Improved DNA constructs for manufacturing messenger RNA by in vitro transcription

Abstract

The present disclosure provides methods and compositions for improving the in vitro transcription (IVT) of messenger RNA (mRNA) using SP6, T7, or KP34 RNA polymerase. In particular, the present disclosure provides in vitro transcription templates with optimized nucleic acid sequences specific for use with SP6, T7, or KP34 RNA polymerase that improve mRNA transcript yield and / or reduce amounts of dsRNA.

Description

Sanofi Ref: PAT24103-WO-PCTIMPROVED DNA CONSTRUCTS FOR MANUFACTURING MESSENGER RNA BY IN VITRO TRANSCRIPTIONRELATED APPLICATIONS

[0001] The present application claims priority from European patent application no. 24306813.7, filed October 28, 2024, the contents of which are herein incorporated by reference in their entirety.SEQUENCE LISTING[2] The present specification makes reference to a Sequence Listing, submitted electronically as an .xml file name “PAT24103-WO-PCT_SL.xml” on October 28, 2025. The .xml file was generated on October 21, 2025, and is 192 KB in size. The entire contents of the sequence listing are herein incorporated by reference.FIELD OF THE INVENTION[3] The present invention relates to improved DNA constructs for the manufacturing of messenger RNA (mRNA) by in vitro transcription (IVT). In particular, the invention provides promoter sequences that are optimized for use with SP6 RNA polymerase, T7 RNA polymerase, or KP34 RNA polymerase.BACKGROUND OF THE INVENTION[4] Messenger RNA (mRNA) is becoming increasingly important as a therapeutic agent. mRNA therapy can be used to restore normal levels of an endogenous protein or provide an exogenous therapeutic protein (e.g., a vaccine antigen or antibody) without permanently altering the genome sequence or entering the nucleus of the cell. mRNA therapy takes advantage of the cell’s own protein production and processing machinery to express a therapeutic peptide, polypeptide, or protein, is flexible to tailored dosing and formulation, and is broadly applicable to any disease or condition that is treatable through the provision of an exogenous protein.[5] The process of manufacturing mRNA for use in therapy typically involves the in vitro transcription (IVT) of mRNA from a DNA template using a phage-derived DNA- dependent RNA polymerase. The DNA template can be prepared by standard molecularSanofi Ref: PAT24103-WO-PCT biology techniques. For example, a nucleic acid sequence encoding a peptide, polypeptide, or protein of interest may be cloned into a multi-copy plasmid which is then propagated in Escherichia coli (E. coll) and purified for use as a template in an IVT reaction.[6] A synthesis process using a phage-derived DNA-dependent RNA polymerase commonly yields transcriptional by-products in addition to the desired mRNA transcripts. For example, T7 RNA polymerase forms double-stranded RNA (dsRNA) during IVT. Although generally producing less dsRNA than T7 RNA polymerase, SP6 RNA polymerase also forms dsRNA (see, e.g., WO 2022 / 082001 and WO 2018 / 157153). The formation of dsRNA is undesirable since it leads to inefficient translation of the administered mRNA product and results in the induction of cytokines, eliciting an interferon (IFN)-mediated inflammatory immune response.[7] As dsRNA is highly immunogenic, it is desirable to eliminate or greatly reduce the amount of dsRNA from the in vitro transcribed mRNA for many reasons including, for example, to limit cytokine induction and reactogenicity in vivo and to avoid time-consuming and expensive purification of the mRNA after in vitro transcription (IVT). Moreover, it is also desirable to maximize mRNA yield to decrease production costs. Therefore, a need remains to improve existing methods for the manufacturing of mRNA by IVT.SUMMARY OF THE INVENTION[8] The present invention is based on the discovery that optimization of the nucleotide sequences 5’- and 3 ’-adjacent to the core promoter utilized by phage-derived DNA-dependent RNA polymerases such as SP6, T7, and KP34 can improve messenger RNA (mRNA) transcript yield while also decreasing the amount of double-stranded RNA (dsRNA) present that is produced during in vitro transcription (IVT).[9] In a first aspect, the invention relates to a DNA construct (e.g., an IVT template for use with SP6 RNA polymerase) comprising the nucleic acid sequence 5’-Pi- ATTTAGGXIGACACTATA-P2-C-3’ (5’-Pi-[SEQ ID NO: l]-P2-C-3’), wherein Pi is an upstream promoter region, Xi is selected from G, A, or T, P2is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and consists of GX2A-Si, wherein X2is selected from A or G, Si is GAGGA or CTGGTGGA and is optional, and wherein the 3’ terminal C is the first nucleotide of the 5’ untranslated regionSanofi Ref: PAT24103-WO-PCT(5’ UTR). In some embodiments, the transcriptional start site of the mRNA transcript is GGA. In some embodiments, the transcriptional start site of the mRNA transcript is GAA. In some embodiments, the sequence located immediately downstream of the 3’ terminal C is AGATCGCC. The DNA construct can be linear or linearized.

[0010] In this first aspect, the amount of dsRNA comprised in the mRNA transcripts obtainable with the DNA construct when used with SP6 RNA polymerase as a template for IVT is decreased and / or the mRNA transcript yield is increased relative to mRNA transcripts obtainable with a reference DNA construct comprising a nucleic acid sequence 5’- ATTTAGGTGACACTATAGGAC-3’ (SEQ ID NO: 2) without an upstream promoter region Pi, wherein the 3’ terminal four nucleic acids of the nucleic acid sequence comprise the transcriptional start site of an mRNA transcript.

[0011] In some embodiments of this first aspect, Pi is 18 nucleotides upstream of the transcriptional start site. In some embodiments, Pi is selected from the group consisting of 5’-CTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 3), 5’-TTATGTATCATACACATACG-3’ (SEQ ID NO: 4), and 5’-TGGACAAATCTGTGTCTCTT-3’ (SEQ ID NO: 5).

[0012] In some embodiments of this first aspect, Si is absent and X2 is G. In some embodiments, Si is GAGGA. In some embodiments, Si is GAGGA and X2 is A. In some embodiments, Si is CTGGTGGA. In some embodiments, Si is CTGGTGGA and X2 is A.

[0013] In some embodiments of this first aspect, Pi is 5’-CTCGCGGTCTTTAATTGCCT-3 ’ (SEQ ID NO: 3) and Xi is T or A. In other embodiments, Pi is 5’-TTATGTATCATACACATACG-3’ (SEQ ID NO: 4) and Xi is T or A. In other embodiments, Pi is 5’-TGGACAAATCTGTGTCTCTT-3’ (SEQ ID NO: 5) and X 1 is G or A.

[0014] In some embodiments of this first aspect, the nucleic acid sequence is selected from the group consisting of 5’-CTCGCGGTCTTTAATTGCCTATTTAGGTGACACTATAGGAC-3’ (SEQ ID NO: 6); 5 ’ -TTATGTATC AT AC AC ATACGATTTAGGTGAC ACT AT AG A AG AGG AC -3 ’ (SEQ ID NO: 7), 5’-TGGACAAATCTGTGTCTCTTATTTAGGGGACACTATAGAAGAGGAC-3’ (SEQ ID NO: 8), 5’-TTATGTATCATACACATACGATTTAGGAGACACTATAGAAGAGGAC- 3’ (SEQ ID NO: 9) and 5’-Sanofi Ref: PAT24103-WO-PCTCTCGCGGTCTTTAATTGCCTATTTAGGAGACACTATAGAAGAGGAC-3’ (SEQ ID NO: 10).

[0015] In a second aspect, the invention relates to a DNA construct (e.g., an IVT template for use with SP6 RNA polymerase) comprising the nucleic acid sequence 5’-Pi- ATTTAGGX1GACACTATA-P2-C-3’ (5’-Pi-[SEQ ID NO: l]-P2-C-3’), wherein Pi is an upstream promoter region, Xi is selected from G, A, or T, P2is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and consists of GAA-Si, wherein Si is GGGAGGTAG, GAGAGAATT, or TACAAGCTT, and wherein the 3’ terminal C is the first nucleotide of the 5’ untranslated region (5’ UTR). In some embodiments, the transcriptional start site of the mRNA transcript is GAA. In some embodiments, the sequence located immediately downstream of the 3’ terminal C is AGATCGCC. The DNA construct can be linear or linearized.

[0016] In this second aspect, the amount of dsRNA comprised in the mRNA transcripts obtainable with the DNA construct when used with SP6 RNA polymerase as a template for IVT is decreased and / or the mRNA transcript yield is increased relative to mRNA transcripts obtainable with a reference DNA construct comprising a nucleic acid sequence 5’-ATTTAGGTGACACTATAGGAC-3’ (SEQ ID NO: 2) without an upstream promoter region Pi, wherein the 3’ terminal four nucleic acids of the nucleic acid sequence comprise the transcriptional start site of an mRNA transcript.

[0017] In some embodiments of this second aspect, Pi is 18 nucleotides upstream of the transcriptional start site. In some embodiments, Pi is selected from the group consisting of 5’-CTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 3), 5’-TGGACAAATCTGTGTCTCTT-3’ (SEQ ID NO: 5), or 5’-GCACGTCGCCGCGCAGGTATGGCTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 147).

[0018] In some embodiments of this second aspect, Si is GGGAGGTAG. In some embodiments, Si is GAGAGAATT.

[0019] In some embodiments of this second aspect, Pi is 5’-CTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 3) and Xi is T. In other embodiments, Pi is 5’-TGGACAAATCTGTGTCTCTT-3’ (SEQ ID NO: 5) and Xi is G. In some embodiments, Pi is 5’-Sanofi Ref: PAT24103-WO-PCTGCACGTCGCCGCGCAGGTATGGCTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 147) and Xi is T.

[0020] In some embodiments of this second aspect, the nucleic acid sequence is selected from the group consisting of 5’- CTCGCGGTCTTTAATTGCCTATTTAGGTGACACTATAGAAGGGAGGTAG-3’(SEQ ID NO: 155), 5’-TGGACAAATCTGTGTCTCTTATTTAGGGGACACTATAGAAGAGAGAATT-3’ (SEQ ID NO: 156), and 5’-GCACGTCGCCGCGCAGGTATGGCTCGCGGTCTTTAATTGCCTATTTAGGTGACA CTATAGAAGGGAGGTAG-3’ (SEQ ID NO: 215).

[0021] The first and second aspects of the invention also relate to a method for manufacturing mRNA by IVT comprising a) providing a DNA construct as described above and b) contacting the DNA construct with an SP6 RNA polymerase under conditions suitable for obtaining mRNA transcripts. As shown herein, using this method, the amount of dsRNA comprised in the mRNA transcripts obtained in step b) is decreased and / or the mRNA transcript yield is increased relative to mRNA transcripts obtained with a reference DNA construct comprising a nucleic acid sequence 5’-ATTTAGGTGACACTATAGGAC-3’ (SEQ ID NO: 2) without an upstream promoter region Pi, wherein the 3’ terminal four nucleic acids of the nucleic acid sequence comprise the transcriptional start site of an mRNA transcript.

[0022] When a method of the first and second aspects of the invention is used, the amount of dsRNA comprised in the mRNA transcripts obtained in step b) may be at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70% lower relative to the mRNA transcripts obtained with the reference DNA construct. The amount of dsRNA can be determined by ELISA using antibodies J2 and KI.

[0023] Alternatively, or in addition, when a method of first and second aspects of the invention is used, the mRNA transcript yield may be increased by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% relative to the mRNA transcript yield obtained with the reference DNA construct. In some embodiments, the mRNA transcript yield may be increased by at least 75%, 100%, 125%, or 150%. The mRNA transcript yield can be determined by UV spectroscopy at 260 nm.Sanofi Ref: PAT24103-WO-PCT

[0024] In a third aspect, the invention relates to a DNA construct (e.g., an IVT template for use with T7 RNA polymerase) comprising the nucleic acid sequence 5’-Pi- TAATACGACTCACTATX1-P2-C-3’ (5’-Pi-[SEQ ID NO: 75]-P2-C-3’), wherein Pi is an upstream promoter region, Xi is selected from T or A, P2is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and consists of X2GGA-SI, wherein X2is selected from A or G and is optional, Si is TAATCGGA and is optional, and the 3’ terminal C is the first nucleotide of the 5’ untranslated region (5’ UTR). In some embodiments, the transcriptional start site of the mRNA transcript is GGA. In other embodiments, the transcriptional start site of the mRNA transcript is AGG. In other embodiments, the transcriptional start site of the mRNA transcript is GGG. In some embodiments, the sequence located immediately downstream of the 3’ terminal C is AGATCGCC. The DNA construct can be linear or linearized.

[0025] In this third aspect, the amount of dsRNA comprised in the mRNA transcripts obtainable with the DNA construct when used with T7 RNA polymerase as a template for IVT is decreased and / or the mRNA transcript yield is increased relative to mRNA transcripts obtainable with a reference DNA construct comprising a nucleic acid sequence 5’-TAATACGACTCACTATAGGAC-3’ (SEQ ID NO: 76) without an upstream promoter region Pi, wherein the 3’ terminal four nucleic acids of the nucleic acid sequence comprise the transcriptional start site of an mRNA transcript.

[0026] In some embodiments of this third aspect, Pi is 18 nucleotides upstream of the transcriptional start site. In some embodiments, Pi is selected from the group consisting of 5’-GAATT-3', 5’-GCCTCGAAAT-3’ (SEQ ID NO: 84), 5’-GTCCCTAAAT-3’ (SEQ ID NO: 85), 5’-AGCACCGAAG-3’ (SEQ ID NO: 79), and 5’-AGCACCAAAT-3’ (SEQ ID NO: 78).

[0027] In some embodiments of this third aspect, Xi is T, X2is A and Si is absent. In some embodiments, Xi is A, X2is G and Si is TAATCGGA.

[0028] In some embodiments of this third aspect, the nucleic acid sequence is selected from the group consisting of 5’-AGCACCGAAGTAATACGACTCACTATTAGGAC-3’ (SEQ ID NO: 81), and 5’-AGCACCAAATTAATACGACTCACTATAGGGATAATCGGAC (SEQ ID NO: 82).

[0029] This third aspect of the invention also relates to a method for manufacturing mRNA by IVT comprising a) providing a DNA construct as described above and b)Sanofi Ref: PAT24103-WO-PCT contacting the DNA construct with a T7 RNA polymerase under conditions suitable for obtaining mRNA transcripts. As shown herein, using this method, the amount of dsRNA comprised in the mRNA transcripts obtained in step b) is decreased and / or the mRNA transcript yield is increased relative to mRNA transcripts obtained with a reference DNA construct comprising a nucleic acid sequence 5’-TAATACGACTCACTATAGGAC-3’ (SEQ ID NO: 76) without an upstream promoter region Pi, wherein the 3’ terminal four nucleic acids of the nucleic acid sequence comprise the transcriptional start site of an mRNA transcript.

[0030] When a method of the third aspect of the invention is used, the amount of dsRNA comprised in the mRNA transcripts obtained in step b) may be at least 20% less, 25% less or 30% lower relative to the mRNA transcripts obtained with the reference DNA construct. The amount of dsRNA can be determined by ELISA using antibodies J2 and KI.

[0031] Alternatively, or in addition, when a method of the third aspect of the invention is used, the mRNA transcript yield may be increased by at least 40%, 45% or 50% relative to the mRNA transcript yield obtained with the reference DNA construct. The mRNA transcript yield can be determined by UV spectroscopy at 260 nm.

[0032] In a fourth aspect, the invention relates to a DNA construct (e.g., an IVT template for use with KP34 RNA polymerase) comprising the nucleic acid sequence 5’-Pi- TAATGTTACAGGAGTA-P2-C-3’ (5’-Pi-[SEQ ID NO: 177]-P2-C-3’), wherein Pi is an upstream promoter region, P2is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and consists of GGX1-S1, wherein Xi is A or G, and Si is GA, GGA, or ATGAAGTTA, and the 3’ terminal C is the first nucleotide of the 5’ untranslated region (5’ UTR). In some embodiments, the transcriptional start site of the mRNA transcript is GGA. In other embodiments, the transcriptional start site of the mRNA transcript is GGG. In some embodiments, the sequence located immediately downstream of the 3’ terminal C is AGATCGCC. The DNA construct can be linear or linearized.

[0033] In this fourth aspect, the amount of dsRNA comprised in the mRNA transcripts obtainable with the DNA construct when used with KP34 RNA polymerase as a template for IVT is decreased and / or the mRNA transcript yield is increased relative to mRNA transcripts obtainable with a reference DNA construct comprising a nucleic acid sequence 5’-CATCTAGATAATGTTACAGGAGTAGGAC-3’ (SEQ ID NO: 178), whereinSanofi Ref: PAT24103-WO-PCT the 3’ terminal four nucleic acids of the nucleic acid sequence comprise the transcriptional start site of an mRNA transcript.

[0034] In some embodiments of this fourth aspect, Pi is 17 nucleotides upstream of the transcriptional start site. In some embodiments, Pi is selected from the group consisting of 5’-CATCTAGA-3’, 5’-TTCTTCTTCTGCTAAAAATT-3’ (SEQ ID NO: 179), and 5’- CCGTCAAGTGCTAGAATCCCCGTTCTTCTTCTGCTAAAAATT-3’ (SEQ ID NO: 180).

[0035] In some embodiments of this fourth aspect, Xi is A, and Si is ATGAAGTTA.

[0036] In some embodiments of this fourth aspect, the nucleic acid sequence is selected from the group consisting of 5’- TTCTTCTTCTGCTAAAAATTTAATGTTACAGGAGTAGGAATGAAGTTA-3 ’ (SEQ ID NO: 185), and 5’-CCGTCAAGTGCTAGAATCCCCGTTCTTCTTCTGCTAAAAATTTAATGTTACAGG AGTAGGAATGAAGTTA-3’ (SEQ ID NO: 186).

