutic protein comprises an HA protein from Group I and Group II influenza A and/or an HA protein from the Victoria and Yamagata.

[123] In some embodiments, the composition of the instant disclosure is a vaccine and the at least one mRNA molecule of the vaccine composition encodes an influenza virus protein selected from Hl, H3, HA from a B/Victoria lineage, and/or HA from a B/Yamagata lineage. In some embodiments, the composition of the instant 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., quadrivalent), such as an Hl from a first standard of care influenza virus strain, an H3 from a second standard of care influenza virus strain, an HA from a third standard of care influenza virus strain from the B/Victoria lineage, and an HA from a fourth standard of care influenza virus strain from the B/Yamagata lineage.

[124] In some embodiments, the composition of the instant disclosure is a vaccine and the at least one mRNA molecule of the vaccine composition encodes an influenza virus protein selected from Nl, N2, NA from a B/Victoria lineage, and/or NA from a B/Yamagata lineage. In some embodiments, the composition of the instant 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., quadrivalent), such as a Nl from a first standard of care influenza virus strain, a N2 from a second standard of care influenza virus strain, an NA from a third standard of care influenza virus strain from the B/Victoria lineage, and an NA from a fourth standard of care influenza virus strain from the B/Yamagata lineage.

[125] In some embodiments, the composition of the instant 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 proteins from the following influenza strains: A/Wisconsin/588/2019, A/Tasmania/503/2020, B/Washington/02/2019 and

B/PHUKET/3073/2013. [126] In some embodiments, the composition of the instant disclosure is a quadrivalent vaccine comprising four mRNA molecules, wherein the four mRNA molecules each encode a different HA protein from each of the following influenza strains: A/Wisconsin/588/2019, A/Tasmama/503/2020, B/Washington/02/2019 and B/PHUKET/3073/2013.

[127] In some embodiments, the at least one mRNA molecule in a composition of the disclosure may be structurally or chemically modified to achieve desired functions or properties. For example, the sequence of a naturally occurring mRNA may be optimized to match codon frequencies in certain tissue targets and/or host organisms to ensure proper folding; biasing G/C content to increase mRNA stability; minimizing tandem repeat codons or base runs that can impair expression; customizing transcriptional and translational control regions; inserting or removing protein trafficking sequences; and removing/adding post translation modification sites in an encoded protein (e.g., glycosylation sites). 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.).

[128] In some embodiments, the at least one mRNA molecule in a composition of the disclosure may 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 ribonucleotide, and a 2'-methoxy ribonucleotide. In some embodiments, every uridine in the ribonucleic acid molecule is replaced by a pseudouridine, e.g., a methylpseudouridine, such as 1N- methylpseudouridine. Other modifications include the incorporation of a synthetic UTR sequence from a-globin or P-globin to increase protein expression or one or more phosphorothioate bonds. Other mRNA modifications are known in the art and described, 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 andKim eta/. “Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency.” Mol Cell Toxicol. 2022;18(l): l-8. doi: 10.1007/s 13273-021 -00171 -4 and WO 2017/070620. Methods of Obtaining and Preparing mRNA Molecules

[129] The at least one mRNA molecule may be obtained by any method known in the art. In some embodiments, the at least one mRNA molecule in a composition of the disclosure is obtained by initially isolating total mRNA from a source as described herein, e.g., cells, tissues, organs or other samples, and reverse transcribing the total mRNA into cDNA to prepare a cDNA library by methods that are well-known in the art (See, e.g., Green, M. and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual. 4th Edition, Vol. II, Cold Spring Harbor Laboratory Press, New York.). Briefly, isolated mRNA may, in some embodiments, be subjected to a first-strand cDNA synthesis reaction as described herein. Double-stranded cDNA may then be prepared using any known method, including, for example, commercially available kits such as Just cDNA doublestranded cDNA Synthesis Kit (Agilent Technologies, Inc.) and 2^nd Strand cDNA synthesis kit (ThermoFisher Scientific, Inc.). For example, second strand cDNA may be generated using the first strand cDNA as a template and catalyzing the reaction using known methods, including, for example, E. coli DNA polymerase I in combination with E. coli RNase H and E. coli DNA ligase. E.coli RNase H inserts nicks into the RNA, which is complementary to the first strand cDNA, providing 3' OH-primers for DNA polymerase I. The 5 '-3' exonuclease activity of E. coli DNA polymerase I removes the RNA strand in the direction of synthesis, while its polymerase activity replaces the RNA with deoxyribonucleotides. E. coli DNA ligase links the gaps to complete the double stranded cDNA strand. The double-stranded cDNAs can be inserted into a plasmid or other vector, transformed into host cells, and the cDNA library is screened for the cDNA of interest. A plasmid or other vector containing a cDNA of interest may be used as a template for e.g., in vitro transcription to obtain the at least one mRNA molecule in a composition of the disclosure as described below.

[130] In other embodiments, the at least one mRNA molecule in a composition of the disclosure is synthesized by e.g. chemical synthesis as 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, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005. For example, the at least one mRNA molecule in of a composition of the disclosure may be manufactured in whole or in part using solid phase techniques. Solid-phase chemical synthesis of nucleic acids is an automated method wherein molecules are immobilized on a solid support and synthesized step by step in a reactant solution.

[131] In other embodiments, the at least one mRNA molecule in a composition of the disclosure may be manufactured in whole or in part using liquid-phase chemical synthesis, i.e., the synthesis of nucleic acids of the present disclosure may be manufactured by the sequential addition of monomer building blocks that may be carried out in a liquid phase. In some embodiments, the use of solid-phase or liquid-phase chemical synthesis in combination with enzymatic ligation provides an efficient way to generate long chain nucleic acids that cannot be obtained by chemical synthesis alone.

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

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

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

[135] In some embodiments, immediately downstream of the poly A tail coding sequence on the plasmid DNA template is a recognition site for a restriction endonuclease to linearize the plasmid. Linearization of the plasmid can mitigate transcriptional readthrough.

[136] The linearized DNA template may 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 molecule(s) of the disclosure. In some embodiments, more than one linearized template is used in an IVT system, each of which corresponds to at least one mRNA molecule in a composition of the disclosure. For example, as described in the examples, linearized templates corresponding to each of the mRNA molecule(s) in a quadrivalent influenza vaccine composition of the disclosure may be used in an IVT system to simultaneously prepare all four of the mRNA molecule(s) of the exemplified quadrivalent influenza vaccine composition.

[137] In some embodiments, the 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 may be selected from natural and unnatural (modified) NTPs. In some embodiments, the RNA polymerase may be selected from, for example, a phage RNA polymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, and an SP6 RNA polymerase.

[138] In some embodiments, dithiothreitol (DTT) and/or 1 mM spermidine is 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 may be generated following each nucleotide incorporation into two units of inorganic phosphate. In this embodiment, the magnesium remains in solution and does not precipitate as magnesium pyrophosphate.

[139] In some embodiments, the in vitro transcription reaction is allowed to proceed, for example, under constant mixing at 37°C for 4 hours. In some embodiments, yields range from, e.g., 1-5 mg of RNA per mL of transcription reaction. After the reaction, the mRNA may be purified by any method known in the art, including, for example, a commercially-available silica- based column systems, such as the Qiagen RNeasy® kit (Qiagen, Inc) or mRNA Ambion's MEGACLEAR™ Kit (ThermoFisher Scientific, Inc.) following the manufacturer's instructions. In some embodiments, purification comprises removing the linearized plasmid DNA template, e.g., the DNA template is separated from the RNA transcript. In one embodiment, the DNA template is removed chromatographically using poly A capture, e.g., an oligo(dT), based affinity purification step. In this embodiment, the RNA transcript binds the affinity substrate while the DNA template flows through and is removed. In other embodiments, DNase I is used to enzymatically digest DNA template immediately following in vitro transcription.

[140] In some embodiments, the RNA transcript is enzymatically capped at the 5'-end after in vitro transcription. Capping can be performed either before or after purification of the RNA transcript. Capping may be performed by any method known in the art. For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-O-methyltransferase enzyme may be used to create a canonical 5'-5'-triphosphate linkage between the 5'-terminal nucleotide of an mRNA and a guanine cap nucleotide, wherein the cap guanine contains an N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2'-O-methyl.

