Optimised rnas

Abstract

The present invention provides methods of producing RNAs optimised to have higher translation efficiency. Also provided are RNAs produced by such methods and their use as medicaments. Also provided are computer implemented methods for designing such RNAs.

Description

[0001] Optimised RNAs

[0002] Field of Invention

[0003] The present invention provides methods of producing RNAs optimised to have higher translation efficiency. Also provided are RNAs produced by such methods and their use as medicaments. Also provided are computer implemented methods for designing such RNAs.

[0004] In vitro transcribed messenger RNA (IVT mRNA) has emerged as a promising avenue for developing novel therapeutic interventions. While IVT mRNA holds great potential for future therapies, efforts to enhance the production of full-length, active proteins are imperative for realising its clinical utility1. Researchers typically employ codon optimization strategies to enhance translation efficiency, recognizing the pivotal role of the open reading frame (ORF) in mRNA vaccine efficacy2. Additionally, optimizing mRNA secondary structure and stability, often achieved through increasing the GC mRNA content of the coding sequence, can bolster mRNA vaccine safety, efficacy, and stability3. However, achieving optimal translation efficiency and in vivo protein expression requires careful consideration of factors such as codon usage frequency and host tRNA availability, which vary across organisms and tissues4. Further optimization efforts must also address host cell-dependent factors, including tRNA complement and dynamics of tRNA processing by the ribosome, to optimize translation rates and mitigate transcript degradation pathways5. Despite advances in optimization algorithms, concerns regarding their effectiveness have been raised. Moreover, the presence of numerous "slow" codons in vaccine sequences, decoded by less abundant tRNAs, may contribute to elongation stalling6. Overall, the wider utility of mRNAs as tools and therapeutics will benefit from technology to increase or tailor mRNA translation efficiency and stability.

[0005] There is a need for improved RNAs, such as therapeutic mRNAs.

[0006] There is a need for improved methods of producing RNAs, such as therapeutic mRNAs.

[0007] There is a need for improved methods of designing RNAs, such as therapeutic mRNAs.

[0008] There is a need for improved methods and means for increasing translation efficiency and / or fidelity of mRNAs, such as therapeutic mRNAs.

[0009] Brief summary of the disclosure

[0010] Draft Claims In a first aspect there is provided, a nucleic acid molecule comprising a nucleic acid sequence encoding one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected based one or more properties of the codon, wherein the one or more properties comprise one or more of: a level of tRNA isoacceptor expression in a target cell; codon decoding speeds; and / or binding affinity between the codon and cognate tRNA anticodon.

[0011] In certain embodiments, at least one codon has been selected based on the level of tRNA isoacceptor expression in a target cell.

[0012] In certain embodiments, all codons for one amino acid have been selected based on the one or more properties of the codon.

[0013] In certain embodiments, all codons for all amino acids have been selected based on the one or more properties of each codon.

[0014] In certain embodiments, the nucleic acid sequence is configured to increase translation efficiency and / or translation fidelity in the target cell.

[0015] In certain embodiments, the target cell comprises an in vivo or in vitro cell.

[0016] In certain embodiments, the target cell comprises a skeletal muscle cell and wherein the codon for: alanine comprises the sequence 5’ GCU 3’; arginine comprises the sequence 5’ AGG 3’; aspartic acid comprises the sequence 5’ GAC 3’; glutamic acid comprises the sequence 5’ GAG 3’; glycine comprises the sequence 5’ GGC 3’; leucine comprises the sequence 5’ CUU 3’; lysine comprises the sequence 5’ AAG 3’; proline comprises the sequence 5’ CCU 3’; serine comprises the sequence 5’ AGC 3’; asparagine comprises the sequence 5’ AAC 3’; glutamine comprises the sequence 5’ CAG 3’; histidine comprises the sequence 5’ CAC 3’; valine comprises the sequence 5’ GUG 3’; and / or methionine comprises the sequence 5’ AUG 3’; or wherein the target cell comprises an HEK293T cell and wherein the codon for: alanine comprises the sequence 5’ GCU 3’; arginine comprises the sequence 5’ CGU 3’; aspartic acid comprises the sequence 5’ GAC 3’; glutamic acid comprises the sequence 5’ CAA 3’; glycine comprises the sequence 5’ GGC 3’; leucine comprises the sequence 5’ CUG 3’; lysine comprises the sequence 5’ AAG 3’; proline comprises the sequence 5’ CCU 3’; serine comprises the sequence 5’ AGC 3’; asparagine comprises the sequence 5’ AAC 3’; glutamine comprises the sequence 5’ CAG 3’; histidine comprises the sequence 5’ CAC 3’; valine comprises the sequence 5’ GUU 3’; isoleucine comprises the sequence 5’ AUA 3’; threonine comprises the sequence 5’ ACU 3’; tryptophan comprises the sequence 5’ UGG 3’; tyrosine comprises the sequence 5’ UAC 3’; phenylalanine comprises the sequence 5’ UUC 3’; and / or methionine comprises the sequence 5’ AUG 3’

[0017] In a second aspect there is provided, a nucleic acid molecule comprising a nucleic acid sequence encoding one or more codons for translation to one or more amino acids and encoding a signal peptide selected from: alpha-1 -antichymotrypsin (AACT) signal peptide; or Human Serum albumin (HSA) signal peptide.

[0018] In certain embodiments, the nucleic acid molecule is for translation in target cell, wherein the target cell is a muscle cell, liver cell, kidney cell, cardiomyocyte and / or antigen presenting cell.

[0019] In certain embodiments, the target cell is a liver cell and the signal peptide comprises alpha- 1 -antichymotrypsin (AACT) signal peptide.

[0020] In certain embodiments, the target cell is a muscle cell and the signal peptide comprises human Serum albumin (HSA) signal peptide.

[0021] In certain embodiments, the alpha-1-antichymotrypsin (AACT) signal peptide comprises an amino acid sequence according to SEQ ID NO: 10 and / or wherein human Serum albumin (HSA) signal peptide comprises an amino acid sequence according to SEQ ID NO: 22.

[0022] In a third aspect, there is provided, a nucleic acid molecule comprising a nucleic acid sequence encoding one or more codons for translation to one or more amino acids in a target cell and encoding a 5’ and 3’ untranslated region (UTR): wherein the 5’ UTR is derived from a MYL1 mRNA and the 3’ UTR is derived from a MYL1 mRNA (MUTR2); wherein the 5’ UTR is derived from a TNNC1 mRNA and the 3’ UTR is derived from a ENO3 mRNA (MUTR4); wherein the 5’ UTR is derived from a TNNC1 mRNA and the 3’ UTR is derived from a TNNC1 mRNA (MUTR5); wherein the 5’ UTR is derived from a ENO3 mRNA and the 3’ UTR is derived from a ENO3 mRNA (MUTR6); wherein the 5’ UTR is derived from a MYH1 mRNA and the 3’ UTR is derived from a MYH1 mRNA (MUTR7); wherein the 5’ UTR is derived from a MYL2 mRNA and the 3’ UTR is derived from a MYL2 mRNA (MUTR8); wherein the 5’ UTR is derived from a CKM mRNA and the 3’ UTR is derived from a CKM mRNA (MUTR10); wherein the 5’ UTR is derived from a ENO3 mRNA and the 3’ UTR is derived from a COX6A2 mRNA (MUTR11); wherein the 5’ UTR is derived from a ORM1 mRNA and the 3’ UTR is derived from a ORM1 mRNA (LUTR1); wherein the 5’ UTR is derived from a SAA2 mRNA and the 3’ UTR is derived from a SAA2 mRNA (LUTR2); wherein the 5’ UTR is derived from a SAA4 mRNA and the 3’ UTR is derived from a SAA4 mRNA (LUTR3); wherein the 5’ UTR is derived from a CFHR2 mRNA and the 3’ UTR is derived from a SAA4 mRNA (LUTR5); wherein the 5’ UTR is derived from a SAA1 mRNA and the 3’ UTR is derived from a SAA1 mRNA (LUTR6); wherein the 5’ UTR is derived from a GC mRNA and the 3’ UTR is derived from a GC mRNA (LUTR7); wherein the 5’ UTR is derived from a AHSG mRNA and the 3’ UTR is derived from a AHSG mRNA (LUTR8); wherein the 5’ UTR is derived from a SAA4 mRNA and the 3’ UTR is derived from a APOA1 mRNA (LUTR9); or wherein the 5’ UTR is derived from a SAA1 mRNA and the 3’ UTR is derived from a GC mRNA (LUTR10).

[0023] In certain embodiments: the 5’ UTR comprises or consists of SEQ ID NO: 37 and the 3’ UTR comprises or consists of SEQ ID NO: 38 (MUTR2); the 5’ UTR comprises or consists of SEQ ID NO: 41 and the 3’ UTR comprises or consists of SEQ ID NO: 42 (MUTR4); the 5’ UTR comprises or consists of SEQ ID NO: 43 and the 3’ UTR comprises or consists of SEQ ID NO: 44 (MUTR5); the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 46 (MUTR6); the 5’ UTR comprises or consists of SEQ ID NO: 47 and the 3’ UTR comprises or consists of SEQ ID NO: 48 (MUTR7); the 5’ UTR comprises or consists of SEQ ID NO: 49 and the 3’ UTR comprises or consists of SEQ ID NO: 50 (MUTR8); the 5’ UTR comprises or consists of SEQ ID NO: 53 and the 3’ UTR comprises or consists of SEQ ID NO: 54 (MUTR10); the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 55 (MUTR11); the 5’ UTR comprises or consists of SEQ ID NO: 56 and the 3’ UTR comprises or consists of SEQ ID NO: 57 (LUTR1); the 5’ UTR comprises or consists of SEQ ID NO: 58 and the 3’ UTR comprises or consists of SEQ ID NO: 59 (LUTR2); the 5’ UTR comprises or consists of SEQ ID NO: 60 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR3); the 5’ UTR comprises or consists of SEQ ID NO: 64 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR5); the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 66 (LUTR6); the 5’ UTR comprises or consists of SEQ ID NO: 67 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR7); the 5’ UTR comprises or consists of SEQ ID NO: 69 and the 3’ UTR comprises or consists of SEQ ID NO: 70 (LUTR8); the 5’ UTR comprises or consists of SEQ ID NO: 60 and the 3’ UTR comprises or consists of SEQ ID NO: 63 (LUTR9) or the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR10).

[0024] In certain embodiments, the target cell is a muscle cell and: the 5’ UTR comprises or consists of SEQ ID NO: 37 and the 3’ UTR comprises or consists of SEQ ID NO: 38 (MUTR2); the 5’ UTR comprises or consists of SEQ ID NO: 41 and the 3’ UTR comprises or consists of SEQ ID NO: 42 (MUTR4); the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 46 (MUTR6); the 5’ UTR comprises or consists of SEQ ID NO: 47 and the 3’ UTR comprises or consists of SEQ ID NO: 48 (MUTR7); the 5’ UTR comprises or consists of SEQ ID NO: 49 and the 3’ UTR comprises or consists of SEQ ID NO: 50 (MUTR8); the 5’ UTR comprises or consists of SEQ ID NO: 53 and the 3’ UTR comprises or consists of SEQ ID NO: 54 (MUTR10); the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 55 (MUTR11); the 5’ UTR comprises or consists of SEQ ID NO: 56 and the 3’ UTR comprises or consists of SEQ ID NO: 57 (LUTR1); the 5’ UTR comprises or consists of SEQ ID NO: 58 and the 3’ UTR comprises or consists of SEQ ID NO: 59 (LUTR2); the 5’ UTR comprises or consists of SEQ ID NO: 60 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR3); the 5’ UTR comprises or consists of SEQ ID NO: 64 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR5); the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 66 (LUTR6); the 5’ UTR comprises or consists of SEQ ID NO: 67 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR7); the 5’ UTR comprises or consists of SEQ ID NO: 69 and the 3’ UTR comprises or consists of SEQ ID NO: 70 (LUTR8); or the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR10).

[0025] In certain embodiments, the target cell is a liver cell and: the 5’ UTR comprises or consists of SEQ ID NO: 37 and the 3’ UTR comprises or consists of SEQ ID NO: 38 (MUTR2); the 5’ UTR comprises or consists of SEQ ID NO: 41 and the 3’ UTR comprises or consists of SEQ ID NO: 42 (MUTR4); the 5’ UTR comprises or consists of SEQ ID NO: 43 and the 3’ UTR comprises or consists of SEQ ID NO: 44 (MUTR5); the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 46 (MUTR6); the 5’ UTR comprises or consists of SEQ ID NO: 47 and the 3’ UTR comprises or consists of SEQ ID NO: 48 (MUTR7); the 5’ UTR comprises or consists of SEQ ID NO: 49 and the 3’ UTR comprises or consists of SEQ ID NO: 50 (MUTR8); the 5’ UTR comprises or consists of SEQ ID NO: 53 and the 3’ UTR comprises or consists of SEQ ID NO: 54 (MUTR10); the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 55 (MUTR11); the 5’ UTR comprises or consists of SEQ ID NO: 56 and the 3’ UTR comprises or consists of SEQ ID NO: 57 (LUTR1); the 5’ UTR comprises or consists of SEQ ID NO: 58 and the 3’ UTR comprises or consists of SEQ ID NO: 59 (LUTR2); the 5’ UTR comprises or consists of SEQ ID NO: 60 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR3); the 5’ UTR comprises or consists of SEQ ID NO: 64 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR5); the 5’ UTR comprises or consists of SEQ ID NO: 67 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR7); the 5’ UTR comprises or consists of SEQ ID NO: 69 and the 3’ UTR comprises or consists of SEQ ID NO: 70 (LUTR8); or the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR10).

[0026] In certain embodiments, the target cell is an HEK293T cell and the 5’ UTR comprises or consists of SEQ ID NO: 41 and the 3’ UTR comprises or consists of SEQ ID NO: 42 (MUTR4); the 5’ UTR comprises or consists of SEQ ID NO: 47 and the 3’ UTR comprises or consists of SEQ ID NO: 48 (MUTR7); the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 55 (MUTR11); the 5’ UTR comprises or consists of SEQ ID NO: 56 and the 3’ UTR comprises or consists of SEQ ID NO: 57 (LUTR1); the 5’ UTR comprises or consists of SEQ ID NO: 58 and the 3’ UTR comprises or consists of SEQ ID NO: 59 (LUTR2); the 5’ UTR comprises or consists of SEQ ID NO: 60 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR3); the 5’ UTR comprises or consists of SEQ ID NO: 64 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR5); the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 66 (LUTR6); the 5’ UTR comprises or consists of SEQ ID NO: 67 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR7); the 5’ UTR comprises or consists of SEQ ID NO: 69 and the 3’ UTR comprises or consists of SEQ ID NO: 70 (LUTR8); the 5’ UTR comprises or consists of SEQ ID NO: 60 and the 3’ UTR comprises or consists of SEQ ID NO: 63 (LUTR9); or the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR10).

[0027] In a fourth aspect there is provided, a molecule comprising a nucleic acid sequence encoding one or more codons for translation to one or more amino acids in a target cell and comprising at least one alternative reading frame sequence which encodes an alternative translation product that differs from the translation product of in-frame translation of the nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence encoding at least two consecutive stop codons (tandem stop codon) for reducing translation of the alternative translation products.

[0028] In certain embodiments, the at least two consecutive stop codons each comprise a nucleic acid sequence consisting of UAA or UAG.

[0029] In certain embodiments, the nucleic acid comprises two consecutive stop codons and wherein the two consecutive stop codons together comprise a nucleic acid sequence comprising or consisting of:

[0030] UAAUAA;

[0031] UAAUAG;

[0032] UAGUAA; or

[0033] UAGUAG.

[0034] In certain embodiments, the at least one tandem stop codon is encoded in a +1 reading frame from the translation product of in-frame translation. In certain embodiments, the nucleic acid comprises an RNA and wherein at least one tandem stop codon comprise at least one N1-methyl pseudouridine.

[0035] In certain embodiments of any aspect, the nucleic acid molecule comprises an mRNA or a DNA molecule encoding an mRNA.

[0036] In a fifth aspect there is provided, a nucleic acid molecule comprising a nucleic acid sequence encoding any one or more of the first to fourth aspects in any combination.

[0037] In a sixth aspect there is provided, an in vitro transcribed mRNA comprising a nucleic acid sequence encoding any one or more of the first to fourth aspects in any combination.

[0038] In certain embodiments, the nucleic acid molecule is an mRNA or encodes an mRNA, or the in vitro transcribed mRNA, and wherein the mRNA comprises a therapeutic mRNA.

[0039] In certain embodiments, the nucleic acid molecule or in vitro transcribed mRNA comprises a nucleic acid sequence encoding a poly(A) tail wherein the poly(A) tail comprises 97 to 135 adenine residues; optionally comprises 97 adenine residues.

[0040] In certain embodiments, the nucleic acid molecule is an mRNA, or the in vitro transcribed mRNA and the mRNA comprises a m7G-5'ppp5’GpG 5’ cap.

[0041] In certain embodiments, the nucleic acid molecule is an mRNA, or the in vitro transcribed mRNA, and the mRNA comprises one or more modified ribonucleotide.

[0042] In a seventh aspect there is provided, a method of producing an optimised nucleic acid molecule for translation, the method comprising; determining preferred codons in a target cell for at least one amino acid; producing a nucleic acid molecule that comprises a nucleic acid sequence encoding one or more codons for translation to one or more amino acids, wherein at least one codon for the first amino acid comprises the preferred codon; wherein determining preferred codons comprises: determining a level of tRNA isoacceptor expression in a target cell for at least one first amino acid; determining codon decoding speed for at least one first amino acid; and / or determining binding affinity between a codon and cognate tRNA anticodon for at least one first amino acid.

[0043] In certain embodiments, determining a level of tRNA isoacceptor expression comprises: determining the most abundantly expressed tRNA anti-codon for the at least one first amino acid and wherein the preferred codon comprises the complement of the most abundantly expressed tRNA anti-codon; or determining the ratio of the level of expression of cognate to near-cognate tRNAs for the at least one first amino acid and wherein the preferred codon comprises the complement of the anti-codon with the lowest ratio of near-cognate to cognate tRNA.

[0044] In certain embodiments, the method further comprises determining the preferred codon for one or more further amino acids.

[0045] In certain embodiments, the nucleic acid molecule comprises a nucleic acid sequence encoding at least one codon for the each of the one or more further amino acids, wherein the at least one codon for each further amino acid comprises the preferred codon for each further amino acid.

[0046] In certain embodiments, all codons for each of the at least one first and / or further amino acids comprise the preferred codon.

[0047] In certain embodiments, the nucleic acid molecule comprises an mRNA or a DNA molecule encoding an mRNA.

[0048] In certain embodiments, the producing further comprises in vitro transcribing the DNA molecule to produce the mRNA.

[0049] In certain embodiments, the mRNA comprises a therapeutic mRNA.

[0050] In certain embodiments, the producing further comprises adding a nucleic acid sequence encoding a poly(A) tail; optionally wherein the poly(A) tail comprises 97 to 135 adenine residues; further optionally wherein the poly(A) tail comprises 97 adenine residues.

[0051] In certain embodiments, the method comprises enzymatically adding a or the poly(A) tail optionally, as described herein.

[0052] In certain embodiments, the producing further comprises adding a m7G-5'ppp5’GpG 5’ cap.

[0053] In certain embodiments, the mRNA comprises one or more modified ribonucleotide.

[0054] In an eighth aspect there is provided, use of a nucleic acid molecule encoding a 5’ and 3’ UTR: wherein the 5’ UTR is derived from a MYLPF mRNA and the 3’ UTR is derived from a MYLPF mRNA (MUTR2); wherein the 5’ UTR is derived from a TNNC1 mRNA and the 3’ UTR is derived from a ENO3 mRNA (MUTR4); wherein the 5’ UTR is derived from a ENO3 mRNA and the 3’ UTR is derived from a ENO3 mRNA (MUTR6); wherein the 5’ UTR is derived from a MYH1 mRNA and the 3’ UTR is derived from a MYH1 mRNA (MUTR7); wherein the 5’ UTR is derived from a MYL2 mRNA and the 3’ UTR is derived from a MYL2 mRNA (MUTR8); wherein the 5’ UTR is derived from a CKM mRNA and the 3’ UTR is derived from a CKM mRNA (MUTR10); wherein the 5’ UTR is derived from a ENO3 mRNA and the 3’ UTR is derived from a COX6A2 mRNA (MUTR11); wherein the 5’ UTR is derived from a ORM1 mRNA and the 3’ UTR is derived from a ORM1 mRNA (LUTR1); wherein the 5’ UTR is derived from a SAA2 mRNA and the 3’ UTR is derived from a SAA2 mRNA (LUTR2); wherein the 5’ UTR is derived from a SAA4 mRNA and the 3’ UTR is derived from a SAA4 mRNA (LUTR3); wherein the 5’ UTR is derived from a CFHR2 mRNA and the 3’ UTR is derived from a SAA4 mRNA (LUTR5); wherein the 5’ UTR is derived from a SAA1 mRNA and the 3’ UTR is derived from a SAA1 mRNA (LUTR6); wherein the 5’ UTR is derived from a GC mRNA and the 3’ UTR is derived from a GC mRNA (LUTR7); wherein the 5’ UTR is derived from a AHSG mRNA and the 3’ UTR is derived from a AHSG mRNA (LUTR8); or wherein the 5’ UTR is derived from a SAA1 mRNA and the 3’ UTR is derived from a GC mRNA (LUTR10); for translation of an mRNA in a muscle cell.

[0055] In a ninth aspect there is provided use of a nucleic acid molecule encoding a UTR: wherein the 5’ UTR is derived from a MYLPF mRNA and the 3’ UTR is derived from a MYLPF mRNA (MUTR2); wherein the 5’ UTR is derived from a TNNC1 mRNA and the 3’ UTR is derived from a ENO3 mRNA (MUTR4); wherein the 5’ UTR is derived from a TNNC1 mRNA and the 3’ UTR is derived from a TNNCI mRNA (MUTR5); wherein the 5’ UTR is derived from a ENO3 mRNA and the 3’ UTR is derived from a ENO3 mRNA (MUTR6); wherein the 5’ UTR is derived from a MYH1 mRNA and the 3’ UTR is derived from a MYH1 mRNA (MUTR7); wherein the 5’ UTR is derived from a MYL2 mRNA and the 3’ UTR is derived from a MYL2 mRNA (MUTR8); wherein the 5’ UTR is derived from a CKM mRNA and the 3’ UTR is derived from a CKM mRNA (MUTR10); wherein the 5’ UTR is derived from a ENO3 mRNA and the 3’ UTR is derived from a COX6A2 mRNA (MUTR11); wherein the 5’ UTR is derived from a ORM1 mRNA and the 3’ UTR is derived from a ORM1 mRNA (LUTR1); wherein the 5’ UTR is derived from a SAA2 mRNA and the 3’ UTR is derived from a SAA2 mRNA (LUTR2); wherein the 5’ UTR is derived from a SAA4 mRNA and the 3’ UTR is derived from a SAA4 mRNA (LUTR3); wherein the 5’ UTR is derived from a CFHR2 mRNA and the 3’ UTR is derived from a SAA4 mRNA (LUTR5); wherein the 5’ UTR is derived from a SAA1 mRNA and the 3’ UTR is derived from a SAA1 mRNA (LUTR6); wherein the 5’ UTR is derived from a GC mRNA and the 3’ UTR is derived from a GC mRNA (LUTR7); wherein the 5’ UTR is derived from a AHSG mRNA and the 3’ UTR is derived from a AHSG mRNA (LUTR8); or wherein the 5’ UTR is derived from a SAA1 mRNA and the 3’ UTR is derived from a GC mRNA (LUTR10; for translation of an mRNA in a liver cell.

[0056] In a tenth aspect there is provided, use of an alpha-1-antichymotrypsin (AACT) signal peptide for translation of an mRNA in a liver cell. In an eleventh aspect there is provided, use of a human Serum albumin (HSA) signal peptide for translation of an mRNA in a muscle cell.

[0057] In certain embodiments, the nucleic acid sequence encoding: the human Serum albumin (HSA) signal peptide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 23 or 24; or the alpha-1 -antichymotrypsin (AACT) signal peptide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 11 or 12.

[0058] In a twelfth aspect there is provided, a nucleic acid molecule or in vitro transcribed mRNA of as described herein or a pharmaceutical composition thereof for use as a medicament.

[0059] In a thirteenth aspect there is provided, a nucleic acid molecule or in vitro transcribed mRNA of as described herein or a pharmaceutical composition thereof for use as a vaccine.

[0060] In a fourteenth aspect there is provided, a nucleic acid molecule or in vitro transcribed mRNA of as described herein or a pharmaceutical composition thereof for use in inducing an immune response in a subject.

[0061] In a fifteenth aspect there is provided, a method of treating or preventing a disease in a subject in need thereof, comprising administering a nucleic acid molecule or in vitro transcribed mRNA as described herein or a pharmaceutical composition thereof to the subject.

[0062] In a sixteenth aspect there is provided, a method of vaccinating a subject in need thereof, comprising administering a nucleic acid molecule or in vitro transcribed mRNA as described herein or a pharmaceutical composition thereof to the subject.

[0063] In a seventeenth aspect there is provided, a method of inducing an immune response in a subject in need thereof, comprising administering a nucleic acid molecule or in vitro transcribed mRNA as described herein or a pharmaceutical composition thereof to the subject.

[0064] In an eighteenth aspect there is provided, a computer-implemented method for designing an optimised RNA sequence for translation, the method comprising:

[0065] (a) receiving a target translated protein sequence, one or more user-defined objectives for the optimised RNA sequence, and one or more parameters associated with each user-defined objective and corresponding to the optimised RNA sequence, the one or more parameters including: codon composition efficiency for translation of the RNA sequence to the target translated protein sequence; presence or absence of specified nucleotide repeats; GC content; desirability of specified nucleotide sub-sequences; and location of one or more stop codons;

[0066] (b) receiving deviation information for each parameter, wherein the deviation information is for processing a nucleotide sequence and is indicative of a degree of deviation of a nucleotide sequence from a target value of the associated parameter;

[0067] (c) determining a score function for each parameter based on the deviation information, wherein each score function is usable for processing a nucleotide sequence to generate a score indicative of the degree of deviation between the nucleotide sequence and the target value of the parameter associated with that score function;

[0068] (d) selecting, from amongst a plurality of nucleotide sequences, a set of random nucleotide sequences, wherein each random nucleotide sequence comprises a nucleotide sequence that encodes the target translated protein sequence;

[0069] (e) generating one or more parameter scores for each random nucleotide sequence based on the score functions determined for the corresponding one or more parameters;

[0070] (f) generating a total score for each random nucleotide sequence based on the one or more parameter scores generated for that random nucleotide sequence;

[0071] (g) assigning a rank to each random nucleotide sequence in the set of random nucleotide sequences based on the generated total scores of the random nucleotide sequences from a lowest rank to a highest rank; and removing, from the plurality of nucleotide sequences, one or more of the lowest ranked random nucleotide sequences based on the assigned ranking of each random nucleotide sequence, to provide a reduced plurality of nucleotide sequences;

[0072] (h) performing a plurality of iterative steps including repeating steps (d) to (g), wherein the plurality of nucleotide sequences in step (d) in an iterative step corresponds to the reduced plurality of nucleotide sequences of step (g) in the previous iterative step; (i) wherein step (h) is performed until a stop condition is met to provide a set of optimised RNA sequences, wherein each optimised RNA sequence has a total score above a score threshold; wherein each optimised RNA sequence comprises at least one different sequence property.