[0037] This fourth aspect of the invention also relates to a method for manufacturing mRNA by IVT comprising a) providing a DNA construct as described above and b) contacting the DNA construct with a KP34 RNA polymerase under conditions suitable for obtaining mRNA transcripts. As shown herein, using this method, the amount of dsRNA comprised in the mRNA transcripts obtained in step b) is decreased and / or the mRNA transcript yield is increased relative to mRNA transcripts obtained with a reference DNA construct comprising a nucleic acid sequence 5’- CATCTAGATAATGTTACAGGAGTAGGAC-3’ (SEQ ID NO: 178), wherein the 3’ terminal four nucleic acids of the nucleic acid sequence comprise the transcriptional start site of an mRNA transcript.

[0038] When a method of the fourth aspect of the invention is used, the amount of dsRNA comprised in the mRNA transcripts obtained in step b) may be at least 20% less, 25% less or 30% lower relative to the mRNA transcripts obtained with the reference DNA construct. The amount of dsRNA can be determined by ELISA using antibodies J2 and KI.

[0039] Alternatively, or in addition, when a method of the fourth aspect of the invention is used, the mRNA transcript yield may be increased by at least 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, or 150% relative to the mRNA transcriptSanofi Ref: PAT24103-WO-PCT yield obtained with the reference DNA construct. The mRNA transcript yield can be determined by UV spectroscopy at 260 nm.

[0040] The DNA construct in a manufacturing method described herein may be at a concentration of 0.05 mg / mL to 0.5 mg / mL. The SP6, T7, or KP34 RNA polymerase in a manufacturing method described herein may be at a concentration of 0.01 mg / mL to 0.5 mg / mL.

[0041] IVT may take place in the presence of a modified ribonucleoside. In some embodiments, the modified ribonucleoside is pseudouridine, N1 -methylpseudouridine, 5- methylcytidine, 5-methoxyuridine, 2’-O-methyladenosine, 2’-O-methyluridine, 2’-O- methylcytidine, or 2 ’-O-m ethylguanosine, e.g., wherein the modified nucleoside is Nl- methylpseudouridine.

[0042] A method for manufacturing mRNA as described herein may further comprise a step of purifying the mRNA transcripts obtained in step b) of the manufacturing method from the enzyme(s) used in the manufacturing of the mRNA (e.g., SP6, T7, or KP34 RNA polymerase). In some embodiments, the step of purifying the mRNA transcripts involves a method other than (i) cellulose chromatography, and / or (ii) high-performance chromatography (HPLC) with a buffer system comprising triethylammonium acetate and / or acetonitrile. In some embodiments, the step of purifying the mRNA transcripts comprises precipitation with guanidinium thiocyanate (GSCN) and ethanol.

[0043] Other features, objects, and advantages of the present invention are apparent in the detailed description, drawings, and embodiments that follow. It should be understood, however, that the detailed description, the drawings, and the embodiments, while indicating embodiments of the present invention, are given by way of illustration only, not limitation. Various changes and modifications will become apparent to those skilled in the art.BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Embodiments of the invention will be described, by way of example, with reference to the following drawings.

[0045] Figure 1 illustrates RNA yields and amount of double-stranded RNA (dsRNA) obtained from in vitro transcription (IVT) reactions using 54 DNA constructs comprising different test promoters as templates for SP6 RNA polymerase. Data is representative of n=4 (RNA yield; dark grey bars) and n=3 (dsRNA; light grey bars). ResultsSanofi Ref: PAT24103-WO-PCT were normalized to a reference DNA construct (denoted as R). Values for dsRNA are reported as percentages of total RNA present in the IVT product. Each data point is represented by a black filled circle, filled bars represent the average value + standard deviation. The average RNA yield and dsRNA / RNA obtained from the reference DNA construct are depicted by the solid black line. The dashed black line represents a 20% increase in RNA yield compared to the reference DNA construct. The dotted black line represents a 40% decrease in dsRNA compared to the reference DNA construct.

[0046] Figure 2 illustrates RNA yields and amount of double-stranded RNA (dsRNA) obtained from in vitro transcription (IVT) reactions using 17 DNA constructs comprising different test promoters as templates for SP6 RNA polymerase. Data is representative of n=2 (RNA yield; dark grey bars) and n=l (dsRNA; light grey bars). Results were normalized to a reference DNA construct (denoted as R). Values for dsRNA are reported as percentages of total RNA present in the IVT product. Each data point for RNA yield is represented by a black filled circle, and filled bars represent the average value + standard deviation. The average RNA yield and dsRNA / RNA obtained from the reference DNA construct are depicted by the solid black line. The dashed black line represents a 20% increase in RNA yield compared to the reference DNA construct. The dotted black line represents a 40% decrease in dsRNA compared to the reference DNA construct.

[0047] Figure 3 illustrates RNA yields, amount of double-stranded RNA (dsRNA), and amount of abortive transcripts obtained from in vitro transcription (IVT) reactions using DNA plasmids comprising different test promoters and one of four different coding sequences (CDS 1, CDS 2, CDS 3, or CDS 4) as templates for SP6 RNA polymerase. CDS 1 encoded an influenza surface protein of 1698bp, CDS 2 encoded a bacterial virulence factor of 855 bp, CDS 3 encoded a coronavirus spike protein of 4032 bp, and CDS 4 encoded a green fluorescence protein (GFP) of 717 bp. Data are representative of n=3 (RNA yield shown as dark grey bars; dsRNA shown as light grey bars) and n=l (abortive transcripts shown as non-filled bars), respectively. Results were normalized to a reference DNA plasmid (denoted as R). Values for dsRNA and abortive transcripts are reported as percentages of total RNA present in the IVT product, and each of RNA yield, dsRNA and abortive transcript are presented as fold change relative to R. Each data point for RNA yield is represented by a non-filled circle, and filled bars represent the average value + standard deviation. The average RNA yield, dsRNA / RNA and abortive transcripts obtained from the reference DNA plasmid are depicted by the solid black line. The dashed black line represents a 1.4 fold increase inSanofi Ref: PAT24103-WO-PCTRNA yield compared to the reference DNA plasmid. The dotted black line represents a 0.5 fold decrease in dsRNA compared to the reference DNA plasmid. ND = not determined.

[0048] Figure 4 illustrates RNA yields and quantification of double-stranded RNA (dsRNA) obtained from in vitro transcription (IVT) reactions using 21 DNA constructs comprising different test promoters as templates for T7 RNA polymerase. Data is representative of n=3 (RNA yield; dark grey bars) and n=2 (dsRNA; light grey bars) Results were normalized to a reference DNA construct (denoted as R). Values for dsRNA are reported as percentages of total RNA present in the IVT product. Each data point is represented by a black filled circle, filled bars represent the average value + standard deviation. The average RNA yield and dsRNA / RNA obtained from the reference DNA construct are depicted by the solid black line. The dashed black line represents a 50% increase in RNA yield compared to the reference DNA construct. The dotted black line represents a 30% decrease in dsRNA compared to the reference DNA construct.

[0049] Figure 5 illustrates RNA yields and amount of double-stranded RNA (dsRNA) obtained from in vitro transcription (IVT) reactions using 19 DNA constructs comprising different test promoters as templates for KP34 RNA polymerase. Data is representative of n=2 (RNA yield; dark grey bars) and n=l (dsRNA; light grey bars). Results were normalized to a reference DNA construct (denoted as R). Values for dsRNA are reported as percentages of total RNA present in the IVT product. Each data point is represented by a black filled circle, filled bars represent the average value + standard deviation. The average RNA yield and dsRNA / RNA obtained from the reference DNA construct are depicted by the solid black line. The dashed black line represents a 20% increase in RNA yield compared to the reference DNA construct. The dotted black line represents a 30% decrease in dsRNA compared to the reference DNA construct.DEFINITIONS

[0050] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification.

[0051] As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. For example, an “mRNA transcript” is understood to represent one or more mRNA transcript(s).Sanofi Ref PAT24103-WO-PCTAs such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.

[0052] As used herein, “and / or” is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include “A and B”, “A or B”, “A” (alone), and “B” (alone). Likewise, the term "and / or" as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0053] Throughout this specification and embodiments, the words “have” and “comprise”, or variations such as “has”, “having”, “comprises”, or “comprising” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. It is further understood that wherever embodiments are described herein with the language “comprising” or “having” of grammatical equivalents thereof, otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided.

[0054] As used herein, the term “about” refers to an interval of accuracy that a person skilled in the art will understand to still ensure the technical effect of the feature in question. The term indicates a deviation from the indicated numerical value of ± 10%. In some embodiments, the deviation is ± 5% of the indicated numerical value. In certain embodiments, the deviation is ± 1% of the indicated numerical value.

[0055] As used herein, the term “core promoter” refers to the minimal nucleic acid sequence that a DNA-dependent RNA polymerase can utilize to initiate transcription from a DNA construct in vitro.

[0056] As used herein, the term “mRNA” refers to a polyribonucleotide that encodes at least one peptide, polypeptide, or protein. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions (e.g., a 5’ untranslated region and a 3’ untranslated region). The disclosure particularly relates to the manufacturing of mRNA by in vitro transcription (IVT). Where appropriate, mRNA can comprise nucleoside analogues such as analogues having chemically modified bases or sugars, backbone modifications, etc. An mRNA sequence is presented in the 5’ to 3’ direction unless otherwise indicated. A typical mRNA comprises a 5’ cap, a 5’ untranslated region (5’ UTR), a protein-coding region, a 3’ untranslated region (3’ UTR),Sanofi Ref: PAT24103-WO-PCT and a 3’ tail. A poly(A) tail is typically added to the RNA by a poly(A) polymerase or is included in the DNA template that is used for in vitro transcription to mimic naturally occurring mRNAs. The presence of a poly(A) tail at the 3’ end of an mRNA molecule prevents degradation inside cells, thereby enhancing its half-life in vivo.

[0057] The terms “peptide”, “polypeptide” or “protein” are used interchangeably herein. For example, the expression “a nucleic acid sequence encoding a polypeptide” refers to any nucleic acid sequence that encodes an amino acid sequence which, upon expression, forms a peptide, polypeptide, or protein. Such a nucleic acid sequence may further comprise a 5 ’-untranslated region (5’ UTR) and 3 ’-untranslated region (3’-UTR).

[0058] As used herein, the term “template” refers to a molecule (typically a DNA construct such as a plasmid) comprising a nucleic acid sequence encoding an mRNA transcript to be synthesized by in vitro transcription (IVT). The template is used for IVT in order to produce the mRNA transcript encoded by the template. The template comprises all elements necessary for IVT, particularly a promoter element for binding of a DNA-dependent RNA polymerase, which is operably linked to the nucleic acid sequence encoding a desired mRNA transcript. The template may be a linear DNA molecule or a circular DNA molecule that is linearized before IVT.

[0059] As used herein, the term “double-stranded RNA” or “dsRNA” refers to RNA produced during IVT comprising two complementary strands of ribonucleic acids basepaired with each other. During IVT, dsRNA is generated in cis by looping of full-length RNA with internal regions of complementarity. In addition, abortive transcripts are generated during the initiation phase of IVT, and the 3' end of the full-length RNA can prime complementary RNA synthesis from the primary transcripts in trans. Promoter-independent transcription of full-length anti-sense RNA is another mechanism of dsRNA generation.

[0060] As used herein, the term “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA (e.g., mRNA) template from a DNA sequence (e.g., by transcription); (2) processing of an RNA (e.g., mRNA) transcript (e.g., by editing, 5’ cap formation, and / or 3’ end formation); (3) translation of an RNA into a peptide, polypeptide, or protein in vitro or in vivo, and / or (4) post-translational modification of a polypeptide or protein. In this application, the terms “expression” and “production,” and grammatical equivalents, are used inter-changeably.Sanofi Ref: PAT24103-WO-PCT

[0061] As used herein, the term “substantially” refers to the qualitative condition of exhibiting a 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 rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0062] As used herein, the term “zzz vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, rather than within a cell or a multi-cellular organism. For example, as used herein, the term “z z vitro transcription” or “z z vitro synthesis” refers to transcription or synthesis of RNA that occurs outside of a cell (and usually in the absence of a cell lysate), typically in a test tube or reaction vessel (e.g., a bioreactor). “ / / / vitro transcription” or “z z vitro synthesis” typically involves the use of recombinantly produced and purified enzyme components (e.g., SP6, T7, or KP34 RNA polymerase).

[0063] As used herein, the terms “Klebsiella phage KP34 RNA polymerase”, “KP34 RNA polymerase”, “KP34 polymerase”, and “KP34” are used interchangeably and all refer to a DNA-dependent RNA polymerase obtainable from a Klebsiella phage (e.g., an RNA polymerase with the amino acid sequence set forth in SEQ ID NO: 187).

[0064] Unless otherwise defined herein, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs and as commonly used in the art to which this application belongs. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure. In case of conflict, the present specification, including definitions, will control.

[0065] Generally, nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, virology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.Sanofi Ref: PAT24103-WO-PCT

[0066] All publications and other reference materials referenced herein are hereby incorporated by reference in their entirety. Although a number of documents are cited herein, this citation does not constitute an admission that any of these documents form part of the common general knowledge in the art.DETAILED DESCRIPTION OF THE INVENTION

[0067] DNA-dependent RNA polymerases initiate transcription by contacting a suitable promoter sequence in a DNA construct. The invention relates to DNA constructs that comprise optimized promoter sequences operably linked to a nucleic acid sequence encoding an mRNA transcript. When used for IVT reactions with an appropriate RNA polymerase, the provided DNA constructs show improved mRNA transcript yield and / or decreased production of dsRNA.DNA-dependent RNA polymerases

[0068] The present disclosure relates in particular to SP6, T7, and KP34 RNA polymerases which are each phage-derived DNA-dependent RNA polymerases. KP34 is only distantly related to SP6 and T7.

[0069] Naturally occurring SP6 RNA polymerase (NCBI Reference Sequence: GenBank: Y00105.1) has the following amino acid sequence:MQDLHAIQLQLEEEMFNGGIRRFEADQQRQIAAGSESDTAWNRRLLSELIAPMAEG IQAYKEEYEGKKGRAPRALAFLQCVENEVAAYITMKVVMDMLNTDATLQAIAMS VAERIEDQVRFSKLEGHAAKYFEKVKKSLKASRTKSYRHAHNVAVVAEKSVAEK DADFDRWEAWPKETQLQIGTTLLEILEGSVFYNGEPVFMRAMRTYGGKTIYYLQT SESVGQWISAFKEHVAQLSPAYAPCVIPPRPWRTPFNGGFHTEKVASRIRLVKGNR EHVRKLTQKQMPKVYKAINALQNTQWQINKDVLAVIEEVIRLDLGYGVPSFKPLID KENKPANPVPVEFQHLRGRELKEMLSPEQWQQFINWKGECARLYTAETKRGSKSA AVVRMVGQARKYSAFESIYFVYAMDSRSRVYVQSSTLSPQSNDLGKALLRFTEGR PVNGVEALKWFCINGANLWGWDKKTFDVRVSNVLDEEFQDMCRDIAADPLTFTQ WAKADAPYEFLAWCFEYAQYLDLVDEGRADEFRTHLPVHQDGSCSGIQHYSAML RDEVGAKAVNLKPSDAPQDIYGAVAQVVIKKNALYMDADDATTFTSGSVTLSGTE LRAMASAWDSIGITRSLTKKPVMTLPYGSTRLTCRESVIDYIVDLEEKEAQKAVAE GRTANKVHPFEDDRQDYLTPGAAYNYMTALIWPSISEVVKAPIVAMKMIRQLARF AAKRNEGLMYTLPTGFILEQKIMATEMLRVRTCLMGDIKMSLQVETDIVDEAAMMSanofi Ref: PAT24103-WO-PCTGAAAPNFVHGHDASHLILTVCELVDKGVTSIAVIHDSFGTHADNTLTLRVALKGQ MVAMYIDGNALQKLLEEHEVRWMVDTGIEVPEQGEFDLNEIMDSEYVFA (SEQ ID NO: 11).

[0070] An SP6 RNA polymerase suitable for use with the present invention can be a modified enzyme having substantially the same or improved polymerase activity as a naturally occurring SP6 RNA polymerase. Thus, in some embodiments, the SP6 RNA polymerase may be modified from SEQ ID NO: 11, e.g., may comprise one or more amino acid substitutions, deletions, insertions, and / or additions relative to SEQ ID NO: 11. In some embodiments, a suitable SP6 RNA polymerase has an amino acid sequence that is about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 11. In particular embodiments, the amino acid sequence of the SP6 RNA polymerase is at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, or 95%) identical to the amino acid sequence of SEQ ID NO: 11.

[0071] Naturally occurring T7 RNA polymerase (NCBI Reference Sequence: NP 041960.1) has the following amino acid sequence:MNTINIAKNDFSDIELAAIPFNTLADHYGERLAREQLALEHESYEMGEARFRKMFE RQLKAGEVADNAAAKPLITTLLPKMIARINDWFEEVKAKRGKRPTAFQFLQEIKPE AVAYITIKTTLACLTSADNTTVQAVASAIGRAIEDEARFGRIRDLEAKHFKKNVEEQ LNKRVGHVYKKAFMQVVEADMLSKGLLGGEAWSSWHKEDSIHVGVRCIEMLIES TGMVSLHRQNAGVVGQDSETIELAPEYAEAIATRAGALAGISPMFQPCVVPPKPWT GITGGGYWANGRRPLALVRTHSKKALMRYEDVYMPEVYKAINIAQNTAWKINKK VLAVANVITKWKHCPVEDIPAIEREELPMKPEDIDMNPEALTAWKRAAAAVYRKD KARKSRRISLEFMLEQANKFANHKAIWFPYNMDWRGRVYAVSMFNPQGNDMTK GLLTLAKGKPIGKEGYYWLKIHGANCAGVDKVPFPERIKFIEENHENIMACAKSPL ENTWWAEQDSPFCFLAFCFEYAGVQHHGLSYNCSLPLAFDGSCSGIQHFSAMLRD EVGGRAVNLLPSETVQDIYGIVAKKVNEILQADAINGTDNEVVTVTDENTGEISEK VKLGTKALAGQWLAYGVTRSVTKRSVMTLAYGSKEFGFRQQVLEDTIQPAIDSGK GLMFTQPNQAAGYMAKLIWESVSVTVVAAVEAMNWLKSAAKLLAAEVKDKKTG EILRKRCAVHWVTPDGFPVWQEYKKPIQTRLNLMFLGQFRLQPTINTNKDSEIDAH KQESGIAPNFVHSQDGSHLRKTVVWAHEKYGIESFALIHDSFGTIPADAANLFKAV RETMVDTYESCDVLADFYDQFADQLHESQLDKMPALPAKGNLNLRDILESDFAFA (SEQ ID NO: 83).Sanofi Ref: PAT24103-WO-PCT

[0072] A T7 RNA polymerase suitable for use with the present invention can be a modified enzyme having substantially the same or improved polymerase activity as a naturally occurring T7 RNA polymerase. Thus, in some embodiments, the T7 RNA polymerase may be modified from SEQ ID NO: 83, e.g., may comprise one or more amino acid substitutions, deletions, insertions, and / or additions relative to SEQ ID NO: 83. In some embodiments, a suitable T7 RNA polymerase has an amino acid sequence that is about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 83. In particular embodiments, the amino acid sequence of the T7 RNA polymerase is at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, or 95%) identical to the amino acid sequence of SEQ ID NO: 83.

