Generation of optimized nucleotide sequences

Abstract

This provides a method for generating optimized nucleotide sequences. [Solution] A method for generating an optimized nucleotide sequence, comprising at least normalization of a codon usage table and selection of codons for a given amino acid sequence based on the usage frequency of codons in the normalized codon usage table, comprising the steps of: generating a list of multiple optimized nucleotide sequences encoding an amino acid sequence 105; filtering the list of optimized nucleotide sequences; synthesizing one or more optimized nucleotide sequences; and / or administering one or more synthesized optimized nucleotide sequences.

Description

[Technical Field]

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

[0002] Sequence List This specification refers 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 is 63.5 KB in size. The entire contents of the sequence listing are incorporated herein by reference.

[0003] Field of Invention The present invention relates to a method for generating optimized nucleotide sequences. In particular, the present invention relates to a method for optimizing nucleotide sequences for in vitro synthesis and cell expression of functional proteins, polypeptides, or peptides encoded by the optimized nucleotide sequences. [Background technology]

[0004] mRNA therapy is becoming increasingly important for treating a variety of diseases, particularly those caused by protein or gene dysfunction. Genetic mutations in an organism's DNA sequence can lead to abnormal gene expression, resulting in defects in protein production or function. For example, underlying DNA sequence mutations can lead to insufficient or excessive protein expression, or the production of dysfunctional proteins. Restoration of normal or healthy protein levels can be achieved through mRNA therapy, which is widely applicable to a range of diseases caused by gene or protein dysfunction.

[0005] In mRNA therapy, mRNA encoding a functional protein that can replace a defective or deficient protein is delivered to a target cell or tissue. Administration of mRNA encoding a therapeutic protein effective in treating or preventing a disease or disorder can also be a cost-effective alternative to therapies using recombinant peptides, polypeptides, or proteins. mRNA therapy can restore normal levels of endogenous proteins or provide exogenous therapeutic proteins without permanently altering the genomic sequence or entering the cell nucleus. mRNA therapy utilizes the cell's own protein production and processing mechanisms to treat diseases or disorders, is adaptable to personalized dosing and formulations, and is broadly applicable to any disease or condition caused by underlying gene or protein defects or treatable by the provision of exogenous proteins.

[0006] The expression level of mRNA-encoded proteins can significantly influence the efficacy and therapeutic benefits of mRNA therapy. Effective expression or production of proteins from mRNA within cells depends on a variety of factors. Optimizing the composition and order of codons within the protein-coding nucleotide sequence ("codon optimization") can lead to higher expression of mRNA-encoded proteins. While various methods for performing codon optimization are known in the art, each has considerable drawbacks and limitations from a computational and / or therapeutic standpoint. In particular, known codon optimization methods are often "optimal." This involves replacing every codon for each amino acid with the best codon available for that amino acid, so that the "modified" sequence contains only one codon that codes for each amino acid (hence sometimes being called a one-to-one sequence).

[0007] Therefore, there is a need for improved codon optimization methods to generate optimized nucleotide sequences for increased protein expression in mRNA therapy. [Overview of the project] [Problems that the invention aims to solve]

[0008] This invention addresses the need for improved nucleic acid optimization methods for effective mRNA therapy by providing a method for analyzing amino acid sequences to construct at least one optimized nucleotide sequence. The optimized nucleotide sequence is designed to increase protein expression compared to the expression of the protein associated with the naturally occurring nucleotide sequence. The nucleic acid optimization method of this invention provides the ability to increase the expression of a target protein in vitro, in settings where it is desirable to synthesize full-length mRNA transcripts and achieve higher protein yields. [Means for solving the problem]

[0009] For example, codon optimization is used to increase the expression of a target protein in mRNA therapy, immunology and vaccination, cancer immunotherapy, biotechnology, and manufacturing. Codon optimization yields a nucleotide sequence that codes for a protein based on various criteria, without altering the translated amino acid sequence of the encoded protein due to the redundancy of the gene code.

[0010] To avoid an imbalance between mRNA codon use and the abundance of congeneral tRNAs, codon optimization can result in a codon composition within the nucleotide sequence that closely matches the naturally occurring abundance of transfer RNA (tRNA) in the host cell, thus avoiding depletion of specific tRNAs. Since tRNA abundance affects the rate of protein translation, codon optimization of the nucleotide sequence can increase protein translation efficiency and yield the encoded protein. For example, a shortage of rare tRNAs can stall or halt protein translation; therefore, by avoiding the use of rare codons, characterized by low codon use, protein translation efficiency and protein yield can be increased. However, the codon optimization process may remove information within the nucleotide sequence that is important for encoding protein translation and ensuring proper folding of the nascent polypeptide chain, and may be done at the cost of reduced functional activity of the encoded protein, potentially leading to loss of efficacy (Mauro and Chappell, Trends Mol). Med., 2014;20(11):604~13). The inventors have found that optimized sequences that retain a certain degree of diversity, that is, do not necessarily contain only one codon encoding each amino acid, can achieve increased protein yields that surpass both naturally occurring sequences and one-to-one sequences.

[0011] In a first aspect, the present invention relates to a computer implementation method for generating an optimized nucleotide sequence, comprising: (i) receiving an amino acid sequence encoding a peptide, polypeptide, or protein; (ii) receiving a first codon usage table, the first codon usage table comprising a list of amino acids, each amino acid in the table being associated with at least one codon, and each codon being associated with a usage frequency; (iii) removing any codons associated with usage frequencies that are below a threshold frequency from the codon usage table; (iv) generating a normalized codon usage table by normalizing the usage frequencies of the codons not removed in step (iii); and (v) selecting a codon for each amino acid in the amino acid sequence based on the usage frequencies of one or more codons associated with the amino acid in the normalized codon usage table. The present invention relates to a method comprising the step of generating an optimized nucleotide sequence encoding an amino acid sequence by selecting a threshold frequency. In some embodiments, the threshold frequency is selectable by the user. In some embodiments, the threshold 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 threshold frequencies having the values ​​described herein can generate an optimized sequence that can achieve increased protein yield.

[0012] In some embodiments, the step of generating a normalized codon usage table includes (a) distributing the usage frequency of each codon associated with a 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 create a normalized codon usage table. In some embodiments, the usage frequencies of the removed codons are distributed equally among the remaining codons. In some embodiments, the usage frequencies of the removed codons are distributed proportionally among the remaining codons based on the usage frequency of each remaining codon.

[0013] In some embodiments, the step of selecting codons for each amino acid comprises: (a) identifying in a 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, 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 codon usage table; and (c) repeating steps (a) and (b) until a codon for each amino acid in the amino acid sequence is selected.

[0014] In some embodiments, the step of generating an optimized nucleotide sequence (step (v) in the above method) by selecting codons for each amino acid in the amino acid sequence is performed n times to generate a list of optimized nucleotide sequences.

[0015] 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, if they are less likely to be effective by not meeting one or more criteria, the method allows a significant number of candidate optimized nucleotide sequences to be excluded from consideration. In other words, since the criteria indicate the actual effectiveness of the optimized nucleotide sequence, nucleotide sequences that do not meet one or more criteria are excluded from further consideration. The one or more criteria may include sequences that do not contain one or more termination signals; sequences having a guanine-cytosine content within a predetermined range; sequences having a codon adaptation index exceeding a threshold value; sequences that do not contain one or more cis elements; sequences that do not contain one or more repetitive elements; and other purpose-specific criteria.

[0016] Thus, the method results in a list of optimized nucleotide sequences that are short or filtered. By reducing the number of optimized nucleotide sequences in the list, additional steps performed on the sequences in the list, such as further algorithmic steps or physical synthesis steps, are also advantageous as the number and complexity are reduced.

[0017] In some embodiments, the step of screening the list of optimized nucleotide sequences comprises determining, for a particular criterion, whether each optimized nucleotide sequence in the list of optimized nucleotide sequences or in the most recently updated list 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.

[0018] In some embodiments, the step of determining whether each optimized nucleotide sequence in the list of optimized nucleotide sequences or in the most recently updated list meets the criterion comprises, for each nucleotide sequence, determining whether a first portion of the nucleotide sequence meets the criterion, and the step of updating the list of optimized nucleotide sequences comprises removing the nucleotide sequence if the first portion does not meet the criterion. In some embodiments, the step of determining whether each optimized nucleotide sequence in the list of optimized nucleotide sequences or in the most recently updated list meets the criterion further comprises, for each nucleotide sequence, determining whether one or more additional portions of the nucleotide sequence that do not overlap with each other and do not overlap with the first portion meet the criterion, and the step of updating the list of optimized sequences comprises removing the nucleotide sequence if any portion does not meet the criterion, and optionally, the step of determining whether the optimized nucleotide sequence meets the criterion is stopped if any portion is determined not to meet the criterion.

[0019] In this way, by filtering the optimized nucleotide sequences, sequences are discarded from the list before computation, and time resources are spent analyzing the entire sequence, making the method computationally advantageous. Therefore, the method is advantageous because it becomes more efficient. Furthermore, for some criteria, part-by-part analysis results in a more detailed and selective screening process. Using guanine-cytosine content as an example, the method is advantageous because it not only removes sequences whose average guanine-cytosine content falls outside a given range, but also removes any sequences that have spikes or troughs in guanine-cytosine content in specific parts, which could hinder efficient transcription or translation. Such peaks or troughs would be missed if only the entire sequence were analyzed simultaneously, because the parts of the sequence other than the analysis portion could yield an acceptable average guanine-cytosine content. By analyzing part by part, not only is computational efficiency improved, but problems within candidate sequences that would be shielded in average by other methods are also identified.

[0020] In this specification, guanine-cytosine content is used as an example, but it is understood that any criteria described herein are analyzed partwise as described above. For some criteria, such as sequences containing termination signals, computational efficiency is increased, but the outcome of partwise screening does not affect the content of the resulting list; that is, evaluation of termination signals in a part removes the same nucleotide sequence from the list as an evaluation of the whole sequence. For other criteria, such as guanine-cytosine content or codon adaptation indices, the screening outcomes may differ; for example, using partwise analysis removes certain sequences that would not have been removed if the sequences were evaluated as a whole.

[0021] The first portion and / or one or more further portions of the nucleotide sequence may contain a predetermined number of nucleotides, which may be 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. This length of portion has been found to provide an optimal balance between codon use of mRNA and the abundance of congeneral tRNAs.

[0022] In some embodiments, the first criterion includes nucleotide sequences that do not contain termination signals, and as a result, the method includes the steps of determining whether each optimized nucleotide sequence in the list of optimized nucleotide sequences or in the most recently updated list contains termination signals; 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.

[0023] Thus, the method yields a list of short or filtered optimized nucleotide sequences. Reducing the number of optimized nucleotide sequences in the list is advantageous because it also reduces the number and complexity of further steps performed on the sequences in the list, such as further algorithmic or physical synthesis steps. In some embodiments, the termination signal has the following nucleotide sequence: 5'-X1ATCTX2TX3-3' [wherein X1, X2, and X3 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 TTTTTT; and / or AAGCTT; and / or GAAGAGC; and / or TCTAGA. In some embodiments, the termination signal has the following nucleotide sequence: 5'-X1AUCUX2UX3-3' [wherein X1, X2, and X3 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.

[0024] In some embodiments, the second criterion includes nucleotide sequences having a guanine-cytosine content within a predetermined guanine-cytosine content range, and consequently, the method includes the steps of determining the guanine-cytosine content of each optimized nucleotide sequence in a list of optimized nucleotide sequences or in a recently updated list, wherein the guanine-cytosine content is the percentage of bases in the nucleotide sequence that are guanine or cytosine; and updating the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or the recently updated list if its guanine-cytosine content falls outside the predetermined guanine-cytosine content range. Reducing the number of optimized nucleotide sequences in the list is advantageous because it also reduces the number and complexity of any further steps performed on the sequences in the list, such as further algorithmic steps or physical synthesis steps. In some embodiments, the predetermined guanine-cytosine content range is 15% to 75%, or 40% to 60%, or in particular, 30% to 70%.

[0025] In some embodiments, a third criterion includes nucleotide sequences whose codon adaptation index exceeds a predetermined codon adaptation index threshold, and as a result, the method includes the steps of determining the codon adaptation index of each optimized nucleotide sequence in the list of optimized nucleotide sequences or in the recently updated list, wherein the codon adaptation index of a sequence is a measure of codon use bias and can be a value between 0 and 1; and updating the list of optimized nucleotide sequences or the recently updated list by removing any nucleotide sequence whose codon adaptation index is less than or equal to a predetermined codon adaptation index threshold. Thus, the method yields a shorter or filtered list of optimized nucleotide sequences. In some embodiments, the codon adaptation index threshold is selectable 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. Reducing the number of optimized nucleotide sequences in the list is advantageous because it also reduces the number and complexity of further steps performed on the sequences in the list, such as further algorithmic or physical synthesis steps.

[0026] In some embodiments, the fourth criterion includes a nucleotide sequence that does not contain at least two, e.g., three adjacent identical codons, and consequently the method includes the steps of determining whether any optimized nucleotide sequence in the list of optimized nucleotide sequences or in the recently updated list contains at least two, e.g., three adjacent identical codons; and updating the list of optimized nucleotide sequences or the recently updated list by removing any nucleotide sequence that contains at least two, e.g., three adjacent identical codons. It has been found that repeating identical codons, in other words, adjacent identical codons, can stall transcription. Therefore, two By removing any optimized nucleotide sequences containing four or more, five or more, six or more, seven or more, eight or more, nine or more, or in particular three or more identical adjacent codons, sequences that would result in a decrease in transcriptional effectiveness are ignored and removed.

[0027] In any embodiment of the present invention, the generation of an updated list of optimized nucleotide sequences is performed by the following steps: (I) The step of determining the presence of termination signals in one or more optimized nucleotide sequences, and if they contain termination signals, removing the nucleotide sequences from the list of optimized nucleotide sequences or the recently updated list; (II) Determining the guanine-cytosine content of one or more optimized nucleotide sequences, and removing the nucleotide sequences from the list of optimized nucleotide sequences or the recently updated list if their guanine-cytosine content falls outside a given range; (III) Determining the codon adaptation index of one or more optimized nucleotide sequences, and removing the nucleotide sequences from the list of optimized nucleotide sequences or the recently updated list if their guanine-cytosine content falls outside a predetermined range. This is performed by removing an optimized sequence from the list based on one of the following, two of the following, or any three of the following.

[0028] In a second aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises carrying out step (I).

[0029] In a third aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises carrying out step (II).

[0030] In a fourth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further includes carrying out step (III).

[0031] In a fifth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises carrying out step (I), and then step (II).

[0032] In a sixth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises carrying out step (I), and then step (III).

[0033] In a seventh aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises performing step (II), and then step (I).

[0034] In an eighth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises carrying out step (II), and then step (III).

[0035] More typically, the method according to the present invention comprises a termination signal-based step (I), a guanine-cytosine content-based step (II), and a codon adaptation index-based step (III) to create a short list of optimized nucleotide sequences, all of which are expected to result in a full-length mRNA transcript when synthesized by in vitro transcription and in vivo in high levels of expression of the mRNA-encoded protein. The termination signal-based step (I), the guanine-cytosine content-based step (II), and the codon adaptation index-based step (III) are performed in any order. It is advantageous for the steps to be performed in a specific order to optimize computation time when determining the short list of optimized nucleotide sequences.

[0036] 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), and then step (III). Filtering in this order may be advantageous because it maximizes the computational efficiency of the filtering steps. The inventors have found that for a list of typical optimized nucleotide sequences and typical input parameters, a motif screen filter followed by a GC content analysis filter followed by a CAI analysis filter removes most sequences from the list. The computational efficiency of the filtering steps is partially determined by the total number of sequences analyzed, i.e., the sum of sequences analyzed within each filtering step, so that more sequences are removed early in the filtering steps, and fewer sequences require analysis later in the filtering steps, thereby increasing the overall computational efficiency of the method. Furthermore, while the CAI analysis filter requires analysis of the entire sequence, in embodiments of the present invention, the motif screen filter and GC content analysis filter may analyze only a portion or part of the sequence. Therefore, a method that emphasizes reducing the number of sequences in the list, which is input into the CAI analysis process, is likely to be more computationally efficient than other methods.

[0037] In a tenth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises carrying out step (I), then step (III), then step (II).

[0038] In an eleventh aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises carrying out step (II), then step (I), then step (III).

[0039] In a twelfth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises carrying out step (II), then step (III), then step (I).

[0040] In a thirteenth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises carrying out step (III), then step (I), then step (II).

[0041] In a fourteenth aspect of the present invention, after generating one or more optimized nucleotide sequences, the method further comprises carrying out step (III), then step (II), then step (I).

[0042] In some embodiments, the amino acid sequence is received from an amino acid sequence database. In some embodiments, the method further includes the step of requesting an amino acid sequence from an amino acid sequence database, and the amino acid sequence is received in response to the request.

[0043] In some embodiments, the first codon usage table is received from a codon usage table database. In some embodiments, the method further includes the step of requesting the first codon usage table from a codon usage table database, and the first codon usage table is received in response to the request.

[0044] In a fifteenth aspect, the present invention relates to a computer program that, when the program is executed by a computer, includes instructions causing the computer to perform a method according to any embodiment of the first aspect.

[0045] In a sixteenth aspect, the present invention relates to a data processing system including means for performing a method according to any embodiment of the first aspect.

[0046] In the seventeenth aspect, the present invention relates to a computer-readable data storage medium on which a computer program of the third aspect is stored.

[0047] In the eighteenth aspect, the present invention relates to a data transport signal for transporting a computer program according to the third aspect.

[0048] In a 19th aspect, the present invention relates to a method for synthesizing nucleotide sequences, comprising the steps of: performing a method according to any embodiment of the first aspect to generate at least one optimized nucleotide sequence; and synthesizing at least one of the generated optimized nucleotide sequences. In some embodiments, the method further comprises the step of inserting at least one of the synthesized optimized sequences into a nucleic acid vector for use in in vitro transcription.