[141] The RNA transcript 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

[142] In some embodiments, the 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 prepare single-stranded cDNA. A reverse transcription (RT) reaction refers to the process in which single-stranded RNA is reverse transcribed into complementary DNA (cDNA) using any known method. In some embodiments, the RT reaction includes a reverse transcriptase enzyme, one or more primers, dNTPs (refers to an equal molar mixture of dATP, dTTP, dCTP, and dGTP), and an optional RNase inhibitor. General methods and kits including reaction components for reverse transcription are known in the art and can be employed with the present methods.

[143] Reverse transcriptases useful in the methods of this 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 Immunovirus reverse transcriptase (HIV-RT), EIAV-RT, RAV2-RT, Tth DNA polymerase, SuperScript I, SuperScript II, SuperScript III (ThermoFisher Scientific, Inc.), and mutants, variants and derivatives thereof.

[144] In some embodiments, the primers for the first strand cDNA synthesis reaction generally rely on the principles of oligo(dT) priming. Oligo(dT) refers to a short single-stranded sequence of deoxythymidine (dT). In some embodiments, the primers include a stretch of at least 12 thymidines. In reverse transcription reactions, the primer binds to the poly(A) tail of mRNA molecules and the oligo(dT) initiates reverse transcription at the 3 '-end of the transcript. In some embodiments, the reverse transcribing 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. [145] Multiple types of oligo(dT) primers are known in the art and commercially available. For example, 01igo(dT)2o is a homogenous mixture of 20-mer thymidines, while oligo(dT)i2-is is a mixture of 12-mer to 18-mer thymidines. The stretch of poly(T) can be any length suitable to hybridize to a mRNA poly(A) tail and be extended by reverse transcriptase during a RT reaction. The primer can also be a mixture of oligo(dT) primers that have poly(T) stretches of different lengths.

[146] In some embodiments, the oligo(dT) primers used in a first strand cDNA synthesis reaction is an anchored oligo(dT). Anchored oligo(dT) primers are designed to avoid polyA slippage by ensuring that they anneal at the 3'-UTR/polyA junction. In some embodiments, an anchored oligo(dT) includes a stretch of poly(T) followed by a nucleotide that is not thymidine (expressed as “V” which can be adenine, cytosine, or guanine). In some embodiments, this primer is used as a mixture so that all species of “V” (adenine, cytosine, and guanine) are represented. In some embodiments, the non-thymidine nucleotide is the 3 '-terminal nucleotide. In other embodiments, the “V” nucleotide is followed by one or more additional nucleotides that can be thymidine, adenine, cytosine, or guanine (referred to “N”). In some embodiments, this primer comprises a mixture so that all species of “N” are represented. Oligo(dT) primers can be expressed 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. Anchored Oligo(dT) primers can be expressed as oligo(dT)_nV, where “n” is the number of thymidines in the poly(T) stretch and “V” is adenine, cytosine, and guanine, or oligo(dT)_nVN, where “n” is the number of thymidines in the poly(T) stretch and “V” is adenine, cytosine, and guanine, and “N” is any nucleotide.

[147] In some embodiments, 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, are first incubated with the primers described above under conditions that denature mRNA secondary structure (e.g., about 65° C). The primer/mRNA mixture is then quickly chilled on ice to let the primer anneal to the mRNA. Next, other components of the RT reaction are added to the mixture including dNTPs, RNase inhibitor, reverse transcriptase and RT buffer, which includes, e.g., Tris-HCl, KC1, MgCh, DTT.

[148] In some embodiments, an extension reaction is carried out under conditions that allow the primer to be extended by reverse transcriptase, e.g., 15, 30, 45, 60, or more minutes at a temperature between about 37° C and 55° C. For some thermal insensitive reverse transcriptase enzymes, the reaction can be carried out at higher temperatures. In one embodiment, SuperScript Reverse Transcriptase III (SSRTIII) is used to generate a full-length cDNA. In some embodiments, the reverse transcriptase can be inactivated after annealing and extension with a short incubation at a high temperature (e.g., 5-10 min at >85° C). In some embodiments, RNA template is removed. In some embodiments, template RNA is destroyed by treating the RT reaction with Rnase H.

[149] In some embodiments, one or more of the first-strand cDNA(s) prepared corresponding to each species of mRNA molecules in a composition of the disclosure as described above are used directly as a template for the Polymerase Chain Reaction (PCR) to create a double-stranded cDNA, which is then amplified. PCR is a technique well-known 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, reaction conditions for amplification comprise therm ocy cling, i.e., alternating the temperature of the reaction mixture to facilitate each of the steps of the PCR cycle. In some embodiments, PCR is extended through mul tiple cycles of denaturation, annealing and replication, and optionally augmented with an initial prolonged denaturation step and a final prolonged extension (polymerization) step. In some embodiments, thermocycling occurs within a temperature range of between about 23° C to about 100° C, such as, between 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 thermocycles may vary, depending upon e.g., the quantity 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.

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

[151] In some embodiments, the amplified double-stranded cDNA is purified. Commercially available purification kits, e.g., QIAquick® PCR purification kit (Qiagen, Inc.), may be used.

Oligonucleotide Primers

[152] In some embodiments, oligonucleotide primers useful for PCR are about 15 to about 30 bases in length, are not palindromic (self-complementary) and are not complementary to other primers that may be used in the reaction mixture. In some embodiments, primers, which serve 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, may also be used to facilitate replication of the nucleic acid (e.g., PCR amplification of DNA). Any primer may be synthesized by a practitioner of ordinary skill in the art or may be purchased from any of a number of commercial vendors.

[153] In some embodiments, the primers used for the PCR are different from the reverse transcription primers. In some embodiments, the PCR primers are sequence specific primers. Desirable sequence specific primers for use with the instant methods may be designed by an ordinary artisan using methods known in the art, including, for example, using primer designing software such as the web-based tool Primer3 WEB.

[154] In some embodiments, a sequence specific PCR primer pair may be used to amplify only one of the cDNAs corresponding to a mRNA molecul e in a composition of the disclosure. In other embodiments, a sequence specific primer pair may be used to amplify at least two of the cDNAs corresponding to at least two of the mRNA molecules of the disclosure. In other embodiments, a sequence specific primer pair may be used to amplify all of the mRNA molecules of interest in a composition of the discl osure.

[155] In some embodiments, primers for amplification of the double-stranded cDN A molecules anneal to cDNA regions corresponding to the 3'- and 5'-UTRs of the mRNA molecule(s) in a composition of the present disclosure. In some embodiments, a single pair of such primers are capable of annealing with more than one of the cDNA’s corresponding to the mRNAs in a composition of the present disclosure.

[156] For example, in some embodiments, e.g., where a composition of the disclosure comprises more than one mRNA molecule, such as four different mRNA molecules, each encoding four different HA antigenic proteins from four different influenza A strains, as described herein, primers which anneal to cDNA nucleotides corresponding to each of the 3'- and 5'-UTRs of the mRNA molecules may be used to obtain and amplify double-stranded cDNA from a singlestranded cDNA molecule. 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 may be paired with a 5'-UTR primer selected from SEQ ID NOs: 1, 6, 7, 8 or 9. In some embodiments, the 3'-UTR primer of SEQ ID NO: 2 may be paired with a 5'-UTR primer selected from SEQ ID NOs: 1, 6, 7, 8 or 9. In some embodiments, the 3'-UTR primer of SEQ ID NO: 2 may be paired with a 5'-UTR primer selected from SEQ ID NOs: 6, 7, 8 or 9. In some embodiments, the primers comprise the 3'-UTR primer as set forth in SEQ ID NO: 2 paired with the 5'-UTR primer set forth in SEQ ID NO: 6. Such sequence specific primers may be beneficially used in PCR to avoid, e.g., spurious background products.

Restriction Fragment Analysis

[157] 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 pattern. This test pattern is then compared with a control cDNA fragment pattern, wherein the at least one mRNA molecule is present in a composition of the disclosure when the test cDNA fragment pattern comprises the control cDNA fragment pattern.