[0073] Naturally occurring Klebsiella phage KP34 RNA polymerase (NCBI Reference Sequence: YP 003347629.1) has the following amino acid sequence:MISALSTVVVPEEALVKRQLELEETYKIRGIERARKLITDALQNGGIMNLPMTQRM LTSAYEVAAAAIDEMRNVKAPGIGGKYRRFLRLIPLDVLTTLSLCTMFEAFSVAPG ESASRRQTAQAVMSALGRNVQSELLSLQLRNVAPAYMDRVYEYLTERRTKSPTHI LRTLRASAENVHYGHEPWTNAQNISVGRLLCAAVFETGLFQWKTGSGNLSMLYPA DDVMEAFQQLVESADTVTMKPPMLVPPVQHTTMWDGGYLTPIDNRGTYHNSHID RTRLREVAEAFI<SADGII<I<ALNI<AQETPYRINI<RILELVQEARALGIGVGMPRSVP EPKPEWYLDGVPKENYTEEELDRFGEWKTRMSLWYSADRKRVSQLRSLLTTLEM AEEFKDEKALYFPTCVDWRYRLYFKSSLHPQGSDLQKALLEFGRGKPLGDRGLFW LKVHVATCFGYDKTLFEDRAAWVDANFAEIEQLTVSPFDCPAFTSADSPWCLLAA AIDLVNAVRSGCPEEHISRIPVAMDATNSGGQHLSALLRDPVGGRLTNLYWEGND KKADLYMDVKRRTDEKVILDLDKEDFIIQSTYWRENEITRSMTKRPSMTYFYSATV RSCSDYIFEGACAEGYEGTDTNSLWNLSCYLAPRMRAAIEEANPAAAAVMGYLQN LARRVPASQHLQWYTPLGGLVMNRYTQREEVRVRIDCMNLSAVLVHNRDFKTCN KRKAASGIAPNFVHSLDSTHLMMVLCAAEGLDIVPIHDSLATHAADVDDMHRHIR EQFVRLYEENDLLGDITRAAAAAGADLTDLDMPEVGTLDIRQVLESPFFFC (SEQ ID NO: 187)

[0074] A Klebsiella phage KP34 RNA polymerase suitable for use with the present invention can be a modified enzyme having substantially the same or improved polymerase activity as a naturally occurring Klebsiella phage KP34 RNA polymerase. Thus, in some embodiments, the KP34 RNA polymerase may be modified from SEQ ID NO: 187, e.g., maySanofi Ref: PAT24103-WO-PCT comprise one or more amino acid substitutions, deletions, insertions, and / or additions relative to SEQ ID NO: 187. In some embodiments, a suitable KP34 RNA polymerase has an amino acid sequence that is about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 187. In particular embodiments, the amino acid sequence of the KP34 RNA polymerase is at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, or 95%) identical to the amino acid sequence of SEQ ID NO: 187.

[0075] In some embodiments, a suitable SP6, T7, or KP34 RNA polymerase may be a truncated protein (from N-terminus, C-terminus, or internally) but retains the polymerase activity. In some embodiments, a suitable SP6, T7, or KP34 RNA polymerase is a fusion protein. For example, an SP6, T7, or KP34 RNA polymerase may include one or more tags to promote isolation, purification, or solubility of the enzyme. A suitable tag may be located at the N-terminus, C-terminus, and / or internally. Typically, the tag is located at the N- terminus. Non-limiting examples of a suitable tag include Calmodulin-binding protein (CBP); Fasciola hepatica 8-kDa antigen (Fh8); FLAG tag peptide; glutathione-5-transferase (GST); Histidine tag (e.g., hexahistidine tag (His6)); maltose-binding protein (MBP); N- utilization substance (NusA); small ubiquitin related modifier (SUMO) fusion tag; Streptavidin binding peptide (STREP); Tandem affinity purification (TAP); and thioredoxin (TrxA). Other tags may be used in the present invention. These and other fusion tags have been described, e.g., in Costa et al., Frontiers in Microbiology, 5(2014): 63 and in PCT / US16 / 57044, the contents of which are incorporated herein by reference in their entireties. In some embodiments, a His tag is located at the N-terminus of SP6, T7, or KP34 RNA polymerase.DNA constructs for use with SP6 RNA polymerase

[0076] Both the regions upstream (Pi) and downstream (P2) of the SP6 core promoter can influence transcriptional activity of SP6 RNA polymerase. Surprisingly, by combining modifications to both regions, the inventors discovered nucleic acid sequences that can improve mRNA transcript yield and the amount of dsRNA that SP6 RNA polymerase generates during IVT.

[0077] Provided herein are optimized DNA construct for use as an IVT template with SP6 RNA polymerase (e.g., a DNA plasmid) that comprises the following nucleic acid sequence:5’-Pi-ATTTAGGXiGACACTATA-P2-C-3’ (5’-PI-[SEQ ID NO: l]-P2-C-3’),Sanofi Ref: PAT24103-WO-PCT wherein:Pi is an upstream promoter region;Xi is selected from G, A, or T;P2 is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and consists of GX2A-S1, wherein:X2 is selected from A or G; andSi is selected from GAGGA, CTGGTGGA, GGGAGGTAG, GAGAGAATT, or TACAAGCTT and is optional; or the 3’ terminal C is the first nucleotide of the 5’ untranslated region (5’ UTR).

[0078] In some aspects, an optimized DNA construct for use as an IVT template with SP6 RNA polymerase (e.g., a DNA plasmid) is provided that comprises the following nucleic acid sequence:5’-Pi-ATTTAGGXiGACACTATA-P2-C-3’ (5’-PI-[SEQ ID NO: l]-P2-C-3’) wherein:Pi is an upstream promoter region;Xi is selected from G, A, or T;P2 is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and consists of GX2A-S1, wherein:X2 is selected from A or G;Si is GAGGA or CTGGTGGA and is optional; and the 3’ terminal C is the first nucloetide of the 5’ untranslated region (5’ UTR).

[0079] In other aspects, an optimized DNA construct for use as an IVT template with SP6 RNA polymerase (e.g., a DNA plasmid) is provided that comprises the following nucleic acid sequence:5’-Pi-ATTTAGGXiGACACTATA-P2-C-3’ (5’-PI-[SEQ ID NO: l]-P2-C-3’) wherein:Pi is an upstream promoter region;Xi is selected from G, A, or T;Sanofi Ref: PAT24103-WO-PCTP2 is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and consists of GAA-Si, wherein:Si is GGGAGGTAG, GAGAGAATT, or TACAAGCTT; and the 3’ terminal C is the first nucleotide of the 5’ untranslated region (5’ UTR).In each of the above nucleic acid sequence, the SP6 promoter sequence is shown in bold.SP6 upstream promoter region

[0080] The upstream promoter region Pi is positioned directly upstream of an SP6 RNA polymerase core promoter z.e., there are no intervening nucleotides between the upstream promoter sequence and a core promoter sequence. The upstream promoter is typically positioned about 18 nucleotides upstream of the transcriptional start site.

[0081] A suitable upstream promoter sequence Pi comprises at least 5 nucleotides. In some embodiments, the upstream promoter Pi comprises at least about 10 nucleotides or at least about 15 nucleotides. In some embodiments, the upstream promoter sequence Pi comprises about 20 nucleotides. In some embodiments, the upstream promoter sequence Pi may be about 30 nucleotides. In some embodiments, the upstream promoter sequence Pi may be about 40 nucleotides. For example, the upstream promoter sequence Pi may be 5-45 nucleotides, e.g., 15-45 nucleotides, or 20-45 nucleotides.

[0082] Exemplary upstream promoter sequences Pi for use with the DNA constructs described herein are provided in Table 1.Table 1. SP6 upstream promoter sequencesSanofi Ref: PAT24103-WO-PCTSP6 core promoter

[0083] Some variation in the SP6 core promoter sequence is possible. Exemplary SP6 core promoter sequences for use in the DNA constructs include those provided in Table 2.Table 2. SP6 core promoter sequences

[0084] A suitable SP6 promoter may be combined with an SP6 upstream promoter sequence from Table 1 to form the following sequences, wherein the core promoter sequence is shown in bold:CTCGCGGTCTTTAATTGCCTATTTAGGTGACACTATA (SEQ ID NO: 16)CTCGCGGTCTTTAATTGCCTATTTAGGAGACACTATA (SEQ ID NO: 17)TTATGTATCATACACATACGATTTAGGTGACACTATA (SEQ ID NO: 18)TTATGTATCATACACATACGATTTAGGAGACACTATA (SEQ ID NO: 19)TGGACAAATCTGTGTCTCTTATTTAGGGGACACTATA (SEQ ID NO: 20)TGGACAAATCTGTGTCTCTTATTTAGGAGACACTATA (SEQ ID NO: 21)GAATTATTTAGGTGACACTATA (SEQ ID NO: 22)GAATTATTTAGGAGACACTATA (SEQ ID NO: 23)CGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGTGAC ACTATA (SEQ ID NO: 152)CGGCTACAATTAATACATAACCTTATGTATCATACACATACGATTTAGGAGAC ACTATA (SEQ ID NO: 153)Sanofi Ref: PAT24103-WO-PCTGCACGTCGCCGCGCAGGTATGGCTCGCGGTCTTTAATTGCCTATTTAGGTGAC ACTATA (SEQ ID NO: 154)

[0085] DNA constructs comprising the upstream promoter and core promoter sequences as set forth in SEQ ID NOs: 16-20 and 154 are particularly useful for IVT of mRNA.SP6 downstream promoter region

[0086] The nucleic acid sequence directly downstream (P2) of the SP6 core promoter, i. e. , 3 ’ adj acent to the core promoter sequence, can also be optimized to improve mRNA yield during IVT. The downstream promoter region P2 typically includes the transcriptional start site for an mRNA transcript that is synthesized during IVT when the DNA construct is contacted with an SP6 RNA polymerase. The transcriptional start site of the mRNA transcript can be or comprise GGA or GAA (e.g., GAAGAGGA). These transcriptional start sites have been shown to provide high transcription activity in the context of the SP6 downstream promoter region of the DNA constructs tested herein. Typically, the first nucleotide of the transcriptional start site is G.

[0087] A suitable nucleic sequence for the SP6 downstream promoter region may be selected from the sequences listed in Table 3:Table 3. SP6 downstream promoter sequences

[0088] The 3 ’-end of a downstream promoter sequence provided in Table 3 may be immediately adjacent to a C nucleotide.Sanofi Ref: PAT24103-WO-PCT

[0089] A downstream promoter sequence provided in Table 3 may be combined with an SP6 upstream promoter sequence from Table 1 and a core promoter sequence from Table 2. For example, each of the SP6 downstream promoter sequences provided in Table 3 may be combined with any one of the sequences set forth in SEQ ID NOs: 16-23 to form an SP6 promoter that can be tested to determine whether the amount of dsRNA comprised in the mRNA transcripts obtainable with a DNA construct comprising the SP6 promoter is decreased and / or the mRNA transcript yield is increased relative to mRNA transcripts obtainable with a reference DNA construct. DNA constructs comprising the upstream promoter and core promoter sequences as set forth in SEQ ID NOs: 16-20 and 154 are particularly useful for IVT of mRNA.Exemplary SP6 DNA constructs

[0090] In particular embodiments, an optimized DNA construct for use as an IVT template with SP6 RNA polymerase (e.g., a DNA plasmid) is provided that comprises the following nucleic acid sequence:5’-Pi-ATTTAGGXiGACACTATA-P2-C-3’ (5’-PI-[SEQ ID NO: l]-P2-C-3’) wherein:Pi is an upstream promoter region and selected fromCTCGCGGTCTTTAATTGCCT (SEQ ID NO: 3),TTATGTATCATACACATACG (SEQ ID NO: 4),TGGACAAATCTGTGTCTCTT (SEQ ID NO: 5), andGCACGTCGCCGCGCAGGTATGGCTCGCGGTCTTTAATTGCCT (SEQID NO: 147);Xi is selected from G, A, or T;P2 is a downstream promoter region that comprises the transcriptional start site of mRNA transcript and P2 is selected from GGA, GAAGAGGA, GAAGGGAGGTAG (SEQ ID NO: 150), and GAAGAGAGAATT (SEQ ID NO: 151); and the 3’ terminal C is the first nucloetide of the 5’ untranslated region (5’ UTR).

[0091] In some embodiments, Pi is CTCGCGGTCTTTAATTGCCT (SEQ ID NO:3) and P2 is GGA. In some embodiments, Pi is TTATGTATCATACACATACG (SEQ ID NO: 4) and P2 is GAAGAGGA. In some embodiments, Pi is CTCGCGGTCTTTAATTGCCT (SEQ ID NO: 3) and P2is GAAGAGGA. In someSanofi Ref: PAT24103-WO-PCT embodiments, Pi is CTCGCGGTCTTTAATTGCCT (SEQ ID NO: 3) and P2is GAAGGGAGGTAG (SEQ ID NO: 150). In some embodiments, Pi is TGGACAAATCTGTGTCTCTT (SEQ ID NO: 5) and P2is GAAGAGAGAATT (SEQ ID NO: 151). In some embodiments, Pi isGCACGTCGCCGCGCAGGTATGGCTCGCGGTCTTTAATTGCCT (SEQ ID NO: 147) and P2is GAAGGGAGGTAG (SEQ ID NO: 150).

[0092] Further exemplary nucleic sequences for use in the DNA constructs described herein are provided in Table 4 below, wherein the core promoter sequence is shown in bold, the downstream promoter region P2is underlined, and the first nucleotide of the transcriptional start site of an mRNA transcript is shown in italic. The 3’ terminal X is the first nucleotide of the 5’ UTR of the mRNA transcript. X can be selected from A, T, C, and G.Table 4. Exemplary SP6 nucleic acid sequencesSanofi Ref PAT24103-WO-PCT

[0093] In particular embodiments, the first nucleotide of the chosen 5’ UTR is C.Accordingly, an exemplary nucleic acid sequence may be selected from SEQ ID NOs: 6-10Sanofi Ref: PAT24103-WO-PCT and 26-42. Alternatively, an exemplary nucleic acid sequence may be selected from SEQ ID NOs: 165, 166, 168, and 214. In one specific embodiment, the exemplary nucleic acid sequence may be selected from SEQ ID NO: 8 or 10.

[0094] Additional exemplary DNA constructs include DNA constructs which comprise the nucleic acid sequences set forth in SEQ ID NOs: 43, 45, 54, 57, 65, and 67.

[0095] As shown herein, DNA constructs comprising the nucleic acid sequences set forth in SEQ ID NOs: 6-10, 27, 29, 30, 31, 33, 34, 35, 37, 40, 42, and 54 yielded more mRNA than a reference DNA construct when used for the production of mRNA by IVT. Similarly, DNA constructs comprising the nucleic acid sequences set forth in SEQ ID NOs: 158, 160, 165, 166, and 168 yielded more mRNA than a reference DNA construct when used for the production of mRNA by IVT. Moreover, DNA constructs comprising the nucleic acid sequences set forth in SEQ ID NOs: 6-10, 26-42, 43, 45, 54, 57, 65, and 67 generated less dsRNA than a reference DNA construct when used for the production of mRNA by IVT. Similarly, DNA constructs comprising the nucleic acid sequences set forth in SEQ ID NOs: 158, 160, and 162-168 generated less dsRNA than a reference DNA construct when used for the production of mRNA by IVT. In addition, a DNA construct comprising the nucleic acid sequence set forth in SEQ ID NO: 214 yielded more mRNA than a reference DNA construct when used for the production of three different mRNAs by IVT. A DNA construct comprising the nucleic acid sequence set forth in SEQ ID NO: 214 also generated less dsRNA than a reference DNA construct when used for the production of three different mRNAs by IVT. Furthermore, a DNA construct comprising the nucleic acid sequence set forth in SEQ ID NO: 214 reduced the amount of abortive transcripts produced compared to a reference DNA construct when used for the production of three different mRNAs by IVT.