[0073] In certain embodiments, the random nucleotide sequence assigned with the lowest rank has a total score indicative of the one or more parameter scores having the greatest degree of deviation from the target values associated with the respective one or more parameters and the random nucleotide sequence assigned with the highest rank has a total score indicative of the one or more parameter scores having the smallest degree of deviation from the target values associated with the respective one or more parameters.

[0074] In certain embodiments, the method further comprises identifying the highest ranked random nucleotide sequence for the set of random nucleotide sequences in a first iterative step and for the set of random nucleotide sequences in a second iterative step, the first and second iterative steps among the plurality of iterative steps; and determining a difference in the total score of the highest ranked random nucleotide sequence in the first iterative step and the second iterative step; comparing the difference in the total score of the highest ranked random nucleotide sequence in the first iterative step and the second iterative step with a difference threshold, wherein the stop condition comprises one or more of: a predetermined number of iterations; based on the comparison, the difference in the total score of the highest ranked random nucleotide sequence in the first iterative step and the second iterative step being below the difference threshold; and at least one selected random nucleotide sequence has a total score above an optimisation threshold.

[0075] In certain embodiments, the user defined objectives comprise one or more of target application, biological activity, expression profile, manufacturability, secretion profile and / or storability.

[0076] In certain embodiments, the method further comprises: selecting one or more sequence elements from a database based on the one or more user defined objectives and the one or more parameters; optionally wherein the sequence elements comprise one or more of a 5’-UTR, a 3’-UTR, a poly(A) tail length, signal peptide sequence, an aptamer sequence, protein binding sequences, tandem stop codon and / or nucleic acid binding sequences. combining the selected sequence elements with each optimised RNA sequence to provide a set of full-length mRNA sequences; and outputting a signal indicative of a set of optimised full-length mRNA sequences, wherein the set of optimised full-length mRNA sequences comprises full-length mRNA sequences which comprise at least one optimised RNA sequence which meets the score threshold of step (i) above.

[0077] In certain embodiments, the set of optimised full-length mRNA sequences comprises full- length mRNA sequences which comprise an optimised RNA sequence which has a total score below the score threshold of step (i), the method further comprises repeating steps (d) to (i) above for the optimised full-length mRNA sequences; and outputting a signal indicative of a set of optimised full-length mRNA sequences, comprising the optimised full-length mRNA sequence identified as having the highest total score.

[0078] In certain embodiments, the: target application comprises one or more of: administration route, end user purpose, target tissue type, target cell type, cellular localisation, and / or cellular processing ; codon composition efficiency is determined using one or more of: codon decoding times, codon preference, codon usage frequency, codon usage patterns, abundance of tRNA isoacceptors and / or ratio of cognate to near-cognate tRNAs; specified nucleotide sub-sequences comprise: frameshifting sequences, ribosomal slippery sequences, transcription terminator sequences, RNA polymerase stalling sequences, aptamer sequences, secondary structure forming sequences, restriction enzyme sites, RNA binding protein binding sites and / or frameshifted premature termination codons; biological activity comprises one or more of: immunogenicity, cellular interactions, adjuvant activity, enzymatic activity and / or cellular effects; expression profile comprises expression duration, expression location and / or expression level; manufacturability comprises yield of encoded protein; secretion profile comprises localisation of the RNA and / or encoded protein in a target cell; and / or storability comprises stability of the RNA at predetermined environmental conditions.

[0079] In certain embodiments, step (g) further comprises combining n nucleotides of the 5' end of at least one first removed nucleotide sequence with L - n nucleotides of the 3’ end of a second removed nucleotide sequence to provide a mixed sequence; and / or randomly mutating at least one of the removed nucleotide sequences to generate a randomly mutated sequence, carrying out steps (e) to (g) for each randomly mutated sequence and selecting at least one optimised randomly mutated sequence based on the score threshold.

[0080] In certain embodiments, in the plurality of iterative steps of step (h), the set of random nucleotide sequences in a given iterative step is selected to include the non-removed nucleotide sequences present from the set of random nucleotide sequences in the previous iterative step and one or more nucleotide sequences that were not included in the set of random nucleotide sequences in the previous iterative step, wherein the set of random nucleotide sequences in the given iterative step further comprises one or more mixed sequences and / or optimised randomly mutated sequences.

[0081] In some embodiments, the set of random nucleotide sequences comprises one or more seeding nucleotide sequences, wherein each seeding nucleotide sequence comprises a nucleotide sequence that has a predetermined parameter score for one of the one or more parameters and encodes the target translated protein sequence.

[0082] In a nineteenth aspect there is provided, computer readable instructions which, when executed by one or more processors, cause the one or more processors to perform the method of the eighteenth aspect.

[0083] In a twentieth aspect there is provided, an apparatus for designing an optimised RNA sequence for translation, the apparatus comprising: a memory arranged to store machine-readable instructions; an input unit arranged to receive an input; and processing circuitry arranged to operably execute the stored machine-readable instructions to: (a) receive, via the input unit, a target translated protein sequence, one or more user- defined objectives for the optimised RNA sequence, and one or more parameters associated with each user-defined objective and corresponding to the target translated protein sequence, the one or more parameters including: codon composition efficiency fortranslation of the RNA sequence to the target translated protein sequence; presence or absence of specified nucleotide repeats;

[0084] GC content; and desirability of specified nucleotide sub-sequences;

[0085] (b) receive, via the input unit, deviation information for each parameter, the deviation information for processing a nucleotide sequence and indicative of a degree of deviation of a nucleotide sequence from a target value of the associated parameter;

[0086] (c) determine a score function for each parameter based on the deviation information, wherein each score function is usable for processing a nucleotide sequence to generate a score indicative of a degree of deviation between the nucleotide sequence and the target value of the parameter associated with that score function;

[0087] (d) select, from amongst a plurality of nucleotide sequences, a set of random nucleotide sequences, wherein each random nucleotide sequence comprises a nucleotide sequence that encodes the target translated protein sequence;

[0088] (e) generate one or more parameter scores for each random nucleotide sequence based on the score functions determined for the corresponding one or more parameters;

[0089] (f) generate a total score for each random nucleotide sequence based on the one or more parameter scores generated for that random nucleotide sequence;

[0090] (g) assign a rank to each random nucleotide sequence in the set of random nucleotide sequences based on the generated total scores of the random nucleotide sequences from a lowest rank to a highest rank; and remove, from the plurality of nucleotide sequences, one or more of the lowest ranked random nucleotide sequences based on the assigned ranking of each random nucleotide sequence, to provide a reduced plurality of nucleotide sequences; (h) perform a plurality of iterative steps including repeating steps (d) to (g), wherein the plurality of nucleotide sequences in step (d) in an iterative step corresponds to the reduced plurality of nucleotide sequences of step (g) in the previous iterative step,

[0091] (i) wherein step (h) is repeated until a stop condition is met to provide a set of optimised RNA sequences, wherein each optimised RNA sequence has a total score above a score threshold. wherein each optimised RNA sequence comprises at least one different sequence property.

[0092] In a twenty-first aspect there is provided, a system for designing an optimised RNA sequence for translation, the system comprising: the apparatus of the twentieth aspect; and a second apparatus comprising a database including one or more sequence elements, wherein the apparatus of the twentieth aspect is configured to retrieve the one or more sequence elements from the second apparatus for providing a set of full-length mRNA sequences.

[0093] In some embodiments, the set of random nucleotide sequences comprises one or more seeding nucleotide sequences wherein each seeding nucleotide sequence comprises a nucleotide sequence that has a predetermined parameter score for one of the one or more parameters and encodes the target translated protein sequence.

[0094] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0095] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0096] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0097] Various aspects of the invention are described in further detail below. Brief description of the Figures

[0098] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0099] Figure 1 shows FQ (FQ RNA corresponds to an RNA where codons have been selected based on near-cognate:cognate ratios using methods described herein) coding sequence variant increases nluc mRNA translation efficiency. STA, FQ, MIN, or GS nLuc mRNAs were transfected into HEK293T cells and translation efficiency assessed by nLuc protein synthesis (n=3 replicated experiments). A shows data for unmodified mRNAs and B, (N)1- methylpseudouridylated mRNAs. Data are mean +SE secreted luciferase activity in cell culture medium normalised to STA nLuc levels.

[0100] Figure 2 shows mRNA coding sequence optimisation increases mRNA vaccination efficacy in mice. A, Schematic showing mRNA construct design containing the receptor binding domain of SARS-CoV-2, the influenza HA transmembrane domain and an MHC class I epitope (SIINFEKL - SEQ ID NO: 72). B, Neutralising antibody titres at 50% inhibition (NT50) against wildtype SARS-CoV-2 1 week after immunisation with a 20 pg dose of ST- RBD mRNA or FQ-RBD mRNA. C, Representative plots (left) and summary data (right) showing production of IFNy produced by CD8+ splenocytes from mice vaccinated with 20 pg mRNA in response to SIINFEKL (SEQ ID NO: 72) stimulation. D, Neutralising antibody titres at 50% inhibition (NT50) against wildtype SARS-CoV-2 1 week after final immunisation from mice immunised with 2 doses of 4 pg ST-RBD mRNA or FQ-RBD mRNA 3 weeks apart. E, Representative plots (left) and summary data (right) showing production of IFNy produced by CD8+ splenocytes from mice vaccinated with 2 doses of 4 pg mRNA 3 weeks apart in response to SIINFEKL (SEQ ID NO: 72) stimulation. Statistically significant differences between groups were determined by one-way ANOVA.

[0101] Figure 3 shows isoacceptor tRNA expression in target cells. A, HEK293T or B, primary human skeletal muscle cells. Data are transcripts per million for aggregated isodecoder tRNA read counts.

[0102] Figure 4 shows isoacceptor tRNA-based sequence optimisation increases nluc mRNA translation efficiency. A, Coding sequence-optimised nLuc mRNA (ISOpt) or GS nLuc mRNA were transfected into HEK293T cells and translation efficiency assessed by nLuc protein synthesis (n=3 replicated experiments). B, An nLuc mRNA optimised for expression in SKMC cells (SKMC-Opt, n=4), or control nLuc mRNA (GS, n=3), were transfected into primary SKMCs and translation efficiency assessed by nLuc protein synthesis. Data are mean secreted luciferase activity in cell culture medium normalised to GS nLuc levels (A), or raw RLU (B). P<0.05, Welch’s unpaired one-tailed T-test. Figure 5 shows effects of ER signal peptide on nLuc mRNA expression in Huh7, HEK293T, and SKMCs. nLuc mRNAs encoding different signal peptide sequences were transfected into A, Huh7, B, HEK293T, or C, SKMCs and expression assessed by nluc protein synthesis (n=6 replicated experiments). Data are mean+SE secreted luciferase activity in cell culture medium normalised to NT nLuc levels.

[0103] Figure 6 shows impact of specific cell-derived UTRs on nLuc expression from / V mRNAs in different cell types. nLuc mRNAs containing different skeletal muscle-origin untranslated region (UTR) sequences were transfected into A, SKMCs, B, HEK293T, or C, HuH7s. nLuc mRNAs containing different liver-origin UTR sequences were transfected into D, Huh7, E, HEK293T, or F, SKMCs. A-F, mRNA translation efficiency was assessed by nluc protein synthesis (n=3 replicated experiments). Data are mean+SE secreted luciferase activity in cell culture medium normalised to control nLuc levels (CON).

[0104] Figure 7 shows Poly(A) tail length influences nLuc protein expression from IVTmRNAs. A, Estimated polyA tail length for E.coli poly(A) polymerase units (U) under standard reaction conditions. B, nLuc mRNAs were polyadenylated using the indicated units of E.coli poly(A) polymerase, purified, were transfected into HEK293T cells, and expression assessed by nluc protein synthesis. Data are mean+SE secreted luciferase activity in cell culture medium normalised to 0 U poly(A) polymerase nLuc levels. C, nLuc mRNAs were synthesised containing either m7G-5'ppp5’GpG or m7G5'ppp5'2'OMeApG, purified, transfected into SKMCs, and expression assessed by nluc protein synthesis (n=4 replicated experiments). Data are mean secreted luciferase activity in cell culture medium after 24 hours. P<0.05, Welch’s unpaired one-tailed T-test.

[0105] Figure 8 shows overview of sequence optimisation method. A, Examples of possible functions relating sequence parameters to scores. In each case the user-defined objective is to meet the parameter value (for example, GC content) indicated by the red line, at which point the score is 1. The different functions define how quickly movement away from the target parameter set by the objective is penalised. B, Flow chart illustrating key processes in assembling high-performing sequences for mRNA sequence optimisation.

[0106] Figure 9 shows a method 900 of designing an optimised RNA sequence for translation according to embodiments of the disclosure.

[0107] Figure 10 shows an apparatus 1002 for designing an optimised RNA sequence for translation in accordance with embodiments of the disclosure.

[0108] Figure 11 shows affinity measurements can identify preferred codons. A) RNA oligonucleotides containing specific anticodon (Top line) and codon (bottom line) sequences coupled to anchor nucleotides (grey) were annealed (UCAGUCGAUAI (SEQ ID NO: 83) - bold underline = anchor), and the affinity between codons and anticodons determined using temperature-dependent CD spectroscopy. B) affinity measurements for modified and unmodified AUU (lie) codons and cognate (UAI) and near-cognate (GAA) tRNA anticodons. The uridine modification, reduces the difference in binding affinity between the cognate and near-cognate anticodon with an AUU codon, indicating that this codon is less preferred in modified sequences compared to unmodified ones. C, expression levels of a model sequence (nanoLuciferase) derived from modified or unmodified, in vitro transcribed RNAs transfected into HEK293 cells. The RNA sequences are identical except for the use of isoleucine encoding codons, which are either AUU or AUG. The results demonstrate that codomanticodon affinity measurements can be used to identify preferred codons for use in sequence design strategies.

[0109] Figure 12 shows UGA stop codon is sufficient to prevent full protein expression but not to prevent T cell antigen presentation, (a) Distribution of stop codons encoded in the +1 frame of SARS-CoV-2 viral spike sequence (top) and Pfizer BioNTech mRNA vaccine spike sequence (bottom), (b) Schematic showing FLuc and FLuc U*GA mRNA constructs and how they are translated in frame or in the +1 frame, (c) Western blot (anti-Flag epitope) of lysate of untransfected cells, cells transfected with FLuc mRNA, or cells transfected with FLuc U*GA mRNA. Asterisk identifies a band at the 65.5 KDa the molecular weight of full length FLuc protein. Blot was also stained for beta actin as a loading control, (d) Luciferase activity from cell lysates of untransfected cells, cells transfected with FLuc or FLuc U*GA constructs. FLuc versus FLuc U*GA, P<0.0001 (one-way ANOVA) (e) Proportion of CD8+ OT-I T cells activated (CD69+) when incubated with untransfected cells or cells transfected with FLuc or FLuc U*GA modified mRNA. (f) Proportion of SIINFEKL (SEQ ID NO: 72) Multimer+ CD8+ splenocytes of mice that were immunised with FLuc mRNA or FLuc U*GA mRNA. FLuc versus FLuc U*GA, / ^0.0004 (one-way ANOVA) (g) Proportion of SIINFEKL (SEQ ID NO: 72) Multimer+ T cells identified in spleen of mice immunised with FLuc mRNA or FLuc- mRNA, that are terminally differentiated KLRG1+CD127_. Representative plots are shown on the left and summary data is shown on the right, (h) IFN-y production of splenocytes when incubated with or without SIINFEKL (SEQ ID NO: 72). Representative are shown on the left and summary statistics of median fluorescence intensity of IFN-y+ cells on the right, (i) Peripheral blood mononuclear cells (PBMC) IFN-y ELISpot responses from human donors vaccinated with AstraZeneca ChAdOxI nCoV-19 or Pfizer BNT162b2 stimulated with peptides covering the Pfizer 3’ UTR sequence.

[0110] Figure 13 shows an vitro system for antigen evaluation from modified mRNA therapeutics, (a) HEK293T cells and sorted HEK293T cells transduced with an H-2Kbexpressing plasmid stained with an anti-H-2Kbantibody. Representative histograms are shown on the left and summary data is shown on the right. HEK293T versus HEK293T H- 2Kb, P<0.0001 (unpaired t test) (b) H-2Kbtransduced HEK293T cells incubated with or without SIINFEKL (SEQ ID NO: 72) stained with the 25-D1.15 antibody clone specific for the SIINFEKL (SEQ ID NO: 72) presented on H-2Kb. Representative histograms are shown on the left and summary data is shown on the right. Untreated versus +SIINFEKL (SEQ ID NO: 72), P<0.0001 (unpaired t test) (c) H-2Kb transduced HEK293T cells transfected with the listed modified mRNA constructs stained with the 25-D1.16 antibody. Representative histograms are shown on the left and summary data is shown on the right. No SIINFEKL (SEQ ID NO: 72) versus In frame SIINFEKL (SEQ ID NO: 72), P<0.0001; No SIINFEKL (SEQ ID NO: 72) versus 3x In frame SIINFEKL (SEQ ID NO: 72), P<0.0001 , In frame SIINFEKL (SEQ ID NO: 72) versus 3x In frame SIINFEKL (SEQ ID NO: 72), P<0.0001 (oneway ANOVA). (d) Schematic of the co-culture system in which OT-I cells are layered 24 hours after transfection of H-2Kbtransduced HEK293T cells with modified mRNA constructs. Intended (in frame) and mistranslated (+1 frame and 3’UTR) T cell antigens can be detected by measuring CD69 upregulation on OT-I CD8+ T cells (e) Proportion of CD8+ OT1 T cells activated (CD69+) when incubated with untransfected cells or cells transfected with listed modified mRNA constructs. Representative plots are shown on the left and summary data is shown on the right. No SIINFEKL (SEQ ID NO: 72) versus In frame SIINFEKL (SEQ ID NO: 72), P<0.0001; No SIINFEKL (SEQ ID NO: 72) versus 3x In frame SIINFEKL (SEQ ID NO: 72), P0.0001, In frame SIINFEKL (SEQ ID NO: 72) versus 3x In frame SIINFEKL (SEQ ID NO: 72), P<0.0001 (one-way ANOVA).

[0111] Figure 14 shows CD8+ T cells specific for +1 encoded antigen can limit repetitive doses. Proportion of SIINFEKL (SEQ ID NO: 72) Multimer+ CD8+ T cells in the draining lymph node (a) or muscle (b), taken from mice 24 hours or 48 hours after vaccination with modified mRNA COVID-19 vaccines with either SIINFEKL (SEQ ID NO: 72) encoded in the +1 frame (+1 SIINFEKL (SEQ ID NO: 72)) or not encoded (No SIINFEKL (SEQ ID NO: 72)). dLN No SIINFEKL (SEQ ID NO: 72) (48h) versus +1 SIINFEKL (SEQ ID NO: 72) (48h), / ^0.0256; muscle +1 SIINFEKL (SEQ ID NO: 72) (24h) vs +1 SIINFEKL (SEQ ID NO: 72) (48h), P=0.0398; No SIINFEKL (SEQ ID NO: 72) (48h) versus +1 SIINFEKL (SEQ ID NO: 72) (48h), P=0.0003 (one-way ANOVA). (c) Comparative phenotype of SIINFEKL (SEQ ID NO: 72) Multimer+ cells in the draining lymph node and muscle from mice 24 hours or 48 hours after vaccination with +1 SIINFEKL (SEQ ID NO: 72) or No SIINFEKL (SEQ ID NO: 72) constructs. Draining lymph node is shown in orange and muscle is shown in purple. Representative plots are shown on the left and data from individual mice is displayed on the right. CD127+KLRG1- 24h dLN versus muscle, / ^0.02; CD127+KLRG1- 48h dLN versus muscle, P=0.0027; CD127-KLRG1+ 24h dLN versus muscle, P=0.0201; CD127-KLRG1 + 48h dLN versus muscle, P=0.0029 (paired t test), (d) Immunofluorescent staining of FFPE sections of muscle taken 48 hours after immunisation, with either No SIINFEKL (SEQ ID NO: 72) (top) or +1 SIINFEKL (SEQ ID NO: 72) (bottom), (e) RBD titre 21 days after transfer of OT-I or WT effector T cells and immunisation with either No SIINFEKL (SEQ ID NO: 72) or +1 SIINFEKL (SEQ ID NO: 72) constructs. No SIINFEKL (SEQ ID NO: 72) (+OT-I transfer) versus +1 SIINFEKL (SEQ ID NO: 72) (+OT-I transfer), P=0.0307; +1 SIINFEKL (SEQ ID NO: 72) (+OT-I transfer) versus +1 SIINFEKL (SEQ ID NO: 72) (+WT transfer), / ^0.0254; +1 SIINFEKL (SEQ ID NO: 72) (+OT-I transfer) versus No SIINFEKL (SEQ ID NO: 72) (WT transfer), P=0.0033 (one-way ANOVA) (f) SARS-CoV-2 live virus neutralisation titre from mice in receipt of 3 weekly doses of either No SIINFEKL (SEQ ID NO: 72) or +1 SIINFEKL (SEQ ID NO: 72) COVID- 19 vaccines. Day 42 No SIINFEKL (SEQ ID NO: 72) versus +1 SIINFEKL (SEQ ID NO: 72), P= 0.0266; day 63 No SIINFEKL (SEQ ID NO: 72) versus +1 SIINFEKL (SEQ ID NO: 72), P=0.00966 (Mann-Whitney tests).

[0112] Figure 15 shows strategies to prevent the expression of antigens encoded in the +1 frame, (a) Proportion of CD8+ OT-I T cells activated when incubated with cells transfected with modified mRNA COVID-19 vaccines with either SIINFEKL not encoded (No SIINFEKL), encoded in the +1 frame (+1 SIINFEKL), or with an equivalent to the U*208C synonymous mutation previously reported to remove the slippery site1(U*925C). (b) Proportion of CD8+ OT1 T cells activated when incubated with cells transfected with modified mRNA COVID-19 vaccines with SIINFEKL encoded in frame immediately after in frame U*GA, U*AG or U*AA stop codons. U*GA versus U*AA, P<0.0001; U*GA versus U*AG, P<0.0001 (one-way ANOVA) (c) Proportion of CD8+ OT1 T cells activated when incubated with cells transfected with modified mRNA COVID-19 vaccines with +1 frame encoded SIINFEKL immediately after +1 frame encoded single or tandem U*AG or U*AA stop codons. U*AA versus U*AAU*AA, P=0.0289; U*AA versus U*AG, P=0.0469; U*AA versus U*AGU*AG, P=0.0026 (one-way ANOVA). (d) Proportion of CD8+ T cells IFN-y+ after SIINFEKL restimulation of splenocytes from mice immunised with modified mRNA COVID-19 vaccines with +1 frame encoded SIINFEKL immediately after +1 frame encoded single or tandem U*AA stop codons compared to the No SIINFEKL vaccine. Representative plots are shown on the left and summary data is shown on the right. +1 SIINFEKL versus U*AA, P=0.0174; +1 SIINFEKL versus U*AAU*AA, P=0.0004 (one-way ANOVA).

[0113] Figure 16 shows a modified mRNA COVID- 19 vaccine without evidence of cryptic antigen expression encoded in either the +1 frame or 3’ UTR. (a) Schematic showing modified mRNA COVID-19 vaccines with SIINFEKL encoded in the +1 frame (+1 SIINFEKL), optimised with the insertion of multiple synonymous changes leading to +1 encoding of single or tandem U*AA, or U*AG stops with SIINFEKL encoded in the +1 frame (Optimised +1 SIINFEKL) or optimised with the insertion of multiple synonymous changes leading to +1 encoding of single or tandem U*AA, or U*AG stops without SIINFEKL encoded (Optimised), (b) Proportion of CD8+ OT-I T cells activated when incubated with cells transfected with PfizerRBD+SHNFEKL, Optimised+SIINFEKL or Optimised modified mRNA COVID-19 vaccines. PfizerRBD+SHNFEKL versus Optimised+SIINFEKL, P<0.0001 ;

[0114] PfizerRBD+SHNFEKL versus Optimised, P<0.0001 (one-way ANOVA). Proportion of CD8+ T cells (c) SIINFEKL Multimer+ in the muscle or spleen or (d) IFN-y+ after restimulation of splenocytes with ± SIINFEKL from mice immunised with PfizerRBD+SHNFEKL, Optimised+SIINFEKL or Optimised modified mRNA COVID-19 vaccines. Muscle PfizerRBD+SHNFEKL versus Optimised+SIINFEKL, / ^0.0235; PfizerRBD+SHNFEKL versus Optimised, P=0.0341 ; Spleen PfizerRBD+SHNFEKL versus Optimised+SIINFEKL, P=0<0.0001 ; PfizerRBD+SHNFEKL versus Optimised, P<0.0001 (one-way ANOVA) (e) Proportion of CD8+ T cells expressing activation markers in an activation induced marker (AIM) assessment after stimulation with an overlapping peptide pool covering the RBD in splenocytes from mice immunised with PfizerRBD+SHNFEKL, Optimised+SIINFEKL or Optimised modified mRNA COVID-19 vaccines. %CD25+ T cells PfizerRBD+SHNFEKL versus Optimised+SIINFEKL, P=0.0007; PfizerRBD+SHNFEKL versus Optimised, P=0.0084; % CD69+ T cells PfizerRBD+SHNFEKL versus Optimised+SIINFEKL, P=0.0017 (one-way ANOVA) (f) SARS-CoV-2 live virus neutralisation titre from mice immunised with PfizerRBD+SHNFEKL or Optimised+SIINFEKL modified mRNA COVID-19 vaccines. IFN-y ELISpot responses from splenocytes immunised with BNT162b2 or Optimised+SIINFEKL modified mRNA COVID-19 vaccines stimulated with overlapping peptide pools covering (g) the +1 frame of the RBD sequence or (h) 3’UTR of the Pfizer sequence, (g) BNT162b2 versus Optimised+SL8, P=0.0238 (unpaired t test); (h) BNT162b2 versus Optimised+SIINFEKL, P=0.0246 (unpaired t test).

[0115] Figure 17 shows schematics of the mRNA non-coding and coding sequences of SARS- CoV-2 RDB domain containing different UTRs derived from muscle or liver and containing an MHC class I epitope (SIINFEKL - SEQ ID NO: 72).

