RU2847257C1 - Creation of optimized nucleotide sequences - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: described is a computerized method of creating an optimized nucleotide sequence, involving: (i) obtaining an amino acid sequence, wherein the amino acid sequence encodes a peptide, polypeptide or protein; (ii) obtaining a first codon usage table, where the first codon usage table comprises a list of amino acids, where each amino acid in the table is associated with at least one codon and each codon is associated with a usage frequency; (iii) deleting any codons associated with a usage frequency that is less than a threshold frequency from the codon usage frequency table; (iv) creating a normalized codon frequency table by normalizing the codon usage frequencies not removed in step (iii); and (v) creating an optimized nucleotide sequence encoding an amino acid sequence, by selecting a codon for each amino acid in the amino acid sequence based on the frequency of use of one or more codons associated with the amino acid in a normalized table of codon usage frequencies. Also described is a method for synthesis of nucleic acid having a nucleotide sequence, involving: performing the described computerized method to create at least one optimized nucleotide sequence; and synthesis of at least one nucleic acid comprising one of the generated optimized nucleotide sequences.

EFFECT: invention widens the range of means of optimizing nucleic acid for synthesis in vitro and for expression of a functional protein, polypeptide or peptide encoded by such an optimized acid in a cell.

54 cl, 15 dwg, 3 tbl

Description

RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 63/021,345, filed May 7, 2020, the disclosure of which is incorporated herein by reference in its entirety. U.S. Provisional Patent Application Ser. No. 62/978,180, filed February 18, 2020, is incorporated herein by reference in its entirety.

SEQUENCE LIST

[0002] This disclosure makes reference to the Sequence Listing (submitted electronically on May 7, 2021, as a .txt file named MRT-2131WO_SL). This .txt file was created on April 27, 2021, and has a size of 63.5 KB. The entire contents of the sequence listing are incorporated herein by reference.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0003] The present invention relates to methods for creating an optimized nucleotide sequence. In particular, the present invention relates to methods in which a nucleotide sequence is optimized for in vitro synthesis and for expression of a functional protein, polypeptide or peptide encoded by the optimized nucleotide sequence in a cell.

BACKGROUND OF THE INVENTION

[0004] mRNA-based therapy is becoming increasingly important in the treatment of various diseases, particularly those caused by protein or gene dysfunction. Genetic mutations in an organism's DNA sequence can lead to aberrant gene expression, resulting in defects in protein production or function. For example, mutations in the underlying DNA sequence can lead to underexpression or overexpression of a protein or the production of dysfunctional proteins. Restoration of normal or healthy protein levels can be achieved through mRNA-based therapy, which is widely applicable to a number of diseases caused by gene or protein dysfunction.

[0005] In mRNA-based therapy, an mRNA molecule encoding a functional protein that can replace a defective or missing protein is delivered to a target cell or tissue. Administration of mRNA encoding a therapeutic protein effective in the treatment or prevention of a disease or disorder can also provide a cost-effective alternative to recombinantly produced peptide, polypeptide, or protein therapy. mRNA-based therapy can restore normal levels of an endogenous protein or provide an exogenous therapeutic protein without irreversibly altering the genomic sequence or entering the cell nucleus. mRNA-based therapy takes advantage of a cell's own protein production and processing machinery to treat diseases or disorders, is flexible in dosage and composition, and is broadly applicable to any disease or condition caused by a defect in the underlying gene or protein or treatable by providing an exogenous protein.

[0006] Expression levels of a protein encoded by mRNA can significantly impact the efficacy and therapeutic benefits of mRNA-based therapies. Efficient expression or production of a protein from mRNA within a cell depends on a variety of factors. Optimization of the composition and order of codons in a nucleotide sequence encoding a protein ("codon optimization") can result in higher expression of the protein encoded by mRNA. Various methods of codon optimization are known in the art; however, each has significant drawbacks and limitations from a computational and/or therapeutic standpoint. In particular, known methods of codon optimization often involve replacing each codon with the most frequently used codon for that amino acid for each amino acid, so that the "optimized" sequence contains only one codon encoding each amino acid (therefore, such a sequence can be referred to as a one-to-one sequence).

[0007] Accordingly, there is a need for improved codon optimization methods that provide an optimized nucleotide sequence to enhance protein expression in mRNA-based therapy.

ESSENCE OF THE INVENTION

[0008] The present invention addresses the need for improved methods for optimizing nucleic acids for effective mRNA-based therapy by providing a method for analyzing an amino acid sequence to obtain at least one optimized nucleotide sequence. The optimized nucleotide sequence is designed to increase protein expression compared to the expression of a protein associated with the nucleotide sequence found in nature. The nucleic acid optimization methods of the present invention provide the ability to synthesize full-length mRNA transcripts in vitro and increase expression of a protein of interest under conditions where it is desirable to achieve a higher protein yield.

[0009] For example, codon optimization can be used to increase the expression of a protein of interest in mRNA-based therapy, immunology and vaccination, cancer immunotherapy, biotechnology, and manufacturing. Codon optimization provides a protein-coding nucleotide sequence based on various criteria without changing the sequence of translated amino acids of the encoded protein due to redundancy in the genetic code.

[00010] To avoid an imbalance between the frequency of mRNA codon usage and the number of cognate tRNAs, codon optimization can ensure a codon composition in the nucleotide sequence that better matches the naturally occurring number of transfer RNAs (tRNAs) in the host cell and avoids depletion of a particular tRNA. Because the number of tRNAs affects the rate of protein translation, optimization of the nucleotide sequence codons can improve protein translation efficiency and the yield of the encoded protein. For example, avoiding rare codons, which are characterized by a low codon usage frequency, can improve protein translation efficiency and protein yield, since a shortage of rare tRNAs can halt or abort protein translation. However, codon optimization may come at the expense of reduced functional activity of the encoded protein and associated loss of efficiency, as the process can remove information encoded in the nucleotide sequence that is important for controlling protein translation and ensuring the correct folding of the synthesized polypeptide chain (Mauro & Chappell, Trends Mol Med. 2014; 20(11):604-13). The present inventors have found that optimized sequences that retain some diversity, i.e., do not necessarily include only one codon encoding each amino acid, can provide increased protein yield compared to both naturally occurring sequences and one-to-one sequences.

[00011] In a first aspect, the present invention relates to a computerized method for generating an optimized nucleotide sequence, comprising: (i) obtaining an amino acid sequence, wherein the amino acid sequence encodes a peptide, polypeptide, or protein; (ii) obtaining a first table of codon usage frequencies, wherein the first table of codon usage frequencies comprises a list of amino acids, wherein each amino acid in the table is associated with at least one codon, and each codon is associated with a usage frequency; (iii) removing from the table of codon usage frequencies any codons associated with a usage frequency that is less than a cutoff frequency; (iv) creating a normalized table of codon usage frequencies by normalizing the frequencies of codons not removed in step (iii); and (v) creating an optimized nucleotide sequence encoding the amino acid sequence by selecting a codon for each amino acid in the amino acid sequence based on the frequency of usage of one or more codons associated with the amino acid in the normalized table of codon usage frequencies. In some embodiments, the cutoff frequency is selected by the user. In some embodiments, the cutoff frequency is in the range of 5% to 30%, in particular 5%, or 15%, or 20%, or 25%, or 30%, or in particular 10%. The inventors have found that cutoff frequencies having the values described herein can result in the formation of optimized sequences that are capable of providing increased protein yield.

[00012] In some embodiments, the step of creating a normalized codon usage frequency table comprises: (a) distributing the usage frequencies of each codon associated with the first amino acid and removed in step (iii) to the remaining codons associated with the first amino acid; and (b) repeating step (a) for each amino acid to obtain a normalized codon usage frequency table. In some embodiments, the usage frequencies of the removed codons are distributed evenly among the remaining codons. In some embodiments, the usage frequencies of the removed codons are distributed among the remaining codons proportionally to the usage frequencies of each remaining codon.

[00013] In some embodiments, selecting a codon for each amino acid comprises: (a) identifying in a normalized codon usage table one or more codons associated with a first amino acid of an amino acid sequence; (b) selecting a codon associated with the first amino acid, wherein the probability of selecting a particular codon is equal to the usage frequency associated with the codon associated with the first amino acid in the normalized codon usage table; and (c) repeating steps (a) and (b) until a codon for each amino acid in the amino acid sequence is selected.

[00014] In some embodiments, the step of generating an optimized nucleotide sequence by selecting a codon for each amino acid in the amino acid sequence (step (v) in the above method) is performed n times to generate a list of optimized nucleotide sequences.

[00015] In some embodiments, the method further comprises: screening the list of optimized nucleotide sequences to identify and remove optimized nucleotide sequences that do not meet one or more criteria. Thus, the method allows for the exclusion from consideration of a significant number of candidate optimized nucleotide sequences if the likelihood that they are effective is reduced due to non-compliance with one or more criteria. In other words, the criteria indicate the practical effectiveness of the optimized nucleotide sequence, therefore, nucleotide sequences that do not meet one or more criteria can be excluded from further consideration. The one or more criteria can include: a sequence that does not contain one or more termination signals; a sequence characterized by a guanine-cytosine content in a predetermined range; a sequence that has a codon adaptation index above a threshold value; a sequence that does not contain one or more cis-elements; a sequence that does not contain one or more repeating elements; and other criteria of interest.

[00016] Thus, the method provides a shorter or filtered list of optimized nucleotide sequences. By reducing the number of optimized nucleotide sequences in the list, additional steps performed on the sequences in the list, such as additional algorithmic steps or physical synthesis steps, are advantageously reduced in number and complexity.

[00017] In some embodiments, screening a list of optimized nucleotide sequences comprises, for a certain criterion: determining whether each optimized nucleotide sequence in the list or the most recently updated list of optimized nucleotide sequences meets the criterion; and updating the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or the most recently updated list if the nucleotide sequence does not meet the criterion.

[00018] In some embodiments, determining whether each optimized nucleotide sequence in the list or the most recently updated list of optimized nucleotide sequences meets the criterion includes, for each nucleotide sequence, determining whether a first portion of the nucleotide sequence meets the criterion, and where updating the list of optimized nucleotide sequences includes deleting the nucleotide sequence if the first portion does not meet the criterion. In some embodiments, determining whether each optimized nucleotide sequence in the list or the most recently updated list of optimized nucleotide sequences meets the criterion further comprises, for each nucleotide sequence, determining whether one or more additional portions of the nucleotide sequence meet the criterion, wherein the additional portions do not overlap with each other and with the first portion, and updating the list of optimized sequences comprises deleting the nucleotide sequence if any portion does not meet the criterion, optionally, wherein the determination of whether the optimized nucleotide sequence meets the criterion is stopped when it is determined that any portion does not meet the criterion.

[00019] By filtering optimized nucleotide sequences in this manner, the method is computationally efficient because sequences can be eliminated from the list before the computational and time resources are spent analyzing the entire sequence. Thus, the method is advantageously more efficient. Furthermore, for some criteria, portion-by-portion analysis provides a more detailed and selective screening process. Using guanine-cytosine content as an example, the method not only removes sequences for which the average guanine-cytosine content falls outside a predetermined range, but also advantageously removes any sequence that has a sharp rise or fall in guanine-cytosine content in a particular portion, which may interfere with efficient transcription or translation. Such peaks or falls might be missed if the entire sequence were analyzed only at once, because portions of the sequence outside the portion being analyzed could bring the average guanine-cytosine content into the acceptable range. By analyzing in parts, one can not only improve computational efficiency, but also identify problems in candidate sequences that are otherwise masked in the average.

[00020] Although guanine-cytosine content has been used as an example in this document, it should be understood that any criterion described in this document can be analyzed in a partial manner, as described above. For some criteria, such as a sequence containing a termination signal, computational efficiency will be increased, but the result of screening in a partial manner will not affect the content of the resulting list, i.e., evaluating termination signals in a partial manner will result in the removal of the same nucleotide sequences from the list as if the entire sequence were evaluated. For others, such as guanine-cytosine content or codon adaptation index, the screening results may differ, for example, certain sequences may be removed when using a partial analysis that would not be removed when evaluating the sequences in their entirety.

[00021] The first portion and/or one or more additional portions of the nucleotide sequence may comprise a predetermined number of nucleotides, optionally the predetermined number of nucleotides is in the range of 5 to 300 nucleotides, or 10 to 200 nucleotides, or 15 to 100 nucleotides, or 20 to 50 nucleotides, for example 30 nucleotides, for example 100 nucleotides. It has been found that portions of this length provide an optimal balance.

[00022] In some embodiments, the first criterion provides a nucleotide sequence that does not contain a termination signal, so that the method includes: determining whether each optimized nucleotide sequence in the list or the most recently updated list of optimized nucleotide sequences contains a termination signal; and updating the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or the most recently updated list if the nucleotide sequence contains one or more termination signals.

[00023] Thus, the method provides a shorter or filtered list of optimized nucleotide sequences. By reducing the number of optimized nucleotide sequences in the list, additional steps performed on the sequences in the list, such as additional algorithmic steps or physical synthesis steps, are advantageously reduced in number and complexity.

In some embodiments, the termination signal has the following nucleotide sequence: 5'-X ₁ AUCUX ₂ UX ₃ -3', wherein X ₁ , X ₂ and X ₃ are independently selected from A, C, T or G. In some embodiments, the termination signal has one of the following nucleotide sequences: TATCTGTT; and/or TTTTTTT; and/or AAGCTT; and/or GAAGAGC; and/or TCTAGA. In some embodiments, the termination signal has the following nucleotide sequence: 5'-X ₁ AUCUX ₂ UX ₃ -3', wherein X ₁ , X ₂ and X ₃ are independently selected from A, C, U or G. In some embodiments, the termination signal has one of the following nucleotide sequences: UAUCUGUU; and/or UUUUUU; and/or AAGCUU; and/or GAAGAGC; and/or UCUAGA.

[00024] In some embodiments, the second criterion provides a nucleotide sequence characterized by a guanine-cytosine content within a predetermined range of guanine-cytosine content, such that the method comprises: determining the guanine-cytosine content of each of the optimized nucleotide sequences in a list or the most recently updated list of optimized nucleotide sequences, wherein the guanine-cytosine content of a sequence is the percentage of bases in a nucleotide sequence that are guanine or cytosine; updating the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or the most recently updated list if its guanine-cytosine content is outside the predetermined range of guanine-cytosine content. By reducing the number of optimized nucleotide sequences in the list, additional steps performed on the sequences in the list, such as additional algorithm steps or physical synthesis steps, are advantageously reduced in number and complexity. In some embodiments, the desired range of guanine-cytosine content is from 15% to 75%, or from 40% to 60%, or, in particular, from 30% to 70%.

[00025] In some embodiments, the third criterion provides a nucleotide sequence having a codon adaptation index greater than a predetermined codon adaptation index threshold, so that the method comprises: determining the codon adaptation index of each of the optimized nucleotide sequences in the list or the most recently updated list of optimized nucleotide sequences, wherein the codon adaptation index of a sequence is a measure of the preference for the frequency of codon usage and can have a value from 0 to 1; updating the list or the most recently updated list of optimized nucleotide sequences by removing any nucleotide sequence if its codon adaptation index is less than or equal to the predetermined codon adaptation index threshold. Thus, the method provides a shorter or filtered list of optimized nucleotide sequences. In some embodiments, the codon adaptation index threshold is selected by the user. In some embodiments, the codon adaptation index threshold is 0.7, or 0.75, or 0.85, or 0.9, or, in particular, 0.8. By reducing the number of optimized nucleotide sequences in the list, additional steps performed on the sequences in the list, such as additional algorithm steps or physical synthesis steps, are advantageously reduced in number and complexity.

[00026] In some embodiments, the fourth criterion provides for a nucleotide sequence that does not contain at least 2, such as 3, contiguous identical codons, so that the method further comprises: determining whether any optimized nucleotide sequence in the list or the most recently updated list of optimized nucleotide sequences contains at least 2, such as 3, contiguous identical codons; and updating the list or the most recently updated list of optimized nucleotide sequences by removing any nucleotide sequence if it contains at least 2, such as 3, contiguous identical codons. It has been found that repeated identical codons, in other words, contiguous identical codons, can stop transcription. Thus, by removing from the list any optimized nucleotide sequence containing 2 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or in particular 3 or more identical contiguous codons, it is possible to disregard and remove sequences that provide less efficient transcription.

[00027] In any aspect of the present invention, the creation of an updated list of optimized nucleotide sequences may be accomplished by removing optimized sequences from the list based on any one, any two, or any three of the following steps:

(I) determining the presence of a termination signal in one or more optimized nucleotide sequences and removing the nucleotide sequences from the list or the most recently updated list of optimized nucleotide sequences if they contain a termination signal;

(II) determining the guanine-cytosine content of one or more optimized nucleotide sequences and removing nucleotide sequences from the list or the most recently updated list of optimized nucleotide sequences if their guanine-cytosine content is outside a predetermined range;

(III) determining the codon adaptation index of one or more optimized nucleotide sequences and removing nucleotide sequences from the list or the most recently updated list of optimized nucleotide sequences if their guanine-cytosine content is outside a specified range.

[00028] In a second aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (I).

[00029] In a third aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (II).

[00030] In a fourth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (III).

[00031] In a fifth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (I), then step (II).

[00032] In a sixth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (I), then step (III).

[00033] In a seventh aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (II), then step (I).

[00034] In an eighth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (II), then step (III).

[00035] More typically, the method according to the present invention comprises termination signal-based steps (I), guanine-cytosine content-based steps (II), and codon adaptation index-based steps (III) to obtain a shortlist of optimized nucleotide sequences, all of which are expected to provide a full-length mRNA transcript when synthesized by transcription in vitro and to provide high levels of expression of the protein encoded by the mRNA in vivo. The termination signal-based steps (I), the guanine-cytosine content-based steps (II), and the codon adaptation index-based steps (III) can be performed in any order. Advantageously, these steps can be performed in a certain order in order to optimize the computing time in determining the shortlist of optimized nucleotide sequences.

[00036] In a ninth specific aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (I), then step (II), then step (III). By filtering in this order, the computational efficiency of the filtering steps can be advantageously maximized. The inventors of the present invention have found that for a typical list of optimized nucleotide sequences and typical input parameters, filtering based on motif selection removes the majority of sequences from the list, followed by filtering based on GC content analysis, and then filtering based on CAI analysis. Since the computational efficiency of the filtering process is determined in part by the total number of sequences analyzed, i.e., the sum of the sequences analyzed in each filtering step, the more sequences that can be removed at the beginning of the filtering process, the fewer sequences that need to be analyzed later in the filtering process, which increases the overall computational efficiency of the method. Furthermore, CAI-based filtering requires analysis of the entire sequence, whereas in embodiments of the present invention, motif-based filtering and GC-content-based filtering can analyze only components or portions of the sequence. Thus, a method that emphasizes reducing the number of sequences in the list input during the CAI analysis stage is likely to be more computationally efficient than other methods.

[00037] In a tenth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (I), then step (III), then step (II).

[00038] In an eleventh aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (II), then step (I), then step (III).

[00039] In a twelfth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (II), then step (III), then step (I).

[00040] In a thirteenth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (III), then step (I), then step (II).