[0049] In some embodiments, the method further includes the step of inserting one or more termination signals at the 3' end of the synthetically optimized nucleotide sequence. In some embodiments, more than one termination signal is inserted, and the termination signals are separated by 10 base pairs or less, for example, 5 to 10 base pairs. In some embodiments, one or more termination signals have the following nucleotide sequence: 5'-X1ATCTX2TX3-3' [wherein X1, X2, and X3 are independently selected from A, C, T, or G]. In some embodiments, one or more termination signals have one of the following nucleotide sequences: TATCTGTT;TTTTTT;AAGCTT;GAAGAGC; and / or TCTAGA. In some embodiments, one or more termination signals have the following nucleotide sequence: (a) 5'-X1ATCTX2TX3-(Z N )-X4ATCTX5TX6-3', or (b)5'-X1ATCTX2TX3-(Z N )-X4ATCTX5TX6-(Z M )-X7ATCTX8TX9-3'[In array, X1, X2, X3, X4, 5、 X6, X7, X8, and X9 are independently selected from A, C, T, or G, and Z N This represents the spacer sequence of the N nucleotide, Z M Each of these represents a spacer sequence of M nucleotides, each independently selected from A, C, T, or G, and is coded by [N and / or M independently being 10 or less].

[0050] In some embodiments, the nucleic acid vector includes an RNA polymerase promoter operably ligated to the optimized nucleotide sequence, and optionally the RNA polymerase promoter is the SP6 RNA polymerase promoter or the T7 RNA polymerase promoter. In some embodiments, the nucleic acid vector includes a nucleotide sequence encoding a 5'UTR operably ligated to the optimized nucleotide sequence. In some embodiments, the 5'UTR is different from the 5'UTR of 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 includes a nucleotide sequence encoding a 3'UTR operably ligated to the optimized nucleotide sequence. In some embodiments, the 3'UTR is different from the 3'UTR of 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 before in vitro transcription. In some embodiments, the plasmid is not linearized before in vitro transcription. In some embodiments, the plasmid is superhelical.

[0051] In some embodiments, the method further includes the step of synthesizing mRNA using at least one of the synthetically optimized nucleotide sequences in in vitro transcription. In some embodiments, mRNA is synthesized by SP6 RNA polymerase. Morphologically, 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 includes a tag. In some embodiments, the tag is a His tag. In some embodiments, the mRNA is synthesized by T7 RNA polymerase.

[0052] In some embodiments, the method further includes a separate step of capping and / or tailing the synthesized mRNA. In some embodiments, capping and tailing occur during in vitro transcription.

[0053] In some embodiments, mRNA is synthesized in a reaction mixture containing NTPs at concentrations ranging from 1 to 10 mM for each NTP, a DNA template at a concentration ranging from 0.01 to 0.5 mg / ml, and SP6 RNA polymerase at a concentration ranging from 0.01 to 0.1 mg / ml. In some embodiments, the reaction mixture contains NTPs at concentrations ranging from 5 mM for each NTP, a DNA template at a concentration of 0.1 mg / ml, and SP6 RNA polymerase at a concentration of 0.05 mg / ml.

[0054] In some embodiments, mRNA is synthesized at temperatures ranging from 37 to 56°C.

[0055] In some embodiments, the NTP is a naturally occurring NTP. In some embodiments, the NTP includes a modified NTP.

[0056] In some embodiments, the method further comprises the steps of synthesizing a reference nucleotide sequence encoding an amino acid sequence and at least one synthetically optimized nucleotide sequence according to the method of the present invention, and contacting the reference nucleotide sequence and at least one optimized nucleotide sequence with a separate cell or organism. In a typical embodiment, the cell or organism contacted with at least one synthetically optimized nucleotide sequence produces the protein encoded by the optimized nucleotide sequence in an increased yield compared to the yield of the protein encoded by the reference nucleotide sequence produced by the cell or organism contacted with the synthetic reference nucleotide sequence. In any aspect of the present invention, the at least one optimized nucleotide sequence, when synthesized, is configured to increase protein expression compared to the expression of the 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 produced by a method other than the method according to the first aspect of the present invention.

[0057] In some embodiments, the method further includes the step of transfecting cells with a synthetically optimized nucleotide sequence in vitro or in vivo. In some embodiments, the expression level of the protein encoded by the synthetically optimized nucleotide sequence is determined in the transfected cells. In some embodiments, the functional activity of the protein encoded by the synthetically optimized nucleotide sequence is determined in the transfected cells.

[0058] In a 20th aspect of the present invention, the present invention provides a synthetically optimized nucleotide sequence produced according to the method of the present invention for therapeutic use. This aspect of the present invention includes a treatment method comprising the step of administering the synthetically optimized nucleotide sequence produced according to the method of the present invention to a human subject requiring such treatment. In some embodiments, the methods described herein provide a therapeutic composition comprising mRNA encoding a therapeutic peptide, polypeptide, or protein for delivery to a subject or for use in the treatment of a subject. In some embodiments, the mRNA is a cystic fibrosis transmembrane conductance regulator (CFTR). ) Codes for proteins.

[0059] In the 21st aspect, the present invention includes an optimized nucleotide sequence comprising codons associated with a usage frequency of 10% or more, wherein the optimized nucleotide sequence is (i) The following nucleotide sequence: 5'-X1AUCUX2UX3-3'[in the sequence, X1, X2, and X3 are independently selected from A, C, U, or G]; and not containing a termination signal having one of 5'-X1AUCUX2UX3-3'[in the sequence, X1, X2, and X3 are independently selected from A, C, U, or G]; (ii) Not containing negative cis-adjusting elements and negative repeating elements; (iii) Having a codon adaptation index greater than 0.8; When divided into non-overlapping 30-nucleotide segments, each segment of the optimized nucleotide sequence has a guanine-cytosine content ranging from 30% to 70%. This yields in vitro synthetic nucleic acids. In some embodiments, the optimized nucleotide sequence does not contain 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 in vitro synthetic nucleic acid is an in vitro synthetic nucleic acid for therapeutic use.

[0060] Embodiments of the present invention will be described with reference to the following drawings, as an example. [Brief explanation of the drawing]

[0061] [Figure 1] This figure shows a codon optimization method according to one embodiment of the present invention. [Figure 2A] Figure 2A shows an exemplary codon usage table for humans (Homo sapiens) generated from one or more experimentally obtained 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 data from the NCBI GenBank database (Flat File Release 160.0). [Figure 2B] Figure 2B shows the normalized codon usage table generated by normalizing the codon usage frequencies of the exemplary codon usage table in Figure 2A. [Figure 3] This figure shows a section of a constructed codon usage table for use in an exemplary method for codon usage table normalization. [Figure 4A] Figure 4A shows an exemplary table from Figure 3, normalized to an equal frequency distribution. [Figure 4B] Figure 4B shows an exemplary table from Figure 3, normalized by a proportional frequency distribution. [Figure 5] This figure shows a constructed section of an amino acid sequence for use in an exemplary method for codon optimization. [Figure 6] This figure shows an exemplary repository of nucleotide sequence motifs containing stop signals, suitable for use when removing nucleotide sequences containing one or more stop signals. [Figure 7]This figure shows a method for applying further algorithmic or filtering steps to a list of optimized nucleotide sequences. In a particular embodiment, the list of optimized nucleotide sequences for filtering is generated according to the method shown in Figure 1. [Figure 8] This figure shows an embodiment of the present invention in which a guanine-cytosine (GC) content analysis filter is applied to a list of optimized nucleotide sequences. In a particular embodiment, the list of optimized nucleotide sequences for filtering is generated according to the method shown in Figure 1. [Figure 9] This figure shows embodiments of the present invention in which a motif screen filter and a codon adaptation index (CAI) analysis filter are applied to a list of optimized nucleotide sequences. In certain embodiments, the list of optimized nucleotide sequences for filtering is generated according to the method shown in Figure 1. [Figure 10] This figure shows a specific embodiment of the present invention in which a motif screen filter, a guanine-cytosine (GC) content analysis filter, and a codon adaptation index (CAI) analysis filter are applied in this order to a list of optimized nucleotide sequences. In this specific embodiment, the list of optimized nucleotide sequences for filtering is generated according to the method shown in Figure 1. [Figure 11] This figure shows examples of analysis of guanine-cytosine (GC) content in unoptimized and optimized nucleotide sequences, and the guanine-cytosine (GC) content of the portion of the nucleotide sequence encoding EPO is determined for adjacent non-overlapping regions of 30 nucleotides in length. [Figure 12] This figure shows an exemplary bar graph illustrating the yield of proteins produced from various codon-optimized nucleotide sequences, as determined by an ELISA assay of EPO. [Figure 13A]Figure 13A shows an example of a Western blot used to determine the protein expression yield of the CFTR protein encoded by the optimized nucleotide sequence generated according to the method of the present invention in a time-course experiment after the optimized nucleotide sequence has been transfected into human cells. [Figure 13B] Figure 13B shows an exemplary line plot illustrating the quantification of the Western blot data shown in Figure 13A. [Figure 14A] Figure 14A shows an exemplary plot of data obtained from a bioassay to test mRNA containing an optimized nucleotide sequence encoding hCFTR. The short-circuit current (ISC) output in the Ussing epithelial voltage clamp device for each test mRNA is shown. [Figure 14B] Figure 14B is an exemplary bar plot showing the change in hCFTR activity, as shown in Figure 14A, expressed as a percentage of the activity of the reference mRNA encoding hCFTR. [Figure 15A] Figure 15A shows an exemplary Western blot illustrating the translation and expression of codon-optimized DNAI1 mRNA in HEK293T cells. Western blotting was performed using anti-DNAI1 antibody and anti-vinculin antibody (loading control). [Figure 15B] Figure 15B shows an exemplary bar graph illustrating the levels of DNAI1 protein expression normalized to vinculin protein (loading control), quantified from the exemplary Western blot in Figure 15A. DNAI1 protein expression yield is graphed as an increase factor relative to the reference level achieved with mRNA encoding a non-codon-optimized DNAI1 sequence. [Modes for carrying out the invention]

[0062] definition To facilitate understanding of the present invention, certain terms are first defined below. Further definitions of the following terms and other terms are provided herein.

[0063] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly indicates otherwise.

[0064] Unless otherwise specified or evident from the context, the term “or” as used herein is understood to be inclusive and encompasses both “or” and “and.”

[0065] When used herein, the terms “for example” and “that is” are used merely as examples and not intended to be limiting, and should not be construed as referring only to items explicitly listed herein.

[0066] Terms like "at least," "at least," and "more than," for example, "at least one," can mean 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, 4 2, 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 It is understood that this includes, but is not limited to, values ​​greater than, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or any larger number or fractions in between.

[0067] Conversely, the term "less than or equal to" includes each value smaller than the listed value. For example, "less than or equal to 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 This includes nucleotides with 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. It also includes any smaller number or fractions in between.

[0068] Terms such as "multiple," "at least two," "two or more," and "at least the second" are used to mean 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 ,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,9 3, 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 It is understood that this includes, but is not limited to, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000 and above. It also includes any larger number or fractions in between.

[0069] Unless otherwise specified or evident from the context, the term “about” as used herein is understood to mean within the normal tolerance in the art, e.g., within two standard deviations from 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 otherwise evident from the context, all numerical values ​​provided herein reflect the normal variation that a person skilled in the art would recognize.

[0070] As used herein, terms such as “interrupted transcript” or “pre-interrupted transcript” refer to any transcript shorter than the full-length mRNA molecule encoded by the DNA template, resulting from sequence-independent premature release of RNA polymerase from the template DNA. In some embodiments, the interrupted transcript may be less than 90% of the length of the full-length mRNA molecule transcribed from the target DNA molecule, for example, less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% of the full-length mRNA molecule.

[0071] As used herein, the term “codon” refers 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 in the process of translation or protein synthesis. The genetic code is degenerate, and more than one codon may encode a particular amino acid residue. For example, a codon may contain DNA or RNA nucleotides.

[0072] As used herein, the terms “codon optimization” and “codon optimized” refer to improving the protein expression of a nucleic acid by modifying the codon composition of a spontaneously occurring or wild-type nucleic acid encoding a peptide, polypeptide, or protein, without altering its amino acid sequence. In connection with the present invention, “codon optimization” may also refer to the process of arriving at one or more optimized nucleotide sequences by removing suboptimal nucleotide sequences from a list of nucleotide sequences using filters such as guanine-cytosine content, codon adaptation indices, the presence of destabilized nucleic acid sequences or motifs, and / or the presence of rest sites and / or terminator signals.

[0073] As used herein, “full-length mRNA” is characterized by the use of a specific assay, e.g., detection using gel electrophoresis and UV, and UV absorption spectroscopy with separation by capillary electrophoresis. The length of the mRNA molecule encoding a full-length polypeptide is at least 50% of the length of the full-length mRNA molecule transcribed from the target DNA, e.g., 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%, or 99.9% of the length of the full-length mRNA molecule transcribed from the target DNA.

[0074] As used herein, the term "in vitro" refers to events occurring in an artificial environment, such as in a test tube or reactor, or in a cell culture, rather than within a multicellular organism.

[0075] As used herein, the term "in vivo" refers to events occurring within multicellular organisms such as humans and non-human animals. In relation to cell-based systems, the term may be used to refer to events occurring within living cells (as opposed to, for example, in vitro systems).

[0076] As used herein, the term “messenger RNA (mRNA)” refers to a polyribonucleotide that encodes at least one polypeptide. As used herein, mRNA encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions. mRNA may be purified from natural sources, produced using recombinant expression systems and optionally purified, transcribed in vitro, or chemically synthesized. Where appropriate, for example, in chemically synthesized molecules, mRNA may contain nucleoside analogs such as those with chemically modified bases or sugars, skeletal modifications, etc. Unless otherwise specified, mRNA sequences are presented in the 5' to 3' direction.

[0077] 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 residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to a polynucleotide chain containing individual nucleic acid residues. In some embodiments, “nucleic acid” includes not only RNA but also single-stranded and / or double-stranded DNA and / or cDNA. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e., analogs having something other than a phosphodiester backbone. Unless otherwise specified, nucleic acid sequences are presented in the 5' to 3' direction.

[0078] As used herein, the term “nucleotide sequence” refers, in its broadest sense, to the order of nucleic acid bases within a nucleic acid. In some embodiments, “nucleotide sequence” refers to the order of individual nucleic acid bases within a gene. In some embodiments, “nucleotide sequence” refers to the order of individual nucleic acid bases within a protein-coding gene. In some embodiments, “nucleotide sequence” refers to the order of individual nucleic acid bases within single-stranded and / or double-stranded DNA and / or cDNA. In some embodiments, “nucleotide sequence” refers to the order of individual nucleic acid bases within RNA. In some embodiments, “nucleotide sequence” refers to the order of individual nucleic acid bases within mRNA. In certain embodiments, “nucleotide sequence” refers to the order of individual nucleic acid bases within a protein-coding sequence of RNA or DNA. Unless otherwise specified, nucleotide sequences are typically presented in the 5' to 3' direction.

[0079] As used herein, the term “interruption” refers to the termination of transcription before the full-length DNA template is transcribed. As used herein, interruption can occur due to the presence of a nucleotide sequence motif (also referred to herein simply as “motif”), such as a termination signal, within the DNA template, resulting in an mRNA transcript shorter than the full-length mRNA (“interrupted transcript” or “cleaved mRNA transcript”). Examples of termination signals include the Escherichia coli (E. coli) rrnB terminator t1 signal (consensus sequence: ATCTGTT) and its variants, as described herein.

[0080] As used herein, the term “template DNA” (or “DNA template”) refers to a DNA molecule containing a nucleic acid sequence that encodes 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, particularly T3, T7, and SP6, operably linked to the DNA sequence encoding the desired mRNA transcript. It contains a promoter element for binding of DNA-dependent RNA polymerases such as RNA polymerase. Furthermore, the template DNA contains a DNA component encoding the mRNA transcript. To determine the identity of the sequence, for example by PCR or DNA sequencing, primer binding sites may be included at the 5' and / or 3' of the DNA sequence encoding the mRNA transcript. “Template DNA” can be a linear or circular DNA molecule in relation to the present invention. As used herein, the term “Template DNA” may refer to a DNA vector, such as plasmid DNA, containing a nucleic acid sequence encoding a desired mRNA transcript.

[0081] All technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which this application belongs, and as commonly used in the art to which this application belongs. Publications and other reference materials referenced herein to explain the background of the invention and to provide further details regarding its implementation are incorporated herein by reference.

[0082] Detailed description of the invention Codon optimization feature In the process of gene expression, nucleotide sequences encoded in DNA sequences are transcribed into RNA molecules, which are then translated into proteins containing polypeptide chains. Sequence information that specifies the exact order of amino acid residues that will be incorporated into the protein product is encoded in DNA and / or mRNA sequences as "codons." A codon consists of a sequence of three nucleotides that together form a unit of the genetic code, with each codon corresponding to a specific amino acid or stop codon signal. The genetic code is degenerate, meaning that more than one codon may encode a particular amino acid residue.

[0083] mRNA is typically considered a type of RNA that carries information from DNA to ribosomes. The presence of mRNA is usually very short-lived, involving processing and translation followed by degradation. Typically in eukaryotes, mRNA processing involves the addition of a “cap” to the N-(5') end and a “tail” to the C-(3') end. A typical cap is a 7-methylguanosine cap, which is guanosine linked to the initially transcribed nucleotide via a 5'-5'-triphosphate bond. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The tail is typically a polyadenylation event, where a poly(A) moiety is added to the 3' end of the mRNA molecule. The presence of this “tail” functions to protect mRNA from exonuclease degradation. Messenger RNA is typically translated by ribosomes into a set of amino acids that make up proteins.

[0084] Numerous factors can influence the level at which a particular protein is expressed or produced at various stages of gene expression. For example, since DNA sequences are transcribed into mRNA by the RNA polymerase enzyme, the presence of a specific nucleotide sequence motif can cause transcription to terminate prematurely. The specific composition and order of codons within the protein-coding region ("coding sequence") of a gene can also positively or negatively affect the efficiency and yield of protein expression. For instance, the presence of rare codons, characterized by low codon usage frequency, can negatively impact the yield of protein expression due to the low amount of syngeneic transfer RNA encoding a particular amino acid. In biotechnology and therapeutic applications, such as in therapies including mRNA therapy, it is often desirable to increase or maximize the yield of a protein when expressing that protein from a protein-coding nucleotide sequence. Codon optimization produces 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, numerous nucleotide sequences can encode the same amino acid sequence. Codon optimization aims to produce one or more nucleotide sequences that will result in increased protein yield.