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

[159] In some embodiments, based on the nucleic acid sequence of the template from which the at least one mRNA was transcribed, the predicted nucleic acid sequence of the at least one amplified double-stranded cDNA molecule of the present 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 to obtain test and/or control cDNA fragments. In some embodiments, it may be desirable to identify the absence of a restriction site, e.g., the absence of an Alul or EcoRI site.

[160] The double-stranded cDNA molecule may be digested with the appropriate restriction enzyme and the resulting fragments subjected to separation as described herein. One of skill in the art will understand how to analyze a nucleic acid sequence of interest, such as the amplified double-stranded cDNA molecules of the present disclosure, to identify a suitable restriction enzyme or combination 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.

[161] For example, in some embodiments, software, such as the Geneious Prime software (Biomatters, Inc.) or other methods known to those of skill in the art, may be used to identify one or more restriction enzyme sites in a cDNA of the disclosure. In some embodiments, the software or other methods known to those of skill in the art may be used to select a set of desirable restriction enzymes e.g., all known commercially available restriction enzymes or a desired subset thereof. Restriction enzymes, which cut 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 sites, such as at 1 or more restriction sites may be identified. In some embodiments, in order to obtain a specific number of fragments of distinguishable length, cut regions are identified. For example, in some embodiments, only restriction enzymes are identified, which cleave the cDNA within a specified region, e.g., a specified internal region of the cDNA or any other cDNA region. In other embodiments, restriction enzymes are identified, which do not cleave the cDNA of the disclosure within a specified region, but which cleave the cDNA outside of the specified region. In other embodiments, enzymes may be identified that cut anywhere within the cDNA of the disclosure. In some embodiments, the software displays a list of fragments that are produced by a selected restriction enzyme.

[162] For multivalent compositions, each of the different amplified double-stranded cDNA molecules may be analyzed to identify at least one restriction site for each of the different amplified double-stranded cDNA molecules. In some embodiments, based on the nucleic acid sequences of the templates from which the different mRNAs were transcribed, the predicted nucleic acid sequences of the different amplified double-stranded cDNA molecules are analyzed to identify at least one restriction site for each 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 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.

[163] In some embodiments, a restriction enzyme is used that generates clearly distinguishable cDNA fragments for each of the different amplified double-stranded cDNA molecules. For example, the generated DNA fragments for each of the different amplified double-stranded cDNA molecules may differ in length by at least 50 bases, e.g., 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 generated DNA fragments for each of the different amplified double-stranded cDNA molecules differ in length by at least 400 bases, e.g., 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 capable of digesting only one (or two) cDNA molecule(s), corresponding to only one (or two) mRNA molecule(s) in a composition containing a plurality of mRNA molecules, are more desirable to more clearly distinguish and/or quantify the generated cDNA fragments.

[164] In some embodiments, a restriction enzyme resulting in a limited number of fragments is more desirable, such as a restriction enzyme that produces 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. [165] Suitable restriction enzymes include, for example, Aarl, Aatll, AbsI, AccI, Acell, Acelll, Acil, Acll, Acyl, Affel, Aflll, AfUII, Agel, Agsl, Ahalll, Alul, AlwNI, AmaCSI, ApaBI, Apal, ApaLI, Apol, ApyPI, AquII, AquIII, AquIV, Asci, Asi256I, Asp718I, Asul, AsuII, Aval, Avail, Avril, Ball, BamHI, Bbel, BbvCI, Bbvl, BbvII, BccI, Bce83I, BceAI, BceAI, Bcefl, BciVI, Bell, Betl, Bfil, Bgll, Bglll, Bini, BpulOI, BsaAI, BsaBI, Bsbl, BscAI, BscXI, BscXI, BscXI, BscXI, BseMII, BseMII, BsePI, BsePI, BsePI, BseRI, BseSI, BseYI, BseYI, Bsgl, Bsil, BsiYI, BsmAI, BsmFI, BsmI, BsmI, BsmI, BsmI, BsmI, BsmI, Bspl20I, Bspl407I, BspCNI, BspD6I, BspHI, BspKT6I, BspLUl lI, BspMI, BspMII, BsrBI, BsrDI, BsrI, BstAPI, BstEII, BstF5I, BstNI, BstUI, BstXI, BtgZI, BthCI, Btrl, BtsI, BtsIMutl, Cac8I, CauII, Cchll, CdpI, CfrlOI, CfrI, Chai, CjeNIII, Clal, CstMI, CviAII, CviJI, CviQI, CviRI, Ddel, Drall, Dralll, DraRI, DraRI, DrdI, DrdIV, DrdV, Dsal, EamllO5I, Ecil, Eco31I, Eco47III, Eco56I, Eco57I, Eco57MI, Eco78I, EcoHI, EcoICRI, EcoNI, EcoRI, EcoRII, EcoRV, EcoT22I, EsaBC3I, Esp3I, EspI, Fail, Fatl, Faul, Fmul, Fnu4HI, FnuDII, FokI, Fsel, Fsel, FspAI, Gdill, Gsal, Gsul, Hael, Haell, Haelll, HauII, Hgal, HgiAI, HgiCI, Hhal, Hindi, Hindll, HmdIII, Hinfl, HinPlI, Hpal, Hpall, HphI, Hpyl78III, Hpyl88I, Hpy8I, Hpy99I, KasI, Kpnl, Ksp632I, LmnI, Lpnl, Mael, Maell, Maelll, MaqI, MauBI, Mbol, MboII, McaTI, McrI, Mfel, Mid, Mlyl, Mmel, Mnll, Msel, MslI, MspGI, MstI, Mwol, Nael, Narl, Ne , Ndel, NgoAVII, Nhel, NlaCI, Nlalll, NlalV, N113877I, NmeA6CIII, NmeAIII, Notl, Nrul, NspBII, NspI, Ohl, PabI, Pad, PasI, PasI, PflMI, Pfol, PlaDI, Piel, PmaCI, Pmel, PpulOI, PpuMI, PshAI, Psil, PspO3I, PspOMII, PspPRI, PspXI, Pssl, PstI, Pvul, PvuII, Reel, RdeGBII, Rlall, RleAI, RpaB5I, RpaBI, Rpal, Rsal, RsrII, SacI, SacII, Sall, SanDI, SapI, Saul, Seal, Sell, ScrFI, SdeAI, Sdul, Seel, Sell, Setl, SexAI, SfaNI, Sfel, Sfil, Sgfl, SgrAI, SgrDI, SgrAI, SgrDI, Siml, Smal, Smll, SnaBI, Spel, SphI, SplI, Srfl, Sse232I, Sse8387I, Sse8647I, SsoII, SspD5I, SspI, SstE37I, Sthl32I, Sth302II, StsI, Stul, Styl, Swal, Tail, TaqI, TaqII, Taqlll, Tati, Taul, Tfil, Tkol, TkoII, Tsel, Tsoi, Tsp45I, Tsp4CI, TspDTI, TspEI, TspGWI, TspRI, Tthl l ll, Tthl l lll, Unbl, VpaKl 1 Al, VspI, Wvil, Xbal, Xcal, Xcml, Xhol, XhoII, Xmal, Xmalll, XmnI, and/or Zral.

[166] In some embodiments, the at least one restriction enzyme comprises BstUI. In some embodiments, the at least one restriction enzyme comprises Agel, Hinll, Affel and Sac I. In some embodiments, the at least one restriction enzyme comprises Aval, AccI, PflMI and Stul.

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

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

[169] In some embodiments, the composition of the present disclosure is a quadrivalent 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 an mRNA molecule in the quadrivalent vaccine, a second amplified double-stranded DNA molecule corresponding to a second species of an mRNA molecule in the quadrivalent vaccine, a third amplified double-stranded DNA molecule that corresponds to a third species of an mRNA molecule in the quadrivalent vaccine, and a fourth amplified double-stranded DNA molecule corresponding to a fourth species of an mRNA molecule in the quadrivalent 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.