[00041] In a fourteenth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (III), then step (II), then step (I).

[00042] In some embodiments, the amino acid sequence is obtained from an amino acid sequence database. In some embodiments, the method further comprises querying the amino acid sequence from the amino acid sequence database, wherein the amino acid sequence is obtained in response to the query.

[00043] In some embodiments, the first table of codon usage frequencies is obtained from a database of codon usage tables. In some embodiments, the method further comprises querying the first table of codon usage frequencies from the database of codon usage tables, wherein the first table of codon usage frequencies is obtained in response to the query.

[00044] In a fifteenth aspect, the present invention relates to a computer program comprising instructions that, when the program is executed by a computer, cause the computer to perform a method according to any of the embodiments of the first aspect.

[00045] In a sixteenth aspect, the present invention relates to a data processing system comprising means for implementing the method according to any of the embodiments of the first aspect.

[00046] In a seventeenth aspect, the present invention relates to a computer-readable storage medium on which the computer program of the third aspect is stored.

[00047] In an eighteenth aspect, the present invention relates to a data carrier signal carrying the computer program of the third aspect.

[00048] In a nineteenth aspect, the present invention relates to a method for synthesizing a nucleotide sequence, comprising: performing the method according to any embodiment of the first aspect to create at least one optimized nucleotide sequence; and synthesizing at least one of the created optimized nucleotide sequences. In some embodiments, the method further comprises inserting at least one of the synthesized optimized sequences into a nucleic acid vector for use in in vitro transcription.

[00049] In some embodiments, the method further comprises inserting one or more termination signals at the 3' end of the synthesized optimized nucleotide sequences. In some embodiments, more than one termination signal is inserted and the termination signals are separated by 10 base pairs or less, such as separated by 5-10 base pairs. In some embodiments, the one or more termination signals have the following nucleotide sequence: 5'-X ₁ ATCTX ₂ TX ₃ -3', where X ₁ , X ₂ and X ₃ are independently selected from A, C, T or G. In some embodiments, the one or more termination signals have one of the following nucleotide sequences: TATCTGTT; TTTTTTT; AAGCTT; GAAGAGC and/or TCTAGA. In some embodiments, more than one termination signal is encoded by the following nucleotide sequence: (a) 5'-X ₁ ATCTX ₂ TX ₃ -(Z _N )-X ₄ ATCTX ₅ TX ₆ -3' or (b) 5'-X ₁ ATCTX ₂ TX ₃ -(Z _N )-X ₄ ATCTX ₅ TX ₆ -(Z _M )-X ₇ ATCTX ₈ TX ₉ -3', wherein X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ , X ₈ and X ₉ are independently selected from A, C, T or G, Z _N is a spacer sequence of N nucleotides, and Z _M is a spacer sequence of M nucleotides, each of which is independently selected from A, C, T or G, and wherein N and/or M are independently 10 or less.

[00050] In some embodiments, the nucleic acid vector comprises a promoter for RNA polymerase operably linked to an optimized nucleotide sequence, wherein optionally the promoter for RNA polymerase is a promoter for SP6 RNA polymerase or a promoter for T7 RNA polymerase. In some embodiments, the nucleic acid vector comprises a nucleotide sequence encoding a 5' UTR operably linked to an optimized nucleotide sequence. In some embodiments, the 5' UTR is different from the 5' UTR of a naturally occurring mRNA encoding an amino acid sequence. In some embodiments, the 5' UTR has the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the nucleic acid vector comprises a nucleotide sequence encoding a 3' UTR operably linked to an optimized nucleotide sequence. In some embodiments, the 3' UTR is different from the 3' UTR of a naturally occurring mRNA encoding an amino acid sequence. In some embodiments, the 3'-UTR has the nucleotide sequence of SEQ ID NO: 17 or SEQ ID NO: 18. In some embodiments, the nucleic acid vector is a plasmid. In some embodiments, the plasmid is linearized prior to in vitro transcription. In some embodiments, the plasmid is not linearized prior to in vitro transcription. In some embodiments, the plasmid is supercoiled.

[00051] In some embodiments, the method further comprises using at least one of the synthesized optimized nucleotide sequences in in vitro transcription to synthesize mRNA. In some embodiments, mRNA is synthesized using SP6 RNA polymerase. In some embodiments, SP6 RNA polymerase is a naturally occurring SP6 RNA polymerase. In some embodiments, SP6 RNA polymerase is a recombinant SP6 RNA polymerase. In some embodiments, SP6 RNA polymerase comprises a tag. In some embodiments, the tag is a histidine tag. In some embodiments, mRNA is synthesized using T7 RNA polymerase.

[00052] In some embodiments, the method further comprises a separate step of capping and/or extending the synthesized mRNA. In some embodiments, capping and extending occur during in vitro transcription.

[00053] In some embodiments, mRNA is synthesized in a reaction mixture comprising NTPs in a concentration range of 1-10 mM of each NTP, DNA template in a concentration range of 0.01-0.5 mg/mL, and SP6 RNA polymerase in a concentration range of 0.01-0.1 mg/mL. In some embodiments, the reaction mixture comprises NTPs at a concentration of 5 mM of each NTP, DNA template at a concentration of 0.1 mg/mL, and SP6 RNA polymerase at a concentration of 0.05 mg/mL.

[00054] In some embodiments, mRNA is synthesized at a temperature in the range of 37-56°C.

[00055] In some embodiments, the NTPs are naturally occurring NTPs. In some embodiments, the NTPs include modified NTPs.

[00056] In some embodiments, the method further comprises synthesizing a reference nucleotide sequence encoding an amino acid sequence and at least one synthesized optimized nucleotide sequence in accordance with the method of the present invention, and contacting the reference nucleotide sequence and the at least one optimized nucleotide sequence with a single cell or organism. In a typical embodiment, a cell or organism contacted with at least one synthesized optimized nucleotide sequence provides an increased yield of a protein encoded by the optimized nucleotide sequence, compared to the yield of a protein encoded by the reference nucleotide sequence produced by a cell or organism contacted with the synthesized reference nucleotide sequence. In any aspect of the present invention, at least one optimized nucleotide sequence, when synthesized, can be configured to increase the expression of a protein, compared to the expression of a protein encoded by the reference nucleotide sequence, when synthesized. The reference nucleotide sequence may be: (a) a naturally occurring nucleotide sequence encoding an amino acid sequence; or (b) a nucleotide sequence encoding an amino acid sequence created by a method other than the method according to the first aspect of the present invention.

[00057] In some embodiments, the method further comprises transfecting the synthesized optimized nucleotide sequence into a cell either in vitro or in vivo. In some embodiments, the expression level of the protein encoded by the synthesized optimized nucleotide sequence is determined in the transfected cell. In some embodiments, the functional activity of the protein encoded by the synthesized optimized nucleotide sequence is determined in the transfected cell.

[00058] In a twentieth aspect, the present invention provides a synthesized optimized nucleotide sequence created according to the method of the present invention for use in therapy. Included in this aspect of the present invention are methods of treatment comprising administering a synthesized optimized nucleotide sequence created according to the method of the present invention to a human subject in need of such treatment. In some embodiments, the methods described herein provide a therapeutic composition comprising mRNA that encodes a therapeutic peptide, polypeptide, or protein for use in delivering to a subject or in treating a subject. In some embodiments, the mRNA encodes a protein that is a cystic fibrosis transmembrane conductance regulator (CFTR).

[00059] In the twenty-first aspect, the present invention provides an in vitro synthesized nucleic acid comprising an optimized nucleotide sequence consisting of codons associated with a usage frequency that is equal to or greater than 10%; wherein the optimized nucleotide sequence:

does not contain a termination signal having one of the following nucleotide sequences:

5'-X ₁ AUCUX ₂ UX ₃ -3', where X ₁ , X ₂ and X ₃ are independently selected from A, C, U or G; and 5'-X ₁ AUCUX ₂ UX ₃ -3', where X ₁ , X ₂ and X ₃ are independently selected from A, C, U or G;

does not contain any negative cis-regulatory elements or negative repeat elements; and

has a codon adaptation index greater than 0.8;

wherein, when divided into non-overlapping portions of 30 nucleotides in length, each portion of the optimized nucleotide sequence is characterized by a guanine-cytosine content range from 30% to 70%. In some embodiments, the optimized nucleotide sequence does not comprise a termination signal having one of the following sequences: TATCTGTT; TTTTTT; AAGCTT; GAAGAGC; TCTAGA; UAUCUGUU; UUUUUU; AAGCUU; GAAGAGC; UCUAGA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid synthesized in vitro is intended for use in therapy.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[00060] Embodiments of the present invention will be described by way of example with reference to the following drawings, in which:

[00061] Figure 1 depicts a method for codon optimization according to an embodiment of the present invention.

[00062] Figure 2A depicts an illustrative table of codon usage frequencies for Homo sapiens, generated from one or more experimentally determined codon usage frequencies. The values in the table were obtained from data accessed through the Codon Usage Database, which is based on publicly available codon usage frequency data from the NCBI GenBank database (Flat File Release 160.0).

[00063] Figure 2B depicts a normalized codon usage frequency table created by normalizing the codon usage frequencies from the exemplary codon usage frequency table in Figure 2A.

[00064] Figure 3 depicts a constructed section of a codon usage table for use with an exemplary method for normalizing a codon usage table.

[00065] Figure 4A depicts the illustrative table from Figure 3 normalized to an equal distribution of usage frequency.

[00066] Figure 4B depicts the illustrative table from Figure 3 normalized with a proportional distribution of frequency of use.

[00067] Figure 5 depicts a designed section of an amino acid sequence for use in an exemplary method for codon optimization.

[00068] Figure 6 depicts an example of a repository of nucleotide sequence motifs that includes a termination signal suitable for use in removing nucleotide sequences containing another termination signal.

[00069] Figure 7 depicts a method for applying additional algorithm steps or filtering steps to a list of optimized nucleotide sequences. In a particular embodiment, the list of optimized nucleotide sequences for filtering was created in accordance with the method shown in Figure 1.

[00070] Figure 8 depicts an embodiment of the present invention in which a list of optimized nucleotide sequences is filtered based on guanine-cytosine (GC) content analysis. In a particular embodiment, the list of optimized nucleotide sequences for filtering was generated in accordance with the method shown in Figure 1.

[00071] Figure 9 depicts an embodiment of the present invention in which motif selection-based filtering and codon adaptation index (CAI) analysis-based filtering are applied to a list of optimized nucleotide sequences. In a particular embodiment, the list of optimized nucleotide sequences for filtering was generated in accordance with the method shown in Figure 1.

[00072] Figure 10 depicts a specific embodiment of the present invention, in which a filtering based on motif selection, a filtering based on guanine-cytosine (GC) content analysis, and a filtering based on codon adaptation index (CAI) analysis were applied to a list of optimized nucleotide sequences, in that order. In a specific embodiment, the list of optimized nucleotide sequences for filtering was generated in accordance with the method shown in Figure 1.

[00073] Figure 11 depicts an illustrative analysis of the guanine-cytosine (GC) content of unoptimized and optimized nucleotide sequences, wherein the guanine-cytosine (GC) content of portions of the nucleotide sequence encoding EPO is determined for contiguous, non-overlapping portions of 30 nucleotides in length.

[00074] Figure 12 depicts an illustrative histogram showing the yield of protein obtained from various codon-optimized nucleotide sequences as determined by an EPO ELISA assay.

[00075] Figure 13A depicts an exemplary Western blot used to determine the expression yield of CFTR protein encoded by optimized nucleotide sequences generated according to the method of the present invention in a time-dependent experiment after the optimized nucleotide sequences were transfected into human cells.

[00076] Figure 13B depicts an illustrative line graph showing the quantification of the Western blot data shown in Figure 13A.

[00077] Figure 14A depicts an illustrative graph of data obtained from a bioassay for testing mRNA containing an optimized nucleotide sequence encoding hCFTR. It depicts the output short-circuit current (I _sc ) in the Ussing epithelial voltage clamp apparatus for each mRNA tested.

[00078] Figure 14B depicts an example of a histogram illustrating the change in hCFTR activity shown in Figure 14A, expressed as a percentage of the activity of a reference mRNA encoding hCFTR.

[00079] Figure 15A depicts an illustrative Western blot demonstrating the translation and expression of codon-optimized DNAI1 mRNA in HEK2 93T cells. The Western blot was performed with an anti-DNAI1 antibody and an anti-vinculin antibody (loading control).

[00080] Figure 15B depicts an illustrative histogram showing the expression level of DNAI1 protein normalized to the vinculin protein (loading control), quantified from the illustrative Western blot in Figure 15A. DNAI1 protein expression yields are plotted as fold increases relative to the reference level achieved with mRNA encoding the DNAL1 sequence that was not codon-optimized.

Definitions

[00081] To facilitate understanding of the present invention, certain terms are initially defined below. Additional definitions of the following terms and other terms are provided throughout the description.

[00082] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[00083] Unless specifically stated or obvious from the context, the term "or" as used herein is understood to include and encompass both "or" and "and".

[00084] The terms "for example" and "i.e." as used herein are used merely as examples, without limitation, and should not be construed as referring only to those elements that are expressly listed in the description.

[00085] The terms "or more," "at least," "more than," and the like, such as "at least one," should be understood to include, without limitation, 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, bb, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 14 9 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than the stated value. Any higher number or intermediate fractional number is also included.

[00086] Conversely, the term "not more than" includes every value less than the stated value. For example, the expression "not more than 100 nucleotides" includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, and 0 nucleotides. Any smaller number or intermediate fractional number is also included.

[00087] It should be understood that the terms “plurality”, “at least two”, “two or more”, “at least a second”, etc. include, without limitation, at least 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, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 14 9 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 or more. Any larger number or intermediate fractional number is also included.

[00088] Unless specifically stated or obvious from the context, the term "about" as used herein is understood to mean within the range of normal tolerance in the art, for example, within 2 standard deviations of the mean. "About" can be understood to mean within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% of the stated value. Unless the context otherwise requires, all numerical values given herein reflect normal variations that can be appreciated by one of ordinary skill in the art.

[00089] As used herein, the term "abortive transcript" or "prematurely aborted transcript" or the like is any transcript that is shorter than the full-length mRNA molecule encoded by the DNA template, which results from the premature release of RNA polymerase from the template DNA in a sequence-independent manner. In some embodiments, the abortive transcript may be less than 90% of the length of the full-length mRNA molecule that is transcribed from the target DNA molecule, such as less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1% of the length of the full-length mRNA molecule.

[00090] As used herein, the terms "codon" and "codons" refer to a sequence of three nucleotides that together form a unit of the genetic code. Each codon corresponds to a specific amino acid or stop signal during translation or protein synthesis. The genetic code is degenerate, and a particular amino acid residue can be encoded by more than one codon. For example, codons can contain DNA or RNA nucleotides.

[00091] As used herein, the terms "codon optimization" and "codon-optimized" refer to modifications to the codon composition of a naturally occurring or wild-type nucleic acid encoding a peptide, polypeptide, or protein that do not alter its amino acid sequence, thereby improving protein expression from said nucleic acid. In the context of the present invention, "codon optimization" may also refer to a process by which one or more optimized nucleotide sequences are obtained by filtering out less optimal nucleotide sequences from a list of nucleotide sequences, such as filtering by guanine-cytosine content, codon adaptation index, the presence of destabilizing sequences or nucleic acid motifs, and/or the presence of pause sites and/or terminator signals.

[00092] As used herein, "full-length mRNA" is characterized using a specific assay, such as gel electrophoresis and detection using UV spectroscopy and UV spectrophotometry with separation by capillary electrophoresis. The length of the mRNA molecule encoding the full-length polypeptide is at least 50% of the length of the full-length mRNA molecule that is transcribed from the target DNA, for example, at least 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.01%, 99.05%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% of the length of the full-length mRNA molecule that is transcribed from the target DNA.

[00093] As used herein, the term "in vitro" refers to events that occur in an artificial environment, such as a test tube or reaction vessel, cell culture, etc., rather than in a multicellular organism.

[00094] As used herein, the term "in vivo" refers to events that occur in a multicellular organism, such as a human or non-human animal. In the context of cellular systems, the term may be used in relation to events that occur in a living cell (as opposed to, for example, in vitro systems).

[00095] As used herein, the term "messenger RNA (mRNA)" refers to a polyribonucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may include one or more coding and non-coding regions. mRNA may be obtained by purification from natural sources, produced using recombinant expression systems, and optionally purified, transcribed in vitro, or chemically synthesized. If desired, for example, in the case of chemically synthesized molecules, mRNA may contain nucleoside analogs, such as analogs having chemically modified bases or sugars, backbone modifications, etc. The mRNA sequence is presented in the 5'-3' direction unless otherwise indicated.

[00096] As used herein, the term "nucleic acid" in its broadest sense refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester bond. In some embodiments, "nucleic acid" refers to individual nucleic acid units (e.g., nucleotides and/or nucleosides). In some embodiments, "nucleic acid" refers to a polynucleotide chain comprising individual nucleic acid units. In some embodiments, "nucleic acid" encompasses RNA, as well as single- and/or double-stranded DNA and/or cDNA. Moreover, the terms "nucleic acid," "DNA," "RNA," and/or similar terms include nucleic acid analogs, i.e., analogs having a backbone other than a phosphodiester backbone. Nucleic acid sequences are presented in the 5' to 3' direction unless otherwise indicated.

[00097] As used herein, the term "nucleotide sequence" refers in its broadest sense to the order of nucleobases in a nucleic acid. In some embodiments, "nucleotide sequence" refers to the order of individual nucleobases in a gene. In some embodiments, "nucleotide sequence" refers to the order of individual nucleobases in a gene encoding a protein. In some embodiments, "nucleotide sequence" refers to the order of individual nucleobases in single- and/or double-stranded DNA and/or cDNA. In some embodiments, "nucleotide sequence" refers to the order of individual nucleobases in RNA. In some embodiments, "nucleotide sequence" refers to the order of individual nucleobases in mRNA. In a particular embodiment, "nucleotide sequence" refers to the order of individual nucleobases in a protein-coding RNA or DNA sequence. The nucleotide sequence is typically presented in the 5' to 3' direction, unless otherwise indicated.

[00098] As used herein, the term "premature termination" refers to the termination of transcription before the full length of the DNA template has been transcribed. As used herein, premature termination may be caused by the presence of a nucleotide sequence motif (also referred to herein simply as a "motif"), such as a termination signal, in the DNA template, which results in the formation of mRNA transcripts that are shorter than the full length mRNA ("prematurely terminated transcripts" or "truncated mRNA transcripts"). Examples of termination signals include the E. coli rrnB tl terminator signal (consensus sequence: ATCTGTT) and its variants, as described herein.

[00099] As used herein, the term "template DNA" (or "template DNA") refers to a DNA molecule containing a nucleic acid sequence encoding an mRNA transcript to be synthesized by in vitro transcription. The template DNA is used as a template for in vitro transcription to produce the mRNA transcript encoded by the template DNA. The template DNA contains all the elements necessary for in vitro transcription, in particular, a promoter element for binding a DNA-dependent RNA polymerase, such as, for example, T3, T7 and SP6 RNA polymerases, which is operably linked to a DNA sequence encoding the desired mRNA transcript. In addition, the template DNA may contain primer binding sites 5' and/or 3' relative to the DNA sequence encoding the mRNA transcript to determine the identity of the DNA sequence encoding the mRNA transcript, for example, by PCR or DNA sequencing. "Matrix DNA" in the context of the present invention may be a linear or circular DNA molecule. As used herein, the term "matrix DNA" may refer to a DNA vector, such as plasmid DNA, that contains a nucleic acid sequence encoding the desired mRNA transcript.