[0096] In particular, the inventors observed greater mRNA yields and lower amounts of dsRNA compared to a reference DNA template when DNA constructs comprising the nucleic acid sequences set forth in SEQ ID NOs: 6-10 and 27, 29, 30, 31, 33, 34, 35, 37, 40, 42 and 54 were used for the production of mRNA by IVT. Accordingly, in some embodiments, a DNA construct (e.g., a template for IVT of mRNA) is provided that comprises an optimized SP6 promoter having the nucleic acid sequence as set forth in SEQ ID NOs: 6-10 and 27, 29, 30, 31, 33, 34, 35, 37, 40, 42 and 54. Similarly, the inventors observed greater mRNA yields and lower amounts of dsRNA compared to a reference DNA template when DNA constructs comprising the nucleic acid sequences set forth in SEQ IDSanofi Ref: PAT24103-WO-PCTNOs: 158, 160, 165, 166 and 168 were used for the production of mRNA by IVT. Accordingly, in some embodiments, a DNA construct (e.g., a template for IVT of mRNA) is provided that comprises an optimized SP6 promoter having the nucleic acid sequence as set forth in SEQ ID NOs: 165, 166, and 168. The inventors also observed greater mRNA yields and lower amounts of dsRNA compared to a reference DNA construct when a DNA construct comprising the nucleic acid sequence set forth in SEQ ID NO: 214 was used for the production of three different mRNAs by IVT. Accordingly, in some embodiments, a DNA construct (e.g., a template for IVT of mRNA) is provided that comprises an optimized SP6 promoter having the nucleic acid sequence as set forth in SEQ ID NO: 214.

[0097] The inventors have observed particularly good performances of SP6 RNA polymerase when the nucleic sequences set forth in SEQ ID NOs: 6-10 were used as promoter sequences for the production of mRNA by IVT. Accordingly, in particular embodiments, a DNA construct (e.g., a template for IVT of mRNA) is provided that comprises an optimized SP6 promoter having the nucleic acid sequence as set forth in SEQ ID NOs: 6-10. In specific embodiments, a DNA construct is provided that comprises an optimized SP6 promoter having the nucleic acid sequence as set forth in SEQ ID NOs: 8 or 10. Similarly, the inventors have observed particularly good performances of SP6 RNA polymerase when the nucleic sequences set forth in SEQ ID NOs: 166 and 168 were used as promoter sequences for the production of mRNA by IVT. Accordingly, in further particular embodiments, a DNA construct (e.g., a template for IVT of mRNA) is provided that comprises an optimized SP6 promoter having the nucleic acid sequence as set forth in SEQ ID NOs: 166 and 168. The inventors also observed particularly good performances of SP6 RNA polymerase when the nucleic acid sequence set forth in SEQ ID NO: 214 was used as a promoter sequence for the production of mRNA by IVT. Thus, in further particular embodiments, a DNA construct (e.g., a template for IVT of mRNA) is provided that comprises an optimized SP6 promoter having the nucleic acid sequence as set forth in SEQ ID NO: 214.DNA constructs for use with T7 RNA polymerase

[0098] Similar to SP6, both the regions upstream Pi and downstream P2 of the T7 core promoter sequence can influence transcriptional activity of T7 RNA polymerase. Surprisingly, by combining modifications to both regions, the inventors further discovered nucleic acid sequences that can improve mRNA transcript yield and the amount of dsRNA that T7 RNA polymerase generates during IVT.Sanofi Ref: PAT24103-WO-PCT

[0099] In some aspects, an optimized DNA construct for use as an IVT template with T7 RNA polymerase (e.g., a DNA plasmid) is provided that comprises the following nucleic acid sequence:5’-Pi-TAATACGACTCACTATXi-P2-C-3’ (5’-P-[SEQ ID NO: 75]-P2-C-3’) wherein:Pi is an upstream promoter region;Xi is selected from T or A;P2is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and consists of X2GGA-SI, wherein:X2is selected from A or G, and is optional;Si is TAATCGGA and is optional; and the 3’ terminal C is the first nucleotide of the 5’ untranslated region (5’ UTR). The T7 core promoter sequence is shown in bold.7'7 upstream promoter region

[0100] The upstream promoter region Pi is positioned directly upstream of a T7 RNA polymerase core promoter z.e., there are no intervening nucleotides between the upstream promoter sequence and a core promoter sequence. The upstream promoter is typically positioned about 18 nucleotides upstream of the transcriptional start site.

[0101] A suitable upstream promoter sequence Pi comprises at least 5 nucleotides. In some embodiments, the upstream promoter comprises at least 5 nucleotides, at least 7 nucleotides, at least 10 nucleotides. In some embodiments, the upstream promoter sequence comprises 5 nucleotides. In some embodiments, the upstream promoter sequence comprises9 nucleotides. In some embodiments, the upstream promoter sequence comprises10 nucleotides.

[0102] Exemplary upstream promoter sequences Pi for use with the DNA constructs described herein are provided in Table 5.Table 5. T7 upstream promoter sequencesSanofi Ref: PAT24103-WO-PCTT7 core promoter

[0103] Some variation in the T7 core promoter is possible. Exemplary T7 core promoter sequences for use in the DNA constructs include those provided in Table 6.Table 6. T7 core promoter sequences

[0104] A suitable T7 promoter may be combined with a T7 upstream promoter sequence from Table 5 to form the following sequences, wherein the core promoter sequence is shown in bold:GAATTTAATACGACTCACTATA (SEQ ID NO: 88)AGCACCGAAGTAATACGACTCACTATT (SEQ ID NO: 89)AGCACCAAATTAATACGACTCACTATA (SEQ ID NO: 90)GCCTCGAAATTAATACGACTCACTATA (SEQ ID NO: 91)GTCCCTAAATTAATACGACTCACTATA (SEQ ID NO: 92)

[0105] DNA constructs comprising the upstream promoter and core promoter sequences as set forth in SEQ ID NOs: 89 and 90 are particularly useful for IVT of mRNA.7'7 downstream promoter region

[0106] The nucleic acid sequence 3’ directly downstream (P2) of the T7 core promoter, z.e., 3’ adjacent to the core promoter sequence, can also be optimized to improve mRNA yield during IVT. The downstream promoter region P2 typically includes the transcriptional start site for an mRNA transcript that is synthesized during IVT when theSanofi Ref: PAT24103-WO-PCTDNA construct is contacted with a T7 RNA polymerase. The transcriptional start site of the mRNA transcript can be or comprise AGG, AGGA, GGA, or GGG (e.g., GGGATAATCGGA). These transcriptional start sites have been shown to provide high transcription activity in the context of the T7 downstream promoter region of the DNA constructs tested herein. Typically, the first nucleotide of the transcriptional start site is G or A.

[0107] A suitable nucleic sequence for the T7 downstream promoter region may be selected from the sequences listed in Table 7:Table 7. T7 downstream promoter sequences

[0108] The 3 ’-end of a downstream promoter sequence provided in Table 7 may be immediately adjacent to a C nucleotide.

[0109] A downstream promoter sequence provided in Table 7 may be combined with a T7 upstream promoter sequence from Table 5 and a core promoter sequence from Table 6. For example, each of the T7 downstream promoter sequences provided in Table 7 may be combined with any one of the sequences set forth in SEQ ID NOs: 88-92 to form a T7 promoter that can be tested to determine whether the amount of dsRNA comprised in the mRNA transcripts obtainable with a DNA construct comprising the T7 promoter is decreased and / or the mRNA transcript yield is increased relative to mRNA transcripts obtainable with a reference DNA construct. DNA constructs comprising the upstream promoter and core promoter sequences as set forth in SEQ ID NOs: 89 and 90 are particularly useful for IVT of mRNA.Sanofi Ref: PAT24103-WO-PCTExemplary T7 DNA constructs[HO] Further exemplary nucleic sequences for use in the DNA constructs described herein are provided in Table 8 below, wherein the core promoter sequence is shown in bold, the downstream promoter region P2 is underlined, and the first nucleotide of the transcriptional start site of an mRNA transcript is shown in italic. The 3’ terminal X is the first nucleotide of the 5’ UTR of the mRNA transcript. X can be selected from A, T, C, and G.Table 8. Exemplary T7 nucleic acid sequences[Hl] In particular embodiments, the first nucleotide of the chosen 5’ UTR is C.Accordingly, an exemplary nucleic acid sequence may be selected from SEQ ID NOs: 81, 82 and 96-105.

[0112] As shown herein, DNA constructs comprising the nucleic acid sequences set forth in SEQ ID NOs: 81, 82, 96, 97, 99, 100, 103 and 104 yielded more mRNA than a reference DNA construct when used for the production of mRNA by IVT. Moreover, DNA constructs comprising the nucleic acid sequences set forth in SEQ ID NOs: 81, 82, and 96-Sanofi Ref: PAT24103-WO-PCT102, 104 and 105 generated less dsRNA than a reference DNA construct when used for the production of mRNA by IVT.

[0113] In particular, the inventors observed greater mRNA yields and lower amounts of dsRNA compared to a reference DNA template when DNA constructs comprising the nucleic acid sequences set forth in SEQ ID NOs: 81, 82, 96, 97, 99, 100, and 104 were used for the production of mRNA by IVT. Accordingly, in some embodiments, a DNA construct (e.g., a template for IVT of mRNA) is provided that comprises an optimized T7 promoter having the nucleic acid sequence as set forth in SEQ ID NOs: 81, 82, 96, 97, 99, 100, and 104.

[0114] The inventors have observed particularly good performances of T7 RNA polymerase when the nucleic acid sequences set forth in SEQ ID NOs: 81 and 82 were used as promoter sequences for the production of mRNA by IVT. Accordingly, in particular embodiments, a DNA construct (e.g., a template for IVT of mRNA) is provided that comprises an optimized T7 promoter having the nucleic acid sequence as set forth in SEQ ID NOs: 81 and 82.DNA constructs for use with KP34 RNA polymerase

[0115] Similar to both SP6 and T7, both the regions upstream (Pi) and downstream (P2) of the KP34 core promoter sequence can influence transcriptional activity of KP34 RNA polymerase. Surprisingly, by combining modifications to both regions, the inventors discovered nucleic acid sequences that can improve mRNA transcript yield and the amount of dsRNA that KP34 RNA polymerase generates during IVT.

[0116] In some aspects, an optimized DNA construct for use as an IVT template with KP34 RNA polymerase (e.g., a DNA plasmid) is provided that comprises the following nucleic acid sequence:5’-Pi-TAATGTTACAGGAGTA-P2-C-3’ (5’-PI-[SEQ ID NO: 177]-P2-C-3’), wherein:Pi is an upstream promoter region;P2is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and consists of GGX1-S1, wherein:Xi is selected from A or G;Sanofi Ref PAT24103-WO-PCTS1 is GA, GGA, or ATGAAGTTA; and the 3’ terminal C is the first nucleotide of the 5’ untranslated region (5’ UTR).The KP34 core promoter sequence is shown in bold.KP34 upstream promoter region

[0117] The upstream promoter region Pi is positioned directly upstream of a KP34 RNA polymerase core promoter z.e., there are no intervening nucleotides between the upstream promoter sequence and a core promoter sequence. The upstream promoter is typically positioned about 17 nucleotides upstream of the transcriptional start site.

[0118] A suitable upstream promoter sequence Pi comprises at least 5 nucleotides. In some embodiments, the upstream promoter Pi comprises at least about 10 nucleotides or at least about 15 nucleotides. In some embodiments, the upstream promoter sequence Pi comprises about 20 nucleotides. In some embodiments, the upstream promoter sequence Pi comprises about 30 nucleotides. In some embodiments, the upstream promoter sequence Pi comprises about 40 nucleotides. For example, the upstream promoter sequence Pi may be 5- 45 nucleotides, e.g., 15-45 nucleotide, or 20-45 nucleotides.

[0119] Exemplary upstream promoter sequences Pi for use with the DNA constructs described herein are provided in Table 9.Table 9. KP34 upstream promoter sequencesKPS 4 core promoter

[0120] An exemplary KP34 core promoter comprises the following sequence: 5’-TAATGTTACAGGAGTA-3’ (SEQ ID NO: 177).Sanofi Ref: PAT24103-WO-PCT

[0121] A suitable KP34 promoter may be combined with an SP6 upstream promoter sequence from Table 9 to form the following sequences, wherein the core promoter sequence is shown in bold:CATCTAGATAATGTTACAGGAGTA (SEQ ID NO: 182)TTCTTCTTCTGCTAAAAATTTAATGTTACAGGAGTA (SEQ ID NO: 183)CCGTCAAGTGCTAGAATCCCCGTTCTTCTTCTGCTAAAAATTTAATGTTACAGG AGTA (SEQ ID NO: 184)

[0122] DNA constructs comprising the upstream promoter and core promoter sequences as set forth in SEQ ID NOs: 201 and 206 are particularly useful for IVT of mRNA.KP34 downstream promoter region

[0123] The nucleic acid sequence directly downstream (P2) of the KP34 core promoter, z.e., 3’ adjacent to the core promoter sequence, can also be optimized to improve mRNA yield during IVT. The downstream promoter region P2 typically includes the transcriptional start site for an mRNA transcript that is synthesized during IVT when the DNA construct is contacted with an KP34 RNA polymerase. The transcriptional start site of the mRNA transcript can be or comprise GGG or GGA. These transcriptional start sites have been shown to provide high transcription activity in the context of the KP34 downstream promoter region of the DNA constructs tested herein. Typically, the first nucleotide of the transcriptional start site is G.

[0124] A suitable nucleic sequence for the KP34 downstream promoter region may be selected from the sequences listed in Table 10:Table 10. KP34 downstream promoter sequencesSanofi Ref: PAT24103-WO-PCT

[0125] The 3 ’-end of a downstream promoter sequence provided in Table 10 may be immediately adjacent to a C nucleotide.

[0126] A downstream promoter sequence provided in Table 10 may be combined with a KP34 upstream promoter sequence from Table 9 and a core promoter sequence (e.g., TAATGTTACAGGAGTA (SEQ ID NO: 177). For example, each of the KP34 downstream promoter sequences provided in Table 10 may be combined with any one of the sequences set forth in SEQ ID NOs: 182-184 to form a KP34 promoter that can be tested to determine whether the amount of dsRNA comprised in the mRNA transcripts obtainable with a DNA construct comprising the KP34 promoter is decreased and / or the mRNA transcript yield is increased relative to mRNA transcripts obtainable with a reference DNA construct. DNA constructs comprising the upstream promoter and core promoter sequences as set forth in SEQ ID NOs: 201 and 206 are particularly useful for IVT of mRNA.Exemplary KP34 DNA constructs

[0127] In some embodiments, an optimized DNA construct for use as an IVT template with KP34 RNA polymerase (e.g., a DNA plasmid) is provided that comprises the following nucleic acid sequence:5’-Pi-TAATGTTACAGGAGTA-P2-C-3’ (5’-PI-[SEQ ID NO: 177]-P2-C-3’), wherein:Pi is an upstream promoter region is selected from CATCTAGA, TTCTTCTTCTGCTAAAAATT (SEQ ID NO: 179), andCCGTCAAGTGCTAGAATCCCCGTTCTTCTTCTGCTAAAAATT (SEQ ID NO: 180), and P2is selected from GGGGA and GGAATGAAGTTA (SEQ ID NO: 181);P2is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and is selected from GGGGA and GGAATGAAGTTA (SEQ ID NO: 181); and the 3’ terminal C is the first nucleotide of the 5’ untranslated region (5’ UTR).

[0128] In some embodiments, Pi is CATCTAGA and P2is GGGGA. In some embodiments, Pi is TTCTTCTTCTGCTAAAAATT (SEQ ID NO: 179) and P2is GGAATGAAGTTA (SEQ ID NO: 181). In some embodiments, Pi is CCGTCAAGTGCTAGAATCCCCGTTCTTCTTCTGCTAAAAATT (SEQ ID NO: 180) and P2is GGAATGAAGTTA (SEQ ID NO: 181).Sanofi Ref: PAT24103-WO-PCT

[0129] Further exemplary nucleic sequences for use in the DNA constructs described herein are provided in Table 11 below, wherein the core promoter sequence is shown in bold, the downstream promoter region P2 is underlined, and the first nucleotide of the transcriptional start site of an mRNA transcript is shown in italic. The 3’ terminal X is the first nucleotide of the 5’ UTR of the mRNA transcript. X can be selected from A, T, C, and G.Table 11. Exemplary KP34 nucleic acid sequences

[0130] In particular embodiments, the first nucleotide of the chosen 5’ UTR is C. Accordingly, an exemplary nucleic acid sequence may be selected from SEQ ID NOs: 192, 193, 196, 198, 201, 203, and 206.

[0131] Additional exemplary nucleic DNA constructs include DNA constructs which comprise the nucleic acid sequences set forth in SEQ ID NOs: 197.

[0132] As shown herein, DNA constructs comprising the nucleic acid sequences set forth in SEQ ID NOs: 192, 193, 194, 197, 201 and 206 yielded more mRNA than a reference DNA construct when used for the production of mRNA by IVT. Moreover, each of these DNA constructs generated less dsRNA than a reference DNA construct when used for the production of mRNA by IVT.Sanofi Ref: PAT24103-WO-PCT

[0133] In particular, the inventors observed greater mRNA yields and lower amounts of dsRNA compared to a reference DNA template when DNA constructs comprising the nucleic acid sequences set forth in SEQ ID NOs: 192, 193, 201, and 206were used for the production of mRNA by IVT. Accordingly, in some embodiments, a DNA construct (e.g., a template for IVT of mRNA) is provided that comprises an optimized KP34 promoter having the nucleic acid sequence as set forth in SEQ ID NOs: 192, 193, 201, and 206.

[0134] The inventors have observed particularly good performances of KP34 RNA polymerase when the nucleic sequences set forth in SEQ ID NOs: 201 and 206 were used as promoter sequences for the production of mRNA by IVT. Accordingly, in particular embodiments, a DNA construct (e.g., a template for IVT of mRNA) is provided that comprises an optimized KP34 promoter having the nucleic acid sequence as set forth in SEQ ID NOs: 201 and 206. mRNA transcript coding region

[0135] The promoter sequence in a DNA construct as described herein is typically operably linked to a nucleic acid sequence encoding an mRNA transcript. The nucleotide sequence encoding an mRNA transcript usually comprises a 5’ untranslated region (5’ UTR), a coding region for a peptide, polypeptide, or protein of interest, and a 3’ untranslated region (3’ UTR).

[0136] In some embodiments, the 5’ untranslated region includes one or more elements that affect stability or translation of the mRNA transcript, for example, an iron responsive element. In some embodiments, a 5’ untranslated region may be between about 50 and 500 nucleotides in length.