[170] In some instances, it may not be possible or may be challenging to identify a single restriction enzyme that digests each of a plurality of amplified double-stranded cDNA molecules. Alternatively, a restriction enzyme may result in fragments for two or more of a plurality of amplified double-stranded cDNA molecules that are of a similar size and, consequently, challenging to distinguish upon separation. In yet a further alternative, a restriction enzyme may result in small fragments for one or more of the plurality of the at least one amplified double- stranded cDNA molecule of the disclosure that are challenging to quantify. In such embodiments, two or more restriction enzymes may 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 may be obtained for each species of amplified double-stranded cDNA molecules of a plurality, which are distinguishable in size and readily quantifiable.

[171] For example, in some embodiments, the composition of the present disclosure 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 species of mRNA molecule in the quadrivalent vaccine, a second amplified double-stranded cDNA molecule corresponding to a second species of mRNA molecule in the quadrivalent vaccine, a third amplified double-stranded cDNA molecule corresponding to a third species of mRNA molecule in the quadrivalent vaccine, and a fourth amplified double-stranded cDNA molecule corresponding to a fourth species of mRNA molecule in the quadrivalent vaccine, wherein the at least one restriction enzyme comprises a first, second, third, and 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 Agel, Hindi, Affel and Sacl.

[172] In other embodiments, the composition of the disclosure may be a quadrivalent vaccine and the plurality of amplified double-stranded DNA molecules comprises a first amplified doublestranded cDNA molecule corresponding to a first species of mRNA molecule in the quadrivalent vaccine, a second amplified double-stranded cDNA molecule corresponding to a second species of mRNA molecule in the quadrivalent vaccine, a third amplified double-stranded cDNA molecule corresponding to a third species of mRNA molecule in the quadrivalent vaccine, and a fourth amplified double-stranded cDNA molecule corresponding to a fourth species of mRNA molecule in the quadrivalent 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. [173] In other embodiments, the composition of the disclosure may be a quadrivalent vaccine and the plurality of amplified double-stranded DNA molecules comprises a first amplified doublestranded cDNA molecule corresponding to a first species of mRNA molecule in the quadrivalent vaccine, a second amplified double-stranded cDNA molecule corresponding to a second species of mRNA molecule in the quadrivalent vaccine, a third amplified double-stranded cDNA molecule corresponding to a third species of mRNA molecule in the quadrivalent vaccine, and a fourth amplified double-stranded cDNA molecule corresponding to a fourth species of mRNA molecule in the quadrivalent 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.

[174] In other embodiments, the composition of the disclosure may be a quadrivalent vaccine and the plurality of amplified double-stranded DNA molecules comprises a first amplified doublestranded cDNA molecule corresponding to a first species of mRNA molecule in the quadrivalent vaccine, a second amplified double-stranded cDNA molecule corresponding to a second species of mRNA molecule in the quadrivalent vaccine, a third amplified double-stranded cDNA molecule corresponding to a third species of mRNA molecule in the quadrivalent vaccine, and a fourth amplified double-stranded cDNA molecule corresponding to a fourth species of mRNA molecule in the quadrivalent 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 doublestranded cDNA molecule.

Separation

[175] In some embodiments, the test cDNA fragments resulting from the restriction enzyme digests described above are separated. Separation may be accomplished by various methods known to those of skill in the art. For example, methods of liquid chromatography, such as HPLC or UPLC, may be used to separate the test cDNA fragments of the disclosure. HPLC and UPLC rely on pumps to pass a pressurized liquid solvent containing a sample mixture through a column filled with a solid absorbent material. Each component in the sample interacts differently with the adsorbent material, causing different flow rates for the different components, leading to the separation of the components as they flow out of the column.

[176] In some embodiments, electrophoresis may be used to separate the test cDNA fragments of the disclosure. 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 electrophoretic apparatus contains a gel casting tray to prepare a gel, casting combs to prepare wells, a buffer tank, positive and negative electrodes, and a voltage supply unit. Molecules, e.g., nucleic acids, which are negatively charged, move from the cathode to the anode. If high resolution or separation of molecules is desired, a higher concentration gel with a lesser pore size can be prepared. Nucleic acids separated on a gel matrix may be observed with an ultraviolet transilluminator after staining with an intercalating dye, such as ethidium bromide, SYBR® Green, or acridine orange. The separated molecules appear as bands on the gel matrix. In some embodiments, the electrophoresis separation method of the disclosure comprises agarose gel electrophoresis.

[177] In some embodiments, capillary electrophoresis (CE) is used to separate the test cDNA fragments of the disclosure. CE is a modification of gel electrophoresis. In CE, similarly to gel electrophoresis, separation of molecules, e.g., the cDNA test fragments of the disclosure, is based on charge, size and shape of the molecules. However, CE is performed in a capillary tube with either a gel substance, such as polyacrylamide, or a liquid polymer, such as hydroxymethyl cellulose. In some embodiments, capillary tubes may be made of fused silica with an internal diameter ranging from 50-100 pm and a length ranging from 25-100 cm. Samples are injected into the capillary tube containing the polymer material and are separated more rapidly than conventional gel electrophoresis. CE can provide greater resolution than gel electrophoresis and the separation may be more accurate. In some embodiments, the molecules separated using CE are detected via spectrophotometric automated detectors. In other embodiments, the 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 may be used to separate the test and/or control cDNA fragments of the disclosure, e.g., Agilent 5200, 5300, and 5400 Fragment Analyzer systems (Agilent, Inc.). In some embodiments, the cDNA fragments may be visualized as an electropherogram using e.g., Agilent 5200, 5300, and/or 5400 Fragment Analyzer system software. In other embodiments, the cDNA fragments may be visualized as a digital or “virtual” electrophoresis agarose gel using, e.g., 5200, 5300, and 5400 Fragment Analyzer systems (Agilent, Inc.).

[178] In some embodiments, the separated test cDNA fragments comprise a 5 '-test cDNA fragment and a 3 '-test cDNA fragment. In some embodiments, the separated cDNA test fragments further comprise one or more fragments that are located internally to the 5 '-test cDNA fragment and the 3 '-test cDNA fragment in the intact double-stranded cDNA molecule prior to restriction enzyme digestion and separation.

[179] In some embodiments, the test cDNA fragments and the control cDNA fragment in the control cDNA fragment pattern are labeled before or after separation. 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

[180] In one aspect, this disclosure provides methods of assessing the integrity or degradation of the at least one test mRNA molecule disclosed herein. In some embodiments, at least one of the test cDNA fragments in the test cDNA fragment pattern is quantified as described herein, e.g., to assess the extent of degradation, if any, of the at least one mRNA molecule disclosed herein. The amount of the quantified test cDNA fragment in the test cDNA fragment pattern is then compared to an amount of a corresponding control cDNA fragment in a control cDNA fragment pattern. A reduced amount of the at least one test cDNA fragment in comparison to the amount of the corresponding control cDNA fragment indicates degradation and thereby reduced 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, quantitation is determined as described herein by determining the intensity of the labeled control cDNA fragment and the intensity of the labeled test cDNA fragment. A decrease in intensity of the labeled test cDNA fragment in comparison to the intensity of the labeled control cDNA fragment indicates degradation of the corresponding at least one mRNA molecule in a 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), indicates degradation. In some embodiments, the corresponding mRNA molecule is discarded if a decrease in intensity of at least 10% between the test cDNA and control cDNA fragment is detected. In some embodiments, the control cDNA fragment corresponds to an mRNA molecule obtained at time period 0 (TPo) and the test cDNA fragment corresponds to an mRNA molecule obtained at later time period X (TPx). In some embodiments, an mRNA molecule is prepared at time period 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 was prepared. This embodiment may be used to assess, e.g., mRNA storage conditions, to determine, for example, if degradation of the mRNA molecule is occurring over time.

[181] In some embodiments, the quantified test cDNA fragment is a quantified 3 '-test cDNA fragment and the quantified control cDNA fragment is a 3'-quantified control cDNA fragment. In some embodiments, the quantified test cDNA fragment is a quantified 5 '-test cDNA fragment and the quantified control cDNA fragment is a 5'-quantified control cDNA fragment. In some embodiments, the quantified test cDNA fragment is a fragment internal to the 3 '-test cDNA fragment and the 5 '-test cDNA fragment and the quantified control cDNA fragment is a corresponding fragment internal to the 3 '-control cDNA fragment and the 5 '-control cDNA fragment.