[0085] Amino acid sequence for generating optimized nucleotide sequences Spontaneously occurring nucleotide sequences may be used to provide the amino acid sequences encoding proteins, polypeptides, or peptides of interest. Nucleotide sequences can be obtained by isolating nucleic acid molecules from organisms of interest and determining the precise order of their nucleic acid bases (e.g., guanine, thymine, uracil, adenine, and cytosine). Several methods known in the art are suitable for obtaining spontaneously occurring nucleotide sequences. Nucleotide sequences of protein-coding genes can be obtained by various well-known DNA or RNA sequencing methods.

[0086] For example, DNA from human cells can be extracted, isolated, and then fragmented. The fragmented DNA can be placed in a DNA vector, cloned, and amplified in a bacterial host to generate a "library" of short DNA fragments. Alternatively, polymerase chain reaction (PCR) can be used to amplify the fragmented DNA and incorporate it into a library suitable for high-throughput sequencing. Short DNA fragments derived from the original DNA material of the organism can be individually sequenced and then assembled into one or more longer continuous sequences by sequence assembly. Sequence assembly is a bioinformatics approach that aligns and merges short fragments of nucleotide sequences derived from longer nucleotide sequences to reconstruct the original nucleotide sequence or consensus nucleotide sequence.

[0087] The nucleotide sequences thus generated, i.e., sequences that are experimentally derived and known to accurately describe spontaneously occurring sequences, are typically stored in publicly accessible repositories or databases. For example, nucleotide sequences that can be processed by the method of the present invention can be obtained from the GenBank database of the National Center for Biotechnology Information (NCBI). GenBank is an open-access, annotated collection of publicly available nucleotide sequences and the protein sequences to which they are translated.

[0088] Generating a codon usage table The genetic code has 64 possible codons. Each codon contains a sequence of three nucleotides. The frequency of use for each codon in the protein-coding region of the genome can be calculated by determining the number of occurrences of a particular codon within the protein-coding region of the genome, and then dividing the resulting value by the total number of codons encoding the same amino acid within the protein-coding region of the genome. For example, these calculations can be performed on nucleotide sequences found in publicly accessible repositories and / or databases, and thus can also represent experimentally derived data.

[0089] Codon usage tables identify 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 frequency of use. Codon usage tables are stored in publicly available databases such as the Codon Usage Database (Nakamura et 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 (Athey et al., (2017) BMC Bioinformatics 18(1), 391; available online at http: / / hive.biochemistry.gwu.edu / review / codon).

[0090] Codon optimization Figure 1 shows the codon optimization method according to the present invention. In the first step 101, an amino acid sequence is received. The amino acid sequence may be received from a remote system, server, and / or a publicly accessible database, and may be received wirelessly, for example, via the Internet. Alternatively, the amino acid sequence may be received from a local system, for example, via a wired connection. The amino acid sequence contains multiple amino acids.

[0091] In the second step 102, a first codon usage table is received. The first codon usage table may be received from a remote system, server and / or a publicly accessible database, as well as wirelessly, for example, via the Internet. Alternatively, the first codon usage table may be received from a local system, for example, via a wired connection. The first codon usage table includes 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.

[0092] In the third step 103, if codons are associated with codon usage frequencies below a threshold frequency, those codons are removed from the first codon usage table.

[0093] In the fourth step 104, the codon usage frequencies of codons that were not removed in the third step 103 are normalized, and a normalized codon usage table is generated.

[0094] In step 5, step 105, an optimized nucleotide sequence is generated 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 acids in the normalized codon usage table.

[0095] Normalization of codon usage tables Refer to Figure 2A, which shows a codon usage table that may be found in the codon usage table database. It should be recognized that the codon usage table shown is merely an example, and any codon usage table, for example, any codon usage table available from the database, can be used by the present invention to produce an optimized nucleotide sequence. The data used to produce Figure 2A was obtained from data accessed via the Codon Usage Database, NCBI. This was derived from publicly available codon usage data via the GenBank database (Flat File Release 160.0).

[0096] The codon usage table contains experimentally derived data regarding the frequency with which each codon is used to code a particular amino acid for the specific biological origin from which the table was generated. This information is expressed for each codon as a percentage (0-100%) or proportion (0-1) of the frequency with which that codon is used to code a particular amino acid, relative to the total number of times the codon codes for an amino acid.

[0097] Figure 2B shows a normalized codon usage table generated from the table in Figure 2A by the method of the present invention. In the example in Figure 2B, the threshold frequency for performing normalization was 10%. It will be recognized that this is merely an example and that embodiments of the present invention may use any other suitable threshold frequencies described herein.

[0098] A method for providing a normalized codon usage table is shown in Figure 3, as provided in the case of Figure 2B. Figure 3 uses exemplary amino acids "X" and "Y". When generating a normalized codon usage table, it will be recognized that any number of amino acids, from one amino acid to any amino acid in the codon usage table, may be normalized. In the example in Figure 3, amino acid X is encoded by codons A, B, C, D, E, and F at frequencies defined in the figure (each codon is represented by a nucleotide triplet, hence AAA, B in the figure). (As shown in BB, etc.). Amino acid Y is coded by codons G and H at the frequencies defined in the figure. In the first step, any codons with usage frequencies below the threshold frequency are removed from the table. It should be recognized that although the method shown in Figure 3 uses a 10% threshold frequency, this is merely an example and is not intended to limit the scope of the invention. Threshold frequencies can range from 5% to 30%, for example, 5%, or 15%, or 20%, or 25%, or 30%, or especially 10%. These threshold frequency values ​​have been found to provide an effective balance between increasing protein yield and retaining information important for controlling translation of nascent polypeptide chains and ensuring intrinsic folding. It should be recognized that the codon usage table in Figure 3, in particular, consists of only two amino acids and therefore does not accurately describe actual spontaneous codon usage. The table in Figure 3 is intended to be merely an illustration of a method for normalizing codon usage tables.

[0099] In the example in Figure 3, codons C and E have usage frequencies below the 10% threshold frequency and are therefore removed from the table. The combined usage frequency of the removed codons, C and E, is 16%. This combined usage frequency is then distributed among the remaining codons encoding amino acid X. It is important to note that the combined usage frequency removed from amino acid X is similarly distributed among the remaining codons encoding amino acid X; that is, in the examples in Figures 4A and 4B, the usage frequencies of codons G and H encoding amino acid Y remain unchanged.

[0100] In some embodiments, the removed aggregate usage frequency is equally distributed among the remaining codons encoding amino acid X. Such an embodiment is shown in Figure 4A. The removed aggregate usage frequency of 16% is equally distributed among the remaining codons A, B, D, and F, so that each of the remaining codons receives an additional usage frequency of 4%. The codon usage frequency of amino acid X is now normalized.

[0101] In some embodiments, the removed aggregate usage frequency is proportionally distributed among the remaining codons encoding amino acid X. Such embodiments are shown in Figure 4B. The removed aggregate usage frequency of 16% is distributed among the remaining codons A, B, D, and F in proportion to the usage frequencies of the remaining codons A, B, D, and F. In this example, the usage frequency ratios of codons A, B, D, and F are 15:20:38:11, or 0.18:0.24:0.45:0.13. Codon A receives 0.18 times (3%) of 16%, B receives 0.24 times (4%) of 16%, D receives 0.45 times (7%) of 16%, and F receives 0.13 times (2%) of 16%. The codon usage frequency of amino acid X is now normalized.

[0102] Thus, the structure and content of the received codon usage table, or the first codon usage table, teach the generation of a normalized codon usage table. The number of codons associated with each amino acid teaches the redistribution of the removed codon usage frequencies, and the codon usage frequencies themselves teach which codons are removed and, in some embodiments, the proportionality of the distribution.

[0103] Generation of optimized nucleotide sequences The optimized nucleotide sequence is generated 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 the normalized codon usage table. The optimized nucleotide sequence is then generated by arranging the selected codons in the order in which the amino acids associated with them appear in the amino acid sequence.

[0104] Refer to Figure 5 for an illustration of the generation of an optimized nucleotide sequence using codons A, B, C, D, E, and F from Figures 3, 4A, and 4B. In the illustration in Figure 5, each codon may be represented by three nucleotides, such as codon A being represented by nucleotide AAA, codon B being represented by nucleotide BBB, and so on.

[0105] An exemplary amino acid sequence, XYYXXX, is received. For this example, the inventors 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 table in Figure 3 is normalized on a probability basis, resulting in the normalized codon usage table in Figure 4B. In 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 table. For example, for the first amino acid X in the sequence, there is an 18% chance that codon A is selected, a 24% chance that codon B is selected, a 45% chance that codon D is selected, and a 13% chance that codon F is selected. This is because amino acid X is encoded by codons A, B, D, and F and is therefore associated with these codons in the normalized codon usage table, and thus the codon selected for amino acid X will be one of codons A, B, D, and F.

[0106] This process is repeated for each amino acid, using a normalized codon usage table to teach the probability of selecting a particular codon. Thus, for the second amino acid Y in the sequence, codon G is selected with a 60% probability, and codon H is selected with a 40% probability. After codon selection for each amino acid, the resulting codon sequence, composed of nucleotides, is sometimes referred to as the optimized nucleotide sequence.

[0107] Figure 5 is illustrative and intended solely to aid in understanding the generation of optimized nucleotide sequences. Figure 5 may not show the actual length, content, or structure of the received amino acid sequence or optimized nucleotide sequence, and is merely a schematic diagram illustrating the method.

[0108] Generation of multiple optimized nucleotide sequences The generation of optimized nucleotide sequences using amino acid sequences and normalized codon usage tables may be performed more than once to generate a list of optimized nucleotide sequences.

[0109] Since the generation of optimized nucleotide sequences is based on the probabilistic selection of codons, the list can contain any number of different optimized nucleotide sequences. Furthermore, since the generation of optimized nucleotide sequences is based on the probabilistic selection of substitution codons, the list can contain any number of pairs of optimized nucleotide sequences, i.e., identical optimized nucleotide sequences. When generating a list of optimized nucleotide sequences, identical optimized sequences are typically removed.

[0110] In some embodiments, one or more, or all, of the optimized nucleotide sequences in the list of optimized nucleotide sequences are synthesized for transfection testing, therapeutic use, or any other use of the synthetic optimized nucleotide sequences described herein.

[0111] Filtering the list of optimized nucleotide sequences The number of optimized nucleotide sequences in a list of optimized nucleotide sequences depends at least on the length and content of the amino acid sequence, the threshold codon usage frequency value, the contents of the first codon usage table, and the number of times the codon optimization algorithm is run, i.e., the number of times optimized nucleotide sequences are generated. For example, a list of optimized nucleotide sequences may contain more than 10,000 optimized nucleotide sequences. Synthesizing and testing each optimized nucleotide sequence in the list in cells, tissues, or organisms may be advantageous in some scenarios, for example, for certain algorithm input parameters, such as relatively short amino acid sequences. Similarly, in certain scenarios, it may not be advantageous, for example, if it is desirable to reduce the complexity of the computer process or to reduce the number of sequences synthesized and tested in cells, tissues, or organisms. Therefore, it may be desirable to reduce the number of optimized nucleotide sequences in the list of nucleotide sequences before synthesis. This can advantageously reduce the time and resources required to compose any array of lists.

[0112] Therefore, in a typical embodiment, one or more further algorithmic steps are performed on the list of optimized nucleotide sequences to filter the list or to remove optimized nucleotide sequences from the list. One or more further algorithmic steps may be referred to as motif screening, GC content analysis, and codon adaptation index (CAI) analysis. Although certain further algorithmic steps are described in detail herein, it will be recognized that these may not be filtering steps performed in isolation, and that additional steps may be performed to further filter the list of optimized nucleotide sequences within the scope of these claims.

[0113] The inventors have found that these further algorithmic steps, as well as the associated motifs, ranges, and thresholds, favorably filter the list of optimized nucleotide sequences by removing sequences from the list that may be less effective than those remaining on the list. Thus, filtering the list is not simply a matter of discretion. In other words, filtering the list to a certain number of sequences using the method described herein will produce an updated list of sequences containing more effective sequences than would be possible if the same certain number of sequences were randomly selected from the list. Thus, efficiency and the reduction in complexity achieved in the synthesis process are obtained without sacrificing a large number of effective optimized nucleotide sequences. For example, the optimized nucleotide sequences produced by the method of the present invention do not contain stop signals. The absence of stop signals facilitates the synthesis of full-length mRNA molecules from the encoded optimized nucleotide sequences using in vitro transcription. The presence of stop signals results in premature termination of in vitro transcription. Therefore, filtering the list using the method described herein produces an updated list of sequences containing more effective sequences.

[0114] 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 further algorithmic step, as detailed herein. In other words, the criteria may include: optimized nucleotide sequences that do not contain a stop signal (Criterion 1), optimized nucleotide sequences that have a guanine-cytosine content within a predetermined guanine-cytosine content range (Criterion 2), optimized nucleotide sequences that have a codon adaptation index greater than a predetermined threshold for codon adaptation index (Criterion 3), and optimized nucleotide sequences that do not have a stop signal. It should be noted that the numbering of the criteria used is for clarity only and is not intended to limit the order of the steps. The order of the steps is described in detail elsewhere herein.

[0115] Although certain criteria are described in detail herein, it should be recognized that these may not be the only criteria used to screen for optimized nucleotide sequences, and that additional criteria may be used to further filter the list of optimized nucleotide sequences within the scope of these claims.

[0116] When screening each optimized nucleotide sequence, the entire sequence may be analyzed before determining whether it meets the criteria. Alternatively, each optimized nucleotide sequence may be analyzed in parts. These parts are sometimes referred to as windows.

[0117] For example, in a list of optimized nucleotide sequences with a length of 600 nucleotides, the length of a portion of an optimized nucleotide sequence may be selected to be 30 nucleotides. The first 30 nucleotides of an optimized nucleotide sequence, i.e., nucleotides 1-30 of the optimized nucleotide sequence, may be the first to be analyzed for compliance with certain criteria. If the first portion does not meet the criteria, the optimized nucleotide sequence may be removed from the list of optimized nucleotide sequences.

[0118] If the first portion meets the criteria, the filter may then analyze a second portion of the optimized nucleotide sequence. In this example, this could be the second 30 nucleotides of the optimized nucleotide sequence, i.e., nucleotides 31-60. Partial analysis may be repeated for each portion until either of the following occurs: the portion is found not to meet the criteria (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 the filter may retain the optimized nucleotide sequence in the list and move on to the next optimized nucleotide sequence in the list). In this example, if the filter reaches the final portion of the optimized nucleotide sequence, i.e., nucleotides 571-600, and this final portion meets the criteria, the filter may retain the optimized nucleotide sequence in the list and move on to the next optimized nucleotide sequence in the list. Alternatively, each portion may be 100 nucleotides long.

[0119] Although the above example illustrates a segmental filter starting from the first nucleotide and progressing to the final nucleotide, it will be recognized that this is merely an example, and the order in which the segments of the optimized nucleotide sequence are analyzed can be any order that is apparent to those skilled in the art. The filter may, for example, start at the segment containing the final nucleotide (nucleotide 600 in the working example) and work backward towards nucleotide 1, which is the first nucleotide, or start at any segment between the first and final nucleotides.

[0120] There may be first, final, or intermediate parts of the optimized nucleotide sequence that have different lengths from the rest of the sequence. This can occur, for example, if the nucleotide length of the optimized nucleotide sequence is not divisible by the nucleotide length of the part.

[0121] As detailed elsewhere in this specification, partwise analysis may be advantageous not only in terms of computational efficiency but also in more effectively identifying less desirable sequences. Less desirable sequences may include sections that, on average, meet the criteria but do not, such as peaks or troughs in GC content or CAI score.

[0122] 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 does not meet any of them; or, in particular, all sequences in the list may be screened for a specific criterion, and the resulting reduced, filtered list may be screened for further criteria of interest.

[0123] Motif screening In some embodiments, a motif screen filter may be applied to a 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 stop signal. The list of optimized nucleotide sequences may be a list of optimized nucleotide sequences originally generated by the codon optimization algorithm, or a list of optimized nucleotide sequences that have already been filtered by one or more further algorithmic steps. A list of optimized nucleotide sequences that has already been filtered or updated by one or more additional algorithmic steps may be referred to as an updated list of optimized nucleotide sequences or a recently updated list. Any optimized nucleotide sequence containing the termination signal may be removed from the list, resulting in an updated list.

[0124] Referring to Figure 6, the stop signals may have the following nucleotide sequences: 5'-X1ATCTX2TX3-3' [wherein X1, X2, and X3 are independently selected from A, C, T, or G]; TATCTGTT; TTTTTT; AAGCTT; GAAGAGC; TCTAGA; UAUCUGUU; UUUUUU; AAGCUU; GAAGAGC; UCUAGA; and / or 5'-X1AUCUX2UX3-3' [wherein X1, X2, and X3 are independently selected from A, C, U, or G]. A motif screen filter may determine whether each optimized nucleotide sequence contains one, some, or all of these stop signals.

[0125] Each optimized nucleotide sequence may be analyzed in its entirety, i.e., from the first nucleotide to the last nucleotide. In certain embodiments, if it is determined that a stop signal exists in a particular optimized nucleotide sequence, the analysis of that sequence may be stopped; and the sequence may then be removed from the list without analyzing each of its nucleotides. In certain embodiments, this form of analysis may be applied to each optimized nucleotide sequence in the list. If it has already been determined that a stop signal exists in a sequence, this type of analysis may be advantageous because it is computationally efficient not to analyze the entire sequence.

[0126] As will be explained in more detail with respect to GC content analysis, each optimized nucleotide sequence may be analyzed in parts. Analysis of an optimized nucleotide sequence may be stopped when it is determined that a part contains a stop signal. This may be advantageous because it is computationally efficient not to analyze the entire sequence if it has already been determined that a stop signal is present in the sequence. With respect to the subsequent GC content analysis, the parts may or may not overlap and may be of any length, e.g., 5 to 300 nucleotides, or 10 to 200 nucleotides, or 15 to 100 nucleotides, or 20 to 50 nucleotides, or in particular 30 or 100 nucleotides. Each part of an optimized nucleotide sequence may be of the same length, or, for example, the nucleotide length of an optimized nucleotide sequence may not be divisible by the nucleotide length of a part, for example, the first, last, or intermediate part of an optimized nucleotide sequence may be of a different length from the other parts.