[0137] In some embodiments, a 3’ untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect stability or location of the mRNA transcript in a cell, or one or more binding sites for miRNAs. In some embodiments, a 3’ untranslated region may be between 50 and 500 nucleotides in length or longer.

[0138] In some embodiments, the nucleotide sequence comprises a 5’ UTR different from the 5’ UTR present in a naturally occurring mRNA encoding the polypeptide of interest.

[0139] In some embodiments, the nucleotide sequence comprises a 3’ UTR different from the 3’ UTR present in a naturally occurring mRNA encoding the polypeptide of interest.Sanofi Ref: PAT24103-WO-PCT

[0140] For example, suitable 5’ and 3’ UTRs are described in W02012 / 075040, which is incorporated herein by reference.

[0141] In certain embodiments, the 5’ and / or 3’ UTR sequences can be derived from a naturally occurring mRNA transcript which are stable (e.g., globin, actin, GAPDH, tubulin, histone, or citric acid cycle enzymes) to increase the stability of the mRNA transcript. For example, a 5’ UTR sequence may include a partial sequence of a CMV immediate-early 1 (IE1) gene, or a fragment thereof, to improve the nuclease resistance and / or improve the halflife of the mRNA transcript. Also contemplated is the inclusion of a sequence encoding human growth hormone (hGH), or a fragment thereof, to the 3’ end or untranslated region of the mRNA transcript. Exemplary 5’ UTRs include a sequence derived from a CMV immediate-early 1 (IE1) gene (U.S. Publication Nos. 2014 / 0206753 and 2015 / 0157565, each of which is incorporated herein by reference), or the sequences provided in Example 1 of U.S. Publication No. 2016 / 0151409, incorporated herein by reference.

[0142] In various embodiments, the 5’ UTR may be derived from the 5’ UTR of a TOP gene. TOP genes are typically characterized by the presence of a 5 ’-terminal oligopyrimidine (TOP) tract. Furthermore, most TOP genes are characterized by growth- associated translational regulation. However, TOP genes with a tissue specific translational regulation are also known. In certain embodiments, the 5’ UTR derived from the 5’ UTR of a TOP gene lacks the 5’ TOP motif (the oligopyrimidine tract) (e.g., U.S. Publication Nos. 2017 / 0029847, 2016 / 0304883, 2016 / 0235864, and 2016 / 0166710, each of which is incorporated herein by reference).

[0143] In certain embodiments, the 5’ UTR is derived from a ribosomal protein Large 32 (L32) gene (U.S. Publication No. 2017 / 0029847, supra).

[0144] In certain embodiments, the 5’ UTR is derived from the 5’ UTR of a hydroxysteroid (17-b) dehydrogenase 4 gene (HSD17B4) (U.S. Publication No. 2016 / 0166710, supra).

[0145] In certain embodiments, the 5’ UTR is derived from the 5’ UTR of an ATP5A1 gene (U.S. Publication No. 2016 / 0166710, supra).

[0146] In some embodiments, an internal ribosome entry site (IRES) is used instead of a 5’ UTR.Sanofi Ref: PAT24103-WO-PCT

[0147] The DNA constructs described herein may comprise a 5’ UTR sequence wherein the first base of the 5’ UTR is C, e.g., a partial sequence of a CMV IE1 gene. In a particular embodiment, the promoter sequence in a DNA construct described herein further comprises the nucleic acid sequence CAGATCGCCTGGAGACGC (SEQ ID NO: 116) 3’ to the downstream promoter region P2. The skilled person is aware that alternative 5’ UTRs may be used in accordance with the present invention.

[0148] An exemplary 5’ UTR for use with the DNA construct disclosed herein has the following nucleic acid sequence:CAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCG GGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCC GTGCCAAGAGTGACTCACCGTCCTTGACACG (SEQ ID NO: 117).Manufacturing mRNA by in vitro transcription (IVT)

[0149] The invention also relates to methods for manufacturing mRNA by IVT. Such methods comprise a) providing a DNA construct as described herein and b) contacting the DNA construct with an RNA polymerase under conditions suitable for obtaining mRNA transcripts.

[0150] Suitable conditions for synthesizing mRNA transcripts via IVT are well- known - see, e.g., US Patent Publication No. US 2018 / 0258423 and International Patent Publication No. WO 2021 / 168052A1, which are incorporated herein by reference - and can be used to practice the present invention. Briefly, IVT is typically performed with a reaction mixture comprising a DNA construct as template, a pool of ribonucleotide triphosphates, a buffering reagent (that may include DTT), and one or more salts (e.g., MgCh and NaCl). A typical IVT reaction buffer may also include spermidine. The exact conditions will vary according to the specific application.Linearization

[0151] mRNA transcripts are typically transcribed from DNA constructs such as plasmids which may be linearized before IVT using a restriction enzyme. Any suitable restriction enzyme may be used. Generally, the restriction enzyme is a type II restriction enzyme, such as a type IIP or type IIS restriction enzyme. In some embodiments, the restriction enzyme is EcoRI, BciVI, Spel, Xbal, Ndel, Aflll, Sacl, Kpnl, Smal, BamHI, Sail,Sanofi Ref: PAT24103-WO-PCTSbfl, Pstl, BspQI, SapI, or Hindlll. In some embodiments, the linearized DNA template has blunt ends.Template concentration

[0152] The concentration of the DNA construct in an IVT reaction may range from 0.05 mg / mL to 0.5 mg / mL. In some embodiments, the concentration of the DNA construct is 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL, 0.2 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.23 mg / mL, 0.24 mg / mL, 0.25 mg / mL, 0.26 mg / mL, 0.27 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.3 mg / mL, 0.31 mg / mL, 0.32 mg / mL, 0.33 mg / mL, 0.34 mg / mL, 0.35 mg / mL, 0.36 mg / mL, 0.37 mg / mL, 0.38 mg / mL, 0.39 mg / mL, 0.4 mg / mL, 0.41 mg / mL, 0.42 mg / mL, 0.43 mg / mL, 0.44 mg / mL, 0.45 mg / mL, 0.46 mg / mL, 0.47 mg / mL, 0.48 mg / mL, 0.49 mg / mL, or 0.5 mg / mL.Polymerase concentration

[0153] The concentration of the SP6 RNA polymerase in an IVT reaction may range from 0.01 to 0.5 mg / mL. In some embodiments, the concentration of the SP6 RNA polymerase is 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL, 0.2 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.23 mg / mL, 0.24 mg / mL, 0.25 mg / mL, 0.26 mg / mL, 0.27 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.3 mg / mL, 0.31 mg / mL, 0.32 mg / mL, 0.33 mg / mL, 0.34 mg / mL, 0.35 mg / mL, 0.36 mg / mL, 0.37 mg / mL, 0.38 mg / mL, 0.39 mg / mL, 0.4 mg / mL, 0.41 mg / mL, 0.42 mg / mL, 0.43 mg / mL, 0.44 mg / mL, 0.45 mg / mL, 0.46 mg / mL, 0.47 mg / mL, 0.48 mg / mL, 0.49 mg / mL, or 0.5 mg / mL.

[0154] The concentration of the T7 RNA polymerase in an IVT reaction may range from 0.01 to 0.5 mg / mL. In some embodiments, the concentration of the T7 RNA polymerase is 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL, 0.2 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.23 mg / mL, 0.24 mg / mL, 0.25 mg / mL, 0.26 mg / mL, 0.27 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.3 mg / mL, 0.31 mg / mL, 0.32 mg / mL, 0.33 mg / mL, 0.34 mg / mL, 0.35 mg / mL, 0.36 mg / mL, 0.37 mg / mL, 0.38 mg / mL, 0.39 mg / mL, 0.4 mg / mL, 0.41 mg / mL,Sanofi Ref: PAT24103-WO-PCT0.42 mg / mL, 0.43 mg / mL, 0.44 mg / mL, 0.45 mg / mL, 0.46 mg / mL, 0.47 mg / mL, 0.48 mg / mL, 0.49 mg / mL, or 0.5 mg / mL.

[0155] The concentration of the KP34 RNA polymerase in an IVT reaction may range from 0.01 to 0.5 mg / mL. In some embodiments, the concentration of the KP34 RNA polymerase is 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, 0.04 mg / mL, 0.05 mg / mL, 0.06 mg / mL, 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, 0.1 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, 0.14 mg / mL, 0.15 mg / mL, 0.16 mg / mL, 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL, 0.2 mg / mL, 0.21 mg / mL, 0.22 mg / mL, 0.23 mg / mL, 0.24 mg / mL, 0.25 mg / mL, 0.26 mg / mL, 0.27 mg / mL, 0.28 mg / mL, 0.29 mg / mL, 0.3 mg / mL, 0.31 mg / mL, 0.32 mg / mL, 0.33 mg / mL, 0.34 mg / mL, 0.35 mg / mL, 0.36 mg / mL, 0.37 mg / mL, 0.38 mg / mL, 0.39 mg / mL, 0.4 mg / mL, 0.41 mg / mL, 0.42 mg / mL, 0.43 mg / mL, 0.44 mg / mL, 0.45 mg / mL, 0.46 mg / mL, 0.47 mg / mL, 0.48 mg / mL, 0.49 mg / mL, or 0.5 mg / mL.Buffering reagent

[0156] IVT typically takes place in the presence of a buffering agent. In some embodiments, the buffering agent is selected from Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, and sodium phosphate. In one embodiment, the buffering agent is Tris-HCl. In a further embodiment, Tris-HCl is present at a concentration of less than 40 mM. In some embodiments, Tris-HCl is present at a concentration of about 25 mM. pH

[0157] The pH of the reaction mixture may be between about 6 to 8.5, from 6.5 to 8.0, from 7.0 to 7.5, and in some embodiments, the pH is 7.5.Temperature

[0158] IVT may take place at a temperature from about 37°C to about 42°C. In some embodiments, IVT takes place at a temperature of about 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C.Co-factor

[0159] Most polymerases include a divalent cation as a co-factor. Accordingly, in some embodiments, IVT takes place in the presence of divalent cation, e.g., Mn2+or Mg2+.Sanofi Ref: PAT24103-WO-PCT

[0160] In particular embodiments, IVT takes place in the presence of magnesium chloride (MgCh). In some embodiments, the MgCh concentration is greater than 20 mM and less than 40 mM (e.g., less than 30 mM). In some embodiments, the MgCh concentration is about 25 mM.Salt

[0161] To provide optimal conditions for IVT, a salt is typically included in the reaction mixture. In some embodiments, IVT takes place in the presence of sodium chloride (NaCl). In some embodiments, the NaCl concentration is less than 20 mM. In some embodiments, the NaCl concentration is between 0.05 mM and 15 mM. In some embodiments, the NaCl concentration is between 0.1 mM and 10 mM. In some embodiments, the NaCl concentration is between 0.1 mM and 5 mM. In some embodiments, the NaCl concentration is between 0.1 mM and 2 mM. In some embodiments, the NaCl concentration is between 0.1 mM and 1 mM. In some embodiments, the NaCl concentration is about 0.5 mM.Ribonucleotides

[0162] The concentration of each ribonucleotide (e.g., ATP, UTP, GTP, and CTP) in an IVT reaction may be between about 0.1 mM and about 10 mM, e.g., between about 1 mM and about 10 mM, between about 2 mM and about 10 mM, between about 3 mM and about 10 mM, between about 1 mM and about 8 mM, between about 1 mM and about 6 mM, between about 3 mM and about 10 mM, between about 3 mM and about 8 mM, between about 3 mM and about 6 mM, between about 4 mM and about 7 mM, between about 4 mM and about 6 mM, or between about 4 mM and about 5 mM. In some embodiments, each ribonucleotide is at about 5 mM in a reaction mixture.

[0163] In some embodiments, the total concentration of ribonucleotide (for example, ATP, GTP, CTP, and UTPs combined) used in the reaction is between about 1 mM and about 40 mM. In some embodiments, the total concentration of ribonucleotide (for example, ATP, GTP, CTP, and UTPs combined) used in the reaction is between about 1 mM and about 30 mM, between about 1 mM and about 28 mM, between about 1 mM and about 25 mM, or between about 1 mM and about 20 mM.

[0164] In some embodiments, the total ribonucleotide concentration is less than about 30 mM. In some embodiments, the total ribonucleotide concentration is less than about 25Sanofi Ref PAT24103-WO-PCT mM. In some embodiments, the total ribonucleotide concentration is less than about 20 mM. In some embodiments, the total ribonucleotide concentration is less than about 15 mM. In some embodiments, the total ribonucleotide concentration is less than about 10 mM.Modified RNA

[0165] In some embodiments, mRNA transcripts are synthesized with one or more modifications (i.e., as modified mRNA), wherein the modification refers to chemical or biological modifications comprising backbone modifications, sugar modifications, or base modifications. A backbone modification is a modification in which phosphates of the backbone of the nucleotides of the RNA are chemically modified (e.g., phosphorothioates and 5'-A-phosphoramidite linkages). A sugar modification is a chemical modification of the sugar of the nucleotides of the RNA (e.g., 2’-fluororibose, ribose, 2’ -deoxyribose, arabinose, and hexose). A base modification is a chemical modification of the base moiety of the nucleotides of the RNA.

[0166] In a particular embodiment, modified mRNA comprises a modified ribonucleotide, such as ribonucleotide analogue (e.g., adenosine analogue, guanosine analogue, cytidine analogue, and / or uridine analogue). The presence of a modified ribonucleotide may render the mRNA more stable and / or less reactogenic than a control mRNA with the same sequence but containing only naturally occurring ribonucleotides.

[0167] The modified ribonucleotide typically takes the place of a naturally occurring nucleotide. Accordingly, mRNA transcripts produced by the methods described herein may comprise both unmodified and modified ribonucleotides. Such mRNA transcripts can be prepared by including a modified ribonucleoside in the IVT reaction mixture, typically in place of a naturally occurring ribonucleoside (e.g., N1 -methylpseudouridine in place of uridine). This results in mRNA transcripts in which 100% of the naturally occurring ribonucleotide are replaced by a corresponding modified ribonucleotide (e.g., 100% of the uridines are replaced with Nl-methyl-pseudouri dine). In some embodiments, only a portion of the naturally occurring ribonucleoside (e.g., at least 1%, 5%, 10%, 15%, 20% or 25% of the naturally occurring ribonucleoside) is replaced with a modified ribonucleoside. In some embodiments, one or more naturally occurring ribonucleosides is replaced with a modified ribonucleoside. For example, two or more ribonucleosides may be modified ribonucleosides (e.g., uridines may be replaced with 2-thio-uridine and cytidines may be replaced with 5-Sanofi Ref: PAT24103-WO-PCT methylcytidine). For example, 25% of the uridines may be replaced with 2-thio-uridine and / or 25% of cytidine residues may be replaced with 5-methylcytidine.

[0168] In some embodiments, the modified ribonucleoside comprises at least one modification selected from a modified sugar, and a modified nucleobase relative to the corresponding naturally occurring ribonucleoside.

[0169] The modified ribonucleoside can be a modified uridine, cytidine, adenosine, or guanosine. Some exemplary chemical modifications of ribonucleosides in mRNA include, e.g., pyridine-4-one ribonucleoside, 5 -aza-uridine, 2-thio-5 -aza-uridine, 2-thiouridine, 4-thio pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3 -methyluridine, 5 -carb oxy methyl uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2 -thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio- 1 -methylpseudouridine, 2-thio-l-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 2-thio-l- methyl-l-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2- thio dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio- uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, 5-aza- cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4- methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1-deaza- pseudoisocytidine, 1 -methyl- 1 -deaza pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl -cytidine, 4-methoxy -pseudoisocytidine, 4-m ethoxy- 1-methyl- pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza adenine, 7-deaza-8 -azaadenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1 -methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyladenosine, 7-methyladenine, 2-methylthioadenine, 2-methoxyadenine, inosine, 1- methyl-inosine, wyosine, wybutosine, 7-deaza guanosine, 7-deaza-8 -aza-guanosine, 6-thio guanosine, 6-thio-7-deazaguanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-m ethylguanosine, 7-methylinosine, 6-methoxy guanosine, 1 -methylguanosine, N2-Sanofi Ref PAT24103-WO-PCT methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio- guanosine.

[0170] In some embodiments, the modified ribonucleoside is a modified uridine selected from pseudouridine, pyridine-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2- thio-5-aza-uridine, 2-thio-uridine, 4-thio-uridine, 4-thio-pseudouridine, 2-thio- pseudouridine, 5-hydroxy uridine, 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodom uridine or 5-bromo uridine), 3-methyl uridine, 5-methoxy-uridine, uridine-5-oxyacetic acid, uridine-5-oxyacetic acid methyl ester, 5-carboxymethyl-uridine, 1 -carboxymethylpseudouridine, 5-carboxyhydroxymethyl uridine, 5-carboxyhydroxymethyl-uridine methyl ester, 5-methoxycarbonylmethyluridine, 5-methoxycarbonylmethyl-2-thio-uridine, 5- aminomethyl-2-thiouridine, 5-methylaminomethyl uridine, 5-methylaminomethyl-2-thio- uridine, 5-methylaminomethyl-2-selenouridine, 5-carbamoylmethyl-uridine, 5- carboxymethylaminomethyl-uridine, 5-carboxymethylaminomethyl-2 -thio-uridine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine, 1-taurinom ethyl pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-pseudouridine, 5- methyl-uridine (m5U, e.g., having the nucleobase deoxythymine), 1-methyl-pseudouridine, 5-methyl-2-thio-uridine, l-methyl-4-thio-pseudouridine, 4-thio- 1-methyl-pseudouridine, 3- methyl-pseudouridine), 2 -thio- 1-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouri dine, 2- thio-l-methyl-l-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine, 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, 4-m ethoxy -2 -thio-pseudouri dine, Nl-methyl-pseudouridine, 3-(3-amino-3- carboxypropyl) uridine, l-methyl-3-(3-amino-3-carboxypropyl) pseudouridine, 5- (isopentenylaminomethyl) uridine, 5-(isopentenylaminomethyl)-2-thio-uridine, alpha-thio- uridine, 2’-O-methyl uridine, 5,2’-O-dimethyl uridine, 2’-O-methyl-pseudouridine, 2-thio- 2’-O-methyl uridine, 5-methoxycarbonylmethyl-2’-O-methyl uridine, 5-carbamoylmethyl- 2’-O-methyl uridine, 5-carboxymethylaminomethyl-2’-O-methyl uridine, 3,2’-O- dimethyl uridine, 5-(isopentenylaminomethyl)-2’-O-methyl uridine, 1 -thiouridine, deoxythymidine, 2’-F-ara-uridine, 2’-F-uridine, 2’-OH-ara-uridine, 5-(2- carbomethoxyvinyl) uridine, and 5-[3-(l-E-propenylamino) uridine.