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

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

[184] 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 fragment indicates degradation of the corresponding mRNA molecule of a composition of the disclosure. In some embodiments, the corresponding mRNA molecule is discarded if a decrease of at least 10% between the test cDNA and control cDNA fragment is detected. In certain embodiments, the test cDNA and control cDNA fragments are labeled as described herein.

[185] In other embodiments, a ratio of an amount of a quantified 3 '-test cDNA fragment to an amount of a quantified 5'-test cDNA fragment is determined (“test ratio”), wherein each of the test cDNA fragments are included in a cDNA test fragment pattern. In this embodiment, the test ratio is compared to a ratio of an amount of a quantified 3 '-control cDNA fragment to an amount of a quantified 5 '-control cDNA fragment (“control ratio”). In this embodiment, a change in the 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) between the ratio and control ratio indicates degradation of the at least one mRNA molecule in a composition of the present disclosure.

[186] An example of the foregoing embodiment is illustrated in FIG. 1. As shown in FIG. 1, two lanes containing cDNAs labeled with ethidium bromide and illuminated on an agarose gel, are depicted. The lane designated TPo depicts a control fragment pattern containing two fragments, a 5'-control cDNA fragment and a 3 '-control cDNA fragment, which were obtained from a restriction digest of a double-stranded cDNA corresponding to a template control mRNA molecule prepared using in vitro transcription (IVT) at time period 0 (TPo). The lane designated TPx depicts a cDNA fragment pattern containing two fragments, a 5'-test cDNA fragment and a 3'-test cDNA fragment, obtained from a restriction digest of a double-stranded cDNA corresponding to the same template mRNA molecule used to prepare the control cDNA fragments, but this template mRNA molecule had been in storage for a period of time (TPx).

[187] The relative intensity of each of the illuminated fragments was determined. The ratio of the relative fluorescent intensity of the 5'-control fragment to the 3'-control fragment (RFTPO) was 1.5. The ratio of the relative fluorescent intensity of the 5'-test cDNA fragment to the 3'-test cDNA fragment was 2.5 (RFTPX). Accordingly, the difference between RFTPO and RFTPX (ARF) is 1 and the percent change ( F/RFTPO) is 66.7%. Since this change between the test and control ratios is 10% or greater, degradation of the mRNA molecule, at least at the 3 '-end, is indicated.

[188] 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.

Quantitation

[189] The test cDNA and control cDNA fragments of the disclosure may be quantified by any method known in the art. The quantitation may be relative and/or absolute. For example, in some embodiments, relative quantitation of cDNA fragments may be determined by labeling cDNA fragments on an agarose gel, for example, using an intercalating dye (e.g., staining the gel) or a fluorescent dye, visualizing the cDNA fragments with a transilluminator and assessing the relative fluorescent intensity of the fragments using e.g., ImageJ software (National Institutes of Health (NIH at imagej.nih.gov/ij/download.html). In other embodiments, the relative fluorescent intensity of the fragments may be assessed by comparing the intensity of the test cDNA and/or control cDNA fragments to a DNA ladder comprising fragments of known quantity, e.g., Invitrogen DNA ladders (ThermoFisher Scientific Inc.). Relative quantitation of the test cDNA and control cDNA fragments of the disclosure may also be determined during fragment separation using known techniques. For example, in some embodiments, the amount of test cDNA and control cDNA fragments may be quantified using capillary electrophoresis, e.g. using the commercially available 5300 Analyzer System (Agilent Technologies, Inc.)

[190] In some embodiments, absolute quantitation of the test cDNA and control cDNA fragments of the disclosure may be determined during fragment separation. For example, in some embodiments, the amount of test cDNA and control cDNA fragments may be quantified using liquid chromatography, such as high-performance liquid chromatography (HPLC). In an alternative embodiment, fragments may be quantified by purifying a fragment of interest from, e.g., an agarose gel, for example using a commercially available kit, such as QIAquick® Gel Extraction Kit (Qiagen, Inc) and determining the absolute quantitation of the purified cDNA fragments using e.g., a spectrophotometer, such as the NanoDrop™ spectrophotometer (ThermoFisher Scientific, Inc).

Compositions

[191] In some embodiments, the present disclosure is directed to a primer pair comprising a 5'- UTR primer and 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 3'-UTR primer that may be suitable for the methods described in the present disclosure can be used.

[192] In some embodiments, the present disclosure is directed 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.

[193] In some embodiments, the primers are detectably labeled. Any detectable label used in conjunction with nucleic acid technology can be used, including for example, a label that generates a signal that results from a chemical reaction, such as a change in optical absorbance, a change in fluorescence, the generation of chemiluminescence or electrochemiluminescence, a change in reflectivity, refractive index or light scattering, the accumulation or release of detectable labels from the surface, the oxidation or reduction or redox species, an electrical current or potential, changes in magnetic fields, etc. In some embodiments, the detectable label comprises a DNA intercalating label, including, for example, ethidium bromide, SYBR® Green, or acridine orange. In some embodiments, the labels are fluorescent dyes, such, for example, 6-FAM™ (blue), VIC (green), NED™ (Yellow/Black), PET (Red), and LIZ (orange).

[194] The labelled primers are not naturally occurring molecules; that is the combination of the nucleic acid primer coupled to the label does not exist in nature.

[195] The primers may 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 vendors.

EXAMPLES

Example 1. Selection of mRNAs

[196] A quadrivalent (QIV) mRNA vaccine containing four different species of mRNA molecules, each of which encodes a hemagglutinin (HA) antigen from a different influenza strain, as summarized in Table 1, was generated and characterized.

Table 1. QIV mRNA vaccine targets

[197] FIG. 2A depicts a sequence alignment of HA genes for each of the four influenza strains described in Table 1. As shown in FIG. 2B, the HA genes share percent identities ranging from 47.2% to as high as 76.4%. As noted in the instant disclosure, the present methods, as exemplified herein below, may be used to readily distinguish between cDNAs representing mRNA molecules having high sequence identities, e.g., greater than 75%. The present methods exemplified herein may also be used to determine the presence, integrity and quantity of different mRNA molecules in a mRNA therapeutic composition, such as the QIV mRNA vaccine used in these examples. Example 2. In Vitro Transcription

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

[199] Concentrations of each of the four IVT mRNAs were determined using a NanoDrop™ Spectrophotometer (ThermoFisher Scientific, Inc.). As shown below in Table 2, the concentrations ranged from 1222 ng/pl to 2587 ng/pl.

Table 2. Quantitation of IVT mRNAs

[200] The quality of the IVT mRNAs was assessed by capillary electrophoresis (CE) using an Agilent 5300 Fragment Analyzer (Agilent Technologies, Inc.) with the 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 an RNA separation gel (DNF-265-0240) mixed with an intercalating dye (DNF-600-U030). The samples were heat-denatured, and added to wells in a sample plate containing diluent marker (DNF-369-0004). An RNA ladder (DNF- 382-U020) was used as a control. The plate was loaded into the fragment analyzer, the fragments were then separated, and visualized on a digital gel image as shown in FIG. 3. [201] FIG. 3 shows that the IVT mRNAs ranged from about 1950 to 2000 nucleotides as expected. Moreover, additional smaller fragments, an indicator of problematic transcription, or larger fragments, an indicator of DNA template contamination, were not detected. Accordingly, the IVT mRNAs were considered to be of high quality.

Example 2A. 1^st Strand cDNA synthesis

[202] Reverse transcription (RT) was used to synthesize 1^st strand complementary DNA (cDNA) from each of the four IVT mRNA samples. RT was performed using Invitrogen SuperScript™ III First-Strand Synthesis reagents according to the manufacturer’s protocol (ThermoFisher Scientific, Inc). Briefly, IVT mRNA (1000 ng) from each sample was used as a template for different oligo(dT) primers (100 pM). Messenger RNA primer annealing was performed by incubating the mRNAZoligo(dT) mixtures at 65 °C for five minutes followed by rapid cooling on ice. cDNA master mix, containing RNase inhibitor, SuperS cript™ 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 of the mRNA templates was removed by adding RNase H to the terminated reaction, followed by incubation at 37 °C for 20 minutes.