[0127] Analysis of GC content In some embodiments, a guanine-cytosine (GC) content filter may 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 optimized nucleotide sequence in the list, where the GC content of a sequence is the percentage of bases that are guanine (G) or cytosine (C) in the nucleotide sequence. The list of optimized nucleotide sequences may be a list of optimized nucleotide sequences originally generated by the codon optimization algorithm, or a list of optimized nucleotide sequences that have already been filtered by one or more further algorithmic steps. A list of optimized nucleotide sequences that have already been filtered or updated by one or more additional algorithmic steps may be referred to as an updated list of optimized nucleotide sequences, or a recently updated list. Any optimized nucleotide sequences with GC content outside a predetermined GC content range may be removed from the list to produce an updated list.

[0128] Each optimized nucleotide sequence may be analyzed in its entirety, i.e., from the first nucleotide to the last nucleotide of the sequence. Then, the optimized nucleotide sequence The overall GC content is determined, and sequences may be removed accordingly.

[0129] 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 falls outside a predetermined range of GC content, the optimized nucleotide sequence containing that portion is removed from the list.

[0130] In certain embodiments, a GC content filter is applied to each optimized nucleotide sequence segment by segment, and if it is determined that a segment has a GC content outside a predetermined range, the filter stops and the sequence is removed. Such an analysis may be advantageous because it is computationally efficient not to analyze the entire sequence if it has already been found that a segment with a GC content outside the predetermined range exists in that sequence.

[0131] In certain embodiments, the portions do not overlap, but in other embodiments, the portions may overlap. It will be recognized that this particular embodiment can be performed with portions of any length, for example, 5 to 300 nucleotides, or 10 to 200 nucleotides, or 15 to 100 nucleotides, or 20 to 50 nucleotides, or in particular 30 or 100 nucleotides. In some embodiments, a predetermined range of GC content may be selectable by the user. It will also be recognized that this particular embodiment can be performed with optimized nucleotide sequences of any length.

[0132] For example, the guanine-cytosine (GC) content of non-optimized and optimized nucleotide sequences can be analyzed in the portion of the nucleotide sequence encoding EPO, in which case the guanine-cytosine (GC) content of the portion of the nucleotide sequence encoding EPO can be determined for adjacent non-overlapping portions of 30 nucleotides in length. An exemplary analysis of this is shown in Figure 11.

[0133] An exemplary GC content filter is described herein. This is merely an example, and it will be apparent to any person skilled in the art that the methods described herein may be performed using optimized nucleotide sequences and / or portions of any length. As an example, for an optimized nucleotide sequence in a list of optimized nucleotide sequences having a length of 600 nucleotides, a portion of 30 nucleotides may be selected. The GC content filter may first analyze the first 30 nucleotides of the optimized nucleotide sequence, i.e., nucleotides 1-30 of the optimized nucleotide sequence. The analysis may include determining the number of either G or C nucleotides in the portion, and determining the GC content of the portion may include dividing the number of G or C nucleotides in that portion by the total number of nucleotides in that portion. The result of this analysis provides a value that describes the proportion of G or C nucleotides in that portion, which may be a percentage, e.g., 50%, or a decimal, e.g., 0.5. If the GC content of the first portion falls outside a predetermined range of GC content, the optimized nucleotide sequence may be removed from the list of optimized nucleotide sequences.

[0134] If the GC content of the first portion falls within a predetermined range of GC content, then the GC content filter may analyze the second portion of the optimized nucleotide sequence. In this example, this could be the second 30 nucleotides of the optimized nucleotide sequence, i.e., nucleotides 31-60. The analysis of portions may be repeated for each portion until one of the following occurs: the portion is found to have a GC content outside the predetermined range of GC content (in this 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 this case, the GC content filter may keep the optimized nucleotide sequence in the list and move to the next optimized nucleotide sequence in the list). In this example, the GC content filter may move to the next optimized nucleotide sequence. If the rectoid sequence reaches its final part, i.e., nucleotides 571-600, and this final part has a GC content that falls within a predetermined range of GC content, the GC content filter may retain the optimized nucleotide sequence in its list and move to the next optimized nucleotide sequence in the list. Alternatively, each part may be 100 nucleotides long.

[0135] Although the above embodiment describes a segmental GC content filter starting from the first nucleotide and progressing to the final nucleotide, it will be recognized that this is merely an example, and the order in which the segments of the optimized nucleotide sequence are analyzed may be any order that is apparent to those skilled in the art. The GC content filter may, for example, start at the segment containing the final nucleotide (nucleotide 600 in the working example) and work backward towards the first nucleotide, nucleotide 1, or start at any position between the first and final nucleotides.

[0136] The first, final, or intermediate portion of an optimized nucleotide sequence may have a different length from other portions. This can occur, for example, if the nucleotide length of the optimized nucleotide sequence is not divisible by the nucleotide length of the portion.

[0137] Codon Adaptation Index (CAI) Analysis In some embodiments, codon adaptation index (CAI) analysis may be performed on some or all of the optimized nucleotide sequences in the 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 CAI is a measure of codon use bias and can take values ​​between 0 and 1. The list of optimized nucleotide sequences may be a list of optimized nucleotide sequences originally generated by the codon optimization algorithm, or a list of optimized nucleotide sequences that have already been filtered by one or more further algorithmic steps. A list of optimized nucleotide sequences that have already been filtered or updated by one or more additional algorithmic steps may be referred to as an updated list of optimized nucleotide sequences, or a recently updated list. Any optimized nucleotide sequences with a CAI below a predetermined CAI threshold may be removed from the list to produce an updated list.

[0138] In some embodiments, the CAI threshold is user-selectable. In some embodiments, the CAI threshold is 0.7, 0.75, 0.85, or 0.9. In certain embodiments, the CAI threshold is 0.8.

[0139] CAI is calculated for each optimized nucleotide sequence in any way that would be apparent to those skilled in the art, for example, "The codon adaptation index -- a measure of directional synonymous codon usage." The calculation may also be performed as described in "bias, and its potential applications" (Sharp and Li, 1987. Nucleic Acids Research 15(3), pp. 1281-1295); (available online from https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC340524 / ).

[0140] Performing the calculation of the codon adaptation index may involve the following or similar methods. For each amino acid of the sequence, the weight of each codon in the sequence may be represented by a parameter named relative fitness (w i ). The relative fitness may be calculated as the ratio between the observed frequency of the codon f i for that amino acid from a reference sequence set and the frequency of the most frequent synonymous codon f j . Then, the codon adaptation index of the sequence may be calculated over the length of the sequence (measured as codons) as the geometric mean of the weights associated with each codon . The reference sequence set used to calculate the codon adaptation index may be the same reference sequence set from which the codon usage table used in the method of the present invention is derived.

[0141] As described above, the CAI analysis filter may be applied as a per-part analysis as detailed herein. In other words, the CAI measure of each part of each optimized nucleotide sequence is determined, and if there is a part having a CAI below a predetermined CAI threshold, that sequence may be removed from consideration (i.e., removed from the list). Thus, by performing this method, both increased computational efficiency and a more selective filter are achieved.

[0142] Combination of further algorithmic steps Figure 7 shows that 0, 1, 2, or 3 of the motif screen filter, GC content analysis filter, and CAI analysis filter can be applied to the list of optimized nucleotide sequences in any order. Each filter may be used only once, as each filter has the same effect on the list when applied to the same list of optimized nucleotide sequences with the same input parameters. For example, if the motif screen filter and GC content analysis filter have been applied to the list of optimized nucleotide sequences, applying an additional motif screen filter or an additional GC content analysis filter to the updated list of optimized nucleotide sequences would have no effect, because any sequences in the list that have been filtered have already been removed. Figure 7 also shows that there are embodiments of the invention in which no filters are applied to the list of optimized nucleotide sequences.

[0143] Figure 8 shows an embodiment of the present invention in which only one filter is applied to the list of optimized nucleotide sequences. In this embodiment, a GC content analysis filter is selected, but it will be clear that this is illustrative, and that if only one filter is desired, a motif screen filter or a CAI filter can be selected instead.

[0144] Figure 9 shows an embodiment of the present invention in which only two filters are applied to a list of optimized nucleotide sequences. In this embodiment, the motif screen filter and the CAI analysis filter are applied in that order, but it will be clear that this is illustrative, and that any two of the motif screen filter, GC content analysis filter, and CAI analysis filter can be applied in any order if only two filters are desired. In the example in Figure 9, the motif screen filter is applied to the list of optimized nucleotide sequences to produce an updated list of optimized nucleotide sequences. Before the updated list of optimized nucleotide sequences is further filtered by the CAI analysis filter, the list may be referred to as the recently updated list of optimized nucleotide sequences. The CAI analysis filter is then applied to the recently updated list of optimized nucleotide sequences to produce an updated list of optimized nucleotide sequences or a further updated list.

[0145] Figure 10 shows a particular embodiment of the present invention in which three filters are applied to a list of optimized nucleotide sequences. In this particular embodiment, a motif screen filter, a GC content analysis filter, and a CAI analysis filter are applied in that order to produce an updated list of optimized nucleotide sequences. In alternative embodiments using three filters, it will become clear that the motif screen filter, the GC content analysis filter, and the CAI analysis filter may be applied in any order. Similar to Figure 9, between each filtering step, i.e., between the motif screen filter and the GC content analysis filter, and between the GC content analysis filter and the CAI analysis filter, the list of optimized nucleotide sequences may be referred to as the recently updated list of optimized nucleotide sequences (not shown in Figure 10). Similar to the exemplary embodiments in Figures 8 and 9, the sequence of the updated list of optimized nucleotide sequences produced at the end of any and all filtering steps is then They may be synthesized by any of the synthesis methods described herein.

[0146] A synergistic benefit may be obtained from using more than one of the further algorithmic steps for filtering. This is achieved because the input to each of the further algorithmic steps is a recently updated list of optimized nucleotide sequences, i.e., a list of already filtered sequences. This reduces the amount of processing and time required for further filtering steps, as there are fewer sequences to analyze than the list of sequences, thereby increasing the efficiency of this method.

[0147] Adjacent identical codons In some embodiments, some or all of the list of optimized nucleotide sequences may be analyzed to determine optimized nucleotide sequences having at least two, for example, three or more adjacent identical codons. This further algorithmic step may be the only further algorithmic step, or it may be performed before or after one or more of the following: motif screening, GC content analysis, and CAI analysis. The analysis may be performed partwise on each optimized nucleotide sequence, as detailed herein.

[0148] For example, a particular optimized nucleotide sequence may be analyzed and determined to contain a section that includes: CAGCAGCAG. Such a section containing a certain repeat codon can potentially halt transcription, so the sequence is removed from the list.

[0149] In some embodiments, an adjacent rarity threshold is used to determine rare codons, where codons below the adjacent rarity threshold are considered rare codons. Rare codons may be identified by comparing their usage frequency in the normalized codon usage table with the adjacent rarity threshold. Thus, the adjacent rarity threshold identifies codons that have usage above the threshold frequency enough to be included in the normalized codon usage table, but are still relatively rare among the codons in the normalized codon usage table. In some embodiments, only rare adjacent identical codons cause the optimized nucleotide sequence to be removed from the list of optimized nucleotide sequences.

[0150] The adjacent rarity threshold can be between 10 and 50%, for example between 15 and 40%, or for example between 20 and 30%, and depends on the threshold frequency used to normalize the codon usage table. Since any codon with a usage frequency below the threshold frequency does not appear in the normalized codon usage table, the adjacent rarity threshold must be greater than the threshold frequency to have an effect.

[0151] Using the same example as above, but filtering only for rare adjacent identical codons, if CAG appears in the normalized codon usage table at a frequency above the adjacent rarity threshold, sequences containing CAGCAGCAG are not removed from the list. Instead, if CAG appears in the normalized codon usage table at a frequency below the adjacent rarity threshold, sequences containing CAGCAGCAG are removed from the list.

[0152] Filters for adjacent identical codons, including, in some cases, filters for rare adjacent identical codons, can be applied at any stage after the list of optimized nucleotide sequences has been created. In other words, filters for adjacent identical codons, including, in some cases, filters for rare adjacent identical codons, can be applied in any order, along with any other further algorithmic steps.

[0153] Synthesis and expression of optimized nucleotide sequences In a further embodiment, the present invention relates to a method for synthesizing a nucleotide sequence, at least The present invention provides a method comprising: performing a computer implementation method to generate 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 which 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 vary depending on the specific application.

[0154] In some embodiments, the synthesized optimized nucleotide sequence produced by 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 terms “plasmid” or “plasmid nucleic acid vector” refer to a circular nucleic acid molecule, e.g., an artificial nucleic acid molecule. Plasmid DNA in the context of the present invention is suitable for incorporating or containing a desired nucleic acid sequence, e.g., a sequence encoding an mRNA transcript and / or an open reading frame encoding at least one protein, polypeptide, or peptide. Such plasmid DNA constructs / vectors may be expression vectors, cloning vectors, transfer vectors, etc.

[0155] A nucleic acid vector typically includes a sequence, or a portion thereof, that corresponds to (encodes) a desired mRNA transcript, for example, the open reading frame of the mRNA and a sequence corresponding to the 5'- and / or 3'UTR. 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 the 5' / 3'UTR and the 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 include a poly-A tail. The poly-A tail may be added to the mRNA transcript in a post-synthesis processing step.

[0156] In some embodiments, the nucleic acid vector includes a nucleotide sequence encoding a 5'UTR operably ligated to an optimized nucleotide sequence. In certain embodiments, the 5'UTR differs from the 5'UTR of naturally occurring mRNA encoding an amino acid sequence. In certain embodiments, the 5'UTR has the nucleotide sequence of SEQ ID NO: 19.

[0157] In some embodiments, the nucleic acid vector includes a nucleotide sequence encoding a 3'UTR operably ligated to an optimized nucleotide sequence. In certain embodiments, the 3'UTR differs from the 3'UTR of a naturally occurring mRNA encoding an amino acid sequence. In certain embodiments, the 3'UTR has the nucleotide sequence of SEQ ID NO: 20 or SEQ ID NO: 21.

[0158] For example, the nucleotide sequence of the present invention can be synthesized from a nucleic acid vector containing a 5'UTR, an optimized nucleotide sequence, and a 3'UTR (and optionally one or more stop signals at the 3' end of the optimized nucleotide sequence) to produce mRNA containing the 5'UTR, the optimized nucleotide sequence, and the 3'UTR.

[0159] In some embodiments, the nucleic acid vector includes a promoter sequence, such as an RNA polymerase promoter sequence like a T3, T7, or SP6 RNA polymerase promoter sequence.

[0160] In some embodiments, the nucleic acid vector has one or more termination signals (e.g., two or three termination signals) downstream of the 3' end of the synthesized optimized nucleotide sequence. This includes, in some embodiments, the method further includes inserting one or more termination signals at the 3' end of the synthesized optimized nucleotide sequence. In some embodiments, two or more termination signals are inserted, and the termination signals are separated by 10 base pairs or less, for example, 5 to 10 base pairs. By adding one or more termination signals downstream of the optimized nucleotide sequence, efficient termination of transcription is promoted when RNA is transcribed from plasmid DNA containing the optimized nucleotide sequence, resulting in targeted termination of in vitro transcription by one or more termination signals, thereby limiting abnormal runon transcription. In some embodiments, the nucleic acid vector includes two or more termination signals, for example, two or more, three or more, or four or more termination signals. The presence of multiple termination signals improves the efficiency of in vitro transcription termination at the targeting site.

[0161] In some embodiments, one or more stop signals have the following nucleotide sequence: 5'-X1ATCTX2TX3-3', where X1, X2, and X3 are independently selected from A, C, T, or G. In some embodiments, one or more stop signals have one of the following nucleotide sequences: TATCTGTT; and / or TTTTTT; and / or AAGCTT; and / or GAAGAGC; and / or TCTAGA. In some embodiments, one or more stop signals have the following nucleotide sequence: 5'-X1AUCUX2UX3-3', where X1, X2, and X3 are independently selected from A, C, U, or G. In some embodiments, one or more stop signals have one of the following nucleotide sequences: UAUCUGUU; and / or UUUUU; and / or AAGCUU; and / or GAAGAGC; and / or UCUAGA. In some embodiments, two or more stop signals are the following nucleotide sequences: (a) 5'-X1ATCTX2TX3-(Z N )-X4ATCTX5TX6-3' or (b)5'-X 11 ATCTX2TX3-(Z N )-X4ATCTX5TX6-(Z M )-X7ATCTX8TX9-3' is coded by, where X1, X2, X3, X4, X5, X6, X7, X8 and X9 are independently selected from A, C, T or G, and Z N This represents a spacer sequence of N nucleotides, Z M represents a spacer sequence of M nucleotides, each independently selected from A, C, T, or G, where N and / or M are independently less than or equal to 10.

[0162] Therefore, in certain embodiments of the present invention, plasmid DNA containing one or more stop signals (e.g., two or three stop signals) downstream of the 3' end of the synthesized optimized nucleotide sequence does not require linearization for in vitro transcription. Specifically, the present invention makes it possible to produce mRNA transcripts from circular nucleic acid vectors (typically superhelical), such as plasmid DNA, using SP6 / T7 RNA polymerase for in vitro transcription.

[0163] SP6 RNA polymerase In some embodiments, mRNA is synthesized by SP6 RNA polymerase. In some embodiments, the SP6 RNA polymerase is spontaneously occurring SP6 RNA polymerase. In some embodiments, the SP6 RNA polymerase is recombinant SP6 RNA polymerase. In some embodiments, the SP6 RNA polymerase includes a tag. The tag may be used to facilitate the detection or purification of the protein. In some embodiments, the tag is a his-tag, which can be used, for example, for purification by Ni-NTA affinity chromatography.