[0116] Figure 18 shows B cell antigen response determined by flow cytometry using the different UTRs and RBD-SIINFEKL constructs described in Figure x and the SIINFEKL antigen assay.

[0117] Figure 19 shows the percentage of cells showing a positive T cell antigen response using the different UTRs and RBD-SIINFEKL constructs described in Figure 17.

[0118] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail.

[0119] Various aspects of the invention are described in further detail below.

[0120] Detailed Description

[0121] Provided herein are nucleic acid molecules that encode optimized mRNAs. The nucleic acid molecules may be DNA (e.g. a transcription template) or RNA. The term mRNA refers to an RNA molecule that encodes a protein. mRNA may refer to a ribonucleic acid molecule (RNA) that has been transcribed from a DNA sequence by an RNA polymerase enzyme, and interacts with a ribosome to synthesize protein encoded by DNA. Generally, mRNA is classified into two sub-classes: pre-mRNA and mature mRNA. Precursor mRNA (pre-mRNA) is mRNA that has been transcribed by RNA polymerase but has not undergone any post- transcriptional processing (e.g., 5'capping, splicing, editing, and polyadenylation) and may therefore include 5’ untranslated region (UTR), introns and / or one or more 3’ UTRs. Mature mRNA has been modified via post-transcriptional processing (e.g., spliced to remove introns and polyadenylated region) and is capable of interacting with ribosomes to perform protein synthesis. The particular nucleic acid molecule sequence composition and length of an mRNA will depend on the protein encoded by the mRNA. Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5'IITR, a 3'IITR, a 5' cap and a poly-A tail. In vitro transcribed (IVT) mRNA may function as mRNA but is distinguished from wild-type mRNA in their functional and / or structural design features, which serve to overcome existing problems of effective polypeptide production using nucleic- acid based therapeutics. For example, IVT mRNA may be chemically modified. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”.

[0122] The optimized mRNAs described herein may be referred to as “modified”. In the context of the invention, a modified optimized mRNA refers to an optimized mRNA that has been modified to introduce chemically modified nucleotides and / or genetic (nucleic acid molecule sequence) modifications.

[0123] The optimized mRNAs described herein may comprise naturally occurring ribonucleotides and / or non-naturally occurring ribonucleotides (e.g. canonical nucleotides) such as chemically modified nucleotides. In some examples, the optimized mRNAs provided herein may include at least one chemically modified ribonucleotide. In some examples, the chemically modified ribonucleotide may be selected from the group consisting of pseudouridine, N1-methylpseudouridine (l-methyl^P), 2-thiouridine, 4'-thiouridine, 5- methylcytosine, 2-thio-l-methyl-1-deaza-pseudouridine, 2-thio-1-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-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and 2 -0- methyl uridine. Other exemplary chemical modifications useful in optimized mRNAs described herein include those listed in US Published patent application 2015 / 0064235 which is incorporated herein.

[0124] In some examples, the optimized mRNAs provided herein include at least one N1- methylpseudouridine.

[0125] The optimized mRNAs provided herein may be pre-mRNAs or mature mRNAs. In some examples, the optimized mRNAs provided herein may include one or more features of a pre- mRNA but not all features of a pre-mRNA. For example, the optimized mRNAs provided herein may include a poly-adenylation, a 5’UTR and a 3’ UTR sequence but not include introns. In some examples, the optimized mRNAs provided herein may include one or more features of a mature mRNA, but not all features of a mature mRNA

[0126] In some examples, the optimized mRNAs provided herein (or nucleic acid molecules encoding said optimized mRNAs) are therapeutic mRNAs (i.e. therapeutic optimized mRNAs). “Therapeutic mRNA” refers to an mRNA molecule (e.g., an in vitro transcribed (IVT) mRNA) that encodes a therapeutic protein. Therapeutic proteins mediate a variety of effects in a host cell or a subject in order to treat a disease or ameliorate the signs and symptoms of a disease. For example, a therapeutic protein can replace a protein that is deficient or abnormal, augment the function of an endogenous protein, provide a novel function to a cell (e.g., inhibit or activate an endogenous cellular activity, or act as a delivery agent for another therapeutic compound (e.g., an antibody-drug conjugate). Therapeutic mRNA may be useful for the treatment or prevention of the following diseases and conditions: infectious diseases (such as bacterial infections, viral infections, parasitic infections), cell proliferation disorders (such as cancer), genetic disorders, inflammatory disease, cardiovascular disorders, metabolic diseases, allergic disease, neurodegenerative diseases, protein or enzyme deficiency disorder and / or autoimmune diseases.

[0127] Examples of therapeutic mRNAs are Pfizer and BioNtech’s BNT162b2 (Covid-19), Moderna’s mRNA-1273 (Covid-19), mRNA-2416 (solid tumour or lymphoma), MRT5005 (cystic fibrosis), mRNA-2752 (solid tumour or lymphoma), AZD-8601 (heart failure), NY- ESO-1 (multiple myeloma, synovial sarcoma, melanoma), CTX001 (P-thalassemia), SB- 728mR-HSPC (HIV ), SB-728mR-T (HIV), BNT163 (HSV2), BNT164 (tuberculosis), BNT165 (malaria), BNT167 (shingles), BNT161 (influenza), BNT153 (undisclosed cancers), BNT152 (undisclosed cancers), BNT142 (undisclosed cancers), BNT141 (undisclosed cancers), BNT131 (undisclosed cancers), BNT122 (melanoma), colorectal cancer, BNT116 (non-small cell lung carcinoma), BNT115 (ovarian cancer), BNT113 (head and neck cancer), BNT112 (prostate cancer), BNT111 (melanoma), mRNA-1345 (Respiratory syncytial virus), mRNA- 1010 (influenza), mRNA-1647 (cytomegalovirus), mRNA-4157 / V940 (melanoma), mRNA- 3927 (Propionic acidemia), mRNA-0184 (heart failure), VX-522 (cystic fibrosis). The uses of each mRNA therapy are shown in brackets.

[0128] Therapeutic mRNA molecules are generally synthesized in a laboratory (e.g., by in vitro transcription). mRNA can be isolated from tissues or cells by a variety of methods. For example, a total RNA extraction can be performed on cells or a cell lysate, and the resulting extracted total RNA can be purified (e.g., on a column comprising oligo-dT beads) to obtain extracted mRNA. Alternatively, mRNA can be synthesized in a cell-free environment, for example, by in vitro transcription (IVT). IVT is a process that permits template-directed (e.g. via an IVT DNA template) synthesis of a ribonucleic acid molecule (RNA) (e.g., messenger RNA (mRNA)). It is based, generally, on the engineering of a DNA template that includes a bacteriophage promoter sequence upstream of the sequence of interest, followed by transcription using a corresponding RNA polymerase. In vitro mRNA transcripts, for example, may be used as therapeutics in vivo to direct ribosomes to express protein therapeutics within targeted tissues.

[0129] An “in vitro transcription template (IVT),” as used herein, refers to deoxyribonucleic acid molecule (DNA) suitable for use in an IVT reaction for the production of messenger RNA (mRNA). In some examples, an IVT template encodes a 5' untranslated region, contains an open reading frame, and encodes a 3' untranslated region and a poly(A) tail. The particular nucleotide sequence composition and length of an IVT template will depend on the mRNA of interest encoded by the template. IVT mature mRNA preparation includes several steps, linear DNA template obtainment, IVT, 5' capping, and poly(A) tail adding.

[0130] In some examples, the nucleic acid molecules provided herein are in vitro transcription templates (i.e. DNA suitable for IVT) or transcription templates (i.e. DNA suitable for transcription in a subject).

[0131] A “5' untranslated region (UTR)” refers to a region of an mRNA that is directly upstream (i.e., 5') from the start codon (i.e., the first codon of an mRNA transcript translated by a ribosome) that does not encode a protein or peptide. IVT is performed by linearizing plasmid DNA templates or PCR templates requiring at least a promoter and the corresponding mRNA construct sequence. IVT may be carried out by adding polymerases (T7, T3, or SP6) but requires additional capping. Uncapped mRNA is rapidly degraded by RNase and contains a 5'-ppp group, which causes greater immune stimulation and can be treated with phosphatase to reduce undesirable efficacy. Two methods may be implemented for the capping of IVT mRNA: co-transcriptional capping and posttranscriptional capping. Cap dinucleotide mixtures containing four nucleoside triphosphates (NTPs) are incorporated at the 5' end of the RNA with RNA polymerase during co-transcriptional capping. Co- transcriptional capping processing has permitted coordinated transcription with mRNA capping. Poly(A) tails of IVT mRNAs are normally encoded in the DNA template or attached to IVT mRNA by enzymatic polyadenylation. The former may have more precise control of the length of the poly(A) tail. IVT mRNAs may be mixed with RNA polymerase and DNA templates after synthesis; thus, purification of IVT mRNA may be needed, including removing immunostimulatory contaminants, free ribonucleotides, short mRNA and DNA templates. Generally, Dnase is used to degrade excess DNA templates. Commercial purification kits may be used to purify and separate the synthesized mRNA, followed by precipitation using ethanol or isopropanol, which can remove most contaminants and obtain high-purity mRNA, and then the mRNA may be precipitated with high concentrations of LiCI or alcohol-based precipitation, chromatographic methods (molecular exclusion chromatography, ion-exchange chromatography, or affinity chromatography with immobilized oligo-dT), or elution from a silica membrane column, which removes proteins, free nucleotides or other components but not dsRNA impurities. Reversed-phase HPLC may be used to remove dsRNA contaminants from the transcription reaction solution.

[0132] A “3' untranslated region (UTR)” refers to a region of an mRNA that is directly downstream (i.e. , 3') from the stop codon (i.e. , the codon of an mRNA transcript that signals a termination of translation) that does not encode a protein or peptide.

[0133] A “polyA tail” is a region of mRNA that is downstream, e.g., directly downstream (i.e., 3'), from the 3' UTR that contains multiple, consecutive adenosine monophosphates. A polyA tail may contain 10 to 300 adenosine monophosphates. For example, a polyA tail may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosine monophosphates. In some examples, a polyA tail contains 50 to 250 adenosine monophosphates. In a relevant biological setting (e.g., in cells, in vivo, etc.), the poly(A) tail functions to protect mRNA from enzymatic degradation, e.g., in the cytoplasm, and aids in transcription termination, export of the mRNA from the nucleus, and translation. However, in some examples, mRNA molecules provided herein do not comprise a polyA tail (such molecules are referred to as “tailless”). An “open reading frame” is a continuous stretch of DNA or RNA beginning with a start codon e.g., methionine (ATG) and ending with a stop codon (e.g., TAA, TAG or TGA) and encodes a protein or peptide.

[0134] In some examples, the optimised mRNAs provided herein may provide an improved translation efficiency in comparison to a control mRNA. The control mRNA may be any mRNA that does not include at least one of the preferred codons, signal peptides, UTR pairs, optimised poly(A) tail and / or 5’ cap as described herein.

[0135] In some examples, the optimised mRNAs provided including any one or more of the preferred codons, signal peptides, UTR pairs, optimised poly(A) tail and / or 5’ cap as described herein may have an increased translation efficiency in a target cell as described herein of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 130%, 140%, 150% or 200% in comparison to a control mRNA.

[0136] For example, an mRNA comprising at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 or more amino acid codons which are preferred codons as described herein may have an increased translation efficiency in a target cell as described herein of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 130%, 140%, 150% or 200% in comparison to a control mRNA.

[0137] For example, an mRNA comprising a 5’ and 3’ UTR pair as described herein may have an increased translation efficiency in a target cell as described herein of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 130%, 140%, 150% or 200% in comparison to a control mRNA.

[0138] For example, an mRNA comprising an optimised poly(a) tail as described herein may have an increased translation efficiency in a target cell as described herein of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 130%, 140%, 150% or 200% in comparison to a control mRNA.

[0139] For example, an mRNA comprising a nucleic acid molecule sequence encoding a signal peptide as described herein may have an increased translation efficiency in a target cell as described herein of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,

[0140] 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,

[0141] 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%,

[0142] 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,

[0143] 120%, 130%, 140%, 150% or 200% in comparison to a control mRNA.

[0144] For example, an mRNA comprising an optimised 5’ cap as described herein may have an increased translation efficiency in a target cell as described herein of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%,

[0145] 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%,

[0146] 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 130%, 140%, 150% or 200% in comparison to a control mRNA.

[0147] In some examples, the optimised mRNAs provided herein may provide an improved translation fidelity in comparison to a control mRNA. The control mRNA may be any mRNA that does not include at least one of the preferred codons, signal peptides, UTR pairs, optimised poly(A) tail and / or 5’ cap as described herein.

[0148] In some examples, the optimised mRNAs provided including any one or more of the preferred codons, signal peptides, UTR pairs, optimised poly(A) tail and / or 5’ cap as described herein may have an increased translation fidelity in a target cell as described herein of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%,

[0149] 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,

[0150] 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,

[0151] 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 130%,

[0152] 140%, 150% or 200% in comparison to a control mRNA.

[0153] For example, an mRNA comprising at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150 or more amino acid codons which are preferred codons as described herein may have an increased translation fidelity in target cell as described herein of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 130%, 140%, 150% or 200% in comparison to a control mRNA.

[0154] For example, an mRNA comprising a 5’ and 3’ UTR pair as described herein may have an increased translation fidelity in a target cell as described herein of at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 130%, 140%, 150% or 200% in comparison to a control mRNA.

[0155] For example, an mRNA comprising an optimised poly(a) tail as described herein may have an increased translation fidelity in a target cell as described herein of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 130%, 140%, 150% or 200% in comparison to a control mRNA.

[0156] For example, an mRNA comprising a nucleic acid molecule sequence encoding a signal peptide as described herein may have an increased translation fidelity in a target cell as described herein of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%,

[0157] 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,

[0158] 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%,

[0159] 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%,

[0160] 120%, 130%, 140%, 150% or 200% in comparison to a control mRNA.

[0161] For example, an mRNA comprising an optimised 5’ cap as described herein may have an increased translation fidelity in a target cell as described herein of at least 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120%, 130%, 140%, 150% or 200% in comparison to a control mRNA.

[0162] The nucleic acid molecules described herein may be isolated nucleic acid molecules. An “isolated” nucleic acid is substantially separated away from other nucleic acid sequences with which the nucleic acid is normally associated, such as from the chromosomal or extrachromosomal DNA of a cell in which the polynucleotide naturally occurs. The term also embraces nucleic acids that are biochemically purified so as to substantially remove contaminating nucleic acids and other cellular components. Isolated nucleic acids are substantially free of sequences (such as protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the polynucleotide) in the genomic DNA of the organism from which the nucleic acid is derived. For example, the isolated nucleic acid can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid in genomic DNA of the cell from which the nucleic acid is derived.

[0163] In some examples, the nucleic acid molecules are purified nucleic acid molecules. The term "purified," relates to the isolation of a molecule or compound in a form that is substantially free of contaminants normally associated with the molecule or compound in a native or natural environment, or substantially enriched in concentration relative to other compounds present when the compound is first formed, and means having been increased in purity as a result of being separated from other components of the original composition. The term "purified nucleic acid" is used herein to describe a nucleic acid sequence which has been separated, produced apart from, or purified away from other biological compounds including, but not limited to polypeptides, lipids and carbohydrates, while affecting a chemical or functional change in the component (e.g. a nucleic acid may be purified from a chromosome by removing protein contaminants and breaking chemical bonds connecting the nucleic acid to the remaining DNA in the chromosome).

[0164] Target Cells

[0165] In some examples, the target cell is a human cell. In some examples, the target cell is an in vitro cell, an ex vivo cell or an in vivo cell. For example, the levels of tRNA isoacceptors or anticodons thereof may be determined from a cell that has been obtained from a subject and is maintained in vitro or ex vivo. In some examples, the levels of tRNA isoacceptors or anticodons thereof may be determined from a sample obtained from a subject’s in vivo cell.

[0166] In some examples, the target cell may be selected from muscle cells, liver cells, kidney cells, immune cells, nervous system cells, fat cells, bone cells, blood cells and stem cells.

[0167] In some examples, the target cell may be a muscle cell such as a skeletal muscle cell, smooth muscle cell or cardiac cell. In some examples, the target cell is a skeletal muscle cell.

[0168] In some examples, the target cell may be a liver cell such as hepatocytes, parenchymal cells, non-parenchymal cells, sinusoidal endothelial cells, Kupffer cells, stellate cells, and lymphocytes. In some examples, the target cell is a hepatocyte. In some examples, the target cell may be a kidney cell, such as an embryonic kidney cell (such as HEK293 cell), specialized filtration cells (e.g., mesangial cells and podocytes), epithelial cells (e.g., tubule brush border cells, thin segment cells, thick ascending limb cells, distal tubule cells), specialized resorption cells (e.g., collecting duct principal cells, collecting duct intercalated cells), and renal interstitial cells (e.g., fibroblasts, immune cells, macrophages, dendritic cells, and perivascular cells). In some examples, the target cell is a human embryonic kidney cell.

[0169] In some examples, the target cell may be an immune cell, “immune cell” includes any cell within the immune system. Examples include white blood cells, such as lymphocytes (e.g. T lymphocytes or T cells, B cells or natural killer cells), dendritic cells, follicular dendritic cells and granulocytes.

[0170] In some examples, the target cell is an antigen-presenting cell. “Antigen-presenting cell” refers to cells capable of acquiring, processing, presenting, or displaying at least one antigen or antigenic fragment on (or at) its cell surface. In general, the term “antigen-presenting cell” can be any cell that aids the enhancement of an immune response or immune tolerance (e.g., from the T-cell or B-cell arms of the immune system) to an antigen. For example, a cell that displays or presents an antigen normally or preferentially with a class II major histocompatibility molecule or complex to an immune cell may be denoted as an “antigen-presenting cell”. Antigen-presenting cells include but are not limited to astrocytes, oligodendrocytes, microglia, macrophages, B cells, dendritic cells and precursors thereof.

[0171] In some examples, the target cell is a nervous system cell. Nervous system cells include nerve cells, neural stem cells and neural progenitor cells. Examples of nerve cells include inhibitory nerve cells, excitatory nerve cells, dopamin-producing nerve cells, cranial nerves, intervening nerves, and optic nerves. Alternatively, nervous system cells may be motor nerve cells, oligodendrocyte progenitor cells, astrocytes, oligodendrocytes or the like.

[0172] In some examples, the target cell is a bone cell such as osteoblasts, osteoclasts, and osteocytes.

[0173] In some examples, the target cell is a blood cell, such as red blood cells, platelets, monocytes, lymphocytes, neutrophils, eosinophils, basophils, and macrophages.

[0174] In some examples, the target cell is a stem cell. “Stem cell” refers to a multipotent cell having the capacity to self renew and to differentiate into multiple cell lineages. Stem cells include pluripotent stem cells, somatic stem cells, embryonic stem cells, induced pluripotent stem, hematopoietic stem cells, mesenchymal stem cells, neural stem cells, epithelial stem cells.

[0175] In some examples, the target cell is not a totipotent cell. In some examples, the target cell is a cardiac cell, such as a cardiomyocyte. “Cardiomyocyte” refers to heart muscle cells. Cardiomyocytes may be ventricular-, atrial-, and / or nodal-type cardiomyocytes, or a mixed population of cardiomyocytes.

[0176] Cardiomyocytes may exhibit one or more features including, capacity to beat spontaneously, are electrically mature, metabolically mature, contractility mature, exhibit appropriate expression of one or more gene markers (e.g., TNNI3, TNNT1, MYH6, MYH7, KCNJ2, RyR, and REST), exhibit appropriate expression of one or more quiescence markers (e.g., p16 and p130), and / or exhibit appropriate morphological characteristics (e.g., rod shaped cells and organized sarcomeres).

[0177] Optimised Codons

[0178] The nucleic acid molecules provided herein encode one or more codons that have been optimised using the methods described herein in order to provide improved translation efficiency and / or fidelity when translated in a cell.

[0179] Also provided are methods of producing nucleic acid molecules including preferred codons.

[0180] “Fidelity of translation” refers to the accuracy of translation of an mRNA. For example, the translation of the desired protein (comprising the amino acids encoded by the in-frame codons) which is produced by translation occurring in the intended reading frame (i.e. correct or in-frame translation). “Efficiency of translation” refers to the speed at which a protein is translated from an RNA molecule and / or the amount of protein translated from an RNA molecule.

[0181] The codons encoded by the nucleic acid molecules described herein may be selected using various methods described below. Codons that may have been selected for improved properties may be referred to herein as “preferred codons”. Preferred codon refers to a codon that encodes for the transcription of a desired amino acid that has been selected from other codons that encode for the transcription of the same amino acid.

[0182] The preferred codons may be selected based on a level of tRNA isoacceptor expression in a target cell. Isoacceptor tRNAs refers to tRNAs that have different anticodons but still carry the same amino acid. A particular tRNA may be denoted herein according to its aminoacylating amino acid, which is indicated such as in tRNALeuor tRNA-Leu. A particular tRNA may additionally be denoted according to its anticodon, indicated, such as tRNALeu(CAG) or tRNA-Leu-CAG.

[0183] For example, the preferred codon encoding an amino acid may be selected based on the amount or abundance of each isoacceptor tRNA for the amino acid in the target cell. The level of tRNA isoacceptor for at least one or each amino acid may be determined using any suitable method. For example, using mRNA sequencing methods such as RNA sequencing (e.g. small RNA sequencing).

[0184] RNA sequencing may include the use of methods such as amplification (PCR) based methods (reverse transcriptase PCR (RT-PCR) and quantitative reverse transcriptase PCR (qRT- PCR), or RNAseq (next generation sequencing, also referred to as second generation sequencing or massively parallel sequencing). Methods of DNA sequencing are known and include methods similar to those used for determining mRNA sequences.

[0185] Next Generation Sequencing (NGS) (second generation sequencing or massively parallel sequencing; Mardis, E. R. (2008). As there are many NGS technologies available, there are small differences in the methodology for RNA sequencing. The following is a description of how RNA sequencing using NGS works in general. Total RNA is extracted from the sample of interest using a common RNA extraction method. Post-extraction processes can be used to enrich the RNA sample. Complimentary DNA (cDNA) is then synthesised using extracted RNA. cDNA is then used as the template for RNA sequencing. NGS uses variations of sequencing by synthesis (SBS) chemistry (Fuller, C. W., et al. (2009). The challenges of sequencing by synthesis. Nature biotechnology, 27(11), 1013-1023). With cDNA as a template, new nucleotide fragments, known as reads, are synthesised base by base, with each incorporated base recorded during sequencing (Fuller, 2009). The data output from RNA sequencing is a list of all the reads generated, and their sequence (Fuller, 2009 and Metzker, 2010). This data undergoes quality assessment (Patel, R. K., & Jain, M. (2012). NGS QC Toolkit: a toolkit for quality control of next generation sequencing data. PloS one, 7(2), e30619).

[0186] Small RNA sequencing is a technique to isolate and sequence small RNA species, such as tRNAs.

[0187] In some examples, the preferred codons are determined by determining the levels of expression of tRNA anticodons for at least one or each amino acid in a target cell. Determining the level of expression of tRNA anticodons may be done using any suitable methods known in the field. For example, determining levels of expression may utilise RNA sequencing methods described above. In addition, or alternatively, tRNA anticodon expression levels may be determined using RT-PCR, real time RT-PCR, next generation sequencing, western blot, dot blot, enzyme linked immunosorbent assay (ELISA). Differential expression of genes may be determined by performing RNA expression analysis. RNA may be extracted from a target cell and the level of RNA may be quantified by hybridisation of probes to provide a gene count. The level of expression or gene count, of each gene may then be normalised based on the expression levels of a number of housekeeping genes.

[0188] Determination of the amount of tRNA isoacceptors or expression levels of tRNA anticodons for tRNA isoacceptors may be used to select specific codons to be encoded in the nucleic acid. For example, the nucleic acid molecule may encode the codon for an amino acid that corresponds to the most abundant isoacceptor tRNA and anticodon thereof for the same amino acid detected in a target cell. For example, the nucleic acid molecule may encode the codon that corresponds to the most abundantly expressed tRNA anticodon for the same amino acid detected in a target cell.

[0189] In some examples, preferred codons may be determined by analysis of codon decoding speed or rate in a target cell. Codon decoding rate may be determined using methods such as ribosome profiling. Ribosome profiling may be used to determine the time, extent, rate and / or fidelity of ribosome decoding of a particular codon of an RNA, which in turn is determined by the amount of time a ribosome spends at a particular codon (also referred to as dwell time). The latter may be referred to as a "codon elongation rate" or a "codon decoding rate". Relative dwell time of ribosomes between two locations in an RNA, instead of the actual or absolute dwell time at a single location, can also be determined by comparing the number of sequencing reads of protected mRNA fragments at each location (e.g., a codon) (see O'Connor et al., (2016) Nature Commun 7: 12915).

[0190] In some examples, preferred codon selection may be determined using the ratio of nearcognate to cognate tRNA. Methods of determining the ratio of near-cognate to cognate tRNA can be found in “Chu, Dominique, David J. Barnes, and Tobias Von Der Haar. "The role of tRNA and ribosome competition in coupling the expression of different mRNAs in Saccharomyces cerevisiae." Nucleic acids research 39.15 (2011): 6705-6714”. In some examples, the preferred codon may be the codon with the lowest ratio of near-cognate to cognate tRNAs. The ratio might vary from zero to ~50, and the preferred codon may be the codon which has the lowest ratio of all codons encoding the same amino acid.

[0191] In some examples, preferred codons may be determined by analysis of the binding affinity between a codon and its cognate tRNA anticodon. For example, RNA oligonucleotides may be chemically synthesized using standard phosphoramidite chemistry. mRNA codon and tRNA anticodon sequence may be synthesised fused to standard anchor sequences (see Figure 11 A). A set of oligonucleotides comprising only the anchor sequences, without added codon / anticodon sequences, may be used as controls. Oligos may then be resuspended in buffer, and pairs of oligos containing codon and anticodon sequences are mixed. The mixed oligos are heated in defined temperature increments, and circular dichroism (CD) spectra are recorded after each temperature increase. Melting points are determined from the temperature-dependent CD spectra, by plotting the spectral shift at 265 nm against the temperature. For more details see “Wang, Xiaoyu, et al. "Biophysics of artificially expanded genetic information systems. Thermodynamics of DNA duplexes containing matches and mismatches involving 2-amino-3-nitropyridin-6-one (Z) and imidazo [1 , 2-a]-1 , 3, 5-triazin-4 (8H) one (P)." ACS Synthetic Biology 6.5 (2017): 782-792”.

[0192] In some examples, preferred codons may be determined by analysis of frequency with which codons are used in a genome. For example, the relative usage of all codons in the human genome is known, and more frequently used codons are usually found in more highly expressed mRNAs. Preferred codons may thus be determined by analysing which codons are most frequently used in natural genes.