Example 2B. PCR evaluation of 1st strand cDNAs i. PCR

[203] The first strand cDNAs obtained as described above were used as templates for Polymerase Chain Reactions (PCRs). 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, which contained the plasmid encoding Sample I.D. No. 1 as the DNA template with primers SEQ ID NOs: 1 and 2.

[204] The SEQ ID NOs: 1 and 2 primers were designed by evaluating each of the 4 individual cDNA sequences corresponding to mRNA molecule Sample ID Nos. 1-4 using the web-based tool Primer3 WEB, which allows for the selection of multiple input parameters such as primer length, melting temperature (Tm) and GC content. The resulting PCR primers were evaluated for specificity by performing cDNA alignments with the other 3 cDNA sequences in the QIV sample. [205] PCRs were performed using the Phusion® HotStart Flex 2X master mix according to the manufacturer’s protocol (New England BioLabs®, Inc.). After 30 cycles of the PCR, the reaction products were separated by electrophoresis on a 1.2% agarose gel and photographed.

[206] FIG. 4 shows four of the PCR reaction products, which were all produced from the first strand cDNA template representing Sample I.D. No. 1, and the four different primer pairs noted above. As shown in FIG. 4, all of the PCR reactions produced a full-length amplicon. As described further below, the PCR primers were then optimized to mitigate background products.

Table 3. Primers for 1^st strand cDNA evaluation

ii.. PCR Optimization

[207] A series of truncated 5'-UTR PCR primers were designed to improve the specificity of the PCR amplification reaction. The primers truncated from SEQ ID NO: 1 are shown below in Table 4. 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 of SEQ ID NO: 2 (3'-UTR- 12) depicted in Table 3. For direct comparison, 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. The 1^st strand cDNA, which was synthesized from the IVT mRNA of Sample I.D. No. 1, was used as the template in all five of the PCR reactions. DNA polymerase Hot Start Taq 2X Master mix (Intact® Genomics, Inc.) was included in each of the PCR reaction mixtures. [208] After 30 cycles of PCR, the resulting products were separated by electrophoresis on a 1.2% agarose gel and photographed. As seen in FIG. 5, the desired full-length amplicon was observed in all products. However, less background product was observed from the PCR reaction produced with truncated 5'-UTR primers (lanes 2-5) in comparison to the PCR reaction produced with 5'- UTR untruncated primer (lane 1).

Table 4. 5' PCR primers for PCR optimization

Hi. PCR evaluation of individual first strand cDNAs and a mix of the first strand cDNAs

[209] 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 of the first strand cDNA templates (“QIV cDNA template mix”) was also prepared. DNA polymerase Hot Start Taq 2X Master mix (Intact® Genomics, Inc.) was included in each of the 5 PCR reaction mixtures.

[210] After 30 cycles of PCR, the resulting products were separated by electrophoresis on a 1.2% agarose gel and photographed. As seen in FIG. 6, each of the PCR reaction products obtained using the individual 1^st strand synthesis cDNAs as templates (lanes 1-4) or the QIV cDNA template mix (lane 5) resulted in the desired amplicons with little apparent non-specific amplification.

Example 3. Restriction Fragment Length Polymorphisms (RFLP) i. Restriction Enzymes (REs) recognizing one cDNA species in a mixture of 4 cDNA species

[211] PCR reaction products obtained using the individual first strand cDNA templates representing IVT mRNAs Sample I.D. Nos. 1-4 as described above in Example 2Bii were sequenced to identify restriction enzyme (RE) sites for Restriction Fragment Length Polymorphism (RFLP) analyses. RE sites were identified for four REs, which each recognized only one of the four template cDNAs, i.e., Agel, Hindi, Afel, and SacI, respectively.

[212] Subsequently, PCR reaction products, obtained using the QIV cDNA template mix, were digested with Agel, Hindi, Afel, or SacI according to the manufacturer’s protocol (New England BioLabs®, Inc.). Equivalent amounts of the PCR reaction products were used for each RE digestion (1500 ng/50 pl final volume). Following RE digestion, the PCR reaction products were purified using QIAGEN® PCR purification reagents according to the manufacturer’s protocol and then eluted with 100 pl of water. The restriction enzyme digestion products (60 ng) were separated by electrophoresis on a 1.2% agarose gel and photographed. For comparison, an undigested PCR reaction product prepared from a mixture of the four cDNAs was also loaded on the gel.

[213] As shown in FIG. 7, and Table 5, below, Agel cut cDNA representing Sample I.D. No. 1 only, resulting in two fragments (1137 bp and 804 bp). Similarly, Hindi, Afel and SacI, cut cDNA representing only Sample I.D. Nos. 2, 3 and 4, respectively, each resulting in two fragments.

Table 5. Restriction Enzymes targeting one of four PCR Reaction Products

[214] As an alternative to separating the cDNA fragments by agarose gel electrophoresis, Capillary Electrophoresis (CE) was used to both separate and quantify the cDNA fragments produced by RE digestion. Initially, serial 2-fold dilutions of the PCR reaction products digested with Agel, Hindi, Afel, and SacI were prepared in Tris-EDTA (TE) buffer (6, 3, 1.5 and 0.75 ng/pl) and then separated by CE using an Agilent 5300 Fragment Analyzer.

[215] FIG. 8 depicts a digital gel image of the digested PCR products obtained from the QIV cDNA template mix and separated by CE. Similarly to agarose gel electrophoresis, the digital gel image of the digested PCR reaction product depicts two fragments cleaved with Agel, Hindi, Afel and SacI, respectively. [216] FIG. 9A depicts an electropherogram obtained with the Agilent 5300 Fragment Analyzer. Two peaks corresponding to the 1165 bp and 824 bp fragments obtained from the Afel digestion, which only recognizes the cDNA in the PCR reaction product corresponding to mRNA Sample I.D. No. 3, was observed as well as a peak corresponding to the uncut cDNA. FIG. 9B depicts a photograph of an agarose gel showing the same digestion products for comparison.

[217] The Agilent Fragment Analyzer also reported that 16.3% of the total sample was attributed to the 824 bp 5'-test cDNA fragment, 20.9% of the total sample was attributed to the 1165 bp 3'- test cDNA fragment and 58.2 % of the total sample was attributed to the remaining three uncut cDNAs. As described herein above in the detailed description, relative concentrations of 5 '-test and 3 '-test cDNA fragments may be used to assess the integrity of an mRNA sample by comparing the relative concentrations of each fragment to e.g., the relative concentration of a control fragment. For example, the relative amount of the 3'-test cDNA fragment may 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 is 4.6% (20.9%-16.3%). Since this difference does not reach at least 10%, degradation of mRNA Sample No. 3 is not indicated. ii. REs recognizing two cDNA species in a mixture of cDNA species

[218] Additional RE sites recognized by Aval, AccI, Stul and PfIMI were identified in the cDNAs corresponding to mRNA Sample I.D. Nos. 1-4. Aval recognized RE sites in cDNAs corresponding to Sample I.D. Nos. 1 and 2 only. Aval cut the cDNA corresponding to Sample I.D. No. 2 at two sites located in the coding portion of the sequence, i.e., at position 198 from the 5’ end and at position 783 from the 3’ end. Therefore, after digestion with Aval, three fragments were produced (starting at the 5’ end) having lengths of 198 bp, 960 bp and 783 bp, respectively. Accordingly, the 960 bp fragment was internal to the 5 ’-test cDNA fragment of 198 bp and the 3’- test cDNA fragment of 783 bp. Aval cut the cDNA corresponding to Sample I.D. No. 1 at one site resulting in two fragments, a 5 ’test cDNA fragment and a 3 ’-test cDNA fragment. AccI and Stul recognized one RE site in each of the cDNAs corresponding to Sample I.D. Nos. 3 and 4 only, while PfIMI recognized one RE site in each of the cDNAs corresponding to Sample I.D. Nos. 2 and 4 only. [219] Four PCR reactions products, obtained using the QIV cDNA template mix as described above in Example 2Bii, were each digested with Aval, AccI, Stul or PfIMI. RE reactions were performed as described above in Example 3ii. Restriction enzyme digestion products (60 ng) were separated by electrophoresis on a 1.2% agarose gel and photographed. For comparison, an undigested mixture of the four cDNAs was also loaded on the gel.