[0164] SP6 RNA polymerase is a DNA-dependent RNA polymerase with high sequence specificity for the SP6 promoter sequence. Typically, this polymerase polymerizes either RNA on single-stranded or double-stranded DNA downstream of its promoter from 5' to 3'. It catalyzes in vitro synthesis; it incorporates native and / or modified ribonucleotides into the polymerized transcript.

[0165] The sequence of bacteriophage SP6 RNA polymerase was initially described as having the following amino acid sequence (GenBank: Y00105.1):

[0166] (query number 1)

[0167] A suitable SP6 RNA polymerase for the present invention may be any enzyme having substantially the same polymerase activity as bacteriophage SP6 RNA polymerase. Therefore, in some embodiments, a suitable SP6 RNA polymerase for the present invention may be modified 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 approximately 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 cleaved protein (from the N-terminus, C-terminus, or internally), but the polymerase activity is retained. In some embodiments, the suitable SP6 RNA polymerase is a fusion protein.

[0168] In some embodiments, SP6 RNA polymerase is encoded by a gene having the following nucleotide sequence: ATGCAAGATTTACACGCTATCCAGCTCAATTAGAAGAAGAGATGTTTAATGGTGGCATTCGTCGCTTCGAAGCAGATCAACAACGCCAGATTGCAGCAGGTAGCGAGAGCGACACAGCATGGAACCGCCGCCTGTTGTCAGAACTTATTGCACCTATGGCTGAAGGCATTCAGGCTTATAAAGAAGAGTACGAAGGTAA AACAGAAGATCATGGCAACCGAGATGCTACGCGTGCGTACCTGTCTGATGGGTGATATCAAGATGTCCCTTCAGGTTGAAACGGATATCGTAGATGAAGCCGCTATGATGGGAGCAGCAGCACCTAATTTCGTACACGGTCATGACGCAAGTCACCTTATCCTTACCGTATGTGAATTGGTAGACAAGGGCGTAACTAGTATCGCTGTAATCCACG ACTCTTTTGGTACTCATGCAGACAACACCCTCACTCTTAGAGTGGCACTTAAAGGGCAGATGGTTGCAATGTATATTGATGGTAATGCGCTTCAGAAACTACTGGAGGAGCATGAAGTGCGCTGGATGGTTGATACAGGTATCGAAGTACCTGAGCAAGGGGAGTTCGACCTTAACGAAATCATGGATTCTGAATACGTATTTGCCTAA (SEQ ID NO: 2).

[0169] Suitable genes encoding SP6 RNA polymerase suitable for 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 Sequence ID No. 2.

[0170] SP6 RNA polymerases suitable for the present invention may be commercially available products from, for example, Ambion, New England Biolabs (NEB), Promega, and Roche. SP6 may be ordered and / or custom-designed from commercial or non-commercial sources according to the amino acid sequence of SEQ ID NO: 1 described herein or a variant of SEQ ID NO: 1. The SP6 RNA polymerase may be modified to enhance RNA polymerase activity, resulting in high fidelity / high efficiency / high capacity, such as mutations in the SP6 RNA polymerase gene or post-translational modifications of the SP6 RNA polymerase itself. Examples of such modified SP6 include Ambion's SP6 RNA Polymerase-Plus®, NEB's HiScribe SP6, and Promega's RiboMAX® and Riboprobe® Systems.

[0171] In some embodiments, the SP6 RNA polymerase is heat-stable. In certain embodiments, the amino acid sequence of the SP6 RNA polymerase for use with the present invention includes one or more mutations from wild-type SP6 polymerase that activate the enzyme at temperatures in the range of 37°C to 56°C. In some embodiments, the SP6 RNA polymerase for use with the present invention functions at an optimal temperature of 50°C to 52°C. In other embodiments, the SP6 RNA polymerase for use with 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 with the present invention has a half-life of 60 to 120 minutes (e.g., 70 to 100 minutes, or 80 to 90 minutes) at 50°C.

[0172] In some embodiments, the suitable SP6 RNA polymerase is a fusion protein. For example, SP6 RNA polymerase may contain one or more tags to facilitate the isolation, purification, or solubility of the enzyme. Suitable tags may be located at the N-terminus, C-terminus, and / or internally. Non-limiting examples of suitable tags include calmodulin-binding protein (CBP); Fasciola hepatica 8kDa antigen (Fh8); FLAG tag peptide; glutathione-S-transferase (GST); histidine tag (e.g., hexahistidine tag (His6)); maltose-binding protein (MBP); N-utilizing agent (NusA); small ubiquitin-like modifier (SUMO) fusion tag; streptavidin-binding peptide (STREP); tandem affinity purification (TAP); and thioredoxin (TrxA). Other tags may also be used in this invention. These and other fusion tags are, for example, those described by Costa et al. This is described in Frontiers in Microbiology 5 (2014): 63 and 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.

[0173] SP6 Promoter Any promoter recognizable by SP6 RNA polymerase may be used in the present invention. Typically, the SP6 promoter contains 5'ATTTAGTGACACTATAG-3' (SEQ ID NO: 3). Variants of the SP6 promoter have been discovered and / or created to optimize the recognition and / or binding of that promoter to SP6. Non-limiting variants include, but are not limited to, the following: 5'-ATTTAGGGGACACTATAGAAGAG-3';5'-ATTTAGGGGACACTATAGAAGG-3';5'-ATTTAGGGGACACTATAGAAGGG-3';5'-ATTTAGGTGACACTATAGAA-3'; 5'-ATTTAGGTGACACTATAGAAGA-3'; 5'-ATTTAGGTGACACTATAGAAGAG-3'; 5'-ATTTAGGTGACACTATAGAAGG-3'; 5'-CATACGATTTAGGTGACACTATAG-3' (Sequences 4 to 13) When N is used in a nucleotide sequence, N can be A, C, T, or G.

[0174] Furthermore, an SP6 promoter suitable for the present invention may be approximately 95%, 90%, 85%, 80%, 75%, or 70% identical or homologous to any one of SEQ ID NOs: 4 to SEQ ID NOs: 13. Additionally, an SP6 promoter suitable for the present invention may contain one or more additional nucleotides at the 5' and / or 3' positions relative to any of the promoter sequences described herein.

[0175] T7 RNA polymerase In some embodiments, mRNA is synthesized by T7 RNA polymerase.

[0176] T7 RNA polymerase is a DNA-dependent RNA polymerase with high sequence specificity for the T7 promoter sequence. Typically, this polymerase catalyzes the 5'→3' in vitro synthesis of RNA on either single-stranded or double-stranded DNA downstream of its promoter; and incorporates native and / or modified ribonucleotides into the polymerized transcript.

[0177] In some embodiments, the T7 RNA polymerase is thermally stable. In certain embodiments, the amino acid sequence of the T7 RNA polymerase for use with the present invention includes one or more mutations from wild-type T7 polymerase that activate the enzyme at temperatures in the range of 37°C to 56°C. An example of a suitable RNA polymerase is NEB's Hi-T7® RNA polymerase. In some embodiments, the T7 RNA polymerase for use with the present invention functions at an optimal temperature of 50°C to 52°C. In other embodiments, the T7 RNA polymerase for use with 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 with the present invention has a half-life of 60 to 120 minutes (e.g., 70 to 100 minutes, or 80 to 90 minutes) at 50°C.

[0178] T7 promoter Any promoter that can be recognized by T7 RNA polymerase may be used in the methods described herein. Typically, the T7 promoter is Contains 5'-TAATACGACTCACTATAG-3' (SEQ ID NO: 14).

[0179] Post-synthesis processing In some embodiments, the method of the present invention further includes other steps of capping and / or tailing the synthesized mRNA.

[0180] Typically, a 5' cap and / or 3' tail may be added after synthesis. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect mRNA from exonuclease degradation.

[0181] The 5' cap is typically added as follows: First, an RNA terminal 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 phosphate via guanylyltransferase, thereby creating a 5'5'5' triphosphate bond; and then the 7-nitrogen of guanine is methylated by a 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 certain embodiments, the cap structure is m7G(5')ppp(5')(2'OMeG). Additional cap structures are described in U.S. Patent Publication No. 2016 / 0032356 and U.S. Provisional Patent Application No. 62 / 464,327, filed on February 27, 2017, and are incorporated herein by reference.

[0182] Typically, the tail structure includes a poly(A) and / or poly(C) tail. The polyA or polyC tail on the 3' end of the mRNA typically contains, respectively, 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, and at least 500 adenosine or cytosine nucleotides. It contains 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 kb of adenosine or cytosine nucleotides. In some embodiments, the poly-A or poly-C tail contains about 10 to 800 adenosine or cytosine nucleotides (e.g., about 10 to 200 adenosine or cytosine nucleotides, about 10 to 300 adenosine or cytosine nucleotides, about 10 to 400 adenosine or cytosine nucleotides, about 10 to 500 adenosine or cytosine nucleotides, about 10 to 550 adenosine or cytosine nucleotides, about 10 to 600 adenosine or cytosine nucleotides, about 50 to 600 adenosine or cytosine nucleotides, about 100 to 600 adenosine or cytosine nucleotides, about 150 to 600 adenosine or cytosine nucleotides, about 200 to 60 Each comprises (0 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). In some embodiments, the tail structure includes combinations of poly(A) tails and poly(C) tails having various lengths as described herein. In some embodiments, the tail structure contains 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 contains at least 50%, 55%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, or 99% cytosine nucleotides.

[0183] As described herein, the addition of a 5' cap and / or 3' tail is in Capping and / or tailing facilitates the detection of interrupted transcripts generated during in vitro synthesis, as without them, these immature and interrupted mRNA transcripts may be too small to be detected. Therefore, in some embodiments, a 5' cap and / or 3' tail are attached to the synthesized mRNA before the mRNA is tested for purity (e.g., the level of interrupted transcripts present in the mRNA). In some embodiments, the 5' cap and / or 3' tail are attached to the synthesized mRNA before the mRNA is purified as described herein. In other embodiments, the 5' cap and / or 3' tail are attached to the synthesized mRNA after the mRNA has been purified as described herein.

[0184] In some embodiments, capping and tailing occur during in vitro transcription.

[0185] Conditions for mRNA synthesis reaction mixture In some embodiments, the concentration of RNA polymerase in the reaction mixture may be about 1–100 nM, 1–90 nM, 1–80 nM, 1–70 nM, 1–60 nM, 1–50 nM, 1–40 nM, 1–30 nM, 1–20 nM, or about 1–10 nM. In certain embodiments, the concentration of RNA polymerase is about 10–50 nM, 20–50 nM, or 30–50 nM. Concentrations of RNA polymerase ranging from 100 to 10000 Units / ml may be used, for example, concentrations of 100-9000 Units / ml, 100-8000 Units / ml, 100-7000 Units / ml, 100-6000 Units / ml, 100-5000 Units / ml, 100-1000 Units / ml, 200-2000 Units / ml, 500-1000 Units / ml, 500-2000 Units / ml, 500-3000 Units / ml, 500-4000 Units / ml, 500-5000 Units / ml, 500-6000 Units / ml, 1000-7500 Units / ml, and 2500-5000 Units / ml can be used.

[0186] The concentrations of each ribonucleotide (e.g., ATP, UTP, GTP, and CTP) in the reaction mixture range from approximately 0.1 mM to approximately 10 mM, for example, approximately 1 mM to approximately 10 mM, approximately 2 mM to approximately 10 mM, approximately 3 mM to approximately 10 mM, approximately 1 mM to approximately 8 mM, approximately 1 mM to approximately 6 mM, approximately 3 mM to approximately 10 mM, approximately 3 mM to approximately 8 mM, approximately 3 mM to approximately 6 mM, and approximately 4 mM to approximately 5 mM. In some embodiments, each ribonucleotide is present in approximately 5 mM of the reaction mixture. In some embodiments, rNTPs (e.g., ATP, GTP, CTP) used in the reaction are also present. The total concentration of rNTPs (combinations of ATP, GTP, CTP, and UTP) is in the range of 1 mM to 40 mM. In some embodiments, the total concentration of rNTPs used in the reaction (e.g., combinations of ATP, GTP, CTP, and UTP) 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 rNTP concentration is less than 30 mM. In some embodiments, the total rNTP concentration is less than 25 mM. In some embodiments, the total rNTP concentration is less than 20 mM. In some embodiments, the total rNTP concentration is less than 15 mM. In some embodiments, the total rNTP concentration is less than 10 mM.

[0187] In certain embodiments, the concentration of each rNTP in the reaction mixture is optimized based on the frequency of each nucleic acid in the nucleic acid sequence encoding a given mRNA transcript. Specifically, such a sequence-optimized reaction mixture contains the respective ratios of four rNTPs (e.g., ATP, GTP, CTP, and UTP) corresponding to the ratios of these four nucleic acids (A, G, C, and U) in the mRNA transcript.

[0188] In some embodiments, an initiation nucleotide is added to the reaction mixture before the initiation of in vitro transcription. The initiation nucleotide is the nucleotide corresponding to the first nucleotide (+1 position) of the mRNA transcript. The initiation nucleotide may be added, in particular, to improve the initiation rate of RNA polymerase. The initiation nucleotide can be a nucleoside monophosphate, a nucleoside diphosphate, or a nucleoside triphosphate. The initiation nucleotide can be a mononucleotide, a dinucleotide, or a trinucleotide. In embodiments where the first nucleotide of the mRNA transcript is G, the initiation nucleotide is typically GTP or GMP. In certain embodiments, the initiation nucleotide is a cap analog. The cap analog is G[5']ppp[5']G, m 7 G[5']ppp[5']G, m3 2,2,7 G[5']ppp[5']G, m2 7,3’-O G[5']ppp[5']G (3'-ARCA), m2 7,2’-OGpppG(2'-ARCA), m2 7,2’-O GppspG D1 (β-S-ARCA D1) and m2 7,2’-O It can be selected from GppspG D2 (β-S-ARCA D2).

[0189] In certain embodiments, the first nucleotide of the RNA transcript is G, the start nucleotide is a cap analog of G, and the corresponding rNTP is GTP. In such embodiments, the cap analog is present in excess in the reaction mixture compared to GTP. In some embodiments, the cap analog is added at starting concentrations ranging from 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.

[0190] More typically, in the context of the present invention, the cap structure, such as a cap analog, is added to the mRNA transcript obtained during in vitro transcription only after the mRNA transcript has been synthesized, for example, in a post-synthesis processing step. Typically, in such embodiments, the mRNA transcript is first purified (for example, by tangential flow filtration) before the cap structure is added.

[0191] RNA polymerase reaction buffers typically contain salts / buffers, such as Tris, HEPES, ammonium sulfate, sodium bicarbonate, sodium citrate, sodium acetate, potassium phosphate, sodium phosphate, sodium chloride, and magnesium chloride.

[0192] The pH of the reaction mixture may be approximately 6–8.5, 6.5–8.0, or 7.0–7.5, and in some embodiments, the pH is 7.5.

[0193] A reaction mixture is formed by combining a DNA template (e.g., as described above, in an amount / concentration sufficient to provide the desired amount of RNA), RNA polymerase reaction buffer, and RNA polymerase. The reaction mixture is incubated at approximately 37°C to approximately 56°C for 30 minutes to 6 hours, for example, approximately 60 minutes to approximately 90 minutes. In some embodiments, incubation is carried out at approximately 37°C to approximately 42°C. In other embodiments, incubation is carried out at approximately 43°C to approximately 56°C, for example, approximately 50°C to approximately 52°C. As demonstrated herein, in The yield of precisely terminated mRNA transcripts obtained in a vitro transcription reaction can be significantly increased by using a template containing the DNA sequence, with one or more termination signals described herein at the end of the DNA sequence encoding the mRNA transcript of interest, and carrying out the reaction at a temperature of approximately 50°C to approximately 52°C.

[0194] In some embodiments, approximately 5 mM NTP, approximately 0.05 mg / mL RNA polymerase, and approximately 0.1 mg / mL DNA template are incubated in a suitable RNA polymerase reaction buffer (final reaction mixture pH approximately 7.5) at approximately 37°C to approximately 42°C for 60 to 90 minutes. In other embodiments, approximately 5 mM NTP, approximately 0.05 mg / mL RNA polymerase, and approximately 0.1 mg / mL DNA template are incubated in a suitable RNA polymerase reaction buffer (final reaction mixture pH approximately 7.5) at approximately 50°C to approximately 52°C for 60 to 90 minutes.

[0195] In some embodiments, the reaction mixture contains a double-stranded DNA template having an RNA polymerase-specific promoter, RNA polymerase, an RNase inhibitor, pyrophosphatase, 29 mM NTP, 10 mM DTT, and reaction buffer (800 mM HEPES, 20 mM spermidine, 250 mM MgCl2, pH 7.7 for 10x), and a sufficient amount (QS) of RNase-free water to bring the reaction to the desired volume; the reaction mixture is then incubated at 37°C for 60 minutes. The polymerase reaction is then quenched by adding DNase I and DNase I buffer (100 mM Tris-HCl, 5 mM MgCl2, and 25 mM CaCl2, pH 7.6 for 10x) to facilitate the digestion of the double-stranded DNA template in preparation for purification. This embodiment has been shown to be sufficient to produce 100 grams of mRNA.

[0196] In some embodiments, the reaction mixture comprises NTP at a concentration in the range of 1 to 10 mM, DNA template at a concentration in the range of 0.01 to 0.5 mg / ml, and RNA polymerase at a concentration in the range of 0.01 to 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.

[0197] nucleotide 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, the mRNA transcript according to the present invention is synthesized using natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine) and the following: nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynylcytidine, C-5 propynyluridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5 - Iodouridine, C5-propynyluridine, C5-propynylcytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxyadenosine, 8-oxoguanosine, O(6)-methylguanine, pseudouridine (e.g., N-1-methylpseudridine), 2-thiouridine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inter - Synthesized using one or more kalate 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 bonds).