[000100] All technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this application belongs and as commonly used in the field to which this application belongs. Publications and other references cited in this document to describe the prior art of the present invention and to provide additional details regarding its implementation are incorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION

Codon optimization functions

[000101] During gene expression, the nucleotide sequence encoded in the DNA sequence is transcribed into RNA molecules and then translated into proteins containing polypeptide chains. The sequence information that determines the precise order of amino acid residues to be included in the protein product is encoded in "codons" in the DNA and/or mRNA sequence. Codons contain a sequence of three nucleotides that together form a unit of genetic code, and each codon corresponds to a specific amino acid or stop codon signal. The genetic code is degenerate, and a particular amino acid residue may be encoded by more than one codon.

[000102] mRNA is generally considered a type of RNA that carries information from DNA to the ribosome. The lifespan of mRNA is typically very short and involves processing and translation, followed by degradation. Typically, in eukaryotic organisms, mRNA processing involves the addition of a cap at the N-terminus (5') and a tail at the C-terminus (3'). A typical cap is the 7-methylguanosine cap, which is a guanosine linked via a 5'-5'-triphosphate linkage to the first nucleotide to be transcribed. The presence of the cap is important for resistance to nucleases, which are found in most eukaryotic cells. The presence of the tail typically represents a polyadenylation event, in which a poly-A moiety is added to the 3' end of the mRNA molecule. The presence of this tail serves to protect mRNA from degradation by exonucleases. Messenger RNA is typically translated by ribosomes into the series of amino acids that make up protein.

[000103] At various stages of gene expression, numerous factors can influence the level of expression or production of a particular protein. For example, the presence of certain nucleotide sequence motifs can cause premature termination of transcription as the DNA sequence is transcribed into mRNA by the enzyme RNA polymerase. The specific composition and order of codons in the protein-coding region ("coding sequence") of a gene can also positively or negatively influence the efficiency and yield of protein expression. For example, the presence of rare codons characterized by a low codon usage frequency can negatively influence the yield of protein expression due to the low number of cognate transfer RNAs encoding a particular amino acid. In biotechnological and therapeutic applications, it is often desirable to increase or maximize the yield of protein upon expression of said protein from the nucleotide sequence encoding it, for example, in therapeutic applications including mRNA-based therapy. Codon optimization provides protein-coding nucleotide sequences based on various criteria without altering the encoded amino acid sequence due to the redundancy of the genetic code. In other words, since multiple codons encode a single amino acid, a large number of nucleotide sequences can encode the same amino acid sequence. Codon optimization aims to create one or more nucleotide sequences that will increase protein yield.

Amino acid sequences for the creation of optimized nucleotide sequences

[000104] Naturally occurring nucleotide sequences can be used to obtain

The amino acid sequence encoding a protein, polypeptide, or peptide of interest. Nucleotide sequences can be obtained by isolating a nucleic acid molecule from the organism of interest and determining the precise order of its nitrogenous bases (e.g., guanine, thymine, uracil, adenine, and cytosine). Numerous methods suitable for obtaining naturally occurring nucleotide sequences are known in the art. The nucleotide sequence of protein-coding genes can be obtained using a variety of well-known DNA or RNA sequencing methods.

[000105] For example, DNA from a human cell can be extracted, isolated, and subsequently fragmented. The fragmented DNA can be cloned into DNA vectors and amplified in bacterial hosts, creating “libraries” of short DNA fragments. Alternatively, the fragmented DNA can be amplified using polymerase chain reaction (PCR) and included in libraries suitable for high-throughput sequencing-based methods. Short DNA fragments derived from the starting DNA material of the source organism can be sequenced individually and then assembled into a long contiguous sequence or sequences by sequence assembly. Sequence assembly is a bioinformatics approach that aligns and joins short fragments of nucleotide sequences derived from a longer nucleotide sequence to reconstruct the original or consensus nucleotide sequence.

[000106] The nucleotide sequences generated in this manner, i.e., sequences obtained experimentally and known to accurately describe naturally occurring sequences, are typically stored in publicly available repositories or databases. For example, nucleotide sequences that can be processed according to the method of the present invention can be obtained from the GenBank database of the National Center for Biotechnology Information (NCBI). GenBank is an annotated collection of publicly available nucleotide sequences and their translated protein sequences with open access.

Creating codon frequency tables

[000107] The genetic code has 64 possible codons. Each codon contains a sequence of three nucleotides. The frequency of each codon's usage in protein-coding regions of the genome can be calculated by determining the number of times a particular codon appears in protein-coding regions of the genome and then dividing this value by the total number of codons encoding the same amino acid in protein-coding regions of the genome. These calculations can be performed on nucleotide sequences found, for example, in publicly available repositories and/or databases, and therefore also represent experimentally obtained data.

[000108] A codon usage table lists the frequency of use of each codon in a given organism. Each amino acid in the table is associated with at least one codon, and each codon is associated with a usage frequency. Codon usage tables are stored in publicly available databases such as the Codon Usage Frequency Database (Nakamura efc al. (2000) Nucleic Acids Research 28(1), 292; available online at https://www.kazusa.or.jp/codon/) and the High-performance Integrated Virtual Environment-Codon Usage Tables (HIVE-CUTs) database of codon usage tables (Athey efc al., (2017) BMC Bioinformatics 18(1), 391; available online at http://hive.biochemistry.gwu.edu/review/codon).

Codon optimization

[000109] Figure 1 depicts a method for codon optimization according to the present invention. In a first step 101, an amino acid sequence is obtained. The amino acid sequence may be obtained from a remote system, server, and/or public database, and may be obtained wirelessly, such as via the Internet. Alternatively, the amino acid sequence may be obtained from a local system, such as via a wired connection.

The amino acid sequence includes many amino acids.

[000110] In the second step 102, a first table of codon usage frequencies is obtained. The first table of codon usage frequencies can be obtained from a remote system, server, and/or public database, and can be obtained wirelessly, for example, via the Internet. Alternatively, the first table of codon usage frequencies can be obtained from a local system, for example, via a wired connection. The first table of codon usage frequencies contains a list of amino acids, where each amino acid in the table is associated with at least one codon, and each codon is associated with a usage frequency.

[000111] In a third step, 103 codons are removed from the first codon usage frequency table if they are associated with a codon usage frequency that is less than a threshold frequency.

[000112] In the fourth step 104, the codon usage frequencies for the codons not removed in the third step 103 are normalized to create a normalized codon usage frequency table.

[000113] In a fifth step 105, an optimized nucleotide sequence is created by selecting a codon for each amino acid in the amino acid sequence based on the frequency of use of one or more codons associated with the amino acid in a normalized codon frequency table.

Normalization of the codon frequency table

[000114] Figure 2A depicts a codon usage table that can be found in the codon usage table database. The depicted codon usage table is only an example, and it should be understood that any codon usage table, for example, any codon usage table available in the database, can be used in the present invention to obtain an optimized nucleotide sequence. The data used to create Figure 2A were obtained from data accessible through the Codon Usage Database, based on publicly available codon usage data in the NCBI GenBank database (Flat File Release 160.0).

[000115] A codon frequency table contains experimentally obtained data on how often, for a specific biological source from which the table was created, each codon is used to encode a particular amino acid. This information is expressed for each codon as a percentage (from 0 to 100%) or a fraction (from 0 to 1) of how often that codon is used to encode a particular amino acid relative to the total number of times the codon encodes that amino acid.

[000116] Figure 2B illustrates a normalized table of codon usage frequencies that was created from the table in Figure 2A in accordance with the method of the present invention. In the example in Figure 2B, a cutoff frequency of 10% was used to perform the normalization. It should be understood that this is only an example, and that any other suitable cutoff frequency, as described herein, can be used in embodiments of the present invention.

[000117] A method by which a normalized codon usage table can be provided, and which was provided in the case of Figure 2B, is depicted in Figure 3, where the exemplary amino acids "X" and "Y" are used. It should be understood that in creating a normalized codon usage table, any number of amino acids can be normalized, from one amino acid to every amino acid in the codon usage table. In the example of Figure 3, amino acid X is encoded by codons A, B, C, D, E, and F (each codon is represented by a triplet of nucleotides and, thus, is designated in the figure as AAA, BBB, etc.) with the frequencies indicated in the figure. Amino acid Y is encoded by codons G and H with the frequencies indicated in the figure. In the first step, any codons whose usage frequency is below the cutoff frequency are removed from the table. It should be understood that although a cutoff frequency of 10% is used in the method depicted in Figure 3, this is provided only as an example and is not intended to limit the scope of the present invention. The cutoff frequency may be in the range of 5% to 30%, for example, 5%, or 15%, or 20%, or 25%, or 30%, or, in particular, 10%. These cutoff frequency values have been found to provide an effective balance between increased protein yield and the preservation of information important for translation control and ensuring the correct folding of the nascent polypeptide chain. It should be understood that the codon usage table in Figure 3 does not accurately describe the actual naturally occurring codon usage frequency, not least because it consists of only two amino acids. The table in Figure 3 is intended only to illustrate the method for normalizing the codon usage table.

[000118] In the example of Figure 3, codons C and E have a usage frequency below the 10% cutoff frequency and are thus removed from the table. The cumulative usage frequency of the removed codons C and E is 16%. This cumulative usage frequency is then distributed among the remaining codons that encode amino acid X. It is important to note that the cumulative usage frequency removed from amino acid X is distributed only among the remaining codons that also encode amino acid X, i.e., in the example of Figures 4A and 4B, the usage frequencies of codons G and H, which encode amino acid Y, remain unchanged.

[000119] In some embodiments, the removed cumulative usage frequency is evenly distributed among the remaining codons that encode amino acid X. Such an embodiment is depicted in Figure 4A. The removed cumulative usage frequency of 16% is evenly distributed among the remaining codons A, B, D, and F, such that each remaining codon receives an additional 4% usage frequency. The codon usage frequencies of amino acid X are now normalized.

[000120] In some embodiments, the removed cumulative usage rate is proportionally distributed among the remaining codons that encode amino acid X. Such an embodiment is depicted in Figure 4B. The removed cumulative usage rate of 16% is distributed among the remaining codons A, B, D, and F in proportion to the usage rate of the remaining codons A, B, D, and F. In this example, the ratio of codon usage rates of A, B, D, and F is 15:20:38:11, or 0.18:0.24:0.45:0.13. Codon A receives 0.18 of 16% (3%), B receives 0.24 of 16% (4%), D receives 0.45 of 16% (7%), and F receives 0.13 of 16% (2%). The codon usage rates of amino acid X are now normalized.

[000121] Thus, the structure and content of the resulting codon usage frequency table or the first codon usage frequency table indicate the creation of a normalized codon usage frequency table. The number of codons associated with each amino acid determines the redistribution of the frequencies of usage of the deleted codons, and the codon usage frequencies themselves determine which codons are deleted and, in some embodiments, the proportionality of the distribution.

Creation of an optimized nucleotide sequence

[000122] An optimized nucleotide sequence is created by selecting a codon for each amino acid in the amino acid sequence based on the frequency of usage of one or more codons associated with the amino acid in a normalized codon frequency table. The optimized nucleotide sequence is created by arranging the selected codons in the order in which the amino acid associated with them appears in the amino acid sequence.

[000123] Figure 5 is an illustration of the creation of an optimized nucleotide sequence using codons A, B, C, D, E, and F from Figures 3, 4A, and 4B. Each codon can be represented by three nucleotides; in the illustration of Figure 5, codon A is represented by nucleotides AAA, codon B by nucleotides BBB, and so on.

[000124] An illustrative amino acid sequence is obtained

sequence X Y Y X X X. For this example, we assume that amino acids X and Y are associated with codons A, B, C, D, E, F, G, and H, as defined with respect to Figures 3, 4A, and 4B. In this example, the codon usage frequency table in Figure 3 was probabilistically normalized, resulting in the normalized codon usage frequency table in Figure 4B. At step 501, for each amino acid, a codon is selected with a probability equal to the usage frequency associated with the codon in the normalized codon usage frequency table. For example, for the first amino acid in the sequence, X, the probability that codon A will be selected is 18%, the probability that codon B will be selected is 24%, the probability that codon D will be selected is 45%, and the probability that codon F will be selected is 13%. This is because amino acid X is encoded by codons A, B, D, and F, and is thus associated with these codons in the normalized codon usage table, so the codon chosen for amino acid X will be one of codons A, B, D, and F.

[000125] This process is repeated for each amino acid using a normalized codon usage table to indicate the probability of selecting a particular codon. Thus, for the second amino acid in the sequence, Y, the G codon is selected with a probability of 60%, and the H codon is selected with a probability of 40%. After selecting a codon for each amino acid, the resulting codon sequence, consisting of nucleotides, can be called an optimized nucleotide sequence.

[000126] Figure 5 is illustrative and is intended only to aid in understanding the creation of optimized nucleotide sequences. Figure 5 may not show the length, composition, or structure of the actual amino acid sequence or optimized nucleotide sequence obtained; it merely schematically illustrates the method.

Creation of a set of optimized nucleotide sequences

[000127] The creation of an optimized nucleotide sequence using the amino acid sequence and the normalized codon usage table may be performed more than once to create a list of optimized nucleotide sequences.

[000128] The list may include any number of different optimized nucleotide sequences, since the creation of an optimized nucleotide sequence is based on the probabilistic choice of codons. The list may include any number of repeated optimized nucleotide sequences, i.e., identical optimized nucleotide sequences, again, since the creation of an optimized nucleotide sequence is based on the probabilistic choice of replacement codons. Identical optimized sequences are typically removed when creating a list of optimized nucleotide sequences.

[000129] In some embodiments, one or more or all of the optimized nucleotide sequences in the list of optimized nucleotide sequences are synthesized for testing by transfection, use in therapy, or for any other use of the synthesized optimized nucleotide sequence described herein.

Filtering the list of optimized nucleotide sequences

[000130] The number of optimized nucleotide sequences in the list of optimized nucleotide sequences depends at least on the length and composition of the amino acid sequence, the value of the threshold codon usage frequency, the composition of the first table of codon usage frequencies, and the number of cycles of the codon optimization algorithm, i.e., the number of times the optimized nucleotide sequence is created. For example, the list of optimized nucleotide sequences may contain 10,000 optimized nucleotide sequences or more. Synthesizing and testing each optimized nucleotide sequence from the list in a cell, tissue, or organism may be advantageous in some situations, for example, for certain input parameters of the algorithm, such as a relatively short amino acid sequence. Likewise, this may be disadvantageous in certain situations, for example, if it is necessary to reduce the complexity of the computer process or the number of sequences that are synthesized and tested in a cell, tissue, or organism. Therefore, it may be necessary to reduce the number of optimized nucleotide sequences in the list of nucleotide sequences, for example, before synthesis. This could advantageously reduce the time required to synthesize each sequence in the list and the resources required to do so.

[000131] Accordingly, in an exemplary embodiment, one or more additional algorithm steps are performed on the list of optimized nucleotide sequences to filter the list or remove optimized nucleotide sequences from the list. The one or more additional algorithm steps may be referred to as motif selection, GC content analysis, and codon adaptation index (CAI) analysis. It should be understood that, although specific additional algorithm steps are described in detail herein, they may not be the only filtering steps performed, and additional steps may be performed to further filter the list of optimized nucleotide sequences within the scope of the claims of the present invention.

[000132] The inventors of the present invention have found that these additional algorithm steps and their associated motifs, ranges, and thresholds advantageously filter the list of optimized nucleotide sequences by removing from the list those sequences that are likely to be less effective than the sequences retained in the list. Thus, the filtering of the list is not simply arbitrary. In other words, filtering the list to a certain number of sequences using the methods described herein will result in an updated sequence list containing more effective sequences than if the same certain number of sequences were selected from the list at random. Thus, the efficiency and complexity reduction achieved in the synthesis process are not achieved at the expense of losing a large number of effective optimized nucleotide sequences. For example, the optimized nucleotide sequences generated by the methods of the present invention do not contain termination signals. The absence of termination signals facilitates the synthesis of full-length mRNA molecules from encoded optimized nucleotide sequences using in vitro transcription. The presence of termination signals leads to premature termination of transcription in vitro, so filtering the list using the methods described in this document provides an updated list of sequences containing more efficient sequences.

[000133] Filtering the list of optimized nucleotide sequences may be referred to as screening the list of optimized nucleotide sequences to identify and remove optimized nucleotide sequences that do not meet one or more criteria. Each criterion may relate to a specific additional step of the algorithm, as described in detail herein. In other words, the criteria may include: an optimized nucleotide sequence that does not contain a termination signal (the first criterion), an optimized nucleotide sequence that has a guanine-cytosine content within a predetermined guanine-cytosine content range (the second criterion), an optimized nucleotide sequence that has a codon adaptation index that is greater than a predetermined codon adaptation index threshold (the third criterion), and an optimized nucleotide sequence that does not. It should be understood that the numbering of the criteria used is for clarity only and is not intended to limit the order of the steps, which is described in more detail elsewhere in this document.

[000134] It should be understood that, although specific criteria are described in detail herein, they may not be the only criteria against which optimized nucleotide sequences are screened, and screening may be conducted against additional criteria to further filter the list of optimized nucleotide sequences within the scope of the claims of the present invention.

[000135] When screening each optimized nucleotide sequence, the optimized nucleotide sequence may be analyzed in its entirety before determining whether it meets the criteria. Alternatively, each optimized nucleotide sequence may be analyzed in portions. A portion may be referred to as a window.

[000136] For example, for an optimized nucleotide sequence in a list of optimized nucleotide sequences that is 600 nucleotides long, the length of the portion may be selected to be 30 nucleotides. The first 30 nucleotides of the optimized nucleotide sequence may first be analyzed for compliance with a certain criterion, i.e., nucleotides 1 through 30 of the optimized nucleotide sequence. If the first portion does not meet the criterion, the optimized nucleotide sequence may be removed from the list of optimized nucleotide sequences.

[000137] If the first portion meets the criterion, the second portion of the optimized nucleotide sequence can then be analyzed with this filtering. In this example, this could be the second 30 nucleotides, i.e., nucleotides 31 through 60 of the optimized nucleotide sequence. The analysis of portions can be repeated for each portion until either: a portion that does not meet the criterion is found, in which case the optimized nucleotide sequence can be removed from the list, or the entire optimized nucleotide sequence is analyzed and no such portion is found, in which case the filtering keeps the optimized nucleotide sequence in the list and can move on to the next optimized nucleotide sequence in the list. In this example, if the filtering reaches the last portion of the optimized nucleotide sequence, i.e., If nucleotides 571 through 600 are found, and this last portion satisfies the criterion, this filtering retains the optimized nucleotide sequence in the list and can move on to the next optimized nucleotide sequence in the list. Alternatively, and specifically, each portion can be 100 nucleotides long.