[0171] In some embodiments, the modified uridine is selected from Nl- methylpseudouridine, pseudouridine, 2-thiouridine, 4’ -thiouridine, 2-thio-l -methyl- 1-deaza-Sanofi Ref PAT24103-WO-PCT pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy -pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5 -methyluridine, 5- methoxyuridine, and 2’-O-methyl uridine. In some embodiments, the modified uridine is Nl- methylpseudouridine.

[0172] In some embodiments, the modified ribonucleoside is a modified cytidine selected from 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3 -methylcytidine, N4-acetyl cytidine, 5-formyl-cytidine, N4-methylcytidine, 5-methylcytidine, 5-halo cytidine (e.g., 5- iodo cytidine), 5-hydroxy methylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methylcytidine, 4-thio- pseudoisocytidine, 4-thio-l-methyl-pseudoisocytidine, 4-thio-l -methyl- 1-deaza- pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio zebularine, 2-thio-zebularine, 2-methoxy cytidine, 2- methoxy-5-methylcytidine, 4-methoxy pseudoisocytidine, 4-methoxy- 1-methyl- pseudoisocytidine, lysidine, alpha-thio-cytidine, 2’-O-methylcytidine, 5,2’-O-dimethyl cytidine, N4-acetyl-2’-O-methylcytidine, N4,2’-O-dimethyl cytidine, 5-formyl-2’-O- methylcytidine, N4,N4,2’-O-trimethyl cytidine, 1 -thio-cytidine, 2’-F-ara-cytidine, 2’- F cytidine, and 2’-OH-ara-cytidine.

[0173] In some embodiments, the modified ribonucleoside is a modified pyrimidine ribonucleoside. In some embodiments, the modified ribonucleoside is selected from pseudouridine, N1 -methylpseudouridine, 5-methylcytidine, 5-methoxyuridine, and any combination thereof. In some embodiments, both cytidine and uridine are replaced with modified nucleosides (e.g., N1 -methylpseudouridine and 5-methylcytidine).

[0174] In some embodiments, the modified ribonucleoside is a modified purine ribonucleoside. In some embodiments, the modified ribonucleoside is a modified adenosine selected from 2-amino purine, 2,6-diamino purine, 2-amino-6-halo purine (e.g., 2-amino-6- chloro purine), 6-halo purine (e.g., 6-chloro purine), 2-amino-6-m ethyl purine, 8-azido adenosine, 7-deaza-adenine, 7-deaza-8-aza adenine, 7-deaza-2-amino purine, 7-deaza-8-aza- 2-amino purine, 7-deaza-2,6-diamino purine, 7-deaza-8-aza-2,6-diamino purine, 1- methyladenosine, 2-methyl adenine, N6-methyladenosine, 2-methylthio-N6- methyladenosine, N6-isopentenyl adenosine, 2-methylthio-N6-isopentenyl adenosine, N6-Sanofi Ref PAT24103-WO-PCT(cis-hydroxyisopentenyl) adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyl adenosine, N6-threonylcarbamoyl adenosine, N6-methyl-N6- threonylcarbamoyl adenosine, 2-methylthio-N6-threonylcarbamoyl adenosine, N6,N6- dimethyl adenosine, N6-hydroxynorvalylcarbamoyl adenosine, 2-methylthio-N6- hydroxynorvalylcarbamoyl adenosine, N6-acetyl adenosine, 7-methyladenine, 2-methylthio- adenine, 2-methoxyadenine, alpha-thio-adenosine, 2’-O-methyladenosine, N6,2’-O-dimethyl adenosine, N6,N6,2’-O-trimethyl adenosine, l,2’-O-dimethyl adenosine, 2’-O- ribosyl adenosine (phosphate), 2-amino-N6-methyl purine, 1 -thio-adenosine, 8-azido- adenosine, 2’-F-ara-adenosine, 2’-F adenosine, 2’-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl) adenosine.

[0175] In some embodiments, the modified ribonucleoside is a modified guanosine selected from inosine, 1 -methyl inosine, wyosine, methylwyosine, 4-dem ethyl wyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, undermodified hydroxywybutosine, 7-deaza-guanosine, queuosine, epoxyqueuosine, galactosyl queuosine, mannosyl queuosine, 7-cyano-7-deaza-guanosine, 7-aminomethyl-7-deaza-guanosine, archaeosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio- 7-deaza-8 -aza-guanosine, 7-m ethylguanosine, 6-thio-7-methylguanosine, 7-methyl inosine, 6-m ethoxy guanosine, 1 -methylguanosine, N2-methyl-guanosine, N2,N2-dimethyl guanosine, N2,7-dimethyl guanosine, N2,N2,7-dimethyl guanosine, 8-oxo-guanosine, 7-methyl-8-oxo- guanosine, 1 -methylguanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio- guanosine, alpha-thio-guanosine, 2 ’-O-m ethylguanosine, N2-methyl-2’-O-methylguanosine, N2,N2-dimethyl-2’-O-methylguanosine, l-methyl-2’-O-methylguanosine, N2,7-dimethyl-2’- O-methylguanosine, 2’-O-methyl inosine, l,2’-O-dimethyl inosine, 2’-O-ribosyl guanosine (phosphate), 1 -thio-guanosine, O6-methylguanosine, 2’ -F-ara guanosine, and 2’- F guanosine.

[0176] In some embodiments, the modified ribonucleoside is a ribonucleoside analogue selected from 2-aminoadenosine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl- uridine, C5-propynyl-cytidine, C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., Nl- methylpseudouridine), 2-thiouridine, and 2-thiocytidine. See, e.g., U.S. Patent No. 8,278,036 or WO 2011 / 012316 for a discussion of 5-methylcytidine, pseudouridine, and 2-thio-uridine and their incorporation into mRNA.Sanofi Ref PAT24103-WO-PCT

[0177] In some embodiments, the modified ribonucleoside is selected from pseudouridine, N1 -methylpseudouridine, 2-thiouridine, 4’ -thiouridine, 5-methylcytidine, 2- thio-1 -m ethyl- 1 -deaza-pseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5 -aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2- thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-l-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methyluridine, 5-methoxyuridine, and 2’-O-methyl uridine.

[0178] In some embodiments, the modified ribonucleoside is selected from pseudouridine, N1 -methylpseudouridine, 5-methylcytidine, 5-methoxyuridine, 2’-O- methyladenosine, 2’-O-methyluridine, 2’-O-methylcytidine, and 2’-O-methylguanosine.

[0179] In some embodiments, mRNA transcripts are provided in which 25% of uracil residues are 2-thio-uracil and 25% of cytosine residues are 5-methylcytosine. Teachings for the use of such modified RNA are disclosed in US Patent Publication US 2012 / 0195936 and international publication WO 2011 / 012316, both of which are hereby incorporated by reference in their entirety. In some embodiments, the in vitro transcribed mRNA may be RNA where 100% of uracil residues are N1 -methylpseudouracil (also occasionally referred to as 1 -methylpseudouracil).Time

[0180] The length of an IVT reaction may depend on the length of the mRNA transcript. In a typical embodiment, the mRNA transcript comprises at least 500 ribonucleotides. In some embodiments, the mRNA transcript comprises about 500 to about 20,000 ribonucleotides. In some embodiments, the mRNA transcript comprises about 700 to about 15,000 ribonucleotides. In some embodiments, the mRNA transcript comprises about 800 to about 12,000 ribonucleotides. In some embodiments, the mRNA transcript comprises about 1,000 to about 10,000 ribonucleotides. In some embodiments, the mRNA transcript comprises about 1,500 to about 7,000 ribonucleotides. In some embodiments, the mRNA transcript comprises about 2,000 to about 5,000 ribonucleotides.

[0181] Accordingly, the period over which IVT may take place to synthesize mRNA can vary widely. In some embodiments, IVT takes place over a period of about thirty minutes to about six hours. In some embodiments, IVT takes place over a period of about sixty to about ninety minutes.Sanofi Ref: PAT24103-WO-PCT

[0182] IVT can be terminated by removing the DNA template, e.g., through the addition of DNase I and a suitable buffer. For example, the polymerase reaction can be quenched by addition of DNase I and a DNase I buffer (100 mM Tris-HCl, 5 mM MgCh and 25 mM CaCh, pH 7.6 at lOx) to facilitate digestion of the double-stranded DNA template in preparation for purification. mRNA transcript yield and amount o f dsRNA

[0183] The present invention is based on the identification of optimized SP6, T7, and KP34 promoter sequences that increase mRNA transcript yield and / or decrease the amount of dsRNA contamination in the mRNA transcripts when used in DNA constructs for the manufacture of mRNA by IVT. Typically, the DNA constructs described herein advantageously yield more mRNA comprising less dsRNA.

[0184] The advantages of the DNA constructs described herein are particularly noticeable in comparison to reference DNA constructs that only include a core promoter sequence to drive transcription. A suitable reference DNA construct to assess the performance of the DNA constructs with optimized SP6 promoter sequences described herein may comprise the nucleic acid sequence 5’-ATTTAGGTGACACTATAGGAC-3’ (SEQ ID NO: 2; the core promoter is shown in bold), z.e., without an upstream promoter region Pi, wherein the 3’ terminal four nucleic acids of the nucleic acid sequence comprise the transcriptional start site of the mRNA transcript, wherein the first nucleotide of the transcriptional start site of an mRNA transcript is in italics. A suitable reference DNA construct to assess the performance of the DNA constructs with optimized T7 promoter sequences described herein may comprise the nucleic acid sequence 5’-TAATACGACTCACTATAGGAC-3’ (SEQ ID NO: 76; the core promoter is shown in bold), i.e. , without an upstream promoter region Pi, wherein the 3 ’ terminal four nucleic acids of the nucleic acid sequence comprise the transcriptional start site of the mRNA transcript, wherein the first nucleotide of the transcriptional start site of an mRNA transcript is in italics. A suitable reference DNA construct to assess the performance of the DNA constructs with optimized KP34 promoter sequences described herein may comprise the nucleic acid sequence 5 -CATCTAGATAATGTTACAGGAGTAGGAC-3’ (SEQ ID NO: 178; the core promoter is shown in bold), z.e., a known KP34 promoter, wherein the 3’ terminal four nucleic acids of the nucleic acid sequence comprise the transcriptional start site of the mRNASanofi Ref: PAT24103-WO-PCT transcript, wherein the first nucleotide of the transcriptional start site of an mRNA transcript is in italics.

[0185] In some embodiments, mRNA transcript yield is increased by at least 10%, at least 15%, at least 20%, at least 30%, at least 40%, or at least 50% relative to the mRNA transcript yield obtained with the reference DNA construct. In some embodiments, the mRNA transcript yield may be increased by at least 75%, 100%, 125%, or 150%. In some embodiments, mRNA transcript yield obtained by an SP6 DNA construct disclosed herein is increased by at least 15% relative to the mRNA transcript yield obtained with the reference DNA construct (e.g., as set forth in SEQ ID NO: 2). In some embodiments, mRNA transcript yield obtained by an SP6 DNA construct disclosed herein is increased by at least 20% relative to the mRNA transcript yield obtained with the reference DNA construct (e.g., as set forth in SEQ ID NO: 2). In some embodiments, the amount of abortive transcripts is reduced relative the amount of abortive transcripts obtained with a reference DNA construct.

[0186] In some embodiments, mRNA transcript yield obtained by an T7 DNA construct disclosed herein is increased by at least 40% relative to the mRNA transcript yield obtained with the reference DNA construct (e.g., as set forth in SEQ ID NO: 76). In some embodiments, mRNA transcript yield obtained by an T7 DNA construct disclosed herein is increased by at least 50% relative to the mRNA transcript yield obtained with the reference DNA construct (e.g., as set forth in SEQ ID NO: 76).

[0187] In some embodiments, mRNA transcript yield obtained by a KP34 DNA construct disclosed herein is increased by at least 20% relative to the mRNA transcript yield obtained with the reference DNA construct (e.g., as set forth in SEQ ID NO: 178). In some embodiments, mRNA transcript yield obtained by a KP34 DNA construct disclosed herein is increased by at least 30% relative to the mRNA transcript yield obtained with the reference DNA construct (e.g., as set forth in SEQ ID NO: 178).

[0188] In some embodiments, mRNA transcript yield obtained by a KP34 DNA construct disclosed herein is increased by at least 40% relative to the mRNA transcript yield obtained with the reference DNA construct (e.g, as set forth in SEQ ID NO: 178). In some embodiments, mRNA transcript yield obtained by a KP34 DNA construct disclosed herein is increased by at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% relative to the mRNA transcript yield obtained with the reference DNA construct (e.g, as set forth in SEQ ID NO: 178). In some embodiments, mRNA transcript yield obtained by a KP34 DNASanofi Ref: PAT24103-WO-PCT construct disclosed herein is increased by at least 100% (z.e., a 2-fold increase) relative to the mRNA transcript yield obtained with the reference DNA construct (e.g., as set forth in SEQ ID NO: 178). In some embodiments, mRNA transcript yield obtained by a KP34 DNA construct disclosed herein is increased by at least 150% (z.e., a 3-fold increase) relative to the mRNA transcript yield obtained with the reference DNA construct (e.g., as set forth in SEQ ID NO: 178).

[0189] Various methods can be used to characterize in vitro synthesized mRNA transcripts. mRNA transcripts may be detected and quantified using any methods available in the art, e.g., using blotting (e.g., dot blot), capillary electrophoresis, chromatography, fluorescence, gel electrophoresis, HPLC, silver stain, spectroscopy, ultraviolet (UV), or UPLC, UV absorption spectroscopy with separation by capillary electrophoresis, or any combination thereof. In some embodiments, mRNA transcripts are first denatured by a glyoxal dye before analysis by gel electrophoresis (“glyoxal gel electrophoresis”). In some embodiments, mRNA transcript yield is determined by UV spectroscopy at 260 nm.

[0190] In some embodiments, the amount of dsRNA comprised in mRNA transcripts is decreased by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, or at least 40% relative to the mRNA transcript yield obtained with the reference DNA construct. In some embodiments, the amount of dsRNA comprised in mRNA transcripts obtained by an SP6 DNA construct disclosed herein is decreased by at least 25% relative to the amount of dsRNA comprised in mRNA transcripts obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 2). In some embodiments, the amount of dsRNA comprised in mRNA transcripts obtained by an SP6 DNA construct disclosed herein is decreased by at least 30% relative to the amount of dsRNA comprised in mRNA transcripts obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 2). In some embodiments, the amount of dsRNA comprised in mRNA transcripts obtained by an SP6 DNA construct disclosed herein is decreased by at least 35% relative to the amount of dsRNA comprised in mRNA transcripts obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 2). In some embodiments, the amount of dsRNA comprised in mRNA transcripts obtained by an SP6 DNA construct disclosed herein is decreased by at least 40% relative to the amount of dsRNA comprised in mRNA transcripts obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 2). In some embodiments, the amount of dsRNA comprised in mRNA transcripts obtained by an SP6 DNA construct disclosed herein is decreased by at least 50% relative to the amount of dsRNA comprised in mRNA transcriptsSanofi Ref: PAT24103-WO-PCT obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 2). In some embodiments, the amount of dsRNA comprised in mRNA transcripts obtained by an SP6 DNA construct disclosed herein is decreased by at least 60% relative to the amount of dsRNA comprised in mRNA transcripts obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 2). In some embodiments, the amount of dsRNA comprised in mRNA transcripts obtained by an SP6 DNA construct disclosed herein is decreased by at least 70% relative to the amount of dsRNA comprised in mRNA transcripts obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 2).

[0191] In some embodiments, the amount of dsRNA comprised in mRNA transcripts obtained by a T7 DNA construct disclosed herein is decreased by at least 20% relative to the amount of dsRNA comprised in mRNA transcripts obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 76). In some embodiments, the amount of dsRNA comprised in mRNA transcripts obtained by a T7 DNA construct disclosed herein is decreased by at least 25% relative to the amount of dsRNA comprised in mRNA transcripts obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 76). The amount of dsRNA comprised in mRNA transcripts obtained by a T7 DNA construct disclosed herein is decreased by at least 30% relative to the amount of dsRNA comprised in mRNA transcripts obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 76).

[0192] In some embodiments, the amount of dsRNA comprised in mRNA transcripts obtained by a KP34 DNA construct disclosed herein is decreased by at least 20% relative to the amount of dsRNA comprised in mRNA transcripts obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 178). In some embodiments, the amount of dsRNA comprised in mRNA transcripts obtained by a KP34 DNA construct disclosed herein is decreased by at least 25% relative to the amount of dsRNA comprised in mRNA transcripts obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 178). The amount of dsRNA comprised in mRNA transcripts obtained by a KP34 DNA construct disclosed herein is decreased by at least 30% relative to the amount of dsRNA comprised in mRNA transcripts obtained with a reference DNA construct (e.g., as set forth in SEQ ID NO: 178).