[0198] In some embodiments, the mRNA contains one or more non-standard nucleotide residues. These non-standard nucleotide residues may 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 International Publication No. 2011012316. The mRNA may be defined as RNA in which 25% of the U residues are 2-thiouridine and 25% of the C residues are 5-methylcytidine. Teachings relating to the use of RNA are disclosed in U.S. Patent Application Publication No. 20120195936 and International Publication No. 2011012316, both of which are incorporated herein by reference in their entirety. The presence of non-standard nucleotide residues may make mRNA more stable and / or less immunogenic than control mRNA having the same sequence but containing only standard residues. In further embodiments, mRNA may contain 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 nucleic acid base modifications. Some embodiments may further include additional modifications to the furanose ring or nucleic acid bases. Additional modifications may include, for example, sugar modifications or substitutions (e.g., one or more of 2'-O-alkyl modifications, locked nucleic acids (LNAs)). In some embodiments, RNA may be complexed or hybridized with additional polynucleotides and / or peptide polynucleotides (PNAs). In some embodiments where the sugar modification is a 2'-O-alkyl modification, such modifications may include, but are not limited to, 2'-deoxy-2'-fluoro modifications, 2'-O-methyl modifications, 2'-O-methoxyethyl modifications, and 2'-deoxy modifications.In some embodiments, any of these modifications may be present individually or in combination in amounts exceeding 0-100% of the nucleotides, for example, 0%, 1%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, or 100% of the constituent nucleotides.

[0199] Transfection and screening of optimized nucleotide sequences in cells In some embodiments, the method of the present invention further includes transfecting cells with the synthesized optimized nucleotide sequence 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 includes synthesizing a reference nucleotide sequence and at least one synthesized optimized nucleotide sequence produced according to the method of the present invention, and contacting each nucleotide sequence with separate cells or organisms. In a typical embodiment, cells or organisms contacted with at least one synthesized optimized nucleotide sequence increase the 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 cells or organisms 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 produced by a method other than the method of the present invention.

[0200] It may be desirable to confirm that the synthesized optimized nucleotide sequence, generated according to the method of the present invention, increases the expression of the encoded protein when transfected into cells. To experimentally verify the increase in protein expression and production, well-known methods in the art, such as Western blotting, are suitable. Furthermore, multiple synthetically optimized nucleotide sequences produced by the method of the present invention can be screened to identify the optimized nucleotide sequence that produces the highest protein yield. In some embodiments, the expression level of the protein encoded by the synthetically optimized nucleotide sequence increases by at least twofold, for example, at least threefold or fourfold.

[0201] 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 range of established methods. These methods may vary depending on the characteristics of the encoded protein of interest. In the context of codon optimization, it may be important to experimentally verify the functional activity of the protein encoded by the optimized nucleotide sequence synthesized in vitro or in vivo to confirm that the expression of the encoded protein produces the desired functional effect. For example, enzyme activity assays 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 evaluate the activity of the human cystic fibrosis transmembrane conductance regulator (hCFTR) protein expressed from mRNA encoding the codon-optimized hCFTR sequence produced by the method of the present invention. This assay monitors the chloride transport function of epithelial cells transfected with hCFTR mRNA.

[0202] Therapeutic applications The present invention provides a synthesized, optimized nucleotide sequence, generated according to the method of the present invention, for use in therapeutic applications.

[0203] In the field of mRNA therapy, codon optimization can be used to increase the expression of mRNA-encoded functional proteins in target cells, thereby improving protein deficiencies in various disorders such as cystic fibrosis (CF), primary ciliary dysplasia (PCD), pulmonary arterial hypertension (PAH), and idiopathic pulmonary fibrosis (IPF).

[0204] In certain aspects of the present invention, the optimized nucleotide sequence encodes the human cystic fibrosis transmembrane conductance regulator (hCFTR) protein: MQRSPLEKASVVSKLFFSWTRPILRKGYRQRLELSDIYQIPSVDSADNLSEKLEREWDRELASKKNPKLINALRRCFFWRFMFYGIFLYLGEVTKAVQPLLLGRIIASYDPDNKEERSIAIYLGIGLCLLFIVRTLLLHPAIFGLHHIGMQMRIAMFSLIYKKTLKLSSRVLDKISIGQL VSLLSNNLNKFDEGLALAHFVWIAPLQVALLMGLIWELLQASAFCGLGFLIVLALFQAGLGRMMMKYRDQRAGKISERLVITSEMIENIQSVKAYCWEEAMEKMIENLRQTELKLTRKAAYVRYFNSSAFFFSGFFVVFLSVLPYALIKGIILRKIFTTISFCIVLRMAVTRQFPWAVQT WYDSLGAINKIQDFLQKQEYKTLEYNLTTTEVVMENVTAFWEEGFGELFEKAKQNNNNRKTSNGDDSLFFSNFSLLGTPVLKDINFKIERGQLLAVAGSTGAGKTSLLMVIMGELEPSEGKIKHSGRISFCSQFSWIMPGTIKENIIFGVSYDEYRYRSVIKACQLEEDISKFAEKDNIV LGEGGITLSGGQRARISLARAVYKDADLYLLDSPFGYLDVLTEKEIFESCVCKLMANKTRILVTSKMEHLKKADKILILHEGSSYFYGTFSELQNLQPDFSSKLMGCDSFDQFSAERRNSILTETLHRFSLEGDAPVSWTETKKQSFKQTGEFGEKRKNSILNPINSIRKFSIVQKTPLQ (Sequence ID 15)

[0205] In a particular embodiment, the optimized nucleotide sequence encoding the hCFTR protein according to the present invention shares at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 26 and encodes a CFTR protein having the amino acid sequence of SEQ ID NO: 15. In a particular embodiment, the optimized nucleotide sequence encoding the hCFTR protein according to the present invention is SEQ ID NO: 26. In a particular embodiment, the optimized nucleotide sequence encoding the hCFTR protein according to the present invention shares at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 27 and encodes an hCFTR protein having the amino acid sequence of SEQ ID NO: 15. In a particular embodiment, the optimized nucleotide sequence encoding the hCFTR protein according to the present invention is SEQ ID NO: 27. In a particular embodiment, the optimized nucleotide sequence encoding the hCFTR protein according to the present invention shares at least 85%, 88%, 90%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 28 and encodes an hCFTR protein having the amino acid sequence of SEQ ID NO: 15. In a particular embodiment, the optimized nucleotide sequence encoding the hCFTR protein according to the present invention is SEQ ID NO: 28.

[0206] In certain embodiments, the present invention provides nucleic acids comprising an optimized nucleotide sequence encoding the hCFTR protein according to the present invention. In certain embodiments, the present invention provides mRNA comprising an optimized nucleotide sequence encoding the hCFTR protein according to the present invention. In some embodiments, the mRNA comprising an optimized nucleotide sequence encoding the hCFTR protein according to the present invention also comprises 5' and 3' UTR sequences. Exemplary 5' and 3' UTR sequences are shown below: Exemplary 5' UTR sequence GGACAGAUCGCCUGGAGACGCCAUCCACGCUGUUUUGACCUCCAUAGAAGACACCGGGACCGAUCCAGCCUCCGCGGCCGGGAACGGUGCAUUGGAACGCGGAUUCCCCGUGCCAAGAGUGACUCACCGUCCUUGACACG (SEQ ID NO: 16) Exemplary 3'UTR sequence CGGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUG GCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAGCU (Sequence No. 17) or GGGUGGCAUCCCUGUGACCCCUCCCCAGUGCCUCUCCUGGCCCUGGAAGUUGCCACUCCAGUGCCCACCAGCCUUGUCCUAAUAAAAUUAAGUUGCAUCAAAGCU (SEQ ID NO: 18)

[0207] The synthesized optimized nucleotide sequences produced according to the method of the present invention have also been found to be used in mRNA vaccines. In the context of prophylactic mRNA vaccines, codon optimization can be used to maximize the expression of recombinant antigens encoded by mRNA delivered to the target for optimal antigenic activity, thereby generating protective immunity against pathogens.

[0208] Similarly, in the field of cancer immunotherapy, codon optimization can be used to maximize the expression of recombinant tumor neoantigens encoded by mRNA delivered to the target, thereby generating an adaptive immune response against abnormal tumor cells expressing the neoantigen.

[0209] Biotechnology applications In the field of biotechnology, particularly in the context of recombinant protein production, codon optimization can be used to increase the production of a target protein within host cells such as bacteria, yeast, insects, plants, or mammalian cells.

[0210] For example, the method of the present invention can be used to optimize the protein expression yield of recombinant insulin protein produced in Escherichia coli. Recombinant protein expression can also occur, for example, within host cells or in cell-free protein extracts 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]

[0211] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention. [Examples]

[0212] Example 1. Generation of optimized nucleotide sequences This embodiment illustrates a process that yields an optimized nucleotide sequence according to the present invention, which is optimized to produce a full-length transcript during in vitro synthesis and results in high levels of expression of the encoded protein.

[0213] This process combines the codon optimization method shown in Figure 1 with a series of filtering steps illustrated in Figure 10 to generate a list of optimized nucleotide sequences. Specifically, as shown in Figure 1, the process receives an amino acid sequence of interest and a first codon usage table that reflects the frequency of each codon in a given organism (i.e., human codon usage priority in the context of this embodiment). The process then removes codons from the first codon usage table if their usage frequency is below a threshold frequency (10%). The codon usage frequencies of codons not removed in the first step are normalized to generate a normalized codon usage table.

[0214] Normalization of the codon usage table involves redistributing the frequency values ​​for each removed codon; the frequency of a particular removed codon is added to the frequency of other codons that share an amino acid with the removed codon. In this example, the redistribution is performed on the codons that were not removed from the table. The process can be carried out according to exemplary methods, such as those described in relation to Figures 3 and 4B, in proportion to the frequency of codon use. The process generates a list of optimized nucleotide sequences using a normalized codon usage table. Each of the optimized nucleotide sequences codes for the desired amino acid sequence.

[0215] As illustrated in Figure 10, the list of optimized nucleotide sequences is further processed by applying a motif screening filter, a guanine-cytosine (GC) content analysis filter, and a codon adaptation index (CAI) analysis filter in that order to generate an updated list of optimized nucleotide sequences. The motif screening filter, illustrated in Figure 6, is used to remove sequences that may inhibit transcription or translation. The GC content analysis filter performs the process illustrated in Figure 11.

[0216] As shown in the following examples, this process yields an optimized nucleotide sequence encoding the desired amino acid sequence. The nucleotide sequence yields a full-length transcript during in vitro synthesis, resulting 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. [Examples]

[0217] Example 2. Codon optimization for generating nucleotide sequences with high CAI scores improves protein yield. This example demonstrates that codon-optimized protein-coding sequences with a codon adaptation index (CAI) of approximately 0.8 or higher are superior to codon-optimized protein-coding sequences with a CAI of less than 0.8.

[0218] Codon optimization was performed on the wild-type amino acid sequence of human erythropoietin (hEPO). hEPO is a protein hormone secreted by the kidney in response to low levels of oxygen in cells (hypoxia). hEPO is essential for erythropoiesis, the production of red blood cells. Recombinant hEPO is commonly used to treat anemia, a condition characterized by a decrease in red blood cell or hemoglobin counts, which can occur in patients with chronic kidney disease or those undergoing cancer chemotherapy.

[0219] Using different codon optimization algorithms, a total of five novel codon-optimized nucleotide sequences (#1-#5) encoding hEPO were generated. Nucleotide sequences #4 and #5 were generated according to the method of the present invention, as shown in Example 1. For reference, nucleotide sequences having codon-optimized hEPO coding sequences are provided as those previously experimentally validated both in vitro and in vivo. The reference nucleotide sequence (SEQ ID NO: 19) was found to provide superior protein yield compared to wild-type nucleotide sequences and other codon-optimized nucleotide sequences encoding the hEPO protein. The characteristics of each of the five nucleotide sequences regarding CAI, GC content, codon frequency distribution (CFD), and the presence of negative CIS elements and negative repeat elements are summarized in Table 1.

[0220] [Table 1]

[0221] To test the protein yield from each codon-optimized sequence, six nucleic acid vectors were prepared containing one of the six nucleotide sequences encoding the hEPO protein, flanked by identical 3' and 5' untranslated sequences (3'UTR), and preceded by an RNA polymerase promoter. These nucleic acid vectors served as templates for in vitro transcription reactions to provide six batches of mRNA containing the six codon-optimized nucleotide sequences (reference and nucleotide sequences #1-#5). Capping and tailing were performed separately. Each of the capped and tailed mRNAs was transfected separately into the cell line (HEK293). The expression levels of the encoded hEPO protein were evaluated by ELISA. The results of this experiment are summarized in Figure 12.

[0222] As can be seen in Figure 12, the highest level of expression was observed with nucleotide sequence #3 (SEQ ID NO: 22), which produced nearly twice the amount of hEPO protein as the experimentally validated reference nucleotide sequence. A trend toward higher protein yield could be observed for sequences that depended on their CAI (see Table 1). Nucleotide sequence #3, which had the highest protein yield, also had the highest CAI. Nucleotide sequences #4 (SEQ ID NO: 23) and #5 (SEQ ID NO: 24), which had the second and third highest yields, also had the second and third highest CAIs. Nucleotide sequences #1 (SEQ ID NO: 20) and #2 (SEQ ID NO: 21), which had the lowest performance, also had the lowest CAI. Incidentally, 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 the codon-optimized sequences tested, but all did not perform as well as nucleotide sequences #3, #4, and #5, which had low GC content. Notably, the nucleotide sequences #1 and #2, which had the lowest performance, also exhibited higher CFD (Computational Fluid Dynamics).

[0223] In summary, the data from this embodiment demonstrate that codon optimization of therapeutically relevant nucleotide sequences to achieve a CAI of approximately 0.8 or higher results in a greater protein yield than, for example, codon optimization to achieve a nucleotide sequence with the highest possible GC content. [Examples]

[0224] Example 3. Codon optimization of the CFTR mRNA sequence to increase CAI results in higher protein expression. This example confirms that codon-optimized protein coding sequences with a codon adaptation index (CAI) of approximately 0.8 or higher are superior to codon-optimized protein coding sequences with a CAI of less than 0.8.

[0225] The hEPO protein tested in Example 1 is a relatively short polypeptide whose amino acid sequence is encoded by a 495-nucleotide sequence. To determine whether the findings from Example 1 apply to much longer nucleotide sequences encoding larger proteins, codon optimization was performed on human cystic fibrosis transmembrane conductance regulator (hCFTR). hCFTR is encoded by a 4440-nucleotide sequence, meaning its sequence is approximately 10 times longer than the hEPO coding sequence.

[0226] Mutations in the gene encoding the hCFTR protein cause cystic fibrosis (CF), the most common genetic disorder in the Caucasian population. This is characterized by abnormal transport of chloride and sodium through the epithelium, which most critically affects the lungs, and thick, viscous secretions affecting the pancreas, liver, and intestines. mRNA encoding a codon-optimized hCFTR coding sequence is being developed as a novel therapeutic for treating CF.

[0227] Codon optimization was performed on native hCFTR amino acid sequences 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. As a reference, a nucleotide sequence having a codon-optimized hCFTR coding sequence using a different algorithm was provided (SEQ ID NO: 25). This reference nucleotide sequence (SEQ ID NO: 25) had been previously experimentally validated both in vitro and in vivo. The reference nucleotide sequence had been found to provide superior protein yield compared to other previously tested codon-optimized nucleotide sequences encoding hCFTR proteins. Compared to the reference nucleotide sequence, the CAI and GC content % of the codon-optimized hCFTR#2 and hCFTR#3 sequences were significantly increased. Furthermore, the codon frequency distribution (CFD) % was 0% compared to 6% for the reference nucleotide sequence, indicating successful removal of rare codon clusters detrimental to translation efficiency. Additional filtering to remove negative regulatory motifs significantly reduced the number of negative cis-regulation (CIS) elements in hCFTR #2 and hCFTR #3 (see Table 2).

[0228] [Table 2]

[0229] To test the protein yield from each codon-optimized sequence, four nucleic acid vectors were prepared, each containing an expression cassette preceded by an RNA polymerase promoter, with one of the four nucleotide sequences encoding the hCFTR protein adjacent to identical 3' and 5' untranslated sequences (3' and 5' UTR). These nucleic acid vectors served as templates for in vitro transcription reactions to provide four batches of mRNA containing the four codon-optimized nucleotide sequences (reference and hCFTR #1-#3). Capping and tailing were performed separately.

[0230] Each of the capped and tailed mRNAs was transfected separately into the cell line (HEK293). 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 developed 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. Codon-optimized nucleotide sequences hCFTR #2 and hCFTR #3, both with a CAI of 0.89, resulted in significantly higher yields of encoded hCFTR protein compared to the reference nucleotide sequence and hCFTR #1, both with a CAI of 0.7. This effect was more pronounced at 24 hours (see Figure 13B), which is thought to be due to the relatively rapid degradation of mRNA in HEK293 cells after transfection.

[0231] The data from this embodiment demonstrate that codon optimization of therapeutically relevant nucleotide sequences (hCFTRs) to achieve a CAI of approximately 0.8 or higher yields greater protein yields, especially when combined with optimization of their CFD and GC content, as well as removal of any negative CIS elements from the nucleic acid sequence. The data from this embodiment also confirm that codon optimization of hCFTR mRNA by the present invention yields significantly higher hCFTR protein yields in human cells compared to nucleotide sequences codon-optimized using different algorithms. [Examples]

[0232] Example 4. Codon optimization of CFTR nucleotide sequences leads to increased functional activity in cells. This example demonstrates that codon optimization of the hCFTR nucleotide sequence by the method of the present invention does not affect the functional activity of hCFTR in human cells.

[0233] The administration of hCFTR mRNA is intended to induce its uptake by airway epithelial cells in CF patients, followed by its internalization into the cytoplasm of target cells. Once cellular uptake is achieved, the hCFTR mRNA is translated into the normal hCFTR protein, which is then processed by the cell's endocrine pathway, resulting in the intracellular localization of the hCFTR protein to the apical membrane. This approach improves the functional CFTR deficiency in the lungs of CF patients by producing a functional hCFTR protein in the airway epithelium upon administration of hCFTR mRNA. It is thought that codon optimization of the nucleotide sequence of hCFTR mRNA can increase the expression level of the functional hCFTR protein, thereby enabling greater expression of the functional hCFTR protein in target airway epithelial cells of CF patients.