[000138] Although the above example describes filtering in parts, starting from the first nucleotide and ending with the last nucleotide, it should be understood that this is only an example, and the order in which the parts of the optimized nucleotide sequence are analyzed can be any order obvious to a person skilled in the art. Filtering can, for example, start with the part including the last nucleotide (in the working example, nucleotide 600) and return to the first nucleotide, nucleotide 1, or it can start with a part at any position between the first and last nucleotides.

[000139] There may be a first, last, or intermediate portion of an optimized nucleotide sequence whose length differs from the length of the other portions. This may occur, for example, if the length in nucleotides of the optimized nucleotide sequence is not exactly divisible by the length in nucleotides of its portions.

[000140] As discussed in detail elsewhere in this document, part-by-part analysis may be advantageous at least for computational efficiency, but also for more efficiently identifying less desirable sequences that may meet the criterion on average, but which contain regions that do not meet the criterion, such as peaks or dips in GC content or CAI score.

[000141] The optimized nucleotide sequences in the list may be screened for compliance with one or more criteria in one of two ways: each sequence may be screened for all relevant criteria and removed from the list if it fails to meet any of them; or, in particular, all sequences in the list may be screened for certain criteria and a reduced filtered list may be screened for additional criteria of interest.

Selection of motives

[000142] In some embodiments, motif-based filtering can be applied to the list of optimized nucleotide sequences. In such embodiments, the list of optimized nucleotide sequences is analyzed to determine whether each optimized nucleotide sequence in the list contains a termination signal. The list of optimized nucleotide sequences can be a list of optimized nucleotide sequences initially generated using a codon optimization algorithm, or can be a list of optimized nucleotide sequences that has already been filtered in one or more additional steps of the algorithm. A list of optimized nucleotide sequences that has already been filtered or updated in one or more additional steps of the algorithm can be referred to as an updated list or the most recently updated list of optimized nucleotide sequences. Any optimized nucleotide sequence that contains one or more termination signals can be removed from the list to obtain an updated list.

[000143] Referring to Figure 6, the termination signal may have the following nucleotide sequences: 5'-X ₁ ATCTX ₂ TX ₃ -3', where X ₁ , X ₂ and X ₃ are independently selected from A, C, T or G; TATCTGTT; TTTTTTT; AAGCTT; GAAGAGC; TCTAGA; UAUCUGUU; UUUUUU; AAGCUU; GAAGAGC; UCUAGA; and/or 5'-X ₁ AUCUX ₂ UX ₃ -3', where X 1 , X ₂ and X ₃ are independently selected from A, C, U or G. Motif selection filtering can determine whether each optimized nucleotide sequence contains one, more or all of these termination signals.

[000144] Each optimized nucleotide sequence can be analyzed in its entirety, i.e., from the first nucleotide in the sequence to the last nucleotide in the sequence. In a particular embodiment, the analysis of a particular optimized nucleotide sequence can be stopped when the presence of a termination signal in this sequence is determined; this sequence can then be removed from the list without analyzing each of its nucleotides. In a particular embodiment, this form of analysis can be applied to each optimized nucleotide sequence in the list. Such analysis can be advantageous since it is computationally efficient not to analyze the entire sequence if the presence of a termination signal in this sequence has already been determined.

[000145] Each optimized nucleotide sequence can be analyzed in portions, as will be described in more detail with respect to the GC content analysis. The analysis of the optimized nucleotide sequence can be stopped after determining that a portion contains a termination signal. This can be advantageous since it is computationally efficient not to analyze the entire sequence if the presence of a termination signal in this sequence has already been determined. With respect to the subsequent GC content analysis, the portions may or may not overlap and may be of any length, for example, from 5 to 300 nucleotides, or from 10 to 200 nucleotides, or from 15 to 100 nucleotides, or from 20 to 50 nucleotides, or, in particular, 30 nucleotides or 100 nucleotides. Each of the parts of the optimized nucleotide sequence may be the same length, or, for example, the first, last, or intermediate part of the optimized nucleotide sequence may have a length that differs from the other parts, for example, if the length in nucleotides of the optimized nucleotide sequence is not exactly divisible by the length in nucleotides of these parts.

GC content analysis

[000146] In some embodiments, filtering based on guanine-cytosine (GC) content can be applied to the list of optimized nucleotide sequences. In such embodiments, the list of optimized nucleotide sequences is analyzed to determine the GC content of each of the optimized nucleotide sequences in the list of optimized nucleotide sequences, where the GC content of a sequence is the percentage of bases in a nucleotide sequence that are guanine (G) or cytosine (C). The list of optimized nucleotide sequences may be a list of optimized nucleotide sequences initially generated using a codon optimization algorithm, or may be a list of optimized nucleotide sequences that has already been filtered in one or more additional steps of the algorithm. A list of optimized nucleotide sequences that has already been filtered or updated in one or more additional steps of the algorithm may be referred to as an updated list or the most recently updated list of optimized nucleotide sequences. Any optimized nucleotide sequence whose GC content falls outside the pre-defined GC content range can be removed from the list to produce an updated list.

[000147] Each optimized nucleotide sequence can be analyzed in its entirety, i.e., from the first nucleotide in the sequence to the last nucleotide in the sequence. The GC content of the entire optimized nucleotide sequence can then be determined and sequences removed accordingly.

[000148] In some embodiments, only a portion of each optimized nucleotide sequence is analyzed and the GC content of that portion is determined. In such embodiments, if the GC content of the analyzed portion is outside a predetermined GC content range, the optimized nucleotide sequence containing that portion is removed from the list.

[000149] In a particular embodiment, GC content-based filtering is applied to each optimized nucleotide sequence in portions, with the filtering being stopped and the sequence being removed if a portion is determined to have a GC content outside a predetermined range. Such analysis may be advantageous because it is computationally efficient to not analyze the entire sequence if the presence of a portion in that sequence having a GC content outside a predetermined GC content range has already been detected.

[000150] In a particular embodiment, the portions do not overlap, but in other embodiments, the portions may overlap. It should be understood that this particular embodiment can be performed with a portion of any length, such as from 5 to 300 nucleotides, or from 10 to 200 nucleotides, or from 15 to 100 nucleotides, or from 20 to 50 nucleotides, or, in particular, 30 nucleotides or 100 nucleotides. In some embodiments, a predetermined range of GC content can be selected by the user. It should also be understood that this particular embodiment can be performed with any length of the optimized nucleotide sequence.

[000151] For example, an analysis of the guanine-cytosine (GC) content of non-optimized and optimized nucleotide sequences can be performed on portions of the nucleotide sequence encoding EPO, where the guanine-cytosine (GC) content of portions of the nucleotide sequence encoding EPO is determined for contiguous, non-overlapping portions of 30 nucleotides in length. This illustrative analysis is depicted in Figure 11.

[000152] This document describes an exemplary filtering based on GC content. It will be obvious to anyone skilled in the art that this is only an example, and that the methods described herein can be performed with an optimized nucleotide sequence and/or a portion of any length. For example, for an optimized nucleotide sequence in a list of optimized nucleotide sequences that has a length of 600 nucleotides, the length of the portion can be selected equal to 30 nucleotides. When filtering based on GC content, the first 30 nucleotides of the optimized nucleotide sequence, i.e., nucleotides 1 through 30 of the optimized nucleotide sequence, can be analyzed first. The analysis can include determining the number of nucleotides in the portion containing either G or C, and determining the GC content of the portion can involve dividing the number of G or C nucleotides in the portion by the total number of nucleotides in the portion. The result of this analysis will yield a value describing the proportion of nucleotides in the portion that are G or C, and can be a percentage, such as 50%, or a decimal fraction, such as 0.5. If the GC content of the first portion falls outside the predetermined GC content range, the optimized nucleotide sequence can be removed from the list of optimized nucleotide sequences.

[000153] If the GC content of the first portion falls within a predetermined GC content range, the second portion of the optimized nucleotide sequence may then be analyzed using GC content filtering. In this example, this may be the second 30 nucleotides, i.e., nucleotides 31 through 60 of the optimized nucleotide sequence. The analysis of the portions may be repeated for each portion until either: a portion characterized by a GC content outside the predetermined GC content range is found, in which case the optimized nucleotide sequence may be removed from the list, or the entire optimized nucleotide sequence is analyzed and no such portion is found, in which case GC content filtering keeps the optimized nucleotide sequence in the list and may move on to the next optimized nucleotide sequence in the list. In this example, if GC content filtering reaches the last portion of the optimized nucleotide sequence, i.e., If the GC content of this last portion falls within the specified GC content range, GC content-based filtering retains the optimized nucleotide sequence in the list and can move on to the next optimized nucleotide sequence in the list. Alternatively, and specifically, each portion can be 100 nucleotides long.

[000154] Although the above example describes filtering by portions based on GC content, starting from the first nucleotide and ending with the last nucleotide, it should be understood that this is only an example, and the order in which the portions of the optimized nucleotide sequence are analyzed can be any order obvious to one skilled in the art. Filtering based on GC content can, for example, start with the portion including the last nucleotide (in the working example, nucleotide 600) and return to the first nucleotide, nucleotide 1, or can start with the portion at any position between the first and last nucleotides.

[000155] There may be a first, last, or intermediate portion of an optimized nucleotide sequence whose length differs from the length of the other portions. This may occur, for example, if the length in nucleotides of the optimized nucleotide sequence is not exactly divisible by the length in nucleotides of its portions.

Codon Adaptation Index (CAI) analysis

[000156] In some embodiments, a codon adaptation index (CAI) analysis can be performed for some or all of the optimized nucleotide sequences in a list of optimized nucleotide sequences. In such embodiments, one or more optimized nucleotide sequences in the list of optimized nucleotide sequences are analyzed to determine the CAI of each sequence, where the CAI is a measure of codon usage preference and can have a value from 0 to 1. The list of optimized nucleotide sequences can be a list of optimized nucleotide sequences initially generated using a codon optimization algorithm, or can be a list of optimized nucleotide sequences that has already been filtered in one or more additional steps of the algorithm. A list of optimized nucleotide sequences that has already been filtered or updated in one or more additional steps of the algorithm can be referred to as an updated list or the most recently updated list of optimized nucleotide sequences. Any optimized nucleotide sequence that has a CAI less than or equal to a given CAI threshold can be removed from the list to produce an updated list.

[000157] In some embodiments, the CAI threshold is selected by the user. In some embodiments, the CAI threshold is 0.7, 0.75, 0.85, or 0.9. In a particular embodiment, the CAI threshold is 0.8.

[000158] The CAI can be calculated for each optimized nucleotide sequence in any manner obvious to one skilled in the art, for example, as described in "The codon adaptation index—a measure of directional synonymous codon usage bias, and its potential applications" (Sharp and Li, 1987. Nucleic Acids Research 15(3), p.1281-1295); available at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC34052 4/.

[000159] The implementation of calculating the codon adaptation index may include a method corresponding to or similar to the following. For each amino acid in a sequence, the weight of each codon in the sequence may be represented by a parameter called the relative adaptivity (W _i ). The relative adaptivity may be calculated from a set of reference sequences as the ratio between the observed codon frequency f _i and the frequency of the most frequently occurring synonymous codon f _j for this amino acid. The codon adaptation index of the sequence may then be calculated as the geometric mean of the weight associated with each codon over the length of the sequence (measured in codons). The set of reference sequences used to calculate the codon adaptation index may be the same set of reference sequences from which the codon usage frequency table used in the methods of the present invention is derived.

[000160] As noted previously, filtering based on CAI analysis can be applied as a part-by-part analysis, as described in detail herein. In other words, a CAI measure can be determined for parts of each optimized nucleotide sequence, and a sequence can be removed from consideration (i.e., removed from the list) if the CAI of any part is equal to or less than a specified CAI threshold. This implementation of the method provides both increased computational efficiency and more selective filtering.

Combining additional stages of the algorithm

[000161] Figure 7 shows that one, two, or three of the filtering based on motif selection, filtering based on GC content analysis, and filtering based on CAI analysis, and in any order, or none of them, can be applied to the list of optimized nucleotide sequences. Since each filtering applied to the same list of optimized nucleotide sequences and with the same input parameters has the same effect on the list, each filtering can be used only once. For example, if filtering based on motif selection and filtering based on GC content analysis were applied to the list of optimized nucleotide sequences, applying additional filtering based on motif selection or additional filtering based on GC content analysis to the updated list of optimized nucleotide sequences would have no effect. This is because any sequences in the list that do not meet the requirements of any of these filters have already been removed. Figure 7 also shows that there are embodiments of the present invention in which no filtering is applied to the list of optimized nucleotide sequences.

[000162] Figure 8 depicts an embodiment of the present invention in which only one filtering is applied to the list of optimized nucleotide sequences. In this embodiment, filtering based on GC content analysis was selected, however, it will be clear that this is an example, and that if only one filtering is needed, filtering based on motif selection or filtering based on CAI can be selected as an alternative.

[000163] Figure 9 depicts an embodiment of the present invention in which only two filters are applied to the list of optimized nucleotide sequences. In this embodiment, motif selection-based filtering and CAI analysis-based filtering were applied in the order shown, however, it will be understood that this is an example and that if only two filters are desired, any two of motif selection-based filtering, GC content-based filtering, and CAI analysis-based filtering can be applied in any order. In the example of Figure 9, motif selection-based filtering is applied to the list of optimized nucleotide sequences to obtain an updated list of optimized nucleotide sequences. Before the updated list of optimized nucleotide sequences is further filtered using CAI analysis-based filtering, this list may be referred to as the most recently updated list of optimized nucleotide sequences. The CAI analysis-based filtering is then applied to the most recently updated list of optimized nucleotide sequences to produce an updated or further updated list of optimized nucleotide sequences.

[000164] Figure 10 depicts a specific embodiment of the present invention in which three filters are applied to a list of optimized nucleotide sequences. In this specific embodiment, filtering based on motif selection, filtering based on GC content analysis, and filtering based on CAI analysis were applied in that order to obtain an updated list of optimized nucleotide sequences. It will be appreciated that in alternative embodiments using three filters, filtering based on motif selection, filtering based on GC content analysis, and filtering based on CAI analysis can be applied in any order. Similar to Figure 9, after each filtering step, i.e., between filtering based on motif selection and GC content analysis, and between filtering based on GC content analysis and CAI analysis, the list of optimized nucleotide sequences may be referred to as the latest updated list of optimized nucleotide sequences (not shown in Figure 10). As in the illustrative embodiments shown in Figures 8 and 9, the sequences from the updated list of optimized nucleotide sequences obtained at the end of any and all filtering steps can then be synthesized according to any of the synthetic methods described herein.

[000165] Filtering with more than one additional algorithm stage can have a synergistic beneficial effect. This is achieved by having the input data for each additional algorithm stage be the most recently updated list of optimized nucleotide sequences, i.e., it can be a list of sequences that have already been filtered. This reduces the processing and time requirements for performing the additional filtering stage, since there are fewer sequences to analyze in the list, increasing the efficiency of the method.

Adjacent identical codons

[000166] In some embodiments, some or all of the optimized nucleotide sequences in the list of optimized nucleotide sequences can be analyzed to determine optimized nucleotide sequences that have at least 2, such as 3 or more contiguous identical codons. This additional step of the algorithm can be the only additional step of the algorithm or can be performed before or after one or more of motif selection, GC content analysis, and CAI analysis. The analysis can be performed for each optimized nucleotide sequence in parts, as described in detail herein.

[000167] For example, a particular optimized nucleotide sequence may be analyzed and determined to contain a region including: CAGCAGCAG. Such a region, including a particular repeating codon, may stop transcription, so the sequence is removed from the list.

[000168] In some embodiments, a contiguity rarity threshold is used to determine rare codons, where codons with a value below the contiguity rarity threshold are considered rare codons. Rare codons can be identified by comparing the usage frequencies in a normalized codon usage frequency table with the contiguity rarity threshold. Thus, using the contiguity rarity threshold, codons are identified whose usage frequency exceeds the threshold frequency so that they can be included in the normalized codon usage frequency table, but, nevertheless, they are relatively rare among the codons in the normalized codon usage frequency table. In some embodiments, only rare contiguous identical codons cause the removal of the optimized nucleotide sequence from the list of optimized nucleotide sequences.

[000169] The contiguity rarity threshold may range from 10 to 50%, for example, from 15 to 40%, for example, from 20 to 30%, and will depend on the cutoff frequency used to normalize the codon usage frequency table. The contiguity rarity threshold must be greater than the cutoff frequency to have an effect, since any codon with a usage frequency below the cutoff frequency will not appear in the normalized codon usage frequency table.

[000170] Using the same example as above, but filtering only for rare contiguous identical codons, if CAG appears in the normalized codon usage table with a frequency equal to or greater than the contiguous rarity threshold, the sequence containing CAGCAGCAG will not be removed from the list. If instead CAG appears in the normalized codon usage table with a frequency less than the contiguous rarity threshold, the sequence containing CAGCAGCAG will be removed from the list.

[000171] Filtering for adjacent identical codons, including, optionally, for rare adjacent identical codons, may be applied at any stage after the creation of the list of optimized nucleotide sequences. In other words, filtering for adjacent identical codons, including, optionally, for rare adjacent identical codons, may be applied with any other additional stage of the algorithm, and the stages are performed in any order.

Synthesis and expression of optimized nucleotide sequences

[000172] In a further aspect, the present invention provides a method for synthesizing a nucleotide sequence, comprising: performing a computerized method of the present invention to generate at least one optimized nucleotide sequence; and synthesizing at least one of the generated optimized nucleotide sequences. In vitro synthesis (also commonly referred to as "in vitro transcription") is typically performed using a nucleic acid vector, such as a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and a suitable RNA polymerase (e.g., T3, T7, or SP6 RNA polymerase), DNase I, pyrophosphatase, and/or RNase inhibitor. The exact conditions will vary depending on the specific application.

[000173] In some embodiments, the synthesized optimized nucleotide sequence generated according to the method of the present invention is inserted into a nucleic acid vector for use in in vitro transcription. In some embodiments, the nucleic acid vector is a plasmid. The term "plasmid" or "plasmid-based nucleic acid vector" refers to a circular nucleic acid molecule, such as an artificial nucleic acid molecule. Plasmid DNA in the context of the present invention is suitable for including or containing a desired nucleic acid sequence, such as a nucleic acid sequence comprising a sequence encoding an mRNA transcript and/or an open reading frame encoding at least one protein, polypeptide, or peptide. Such plasmid DNA-based constructs/vectors may be expression vectors, cloning vectors, transfer vectors, etc.

[000174] A nucleic acid vector typically comprises a sequence corresponding to (encoding for) a desired mRNA transcript or a portion thereof, such as a sequence corresponding to an open reading frame and 5' and/or 3' UTR of the mRNA. In some embodiments, the sequence corresponding to the desired mRNA transcript may also encode a poly(A) tail after the 3' UTR, so that the poly(A) tail is included in the mRNA transcript. More typically, in the context of the present invention, the sequence corresponding to the desired mRNA transcript consists of a 5'/3' UTR and an open reading frame. In some embodiments of the present invention, the mRNA transcript synthesized from the nucleic acid vector during in vitro transcription does not comprise a poly(A) tail. The poly(A) tail can be added to the mRNA transcript during a processing step after synthesis.