[220] As is evident from FIG. 10, digestion with Aval, AccI, Stul or PfIMI each resulted in different fragment patterns. Aval, produced five fragments, while AccI, Stul and PfIMI each produced a pattern having four fragments of different sizes as shown in the Table below.

Table 6. Restriction Enzymes targeting two of four PCR Reaction Products

[221] As described above in Example 3i for restriction enzymes Agel, Hindi, Afel and SacI, CE was used to separate and quantify the DNA fragments produced by Aval, AccI and Stul digestion. FIG. 11 depicts a digital gel image of the digested diluted PCR products obtained from the QIV cDNA template mix. As is evident from FIG. 11, the digital gel image resulted in the same fragment pattern as shown in the agarose gel of FIG. 10.

[222] FIG. 12A depicts an electropherogram of a PCR reaction product digested with AccI as described for FIG. 11, separated by CE and showing five large peaks from left to right corresponding to a first 5'-test cDNA fragment (543 bp, corresponding to Sample I.D. No. 4), a first 3'-test fragment (654 bp, corresponding to Sample I.D. No. 3), a second 5'-test fragment (1335 bp, corresponding to Sample I.D. No. 3), a second 3'-test fragment (1452 bp, corresponding to Sample I.D. No. 4), and uncut cDNA (corresponding to Sample I.D. Nos. 1-4).

[223] The Agilent Fragment Analyzer also reported that 17.1% of the total sample was attributed to the 1335 bp 5'-test cDNA fragment (Sample I.D. No. 3), 10.6% of the total sample was attributed to the 654 bp 3'-test cDNA fragment (Sample I.D. No. 3), 7.9% of the total sample was attributed to the 543 bp 5'-test cDNA fragment (Sample I.D. No. 4), 17.4% of the total sample was attributed to the 1452 bp 3 '-test cDNA fragment (Sample I.D. No. 4) and 44.0 percent of the total sample was attributed to the remaining 2 uncut cDNAs. Accordingly, the fragment pattern produced by Agel, Hindi, Afel and SacI may be used to confirm the presence of each of the mRNAs in the QIV influenza vaccine.

[224] Further, the relative amounts of the 5'-test cDNA fragment and the 3 '-test cDNA fragment may 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 for Sample I.D. No. 3 may be used as a control. In this example, the difference between the relative amounts of the 5 '-test cDNA fragment (as a control) and the 3'-test cDNA fragment for Sample I.D. No. 3 is 6.5% (17.1%-10.6%). Since this difference does not reach at least 10%, degradation of mRNA Sample No. 3 is not indicated. Similarly, the relative amount of the 3 '-test fragment for Sample I.D. No. 4 may be used as a control. Since the difference between the relative amounts of the 3 '-test cDNA fragment (as a control) and the 5'-test fragment is 9.5% (17.4%-7.9%), degradation for the mRNA of Sample I.D. No. 4 is not indicated.

Hi. Restriction Enzyme (RE) recognizing 4 cDNAs in a PCR reaction product

[225] PCR reaction products obtained using four individual first-strand synthesis cDNA templates representing IVT mRNAs from four additional samples (Samples A-D) were prepared as described for Sample I.D. Nos. 1-4 above. The PCR products were sequenced to identify restriction enzyme (RE) sites for Restriction Fragment Length Polymorphism (RFLP) analyses. RE sites were identified in all four cDNAs that are recognized by BstUI.

[226] The lengths of the DNA fragments which would be produced from the PCR reaction products if the products were digested with BstUI were predicted using Geneious Prime software (Biomatters, Inc.). Geneious Prime software uses DNA sequence files to enable the generation of virtual gels for restriction enzymes. The predicted fragments for each of the PCR products after theoretical digestion with BstUI were visualized on a virtual agarose gel. FIG. 13 depicts the predicted fragment patterns for each of the theoretically digested PCR products. As shown in Table 6, below, the predicted fragment lengths of the theoretically digested PCR product, representing Sample A, was predicted to produce 3 fragments with lengths of 318 bp, 670 bp and 713 bp. The theoretically BstUI digested PCR product, representing Sample B, was predicted to produce 3 fragments with lengths of 1262 bp, 153 bp and 286 bp. The theoretically BstUI digested PCR product representing Sample C and Sample D also were predicted to produce three fragments with lengths of 830 bp, 704 bp, 215 bp (Sample C) and 380, 702 and 673 bp (Sample D).

Table 7. Fragment lengths of PCR Products representing IVT mRNA (Samples A- D) cut with BstUI.

*5’ fragment; ** 3’ fragment

[227] FIG. 14 depicts the fragment pattern for all of the theoretically BstUI digested PCR products as described above, loaded onto a single lane. As is evident from Table 6, above, and FIG. 14, some of the predicted fragment lengths are nearly identical, i.e., the theoretically digested PCR product representing Sample A has a predicted fragment length of 670 bp and the theoretically digested PCR product representing Sample D has a similarly predicted sized fragment of 673 bp in length. In addition, the theoretically digested PCR products representing Sample D, Sample C and Sample A each include similarly predicted sized fragments of 702, 704 and 713 bp, respectively. Further, two of the theoretically BstUI digested PCR reaction products representing Sample B and Sample A are predicted to produce smaller fragments, i.e., 286 and 318 bp, respectively.

[228] As discussed in the present disclosure, similarly sized fragments may be more challenging to distinguish, making pattern determination for a given mRNA difficult. Further, as also noted herein, shorter fragments, such as those less than 400 bp, may be more difficult to quantify. Accordingly, although a single restriction enzyme was identified in this example, which theoretically cut each of the PCR products, representing four different mRNAs, restriction enzymes capable of digesting, e.g., only one or two cDNAs corresponding to only one or two mRNA molecules in a composition containing a plurality of mRNA molecules, as described in the detailed description, may be more desirable in some embodiments to more clearly distinguish and/or quantify the generated cDNA fragments.

[229] While the foregoing disclosure has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be clear to one of ordinary skill in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the disclosure and may be practiced within the scope of the appended claims. For example, all constructs, methods, and/or component features, steps, elements, or other aspects thereof can be used in various combinations.

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

Claims

CLAIMS We claim:

1. A method of determining the presence of at least one messenger RNA (mRNA) molecule in a composition, which method 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, to thereby form a test cDNA fragment pattern; and

(f) comparing the test cDNA fragment pattern with a control cDNA fragment pattern, wherein the at least one mRNA molecule is present in the composition when the test cDNA fragment pattern comprises the control cDNA fragment pattern.

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) quantifying an amount of at least one test cDNA fragment in the test cDNA fragment pattern; and

(h) comparing the amount of the at least one test cDNA fragment to an amount of a control cDNA fragment in the control cDNA fragment pattern, wherein a reduced amount of the at least one test cDNA fragment in comparison to the amount of the control cDNA fragment indicates degradation and thereby reduced integrity of the at least one mRNA molecule in the composition.

3. A method of quantifying at least one messenger RNA (mRNA) molecule in a composition, which method comprises:

(b) generating at least one double-stranded cDNA molecule from the template; (c) amplifying the at least one double-stranded cDNA molecule;

(e) separating the test cDNA fragments, to thereby form a test cDNA fragment pattern;

(f) determining an amount of at least one test cDNA fragment in the test cDNA fragment pattern;

(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.

4. A process of manufacturing a composition comprising at least one messenger RNA (mRNA) molecule, wherein the process comprises:

(b) generating at least one double-stranded cDNA molecule from the template;

(c) amplifying the at least one double-stranded cDNA molecule;

(g) comparing the amount of the at least one test cDNA fragment to an amount of a control cDNA fragment, and

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

6. The method of any one of the preceding claims, wherein the at least one mRNA molecule is a product of in vitro transcription of a non-amplified DNA template.

7. The method of any one of the preceding claims, wherein the test cDNA fragments are separated by liquid chromatography or electrophoresis.

8. The method of claim 7, wherein the liquid chromatography comprises High Performance Liquid Chromatography (HPLC) or Ultra 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 3'-test cDNA fragment comprise a detectable label.