[0234] Codon optimization has been reported to come at the cost of reduced functional activity and consequent loss of efficacy of the encoded protein, as the process may remove information encoded in nucleotide sequences that are crucial for controlling protein translation and ensuring proper folding of the nascent polypeptide chain (Mauro & Chappe). (Ill, Trends Mol Med. 2014; 20(11):604-13). To test the functional activity of the hCFTR protein expressed from the codon-optimized sequence generated using the codon optimization method as shown in Example 1, the hCFTR mRNA produced in Example 2 was tested using the Ussing chamber assay. This assay evaluates the functional activity of the protein expressed from the hCFTR mRNA by monitoring the chloride transport function of epithelial cells transfected with the mRNA using epithelial voltage clamp. Specifically, the functional activity of the hCFTR protein expressed from mRNA containing the control hCFTR coding sequence (SEQ ID NO: 25) or the coding sequences 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 (FRT) epithelial cells. FRT epithelial cells are commonly used as a model for studying human airway epithelial cell function. FRT epithelial cells were cultured in a monolayer on a Snapwell® filter insert and transfected with four hCFTR mRNAs. The four hCFTR mRNAs were produced as described in Example 2. A control mRNA, previously validated in this assay, was used as a reference standard.

[0235] Correctly translated and localized hCFTR proteins produced from hCFTR mRNA increase the short-circuit current (ISC) output in a Ussing epithelial voltage clamp device when CFTR agonists (forskolin and VX-770 [Kalydeco®]) are applied. Application of the CFTR antagonist CFTRinh-172 blocks hCFTR. The ISC current polarity agreement in this assay records the sodium current from apex to basolateral and the chloride current from basolateral to apex as negative values; therefore, if transfection of the test hCFTR mRNA produces a high negative value, it can be concluded that the encoded hCFTR protein is functional (Figure 14A). Furthermore, by transfecting with equal amounts of mRNA, it is possible to evaluate whether the mRNA produces a higher yield of hCFTR protein, as there is a correlation between protein yield and activity. Transfecting FRT epithelial cells with mRNA containing the hCFTR #1 coding sequence resulted in activity equivalent to that obtained when transfected with control mRNA containing the hCFTR coding sequence (Figure 14B). mRNA containing the nucleotide sequence encoding hCFTR produced by the method of the present invention showed a significant increase in activity. Consistent with the higher protein yield observed in Example 2, the hCFTR protein produced from mRNA encoding hCFTR #2 exhibited more than twice the activity of the control mRNA, and the hCFTR protein produced from mRNA encoding hCFTR #3 exhibited three times the activity of the control mRNA. This confirms that the higher protein yields obtained from hCFTR #2 and hCFTR #3 observed in Example 2 directly correlate with higher functional activity, demonstrating that codon optimization by the method of the present invention does not negatively affect the functional activity of the encoded protein.

[0236] In summary, codon optimization by the present invention results in higher expression of encoded proteins in human cells, and the expressed proteins provide full functional activity in model systems highly relevant to human therapeutics.

Example

[0237] Example 5. Codon optimization of the DNAI1 mRNA sequence to increase CAI results in higher protein expression. The data of this example demonstrate that codon optimization of additional therapeutically relevant nucleotide sequences (DNAI1) to achieve a CAI of about 0.8 or higher, especially in combination with optimization of its CFD and GC content and removal of any negative CIS elements from the nucleic acid sequence, results in a greater protein yield in cells. The data of this example also confirm that the CAI value is positively correlated with the protein expression yield for codon-optimized mRNA generated according to the method of the present invention.

[0238] Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder characterized by abnormal cilia and flagella lining the airways, genital tract, and other organs and tissues. Symptoms can appear as early as birth, with respiratory difficulties, and affected individuals develop frequent respiratory infections starting from infancy. Also, people with PCD experience nasal congestion and chronic cough throughout the year. Chronic respiratory infections cause a condition called bronchiectasis, which damages the passages called bronchi and can lead to life-threatening respiratory problems. Some individuals with PCD also have infertility, recurrent ear infections, and abnormal positions of the chest and abdominal organs. Among several genes that have been confirmed to be directly involved in PCD pathogenesis, a significant number of mutations are found in two genes: DNAI1 and DNAH5, which encode the intermediate and heavy chains of axonemal dynein, respectively.

[0239] mRNA encoding the codon-optimized DNAI1 coding sequence has been developed as a novel therapeutic agent for treating PCD.

[0240] Codon optimization was performed using the native DNAI1 amino acid sequence according to the method of the present invention, as shown in Example 1, to generate three sequences named DNAI1#1 (SEQ ID NO: 29), DNAI1#2 (SEQ ID NO: 30), and DNAI1#3 (SEQ ID NO: 31). The 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 motif screen filters, guanine-cytosine (GC) content analysis filters, and codon adaptation index (CAI) analysis filters. The codon-optimized nucleotide sequences generated according to the method of the present invention had CAI values ​​of 0.8 or higher, as shown in Table 3.

[0241] [Table 3]

[0242] To test the protein yield from each codon-optimized sequence, four nucleic acid vectors were prepared, each containing an expression cassette preceded by an RNA polymerase promoter, with one of the four nucleotide sequences encoding the DNAI1 protein adjacent to the same 5' and 3' UTR. These nucleic acid vectors served as templates for in vitro transcription reactions to provide four batches of mRNA containing the four codon-optimized nucleotide sequences (DNAI1#1-#4). Capping and tailing were performed separately.

[0243] 2 μg each of capped and tailed mRNA was transfected into 10 5 Individual HEK293T cells were used to transfect. Untransfected HEK293T cells were also included to provide a negative control. Cell lysates were collected 24 hours after transfection, protein samples were extracted, and SDS-PAGE was performed. The cells were processed for this purpose. Two samples from each batch of cells were processed and analyzed. The expression level of the encoded DNAI1 protein was assessed by Western blotting using an anti-DNAI1 primary antibody (αDNAI1). The expression level of vinculin was also measured using an anti-vinculin primary antibody (αvinculin) and used as a loading control. The signals were developed and quantified using the LI-COR imaging system, and the DNAI1 protein yield normalized to vinculin was graphed in Figure 15B as a multiple increase relative to the reference level achieved with mRNA encoding a non-codon-optimized DNAI1 sequence. The results of this experiment are summarized in Figure 15. The codon-optimized nucleotide sequence DNAI1#1, with the highest CAI (0.90), produced the highest level of DNAI1 protein compared to the reference (DNAI1#4). Codon-optimized sequences DNAI1#2 and DNAI1#3 both had a CAI of 0.87 and produced comparable levels of DNAI1 protein despite the differences in nucleotide sequences, demonstrating that CAI is closely related to protein expression levels. The codon-optimized sequence DNAI1#4, with a CAI of 0.83, produced the lowest amount of protein compared to optimized nucleotide sequences with higher CAIs, but still showed a significant increase compared to the reference level.

[0244] Combined, these data indicate that for mRNA containing the codon-optimized nucleotide sequence of the present invention, higher CAI strongly correlates with protein expression yield, and that different codon-optimized nucleotide sequences with similar CAI values ​​produce similar levels of encoded protein in the cell.

[0245] Numbered Embodiments of the Invention 1. A computer implementation method for generating an optimized nucleotide sequence, (i) A step of receiving an amino acid sequence that codes for a peptide, polypeptide, or protein; (ii) A step of receiving a first codon usage table, the first codon usage table comprising a list of amino acids, each amino acid in the table being associated with at least one codon, and each codon being associated with a frequency of use; (iii) The step of removing any codons associated with usage frequency that are below a threshold frequency from the codon usage table; (iv) A step of generating a normalized codon usage table by normalizing the usage frequency of codons that were not removed in step (iii); and (v) The process of generating 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 an amino acid in the normalized codon usage table. A method that includes this. 2. The normalization process is: (a) A step of distributing the usage frequency 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) For each amino acid, the process of creating a normalized codon usage table is repeated by repeating step (a). The method according to Embodiment 1, including the method described above. 3. The method according to Embodiment 2, wherein the usage frequency of the removed codons is equally distributed among the remaining codons. 4. The method according to Embodiment 2, wherein the usage frequency of the removed codons is distributed proportionally among the remaining codons based on the usage frequency of each remaining codon. 5. The process of selecting codons for each amino acid is: (a) The step of identifying one or more codons associated with the first amino acid of an amino acid sequence in a normalized codon usage table; (b) A step of selecting a codon associated with a first amino acid, wherein a particular codon The probability of selection is equal to the frequency of use associated with the codon associated with the first amino acid in the normalized codon usage table; and (c) Repeat steps (a) and (b) until a codon is selected for each amino acid in the amino acid sequence. The method according to any one of Embodiments 1 to 4, comprising 6. The method according to any one of Embodiments 1 to 5, wherein step (v) is performed multiple times to generate a list of optimized nucleotide sequences. 7. The method according to any one of Embodiments 1 to 6, wherein the threshold frequency is selectable by a user. 8. The method according to any one of Embodiments 1 to 7, wherein the threshold frequency is in the range of 5% to 30%, particularly 5%, 10%, or 15%, or 20%, or 25%, or 30%, or particularly 10%. 9. The method according to any one of Embodiments 6 to 8, further comprising: determining whether each optimized nucleotide sequence in the list of optimized nucleotide sequences or the most recently updated list 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 when the nucleotide sequence contains one or more termination signals. The method according to any one of Embodiments 6 to 8, further comprising 10. One or more termination signals are the following nucleotide sequences: 5’-X1ATCTX2TX3-3’ [wherein, in the sequence, X1, X2, and X3 are independently selected from A, C, T, or G], the method according to Embodiment 9. 11. One or more termination signals are the following nucleotide sequences: TATCTGTT; and / or TTTTTT; and / or AAGCTT; and / or GAAGAGC; and / or TCTAGA The method according to Embodiment 10, having one or more of the above. 12. One or more termination signals are the following nucleotide sequences: 5’-X1AUCUX2UX3-3’ The method according to Embodiment 9, wherein [in the array, X1, X2, and X3 are independently selected from A, C, U, or G]. 13. One or more termination signals are the following nucleotide sequences: UAUCUGUU; and / or UUUUUU; and / or AAGCUU; and / or GAAGAGC; and / or UCUAGA The method according to Embodiment 12, having one of the following. 14. The step of determining the guanine-cytosine content of each optimized nucleotide sequence in the list of optimized nucleotide sequences, or in the recently updated list, which is the percentage of bases in the nucleotide sequence that are guanine or cytosine; The process of updating the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or the recently updated list if the guanine-cytosine content falls outside a predetermined guanine-cytosine content range. The method according to any one of embodiments 6 to 13, further comprising: 15. The step of determining the guanine-cytosine content of each nucleotide sequence is performed for each nucleotide sequence, A process for determining the guanine-cytosine content of the first portion of a nucleotide sequence. The step of updating the list of optimized nucleotide sequences, which includes this step, If the guanine-cytosine content of the first portion falls outside a predetermined guanine-cytosine content range, a step is taken to remove the nucleotide sequence. The method according to Embodiment 14, including the method described in Embodiment 14. 16. The step of determining the guanine-cytosine content of each nucleotide sequence is performed for each nucleotide sequence. A process for determining the guanine-cytosine content of one or more additional portions of a nucleotide sequence. The process of updating the list of optimized sequences, further including the following, where the further parts do not overlap with each other and do not overlap with the first part, is as follows: A step of removing a nucleotide sequence if the guanine-cytosine content of any portion falls outside a predetermined guanine-cytosine content range. The method according to Embodiment 15, wherein the step of determining the guanine-cytosine content of a nucleotide sequence is stopped if it is determined that the guanine-cytosine content of 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 further portions of the nucleotide sequence comprises a predetermined number of nucleotides, which may be 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. 18. The method according to Embodiment 17, wherein a predetermined guanine-cytosine content range is selectable by the user. 19. The method according to Embodiment 17 or 18, wherein the specified guanine-cytosine content range is 15% to 75%, or 40% to 60%, or in particular 30% to 70%. 20. The process of determining a codon adaptation index for each optimized nucleotide sequence in the list of optimized nucleotide sequences, or in the most recently updated list, which is a measure of codon use bias and may have a value between 0 and 1; The process of updating the list of optimized nucleotide sequences, or the recently updated list, by removing any nucleotide sequence if the codon adaptation index is below a predetermined codon adaptation index threshold. The method according to any one of embodiments 6 to 19, further comprising: 21. The method according to Embodiment 20, wherein the codon adaptation index threshold is selectable 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 one of Embodiments 1 to 22, wherein the amino acid sequence is obtained from an amino acid sequence database. 24. The method according to Embodiment 23, further comprising the step of requesting an amino acid sequence from an amino acid sequence database, wherein the amino acid sequence is received in response to the request. 25. The first codon usage table is obtained from a codon usage table database, as described in any one of Embodiments 1 to 24. 26. The method of Embodiment 24, further comprising the step of requesting a first codon usage table from a codon usage table database, wherein the first codon usage table is received in response to the request. 27. The method according to any one of embodiments 1 to 26, further comprising the step of displaying at least one optimized nucleotide sequence on a screen. 28. A computer program that, when the program is executed by a computer, includes instructions that cause the computer to perform the method described in any one of Embodiments 1 to 27. 29. A data processing system comprising means for performing the method described in any one of Embodiments 1 to 27. 30. A computer-readable data storage medium storing the computer program described in Embodiment 28. 31. A data transport signal for transporting the computer program described in Embodiment 28. 32. A method for synthesizing a nucleotide sequence, A step of generating at least one optimized nucleotide sequence by performing the computer implementation method described in any one of Embodiments 1 to 27; and The process of synthesizing at least one of the generated optimized nucleotide sequences. A method that includes this. 33. The method according to Embodiment 32, further comprising the step of 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, further comprising the step of inserting one or more termination signals at the 3' end of a synthetically optimized nucleotide sequence. 35. One or more termination signals are the following nucleotide sequences: 5'-X1ATCTX2TX3-3' The method according to Embodiment 34, coded by [in the array, X1, X2, and X3 are independently selected from A, C, T, or G]. 36. One or more termination signals are the following nucleotide sequences: TATCTGTT; TTTTTT; AAGCTT; GAAGAGC; and / or TCTAGA The method according to embodiment 34 or 35, as coded by one or more of the above. 37. The method according to any one of embodiments 34 to 36, wherein more than one termination signal is inserted, and the termination signals are separated by 10 base pairs or less, for example, 5 to 10 base pairs. 38. Termination signals exceeding 1 include the following nucleotide sequence: (a) 5'-X1ATCTX2TX3-(Z N )-X4ATCTX5TX6-3', or (b)5'-X1ATCTX2TX3-(Z N )-X4ATCTX5TX6-(Z M )-X7ATCTX8TX9-3'[In array, X1, X2, X3, X4, 5、 X6, X7, X8, and X9 are independently selected from A, C, T, or G, and Z N This represents the spacer sequence of the N nucleotide, Z M The method according to Embodiment 36, wherein each of them independently represents a spacer sequence of M nucleotides, selected from A, C, T, or G, and N and / or M are independently 10 or less. 39. The method according to any one of embodiments 33 to 38, wherein the nucleic acid vector comprises an RNA polymerase promoter operably linked to an optimized nucleotide sequence, and optionally the RNA polymerase promoter is an SP6 RNA polymerase promoter or a T7 RNA polymerase promoter. 40. The method according to any one of embodiments 33 to 39, wherein the nucleic acid vector is a plasmid. 41. The method according to embodiment 40, wherein the plasmid is linearized before in vitro transcription. 42. The method according to Embodiment 40, wherein the plasmid is not linearized before in vitro transcription. 43. The plasmid is superhelical, as described in Embodiment 42. 44. The method according to any one of embodiments 32 to 43, further comprising the step of synthesizing mRNA by using at least one of the synthetically optimized nucleotide sequences in in vitro transcription. 45. mRNA is synthesized by SP6 RNA polymerase, as described in Embodiment 44. Method of loading. 46. ​​The method according to embodiment 45, wherein the SP6 RNA polymerase is a naturally occurring SP6 RNA polymerase. 47. The method according to embodiment 45, wherein the SP6 RNA polymerase is recombinant SP6 RNA polymerase. 48. The method according to embodiment 47, wherein SP6 RNA polymerase includes a tag. 49. The method according to Embodiment 48, wherein the tag is the His tag. 50. mRNA is synthesized by T7 RNA polymerase, as described in Embodiment 44. 51. The method according to any one of embodiments 44 to 50, further comprising a separate step of capping and / or tailing the synthesized mRNA. 52. The method according to any one of Embodiments 44-50, wherein capping and tailing occur during in vitro transfer. 53. The method according to any one of Embodiments 44 to 52, wherein mRNA is synthesized in a reaction mixture comprising NTPs in concentrations ranging from 1 to 10 mM, a DNA template in concentrations ranging from 0.01 to 0.5 mg / ml, and SP6 RNA polymerase in concentrations ranging from 0.01 to 0.1 mg / ml. 54. The method according to Embodiment 53, wherein the reaction mixture comprises NTPs at a concentration of 5 mM each, a DNA template at a concentration of 0.1 mg / ml, and SP6 RNA polymerase at a concentration of 0.05 mg / ml. 55. The mRNA is synthesized at a temperature in the range of 37 to 56°C, according to any one of Embodiments 44 to 54. 56. The method according to any one of embodiments 53 to 55, wherein the NTP is a naturally occurring NTP. 57. The method according to any one of embodiments 53 to 55, including a modified NTP. 58. The method according to any one of Embodiments 32 to 57, further comprising the step of transfecting cells with a synthetically optimized nucleotide sequence in vitro or in vivo. 59. The method according to Embodiment 58, wherein the expression level of the protein encoded by the synthetically optimized nucleotide sequence in the transfected cell is determined. 60. The method according to Embodiment 58 or 59, wherein the functional activity of a protein encoded by a synthetically optimized nucleotide sequence is determined. 61. The method according to any one of Embodiments 1 to 27, further comprising the steps of 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 to 60, and contacting the reference nucleotide sequence and at least one optimized nucleotide sequence with a separate cell or organism, wherein the cell or organism contacted with at least one synthetic optimized nucleotide sequence produces a protein encoded by the optimized nucleotide sequence in a yield increased compared to the yield of the protein encoded by the reference nucleotide sequence produced by the cell or organism contacted with the synthetic reference nucleotide sequence. 62. The method according to any one of Embodiments 32 to 60, further comprising the step of preparing a therapeutic composition comprising mRNA encoding a therapeutic peptide, polypeptide, or protein for use in delivery to or treatment of a subject. 63. The method according to Embodiment 62, wherein mRNA encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein. 64. The method according to any one of Embodiments 1 to 27, wherein 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 according to any one of Embodiments 61 to 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 produced by a method other than the method described in any one of Embodiments 1 to 27. 66. A synthetically optimized nucleotide sequence produced according to the method described in any one of embodiments 32-57 and 62-65 for use in therapeutic applications. 67. A treatment method comprising the step of administering a synthetically optimized nucleotide sequence, generated according to any one of embodiments 32-57 and 62-65, to a human subject requiring such treatment. An in vitro synthetic nucleic acid comprising an optimized nucleotide sequence consisting of codons associated with a usage frequency of 68.10% or more, wherein the optimized nucleotide sequence is (i) The following nucleotide sequence: 5'-X1AUCUX2UX3-3'[in the sequence, X1, X2, and X3 are independently selected from A, C, U, or G]; and not containing a termination signal having one of 5'-X1AUCUX2UX3-3'[in the sequence, X1, X2, and X3 are independently selected from A, C, U, or G]; (ii) Not containing negative cis-adjusting elements and negative repeating elements; (iii) Having a codon adaptation index greater than 0.8; When divided into non-overlapping 30-nucleotide segments, each segment of the optimized nucleotide sequence has a guanine-cytosine content ranging from 30% to 70%. In vitro synthesized nucleic acids. 69. The in vitro synthetic nucleic acid according to Embodiment 68, wherein the optimized nucleotide sequence does not contain 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 according to embodiment 68 or 69, wherein the nucleic acid is mRNA. 71. In vitro synthetic nucleic acid according to any one of embodiments 68 to 70 for therapeutic use.