[0193] It will be understood that the methods of determining preferred codons for a target cell may be carried out for at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids encoded by a nucleic acid molecule as described herein. For example, all the amino acids encoded by a nucleic acid molecule may be encoded by a preferred codon determined by one or more of the methods described above. In some examples, only a portion of the codons are preferred codons. In some examples, a target cell may not have a preferred codon for one or more amino acids and as such, any codon for that amino acid may be used. In some examples, an amino acid may have more than one preferred codon and the preferred codons encoded for each occurrence of the codon may include a single preferred codon or a mixture of preferred codons. In some examples, where there is no preferred codon or multiple preferred codons determined, the codon for the amino acid encoded by the nucleic acid molecule may selected based on other factors or properties. For example, selected based on one or more of the parameters such as target application or specified nucleotide subsequences as described herein.

[0194] After determination of preferred codons, the nucleic acid molecule may be produced incorporating the preferred codons using methods known in the field. For example, for the production of DNA molecules encoding optimised mRNAs as described herein, such as solidphase DNA synthesis and ligation methods, DNA assembly methods (e.g. using BioBricks, and Golden Gate cloning methods), long-overlap based assembly methods (such as Gibson assembly, MODAL, or BASIC).

[0195] Production of optimised mRNAs as described herein may done using the methods described above (i.e. de novo synthesis) or may be produced by in vitro transcription as described herein. For example, a DNA molecule encoding the optimised mRNA may be produced and used as a template for in vitro transcription. In some examples, the methods include adding a poly(A) tail, as described herein. For example, an optimised poly(A) tail as described herein (e.g. comprising about 97 to 100 adenine residues). In particular, the methods may include enzymatically adding a poly(A) tail, such as an optimised poly(A) tail as described herein. For example, the poly(A) tail may be added by contacting the mRNA with E.Coli poly(A) polymerase.

[0196] In some examples, the methods include capping the optimised mRNA molecule with a 5’ capping agent. For example, capping the optimised mRNA molecule using a m7G-5'ppp5’GpG 5’ cap or m7G5'ppp5'2'OMeApG. In particular, the optimised mRNA may be capped using a m7G-5'ppp5’GpG 5’ cap.

[0197] The methods may further include purification of the nucleic acid. For example, purification of the optimised mRNA. Methods of purifying nucleic acid molecules are well known and purification can be carried out using suitable commercially available kits. In some examples, purification is carried out using methods based on phenol-chloroform extraction, spin column purification, and magnetic bead-based method.

[0198] Muscle Cell Preferred Codons

[0199] As shown in the examples provided herein, preferred codons may be determined for specific target cell types. For example, the target cell may be a muscle cell, in such examples, the set of preferred codons may be referred to herein as “muscle cell preferred codons”. Muscle cell preferred codons are not to be construed as limited to muscle cells and may be preferred codons in other cell types.

[0200] In some examples, the target cell is a muscle cell (such as a human skeletal muscle cell) and the preferred codon for alanine is 5’ GCU 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one alanine residue is encoded by the codon 5’ GCU 3’. In some examples, all alanine residues are encoded by the codon 5’ GCU 3’.

[0201] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for arginine is 5’ AGG 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one arginine residue is encoded by the codon 5’ AGG 3’. In some examples, all arginine residues are encoded by the codon 5’ AGG 3’.

[0202] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for aspartic acid is 5’ GAC 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one aspartic acid residue is encoded by the codon 5’ GAC 3’. In some examples, all aspartic acid residues are encoded by the codon 5’ GAC 3’.

[0203] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for a glutamic acid is 5’ GAG 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one glutamic acid residue is encoded by the codon 5’ GAG 3’. In some examples, all glutamic acid residues are encoded by the codon 5’ GAG 3’.

[0204] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for a glycine is 5’ GGC mRNA 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one glycine residue is encoded by the codon 5’ GGC mRNA 3’. In some examples, all glycine residues are encoded by the codon 5’ GGC mRNA 3’.

[0205] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for a leucine is 5’ CUU 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one leucine residue is encoded by the codon 5’ CUU 3’. In some examples, all leucine residues are encoded by the codon 5’ CUU 3’.

[0206] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for a lysine is 5’ AAG 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one lysine residue is encoded by the codon 5’ AAG 3’. In some examples, all lysine residues are encoded by the codon 5’ AAG 3’.

[0207] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for a proline is 5’ CCU 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one proline residue is encoded by the codon 5’ CCU 3’. In some examples, all proline residues are encoded by the codon 5’ CCU 3’.

[0208] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for a serine is 5’ AGC mRNA 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one serine residue is encoded by the codon 5’ AGC mRNA 3’. In some examples, all serine residues are encoded by the codon 5’ AGC mRNA 3’.

[0209] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for a asparagine is 5’ AAC 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one asparagine residue is encoded by the codon 5’ AAC 3’. In some examples, all asparagine residues are encoded by the codon 5’ AAC 3’.

[0210] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for a glutamine is 5’ CAG 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one glutamine residue is encoded by the codon 5’ CAG 3’. In some examples, all glutamine residues are encoded by the codon 5’ CAG 3’.

[0211] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for a histidine is 5’ CAC 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one histidine residue is encoded by the codon 5’ CAC 3’. In some examples, all histidine residues are encoded by the codon 5’ CAC 3’.

[0212] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for a valine is 5’ GUG 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one valine residue is encoded by the codon 5’ GUG 3’. In some examples, all valine residues are encoded by the codon 5’ GUG 3’.

[0213] In some examples, the target cell is a skeletal muscle cell (such as a human skeletal muscle cell) and the preferred codon for a methionine is 5’ AUG 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a skeletal muscle cell wherein at least one methionine residue is encoded by the codon 5’ AUG 3’. In some examples, all methionine residues are encoded by the codon 5’ AUG 3’. Kidney Cell Preferred Codons

[0214] As shown in the examples provided herein, preferred codons may be determined for specific target cell types. For example, the target cell may be a kidney cell, in such examples, the set of preferred codons may be referred to herein as “kidney cell preferred codons”. It is well known that human embryonic kidney cells are used in the field as a model for a number of different cells. As such, kidney cell preferred codons are not to be construed as limited to kidney cells and may be preferred codons in other cell types.

[0215] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for alanine is 5’ GCU 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one alanine residue is encoded by the codon 5’ GCU 3’. In some examples, all alanine residues are encoded by the codon 5’ GCU 3’.

[0216] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for arginine is 5’ CGU 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one arginine residue is encoded by the codon 5’ CGU 3’. In some examples, all arginine residues are encoded by the codon 5’ CGU 3’.

[0217] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for aspartic acid is 5’ GAC 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one aspartic acid residue is encoded by the codon 5’ GAC 3’. In some examples, all aspartic acid residues are encoded by the codon 5’ GAC 3’.

[0218] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for glutamic acid is 5’ CAA 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one glutamic acid residue is encoded by the codon 5’ CAA 3’. In some examples, all glutamic acid residues are encoded by the codon 5’ CAA 3’.

[0219] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for glycine is 5’ GGC mRNA 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one glycine residue is encoded by the codon 5’ GGC mRNA 3’. In some examples, all glycine residues are encoded by the codon 5’ GGC mRNA 3’.

[0220] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for leucine is 5’ CUG 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one leucine residue is encoded by the codon 5’ CUG 3’. In some examples, all leucine residues are encoded by the codon 5’ CUG 3’.

[0221] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for lysine is 5’ AAG 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one lysine residue is encoded by the codon 5’ AAG 3’. In some examples, all lysine residues are encoded by the codon 5’ AAG 3’.

[0222] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for proline is 5’ CCU 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one proline residue is encoded by the codon 5’ CCU 3’. In some examples, all proline residues are encoded by the codon 5’ CCU 3’.

[0223] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for serine is 5’ AGC mRNA 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one serine residue is encoded by the codon 5’ AGC mRNA 3’. In some examples, all serine residues are encoded by the codon 5’ AGC mRNA 3’.

[0224] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for asparagine is 5’ AAC 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one asparagine residue is encoded by the codon 5’ AAC 3’. In some examples, all asparagine residues are encoded by the codon 5’ AAC 3’.

[0225] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for glutamine is 5’ CAG 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one glutamine residue is encoded by the codon 5’ CAG 3’. In some examples, all glutamine residues are encoded by the codon 5’ CAG 3’. In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for histidine is 5’ CAC 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one histidine residue is encoded by the codon 5’ CAC 3’. In some examples, all histidine residues are encoded by the codon 5’ CAC 3’.

[0226] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for valine is 5’ GUU 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one valine residue is encoded by the codon 5’ GUU 3’. In some examples, all valine residues are encoded by the codon 5’ GUU 3’.

[0227] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for isoleucine is 5’ AUA 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one isoleucine residue is encoded by the codon 5’ AUA 3’. In some examples, all isoleucine residues are encoded by the codon 5’ AUA 3’.

[0228] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for threonine is 5’ ACU 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one threonine residue is encoded by the codon 5’ ACU 3’. In some examples, all threonine residues are encoded by the codon 5’ ACU 3’.

[0229] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for tryptophan is 5’ UGG 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one tryptophan residue is encoded by the codon 5’ UGG 3’. In some examples, all tryptophan residues are encoded by the codon 5’ UGG 3’.

[0230] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for tyrosine is 5’ UAC 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one tyrosine residue is encoded by the codon 5’ UAC 3’. In some examples, all tyrosine residues are encoded by the codon 5’ UAC 3’. In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for phenylalanine is 5’ UUC 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one phenylalanine residue is encoded by the codon 5’ UUC 3’. In some examples, all phenylalanine residues are encoded by the codon 5’ UUC 3’.

[0231] In some examples, the target cell is a kidney cell (such as a human embryonic kidney cell, e.g., HEK293T cell) and the preferred codon for methionine is 5’ AUG 3’. As such, in one example, there is provided a nucleic acid molecule such as an mRNA (or DNA encoding the mRNA) for translation in a kidney cell wherein at least one methionine residue is encoded by the codon 5’ AUG 3’. In some examples, all methionine residues are encoded by the codon 5’ AUG 3’.

[0232] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0233] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0234] In some examples, the optimised mRNA is a therapeutic optimised mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0235] Signal Peptides

[0236] Provided herein are nucleic acid molecules that encode a signal peptide derived from alpha- 1 -antichymotrypsin (AACT) signal peptide or Human Serum albumin (HSA) signal peptide.

[0237] In particular, the nucleic acid molecule may be an mRNA (such as a therapeutic optimised mRNA as described herein) or a DNA molecule encoding an optimised mRNA.

[0238] In some examples, the encoded signal peptide is an endoplasmic reticulum (ER) signal peptide. The nucleic acid molecule sequence encoding the ER signal peptide may be preferred to as an ER secretion signal sequence. As such, in some examples, the nucleic acid molecules described herein may include an ER secretion signal sequence. The ER signal peptide may be encoded so that the translated peptide includes the ER signal peptide at the N-terminus of the peptide. Therefore, the nucleic acid molecules described herein may include a nucleic acid molecule sequence located 5’ of a protein encoded by the nucleic acid. In some examples, the ER signal peptide is suitable for binding by a signal recognition particle (SRP) in a target cell and for translocation of the peptide during translation to the endoplasmic reticulum.

[0239] The signal peptide may have effects on the efficiency of translation as well as provide secretion of translated proteins. For example, the nucleic acid molecule may be a therapeutic optimised mRNA encoding an antigen (such as a viral antigen) that needs to be secreted from the target cell to provide a therapeutic effect. In addition, factors such as protein yield, cellular localisation, secretion efficiency, and post-translational modifications may vary depending on the signal peptide used.

[0240] AACT is a member of the serpin family of proteins, a group of proteins that inhibit serine proteases. The gene encoding AACT (SERPINA3) is overexpressed in the liver. This gene is one in a cluster of serpin genes located on the q arm of chromosome 14. Although its physiological function is unclear, it can inhibit neutrophil cathepsin G and mast cell chymase, both of which can convert angiotensin-1 to the active angiotensin-2. Human AACT is represented by UniProtKB ID P01011.

[0241] The signal sequence of AACT is considered to comprise amino acid residues 1 to 23 of the sequence provided by UniProtKB number P01011. In some examples, the AACT signal peptide provided herein comprises at least residues 1 to 23 of human AACT:

[0242] MERMLPLLALGLLAAGFCPAVLC (SEQ ID NO: 71)

[0243] In some examples, the AACT signal peptide provided herein comprises at least residues 1 to 25 of human AACT:

[0244] MERMLPLLALGLLAAGFCPAVLCHP (SEQ ID NO: 10)

[0245] In some examples, the nucleic acid molecule encodes a signal peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 10.

[0246] In some examples, the nucleic acid molecule encodes a signal peptide comprising SEQ ID NO: 10. In some examples, the nucleic acid molecule encodes a signal peptide comprising SEQ ID NO: 71.

[0247] In some examples, the nucleic acid molecule includes a nucleic acid molecule sequence encoding a signal peptide comprising SEQ ID NO: 10. In some examples, the nucleic acid molecule includes a nucleic acid molecule sequence encoding a signal peptide consisting of SEQ ID NO: 10.

[0248] In some examples, the nucleic acid molecule includes a nucleic acid molecule sequence encoding a signal peptide comprising of SEQ ID NO: 10 and a nucleic acid molecule sequence encoding a protein (such as a therapeutic protein) wherein the nucleic acid molecule sequence encoding the signal peptide is located upstream (5’) of the nucleic acid molecule sequence encoding a protein.

[0249] In some examples, the nucleic acid molecule includes a nucleic acid molecule sequence encoding a signal peptide consisting of SEQ ID NO: 10 and a nucleic acid molecule sequence encoding a protein (such as a therapeutic protein) wherein the nucleic acid molecule sequence encoding the signal peptide is located upstream (5’) of the nucleic acid molecule sequence encoding a protein.

[0250] In some examples, the nucleic acid molecule sequence encoding the signal peptide according to SEQ ID NO: 10 comprises a nucleic acid molecule sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:

[0251] 11.

[0252] In some examples, the nucleic acid molecule sequence encoding the signal peptide according to SEQ ID NO: 10 comprises a nucleic acid molecule sequence comprising SEQ ID NO: 11.

[0253] In some examples, the nucleic acid molecule sequence encoding the signal peptide according to SEQ ID NO: 10 comprises a nucleic acid molecule sequence consisting of SEQ ID NO: 11.

[0254] In some examples, the nucleic acid molecule sequence encoding the AACT signal peptide may comprise one or more preferred codons as described herein (i.e. one or more codons that have been optimised using the methods described herein).

[0255] In some examples, the nucleic acid molecule sequence encoding the signal peptide according to SEQ ID NO: 10 comprises a nucleic acid molecule sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO:

[0256] 12.

[0257] In some examples, the nucleic acid molecule sequence encoding the signal peptide according to SEQ ID NO: 10 comprises a nucleic acid molecule sequence comprising SEQ ID NO: 12.

[0258] In some examples, the nucleic acid molecule sequence encoding the signal peptide according to SEQ ID NO: 10 comprises a nucleic acid molecule sequence consisting of SEQ ID NO: 12.

[0259] HSA is the serum albumin found in human blood. It is the most abundant protein in human blood plasma. The gene for HSA is located on chromosome 4 in locus 4q13.3. HSA functions in the regulation of blood plasma colloid osmotic pressure and acts as a carrier protein for a wide range of endogenous molecules including hormones, fatty acids, and metabolites, as well as exogenous drugs. Additionally, HSA exhibits an esterase-like activity with broad substrate specificity. The encoded preproprotein is proteolytically processed to generate the mature protein. Human AACT is represented by UniProtKB ID P02768.

[0260] The signal sequence of HSA is considered to comprise amino acid residues 1 to 18 of the sequence provided by UniProtKB ID P02768. In some examples, the HSA signal peptide provided herein comprises at least residues 1 to 18 of human HSA:

[0261] MKWVUFISLLFLFSSAYS (SED ID NO: 22)

[0262] In some examples, the nucleic acid molecule encodes a signal peptide having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 22.

[0263] In some examples, the nucleic acid molecule encodes a signal peptide comprising SEQ ID NO: 22.

[0264] In some examples, the nucleic acid molecule includes a nucleic acid molecule sequence encoding a signal peptide comprising SEQ ID NO: 22.

[0265] In some examples, the nucleic acid molecule includes a nucleic acid molecule sequence encoding a signal peptide consisting SEQ ID NO: 22.

[0266] In some examples, the nucleic acid molecule includes a nucleic acid molecule sequence encoding a signal peptide comprising of SEQ ID NO: 22 and a nucleic acid molecule sequence encoding a protein (such as a therapeutic protein) wherein the nucleic acid molecule sequence encoding the signal peptide is located upstream (5’) of the nucleic acid molecule sequence encoding a protein.

[0267] In some examples, the nucleic acid molecule includes a nucleic acid molecule sequence encoding a signal peptide consisting of SEQ ID NO: 22 and a nucleic acid molecule sequence encoding a protein (such as a therapeutic protein) wherein the nucleic acid molecule sequence encoding the signal peptide is located upstream (5’) of the nucleic acid molecule sequence encoding a protein.

[0268] In some examples, the nucleic acid molecule sequence encoding the signal peptide according to SEQ ID NO: 22 comprises a nucleic acid molecule sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 23.

[0269] In some examples, the nucleic acid molecule sequence encoding the signal peptide according to SEQ ID NO: 22 comprises a nucleic acid molecule sequence comprising SEQ ID NO: 23. In some examples, the nucleic acid molecule sequence encoding the signal peptide according to SEQ ID NO: 22 comprises a nucleic acid molecule sequence consisting of SEQ ID NO: 23.

[0270] In some examples, the nucleic acid molecule sequence encoding the HSA signal peptide may comprise one or more preferred codons as described herein (i.e. one or more codons that have been optimised using the methods described herein).

[0271] In some examples, the nucleic acid molecule sequence encoding the signal peptide according to SEQ ID NO: 22 comprises a nucleic acid molecule sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 24.

[0272] In some examples, the nucleic acid molecule sequence encoding the signal peptide according to SEQ ID NO: 22 comprises a nucleic acid molecule sequence comprising SEQ ID NO: 24.

[0273] In some examples, the nucleic acid molecule sequence encoding the signal peptide according to SEQ ID NO: 22 comprises a nucleic acid molecule sequence consisting of SEQ ID NO: 24.

[0274] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding an AACT signal peptide as described above. In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding an HSA signal peptide as described above.

[0275] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding an AACT signal peptide as described above. In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding an HSA signal peptide as described above. In some examples, one or more codons of the encoded signal peptide comprise a preferred codon.

[0276] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding an AACT signal peptide as described above. In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding an HSA signal peptide as described above. In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding an AACT signal peptide as described above. In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding an HSA signal peptide as described above. In some examples, one or more codons of the encoded signal peptide comprise a preferred codon.

[0277] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide, as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P).

[0278] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0279] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0280] In some examples, an AACT signal peptide as described herein may provide improved translation efficiency in liver cells in particular. As such, there is provided use of an AACT signal peptide as described herein for translation of optimised mRNA in a liver cell. For example, use of an AACT signal peptide comprising or consisting of an amino acid sequence according to SEQ ID NO: 10. For example, use of a nucleic acid molecule comprising a nucleic acid molecule sequence encoding an AACT signal peptide comprising or consisting of an amino acid sequence according to SEQ ID NO: 10. For example, use of a nucleic acid molecule comprising a nucleic acid molecule sequence according to SEQ ID NO: Optimised UTR Pairs

[0281] Provided herein are untranslated regions (UTRs) that may improve the efficiency of translation of an optimised mRNA, as described herein. The UTRs provided herein may be provided as pairs including one 5’ UTR and one 3’ UTR which may both be included or encoded by a nucleic acid molecule as described herein at respective ends of the nucleic acid molecules described herein. The pairs of UTRs may be referred to using a designation based on the cell type from which the UTRs are derived. For example, MUTR1 refers to a pair of UTRs derived from genes expressed in muscle (M) cells. As such, use of the designations refers to a specific pair of UTRs as detailed in Tables 1 and 2 below:

[0282] Table 1 - Muscle-specific UTR combinations

[0283] Table 2 - Liver-specific UTR combinations

[0284]

[0285] “Untranslated region” refers to a region positioned at both sides of a coding region and is not to be translated into a protein. mRNAs may include a 5'-untranslated region (5-UTR) at the 5' terminal end and a 3'-untranslated region (3-UTR) at the 3' terminal end of a mRNA. “Coding region” refers to a region to be translated into a protein in mRNA.

[0286] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from Myosin Light Chain, Phosphorylatable, Fast Skeletal Muscle (MYLPF) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from Myosin Light Chain, Phosphorylatable, Fast Skeletal Muscle (MYLPF) mRNA (MUTR1). In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from MYLPF mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from MYLPF mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from MYLPF mRNA and a 3’ UTR derived from MYLPF mRNA. In some examples, the 5’ UTR derived from MYLPF mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 35. In some examples, the 5’ UTR derived from MYLPF mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 35. In some examples, the 5’ UTR derived from MYLPF mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 35.

[0287] In some examples, the 3’ UTR derived from MYLPF mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 36. In some examples, the 3’ UTR derived from MYLPF mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 36. In some examples, the 3’ UTR derived from MYLPF mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 36.

[0288] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYLPF mRNA and a 3’ UTR derived from MYLPF mRNA as described above.

[0289] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYLPF mRNA and a 3’ UTR derived from MYLPF mRNA as described above.

[0290] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYLPF mRNA and a 3’ UTR derived from MYLPF mRNA as described above.

[0291] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYLPF mRNA and a 3’ UTR derived from MYLPF mRNA as described above.

[0292] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0293] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0294] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0295] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from Myosin Light Chain (MYL1) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from Myosin Light Chain (MYL1) mRNA (MUTR2).

[0296] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from MYL1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from MYL1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from MYL1 mRNA and a 3’ UTR derived from MYL1 mRNA.

[0297] In some examples, the 5’ UTR derived from MYL1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 37. In some examples, the 5’ UTR derived from MYL1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 37. In some examples, the 5’ UTR derived from MYL1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 37.

[0298] In some examples, the 3’ UTR derived from MYL1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 38. In some examples, the 3’ UTR derived from MYL1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 38. In some examples, the 3’ UTR derived from MYL1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 38.

[0299] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYL1 mRNA and a 3’ UTR derived from MYL1 mRNA as described above.

[0300] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYL1 mRNA and a 3’ UTR derived from MYL1 mRNA as described above.

[0301] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYL1 mRNA and a 3’ UTR derived from MYL1 mRNA as described above.

[0302] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYL1 mRNA and a 3’ UTR derived from MYL1 mRNA as described above.

[0303] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0304] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0305] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0306] In some examples, the UTR pair of MUTR2 (i.e. SEQ ID NOs: 37 and 38) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from MYL1 mRNA (SEQ ID NO: 37) and a 3’ UTR derived from MYL1 mRNA (SEQ ID NO: 38) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from MYL1 mRNA (SEQ ID NO: 37) and a 3’ UTR derived from MYL1 mRNA (SEQ ID NO: 38) as described herein in muscle cells. In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from MYL1 mRNA (SEQ ID NO: 37) and a 3’ UTR derived from MYL1 mRNA (SEQ ID NO: 38) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from MYL1 mRNA (SEQ ID NO: 37) and a 3’ UTR derived from MYL1 mRNA (SEQ ID NO: 38) as described herein in muscle cells.

[0307] In some examples, the UTR pair of MUTR2 (i.e. SEQ ID NOs: 37 and 38) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from MYL1 mRNA (SEQ ID NO: 37) and a 3’ UTR derived from MYL1 mRNA (SEQ ID NO: 38) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from MYL1 mRNA (SEQ ID NO: 37) and a 3’ UTR derived from MYL1 mRNA (SEQ ID NO: 38) as described herein in liver cells.

[0308] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from MYL1 mRNA (SEQ ID NO: 37) and a 3’ UTR derived from MYL1 mRNA (SEQ ID NO: 38) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from MYL1 mRNA (SEQ ID NO: 37) and a 3’ UTR derived from MYL1 mRNA (SEQ ID NO: 38) as described herein in liver cells.

[0309] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from actin, alpha"! , skeletal muscle (ACTA1) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from actin, alpha"!, skeletal muscle (ACTA1) mRNA (MUTR3).

[0310] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from ACTA1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from ACTA1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from ACTA1 mRNA and a 3’ UTR derived from ACTA1 mRNA.

[0311] In some examples, the 5’ UTR derived from ACTA1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 39. In some examples, the 5’ UTR derived from ACTA1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 39. In some examples, the 5’ UTR derived from ACTA1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 39.

[0312] In some examples, the 3’ UTR derived from ACTA1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 40. In some examples, the 3’ UTR derived from ACTA1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 40 In some examples, the 3’ UTR derived from ACTA1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 40.

[0313] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ACTA1 mRNA and a 3’ UTR derived from ACTA1 mRNA as described above.

[0314] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ACTA1 mRNA and a 3’ UTR derived from ACTA1 mRNA as described above.

[0315] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ACTA1 mRNA and a 3’ UTR derived from ACTA1 mRNA as described above.

[0316] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ACTA1 mRNA and a 3’ UTR derived from ACTA1 mRNA as described above.

[0317] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0318] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0319] In some examples, the optimised mRNA is a therapeutic optimised mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0320] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from Troponin C1 , Slow Skeletal And Cardiac Type (TNNC1) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from enolase 3 (ENO3) mRNA (MUTR4).

[0321] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from TNNC1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from TNNC1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from TNNC1 mRNA and a 3’ UTR derived from TNNC1 mRNA.

[0322] In some examples, the 5’ UTR derived from TNNC1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 41. In some examples, the 5’ UTR derived from TNNC1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 41. In some examples, the 5’ UTR derived from TNNC1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 41.

[0323] In some examples, the 3’ UTR derived from ENO3 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 42. In some examples, the 3’ UTR derived from ENO3 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 42. In some examples, the 3’ UTR derived from ENO3 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 42.

[0324] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from TNNC1 mRNA and a 3’ UTR derived from ENO3 mRNA as described above.

[0325] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from TNNC1 mRNA and a 3’ UTR derived from ENO3 mRNA as described above.

[0326] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from TNNC1 mRNA and a 3’ UTR derived from ENO3 mRNA as described above.

[0327] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from TNNC1 mRNA and a 3’ UTR derived from ENO3 mRNA as described above.

[0328] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0329] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0330] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0331] In some examples, the UTR pair of MUTR4 (i.e. SEQ ID NOs: 41 and 42) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from TNNC1 mRNA (SEQ ID NO: 41) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 42) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from TNNC1 mRNA (SEQ ID NO: 41) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 42) as described herein in muscle cells. In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from TNNC1 mRNA (SEQ ID NO: 41) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 42) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from TNNC1 mRNA (SEQ ID NO: 41) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 42) as described herein in muscle cells.