[0193] In some embodiments, mRNA transcripts obtained by a method of the invention are substantially free of dsRNA. In some embodiments, the amount of dsRNA is below the limit of detection. In some embodiments, the presence of dsRNA is determined by dot blot using antibody J2, KI, orK2 (e.g., J2). In some embodiments, the presence of dsRNASanofi Ref: PAT24103-WO-PCT is determined by ELISA, e.g., a sandwich ELISA using antibodies J2 and KI, or antibodies KI and K2. In some embodiments, the amount of dsRNA by weight is below the limit of detection in a 200 ng sample of in vitro synthesized mRNA. In some embodiments, the amount of dsRNA by weight is below the limit of detection in a 200 ng sample of in vitro synthesized mRNA as determined by dot blot, e.g., using monoclonal antibody J2.

[0194] Also provided herein are compositions that comprise mRNA transcripts, an SP6, T7, or KP34 RNA polymerase, and less than 5% or less than 1% of dsRNA by weight. Such compositions are obtainable by the manufacturing methods described herein without any purification of the mRNA transcripts. In some embodiments, the mRNA transcripts comprise less than about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2% 0.1%, 0.05%, or 0.01% of dsRNA by weight, e.g., less than 0.5%. The amount of dsRNA may be determined by ELISA, e.g., a sandwich ELISA using antibodies J2 and KI, or antibodies KI and K2.Exemplary IVT conditions

[0195] In some embodiments, a suitable reaction mixture comprises a DNA construct with an SP6 RNA polymerase-specific promoter, SP6 RNA polymerase, RNase inhibitor, pyrophosphatase, NTPs, lO mM DTT and a reaction buffer (25 mM Tris-HCl, 2 mM spermidine, 25 mM MgCh, 0.5 mM NaCl, and pH 7.5). In some embodiments, this reaction mixture is incubated at 37°C for the length of time needed to complete IVT of the mRNA transcript encoded by the DNA template.

[0196] In some embodiments, a reaction mixture includes each NTP at a concentration ranging from 1-10 mM, a DNA construct at a concentration ranging from 0.01-0.5 mg / mL, and SP6 RNA polymerase at a concentration ranging from 0.01-0.1 mg / mL.

[0197] In some embodiments, a suitable reaction mixture comprises a DNA construct with a T7 RNA polymerase-specific promoter, T7 RNA polymerase, RNase inhibitor, pyrophosphatase, NTPs, lO mM DTT and a reaction buffer (25 mM Tris-HCl, 2 mM spermidine, 25 mM MgCh, 0.5 mM NaCl, and pH 7.5). In some embodiments, this reaction mixture is incubated at 37°C for the length of time needed to complete IVT of the mRNA transcript encoded by the DNA template.

[0198] In some embodiments, a reaction mixture includes each NTP at a concentration ranging from 1-10 mM, a DNA construct at a concentration ranging from 0.01-0.5 mg / mL, and T7 RNA polymerase at a concentration ranging from 0.01-0.1 mg / mL.Sanofi Ref PAT24103-WO-PCT

[0199] In some embodiments, a suitable reaction mixture comprises a DNA construct with a KP34 RNA polymerase-specific promoter, KP34 RNA polymerase, RNase inhibitor, pyrophosphatase, NTPs, lO mM DTT and a reaction buffer (25 mM Tris-HCl, 2 mM spermidine, 25 mM MgCh, 0.5 mM NaCl, and pH 7.5). In some embodiments, this reaction mixture is incubated at 37°C for the length of time needed to complete IVT of the mRNA transcript encoded by the DNA template.

[0200] In some embodiments, a reaction mixture includes each NTP at a concentration ranging from 1-10 mM, a DNA construct at a concentration ranging from 0.01-0.5 mg / mL, and KP34 RNA polymerase at a concentration ranging from 0.01-0.1 mg / mL.Purification

[0201] The inventors of the present invention have surprisingly discovered that the use of DNA templates as disclosed herein reduces the presence of dsRNA in in vitro synthesized mRNA. This simplifies the post-synthesis processing of the in vitro synthesized mRNA greatly.

[0202] For example, a method of the invention may further comprise a step of purifying the mRNA transcripts obtained from the IVT reaction performed from the SP6, T7, or KP34 RNA polymerase. In some embodiments, the step of purifying the mRNA transcripts involves a method other than cellulose chromatography. In some embodiments, the step of purifying the mRNA transcripts involves a method other than HPLC. In some embodiments, the step of purifying the mRNA transcripts involves a method other than HPLC with a buffer system comprising triethylammonium acetate and / or acetonitrile. In some embodiments, the step of purifying the mRNA transcripts involves a method other than anion-exchange fast performance liquid chromatography.

[0203] In some embodiments, the mRNA transcripts are purified without a chaotropic agent. In some embodiments, the mRNA transcripts are purified under non-denaturing conditions. In some embodiments, the mRNA transcripts are purified without use of lithium chloride, sodium chloride, potassium chloride, guanidium chloride, guanidium thiocyanate, guanidium isothiocyanate, ammonium acetate, and combinations thereof.

[0204] Various methods may be used to purify mRNA. In some embodiments, the mRNA is purified by precipitation and centrifugation. In some embodiments, the mRNA isSanofi Ref PAT24103-WO-PCT purified by filtration using, e.g., Normal Flow Filtration (NFF) or Tangential Flow Filtration (TFF).

[0205] Suitable purification methods include those described in published U.S. Application Nos. US 2016 / 0040154, US 2015 / 0376220, US 2018 / 0251755, US 2018 / 0251754, US 2020 / 0095571, US 2021 / 0388338, and US 2021 / 0002635, and in International Patent Publication No. WO 2022 / 072836, all of which are incorporated by reference herein.

[0206] The mRNA transcripts obtained by the manufacturing methods described herein and purified, e.g., to remove enzyme components used during IVT such as an SP6, T7, or KP34 RNA polymerase, can be formulated as a pharmaceutical composition. Such mRNA transcripts find utility in methods of treating or preventing a disease or disorder in a subject. These methods typically comprise administering a pharmaceutical composition comprising the mRNA transcripts to the subject.EQUIVALENTS

[0207] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below.EXAMPLESExample 1. Preparing and assessing in vitro transcribed mRNA

[0208] This example illustrates the method used for SP6 and T7 RNA polymerase- based mRNA synthesis. It also describes how mRNA yield was assessed and how the amount of dsRNA was determined.

[0209] A double-stranded linear DNA fragment (LDF) encoding a protein coding sequence of interest operably linked to an RNA polymerase-specific promoter was used for the in vitro transcription (IVT) reaction. The reaction buffer was prepared with RNase-free water and contained an RNA polymerase (e.g., SP6 or T7 RNA polymerase, as indicated), RNase inhibitor, pyrophosphatase, NTPs, DTT, and a buffering agent.

[0210] Unless indicated otherwise, the composition of the reaction buffer was the same for both SP6 RNA polymerase and T7 RNA polymerase (25 mM Tris-HCl, 2 mM spermidine, 25 mM MgCh, 0.5 mM NaCl, and pH 7.5). The reaction mixtures wereSanofi Ref: PAT24103-WO-PCT incubated at 37°C for 90 min. In vitro transcribed mRNA was prepared with ATP, CTP, GTP and N1 -methylpseudouridine (a modified UTP) to prepare in vitro transcribed mRNA comprising a modified ribonucleotide. DNase I was added to stop the reaction, and the reaction mixture was incubated for 15 more minutes at 37°C. The resulting in vitro transcribed mRNA was purified. RNA was quantified with a Lunatic Spectrophotometer using a 2 pL aliquot of the purified in vitro transcribed mRNA.

[0211] dsRNA was detected by ELISA using a 50 pL aliquot of the purified in vitro transcribed mRNA. Data was normalized relative to the total RNA concentration. ELISA- based quantification of dsRNA was performed using two specific commercially available mouse monoclonal antibodies (mAbs). The KI monoclonal antibody (Jena Bioscience) was used as the capture antibody (z.e., adsorbed to the surface of wells in a 96-well plate to bind any dsRNA present in the sample). The J2 monoclonal antibody (Jena Bioscience) was used as the detection antibody coupled with horseradish peroxidase (HRP). Following the addition of the chromogenic peroxidase substrate TMB, spectrophotometry was used to measure the intensity of the yellow color associated with the presence of the bound antibodies. Color intensity is proportional to the amount of dsRNA present in the sample.Example 2. Promoter sequence optimization improves SP6 RNA polymerase activity

[0212] This example illustrates that optimization of the SP6 promoter sequence can improve mRNA transcript yield and decrease the amount of dsRNA that SP6 RNA polymerase generates during IVT.

[0213] 53 different DNA constructs comprising SP6 promoters were tested to investigate SP6 RNA polymerase activity. A DNA construct comprising a non-optimized SP6 promoter having the nucleic acid sequence of SEQ ID NO: 74 was included as a control. IVT was performed and mRNA transcripts were purified as described in Example 1. The transcript yield and amount of dsRNA were normalized relative to the yield obtained with a reference DNA construct comprising a nucleic acid sequence 5’-ATTTAGGTGACACTATAGGAC-3’ (SEQ ID NO: 2) as a promoter. The SP6 promoter of the reference DNA construct did not include an upstream promoter region (referred to herein as “Pi”). The 3’ terminal four nucleic acids of the promoter comprised the transcriptional start site of the mRNA transcript.

[0214] As shown in Figure 1, both the yield of and the amount of dsRNA in the mRNA transcripts obtained with the reference DNA construct (denoted as “R”) was set toSanofi Ref: PAT24103-WO-PCT100%, as depicted by the solid black line. The dashed line in Figure 1 represents a threshold of a 20% increase in mRNA yield compared to the reference DNA construct. The dotted line in Figure 1 represents a threshold of a 40% decrease in dsRNA compared to the reference DNA construct. Representative mRNA yield data (n=4) and dsRNA data (n=3) are illustrated by the dark grey and light grey bars, respectively. The 54 tested SP6 promoters are provided in Table 12. The core promoter is shown in bold, the downstream promoter region P2 is underlined and the first nucleotide of the transcriptional start site of the mRNA transcript is in italic.Table 12. Tested DNA constructs comprising an SP6 promoter sequenceSanofi Ref: PAT24103-WO-PCTSanofi Ref: PAT24103-WO-PCTSanofi Ref: PAT24103-WO-PCT

[0215] The 3’ C nucleotide at the end of SEQ ID NOs: 1-53 corresponds to the start of a 5’ UTR sequence. Each of the promoter sequences tested comprised the 5’ UTR sequence from a CMV IE1 gene comprising the nucleic acid sequence CAGATCGCCT (SEQ ID NO: 115).

[0216] Of the 54 tested DNA constructs comprising an SP6 promoter, 18 nucleic acid sequences provided an improved mRNA yield. These 18 nucleic acid sequences are set forth in SEQ ID NOs: 6-10, 27, 29-31, 33-35, 37, 40, 42, 53, 54, and 56. Out of these 18 nucleic acid sequences, 9 had a more than 20% increase in mRNA yield compared to the reference DNA construct. These 9 had the sequences set forth in SEQ ID NOs: 6-10, 31, 35 53, and 56.

[0217] Of the 54 tested DNA constructs comprising an SP6 promoter, 32 nucleic acid sequences decreased the amount of dsRNA. The 32 nucleic acid sequences are set forth in SEQ ID NOs: 6-10, 26-45, 53, 54, 56, 57, 64, 65, and 67. Out of these 32 nucleic acid sequences, 9 had a more than 40% decrease in the amount of dsRNA. These 9 had the nucleic acid sequences set forth in SEQ ID NOs: 6-10, 32, 37, 38, and 41.

[0218] Of the 54 tested DNA constructs comprising an SP6 promoter, 5 scored above a 20% threshold increase for mRNA yield compared to the reference DNA construct and below a 40% threshold for amount of dsRNA compared to the reference DNA construct. These 5 had the sequences set forth in SEQ ID NOs: 6-10.

[0219] This example illustrates that optimization of the SP6 promoter sequence can improve mRNA transcript yield and reduce the amount of dsRNA that is generated by SP6 RNA polymerase during IVT.Sanofi Ref: PAT24103-WO-PCTExample 3. Identification of further improved SP6 RNA polymerase activity with optimized promoter sequence

[0220] This example illustrates that optimization of the SP6 promoter sequence can further improve mRNA transcript yield and decrease the amount of dsRNA that SP6 RNA polymerase generates during IVT.

[0221] 12 different DNA constructs comprising SP6 promoters were tested to investigate SP6 RNA polymerase activity. Of the 12 DNA constructs, four used a known downstream promoter sequence in the presence or absence of an upstream promoter sequence, as set forth in SEQ ID NOs: 158-161. Further, the highest-performing optimized SP6 promoters (SEQ ID NOs: 6-10) that were identified in Example 2 were also included. IVT was performed and mRNA transcripts were purified as described in Example 1. The transcript yield and amount of dsRNA were normalized relative to the yield obtained with a reference DNA construct comprising a nucleic acid sequence 5’-ATTTAGGTGACACTATAGGAC-3’ (SEQ ID NO: 2) as a promoter. The SP6 promoter of the reference DNA construct did not include an upstream promoter region (referred to herein as “Pi”). The 3’ terminal four nucleic acids of the promoter comprised the transcriptional start site of the mRNA transcript.

[0222] As shown in Figure 2, both the yield of and the amount of dsRNA in the mRNA transcripts obtained with the reference DNA construct (denoted as “R”) was set to 100%, as depicted by the solid black line. The dashed line in Figure 2 represents a threshold of a 20% increase in mRNA yield compared to the reference DNA construct. The dotted line in Figure 2 represents a threshold of a 40% decrease in dsRNA compared to the reference DNA construct. Representative mRNA yield data (n=2) and dsRNA data (n=l) are illustrated by the dark grey and light grey bars, respectively. The tested SP6 promoters are provided in Table 13. The core promoter is shown in bold, the downstream promoter region P2 is underlined and the first nucleotide of the transcriptional start site of the mRNA transcript is in italic.Table 13. Tested DNA constructs comprising an SP6 promoter sequenceSanofi Ref: PAT24103-WO-PCTSanofi Ref: PAT24103-WO-PCT

[0223] The 3’ C nucleotide at the end of SEQ ID NOs: 6-10 and 157-168 corresponds to the start of a 5’ UTR sequence. Each of the promoter sequences tested comprised the 5’ UTR sequence from a CMV IE1 gene comprising the nucleic acid sequence CAGATCGCCT (SEQ ID NO: 115).

[0224] As previously observed, DNA constructs comprising an SP6 promoter sequence as set forth in SEQ ID NOs: 6-10 (z.e., SP6 promoter numbers 1-5 on Figure 2) exceeded the 20% threshold increase for mRNA yield and exhibited around 40% lower dsRNA compared to the reference DNA construct. None of the sequences comprising the known downstream promoter (z.e., SEQ ID NOs: 158-161; denoted as 7-10 on Figure 2) outperformed the previously tested highest performing optimized SP6 promoters.

[0225] Of the 7 DNA constructs comprising a newly optimized SP6 promoter, 3 nucleic acid sequences provided an improved mRNA yield. These 3 nucleic acid sequences are set forth in SEQ ID NOs: 165, 166, and 168. Out of these 3 nucleic acid sequences, 2 had a more than 20% increase in mRNA yield compared to the reference DNA construct. These 2 had the sequences set forth in SEQ ID NOs: 166 and 168. Specifically, the optimized SP6 promoter comprising the nucleic acid sequences set forth in SEQ ID NO: 166 and SEQ ID NO: 168 achieved a 50% increase and 35% increase in mRNA yield, respectively, compared to the reference DNA construct.

[0226] Of the 7 DNA constructs comprising a newly optimized SP6 promoter, all 7 nucleic acid sequences decreased the amount of dsRNA. The 7 nucleic acid sequences are set forth in SEQ ID NOs: 162-168. Out of these 7 nucleic acid sequences, 6 had more than a 40% decrease in the amount of dsRNA. These 6 had the nucleic acid sequences set forth in SEQ ID NOs: 162, and 164-168. Surprisingly, 3 of the 6 nucleic acid sequences had at least a 70% decrease in the amount of dsRNA. These 3 nucleic acid sequences had the nucleic acid sequences set forth in SEQ ID NOs: 166-168.Sanofi Ref: PAT24103-WO-PCT

[0227] Of the new tested sequences, 2 scored above a 20% threshold increase for mRNA yield compared to the reference DNA construct and below a 40% threshold for amount of dsRNA compared to the reference DNA construct. These 2 had the sequences set forth in SEQ ID NOs: 166 and 168. Specifically, the DNA construct comprising SEQ ID NO: 166 demonstrated over 50% increase in mRNA yield and around a 70% decrease in the amount of dsRNA compared to the reference DNA construct. Similarly, the DNA construct comprising SEQ ID NO: 168 demonstrated around a 35% increase in mRNA yield and around a 70% decrease in the amount of dsRNA compared to the reference DNA construct.

[0228] This example illustrates that optimization of the SP6 promoter sequence can further improve mRNA transcript yield and reduce the amount of dsRNA that is generated by SP6 RNA polymerase during IVT.Example 4. Promoter sequence optimization improves the transcription of different mRNAs

[0229] This example illustrates that optimization of the SP6 promoter sequence can improve mRNA transcript yield and decrease the amount of dsRNA that SP6 RNA polymerase generates during IVT of mRNAs of various lengths and nucleic acid compositions.

[0230] Plasmids were designed to encode a different protein coding DNA sequence (CDS) of interest operably linked to an SP6 polymerase-specific optimized promoter and used for the IVT reaction. The encoded proteins were an influenza surface protein of 1698bp (CDS 1), a bacterial virulence factor of 855 bp (CDS 2), a coronavirus spike protein of 4032 bp (CDS 3), and green fluorescence protein (GFP) of 717 bp (CDS 4). IVT was performed and mRNA transcripts were purified as described in Example 1. Abortive transcripts were detected by High-Performance Liquid Chromatography with Ultraviolet detection (HPLC- UV).