Claims

1. A computer implementation method for generating an optimized nucleotide sequence, (i) A step of receiving an amino acid sequence that encodes a peptide, polypeptide, or protein; (ii) A step of receiving a first codon usage table, the first codon usage table comprising a list of amino acids, each amino acid in the table being associated with at least one codon, and each codon being associated with a usage frequency; (iii) The step of removing any codons associated with usage frequency that are below a threshold frequency from the codon usage table; (iv) A step of generating a normalized codon usage table by normalizing the usage frequency of codons that were not removed in step (iii); and (v) The process of generating 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 an amino acid in the normalized codon usage table. A method that includes this.

2. The normalization process is, (a) a step of distributing the usage frequency 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) For each amino acid, the process of creating a normalized codon usage table is repeated by repeating step (a). The method according to claim 1, including the method described in claim 1.

3. The method according to claim 2, wherein the usage frequency of the removed codons is equally distributed among the remaining codons.

4. The method according to claim 2, wherein the usage frequency of the removed codons is distributed proportionally among the remaining codons based on the usage frequency of each remaining codon.

5. The process of selecting codons for each amino acid is, (a) The step of identifying one or more codons associated with the first amino acid of an amino acid sequence in a normalized codon usage table; (b) A step of selecting a codon associated with a first amino acid, wherein the probability of selecting a particular codon is equal to the frequency of use associated with the codon associated with the first amino acid in the normalized codon usage table; and (c) Repeat steps (a) and (b) until a codon is selected for each amino acid in the amino acid sequence. The method according to any one of claims 1 to 4, including the method described in any one of claims 1 to 4.

6. The method according to any one of claims 1 to 5, wherein step (v) is performed multiple times to generate a list of optimized nucleotide sequences.

7. The method according to any one of claims 1 to 6, wherein the threshold frequency is selectable by the user.

8. The method according to any one of claims 1 to 7, wherein the threshold frequency is in the range of 5% to 30%, particularly 5%, 10%, or 15%, or 20%, or 25%, or 30%, or particularly 10%.

9. A process of screening a list of optimized nucleotide sequences to identify and remove those that do not meet one or more criteria. The method according to any one of claims 6 to 8, further comprising:

10. The process of screening the list of optimized nucleotide sequences involves, for each of one or more criteria, The process of determining whether each optimized nucleotide sequence in the list of optimized nucleotide sequences, or in the most recently updated list, meets the criteria; If a nucleotide sequence does not meet the criteria, the process of updating the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or from the recently updated list. The method according to claim 9, including the method described in claim 9.

11. The process of determining whether each optimized nucleotide sequence in the list of optimized nucleotide sequences, or in the recently updated list, meets the criteria is as follows for each nucleotide sequence: The first part of the nucleotide sequence is the process of determining whether it meets the criteria. The step of updating the list of optimized nucleotide sequences, which includes the following steps, If the first part does not meet the criteria, the step of removing the nucleotide sequence. The method according to claim 10, including the method described in claim 10.

12. The process of determining whether each optimized nucleotide sequence in the list of optimized nucleotide sequences, or in the recently updated list, meets the criteria is as follows for each nucleotide sequence: The process of determining whether one or more additional parts of a nucleotide sequence that do not overlap with each other and do not overlap with the first part meet the criteria. The process of updating the list of optimized sequences, which further includes, A process to remove nucleotide sequences if any part does not meet the criteria. The method according to claim 11, wherein the step of determining whether the optimized nucleotide sequence meets the criteria is terminated if, in some cases, any part is determined not to meet the criteria.

13. The method according to claim 11 or 12, wherein a first portion and / or one or more further portions of a nucleotide sequence comprises a predetermined number of nucleotides, which optionally range from 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.

14. The first criterion is that the nucleotide sequence does not contain a termination signal, and as a result, the steps for determining and updating are as follows: The steps include determining whether each optimized nucleotide sequence in the list of optimized nucleotide sequences, or in the recently updated list, contains a termination signal; and A step to update the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or the recently updated list if the nucleotide sequence contains one or more termination signals. The method according to any one of claims 9 to 13, including the method described in any one of claims 9 to 13.

15. One or more termination signals are the following nucleotide sequences: 5’-X １ ATCTX ２ TX ３ -3’ [In the array, X １ , X ２ , and X ３ [These are independently selected from A, C, T, or G.] The method according to claim 14, having the following characteristics.

16. One or more termination signals are the following nucleotide sequences: TATCTGTT; and / or TTTTTT; and / or AAGCTT; and / or GAAGAGC; and / or TCTAGA The method according to claim 15, comprising one or more of the above.

17. One or more termination signals are the following nucleotide sequences: 5’-X １ AUCUX ２ UX ３ -3’ [In the array, X １ , X ２ , and X ３ The method according to claim 16, wherein [ is independently selected from A, C, U, or G].

18. One or more termination signals are the following nucleotide sequences: UAUCUGUU; and / or UUUUUU; and / or AAGCUU; and / or GAAGAGC; and / or UCUAGA The method according to claim 17, comprising one of the above.

19. The second criterion includes a nucleotide sequence having a guanine-cytosine content within a predetermined guanine-cytosine content range, and as a result, the steps of determining and updating are as follows: A step of determining the guanine-cytosine content of each optimized nucleotide sequence in a list of optimized nucleotide sequences, or in a recently updated list, wherein the guanine-cytosine content is the percentage of bases in the nucleotide sequence that are guanine or cytosine; The process of updating the list of optimized nucleotide sequences by removing any nucleotide sequence from the list or the recently updated list if the guanine-cytosine content falls outside a predetermined guanine-cytosine content range. The method according to any one of claims 9 to 18, including the method described in any one of claims 9 to 18.

20. The method according to claim 19, wherein a predetermined guanine-cytosine content range is selectable by the user.

21. The method according to claim 19 or 20, wherein the predetermined guanine-cytosine content range is 15% to 75%, or 40% to 60%, or in particular 30% to 70%.

22. The third criterion is that the codon adaptation index includes a nucleotide sequence in which the codon adaptation index exceeds a predetermined codon adaptation index threshold, and as a result, the steps for determining and updating are as follows: A step of determining a codon adaptation index for each optimized nucleotide sequence in a list of optimized nucleotide sequences, or in a recently updated list, wherein the codon adaptation index of the sequence is a measure of codon use bias and may have a value between 0 and 1; The process of updating the list of optimized nucleotide sequences, or the recently updated list, by removing any nucleotide sequence if the codon adaptation index is below a predetermined codon adaptation index threshold. The method according to any one of claims 9 to 21, including the method described in any one of claims 9 to 21.

23. The method according to claim 22, wherein the codon adaptation index threshold is selectable by the user.

24. The method according to claim 22 or 23, wherein the codon adaptation index threshold is 0.7, or 0.75, or 0.85, or 0.9, or in particular 0.

8.

25. The fourth criterion is that the nucleotide sequence must contain at least two, for example, three, adjacent identical codons, and as a result, the steps of determining and updating are as follows: The steps include determining whether any optimized nucleotide sequence in the list of optimized nucleotide sequences, or in the recently updated list, contains at least two, for example, three or more adjacent identical codons; and The process of updating the list of optimized nucleotide sequences, or the recently updated list, by removing any nucleotide sequence that contains at least two, for example, three or more adjacent identical codons. The method according to any one of claims 9 to 24, including the method described in any one of claims 9 to 24.

26. The method according to claim 25, wherein the fourth criterion applies only to codons whose frequency in the normalized codon usage table is less than the adjacent scarcity threshold, and the adjacent scarcity threshold is between 10 and 50%, for example between 15 and 40%, for example between 20 and 30%.

27. The method according to any one of claims 1 to 26, wherein the amino acid sequence is obtained from an amino acid sequence database.

28. The method according to claim 26, further comprising the step of requesting an amino acid sequence from an amino acid sequence database, wherein the amino acid sequence is received in response to the request.

29. The method according to any one of claims 1 to 28, wherein the first codon usage table is received from a database of codon usage tables.

30. The method according to claim 29, further comprising the step of requesting a first codon usage table from a database of codon usage tables, wherein the first codon usage table is received in response to the request.

31. The method according to any one of claims 1 to 30, further comprising the step of displaying at least one optimized nucleotide sequence on a screen.

32. A computer program that, when the program is executed by a computer, includes an instruction that causes the computer to perform the method described in any one of claims 1 to 31.

33. A data processing system comprising means for performing the method described in any one of claims 1 to 31.

34. A computer-readable data storage medium storing the computer program described in claim 32.

35. A data transport signal for transporting the computer program described in claim 32.

36. A method for synthesizing nucleotide sequences, A step of generating at least one optimized nucleotide sequence by performing the computer implementation method according to any one of claims 1 to 31; and The process of synthesizing at least one of the generated optimized nucleotide sequences. A method that includes this.

37. The method according to claim 36, further comprising the step of inserting the synthesized optimized sequence into a nucleic acid vector for use in in vitro transcription.

38. The method according to claim 36 or 37, further comprising the step of inserting one or more termination signals at the 3' end of a synthetically optimized nucleotide sequence.

39. One or more termination signals are the following nucleotide sequences: 5’-X １ ATCTX ２ TX ３ -3’ [In the array, X １ , X ２ , and X ３ The method according to claim 38, wherein is independently coded by [A, C, T, or G].

40. One or more termination signals are the following nucleotide sequences: TATCTGTT; TTTTTT; AAGCT; GAAGAGC; and / or TCTAGA The method according to claim 38 or 39, as coded by one or more of the above.

41. The method according to any one of claims 38 to 40, wherein one or more termination signals are inserted, and the termination signals are separated by 10 base pairs or less, for example, 5 to 10 base pairs.

42. More than one termination signal is the following nucleotide sequence: (a) 5'-X １ ACTTX ２ TX ３ - (Z Ｎ )-X ４ ACTTX ５ TX ６ -3', or (b) 5'-X １ ACTTX ２ TX ３ - (Z Ｎ )-X ４ ACTTX ５ TX ６ - (Z Ｍ )-X ７ ACTTX ８ TX ９ -3'[In the array, X １ , X ２ , X ３ , X ４ , X ５、 X ６ , X ７ , X ８ , and X ９ The elements are independently selected from A, C, T, or G, and Z Ｎ This represents the spacer sequence of the N nucleotide, Z Ｍ The method according to claim 40, wherein each of them independently represents a spacer sequence of M nucleotides selected from A, C, T, or G, and N and / or M are independently less than 10.

43. The method according to any one of claims 37 to 42, wherein the nucleic acid vector comprises an RNA polymerase promoter operably linked to an optimized nucleotide sequence, and optionally the RNA polymerase promoter is an SP6 RNA polymerase promoter or a T7 RNA polymerase promoter.

44. The method according to any one of claims 37 to 43, wherein the nucleic acid vector comprises a nucleotide sequence encoding a 5'UTR operably ligated to an optimized nucleotide sequence.

45. The method according to claim 44, wherein the 5'UTR is different from the 5'UTR of a naturally occurring mRNA encoding an amino acid sequence.

46. The method according to claim 42, wherein the 5'UTR has the nucleotide sequence of SEQ ID NO:

16.

47. The method according to any one of claims 37 to 46, wherein the nucleic acid vector comprises a nucleotide sequence encoding a 3'UTR operably ligated to an optimized nucleotide sequence.

48. The method according to claim 46, wherein the 3'UTR is different from the 3'UTR of spontaneously occurring mRNA encoding an amino acid sequence.

49. The method according to claim 48, wherein the 3'UTR has the nucleotide sequence of SEQ ID NO: 17 or SEQ ID NO:

18.

50. The method according to any one of claims 37 to 49, wherein the nucleic acid vector is a plasmid.

51. The method according to claim 50, wherein the plasmid is linearized before in vitro transcription.

52. The method according to claim 50, wherein the plasmid is not linearized before in vitro transcription.

53. The method according to claim 52, wherein the plasmid is superhelical.

54. The method according to any one of claims 36 to 53, further comprising the step of synthesizing mRNA by using at least one of the synthetically optimized nucleotide sequences in in vitro transcription.

55. The method according to claim 54, wherein the mRNA is synthesized by SP6 RNA polymerase.

56. The method according to claim 55, wherein the SP6 RNA polymerase is a naturally occurring SP6 RNA polymerase.

57. The method according to claim 55, wherein the SP6 RNA polymerase is recombinant SP6 RNA polymerase.

58. The method according to claim 57, wherein SP6 RNA polymerase includes a tag.

59. The method according to claim 58, wherein the tag is a His tag.

60. The method according to claim 54, wherein the mRNA is synthesized by T7 RNA polymerase.

61. The method according to any one of claims 54 to 60, further comprising a separate step of capping and / or tailing the synthesized mRNA.

62. The method according to any one of claims 54 to 60, wherein capping and tailing occur during in vitro transfer.

63. The method according to any one of claims 54 to 62, wherein mRNA is synthesized in a reaction mixture comprising NTPs in concentrations ranging from 1 to 10 mM for each NTP, a DNA template in concentrations ranging from 0.01 to 0.5 mg / ml, and SP6 RNA polymerase in concentrations ranging from 0.01 to 0.1 mg / ml.

64. The method according to claim 63, wherein the reaction mixture comprises NTPs at a concentration of 5 mM of each NTP, a DNA template at a concentration of 0.1 mg / ml, and SP6 RNA polymerase at a concentration of 0.05 mg / ml.

65. The mRNA is synthesized at a temperature in the range of 37 to 56°C, any one of claims 54 to 64. The method described in section [section number].

66. The method according to any one of claims 63 to 65, wherein the NTP is a naturally occurring NTP.

67. The method according to any one of claims 63 to 65, wherein the NTP includes a modified NTP.

68. The method according to any one of claims 36 to 67, further comprising the step of transfecting cells with a synthetically optimized nucleotide sequence in vitro or in vivo.

69. The method according to claim 68, wherein the expression level of a protein encoded by a synthetically optimized nucleotide sequence in a transfected cell is determined.

70. The method according to claim 68 or 69, wherein the functional activity of the protein encoded by the synthetically optimized nucleotide sequence is determined.

71. The method according to any one of claims 1 to 31, further comprising the steps of 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 claims 36 to 70, and further the steps of contacting the reference nucleotide sequence and at least one optimized nucleotide sequence with a separate cell or organism, wherein the cell or organism contacted with at least one synthetic optimized nucleotide sequence produces a protein encoded by the optimized nucleotide sequence in a yield increased compared to the yield of protein encoded by the reference nucleotide sequence produced by the cell or organism contacted with the synthetic reference nucleotide sequence.

72. The method according to any one of claims 36 to 70, further comprising the step of preparing a therapeutic composition comprising mRNA encoding a therapeutic peptide, polypeptide, or protein for delivery to a target or use in treatment of a target.

73. The method according to claim 72, wherein the mRNA encodes a cystic fibrosis transmembrane conductance regulator (CFTR) protein.

74. The method according to any one of claims 1 to 31, wherein 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.

75. The method according to any one of claims 71 to 74, 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 produced by a method other than the method according to any one of claims 1 to 31.

76. A synthetically optimized nucleotide sequence produced according to the method of any one of claims 36-67 and 72-75 for use in therapeutic purposes.

77. A treatment method comprising the step of administering a synthetically optimized nucleotide sequence, generated according to any one of claims 36 to 67 and 72 to 75, to a human subject requiring such treatment.

78. In In vitro synthesized nucleic acids, the optimized nucleotide sequence is (iv) The following nucleotide sequence: 5'-X １ AUCUX ２ UX ３ -3'[In the array, X １ , X ２ , and X ３ [ is independently selected from A, C, U, or G]; and 5'-X １ AUCUX ２ UX ３ -3'[In the array, X １ , X ２ , and X ３ It does not contain a termination signal having one of A, C, U, or G independently; (v) Not containing negative cis adjustment elements and negative repeating elements; (vi) Having a codon adaptation index greater than 0.8; When divided into non-overlapping 30-nucleotide segments, each segment of the optimized nucleotide sequence has a guanine-cytosine content ranging from 30% to 70%. In vitro synthesized nucleic acids.

79. The in vitro synthetic nucleic acid according to claim 78, wherein the optimized nucleotide sequence does not contain a termination signal having one of the following sequences: TATCTGTT;TTTTTT;AAGCTT;GAAGAGC;TCTAGA;UAUCUGUU;UUUUUU;AAGCUU;GAAGAGC;UCUAGA.

80. The in vitro synthetic nucleic acid according to claim 78 or 79, wherein the nucleic acid is mRNA.

81. An in vitro synthetic nucleic acid according to any one of claims 78 to 80 for use in the treatment of a disease.

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