[000175] In some embodiments, the nucleic acid vector comprises a nucleotide sequence encoding a 5'-UTR operably linked to an optimized nucleotide sequence. In specific embodiments, the 5'-UTR differs from the 5'-UTR of a naturally occurring mRNA encoding an amino acid sequence. In a specific embodiment, the 5'-UTR has the nucleotide sequence of SEQ ID NO: 19.

[000176] In some embodiments, the nucleic acid vector comprises a nucleotide sequence encoding a 3'-UTR operably linked to an optimized nucleotide sequence. In specific embodiments, the 3'-UTR differs from the 3'-UTR of a naturally occurring mRNA encoding an amino acid sequence. In a specific embodiment, the 3'-UTR has the nucleotide sequence of SEQ ID NO: 20 or SEQ ID NO: 21.

[000177] For example, a nucleotide sequence of the present invention can be synthesized from a nucleic acid vector comprising a 5' UTR, an optimized nucleotide sequence, and a 3' UTR (and optionally one or more termination signals at the 3' end of the optimized nucleotide sequence) to generate an mRNA comprising the 5' UTR, the optimized nucleotide sequence, and the 3' UTR.

[000178] In some embodiments, the nucleic acid vector comprises a promoter sequence, such as an RNA polymerase promoter sequence, such as a T3, T7, or SP6 RNA polymerase promoter sequence.

[000179] In some embodiments, the nucleic acid vector comprises one or more termination signals (e.g., two or three termination signals) downstream of the 3' end of the synthesized optimized nucleotide sequence. In some embodiments, the method further comprises inserting one or more termination signals at the 3' end of the synthesized optimized nucleotide sequences. In some embodiments, more than one termination signal is inserted, and the termination signals are separated by 10 base pairs or less, such as separated by 5-10 base pairs. The addition of one or more termination signals downstream of the optimized nucleotide sequence promotes efficient transcription termination since RNA is transcribed from plasmid DNA containing the optimized nucleotide sequence, resulting in targeted transcription termination in vitro at one or more termination signals and thus limiting aberrant transcription continuation. In some embodiments, the nucleic acid vector comprises more than one termination signal, such as two or more, three or more, or four or more. The presence of multiple termination signals increases the efficiency of transcription termination in vitro at the target site.

[000180] In some embodiments, the one or more termination signals have the following nucleotide sequence: 5'-X ₁ ATCTX ₂ TX ₃ -3', wherein X ₁ , X ₂ and X ₃ are independently selected from A, C, T or G. In some embodiments, the one or more termination signals have one of the following nucleotide sequences: TATCTGTT; and/or TTTTTTT; and/or AAGCTT; and/or GAAGAGC; and/or TCTAGA. In some embodiments, the one or more termination signals have the following nucleotide sequence: 5'-X ₁ AUCUX ₂ UX ₃ -3', wherein X ₁ , X ₂ and X ₃ are independently selected from A, C, U or G. In some embodiments, the one or more termination signals have one of the following nucleotide sequences: UAUCUGUU; and/or UUUUUU; and/or AAGCUU; and/or GAAGAGC; and/or UCUAGA. In some embodiments, more than one termination signal is encoded by the following nucleotide sequence: (a) 5'-X ₁ ATCTX ₂ TX ₃ -(Z _N )- X ₄ ATCTX ₅ TX ₆ -3' or (b) 5'-X ₁ ATCTX ₂ TX ₃ -(Z _N )-X ₄ ATCTX ₅ TX ₆ -(Z _M )- X ₇ ATCTX ₈ TX ₉ -3', wherein X ₁ , X ₂ , X ₃ , X ₄ , X ₅ , X ₆ , X ₇ , X ₈ and X ₉ are independently selected from A, C, T or G, ZN is a spacer sequence of N nucleotides, and Z _M is a spacer sequence of M nucleotides, each of which is independently selected from A, C, T or G, and wherein N and/or M are independently 10 or less.

[000181] Accordingly, in a particular embodiment of the present invention, plasmid DNA containing one or more termination signals (e.g., two or three termination signals) downstream of the 3' end of the synthesized optimized nucleotide sequence does not require linearization for in vitro transcription. In particular, the present invention makes it possible to produce mRNA transcripts from circular nucleic acid vectors, such as plasmid DNA (which is typically supercoiled), using SP6/T7 RNA polymerase for in vitro transcription.

RNA polymerase SP6

[000182] In some embodiments, mRNA is synthesized using SP6 RNA polymerase. In some embodiments, the SP6 RNA polymerase is a naturally occurring SP6 RNA polymerase. In some embodiments, the SP6 RNA polymerase is a recombinant SP6 RNA polymerase. In some embodiments, the SP6 RNA polymerase comprises a tag. Tags can be used to facilitate detection or purification of the protein. In some embodiments, the tag is a histidine tag, which, for example, can be used for purification using Ni-NTA affinity chromatography.

[000183] SP6 RNA polymerase is a DNA-dependent RNA polymerase with high sequence specificity for sequences with the SP6 promoter. Typically, this polymerase catalyzes RNA synthesis in the 5'-3' direction in vitro on either single-stranded DNA or double-stranded DNA downstream of its promoter; it incorporates native ribonucleotides and/or modified ribonucleotides into the polymerized transcript.

[000184] The sequence of bacteriophage SP6 RNA polymerase was originally described (GenBank: Y00105.1) as having the following amino acid sequence:

[000185]

[000186] An SP6 RNA polymerase suitable for the present invention may be any enzyme that has substantially the same polymerase activity as bacteriophage SP6 RNA polymerase. Thus, in some embodiments, an SP6 RNA polymerase suitable for the present invention can be obtained by modification from SEQ ID NO: 1. For example, a suitable SP6 RNA polymerase may contain one or more amino acid substitutions, deletions, or additions. In some embodiments, a suitable SP6 RNA polymerase has an amino acid sequence that is about 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75%, 70%, 65%, or 60% identical or homologous to SEQ ID NO: 1. In some embodiments, a suitable SP6 RNA polymerase may be a truncated protein (from the N-terminus, C-terminus, or internally), but retains polymerase activity. In some embodiments, a suitable SP6 RNA polymerase is a fusion protein.

[000187] In some embodiments, SP6 RNA polymerase is encoded by a gene having the following nucleotide sequence:

[000188] A suitable gene encoding an SP6 RNA polymerase useful in the present invention may be approximately 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, or 80% identical or homologous to SEQ ID NO: 2.

[000189] An SP6 RNA polymerase suitable for the present invention may be a commercially available product, such as from Ambion, New England Biolabs (NEB), Promega, and Roche. SP6 may be ordered and/or custom-designed from a commercial or non-commercial source according to the amino acid sequence of SEQ ID NO: 1 or a variant of SEQ ID NO: 1, as described herein. The SP6 RNA polymerase may be a standard-fidelity polymerase or may be a high-fidelity/high-efficiency/high-capacity polymerase that has been modified to increase RNA polymerase activity, such as through mutations in the SP6 RNA polymerase gene or post-translational modifications of the SP6 RNA polymerase itself. Examples of such modified SP6 include SP6 RNA Polymerase-Plus™ from Ambion, HiScribe SP6 from NEB, and the RiboMAX™ and Riboprobe® systems from Promega.

[000190] In some embodiments, the SP6 RNA polymerase is thermostable. In a particular embodiment, the amino acid sequence of the SP6 RNA polymerase for use in the present invention comprises one or more mutations compared to the wild-type SP6 polymerase that render the enzyme active at a temperature in the range of 37°C to 56°C. In some embodiments, the SP6 RNA polymerase for use in the present invention functions at an optimal temperature of 50°C to 52°C. In another embodiment, the SP6 RNA polymerase for use in the present invention has a half-life of at least 60 minutes at 50°C. For example, a particularly suitable SP6 RNA polymerase for use in the present invention has a half-life of 60 minutes to 120 minutes (e.g., 70 minutes to 100 minutes or 80 minutes to 90 minutes) at 50°C.

[000191] In some embodiments, a suitable SP6 RNA polymerase is a fusion protein. For example, an SP6 RNA polymerase may include one or more tags that facilitate the isolation, purification, or solubility of the enzyme. A suitable tag may be located at the N-terminus, C-terminus, and/or internally. Non-limiting examples of a suitable tag include calmodulin binding protein (CBP); Fasciola hepatica 8-kDa antigen (Fh8); a peptide that is a FLAG tag; glutathione S-transferase (GST); a histidine tag (e.g., a hexahistidine tag (His6)); maltose binding protein (MBP); a substance utilized with protein N (NusA); a fusion tag that is a small ubiquitin-like modifier (SUMO); streptavidin-binding peptide (STREP); a tandem affinity purification (TAP) tag, and thioredoxin (TrxA). Other tags may be used in the present invention. These and other fusion tags have been described, for example, by Costa et al. Frontiers in Microbiology 5 (2014): 63 and in PCT/US16/57044, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the His-tag is located at the N-terminus of SP6.

Promoter for SP6

[000192] Any promoter that can be recognized by SP6 RNA polymerase can be used in the present invention. Typically, a promoter for SP6 comprises 5'-ATTTAGGTGACACTATAG-3' (SEQ ID NO: 3). Variants of the promoter for SP6 have been discovered and/or created to optimize the recognition and/or binding of SP6 to its promoter. Non-limiting variants include, but are not limited to:

When N is used in nucleotide sequences, N represents A, C, T, or G.

[000193] Furthermore, a suitable promoter for SP6 for the present invention may be about 95%, 90%, 85%, 80%, 75%, or 70% identical or homologous to any of SEQ ID NO: 4 to SEQ ID NO: 13. Moreover, a promoter for SP6 suitable for the present invention may comprise one or more additional nucleotides at the 5' and/or 3' end of any of the promoter sequences described herein.

T7 RNA polymerase

[000194] In some embodiments, mRNA is synthesized using T7 RNA polymerase.

[000195] T7 RNA polymerase is a DNA-dependent RNA polymerase with high sequence specificity for T7 promoter sequences. Typically, this polymerase catalyzes RNA synthesis in the 5'-3' direction in vitro on either single-stranded DNA or double-stranded DNA downstream of its promoter; it incorporates native ribonucleotides and/or modified ribonucleotides into the polymerized transcript.

[000196] In some embodiments, the T7 RNA polymerase is thermostable. In a particular embodiment, the amino acid sequence of the T7 RNA polymerase for use in the present invention comprises one or more mutations compared to the wild-type T7 polymerase that render the enzyme active at a temperature in the range of 37°C to 56°C. An example of a suitable RNA polymerase is Hi-T7® RNA polymerase from NEB. In some embodiments, the T7 RNA polymerase for use in the present invention functions at an optimal temperature of 50°C to 52°C. In another embodiment, the T7 RNA polymerase for use in the present invention has a half-life of at least 60 minutes at 50°C. For example, a particularly suitable T7 RNA polymerase for use in the present invention has a half-life of 60 minutes to 120 minutes (e.g., 70 minutes to 100 minutes or 80 minutes to 90 minutes) at 50°C.

Promoter for T7

[000197] Any promoter that can be recognized by T7 RNA polymerase can be used in the methods described herein. Typically, a promoter for T7 comprises 5'-TAATACGACTCACTATAG-3' (SEQ ID NO: 14).

Post-synthetic processing

[000198] In some embodiments, the method of the present invention further comprises a separate step of copying and/or extending the synthesized mRNA.

[000199] Typically, a 5' cap and/or 3' tail may be added after synthesis. The presence of the cap is important for providing resistance to nucleases found in most eukaryotic cells. The presence of the tail serves to protect the mRNA from degradation by exonucleases.

[000200] The 5' cap is typically added as follows: first, terminal RNA phosphatase removes one of the terminal phosphate groups from the 5' nucleotide, leaving two terminal phosphates, then guanosine triphosphate (GTP) is added to the terminal phosphates by guanylyltransferase, forming a 5',5',5-triphosphate linkage; and then the nitrogen atom at position 7 of guanine is methylated by methyltransferase. Examples of cap structures include, but are not limited to, tom7G(5')ppp(5')(2'OMeG), m7G(5')ppp(5')(2'OMeA), m7(3'OMeG)(5')ppp(5')(2'OMeG), m7(3'OMeG)(5')ppp(5')(2'OMeA), m7G(5')ppp(5')(A), G(5')ppp(5')A, and G(5')ppp(5')G. In a particular embodiment, the cap structure is r7G(5')ppp(5')(2'OMeG). Additional cap structures are described in U.S. Published Application No. US 2016/0032356 and U.S. Provisional Application No. 62/464,327, filed on 27 February 2017, which are incorporated herein by reference.

[000201] Typically, the tail structure includes a poly(A) and/or poly(C) tail. The poly(A) or poly(C) tail at the 3' end of the mRNA typically contains at least 50 adenosine or cytosine nucleotides, at least 150 adenosine or cytosine nucleotides, at least 200 adenosine or cytosine nucleotides, at least 250 adenosine or cytosine nucleotides, at least 300 adenosine or cytosine nucleotides, at least 350 adenosine or cytosine nucleotides, at least 400 adenosine or cytosine nucleotides, at least 450 adenosine or cytosine nucleotides, at least 500 adenosine or cytosine nucleotides, at least 550 adenosine or cytosine nucleotides, at least 600 adenosine or cytosine nucleotides, at least 650 adenosine or cytosine nucleotides, at least 700 adenosine or cytosine nucleotides, at least 750 adenosine or cytosine nucleotides, at least 800 adenosine or cytosine nucleotides, at least 850 adenosine or cytosine nucleotides, at least 900 adenosine or cytosine nucleotides, at least 950 adenosine or cytosine nucleotides, or at least 1,000 adenosine or cytosine nucleotides, respectively. In some embodiments, the poly(A) or poly(C) tail may be about 10-800 adenosine or cytosine nucleotides (e.g., about 10-200 adenosine or cytosine nucleotides, about 10-300 adenosine or cytosine nucleotides, about 10-400 adenosine or cytosine nucleotides, about 10-500 adenosine or cytosine nucleotides, about 10-550 adenosine or cytosine nucleotides, about 10-600 adenosine or cytosine nucleotides, about 50-600 adenosine or cytosine nucleotides, about 100-600 adenosine or cytosine nucleotides, about 150-600 adenosine or cytosine nucleotides, about 200-600 adenosine or cytosine nucleotides, about 250-600 adenosine or cytosine nucleotides, about 300-600 adenosine or cytosine nucleotides, about 350-600 adenosine or cytosine nucleotides, about 400-600 adenosine or cytosine nucleotides, about 450-600 adenosine or cytosine nucleotides, about 500-600 adenosine or cytosine nucleotides, about 10-150 adenosine or cytosine nucleotides, about 10-100 adenosine or cytosine nucleotides, about 20-70 adenosine or cytosine nucleotides, or about 20-60 adenosine or cytosine nucleotides), respectively. In some embodiments, the tail structure comprises a combination of poly(A) and poly(C) tails of varying lengths as described herein. In some embodiments, the tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% adenosine nucleotides. In some embodiments, the tail structure comprises at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[000202] As described herein, the addition of a 5' cap and/or 3' tail facilitates the detection of abortive transcripts formed during in vitro synthesis because without capping and/or tailing, the size of these prematurely terminated mRNA transcripts may be too small to be detected. Thus, in some embodiments, a 5' cap and/or 3' tail is added to the synthesized mRNA before testing the mRNA for purity (e.g., the level of abortive transcripts present in the mRNA). In some embodiments, a 5' cap and/or 3' tail is added to the synthesized mRNA before purifying the mRNA as described herein. In other embodiments, a 5' cap and/or 3' tail is added to the synthesized mRNA after purifying the mRNA as described herein.

[000203] In some embodiments, copying and amplification occur during in vitro transcription.

Reaction mixture conditions for mRNA synthesis

[000204] In some embodiments, the concentration of RNA polymerase in the reaction mixture can be from about 1 to 100 nM, from 1 to 90 nM, from 1 to 80 nM, from 1 to 70 nM, from 1 to 60 nM, from 1 to 50 nM, from 1 to 40 nM, from 1 to 30 nM, from 1 to 20 nM, or from about 1 to 10 nM. In certain embodiments, the concentration of RNA polymerase is from about 10 to 50 nM, from 20 to 50 nM, or from 30 to 50 nM. RNA polymerase can be used at a concentration of 100 to 10,000 U/ml, for example, concentrations of 100 to 9,000 U/ml, 100 to 8,000 U/ml, 100 to 7,000 U/ml, 100 to 6,000 U/ml, 100 to 5,000 U/ml, 100 to 1,000 U/ml, 200 to 2,000 U/ml, 500 to 1,000 U/ml, 500 to 2,000 U/ml, 500 to 3,000 U/ml, 500 to 4,000 U/ml, 500 to 5,000 U/ml, 500 to 6000 U/ml, 1000 to 7500 U/ml and 2500 to 5000 U/ml.

[000205] The concentration of each ribonucleotide (e.g., ATP, UTP, GTP, and CTP) in the reaction mixture is from about 0.1 mM to about 10 mM, such as from about 1 mM to about 10 mM, from about 2 mM to about 10 mM, from about 3 mM to about 10 mM, from about 1 mM to about 8 mM, from about 1 mM to about 6 mM, from about 3 mM to about 10 mM, from about 3 mM to about 8 mM, from about 3 mM to about 6 mM, from about 4 mM to about 5 mM. In some embodiments, the concentration of each ribonucleotide in the reaction mixture is about 5 mM. In some embodiments, the total concentration of rNTPs (e.g., ATP, GTP, CTP, and UTP together) used in the reaction is in the range of 1 mM to 40 mM. In some embodiments, the total concentration of rNTPs (e.g., ATP, GTP, CTP, and UTP together) used in the reaction is in the range of 1 mM to 30 mM, or 1 mM to 28 mM, or 1 mM to 25 mM, or 1 mM to 20 mM. In some embodiments, the total concentration of rNTPs is less than 30 mM. In some embodiments, the total concentration of rNTPs is less than 25 mM. In some embodiments, the total concentration of rNTPs is less than 20 mM. In some embodiments, the total concentration of rNTPs is less than 15 mM. In some embodiments, the total concentration of rNTPs is less than 10 mM.

[000206] In a specific embodiment, the concentration of each rNTP in the reaction mixture is optimized based on the frequency of occurrence of each nucleic acid in the nucleic acid sequence that encodes the mRNA transcript. In particular, such a sequence-optimized reaction mixture contains each of the four rNTPs (e.g., ATP, GTP, CTP, and UTP) in a ratio that corresponds to the ratio of these four nucleic acids (A, G, C, and U) in the mRNA transcript.