[0332] In some examples, the UTR pair of MUTR4 (i.e. SEQ ID NOs: 41 and 42) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from TNNC1 mRNA (SEQ ID NO: 41) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 42) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from TNNC1 mRNA (SEQ ID NO: 41) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 42) as described herein in liver cells.

[0333] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from TNNC1 mRNA (SEQ ID NO: 41) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 42) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from TNNC1 mRNA (SEQ ID NO: 41) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 42) as described herein in liver cells.

[0334] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from Troponin T1, Slow Skeletal Type (TNNT1) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from Troponin T1 , Slow Skeletal Type (TNNT1) mRNA (MUTR5).

[0335] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from TNNT1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from TNNT1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from TNNT1 mRNA and a 3’ UTR derived from TNNT1 mRNA.

[0336] In some examples, the 5’ UTR derived from TNNT1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 43. In some examples, the 5’ UTR derived from TNNT1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 43. In some examples, the 5’ UTR derived from TNNT1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 43. In some examples, the 3’ UTR derived from TNNT1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 44. In some examples, the 3’ UTR derived from TNNT1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 44. In some examples, the 3’ UTR derived from TNNT1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 44.

[0337] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from TNNT1 mRNA and a 3’ UTR derived from TNNT1 mRNA as described above.

[0338] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from TNNT1 mRNA and a 3’ UTR derived from TNNT1 mRNA as described above.

[0339] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from TNNT 1 mRNA and a 3’ UTR derived from TNNT1 mRNA as described above.

[0340] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from TNNT1 mRNA and a 3’ UTR derived from TNNT1 mRNA as described above.

[0341] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0342] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above. In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0343] In some examples, the UTR pair of MLITR5 (i.e. SEQ ID NOs: 43 and 44) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from TNNC1 mRNA (SEQ ID NO: 43) and a 3’ UTR derived from TNNC1 mRNA (SEQ ID NO: 44) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from TNNC1 mRNA (SEQ ID NO: 43) and a 3’ UTR derived from TNNC1 mRNA (SEQ ID NO: 44) as described herein in liver cells.

[0344] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from TNNC1 mRNA (SEQ ID NO: 43) and a 3’ UTR derived from TNNC1 mRNA (SEQ ID NO: 44) as described herein may be for use in liver cells. As such, there is provided use of an mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from TNNC1 mRNA (SEQ ID NO: 43) and a 3’ UTR derived from TNNC1 mRNA (SEQ ID NO: 44) as described herein in liver cells.

[0345] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from enolase 3 (ENO3) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from enolase 3 (ENO3) mRNA (MUTR6).

[0346] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from ENO3 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from ENO3 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from ENO3 mRNA and a 3’ UTR derived from ENO3 mRNA (MUTR6).

[0347] In some examples, the 5’ UTR derived from ENO3 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 45. In some examples, the 5’ UTR derived from ENO3 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 45. In some examples, the 5’ UTR derived from ENO3 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 45.

[0348] In some examples, the 3’ UTR derived from ENO3 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 46. In some examples, the 3’ UTR derived from ENO3 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 46. In some examples, the 3’ UTR derived from ENO3 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 46.

[0349] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ENO3 mRNA and a 3’ UTR derived from ENO3 mRNA as described above.

[0350] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ENO3 mRNA and a 3’ UTR derived from ENO3 mRNA as described above.

[0351] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ENO3 mRNA and a 3’ UTR derived from ENO3 mRNA as described above.

[0352] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ENO3 mRNA and a 3’ UTR derived from ENO3 mRNA as described above.

[0353] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0354] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0355] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0356] In some examples, the UTR pair of MLITR6 (i.e. SEQ ID NOs: 45 and 46) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 46) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 46) as described herein in muscle cells.

[0357] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 46) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 46) as described herein in muscle cells.

[0358] In some examples, the UTR pair of MUTR6 (i.e. SEQ ID NOs: 45 and 46) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 46) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 46) as described herein in liver cells.

[0359] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 46) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from ENO3 mRNA (SEQ ID NO: 46) as described herein in liver cells.

[0360] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from myosin heavy chain 1 (MYH1) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from myosin heavy chain 1 (MYH1) mRNA (MUTR7).

[0361] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from MYH1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from MYH1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from MYH1 mRNA and a 3’ UTR derived from MYH1 mRNA (MUTR7).

[0362] In some examples, the 5’ UTR derived from MYH1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 47. In some examples, the 5’ UTR derived from MYH1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 47. In some examples, the 5’ UTR derived from MYH1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 47.

[0363] In some examples, the 3’ UTR derived from MYH1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 48. In some examples, the 3’ UTR derived from MYH1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 48. In some examples, the 3’ UTR derived from MYH1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 48.

[0364] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYH1 mRNA and a 3’ UTR derived from MYH1 mRNA as described above.

[0365] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYH1 mRNA and a 3’ UTR derived from MYH1 mRNA as described above.

[0366] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYH1 mRNA and a 3’ UTR derived from MYH1 mRNA as described above.

[0367] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYH1 mRNA and a 3’ UTR derived from MYH1 mRNA as described above.

[0368] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0369] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0370] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0371] In some examples, the UTR pair of MLITR7 (i.e. SEQ ID NOs: 47 and 48) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from MYH1 mRNA (SEQ ID NO: 47) and a 3’ UTR derived from MYH1 mRNA (SEQ ID NO: 48) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from MYH1 mRNA (SEQ ID NO: 47) and a 3’ UTR derived from MYH1 mRNA (SEQ ID NO: 48) as described herein in muscle cells.

[0372] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from MYH1 mRNA (SEQ ID NO: 47) and a 3’ UTR derived from MYH1 mRNA (SEQ ID NO: 48) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from MYH1 mRNA (SEQ ID NO: 47) and a 3’ UTR derived from MYH1 mRNA (SEQ ID NO: 48) as described herein in muscle cells.

[0373] In some examples, the UTR pair of MUTR7 (i.e. SEQ ID NOs: 47 and 48) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from MYH1 mRNA (SEQ ID NO: 47) and a 3’ UTR derived from MYH1 mRNA (SEQ ID NO: 48) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from MYH1 mRNA (SEQ ID NO: 47) and a 3’ UTR derived from MYH1 mRNA (SEQ ID NO: 48) as described herein in liver cells.

[0374] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from MYH1 mRNA (SEQ ID NO: 47) and a 3’ UTR derived from MYH1 mRNA (SEQ ID NO: 48) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from MYH1 mRNA (SEQ ID NO: 47) and a 3’ UTR derived from MYH1 mRNA (SEQ ID NO: 48) as described herein in liver cells.

[0375] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from myosin light chain 2 (MYL2) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from myosin light chain 2 (MYL2) mRNA (MUTR8).

[0376] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from MYL2 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from MYL2 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from MYL2 mRNA and a 3’ UTR derived from MYL2 mRNA (MUTR8).

[0377] In some examples, the 5’ UTR derived from MYL2 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 49. In some examples, the 5’ UTR derived from MYL2 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 49. In some examples, the 5’ UTR derived from MYL2 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 49.

[0378] In some examples, the 3’ UTR derived from MYL2 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 50. In some examples, the 3’ UTR derived from MYL2 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 50. In some examples, the 3’ UTR derived from MYL2 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 50.

[0379] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYL2 mRNA and a 3’ UTR derived from MYL2 mRNA as described above.

[0380] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYL2 mRNA and a 3’ UTR derived from MYL2 mRNA as described above. In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYL2 mRNA and a 3’ UTR derived from MYL2 mRNA as described above.

[0381] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from MYL2 mRNA and a 3’ UTR derived from MYL2 mRNA as described above.

[0382] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0383] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0384] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P).

[0385] In some examples, the UTR pair of MUTR8 (i.e. SEQ ID NOs: 49 and 50) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from MYL2 mRNA (SEQ ID NO: 49) and a 3’ UTR derived from MYL2 mRNA (SEQ ID NO: 50) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from MYL2 mRNA (SEQ ID NO: 49) and a 3’ UTR derived from MYL2 mRNA (SEQ ID NO: 50) as described herein in muscle cells.

[0386] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from MYL2 mRNA (SEQ ID NO: 49) and a 3’ UTR derived from MYL2 mRNA (SEQ ID NO: 50) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from MYL2 mRNA (SEQ ID NO: 49) and a 3’ UTR derived from MYL2 mRNA (SEQ ID NO: 50) as described herein in muscle cells.

[0387] In some examples, the UTR pair of MUTR8 (i.e. SEQ ID NOs: 49 and 50) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from MYL2 mRNA (SEQ ID NO: 49) and a 3’ UTR derived from MYL2 mRNA (SEQ ID NO: 50) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from MYL2 mRNA (SEQ ID NO: 49) and a 3’ UTR derived from MYL2 mRNA (SEQ ID NO: 50) as described herein in liver cells.

[0388] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from MYL2 mRNA (SEQ ID NO: 49) and a 3’ UTR derived from MYL2 mRNA (SEQ ID NO: 50) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from MYL2 mRNA (SEQ ID NO: 49) and a 3’ UTR derived from MYL2 mRNA (SEQ ID NO: 50) as described herein in liver cells.

[0389] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from troponin 12, fast skeletal type (TNNI2) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from troponin 12, fast skeletal type (TNNI2) mRNA (MUTR9).

[0390] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from TNNI2 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from TNNI2 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from TNNI2and a 3’ UTR derived from TNNI2 mRNA (MUTR9).

[0391] In some examples, the 5’ UTR derived from TNNI2 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 51. In some examples, the 5’ UTR derived from TNNI2 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 51. In some examples, the 5’ UTR derived from TNNI2 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 51.

[0392] In some examples, the 3’ UTR derived from TNNI2 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 52. In some examples, the 3’ UTR derived from TNNI2 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 52. In some examples, the 3’ UTR derived from TNNI2 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 52.

[0393] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from TNNI2 mRNA and a 3’ UTR derived from TNNI2 mRNA as described above.

[0394] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from TNNI2 mRNA and a 3’ UTR derived from TNNI2 mRNA as described above.

[0395] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from TNNI2 mRNA and a 3’ UTR derived from TNNI2 mRNA as described above.

[0396] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from TNNI2 mRNA and a 3’ UTR derived from TNNI2 mRNA as described above.

[0397] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0398] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0399] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P).

[0400] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from creatine kinase, M-type (CKM) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from creatine kinase, M-type (CKM) mRNA (MUTR10).

[0401] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from CKM mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from CKM mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from CKM mRNA and a 3’ UTR derived from CKM mRNA (MUTR10).

[0402] In some examples, the 5’ UTR derived from CKM mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 53. In some examples, the 5’ UTR derived from CKM mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 53. In some examples, the 5’ UTR derived from CKM mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 53.

[0403] In some examples, the 3’ UTR derived from CKM mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 54. In some examples, the 3’ UTR derived from CKM mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 54. In some examples, the 3’ UTR derived from CKM mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 54.

[0404] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from CKM mRNA and a 3’ UTR derived from CKM mRNA as described above.

[0405] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from CKM mRNA and a 3’ UTR derived from CKM mRNA as described above.

[0406] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from CKM mRNA and a 3’ UTR derived from CKM mRNA as described above. In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from CKM mRNA and a 3’ UTR derived from CKM mRNA as described above.

[0407] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0408] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0409] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P).

[0410] In some examples, the UTR pair of MUTR10 (i.e. SEQ ID NOs: 53 and 54) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from CKM mRNA (SEQ ID NO: 53) and a 3’ UTR derived from CKM mRNA (SEQ ID NO: 54) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from CKM mRNA (SEQ ID NO: 53) and a 3’ UTR derived from CKM mRNA (SEQ ID NO: 54) as described herein in muscle cells.

[0411] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from CKM mRNA (SEQ ID NO: 53) and a 3’ UTR derived from CKM mRNA (SEQ ID NO: 54) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from CKM mRNA (SEQ ID NO: 53) and a 3’ UTR derived from CKM mRNA (SEQ ID NO: 54) as described herein in muscle cells. In some examples, the UTR pair of MLITR10 (i.e. SEQ ID NOs: 53 and 54) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from CKM mRNA (SEQ ID NO: 53) and a 3’ UTR derived from CKM mRNA (SEQ ID NO: 54) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from CKM mRNA (SEQ ID NO: 53) and a 3’ UTR derived from CKM mRNA (SEQ ID NO: 54) as described herein in liver cells.

[0412] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from CKM mRNA (SEQ ID NO: 53) and a 3’ UTR derived from CKM mRNA (SEQ ID NO: 54) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from CKM mRNA (SEQ ID NO: 53) and a 3’ UTR derived from CKM mRNA (SEQ ID NO: 54) as described herein in liver cells.

[0413] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from enolase 3 (ENO3) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from cytochrome c oxidase subunit 6A2 (COX6A2) mRNA (MUTR11).

[0414] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from ENO3 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from COX6A2 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from ENO3 mRNA and a 3’ UTR derived from COX6A2 mRNA (MUTR11).

[0415] In some examples, the 5’ UTR derived from ENO3 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 45. In some examples, the 5’ UTR derived from ENO3 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 45. In some examples, the 5’ UTR derived from ENO3 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 45.

[0416] In some examples, the 3’ UTR derived from COX6A2 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 55. In some examples, the 3’ UTR derived from COX6A2 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 55. In some examples, the 3’ UTR derived from COX6A2 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 55. In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ENO3 mRNA and a 3’ UTR derived from COX6A2 mRNA as described above.

[0417] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ENO3 mRNA and a 3’ UTR derived from COX6A2 mRNA as described above.

[0418] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ENO3 mRNA and a 3’ UTR derived from COX6A2 mRNA as described above.

[0419] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ENO3 mRNA and a 3’ UTR derived from COX6A2 mRNA as described above.

[0420] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0421] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0422] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P). In some examples, the UTR pair of MLITR11 (i.e. SEQ ID NOs: 45 and 55) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from COX6A2 mRNA (SEQ ID NO: 55) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from COX6A2 mRNA (SEQ ID NO: 55) as described herein in muscle cells.

[0423] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from COX6A2 mRNA (SEQ ID NO: 55) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from COX6A2 mRNA (SEQ ID NO: 55) as described herein in muscle cells.

[0424] In some examples, the UTR pair of MUTR11 (i.e. SEQ ID NOs: 45 and 50) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from COX6A2 mRNA (SEQ ID NO: 55) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from COX6A2 mRNA (SEQ ID NO: 55) as described herein in liver cells.

[0425] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from COX6A2 mRNA (SEQ ID NO: 55) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from ENO3 mRNA (SEQ ID NO: 45) and a 3’ UTR derived from COX6A2 mRNA (SEQ ID NO: 55) as described herein in liver cells.

[0426] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from orosomucoid 1 (ORM1) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from orosomucoid 1 (ORM1) mRNA (LUTR1).

[0427] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from ORM1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from ORM1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from ORMIand a 3’ UTR derived from ORM1 mRNA (LUTR1). In some examples, the 5’ UTR derived from ORM1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 56. In some examples, the 5’ UTR derived from ORM1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 56. In some examples, the 5’ UTR derived from ORM1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 56.

[0428] In some examples, the 3’ UTR derived from ORM1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 57. In some examples, the 3’ UTR derived from ORM1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 57. In some examples, the 3’ UTR derived from ORM1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 57.

[0429] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ORM1 mRNA and a 3’ UTR derived from ORM1 mRNA as described above.

[0430] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ORM1 mRNA and a 3’ UTR derived from ORM1 mRNA as described above.

[0431] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ORM1 mRNA and a 3’ UTR derived from ORM1 mRNA as described above.

[0432] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from ORM1 mRNA and a 3’ UTR derived from ORM1 mRNA as described above.

[0433] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P). In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0434] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P).

[0435] In some examples, the UTR pair of LLITR1 (i.e. SEQ ID NOs: 56 and 57) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from ORM1 mRNA (SEQ ID NO: 56) and a 3’ UTR derived from ORM1 mRNA (SEQ ID NO: 57) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from ORM1 mRNA (SEQ ID NO: 56) and a 3’ UTR derived from ORM1 mRNA (SEQ ID NO: 57) as described herein in muscle cells.

[0436] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from ORM1 mRNA (SEQ ID NO: 56) and a 3’ UTR derived from ORM1 mRNA (SEQ ID NO: 57) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from ORM1 mRNA (SEQ ID NO: 56) and a 3’ UTR derived from ORM1 mRNA (SEQ ID NO: 57) as described herein in muscle cells.

[0437] In some examples, the UTR pair of LUTR1 (i.e. SEQ ID NOs: 56 and 57) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from ORM1 mRNA (SEQ ID NO: 56) and a 3’ UTR derived from ORM1 mRNA (SEQ ID NO: 57) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from ORM1 mRNA (SEQ ID NO: 56) and a 3’ UTR derived from ORM1 mRNA (SEQ ID NO: 57) as described herein in liver cells.

[0438] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from ORM1 mRNA (SEQ ID NO: 56) and a 3’ UTR derived from ORM1 mRNA (SEQ ID NO: 57) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from ORM1 mRNA (SEQ ID NO: 56) and a 3’ UTR derived from ORM1 mRNA (SEQ ID NO: 57) as described herein in liver cells.

[0439] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from serum amyloid A2 (SAA2) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from serum amyloid A2 (SAA2) mRNA (LUTR2).

[0440] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from SAA2 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from SAA2 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from SAA2 mRNA and a 3’ UTR derived from SAA2 mRNA (LUTR2).

[0441] In some examples, the 5’ UTR derived from SAA2 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 58. In some examples, the 5’ UTR derived from SAA2 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 58. In some examples, the 5’ UTR derived from SAA2 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 58.

[0442] In some examples, the 3’ UTR derived from SAA2 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 59. In some examples, the 3’ UTR derived from SAA2 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 59. In some examples, the 3’ UTR derived from SAA2 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 59.

[0443] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA2 mRNA and a 3’ UTR derived from SAA2 mRNA as described above.

[0444] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA2 mRNA and a 3’ UTR derived from SAA2 mRNA as described above.

[0445] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA2 mRNA and a 3’ UTR derived from SAA2 mRNA as described above. In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA2 mRNA and a 3’ UTR derived from SAA2 mRNA as described above.

[0446] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0447] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0448] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P).

[0449] In some examples, the UTR pair of LUTR2 (i.e. SEQ ID NOs: 58 and 59) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from SAA2 mRNA (SEQ ID NO: 58) and a 3’ UTR derived from SAA2 mRNA (SEQ ID NO: 59) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from SAA2 mRNA (SEQ ID NO: 58) and a 3’ UTR derived from SAA2 mRNA (SEQ ID NO: 59) as described herein in muscle cells.

[0450] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA2 mRNA (SEQ ID NO: 58) and a 3’ UTR derived from SAA2 mRNA (SEQ ID NO: 59) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA2 mRNA (SEQ ID NO: 58) and a 3’ UTR derived from SAA2 mRNA (SEQ ID NO: 59) as described herein in muscle cells. In some examples, the UTR pair of LLITR2 (i.e. SEQ ID NOs: 58 and 59) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from SAA2 mRNA (SEQ ID NO: 58) and a 3’ UTR derived from SAA2 mRNA (SEQ ID NO: 59) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from SAA2 mRNA (SEQ ID NO: 58) and a 3’ UTR derived from SAA2 mRNA (SEQ ID NO: 59) as described herein in liver cells.

[0451] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA2 mRNA (SEQ ID NO: 58) and a 3’ UTR derived from SAA2 mRNA (SEQ ID NO: 59) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA2 mRNA (SEQ ID NO: 58) and a 3’ UTR derived from SAA2 mRNA (SEQ ID NO: 59) as described herein in liver cells.

[0452] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from serum amyloid A4, constitutive (SAA4) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from serum amyloid A4, constitutive (SAA4) mRNA (LUTR3).

[0453] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from SAA4 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from SAA4 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from SAA4 mRNA and a 3’ UTR derived from SAA4 mRNA (LUTR3).

[0454] In some examples, the 5’ UTR derived from SAA4 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 60. In some examples, the 5’ UTR derived from SAA4 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 60. In some examples, the 5’ UTR derived from SAA4 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 60.

[0455] In some examples, the 3’ UTR derived from SAA4 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 61. In some examples, the 3’ UTR derived from SAA4 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 61. In some examples, the 3’ UTR derived from SAA4 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 61. In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA4 mRNA and a 3’ UTR derived from SAA4 mRNA as described above.

[0456] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA4 mRNA and a 3’ UTR derived from SAA4 mRNA as described above.

[0457] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA4 mRNA and a 3’ UTR derived from SAA4 mRNA as described above.

[0458] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA4 mRNA and a 3’ UTR derived from SAA4 mRNA as described above.

[0459] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0460] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0461] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P). In some examples, the UTR pair of LLITR3 (i.e. SEQ ID NOs: 60 and 61) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from SAA4 mRNA (SEQ ID NO: 60) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from SAA4 mRNA (SEQ ID NO: 60) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein in muscle cells.

[0462] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA4 mRNA (SEQ ID NO: 60) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA4 mRNA (SEQ ID NO: 60) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein in muscle cells.

[0463] In some examples, the UTR pair of LUTR3 (i.e. SEQ ID NOs: 60 and 61) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from SAA4 mRNA (SEQ ID NO: 60) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from SAA4 mRNA (SEQ ID NO: 60) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein in liver cells.

[0464] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA4 mRNA (SEQ ID NO: 60) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA4 mRNA (SEQ ID NO: 60) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein in liver cells.

[0465] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from apolipoprotein A1 (APOA1) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from apolipoprotein A1 (APOA1) mRNA (LUTR4).

[0466] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from APOA1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from APOA1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from APOA1 mRNA and a 3’ UTR derived from APOA1 mRNA (LUTR4). In some examples, the 5’ UTR derived from APOA1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 62. In some examples, the 5’ UTR derived from APOA1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 62. In some examples, the 5’ UTR derived from APOA1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 62.

[0467] In some examples, the 3’ UTR derived from APOA1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 63. In some examples, the 3’ UTR derived from APOA1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 63. In some examples, the 3’ UTR derived from APOA1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 63.

[0468] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from APOA1 mRNA and a 3’ UTR derived from APOA1 mRNA as described above.

[0469] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from APOA1 mRNA and a 3’ UTR derived from APOA1 mRNA as described above.

[0470] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from APOA1 mRNA and a 3’ UTR derived from APOA1 mRNA as described above.

[0471] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from APOA1 mRNA and a 3’ UTR derived from APOA1 mRNA as described above.

[0472] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0473] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0474] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P).

[0475] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from complement factor H related 2 (CFHR2) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from serum amyloid A4, constitutive (SAA4) mRNA (LUTR5).

[0476] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from CFHR2 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from SAA4 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from CFHR2 mRNA and a 3’ UTR derived from SAA4 mRNA (LUTR5).

[0477] In some examples, the 5’ UTR derived from CFHR2 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 64. In some examples, the 5’ UTR derived from CFHR2 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 64. In some examples, the 5’ UTR derived from CFHR2 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 64.

[0478] In some examples, the 3’ UTR derived from SAA4 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 61. In some examples, the 3’ UTR derived from SAA4 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 61. In some examples, the 3’ UTR derived from SAA4 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 61. In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from CFHR2 mRNA and a 3’ UTR derived from SAA4 mRNA as described above.

[0479] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from CFHR2 mRNA and a 3’ UTR derived from SAA4 mRNA as described above.

[0480] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from CFHR2 mRNA and a 3’ UTR derived from SAA4 mRNA as described above.

[0481] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from CFHR2 mRNA and a 3’ UTR derived from SAA4 mRNA as described above.

[0482] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0483] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0484] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P). In some examples, the UTR pair of LLITR5 (i.e. SEQ ID NOs: 64 and 61) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from CFHR2 mRNA (SEQ ID NO: 64) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from CFHR2 mRNA (SEQ ID NO: 64) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein in muscle cells.

[0485] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from CFHR2 mRNA (SEQ ID NO: 64) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from CFHR2 mRNA (SEQ ID NO: 64) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein in muscle cells.

[0486] In some examples, the UTR pair of LUTR5 (i.e. SEQ ID NOs: 64 and 61) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from CFHR2 mRNA (SEQ ID NO: 64) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from CFHR2 mRNA (SEQ ID NO: 64) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein in liver cells.

[0487] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from CFHR2 mRNA (SEQ ID NO: 64) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from CFHR2 mRNA (SEQ ID NO: 64) and a 3’ UTR derived from SAA4 mRNA (SEQ ID NO: 61) as described herein in liver cells.

[0488] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from serum amyloid A1 (SAA1) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from serum amyloid A1 (SAA1) mRNA (LUTR6).

[0489] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from SAA1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from SAA1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from SAAIand a 3’ UTR derived from SAA1 mRNA (LUTR6). In some examples, the 5’ UTR derived from SAA1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 65. In some examples, the 5’ UTR derived from SAA1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 65. In some examples, the 5’ UTR derived from SAA1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 65.

[0490] In some examples, the 3’ UTR derived from SAA1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 66. In some examples, the 3’ UTR derived from SAA1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 66. In some examples, the 3’ UTR derived from SAA1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 66.

[0491] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA1 mRNA and a 3’ UTR derived from SAA1 mRNA as described above.

[0492] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA1 mRNA and a 3’ UTR derived from SAA1 mRNA as described above.

[0493] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA1 mRNA and a 3’ UTR derived from SAA1 mRNA as described above.

[0494] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA1 mRNA and a 3’ UTR derived from SAA1 mRNA as described above.

[0495] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P). In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0496] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P).

[0497] In some examples, the UTR pair of LLITR6 (i.e. SEQ ID NOs: 65 and 66) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from SAA1 mRNA (SEQ ID NO: 65) and a 3’ UTR derived from SAA1 mRNA (SEQ ID NO: 66) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from SAA1 mRNA (SEQ ID NO: 65) and a 3’ UTR derived from SAA1 mRNA (SEQ ID NO: 66) as described herein in muscle cells.

[0498] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA1 mRNA (SEQ ID NO: 65) and a 3’ UTR derived from SAA1 mRNA (SEQ ID NO: 66) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA1 mRNA (SEQ ID NO: 65) and a 3’ UTR derived from SAA1 mRNA (SEQ ID NO: 66) as described herein in muscle cells.

[0499] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from GC, vitamin D binding protein (GC) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from GC, vitamin D binding protein (GC) mRNA (LUTR7).

[0500] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from GC mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from GC mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from GC mRNA and a 3’ UTR derived from GC mRNA (LUTR7).

[0501] In some examples, the 5’ UTR derived from GC mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 67. In some examples, the 5’ UTR derived from GC mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 67. In some examples, the 5’ UTR derived from GC mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 67.

[0502] In some examples, the 3’ UTR derived from GC mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 68. In some examples, the 3’ UTR derived from GC mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 68. In some examples, the 3’ UTR derived from GC mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 68.