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

16. The method of claim 15, wherein the detectable label is a DNA intercalating label.

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

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

19. The method of claim 18, wherein a reduction of the intensity of the labeled 3 '-test cDNA fragment in comparison to the intensity of the labeled 5 '-test cDNA fragment or a reduction of the intensity of the labeled 5'-test cDNA fragment in comparison 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 a ratio of the intensity of the labeled 5'-test cDNA fragment to the intensity of the labeled 3'-test cDNA fragment in comparison 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 fragments of the control ratio correspond to an mRNA molecule obtained at time period 0 (TPo) and the cDNA fragments of the test ratio correspond to an mRNA molecule obtained at later time period X (TPx).

22. The method of claim 20 or 21, wherein the change in the ratio of the intensity of the labeled 5'-test cDNA fragment to the intensity of the labeled 3'-test cDNA fragment in comparison to a control ratio is at least 10%.

23. The method of any one of claims 2-12, wherein the control cDNA fragment and the at least one test cDNA fragment comprise a detectable label, wherein the quantifying comprises comparing an intensity of the at least one labeled test cDNA fragment with the intensity of the labeled control cDNA fragment, wherein a reduction in the intensity of the at least one labeled test cDNA fragment in comparison to the intensity of the labeled control cDNA fragment indicates 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 label is a fluorescent dye.

26. The method of claim 25, wherein the detectable label is a DNA intercalating label.

27. The method of any one of claims 2-12 and 23-26, wherein the control cDNA fragment corresponds to an mRNA molecule obtained at time period 0 (TPo) and the test cDNA fragment corresponds to an mRNA molecule obtained at later time period X (TPx).

28. The method of any one of the preceding claims, wherein the composition is a pharmaceutical composition.

29. The method of any one 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 species of mRNA molecules, a trivalent vaccine comprising three different species of mRNA molecules, and a quadrivalent vaccine comprising four different species 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 species of mRNA molecules share at least 50% sequence identity.

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

38. The method of any one of claims 30-37, wherein the different species of mRNA molecules differ in length 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.

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

40. The method of any one of the preceding claims, wherein the amplifying of the double-stranded cDNA comprises annealing a 5'-UTR primer and a 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 one 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 each of the different mRNA molecules differ in length from each other 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.

44. The method of any one 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 each of the different mRNA molecules differ in length from each other 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.

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 doublestranded cDNA molecule corresponding to a first species of mRNA molecule in the quadrivalent vaccine, a second amplified double-stranded cDNA molecule corresponding to a second species of mRNA molecule in the quadrivalent vaccine, a third amplified double-stranded cDNA molecule corresponding to a third species of mRNA molecule in the quadrivalent vaccine, and a fourth amplified double-stranded cDNA molecule corresponding to a fourth species 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 doublestranded DNA 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 species of mRNA molecule in the quadrivalent vaccine, a second amplified double-stranded cDNA molecule corresponding to a second species of mRNA molecule in the quadrivalent vaccine, a third amplified double-stranded cDNA molecule corresponding to a third species of mRNA molecule in the quadrivalent vaccine, and a fourth amplified double-stranded cDNA molecule corresponding to a fourth species 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 doublestranded cDNA molecule corresponding to a first species of mRNA molecule in the quadrivalent vaccine, a second amplified double-stranded cDNA molecule corresponding to a second species of mRNA molecule in the quadrivalent vaccine, a third amplified double-stranded cDNA molecule corresponding to a third species of mRNA molecule in the quadrivalent vaccine, and a fourth amplified double-stranded cDNA molecule corresponding to a fourth species of mRNA molecule in the quadrivalent vaccine, and wherein the at least one restriction enzyme comprises a first, second, third, and 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 Aarl, Aatll, AbsI, AccI, Acell, Acelll, Acil, Acll, Acyl, Affel, Aflll, AfUII, Agel, Agsl, Ahalll, Alul, AlwNI, AmaCSI, ApaBI, Apal, ApaLI, Apol, ApyPI, AquII, AquIII, AquIV, Asci, Asi256I, Asp718I, Asul, AsuII, Aval, Avail, Avril, Ball, BamHI, Bbel, BbvCI, Bbvl, BbvII, BccI, Bce83I, BceAI, BceAI, Bcefl, BciVI, Bell, Betl, Bfil, Bgll, Bglll, Bini, BpulOI, BsaAI, BsaBI, Bsbl, BscAI, BscXI, BscXI, BscXI, BscXI, BseMII, BseMII, BsePI, BsePI, BsePI, BseRI, BseSI, BseYI, BseYI, Bsgl, Bsil, BsiYI, BsmAI, BsmFI, BsmI, BsmI, BsmI, BsmI, BsmI, BsmI, Bspl20I, Bspl407I, BspCNI, BspD6I, BspHI, BspKT6I, BspLUl lI, BspMI, BspMII, BsrBI, BsrDI, BsrI, BstAPI, BstEII, BstF5I, BstNI, BstUI, BstXI, BtgZI, BthCI, Btrl, BtsI, BtsIMutl, Cac8I, CauII, Cchll, CdpI, CfrlOI, CfrI, Chai, CjeNIII, Clal, CstMI, CviAII, CviJI, CviQI, CviRI, Ddel, Drall, Dralll, DraRI, DraRI, DrdI, DrdIV, DrdV, Dsal, Eaml lO5I, Ecil, Eco31I, Eco47III, Eco56I, Eco57I, Eco57MI, Eco78I, EcoHI, EcoICRI, EcoNI, EcoRI, EcoRII, EcoRV, EcoT22I, EsaBC3I, Esp3I, EspI, Fail, Fatl, Faul, Fmul, Fnu4HI, FnuDII, FokI, Fsel, Fsel, FspAI, Gdill, Gsal, Gsul, Hael, Haell, Haelll, HauII, Hgal, HgiAI, HgiCI, Hhal, Hindi, Hindll, HmdIII, Hinfl, HinPlI, Hpal, Hpall, HphI, Hpyl78III, Hpyl88I, Hpy8I, Hpy99I, KasI, Kpnl, Ksp632I, LmnI, Lpnl, Mad, Madl, Maelll, MaqI, MauBI, Mbol, MboII, McaTI, McrI, Mfd, Mini, Mlyl, Mmel, Mnll, Msd, MslI, MspGI, MstI, Mwol, Nad, Narl, Ncol, Ndd, NgoAVII, Nhd, NlaCI, Nlalll, NlalV, N113877I, NmeA6CIII, NmeAIII, Notl, Nrul, NspBII, NspI, Olil, PabI, Pad, PasI, PasI, PflMI, Pfol, PlaDI, Piel, PmaCI, Pmel, PpulOI, PpuMI, PshAI, Psil, PspO3I, PspOMII, PspPRI, PspXI, Pssl, PstI, Pvul, PvuII, Reel, RdeGBII, Rlall, RleAI, RpaB5I, RpaBI, Rpal, Rsal, RsrII, Sad, SacII, Sall, SanDI, SapI, Saul, Seal, Scil, ScrFI, SdeAI, Sdul, Seel, Sell, Setl, SexAI, SfaNI, Sfel, Sfil, Sgfl, SgrAI, SgrDI, SgrAI, SgrDI, Siml, Smal, Smll, SnaBI, Spel, SphI, SplI, Srfl, Sse232I, Sse8387I, Sse8647I, SsoII, SspD5I, SspI, SstE37I, Sthl32I, Sth302II, StsI, Stul, Styl, Swal, Tail, TaqI, TaqII, Taqlll, Tati, Taul, Tfil, Tkol, TkoII, Tsel, Tsoi, Tsp45I, Tsp4CI, TspDTI, TspEI, TspGWI, TspRI, Tthl 1 II, Tthl 1 III, Unbl, VpaKl 1 Al, VspI, Wvil, Xbal, Xcal, Xcml, Xhol, XhoII, Xmal, Xmalll, XmnI, and/or Zral.

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

53. The method of any one of the preceding claims, wherein the at least one restriction enzyme comprises Agel, Hindi, Affel and Sac I.

54. The method of any one of the preceding claims, wherein the at least one restriction enzyme comprises Aval, AccI, PflMI and Stul.

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

56. A primer pair comprising a 5'-UTR primer and 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 claims 56 or 57, wherein the primers are labeled with a detectable label.