[000207] In some embodiments, a start nucleotide is added to the reaction mixture before in vitro transcription begins. The start nucleotide is a nucleotide that corresponds to the first nucleotide of the mRNA transcript (position +1). The start nucleotide can be specifically added to increase the rate of RNA polymerase initiation. The start nucleotide can be a nucleoside monophosphate, a nucleoside diphosphate, or a nucleoside triphosphate. The start nucleotide can be a mononucleotide, a dinucleotide, or a trinucleotide. In embodiments where the first nucleotide of the mRNA transcript is G, the start nucleotide is typically GTP or GMP. In a particular embodiment, the start nucleotide is an analog of a cap structure. The analog of a cap structure can be selected from the group consisting of

[000208] In particular embodiments, the first nucleotide of the RNA transcript is G, the starting nucleotide is a G cap structure analog, and the corresponding rNTP is GTP. In such embodiments, the cap structure analog is present in the reaction mixture in excess of GTP. In some embodiments, the cap structure analog is added at an initial concentration in the range of about 1 mM to about 20 mM, about 1 mM to about 17.5 mM, about 1 mM to about 15 mM, about 1 mM to about 12.5 mM, about 1 mM to about 10 mM, about 1 mM to about 7.5 mM, about 1 mM to about 5 mM, or about 1 mM to about 2.5 mM.

[000209] More typically, in the context of the present invention, a cap structure, such as a cap structure analog, is added to mRNA transcripts produced during in vitro transcription only after the synthesis of the mRNA transcripts, for example, during a post-synthesis processing step. Typically, in such embodiments, the mRNA transcripts are first purified (for example, by tangential flow filtration) before the addition of the cap structure.

[000210] The reaction buffer for RNA polymerase typically contains a salt/buffering agent such as Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, sodium phosphate, sodium chloride, and magnesium chloride.

[000211] The pH of the reaction mixture can be from about 6 to 8.5, from 6.5 to 8.0, from 7.0 to 7.5, and in some embodiments, the pH is 7.5.

[000212] A DNA template (e.g., as described above and in an amount/concentration sufficient to produce the desired amount of RNA), an RNA polymerase reaction buffer, and an RNA polymerase are combined to form a reaction mixture. The reaction mixture is incubated at a temperature of from about 37°C to about 56°C for from thirty minutes to six hours, such as from about sixty to about ninety minutes. In some embodiments, the incubation occurs at a temperature of from about 37°C to about 42°C. In another embodiment, the incubation is conducted at a temperature of from about 43°C to about 56°C, such as from about 50°C to about 52°C. As shown herein, the yield of precisely terminated mRNA transcripts obtained in an in vitro transcription reaction can be significantly increased by incorporating one or more termination signals described herein at the end of a DNA sequence encoding an mRNA transcript of interest and performing the reaction with a template comprising the DNA sequences at a temperature of from about 50°C to about 52°C.

[000213] In some embodiments, about 5 mM NTP, about 0.05 mg/ml polymerase, and about 0.1 mg/ml DNA template in a suitable RNA polymerase reaction buffer (the pH of the final reaction mixture is about 7.5) are incubated at a temperature of about 37°C to about 42°C for sixty to ninety minutes. In other embodiments, about 5 mM NTP, about 0.05 mg/ml polymerase, and about 0.1 mg/ml DNA template in a suitable RNA polymerase reaction buffer (the pH of the final reaction mixture is about 7.5) are incubated at a temperature of about 50°C to about 52°C for sixty to ninety minutes.

[000214] In some embodiments, the reaction mixture comprises a double-stranded DNA template with an RNA polymerase-specific promoter, RNA polymerase, RNase inhibitor, pyrophosphatase, 29 mM NTP, 10 mM DTT, and reaction buffer (at 10-fold concentration: 800 mM HEPES, 20 mM spermidine, 250 mM MgCl2, pH 7.7) and RNase-free water at a quantity sufficient (QS) to obtain the required reaction volume; the reaction mixture is then incubated at 37°C for 60 minutes. The polymerase reaction is then quenched by the addition of DNase I and DNase I buffer (at 10x concentration: 100 mM Tris-HCl, 5 mM MgCl ₂ , and 25 mM CaCl ₂ , pH 7.6) to facilitate cleavage of the double-stranded DNA template in preparation for purification. This embodiment has been shown to be sufficient to produce 100 grams of mRNA.

[000215] In some embodiments, the reaction mixture comprises NTP at a concentration in the range of 1-10 mM, DNA template at a concentration in the range of 0.01-0.5 mg/mL, and RNA polymerase at a concentration in the range of 0.01-0.1 mg/mL, for example, the reaction mixture comprises NTP at a concentration of 5 mM, DNA template at a concentration of 0.1 mg/mL, and RNA polymerase at a concentration of 0.05 mg/mL.

Nucleotides

[000216] Various naturally occurring or modified nucleosides can be used to produce mRNA according to the present invention. In some embodiments, the mRNA transcript according to the present invention is synthesized using natural nucleosides (i.e., adenosine, guanosine, cytidine, uridine). In other embodiments, an mRNA transcript according to the present invention is synthesized using naturally occurring nucleosides (e.g., adenosine, guanosine, cytidine, uridine) and one or more of the following: nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C5-propynylcytidine, C5-propynyluridine, 2-aminoadenosine, C5-bromuridine, C5-fluorouridine, C5-ioduridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-1-methyl-pseudouridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) and/or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages).

[000217] In some embodiments, the mRNA provides one or more non-standard nucleotide residues. Non-standard nucleotide residues can include, for example, 5-methylcytidine ("5mC"), pseudouridine ("ΨU"), and/or 2-thiouridine ("2sU"). For a discussion of such residues and their incorporation into mRNA, see, for example, U.S. Patent No. 8,278,036 or WO2011012316. The mRNA can be an RNA, which is defined as an RNA in which 25% of the U residues are 2-thiouridine and 25% of the C residues are 5-methylcytidine. Directions for using RNA are disclosed in U.S. Patent Publication US20120195936 and International Publication WO2011012316, both of which are incorporated herein by reference in their entireties. The presence of non-standard nucleotide residues may make the mRNA more stable and/or less immunogenic than a control mRNA with the same sequence but containing only standard residues. In additional embodiments, the mRNA may comprise one or more non-standard nucleotide residues selected from isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine, and 2-chloro-6-aminopurinecytosine, as well as combinations of these modifications and other nucleobase modifications. Some embodiments may also provide for additional modifications of the furanose ring or nucleobase. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more of a 2'-O-alkyl, locked nucleic acid (LNA) modification). In some embodiments, RNAs may form complexes or hybridize with additional polynucleotides and/or peptide polynucleotides (PNAs). In some embodiments, where the sugar modification is a 2'-O-alkyl modification, such modification may include, but is not limited to, 2'-deoxy-2'-fluoro modification, 2'-O-methyl modification, 2'-O-methoxyethyl modification, and 2'-deoxy modification. In some embodiments, any of these modifications may be present in 0-100% of the nucleotides, such as greater than 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotides, individually or in combination.

Transfection and screening of optimized nucleotide sequences in cells

[000218] In some embodiments, the method of the present invention further comprises transfecting the synthesized optimized nucleotide sequence into a cell either in vivo or in vitro. In some embodiments, the expression level of the protein encoded by the synthesized optimized nucleotide sequence is determined. In some embodiments, the method further comprises synthesizing a reference nucleotide sequence and at least one synthesized optimized nucleotide sequence created in accordance with the method of the present invention, and contacting each nucleotide sequence with an individual cell or organism. In a typical embodiment, a cell or organism contacted with at least one synthesized optimized nucleotide sequence provides an increased yield of the protein encoded by the optimized nucleotide sequence compared to the yield of the protein encoded by the reference nucleotide sequence produced by the cell or organism contacted with the synthesized reference nucleotide sequence. The reference nucleotide sequence may be: (a) a naturally occurring nucleotide sequence encoding an amino acid sequence; or (b) a nucleotide sequence encoding an amino acid sequence generated by a method other than the method of the present invention.

[000219] It may be necessary to verify that the synthesized optimized nucleotide sequences created in accordance with the methods of the present invention increase the expression of the encoded protein when transfected into a cell. Methods well known in the art, such as Western blotting, are suitable for experimentally verifying that the optimization of the codons of a given nucleotide sequence results in increased expression and production of the encoded protein. In addition, a plurality of synthesized optimized nucleotide sequences created by the methods of the present invention can be screened to identify the optimized nucleotide sequence(s) that provide(s) the highest protein yield. In some embodiments, the expression level of the protein encoded by the synthesized optimized nucleotide sequence is increased by at least 2-fold, such as at least 3-fold or 4-fold.

[000220] In some embodiments, the functional activity of the protein encoded by the synthesized optimized nucleotide sequence is determined. The functional activity of the protein encoded by the optimized nucleotide sequence can be determined using a number of conventional methods. These methods may vary depending on the properties of the encoded protein of interest. In the context of codon optimization, it may be important to experimentally test the functional activity of the protein encoded by the synthesized optimized nucleotide sequence(s) in vitro or in vivo to ensure that expression of the encoded protein(s) causes the desired functional effect(s). For example, an enzyme activity assay can be used to determine the functional enzymatic activity of the enzyme encoded by the optimized nucleotide sequence in cells. For example, the Ussing epithelial voltage-clamp assay can be used to assess the activity of the human cystic fibrosis transmembrane conductance regulator (hCFTR) protein expressed from mRNA encoding a codon-optimized hCFTR sequence generated using the methods of the present invention. This assay monitors the chloride transport function of epithelial cells transfected with hCFTR mRNA.

Therapeutic applications

[000221] The present invention provides a synthesized optimized nucleotide sequence created according to the method of the present invention for use in therapy.

[000222] In the field of mRNA-based therapeutics, codon optimization can be used to increase the expression of a functional protein encoded by the mRNA in a target cell, thereby correcting protein deficiencies in various disorders, including cystic fibrosis (CF), primary ciliary dyskinesia (PCD), pulmonary arterial hypertension (PAH), and idiopathic pulmonary fibrosis (IPF).

[000223] In certain aspects of the present invention, the optimized nucleotide sequence encodes a protein that is a human cystic fibrosis transmembrane conductance regulator (hCFTR):

[000224] In one specific embodiment, the optimized nucleotide sequence encoding the hCFTR protein according to the present invention is at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 26 and encodes a CFTR protein having the amino acid sequence of SEQ ID NO: 15. In a specific embodiment, the optimized nucleotide sequence encoding the hCFTR protein according to the present invention is SEQ ID NO: 26. In one specific embodiment, the optimized nucleotide sequence encoding the hCFTR protein according to the present invention is at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 27 and encodes a hCFTR protein having the amino acid sequence of SEQ ID NO: 15. In a specific embodiment, the optimized nucleotide sequence encoding the hCFTR protein according to the present invention is SEQ ID NO: 27. In one specific embodiment, the optimized nucleotide sequence encoding the hCFTR protein according to the present invention is at least 85%, 88%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 28 and encodes the hCFTR protein having the amino acid sequence of SEQ ID NO: 15. In a specific embodiment, the optimized nucleotide sequence encoding the hCFTR protein according to the present invention is SEQ ID NO: 28.

[000225] In certain aspects, the present invention provides a nucleic acid comprising an optimized nucleotide sequence encoding an hCFTR protein according to the present invention. In particular embodiments, the present invention provides an mRNA comprising an optimized nucleotide sequence encoding an hCFTR protein according to the present invention. In some embodiments, an mRNA comprising an optimized nucleotide sequence encoding an hCFTR protein according to the present invention also comprises 5'- and 3'-UTR sequences. Exemplary 5'- and 3'-UTR sequences are shown below.

Illustrative 5'-UTR sequence

Illustrative 3'-UTR sequence or

[000226] The synthesized optimized nucleotide sequences created according to the method of the present invention also find use in mRNA-based vaccines. In the context of prophylactic mRNA-based vaccines, codon optimization can be used to maximize the expression of the recombinant antigen encoded by the mRNA delivered to the subject for optimal antigenic activity, thereby creating protective immunity against the pathogen.

[000227] Similarly, in the field of cancer immunotherapy, codon optimization can be used to maximize the expression of a recombinant tumor neoantigen encoded by mRNA delivered to a subject, thereby generating an adaptive immune response against aberrant tumor cells expressing the neoantigen.

Biotechnological applications

[000228] In the field of biotechnology, particularly in the context of recombinant protein production, codon optimization can be used to increase the production of a protein of interest in a host cell, such as a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell.

[000229] For example, the method of the present invention can be used to optimize the protein expression yield for a recombinant insulin protein produced in E. coli. Expression of recombinant proteins can also occur, for example, in a host cell or in a cell-free protein extract suitable for protein expression. Codon optimization can also be used to increase the production of industrially useful enzymes suitable for use in biotechnology, manufacturing, diagnostics, and/or research.

EXAMPLES

[000230] The following examples are included for illustrative purposes only and are not intended to limit the scope of the present invention.

Example 1. Creation of optimized nucleotide sequences.

[000231] This example illustrates a method that provides optimized nucleotide sequences according to the present invention that are optimized to produce full-length transcripts during in vitro synthesis and result in high levels of expression of the encoded protein.

[000232] This process combines the codon optimization method shown in Figure 1 with the sequence of filtering steps depicted in Figure 10 to create a list of optimized nucleotide sequences. Specifically, as depicted in Figure 1, the method provides an amino acid sequence of interest and a first codon usage table that reflects the frequency of each codon in a given organism (namely, human codon usage preferences in the context of the present example). The method then removes codons from the first codon usage table if they are associated with a codon usage frequency that is less than a threshold frequency (10%). The codon usage frequencies for the codons not removed in the first step are normalized to create a normalized codon usage table.

[000233] Normalization of a codon usage table involves redistributing the usage value for each deleted codon; the usage frequency for a particular deleted codon is added to the usage frequencies of other codons with which the deleted codon shares an amino acid. In this example, the redistribution is proportional to the magnitude of the usage frequencies of codons not deleted from the table and can be performed according to an illustrative method as described in connection with Figures 3 and 4B. In this method, a normalized codon usage table is used to create a list of optimized nucleotide sequences. Each of the optimized nucleotide sequences encodes an amino acid sequence of interest.

[000234] As shown in Figure 10, the list of optimized nucleotide sequences is further processed by applying motif-based filtering, guanine-cytosine (GC) content analysis-based filtering, and codon adaptation index (CAI) analysis-based filtering, in that order, to create an updated list of optimized nucleotide sequences. Motif-based filtering, shown in Figure 6, is used to remove sequences that may interfere with transcription or translation. GC content analysis-based filtering follows the process shown in Figure 11.

[000235] As shown in the following examples, this process provides optimized nucleotide sequences encoding the amino acid sequence of interest. The nucleotide sequences provide full-length transcripts during in vitro synthesis and result in high levels of expression of the encoded protein (see Examples 2 and 3). As shown in Example 4, the expressed protein is fully functional.

Example 2: Optimizing codons to create nucleotide sequences with a high CAI score improves protein yield.

[000236] This example demonstrates that codon-optimized protein-coding sequences with a codon adaptation index (CAI) of approximately 0.8 or greater are superior to codon-optimized protein-coding sequences with a CAI below 0.8.

[000237] Codon optimization was performed on the amino acid sequence of wild-type human erythropoietin (hEPO). hEPO is a protein hormone secreted by the kidneys in response to low cellular oxygen levels (hypoxia). hEPO is necessary for erythropoiesis, the production of red blood cells. Recombinant hEPO is commonly used in the treatment of anemia, a condition characterized by low red blood cell or hemoglobin levels that can occur in subjects with chronic kidney disease or in subjects undergoing cancer chemotherapy.

[000238] Using various codon optimization algorithms, a total of 5 new codon-optimized nucleotide sequences encoding hEPO (No. 1 through No. 5) were generated. Nucleotide sequences No. 4 and No. 5 were generated according to the method of the present invention, as shown in Example 1. A nucleotide sequence with a codon-optimized hEPO coding sequence, which had previously been experimentally validated both in vitro and in vivo, was provided as a reference. The reference nucleotide sequence (SEQ ID NO: 19) was found to provide superior protein yield compared to the wild-type nucleotide sequence and other codon-optimized nucleotide sequences encoding the hEPO protein. The characteristics of each of the 5 nucleotide sequences in terms of CAI, GC content, codon frequency distribution (CFD), and the presence of negative cis-elements and negative repeat elements are summarized in Table 1.

[000239] To test the protein yield for each codon-optimized sequence, six nucleic acid vectors were prepared, each containing an expression cassette containing one of the six nucleotide sequences encoding the hEPO protein, flanked by identical 3'- and 5'-untranslated sequences (3'- and 5'-UTRs) preceded by a promoter for RNA polymerase. These nucleic acid vectors served as templates for in vitro transcription reactions to produce six batches of mRNA containing the six codon-optimized nucleotide sequences (reference and nucleotide sequences #1 through #5). Copying and extension were performed separately. Each of the copied and tailed mRNAs was separately transfected into a cell line (HEK2 93). Expression levels of the encoded hEPO protein were assessed by ELISA. The results of this experiment are summarized in Figure 12.

[000240] As can be seen in Figure 12, the highest expression level was observed with nucleotide sequence #3 (SEQ ID NO: 22), which provided almost twice as much hEPO protein as the experimentally validated reference nucleotide sequence. A trend toward higher protein yield can be observed for sequences depending on their CAI (see Table 1). Nucleotide sequence #3 with the highest protein yield had the highest CAI. The second and third nucleotide sequences with the highest yield, #4 (SEQ ID NO: 23) and #5 (SEQ ID NO: 24), had the second and third highest CAI. Nucleotide sequences #1 (SEQ ID NO: 20) and #2 (SEQ ID NO: 21), with the lowest efficiency, also had the lowest CAI. Furthermore, these were also the nucleotide sequences with the lowest GC content. However, GC content alone was not decisive. The reference nucleotide sequence had the highest GC content (61%) among all codon-optimized sequences tested, but it was not as efficient as nucleotide sequences #3, #4, and #5, each of which had lower GC content. Notably, nucleotide sequences #1 and #2, with the lowest efficiency, also had higher CFD values.

[000241] Taken together, the data in this example demonstrate that codon optimization of a therapeutically relevant nucleotide sequence to produce a CAI of approximately 0.8 or greater results in higher protein yield than, for example, codon optimization to produce a nucleotide sequence with the highest possible GC content.

Example 3. Codon optimization of the CFTR mRNA sequence to increase CAI results in higher protein expression.

[000242] This example confirms that codon-optimized protein-coding sequences with a codon adaptation index (CAI) of approximately 0.8 or greater are superior to codon-optimized protein-coding sequences with a CAI below 0.8.

[000243] The hEPO protein tested in Example 1 is a relatively short polypeptide, the amino acid sequence of which is encoded by a sequence of 495 nucleotides. To determine whether the results obtained in Example 1 are applicable to much longer nucleotide sequences encoding a large protein, codon optimization was performed on the human cystic fibrosis transmembrane conductance regulator (hCFTR). hCFTR is encoded by a sequence of 4440 nucleotides, i.e., its sequence is approximately 10 times longer than the hEPO coding sequence.

Mutations in the gene encoding the hCFTR protein cause cystic fibrosis (CF), the most common genetic disorder in the Caucasian population. It is characterized by abnormal transport of chloride and sodium ions across epithelia, leading to the formation of thick, sticky secretions that particularly affect the lungs, as well as the pancreas, liver, and intestines. mRNA encoding a codon-optimized hCFTR coding sequence is being developed as a new therapeutic agent for the treatment of cystic fibrosis.