[0231] Eight different DNA constructs (labelled 1-8 in Figure 3) comprising eight different SP6 promoters (labelled correspondingly 1-8 in Table 14 below) were tested to investigate SP6 RNA polymerase activity for the IVT of different mRNAs. They included the seven high performing DNA constructs identified in Examples 2 and 3 (promoters 1-6 and 8 in Table 14) and one additional test promoter with a longer upstream promoter sequence (SP6 promoter 7 in Table 14). The transcript yield, amount of dsRNA and amount of abortive transcripts were normalized relative to the yield obtained with a reference DNA constructSanofi Ref: PAT24103-WO-PCT comprising a nucleic acid sequence 5’-ATTTAGGTGACACTATAGGAC-3’ (SEQ ID NO: 2) as a promoter (labelled “R” in Figure 3). The SP6 promoter of the reference DNA construct did not include an upstream promoter region (referred to as “Pi” elsewhere herein). The 3’ terminal four nucleic acids of the promoter comprised the transcriptional start site of the mRNA transcript.

[0232] As shown in Figure 3, each of the yield of and the amounts of dsRNA and abortive transcripts in the mRNA transcripts obtained with the reference DNA plasmid (denoted as “R”) was set to 100%, as depicted by the solid black line. The dashed line in Figure 3 represents a threshold of a 1.4 fold increase in mRNA yield compared to the reference DNA plasmid. The dotted line in Figure 3 represents a threshold of a 0.5 fold decrease in dsRNA compared to the reference DNA plasmid. Representative mRNA yield data (n=3), dsRNA data (n=3) and abortive transcript data (n=l) are illustrated by the dark grey, light grey and unfilled bars, respectively. The tested SP6 promoters are provided in Table 14. The core promoter is shown in bold, the downstream promoter region (referred to as P2 elsewhere herein) is underlined and the first nucleotide of the transcriptional start site of the mRNA transcript is in italic.Table 14. Tested DNA plasmids comprising an SP6 promoter sequenceSanofi Ref: PAT24103-WO-PCT

[0233] The 3’ C nucleotide at the end of SEQ ID NOs: 6-10, 166, 168, and 214 corresponds to the start of a 5’ UTR sequence. Each of the promoter sequences tested comprised the 5’ UTR sequence from a CMV IE1 gene comprising the nucleic acid sequence CAGATCGCCT (SEQ ID NO: 115).

[0234] Consistent with the previous findings, DNA plasmids comprising one of the SP6 promoter sequences set forth in SEQ ID NOs: 6-10 exceeded a 1.4 fold increase for mRNA yield irrespective of which coding sequence was included compared to the reference DNA plasmid. Similarly, DNA plasmids comprising an SP6 promoter sequence as set forth in SEQ ID NOs: 6-10 exhibited lower dsRNA compared to the reference DNA plasmid. Specifically, DNA plasmids comprising one of the SP6 promoter sequences set forth in SEQ ID NOs: 7-10 exhibited more than a 0.5 fold decrease in the amount of dsRNA compared to the reference DNA plasmid for at least 3 of the 4 tested coding sequences.

[0235] Consistent with the previous findings, DNA plasmids comprising one of the P6 promoter sequences set forth in SEQ ID NO: 166 and 168 provided an improved mRNA yield compared to the reference DNA plasmid. Specifically, DNA plasmids comprising one of the SP6 promoter sequences set forth in SEQ ID NOs: 166 and 168 exceeded a 1.4 fold increase for mRNA yield for at least 2 of the tested coding sequences compared to the reference DNA plasmid. Similarly, DNA plasmids comprising one of the SP6 promoter sequence set forth in SEQ ID NOs: 166 and 168 exhibited lower dsRNA compared to the reference DNA plasmid. Specifically, DNA plasmids comprising one of the SP6 promoter sequences set forth in SEQ ID NOs: 166 and 168 exhibited more than a 0.5 fold decrease in the amount of dsRNA compared to the reference DNA plasmid for at least 2 of the 4 tested coding sequences.

[0236] DNA plasmids comprising one of the SP6 promoter sequences set forth in SEQ ID NO: 214 also provided an improved mRNA yield compared to the reference DNA plasmid. Specifically, DNA plasmids comprising the SP6 promoter sequence set forth in SEQSanofi Ref: PAT24103-WO-PCTID NO: 214 exceeded a 1.4 fold increase for mRNA yield for 3 of the tested coding sequences compared to the reference DNA construct. Similarly, DNA plasmids comprising the SP6 promoter sequence set forth in SEQ ID NO: 214 exhibited lower dsRNA compared to the reference DNA plasmid. Specifically, the DNA construct comprising the SP6 promoter sequence set forth in SEQ ID NO: 214 and a coding sequence for GFP exhibited more than a 0.5 fold decrease in the amount of dsRNA compared to the reference DNA plasmid.

[0237] Notably, of the 32 tested DNA plasmids, 26 displayed a comparable or reduced amount of abortive transcripts compared to the reference DNA plasmid.

[0238] This example illustrates that optimization of the SP6 promoter sequence can improve mRNA transcript yield and reduce the amount of dsRNA that is generated by SP6 RNA polymerase during IVT of different mRNAs.Example 5. Promoter sequence optimization improves T7 RNA polymerase activity

[0239] This example illustrates that optimization of the T7 promoter sequence can improve mRNA transcript yield and decrease the amount of dsRNA that T7 RNA polymerase generates during IVT.

[0240] 21 different DNA constructs comprising a T7 promoter were tested to investigate T7 RNA polymerase activity. IVT was performed and mRNA transcripts were purified as described in Example 1. The transcript yield and amount of dsRNA were normalized relative to the yield obtained with a reference DNA construct comprising a nucleic acid sequence 5’- TAATACGACTCACTATAGGAC -3’ (SEQ ID NO: 76) as a promoter. The T7 promoter of the reference DNA construct did not include an upstream promoter region (referred to herein as “Pi”). The 3’ terminal four nucleic acids of the promoter comprised the transcriptional start site of the mRNA transcript.

[0241] As shown in Figure 4, both the yield and the amount of dsRNA in the mRNA transcripts obtained with the reference DNA construct (denoted as “R) was set to 100%, as depicted by the solid black line). The dashed line in Figure 4 represents a threshold of a 50% increase in mRNA yield compared to the reference DNA construct. The dotted line in Figure 4 represents a threshold of a 30% decrease in dsRNA compared to the reference DNA construct. Representative mRNA yield data (n=3) and dsRNA data (n=2) are illustrated by the dark grey and light grey bars, respectively. The 21 tested T7 promoters are provided in Table 15. The core promoter is in bold, the downstream promoter region P2 is underlined and the first nucleotide of the transcriptional start site of the mRNA transcript is in italic.Sanofi Ref: PAT24103-WO-PCTTable 15. Tested DNA constructs comprising a T7 promoter sequenceSanofi Ref: PAT24103-WO-PCT

[0242] The 3’ C nucleotide at the end of SEQ ID NOs: 1-21 corresponds to the start of a 5’ UTR sequence. Each of the promoter sequences tested comprised the 5’ UTR sequence from a CMV IE1 gene comprising the nucleic acid sequence CAGATCGCCT (SEQ ID NO: 115).

[0243] Of the 21 tested T7 promoters, 11 sequences provided an improved mRNA yield relative to the reference DNA construct. These 11 sequences are set forth in SEQ ID NOs: 81, 82, 96, 97, 99, 100, 103, 104, 108, 109 and 110. Out of these 11 sequences, 3 provided a more than 50% increase in mRNA yield compared to the reference DNA construct. These 3 had the sequences set forth in SEQ ID NOs: 81, 82, and 100.

[0244] Of the 21 tested T7 promoters, 13 nucleic acid sequences decreased the amount of dsRNA. These 13 nucleic acid sequences are set forth in SEQ ID NOs: 81, 82, 96- 105 and 109. Out of these 13 nucleic acid sequences, 6 had a more than 30% decrease in the amount of dsRNA. These 6 had the nucleic acid sequence set forth in SEQ ID NOs: 81, 82, 98, 101, 102, and 105.

[0245] Of the 21 tested T7 promoters, 2 provided a more than 50% increase in mRNA yield compared to the reference DNA construct and had a more than 30% decrease in the amount of dsRNA compared to the reference DNA construct. These 2 had the nucleic acid sequences set forth in SEQ ID NOs: 81 and 82.

[0246] This example illustrates that optimization of the T7 promoter sequence can improve mRNA transcript yield and reduce the amount of dsRNA that is generated by T7 RNA polymerase during IVT.Example 6. Promoter sequence optimization improves KP34 RNA polymerase activity

[0247] This example illustrates that optimization of the KP34 promoter sequence can improve mRNA transcript yield and decrease the amount of dsRNA that KP34 RNA polymerase generates during IVT.

[0248] 19 different DNA constructs comprising a KP34 promoter were tested to investigate KP34 RNA polymerase activity. IVT was performed using KP34 polymerase and mRNA transcripts were purified as described in Example 1. The transcript yield and amount of dsRNA were normalized relative to the yield obtained with a reference DNA construct comprising a nucleic acid sequence 5’-CATCTAGATAATGTTACAGGAGTAGGAC-3’ (SEQ ID NO: 178) as a promoter. The 3’ terminal four nucleic acids of the promoterSanofi Ref: PAT24103-WO-PCT comprised the transcriptional start site of the mRNA transcript. Of the 18 DNA constructs, eight used a known downstream promoter sequence in the presence or absence of an upstream promoter, as set forth in SEQ ID NOs: 190, 191, 194, 195, 199, 200, 204, 205. Further, 4 of the 18 DNA constructs did not comprise an upstream promoter, as set forth in SEQ ID NOs: 188-191.

[0249] As shown in Figure 5, both the yield and the amount of dsRNA in the mRNA transcripts obtained with the reference DNA construct (denoted as “R) was set to 100%, as depicted by the solid black line). The dashed line in Figure 5 represents a threshold of a 20% increase in mRNA yield compared to the reference DNA construct. The dotted line in Figure 5 represents a threshold of a 30% decrease in dsRNA compared to the reference DNA construct. Representative mRNA yield data (n=2) and dsRNA data (n=l) are illustrated by the dark grey and light grey bars, respectively. The 19 tested KP34 promoters are provided in Table 16. The core promoter is shown in bold, the downstream promoter region P2 is underlined and the first nucleotide of the transcriptional start site of the mRNA transcript is in italic.Table 16. Tested DNA constructs comprising a KP34 promoter sequenceSanofi Ref: PAT24103-WO-PCT

[0250] The 3’ C nucleotide at the end of SEQ ID NOs: 188-206 corresponds to the start of a 5’ UTR sequence. Each of the promoter sequences tested comprised the 5’ UTR sequence from a CMV IE1 gene comprising the nucleic acid sequence CAGATCGCCT (SEQ ID NO: 115).

[0251] Of the 19 tested KP34 promoters, 14 sequences provided an improved mRNA yield relative to the reference DNA construct. These 14 sequences are set forth in SEQ ID NOs: 192-204 and 206. Out of these 14 sequences, 12 provided more than a 20% increase in mRNA yield compared to the reference DNA construct. These 12 had the sequences set forth in SEQ ID NOs: 192, 194-201, 203 and 206. Further out of these 12 sequences, 8 provided more than a 60% increase in mRNA yield compared to the reference DNA construct. These 8 had the sequences set forth in SEQ ID NOs: 195, 196, 198, 199, 200, 201, 204 and 206. Further, out of these 8 sequences, 4 provided more than a 100% (i.e., 2-fold) increase inSanofi Ref: PAT24103-WO-PCT mRNA yield compared to the reference DNA construct. These 4 had the sequences set forth in SEQ ID NOs: 196, 199, 204 and 206.

[0252] Of the 19 tested KP34 promoters, 6 nucleic acid sequences decreased the amount of dsRNA. These 6 nucleic acid sequences are set forth in SEQ ID NOs: 192-194, 197, 201 and 206. Out of these 6 nucleic acid sequences, 4 had around a 30% or more decrease in the amount of dsRNA. These 4 had the nucleic acid sequence set forth in SEQ ID NOs: 192, 193, 201 and 206.

[0253] Of the 19 tested KP34 promoters, 3 provided a more than 20% increase in mRNA yield compared to the reference DNA construct and around, or more than a 30% decrease in the amount of dsRNA compared to the reference DNA construct. These 3 had the nucleic acid sequences set forth in SEQ ID NOs: 192, 201, and 206.

[0254] This example illustrates that optimization of the KP34 promoter sequence can improve mRNA transcript yield and reduce the amount of dsRNA that is generated by KP34 RNA polymerase during IVT.

Claims

Sanofi Ref: PAT24103-WO-PCTCLAIMS1. A DNA construct comprising the nucleic acid sequence 5’-Pi- ATTTAGGX1GACACTATA-P2-C-3’, wherein:Pi is an upstream promoter region;Xi is selected from G, A, or T;P2 is a downstream promoter region that comprises the transcriptional start site of a messenger RNA (mRNA) transcript and consists of GX2A-S1, wherein:X2 is selected from A or G; andSi is selected from GAGGA, CTGGTGGA, GGGAGGTAG, GAGAGAATT, or TACAAGCTT and is optional; or the 3’ terminal C is the first nucleotide of the 5’ untranslated region (5’ UTR).

2. The DNA construct of claim 1, wherein the transcriptional start site is GGA or GAA.

3. The DNA construct of claim 1 or 2, wherein the sequence located immediately downstream of P2-C is AGATCGCC.

4. The DNA construct of any one of the preceding claims, wherein Pi is 18 nucleotides upstream of the transcriptional start site.

5. The DNA construct of any one of the preceding claims, wherein Pi is selected from the group consisting of 5’-CTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 3), 5’- TTATGTATCATACACATACG-3’ (SEQ ID NO: 4), 5’- TGGACAAATCTGTGTCTCTT-3’ (SEQ ID NO: 5) and 5’-GCACGTCGCCGCGCAGGTATGGCTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 147).

6. The DNA construct of any one of the preceding claims, wherein:(a) Si is GAGGA, optionally wherein X2 is A; or(b) Si is CTGGTGGA and X2is A; or(c) Si is GGGAGGTAG and X2is A; or(d) Si is GAGAGAATT and X2is A.73Sanofi Ref: PAT24103-WO-PCT7. The DNA construct of any one of claims 1-5, wherein Pi is 5’- CTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 3) and Xi is T or A.

8. The DNA construct of any one of claims 1-5, wherein Pi is 5’- TTATGTATCATACACATACG-3’ (SEQ ID NO: 4) and Xi is T or A.

9. The DNA construct of any one of claims 1-5, wherein Pi is 5’- TGGACAAATCTGTGTCTCTT-3’ (SEQ ID NO: 5) and Xi is G or A.

10. The DNA construct of any one of claims 1-5, wherein Pi is 5’- GCACGTCGCCGCGCAGGTATGGCTCGCGGTCTTTAATTGCCT-3’ (SEQ ID NO: 147) and Xi is T.

11. The DNA construct of any one of the preceding claims, wherein the nucleic acid sequence is selected from the group consisting of:(a) 5 ’ -CTCGCGGTCTTTAATTGCCT ATTT AGGTGAC ACT AT AGGAC-3 ’ (SEQ ID NO: 6);(b) 5’- TTATGTATCATACACATACGATTTAGGTGACACTATAGAAGAGGAC-3’ (SEQ ID NO: 7);(c) 5’- TGGACAAATCTGTGTCTCTTATTTAGGGGACACTATAGAAGAGGAC-3’ (SEQ ID NO: 8);(d) 5’- TTATGTATCATACACATACGATTTAGGAGACACTATAGAAGAGGAC-3’ (SEQ ID NO: 9);(e) 5’- CTCGCGGTCTTTAATTGCCTATTTAGGAGACACTATAGAAGAGGAC-3’ (SEQ ID NO: 10);(f) 5’-CTCGCGGTCTTTAATTGCCTATTTAGGTGACACTATAGAAGGGAGGTAG-3’ (SEQ ID NO: 155);74Sanofi Ref: PAT24103-WO-PCT(g) 5 ’ TGGAC AAATCTGTGTCTCTT ATTTAGGGGAC ACT ATAGAAGAGA GAATT-3’ (SEQ ID NO: 156); and(h) 5’- GCACGTCGCCGCGCAGGTATGGCTCGCGGTCTTTAATTGCCTATTTAGGTGACA CTATAGAAGGGAGGTAG-3’ (SEQ ID NO: 215).

12. A method for manufacturing messenger RNA (mRNA) by in vitro transcription (IVT) comprising:(a) providing the DNA construct of any one of the preceding claims;(b) contacting the DNA construct with an SP6 RNA polymerase under conditions suitable for obtaining mRNA transcripts.

13. The method of claim 12, wherein the amount of double-stranded RNA (dsRNA) comprised in the mRNA transcripts obtained in step b) is decreased and / or the mRNA transcript yield is increased relative to mRNA transcripts obtained with a reference DNA construct comprising a nucleic acid sequence 5’-ATTTAGGTGACACTATAGGAC-3’ (SEQ ID NO: 2) without an upstream promoter region Pi, wherein the 3’ terminal four nucleic acids of the nucleic acid sequence comprise the transcriptional start site of an mRNA transcript.

14. The method of claim 13, wherein the amount of dsRNA comprised in the mRNA transcripts obtained in step b) is at least 25%, 30% or 35% lower relative to the mRNA transcripts obtained with the reference DNA construct, optionally wherein the amount of dsRNA is determined by ELISA using antibodies J2 and KI.

15. The method of any one of claims 12-14, wherein the mRNA transcript yield is increased by at least 15% or 20% relative to the mRNA transcript yield obtained with the reference DNA construct, optionally wherein the mRNA transcript yield is determined by UV spectroscopy at 260 nm.

16. The method of any one of claims 12-15, further comprising a step of purifying the mRNA transcripts obtained in step b) from the SP6 RNA polymerase, optionally wherein the step of purifying the mRNA transcripts involves a method other than (i) cellulose75Sanofi Ref: PAT24103-WO-PCT chromatography, and / or (ii) high-performance chromatography (HPLC) with a buffer system comprising triethylammonium acetate and / or acetonitrile.76

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