[0503] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from GC mRNA and a 3’ UTR derived from GC mRNA as described above.

[0504] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from GC mRNA and a 3’ UTR derived from GC mRNA as described above.

[0505] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from GC mRNA and a 3’ UTR derived from GC mRNA as described above.

[0506] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from GC mRNA and a 3’ UTR derived from GC mRNA as described above.

[0507] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0508] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0509] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P).

[0510] In some examples, the UTR pair of LLITR7 (i.e. SEQ ID NOs: 67 and 68) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from GC mRNA (SEQ ID NO: 67) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from GC mRNA (SEQ ID NO: 67) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein in muscle cells.

[0511] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from GC mRNA (SEQ ID NO: 67) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from GC mRNA (SEQ ID NO: 67) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein in muscle cells.

[0512] In some examples, the UTR pair of LUTR7 (i.e. SEQ ID NOs: 67 and 68) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from GC mRNA (SEQ ID NO: 67) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from GC mRNA (SEQ ID NO: 67) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein in liver cells.

[0513] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from GC mRNA (SEQ ID NO: 67) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from GC mRNA (SEQ ID NO: 67) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein in liver cells. In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from alpha 2-HS glycoprotein (AHSG) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from alpha 2-HS glycoprotein (AHSG) mRNA (LUTR8).

[0514] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from AHSG mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from AHSG mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from AHSG mRNA and a 3’ UTR derived from AHSG mRNA (LUTR8).

[0515] In some examples, the 5’ UTR derived from AHSG mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 69. In some examples, the 5’ UTR derived from AHSG mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 69. In some examples, the 5’ UTR derived from AHSG mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 69.

[0516] In some examples, the 3’ UTR derived from AHSG mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 70. In some examples, the 3’ UTR derived from AHSG mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 70. In some examples, the 3’ UTR derived from AHSG mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 70.

[0517] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from AHSG mRNA and a 3’ UTR derived from AHSG mRNA as described above.

[0518] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from AHSG mRNA and a 3’ UTR derived from AHSG mRNA as described above.

[0519] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from AHSG mRNA and a 3’ UTR derived from AHSG mRNA as described above.

[0520] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from AHSG mRNA and a 3’ UTR derived from AHSG mRNA as described above.

[0521] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0522] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0523] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P).

[0524] In some examples, the UTR pair of LUTR8 (i.e. SEQ ID NOs: 69 and 70) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from AHSG mRNA (SEQ ID NO: 69) and a 3’ UTR derived from AHSG mRNA (SEQ ID NO: 70) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from AHSG mRNA (SEQ ID NO: 69) and a 3’ UTR derived from AHSG mRNA (SEQ ID NO: 70) as described herein in muscle cells.

[0525] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from AHSG mRNA (SEQ ID NO: 69) and a 3’ UTR derived from AHSG mRNA (SEQ ID NO: 70) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from AHSG mRNA (SEQ ID NO: 69) and a 3’ UTR derived from AHSG mRNA (SEQ ID NO: 70) as described herein in muscle cells.

[0526] In some examples, the UTR pair of LUTR8 (i.e. SEQ ID NOs: 69 and 70) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from AHSG mRNA (SEQ ID NO: 69) and a 3’ UTR derived from AHSG mRNA (SEQ ID NO: 70) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from AHSG mRNA (SEQ ID NO: 69) and a 3’ UTR derived from AHSG mRNA (SEQ ID NO: 70) as described herein in liver cells.

[0527] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from AHSG mRNA (SEQ ID NO: 69) and a 3’ UTR derived from AHSG mRNA (SEQ ID NO: 70) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from AHSG mRNA (SEQ ID NO: 69) and a 3’ UTR derived from AHSG mRNA (SEQ ID NO: 70) as described herein in liver cells.

[0528] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from serum amyloid A4, constitutive (SAA4) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from apolipoprotein A1 (APOA1) mRNA (LUTR9).

[0529] In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from SAA4 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from APOA1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from SAA4 mRNA and a 3’ UTR derived from APOA1 mRNA (LUTR9).

[0530] In some examples, the 5’ UTR derived from SAA4 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 60. In some examples, the 5’ UTR derived from SAA4 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 60. In some examples, the 5’ UTR derived from SAA4 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 60.

[0531] In some examples, the 3’ UTR derived from APOA1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 63. In some examples, the 3’ UTR derived from APOA1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 63. In some examples, the 3’ UTR derived from APOA1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 63.

[0532] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA4 mRNA and a 3’ UTR derived from APOA1 mRNA as described above. In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA4 mRNA and a 3’ UTR derived from APOA1 mRNA as described above.

[0533] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA4 mRNA and a 3’ UTR derived from APOA1 mRNA as described above.

[0534] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA4 mRNA and a 3’ UTR derived from APOA1 mRNA as described above.

[0535] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0536] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0537] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P).

[0538] In some examples, there is provided a UTR pair comprising a nucleic acid molecule encoding a 5’ UTR derived from serum amyloid 1 (SAA1) mRNA and a nucleic acid molecule encoding a 3’ UTR derived from GC, vitamin D binding protein (GC) mRNA (LUTR10). In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from SAA1 mRNA. In some examples, there is provided a nucleic acid molecule comprising a 3’ UTR derived from GC mRNA. In some examples, there is provided a nucleic acid molecule comprising a 5’ UTR derived from SAA1 mRNA and a 3’ UTR derived from GC mRNA (LUTR10).

[0539] In some examples, the 5’ UTR derived from SAA1 mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 65. In some examples, the 5’ UTR derived from SAA1 mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 65. In some examples, the 5’ UTR derived from SAA1 mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 65.

[0540] In some examples, the 3’ UTR derived from GC mRNA comprises a nucleic acid molecule sequence comprising a nucleic acid molecule sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity to SEQ ID NO: 68. In some examples, the 3’ UTR derived from GC mRNA comprises a nucleic acid molecule sequence according to SEQ ID NO: 68. In some examples, the 3’ UTR derived from GC mRNA consists of a nucleic acid molecule sequence according to SEQ ID NO: 68.

[0541] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA1 mRNA and a 3’ UTR derived from GC mRNA as described above.

[0542] In some examples, there is provided a DNA molecule encoding an optimised mRNA comprises one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA1 mRNA and a 3’ UTR derived from GC mRNA as described above.

[0543] In some examples, there is provided an optimised mRNA molecule comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA1 mRNA and a 3’ UTR derived from GC mRNA as described above.

[0544] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above and comprising a nucleic acid molecule sequence encoding a 5’ UTR derived from SAA1 mRNA and a 3’ UTR derived from GC mRNA as described above. In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)1- methylpseudouridine (l-methyl^P).

[0545] In some examples, there is provided an optimised mRNA molecule comprising one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected using one or more of the methods described above.

[0546] In some examples, the optimised mRNA is a therapeutic mRNA. In some examples, the optimised mRNA has been transcribed by in vitro transcription. In some examples, the optimised mRNA comprises one or more modified ribonucleotides. That is to say that one or more of the ribonucleotides of the optimised mRNA are replaced with a modified ribonucleotide as described herein. For example, one or more uracils in the optimised mRNA may be (N)l-methylpseudouridine (l-methyl^P).

[0547] In some examples, the UTR pair of LLITR10 (i.e. SEQ ID NOs: 65 and 68) as described above may increase efficiency of translation in muscle cells in particular. As such, a 5’ UTR derived from SAA1 mRNA (SEQ ID NO: 65) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein may be for use in muscle cells. As such, there is provided use of a 5’ UTR derived from SAA1 mRNA (SEQ ID NO: 65) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein in muscle cells.

[0548] In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA1 mRNA (SEQ ID NO: 65) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein may be for use in muscle cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA1 mRNA (SEQ ID NO: 65) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein in muscle cells.

[0549] In some examples, the UTR pair of LUTR10 (i.e. SEQ ID NOs: 65 and 68) as described above may increase efficiency of translation in liver cells in particular. As such, a 5’ UTR derived from SAA1 mRNA (SEQ ID NO: 65) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein may be for use in liver cells. As such, there is provided use of a 5’ UTR derived from SAA1 mRNA (SEQ ID NO: 65) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein in liver cells. In some examples, an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA1 mRNA (SEQ ID NO: 65) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein may be for use in liver cells. As such, there is provided use of an optimised mRNA or DNA encoding such an optimised mRNA comprising a nucleic acid molecule sequence comprising a 5’ UTR derived from SAA1 mRNA (SEQ ID NO: 65) and a 3’ UTR derived from GC mRNA (SEQ ID NO: 68) as described herein in liver cells.

[0550] Optimised Poly(A) Tail and 5’ cap

[0551] The optimised mRNAs or DNAs encoding such mRNAs provided herein may include an optimised poly (A) tail. For example, the optimised poly(A) may comprise at least about 90 adenine residues. In some examples, the optimised poly(A) tail comprises at least about 95 adenine residues. In some examples, the optimised poly(A) tail comprises at least about 97 adenine residues. In some examples, the optimised poly(A) tail comprises from about 95 to about 135 adenine residues. In some examples, the optimised poly(A) tail comprises from about 97 to about 135 adenine residues. In some examples, the optimised poly(A) tail comprises from about 95 to about 100 adenine residues. In some examples, the optimised poly(A) tail comprises from about 97 to about 100 adenine residues. In some examples, the optimised poly(A) tail comprises about 97 adenine residues.

[0552] In some examples, the optimised poly(A) tail may be added using E.coli poly(A) polymerase. In such cases, the poly(A) tail length may be defined by the number of E.coli poly(A) polymerase units. For example, the optimised poly(A) tail comprises from 5 to 10 E.coli poly(A) polymerase units. In some examples, the optimised poly(A) tail comprises 5 E.coli poly(A) polymerase units.

[0553] In some examples, the methods described herein comprise enzymatically adding a poly(A) tail to the optimised mRNAs as described herein. As such, the optimised mRNAs as described herein may include an enzymatically added poly(A) tail.

[0554] In some examples, the optimised mRNAs as described herein may include a 5’ cap. In particular, the 5’ cap may be m7G-5'ppp5’GpG. In particular, a m7G-5'ppp5’GpG 5’ cap may provide improved translation efficiency and / or fidelity in muscle cells. As such, an mRNA including the 5’ cap m7G-5'ppp5’GpG may be for use in muscle cells. For example, in muscle cells of a subject.

[0555] Tandem (Consecutive) Stop Codons The optimised mRNAs or DNAs encoding said mRNAs provided herein may include at least two consecutively arranged stop codons. That is to say the optimised mRNA or DNA encoding said mRNA may include two stop codons adjacent to each other. Two adjacent stop codons may be referred to herein as a tandem stop codon. Two consecutive stop codons may be referred to herein as a tandem stop codon.

[0556] The tandem stop codon may be encoded in an alternative reading frame to a protein encoded by the optimised mRNA or DNA encoding said mRNA. Proteins are translated by reading tri-nucleotides (codons) from the 5’ to the 3’ end, starting with the amino acid methionine as the start (initiation) codon. Each codon is translated into a single amino acid. The code itself is degenerate, meaning that a particular amino acid can be specified by more than one codon. A shift of any number of nucleotides that is not divisible by 3 in the reading frame will cause subsequent codons to be read differently from that of the intended or correct reading frame. This effectively changes the ribosomal reading frame leading to the production of alternative polypeptides encoded by mRNA. The translation of a frameshifted codon may be referred to as out-of-frame translation, and the products (i.e. alternative proteins or polypeptides) may be referred to as out-of-frame products or proteins or as alternative products or proteins (i.e. alternative to the product encoded by the non- frameshifted (correct or in frame translation product) open reading frame). Out-of-frame and / or alternative translation products may be more immunogenic in comparison to the inframe translation product. The out-of-frame and / or alternative translation products may also have a reduced efficacy in comparison to the in-frame translation product.

[0557] The tandem stop codon may be encoded in a frameshifted reading frame in comparison to the reading frame for production of the desired protein encoded by the optimised mRNA. For example, the tandem stop codon may be encoded in a -1, -2, +1, or +2 frameshifted reading frame. In some examples, the tandem stop codon may be encoded in a +1 frameshifted reading frame.

[0558] The tandem stop codon may include any two of the three stop codons: UAA; UAG; or UGA. In some examples, the tandem stop codon comprise a nucleic acid sequence selected from one of UAAUAA, UAAUAG, UAGUA, or UAGUAG.

[0559] In some examples, the optimised mRNAs or DNAs encoding said mRNAs provided herein comprise more than one tandem stop codon. For example, 1, 2, 3, 4, or 5 tandem stop codons. Tandem stop codons may be encoded out-of-frame at any point in the nucleic acid sequence where an alternative product may be produced due to frameshifted translation.

[0560] In some examples, the tandem stop codon comprises one or more N1-methyl pseudouridine. In some examples, only one of the consecutive stop codons comprises N1-methyl pseudouridine. In some examples, both consecutive stop codons comprise at least one N1- methyl pseudouridine.

[0561] The tandem stop codons may reduce the production of unwanted polypeptides due to ribosomal slippage and / or frameshifting. As such, the tandem stop codons may help to reduce the immunogenicity of the nucleic acid, such as an mRNA. In some examples, the tandem stop codons may reduce the production of polypeptides that elicit a T-cell response. In some examples, the tandem stop codons may reduce the production of functional T cell antigens.

[0562] Combinations

[0563] It will be understood that optimised mRNAs or nucleic acid molecules encoding such mRNAs may include one or more of the preferred codons, signal peptides, UTR pairs, optimised poly(A) tail, tandem stop codons and / or 5’ cap as described herein.

[0564] For example, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein and one or more of the signal peptides, UTR pairs, optimised poly(A) tail and / or 5’ cap as described herein.

[0565] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein and a nucleic acid sequence encoding signal peptide as described herein.

[0566] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein and an UTR pair as described herein.

[0567] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein and an optimised poly(A) tail as described herein.

[0568] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein and a 5’ cap as described herein.

[0569] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein and a 5’ cap as described herein.

[0570] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein and at least one tandem stop codon as described herein. In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein and an UTR pair as described herein.

[0571] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein and an optimised poly(A) tail as described herein.

[0572] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein and a 5’ cap as described herein.

[0573] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein and at least one tandem stop codon as described herein. In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include an UTR pair as described herein and a 5’ cap as described herein.

[0574] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include an UTR pair as described herein and an optimised poly(A) tail as described herein.

[0575] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include an UTR pair as described herein and at least one tandem stop codon as described herein. In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include an optimised poly(A) tail as described herein and a 5’ cap as described herein.

[0576] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include an optimised poly(A) tail as described herein and at least one tandem stop codon as described herein.

[0577] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include at least one tandem stop codon and a 5’ cap as described herein as described herein. In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein and an UTR pair as described herein. In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein and optimised poly(A) tail as described herein.

[0578] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein and at least one tandem stop codon as described herein. In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule)) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein and a 5’ cap as described herein.

[0579] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, an UTR pair as described herein and optimised poly(A) tail as described herein.

[0580] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, an UTR pair as described herein and at least one tandem stop codon as described herein.

[0581] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, an UTR pair as described herein and a 5’ cap as described herein.

[0582] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, optimised poly(A) tail as described herein and a 5’ cap as described herein.

[0583] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, optimised poly(A) tail as described herein and at least one tandem stop codon as described herein.

[0584] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, at least one tandem stop codon as described herein and a 5’ cap as described herein.

[0585] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein and optimised poly(A) tail as described herein.

[0586] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein and at least one tandem stop codon as described herein.

[0587] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein and a 5’ cap as described herein.

[0588] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein, optimised poly(A) tail as described herein and at least one tandem stop codon as described herein.

[0589] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein, optimised poly(A) tail as described herein and a 5’ cap as described herein.

[0590] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein, at least one tandem stop codon as described herein and a 5’ cap as described herein.

[0591] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include an UTR pair as described herein, optimised poly(A) tail as described herein and at least one tandem stop codon as described herein.

[0592] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include an UTR pair as described herein, optimised poly(A) tail as described herein and a 5’ cap as described herein.

[0593] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include an UTR pair as described herein, at least one tandem stop codon as described herein and a 5’ cap as described herein.

[0594] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include optimised poly(A) tail as described herein, at least one tandem stop codon described herein and a 5’ cap as described herein. In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein and optimised poly(A) tail as described herein.

[0595] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein and at least one tandem stop codon as described herein.

[0596] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein and a 5’ cap as described herein.

[0597] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein, optimised poly(A) tail as described herein and at least one tandem stop codon as described herein.

[0598] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein, optimised poly(A) tail as described herein and a 5’ cap as described herein.

[0599] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein, at least one tandem stop codon as described herein and a 5’ cap as described herein.

[0600] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, an UTR pair as described herein, optimised poly(A) tail as described herein and at least one tandem stop codon as described herein.

[0601] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, an UTR pair as described herein, optimised poly(A) tail as described herein and a 5’ cap as described herein.

[0602] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, an UTR pair as described herein, at least one tandem stop codon as described herein and a 5’ cap as described herein.

[0603] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, at least one tandem stop codon described herein, optimised poly(A) tail as described herein and a 5’ cap as described herein.

[0604] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein, optimised poly(A) tail and at least one tandem stop codon as described herein.

[0605] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein, optimised poly(A) tail and a 5’ cap as described herein.

[0606] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein, at least one tandem stop codon and a 5’ cap as described herein.

[0607] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include a nucleic acid sequence encoding signal peptide as described herein, optimised poly(A) tail as described herein, at least one tandem stop codon and a 5’ cap as described herein.

[0608] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include an UTR pair as described herein, optimised poly(A) tail as described herein, at least one tandem stop codon and a 5’ cap as described herein.

[0609] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein an UTR pair as described herein, optimised poly(A) tail as described herein and at least one tandem stop codon as described herein.

[0610] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein an UTR pair as described herein, optimised poly(A) tail as described herein and a 5’ cap as described herein.

[0611] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein, at least one tandem stop codon as described herein and a 5’ cap as described herein.

[0612] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein, optimised poly(A) tail, at least one tandem stop codon as described herein and a 5’ cap as described herein.

[0613] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, an UTR pair as described herein, optimised poly(A) tail, at least one tandem stop codon as described herein and a 5’ cap as described herein.

[0614] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that includes a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein, optimised poly(A) tail, at least one tandem stop codon as described herein and a 5’ cap as described herein.

[0615] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein, optimised poly(A) tail, at least one tandem stop codon as described herein and a 5’ cap as described herein.

[0616] In some examples, there is provided nucleic acid molecules encoding an optimised mRNA (e.g. a DNA or RNA molecule) that include one or more preferred codons as described herein, a nucleic acid sequence encoding signal peptide as described herein, an UTR pair as described herein, optimised poly(A) tail as described herein and a 5’ cap as described herein.

[0617] Medical Uses

[0618] The nucleic acid molecules described herein (e.g. DNA transcription templates or optimized mRNAs) may be used for a number of in vitro and in vivo uses. Reference to medicals uses, nucleic acid molecules described herein for use in methods of treatment and methods of manufacturing a medicament using the nucleic acid molecules described herein are all to be understood to relate to methods of treating a subject using the nucleic acid molecules described herein. Thus in one example, there is provided a nucleic acid molecule as described herein for manufacture of a medicament for treating any of the disorders, conditions or diseases described herein. In one example, there is provided a nucleic acid molecule as described herein for use in treating any of the disorders, conditions or diseases described herein. In one example, there is provided a nucleic acid molecule as described herein for treating any of the disorders, conditions or diseases described herein. In one example, there is provided a method of treating a subject in need thereof comprising administering a nucleic acid molecule as described herein. For example, the method is a method for treating any of the disorders, conditions or diseases described herein.

[0619] The nucleic acid molecules described herein may be for use as medicaments. For example, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use in methods of preventing or treating a disease or condition in a subject. The disease or condition treated may depend on the protein encoded by the mRNA or DNA template described herein. In general, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use in treating any diseases or conditions that may benefit from the administration of an mRNA or protein translated therefrom. The optimized mRNAs or DNA templates encoding said mRNAs may be for use or used in methods of treating infectious diseases (such as bacterial infections, viral infections, parasitic infections), cell proliferation disorders (such as cancer), genetic disorders, inflammatory disease, cardiovascular disorders, metabolic diseases, allergic disease, neurodegenerative diseases, protein or enzyme deficiency disorder and / or autoimmune diseases.

[0620] For example, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use in methods of treating a genetic disorder. “Genetic disorder” refers to a congenital or acquired disease caused by chromosomal or mitochondrial DNA abnormalities, and examples thereof include Down syndrome, Wilson disease, Edwards syndrome, Patau syndrome, Turner syndrome, Klinefelter syndrome, Apert syndrome, Crouzon syndrome, 22q11.2 deletion syndrome, Williams syndrome, Laurence-Moon-Biedl syndrome, Prader-Willi syndrome, Angelman syndrome, Kallmann syndrome, Aicardi- Goutieres syndrome, Miller-Dieker syndrome, Rubinstein-Taybi syndrome, Cornelia de Lange syndrome, cri-du-chat syndrome, super female, super male, and mitochondrial disease.

[0621] For example, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use in methods of treating a protein or enzyme deficiency disorder. “Protein or enzyme deficiency disorders” refers to any disease or disorder that is associated with a subject lacking one or more proteins or enzymes or lacking sufficient activity of a protein or enzyme leading to symptoms and adverse effects in the subject. Examples of protein or enzyme deficiency disorders include Pompe disease, mucopolysaccharidosis types 1, 11, and VI, hemophilias A and B hypercystinia, Danon’s disease, myoclonic renal failure syndrome, sialic acid storage disorders such as ISSD, Salla disease and moderately severe Salla disease, Niemann-Pick disease C1 and C2, mucolipid accumulation disease type IV; neuronal ceroid lipofuscinosis includes but not limited to ceroid lipofuscinosis 1 Types (Haltia-Santavuori disease and INCL), neuronal ceroid lipofuscinosis type 2 (Jansky- Bielschowsky disease), cereoid lipofuscinosis type 3 (Batten-Spielmeyer-Sjogren disease), waxy Lipofuscinosis type 4 (Parry’s disease and Kufs A and B), cereoid lipofuscinosis type 5 (late infant Finnish type), cereofuscinosis type 6 (Lake-Cavanagh) Or Indian type), cereoid lipofuscinosis type 7 (Turkish type), cereoid lipofuscinosis type 8 ( Northern epilepsy, epilepsy mental disorder), cereoid lipofuscinosis 9, cereoid lipofuscinosis 10, cereoid lipofuscinosis 11, cereoid lipofuscinosis 12, cereoid Lipofuscinosis 13, Cereofuscinosis 14; Lysosomal-related organelle disorders including but not limited to Hermansky-Pudlak disease type 1 , Hermansky-Pudlak disease type 2, Hermansky-Pudlak disease type 3, Hermansky-Pudlak disease type 4, Hermansky-Pudlak disease type 5, Hermansky-Pudlak disease type 6, Hermansky-Pudlak disease type 7, Hermansky-Pudlak disease type 8, Hermansky-Pudlak disease type 9, Griscelli syndrome 1 (Elejalde syndrome) ), Griscelli syndrome 2, Chediak-Higashi disease, lysosomal storage disorders, e.g. Hurler syndrome, Niemann-Pick disease, Tay-Sachs disease, Gaucher disease, Fabry disease or Krabbe disease; Phenylketonuria; mitochondrial disorders; Friedreich ataxia; peroxisomal disorders, e.g. Zellweger syndrome or Adrenoleukodystrophy; metal metabolism disorders, e.g. Wilson disease or hemochromatosis; organic acidemias, e.g. methylmalonic acidemia or propionic acidemia; urea cycle disorders, e.g. ornithine transcarbamylase deficiency or citrullinemia and / or p-thalassemia. Other examples of enzyme deficiency disorders include Type I diabetes mellitus, which results from the patient’s failure to produce insulin.

[0622] For example, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use in methods of treating cardiovascular disorders. "Cardiovascular disease" or "cardiovascular disorder" refers to diseases affecting the heart or blood vessels or both. For example, cardiovascular disease includes arrhythmia (atrial or ventricular or both); atherosclerosis and its sequelae; angina; cardiac rhythm disturbances; myocardial ischemia; myocardial infarction; cardiac or vascular aneurysm; vasculitis, stroke; peripheral obstructive arteriopathy of a limb, an organ, or a tissue; reperfusion injury following ischemia of the brain, heart, kidney or other organ or tissue; endotoxic, surgical, or traumatic shock; hypertension, valvular heart disease, heart failure, abnormal blood pressure; shock; vasoconstriction (including that associated with migraines); vascular abnormality, insufficiency limited to a single organ or tissue.

[0623] For example, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use in methods of treating an autoimmune disease. “Autoimmune disease” refers to a disease or condition in which a subject's immune system has an aberrant immune response against a substance that does not normally elicit an immune response in a healthy subject. Examples of autoimmune diseases that may be treated include Acute Disseminated Encephalomyelitis (ADEM), Acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, Agammaglobulinemia, Alopecia areata, Amyloidosis, Ankylosing spondylitis, Anti- GBM / Anti-TBM nephritis, Antiphospholipid syndrome (APS), Autoimmune angioedema, Autoimmune aplastic anemia, Autoimmune dysautonomia, Autoimmune hepatitis, Autoimmune hyperlipidemia, Autoimmune immunodeficiency, Autoimmune inner ear disease (Al ED), Autoimmune myocarditis, Autoimmune oophoritis, Autoimmune pancreatitis, Autoimmune retinopathy, Autoimmune thrombocytopenic purpura (ATP), Autoimmune thyroid disease, Autoimmune urticaria, Axonal or neuronal neuropathies, Balo disease, Behcet's disease, Bullous pemphigoid, Cardiomyopathy, Castleman disease, Celiac disease, Chagas disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic recurrent multifocal ostomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid / benign mucosal pemphigoid, Crohn's disease, Cogans syndrome, Cold agglutinin disease, Congenital heart block, Coxsackie myocarditis, CREST disease, Essential mixed cryoglobulinemia, Demyelinating neuropathies, Dermatitis herpetiformis, Dermatomyositis, Devic's disease (neuromyelitis optica), Discoid lupus, Dressier's syndrome, Endometriosis, Eosinophilic esophagitis, Eosinophilic fasciitis, Erythema nodosum, Experimental allergic encephalomyelitis, Evans syndrome, Fibromyalgia, Fibrosing alveolitis, Giant cell arteritis (temporal arteritis), Giant cell myocarditis, Glomerulonephritis, Goodpasture's syndrome, Granulomatosis with Polyangiitis (GPA) (formerly called Wegener's Granulomatosis), Graves' disease, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, Hemolytic anemia, Henoch- Schonlein purpura, Herpes gestationis, Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, lgG4-related sclerosing disease, Immunoregulatory lipoproteins, Inclusion body myositis, Interstitial cystitis, Juvenile arthritis, Juvenile diabetes (Type 1 diabetes), Juvenile myositis, Kawasaki syndrome, Lambert-Eaton syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Ligneous conjunctivitis, Linear IgA disease (LAD), Lupus (SLE), Lyme disease, chronic, Meniere's disease, Microscopic polyangiitis, Mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy, Neuromyelitis optica (Devic's), Neutropenia, Ocular cicatricial pemphigoid, Optic neuritis, Palindromic rheumatism, PANDAS (Pediatric Autoimmune Neuropsychiatric Disorders Associated with Streptococcus), Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonnage-Turner syndrome, Pars planitis (peripheral uveitis), Pemphigus, Peripheral neuropathy, Perivenous encephalomyelitis, Pernicious anemia, POEMS syndrome, Polyarteritis nodosa, Type I, II, & III autoimmune polyglandular syndromes, Polymyalgia rheumatica, Polymyositis, Postmyocardial infarction syndrome, Postpericardiotomy syndrome, Progesterone dermatitis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Psoriasis, Psoriatic arthritis, Idiopathic pulmonary fibrosis, Pyoderma gangrenosum, Pure red cell aplasia, Raynauds phenomenon, Reactive Arthritis, Reflex sympathetic dystrophy, Reiter's syndrome, Relapsing polychondritis, Restless legs syndrome, Retroperitoneal fibrosis, Rheumatic fever, Rheumatoid arthritis, Sarcoidosis, Schmidt syndrome, Scleritis, Scleroderma, Sjogren's syndrome, Sperm & testicular autoimmunity, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac's syndrome, Sympathetic ophthalmia, Takayasu's arteritis, Temporal arteritis / Giant cell arteritis, Thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, Transverse myelitis, Type 1 diabetes, Ulcerative colitis, Undifferentiated connective tissue disease (UCTD), Uveitis, Vasculitis, Vesiculobullous dermatosis, Vitiligo, or Wegener's granulomatosis (i.e., Granulomatosis with Polyangiitis (GPA).