[000245] Codon optimization was performed for the native amino acid sequence of hCFTR according to the method of the present invention, as shown in Example 1. Three sequences, designated hCFTR #1 (SEQ ID NO:26), hCFTR #2 (SEQ ID NO:27), and hCFTR #3 (SEQ ID NO:28), were selected for further analysis. A nucleotide sequence with the codon-optimized hCFTR encoding sequence using a different algorithm (SEQ ID NO:25) was provided as a reference. This reference nucleotide sequence (SEQ ID NO:25) had previously been experimentally validated both in vitro and in vivo. The reference nucleotide sequence was found to provide superior protein yield compared to other previously tested codon-optimized nucleotide sequences encoding the hCFTR protein. Compared with the reference nucleotide sequence, the CAI and GC content in % were significantly increased in the codon-optimized hCFTR#2 and hCFTR#3 sequences. Furthermore, their codon frequency distribution (CFD) percentage was 0% compared to 6% for the reference nucleotide sequence, indicating that clusters of rare codons detrimental to translation efficiency were successfully removed. Additional filtering to remove negative regulatory motifs resulted in a significant reduction in the number of negative cis-regulatory (CIS) elements in hCFTR#2 and hCFTR#3 (see Table 2).

[000246] To test the protein yield for each codon-optimized sequence, four nucleic acid vectors were generated, each containing an expression cassette containing one of the four nucleotide sequences encoding the hCFTR protein, flanked by identical 3'- and 5'-untranslated sequences (3'- and 5'-UTRs) preceded by a promoter for RNA polymerase. These nucleic acid vectors served as templates for in vitro transcription reactions to produce four batches of mRNA containing the four codon-optimized nucleotide sequences (reference and hCFTR #1-#3). Copying and extension were performed separately.

[000247] Each of the replicated and tailed mRNAs was separately transfected into a cell line (HEK2 93). Cell lysates were collected 24 and 48 hours after transfection. Protein samples were extracted and processed for SDS-PAGE. Expression levels of the encoded hCFTR protein were assessed by Western blotting. Protein bands were detected and quantified using the LI-COR system. Protein yield was expressed as relative fluorescence units (RFU). The results of this experiment are summarized in Figure 13. The codon-optimized hCFTR#2 and hCFTR#3 nucleotide sequences, both of which had a CAI value of 0.89, produced significantly higher yields of encoded hCFTR protein compared to the reference nucleotide sequence and hCFTR#1, both of which had a CAI value of 0.7. This effect was more pronounced after 24 hours (see Figure 13B), presumably due to the relatively rapid degradation of mRNA in HEK2 93 cells after transfection.

[000248] The data in this example demonstrate that codon optimization of a therapeutically relevant nucleotide sequence (hCFTR) to obtain a CAI of approximately 0.8 or greater results in higher protein yield, particularly in combination with optimization of its CFD and its GC content, as well as removal of any negative cis-elements from the nucleic acid sequence. The data in this example also confirm that codon optimization of hCFTR mRNA according to the methods of the present invention results in a very high yield of hCFTR protein in human cells compared to nucleotide sequences codon-optimized using a different algorithm.

Example 4. Optimization of the CFTR nucleotide sequence codons leads to increased functional activity in cells.

[000249] This example demonstrates that codon optimization of the hCFTR nucleotide sequence in accordance with the method of the present invention does not adversely affect the functional activity of hCFTR in human cells.

[000250] Administration of hCFTR mRNA should result in its uptake by airway epithelial cells in patients with CF, followed by internalization into the cytoplasm of target cells. Once cellular uptake occurs, hCFTR mRNA is translated into normal hCFTR protein, which is then processed in the endogenous secretory pathway of the cell, resulting in localization of the hCFTR protein to the apical surface of the cell membrane. Using this approach, administration of hCFTR mRNA produces functional hCFTR protein in the airway epithelium, thereby correcting the deficiency of functional CFTR in the lungs of patients with CF. Codon optimization of the hCFTR mRNA nucleotide sequence can increase the expression of functional hCFTR protein, which is believed to lead to increased amounts of functional hCFTR protein in airway epithelial target cells in patients with CF.

[000251] It has been reported that codon optimization can occur at the expense of a decrease in the functional activity of the encoded protein and an associated loss of efficiency, since the process can result in the removal of information encoded in the nucleotide sequence that is important for controlling protein translation and ensuring the correct folding of the synthesized polypeptide chain (Mauro & Chappell, Trends Mol Med. 2014; 20(11):604-13). To test the functional activity of the hCFTR protein expressed from the codon-optimized sequences generated using the codon optimization method shown in Example 1, the hCFTR mRNAs obtained in Example 2 were tested in a Ussing chamber assay. This assay uses epithelial voltage clamp to assess the functional activity of the protein expressed from hCFTR mRNA by monitoring the chloride transport function of epithelial cells that have been transfected with the mRNA. Specifically, the functional activity of hCFTR protein expressed from mRNA with the hCFTR control coding sequence (SEQ ID NO: 25) or the coding sequence of hCFTR #1 (SEQ ID NO: 26), hCFTR #2 (SEQ ID NO: 27), or hCFTR #3 (SEQ ID NO: 28) was measured in Fischer rat thyroid epithelial cells (FRT). FRT epithelial cells are commonly used as a model for studying human airway epithelial cell function. FRT epithelial cells were grown in monolayers on Snapwell™ filter inserts and transfected with 4 hCFTR mRNA. 4 hCFTR mRNA was prepared as described in Example 2. The control mRNA was previously validated in this assay and used as a reference standard.

[000252] Properly translated and localized hCFTR protein, produced from hCFTR mRNA, increases the short-circuit current (Isc) output in the Ussing apparatus for epithelial voltage clamp when treated with CFTR agonists (forskolin and VX-770 [Kalydeco]). Treatment with the CFTR antagonist, CFTRinh-172, locks hCFTR into a locked state. The Isc current polarity measurement method in this assay records the apical-basolateral sodium current and the basolateral-apical chloride current as negative values, and therefore, if transfection of the test hCFTR mRNA yields a high negative value, it can be concluded that the encoded hCFTR protein is functional (Figure 14A). Furthermore, by transfecting equal amounts of mRNA, it is possible to assess whether mRNA yields a higher hCFTR protein yield, since protein yield and activity correlate. Transfection of FRT epithelial cells with mRNA containing the hCFTR #1 coding sequence resulted in activity comparable to that achieved by transfection with mRNA containing the control hCFTR coding sequence (Figure 14B). mRNAs encoding the nucleotide sequence encoding hCFTR, generated by the method of the present invention, resulted in a significant increase in activity. In accordance with the higher protein yields observed in Example 2, the hCFTR protein obtained from mRNA encoding hCFTR #2 resulted in a greater than 2-fold increase in activity compared to the control mRNA, and the hCFTR protein obtained from mRNA encoding hCFTR #3 resulted in a 3-fold increase in activity compared to the control mRNA. This confirms that the higher protein yield obtained from hCFTR #2 and hCFTR #3 observed in Example 2 directly correlates with higher functional activity, demonstrating that codon optimization according to the method of the present invention does not negatively impact the functional activity of the encoded proteins.

[000253] Thus, codon optimization according to the method of the present invention results in higher expression of the encoded protein in human cells, and the expressed protein provides full functional activity in a model system that is a well-established model for human therapy.

Example 5. Codon optimization of DNAI1 mRNA sequence to increase CAI results in higher protein expression.

[000254] The data in this example demonstrate that codon optimization of an additional therapeutically significant nucleotide sequence (DNAI1) to obtain a CAI of approximately 0.8 or greater results in higher protein yield in cells, particularly in combination with optimization of its CFD and its GC content, as well as removal of any negative cis-elements from the nucleic acid sequence. The data in this example also confirm that CAI values positively correlate with protein expression yield for codon-optimized mRNAs generated according to the methods of the present invention.

Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder characterized by the presence of abnormal cilia and flagella found in the lining of the respiratory tract, reproductive system, and other organs and tissues. Symptoms are present at birth due to breathing problems, and affected individuals develop frequent respiratory tract infections beginning in early childhood. People with PCD also experience year-round nasal congestion and a chronic cough. Chronic respiratory tract infections can lead to a condition called bronchiectasis, which damages the airways called bronchi and can cause life-threatening breathing problems. Some individuals with PCD also experience infertility, recurrent ear infections, and abnormal organ placement in the chest and abdomen. Among several genes confirmed to be directly involved in the pathogenesis of PCD, a significant number of mutations have been found in two genes: DNAI1 and DNAH5, encoding the intermediate and heavy chains of axonemal dynein, respectively.

[000256] mRNA encoding the codon-optimized DNAI1 coding sequence is being developed as a new therapeutic agent for the treatment of PCD.

[000257] Codon optimization was performed using the native amino acid sequence of DNAI1 according to the methods of the present invention, as shown in Example 1, to generate three sequences designated DNAI1#1 (SEQ ID NO: 29), DNAI1#2 (SEQ ID NO: 30), DNAI1#3 (SEQ ID NO: 31). A codon-optimized DNAI1 sequence, DNAI1#4 (SEQ ID NO: 32), was also included as a reference. DNAI1#4 was codon-optimized, but was not further processed by applying filtering based on motif selection, filtering based on guanine-cytosine (GC) content analysis, and filtering based on codon adaptation index (CAI) analysis. The resulting codon-optimized nucleotide sequences generated according to the methods of the present invention had CAI values of 0.8 or

[000258] To test the protein yield for each codon-optimized sequence, four nucleic acid vectors were generated, each containing an expression cassette containing one of the four DNAI1 protein-encoding nucleotide sequences flanked by identical 5' and 3' UTRs preceded by a promoter for RNA polymerase. These nucleic acid vectors served as templates for in vitro transcription reactions to produce four batches of mRNA containing the four codon-optimized nucleotide sequences (DNAI1 #1-#4). Copying and extension were performed separately.

[000259] 2 μg of each replicated and tailed mRNA were used to transfect 105 transfected HEK2 93T cells. Untransfected HEK2 93T cells were also included to provide a negative control. Cell lysates were collected 24 hours after transfection, and protein samples were extracted and processed for SDS-PAGE. Two samples from each batch of cells were processed and analyzed. Expression levels of the encoded DNAI1 protein were assessed by Western blotting using the primary antibody to DNAI1 (αDNAI1). Vinculin expression levels were also measured using the primary antibody to vinculin (aVinculin) to provide a loading control. Signals were developed and quantified using the LI-COR imaging system, and DNAI1 protein yields normalized to vinculin were plotted in Figure 15B as a fold increase relative to the reference level achieved with mRNA encoding the DNAL1 sequence that was not codon-optimized. The results of this experiment are summarized in Figure 15. The codon-optimized DNAI1 sequence #1, which had the highest CAI (0.90), produced the highest DNAI1 protein level compared to the reference (DNAI1 #4). The codon-optimized DNAI1 #2 and DNAI1 #3 sequences had a CAI of 0.87 and produced comparable DNAI1 protein levels despite the differences in nucleotide sequence, indicating that CAI is closely related to protein expression yield. The codon-optimized DNAI1 #4 sequence with a CAI of 0.83 produced the lowest amount of protein compared to the optimized nucleotide sequences with a higher CAI, but still significantly higher than the reference level.

[000260] Taken together, these data show that for mRNAs containing the codon-optimized nucleotide sequence of the present invention, a higher CAI is a strong indicator of protein expression yield, and also show that different codon-optimized nucleotide sequences with the same CAI values yield similar levels of the encoded protein in cells.

Numbered embodiments of the invention

1. A computerized method for generating an optimized nucleotide sequence, comprising: (i) obtaining an amino acid sequence, wherein the amino acid sequence encodes a peptide, polypeptide, or protein; (ii) obtaining a first table of codon usage frequencies, wherein the first table of codon usage frequencies comprises a list of amino acids, wherein each amino acid in the table is associated with at least one codon and each codon is associated with a usage frequency; (iii) removing from the table of codon usage frequencies any codons associated with a usage frequency that is less than a threshold frequency; (iv) creating a normalized table of codon usage frequencies by normalizing the frequencies of codons not removed in step (iii); and (v) creating an optimized nucleotide sequence encoding the amino acid sequence by selecting a codon for each amino acid in the amino acid sequence based on the usage frequency of one or more codons associated with the amino acid in the normalized table of codon usage frequencies.

2. The method according to embodiment 1, wherein the normalization comprises:

(a) distributing the usage frequencies of each codon associated with the first amino acid and removed in step (iii) to the remaining codons associated with the first amino acid; and (b) repeating step (a) for each amino acid to obtain a normalized codon usage frequency table.

3. The method according to embodiment 2, wherein the frequency of usage of the deleted codons is uniformly distributed among the remaining codons.

4. The method according to embodiment 2, wherein the frequency of usage of the deleted codons is distributed among the remaining codons in proportion to the frequency of usage of each remaining codon.

5. The method according to any of the previous embodiments, wherein selecting a codon for each amino acid comprises:

(a) identifying in a normalized codon usage table one or more codons associated with the first amino acid of the amino acid sequence; (b) selecting a codon associated with the first amino acid, where the probability of selecting a particular codon is equal to the usage frequency associated with the codon associated with the first amino acid in the normalized codon usage table; and

(c) repeating steps (a) and (b) until a codon for each amino acid in the amino acid sequence has been selected.

6. The method according to any of the previous embodiments, wherein step (v) is performed multiple times to generate a list of optimized nucleotide sequences.

7. The method according to any of the previous embodiments, wherein the threshold frequency is selected by the user.

8. The method according to any of the previous embodiments, wherein the threshold frequency is in the range from 5% to 30%, in particular 5%, 10% or 15%, or 20%, or 25%, or 30%, or in particular 10%.

9. The method according to any one of embodiments 6-8, further comprising:

determining whether each optimized nucleotide sequence in the list or the most recently updated list of optimized nucleotides contains

termination signal sequences; and

updating the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or the most recently updated list if the nucleotide sequence contains one or more termination signals.

10. The method of embodiment 9, wherein the one or more termination signals have the following nucleotide sequence:

5'-X ₁ ATCTX ₂ TX ₃ -3', where X ₁ , X ₂ and X ₃ are independently selected from A, C, T or G.

11. The method of embodiment 10, wherein the one or more termination signals have one or more of the following nucleotide sequences:

TATCTGTT; and/or

TTTTTTT; and/or

AAGCTT; and/or

GAAGAGC and/or

TCTAGA.

12. The method of embodiment 9, wherein the one or more termination signals have the following nucleotide sequence:

5'-X ₁ AUCTX ₂ UX ₃ -3', where X ₁ , X ₂ and X ₃ are independently selected from A, C, U or G.

13. The method of embodiment 12, wherein the one or more termination signals have one of the following nucleotide sequences:

UAUCUGUU; and/or

UUUUUU; and/or

AAGCUU; and/or

GAAGAGC and/or

UCUAGA.

14. The method according to any one of embodiments 6-13, further comprising:

determining the guanine-cytosine content of each of the optimized nucleotide sequences in the list or in the most recently updated list of optimized nucleotide sequences, where the guanine-cytosine content of a sequence is the percentage of bases in the nucleotide sequence that are guanine or cytosine;

updating the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or the most recently updated list if the guanine-cytosine content is outside the specified guanine-cytosine content range.

15. The method of embodiment 14, wherein determining the guanine-cytosine content in each of the optimized nucleotide sequences for each nucleotide sequence comprises:

determination of the guanine-cytosine content in the first part of the nucleotide sequence, and where the update of the list of optimized nucleotide sequences includes:

deletion of a nucleotide sequence if the guanine-cytosine content in the first part is outside the specified guanine-cytosine content range.

16. The method according to embodiment 15, wherein determining the guanine-cytosine content in each of the optimized nucleotide sequences further for each nucleotide sequence comprises:

determining the guanine-cytosine content of one or more additional portions of the nucleotide sequence, where the additional portions do not overlap with each other and with the first portion, and where the update of the list of optimized sequences includes:

deleting the nucleotide sequence if the guanine-cytosine content in any portion is outside a predetermined guanine-cytosine content range, optionally wherein the determination of the guanine-cytosine content in the nucleotide sequence is stopped when it is determined that the guanine-cytosine content in any portion is outside a predetermined guanine-cytosine content range.

17. The method according to embodiment 15 or 16, wherein the first portion and/or one or more additional portions of the nucleotide sequence comprise a predetermined number of nucleotides, wherein the optionally predetermined number of nucleotides is in the range of 5 to 300 nucleotides, or 10 to 200 nucleotides, or 15 to 100 nucleotides, or 20 to 50 nucleotides, such as 30 nucleotides.

18. The method of embodiment 17, wherein the predetermined range of guanine-cytosine content is selected by the user.

19. The method according to embodiment 17 or 18, wherein the predetermined range of guanine-cytosine content is from 15% to 75%, or from 40% to 60%, or, in particular, from 30% to 70%.

20. The method according to any one of embodiments 6-19, further comprising:

determining the codon adaptation index of each of the optimized nucleotide sequences in the list or the most recently updated list of optimized nucleotide sequences, where the codon adaptation index of a sequence is a measure of the preference for the frequency of codon usage and can take a value from 0 to 1;

updating the list or the most recently updated list of optimized nucleotide sequences by deleting any nucleotide sequence if its codon adaptation index is less than or equal to a predetermined codon adaptation index threshold.

21. The method of embodiment 20, wherein the codon adaptation index threshold is selected by the user.

22. The method according to embodiment 20 or 21, wherein the codon adaptation index threshold is 0.7, or 0.75, or 0.85, or 0.9, or, in particular, 0.8.

23. The method according to any of the previous embodiments, wherein the amino acid sequence is obtained from an amino acid sequence database.

24. The method of embodiment 23, further comprising querying the amino acid sequence from a database of amino acid sequences, wherein the amino acid sequence is obtained in response to the query.

25. The method according to any of the previous embodiments, wherein the first table of codon usage frequencies is obtained from a database of codon usage frequency tables.

26. The method of embodiment 24, further comprising requesting the first table of codon usage frequencies from a database of codon usage frequency tables, wherein the first table of codon usage frequencies is obtained in response to the request.

27. The method according to any of the previous embodiments, further comprising displaying at least one optimized nucleotide sequence on a screen.

28. A computer program comprising instructions that, when executed by a computer, cause the computer to perform the method of any of the preceding embodiments.

29. A data processing system comprising means for performing the method according to any of the previous embodiments.

30. A machine-readable storage medium on which the computer program according to embodiment 28 is stored.

31. A data carrier signal carrying a computer program according to embodiment 28.

32. A method for synthesizing a nucleotide sequence, comprising:

performing the computerized method of any one of embodiments 1-27 to generate at least one optimized nucleotide sequence; and

synthesis of at least one of the created optimized nucleotide sequences.

33. The method of embodiment 32, wherein the method further comprises inserting the synthesized optimized sequence into a nucleic acid vector for use in in vitro transcription.

34. The method according to embodiment 32 or 33, wherein the method further comprises inserting one or more termination signals at the 3' end of the synthesized optimized nucleotide sequence.

35. The method of embodiment 34, wherein the one or more termination signals are encoded by the following nucleotide sequence: 5'-X ₁ ATCTX ₂ TX ₃ -3', where X ₁ , X ₂ and X ₃ are independently selected from A, C, T or G.

36. The method of embodiment 34 or 35, wherein the one or more termination signals are encoded by one or more of the following nucleotide sequences:

TATCTGTT;

TTTTTT;

AAGCTT;

GAAGAGC and/or

TCTAGA.

37. The method according to any one of embodiments 34-36, wherein more than one termination signal is inserted, and said termination signals are separated by 10 base pairs or less, such as separated by 5-10 base pairs.

38. The method of embodiment 36, wherein more than one termination signal is encoded by the following nucleotide sequence: (a) 5'-X ₁ ATCTX ₂ TX ₃ -(Z _N )-X ₄ ATCTX ₅ TX ₆ -3' or (b) 5'-X ₁ ATCTX ₂ TX ₃ -(Z N)-X ₄ ATCTX ₅ TX ₆ -(Z _M )-X ₇ ATCTX ₈ TX ₉ -3', where X ₁ , X ₂ , X ₃ , X ₄ , X ₅ ; X ₆ , X ₇ , X ₈ and X ₉ are independently selected from A, C, T or G, Z _N is a spacer sequence of N nucleotides, and Z _M is a spacer sequence of M nucleotides, each of which is independently selected from A, C, T or G, and where N and/or M are independently 10 or less.

39. The method according to any one of embodiments 33-38, wherein the nucleic acid vector comprises a promoter for RNA polymerase operably linked to an optimized nucleotide sequence, wherein optionally the promoter for RNA polymerase is a promoter for SP6 RNA polymerase or a promoter for T7 RNA polymerase.

40. The method according to any one of embodiments 33-39, wherein the nucleic acid vector is a plasmid.

41. The method of embodiment 40, wherein the plasmid is linearized prior to in vitro transcription.

42. The method of embodiment 40, wherein the plasmid is not linearized prior to in vitro transcription.

43. The method of embodiment 42, wherein the plasmid is supercoiled.

44. The method according to any one of embodiments 32-43, wherein the method further comprises using at least one of the synthesized optimized nucleotide sequences in in vitro transcription to synthesize mRNA.

45. The method according to embodiment 44, wherein the mRNA is synthesized using RNA polymerase SP6.

46. The method of embodiment 45, wherein the SP6 RNA polymerase is a naturally occurring SP6 RNA polymerase.

47. The method of embodiment 45, wherein the SP6 RNA polymerase is recombinant SP6 RNA polymerase.

48. The method of embodiment 47, wherein the SP6 RNA polymerase comprises a label.

49. The method of embodiment 48, wherein the tag is a histidine tag.

50. The method of embodiment 44, wherein the mRNA is synthesized using T7 RNA polymerase.

51. The method according to any one of embodiments 44-50, wherein the method further comprises a separate step of copying and/or extending the synthesized mRNA.

52. The method according to any one of embodiments 44-50, wherein the copying and extension occurs during in vitro transcription.

53. The method according to any one of embodiments 44-52, wherein mRNA is synthesized in a reaction mixture containing NTPs in a concentration range of 1-10 mM each NTP, DNA template in a concentration range of 0.01-0.5 mg/ml, and SP6 RNA polymerase in a concentration range of 0.01-0.1 mg/ml.

54. The method of embodiment 53, wherein the reaction mixture comprises NTPs at a concentration of 5 mM of each NTP, DNA template at a concentration of 0.1 mg/ml, and RNA polymerase SP6 at a concentration of 0.05 mg/ml.

55. The method according to any one of embodiments 4 4-54, wherein the mRNA is synthesized at a temperature in the range of 37-56°C.

56. The method according to any one of embodiments 53-55, wherein the NTPs are naturally occurring NTPs.

57. The method of any one of embodiments 53-55, wherein the NTPs include modified NTPs.

58. The method according to any one of embodiments 32-57, wherein the method further comprises transfecting the synthesized optimized nucleotide sequence into a cell either in vitro or in vivo.

59. The method according to embodiment 58, wherein the expression level of the protein encoded by the synthesized optimized nucleotide sequence is determined in the transfected cell.

60. The method according to embodiment 58 or 59, wherein the functional activity of the protein encoded by the synthesized optimized nucleotide sequence is determined.

61. The method of any one of embodiments 1-27, further comprising synthesizing a reference nucleotide sequence encoding an amino acid sequence and at least one optimized nucleotide sequence according to the method of any one of embodiments 32-60, and contacting the reference nucleotide sequence and the at least one optimized nucleotide sequence with a single cell or organism, wherein the cell or organism contacted with the at least one synthesized optimized nucleotide sequence produces an increased yield of a protein encoded by the optimized nucleotide sequence, compared to the yield of a protein encoded by the reference nucleotide sequence produced by the cell or organism contacted with the synthesized reference nucleotide sequence.

62. The method of any one of embodiments 32-60, wherein the method further comprises providing a therapeutic composition comprising mRNA that encodes a therapeutic peptide, polypeptide, or protein for use in delivering to a subject or in treating a subject.

63. The method of embodiment 62, wherein the mRNA encodes a protein that is a cystic fibrosis transmembrane conductance regulator (CFTR).

64. The method according to any one of embodiments 1-27, wherein the at least one optimized nucleotide sequence, when synthesized, is configured to increase the expression of the protein encoded by the at least one optimized nucleotide sequence, compared to the expression of the protein encoded by the reference nucleotide sequence, when synthesized.

65. The method of any one of embodiments 61-64, wherein the reference nucleotide sequence is (a) a naturally occurring nucleotide sequence encoding an amino acid sequence; or (b) a nucleotide sequence encoding an amino acid sequence generated by a method other than the method of any one of embodiments 1-27.

66. A synthesized optimized nucleotide sequence created according to the methods of any one of embodiments 32-57 and 62-65, for use in therapy.

67. A method of treatment comprising administering a synthesized optimized nucleotide sequence created in accordance with the method of any one of embodiments 32-57 and 62-65 to a human subject in need of such treatment.

68. An in vitro synthesized nucleic acid comprising an optimized nucleotide sequence consisting of codons associated with a usage frequency that is 10% or greater; wherein the optimized nucleotide sequence:

(i) does not contain a termination signal having one of the following nucleotide sequences:

5'-X ₁ AUCUX ₂ UX ₃ -3', where X ₁ , X ₂ and X ₃ are independently selected from A, C, U or G; and 5'-X ₁ AUCUX ₂ UX ₃ -3', where X ₁ , X ₂ and X ₃ are independently selected from A, C, U or G;

(ii) does not contain any negative cis-regulatory elements and negative repeat elements; and

(iii) has a codon adaptation index greater than 0.8;

where, when divided into non-overlapping parts of 30 nucleotides in length, each part of the optimized nucleotide sequence is characterized by a range of guanine-cytosine content from 30% to 70%.

69. The in vitro synthesized nucleic acid of embodiment 67, wherein the optimized nucleotide sequence does not comprise a termination signal having one of the following sequences: TATCTGTT; TTTTTT; AAGCTT; GAAGAGC; TCTAGA; UAUCUGUU; UUUUUU; AAGCUU; GAAGAGC; UCUAGA.

70. The in vitro synthesized nucleic acid of embodiment 68 or 69, wherein the nucleic acid is mRNA.

71. An in vitro synthesized nucleic acid according to any one of embodiments 68-70 for use in therapy.

Claims (97)

1. A computerized method for creating an optimized nucleotide sequence, comprising: (i) obtaining an amino acid sequence, wherein the amino acid sequence encodes a peptide, polypeptide or protein; (ii) obtaining a first table of codon usage frequencies, wherein the first table of codon usage frequencies comprises a list of amino acids, wherein each amino acid in the table is associated with at least one codon and each codon is associated with a usage frequency; (iii) removing from the codon usage frequency table any codons associated with a usage frequency that is less than the threshold frequency; (iv) creating a normalized codon usage frequency table by normalizing the codon usage frequencies not removed in step (iii); and (v) creating an optimized nucleotide sequence encoding an amino acid sequence by selecting a codon for each amino acid in the amino acid sequence based on the frequency of usage of one or more codons associated with the amino acid in a normalized codon frequency table. 2. The method according to paragraph 1, wherein the normalization includes: (a) distributing the frequency of usage of each codon associated with the first amino acid and removed in step (iii) to the remaining codons associated with the first amino acid; and (b) repeating step (a) for each amino acid to obtain a normalized codon usage frequency table. 3. The method according to claim 2, wherein the frequency of use of the deleted codons is uniformly distributed among the remaining codons. 4. The method according to claim 2, wherein the frequency of use of the deleted codons is distributed among the remaining codons in proportion to the frequency of use of each remaining codon. 5. The method according to any of the preceding claims, wherein the selection of a codon for each amino acid comprises: (a) identifying in a normalized codon usage table one or more codons associated with the first amino acid in the sequence; (b) selecting the codon associated with the first amino acid, where the probability of selecting a particular codon is equal to the usage frequency associated with the codon associated with the first amino acid in a normalized table of codon usage frequencies; and (c) repeating steps (a) and (b) until a codon for each amino acid in the amino acid sequence has been selected. 6. The method according to any of the preceding claims, wherein step (v) is performed several times to generate a list of optimized nucleotide sequences. 7. A method according to any of the previous points, where the threshold frequency is selected by the user. 8. The method according to any of the preceding claims, wherein the threshold frequency is in the range from 5% to 30%, in particular 5%, 10% or 15%, or 20%, or 25%, or 30%, or in particular 10%. 9. The method according to any one of paragraphs 6-8, further comprising: screening the list of optimized nucleotide sequences to identify and remove optimized nucleotide sequences that do not meet one or more criteria. 10. The method of claim 9, wherein screening the list of optimized nucleotide sequences for each of one or more criteria comprises: determining whether each optimized nucleotide sequence in the list or the most recently updated list of optimized nucleotide sequences meets the criterion; and updating the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or the most recently updated list if the nucleotide sequence does not meet the criterion. 11. The method of claim 10, wherein determining whether each optimized nucleotide sequence in the list or the most recently updated list of optimized nucleotide sequences meets the criterion, for each nucleotide sequence, comprises: determining whether the first part of the nucleotide sequence meets the criterion, and where the update of the list of optimized nucleotide sequences includes: deletion of a nucleotide sequence if the first part does not meet the criterion. 12. The method according to claim 11, wherein determining whether each optimized nucleotide sequence in the list or the most recently updated list of optimized nucleotide sequences meets the criterion, for each nucleotide sequence, further comprises: determining whether one or more additional portions of the nucleotide sequence meet the criterion, where the additional portions do not overlap with each other or with the first portion, and where the update of the list of optimized sequences includes: deleting the nucleotide sequence if any portion does not meet the criterion, optionally, where the determination of whether the optimized nucleotide sequence meets the criterion is stopped when it is determined that any portion does not meet the criterion. 13. The method according to claim 11 or 12, wherein the first part and/or one or more additional parts of the nucleotide sequence comprise a predetermined number of nucleotides, wherein the optionally predetermined number of nucleotides is in the range from 5 to 300 nucleotides, or from 10 to 200 nucleotides, or from 15 to 100 nucleotides, or from 20 to 50 nucleotides, for example 30 nucleotides, for example 100 nucleotides. 14. The method according to any one of paragraphs 9-13, wherein the first criterion provides for a nucleotide sequence that does not contain a termination signal, so that the determination and updating include: determining whether each optimized nucleotide sequence in the list or the most recently updated list of optimized nucleotide sequences contains a termination signal; and updating the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or the most recently updated list if the nucleotide sequence contains one or more termination signals. 15. The method according to claim 14, wherein one or more termination signals have the following nucleotide sequence: 5'-X1ATCTX2TX3-3', where X1, X2 and X3 are independently selected from A, C, T or G. 16. The method according to claim 15, wherein one or more termination signals have one or more of the following nucleotide sequences: TATCTGTT; and/or TTTTTT; and/or AAGCTT; and/or GAAGAGC; and/or TCTAGA. 17. The method according to claim 14, wherein one or more termination signals have the following nucleotide sequence: 5'-X1AUCUX2UX3-3', where X1, X2 and X3 are independently selected from A, C, U or G. 18. The method according to claim 17, wherein one or more termination signals have one of the following nucleotide sequences: UAUCUGUU; and/or UUUUUU; and/or AAGCUU; and/or GAAGAGC; and/or UCUAGA. 19. The method according to any one of claims 9 to 18, wherein the second criterion provides a nucleotide sequence characterized by a guanine-cytosine content within a predetermined range of guanine-cytosine content, such that the determination and updating include: determining the guanine-cytosine content of each of the optimized nucleotide sequences in the list or in the most recently updated list of optimized nucleotide sequences, where the guanine-cytosine content of a sequence is the percentage of bases in the nucleotide sequence that are guanine or cytosine; updating the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or the most recently updated list if the guanine-cytosine content is outside the predetermined guanine-cytosine content range. 20. The method according to claim 19, wherein the predetermined range of guanine-cytosine content is selected by the user. 21. The method according to claim 19 or 20, wherein the predetermined range of guanine-cytosine content is from 15% to 75%, or from 40% to 60%, or, in particular, from 30% to 70%. 22. The method according to any one of paragraphs 9-21, wherein the third criterion provides a nucleotide sequence having a codon adaptation index greater than a predetermined codon adaptation index threshold value, such that the determination and updating include: determining the codon adaptation index of each of the optimized nucleotide sequences in the list or the most recently updated list of optimized nucleotide sequences, where the codon adaptation index of a sequence is a measure of the preference for the frequency of codon usage and can take a value from 0 to 1; updating the list or the most recently updated list of optimized nucleotide sequences by deleting any nucleotide sequence if its codon adaptation index is less than or equal to a predetermined codon adaptation index threshold. 23. The method according to claim 22, wherein the threshold value of the codon adaptation index is selected by the user. 24. The method according to claim 22 or 23, wherein the codon adaptation index threshold value is 0.7, or 0.75, or 0.85, or 0.9, or, in particular, 0.8. 25. The method according to any one of claims 9 to 24, wherein the fourth criterion provides for a nucleotide sequence that does not contain at least 2, such as 3, contiguous identical codons, so that determining and updating comprises: determining whether any optimized nucleotide sequence in the list or the most recently updated list of optimized nucleotide sequences contains at least 2, such as 3 or more, contiguous identical codons; and updating the list or the most recently updated list of optimized nucleotide sequences by deleting any nucleotide sequence if it contains at least 2, such as 3 or more, contiguous identical codons. 26. The method according to claim 25, wherein the fourth criterion is applied only to codons whose frequency in the normalized table of codon usage frequencies is less than the threshold value of contiguity rarity, where the threshold value of contiguity rarity is in the range of 10-50%, for example 15-40%, for example 20-30%. 27. The method according to any of the preceding claims, wherein the amino acid sequence is obtained from an amino acid sequence database. 28. The method of claim 26, further comprising requesting an amino acid sequence from a database of amino acid sequences, wherein the amino acid sequence is obtained in response to the request. 29. The method according to any one of the preceding claims, wherein the first table of codon usage frequencies is obtained from a database of codon usage frequency tables. 30. The method of claim 29, further comprising requesting a first table of codon usage frequencies from a database of codon usage frequency tables, wherein the first table of codon usage frequencies is obtained in response to the request. 31. The method according to any of the preceding claims, further comprising displaying at least one optimized nucleotide sequence on a screen. 32. A method for synthesizing a nucleic acid having a nucleotide sequence, comprising: performing the computerized method of any one of claims 1-31 to create at least one optimized nucleotide sequence; and synthesis of at least one nucleic acid containing one of the created optimized nucleotide sequences. 33. The method of claim 32, wherein the method further comprises inserting the synthesized optimized nucleic acid into a nucleic acid vector for use in in vitro transcription. 34. The method according to claim 32 or 33, wherein the method further comprises inserting one or more termination signals at the 3'-end of the synthesized optimized nucleotide sequence. 35. The method according to claim 34, wherein one or more termination signals are encoded by the following nucleotide sequence: 5'-X1ATCTX2TX3-3', where X1, X2 and X3 are independently selected from A, C, T or G. 36. The method according to claim 34 or 35, wherein one or more termination signals are encoded by one or more of the following nucleotide sequences: TATCTGTT; TTTTTT; AAGCTT; GAAGAGC; and/or TCTAGA. 37. The method according to any one of paragraphs 34-36, wherein more than one termination signal is inserted, and said termination signals are separated by 10 base pairs or less, for example separated by 5-10 base pairs. 38. The method according to claim 37, wherein more than one termination signal is encoded by the following nucleotide sequence: (a) 5'-X1ATCTX2TX3-(ZN)-X4ATCTX5TX6-3' or (b) 5'-X1ATCTX2TX3-(ZN)-X4ATCTX5TX6-(ZM)-X7ATCTX8TX9-3', where X1, X2, X3, X4, X5, X6, X7, X8 and X9 are independently selected from A, C, T or G, ZN is a spacer sequence of N nucleotides, and ZM is a spacer sequence of M nucleotides, each of which is independently selected from A, C, T or G, and where N and/or M are independently 10 or less. 39. The method according to any one of claims 33-38, wherein the nucleic acid vector comprises a promoter for RNA polymerase operably linked to an optimized nucleotide sequence, wherein optionally the promoter for RNA polymerase is a promoter for SP6 RNA polymerase or a promoter for T7 RNA polymerase. 40. The method according to any one of claims 33-39, wherein the nucleic acid vector comprises a nucleotide sequence encoding a 5'-UTR operably linked to an optimized nucleotide sequence. 41. The method of claim 40, wherein the 5'-UTR is different from the 5'-UTR of a naturally occurring mRNA encoding the amino acid sequence. 42. The method according to claim 41, wherein the 5’-UTR has the nucleotide sequence of SEQ ID NO: 16. 43. The method according to any one of claims 33-42, wherein the nucleic acid vector comprises a nucleotide sequence encoding a 3'-UTR operably linked to an optimized nucleotide sequence. 44. The method of claim 43, wherein the 3'-UTR is different from the 3'-UTR of a naturally occurring mRNA encoding the amino acid sequence. 45. The method according to claim 44, wherein the 3’-UTR has the nucleotide sequence of SEQ ID NO: 17 or SEQ ID NO: 18. 46. The method according to any one of claims 33-45, wherein the nucleic acid vector is a plasmid. 47. The method according to claim 46, wherein the plasmid is linearized prior to in vitro transcription. 48. The method of claim 47, wherein the plasmid is not linearized prior to in vitro transcription. 49. The method of claim 48, wherein the plasmid is supercoiled. 50. The method according to any one of claims 32-49, wherein the method further comprises using at least one of the synthesized optimized nucleic acids in in vitro transcription to synthesize mRNA. 51. The method according to any one of claims 32-50, wherein the method further comprises transfecting the synthesized optimized nucleic acid into a cell either in vitro or in vivo. 52. The method according to claim 51, wherein the expression level of the protein encoded by the synthesized optimized nucleic acid is determined in the transfected cell. 53. The method according to claim 51 or 52, wherein the functional activity of the protein encoded by the synthesized optimized nucleic acid is determined. 54. The method of any one of paragraphs 32-53, wherein the method further comprises providing a therapeutic composition comprising mRNA that encodes a therapeutic peptide, polypeptide or protein for use in delivering to a subject or in treating a subject.