[0624] For example, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use in methods of treating a neurodegenerative diseases. “Neurodegenerative disease” refers to a disease or condition in which the function of a subject's nervous system becomes impaired. Examples of neurodegenerative diseases that may be treated include Alexander's disease, Alper's disease, Alzheimer's disease, Amyotrophic lateral sclerosis, Ataxia telangiectasia, Batten disease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), Bovine spongiform encephalopathy (BSE), Canavan disease, Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease, frontotemporal dementia, Gerstmann-Straussler-Scheinker syndrome, Huntington's disease, HIV-associated dementia, Kennedy's disease, Krabbe's disease, kuru, Lewy body dementia, Machado-Joseph disease (Spinocerebellar ataxia type 3), Multiple sclerosis, Multiple System Atrophy, Narcolepsy, Neuroborreliosis, Parkinson's disease, Pelizaeus- Merzbacher Disease, Pick's disease, Primary lateral sclerosis, Prion diseases, Refsum's disease, Sandhoffs disease, Schilder's disease, Subacute combined degeneration of spinal cord secondary to Pernicious Anaemia, Schizophrenia, Spinocerebellar ataxia (multiple types with varying characteristics), Spinal muscular atrophy, Steele-Richardson-Olszewski disease, or Tabes dorsalis.

[0625] For example, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use in methods of treating a metabolic disease. “Metabolic disease” refers to a disease or condition in which a subject's metabolism or metabolic system (e.g., function of storing or utilizing energy) becomes impaired. Examples of metabolic diseases that may be treated include diabetes (e.g., type I or type II), obesity, metabolic syndrome, or a mitochondrial disease (e.g., dysfunction of mitochondria or aberrant mitochondrial function).

[0626] For example, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use in methods of treating an inflammatory disease. “Inflammatory disease” refers to a disease or condition characterized by aberrant inflammation (e.g. an increased level of inflammation compared to a control, such as a healthy person not suffering from a disease). Examples of inflammatory diseases include traumatic brain injury, arthritis, rheumatoid arthritis, psoriatic arthritis, juvenile idiopathic arthritis, multiple sclerosis, systemic lupus erythematosus (SLE), myasthenia gravis, juvenile-onset diabetes, diabetes mellitus type 1, Guillain-Barre syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, ankylosing spondylitis, psoriasis, Sjogren's syndrome, vasculitis, glomerulonephritis, autoimmune thyroiditis, Behcet's disease, Crohn's disease, ulcerative colitis, bullous pemphigoid, sarcoidosis, ichthyosis, Graves ophthalmopathy, inflammatory bowel disease, Addison's disease, Vitiligo, asthma, allergic asthma, acne vulgaris, celiac disease, chronic prostatitis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, sarcoidosis, transplant rejection, interstitial cystitis, atherosclerosis, and atopic dermatitis.

[0627] For example, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use in methods of treating cancer. The optimized mRNAs or DNA templates encoding said mRNAs provided herein may help to induce effective tumour- reactive T-cell responses to a tumour. When for use in treating cancer, the optimized mRNAs or DNA templates encoding said mRNAs encode a tumour-associated epitope. The optimized mRNAs or DNA templates encoding said mRNAs described herein may effectively help generate a population of immune cells, in particular of CD8+ effector T cells (also known as cytotoxic T lymphocytes (CTLs)). The immune cells induced by administration of the optimized mRNAs or DNA templates encoding said mRNAs described herein may be reactive to the epitope, or epitopes, translated from the optimized mRNAs or DNA templates encoding said mRNAs described herein. These immune cells are then primed for the killing of cancer cells that present the same or similar epitopes. Such optimized mRNAs or DNA templates encoding said mRNAs for use in treating cancer may be referred to as “cancer vaccines” or “cancer immunotherapy vaccines”. The medical uses and methods of treating cancer may include administering to a subject in need thereof a therapeutically effective amount of an optimized mRNAs or DNA templates encoding said mRNAs as described herein.

[0628] The medical uses and methods of treatment described herein may be used in the treatment of a wide range of cancers. T umours may be of mesenchymal or epithelial origin. Cancers include cancers of the colon, rectum, cervix, breast, lung, stomach, uterus, skin, mouth, tung, lips, larynx, kidney, bladder, prostate, brain, and blood cells. The medical uses and methods of treatment described herein may be used in the treatment of solid tumours.

[0629] Suitably, a cancer to be treated by a medical use or method of treatment described herein may be a solid tumour selected from but not limited to the group consisting of: pancreatic ductal adenocarcinoma, pancreatic cancer; breast cancer; melanoma; non-small cell lung cancer; small cell lung cancer; nasopharyngeal cancer; hepatocellular cancer; colorectal cancer; oesophageal cancer; gastric cancer; anal cancer; small intestine cancer; mesothelioma; kidney cancer; renal cell carcinoma; bladder cancer; prostate cancer; ovarian cancer; vulval cancer; cervical cancer; penile cancer; uveal melanoma; retinoblastoma; sarcoma; osteosarcoma; glioblastoma; adrenocortical carcinoma; neuroblastoma; Wilms tumour; endometrial cancer; and thyroid cancer.

[0630] In reference to cancer, the terms " treatment" and " treating" should be taken as encompassing therapy undertaken in order to prevent, slow down, or reduce an undesired physiological change or disorder, such as the growth, development or spread of cancer. Beneficial or desired results include but are not limited to the alleviation of symptoms, diminishment of the extent of disease, stabilized state of disease (which is to say, a disease that is not worsening), delay or slowing of disease progression, de-staging the tumour (e.g., changing from borderline resectable to amendable for surgical resection), amelioration or palliation of the disease state, and remission (either partial or total).

[0631] Treatment may bring about prolonged survival as compared to expected survival if not receiving treatment. Alternatively, or additionally, treatment may provide a patient with an improved standard of life as compared to that which would be expected if not receiving treatment.

[0632] For example, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use in methods of treating allergic disease. An “allergic disease” refers to a condition caused by hypersensitivity of the immune system to typically harmless substances in the environment. Allergic diseases include but are not limited to, asthma, hypersensitivity lung diseases, rhinitis, rhino-conjunctivitis, rhinosinusitis, atopic eczema, contact dermatitis, allergic conjunctivitis (intermittent and persistent), vernal conjunctivitis (hay fever), atopic keratoconjunctivitis, giant papillary conjunctivitis, urticaria (hives), angioedema, hypersensitivity pneumonitis, eosinophilic bronchitis, vasculitis, hypersensitivity vasculitis, antineutrophil cytoplasmic antibody (ANCA) associated vasculitis, Wegner's granulomatosis, Churg Strauss vasculitis, microscopic polyangiitis, temporal arteritis, celiac disease, mastocytosis, and anaphylaxis.

[0633] In particular, the optimized mRNAs or DNA templates encoding said mRNAs described herein may encode a protein that includes an allergenic epitope. It will be apparent that in the majority of cases that the allergenic epitope is an epitope from or derived from an allergen that causes allergy symptoms or allergic reaction in a subject.

[0634] In some examples, the optimized mRNAs or DNA templates encoding said mRNAs described herein that encode a protein that includes an allergenic epitope may be for use in methods of allergy immunotherapy (AIT). In some examples, subcutaneous allergy immunotherapy (SCIT). Optimized mRNAs or DNA templates encoding said mRNAs for use in AIT and / or SCIT may be referred to as allergy vaccines.

[0635] In general, AIT comprises administering an allergen to the patient in order to treat an allergy to that allergen of the patient, i.e. , reducing current or future immune response, such as an allergen-specific IgE response and / or histamine release by mastocytes and / or granulocytes induced by the allergen, and / or manifestation of clinical symptoms of allergy. Immunotherapy is conventionally carried out by repeatedly administering a mono-dose or incremental doses of an allergen to a patient in need thereof, thereby resulting in an adaptive immune response of the patient who becomes desensitised to the allergen.

[0636] During AIT or SCIT, increasing doses of the allergen or allergenic epitope are administered, followed by a maintenance dose for several years, with the goal of inducing immunological changes leading to symptom amelioration while on therapy, as well as sustained desensitization off AIT or SCIT (immune tolerance).

[0637] Typically, at the start of AIT or SCIT, subjects receive increasing doses of the allergen or allergenic epitope at weekly intervals over several weeks to months, under tightly monitored medical supervision. The gradual dose escalation enables tolerability to therapy and mitigates the risk of severe hypersensitivity reactions related to allergen administration.

[0638] For example, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use in methods of treating infectious diseases. “Infectious disease” refers to a disease which results from an infection. Infection is a condition caused by the invasion of an organism by a foreign agent (i.e., an infectious agent). Infectious agents include but are not limited to, bacteria, fungi, viruses, viroids, nematodes (e.g., parasites such as roundworms and pinworms), arthropods (e.g., mites, fleas, lice, ticks), and macroparasites (e.g., tapeworms). Common infectious diseases include bacterial and viral infections. The optimized mRNAs or DNA templates encoding said mRNAs described herein, when for use in methods of treating infectious disease, may include an epitope or antigen from or derived from the pathogen causing the infectious disease.

[0639] The optimized mRNAs or DNA templates encoding said mRNAs described herein may be particularly useful in prevention or treatment of infectious diseases caused by intracellular pathogens. For example, viruses (e.g., CMV, HIV, SARS viruses, such as COVID-19, coronaviruses), bacteria (e.g., Listeria, Mycobacteria, Salmonella (e.g., S. typhi) enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic Escherichia coli (EHEC), Yersinia, Shigella, Chlamydia, Chlamydophila, Staphylococcus, Legionella), protozoa (e.g., Taxoplasma), fungi, and intracellular parasites (e.g., Plasmodium (e.g., P. vivax, P. falciparum, P. ovale, and P. malariae). The compositions and formulations described herein may reduce humoral response against an immunogenic immunomodulator, including epitopes derived from such intracellular pathogens and increase cellular mediated response.

[0640] "Treatment” in relation to infectious diseases refers to any administration of a therapeutic optimized mRNA as described herein that partially or completely alleviates, ameliorates, relieves, inhibits, delays the onset of, reduces the severity of and / or reduces the incidence of one or more symptoms or features of an infectious disease or the predisposition toward the disease. Such treatment may be of a subject who does not exhibit signs of the relevant disease, and / or of a subject who exhibits only early signs of the disease. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease. As such the term "treating" in reference to infectious diseases refers to the vaccination of a subject. “Prevention" refers to a delay of onset of an infectious disease. Prevention may be considered complete when onset of an infectious disease or disorder has been delayed for a predefined period of time.

[0641] In some examples, the optimized mRNAs or DNA templates encoding said mRNAs described herein may be for use as a vaccine. As such, in some examples provided herein are immunogenic compositions comprising a therapeutic optimized mRNA as described. In some examples, the compositions are vaccine compositions. The terms "immunogenic composition" and "immunological composition" and "immunogenic or immunological composition" refer to compositions that elicit an immune response against an antigen or immunogen after administration into a subject. The terms "vaccine" and "vaccine composition" refers to compositions that induce a protective immune response against the antigen of interest or which efficaciously protects against the antigen; for instance, after administration to the subject, elicits a protective immune response against the targeted antigen or immunogen. In some examples, the methods of preventing and / or treating infectious disease is a method of vaccination. "Vaccination" refers to the administration of a therapeutic optimized mRNA as described herein intended to generate an immune response, for example to a diseasecausing pathogen. Vaccination can be administered before, during, and / or after exposure to a disease-causing pathogen, and in some examples, before, during, and / or shortly after exposure to the agent. In some examples, vaccination includes multiple administrations, appropriately spaced in time, of a therapeutic optimized mRNA as described herein.

[0642] In some examples, the optimized mRNAs or DNA templates encoding said mRNAs may be for use in vaccination against a virus. For example, for use in vaccination of a subject against viruses of the retroviridae, orthmyxoviridae, paramyxoviridae, arenaviridae, bunyaviridae, flaviviridae, filoviridae, togaviridae, picornaviridae, caliciviridae and coronaviridae families. Examples of such viruses include, but are not limited to, adenovirus, rhinovirus, hepatitis, immunodeficiency virus, polio, measles, Ebola, Coxsackie, Rhino, West Nile, small pox, encephalitis, yellow fever, Dengue fever, influenza (including human, avian, and swine), lassa, lymphocytic choriomeningitis, junin, machuppo, guanarito, hantavirus, Rift Valley Fever, La Crosse, California encephalitis, Crimean-Congo, Marburg, Japanese Encephalitis, Kyasanur Forest, Venezuelan equine encephalitis, Eastern equine encephalitis, Western equine encephalitis, severe acute respiratory syndrome (SARS), parainfluenza, respiratory syncytial, Punta Toro, Tacaribe and pachindae.

[0643] In some examples, the virus is an influenza (including human, avian, and swine) or a severe acute respiratory syndrome (SARS) virus. In some examples, the virus is a coronaviridae virus. In some examples, the virus is Covid-19.

[0644] As used herein, the terms “treat”, “treating” and "treatment" generally are taken to include an intervention performed with the intention of preventing the development or altering the pathology of a condition, disorder or symptom (e.g., an allergic disease, infectious disease, etc.). Accordingly, "treatment" refers to both therapeutic treatment and prophylactic or preventative measures (such as vaccination), wherein the object is to prevent or slow down (lessen) the targeted condition, disorder or symptom. “Treatment” therefore encompasses a reduction, slowing or inhibition of disease symptoms, for example, at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% when compared to before treatment.

[0645] As used herein the term “subject” generally refers to an individual, e.g., a human, having or at risk of having a specified condition, disorder or symptom. The subject may be a patient, i.e. , a subject in need of treatment in accordance with the invention. The subject may have received treatment for the condition, disorder or symptom. Alternatively, the subject has not been treated prior to treatment in accordance with the present invention. The optimized mRNAs or DNA templates encoding said mRNAs described herein generally can be administered to the subject by any conventional route, including injection or by gradual infusion over time. The administration may, for example, by intramuscular, intravascular, intracavity, intracerebral, intralesional, rectal, subcutaneous, intradermal, epidural, intrathecal, and percutaneous administration.

[0646] The methods of treatment and medical uses described herein may provide optimized mRNAs or DNA templates encoding said mRNAs as described herein to a recipient via any suitable route of administration.

[0647] The optimized mRNAs or DNA templates encoding said mRNAs can be administered via any desired route of administration. The therapeutic optimized mRNAs, or medical uses, may make use of a route of administration selected from the group consisting of: intravenous (iv) administration; subcutaneous (sc) administration; intramuscular (im) administration; intradermal (id) administration; sublingual (si) administration; and intranasal administration.

[0648] The skilled person will be able to determine suitable forms of the optimized mRNAs or DNA templates encoding said mRNAs of the invention for use with the desired route of administration.

[0649] In some examples, the optimized mRNAs or DNA templates encoding said mRNAs as described may be administered via a route selected from intratumoral, inhalation, or intracardiac injection.

[0650] The optimized mRNAs or DNA templates encoding said mRNAs described herein are for administration in an effective amount. An “effective amount” is an amount that alone, or together with further doses, produces the desired (therapeutic or non-therapeutic) response. The effective amount to be used will depend, for example, upon the therapeutic (or non- therapeutic) objectives, the route of administration, and the condition of the patient / subject. For example, the suitable dosage of a therapeutic optimized mRNA of the invention for a given patient / subject will be determined by the attending physician (or person administering the composition), taking into consideration various factors known to modify the action of the optimized mRNAs or DNA templates encoding said mRNAs of the invention for example severity and type of disease, body weight, sex, diet, time and route of administration, other medications and other relevant clinical factors. The dosages and schedules may be varied according to the particular condition, disorder or symptom of the overall condition of the patient / subject. Effective dosages may be determined by either in vitro or in vivo methods.

[0651] As detailed above, certain preferred codons, signal peptides and / or UTR pairs may be more effective in certain cell types. As such, it will be understood that administration of the nucleic acid molecules described herein may be to particular target cells depending on which cell the preferred codons, signal peptides and / or UTR pairs provide the greatest increase in translation efficiency and / or fidelity.

[0652] As such, there is provided herein a nucleic acid molecule comprising a nucleic acid molecule sequence encoding one or more codons for translation to one or more amino acids, wherein at least one codon comprises a muscle cell preferred codon as described herein for use as described herein wherein the nucleic acid molecule is administered to a subject’s muscle cells.

[0653] As such, there is provided herein a nucleic acid molecule comprising a nucleic acid molecule sequence encoding one or more codons for translation to one or more amino acids, wherein at least one codon comprises a kidney cell preferred codon as described herein for use as described herein wherein the nucleic acid molecule is administered to a subject’s kidney cells.

[0654] As such, there is provided herein a nucleic acid molecule comprising a nucleic acid molecule sequence encoding an AACT ...

Claims

Claims1. A nucleic acid molecule comprising a nucleic acid sequence encoding one or more codons for translation to one or more amino acids, wherein at least one codon comprises a preferred codon, wherein the preferred codon has been selected based on one or more properties of the codon, wherein the one or more properties comprise one or more of: a. a level of tRNA isoacceptor expression in a target cell; b. codon decoding speeds; and / or c. binding affinity between the codon and cognate tRNA anticodon.

2. The nucleic acid of claim 1, wherein at least one codon has been selected based on the level of tRNA isoacceptor expression in a target cell.

3. The nucleic acid of claim 1 or 2, wherein all codons for one amino acid have been selected based on the one or more properties of the codon.

4. The nucleic acid of any preceding claim, wherein all codons for all amino acids have been selected based on the one or more properties of each codon.

5. The nucleic acid of any preceding claim, wherein the nucleic acid sequence is configured to increase translation efficiency and / or translation fidelity in the target cell.

6. The nucleic acid of any preceding claim, wherein the target cell comprises an in vivo or in vitro cell.

7. The nucleic acid of any preceding claim, wherein the target cell comprises a skeletal muscle cell and wherein the codon for: a. alanine comprises the sequence 5’ GCU 3’; b. arginine comprises the sequence 5’ AGG 3’; c. aspartic acid comprises the sequence 5’ GAC 3’; d. glutamic acid comprises the sequence 5’ GAG 3’; e. glycine comprises the sequence 5’ GGC 3’; f. leucine comprises the sequence 5’ CUU 3’; g. lysine comprises the sequence 5’ AAG 3’; h. proline comprises the sequence 5’ CCU 3’; i. serine comprises the sequence 5’ AGC 3’;j. asparagine comprises the sequence 5’ AAC 3’; k. glutamine comprises the sequence 5’ CAG 3’; l. histidine comprises the sequence 5’ CAC 3’; m. valine comprises the sequence 5’ GUG 3’; and / or n. methionine comprises the sequence 5’ AUG 3’; or wherein the target cell comprises an HEK293T cell and wherein the codon for: a. alanine comprises the sequence 5’ GCU 3’; b. arginine comprises the sequence 5’ CGU 3’; c. aspartic acid comprises the sequence 5’ GAC 3’; d. glutamic acid comprises the sequence 5’ CAA 3’; e. glycine comprises the sequence 5’ GGC 3’; f. leucine comprises the sequence 5’ CUG 3’; g. lysine comprises the sequence 5’ AAG 3’; h. proline comprises the sequence 5’ CCU 3’; i. serine comprises the sequence 5’ AGC 3’; j. asparagine comprises the sequence 5’ AAC 3’; k. glutamine comprises the sequence 5’ CAG 3’; l. histidine comprises the sequence 5’ CAC 3’; m. valine comprises the sequence 5’ GUU 3’; n. isoleucine comprises the sequence 5’ AUA 3’; o. threonine comprises the sequence 5’ ACU 3’; p. tryptophan comprises the sequence 5’ UGG 3’; q. tyrosine comprises the sequence 5’ UAC 3’; r. phenylalanine comprises the sequence 5’ UUC 3’; and / or s. methionine comprises the sequence 5’ AUG 3’8. A nucleic acid molecule comprising a nucleic acid sequence encoding one or more codons for translation to one or more amino acids and encoding a signal peptide selected from:a. alpha-1 -antichymotrypsin (AACT) signal peptide; or b. Human Serum albumin (HSA) signal peptide.

9. The nucleic acid molecule of claim 8, wherein the nucleic acid molecule is for translation in the target cell, wherein the target cell is a muscle cell, liver cell, kidney cell, cardiomyocyte, and / or antigen presenting cell.

10. The nucleic acid molecule of claim 9, wherein a. the target cell is a liver cell and the signal peptide comprises alpha-1 - antichymotrypsin (AACT) signal peptide; or b. the target cell is a muscle cell and the signal peptide comprises human Serum albumin (HSA) signal peptide.

11. The nucleic acid molecule of any of claims 8 to 10, wherein the alpha-1 - antichymotrypsin (AACT) signal peptide comprises an amino acid sequence according to SEQ ID NO: 10 and / or wherein human Serum albumin (HSA) signal peptide comprises an amino acid sequence according to SEQ ID NO: 22.

12. A nucleic acid molecule comprising a nucleic acid sequence encoding one or more codons for translation to one or more amino acids in a target cell and encoding a 5’ and 3’ untranslated region (UTR): a. wherein the 5’ UTR is derived from a MYL1 mRNA and the 3’ UTR is derived from a MYL1 mRNA (MUTR2); b. wherein the 5’ UTR is derived from a TNNC1 mRNA and the 3’ UTR is derived from a ENO3 mRNA (MUTR4); c. wherein the 5’ UTR is derived from a TNNC1 mRNA and the 3’ UTR is derived from a TNNC1 mRNA (MUTR5); d. wherein the 5’ UTR is derived from a ENO3 mRNA and the 3’ UTR is derived from a ENO3 mRNA (MUTR6); e. wherein the 5’ UTR is derived from a MYH1 mRNA and the 3’ UTR is derived from a MYH1 mRNA (MUTR7); f. wherein the 5’ UTR is derived from a MYL2 mRNA and the 3’ UTR is derived from a MYL2 mRNA (MUTR8); g. wherein the 5’ UTR is derived from a CKM mRNA and the 3’ UTR is derived from a CKM mRNA (MUTR10);h. wherein the 5’ UTR is derived from a ENO3 mRNA and the 3’ UTR is derived from a COX6A2 mRNA (MUTR11); i. wherein the 5’ UTR is derived from a ORM1 mRNA and the 3’ UTR is derived from a ORM1 mRNA (LUTR1); j. wherein the 5’ UTR is derived from a SAA2 mRNA and the 3’ UTR is derived from a SAA2 mRNA (LUTR2); k. wherein the 5’ UTR is derived from a SAA4 mRNA and the 3’ UTR is derived from a SAA4 mRNA (LUTR3); l. wherein the 5’ UTR is derived from a CFHR2 mRNA and the 3’ UTR is derived from a SAA4 mRNA (LUTR5); m. wherein the 5’ UTR is derived from a SAA1 mRNA and the 3’ UTR is derived from a SAA1 mRNA (LUTR6); n. wherein the 5’ UTR is derived from a GC mRNA and the 3’ UTR is derived from a GC mRNA (LUTR7); o. wherein the 5’ UTR is derived from a AHSG mRNA and the 3’ UTR is derived from a AHSG mRNA (LUTR8); p. wherein the 5’ UTR is derived from a SAA4 mRNA and the 3’ UTR is derived from a APOA1 mRNA (LUTR9); or q. wherein the 5’ UTR is derived from a SAA1 mRNA and the 3’ UTR is derived from a GC mRNA (LUTR10).

13. The nucleic acid molecule of claim 12, wherein: a. the 5’ UTR comprises or consists of SEQ ID NO: 37 and the 3’ UTR comprises or consists of SEQ ID NO: 38 (MUTR2); b. the 5’ UTR comprises or consists of SEQ ID NO: 41 and the 3’ UTR comprises or consists of SEQ ID NO: 42 (MUTR4); c. the 5’ UTR comprises or consists of SEQ ID NO: 43 and the 3’ UTR comprises or consists of SEQ ID NO: 44 (MUTR5); d. the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 46 (MUTR6); e. the 5’ UTR comprises or consists of SEQ ID NO: 47 and the 3’ UTR comprises or consists of SEQ ID NO: 48 (MUTR7);f. the 5’ UTR comprises or consists of SEQ ID NO: 49 and the 3’ UTR comprises or consists of SEQ ID NO: 50 (MLITR8); g. the 5’ UTR comprises or consists of SEQ ID NO: 53 and the 3’ UTR comprises or consists of SEQ ID NO: 54 (MUTR10); h. the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 55 (MUTR11); i. the 5’ UTR comprises or consists of SEQ ID NO: 56 and the 3’ UTR comprises or consists of SEQ ID NO: 57 (LUTR1); j. the 5’ UTR comprises or consists of SEQ ID NO: 58 and the 3’ UTR comprises or consists of SEQ ID NO: 59 (LUTR2); k. the 5’ UTR comprises or consists of SEQ ID NO: 60 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR3); l. the 5’ UTR comprises or consists of SEQ ID NO: 64 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR5); m. the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 66 (LUTR6); n. the 5’ UTR comprises or consists of SEQ ID NO: 67 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR7); o. the 5’ UTR comprises or consists of SEQ ID NO: 69 and the 3’ UTR comprises or consists of SEQ ID NO: 70 (LUTR8); p. the 5’ UTR comprises or consists of SEQ ID NO: 60 and the 3’ UTR comprises or consists of SEQ ID NO: 63 (LUTR9) or q. the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR10).

14. The nucleic acid molecule of claim 12 or 13, wherein the target cell is a muscle cell and: a. the 5’ UTR comprises or consists of SEQ ID NO: 37 and the 3’ UTR comprises or consists of SEQ ID NO: 38 (MUTR2); b. the 5’ UTR comprises or consists of SEQ ID NO: 41 and the 3’ UTR comprises or consists of SEQ ID NO: 42 (MUTR4); c. the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 46 (MUTR6);d. the 5’ UTR comprises or consists of SEQ ID NO: 47 and the 3’ UTR comprises or consists of SEQ ID NO: 48 (MLITR7); e. the 5’ UTR comprises or consists of SEQ ID NO: 49 and the 3’ UTR comprises or consists of SEQ ID NO: 50 (MUTR8); f. the 5’ UTR comprises or consists of SEQ ID NO: 53 and the 3’ UTR comprises or consists of SEQ ID NO: 54 (MUTR10); g. the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 55 (MUTR11); h. the 5’ UTR comprises or consists of SEQ ID NO: 56 and the 3’ UTR comprises or consists of SEQ ID NO: 57 (LUTR1); i. the 5’ UTR comprises or consists of SEQ ID NO: 58 and the 3’ UTR comprises or consists of SEQ ID NO: 59 (LUTR2); j. the 5’ UTR comprises or consists of SEQ ID NO: 60 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR3); k. the 5’ UTR comprises or consists of SEQ ID NO: 64 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR5); l. the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 66 (LUTR6); m. the 5’ UTR comprises or consists of SEQ ID NO: 67 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR7); n. the 5’ UTR comprises or consists of SEQ ID NO: 69 and the 3’ UTR comprises or consists of SEQ ID NO: 70 (LUTR8); o. the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR10); or wherein the target cell is a liver cell and: a. the 5’ UTR comprises or consists of SEQ ID NO: 37 and the 3’ UTR comprises or consists of SEQ ID NO: 38 (MUTR2); b. the 5’ UTR comprises or consists of SEQ ID NO: 41 and the 3’ UTR comprises or consists of SEQ ID NO: 42 (MUTR4); c. the 5’ UTR comprises or consists of SEQ ID NO: 43 and the 3’ UTR comprises or consists of SEQ ID NO: 44 (MUTR5);d. the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 46 (MLITR6); e. the 5’ UTR comprises or consists of SEQ ID NO: 47 and the 3’ UTR comprises or consists of SEQ ID NO: 48 (MUTR7); f. the 5’ UTR comprises or consists of SEQ ID NO: 49 and the 3’ UTR comprises or consists of SEQ ID NO: 50 (MUTR8); g. the 5’ UTR comprises or consists of SEQ ID NO: 53 and the 3’ UTR comprises or consists of SEQ ID NO: 54 (MUTR10); h. the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 55 (MUTR11); i. the 5’ UTR comprises or consists of SEQ ID NO: 56 and the 3’ UTR comprises or consists of SEQ ID NO: 57 (LUTR1); j. the 5’ UTR comprises or consists of SEQ ID NO: 58 and the 3’ UTR comprises or consists of SEQ ID NO: 59 (LUTR2); k. the 5’ UTR comprises or consists of SEQ ID NO: 60 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR3); l. the 5’ UTR comprises or consists of SEQ ID NO: 64 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR5); m. the 5’ UTR comprises or consists of SEQ ID NO: 67 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR7); n. the 5’ UTR comprises or consists of SEQ ID NO: 69 and the 3’ UTR comprises or consists of SEQ ID NO: 70 (LUTR8); o. the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR10); or wherein the target cell is an HEK293T cell and a. the 5’ UTR comprises or consists of SEQ ID NO: 41 and the 3’ UTR comprises or consists of SEQ ID NO: 42 (MUTR4); b. the 5’ UTR comprises or consists of SEQ ID NO: 47 and the 3’ UTR comprises or consists of SEQ ID NO: 48 (MUTR7); c. the 5’ UTR comprises or consists of SEQ ID NO: 45 and the 3’ UTR comprises or consists of SEQ ID NO: 55 (MUTR11);d. the 5’ UTR comprises or consists of SEQ ID NO: 56 and the 3’ UTR comprises or consists of SEQ ID NO: 57 (LLITR1); e. the 5’ UTR comprises or consists of SEQ ID NO: 58 and the 3’ UTR comprises or consists of SEQ ID NO: 59 (LUTR2); f. the 5’ UTR comprises or consists of SEQ ID NO: 60 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR3); g. the 5’ UTR comprises or consists of SEQ ID NO: 64 and the 3’ UTR comprises or consists of SEQ ID NO: 61 (LUTR5); h. the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 66 (LUTR6); i. the 5’ UTR comprises or consists of SEQ ID NO: 67 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR7); j. the 5’ UTR comprises or consists of SEQ ID NO: 69 and the 3’ UTR comprises or consists of SEQ ID NO: 70 (LUTR8); k. the 5’ UTR comprises or consists of SEQ ID NO: 60 and the 3’ UTR comprises or consists of SEQ ID NO: 63 (LUTR9); or l. the 5’ UTR comprises or consists of SEQ ID NO: 65 and the 3’ UTR comprises or consists of SEQ ID NO: 68 (LUTR10).

15. A nucleic acid molecule comprising a nucleic acid sequence encoding one or more codons for translation to one or more amino acids in a target cell and comprising at least one alternative reading frame sequence which encodes an alternative translation product that differs from the translation product of in-frame translation of the nucleic acid, wherein the nucleic acid comprises a nucleic acid sequence encoding at least two consecutive stop codons (tandem stop codon) for reducing translation of the alternative translation products.

16. The nucleic acid molecule of claim 15, wherein the at least two consecutive stop codons each comprise a nucleic acid sequence consisting of UAA or UAG.

17. The nucleic acid molecule of claim 15 or 16, comprising two consecutive stop codons and wherein the two consecutive stop codons together comprise a nucleic acid sequence comprising or consisting of: a. UAAUAA; b. UAAUAG;c. UAGUAA; or d. UAGUAG.

18. The nucleic acid molecule of any of claims 15 to 17, wherein the at least one tandem stop codon is encoded in a +1 reading frame from the translation product of in-frame translation.

19. The nucleic acid molecule of any of claims 15 to 18, wherein the nucleic acid comprises an RNA and wherein at least one tandem stop codon comprise at least one N1 -methyl pseudouridine.

20. The nucleic acid molecule of any preceding claim, wherein the nucleic acid molecule comprises an mRNA or a DNA molecule encoding an mRNA.

21. A nucleic acid molecule comprising a nucleic acid sequence encoding: a. a nucleic acid sequence according to any of claims 1 to 7 and 20; b. a nucleic acid sequence according to any of claims 8 to 11 and 20; c. a nucleic acid sequence according to any of claims 12 to 14 an 20; and / or d. a nucleic acid sequence according to any of claims 15 to 19 or 20.

22. An in vitro transcribed mRNA comprising a nucleic acid sequence encoding: a. a nucleic acid sequence according to any of claims 1 to 7; b. a nucleic acid sequence according to any of claims 8 to 11 ; c. a nucleic acid sequence according to any of claims 12 to 14; and / or d. a nucleic acid sequence according to any of claims 15 to 19.

23. The nucleic acid molecule any of claims 1 to 21 , wherein the nucleic acid molecule is an mRNA or encodes an mRNA, or the in vitro transcribed mRNA of claim 22, wherein the mRNA comprises a therapeutic mRNA.

24. The nucleic acid molecule of any of claims 1 to 21 and 23 or the in vitro transcribed mRNA of any of claims 22 or 23, wherein the nucleic acid molecule or in vitro transcribed mRNA comprises a nucleic acid sequence encoding a poly(A) tail wherein the poly(A) tail comprises 97 to 135 adenine residues; optionally comprises 97 adenine residues.

25. The nucleic acid molecule of any of claims 1 to 21 and 23 to 24, wherein the nucleic acid molecule is an mRNA, or the in vitro transcribed mRNA of any of claims 22 to 24, wherein the mRNA comprises a m7G-5'ppp5’GpG 5’ cap.

26. The nucleic acid molecule of any of claims 1 to 21 and 23 to 25, wherein the nucleic acid molecule is an mRNA, or the in vitro transcribed mRNA of any of claims 22 to 26, wherein the mRNA comprises one or more modified ribonucleotide.

27. A method of producing an optimised nucleic acid molecule for translation, the method comprising; a. determining preferred codons in a target cell for at least one amino acid; b. producing a nucleic acid molecule that comprises a nucleic acid sequence encoding one or more codons for translation to one or more amino acids, wherein at least one codon for the first amino acid comprises the preferred codon; wherein determining preferred codons comprises: i. determining a level of tRNA isoacceptor expression in a target cell for at least one first amino acid; ii. determining codon decoding speed for at least one first amino acid; and / or iii. determining binding affinity between a codon and cognate tRNA anticodon for at least one first amino acid.

28. The method of claim 27, wherein determining a level of tRNA isoacceptor expression comprises: a. determining the most abundantly expressed tRNA anti-codon for the at least one first amino acid and wherein the preferred codon comprises the complement of the most abundantly expressed tRNA anti-codon; or b. determining the ratio of the level of expression of cognate to near-cognate tRNAs for the at least one first amino acid and wherein the preferred codon comprises the complement of the anti-codon with the lowest ratio of nearcognate to cognate tRNAs .

29. The method of claim 27 or 28, wherein the method further comprises determining the preferred codon for one or more further amino acids.

30. The method of claim 29, wherein the nucleic acid molecule comprises a nucleic acid sequence encoding at least one codon for the each of the one or more further amino acids, wherein the at least one codon for each further amino acid comprises the preferred codon for each further amino acid.

31. The method of any of claims 27 to 30, wherein all codons for each of the at least one first and / or further amino acids comprise the preferred codon.

32. The method of any of claims 27 to 31, wherein the nucleic acid molecule comprises an mRNA or a DNA molecule encoding an mRNA.

33. The method of claim 32, wherein the producing further comprises in vitro transcribing the DNA molecule to produce the mRNA.

34. The method of claim 32 or 33, wherein the mRNA comprises a therapeutic mRNA.

35. The method of any of claims 27 to 34, wherein the producing further comprises adding a nucleic acid sequence encoding a poly(A) tail; optionally wherein the poly(A) tail comprises 97 to 135 adenine residues; further optionally wherein the poly(A) tail comprises 97 adenine residues.

36. The method of claim 32 to 35, wherein the method comprises enzymatically adding a or the poly(A) tail optionally, according to claim 35.

37. The method of any of claims 32 to 36, wherein the producing further comprises adding a m7G-5'ppp5’GpG 5’ cap.

38. The method of any of claims 32 to 37, wherein the mRNA comprises one or more modified ribonucleotide.

39. Use of a nucleic acid molecule encoding a 5’ and 3’ UTR: a. wherein the 5’ UTR is derived from a MYLPF mRNA and the 3’ UTR is derived from a MYLPF mRNA (MUTR2); b. wherein the 5’ UTR is derived from a TNNC1 mRNA and the 3’ UTR is derived from a ENO3 mRNA (MUTR4); c. wherein the 5’ UTR is derived from a ENO3 mRNA and the 3’ UTR is derived from a ENO3 mRNA (MUTR6); d. wherein the 5’ UTR is derived from a MYH1 mRNA and the 3’ UTR is derived from a MYH1 mRNA (MUTR7); e. wherein the 5’ UTR is derived from a MYL2 mRNA and the 3’ UTR is derived from a MYL2 mRNA (MUTR8); f. wherein the 5’ UTR is derived from a CKM mRNA and the 3’ UTR is derived from a CKM mRNA (MUTR10); g. wherein the 5’ UTR is derived from a ENO3 mRNA and the 3’ UTR is derived from a COX6A2 mRNA (MUTR11);h. wherein the 5’ UTR is derived from a ORM1 mRNA and the 3’ UTR is derived from a ORM1 mRNA (LUTR1); i. wherein the 5’ UTR is derived from a SAA2 mRNA and the 3’ UTR is derived from a SAA2 mRNA (LUTR2); j. wherein the 5’ UTR is derived from a SAA4 mRNA and the 3’ UTR is derived from a SAA4 mRNA (LUTR3); k. wherein the 5’ UTR is derived from a CFHR2 mRNA and the 3’ UTR is derived from a SAA4 mRNA (LUTR5); l. wherein the 5’ UTR is derived from a SAA1 mRNA and the 3’ UTR is derived from a SAA1 mRNA (LUTR6); m. wherein the 5’ UTR is derived from a GC mRNA and the 3’ UTR is derived from a GC mRNA (LUTR7); n. wherein the 5’ UTR is derived from a AHSG mRNA and the 3’ UTR is derived from a AHSG mRNA (LUTR8); or o. wherein the 5’ UTR is derived from a SAA1 mRNA and the 3’ UTR is derived from a GC mRNA (LUTR10); for translation of an mRNA in a muscle cell.

40. Use of a nucleic acid molecule encoding a UTR: a. wherein the 5’ UTR is derived from a MYLPF mRNA and the 3’ UTR is derived from a MYLPF mRNA (MUTR2); b. wherein the 5’ UTR is derived from a TNNC1 mRNA and the 3’ UTR is derived from a ENO3 mRNA (MUTR4); c. wherein the 5’ UTR is derived from a TNNC1 mRNA and the 3’ UTR is derived from a TNNC1 mRNA (MUTR5); d. wherein the 5’ UTR is derived from a ENO3 mRNA and the 3’ UTR is derived from a ENO3 mRNA (MUTR6); e. wherein the 5’ UTR is derived from a MYH1 mRNA and the 3’ UTR is derived from a MYH1 mRNA (MUTR7); f. wherein the 5’ UTR is derived from a MYL2 mRNA and the 3’ UTR is derived from a MYL2 mRNA (MUTR8); g. wherein the 5’ UTR is derived from a CKM mRNA and the 3’ UTR is derived from a CKM mRNA (MUTR10);h. wherein the 5’ UTR is derived from a ENO3 mRNA and the 3’ UTR is derived from a COX6A2 mRNA (MUTR11); i. wherein the 5’ UTR is derived from a ORM1 mRNA and the 3’ UTR is derived from a ORM1 mRNA (LUTR1); j. wherein the 5’ UTR is derived from a SAA2 mRNA and the 3’ UTR is derived from a SAA2 mRNA (LUTR2); k. wherein the 5’ UTR is derived from a SAA4 mRNA and the 3’ UTR is derived from a SAA4 mRNA (LUTR3); l. wherein the 5’ UTR is derived from a CFHR2 mRNA and the 3’ UTR is derived from a SAA4 mRNA (LUTR5); m. wherein the 5’ UTR is derived from a SAA1 mRNA and the 3’ UTR is derived from a SAA1 mRNA (LUTR6); n. wherein the 5’ UTR is derived from a GC mRNA and the 3’ UTR is derived from a GC mRNA (LUTR7); o. wherein the 5’ UTR is derived from a AHSG mRNA and the 3’ UTR is derived from a AHSG mRNA (LUTR8); or p. wherein the 5’ UTR is derived from a SAA1 mRNA and the 3’ UTR is derived from a GC mRNA (LUTR10; for translation of an mRNA in a liver cell.

41. Use of an alpha- 1 -antichymotrypsin (AACT) signal peptide for translation of an mRNA in a liver cell.

42. Use of a human Serum albumin (HSA) signal peptide for translation of an mRNA in a muscle cell.

43. The nucleic acid molecule according to any of claims 8 to 11 , 21 and 23 to 26, or in vitro transcribed mRNA according to any of claims 22 to 26, or use according to claims 41 or 42, wherein the nucleic acid sequence encoding: a. the human Serum albumin (HSA) signal peptide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 23 or 24; or b. the alpha-1-antichymotrypsin (AACT) signal peptide comprises or consists of a nucleic acid sequence according to SEQ ID NO: 11 or 12.

44. A nucleic acid molecule or in vitro transcribed mRNA of any of claims 21 to 26 or a pharmaceutical composition thereof for use as a medicament.

45. A nucleic acid molecule or in vitro transcribed mRNA of any of claims 21 to 26 or a pharmaceutical composition thereof for use as a vaccine.

46. A nucleic acid molecule or in vitro transcribed mRNA of any of claims 21 to 26 or a pharmaceutical composition thereof for use in inducing an immune response in a subject.

47. A method of treating or preventing a disease in a subject in need thereof, comprising administering a nucleic acid molecule or in vitro transcribed mRNA of any of claims 21 to 26 or a pharmaceutical composition thereof to the subject.

48. A method of vaccinating a subject in need thereof, comprising administering a nucleic acid molecule or in vitro transcribed mRNA of any of claims 21 to 26 or a pharmaceutical composition thereof to the subject.

49. A method of inducing an immune response in a subject in need thereof, comprising administering a nucleic acid molecule or in vitro transcribed mRNA of any of claims 21 to 26 or a pharmaceutical composition thereof to the subject.

50. A computer-implemented method for designing an optimised RNA sequence for translation, the method comprising: a. receiving a target translated protein sequence, one or more user-defined objectives for the optimised RNA sequence, and one or more parameters associated with each user-defined objective and corresponding to the optimised RNA sequence, the one or more parameters including: i. codon composition efficiency for translation of the RNA sequence to the target translated protein sequence; ii. presence or absence of specified nucleotide repeats; iii. GC content; iv. desirability of specified nucleotide sub-sequences; and v. location of one or more stop codons; b. receiving deviation information for each parameter, wherein the deviation information is for processing a nucleotide sequence and is indicative of a degree of deviation of a nucleotide sequence from a target value of the associated parameter; c. determining a score function for each parameter based on the deviation information, wherein each score function is usable for processing a nucleotide sequence to generate a score indicative of the degree of deviation betweenthe nucleotide sequence and the target value of the parameter associated with that score function; d. selecting, from amongst a plurality of nucleotide sequences, a set of random nucleotide sequences, wherein each random nucleotide sequence comprises a nucleotide sequence that encodes the target translated protein sequence; e. generating one or more parameter scores for each random nucleotide sequence based on the score functions determined for the corresponding one or more parameters; f. generating a total score for each random nucleotide sequence based on the one or more parameter scores generated for that random nucleotide sequence; g. assigning a rank to each random nucleotide sequence in the set of random nucleotide sequences based on the generated total scores of the random nucleotide sequences from a lowest rank to a highest rank; and removing, from the plurality of nucleotide sequences, one or more of the lowest ranked random nucleotide sequences based on the assigned ranking of each random nucleotide sequence, to provide a reduced plurality of nucleotide sequences; h. performing a plurality of iterative steps including repeating steps (d) to (g), wherein the plurality of nucleotide sequences in step (d) in an iterative step corresponds to the reduced plurality of nucleotide sequences of step (g) in the previous iterative step; i. wherein step (h) is performed until a stop condition is met to provide a set of optimised RNA sequences, wherein each optimised RNA sequence has a total score above a score threshold. wherein each optimised RNA sequence comprises at least one different sequence property.

51. The method of claim 50, wherein the random nucleotide sequence assigned with the lowest rank has a total score indicative of the one or more parameter scores having the greatest degree of deviation from the target values associated with the respective one or more parameters and the random nucleotide sequence assigned with the highest rank has a total score indicative of the one or more parameter scores having the smallest degree of deviation from the target values associated with the respective one or more parameters.

52. The method of claim 50 or 51 , further comprising identifying the highest ranked random nucleotide sequence for the set of random nucleotide sequences in a first iterative step and for the set of random nucleotide sequences in a second iterative step, the first and second iterative steps among the plurality of iterative steps; and determining a difference in the total score of the highest ranked random nucleotide sequence in the first iterative step and the second iterative step; comparing the difference in the total score of the highest ranked random nucleotide sequence in the first iterative step and the second iterative step with a difference threshold, wherein the stop condition comprises one or more of: a. a predetermined number of iterations; b. based on the comparison, the difference in the total score of the highest ranked random nucleotide sequence in the first iterative step and the second iterative step being below the difference threshold; and c. at least one selected random nucleotide sequence has a total score above an optimisation threshold.

53. The method of any one of claims 50 to 52, wherein the user defined objectives comprise one or more of target application, biological activity, expression profile, manufacturability, secretion profile and / or storability.

54. The method of any one of claims 50 to 53, further comprising: a. selecting one or more sequence elements from a database based on the one or more user defined objectives and the one or more parameters; optionally wherein the sequence elements comprise one or more of a 5’-UTR, a 3’-UTR, a poly(A) tail length, signal peptide sequence, an aptamer sequence, protein binding sequences, tandem stop codon and / or nucleic acid binding sequences. b. combining the selected sequence elements with each optimised RNA sequence to provide a set of full-length mRNA sequences; and c. outputting a signal indicative of a set of optimised full-length mRNA sequences, wherein the set of optimised full-length mRNA sequences comprises full-length mRNA sequences which comprise at least one optimised RNA sequence which meets the score threshold of claim 50 (i).

55. The method of claim 54, wherein the set of optimised full-length mRNA sequences comprises full-length mRNA sequences which comprise an optimised RNA sequence which has a total score below the score threshold of claim 50 (i), the method further comprises a. repeating steps (d) to (i) of claim 50 for the optimised full-length mRNA sequences; and b. outputting a signal indicative of a set of optimised full-length mRNA sequences, comprising the optimised full-length mRNA sequence identified as having the highest total score.

56. The method of any of claims 50 to 55, wherein: a. target application comprises one or more of: administration route, end user purpose, target tissue type, target cell type, cellular localisation, and / or cellular processing ; b. codon composition efficiency is determined using one or more of: codon decoding times, codon preference, codon usage frequency, codon usage patterns, abundance of tRNA isoacceptors and / or ratio of cognate to nearcognate tRNAs; c. specified nucleotide sub-sequences comprise: frameshifting sequences, ribosomal slippery sequences, transcription terminator sequences, RNA polymerase stalling sequences, aptamer sequences, secondary structure forming sequences, restriction enzyme sites, RNA binding protein binding sites and / or frameshifted premature termination codons; d. biological activity comprises one or more of: immunogenicity, cellular interactions, adjuvant activity, enzymatic activity and / or cellular effects; e. expression profile comprises expression duration, expression location and / or expression level; f. manufacturability comprises yield of encoded protein; g. secretion profile comprises localisation of the RNA and / or encoded protein in a target cell; and / or h. storability comprises stability of the RNA at predetermined environmental conditions.

57. The method of any of claims 50 to 56,wherein step (g) of claim 50 further comprises combining n nucleotides of the 5' end of at least one first removed nucleotide sequence with L - n nucleotides of the 3’ end of a second removed nucleotide sequence to provide a mixed sequence; and / or randomly mutating at least one of the removed nucleotide sequences to generate a randomly mutated sequence, carrying out steps (e) to (g) for each randomly mutated sequence and selecting at least one optimised randomly mutated sequence based on the score threshold.

58. The method of any of claims 50 to 57, wherein, in the plurality of iterative steps of step (h) of claim 50, the set of random nucleotide sequences in a given iterative step is selected to include the non-removed nucleotide sequences present from the set of random nucleotide sequences in the previous iterative step and one or more nucleotide sequences that were not included in the set of random nucleotide sequences in the previous iterative step, wherein the set of random nucleotide sequences in the given iterative step further comprises one or more mixed sequences and / or optimised randomly mutated sequences.

59. The method of any of claims 50 to 58, wherein the set of random nucleotide sequences comprises one or more seeding nucleotide sequences wherein each seeding nucleotide sequence comprises a nucleotide sequence that has a predetermined parameter score for one of the one or more parameters and encodes the target translated protein sequence.

60. Computer readable instructions which, when executed by one or more processors, cause the one or more processors to perform the method of any one of claims 50 to 59.

61. An apparatus for designing an optimised RNA sequence for translation, the apparatus comprising: a memory arranged to store machine-readable instructions; an input unit arranged to receive an input; and processing circuitry arranged to operably execute the stored machine-readable instructions to: a. receive, via the input unit, a target translated protein sequence, one or more user-defined objectives for the optimised RNA sequence, and one or more parameters associated with each user-defined objective and corresponding to the target translated protein sequence, the one or more parameters including:i. codon composition efficiency for translation of the RNA sequence to the target translated protein sequence; ii. presence or absence of specified nucleotide repeats; iii. GC content; and iv. desirability of specified nucleotide sub-sequences; b. receive, via the input unit, deviation information for each parameter, the deviation information for processing a nucleotide sequence and indicative of a degree of deviation of a nucleotide sequence from a target value of the associated parameter; c. determine a score function for each parameter based on the deviation information, wherein each score function is usable for processing a nucleotide sequence to generate a score indicative of a degree of deviation between the nucleotide sequence and the target value of the parameter associated with that score function; d. select, from amongst a plurality of nucleotide sequences, a set of random nucleotide sequences, wherein each random nucleotide sequence comprises a nucleotide sequence that encodes the target translated protein sequence; e. generate one or more parameter scores for each random nucleotide sequence based on the score functions determined for the corresponding one or more parameters; f. generate a total score for each random nucleotide sequence based on the one or more parameter scores generated for that random nucleotide sequence; g. assign a rank to each random nucleotide sequence in the set of random nucleotide sequences based on the generated total scores of the random nucleotide sequences from a lowest rank to a highest rank; and remove, from the plurality of nucleotide sequences, one or more of the lowest ranked random nucleotide sequences based on the assigned ranking of each random nucleotide sequence, to provide a reduced plurality of nucleotide sequences; h. perform a plurality of iterative steps including repeating steps (d) to (g), wherein the plurality of nucleotide sequences in step (d) in aniterative step corresponds to the reduced plurality of nucleotide sequences of step (g) in the previous iterative step, i. wherein step (h) is repeated until a stop condition is met to provide a set of optimised RNA sequences, wherein each optimised RNA sequence has a total score above a score threshold. wherein each optimised RNA sequence comprises at least one different sequence property.

62. The method of claim 61 , wherein the set of random nucleotide sequences comprises one or more seeding nucleotide sequences wherein each seeding nucleotide sequence comprises a nucleotide sequence that has a predetermined parameter score for one of the one or more parameters and encodes the target translated protein sequence.

63. A system for designing an optimised RNA sequence for translation, the system comprising: the apparatus of claim 61 or 62; and a second apparatus comprising a database including one or more sequence elements, wherein the apparatus of claim 61 or 62 is configured to retrieve the one or more sequence elements from the second apparatus for providing a set of full-length mRNA sequences.

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